Triterpene saponins with hyaluronidase inhibitory activity from the seeds of Camellia sinensis

Article abstract of DOI:10.1248/cpb.60.612

The MeOH extract of the seeds of Camellia sinensis (L.) KUNTZE gave twelve new saponins (1-12) along with ten known saponins (13-22). These saponins (1-22) showed stronger hyaluronidase inhibitory activity than the positive control, rosmarinic acid.

Full text of DOI:10.1248/cpb.60.612

612
Regular Article
Chem. Pharm. Bull. 60(5) 612–623 (2012)
Vol. 60, No. 5
Triterpene Saponins with Hyaluronidase Inhibitory Activity from the
Seeds of Camellia sinensis
,b
Michiko Myose,a Tsutomu Warashina,* and Toshio Miyasea
b
a School of Pharmaceutical Sciences, University of Shizuoka; and Institute for Environmental Sciences, University of
Shizuoka; 52–1 Yada, Suruga-ku, Shizuoka 422–8526, Japan.
Received December 10, 2011; accepted March 1, 2012; published online March 2, 2012
The MeOH extract of the seeds of Camellia sinensis (L.) KUNTZE gave twelve new saponins (1–12) along
with ten known saponins (13–22). These saponins (1–22) showed stronger hyaluronidase inhibitory activity
than the positive control, rosmarinic acid.
Key words triterpene saponin; Camellia sinensis; hyaluronidase inhibitor
Tea is cultivated widely in Asia and has a longstanding
reputation for its health-promoting properties. The bioactiv-
Results and Discussion
The seeds of C. sinensis cultivated in Shizuoka prefecture,
ity of the constituents of Camellia species has been reported Japan, were extracted with MeOH, and the extract was con-
previously, including for antisweet, gastroprotective, gastric- centrated under reduced pressure and then dissolved in water
emptying inhibitory, gastrointestinal transit accelerating, an- and extracted with EtOAc. The MeOH eluate, obtained from a
tioxidative and antimicrobial activity.1–3) Although studies on Mitsubishi Diaion HP-20 column for the water layer, was sub-
hyaluronidase inhibitory activity of tea saponins have been jected to preparative HPLC, affording twelve new compounds
reported, detailed analysis of these compounds is still lack- (1–12), together with ten known compounds (13–22), which
ing.4) The hyaluronidase inhibitor rosmarinic acid is already were identified by comparison of NMR data with reported
known.5) In the course of the studies on the constituents of the data.
seeds of Camellia sinensis, we obtained twelve new saponins
Teaseedsaponin A (1) was isolated as a colorless amorphous
(1–12) together with ten known saponins (13–22).1,2,6–10) The powder. The molecular formula was established as C62H96O28
present report deals with the isolation of these saponins, struc- on the basis of the high resolution (HR)-FAB-MS data
1
tural elucidation of the new compounds, and analysis of their ([M−H]− ion at m/z 1287.6034). The H- and 13C-NMR data
1
hyaluronidase inhibitory activities. As a result, compounds were similar to those of foliatheasaponin III (14).7) The H-
(1–22) were found to have stronger inhibitory activities (IC50: NMR spectrum of 1 indicated the presence of the following
19.3–55.6µM) than rosmarinic acid (IC50: 240.1µM).
functions, seven singlet methyl groups at δ 0.81, 0.92, 1.08,
1.11, 1.24, 1.28, 1.50 (each 3H, s, 25, 26, 24, 29, 30, 23, 27), a
Chart 1
*To whom correspondence should be addressed. e-mail: warashin@u-shizuoka-ken.ac.jp
© 2012 The Pharmaceutical Society of Japan

May 2012
613
methylene and three methines bearing an oxygen function at δ similar to those of theasaponin A3 (15).8) Acid hydrolysis gave
3.73, 4.22 (each 1H, d, J=11.0Hz, 28), 4.40 (1H, d, J=10.0Hz, D-xylose, D-galactose, L-arabinose and D-glucuronic acid, and
22), 5.84 (1H, brs, 16), 5.93 (1H, d, J=10.0Hz, 21), an olefinic the HMBC experiment showed that the sugar chain at C-3 was
proton at δ 5.44 (1H, brs, 12), four anomeric protons at δ 4.91 decided to be β-D-xylopyranosyl-(1
→2)-α-L-arabinopyranosyl-
(1H, d, J=7.5Hz, GlcA-1), 5.11 (1H, d, J=7.5Hz, Glc-1), 5.65 (1 3)-[β-D-galactopyranosyl-(1 2)]-β-D-glucuronopyranosyl.
→
→
(1H, d, J=7.5Hz, Gal-1), 5.77 (1H, d, J=5.0Hz, Ara-1), two Consequently, the structure of teaseedsaponin C was deter-
acetyl methyl at δ 1.97, 2.50 (each 3H, s, 28-O-Ac-2, 16- mined to be 3.
O-Ac-2), one angeloyl unit at δ 1.92 (3H, dq, J=1.5, 1.5Hz,
The molecular formula C63H98O28 was concluded for tea-
21-O-Ang-5), 2.01 (3H, dq, J=7.0, 1.5Hz, 21-O-Ang-4), 5.91 seedsaponin D (4) on the basis of the molecular peak at m/z
(1H, qq, J=7.0, 1.5Hz, 21-O-Ang-3). The 13C-NMR spec- 1301.6156 in the HR-FAB-MS. The 1H- and 13C-NMR data
trum of 1 showed signals owing to four carboxyl groups at δ for 4 were similar to those of floratheasaponin B,2) which sug-
172.1 (GlcA-6), 168.2 (21-O-Ang-1), 169.8 (16-O-Ac-1), 170.4 gested that 4 had two angeloyl groups. In addition, in the 13C-
(22-O-Ac-1). In the 13C-NMR spectrum, the carbon signals NMR spectrum, C-15 shifted to a higher field at δ 34.8 (15),
resulting from a sugar chain at C-3 were similar to those of compared with those of floratheasaponin B,2) suggesting that
1
camelliasaponin A1 (13).6) Acid hydrolysis gave D-glucose, 4 had a methylene group at C-15. In the H-NMR spectrum
D-galactose, L-arabinose and D-glucuronic acid, and the 1H- at δ 4.14 (1H, dd, J=12.5, 4.5Hz, 3) and 13C-NMR at δ 83.0
detected heteronuclear multiple-bond connectivity (HMBC) (3) data for C-3 were similar to those of 3, which suggested
experiment showed that the sugar chain at C-3 was decided to that 4 had a hydroxyl methyl group at C-23. In the 13C-NMR
be β-D-glucopyranosyl-(1
→2)-α-L-arabinopyranosyl-(1→3)-[β- spectrum, the carbon signals resulting from the sugar chain at
D-galactopyranosyl-(1 2)]-β-D-glucuronopyranosyl. Further- C-3 were superimposable on those of 1, and as a result of acid
→
more, an HMBC experiment with 1 showed long-range cor- hydrolysis, the structure of teaseedsaponin D was determined
relations between H-16 and the carboxyl carbon of the acetyl to be 4.
unit (δC 169.8), H-21 and the carboxyl carbon of the angeloyl
Teaseedsaponin E (5) was established as C62H96O27 on the
unit (δC 168.2), and H-28 and the carboxyl carbon of the acetyl basis of the HR-FAB-MS data ([M−H]− ion at m/z 1271.6079).
unit (δC 170.4). Consequently, the structure of teaseedsaponin The H- and 13C-NMR data were superimposable on those of
A was determined to be 1.
1
4 except for the sugar moiety. The 13C-NMR data of the sugar
Teaseedsaponin B (2) was established as C62H96O28 on the chain at C-3 were similar to that of 3, and the result of acid
basis of HR-FAB-MS data ([M−H]− ion at m/z 1287.5997), hydrolysis showed that the structure of teaseedsaponin E was
which was the same as that of 1. The 1H- and 13C-NMR determined to be 5.
data of 2 were similar to those of 1, which suggested that
Teaseedsaponin F (6) had a molecular formula C62H98O27,
1
2 had two acetyl groups and one angeloyl group. In the H- as determined by negative-mode HR-FAB-MS data at m/z
NMR spectrum, H-22 shifted to a lower field at δ 6.12 (1H, 1273.6219. The H- and 13C-NMR spectra of 6 were similar
1
d, J=10.0Hz, 22) compared with that of 1, suggesting that 2 to those of 5, which suggested that 6 had two acyl groups.
1
had an acetyl group at C-22. Furthermore, the positions of The H-NMR spectrum of 6 showed signals resulting from a
three acyl units in 2 were decided by the HMBC experiment, 2-metyl-butyroyl unit at δ 0.80 (3H, t, J=7.5Hz, 2Mb-4), 1.10
which exhibited long-range correlations between H-16 and the (3H, d, J=7.0Hz, 2Mb-5), 1.71 (2H, dq, J=7.0, 7.5Hz, 2Mb-
carboxyl carbon of the acetyl unit (δC 169.8), H-21 and the car- 3), 2.27 (1H, sext, J=7.0Hz, 2Mb-2), which were similar to
boxyl carbon of the angeloyl unit (δC 167.8), and H-22 and the those of floratheasaponin C.2) Finally, the HMBC spectrum
carboxyl carbon of the acetyl unit (δC 170.4). Consequently the of 6 showed long-range correlations between H-21 and the
structure of teaseedsaponin B was determined to be 2.
carboxyl carbon of the angeloyl unit (δC 167.6), and H-22 and
Teaseedsaponin C (3) showed a molecular peak at m/z the carboxyl carbon of the 2-methyl-butyroyl unit (δC 176.7).
1187.5849 in negative mode HR-FAB-MS, suggesting a mo- It was determined that 6 had an angeloyl unit at C-21 and a
1
lecular formula of C58H92O25. In the H-NMR spectrum, H-3 2-methyl-butyroyl unit at C-22. Consequently, the structure of
shifted to a lower field at δ 4.12 (1H, dd, J=11.5, 3.5Hz, 3), teaseedsaponin F was determined to be 6.
and in the 13C-NMR spectrum, C-3 shifted to a higher field
For teaseedsaponin G (7), the molecular formula C58H90O25
at δ 83.3 (3), compared with those of 1, suggesting that 3 had was deduced from HR-FAB-MS data ([M−H]− ion at m/z
1
1
a hydroxyl methyl group at C-23. In the H-NMR spectrum, 1185.5696). The H- and 13C-NMR data were very similar to
1
H-16 shifted to a higher field at δ 4.59 (1H, brs), and in the those of 3. In the H-NMR spectrum, H-23 shifted to a lower
13C-NMR spectrum, C-21 shifted to a higher field at δ 41.8 field at δ 9.85 (1H, s, 23), and in the 13C-NMR spectrum, C-23
(21), compared with those of 2, suggesting that C-16 was not shifted to a lower field at δ 209.8 (23), compared with those
1
acylated and C-21 had a methylene group. The H-NMR spec- of 3, suggesting that 7 had an aldehyde group at C-23. Con-
trum of 3 showed signals owing to a cis-(2)-hexenoic acid unit sequently, the structure of teaseedsaponin G was determined
at δ 0.85 (3H, t, J=7.5Hz, Hex-6), 1.37 (2H, sext, J=7.5Hz, to be 7.
Hex-5), 2.75 (2H, m, Hex-4), 5.93 (1H, dt, J=11.5, 2.0Hz, Hex-
Teaseedsaponin H (8) showed an [M−H]− ion peak in HR-
2), 6.12 (1H, dt, J=11.5, 7.5Hz, Hex-3), on the basis of the ho- FAB-MS at m/z 1273.5846 corresponding to a molecular for-
monuclear Hartman–Hahn (HOHAHA) difference spectrum. mula of C61H94O28. The H- and 13C-NMR spectra of 8 were
1
The HMBC experiment for 3 showed a long-range correlation similar to those of assamsaponin H,9) which suggested that
1
between H-22 and the carboxyl carbon of the cis-(2)-hexenoic 8 had two acyl groups. The H-NMR spectrum of 8 showed
acid unit (δC 166.7), and it was determined that 3 had the cis- signals resulting from a cis-(2)-hexenoic acid unit at δ 0.85
(2)-hexenoic acid unit at C-22. In the 13C-NMR spectrum, (3H, t, J=7.5Hz, Hex-6), 1.38 (2H, sext, J=7.5Hz, Hex-5),
the carbon signals resulting from the sugar chain at C-3 were 2.74 (2H, m, Hex-4), 6.05 (1H, dt, J=11.0, 1.0Hz, Hex-2), 6.15

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Vol. 60, No. 5
(1H, dt, J=11.0, 7.5Hz, Hex-3), and the positions of two acyl inhibitory activities were measured. Table 3 shows the IC50
groups in 8 were clarified by the HMBC experiment. Thus, values. As a result, the inhibitory activities of compounds
long-range correlations were observed between H-21 and the 1–22 were found to be stronger than rosmarinic acid, used as
carboxyl carbon of the cis-(2)-hexenoic acid part (δC 167.1) and a positive control. Furthermore the inhibitory activity of 5, 6,
between H-28 and the carboxyl carbon of the acetyl part (δC camelliasaponin A1 (13), camelliasaponin B1 (16) and assa-
170.6), and it was determined that 8 had the cis-(2)-hexenoic msaponin F (21) had nearly ten-times stronger hyaluronidase
acid part at C-21 and the acetyl part at C-28. Consequently, inhibitory activities than rosmarinic acid. As far as we know,
the structure of teaseedsaponin H was determined to be 8.
this is the first report on oleanane-type triterpene saponin
Teaseedsaponin I (9) showed a molecular peak at m/z having hyaluronidase inhibitory activity. Moreover, structure–
1299.6010 in the negative mode HR-FAB-MS, suggesting a activity relationship was not able to be acquired, there were
1
molecular formula C63H96O28. The H- and 13C-NMR spectra few data obtained this time. So, we will research the saponin
of 9 were similar to those of 4, which suggested that 9 had from other plants.
two angeloyl groups. Furthermore, the 1H- and 13C-NMR
spectra were shifted to a lower field at δH 9.91 (1H, s, 23), δC
209.9 (23), compared with those of 4, suggesting that 9 had
Experimental
General Procedures Optical rotations were measured on
an aldehyde group at C-23. Consequently, the structure of tea- a JASCO DIP-1000 digital polarimeter. UV spectra were mea-
1
seedsaponin I was determined to be 9.
sured in methanol on a JASCO V-630 spectrophotometer. H-
Teaseedsaponin J (10), whose molecular formula C62H94O27 (400MHz) and 13C- (100MHz) NMR spectra were recorded
was determined by negative-mode HR-FAB-MS data at m/z on an α-400 FT-NMR spectrometer, and chemical shifts are
1269.5904. The H- and 13C-NMR data were very similar to given as δ values, with tetramethylsilane (TMS) as an inter-
1
those of 9 except for the sugar moiety. 13C-NMR data of the nal standard at 35 C in pyridine-d5. Inverse-detected hetero-
°
1
sugar chain at C-3 were similar to those of 7, and the results nuclear correlations were measured using H-detected hetero-
of acid hydrolysis showed that the structure of teaseedsaponin nuclear multiple quantum coherency (HMQC) (optimized for
n
J was determined to be 10.
1JC–H=145Hz) and HMBC (optimized for JC–H=8Hz) pulse
The molecular formula, C62H94O27 was concluded for tea- sequences with a pulse field gradient. HR-FAB-MS data were
seedsaponin K (11), on the basis of the molecular peak at m/z obtained on a JEOL JMS 700 mass spectrometer in the nega-
1
1269.5923, which was the same as that of 10. The H- and 13C- tive mode using m-nitrobenzyl alcohol as the matrix. Prepara-
NMR data of 11 were similar to those of 10, which suggested tive HPLC was performed on a JASCO 800 instrument.
that 11 had two acyl groups. In the 13C-NMR spectrum, acyl-4
Plant Material The seeds of C. sinensis were collected
and acyl-5 shifted to a higher field at δ 14.0 (Tig-4), 12.3 (Tig- at Takakusayama in Shizuoka prefecture, Japan, in October
5), compared with the angeloyl group, suggesting that 11 had 2010.
a tigloyl group. In the HMBC of 11, long-range correlations
Extraction and Isolation The cut seeds of C. sinensis
observed between H-21 and the angeloyl carboxyl carbon (δC (920g) were extracted twice with methanol (5L) under reflux
167.9) and between H-22 and the tigloyl carboxyl carbon (δC for 3h. The extract was concentrated under reduced pressure
169.4), and it was determined that 11 had an angeloyl group at to give a residue (111g). The residue was suspended in hot
C-21 and a tigloyl group at C-22. Consequently, the structure water (1.5L) and extracted with ethyl acetate continuously for
of Teaseedsaponin K was determined to be 11.
8h. After removing EtOAc, the water layer was fractionated
Teaseedsaponin L (12) showed an [M−H]− ion peak in using a Mitsubishi Diaion HP-20 column (6.5×28cm), elut-
HR-FAB-MS at m/z 1285.5851 corresponding to a molecular ing with water (10L), H2O–MeOH (50:50) (5L) and MeOH
formula of C62H94O28. In the 1H-NMR spectrum, H-16 and (3L). The fraction eluted with MeOH was concentrated under
H-22 shifted to a lower field at δ 5.59 (1H, brs, 16) and 6.12 reduced pressure to give a brown residue (13.3g). The MeOH
(1H, d, J=10.0Hz, 22), respectively, compared with those of 8, eluate (4.0g) was subjected to preparative HPLC [column,
suggesting that 12 had an acetyl group at C-16 and C-22. Fur- Tosoh TSKgel ODS-80Ts, 5.5×180cm; solvent, 0.1% trifluoro-
1
thermore, in the H-NMR spectrum, H-28 shifted to a higher acetic acid (TFA)–CH3CN (65:35–57:43) linear gradient, UV
field at δ 3.46, 3.57 (each 1H, d, J=10.5Hz, 28), compared 205nm] to give 30 fractions (Frs. A–Z and a–d). The 61:39
with that of 8, suggesting that 12 had a hydroxymethyl group solvent gave Fr. F [theasaponin A3 (15) (142.3mg)], the 59:41
at C-28. In addition, the 1H-NMR spectrum of 12 showed solvent gave Fr. R (3) (13.1mg), the 58:42 solvent gave Fr. V
signals resulting from a cis-(2)-hexenoic acid unit at δ 0.85 (7) (31.7mg), the 57:43 solvent gave Fr. Y (5) (49.8mg) and
(3H, t, J=7.5Hz, Hex-6), 1.39 (2H, sext, J=7.5Hz, Hex-5), Fr. a (9) (13.3mg), the 56:44 solvent gave Fr. b (10) (54.1mg).
2.74 (2H, m, Hex-4), 5.98 (1H, dt, J=11.0, 1.5Hz, Hex-2), 6.19 Then, Fr. M (37.9mg) was subjected to semipreparative HPLC
(1H, dt, J=11.0, 7.5Hz, Hex-3). The HMBC experiment for 12 [column, Cosmosil 5PE-MS, 2×25cm; solvent, H2O–CH3CN
showed long-range correlations between H-16 and the acetyl (60:40)+TFA (0.05%), UV 205nm] to give 1 (4.2mg), 2
carbon (δC 169.8), H-21 and the cis-(2)-hexenoic acid carbon (8.7mg) and 8 (4.3mg). Fr. X (15.8mg) was subjected to semi-
(δC 166.6), and H-22 and the acetyl carbon (δC 170.5). It was preparative HPLC [column, Cosmosil 5PE-MS, 2×25cm; sol-
determined that 12 had acetyl groups at C-16 and C-22 and a vent, H2O–CH3CN (57.5:42.5)+TFA (0.05%), UV 205nm] to
cis-(2)-hexenoic acid group at C-21. Furthermore, in the 13C- give 4 (5.3mg). Fr. Z (20.6mg) was subjected to semiprepara-
NMR spectrum, the carbon signals resulting from the sugar tive HPLC [column, Cosmosil 5PE-MS, 2×25cm; solvent,
chain at C-3 were similar to those of 3, and by acid hydrolysis, H2O–CH3CN (57.5:42.5)+TFA (0.05%), UV 205nm] to give 6
the structure of teaseedsaponin L was determined to be 12.
(4.9mg) and 11 (9.4mg). Fr. U (26mg) was subjected to semi-
In this study, in order to investigate the biological activities preparative HPLC [column, Develosil C30-UG-5, 2×25cm;
of isolated compounds 1–22, their effects on hyaluronidase solvent, H2O–CH3CN (55:45)+TFA (0.05%), UV 205nm]

May 2012
615
to give 12 (16.7mg). Fr. N (34.0mg) was subjected to semi- +12 (c=1.09, MeOH). UV λmax (MeOH) nm (logε): 216 (4.25).
preparative HPLC [column, Cosmosil Cholester, 2×25cm; 1H-NMR: see Table 1. 13C-NMR: see Table 2. HR-FAB-MS
solvent, H2O–CH3CN (57.5:42.5)+TFA (0.05%), UV 205nm] m/z: 1269.5904 (Calcd for C62H93O27: 1269.5905).
to give camelliasaponin A1 (13) (12.9mg) and theasaponin E5
Teaseedsaponin K (11): Colorless amorphous powder. [α]D25
(20) (6.4mg). Fr. O (29mg) was subjected to semipreparative +5.8 (c=0.85, MeOH). UV λmax (MeOH) nm (logε): 207
1
HPLC [column, Cosmosil 5PE-MS, 2×25cm; solvent, H2O– (4.31). H-NMR: see Table 1. 13C-NMR: see Table 2. HR-FAB-
CH3CN (60:40) + TFA (0.05%), UV 205nm] to give foliath- MS m/z: 1269.5923 (Calcd for C62H93O27: 1269.5905).
easaponin III (14) (5.2mg). Fr. G (33.0mg) was subjected to
Teaseedsaponin L (12): Colorless amorphous powder. [α]D25
semipreparative HPLC [column, Cosmosil 5PE-MS, 2×25cm; +5.8 (c=1.45, MeOH). UV λmax (MeOH) nm (logε): 210 (4.30),
1
solvent, H2O–CH3CN (62.5:37.5)+TFA (0.05%), UV 205nm] 250 (3.18). H-NMR: see Table 1. 13C-NMR: see Table 2. HR-
to give camelliasaponin B1 (16) (7.1mg) and assamsaponin FAB-MS m/z: 1285.5851 (Calcd for C62H93O28: 1285.5854).
F (21) (6.9mg). Fr. H (30mg) was subjected to semiprepara-
Acid Hydrolysis and Sugar Identification11) Compounds
tive HPLC [column, Cosmosil 5PE-MS, 2×25cm; solvent, 1–12 (ca. 1mg) were dissolved separately in 50% TFA (50µL).
°
H2O–CH3CN (62.5:37.5)+TFA (0.05%), UV 205nm] to give The solutions were heated at 100 C for 1h. The reaction mix-
teasaponin E2 (17) (4.1mg) and assamsaponin B (22) (8.3mg). tures were dried, diluted with H2O, and extracted with EtOAc.
Fr. D (20mg) was subjected to semipreparative HPLC [col- The H2O layers were concentrated to dryness. The residues
umn, Cosmosil 5PE-MS, 2×25cm; solvent, H2O–CH3CN were stirred with L-cysteine methyl ester hydrochloride in
°
(62.5:37.5)+TFA (0.05%), UV 205nm] to give assamsaponin pyridine (20mg/mL, 50µL) at 60 C for 1h. o-Tolyl isthiocya-
°
G (18) (8.0mg). Fr. E (23mg) was subjected to semiprepara- nate (5µL) was added and heated at 60 C for another 1h. The
tive HPLC [column, Cosmosil 5PE-MS, 2×25cm; solvent, reaction mixtures were subjected to HPLC analysis [column:
H2O–CH3CN (62.5:37.5)+TFA (0.05%), UV 205nm] to give Cosmosil 5C18-AR-II, 4.6×250mm; solvent, H2O–CH3CN
teasaponin E1 (19) (16.7mg).
(78:22)+TFA (0.05%), UV 250nm] to identify the derivatives
Teaseedsaponin A (1): Colorless amorphous powder. [α]D26 of D-glucuronic acid (tR 62.8min), D-galactose (tR 19.2min),
−6.1 (c=0.33, MeOH). UV λmax (MeOH) nm (logε): 221.5 and L-arabinose (tR 25.0min) from 1–12, D-xylose (tR 25.8min)
(3.95). 1H-NMR: see Table 1. 13C-NMR: see Table 2. HR- from 3, 5, 6, 7, 10, 11 and 12, D-glucose (tR 22.2min) from 1,
FAB-MS m/z: 1287.6034 (Calcd for C62H95O28: 1287.6011).
2, 4, 8 and 9.
Teaseedsaponin B (2): Colorless amorphous powder. [α]D25
Assay of Inhibitory Effect on Hyaluronidase Inhibitory
−8.5 (c=0.74, MeOH). UV λmax (MeOH) nm (logε): 216.5 effects on hyaluronidase were determined by the Morgan–El-
(4.03). 1H-NMR: see Table 1. 13C-NMR: see Table 2. HR- son method12) as modified by Kakegawa et al.13) Thus, samples
FAB-MS m/z: 1287.5997 (Calcd for C62H95O28: 1287.6011).
dissolved in 0.1mol/L acetate buffer (pH 4.0) (40µL) and hy-
Teaseedsaponin C (3): Colorless amorphous powder. [α]D25 aluronidase (Type IV-S: derived from bovine testis, 2140units/
−0.2 (c=1.09, MeOH). UV λmax (MeOH) nm (logε): 218 (4.11), mg solid, Sigma Chemical Co., St. Louis, U.S.A.) in buffer
1
247 (3.27). H-NMR: see Table 1. 13C-NMR: see Table 2. HR- (final concentration: 400 NF units/mL, 20µL) were mixed,
°
and the mixtures were incubated at 37 C for 20min. Next,
FAB-MS m/z: 1187.5849 (Calcd for C58H91O25: 1187.5850).
Teaseedsaponin D (4): Colorless amorphous powder. [α]D25 the compound 48/80 (Sigma Chemical Co., St. Louis, U.S.A.)
−1.7 (c=0.54, MeOH). UV λmax (MeOH) nm (logε): 215.5 in buffer (0.1mg/mL, 40µL) was added as an activator, and
(4.08), 254.5 (3.13). 1H-NMR: see Table 1. 13C-NMR: see the mixtures were allowed to react for 20min at 37 C. After
°
Table 2. HR-FAB-MS m/z: 1301.6156 (Calcd for C63H97O28: hyaluronic acid potassium salt, derived from rooster comb
1301.6167).
(Wako Pure Chemical Industries Ltd., Osaka, Japan) in buffer
Teaseedsaponin E (5): Colorless amorphous powder. [α]D25 (final concentration: 0.4mg/mL, 100µL had been added, the
°
−0.3 (c=1.17, MeOH). UV λmax (MeOH) nm (logε): 212 (4.23). mixture was incubated at 37 C for 40min. Then, the reac-
1H-NMR: see Table 1. 13C-NMR: see Table 2. HR-FAB-MS tions were stopped by adding 40µL of 0.4mol/L NaOH, and
m/z: 1271.6079 (Calcd for C62H95O27: 1271.6062).
the mixtures were cooled. Boric acid solution (pH 9.1) (40µL)
Teaseedsaponin F (6): Colorless amorphous powder. [α]D26 was added to each solution, and the mixtures were boiled for
+3.4 (c=0.40, MeOH). UV λmax (MeOH) nm (logε): 225 3min. The mixtures were cooled using ice, 1.2mL of a p-
(3.98), 255 (3.18). 1H-NMR: see Table 1. 13C-NMR: see dimethylaminobenzaldehyde reagent (40µL) was added, and
°
Table 2. HR-FAB-MS m/z: 1273.6219 (Calcd for C62H97O27: the mixtures were allowed to react for 20min at 37 C. After
1273.6218).
the reactions had been completed, absorbance at λ 595nm was
Teaseedsaponin G (7): Colorless amorphous powder. [α]D26 measured. A blank test was performed in the same fashion as
+7.3 (c=1.0, MeOH). UV λmax (MeOH) nm (logε): 259.5 a negative control. The following formula was used to calcu-
(3.03). 1H-NMR: see Table 1. 13C-NMR: see Table 2. HR- late the inhibitory effects on hyaluronidase activity:
FAB-MS m/z: 1185.5696 (Calcd for C58H89O25: 1185.5694).
rate of inhibiting hyaluronidase activity (%)
Teaseedsaponin H (8): Colorless amorphous powder. [α]D25
=[{(St  Sb )  (Ct  Cb )}/ (St  Sb )]×100
+19.9 (c=0.37, MeOH). UV λmax (MeOH) nm (logε): 208
(4.25). 1H-NMR: see Table 1. 13C-NMR: see Table 2. HR- where St is absorbance of the solution containing a study
FAB-MS m/z: 1273.5846 (Calcd for C61H93O28: 1273.5854).
sample at λ 595nm, Sb is absorbance of the blank solution at
Teaseedsaponin I (9): Colorless amorphous powder. [α]D25 λ 595nm, Ct is absorbance of the control solution at λ 595nm,
+7.1 (c=1.31, MeOH). UV (MeOH) λmax (logε) nm: 211.5 and Cb is absorbance of the blank control solution at λ 595nm.
(4.27). 1H-NMR: see Table 1. 13C-NMR: see Table 2. HR- The IC50 value of inhibitory effects on hyaluronidase was de-
FAB-MS m/z: 1299.6010 (Calcd for C63H95O28: 1299.6011).
termined within the range of the concentrations tested.
Teaseedsaponin J (10): Colorless amorphous powder. [α]D25

616
Vol. 60, No. 5
Table 1. 1H-NMR Spectroscopic Data (400MHz, Pyridine-d5) of Compounds 1–12
1
2
3
4
Position
δH (J in Hz) HMBC (H to C) δH (J in Hz) HMBC (H to C) δH (J in Hz) HMBC (H to C) δH (J in Hz) HMBC (H to C)
3
5
3.23 (dd, 12.0,
4.0)
3.24 (dd, 11.5,
3.5)
4.12 (dd, 11.5,
3.5)
4.14 (dd, 12.5,
4.5)
0.71 (brd,
10.0)
0.72 (brd,
12.0)
1.58 (brd,
11.0)
1.57 (brd,
11.0)
9
1.64 (brd,
10.0)
1.64 (over-
lapped)
1.84 (over-
lapped)
1.85 (over-
lapped)
11
1.84 (over-
lapped)
1.85 (over-
lapped)
1.85 (over-
lapped)
1.86 (over-
lapped)
1.90 (over-
lapped)
1.91 (over-
lapped)
1.93 (over-
lapped)
1.94 (over-
lapped)
12
15
5.44 (brs)
9, 14
5.40 (brs)
9, 14
5.39 (brs)
14
5.43 (brs)
9, 14
1.64 (over-
lapped)
1.62 (over-
lapped)
1.59 (over-
lapped)
1.59 (over-
lapped)
1.92 (over-
lapped)
1.89 (over-
lapped)
16
18
5.84 (brs)
1'
5.61 (brs)
1'
4.59 (brs)
4.47 (brs)
2.77 (over-
lapped)
3.00 (dd, 13.5,
5.0)
3.04 (dd, 14.0,
4.0)
3.08 (over-
lapped)
19
1.42 (over-
lapped)
1.41 (over-
lapped)
1.32 (over-
lapped)
1.40 (over-
lapped)
1.86 (over-
lapped)
1.86 (over-
lapped)
21
22
23
5.93 (d, 10.0)
4.40 (d, 10.0)
1.28 (s)
20, 22, 29,
30, 1″
16, 17, 21
5.87 (d, 10.0) 22, 29, 30, 1″
6.66 (d, 10.0)
20, 22, 29,
30, 1″
6.12 (d, 10.0) 16, 17, 21, 28,
6.19 (dd, 12.0,
5.5)
1‴
6.28 (d, 10.0) 16, 17, 22, 28,
1‴
1‴
3, 5
3, 4, 5, 24
1.29 (s)
3, 4, 5, 24
3.76 (d, 10.5)
4.37 (d, 10.5)
1.07 (s)
3.75 (d, 10.0)
4.40 (d, 10.0)
1.04 (s)
24
25
26
27
28
1.08 (s)
0.81 (s)
0.92 (s)
1.50 (s)
3.73 (d, 11.0)
4.22 (d, 11.0)
1.11 (s)
1.24 (s)
(Ac)
3, 4, 5, 23
5, 9, 10
1.10 (s)
0.79 (s)
0.78 (s)
1.50 (s)
3.46 (d, 11.0)
3.60 (d, 11.0)
1.07 (s)
1.29 (s)
(Ac)
3, 4, 5, 23
1, 5, 9, 10
7, 8, 9, 14
8, 13, 14, 15
22
3, 4, 5, 23
9, 10
3, 4, 5, 23
1, 5, 10
0.91 (s)
0.91 (s)
7, 8, 9, 14
8, 13, 14, 15
1″″
0.91 (s)
7, 8, 14
0.90 (s)
7, 8, 9, 14
8, 13, 14, 15
18, 22
1.82 (s)
8, 13, 14, 15
1.79 (s)
3.51 (d, 10.7)
3.68 (d, 10.7)
1.03 (s)
3.40 (d, 11.0)
3.66 (d, 11.0)
1.07 (s)
29
19, 20, 21, 30
19, 20, 21, 29
19, 20, 21, 30
19, 20, 21, 29
19, 20, 21, 30
19, 20, 21, 29
19, 20, 21, 30
19, 20, 21, 29
30
1.27 (s)
1.32 (s)
16-O-
2′
21-O-
2.50 (s)
(Ang)
1′
2.51 (s)
(Ang)
1′
(Ang)
2″
3″
5.91 (qq, 7.0,
1.5)
5.99 (qq, 7.0,
2.0)
5″
5.97 (qq, 7.0,
1.5)
5″
4″
5″
2.01 (dq, 7.0,
1.5)
2″, 3″
2.06 (dq, 7.0,
2.0)
2″, 3″
2.08 (dq, 7.0,
1.5)
2″, 3″
1.92 (dq, 1.5,
1.5)
1″, 2″, 3″
1.98 (dq, 2.0,
2.0)
1″, 2″, 3″
2.01 (dq, 1.5,
1.5)
1″, 2″, 3″
6″
22-O-
2‴
(Ac)
(Hex)
(Ang)
2.04 (s)
1‴
5.93 (dt, 11.5,
2.0)
3‴, 4‴
2‴
3‴
4‴
5‴
6.12 (dt, 11.5,
7.5)
5.92 (qq, 7.0,
1.5)
5‴
2.75 (m)
2‴, 5‴, 6‴
4‴, 6‴
4‴, 5‴
2.05 (dq, 7.0,
1.5)
2‴, 3‴
1.37 (sext,
7.5)
1.91 (dq, 1.5,
1.5)
1‴, 2‴, 3‴
6‴
28-O-
2″″
0.85 (t, 7.5)
(Ac)
1.97 (s)
1″″

May 2012
617
Table 1. (Continued)
1
2
3
4
Position
δH (J in Hz) HMBC (H to C) δH (J in Hz) HMBC (H to C) δH (J in Hz) HMBC (H to C) δH (J in Hz) HMBC (H to C)
GlcA
1
4.91 (d, 7.5)
3
4.92 (d, 7.5)
3
5.04 (d, 7.5)
3
5.06 (d, 7.5)
3
2
3
4
5
4.65 (dd, 8.5,
7.5)
4.68 (dd, 8.5,
7.5)
4.59 (over-
lapped)
4.63 (dd, 8.0,
7.5)
4.41 (over-
lapped)
4.41 (over-
lapped)
4.27 (over-
lapped)
4.28 (over-
lapped)
4.54 (over-
lapped)
4.52 (over-
lapped)
4.45 (over-
lapped)
4.48 (over-
lapped)
4.51 (over-
lapped)
4.51 (over-
lapped)
4.41 (over-
lapped)
4.40 (over-
lapped)
Gal
1
2
5.65 (d, 7.5)
GlcA-2
5.67 (d, 7.5)
GlcA-2
5.83 (d, 7.5)
GlcA-2
5.76 (d, 7.5)
GlcA-2
4.48 (over-
lapped)
4.51 (over-
lapped)
4.49 (over-
lapped)
4.50 (over-
lapped)
3
4.22 (over-
lapped)
4.24 (over-
lapped)
4.31 (over-
lapped)
4.23 (over-
lapped)
4
5
4.50 (brs)
4.52 (brs)
4.52 (brs)
4.50 (brs)
4.18 (over-
lapped)
4.20 (over-
lapped)
4.33 (over-
lapped)
4.27 (over-
lapped)
6
4.42 (over-
lapped)
4.41 (over-
lapped)
4.35 (over-
lapped)
4.34 (over-
lapped)
4.47 (over-
lapped)
4.46 (over-
lapped)
4.47 (over-
lapped)
4.48 (over-
lapped)
Ara
1
2
5.77 (d, 5.0)
GlcA-3
5.78 (d, 5.0)
GlcA-3
5.72 (d, 6.5)
GlcA-3
5.78 (d, 5.0)
GlcA-3
4.72 (dd, 7.0,
5.0)
4.74 (dd, 7.5,
5.0)
4.56 (over-
lapped)
4.77 (dd, 6.5,
5.0)
3
4
5
4.41 (over-
lapped)
4.43 (over-
lapped)
4.36 (over-
lapped)
4.47 (over-
lapped)
4.36 (over-
lapped)
4.37 (over-
lapped)
4.32 (over-
lapped)
4.49 (over-
lapped)
3.73 (over-
lapped)
3.74 (over-
lapped)
3.76 (dd, 12.0,
2.0)
3.75 (over-
lapped)
4.35 (over-
lapped)
4.39 (over-
lapped)
4.38 (over-
lapped)
4.37 (over-
lapped)
Xyl
1
2
5.02 (d, 7.5)
Ara-2
4.12 (dd, 8.0,
5.0)
3
4.02 (dd, 8.0,
5.0)
4
5
4.24 (m)
3.52 (dd, 11.0,
11.0)
4.42 (over-
lapped)
Glc
1
2
5.11 (d, 7.5)
Ara-2
5.13 (d, 7.5)
Ara-2
5.12 (d, 7.5)
Ara-2
4.11 (dd, 11.0,
7.5)
4.12 (dd, 10.0,
7.5)
4.13 (over-
lapped)
3
4
4.09 (over-
lapped)
4.11 (over-
lapped)
4.12 (over-
lapped)
4.29 (over-
lapped)
4.21 (over-
lapped)
4.18 (over-
lapped)
5
6
3.78 (m)
3.82 (m)
3.82 (m)
4.29 (dd, 12.0,
5.0)
4.30 (dd, 12.0,
5.0)
4.28 (over-
lapped)
4.41 (over-
lapped)
4.41 (over-
lapped)
4.47 (over-
lapped)

618
Vol. 60, No. 5
Table 1. (Continued)
5
6
7
8
Position
δH (J in Hz) HMBC (H to C) δH (J in Hz) HMBC (H to C) δH (J in Hz) HMBC (H to C) δH (J in Hz) HMBC (H to C)
3
5
4.12 (over-
lapped)
4.12 (dd, 10.5,
5.0)
4.01 (over-
lapped)
4.03 (dd, 11.5,
4.5)
1.55 (brd,
11.0)
1.56 (brd,
11.0)
1.38 (over-
lapped)
1.39 (over-
lapped)
9
1.82 (over-
lapped)
1.83 (over-
lapped)
1.78 (over-
lapped)
1.77 (over-
lapped)
11
1.85 (over-
lapped)
1.85 (over-
lapped)
1.77 (over-
lapped)
1.74 (over-
lapped)
1.97 (over-
lapped)
1.95 (over-
lapped)
1.98 (over-
lapped)
1.96 (over-
lapped)
12
15
5.43 (brs)
9, 14
5.42 (brs)
14
5.37 (brs)
9, 14
5.43 (brs)
9, 14
1.56 (over-
lapped)
1.54 (over-
lapped)
1.54 (over-
lapped)
1.58 (brd,
15.0)
1.87 (over-
lapped)
1.85 (over-
lapped)
1.87 (over-
lapped)
1.85 (brd,
15.0)
16
18
4.45 (brs)
4.45 (brs)
4.57 (brs)
4.70 (brs)
3.08 (over-
lapped)
3.07 (over-
lapped)
3.08 (dd, 14.0,
3.0)
2.82 (dd, 14.0,
4.0)
19
1.41 (over-
lapped)
1.38 (over-
lapped)
1.33 (over-
lapped)
1.84 (over-
lapped)
1.84 (over-
lapped)
1.78 (over-
lapped)
21
22
23
6.66 (d, 10.0)
29, 30, 1″
6.62 (d, 10.5)
20, 22, 29,
30, 1″
1.99 (dd, 11.5,
5.5)
6.42 (d, 10.0) 22, 29, 30, 1″
6.28 (d, 10.0) 16, 17, 28, 1‴
6.21 (d, 10.5) 16, 17, 21, 28,
6.10 (dd, 11.5, 16, 17, 28, 1‴
5.5)
4.45 (d, 10.0)
9.90 (s)
17, 16, 21
4
1‴
3
3.74 (d, 10.5)
4.33 (d, 10.5)
1.04 (s)
3
3.77 (d, 10.5)
4.38 (d, 10.5)
1.07 (s)
9.85 (s)
4, 24
24
25
26
27
28
3, 4, 5, 23
9, 10
3, 4, 5, 23
1, 10
1.44 (s)
3, 4, 5, 23
1, 5, 10
1.44 (s)
3, 4, 5, 23
1, 5, 9, 10
7, 8, 9, 14
8, 14, 15
18, 1″″
0.91 (s)
0.91 (s)
0.84 (s)
0.82 (s)
0.90 (s)
7, 8, 14
8, 14, 15
18, 22
0.90 (s)
7, 8, 9, 14
8, 13, 14, 15
18, 22
0.82 (s)
7, 8, 9, 14
8, 13, 14
17, 18, 22
0.94 (s)
1.79 (s)
1.77 (s)
1.81 (s)
1.77 (s)
3.40 (d, 10.5)
3.66 (d, 10.5)
1.07 (s)
3.39 (d, 11.0)
3.66 (d, 11.0)
1.06 (s)
3.49 (d, 10.5)
3.63 (d, 10.5)
1.05 (s)
4.21 (d, 10.0)
4.30 (d, 10.0)
1.13 (s)
29
19, 20, 21, 30
19, 20, 21, 29
19, 20, 21, 30
19, 20, 21, 29
19, 20, 21, 30
19, 20, 21, 29
19, 20, 21, 30
19, 20, 21, 29
30
1.32 (s)
1.30 (s)
1.27 (s)
1.30 (s)
16-O-
2′
21-O-
(Ang)
(Ang)
(Hex)
2″
6.05 (dt, 11.0,
1.0)
1″, 4″
1″
3″
4″
5″
5.97 (qq, 7.0,
1.5)
1″, 4″, 5″
1″, 2″, 3″
1″, 2″, 3″
6.06 (qq, 7.5,
1.5)
5″
6.15 (dt, 11.0,
7.5)
2.08 (dq, 7.0,
1.5)
2.15 (dq, 7.0,
1.5)
2″, 3″
2.74 (m)
2.00 (brs)
2.03 (dq, 1.5,
1.5)
1″, 2″, 3″
1.38 (sext,
7.5)
3″, 4″, 6″
4″, 5″
6″
22-O-
2‴
0.85 (t, 7.5)
(Ang)
(2Mb)
(Hex)
2.27 (sext,
7.0)
1‴, 3‴
4‴, 5‴
5.94 (dt, 11.5,
2.0)
3‴, 4‴
2‴, 4‴, 5‴
3‴, 5‴, 6‴
3‴, 4‴, 6‴
4‴, 5‴
3‴
4‴
5‴
5.92 (qq, 7.0,
1.5)
1‴, 4‴, 5‴
1‴, 2‴, 3‴
1‴, 2‴, 3‴
1.71 (dq, 7.0,
7.5)
6.14 (dt, 11.5,
7.5)
2.04 (dq, 7.0,
1.5)
0.80 (t, 7.5)
2‴, 3‴
2.76 (m)
1.91 (br s)
1.10 (d, 7.0)
1‴, 2‴, 3‴
1.39 (sext,
7.5)
6‴
28-O-
2″″
0.86 (t, 7.5)
(Ac)
1.99 (s)
1″″

May 2012
619
Table 1. (Continued)
5
6
7
8
Position
δH (J in Hz) HMBC (H to C) δH (J in Hz) HMBC (H to C) δH (J in Hz) HMBC (H to C) δH (J in Hz) HMBC (H to C)
GlcA
1
5.03 (d, 7.0)
3
5.03 (d, 8.0)
3
4.82 (d, 7.0)
3
4.84 (d, 7.5)
3
2
3
4
5
4.57 (dd, 9.0,
7.0)
4.59 (dd, 8.0,
8.0)
4.47 (over-
lapped)
4.50 (over-
lapped)
4.27 (over-
lapped)
4.26 (over-
lapped)
4.30 (over-
lapped)
4.31 (over-
lapped)
4.38 (over-
lapped)
4.43(over-
lapped)
4.43 (over-
lapped)
4.47 (over-
lapped)
4.37 (over-
lapped)
4.40 (over-
lapped)
4.45 (over-
lapped)
4.47 (over-
lapped)
Gal
1
2
5.80 (d, 7.0)
GlcA-2
5.84 (d, 8.0)
GlcA-2
5.70 (d, 7.0)
GlcA-2
5.63 (d, 7.5)
GlcA-2
4.50 (over-
lapped)
4.48 (over-
lapped)
4.40 (over-
lapped)
4.43 (over-
lapped)
3
4.30 (over-
lapped)
4.29 (over-
lapped)
4.26 (over-
lapped)
4.23 (over-
lapped)
4
5
4.52 (brs)
4.51 (brs)
4.48 (brs)
4.52 (brs)
4.29 (over-
lapped)
4.32 (over-
lapped)
4.32 (over-
lapped))
4.28 (over-
lapped)
6
4.44 (over-
lapped)
4.35 (over-
lapped)
4.31 (over-
lapped)
4.31 (over-
lapped)
4.47 (over-
lapped)
4.45 (over-
lapped)
4.48 (over-
lapped)
Ara
1
2
5.68 (d, 5.0)
GlcA-3
5.72 (d, 6.0)
GlcA-3
5.69 (d, 5.0)
GlcA-3
5.78 (d, 5.0)
GlcA-3
4.53 (dd, 9.0,
5.0)
4.55 (dd, 8.0,
6.0)
4.53 (dd, 8.0,
6.0)
4.73 (dd, 7.0,
5.0)
3
4
5
4.33 (over-
lapped)
4.35 (over-
lapped)
4.34 (over-
lapped)
4.44 (over-
lapped)
4.29 (over-
lapped)
4.31 (over-
lapped)
4.29 (over-
lapped)
4.38 (over-
lapped)
3.75 (over-
lapped)
3.75 (dd, 15.0,
9.0)
4.34 (over-
lapped)
3.75 (dd, 11.5,
2.0)
4.35 (over-
lapped)
4.38 (over-
lapped)
4.73 (brd,
10.0)
4.43 (over-
lapped)
Xyl
1
2
5.03 (d, 7.0)
Ara-2
5.01 (d, 8.0)
Ara-2
4.99 (d, 7.0)
Ara-2
4.11 (dd, 8.0,
7.0)
4.11 (dd, 8.0,
9.0)
4.10 (dd, 8.5,
7.5)
3
4.03 (dd, 8.0,
8.0)
4.01 (dd, 9.0,
9.0)
4.02 (dd, 8.5,
8.5)
4
5
4.21 (m)
4.22 (m)
4.19 (m)
3.49 (dd, 10.5,
10.5)
4.42 (over-
lapped)
3.51 (brt,
10.0)
4.37 (over-
lapped)
3.51 (dd, 11.0,
10.0)
4.42 (over-
lapped)
Glc
1
2
5.10 (d, 7.0)
Ara-2
4.11 (dd, 7.0,
7.0)
3
4
4.11 (over-
lapped)
4.21 (over-
lapped)
5
6
3.82 (ddd. 8.0, 6.0, 3.0)
4.28 (over-
lapped)
4.41 (over-
lapped)

620
Vol. 60, No. 5
Table 1. (Continued)
9
10
11
12
Position
δH (J in Hz) HMBC (H to C) δH (J in Hz) HMBC (H to C) δH (J in Hz) HMBC (H to C) δH (J in Hz) HMBC (H to C)
3
5
4.05 (dd, 12.0,
4.5)
4.01 (over-
lapped)
4.03 (over-
lapped)
3.96 (dd, 11.9,
4.5)
1.37 (over-
lapped)
1.38 (over-
lapped)
1.37 (over-
lapped)
1.37 (over-
lapped)
9
1.77 (over-
lapped)
1.77 (over-
lapped)
1.80 (over-
lapped)
1.76 (over-
lapped)
11
1.75 (over-
lapped)
1.78 (over-
lapped)
1.78 (over-
lapped)
1.95 (over-
lapped)
12
15
5.41 (brs)
9, 14
5.41 (brs)
9, 14
5.41 (brs)
9, 14
5.38 (brs)
9, 14
1.52 (over-
lapped)
1.51 (over-
lapped)
1.54 (over-
lapped)
1.53 (brd,
15.0)
1.78 (over-
lapped)
1.83 (over-
lapped)
1.85 (over-
lapped)
1.81 (brd,
15.0)
16
18
4.46 (brs)
4.45 (brs)
4.44 (brs)
5.59 (brs)
1′
3.11 (over-
lapped)
3.06 (brd,
10.0)
3.09 (brd,
10.0)
2.99 (dd, 14.0,
4.0)
19
1.42 (brd,
10.0)
1.42 (over-
lapped)
1.42 (brd,
10.0)
1.45(over-
lapped)
1.80 (over-
lapped)
1.80 (over-
lapped)
21
22
23
6.66 (d, 10.0) 22, 29, 30, 1″
6.66 (d, 10.0)
20, 22, 29,
30, 1″
6.72 (d, 10.5) 22, 29, 30, 1″
5.83 (d, 10.0) 22, 29, 30, 1″
6.12 (d, 10.0) 16, 21, 28, 1‴
6.28 (d, 10.0) 16, 17, 21, 28,
6.27 (d, 10.0) 16, 17, 21, 28,
6.23 (d, 10.5) 16, 17, 21, 28,
1‴
1‴
4
1‴
9.91 (s)
3, 24
9.87 (s)
9.91 (s)
4
9.95 (s)
4, 24
24
25
26
27
28
1.45 (s)
3, 4, 5, 23
5, 9, 10
1.44 (s)
3, 4, 5, 23
1, 9, 10
7, 14
1.46 (s)
3, 4, 23
9, 10
1.48 (s)
0.78 (s)
0.72 (s)
1.43 (s)
3.46 (d, 10.5)
3.57 (d, 10.5)
1.08 (s)
1.27 (s)
(Ac)
3, 4, 5, 23
1, 5, 9, 10
7, 9, 14
0.82 (s)
0.88 (s)
0.82 (s)
0.83 (s)
7, 8, 9, 14
8, 14, 15
17, 18, 22
0.89 (s)
0.82 (s)
7, 8, 14
13, 14, 15
22
1.79 (s)
1.79 (s)
13, 14, 15
1.79 (s)
13, 14, 15
3.39 (d, 11.0)
3.62 (d, 11.0)
1.10 (s)
3.38 (d, 11.0)
3.62 (d, 11.0)
1.08 (s)
3.36 (d, 11.0)
3.61 (d, 11.0)
1.11 (s)
29
19, 20, 21, 30
19, 20, 21, 29
19, 20, 21, 30
19, 20, 21, 29
19, 20, 21, 30
19, 20, 21,29
19, 20, 21, 30
19, 20, 21, 29
30
1.33 (s)
1.33 (s)
1.33 (s)
16-O-
2′
21-O-
2.57 (s)
(Hex)
1′
(Ang)
(Ang)
(Ang)
2″
5.98 (dt, 11.0,
1.5)
1″, 4″
3″
4″
5″
5.98 (qq, 7.0,
1.5)
5″
5.97 (qq, 7.0,
2.0)
5″
5.93 (qq, 7.0,
1.5)
4″, 5″
2″, 3″
6.19 (dt, 11.0,
7.5)
2.04 (dq, 7.0,
1.5)
2″, 3″
2.08 (dq, 7.0,
2.0)
2″, 3″
2.05 (dq, 7.0,
1.5)
2.74 (m)
2″, 3″, 5″, 6″
3″, 4″, 6″
4″, 5″
2.01 (dq, 1.5,
1.5)
1″, 2″, 3″
2.01 (dq, 2.0,
2.0)
1″, 2″, 3″
1.99 (dq, 1.5,
1.5)
1″, 2″, 3″
1.39 (sext,
7.5)
6″
22-O-
2‴
0.85 (t, 7.5)
(Ac)
(Ang)
(Ang)
(Tig)
2.05 (s)
1‴
3‴
5.92 (qq, 7.0,
1.5)
5‴
5.91 (qq, 7.0,
2.0)
5‴
6.95 (qq, 7.5,
1.5)
1‴
4‴
5‴
2.09 (dq, 7.0,
1.5)
2‴, 3‴
1‴, 3‴
2.04 (dq, 7.0,
2.0)
2‴, 3‴
1.47 (over-
lapped)
2‴, 3‴
1.90 (dq, 1.5,
1.5)
1.90 (dq, 2.0,
2.0)
1‴, 2‴, 3‴
1.84 (s)
1‴, 2‴, 3‴
6‴
28-O-
2″″

May 2012
621
Table 1. (Continued)
9
10
11
12
Position
δH (J in Hz) HMBC (H to C) δH (J in Hz) HMBC (H to C) δH (J in Hz) HMBC (H to C) δH (J in Hz) HMBC (H to C)
GlcA
1
4.85 (d, 7.5)
3
4.82 (d, 7.5)
3
4.84 (d, 7.5)
3
4.82 (d, 7.5)
3
2
3
4
5
4.50 (over-
lapped)
4.47 (over-
lapped)
4.51 (over-
lapped)
4.51 (over-
lapped)
4.30 (over-
lapped)
4.30 (over-
lapped)
4.32 (over-
lapped)
4.33 (over-
lapped)
4.47 (over-
lapped)
4.42 (over-
lapped)
4.46 (over-
lapped)
4.47 (over-
lapped)
4.50 (over-
lapped)
4.44 (over-
lapped)
4.48 (over-
lapped)
4.49 (over-
lapped)
Gal
1
2
5.63 (d, 7.5)
GlcA-2
5.70 (d, 7.5)
GlcA-2
5.73 (d, 7.5)
GlcA-2
5.73 (d, 7.5)
GlcA-2
4.41 (over-
lapped)
4.41 (over-
lapped)
4.44 (over-
lapped)
4.45 (over-
lapped)
3
4.32 (over-
lapped)
4.27 (over-
lapped)
4.29 (over-
lapped)
4.30 (over-
lapped)
4
5
4.52 (brs)
4.49 (brs)
4.50 (brs)
4.50 (brs)
4.30 (over-
lapped)
4.32 (over-
lapped)
4.35 (over-
lapped)
4.36 (over-
lapped)
6
4.31 (over-
lapped)
4.46 (over-
lapped)
4.47 (over-
lapped)
4.35 (over-
lapped)
4.50 (over-
lapped)
4.50 (over-
lapped)
Ara
1
2
5.78 (d, 5.0)
GlcA-3
5.69 (d, 6.0)
GlcA-3
5.72 (d, 6.0)
GlcA-3
5.74 (d, 5.0)
GlcA-3
4.72 (dd, 5.0,
7.0)
4.52 (dd, 6.0,
8.0)
4.54 (dd, 7.5,
6.0)
4.55 (dd, 7.0,
5.0)
3
4
5
4.48 (over-
lapped)
4.35 (over-
lapped)
4.36 (dd, 7.5,
3.5)
4.38 (over-
lapped)
4.42 (over-
lapped)
4.30 (over-
lapped)
4.31 (over-
lapped)
4.32 (over-
lapped)
3.74 (dd, 11.5,
2.0)
3.73 (brd,
10.0)
3.75 (dd, 12.5,
2.0)
3.75 (dd, 11.5,
1.0)
4.36 (over-
lapped)
4.36 (over-
lapped)
4.37 (over-
lapped)
4.38 (over-
lapped)
Xyl
1
2
4.99 (d, 7.5)
Ara-2
5.00 (d, 7.5)
Ara-2
5.01 (d, 7.5)
Ara-2
4.09 (dd, 8.5,
7.5)
4.11 (dd, 9.0,
7.5)
4.11 (dd, 8.5,
7.5)
3
4.00 (dd, 8.5,
8.5)
4.01 (dd, 9.0,
9.0)
4.02 (dd, 8.5,
8.5)
4
5
4.21 (m)
4.20 (m)
4.24 (m)
3.50 (brt,
11.0)
3.52 (brt,
11.0)
3.52 (brt,
11.0)
4.42 (over-
lapped)
4.42 (over-
lapped)
4.43 (over-
lapped)
Glc
1
2
5.09 (d, 7.5)
Ara-2
4.09 (dd, 8.0,
7.5)
3
4
5
6
4.11 (over-
lapped)
4.21 (dd, 10.0,
9.0)
3.81 (ddd, 9.0,
8.0, 5.0)
4.28 (over-
lapped)
4.40 (over-
lapped)

622
Vol. 60, No. 5
Table 2. 13C-NMR Spectroscopic Data of Compounds 1–12 (100MHz, Pyridine-d5)
Carbon
1
2
3
4
5
6
7
8
9
10
11
12
1
2
38.8
26.6
89.5
40.1
55.8
18.2
33.0
39.6
46.2
36.8
23.9
123.2
141.0
41.4
31.0
71.0
47.0
40.2
47.3
35.9
80.1
70.0
28.0
16.8
15.7
17.0
27.1
65.0
30.0
19.9
(Ac)
169.8
22.1
(Ang)
168.2
129.1
136.9
16.1
21.0
38.8
26.5
89.5
40.0
55.8
18.3
33.1
39.6
46.8
26.8
23.8
125.2
141.0
41.2
31.0
71.5
46.9
39.6
47.3
36.0
78.5
73.4
28.0
16.8
15.6
16.7
27.0
63.8
29.4
19.7
(Ac)
169.8
22.0
(Ang)
167.8
128.5
138.0
16.0
21.0
38.8
25.5
83.3
43.5
48.4
18.3
32.9
40.2
47.1
36.8
23.9
122.9
143.8
41.8
35.1
70.0
44.9
41.0
47.5
32.1
41.8
72.8
65.0
13.6
16.3
17.0
27.6
63.7
33.5
25.2
38.8
25.6
83.0
43.5
48.3
18.2
32.9
40.2
47.0
36.8
23.9
122.9
142.8
41.7
34.8
68.8
48.1
40.2
47.2
36.4
78.8
73.8
64.9
13.6
16.2
17.0
27.6
63.7
29.5
20.3
38.8
25.6
83.4
43.5
48.3
18.3
32.9
40.2
47.3
36.8
24.0
122.4
142.8
41.7
34.9
68.8
48.1
40.2
47.1
36.4
78.9
73.8
65.0
13.5
16.3
17.0
27.6
63.7
29.6
20.3
38.8
25.5
83.2
43.5
48.2
18.2
33.0
40.2
47.0
36.8
23.9
123.0
142.9
41.7
34.9
68.3
48.3
40.2
47.2
36.4
78.7
73.6
65.0
13.0
16.0
17.0
27.5
63.7
29.5
20.3
38.3
25.3
84.4
55.2
48.4
20.4
32.5
40.4
46.9
36.1
23.8
123.0
143.9
41.9
35.0
69.9
44.9
41.0
47.5
32.1
41.9
72.7
209.8
11.1
15.9
16.9
27.6
63.7
33.5
25.3
38.2
25.2
84.3
55.1
48.5
20.5
32.5
40.6
46.9
36.0
23.9
122.7
142.8
41.9
34.7
67.7
47.1
40.3
47.4
36.1
81.2
71.2
209.8
11.1
15.9
17.1
27.3
66.8
29.8
20.2
38.3
25.2
84.3
55.1
48.5
20.4
32.4
40.8
46.8
36.1
23.8
123.0
142.9
41.7
34.8
68.7
48.1
40.2
47.2
36.4
78.8
73.7
209.9
11.1
15.8
16.9
27.6
63.7
29.6
20.3
38.3
25.3
84.4
55.2
48.4
20.4
32.5
40.4
46.8
36.1
23.8
123.0
142.9
41.7
34.8
68.7
48.1
40.2
47.2
36.4
78.7
73.7
209.8
11.1
15.9
16.9
27.6
63.7
29.6
20.3
38.2
25.2
84.4
55.2
48.4
20.4
32.4
40.3
46.8
36.1
23.8
123.0
143.9
41.7
34.8
68.4
48.3
40.1
47.2
36.3
78.7
74.1
209.8
11.1
15.8
16.9
27.5
63.6
29.5
20.2
38.1
25.3
84.6
55.1
48.5
20.3
32.4
40.3
46.9
36.0
23.7
124.8
141.1
41.4
31.0
71.3
46.7
39.6
47.2
35.9
78.3
73.4
210.1
11.1
15.8
16.8
26.9
63.8
29.4
19.8
(Ac)
169.8
22.0
(Hex)
166.6
120.3
149.3
31.2
22.5
13.8
(Ac)
170.5
20.8
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
16-O-
1′
2′
21-O-
(Ang)
167.7
129.1
137.0
15.9
(Ang)
167.7
129.0
137.1
15.9
(Ang)
167.6
128.8
138.3
16.2
(Hex)
167.1
121.3
149.0
30.0
(Ang)
167.7
129.1
137.1
15.8
(Ang)
167.7
129.1
137.2
15.9
(Ang)
167.9
129.2
138.5
15.8
1″
2″
3″
4″
5″
6″
22-O-
1‴
2‴
3‴
4‴
5‴
6‴
28-O-
1″″
2″″
21.0
21.0
21.0
22.6
21.0
21.0
20.9
13.9
(Ac)
170.4
20.9
(Hex)
166.7
121.3
149.3
31.1
(Ang)
168.3
129.1
137.1
15.8
(Ang)
168.3
129.2
137.2
15.9
(2Mb)
176.7
41.7
(Hex)
166.7
121.3
149.3
31.2
(Ang)
168.3
129.1
137.1
15.9
(Ang)
168.3
129.1
137.2
15.9
(Tig)
169.4
129.2
137.0
14.0
27.1
11.9
22.6
20.8
20.8
16.7
22.6
20.8
20.8
12.3
13.9
13.9
(Ac)
170.4
20.7
(Ac)
170.6
20.7

May 2012
623
Table 2. (Continued)
Carbon
1
2
3
4
5
6
7
8
9
10
11
12
GlcA
1
105.7
79.2
84.2
71.3
77.2
172.1
105.7
79.2
84.3
71.3
77.2
172.2
104.1
78.6
84.7
71.0
77.4
171.9
104.6
78.8
85.1
71.2
77.3
172.0
103.9
78.9
84.0
71.0
77.0
173.0
104.1
78.6
84.7
71.0
77.4
171.9
104.1
78.3
84.1
70.8
77.3
172.0
104.0
78.5
84.5
70.9
77.3
172.0
104.0
78.5
84.5
71.0
77.3
171.9
104.1
78.2
84.1
70.5
77.5
172.0
104.1
78.3
84.2
70.8
77.3
171.9
104.3
78.3
84.7
70.8
77.4
171.9
2
3
4
5
6
Gal
1
103.8
73.9
75.0
69.9
76.5
61.9
103.7
73.9
75.1
69.9
76.5
61.9
103.1
73.8
75.3
70.2
76.5
62.0
103.5
73.8
75.1
70.0
76.6
62.0
103.0
73.8
75.0
70.3
76.4
62.0
103.2
73.8
75.3
70.2
76.5
62.0
103.3
73.7
75.3
70.6
76.5
62.1
103.6
73.8
75.2
70.3
76.6
62.1
103.6
73.7
75.2
70.3
76.6
62.2
103.3
73.7
75.3
70.8
76.5
62.1
103.3
73.7
75.4
70.6
76.5
62.1
103.1
73.7
75.4
70.5
76.5
62.2
2
3
4
5
6
Ara
1
101.8
81.2
72.5
67.8
66.1
101.8
81.2
72.6
67.7
65.0
101.7
82.3
73.4
68.4
66.1
101.8
81.2
72.4
67.6
64.9
101.8
82.1
73.5
68.4
66.4
101.7
82.3
73.4
68.3
66.0
101.7
82.3
73.4
68.4
66.1
101.8
81.2
72.3
67.8
64.9
101.7
81.3
72.4
67.6
64.9
101.7
82.3
73.4
68.4
66.2
101.7
82.3
73.4
68.4
66.0
101.7
82.3
73.4
68.4
66.1
2
3
4
5
Xyl
1
107.0
75.9
78.3
70.8
67.5
106.7
75.6
78.7
70.8
67.4
107.0
75.9
78.3
70.8
67.5
107.0
75.9
78.2
70.8
67.5
107.0
75.8
78.2
70.8
67.5
107.1
75.9
78.3
70.8
67.5
107.1
75.9
78.3
70.8
67.5
2
3
4
5
Glc
1
105.9
75.8
78.4
71.4
78.5
62.6
105.9
75.8
78.4
71.5
78.4
62.6
105.9
75.8
78.4
71.6
78.4
62.7
106.0
75.9
78.5
71.7
78.5
62.8
106.0
75.9
78.4
71.6
78.5
62.7
2
3
4
5
6
Table 3. Hyaluronidase Inhibitory Activities (IC50) of Isolated Com-
pounds (1–22)
References
1) Kitagawa I., Hori K., Motozawa T., Murakami T., Yoshikawa M.,
Chem. Pharm. Bull., 46, 1901–1906 (1998).
Compound
IC50 (µM)
2) Yoshikawa M., Morikawa T., Yamamoto K., Kato Y., Nagatomo A.,
Matsuda H., J. Nat. Prod., 68, 1360–1365 (2005).
3) Dogra D., Ahuja S., Krishnan S., Kohli S., Ramteka A., Atale N.,
Rani V., J. Pharm. Res., 4, 1833–1835 (2011).
1
55.2
36.4
26.6
30.2
20.0
24.2
28.8
48.7
44.9
36.8
40.5
28.5
24.9
52.0
26.4
24.1
54.9
29.7
38.7
51.5
19.3
55.6
240.1
2
3
4) Sagesaka Y. M., Uemura T., Suzuki Y., Sugiura T., Yoshida M.,
Yamaguchi K., Kyuki K., Yakugaku Zasshi, 116, 238–243 (1996).
5) Ippoushi K., Yamaguchi Y., Itou H., Azuma K., Higashio H., Food
Sci. Technol. Res., 6, 74–77 (2000).
6) Yoshikawa M., Murakami T., Yoshizumi S., Murakami N., Yama-
hara J., Matsuda H., Chem. Pharm. Bull., 44, 1899–1907 (1996).
7) Morikawa T., Nakamura S., Kato Y., Muraoka O., Matsuda H., Yo-
shikawa M., Chem. Pharm. Bull., 55, 293–298 (2007).
8) Morikawa T., Li N., Nagatomo A., Matsuda H., Li X., Yoshikawa
M., J. Nat. Prod., 69, 185–190 (2006).
9) Murakami T., Nakamura J., Kageura T., Matsuda H., Yoshikawa M.,
Chem. Pharm. Bull., 48, 1720–1725 (2000).
10) Murakami T., Nakamura J., Matsuda H., Yoshikawa M., Chem.
Pharm. Bull., 47, 1759–1764 (1999).
11) Tanaka T., Nakashima T., Ueda T., Tomii K., Kouno I., Chem.
Pharm. Bull., 55, 899–901 (2007).
12) Reissig J. L., Strominger J. L., Leloir L. F., J. Biol. Chem., 217,
959–966 (1955).
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
13) Kakegawa H., Matsumoto H., Satoh T., Chem. Pharm. Bull., 40,
1439–1442 (1992).
21
22
Rosmarinic acid