BR102018077523B1 - MINIEMULSIONS OF BIOACTIVE FRACTIONS OF PASSIFLORA AND THEIR FORMULATIONS - Google Patents

Abstract

MINIEMULSIONS OF BIOACTIVE FRACTIONS OF PASSIFLORA, COMPOSITIONS COMPRISING SUCH MINIEMULSIONS AND FORMULATIONSThe present invention relates to miniemulsions comprising one or more bioactive fractions of Passiflora (passion fruit) selected from hydrophilic, lipophilic ) obtained by pressurized liquid extraction of Passiflora (passion fruit) with proof of the benefits of the 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 cosmetic, nutraceutical, nutracosmetic, pharmaceutical 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

FIELD OF INVENTION

[1] The present invention refers to miniemulsions comprising one or more bioactive fractions of Passiflora (passion fruit) selected from hydrophilic, lipophilic fractions obtained by supercritical extraction or concentrated hydrophilic bioactive fractions of Passiflora (passion fruit) obtained by pressurized liquid extraction with proof of the benefits of enriched bioactive fractions isolated from the present invention, from miniemulsions as well as products comprising such miniemulsions with antioxidant properties that act to prevent and correct the different factors and mechanisms responsible for skin aging. The miniemulsion of the present invention can be used in cosmetic, nutraceutical, nutracosmetic, pharmaceutical 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.

BASICS OF THE INVENTION

[4] Phytochemical compounds have conquered an important share of the cosmetics and pharmaceuticals market in recent years. It is estimated that this class of compounds will reach annual sales worth US$4.63 billion in 2020. The main factors that affect this market are health problems such as: cardiovascular diseases; cancer; and type two diabetes. At the same time, the aging of the population and increased awareness about 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 pomace, 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 supercritical fluid extraction (SFE) and pressurized liquid extraction (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 using the DPPH, FRAP and ORAC methods. Therefore, such extracts can be explored to formulate new products with antioxidant properties.

[6] However, technological paths to enable the availability of extracts need to be developed, with a view to meeting the inherent needs of the formulations, such as stability and bioavailability. A possible way to overcome these limitations is the development of 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 dispersed phase particles have a diameter in the range of 50 nm to 1 μm (SLOMKOWSKI et al., 2011). Depending on the diameter range of the emulsion droplets, the system may have the ability to increase the bioavailability of highly lipophilic substances, have high stability characteristics regarding particle aggregation and gravitational separation (MCCLEMENTS, 2011). The applications of emulsions have been the target 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 for the active compounds in the cosmetic formula to permeate the skin. The penetration of active compounds is determined by a series of factors, such as: 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 bioactives in the oil and water phases, seem to be one of the most suitable forms for the topical application of active compounds.

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

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

STATE OF THE TECHNIQUE

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

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

[13] These seeds were chosen because they contain 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. Furthermore, only the hydrophilic extract containing some markers (sugars and peptides), which are different from the markers of the present invention, was evaluated. The present invention evaluated the benefits of an emulsified complex, stable and with low polydispersity, containing active compounds (extracts) in both hydro and lipophilic phases. Furthermore, application examples were developed for the emulsified complex

[15] Document 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 treatments, among others.

[16] These seeds were chosen because they contain 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 unsaponifiable fraction, containing between 3 and 100% by weight of unsaponifiable matter in relation to the total weight of the extract.

[18] Document 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] Active ingredients can be formulated into emulsions to treat skin, such as skin irritations.

[20] In the article published in Brazilian Journal of Pharmaceutical Science 2017;53(1):e16116, passion fruit seed extract was enriched with other phenolic substances and formulated in makeup with sun protection, such as concealers and foundations, in order to inhibit premature aging or photoaging.

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

[22] The aforementioned article does not address the bioactives responsible for the benefits to the skin. Furthermore, it deals with the production and study of the stability of specific commercial formulations, and the present invention was 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 mentioned above in that it presents a stable emulsified complex with low polydispersity comprising lipophilic and hydrophilic bioactive fractions of passion fruit residue, obtained through clean technologies.

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

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

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

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

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

[29] The article by Matsui et al (2010) addresses the potential of using piceatannol from passion fruit seeds 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, from passion fruit seeds, regulates glutathione (GSH) levels in keratinocytes, suppresses UVB-induced generation of reactive oxygen species (ROS) and reduces MMP-1 induction in keratinocytes pre-treated with piceatannol.

[31] The article by Yokozawa et al (2007) demonstrates the benefit of using piceatannol as a melanogenesis inhibition 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 compound resveratrol, analogous to piceatannol, and alpha-tocopherol in oil-in-water emulsions.

[35] The advantage of the present invention in relation to the documents mentioned above is the fact 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 isolated compounds, not taking advantage of the synergy of these compounds initially present in the fruit seed. Furthermore, the current invention uses a clean and mild 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 from both 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 of 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 oily phase comprises a hydrophobic bioactive fraction with compounds belonging to the tocol group. An additional object of the present invention is cosmetic, nutraceutical, nutracosmetic, pharmaceutical and food products comprising 1% to 3% by weight of the emulsion of the present invention, for application to the skin. The following is a brief description of the objects of the present invention.

[38] Miniemulsion of Passiflora bioactive fractions being an oil-in-water emulsion comprising: a. 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 c. 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] Previously described miniemulsion in which the hydrophobic phase:aqueous phase ratio is 1:3.

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

[43] Previously described miniemulsion in which the lipophilic bioactive fraction (oil phase) comprises compounds belonging to the tocol group.

[44] Previously described miniemulsion in which compounds belonging to the tocol group comprise α-tocopherol, β-tocopherol, Y—tocopherol, δ-tocopherol, α-tocotrienol, Y—tocotriene, δ-tocotrienol and combinations thereof.

[45] Previously described miniemulsion in which the hydrophilic bioactive fraction is obtained through supercritical extraction.

[46] Previously described miniemulsion in which the hydrophilic bioactive fraction is obtained through supercritical extraction with carbon dioxide as solvent.

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

[48] Previously described miniemulsion in which the lipophilic bioactive fraction is obtained through supercritical extraction (SFE) with carbon dioxide as solvent, sequentially, after obtaining the hydrophilic bioactive fraction.

[49] Previously described miniemulsion in which the lipophilic bioactive fraction is obtained through supercritical extraction (SFE) with carbon dioxide as solvent, sequentially, preferably at 40°C and 34 MPa (Step 2).

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

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

[52] Previously described miniemulsion in which the concentrated hydrophilic bioactive fraction is obtained through 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 being a cosmetic, nutraceutical, nutracosmetic, pharmaceutical and food composition.

[55] Cosmetic formulation comprising: - 1.00% miniemulsion of bioactive Passiflora fractions; - 83.5% purified water; - 0.05% Edeta® BD; - 1.50% Carbopol® Aqua SF-1 Polymer; - 0.30% Aculyn 22; - 2.52% Sodium Laureth Sulfate; - 0.18% Sodium Hydroxide; - 2.00% Glucam™ E-20; - 3.60% Disodium Cocoamphodiacetate; - 0.50% Coco-Glucoside; - 1.60% 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.

[56] Cosmetic formulation comprising: - 3.00% miniemulsion of bioactive Passiflora fractions; - 26.70 glycerin; - 39.17% purified water; - 0.50% Triethanolamine; - 0.30% Pemulen TR-1; - 29.83% of Dow Corning 7-3101; - 0.50% Neolone™ PE; to be used as a facial moisturizing serum.

[57] Cosmetic formulation comprising: - 3.00% miniemulsion of bioactive Passiflora fractions; - 3.00 of Procetyl 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 Glycerin - 58.70 Purified Water - 0.55 Neolone™ PE to be used as a facial moisturizer with sun protection.

BRIEF DESCRIPTION OF FIGURES

[58] Figure 1 - Flowchart of the laboratory unit with cells of two 5-liter cells (C-1 and C-2). V-1 to V-14: Block valves; VB-A: Automated back-pressure valve; VB-1 to VB-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: Pressurized column with adsorbent material for CO2 filtration.

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

[60] Figure 3 - Extraction kinetics of passion fruit pomace 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: 280 g/min.

[61] Figure 4 - Extraction bed with pressurized liquid from defatted passion fruit pomace and the diagram of an extraction bed demonstrating the finite element volume (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 velocity of the fluid at the inlet and outlet of the extractor and u is the velocity within the porous particle bed (^/ε) .

[62] Figure 5 - Emulsification process and the emulsions obtained for the first phase of 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 emulsification tests, with Montanov L® as emulsifier and xanthan gum as stabilizer. Images after 7 days of storage at 20°C.

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

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

[66] Figure 9 - Kinetic stability of emulsions 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), SFE Global bioactive fraction (2U and 2H), lipophilic bioactive fraction SFE Phase 2 (3U and 3H) and hydrophilic bioactive fraction SFE Phase 1 (4U and 4H) of passion fruit pomace.

[68] Figure 11 - Microphotographs of 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 2U sample condition, where the lipophilic phase was stained with Nile Red. Scale bar of (A) and (B) 10 μm and (C) 100 μm.

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

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

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

[72] Figure 15 - Dose-response curve for different elastase inhibitors, the concentrated hydrophilic bioactive fraction (concentrated aqueous) (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 (Lot 1) of bioactive fractions (B ) from passion fruit pomace. 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 bioactive fractions from passion fruit pomace (Emulsion at 25 μg/μl), standard inhibitor (Inhibitor at 10 mM), and the commercial product (Product 14 μg/μl).

[75] Figure 18 - Colony-forming units for the substances: (a) bioactive fraction SFE Global, (b) hydrophilic bioactive fraction SFE Phase 1 of passion fruit pomace, (c) concentrated hydrophilic bioactive fraction (aqueous) of passion fruit pomace Batch 1 and (d) concentrated (aqueous) hydrophilic bioactive fraction of passion fruit pomace Batch 2, compared to the basal 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 (aqueous) hydrophilic bioactive fraction of passion fruit pomace Lot 1 (Table 13: 2U) and; Emulsion with concentrated (aqueous) hydrophilic bioactive fraction of passion fruit pomace Lot 2, compared to the basal control and the 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 color, odor and appearance characteristics 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 easy cleansing lotion cosmetic formulation. Data were obtained in triplicate and results expressed as mean ± standard deviation.

[79] Figure 22 - shows the color, odor and appearance characteristics 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 moisturizing serum formulation. The results were expressed as mean ± standard deviation

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

[82] Figure 25 - shows the odor and appearance characteristics 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. Data were obtained in triplicate and results expressed as mean ± standard deviation.

DETAILED DESCRIPTION OF THE INVENTION

[85] The present invention refers to miniemulsions and microemulsions comprising one or more bioactive fractions of Passiflora (passion fruit) selected from hydrophilic, lipophilic fractions obtained by supercritical extraction or concentrated hydrophilic bioactive fractions of Passiflora (passion fruit) obtained by pressurized liquid extraction with proof of benefits of the isolated enriched bioactive fractions of 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 cosmetic, nutraceutical, nutracosmetic, pharmaceutical 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 for its ability to inhibit elastase and collagenase, collagen synthesis and cell colony formation.

[88] The bioactive fractions of dry passion fruit seeds used in miniemulsions were obtained by two extraction steps. The first extractive stage consisted of extracting the non-polar 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 based on the fatty acid profile, tocopherol and tocotrienol profile, acidity, total carotenoids and antioxidant activity, and used as the lipophilic phase (oil) of the prepared miniemulsion. In the second extraction 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 ethanolic bioactive fraction was rota-evaporated, characterized based on 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 high antioxidant activity and zero cellular toxicity.

[89] The present invention uses a clean and mild 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 products obtained. The bioactive fractions of the oily and aqueous phases obtained in the two-step extractive process are disclosed in the publications in Food Research International 85 (2016) 51-58, The Journal of Supercritical Fluids, Volume 110, 2016, Pages 1-10, and in the application 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 prolonged kinetic stability, which was maintained after incorporating the emulsions into cosmetic products at 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 obtaining the emulsion with the correct and stable droplet size.

[94] A person skilled in the art will know how to recognize an emulsifier available in the prior art to apply it in the present invention. The same reasoning applies to the stabilizer. Preferably the stabilizer is a gum. Most preferably the stabilizer is xanthan gum.

EXAMPLES Characterization of the plant matrix/raw material

[95] After receiving the passion fruit pomace, 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 pomace was ground in a knife mill, and stored, protected from light, at -18° C until the moment of extraction.

[96] Determination of Moisture Content: 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, and were then weighed. This operation was repeated until the samples had a constant weight.

[97] Lipid Content: Approximately five grams of dried and ground plant matrix were weighed into a filter paper cartridge, which was inserted into a Soxhlet extractor. The mass ratio between solvent and raw material was 1:30 and the extraction time was six hours. Hexane (Synth, São Paulo, Brazil) was used as the solvent. At the end of six hours, the extract was collected and the residual solvent was evaporated under vacuum in 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 specific mass, real specific mass and bed porosity. 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 ith sieve (mm); di+1 is the nominal opening of the sieve larger than the ith sieve (mm); wi is mass retained on the ith sieve; n is the total number of fractions.

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

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

[103] The porosity of the extraction bed was determined through 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 bioactive fractions were obtained as previously described by Viganó et al., (2016b) and by Viganó et al., (2016a). The passion fruit pomace 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 tocopherols and tocotrienols, called Step/Phase 1, was extracted under conditions of 60°C and 17 MPa, as determined by Viganó et al., (2016b). Then, this mass of plant matrix was subjected to a second extraction stage, with the aim of removing all lipids from the matrix, under conditions of 40°C and 34 MPa, which was called Stage/Phase 2. A third fraction bioactive fraction was also obtained through supercritical extraction, but the process occurred under a single temperature and pressure condition, 40° and 34 MPa, respectively, this bioactive fraction was called Global. The bioactive fractions were obtained using a laboratory unit (Thar Technologies, model SFE-2X5LF-2-FMC, Pittsburgh, USA), according to the schematic diagram presented in Figure 1. All lipophilic bioactive fractions were stored in an amber packaging, under refrigeration, and chemically characterized for subsequent formulation of the oily, or lipophilic, phase that makes up the emulsions.

[105] The supercritical extraction system, used to implement the present invention, without however restricting the scope thereof, consists of two extractors (C-1 and C-2), with a volume of 5 liters each. They are surrounded by 2000 W heating blankets, 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. The CO2 is cooled by thermal exchange (heat exchanger - TR) with an ethylene glycol and water bath (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 F C Mass 6000, Munich, Germany). The raw material is packaged in a nylon cell of the same dimensions as the extractor and inserted inside. After passing through the extractor, the solvent/extract system passes through cyclone-type 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 block valves and the three separator valves. The automatic valve (VB-A) is responsible for controlling the system pressure, while the pump controls the flow, and a system of thermocouples and pressure gauges monitor the system temperatures and pressures, respectively. All of this equipment is connected to a computer that controls the parameters based on values defined by the user. The laboratory unit also has a pressurized column with adsorbent material and a buffer 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 pressurized liquid extraction process (PLE - Pressurized Fluid Extraction) to obtain the phenolic compounds, as shown in Figure 2, which demonstrates the schematic diagram of the PLE unit used in these experiments. In this step, a mixture of ethanol and water was used with solvent, at 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 rotaevaporation process (MA120, Marconi, Piracicaba, Brazil) to remove the ethanol, under vacuum at 40° C. The concentrated hydrophilic bioactive fraction, which was called concentrate, was duly stored for future use as an aqueous, or hydrophilic, phase of 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 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 maintain it at the process temperature; a manometer (Zurich, São Paulo, Brazil) to indicate the pressure in the extraction cell; block valves (V-1 and V-3) (Autoclave Engineers, Ohio, USA) to control fluid passage; temperature indicator and controller (IC-1); a micrometric valve (VM) used mainly to control pressure by regulating the flow of the solvent and a container for collecting the extract

Characterization of bioactive fractions

[109] Knowledge of the concentrations of target compounds, contained in the extracts, is of fundamental importance for the emulsion formulation stage. Because of this, the extracts were subjected to the following analyses:

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

[111] Quantification of tocols: Quantification was carried out by gas chromatography (CGC AGILENT 68650), with an Agilent DB-23 capillary chromatographic column (50% cyanopropyl and methylpolysiloxan), and dimensions: length of 60m, internal diameter: 0.25mm , and 0.25 μm film. The operating conditions were: column flow of 1.00mL/min, linear speed of 24 cm/s; detector temperature 280°C; injector temperature: 250°C; and an oven temperature ramp: 110°C for 5 minutes.; 110 to 215°C, with a heating rate of 5°C/min, and 215°C for 24 min. Helium with carrier gas and hexane were 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. method. (2009);

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

[113] Quantification of total carotenoids: The lipophilic extracts, as well as the commercial passion fruit seed oil, 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 as milligrams of total carotenoids per gram of lipophilic extract (VIGANÓ et al., 2016b) .

[114] Quantification of acid value: Approximately 2 g of sample was weighed in a 125 ml Erlenmeyer flask. 25 ml of neutral ethyl ether-alcohol solution (2:1) (Synth, São Paulo, Brazil) and two drops of phenolphthalein indicator were added to the sample. It was titrated with 0.1 M sodium hydroxide solution until a pink color appeared. The acid value was expressed as oleic acid equivalent per gram of lipophilic extract (%). According to methodology 325/IV 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 a constant mass. 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 modifications. 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 in test tubes, where 500 μl of extract and 500 μl of Folin-Ciocalteau reagent (Dinâmica, SP, Brazil) were mixed. After 3 minutes, 500 μl 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 protected from light, at room temperature (25° C). Finally, absorbance was measured at a wavelength of 760 nm using a quartz cuvette, in 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 as 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). Firstly, 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 phenol melted at 50° C, in 200 ml of distilled water. Then, a solution of sodium and potassium tartrate was prepared by diluting 15.1 g of sodium and potassium tartrate tetrahydrate (KNaC4H4O6 4H2O) 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 glucose standard 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 (blank) 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 tartrate solution was added to each tube and shaken. Finally, absorbance was measured at a wavelength of 540 nm on a UV-Vis spectrophotometer (Hach, DR/4000U, Colorado, USA). The amount of sugars 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 into a beaker with 100 ml of distilled water. Subsequently, the solution was titrated with 0.1 M sodium hydroxide until pH 8.4, measured with the aid of a bench pH meter (Quimis Q400AS, São Paulo, Brazil). The titratable acidity results were expressed in grams of acetic acid per ml of extract (%), according to methodology 253/IV of the Adolfo Lutz Institute (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 a 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 with a nylon membrane with porosity 0.22 μm. 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 assays were carried out in accordance with the procedures described in the BALB/C Cytotoxicity Assay 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 confluency of 3000 cells per well, in DMEM basal medium containing 10% NBCS, and maintained in a CO2 incubator for 24 hours. After the adaptation period, the cells were exposed to 8 concentrations of passion fruit pomace extracts in DMEM medium with reduced animal serum (5% NBCS) for a period of 48 hours. One plate was reserved for testing the positive control (SDS). The compound Sodium Dodecyl Sulfate (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 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 value (50% Lethal Dose), corresponding to the dose capable of killing 50% of individuals in a test population. The extracts obtained in Phase 1 (tocols), in Phase 2 (fatty acids), the overall yield obtained by extraction with supercritical CO2 (Global), the phenolic extract obtained by extraction with pressurized liquid, and the commercial passion fruit seed oil were analyzed. .

[122] Analysis of reactive oxygen species - ORAC (Oxygen Radical Absorbance Capacity): The fractions enriched with bioactives were also analyzed for antioxidant capacity via ORAC assays. Such tests were carried out in accordance with 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 to dark microplates. Loss of fluorescence was read under excitation at 485 nm and emission at 520 nm. An analytical curve was obtained using Trolox.

Emulsion formulation

[123] Firstly, previous studies were carried out with commercial passion fruit seed oil 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 The amount of bioactive fractions obtained by SFE is limited. Subsequently, extracts from supercritical CO2 extractions 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, were used to compose the water phase. Water/oil 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 emulsion ingredients, some formulations were made, as shown in Table 1. Formulation 1 was taken as the first object of study, being formed by commercial oil from the seed and hydrophilic bioactive fraction from the bagasse degreased passion fruit. Formulations 2 and 3 differ in that they contain a fraction of lipophilic passion fruit bioactives obtained under different process conditions, oil from the Global phase and the bioactive fraction from Phase 2. With these treatments it will be possible to verify the effect of the oil phase on the emulsions. Formulation 4, which represents the formulation in which the project hypothesis is based, 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 preferably coming from passion fruit pomace. Formulation 5, in turn, differs from the others by containing only water in the hydrophilic phase and oleic acid in the lipophilic phase. Table 1 - Components of the emulsion phases.

[125] For each experiment, about 100 ml of emulsion were 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 passion fruit pomace extract; 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 subjected to an ultrasound probe (Grupo Unique, 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 aid of a jacketed beaker coupled to a cooling bath, set at 15° C, under mechanical agitation 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 analysis of droplet size, optical microscopy, kinetic stability, collagen synthesis, cell colony formation assays and activation/inhibition capacity of collagenase and elastase enzymes.

[126] The variables tested 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] The emulsions were characterized in terms of their physical and chemical aspects. To this end, the analyzes listed below were carried out:

[128] Stability by light scattering (Turbiscan Lab®): The emulsions were subjected to analysis in Turbiscan Lab® to verify possible instability phenomena inherent to emulsified systems. This equipment allows you to determine the occurrence of phenomena such as creaming, sedimentation, coalescence and also evaluate the homogeneity of the samples. The equipment consists of a near-infrared light source (880 nm) and two detectors that act synchronously. In this way, the transmission detector receives information from 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, which were subsequently analyzed on day 1, 7, and/or 10 at a temperature of 25°C. The stability of the emulsions was measured using the TSI (Turbiscan Stability Index) parameter, which takes into account all the processes that occur in the sample, as described by Kang et al., (2011).

[129] Mean droplet size: The mean droplet size, I define 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 carried out 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). Images were captured with AxioVision Rel. 4.8 software (Carl Zeiss, Germany). The analysis was carried out 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 using 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-collagenous proteins. After labeling the proteins with Sirius Red/Fast Green dyes, relative quantification of collagen was performed by spectrophotometry. The doses of the test substance (3 non-toxic concentrations) used in the relative collagen quantification assay were determined from an initial cell viability assay. Primary cultures of normal human fibroblasts were used in the tests, with all experiments carried out in biological duplicates (3 independent tests). The incubation times for cell viability and collagen synthesis assays were 48 hours and 72 hours, respectively.

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

[133] In order to determine the concentration at which 50% of elastase is inhibited, the experimental data on inhibition (%) and concentration of inhibitors (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: inhibition of the collagenase enzyme 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. Aliquots of DQ-gelatin or DQ-collagen type IV substrate and active enzyme were added to this plate. The remaining details of the analysis were carried out as previously described for the analysis of elastase enzyme activity.

[136] In vitro cell colony formation study: initially a cell viability test was carried out to determine the highest non-cytotoxic dose. In all experiments, cells were treated with 3 different concentrations of the test substance. To this end, to date, tests have only been carried out with miniemulsion samples. 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 counting and subsequent assessment of the cells' clonogenic potential. After the treatment period, cells were fixed (4% paraformaldehyde) and stained with crystal violet. Colonies were counted by visual assessment. Colony formation capacity was expressed as the number of colonies observed for each treatment.

Results obtained

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

Raw material characterization

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

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

Obtaining enriched bioactive fractions, 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 passion fruit pomace in two distinct phases, based on the results obtained by Viganó et al, (2016b). The first stage aimed to obtain a hydrophilic bioactive fraction enriched with tocols, under conditions of 60°C and 17 MPa, and a second stage aimed to degrease the plant matrix and obtain a lipophilic bioactive fraction with a mixture of water and ethanol. at high pressures. Thus, in the second stage, the supercritical CO2 conditions were adjusted to a high solvent density, 40°C and 34 MPa, in which a high solvation capacity for fatty acids was obtained. Figure 3 demonstrates the extraction kinetics for both steps, Step/Phase 1 and Step/Phase 2.

[141] It can be seen in Figure 3 that the values of the ratio between solvent mass (S) and feed mass, or the amount of plant matrix to be extracted (F), were 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 supercritical CO2 extraction of passion fruit pomace and obtained S/F values of 150 for obtaining tocols and 400 for the extraction phase of fatty acids and carotenoids (defatted), with yields of 6.56 and 23.55% for Stage/Phase 1 and Stage/Phase2, respectively. Barrales et al., (2015), studied the extraction with supercritical CO2 of passion fruit pomace 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 laboratory-scale equipment was used, with extractor volumes between 10 and 300 ml. It is believed that this difference is linked to the increase in scale carried out by this project, since pilot-scale laboratory equipment was used, 5-liter extractors and 1-liter separators. Another factor that influenced this difference in yield was the characteristics of the material coming from the industry. The material used in the present project had a higher content of fruit peels and pulp, reflecting, as previously stated, the lipid yield, consequently, the overall yield of supercritical extraction.

[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 greater the yield, since more solvent was used in the process. The S/F values were set for the first supercritical extraction stage 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 necessary 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 smaller. Table 3 presents global yield results for the two extraction stages with supercritical CO2, as well as the yield for a single condition, called global extraction, and for the pressurized fluid extraction (PLE) stage. Table 3 - Yields of enriched bioactive fractions obtained for each extraction step from passion fruit pomace. *Yield (%) calculated based on the concentration of solids obtained (g) per mass of degreased 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 pressurized fluid with ethanol and water.

[143] After the two supercritical extraction stages, the remaining material 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 the 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 work was set at 60 kg/kg, which corresponds to the end of the constant period extraction stage (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 hydro-alcoholic bioactive fraction was rotary evaporated, under vacuum (760 mmHg) at 40°C, to separate the ethanol. The final bioactive fraction in aqueous medium (3), identified as a concentrated hydrophilic bioactive fraction, was properly characterized and used in all emulsification tests of the present project.

[144] It can be observed that the value presented in Table 3 for total phenolic yield was lower than that cited by Viganó et al., (2016a), 55 mg AGE/g of defatted passion fruit pomace. 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 plant matrix refers to variables linked to fruit production, such as climate, water stress, fertilization, harvest time, among others, and as it is 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 vegetable matrix and a greater yield of total sugars in the hydrophilic extract. Therefore, the standardization of the plant matrix is extremely important for the efficiency of the extraction process of bioactive compounds.

[145] In turn, the increase in scale promoted to implement the present invention was a determining factor in the difference in the chemical characteristics of the hydrophilic extract, for example, the yield of the piceatannol compound was 2.2 mg/g of degreased bagasse, while Viganó et al., (2016a), obtained 17.2 mg/g. This research carried out the extraction process in 10 ml pressure vessels, however in this work scales ten times larger (100 ml) were used. The scale-up carried out in this invention was based on the S/F factor, the ratio between mass of solvent (S) and mass of plant matrix (F), which, physically, is linked to the solubility factor of the extract/compound 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 increasing scale in the extractive process with pressurized liquid, Figure 4 shows the plant matrix bed subjected to the extractive process on a scale of 100 ml.

[146] It can be seen, in Figure 4, the difference in color of the defatted passion fruit pomace, in relation to the position in the extraction bed (z) in the tests with pressurized liquid. This difference becomes evident when the first layers of plant matrix at the input (z = 0) are compared to the last layers at the extractor output (z = L). This behavior may be related to the residence time of the solvent inside the extractor, that is, with the velocity within the porous bed of particles (^/ε), since this factor controls the mass transfer inside the particle (PRONYK and MAZZA, 2009). According to Anekpankul et al., (2007) the extraction rate of noni root (Morinda citrifolia) with pressurized water is controlled by 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), carried out extraction of piceatannol from passion fruit pomace in a 10 ml extraction vessel, with a flow rate set at 3.52 ml/min, while the present work used a flow rate of 23.5 ml/min. min. Therefore, it is noted that there is a large difference between the works, making the scale-up study necessary to improve the yield of piceatannol.

[147] The influence of the quality of the plant matrix/raw material and the drying process were analyzed in relation to the extraction of the target compound piceatannol. To this end, passion fruit pomace was purchased containing small amounts of peels, arils and pulp, that is, the pomace basically formed by passion fruit seeds. This batch of raw material, called Passion Fruit Pomace Batch 2, was dehydrated by freeze-drying, in order to reduce the degradation of bioactive compounds. Subsequently, the dried material was subjected to the extraction process with supercritical fluid to remove the non-polar compounds - fraction of hydrophobic bioactives and then extraction with pressurized liquids was carried out to extract the phenolic compounds, specifically piceatannol. Table 4 presents 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 piceatannol compound, for both lots, Lot 1 and Lot 2. Table 4 - Bioactive yields obtained for each passion fruit pomace extraction stage, as well as for the different lots. *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 observed that all yields from the two extraction stages were higher for the plant matrix with higher seed content, especially for the amount of lipophilic bioactive fraction, SFE yield, 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 may be associated with two factors, the quality of the plant matrix, and/or due to the mild conditions of the drying process, since Batch 1 was dehydrated in an oven with air circulation at 60° C and Batch 2 by freeze drying. It is believed that the quality of the plant matrix is a determining factor in yield behavior, since the peel, aril and pulp of the fruit contain small amounts of non-polar compounds, such as fatty acids and tocols, and the compound piceatannol. Matsui et al. (2010), demonstrated that extracts obtained from passion fruit peel and pulp have non-significant effects on increasing collagen synthesis and decreasing melanin synthesis or, specifically, negative effects, such as decreasing collagen synthesis for the extract. originating from the fruit skin in concentrations greater than 50 μg/ml.

Characterization of lipophilic and hydrophilic bioactive fractions

[149] The lipophilic bioactive fractions obtained through extraction with supercritical CO2 were characterized based on the fatty acid profile, amount of tocopherols and tocotrienols, total carotenoids, acidity index and antioxidant capacity (ORAC). The results obtained for the content of fatty acids 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 passion fruit oil samples, 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 fatty acids (PUFA) and saturated fatty acids (SFA). PUFA represent around 65 to 67%, due to the high concentration of linoleic acid (o-6), followed by monounsaturated acids (MUFA), represented mainly by oleic acid (o-9). Thus, 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 membrane integrity and cell division in the body, and their use is recommended. Table 5 - Fatty acid profile (% g/g) for lipophilic bioactive fractions obtained by supercritical CO2 extraction from passion fruit pomace, for commercial passion fruit seed oil and comparison with published works. nd - 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 S/F of 160 kg/kg; Barrales et al., (2015) - 26 MPa, 40°C and S/F 210 kg/kg.

[150] It is also noted in Table 5 that the profile of all supercritical extraction stages had values close to those determined by other scientific works, such as Viganó et al. (2016b), and Barrales et al. (2015). Furthermore, 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 noted 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 the commercial oil. Table 6 - Profile of tocopherols and tocotrienols (mg/100g) for the bioactive extract fractions obtained by supercritical CO2 extraction of passion fruit pomace, for commercial passion fruit seed oil. Values expressed in mg of the respective component per 100 g of oil (bioactive extract-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. It is observed, in Table 6, that the majority tocols present in the bioactive fractions of passion fruit are δ-tocotrienol, Y—tocotrienol and α-tocotrienol, followed by α-tocopherol and y-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 under conditions of 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, the commercial oil presented the lowest values of total tocols, with α-tocopherol, β-tocopherol and α-tocotrienol not being detected. However, only α-tocopherol meets human vitamin E requirements, as the different tocols are poorly recognized by the α-tocopherol transport protein (α-TTP) (HOSOMI et al., 1997). Despite this, essentially the four tocopherols and tocotrienols have the same antioxidant capacity (TRABER and ATKINSON, 2007). This statement becomes 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 the 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 passion fruit seed commercial. Furthermore, the concentration of carotenoids was 20 times higher than the results obtained by Viganó et al. (2016b). According to the authors, the main 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 must be highlighted is the selectivity of the supercritical solvent, it is noted that in conditions of lower solvation power, that is, lower density (Phase 1, 60° C and 17 MPa), the extraction of carotenoids was lower than under high density conditions (Global, 40° C and 34 MPa).

[153] It is also noted that the antioxidant capacity of the bioactive fractions enriched in tocotrienols and tocopherols were greater than the values obtained for the other supercritical extraction stages, both the global extract and the bioactive fraction from the second stage. The oxygen radical absorption capacity (ORAC) for the first phase presented a higher value 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. Furthermore, the antioxidant capacity of the first stage was greater than the results obtained by Viganó et al. (2016b), in which they obtained values close to 200 μmol TE/g of lipophilic extract for different supercritical extraction conditions. According to the results presented by Huang et al. (2002), tocols, especially δ-tocopherol, have a capacity to absorb oxygenated radicals close to the standard used in ORAC analysis, Trolox. In general, the ORAC method has been reported to be the most relevant for in vitro testing, due to the use of a biologically relevant radial (THAIPONG et al., 2006). However, the method presents 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 noted in Table 7 that the bioactive fractions obtained in Phase 1 of SFE presented high acidity index values compared to the other bioactive fractions and commercial passion fruit seed oil. This 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. others, and low solubility of 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 obtainment increased, causing a dilution effect of the free fatty acids, and a dilution of the acid value 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 pressurized liquid extraction (PLE) were determined for Batch 1 of plant matrix. Table 8 presents the results obtained for the analyzes of solids, sugars and total phenolics, antioxidant capacity value, ORAC, concentration of the piceatannol compound, pH and titratable acidity of the concentrated hydrophilic bioactive fraction. Table 8 - Characteristics of the concentrated hydrophilic bioactive fraction (step 3) obtained from passion fruit pomace defatted by extraction with pressurized fluids (Batch 1).

[156] It can be noted in Table 8 that the concentrated hydrophilic bioactive fraction (step 3) of passion fruit pomace comprises high levels of total solids, represented by sugars and phenolic compounds. It should be noted that some phenolic compounds are glycosylated, that is, linked to glucose molecules, meaning that they are also 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 ORAC antioxidant capacity value.

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

Cytotoxicity Assays

[158] The bioactive fractions obtained in all extraction steps, lipophilic bioactive fraction from Step/Phase 1 (comprising tocols), lipophilic bioactive fraction from Step/Phase 2 (comprising fatty acids), overall lipophilic bioactive fraction and the commercial hydrophilic extract from bagasse (EHC - Lot 1), as well as commercial passion fruit seed oil were subjected to cytotoxicity testing, using the method described by OECD 129 (OECD, 2010). The results of the tests are presented in Table 9. It can be seen that only the bioactive feed from Step/Phase 2 of supercritical extraction showed a toxic response to cellular tests, with IC50 values and the initial dose for LD50 studies were 61. 3 μg/ml and 488.7 mg/kg, respectively. The other bioactive fractions, Phase 1 and global supercritical extraction, the commercial passion fruit seed oil and the concentrated hydrophilic extract of passion fruit pomace did not present detectable IC50 and LD50 values. Table 9 - Results relating to cytotoxicity tests obtained from the bioactive fractions of the present invention, commercial oil and commercial hydrophilic extract (EHC) from passion fruit pomace and commercial passion fruit seed oil. xpH of the highest concentration; zInitial dose for the LD50 test.

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

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

Emulsification tests

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

[162] In the first stage of the emulsification tests, different concentrations of the Easynov® emulsifier (EM) and ratios between the hydrophilic phase (FH), concentrated hydrophilic bioactive fraction - obtained by step 3, and the lipophilic phase (FL), composed by commercial passion fruit seed oil. Furthermore, 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 stage are presented in Table 11. Figure 5, in addition to demonstrating the emulsions obtained for each formulation tested in this stage, demonstrates the emulsification process. Table 11 - TSI experimental results for different concentrations of hydro and lipophilic phases, and amount of emulsifier tested for O/W emulsions. n° - experiment number, correlated with Figure 5; 1FL - Lipophilic phase; 2FH — Hydrophilic phase; 3TSI - Emulsion stability index in 24 hours.

[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, with 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, creamation or sedimentation. It can be seen in Figure 7 that the tested formulations and the process applied to emulsify passion fruit oil and hydrophilic extract were not suitable, as all emulsions were kinetically unstable. Experiment number 9 was the one that showed the least instability, due to the high concentration of emulsifier and oil used in this formulation, which increased viscosity, consequently promoting a barrier to coalescence, favoring the stability of the emulsion. Furthermore, 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. Therefore, it was decided that the emulsifier would be changed to Montanov L® (C1422 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 studies, found in the literature, observed that the application of ultrasonic waves, especially their excess, can result in an increase in the diameter of the emulsion droplets, consequently increasing the possibility of the emulsion coalescing and phase separation occurring. This instability is typically attributed to the effects of Bjerknes forces (LEIGHTON, 1994) that drive emulsion droplets towards nodes in the acoustic field, where the proximity of the droplets results in increased coalescence (PATIL and GOGATE, 2018). Therefore, the initial hypothesis of using ultrasound to reduce droplet size was discarded.

[165] The second proposed emulsification process was tested, fixing the composition of both aqueous and oily phases at a ratio of 3:1 hydrophilic phase (FH) to lipophilic phase (FL), and varying the amount of emulsifier (EM) in relation to the lipophilic phase (FL). Furthermore, based on a recommendation from one of the FAPESP referees, the hypothesis regarding the use of a stabilizer in the emulsion was tested. The stabilizer chosen for this stage was xanthan gum. Several studies have demonstrated the advantages of adding xanthan gum to the physicochemical properties of emulsions, such as droplet size, viscosity and stability (HEMAR et al., 2001; KRSTONOSIC 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 kinetic stability (TSI) results, with the amount of stabilizer (ES) being determined based on the amount of the hydrophilic phase (FH) Table 12 - TSI experimental results for different concentrations of hydro and lipophilic phases, and amount of emulsifier tested for O/W emulsions. n° - experiment number, correlated with Figure 7; 1EM - Emulsifier concentration; 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% 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 that of emulsions without xanthan gum, in the same amount of emulsifier (20%). Furthermore, it was observed that the stabilities 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 generally observed that the addition of xanthan gum was beneficial to the stability of the emulsions. According to Hayati et al. (2009), dispersions with polysaccharides provide a sufficiently thick continuous phase that hinders the tendency of dispersed oils to migrate, i.e., reduces droplet mobility and collision frequency, and as a result, coalescence or flocculation of 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 droplet aggregation.

[167] Based on the results of this stage, it was possible to develop a new plan for the emulsification process of the hydrophilic bioactive fraction from bagasse and commercial passion fruit seed oil. At this stage, 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% xanthan gum solution (g/ml) 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) present 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 noted in Figure 7 that the increase in emulsifier concentration provided a decrease in the emulsion droplet size, for the three levels of stabilizer concentration. 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. Furthermore, the increase in the concentration of stabilizer, xanthan gum, promoted an increase in droplet diameter for the three emulsifier concentration levels. This behavior may be related to the increase in the viscosity of the emulsion with the addition of 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 storage time for all conditions tested, with the means statistically different at the 95% confidence level (p < 0.05) . However, the smallest differences between days 1 and 10 were observed at low concentrations of emulsifier and intermediate to high concentrations of stabilizer. Polydispersity (PDI), in turn, decreased with increasing concentrations of emulsifier and stabilizer, but in the higher concentration condition, due to high viscosity, the emulsifier process generated emulsions with a large droplet size distribution. Furthermore, droplet size dispersion increased with storage time. 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 may indicate a uniform size distribution. Figure 8 demonstrates the droplet size distribution profile as a function of stabilizer (A) and emulsifier (B) concentrations.

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

[171] In addition to droplet size and polydispersity, another important factor is the kinetic stability of the emulsions, that is, the evaluation of the phase separation of the emulsions prepared. Therefore, Figure 9 presents 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 during a period of 7 to 10 days of storage, however conditions E-9, E-8 and E-6 showed an increase in the stability index after the 7th 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 lower concentration of the components. Such a difference between the values of the experimental conditions does not necessarily reflect 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 result in such differences.

[172] For a better interpretation of TSI values, polydispersity and average droplet diameter data, as well as the physical appearance of the emulsions, must be taken into consideration. Therefore, based on the values of such responses, it can be said that the experimental condition with an emulsifier concentration of 10% (g/ml FL) and a stabilizer concentration of 0.5% (g/ml FH), Compared to the other conditions, it obtained lower droplet diameter, polydispersity and TSI values, using smaller quantities of components in the formulation (stabilizer and emulsifier). Therefore, this experimental condition was taken as a basis for the formulation of the other emulsions with the bioactive fractions obtained by SFE (Stage/Phase 1, Stage/Phase 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).

[173] Furthermore, the hypothesis of using a high pressure homogenizer to reduce droplet size and polydispersity was tested and the results compared with those obtained for the method using rotor-stator. Table 13 demonstrates 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, on two different days of storage. Table 13 - Average droplet diameter, polydispersity index and stability for emulsions obtained with passion fruit pomace extracts. n° - experiment number, correlated with Figure 10; *High pressure homogenizer with three passes at 50 MPa; 1Turbiscan Stability Index evaluated by 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 from Step/Phase 1 of supercritical fluid extraction (SFE) showed droplets with sizes of 8.2 μm, despite the same phase concentration and processing conditions. This behavior may be related to the high viscosity of the bioactive fraction obtained in this extraction stage, which makes flow difficult and, consequently, reduces the efficiency of the emulsification process. In turn, the samples submitted to the high pressure homogenizer, after the rotor-stator, generally presented a larger droplet size than the other samples, this increase was 50% in relation to the samples prepared using only the rotor -stator. Furthermore, the stability indices 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 SFE Global bioactive fraction (2U and 2H), SFE Ease 2 bioactive fraction (3U and 3H) and SFE Ease bioactive fraction 1 (4U and 4H) of passion fruit pomace.

[175] It is also noted in Figure 10 that the change in color of the samples subjected to the high pressure homogenization process compared to 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 on the sample, greater friction 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, 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 increase in droplet size, samples 2U and 2H were subjected to optical microscopy after 30 days of storage, as shown in Figure 11. In addition, fluorescence microscopy was performed in the 2U condition to confirmation of the type of emulsion obtained.

[176] It can be seen, in Figure 11, that the emulsions obtained using the rotor-stator (A) presented smaller and less polydisperse droplets than the emulsions subjected 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 Montanov L® emulsifier and xanthan gum as 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 passion fruit pomace obtained via supercritical extraction. Based on the results presented, the use of the high pressure homogenizer was discarded, and the 2U formulation, global bioactive fraction (SFE) and concentrated hydrophilic bioactive fraction (PLE) of passion fruit pomace, as well as the emulsification method, were selected for future project steps. It is worth noting that, to date, droplets with sizes close to 1 μm, that is, close to the concept of miniemulsions, have been obtained under experimental conditions with an emulsifier concentration of 10% (g/ml FL) and a stabilizer concentration of 0 .5% (g/ml FH), using only rotor-stator, with values of 2.8 μm. This 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 through the dispersion system, avoiding the formation of a region with a low shear rate.

Collagen synthesis

[177] Firstly, at this stage, cell viability was evaluated in 3T3 cell cultures 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 at the concentrations tested, 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 Stage/Phase 1 bioactive fraction. collagen synthesis using human fibroblast cells. Figure 12 demonstrates the results obtained for the tested products.

[178] It is observed, in Figure 12, that the passion fruit pomace products 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 in collagen synthesis, although not significant. Another point to be highlighted is for the aqueous extract of passion fruit pomace Lot 2, Figure 12 (b), where a trend towards increased collagen production was observed, although not significant at the 5% level. In general, passion fruit pomace products showed a behavior of maintaining/promoting collagen synthesis, making their use in cosmetic products possible.

[179] Emulsions with the lipophilic bioactive fraction of SFE (Global Phase) and with the aqueous extract of passion fruit pomace, both from Batch 1 (c) and Batch 2 (d), were tested for this assay. Furthermore, two control samples were subjected to such tests, an emulsion with water and oleic acid (a) and a commercial product (Serum) from one of the market's leading companies (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 presents the results obtained for the synthesis of total collagen in human fibroblasts for the emulsions tested.

[180] The emulsion comprising SFE Global bioactive fraction and concentrated hydrophilic bioactive fraction (PLE) Lot 2 showed a trend towards increased collagen synthesis, when compared to the basal control, with concentrations between 1 and 0.01 μgm/ml. Furthermore, it is generally noted that the emulsions obtained with the bioactive fractions of passion fruit pomace, the control emulsion and the commercial product did not reduce and/or inhibit collagen production. According to Matsui et al. (2010), this protein is produced in fibroblast cells and is of fundamental importance, as 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 collagen synthesis 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 transforming growth factor β1 (TGF-β1), which stimulates the accumulation of type I collagen mRNA. procollagen in human fibroblast cells (KOYA-MIYATA et al., 2004). According to Kim et al. (2008), piceatannol, an active compound present in the hydrophilic bioactive fraction and concentrated hydrophilic bioactive fraction of passion fruit pomace of the present invention and in said emulsions of the present invention, was shown to be an inhibitor of the JAK1/STAT-1 pathway, which induces MMP-1 Gene expression 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 (Batch 2) may interfere with the inhibition of matrix metalloproteinases (MMPs) or the activity of polyphenols present in aqueous extracts and emulsions. .

Inhibition of metalloproteinases (MMPs): Elastase and Collagenase

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

Concentration in μg of the compound per μl of reaction medium

[182] It can be observed, in Figure 14, both for the reaction kinetics with the bioactive fraction and for the emulsion of the present invention, a characteristic behavior of an enzymatic reaction, especially for the controls and the lowest concentrations of inhibitors (A ) and (B). It is also noted that, for the concentrated hydrophilic bioactive fraction of passion fruit pomace, concentrations below 2.5 μg of extract per μl of reaction medium led to product generation rates (fluorescence) close to the rates of the control reaction, indicating that In such conditions, complete inhibition of elastase was not possible. The same behavior was observed for reactions with the emulsion as 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 said bioactive fraction and emulsion, respectively, were able to reduce the emission of fluorescence in the reaction medium, indicating that the presence of the compounds at these concentrations inhibited the catalysis capacity of elastase in the reaction. of elastin degradation. Based on the fluorescence data, the elastase inhibition values were determined for the different concentrations of inhibitors, as shown in Figure 15.

[183] It is observed, Figure 15, that the increase in the concentration of the emulsion (Batch 1) and the concentrated aqueous hydrophilic bioactive fraction of passion fruit pomace (Batch 1) led to an increase in the inhibition (%) of Elastase activity. Complete inhibition of the enzyme occurred at different concentrations for the emulsion and the aqueous extract, with values of 90 and 50 μg/μl, respectively. This behavior is possibly associated with the dilution effect in the preparation of emulsions, since it was necessary to add the aqueous xanthan gum solution to promote stability. Furthermore, with the aim of determining the concentration at which 50% of elastase is inhibited (/C50), the experimental data on inhibition (%) and inhibitor concentration (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 Hyil Curve parameters, as well as the IC50 values and the model determination coefficient (r2). Table 14 - Values of Hiil Curve parameters adjusted to dose-response data for different concentrations of inhibitors. 1 Concentrated hydrophilic bioactive fraction (aqueous) of defatted passion fruit pomace Batch 1 obtained by extraction with pressurized liquid; 2 Emulsion produced with a concentrated hydrophilic bioactive fraction (aqueous) and a global lipophilic bioactive fraction obtained by supercritical extraction (34 MPa and 40°C).

[184] The IC50 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 of three different fruits, Sarandi or Emblica mirobalam (Phyllanthus emblica), Sapoti or Sapota (Manilkara zapota) and Milk Thistle (Silybum marianum) in inhibiting elastase and obtained IC50 values of approximately 387. 35 and 38 μg/ml, respectively. Subsequently, tests were carried out to determine the inhibition of the collagenase metalloproteinase enzyme (EnzChek® Gelatinase/Gollagenase Assay Kit, Molecular Probes), for the emulsion (2U, Lot 1 - Table 13) comprising the overall global bioactive fraction of the supercritical extraction as lipophilic phase and the concentrated hydrophilic bioactive fraction obtained via pressurized liquid extraction from Batch 1 passion fruit pomace. Furthermore, this same bioactive fraction was also tested as 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 bioactive fractions (B) from passion fruit pomace.

[185] It is observed in Figure 16 that, specifically for the controls, a behavior characteristic 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 concentrations tested led to a total reduction in the fluorescence of the enzymatic reaction, indicating that in all concentration conditions tested, both the concentrated hydrophilic bioactive fraction of passion fruit pomace and the emulsion of bioactive fractions of passion fruit pomace were capable of inhibiting the action of degradation of the collagen substrate by the metalloproteinase collagenase. Therefore, it was not possible to determine the values of the Hyil Curve parameters and the IC50 values. However, based on fluorescence data, collagenase inhibition values were determined for the concentrated hydrophilic bioactive fraction of passion fruit pomace (bioactive fraction at 25 μg/μl), the emulsion of bioactive fractions of passion fruit pomace (Emulsion at 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 noted in Figure 17 that the emulsion with bioactive fractions from passion fruit pomace, as well as the concentrated hydrophilic bioactive fraction were able to inhibit the enzyme that degrades collagen by 100%, whereas the commercial product obtained values inhibition of approximately 20%, i.e. a 4-fold difference in inhibitory capacity. The metalloproteinases (MMPs) elastase and collagenase are directly responsible for the degradation of elastin and collagen types I, II and III, which are fundamental components for the maintenance and quality of dermal-epidermal fibers (CHANVORACHOTE et al., 2009). The degradation of these structural fibers results in a decrease in integrity and elasticity, contributing to the formation of wrinkles and skin aging. Therefore, the potential application of the concentrated hydrophilic bioactive fraction and the emulsion as a cosmetic ingredient can be noted. Cell Colony Formation Assays

[187] The proliferative capacity of human keratinocytes was evaluated, firstly, 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 pomace, Lot 1 and Lot 2. The results obtained in these tests are presented in Figure 18.

[188] It can be seen in Figure 18 that the lipophilic bioactive fractions of passion fruit pomace, Global bioactive fraction and hydrophilic bioactive fraction Stage/Phase 1, promoted the cellular reproduction of human keratinocytes compared to the basal control and the compound β-Estradiol , at the 5% level. Furthermore, it is observed that the concentrated hydrophilic bioactive fractions Lot 1 and Lot 2 from defatted passion fruit pomace did not significantly promote colony formation compared to the basal control, at the level of 5%. Similar results were found by ASLAM et al. (2006), in which they demonstrated that lipophilic extracts from the pomegranate seed (Punica granatum) promoted the formation of human keratinocyte cell colonies, but aqueous extracts from the peel, pulp and seed did not stimulate the cells' proliferation capacity and, in certain concentrations, they 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 claims. Figure 19 demonstrates the results obtained for the quantity 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 pomace Lot 1 (Table 13: 2U) and; Emulsion with concentrated hydrophilic bioactive fraction from passion fruit pomace Lot 2, compared to the basal control and the 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 make 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. Furthermore, it can be noted, Figure 19 (c) and (d), that the emulsions comprising the bioactive fractions of passion fruit pomace, Lot 1 and Lot 2, in different concentrations, were able to promote cell colony formation, that is, the presence of bioactive compounds, originating from the bioactive fractions obtained by SFE and PLE in the emulsion, increased the proliferation of human keratinocyte cells.

[191] Hou et al. (2012), evaluated 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-Y , peroxisome proliferator-activated receptors, a group of nuclear receptor proteins that function as transcription factors that regulate gene expression. PPARs play an essential role in regulating cell differentiation and keratinocyte development. Green tea polyphenols, 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, carotenoids. Several authors have demonstrated the ability to promote the proliferation of keratinocytes in the presence of retinoic acid (oxidized form of Vitamin A), which 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, these results demonstrate a positive perspective for the use of this input in cosmetic products, since emulsions use hydro and lipophilic compounds in their composition, with positive effects on cell proliferation, indicating that their use may bring benefits such as increased rate of renewal of epidermal cells, decreased desquamation 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, supercritical fluid extraction (SFE) and pressurized liquid extraction (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. Furthermore, the extraction techniques used have proven to be technically effective, however, specifically, the technique with pressurized fluids, more studies must be carried out in order to understand the phenomenological effects of the increase in scale. Likewise, the emulsification process proved to be effective in obtaining stable emulsions with low polydispersity, but such data are insufficient to promote the scale-up of this process. Finally, it was demonstrated that miniemulsions comprising bioactive compounds from passion fruit pomace were capable of promoting cellular proliferation of human keratinocytes, inhibiting the enzyme that degrades elastin (elastase) and maintaining/promoting collagen synthesis. Therefore, based on these statements, the proposed emulsions present scientific feasibility for application as an input in cosmetic products.

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

[195] The invention also 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 skin cell renewal. Furthermore, the present invention proposes the deconstruction of the plant matrix through extraction and reconstruction through 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 mini-emulsion of Passiflora bioactive fractions, obtained as previously described, as an active ingredient associated with one or more vehicles ( excipients, adjuvants, carriers, etc.) and/or cosmetically acceptable ingredients.

[197] Consequently, the present invention relates to a cosmetic formulation of facial cleansing lotion comprising: - 1.00% miniemulsion of bioactive Passiflora fractions; - 83.5% purified water; - 0.05% Edeta® BD; - 1.50% Carbopol® Aqua SF-1 Polymer; - 0.30% Aculyn 22; - 2.52% Sodium Laureth Sulfate; - 0.18% Sodium Hydroxide; - 2.00% Glucam™ E-20; - 3.60% Disodium Cocoamphodiacetate; - 0.50% Coco-Glucoside; - 1.60% 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 yellowish, with a characteristic odor of the base, without fragrance. Table 15 - Cosmetic formulation of facial cleansing lotion.

[199] Additionally, the invention relates to a cosmetic formulation of facial moisturizing serum comprising: - 3.00% miniemulsion of bioactive Passiflora fractions; - 26.70 glycerin; - 39.17% purified water; - 0.50% Triethanolamine; - 0.30% Pemulen TR-1; - 29.83% of Dow Corning 7-3101; - 0.50% Neolone™ PE;

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

[201] The invention also relates to a cosmetic formulation of easy moisturizer with sunscreen comprising: - 3.00% mini-emulsion of bioactive Passiflora fractions; - 3.00 of Procetyl 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 Glycerin - 58.70 Purified Water - 0.55 Neolone™ PE

[202] The aforementioned formulation has the following characteristics: homogeneous, shiny, cream-colored emulsion, with a 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 the ANVISA Cosmetic Product Stability Guide. The samples were kept for three months at temperatures 5°C, room temperature (RT) and 40°C. Data on quantitative (pH and viscosity) and qualitative (color, odor and appearance) variables were collected at the following times: initial (right after production), 2 weeks, 1 month, 2 months and 3 months.

Formulations analyzed:

[204] Cosmetic formulation of Facial Cleansing lotion - LLFR221117, batch 281117, packaged in a transparent plastic bottle with a 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 lotion formulation easy to clean. 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 presented in Table 18 and Figure 21.

[206] Cosmetic formulation of Facial Moisturizing serum - SHFR291117, batch 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 change in color, as shown in Figure 22. Table 19 - pH values for the cosmetic moisturizing serum formulation. Test conditions: 25°C, Gehaka pH meter, model PG1800.

[207] 10% (w/w) dispersions were prepared in distilled water, in triplicate, to obtain data. The results were expressed as mean ± standard deviation, as presented in Table 19 and Figure 23. Table 20 - Viscosity Values (mPas): Test conditions: 25°C, Brookfield DV-III viscometer + 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 presented in Table 20 and Figure 24.

[209] Facial moisturizing cosmetic formulation with Sun Protection - HFPSR051217, lot 121217, packaged in an opaque white tube with flip-top 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 change in color from the first month of conducting the stability study, as shown in Figure 25. The sample kept at RT showed lower viscosity when compared to 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.

[210]

[211] 10% (w/w) dispersions were prepared in distilled water, in triplicate, to obtain data. The results were expressed as mean ± standard deviation, as presented 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 viscometer -III + Rheometer, 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.

Test conclusions for cosmetic formulations

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

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

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

BIBLIOGRAPHY

[216] A.O.A.C. Official methods of analysis of AOAC international. Association of Official Analytical Chemists. Arlington 1998.

[217] A.O.C.S. Official methods and recommended practices of the American Oil Chemists' Society. American Oil Chemists' Society. Champaign, USA: AOCS press. 5th 2009.

[218] A.S.A.E. Method of Determining and Expressing Fineness of Feed Materials by Sieving. American Society of Agricultural EngineersStandards. S319.3: 447-550 p. 1998.

[219] ANEKPANKUL, T.; GOTO, M.; SASAKI, M.; PAVASANT, P. and SHOTIPRUK, A.

[220] Extraction of anti-cancer damnacanthal from roots of Morinda citrifolia by subcritical water. Separation and Purification Technology, vol. 55, no. 3, p. 343-349, 2007.

[221] ASLAM, M. N.; LANSKY, E. P. and VARANI, J. Pomegranate as a cosmeceutical source: Pomegranate fractions promote proliferation and procollagen synthesis and inhibit matrix metalloproteinase-1 production in human skin cells. Journal of Ethnopharmacology, vol. 103, no. 3, p. 311-318, 2006.

[222] BABAMIRI, K. and NASSAB, R. Cosmeceuticals: The Evidence Behind the Retinoids.

[223] Aesthetic Surgery Journal, vol. 30, no. 1, p. 74-77, 2010.

[224] BARRALES, F. M.; REZENDE, C. A. and MARTÍNEZ, J. Supercritical CO2 extraction of passion fruit (Passiflora edulis sp.) seed oil assisted by ultrasound. The Journal of Supercritical Fluids, vol. 104, p. 183-192, 2015.

[225] BELLEMÈRE, G.; STAMATAS, G. N.; BRUÈRE, V.; BERTIN, C.; ISSACHAR, N. and ODDOS, T. Antiaging Action of Retinol: From Molecular to Clinical. Skin Pharmacology and Physiology, vol. 22, no. 4, p. 200-209, 2009.

[226] CHANVORACHOTE, P.; PONGRAKHANANON, V.; LUANPITPONG, S.; CHANVORACHOTE, B.; WANNACHAIYASIT, S. and NIMMANNIT, U. Type I pro-collagen promoting and anti-collagenase activities of Phyllanthus emblica extract in mouse fibroblasts. J Cosmet Sci, vol. 60, no. 4, p. 395-403, 2009.

[227] DÁVALOS, A.; GÓMEZ-CORDOVÉS, C. and BARTOLOMÉ, B. Extending Applicability of the Oxygen Radical Absorbance Capacity (ORAC-Fluorescein) Assay.

[228] Journal of Agricultural and Food Chemistry, vol. 52, no. 1, p. 48-54, 2004.

[229] DOUCET, L.; ASCANIO, G. and TANGUY, P. A. Hydrodynamics Characterization of Rotor-Stator Mixer with Viscous Fluids. Chemical Engineering Research and Design, vol. 83, no. 10, p. 1186-1195, 2005.

[230] EPA, U. S. E. P. A. Chemical Hazard Classification And Labeling: Comparison Of Opp Requirements And The GHS 2004.

[231] FLOURY, J.; DESRUMAUX, A. and LARDIÈRES, J. Effect of high-pressure homogenization on droplet size distributions and rheological properties of model oil-in-water emulsions. Innovative Food Science & Emerging Technologies, v. 1, no. 2, p. 127- 134, 2000.

[232] FUJISHITA, K.; KOIZUMI, S. and INOUE, K. Upregulation of P2Y2 receptors by retinoids in normal human epidermal keratinocytes. Purinergic Signaling, vol. 2, no. 3, p. 491, 2006.

[233] GOTTLIEB, N. and SCHWARTZBACH, C. Development of an internal mixing two-fluid nozzle by systematic variation of internal parts. 2004.

[234] HAYATI, I. N.; CHE MAN, Y. B.; TAN, C. P. and NOR AINI, I. Droplet characterization and stability of soybean oil/palm kernel olein O/W emulsions with the presence of selected polysaccharides. Food Hydrocolloids, v. 23, no. 2, p. 233-243, 2009.

[235] HEMAR, Y.; TAMEHANA, M.; MUNRO, P. A. and SINGH, H. Influence of xanthan gum on the formation and stability of sodium caseinate oil-in-water emulsions. Food Hydrocolloids, v. 15, no. 4, p. 513-519, 2001.

[236] HOSOMI, A.; ARITA, M.; SATO, Y.; KIYOSE, C.; UEDA, T.; IGARASHI, O.; ARAI, H. and INOUE, K. Affinity for α-tocopherol transfer protein as a determinant of the biological activities of vitamin E analogs. FEBS Letters, vol. 409, no. 1, p. 105-108, 1997.

[237] HOU, M.; MAN, M.; MAN, W.; HU, W.; HUPE, M.; PARK, K.; CRUMRINE, D.; ELIAS, P. M. and MAN, M.-Q. Topical hesperidin improves epidermal permeability barrier function and epidermal differentiation in normal murine skin. Experimental Dermatology, vol. 21, no. 5, p. 337-340, 2012.

[238] HSU, S.; BOLLAG, W. B.; LEWIS, J.; HUANG, Q.; SINGH, B.; SHARAWY, M.; YAMAMOTO, T. and SCHUSTER, G. Green tea polyphenols induce differentiation and proliferation in epidermal keratinocytes. J Pharmacol Exp Ther, v. 306, no. 1, p. 29-34, 2003.

[239] HUANG, D.; OR, B.; HAMPSCH-WOODILL, M.; FLANAGAN, J. A. and DEEMER, E. K. Development and Validation of Oxygen Radical Absorbance Capacity Assay for Lipophilic Antioxidants Using Randomly Methylated β-Cyclodextrin as the Solubility Enhancer. Journal of Agricultural and Food Chemistry, vol. 50, no. 7, p. 1815-1821, 2002.

[240] I.A.L. Physicochemical methods for food analysis. Adolfo Lutz Institute. Brasilia 2005.

[241] JENKINS, D. J.; KENDALL, C.W.; MARCHIE, A.; PARKER, T. L.; CONNELLY, P.W.; QIAN, W.; HAIGHT, J. S.; FAULKNER, D.; VIDGEN, E.; LAPSLEY, K.G. and

[242] SPILLER, G. A. Dose response of almonds on coronary heart disease risk factors: blood lipids, oxidized low-density lipoproteins, lipoprotein(a), homocysteine, and pulmonary nitric oxide: a randomized, controlled, crossover trial. Circulation, vol. 106, no. 11, p. 1327- 32, 2002.

[243] KANG, W.; Xu, B.; WANG, Y.; LI, Y.; SHAN, X.; AN, F. and LIU, J. Stability mechanism of W/O crude oil emulsion stabilized by polymer and surfactant. Colloids and Surfaces A: Physicochemical and Engineering Aspects, vol. 384, n. 1, p. 555-560, 2011.

[244] KIM, S.; KIM, Y.; LEE, Y. and CHUNG, J. H. Ceramide accelerates ultraviolet-induced MMP-1 expression through JAK1/STAT-1 pathway in cultured human dermal fibroblasts. J Lipid Res, vol. 49, no. 12, p. 2571-81, 2008.

[245] KOYA-MIYATA, S.; OKAMOTO, I.; USHIO, S.; IWAKI, K.; IKEDA, M. and KURIMOTO, M. Identification of a collagen production-promoting factor from an extract of royal jelly and its possible mechanism. Biosci Biotechnol Biochem, v. 68, no. 4, p. 767-73, 2004.

[246] KRSTONOSIC, V.; DOKIC, L.; DOKIC, P. and DAPCEVIC, T. Effects of xanthan gum on physicochemical properties and stability of corn oil-in-water emulsions stabilized by polyoxyethylene (20) sorbitan monooleate. Food Hydrocolloids, v. 23, no. 8, p. 2212-2218, 2009.

[247] LEIGHTON, T. G. 4 - The Forced Bubble. In: (Ed.). The Acoustic Bubble: Academic Press, 1994. p.287-438. ISBN 978-0-12-441920-9.

[248] LIVERSIDGE, G. G. and CUNDY, K. C. Particle size reduction for improvement of oral bioavailability of hydrophobic drugs: I. Absolute oral bioavailability of nanocrystalline danazol in beagle dogs. International journal of pharmaceuticals, vol. 125, n. 1, p. 91-97, 1995.

[249] LORENCINI, M.; BROHEM, C. A.; DIEAMANT, G. C.; ZANCHIN, N. I. T. and MAIBACH, H. I. Active ingredients against human epidermal aging. Aging Research Reviews, vol. 15, no. Supplement C, p. 100-115, 2014.

[250] MÁRQUEZ, A. L.; MEDRANO, A.; PANIZZOLO, L. A. and WAGNER, J. R. Effect of calcium salts and surfactant concentration on the stability of water-in-oil (w/o) emulsions prepared with polyglycerol polyricinoleate. Journal of colloid and interface science, vol. 341, no. 1, p. 101-108, 2010.

[251] MATSUI, Y.; SUGIYAMA, K.; KAMEI, M.; TAKAHASHI, T.; SUZUKI, T.;

[252] KATAGATA, Y. and ITO, T. Extract of Passion Fruit (Passiflora edulis) Seed Containing High Amounts of Piceatannol Inhibits Melanogenesis and Promotes Collagen Synthesis. Journal of Agricultural and Food Chemistry, vol. 58, no. 20, p. 11112-11118, 2010.

[253] MCCLEMENTS, D. J. Edible nanoemulsions: fabrication, properties, and functional performance. Soft Matter, vol. 7, no. 6, p. 2297-2316, 2011.

[254] MCCLEMENTS, D. J. Food emulsions: principles, practices, and techniques. CRC press, 2015. ISBN 1498726690.

[255] MEIRELES, M. A. A. Extraction of bioactive compounds from Latin American plants. In: (Ed.). Supercritical fluid extraction of nutraceuticals and bioactive compounds: CRC Press—Taylor & Francis Group, Boca Raton, 2008. p.243-274.

[256] MILLER, G. L. Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar. Analytical Chemistry, vol. 31, no. 3, p. 426-428, 1959.

[257] OECD. OECD GUIDELINE FOR TESTING OF CHEMICALS: Acute Oral

[258] Toxicity - Acute Toxic Class Method. No 423 2001.

[259] OECD. Guidance Document On Using Cytotoxicity Tests To Estimate Starting Doses For Acute Oral Systemic Toxicity Tests N°129. Series on Testing and Assessment. ENV/JM/MONO 20. 2010

[260] PATIL, L. and GOGATE, P. R. Ultrasound assisted synthesis of stable oil in milk emulsion: Study of operating parameters and scale-up aspects. Ultrasonics Sonochemistry, v. 40, no. Part A, p. 135-146, 2018.

[261] PEPE, S. Effect of dietary polyunsaturated fatty acids on age-related changes in cardiac mitochondrial membranes. Experimental Gerontology, vol. 40, no. 5, p. 369- 376, 2005.

[262] PIENTAWEERATCH, S.; PANAPISAL, V. and TANSIRIKONGKOL, A. Antioxidant, anti-collagenase and anti-elastase activities of Phyllanthus emblica, Manilkara zapota and silymarin: an in vitro comparative study for anti-aging applications. Pharm Biol, vol. 54, no. 9, p. 1865-72, 2016.

[263] PRIOR, R. L.; HOANG, H.; GU, L.; WU, X.; BACCHIOCCA, M.; HOWARD, L.; HAMPSCH-WOODILL, M.; HUANG, D.; OU, B. and JACOB, R. Assays for Hydrophilic and Lipophilic Antioxidant Capacity (oxygen radical absorbance capacity (ORACFL)) of Plasma and Other Biological and Food Samples. Journal of Agricultural and Food Chemistry, vol. 51, no. 11, p. 3273-3279, 2003.

[264] PRONYK, C. and MAZZA, G. Design and scale-up of pressurized fluid extractors for food and bioproducts. Journal of Food Engineering, vol. 95, no. 2, p. 215-226, 2009.

[265] REUTERS. Research and Markets: Phytonutrients Market 2015: Carotenoids, Phytosterols, Flavonoids, Phenolic Compounds, and Vitamin - Global Trends and Forecast to 2020. 2015.

[266] SINGLETON, V. L.; ORTHOFER, R. and LAMUELA-RAVENTÓS, R. M. Analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent. In: LESTER, P. (Ed.). Methods in Enzymology: Academic Press, v.Volume 299, 1999. p.152-178. ISBN 00766879.

[267] SLOMKOWSKI, S.; ALEMÁN, J. V.; GILBERT, R. G.; HESS, M.; HORIE, K.; JONES, R. G.; KUBISA, P.; MEISEL, I.; MORMANN, W. and PENCZEK, S. Terminology of polymers and polymerization processes in dispersed systems (IUPAC Recommendations 2011). Pure and Applied Chemistry, vol. 83, no. 12, p. 2229-2259, 2011.

[268] SPERANZA, P.; RIBEIRO, A. P. B.; CUNHA, R. L.; MACEDO, J. A. and MACEDO, G. A. Influence of emulsion droplet size on antimicrobial activity of interesterified Amazonian oils. LWT-Food Science and Technology, v. 60, no. 1, p. 207-212, 2015.

[269] SUN, C.; GUNASEKARAN, S. and RICHARDS, M. P. Effect of xanthan gum on physicochemical properties of whey protein isolate stabilized oil-in-water emulsions. Food Hydrocolloids, v. 21, no. 4, p. 555-564, 2007.

[270] TEMELLI, F. Perspectives on supercritical fluid processing of fats and oils. The Journal of Supercritical Fluids, vol. 47, no. 3, p. 583-590, 2009.

[271] THAIPONG, K.; BOONPRAKOB, U.; CROSBY, K.; CISNEROS-ZEVALLOS, L. and HAWKINS BYRNE, D. Comparison of ABTS, DPPH, FRAP, and ORAC assays for estimating antioxidant activity from guava fruit extracts. Journal of Food Composition and Analysis, vol. 19, no. 6, p. 669-675, 2006.

[272] TRABER, M. G. and ATKINSON, J. Vitamin E, antioxidant and nothing more. Free Radical Biology and Medicine, vol. 43, no. 1, p. 4-15, 2007.

[273] VIGANÓ, J.; AGUIAR, A. C.; MORAES, D. R.; JARA, J. L. P.; EBERLIN, M. N.; CAZARIN, C. B. B.; MARÓSTICA, M. R. and MARTÍNEZ, J. Sequential high pressure extractions applied to recover piceatannol and scirpusin B from passion fruit bagasse. Food Research International, vol. 85, p. 51-58, 2016a.

[274] VIGANÓ, J.; COUTINHO, J. P.; SOUZA, D. S.; BARONI, N. A. F.; GODOY, H.T.; MACEDO, J. A. and MARTÍNEZ, J. Exploring the selectivity of supercritical CO2 to obtain nonpolar fractions of passion fruit bagasse extracts. The Journal of Supercritical Fluids, vol. 110, p. 1-10, 2016b.

[275] WU, X.; BEECHER, G. R.; HOLDEN, J.M.; HAYTOWITZ, D. B.; GEBHARDT, S. E. and PRIOR, R. L. Lipophilic and Hydrophilic Antioxidant Capacities of Common Foods in the United States. Journal of Agricultural and Food Chemistry, vol. 52, no. 12, p. 4026- 4037, 2004.

[276] Ye, A.; HEMAR, Y. and SINGH, H. Enhancement of coalescence by xanthan addition to oil-in-water emulsions formed with extensively hydrolysed whey proteins. Food Hydrocolloids, v. 18, no. 5, p. 737-746, 2004.

[277] YEHUDA, S.; RABINOVITZ, S.; L. CARASSO, R. and I. MOSTOFSKY, D. The role of polyunsaturated fatty acids in restoring the aging neuronal membrane. Neurobiology of Aging, vol. 23, no. 5, p. 843-853, 2002.

[278] YOUDIM, K. A.; MARTIN, A. and JOSEPH, J. A. Essential fatty acids and the brain: possible health implications. International Journal of Developmental Neuroscience, vol. 18, no. 4, p. 383-399, 2000.

[279] YUKUYAMA, M. N.; GHISLENI, D. D. M.; PINTO, T. J. A. and BOU-CHACRA, N. A. Nanoemulsion: process selection and application in cosmetics - a review. International Journal of Cosmetic Science, vol. 38, no. 1, p. 13-24, 2016.

[280] ZHANG, T.; ZHANG, Q.; CHEN, J.; FANG, K.; DOU, J. and GU, N. The controllable preparation of porous PLGA microspheres by the oil/water emulsion method and its application in 3D culture of ovarian cancer cells. Colloids and Surfaces A: Physicochemical and Engineering Aspects, vol. 452, p. 115-124, 2014.

Claims (19)

1. Mini-emulsion characterized by being an oil-in-water emulsion and containing bioactive Passiflora fractions comprising: a. 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 c. from 0.05% to 2% w/v of a stabilizer. 2. Miniemulsion, according to claim 1, characterized by the raw material belonging to the genus Passiflora being Passiflora edulis. 3. Miniemulsion, according to any of the previous claims, characterized in that the part of the plant to be used as raw material is preferably selected from seed or bagasse. 4. Miniemulsion, according to one or more claims 1-3, characterized in that the hydrophobic phase:aqueous phase ratio is 1:3. 5. Miniemulsion, according to one or more claims 1-4, characterized by the hydrophilic bioactive fraction (aqueous phase) comprising piceatannol. 6. Miniemulsion, according to one or more claims 1-3, characterized by the lipophilic bioactive fraction (oil phase) comprising compounds belonging to the tocol group. 7. Miniemulsion, according to claim 6, characterized by the compounds belonging to the group of tocols comprising α-tocopherol, β-tocopherol, Y—tocopherol, δ-tocopherol, α-tocotrienol, Y—tocotriene, δ-tocotrienol and combinations thereof same. 8. Miniemulsion, according to claim 5, characterized in that the hydrophilic bioactive fraction is obtained through supercritical extraction. 9. Miniemulsion, according to claim 5, characterized in that the hydrophilic bioactive fraction is obtained through supercritical extraction with carbon dioxide as solvent. 10. Miniemulsion, according to claim 9, characterized in that the hydrophilic bioactive fraction is obtained through supercritical extraction with carbon dioxide as solvent preferably at 60 °C and 17 MPa (Step 1). 11. Miniemulsion, according to claim 6, characterized in that the lipophilic bioactive fraction is obtained through supercritical extraction (SFE) with carbon dioxide as solvent, sequentially, after obtaining the hydrophilic bioactive fraction. 12. Miniemulsion, according to claim 11, characterized in that the lipophilic bioactive fraction is obtained through supercritical extraction (SFE) with carbon dioxide as solvent, sequentially, preferably at 40°C and 34 MPa (Step 2). 13. Miniemulsion, according to any one of claims 1-4, characterized in that the bioactive fraction obtained through supercritical extraction (SFE) is in a single step (global) with carbon dioxide as solvent preferably at 40°C and 34 MPa . 14. Miniemulsion, according to claim 5, characterized in that the concentrated hydrophilic bioactive fraction is obtained through pressurized liquid extraction (PLE) preferably with a mixture of solvent selected from ethanol and water, sequentially, after obtaining the hydrophilic bioactive fractions and lipophilic (Stage 3 - PLE). 15. Miniemulsion, according to claim 14, characterized in that the concentrated hydrophilic bioactive fraction is obtained through extraction with pressurized liquid preferably at 70°C and 10 MPa. 16. Cosmetic formulation characterized by comprising a miniemulsion of bioactive Passiflora fractions, defined according to previous claims, associated with one or more vehicles (excipients, adjuvants, carriers, etc.) and/or cosmetically acceptable ingredients. 17. Cosmetic formulation characterized by comprising: - 1.00% miniemulsion of bioactive Passiflora fractions; - 83.5% purified water; - 0.05% Disodium EDTA; - 1.50% Acrylates copolymer; - 0.30% Acrylates/STEARETH-20 Methacrylate copolymer; - 2.52% Sodium Laureth Sulfate; - 0.18% Sodium Hydroxide; - 2.00% Methyl GLUCETH-20; - 3.60% Disodium Cocoamphodiacetate; - 0.50% Coco-Glucoside; - 1.60% Cocamidopropyl Betaine; - 3.00% PEG-40 Hydrogenated Castor Oil; - 0.70% Methylisothiazolinone (and) Phenoxyethanol; - Citric Acid, qsq to adjust the pH between 6.5 and 6.7, to be used as a facial cleansing lotion. 18. Cosmetic formulation characterized by comprising: - 3.00% miniemulsion of bioactive Passiflora fractions; - 26.70 glycerin; - 39.17% purified water; - 0.50% Triethanolamine; - 0.30% Acrylates/ C10-30 Alkyl acrylate crosspolymer; - 29.83% Cyclopentasiloxane (and) Dimethicone crosspolymer (and) Dimethicone (and) Laureth- 23 (and) Laureth-4 (and) aqua; - 0.50% Methylisothiazolinone (and) Phenoxyethanol; to be used as a facial moisturizing serum. 19. Cosmetic formulation characterized by comprising: - 3.00% miniemulsion of bioactive Passiflora fractions; - 3.00 of PPG-5 CETETHE-20; - 5.00 Glyceryl Stearate; - 5.00 Stearic Acid; - 2.00 Cetearyl alcohol (and) Cocoglucoside; - 1.00 Arachidyl alcohol (and) Behenyl alcohol (and) Arachidyl glucoside; - 1.00 Stearyl Alcohol; - 2.50 Zinc Oxide; - 15.00 Capric/Caprylic Triglyceride; - 0.25 Hydroxypropyl Guar; - 3.00 Glycerin; - 58.70 Purified Water; - 0.55 Methylisothiazolinone (and) Phenoxyethanol; to be used as a facial moisturizer with sun protection.