Multidirectional Activity Of Bakuchiol Against Cellular Mechanisms Of Facial Aging - Experimental Evidence For A Holistic Treatment Approach Part 2

Antioxidative power

Electron spin resonance was applied to assay the AP of test substances, whereby a solution of 1 ppm vitamin C is defined as 1 AU. Retinol had a longer reaction time (2.59min) than bakuchiol (0.99min) or vitamin C (0.24min) indicating a lower reactivity of retinol with free radicals. Further, the wc values showed that bakuchiol (0.028mg) had an increased capacity to react with free radicals compared to retinol (0.151mg). Both these characteristics lead to an AP value of 12 125AU for bakuchiol and 848AU for retinol (Figure 2b).

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Determination of anti-inflammatory effects

For the investigation of the (ii) anti-inflammatory effects of bakuchiol and retinol, we determined the level of the proinflammatory cytokines PGE2 and MIF.

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PGE2 levels

Prostaglandin E2 levels of LPS-treated HDFs were significantly elevated relative to the untreated control (p=0.0005) indicating successful stress induction (Figure 2c). As expected, the pharmacologically well-known high-standard diclofenac induced significantly decreased PGE2 levels in LPS-treated HDFs compared to the stressed control (p=0.0005). In LPS- and bakuchiol-treated cells, PGE2 levels were significantly reduced relative to HDFs only treated with LPS (p=0.0005 for all indicated concentrations). Application of retinol at concentrations equal to or higher than 2.5 μM also significantly diminished PGE2 levels about the stressed control (2.5 μM: p=0.0024; 5 and 10 μM: p=0.0005).

MIF protein levels

As depicted in Figure 2d, stressed control HDFs displayed a significant increase in MIF protein levels relative to the unstressed control (p=0.0020) demonstrating efficient stress induction. Treatment of stressed HDFs with bakuchiol resulted in significantly decreased MIF protein levels (1 μM: p=0.0020; 10 μM: p=0.0039) compared to the stressed control. The application of retinol also significantly lowered MIF protein levels (1 and 10 μM: p=0.0020).

Analysis of cell activity

To examine the impact of bakuchiol and retinol on (iii) cell activity, FGF7 protein levels and WST-1 metabolization were measured.

Determination of FGF7 protein levels

Treatment of HDFs with 10 μM bakuchiol significantly increased FGF7 protein levels relative to control cells (p = 0.0396), while 10 μM retinol displayed no significant effect (Figure 3a).

Determination of WST-1 metabolization

As illustrated in Figure 3b, triton-X significantly lowered WST-1 metabolization levels in HDFs relative to control cells (p = 0.0000) indicating proper assay implementation. HDFs treated with 1 and 10 μM bakuchiol displayed significantly increased WST-1 metabolization levels compared to the control (p = 0.0000 for both concentrations). Treatment with 1 μM retinol also caused a significant augmentation of WST-1 metabolization levels (p = 0.0066) relative to control cells, while 10 μM retinol had no significant effect.

Expression of ECM components

To assess bakuchiol- and retinol-mediated effects on the expression of (iv) ECM components, we determined COL7A1, COL1A1, and FN protein expression.

Determination of COL7A1 and COL1A1 protein levels

COL7A1 protein levels in cells treated with the high standard TGF-β and sodium ascorbate were significantly higher relative to control cells (4 h: p=0.0020, 72 or 96h: p=0.0010) as displayed in Figure 3c. Treatment of cells with bakuchiol or retinol in a concentration of 1 μM (p=0.0020 for both test substances) and 10 μM (bakuchiol: p=0.0195, retinol: p=0.0059) significantly augmented COL7A1 protein levels already after 4 h compared to the control. After an extended incubation time, HDFs stimulated with 10 μM bakuchiol or retinol also displayed a significant increase in COL7A1 protein levels(bakuchiol: p=0.0029, retinol: p = 0.0420) relative to control cells.

HDFs treated with the high standard TGF-β and sodium ascorbate additionally showed a significant increase in COL1A1 protein levels (p = 0.0010) as shown in Figure 3d. Similarly, COL1A1 protein levels were significantly increased after 4 h of stimulation with bakuchiol (1 μM: p = 0.0020, 10 μM: p = 0.0322) or retinol (1 μM: p = 0.0244, 10 μM: p = 0.0098) relative to control cells.

Determination of FN protein levels

Figure 3e illustrates that HDFs treated with 10 μM bakuchiol or retinol demonstrated a significant increase in FN protein levels (bakuchiol: p = 0.0090, retinol: p = 0.0302) relative to control cells.

Study I: Ex vivo determination of FN protein levels

An ex vivo study was carried out to investigate whether the previous data translate into ex vivo results. As depicted in Figure 4a, bakuchiol-treated sites showed a statistically significant increase in FN protein levels in untreated control areas (p = 0.0340) and areas treated with vehicle (p = 0.0088). Retinol-treated sites displayed no significant alteration of FN protein levels in untreated or vehicle-treated areas. However, incompatibility reactions caused a lower number of subjects tested for retinol treatment (untreated: n = 26, vehicle: n = 29, bakuchiol: n = 30, retinol: n = 19). Additional minor deviations in the number of subjects tested were caused by sampling issues.

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Improvement of the epidermal regeneration and re-epithelization

To study the effects of bakuchiol and retinol on (v) epidermal regeneration and re-epithelization, an in vitro wound healing model was applied. Figure 4b illustrates that bakuchiol-treated wounds showed a significant increase in the length of the regenerated epidermis about untreated (p=0.0251) and control wounds (p=0.0102). In contrast, wounds supplemented with retinol displayed no significant change in the length of the regenerated epidermis compared to both controls. Figure 4c exemplifies the progress of wound healing 43h after bakuchiol or retinol treatment as well as in control and untreated wounds.

Study II: In vivo determination of skin condition improvement

After 12 weeks of treatment with the bakuchiol-containing formulation (t1), subjects(n = 34)rated the difference in the youthful appearance of their skin to the baseline determination (t0) with a mean t1-t0 value of 2.57±2.14. Compared to the t1-t0 value of the vehicle-treated site (2.06±1.89), the youthfulness of the bakuchiol-treated area was rated as significantly improved (p = 0.0275). Both treatments were rated as significantly better than the baseline (p = 0.0000).

In vivo studies: Tolerability

Results showed that the bakuchiol-containing formulation in both in vivo studies was well tolerated. Over the entire duration of usage, one adverse skin reaction was observed, which was documented for both the bakuchiol-containing formulation and the vehicle. After treatment with retinol-containing formulations in Study I, 23% of the entire panel of 52 subjects reported incompatibility reactions such as erythema, desquamation, dryness, and itching, which led to the dropout of five subjects.

DISCUSSION

Previous studies have implied that bakuchiol acts as a functional analog of retinol [19–21]. Bakuchiol, thus, appears to be a promising alternative to retinol for facial anti-aging treatments. As cellular aging is multifactorial, we investigated the effects of bakuchiol in comparison to retinol on different key processes to analyze its potential for a holistic treatment approach.

As depicted in (Table S1), we determined the (i) antioxidative and (ii) anti-inflammatory capacities of bakuchiol and retinol. We further analyzed how they influence (iii) cell activation, impact the formation of (iv) ECM components, and (v) skin regeneration. In our investigation, we determined that bakuchiol shares functional similarities with retinol and at the same time exhibits unique, beneficial characteristics (Figure 5).

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Our data demonstrated that bakuchiol but not retinol showed a high (i) antioxidative capacity and power. These data are in line with previous studies illustrating that bakuchiol decreases oxidative stress, prevents mitochondrial lipid peroxidation, and protects mitochondrial function [22, 24, 38]. Retinol, however, has not been reported to exert antioxidative actions.

As the induction of ROS leads to inflammatory stress, we investigated the effects of bakuchiol and retinol on the expression of the two (ii) proinflammatory cytokines PGE2 and MIF.

We first analyzed PGE2, which is a major prostaglandin generated in the human skin. PGE2 reduces collagen production and induces matrix metalloproteinase 1 (MMP-1) expression in fibroblasts in vitro [39]. These PGE2-mediated processes are cutaneous aging mechanisms [39]. Thus, targeting PGE2 might be a promising strategy to oppose age-associated collagen depletion [40]. Normally, low amounts of PGE2 are synthesized. However, in skin aging, fibroblasts show elevated PGE2 levels [40, 41]. Herein, we show for the first time that bakuchiol and retinol significantly reduce PGE2 levels in HDFs in a dose-dependent fashion. However, the effect induced by retinol was less pronounced than by bakuchiol. Our results are supported by a previous study using an in vivo inflammation model in which topically applied bakuchiol significantly reduced the PGE2 content in the arachidonic acid-induced response [42]. Similarly, retinoids were shown to suppress PGE2 expression in human oral epithelial cells [43] and in human oral squamous carcinoma cells [44].

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MIF is another proinflammatory cytokine that is ubiquitously expressed in various organs including the skin [45]. It is crucial for cell proliferation, angiogenesis, and differentiation [46]. In the context of photoaging, both UVA and UVB irradiation increases MIF secretion by keratinocytes and dermal fibroblasts[46, 47]. Urschitz et al. reported a 4-fold upregulation of MIF mRNA in photoaged preauricular skin [48]. Our results showed a significant, similar reduction of MIF protein levels in HDFs induced by bakuchiol and retinol indicating anti-inflammatory properties. Indeed, it has been evidenced by earlier studies that bakuchiol exerts anti-inflammatory actions [19, 25– 27, 38]. However, the regulation of MIF protein levels by bakuchiol or retinol has not yet been documented.

Although PGE2 and MIF are both increased in cutaneous aging [46, 49], their regulation occurs via two different signaling pathways. Therefore, the bakuchiol- and retinol-induced decreases of both factors represent a broad anti-inflammatory approach in antiageing treatment.

Oxidative and inflammatory stresses put the regenerative capacity of the skin at serious risk. Further, cutaneous regeneration diminishes with age. We, therefore, investigated the effect of bakuchiol and retinol on the cutaneous regenerative capacity by analyzing (iii) cell activity.

Keratinocytes are proposed to stimulate fibroblasts to synthesize growth factors, which, in turn, stimulate keratinocyte proliferation in a double paracrine manner [50]. The growth factor FGF7 is an example of such a mitogen [51]. It is also referred to as keratinocyte growth factor-1 [51] and enhances the proliferation of keratinocytes [52] as well as their interaction with ECM components [53]. Our study demonstrates that bakuchiol-treated HDFs showed significantly increased FGF7 protein levels. In contrast, FGF7 protein levels were slightly reduced by retinol treatment. This novel finding indicates that bakuchiol might support skin regeneration and repair processes by directly upregulating keratinocytes and indirectly increasing fibroblast proliferation. Bakuchiol thereby acts against the decline of growth factor levels that occurs during aging [54].

Another factor that impacts the regenerative potential of the skin is the age-associated reduction in the number [55] and growth rate [56] of dermal fibroblasts. Since an increase in WST-1 metabolism indicates improved cell viability [57], proliferation [58], and metabolic activity [59], we analyzed WST-1 metabolization after the application of bakuchiol or retinol. Our results suggest that bakuchiol and to a certain extent also retinol can stimulate these cell activity-related characteristics in HDFs.

In line with the reduction of cell activity, aging skin is characterized by diminished production of collagen and other ECM components as well as an augmented MMP expression [60–65]. These alterations result in ECM damage, disturbed skin functions, and subsequently the formation of wrinkles. We hypothesized that the increased fibroblast activity and decreased PGE2 and MIF levels mediated by bakuchiol could promote ECM components. Indeed, Chaudhuri and co-workers showed that bakuchiol upregulates COL1A1 on gene and protein levels [19]. To investigate the effects of bakuchiol and retinol on the ECM of HDFs, we analyzed protein expression of the (iv) structural ECM factors COL1A1 and COL7A1 and the ECM adhesion factor FN.

COL1A1 is the most abundant structural protein in the skin [66]. However, aged fibroblasts display a reduced capacity for collagen synthesis [67]. COL7A1 forms anchoring fibrils in dermo-epidermal junctions and enhance mechanical skin stability [68]. During photo-aging, COL7A1 levels decrease causing a weakened bond between the dermis and epidermis [69–71].

Our data demonstrate that bakuchiol and retinol increase COL1A1 levels confirming earlier observations. A previous study found that bakuchiol significantly enhances expression levels of COL1 mRNA and significantly reduces MMP-1 mRNA levels [72]. COL1A1 gene expression was shown to be augmented in vivo after 4 weeks of 0.1% retinol treatment [73]. Topical application of 0.4% retinol also significantly increased COL1A1 protein expression in the ECM in aged human skin in vivo [74]. However, our data clarify that in HDFs, COL1A1, and COL7A1 protein expression are increased already 4 h after stimulation with bakuchiol and retinol. We further show that COL7A1 protein expression persists at least for 72h.

Another factor we investigated was the ubiquitous ECM adhesion protein FN found in two isoforms, namely plasma and cellular FN. It plays a crucial role in developmental processes, cell adhesion, migration, and differentiation [75, 76]. Cellular FN is generated and assembled into fibril networks, impacting ECM homeostasis and ECM-cell interactions [77]. Chronic UV exposure leads to a down-regulation of FN gene expression in human skin biopsies [78]. Our data revealed a significant upregulation of cellular FN protein expression in HDFs after stimulation with bakuchiol and retinol. A previous in vivo study shows that topical treatment with 0.4% retinol leads to significantly increased FN protein levels in the ECM of aged human skin [74]. It has not yet been reported, though, that application of bakuchiol can induce enhanced FN protein expression in HDFs. To analyze whether these in vitro data translate into in vivo results, we determined the effect of bakuchiol and retinol on FN protein levels in an ex vivo study. After a 4-week application, bakuchiol-treated areas showed a significant increase in FN protein values compared to the vehicle. Retinol application also resulted in augmented FN protein levels; however, this effect was not significant. This might be caused by retinol-mediated incompatibility reactions that reduced the number of subjects tested.

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As a major component of the ECM, FN plays a crucial role in wound healing, being essential for tissue formation and connective tissue repair. FN functions in all phases of wound healing and thereby interacts with different cell types to build the ECM [79]. FGF7 is another important factor for wound healing. In acute human wounds, FGF7 gene expression is rapidly up-regulated. FGF7 mostly locates in dermal fibroblasts adjacent to the wound and in fibroblasts of the granulation tissue [52]. The wound healing process is delayed with aging [80]. This is due to impaired cell proliferation and migration of fibroblasts and keratinocytes, a diminished reaction to growth factors, and a decreased synthesis of ECM components [80]. These observations correlate with the general changes occurring during skin aging [81]. Following aesthetic procedures such as Fraxel laser treatment the generation of micro-wounds initiates microscopic wound healing processes leading to improved skin structure and rejuvenation [82]. Therefore, the ability of antiageing compounds to stimulate regenerative processes can indicate their skin rejuvenating potential. Considering the involvement of FN and FGF7 in wound healing and the bakuchiol-induced upregulation of these factors in vitro, we next determined the effects of bakuchiol and retinol on (v) epithelial regeneration. Therefore, an in vitro wound healing model was applied [34]. The length of the regenerated epidermis of bakuchiol-treated wounds was significantly increased, while retinol had no effect. These data reflect the more pronounced in vitro effect of bakuchiol on the wound healing-associated parameters FGF7, FN, and cellular metabolic activity when compared to retinol.

To determine whether bakuchiol, besides its positive activities, also improves the perceived skin appearance, a second self-grading based in vivo study was performed. Study participants graded the youthfulness of their facial skin. When compared to baseline self-grading at t0, treatment with both the vehicle and the bakuchiol-containing formulation for 12 weeks significantly enhanced the perceived skin appearance. The vehicle was selected to be as little nourishing as possible. However, a certain improvement in self-grading, especially regarding measurement at t0 after 3days of not using any skin care products, cannot be excluded. Nonetheless, after the application of the bakuchiol-containing formulation, subjective grading of the youthful skin appearance was significantly increased compared to the corresponding vehicle about t1-t0 values.

In our in vivo studies, bakuchiol had good skin compatibility. This is in line with a previous study showing that a bakuchiol-containing moisturizer was well tolerated in subjects with sensitive skin [18]. In contrast, the retinol application performed in the study caused skin irritations in several volunteers. It is well-documented that retinol can induce various skin issues including erythema, itching, desquamation, or papules [6, 14]. Further, retinoids are associated with photosensitization and are degraded by exposure to air or light to biologically inactive substances [11]. Hence, the efficacy of retinol in an antiageing treatment strongly depends on its delivery mode. Bakuchiol, on the other hand, is photostable and can be applied diurnally. The photostabilizing effect of bakuchiol on retinol, as demonstrated by Chaudhuri et al. [83], provides a promising rationale for the combination of both compounds.

Our results expand the scientific knowledge about bakuchiol and advance our understanding of cutaneous effects exerted by retinol. Figure 5 summarizes the proposed actions of Bakuchiol. Moreover, our data provide evidence for the multidirectional efficacy of bakuchiol against several cellular hallmarks of skin aging, exceeding the effects of plant-derived functional retinoid analogs.

CONCLUSION

Treatment with bakuchiol provides an advanced, holistic, and multidirectional treatment approach for skin aging as it acts (i) antioxidative, (ii) anti-inflammatory, impacts (iii) cell activity, increases the expression of critical (iv) ECM components and improves (v) epidermal regeneration and re-epithelization.

ACKNOWLEDGEMENTS

The authors would like to thank Dr. Silke Gallinat for her support in preparing the manuscript.

CONFLICT OF INTEREST

Anika Bluemke, Annika P. Ring, Jeannine Immeyer, Anke Hoff, Tanya Eisenberg, Wolfram Gerwat, Franziska Meyer, SabrinaBreitkreutz,LinaM. Klinger, FrankRippke, and Dorothea Schweiger are employees of Beiersdorf AG. Grit Sandig and Marietta Seifert are employees of the Gematria Test Lab GmbH. Doerte Segger is an employee of the SGS Institute Fresenius GmbH. None of the authors state a conflict of interest.

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