Phenols displaying tyrosinase inhibition from Humulus lupulus

Abstract

Tyrosinase is the rate-limiting enzyme for the production of melanin and other pigments via the oxidation of l-tyrosine. The methanol extract from Humulus lupulus showed potent inhibition against mushroom tyrosinase. The bioactivity-guided fractionation of this methanol extract resulted in the isolation of seven flavonoids (1–7), identified as xanthohumol (1), 4′-O-methylxanthohumol (2), xanthohumol C (3), flavokawain C (4), xanthoumol B (5), 6-prenylnaringenin (6) and isoxanthohumol (7). All isolated flavonoids (1–7) effectively inhibited the monophenolase (IC₅₀s = 15.4–58.4 µM) and diphenolase (IC₅₀s = 27.1–117.4 µM) activities of tyrosinase. Kinetic studies using Lineweaver–Burk and Dixon-plots revealed that chalcones (1–5) were competitive inhibitors, whereas flavanones (6 and 7) exhibited both mixed and non-competitive inhibitory characteristics. In conclusion, this study is the first to demonstrate that the phenolic phytochemicals of H. lupulus display potent inhibitory activities against tyrosinase.

• Competitive inhibition
• diphenolase
• Hulumuls lupulus
• monophenolase
• tyrosinase inhibitor

Introduction

Tyrosinase (EC 1.14.18.1) is a metalloenzyme widely distributed in nature, which catalyzes two distinct reactions of melanin biosynthesis: the hydroxylation of monophenol (monophenolase activity) and the oxidation of o-diphenol to o-quinone (diphenolase activity)1. These quinone products are spontaneously converted to melanin2. Furthermore, this ubiquitous enzyme initiates the biosynthesis of melanin and is responsible for the browning that occurs upon the bruising or long-term storage of vegetable, fruits and mushrooms. The production of melanin through tyrosinase is essential for the protection of skin from solar radiation3. However, this beneficial trait can show harmful effects under severe conditions in which the overproduction of melanin results in skin hyperpigmentation, characterized by age spots, melasma and chloasma, which are common human maladies3–5. It has also been reported that tyrosinase contributes to the neurodegeneration associated with Parkinson’s disease, particularly in males6. Tyrosinase inhibitors have recently attracted much research attention, reflecting the potent role of these inhibitors in decreasing hyperpigmentation through enzyme inhibition. These findings underline the importance of the discovery and development of tyrosinase inhibitors. Hence, tyrosinase inhibitors show potential for application in medicinal and cosmetic products. Tyrosinase inhibitors typically inactivate the copper within the active site through chelation, obviating the substrate–enzyme interaction, or inhibit oxidation through an electrochemical process7^,8.

Humulus lupulus L. (Cannabaceae) is cultivated widely throughout the world for its female inflorescences (common hop or hop). This plant material represents the main ingredient in beer and valued as a source of bitter flavor and biologically active polyphenols9. Since ancient times, hops have also been used to treat sleep disorders, restlessness and excitability and to promote digestion10. Moreover, hops have been used as a folk remedy to treat a wide range of ailments, including spasms, cough, fever, inflammation, earache and toothache11. The primary bioactive constituents of hops are prenylated acylphloroglucinols and prenylflavonoids. These bioactive metabolites exhibit antimicrobial, antioxidant, estrogenic, anti-inflammatory and antiatherogenic activities and also prevent cancer cell invasion12^,13. Previous studies have shown that prenylated chalcones, specifically xanthohumol, are the major bioactive substances in hops14. Herein, we explore this important plant as a rich source of tyrosinase inhibitors. However, to the best of our knowledge, no report is available concerning the identification of H. lupulus constituents that target tyrosinase inhibition. Thus, this study is the first to highlight the beneficial application of the constituents for targeting tyrosinase enzymes.

In this study, we isolated seven phenolic compounds from the H. lupulus, and the structures of these compounds were identified using spectroscopic methods. All isolated compounds were evaluated for inhibitory activity towards the monophenolase and diphenolase activities of tyrosinase. The underlying inhibition mechanisms were assessed using Lineweaver–Burk and Dixon plots (Figures 2 and 3).

Materials and methods

Chemical and instruments

Methanol, acetone, ethyl acetate, chloroform and n-hexane were purchased from Duksan Co. (Gyeonggi, Korea). Column chromatography was performed using silica gel (230–400 mesh; Merck Co., Darmstadt, Germany), YMC-gel ODS-A (S-75 µm; YMC) and Sephadex LH-20 (GE Healthcare Bio-Science AB, Uppsala, Sweden). Thin-layer chromatography (TLC) was performed on pre-coated silica gel 60 F₂₅₄ (0.25 mm; Merck, Darmstadt, Germany). Spots were detected by UV light (245 nm) and spaying with 10% H₂SO₄ followed by heating. Optical rotations were measured on a Perkin-Elmer 343 polarimeter (Waltham, MA). Circular dichroism (CD) spectra were recorded on a JASCOJ-715 spectropolarimeter. Melting points were measured on a Thomas Scientific capillary apparatus (Swedesboro, NJ). UV spectra were measured on a Beckman DU650 spectrophotometer (Indianapolis, IN). 1D and 2D NMR spectra were recorded on a Bruker (AM 500 MHz) spectrometer (Billerica, MA), using Acetone-d, as solvent and tetramethylsilane (TMS) as an internal standard. Electron ionization (EI) and EI-high resolution (HR) mass spectra were obtained on a JEOL JMS-700 instrument (JEOL, Tokyo, Japan). Enzymatic assay was carried out on a SpectraMaxM3Multi-Mode Microplate Reader (Molecular Devices, Sunnyvale, CA).

Plant material

Humulus lupulus [imported from USA, as permitted by Ministry of Food and Drug Safety (MFDS)] (approximately 1.5 kg) was purchased from a market, and identified by Prof. Y. S. Ju, Woosuk University, Republic of Korea. A voucher specimen (KIOM 130104) has been deposited in the Herbarium of the Menopausal Disorder Research Team, Korea Institute of Oriental Medicine, Republic of Korea. Sample was harvested in 2013 during the hop season (August–September), dried at 55 °C with air flow for 12 h and stored (vacuum-packed) at −20 °C until chemical analysis.

Preparation of extracts

Prior to further analysis, hops were cut into small pieces with the aid of a laboratory blade cutter. Chopped hops (2 g) were extracted (with shaking) into 20 mL of solvent at room temperature for 24 h. Four different solvent extraction systems were used: ethyl acetate, methanol, 50% methanol and distilled water (H₂O). Evaporation of each solvent under reduced pressure yielded the four crude extracts, which were used for biological activities.

Extraction, fractionation and isolation of H. lupulus

The dried hops were ground to powder. The powder (500 g) was extracted by methanol (4 L) at room temperature over 7 days. After removing solvent under reduced pressure, a black gum (162 g) was separated using silica-gel column chromatography (230–400 mesh, 100 × 8 cm) with a gradient solvent system of n-hexane/acetone (200:1. 150:1, 100:1, 50:1, 25:1, 10:1, 1:1, each 1.5 L) to yield five fractions [A (1.5 L, 3.8 g); B (1.5 L, 4.1 g); C (1.5 L, 5.3 g); D (1.5 L, 7.4 g); E (1.5 L, 12.1 g)]. Fractions showed yellow or red spots when visualized by 10% H₂SO₄ in ethanol and were again fractionated by silica gel chromatography (230–400 mesh) with an n-hexane/ACN gradient (0–100%) of increasing polarity, and C-18 column separated or purified by Sephadex LH-20 column chromatography or preparative TLC. Specifically, fraction C (5.3 g) was submitted to MPLC (LC-Forte/R, YMC) with silica gel (45 µm, 350 g) and eluted using a stepwise n-Hexane/ACN gradient (60:1, 40: 1, 20:1, 10:1, 5:1, 1:1, each 0.5 L, 40 mL/min) to give seven sub-fractions [C1 (0.5 L, 0.4 g); C2 (0.5 L, 1.5 g); C3 (0.5 L, 0.3 g); C4 (0.5 L, 0.8 g); C5 (0.5 L, 0.7 g); C6 (0.5 L, 0.3 g); C7 (0.5 L, 0.4 g)]. This MPLC procedure was repeated five times using the same conditions before further isolation. Sub-fractions C2 (1.5 g) were purified over silica gel (45 µm, 120 g) eluting with n-hexane/ACN (30:1) isocratic elution (25 mL/min) to give 10 sub-fractions [C2.1 (0.2 L, 200 mg); C2.2 (0.2 L, 125 mg); C2.3 (0.2 L, 131 mg); C2.4 (0.2 L, 126 mg); C2.5 (0.2 L, 73 mg); C2.6 (0.2 L, 57 mg); C2.7 (0.2 L, 52 mg); C2.8 (0.2 L, 109 mg); C2.9 (0.2 L, 130 mg); C2.10 (0.2 L, 216 mg)]. Sub-fractions C2.3-C2.5 (330 mg) were further purified by repeated silica gel chromatography (45 µm, 80 g) to five 1 (81 mg) and 3 (8 mg). Repeated silica gel CC of C2.10 (216 mg) yielded 2 (16 mg). Fraction D (7.4 g) was separated using C-18 column chromatography (30 µm, 250 g) with gradient elution of H₂O/ACN (10:1, 8:1, 6:1, 4:1, 2:1, 1:1, each 0.5 L, 40 mL/min) to yield six sub-fractions [D1 (0.5 L, 1.2 g); D2 (0.5 L, 0.8 g); D3 (0.5 L, 0.4 g); D4 (0.5 L, 0.5 g); D5 (0.5 L, 2.1 g); D6 (0.5 L, 0.9 g)] Sub-fraction D2 (800 mg) was purified by Sephadex LH-20 (60 × 3.5 cm) using methanol to afford 4 (22 mg). Sub-fraction D3 (400 mg) was purified by preparative TLC, and eluted with 70% acetone to afford 5 (13 mg). Sub-fractions D5 (2.1 g) were fractionated over silica gel (45 µm, 250 g) eluting with n-Hexane/ACN (4:1) isocratic elution (25 mL/min) to give 60 sub-fractions. Repeated silica gel CC of sub-fraction 28–35 (140 mg) yielded 6 (28 mg) and 7 (22 mg). The purity of the isolated compounds range from 95.0% to 99.5% as assessed by analytical HPLC [Agilent 1260 HPLC system; ZORBAX Eclipse Plus C 18 (150 × 4.6 mm, 5 µm) column: ACN/H₂O (40:60); UV detection, 254, 280, 310, 370 nm; flow rate, 1.0 mL/min].

Assay of tyrosinase inhibitory activity

Mushroom tyrosinase (EC1.14.18.1; Sigma Chemical Co., St. Louis, MO) was assayed as described previously with some modification15^,16, using either l-tyrosine (monophenolase) or l-DOPA (diphenolase) as substrate. In spectrophotometric experiments, enzyme activity [initial velocity (v_i)] was monitored by observing dopachrome formation at 475 nm at 30 °C. All test samples were first dissolved in DMSO at 10 mM and diluted to the required concentration. First, 20 µL of 1.8 mM l-tyrosine of 3.6 mM l-DOPA aqueous solution was mixed with 165 µL of 0.05 M phosphate buffer (pH 6.8) and 5 µL of the test sample in 96-well microplates. After the mixture was pre-incubated at 30 °C for 10 min, 10 µL of tyrosinase solution (250 U/mL) was added to the phosphate buffer and incubated for additional 15 min. DMSO without test compounds was used as control, and kojic acid (99% purity; Sigma) was used as a positive control. Each assay was conducted as three separate replicates. The inhibitory effects of the tested compounds were expressed as the concentrations that inhibited 50% of the enzyme activity (IC₅₀). The percentage inhibition ratio (%) was calculated according to the following equation: % inhibition = [(rate of control reaction − rate of sample reaction)/rate of control reaction] × 10017.

Enzyme kinetic assay and progress linear determination

The inhibition kinetic of the enzyme by the isolated compounds was analyzed by Linerweaver–Burk double-reciprocal-plots and compared to data obtained in the absence of inhibitor. Kinetic assays and progress curves were carried out using 250 U/mL tyrosinase, and substrate (l-tyrosine and l-DOPA) in double distilled water at 475 nm and 37 °C. Enzyme activities were measured continuously for 10 min using a spectrophotometer. To determine the kinetic parameters associated with tyrosinase inhibition, progress curves were obtained at several inhibitor concentrations using various substrate concentrations18.

Statistical analysis

All the measurements were made in triplicate. The results were subject to variance analysis using Sigma plot, Version 9.1 (SAS Institute Inc., Cary, NC; 2002). Differences were considered significant at p < 0.05.

Results

This study started from the methanol extract of H. lupulus that showed a potent inhibition against mushroom tyrosinase-catalyzed oxidation of both l-tyrosine and l-DOPA. The activity of the enzyme was assayed according to a standard procedure based on spectrophotometric measurement of the catalyzed l-tyrosine oxidation. As shown in Table 1, all extracts investigated, except for the water extract, exhibited a significant degree of tyrosinase inhibition (IC₅₀ < 131 µg/mL). The high potency of the MeOH extract prompted the identification of the compounds those were responsible for this effect. We conducted phytochemical investigations on the MeOH extract of the target plant via repeated column chromatography over silica gel, octadecyl-functionalized silica gel and Sephadex LH-20. These efforts led to the isolation of seven compounds 1–7 as shown Figure 1. The compounds were identified as xanthohumol (1), 4′-O-methylxanthohumol (2), xanthohumol C (3), flavokawain C (4), xanthoumol B (5), 6-prenylanringenin (6) and isoxanthohumol (7) by comparing their spectroscopic data with those previously reported19–24. The representative chalcone 1 was obtained as a yellowish powder having molecular formula C₂₁H₂₂O₅ and 11 degrees of unsaturation, as established by HREIMS (m/z 354.1487 [M⁺], calcd 354.1467). ¹H and ¹³C NMR data in combination with molecular indicated the α,β-unsaturated ketone moiety with two aromatic rings for a chalcone. 3,3-Dimethlyally group was confirmed and their position was identified by COSY and HMBC correlation. Thus, compound 1 was identified (E)-1-(2-hydroxy-6-methoxy-4-(3-methylbut-2-enyl) phenyl)-3-(4-hydroxyphenyl)prop-2-en-1-one, called xanthohumol.

Figure 1. Chemical structures of compounds 1–7 isolated from the Humulus lupulus L.

Figure 2. (A) Effect of compound 1 and kojic acid on the tyrosinase catalyzed oxidation of l-tyrosine. (B) Relationship between the catalytic activity of tyrosinase and concentration of compound 1. (C) Lineweacer–Burk plots showing the reciprocal of the velocity (1/V) of the mushroom tyrosinase reactions versus the reciprocal of the substrate concentration (1/S) with l-tyrosine as the substrate. (D) Dixon plot for the inhibition of compound 1 on the catalyzed oxidation of tyrosinase in the presence of different concentration of substrate for lines from bottom to: (•) 80 µM; (▾) 160 µM; (○) 320 µM.

Figure 3. (A and B) Effect of compound 6 on the tyrosinase catalyzed oxidation of l-tyrosine and l-DOPA. (C and D) K_M values as a function of the concentration of the compound 6. (Insert) Dependence of the values of V_Max on the concentration of compound 6.

Table 1. Comparison of extraction yield and tyrosinase inhibition of H. lupulus using different solvents.

		Tyrosinase (l-tyrosine)
Extraction solvent	Extraction yield (%)*	IC50 (µg/mL)	Inhibition (%)†
Ethylacetate	42.2	131.2 ± 3.7	22.3 ± 2.6
Methanol	30.6	61.2 ± 2.4	54.7 ± 3.1
50% Methanol	21.4	87.4 ± 1.4	48.2 ± 2.5
Water	9.2	NT‡	ND¶

*Extraction yields are given as g/100 g dry weight.

†Sample concentration was 200 µg/mL.

‡NT in not tested.

¶ND is not detected.

The isolated compounds (1–7) exhibited the inhibitory effects on the monophenolase activity of mushroom tyrosinase with IC₅₀ values of 15.4, 34.3, 20.6, 60.2, 22.1, 38.01 and 77.4 µM, respectively (Table 2). All compounds (1–7) also inhibited the diphenolase activity of mushroom tyrosinase with IC₅₀ values ranging from 31.1 to 157.4 µM (Table 2). The potency of compound 1 (IC₅₀ = 15.4 µM) can be favorably compared with that of commercially available inhibitors currently used in cosmetics, such as kojic acid (IC₅₀ = 11.5 µM).

Table 2. Inhibitory effects of isolated compounds 1–7 on mushroom tyrosinase activities.

	l-Tyrosinase (Monophenloase)		l-DOPA (Diphenolase)
Compounds	IC50* (µM)	Type of inhibition (Ki, µM)	IC50 (µM)	Type of inhibition (Ki, µM)
1	15.4	Competitive (7.9)	31.1	Competitive (15.6)
2	34.3	Competitive (18.1)	70.5	Competitive (27.4)
3	20.6	Competitive (12.4)	41.3	Competitive (19.8)
4	60.2	Competitive (27.3)	106.7	Competitive (60.1)
5	22.1	Competitive (12.9)	46.7	Competitive (27.3)
6	38.1	Mixed (20.2)	77.2	Non-competitive (80.1)
7	77.4	Mixed (40.1)	157.4	Non-competitive (153.2)
Kojic acid†	11.5	NT‡	24.1	NT

*All extracts were examined in a set of experiments repeated three times: Sample concentration which led to 50% enzyme activity loss.

†Kojic acid was used as a positive control.

‡NT is not tested.

In kinetic analysis, compound 1 was ascertained as a reversible inhibitor by plots of the initial velocity versus the concentration of compound 1. Concretely, the residual enzyme activity dramatically decreased with increasing inhibitor 1 concentration as shown in Figure 2(B). All other compounds (2–7) were also found to be reversible inhibitors. The additional information of kinetic behaviors toward l-tyrosine or l-DOPA oxidation was obtained using the Lineweaver–Burk double reciprocal method. The inhibitory behaviors were found to be different according to chemical structures between chalcones (1–5) and flavanone (6 and 7). The chalcones (1–5) were estimated as competitive inhibitors against both monophenolase and diphenolase, due to raising the apparent K_m of the enzyme for its substrate without affecting the value of V_max. While the flavanones (6 and 7) displayed the different inhibition profile against mono- and diphenolase. The both compounds showed mixed-type behavior toward monophenolase, whereas they emerged to be non-competitive inhibitors against diphenolase. A series of lines intersecting to the left of the vertical axis indicated compound 6 was mixed typed inhibitor (Figure 3C). Non-competitive behavior of compound 6 toward diphenolase were estimated by decrease the value of V_max without affecting the apparent K_m for the substrate (Figure 3D). The K_i values of inhibitors (1–7) were easily estimated from Dixon plots (Table 2). The most potent compound 1 inhibited mono- and diphenolase with K_i values of 7.9 and 15.6 µM, respectively.

Discussion

The tyrosinase activity-guided fractionation of the MeOH extract of H. lupulus resulted in the isolation of five chalcones (1–5) and two flavones (6 and 7). All compounds apart from 4 have a characteristic structural feature having isoprenyl group. The positions of the isoprenyl groups and quaternary carbons in each compound were successfully established using HMBC experiments, showing long-range connectivities between protons and carbons. The spectroscopic data of all compounds 1–7 were consistent with the results of previous studies.

The various biological benefits of metabolites from H. lupulus have been documented, but there is no report of anti-tyrosinase inhibitory activity. Thus, we examined the inhibitory potencies and kinetics of the isolated polyphenols (1–7) toward tyrosinase.

Table 2 shows that all isolated compounds exhibited a significant degree of tyrosinase inhibition, with IC₅₀s range from 15.4 to 77.4 µM for monophenolase and 31.1 to 157.4 µM for diphenolase. For example, compound 1 inhibited monophenolase (IC₅₀ = 15.4 µM) and diphenolase (IC₅₀ = 31.1 µM), respectively. The potency of these inhibitors was affected by subtle changes in structure. It appears that better inhibition is observed when the chalcone A-ring bears isoprenyl group. This is apparent from a comparison of prenylated chalcone 2 (IC₅₀ = 34.3 µM) and chalcone 4 (IC₅₀ = 60.2 µM). The hydroxyl group on C-4 was effective in tyrosinase inhibition: compound 1 (IC₅₀ = 15.4 µM) versus compound 2 (IC₅₀ = 34.3 µM). Taken together, these results suggest that the increasing number of isoprenyl and hydroxyl groups increases the potency of these inhibitors. In parallel comparison between chalcones (1–5) and flavanones (6 and 7) established that chalcones were almost tree fold as affective as flavanones. Higher hydroxylated flavanone 6 (IC₅₀ = 38.1 µM) showed better inhibition than flavanone 7 (IC₅₀ = 77.4 µM).

All isolated compounds (1–7) manifested a similar relationship between enzyme activity and concentration by dose-dependently. The inhibition of tyrosinase by compound 1 is illustrated in Figure 2, representatively. Increasing the concentration of the inhibitor drastically lowered residual enzyme activity. The plots of the remaining enzyme activity versus the concentrations of enzyme at different inhibitor concentrations gave a family of straight lines, which all passed through the origin. Increase of inhibitor concentration resulted in a decrease of the slope of the line, indicating that the presence of an inhibitor did not reduce the amount of enzyme but just resulted in the inhibition of enzyme activity. This indicated that compound 1 is a reversible inhibitor. We then further characterized the inhibitory mechanisms of chalcone 1 and flavanone 6 against mono- and diphenolase. The chalcone series (1–5) inhibited both monophenolase (l-tyrosine) and diphenolase (l-DOPA) with competitive manner. Figure 2(C) shows that the most potent compound 1 (IC₅₀ = 15.4 µM) inhibited the oxidation of l-tyrosine competitively. This is because the Lineweaver–Burk plots of 1/V versus 1/[S] result in a family of straight lines with the same y-axis intercept. Hence, the presence of a competitive inhibitor 1 has the kinetic effects of raising the apparent K_m of the enzyme for its substrate without affecting the value of V_max. The compound 1 also showed competitive inhibition toward diphenolase (Table 2 and Supplementary Material). The flavanones 6 and 7 showed mixed type inhibition against monophenolase, while non-competitive inhibition against diphenolase. As shown in Figure 3(C), flavanone 6 has a mixed type of inhibition against monophenolase because increasing inhibitor concentration resulted in a family of lines which intersected at a non-zero point on both x- and y-axis. In contrast, the flavanone 6 showed diphenolase non-competitive inhibitor because V_max (1/y-intercept) decreased whereas the K_m value (−1/x-intercept) remained constant as the inhibitor concentration increased (Figure 4D). The equilibrium constant for inhibitor binding, K_i was obtained from the value at the intersection of three lines from Dixon plots. The K_i values of inhibitors are presented in Table 2.

Figure 4. Dixon plots for the inhibition of compounds (2–7) on the monophenolase activity of tyrosinase. In the presence of different concentrations of substrate for lines from bottom to top: 320, 160, 80 µM.

Conclusion

This study demonstrates that the methanol extract of H. lupulus shows potent inhibitory activity toward tyrosinase. Purification of this fraction gave seven phenolic phytochemicals that displayed good tyrosinase inhibitory activities. The kinetic analysis confirmed that the chalcones (1–5) were competitive inhibitors for both monophenolase as well as diphenolase. Flavanones (6 and 7) were mixed-typed inhibitors for monophenolase and non-competitive inhibitors for diphenolase. These results suggest that some phenolic phytochemicals from H. lupulus might serve as candidates for skin-lightening agents.

Supplementary material available online

Supporting information

Declaration of interest

All students were supported by a scholarship from the BK21 plus. This research was supported by a grant from the Young Scientists personnel expenditure (490009-1297) of National Research Council of Science & Technology (NST) and a grant from the Korea Institute of Oriental Medicine (Grant No. K15290). The authors report no declarations of interest.