Tyrosinase Inhibition Activity of Monoterpene Glucosides From Brugmansia arborea Flowers

Abstract

Three monoterpene glucosides were isolated from the flowers of Brugmansia arborea L. using repeated silica gel and octadecyl SiO₂ column chromatography. Based on spectroscopic data including 1d-NMR (¹H, ¹³C, and distortionless enhancement by polarization transfer (DEPT)), 2D-NMR (gradient correlation spectroscopy (gCOSY), gradient heteronuclear single quantum coherence (gHSQC), and gradient heteronuclear multiple bond coherence (gHMBC)), Infrared Spectroscophy, and Mass Spectroscophy, the glucosides were identified as citronellol O- β-D-glucopyranoside (1), jasminoside N (2), and jasminoside P (3). The EtOAc (Brugmansia arborea Flowers ethyl acetate fraction [BAFE]) and n-BuOH (Brugmansia arborea Flowers n-butanol fraction [BAFB]) fractions showed high inhibition of tyrosinase activity (BAFE: IC₅₀ = 68.0 and BAFB: IC₅₀ = 59.3 μg/mL), and all isolated monoterpenes inhibited tyrosinase activity (1: IC₅₀ = 156.5, 2: IC₅₀ = 198.2, and 3: IC₅₀ = 191.0 μM).

Brugmansia arborea L. (Solanaceae), commonly called angel’s trumpet, is an evergreen shrub distributed in America, Africa, Australia, and Asia. ¹ This plant is commonly used for ornamental purposes ² and also for analgesic, antirheumatic, vulnerary, decongestant, and antispasmodic purposes. ² Recent studies report that B. arborea contains alkaloids such as atropine, scopolamine, hyroscine, hyoscyamine, 3α-tropanol, 3β-tropanol, and cuscohygrine as principal components and has shown anticholinergic activity. ^{3 -5} Despite these practical uses and pharmacological activities, the constituents of B. arborea flowers are rarely reported. Therefore, this study searched for their active materials. We previously reported the isolation of two benzonitriles from B. arborea flowers. ⁶ In this continuing study, three rarely occurring monoterpene glucosides (1-3) were isolated and identified. These compounds are for the first time reported as constituents of B. arborea flowers, and compounds 2 and 3 have been only isolated from the fruits of Gardenia jasminoides. ⁷

Brugmansia arborea flowers were extracted in MeOH, and the concentrated extract was partitioned into EtOAc, n-BuOH, and H₂O fractions. The EtOAc and n-BuOH fractions were subjected to repeated column chromatography (CC) to yield 3 compounds (1-3). Comparison of their NMR and Mass Spectroscophy data with the reported values led to identification of the monoterpene glucosides citronellol O-β-d-glucopyranoside (1), ⁸ geraniol O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranoside (jasminoside N) (2), ⁷ and 3,7-dimethyl-2,6-nonadien-1-oic acid O-β-D-glucopyranosyl-(1→6)-β-D-gluco-pyranoside (jasminoside P) (3) (Figure 1). ⁷ Compound 1 was first isolated from Vitis vinifera Muscat ⁹ and reported to have deodorizing action, ¹⁰ whereas compounds 2 and 3 were first isolated from G. jasminoides fruit. ⁷ The EtOAc and n-BuOH fractions and isolated monoterpene glucosides 1 to 3 were evaluated for their whitening effects by testing for the inhibition of mushroom tyrosinase activity (Table 1). The EtOAc (Brugmansia arborea Flowers ethyl acetate fraction [BAFE]) and n-BuOH (Brugmansia arborea Flowers n-butanol fraction [BAFB]) fractions showed significant inhibition of tyrosinase activity (BAFE: IC₅₀ = 68.0 and BAFB: IC₅₀ = 59.3 μg/mL). Compounds 1 to 3 also inhibited tyrosinase activity (1: IC₅₀ = 156.5, 2: IC₅₀ = 198.2, and 3: IC₅₀ = 191.0 µM). Hence, the EtOAc and n-BuOH fractions of B. arborea flowers and isolated monoterpene glucosides may be valuable because of their therapeutic potential as materials for whitening cosmetics.

Figure 1

Chemical structures of monoterpene glucosides 1 to 3 isolated from Brugmansia arborea flowers.

Table 1

Inhibition of Tyrosinase Activity by EtOAc (BAFE) and n-BuOH (BAFB) Fractions and Isolated Monoterpene Glucosides From Brugmansia arborea Flowers.

Compounds	% Inhibition	IC50 (μM) c
Kojic acid	92.5 ± 0.53 a	28.6 ± 0.61 a
1	51.8 ± 1.13 a	156.5 ± 1.84 a
2	40.8 ± 0.37 a	198.2 ± 1.48 a
3	42.4 ± 0.65 a	191.0 ± 0.99 a
BAFE	76.4 ± 0.60 b	68.0 ± 0.79 b
BAFB	87.5 ± 0.28 b	59.3 ± 0.87 b

All compounds were examined in experiments repeated 3 times.

^aConcentration for measuring inhibition ratio on mushroom tyrosinase activity was 200 µM.

^bConcentration for measuring inhibitiion ratio on mushroom tyrosinase activity was 100 μg/mL.

^cIC50 values are the concentration that caused 50% enzyme activity loss.

Experimental

Plant Materials, Instruments, and Chemicals

Brugmansia arborea flowers were supplied and identified as before. ⁶ Equipment and chromatographic materials were as previously described. ^11,12

Extraction and Isolation

Dried B. arborea flowers (900 g) were extracted with 80% aqueous MeOH (27 L × 3) at room temperature for 24 hours. The concentrated MeOH extract obtained under reduced pressure (160 g) was suspended in water (1 L) and successively extracted with ethyl acetate (EtOAc, 1 L × 3) and n-butanol (n-BuOH, 0.6 L × 3); the extracts were concentrated to produce EtOAc (BAFE, 12 g), n-BuOH (BAFB, 16 g), and H₂O fractions (BAFW, 132 g). The EtOAc fraction (12 g) was used for silica gel (SiO₂) CC (7.0 cm × 15.0 cm) eluting with n-hexane-EtOAc (4:1 → 2:1 → 1:1, 2.0 L each) and CHCl₃-MeOH-H₂O (50:3:1 → 36:3:1 → 25:3:1 → 18:3:1 → 12:3:1 → 9:3:1 → 7:3:1 → 6:4:1, 1.5 L each). The eluates were monitored by Thin Layer Chromatography (TLC) to produce 16 fractions (BAFE-1–BAFE-16). BAFE-10 [966 mg, elution volume/total volume (Ve/Vt) 0.649-0.718] was subjected to octadecyl SiO₂ (ODS) CC (4.5 cm × 10 cm) eluting with acetone-H₂O (1:2 → 1:1, 3.7 L each) to yield 16 fractions (BAFE-10-1–BAFE-10-16). BAFE-10-15 (102.0 mg, Ve/Vt 0.736-0.990) was subjected to SiO₂ CC (3.2 cm × 12 cm) eluting with CHCl₃-MeOH-H₂O (24:3:1, 1.3 L) to yield 14 fractions (BAFE-10-15-1–BAFE-10-15-14). SiO₂ CC (1.7 cm × 6 cm) of BAFE-10-15-9 (27 mg, Ve/Vt 0.385-0.500) eluting with CHCl₃-MeOH-H₂O (27:3:1, 240 mL) produced 12 fractions (BAFE-10-15-9-1–BAFE-10-15-9-12), and purified compound 1 (BAFE-10-15-9-6, 6.5 mg, Ve/Vt 0.163-0.188, TLC [SiO₂ F₂₅₄] R _f 0.54, CHCl₃-MeOH-H₂O = 10:3:10). The n-BuOH fraction (16 g) was separated by SiO₂ CC (7.0 cm × 13.0 cm) eluting with CHCl₃-MeOH-H₂O (12:3:1 → 9:3:1 → 6:4:1, 12.0 L each) to produce 14 fractions (BAFB-1–BAFB-14). BAFB-9 (1.2 g, Ve/Vt 0.163-0.225) was subjected to SiO₂ CC (4.0 cm × 15 cm) eluting with CHCl₃-MeOH-H₂O (10:3:1, 3.2 L) to yield 14 fractions (BAFB-9-1–BAFB-9-14). BAFB-9-8 (488.0 mg, Ve/Vt 0.308-0.607) was fractionated by SiO₂ CC (3.5 cm × 15 cm) eluting with EtOAc-n-BuOH-H₂O (22:3:1, 2.8 L) to yield 17 fractions (BAFB-9-8-1–BAFB-9-8-17). BAFB-9-8-14 (60.0 mg, Ve/Vt 0.587-0.806) was separated by ODS CC (1.5 cm × 5 cm) eluting with MeOH-H₂O (1:1, 150 mL) to produce 11 fractions (BAFB-9-8-14-1–BAFB-9-8-14-11) and purified compound 2 (BAFB-9-8-14-4, 14.3 mg, Ve/Vt 0.100-0.120, TLC [SiO₂ F₂₅₄] R _f 0.58, EtOAc-n-BuOH-H₂O = 4:5:1). BAFB-14 (3.0 g, Ve/Vt 0.471-0.496) was subjected to ODS CC (4.5 cm × 7.0 cm) eluting with MeOH-H₂O (1:2, 3.2 L) to yield 8 fractions (BAFB-14-1–BAFB-14-8). BAFB-14-6 (344.0 mg, Ve/Vt 0.209-0.672) was fractionated by SiO₂ CC (3.0 cm × 18.0 cm) eluting with EtOAc-n-BuOH-H₂O (16:3:1, 6.8 L) to produce 17 fractions (BAFB-14-6-1–BAFB-14-6-17), along with purified compound 3 (BAFB-14-6-15, 5.5 mg, Ve/Vt 0.949-0.962, TLC [SiO₂ F₂₅₄] R _f 0.35, EtOAc-n-BuOH-H₂O = 4:5:1).

Tyrosinase Inhibition Assay

Mushroom tyrosinase (EC 1.14.18.1) (Sigma Chemical Co., St Louis, MO, Uited States) was used for in vitro bioassays, as described previously, with some modifications. ^13,14 l-3,4-Dihydroxyphenylalanine (l-DOPA) was used as a substrate. Enzyme activity was monitored by detecting dopachrome formation at 475 nm with a UV-Vis spectrophotometer (Spectro UV-Vis Double beam; UVD-3500, Labomed, Inc., Los Angeles, CA, United States) at 30°C. All samples were dissolved in EtOH and used for experiments after dilution. First, 200 mL of 2.7 mM l-DOPA (Km = 180 µM) aqueous solution was mixed with 2687 mL of 0.25 M phosphate buffer (pH 6.8). Then, 100 µL sample solution and 13 µL phosphate buffered solution of mushroom tyrosinase (144 units) were added. Assays were conducted in triplicate. Inhibition concentration leading to 50% activity loss (IC₅₀) was obtained by fitting experimental data to the logistic curve by the following equation: Activity (%) =100[1/(1 + ([I]/IC₅₀))], ¹⁵ where [I] is the inhibition concentration.