Potency of bisresorcinol from Heliciopsis terminalis on skin aging: in vitro bioactivities and molecular interactions

Chemicals and instruments

All the chemicals used in this study were of analytical grade. Organic solvents, including 1-butanol, acetone, chloroform (also for NMR), and ethyl acetate, were purchased from KANTO CHEMICAL CO., INC., Japan. Chemicals, such as hydrochloric acid, K₂HPO₄, KH₂PO₄, L-tyrosine, silica gel, octadecyl silica gel, and tris (hydroxymethyl) aminomethane, were bought from NACALAI TESQUE, INC., Japan. Standard substances, including β-arbutin, caffeic acid, and ursolic acid, and tricine were obtained from Tokyo Chemical Industry, Japan. Enzymes, such as collagenase, elastase, and tyrosinase, including N-succinyl-Ala-Ala-Ala-p-nitroanilide (SANA) and dimethyl sulfoxide (DMSO), were acquired from Sigma-Aldrich, Germany. DMSO and methanol (NMR grade) were purchased from ISOTEC^®, Sigma-Aldrich, Germany. A peptide: MOCAc-PRO-Leu-Gly-Leu-A₂pr(Dnp)-Ala-Arg-NH₂, was ordered from Peptide Institute, Inc., Japan. Instruments used included HPLC (SCL-10A sp/RID-6A/c-R3A; SHIMADZU, Kyoto, Japan), Fluorescence microplate reader (Enspire 2300 Multimode reader; PerkinElmer, Waltham, MA, USA), HR-ESI-MS (LTQ orbitrap XL; Thermo Fisher Scientific, Waltham, MA, USA), Infrared Spectrophotometer (FT-720, HORIBA), Nuclear Magnetic Resonance Spectrometer (ECA-500/600, JEOL), and UV-Vis microplate reader (Multiskan Go; Thermo Scientific, Waltham, MA, USA).

Compound purification, identification and ratification

The crude extract in ethanol of H. terminalis’s trunk was generously obtained from Professor P.M. Giang, Faculty of Chemistry, Vietnam National University, Hanoi, Vietnam with voucher specimen number of 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 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 fourths volume, followed by the separation on the ODS column using gradient elution of methanol and acetone. The desired compound, i.e., bisresorcinol (Fig. 1), was obtained from eluents containing 70% acetone and confirmed by MS, NMR and IR spectroscopies in regard to the instrumental library data. Thin layer chromatography was used for estimation of its purity.

Biological activity assays

In vitro experiments that consisted of enzyme-inhibitory assays on collagenase, elastase and tyrosinase for the bisresorcinol 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:

$\mathrm{\%}\mathrm{I}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}=\{1–({\mathrm{F}}_{\mathrm{s}\mathrm{a}\mathrm{m},30}–{\mathrm{F}}_{\mathrm{s}\mathrm{a}\mathrm{m},0})÷({\mathrm{F}}_{\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t},30}–{\mathrm{F}}_{\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t},0})\}\times 100.$

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:

$\mathrm{\%}\mathrm{I}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}=\{1–[({\mathrm{A}}_{\mathrm{s}\mathrm{a}\mathrm{m},\mathrm{o}/\mathrm{n}}–{\mathrm{A}}_{\mathrm{s}\mathrm{a}\mathrm{m},0})÷({\mathrm{A}}_{\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t},\mathrm{o}/\mathrm{n}}–{\mathrm{A}}_{\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t},0})]\}\times 100.$

Tyrosinase inhibition

The used tyrosinase inhibitory assay was adopted 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, 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:

$\mathrm{\%}\mathrm{I}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}=\{1–({\mathrm{A}}_{\mathrm{s}\mathrm{a}\mathrm{m},10}–{\mathrm{A}}_{\mathrm{s}\mathrm{a}\mathrm{m},0})÷({\mathrm{A}}_{\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t},10}–{\mathrm{A}}_{\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t},0})\}\times 100.$

Preparation of ligands and receptors

Three-dimensional (3D) structures of bisresorcinol (CID 8917124) and reference compounds, including substrates and inhibitors, were downloaded from 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 (https://cactus.nci.nih.gov/translate/). The crystal structures of enzymes, such as collagenase (PDB ID 2Y6I), elastase (PDB ID 1BRU), and tyrosinase (PDB ID 2Y9X) were obtained from RCSB protein database (http://www.rcsb.org). The water and the attached molecules were dissected from all selected protein structures. Meanwhile, polar hydrogen atoms were added in 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 spacing of 0.375 Å. The x-y-z dimensions were set to be 126-135-160 ų for collagenase, 130-120-126 ų for elastase, and 80-80-80 ų 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. 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 (ΔG_bind) was analyzed by ADT. Interaction(s) was visualized using BIOVIA Discovery Studio (BIOVIA, 2020). Molecular interaction between the compound and a protein receptor was analyzed and visualized using BIOVIA Discovery Studio software (BIOVIA, 2020). The structure of protein binding site compound was visualized using Visual Molecular Dynamics (VMD) package (Humphrey, Dalke & Schulten, 1996).

Statistical analysis

For in vitro study, each experiment was done in triplicate. Data were expressed as means ± standard error of the mean (SEM). The Student’s t-test was used to evaluate differences between results, and p-values of <0.01 were indicated to be significantly different. The performance regarding statistics was run on Libre Office Calc 5 in Ubuntu 16.04.6 LTS software.

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)-Ala-Arg-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, anti-collagenase activity of the bisresorcinol was significantly stronger than caffeic acid.

Inhibition of elastase enzyme was determined at a concentration range between 10 and 100 μmol L⁻¹. The residual enzyme activity was spectroscopically measured at 410 nm in response to the amount of p-nitroaniline released from the substrate SANA after cleavage. The bisresorcinol strongly inhibited elastase activity in a concentration-dependent manner with the maximum inhibition of 100% at 50 µmol L⁻¹. However, the inhibition decreased rapidly at concentrations less than 30 μmol L⁻¹. Notably, the dose-response curve of ursolic acid was less steep than that of the bisresorcinol by reaching 80% inhibition at 100 µmol L⁻¹. The IC₅₀ values of the bisresorcinol and ursolic acid were respectively 33.2 and 34.3 µmol L⁻¹, which were not significantly different.

In analysis of tyrosinase inhibition, samples were diluted to a concentration range of 10–100 µmol L⁻¹. L-tyrosine was used as a substrate of mushroom tyrosinase and β-arbutin was a known inhibitor. The bisresorcinol showed the IC₅₀ of 22.6 µmol L⁻¹. The IC₅₀ of β-arbutin was 78.5 µmol L⁻¹. Our test compound showed 100% inhibition at 50 µmol L⁻¹. In contrast, the enzyme inhibition of β-arbutin higher than 50% was not determined in the tested range.

Molecular interaction analysis

Molecular docking was applied to predict binding sites of test compounds to protein receptors, such as collagenase, elastase and tyrosinase, in comparison with known inhibitors of a corresponding enzyme (Supplemental Information). Consisting with previous publications, the relative binding affinity was evaluated meanwhile binding interactions were illustrated through the best predicted conformation (Teajaroen et al., 2020; Jewboonchu et al., 2020; Tanawattanasuntorn et al., 2020; Saeloh et al., 2017).

For collagenase enzyme, results of ligand-protein interactions were demonstrated in Fig. 3 and Table S1. The binding energy of −5.89 kcal mol⁻¹ to clostridial collagenase (PDB ID 2Y6I) was presented by the bisresorcinol. A range of the binding energy between −3.68 and −7.90 kcal mol⁻¹ was determined for other known collagenase inhibitors, including caffeic acid.

Both the bisresorcinol and caffeic acid shared the collagenase binding sites (Fig. 3A). Four amino acids responsible for caffeic acid attachment included His524, Trp496, His527, and Trp539 (Fig. 3B). His524 and Trp496 interacted caffeic acid by hydrogen bonds through carboxylic and phenolic hydroxyl group(s). His527 and Trp539 bonded to the phenolic ring by π-π stacking. Other amino acids within the enzyme pocket contributed their binding through Van der Waals force (Fig. 3C). Interactions between the collagenase and the bisresorcinol were shown in the Figs. 3D and 3E, involving Trp496 and Trp539 amino acids that donated π-electrons to bond to the phenolic ring of the bisresorcinol. The presence of hydrogen bonds between the hydroxyl groups of the bisresorcinol and amino acids Asp601 and Ser602 was observed. Moreover, the Zn atom in the enzyme active site might be coordinated to both the bisresorcinol and caffeic acid.

The crystal structure of pig elastase (PDB ID 1BRU) was adopted to determine binding behaviors to elastase enzyme of the bisresorcinol and other known inhibitors, including ursolic acid. Results were shown in Fig. 4 and Table S2. The binding energy of all test compounds ranged between −5.62 and −8.94 kcal mol⁻¹ and that of −5.69 kcal mol⁻¹ was calculated for the bisresorcinol.

There was similarity in binding to the elastase of the bisresorcinol and ursolic acid (Fig. 4A). Regarding ursolic acid, hydrogen bonding was proposed for a cyclic aliphatic hydroxyl group and Ser96, as well as a carboxylic group and Asn192 (Figs. 4B and 4C). Interestingly, structural folding of the bisresorcinol was presumed. This might facilitate interactions between phenolic hydroxyl groups of the compound and amino acids like Asn147, Ser190, Phe215, and Ser217 through hydrogen bonding (Figs. 4D and 4E).

To investigate binding affinity to tyrosinase of compounds, the crystal structure of mushroom tyrosinase (PDB ID 2Y9X) was applied. Results were displayed in Fig. 5 and Table S3, indicating that the binding energy of the bisresorcinol was −6.57 kcal mol⁻¹, being in a range of −4.63 to −8.12 kcal mol⁻¹ for other known inhibitors. In contrast, all known substrates were tightly bound to the tyrosinase according to an enormous decrease of the binding energy to a range from −15.46 and −23.94 kcal mol⁻¹.

The coordinate covalent bonds between metal ions and β-arbutin or the bisresorcinol at the tyrosinase active site likely occurred. However, copper ions (Cu²⁺) were replaced by zinc ions (Zn²⁺) in the AutodockTool, because the force field for Cu²⁺ is unavailable and Cu²⁺ and Zn²⁺ ions are quite identical in charges and sizes (Santos-Martins et al., 2014). For β-arbutin, one zinc ion might covalently bond to the phenolic hydroxyl group, and another zinc ion interacted with the phenol ring through π-cation bond with His259 and His263 residues (Figs. 5B and 5C). In addition, the amino acids, such as Asn260, Ser282, and Val283, are linked to its hydroxyl groups by hydrogen bonding. In view of the bisresorcinol, the phenolic hydroxyl group formed hydrogen bonds with Met280, Ala 246, and Glu239. The π-π stacked interaction between the phenol ring and His263, and the π-cation attraction between another phenol ring and Arg321 were distinctly observed.

In summary, molecular interactions and binding affinity between the bisresorcinol and collagenase, elastase, or tyrosinase were tabulated in Table 2.