Carotenoids from female Grapsus albolineatus as potential anti-ageing compounds

Ethical considerations

The researcher submitted a research permit (ethical clearance) to the UB (Universitas Brawijaya) Ethics committee team. Part of this research was conducted at the university.

Ethical clearance was obtained by including a research proposal, in which the method had a 3R stage, namely: 1) Reduction: using 3 animals, 2) Replacement: using 1 gram of the carapace organ, 3) Refinement: To avoid distress in animals, the researcher used 95% alcohol which functions as an analgesic.

This research was funded by the Institution of Research and Community Service Sam Ratulangi University (UNSRAT) with assignment letter Number 673/UN12.13/LT/2019. which was signed by the chairman of Institution of Research and community service UNSRAT, after being passing evaluation by the university reviewer.

Sampling and handling

Sampling was carried out by exploring the intertidal area at the lowest low tide at night. The sample was put into a container filled with a little seawater, to ensure the crabs did not die. The dorsal carapace of the 3.7 cm crab in that section was smeared with 95% alcohol, when the crab fainted, surgery was carried out and then extraction was carried out.

Total pigment extraction

Crab samples were first injected to obtain blood from G. albolineatus using a five-mile injection and then dissected in order to separate the hepatopancreas and gonads. The next step was to separate the epidermis layer, which is a thin membrane connected to the interior of the carapace, and the black epidermis layer with tweezers and a laboratory knife. The organ carapace was crushed and immersed for five minutes in 2N hydrodrochloric acid. Then we strained it through filter paper into a separator flask,added petroleum ether and distilled water to make two layers. The absorption peak was then calculated using a UV-Vis spectrophotometer with a wavelength range of 380-550 nm.

Quantitative analysis: Total pigment extract (Concentration and quantity)

The absorbance of the carapace pigment extract was measured using a UV-Vis spectrophotometer at 380-550 nm wavelength. The spectrogram’s shape is a curve with the X axis representing wavelength, and the Y axis representing absorbance, and the highest absorption peak at a certain wavelength can be read. Each spectrogram produced can be used to calculate the amount and concentration of G. albolineatus pigments utilized in this investigation.^18,19

Qualitative analysis: pigment separation by column chromatography

After that, the entire pigment extract was separated using column chromatography. The n-hexane-acetone was the solvent employed in the mobile phase (70:30). The X fraction was collected, which was then accommodated and examined for absorption with a UV-Vis spectrophotometer at a wavelength of 380-550 nm, yielding the spectrophotometer’s highest absorption peak.^18,19

High performance liquid chromatography analysis

High performance liquid chromatography (HPLC) type LC.20ADVP with photodiode array detector (PDA; SPD-M20A-Shimadzu). The HPLC column used was C30 (YMC carotenoid, 150 × 4.6 mm I.D) with a column temperature of 30°C and a flow rate of 1 mL/min with a gradient system of H2O, methanol and methyl tertiary butyl ether. The sample used was 20 μL. Carotenoids were separated in a gradient form for 50 min using a mixed solution of methanol, methyl tertiary butyl ether, and water (80:5:15) at a flow rate of 1 mL/min. After 10min, the water content was lowered linearly, and the acetone was added to get the acetone proportion up to 20 percent at 50 min after injection. The final composition of the gradient mixture was methanol: methyl tertiary butyl ether (80:20).

Molecular docking analysis of carotenoids with aging receptors

The binding mechanism to the active site of three enzymes was investigated using molecular docking: fibroblast collagenase (1CGL), porcine pancreatic elastase (4YM9), and hyaluronidase (1FCV).^20–22 These three receptors are available on the Protein DataBank (PDB) website and may be saved as a PDB file.²³

Enzyme preparation was accomplished by eliminating water and cofactors from each enzyme. The protein was first optimized by adding polar hydrogen, followed by the addition of atomic charge with Kolman charge and nonpolar hydrogen. The receptor was then stiffened and stored in PDBQT (Protein DataBank, partial charge (Q), and atom type) format.⁹

The Canonical SmilesY of each ligand utilized in ligand preparation was acquired from Pubchem.²⁴ Subsequently, Canonical Smiles was converted to Chemdraw 2D 16.0 to produce a 2D compound, which was then modified into 3D using Chemdraw 3D and a reduced calculation was done. The file was stored as a PDB file. The ligand’s PDB file was then mixed with polar hydrogen and given the gasteiger atomic charge and torque on each ligand, after which the ligand was stored in PDBQT format.

The AutoDock 4.2.6 software was used to simulate docking.²⁵ At each active site, molecular docking was performed. The active site is determined by verifying the docking of each inhibitor and the corresponding receptor. A grid box was created in the middle of the ligand location to determine the coordinates of the receptor’s active side. Then, for 1CGL and 4YM9 receptors, dimensions of 50×50×50 points were utilized, while for 1FCV, box dimensions of 60×60×60 points were chosen, with a grid point spacing of 0.375 Å. The Lamarckian Genetic Algorithm was the calculation employed in this docking approach. The optimal conformation was chosen based on the lowest bonding energy among the most conformational populations.

To observe the interactions that occur between the ligand and the receptor, docking visualization was performed using Biovia Discovery Studio 2020.²⁶ Interactions are depicted in three and two dimensions, respectively. Hydrogen bonding and hydrophobicity were utilized to analyze intermolecular interactions.

Quantiative analysis: pigment extraction of female G. albolineatus

The highest wavelength of the content and concentration of total pigment extract in G. albolineatus crabs, particularly in the carapace, was 474 nm. As a result, the overall pigment extract content and concentration value was 4.33 μg/g, whereas the pigment content in the carapace organ was 4.46 μg/g.

Qualitative analysis: pigment extraction of female G. albolineatus using column chromatography

The column chromatography separation was repeated twice with an n-hexane-acetone mobile phase (70:30). A total pigment extract divided into four fractions: F1, F2, F3, and F4. A UV-Vis Spectrophotometer with a wavelength range of 380-550 nm was used to examine the four fractions. The presence of x-carotene pigments was indicated by wavelengths of 426, 448, and 475 nm, whereas the presence of zeaxanthin pigments was indicated by wavelengths of 426, 450, and 475 nm in the second fraction; the pigment types in F3 and F4 were unidentified.²⁷

F1 was not processed through the second round. However, F2, F3, and F4 were separated again using column chromatography: F2.1 was characterized as echinenone and F2.2 as astase. Furthermore, the pigment was not identified in F3.1 and F4.1. Krocoxanthin was found in F3.2, alloxanthin was identified in F3.3, and pyrhoxanthin was identified in F4.2.

Pigment type analysis with high performance liquid chromatography

To generate 61 maximum absorption peaks, the findings of one column chromatography fraction were separated using HPLC with a propagation time of 50 minutes. The absorption spectra of each peak from the HPLC chromatogram were identified at 463 nm, and those with an area per-centage greater than 3% were investigated by examining the spectral pattern using an HPLC UV-Vis absorption spectrophotometer

The HPLC fraction one findings showed eleven absorption peaks. Table 1 shows the absorption generated at the highest peak. The greatest absorption peak at 473 nm was determined to be a kind of didehydroastaxanthin pigment, while the maximum absorption peak at 476 nm was determined to be a type of tetrahydroastaxanthin. (Tables 1-3). The HPLC separation was carried out at a propagation time of 50 minutes and generated 65 absorption peaks in the findings of the second fraction column chromatography.

At a wavelength of 436 nm, the absorption spectrum region with a larger percentage results in the formation of a maximum absorption peak. The highest absorption in Table 2 helps determine the kind of carotenoid pigment.²⁸

Six spectrum peaks were identified from the separation of nine absorption peaks by HPLC. Only five types of pigments could be identified using the six peak spectral pattern. The HPLC separation resulted in the formation of an absorption peak with a peak area of 3%in Table 3. The greatest absorption peaks were discovered by the absorption of HPLC UV-Vis spectrophotometer, namely the 8th, 14th, 15th, 16th, 17th, and 34th absorption peaks, allowing the kind of pigment to be determined. Figure 1 presents the visualization of the compound structure of the pigment types produced by the HPLC analysis.

Figure 1. Compounds obtained from G. albolineatus and their structures: (a) didehydroastaxanthin; (b) tetrahydroxanthine; (c) dihydroxyastaxanthin; (d) diatoxanthin; (e)astaxanthin; (f) adonixanthin.

Molecular docking analysis of carotenoids from G. albolineatus on collagenase protein (1CGL)

The value of the bond energy is generated from the total final intermolecular energy, namely from van der Waals bonds, hydrogen bonds, de-solvation energy, and electrostatic energy, then from the final total internal energy, tor sional energy and unbound system’s energy. This summa tion of energy is usually known as the estimated free energy of binding (EFEB). The inhibition constant was also estimated by using Autodock 4.2.6 at 298 K. The low value of the inhibition constant was related to the biological activity.²⁹

The carotenoids discovered by HPLC (Figure 1) were used as test ligands for binding proteins that contribute to skin aging. According to Table 4, after compared to other carotenoid compounds, adonixanthin had the lowest binding energy with -6.61 kcal/mol and the highest inhibition constant with 14.23 mM. The binding energy of these carotenoids, however, is not better than the native ligand (-7.83 kcal/mol with the lowest inhibition constant of 1.81 mM). The visualization of molecular docking results is shown in Figures 2-4.

Figure 2. Visualization results of carotenoids as collagenase (1CGL) enzyme inhibitors: (a) didehydroastaxanthin; (b) tetrahydroxanthine; (c) dihydroxyastaxanthin; (d) diatoxanthin; (e)astaxanthin; (f) adonixanthin.

Figure 3. Visualization results of carotenoids as collagenase (1CGL) enzyme inhibitors: (a) didehydroastaxanthin; (b) tetrahydroxanthine; (c) dihydroxyastaxanthin; (d) diatoxanthin; (e)astaxanthin; (f) adonixanthin.

Figure 4. Visualization results of carotenoids as collagenase (1CGL) enzyme inhibitors: (a). didehydroastaxanthin; (b) tetrahydroxanthine; (c) dihydroxyastaxanthin; (d) diatoxanthin; (e) astaxanthin; (f) adonixanthin.

Molecular docking analysis of carotenoid Compounds from G. albolineatus on protein elastase (4YM9)

According to the results of the interaction between carotenoids and the protein elastase, carotenoid pigments have a higher binding energy than natural ligands, which only have a binding energy of -5.55 kcal/mol and an inhibition constant of 85.31 mM (Table 5). Adonixanthin and astaxanthin have the highest binding energies, with binding energies of -9.03 kcal/mol and -9.01 kcal/mol, respectively.

Molecular Docking Analysis of Carotenoid Compounds from G. albolineatus on Protein elastase (4YM9)

The carotenoid compounds from G. albolineatus had a lower binding energy than the natural hyaluronidase ligand, which had a binding value of -4.17 kcal/mol. Astaxanthin had a binding energy of -2.27 kcal/mol and is only bound to four amino acid residues, whereas didehydroastaxanthin had a binding energy of -7.71 kcal/mol (Table 6).