Molecular Docking Simulation of Phenolics towards Tyrosinase, Phenolic Content, and Radical Scavenging Activity of Some Zingiberaceae Plant Extracts

: In Indonesia, plants have been indigenously used to treat various diseases and as cosmetics. It is always challenging to explore the molecular interactions of phenolic compounds towards the levels of constituents that contribute to the biological activities of plants. This study aimed to select a plant of the Zingiberaceae family with the highest phenolics and ﬂavonoids, the strongest radical scavenging activity, and the best interaction towards tyrosinase in terms of docking score and binding mode. Initially, the total phenolics and radical scavenging capacity of Zingiberaceae plants, namely, Hedychium coronarium , Curcuma zedoaria , Curcuma heyneana , and Alpinia galanga , were determined using the Folin–Cioc â lteu method and the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. The main phytoconstituents of plants with the highest phenolic levels were docked to the binding site of tyrosinase. Three anti-melanogenesis agents commonly used in cosmetics, namely, arbutin, hydroquinone, and kojic acid, were used as the standard. Our study revealed that all the tested plants contain polyphenolic compounds in the range of 17.92 ( C. zedoaria rhizome extract) to 252.36 ( A. galanga rhizome extract) mg GAE/g and have radical scavenging capacity, with IC 50 values in the range of 66.67 ( A. galanga rhizome extract) to 320.0 ( C. heyneana rhizome extract) µ g/mL. A molecular docking simulation demonstrated that four constituents, i.e., kaempferol, galangin, ethyl p -methoxycinnamate, and 6-gingerol, could occupy the binding site of tyrosinase with prominent afﬁnity and interact with essential residues of the enzyme. This study conﬁrms that Alpinia galanga possesses the potential to be further developed as a cosmetic with a radical scavenging and tyrosinase inhibitory activity. However, it may be interesting to carry out further studies of how the plant extract affects the melanogenesis signaling pathway.


Introduction
Despite the vast advances of modern medications, in Asian and African countries, plants are still utilized as the first choice in reducing symptoms of disease.Tropical plants have been indigenously used to treat various disorders.An analysis of articles indexed in the Scopus database described that plants of the families Asteraceae, Fabaceae, Lamiaceae, and Zingiberaceae had been the focus of interest in many drug discovery studies (14,652 articles).From the years of 2015 to 2019, the most-studied topics of medicinal plants were centered on cancer, antidiabetic, and anti-inflammatory activities [1].
The numerous biological activities of medicinal plants are thought to originate from the secondary metabolite contents, among which are the phenolic compounds that contain hydroxylated aromatic rings, the hydroxy group being attached directly to the phenyl or substituted phenyl, as well as the flavonols [2].Flavonols, which rank the highest in the number of the flavonoids class, are subcategorized as 2-benzo-γ-pirone.Quercetin (depicted in Figure 1), contained in almost all plants, is one of the most popular flavonols with its unsaturated double bond C2=C3 that extends pi-conjugation onto the carbonyl group in the C-ring, providing a strong radical scavenging capacity [3].The ultraviolet spectra of flavonols are characterized by two main peaks; the band I belongs to the cinnamoyl moiety at 320-385 nm (showing the electromagnetic radiation absorption of ring B), and band II belongs to the benzoyl moiety at 250-285 nm ring A (showing the electromagnetic radiation absorption of ring A) [4].Moreover, a catechol moiety also donates antioxidant activity [5,6].
The numerous biological activities of medicinal plants are thought to originate from the secondary metabolite contents, among which are the phenolic compounds that contain hydroxylated aromatic rings, the hydroxy group being attached directly to the phenyl or substituted phenyl, as well as the flavonols [2].Flavonols, which rank the highest in the number of the flavonoids class, are subcategorized as 2-benzo-γ-pirone.Quercetin (depicted in Figure 1), contained in almost all plants, is one of the most popular flavonols with its unsaturated double bond C2=C3 that extends pi-conjugation onto the carbonyl group in the C-ring, providing a strong radical scavenging capacity [3].The ultraviolet spectra of flavonols are characterized by two main peaks; the band I belongs to the cinnamoyl moiety at 320-385 nm (showing the electromagnetic radiation absorption of ring B), and band II belongs to the benzoyl moiety at 250-285 nm ring A (showing the electromagnetic radiation absorption of ring A) [4].Moreover, a catechol moiety also donates antioxidant activity [5,6].Plants of the families Zingiberaceae, Asteraceae, Acanthaceae, and Fabaceae contain the highest number of medicinal properties, while the most-used parts are the leaves, roots, fruits, rhizomes, whole plants, flowers, seeds, and barks [7].An in vitro study reported that flavonols from the Heterotheca inuloides rhizome, namely, galangin, kaempferol, and quercetin, could inhibit melanogenesis by suppressing tyrosinase activity via chelation with copper in the catalytic site of the enzyme [8].Moreover, the aqueous acetone extract of Alpinia officinarum rhizomes inhibits melanogenesis in theophylline-stimulated murine B164A5 melanoma cells.Two flavonols (kaempferide and galangin) were reported to reduce melanogenesis with IC50 values in the range of 10-48 µ M [9].Another flavonol of Zingiberaceae plants, namely, 6-shogaol, had shown suppression toward the activity of tyrosinase, melanin formation, and the expression levels of tyrosinase-related protein 1 (TRP-1) and microphthalmia-associated transcriptional factor (MITF) [10].
Molecular docking studies of polyphenolic compounds have been reported towards human pancreatic alpha-amylase for antidiabetics [11,12]; towards alpha-glucosidase for antidiabetics [12]; towards the main protease enzymes for antivirals [13]; towards βlactoglobulin for antioxidants [14]; towards all non-redundant holo-proteins from the Protein Data Bank for antioxidative, anti-inflammatory, anticarcinogenic, and antimicrobial activities [15]; and towards cyclooxygenases for anti-inflammatory activities [16].No studies have been reported on tyrosinase for anti-melanogenesis drug discovery.
Considering this, our study aimed to discover plants with tyrosinase inhibitory activity.This study was started by determining the total phenolics (via the Folin-Ciocalteu method) and the radical scavenging capacity (1,1-diphenyl-2-picrylhydrazyl assay) of Plants of the families Zingiberaceae, Asteraceae, Acanthaceae, and Fabaceae contain the highest number of medicinal properties, while the most-used parts are the leaves, roots, fruits, rhizomes, whole plants, flowers, seeds, and barks [7].An in vitro study reported that flavonols from the Heterotheca inuloides rhizome, namely, galangin, kaempferol, and quercetin, could inhibit melanogenesis by suppressing tyrosinase activity via chelation with copper in the catalytic site of the enzyme [8].Moreover, the aqueous acetone extract of Alpinia officinarum rhizomes inhibits melanogenesis in theophylline-stimulated murine B164A5 melanoma cells.Two flavonols (kaempferide and galangin) were reported to reduce melanogenesis with IC 50 values in the range of 10-48 µM [9].Another flavonol of Zingiberaceae plants, namely, 6-shogaol, had shown suppression toward the activity of tyrosinase, melanin formation, and the expression levels of tyrosinase-related protein 1 (TRP-1) and microphthalmia-associated transcriptional factor (MITF) [10].
Molecular docking studies of polyphenolic compounds have been reported towards human pancreatic alpha-amylase for antidiabetics [11,12]; towards alpha-glucosidase for antidiabetics [12]; towards the main protease enzymes for antivirals [13]; towards βlactoglobulin for antioxidants [14]; towards all non-redundant holo-proteins from the Protein Data Bank for antioxidative, anti-inflammatory, anticarcinogenic, and antimicrobial activities [15]; and towards cyclooxygenases for anti-inflammatory activities [16].No studies have been reported on tyrosinase for anti-melanogenesis drug discovery.
Considering this, our study aimed to discover plants with tyrosinase inhibitory activity.This study was started by determining the total phenolics (via the Folin-Ciocalteu method) and the radical scavenging capacity (1,1-diphenyl-2-picrylhydrazyl assay) of plants of the family Zingiberaceae, i.e., Hedychium coronarium, Curcuma zedoaria, Curcuma heyneana, and Alpinia galanga.The plant with the highest total phenolic level was further studied for its binding mode toward tyrosinase to predict the potential of the anti-melanogenesis activity.

Plant Extraction
Each of the rhizomes of the plants was cleaned from the soil, dust, and other foreign inorganic matter, washed, sliced, and sun-dried.A quantity of 500 g of the dried plant was soaked in ethanol 70% for 24 h at 25 ± 1 • C. Ethanol was selected as the solvent because it is a universal solvent that has the capability to dissolve many secondary metabolites and is a good solvent for flavonol extraction.Furthermore, ethanol is safe for human consumption [19,20].
The extract was filtered using a Whatman paper, and the residue was re-extracted for 2 × 24 h and collected.The solvent was evaporated in a vacuum rotavapor at 45 ± 5 • C, 80 rotations per minute, followed by evaporation in a water bath at 50 ± 1 • C until a thick consistency was obtained.The ethanol extract of C. heyneana (25 g) was further partitioned using a mixture of 25 mL of water and 25 mL of n-hexane in a separatory funnel.The n-hexane phase was collected and was added with 25 mL of ethyl acetate to obtain the ethyl acetate fractions.The same procedure was carried out for C. zedoaria (25 g).Ethyl acetate is a semipolar solvent that is widely used to extract polyphenols or flavonols.Partitioning using ethyl acetate was reported to successfully yield the highest amount of total phenolic metabolites [19].

Phytochemical Screening and Total Phenolics
The thick extract was phytochemically screened by following a previous method [21] as follows:

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Flavonoids were detected by treating the extracts with a few drops of lead acetate solution, which resulted in the formation of a yellow precipitate.

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Phenols were detected by treating the extracts with 3-4 drops of ferric chloride solution, which resulted in the formation of a dark blue color.

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Tannins were detected by treating the extracts with a 1% gelatin solution containing sodium chloride, which resulted in the formation of a white precipitate.

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Alkaloids were detected by treating the extracts with dilute hydrochloric acid and added with the Dragendorff reagent, which resulted in the formation of a red precipitate.

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Glycosides were detected by treating the extracts with sodium nitroprusside in pyridine and sodium hydroxide, which resulted in the formation of a pink to dark red color.

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Saponins were detected by treating the extracts with 20 mL of distilled water and were shaken in a graduated cylinder for 15 min.The formation of a 1 cm layer of foam indicates the presence of saponins.

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Phytosterols were detected by treating the extracts with chloroform and the Liebermann-Burchard reagent, which resulted in the formation of a brown ring at the junction.
The total phenolic compounds were measured as equivalent to gallic acid using the Folin-Ciocâlteu colorimetry method described elsewhere [22,23] with modifications.

Radical Scavenging Capacity Assay Using the DPPH Method
The antioxidant activity of the plant extracts was evaluated using the DPPH method as described by Ayele and co-workers [23] with modifications.Briefly, an accurately weighed 20 mg of DPPH was dissolved in analytical-grade methanol in a 500 mL volumetric flask to obtain a concentration of 40 µg/mL.
The standards used were ascorbic acid and quercetin.A solution of ascorbic acid was prepared by dissolving 10 mg of ascorbic acid in 250 mL of methanol, and a solution of quercetin was prepared by dissolving 10 mg of quercetin in 250 mL of methanol.The standard addition curves were generated by preparing different concentrations, i.e., 0.41, 1.02, 2.56, 6.4, 16, and 40 µg/mL, of the standard solutions.
To each microplate well, 100 µL of standard and 100 µL of DPPH solutions were added; then, the plate was covered with aluminum foil and kept in the dark for 30 min.For the extracts, 100 µL of 5.12, 12.8, 32.0, 80.0, 200.0, and 500 µg/mL of the extracts were mixed with 100 µL of DPPH in a microplate well.The mixture was kept in the dark for 30 min.Finally, the absorbance was measured at 517 nm.
The % radical scavenging capacity (or % inhibition of DPPH radicals) was calculated using Equation (1) as follows: where A DPPH = the absorbance of DPPH in methanol and A sample = the absorbance of the extract.The DPPH radical solution had a purple color before the reaction, and after it was reacted with the antioxidant of the extract, the color changed to pale yellow due to the formation of a stable DPPH-H.IC 50 values were calculated from the plotted graph of the radical scavenging capacity against the various concentrations of extracts using the GraphPad Prism version 8.0 software to obtain a 4PL (4-parameter logistic) written in Equation (2) as follows: a = the minimum value that can be obtained (at 0); d = the maximum value that can be obtained; c = the point of inflection; b = Hill's slope of the curve (the steepness of the cure at point c); y = the dependent variable; x = the independent variable (concentration).IC 50 (in µg/mL) is defined as the capacity to reduce the initial DPPH radical concentration by 50%.

Liquid Chromatography-Mass Spectroscopy (LC-MS) for the Total Flavonoid Content Analysis
Quercetin was used as the standard in the total flavonoid content analysis because it is an important bioactive metabolite found in numerous plants.It is categorized in the class of flavonoids and has been reported for its extensive pharmacology activities [24].Previous studies have described that the commonly used method for the analysis of quercetin is liquid chromatography, e.g., LC-MS [25,26], HPLC [27][28][29][30], and UPLC [31,32].
The quercetin standard solution was prepared by dissolving an accurately weighed 10 mg of quercetin (using a Mettler Toledo MS 204TS digital analytical balance) in 100 mL of analytical-grade ethanol.The solution was diluted to obtain a concentration of 10 µg/mL.
The extract solution was prepared by dissolving 100 mg of extract with the highest phenolic content in 100 mL of analytical-grade ethanol to obtain a concentration of 1000 µg/mL.A volume of 15 mL extract 1000 µg/mL was put into five volumetric flasks (25 mL in capacity).A variation volume of quercetin standard solution was put into the flasks to obtain quercetin concentrations of 0, 4, 8, 16, and 32 µg/mL, respectively.The solutions were filtered using Millipore membrane 0.2 µm, and each 5 µL of the solution was injected into the AcquityTM Ultra Performance LC BEH shield RP18 1.7 µm, 2.1 × 100 mm column (part no.186002352, serial no.01853023815719) embedded in a Waters AcquityTM Ultra Performance LC instrument.The mobile phase system was a mixture of phase A (water and formic acid) and phase B (methanol).The flow rate was set at 0.2 mL/minute.The mass spectroscopy system used in tandem with the LC was a Waters XevoTM Qtof MS in positive ESI ionization mode, scanned at the range of 50-1000 m/z, with the source temperature at 100 • C, the de-solvated temperature at 350 • C, and a cone N 2 gas flow of 15 L/h.The LC chromatogram and the MS spectra were compared to those of the standard quercetin.The hardware used was a MacBook Pro (13-inch, M1, 2020), macOS Ventura, with an Apple M1 processor chip and a memory of 8 GB.

Preparation of the Macromolecule
The X-ray crystallographic 3D structure of the tyrosinase enzyme was isolated from Bacillus megaterium in complex with the kojic acid inhibitor (PDB ID 3NQ1 with a resolution of 2.3 Å; DOI: https://doi.org/10.2210/pdb3NQ1/pdb,accessed on 10 March 2023) deposited by Sendovski et al. (2011) [33].The protein was downloaded from the Protein Data Bank (https://www.rcsb.org/accessed on 10 March 2023).SwissPDBViewer v.4.01 (downloaded from https://spdbv.unil.ch/accessed on 10 March 2023) was used to separate the chains and fix the macromolecule.Hydrogens were added to the macromolecule PDB, followed by the calculation of their partial charges.

Preparation of the Ligand
The 2D structures of the ligands (ethyl p-methoxycinnamate, 6-gingerol, galangin, and kaempferol) and standard ligands (arbutin, kojic acid, and hydroquinone) were built using ChemDraw 12.0 and saved in .cdxformat.The geometry optimization of the ligands was performed by applying the molecular mechanic 2 (MM2), and the optimized ligands were saved in .pdbformat [34,35].

Molecular Docking Simulation
This step was performed using AutoDock Vina by docking the four phenolic compounds and the three standards as the ligands in the catalytic site of tyrosinase.The binding affinity in terms of the docking score (kcal/mol), the hydrogen bond and the hydrophobic interactions, and the close contact residues were recorded and compared.

Phytochemical Screening, Total Phenolics, and Radical Scavenging Capacity
Phytochemical screening of the plant extracts revealed the presence of flavonoids and polyphenols in the rest of the tested plants.The total phenols and the radical scavenging activity of the plants are summarized in Table 1.The ethanol extract of the Alpinia galanga rhizome demonstrated the highest phenolic level (252.36 mg GAE/g extract) and antioxidant activity (IC 50 of 66.67 µg/mL); therefore, it was further analyzed by the LC-MS method.
Furthermore, the association between total phenolics and radical scavenging activity was analyzed using the Pearson correlation, which resulted in a moderate correlation between the two variables (r = 0.6206) (Figure 2).

LC-MS Analysis of A. galanga Extract
The LC chromatogram of A. galanga is depicted in Figure 3a and the MS spectrum of A. galanga is shown in Figure 3b.The total flavonoids calculated as quercetin in the extract were determined using a standard addition method, and the resulting curve, which is a plot between the area under the curve (AUC) and the concentration of standard quercetin, is presented in Figure 4.

LC-MS Analysis of A. galanga Extract
The LC chromatogram of A. galanga is depicted in Figure 3a and the MS spectrum of A. galanga is shown in Figure 3b.The total flavonoids calculated as quercetin in the extract were determined using a standard addition method, and the resulting curve, which is a plot between the area under the curve (AUC) and the concentration of standard quercetin, is presented in Figure 4.

LC-MS Analysis of A. galanga Extract
The LC chromatogram of A. galanga is depicted in Figure 3a and the MS spectrum of A. galanga is shown in Figure 3b.The total flavonoids calculated as quercetin in the extract were determined using a standard addition method, and the resulting curve, which is a plot between the area under the curve (AUC) and the concentration of standard quercetin, is presented in Figure 4.By using the standard addition curve, the amount of quercetin in A. galanga extract was calculated and resulted in 0.378% w/w.

Molecular Docking Simulation
A molecular docking simulation revealed that known constituents of A. galanga could occupy the catalytic site of tyrosinase and interact with essential residues.Although kaempferol showed the strongest binding affinity towards tyrosinase, as proven by its smallest docking score of −7.6 kcal/mol, galangin is considered the best ligand based on its similar binding mode to that of kojic acid.Galangin also demonstrated a strong binding affinity which is comparable with that of kaempferol.The results are summarized in Table 2.The 2D and 3D structure of galangin, the total charge density and the electrostatic potential map, and the molecular interaction of galangin with tyrosinase are presented in Figure 5. Galangin interacts with tyrosinase by building one hydrogen bond to Gly216 at a distance of 1.754 Åand hydrophobic interactions with Asn205, His208, Gly216, and Val218.Hydrophobic interaction with His208 and Gly216 was also shown by kojic acid.The green color indicates the electropositive region, the violet color is electronegative; and (c) the molecular interaction with tyrosinase residues in the catalytic site of the enzyme.The copper ion is labeled as CU501.The pink spheres indicate the hydrogen bond.
Similarly, our study reveals that the levels of phenolics in Zingiberaceae plants are
Similarly, our study reveals that the levels of phenolics in Zingiberaceae plants are highly varied, with the highest contents being found in the Alpinia galanga rhizome (252.36 mg GAE/g extract), the C. heyneana rhizome (83.69 mg GAE/g extract), the H. coronarium rhizome (31.03 mg GAE/g extract), and the C. zedoaria rhizome (17.92 mg GAE/g extract).The phenolic levels are in a moderate correlation (r = 0.6206) with the radical scavenging capacity of the plant extracts, confirming that phenolics are indicators antioxidant properties.Comparable to our results, a previous study reported that the total phenolics of C. zedoaria collected in Kanchanaburi, Thailand, in 2013 was 9.3 ± 0.7 mg GAE/g extract [40].Another study conducted in Saudi Arabia described that the phenolic contents of A. galanga, obtained from the local market in Riyadh, were 53.18 mg GAE/g extract [41], indicating a smaller amount compared to the present study.
The plant with the highest phenolic levels further had its quercetin levels determined (total flavonoids calculated as quercetin) using the liquid chromatography in tandem with mass spectrometry (LC-MS) method, which resulted in the evidence that quercetin is present in A. galanga extract, as proven by a small peak at 9.27 min (similar to standard quercetin eluted at 9.16 min) and confirmed by an MS peak at m/z 303.1593 (the molecular weight of quercetin), in an amount of 0.378% w/w.
Our result is in accordance with, although lower than, that given in a previous work of Suzery et al. (2019), who reported that quercetin in the ethyl acetate fraction of A. galanga collected from Banyumanik, Central Java, Indonesia, was 0.5469% w/w [42].Discovering plants with high levels of phenolics and strong antioxidant properties is beneficial for the development of novel drugs and cosmetics.
When free oxygen radicals are formed in the body, the enzyme superoxide dismutase (SOD) immediately catches the oxygen anions (O 2− ) and converts them to peroxides (H 2 O 2 ).The enzyme then catalyzes the degradation of H 2 O 2 to nontoxic water molecules (H 2 O) with the help of glutathiones (GSH).It was announced that quercetin, a flavonol, could stimulate the production of GSH in animal models [43][44][45].Quercetin has been widely reported to possess the capacity to give its hydrogen atoms and to extinguish the reactivity of oxygen radicals due to its phenolic hydroxyl group and double bond [46].In vitro studies confirmed that quercetin significantly reduced the levels of inflammatory mediators, e.g., NO synthase, COX-2, and C-reactive protein, in human hepatocyte-derived cell lines [47].A combination of quercetin with glutathione was described to show a synergy.The observed synergy was parallel with the glutathione content, elevating up to the 1:16 ratio and then decreasing [48].Studies in humans reported that quercetin significantly decreased plasma concentrations of atherogenic oxidized low-density lipoproteins but did not significantly alter TNF-α and C-reactive protein when compared with placebo [49].A randomized clinical trial to determine the safety of quercetin therapy in patients with chronic obstructive pulmonary disease confirmed that this flavonol is safely tolerated up to a dose of 2000 mg per day [50].Interestingly, a recently published article reported that quercetin treatment in patients with early-stage COVID-19 resulted in speedy clearance of SARS-CoV-2, faster recovery of the acute symptoms, and modulation of the host's hyperinflammatory response [51].However, despite the advantages of quercetin in ameliorating COVID-19 infection and symptoms, it was thought that further clinical trials are needed to strongly establish its clinical efficacy against COVID-19 [52].
Our molecular docking simulation revealed that the phytoconstituents of A. galanga could settle in the catalytic site of tyrosinase by interacting with essential residues.Although kaempferol showed the strongest binding affinity towards tyrosinase, as proven by its smallest docking score of −7.6 kcal/mol, galangin (docking score of −7.1 kcal/mol) is considered the best ligand based on its similar binding mode to that of kojic acid.Galangin also demonstrated a strong binding affinity, which is comparable with that of kaempferol.
Numerous flavonols have been reported for their activity in inhibiting tyrosinase by competitively blocking the oxidation of the substrate L-DOPA (dihydroxyphenylalanine) into dopachrome and melanin by tyrosinase.This inhibitory activity is performed by chelating the copper in the enzyme's catalytic site.The order of the flavonols with respect to their inhibitory activity toward tyrosinase, from the highest to the lowest, is quercetin > myricetin > kaempferol > galangin.However, compared to kojic acid, the inhibitory activity of these flavonols is weaker [53].Kojic acid, a strong whitening ingredient in cosmetics, is a slow-binding inhibitor of tyrosinase in its active form [54]. Kojic acid, co-crystallized in tyrosinase by Sendovski and co-workers [33], binds to the catalytic site of the enzyme by building hydrophobic interactions with Asn205 and Gly196 (Figure 6). to their inhibitory activity toward tyrosinase, from the highest to the lowest, is quercetin > myricetin > kaempferol > galangin.However, compared to kojic acid, the inhibitory activity of these flavonols is weaker [53].Kojic acid, a strong whitening ingredient in cosmetics, is a slow-binding inhibitor of tyrosinase in its active form [54]. Kojic acid, cocrystallized in tyrosinase by Sendovski and co-workers [33], binds to the catalytic site of the enzyme by building hydrophobic interactions with Asn205 and Gly196 (Figure 6).Tyrosinase is a metalloenzyme containing copper ions in its catalytic site, which functions as a rate-limiting enzyme in melanin biosynthesis.The catalytic role of this enzyme comprises (1) the monophenolase activity, where it hydroxylates the monophenols to orto-diphenols, and (2) the diphenolase activity, where tyrosinase oxidizes orto-diphenols to orto-quinones [55].According to our molecular docking simulation, kojic acid does not interact with Asn205, though hydrophobic interactions with His204 (3.7 Å ), His208 (3.6-3.9Å ), and Gly216 (3.8 Å ) were observed.Interestingly, our tested phenolic compound, namely, galangin, also builds hydrophobic interactions with Asn205 (3.6 Å ), His208 (3.9 Å ), Gly216 (3.9 Å ), and Val218 (3.6-3.7 Å ) and one hydrogen bond with Gly216 (1.754 Ȧ), as shown in Figure 5 and summarized in Table 2.

Conclusions
The present study confirms that phenolic compounds and/or flavonols are the main contributors to the radical scavenging capacity of Zingiberaceae plants.The limitation of this study is that the number of plants assayed may not represent the whole plant family, thus opening the chance for the researchers to further investigate medicinal plants belonging to the Zingiberaceae family.It should be noted that, of the four assayed plants, the ethanol extract of the Alpinia galanga rhizome demonstrated the highest phenolic level (252.36 mg GAE/g extract) and a moderate radical scavenging activity (IC50 of 66.67 µg/mL).Quercetin is present in this plant extract in a low amount.Moreover, the phytoconstituents of A. galanga, e.g., kaempferol, galangin, ethyl p-methoxycinnamate, and 6-gingerol, could occupy the binding site of tyrosinase with prominent affinity and Tyrosinase is a metalloenzyme containing copper ions in its catalytic site, which functions as a rate-limiting enzyme in melanin biosynthesis.The catalytic role of this enzyme comprises (1) the monophenolase activity, where it hydroxylates the monophenols to orto-diphenols, and (2) the diphenolase activity, where tyrosinase oxidizes orto-diphenols to orto-quinones [55].According to our molecular docking simulation, kojic acid does not interact with Asn205, though hydrophobic interactions with His204 (3.7 Å), His208 (3.6-3.9Å), and Gly216 (3.8 Å) were observed.Interestingly, our tested phenolic compound, namely, galangin, also builds hydrophobic interactions with Asn205 (3.6 Å), His208 (3.9 Å), Gly216 (3.9 Å), and Val218 (3.6-3.7 Å) and one hydrogen bond with Gly216 (1.754 Å), as shown in Figure 5 and summarized in Table 2.

Conclusions
The present study confirms that phenolic compounds and/or flavonols are the main contributors to the radical scavenging capacity of Zingiberaceae plants.The limitation of this study is that the number of plants assayed may not represent the whole plant family, thus opening the chance for the researchers to further investigate medicinal plants belonging to the Zingiberaceae family.It should be noted that, of the four assayed plants, the ethanol extract of the Alpinia galanga rhizome demonstrated the highest phenolic

Figure 1 .
Figure 1.Two-dimensional chemical structure of quercetin (downloaded from http://www.chemspider.com/with ChemSpider ID 12269344; accessed on 10 August 2023).The C2=C3 double bond is indicated by a curved arrow.Quercetin belongs to the flavonoid class with a general structure of a 15-carbon skeleton, comprised of two phenyl rings (A and B) and a heterocyclic ring (C).

Figure 1 .
Figure 1.Two-dimensional chemical structure of quercetin (downloaded from http://www.chemspider.com/ with ChemSpider ID 12269344; accessed on 10 August 2023).The C2=C3 double bond is indicated by a curved arrow.Quercetin belongs to the flavonoid class with a general structure of a 15-carbon skeleton, comprised of two phenyl rings (A and B) and a heterocyclic ring (C).

Figure 2 .
Figure 2. Pearson correlation analysis graph of the association between total phenolics and radical scavenging activity, resulting in a correlation coefficient of r = 0.6206.The blue dots indicate the data of the two variables (IC50 value and total phenolics)

Figure 2 .
Figure 2. Pearson correlation analysis graph of the association between total phenolics and radical scavenging activity, resulting in a correlation coefficient of r = 0.6206.The blue dots indicate the data of the two variables (IC 50 value and total phenolics).

Figure 2 .
Figure 2. Pearson correlation analysis graph of the association between total phenolics and radical scavenging activity, resulting in a correlation coefficient of r = 0.6206.The blue dots indicate the data of the two variables (IC50 value and total phenolics)

Figure 3 .Figure 3 .Figure 3 .
Figure 3. (a) LC chromatogram and (b) MS spectrum of A. galanga rhizome extract.Quercetin was confirmed to be present in the A. galanga extract, as shown by a small peak at 9.27 min (similar to standard quercetin eluted at 9.16 min) and an MS peak at m/z 303.1593.

Figure 4 .Figure 4 .
Figure 4.The standard addition curve of quercetin spiked to the Alpinia galanga rhizome extract resulted in a linear regression equation of y = 2.5013x + 3.4161, with a coefficient of correlation R 2 of 0.9901.By using the standard addition curve, the amount of quercetin in A. galanga extract was calculated and resulted in 0.378% w/w.

Figure 5 .
Figure 5. Galangin with a molecular weight of 270.24, cLog P of 2.37, and volume of 718.24 Å 3 : (a) the 2D and 3D structure; (b) the total charge density (left) and the electrostatic potential map (right).The green color indicates the electropositive region, the violet color is electronegative; and (c) the molecular interaction with tyrosinase residues in the catalytic site of the enzyme.The copper ion is labeled as CU501.The pink spheres indicate the hydrogen bond.

1 Figure 5 .
Figure 5. Galangin with a molecular weight of 270.24, cLog P of 2.37, and volume of 718.24 Å 3 : (a) the 2D and 3D structure; (b) the total charge density (left) and the electrostatic potential map (right).The green color indicates the electropositive region, the violet color is the electronegative; and (c) the molecular interaction with tyrosinase residues in the catalytic site of the enzyme.The copper ion is labeled as CU501.The pink spheres indicate the hydrogen bond.

Table 1 .
Total phenolics and radical scavenging capacity of the Zingiberaceae plants.

Linear Regression Equation Obtained from the Calibration Graph) DPPH Radical Scavenging Capacity Presented as an IC 50 Value in µg
Cosmetics 2023, 10, x FOR PEER REVIEW 7 of 15

Table 2 .
Molecular docking simulation of kaempferol, galangin, ethyl p-methoxy cinnamate, and 6-gingerol with residues in the catalytic site of tyrosinase.Arbutin, kojic acid, and hydroquinone were employed as the standards.