Flavonoid glycosides from Sedum bulbiferum

Article abstract of DOI:10.1248/cpb.c17-00678

The MeOH extract from dried whole Sedum bulbiferum MAKINO (Crassulaceae) plants yielded 34 compounds, including six new flavonoid glycosides and 28 known compounds. The structures of new compounds were established using NMR, Mass spectroscopic analysis an

Full text of DOI:10.1248/cpb.c17-00678

Vol. 65, No. 12
Chem. Pharm. Bull. 65, 1199–1204 (2017)
1199
Note
Flavonoid Glycosides from Sedum bulbiferum
,a
Tsutomu Warashina* and Toshio Miyaseb
a School of Food and Nutritional Sciences, University of Shizuoka; 52–1 Yada, Suruga-ku, Shizuoka 422–8526,
b
Japan: and School of Pharmaceutical Sciences, University of Shizuoka; 52–1 Yada, Suruga-ku, Shizuoka 422–8526,
Japan.
Received August 27, 2017; accepted September 27, 2017
The MeOH extract from dried whole Sedum bulbiferum MAKINO (Crassulaceae) plants yielded 34 com-
pounds, including six new flavonoid glycosides and 28 known compounds. The structures of new compounds
were established using NMR, Mass spectroscopic analysis and chemical evidence.
Key words Sedum bulbiferum;ꢀCrassulaceae;ꢀflavonoidꢀglycoside;ꢀkaempferol
The genus SedumꢀareꢀclassifiedꢀintoꢀtheꢀCrassulaceausꢀfam- signals were observed at δ 5.61 (1H, d, J=1.5ꢀHz),ꢀ 5.56ꢀ (1H,ꢀ
ily, and recently, some Sedum species were reported to be use- d, J=1.5ꢀHz),ꢀ4.36ꢀ(1H,ꢀd,ꢀJ=8.0ꢀHz)ꢀandꢀδ 107.2, 103.2, 100.0.
fulꢀ inꢀ theꢀ cosmeticꢀ industry,ꢀ forꢀ example,ꢀ skinꢀ moisturizingꢀ Thus, 19ꢀ wasꢀ consideredꢀ toꢀ beꢀ aꢀ kaempferolꢀ triglycoside.ꢀ
and lightening effects, improvement and protection of rough Acid hydrolysis of 19 afforded L-rhamnose and D-quinovose
skin,ꢀandꢀproliferationꢀofꢀskinꢀfibroblasts.1) Sedum bulbiferum withꢀ kaempferol.ꢀ Onꢀ theꢀ basisꢀ ofꢀ 1H-detected heteronuclear
1
MAKINO is also one of the Crassulaceaus plants and is widely multiple quantum coherency (HMQC) and H–1H shift cor-
distributedꢀ inꢀ theꢀ Honshu,ꢀ Shikoku,ꢀ andꢀ Kyushuꢀ Islandsꢀ ofꢀ relation spectroscopy (COSY) measurements, the signals at δ
Japan,ꢀ theꢀ Koreanꢀ Peninsula,ꢀ andꢀ China.ꢀ Thisꢀ plantꢀ hasꢀ notꢀ 5.61, 103.2, δ 5.56, 100.0, and δ 4.36, 107.2 were assigned at
beenꢀusedꢀasꢀaꢀforkꢀmedicine,ꢀbutꢀitsꢀextractꢀwasꢀreportedꢀtoꢀ the anomeric proton and carbon of α-L-rhamnopyranoses and
exhibit the anti-tumor activity.2) There have been no detailed β-D-quinovopyranose, whose conformations were judged from
1
reports about the constituents of S. bulbiferum, so in the the H-chemical shift or coupling constant of each anomeric
course of research to determine the phytochemicals in the protonꢀ signal,ꢀ respectively.ꢀ Inꢀ theꢀ 1H-detected heteronuclear
Crassulaceaus family we investigated the constituents of this multiple-bond connectivity (HMBC) measurements, long-
plant.ꢀ Weꢀ wereꢀ particularlyꢀ interestedꢀ inꢀ efficaciousꢀ agentsꢀ range correlations were exhibited between C-3 of the aglycone
fromꢀthisꢀfamilyꢀinꢀtheꢀcosmeticꢀfield.
(δ 137.1) and H-1 of α-L-rhamnopyranose (δ 5.61), C-2 of α-L-
MeOH extract from dried whole S. bulbiferum plants was rhamnopyranose (δ 83.0) and H-1′ of β-D-quinovopyranose
suspended in 90% MeOH in water. The solution was extracted (δ 4.36), and C-7 of the aglycone (δ 163.7) and H-1″ of α-L-
with n-hexane and partitioned into a n-hexane-soluble fraction rhamnopyranose (δꢀ 5.56).ꢀ Inꢀ addition,ꢀ theꢀ rotatingꢀ frameꢀ
and a 90% MeOH in water-soluble fraction. The 90% MeOH nuclear Overhauser effect (ROE) difference experiment ex-
in water-soluble fraction was dried in vacuo, and the residue hibited ROEs between H-1′ of β-D-quinovopyranose and H-2
was suspended in water. The suspension was extracted with of α-L-rhamnopyranose [δ 4.26 (1H, dd, J=3.5,ꢀ 1.5ꢀHz)],ꢀ andꢀ
diethylether and partitioned into an ether-soluble fraction and H-1‴ of α-L-rhamnopyranose and H-6 (δ 6.48), H-8 (δ 6.74)
a water-soluble fraction. The residues of the water soluble- of the aglycone. Hence, 19ꢀ wasꢀ determinedꢀ toꢀ beꢀ kaempferolꢀ
fraction and the ether-soluble fraction were subjected to silica 3-O-β-D-quinovopyranosyl-(1→2)-α-L-rhamnopyranoside-7-
gel column chromatography and/or semi-preparative HPLC O-α-L-rhamnopyranoside.
toꢀgiveꢀthreeꢀflavonoidsꢀ(1–3),ꢀandꢀthirty-oneꢀflavonoidꢀglyco-
TheꢀmolecularꢀformulaꢀofꢀbulbiferumosideꢀIIꢀ(21) was pro-
sides (4–34). Compounds 1,3) 2,3) 3,3) 4,4) 5,5) 6,5) 7,6) 8,7) 9,8) posed to be C32H38O19, based on HR-FAB-MS. The aglycone
10,9) 11,10) 12,11) 13,4) 14,4) 15,5,12) 16,13) 17,12) 18,14) 20,15) 22,16) of 21 was determined to be quercetin, following observation
23,17) 26,18) 27,19) 28,20) 29,21) 30,21) 31,21) and 3422) were identi- of 15 carbon signals including twelve aromatic carbons (δ
fiedꢀ asꢀ shownꢀ inꢀ Chartꢀ 1ꢀ basedꢀ onꢀ theꢀ 1H and/or 13C-NMR 163.6, 163.0, 158.1, 150.1, 146.5, 122.9, 122.7, 116.9, 116.5,
spectroscopic data.
107.5,ꢀ100.6,ꢀ95.6),ꢀtwoꢀolefinꢀcarbonsꢀ(δ 159.7 and 137.1), one
TheꢀmolecularꢀformulaꢀofꢀbulbiferumosideꢀIꢀ(19), C33H40O18, carbonyl carbon (δ 179.9), and the AX-type aromatic protons
was established based on high resolution (HR)-FAB-MS. The [δ 6.71 (1H, d, J=2.0ꢀHz)ꢀandꢀ6.46ꢀ(1H,ꢀd,ꢀJ=2.0ꢀHz)]ꢀderivedꢀ
aglycone of 19ꢀ wasꢀ identifiedꢀ asꢀ kaempferol,ꢀ accordingꢀ toꢀ from the A-ring and the AMX-type proton signals [δ 7.38 (1H,
observations of 15 carbon signals including twelve aromatic d, J=2.0ꢀHz),ꢀ 7.33ꢀ (1H,ꢀ dd,ꢀ J=8.0,ꢀ 2.0ꢀHz),ꢀ andꢀ 6.93ꢀ (1H,ꢀ d,ꢀ
1
carbons (δ 163.7, 163.1, 161.9, 158.2, 132.0×2, 116.7×2, 122.4, J=8.0ꢀHz)]ꢀ derivedꢀ fromꢀ theꢀ B-ringꢀ inꢀ theꢀ 13C- and H-NMR
1
107.2,ꢀ 100.6,ꢀ 95.7),ꢀ twoꢀ olefinꢀ carbonsꢀ (δ 159.8, 137.1), and spectroscopic data. Moreover, in the H- and 13C-NMR spec-
one carbonyl carbon (δ 179.9), and the AX-type aromatic tra of 21, three anomeric proton and carbon signals were also
proton [δ 6.74 (1H, d, J=2.0ꢀHz),ꢀ andꢀ 6.48ꢀ (1H,ꢀ d,ꢀ J=2.0ꢀHz)]ꢀ observed at δ 5.37 (1H, d, J=1.5ꢀHz),ꢀ 5.56ꢀ (1H,ꢀ d,ꢀ J=1.5ꢀHz),ꢀ
and AA′XX′-type aromatic proton signals [δ 7.82 (2H, brd, 4.27 (1H, d, J=7.5ꢀHz)ꢀ andꢀ δ 107.8, 103.3, 100.0. Thus, 21
J=8.5ꢀHz),ꢀ andꢀ 6.96ꢀ (2H,ꢀ brꢀd,ꢀ J=8.5ꢀHz)]ꢀ inꢀ theꢀ 13C- and was considered to be a quercetin triglycoside. Acid hydroly-
1H-NMR spectroscopic data. Moreover, in the 1H- and sis of 21 afforded L-rhamnose and D-xylose with quercetin.
13C-NMR spectra of 19, three anomeric proton and carbon Because the 13C-NMR spectroscopic data of the sugar moiety
*ꢀToꢀwhomꢀcorrespondenceꢀshouldꢀbeꢀaddressed.ꢀ e-mail:ꢀwarashin@u-shizuoka-ken.ac.jp
© 2017 The Pharmaceutical Society of Japan

1200
Chem. Pharm. Bull.
Vol. 65, No. 12 (2017)
Chart 1. Structure of Compounds 1–34
of 21 were consistent with those of 20, the structure of 21 (1H, dd, J=12.0,ꢀ 2.0ꢀHz)ꢀ andꢀ 4.09ꢀ (1H,ꢀ dd,ꢀ J=12.0,ꢀ 7.0ꢀHz)].ꢀ
wasꢀ identifiedꢀ asꢀ quercetinꢀ 3-O-β-D-xylopyranosyl-(1→2)-α- Moreover, HMBC measurements in 24 revealed long-range
L-rhamnopyranoside-7-O-α-L-rhamnopyranoside. This sugar correlations between H-6′ of β-D-glucopyranose (δ 4.58,
linkageꢀ wasꢀ confirmedꢀ byꢀ consequencesꢀ ofꢀ theꢀ HMBCꢀ mea- 4.09) and C-α′ of the (E)-p-coumaroyl group (δ 168.7). Simi-
surement and ROE difference experiments irradiating each larly, the HOHAHA difference experiments of 25 showed
anomeric proton.
signal enhancement for H-6′ of β-D-glucopyranose [δ 4.29
Theꢀ molecularꢀ formulaeꢀ ofꢀ bulbiferumosidesꢀ IIIꢀ (24) (1H, dd, J=12.0,ꢀ 2.0ꢀHz)ꢀ andꢀ 4.25ꢀ (1H,ꢀ dd,ꢀ J=12.0,ꢀ 6.5ꢀHz)]ꢀ
andꢀ IVꢀ (25)ꢀ wereꢀ identifiedꢀ asꢀ C42H46O21 by HR-FAB-MS. on irradiating H-1′ of the β-D-glucopyranosyl groups [δ
The NMR spectroscopic data for 24 and 25 were similar 4.41 (1H, d, J=8.0ꢀHz)].ꢀ HMBCꢀ correlationsꢀ wereꢀ observedꢀ
to those of 22, and signals due to the (E)-p-coumaroyl and between H-6′ of the β-D-glucopyranosyl group (δ 4.29,
(Z)-p-coumaroyl groups were observed in 24 and 25, re- 4.25) and C-α′ of the (Z)-p-coumaroyl groups (δ 167.8)
spectively.ꢀ Additionally,ꢀ alkalineꢀ hydrolysisꢀ yieldedꢀ (E)-p- in 25. Thus, 24 and 25ꢀ wereꢀ establishedꢀ toꢀ beꢀ kaempferolꢀ
coumaric acid from 24 and (Z)-p-coumaric acid from 25 3-O-β-D-6-O-[4-hydroxy-(E)-cinnamoyl]-glucopyranosyl-
together with 22.ꢀ Inꢀ theꢀ homonuclearꢀ Haltmann–Hahnꢀ (1→2)-β-D-glucopyranoside-7-O-α-L-rhamnopyranoside
(HOHAHA) difference experiment of 24, irradiation of H-1′ andꢀ kaempferolꢀ 3-O-β-D-6-O-[4-hydroxy-(Z)-cinnamoyl]-
of β-D-glucopyranose [δ 4.47 (1H, d, J=8.0ꢀHz)]ꢀ exhibitedꢀ glucopyranosyl-(1→2)-β-D -glucopyranoside-7-O-α-L-
signal enhancement for H-6′ of β-D-glucopyranose [δ 4.58 rhamnopyranoside, respectively.

Vol. 65, No. 12 (2017)
Chem. Pharm. Bull.
1201
Chartꢀ 2.ꢀ KeyꢀROEs,ꢀHOHAHAs,ꢀandꢀHMBCꢀCorrelationsꢀofꢀCompoundsꢀ19 and 33
Bulbiferumosides V (32)ꢀ andꢀ VIꢀ (33) showed the mo- were collected on a JEOL JMS-700 spectrometer in the
lecular formulae, C35H42O21 and C44H48O23 by HR-FAB-MS. positive mode using a m-nitrobenzylꢀ alcoholꢀ matrix.ꢀ Bothꢀ
The NMR spectroscopic data for 32 were similar to those 1H- and 13C-NMR spectra were recorded a JEOL α-400 (400,
of 27, and signals due to the acetyl group were observed 100.40ꢀMHz,ꢀ respectively).ꢀ Chemicalꢀ shiftsꢀ areꢀ givenꢀ inꢀ δ
at δ 172.3, 20.4, and δ 1.75 (3H, s). Two-dimensional (2D) (ppm) with tetramethylsilane (TMS) as an internal standard.
NMR measurements and ROE experiments irradiating each Inverse-detectedꢀ heteronuclearꢀ correlationsꢀ wereꢀ measuredꢀ
anomeric proton were showed the presence of 27 as a part of usingꢀ HMQCꢀ (optimizedꢀ forꢀ 1JC–H=145ꢀHz)ꢀ andꢀ HMBCꢀ (op-
32,ꢀ whichꢀ wasꢀ supportedꢀ byꢀ alkalineꢀ hydrolysisꢀ affordingꢀ 27 timizedꢀ forꢀ nJC–H=8ꢀHz)ꢀ pulseꢀ sequencesꢀ withꢀ aꢀ pulseꢀ fieldꢀ
from 32. HOHAHA difference experiments irradiating H-1 gradient. The ROE difference spectra were recorded with the
(δ 5.41) and H-1′ (δ 4.78) of the β-D-glucopyranosyl groups mixing time set at 250ms. The HOHAHA difference spec-
showed signal enhancement at H-6s (δ 4.18, 4.03) and H-6′s traꢀ wereꢀ recordedꢀ withꢀ theꢀ spinꢀ lockingꢀ timeꢀ setꢀ atꢀ 180ꢀms.ꢀ
(δ 3.82, 3.71) of the β-D-glucopyranosyl groups. Moreover, in Aꢀ Hitachi-G-3000ꢀ gasꢀ chromatographꢀ withꢀ flameꢀ ionizationꢀ
the HMBC measurement, the long-range correlations were detectorꢀ (FID)ꢀ wasꢀ utilizedꢀ forꢀ CG.ꢀ JASCOꢀ 800ꢀ andꢀ 900ꢀ
observed at the above carbonyl carbon signal (δ 172.3) and system instruments were used for HPLC (column: Nomura
H-6s of β-D-glucopyranose. Thus, 32 was determined to be Chemical Co., Ltd., Aichi, Japan, Develosil-ODS 15/30 50mm
kaempferolꢀ 3-O-β-D-glucopyranosyl-(1→2)-β-D-(6-O-acetyl)- i.d.×100ꢀcmꢀ andꢀ YMCꢀ Co.,ꢀ Ltd.,ꢀ Kyoto,ꢀ Japan,ꢀ YMC-ODSꢀ
glucopyranoside-7-O-α-L-rhamnopyranoside. The NMR spec- 20mm i.d.×25 cm).
troscopic data for 33 suggested the presence of an acetyl
Plant Materials Whole S. bulbiferum plants were collect-
group and a (E)-p-coumaroyl group with 27. Based on the edꢀinꢀShizuokaꢀcity,ꢀJapanꢀinꢀMayꢀ2011ꢀandꢀ2012,ꢀandꢀidenti-
results of the HMBC measurement and HOHAHA difference fiedꢀ byꢀ Dr.ꢀ T.ꢀ Warashina.ꢀ Theꢀ driedꢀ materialsꢀ wereꢀ storedꢀ inꢀ
experiments irradiating the anomeric protons of the β-D- aꢀ herbariumꢀ ofꢀ theꢀ Universityꢀ ofꢀ Shizuokaꢀ (voucherꢀ number,ꢀ
glucopyranosyl groups, the acetyl group and the (E)-p-couma- 918-M).
royl group were attached at the C-6 and C-6′ positions of the
Extraction and Isolation Dried whole plants of S. bul-
β-D-glucopyranosyl groups, respectively. Hence, 33 was iden- biferum (390g) were extracted twice with MeOH (3.5L) under
tifiedꢀasꢀkaempferolꢀ3-O-β-D-[6-O-4-hydroxy-(E)-cinnamoyl]- refluxꢀ forꢀ 3ꢀh.ꢀ Theꢀ extractꢀ (29.8ꢀg)ꢀ wasꢀ concentratedꢀ underꢀ
glucopyranosyl-(1→2)-β-D-(6-O-acetyl)-glucopyranoside-7- reduced pressure and the residue was dissolved in the MeOH–
O-α-L-rhamnopyranoside (Chart 2).
H2O (9:1) solution (1L). This solution was successively ex-
The present investigation of the constituents in Sedum bul- tracted with n-hexane (1L). The MeOH–H2O (9:1) extract
biferumꢀ affordedꢀ sixꢀ newꢀ flavonoidꢀ glycosidesꢀ togetherꢀ withꢀ was evaporated dry, and the resulting residue was partitioned
28ꢀ knownꢀ compounds.ꢀ Inꢀ ourꢀ previousꢀ paper,23) we reported between the diethylether-soluble fraction and H2O-soluble
someꢀ kaempferolꢀ glycosidesꢀ fromꢀ Botrychium ternatum, that fraction. The ether-soluble fraction was concentrated, and the
didꢀ notꢀ showꢀ proliferationꢀ onꢀ humanꢀ skinꢀ fibroblastsꢀ orꢀ aꢀ ty- residue (1.47g) was subjected to silica gel column chroma-
rosinaseꢀinhibitoryꢀeffect.ꢀSinceꢀtheꢀflavonoidꢀglycosidesꢀfromꢀ tography with a CHCl3–MeOH (99:1→85:15, v/v) system to
S. bulbiferum are similar to those of B. ternatum, they are obtain eight fractions. (A (188mg), B (82mg), C (65mg), D
notꢀ expectedꢀ toꢀ beꢀ usefulꢀ inꢀ theꢀ cosmeticꢀ field.ꢀ Inꢀ theꢀ litera- (177mg), E (150mg), F (218mg), G (72mg), and H (307mg)).
ture,24–26)ꢀsomeꢀflavonoidꢀglycosidesꢀfromꢀotherꢀSedum species Using semi-preparative HPLC (YMC-ODS 20mm i.d.×25cm
were reported to have lipid accumulation inhibitory, anti- (MeCN–H2O (1:4, 22.5:77.5, 25:75, 3:7, 32.5:67.5, v/v), and
nociceptive,ꢀ andꢀ anti-inflammatoryꢀ activities,ꢀ andꢀ weꢀ wereꢀ MeOH–H2O (35:65, 1:1, v/v), fraction C afforded 2 (10mg).
interestedꢀinꢀtheseꢀeffectsꢀinꢀtheꢀflavonoidꢀglycosidesꢀaffordedꢀ Fraction D yielded 1 (5mg), 3 (5mg), and 34 (8mg). Fractions
by S. bulbiferum.
F and G produced 13 (10mg) and 4 (9mg), respectively.
The H2O layer of the MeOH–H2O (9:1) extract was passed
through a porous polymer gel (Diaion HP-20, Mitsubishi
Experimental
General Procedures Optical rotations and UV spectra Chemicalꢀ Co.,ꢀ Tokyo,ꢀ Japan)ꢀ columnꢀ withꢀ absorbedꢀ mate-
were measured on a JASCO-P2200 polarimeter and JASCO rial being eluted with MeOH–H2O 1:1 (2.7L), 7:3 (2.5L),
V-630 spectrophotometer, respectively. FAB-MS spectra and MeOH (2.0L). The MeOH–H2O 1:1 and 7:3 fractions

1202
Chem. Pharm. Bull.
Vol. 65, No. 12 (2017)
Table 1. 13C-NMR Spectroscopic Data of Compounds 19, 21, 24, 25, 32,
and 33
and MeOH–H2O (4:6, 45:55, v/v)). This residue yielded 4
(15mg), 5 (8mg), 6 (9mg), 7 (18mg), 8 (15mg), 10 (6mg), 11
(16mg), 12 (7mg), 14 (31mg), 15 (7mg), 17 (5mg), 19 (2mg),
20 (35mg), 22 (24mg), 24 (87mg), 25 (9mg), 29 (48mg), and
33 (16mg). The residue of the MeOH–H2O (1:1) fraction
(3.0g) was subjected to semi-preparative HPLC (Develosil-
ODS-15/30 50mm i.d.×100cm (MeCN–H2O 14:86→19:81,
v/v), YMC-ODS 20mm i.d.×25cm (MeCN–H2O (1:9,
12.5:87.5, 15:85, 17.5: 82.5, v/v), MeOH–H2O (25:75, 3:7,
32.5:67.5, 35:65, 4:6, v/v)). This residue provided 9 (14mg),
16 (5mg), 18 (4mg), 21 (10mg), 23 (5mg), 26 (6mg), 27
(30mg), 28 (18mg), 30 (15mg), 31 (5mg), 32 (25mg), and 34
(8mg).
19
21
24
25
32
33
Aglycone moiety
C-2
-3
159.8
137.1
179.9
163.1
100.6
163.7
95.7
159.7
137.1
179.9
163.0
100.6
163.6
95.6
158.9
137.1
179.7
162.9
100.6
163.4
95.6
159.6
137.8
179.9
163.1
100.6
163.5
95.7
159.8
134.9
179.7
162.9
100.6
163.5
95.7
159.0
135.1
179.8
162.7
100.5
163.3
95.5
-4
-5
-6
-7
-8
-9
158.2
107.2
122.4
132.0
116.7
161.9
116.7
132.0
158.1
107.5
122.7
116.9
146.5
150.1
116.5
122.9
157.8
107.7
122.4
132.0
116.5
161.6
116.5
132.0
158.1
108.0
122.4
132.1
116.4
161.6
116.4
132.1
158.0
107.4
122.7
132.4
116.2
161.7
116.2
132.4
157.7
107.3
122.4
132.6
116.3
161.7
116.3
132.6
-10
BulbiferumosideꢀIꢀ(19)
-1′
-2′
-3′
-4′
-5′
-6′
Yellow amorphous powder. [α]D20 −214 (c=0.21, MeOH).
FAB-MS m/z: 747 [M+Na]+, 725 [M+H]+. HR-FAB-MS m/z:
747.2136 (Calcd for C33H40O18Na: 747.2112). UV λmax (MeOH)
1
nm (logε): 266 (4.39), 344 (4.24). H-NMR data (measured
in MeOH-d4 solution at 35°C): aglycone moiety, δ 7.82 (2H,
brd, J=8.5ꢀHz,ꢀ H-2′, 6′), 6.96 (2H, brd, J=8.5ꢀHz,ꢀ H-3′,5′),
6.74 (1H, d, J=2.0ꢀHz,ꢀ H-8),ꢀ 6.48ꢀ (1H,ꢀ d,ꢀ J=2.0ꢀHz,ꢀ H-6).ꢀ
3-O-Sugar moiety, δ 5.61 (1H, d, J=1.5ꢀHz,ꢀRha-1),ꢀ4.36ꢀ(1H,ꢀ
d, J=8.0ꢀHz,ꢀ Qui-1′), 4.26 (1H, dd, J=3.5,ꢀ 1.5ꢀHz,ꢀ Rha-2),ꢀ
3.81 (1H, dd, J=9.5,ꢀ 3.5ꢀHz,ꢀ Rha-3),ꢀ 3.57ꢀ (1H,ꢀ dq,ꢀ J=9.5,
6.0ꢀHz,ꢀ Rha-5),ꢀ 3.33ꢀ (overlapping,ꢀ Rha-4),ꢀ 3.30ꢀ (overlapping,ꢀ
Qui-3′), 3.24 (1H, dq, J=9.5,ꢀ 6.0ꢀHz,ꢀ Qui-5′), 3.22 (1H, dd,
J=9.0,ꢀ 8.0ꢀHz,ꢀ Qui-2′), 2.95 (1H, t, J=9.0ꢀHz,ꢀ Qui-4′), 1.16
(3H, d, J=6.0ꢀHz,ꢀ Qui-6′), 0.98 (3H, d, J=6.0ꢀHz,ꢀ Rha-6).ꢀ
7-O-Sugar moiety, δ 5.56 (1H, d, J=1.5, Rha-1″), 4.02 (1H, dd,
J=3.5,ꢀ 1.5ꢀHz,ꢀ Rha-2″), 3.83 (1H, dd, J=9.5,ꢀ 3.5ꢀHz,ꢀ Rha-3″),
3.60 (1H, dq, J=9.5,ꢀ 6.0ꢀHz,ꢀ Rha-5″), 3.48 (1H, t, J=9.5ꢀHz,ꢀ
Rha-4″), 1.26 (3H, d, J=6.0ꢀHz,ꢀRha-6″).
Sugar moieties
3-O-Sugars
Rha
Rha
103.3
82.6
72.1a)
73.7b)
71.8c)
17.7
Xyl
Rha
102.6
83.6
72.1a)
73.1b)
71.7c)
17.7
Rha
103.5
83.8
Glc
Glc
100.8
84.9
77.5
71.4
75.4
64.0
Glc
-1
-2
-3
-4
-5
-6
103.2
83.0
71.9
73.5a)
71.9
17.6
Qui
100.8
82.3
77.7a)
71.3b)
75.5c)
64.0
71.8a)
73.7
71.8a)
17.7
Glc
Glc
Glc
-1′
-2′
-3′
-4′
-5′
-6′
107.2
75.7
77.7
76.9
73.7a)
18.1
107.8
75.3
77.8
71.0
67.1
—
107.0
75.2d)
77.7
107.1
75.2
104.6
75.4c)
78.3
71.3b)
77.9a)
62.7
106.3
76.2
77.9
72.2a)
75.7
65.0
77.7
71.8a)
72.3
75.4d)
BulbiferumosideꢀIIꢀ(21)
75.6
Yellow amorphous powder. [α]D20 −191 (c=0.92, MeOH).
FAB-MS m/z: 749 [M+Na]+, 727 [M+H]+. HR-FAB-MS
m/z: 749.1923, 727.2088 (Calcd for C32H38O19Na: 749.1905,
C32H39O19: 727.2086). UV λmax (MeOH) nm (logε): 258
(4.37), 267 (sh), 351 (4.22). 1H-NMR data (measured in
MeOH-d4 solution at 35°C): aglycone moiety, δ 7.38 (1H, d,
J=2.0ꢀHz,ꢀH-2′), 7.33 (1H, dd, J=8.0,ꢀ2.0ꢀHz,ꢀH-6′), 6.93 (1H,
d, J=8.0ꢀHz,ꢀ H-5′), 6.71 (1H, d, J=2.0ꢀHz,ꢀ H-8),ꢀ 6.46ꢀ (1H,ꢀ d,ꢀ
J=2.0ꢀHz,ꢀ H-6).ꢀ 3-O-Sugar moiety, δ 5.37 (1H, d, J=1.5ꢀHz,ꢀ
Rha-1), 4.27 (1H, d, J=7.5ꢀHz,ꢀ Xyl-1′), 4.20 (1H, dd, J=3.5,
1.5ꢀHz,ꢀ Rha-2),ꢀ 3.88ꢀ (1H,ꢀ dd,ꢀ J=9.5,ꢀ 3.5ꢀHz,ꢀ Rha-3),ꢀ 3.85ꢀ
(overlapping, Rha-5), 3.65 (1H, dd, J=11.5,ꢀ 5.0ꢀHz,ꢀ Xyl-5′),
3.39 (1H, m, Xyl-4′), 3.34 (1H, t, J=9.5ꢀHz,ꢀ Rha-4),ꢀ 3.29ꢀ
(1H, t, J=9.0ꢀHz,ꢀXyl-3′), 3.19 (1H, dd, J=9.0,ꢀ7.5ꢀHz,ꢀXyl-2′),
3.06 (1H, dd, J=11.5,ꢀ10.5ꢀHz,ꢀXyl-5′), 1.03 (3H, d, J=6.0ꢀHz,ꢀ
Rha-6). 7-O-Sugar moiety, δ 5.56 (1H, J=1.5ꢀHz,ꢀ Rha-1″),
4.02 (1H, J=3.5,ꢀ1.5ꢀHz,ꢀRha-2″), 3.83 (1H, dd, J=9.5,ꢀ3.5ꢀHz,ꢀ
Rha-3″), 3.60 (1H, dq, J=9.5,ꢀ 6.0ꢀHz,ꢀ Rha-5″), 3.48 (1H, t,
J=9.5ꢀHz,ꢀRha-4″), 1.26 (3H, d, J=6.0ꢀHz,ꢀRha-6″).
64.5
64.4
7-O-Sugars
Rha
100.0
71.7
72.1
73.7a)
71.3
18.1
Rha
Rha
Rha
Rha
Rha
-1″
-2″
-3″
-4″
-5″
-6″
100.0
71.7c)
72.0a)
73.6b)
71.3
99.9
99.9
71.7a)
72.1
73.7
71.3
18.1
99.9
71.7
72.1
73.6
71.4b)
18.1
99.8
71.7
72.1a)
73.7
71.2
18.1
71.8c)
72.0a)
73.5c)
71.3
18.1
18.1
Ester moieties
-α
—
—
—
—
—
—
—
—
—
—
—
—
172.3
20.4
—
172.2
20.2
-β
—
—
—
—
—
—
—
—
—
—
-α′
-β′
-γ′
-1′
-2′,6′
-3′,5′
-4′
168.7
114.8
146.5
126.9
131.0
116.7
161.1
167.8
115.8
144.8
127.3
133.7
115.9
160.0
168.9
114.7
146.4
126.8
130.7
116.6
161.0
—
—
—
—
—
—
BulbiferumosideꢀIIIꢀ(24)
Measured in MeOH-d4 solution at 35°C. a–d)ꢀ Interchangeableꢀ inꢀ eachꢀ column.ꢀ
Rha: α-L-rhamnopyranose, Qui: β-D-quinovopyranose, Xyl: β-D-xylopyranose, Glc:
β-D-glucopyranose.
Yellow amorphous powder. [α]D23 −137 (c=0.69, MeOH).
FAB-MS m/z: 909 [M+Na]+. HR-FAB-MS m/z: 909.2417
(Calcd for C42H46O21Na: 909.2429). UV λmax (MeOH) nm
1
were dried in vacuo, yielding 3.0 and 1.5g, respectively. (logε): 226 (4.38), 268 (4.38), 316 (4.48). H-NMR data (mea-
The residue of the MeOH–H2O (7:3) fraction (1.5g) was sured in MeOH-d4 solution at 35°C): aglycone moiety, δ 7.66
subjected to semi-preparative HPLC (Develosil-ODS-15/30 (2H, brd, J=8.5ꢀHz,ꢀH-2′, 6′), 6.91 (2H, brd, J=8.5, H-3′, 5′),
50mm i.d.×100cm (MeCN–H2O 17:83→2:8, v/v), YMC- 6.49 (1H, d, J=2.0ꢀHz、H-8), 6.40 (1H, d, J=2.0ꢀHz,ꢀ H-6).ꢀ
ODS 20mm i.d.×25cm (MeCN–H2O (15: 85, 17.5:82.5, v/v), 3-O-Sugar moiety, δ 5.79 (1H, d, J=1.5ꢀHz,ꢀRha-1),ꢀ4.58ꢀ(1H,ꢀ

Vol. 65, No. 12 (2017)
Chem. Pharm. Bull.
1203
dd, J=12.0,ꢀ 2.0ꢀHz,ꢀ Glc-6′), 4.47 (1H, d, J=8.0ꢀHz,ꢀ Glc-1′), 6.49 (1H, d, J=2.0ꢀHz,ꢀ H-8),ꢀ 6.38ꢀ (1H,ꢀ d,ꢀ J=2.0ꢀHz,ꢀ H-6).ꢀ
4.39 (1H, dd, J=3.5,ꢀ 1.5ꢀHz,ꢀ Rha-2),ꢀ 4.09ꢀ (1H,ꢀ dd,ꢀ J=12.0, 3-O-Sugar moiety, δ 5.17 (1H, d, J=8.0ꢀHz,ꢀ Glc-1),ꢀ 4.72ꢀ (1H,ꢀ
7.0ꢀHz,ꢀ Glc-6′), 3.84 (1H, dd, J=9.5,ꢀ 3.5ꢀHz,ꢀ Rha-3),ꢀ 3.57ꢀ d, J=8.0ꢀHz,ꢀ Glc-1′), 4.46 (1H, dd, J=12.0,ꢀ 2.5ꢀHz,ꢀ Glc-6′),
(1H, dq, J=9.5,ꢀ 6.0ꢀHz,ꢀ Rha-5),ꢀ 3.49ꢀ (overlapping,ꢀ Glc-5′), 4.37 (1H, dd, J=12.0,ꢀ 7.0ꢀHz,ꢀ Glc-6′), 4.13 (1H, dd, J=12.0,
3.43 (1H, t, J=9.0ꢀHz,ꢀ Glc-3′), 3.37 (1H, t, J=9.5ꢀHz,ꢀ Rha-4),ꢀ 2.0ꢀHz,ꢀ Glc-6),ꢀ 4.02ꢀ (1H,ꢀ dd,ꢀ J=12.0,ꢀ 6.0ꢀHz,ꢀ Glc-6),ꢀ 3.69ꢀ
3.30 (overlapping, Glc-2′), 3.28 (1H, t, J=9.0ꢀHz,ꢀ Glc-4′), (1H, dd, J=9.0,ꢀ 8.0ꢀHz,ꢀ Glc-2),ꢀ 3.68ꢀ (overlapping,ꢀ Glc-5′),
1.06 (3H, J=6.0ꢀHz,ꢀRha-6).ꢀ7-O-Sugar moiety, δ 5.51 (1H, d, 3.56 (1H, t, J=9.0ꢀHz,ꢀ Glc-3),ꢀ 3.48ꢀ (1H,ꢀ t,ꢀ J=9.0ꢀHz,ꢀ Glc-3′),
J=1.5ꢀHz,ꢀ Rha-1″), 4.04 (1H, dd, J=3.5,ꢀ 1.5ꢀHz,ꢀ Rha-2″), 3.83 3.41 (1H, dd, J=9.0,ꢀ 8.0ꢀHz,ꢀ Glc-2′), 3.36 (1H, t, J=9.0ꢀHz,ꢀ
(1H, dd, J=9.5,ꢀ 3.5ꢀHz,ꢀ Rha-3″), 3.64 (1H, dq, J=9.5,ꢀ 6.0ꢀHz,ꢀ Glc-4′), 3.29 (overlapping, Glc-4, 5). 7-O-Sugar moiety, δ 5.52
Rha-5″), 3.49 (1H, t, J=9.5ꢀHz,ꢀRha-4″), 1.27 (3H, d, J=6.0ꢀHz,ꢀ (1H, d, J=1.5ꢀHz,ꢀ Rha-1″), 4.04 (overlapping, Rha-2″), 3.83
Rha-6″). Ester moiety, δ 7.38 (1H, d, J=16.0ꢀHz,ꢀ H-γ′), 7.16 (1H, dd, J=9.5,ꢀ 3.5ꢀHz,ꢀ Rha-3″), 3.60 (1H, dq, J=9.5,ꢀ 6.0ꢀHz,ꢀ
(2H, brd, J=8.5ꢀHz,ꢀ H-2′, 6′), 6.66 (2H, brd, J=8.5ꢀHz,ꢀ H-3′, Rha-5″), 3.49 (1H, t, J=9.5ꢀHz,ꢀRha-4″), 1.26 (3H, d, J=6.0ꢀHz,ꢀ
5′), 5.94 (1H, d, J=16.0ꢀHz,ꢀH-β′).
BulbiferumosideꢀIVꢀ(25)
Rha-6″). Ester moiety, δ 7.34 (1H, d, J=16.0ꢀHz,ꢀ H-γ′), 7.06
(2H, brd, J=9.0ꢀHz,ꢀ H-2′, 6′), 6.59 (2H, brd, J=9.0ꢀHz,ꢀ H-3′,
Yellow amorphous powder. [α]D23 −157 (c=0.89, MeOH). 5′), 5.99 (1H, d, J=16.0ꢀHz,ꢀH-β′), 1.66 (3H, s, H-β).
FAB-MS m/z: 909 [M+Na]+. HR-FAB-MS m/z: 909.2430
(Calcd for C42H46O21Na: 909.2429). UV λmax (MeOH) nm 19 and 21 (ca. 1mg) was dissolved in 2M HCl (200µL) and
(logε): 228 (sh), 267 (4.39), 315 (4.38). H-NMR data (mea- 1,4-dioxane (20µL). The solutions were heated at 100°C
Acid Hydrolysis of Compounds 19 and 21 Compounds
1
sured in MeOH-d4 solution at 35°C): aglycone moiety, δ 7.68 for 1h. After hydrolysis, each reaction mixture was diluted
(2H, brd, J=9.0ꢀHz,ꢀH-2′, 6′), 6.90 (2H, brd, J=9.0, H-3′, 5′), with H2O and extracted with EtOAc. The EtOAc layer was
6.68 (1H, d, J=2.0ꢀHz、H-8), 6.43 (1H, d, J=2.0ꢀHz,ꢀ H-6).ꢀ concentrated dry, and the residue from each compound was
3-O-Sugar moiety, δ 5.61 (1H, d, J=1.5ꢀHz,ꢀRha-1),ꢀ4.41ꢀ(1H,ꢀ analyzedꢀ usingꢀ HPLCꢀ throughꢀ comparisonꢀ withꢀ anꢀ authenticꢀ
d, J=8.0ꢀHz,ꢀ Glc-1′), 4.40 (overlapping, Rha-2), 4.29 (1H, sample. HPLC conditions: column, YMC-ODS-AM 4.6mm
dd, J=12.0,ꢀ 2.0ꢀHz,ꢀ Glc-6′), 4.25 (1H, dd, J=12.0,ꢀ 6.5ꢀHz,ꢀ i.d.×25ꢀcm;ꢀ flowꢀ rate,ꢀ 1.0ꢀmL/min;ꢀ 30%ꢀ MeCNꢀ inꢀ waterꢀ
Glc-6′), 3.83 (1H, dd, J=9.5,ꢀ 3.5ꢀHz,ꢀ Rha-3),ꢀ 3.58ꢀ (1H,ꢀ dq,ꢀ +0.05%ꢀ trifluoroaceticꢀ acidꢀ (TFA);ꢀ tR, 15.2min (quercetin
J=9.5,ꢀ 6.0ꢀHz,ꢀ Rha-5),ꢀ 3.44ꢀ (1H,ꢀ m,ꢀ Glc-5′), 3.39 (1H, t, (3)),ꢀ 29.4ꢀminꢀ (kaempferolꢀ (2)). 2 and 3 were detected in 19
J=9.0ꢀHz,ꢀ Glc-3′), 3.36 (1H, t, J=9.5ꢀHz,ꢀ Rha-4),ꢀ 3.26ꢀ (1H,ꢀ and 21, respectively. The H2Oꢀ layerꢀ wasꢀ neutralizedꢀ withꢀ anꢀ
dd, J=9.0,ꢀ 8.0ꢀHz,ꢀ Glc-2′), 3.22 (1H, t, J=9.0ꢀHz,ꢀ Glc-4′), AmberliteꢀIRA-60Eꢀcolumn,ꢀandꢀtheꢀeluateꢀwasꢀconcentratedꢀ
1.02 (3H, J=6.0ꢀHz,ꢀRha-6).ꢀ7-O-Sugar moiety, δ 5.52 (1H, d, dry. The residue was stirred with D-cysteine methyl ester
J=1.5ꢀHz,ꢀ Rha-1″), 4.02 (1H, dd, J=3.5,ꢀ 1.5ꢀHz,ꢀ Rha-2″), 3.83 hydrochloride,ꢀ hexamethyldisilazaneꢀ andꢀ trimethylsilylchlo-
(1H, dd, J=9.5,ꢀ 3.5ꢀHz,ꢀ Rha-3″), 3.61 (1H, dq, J=9.5,ꢀ 6.0ꢀHz,ꢀ ride in pyridine using the same procedures as in previous
Rha-5″), 3.48 (1H, t, J=9.5ꢀHz,ꢀRha-4″), 1.25 (3H, d, J=6.0ꢀHz,ꢀ reports.27,28) After the reactions, the supernatant was subjected
Rha-6″). Ester moiety, δ 7.44 (2H, brd, J=8.5ꢀHz,ꢀ H-2′, 6′), toꢀGC.ꢀGCꢀconditions:ꢀcolumn,ꢀTC-1ꢀ(GLꢀScienceꢀInc.,ꢀTokyo,ꢀ
6.67 (2H, brd, J=8.5ꢀHz,ꢀ H-3′, 5′), 6.36 (1H, d, J=13.0ꢀHz,ꢀ Japan) 0.25mm i.d.×30m; carrier gas, N2; column tempera-
H-γ′), 5.34 (1H, d, J=13.0ꢀHz,ꢀH-β″).
Bulbiferumoside V (32)
ture, 215°C; tR, 22.2min (L-rhamnoseꢀ (Tokyoꢀ Kaseiꢀ Kogyoꢀ
Co.,ꢀ Ltd.,ꢀ Tokyo,ꢀ Japan)),ꢀ 21.7ꢀminꢀ (D-rhamnose), 21.6min
Yellow amorphous powder. [α]D20 −126 (c=0.84, MeOH). (D-quinovose (Sigma Chem. Co., St. Louis, U.S.A.)), 20.5min
FAB-MS m/z: 821 [M+Na]+. HR-FAB-MS m/z: 821.2133 (L-quinovose), 19.1min (D-xyloseꢀ (Tokyoꢀ Kaseiꢀ Kogyoꢀ Co.,ꢀ
(Calcd for C35H42O21Na: 821.2116). UV λmax (MeOH) nm Ltd.), 17.6min (L-xylose). The tRs for D-rhamnose, L-quino-
(logε): 267 (4.32), 349 (4.24). 1H-NMR data (measured in vose and L-xylose were obtained from their enantiomers (L-
MeOH-d4 solution at 35°C): aglycone moiety, δ 8.02 (2H, brd, rhamnose+L-cysteine, D-quinovose+L-cysteine, D-xylose+L-
J=9.0ꢀHz,ꢀH-2′, 6′), 6.90 (2H, brd, J=9.0, H-3′, 5′), 6.74 (1H, cysteine). L-Rhamnose and D-quinovose were found in 19 and
d, J=2.0ꢀHz,ꢀ H-8),ꢀ 6.45ꢀ (1H,ꢀ d,ꢀ J=2.0ꢀHz,ꢀ H-6).ꢀ 3-O-Sugar L-rhamnose and D-xylose were detected in 21.
moiety, δ 5.41 (1H, d, J=8.0ꢀHz,ꢀGlc-1),ꢀ4.78ꢀ(1H,ꢀd,ꢀJ=8.0ꢀHz,ꢀ
Alkaline Hydrolysis of Compounds 24, 25, 32, and 33
Glc-1′), 4.18 (1H, dd, J=12.0,ꢀ 2.0ꢀHz,ꢀ Glc-6),ꢀ 4.03ꢀ (1H,ꢀ dd,ꢀ Compounds 24, 25, 32, and 33 (ca. 1mg) were dissolved in
J=12.0,ꢀ 5.5ꢀHz,ꢀ Glc-6),ꢀ 3.82ꢀ (1H,ꢀ dd,ꢀ J=12.0,ꢀ 2.0ꢀHz,ꢀ Glc-6′), 0.05M NaOH (100µL), and stirred at room temperature for
3.75 (1H, dd, J=9.0,ꢀ 8.0ꢀHz,ꢀ Glc-2),ꢀ 3.71ꢀ (1H,ꢀ dd,ꢀ J=12.0, 3.5h under an N2 atmosphere. After the reactions, each mix-
5.0ꢀHz,ꢀ Glc-6′), 3.62 (1H, t, J=9.0ꢀHz,ꢀ Glc-3),ꢀ 3.42ꢀ (overlap- tureꢀwasꢀneutralizedꢀwithꢀanꢀAmberliteꢀIR-120Bꢀcolumnꢀwithꢀ
ping, Glc-2′), 3.39 (overlapping, Glc-3′, 4′), 3.38 (overlapping, the eluate concentrated dry. The residue was partitioned be-
Glc-5), 3.33 (overlapping, Glc-4, 5′). 7-O-Sugar moiety, δ 5.57 tween EtOAc and H2O, and both layers were concentrated dry.
(1H, d, J=1.5ꢀHz,ꢀ Rha-1″), 4.03 (overlapping, Rha-2″), 3.83 TheꢀresidueꢀfromꢀtheꢀEtOAcꢀlayerꢀwasꢀanalyzedꢀusingꢀHPLCꢀ
(1H, dd, J=9.5,ꢀ 3.5ꢀHz,ꢀ Rha-3″), 3.59 (1H, dq, J=9.5,ꢀ 6.0ꢀHz,ꢀ through comparison with an authentic sample. HPLC condi-
Rha-5″), 3.48 (1H, t, J=9.5ꢀHz,ꢀRha-4″), 1.26 (3H, d, J=6.0ꢀHz,ꢀ tions: column, YMC-ODS-AM 4.6mm i.d.×25ꢀcm;ꢀ flowꢀ rate,ꢀ
Rha-6″). Ester moiety, δ 1.75 (3H, s, H-β).
BulbiferumosideꢀVIꢀ(33)
1.0mL/min; 17.5% MeCN in water +0.05% TFA; tR, 16.0min
[(E)-p-coumaricꢀacidꢀ(TokyoꢀKaseiꢀKogyoꢀCo.,ꢀLtd.)],ꢀ17.6ꢀminꢀ
Yellow amorphous powder. [α]D23 −83 (c=0.79, MeOH). [(Z)-p-coumaric acid (The authentic sample was provided by
FAB-MS m/z: 967 [M+Na]+. HR-FAB-MS m/z: 967.2513 Prof.ꢀ T.ꢀ Miyase.)].ꢀ (E)-p-Coumaric acid was detected in 24
(Calcd for C44H48O23Na: 967.2484). UV λmax (MeOH) nm and 33, and (Z)-p-coumaricꢀ acidꢀ wasꢀ identifiedꢀ inꢀ 25. The
1
(logε): 225 (sh), 267 (4.40), 317 (4.48). H-NMR data (mea- residues from the H2O layers of compounds 24, 25, 32, and
sured in MeOH-d4 solution at 35°C): aglycone moiety, δ 8.05 33ꢀwereꢀalsoꢀanalyzedꢀusingꢀHPLCꢀthroughꢀcomparisonꢀwithꢀ
(2H, brd, J=8.5ꢀHz,ꢀH-2′, 6′), 6.89 (2H, brd, J=8.5, H-3′, 5′), compounds 22 and 27. HPLC conditions: column, YMC-ODS-

1204
Chem. Pharm. Bull.
Vol. 65, No. 12 (2017)
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Conflict of Interest Theꢀ authorsꢀ declareꢀ noꢀ conflictꢀ ofꢀ
interest.
Supplementary Materials The online version of this
1
article contains supplementary materials. Table S1. H-NMR
spectroscopic data of compounds 19, 21, 24, 25, 32, and 33.
1
Figure S1. The 13C- and H-NMR spectra of compounds 19,
21, 24, 25, 32, and 33. Figure S2. The HOHAHA spectra of
compounds 24, 25, 32, and 33.
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