Constituents with tyrosinase inhibitory activities from branches of Ficus erecta var. sieboldii King

Abstract

Phytochemical investigation of the branches of Ficus erecta var. sieboldii King resulted in the isolation of eight constituents: p-hydroxybenzoic acid (1), methyl p-hydroxybenzoate (2), vanillic acid (3), methyl vanillate (4), syringic acid (5), β-sitosterol (6), α-amyrin acetate (7), and ethyl linoleate (8). Their chemical structures were identified via spectroscopic means as well as by comparing their data with literature values. Studies on tyrosinase inhibition activities were conducted for the isolated compounds. Among them, p-hydroxybenzoic acid (1) and methyl p-hydroxybenzoate (2) were identified as active tyrosinase inhibitors with IC₅₀ values of 0.98 ± 0.042 and 0.66 ± 0.025 mM, respectively, showing comparable activities to that of arbutin (IC₅₀ = 0.32 ± 0.015 mM), a standard control. Inhibition kinetics, as analyzed by Lineweaver-Burk plots, indicated that compounds 1 and 2 were competitive inhibitors of diphenolase of mushroom tyrosinase. Notably, isolated compounds 1–8 were reported for the first time as constituents of F. erecta.

Keywords::

• Ficus erecta
• tyrosinase inhibition
• p-hydroxybenzoic acid
• methyl p-hydroxybenzoate
• competitive inhibitor

Introduction

The genus Ficus belongs to the family Moraceae and is represented by more than 800 species worldwide¹. Some of these species, such as F. carica, a fruit tree commonly referred to as fig, are used as foods or as medicinal plants for the treatment of diverse medical disorders². Chemical investigations on the species of the genus have led to the identification of more than 100 compounds, including flavonoids, triterpenoids, coumarins, and phenanthroindolizidine alkaloids². Five plants have been described in Korea as species of the Ficus genus. F. erecta var. sieboldii King is a deciduous tree distributed in the southern seashore areas of Korea that grows up to 5 m in height³. Its roots are often used by the local community as a protective remedy against arthritis and stomach aches⁴. Interestingly, no phytochemical studies have been reported on this plant to date.

Melanin pigment, a biopolymer, is produced by skin epidermal melanocytes on exposure to external ultraviolet (UV) radiation. This is an essential physiological response and protects the inner skin from deleterious UV damage. However, overproduction and accumulation of dark melanin in the skin result in hyperpigmentation disorders such as melasma and solar lentigos⁵. In melanin biosynthesis, the first step is the hydroxylation of a monophenol, l-tyrosine, to generate 3,4-dihydroxyphenylalanine (l-DOPA). The resulting l-DOPA is oxidized again to produce an o-quinone (DOPA quinone). These two steps, which are catalyzed by a copper-containing metalloenzyme (tyrosinase), are the rate-determining steps in melanin synthesis⁶. As tyrosinase plays a key role in melanin synthesis, inhibition of this enzyme is an effective method for downregulating melanin production. Accordingly, tyrosinase inhibitors have become increasingly important in medicinal and cosmetic products for hyperpigmentation treatment⁷. Currently, chemicals such as arbutin, kojic acid, and hydroquinone derivatives are clinically used. However, questions regarding their low activities and cell toxicities have become a matter of public concern⁸. Therefore, development of anti-tyrosinase agents exhibiting high efficiency and low toxicity is still necessary for skin-whitening products. Regarding the use of tyrosinase inhibitors in cosmetics, much effort has focused on natural products⁹.

This laboratory is continuously screening plants from Jeju Island to discover natural ingredients for use in cosmetic preparations. Jeju Island is located in the southernmost part of Korea and is famous for its plant diversity with more than 1,800 plant species¹⁰. As part of our ongoing phytochemical studies^11,12, we became interested in F. erecta var. sieboldii King since ethanol extract prepared from its branches shows considerable inhibition of mushroom tyrosinase. This led us to carry out chemical studies to identify the active constituents in the extract, resulted in the isolation of eight compounds: p-hydroxybenzoic acid (1), methyl p-hydroxybenzoate (2), vanillic acid (3), methyl vanillate (4), syringic acid (5), β-sitosterol (6), α-amyrin acetate (7), and ethyl linoleate (8). Herein, we describe the characterization of the isolated compounds as well as their anti-tyrosinase activities.

Materials and methods

Reagents and equipments. JNM-ECX 400 (JEOL) instrument was used for ¹H (400 MHz), ¹³C (100 MHz), and 2D NMR spectra with chemical shift data in expressed in ppm relative to the solvent used. Vacuum liquid chromatography (VLC) and column chromatography (CC) were performed using silica gel 60 H (15 µm, Merck) and silica gel (0.063–0.2 mm), respectively. Silica gel 60 F₂₅₄ coated on aluminium plates (Merck, Darmstadt, Germany) was used for thin layer chromatography (TLC). Arbutin was purchased from Bioland Ltd (Chungbuk, Korea). All solvents were of analytical grade and used without further purification.

Plant material. Branches of F. erecta were collected from Jeju Botanical Garden, Jeju Island, Korea in February, 2009. A voucher specimen (no. J-224) was deposited at the Natural Product Chemistry Lab, Department of Chemistry, Cheju National University, Korea.

Extraction and isolation. Air-dried and pulverized branches of F. erecta (1.0 kg) were extracted three times with 70% ethanol (v/v) (20.0 L) using a mechanical stirrer at room temperature for 24 h. The resulting ethanol solutions were combined and filtered. The filtrate was concentrated using a rotary evaporator at a temperature not exceeding 45°C. The obtained gummy mass (100.0 g) was suspended in water and successively partitioned to give n-hexane (1.6 g), EtOAc (3.1 g), n-butanol (10.2 g), and water (54.0 g) extracts.

A portion of EtOAc extract (1.6 g) was subjected to vacuum liquid chromatography (VLC) through silica gel using gradient n-hexane-EtOAc-methanol eluents to produce 24 fractions (frs. V1–1 to V1–24). Compound 6 (14.6 mg) was obtained from fraction V1–5. Fraction V1–11 (40.2 mg) was further purified by silica gel column chromatography (CC) with chloroform-methanol (15:1) as the mobile phase to give compounds 2 (2.7 mg) and 4 (4.0 mg). Another portion of EtOAc extract (1.4 g) was also similarly subjected to VLC through silica gel using gradient solvents to afford 16 fractions (frs. V2–1 to V2–16). Compound 1 (9.7 mg) was obtained from fr. V2–5 by silica gel CC with chloroform-methanol (8:1) eluent. Another EtOAc extract (2.3 g) prepared using the same procedure described above from the branches of F. erecta (527.0 g) was also subjected to VLC over silica gel using gradient solvents to afford 16 fractions (frs. V3–1 to V3–16). Compound 8 (23.1 mg) was obtained from fraction V3–2. Compound 4 (9.6 mg) was obtained from fraction V3–3 by recrystallization from methanol. Purification of fraction V3–6 by silica gel CC with chloroform-methanol (8:1) afforded compound 3 (13.6 mg). Similarly, silica gel CC of fraction V3–8 with chloroform-methanol (5:1) provided compound 5 (4.8 mg).

Tyrosinase inhibition assay. Diphenolase activities of mushroom tyrosinase were studied according to the method described in the literature¹³ with some modifications. Briefly, 1250 units/mL of mushroom tyrosinase and 0.07 mL of 2 mM l-tyrosine were added to a solution of 0.09 mL of KH₂PO₄-K₂HPO₄ buffer (0.1 M, pH 6.8) containing the sample. Right after the test mixture (0.2 mL) was incubated for 10 min at 37°C, absorption due to the formation of dopachrome was monitored at 492 nm. The same mixture except the sample was used as a control. Arbutin (hydroquinone-O-β-glucopyranoside) was used as a positive control. Each treatment was replicated three times. The percentage inhibition of tyrosinase activity was calculated as follows:

where Abs_sample is the absorbance of the experimental sample, Abs_blank is the absorbance of the blank, and Abs_control is the absorbance of the control. The concentration of sample at that inhibited enzyme activity by 50% (IC₅₀) was obtained by linear curve fitting.

Kinetic analysis of tyrosinase inhibition. Various concentrations of l-tyrosine (0.2 to 0.6 mM) were added as substrates to a 96-well plate containing 5 μL of aqueous solution of mushroom tyrosinase (1250 units) and 100 mM potassium phosphate buffer (pH 6.8) with (25, 50, and 100 μg/mL of the compounds) or without test sample to a final volume of 200 μL. Using a microplate reader, the initial rate of dopachrome formation in the reaction mixture was determined based on the linear increase in absorbance at 492 nm [δ(OD)]. Michaelis constant (K_m) and maximal velocity (V_max) of tyrosinase were determined by Lineweaver-Burk plots at various concentrations of l-tyrosine.

Results and discussion

The ethanol extract and subsequent n-hexane, ethyl acetate (EtOAc), n-butanol, and water fractions were prepared according to the procedure described in the experimental section from the branches of F. erecta. Among these extracts, the EtOAc-soluble fraction was found to contain potent anti-tyrosinase activity with a IC₅₀ of 75.7 μg/mL (Figure 1). The potency of this activity was similar to that of arbutin (IC₅₀ = 87.4 μg/mL), a standard control currently applied as a whitening ingredient in cosmetic formulations. As shown in Figure 1, the other fractions exhibited very low inhibition activities against tyrosinase, implying that most of the active constituents were partitioned into the EtOAc fraction. Therefore, phytochemical investigation to identify the bioactive compounds was conducted on this EtOAc fraction.

Figure 1.  Tyrosinase inhibition activities of extracts from F. erecta.

Repeated column chromatography of the EtOAc fraction through silica gel resulted in the isolation of eight constituents (Figure 2): p-hydroxybenzoic acid (1)¹⁴, methyl p-hydroxybenzoate (2)¹⁵, vanillic acid (3)¹⁶, methyl vanillate (4)¹⁷, syringic acid (5)¹⁸, β-sitosterol (6)¹⁹, α-amyrin acetate (7)²⁰, and ethyl linoleate (8)²¹. Compounds 1–5 were all p-hydroxybenzoic acid derivatives, which were fully identified by spectroscopic methods, including ¹H and ¹³C NMR data. The non-polar component β-sitosterol (6), frequently encountered in plant extract, was also identified by NMR data. The ester derivatives of the triterpenol α-amyrin and linoleic acid were also characterized by ¹H and ¹³C NMR spectroscopy. The structures of these compounds were confirmed by comparing their spectroscopic data to those in the literature. As far as we know, all of the identified compounds (1–8) were isolated for the first time from this plant.

Figure 2.  Structures of isolated compounds 1–8.

The isolated compounds (1–8) were investigated for their anti-tyrosinase activities using l-tyrosine as the substrate. The test solutions for each compound were prepared at varying concentrations (25 to 100 μg/mL) and examined for their inhibitory effects against mushroom tyrosinase. An increase in absorbance at 492 nm due to the formation of dopachrome was observed using a spectrophotometer, and the results are summarized in Figure 3. Among these compounds, p-hydroxybenzoic acid (1) and methyl p-hydroxybenzoate (2) were found to be potent tyrosinase inhibitors. They inhibited the activity of mushroom tyrosinase with IC₅₀ values of 0.98 ± 0.042 and 0.66 ± 0.025 mM, respectively, which were comparable to that of the positive control arbutin (IC₅₀ = 0.32 ± 0.015 mM). However, the other compounds (3–8) displayed poor inhibitory activities, showing IC₅₀ values of more than 200.0 μg/mL.

Figure 3.  Tyrosinase inhibition activities of compounds 1 and 2. The data represented the mean ± SD of triplicate value for three independent experiments.

Since compounds 1 and 2 exerted strong inhibition against the diphenolase activity of tyrosinase, the mode of inhibition was investigated by employing Lineweaver-Burk plot analysis (Figures 4, 5). p-Hydroxybenzoic acid (1) showed an identical V_max value of 0.048 δ(OD)/min at various concentrations (0, 25, 50, and 100 μg/mL), and its K_m values were 0.38, 0.51, 0.58, and 0.90 mM, respectively. As the concentration of l-tyrosine substrate varied, the K_m value of tyrosinase increased in a dose-dependent manner without changes in V_max. This indicates that p-hydroxybenzoic acid (1) acted as a competitive tyrosinase inhibitor. There is a report that found that compound 1 acts as a competitive inhibitor using l-DOPA as the substrate²². Likewise, methyl p-hydroxybenzoate (2) was also characterized as a competitive tyrosinase inhibitor using l-tyrosine as the substrate at various concentrations (0, 25, 50, and 100 μg/mL). It showed the same V_max value of 0.050 δ(OD)/min, and its K_m values were 0.29, 0.35, 0.44, and 0.71 mM, respectively. The compounds 1 and 2 have inhibition constants (K_i) of 0.52 and 0.40 mM, respectively, indicating their similar binding affinities to the enzyme.

Figure 4.  Lineweaver-Burk plot of mushroom tyrosinase and l-tyrosine without (•) and with 0.18 mM (▴), 0.36 mM (▪), and 0.72 mM (♦) of p-hydroxy benzoic acid (1).

Figure 5.  Lineweaver-Burk plot of mushroom tyrosinase and l-tyrosine without (•) and with 0.16 mM (▴), 0.33 mM (▪), and 0.66 mM (♦) of methyl p-hydroxy benzoate (2).

Tyrosinase belongs to a metalloprotein family possessing a dinuclear copper-binding domain in its active site. Even though human tyrosinase is not characterized yet, the protein from Streptomyces castaneoglobisporus HUT 6202 has been elucidated recently²³. Its crystal structure confirmed the presence of three different dinuclear forms in its active site such as deoxy-form [Cu(I)-Cu(I)], oxy-from [Cu(II)-O₂⁻²-Cu(II)] and met-form [Cu(II)-OH-Cu(II)], where each copper metal is coordinated by three histidine residues. The oxy-tyrosinase was proposed as the active form responsible for the oxidation of monophenol to 1,2-diphenol. Monophenol-type inhibitors are assumed to exert activities upon binding to copper metal as a monophenolase substrate, as evidenced by the competitive inhibitor arbutin²⁴. Considering their competitive inhibition mechanism, it is reasonable to suggest that monophenol compounds 1 and 2 exhibit their activities by the coordination of phenoxy groups to copper in the oxy-form (A) at the active site of mushroom tyrosinase. Alternatively, the inhibition could occur by binding of the inhibitors to the met-tyrosinase as shown in B. Met-tyrosinase is known to have function capable of converting diphenol to orthoquinone but not oxidizing monophenol to catechol. Therefore, binding of the monophenolic inhibitor to the met-tyrosinase should eventually block the enzyme reaction of l-tyrosine leading to the DOPA qinone²⁵.

In conclusion, during the investigation of tyrosinase inhibitory constituents from the branches of F. erecta, eight compounds were identified. Among the isolates, p-hydroxybenzoic acid (1) and methyl p-hydroxybenzoate (2) were identified as the active constituents with competitive tyrosinase inhibition activities against l-tyrosine substrate. Ethyl acetate extracts of F. erecta containing p-hydroxybenzoic acid derivatives as active components may prove to have potential values as skin-whitening cosmetic additives in the future.

Acknowledgment

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2011-0007254).

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.