A clinical evaluation of the efficacy and tolerability of a novel topical antioxidant formulation featuring vitamin C, astaxanthin, and fermented turmeric

Ultraviolet light, visible light, infrared light, and pollution are a few examples of environmental factors that exacerbate the formation of reactive oxygen species (ROS) that cause damage to skin cells' DNA, proteins, and lipids. By supplementing the skin with antioxidants, we can help neutralize ROS formed by these extrinsic factors before they can damage the skin.


| INTRODUC TI ON
Over our lifetime, our skin is exposed to myriad environmental factors that contribute to aging along with both internal and genetic factors.Ultraviolet light, visible light, infrared light, and pollution are only a few examples of environmental factors that exacerbate the formation of reactive oxygen species (ROS) that cause damage to the skin cells' DNA, proteins, and lipids. 1 ROS, colloquially referred to as "free radicals," are naturally formed by mitochondrial metabolism in response to internal and external factors.However, it has been well-researched that skin damage occurs when our innate system that eliminates these ROS gets overwhelmed.ROS can cause hyperpigmentation, wrinkling, dryness, loss of elasticity, and roughness, contributing to the overall aged appearance of the skin. 2 Naturally, rejuvenating skin care is formulated with various factors to combat these signs of skin aging.The use of sunscreen to combat free radical damage is not enough, because sunscreen only protects against ultraviolet light formation of ROS.Further, only approximately 3%-7% of sunlight (at sea level) is composed of UVA and UVB rays, meaning our skin is constantly susceptible to ROS damage from several other sources. 1 By supplementing the skin with antioxidants, we can help neutralize free radicals formed by these additional extrinsic factors before they can damage the skin.Topical antioxidants, such as vitamin C (L-ascorbic acid), vitamin E (tocopherol or alpha-tocopherol), and ferulic acid, are commonly used ingredients in skincare to combat free radical damage.Antioxidants are thought to be more stable and effective when combined.A common blend in cosmeceuticals is vitamin C and vitamin E because the two work in tandem to quench free radicals.Additionally, vitamin C regenerates the oxidized form of vitamin E back to its reduced form, which essentially "resets" its ability to reduce free radicals. 3tamin E (a lipid-soluble molecule) also facilitates the penetration of vitamin C (a water-soluble molecule) through the stratum corneum to act within cell membranes. 3Furthermore, the addition of ferulic acid to this mix improves the chemical stability as well as doubles the photoprotection. 4tamin C plays several roles within the skin.The human body is incapable of synthesizing its own vitamin C; therefore, it must be obtained from outside sources.In addition to acting as an antioxidant that both neutralizes and reduces the formation of ROS, vitamin C is a required cofactor in the collagen synthesis pathway and has a role in the signaling cascade for fibroblast proliferation.Further, vitamin C improves hyperpigmentation by inhibiting tyrosinase, a crucial enzyme in the melanogenesis pathway.L-ascorbic acid is the biologically active form present in human skin; however, L-ascorbic acid has proven to be a challenge to formulate with in cosmeceuticals. 2L-ascorbic acid is unstable and quickly oxidizes if not formulated with either a low pH or anhydrous solution and stored using air-tight packaging. 5Low pH formulations can be a cause of skin irritation and cause symptoms of stinging in those with sensitive skin. 6Thus, several more stable derivatives and alternatives to vitamin C have been established.
Many of these derivatives, however, require conversion in vivo to L-ascorbic acid before they can be utilized by the skin. 5e such vitamin C derivative quickly gaining popularity is tetrahexyldecyl ascorbate (THD ascorbate), which is a stable vitamin C precursor that can be formulated at a pH as high as 6.5.THD ascorbate is converted to L-ascorbic acid through enzymes in the skin and this derivative of vitamin C is oil-soluble, unlike water-soluble L-ascorbic acid.The oil-soluble nature of TDH ascorbate is thought to allow for greater penetration into skin cells because of its ability to pass through the lipid-rich stratum corneum. 7other derivative of L-ascorbic acid is 3-O-Ethyl ascorbic acid.This water-soluble derivative is structurally similar to L-ascorbic acid, but the addition of an ethyl group provides enhanced stability to the molecule. 8This ingredient can be formulated at a pH of 4-5.5, making it more suitable than L-ascorbic acid for sensitive skin that tends to react to products with a lower pH. 6While cells have a lipid barrier, they are primarily water-based intracellularly.Therefore, combining water-soluble and oil-soluble formulations, such as THD ascorbate and 3-O-Ethyl ascorbic acid, creates an ideal solution that works in a complementary fashion to deeply penetrate and permeate through the skin. 7rmeric is another antioxidant known for its health benefits when ingested by mouth and making its way into skincare.This traditional Indian spice is well-studied for its anti-inflammatory properties.Two of the most powerful compounds in turmeric are curcumin and turmerone, both thought to play a large role in promoting skin health.Curcumin has been shown in preclinical studies to have antioxidant and wound-healing properties through inhibiting inflammatory cytokines and promoting collagen formation. 9Fermenting turmeric with lactobacilli has been shown to increase the concentration of curcumin without increasing its cytotoxicity. 10nally, astaxanthin is an antioxidant that has been demonstrated to have numerous skin benefits when ingested orally, but up until now there has been a paucity of data using this ingredient in topical formulations.This powerful carotenoid is naturally synthesized by bacteria, microalgae, and yeast and has a characteristic orange-red color.Ingestion of astaxanthin is thought to be why flamingos turn pink, as the algae and crustaceans in their diet are rich sources of this carotenoid.Astaxanthin produced naturally by H. pluvialis microalgae is considered to be more bioactive than that produced via fermentation or chemical synthesis. 11Studies have shown that astaxanthin has more ROS scavenging capacity than either vitamin C or vitamin E. 12,13 In addition to free radical scavenging, astaxanthin appears to exert its benefits on the skin through photoprotective and anti-inflammatory effects, as well as playing a role in DNA repair.In vitro, astaxanthin has effectively suppressed cell damage following UV irradiation, and inhibited the expression of matrix metalloproteinases, thereby increasing collagen content. 12merous skin protective effects of astaxanthin have been demonstrated in human intervention studies as well.Several studies have evaluated oral supplementation with astaxanthin capsules, two studies examined topical application alone, and one study combined oral supplementation with topical application.These studies have shown increased skin elasticity, reduction in wrinkles, reduction of erythema, suppression of melanogenesis and hyperpigmentation, and improvement in the skin barrier when compared to placebo. 12ven the impressive data behind oral astaxanthin in vitro and in vivo as a supplement for skin health, it shows immense promise as  All subjects provided written informed consent and photo release before participating in the study.

| Human repeat insult patch test
This was a single center, single-blind study that enrolled 57 male and female participants to investigate the potential irritation and sensitization of the test article using repeated cutaneous semiocclusive/occlusive patches.The test article was patched under 10 semiocclusive patches (9 inductive and 1 challenge patch) based on the modified Draize method. 7Induction patches were worn for either 47 or 71 h over 3 weeks.Participants had a rest period of 14 days.Challenge patches were then applied for 48 h and read both 1 h and 48 h post-removal for visible signs of irritation or sensitization.Overall appearance of aging on the face, neck and chest showed a statistically significant improvement as measured by an expert grader at both the 2-week and 12-week time points (Figures 3 and 4).

| Clinical grading and self-perception of skin
One hundred percent of subjects reported that the test article is gentle enough for daily use, is a "game-changer" for skin, and layers well with other skincare products.There was one adverse event reported during the study which was ultimately deemed unrelated to the study test article or procedure.No significant adverse events were reported during the course of the study.clinical evidence or published in peer-reviewed scientific journals, especially vitamin C serums.To date, the very few studies that have been conducted, including those that remain unpublished but are referenced in marketing materials only, are limited to an older cohort of Caucasian subjects.Our study deliberately selected a diverse cohort of participants to ensure representation of every type of consumer; including an age range inclusive of a younger generation, all Fitzpatrick skin types (I through VI), individuals with normal, oily, dry, and combination skin, and those with sensitive skin.Further, studies using topicals to improve the appearance of the skin often focus on the face alone.In this study, subjects were instructed to use the test product on the face, neck, and chest, and all three areas were assessed by independent expert grader.Both topical and procedural interventions aimed at reversing signs of photoaging on the neck and chest have been gaining traction in recent years.There has been a recent movement in focusing on treating these areas as one unit, both in terms of treatment and prevention of photoaging.The authors suspect that these choices reflect recent social media popularity of skincare, the rise of terms like "tech neck" on social media, and a younger demographic that is interested in aging prophylaxis and caring for their skin in a proactive, preventive way.

| DISCUSS ION
Recent advances in vitamin C formulations such as Asta C™ have tackled the challenges of irritation and instability of more traditional L-ascorbic acid formulations.These next-generation vitamin C derivatives are proving to be more stable and better tolerated on sensitive skin without compromising efficacy.Our serum combines multiple antioxidants that work together to deliver a complementary blend of antioxidants into the skin and penetrate all compartments of human skin.A 2019 study of 22 females (average age 56) looked at the impact of a different antioxidant blend on photoaging of the face after 12 weeks of twice daily use.The demographics of that study differ from the current one in several ways; first, that study looked primarily at subjects with Fitzpatrick skin type II.Furthermore, the mean age of the subjects was close to a decade older than that in the current study.Finally, the neck and chest were not evaluated in that study.Similar to the current study, a blend of water-soluble and lipid-soluble antioxidants were utilized, and they did demonstrate statistically significant improvements in fine lines, redness, and hyperpigmentation over a 16-week period. 14Unfortunately, very few other vit C serums have been subjected to clinical studies evaluating objective photoaging parameters and published in peer-reviewed journals.
To our knowledge, this is the first time that topical astaxanthin naturally produced by H. pluvialis has undergone human clinical evaluation as part of a vitamin C antioxidant formulation.Astaxanthin is a powerful and highly bioavailable free radical scavenger.In vitro studies estimate that its antioxidant capacity is 10 times greater than other carotenoids (lutein, zeaxanthin, beta-carotene) and it is estimated to have 100 times more antioxidant power than vitamin E. 11,15 Studies looking specifically at skin benefits of astaxanthin  16 This suggests that the use of oral astaxanthin might compliment the effects of topical sunscreen in protecting the skin from ultraviolet damage. 17Astaxanthin is also anti-inflammatory through inhibition of nitrous oxide production, and can accelerate wound healing by increasing collagen content in the skin. 12,15One randomized controlled trial (RCT) gave either 4 mg oral astaxanthin or a placebo to 23 participants for 9 weeks and studied the change in minimal erythema dose (MED).The astaxanthin group showed a significant increase in MED from baseline when compared to placebo.Another RCT gave either 0, 2, or 8 mg of oral astaxanthin to participants daily for 8 weeks and tested the inflammatory response of participants.They found that by Week 4 there was a dramatic decrease in DNA damage biomarkers, suggesting there was decreased ROS damage to DNA as well as decreased inflammation. 18Lastly, 31 participants taking 4 mg oral astaxanthin for 4 weeks showed significant decreased signs of photoaging on a residual skin surface component scale. 19Theoretically, if oral astaxanthin can have such profound effects on human skin, it likely holds immense promise as a topical product for photoaging if formulated in a way that is stable, potent, and bioavailable upon topical application.One study combined 6 mg oral astaxanthin and 1 mL topical astaxanthin twice daily in 30 women for 8 weeks.They saw statistically significant improvement in the depth of wrinkles, hyperpigmentation, and elasticity of the skin at both Weeks 4 and 8.The same group then did a randomized controlled trial in 36 men using either 6 mg oral astaxanthin or oral placebo for 6 weeks.There was a significant improvement in skin elasticity, wrinkles, and transepidermal water loss in the astaxanthin group versus the placebo group, with this study specifically emphasizing safety and efficacy in males. 20e limitations of our study include a small sample size and the lack of a control group.Additionally, a longer study duration would be desirable, as this study was only 12 weeks and improvements in skin parameters appeared to be progressively improving, thus we do not know at what point the benefits of twice daily use would start to plateau.Randomized controlled trials and longer studies should be performed in the future to further elicit the effects of the test article.

| CON CLUS IONS
Topical vitamin C antioxidant serums remain one of the most in demand and popular skincare products both in the United States and abroad.Patients often approach their dermatologists looking for evidence-based recommendations to help determine which products are effective and well tolerated, but there remains a paucity of peer reviewed publications addressing this topic to help guide physician recommendations.This study showed that treatment with Asta C™ demonstrated early and progressive improvement in signs of facial photoaging, suggesting that this novel topical antioxidant serum has the ability to reduce free radical damage of the skin in all skin tones.We highlighted the use of this product to not only benefit the face, but the neck and chest as well.These areas are notably difficult to treat retroactively, therefore the use of this product may prevent the need

a
topical ingredient added to an antioxidant formulation meant for daily use on the skin.Asta C™ Vitamin C Age Defense Serum (Dr.Whitney Bowe Beauty, Greenwich, CT) combines the above ingredients (THD ascorbate, 3-O-Ethyl ascorbic acid, fermented turmeric, astaxanthin, and vitamin E (tocopherol)) with the aim of providing the skin with a comprehensive blend of water-soluble and lipid-soluble antioxidants in a well-tolerated, stable, and clinically effective topical serum meant for twice daily use.This prospective open-label study explores the safety and efficacy of this novel topical formulation of antioxidants designed to fight free radical damage and improve overall skin quality, as well as the appearance of fine lines, wrinkles, radiance, and hyperpigmentation of the skin.
This research was sponsored by Bowe Glow, Inc. and conducted by contract research organization, Princeton Consumer Research (PCR) Corp., United States, and United Kingdom.These studies conformed to the requirements of the 1964 Declaration of Helsinki and its subsequent amendments and were conducted in accordance with applicable International Council for Harmonization, 2016.Integrated Addendum to ICH E6(R1): Guideline for Good Clinical Practice E6(R2) in as much as they apply to human subjects' research consumer product testing.The protocols were approved by PCR Corp., Tampa, Florida and PCR Corp., Chelmsford, United Kingdom.Subjects provided written informed consent before participation.

3 | RE SULTS 3 . 1 |
This single-center clinical study evaluated the efficacy of twicedaily application of the test article (Asta C™ Vitamin C Age Defense Serum, Dr. Whitney Bowe Beauty) for 12 weeks in 32 subjects.Healthy female subjects aged 35-60 with mild to moderate fine lines, wrinkles, and hyperpigmentation/uneven skin tone were enrolled in this study.Fitzpatrick skin types I-VI were included to ensure that all skin tones were represented.All skin types (dry, normal, combination, oily) were included, and 50% of the subjects had selfperceived sensitive skin.All enrolled subjects had to be able to provide informed consent and be willing to follow the study protocol.Subjects were excluded if they had any skin disease of the face, neck, or chest; known allergies or sensitivity to similar products; were pregnant, breastfeeding, or had plans to become pregnant during the study; history of cosmetic medical procedures in the last year; current use of steroids, Accutane, retinol, and skincare with active ingredients (AHA, BHA, vitamin C) or use of these products within the last 4 months; concurrent medications that may affect the response to the test article; or a medical condition that would either compromise safety of the subject or confound study results.Enrolled subjects completed a 1-week washout period with use of only a gentle cleanser to be applied to their face, neck, and chest.Subjects were instructed to use their own moisturizer and sunscreen as needed throughout the study period, but no other products.A home-use diary was utilized to measure compliance with the test article.Clinical grading assessments were performed for all subjects during Weeks 0, 2, and 12, and the self-perception questionnaires were completed during Weeks 2 and 12.Ten subjects (with at least one representation of each Fitzpatrick skin type) were selected for professional clinical photography of the face, neck, and chest during Weeks 0, 2 and 12. Visual and tactile grading was performed by a clinical expert on a rating scale from 0 = none to 9 = severe in categories such as overall skin appearance, skin aging, fine lines, wrinkles, hyperpigmentation, radiance, texture, elasticity, and redness.Subjects completed a self-perception questionnaire consisting of 26 questions in which they agreed or disagreed with statements pertaining to the appearance of their skin and the qualities of the test article.Adverse events were monitored and recorded throughout the study period.Human repeat insult patch test Fifty-five subjects completed the HRIPT dermatological safety study.No adverse reactions were elicited during the induction phase or throughout the duration of testing.There were also no questionable reactions observed during the challenge phase (Days 38 and 40) by any of the subjects to any of the test articles.These results support the assessment that under the conditions of the study, the test articles have demonstrated a low potential for irritation and sensitization.The test article can be considered as safe for use under the conditions of the study.
Thirty-four female subjects aged 34-60 years (average 47), Fitzpatrick skin types I-VI, with all skin types (normal, dry, oily, and combination) were screened for this study, with 50% of the subjects having self-perceived sensitive skin.Thirty-three subjects were enrolled into the study.Thirty-two subjects completed the study through Week 12. Two subjects dropped out of the study due to scheduling conflicts prior to the follow-up visits; one prior to baseline and one prior to Week 4. Expert-clinical graded assessments of facial, neck, and/or chest skin (10-point scale [0 = none, 1-3 = mild, 4-6 = moderate, 7-9 = severe]) reported visible improvements from baseline to Week 12 in overall skin quality, overall appearance of aging, global fine lines, radiance, texture, redness, firmness, hyperpigmentation, and global wrinkles (Figure 1).Fine lines (face, neck, and chest) over the 12week study period showed progressive improvement of 10.4% at Week 2 and 19.6% at Week 12.These results were statistically significant.One hundred percent (100%) of subjects demonstrated improvement in the appearance of fine lines at Week 12 as objectively measured by the expert clinical grader (Figure 2).Isolating the face, neck, and chest showed an average of 18.9%, 20.9%, and 18.8% improvement from baseline at 12 weeks, respectively.By Week 12, there was an average of 9% improvement from baseline of deeper wrinkles on the face/neck/chest, with 81.3% of subjects showing statistically significant improvement.The overall texture of the skin of the face/neck/chest improved by 10.2% at Week 2 and by 18.7% at Week 12 relative to baseline.The face showed the greatest improvement in texture, with 81.3% of participants displaying a decrease in clinical grade by 1 point or more.Hyperpigmentation/uneven skin tone (face, neck, and chest) over the 12-week study period showed 10.7% improvement at Week 12. Facial redness was also decreased by 20%.Eighty-one percent (81%) of subjects demonstrated significant improvement in the appearance of elasticity/firmness relative to baseline severity.Firmness of the skin increased from baseline by 10.9% over the face, neck, and chest.The category with the greatest improvement over the 12-week study period was the radiance of the skin, with a 26.6% increase from baseline and 100% of participants showing significant improvement.All subjects demonstrated improvement in overall skin quality (face, neck, and chest) by the end of the 12-week study period.
This study evaluated the efficacy and tolerability of a novel topical antioxidant formulation featuring vitamin C, astaxanthin, fermented turmeric, and vitamin E. This study will be one of the first of its kind published in the literature.Very few cosmeceuticals are backed by F I G U R E 1 Average percent change from baseline assessment of facial photodamage based on expert clinical grading for measurements taken at 2 weeks and 12 weeks.

F I G U R E 2
Percent of subjects demonstrating a statistically significant improvement in expert clinical grading from baseline taken at 2 weeks and 12 weeks.F I G U R E 3 Overall appearance of aging assessed by expert clinical grader from Week 0 to Week 12 of the face, neck, and chest.The clinical scale grades on a 10-point scale [0 = none, 1-3 = mild, 4-6 = moderate, 7-9 = severe].shown UV protective as well as rejuvenating effects.One study demonstrated that astaxanthin significantly protected against UVA damage in vivo and was taken up by fibroblasts to a greater extent than other carotenoids.

F I G U R E 4
Top: Hyperpigmentation on a woman's cheek at baseline and Week 12. Bottom: Fine lines, wrinkles, and hyperpigmentation on a woman's neck at baseline and Week 12. *Based on a 12-week independent clinical study of 32 subjects who used Asta C™ twice a day, as measured at baseline + the 12-week time point.for more invasive cosmetic interventions in the future.Additionally, the skin of the neck is more sensitive than that of the face and chest, and consequently products that are potent enough to drive visible, measurable improvement of facial skin are often poorly tolerated on the neck.Asta C™ was shown to be effective yet well-tolerated on this delicate skin.Subjective data from participants noted that this product was seamlessly layered with their daily skincare, which is important in a product meant for daytime use under sunscreen makeup and other color cosmetics.Cosmetically elegant products that integrate well into a person's routine and show early improvements help to encourage consistent daily use.Overall, the current clinical study demonstrates that Asta C™ is safe, well-tolerated, and effective in improving overall skin quality, as well as the appearance of fine lines, wrinkles, radiance, and hyperpigmentation of the skin.AUTH O R CO NTR I B UTI O N S WB conceived the test article.WB and PY planned and executed the clinical study.HK wrote the manuscript and produced the figures.WB, KK, and PY provided expertise and crucial edits to the final manuscript.