Multidirectional Activity Of Bakuchiol Against Cellular Mechanisms Of Facial Aging - Experimental Evidence For A Holistic Treatment Approach Part 1

Abstract

Objective: Skin aging is a multifactorial process involving the formation of reactive oxygen species, consecutive inflammation with reduced epidermal and dermal cell viability, and resulting damage to the extracellular matrix. Effective dermo-cosmetic treatment modalities should ideally address these hallmarks in a holistic approach. Here, we determined the corresponding activity profile of bakuchiol, a plant-derived meroterpene, in an array of in vitro, ex vivo, and in vivo studies and compared it to retinol, currently considered the gold standard in topical anti-aging cosmetics. 

Glycoside of cistanche can also increase the activity of SOD in heart and liver tissues, and significantly reduce the content of lipofuscin and MDA in each tissue, effectively scavenging various reactive oxygen radicals (OH-, H₂O₂, etc.) and protecting against DNA damage caused by OH-radicals. Cistanche phenylethanoid glycosides have a strong scavenging ability of free radicals, a higher reducing ability than vitamin C, improve the activity of SOD in sperm suspension, reduce the content of MDA, and have a certain protective effect on sperm membrane function. Cistanche polysaccharides can enhance the activity of SOD and GSH-Px in erythrocytes and lung tissues of experimentally senescent mice caused by D-galactose, as well as reduce the content of MDA and collagen in lung and plasma, and increase the content of elastin, have a good scavenging effect on DPPH, prolong the time of hypoxia in senescent mice, improve the activity of SOD in serum, and delay the physiological degeneration of lung in experimentally senescent mice With cellular morphological degeneration, experiments have shown that Cistanche has the good antioxidant ability and has the potential to be a drug to prevent and treat skin aging diseases. At the same time, echinacoside in Cistanche has a significant ability to scavenge DPPH free radicals and can scavenge reactive oxygen species, prevent free radical-induced collagen degradation, and also has a good repair effect on thymine free radical anion damage.

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Methods: The antioxidative capacity and power of bakuchiol and retinol were analyzed by measuring 2,2′-diphenyl-1-picrylhydrazyl (DPPH) reduction via its absorption decay and electron spin resonance spectroscopy, respectively. Effects on prostaglandin E2 (PGE2), macrophage migration inhibitory factor(MIF), fibroblast growth factor 7 (FGF7), collagen type I and VII (COL1A1, COL7A1), fibronectin (FN) levels as well as the metabolization of water-soluble tetrazolium 1 (WST-1)  were determined in human dermal fibroblasts. Epidermal regeneration was assessed utilizing an in vitro wound healing model. FN protein levels were analyzed ex vivo after treatment with a formulation containing bakuchiol, retinol, or vehicle using a suction blister fluid. Skin condition improvement was determined in vivo in a split-face comparison study after the application of bakuchiol or vehicle. 

Results: In contrast to retinol, bakuchiol demonstrated high antioxidative efficacy. Levels of PGE2 and MIF were significantly decreased by both bakuchiol and retinol. Bakuchiol but not retinol significantly increased FGF7 protein levels. WST-1 metabolization levels were significantly augmented by bakuchiol and retinol. Bakuchiol and retinol application led to a significant augmentation of COL1A1, COL7A1, and FN protein levels. Wounds supplemented with bakuchiol but not retinol displayed a significant increase in epidermis regeneration. Clinically, areas treated with a bakuchiol-containing formulation showed a statistically significant increase in FN protein values after a 4-week application compared to untreated areas and areas treated with vehicle.

Conclusion: These data provide evidence for the multidirectional efficacy of bakuchiol against cellular hallmarks of skin aging. Its activity profile shares some common features with retinol but demonstrates several hitherto unknown positive effects in our studies, namely stimulation of the critical extracellular matrix component FN, and accelerated epidermal regeneration and wound healing.

KEYWORDS

anti-aging, bakuchiol, claim substantiation in vivo/ex vivo/in vitro, retinol, skin physiology/  structures

INTRODUCTION

Aged skin is characterized by wrinkles, uneven pigmentation, skin roughness, and laxity. These clinical signs are the result of structural and metabolic alterations caused by processes of intrinsic and extrinsic aging. Intrinsic aging has been ascribed to factors including telomere shortening, chronic inflammation, mitochondrial DNA single mutations, and free radicals [1]. Aged skin further comprises a reduction of its antioxidative systems[2]. Also, the rate of cell proliferation declines due to the biological aging process leading to a loss of skin structure and function. Extrinsic aging is primarily triggered by UV irradiation and environmental influences. These insults result in skin damage that reinforces the chronological decline and accelerates cutaneous aging. Human skin further loses the ability to cope with inflammatory conditions throughout aging, resulting in a chronic proinflammatory state.

The topical application of retinoids such as retinoic acid, retinal, or retinol is regarded as the clinical gold standard for effective anti-aging treatment [3, 4]. The molecular mechanisms of retinoids have been extensively described [5–10]. Topical retinoids effectively reduce visible signs of aging like wrinkles, laxity, or roughness [4, 11] and decrease depigmentation of photodamaged skin including livedo reticularis and actinic lentigines [12]. However, topical treatment with retinoids can lead to concentration-dependent skin dryness and irritation [13]. As the application of retinol causes minor adverse reactions compared to other retinoids such as retinoic acid [6, 14], it is a widely used active in the cosmetic treatment of facial aging.

In contrast to retinol which has been applied in skin care products since 1984 [15], bakuchiol has only recently gained attention as a topical anti-aging compound. Bakuchiol is a meroterpene (Figure 1) that is derived from Psoralea corylifolia seeds. It has been used in traditional Indian and Chinese medicine for centuries [16, 17] and is well tolerated [18]. Bakuchiol was suggested to exhibit retinol-like functions, as in a skin substitute model, both substances show similar gene expression patterns in vitro [19] and an improvement of cutaneous photodamage in vivo [20]. Hence, it has also been referred to as a plant-derived functional retinoid analog [21]. Further studies demonstrated antioxidant [19, 22–24], anti-inflammatory [19, 25–27], antibacterial [28] as well as antiproliferative and antitumor effects [29, 30] of bakuchiol.

To effectively ameliorate and delay the multifactorial skin aging processes, different cellular mechanisms need to be addressed in an integrated approach. Bakuchiol simultaneously modulates various targets rendering it a  promising compound in this regard. As oxidative and inflammatory stresses are closely related to cutaneous aging,  their prevention by bakuchiol might promote the overall skin condition. However, the quality of the current scientific evidence has recently been critically appraised [31]. In this context, we determined the (i) antioxidative and (ii)  anti-inflammatory capacities of bakuchiol and retinol and examined their ability to improve the cellular metabolism and synthesis of growth factor 7 summarized as(iii) cell activity. We further analyzed whether bakuchiol and retinol impact the expression of certain (iv) ECM components and improve (v) epidermal regeneration and re-epithelization. Finally, an in vivo study was conducted to verify the clinical anti-aging capacity of bakuchiol in human skin.

MATERIALS AND METHODS 

Test materials

Bakuchiol was obtained from Sytheon Ltd (Boonton, New Jersey, United States). Retinol was purchased from Sigma-Aldrich (St. Louis, Missouri, United States). For cell culture experiments, both test substances were freshly diluted in DMSO (Merck, Darmstadt, Germany). For the DMSO stock solutions, the applied concentration of test substances was 1000-fold above the highest concentration applied in cell culture yielding 0.1% DMSO in media or buffer. Further dilutions were performed using media or buffer with 0.1% DMSO. Hence, all cell culture experiments were carried out in the presence of 0.1% DMSO. For the in vivo studies, topical retinol was formulated in the same vehicle as bakuchiol.

In vitro studies

Determination of the antioxidative capacity

Bakuchiol, retinol (both applied at a final concentration of 100μM), or the high standard trolox (Merck, final concentration of 25μM) were diluted in DMSO. Briefly, 30μL  of these compounds (10-fold higher concentration than the final assay concentration) were added to a 96-well flat bottom plate. Subsequently, 270μL of 39μg/mL DPPH (Merck,  diluted in a 1:1 water/ethanol mixture) were quickly added to each well yielding a final concentration of 10% DMSO. As a control, the DPPH solution was incubated with DMSO  lacking test substances. Further, blank controls were performed by incubating bakuchiol or retinol with water/  ethanol (1:1) or only the water/ethanol (1:1) mixture containing 10% DMSO in the absence of DPPH. After 10, 30, and 60min, the absorbance at 524nm was measured using a Spark multimode microplate reader (Tecan, Männedorf, Switzerland). Signals of blank controls were subtracted.

Determination of the antioxidative power

HDFs were seeded in 6-well plates with 150 000 cells/well in a 2 mL medium containing 10% calf serum and incubated for 24h. Subsequently, the old medium was discarded, and cells were treated with a medium containing 2% calf serum and bakuchiol or retinol in a final concentration of 10 μM. Control HDFs were supplemented with 0.1% DMSO lacking bakuchiol or retinol. Blank controls were performed by incubating a medium without cells. After 24h, the conditioned medium was transferred to Vivaspin protein concentrator spin columns (5000 MWCO; Sartorius, Göttingen, Germany) and concentrated approximately 10-fold. Volumes of all medium supernatants were equalized by re-addition of the flow through. FGF7 protein levels of the concentrated conditioned medium were analyzed using a commercially available ELISA kit following the manufacturer's instructions (Bio-Techne GmbH). Measurements were performed using a Spark multimode microplate reader (Tecan). Signals obtained from blank controls were subtracted and FGF7 protein levels were normalized to the total cell number that was determined utilizing a cell counter (Scepter, Merck).

Determination of WST-1 metabolization

HDFs were seeded in 96-well plates with 3000 cells/well in 100μL medium containing 10% calf serum and incubated for 24h. The depleted medium was discarded, and cells were treated with 100μL medium containing retinol or bakuchiol in a final concentration of 1 μM or 10 μM, respectively. Control HDFs were supplemented with 0.1% DMSO lacking bakuchiol or retinol. As a further control, cells were treated with 10% triton-X (Merck). Blank controls were performed by incubating a medium without cells. After 72h, cells were stained using the commercially available cell proliferation reagent WST-1 according to the manufacturer's instructions (Merck). Absorbance was measured at 450 and 620nm using a Tecan infinity M200  microplate reader (Tecan). The difference in these measurements was used for analysis. Signals of blank controls were subtracted.

Determination of COL1A1 and COL7A1  protein levels

HDFs were seeded in 96-well plates with 10  000 cells/  well in 100μL medium containing 10% calf serum and incubated for 24h. Bakuchiol or retinol was diluted in 100μL medium without calf serum, and supplemented to the present medium in a final concentration of 1 μM or 10 μM, respectively. The control was supplemented with 100μL serum-free medium yielding a concentration of 0.1% DMSO corresponding to the retinol and bakuchiol treatment. As a high standard, 10 ng/mL transforming growth factor-β (TGF-β) and 11μg/mL sodium ascorbate (both Merck) were applied. Blank controls were performed by incubating a medium without cells. After 4  h, COL1A1 and COL7A1 protein levels of the conditioned medium were analyzed using commercially available ELISA kits following the manufacturer's instructions (Novus Biologicals, Littleton, Colorado, United States). Measurements were performed using a Spark multimode microplate reader (Tecan). Signals of blank controls were subtracted. COL1A1 and COL7A1 protein levels were normalized to the total cell lysate protein amount that was determined using a commercially available bicinchoninic acid assay kit (Thermo Fisher Scientific) according to the manufacturer's instructions. A prerequisite for the use of the results was the positive reaction of cells to the high standard TGF-β and sodium ascorbate.

For analysis of COL7A1 protein levels after an extended incubation time, HDFs were seeded in 96-well plates with 3000 cells/well in 100μL medium containing 10% calf serum. All treatments, controls, and the analysis were performed as described above with the exception that bakuchiol and retinol were only used in a final concentration of 10 μM. Cells were harvested when subconfluence was reached, in particular, after 72 or 96h.

Determination of FN protein levels

HDFs were seeded in 96-well plates with 10 000 cells/well in 100μL medium containing 10% calf serum and incubated for 24h. The old medium was replaced by a medium containing 2% calf serum and bakuchiol or retinol in a final concentration of 10  μM. The control was supplemented with 0.1% DMSO lacking bakuchiol or retinol. Blank controls were performed by incubating a medium without cells. After 24h, FN protein levels of the conditioned medium were analyzed using a commercially available ELISA kit following the manufacturer's instructions (R&D Systems, Minneapolis, Minnesota, United States). Measurements were performed using a Spark multimode microplate reader (Tecan). Signals of blank controls were subtracted. FN protein levels were normalized to the total cell lysate protein amount that was determined as aforementioned.

Determination of epidermal regeneration in an  in vitro wound healing model

The experiments were conducted as described previously [34]. For the treatment of wound healing models, bakuchiol or retinol was diluted in DPBS and added in a final concentration of 100μM to the wound region (5  μL per wound). The reference wounds were supplemented with the same amount of DPBS containing 0.1% DMSO without bakuchiol or retinol. Additional wounds were left untreated as a further control. Wound healing models were incubated for 43h at 95% humidity, 5% CO2, and 37°C. Subsequently, the samples were snap-frozen in isopentane pre-cooled with liquid nitrogen, and stored at −80°C. Re-epithelialization was evaluated in hematoxylin and eosin-stained cryostat sections by measuring the length of the regenerated epidermis using a Leica DMLS microscope (10×), a Leica MC 170 HD CCD camera and the Leica LAS V4.9 software (Leica Microsystems, Wetzlar, Germany). Quantification was performed in a blinded fashion.

In vivo studies I and II

For both in vivo studies, the recommendations of the current version of the Declaration of Helsinki and the guideline of the International Conference on Harmonization of Good Clinical Practice were observed as applicable to a  cosmetic study. The protocol of study I was approved by the Independent Ethics Committee Freiburg (feki code 08/2610). In both studies, all volunteers provided written,  informed consent. Subjects had healthy skin, belonged to Fitzpatrick skin type I to III and the start, or change of hormonal medication was an exclusion criterion.

During a 10-day preconditioning period and throughout the entire study period, subjects were required to refrain from UV exposure in the test areas. Sweat-promoting activities were prohibited 24h before scheduled assessments.

An investigator demonstrated the correct application of formulations using an amount, which corresponded to the usual skin care regimen of subjects.

Study I: Ex vivo determination of FN protein levels

Out of 52 female subjects, who were enrolled in this vehicle-controlled study, 33 subjects completed the study, and data of 31 subjects (30–64 years, mean age: 50.9 years)  were included in data analysis. Dropouts occurred due to the SARS-CoV-2 pandemic and personal reasons (13  subjects) as well as due to incompatibility reactions (five subjects, caused by retinol treatment). Retinol-mediated incompatibility reactions as well as sampling issues caused different numbers of subjects tested for each condition (see results for details).

Seven days before scheduled assessments, the use of skincare products, cleansers, and soaps on the forearms was prohibited. On the first study day, four test areas were established on the inner forearms. On two test areas, two verum formulations were applied to contain 0.5% bakuchiol or 0.15% retinol, respectively. In an opinion published by the Scientific Committee on Consumer Safety in 2016, the applied retinol concentration was considered a cosmetic treatment [35]. In the other two test areas, the corresponding vehicle was used, or the area was left untreated. The positioning of treatment locations was permuted. After 4 weeks of twice-daily application of test formulations, volunteers returned to the test institute. In each test area, three suction blisters (7 mm in diameter) were generated as previously described [36, 37]. Briefly, custom-made suction blister cups were put onto the test areas and a  vacuum of 550–850 mbar was applied. After approximately 90–150 min, when suction blisters had formed,  the vacuum was released, and the fluid was aspirated from the blister using a 24-gauge needle. Fluids were frozen immediately at −80°C on dry ice until analysis. FN protein levels were quantified in suction blister fluid samples using a commercially available ELISA kit (R&D Systems). Measurements were performed using a Spark multimode microplate reader (Tecan). FN levels were normalized to total protein levels of suction blister fluid samples that were determined as aforementioned.

Study II: In vivo determination of skin   condition improvement

In total, 43 female volunteers were enrolled in this vehicle-controlled split-face comparison study. According to medical assessments, subjects showed mixed skin types (dry, normal, oily, and combination skin). 34 subjects (39–66 years, mean age: 56.2 years) completed the study and were included in the analysis. Dropouts were caused by technical problems and non-compliance (eight subjects),  as well as incompatibility reactions (one subject, caused by vehicle and bakuchiol treatment).

Two weeks before the beginning of the study and during the entire study period, volunteers were required to refrain from using self-tanning products or intensive facial cosmetic treatments (e.g. removal of superficial skin layers). During the 10-day preconditioning period and throughout the study, subjects were asked to abstain from performing permanent makeup, eyelash and eyebrow treatments, eye masks, and patches. Three days before the first assessment, subjects were asked to refrain from using face care products. On the evening before the scheduled grading, subjects were required to stop the application of decorative cosmetics.

In the first 7 days of the 10-day preconditioning phase,  subjects received a cream jar containing the study vehicle cream (lacking any information on the content) and applied it twice daily to their entire face. At baseline,  volunteers performed a self-grading. In particular, they visually assessed the overall appearance of their facial skin by observing its freshness and radiance as well as any signs of skin aging. Thereby a visual analog scale ranging from 1 (very fatigued, aged) to 10 (very fresh, no signs of aged skin) was applied. Then, subjects received two blinded cream jars containing the verum (vehicle containing 0.5% bakuchiol) or the vehicle, respectively,  without any specification on the content. During the study period of 12 weeks, one facial side was treated twice daily with verum while the other facial side was treated twice daily with the vehicle. The allocation of treatments to the test sites was permuted. After 12 weeks of regular use, volunteers again performed the self-grading as aforementioned.

Statistical analysis

Statistical analyses were performed using Microsoft Excel for Office 365 (Microsoft Corporation, Redmond, Washington, United States), SAS Software Package for Windows V9.4 (SAS Institute GmbH, Heidelberg, Germany), and GraphPad Prism V8 (GraphPad Software, San Diego, California, United States).

The normal distribution of data was assessed using a Shapiro–Wilk's test. If the normal distribution was confirmed,  a repeated measure analysis of variance (RM-ANOVA) with post hoc pairwise comparison was performed. If the normality hypothesis was rejected, Blom-transformed ranks of the original data were assessed using an RM-ANOVA with post-hoc pairwise comparison, or original data were assessed by using a Wilcoxon sign rank test. All statistical tests were two-sided at a significance level of alpha = 0.05.

RESULTS

In vitro studies

Determination of antioxidative effects

To analyze the (i) antioxidative effects of bakuchiol and retinol, we performed two different assays using DPPH as a detector molecule.

Antioxidative capacity

The antioxidative capacity was determined by measuring the reduction of DPPH via its absorption decay. The high-standard Trolox showed a significantly elevated antioxidative capacity relative to the control (p=0.0000 for all indicated time points) verifying proper measurement (Figure 2a). About the control, the absorbance in bakuchiol-treated samples was also significantly reduced at all time points investigated (10 min: p = 0.0003, 30 and 60 min: p = 0.0000) demonstrating an increased antioxidative capacity. In contrast, Retinol showed no significant antioxidative capacity compared to the control.

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