Reinvestigation of structures of robustasides B and C, and isolation of (E)-2,5-dihydroxycinnamic acid esters of arbutin and glucose from the leaves of Grevillea robusta

Article abstract of DOI:10.1248/cpb.c12-00549

From the leaves of Grevillea robusta, compounds whose NMR data were superimposable on those of robustasides B and C were isolated along with two new compounds, (E)-2,5-dihydroxycinnamic acid esters of arbutin and D-glucose, and two known compounds, robust

Full text of DOI:10.1248/cpb.c12-00549

October 2012
Note
Chem. Pharm. Bull. 60(10) 1347–1350 (2012)
1347
Reinvestigation of Structures of Robustasides B and C, and Isolation of
(E)-2,5-Dihydroxycinnamic Acid Esters of Arbutin and Glucose from the
Leaves of Grevillea robusta
,a,b
Yukiko Yamashita-Higuchi,a Sachiko Sugimoto,a Katsuyoshi Matsunami,a and Hideaki Otsuka*
a Department of Pharmacognosy, Graduate School of Biomedical and Health Sciences, Hiroshima University; 1–2–3
b
Kasumi, Minami-ku, Hiroshima 734–8553, Japan: and Department of Natural Products Chemistry, Faculty of
Pharmacy, Yasuda Women’s University; 6–13–1 Yasuhigashi, Asaminami-ku, Hiroshima 731–0153, Japan.
Received June 20, 2012; accepted July 10, 2012
From the leaves of Grevillea robusta, compounds whose NMR data were superimposable on those of ro-
bustasides B and C were isolated along with two new compounds, (E)-2,5-dihydroxycinnamic acid esters of
arbutin and D-glucose, and two known compounds, robustaside A and (E)-2,5-dihydroxycinnamic acid. The
structures of robustasides B and C were not arbutin caffeates, being revised to arbutin (E)-2,5-dihydroxycin-
namic acid esters.
Key words Grevillea robusta; Proteaceae; robustaside B; robustaside C; (E)-2,5-dihydroxycinnamic acid;
grevilloside
Grevillea robusta A. CUNN., which belongs to Proteaceae, resonances with those of methyl (E)-2,5-dihydroxycinnamate
originates from subtropical areas of eastern Australia and has (9) and caffeate (10)7) (Table 1), it is obvious that the acyl moi-
been planted in Japan. It is an evergreen tree between 20–35m ety is (E)-2,5-dihydroxycinnamate. Therefore, the structure of
in height with dark green delicately dented bipinnatifid leaves 1 was elucidated to be arbutin 6′-O-2,5-dihydroxycinnamic
reminiscent of fronds. The leaves are 15–30cm long with acid ester, as shown in Fig. 1, and the structure of robustaside
grey-white or rusty undersides. Phytochemical investigation B′ (7) was revised to 1.
of the same plant, collected in Egypt, has been reported
Robustaside C (2), [α]D26 −75.7, was also isolated as an
and several phenolic glucosides were isolated.1) Cytotoxic amorphous powder and its elemental composition was de-
5-alkylresorcinol metabolites were also isolated from the title termined to be C27H32O15 by HR-ESI-MS. The IR and UV
plant2) and a MeOH extract of its timber exhibited potent spectra were similar to those of 1, and both the H- and
1
leishmanicidal activity.3) In previous papers, the isolation of 13C-NMR spectra also showed good similarity, except for the
glucosides of 5-alkylresorcinol derivatives was reported.4,5)
Further phytochemical work resulted in the isolation of two
known compounds, robustasides B (1) and C (2), and two
new (E)-2,5-dihydroxycinnamic acid esters of arbutin and
D-glucose (3, 4) along with arbutin 6′-O-p-coumarate, ro-
bustaside A (5)1) and (E)-2,5-dihydroxycinnamic acid (6).6)
The structures of robustasides B′ (7) and C′ (8) reported by
Ahmed et al.1) must be revised according to this investigation
(Fig. 1).
Robustaside B (1), [α]D26 −52.7, was isolated as an amor-
phous powder and its elemental composition was determined
to be C21H22O10 by high-resolution electrospray-ionization
mass-spectrometry (HR-ESI-MS). The IR spectrum exhib-
ited absorption bands ascribable to hydroxy (3363cm−1) and
ester carbonyl (1697cm−1) groups, and aromatic rings (1629,
1
1509cm−1). In the H-NMR spectrum, four protons coupled
with an AA′BB′ system, three aromatic protons [δH 6.72 (2H)
and 6.93], two olefinic protons with a trans geometry and
an anomeric proton (δH 4.73) were observed, and D-glucose
was identified by sugar analysis using a chiral detector. The
1
13C-NMR spectral data as well as the H-NMR data were
the same as those reported for robustaside B′ (7), which was
isolated from the same plant. In the heteronuclear multiple
bond correlation (HMBC) spectrum, however, the H-7‴ pro-
ton [δH7.97 (d, J=16Hz)] showed correlation peaks with δC
151.7 (C-2‴, s) and 114.9 (C-6‴, d) (Fig. 2a). Thus, the acyl
moiety may not be a caffeate. In comparison of its 13C-NMR
The authors declare no conflict of interest.
Fig. 1. Structures of Isolated and Reference Compounds
© 2012 The Pharmaceutical Society of Japan
*To whom correspondence should be addressed. e-mail: hotsuka@hiroshima-u.ac.jp

1348
Vol. 60, No. 10
to the hydroxy group at the 6′-position judging from the
HMBC correlation between δH 4.60 and 4.41 on C-6′ and δC
169.2 (C-9‴), whereas the other carbonyl carbon (δC 168.6)
showed a HMBC correlation with the proton shifted downfield
at δH 5.08, which was assigned to H-2′ from the correlation
1
with the anomeric proton (δH 4.97) in the H–1H correlation
spectrum. Therefore, the structure of compound 3 was eluci-
dated to be arbutin 2′,6′-di-O-(E)-2,5-dihydroxycinnamate, as
shown in Fig. 1, and it was given the trivial name, grevilloside
I.
Compound 4, [α]D28 +28.8, was isolated as an amorphous
powder and its elemental composition was determined to be
C15H18O9. Although compound 4 gave distinct two peaks on
HPLC separation, the NMR spectra for these two peaks were
identical. Thus, the compounds isolated from these two peaks
were interconvertible with each other and 4 must exist as an
equilibrium mixture. On acid hydrolysis, 4 gave D-glucose as
a sugar component and in the NMR spectra of 4, two ano-
meric carbons (δC 94.8, 98.3) were observed with the respec-
tive anomeric protons [δH 5.12 (½H, d, J=4Hz, H-1′α), and
4.53 (½H, d, J=8Hz, H-1′β)]. The abundance ratio of α- and
β-isomers was estimated to be nearly 1:1 from the integrals of
1
isolated signals in the H-NMR spectrum.
Experimental
General Experimental Procedures The general experi-
mental procedures used in this study were the same as those
used in a previous paper.4) Methyl (E)-2,5-dihydroxycin-
namate (9) was purchased from Sigma-Aldrich Co., LLC (St.
Louis, MO, U.S.A.).
Fig. 2. Diagnostic HMBC Correlations for 1 (a) and 2 (b)
Plant Material The plant material was the same
as that used in our previous experiment (accession No.
05-GR-Okinawa-0629).4)
The dashed line curve in (b) shows the results of difference NOE experiments.
presence of terminal glucopyranose. Glucose was determined
Extraction and Isolation Dried leaves of G. robusta
to be D-series by the same method as used for 1. Since the (6.35kg) were extracted three times with MeOH (30L) at
13C-NMR resonances of the arbutin moiety were superimpos- 25°C for one week and then concentrated to 3L in vacuo.
able on those of 1, the terminal β-D-glucopyranose must be The extract was washed with n-hexane (3L, 32.6g) and
attached to one of the phenolic hydroxy groups on the acyl then the MeOH layer was concentrated to a gummy mass.
group. In the HMBC spectrum, the anomeric proton (δH 4.79) The latter was suspended in water (3L) and then extracted
showed a cross peak with δC 151.3 (C-2‴), and significantly with EtOAc (3L) to give 160g of an EtOAc-soluble frac-
different nuclear Overhauser effect (NOE) correlations were tion. The aqueous layer was extracted with 1-BuOH (3L) to
observed between the anomeric proton and the H-3‴ (δH give a 1-BuOH-soluble fraction (405g), and the remaining
7.16) aromatic proton (Fig. 2b). Further HMBC correlations water-layer was concentrated to yield 475g of a water-soluble
between H-7‴ (δH 141.9) and C-2‴ and C-6‴, and H-6‴ [δH fraction. A portion (237g) of the 1-BuOH-soluble fraction was
7.04 (d, J=3Hz)] and C-2‴, and three aromatic protons of the applied to a Diaion HP-20 column (Φ=75mm, L=50cm) using
acyl group were well resolved and clearly coupled in an ABX H2O–MeOH (4:1, 8L), (2:3, 8L), (3:2, 8L), and (1:4, 8L),
system. Therefore, the structure of 2 was elucidated to be and MeOH (6L), 1L fractions being collected. The residue
2″-O-β-D-glucopyranoside of robustaside B (1), as shown in (19.9g in fractions 4–6) of the 20% MeOH eluent was sub-
Fig. 1. On the basis of the aforementioned spectroscopic data, jected to silica gel (450g) CC, with elution with CHCl3 (3L)
the structure of robustaside C′ (8) must be revised to 2.
and CHCl3–MeOH [(49:1, 3L), (24:1, 3L), (23:2, 3L), (9:1,
Compound 3, [α]D22 −23.5, was isolated as an amorphous 3L), (7:1, 3L), (17:3, 3L), (4:1, 3L), (3:1, 3L), and (3:2,
powder and its elemental composition was determined to 3L)]. The fractions collected were 500mL.
be C30H28O13 by HR-ESI-MS. The IR and UV spectral data
The residue (45.1g) of fractions 7–12 obtained on Diaion
were similar to those of 1 and 2, and the NMR spectra also HP-20 CC was subjected to silica gel (450g) CC, with elution
showed close resemblance to those of 1. In the 13C-NMR with CHCl3 (4.5L) and CHCl3–MeOH [(49:1, 4.5L), (24:1,
spectrum, signals (δC 168.6, 169.2) for two carbonyl carbons 4.5L), (23:2, 4.5L), (9:1, 4.5L), (7:1, 4.5L), (17:3, 4.5L),
were observed and some of the sp2 carbon signals assignable (4:1, 4.5L), (3:1, 4.5L), and (3:2, 4.5L)]. The fractions col-
to the acyl moieties appeared at two close frequencies. In the lected were 500mL. An aliquot (1.76g) of combined fractions
1H-NMR spectrum, the integrals of the protons assignable to 61–66 (2.61g) of the 12.5–15% MeOH eluate was separated
the acyl moieties also implied that two units of 2,5-dihydroxy- by octadecyl silica (ODS) open CC to give two residues in
cinnate were present in 3. One of the acyl moieties was linked fractions 88–99 (204mg) and fractions 133–159 (435mg).

October 2012
1349
Tab1e 1. 13C-NMR Spectroscopic Data for Robustasides B (1) and C (2), Grevilloside I (3), Compound 4, (E)-2,5-Dihydroxycinnamic Acid (6), Methyl
(E)-2,5-Dihydroxycinnamate (9), and Caffeate (10)
C
1
2
3
4
6
9
10 f)
1
152.5
119.7
116.8
153.9
103.9
75.0
152.4
119.8
116.8
154.0
103.9
75.0
152.3
119.9
116.9
154.1
102.4
75.2
2,6
3,5
4
α
94.0
73.8
75.5
72.1
70.9
64.9
122.94
β
98.3
76.3
78.0
71.8
74.8
65.0
122.96
1′
2′
3′
78.0
78.2
76.1
4′
5′
6′
1″
2″
3″
4″
5″
6″
7″
8″
9″
71.9
71.4
72.0
75.6
75.5
75.7
64.8
64.8
64.7
122.97a)
151.6
118.09b)
120.4
151.35c)
114.86d)
142.6
117.91e)
168.6
123.1
151.4
118.5
120.1
151.4
114.8
142.3
118.0
171.3
123.1
151.6
118.0
120.3
151.4
114.8
142.3
117.9
170.0
52.0
127.3
115.3
149.8
146.8
116.4
123.3
148.2
114.7
168.0
151.6
118.1
120.32
120.34
151.3
114.9
142.46
117.93
169.44
142.5
117.99
169.53
-OMe
1‴
2‴
3‴
4‴
5‴
6‴
7‴
8‴
9‴
1⁗
2⁗
3⁗
4⁗
5⁗
6⁗
123.0
151.7
118.1
120.4
151.4
114.9
142.5
117.9
169.3
126.8
151.3
120.09
120.06
154.1
113.7
141.9
118.7
169.1
104.0
75.5
123.00a)
151.7
118.11b)
120.4
151.36c)
114.95d)
142.7
117.99e)
169.2
78.2
71.7
78.0
62.7
a–e) Maybe exchangeable. f) Data from ref. 5.
The former was separated by DCCC to give 35.6mg of 6 in An aliquot (1.87g) of combined fractions 55–62 (2.80g) of
fractions 39–46. The latter was purified by DCCC to afford the 12.5% MeOH eluate was separated by ODS open CC
227mg of 1 in fractions 24–29. An aliquot (1.85g) of com- and the residue (167mg) in fractions 129–138 by DCCC to
bined fractions 67–73 (2.01g) of the 12.5–15% MeOH eluate give 23.9mg of 1 in fractions 21–27. The residue (170mg) in
was separated by ODS open CC to give a residue in fractions fractions 175–182 was separated by DCCC and the residue
70–88 (273mg), which was then separated by DCCC. The res- (31.6mg) in fractions 28–35 was purified by HPLC (H2O–
idue (130mg) in fractions 12–18 was purified by HPLC (H2O– MeOH, 3:2) to give 13.9mg of 3 from the peak at 24min.
MeOH, 4:1) to give two interconvertible peaks at 10min and
Robustaside B (1): Amorphous powder. [α]D26 −52.7 (c=0.96,
13min (a total of 9.2mg of 4). An aliquot (1.74g) of combined MeOH). IR νmax (film) cm−1: 3363, 2953, 2837, 1697, 1629,
fractions 67–73 (3.85g) of the 12.5–15% MeOH eluate was 1509, 1455, 1341, 1267, 1211, 1074, 1028. UV λmax (MeOH) nm
1
separated by ODS open CC to give 25.3mg of 2 in fractions (logε): 280 (4.15), 220 (4.18). H-NMR (400MHz, CD3OD) δ:
100–115.
7.97 (1H, d, J=16Hz, H-7‴), 6.96 (2H, d, J=9Hz, H-2, 6), 6.93
The residue (39.0g) of fractions 13–18 obtained on Diaion (1H, brs, H-6‴), 6.72 (2H, m, H-3‴, 4‴), 6.67 (2H, d, J=9Hz,
HP-20 CC was subjected to silica gel (400g) CC, and eluted H-3, 5), 6.54 (1H, d, J=16Hz, H-8‴), 4.73 (1H, d, J=7Hz,
with CHCl3 (4.5L) and CHCl3–MeOH [(49:1, 4.5L), (24:1, H-1′), 4.54 (1H, dd, J=12, 2Hz, H-6′a), 4.36 (1H, dd, J=12,
4.5L), (23:2, 4.5L), (9:1, 4.5L), (7:1, 4.5L), (17:3, 4.5L), 6Hz, H-6′b), 3.68–3.33 (4H, m, H-2′, 3′, 4′, 5′). 13C-NMR
(4:1, 4.5L), (3:1, 4.5L), and (3:2, 4.5L)]. Each fraction col- (100MHz, CD3OD): Table 1. HR-ESI-MS (positive-ion mode)
lected was 500mL. An aliquot (1.90g) of combined fractions m/z: 457.1112 [M+Na]+ (Calcd for C21H22O10Na: 457.1105).
48–54 (4.97g) of the 10% MeOH eluate was separated by
Robustaside C (2): Amorphous powder. [α]D26 −75.5 (c=0.75,
ODS open CC and the residue (297mg) in fractions 171–180 MeOH). IR νmax (film) cm−1: 3363, 2921, 1697, 1629, 1558,
was purified by DCCC to give 19.5mg of 5 in fractions 37–40. 1510, 1454, 1392, 1289, 1212, 1072. UV λmax (MeOH) nm

1350
Vol. 60, No. 10
1
(logε): 280 (4.20), 216 (4.27). H-NMR (400MHz, CD3OD)
Sugar Analysis About 500µg of each compound (3, 4)
δ: 8.20 (1H, d, J=16Hz, H-7‴), 7.16 (1H, d, J=9Hz, H-3‴), was hydrolyzed with 1M HCl (0.1mL) at 90°C for 2h. The
7.04 (1H, d, J=3Hz, H-6‴), 6.95 (2H, d, J=9Hz, H-2, 6), reaction mixtures were partitioned with an equal amount of
6.83 (1H, dd, J=9, 3Hz, H-4″), 6.66 (2H, d, J=9Hz, H-3, 5), EtOAc (0.1mL), and the water layers were analyzed with a
6.47 (1H, d, J=16Hz, H-8‴), 4.79 (1H, m, H-1⁗), 4.75 (1H, m, chiral detector (JASCO OR-2090plus) on an amino column
H-1′), 4.53 (1H, dd, J=12, 2Hz, H-6′a), 4.40 (1H, dd, J=12, [Asahipak NH2P-50 4E, Φ=4.6mm, L=25cm, CH3CN–H2O
6Hz, H-6′b), 3.87 (1H, dd, J=12, 2Hz, H-6⁗a), 3.70 (1H, dd, (3:1), 1mL/min]. The hydrolyzates of 3 and 4 each gave a
J=12, 6Hz, H-6⁗b), 3.73–3.35 (8H, m, H-2′, 3′, 4′, 5′, 2⁗, 3⁗, peak for D-glucose at 14.0min with a positive optical rotation
4⁗, 5⁗). 13C-NMR (100MHz, CD3OD): Table 1. HR-ESI-MS sign. The peaks were identified by co-chromatography with
(positive-ion mode) m/z: 619.1636 [M+Na]+ (Calcd for authentic D-glucose.
C27H32O15Na: 619.1633).
Grevilloside I (3): Amorphous powder. [α]D22 −23.5 (c=0.93,
Acknowledgements The authors are grateful for access to
MeOH). IR νmax (film) cm−1: 3354, 2930, 2860, 1699, 1627, the superconducting NMR instrument (JEOL JNM α-400) at
1507, 1206, 1076. UV λmax (MeOH) nm (logε): 359 (4.01), the Analytical Center of Molecular Medicine of the Hiroshima
1
279 (4.34), 213 (4.14). H-NMR (400MHz, CD3OD) δ: 8.01 University Faculty of Medicine, and an Applied Biosystem
(2H, J=16Hz, H-7″, 7‴), 6.94 (2H, brs, H-6″, 6‴), 6.87 (2H, d, QSTAR XL system ESI (Nano Spray)-MS at the Analysis
J=9Hz, H-2, 6), 6.73–6.71 (4H, m, H-3,″ 4″, 3‴, 4‴), 6.65 (2H, Center of Life Science of the Graduate School of Biomedical
d, J=9Hz, H-3, 5), 6.57 (2H, d, J=16Hz, H-8″, 8‴), 5.08 (1H, Sciences, Hiroshima University. This work was supported in
dd, J=8, 8Hz, H-2′), 4.97 (1H, d, J=8Hz, H-1′), 4.60 (1H, dd, part by Grants-in-Aid from the Ministry of Education, Cul-
J=12, 2Hz, H-6′a), 4.41 (1H, d, J=12, 6Hz, H-6′b), 3.78–3.70 ture, Sports, Science and Technology of Japan, and the Japan
(2H, m, H-3′, 5′), 3.56 (1H, dd, J=8Hz, H-4′). 13C-NMR Society for the Promotion of Science. Thanks are also due to
(100MHz, CD3OD): Table 1. HR-ESI-MS (positive-ion mode) the Research Foundation for Pharmaceutical Sciences and the
m/z: 619.1425 [M+Na]+ (Calcd for C30H28O13Na: 619.1422).
Compound 4: Amorphous powder, [α]D28 +28.8 (c=0.91,
MeOH). IR νmax (film) cm−1: 3388, 2924, 2860, 1698, 1630,
1504, 1457, 1188, 1028. UV λmax (MeOH) nm (logε): 358
Takeda Science Foundation for the financial support.
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