BR102018077523A2 - miniemulsions of bioactive fractions of passionflower, compositions comprising such miniemulsions and formulations - Google Patents

Abstract

The present invention relates to miniemulsions comprising one or more bioactive fractions of Passiflora (passion fruit) selected from hydrophilic, lipophilic obtained by supercritical extraction or concentrated hydrophilic fractions of Passiflora (passion fruit) obtained by pressurized liquid extraction from Passiflora (passion fruit) with proof of the benefits enriched bioactive fractions isolated from the present invention, miniemulsions as well as products comprising such miniemulsions with antioxidant properties that act in the prevention and correction of the different factors and mechanisms responsible for skin aging. The miniemulsion of the present invention can be used in cosmetics, nutraceuticals, nutracosmetics, drugs and food products. Additional objects of the present invention are cosmetic formulations of facial cleansing lotion, facial moisturizing serum and facial moisturizer with sun protection.

Description

PASSIFLORA BIOACTIVE FRACTION MINIEMULSIONS, COMPOSITIONS UNDERSTANDING SUCH MINIEMULSIONS AND FORMULATIONS FIELD OF THE INVENTION

[1] The present invention relates to miniemulsions comprising one or more bioactive fractions of Passionflower (passion fruit) selected from hydrophilic, lipophilic obtained by supercritical extraction or concentrated hydrophilic bioactive fractions of Passionflower (passion fruit) obtained by pressurized liquid extraction with proof of the benefits of the enriched bioactive fractions isolated from the present invention, from miniemulsions as well as products comprising such miniemulsions with antioxidant properties that act in the prevention and correction of the different factors and mechanism responsible for skin aging. The miniemulsion of the present invention can be used in cosmetics, nutraceuticals, nutracosmetics, drugs and food products.

[2] Additional objects of the present invention are cosmetic formulations of facial cleansing lotion, facial moisturizing serum and easy moisturizer with sun protection.

[3] The present invention has application in the field of medicinal or cosmetic preparations, with organic active ingredients.

BACKGROUND OF THE INVENTION

[4] Phytochemicals have in recent years conquered an important share of the cosmetics and pharmaceuticals market. It is estimated that this class of compounds will reach an annual commercial value of US $ 4.63 billion in 2020, with an annual growth rate of 7.2%, between 2015 and 2020. The main factors that condition this market are the problems health as: cardiovascular diseases; cancer; and type two diabetes. At the same time, the aging of the population and increased awareness of health and well-being also contribute to the growth of this market (REUTERS, 2015)

[5] Viganó et al., (2016b) and (2016a) developed a sequential extraction process on a laboratory scale to recover four fractions of bioactive compounds from passion fruit bagasse, a residue from the industrialization of this fruit. The fractions were rich in tocotrienols, polyunsaturated fatty acids, carotenoids and phenolic compounds. The extraction techniques used were extraction with supercritical fluid (SFE) and extraction with pressurized liquid (PLE), both environmentally friendly technologies, as they do not harm the environment and produce extracts free of toxic solvents. The works of these authors observed that the extracts have antioxidant capacity. This property was evaluated by the DPPH, FRAP and ORAC methods. Therefore, such extracts can be explored for the formulation of new products with antioxidant properties.

[6] However, the technological paths to make the extracts available must be developed, in order to meet the inherent needs of the formulations, such as stability and bioavailability. One possible way to get around these limitations is to develop emulsified systems.

[7] An emulsion consists of at least two immiscible liquids, one of which is dispersed in the form of small spherical droplets in the other (MCCLEMENTS, 2011). A miniemulsion is defined as the category of emulsion in which the particles of the dispersed phase have a diameter in the range of 50 nm to 1 μm (SLOMKOWSKI et al., 2 011). Depending on the emulsion droplet diameter range, the system may have the ability to increase the bioavailability of highly lipophilic substances, have a characteristic of high stability in terms of particle aggregation and gravitational separation (MCCLEMENTS, 2011). Emulsion applications have been the subject of many studies in recent decades, such as obtaining carriers (LIVERSIDGE and CUNDY, 1995), inhibiting microbial growth (SPERANZA et al., 2015), inhibiting tumor cells (ZHANG et al ., 2014) and in the formulation of cosmetics (YUKUYAMA et al., 2016).

[8] Specifically for cosmetic applications, it is necessary that the active compounds of the cosmetic formula permeate the skin. The penetration of the active compounds is determined by a number of factors, such as: the molecular size; the degree of ionization; lipophilicity; and the synergy between the base of the formula component and the skin (YUKUYAMA et al., 2016). Considering the structural composition of the skin and its barrier properties, miniemulsions, containing bioactive substances in the oil and water phases, seem to be one of the most suitable ways for topical application of active compounds.

[9] The main problem to be solved arose from the need for an antioxidant solution that works to prevent and correct the different factors and mechanisms responsible for skin aging, such as degradation by metalloproteinases and decreased collagen / elastin production rate, and decreased cell turnover. The antioxidant complex was obtained through the reconstruction and regrouping of different selective extracts in a stable antioxidant complex, in a synergistic way, and that enhance the real and histological activity of cellular anti-aging, due to the presence of compounds with different characteristics and mechanism of action.

[10] The technology differential is the obtaining of an antioxidant complex reconstructed with the natural bioactive substances present in the passion fruit seed, for the prevention and correction of the different factors and mechanism responsible for skin aging.

TECHNICAL STATUS

[11] The search for documents that described all or at least some aspects of the invention showed several results, listed and analyzed below.

[12] US 2016/074312 describes cosmetic, pharmaceutical, dermatological or nutraceutical compositions comprising passion fruit seed extract, in which the extract obtained is formulated to obtain the desired compositions, which can be in the form of oil in water or water in oil.

[13] These seeds were chosen because they comprise polyphenols (such as piceatannol), phytosterols, linoleic acid, oleic acid and tocopherols, in addition to sugars and proteins. In a preferred embodiment, the extract obtained is rich in sugars and proteins

[14] This document describes a slow process for obtaining the complex and which requires several steps. In addition, only the hydrophilic extract containing some markers (sugars and peptides) was evaluated, which are different from the markers of the present invention. The present invention evaluated the benefits of an emulsified, stable and low polydispersity complex, containing active compounds (extracts) in both hydro and lipophilic phases. In addition, application examples for the emulsified complex

[15] US 2016/235794 describes a lipid extract obtained from passion fruit seeds, which can be incorporated into numerous cosmetic products, such as oil-in-water emulsions and water-in-oil emulsions, for anti-aging treatment, among others.

[16] These seeds were chosen because they comprise polyphenols (such as piceatannol), phytosterols, linoleic acid, oleic acid and tocopherols, in addition to sugars and proteins.

[17] In a preferred embodiment, the extract is a passion fruit seed oil concentrated in its non-saponifiable fraction, containing between 3 and 100% by weight of non-saponifiable matter in relation to the total weight of the extract.

[18] US 2008/118449 describes cosmetic formulations comprising piceatannol (naturally occurring polyphenol, present, for example, in passion fruit seeds) and retinol. Despite mentioning one of the markers of the present invention, piceatannol, this document does not use natural extracts nor does it mention the source of the marker.

[19] The active ingredients can be formulated into emulsions for treating the skin, such as skin irritations.

[20] In the article published in Brazilian Journal of Pharmaceutical Science 2017; 53 (1): el6116 the passion fruit seed extract was enriched with other phenolic substances and formulated in makeup with sun protection, such as concealers and bases, in order to inhibit premature aging or photoaging.

[21] The extracts were obtained through extraction under reflux using 40% methanolic water and were directly incorporated into the proposed formulations (that is, miniemulsions were not obtained).

[22] This article does not address the bioactive agents responsible for the benefits to the skin. In addition, it deals with the production and study of the stability of specific commercial formulations, the present invention being developed with the aim of being a complete antioxidant input, covering different metabolic routes and a large scope of antioxidant compounds

[23] The present invention differs from the four documents cited above in that it presents a stable emulsified complex with low polydispersity comprising lipophilic and hydrophilic bioactive fractions of the passion fruit residue, obtained through clean technologies.

[24] It also demonstrates a means of stabilizing an emulsion containing both bioactive fractions of passion fruit, aqueous and lipid, producing a complex with a high capacity for protection and cellular renewal of the skin. In addition, the present invention proposes the deconstruction of the plant matrix through extraction and reconstruction through the emulsification of the extracts, instead of focusing only on the benefits of the isolated extracts.

[25] JP2008162937 describes the composition of a skin-care cosmetic to reduce the harmful effects of retinol on the skin. Such formula includes: ethinol, retinyl esters, retinal, retinoic acid, retinoic acid salts, derivatives or analogues, as well as vitamin A, selected from the group consisting of a mixture with any of these, with a concentration of 0.001% to 5%; and piceatannol, salts, esters, amides, prodrugs and related substances, dermatologically acceptable and related, as well as in combinations with any of these, concentration between 0.0001% and 10%.

[26] JP2016088912 describes obtaining an emulsion of the type Water / Oil / Water (W / O / W) for the protection of the active compound piceatannol from plant extracts, such as extracts of Passion Fruit seeds, Rhodomyrtus Tomentosa or Makiba Burashinoki.

[27] JP2010030911 claims the use of the active compound piceatannol from passion fruit seed in cosmetics and products for oral consumption as a promoter of collagen production, in concentrations of 0.00005 to 5% (mass) of active compound

[28] JP2007223919 invalidates the benefits of using glycosylated piceatannol in skin-care cosmetics, such as the benefits of: antioxidant, anti-inflammatory, whitening agent, tyrosinase inhibitor, cell renewal promoter (human keratinocytes) and anti-aging agent

[29] The article by Matsui et al (2010) addresses the potential of using piceatannol from passion fruit seed as an inhibitor of melanin synthesis and as a promoter of collagen synthesis in human fibroblast cells

[30] The article by Uchida et al (2013) demonstrates that piceatannol, derived from passion fruit seed, regulates the levels of glutathione (GSH) in keratinocytes, suppresses UVB-induced generation of reactive oxygen species (ROS) and reduces MMP-1 induction in keratinocytes pretreated with piceatannol.

[31] The article by Yokozawa et al (2007) demonstrates the benefit of using piceatannol as a melanogenesis inhibiting agent.

[32] The article by Matencio et al (2016) demonstrates the encapsulation of the active compound piceatannol in cyclodextrin matrices.

[33] The article by Zhang et al (2014) demonstrates the encapsulation, stability and increased availability of stilbene in nanoemulsions.

[34] The article by Wang et al (2016) demonstrates the encapsulation of the resveratrol compound, analogous to piceatannol, and of alpha-tocopherol in oil-in-water emulsions.

[35] The present invention has the advantage over the documents mentioned above that it uses a cosmetic ingredient made with natural ingredients, comprising natural active compounds: Carotenoids; Tocois; Fatty Acids and Phenolic Compounds and do not use toxic organic solvents.

[36] The state of the art was limited to obtaining and using the isolated compounds without taking advantage of the synergy of these compounds initially present in the seed of the fruit. In addition, the current invention uses a clean and soft technology to obtain the extracts, in which it applies green solvents and GRAS (Generally Recognized as Safe), does not generate any environmental residue, and preserves the unique characteristics of the extracts obtained.

BRIEF DESCRIPTION OF THE INVENTION

[37] In a first aspect, the present invention provides a miniemulsion comprising bioactive compounds of hydrophilic and hydrophobic fractions selected from Carotenoids; Tocois; Fatty Acids and Phenolic Compounds obtained by supercritical extraction without toxic organic solvent from raw material belonging to the genus Passiflora. An object of the present invention is an oil-in-water emulsion comprising a hydrophobic phase: aqueous phase ratio within the range of 1: 2 to 1: 4, 1% to 25% w / v of an emulsifier and 0.05 % to 2% w / v of a stabilizer. In a preferred embodiment, the emulsion comprises a hydrophobic phase: aqueous phase ratio is 1: 3, the stabilizer is xanthan gum and is a miniemulsion. Specifically, the raw material used in the present invention is Passiflora edulis, the aqueous phase comprises a hydrophilic bioactive fraction with piceatannol and the oil phase comprises a hydrophobic bioactive fraction with compounds belonging to the ring group. It is an additional object of the present invention cosmetic, nutraceutical, nutracosmetics, drugs and food products comprising from 1% to 3% by weight of the emulsion of the present invention, for application on the skin. The following is a brief description of the objects of the present invention.

• a. uma razão fase hidrofóbica:fase aquosa dentro da faixa de 1:2 a 1:4;
• b. de 1% a 25% p/v de um emulsificante; e
• c. de 0,05% a 2% p/v de um estabilizante.

[38] Miniemulsion of bioactive fractions of Passiflora being an oil-in-water emulsion comprising:

• The. a hydrophobic phase: aqueous phase ratio within the range of 1: 2 to 1: 4;
• B. from 1% to 25% w / v of an emulsifier; and
• ç. from 0.05% to 2% w / v of a stabilizer.

[39] Miniemulsion whose raw material belonging to the genus Passiflora is preferably Passiflora edulis.

[40] Miniemulsion whose raw material described above is preferably selected from seed or bagasse.

[41] Miniemulsion described earlier in which the hydrophobic phase: aqueous phase ratio is 1: 3.

[42] Miniemulsion previously described in which the hydrophilic bioactive fraction (aqueous phase) comprises piceatannol.

[43] Miniemulsion previously described in which the lipophilic bioactive fraction (oily phase) comprises compounds belonging to the group of tocols.

[44] Miniemulsion described above in which compounds belonging to the group of tocols comprise α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol, α-tocotrienol, γ-tocotriene, δ-tocotrienol and combinations thereof.

[45] Miniemulsion previously described in which the hydrophilic bioactive fraction is obtained by means of supercritical extraction.

[46] Miniemulsion previously described in which the hydrophilic bioactive fraction is obtained by supercritical extraction with carbon dioxide as a solvent.

[47] Miniemulsion previously described in which the hydrophilic bioactive fraction is obtained by means of supercritical extraction with carbon dioxide as solvent preferably at 60 ° C and 17 MPa (Step 1).

[48] Miniemulsion previously described in which the lipophilic bioactive fraction is obtained by means of supercritical extraction (SFE) with carbon dioxide as a solvent, sequentially, after obtaining the hydrophilic bioactive fraction.

[49] Miniemulsion previously described in which the lipophilic bioactive fraction is obtained by means of supercritical extraction (SFE) with carbon dioxide as a solvent, sequentially, preferably at 40 ° C and 34 MPa (Step 2).

[50] Miniemulsion previously described in which the bioactive fraction obtained by means of supercritical extraction (SFE) is in a single step (global) with carbon dioxide as solvent preferably at 40 ° C and 34 MPa.

[51] Miniemulsion previously described in which concentrated hydrophilic bioactive fraction is obtained by means of extraction with pressurized liquid (PLE) preferably with a solvent mixture selected from ethanol and water, sequentially, after obtaining the hydrophilic and lipophilic bioactive fractions (Step 3 - PLE).

[52] Miniemulsion previously described in which the concentrated hydrophilic bioactive fraction is obtained by extraction with pressurized liquid, preferably at 70 ° C and 10 MPa.

[53] Composition comprising the miniemulsion defined in any of the previous descriptions.

[54] Composition previously described as a cosmetic, nutraceutical, nutracosmetic, pharmaceutical and food composition.

• -1,00% de miniemulsão de frações bioativas de Passiflora;
• - 83,5% de água purificada;
• - 0,05% de Edeta® BD;
• - 1,50% de Carbopol® Aqua SF-1 Polymer;
• - 0,30% de Aculyn 22;
• - 2,52% de Sodium Laureth Sulfate;
• - 0,18% de Sodium Hydroxide;
• - 2,00% de Glucam™ E-20;
• - 3,60% de Disodium Cocoamphodiacetate;
• - 0,50% de Coco-Glucoside;
• - 1,60% de Cocamidopropyl Betaine;
• - 3,00% de PEG-40 Hydrogenated Castor Oil;
• - 0,70% de Neolone™ PE;
• - Citric Acid, qsq para ajustar o pH entre 6,5 e 6,7, para ser para uso como loção de limpeza facial.

[55] Cosmetic formulation comprising:

• -1.00% miniemulsion of bioactive fractions of Passiflora;
• - 83.5% of purified water;
• - 0.05% of Edeta® BD;
• - 1.50% of Carbopol® Aqua SF-1 Polymer;
• - 0.30% Aculyn 22;
• - 2.52% of Sodium Laureth Sulfate;
• - 0.18% Sodium Hydroxide;
• - 2.00% Glucam ™ E-20;
• - 3.60% of Disodium Cocoamphodiacetate;
• - 0.50% Coco-Glucoside;
• - 1.60% of Cocamidopropyl Betaine;
• - 3.00% PEG-40 Hydrogenated Castor Oil;
• - 0.70% Neolone ™ PE;
• - Citric Acid, qsq to adjust the pH between 6.5 and 6.7, to be used as a facial cleansing lotion.

• -3,00% de miniemulsão de frações bioativas de Passiflora;
• - 26,70 de glicerina;
• - 39,17% de água purificada;
• - 0,50% de Trietanolamina;
• - 0,30% de Pemulen TR-1;
• - 29,83% de Dow Corning 7-3101;
• - 0,50% de Neolone™ PE;

para ser para uso como sérum hidratante facial.[56] Cosmetic formulation comprising:

• -3.00% miniemulsion of bioactive fractions of Passiflora;
• - 26.70 of glycerin;
• - 39.17% of purified water;
• - 0.50% Triethanolamine;
• - 0.30% Pemulen TR-1;
• - 29.83% Dow Corning 7-3101;
• - 0.50% Neolone ™ PE;

to be for use as a facial moisturizing serum.

• -3,00% de miniemulsão de frações bioativas de Passiflora;
• - 3,00 de Procetil AWS;
• - 5,00 Glyceryl Stearate;
• - 5,00 Stearic Acid;
• - 2,00 Montanov 82;
• - 1,00 Montanov 202;
• - 1,00 Stearyl Alcohol;
• - 2,50 Zinc Oxide;
• - 15,00 Capric/Caprylic Triglyceride;
• - 0,25 Jaguar HP-105
• - 3,00 Glycerina
• - 58,70 Água Purificada
• - 0,55 Neolone™ PE

para ser para uso como hidratante facial com proteção solar.[57] Cosmetic formulation comprising:

• -3.00% miniemulsion of bioactive fractions of Passiflora;
• - 3.00 Procetil AWS;
• - 5.00 Glyceryl Stearate;
• - 5.00 Stearic Acid;
• - 2.00 Montanov 82;
• - 1.00 Montanov 202;
• - 1.00 Stearyl Alcohol;
• - 2.50 Zinc Oxide;
• - 15.00 Capric / Caprylic Triglyceride;
• - 0.25 Jaguar HP-105
• - 3.00 Glycerina
• - 58.70 Purified Water
• - 0.55 Neolone ™ PE

to be for use as a facial moisturizer with sun protection.

DESCRIPTION OF THE FIGURES

[58] Figure 1 - Flowchart of the laboratory unit with two cells of 5 liters cells (C-1 and C-2). V-1 to V-14: Shut-off valves; VB-A: Automated back-pressure valve; GB-1 to GB-3: Back-pressure valves; VS: Safety / relief valves; B: CO2 pump; TA: Heat exchanger (heating); TR: Heat exchanger (cooling); IC-1 to IC-5: Temperature indicators and controller; I-2: Temperature indicator; I-1, I-2 and I-4 to I-7: Pressure indicators; S-1 to S-3: 1 liter separators; FC: CO2 filter; F: Mass flow meter; Tank: Lung tank for CO2 recycling; Filter: Column pressurized with adsorbent material for CO2 filtration.

[59] Figure 2 - Flowchart of the extraction unit with pressurized fluid (PLE); V-1 to V-3: Shut-off valves; VM: Micrometric valve; B - Liquid pump; BA: Heating bath; R: liquid solvent reservoir; I-1 and I-2: Pressure and temperature indicators, respectively; IC-1: Extraction cell jacket temperature controller;

[60] Figure 3 - Passion kernel extraction kinetics with supercritical CO2 in two distinct stages (Phase 1: 60 ° C and 17 MPa (VIGANÓ et al., 2016b); Phase 2: 40 ° C and 34 MPa). CO2 flow rate: 280 g / min.

[61] Figure 4 - Extraction bed with pressurized liquid from defatted passion fruit bagasse and the diagram of an extraction bed showing the volume of finite element (Az). Where z is the axial position along the extractor, L is the height of the extractor bed, Cf is the concentration of solute (extract) in the solvent, U is the speed of the fluid at the inlet and outlet of the extractor and i is the speed within the bed of porous particles (U / Ԑ).

[62] Figure 5 - Emulsification process and the emulsions obtained for the first phase of the emulsification tests, with Easynov® with emulsifier and the application of ultrasound.

[63] Figure 6 - Emulsification process and the emulsions obtained for the second phase of the emulsification tests, with Montanov L® as an emulsifier and xanthan gum as a stabilizer. Images after 7 days of storage at 20 ° C.

[64] Figure 7 - Effect of the concentration of emulsifier and stabilizer on the average droplet diameter (d [3.2]) and the polydispersity index (PDI) of the emulsions with aqueous bioactive fractions of the bagasse and commercial oil from the passion fruit seed. Horizontal axis: first emulsifier concentration number% (g / ml FL), second stabilizer concentration number (%, g / ml FH).

[65] Figure 8 -Effect of stabilizer concentration (A), with emulsifier concentration fixed at 10%, and the effect of emulsifier concentration (B), with stabilizer concentration fixed at 1%, on the size distribution of droplet.

[66] Figure 9 - Emulsions kinetic stability represented by TSI (Turbiscan Stability Index) values, evaluated during 10 days of storage.

[67] Figure 10 - Emulsions made with aqueous bioactive fraction and commercial seed oil (1U and 1H), bioactive fraction SFE Global (2U and 2H), lipophilic bioactive fraction SFE Phase 2 (3U e3H) and hydrophilic bioactive fraction SFE Phase 1 (4U and 4H) of passion fruit bagasse.

[68] Figure 11 - Microphotographs of the emulsions prepared with global bioactive fraction of SFE (lipophilic phase) and concentrated hydrophilic bioactive fraction (phenolic bioactive fraction) of PLE (hydrophilic phase) obtained by (A) rotor-stator (sample 2U) and ( B) high pressure homogenizer (sample 2H), after 30 days. (C) Fluorescence microscopy for the emulsion obtained according to the condition of the 2U sample, where the lipophilic phase was stained with Nile Red. Scale bar of (A) and (B) 10 μm and (C) 100 μm.

[69] Figure 12 - Synthesis of total collagen in human fibroblasts for the substances (a) aqueous bioactive fraction of passion fruit bagasse Lot 1; (b) bioactive fraction of passion fruit bagasse Lot 2; (c) SFE Phase 1 hydrophilic bioactive fraction of passion fruit bagasse e; Global SFE bioactive fraction of passion fruit bagasse.

[70] Figure 13 - Synthesis of total collagen in human fibroblasts for substances (a) Standard emulsion (Table 13: 5U); (b) Commercial product; (c) Emulsion with aqueous bioactive fraction of passion fruit bagasse Lot 1 (Table 13: 2U) e; (d) Emulsion with aqueous bioactive fraction of passion fruit bagasse Lot 2.

[71] Figure 14 - Elastase reaction kinetics from swine pancreas (EnzChek® Elastase Assay Kit, Molecular Probes) in different concentrations of inhibitors, aqueous bioactive fraction (A) and emulsion (Lot 1) of the bioactive fractions (B) of passion fruit bagasse.

[72] Figure 15 - Dose-response curve for different elastase inhibitors, the concentrated hydrophilic bioactive fraction (aqueous concentrate) (PLE) and the emulsion.

[73] Figure 16 - Reaction kinetics of collagenase from Clostridium histolyticum (EnzChek® Gelatinase / Gollagenase Assay Kit, Molecular Probes) in different concentrations of inhibitors, aqueous bioactive fraction (A) and emulsion (Batch 1) of the bioactive fractions (B ) of the passion fruit bagasse. Concentration in μg of the compound per μl of reaction medium.

[74] Figure 17 - Inhibition of the collagenase enzyme for the hydrophilic bioactive fraction (25 μg / μl), the emulsion of the bioactive fractions of the passion fruit bagasse (Emulsion at 25 μg / μl), standard inhibitor (10 mM inhibitor), and the commercial product (Product 14 μg / μl).

[75] Figure 18 - Colony forming units for the substances: (a) SFE Global bioactive fraction, (b) SFE hydrophilic bioactive fraction Phase 1 of passion fruit bagasse, (c) concentrated hydrophilic (aqueous) bioactive fraction of passion fruit bagasse Lot 1 and (d) concentrated hydrophilic (aqueous) bioactive fraction of the passion fruit bagasse Lot 2, compared to the baseline control and the β-Estradiol control (1 μM). Different letters on the same graph represent a significant difference at the 5% level (p <0.05).

[76] Figure 19 - Colony forming units for the substances: (a) Standard emulsion (Table 13: 5U); (b) Commercial product; (c) Emulsion with concentrated hydrophilic (aqueous) bioactive fraction of passion fruit bagasse Lot 1 (Table 13: 2U) e; Emulsion with concentrated hydrophilic (aqueous) bioactive fraction of passion fruit bagasse Lot 2, compared to baseline control and B-Estradiol control (1 μM). Different letters on the same graph represent a significant difference at the 5% level (p <0.05).

[77] Figure 20 - shows the characteristics of color, odor and appearance during three months of conducting the stability study for the cosmetic formulation of facial cleansing lotion.

[78] Figure 21 - shows the pH values for the cosmetic formulation of easy to clean lotion. The data were obtained in triplicate and the results expressed as mean ± standard deviation.

[79] Figure 22 - presents the characteristics of color, odor and appearance during three months of conducting the stability study for the cosmetic formulation of facial moisturizing serum.

[80] Figure 23 - shows the pH values for the cosmetic formulation of moisturizing serum. The results were expressed as mean ± standard deviation

[81] Figure 24 - shows the Viscosity values (mPas) for the cosmetic formulation of moisturizing serum. The data were obtained in triplicate and the results expressed as mean ± standard deviation.

[82] Figure 25 - presents the characteristics of odor and appearance for the Facial moisturizing cosmetic formulation with Sun Protection during the 3 months of conducting the stability study.

[83] Figure 26 - shows the pH values for the cosmetic formulation of Facial moisturizer with Sun Protection. The results were expressed as mean ± standard deviation.

[84] Figure 27 - shows the viscosity values (mPas) for the cosmetic formulation of Facial moisturizer with Sun Protection. The data were obtained in triplicate and the results expressed as mean ± standard deviation.

DETAILED DESCRIPTION OF THE INVENTION

[85] The present invention relates to miniemulsions and microemulsions comprising one or more bioactive fractions of Passiflora (passion fruit) selected from hydrophilic, lipophilic obtained by supercritical extraction or concentrated hydrophilic bioactive fractions of Passiflora (passion fruit) obtained by pressurized liquid extraction with proof of pressurized liquid extraction. benefits of enriched bioactive fractions isolated from the present invention, miniemulsions as well as products comprising such miniemulsions with antioxidant properties that act in the prevention and correction of the different factors and mechanism responsible for skin aging. The miniemulsion of the present invention can be used in cosmetics, nutraceuticals, nutracosmetics, drugs and food products.

[86] Additional objects of the present invention are cosmetic formulations of facial cleansing lotion, facial moisturizing serum and easy moisturizer with sun protection.

[87] The emulsion of the present invention was evaluated against the ability to inhibit elastase and collagenase, collagen synthesis and cell colony formation.

[88] The bioactive fractions of the dried passion fruit seed used in the miniemulsions were obtained by two extraction steps. The first extractive step consisted of extracting the support compounds present in the seed, such as fatty acids and tocols (vitamin E), which can be obtained by different extraction techniques, preferably by extraction with supercritical fluids. This bioactive fraction obtained was characterized against the fatty acid profile, tocopherol and tocotrienol profile, acidity, total carotenoids and antioxidant activity, and used as the lipophilic (oil) phase of the elaborated miniemulsion. The second extractive stage, the defatted passion fruit seed was subjected to different extraction methods, preferably with pressurized liquids and an ethanol / water mixture as an extractive solvent. This bioactive ethanolic fraction was route-evaporated, characterized by the total phenolic content, amount of piceatannol, acidity, total sugars and antioxidant capacity, and used as the hydrophilic (aqueous) phase of the miniemulsions. Subsequently, it was found that the two bioactive fractions obtained had a high concentration of several bioactive compounds such as tocopherols, tocotrienols, carotenoids and phenolic compounds, especially the compound with high technical value, piceatannol, in addition to the high antioxidant activity and null cellular toxicity

[89] The present invention uses a clean and soft technology to obtain the extracts, in which it applies green solvents and GRAS (Generally Recognized as Safe), does not generate any environmental residues, and preserves the unique characteristics of the products obtained. The bioactive fractions of the oil and water phases obtained in the two-stage extraction process are disclosed in publications in Food Research International 85 (2016) 51-58, The Journal of Supercritical Fluids, Volume 110, 2016, Pages 1-10, and in the order of Brazilian patent BR 10 2016 014976 2

[90] The raw material used in the present invention can be any plant selected from the genus Passiflora. Preferably the plant is Passiflora edulis (passion fruit)

[91] The emulsions of the present invention can be applied as an input or final product, for the benefit of the skin, in different concentrations for cosmetic, nutracosmetic, nutraceutical, pharmacological and food formulations.

[92] The emulsions of the present invention have an average droplet size of 1-4 μm and with extended kinetic stability, which was maintained after the incorporation of emulsions in cosmetic products in concentrations of 1 to 3%

[93] The emulsions of the present invention comprise two necessary components: an emulsifier and a stabilizer. The joint action of these two components allows the emulsion to be obtained with the correct and stable droplet size.

[94] One skilled in the art will be able to recognize an emulsifier available in the state of the art to apply it to the present invention. The same reasoning is valid for the stabilizer. Preferably the stabilizer is a gum. Most preferably the stabilizer is xanthan gum.

EXAMPLES Characterization of the plant / raw material matrix

[95] After receiving the passion fruit bagasse, it was subjected to the drying process in a forced air circulation study (Fanem, 320-SE, São Paulo, Brazil) at 60 ° C, for 36 hours, according to the methodology of Viganó et al., (2016b). Subsequently, the dehydrated passion fruit cake was ground in a knife mill, and stored, protected from light, at -18 ° C until extraction.

[96] Determination of Moisture Content: The moisture content was determined according to the AOAC methodology (1998), method 931.04 (Association of Official Analytical Chemists), through gravimetric measurement in a forced air circulation oven (Fanem, model 320 SE, São Paulo, Brazil) at 105 ° C until constant weight is reached. Approximately five grams of samples, placed in porcelain crucibles, were left in the oven at 105 ° C. After 3 hours the samples were placed in a desiccator until they reached room temperature, being then weighed. This operation was repeated until the samples had a constant weight.

[97] Lipid content: Approximately five grams of dried and ground vegetable matrix were weighed in a filter paper cartridge, the same being inserted in a Soxhlet extractor. The mass ratio between solvent and raw material was 1:30 and the extraction time was six hours. It was used as a hexane solvent (Synth, São Paulo, Brazil). At the end of six hours, the extract was collected and the residual solvent evaporated under vacuum on a rotary evaporator (MA120, Marconi, Piracicaba, Brazil). All experiments were performed in duplicate.

Particle bed characterization

[98] The particle bed was characterized by determining the average particle diameter, apparent density, actual density and porosity of the bed. The methodologies used are described below.

[99] The average particle diameter was determined using the model proposed by ASAE (1998), according to Equation 1

[100] where: dmg is average particle diameter (mm); di is the opening of the i-th sieve (mm); di + 1 is the nominal sieve opening greater than the i-th sieve (mm); wi is mass retained in the i-th sieve; n is the total number of fractions.

[101] The apparent specific mass (pa) of the bed of particles was obtained by the relation between the sample mass used in the extractions by the volume of the bed, thus including only the pores of the bed and not the pores of the interior of the particles.

[102] The real specific mass (pr) was obtained by the pycnometry method with helium gas at Unicamp's Analytical Center. The equipment used was the Quantnrome Ultrapyc model 1200e automatic pycnometer.

[103] The porosity of the extraction bed was determined using the real and apparent specific mass of the samples, including the pores of the bed and the interior of the particles, using Equation (2).

Obtaining bioactive compounds: bioactive fractions

[104] Passiflora's bioactive fractions were obtained as previously described by Viganó et al., (2016b) and by Viganó et al., (2016a). The passion fruit bagasse was subjected to the supercritical extraction process (SFE - Supercritical Fluid Extraction), using carbon dioxide (CO2) as a solvent, in two distinct stages. First, the phase rich in tocopherol and tocotrienol tocols, called Stage / Phase 1, was extracted under conditions of 60 ° C and 17 MPa, as determined by Viganó et al. , (2016b). Then, this vegetable matrix mass was subjected to a second extraction step, in order to remove all lipids from the matrix, under the conditions of 40 ° C and 34 MPa, in which it was called Step / Phase 2. A third fraction bioactive was also obtained through supercritical extraction, however the process took place under a single temperature and pressure condition, 40 ° and 34 MPa, respectively, this bioactive fraction was named Global. The bioactive fractions were obtained using a laboratory unit (Thar Technologies, model SFE-2X5LF-2-FMC, Pittsburgh, USA), according to the schematic diagram shown in Figure 1. All lipophilic bioactive fractions were stored in amber packaging, under refrigeration, and chemically characterized for later formulation of the oily or lipophilic phase, which make up the emulsions.

[105] The supercritical extraction system, used to implement the present invention, without however restricting its scope, consists of two extractors (C-1 and C-2), with a volume of 5 liters each. They are surrounded by heating blankets of 2,000 W, and are intended to operate alternately, thus simulating a continuous process. The system has a CO2 pump with a pumping capacity of 300 g / min. CO2 is cooled by heat exchange (heat exchanger - TR) with an ethylene glycol bath and water (Thermo Electron Corporation, model NESLAB RTE10, Newington, USA) at 271 K, then passes through a heat exchanger to heat the solvent and, later, on a flow meter (Siemens, model Sitrans FC Mass 6000, Munich, Germany). The raw material is packed in a nylon cell of the same dimensions as the extractor and inserted inside it. After passing through the extractor, the solvent / extract system passes through cyclone separators (S-1 to S-3) that are connected in series, which have different operating temperatures and pressures.

[106] The entire pressure, flow and temperature control system is automated, with the exception of the blocking valves and the three separator valves. The automatic valve (VB-A) is responsible for the pressure control of the system, while the pump controls the flow, and a system of thermocouples and pressure gauges monitor the temperatures and pressures of the system, respectively. All of this equipment is connected to a computer that controls the parameters from the values defined by the user. The laboratory unit also has a pressurized column with adsorbent material and a lung tank for the filtration and storage of CO2, these components are part of the solvent recycling system.

[107] Subsequently, the defatted bagasse was subjected to the extraction process with pressurized liquids (PLE - Pressurized Fluid Extraction) to obtain the phenolic compounds, according to Figure 2, which shows the schematic diagram of the PLE unit, used in these experiments. In this step, a mixture of ethanol and water was used with solvent, in concentrations of 75 and 25% (mass / mass), respectively. The pressure and temperature conditions were 10 MPa and 70 ° C, respectively, as determined by Viganó et al., (2016a). The hydroalcoholic extract was then subjected to the rotary evaporation process (MA120, Marconi, Piracicaba, Brazil) for the removal of ethanol, under vacuum at 40 ° C. The concentrated hydrophilic bioactive fraction, in which it was termed as concentrate, was properly stored for future use as an aqueous or hydrophilic phase of the emulsions.

[108] The equipment consists of an HPLC pump (PU-208, Jasco, Easton, USA) that operates in a flow range of 0.01-20 mL / min and a maximum pressure of 50 MPa; a 100 mL stainless steel extraction cell with a metal filter at the cell outlet, where the plant material is placed; a thermostatic bath (BA) (Marconi, model MA126 / BD, São Paulo, Brazil) responsible for preheating the fluid before entering the cell; an electric heating jacket to coat the extraction cell and keep it at the process temperature; a manometer (Zurich, São Paulo, Brazil) to indicate the pressure in the extraction cell; blocking valves (V-1 and V-3) (Autoclave Engineers, Ohio, USA) to control the passage of fluid; temperature indicator and controller (IC-1); a micrometric valve (VM) used mainly for pressure control by regulating the flow of the solvent and a container for collecting the extract

Characterization of bioactive fractions

[109] The knowledge of the concentrations of the target compounds, contained in the extracts, is of fundamental importance for the stage of formulation of the emulsions. As a result, the extracts were subjected to the following analyzes:

[110] Global extraction yield: Fractions enriched with bioactives from SFE and PLE were analyzed for overall yield, that is, the dry extract mass divided by the amount of raw material used for extraction;

[111] Tocol quantification: Quantification was performed by gas chromatography (CGC AGILENT 68650), with capillary chromatographic column DB-23 Agilent (50% cyanopropyl and methylpolysiloxan), and dimensions: length 60m, internal diameter: 0.25mm , and 0.25 μm film. The operational conditions were: column flow of 1.00mL / min, linear speed of 24 cm / s; detector temperature of 280 ° C; injector temperature: 250 ° C; and an oven temperature ramp: 110 ° C for 5 minutes .; 110 to 215 ° C, with heating rate of 5 ° C / min, and 215 ° C for 24 min. Helium with carrier gas, hexane was used to dilute the samples, and a volume of 1.0 μL was injected. The proposed methodology is in accordance with the official A.O.C.S. (2009);

[112] Obtaining the fatty acid profile: it was performed by gas chromatography according to the described method of quantification of tocols and the methodology of A.O.C.S. (2009);

[113] Quantification of total carotenoids: The lipophilic extracts, as well as the commercial oil of the passion fruit seed, were diluted in ethyl ether and the absorbance was measured at a wavelength of 450 nm using a quartz cuvette, in spectrophotometer (Hach, DR / 4000U, Colorado, USA). A standard curve using β-carotene (Sigma-Aldrich St. Louis, USA) was constructed (1 to 9 μg / ml) and the results were expressed in milligrams of total carotenoids per gram of lipophilic extract (VIGANÓ et al., 2016b) .

[114] Acidity index quantification: Approximately 2 g of the sample was weighed in a 125 ml Erlenmeyer flask. 25 ml of neutral ether-alcohol solution (2: 1) (Synth, São Paulo, Brazil) was added and two drops of the phenolphthalein indicator were added to the sample. Titrate with 0.1 M sodium hydroxide solution until the pink color appears. The acidity index was expressed in oleic acid equivalent per gram of lipophilic extract (%). According to the 325 / IV methodology of the Adolfo Lutz Institute (I.A.L., 2005).

[115] Determination of total solids: The determination of total solids was carried out by drying 5 ml of extract, in porcelain crucibles, at 60 ° C in an oven with forced air circulation (Fanem, 320-SE, São Paulo , Brazil). The crucibles were weighed until they reached constant mass. The total solids data were used to determine the overall yield of extractions with pressurized liquids.

[116] Quantification of total phenolic compounds: The content of total phenolic compounds was determined by spectrophotometry using the Folin-Ciocalteau method, according to the methodology proposed by Singleton et al. (1999) with changes. First, the concentration of total solids in the samples was adjusted to 1 mg of solid per ml of solution, with the addition of distilled water. Subsequently, the reaction was carried out and test tubes, where 500 μ1 of extract and 500 μ1 of Folin-Ciocalteau reagent (Dinâmica, SP, Brazil) were mixed. After 3 minutes, 500 μ1 of saturated sodium carbonate solution (Synth, São Paulo, Brazil) and 3.5 ml of distilled water were added, the tubes were shaken for 10 seconds and left for 2 hours in the shade, at room temperature (25 ° C). Finally, the absorbance was measured at a wavelength of 760 nm using a quartz cuvette, using a UV-Vis spectrophotometer (Hach, DR / 4000U, Colorado, USA). The calibration curve was constructed (0.01 to 0.075 mg / ml) with gallic acid (Sigma-Aldrich St. Louis, MO, USA) and the results were expressed in milligrams of gallic acid equivalents (AGE) per gram of matrix dehydrated vegetable (mg AGE / g MP), or per ml of concentrated extract (mg AGE / ml).

[117] Quantification of reducing sugars: The total sugar content in the extracts was determined by the dinitrosalicylic acid method, described by Miller (1959). First, the DNS reagent was prepared by mixing 1.4133 g of DNS with 2.64 g of NaOH, 1.1067 g of sodium metabisulfite and 1.01 ml of melted phenol at 50 ° C, in 200 ml of distilled water. Then, a solution of sodium and potassium tartrate was prepared, diluting 15.1 g of sodium and potassium tartrate tetrahydrate (KNaC4H406 4H20) in 1 liter of distilled water. The concentration of the extracts was adjusted to 1 mg of solids per ml of solution, with the addition of distilled water. A standard glucose curve was prepared, with concentrations ranging from 0.01 to 0.1 mg / ml. Then, the reaction was carried out in test tubes, mixing 1 ml of the extracts, distilled water (white) or the dilutions of the calibration curve, with 1 ml of the DNS reagent. The tubes were shaken for 10 seconds and heated for 5 minutes at 100 ° C in boiling water. After that, the tubes were cooled using an ice bath for 5 minutes. Finally, 16 ml of the tartrate solution was added to each tube and stirred. Finally, absorbance was measured at a wavelength of 540 nm on a UV-Vis spectrophotometer (Hach, DR / 4000U, Colorado, USA). The amount of sugar was expressed in mg of glucose equivalent per ml of concentrated hydrophilic extract.

[118] Determination of titratable acidity: Approximately 10 ml of the concentrated extract (hydrophilic extract) was pipetted in a beaker with 100 ml of distilled water. Subsequently, the solution was titrated with sodium hydroxide at 0.1 M to pH 8.4, measured with the aid of a bench pH meter (Quimis Q400AS, São Paulo, Brazil). The results of titratable acidity were expressed in grams of acetic acid per ml of extract (%), according to the methodology 253 / IV of the Instituto Adolfo Lutz (I.A.L., 2005).

[119] pH determination: The pH of the hydrophilic extract was measured with a bench pH meter (Quimis Q400AS, São Paulo, Brazil), at room temperature (25 ° C).

[120] Piceatannol quantification: Piceatannol quantification was performed on HPLC (Thermo Scientic Ultimate 3000 series) equipped with DAD detector (DAD-3000). The mobile phase used was ultrapure water (Solution A) and acetonitrile (Solution B), both acidified with formic acid (1%) and filtered through a 0.22 μm nylon membrane. The samples were diluted in a solution of 25% water and 75% ethanol. The column used was a Kinetex 2.6μ C18 100 x 3.0 mm (Phenomenex, California, USA).

[121] Cytotoxicity: Cytotoxicity tests were performed according to the procedures described in the BALB / C Cytotoxicity Test by OECD 129 and in NORMA-0004 (Guidance document on using cytotoxicity tests to estimate starting doses for acute oral systematic toxicity tests) . Briefly, Balb / C 3T3 cells were seeded in 96-well plates at the confluence of 3000 cells per well, in a DMEM basal medium containing 10% NBCS, and kept in a CO2 incubator for 24 hours. After the adaptation period, the cells were exposed to 8 concentrations of passion fruit bagasse extracts in DMEM medium with reduction of animal serum (5% NBCS) for 48 hours. One plate was reserved for testing positive control (SDS). The compound Dodecyl Sulfate Sodium (SDS) was used as a positive control in order to guarantee the acceptance parameters of the assay. The IC50 value (μg / ml) was calculated using the four-parameter logistic curve (Hill's curve) obtained from the dose-response curve. This value makes it possible to classify the relative cytotoxicity of the test compound. The IC50 was then used to predict the LD50 (Lethal Dose 50%), corresponding to the dose capable of killing 50% of the individuals in a test population. The extracts obtained in Phase 1 (tocols), in Phase 2 (fatty acids), global yield obtained by extraction with supercritical CO2 (Global), the phenolic extract obtained by extraction with pressurized liquid, and the commercial oil of passion fruit were analyzed .

[122] Analysis of reactive oxygen species -ORAC (Oxygen Radical Absorbance Capacity): Bioactive enriched fractions were also analyzed for antioxidant capacity via ORAC assays. Such tests were performed according to what was reported by Prior et al. (2003) and Dávalos et al. (2004). The samples or standard (Trolox), fluorescein in phosphate buffer and AAPH (2,2-azobis (2-amidinopropane) dihydrochloride), were added in dark microplates. The loss of fluorescence was read under excitation at 485 nm and emission at 520 nm. An analytical curve was obtained using Trolox.

Formulation of emulsions

[123] First, previous studies were carried out with the commercial oil of the passion fruit seed and the phenolic extract obtained via pressurized liquids, to evaluate the type of emulsifier, the process and its conditions, and the use of stabilizer, since the quantity of bioactive fractions obtained by SFE is limited. Subsequently, extracts from extractions with supercritical CO2 were used to compose the oil phase of the emulsion. At the same time, the extract rich in phenolic compounds, from extractions with pressurized fluids, was used to compose the water phase. Water / oil type emulsions were produced in a volumetric ratio of 3: 1, using Easynov (Puteaux, France) and Montanov L® as surfactants. The amount of extract in each of the phases was determined based on the cytotoxicity assay, according to the method described above.

[124] In order to compare the performance of the ingredients of the emulsion, some formulations were performed, as shown in Table 1. Formulation 1 was taken with the first object of study, being formed by commercial seed oil and hydrophilic bioactive fraction of bagasse defatted passion fruit. Formulations 2 and 3 differ in that they contain a fraction of passion fruit lipophilic bioactive obtained in different process conditions, Global phase oil and Phase 2 bioactive fraction. With these treatments it will be possible to verify the effect of the oil phase on emulsions. Formulation 4, which represents the formulation in which the project hypothesis is deposited, was compared with the other formulations. It is composed of a hydrophilic bioactive fraction comprising a phenolic group rich in piceatannol in the water phase and a lipophilic bioactive fraction enriched in tocotrienols in the oil phase, both of which preferably come from the passion fruit bagasse. Formulation 5, in turn, differs from the others in that it contains only water in the hydrophilic phase and oleic acid in the lipophilic phase.
Table 1 - Components of the emulsion phases.

• 1) Uma solução foi preparada pela dispersão do emulsificante em óleo;
• 2) Uma solução foi preparada pela dispersão do estabilizante em água destilada;
• 3) Uma certa quantidade da solução de estabilizante foi dispersa em extrato hidrofílico do bagaço de maracujá;
• 4) A fase aquosa foi adicionada à fase óleo e uma emulsão foi formada com auxilio de um homogeneizador do tipo rotor-estator (Ultra Turrax T18, IKA, Alemanha);
• 5) Posteriormente, parte das emulsões foram submetidas a uma sonda de ultrassom (Grupo Unique, modelo DES500, Campinas, Brasil) ou tratadas em um homogeneizador a alta pressão (Panda 2K NS1001L, Niro Soavi, Itália), e a outra parte não passaram por outros processos de homogeneização.
• 6) As emulsões produzidas foram resfriadas com o auxilio de um becker encamisado acoplado ao um banho de refrigeração, ajustado a 15° C, sob agitação mecânica de 200 rpm durante 10 minutos.
• 7) Por fim, as emulsões foram armazenadas a 20° C em incubadora (BOD) durante 30 dias. Alíquotas foram coletadas durante esse período para a análise de tamanho de gotícula, microscopia óptica, estabilidade cinética, síntese de colágeno, ensaios de formação de colônia celular e capacidade de ativação/inibição das enzimas colagenase e elastase.

[125] For each experiment, about 100 ml of emulsion was produced, according to the following steps:

• 1) A solution was prepared by dispersing the emulsifier in oil;
• 2) A solution was prepared by dispersing the stabilizer in distilled water;
• 3) A certain amount of the stabilizer solution was dispersed in hydrophilic extract from the passion fruit bagasse;
• 4) The aqueous phase was added to the oil phase and an emulsion was formed with the aid of a rotor-stator homogenizer (Ultra Turrax T18, IKA, Germany);
• 5) Subsequently, part of the emulsions were submitted to an ultrasound probe (Unique Group, model DES500, Campinas, Brazil) or treated in a high pressure homogenizer (Panda 2K NS1001L, Niro Soavi, Italy), and the other part did not pass by other homogenization processes.
• 6) The emulsions produced were cooled with the help of a jacketed beaker coupled to a cooling bath, adjusted to 15 ° C, under mechanical stirring at 200 rpm for 10 minutes.
• 7) Finally, the emulsions were stored at 20 ° C in an incubator (BOD) for 30 days. Aliquots were collected during this period for droplet size analysis, optical microscopy, kinetic stability, collagen synthesis, cell colony formation assays and the ability to activate / inhibit collagenase and elastase enzymes.

[126] The tested variables were: emulsifier; amount of emulsifier; amount of stabilizer; ratios between the hydro and lipophilic phases (for an emulsifier), and type of processing, rotor-stator homogenizer, high pressure homogenizer and / or ultrasound.

Characterization of emulsions

[127] Emulsions were characterized in terms of their physical and chemical aspects. For this, the analyzes listed below were performed:

[128] Stability by light scattering (Turbiscan Lab®): The emulsions were subjected to analysis in Turbiscan Lab® to check for possible instability phenomena inherent in the emulsified systems. This equipment allows to determine the occurrence of phenomena such as creaming, sedimentation, coalescence and, also, to evaluate the homogeneity of the samples. The equipment consists of a near infrared light source (880 nm) and two detectors that act in a synchronized manner. In this way, the transmission detector receives information about the light transmitted through the product and the backscattering detector measures the light reflected by the product. For each experiment, approximately 20 ml of emulsion were placed in glass tubes suitable for analysis, later analyzed on day 1, 7, and / or 10 at a temperature of 25 ° C. Emulsion stability was measured using the TSI (Turbiscan Stability Index) parameter, which takes into account all processes that occur in the sample, as described by Kang et al. , (2011).

[129] Average droplet size: the average droplet size, I define it as d [3.2], the polydispersity index (PDI) and the droplet size distribution were determined by laser diffraction spectrometry (Mastersizer 2000, Malvern , UK). The analysis was performed at times 1 and 7 or 10 days.

[130] Electron microscopy: the microstructure of the emulsions was analyzed using an optical microscope (Axio Scope.A1, Carl Zeiss, Germany). The images were captured with the software AxioVision Rel. 4.8 (Carl Zeiss, Germany). The analysis was performed after 30 days of preparing the emulsions.

[131] Analysis of total collagen synthesis: the method used for this study was based on the detection of human collagen by the Sirius Red / Fast Green method (Houghton et al., 1996). This method is a relatively simple way to determine the total amount of collagen and non-collagen proteins in cell cultures. Sirius Red binds to all types of collagen, while Fast Green binds to other non-collagen proteins. After labeling the proteins with Sirius Red / Fast Green dyes, the relative quantification of collagen was performed by spectrophotometry. The doses of the test substance (3 non-toxic concentrations) used in the collagen relative quantification assay were determined from an initial cell viability assay. In the tests, primary cultures of normal human fibroblasts were used, and all experiments were carried out in biological duplicates (3 independent tests). The incubation times for cell viability assays and collagen synthesis were 48 hours and 72 hours, respectively.

[132] Analysis of the ability to produce an anti-aging effect via the elastase enzyme activity assay: elastase inhibition was measured using the EnzCheckOElastase kit (Molecular Probes Inc., USA). Sample aliquots (emulsion and extract) and buffer (control) were added to a 96-well plate. On this plate, aliquots of DQ-elastin substrate and active enzyme were added. The fluorescence intensity was measured in a fluorimeter, under excitation at 485 nm and emission at 515 nm, during predetermined intervals. The increase in fluorescence is proportional to the proteolytic activity. Therefore, the absence of fluorescence will be identified as a potential elastase inhibitor. The reactions were carried out in triplicate. The percentage of inhibition will be calculated as shown in Equation 3, in which Fcontrole and Famostra are the fluorescence values at a given time, discounting the values of the respective blank, control and sample, in different concentrations, respectively.

[133] In order to determine the concentration at which 50% of the elastase is inhibited, the experimental data of inhibition (%) and inhibitor concentration (μg / ml) were adjusted to the Hill model, according to Equation (4)

[134] where: min is the lower plateau of the curve; max is the upper plateau; Hill Slop is the slope of the linear part of the Hill curve; IC50 is the concentration at which 50% of the enzymes are inactivated; and [C] is the concentration of the inhibitor.

[135] Analysis of the ability to produce an anti-aging effect via collagenase enzyme activity assay: collagenase enzyme inhibition was measured using the EnzCheck® Gelatinase / Collagenase kit (Molecular Probes Inc., USA). An aliquot of the sample (emulsion and extract) and buffer (control) was added to a 96-well plate. In this plate, aliquots of type IV substrate of DQ-gelatin or DQ-collagen and active enzyme were added. The remaining details of the analysis were carried out according to what was previously described for the analysis of elastase enzyme activity.

[136] Cell colony formation study in vitro: initially a cell viability test was performed to determine the highest non-cytotoxic dose. In all experiments, the cells were treated with 3 different concentrations of the test substance. To this end, tests have only been carried out so far with samples of miniemulsions. From the highest non-cytotoxic dose, 3 doses were defined, varying 10 times between each concentration (Ex: 1, 0.1 and 0.01 mg / ml). All treatments and technical replicates were performed in triplicates. The methods used in the present study were performed as described: To determine the proliferation capacity of human keratinocytes, HaCaT cells were cultured at low density (33 cells / cm2). After 48 hours, the cells were treated with the test substances, in 3 non-cytotoxic concentrations obtained in the cell viability test and maintained in culture for a period of 5 days. This incubation period allows the cells to divide and form separate colonies, which allows their counting and subsequent evaluation of the clonogenic potential of the cells. After the treatment period, the cells were fixed (4% paraformaldehyde) and stained with violet crystal. Colonies were counted by visual assessment. The colony forming capacity was expressed in colony count observed for each treatment.

Results obtained

[137] In this Section, the results of the characterization of the raw material, the results of extractive processes, as well as their chemical composition will be presented. Finally, the results of emulsifying the bioactive fractions and the collagen synthesis assays, elastase and collagenase inhibition and the formation of cell colony will be presented and discussed.

Characterization of the raw material

[138] A fundamental aspect in extractive processes is the maintenance of the quality and physico-chemical characteristics of the raw material. As, the purpose of this project is the use of an agro-industrial residue, the characterization of it is justified. Therefore, Table 2 presents the characterization of the plant matrix, raw material used preferably as an example of the present invention, without however restricting the scope, the dried and ground passion fruit bagasse.
Table 2 - Characterization of the plant matrix and comparison with works published in the literature.

[139] The passion fruit bagasse used in the present invention showed a large amount of peel, which impacted on a lower content of total lipids when compared to other works found in the literature for the same raw material. Another important aspect is the amount of water in the dehydrated matrix, which can negatively affect the extraction process. The values obtained for this parameter were higher than the studies by Viganó et al., (2016b) and Barrales et al., (2015), however they are within the acceptable limit for extraction with supercritical fluid (MEIRELES, 2008).

Obtaining bioactive fractions enriched both lipophilic and hydrophilic, comprising phenolic compounds

[140] To implement the present invention, without however restricting the scope, the bioactive fractions were obtained by supercritical extraction from the passion fruit bagasse in two distinct phases, based on the results obtained by Viganó et al, (2016b). The first stage aimed at obtaining a hydrophilic bioactive fraction enriched with tocols, under the conditions of 60 ° C and 17 MPa, and a second stage with the objective of degreasing the vegetable matrix and obtaining a lipophilic bioactive fraction with a mixture of water and ethanol. at high pressures. Thus, in the second stage the conditions of the supercritical CO2 were adjusted to a high solvent density, 40 ° C and 34 MPa, in which a high fatty acid solvation capacity is obtained. Figure 3 shows the extraction kinetics for both stages, Stage / Stage 1 and Stage / Stage 2.

[141] It can be seen in Figure 3 that the ratio of the solvent mass (S) to the feed mass, or the amount of vegetable matrix to be extracted (F), was 32 kg / kg (S / F) for Phase 1 and 60 kg / kg (S / F) for Phase 2, with yields of 2.6% (kg / kg) and 14% (kg / kg), respectively. Viganó et al., (2016b), studied the extraction with supercritical CO2 of passion fruit bagasse and obtained S / F values of 150 for obtaining tocols and 400 for the extraction phase of fatty and carotenoid acids (defatted), with yields of 6.56 and 23.55% for Stage / Stage 1 and Stage / Stage 2, respectively. Barrales et al., (2015), studied the extraction with supercritical CO2 of passion fruit bagasse from the juice industry and obtained an overall yield of 18.5%, using an S / F value of 210 kg / kg, at 26 MPa and 40 ° C. It is noted that the global yield values obtained by this project were lower than those found in the literature, in which they used equipment on a laboratory scale, with extractor volumes between 10 and 300 ml. It is believed that this difference is linked to the scale increase carried out by this project, since laboratory equipment on a pilot scale, 5-liter extractors and 1-liter separators were used. Another factor that influenced this difference in performance was the characteristics of the material from the industry. The material used in the present project had a higher content of fruit peels and pulp, reflecting, as stated earlier, in the lipid yield, consequently, in the overall yield of the supercritical extraction.

*Rendimento (%) calculado com base na concentração de sólidos obtidos (g) por massa de matéria-prima desengordurada (g); * **Rendimento expresso em mg de ácido gálico equivalente por g de matriz desengordurada; ***Rendimento em mg por g de matriz desengordurada. Sendo PLE: Extração com fluido pressurizado com etanol e água.[142] However, the determining factor in the overall yield is the S / F value, that is, the amount of solvent used per unit of raw material, since the greater the mass of solvent used for the same amount of matrix , the higher the yield, since more solvent was used in the process. The S / F values for the first supercritical extraction stage were fixed based on the extraction kinetics obtained by Viganó et al., (2016b), in which it was decided to use the S / F value that represented the end of the stage constant period extraction (CER). However, for the second stage, the S / F value was determined to be the value needed to degrease the vegetable matrix. It is noted that this value was lower than the studies found in the literature, since the amount of lipids in the matrix is lower. Table 3 presents results of overall yield for the two extraction steps with supercritical CO2, as well as the yield for a single condition, called global extraction, and for the extraction step with pressurized fluid (PLE).
Table 3 - Yields of enriched bioactive fractions obtained for each stage of extraction from passion fruit bagasse.

* Yield (%) calculated based on the concentration of solids obtained (g) per mass of defatted raw material (g); * ** Yield expressed in mg of gallic acid equivalent per g of defatted matrix; *** Yield in mg per g of defatted matrix. Being PLE: Extraction with fluid pressurized with ethanol and water.

[143] After the two stages of supercritical extraction, the material remaining inside the extractor was collected, weighed and stored for later extraction with pressurized liquid. In this step (3), a mixture of ethanol and water was used with solvent under optimal conditions previously determined by Viganó et al., (2016a), 70 ° C, 10 MPa, a water-to-ethanol concentration ratio of 25% ( g of water / g of total solvent) and an S / F of 300 kg / kg. Based on the kinetic results of Viganó et al., (2016a), the S / F ratio used in the present study was set at 60 kg / kg, which corresponds to the end of the constant period extraction step (CER), in which approximately 60% of the bioactive compound (piceatannol) is extracted. The overall yield in this extraction stage was approximately 38%, which is higher than the values found in the literature (VIGANÓ et al., 2016a). At the end of this step (3), the bioactive hydro alcoholic fraction was rotated, under vacuum (760 mmHg) at 40 ° C, to separate ethanol. The final bioactive fraction in aqueous medium (3), identified as concentrated hydrophilic bioactive fraction, was properly characterized and used in all emulsification tests of the present project.

[144] It can be seen that the value presented in Table 3 of total phenolic yield was lower than that mentioned by Viganó et al., (2016a), 55 mg AGE / g of defatted passion fruit bagasse. This difference is associated with two main factors, the quality of the plant matrix and the scale of the extractive process. The quality of the vegetable matrix refers to the variables linked to fruit production, such as climate, water stress, fertilization, harvest time, among others, and because it is a waste from the juice industry, the separation efficiency between the pulp and seed , since the smaller this separation, the greater the amount of pulp, consequently the greater the amount of sugars in the plant matrix and the higher yield of total sugars in the hydrophilic extract. Therefore, the standardization of the plant matrix is extremely important for the efficiency of the extractive process of bioactive compounds.

[145] In turn, the scale increase promoted for the realization of the present invention was a determining factor in the difference in the chemical characteristics of the hydrophilic extract, for example, the yield of the compound piceatannol was 2.2 mg / g of bagasse degreasing, while Viganó et al., (2016a), obtained 17.2 mg / g. Such research carried out the extraction process in pressure vessels of 10 ml, but in this work, scales ten times larger (100 ml) were used. The scale-up carried out in this invention was based on the factor S / F, ratio between mass of solvent (S) and mass of plant matrix (F), in which, physically, it is linked to the extract / compound solubility factor in the solvent used , not taking into account other factors that influence the mass transfer process, such as the residence time of the solvent inside the extractor. In order to demonstrate the influence of the scale increase in the extraction process with pressurized liquid, Figure 4 shows the bed of plant matrix submitted to the extraction process on a 100 ml scale.

[146] Figure 4 shows the difference in the color of the passion fruit defatted bagasse, in relation to the position in the extraction bed (z) in the pressurized liquid tests. This difference is evident when the first layers of plant matrix at the entrance (z = 0) are compared to the last layers at the extractor outlet (z = L). Such behavior may be related to the residence time of the solvent inside the extractor, that is, the speed within the porous bed of particles (U / Ԑ), since this factor controls the mass transfer inside the particle (PRONYK and MAZZA, 2009). According to Anekpankul et al., (2007) the extraction rate of the noni root (Morinda citrifolia) with pressurized water is controlled by the mass transfer inside the particle, for flow rates above 2.4 ml / min. On the other hand, according to the authors, below this value, the extraction rate is controlled by the resistance to mass transfer in the film around the vegetable matrix particle, that is, an external resistance. Viganó et al., (2016a), extracted piceatannol from the passion fruit bagasse in a 10 ml extraction vessel, with a flow rate fixed at 3.52 ml / min, while the present one used a flow rate of 23.5 ml / min. min. Therefore, it is noted that, a great difference between the works, making the study of scale increase necessary for the improvement of the yield of piceatannol.

*Rendimento expresso em mg de ácido gálico equivalente por g de matriz desengordurada; **Rendimento em mg por g de matriz desengordurada.[147] The influence of the quality of the plant / raw material matrix and the drying process were analyzed in view of the extraction of the target compound piceatannol. For this purpose, passion fruit bagasse containing small amounts of peels, aryl and pulp was purchased, that is, the bagasse formed basically by passion fruit seed. Such batch of raw material, called Bachaço de Maracujá Lote 2, was dehydrated by lyophilization, in order to decrease the degradation of bioactive compounds. Subsequently, the dry material was subjected to the extraction process with supercritical fluid to remove the supporting compounds - fraction of hydrophobic bioactive substances, and then, the extraction was carried out with pressurized liquids, for the extraction of phenolic compounds, specifically the piceatannol. Table 4 shows the comparison of the yields of the SFE extraction (40 ° C, 34 MPa, CO2) and the PLE extraction (70 ° C, 10 MPa, Ethanol / Water), specifically, the yield of total phenolics and the compound piceatannol, for both lots, Lot 1 and Lot 2.
Table 4 - Bioactive yields obtained for each stage of extraction of passion fruit bagasse, as well as for the different batches.

* Yield expressed in mg of gallic acid equivalent per g of defatted matrix; ** Yield in mg per g of defatted matrix.

[148] It can be seen that all yields, from the two extraction stages, were higher for the vegetable matrix with higher seed content, especially for the amount of bioactive lipophilic fraction, yield of the SFE, from 15.7 to 21, 8%, and for piceatannol, from 2.2 to 5.2 mg / g, an increase of approximately 230% in obtaining this compound. This behavior can be associated with two factors, the quality of the plant matrix, and / or due to the mild conditions of the drying process, since Lot 1 was dehydrated in an oven with air circulation at 60 ° C and Lot 2 by lyophilization. It is believed that the quality of the plant matrix is a determining factor in the performance of yields, since the peel, aryl and pulp of the fruit have small amounts of support compounds, such as fatty acids and tocols, and the compound piceatannol. Matsui et al. (2010), demonstrated that the extracts obtained from the passion fruit peel and pulp have non-significant effects in increasing collagen synthesis and decreasing melanin synthesis or, specifically, negative effects, such as the reduction of collagen synthesis for the extract from the peel of the fruit in concentrations greater than 50 μg / ml.

Characterization of lipophilic and hydrophilic bioactive fractions

n.d. - Não detectado pelo método analítico; MUFA - Ácidos graxos monoinsaturado; PUFA - Ácidos graxos poli-insaturados; SFA - Ácidos graxos saturados; *Declarado pela empresa fornecedora; Condições de extração supercrítica: Viganó et al., (2016b) - 17 MPa, 60°C e S/F de 160 kg/kg; Barrales et al., (2015) - 26 MPa, 40°C e S/F 210 kg/kg.[149] The lipophilic bioactive fractions obtained through extraction with supercritical CO2 were characterized against the fatty acid profile, amount of tocopherols and tocotrienols, total carotenoids, acidity index and antioxidant capacity (ORAC). The results obtained for the fatty acid content were compared to a commercial passion fruit seed oil, as shown in the data shown in Table 5. It is observed that the predominant fatty acid, in all samples of passion fruit oil, was the acid linoleic, ranging from approximately 61 to 66% (g / g). Other fatty acids with relevant values were oleic acid, from 16 to 18% (g / g), followed by palmitic acid, from 11 to 14% (g / g). Table 5 also shows the values for monounsaturated fatty acids (MUFA), polyunsaturated (PUFA) and saturated fatty acids (SFA). PUFAs represent about 65 to 67%, due to the high concentration of linoleic acid (ω-6), followed by monounsaturated acids (MUFA), mainly represented by oleic acid (ω-9). Thus, the passion fruit oil obtained by supercritical extraction is rich in unsaturated fatty acids (MUFA and PUFA). According to Yehuda et al. (2002), Youdim et al. (2000), Pepe (2005) and Jenkins et al. (2002) linoleic (18: 2n-6) and linolenic (18: 3n-3) acids are essential for maintaining the integrity of the membrane and cell division in the body, and its use is recommended.
Table 5 - Fatty acid profile (% g / g) for the bioactive lipophilic fractions obtained by extraction with supercritical CO2 from the passion fruit bagasse, for the commercial oil of the passion fruit seed and the comparison with published works.

na - Not detected by the analytical method; MUFA - Monounsaturated fatty acids; PUFA - Polyunsaturated fatty acids; SFA - Saturated fatty acids; * Declared by the supplier company; Supercritical extraction conditions: Viganó et al., (2016b) - 17 MPa, 60 ° C and 160 kg / kg S / F; Barrales et al., (2015) - 26 MPa, 40 ° C and S / F 210 kg / kg.

Valores expressos em mg do respectivo componente por 100 g de óleo (extrato-fração bioativa); Comercial - Óleo comercial da semente de maracujá;[150] It is also noted, in Table 5, that the profile of all stages of supercritical extraction had values close to those determined by other scientific works, such as Viganó et al. (2016b), and Barrales et al. (2015). In addition, the fatty acid profile was also determined for a commercial passion fruit seed oil, obtained by the cold pressing process. In general, similar profiles of monounsaturated, polyunsaturated and saturated fatty acids were observed for oils from supercritical extraction and commercial oil. Despite this similarity, the extracts obtained by supercritical extraction showed high levels of tocopherols and tocotrienols, total carotenoids and greater antioxidant capacity than commercial oil.
Table 6 - Profile of tocopherols and tocotrienols (mg / 100g) for the bioactive fractions extracts obtained by extraction with supercritical CO2 from the passion fruit bagasse, for the commercial oil of the passion fruit seed.

Values expressed in mg of the respective component per 100 g of oil (extract-bioactive fraction); Commercial - Commercial passion fruit seed oil;

[151] Table 6 presents the profiles of tocopherols and tocotrienols obtained for different bioactive fractions from supercritical extraction, as well as for commercial oil. Table 6 shows that the major tocols present in the bioactive fractions of passion fruit are δ-tocotrienol, γ-tocotrienol and α-tocotrienol, followed by α-tocopherol and γ- tocopherol, with values of approximately 106, 81, 59 , 45 and 41 mg / 100g, respectively. The highest concentration of total tocols was obtained in the supercritical extraction process at 60 ° C and 17 MPa, that is, for the first extraction phase, with a total of 350 mg of tocols per 100 g of extract. For the other bioactive fractions, this value was approximately 3 times lower, ranging from 86 to 95 mg / 100g of extract. In turn, commercial oil had the lowest total tocolysis values, with α-tocopherol, β-tocopherol and α-tocotrienol not being detected. However, only α-tocopherol meets the human requirements for vitamin E, as the different tocols are poorly recognized by the α-tocopherol transport protein (a-TTP) (HOSOMI et al., 1997). Despite this, essentially, the four tocopherols and tocotrienols, have the same antioxidant capacity (TRABER and ATKINSON, 2007). This statement is evident when we compare the ORAC antioxidant capacity of the different extracts obtained, in the different extraction stages, as shown in Table 7.
Table 7 - Comparison between antioxidant capacity, amount of total carotenoids and acidity index of the different bioactive fractions obtained by supercritical extraction and commercial oil.

[152] The concentration of carotenoids for the bioactive fractions of the second extraction stage and for the global yield experiment (Global) were higher than the value of the first phase (tocols), and approximately 90 times greater than the concentration of carotenoids obtained for the oil commercial passion fruit seed. In addition, the carotenoid concentration was 20 times higher than the results obtained by Viganó et al. (2016b). According to the authors, the major carotenoids are β-carotene and β-cryptoxanthin, with concentrations of 16.77 ± 0.88 and 6.29 μg / g of extract, respectively. Another important factor that should be highlighted is the selectivity of the supercritical solvent, it is noted that under conditions of lower solvation power, that is, lower density (Phase 1, 60 ° C and 17 MPa), the extraction of carotenoids was less than in high density conditions (Global, 40 ° C and 34 MPa).

[153] It is also noted that the antioxidant capacity of bioactive fractions enriched in tocotrienols and tocopherols were greater than the values obtained for the other stages of supercritical extraction, both the global extract and the bioactive fraction of the second stage. The capacity for absorption of oxygenated radicals (ORAC) for the first phase was higher than those found by Wu et al. (2004), for the lipophilic phase of different products of plant origin, such as fruits, seeds, cereals, dehydrated fruits and spices. In addition, the antioxidant capacity of the first stage was greater than the results obtained by Viganó et al. (2016b), which obtained values close to 200 μmol TE / g of lipophilic extract for different conditions of supercritical extraction. According to the results presented by Huang et al. (2002), tocols, especially δ-tocopherol, have the capacity to absorb oxygenated radicals close to the standard used in the analysis of ORAC, the Trolox. In general, the ORAC method has been reported as the most relevant for in vitro testing, due to the use of a biologically relevant radial (THAIPONG et al., 2006). However, the method has critical points, such as the solubility of the lipophilic (oily) bioactive fraction.

[154] As it is a composition comprising free fatty acids, triacylglycerols, tocopherols and tocotrienols, phytosterols, carotenoids, among other compounds .. Therefore, a fundamental characteristic in the quality of the bioactive fractions obtained by SFE is the acidity index. It can be seen in Table 7 that the bioactive fractions obtained in Phase 1 of the SFE showed high values of the acidity index in comparison with the other bioactive fractions and the commercial oil of the passion fruit seed. Such behavior is associated with the selectivity of supercritical extraction in such specific process conditions (60 ° C and 17 MPa), since in this condition there is a high solubility of free fatty acids, such as oleic, linoleic, palmitic acid, among others, and low solubility of the other compounds in the extract, such as carotenoids and triacylglycerols (TEMELLI, 2009). When the process conditions were changed (40 ° C and 34 MPa), the solubility of these other compounds was increased, consequently, their obtaining increased, causing a dilution effect of free fatty acids, and a dilution of the acidity index of 15% to 1% (g oleic acid / g oil), for Phase 1 and Global, respectively.

[155] In addition to the characterization of the lipophilic bioactive fraction, the characteristics of the concentrated hydrophilic bioactive fraction (3) obtained through extraction with pressurized liquid (PLE) for Batch 1 of plant matrix were carried out. Table 8 presents the results obtained for the analysis of solids, sugars and total phenolics, value of antioxidant capacity, ORAC, the concentration of the compound piceatannol, pH and titratable acidity of the concentrated hydrophilic bioactive fraction.
Table 8 - Characteristics of the concentrated hydrophilic bioactive fraction (step 3) obtained from the defatted passion fruit bagasse by extraction with pressurized fluids (Lot 1).

[156] It can be seen in Table 8 that the concentrated hydrophilic bioactive fraction (step 3) of the passion fruit bagasse comprises high levels of total solids, represented by sugars and phenolic compounds. Each point out that some phenolic compounds are glycosylated, that is, linked to glucose molecules, causing them to also be quantified by the colorimetric method used to determine total sugars. Despite the high concentration of sugars, it can be said that the extract contains high levels of phenolic compounds, and consequently high antioxidant capacity, which is reflected in the value of the ORAC antioxidant capacity.

[157] The values presented in Table 7 were also expressed in the form of income, that is, per unit of mass of passion fruit bagasse. The yield values for solids, sugars and total phenolics were 38.4 ± 0.01% (m / m), 215.4+ 7.80 mg G / g and 21.3 ± 0.33 mg AGE / g , respectively. The global yield of solids is above that reported in the literature, approximately 38% (w / w), but the yield of total phenolics was lower than that mentioned by Viganó et al. (2016a), in which I obtained a value of 55 mg AGE / g of defatted passion fruit bagasse. Such differences are associated with two main factors, the quality of the plant matrix and the scale of the extractive process, as previously discussed.

Cytotoxicity assays

1pH da maior concentração; 2Dose inicial para o teste de LD50.[158] The bioactive fractions obtained at all extraction stages, lipophilic bioactive fraction from Step / Phase 1 (including tocols), lipophilic bioactive fraction from Step / Phase 2 (including fatty acids), global lipophilic bioactive fraction and commercial hydrophilic extract bagasse (EHC - Lote 1), as well as the commercial oil of passion fruit seed were submitted to the cytotoxicity test, by the method described by OECD 129 (OECD, 2010). The results of the tests are shown in Table 9. It can be seen that only the bioactive ration from Step / Phase 2 of supercritical extraction showed a toxic response to cell tests, with IC 50 values and the initial dose for LD 50 studies was 61, 3 μg / ml and 488.7 mg / kg, respectively. The other bioactive fractions, Phase 1 eglobal of supercritical extraction, the commercial oil of the passion fruit seed and the concentrated hydrophilic extract of the passion fruit bagasse did not present detectable IC50 and LD50 values.
Table 9 - Results related to cytotoxicity tests obtained from the bioactive fractions of the present invention, commercial oil and commercial hydrophilic extract (EHC) from the passion fruit bagasse and the commercial oil from the passion fruit seed.

1pH of the highest concentration; 2 Initial dose for LD50 test.

1Sem categoria.[159] According to OECD 423 (OECD, 2001), for acute oral toxicity studies in animals, substances with initial doses between 2000 and 5000 mg / kg (Category 5 GHS) can be exempted from the test of acute oral toxicity depending on the purpose of the product. The limit dose 5000 mg / kg should only be performed under exceptional conditions, where there are specific regulatory justifications. However, the labeling of products containing an informative description regarding the potential for acute oral toxicity and its category is necessary. Table 10 presents the category and description of product safety on labels, according to the classification of the Globally Harmonized System of Classification and Labeling of Chemicals (Globally Harmonized System of Classification and Labeling of Chemicals - GHS), and adopted by the Economic Council and United Nations (UN).
Table 10 - Classification of chemical substances according to the potential for acute oral toxicity according to UM-GHS (EPA, 2004)

1No category.

[160] However, this classification and labeling is carried out based on results obtained in acute oral toxicity studies in animals. The bioactive lipophilic fraction obtained in Step / Phase 2 of the supercritical extraction obtained the predictive value of initial doses for the study of acute oral toxicity LD50 of 488.7 mg / kg. It is concluded that the test substance could be classified in categories 4, according to the classification of UM-GSH (EPA, 2004). On the other hand, for the other products (bioactive fraction, commercial oil and commercial hydrophilic extract EHC) it was not possible to determine a predictive value of initial doses for the study of acute oral toxicity LD50. Therefore, it is concluded that, there is no need to carry out studies of acute oral toxicity in animals since the test substance would probably be classified as "without category (S.C.)", according to the classification of UM-GSH (EPA, 2004). Therefore, because they do not present toxicity, in all emulsification tests, the concentrated hydrophilic bioactive fraction and the lipophilic bioactive fractions (Stage / Stage 1 and Global) of the passion fruit bagasse were used in an integral way, without previous dilution, taking into account , to maintain the limits of the LD50 values, the dilution of the extracts with the use of emulsifiers and stabilizing solutions in the emulsification process.

Emulsification tests

[161] Emulsification tests take place in three distinct stages. First, the initial hypothesis of using the Easynov® emulsifier (INCI Name: Octyldodecanol & octyldodecyl xyloside & PEG -30 dipolyhydroxystearate) and the application of ultrasound was properly tested, for emulsions between concentrated hydrophilic bioactive fraction - step 3 (Lot 1) and oil commercial passion fruit seed. Later, with the results of the previous phase, the process conditions and the emulsifier were redefined, and new plans were executed for the same phase composition of the emulsion. Finally, the best condition of stability and droplet size was selected and reproduced for the emulsions with the lipophilic bioactive fractions, Stage / Stage 1, Stage / Stage 2 and Global, obtained by supercritical extraction.

n° - número do experimento, correlacionado com a Figura 5; 1FL - Fase lipofílica; 2FH - Fase hidrofílica; 3TSI - ĺndice de estabilidade das emulsões em 24 horas.[162] In the first stage of the emulsification tests, different concentrations of the Easynov® (EM) emulsifier were tested and ratios between the hydrophilic phase (FH), concentrated hydrophilic bioactive fraction - obtained by step 3, and the lipophilic phase (FL), composed by the commercial oil of passion fruit seed. In addition, the hypothesis of using ultrasound in the emulsification process was also evaluated as a way of expanding the scope without restricting it. The TSI results obtained in this step are shown in Table 11. Figure 5, in addition to demonstrating the emulsions obtained for each formulation tested in this step, demonstrates the emulsification process.
Table 11 - Experimental results of TSI for different concentrations of the hydro and lipophilic phases, and amount of emulsifier tested for O / W emulsions.

n ° - number of the experiment, correlated with Figure 5; 1FL - Lipophilic phase; 2FH - Hydrophilic phase; 3TSI - 24 hour emulsion stability index.

[163] It is observed that the TSI values shown in Table 11 vary between 4.5 and 6.7 for the 24-hour test period. The values directly reflect the stability of the emulsions, and the greater the magnitude of TSI the greater its instability, or even, the greater the TSI the greater the phase separation in the emulsion, be it coalescence, flocculation, creaming or sedimentation. It can be seen in Figure 7 that the tested formulations and the process applied to emulsify oil and hydrophilic passion fruit extract were not suitable, as all emulsions were kinetically unstable. Experiment number 9 showed the least instability, due to the high concentration of emulsifier and oil used in this formulation, which increased the viscosity, consequently promoting a barrier to coalescence, favoring the stability of the emulsion. In addition, the high oil concentration favored stability because, according to the supplier, although the EasyNov® emulsifier is effective for oil in water (O / W) emulsions, it is recommended for water in oil (W / O) emulsions. Thus, it was decided that the emulsifier would be exchanged for Montanov L® (C14-22 alcohols & C12-20 alkyl glucoside), from the same supplier company, which is recommended for oil-in-water (O / W) emulsions.

[164] Another factor that favored the instability of emulsions was the application of ultrasonic waves. Several works, found in the literature, have observed that the application of ultrasonic waves, mainly the excess of them, can cause an increase in the emulsion droplet diameter, consequently, it increases the possibility of the coalescing emulsion and the phase separation occurs. This instability is typically attributed to the effects of Bjerknes' forces (LEIGHTON, 1994) that propel the emulsion droplets to the acoustic field nodes, where the proximity of the droplets results in an increase in coalescence (PATIL and GOGATE, 2018). Therefore, the initial hypothesis of using ultrasound to decrease the size of the drop was discarded.

n° - número do experimento, correlacionado com a Figura 7; 1EM - Concentração de emulsificante; 2ES - Concentração de estabilizante; 3TSI - ĺndice de estabilidade das emulsões em 7 dias.[165] The proposed second emulsification process was tested, fixing the composition of both phases, aqueous and oily, in a ratio of 3: 1 from hydrophilic phase (FH) to lipophilic phase (FL), and varying the amount of emulsifier (EM) in relation to the lipophilic (FL) phase. In addition, based on a recommendation from one of FAPESP's reviewers, the hypothesis about the use of a stabilizer in the emulsion was tested. The stabilizer chosen for this step was xanthan gum. Several studies have demonstrated the advantages of adding xanthan gum in the physicochemical properties of emulsions, such as the size of the droplets, their viscosity and stability (HEMAR et al., 2001; KRSTONOŠIC et al., 2009; SUN et al ., 2007; YE et al., 2004). Table 12 presents the concentrations of the formulations tested for this emulsification step, as well as the results of kinetic stability (TSI), and the amount of stabilizer (ES) was determined based on the amount of the hydrophilic phase (FH)
Table 12 - Experimental TSI results for different concentrations of the hydro and lipophilic phases, and amount of emulsifier tested for O / W emulsions.

n ° - number of the experiment, correlated with Figure 7; 1EM - Concentration of emulsifier; 2ES - Stabilizer concentration; 3TSI - Emulsion stability index in 7 days.

[166] Note that the stability index (TSI) of the emulsions increased with the increase in the amount of emulsifier, from 35.1 to 13.4 with 5 and 20% in grams of emulsifier per milliliter of lipophilic phase, respectively. The addition of stabilizer, xanthan gum, also promoted an improvement in the kinetic stability of the emulsions, with TSI values ranging from 19.3 to 0.3. The TSI value for emulsions with stabilizer was approximately ten times lower than for emulsions without xanthan gum, in the same amount of emulsifier (20%). In addition, it is observed that the stability of the emulsions also depended on the amount of gum added, and the addition of small concentrations was not possible to promote stability over seven days of analysis. The emulsification process, as well as the images of the emulsions obtained for this step are shown in Figure 7. It was observed, in general, that the addition of xanthan gum was beneficial to the stability of the emulsions. According to Hayati et al. (2009), polysaccharide dispersions provide a sufficiently thick continuous phase that hinders the tendency of dispersed oils to migrate, that is, it reduces the mobility of the droplets and the frequency of collision, and as a result, the coalescence or flocculation of the emulsion droplets can be delayed. According to McClements (2015), in addition to increasing the viscosity of the continuous phase, stabilizers can even cause gelation of the continuous phase, thus preventing the aggregation of droplets.

[167] Based on the results of this stage, it was possible to develop a new plan for the emulsification process of the bioactive hydrophilic fraction of the bagasse and the commercial oil of the passion fruit seed. In this step, the following variables were evaluated: concentration of emulsifier in the lipophilic phase (FL), expressed in grams of emulsifier (EM) per ml of oil (%, g / ml), and concentration of xanthan gum (ES) in the hydrophilic phase ( FH), expressed in grams of 2% (g / ml) xanthan gum solution per ml of aqueous hydrophilic bioactive fraction (%, g / ml). The experiments were conducted with a ratio between the hydro and lipophilic phases set at 3/1. The emulsification process was identical to that proposed in Figure 7. Figure 7 (A) and (B), show the results of average diameter (d [3.2]) and the polydispersity index (PDI) of the emulsions on day 1 and after 10 days of storage.

[168] It can be seen in Figure 7 that the increase in the concentration of emulsifier provided a decrease in the droplet size of the emulsion, for the three levels of concentration of stabilizer. The smallest droplet diameters were obtained in the experimental conditions of 10 and 20% emulsifier in relation to the lipophilic phase, and in the lowest concentrations of stabilizer, 0.5 to 1% in relation to the hydrophilic phase. In addition, the increase in the concentration of stabilizer, xanthan gum, promoted an increase in the diameter of the droplet, for the three levels of concentration of emulsifier. Such behavior may be related to the increase in the emulsion viscosity with the increase in the amount of xanthan gum, which can significantly interfere with the shear rate during the emulsification process.

[169] It is also noted, in Figure 7, that the droplet size increased with the storage time for all conditions tested, and the means differed statistically at the 95% confidence level (p <0.05) . However, the smallest differences between day 1 and 10 were observed in low concentrations of emulsifier and intermediate concentrations for high stabilizer. The polydispersity (PDI), in turn, decreased with increasing concentrations of emulsifier and stabilizer, however in the condition of greater concentration, due to the high viscosity, the emulsifier process generated emulsions with a large droplet size distribution. In addition, droplet size dispersion increased with time of storage. The polydispersity index indicates the size of the droplet size dispersion, that is, the lower this value, the smaller the amplitude of the curve, indicating uniform emulsions. According to Gottlieb and Schwartzbach (2004), dispersed systems with PDI values less than 2 can indicate a uniform size distribution. Figure 8 shows the droplet size distribution profile as a function of the stabilizer (A) and emulsifier (B) concentrations.

[170] It is observed that the droplet size distribution profile varied with the concentrations of emulsifier and stabilizer, and the decrease in the concentration of xanthan gum resulted in less polydispersity, tending to a uniform peak, or a modal distribution, around 3 μm. However, the decrease in the concentration of emulsifier led to an increase in polydispersity and a displacement of the peaks, with the highest concentration occurring a dispersion with two distinct peaks (bimodal). According to Márquez et al. (2010), the droplet size depends on the concentration of emulsifier, that is, in the same fraction of dispersed phase, the size of the drops tends to decrease with the increase of the concentration of emulsifier. However, its increase can lead to an increase in viscosity, making the flow more difficult and, consequently, reducing the emulsification efficiency. According to Doucet et al. (2005), the rotor-stator equipment is commonly used in highly viscous fluids, however it was observed that a difference in the flow of these fluids inside the vessel. Still, according to the authors, in regions close to the rotor-stator, high turbulence and shear rate were observed, surrounded by regions where the viscosity is high and the movement of the fluid becomes laminar, until the region near the vessel wall where the fluid is almost stagnant.

[171] In addition to the droplet size and polydispersity, another important factor is the kinetic stability of the emulsions, that is, the evaluation of the phase separation of the elaborated emulsions. Thus, Figure 9 shows the experimental results of the stability of the emulsions over 10 days of storage, at a controlled temperature of 20 ° C. It can be seen that most of the experimental conditions were stable over a period of 7 to 10 storage days, however the conditions E-9, E-8 and E-6 showed an increase in the stability index after the 7 day of storage. The maximum value for TSI was 2.3 for the condition of high concentrations of emulsifier and stabilizer, and the minimum value was 0.3, for the condition of lowest concentration of the components. Such difference between the values of the experimental conditions does not necessarily reflect a greater or lesser stability, in fact, factors such as the high viscosity of the formulations and the presence of small air bubbles inside the samples, can cause such differences.

[172] For a better interpretation of the TSI values, the polydispersity data and average droplet diameter, as well as the physical appearance of the emulsions, must be taken into account. Therefore, based on the values of such responses, it can be said that the experimental condition with emulsifier concentration of 10% (g / ml FL) and the stabilizer concentration of 0.5% (g / ml FH), comparing with the other conditions, it obtained lower values of droplet diameter, polydispersity and TSI, using smaller amounts of components in the formulation (stabilizer and emulsifier). Therefore, such an experimental condition was taken based on the formulation of the other emulsions with the bioactive fractions obtained by SFE (Stage / Stage 1, Stage / Stage 2 and Global), as well as two model emulsions, one containing commercial oil and hydrophilic bagasse extract. passion fruit (Commercial - EHC) and another containing only oleic acid and water (White / Control).

n° - número do experimento, correlacionado com a Figura 10; *Homogeneizador a alta pressão com três passes a 50 MPa; 1Turbiscan Stability Index avaliada por 7 dias de armazenamento a 20° C.[173] In addition, the hypothesis of using a high pressure homogenizer for decreasing droplet size and polydispersity was tested and the results compared with those obtained for the method using rotor-stator. Table 13 shows the results of droplet size (d [3,2]) and polydispersity (PDI) for both methods (H: high pressure homogenizer and U: rotor-stator), as well as for the different lipophilic extracts obtained via SFE, in two different days of storage.
Table 13 - Average droplet diameter, polydispersity index and stability for emulsions obtained with extracts of passion fruit bagasse.

n ° - number of the experiment, correlated with Figure 10; * High pressure homogenizer with three passes at 50 MPa; 1Turbiscan Stability Index assessed for 7 days of storage at 20 ° C.

[174] The average droplet size of the emulsions obtained by rotor-stator (1U to 5U), with and without the bioactive fractions of SFE, presented values around 2.8 μm and polydispersity of 1.7, however the emulsions formulated with the lipophilic bioactive fraction of Step / Phase 1 of the extraction with supercritical fluid (SFE) they presented droplets with sizes of 8.2 μm, despite the same conditions of concentration of the phases and processing. Such behavior may be related to the high viscosity of the bioactive fraction obtained in this extraction stage, in which it made flow difficult and, consequently, the emulsification process decreased efficiency. In turn, the samples submitted to the high pressure homogenizer, after the rotor-stator, presented, in general, a droplet size larger than the other samples, such increase was 50% in relation to the samples prepared using only the rotor. -stator. In addition, the stability indexes of these emulsions were higher than the others, that is, the high pressure homogenization process caused an increase in the instability of the dispersed system, consequently, an increase in the droplet coalescence rate and, finally, the phase separation. Figure 10 shows the emulsions made with commercial aqueous extract and commercial seed oil (1U and 1H), and with the bioactive fraction SFE Global (2U and 2H), bioactive fraction SFE Ease 2 (3U e3H) and bioactive fraction SFE Ease 1 (4U and 4H) of passion fruit bagasse.

[175] It is also noted, in Figure 10, that the color change of the samples submitted to the high pressure homogenization process in comparison with the samples submitted only to the rotor-stator (Ultra Turaxx). This change in color can be associated with the fact that the high pressure process generates greater shear in the sample, greater frictional force between the surfaces of the equipment and the sample and, consequently, part of this energy is transformed into heat, causing a heating, and consequently, a degradation of the compounds present in the samples (FLOURY et al., 2000). Possibly, the increase in droplet size, polydispersity and instability are also associated with this behavior. To confirm the effects of phase separation and increased droplet size, samples 2U and 2H were subjected to optical microscopy after 30 days of storage, as shown in Figure 11. In addition, a fluorescence microscopy was performed in the 2U condition for confirmation of the type of emulsion obtained.

[176] It can be seen, in Figure 11, that the emulsions obtained using the rotor-stator (A) had smaller and less polydispersed droplets than the emulsions submitted to the high pressure homogenizer (B), confirming the data obtained through spectrometry laser diffraction. Furthermore, it can be seen in Figure 11 (C) that the emulsions obtained in this research, using the emulsifier Montanov L® and xanthan gum as a stabilizer, were oil-in-water emulsions, that is, the continuous phase is represented by the fraction bioactive comprising phenolic compounds and the dispersed phase (droplets) is represented by the bioactive fraction of the passion fruit bagasse obtained via supercritical extraction. Based on the results presented, the use of the high-pressure homogenizer was discarded, and the formulation 2U, bioactive agglomerate fraction (SFE) and concentrated hydrophilic bioactive fraction (PLE) of the passion fruit bagasse, as well as the emulsification method, were selected for future ones. project steps. It should be noted that, until now, droplets with sizes close to 1 μm, that is, close to the concept of miniemulsions, were obtained under experimental conditions with emulsifier concentration of 10% (g / ml FL) and stabilizer concentration of 0 , 5% (g / ml FH), using only rotor-stator, with values of 2.8 μm. Such behavior is directly associated with the equipment used in the emulsification process (rotor-stator). Therefore, in order to optimize the droplet size to values close to 1 μm and standardize the emulsions, in the next phase of this research (PIPE Phase 2), the use of a rotor-stator system with perpendicular flow will be proposed, in which it forces the passage of the sample in the dispersion system, avoiding the formation of a region with a low shear rate.

Collagen synthesis

[177] Firstly, at this stage, cell viability in 3T3 cell cultures was evaluated to determine the highest non-toxic concentration of the bioactive fractions of the present invention and the emulsions to be used in collagen synthesis assays. The results of this stage demonstrated that the hydrophilic bioactive fractions, from Lot 1 and Lot 2, and the lipophilic bioactive fractions, SFE Phase 1 bioactive fraction and Global bioactive fraction, did not present cellular toxicity in the tested concentrations, and in the synthesis tests of collagen, concentrations of 1000, 100 and 10 μg / ml were used for the hydrophilic bioactive fractions and SFE Global bioactive fraction and 100, 10 and 1 μg / ml for the SFE bioactive fraction Step / Phase 1. Thus, it was possible to evaluate collagen synthesis using human fibroblast cells. Figure 12 shows the results obtained for the tested products.

[178] It can be seen, in Figure 12, that the products of the passion fruit bagasse did not significantly alter the collagen synthesis of human fibroblasts, at the level of 5%. It is noted, specifically in Figure 12 (c) and (d), that the lipophilic bioactive fractions of SFE showed a downward trend, but not significant. Another point to be highlighted is the aqueous extract of the passion fruit bagasse Lot 2, Figure 12 (b), where there was a tendency to increase collagen production, although not significant at the 5% level. In general, the products of the passion fruit bagasse showed behavior of maintenance / promotion of collagen synthesis, being possible its use in cosmetic products.

[179] Emulsions with the lipophilic bioactive fraction of SFE (Global Phase) and with the aqueous extract of passion fruit bagasse, both from Lot 1 (c) and from Lot 2 (d), were tested for this test. In addition, two control samples were submitted to such tests, an emulsion with water and oleic acid (a) and a commercial product (Serum) from one of the leading companies in the market (b), which contains hyaluronic acid and resveratrol, a polyphenol similar to piceatannol (MATSUI et al., 2010), widely known for its anti-aging properties. Figure 13 shows the results obtained for the synthesis of total collagen in human fibroblasts for the tested emulsions.

[180] The emulsion comprising SFE Global bioactive fraction and concentrated hydrophilic bioactive fraction (PLE) Lot 2 showed an increasing trend in collagen synthesis, when compared to baseline control, with concentrations between 1 and 0.01 μgm / ml. In addition, it is noted, in general, that the emulsions obtained with the bioactive fractions of the passion fruit bagasse, the control emulsion and the commercial product did not decrease and / or inhibit the production of collagen. According to Matsui et al. (2010), this protein is produced in fibroblast cells and is of fundamental importance, since approximately 70% of the dermis consists of collagen and exhibits different functions in the body, including cell proliferation and differentiation. The functional properties of the skin depend on the quality and condition of the collagen present in the dermis. Some food components effectively promote the synthesis of collagen in the skin. However, other substances act as cofactors of prolyl hydroxylases and lysyl hydroxylase, which are the main enzymes responsible for collagen synthesis, and some substances induce the transforming growth factor β1 (TGF-β1), which stimulates the accumulation of type I mRNA of procollagen in human fibroblast cells (KOYA-MIYATA et al., 2004). According to Kim et al. (2008), the piceatannol, active compound present in the hydrophilic bioactive fraction and concentrated hydrophilic bioactive fraction of the passion fruit bagasse of the present invention and in the said emulsions of the present invention presented itself as an inhibitor of the JAK1 / STAT-1 pathway, which induces expression of the MMP-1 gene in cultured human dermal fibroblasts. These studies lead us to believe that the induced increase and maintenance of collagen levels by the bioactive fraction enriched in piceatannol (Lot 2) may interfere with the inhibition of matrix metalloproteinase (MMPs) or by the activity of polyphenols present in aqueous extracts and emulsions .

Inhibition of metalloproteinases (MMPs): Elastase and Collagenase

[181] The inhibition tests of the enzyme Elastase from swine pancreas (EnzChek® Elastase Assay Kit, Molecular Probes) were performed for the emulsion (2U, Lot 1 - Table 13) comprising the global bioactive fraction of the supercritical extraction as a lipophilic phase and, the hydrophilic bioactive fraction concentrated via pressurized extraction of passion fruit bagasse Lot 1. In addition, this same bioactive fraction was also tested with an inhibitor. Figure 14 demonstrates the kinetics of fluorescence emission of the reaction with Elastase in different concentrations of inhibitors, aqueous extract (A) and emulsion of the bioactive fractions (B) of the passion fruit bagasse.

Concentration in μg of the compound per μl of reaction medium

[182] In Figure 14, one can observe, both for the reaction kinetics with bioactive fraction and for the emulsion of the present invention, a characteristic behavior of an enzymatic reaction, especially for controls and, the lowest concentrations of inhibitors (A ) and (B). It should also be noted that, for the concentrated hydrophilic bioactive fraction of the passion fruit bagasse, concentrations below 2.5 μg of extract per μl of reaction medium led the product generation rates (fluorescence) close to the control reaction rates, indicating that for such conditions it was not possible to completely inhibit elastase. The same behavior was observed for reactions with the emulsion as an inhibitor, but at higher concentrations, for example, below 7 μg of emulsion per μl of reaction medium. Concentrations from 50 and 57 μg / μl for the said bioactive fraction and emulsion, respectively, were able to reduce the fluorescence emission in the reaction medium, indicating that the presence of the compounds in these concentrations inhibited the ability to catalyze the elastase in the reaction degradation of elastin. Based on the fluorescence data, the elastase inhibition values were determined for the different concentrations of inhibitors, as shown in Figure 15.

1 Fração bioativa hidrofílica concentrada (aquosa) do bagaço desengordurado do maracujá Lote 1 obtido por extração com liquido pressurizado; 2 Emulsão produzida com fração bioativa hidrofílica concentrada (aquosa) e fração bioativa lipofílica global obtida por extração supercrítica (34 MPa e 40°C).[183] It can be seen in Figure 15 that the increase in the concentration of the emulsion (Lot 1) and the hydrophilic bioactive fraction concentrated in the passion fruit bagasse (Lot 1) caused an increase in the inhibition (%) of Elastase activity. The complete inhibition of the enzyme occurred in different concentrations for the emulsion and the aqueous extract, with values of 90 and 50 pg / pl, respectively. Such behavior, possibly, is associated with the dilution effect in the preparation of the emulsions, since there was a need to add the aqueous solution of xanthan gum to promote stability. In addition, in order to determine the concentration at which 50% of the elastase is inhibited (7C50), the experimental data of inhibition (%) and concentration of inhibitors (μg / ml) were adjusted to the Hill model, according to Equation ( 4), described in section 4.1.6. Table 14 shows the values of the Hiil Curve parameters, as well as the values of 7C50 and the model determination coefficient (r2).
Table 14 - Values of the Hiil Curve parameters adjusted to dose-response data for different concentrations of inhibitors.

1 Concentrated hydrophilic (aqueous) bioactive fraction of the defatted bagasse of passion fruit Lot 1 obtained by extraction with pressurized liquid; 2 Emulsion produced with concentrated hydrophilic bioactive fraction (aqueous) and global lipophilic bioactive fraction obtained by supercritical extraction (34 MPa and 40 ° C).

[184] The 7C50 values obtained for the enriched fraction and for the emulsion were 9.14 and 22.1 μg / μl, respectively. Pientaweeratch et al. (2016), evaluated ethanolic extracts from three different fruits, Sarandi or Emblica mirobalam (Phyllanthus emblica), Sapoti or Sapota (Manilkara zapota) and Thistle (Silybum marianum) in the inhibition of elastase and obtained values of 7C50 of approximately 387, 35 and 38 μg / ml, respectively. Subsequently, tests were performed to determine the inhibition of the enzyme metalloproteinase collagenase (EnzChek® Gelatinase / Gollagenase Assay Kit, Molecular Probes), for the emulsion (2U, Lot 1 - Table 13) comprising the global global bioactive fraction of the supercritical extraction as lipophilic phase and, the concentrated hydrophilic bioactive fraction obtained via extraction with pressurized liquids from the passion fruit bagasse Lot 1. In addition, this same bioactive fraction was also tested with inhibited. Figure 16 demonstrates the fluorescence emission kinetics of the reaction with collagenase at different concentrations of inhibitors, concentrated hydrophilic bioactive fraction (A) and emulsion of the bioactive fractions (B) of passion fruit bagasse.

[185] It can be seen in Figure 16 that, specifically for controls, a characteristic behavior of an enzymatic reaction, however for the concentrations of inhibitors (A) and (B) it is noted that there was no similar behavior, since all the tested concentrations led to a total reduction of the fluorescence of the enzymatic reaction, indicating that in all tested concentration conditions both the concentrated hydrophilic bioactive fraction of the passion fruit bagasse and the emulsion of the bioactive fractions of the passion fruit bagasse were able to inhibit the action of degradation of the collagen substrate by the metalloproteinase collagenase. Therefore, it was not possible to determine the values of the Hiil Curve parameters and the IC50 values. However, based on the fluorescence data, the collagenase inhibition values were determined for the concentrated hydrophilic bioactive fraction of the passion fruit bagasse (25 μg / μl bioactive fraction), the emulsion of the bioactive fractions of the passion fruit bagasse (Emulsion 25 μg / μl), as well as for a standard inhibitor (10 mM inhibitor), 1,10-phenanthroline monohydrate, and the commercial product, which contains hyaluronic acid and resveratrol, a polyphenol similar to piceatannol (Product 14 μg / μl) . Figure 17 shows the inhibition values for the bioactive products / fractions of the present invention mentioned above.

[186] It can be seen in Figure 17 that the emulsion with bioactive fractions of the passion fruit bagasse, as well as the concentrated hydrophilic bioactive fraction were able to inhibit the enzyme that degrades collagen by 100%, while the commercial product obtained values of inhibition of approximately 20%, that is, a difference of 4 times in the inhibitory capacity. Metalloproteinases (MMPs) elastase and collagenase are directly responsible for the degradation of elastin and collagen type I, II and III, in which they are fundamental components for the maintenance and quality of dermo-epidermal fibers (CHANVORACHOTE et al., 2009). The degradation of these structural fibers results in decreased integrity and elasticity, contributing to the formation of wrinkles and skin aging. Thus, it is noted the potential application of the concentrated hydrophilic bioactive fraction and the emulsion with cosmetic input.

Cell Colony Formation Assays

[187] The proliferative capacity of human keratinocytes was evaluated, first, for the bioactive fractions obtained by supercritical extraction, Global bioactive fraction and hydrophilic bioactive fraction Step / Phase 1, and the concentrated hydrophilic bioactive fractions obtained with pressurized liquid (ethanol and water) of passion fruit bagasse, Lot 1 and Lot 2. The results obtained in these tests are shown in Figure 18.

[188] It can be seen in Figure 18 that the lipophilic bioactive fractions of the passion fruit bagasse, Global bioactive fraction and hydrophilic bioactive fraction Stage / Phase 1, promoted the cell reproduction of human keratinocytes in comparison to the basal control and the compound β-Estradiol , at the 5% level. In addition, it is observed that the concentrated hydrophilic bioactive fractions Lot 1 and Lot 2 from the defatted bagasse of passion fruit did not significantly promote colony formation compared to baseline control, at the level of 5%. Similar results were found by ASLAM et al. (2006), in which they demonstrate that the lipophilic extracts of the rome seed (Punica granatum) promoted the formation of colony of human keratinocyte cells, however the aqueous extracts of the bark, pulp and seed did not stimulate the capacity of cell proliferation and, in certain concentrations, decreased and / or inactivated human keratinocyte cells.

[189] The proliferative capacity of human keratinocytes was also tested for four (4) different emulsions, emulsions comprising SFE Global bioactive fraction and concentrated hydrophilic bioactive fraction Lot 1 (2U - Table 13) and Lot 2, the standard emulsion (5U - Table 13) containing only water, oleic acid, emulsifier and stabilizer, and a commercial product known for its anti-aging appeals. Figure 19 shows the results obtained for the number of colony forming units of the following test substances: (a) Standard emulsion (Table 13: 5U); (b) Commercial product; (c) Emulsion with concentrated hydrophilic bioactive fraction of passion fruit bagasse Lot 1 (Table 13: 2U) e; Emulsion with concentrated hydrophilic bioactive fraction of passion fruit bagasse Lot 2, compared to baseline control and B-Estradiol control (1 μM).

[190] It can be seen in Figure 19 that the emulsion composed of standard substances (a), distilled water and oleic acid, in different concentrations did not show a significant difference in the formation or cell proliferation of human keratinocytes. The same behavior was observed for the commercial product (b) containing hyaluronic acid and resveratrol. In addition, it can be noted, Figure 19 (c) and (d), that the emulsions comprising the bioactive fractions of the passion fruit bagasse, Lot 1 and Lot 2, in different concentrations, were able to promote the formation of cell colony, that is, the presence of bioactive compounds, derived from the bioactive fractions obtained by SFE and PLE in the emulsion, increased the proliferation of human keratinocyte cells.

[191] Hou et al. (2012), assessed topical application of hesperidin, a polyphenol found in orange peel extract, in mice and found that the compound stimulated the proliferation and differentiation of lamellar bodies in the epidermis, as well as the activation of PPAR-α and PPAR-γ , peroxisome proliferator-activated receptors (Peroxisome proliferator-activated receptor), a group of nuclear receptor proteins that function as transcription factors that regulate gene expression. PPARs play an essential role in the regulation of cell differentiation and keratinocyte development. Polyphenols in green tea, especially epigallocatechin-3-gallate (EGCG), were tested by Hsu et al. (2003), in primary human keratinocytes. The authors found that polyphenols stimulated the proliferation and differentiation of keratinocytes. In elderly keratinocytes, with reduced rates of cellular activity, the authors demonstrate that treatment with green tea polyphenols renewed DNA synthesis and the activation of succinate dehydrogenase.

[192] In addition to the beneficial effects of polyphenols (hydrophilic) on the proliferation of human keratinocytes, we can mention the properties of lipophilic compounds, with the precursors of Vitamin A, the carotenoids. Several authors have demonstrated the ability to promote the proliferation of keratinocytes in the presence of retinoic acid (oxidized form of Vitamin A), in which it modulates the expression of genes involved in cell differentiation and proliferation through nuclear receptors (BABAMIRI and NASSAB, 2010; BELLEMÈRE et al., 2009; FUJISHITA et al., 2006). Therefore, such results demonstrate a positive perspective for the use of this input in cosmetic products, since the emulsions use hydro and lipophilic compounds in their composition, with positive effects on cell proliferation, indicating that its use may bring benefits such as increased renewal rate of epidermal cells, decreased peeling and increased epidermal thickness (LORENCINI et al., 2014).

[193] In general, based on the results obtained, it can be concluded that the bioactive fractions of the present invention obtained through clean extraction techniques, extraction with supercritical fluids (SFE) and extraction with pressurized liquids (PLE), as well as compositions comprising said bioactive fractions and said emulsions have high antioxidant capacity and high concentrations of bioactive compounds, such as piceatannol, tocols, fatty acids and carotenoids. In addition, the extraction techniques used prove to be technically effective, however, specifically, the technique with pressurized fluids, more studies should be carried out in order to understand the phenomenological effects of the scale increase. Likewise, the emulsification process proved to be effective for obtaining stable emulsions with low polydispersity, however such data are insufficient to promote the increase in scale of this process. Finally, it was demonstrated that miniemulsions comprising bioactive compounds from passion fruit bagasse were able to promote cell proliferation of human keratinocytes, inhibit the enzyme that degrades elastin (elastase) and maintain / promote collagen synthesis. Thus being based on these statements, the proposed emulsions have scientific feasibility for application as an input in cosmetic products.

[194] The present invention is unprecedented and advantageous in relation to the state of the art as it presents a stable emulsified complex with low polydispersity comprising bioactive lipophilic and hydrophilic fractions of the passion fruit residue, obtained through clean technologies.

[195] The invention further demonstrates a means of stabilizing an emulsion containing both bioactive fractions of passion fruit, hydrophilic and lipid, producing a complex with a high capacity for protection and cellular renewal of the skin. In addition, the present invention proposes the deconstruction of the plant matrix through extraction and reconstruction through the emulsification of bioactive fractions, instead of focusing only on the benefits of extracts isolated from the literature.

COSMETIC FORMULATIONS

[196] Additional objects of the present invention are cosmetic formulations of facial cleansing lotion, facial moisturizing serum and facial moisturizer with sun protection comprising the miniemulsion of bioactive fractions of Passiflora, obtained as described above, as an active ingredient associated with one or more vehicles ( excipients, adjuvants, carriers, etc.) and / or cosmetically acceptable ingredients.

• -1,00% de miniemulsão de frações bioativas de Passiflora;
• - 83,5% de água purificada;
• - 0,05% de Edeta® BD;
• - 1,50% de Carbopol® Aqua SF-1 Polymer;
• - 0,30% de Aculyn 22;
• - 2,52% de Sodium Laureth Sulfate;
• - 0,18% de Sodium Hydroxide;
• - 2,00% de Glucam™ E-20;
• - 3,60% de Disodium Cocoamphodiacetate;
• - 0,50% de Coco-Glucoside;
• - 1,60% de Cocamidopropyl Betaine;
• - 3,00% de PEG-40 Hydrogenated Castor Oil;
• - 0,70% de Neolone™ PE;
• - Citric Acid, qsq para ajustar o pH entre 6,5 e 6,7.

[197] Consequently, the present invention relates to a cosmetic formulation of facial cleansing lotion comprising:

• -1.00% miniemulsion of bioactive fractions of Passiflora;
• - 83.5% of purified water;
• - 0.05% of Edeta® BD;
• - 1.50% of Carbopol® Aqua SF-1 Polymer;
• - 0.30% Aculyn 22;
• - 2.52% of Sodium Laureth Sulfate;
• - 0.18% Sodium Hydroxide;
• - 2.00% Glucam ™ E-20;
• - 3.60% of Disodium Cocoamphodiacetate;
• - 0.50% Coco-Glucoside;
• - 1.60% of Cocamidopropyl Betaine;
• - 3.00% PEG-40 Hydrogenated Castor Oil;
• - 0.70% Neolone ™ PE;
• - Citric Acid, qsq to adjust the pH between 6.5 and 6.7.

[198] The aforementioned formulation has the following characteristics: transparent fluid lotion, slightly yellow, with a characteristic odor of the base, without fragrance.
Table 15 - Cosmetic formulation of facial cleansing lotion.

• -3,00% de miniemulsão de frações bioativas de Passiflora;
• - 26,70 de glicerina;
• - 39,17% de água purificada;
• - 0,50% de Trietanolamina;
• - 0,30% de Pemulen TR-1;
• - 29,83% de Dow Corning 7-3101;
• - 0,50% de Neolone™ PE;

[199] Additionally, the invention relates to a cosmetic formulation of facial moisturizing serum comprising:

• -3.00% miniemulsion of bioactive fractions of Passiflora;
• - 26.70 of glycerin;
• - 39.17% of purified water;
• - 0.50% Triethanolamine;
• - 0.30% Pemulen TR-1;
• - 29.83% Dow Corning 7-3101;
• - 0.50% Neolone ™ PE;

[200] The aforementioned formulation has as characteristics: bright translucent gel, yellow gold color, with characteristic odor of the base, without fragrance.
Table 16 - Cosmetic formulation of facial moisturizing serum.

• -3,00% de miniemulsão de frações bioativas de Passiflora;
• - 3,00 de Procetil AWS;
• - 5,00 Glyceryl Stearate;
• - 5,00 Stearic Acid;
• - 2,00 Montanov 82;
• - 1,00 Montanov 202;
• - 1,00 Stearyl Alcohol;
• - 2,50 Zinc Oxide;
• - 15,00 Capric/Caprylic Triglyceride;
• - 0,25 Jaguar HP-105
• - 3,00 Glycerina
• - 58,70 Água Purificada
• - 0,55 Neolone™ PE

[201] The invention also relates to a cosmetic formulation of easy moisturizer with sunscreen that comprises:

• -3.00% miniemulsion of bioactive fractions of Passiflora;
• - 3.00 Procetil AWS;
• - 5.00 Glyceryl Stearate;
• - 5.00 Stearic Acid;
• - 2.00 Montanov 82;
• - 1.00 Montanov 202;
• - 1.00 Stearyl Alcohol;
• - 2.50 Zinc Oxide;
• - 15.00 Capric / Caprylic Triglyceride;
• - 0.25 Jaguar HP-105
• - 3.00 Glycerina
• - 58.70 Purified Water
• - 0.55 Neolone ™ PE

[202] The aforementioned formulation has the following characteristics: homogeneous emulsion, bright, cream color, with characteristic odor of the base, without fragrance
Table 17 - Cosmetic formulation of facial moisturizer with sunscreen.

Results of stability tests for cosmetic formulations

[203] The study was conducted based on ANVISA's Cosmetic Product Stability Guide. The samples were kept for three months at 5 ° C, ambient (RT) and 40 ° C. Data on quantitative (pH and viscosity) and qualitative (color, odor and appearance) variables were collected at the following times: initial (just after production), 2 weeks, 1 month, 2 months and 3 months.

Analyzed formulations:

[204] Cosmetic formulation of Facial Cleansing Lotion - LLFR221117, lot 281117, packaged in a transparent plastic bottle with flip-top lid. The color, odor and appearance characteristics remained unchanged during the three months of conducting the stability study at 5 ° C, RT and 40 ° C, as shown in Figure 20.
Table 18 - pH values for the cosmetic formulation of easy-to-clean lotion. Test conditions: 25 ° C, Gehaka pH meter, model PG1800.

[205] The data were obtained in triplicate and the results expressed as mean ± standard deviation, as shown in Table 18 and Figure 21.

[206] Cosmetic formulation of Facial Moisturizing Serum - SHFR291117, lot 291117, packaged in a wide mouth transparent plastic bottle with screw cap. The odor and appearance characteristics remained unchanged during the 3 months of conducting the stability study at 5 ° C, RT and 40 ° C. The samples kept at RT and 40 ° C showed a slight color change, as shown in Figure 22.
Table 19 - pH values for the cosmetic formulation of moisturizing serum. Test conditions: 25 ° C, Gehaka pH meter, model PG1800.

[207] Dispersions at 10% (w / w) in distilled water, in triplicate, were prepared to obtain the data. The results were expressed as mean ± standard deviation, as shown in Table 19 and Figure 23.
Table 20 - Viscosity Values (mPas): Test conditions: 25 ° C, Brookfield DV-III + Rheometer, Spindle TE, 15 rpm, 1 minute, with Helipath.

[208] The data were obtained in triplicate and the results expressed as mean ± standard deviation, as shown in Table 20 and Figure 24.

[209] Facial moisturizing cosmetic formulation with Sun Protection - HFPSR051217, lot 121217, packed in opaque white tube with flip-top lid. The odor and appearance characteristics remained unchanged during the 3 months of conducting the stability study at 5 ° C, RT and 40 ° C. The samples kept at RT and 40 ° C showed slight color change from the first month of conducting the stability study, as shown in Figure 25. The sample kept at RT showed lower viscosity, when compared with the other samples.
Table 21 - pH values for the cosmetic formulation of facial moisturizer with sunscreen. Test conditions: 25 ° C, Gehaka pH meter, model PG1800.

[211] Dispersions at 10% (w / w) and distilled water, in triplicate, were prepared to obtain the data. The results were expressed as mean ± standard deviation, as shown in Table 21 and Figure 26.
Table 22 - Viscosity values (mPas) for the cosmetic formulation of easy moisturizer with sunscreen: Test conditions: 25 ° C, Brookfield DV-III + Rheometer viscometer, Spindle TC, 5 rpm, 1 minute, with Helipath.

[212] The data were obtained in triplicate and the results expressed as mean ± standard deviation, as shown in Table 22 and Figure 27.

Conclusions of tests for cosmetic formulations

[213] The cosmetic formulation of Facial Cleansing Lotion showed pH values between 6.39 and 6.66 and kept its appearance, color and odor characteristics unchanged during the 3 months of stability study.

[214] The cosmetic formulation of Facial Moisturizing Serum showed pH values between 6.41 and 7.30, viscosity values between 14000 and 19000 mPas and kept its appearance, color and odor characteristics unchanged during the 3 months of study. stability. The formulations maintained at RT and 40 ° C showed a slight color change, which does not compromise their stability.

[215] Finally, the cosmetic formulation of Facial Moisturizer with Sun Protection showed pH values between 6.72 and 7.45 and kept its appearance, color and odor characteristics unchanged during the 3 months of stability study. The formulations maintained at RT and 40 ° C showed a slight color change, which does not compromise their stability. The viscosity values were different for each storage temperature of the samples, varying between 11133-19867 mPas (5 ° C), 5800-34733 mPas (TA) and 27000-41000 mPas (40 ° C).

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Claims (21)

Miniemulsion of bioactive fractions of Passiflora characterized by being an oil-in-water emulsion comprising:

• The. a hydrophobic phase: aqueous phase ratio within the range of 1: 2 to 1: 4;
• B. from 1% to 25% w / v of an emulsifier; and
• ç. from 0.05% to 2% w / v of a stabilizer.

Miniemulsion, according to claim 1, characterized by the raw material belonging to the genus Passiflora being Passiflora edulis. Miniemulsion, according to any one of the preceding claims, characterized in that the part of the plant to be used as raw material is preferably selected from seed or bagasse. Miniemulsion according to one or more of the claims 1-3, characterized in that the hydrophobic phase: aqueous phase ratio is 1: 3. Miniemulsion according to one or more of claims 1-4, characterized by the hydrophilic bioactive fraction (aqueous phase) comprising piceatannol. Miniemulsion, according to one or more of the claims 1-3, characterized by the lipophilic bioactive fraction (oily phase) comprising compounds belonging to the group of tocols. Miniemulsion, according to claim 6, characterized by compounds belonging to the group of tocols comprising α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol, α-tocotrienol, γ-tocotriene, δ-tocotrienol and combinations thereof. Miniemulsion, according to claim 5, characterized in that the hydrophilic bioactive fraction is obtained by means of supercritical extraction. Miniemulsion, according to claim 5, characterized in that the hydrophilic bioactive fraction is obtained by means of supercritical extraction with carbon dioxide as a solvent. Miniemulsion, according to claim 9, characterized by the hydrophilic bioactive fraction being obtained by means of supercritical extraction with carbon dioxide as solvent preferably at 60 ° C and 17 MPa (Step 1). Miniemulsion, according to claim 6, characterized by the bioactive lipophilic fraction being obtained by means of supercritical extraction (SFE) with carbon dioxide as a solvent, sequentially, after obtaining the hydrophilic bioactive fraction. Miniemulsion, according to claim 11, characterized in that the bioactive lipophilic fraction is obtained by means of supercritical extraction (SFE) with carbon dioxide as solvent, sequentially, preferably at 40 ° C and 34 MPa (Step 2). Miniemulsion according to any one of claims 1-4, characterized by the bioactive fraction obtained by means of supercritical extraction (SFE) being in a single step (global) with carbon dioxide as solvent preferably at 40 ° C and 34 MPa. Miniemulsion, according to claim 5, characterized in that the concentrated hydrophilic bioactive fraction is obtained by means of extraction with pressurized liquid (PLE) preferably with a mixture of solvent selected from ethanol and water, sequentially, after obtaining the hydrophilic and lipophilic bioactive fractions (Step 3 - PLE). Miniemulsion, according to claim 14, characterized in that the concentrated hydrophilic bioactive fraction is obtained by means of extraction with pressurized liquid preferably at 70 ° C and 10 MPa. Composition characterized by comprising the miniemulsion defined in any one of claims 1 to 6. Composition according to claim 7, characterized in that it is a cosmetic, nutraceutical, nutracosmetic, pharmaceutical and food composition. Cosmetic formulation characterized by comprising the miniemulsion of bioactive fractions of Passiflora, defined according to previous claims, associated with one or more vehicles (excipients, adjuvants, carriers, etc.) and / or cosmetically acceptable ingredients. Cosmetic formulation characterized by comprising:

• - 1.00% miniemulsion of bioactive fractions of Passiflora;
• - 83.5% of purified water;
• - 0.05% of Edeta® BD;
• - 1.50% of Carbopol® Aqua SF-1 Polymer;
• - 0.30% Aculyn 22;
• - 2.52% of Sodium Laureth Sulfate;
• - 0.18% Sodium Hydroxide;
• - 2.00% GlucamTM E-20;
• - 3.60% of Disodium Cocoamphodiacetate;
• - 0.50% Coco-Glucoside;
• - 1.60% of Cocamidopropyl Betaine;
• - 3.00% PEG-40 Hydrogenated Castor Oil;
• - 0.70% NeoloneTM PE;
• - Citric Acid, qsq to adjust the pH between 6.5 and 6.7, to be used as a facial cleansing lotion.

Cosmetic formulation characterized by comprising:

• - 3.00% miniemulsion of bioactive fractions of Passiflora;
• - 26.70 of glycerin;
• - 39.17% of purified water;
• - 0.50% Triethanolamine;
• - 0.30% Pemulen TR-1;
• - 29.83% Dow Corning 7-3101;
• - 0.50% NeoloneTM PE;

to be for use as a facial moisturizing serum. Cosmetic formulation characterized by comprising:

• - 3.00% miniemulsion of bioactive fractions of Passiflora;
• - 3.00 Procetil AWS;
• - 5.00 Glyceryl Stearate;
• - 5.00 Stearic Acid;
• - 2.00 Montanov 82;
• - 1.00 Montanov 202;
• - 1.00 Stearyl Alcohol;
• - 2.50 Zinc Oxide;
• - 15.00 Capric / Caprylic Triglyceride;
• - 0.25 Jaguar HP-105
• - 3.00 Glycerina
• - 58.70 Purified Water
• - 0.55 Neolone ™ PE

to be for use as a facial moisturizer with sun protection.

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