ABSTRACT

Background: A bis resorcinol was isolated as the main constituent of the Heliciopsis terminals’ trunk (Proteaceae). Recently, resorcinol is applied as an active whitening agent in various cosmetic products. Because of the structural mimic of resorcinol, benefits of the bis resorcinol as an aging-enzyme antagonist were demonstrated in this study.

According to relevant studies,cistanche is a common herb that is known as "the miracle herb that prolongs life". Its main component is cistanoside, which has various effects such as antioxidant, anti-inflammatory, and immune function promotion. The mechanism between cistanche and skin whitening lies in the antioxidant effect of cistanche glycosides. Melanin in human skin is produced by the oxidation of tyrosine catalyzed by tyrosinase, and the oxidation reaction requires the participation of oxygen, so the oxygen-free radicals in the body become an important factor affecting melanin production. Cistanche contains cistanoside, which is an antioxidant and can reduce the generation of free radicals in the body, thus inhibiting melanin production.

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Methods: The bis resorcinol was purified from the crude ethanolic extract of H. terminals’ trunk by solvent extraction and preparative chromatography, respectively. Inhibitory activity on collagenase, elastase, and tyrosinase of the compound was investigated by using a different spectroscopic technique. Molecular docking was carried out to predict possible interactions of the substance around the enzyme active sites.

Results: The IC50 values on collagenase of the bis resorcinol and caffeic acid were 156.7 ± 0.7 and 308.9 ± 1.6 µmole L−1, respectively. For elastase activity, the IC50 of 33.2 ± 0.5 and 34.3 ± 0.3 µmole L−1 were respectively determined for the bis resorcinol and ursolic acid. The bis resorcinol was inhibitory to tyrosinase by exhibiting the IC50 of 22.8 µmole L−1, and that of 78.4 µmole L−1 was present for β-arbutin. The bis resorcinol bound to collagenase, elastase, and tyrosinase with the respective binding energies of −5.89, −5.69, and −6.57 kcal mol−1. These binding energies were in the same ranges as tested inhibitors. The aromatic phenol groups in the structure were responsible for principle as well as supporting binding interactions with enzymes. Hydrogen binding due to hydroxyl groups and π-related attractive forces from an aromatic ring(s) provided binding versatility to bis resorcinol. 

Conclusion: The bis resorcinol purified from H. terminals might be useful for inclusion in cosmetic products as an aging-enzyme antagonist.

Subjects Biochemistry, Bioinformatics, Cell Biology, Dermatology, Data Mining, and Machine Learning

Keywords Heliciopsis terminals, Bisresorcinol, Collagenase, Elastase, Tyrosinase, Anti-aging property, Molecular docking, Enzyme inhibition

INTRODUCTION

A bis resorcinol, (8′Z)-3,5-dihydroxy-1-[16′-(3″, 5″-dihydroxy phenyl)-8′-hexadecen-1′-yl] benzene, has been isolated as an abundant constituent from the trunk of Heliciopsis terminals (Kurz) Sleumer, which is a plant of Proteaceae family and has been used by ethnic people in Vietnam for hepatoprotective effect. To date, the medical benefits of this compound as an agent for anti-inflammation, inhibition of malondialdehyde (MDA) production, and hepatoprotection have been clarified in vitro and in vivo (Giang et al., 2019). It is apparent in that a molecule of the bis resorcinol contains two resorcinol molecules (EC No 203-585-2), bonded together with meta-C7H14C = CC7H14 linkage (Fig. 1A). Resorcinol is recently applied in a form of facial concentrated serum for white therapy. Based on the Scientific Committee on Consumer Safety, Brussels, Belgium (SCCS 1270/09, European Commission, Scientific Committee on Consumer Safety, 2010), resorcinol has been regulated as an oxidant in products intended for coloring eyelashes at concentration ranges up to 1.25%, or in hair lotions and shampoos at concentration ranges up to 0.5%. Cosmetical applications of resorcinol are thus diverse. For whitening agents, inhibition of melanin synthesis by melanocytes that reside in the basal cell layer of the epidermis has been assumed. Tyrosinase (EC 1.14.18.1) is a key enzyme for catalyzing melanogenesis in which L-tyrosine is used as the substrate. Although melanin helps protect skin from damage from UV rays, excess melanin production can cause hyperpigmentation, freckle, and age spots. In addition to resorcinol, various synthetic and natural compounds with distinct chemical structures have been demonstrated to exhibit anti-tyrosinase activity (Dobos et al., 2015).

UV radiation is a major external factor that causes skin aging. Instead, skins can age over time by reactive oxygen species (ROS) and lipid peroxides that are internally produced after UV exposure. Secondary products from lipid peroxide metabolisms can damage elastin and collagen fibers of extracellular matrices (ECMs). Therefore, exposure to UV rays can result in dehydration, loss of elasticity, as well as wrinkles of the skin. To our knowledge, the ECMs are periodically remodeled to maintain normal tissue structures in accompany with that expired tissue proteins are properly degraded by matrix metalloproteinases, such as collagenases, and serine proteases like elastases (Thring, Hili & Naughton, 2009). In addition, both quantities and activities of aging enzymes have been increased by oxidative stress, leading to extensive ECM degradation and progressive aging (Sherratt et al., 2019). Therefore, compounds that can form complexes with metal ions are anticipated to be inhibitory to collagenases, elastases, and tyrosinase accordingly (Selvaraj et al., 2014). This project aims to determine the biological effects of bis resorcinol on tyrosinase, collagenase, and elastase in vitro, and a docking technique was carried out to clarify their interactions at molecular levels. It is hopeful to delay the aging process and improve skin appearance with bis resorcinol.

MATERIALS & METHODS

Chemicals and instruments

Compound purification, identification, and ratification

The crude extract in ethanol of H. terminals’ trunk was generously obtained from Professor P.M. Giang, Faculty of Chemistry, Vietnam National University, Hanoi, Vietnam with voucher specimen number HNIP-18473. Then, it was partitioned in methanol/n-hexane by three times repeated. The methanol layer was evaporated to nearly dry and subsequently partitioned in ethyl acetate/1-butane. The ethyl acetate layer was vaporized until its volume was reduced by 80%. The resulting viscous liquid was then purified on a silica gel column using chloroform/methanol gradient elution as follows: 100% chloroform, 30:1, 20:1, 10:1, 7:1, 5:1, 3:1, 3:2, and 2:1 of chloroform/methanol, and 100% methanol, respectively. The eluent from the 5:1 mixed solvent was collected and evaporated to one-fourth volume, followed by the separation on the ODS column using a gradient elution of methanol and acetone. The desired compound, i.e., bis resorcinol (Fig. 1), was obtained from eluents containing 70% acetone and confirmed by MS, NMR, and IR spectroscopies regarding the instrumental library data. Thin-layer chromatography was used for the estimation of its purity.

Biological activity assays

In vitro, experiments that consisted of enzyme-inhibitory assays on collagenase, elastase, and tyrosinase for the bis resorcinol and known inhibitors were carried out as follows.

Collagenase inhibition

The recent anti-collagenase assay was modified from that previously described by Widyowati et al. (2016). In brief, 50 mmol L−1 tricine buffer pH 7.5 supplemented with 400 mmol L−1 NaCl and 10 mmol L−1 CaCl2 was used as the buffering diluent. Collagenase from Clostridium histolyticum (EC.3.4.24.3) was dissolved in the buffer to a concentration of 1 mg L−1. The enzyme-substrate, MOCAc-PRO-Leu-Gly-Leu-A2pr (Dnp)-Ala-Arg-NH2, was prior dissolved in DMSO and subsequently diluted in the buffer to a concentration of 1 mmol L−1. A sample dissolved in DMSO was diluted to different concentrations by using the buffer. A 2-µl sample solution was incubated with 100 µl enzyme solution at 37 ○C for 10 min. Then, a 50-µl substrate was added and thoroughly mixed. Fluorescence emission (F) at 405 nm was immediately recorded and continually monitored for 30 min using a wavelength of 320 nm for excitation. Caffeic acid was used as a position control (Fig. 1). Negative control was performed with water. The percent inhibition (%) was calculated from the equation below:

%Inhibition ={1 - (Fsam; 30 - Fsam; 0)÷(Fcont; 30 - Fcont; 0)} ×100.

Elastase inhibition

The assay according to Abhijit & Manjushree (2010) was applied with some modifications. Briefly, the buffer system was 0.2 mM Tris-HCl buffer (pH 8.0). A solution of 1 µg mL−1 porcine pancreatic elastase (EC.3.4.21.36) was prepared in sterile water. The enzyme substrate, N-Succinyl-Ala-Ala-Ala-p-nitroanilide (SANA), was dissolved in the buffer to a concentration of 80 mmol L−1. A sample dissolved in DMSO was diluted to various concentrations by using the buffer. A 100-µl sample solution was mixed with 50 µl enzyme solution and incubated for 15 min. After that 50 µl of the enzyme-substrate solution was added and thoroughly mixed. The OD410 was measured immediately (0 min) and after incubation overnight (o/n) at 37 °C by using a microplate reader. Ursolic acid was used as a positive control (Fig. 1), and water was used as a negative control. The percentage inhibition (%) was calculated by the following equation:

%Inhibition = {1 - [(Asam; o/n - Asam; 0)÷(Acont; o/n - Acont; 0)]} ×100:

Tyrosinase inhibition

The used tyrosinase inhibitory assay was adapted from the previously described method (Jiratchayamaethasakul et al., 2020). In brief, the assay was carried out in 0.05 mol L−1 phosphate buffer pH 6.8. Mushroom tyrosinase (EC.1.14.18.1) was dissolved in the buffer to a concentration of 100 units mL−1. L-tyrosine, an enzyme substrate, was prepared to a concentration of 0.25 mg mL−1 in the buffer. A test compound dissolved in DMSO was diluted to concentrations ranging between 0.625 and 10 mg mL−1. Into a well of 96-well plates, a 10 µl sample and 40 µl L-tyrosine solution were mixed and incubated for 10 min at room temperature. After that 50 µl enzyme solution was inoculated and mixed thoroughly. The reaction was stopped afterward by incubation on ice for 1 min. The OD475 was measured using a microplate reader. β-Arbutin and water were used as a positive control (Fig. 1) and a negative counterpart, respectively. The percentage inhibition (%) was calculated by the equation as follows:

MOLECULAR DOCKING STUDY

Preparation of ligands and receptors

Three-dimensional (3D) structures of bis resorcinol (CID 8917124) and reference compounds, including substrates and inhibitors, were downloaded from the PubChem database for molecular docking study. A ligand structure was prepared in a Protein Data Bank (PDB) format file using Online SMILES Translator and Structure File Generator. The crystal structures of enzymes, such as collagenase (PDB ID 2Y6I), elastase (PDB ID 1BRU), and tyrosinase (PDB ID 2Y9X) were obtained from the RCSB protein database. The water and the attached molecules were dissected from all selected protein structures. Meanwhile, polar hydrogen atoms were added to the crystallized protein structures by AutoDockTools 1.5.6. Files of target proteins and other used compounds were saved in PDBQT format before performing the molecular docking.

Selection of active site residues and molecular docking

Grid and docking protocols of the active site predictions were prepared using AutoDockTools 1.5.6. Grid sites were set with a spacing of 0.375 Å. The x-y-z dimensions were set to be 126-135-160 Å3 for collagenase, 130-120-126 Å3 for elastase, and 80-80-80 Å3 for tyrosinase. Grid box centers (with offset values in AutoDockTools) were 31.869 (5.000), −19.41 (−25.000), and 17.815 for collagenase, 30.048 (−0.972), 51.253 (−2.722), 17.6 for elastase, and −8.407 (−1.000), −23.795 (−0.250), −36.019 (−3.500) for tyrosinase, respectively. The protein structure was used as a rigid entity while the ligand compound was set as a flexible molecule. The docking study was performed using the Lamarckian genetic algorithm (GA) implemented by AutoDock4 version 4.2. The number of GA runs was 50 with a popular size of 200. The binding energy (ΔGbind) was analyzed by ADT. Interaction(s) was visualized using BIOVIA Discovery Studio (BIOVIA, 2020). The molecular interaction between the compound and a protein receptor was analyzed and visualized using BIOVIA Discovery Studio software (BIOVIA, 2020). The structure of the protein binding site compound was visualized using the Visual Molecular Dynamics (VMD) package (Humphrey, Dalke & Schulten, 1996).

Statistical analysis

RESULTS

A major constituent was successfully purified from H. terminals’ trunk by using solvent extraction and reverse phase HPLC techniques, respectively. The acquired 1 H-NMR and 13C-NMR spectra were compared to those of reference substances through the SciFinder (https://scififinder.cas.org/) and found to correspond with (8′Z)-1,3-dihydroxy-5- [16′-(3″,5″-dihyroxyphenyl)-8′-hexadecen-1′-yl] benzene (Chaturvedula et al., 2002). It was classified as one of the bisresorcinols. This recent bis resorcinol had the molecular formula of C28H40O4 with a molecular weight of 463.2819 Dalton. The extraction yield was calculated to be 0.086%, based on the trunk’ dried weight. Its purity was more than 98%, concerning the 1 H and 13C-NMR spectra acquired (see Figs. S1 and S2).

In vitro bioactivity analysis

Antagonistic effects of the bisresorcinol on aging enzymes, such as collagenase, elastase, and tyrosinase were determined in vitro by using a distinct spectroscopic method. Results were summarized in Table 1 and Fig. 2. For collagenase inhibition, test samples in a range of 50–550 µmol L−1 were prepared using MOCAc-PRO-Leu-Gly-Leu-A2pr (Dnp)-AlaArg-NH2 as the enzyme-substrate and caffeic acid as an enzyme inhibitor. It was indicated that the bisresorcinol at a concentration of 156.7 µmol L−1 decreased the enzyme activity by 50% (IC50). Instead, the IC50 of caffeic acid was 308.9 ± 13.7 µmol L−1. Thus, the anti-collagenase activity of the bisresorcinol was significantly stronger than caffeic acid.

For more info: david.deng@wecistanche.com  WhatApp:86 13632399501