Flavonoid glycosides from Botrychium ternatum

Article abstract of DOI:10.1248/cpb.c12-00744

The MeOH extract from dried whole Botrychium ternatum plants yielded 33 compounds, including seventeen new flavonoid glycosides and sixteen known compounds. The structures of new compounds were established using NMR spectroscopic analysis and chemical evi

Full text of DOI:10.1248/cpb.c12-00744

December 2012
Regular Article
Chem. Pharm. Bull. 60(12) 1561–1573 (2012)
1561
Flavonoid Glycosides from Botrychium ternatum
,a
Tsutomu Warashina,* Kaoru Umehara,b and Toshio Miyaseb
a Institute for Environmental Sciences, University of Shizuoka; and b School of Pharmaceutical Sciences, University of
Shizuoka; 52–1 Yada, Suruga-ku, Shizuoka 422–8526, Japan.
Received August 21, 2012; accepted September 24, 2012
The MeOH extract from dried whole Botrychium ternatum plants yielded 33 compounds, including
seventeen new flavonoid glycosides and sixteen known compounds. The structures of new compounds were
established using NMR spectroscopic analysis and chemical evidence.
Key words Botrychium ternatum; Ophiolossaceae; flavonoid glycoside; kaempferol; ternatumoside
Recently, plants in the Ophiolossaceaus family were re- the signals at δ 5.59, 103.2 and δ 4.36, 107.2 were assigned
ported to be effective in the cosmetic field, for example, skin at the anomeric proton and carbon of α-L-rhamnopyranose
moisturizing and lightning actions, improvement and protec- and β-D-quinovopyranose, whose conformations were judged
tion of rough skin, and proliferation of skin fibroblasts.1,2) from the 1H-chemical shift or coupling constant of each
Botrychium ternatum is one of the Ophiolossaceaus plants and anomeric proton signal, respectively.16) In the 1H-detected
distributed to the Honshu, Shikoku, and Kyushu Islands in heteronuclear multiple-bond connectivity (HMBC) measure-
Japan, the Korean Peninsula, and China. In Japan, this plant is ments, long-range correlations were exhibited between C-3
called “Fuyunohanawarabi” and used to treat dizziness, head- of the aglycone (δ 136.8) and H-1 of α-L-rhamnopyranose (δ
ache, cough, and fever as a folk medicine. In the course of 5.59), and C-2 of α-L-rhamnopyranose (δ 83.0) and H-1′ of
research to find the efficient agents in the cosmetic field from β-D-quinovopyranose (δ 4.36). In addition, the rotating frame
the Ophiolossaceaus family, we investigated the constituents nuclear Overhauser effect (ROE) difference experiment exhib-
of B. ternatum and their biological activities.
ited a ROE between H-1′ of β-D-quinovopyranose and H-2 of
A MeOH extract from dried whole B. ternatum plants was α-L-rhamnopyranose [δ 4.26 (1H, dd, J=3.5, 1.5Hz)]. Hence, 4
suspended in water. The suspension was extracted with diethyl was determined as kaempferol 3-O-β-D-quinovopyranosyl-(1→
ether and partitioned into an ether-soluble fraction and a H2O- 2)-α-L-rhamnopyranoside.
soluble fraction. The ether-soluble fraction was then separated
The molecular formula of ternatumoside II (6) was sug-
into a n-hexane-soluble fraction and a MeOH–H2O (9:1)-sol- gested to be C27H30O15 based on high resolution (HR)-FAB-
uble fraction. The residue of the water soluble-fraction and MS, and 6 was also identified as a kaempferol diglycoside
the MeOH–H2O (9:1)-soluble fraction was subjected to both whose component sugars were α-L-rhamnopyranose and
silica gel column chromatography and semi-preparative HPLC β-D-glucopyranose from the NMR spectroscopic data and
to give thirty flavonoid glycosides (1–30), two flavonoids (31, acid hydrolysis. The sugar linkage of 6 was determined by a
32), and a bibenzyl derivative (33). Compounds 1,3) 2,4) 3,5) ROE difference experiment irradiating the anomeric proton
5,5) 7,6) 13,7) 14,8) 15,9) 16,10) 17,11) 21,12) 24,13) 25,13) 31,14) 32,14) of β-D-glucopyranose and HMBC measurements. A ROE was
1
and 3315) were identified as shown in Chart 1 based on the H- observed between H-1″ of β-D-glucopyranose [δ 4.49 (1H, d,
and/or 13C-NMR spectroscopic data.
J=8.0Hz)] and H-3 of α-L-rhamnopyranose [δ 3.80 (1H, dd,
The molecular formula of ternatumoside I (4), C27H30O14, J=9.0, 3.0Hz)]. The HMBC correlations were shown to be
was established based on a high resolution (HR)-FAB-MS between H-1 of α-L-rhamnopyranose [δ 5.39 (1H, d, J=1.5Hz)]
molecular ion at m/z 601.1539 ([M+Na]+). The aglycone of 4 and C-3 of the aglycone (δ 136.0), H-1″ of β-D-glucopyranose
was identified as kaempferol, according to observations of 15 (δ 4.49) and C-3 of α-L-rhamnopyranose (δ 82.7), and H-3 of
carbon signals including twelve aromatic carbons (δ 166.2, α-L-rhamnopyranose (δ 3.80) and C-1″ of β-D-glucopyranose
163.3, 161.7, 158.6, 131.9×2, 116.6×2, 122.6, 105.9, 100.0, (δ 106.0). Thus, 6 was established to be kaempferol 3-O-β-D-
94.9), two olefin carbons (δ 159.2, 136.8), and one carbonyl glucopyranosyl-(1→3)-α-L-rhamnopyranoside.
carbon (δ 179.7), and the AX-type aromatic proton [δ 6.38
The molecular formulae of ternatumosides III (8) and IV
(1H, d, J=2.0Hz) and 6.21 (1H, d, J=2.0Hz)] and AA′XX′- (9) were identified as C42H46O22 and C51H52O24, respectively,
type aromatic proton signals [δ 7.78 (2H, brd, J=9.0Hz), and by HR-FAB-MS. The NMR spectroscopic data for 8 and 9
1
6.95 (2H, brd, J=9.0Hz)] in the 13C- and H-NMR spectro- were similar to those of 7, and signals due to one and two
1
scopic data. Moreover, in the H- and 13C-NMR spectra of sets of (trans)-p-coumaroyl groups were observed in 8 and
4, two anomeric proton and carbon signals were observed 9, respectively. Additionally, both compounds yielded 7
at δ 5.59 (1H, d, J=1.5Hz), 4.36 (1H, d, J=8.0Hz) and δ and (trans)-p-coumaric acid by alkaline hydrolysis. In the
107.2, 103.2. Thus, 4 was considered to be a kaempferol di- homonuclear Haltmann–Hahn (HOHAHA) difference ex-
glycoside. Acid hydrolysis of 4 afforded L-rhamnose and D- periment of 8, irradiation of H-1″ of β-D-glucopyranose [δ
1
quinovose with kaempferol, and on the basis of H-detected 4.54 (1H, d, J=8.0Hz)] exhibited signal enhancement for
heteronuclear multiple quantum coherency (HMQC) and H-6″ of β-D-glucopyranose [δ 4.51 (2H, overlapping)]. More-
1H–1H shift correlation spectroscopy (COSY) measurements, over, HMBC measurements in 8 revealed long-range cor-
relations between H-6″ of β-D-glucopyranose (δ 4.51) and
C-α′ of (trans)-p-coumaroyl group (δ 169.0). Similarly, the
The authors declare no conflict of interest.
*To whom correspondence should be addressed. e-mail: warashin@u-shizuoka-ken.ac.jp

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Chart 1. Structure of 1–33
HOHAHA difference experiments of 9 showed signal en- 3-O-[β-D-6-O-[4-hydroxy-(E)-cinnamoyl]-glucopyranosyl-(1→
hancement for H-6′ and H-6″ of β-D-glucopyranose [δ 4.50 3)]-β-D-6-O-[4-hydroxy-(E)-cinnamoyl]-glucopyranosyl-(1→
(1H, overlapping), 4.22 (1H, dd, J=12.0, 5.5Hz) and δ 4.52 2)-α-L-rhamnopyranoside, respectively.
(1H, dd, J=12.0, 2.5Hz), 4.48 (1H, dd, J=12.0, 8.0Hz)] on ir-
Ternatumoside V (10) was shown to have the molecu-
radiating H-1′ and H-1″ of the β-D-glucopyranosyl groups [δ lar formula C39H50O25 by the HR-FAB-MS measurements,
4.58 (1H, d, J=8.0Hz) and δ 4.57 (1H, d, J=8.0Hz)]. HMBC and deduced to be a kaempferol tetraglycoside based on
correlations were observed between H-6′, H-6″ of the β-D- the observation of four anomeric carbon and proton signals
glucopyranosyl groups (δ 4.22 and δ 4.52) and C-α, C-α′ of [δ 106.2, 105.4, 104.3, 102.6 and δ 4.67 (1H, d, J=8.0Hz),
the (trans)-p-coumaroyl groups (δ 169.1 and δ 169.0) in 9. 4.63 (1H, d, J=8.0Hz), 4.56 (1H, d, J=8.0Hz), 5.62 (1H,
Thus, 8 and 9 were established to be kaempferol 3-O-[β- d, J=1.5Hz)] with signals due to kaempferol in the NMR
D-6-O-[4-hydroxy-(E)-cinnamoyl]-glucopyranosyl-(1→3)]-β-D- spectra. Because acid hydrolysis of 10 yielded D-glucose and
glucopyranosyl-(1→2)-α-L-rhamnopyranoside and kaempferol L-rhamnose with kaempferol, 10 consisted of kaempferol, one

December 2012
1563
α-L-rhamnopyranosyl group, and three β-D-glucopyranosyl
groups. Comparison of the 13C-NMR spectroscopic data of
10 with those of 7 revealed glycosylation shifts around C-4
of L-rhamnopyranose [C-3 (−0.4ppm), C-4 (+7.3ppm), C-5
(−0.4 ppm)],16) suggesting that the β-D-glucopyranosyl group
was attached at this position, which was confirmed by a ROE
between H-1‴ of β-D-glucopyranose (δ 4.67) and H-4 of α-L-
rhamnopyranose [δ 3.75 (1H, t, J=9.0Hz)]. Thus, 10 was de-
termined as kaempferol 3-O-(2,3,4-tri-O-β-D-glucopyranosyl)-
α-L-rhamnopyranoside.
Ternatumoside VI (11) was assigned the molecular formula
C57H62O29 based on HR-FAB-MS. The NMR spectroscopic
data and alkaline hydrolysis suggested that 11 consisted of
10 and two (trans)-p-coumaroyl groups. As acylation shifts
were observed at H-6′ and H-6″ of the β-D-glucopyranosyl
groups on comparison of the 1H-NMR spectroscopic data
with those of 10, 11 was determined as kaempferol 3-O-[β-D-
glucopyranosyl-(1→4 ) ] - [ β-D-6-O-[4-hydroxy-(E)-cinnamoyl]-
glucopyranosyl-(1→3)]-β-D-6-O-[4-hydroxy-(E)-cinnamoyl]-
glucopyranosyl-(1→2)-α-L-rhamnopyranoside. These esterifi-
cations were supported by the observation of long-range cor-
relations between H-6′, H-6″ of the β-D-glucopyranosyl groups
[δ 4.47 (1H, dd, J=12.0, 2.5Hz) and δ 4.50 (2H, overlapping)]
and C-α, C-α′ of the (trans)-p-coumaroyl groups (δ 169.1 and
δ 169.0) in the HMBC measurements, the same as 9.
The molecular formula of ternatumoside VII (12) was
proposed to be C47H54O26 based on HR-FAB-MS. Com-
parison of the NMR spectroscopic data with those of 8 sug-
gested that 12 had one more pentose in its structure. Acid
hydrolysis following alkaline hydrolysis yielded L-rhamnose,
D-glucose, and D-xylose with kaempferol. On the basis of
1H–1H COSY, HMBC measurements and HOHAHA differ-
1
ence experiments, the assignments of the 13C- and H-NMR
spectroscopic data were carried out as shown in Tables 1 and
2. In the ROE difference experiment, irradiation of H-1‴ of
β-D-xylopyranose [δ 4.64 (1H, d, J=8.0Hz)] showed a ROE
to H-4 of α-L-rhamnopyranose [δ 3.62 (1H, t, J=9.0Hz)]. The
other sugar linkages and the ester function were also con-
firmed by the ROE difference experiment on irradiation of
each anomeric proton and HMBC measurements. Therefore,
12 was established as kaempferol 3-O-[β-D-xylopyranosyl-(1→
4)]-[β-D-6-O-[4-hydroxy-(E)-cinnamoyl]-glucopyranosyl-(1→
3)]-β-D-glucopyranosyl-(1→2)-α-L-rhamnopyranoside.
Chart 2. Important ROEs, HOHAHAs, and HMBC Correlations in Ter-
natumosides III (8) and XII (23)
The molecular formulae of ternatumosides VIII (18) and
IX (19) were proposed to be C39H50O24 and C39H50O25, re- ROEs between H-1⁗ of this α-L-rhamnopyranose and H-6 [δ
spectively, based on HR-FAB-MS. The NMR spectroscopic 6.47 (1H, d, J=2.0Hz)] and H-8 [δ 6.72 (1H, d, J=2.0Hz)]
data for 18 and 19 suggested that these compounds were also of kaempferol. Thus, 18 was elucidated as kaempferol
kaempferol bisdesmosides the same as 13, 14, and 16. The 3-O-(2,3-di-O-β-D-glucopyranosyl)-α-L-rhamnopyranoside-7-
component sugars were identified as two α-L-rhamnopyranosyl O-α-L-rhamnopyranoside. The NMR spectroscopic data of
groups and two β-D-glucopyranosyl groups for 18, and one 19 showed similarity to those of 16, but observation of the
α-L-rhamnopyranosyl group and three β-D-glucopyranosyl glycosylation shifts around the C-2⁗ position of the 7-O-β-D-
groups for 19, because of assignments of the 1H- and glucopyranosyl unit [C-1⁗ (−1.4ppm), C-2⁗ (+8.8ppm), C-3⁗
13C-NMR spectroscopic data on the basis of HOHAHA dif- (−0.4ppm)] indicated that the remaining β-D-glucopyranose
ference experiments and the second dimensional (2D)-NMR was connected to this position. The HMBC correlation be-
(HMQC, HMBC, and 1H–1H COSY) measurements. The tween H-1″‴ of β-D-glucopyranose [δ 4.67 (1H, d, J=8.0Hz)]
NMR spectroscopic data of 18 showed the sugar sequence and C-2⁗ of the 7-O-β-D-glucopyranosyl unit (δ 83.6) support-
attached at the C-3 position of kaempferol to be the same as ed this sugar linkage. Hence, 19 was determined as kaemp-
that in 7. The remaining α-L-rhamnopyranose was considered ferol 3-O-β-D-glucopyranosyl-(1→2)-α-L-rhamnopyranoside-7-
to be connected to the C-7 position of kaempferol based on O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranoside.
the HMBC correlation from H-1⁗ of this α-L-rhamnopyranose
The molecular formulae of ternatumoside X (20) and 21
[δ 5.57 (1H, d, J=1.5Hz)] to C-7 of kaempferol (δ 163.6) and were identified as C48H56O26 and C57H62O28, respectively,

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Vol. 60, No. 12
based on HR-FAB-MS. Alkaline hydrolysis of these com- cinnamoyl]-glucopyranosyl-(1→3)]-β-D-6-O-[4-hydroxy-(E)-
pounds afforded 18 and (trans)-p-coumaric acid. Similarly, cinnamoyl]-glucopyranosyl-(1→2)-α-L-rhamnopyranoside-7-O-
the NMR spectra exhibited signals due to one set and two α-L-rhamnopyranoside, respectively.
sets of (trans)-p-coumaroyl groups in 20 and 21, respec-
The molecular formulae of ternatumosides XV (28) and
tively, together with those of 18. The NMR spectroscopic XVI (29) were established as C62H70O33 and C63H72O34,
data of the 3-O-linked sugar and ester moieties in 20 and respectively. Comparing the NMR spectroscopic data of
21 resemble those of 8 and 9. 20 showed a long-range cor- 28 with those of 23 and 27, 28 was presumed to have a
relation between H-6″ of β-D-glucopyranose [δ 4.45 (1H, dd, β-D-glucopyranosyl group instead of α-L-rhamnopyranosyl
J=12.0, 2.0Hz)] and C-α′ of the (trans)-p-coumaroyl group (δ group at the C-7 position of aglycone in 27. It was as-
168.9), and 21 showed correlations between H-6′, H-6″ of the sumed that the β-D-glucopyranosyl group in 29 was replaced
β-D-glucopyranosyl groups [δ 4.20 (1H, dd, J=12.0, 6.0Hz) with a β-D-xylopyranosyl group at the C-4 position of α-L-
and δ 4.48 (1H, dd, J=12.0, 2.5Hz)] and C-α, C-α′ of the rhamnopyranose in 28 on comparison of the NMR spectro-
(trans)-p-coumaroyl groups (δ 169.0×2) in the HMBC mea- scopic data with those of 25 and 28. These structures were
surements. Thus, 20 and 21 were established as kaempferol confirmed based on the ROE and HOHAHA difference exper-
3-O-[β-D-6-O-[4-hydroxy-(E)-cinnamoyl]-glucopyranosyl-(1→ iments and the 2D-NMR measurements. 28 and 29 were estab-
3)]-β-D-glucopyranosyl-(1→2)-α-L-rhamnopyranoside-7- lished as kaempferol 3-O-[β-D-xylopyranosyl-(1→4 ) ] - [ β-D-6-
O-α-L-rhamnopyranoside and kaempferol 3-O-[β-D-6-O- O-[4-hydroxy-(E)-cinnamoyl]-glucopyranosyl-(1→3)]-β-D-6-
[4-hydroxy-(E)-cinnamoyl]-glucopyranosyl-(1→3)]-β-D-6- O-[4-hydroxy-(E)-cinnamoyl]-glucopyranosyl-(1→2)-α-L-
O-[4-hydroxy-(E)-cinnamoyl]-glucopyranosyl-(1→2)-α-L- rhamnopyranoside-7-O-β-D-glucopyranoside and kaempferol
rhamnopyranoside-7-O-α-L-rhamnopyranoside,
respectively. 3-O-[β-D-glucopyranosyl-(1→4 ) ] - [ β-D-6-O-[4-hydroxy-(E)-
21 has been reported previously, but only LC-MS data were cinnamoyl]-glucopyranosyl-(1→3)]-β-D-6-O-[4-hydroxy-(E)-
described. Accordingly, other physical data including NMR cinnamoyl]-glucopyranosyl-(1→2)-α-L-rhamnopyranoside-7-O-
spectroscopic data are mentioned in this report.
β-D-glucopyranoside, respectively.
Ternatumosides XI (22) and XII (23) were proposed to
Ternatumoside XVII (30) was found to have the molecular
have the molecular formulae C48H56O27 and C57H62O29, based formula, C63H72O34, based on HR-FAB-MS. The NMR spec-
on HR-FAB-MS, and were larger than 20 and 21 by an oxy- troscopic data suggested that 30 contained kaempferol, two
gen atom, respectively. The NMR spectroscopic data for the p-coumaroyl groups, and five sugars. Because acid hydrolysis
aglycone and 3-O-linked sugar and ester moieties of 22 and following alkaline hydrolysis of 30 yielded L-rhamnose and
23 were consistent with those of 20 and 21. In both com- D-glucose and the 13C-NMR spectrum showed one signal due
pounds, each sugar unit connected to C-7 of the aglycone was to C-6 of L-rhamnopyranose at δ 17.8, these sugars consisted
determined to be β-D-glucopyranose, corresponding to the of one α-L-rhamnopyranose and four D-glucopyranoses. In the
NMR spectroscopic data of 16. So, 22 and 23 were identi- 1H-NMR spectrum, three anomeric proton signals due to β-D-
fied as kaempferol 3-O-[β-D-6-O-[4-hydroxy-(E)-cinnamoyl]- glucopyranoses were observed at δ 5.05 (1H, d-like, J=8.0Hz),
glucopyranosyl-(1→3)]-β-D-glucopyranosyl-(1→2)-α-L- 4.58 (1H, d, J=8.0Hz), and 4.57 (1H, d, J=8.0Hz) with that
rhamnopyranoside-7-O-β-D-glucopyranoside and kaempferol of α-L-rhamnopyranose at δ 5.81 (1H, brs), which were simi-
3-O-[β-D-6-O-[4-hydroxy-(E)-cinnamoyl]-glucopyranosyl-(1→ lar to those of 23. The J value of the anomeric proton signal
3)]-β-D-6-O-[4-hydroxy-(E)-cinnamoyl]-glucopyranosyl-(1→ due to the remaining D-glucopyranosyl unit at δ 4.90 (1H, d,
2)-α-L-rhamnopyranoside-7-O-β-D-glucopyranoside,
respec- J=4.0Hz) verified that this D-glucopyranose has an α-form.
tively. These structures were supported by the HMBC mea- The similarity of the NMR spectroscopic data of 30 to those
surements, ROE, and HOHAHA difference experiments ir- of 23 indicated that 30 contained 23. In the ROE difference
radiating the anomeric protons (see Chart 2).
experiment, irradiation of the above anomeric proton due to
HR-FAB-MS revealed the molecular formulae of ter- α-D-glucopyranose exhibited a ROE to H-6⁗ of the 7-O-β-D-
natumosides XIII (26) and XIV (27) to be C53H64O30 and glucopyranosyl unit [δ 3.86 (1H, dd, J=11.5, 2.0Hz)]. Thus, 30
1
C62H70O32, respectively. The 13C- and H-NMR spectroscopic was determined as kaempferol 3-O-[β-D-6-O-[4-hydroxy-(E)-
data of 26 and 27 were similar to those of 24 and 25, but the cinnamoyl]-glucopyranosyl-(1→3)]-β-D-6-O-[4-hydroxy-(E)-
characteristic C-5 and H-5 signals of β-D-xylopyranose were cinnamoyl]-glucopyranosyl-(1→2)-α-L-rhamnopyranoside-7-O-
observed at δ 66.9, δ 3.76 (1H, dd, J=11.5, 5.5Hz), 3.13 (1H, α-D-glucopyranosyl-(1→6)-β-D-glucopyranoside. The 13C- and
t, J=11.5Hz) in 26 and δ 67.0, δ 3.78 (1H, dd, J=11.5, 5.5Hz), 1H-NMR spectroscopic data of the 7-O-sugar chain were
3.15 (1H, t, J=11.5Hz) in 27 the same as in 12. Assignments of consistent with those of rhodioloside B17) and genipin 1-O-
the 13C- and 1H-NMR spectroscopic data (Tables 1 and 2) were isomaltoside.18) This structure was supported by the HMBC
made on the basis of 2D-NMR measurements and HOHAHA measurements and ROE difference experiments irradiating
difference experiments. The above β-D-xylopyranosyl unit was other anomeric protons.
connected to the C-4 position of α-L-rhamnopyranose, which
Some p-coumarated flavonoid glycosides were afforded in
was supported by the ROE difference experiment on irradia- this investigation of the constituents from B. ternatum. Be-
tion of H-1‴ of β-D-xylopyranose [26: δ 4.65 (1H, d, J=8.0Hz); cause these compounds are found in various families includ-
27: δ 4.68 (1H, d, J=8.0Hz)] and HMBC measurements. ing Apocynaceae, Lamiaceae, Lecythidaceae, Leguminosae,
Hence, 26 and 27 were identified as kaempferol 3-O-[β-D- Liliaceae, Primulaceae, Ranunculaceae, Resedaceae, Sapinda-
xylopyranosyl-(1→4)]-[β-D-6-O-[4-hydroxy-(E)-cinnamoyl]- ceae, and Teaceae, they are not considered to be characteristic
glucopyranosyl-(1→3)]-β-D-glucopyranosyl-(1→2)-α-L- constituents in the Ophiolossaceaus family. However, to the
rhamnopyranoside-7-O-α-L-rhamnopyranoside and kaempferol best of our knowledge, this is the first report about p-couma-
3-O-[β-D-xylopyranosyl-(1→4 ) ] - [ β-D-6-O- [ 4 - h y d r o x y - ( E)- rated flavonoid glycosides from this family.

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1567
Previous reports described that Botrychium virginianum (MeCN–H2O (17.5:82.5 and 2:8, v/v), MeOH–H2O (4:6,
(Japanobotrychium virginianum) which belongs to the Ophio- 45:55, and 1:1, v/v)). This residue yielded 1 (8mg), 2 (13mg),
lossaceaus family caused the proliferation of normal human 3 (50mg), 4 (4mg), 5 (51mg), 6 (6mg), 7 (49mg), 8 (14mg),
skin fibroblasts and inhibition of melanin synthesis.1) B. ter- 9 (10mg), 11 (3mg), 12 (5mg), 13 (3mg), 14 (21mg), 21
natum also belongs to the Ophiolossaceaus family. So, we (12mg), 23 (131mg), 25 (30mg), and 27 (29mg). The residue
examined whether the MeOH extract, the MeOH–H2O (9:1) of the MeOH–H2O (1:1) fraction (8.5g) was subjected to
layer from the Et2O-soluble fraction, the MeOH–H2O (7:3) silica gel column chromatography with the CHCl3–MeOH–
eluate from the porous polymer gel, and the flavonoid gly- H2O (90:10:1→75:25:1, v/v) system to obtain nine fractions
cosides 2, 3, 5, 7, 8, 14, 18, 21, 23, and 25 from B. ternatum (A (606mg), B (595mg), C (131mg) D (745mg), E (350mg),
cause the proliferation of normal human skin fibroblasts. The F (885mg), G (667mg), H (1.88g), and I (1.57g)). Using
compounds and the MeOH–H2O (7:3) eluate had no effect on semi-preparative HPLC (Inertsil ODS-3 30mm i.d.×50cm,
the proliferation of fibroblasts at 100–10µg/mL. The MeOH Capcellpak ODS-UG80 30mm i.d.×25cm, YMC-ODS
extract and the MeOH–H2O (9:1) layer showed cytotoxicity 20mm i.d.×25cm (MeCN–H2O (15:85, 17.5:82.5, 2:8, and
against fibroblasts at 100 – 1.0µg/mL. Secondly, in the assay 22.5:77.5, v/v), and MeOH–H2O (4:6, and 45:55, v/v)), frac-
of the cosmetic field, we examined the tyrosinase inhibition tion D (86mg) afforded 3 (55mg) and 5 (4mg). Fraction F
of the MeOH extract, the MeOH–H2O (1:1 and 7:3) eluates (112mg) yielded 5 (35mg). Fraction G (333mg) gave 5 (3mg),
from the porous polymer gel, and the flavonoid glycosides 5, 7 (54mg), and 14 (30mg). Fraction H (300mg) produced 15
7, 22, 23, 25, 27, and 29 from B. ternatum at 400µg/mL. But, (3mg), 16 (6mg), 18 (25mg), 20 (6mg), and 23 (55mg). Frac-
they did not show inhibitory activity against tyrosinase. The tion I (942mg) provided 10 (9mg), 17 (6mg), 18 (10mg), 19
compounds and fractions derived from B. ternatum showed no (53mg), 22 (23mg), 24 (10mg), 26 (16mg), 28 (12mg), 29
effects in our assays of the cosmetic field. Thus, we are inter- (17mg), and 30 (8mg).
ested in the difference of the constituents in B. ternatum and
Ternatumoside I (4): Yellow amorphous powder. [α]D20 −119
B. virginianum from the viewpoints of the biological activities (c=0.39, MeOH). FAB-MS m/z: 601 [M+Na]+. HR-FAB-MS
and chemotaxonomy. Because p-coumarated flavonoid glyco- m/z: 601.1539 (Calcd for C27H30O14Na: 601.1533). UV λmax
sides from Aconitum anthora are reported to have the antioxi- (MeOH) nm (logε): 266 (4.26), 340 (4.10).
dant activity,19) the compounds derived from B. ternatum may
also reveal this activity.
Ternatumoside II (6): Yellow amorphous powder. [α]D19 −110
(c=0.54, MeOH). FAB-MS m/z: 617 [M+Na]+, 595 [M+H]+.
HR-FAB-MS m/z: 617.1511, 595.1675 (Calcd for C27H30O15Na:
617.1482 and C27H31O15: 595.1663). UV λmax (MeOH) nm
Experimental
General Procedures The instrumental analysis was de- (logε): 266 (4.25), 344 (4.08).
scribed previously.20)
Ternatumoside III (8): Yellow amorphous powder. [α]D19 +11
Plant Materials The whole B. ternatum plants were pur- (c=0.68, MeOH). FAB-MS m/z: 925 [M+Na]+, 903 [M+H]+.
chased from Anguo City Ruikang Herb Co., Ltd. in China in HR-FAB-MS m/z: 925.2371, 903.2583 (Calcd for C42H46O22Na:
2010. The dried materials were stored in a herbarium of the 925.2378 and C42H47O22: 903.2559). UV λmax (MeOH) nm
University of Shizuoka (voucher number, M-4396).
Extraction and Isolation The dried whole plants of B.
(logε): 268 (4.38), 315 (4.45).
Ternatumoside IV (9): Yellow amorphous powder. [α]D20 −7.7
ternatum (2.7kg) were extracted twice with MeOH under (c=0.97, MeOH). FAB-MS m/z: 1071 [M+Na]+. HR-FAB-MS
reflux for 3h. The extract was concentrated under reduced m/z: 1071.2783 (Calcd for C51H52O24Na: 1071.2745). UV λmax
pressure and the residue was suspended in water (2L). This (MeOH) nm (logε): 223 (sh), 270 (4.47), 303 (sh), 314 (4.68).
suspension was successively extracted with Et2O (2L). The
Ternatumoside V (10): Yellow amorphous powder. [α]D26 −89
Et2O extract was evaporated dry, and the resulting residue was (c=0.91, MeOH). FAB-MS m/z: 941 [M+Na]+. HR-FAB-MS
partitioned between the n-hexane-soluble fraction and MeOH– m/z: 941.2563 (Calcd for C39H50O25Na: 941.2539). UV λmax
H2O (9:1)-soluble fraction. The MeOH–H2O (9:1) fraction (MeOH) nm (logε): 266 (4.28), 345 (4.09).
was concentrated, and the residue was subjected to silica gel
Ternatumoside VI (11): Yellow amorphous powder. [α]D20
column chromatography with a CHCl3–MeOH (98:2→85:15, −20 (c=0.32, MeOH). FAB-MS m/z: 1233 [M+Na]+. HR-
v/v) system to obtain seven fractions. (A (1.70g), B (397mg), FAB-MS m/z: 1233.3270 (Calcd for C57H62O29Na: 1233.3274).
C (1.16g), D (199mg), E (1.59g), F (602mg), and G (739mg)). UV λmax (MeOH) nm (logε): 222 (sh), 270 (4.51), 305 (sh), 314
Using semi-preparative HPLC (Capcellpak ODS-UG80 30mm (4.69).
i.d.×25cm, YMC-ODS 20mm i.d.×25cm (MeCN–H2O
Ternatumoside VII (12): Yellow amorphous powder. [α]D19
(25:75, and 1:1, v/v), and MeOH–H2O (1:1, and 75:25, v/v) −21 (c=0.47, MeOH). FAB-MS m/z: 1057 [M+Na]+. HR-FAB-
and recrystallization (MeOH), fraction B (397mg) afforded 33 MS m/z: 1057.2799 (Calcd for C47H54O26Na: 1057.2800). UV
(19mg). Fraction D (199mg) yielded 31 (4mg) and 32 (5mg).
The H2O layer of the MeOH extract was passed through a
λmax (MeOH) nm (logε): 268 (4.42), 315 (4.47).
Ternatumoside VIII (18): Yellow amorphous powder. [α]D18
porous polymer gel (Mitsubishi Chemical Co., Diaion HP-20) −146 (c=1.00, MeOH). FAB-MS m/z: 925 [M+Na]+, 903
column with absorbed material being eluted with MeOH– [M+H]+. HR-FAB-MS m/z: 925.2612 (Calcd for C39H50O24Na:
H2O 1:1 (5L), 7:3 (5L), and MeOH (5L). The MeOH–H2O 925.2590). UV λmax (MeOH) nm (logε): 266 (4.32), 322 (sh),
1:1 and 7:3 fractions were dried in vacuo, respectively 345 (4.17).
(8.5g and 4.2g). The residue of the 7:3 fraction (2.0g) was
Ternatumoside IX (19): Yellow amorphous powder. [α]D24
subjected to semi-preparative HPLC (Develosil-ODS-15/30 −134 (c=0.84, MeOH). FAB-MS m/z: 941 [M+Na]+, 919
50mm i.d.×100cm (MeCN–H2O 2:8→25:75, v/v), Capcellpak [M+H]+. HR-FAB-MS m/z: 941.2541, 919.2714 (Calcd for
ODS-UG80 30mm i.d.×25cm, YMC-ODS 20mm i.d.×25cm C39H50O25Na: 941.2539 and C39H51O25: 919.2719). UV λmax

1568
Vol. 60, No. 12
Table 2. 1H-NMR Spectroscopic Data of Compounds 4, 6, 8–12, 18–23, 26–29, and 30
4
6
8
9
10
Aglycone moiety
H-6
6.21 (d, 2.0)
6.38 (d, 2.0)
7.78 (brd, 9.0)
6.95 (brd, 9.0)
6.22 (d, 2.0)
6.39 (d, 2.0)
7.79 (brd, 9.0)
6.95 (brd, 9.0)
6.17 (d, 2.0)
6.24 (d, 2.0)
7.75 (brd, 9.0)
6.94 (brd, 9.0)
6.12 (d, 2.0)
6.11 (d, 2.0)
7.68 (brd, 9.0)
6.91 (brd, 9.0)
6.22 (d, 2.0)
-8
6.39 (d, 2.0)
-2′,6′
-3′,5′
3-O-Sugars
7.79 (brd, 9.0)
6.96 (brd, 9.0)
Rha
Rha
Rha
Rha
Rha
H-1
-2
5.59 (d, 1.5)
4.26 (dd, 3.5, 1.5)
3.80 (dd, 9.5, 3.5)
3.23*
5.39 (d, 1.5)
4.42 (dd, 3.0, 1.5)
3.80 (dd, 9.0, 3.0)
3.51 (t, 9.0)
3.47*
5.82 (d, 1.5)
4.51*
5.81 (d, 1.5)
4.57*
5.62 (d, 1.5)
4.57 (dd, 3.5, 1.5)
4.15 (dd, 9.0, 3.5)
3.75 (t, 9.0)
3.53 (dq, 9.0, 6.0)
1.04 (d, 6.0)
Glc
-3
3.82 (dd, 9.5, 3.0)
3.49 (t, 9.5)
3.32 (m)
3.88 (dd, 9.5, 3.5)
3.52 (t, 9.5)
3.41*
-4
-5
3.54 (dq, 9.5, 6.0)
0.97 (d, 6.0)
Qui
-6
0.99 (d, 6.0)
0.93 (d, 6.0)
Glc
1.00 (d, 6.0)
Glc
H-1′
-2′
-3′
-4′
-5′
4.36 (d, 8.0)
3.22 (dd, 9.0, 8.0)
3.30*
—
—
—
—
—
—
4.57 (d, 8.0)
3.21 (dd, 9.0, 8.0)
3.39 (t, 9.0)
3.32*
4.58 (d, 8.0)
3.26 (dd, 9.0, 8.0)
3.42 (t, 9.0)
3.33 (t, 9.0)
3.52*
4.56 (d, 8.0)
3.22 (dd, 9.0, 8.0)
3.38 (t, 9.0)
3.32*
2.95 (t, 9.0)
3.25 (dq, 9.0, 6.0)
1.17 (d, 6.0)
—
3.32*
3.22*
-6′
3.81 (dd, 12.0, 2.0)
3.69 (dd, 12.0, 5.0)
Glc
4.50*
3.71 (dd, 12.0, 2.0)
3.66 (dd, 12.0, 5.0)
Glc
4.22 (dd, 12.0, 5.5)
Glc
Glc
H-1″
-2″
-3″
-4″
-5″
—
—
—
—
—
—
—
4.49 (d, 8.0)
3.31*
4.54 (d, 8.0)
3.35 (dd, 9.0, 8.0)
3.46 (t, 9.0)
3.30 (t, 9.0)
3.71 (m)
4.57 (d, 8.0)
3.36 (dd, 9.0, 8.0)
3.46 (t, 9.0)
3.31*
4.63 (d, 8.0)
3.29*
3.41 (m)
3.40*
3.35*
3.40*
3.35*
3.70 (m)
3.40*
-6″
3.86 (dd, 12.0, 2.0)
3.76 (dd, 12.0, 4.5)
4.51 (2H, *)
—
4.52 (dd, 12.0, 2.5)
4.48 (dd, 12.0, 8.0)
3.92 (brd, 12.0)
3.79 (brd, 12.0)
Glc
H-1‴
-2‴
-3‴
-4‴
-5‴
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
4.67 (d, 8.0)
3.12 (t, 8.0)
3.31*
3.27*
3.25*
—
-6‴
3.82 (dd, 12.0, 2.0)
3.64 (dd, 12.0, 5.0)
7-O-Sugar
H-1⁗
-2⁗
-3⁗
-4⁗
-5⁗
-6⁗
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
H-1″‴
-2″‴
-3″‴
-4″‴
-5″‴
-6″‴
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
Ester moieties
(R
2) H-β
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
6.12 (d, 16.0)
7.48 (d, 16.0)
7.26 (brd, 8.5)
6.69 (brd, 8.5)
6.24 (d, 16.0)
7.54 (d, 16.0)
7.11 (brd, 8.5)
6.42 (brd, 8.5)
—
—
—
—
—
—
—
—
-γ
-2,6
-3,5
—
—
(R3) H-β′
-γ′
6.26 (d, 16.0)
7.56 (d, 16.0)
7.12 (brd, 8.5)
6.40 (brd, 8.5)
-2′,6′
-3′,5′

December 2012
1569
Table 2. (Continued)
11
12
18
19
20
Aglycone moiety
H-6
6.08 (d, 2.0)
6.16 (d, 2.0)
6.47 (d, 2.0)
6.50 (d, 2.0)
6.39 (d, 2.0)
-8
6.11 (d, 2.0)
6.21 (d, 2.0)
6.72 (d, 2.0)
6.74 (d, 2.0)
6.56 (d, 2.0)
-2′,6′
-3′,5′
3-O-Sugars
7.66 (brd, 8.5)
6.90 (brd, 8.5)
7.72 (brd, 8.5)
6.93 (brd, 8.5)
7.81 (brd, 9.0)
6.97 (brd, 9.0)
7.80 (brd, 9.0)
6.94 (brd, 9.0)
7.79 (brd, 8.5)
6.96 (brd, 8.5)
Rha
Rha
Rha
Rha
Rha
H-1
-2
5.69 (d, 1.5)
4.52 (dd, 3.5, 1.5)
4.12 (dd, 9.5, 3.5)
3.75 (t, 9.5)
3.47*
5.71 (d, 1.5)
4.46 (dd, 3.5, 1.5)
4.06 (dd, 9.0, 3.5)
3.62 (t, 9.0)
3.25 *
5.67 (d, 1.5)
4.55*
5.73 (d, 1.5)
4.31 (dd, 3.5, 1.5)
3.81 (dd, 9.5, 3.5)
3.33 (t, 9.5)
3.41*
5.85 (d, 1.5)
4.50 (dd, 3.5, 1.5)
3.82 (dd, 9.5, 3.5)
3.49 (t, 9.5)
3.23*
-3
3.93*
-4
3.54*
-5
3.54*
-6
1.04 (d, 6.0)
Glc
0.90 (d, 6.5)
Glc
1.00 (d, 6.0)
Glc
0.95 (d, 6.0)
Glc
0.93 (d, 6.0)
Glc
H-1′
-2′
-3′
4.57 (d, 8.0)
3.26 (t, 8.0)
3.41 (t, 8.0)
3.31*
4.55 (d, 8.0)
3.21 (dd, 9.0, 8.0)
3.40 (t, 9.0)
3.36*
4.55 (d, 8.0)
3.21 (dd, 9.0, 8.0)
3.39 (t, 9.0)
3.32*
4.44 (d, 8.0)
3.24 (dd, 9.0, 8.0)
3.36 (t, 9.0)
4.57 (d, 8.0)
3.21 (dd, 9.0, 8.0)
3.39 (t, 9.0)
3.32*
-4′
-5′
-6′
3.48*
3.32*
3.23 (m)
3.71 (dd, 12.0, 2.5)
3.66 (dd, 12.0, 5.0)
Glc
3.24*
3.73*
3.67*
3.32*
4.47 (dd, 12.0, 2.5)
4.21 (dd, 12.0, 5.5)
Glc
3.78 (dd, 12.0, 2.0)
3.68 (dd, 12.0, 5.0)
Glc
3.84*
3.71*
Glc
H-1″
-2″
-3″
-4″
-5″
4.73 (d, 8.0)
3.32*
4.71 (d. 8.0)
3.32*
4.56 (d, 8.0)
3.29*
—
—
—
—
—
—
—
4.54 (d, 8.0)
3.35 (dd, 9.0, 8.0)
3.46 (t, 9.0)
3.29*
3.42 (t, 9.0)
3.31*
3.42 (t, 8.0)
3.28*
3.42*
3.42*
3.71 (m)
3.73*
3.42*
3.72*
-6″
4.50 (2H, *)
—
4.55 (dd, 12.0, 9.0)
4.46 (dd, 12.0, 2.0)
Xyl
3.93*
4.56 (dd, 12.0, 9.0)
4.45 (dd, 12.0, 2.0)
3.80 (dd, 12.0, 4.0)
Glc
H-1‴
-2‴
-3‴
-4‴
-5‴
4.71 (d, 8.0)
3.08 (t, 8.0)
3.28*
4.64 (d, 8.0)
3.03 (dd, 9.0, 8.0)
3.23 (t, 9.0)
3.45 (m)
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
3.26*
3.24*
3.76 (dd, 11.5, 5.0)
3.13 (t, 11.0)
—
—
-6‴
3.82 (dd, 12.0, 2.0)
3.63 (dd, 12.0, 5.0)
—
7-O-Sugar
Rha
Glc
Rha
H-1⁗
-2⁗
-3⁗
-4⁗
-5⁗
-6⁗
—
—
—
—
—
—
—
—
—
—
—
—
—
—
5.57 (d, 1.5)
4.03 (dd, 3.5, 1.5)
3.83 (dd, 9.5, 3.5)
3.48 (t, 9.5)
3.61 (dq, 9.5, 6.0)
1.21 (d, 6.0)
—
5.24 (d-like, 7.5)
3.73*
5.55 (d, 1.5)
4.04 (dd, 3.5, 1.5)
3.84 (dd, 9.5, 3.5)
3.49 (t, 9.5)
3.65*
3.73*
3.46 (t, 9.0)
3.54 (m)
3.92 (dd, 12.0, 2.0)
3.71*
1.29 (d, 6.0)
—
Glc
H-1″‴
-2″‴
-3″‴
-4″‴
-5″‴
-6″‴
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
4.67 (d, 8.0)
3.26 (t, 8.0)
3.40 (t, 8.0)
—
—
—
—
—
—
—
3.26*
3.67 (dd, 12.0, 4.0)
3.61 (dd, 12.0, 2.0)
Ester moieties
(R
2) H-β
6.16 (d, 16.0)
7.49 (d, 16.0)
7.27 (brd, 8.5)
6.68 (brd, 8.5)
6.19 (d, 16.0)
7.50 (d, 16.0)
7.04 (brd, 8.5)
6.38 (brd, 8.5)
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
-γ
-2,6
-3,5
—
—
—
—
(R3) H-β′
-γ′
6.22 (d, 16.0)
7.53 (d, 16.0)
7.07 (brd, 8.5)
6.37 (brd, 8.5)
6.24 (d, 16.0)
7.53 (d, 16.0)
7.08 (brd, 8.5)
6.35 (brd, 8.5)
-2′,6′
-3′,5′

1570
Vol. 60, No. 12
Table 2. (Continued)
21
22
23
26
27
Aglycone moiety
H-6
6.34 (d, 2.0)
6.43 (d, 2.0)
6.38 (d, 2.0)
6.38 (d, 2.0)
6.33 (d, 2.0)
-8
6.41 (d, 2.0)
6.57 (d, 2.0)
6.42 (d, 2.0)
6.54 (d, 2.0)
6.37 (d, 2.0)
-2′,6′
-3′,5′
3-O-Sugars
7.69 (brd, 9.0)
6.92 (brd, 9.0)
7.78 (brd, 9.0)
6.96 (brd, 9.0)
7.69 (brd, 9.0)
6.93 (brd, 9.0)
7.76 (brd, 9.0)
6.96 (brd, 9.0)
7.67 (brd, 8.5)
6.92 (brd, 8.5)
Rha
Rha
Rha
Rha
Rha
H-1
-2
5.82 (d, 1.5)
4.55*
5.84 (d, 1.5)
4.51 (dd, 3.5, 1.5)
3.82 (dd, 9.5, 3.5)
3.49 (t, 9.5)
3.22*
5.82 (brs)
4.55*
5.73 (d, 1.5)
4.45 (dd, 3.0, 1.5)
4.07 (dd, 10.0, 3.0)
3.63 (t, 10.0)
3.24*
5.69 (d, 1.5)
4.49 (dd, 3.5, 1.5)
4.13 (dd, 9.5, 3.5)
3.67 (t, 9.5)
3.48*
-3
3.88 (dd, 9.5, 3.5)
3.53 (t, 9.5)
3.44*
3.87 (dd, 9.5, 3.5)
3.53 (t, 9.5)
3.40*
-4
-5
-6
1.01 (d, 6.0)
Glc
0.92 (d, 6.0)
Glc
1.00 (d, 6.0)
Glc
0.91 (d, 6.0)
Glc
0.99 (d, 6.0)
Glc
H-1′
-2′
-3′
4.56 (d, 8.0)
3.26 (dd, 9.0, 8.0)
3.42 (t, 9.0)
3.33*
4.57 (d, 8.0)
3.21 (t, 9.0)
3.38*
4.55 (d, 8.5)
3.26 (t, 8.5)
3.43 (t, 8.5)
3.33*
4.55 (d, 8.0)
3.22 (dd, 9.0, 8.0)
3.40 (t, 9.0)
3.36*
4.52 (d, 8.0)
3.27 (dd, 9.0, 8.0)
3.42 (t, 9.0)
3.32*
-4′
3.33*
-5′
3.53*
3.33*
3.53*
3.32*
3.47*
-6′
4.53*
3.83 (dd, 12.0, 2.0)
3.71*
4.53*
3.80 (dd, 12.0, 2.0)
3.70 (dd, 12.0, 5.0)
Glc
4.49 (dd, 12.0, 2.5)
4.19 (dd, 12.0, 6.0)
Glc
4.20 (dd, 12.0, 6.0)
Glc
4.21 (dd.12.0, 6.0)
Glc
Glc
H-1″
-2″
-3″
-4″
-5″
4.58 (d, 8.0)
3.36 (dd, 9.0, 8.0)
3.46 (t, 9.0)
3.30*
4.54 (d, 8.0)
3.36 (dd, 9.0, 8.0)
3.46 (t, 9.0)
3.29 (t, 9.0)
3.73*
4.58 (d, 8.0)
3.36 (dd, 9.0, 8.0)
3.46 (t, 9.0)
3.31*
4.72 (d, 8.0)
3.32*
4.75**
3.33*
3.43 (t, 9.0)
3.28*
3.43 (t, 9.0)
3.29*
3.71*
3.72 (m)
3.74*
3.72 (m)
-6″
4.53*
4.56*
4.53*
4.59 (dd, 12.0, 9.0)
4.42 (dd, 12.0, 2.0)
Xyl
4.54 (dd, 12.0, 9.0)
4.44 (dd, 12.0, 2.0)
Xyl
4.48 (dd, 12.0, 2.5)
4.45 (dd, 12.0, 2.0)
4.48 (dd, 12.0, 2.0)
H-1‴
-2‴
-3‴
-4‴
-5‴
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
4.65 (d, 8.0)
3.03 (dd, 9.0, 8.0)
3.24 (t, 9.0)
3.43*
4.68 (d, 8.0)
3.06 (dd, 9.0, 8.0)
3.26 (t, 9.0)
3.45*
3.76 (dd, 11.5, 5.5)
3.13 (t, 11.5)
—
3.78 (dd, 11.5, 5.5)
3.15 (t, 11.5)
—
-6‴
—
—
7-O-Sugar
Rha
Glc
Glc
Rha
Rha
H-1⁗
-2⁗
-3⁗
-4⁗
-5⁗
-6⁗
5.51 (d, 1.5)
4.04 (dd, 3.5, 1.5)
3.84 (dd, 9.5, 3.5)
3.49 (t, 9.5)
3.68 (dq, 9.5, 6.0)
1.30 (d, 6.0)
—
5.07 (d-like, 8.0)
3.50*
5.05 (d-like, 8.0)
3.52*
5.56 (d, 2.0)
4.04 (dd, 3.5, 2.0)
3.84 (dd, 9.5, 3.5)
3.49 (t, 9.5)
3.66 (m)
5.51 (d, 1.5)
4.06 (dd, 3.5, 1.5)
3.85 (dd, 9.5, 3.5)
3.49 (t, 9.5)
3.68*
3.51*
3.52*
3.40*
3.42*
3.55 (m)
3.96 (dd, 12.0, 2.5)
3.71*
3.55*
3.98 (dd, 12.0, 2.0)
3.75 (dd, 12.0, 6.0)
1.29 (d, 6.0)
—
1.30 (d, 6.0)
—
H-1″‴
-2″‴
-3″‴
-4″‴
-5″‴
-6″‴
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
Ester moieties
(R
2) H-β
6.10 (d, 16.0)
7.67 (d, 16.0)
7.24 (brd, 9.0)
6.68 (brd, 9.0)
6.20 (d, 16.0)
7.50 (d, 16.0)
7.05 (brd, 9.0)
6.35 (brd, 9.0)
—
—
6.12 (d, 16.0)
7.48 (d, 16.0)
7.23 (brd, 8.5)
6.68 (brd, 8.5)
6.21 (d, 16.0)
7.50 (d, 16.0)
7.04 (brd, 8.5)
6.37 (brd, 8.5)
—
—
6.12 (d, 16.0)
7.47 (d, 16.0)
7.26 (brd, 9.0)
6.67 (brd, 9.0)
6.16 (d, 16.0)
7.46 (d, 16.0)
6.98 (brd, 9.0)
6.32 (brd, 9.0)
-γ
-2,6
-3,5
—
—
—
—
(R3) H-β′
-γ′
6.25 (d, 16.0)
7.53 (d, 16.0)
7.07 (brd, 8.5)
6.36 (brd, 8.5)
6.20 (d, 16.0)
7.50 (d, 16.0)
7.03 (brd, 8.5)
6.32 (brd, 8.5)
-2′,6′
-3′,5′

December 2012
1571
Table 2. (Continued)
28
29
30
Aglycone moiety
H-6
6.36 (d, 2.0)
6.38 (d, 2.0)
7.67 (brd, 9.0)
6.92 (brd, 9.0)
6.36 (d, 2.0)
6.39 (d, 2.0)
7.68 (brd, 9.0)
6.92 (brd, 9.0)
6.44 (d, 2.0)
6.41 (d, 2.0)
7.71 (brd, 9.0)
6.93 (brd, 9.0)
-8
-2′,6′
-3′,5′
3-O-Sugars
Rha
Rha
Rha
H-1
-2
5.68 (d, 1.5)
4.48 (dd, 3.5, 1.5)
4.12 (dd, 9.5, 3.5)
3.66 (t, 9.5)
3.48*
5.69 (brs)
4.49 (brd, 3.5)
4.12 (dd, 9.5, 3.5)
3.76 (t, 9.5)
3.48*
5.81 (brs)
4.56*
-3
3.86 (dd, 9.5, 3.5)
3.52*
-4
-5
3.39*
-6
0.99 (d, 6.0)
Glc
1.04 (d, 6.0)
Glc
1.00 (d, 6.0)
Glc
H-1′
-2′
-3′
4.51 (d, 8.0)
3.26 (dd, 9.0, 8.0)
3.42 (t, 9.0)
3.32*
4.53 (d, 8.0)
3.26 (dd, 9.0, 8.0)
3.42 (t, 9.0)
3.32*
4.57 (d, 8.0)
3.26 (dd, 9.0, 8.0)
3.44 (t, 9.0)
3.34*
-4′
-5′
3.47*
3.48*
3.54*
-6′
4.50 (dd, 12.0, 2.0)
4.19 (dd, 12.0, 6.0)
Glc
4.51 (dd, 12.0, 2.0)
4.20 (dd, 12.0, 6.0)
Glc
4.54*
4.21 (dd, 12.0, 6.0)
Glc
H-1″
-2″
-3″
4.76**
4.75**
4.58 (d, 8.0)
3.36 (dd, 9.0, 8.0)
3.46 (t, 9.0)
3.31*
3.33 (dd, 9.0, 8.0)
3.42 (t, 9.0)
3.29*
3.33*
3.43 (t, 9.0)
3.29*
-4″
-5″
3.73 (m)
3.73*
3.71*
-6″
4.55 (dd, 12.0, 9.0)
4.43 (dd, 12.0, 2.0)
Xyl
4.56 (dd, 12.0, 9.0)
4.43 (dd, 12.0, 2.0)
Glc
4.52 (dd, 12.0, 8.0)
4.48 (dd, 12.0, 2.0)
H-1‴
-2‴
-3‴
-4‴
-5‴
4.68 (d, 8.0)
3.06 (dd, 9.0, 8.0)
3.25 (t, 9.0)
3.46*
4.72 (d, 8.0)
3.08 (t, 8.0)
3.31*
—
—
—
—
—
—
—
—
3.24*
3.78 (dd, 11.5, 5.5)
3.14 (t, 11.5)
—
3.24*
—
-6‴
3.82 (dd, 12.0, 2.0)
3.63 (dd, 12.0, 5.0)
—
7-O-Sugar
Glc
Glc
Glc
H-1⁗
-2⁗
-3⁗
-4⁗
-5⁗
-6⁗
5.05 (d-like, 8.0)
3.51*
5.05 (d-like, 8.0)
3.52*
5.05 (d-like, 8.0)
3.52*
3.51*
3.52*
3.52*
3.42*
3.42*
3.52*
3.55*
3.55*
3.76*
3.98 (dd, 12.0, 2.5)
3.75 (dd, 12.0, 6.0)
3.98 (dd, 12.0, 2.5)
3.75*
4.40 (dd, 11.5, 5.0)
3.86 (dd, 11.5, 2.0)
α-Glc
H-1″‴
-2″‴
-3″‴
-4″‴
-5″‴
-6″‴
—
—
—
—
—
—
—
—
—
—
—
—
—
—
4.90 (d, 4.0)
3.40 (dd, 9.0, 4.0)
3.37 (t, 9.0)
3.31*
3.66*
3.77*
3.64*
Ester moieties
(R2) H-β
-γ
6.14 (d, 16.0)
7.46 (d, 16.0)
7.24 (brd, 8.5)
6.67 (brd, 8.5)
6.16 (d, 16.0)
7.47 (d, 16.0)
6.97 (brd, 8.5)
6.33 (brd, 8.5)
6.16 (d, 16.0)
7.46 (d, 16.0)
7.24 (brd, 8.5)
6.67 (brd, 8.5)
6.17 (d, 16.0)
7.48 (d, 16.0)
6.97 (brd, 8.5)
6.33 (brd, 8.5)
6.13 (d, 16.0)
7.48 (d, 16.0)
7.25 (brd, 8.5)
6.68 (brd, 8.5)
6.22 (d, 16.0)
7.51 (d, 16.0)
7.06 (brd, 8.5)
6.38 (brd, 8.5)
-2,6
-3,5
(R3) H-β′
-γ′
-2′,6′
-3′,5′
Measured in MeOH-d4 solution at 35°C. *: Overlapped with other signals. **: Overlapped with the H2O signal. Data of the ester moieties may be interchangeable in each
column. Rha:α-L-rhamnopyranose, Qui: β-D-quinovopyranose, Glc: β-D-glucopyranose, α-Glc: α-D-glucopyranose, Xyl: β-D-xylopyranose.

1572
Vol. 60, No. 12
(MeOH) nm (logε): 266 (4.34), 327 (sh), 344 (4.18).
(Tokyo Kasei Kogyo Co., Ltd.)), 15.0min (D-rhamnose),
Ternatumoside X (20): Yellow amorphous powder. [α]D18 −41 13.2min (D-xylose (Tokyo Kasei Kogyo Co., Ltd.)), 12.4min
(c=0.39, MeOH). FAB-MS m/z: 1071 [M+Na]+. HR-FAB-MS (L-xylose), 14.8min (D-quinovose (Sigma Chem. Co.)), 14.3min
m/z: 1071.2996 (Calcd for C48H56O26Na: 1071.2958). UV λmax (L-quinovose). The tRs for L-glucose, D-rhamnose, L-xylose,
(MeOH) nm (logε): 220 (sh), 269 (4.29), 316 (4.34).
and L-quinovose were obtained from their enantiomers (D-
Compound 21: Yellow amorphous powder. [α]D19 −65 glucose+L-cysteine, L-rhamnose+L-cysteine, D-xylose+L-cys-
(c=0.39, MeOH). FAB-MS m/z: 1217 [M+Na]+, 1195 teine, and D-quinovose+L-cysteine). L-Rhamnose was found in
[M+H]+. HR-FAB-MS m/z: 1217.3307 (Calcd for C57H62O28Na: all compounds, and D-glucose was identified in 6, 10, 18 and
1217.3325). UV λmax (MeOH) nm (logε): 226 (4.55), 271 (4.51), 19. D-Quinovose was detected in 4.
314 (4.70).
Alkaline and Acid Hydrolysis of Compounds 8, 9, 11,
Ternatumoside XI (22): Yellow amorphous powder. [α]D24 12, 20–23, 26–29, and 30 Compounds 8, 9, 11, 12, 20–23,
−22 (c=1.06, MeOH). FAB-MS m/z: 1087 [M+Na]+, 1065 26–29, and 30 were dissolved in 0.05 M NaOH (100µL), and
[M+H]+. HR-FAB-MS m/z: 1087.2911 (Calcd for C48H56O27Na: stirred at room temperature for 3–4h under an N2 atmo-
1087.2907). UV λmax (MeOH) nm (logε): 269 (4.42), 316 (4.48). sphere. After the reactions, the mixture was neutralized with
Ternatumoside XII (23): Yellow amorphous powder. [α]D20 an Amberlite IR-120B column with the eluate concentrated
−30 (c=1.62, MeOH). FAB-MS m/z: 1233 [M+Na]+. HR- dry. The residue was partitioned between EtOAc and H2O,
FAB-MS m/z: 1233.3320 (Calcd for C57H62O29Na: 1233.3274). and both layers were concentrated dry. The residue from the
UV λmax (MeOH) nm (logε): 225 (4.44), 271 (4.38), 314 (4.57). EtOAc layer was analyzed using HPLC through a comparison
Ternatumoside XIII (26): Yellow amorphous powder. [α]D24 with the authentic sample. HPLC conditions: column, YMC-
−76 (c=0.52, MeOH). FAB-MS m/z: 1203 [M+Na]+. HR- ODS-AM 4.6mm i.d.×25cm; flow rate, 1.0mL/min; 17.5%
FAB-MS m/z: 1203.3372 (Calcd for C53H64O30Na: 1203.3381). MeCN in water+0.05% trifluoroacetic acid (TFA); tR, 14.0min
UV λmax (MeOH) nm (logε): 269 (4.45), 315 (4.50).
[(trans)-p-coumaric acid (Tokyo Kasei Kogyo Co., Ltd.)].
Ternatumoside XIV (27): Yellow amorphous powder. [α]D20 (trans)-p-Coumaric acid was detected in all compounds. The
−72 (c=0.59, MeOH). FAB-MS m/z: 1349 [M+Na]+. HR- residues from the H2O layers of compounds 8, 9, 11, 20, and
FAB-MS m/z: 1349.3755 (Calcd for C62H70O32Na: 1349.3748). 21 were also analyzed using HPLC through a comparison with
UV λmax (MeOH) nm (logε): 225 (4.53), 271 (4.48), 314 (4.67). compounds 7, 10 and 18. HPLC conditions: column, YMC-
Ternatumoside XV (28): Yellow amorphous powder. [α]D26 ODS-AM 4.6mm i.d.×25cm; flow rate, 1.0mL/min; 17.5%
−43 (c=1.08, MeOH). FAB-MS m/z: 1365 [M+Na]+. HR- MeCN; tR, 11.2min (18), 19.0min (10), 25.2min (7). 7 was
FAB-MS m/z: 1365.3710 (Calcd for C62H70O33Na: 1365.3697). detected in 8 and 9, and 10 was found in 11. 18 was identi-
UV λmax (MeOH) nm (logε): 226 (4.54), 271 (4.49), 314 (4.67). fied in 20 and 21. Other residues were hydrolyzed with 2M
Ternatumoside XVI (29): Yellow amorphous powder. [α]D26 HCl, and the procedures were described above. Kaempferol,
−39 (c=1.12, MeOH). FAB-MS m/z: 1395 [M+Na]+. HR-FAB- L-rhamnose, and D-glucose were detected in all compounds.
MS m/z: 1395.3812 (Calcd for C63H72O34Na: 1395.3803). UV D-Xylose was found in 12, 26, 27, and 28.
λmax (MeOH) nm (logε): 225 (4.52), 271 (4.47), 314 (4.65).
Ternatumoside XVII (30): Yellow amorphous powder. [α]D26
References
−31 (c=0.75, MeOH). FAB-MS m/z: 1395 [M+Na]+. HR-FAB-
1) Koshimizu R., Okumura Y., Hanano A., Jpn. Kokai Tokkyo Koho,
JP 2004043348 A (2004).
2) Aramaki S., Nakajima N., Yoshida H., Jpn. Kokai Tokkyo Koho, JP
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6) Nørbaek R., Kondo T., Phytochemistry, 51, 1113–1119 (1999).
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8) Küçükislamoglu M., Yayli N., Sentürk H. B., Genç H., Ozden S.,
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1
The 13C- and H-NMR spectroscopic data for compounds
4, 6, 8–12, 18–23, 26–29, and 30 were presented in Tables 1
and 2.
Acid Hydrolysis of Compounds 4, 6, 10, 18 and 19
Compounds 4, 6, 10, 18 and 19 (ca. 1mg) were dissolved in
2M HCl (200µL). The solutions were heated at 100°C for 1h.
After hydrolysis, this reaction mixture was diluted with H2O
and extracted with EtOAc. The EtOAc layer was concentrated
dry, and the residue from each compound was analyzed using
HPLC through a comparison with the authentic sample. HPLC
conditions: column, YMC-ODS-AM 4.6mm i.d.×25cm; flow
rate, 1.0mL/min; 35% MeCN in water; tR, 14.0min (kaemp-
ferol: The authentic sample was provided by Prof. T. Miyase.).
Kaempferol was detected in all compounds. The H2O layer
was neutralized with an Amberlite IRA-60E column, and the
eluate was concentrated dry. The residue was stirred with D-
cysteine methyl ester hydrochloride, hexamethyldisilazane and
11) Nørbæk R., Nielsen J. K., Kondo T., Phytochemistry, 51, 1139–1146
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Science Inc.) 0.25mm i.d.×30m; carrier gas, N2; column
temperature, 230°C; tR, 22.8min (D-glucose (Tokyo Kasei
1799–1802 (1976).
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December 2012
1573
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