INTRODUCTION:

Melanin is a pigment found in the skin, hair, choroid layer of the eye, and tumor cells. Eumelanin (brown-black pigment) and pheomelanin (yellow-red pigment) are the two types of melanin pigments that are produced in melanocyte cells¹.

Under normal conditions, melanin production is stable; however, melanin production can change due to exposure to sunlight, hormonal changes, or the influence of cigarettes and alcohol. Uncontrolled melanin production can also lead to melanoma or skin cancer². Antioxidants, tyrosinase enzyme inhibitors, and hormonal activity are just a few of the mechanisms that can slow down the melanin-formation process (also known as melanogenesis) in the human body³. This appropriate inhibition of melanogenesis can be beneficial both for the prevention of skin tan and for the prevention of skin cancer^4,5. Tyrosinase inhibition can occur via two molecular oxygen-based mechanisms: the hydroxylation reaction of L-tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA), known as monophenolase, and the oxidation reaction of L-DOPA to DOPAquinone, known as diphenolase^6.

There are several synthetic compounds that have been shown inhibitory effect on the enzyme, such as mercury, kojic acid, hydroquinone, and arbutin. However, in long-term use, these synthetic compounds have dangerous side effects. Therefore, tyrosinase inhibitory from natural product are needed because the safety and wide range bioactivity^7,8. Marine natural products that can be used as the source of tyrosinase inhibitors is Padina sp^9.

Padina sp is an abundance brown seaweed in Indonesian oceans (about 30%) but has not been used optimally¹⁰. Padina sp composition is dominated by ash content of about 30-48%, carbohydrates of about 25-39%, total dietary fiber of about 27-39% on a dry basis, and a small amount of protein and lipid of about 5-7 and 1.6-1.8%, respectively¹¹. Padina sp has been reported to contain 19 types of terpenes and 5 types of sterols¹². Padina sp also contains phlorotannin compounds. The presence of these compounds in natural products can play a role in protecting the skin from free radical damage and UV exposure, so it can inhibit the formation of melanin¹³. Free radicals, unstable oxygen molecules that degrade skin cells and contribute to wrinkles, are also neutralized by the substance, which also protects cells from cellular damage¹⁴. This study aimed to evaluate the antioxidant activity and tyrosinase inhibitory monophenolase and diphenolase activity extracts of Padina sp both in vitro and in silico analysis.

MATERIALS AND METHODS:

Materials:

The solvents used for phytochemical extraction were n-hexane, ethyl acetate, and methanol with technical grade purchased from local suppliers. The solvents were purified first through a distillation before use. The phytochemical assay used FeCl_3,Pb(CH₃COO)₂, Wagner, Meyer, Dragendorf and Lieberman Burchard reagent. The in vitro analysis used DPPH (Merck), methanol, tyrosinase enzyme, enzyme substrates (L-tyrosine and L-DOPA), and kojic acid purchased from Sigma-Aldrich.

Methods:

a. Sample preparation and extraction:

Padina sp (taken from Selayar Island, South Sulawesi) cleaned and rinsed with running water. Then dried and grind to obtain Padina sp powder. The sample then extracted by graded maceration technique using
three different solvents, namely n-Hx, Et-OAc, and Me-OH each with 5-15 times of remaceration. Then the extract was filtered and the evaporated using a rotary evaporator to obtain dry/thick extracts.

b. Phytochemical screening:

Phytochemical screening was carried out to determine the presence of secondary metabolites such as alkaloids, flavonoids, terpenoids, phenolics, and saponins. Extracts of n-Hx, Et-OAc, and Me-OH were tested for phytochemicals using the modified Harborne standard method^15.

(i) Alkaloids Test. A sample of 1mL was put into 2 different tubes. Tube (I) was added a few drops of Dragendorff's reagent (a positive test result was indicated by the appearance of an orange/red/yellow precipitate). Tube (II) was added a few drops of Mayer's reagent (a positive test result was indicated by the appearance of a yellowish cream-colored precipitate).

(ii) Flavanoids Test. A sample of 1mL was put into 2 different tubes. Tube (I) added a few drops of 1% Pb(CH₃COO)₂ solution (a positive test result was indicated by the appearance of a yellow precipitate). Tube (II) was added a little Mg powder (the tip of a horn spoon) then added a few drops of concentrated HCl then shaken vigorously and allowed to stand for 2minutes (positive test results are indicated by a red color change).

(iii) Terpenoids Test (Triterpenoids or Steroids). A sample of 1mL was added with 0.5mL of Lieberman Burchard's reagent and allowed to stand for 5minutes (a positive test result is indicated by a red/purple color change for triterpenoids and a green/blue color change for steroids).

(iv) Saponins Test. A sample of 1mL was added with 0.5mL of distilled water and then shaken vigorously (a positive test result is indicated by the appearance of foam/foam that lasts more than 30seconds).

(v) Phenolics Test. A sample of 1mL was added to 0.5 mL of distilled water and then a few drops of 1% FeCl₃ were added (a positive test result was indicated by the appearance of a red/purple/green/blue/black precipitate).

c. DPPH method of antioxidant activity test:

The antioxidant activity was carried out using a slightly modified of Brand-Williams et al method. The sample was prepared to obtain a concentration of 500ppm. Then transfer into different test tubes as much as 0.025; 0.05; 0.1; 0.2; and 0.4mL. The sample was then added 1mL of 0.4 mM DPPH and made up to 5mL with methanol to get the final concentrations of 2.5; 5.0; 10.0; 20.0; and 40.0 ppm. Following that, the mixture was left to stand at room temperature for 30 minutes. The absorbance was then measured with a spectrophotometer at 515nm¹⁶.

d. Tyrosinase inhibitor activity test:

Test of tyrosinase inhibitor activity was carried out using the method of Dolorossa et al¹⁷. This test consists of two tests using different substrates, namely L-tyrosine substrate for monophenolase test and L-DOPA substrate for diphenolase test. Test samples (concentration series 62.5; 125; 250; 500; and 1000ppm) of 70μL each were added with 30μL of tyrosinase enzyme, then incubated at room temperature for 5minutes. The mixture was added 110μL of substrate (2mM L-tyrosine or 12mM L-DOPA) into each well of the multi-well plate, incubated for 30minutes at room temperature. The mixture was measured using a multi-well plate reader at 475nm. Kojic acid (concentration series 2.5; 5; 10; 20; and 40 ppm) was used as a positive control.

e. GC-MS analysis:

The promising extract was then analyzed for its phytochemical. The phytochemical identified by Shimadzu GC-MS 2010 Mass Spectrometry plus and NIST and WILEY 9 mass spectra library as structure reference. The column used was SH-Rxi-5Sil MS (30m x 0.25mm) with a flame ionization detector (FID). The splitless mode was used to inject the sample with a temperature of 250^oC, a pressure of 76.9 kPa, and a flow rate of 14mL. min^-1, generally the method refers to Bahrun et al¹⁸.

f. Molecular Docking:

The tyrosinase protein (PDB ID: 4P6S) obtained from RCSB PDB (https://www.rcsb.org/) was subjected to a molecular docking analysis¹⁹. The protein was isolated from the native ligand and charged with polar hydrogen atoms and a Kollman charger. Furthermore, native ligand redocking was performed to validate the method. Docking was accomplished using a grid size of 14 x 20 x 24 Å, spacing at 0.375 Å, and coordinate central grid point at -22.798, 8.306, -8.783. The docking parameters were determined by the Genetic Algorithm (GA) with the population size: 300, number of runs: 50, and the output of Lamarckian GA 4.2²⁰. All of these steps were completed with AutoDock4 with the help of Auto Dock Tools 1.5.6²¹. Ligand-protein interactions were visualized with the Discovery Studio Visualizer. The phytochemical obtained from the GC-MS analysis was used as the test ligand and the kojic acid compound to compare.

RESULT AND DISCUSSION:

Sample preparation, extraction, and phytochemical screening:

The sample was prepared by washing with running water and then by rinsing with distilled water to discard impurities such as salt, sand, and other macro-contaminants, then dried so that the sample was easier to grind. Grinded samples will increase surface area and maximize the extraction. The extraction method used was gradient maceration so that the compounds separated based on the difference in polarity. Maceration was carried out using n-Hx (5 x 24hours of remaceration), Et-OAc (15 x 24 hours of remaceration), then Me-OH (10 x 24 hours of remaceration). The n-Hx, Et-OAc, and Me-OH macerates were dried under vacuum evaporation to obtain thick or dry extracts. Then calculated the yield produced in each extract and obtained a total yield of about 5.50%. The yield of each extract is shown in Table 1. The result indicated that the semipolar compound dominated the sample.

The extracts were tested phytochemically to identify the class of secondary metabolites such as alkaloids, flavonoids, terpenoids/steroids, saponins, and phenolics. The phytochemical of each extract was shown in Table 1. The alkaloids test was carried out using Dragendorff's reagent with positive results indicated by the formation of an orange precipitate. The flavonoids test was carried out using Pb(CH₃COO)₂ reagent with positive results indicated by the formation of a white precipitate. The terpenoid/steroid test was carried out using Liebermann-Bourchard reagent with positive results indicated by the formation of a red-purple (triterpenoid) or blue-green (steroid) solution. The saponins test was carried out by adding some distilled water to the extract and then shaking it vigorously with positive results indicated by the formation of foam that could last 30 seconds. Phenolics test was carried out using FeCl₃ reagent with positive results indicated by the formation of green/black precipitate¹⁵.

Table 1: The results of phytochemical screening and yield of Padina sp extracts

Secondary metabolites	Result
Secondary metabolites	n-Hx	Et-OAc	Me-OH
Phenolics	+	+	+
Flavonoids	+	+	+
Alkaloids	+	+	-
Saponins	-	+	+
Steroids	+	+	-
Yield	0.05%	4.45%	1.00%

Antioxidant activity test:

An antioxidant is a molecule that can prevent other molecules from oxidizing²² and plays a major role in maintaining of immune system²³. Studying antioxidants in drug discovery produces a promising medical revolution in a new health and disease management era^24,25. Antioxidant activity test of Padina sp extract carried out using the DPPH method. DPPH is an easy, rapid, and sensitive method for the antioxidant screening of plant extract compared to other methods^26,27. DPPH was a hydrogen or free electron acceptor from the substrate or antioxidant compounds in the sample to become a stable diamagnetic molecule. The stable form of DPPH was characterized by the colorimetric method (color change from purple to pale yellow)^28,29. The antioxidant activity expressed by the IC₅₀ value is shown in Table 2. The IC₅₀ indicates the concentration of a sample to inhibit 50% activity of free radicals. The lower the IC₅₀ value of a sample extract or compound, the stronger the antioxidant activity.

Table 2: Antioxidant of Padina sp extract

Sample	IC₅₀ (µg/mL)
n-Hx	135.01
Et-OAc	104.71
Me-OH	120.54
Vit. C	2.07

The order of IC₅₀ values from lowest to highest was Et-OAc then Me-OH and n-Hx. This shows that the Et-OAc extract has better antioxidant activity than Me-OH and n-Hx. These data were supported by phytochemical test data. This was in accordance with the research of Huyut et al (2017) who said that the presence of phenolic compounds and flavonoids was directly proportional to the antioxidant activity^30,31. IC₅₀ value of antioxidant extract of n-Hx, Et-OAc and Me-OH Padina sp belonging to the same category of antioxidants, namely moderate category antioxidants. Akbar et al (2021) reported antioxidant activity using the ABTS method, which was similar to the findings in this study³².

Monophenolase tyrosinase inhibitor activity test:

Tyrosinase monophenolase inhibitory activity was carried out using L-tyrosine as a substrate. The reaction was the hydroxylation of L-tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA) with the help of the tyrosinase enzyme. However, in the presence of compounds that were inhibitors of these enzymes, the production of L-DOPA was inhibited/reduced. The results of the inhibition test were indicated by the IC₅₀ value of monophenolase. This value describes the concentration of certain extracts or compounds needed to inhibit 50% of the tyrosinase enzyme activity. The lower the IC₅₀ value of a sample extract or compound, the stronger/better the inhibitory activity. The IC₅₀ value of Padina sp extract monophenolase seen in Table 3.

Table 3: The IC₅₀ value monophenolase of Padina sp extract

Sample	IC₅₀ (µg/mL)
n-Hx	937.68
Et-OAc	132.92
Me-OH	268.68
Kojic Acid	20.99

The order of IC₅₀ monophenolase values of Padina sp extracts from lowest to highest was Et-OAc then Me-OH and n-Hx, the inhibitory activity all of the extracts were categorized as intermediate activity (IC₅₀ 100-450 µg/mL). This shows that the Et-OAc extract has a better inhibitory activity than Me-OH and n-Hx. These data were supported by the antioxidant activity which has the same pattern as this monophenolase tyrosinase inhibitor test. Therefore, it can be concluded that the extract or certain compounds that have antioxidant activity correlate with the activity of the tyrosinase monophenolase inhibitor, which was directly proportional. The IC₅₀ value of Kojic acid (positive control) was lower than the Et-OAc extract of Padina sp. This was because the condition of the sample Padina sp tested was still in the form of crude extract and has not been further purified. However, the extract of Padina sp proven to be potential as a tyrosinase inhibitor through the monophenolase mechanism.

Diphenolase tyrosinase inhibitor activity test:

Diphenolase tyrosinase inhibitor activity test was carried out using L-DOPA as a substrate. The reaction that occurs in the test was the oxidation of L-DOPA to DOPAquinone with the help of the tyrosinase enzyme. However, in the presence of compounds that were inhibitors of these enzymes, the production of DOPAquinone was inhibited/reduced. The results of the inhibition test were indicated by the IC₅₀ value of diphenolase. This value describes the concentration of certain extracts or compounds required to inhibit 50% of the tyrosinase enzyme activity. The lower the IC₅₀ value of a sample extract or compound, the stronger/better the inhibitory activity. The IC₅₀ value diphenolase of Padina sp extract seen in Table 4.

Table 4: The IC₅₀value diphenolase of Padina sp extract

Sample	IC₅₀ (µg/mL)
n-Hx	989.73
Et-OAc	178.33
Me-OH	356.86
Kojic Acid	31.76

Diphenolase activity of Et-OAc and Me-OH extracts of Padina sp had intermediate activity (IC₅₀ 100-450 µg/mL) while n-Hx extract has weak activity (IC₅₀451-700µg/mL). Based on the IC₅₀value (Table 4), Et-OAc extract has a better inhibitory activity than Me-OH and n-Hx. These data were supported by antioxidant test data which has the same pattern as the diphenolase tyrosinase inhibitor test. The IC₅₀ value of Kojic acid (positive control) was categorized as strong activity (<100 µg/mL) as expected compare to Padina sp extract. This was because the condition of the sample Padina sp tested was still in the form of crude extract and has not been further purified. However, the extract of Padina sp proven to be a potential tyrosinase inhibitor through the diphenolase mechanism. Based on the test results of the tyrosinase inhibitor activity of monophenolase and diphenolase, it was seen that each extract and Kojic acid as a positive control had more active inhibitory activity against monophenolase. This is indicated by the IC₅₀ value of monophenolase for each extract and kojic acid is lower than the IC₅₀ value of diphenolase. But overall, it can be concluded that the extract of Padina sp proven to have potential as a tyrosinase inhibitor either through monophenolase or diphenolase mechanisms.

Previously, the ethanolic extract of Padina pavonica was inactive at 200mg/mL for tyrosinase inhibition³³. Padina australis ethanolic extract also has an IC₅₀ of 1.09±0.03mg/Ml³⁴, whereas methanolic extracts of Padina australis at 500mg/mL only have inhibitory activity 2.05±0.13%³⁵. Padina arborescens aqueous extract also demonstrated a low percent inhibitory activity³⁶. Padina australis, Padina boergesenii, Padina distromatica, and Padina tetrastromatica inhibitory effects at 500g/mL were 12.63±0.82, 51.75±0.69, 10.02±0.54, and 18.48±1.24%, respectively³⁷. Preliminary research found that an ethanol extract of Padina boryana has the potential to inhibit tyrosinase activity³⁸. Based on the description of the previous research, the tyrosinase inhibitory was quite diverse and, of course, strongly influenced by the composition of the extract being analyzed. To figure out the tyrosinase inhibition, one of the existing pathways that have been proposed was the inhibitor complex of tyrosinase lead to conformational changes of the 3D structures^39,40

GC-MS analysis:

Figure 1: GC-MS spectra of Et-OAc extract

GC-MS was a technique used to analyze thermally stable, volatile, and lipophilic or nonpolar organic compounds⁴¹. This method used to reveal the phytochemical of the most promising extract based on in vitro analysis, in this case, Et-OAc extract. Analysis of the phytochemical content of the Et-OAc extract with the GC-MS instrument showed 30 peaks representing each compound in the extract, as shown in Figure 1. A total of 6 compounds representing 67.59% of the extract composition were listed in Table 5. Previous research shows these compounds have been scientifically proven to have a wide range of bioactivities.

Molecular Docking

The validation of the docking method by superimposing the native ligands before and after redocking as the first stage in this analysis obtained an RMSD of less than 2 Å (1.57 Å). These findings indicate that the method was valid. The comparison of the poses of these ligands is shown in Figure 2.

Figure 2: Superimpose native ligan before (grey) and after redocking (blue)

Table 5: Main phytochemicals of Et-OAc extract, bioactivity and binding energy

Ligand	PubChem ID	Bioactivity	Binding Energy (kcal/mol)
Spiro(tetrahydrofuryl)2.1'(decalin, 5',5',8'a-trimethyl (1)	566475	-	-6.86
Tetradecanoic acid (2)	11005	Antiurease, antielastase and antioxidant activities⁴²	-3.39
Neophytadiene (3)	10446	Anti-inflammatory, antioxidant, cardioprotective⁴³ and tyrosinase inhibitor⁴⁴	-4.12
Hexadecanoic acid, methyl ester (4)	8181	Anticancer⁴⁵	-3.25
n-Hexadecanoic acid (5)	985	Antioxidant and tyrosinase inhibitor⁴⁶	-3.10
Hexadecanoic acid, ethyl ester (6)	12366	Anti-infammatory⁴⁷	-2.97
Kojic acid* (Standard Compound)	3840	Tyrosinase Inhibitor⁴⁸	-3.73

CONCLUSION:

The results showed that the total yield of the three extracts was 5.50%. The three extracts had moderate antioxidant activity. The IC₅₀ values monophenolase of n-Hx, Et-OAc, Me-OH, and kojic acid extracts were 937.6757; 132,9197; 268.6807; and 20.9959μg/mL. The IC₅₀ values diphenolase of n-Hx, Et-OAc, Me-OH extracts, and Kojic acid were 989.7349; 178.3265; 356,8647; and 31.7596μg/mL. The bioactivity was supported by the presence of phytochemical identified, which has a wide range of bioactivity. The molecular docking also supported this study, the compound Spiro(tetrahydrofuryl)2.1'(decalin), 5',5',8'a-trimethyl has lower binding energy than Kojic acid. Extract of Padina sp proven to have potential as a tyrosinase inhibitor either through monophenolase or diphenolase mechanisms so that it can be further developed as a bioactive compound in cosmetics for skin brightening agent.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this study.

ACKNOWLEDGMENTS:

Thank you to the Ministry of Research, Technology, and Higher Education of the Republic of Indonesia (Ristekdikti RI) for funding this research through the PMDSU scheme. Thanks also to Hasanuddin University for facilitating this research through the Institute for Research and Community Service.

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Received on 13.03.2023 Modified on 04.07.2023

Research J. Pharm. and Tech 2024; 17(3):1173-1180.

DOI: 10.52711/0974-360X.2024.00182

Andi Akbar1, Herlina Rasyid2, Hasnah Natsir2, Bahrun1, Nunuk Hariani Soekamto2*