Study of the Effects of Novel Analogs of Calebin-A on Melanogenesis

: In our previous study, we documented the anti-melanogenic efficacy of calebin-A (CBA), which is a curcuminoid analog. The effects of its newly synthesized analogs, i


Introduction
The abnormal levels of melanin synthesis in the melanocytes of the skin contribute to hyperpigmentation disorders that comprise age spots, melasma, and post-inflammatory hyperpigmentation (PIH) [1,2].Hyperpigmentation, which is a prevalent aesthetic concern across various skin types, mainly manifests itself in middle-aged women and individuals with Fitzpatrick skin types III-VI [3].Although hyperpigmentation is not considered a life-threatening disorder, it may significantly impact the overall well-being of individuals by compromising their appearance and causing emotional and psychological distress.Melanin, which is a remarkable biopolymeric pigment, undergoes synthesis within specialized cellular organelles known as melanosomes contained within melanocytes [4].The tyrosinase enzyme is involved in the production of both eumelanin and pheomelanin according to the mixed melanogenesis model [5].The initial and key step of melanogenesis involves the catalytic conversion of tyrosine to Dopaquinone (DQ) by tyrosinase [6].Tyrosinase inhibitors have maintained their appeal as agents for skin depigmentation.Within the cosmetics industry, various widely employed skin-lightening agents fall under the category of tyrosinase inhibitors.Examples of such inhibitors include kojic acid (KA), hydroquinone, and arbutin [7].Nevertheless, it was demonstrated that these substances possess the capability to induce unfavorable responses, including the manifestation of contact dermatitis, genotoxic effects, and the potential to promote carcinogenesis [8].Therefore, the utilization of innovative nature-derived compounds that can regulate hyperpigmentation disorders without eliciting any detrimental effects continues to attract considerable interest [9][10][11].
Calebin-A (CBA) is a curcuminoid analog of natural origin that was first discovered by Kim et al. [12] and shares structural similarities with curcumin but lacks the β-diketone group at the 1,3-carbon position that makes curcumin's structure unique.Contrary to curcumin's recognized instability at higher pH levels, CBA exhibits superior chemical stability in acidic and basic conditions while preserving curcumin's numerous physiological characteristics [13].CBA was shown to demonstrate efficacy in the control of obesity and inflammation linked to metabolic disorders [14].In another study, CBA showed promise for use in the management of tendinitis due to its potential anti-inflammatory activity [15].The synthesis of various analogs of CBA has also been reported in the literature.For example, the CBA analog Demethoxycalebin-A1 (DA1; Feruloylmethyl 4-hydroxycinnamate) was synthesized using a tedious five-step chemical route, as reported previously [12].Another analog, Demethoxycalebin-A2 (DA2; 4-Hydroxycinnamoylmethyl ferulate), was isolated as a mixture with DA1 [16].Subsequently, the synthesis of another analog, Bisdemethoxycalebin-A (BD; 4-Hydroxycinnamoylmethyl 4-hydroxycinnamate), along with DA1 and DA2, has been reported using a more efficient and economical singlestep, scalable green technique [17,18].Tetrahydrocalebin-A (THCBA), which is another analog of CBA, was obtained by the hydrogenation of CBA using palladium/carbon (Pd/C) as a catalyst, which is a well-known method that is similar to the chemical hydrogenation of the curcumin to obtain tetrahydrocurcumin [19][20][21].The structures of curcumin, CBA, and the four analogs of CBA are depicted in Figure 1.
DA1 was shown to impart protective benefits in β-amyloid-challenged rat and human neuroblastoma cells [12].The biological activities of other analogs have remained underexplored.Our previous study has elucidated the inhibitory effects of CBA on melanogenesis using the B16F10 cell model [22].Nevertheless, based on the current body of scientific literature, there appears to be a dearth of research pertaining to the impact of CBA analogs on the process of melanogenesis.In our ongoing pursuit of discovering innovative bioactive compounds with depigmentation potential, we expanded our investigations to assess the impacts of four CBA analogs on the enzymatic activity of tyrosinase.Additionally, we performed cellular assays that utilized B16F10 murine and MNT-1 human melanoma cells to explore their effects further.The novelty of this study was the evaluation of the four structurally related analogs (BD, DA1, DA2, and THCBA) of the parent molecule CBA on melanogenesis for the first time utilizing in vitro studies.Moreover, as these analogs bear a similarity to the various analogs of curcumin, their study could provide an overview of the structure-activity relationship (SAR) and help to advance the field of curcumin-based therapeutics for treating pigmentation disorders.

Effects of CBA Analogs on Antioxidant Activity
According to the results, BD was the only CBA analog that did not exhibit any antioxidant activity at any concentration.In contrast, the antioxidant activity of the other three analogs was shown to be concentration dependent (Figure 2).DA1 scavenged DPPH radicals by 9.9%, 23.34%, 29.71%, and 42.26% at 10, 20, 35, and 50 µM concentrations, respectively, while DA2 scavenged DPPH radicals by 13.34%, 30.77%, 32.63%, and 45.09% at 10, 20, 35, and 50 µM concentrations, respectively (Figure 2).THCBA scavenged DPPH radicals by 28.74%, 53.85%, 56.41%, and 70.03% at 10, 20, 35, and 50 µM concentrations, respectively (Figure 2).The IC 50 value for the antioxidant activity for THCBA was determined to be 22.00 ± 1.10 µM, while the analogs DA1 and DA2 did not achieve 50% inhibition within the tested concentration ranges.Consequently, the values were not determined.Overall, these data show that THCBA exhibited potent antioxidant activity while DA1 and DA2 showed comparable antioxidant activities that were lower than that of THCBA.

Effects of CBA Analogs on Mushroom Tyrosinase Activity
Our results of the effects of CBA analogs on the activity of tyrosinase enzyme in an in vitro cell-free system using L-Tyr (monophenolase) substrate show that BD caused robust inhibition of the monophenolase activity, with inhibitions of 45.46%, 45.30%, 50.91%, and 55.34% at concentrations of 10, 20, 35, and 50 µM, respectively (Figure 3A).DA1 potently inhibited the monophenolase activity by 65.18% at 10 µM, while at greater concentrations of 20, 35, and 50 µM, the activities were inhibited by 59.81%, 61.14%, and 61.48%, with no concentration dependency in particular (Figure 3A).Next, DA2 inhibited the enzymatic activities by 46.51%, 40.57%, 45.02%, and 47.64% at 10, 20, 35, and 50 µM, respectively (Figure 3A).THCBA inhibited the enzymatic activities by 14.07%, 27.15%, 41.69%, and 41.30% at 10, 20, 35, and 50 µM, respectively (Figure 3A).The IC50 value for the antioxidant activity for THCBA was determined to be 22.00 ± 1.10 µM, while the analogs DA1 and DA2 did not achieve 50% inhibition within the tested concentration ranges.Consequently, the values were not determined.Overall, these data show that THCBA exhibited potent antioxidant activity while DA1 and DA2 showed comparable antioxidant activities that were lower than that of THCBA.

Effects of CBA Analogs on B16F10 and HaCaT Cell Viabilities
We next evaluated the analogs in cellular assays by employing the well-validated B16F10 mouse melanoma cell model; the compounds were evaluated to exclude contributions of cytotoxicity on melanin assay results.With the exception of DA1, which induced considerable cytotoxicity at 50 µM with a diminution in viability of 56.81%, our findings from the MTS assay in B16F10 cells (Figure 4A) demonstrated that all of the other three analogs were nontoxic across the concentration range (10-50 µM).

Effects of CBA Analogs on B16F10 and HaCaT Cell Viabilities
We next evaluated the analogs in cellular assays by employing the well-validated B16F10 mouse melanoma cell model; the compounds were evaluated to exclude contributions of cytotoxicity on melanin assay results.With the exception of DA1, which induced considerable cytotoxicity at 50 µM with a diminution in viability of 56.81%, our findings from the MTS assay in B16F10 cells (Figure 4A) demonstrated that all of the other three analogs were nontoxic across the concentration range (10-50 µM).Moreover, the cytotoxicity of the CBA analogs to keratinocytes were also examined; the results reveal that none of the four compounds displayed any cytotoxicity to HaCaT cells at any concentration after 48 h of treatment (Figure 4B).Based on this data, we selected BD, DA2, and THCBA over the 10-50 µM concentration ranges and DA1 over the 10-35 µM concentration range for the subsequent experiments.Moreover, the cytotoxicity of the CBA analogs to keratinocytes were also examined; the results reveal that none of the four compounds displayed any cytotoxicity to HaCaT cells at any concentration after 48 h of treatment (Figure 4B).Based on this data, we selected BD, DA2, and THCBA over the 10-50 µM concentration ranges and DA1 over the 10-35 µM concentration range for the subsequent experiments.

Effects of CBA Analogs on Melanin Synthesis in B16F10 Cells
The administration of αMSH exhibited a pronounced effect on the augmentation of melanin biosynthesis in the B16F10 cells, as evidenced by visual inspection (Figure 5A).After co-treatment with varying concentrations of the four CBA analogs, only the cells treated with BD were consistently lighter in color at different concentrations, while the cells treated with DA1 or DA2 were lighter in color only at the highest concentrations (Figure 5A).On the other hand, THCBA resulted in a deeper-colored pellet at the highest concentrations, which was unexpected (Figure 5A).

Effects of CBA Analogs on Melanin Synthesis in B16F10 Cells
The administration of αMSH exhibited a pronounced effect on the augmentation of melanin biosynthesis in the B16F10 cells, as evidenced by visual inspection (Figure 5A).After co-treatment with varying concentrations of the four CBA analogs, only the cells treated with BD were consistently lighter in color at different concentrations, while the cells treated with DA1 or DA2 were lighter in color only at the highest concentrations (Figure 5A).On the other hand, THCBA resulted in a deeper-colored pellet at the highest concentrations, which was unexpected (Figure 5A).Quantitation of the intracellular melanin content by spectrophotometric analysis revealed that the BD treatment exhibited a pronounced diminution in the cellular melanin content with a concentration-dependent pattern that showed significant diminutions of 11.18%, 26.08%, 40.14%, and 40.46% at 10, 20, 35, and 50 µM, respectively (Figure 5B).Interestingly, BD at both concentrations of 35 and 50 µM was capable of decreasing the melanin levels robustly such that the residual levels were comparable with the basal negative control group levels (Figure 5B).At the same time, the treatment with DA1 resulted in significant suppression of the melanin content by 29.25% at 35 µM (Figure 5C).In contrast, the treatment with DA2 resulted in a notable reduction in the melanin levels, amounting to a decrease of 23.54% at 50 µM (Figure 5D).Lastly, the treatment with THCBA led to a significant increase by 18.87% at 50 µM concentration, with no significant change at concentrations lower than this (Figure 5E).Quantitation of the intracellular melanin content by spectrophotometric analysis revealed that the BD treatment exhibited a pronounced diminution in the cellular melanin content with a concentration-dependent pattern that showed significant diminutions of 11.18%, 26.08%, 40.14%, and 40.46% at 10, 20, 35, and 50 µM, respectively (Figure 5B).Interestingly, BD at both concentrations of 35 and 50 µM was capable of decreasing the melanin levels robustly such that the residual levels were comparable with the basal negative control group levels (Figure 5B).At the same time, the treatment with DA1 resulted in significant suppression of the melanin content by 29.25% at 35 µM (Figure 5C).In contrast, the treatment with DA2 resulted in a notable reduction in the melanin levels, amounting to a decrease of 23.54% at 50 µM (Figure 5D).Lastly, the treatment with THCBA led to a significant increase by 18.87% at 50 µM concentration, with no significant change at concentrations lower than this (Figure 5E).

Effects of CBA Analogs on Tyrosinase Activity of B16F10 Cells
The B16F10 cellular tyrosinase activity after αMSH treatment was significantly higher compared with the negative control, and the co-treatment with BD showed attenuation of the tyrosinase activity only at the maximum concentration (50 µM), where the activity was significantly attenuated by 35.26% (Figure 6A).Next, the co-treatment of hormonestimulated cells with DA1 analog showed a similar effect to that of BD but at a lower concentration of 35 µM, with significant suppression of tyrosinase activity by 32.74% (Figure 6B).
The tyrosinase activity was unaltered either by co-treatment with DA2 (Figure 6C) or THCBA across the entire concentration range (Figure 6D).
The B16F10 cellular tyrosinase activity after αMSH treatment was significantly higher compared with the negative control, and the co-treatment with BD showed attenuation of the tyrosinase activity only at the maximum concentration (50 µM), where the activity was significantly attenuated by 35.26% (Figure 6A).Next, the co-treatment of hormone-stimulated cells with DA1 analog showed a similar effect to that of BD but at a lower concentration of 35 µM, with significant suppression of tyrosinase activity by 32.74% (Figure 6B).The tyrosinase activity was unaltered either by co-treatment with DA2 (Figure 6C) or THCBA across the entire concentration range (Figure 6D).Overall, these findings suggest that DA1 suppressed the B16F10 cellular tyrosinase activity, but DA2 did not impact the tyrosinase activity in these cells.Consequently, it can be postulated that the mechanism by which DA2 suppresses melanogenesis in cells operates independently of its impact on tyrosinase activity, potentially involving alternative pathways.Furthermore, it is noteworthy that the administration of THCBA failed to elicit any significant augmentation in tyrosinase activity, thus indicating a lack of correlation with the previously observed elevation in melanin levels achieved at a concentration of 50 µM.

Effects of CBA Analogs on Melanin Synthesis in MNT-1 Cells
We next evaluated the impact of the four CBA analogs in MNT-1 human melanoma cells that are closely related to human melanocytes.To this end, the four analogs were first assessed for MNT-1 cell viability over the 10-50 µM concentration range.The results show that none of the four analogs affected the cell viability at any tested concentrations (Figure 7A).Accordingly, all four analogs were evaluated over the entire 10-50 µM concentration range in the further experiments.Overall, these findings suggest that DA1 suppressed the B16F10 cellular tyrosinase activity, but DA2 did not impact the tyrosinase activity in these cells.Consequently, it can be postulated that the mechanism by which DA2 suppresses melanogenesis in cells operates independently of its impact on tyrosinase activity, potentially involving alternative pathways.Furthermore, it is noteworthy that the administration of THCBA failed to elicit any significant augmentation in tyrosinase activity, thus indicating a lack of correlation with the previously observed elevation in melanin levels achieved at a concentration of 50 µM.

Effects of CBA Analogs on Melanin Synthesis in MNT-1 Cells
We next evaluated the impact of the four CBA analogs in MNT-1 human melanoma cells that are closely related to human melanocytes.To this end, the four analogs were first assessed for MNT-1 cell viability over the 10-50 µM concentration range.The results show that none of the four analogs affected the cell viability at any tested concentrations (Figure 7A).Accordingly, all four analogs were evaluated over the entire 10-50 µM concentration range in the further experiments.
Our findings of the cellular melanin content show that, surprisingly, BD did not elicit any discernible impact on the melanin content across all the tested concentrations (Figure 7B).However, DA1 led to significant diminutions of melanin content by 13.69% and 10.71% at 35 and 50 µM, respectively (Figure 7C).DA2 did not alter the melanin content of the cells at any concentration (Figure 7D).In the case of THCBA, no stimulation of melanin content was obtained at any concentration.Instead, the levels remained similar to the untreated control group (Figure 7E).Taken together, these results show that over the duration of 72 h, only DA1 exhibited the capacity to suppress the melanin production of MNT-1 cells.In contrast, BD, DA2, and THCBA showed no effect.Our findings of the cellular melanin content show that, surprisingly, BD did not elicit any discernible impact on the melanin content across all the tested concentrations (Figure 7B).However, DA1 led to significant diminutions of melanin content by 13.69% and 10.71% at 35 and 50 µM, respectively (Figure 7C).DA2 did not alter the melanin content of the cells at any concentration (Figure 7D).In the case of THCBA, no stimulation of melanin content was obtained at any concentration.Instead, the levels remained similar to the untreated control group (Figure 7E).Taken together, these results show that over the duration of 72 h, only DA1 exhibited the capacity to suppress the melanin production of MNT-1 cells.In contrast, BD, DA2, and THCBA showed no effect.

Effects of CBA Analogs on Tyrosinase Activity of MNT-1 Cells
The treatment with BD significantly inhibited the tyrosinase activities of MNT-1 cells by 11.07%, 14.76%, and 25.61% at concentrations of 20, 35, and 50 µM, respectively (Figure 8A).At the same time, the treatment with DA1 significantly inhibited the tyrosinase activities of the MNT-1 cells by 21.93% and 25.30% at 35 and 50 µM concentrations, respectively (Figure 8B).However, no alteration in the tyrosinase activity was obtained by any concentration of either DA2 (Figure 8C) or THCBA (Figure 8D).These results show that although BD did not alter the melanin content, it significantly inhibited the tyrosinase activity.In addition, the suppression of the tyrosinase activity was associated, at least in part, with the anti-melanogenic impact that DA1 had in the MNT-1 cells.
tivities of the MNT-1 cells by 21.93% and 25.30% at 35 and 50 µM concentrations, respectively (Figure 8B).However, no alteration in the tyrosinase activity was obtained by any concentration of either DA2 (Figure 8C) or THCBA (Figure 8D).These results show that although BD did not alter the melanin content, it significantly inhibited the tyrosinase activity.In addition, the suppression of the tyrosinase activity was associated, at least in part, with the anti-melanogenic impact that DA1 had in the MNT-1 cells.

Discussion
The presence of ortho-methoxy groups contributes to the increased stability of the phenoxy radical, thereby enhancing the antioxidant activity [23].This finding aligns with the observed antioxidant properties of DA1, DA2, and THCBA in our study.In contrast, BD, which lacks ortho-methoxy groups, did not exhibit any antioxidant activity.This phenomenon is comparable with the lack of antioxidant activity in bisdemethoxycurcumin, which is an analog of curcumin that has a similar substitution pattern [24][25][26].Based on our prior study [22], where we reported the IC50 values pertaining to DPPH radical scavenging activity exhibited by CBA to be 30.55± 4.82 µM, the results of our current study suggest that THCBA (with an IC50 value of 22.00 ± 1.10 µM) exhibited a greater level of antioxidant activity in comparison with its parent molecule CBA.This result is comparable with the case of tetrahydrocurcumin (THC), which is a hydrogenated derivative of curcumin and was shown to possess higher antioxidant activity than its parent compound curcumin [27].The authors of the study have documented that despite undergoing

Discussion
The presence of ortho-methoxy groups contributes to the increased stability of the phenoxy radical, thereby enhancing the antioxidant activity [23].This finding aligns with the observed antioxidant properties of DA1, DA2, and THCBA in our study.In contrast, BD, which lacks ortho-methoxy groups, did not exhibit any antioxidant activity.This phenomenon is comparable with the lack of antioxidant activity in bisdemethoxycurcumin, which is an analog of curcumin that has a similar substitution pattern [24][25][26].Based on our prior study [22], where we reported the IC 50 values pertaining to DPPH radical scavenging activity exhibited by CBA to be 30.55± 4.82 µM, the results of our current study suggest that THCBA (with an IC 50 value of 22.00 ± 1.10 µM) exhibited a greater level of antioxidant activity in comparison with its parent molecule CBA.This result is comparable with the case of tetrahydrocurcumin (THC), which is a hydrogenated derivative of curcumin and was shown to possess higher antioxidant activity than its parent compound curcumin [27].The authors of the study have documented that despite undergoing chemical reduction to generate THC, the antioxidant effectiveness of curcumin remains intact and, in fact, becomes enhanced [27].
The results of our study reveal that DA1 exhibited superior efficacy in cell-based biological assays compared with other CBA analogs.This activity could be attributed to the 4-hydroxy group located in the para position in the DA1 analog, which was demonstrated to be essential for the manifestation of the biological activities of CBA analogs [12].Our findings highlight a significant contrast in the biological activity of the two analogs DA1 and DA2, with DA1 showing much greater potency compared with the DA2 analog, which was ineffective.DA1 and DA2 are both derivatives of CBA, with one methoxy group removed, and are typically generated simultaneously during synthesis and subsequently isolated.Intriguingly, DA1 and DA2 share similarities with demethoxycurcumin, which was found to exist in two isomeric forms (form A and form B, with one form being more prevalent) based on the position of the methoxy group on the benzene ring, as shown in a previous study on the metabolic fate of demethoxycurcumin in rats [28].Our results of much greater activity of one isomer than the other are consistent with prior studies that showed distinct biological responses of different isomers of curcumin derivatives [29,30] or other compounds [31,32].In our study, we did not investigate the reason for the increased potency of DA1 compared with DA2, as it was not the primary focus.However, we speculate that DA1 may have higher hydrophobicity and lipophilicity than DA2, enabling it to penetrate cell membranes more effectively and reach the melanosomes to exhibit strong activity.This is likely given our MTS results of the DA1 analog in B16F10 and MNT-1 cells, which showed cytotoxicity of DA1 within the concentration ranges assessed.LogP values play a crucial role in SAR studies by revealing the drug's lipophilicity [33].Alternatively, future studies that involve computational chemistry approaches that incorporate molecular docking simulations to dissect the differences in the binding modes of DA1, DA2, and BD analogs to human tyrosinase protein, similar to the methods described in previous reports [34,35], will help in unraveling the differential effects of the two analogs.
Our results highlight that the CBA analogs BD, DA1, and DA2 suppress melanogenesis in B16F10 cells by a unique mechanism that is distinct from CBA, which was not a tyrosinase inhibitor in a cell-free system when tested at concentrations of 10 and 20 µM, as reported in our prior report [22].The impact of the four analogs was also examined in B16F10 cells in an unstimulated state without the use of αMSH.The results show that BD significantly suppressed the cellular melanin contents by 29.11%, 36.02%, and 35.99% at 20, 35, and 50 µM, respectively (Figure S1A).On the other hand, DA1 showed much more potent efficacy at diminishing the melanin content under basal conditions compared with stimulated conditions, as the levels were suppressed by 24.12%, 36.82%, and 29.24% at 20, 35, and 50 µM, respectively (Figure S1B).The treatment with DA2 did not result in significantly different levels at any concentration compared with the untreated control (Figure S1C).Finally, THCBA showed a similar trend to the stimulated condition by significantly increasing the melanin content by 19.45% at 50 µM (Figure S1D).Together, these findings suggest that the anti-melanogenic effectiveness of DA1 is the greatest under unstimulated conditions, while DA2 showed an anti-melanogenic effect only under hormone-stimulated conditions.At the same time, BD showed similar efficacy in diminishing melanogenesis under basal and hormone-stimulated conditions, while THCBA showed similar efficacy in enhancing melanogenesis under both conditions.A comparison of the results of the four CBA analogs alongside the results of the compound CBA in our previous study [22] is summarized in Table 1.It should be noted that the parent compound CBA has not yet been tested in human MNT-1 cells, and hence, the results of CBA analogs obtained in MNT-1 cells cannot be compared with the parent CBA compound.
Table 1.Comparative summary of the results of CBA analogs (BD, DA1, DA2, and THCBA) of the current study with the parent compound CBA of our prior study [22]  N.D. refers to "not determined"; ↓ symbol refers to "decrease"; × refers to "no effect"; B16F10 + refers to hormonestimulated B16F10 cells; TX refers to "cytotoxic"; CBA: calebin-A.
In the diphenolase activity assay, it was observed that the inhibitory effect of the DA1 and DA2 analogs appeared to show saturation at 10 µM, with no additional inhibition seen at higher concentrations of 20, 35, or 50 µM.Similarly, a non-linear concentration response for the DA1 and DA2 analogs was also noted in the monophenolase activity assay.Intriguingly, this non-linear response was not observed in cell-based tyrosinase activity assays in B16F10 or MNT-1 cells, although DA1 showed an inconsistent effect at suppressing the melanin content in MNT-1 cells at concentrations of 35 and 50 µM.It is important to highlight that the cell-free tyrosinase assays utilized a purified mushroom tyrosinase as the enzyme source, which varies from mammalian tyrosinase in structure and molecular motifs [36,37].Furthermore, the assays were performed using a fixed concentration of the L-DOPA substrate at 3 mM, whereas the final substrate concentrations in the B16F10 and MNT-1 cellular tyrosinase experiments were 2.25 mM and 1 mM, respectively.While the exact reason for this non-linear response remains unclear, it might be attributed to the enzyme source, specific substrate concentration, or the absence of cells.To fully understand this, it is essential to conduct kinetic studies on the inhibition mechanism using various substrate concentrations rather than just one concentration.Furthermore, this can assist in determining whether the inhibitory effects of DA1 or DA2 on the enzyme are due to competitive, uncompetitive, noncompetitive, or mixed binding modes.Interestingly, a previous study [38] also reported a non-linear concentration-response relationship of the parent compound CBA at concentrations of 10 and 50 µM on the inhibitory activity of specific cytochrome P450 (CYP450) enzymes: CYP1A2 and CYP2D6.While DA1 and DA2 exhibited a non-linear response in mushroom tyrosinase activity assays, we did not investigate this effect further by broadening the range of concentrations tested in these assays to determine the minimal and maximal effect or saturation of the response, as it was not the primary focus of this study.Moreover, the concentration range could not be expanded to encompass values >50 µM owing to the limited sample availability of the analogs.
Melanocytes constitute an epidermal melanin unit with keratinocytes in a symbiotic relationship in the epidermis [39,40].Hence, we tested the analogs for any cytotoxicity to keratinocytes.We used HaCaT cells, which is an immortalized nontumorigenic cell line, as it demonstrates typical structural development and similar surface markers and functional capabilities to those of primary human keratinocytes [41].Our findings of the opposite effect of THCBA on the stimulation of melanin production in B16F10 cells are reminiscent of the effects of THC formed after the chemical reduction of curcumin.We previously showed that the hydrogenation of curcumin led to the loss of curcumin's anti-melanogenic effect, as THC stimulated melanin production in B16F10 cells and MNT-1 cells [42].In our present study, it was observed that THCBA did not manifest any discernible impact subsequent to the 72 h treatment in the MNT-1 cells.This outcome diverged from the treatment duration employed in our previous study [42], wherein MNT-1 cells were subjected to 5-day treatments with THC.Furthermore, the outcomes obtained from our current study on the mushroom tyrosinase assay reveal that THCBA exhibited inhibitory effects on monophenolase activity while having no impact on the diphenolase activity.These findings closely resemble the observed effects of THC on mushroom tyrosinase activity using both substrates, as documented in our previous study [42].Although BD potently inhibited tyrosinase activity in MNT-1 cells, it failed to elicit any diminution of MNT-1 cells' melanin production after 72 h.Long durations with multiple applications of test compounds may be necessary to observe the effects in the case of BD.Alternatively, BD might act on the subsequent steps of melanogenesis and suppress melanin export.Previous research showed that certain compounds do not impact cellular melanin levels but influence dendrite formation or melanin export [43].A rigorous study to test this hypothesis needs future investigation.It should be noted, however, that KA, which is a known skin-whitener that has been commercialized for use in skin bleaching creams, also failed to elicit any changes in melanin production in MNT-1 cells when tested at a concentration of 703 µM (which is somewhat higher than the 500 µM that was used in our experiments in B16F10 cells as a positive control), although it suppressed tyrosinase activity and also attenuated the gene expression of the melanogenic proteins tyrosinase, TRP-1, and TRP-2 [44].
Our findings show that DA1 potently inhibited tyrosinase activity in B16F10 and MNT-1 cells, where it also consistently diminished the melanin production without any cytotoxicity to both the cells and keratinocytes.According to our results, DA1 exerted a higher cytotoxicity to the B16F10 cells than the MNT-1 cells.As our MTS viability results in MNT-1 cells showed no cytotoxicity of the four analogs across the 10-50 µM concentration range, we also examined higher concentrations (80, 140, and 200 µM) of the four compounds.The results (Figure S2) show that DA1 was the most cytotoxic, followed by BD, DA2, and THCBA.Interestingly, THCBA exerted no loss of cell viability at the maximum concentration of 200 µM.The IC 50 values for cell viability of DA1 were 47.78 ± 2.10 µM and 69.27 ± 6 µM in the B16F10 and MNT-1 cells, respectively.Melanogenesis inhibitors have the potential to be used as adjuvants in immunotherapy, phototherapy, chemotherapy, and/or radiation treatment with the purpose of sensitizing melanoma and, as a result, improving the prognosis of the illness [45][46][47].These show that DA1 might also hold potential for use in anti-melanoma therapeutics, as it potently suppresses tyrosinase activity and melanin, both of which are necessary for candidates that can diminish melanoma cells' melanogenesis to enhance their response to other melanoma treatment modalities.
So far, research on the pharmacokinetics, safety, and metabolic stability has primarily centered on the original compound CBA, with studies conducted in pre-clinical models [48][49][50].Thus, there is a dearth of studies on the stability, lipophilicity, pharmacokinetics, and metabolic stability of the four CBA analogs studied in our current research.Exploring these parameters and conducting in vivo administration studies of these analogs are still in their infancy.Thus, in the absence of information on the metabolism of these analogs, it is difficult to ascertain the plasma concentration of these analogs after oral intake.Nevertheless, the online program SwissADME [51] was used for the evaluation of the physicochemical properties and pharmacokinetics of the four CBA analogs (BD, DA1, DA2, and THCBA).The analysis we conducted focused on the lipophilicity, druglikeness, and pharmacokinetics by the BOILED-Egg model and bioavailability radar [51].The comparative results of the lipophilicity properties are summarized in Table S1.None of the four analogs exerted toxicity since they did not penetrate the blood-brain barrier (BBB), although there was high gastrointestinal absorption (Table S2).Moreover, THCBA showed CYP2D6 inhibition, which contrasted with the other three analogs that did not inhibit the activity of CYP2D6 (Table S2).Conversely, BD, DA1, and DA2 were CYP2C9 inhibitors, while THCBA did not inhibit the activity of CYP2C9 (Table S2).All four analogs followed the Lipinski rule, with no violation that confirmed their drug-likeness property and exhibited a bioavailability score of 0.55 (Table S3).The bioavailability radar plots (Figure S3) and the BOILED-Egg plot (Figure S4) for the four analogs are also provided for further validation of the results.It is important to note that these analogs might be delivered via topical formulations for direct application to the skin to target the suprabasal melanocytes.In addition, future research will need in vivo studies to evaluate the safety and efficacy of the most potent CBA analog when administered topically in a pre-clinical model.This will assist in establishing its anti-melanogenic efficacy and establishing the most effective dosage.

Materials
The four analogs of CBA, namely, BD (97.84% purity), DA1 (97.9% purity), DA2 (97.62% purity), and THCBA (93.05% purity) were supplied by the Sabinsa Corporation (East Windsor, NJ, USA).The synthesis of the three analogs BD, DA1, and DA2 was previously detailed in our earlier reports [17,18].The THCBA analog was produced through the hydrogenation of the parent CBA using a standard procedure that involved a palladium/carbon (Pd/C) catalyst, similar to the standard method used for chemically hydrogenating curcumin to yield tetrahydrocurcumin [19][20][21].The 1 H-NMR and 13 C-NMR spectra information for the DA1 and DA2 analogs is described in our earlier publication [17].The 1 H-NMR and 13 C-NMR spectra of the BD analog are shown in Figure S5 and Figure S6, respectively.The 1 H-NMR spectra with peak resolutions of the THCBA analog are shown in Figure S7 and Figure S8, respectively.In addition, the 13 C-NMR spectra of THCBA are shown in Figure S9.The stock solution of the four CBA analogs was prepared by reconstituting the powders in dimethyl sulfoxide (DMSO) at 50 mM and was stored at −20 • C until use.L-DOPA, α-MSH, L-tyrosine, kojic acid (KA), and mushroom tyrosinase were acquired from Sigma Aldrich (St. Louis, MO, USA).The Bicinchoninic acid (BCA) assay (Pierce BCA kit) and 2,2-Diphenyl-1-picrylhydrazyl (DPPH) were purchased from Thermo Fisher Scientific (Waltham, MA, USA).The MTS cytotoxicity assay was procured from Promega Corporation (Madison, WI, USA).

Monophenolase Activity
To assess the monophenolase activity, various concentrations of CBA analogs (80 µL of each) prepared in 50 mM sodium phosphate (pH 6.8) buffer were dispensed into a 96-well plate, followed by 100 µL of a 2 mM L-TYR solution.The concentration of DMSO within the individual wells did not exceed 0.4%.At this point, a volume of 20 µL of mushroom tyrosinase enzyme at a concentration of 12.5 µg/mL was introduced.The progress of the reaction was assessed by monitoring the absorbance kinetics at a wavelength of 475 nm, with measurements taken every 30 s for a duration of 20 min using a microplate reader.The slopes of the linear range were computed in order to determine the tyrosinase activity of all the groups and expressed as a percentage normalized to the control.

Diphenolase Activity
CBA analogs (80 µL of each concentration) were aliquoted into a 96-well microplate, followed by the addition of 100 µL of 3 mM L-DOPA substrate solution.The reaction was commenced by adding 20 µL of an enzyme solution containing 35 µg/mL of mushroom tyrosinase, and the formation of DOPAchrome was tracked by measuring the rate of absorbance at 475 nm (for 10 min at intervals of 30 s).The tyrosinase activity was reported to be similar to that described above.

DPPH Radical Scavenging Assay
The antioxidant activity of the compounds was assayed based on the widely used DPPH radical scavenging assay that involved a transition from purple to yellow color upon a hydrogen atom transfer (HAT) from the compounds.A volume of 20 µL aliquots of various concentrations of the four CBA analogs or ascorbic acid (AA) included as a positive control was mixed with 180 µL of a methanol solution containing DPPH in a 96-well plate (the final DPPH concentration was 60 µM in all wells).The plate was sealed and incubated at ambient temperature.The absorbance was measured (at a wavelength of 517 nm) after a duration of 30 min.The percentage of DPPH radical scavenged was reported as [(A t − A c )/A c ] × 100, where A c refers to the absorbance value of the control and A t refers to the absorbance value of the sample.

Cell Culture
The B16F10 mouse melanoma cells were procured from the American Type Culture Collection (ATCC) in Manassas, VA, USA.These cells were cultivated using Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (HI-FBS) and 1% antibiotics (penicillin-streptomycin). HaCaT cells were purchased from AddexBio (San Diego, CA, USA) and were grown in DMEM supplemented with 1% antibiotics.MNT-1 human melanoma cells, which were generously provided by Dr. Michael Marks from the University of Pennsylvania, were cultivated utilizing DMEM enriched with 10% AIM-V medium (Gibco, Grand Island, NY, USA), 1% minimum essential medium (MEM), 18% HI-FBS, and 1% antibiotics.All the cells were maintained at 37 • C in a humidified environment in an incubator (95% air-5% CO 2 ).

Cytotoxicity Assay
B16F10 cells (1 × 10 4 cells/well in 0.2 mL complete medium) were seeded in a 96-well plate for 24 h, after which the medium was renewed with a medium containing CBA analogs at various concentrations (diluted such that the final DMSO did not exceed 0.1% in all groups), and the cultures were maintained for 48 h.After 48 h, the culture medium was discarded and replaced with 100 µL of new media containing 20 µL of MTS reagent.The mixture was then incubated for 40 min.Subsequently, 100 µL was aliquoted in a new 96-well plate, and the absorbance was determined at 490 nm using a microplate reader.
To evaluate the cytotoxicity of CBA analogs to keratinocytes, HaCaT cells (2.5 × 10 4 cells/mL) were seeded in a 96-well plate and cultured for 24 h before the medium was replaced with CBA analogs, and the cultures were maintained for another 48 h, while in the case of the MNT-1 cells, a total of 2 × 10 4 MNT-1 cells were plated in a 96-well plate and the medium was replaced with the test compounds after 48 h, followed by 72 h incubation with compounds.The viability was determined by the MTS assay similar to that described above.

Melanogenesis Assay
The assay used to evaluate the melanogenic effects of CBA analogs in B16F10 cells was conducted similarly to the method outlined in our prior study [22].Briefly, the B16F10 cells (1.25 × 10 5 cells/well in 1.5 mL complete medium) were inoculated in 12-well plates and incubated for 24 h.Following this, the culture medium was renewed with a fresh medium that contained various concentrations of CBA analogs in the presence of 150 nM αMSH, and the cultures were incubated for 48 h.To determine the MNT-1 cell's melanin contents, a total of 2.3 × 10 5 MNT-1 cells were plated in a 12-well plate, and the medium was replaced with the test compounds after 48 h, followed by 72 h of incubation with the compounds.Following incubation, the cells were dissociated and subjected to a thorough rinsing process.Subsequently, a solution of sodium hydroxide was added and subjected to a temperature of 70 • C in order to effectively dissolve the melanin pigment.Next, aliquots were transferred into a 96-well plate, and the absorbance values were measured at a wavelength of 475 nm.These values were standardized by dividing them by the total protein content (TPC), resulting in the expression of Abs/µg protein as a percentage of the control.

Intracellular Tyrosinase Activity
The B16F10 cells (4 × 10 4 cells/well in 0.5 mL medium) were seeded onto 24-well plates.After 24 h, the culture medium was substituted with medium that consisted of various doses of CBA analogs with or without 150 nM αMSH and incubated for 48 h.After all the treatments were complete, the cells were detached, washed with PBS, and then lysed in a cell lysis buffer.Lysates (25 µL) were mixed with 75 µL of 3 mM L-DOPA solution (freshly prepared in 50 mM phosphate buffer, pH 6.8) in a 96-well microplate, and the rate of absorbance was measured at 475 nm for 10 min at 30 • C using a microplate reader.To determine the tyrosinase activity, the slope of the linear range of the inhibition velocities was recorded and normalized to TPC and expressed as a percentage of the control group.
MNT-1 cells (2.3 × 10 5 cells/well in a 12-well plate) were grown for 48 h.After 48 h, the cells were subjected to a compound treatment for 72 h.Following the detachment, lysis, and centrifugation of the cells, 50 µL of the resulting lysates were combined with 100 µL of the L-DOPA solution (3 mM).Subsequently, the absorbance was recorded, and the obtained data were processed using a methodology similar to the one previously outlined.

Statistical Analysis
GraphPad Prism software (version 8.0, San Diego, CA, USA) was used in order to first check the normality of the data distribution (using the Shapiro-Wilk test) followed by performing a one-way analysis of variance (ANOVA) with Dunnett's multiple comparison test for all analyses.For the comparison of two groups, Student's t-test was used.When p was less than 0.05, we considered the differences to be statistically significant.Data for the experiments in a cell-free system are given as the mean ± SD of triplicate determinations (n = 3 per group).All the data for cell culture experiments are given as the mean ± SD of at least three independent experiments (n = 3), except the data for melanin content and cellular tyrosinase in MNT-1 cells, which are given as values combined from two independent experiments each in duplicates (n = 4).

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Figure 3 .
Figure 3. (A) Monophenolase activity of tyrosinase enzyme measured with different concentrations of four CBA analogs (BD, DA1, DA2, and THCBA); (B) Diphenolase activity of tyrosinase enzyme measured with different concentrations of CBA analogs; kojic acid (KA) was used at 0.5 mM.

Figure 4 .
Figure 4. Cell viability of (A) B16F10 cells and (B) HaCaT cells treated with CBA analogs at various concentrations in 0.1% DMSO for 48 h; # p < 0.0001 vs. Control.

Figure 4 .
Figure 4. Cell viability of (A) B16F10 cells and (B) HaCaT cells treated with CBA analogs at various concentrations in 0.1% DMSO for 48 h; # p < 0.0001 vs. Control.

Figure 5 .
Figure 5. B16F10 cells after a 48 h treatment with CBA analogs at varying concentrations with or without 150 nM melanocyte stimulating hormone (αMSH); (A) representative photos of cell pellets corresponding to the groups; melanin contents were determined spectrophotometrically for the groups where cells were treated with (B) BD, (C) DA1, (D) DA2, and (E) THCBA; * p < 0.05 vs. Control, ** p < 0.01 vs. Control.

Figure 5 .
Figure 5. B16F10 cells after a 48 h treatment with CBA analogs at varying concentrations with or without 150 nM melanocyte stimulating hormone (αMSH); (A) representative photos of cell pellets corresponding to the groups; melanin contents were determined spectrophotometrically for the groups where cells were treated with (B) BD, (C) DA1, (D) DA2, and (E) THCBA; * p < 0.05 vs. Control, ** p < 0.01 vs. Control.