Prospective randomized double‐blind comparative study of topical acetyl zingerone with tetrahexyldecyl ascorbate versus tetrahexyldecyl ascorbate alone on facial photoaging

Tetrahexydecyl ascorbate (THDA) is a lipophilic precursor to ascorbic acid that may be stabilized by acetyl zingerone (AZ). Studies have shown that the topical application of THDA may have photoprotective effects. Similarly, AZ has been shown to mitigate oxidative and inflammatory stress, thereby improving the appearance of photoaging.


| INTRODUC TI ON
As the first line of defense against the external environment, the skin is perpetually exposed to noxious stimuli, particularly oxidative stress induced by ultraviolet (UV) radiation.The skin has a natural antioxidant system designed to combat free radicals. 1However, as oxidative damage accumulates due to chronic UV exposure, this innate antioxidant system may become overburdened, reducing the skin's ability to fight against reactive oxygen species (ROS).For example, healthy skin contains high levels of vitamin C, and some studies have reported that vitamin C levels in the skin decrease with age and solar exposure. 2,3Moreover, there are a variety of enzymic and non-enzymic antioxidants in the skin.Enzymic antioxidants include superoxide dismutase, glutathione peroxidase, catalase, glutathione reductase, and more. 3Lipophilic antioxidants include alphatocopherol and ubiquinol 10, and hydrophilic antioxidants include vitamin C (ascorbic acid) and uric acid. 3Acute and chronic UV exposure depletes these concentrations of enzymic and non-enzymic antioxidants in the skin, thereby accelerating photodamage and aging.
ROS are also generally implicated in the exacerbation of numerous dermatoses as well. 4e topical application of several antioxidants has been studied as a method of augmenting the skin's ability to combat oxidative stress.For example, tocopherol (vitamin E) is a phenolic antioxidant that has been shown to reduce hyperpigmentation and act as an antiinflammatory agent. 5,6Tocopherol is also lipophilic which allows it to pass the lipid-rich stratum corneum (SC) and exert its antioxidative effects into the deeper layers of the skin. 5,7Vitamin C, also known as ascorbic acid, has been shown to mediate melanogenesis and collagen synthesis in addition to its antioxidative properties. 8,9Thus, topical vitamin C has been used for wound healing, 10 scar formation, 11 contact and atopic dermatitis, 12,13 dyspigmentation, 14,15 and more.
However, vitamin C is hydrophilic in its natural form, which hinders its ability to penetrate through the SC. 16Due to its high polarity, vitamin C is further repelled by the SC and can only penetrate the skin in low pH environments (pH <3.5). 17Another challenge is that vitamin C is susceptible to photolysis which can also be influenced by characteristics of formulation such as pH, other active ingredients, and viscosity. 18erging formulations of topical vitamin C aim to increase penetration and photostability.One approach to this has been adding stabilizing ingredients such as ferulic acid or pyridoxine. 19,20other approach has been to develop derivatives of vitamin C that have better penetrating and stabilizing properties.For example, tetrahexyldecyl ascorbate (THDA) is a lipid soluble, esterified precursor to L-ascorbic acid, thereby improving its ability to penetrate the SC (Figure 1). 21Additionally, THDA does not require to be formulated at pH 3.5 or below as in the case with pure ascorbic acid. 17ce it has bypassed the SC, it undergoes enzymatic conversion to ascorbic acid, which then confers benefits to the skin.
Clinical studies with THDA have shown that when applied topically, it can decrease signs of photodamage and reduce hyperpigmentation. 22,23However, neither of these studies studied the isolated effects of THDA as it was part of a multi-ingredient regimen.
Despite its improved penetrability, THDA has been shown to have limited antioxidant capacity and degrades rapidly when exposed to singlet oxygen. 24Moreover, application of THDA alone to skin substitute cultures triggered activation of type I interferon pathways, like patterns observed in unhealthy or diseased skin (e.g., psoriasis, atopic dermatitis, seborrheic dermatitis, and acne). 24,25Overall, it is unclear if THDA may actually improve features of photoaging in the absence of other stabilizing antioxidants and is evaluated in this clinical study.
The potentially negative effects of THDA on type I interferon pathways can be prevented by using acetyl zingerone (AZ) as a stabilizing antioxidant. 24AZ inhibits THDA degradation and abrogates Conclusions: Daily use of combined THDA and AZ may improve facial wrinkle severity, pigment intensity, and erythema to a greater extent than THDA.While THDA alone increases facial wrinkle severity and erythema, the addition of AZ reduces both.the pro-inflammatory type I interferon response seen when THDA is used as a standalone ingredient.AZ (Figure 2) is a plant-derived phenolic antioxidant that has been shown to have numerous skin benefits on its own.[28][29][30][31] For example, AZ has been shown to inhibit the production of cyclobutane pyrimidine dimers which are UV-induced signature mutations found in melanoma. 30AZ also acts as a selective chelator to mediate oxidative stress by ROS and strengthens the extracellular matrix (ECM), thereby improving the appearance of photoaged skin and strengthening the skin's antioxidative potential. 26Furthermore, AZ is photostable, allowing it to exert its beneficial effects regardless of UV exposure.When used in conjunction with THDA, AZ was able to neutralize singlet oxygens that accelerate THDA degradation, ultimately stabilizing THDA and improving its bioavailability. 26gether, THDA and AZ have been shown to promote the expression of genes associated with lipid homeostasis and keratinocyte differentiation, elevated production of collagen I, IV, and VI proteins, and improved the viability of keratinocytes exposed to oxidative stress. 24Collectively, these findings support the use of AZ as an antioxidant stabilizer of THDA to maximize the benefits of ascorbic acid delivery in topical preparations.
The subjective assessment of skin by clinicians utilizing various scales has been long established for evaluating disease and signs of photoaging.However, emerging evidence suggests that the consistency in subjective grading from one clinician to another may vary considerably.Thus, several studies have evaluated the reliability of objective grading measures compared to subjective techniques.For example, one study found that the use of image-based analysis to grade bulbar hyperemia was 16 times more reliable than subjective analysis by practitioners. 32Another study found that the utilization of a computer-based facial asymmetry assessment program was significantly more consistent in assessing facial asymmetric in facial nerve palsy than subjective regional scoring by otologists. 33Moreover, another study found that there was a strong correlation between image analysis based quantification of facial erythema to subjective clinical grading for erythema, 34 thereby further supporting the potential of imagebased analyses in clinical studies.
One previous clinical study on the topical application of AZ showed that it can improve the appearance of wrinkles, pigmentation, and redness. 28The objective of this double-blinded clinical study is to assess how the addition of AZ to THDA affects the appearance of facial photoaging including facial erythema, uneven pigmentation, and appearance of fine lines in comparison to THDA alone.

| Materials
The AZ used in this study (trade name: Synoxyl® AZ; CAS index name:   F I G U R E 2 Functional groups and roles of acetyl zingerone (adapted from Chaudhuri et al. 26 ).

| Inclusion and exclusion criteria
Healthy male and females with facial fine lines and wrinkles be-  tropical products prior the baseline visit and to refrain from using these products for the duration of the study.Any participants who had facial cosmetic treatments (botulinum toxin, chemical peels, intense pulsed light, facial plastic surgery, etc.) 3 months prior to the baseline visit as well as those who were unwilling to hold facial treatments for the duration of the study were excluded.Adults unable to provide consent and prisoners were also excluded from participating in the study.

| Study visits and procedures
There were a total of 4 visits in this study; there was a screening visit to assess eligibility, a baseline visit, a visit at Week 4, and a visit at Week 8. High-resolution facial photographs were taken and analyzed to assess wrinkle severity, pigment intensity, and redness intensity using the BTBP 3D Clarity Pro® Facial Modeling and Analysis System 34 (Brigh-Tex BioPhotonics, San Jose, CA, USA) at baseline, Week 4, and Week 8. Facial measurements were obtained after participants acclimated to ambient conditions for 15 minutes in a temperature and humidity-controlled room.The facial measurements were performed on the entire face.Skin biophysical features such as facial erythema and melanin index were measured using the SkinColorCatch® (Delfin Technologies Ltd., Kuopio, Finland) at baseline, Week 4, and Week 8 to the infraorbital area.Tolerability was assessed during Week 4 and Week 8 using a self-rated questionnaire that assessed potential symptoms of product use such as burning, itching, stinging, scaling, and redness.Participants were required to rate the symptoms on a 3-point scale: "0" for none, "1" for mild symptoms, "2" for moderate symptoms, and "3" for severe symptoms.

| Statistical analysis
The primary endpoint of this study was to evaluate whether THDA and AZ together could decrease wrinkle severity, pigment intensity, and redness intensity after 8 weeks.Secondary assessments included skin melanin and erythema content following THDA and AZ use.Within group (paired, two-tailed) and between group (unpaired, two-tailed) Student's t-test were utilized to evaluate the differences before and after product use.A p-value less than or equal to 0.05 was considered statistically significant.Subjects served as their own controls using baseline data for within group analyses.Prism v.10 (GraphPad Software LLC, San Diego, CA, USA) was utilized to visualize the data. 35o 65).The difference of age between the two study groups was not statistically significant.There were 20 females and 3 males in the THDA-AZ group and 20 females and 1 male in the THDA only group.There was no significant difference in the distribution of Fitzpatrick skin types between the THDA only group and the THDA-AZ group (Table 1).

| Image system-based photographic analysis of wrinkle severity and pigment intensity
Average wrinkle severity in the THDA-AZ group was reduced by 0.75% and 3.72% at Weeks 4 and 8, respectively.Average wrinkle severity was significantly reduced in the THDA-AZ group compared to the THDA group where wrinkle severity increased by 7.88% and 4.48% at Weeks 4 (p = 0.023) and 8 (p = 0.048), respectively (Figure 4).Average pigment intensity in the THDA and AZ group was reduced by 1.68% and 4.10% at Weeks 4 and 8, respectively.The average pigment intensity was significantly reduced in the THDA and AZ group compared to the THDA group where average pigment intensity decreased by 0.71% and 0.69% at Week 4 and Week 8 (p = 0.0002), respectively (Figure 5).The average redness intensity in the THDA and AZ group was reduced by 3.73% and 14.25% at Weeks 4 and 8, respectively.The average redness intensity in the THDA group was increased by 27.5% and 8.34% at Weeks 4 and 8, respectively.Compared to the THDA group, there was a significant reduction in redness intensity in the THDA and AZ group at Week 4 (p < 0.016) and Week 8 (p < 0.045) (Figure 6).

| Skin colormetric indices (melanin and erythema)
There were no statistically significant differences in melanin content or erythema in the infraorbital area for either cohort after 4 and 8 weeks of product use.*Chi-squared assessment: χ 2 (5) = 8.73, p = 0.120, no statistical difference between the two groups.

| Tolerability
The products were well tolerated in both groups.The questionnaire included five questions assessing whether product use induced any itching, burning, stinging, scaling, and redness respectively.At Weeks 4 and 8, the majority of subjects did not experience any adverse symptoms in either cohort (Tables 2 and 3).

| Facial photography
High-resolution facial photography was taken for both intervention groups at baseline, Week 4, and Week 8 (Figure 7).

| Adverse events
Adverse events beyond tolerability were assessed, and there were no adverse events noted throughout the study.

| DISCUSS ION
The results of this study showed that there was a significant improvement in facial photoaging characteristics in the group that used both THDA and AZ compared to the THDA only group.
Previous studies have shown that formulations with the L-ascorbic acid version of vitamin C and AZ individually have measurable benefits for the skin, including reducing photodamage and hyperpigmentation. 28,36While THDA has been studied in a formulation that also included the ascorbic acid version of vitamin C, 21 the THDA form of vitamin C has not been studied in isolation and its topical delivery has been a challenge due to poor stability and dermal penetration.AZ also has been shown to improve the appearance of uneven pigmentation and the presence of fine lines. 28As an added benefit, AZ can stabilize THDA and improve its bioavailability as demonstrated in chemical and gene expression analyses. 24Our study shows that TDHA on its own may actually lead to no effect or worsening of photoaging parameters on the skin, but the addition of AZ to the THDA leads to improvement of wrinkles, erythema, and pigmentation.
There were several notable findings from this study.The use of THDA alone lead to a significant increase in wrinkle severity after 4 weeks of use and remained increased after 8 weeks.A previous study has shown that THDA increased the expression of genes related to type I interferon signaling such as MX1, MX2, and STAT2 24 as well as the STAT1-57 gene model, which has been implicated in compromised skin such as in wounds and skin cancer. 37DA and ascorbic acid (Figure 6) have both been shown to react with singlet oxygen to produce hydrogen peroxide, 24,38 thereby disrupting the homeostasis of anti-and pro-oxidant factors and leading to the activation of the type I interferon pathway.Excess formation of hydrogen peroxide is normally reduced by catalase, an antioxidant enzyme that converts hydrogen peroxide into water and molecular oxygen. 39However, studies have shown that catalase is easily depleted upon UV exposure. 40,41Moreover, catalase activity was slower to recover following acute UV exposure in older adults, signifying that catalase activity may be related to skin aging.Ultimately, excess hydrogen peroxide formation may be depleting natural antioxidant enzymes found in the skin, which may be contributing to the increase in wrinkle severity seen when using THDA alone.When adding AZ, wrinkle severity was significantly decreased, which is in agreement with a study showing that the two combined ameliorate THDA's pro-inflammatory effect and increase collagen production. 24milarly, topical THDA in the absence of AZ lead to significant increase of erythema within the first 4 weeks of use that remained increased after 8 weeks.Interestingly, our tolerability assessments did not include an increase in erythema and this may reflect that image-based grading has a higher resolution than Likert-based grading scales that require much higher shifts in erythema before they can be assessed in clinical grading scales.This flare of erythema with vitamin C has been previously noted with high concentrations for L-ascorbic acid but this is the first study to isolate the effects of THDA and show that erythema is increased because of topical application.THDA's stimulatory effect on the pro-inflammatory type I interferon pathway may also contribute to the increased erythema seen in our study. 24Previous studies of THDA have utilized it as part of many ingredients in the topical application but no previous studies have isolated the effect of THDA alone as evaluated in this study.Our findings suggest that THDA requires the presence of other antioxidant ingredients, or it may aggravate erythema and wrinkle severity.
Despite the increased erythema seen with THDA, subjective tolerability was similar between the THDA and the THDA-AZ groups suggesting that the erythema may not be related to superficial irritation and may be related to alteration of the local vasculature.
Further studies on the mechanism of action of THDA on capillaries are needed to further explore this further.Our study found that THDA alone lead to a decrease in pigment intensity.However, the addition of AZ decreased pigment intensity to a much greater extent than THDA alone.Pigment intensity also continued to decrease from Week 4 to Week 8 in the THDA and AZ group.Our findings are in agreement with studies showing that THDA and AZ can each modulate pigment intensity.For example, several studies have shown that THDA in combination with various other ingredients may improve parameters of skin photoaging and pigmentation. 21,22,42,43This is likely due to the inhibition of melanin synthesis via downregulation of tyrosinase enzyme activity. 447][28] Moreover, the ability of AZ to stabilize THDA may improve the bioavailability and actions of THDA on dyspigmentation.
There were no significant changes in infraorbital measures of skin colorimetric indices of melanin and erythema after 4 or 8 weeks of product use in either cohort.Skin colorimetric data were derived from the infraorbital regions of the face, which is distinct from the full facial image-based analysis that we performed to quantify wrinkles, pigmentation, and erythema.Although significant results were seen when analyzing the entirety of the face, changes in just the infraorbital areas did not seem to correlate.This may be due to the fact that a single point biophysical measurement may not be as comprehensive as the full-face analysis.Furthermore the color changes noted in the infraorbital area may have a different pathophysiology and origin compared to pigment on the rest of the face. 45Also, studies have shown that there are gradients in skin biophysical measurements across different regions of the face such as in measurements of skin hydration, TEWL, pH, and sebum production. 46,47This may explain the variations in pigment and erythema seen with colorimetric indices that did not correlate with the changes seen when integrating all regions of the face.Furthermore, future studies should be aware of this potential discrepancy when analyzing the differences in skin biophysical measures across different regions of the face and body.
Our findings offer further support for the use of topical AZ since they agree with a previous clinical study that showed improved photoaging parameters with topical treatment. 28However, our findings here extend that utility of AZ to be used alone or in conjunction with THDA.Our results should not be surprising in light of previous findings that THDA is not stable by itself but is stabilized in the presence of AZ. 24 AZ has also been shown to be photostable in the presence of UV exposure, which allows it to maintain its antioxidant capacity in non-extreme conditions. 26e production of reactive oxygen species by UV light exposure is well-established. 481][52][53][54] Thus, the antioxidative properties of AZ may be helpful in scavenging free radicals induced by blue light exposure, 26 and the expansion of studies to include blue light related evaluations are warranted.

| Limiting factors
This study included a relatively small population size of 44 subjects divided into two groups with a relatively large age range.However, the double-blind design increases the validity of the findings, especially in light of agreement with previously published topical AZ studies.While a split-face study design would allow us to reduce confounding due to differences between participants, we elected to avoid a split-face design as they may suffer from accidental application of creams to the wrong side and confounding on data integrity.The duration of the clinical study was limited to 8 weeks, and conclusions in regard to longer term use will require a future study with a longer assessment period.
Although this study included all Fitzpatrick skin types and no difference was seen between the groups, future studies may focus more specifically on certain skin types, since lower skin types are more susceptible to the development of facial wrinkles.The study results here are limited to the combination of 1% AZ with 5% THDA and not with other formulations.While our study shows the comparative efficacy of AZ/THDA vs THDA alone, we cannot make any comparisons against AZ alone in this study.We have shown the efficacy of AZ alone in a separate clinical study, 28 future studies to compare AZ/THDA against AZ alone would be necessary to understand how AZ compares to AZ/ THDA.
In conclusion, the daily use of combined THDA and AZ may improve facial wrinkle severity, pigment intensity, and redness intensity compared to THDA alone in people with photoaged skin.In fact, the use of THDA alone increased facial erythema whereas the combination of THDA and AZ reduced facial erythema.Moreover, future studies assessing the combined use of THDA and AZ in an expanded population are warranted.

FU N D I N G I N FO R M ATI O N
Funding for this study was provided by Sytheon.The sponsor had no role in the design or conduct of the study, the data analysis, or the decision to publish.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.

E TH I C S S TATEM ENT
The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Allendale Institutional Review Board (protocol # TOP_AZ_THDA) on March 16, 2023.

I N FO R M ED CO N S ENT S TATEM ENT
Written informed consent was obtained from all subjects involved in the study.

K
E Y W O R D S acetyl zingerone, photoaging, pigmentation, tetrahexyldecyl ascorbate, vitamin C F I G U R E 1 (A) Tetrahexyldecyl ascorbate and (B) L-ascorbic acid.

This 8 -
week double-blind randomized clinical study was conducted at Integrative Skin Science and Research between April 2023 and June 2023.The study protocol and consent were approved by the Allendale Institutional Review Board and was registered at www. clini caltr ials.gov (NCT05779280).Written informed consent was obtained from all participants involved in the study.Enrolled participants were randomized to receive either topical AZ combined with tetrahexyldecyl ascorbate (THDA 5% + AZ 1%) or tetrahexyldecyl ascorbate (THDA 5%) alone twice daily for 8 weeks.The clinical research coordinator achieved blocked randomization using a computer-based randomization generator and allocation was done via blinded sealed envelopes.Clinical research coordinators, investigators, and the participants were all blinded to the study intervention.In this study, 201 individuals were assessed for eligibility.Out of this group, 157 did not meet the inclusion criteria and 47 declined participation.Participants were recruited from nearby dermatology clinics and through social media from the Sacramento area.There were 44 participants who met inclusion criteria and none of the exclusion criteria.These participants were randomized and allocated into the following two groups: THDA + AZ (THDA-AZ) group or THDA group.In total, 38 participants completed all visits per protocol; 6 withdrew due to failure to meet the time commitment.

Figure 3
Figure 3 displays a CONSORT diagram depicting the flow of participants throughout the study.
tween the ages of 30-65 years old were screened and assessed for eligibility.Exclusion criteria included those who were pregnant, preparing to be pregnant, or breastfeeding.Individuals who had made changes to their hormonal based contraception within 3 months prior to enrollment were excluded.Current tobacco smokers and those with a smoking history greater than 10 packyears were excluded from the study.Participants were instructed to perform a two-week washout of topical retinoid, AZ, vitamin C, bakuchiol, hyaluronic acid, and benzoyl peroxide containing

F
I G U R E 3 CONSORT (Consolidated Standards of Reporting Trials) flow diagram.
Forty-four participants were enrolled into the study, and 38 participants completed the study per protocol.Twenty-one participants were allocated to THDA-AZ, and 23 participants were allocated to THDA alone.The average age of all participants was 51.3 ± 8.9 (range, 31 to 65).The average age of participants in the THDA-AZ group was 53.1 ± 7.8 (range, 38 to 62).The average age of participants in the THDA only group was 49.6 ± 9.8 (range, 31

F I G U R E 4 F I G U R E 6
Abbreviations: AZ, acetyl zingerone; THDA, tetrahexydecyl ascorbate.