Antiaging effects of a skin care formulation containing nanoencapsulated antioxidants: A clinical, in vitro, and ex vivo study

The development of effective cosmetic products for the reduction of the signs of skin aging is a complex process which requires an optimized combination of ingredients and specialized systems to deliver the actives to the skin layers.


| INTRODUC TI ON
Skin aging represents a multifactorial progressive process, with an inevitable and practically irreversible course.As the most external barrier, the skin is in direct contact with numerous environmental factors that damage the morphological structures of the epidermis and dermis, rapidly leading to aesthetic changes.Macroscopically, photoaging leads to significant changes that include the formation of wrinkles, blemishes, dryness, increased barrier fragility, loss of elasticity and firmness, and subcutaneous fat accumulation. 1,2ssibly, the formation of wrinkles is the most obnoxious clinical sign during the facial aging process, induced by the loss of skin support because of the decline in collagen production.This essential scaffold protein constitutes about 90% of the dermal fibers, mainly collagen type III, conferring firmness, elasticity, and mechanical resistance to the skin. 1 The primary mechanism involved in the skin aging process is the overproduction of free radicals after chronic skin exposure to various external aggressions such as sun exposure, pollution and physical, chemical and biological insults. 3Free radicals are unstable and highly reactive molecules that adversely alter lipid, protein and DNA structures, triggering a series of signaling leading, for example, to increased production of dermal proteolytic enzymes such as matrix metalloproteinase-1 (MMP-1). 3[5] Pro-oxidant species also affect epidermal melanocytes, compromising melanin synthesis and inducing malignant transformation. 6spite the crucial role of melanin in protecting cellular components from oxidative damage, the abnormal pro-oxidant accumulation due to photoexposure as well as chronic inflammatory processes may increase the synthesis of factors such as melanocyte stimulating hormone and endothelin-1, which trigger additional melanization of the skin. 7Moreover, photoexposure favors the transfer of melanosomes from melanocytes to keratinocytes, intensifying the aesthetic discomfort of skin aging such as formation of age spots and melasma. 8ncurrently, chronological aging promotes disturbances in the epidermis, reducing the agility of prompt restoration mechanisms.In aged skin, the following structures and functions of the skin barrier are disrupted: skin barrier structure, permeability barrier function, epidermal calcium gradient, epidermal lipid synthesis, cytokine production and response after insults, antimicrobial barrier, and stratum corneum (SC) (hydration. 9Maintenance of optimal SC hydration is an important function of the epidermis and is dependent on some factors such as the amount of natural moisturizing factors (NMFs), which are a complex mixture of low molecular-weight substances produced within corneocytes by filaggrin degradation.The epidermal protein profilaggrin is lately synthesized during epidermal differentiation and plays a key role in generating and maintaining SC flexibility and moisturizing. 9e development of effective products for the prevention of aging and for skin rejuvenation is a complex process that requires an optimized combination of ingredients and specialized systems to deliver the actives to the skin layers.In the present study, we investigated the antiaging efficacy of a cosmetic formulation (AHHF-010) containing a blend of nanoencapsulated ingredients-ascorbyl palmitate, resveratrol, tocopherol, caffeine, carnosine, and niacinamide in improving skin functions, through in vitro and clinical trials.
The clinical efficacy of AHHF-010 was demonstrated by subjective and instrumental analyses of increased collagen synthesis, reduction of wrinkles, and improvement of skin tone and hydration.
Cutaneous and ocular tolerability of this formulation were also clinically determined.Additionally, we conducted a preclinical exploratory study to identify the mechanisms of AHHF-010 in containing oxidative stress, reducing melanin production and increasing the production of type I procollagen and of the epidermal cohesion protein filaggrin, using the experimental model of human cell and skin cultures.
Cosmetic products that alleviate the signs of skin aging are necessary to maintain the structure and healthy function of the skin throughout the years.As people are attacked by oxidative agents such as UV radiation and pollution on a daily basis, cosmetics with effective delivery of antioxidants are allies in maintaining a healthier skin.

| Evaluation of antioxidant activity against IR-A in a human fibroblast culture
Primary dermal fibroblasts isolated from elective abdominoplasty were maintained at 37°C, 5% CO 2 with high glucose DMEM (Corning) supplemented with 10% Bovine Fetal Serum (Gibco) and 0.1% gentamicin (Gibco).After 80%-90% confluence, cells were seeded into chamber slides (Nunc Lab-Tek) and incubated with 3 noncytotoxic concentrations of AHHF-010 (10.01, 3.17 and 1.00 mg/ mL), based on previous results of cytotoxicity assays (data not shown).After a 48-h treatment, cells were exposed to a 360 J/cm 2 dose of IR-A radiation using the Hydrosun 750 and HBM1 devices (Hydrosun Medizintechnik GmbH).After an additional 24 h, cells were incubated with MitoSOX™ Red (ThermoFisher Scientific) for further semi-quantitation of mitochondrial reactive oxygen species (ROSmit) synthesis.Cell images were analyzed with a fluorescence microscope (Olympus Corporation) with cellSens Standard software (Olympus Corporation).After obtaining the images, fluorescence intensity was semi-quantified with ImageJ software (Arbitrary Units -A.U.). 10 2.2.2 | Evaluation of melanin, type I procollagen, and filaggrin production in human skin explants Skin fragments were originating from one (01) healthy subject, female, skin type II, 44 years, who underwent elective plastic surgery in the abdominal region (abdominoplasty).After the surgical procedure, the skin fragments were collected in plastic vials containing 0.9% saline and kept in refrigeration for up to 24 h.Skin fragments were dermatomized (Dermatome D-80; Humeca), ensuring an accurate thickness of 600 μM and circular cuts were made using a 1.5 mm diameter puncture.Fragments were maintained for 1 h at 4°C in DMEM high glucose (Corning) supplemented with Gentamicin (200 mg/500 mL-Gibco) and Amphotericin B (50 μg/500 mL-Sigma) for asepsis.Following the asepsis process, fragments were fit on 0.4 μM pore cell culture inserts (Corning) establishing an air-liquid culture model.The test system was maintained for 2 h in a humid incubator at 37°C, with 5% CO 2 , for later treatment with AHHF-010 at a ratio of 25-30 mg/cm 2 .The use of human skin fragments from elective surgeries for this study was approved by the Ethics Committee of the University São Francisco-SP-CAAE 82685618.9.0000.5514,opinion no 2.493.285.
For melanin and type I procollagen, the fragments were treated with AHHF-010 for 7 consecutive days and exposed to 4 doses of 10 J/cm 2 UV radiation using UVA Cube 400, SOL 500 H2 filter and UV Meter (Honle UV America Inc.).Twenty-four hours after the last exposure to UV radiation and treatment, fragments were collected and submitted to histological procedures.For filaggrin measurement, fragments were subjected to barrier disruption with sodium lauryl sulfate (5% SLS), treated with AHHF-010 at the proportion of 25-30 mg/cm 2 , and incubated for 48 h.After this period, they were submitted to the immunofluorescence assay for filaggrin.
Skin fragments were fixed in 4% buffered paraformaldehyde, cryoprotected, submitted to serial cuts (10 μm) in a Cryostat (Leica-CRYOCUT 1800), and collected directly on silanized slides.The sections were stained by the Fontana-Masson technique, and melanin density was analyzed with a light microscope (Olympus BX53; Olympus Optical Co., Ltd) using the cellSens Standard software (Olympus).
The parameter evaluated was the percentage of melanin present per corresponding area of the basal layer and thorny layer in the epidermis.The percentage values were obtained using the ImageJ Software (version 1.47) through the conversion of the images obtained into binary images followed by the quantification of the black pixels present in the basal layer and in the area of the spinous layer.
For immunofluorescence evaluation, slides were incubated overnight with the anti-filaggrin or procollagen type I primary antibody (Abcam).Subsequently, the sections were washed and incubated with the Goat anti-Rabbit Alexa Fluor 488 secondary antibody (Thermo Fisher Scientific).DAPI (4′-6-Diamidino-2-Phenylindole, Sigma-Aldrich) was used as a DNA marker.The slides were mounted using a specific mounting medium and analyzed with a Fluorescence Microscope (OLYMPUS) with the aid of the cellSens Standard software (© 2010 OLYMPUS CORPORATION).The evaluated parameter was the fluorescence intensity emitted by the specific antibody labeling.After obtaining the images, the fluorescence intensity was quantified with the aid of the ImageJ software (Arbitrary Units-A.U.). 10

| Clinical study
This study was approved by the Ethics Committee for Research Involving Human Subjects.

| Inclusion criteria
Thirty-three female participants aged 40-55 years, skin types I to IV according to Fitzpatrick, 11 not pregnant or nursing and regular users of facial cosmetic were selected for this study.Additional requirements were as follows: absence of skin disease, no medical treatment during the study, no use of retinoic acid, skin peels or similar procedures within 6 months prior to the onset of the study, non-exposure to intensive sun or any tanning methods, and absence of unusual emotional or physical stress.No changes in contraceptive method, dietary or cosmetic habits were allowed during the study.Any failure in this scheduled follow-up resulted in volunteer exclusion.

| Treatment protocol for AHHF-010
For the in-use test, each participant was instructed to apply the product around the eyes and all over the face up to the neck once a day in the morning, massaging in circular motions until the product was fully absorbed.The study duration was up to 84 days, with intermediate evaluations for each parameter investigated.
Cutaneous and ocular acceptability was evaluated for 28 days considering measurements taken before (T0) and after 28 days (T28d).Assessment of perceived efficacy was performed at T0 and after 84 days (T84d) of daily treatment.Reduction of suborbital edema was evaluated after 28 days of treatment, with partials (partial determinations?) after 1 hour (T1h) and 14 days (T14d).Collagen production was determined by spectroscopy at T0 and after 28 (T28d), 56 (T56d), and 84 (T84d) days of use.
Hydration kinetics was determined at previously delimited sites on the forearm at times T0, and 1 (T1h), 4 (T4h), 8 (T8h), 12 (T12h), and 24 (T24h) hours after a single application.A site on the contralateral forearm was also delimited and considered as the control of the experiment.To evaluate the action of the product on skin oiliness, a kinetic curve of sebum production was also constructed at times T0 and at 1 (T1h), 4 (T4h), 8 (T8h), and 12 (T12h) hours after a single application at defined sites in the frontal region (forehead) of the face.
Table 1 summarizes the treatment protocol for each parameter evaluated.

| Cutaneous and ocular acceptability
The assessment of cutaneous and ocular acceptability aimed to verify the potential for sensations of discomfort, irritation or allergies in the skin and eyes, resulting from the use of the investigational product applied under real conditions of use.Research participants were clinically evaluated and monitored by both a dermatologist and an ophthalmologist at the beginning of the study (T0) and after 28 days (T28d) of home use of the investigated product.Physicians evaluated and classified the adverse events presented by the participants, their location, duration, onset of appearance, frequency, intensity, evolution, and eventually adopted drug treatment.

| Perceived efficacy
The perception of efficacy was established by applying a questionnaire according to Table 2, answered by the research participants after 84 days (T84d) of treatment with the investigational product using a five-point scale.Based on the results of this evaluation, an analysis of the frequency of grades was performed for each attribute evaluated.

| Suborbital edema/swelling
The effects of the AHHF-010 product in reducing suborbital edema/ swelling were determined by image analysis using the VECTRA XT 3D Imaging system (Canfield Scientific) whose advanced technology system uses six digital cameras to capture and build a 3D model from the face of the research participant.The images were obtained under controlled conditions by the researcher at the beginning of the study (T0), after 1 h (T1h) of application of the investigational product and after 14 (T14d) and 28 (T28d) days of home use.According to the morphology of the face of each participant, size and location of the region with suborbital swelling, a region was determined for analysis of relative volume (arbitrary units-A.U./μm 2 ), defined as swelling intensity (SI).The percentage of suborbital swelling reduction (SIR%) was calculated according to the equation: SIR% = 100 (SIR-SIf)/SIi, where SI = suborbital swelling intensity; i = initial; f = final.

| Collagen production
This study was based on the hypothesis that the application of the AHHF-010 product could increase collagen synthesis in the skin by means of fluorescence spectroscopy (Fluoromax-Spectrofluometer 4C-TCSPC, Horiba Scientific).In this technique, the light from a xenon lamp passes through a monochromator, where the wavelength is selected, and with the aid of a bifurcated optical fiber, it is directed to the skin.This light comes into contact with the skin and is reflected through another fiber optic guide that passes through another monochromator and is finally directed to the photomultiplier cell.The excitation spectra obtained in this study were as follows: maximum excitation of cross-link collagen at 340 nm and maximum excitation of tryptophan at 295 nm.The efficacy of the investigational product in increasing collagen synthesis can be evidenced when there is an increase in the I 340 /I 295 ratio after treatment (I-intensity).The spectra were acquired on the hemiface (right or left) of the research participants at the beginning of the study (T0) and after 28 (T28d), 56 (T56d), and 84 days (T84d) of home use of AHHF-010.

| Statistical analysis
For preclinical statistical evaluation, the ANOVA test was used to measure the variation of the results by comparing the data of all the groups.We applied the Bonferroni post-test, which strengthened and made more precise the result presented obtained with ANOVA (GraphPad Prism6, Version 6.01, GraphPad Software, Inc.).Significance level was set at 5%.For data analysis and statistics obtained in clinical studies was used the paired bimodal Student t-test, considering a 95% confidence interval (GraphPad™ Prism® 6.0, GraphPad Software).

| Formula stability study
Color: In the initial evaluation (T0), the color of the sample was determined to be beige.In the evaluations carried out at 15 and 30 days, the sample conditioned under sunlight was slightly modified.The other samples remained as initially evaluated.In the evaluations carried out after 60 and 90 days, the sample conditioned under sunlight was clearly modified.The other samples remained as initially evaluated.
Odor: In the initial evaluation (T0), the odor of the sample was determined to be characteristic.
In the evaluations conducted after 15 and 30 days of study, the sample conditioned at 45°C showed a very small loss of intensity

| Formulation AHHF-010 prevents IR-Ainduced oxidative stress
Primary dermal fibroblast cultures were treated with three noncytotoxic concentrations of AHHF-010 (10.01, 3.17, and 1.00 mg/ mL) and submitted to 360 J/cm 2 of IR-A radiation.After 24 h, mitochondrial ROS staining was quantified directly on the cells.
Figure 1 represents the effects of treatment with AHHF-010 on the production of mitochondrial ROS (ROSmit).Fluorescence is revealed in red by the oxidation of the MitoSOX™ Red probe in the cytoplasm and cell nucleus (Figure 1A).As expected, exposure to IR-A radiation produced a marked increase in the production of ROSmit when compared to the unexposed basal control, contributing to the induction of oxidative stress.On the contrary, AHHF-010 demonstrated a protective effect on the cell cultures, reducing the ROSmit labeling at all concentrations evaluated when compared to cells only submitted to IR-A radiation.The semiquantification of these results was obtained using image analysis software which revealed that IR-A radiation promoted a 3.54-fold increase in ROSmit fluorescence (Figure 1B).Pretreatment with AHHF-010 induced a reduction of 73.13, 64.58, and 59.88%, respectively, at concentrations of 010.01, 3.17, and 1.00 mg/mL, when compared to the IR-A group (p < 0.001).According to the results obtained in the statistical analysis, there was no significant increase in I 340 /I 295 values after 28 days of treatment; however, after 56 and 84 days, increases in collagen synthesis were observed (p < 0.05).Concurrently, 93% of these research participants noticed a reduction in expression lines and wrinkles, and the entire panel evaluated (100% of the participants) noticed that the skin became firmer and stronger after 84 days of home use of AHHF-010.This result is supported by the results of microscopic analysis and semi-quantification by image analysis of the increase in filaggrin synthesis in human skin culture (Figure 5).Skin fragments were subjected to barrier disruption with 5% SLS, treated with AHHF-010 at the proportion of 25-30 mg/cm 2 , and incubated for 48 h.The signal for filaggrin is revealed in green in the histological sections from ex vivo skin fragments and has localized labeling particularly in the granular layer (Figure 5A).The results obtained in this evaluation demonstrated that barrier disruption with 5% SLS decreased filaggrin production by 28.28% compared to baseline control (p < 0.001-Figure 5B).

| AHHF-010 provides skin hydration by increasing filaggrin production
Pretreatment with AHHF-010 formulation was able to significantly prevent the drop in filaggrin production, with 23.12% more protein labeling when compared to the 5% SLS group.

| AHHF-010 improves skin tone and reduces melanin production
The results of perceived efficacy (n = 14) after 84 days of home use of AHHF-010 demonstrated that 86% of the panel noticed a more  with AHHF-010 showed a significant reduction in melanic pigmentation when compared to the only UV exposed group (81.53% reduction; p < 0.001).

| AHHF-010 reduces suborbital swelling and dark circles
Nineteen research participants were considered for assessment of suborbital swelling reduction.showed that 79% of the research participants noticed an attenuation of dark circles, confirming the action of this product on the attenuation of aesthetic discomforts in the suborbital region.

| DISCUSS ION
Considering the importance of collagen in maintaining the structural integrity of the dermis, we conducted an ex vivo study using human skin fragments to evaluate the ability of the AHHF-010 formulation to stimulate the synthesis of procollagen type I. Our results demonstrated a substantial action of this formulation, not only in protecting, but also in stimulating the synthesis of procollagen type I, by 3.6 times, in skin cultures subjected to oxidative stress by UV radiation.This result is aligned with the composition of the product that contains ingredients with known antiaging properties.3][14] In addition to its potent antioxidant action, tocopherol plays a fundamental role in protecting DNA by preventing the formation of pyrimidine photoproducts, with anti-inflammatory effects based on the inhibition of eicosanoid and cyclooxygenase-2 secretion and the suppression of the pro-inflammatory signaling pathways NF-κB and STAT-3. 13Carnosine complements these actions, suppressing telomere shortening, preventing advanced glycation end product formation, preserving sirtuin signaling, and rejuvenating senescent cells. 14e clinical response to AHHF-010 regarding collagen production was also evaluated using a noninvasive method based on fluorescence spectroscopy.The characterization of skin fluorescence in terms of native fluorophores such as NADH, collagen, elastin, tryptophan, and porphyrins has been proposed as a way of differentiating healthy skin from diseased skin and intrinsic skin aging from skin photoaging. 15Therefore, this method constitutes an effective way to assess the amount of dermal collagen.In our study, research participants were instructed to use AHHF-010 for 84 consecutive days and the increase of collagen was evaluated by calculating the ratio between the excitation spectra obtained for collagen and tryptophan (I 340 /I 295 ).Corroborating the preclinical findings, treatment efficacy was evidenced by the significant increase in the I 340 /I 295 ratio after 56 and 84 days of use, reaching a 13.44% and 36.42%increase, respectively, compared to baseline (T0).Complementing the approach to collagen production, the evaluation of perceived efficacy demonstrated that 93% of participants reported a reduction in intensity and an improvement in the perception of fine lines and wrinkles after 84 days of home use of AHHF-010.Moreover, 100% of the panel stated that the treatment left the skin firmer and strengthened.
Another point addressed during the subjective study was the improvement in facial pigmentation, a classic and unaesthetic sign of extrinsic aging.In this regard, 86% of the participants reported an improvement in tone uniformity and blemish attenuation.In this sense, preclinical studies have endorsed this effect through the reduction of 81.5% in melanin production induced by UV radiation after 7 days treatment of human skin culture with AHHF-010.This mechanism of action can be attributed to the presence of resveratrol and niacinamide in the cosmetic formulation.Resveratrol has inhibitory effects on catalytic activity, gene expression, and post-translational tyrosinase modifications, providing a mechanistic basis for these antimelanogenic effects. 16Topical niacinamide has beneficial effects in preventing photoimmunosuppression, reducing hyperpigmentation of spots by mechanisms involving inhibition of melanosome transfer from melanocytes to keratinocytes. 17 the intensity of periorbital hyperchromia in 79% of the panel compared to baseline.As is well known, the skin of the periorbital region is the area most susceptible to the deleterious effects of the skin aging process and, even with a healthy skin, a large number of individuals exhibit these effects in large proportions of this region.
Known generically as "dark circles," changes in eyelid color and shape can manifest as hyperpigmentation or hyperchromia, lower eyelid bag, eyelid edema, and very early onset of fine wrinkles. 18 reported by the participants, AHHF-010 favored a reduction in the appearance of fine lines and the tone of dark circles, suggesting its use for eyecare as well.Complementarily, we conducted an instrumental study to evaluate the effectiveness of this treatment in reducing swelling in the suborbital region using a system of coupled cameras that capture images and build a three-dimensional face model of the participants before and after using the product.
This study, conducted over 28 days, resulted in a significant and gradual attenuation in swelling intensity, reaching up to a 3.2% reduction at the end of the study.Importantly, 100% of the participants noticed a reduction in the intensity of suborbital swelling after 14 and 28 days, demonstrating that the AHHF-010 product exerted a beneficial effect in improving this unaesthetic disorder.
The mechanism associated with the effect of reducing swelling and improving appearance in the periorbital region can be attributed to caffeine present in the AHHF-010 formulation.Caffeine, traditionally used in cosmetic formulations to treat gynoid lipodystrophy, also has exceptional antioxidant activity.This alkaloid, found mainly in coffee and teas, helps to protect cells against the action of UV radiation, mitigating the photoaging process, favoring cutaneous microcirculation, and reducing swelling around the eyes. 19e effects of photoaging also reflect on the epidermis.As the outermost region of the skin, the epidermis establishes and maintains an effective barrier that controls water exchange and protects against dehydration, in addition to protecting from physical, chemical, and infectious aggressions, and is therefore essential for physiological homeostasis.Over the years, the functional and structural changes due to the combined and cumulative effects of intrinsic and extrinsic factors result in a reduction in thickness, permeability, calcium gradient, lipid synthesis, and processing, as well as changes in pH, immunocompetent function, and SC hydration. 2,9This pathophysiological aging process translates into important clinical signs that contribute to the unsightly appearance of the skin surface such as dryness, roughness, scaling, erythema, and dullness. 20equate hydration, consequently, is crucial for maintaining a healthy epidermis, and moisturizing ingredients are key components in skin care formulations.SC water retention depends on two main components: the presence of natural hygroscopic agents within the corneocytes (natural moisturizing factors-NMFs) and SC intercellular lipids organized to form a barrier that prevents transepidermal water loss. 9In the outer SC layer, filaggrin is the major source of NMFs.Filaggrin is a histidine-rich protein degraded into amino acid metabolites such as trans-urocanic acid and pyrrolidone carboxylic acid which, combined with other hydrophilic components, constitute the NMF, conferring and preserving moisture (humectancy) in the skin. 21In this study, we evaluated the effects of the AHHF-010 formulation on the synthesis of filaggrin in human skin culture by protein immunostaining.Skin fragments were subjected to barrier disruption with SLS, which resulted in a reduction in filaggrin production.In turn, pretreatment with AHHF-010 preserved the integrity of skin, maintaining the levels of filaggrin similar to those of the control group not subjected to barrier disruption.
Besides, the evaluation of cutaneous water content is of great importance in studies of the effectiveness of cosmetic products intended for this purpose.Corneometry is one of the most used techniques for determining the hydration of the surface layer of the epidermis and for quantifying the moisturizing effect.Skin hydration provided by the application of a moisturizing product is evidenced by the increase in the capacitance value generated between the base of the Corneometer® probe and the skin.The higher the capacitance value, the greater the amount of water in the skin and, therefore, the greater its hydration level. 22In this direction, we evaluated the hydration kinetics produced after a single application of AHHF-010 to the forearm of the research participants.Capacitance measurements obtained up to 24 h of application indicated that the product provided a significant increase in skin hydration over time, reaching a 36.3% increase compared to the untreated site.It is important to note that 100% of the participants showed an increase in corneometric indexes after 1, 4, and 8 h of application and 95% of them after 12 and 24 h, compared to the respective control site.
The effect of AHHF-010 on barrier protection and hydration evaluated by clinical and preclinical studies is provided by the hyaluronic acid (HA) present in the formulation.HA has become one of the most important ingredients in cosmetic products due to its moisturizing, skin protective, and antiaging properties.It has been acknowledged for its ability to retain and replenish moisture in the skin, resulting in a softer, smoother, and radiant appearance.Skin hydration also leads to delayed formation of wrinkles and improves deep fine lines and already developed wrinkles that usually appear with age.In addition to having antioxidant effects, HA also promotes cell regeneration and stimulates collagen production, showing the multifaceted mechanism of this macromolecule. 23ncluding the clinical efficacy studies, the kinetics of sebum production was measured by photometric analysis (sebumeter) with the purpose of making the product available to users with oily and/ or acne-prone skin.The results after a single application of AHHF-010 to the face showed a significant seboregulatory action of the product over the 12 h of the study when compared to the respective contralateral control site.Despite its physiological importance, excess sebum can cause aesthetic discomfort in addition to culminating in the development of comedones and acne.On the contrary, a drastic reduction in sebum production can cause dryness and disturbance of the skin barrier. 24Thus, the ideal condition for the action of a sebum-regulating product is a modulation in sebum production, an effect observed in our results where the kinetics induced by the application of AHHF-010 over 12 h was slightly lower compared to the respective untreated control site.Sebaceous modulation can be associated with caffeine, present in the evaluated formulation, due to its action in stimulating insulin growth factor 1 (IGF-1) and inhibiting 5 alpha reductase enzyme, controlling sebum secretion and also hair loss. 25in aging does not result from an isolated mechanism, but from a set of persistent deleterious effects that eventually compromise cellular homeostasis.When analyzed from a biochemical perspective, aging is considered multifactorial both in its causality and in its final result.Macromolecular dysfunction is observed, in particular deleterious changes in nucleic acids, proteins and lipids which accumulate in tissues, inducing and accelerating tissue aging.Aging is associated with increased somatic mutation, progressive homeostatic dysfunction, protein modification, and lipid peroxidation, which can be attributed to the effects of exogenous and/or endogenous agents. 24 this context, an effective antiaging treatment must have a pluripotent action in order to neutralize the various molecular changes underlying the cellular dysfunction related to aging.Thus, in this study, we present a formulation based on combined antioxidants for the control of the clinical signs of skin aging.
In order to highlight and expand the antioxidant studies of the combination of actives that make up the formulation of AHHF-010, a preclinical study was also conducted using a culture of human fibroblasts exposed to infrared-A radiation (IR-A).The Skin hydration was assessed by capacitance measurements using the Corneometer® 825 probe coupled to the Multi Probe Adapter, MPA 5 equipment (CKeletronics).Two evaluation areas were randomly delimited on the right or left forearm of each research participant, one side being considered the control without product application and the other site receiving the application of AHHF-010.After the research participants were acclimatized, baseline capacitance measurements were obtained at both delimited sites.Then, 20 μL TA B L E 1 Treatment protocol for the assessment of the clinical safety and efficacy of each endpoint evaluated after treatment with AHHF-010.010 was spread evenly on the respective site with the aid of a disposable latex finger.Research participants remained in the laboratory to perform capacitance measurements after 1, 4, 8, 12, and 24 h of product application.The variation of capacitance measurements (Δh) and the percentage of increase in skin hydration (%Hti) obtained at each evaluation time were calculated in relation to baseline measurements according to the following equations: Equation 1: Δh = h ti -h t0 , where h ti = average capacitance values obtained after i hours of application of the investigational product (i = 1, 4, 8, 12, and 24 h); h t0 = mean capacitance values obtained at the beginning of the study (baseline).Equation 2: %Hti = 100 × (Δ hti (investigational product) -Δh ti (control) )/ht0, where Δhti = hydration difference values calculated for the investigational product and control after i hours of application; ht0 = mean capacitance values obtained for the investigational product at the beginning of the study (baseline).

2. 3 . 8 |
Skin sebum content Two evaluation areas were randomly delimited on the forehead of each research participant, one side being considered control without product application and the other site receiving application of AHHF-010.Sebum content was measured using the MPA 5-Sebumeter® 815 equipment (CKeletronics).The working principle of the Sebumeter is based on the direct photometric measurement of sebaceous secretion, not being influenced by humidity.The sebum secretion is collected on the translucent plastic tape of the Sebumeter SM 810 Cassette, which becomes transparent, and the result is obtained by measuring the difference in light transmittance value through the tape before and after impregnation with fat.The amount of sebaceous secretion is given in mg/cm 2 .After acclimatization of the research participants, baseline measurements were obtained.Then, the sites were cleaned with cotton, and 20 μL of the AHHF-010 formulation was applied with the aid of a disposable latex finger.Research participants remained in the laboratory to perform the oiliness measurements after 1, 4, 8, and 12 h of product application.Using the values of sebum amount, called S, the Oil Variation (ΔO) was calculated by the following Equation: ∆O = S t0 -S ti , where S = sebaceous secretion values obtained at the product or control application site; t0 = values obtained at baseline; ti = values obtained after i hours of application (i = 1, 4, 8, and 12 h).The percentage of reduction in skin oiliness (%RO) provided by the investigational product in relation to the initial condition was calculated according to the following Equation: %RO ti = 100 × (S t0 -S ti )/S t0 , where S = mean values of sebum secretion; t0 = values obtained at baseline (baseline); ti = values obtained after i hours of application (i = 1, 4, 8, and 12 h).
in the odor characteristic.The sample conditioned under sunlight showed very little loss of intensity and very slight changes in the odor characteristic.The other samples remained as initially evaluated.In the evaluations carried out after 60 and 90 days of study, the samples conditioned under Sunlight showed a very small loss of intensity and slight changes in the characteristic of the odor.The other samples remained as previously evaluated.pH: The pH analysis was performed through a potentiometric test, defined by the potential difference between two Questions Grade I noticed that my expression lines/wrinkles are less apparent I noticed that my dark circles are less dark I noticed that my skin is more even (dark spots are smoother) I noticed that the product made my skin firmer, stronger Rating scale: 1-Strongly Agree, 2-Partially Agree, 3-Indifferent, 4-Partially Disagree, 5-Strongly Disagree.TA B L E 2 Questions applied (Statements reported?) in the subjective analysis of perceived efficacy after 84 days of treatment with AHHF-010.electrodes-the reference and the measurement-immersed in the analyzed sample and dependent on the activity of hydrogen ions in the medium in which the electrodes were inserted.By definition, pH is the negative logarithm of the concentration of hydrogen ions, through which the acidity and alkalinity of the sample is determined.The study lasted 90 days, and the samples were divided and conditioned in refrigerator, dark room, at 37°C, at 45°C and under sunlight).The pH varied from 5.16 (sample conditioned at 45°C, D90) to 5.58 (sample conditioned in a dark room, D15).Viscosity: Viscosity is determined by the sample's resistance to deformation or flow.Viscosity depends on the physical-chemical characteristics and temperature conditions of the material.The study lasted 90 days, and the samples were divided and conditioned in refrigerator, dark room, at 37°C, at 45°C and under sunlight).The viscosity varied from 20 080 (sample conditioned under sunlight, D30) to 23 880 (sample conditioned in a dark room, D90).Density: Density is determined by the mass to volume ratio.For this parameter, the density was performed with the aid of a pycnometer and calculated from different weights.At first, the empty pycnometer was passed.Then, the same pycnometer filled with purified water was weighed.Finally, the purified water is discarded, with the pycnometer completely dry, the sample is placed inside the pycnometer and a new weighing is performed.The study lasted 90 days, and the samples were divided and conditioned in refrigerator, dark room, at 37°C, at 45°C and under sunlight).The density varied from 1.0219 (sample conditioned at 45°C, D60) to 1.0318 (sample conditioned at 37°C, D60).

Figure 2
Figure 2 illustrates the results of microscopic analysis (A) and semiquantification by image analysis (B) of procollagen synthesis in human skin culture.Skin fragments were treated with AHHF-010 for 7 consecutive days and exposed to 4 doses of 10 J/cm 2 UV radiation.On the eighth day, fragments were collected and submitted to histological procedures.As shown by the semi-quantification of the images obtained, exposure to UV radiation decreased the concentration of procollagen type I by 49.64% in relation to the baseline control (p < 0.001).Conversely, pretreatment with the AHHF-010 formulation was able not only to contain the sharp drop in procollagen type I production induced by UV radiation, but also to increase beyond baseline control values the synthesis of this essential protein for dermal support.Procollagen type I production in the group treated and exposed to UV radiation was 260% (3.6 fold) higher compared to the only irradiated group (p < 0.001).Similar outcomes were observed in a clinical trial assessing increased collagen synthesis in 14 research participants by noninvasive diffuse reflectance spectroscopy.Figure 3 illustrates the mean values of the ratio between collagen and tryptophan intensity (I 340 / I 295 ) obtained before and after 28, 56, and 84 days of home use of AHHF-010.The increase in collagen synthesis induced by AHHF-010 was verified by applying the Student t-test to the I 340 /I 295 data, compared to the values obtained at the beginning of the study.

Figure 4
Figure4represents the average capacitance values (h) obtained throughout the study after the application of AHHF-010, using the corneometry technique.According to the results obtained, there was a significant increase (p < 0.001) in capacitance values after 1, 4, 8, 12, and 24 h of AHHF-010 application, indicating that this product provided an increase in skin hydration.These significant increases were obtained by comparing the treated sites with both the initial baseline control (T0) and the respective controls at T1, T4, T8, T12, and T24 h.It was also observed that 100% of research panel showed improvement in skin hydration after 1, 4, and 8 h of application and 95% of participants showed improvement in skin hydration after 12 and 24 h of application.

F I G U R E 1
IR-A-induced mitochondrial ROS (ROSmit) production is downregulated by treatment with the cosmetic formulation AHHF-010.(A) Microscopic evaluation of ROSmit labeled in red by the oxidation of the MitoSOX™ Red probe in both the cytoplasm and nucleus from human primary dermal fibroblasts exposed to IR-A radiation (360 J/cm 2 ) and incubated with AHHF-010 at three concentrations (10.01, 3.17, and 1.00 mg/mL).The reference bar corresponds to 10 μm.(B) Semi-quantification of the fluorescence intensity (Arbitrary Units-A.U.) of ROSmit production.Data represent the mean ± standard deviation of 10 experimental areas (ANOVA, Bonferroni).F I G U R E 2 (A) Immunofluorescence evaluation of procollagen type I in human skin fragments treated with AHHF-010 (25-30 mg/cm 2 ) and submitted to UV radiation (10 J/cm 2 ).Procollagen type I is labeled in green, and the blue mark represents the cell nucleus (DNA).The reference bar corresponds to 20 μm.(B) Semi-quantification of the fluorescence intensity (Arbitrary Units-A.U.) of procollagen type I immunostaining.The data represent the mean ± standard deviation of 12 areas (ANOVA-Bonferroni).F I G U R E 3 Mean values of the ratio between collagen and tryptophan intensity (I 340 /I 295 ) obtained before and after 28, 56, and 84 days of home use of AHHF-010.Data represent the mean ± standard deviation (n = 14; Student t-test).uniform skin and smoother dark spots.Likewise, preclinical treatment of a skin explant validated this effect by reduction in melanin synthesis.

Figure 6 F I G U R E 7
Figure 6 illustrates the microscopic analysis (A) and the percentage of melanin density (B) in the basal and spinous layers of the epidermis (dark spots).Human skin fragments were treated with

Figure 7
illustrates the mean suborbital SI values obtained initially (T0) and after 1-h (T1h) and 14 (T14d) and 28 (T28d) days of AHHF-010 application.The SI obtained at T1h, T14d, and T28d showed a significant reduction (p < 0.001) of 1.8, 3.0, and 3.2%, respectively, compared to the value obtained at T0.It is important to point out that 100% of the research participants showed a reduction in the intensity of suborbital swelling.

Figure 8
Figure 8 illustrates an example of a result obtained for one of the research participants.Concurrently, the results obtained with the evaluation of perceived efficacy (n = 14) after 84 days of home use of AHHF-010

Figure 9
Figure 9 illustrates the mean values of sebaceous secretion obtained throughout the study with the application of AHHF-010 using the sebumeter technique.At T0, sebum production was similar at the control and treated sites.After 1 h of study, we observed a significant reduction (p < 0.05) in sebaceous secretion at the control site, ) were clinically evaluated by a dermatologist and an ophthalmologist at baseline and after 28 days of home use of AHHF-010.After 28 days of use, it was clinically verified that 97% of the research participants did not show adverse reactions or any signs or symptoms of skin discomfort and did not report irritative or allergenic reactions and/or sensations of discomfort resulting from the use of the product.Only one participant had mild pruritus and scaling.Regarding ocular acceptability, 100% of the research participants did not experience adverse reactions or any signs or symptoms of ocular discomfort and did not report irritative or allergenic F I G U R E 8 Illustration of the results in the suborbital swelling intensity (SI) obtained before (T0) and after 1-h (T1h), 14 (T14d), and 28 (T28d) days of home use of AHHF-010.reactions and/or sensations of ocular discomfort resulting from the use of AHHF-010.There were no reports/records of adverse events during the study.
Furthermore, self-assessment of efficacy revealed positive results of treatment with AHHF-010 in reducing F I G U R E 9 Sebum content determinated by sebumeter in human subjects at baseline (T0) and after T1, T4, T8 and T12 of application of AHHF-010.

5 |
results obtained allowed us to infer that AHHF-010 has a prophylactic action against oxidative stress induced by IR-A, significantly reducing the production of ROSmit to baseline levels and thus contributing to maintaining the metabolic health of the skin.IR-A has been identified as an aggravating factor of tissue damage and of the skin aging process, corroborating the physiological skin changes induced by UV radiation and visible light.24Preliminary studies conducted by our group based on large-scale gene expression using mRNA sequencing (RNA-seq-next generation sequencing) have confirmed that IR-A radiation induces functional changes in the skin by mechanisms that involve an increased inflammatory response, induction of senescence tissue damage, a decreased cell adhesion process, and exacerbation of oxidative stress.24Disturbances in mitochondrial electron transport lead to a decrease in energy production and an increase in the formation of ROS, which result in changes in gene expression and dermal metabolism translated by increased expression of metalloproteinase 1 and decreased collagen synthesis.25Thus, the signs and mediators involved in the regulation of mitochondrial biogenesis are of great importance in the metabolic homeostasis of the skin and the consequent prevention of premature aging.In view of the results presented, the investigational product evaluated is a "high performance cosmetic," with an association of nanoencapsulated antioxidants.Therefore, our goal was to investigate the action mechanisms of this cosmetic, besides the safety and efficacy of the final product (the synergistic action of the active ingredients).It was aimed to demonstrate that changes caused at histological level, inside a research laboratory, were reproducible in a real life use, by measuring biophysical properties of skin with the instrumental evaluation techniques and also through the selfassessment questionnaire.A cosmetic product needs to improve the quality of life of people, they need to perceive its efficacy and appreciate all the comfort and good sensations they bring along.It is very important to obtain beneficial results in skin improvement in ex vivo studies translated into beneficial clinical trials results, which must be measured and quantified, but also perceived by the people.In summary, the results showed clinical improvement of wrinkles, skin tone, periorbital swelling, hydration, and sebum production after the use of the formulation.In addition, preclinical mechanisms proved the depigmenting, collagen stimulating, skin barrier protective, and antioxidant action of the product.It is worth mentioning that not only the rich and effective composition of AHHF-010, but also the nanotechnological strategy applied in the formulation, were responsible for the multiple beneficial actions observed.CON CLUS IONThe proposed cosmetic product has been shown to be safe and effective in improving the condition and appearance of the skin by reducing the intensity of fine lines, wrinkles, blemishes, and swelling, and enhancing skin hydration.The benefits obtained in the evaluated parameters, described in the present study, result from the combination of the established effectiveness of the ingredients in the formulation and the nanoencapsulation-based delivery system, which favors solubility, safety, efficacy, and bioavailability through the skin.AUTH O R CO NTR I B UTI O N SAna Paula Fonseca was responsible for the conceptualization and manuscript structuration.Gustavo Silva, Gustavo Facchini, Ana Lúcia Tabarini, and Samara Eberlin were responsible for data collection, formal analysis, and paper structuration.Prof. Dr. Patrícia Campos, Dr. Carine Dal Pizzol, and Antonio Vanzo were responsible for supervision, paper structuration, and formal analysis.CO N FLI C T O F I NTER E S T S TATEM ENTAna Paula Fonseca, Dr. Carine dal Pizzol, and Antônio Vanzo are employees of Sallve Comércio de Cosméticos LTDA.DATA AVA I L A B I L I T Y S TAT E M E N TResearch data are not shared.E TH I C A L S TATEM ENTThis study was approved by the Committee of Ethics in Research involving Human Subjects (CEP/University São Francisco-SP-CAAE 46036521.5.0000.5514,opinion no 4.697.976).