Article Tetrahexyldecyl ascorbate (THDC) degrades rapidly

Int. J. Mol. Sci. 2021, 22, x. https://doi.org/10.3390/xxxxx www.mdpi.com/journal/ijms

Article 1

Tetrahexyldecyl ascorbate (THDC) degrades rapidly under oxi- 2

dative stress but can be stabilized by acetyl zingerone to en- 3

hance collagen production and antioxidant effects 4

William R. Swindell1*, Manpreet Randhawa2, Geovani Quijas3, Krzysztof Bojanowski4, Ratan K. Chaudhuri5 5

1 The Jewish Hospital, Department of Internal Medicine, Cincinnati, OH, USA; [email protected] 6 2 Sytheon Ltd., Boonton, NJ, USA; [email protected] 7 3 Sunny BioDiscovery Inc., Santa Paula, CA, USA; [email protected] 8 4 Sunny BioDiscovery Inc., Santa Paula, CA, USA; [email protected] 9 5 Sytheon Ltd., Boonton, NJ, USA; [email protected] 10 * Correspondence: [email protected] 11

Abstract: Tetrahexyldecyl Ascorbate (THDC) is an L-ascorbic acid precursor with improved stabil- 12

ity and ability to penetrate the epidermis. The stability and transdermal penetration of THDC, how- 13

ever, may be compromised by the oxidant-rich environment of human skin. In this study, we show 14

that THDC is a poor antioxidant that degrades rapidly when exposed to singlet oxygen. This deg- 15

radation, however, was prevented by combination with acetyl zingerone (AZ) as a stabilizing anti- 16

oxidant. As a standalone ingredient, THDC led to unexpected activation of type I interferon signal- 17

ing, but this pro-inflammatory effect was blunted in the presence of AZ. Moreover, the combination 18

of THDC and AZ increased expression of genes associated with phospholipid homeostasis and 19

keratinocyte differentiation, along with repression of MMP1 and MMP7 expression, inhibition of 20

MMP enzyme activity, and increased production of collagen proteins by dermal fibroblasts. Lastly, 21

whereas THDC alone reduced viability of keratinocytes exposed to oxidative stress, this effect was 22

completely abrogated by the addition of AZ to THDC. These results show that AZ is an effective 23

antioxidant stabilizer of THDC and that combination of these products may improve ascorbic acid 24

delivery. This provides a step towards reaching the full potential of ascorbate as an active ingredient 25

in topical preparations. 26

Keywords: Acetyl zingerone; Ascorbate; Collagen; Skin aging; Tetrahexyldecyl ascorbate; Vitamin 27

C 28

29

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 2 of 22

1. Introduction 30

L-Ascorbic acid (AA) is essential for skin health and nutritional deficiency of this 31

vitamin leads to the well-characterized skin fragility and bruising seen in scurvy (hypo- 32

vitaminosis C). Within the dermis, AA is required for the synthesis and post-translational 33

modification of collagen [1], with functions that include hydroxylation of proline and ly- 34

sine as well as stimulation of collagen gene expression [2, 3]. Interestingly, AA is more 35

abundant in the epidermal layer [4], and in fact the epidermis expresses a unique AA 36

transporter (SVCT1) not expressed in other organs [5]. Within the epidermis, the role of 37

AA may parallel that of calcium [6], with pro-differentiation effects that improve barrier 38

function through elevation of filaggrin and normalization of stratum corneum structure 39

[7]. Such effects are accompanied by an increased abundance of stratum corneum barrier 40

lipids, including glucosylceramides, ceramides and lipid lamellar structures [8]. These ob- 41

servations have prompted development of topical formulations with the goal of directly 42

delivering AA to skin to achieve cosmetic or therapeutic effects. Topical AA delivery has 43

been reported to improve some aspects of intrinsic skin aging [9], although stronger evi- 44

dence supports a role for AA in the antioxidant-mediated prevention of extrinsic aging 45

due to UV radiation exposure [10]. Further evidence has supported a role for topically- 46

applied AA or AA-derivatives for treatment of wound healing and scar formation [11], 47

allergic contact dermatitis [12], hyperpigmentation [13], atopic dermatitis [14], and psori- 48

asis [15]. 49

Longstanding challenges of topical AA formulations have been related to the skin- 50

penetration and stability of AA as an active ingredient. AA is a water-soluble hydrophilic 51

anion with high polarity [16], which is repelled by the stratum corneum and can only 52

penetrate skin under acidic conditions (pH < 3.5) [17]. Moreover, upon exposure to ambi- 53

ent air and UV light, AA undergoes oxidation to dehydro-L-ascorbic acid in a reaction 54

facilitated by high temperatures and potentially by vehicle solution properties (e.g., high 55

pH, metal ions, presence of dissolved oxygen) [18]. Additionally, AA can react with sin- 56

glet oxygen to generate more persistent reactive oxygen species, such as H2O2, such that 57

AA may function as a pro-oxidant in some circumstances [19, 20]. To improve efficacy of 58

topical delivery, ascorbyl phosphate salts have been developed, which feature an added 59

phosphate group to prevent oxidation [21]. These products, however, must undergo an 60

in vivo conversion to AA and tend to have poor skin penetration due to their higher 61

charge density [17]. Another approach has been to develop combination products to im- 62

prove the stability and/or transdermal penetration of AA or AA-derivatives. For example, 63

a formulation that combined AA with ferulic acid was shown to have improved stability 64

[22], and skin permeability was bolstered by combination of AA with pyridoxine (vitamin 65

B6) [23]. 66

Tetrahexyldecyl Ascorbate (THDC) is a lipid-soluble AA precursor esterified with 67

branched chain fatty acid (2-hexyldecanoic acid), which is reported to have an improved 68

stability and ability to penetrate the lipophilic stratum corneum [13, 24]. It is essentially 69

an AA pro-drug that can penetrate the dermis where it may then undergo intracellular 70

enzymatic conversion to AA. Moreover, the lipid-soluble property of THDC may facilitate 71

its incorporation into cell membranes to confer added protective benefit [24]. When com- 72

bined with other ingredients, THDC has been shown to decrease signs of photodamage 73

such as skin wrinkling [24, 25] and improve hyperpigmentation [13, 26]. As such, THDC 74

has become the most popular lipophilic ascorbic acid derivative in the skin care market. 75

Despite its widespread use, however, the stability of THDC in the presence of reactive 76

oxygen species has not been fully demonstrated, and stabilizing antioxidants may be 77

needed to prolong the effective in vivo half-life of THDC. In this respect, acetyl zingerone 78

(AZ) is a previously described antioxidant compound able to neutralize non-radicals such 79

as singlet oxygen and peroxynitrite anion and radicals such as hydroxyl and peroxyl [27- 80

29]. The ability of AZ to physically quench singlet oxygen, in particular, may prolong 81

THDC half-life and impede production of H2O2 generated from the reaction between 82

THDC-derived AA and singlet oxygen [19]. The combination of THDC with AZ may 83

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therefore have synergistic effects above and beyond those of each compound individually, 84

although interactions between these two antioxidant compounds have not been investi- 85

gated. 86

The goals of this study were to investigate the effects of AZ on THDC stability and 87

mechanism of action. We evaluated the effects of THDC, AZ, and THDC + AZ using cell- 88

free in vitro assays and reconstituted human epidermis (RHE) tissues (EpiDermFT™). The 89

effects of both compounds on RHE tissue cultures were evaluated using in situ oligonu- 90

cleotide microarrays (Affymetrix Clariom S assays). Our results provide new insights into 91

the effects of THDC in a human skin model and show how these effects can be modulated 92

through combination with AZ as a stabilizing antioxidant. 93

2. Results 94

2.1. THDC is a poor antioxidant that degrades rapidly in the presence of singlet oxygen 95

The antioxidant profile of THDC against various reactive oxygen species was evalu- 96

ated (Figure 1A). This showed the strongest activity against singlet oxygen, with a total 97

oxygen radical absorbance capacity (ORAC) of 1035 μM trolox equivalents per gram (Fig- 98

ure 1A). This total ORAC value is less than 1% that of AA reported previously (Figure 1B) 99

[30]. THDC was also able to effectively inhibit lipid peroxidation (EC50 38.7 mg/mL; Fig- 100

ure 1C), although its potency in this regard was much less compared to AZ (EC50 = 0.46 101

µg/mL; Figure 1D). We next used HPLC to monitor THDC degradation under singlet ox- 102

ygen (Figure 1E). In the absence of AZ, THDC degraded completely by 6 minutes (Figure 103

1E). However, in the presence of AZ, there was only 25% degradation of THDC after 10 104

minutes (Figure 1E). Photooxidation analyses showed that AZ was stable under 105

UVA/UVB light exposure, with > 80% of AZ remaining following 4 hours of exposure 106

(Figure 1F). 107

Since the reaction between THDC and singlet oxygen generates H2O2 [19], we next 108

evaluated H2O2 scavenging capacity of THDC, AZ and THDC+AZ (1:1). This showed that 109

THDC alone has weak H2O2 neutralization capacity (EC50 850 mcg/mL), whereas AZ or 110

THDC+AZ neutralized H2O2 more effectively (EC50 ≤ 62.6 μg/mL) (Figure 1G). We further 111

evaluated the conversion of THDC to ascorbic acid in the presence of carboxylesterase-2 112

(CES2), which is the main carboxylesterase expressed by KCs with a role in epidermal 113

prodrug metabolism [31]. From 0 to 240 minutes, the percentage of THDC remaining was 114

similar in AZ(-) and AZ(+) conditions, with slightly greater accumulation of ascorbic acid 115

under AZ(+) conditions (Figure 1H and 1I). 116

117


Figure 1. THDC degradation, inhibition of lipid peroxidation, hydrolysis, and antioxidant activity. (A) THDC ORAC 118 profile. (B) AA ORAC profile. The antioxidant activity against each reactive oxygen species was determined (μmole trolox 119 equivalents (TE) per gram). The total ORAC-MR5 score is equal to the sum of individual activity estimates. The AA ORAC 120 profile shown in (B) was reported previously [30]. (B, C) Inhibition of lipid peroxidation. Lipid peroxidation was assessed 121 by colorimetrically estimating malondialdehyde (MDA) formation. The degree of inhibition is shown at different THDC 122 or AZ concentrations. The estimated IC50 concentration is shown in the top margin. (D) THDC degradation under singlet 123 oxygen. The percentage of remaining THDC is shown under AZ(+) and AZ(-) conditions. Degradation was monitored by 124 HPLC. (E) AZ stability under photooxidation. AZ was exposed to UVA and UVB light for 4 hours with the cumulative 125 dose indicated. The percentage of AZ remaining at each time point is shown (absolute quantities in parentheses, μmole 126 TE/gram). (F) H2O2 scavenging activity. The neutralization of H2O2 was assessed at varying THDC, AZ and THDC+AZ 127 (1:1) concentrations (n = 2 replicates). The estimated EC50 value is shown (top margin). (G, H) THDC fatty acid ester 128 hydrolysis with carboxylesterase-2 (CES2). Part (G) shows the percentage of THDC remaining at each time point, and (H) 129 shows the accumulation of ascorbic acid product (mM). 130

2.2. THDC activates type I interferon signaling in the absence of AZ 131

Microarrays were used to evaluate the effects of THDC on gene expression in the 132

absence of AZ (THDC vs. CTL comparison). Under AZ(-) conditions, THDC altered the 133

expression of 406 DEGs, including 236 THDC-increased DEGs (P < 0.05, FC > 1.25) and 134

170 THDC-decreased DEGs (P < 0.05, FC < 0.80). Genes most strongly increased by THDC 135

included zinc finger and SCAN domain containing 26 (ZSCAN26), MX dynamin like 136

GTPase 2 (MX2), ALG11 alpha-1,2-mannosyltransferase (ALG11), and transmembrane 137

protein 140 (TMEM140) (Figure 2B, 2C and 2E). Genes most strongly decreased by THDC 138

included solute carrier family 35 member G3 (SLC35G3), troponin I1 slow skeletal type 139

(TNNI1), progestin and adipoQ receptor family member 6 (PAQR6), and C2 calcium 140


dependent domain containing 4D (C2CD4D) (Figure 2B, 2D and 2F). As a group, THDC- 141

increased genes were most strongly associated with type I interferon signaling, response 142

to virus, and P450 xenobiotic metabolism (Figure 2G and 2I). Likewise, THDC-decreased 143

genes were most strongly associated with motor neuron differentiation, inflammatory 144

leukocyte activation, and O-glycan biosynthesis (Figure 2H and 2J). 145

146

Figure 2. THDC vs. CTL DEG summary. (A) THDC structure. (B) Top-ranked DEGs. The 44 genes with lowest p-value 147 are shown (ranked by FC). (C, D) DEG clouds. The 100 increased (red) or decreased (blue) genes with lowest p-value are 148 shown. Genes with lower p-values are shown with larger font. (E, F) Average expression. Expression of each gene is Z- 149 score normalized (mean = 0; standard deviation = 1). The average expression ± 1 standard error is shown for each gene 150 and treatment. An asterisk (*) denotes a significant expression difference (THDC vs. CTL or THDC+AZ vs. AZ; P < 0.05; 151 moderated t-statistic). (G, H) GO BP terms. GO BP terms most strongly enriched among (G) increased DEGs and (H) 152 decreased DEGs is shown. (I, J) KEGG terms. KEGG terms most strongly enriched among (I) increased and (J) decreased 153


DEGs is shown. In (G) - (J), the number of genes associated with each term is given in parentheses and exemplar DEGs 154 are listed within the figure. 155

2.3. THDC in the presence of AZ up-regulates phospholipid homeostasis genes while repressing 156

chemokine signaling genes 157

We next used microarrays to evaluate THDC responses in the presence of AZ (THDC 158

+ AZ vs. AZ comparison). Under AZ(+) conditions, THDC altered expression of 347 159

DEGs, including 168 THDC-increased DEGs (P < 0.05, FC > 1.25) and 179 THDC-decreased 160

DEGs (P < 0.05, FC < 0.80). Genes most strongly increased by THDC included ATP binding 161

cassette subfamily B member 11 (ABCB11), galectin 9B (LGALS9B), CEA cell adhesion mol- 162

ecule 1 (CEACAM1), and sex determining region Y (SRY) (Figure 3B, 3C and 3E). Genes 163

most strongly decreased by THDC included cholesterol 25-hydroxylase (CH25H), matrix 164

metallopeptidase 7 (MMP7), nucleoside-triphosphatase cancer-related (NTPCR), and 165

methylenetetrahydrofolate dehydrogenase 1 like (MTHFD1L) (Figure 3B, 3D and 3F). As 166

a group, THDC-increased DEGs were associated with phospholipid homeostasis, ion 167

transmembrane transport, and type 2 diabetes mellitus (Figure 3G and 3I). Likewise, 168

THDC-decreased DEGs were associated with response to chemokine, positive regulation 169

of lipid localization, and chemokine signaling (Figure 3H and 3J). 170

171


172 173

Figure 3. THDC+AZ vs. AZ DEG summary. (A) THDC and AZ structures. (B) Top-ranked DEGs. The 44 genes with 174 lowest p-value are shown (ranked by FC). (C, D) DEG clouds. The 100 increased (red) or decreased (blue) genes with 175 lowest p-value are shown. Genes with lower p-values are shown with larger font. (E, F) Average expression. Expression 176 of each gene is Z-score normalized (mean = 0; standard deviation = 1). The average expression ± 1 standard error is shown 177 for each gene and treatment. An asterisk (*) denotes a significant expression difference (THDC vs. CTL or THDC+AZ vs. 178 AZ; P < 0.05; moderated t-statistic). (G, H) GO BP terms. GO BP terms most strongly enriched among (G) increased DEGs 179 and (H) decreased DEGs is shown. (I, J) KEGG terms. KEGG terms most strongly enriched among (I) increased and (J) 180 decreased DEGs is shown. In (G) - (J), the number of genes associated with each term is given in parentheses and exemplar 181 DEGs are listed within the figure. 182

2.4. MMP7 and NQO1 have differential responses to THDC in AZ(-) and AZ(+) conditions 183

Further analyses were performed to identify genes differentially regulated by THDC 184

in AZ(+) and AZ(-) conditions. This identified 302 genes with a positive interaction pattern 185

(P < 0.05) and 392 genes with a negative interaction pattern (P < 0.05). A positive interac- 186

tion pattern is characterized by genes for which expression is up-regulated more strongly 187

by THDC in AZ(+) compared to AZ(-) conditions (Figure 4A). A negative interaction 188


pattern is characterized by genes for which expression is down-regulated more strongly 189

by THDC in AZ(+) conditions compared to AZ(-) conditions (Figure 4B). 190

Examples of genes with a positive interaction pattern included VPS37C subunit of 191

ESCRT-I (VPS37C), glycine amidinotransferase (GATM), fibrillin 3 (FBN3), and early 192

growth response 3 (EGR3) (Figure 4A). Such genes were most strongly associated with 193

transcription, methylation, IL-13 production and oxidative demethylation (Figure 4C). Ex- 194

amples of genes with a negative interaction pattern included superoxide dismutase 2 195

(SOD2), TNF alpha induced protein 6 (TNFAIP6), NAD(P)H dehydrogenase quinone 1 196

(NQO1), and matrix metallopeptidase 7 (MMP7) (Figure 4B). These genes were most 197

strongly associated with response to stress and leukocyte/epithelium migration (Figure 198

4D). The negative interaction pattern was confirmed for a subset of genes using RT-PCR 199

assays ( i.e., MMP1, MMP7, IRF1, SOD2 and CES1; Figure 4E – 4J). 200

201

202 203

Figure 4. Genes with differential responses to THDC in AZ(-) and AZ(+) conditions. (A) Top-ranked genes with a pos- 204 itive interaction effect (i.e., up-regulated by THDC in presence of AZ but not in the absence of AZ). (B) Top-ranked genes 205


with a negative interaction effect (i.e., down-regulated in the presence of AZ but not in the absence of AZ). In (A) and (B), 206 average gene expression is shown for each gene (± 1 standard error of the mean). (C) GO BP terms enriched among genes 207 with a positive interaction pattern. (D) GO BP terms enriched among genes with a negative interaction pattern. In (C) and 208 (D), the number of genes associated with each GO BP term is given in parentheses (left margin) and exemplar genes are 209 listed within each figure. (E – J) Real-time quantitative PCR (RT-PCR) results. Average expression is shown for each group 210 ± 1 standard error (n = 2 per group). Groups without the same letter differ significantly (P < 0.05, Fisher's least significant 211 difference). Relative expression was calculated using hypoxanthine phosphoribosyltransferase 1 (HPRT1) as a reference 212 and then normalized to the CTL treatment. 213

2.5. AZ moderates pro-inflammatory gene expression changes observed with THDC treatment 214

The effects of THDC on pre-defined gene sets were evaluated (Figure 5). In AZ(-) 215

conditions, THDC up-regulated genes increased as part of the unhealthy skin signature 216

(P < 0.01), which is a set of genes increased in diverse types of inflammatory skin disease 217

(e.g., SOD2, PRMT1, RCC1; Figure 5A and 5D) [32]. However, this effect was absent under 218

AZ(+) conditions (Figure 5A). Likewise, under AZ(-) conditions, THDC increased expres- 219

sion of genes belonging to the STAT1-57 module (P < 0.01), which is a set of genes acti- 220

vated by interferon signaling with elevated expression in inflammatory skin disease (e.g., 221

MX2, IFIT3, IFI44; Figure 5K and 5L) [33]. The effect, however, was not seen under AZ(+) 222

conditions (Figure 5K). Otherwise, THDC decreased expression of genes belonging to 223

some matrisome categories [34] in the AZ(+) condition (e.g., collagen genes, ECM glyco- 224

proteins, ECM regulator genes; Figure 5C, 5G and 5I) but not in the AZ(-) condition. Ex- 225

amples of ECM glycoprotein genes with increased expression in the AZ(-) condition only 226

included TNF alpha induced protein 6 (TNFAIP6), hemicentin 1 (HMCN1), and tenascin 227

C (TNC) (Figure 5H). 228

229

230 231

Figure 5. Unhealthy skin signature (USS), matrisome category, and STAT1-57 gene expression responses. (A-C, E-G, I- 232 K) Boxplots show the median and interquartile range of fold-change estimates in AZ(-) and AZ(+) conditions for genes in 233


each category (whiskers: 10th to 90th percentile). The number of genes in each category is indicated in the top margin. For 234 each boxplot, the colored region outlines the interquartile range observed for all other expressed genes not included within 235 the indicated category (p-value: Mann Whitney U test). (D, H, L) Example genes from selected categories with differential 236 responses to THDC in AZ(-) and AZ(+) conditions. Genes are ranked based upon fold-change (THDC/CTL). Genes signif- 237 icantly altered by THDC in AZ(-) or AZ(+) conditions are shown in magenta font (P < 0.05, bottom margin). 238

2.6. The THDC+AZ combination triggers gene expression shifts similar to those seen during KC 239

differentiation 240

We next evaluated the effects of THDC on the expression of genes regulated during 241

KC differentiation. Genes with expression altered during KC differentiation were identi- 242

fied from a prior microarray study that compared differentiating KCs (high calcium me- 243

dium, 7 days) to proliferating KCs (sub-confluent cells) (GSE21413). In the absence of AZ, 244

effects of THDC on gene expression were negatively correlated with those observed dur- 245

ing KC differentiation (r = -0.064, P = 3.67e-10; Figure 6A). Consistent with this, the set of 246

THDC-increased genes evaluated in both studies tended to be decreased by high-calcium 247

medium (Figure 6B), whereas no significant trend was observed for THDC-decreased 248

genes (Figure 6C). These effects of THDC, however, differed in the presence of AZ. Under 249

the AZ(+) condition, effects of THDC on gene expression were positively correlated with 250

those seen during KC differentiation (r = 0.203, P = 6.33e-90; Figure 6D). Consistent with 251

this, THDC-increased genes [AZ(+) condition] tended to be up-regulated in high-calcium 252

medium (P < 0.01; Figure 6E), and THDC-decreased genes [AZ(+) condition] tended to be 253

down-regulated in high-calcium medium (P < 0.01; Figure 6F). Examples of calcium-in- 254

creased genes up-regulated by THDC + AZ included glycine amidinotransferase (GATM), 255

nuclear receptor subfamily 4 group A member 1 (NR4A1), distal-less homeobox 5 (DLX5) 256

(Figure 6G), and examples of calcium-decreased genes down-regulated by THDC + AZ 257

included popeye domain containing 3 (POPDC3), AKT serine/threonine kinase 3 (AKT3), 258

and ATRX chromatin remodeler (ATRX) (Figure 6H). 259


260

Figure 6. Comparison to gene expression responses in calcium-treated KCs (GSE21413). (A, D) FC scatterplots. Each 261 point represents an individual gene. The proportion of genes in each quadrant is shown (top margin; red font: P < 0.05, 262 Fisher's exact test). Proportions are also represented by the right sidebar. The yellow ellipse outlines the middle 90% of 263 genes (Mahalanobis distance). The least square regression estimate is shown (yellow line) with Spearman correlation and 264 corresponding p-value. (B, C, E, F) GSEA analyses. Figures show that cumulative overlap between THDC-regulated genes 265 (± AZ) (P < 0.05) and a list of genes ranked based upon response to calcium (GSE21413). The area between the curve and 266 diagonal is shown with corresponding p-value (Mann–Whitney U test). (G) Calcium-increased genes (FDR < 0.10) de- 267 creased by THDC+AZ. (H) Radiation-decreased genes (FDR < 0.10) increased by THDC+AZ. In (G) and (H), FC estimates 268 are shown for each gene (THDC/CTL) with and without AZ. 269

2.7. The addition of AZ to THDC augments collagen protein and inhibits MMP expression and 270

activity 271

We next evaluated effects of THDC, AZ and THDC+AZ on production of COL I pro- 272

tein in human dermal fibroblasts (Figure 7A). AZ elicited a significant 15-31% extracellu- 273

lar increase in COL I production at all concentrations tested in adult fibroblasts, whereas 274

the combination THDC+AZ increased COL I protein by 12-27% at the highest concentra- 275

tions tested (50 and 100 µg/mL; Figure 7A). In contrast, THDC alone not significantly alter 276

COL I protein levels (Figure 7A). THDC also did not increase extracellular COL I protein 277

in neonatal fibroblasts, although in neonatal cells there was only a modest 7% increase in 278

COL I seen with AZ treatment (50 µg/mL; P < 0.05), without significant effect of 279

THDC+AZ treatment (25 – 100 µg/mL; data not shown). 280

THDC alone did yield a small (< 10%) but significant increase in cellular (but not 281

extracellular) levels of COL IV and COL VI proteins in neonatal and adult fibroblasts (Fig- 282

ure S1). However, AZ significantly increased both cellular and extracellular COL IV and 283


VI proteins in adult and neonatal fibroblasts (Figure S1), with a substantial (> 50%) in- 284

crease observed in extracellular COL IV from neonatal cells (Figure S1B). The effects of 285

THDC+AZ were intermediate to those of THDC and AZ alone, but in some cases 286

THDC+AZ increased extracellular COL IV or VI abundance when THDC alone had no 287

effect (e.g., see Figure S1B and S1H). We next used cell-free assays to evaluate the relative 288

effects of THDC and THDC+AZ on matrix metallopeptidase (MMP) enzyme activity. This 289

showed that the combination THDC+AZ led to more potent inhibition of MMP-1, MMP- 290

2, and MMP-3 activity as compared to AZ alone (Figure 7B - 7E). 291

292 293

Figure 7. Effects of THDC (± AZ) on collagen I protein and matrix metalloproteinase (MMP) enzymes. (A) Collagen I 294 protein (COL I). Collagen I abundance was measured using sandwich ELISA assays with magnesium ascorbyl phosphate 295 (MAP) as a positive control (n = 6-16 per group). Colorimetric signals were first normalized to cell numbers (sulforhoda- 296 mine B assay) and then normalized to the non-treated CTL group. Treatments not sharing the same letter differ signifi- 297 cantly (P < 0.05, Fisher's least significant difference; *P < 0.05, compared to CTL group). (B - E) MMP inhibition. Inhibition 298 of each MMP enzyme is shown at various THDC concentrations. Experiments were performed with AZ (red) and without 299 AZ (black). Estimated EC50 values are shown (top margin). Results were averaged over two replicate studies. 300

2.8. The combination AZ+THDC improves survival of KCs treated with oxidative stress 301


We treated HaCaT KCs with hydrogen peroxide (H2O2) for 0.5 or 3 hours and evalu- 302

ated effects on KC viability (Figure 8). Both treatment periods reduced KC viability alt- 303

hough the reduction was only significant after 3 hours (P < 0.05, Figure 8B). After 0.5 hours 304

of H2O2 treatment, the addition of THDC reduced viability further, whereas AZ restored 305

viability, and the highest overall survival was seen in KCs treated with the THDC+AZ 306

combination (at doses of 200 µg/mL each; Figure 8A). After 3 hours of H2O2 treatment, the 307

addition of THDC did promote a non-significant increase in viability, although a stronger 308

a significant increase was seen with AZ, and again the highest overall survival was ob- 309

served in cells treated with the THDC+AZ combination (at doses of 100 µg/mL each; Fig- 310

ure 8B). 311

312

Figure 8. Effects of THDC (± AZ) on viability of KCs exposed to oxidative stress. (A) 0.5-hour H2O2 exposure (n = 3-7 per 313 group). (B) 3-hour H2O2 exposure (n = 2-4 per group). Cells were incubated with H2O2 (10 mM) for 0.5 or 3 hours and 314 viability was assessed using the neutral red uptake assay. Colorimetric signals are normalized to the non-treated CTL. 315 Groups not sharing the same letter differ significantly (P < 0.05, Fisher's least significant difference; *P < 0.05, comparison 316 to non-treated CTL group). 317

3. Discussion 318

The topical delivery of L-Ascorbic acid (AA) has been a challenge due to its poor 319

stability and dermal penetration. Formulations that include the AA pre-cursor tetrahex- 320

yldecyl ascorbate (THDC) have gained widespread use [13, 24-26], but THDC may itself 321

lack stability within the dermal microenvironment. This study showed that THDC has 322

limited oxygen radical absorbance capacity and undergoes rapid degradation when ex- 323

posed to singlet oxygen (Figure 1D). This degradation, however, could be prevented by 324

the addition of acetyl zingerone (AZ) as a stabilizing antioxidant (Figure 1D). Moreover, 325

whereas treatment of RHE cultures with THDC alone stimulated expression of type I in- 326

terferon genes [35], the unhealthy skin gene signature [32], and the pro-inflammatory 327

STAT1-57 gene module [33], all these effects were abrogated in the presence of AZ. Finally, 328

although THDC did not prevent loss of viability in HaCaT KCs exposed to oxidative 329


stress, the combination THDC+AZ was fully protective (Figure 8). These results support 330

a synergistic mechanism by which AZ can stabilize THDC to facilitate delivery of AA into 331

the dermis. This suggests a novel strategy that can be used to maximize the potential of 332

AA as a topical ingredient in skin care formulations. 333

The current study showed that AZ was more effective than THDC at directly neu- 334

tralizing hydrogen peroxide (Figure 1F). This most likely explains why THDC alone did 335

not rescue the viability of H2O2-treated KCs, whereas the combination of THDC+AZ re- 336

stored KC viability (Figure 8). Under normal conditions, excess formation of H2O2 within 337

cells is removed by catalase, which converts H2O2 into water and molecular oxygen [36]. 338

However, catalase becomes depleted due to sun exposure, with seasonal variation corre- 339

sponding to low activity in summer and higher activity in winter [37, 38]. Recovery of 340

catalase following UVA exposure is also diminished in the skin of older subjects, which 341

contributes to extrinsic skin aging [39]. To compensate for this decline in antioxidant de- 342

fense, the use of topical AA or AA precursors has been proposed [40], but THDC alone 343

may be counterproductive since even more H2O2 may be generated as a degradation prod- 344

uct [19]. Our results suggest that the use of combined THDC-AZ formulations may pro- 345

vide a more stable formulation to maximize ascorbate release. Moreover, our results show 346

that AZ maintained its antioxidant capacity over 4 hours of continual exposure to simu- 347

lated day light (Figure 1E), which is tantamount to a full day of intermittent sun exposure 348

under non-extreme conditions [41]. This long-lasting antioxidant function may be needed 349

to ensure adequate AA release and free radical scavenging activity for THDC+AZ combi- 350

nation products applied to sun-exposed areas of skin (e.g., arms, hands and face). 351

The effects of THDC on gene expression have not been evaluated previously, but our 352

study has demonstrated differences in THDC bioactivity depending upon whether AZ is 353

present or absent as a co-ingredient. For example, treatment of RHE with the combination 354

THDC+AZ elicited gene expression responses similar to those observed in calcium-stim- 355

ulated KCs (Figure 6D - 6E) and further increased expression of genes associated with 356

phospholipid homeostasis (Figure 3G). On the other hand, treatment of RHE with THDC 357

alone tended to elicit gene expression changes opposite to those seen in calcium-treated 358

KCs (Figure 6A - 6C). Pro-differentiation effects of AA in KCs have been described previ- 359

ously [8]. It has been noted that effects of AA mimic those of calcium [6] and the addition 360

of AA to cell culture medium does indeed improve lipogenesis to support maintenance of 361

the stratum corneum barrier [8]. The differential effects of THDC we observed may there- 362

fore be explained by stabilization of THDC in AZ(+) conditions, leading to enhanced re- 363

lease of THDC-derived AA into the epidermal and dermal microenvironments, thereby 364

favoring a pro-differentiation response similar to that seen with calcium treatment of KCs 365

[42]. This suggests that the THDC+AZ combination may have practical benefits promoting 366

epidermal barrier recovery, which may be useful for applications such as xerosis and 367

wound healing [43]. 368

A surprising result from this study was that treatment of RHE tissue with THDC 369

alone led to up-regulation of type I interferon genes such as MX1, MX2 and STAT2 (Figure 370

2G). This was not seen, however, when THDC was combined with AZ (Figure 3G). The 371

type I interferon pathway plays an important role in antiviral defense responses and ap- 372

pears to be non-specifically activated in a broad spectrum of skin diseases [44]. Consistent 373

with this, treatment of RHE with THDC alone (but not THDC+AZ) led to up-regulation 374

of genes belonging to the “unhealthy skin signature” [32] (Figure 5A) as well as the 375

STAT1-57 gene module (Figure 5K), which is also activated under pathological condi- 376

tions such as wounding and skin cancers [33]. To our knowledge, no prior study has 377

demonstrated a detrimental pro-inflammatory effect of topically-applied THDC as a 378

standalone ingredient [13, 24-26]. It is possible that degradation of THDC leads to in- 379

creased H2O2 production, which contributes to an overall imbalance between antioxidant 380

and prooxidant factors that results in downstream activation of the type I interferon path- 381

way [45]. This may have reciprocal interactions with the process of KC differentiation [46], 382

with type I interferon pathway activation suppressing differentiation [47], and 383


differentiation in turn suppressing type I interferon pathway activation [48, 49]. Through 384

these mechanisms, the combination of AZ with THDC may buffer against aberrant acti- 385

vation of the type I interferon pathway that would otherwise be triggered by THDC alone. 386

The link between AA and tertiary collagen structure has been known for decades [1] 387

and it is now recognized that AA can also stimulate collagen mRNA synthesis [2, 3]. In 388

this study, THDC alone had no significant effect on synthesis of extracellular COL I pro- 389

tein in adult fibroblasts, whereas AZ and the combination THDC+AZ significantly in- 390

creased COL I protein levels (Figure 7A). Likewise, cellular and extracellular COL IV and 391

VI levels were generally higher in AZ-treated compared to THDC-treated fibroblasts, with 392

THDC+AZ treatment leading to an intermediate increase in COL IV and VI proteins (Fig- 393

ure S1). THDC also had inhibitory activity against MMP-1, MMP-2, MMP-3 and MMP-12 394

that was bolstered by AZ (Figure 7B - 7E). Stabilization of THDC by AZ may favor im- 395

proved collagen synthesis through increased bioavailability of AA. This would increase 396

collagen mRNA synthesis [2, 3, 50, 51] and prevent accumulation of underhydroxylated 397

procollagen protein to bolster procollagen mRNA translation [51]. Likewise, AA deriva- 398

tives inhibit matrix metalloproteinase-1 (MMP-1) activity in normal human fibroblasts 399

[52], and in one study AA decreased MMP-1 and MMP-2 protein abundance by 20% in 400

UVA-treated human dermal fibroblasts [53]. A nutrient mixture containing AA also in- 401

hibited secretion of MMP-9 monomer and dimer in a broad set of cancer cell lines [54], 402

and similar MMP inhibitory activity has been observed in other human and animal mod- 403

els [55, 56]. The effects of AZ-stabilized THDC observed in this study thus appear con- 404

sistent with prior work demonstrating links between AA release, collagen synthesis, and 405

MMP inhibition [2, 3, 50-56]. 406

THDC has become one of the most widely used L-ascorbic acid precursors, but evi- 407

dence demonstrating its stability under realistic in vivo conditions is lacking. This study 408

shows that THDC alone is a weak antioxidant with limited capacity for sustained AA 409

release when applied topically. However, through combination with AZ as a stabilizing 410

antioxidant, THDC degradation is slowed, leading to improved antioxidant activity, acti- 411

vation of KC differentiation pathways, and endogenous collagen production. The ob- 412

served synergy between THDC and AZ as co-ingredients supports their combination in 413

topical formulations. 414

4. Materials and Methods 415

4.1. THDC antioxidant capacity 416

The oxygen radical absorbance capacity (ORAC) of THDC was evaluated using pre- 417

viously described methods [57]. Fluorescent markers were used to monitor the extent of 418

damage resulting from exposure to peroxyl radicals, hydroxyl radicals, peroxynitrite, and 419

singlet oxygen. Results were quantified in terms of μM trolox equivalent per gram (μM 420

TE/g). The total ORAC was calculated by summing antioxidant activities obtained from 421

assays using each of the four free radical types. 422

4.2. Effects of THDC and AZ on lipid peroxidation 423

The effects of THDC on lipid peroxidation were evaluated by first adding 100 μL of 424

100 mM AAPH (2,2′-azobis(2-amidinopropane) dihydrochloride) into mixtures of squa- 425

lene and sample at different concentrations. Mixtures were then incubated at 37ºC over- 426

night. AAPH is an azo compound that undergoes thermo decomposition to generate per- 427

oxyl radicals, which then oxidizes fatty acid substrates to generate lipid peroxides. De- 428

composition of these unstable peroxides results in the formation of malondialdehyde 429

(MDA), which was quantified colorimetrically at 532 nm. MDA is a well-established index 430

of lipid peroxidation on fatty acids [58]. The effects of AZ on lipid peroxidation were eval- 431

uated using similar methods, except the first step was performed using AMVN (2,2'-azo- 432

bis (2,4-dimethylvaleronitrile)) rather than AAPH. The EC50 estimates were calculated 433

using robust regression as described previously [59]. 434


4.3. THDC degradation under singlet oxygen 435

Experiments were performed using 5 mL of 1 mg/mL THDC mixed with 5 mL 0.16 436

mg/mL lithium molybdate, 4 mL 0.01M NaOH, and 5 mL of 0.015% H2O2. The final pH of 437

the mixed solution was approximately 5.0. A 500 μL sample was taken from the mixture 438

every 2 minutes and added to 1 mL of curcumin (1 mg/mL) to terminate the reaction. The 439

sample was then analyzed by high-performance liquid chromatography (HPLC) to mon- 440

itor degradation. To evaluate the effect of AZ, the experiment was repeated in identical 441

fashion, expect the first step was performed using a 1:1 mixture of 5 mL THDC (1 mg/mL) 442

and AZ (1 mg/mL). 443

4.4. Stability of AZ under photooxidation 444

The AZ test sample was prepared in 50% ethanol within a quartz cuvette (Hellma 445

Analytics). The cuvette was then placed in a Rayonet RPR-100 photochemical reactor 446

equipped with four RMR 3500 (UVA) and four RMR-3000 (UVB) lamps (Southern New 447

England Ultraviolet Company, Branford, CT) to simulate daylight. A total of 100 μg/mL 448

of sample was irradiated at 31oC at a dose of 6.35 mW/cm2. 449

4.5. H2O2 scavenging activity 450

H2O2 scavenging assays were performed as described previously [60, 61]. Test sam- 451

ples were dissolved in ethanol and subsequently diluted with PBS (pH 7.40) to reach the 452

desired concentration. The reaction was then initiated by adding 4 mM of H2O2 to the 453

sample. The reaction was allowed to proceed for 10 minutes and absorbance was then 454

recorded at 230 nm. The EC50 estimates were calculated using robust regression as de- 455

scribed previously [59]. 456

4.6. THDC fatty acid ester hydrolysis 457

The fatty acid ester hydrolysis of THDC with carboxylesterase-2 (CES2) was evalu- 458

ated using previously described methods [62]. 1 mM THDC was dissolved in a mixture of 459

DMSO (2%) and ethylene glycol (10%) in 20 mM HEPES (pH 7.4). A 20 μL enzyme solu- 460

tion (1 mg/mL) was then added and incubated at 37oC for 15, 60, 120, and 240 minutes. 461

The reaction was stopped with methanol and THDC and AA concentrations were meas- 462

ured using a charged aerosol detector. The HPLC mobile phase consisted of 0.1% formic 463

acid in water, 0.1% TFA in methanol, and isopropanol. A Phenomenex (Torrance, CA) 464

Kinetex C18 column was used (4.6 mm x 100 mm, 2.6 μ). To evaluate the effect of AZ on 465

the reaction, the experiment was repeated with 1 mM THDC and AZ dissolved in the 466

DMSO (2%) / ethylene glycol (10%) / 20 mM HEPES solution (pH 7.4). 467

4.7. Microarray profiling of THDC, AZ, and THDC+AZ expression responses 468

Gene expression profiling was performed using EpiDermFT™ tissues (MatTek, Ash- 469

land, MA; cat no. EFT-400, lot no. 29376). The experiment included 4 treatments with 5 470

replicates each (CTL = control, THDC = tetrahexyldecyl ascorbate, AZ = acetyl zingerone; 471

THDC+AZ = tetrahexyldecyl ascorbate with acetyl zingerone in a 1:1 ratio). Test materials 472

were dissolved in DMSO yielding a 20 mg/ml stock solution. Subsequent dilutions of stock 473

solution were performed in distilled water. Samples of test material were added at a con- 474

centration of 10 µg/cm2 to the top surface of EpiDermFT tissues. After 24 hours, tissues 475

were rinsed and RNA was extracted and purified using the RNeasy Plus Mini kit (Qiagen, 476

Germantown, MD; cat. no. 74134) with QiaCube Connect robotic station. Purified total 477

RNA was assessed at 260 and 280 nm using the NanoDrop Lite (Thermo Fisher Scientific, 478

Waltham, MA). The Affymetrix Clariom S array platform was used to quantify genome- 479

wide expression following standard protocols. 480

Microarray pseudoimages were inspected for evidence of spatial artifact (Figure S2) 481

[63]. No major spatial artifacts were identified besides the expected area of increased in- 482

tensity arising from Affymetrix internal control probes (Figure S2). Among the 20 483


samples, 260/280 absorbance ratios were consistent with high purity RNA and ranged 484

from 1.88 to 2.07 (Figure S3A). Probe-level model residuals [64] were centered at zero for 485

each array, with sample THDC-5 notable for higher residual variation (Figure S3B). Nor- 486

malized unscaled standard errors (NUSE) and relative log expression (RLE) metrics were 487

calculated for each array (Figure S3C – S3F) [64]. We noted that two samples (THDC-5, 488

AZ-2) had higher NUSE median and IQR values (Figure S3C and S3D), whereas two other 489

samples (THDC-1, AZ-2) had elevated RLE median and RLE IQR (Figure S3E and S3F). 490

The 20 raw microarray data files (CEL files) were normalized using Robust Multichip 491

Average (R package: Oligo) [65]. A batch correction was applied using the ComBat algo- 492

rithm [66]. This correction was made based upon a division of samples into two batches 493

(n = 8 and n = 12, respectively) during the RNA extraction step. These normalization and 494

batch correction steps yielded expression intensities for 27189 probes. Of these, 21448 495

probes were annotated with a human gene symbol, which together were associated with 496

19525 unique genes. A subset of 20289 annotated probes associated with 18411 unique 497

gene names were linked to protein-coding genes (i.e., those genes having an “NP_” or 498

“NM_” prefix in their Refseq identifier). To limit redundancy in the analysis, when mul- 499

tiple probes were associated with the same human gene, the probe with highest average 500

expression among the 20 samples was selected to include in the analysis. Applying this 501

filter yielded 18411 probes having a one-to-one relationship with 18411 unique protein- 502

coding genes. For each sample, the 20% of genes with lowest overall expression were con- 503

sidered to have absent expression (3682 genes per sample). 504

The outForest algorithm [67] was applied to stratify samples in terms of the number 505

of outlier genes identified (Figure S3G). Fewer than 1% of genes were associated with 506

outlying values in each sample, with a maximal outlier percentage of 0.88% for CTL-4 507

(Figure S3G). A cluster analysis was performed and suggested that THDC-5 may be re- 508

garded as an outlying data sample (Figure S3H). THDC-5 also appeared to be an outlier 509

when the 20 samples were plotted with respect to the first 2 principal component axes 510

(Figure S3I). Together, these analyses suggest that sample THDC-5 could be excluded 511

from analyses, based upon elevated NUSE median and IQR (Figures S3C and S3D) and 512

the outlying pattern seen in cluster and principal component analyses (Figures S3H and 513

S3I). Subsequent differential expression analyses were therefore performed excluding 514

THDC-5 (n = 19 samples total). 515

4.8. Differential expression analyses 516

Differential expression analyses focused on two independent treatment comparisons 517

to evaluate the effects of THDC in AZ(-) conditions (i.e., THDC vs. CTL) and the effects of 518

THDC in AZ(+) conditions (i.e., THDC+AZ vs. AZ). The THDC vs. CTL two-group com- 519

parison was performed for 15044 protein-coding genes with detectable expression in at 520

least 3 of 9 samples. Likewise, the THDC+AZ vs. AZ two-group comparison was per- 521

formed for 15105 protein-coding genes with detectable expression in at least 3 of 10 sam- 522

ples. Differential expression tests were performed using moderated t-statistics with em- 523

pirical Bayes moderation of gene-specific standard errors (R package: limma; function: 524

eBayes). 525

A second set of analyses was performed in which the data were treated as a 2×2 fac- 526

torial design, with presence/absence of THDC as one factor (1 = THDC present; 0 = THDC 527

absent) and presence/absence of AZ as a second factor (1 = AZ present; 0 = AZ absent). 528

These analyses were carried out for 15130 genes with detectable expression in at least 5 of 529

19 samples. This allowed us to evaluate genes for which effects of THDC differ in AZ(-) 530

conditions as compared to AZ(+) conditions (i.e., a significant THDC×AZ interaction ef- 531

fect). Statistical tests were performed using moderated t-statistics (R package: limma; 532

function: eBayes) with a nested interaction formula and estimation of the interaction term 533

using a contrast statement (R package: limma; function: contrasts.fit). 534

For both sets of analyses described above (differential expression and interaction 535

tests), raw p-values were corrected for multiple hypothesis testing using the Benjamini- 536


Hochberg method. Raw p-value distributions obtained from differential expression tests 537

were approximately uniform with no discernible bias among genes having low or high 538

expression (Figure S4). 539

4.9. Real-time quantitative PCR 540

cDNA was prepared using the AzuraQuant cDNA kit (Azura Genomics, Raynham, 541

MA) (CTL, THDC, AZ, THDC+AZ, n = 2 samples per group). The expression of 9 target 542

genes was evaluated using real-time quantitative PCR and the BioRad iCycler iQ detec- 543

tion system. The chosen target genes were MMP1, MMP2, MMP7, MMP14, IRF1, IL11B, 544

SOD2, NQO1, and CES1. PCR primers were purchased from Realtimeprimers (Elkins 545

Park, PA) and reactions were performed using AzuraQuant Green Fast qPCR Mix Fluor 546

(Azura Genomics). The ΔΔCt method was used to estimate relative expression with hy- 547

poxanthine phosphoribosyltransferase 1 (HPRT1) as a reference gene [68]. 548

4.10. Effects of THDC (± AZ) on collagen production 549

The effect of THDC (± AZ) on collagen production was evaluated in exponentially 550

growing neonatal and adult human adult dermal fibroblasts (nHDF and aHDF). Test ma- 551

terials were dissolved in DMSO at 20 mg/ml. All further dilutions of stock solution were 552

made in sterile distilled water. Samples were added to nHDF (Cell Applications, San Di- 553

ego, CA, cat. no. 106K-05n) or aHDF (Cell Applications, San Diego, CA, cat. no. 106K-05a). 554

Cells were cultured in DMEM and 10% FBS. At the end of the experiment, biomarkers 555

were quantified in cells and/or cell culture media. Collagen I quantification was per- 556

formed using reagents from Southern Biotechnology (Birmingham, AL) following a pre- 557

viously described sandwich ELISA protocol [69, 70]. Collagens IV and VI were quantified 558

in formalin-fixed cultures by direct ELISA assays using Southern Biotechnology (Birming- 559

ham, AL) biotinylated anti-type IV collagen antibody/streptavidin-HRP (cat. no. 1340-08) 560

and Santa Cruz Biotechnology (Dallas, TX) HRP-conjugated anti-type VI collagen mono- 561

clonal antibody (cat. no. Sc-377143HRP). Collagen IV was quantified in cell culture condi- 562

tioned medium by sandwich ELISA assay using an R&D systems (Minneapolis, MN) anti- 563

collagen IV α1 antibody for capture (cat. no. AF6308) and Novus (Centennial, CO) poly- 564

clonal biotinylated anti-collagen IV antibody (cat. no. NBP1-26550) followed by streptav- 565

idin-HRP. Collagen VI was quantified using a Santa Cruz HRP-conjugated anti-type VI 566

collagen monoclonal antibody (cat. no. Sc-377143HRP). Tetramethylbenzidine (TMB) rea- 567

gent was used for detection. Total insoluble (cytoskeletal) proteins were quantified using 568

the sulforhodamine B method to standardize collagen signals to cell numbers [71]. Mag- 569

nesium ascorbyl phosphate (MAP) was used as a positive control and sterile distilled wa- 570

ter was the negative control. All colorimetric measurements were performed using the 571

Molecular Devices (San Jose, CA) microplate reader MAX190 and SoftMax3.1.2PRO soft- 572

ware. 573

4.11. Effect of THDC (± AZ) on MMP activity 574

The effects of THDC (± AZ) on the activity of MMP-1, MMP-2, MMP-3 and MMP-12 575

were evaluated. 0.1 gram of sample was dissolved in 1 mL DMSO and diluted 100-fold 576

with MMP buffer (50mM Tris, pH 7.5, 150 mM NaCl, 2mM CaCl2, 5μM ZnSO4, 0.01% 577

Brij-35) to make stock solution. Serial dilutions of the sample (1-to-1) were then made to 578

determine the IC50. Sample and 5 ng/well MMP-1 enzyme (Anaspec, cat. no. AS-72004), 579

MMP-2 enzyme (Anaspec, cat. no. AS-72005), or MMP-3 enzyme (Anaspec, cat. no. AS- 580

72006) were incubated at 37oC for 10 minutes. After 10 minutes, we added 0.2 μM/well 581

520 MMP FRET Substrate XIV (QXL® 520 - γ - Abu - P - Cha - Abu - Smc - HA - Dab(5 - 582

FAM) - AK - NH2 (Smc=S - Methyl - L - cysteine)) (Anaspec, cat. no. AS-60581). The fluo- 583

rescence signal was monitored at an excitation of 485 nm and emission of 530 nm. To 584

evaluate MMP-12 activity, sample was mixed with 0.2 mM/well AAAPVN (N-Succinyl- 585

Ala-Ala-Ala-p-nitroanilide) substrate (Sigma, cat. no. S4760), 139 ng/well elastase (Alfa 586


Aesar, cat. no. J61874) and readings were obtained following previously described meth- 587

ods [72]. 588

4.12. Effect of THDC (± AZ) on survival of H2O2-stressed KCs 589

We evaluated effects of THDC (± AZ) on the viability of HaCaT KCs incubated with 590

hydrogen peroxide. The project design was based on previously described methods [73]. 591

Test materials were dissolved in DMSO at 20 mg/ml with further dilutions made using 592

sterile distilled water. Samples were added to exponentially growing adult HaCaT 593

keratinocytes (Addexbio, San Diego) cultured in a 96 well plate in DMEM and 10% FBS. 594

Test materials were added 1 hour before H2O2 (10 mM) and the incubation period in the 595

presence of test substances was an additional 1 hour. After this time, cells were rinsed and 596

viability was determined by the neutral red uptake assay with Molecular Devices micro- 597

plate reader MAX190 and SoftMax3.1.2PRO software. 598

Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, Figure S1. 599 Effects of THDC (± AZ) on collagen IV and VI proteins. Figure S2. Microarray fluorescent 600 pseudoimages. Figure S3. Affymetrix quality control metrics. Figure S4. Differential expression 601 analyses. 602

Author Contributions: Conceptualization, M.R., K.B. and R.K.C.; Methodology, W.R.S., G.Q. and 603 K.B.; Software, W.R.S.; Validation, G.Q. and K.B.; Formal Analysis, W.R.S., G.Q. and K.B.; Investi- 604 gation, W.R.S., G.Q. and K.B.; Resources, R.K.C.; Data Curation, W.R.S.; Writing – Original Draft 605 Preparation, W.R.S.; Writing – Review & Editing, W.R.S., M.R., G.Q., K.B., R.K.C.; Visualization, 606 W.R.S.; Supervision, R.K.C.; Project Administration, M.R. and R.K.C.; Funding Acquisition, R.K.C. 607 All authors have read and agreed to the published version of the manuscript. 608

Funding: This work was funded by Synthon, Ltd. (Boonton, NJ, USA). 609

Institutional Review Board Statement: Not applicable. 610

Informed Consent Statement: Not applicable. 611 Data Availability Statement: Raw and processed microarray data will be submitted to the Gene 612 Expression Omnibus (GEO) database upon acceptance of this manuscript. All other raw data are 613 available from the corresponding author upon request. 614

Acknowledgments: The authors would like to thank Dr. Tony Chang (International Chemistry Test- 615 ing, Milford, MA) for technical assistance. 616

Conflicts of Interest: THDC and AZ are the active ingredients of Vitasynol® C and Synoxyl® AZ, 617 respectively, which have been developed and patented by Sytheon Ltd (Boonton, NJ). RKC is pres- 618 ident and CEO of Sytheon with ownership interest. MR is the Director of Research & Innovation at 619 Sytheon. KB is CEO of Sunny BioDiscovery, Inc. (Santa Paula, CA) and has received consulting re- 620 imbursement from Sytheon. WRS has received consulting reimbursement from Sytheon. 621

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