Grevillosides J–Q, Arbutin Derivatives from the Leaves of Grevillea robusta and Their Melanogenesis Inhibitory Activity

Abstract

From the 1-BuOH-soluble fraction of a MeOH extract of the leaves of Grevillea robusta, new arbutin derivatives and related compounds, named grevillosides J–Q (1–8), together with eight known compounds (9–18) were isolated. Various kind of acyl groups were attached to β-D-glucose at the 6-position through an ester linkage. Their structures were mainly elucidated from one- and two-dimensional NMR spectroscopic data. For exploitation as skin-lightening and anti-chloasma agents, the inhibitory activities of the isolated compounds as well as ones isolated in previous experiments (19–31) toward tyrosinase and melanin-producing B16 cells were assayed. Several compounds showed promising activity.

Grevillea robusta ALLAN CUNNINGHAM, which belongs to the Proteaceae, originates from subtropical areas of eastern Australia and is planted in Japan for ornamental purposes. It is an evergreen tree of between 20–35 m in height with dark green delicately dented bipinnatifid leaves reminiscent of fronds. The leaves are 15–30 cm long with grey-white or rusty undersides. In previous papers, the isolation of glucosides of 5-alkylresorcinol derivatives (grevillosides A–H) from this plant was reported.^1,2) The structures of robustasides B and C isolated from the same plant³⁾ have been revised based on precise inspection of NMR spectra, and the 2,5-dihydroxycinnamate of arbutin, named grevilloside I, was also isolated.⁴⁾ Further phytochemical work resulted in the isolation of eight new compounds, named grevillosides (J–Q) (1–8) (Fig. 1), and ten known compounds (9–18) (Fig. 2). We also describe the inhibitory effects of constituents of G. robusta isolated in our series of research (1–31)^1,2,4) (Figs. 1, 2) toward mushroom tyrosinase activity and melanogenesis in theophylline-stimulated B16 melanoma cells.

Fig. 1. Structures of New Compounds Isolated

Fig. 2. Structures of Known Compounds Used for Biological Assaying

Compounds 9–18 were isolated in the present study and compounds (19–31) in the previous study.

Results and Discussion

Air-dried leaves of G. robusta were extracted with MeOH three times, followed by concentration. The MeOH extract was then partitioned with solvents of increasing polarity. The 1-BuOH-soluble fraction was separated by means of various chromatographic procedures comprising column chromatography (CC) procedures including a highly-porous synthetic resin CC (Diaion HP-20), normal silica gel CC, reversed-phase (octadecyl silica (ODS)) CC, droplet counter-current chromatography (DCCC), and HPLC to afford eight new compounds, named grevillosides J–Q (1–8), together with ten known compounds (9–18). The isolation details and yields are given in Experimental. The structures of the new compounds were elucidated mainly by spectroscopic evidence, and those of known phenolic compounds were identified as graviquinone (9),⁵⁾ robustasides D (10)³⁾ and G (11),⁶⁾ p-hydroxybenzoic acid glucoside (12),⁷⁾ icariside D₂ (13),⁸⁾ 4-O-β-D-glucopyranoside-3-hydroxy methyl benzoate (14),⁹⁾ rutin (15),¹⁰⁾ and rhamnocitrin (16),¹¹⁾ rhamnetin (17)¹²⁾ and kaempferol 3-O-rutinosides (18)¹⁰⁾ by comparison with data reported in the literature.

Grevilloside J (1), [α]_D −45.8, was isolated as an amorphous powder and its elemental composition was determined to be C₁₇H₂₄O₈ by high-resolution electrospray-ionization mass-spectrometry (HR-ESI-MS). The IR spectrum exhibited absorption bands ascribable to hydroxy groups (3381 cm⁻¹) and a carbonyl functional group (1714 cm⁻¹). In the ¹H-NMR spectrum, two deshielded protons for an AA′BB′ spin–spin coupling system [δ_H 6.94 (2H, d, J=9 Hz) and 6.68 (2H, d, J=9 Hz) were observed. The heteronuclear single-quantum correlation (HSQC) spectrum revealed that these protons were at δ_C 119.8 and 116.6, respectively. Thus, these proton and carbon signals together with two other aromatic carbon signals (δ_C 152.3, 154.0) represented a hydroquinone skeleton that is present in robustaside D (10). An anomeric proton (δ_H 4.71) at δ_C 103.7 and five other oxygenated carbon signals (δ_C 78.0, 75.5, 75.0, 71.9, 64.9) indicated a sugar moiety. Acid hydrolysis of 1 gave D-glucose and the mode of sugar linkage was determined to be β from the coupling constant of the anomeric proton (J=8 Hz). A carbonyl carbon (δ_C 178.2) and the relatively deshielded methylene carbon (δ_C 64.9) of glucose indicated that 1 was an acylated arbutin derivative and the acyl moiety was expected to be a simpler compound with five carbon atoms. Other than the carbonyl carbon, the acyl moiety comprised two methyl, one methylene and one methine carbons. In the ¹H-NMR spectrum, one methyl appeared as a triplet signal and another as a doublet signal. Based on the ¹H–¹H correlation spectroscopy (COSY) and the heteronuclear multiple-bond correlation (HMBC) spectra, the acyl moiety was assigned as 2-methylbutyrate and the esterification site was determined to be the hydroxy group at the 6′-position of the sugar moiety. The absolute configuration of the chiral center of methyl 2-methlybutyrate obtained on mild alkaline hydrolysis was confirmed to be S on HPLC analysis involving authentic methyl (+)-(2S)-2-methylbutyrate and a chiral detector. Therefore, the structure of 1 was elucidated to be that shown in Fig. 1.

Grevilloside K (2), [α]_D −19.3, was isolated as an amorphous powder and its elemental composition was determined to be C₃₀H₂₈O₁₃ by HR-ESI-MS. Grevilloside K (2) was expected to be an arbutin derivative with different acyl groups and thus the IR spectrum exhibited absorption bands ascribable to two types of carbonyl functional groups (1669, 1704 cm⁻¹). From the NMR spectroscopic data, the acyl groups were assigned to be 2,5-dihydroxycinnamic acid, which is present in robustasides B and C⁴⁾ (B in Fig. 1) and (E)-3-(1-hydroxy-4-oxocyclohexa-2,5-dien-1-yl)acrylate which is present in robustasides D³⁾ (C in Fig. 1). Acid hydrolysis of 2 gave D-glucose and the mode of sugar linkage was determined to be β from the coupling constant of the anomeric proton (J=8 Hz). The positions of acyl substituents were confirmed to be the hydroxy groups at the 2′- and 6′-positions on two-dimensional NMR spectroscopy. In the COSY spectrum, the anomeric proton signal (H-1′) was correlated with a deshielded signal at δ_H 5.05 (H-2′), which showed a correlation cross peak with δ_C 168.6 in the HMBC spectrum. Further HMBC correlations shown in Fig. 3 established the carbonyl carbon at δ_C 168.6 was that of the 2,5-dihydroxycinnamate, and another carbonyl carbon at δ_C 167.2 showed HMBC correlation peaks with H₂-6′ (δ_H 4.37, 4.54). Therefore, the structure of 2 was elucidated to be robustaside D 2′-O-2,5-dihydroxycinnamate, as shown in Fig. 1.

Fig. 3. ¹H–¹H COSY and HMBC Correlations for 2

Grevillosides L (3), [α]_D −44.6, was isolated as an amorphous powder and its elemental composition was determined to be C₂₁H₂₄O₁₁ by HR-ESI-MS. The IR spectrum exhibited absorption assignable to hydroxyl groups (3391 cm⁻¹), two types of carbonyl functional groups (1712, 1674 cm⁻¹), and an aromatic ring (1509 cm^–1). Of the 19 significant signals observed in the ¹³C-NMR spectrum, some appeared as two extremely close resonances. An arbutin moiety (C-1 to C-6 and C-1′ to C-6′) was obviously present in the molecule and in the ¹³C-NMR spectrum, the C₆–C₃ acyl moiety comprised trans and cis double bonds, two carbonyl, methylene, oxygenated methylene and methine carbons. In the ¹H–¹H COSY spectrum, methylene protons and an oxymethine proton showed a cross peak, and the oxymethine proton further correlated with one of the olefinic protons at the cis double bond due to a W-figure formation. Based on the latter together with the evidence obtained from the HMBC spectrum (Fig. 4), the structure of the acyl moiety was elucidated to be a H₂O adduct of (E)-3-(1-hydroxy-4-oxocyclohexa-2,5-dien-1-yl)acrylate (C in Fig. 1), namely (E)-3-(1,6-dihydroxy-4-oxocyclohex-2-en-1-yl)acrylate (D in Fig. 1). There are two hydroxylation sites, i.e., the 2″- and 6″-positions of C, and the regioselectivity between these positions seemed to be low. Judging from the formation of the W-figure between H-2″ and H-6″, and the significant nuclear Overhauser effect (NOE) correlation between H₂-5″ and H-7″ (Fig. 5), two hydroxy groups at the 1″- and 6″-positions of D must be directed in the same orientations. Therefore, the hydroxylation must occur at either the si-face of the pro-R (2″)-position or the re-face of the pro-S (6″)-position, enantiomeric 1-R, 6-R and 1-S, 6-S isomers being formed, respectively. As a result, grevillosides L (3) was isolated as an inseparable diastereomeric mixture. Although several different columns [Cosmosil-Cholester and -Hilic (Nakalai Tesque), Develosil C30 (Nomura Chemical), etc.] and solvent systems were tried to separate them, probably due to the remoteness of the chiral centers in the acyl group and the 5′-position, this mixture could not be separated into single compounds.

Fig. 4. ¹H–¹H COSY and HMBC Correlations for 3

Fig. 5. The Solid Lines Indicate NOE Correlations and the Dotted Line a Long-Range Correlation of 3

Grevillosides M (4), [α]_D −38.5, was also isolated as an inseparable mixture like 3 and its elemental composition was determined to be C₂₂H₂₆O₁₁ by HR-ESI-MS. The NMR spectroscopic data showed closely resembled those of 3, except for the appearance of methoxy signals at δ_H 3.41 and 3.42 at δ_C 58.44, which corresponds the increase of 14 mass unit, when compared with 3 (Table 1). Other spectroscopic data were essentially the same as those of 3. The stereochemistry of C-1″ and 6″ was the same as that in 3, from a W-figure long-range coupling between H-2″ and H-6″, observed in the ¹H-NMR spectrum and also a NOE correlation, observed between H₂-5″ and H-7″. Therefore, the structure of 4 was elucidated to be as shown in Fig. 1. This methoxy derivatives may be formed during extraction and separation procedures.

Table 1. ¹³C-NMR Spectroscopic Data for Grevillosides J–Q (1–8) and Robustaside D (10) (100 MHz, CD₃OD)

	1	2	3		4		5	6	7	8		10
1	152.3	152.1	152.29	152.28	152.24		152.2	152.6	152.3			152.3
2	119.8	119.8	119.66	119.62	119.63		119.6	106.9	119.3			119.7
3	116.6	116.8	116.75		116.74		116.7	146.8	116.8			116.8
4	154.0	154.3	153.96		153.99		154.0	142.0	153.8			154.0
5	116.6	116.8	116.75		116.74		116.7	116.4	116.8			116.8
6	119.8	119.8	119.66	119.62	119.63		119.6	109.2	119.3	α	β	119.7
1′	103.7	102.2	103.70	103.67	103.68	103.64	103.7	103.5	102.2	94.1	98.3	103.7
2′	75.0	75.2	74.98		75.0		75.0	74.9	83.9	73.9	76.3	75.0
3′	78.0	76.1	77.95		78.0		77.9	78.0	77.1	74.9	78.0	78.0
4′	71.9	71.9	71.83		71.9		71.7	71.8	71.2	72.1	71.8	71.8
5′	75.5	75.5	75.37	75.34	75.35	75.31	75.3	75.3	77.75	70.7	75.4	75.3
6′	64.9	65.0	65.07	65.04	65.1		65.0	65.1	67.7	65.3	65.4	65.1
1″	178.2	123.0	73.78	73.76	73.94		80.1	70.4	105.5	70.4		70.4
2″	42.5	151.6	150.19	150.17	150.55	150.49	146.8	151.2	76.1	151.1		151.2
3″	27.9	118.1	130.55	130.53	130.43	130.38	130.7	128.7	77.81	128.7		128.7
4″	12.0	120.4	199.44	199.42	198.73		196.3	187.1	71.3	187.0		187.7
5″	16.9	151.4	43.40		40.06		32.3	128.7	77.9	128.7		128.7
6″		114.9	73.78	73.76	83.04	82.99	49.9	151.2	62.6	151.1		151.2
7″		142.7	150.15		149.50	149.41	148.2	148.2		148.14 or 148.19		148.2
8″		118.0	123.32	123.30	123.38	123.35	122.5	122.9		122.87 or 122.93		122.9
9″		168.6	167.46		167.37		167.1	167.3		167.3		167.2
–OCH3					58.44
1‴		70.4					177.4		102.3
2‴		151.2					79.1		72.1
3‴		128.7					106.9		72.4
4‴		187.0					84.8		74.2
5‴		128.7					70.9		69.8
6‴		151.2					62.8		18.1
7‴		148.2
8‴		122.9
9‴		167.2

Grevilloside N (5), [α]_D −76.0, was isolated as an amorphous powder and its elemental composition was determined to be C₂₇H₃₀O₁₆. The IR spectrum exhibited absorption assignable to hydroxy groups (3346 cm⁻¹), γ-lactone (1775 cm⁻¹), and two types of conjugated ketones (1715, 1677 cm⁻¹). NMR spectroscopic data indicated that grevilloside N (5) was an analogous compound to previous ones, namely, an arbutin moiety is present in the molecule and the hydroxy group at the 6′-position was also acylated. However, the acyl moiety comprised not nine but 15 carbons. Signals for a trans double bond [δ_H 6.17 (d, J=16 Hz) and 7.06 (d, J=16 Hz)] and an AB doublet [δ_H 5.95 (d, J=10 Hz) and 6.49 (dd, J=10, 2 Hz)] were observed in the ¹H-NMR spectrum, and the ¹³C-NMR spectrum exhibited an oxygenated tertiary carbon at a fairly deshielded position (δ_C 80.1) and a highly deshielded carbonyl carbon signal (δ_C 196.3). Although these signals were assignable to the partial structure of (E)-3-(1-hydroxy-4-oxocyclohexa-2,5-dien-1-yl)acrylate, the AA′BB′ doublet was replaced by an AB doublet. The ¹³C-NMR spectrum further revealed that the remaining signals comprised those for methylene, methine, ketal, carbonyl, oxygenated tertiary, two oxygenated secondary and oxygenated primary carbons. These signals indicated that one of the double bonds in the ring of (E)-3-(1-hydroxy-4-oxocyclohexa-2,5-dien-1-yl)acrylic acid was involved in the modification with six carbon atoms. In the ¹H–¹H COSY spectrum, two prominent proton sequences, i.e., H₂-5″ to H-6″ and H-4‴ to H-6‴ via H-5‴, were observed along with long-range coupling between H-2″ and H-6″ (Fig. 6), which indicated that one of the double bonds in the ring was converted to be one methylene and one methine during the building of a new skeleton. The HMBC correlations from H-4‴ (δ_H 4.67) to C-1‴ (δ_C 177.4), C-2‴ (δ_C 79.1), and C-3‴ (δ_C 106.9) established the involvement of C-1‴ to C-4‴ in the γ-lactone ring formation, and those from H-5″ (δ_H 2.68, 3.09) to C-2‴ and H-6″ (δ_H 3.18) to C-1‴ substantiated the connectivity of the γ-lactone ring and the six-membered ring at the C-6″ and C-2‴ positions. The elemental composition demands one more degree of unsaturation and this was satisfied by the formation of a cyclic system sharing an oxygen atom between C-1″ and C-3‴. The significant downfield shift of C-1″ (δ_C 80.1) from δ_C 70.4 in 2 in the ¹³C-NMR spectra and the presence of a stable hemiketal also supported this finding. From the biosynthetic point of view, grevilloside N (5) was probably formed through the cycloaddition of ascorbic acid toward the (E)-3-(1-hydroxy-4-oxocyclohexa-2,5-dien-1-yl)acrylate of robustaside D (10) (Fig. 2). As to the stereochemistry, the C-4‴ and C-5‴ positions may be the same as those of ascorbic acid and the hydroxy groups at the ring junctions of five-membered rings may be in anti-parallel positions, but the absolute stereochemistry is remained uncertain. The bonds, 1″ to the ketal oxygen and 6″ to 2‴, must be orientated in the same direction, otherwise, H-2″ and H-6″ could not adopt a W-figure plain to induce a long-range coupling. Therefore, the structure of grevilloside N was tentatively elucidated to be 5 shown in Fig. 1.

Fig. 6. ¹H–¹H COSY and HMBC Correlations for 5

The dotted line indicates a long-range coupling in a W-figure.

Grevilloside O (6), [α]_D −15.4, was isolated as an amorphous powder and its elemental composition was determined to be C₂₁H₂₂O₁₁. In the NMR spectra, typical signals assignable to (E)-3-(1-hydroxy-4-oxocyclohexa-2,5-dien-1-yl)acrylate and those of 6-acylated β-D-glucopyranoside were observed, as seen in previous compounds. Although the aglycone moiety comprised six carbons, it was asymmetrically substituted by three oxygen atoms. The three aromatic proton signals were coupled in an ABX system, and in the difference NOE experiment, on irradiation of the anomeric proton at δ_H 4.70 (d, J=7 Hz), significant signal enhancements were observed for H-2 [δ_H 6.58 (d, J=3 Hz) and H-6 [δ_H 6.42 (dd, J=9, 3 Hz)]. Therefore, the structure of 6 was elucidated to be 1,3,4-trihydroxybenzene 1-O-β-D-glucopyranoside 6′-O-(E)-3-(1-hydroxy-4-oxocyclohexa-2,5-dien-1-yl)acrylate, as shown in Fig. 1.

Grevilloside P (7), [α]_D −47.1 was isolated as an amorphous powder and its elemental composition was determined to be C₂₄H₃₆O₁₆ by HR-ESI-MS. The IR spectrum exhibited absorption bands ascribable to hydroxy groups (3357 cm⁻¹) and an aromatic ring (1510 cm⁻¹). NMR spectral data suggested that 7 is also a congeneric compound to arbutin. Three anomeric protons signals (δ_H 4.62, 4.67, 4.91) observed in the ¹H-NMR spectrum showed correlation cross peaks with the respective anomeric carbon signals (δ_C 105.5, 102.3, 102.2) in the HSQC spectrum. Since the ¹³C-NMR spectrum exhibited twelve signals ascribable to terminal α-rhamnopyranoside and β-glucopyranoside, and the sugar analysis revealed the presence of L-rhamnose and D-glucose, the inner hexose was expected to be β-D-glucopyranose attached to p-hydroquinone aglycone. Acetylation of 7 gave decaacetate (7a), and thus the enormously overlapping ¹H-NMR signals observed in the sugar region became clear as well-resolved peaks. One of the anomeric protons at δ_H 5.19 (d, J=8 Hz) showed correlation with the highly deshielded aromatic carbon at δ_C 156.1 (C-1) in the HMBC spectrum and it also showed a correlation peak signal with intact δ_H 3.90 at δ_C 78.7 (C-2′) in the ¹H–¹H COSY spectrum. The anomeric proton signal at δ_H 4.93 (1H, d, J=8 Hz) was expected to be that of the terminal glucopyranoside and its HMBC correlation to C-2″ established one of the sugar linkages to be Glc(1″→2′)Glc. The HMBC correlation between the anomeric proton of the terminal rhamnopyranoside at δ_H 5.35 (1H, d, J=2 Hz, H-1‴) and δ_C 66.5 with intact methylene protons (δ_H 3.63, 3.72) revealed the remaining sugar linkage to between rhamnopyranose and the 6-position of either glucopyranose. Since the ¹H–¹H COSY spectrum clearly demonstrated the sequences of the sugar ring protons and those at the 6-positions of both glucopyranoses, attachment of the terminal rhamnopyranose was determined to be at the 6′-position of the inner glucopyranose, as shown in Fig. 1.

Grevilloside Q (8), [α]_D +16.3, was isolated as an amorphous powder and its elemental composition was determined to be C₁₅H₁₈O₉ by HR-ESI-MS. On HPLC separation, 8 appeared as two interconvertible peaks and the presence of D-glucose was confirmed by HPLC analysis of the hydrolyzate. In the NMR spectra, two anomeric resonances were observed at δ_H 4.48 on δ_C 98.3 and δ_H 5.08 on δ_C 94.1, the former being assigned as that of the β-anomer and the latter as that of the α-anomer from their coupling constants, 8 Hz and 4 Hz, respectively. Some of the NMR signals for the acyl group appeared as two close signals and the acyl moiety was assigned as (E)-3-(1-hydroxy-4-oxocyclohexa-2,5-dien-1-yl)acrylate (C in Fig. 1). The abundance ratio of α- and β-anomers was estimated to be nearly 1 : 1 from the integrals of isolated signals in the ¹H-NMR spectrum.

Sun burn and chloasma are caused by the deposition of melanin pigment, which is formed on sun light stimulation of melanocytes located in the basement membrane. Arbutin is a natural skin-whitening agent originally isolated from bearberry. Arbutin itself and synthetic α-arbutin (hydroquinone α-D-glucopyranoside) are available on the market as skin-whitening agents and they inhibit tyrosinase, which is involved in the early stage of melanin production.¹³⁾ G. robusta was found to be a rich source of arbutin derivatives in our study. The compounds (1–18) isolated in this study together with those (19–31) in our previous study^1,2,4) were assayed as to their tyrosinase and melanogenesis inhibitory activity by using mouse melanoma cells (B16). However, none of the compounds tested showed any tyrosinase inhibitory activity (positive control, kojic acid: IC₅₀=17.9±2.4 µM). Chuang et al. reported a similar result for graviquinone (9), i.e., it exhibited almost no tyrosinase inhibitory activity (32.78±0.54% inhibition at the concentration of 16.7 g/mL).⁵⁾ On the other hand, of the 31 compounds, 5-alkylresorcinol derivatives (25–30), phenolic compounds (12, 14, 23, 24, 31), and flavonoid glycosides (15–18) were also inactive as to B16 melanogenesis, exception being 13 (Icariside D₂) (59.8% inhibition at 100 µM). The melanogenesis inhibitory activity of icariside D₂ has already been reported in a patent document.¹⁴⁾ Significant activity was exclusively observed for arbutin derivatives (1–11, 19–22) using B16 melanoma cells (2, 59.9±3.0%: 4, 74.3±4.6%: 10, 45.9±6.5%: 19, 55.2±4.0%, and arbutin, 34.3±2.5% inhibition at 100 µM (3, 91.9% at 25 µM, and 9, 56.6% at 1.6 µM). These preliminary data were further confirmed using a high-melanin producing clone, B16Y24, established in this study (Table 2). Although B16Y24 is a potent melanin producer, grevillosides K and O (2, 6), and robustasides D (10) and G (11) could inhibit melanogenesis moderately, and grevillosides M (4) and graviquinone (9) possessed potent inhibitory activity toward it (Table 2). It is noteworthy that their strong melanogenesis inhibitory activity showed almost no association with cytotoxicity (Table 2). Considering the structures and activity relationship, these compounds possessed a common ester moiety i.e., 3-(1-hydroxy-4-oxocyclohexa-2,5-dien-1-yl)acrylate or (E)-3-(1,6-dihydroxy-4-oxocyclohex-2-en-1-yl)acrylate. Therefore, graviquinone (9), the ester moiety of grevillosides M (4), and presumably its methyl ester are relatively small molecules that can be produced with synthetic easiness, and thus they are promising compounds for skin-lightening and anti-chloasma agents.

Table 2. Melanogenesis Inhibitory Activity (B16Y24)

	Melanogenensis IC50 (µM)	Cytotoxicity IC50 (µM)
2	>30a)	>30
4	7.5±3.1	>30
6	52.9±2.5b)	>100b)
9	11.3±0.1	>30
10	20.7±1.8	>30
11	>30c)	>30
Arbutin	175.1±3.4b)	>300b)

Each value represents the mean±S.D. for quadruple experiments. a, c) 45.2±7.3 and 34.7±3.0%, respectively, with the highest dose of 30 µM, due to the limited quantities of samples available. b) The dose was increased up to 100 or 300 µM to determine the IC₅₀ values.

Experimental

General Experimental Procedure

Optical rotations were measured on a JASCO P-1030 digital polarimeter. IR and UV spectra were measured on Horiba FT-710 and JASCO V-520 UV/Vis spectrophotometers, respectively. ¹H- and ¹³C-NMR spectra were taken on a JEOL JNM α-400 at 400 MHz and 100 MHz, respectively, with tetramethylsilane as an internal standard. HR-ESI-MS was performed with an Applied Biosystems QSTAR XL NanoSpray™ System.

A highly-porous synthetic resin (Diaion HP-20) was purchased from Mitsubishi Kagaku (Tokyo, Japan). Silica gel CC was performed on silica gel 60 (E. Merck, Darmstadt, Germany), and ODS open CC on Cosmosil 75C₁₈-OPN (Nacalai Tesque, Kyoto, Japan) [Φ=50 mm, L=25 cm, linear gradient: MeOH–H₂O (1 : 9, 1 L)→(1 : 1, 1 L), fractions of 10 g being collected]. The DCCC (Tokyo Rikakikai, Tokyo, Japan) was equipped with 500 glass columns (Φ=2 mm, L=40 cm), the lower and upper layers of a solvent mixture of CHCl₃–MeOH–H₂O–1-PrOH (9 : 12 : 8 : 2) being used as the stationary and mobile phases, respectively. Five-gram fractions were collected and numbered according to their order of elution with the mobile phase. HPLC was performed on an ODS column (Inertsil; GL Science, Tokyo, Japan; d=6 mm, L=250 mm, 1.6 mL/min), and the eluate was monitored with a UV detector at 254 nm and a refractive index monitor. (S)-(+)-2-Methylbutanoic acid was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) and mushroom tyrosinase was obtained from Sigma-Aldrich Corporation (St. Louis, MO, U.S.A.).

Plant Material

The leaves of G. robusta were collected in Nakagami-gun, Okinawa in June 2005, A voucher specimen was deposited in the Herbarium of the Department of Pharmacognosy, Graduate School of Biomedical and Health Sciences, Hiroshima University (05-GR-Okinawa-0629).

Extraction and Isolation

Dried leaves of G. robusta (6.37 kg) were extracted three times with MeOH (30 L) at 25°C for one week, followed by concentration to 3 L in vacuo. The extract was washed with n-hexane (3 L, 32.6 g) and then the MeOH layer was concentrated to a gummy mass. The latter was suspended in water (3 L) and then extracted with EtOAc (3 L) to give 160 g of an EtOAc-soluble fraction. The aqueous layer was extracted with 1-BuOH (3 L) to give a 1-BuOH-soluble fraction (405 g), and the remaining water layer was concentrated to yield 475 g of a water-soluble fraction. A portion (237 g) of the 1-BuOH-soluble fraction was applied to a Diaion HP-20 column (Φ=75 mm, L=50 cm) using H₂O–MeOH (4 : 1, 6 L), (2 : 3, 6 L), (3 : 2, 6 L), (1 : 4, 6 L), and MeOH (6 L), 1 L fractions being collected. The residue (19.9 g) in fractions 4–6 of the 20% MeOH eluent was subjected to silica gel (450 g) CC, with elution with CHCl₃ (3 L) and CHCl₃–MeOH [(49 : 1, 3 L), (24 : 1, 3 L), (23 : 2, 3 L), (9 : 1, 3 L), (7 : 1, 3 L), (17 : 3, 3 L), (4 : 1, 3 L), (3 : 1, 3 L), and (3 : 2, 3 L)], 500 mL fractions being collected. The residue (2.50 g) in fractions 42–45 of the 15–20% MeOH eluate was separated by ODS open CC to give a residue in fractions 34–38 (461 mg), followed by separation by DCCC to yield a residue (198 mg) in fractions 20–27, which was purified by HPLC (H₂O–MeOH, 20 : 3) to give 8 (6.8 mg) and 13 (4.0 mg). Combined fractions 46–52 (3.84 g) of the 20–25% MeOH eluate were separated by ODS open CC and then DCCC to afford 12 (9.7 mg) and 14 (12.8 mg). An aliquot (2.19 g) of combined fractions 53–66 (3.87 g) of the 25–40% MeOH eluate was separated by ODS open CC and the residue (81.8 mg) in fractions 35–44 by DCCC to give 31.0 mg of a residue in fractions 10–15. The latter was purified by HPLC (H₂O–MeOH, 9 : 1) to give 11.8 mg of 7 from the peak at 11.5 min.

The precipitate in fractions 7–12 of the 20–40% MeOH eluent on Diaion HP-20 CC was collected by filtration to afford 642 mg of 16. The remaining residue (44.5 g) was subjected to silica gel (500 g) CC, with elution with CHCl₃ (4.5 L) and CHCl₃–MeOH [(49 : 1, 4.5 L), (24 : 1, 4.5 L), (19 : 1, 4.5 L), (23 : 2, 4.5 L), (9 : 1, 4.5 L), (7 : 1, 4.5 L), (17 : 3, 4.5 L), (33 : 7, 4.5 L), (4 : 1, 4.5 L), (3 : 1, 4.5 L), (7 : 3, 4.5 L)], 500 mL fractions being collected. An aliquot (1.87 g) of combined fractions 48–52 (13.1 g) of the 8–10% MeOH eluate was separated by ODS open CC to give two residues in fractions 83–96 (682 mg) and 312 mg of 10 in fractions 109–124. The former was separated by DCCC to give 620 mg of 10 in fractions 41–51 and 75.4 mg of 4 in fractions 52–58. An aliquot (1.80 g) of combined fractions 53–60 (8.79 g) of the 10–12.5% MeOH eluate was separated by ODS open CC to give two residues in fractions 66–80 (164 mg) and fractions 89–99 (544 mg). The former was separated by DCCC to give 43.8 mg of 4 in fractions 17–25 and the latter to afford 433 mg of 10 in fractions 35–43. An aliquot (1.76 g) of combined fractions 61–66 (2.61 g) of the 12.5–15% MeOH eluate was separated by ODS open CC to give two residues in fractions 69–87 (176 mg) and 88–99 (204 mg). The former was separated by DCCC to give 70.0 mg of 3 in fractions 17–22, and the latter to give a residue in fractions 25–28 (38.5 mg), which was purified by HPLC by (H₂O–MeOH, 3 : 1) to give 8.8 mg of 6 from the peak at 13 min. An aliquot (1.85 g) of combined fractions 67–73 (2.01 g) of the 15–17.5% MeOH eluate was separated by ODS open CC and the residue (458 mg) in fractions 100–115 by DCCC to give 130 mg in fractions 15–22 and the latter was then purified by HPLC (H₂O–MeOH, 4 : 1) to give 18.8 mg of 5 from the peak at 45 min.

The residue (39.0 g in fractions 13–18) of the 40–60% MeOH eluate obtainedon Diaion HP-20 CC was subjected to silica gel (450 g) CC, with elution with CHCl₃ (4.5 L) and CHCl₃–MeOH [(49 : 1, 4.5 L), (24 : 1, 4.5 L), (19 : 1, 4.5 L), (23 : 2, 4.5 L), (9 : 1, 4.5 L), (7 : 1, 4.5 L), (17 : 3, 4.5 L), (33 : 7, 4.5 L), (4 : 1, 4.5 L), (3 : 1, 4.5 L), (7 : 3, 4.5 L)], 500 mL fractions being collected. An aliquot (1.80 g) of combined fractions 40–47 (10.2 g) of the 8–10% MeOH eluate was separated by ODS open CC to give a residue in fractions 147–164 (1.04 g). The latter was separated by DCCC to give a residue in fractions 83–93 (20.3 mg) and 224 mg of 11 in fractions 99–108. The former was purified by HPLC (H₂O–MeOH, 1 : 1) to give 9.3 mg of 1 from the peak at 9 min. The residue (1.90 g) in fractions 48–54 of the 10% MeOH eluate was separated by ODS open CC to give two residues in fractions 158–170 (525 mg) and fractions 171–180 (297 mg). The former was separated by DCCC to give a residue in fraction of 92–114 (264 mg), which was then purified by HPLC (H₂O–MeOH, 7 : 3) to give 2.0 mg of 9 from the peak at 12 min. The latter was separated by DCCC to give a residue in fractions 41–51 (153 mg), which was then purified by HPLC (H₂O–MeOH, 11 : 3) to give 11.8 mg of 2 from the peak at 8 min. An aliquot (1.82 g) of combined fractions 63–72 (3.58 g) of the 15% MeOH eluate was separated by ODS open CC to give 15 in fractions 175–189 (399 mg). The latter was separated by DCCC to give a residue in fractions 83–93 (20.3 mg) and 224 mg of 11 in fractions 99–108.

The residue (23.9 g in fractions 19–24) of the 60–80% MeOH eluate obtained on Diaion HP-20 CC was subjected to silica gel (400 g) CC, with elution with CHCl₃ (3 L) and CHCl₃–MeOH [(98 : 2, 3 L), (24 : 1, 3 L), (19 : 1, 3 L), (23 : 2, 3 L), (9 : 1, 3 L), (7 : 1, 3 L), (17 : 3, 3 L), (33 : 7, 3 L), (4 : 1, 3 L), (3 : 1, 3 L), (7 : 3, 3 L)], 500 mL fractions being collected. An aliquot (1.83 g) of combined fractions 40–49 (4.08 g) of the 15–20% MeOH eluate was separated by ODS open CC to give 17 in fractions 227–243 (111 mg). An aliquot (2.75 g) of combined fractions 50–57 (2.93 g) of the 20–30% MeOH eluate was separated by ODS open CC to give a residue in fractions 224–241 (601 mg). The latter was separated by DCCC to give 81.4 mg of 18 in fractions 41–51.

Grevilloside J (1)

Amorphous powder; [α]_D²⁷ −45.8 (c=0.62, MeOH); IR ν_max (film) cm⁻¹: 3381, 2933, 1714, 1649, 1510, 1456, 1361, 1213, 1072, 1016; UV λ_max (MeOH) nm (log ε): 285 (3.43), 222 (3.89); ¹H-NMR (400 MHz, CD₃OD) δ: 6.94 (2H, d, J=9 Hz, H-2, 6), 6.68 (2H, d, J=9 Hz, H-3, 5), 4.71 (1H, d, J=8 Hz, H-1′), 4.46 (1H, dd, J=12, 2 Hz, H-6′a), 4.18 (1H, dd, J=12, 6 Hz, H-6′b), 3.57 (1H, ddd, J=9, 6, 2 Hz, H-5′), 3.44 (1H, dd, J=9, 9 Hz, H-3′), 3.41 (1H, dd, J=9, 8 Hz, H-2′), 3.33 (1H, dd, J=9, 9 Hz, H-4′), 2.40 (1H, sextet, J=7 Hz, H-2″), 1.66 (1H, dqui, J=14, 7 Hz, H-3″a), 1.47 (1H, dqui, J=14, 7 Hz, H-3″b), 1.13 (3H, d, J=7 Hz, H₃-5″), 0.89 (3H, t, J=7 Hz, H₃-4″); ¹³C-NMR (100 MHz, CD₃OD): Table 1; HR-ESI-MS (positive-ion mode) m/z: 379.1364 [M+Na]⁺ (Calcd for C₁₇H₂₄O₈Na: 379.1363).

Grevilloside K (2)

Amorphous powder. [α]_D²⁴ −19.3 (c=0.79, MeOH). IR ν_max (film) cm⁻¹: 3354, 2955, 1704, 1669, 1207, 1175, 1077. UV λ_max (MeOH) nm (log ε): 279 (4.27), 222 (4.49). ¹H-NMR (400 MHz, CD₃OD) δ: 8.00 (1H, d, J=16 Hz, H-7″), 6.92 (1H, br s, H-6″), 6.85 (2H, d, J=10 Hz, H-2‴, 6‴), 6.83 (2H, d, J=9 Hz, H-2, 6), 6.71 (1H, d, J=16 Hz, H-7‴), 6.71 (2H, m, H-2″, 3″), 6.66 (1H, d, J=9 Hz, H-3, 5), 6.56 (1H, d, J=16 Hz, H-8″), 6.30 (1H, d, J=16 Hz, H-8‴), 6.23 (2H, d, J=10 Hz, H-3‴, 5‴), 5.05 (1H, dd, J=9, 8 Hz, H-2′), 4.95 (1H, d, J=8 Hz, H-1′), 4.54 (1H, dd, J=12, 2 Hz, H-6′a), 4.37 (1H, dd, J=12, 6 Hz, H-6′b), 3.71 (1H, ddd, J=9, 6, 2 Hz, H-5′), 3.70 (1H, dd, J=9, 9 Hz, H-3′), 3.49 (1H, dd, J=9, 9 Hz, H-4′). ¹³C-NMR (100 MHz, CD₃OD): Table 1. HR-ESI-MS (positive-ion mode) m/z: 619.1396 [M+Na]⁺ (Calcd for C₃₀H₂₈O₁₃Na: 619.1422).

Grevillosides L (3)

Amorphous powder; [α]_D²² −44.6 (c=0.28, MeOH); IR ν_max (film) cm⁻¹: 3391, 2920, 2868, 1712, 1674, 1509, 1214, 1062, 1016; UV λ_max (MeOH) nm (log ε): 280 (3.45), 219 (4.18); ¹H-NMR (400 MHz, CD₃OD) δ: 7.03 (2H, d, J=16 Hz, H₂-7″), 6.915 (2H, d, J=9 Hz, H-2a, 6a); 6.910 (2H, d, J=9 Hz, H-2b, 6b), 6.693 (2H, d, J=9 Hz, H-3a, 5a), 6.688 (2H, d, J=9 Hz, H-3b, 5b), 6.63 (2H, dd, J=10, 1 Hz, H₂-2″), 6.24 (2H, d, J=16 Hz, H₂-8″), 6.07 (2H, d, J=10 Hz, H₂-3″), 4.72 (2H, d, J=7 Hz, H₂-1′), 4.51 (2H, dd, J=12, 2 Hz, H₂-6′a), 4.334 (1H, dd, J=12, 6 Hz, H-6′ba), 4.327 (1H, dd, J=12, 6 Hz, H-6′bb), 4.04 (2H, td, J=5, 1 Hz, H₂-6″), 3.624 (1H, ddd, J=9, 6, 2 Hz, H-5′a), 3.619 (1H, ddd, J=9, 6, 2 Hz, H-5′b), 3.45 (2H, dd, J=9, 9 Hz, H-3′), 3.41 (2H, dd, J=9, 7 Hz, H-2′), 3.37 (2H, dd, J=9, 9 Hz, H-4′), 2.69 (4H, d, J=5 Hz, H₄-5″); ¹³C-NMR (100 MHz, CD₃OD): Table 1; HR-ESI-MS (positive-ion mode) m/z: 475.1204 [M+Na]⁺ (Calcd for C₂₁H₂₄O₁₁Na: 475.1210).

Grevillosides M (4)

[α]_D²² −38.5 (c=0.47, MeOH); IR ν_max (film) cm⁻¹:3398, 2912, 2838, 1712, 1673, 1509, 1216, 1076, 1017; UV λ_max (MeOH) nm (log ε): 282 (3.45), 219 (4.39); ¹H-NMR (400 MHz, CD₃OD) δ: 7.03 (2H, d, J=16 Hz, H₂-7″), 6.913 (2H, d, J=9 Hz, H-2a, 6a); 6.908 (2H, d, J=9 Hz, H-2b, 6b), 6.690 (2H, d, J=9 Hz, H-3a, 5a), 6.685 (2H, d, J=9 Hz, H-3b, 5b), 6.59 (2H, dd, J=10, 1 Hz, H₂-2″), 6.25 (2H, d, J=16 Hz, H₂-8″), 6.04 (2H, d, J=10 Hz, H₂-3″), 4.72 (2H, d, J=7 Hz, H₂-1′), 4.51 (2H, dd, J=12, 2 Hz, H₂-6′a), 4.334 (12H, dd, J=12, 6 Hz, H₂-6′ba), 4.327 (1H, dd, J=12, 6 Hz, H-6′bb), 3.68 (2H, td, J=3, 1 Hz, H₂-6″), 3.624 (1H, dd, J=9, 6, 2 Hz, H-5′a), 3.619 (1H, ddd, J=9, 6, 2 Hz, H-5′b), 3.42 (2H, dd, J=9, 9 Hz, H-3′), 3.42 (3H, s, CH₃O-), 3.41 (2H, dd, J=9, 7 Hz, H-2′), 3.41 (3H, s, CH₃O-), 3.37 (2H, dd, J=9, 9 Hz, H-4′), 2.75 (4H, d, J=3 Hz, H₄-5″); ¹³C-NMR (100 MHz, CD₃OD): Table 1; HR-ESI-MS (positive-ion mode) m/z: 489.1369 [M+Na]⁺ (Calcd for C₂₂H₂₆O₁₁Na: 489.1367).

Grevilloside N (5)

Amorphous powder; [α]_D²³ −76.0 (c=0.53, MeOH); IR ν_max (film) cm⁻¹: 3346, 2906, 1775, 1715, 1677, 1509, 1214, 1072, 1047; UV λ_max (MeOH) nm (log ε): 279 (3.66), 213 (4.42); ¹H-NMR (400 MHz, CD₃OD) δ: 7.06 (1H, d, J=16 Hz, H-7″), 6.91 (2H, J=9 Hz, H-2, 6), 6.68 (2H, d, J=9 Hz, H-3, 6), 6.49 (1H, dd, J=10, 2 Hz, H-2″), 6.17 (1H, d, J=16 Hz, H-8″), 5.95 (1H, d, J=10 Hz, H-3″), 4.71 (1H, d, J=7 Hz, H-1′), 4.67 (1H, d, J=1 Hz, H-4‴), 4.48 (1H, dd, J=12, 2 Hz, H-6′a), 4.37 (1H, dd, J=12, 6 Hz, H-6′b), 4.02 (1H, td, J=7, 1 Hz, H-5‴), 3.61 (1H, ddd, J=9, 6, 2 Hz, H-5′), 3.58 (2H, d, J=7 Hz, H₂-6‴), 3.43 (1H, dd, J=9, 9 Hz, H-3′), 3.41 (1H, dd, J=9, 7 Hz, H-2′), 3.32 (1H, dd, J=9, 9 Hz, H-4′), 3.18 (1H, dd, J=8, 2 Hz, H-6″), 3.09 (1H, d, J=18 Hz, H-5″a), 2.68 (1H, dd, J=18, 8 Hz, H-5″b); ¹³C-NMR (100 MHz, CD₃OD): Table 1; HR-ESI-MS (positive-ion mode) m/z: 633.1430 [M+Na]⁺ (Calcd for C₂₇H₃₀O₁₆Na: 633.1426).

Grevilloside O (6)

Amorphous powder; [α]_D²⁷ −15.4 (c=0.59, MeOH); IR ν_max (film) cm⁻¹: 3360, 2924, 1712, 1668, 1621, 1511, 1452, 1392, 1282, 1187, 1071; UV λ_max (MeOH) nm (log ε): 212 (4.39); ¹H-NMR (400 MHz, CD₃OD) δ: 6.85 (2H, d, J=10 Hz, H-2″, 6″), 6.68 (1H, d, J=16 Hz, H-7″), 6.65 (1H, d, J=9 Hz, H-5), 6.58 (1H, d, J=3 Hz, H-2), 6.42 (1H, dd, J=9, 3 Hz, H-6), 6.27 (1H, d, J=16 Hz, H-8″), 6.22 (2H, d, J=10 Hz, H-3″, 5″), 4.70 (1H, d, J=7 Hz, H-1′), 4.49 (1H, dd, J=12, 2 Hz, H-6′a), 4.32 (1H, dd, J=12, 6 Hz, H-6′b), 3.60 (1H, ddd, J=8, 6, 2 Hz, H-5′), 3.43 (1H, dd, J=9, 8 Hz, H-3′), 3.38 (1H, dd, J=9, 7 Hz, H-2′), 3.34 (1H, m, H-4′); ¹³C-NMR (100 MHz, CD₃OD): Table 1; HR-ESI-MS (positive-ion mode) m/z: 473.1059 [M+Na]⁺ (Calcd for C₂₁H₂₂O₁₁Na: 473.1054).

Grevilloside P (7)

Amorphous powder; [α]_D²⁵ −47.1 (c=0.34, MeOH); IR ν_max (film) cm⁻¹: 3357, 2923, 1510, 1219, 1064, 1052; UV λ_max (MeOH) nm (log ε): 285 (3.41), 222 (4.01); ¹H-NMR (400 MHz, CD₃OD) δ: (6.97 (2H, d, J=9 Hz, H-2, 6), 6.71 (2H, d, J=9 Hz, H-3, 5), 4.91 (1H, d, J=7 Hz, H-1′), 4.67 (1H, d, J=2 Hz, H-1‴), 4.62 (1H, d, J=8 Hz, H-1″), 3.88 (1H, dd, J=12, 2 Hz, H-6″a), 3.81 (1H, dd, J=3, 2 Hz, H-2‴), 3.70 (1H, dd, J=12, 5 Hz, H-6″b), 3.67 (1H, dd, J=11, 2 Hz, H-6′a), 3.67 (1H, dd, J=8, 3 Hz, H-3‴), 3.63 (1H, dd, J=8, 7 Hz, H-2′), 3.58 (1H, dq, J=9, 6 Hz, H-5‴), 3.51 (1H, dd, J=11, 5 Hz, H-6′b), 3.41 (1H, dd, J=8, 8 Hz, H-4″), 3.37 (4H, m, H-3′, 5′, 3″, 5″), 3.34 (1H, m, H-4‴), 3.33 (1H, m, H-4′), 3.25 (1H, dd, J=8, 8 Hz, H-2″), 1.23 (3H, d, J=6 Hz, H₃-6‴); ¹³C-NMR (100 MHz, CD₃OD): Table 1; HR-ESI-MS (positive-ion mode) m/z: 603.1900 [M+Na]⁺ (Calcd for C₂₄H₃₆O₁₆Na: 603.1896).

Grevilloside Q (8)

Amorphous powder; [α]_D²³ +16.3 (c=0.43, MeOH); IR ν_max (film) cm⁻¹: 3370, 2926, 1710, 1667, 1512, 1313, 1274, 1054; UV λ_max (MeOH) nm (log ε): 219 (4.22); ¹H-NMR (400 MHz, CD₃OD) δ: 6.83 (2H, d, J=10 Hz, H-2″, 6″), 6.69 (1H, d, J=16 Hz, H-7″), 6.25 (1H, d, J=16 Hz, H-8,″ 6.21 (2H, d, J=10 Hz, H-3″, 5″), 5.08 (1/2 H, d, J=4 Hz, H-1′α), 4.48 (1/2 H, d, J=8 Hz, H-1′β), 4.48 (1/2 H, dd, J=12, 2 Hz, H-6′aα), 4.43 (1/2 H, dd, J=12, 2 Hz, H-6′aβ), 4.28 (1/2 H, dd, J=12, 6 Hz, H-6′bα), 4.25 (1/2 H, dd, J=12, 6 Hz, H-6′bβ), 4.00 (1/2 H, ddd, J=10, 6, 2 Hz, H-5α), 3.67 (1/2 H, dd J=9, 9 Hz, H-3′α), 3.51 (1/2 H, ddd, J=9, 6, 2 Hz, H-5′β), 3.37 (1/2 H, dd, J=9, 4 Hz, H-2′α), 3.32 (1/2 H, m, H-3′β), 3.14 (1/2 H, dd, J=9, 8 Hz, H-2′β), H-4′α, H-4′β signals overlapped by the HDO signal; ¹³C-NMR (100 MHz, CD₃OD): Table 1; HR-ESI-MS (positive-ion mode) m/z: 365.0859 [M+Na]⁺ (Calcd for C₁₅H₁₈O₉Na: 365.0843).

Sugar Analysis

About 500 µg of each compound (1–8) was hydrolyzed with 1 M HCl (1.0 mL) at 90°C for 2 h. The reaction mixtures were partitioned with an equal amount of EtOAc (1.0 mL), and the water layers were analyzed by HPLC with a chiral detector (JASCO OR-2090plus) on an amino column [Asahipak NH₂P-50 4E, Φ=4.6 mm, L=25 cm, CH₃CN–H₂O (3 : 1), 1 mL/min]. The hydrolyzates of (1–6, 8) each gave a peak for D-glucose at 14.2 min with a positive optical rotation sign. The hydrolyzate of 7 gave peaks for L-rhamnose and D-glucose at 6.8 min and 14.2 min with negative and positive optical rotation signs. The peaks were identified by co-chromatography with authentic L-rhamnose and D-glucose.

Absolute Configuration of 2-Methylbutyric Acid in Grevilloside J (1)

Grevilloside J (1) (1.7 mg) and (S)-(+)-2-methylbutyric acid (50 µL) were dissolved in 1 mL of a 1 : 1 mixture of 10% KOH in H₂O and 50% aqueous dioxane, and then heated for 1 h at 40°C. The cooled reaction mixtures were neutralized with Amberlite IR-120B (H⁺) and then the filtrates were evaporated. The residues were analyzed by HPLC (column: Inertsil ODS-3, 6 mm×250 mm; solvent: 20% CH₃CN in H₂O containing 0.5% trifluoroacetic acid; flow rate: 1.6 mL/min) with a chiral detector (JASCO OR-2090plus) to give a peak of (S)-(+)-2-methylbutyric acid at 17.0 min with a positive optical rotation sign.

Acetylation of Grevilloside P (7)

Grevilloside P (7) (5.9 mg) was acetylated with acetic anhydride (500 µL) and pyridine (500 µL) at 25°C overnight. Evaporation of the reagents gave 9.6 mg of grevilloside Q decaacetate (7a). Grevilloside Q decaacetate (7a) Amorphous powder; [α]_D²¹ −38.5 (c=0.20, MeOH); IR ν_max (film) cm⁻¹: 2960, 2932, 1752, 1682, 1649, 1247, 1222, 1075, 1046; UV λ_max (MeOH) nm (log ε): 267 (3.18), 220 (3.91); ¹H-NMR (400 MHz, CD₃OD) δ: 7.15 (2H, d, J=9 Hz, H-2, 6), 7.03 (2H, d, J=9 Hz, H-3, 5), 5.35 (1H, d, J=2 Hz, H-1‴), 5.33 (1H, dd, J=9, 3 Hz, H-3‴), 5.32 (1H, dd, J=3, 2 Hz, H-2‴), 5.25 (1H, dd, J=9, 9 Hz, H-3″), 5.20 (1H, m, H-3′), 5.19 (1H, d, J=8 Hz, H-1′), 4.99 (1H, dd, J=9, 9 Hz, H-4″), 4.98 (1H, dd, J=9, 9 Hz, H-4‴), 4.93 (1H, d, J=8 Hz, H-1″), 4.90 (1H, dd, J=10, 9 Hz, H-4′), 4.85 (1H, dd, J=9, 8 Hz, H-2″), 4.33 (1H, dd, J=12, 5 Hz, H-6″a), 4.10 (1H, dd, J=12, 2 Hz, H-6″b), 4.02 (1H, ddd, J=9, 5, 2 Hz, H-5″), 3.99 (1H, ddd, J=10, 7, 2 Hz, H-5′), 3.93 (1H, dq, J=9, 6 Hz, H-5‴), 3.90 (1H, dd, J=9, 8 Hz, H-2′), 3.72 (1H, dd, J=13, 7 Hz, H-6′a), 3.63 (1H, dd, J=13, 2 Hz, H-6′b), 2.25 (6H, s, CH₃CO– ×2), 2.15 (3H, s, CH₃CO–), 2.03–1.93 (21H, m, CH₃CO– ×7), 1.20 (3H, d, J=6 Hz, H₃-6‴); ¹³C-NMR (100 MHz, CD₃OD) δ: 172.32, 171.91, 171.88, 171.86, 171.81, 171.71, 171.50, 171.49, 171.47, 171.36 (CH₃CO– ×10), 156.1 (C-1), 147.3 (C-4), 123.5 (C-3, 5), 118.5 (C-2, 6), 101.7 (C-1″), 99.9 (C-1′), 99.1 (C-1‴), 78.7 (C-2′), 76.0 (C-3‴), 75.8 (C-5′), 74.4 (C-3″), 73.0 (C-2″), 72.9 (C-5″), 72.4 (C-4‴), 70.7 (C-2‴), 70.6 (C-3′), 70.4 (C-4′), 70.1 (C-4″), 67.9 (C-5‴), 66.5 (C-6′), 63.3 (C-6″), 21.04, 20.92, 20.85, 20.74, 20.72, 20.71, 20.68, 20.65, 20.64, 20.54 (CH₃CO– ×10), 18.0 (C-6‴); HR-ESI-MS (positive-ion mode) m/z : 1023.2958 [M+Na]⁺ (Calcd for C₄₄H₅₆O₂₆Na : 1023.2952).

Tyrosinase Inhibitory Activity

To the sample solution (20 µL) and 80 µL of 2.5 mM L-dihydroxyphenylalanine solution in 0.1 M phosphate buffer (pH 6.80) 100 µL of mushroom tyrosinase (100 U/mL) in the same buffer was added followed by incubation at 25°C for 5 min. The amounts of dopachrom, formed, were photometrically determined by measuring the optical density (OD) at 405 nm using a microplate reader. Kojic acid was used as a positive control. The inhibition of tyrosinase was calculated as:

  

A_control: without test sample. A_blank: without test sample and tyrosinase.

Cloning of High Melanin Pigment Producing Cells

Mouse melanoma cells (B16, TKG 0144) provided by the Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University were incubated in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% heat inactivated fetal calf serum supplemented with 100 µg/mL kanamycin (Wako Pure Chemical Industries, Ltd. (Osaka, Japan)) and 5.6 µg/mL of amphotericin B (Sigma) at 37°C under a 5% CO₂ atmosphere. From the above mentioned culture, a high melanin-pigment producing cell was cloned by the limiting dilution method and the cloned cell was named B16Y24.

Melanogenesis Inhibitory Activity

B16 and B16Y24 cells (5×10³ cells/well) in 400 µL medium were cultured in a 24-multiwell plate containing 40 µL aliquots of sample solutions and 40 µL of 12 mM theophylline solution in DMEM,¹¹⁾ and then the plate was incubated at 37°C under a 5% CO₂ atmosphere for 72 h. After removal of the medium, 120 µL of 1 M NaOH was added and the hermetically sealed multiwell plate was stood for 24 h at 37°C. The alkaline solutions (100 µL) were transferred to a 96-well plate and the absorbance of each well was measured at 405 nm using a microplate reader. Arbutin was used as a positive control. The inhibition of melanogenesis was calculated as:

  

A_control: without test sample. A_blank: without test sample and theophylline.

Cytotoxic Activity

Cytotoxic activity toward B16 and B16Y24 cells was determined by colorimetric cell viability assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT).¹³⁾ B16 and B16Y24 cells (5×10³ cells/well) in 100 µL of DMEM were cultured in the above mentioned medium in a 96-well plate, a 10 µL aliquot of a sample solution, 10 µL of a 12 mM theophylline solution in DMEM were added to each well, and then the plate was incubated at 37°C under a 5% CO₂ atmosphere for 72 h. After removal of the medium, a solution of MTT (100 µL, 0.5 mg/mL) was added to each well and the incubation was continued for a further 1 h. The absorbance of each well was measured at 540 nm using a microplate reader. The cytotoxic activity was calculated as:

  

A_control: without test sample. A_blank: without test sample and theophylline.

Acknowledgment

The authors are grateful for access to the superconducting NMR instrument and ESI-TOF-MS at the Analytical Center of Molecular Medicine and the Analysis Center of Life Science, respectively, of the Hiroshima University Faculty of Medicine. This work was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan, the Japan Society for the Promotion of Science, and the Ministry of Health, Labour and Welfare. Thanks are also due to the Astellas Foundation for Research on Medicinal Resources and the Takeda Science Foundation for the financial support.

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