Formulation

Skin care

KEYWORDS

HOMEOSTASIS;

OXY-AGING;

FORMULATION;

ASCORBIC ACID;

PEER REVIEWED.

peer-reviewed

Skin homeostasis & oxy-aging: A challenge in formulation

GINA PUIG, MERITXELL LLINÀS, EVA MARTIN, PATRICIA FISAS

R&D+i Department, Natura Bissé Int. S.A. Spain

ABSTRACT: To find a formula that protects the skin against oxidative aging (known as oxy-aging) since this is one of the best strategies to maintain cutaneous homeostasis is the goal of the present study. This theory holds that supplying exogenous antioxidants to the skin, reduces oxidative stress and combats the signs of aging. With that in mind, in this paper we present a formula that provides the skin with maximum efficacy exogenous antioxidants in a high, safe and stable concentration. Active ingredients were combined in concentrations of up to 20% in a non-degrading formulation containing high concentration of ascorbic acid (12%)-a hero ingredient for the cosmetic industry, with a completely anhydrous excipient to achieve a greater antioxidant and rejuvenating benefit for the skin.

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“A study in healthy women providing probiotic yogurt for four weeks showed an improvement in emotional responses as measured by brain scans”

Figure 1. Skin Section with Microbiome. Most microorganisms live in the superficial layers of the stratum corneum and in the upper parts of the hair follicles. Some reside in the deeper areas of the hair follicles and are beyond the reach of ordinary disinfection procedures. There bacteria are a reservoir for recolonization after the surface bacteria are removed.

Materials and methods

Studies of major depressive disorder have been correlated with reduced Lactobacillus and Bifidobacteria and symptom severity has been correlated to changes in Firmicutes, Actinobacteria, and Bacteriodes. Gut microbiota that contain more butyrate producers have been correlated with improved quality of life (1).

A study in healthy women providing probiotic yogurt for four weeks showed an improvement in emotional responses as measured by brain scans (2). A subsequent study by Mohammadi et al. (3) investigated the impacts of probiotic yogurt and probiotic capsules over 6 weeks and found a significant improvement in depression-anxiety-stress scores in subjects taking the specific strains of probiotics contained in the yogurt or capsules. Other studies with probiotics have indicated improvements in depression scores, anxiety, postpartum depression and mood rating in an elderly population (4-7).

Other studies have indicated a benefit of probiotic supplementation in alleviating symptoms of stress. In particular, researchers have looked at stress in students as they prepared for exams, while also evaluating other health indicators such as flu and cold symptoms (1). In healthy people, there is an indication that probiotic supplementation may help to maintain memory function under conditions of acute stress.

Introduction

The skin, as the largest organ of the human body, serves as a vital barrier and interface between the internal and external environments. Its primary functions include protection, thermoregulation, sensory reception, and immune surveillance. To maintain these roles, the skin relies on homeostasis, a dynamic process that balances skin cell proliferation, differentiation, and repair mechanisms. Skin homeostasis is critical for preserving its structural integrity and physiological functions, even in the face of environmental stressors and chronological aging. One emerging area of interest in skin biology is oxy-aging, a term derived from "oxidative aging," that refers to the aging processes driven by oxidative stress, a state in which the balance between reactive oxygen species (ROS) and the skin's antioxidant defense mechanisms is disrupted. ROS, which include free radicals like superoxide anions (O₂⁻) and hydroxyl radicals (OH·), are naturally produced as byproducts of cellular metabolism, particularly within the mitochondria. Environmental factors, such as ultraviolet (UV) radiation, pollution, smoking, and exposure to chemicals, can exacerbate ROS production, overwhelming the skin’s antioxidant systems (superoxide dismutase (SOD), catalase, and glutathione peroxidase that neutralize ROS under normal conditions) leading to oxidative stress. Their overproduction or insufficient neutralization can result in cellular damage. This includes lipid peroxidation, protein oxidation, DNA damage, mitochondria dysfunction and chronic inflammation as ROS can activate signalling pathways such as the nuclear factor-kappa B (NF-κB) pathway, triggering the release of pro-inflammatory cytokines which all contribute to skin aging and compromise homeostasis. The interplay between these oxidative processes and the skin's ability to adapt and repair is central to understanding oxy-aging. The visible effects of these oxidative reactions (exposure of the skin to extrinsic factors such as UV, HEV and IR radiation or pollution or due to chronological aging) include wrinkles, loss of firmness, and loss of radiance thus demonstrating the relationship between oxidative stress and aging (oxy-aging) (1).

Delivering topical exogenous antioxidants is one of the strategies used in the cosmetic industry to reduces oxidative stress and in turn to improve the immune function and consequently the inflammation of the organism (2),(3), (4), (5) while also preventing the visible signs of aging. Therefore, in order to address the oxidative activity that takes place in the skin, it is essential to formulate a product that contains a high concentration of antioxidant ingredients (6).

However, formulating skincare products with these compounds presents several challenges as it is essential to keep them stable during the shelf life of the product. The incorporation of ascorbic acid into a cosmetic formulation is crucial for achieving a potent antioxidative effect on the skin, effectively reducing oxidative stress and supporting the preservation of dermal integrity and functionality. However, ascorbic acid is highly unstable, being prone to oxidation when exposed to oxygen, light, and heat, which poses challenges for its incorporation into topical formulations (7). The appropriate carrier plays a critical role in preserving the stability of vitamin C and facilitating its effective penetration into the skin.

Moreover, the potent antioxidant activity of vitamin C can be further potentiated by combining it with other molecules, such as Pine bark extract, Carnosine, or Turmeric extract, thereby enhancing its antioxidative effect and being an excellent holistic solution capable to maintain skin homeostasis while combating oxidative damage in order to prevent the oxy-aging in multiple biological pathways.

Ascorbic acid (8), (9) is a well-known antioxidant recognized for its ability to neutralize free radicals and play a vital role in collagen biosynthesis, essential for skin and tissue repair. Additionally, it has the capacity to regenerate other antioxidants, thereby enhancing its protective effects. When combined with Curcuma longa extract , an antioxidant and anti-inflammatory agent (10),(11), it becomes a promising candidate for research on oxidative stress, aging, and chronic diseases. Furthermore, carnosine (12),(13) is particularly valuable in studies focused on age-related oxidative damage, neurodegeneration, and mitochondrial dysfunction. Its unique ability to prevent both oxidative damage and glycation distinguishes it from other antioxidants. Moreover, a standardized extract of Pinus pinaster(14), rich in proanthocyanidins, could enhance skin elasticity and reduce oxidative stress in skin tissues.

Materials and methods

Anchor Formula development and stability assessment

L-Ascorbic acid is a carboxylic acid, which is pKa is 4.2, that, upon deprotonation of its hydroxyl group, forms the ascorbate anion. When deprotonated, ascorbate becomes highly susceptible to oxidation, particularly at the hydroxyl group located at position 2. To maintain the reduced form of ascorbic acid for an extended period, it is essential to keep the pH of the vehicle below 3.5–4.0 and avoid the reaction with various components in the environment such as oxygen, water or light.

When the ratio of ascorbic acid to water is too low it can lead to unstable ascorbic acid. On the other hand, ascorbic acid has limited solubility in non-aqueous solutions, requiring solvents like ethanol to dissolve small amounts of the compound.

Dissolving ascorbic acid in polyhydric alcohols was one of the strategies to follow, but not all of them have the same efficiency when it comes to dissolving ascorbic acid.

To achieve the maximum concentration of pure, stabilized ascorbic acid, in a skincare formulation, tests were conducted using various concentrations and different excipients, though always maintaining an anhydrous medium.

To achieve a high concentration of ascorbic acid in the formulation, 10% of this active ingredient was initially dissolved in various solvents such as ethanol, glycerine, propylene glycol, butylene glycol, and propanediol.

After observing that 10% of ascorbic acid dissolves perfectly in propanediol, the concentration of the active ingredient was increased to 15%, but this concentration could not be dissolved in any of the media.

However, polyhydric alcohols have a sensory profile that can be unpleasant on the skin, which makes products formulated with these ingredients feel greasy or sticky, affecting the user experience. The success of a cosmetic preparation is largely determined by the product's sensory characteristics, making it essential to incorporate ingredients that enhance the sensory profile without compromising the solubility of the active ingredient. Consequently, emollients with high polarity were added, which influenced the maximum solubilized concentration of ascorbic acid, ultimately resulting in a final concentration more than 10%.

After obtaining the maximum solubility concentration of ascorbic acid in the anhydrous vehicle carnosine, Curcuma longa extract, and Pinus pinaster extract, along with other antioxidants were added.

During the stability study, chemical stability was evaluated at 25 ºC and 40 ºC by quantifying the concentration of ascorbic acid in the samples using HPLC. To quantify ascorbic acid by HPLC, a solution curve of ascorbic acid of known concentrations was prepared in situ in 95% buffer pH 3.2/5% methanol, and the samples to be analysed were then prepared. At the HPLC condition level, a flow rate of 1ml/min was used for five minutes, and the wavelength was adjusted to 254 nm. The concentration of ascorbic acid in each sample was determined in duplicate by comparing the chromatograms obtained in the samples to those obtained with the standard solution,

Following completion of the stability study, the composition with the best stability results and best sensory acceptance was evaluated and selected by a panel of experts.

Determination of antioxidant capacity

Antioxidant capacity was determined in tubo via acellular ABTS (2,2'-azino-bis (3-3-ethylbenzothiazoline-6-sulfonic acid) assay (ABTS Antioxidant Assay Kit, Zenbio). This assay is based on the ability of a sample to inhibit the oxidation of the compound ABTS to ABTS⁺ and measure spectrophotometrically at 405 nm (or 750 nm, optionally) (15),(16). This ability to inhibit the oxidation of ABTS in the sample will be compared to that of the reference antioxidant, Trolox C (a vitamin E analogue), and will be expressed as millimoles of Trolox equivalents. The percentage of inhibition of the sample relative to the Trolox C control, considering the inhibitory action of the latter as 100%, can also be calculated. As a first step, a stock solution of each of the substances to be tested and a bank of various concentrations of each product to be evaluated in the ABTS test were prepared (Table I).

Table 1. ABTS Test – concentrations tested individually.

*Check values of the combination in Table 2.

The combination of the four active ingredients was evaluated following the same range of concentrations as the ingredients tested individually, based on a ratio of 3:2:1:0.5; the same proportions as in the chosen formulation (Table 2).

Table 2. Test ABTS – Proportions tested in combination*

As a preventive step, any possible interaction of the study substances with the proposed spectrophotometric method was evaluated individually, and for this purpose the spectrophotometric profile of the carried product was explored by measuring the absorbance, especially in the reading wavelength range of the ABTS assay (from 750 to 405 nm) (17).

All the measurements were carried out at least three times (n=3) and then averaged. Percentage inhibition of absorbance was calculated using the following formula (18) :

ABTS·⁺ scavenging effect (%) = ((AB–AA)/AB) ×100

Where:

AB is absorbance of ABTS radical + methanol

AA is absorbance of ABTS radical + sample/standard.

Determination of skin antioxidant potential

Ferric Reducing Antioxidant Power (FRAP) measures the antioxidant capacity of a sample. The assay is based on the reduction of ferric to ferrous ions at acidic pH; a reaction that results in the formation of an intense blue ferrous-tripyridyl triazine complex (TPTZ) with an absorption maximum at 595 nm. The increase in absorbance indicates a higher reducing power, and therefore a higher antioxidant action (19).

At the chemical level, this assay measures a single electron transfer reaction through the color change that occurs as the oxidant is reduced, according to the following scheme (Figure 1):

Figure 1. ABTS test reaction scheme.

The antioxidant potential of the skin was assessed by quantifying antioxidants present in the outermost layers of the skin (stratum corneum). To carry out the procedure, clean skin samples were collected from the cheeks of 30 volunteers after they had used the antioxidant-rich anhydrous formulation twice daily for 28 days. This collection was performed by tape extraction using Corneofix^®. The third strips were collected and then stored at -80 °C in FRAP reagent (Merck®) (0.3M Acetate Buffer pH 3.6, 25 ml; TPTZ Solution, 2.5 ml; FeCl₃ Solution, 2.5 ml). The samples were incubated at 37 °C using a microplate incubator shaker, with 30 minutes of continuous shaking. Absorbance was read at 595 nm. Antioxidant activity was determined by the increase in absorbance at 595 nm (20) and the results were expressed as micromolar Fe (II) equivalents of the samples both before and after treatment.

Clinical assessment of anti-oxidative aging effects

The study aimed to evaluate the brightening and firming efficacy, visible effects of skin with less oxidative stress, of the antioxidant-rich anhydrous formula for facial application. To achieve this objective, an instrumental study was conducted on 30 healthy Caucasian women over 35 years of age and who presented loss of elasticity and poor skin radiance. All the study procedure was carried out in compliance with the ethical principles for the medical research. An informed consent was obtained by the volunteers to start the topical experimental study (21). Assessments were carried out at baseline (T0) and after 28 days of treatment (T28), using non-invasive techniques capable of measuring skin radiance and skin elasticity/firmness. The evaluations were conducted initially (T0) and at the end (T28) of the treatment in a temperature and humidity-controlled environment (respectively T = 18–26°C and RH = 50±10%). Digital photographs of the face were taken using VISIA-CR^® (Canfield Scientific). The radiance measurement was taken with the CM-700d spectrophotometer/colorimeter (Konica Minolta) and the ratio between directional and diffuse reflection enabled the calculation of the shine component. Meanwhile, skin firmness measurements were taken using the Cutometer^® MPA 580 (Courage + Khazaka), an optical system used to measure the skin’s penetration depth inside the probe in the two phases of the measurement. The data obtained were processed and displayed numerically for the purpose of calculating the viscoelastic properties of the skin. The parameter R2, gross elasticity, was measured; the closer the value is to 1, the more elastic the skin is. The R0 parameter, skin distension, was also measured, with a reduction in the R0 indicating an improvement in skin firmness.

Statistical analysis of instrumental measures was subjected to paired Student t-test (within-group analysis vs T0). Variations are considered statistically significant when the p value is <0.05.

Result and discussion

Demonstration of formula development and stability assessment

Of all the formulations tested, respecting the fact that an anhydrous excipient was used, with a high concentration of antioxidants and a pleasant sensory profile, the one yielding the best result in initial and long-term stability was selected. In the end, the mixture of ingredients with a high concentration of antioxidants turned out to have the following composition: Propanediol, Ascorbic Acid, Dimethyl Isosorbide, Alcohol Denat., PEG-12 Dimethicone, Alpha-Arbutin, Carnosine, Curcuma Longa (Turmeric) Root Extract, Diglycerin, Pinus Pinaster Bark/Bud Extract.

The stability studies conducted with the antioxidant-rich anhydrous formula showed satisfactory results for all the parameters studied, both organoleptic and physicochemical. It was possible to solubilize and chemically stabilize all the ascorbic acid, 12%. To ensure that the excipient of the formulation, as well as the operating method, were able to guarantee the stability of ascorbic acid in the formula, the ascorbic acid was determined via HPLC. It was found that there was no alteration in the concentration of the main antioxidant, i.e. ascorbic acid as shown in Table 3.

Table 3. Ascorbic Acid Content – HPLC determination.

The ascorbic acid content remained stable both in the samples at 25ºC for one year and in the samples subjected to accelerated aging at 40ºC during the three months of the study. The ascorbic acid value remained within the expected specifications, yielding values of between 12±0.5%.

These results showed that the water-free excipient of the formulation successfully prevented hydrolysis of the ascorbic acid in solution, ensuring the stability of the main antioxidant throughout the product's shelf life. This demonstrates the significant role of excipients in maintaining the stability of active ingredients in a formulation.

Demonstration of the antioxidant capacity

Using the ABTS assay described above in the materials and methods section, the antioxidant strength of each active ingredient under study and their combinations were evaluated. The results are shown in Table 4. The combination of the four ingredients (ascorbic acid, Curcuma longa, Pinus pinaster and carnosine) was evaluated following the same range of concentrations as the ingredients tested individually (Table 1), and also following the same proportions as those present in the formula (Table 2).

Table 4. Results of antioxidant capacity ABTS, µm Trolox Equivalent.

All individually tested ingredients demonstrated antioxidant activity in the ABTS assay. Furthermore, except for Carnosine, all ingredients showed a dose-dependent antioxidant effect, as shown in Figure 2. The combination of the four ingredients (Ascorbic Acid, Turmeric, Pinus pinaster, and Carnosine) demonstrated clear antioxidant power in the ABTS test, showing a concentration-dependent response thus confirming the enhancing effect that these ingredients deliver in the combination, although it is not a sum of the antioxidant potential of all of them.

Figure 2. Antioxidant activity of the substances indicated as µm Trolox equivalents (te) (n=3). Error bars represent the standard deviation of the mean of three independent assays (n=3).

Evaluation of skin antioxidant potential

Theantioxidant activity of the product when comparing the FRAP values before and after cumulative topical application with the antioxidant-rich anhydrous solution are shown in Figure 3. The value significantly increases by an average of 47.8% (p<0.001) of the FRAP-Fe (II) value (µm).

Figure 3. The graphic shows the mean data obtained at each experimental time. Data are expressed as a mean ± SE. Above the error bar the intragroup statistical analysis is reported as follows: * p<0.05; **p<0.01; ***p<0.001.

As it is possible to notice, after 28 days of use, the product demonstrated a statistically significant increase of FRAP Fe2+ μm by 47.8%. FRAP assay is a direct measure of the total reducing power of a biological matrix and an indirect index of the ability of the system under consideration to resist oxidative damage. An increase of this parameter indicates an increase of skin antioxidant potential. The antioxidant activity of the skin was evaluated after applying the antioxidant-rich anhydrous solution via the FRAP method to the outermost layers of the skin (stratum corneum). It was found to restore the skin by improving its ability to resist oxidative stress.

Results of clinical evaluation for oxy-Aging

Efficacy studies in volunteers have the advantage that they reflect the real conditions of use (skin benefit) of the product. A non-invasive efficacy study was conducted on 30 volunteers over the age of 35, all presenting with loss of skin elasticity and radiance, to evaluate the effectiveness of the antioxidant-rich anhydrous formulation.

The results in Figure 4 show skin radiance before (T=0) and after (T=28) application of the antioxidant-rich anhydrous formula. Skin radiance can be seen to increase significantly by an average of 18.9% (p<0.001).

Figure 4. The graphic shows data obtained at each experimental time for the radiance, expressed as Gloss. Data are expressed as a mean ± SE. Above the error bar the inter-group statistical analysis vs. T0 is reported as follow: * p<0.05, ** p<0.01, *** p<0.001.

After applying the solution for 28 days, a significant increase in skin elasticity (increase of R2 parameter) and firmness (decrease of R0 parameter) of 7.4% (p<0.001) and 7.9% (p<0.001), respectively, was observed (Figure 5 A-B).

Figure 5. The graphics show the data obtained at each experimental time for the elasticity (A) and firmness (B). Data are expressed as a mean ± SE. Above the error bar the inter-group statistical analysis vs. T0 is reported as follow: * p<0.05, ** p<0.01, *** p<0.001.

Also, as shown in Figure 6 shows a general improvement in terms of wrinkles and radiance of a representative volunteer after applying the formula before (left) and after 28 days (right).

Figure 6. Images of 49 old volunteer who experienced an improvement in radiance, elasticity and firmness T0: pre-treatment evaluation; T28: post-treatment evaluation, after 28 days of formulation application.

These results revealed a statistically significant increase in the parameters of skin radiance, elasticity and firmness following topical application of this formulation for 28 days. Topical application of antioxidants decreases the rate at which skin elasticity (22) and firmness are modified. These improvements were also perceived subjectively among the panellists and may be due to the topical action of the antioxidants, which augments and complements the natural skin defence mechanisms, thus making the skin more effective at combating oxidative stress and consequently oxidative aging, what it is called oxy-aging.

Conclusion

Following extensive research and testing, a novel topical formulation was developed, demonstrating efficacy in restoring and maintaining skin homeostasis by protecting the skin from oxy-aging. The formulation incorporates a high-concentration antioxidant cocktail, designed to ensure both the stability and bioactivity of its ingredients, irrespective of the packaging utilized.

The anhydrous (waterless) formula developed maintains the stability of the main active antioxidant ingredients, particularly ascorbic acid, over time. The combination of ingredients in the formula provides greater antioxidant power than ascorbic acid alone. This combination also enhances the skin's inherent antioxidant defence mechanisms by nearly 50%, effectively combating oxidative damage in the epidermis while preserving cutaneous homeostasis. The outcome observed in the volunteers is rejuvenated, firmer, and more radiant skin.

Conflict of Interest Statement

This article was created by employees of Natura Bisse Int. S.A. in the course of their employment.

Conclusion

The future of cosmetics lies in the continued evolution of holistic approaches which represents a transformative shift in the industry, merging scientific advancements, natural ingredients, and wellness principles. By understanding and embracing the interconnectedness of these elements, the cosmetics industry can cultivate products that not only enhance external beauty but also contribute to the overall well-being of individuals and the planet.

The interplay between beauty from within and topical cosmetics is the key for future products. The integration of biotechnology and green chemistry is revolutionizing cosmetic formulations, offering sustainable and biocompatible alternatives.

Developers can implement blockchain to trace the journey of ingredients from source to product. Nevertheless, the efficacy of the natural products should be scientifically proven. Marketers can communicate transparency as a brand value, and parallelly educate consumers by highlighting how specific ingredients contribute to radiant and healthy skin.

By embracing the synergy between these approaches and leveraging scientific advancements, the cosmetics industry can provide consumers with comprehensive beauty solutions that cater to both internal and external dimensions of beauty.

References and notes

Surfactant Applications

The application area lends itself particularly well to the use of AI. Active today in this area is the US company Potion AI (6). The company provides AI-powered formulation tools for beauty and personal care R&D. Their offerings include Potion GPT, next generation ingredient and formula databases and AI document processing. Potion’s work could have a significant impact on the entire surfactant value chain, from raw material suppliers to end consumers. By using their GPT technology, they can help target work toward novel surfactant molecules that have optimal properties for specific applications. By using their ingredient and formula databases, they can access and analyze a vast amount of data on surfactant performance, safety, and sustainability. By using their AI document processing, they can extract and organize relevant information from patents, scientific papers, and regulatory documents. These capabilities could enable Potion AI's customers to design and optimize surfactant formulations that are more effective, eco-friendly, and cost-efficient. A particularly interesting application for this type of capability is deformulation.

Deformulation is the process of reverse engineering a product's formulation by identifying and quantifying its ingredients. Deformulation can be used for various purposes, such as quality control, competitive analysis, patent infringement, or product improvement. However, deformulation can be challenging, time-consuming, and costly, as it requires sophisticated analytical techniques, expert knowledge, and access to large databases of ingredients and formulas.

AI can potentially enhance and simplify the deformulation process by using data-driven methods to infer the composition and structure of a product from its properties and performance. For example, AI can use machine learning to learn the relationships between ingredients and their effects on the product's characteristics, such as color, texture, fragrance, stability, or efficacy. AI can also use natural language processing to extract and analyze information from various sources, such as labels, patents, literature, or online reviews, to identify the possible ingredients and their concentrations in a product.

Figure 2. Skin Section with Microbiome. Most microorganisms live in the superficial layers of the stratum corneum and in the upper parts of the hair follicles. Some reside in the deeper areas of the hair follicles and are beyond the reach of ordinary disinfection procedures. There bacteria are a reservoir for recolonization after the surface bacteria are removed.

About the Author

Gina Puig

Gina Puig, Pharmacy graduate, began her career as a Cosmetic Development Technician. During more than 20 years, she has created and developed cutting-edge formulas. For the past three years, she has been the Head of R&D at Natura Bissé. Transitioning from a lab technician to leading product development, she is passionate about cosmetics, excelling in innovation and meeting consumer needs.

Gina Puig

R&D+i Department, Natura Bissé Int. S.A. Spain

References and notes

05/27/2025 08:28:48