The skin is the outermost part of the human body that plays a vital role in body protection. One of the functions of the skin is to protect the body from UV rays1. Excessive ultraviolet (UV) light exposure increases the contribution of free radicals known as reactive oxygen species (ROS)2. Reactive oxygen species increase pigmentation and cause oxidative stress-induced damage to the melanocytes3. Hyperpigmentation disorders are diseases in which patches of skin are darker than the normal surrounding skin, resulting from the upregulated activity of melanin synthesis, increased numbers of melanocytes, and decreased decomposition of melanosomes4. Hyperpigmentation conditions can also be caused by certain drugs, hormonal changes, or autoimmune conditions5.

The enzyme that plays an essential role in the melanin synthesis pathway is tyrosinase. Tyrosinase has the activity of tyrosine hydroxylation, oxidation of L-DOPA (3,4-dihydroxyphenylalanine), and oxidation of hydroxyindole6. In the process of melanogenesis, tyrosinase acts as a catalyst in two different reactions, the hydroxylation of tyrosine to L-DOPA and the oxidation of L-DOPA to dopaquinone7. Dopaquinone is a highly reactive compound that can spontaneously polarize to form melanin8.

Bleach acts as an inhibitor of melanin production and is a competitive tyrosinase inhibitor. Various tyrosinase inhibitors are found in cosmetic ingredients to prevent hyperpigmentation, including hyaluronic acid, arbutin, kojic acid, mercury, and hydroquinone9. This compound has immense whitening power despite having severe side effects such as carcinogenesis, hepatotoxicity, and dermatitis10. Hydroquinone not only inhibits tyrosinase activity and destroys melanosomes but also causes necrosis of melanocytes by modifying the membrane structure11. This is the potential mechanism of action of hydroquinone as a skin-lightening agent and its toxicity mechanism. The use of this ingredient in cosmetics has been banned since 2001 because of the high risk of carcinogenesis in case of prolonged exposure to hydroquinone12.

Several researchers prefer to identify inhibitors from natural sources due to their less toxicity and better bioavailability, especially for food, cosmetic and medicinal applications13. The class of flavonoid compounds that have tyrosinase inhibitory activity is quercetin from the flavonol group. Flavonoid compounds have tyrosinase inhibitor and chelating activity Cu, where the hydroxyl groups on the A and rings B inhibit the action of tyrosinase14. Besides flavonoids, other polyphenols, also known as tyrosinase inhibitors, include coumarin, stilbenes derivatives15, terpenoid derivatives16, and lignans17.

In previous studies, the antioxidant activity test of Melaleuca leucadendron L. leaves was extracted with methanol and then fractionated with chloroform and butanol. The total phenolic that has been carried out in previous studies was 289.23 ± 5.21 μg GAE/g in methanol extract, 107.36 ± 1.88 μg GAE/g in chloroform extract, and 508.43 ± 2.33 μg GAE/g in butanol extract. While the IC50­ obtained in the M. leucadendron extract as an antioxidant was 14.5 µg/mL in methanol extract, 50.3 µg/mL in chloroform extract, and 10.1 µg/mL in butanol extract18. Based on previous studies, the tyrosinase inhibition test on M. leucadendron leaves has never been carried out. This test is necessary because a high total phenolic and a low IC50 value of antioxidants can act as anti-tyrosinase. We hope this research can contribute to developing new safe, efficient anti-tyrosinase agents to prevent hyperpigmentation disorders.

Materials

Melaleuca leucadendron dry leaves were collected from Balai Penelitian Tanaman Rempah dan Obat (Balitro), which has been identified at the Botanical Garden Plant Conservation Center, Lembaga Ilmu Pengetahuan Indonesia (LIPI), number of the certificate B-1222/IPH3/KS/X/2020 (Figure 1). Chemical reagents such as methanol 75%, L-DOPA (Sigma), tyrosinase (Sigma), kojic acid (Sigma), chloroform, butanol, distilled water, HCl 2 N, Dragendorff reagent, Mayer reagent, Wagner reagent, Bouchardat reagent, HCl, NaNO2 5%, AlCl3 10%, NaOH 1 N, FeCl3 1%, NaOH 2 N, ether, H2SO4, potassium dihydrogen phosphate, dimethyl sulfoxide (DMSO) (Merck), and phosphate buffer (pH 6.5). At the same time, the equipment used includes a digital analytical scale, rotary vacuum evaporator, multi-well plate reader (ELISA), multilevel fractionation device, pH meter, and incubator.

a b

Figure1. Melaleuca leucadendron leaves (a) simplicia powder of M. leucadendron leaves (b).

Methods

Extraction and fractination

Melaleuca leucadendron dry leaves were ground to obtain 500 g of sample powder for extraction. Methanol 75% (5 L) was used as the solvent in the maceration extraction of the samples for 3 x 24 hours. The crude methanol extracts were then dried using a rotary evaporator. Liquid-liquid fractionation was conducted using distilled water, butanol, and chloroform to the methanol extract to obtain fractions with different polarities, then dried using a rotary evaporator.

Phytochemical screening

Extracts and three fractions were carried out in a phytochemical screening test to identify alkaloids, flavonoids, tannins, saponins, steroids/triterpenoids using the classical method19.

Tyrosinase inhibitory assay

Tyrosinase inhibitory activity was evaluated based on inhibition of the sample (diluted in DMSO) to diphenolase activity. The assay was carried out using an ELISA plate well reader with tyrosinase enzyme, L-DOPA as the substrates, phosphate buffer pH 6.5, with three repetitions20. Kojic acid was used as a positive control. The following Equation 1 can calculate the percentage of tyrosinase inhibitory activity:

Inhibition (%) = ] x 100% … [1]

In which,

A: Absorbance of the sample

B: Absorbance of blank

C: Absorbance of sampel control

D: Absorbance of blank control

The IC50 value can be calculated using a linear regression equation, sample concentration (x-axis), and %-inhibition (y-axis). From the equation y = a ln (x) + b, the IC50 value can be calculated using the Equation 2.

ln IC50 = … [2]

The yields of the extracts and their respective fractions are presented in Table I. The extraction method is maceration because the equipment used is simple and easy. Methanol 75% was used as a solvent because it can attract the highest phenolic compounds and has a low antioxidant IC50 value18. During maceration, stirring is carried out so that the pollen liquid penetrates the cell wall and enters the cell cavity containing the active substance. The difference in concentration between the solution inside and outside the cell causes a more concentrated solution to be pushed out so that the metabolite compound can be extracted entirely21.

Based on the results of phytochemical screening obtained on M. leucadendron leaves extract containing flavonoids, saponins, tannins, and steroids/triterpenoids and negative results in the alkaloid test (Table II). Previous research22 showed that M. leucadendron's methanol fraction contains alkaloid compounds, flavonoids, saponins, tannins, steroids, and triterpenoids. The difference in results obtained is due to the use of hexane solvent when maceration. The water fraction shows negative results in the steroid/triterpenoid test; this is because terpenoids can be extracted using non-polar solvents (ether, hexane, chloroform), while in the form of glycosides (generally from triterpenes) the solubility is more remarkable in polar solvents (ethanol, methanol).

TableI. The yield of the extract and fraction of M. leucadendron leaves

TableII. Phytochemical screening test results of M. leucadendron leaves

ME: methanol extract; BF: butanol fraction; CF: chloroform fraction; WF: water fraction

The activity of tyrosinase inhibitors is one of the parameters of skin lightening agents. The mechanism of tyrosinase inhibitors is to decrease skin pigmentation by inhibiting the catalytic of the enzyme to the pigmentation associated with melanin production in the melanogenesis pathway23. Kojic acid is used as a positive control because it is one of the tyrosinase inhibitors used as a cosmetic ingredient. It is a skin protector from the ultraviolet sun and can whiten the skin. Kojic acid prevents the formation of melanin in human melanocytes due to the reversible inhibition of tyrosinase, but it has some side effects, such as skin irritability and instability24.

The butanol fraction had the highest tyrosinase inhibition value (94.737±0.767%) at 1000 μg/mL concentration. However, kojic acid as a positive control show better inhibition at a concentration of 500 μg/mL with tyrosinase inhibition value of 91.155±0.228%. Measurement of the IC50 value was carried out on methanol extract, butanol fraction, water fraction, and kojic acid, while the chloroform fraction was not carried out because of the low percentage value of inhibition at a concentration of 1000 μg/mL (Table III).

TableIII. Tyrosinase inhibitory of M. leucadendron leaves

Values are expressed as mean ± SD of triplicate measurements

The IC50 values ​​of tyrosinase inhibition of all samples are presented in Table IV. Butanol fraction had the lowest IC50 value with 517.94 μg/mL. Nevertheless, kojic acid as a positive control was more potent with IC50 50.06 μg/mL. The butanol fraction is more active than other extracts and fractions, while in previous research18, the total phenolic content was reported to be more significant, 508.43±2.33 μg GAE/g extract, and the antioxidant IC50 value of 4.8 μg/mL. From the research results, it can be seen that there is a correlation between total phenolic and antioxidant activity with tyrosinase activity.

TableIV. IC50 of tyrosinase inhibitory of M. leucadendron leaves

Values are expressed as mean ± SD of triplicate measurements

Inhibition of the tyrosinase enzyme is shown through the IC50 value from methanol extract, butanol fraction, and water fraction was 645.44 μg/mL, 517.94 μg/mL, 669.40 μg/mL, respectively. As a positive control, the IC50 value of kojic acid was 50.06 μg/mL. Phytochemical screening showed that the extract and fraction of M. leucadendron leaves contained tannins, flavonoids, saponins, terpenes, and steroids. These results indicate that the butanol fraction of M. leucadendron leaves is the most potent anti-tyrosinase agent compared to the others.

The authors are thankful to acknowledge the Ministry of Research, Technology, and Higher Education, Republic of Indonesia, for support via Research Grant (26/E1/KPT/2020).

Munawarohthus Sholikha: conceptualization, methodology, get a grant for funding, supervised the experimental works, writing and review. Ainun Wulandari: contributed in the experimental works and assisted for manuscript writing.

None.

The authors declare there is no conflict of interest.

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