Synthesis and new skin-relevant properties of the salicylic acid ester of bakuchiol

Abstract Bakusylan (bakuchiol salicylate) is a bipartite compound obtained by merging two skin-active entities with complementary bioactivities—bakuchiol and salicylic acid—for the purpose of generating a new class of functional retinoids with enhanced skin benefits. Here, we describe its preparation process and report that pure bakusylan exhibits potential for an improved permeation through the stratum corneum, enhances type IV collagen gene expression in organotypic skin substitutes containing both epidermal and dermal layers, and upregulates this protein in adult human dermal fibroblast cultures. The mechanism of action underlying these effects appears to involve the components of the IP3K/Akt signaling pathway selectively implicated in the maintenance of skin integrity, further underlying the suitability of this ester for skin care applications requiring enhanced cutaneous permeation targeting the dermal-epidermal junction. Graphical Abstract


Introduction
Bakuchiol is a meroterpenoid phenol and resveratrol analogue, primarily obtained from the seeds of the Cullen corylifolium species Psoralea corylifolia. In its pure form, this natural product was reported to have skin-beneficial effects, such as anti-ageing, anti-pigmentation and anti-acne (Xin et al. 2019). At least some of these effects are believed to stem from its retinoid functionality (Chaudhuri and Bojanowski 2014) and many have been validated in clinical case studies (Draelos et al. 2020;Lyons et al. 2020;Dhaliwal et al. 2019;Jafernik et al. 2020).
It has been recently reported that certain bakuchiol dimers have unique anti-inflammatory effects (Xu et al. 2021), while other analogues show improved outcomes in anti-cancer and anti-microbial assays (Krishna et al. 2022). These results underscore the bioactive potential of bakuchiol derivatives. Following this rationale, in an attempt to incorporate elements of hydroxy acid functionality in its retinoid-like mechanism of action, we esterified bakuchiol with another natural product-salicylic acid. Salicylic acid is a plant hormone isolated from the willow tree (Salix alba) bark. This beta-hydroxy acid is commonly used in skincare for exfoliation and treatment of a broad array of skin conditions, including dermatitis, acne, psoriasis, calluses, keratosis pilaris, acanthosis nigricans, ichthyosis, and viral warts (Yeoh and Goh 2021). The resulting bipartite molecule-bakusylan-was found to have retinoid-like normalizing properties in two in vitro psoriasiform-surrogate models, without, however, the proinflammatory and teratogenic signature characteristic of retinol (Ma et al. 2017). Here we provide improved preparation process, report skin-beneficial activities in a broader, regenerative and anti-aging context, and propose a PI3K/Aktbased mechanism of action responsible for these effects.

Purification of bakuchiol and synthesis of bakusylan
Bakuchiol, as a natural extract obtained from seeds of the plant Psoralea corylifolia, comes in a variety of grades containing from a few to 98 percent of pure substance, the remaining mass being a collection of poorly defined compounds (Chopra et al. 2013). Some of these impurities are responsible for the dark colour of bakuchiol preparations, which is undesirable in products intended for certain applications, such as skin care. For the purpose of this study, the starting material was a bakuchiol extract of dark brown colour and 75% of purity (per manufacturer's information). Its refinement by silica chromatography resulted in the improvement of purity and much lighter colour ( Figure S1). The improvement of purity was of 23% and was calculated by measuring the area under the HPLC elution peaks of starting and purified bakuchiol samples of the same weight (results not shown). The achievement of substantial discoloration is an important step in the physicochemical characterization of bakuchiol, whose highly pure standards from reputed commercial reagent providers, such as Sigma-Aldrich (68612), Abcam (ab141036) and Santa Cruz Laboratories (sc-202075) often are dark-coloured.
This purified material was then used for the synthesis of its salicylic acid ester through the modified method described by Ma and collaborators (2017), which requires DCC as condensing agent but no catalyst ( Figure 1). This is considered to be an improvement upon earlier esterification and other bakuchiol derivatization approaches, which require a skin-permeable, cytotoxic catalyst DMAP (4-dimethylaminopyridine; Ma et al. 2022;Wu et al. 2018). Of note is one recently published method by Fattahi et al. (2018) offers an interesting alternative approach for the synthesis of esters based on coupling carboxylic acids with phenols without DMAP, in the presence of DIC (N,N-diisopropylcarbodiimide), as the cupling agent. The application of this method is, however, limited to hydrosoluble compounds.
Gas chromatograms and mass spectra (inserts) from GC-MS analysis of purified bakuchiol and bakusylan as their TMSi derivatives are shown respectively in Figure S2a and Figure S2b. Mass spectra confirmed the suggested structure of both substances. The structure of the synthesized product was further confirmed by NMR ( Figure S2c).

Permeation in Strat M-skin approximation model
Visual examination of the Strat M membranes incubated with bakuchiol or bakusylan showed a difference in appearance consistent with the more complete permeation of the latter vs. former ( Figure S3). This qualitative observation was confirmed by the HPLC analysis of the permeates ( Figure S4). The permeation of bakusylan into Strat M membrane was $17% of the topically applied material (3 mg) after 2 h incubation, while the permeation of bakuchiol in the same conditions was 4% (Table SI in the  Supplementary Material). HPLC chromatograms of the extracted permeates displayed peaks at the expected elution times and absorption maxima for both compounds, confirming their identity.

Assays on reconstituted skin substitutes and human dermal fibroblasts
Microscopic observation and the quantities of extracted RNA revealed no differences between the controls and the bakusylan-treated tissues. Comparative transcriptome profiling identified PI3K/Akt as the signal transduction pathway most affected in reconstituted skin tissues treated by bakusylan vs. bakusylan's solvent (0.25% DMSO in water). In that pathway, the components modulated by bakusylan belonged predominantly to the extracellular matrix (ECM) category ( Figure S5A). For further validation, we focused on one of those proteins-type IV collagen (Fold Change bakusylan vs. solvent: 1.6, p value ¼ 0.047 for COL4A3 in RNA-seq)-because it plays a key role in the maintenance of skin integrity, as the main collagen component of the basement membrane that anchors epidermis to dermis (Roig-Rosello and Rousselle 2020). As reported on Figure S5B, the stimulatory effect of bakusylan on type IV collagen could be confirmed at the protein level in normal adult human dermal fibroblast cultures over a three-day treatment period by ELISA assay.
Bakusylan (bakuchiol salicylate) is an in silico designed bipartite molecule intended to provide improved retinol-like normalizing activity to psoriatic skin (Ma et al. 2017). For this project, lower grade starting material bakuchiol (75% purity) was first discoloured by purification on silica gel, which yielded a much lighter and purer substrate. Although the identification of the coloured impurity was not part of this project, it was determined to be more hydrophilic than bakuchiol, as it eluted only with polar solvents, such as methanol or acetone. Determination of other components in the common comercial preparations of bakuchiol is currently ongoing in our laboratory.
The reaction of bakuchiol with pure salicylic acid in the presence of DCC condensing agent was successful without a catalyst. The resulting ester has one free hydroxyl group less, making it less hydrophilic than bakuchiol, as confirmed by reverse phase chromatography [ Figure S4, (Ma et al. 2017)]. The lower polarity of bakusylan raised the possibility of a better miscibility with lipid layers and thus potential for improved skin penetration compared with bakuchiol. The quantification of permeated fractions of both compounds in the skin-approximating Strat M membrane model suggests that this may indeed be the case. While limited penetration through stratum corneum is advantageous for sunscreens and from purely regulatory viewpoint, some degrees of access to the deeper epidermal layers are desirable if a skin active is intended to have truly regenerative and anti-aging effects. Bakuchiol's topical effects have been validated in several clinical case studies (Draelos et al. 2020;Lyons et al. 2020;Dhaliwal et al. 2019;Jafernik et al. 2020), which suggests that intradermal penetration of a small fraction of the 0.5% to 1% dose customarily formulated in skin care products is sufficient for its bioactivity and/or that penetration enhancers can increase the penetrated share (Lewi nska et al. 2021). Furthermore, bakuchiol was found not to penetrate beyond skin when applied topically (Alalaiwe et al. 2018). Accordingly, it may be hypothesized that bakusylan, being a $4 times better penetrant, may be formulated at 0.25%-0.1% to achieve comparable effects. Studies of intradermal hydrolysis of bakusylan and clinical case studies with formulated products will put this hypothesis to test.
In agreement with the skin permeation potential identified above, bakusylan was found to be bioactive in the reconstituted human skin model. Transcriptome Analysis Console (TAC) Software analysis determined that this bioactivity was especially concentrated along the PI3K/Akt signaling pathway axis. Specifically, bakusylan transcriptionally upregulated some ECM components (tenascin C, certain laminins and collagens) implicated in the maintenance of skin integrity, associated with the PI3K/Akt signaling pathway ( Figure S5A). Importantly, the Toll-like Receptor (TLR) and other proinflammatory effectors associated with the PI3K/Akt activation [as reviewed in (Hawkins and Stephens 2015)] were not affected (results not shown, to be published separately). The activation of the PI3K/Akt signal transduction by bakusylan is in agreement with this pathway being a downstream enabler of retinoid signaling (So et al. 2014) and further supports this bakuchiol ester retinol-like functionality identified previously (Ma et al. 2017).
The transcriptional activation of PI3K/Akt signaling should result in the increase of type IV collagen genes expression (Li et al. 2001;Kim et al. 2020). This indeed turned out to be the case at the transcriptional level in human full thickness skin substitutes and was further confirmed at the protein level in normal adult human dermal fibroblast culture ( Figure S5B). Type IV collagen is an essential component of the dermalepidermal junction, shown to be decreased in aged (Roig-Rosello and Rousselle 2020; Feru et al. 2016) and diseased (atopic dermatitis; Kim et al. 2018) skin. Bakusylan, which was found to upregulate this basement membrane component, could potentially provide relief from both conditions, although additional studies are necessary to determine whether these in vitro findings translate into clinical anti-aging and skinregenerative benefits.

Synthesis of bakusylan
Bakuchiol (75% purity; from Flavour Trove, Bangalore, India), was first purified to nearhomogeneity ($98%) through silica gel (high-purity Fluka SiO 2 , pore size 60 Å, 70-230 mesh) chromatography using toluene and cyclohexane (1:1 v/v) as a mobile phase. The improvement on bakuchiol purity was quantified by the comparison of the area under the peak of the starting and purified material using the HPLC system and running conditions described by (Ma et al. 2017). Bakuchiol salicylate (bakusylan) was synthesized by the modified Steglich esterification method as described before (Ma et al. 2017) but without catalyser. Briefly, the reaction was carried out in diethyl ether (anhydrous, cat.#673811, Sigma Aldrich, St. Louis, MO) solvent, with DCC (N,N-dicyclohexylcarbodiimide, cat.# 36650, Sigma Aldrich, St. Louis, MO) as condensing agent. The solvent was removed by evaporation, yielding the oily residue of light yellowish colour and two minor compounds, subsequentially separated from the final bakuchiol ester preparation by silica chromatography. Bakusylan was analyzed by infrared detector (Satellite FT-IR spectrophotometer), NMR (Varian Gemini À 200 MHz), diode array spectrophotometer HP-8452A (Agilent, Palo Alto, CA) and HPLC. The HPLC analysis was performed using an Agilent series 1100 instrument equipped with Agilent G1322A degasser, RheodyneV R 7725 injector, binary pump, diode array-UV detection module, Xterra MS Waters (Milford, MA) C18 column and ChemStation software. Samples dissolved in ethanol were eluted with 0.1% formic acid/acetonitrile gradient at 0.3 ml/ min. The structure and purity of bakuchiol and bakusylan were confirmed by gas chromatography-mass spectrometry (GC-MS; EI 70 eV) using Shimadzu GCMS-QP2010 SE (Shimadzu, Kyoto, Japan) instrument, equipped with a 30 Â 0.25 mm i.d., film thickness 0.25 lm, Zebron-5MS capillary column (Phenomenex, Torrance, California, USA). Helium was used as carrier gas at a flow rate of 1 mL min À1 . Oven temperature was programmed from 100 C to 310 C at 4 C min À1 and then kept for 5 min at 310 C; injector and transfer line temperatures were set at 310 C; ion source temperature was 200 C. Compounds were analyzed as respective trimethylsilyl derivatives (TMSi), which were synthesized by adding 0.1 mL N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS) to the aliquot of the samples (ca. 0.5 mg of purified bakuchiol and reaction product after fractionation). Reaction was performed at 70 C for 30 min.

Permeation in Strat M-skin approximation model
Strat-M membrane (Millipore, Burlington, MA cat. SKBM02560A) was used as the model for the intra-epidermal/intradermal penetration of bakuchiol and bakusylan. This human skin-approximating device is composed of a water-repellent thin waxy layer mimicking the epithelial stratum corneum and an underlying fibrous layer resembling the dermis. It has been developed for estimating the passage of pharmacological entities through the skin and into the blood stream, but here we developed a novel use for it, consisting in extraction and quantification of compounds, which permeated inside the membrane and remained trapped in it, approximating the intra-skin accumulation.
Undiluted samples (3 ll) were placed on top of the membrane in an airtight humidified container and incubated at 37 C for 2 h. The membrane was then wiped with a cotton makeup remover pad, rinsed four times with 1 ml of the 90% molecular biology grade ethanol, minced, and the permeated fraction of the topically applied substances was extracted with 500 ml of 90% ethanol for 2 h. Samples were then analyzed using Agilent HPLC series 1100 (Agilent Technologies, Palo Alto, CA) station equipped with the Autosampler G1313A, Hypersil Gold column (Thermo Fisher, Waltham, MA), diode array UV/Vis detector and ChemStation software, at 210 nm (A 210nm ), 256 nm (A 256nm ) and 325 nm (A 325nm ). [Ethanol (90%): H 3 PO 4 (10%), pH 2.5] solvent combination was used as mobile phase. Injection volume was 1 ml.
3.3. Assay on reconstituted skin substitutes 3D full thickness human skin model (EpiDerm FT from MatTek, Ashland, MA) tissues were topically treated with bakusylan (50 mg/ml in 0.25% DMSO/99.75% H 2 O) or with vehicle (0.25% DMSO) at 3 ml/cm 2 in triplicates for 24 h at optimal culture conditions provided by tissue supplier, and total RNA was isolated using RNeasy Mini kit and QIAcube Connect robotic station (Qiagen, Germantown, MD). RNA quantity and 260 nm/280 nm absorbance ratio were assessed using the NanoDrop Lite (Thermo Fisher Scientific, Waltham, MA).
Isolated RNA samples were sent on dry ice to Thermo Fisher Scientific Microarray Research Services Laboratory (Santa Clara, CA) for transcriptome profiling using the Clariom S GeneChipV R Pico Assay platform. The resulting CHP files containing probe set analysis results generated with Affymetrix software were uploaded and differential gene expression, as well as functional interaction networks were analyzed using the TAC software version 4.0.2.15 (Applied Biosystems by Thermo Fisher).

Assay on fibroblasts
Bakusylan stock solution prepared in DMSO at 20 mg/ml was further diluted in sterile distilled water and samples were added at two doses (2 mg/ml and 5 mg/ml) in six repeats to exponentially growing adult human dermal fibroblasts (aHDF; Cell Applications, San Diego, CA, cat.# 106 K-05a, cultured in DMEM/10%FBS) in a 96 well plate format. The negative control was the equivalent amount of water (solvent) added in 16 replicates. At the end of the experiment (72 h after adding test compounds and solvent control), collagen IV was quantified in the cell culture conditioned medium by sandwich ELISA assay using Southern Biotechnology (Birmingham, AL) unlabelled and biotinylated anti-type IV collagen antibodies, streptavidin/horseradish peroxidase and TMB reagents. Colorimetric measurements were performed using Molecular Devices (Sunnyvale, CA) microplate reader MAX190 and SoftMax3.1.2PRO software. The type IV collagen signal was standardized to the metabolic activity measured with the MTT assay (Berridge and Tan 1993).
Statistical significance was assessed with paired Student test. Deviations of >20% as compared to bakusylan's solvent-treated control with p values below 0.05 were considered statistically significant.

Conclusions
Considering a simple synthesis protocol, enhanced intradermal penetration potential and stimulation of type IV collagen synthesis, bakuchiol salicylate appears to be a functional retinoid and bakuchiol derivative suitable not only for psoriatic but also for general skin care applications.