Antioxidant cosmetic compound, cosmetic composition, application and cosmetic care method

Abstract

The present invention relates to an antioxidant cosmetic composition which counteracts damage caused by oxidative stress and free radicals on the skin and / or scalp, which contains bioactive ingredients from Euteroleaceae (Acai), Thobroma cacao (cocoa) and Inga edulis (Inga), as well as to a cosmetic composition containing the same, to the use and to a method thereof.

Description

Technical Field

[0001] This invention relates to an antioxidant cosmetic complex that combats damage caused by oxidative stress and free radicals on the skin and / or scalp, comprising extracts from Amazonian berries ( Euterpe oleracea (açaí) , cocoa Theobroma cacao (cacau) and Inca beans ( Inga edulis Bioactive components of Inca Inca (ingá), and cosmetic compositions containing them, their uses and methods. Background Technology

[0002] The concept of nourishing tissue implies providing sufficient metabolic balance and redox homeostasis so that the tissue can function in the best possible way. In terms of photoprotection, it is important to assess whether active ingredients help maintain the redox balance of key cells to preserve skin homeostasis, such as keratinocytes, and how much of these active ingredients can prevent the formation of structures that reduce cell viability.

[0003] One of the most widely accepted theories of human aging proposes that aging is the result of cellular senescence, in which an increased number of cells reach the end of their lifespan, ceasing to divide. This limits the body's or tissue's ability to regenerate itself and respond to injury or stress. This process occurs naturally over time and with the number of cell divisions, which tends to gradually decrease with each successive division until the point of senescence is reached.

[0004] Cellular aging can also be externally activated, especially in surviving skin exposed to environmental factors. Completely avoiding sun exposure has never been a viable alternative for humans, as skin and individual health depend on continuous sun stimulation. On the other hand, sun exposure is a major factor in causing redox imbalance, which leads to cell death or aging, or, in the worst case, induces malignant transformation. Therefore, avoiding redox imbalance through antioxidant alternatives is key to developing anti-aging skin products.

[0005] Cellular homeostasis is maintained by a balance between the rates of formation and inhibition of reactive oxygen species (ROS) and reactive nitrogen species (RNS), a balance finely controlled by redox signaling mechanisms. However, the rate of oxidative stress production can significantly exceed the capacity of the maintenance system, leading to an imbalance. Redox imbalance involves a range of physiological conditions and can result in the formation of late-stage glycation or lipid peroxidation products, which hinder cell survival and promote cellular aging.

[0006] The recognition of free radical reactions as promoters of the aging process and the appearance of the first signs of aging (including premature graying of hair) implies interventions aimed at limiting or inhibiting them.

[0007] Plant bioactive substances have been studied because, in addition to their direct antioxidant effects, they can also exert antioxidant protective effects through protective membranes. The relationship between antioxidant capacity and membrane protective efficacy can be used to measure the performance of plant bioactive substances. The ability of a given compound to protect biomembranes is related not only to the antioxidant capacity of the bioactive substance but also to the level of interaction with the biomembrane.

[0008] Therefore, there is still a search in the field for new bioactive agents or bioactive complexes that can effectively combat damage caused by oxidative stress and free radicals in the skin and / or scalp. Attached Figure Description

[0009] Figures 1-3 The result of the percentage of antioxidant protection of the test sample.

[0010] Figure 4 Absorption transients of triplet-state material (derived from methylene blue) after 660 nm laser photon excitation in the absence and with gradually increasing concentrations of β-carotene. All samples were purged with argon for 5 minutes to reduce dissolved oxygen concentration.

[0011] Figure 5 The lifetime of the triplet state is a function of β-carotene concentration.

[0012] Figure 6 The inhibition constant was calculated using the Stern-Volver model, and only the lifetime at the four lowest concentrations of β-carotene was used.

[0013] Figure 7 The absorption transient of the triplet state (derived from methylene blue) at 470 nm after 660 nm laser photon excitation, under conditions of absence and with gradually increasing concentrations of the active substance 6714. All samples were purged with argon for 5 minutes to reduce dissolved oxygen concentration.

[0014] Figure 8 The inhibition constant calculated using the Stern-Volver model in the presence of gradually increasing concentrations of active substance 6714.

[0015] Figure 9 The absorption transient of the triplet state (derived from methylene blue) at 470 nm after 660 nm laser photon excitation was observed in the absence and with gradually increasing concentrations of the active substance 55107. All samples were purged with argon for 5 minutes to reduce dissolved oxygen concentration.

[0016] Figure 10 The inhibition constant calculated using the Stern-Volver model in the presence of gradually increasing concentrations of active substance 55107.

[0017] Figure 11 The absorption transient of the triplet state (derived from methylene blue) at 470 nm after 660 nm laser photon excitation, under conditions of absence and with gradually increasing concentrations of the active substance 55825. All samples were purged with argon for 5 minutes to reduce dissolved oxygen concentration.

[0018] Figure 12 The inhibition constant calculated using the Stern-Volver model in the presence of gradually increasing concentrations of active substance 55825.

[0019] Figure 13 The absorption transient of the triplet state (derived from methylene blue) at 400 nm after 660 nm laser photon excitation, under conditions of absence and with gradually increasing concentrations of the active substance 9617. All samples were purged with argon for 5 minutes to reduce dissolved oxygen concentration.

[0020] Figure 14 The inhibition constant calculated using the Stern-Volver model in the presence of gradually increasing concentrations of active substance 9617.

[0021] Figure 15 The absorption transient of the triplet state material (derived from methylene blue) at 470 nm after 660 nm laser photon excitation, with and without blend 1 at gradually increasing concentrations. All samples were purged with argon for 5 minutes to reduce dissolved oxygen concentration.

[0022] Figure 16 The inhibition constant calculated using the Stern-Volver model in the presence of gradually increasing concentrations of blend 1. The inhibition constant is expressed as % (volume:volume).

[0023] Figure 17 The absorption transient of the triplet state material (derived from methylene blue) at 470 nm after 660 nm laser photon excitation, with and without blend 2 at gradually increasing concentrations. All samples were purged with argon for 5 minutes to reduce dissolved oxygen concentration.

[0024] Figure 18 The inhibition constant calculated using the Stern-Volver model in the presence of gradually increasing concentrations of blend 2. The inhibition constant is expressed as % (volume:volume).

[0025] Figure 19 Transient phosphorescence emission at 1270 nm in ethanol in the absence and with gradually increasing concentrations of β-carotene.

[0026] Figure 20 : The 1O2 inhibition constant calculated using the Stern-Volver model in the presence of gradually increasing concentrations of β-carotene.

[0027] Figure 21 Transient phosphorescence emission at 1270 nm in ethanol in the absence and with gradually increasing concentrations of active substance 6714.

[0028] Figure 22 The 1O2 inhibition constant calculated using the Stern-Volver model in the presence of gradually increasing concentrations of active substance 6714.

[0029] Figure 23 Transient phosphorescence emission at 1270 nm in ethanol in the absence and with gradually increasing concentrations of active substance 55107.

[0030] Figure 24 : The 1O2 inhibition constant calculated using the Stern-Volver model in the presence of gradually increasing concentrations of active substance 55107.

[0031] Figure 25 Transient phosphorescence emission at 1270 nm in ethanol in the absence and with gradually increasing concentrations of active substance 55825.

[0032] Figure 26 The 1O2 inhibition constant calculated using the Stern-Volver model in the presence of gradually increasing concentrations of active substance 55825.

[0033] Figure 27 Soy lecithin liposomes in 10 mM Tris buffer, 0.3 M sodium chloride (pH=8), 15 μM photosensitizer (DMMB), and different samples were used. %CF release as a function of irradiation time was measured. (A and B) Dark plates and (C and D) Irradiated plates.

[0034] Figure 28 Viability is expressed as no irradiation and UVA irradiation (12 J cm⁻¹). -2 The percentage of MTT reduction in HaCat cells after 24 hours. EtOH1 and EtOH2 controls correspond to the amounts of ethanol in blend 1 and blend 2, respectively. Blend 1: 6714 (0.01 mg / mL) + 55107 (0.02 mg / mL) + 55285 (0.02 mg / mL); Blend 2: 6714 (0.01 mg / mL) + 9617 (0.01 mg / mL) + α-Tocopherol (0.01 mg / mL).

[0035] Figure 29 UVA irradiation (12J cm) -248 hours later, the accumulation of lipofuscin in HaCaT cells was detected by Sudan Black B staining.

[0036] Figure 30 UVA irradiation (12J cm) -2 Quantification of Sudan Black B-stained granules in HaCaT cells 48 hours later. Summary of the Invention

[0037] This invention relates to an antioxidant cosmetic complex that combats damage caused by oxidative stress and free radicals on the skin and / or scalp, comprising extracts from Amazonian berries ( Euterpe oleracea (açaí) , cocoa Theobroma cacao (cacau) and Inca beans ( Inga edulis Bioactive components of Inca Inchi (ingá).

[0038] The bioactive components of the present invention can be obtained from specific parts of plant species by methods known in the art. Without any limitation, for example, they can be obtained from epiphytic plant parts, such as stems, leaves and flowers, or only from leaves and from agricultural industrial residues, such as seeds, mesocarp fibers or mucilage.

[0039] In one specific embodiment, the present invention relates to an antioxidant cosmetic complex that combats damage caused by oxidative stress and free radicals in the skin and / or scalp, comprising, based on the total weight of the cosmetic composition: (a) Approximately 0.0002% to approximately 20% of Amazonberries ( Euterpe oleracea ); (b) Approximately 0.0001% to approximately 20% cocoa ( Theobroma cacao ); (c) Approximately 0.0002% to approximately 20% Inca beans ( Inga edulis ).

[0040] The percentages mentioned above and in the following examples are based on the total weight of the final cosmetic composition in which the complex is delivered.

[0041] In another embodiment, the present invention further encompasses cosmetic compositions comprising the antioxidant cosmetic complex of the present invention and cosmetically acceptable excipients.

[0042] The cosmetically acceptable excipients of this invention are those known to those skilled in the art for preparing various forms of cosmetic matrices, such as lotions, creams, gels, serums, and other forms known to those skilled in the art. For example, without any limitation, cosmetically acceptable excipients may be selected from those cited in the International Dictionary and Handbook of Cosmetic Ingredients, 16th edition.

[0043] In another embodiment, the invention further relates to the use of antioxidant cosmetic complexes in the preparation of cosmetic compositions for combating damage caused by oxidative stress and free radicals of the skin and / or scalp.

[0044] Furthermore, in another embodiment, the present invention relates to a method for topical cosmetic treatment of damage to the skin and / or scalp caused by oxidative stress and free radicals, comprising applying an effective amount of the compound or cosmetic composition of the present invention to the skin and / or scalp, whether or not it is needed. Therefore, the methods discussed herein can be cosmetic prevention or treatment.

[0045] The following non-limiting examples illustrate specific embodiments of the invention and demonstrate the effectiveness of the complexes of the invention in treating damage caused by oxidative stress and free radicals. Example

[0046] The antioxidant potential of different bioactive substances and their potential synergistic effects when evaluated together are compared and evaluated in order to develop new ingredients to effectively combat damage caused by oxidative stress in cosmetic compositions.

[0047] In this sense, the antioxidant potential of six plant components was assessed by neutralizing DPPH molecules in the proposed biochemical evaluation and in primary fibroblast cell cultures.

[0048] Furthermore, inhibition of excited states (triple and singlet oxygen) was measured by comparing the ability to assess the inhibitory activity state, and membrane protection was evaluated (using liposomes for spectroscopic testing). The results were compared with gallantanine (the most potent membrane protectant known to date).

[0049] The resulting anti-aging activity of the test components was evaluated in human keratinocytes (HaCaT). Viability protection and anti-aging activity were tested in a UVA-induced damage model. The endpoint was the level of accumulated lipofuscin with and without treatment. Cell viability was monitored by MTT assay, and anti-aging capacity was assessed by the reduction in lipofuscin accumulation.

[0050] The analyzed sample was identified as follows: 55181: Brazilian Pepper Tree (aroeira) Extract - Dissolved in ethanol 53797: Guaçatonga extract - soluble in water 57713: Jambu extract - dissolves in water 6714: Cocoa extract - dissolved in ethanol 55107: Inca Ingá (Inca) Bean Extract - Dissolves in water 55285: Acai berry (açaí) bioactive substances - soluble in water 9617: Green tea - dissolves in water α-Tocopherol Sample 9617 and α-tocopherol were considered only in the comparison of blend 1 x blend 2.

[0051] In the following examples, "blend" means a test mixture or complex containing more than one bioactive ingredient.

[0052] The concentrations used in the excited-state inhibition, membrane protection, and cell viability (MTT) assays were: 6714: 0.01 mg / mL 55107: 0.02 mg / mL) 55285: 0.02 mg / mL Blend 1 - 6714 (0.01 mg / mL) + 55107 (0.02 mg / mL) + 55285 (0.02 mg / mL) Blend 2 - 6714 (0.01 mg / mL) + 9617 (0.01 mg / mL) + α-Tocopherol (0.01 mg / mL) Concentration in anti-aging experiment (lipofuscin accumulation): Blend 1 - 6714 (0.01 mg / mL) + 55107 (0.02 mg / mL) + 55285 (0.02 mg / mL) Blend 2 - 6714 (0.01 mg / mL) + 9617 (0.01 mg / mL) + α-Tocopherol (0.01 mg / mL)

[0053] Example 1 Biochemical DPPH

[0054] The biochemical assay using DPPH (2,2-diphenyl-1-picrylhydrazine) is based on the reduction of stable free radicals in an ethanol solution by a substance with antioxidant activity. DPPH reduction is observed by a decrease in absorbance, as DPPH exhibits a purple color with absorption at 517 nm in its free radical form. In the presence of antioxidants or other free radical substances, the absorption disappears or decreases. Therefore, the use of DPPH allows for the evaluation of the antioxidant potential present in various compounds and plant extracts.

[0055] Results were obtained via IC50, which showed the minimum concentration required to neutralize 50% of DPPH radicals in the solution.

[0056] Based on these values, use IC 50 Concentrations were tested again on all samples, with samples combined in three groups to evaluate the protective synergy of the combination.

[0057] It is possible Figures 1-3 It was observed in IC 50 At the concentrations, all combinations exceeded the 50% protection achieved with separate samples, and blends of active ingredients provided up to 20% more antioxidant protection compared to each active ingredient alone.

[0058] Example 2 Cellular DPPH

[0059] The DPPH reduction assay using endogenous antioxidants was performed on human skin fibroblasts. The basis of this assay (a modification of the conventional DPPH assay) is to assess the potential production of endogenous antioxidants (generated by cells) after stimulating cells with different concentrations of the test compound. After exposing cells to the active ingredient, a solution containing DPPH was added to the plate. Therefore, DPPH reduction is a result of cellular synthesis of endogenous antioxidants under the influence of the active substance.

[0060] In addition to individual components, the top five results from combinations of active ingredients from biochemical DPPH were selected for cellular DPPH testing.

[0061] In IC 50 At the concentrations, all combinations were found to outperform the protection achieved by the isolated samples. Considering the optimal results from the combination of ingredients, the combination of acai berry + cocoa + Sacha Inca was the most effective in increasing antioxidant protection against free radicals compared to each of the individual active ingredients.

[0062] Example 3: Suppression of Excited States

[0063] The ability to suppress excited states was assessed by measuring the transient absorption of the triplet state using flash photolysis and by evaluating the oxygen emission of the singlet state in the near-infrared region.

[0064] Singlet oxygen suppression: The detector (PMT Hamamatsu R5509 - model PC176TSCE005) was cooled to -80°C and set to 1500 V, and the signal was acquired using proprietary software from SHB Analytics GmbH (Germany). Excitation was performed using a Quantel Brilliant laser (2W-3W-Rainbow). The emission from the sample was filtered to detect irradiation in the range of 1270 ± 20 nm. A microcomputer was connected to the system, and transient analysis was performed using an ultrafast acquisition plate. It is important to remember that emission at 1270 nm is characteristic of singlet oxygen and is the safest method for detecting the presence and reactivity of this substance. Decay kinetics provide direct information about the inhibitory efficacy of the active ingredient against singlet oxygen.

[0065] Triple state suppression: using an SHB laser flash photolysis system ( Figure 14 The sample is irradiated with a laser pulse (10 mJ / pulse, 10-20 ns) to provide electronic excitation. To assess the formation and lifetime of excited states or other reactive intermediates, the absorbance of the sample before and after electronic excitation is monitored via an LED beam. It monitors the disappearance of substances in the ground state or the appearance of excited or free radical substances. The laser beam is positioned at 90° to the monitoring beam. A shutter is used in both the excitation and monitoring beams to protect the sample from photolysis. The detector in this technique consists of a combination of a filter and a diode detector. The diode response is read on a digital oscilloscope (Tektronix 2230), and the data is then transmitted to a microcomputer. The entire setup is controlled by a microcomputer, and the instrument has a time resolution of approximately 50 ns. A Quantel Nd:YAG laser (2W, 3W Rainbow, 3ns pulse, 80.1% power) is used. An excitation wavelength of 660 nm is selected in the OPOTEK software. Transient absorption after the laser pulse is measured in a simple photometer based on LEDs (blue, green, and red) and filters. Readings are taken in a 2 mm × 10 mm quartz cuvette. The methylene blue (photosensitizer) sample is dissolved in the specified solvent and bubbled with argon gas to remove oxygen.

[0066] Suppression of triplet substances

[0067] Measurements were performed on a laser flash photolysis system from SHB Analytics GmbH. Quantel Nd:YAG lasers (2W, 3W, Rainbow, with 3ns pulses) were used to excite the sample. Triplets were generated by exciting a 10 μM methylene blue solution in a solution purged with argon to remove oxygen. Oxygen was removed to increase the lifetime of the triplet and allow us to evaluate its reactions with other compounds. In the presence of oxygen (in a saturated solution), the triplet reacts rapidly with oxygen, preventing inhibition of other active ingredients. The formation of the triplet was observed through the transient absorption of this substance, i.e., by the increase and subsequent decrease in absorption at a wavelength of 470 nm. Figure 4 ).

[0068] Note that in Figure 4 In the diagram, the orange line represents the result obtained without the addition of inhibitors. The increased uptake is almost instantaneous (ICS processes occur on a sub-nanosecond scale), and due to the residual oxygen concentration in the solution (approximately 500 times lower than in an air-equilibrium solution), the uptake of this substance decays over time in the range of hundreds of microseconds. In the presence of an air-equilibrium solution (saturated oxygen concentration), the decay of the triplet substance occurs in the sub-microsecond range. It should also be noted that in... Figure 4 In this study, adding progressively increasing concentrations of β-carotene resulted in a decrease in the lifetime of the triplet state substance; that is, the triplet state substance lasted for a shorter period due to the inhibition by β-carotene. It is worth noting that β-carotene was used here as a standard inhibitor because it effectively inhibits both the triplet state substance and singlet oxygen.

[0069] The lifetime of triplet matter is estimated by fitting a decay function to the exponential decay. Note that in Figure 5 In this study, the lifetime of the triplet state decreased with increasing β-carotene concentration. This decay was linear only at low concentrations of the inhibitor, therefore we used only points to estimate the inhibition constant. Figure 6 The inhibition constant was calculated using the Stern-Volmer model, where the decay time in the presence of the active substance was divided by the time in its absence, and this ratio (τ / τ0) was plotted as a function of the inhibitor concentration. Figure 6 ).

[0070] All tested active substances showed some degree of triplet inhibition, with decreasing potency following the order: β-carotene > 6714 > 55107 > 55825 > 9617 (Table 1). Both blends showed high triplet inhibition potency, providing very clear and statistically robust Stern-Volmer plots. Individual inhibition constants and inhibition constants in the blends are not comparable because they are expressed in units of difference.

[0071] Singlet oxygen suppression

[0072] To assess the ability of the active substance to inhibit singlet oxygen (1O2), near-infrared emission (1270 nm) was measured, which is a characteristic of 1O2 and the gold standard used for this detection. β-carotene was also used as a positive inhibition standard.

[0073] The detector (PMT Hamamatsu - Model H10330-45) was cooled to -80°C and set to -800 V, and the signal was acquired using proprietary software from SHB Analytics GmbH (Germany). Excitation was performed using a Quantel Brilliant Nd:YAG laser (2W-3W-Rainbow). Sample emission was detected in the range of 1270 ± 20 nm. A microcomputer was connected to the system, and transient analysis was performed using an ultrafast acquisition plate. It is important to remember that emission at 1270 nm is characteristic of singlet oxygen and is the safest method for detecting the presence and reactivity of this substance. Decay kinetics provide direct information about the inhibitory efficacy of the active ingredient against singlet oxygen.

[0074] In addition to β-carotene, singlet oxygen inhibition was unconditionally observed for compound 6714. The inhibition constant of this active substance is about an order of magnitude lower than that of the β-carotene standard (Table 4). Active substances 55107 and 55825 were ineffective in inhibiting 1O2. Inhibition constants were calculated for illustrative purposes, but no correlation was found between inhibition and active substance concentration. Note the poor linear fit and very low R².

[0075] Example 4: Membrane Protection

[0076] Soybean lecithin liposomes were used as a membrane mimicry model. The protection against oxidative damage and membrane integrity was quantified using methods known in the art.

[0077] Measurement of light-induced carboxyfluorescein (CF) release from liposomes

[0078] Liposome suspensions were prepared using 30 mg soybean lecithin and 1 mL of 50 mM CF membrane, hydrated in Tris buffer (pH=8), to produce liposome suspensions with CF encapsulated in internal compartments. Unencapsulated CF was removed by size exclusion chromatography on a Sephadex G-50 column equilibrated with 0.3 M sodium chloride in 10 mM Tris buffer (pH=8). The liposome-containing fractions were visually identified and collected in clean vials. Membrane damage was quantified in 96-well fluorescent microplates. The volume of the liposome suspension was always 7 μL, and the concentration of the photosensitizer 1,9-dimethylmethylene blue (DMMB) was set at 15 μM (except for the control without photosensitizer). Each well was then filled with 0.3 M sodium chloride, DMMB, and sample solution in 10 mM Tris buffer (pH=8) to ensure a final volume of 300 μL. LED light was used at a maximum emission wavelength of 650 nm and a wavelength of 13.5 mW / cm². -2 The entire microplate was irradiated with an irradiance of (Ethik Technology©, São Paulo, Brazil). Fluorescence at 517 nm (ICF) was monitored as a function of irradiation time using a microplate reader operated at 480 nm excitation. At the end of the experiment, 10% (v / v) Triton X-100 was added to each well, and the fluorescence intensity (ICFT) was recorded again. For each ICF value, the percentage of CF released (released %CF) was calculated.

[0079] Correlation of carboxyfluorescein release with irradiation duration ( Figure 27 Note the contrast in the dark. Figure 27 A and Figure 27 B) exhibited low levels of CF release (approximately 12%), while liposomes irradiated with red light ( Figure 27 C) A significant increase in CF release was rapidly observed after the initial minutes of irradiation, reaching 100% leakage within 30 minutes. This was due to the generation of triplet substances and singlet oxygen in the membrane, leading to the formation of several lipid peroxidation products. Particularly associated with membrane leakage was the formation of truncated lipid aldehydes, which caused membrane leakage.

[0080] The aim of the experiment was to quantify liposome leakage in the presence of Natura's active ingredients. Note that, except for active ingredient 6714, almost all tested active ingredients provided measurable levels of protection. For individual active ingredients, the level of protection decreased in the following order: 55285 > 55107 > 6714. In the blends, note that after 40 minutes of irradiation, blends 1 and 2 protected approximately 50% of the vesicles (…). Figure 27 D).

[0081] Termination of membrane protection method

[0082] The ability of an extract to protect the membrane from oxidative leakage depends on the antioxidant capacity of the active ingredient and its ability to interact with membrane lipids.

[0083] As the skin's first line of defense, corneal extract (CE) undergoes several changes after sun exposure. Lipid oxidation in CE was investigated using molecular dynamics simulations. The behavior of lipids oxidized in the CE bilayer is similar to that observed in less complex model systems (e.g., vesicles), exhibiting a significantly increased permeability to reactive oxygen species, thus generating a self-feeding oxidation cycle.

[0084] Protecting skin lipids from photo-induced oxidation is an interesting approach to prevent damage from the spread of light exposure. However, changes in CE (crease-forming epidermal keratinocytes) linked to increased TWEL (transepidermal water loss) 48 hours after sun exposure appear to be associated with excessive proliferation of keratinocytes that no longer differentiate properly, resulting in abnormal lamellar structures within the CE. Therefore, assessing the effects of active ingredients on keratinocytes is also important to provide a comprehensive hypothesis on how they play a role in maintaining skin homeostasis.

[0085] Example 5: Measurement of anti-aging activity in human keratinocytes

[0086] Two experiments were conducted in this model. The first involved evaluating the effects of the extracts on HaCaT keratinocyte viability after darkness (cytotoxicity) and UVA irradiation (photoprotection) using the MTT assay. In this experiment, the effects of pure extracts 6714, 55107, and 55285, as well as mixtures labeled Blend 1 and Blend 2, were evaluated. In the second experiment, using lipofuscin accumulation as the cellular aging endpoint, we assessed the ability of Blend 1 and Blend 2 to promote anti-aging effects in UVA-exposed HaCaT cells.

[0087] HaCaT cell viability as measured by MTT assay Immortized human skin keratinocytes (HaCaT) were seeded in 48-well cell culture plates (2 x 10⁻⁶ cells / well). 4 Cells / well). The next day, cells were washed with PBS (2x) and incubated for 1 hour in serum-free DMEM medium containing the different samples under study. Since samples 6714 and α-tocopherol (blend 2) were prepared in ethanol, a series of controls were also analyzed using the corresponding amounts of ethanol used in the studies of these samples. After 1 hour of treatment, cells were washed and incubated in PBS with UVA (12 J / cm²). -2 Irradiation. 24 hours after irradiation, a colorimetric determination based on MTT reduction was performed. Figure 28HaCaT cells were incubated for 2 hours in a 37°C culture oven with a solution of 50 μg / mL MTT in 1% DMEM medium, and then the formazan was dissolved in 300 μL / well dimethyl sulfoxide (DMSO). The absorbance was measured at 550 nm using a Spectramax plate reader.

[0088] exist Figure 28 In the results presented, it was noted that all black bars were similar, and the activity values ​​of the control and bioactive substances were close to 100%, indicating that no bioactive substance exhibited measurable levels of toxicity in the dark; that is, no active substance showed cytotoxicity to HaCaT cells. It should also be noted that in the control (no treatment with the active ingredient or alcohol was performed), Figure 28 (See the bar on the left). UVA irradiation caused keratinocyte viability to decrease from 100% to 67%. It should also be noted that keratinocytes previously treated with the active ingredient and even with ethanol showed increased viability (approximately 20%) compared to keratinocytes irradiated with UVA. Keratinocytes previously treated and irradiated with blends 1 and 2 showed approximately 90% viability, representing a 25% increase in protection against UVA effects. Figure 28 It should also be noted that there was no significant difference in the photoprotective effect of the two blends on keratinocyte viability; however, both showed greater protective efficacy compared to the untreated control. Figure 28 ).

[0089] Example 5: Testing the anti-aging properties of lipofuscin

[0090] These procedures follow the best practices of the most current technology. For aging that can be measured within 48 hours, UVA can be considered a substance that increases the rate of cellular aging, which naturally occurs after dozens of mitotic divisions. Oxidative damage caused by UVA in mitochondria and lysosomes disrupts the autophagy flux in the homeostatic axis between these two organelles, which peaks in lipofuscin accumulation.

[0091] Immortalized human keratinocyte (HaCaT) cell lines (passages 11 and 12) were grown in Dulbecco modified Eagle medium (DMEM) supplemented with 10% (v / v) fetal bovine serum, 4 mM L-glutamine, 100 U / mL penicillin, and 100 pg / mL streptomycin, and incubated at 37°C and 5% CO2. HaCaT cells (2 x 10⁻⁶ cells) were then cultured in 37°C and 5% CO₂. 5 Cells were seeded (per well) on coverslips in 6-well plates. After 24 hours, the cells were washed in PBS and treated with blend 1 and blend 2 for 1 hour. The cells were washed again and treated with UVA at 12 J / cm². -2Cells were irradiated with a dose of PBS. After irradiation, the cells were washed and incubated with culture medium for 48 hours. After 48 hours, the cells were washed twice with PBS, fixed with 4% (w / v) formaldehyde for 15 minutes, and washed again with PBS. The cells were then stained with Sudan Black B solution (SBB, 0.7%, in 70% ethanol) for 30 minutes, washed with distilled water, and visualized using coverslips on a Zeiss Axiovert 200 inverted microscope with Plan-APOCHROMAT unit structures. Images were obtained using bright-field transmitted light microscopy. Images were quantized using Image-J software (NIH, Bethesda, MA, USA) by converting TIF images to 8-bit grayscale and adjusting the brightness / darkness levels. SBB-marked regions were defined by selecting regions of units using polygonal selection, obtaining data on the regions, average grayscale values, and integrated image density. Values ​​in the figure were obtained by calculating the mean ± standard deviation by dividing the integrated intensity by the area.

[0092] Note that control cells kept in the dark showed much lower levels of SBB staining than those irradiated with UVA. Figure 29 As mentioned above, this level of SBB contrast is due to lipofuscin accumulation (the SBB staining level in irradiated cells is actually twice the value in irradiated cells). It should also be noted that blends 1 and 2 showed lower contrast levels than the control, indicating that both blends are able to reduce UVA-induced aging. Figure 29 and Figure 30 When comparing the SBB contrast in keratinocytes previously treated with blend 1 or blend 2, observed individually in each experiment, blend 1 showed a smaller change in lipofuscin accumulation compared to its unirradiated control, suggesting a greater protective effect against this pigment accumulation. Figure 30 ).

[0093] Partial conclusions regarding anti-aging effects

[0094] The anti-aging effect can be explained by the antioxidant and free radical scavenging activities and the protection of membranes from reactive substances, which prevents the accumulation of oxidative damage, maintains biomolecules and organelles, and reduces the accumulation of lipofuscin.

[0095] Biochemical and cellular DPPH results indicate that the antioxidant cosmetic complex of the present invention has a greater ability to neutralize free radicals compared to individual ingredients.

[0096] Furthermore, the results of excited-state inhibition, membrane protection, and anti-aging experiments show that the antioxidant cosmetic complex of the present invention has the ability to protect the membrane and increase cell vitality and reduce cell aging.

[0097] The antioxidant cosmetic complex of the present invention has a greater ability to provide protection against the accumulation of oxidative damage, maintain biomolecules and organelles, and inhibit lipofuscin accumulation to a greater extent compared with its irradiated control.

[0098] Therefore, taking into account all the results presented together, the antioxidant cosmetic complex of the present invention proves to be surprisingly more effective in neutralizing free radicals and inhibiting the accumulation of oxidative damage.

[0099] Regarding the antioxidant effects, the test results presented herein for the antioxidant cosmetic complex of the present invention show a 20% increase in antioxidant protection, a 36% increase in the synthesis of endogenous antioxidants, and a 60% increase in protection against oxidative damage to membranes.

[0100] Therefore, the complexes of the present invention can be used in emulsions, nanoemulsions, suspensions, sunscreens, powders, waxes, oils, or any other cosmetic form suitable for cosmetics.

[0101] Those skilled in the art can readily assess the advantages of the invention through its teachings and examples, and can provide variations and equivalent alternatives to the embodiments without departing from the scope of the invention as defined in the appended claims.

Claims

1. An antioxidant cosmetic complex, characterized in that, It contains Amazonian berry ( Euterpe oleracea ),cocoa( Theobroma cacao ) and Inca beans ( Inga edulis ).

2. The complex according to claim 1, characterized in that, Based on the total weight of the cosmetic composition, it comprises: (a) Approximately 0.0002% to approximately 20% of Amazonberries ( Euterpe oleracea ); (b) Approximately 0.0001% to approximately 20% cocoa ( Theobroma cacao ); (c) Approximately 0.0002% to approximately 20% Inca beans ( Inga edulis ).

3. A cosmetic composition, characterized in that, It comprises an antioxidant cosmetic complex as defined in any one of claims 1 or 2, and a cosmetically acceptable excipient.

4. Use of the complex as defined in any one of claims 1 or 2, characterized in that, It is used to prepare cosmetic compositions for combating oxidative stress and free radical damage to the skin and / or scalp.

5. A cosmetic care method, characterized in that, It includes applying the complex as defined in any one of claims 1 or 2 to the skin and / or scalp.

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