article tetrahexyldecyl ascorbate (thdc) degrades rapidly
TRANSCRIPT
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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Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 3 of 22
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
Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 4 of 22
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
Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 5 of 22
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
Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 6 of 22
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
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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
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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
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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
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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
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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
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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
Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 13 of 22
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
Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 14 of 22
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
Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 15 of 22
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
Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 16 of 22
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
Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 17 of 22
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
Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 18 of 22
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
Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 19 of 22
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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