Characterization of new sweet triterpene saponins from Albizia myriophylla

Article abstract of DOI:10.1021/np020220l

Five new oleanane-type triterpene saponins, albiziasaponins A-E (1-5), were isolated from the stems of Albizia myriophylla collected in Thailand, together with two known triterpene saponins, licorice-saponin F3 and yunganoside B₁. The structures of the new compounds were elucidated on the basis of chemical and physicochemical evidence. In addition, when sensory tests were performed on sweetness, albiziasaponin B (2), with a carbonyl group at the C-30 position, was found to show a potent sweetness intensity relative to sucrose (600 times).

Full text of DOI:10.1021/np020220l

1638
J . Nat. Prod. 2002, 65, 1638-1642
Ch a r a cter iza tion of New Sw eet Tr iter p en e Sa p on in s fr om Albizia m yr ioph ylla 1
Masayuki Yoshikawa,* Toshio Morikawa, Kyoko Nakano, Yutana Pongpiriyadacha, Toshiyuki Murakami, and
Hisashi Matsuda
Kyoto Pharmaceutical University, Misasagi, Yamashina-ku, Kyoto 607-8412, J apan
Received May 17, 2002
Five new oleanane-type triterpene saponins, albiziasaponins A-E (1-5), were isolated from the stems of
Albizia myriophylla collected in Thailand, together with two known triterpene saponins, licorice-saponin
F3 and yunganoside B1. The structures of the new compounds were elucidated on the basis of chemical
and physicochemical evidence. In addition, when sensory tests were performed on sweetness, albiziasa-
ponin B (2), with a carbonyl group at the C-30 position, was found to show a potent sweetness intensity
relative to sucrose (600 times).
Albizia myriophylla Benth. (Leguminosae) is widely
distributed in southeastern Asian countries such as Thai-
land (local name “Cha Em Thai”) and Vietnam (“Cay Song
Ran”). In the Thai and Vietnamese systems of traditional
medicine, the stems of A. myriophylla are used to substitute
for licorice due to their sweet taste. In chemical studies on
A. myriophylla, several lignan glycosides and alkaloids
were isolated from the bark of Vietnamese A. myriophylla.2
However, the sweet-tasting constituents of this natural
medicine were left undetermined.
In the course of our characterization studies on bioactive
saponins and glycosides,3 five new oleanane-type triterpene
saponins, albiziasaponins A-E (1-5), were isolated from
the stems of A. myriophylla collected in Thailand. In
addition, the two principal saponins, albiziasaponins A (1)
and B (2), were examined for relative sweetness compared
with sucrose. This paper deals with the isolation, structural
elucidation, and sensory evaluation of the new saponin
constituents of A. myriophylla.
tively, were observed in the negative-ion FABMS of 1. On
enzymatic hydrolysis of 1 with glycyrrhizic acid hydrolase,
24-hydroxy-11-deoxoglabrolide (6)6 was obtained as the
aglycon. Acid hydrolysis with 5% aqueous sulfuric acid
(H2SO4)-1,4-dioxane (1:1) of 1 liberated D-glucuronic acid
and L-rhamnose, which were identified by GLC analysis
of their trimethylsilyl thiazolidine derivatives.7 The 1H and
13C NMR (pyridine-d5, Table 1) spectra of 1, which were
assigned by various NMR experiments,8 showed signals
assignable to two glucuronopyranosyl moieties [δ 5.06 (d,
J ) 7.6 Hz, H-1′) and 5.87 (d, J ) 7.6 Hz, H-1′′)] and a
rhamnopyranosyl moiety [δ 1.81 (d, J ) 6.4 Hz, H-6′′′) and
6.27 (br s, H-1′′′)], together with an aglycon moiety [δ 0.71,
0.82, 0.98, 1.20, 1.22, 1.51 (all s, H3-25, 26, 28, 29, 27, and
23), 2.13 (dd, J ) 6.4, 13.3 Hz, H-18), 3.45 (dd, J ) 4.3,
11.6 Hz, H-3), 3.48, 4.40 (both d, J ) 11.6 Hz, H2-24), 4.20
(br d, J ) ca. 6 Hz, H-22), 5.13 (br s, H-12)]. The
oligoglycoside structure and its connectivity to the aglycon
in 1 were confirmed by a HMBC experiment. Thus, long-
range correlations were observed between the signals of
H-1′ and C-3, H-1′′ and C-2′, and H-1′′′ and C-2′′. On the
basis of this evidence, the structure of albiziasaponin A (1)
was elucidated as shown. The 6′-methyl ester of 1,
albiziasaponin A monomethyl ester (1a ), was also isolated.
The position of the methyl ester of 1a was determined from
the HMBC correlation between the methyl ester proton
[δ 3.77 (s, -OCH3)] and C-6′ (δC 169.6).
Resu lts a n d Discu ssion
The stems of A. myriophylla (collected in Kanchanaburi
Province, Thailand) were cut and extracted with methanol
under reflux. The methanolic extract was partitioned with
a mixture of EtOAc and water to furnish the EtOAc-soluble
and H2O-soluble fractions. The H2O-soluble fraction was
separated by silica gel and octadecyl silica gel (ODS)
column chromatography and finally HPLC to give
albiziasaponins A [1, 1.49% (w/w) from the natural medi-
cine], B (2, 0.13%), C (3, 0.007%), D (4, 0.021%), and E (5,
0.016%) together with albiziasaponin A monomethyl ester
(1a , 0.012%), licorice-saponin F34 (0.008%), and yungano-
24
Albiziasaponin B (2), [R]D +4.6° (MeOH), was also
obtained as colorless fine crystals from CHCl3-MeOH with
mp 220-223 °C. The IR spectrum of 2 showed absorption
bands at 3453, 1730, 1645, and 1047 cm-1, ascribable to
hydroxyl, carboxyl, olefin, and ether functions. The molec-
ular formula, C48H74O20, of 2 was determined from the
positive- and negative-ion FABMS (m/z 993 [M + Na]+ and
969 [M - H]-) and by HRFABMS. Furthermore, fragment
ion peaks at m/z 823 [M - C6H11O4]-, 647 [M - C12H19O10]-,
and 471 [M - C18H27O16]-, which were derived by cleavage
of the glycosidic linkage at the C-2′′, C-2′, and C-3 positions,
were observed in the negative-ion FABMS of 2. Enzymatic
hydrolysis of 2 with glycyrrhizic acid hydrolase afforded
azukisapogenol (7),9 while acid hydrolysis of 2 with 5%
aqueous H2SO4-1,4-dioxane (1:1) liberated D-glucuronic
5
24
side B1 (0.003%). Albiziasaponin A (1), [R]D -28.2°
(MeOH), was obtained as colorless fine crystals from
CHCl3-MeOH with mp 226-229 °C. The IR spectrum of
1 showed absorption bands at 1760 and 1640 cm-1 ascrib-
able to γ-lactone and olefin functions and broad bands at
3453 and 1047 cm-1 suggestive of an oligoglycoside struc-
ture. In the positive- and negative-ion FABMS of 1,
quasimolecular ion peaks were observed at m/z 969 [M +
H]+, 991 [M + Na]+, and 967 [M - H]-, and HRFABMS
analysis revealed the molecular formula of 1 to be C48H72O20.
1
acid and L-rhamnose.7 The H and 13C NMR (pyridine-d5,
Furthermore, fragment ion peaks at m/z 821 [M - C6H11O4]-
-
Table 1) spectra of 2 showed signals assignable to an
azukisapogenol moiety [δ 0.72, 0.88, 0.90, 1.31, 1.37, 1.52
(all s, H3-25, 28, 26, 27, 29, and 23), 2.39 (dd, J ) 3.1, 12.8
Hz, H-18), 3.45 (dd, J ) 4.0, 12.8 Hz, H-3), 3.49, 4.39 (both
d, J ) 11.6 Hz, H2-24), 5.44 (br s, H-12)], two glucurono-
and 645 [M - C12H19O10
]
derived by cleavage of the
glycosidic linkages at the C-2′′ and C-2′ positions, respec-
* To whom correspondence should be addressed. Tel: +81-75-595-4633.
Fax: +81-75-595-4768. E-mail: shoyaku@mb.kyoto-phu.ac.jp.
10.1021/np020220l CCC: $22.00
© 2002 American Chemical Society and American Society of Pharmacognosy
Published on Web 09/28/2002

New Sweet Triterpene Saponins from Albizia
J ournal of Natural Products, 2002, Vol. 65, No. 11 1639
Ch a r t 1
pyranosyl moieties [δ 5.07 (d, J ) 7.6 Hz, H-1′) and 5.90
(d, J ) 7.9 Hz, H-1′′)], and a rhamnopyranosyl moiety
[δ 1.80 (d, J ) 6.1 Hz, H-6′′′) and 6.31 (br s, H-1′′′)]. The
proton and carbon signals assignable to the oligoglycoside
moiety of 2 were superimposable on those of 1. The
oligoglycoside structure and its connectivity were confirmed
by HMBC experiment, which showed long-range correla-
tions between the signals of H-1′ and C-3, H-1′′ and C-2′,
and H-1′′′ and C-2′′. Consequently, the structure of
albiziasaponin B (2) was determined as shown.
1.19, 1.46 (all s, H3-25, 26, 28, 29, 27, and 23), 2.13 (dd, J
) 6.7, 12.8 Hz, H-18), 3.33, 4.28 (both d-like, H2-24), 3.42
(dd, J ) 4.6, 11.9 Hz, H-3), 4.15 (br d, J ) ca. 6 Hz, H-22),
5.12 (br s, H-12)], a glucuronopyranosyl moiety [δ 4.97 (d,
J ) 7.6 Hz, H-1′)], two glucopyranosyl moieties [δ 5.01 (d,
J ) 7.6 Hz, H-1′′′′) and 5.75 (d, J ) 7.6 Hz, H-1′′)], and a
rhamnopyranosyl moiety [δ 1.75 (d, J ) 6.4 Hz, H-6′′′) and
6.33 (br s, H-1′′′)]. In the HMBC experiment on 3, long-
range correlations were observed between the following
proton and carbon signals: H-1′ and C-3, H-1′′ and C-2′,
H-1′′′ and C-2′′, and H-1′′′′ and C-3′′. These findings led to
the elucidation the structure of albiziasaponin C (3) as
shown.
Albiziasaponins D (4) and E (5) were isolated as colorless
fine crystals from CHCl3-MeOH (4: mp 238-240 °C; 5:
mp 240-242 °C). Their IR spectra showed absorption bands
assignable to hydroxyl, γ-lactone, olefin, and ether func-
tions (4: 3445, 1765, 1655, and 1043 cm-1; 5: 3445, 1752,
1655, 1078, and 1047 cm-1), which were similar to those
of 1 and 3. The positive- and negative-ion FABMS of 4
showed quasimolecular ion peaks at m/z 1087 [M + H]+,
1109 [M + Na]+, and 1085 [M - H]-, in addition to
fragment ion peaks at m/z 953 [M - C5H9O4]-, 939 [M -
C6H11O4]-, and 645 [M - C17H29O13]-. In turn, quasi-
molecular ion peaks at m/z 1101 [M + H]+, 1123 [M + Na]+,
and 1099 [M - H]- and fragment ion peaks at m/z 967
24
Albiziasaponin C (3), [R]D -12.6° (MeOH), was also
obtained as colorless fine crystals from CHCl3-MeOH with
mp 269-272 °C. The positive- and negative-ion FABMS of
3 showed quasimolecular and fragment ion peaks at m/z
1139 [M + Na]+, 1115 [M - H]-, 969 [M - C6H11O4]-, 953
[M - C6H11O5]-, and m/z 645 [M - C18H31O14]-. The
HRFABMS revealed the molecular formula of 3 to be
C
54H84O24, and the IR spectrum showed absorption bands
at 3453, 1765, 1632, 1078, and 1048 cm-1, ascribable to
hydroxyl, γ-lactone, olefin, and ether functions. On acid
hydrolysis of 3 with 5% aqueous H2SO4-1,4-dioxane (1:1)
D-glucuronic acid, D-glucose, and L-rhamnose were de-
tected.7 On enzymatic hydrolysis of 3 with glycyrrhizic acid
hydrolase, the aglycon 6 was obtained. The 1H and 13C
NMR (pyridine-d5, Table 1) spectra of 3 showed signals
assignable to an aglycon moiety [δ 0.68, 0.82, 0.98, 1.18,

1640 J ournal of Natural Products, 2002, Vol. 65, No. 11
Yoshikawa et al.
Ta ble 1. 13C NMR Data for Albiziasaponins A-E (1-5) and Albiziasaponin A Monomethyl Ester (1a )a
1
2
3
4
5
1a
1
2
3
4
5
1a
C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
38.8
26.5
90.4
44.1
56.3
18.7
33.4
42.9
47.6
36.6
23.9
125.1
140.9
39.7
25.1
26.6
36.1
45.2
42.6
42.3
38.5
84.7
23.1
63.2
15.5
16.8
24.9
23.5
20.5
180.6
38.8
26.7
90.5
44.1
56.3
18.8
33.1
41.9
47.8
36.6
24.0
122.8
145.1
40.1
27.3
26.6
32.4
48.7
43.5
44.3
31.9
39.1
23.1
63.3
15.5
16.9
26.2
28.5
29.1
179.6
38.5
26.5
91.8
43.8
56.3
18.6
33.3
42.9
47.6
36.5
24.0
125.2
140.8
39.7
25.2
26.6
36.1
45.2
42.7
42.3
38.5
84.5
22.9
63.5
15.6
16.8
25.0
23.6
20.6
180.2
38.5
26.5
91.8
43.8
56.3
19.0
33.3
42.8
47.6
36.5
24.0
125.1
140.8
39.7
25.2
26.6
36.1
45.2
42.7
42.3
38.5
84.5
22.8
63.4
15.6
16.8
24.9
23.5
20.6
180.2
38.5
26.5
90.4
44.2
56.3
19.0
33.5
42.9
47.7
36.6
24.0
125.2
140.8
39.7
25.2
26.7
36.1
45.2
42.7
42.3
38.8
84.5
23.2
63.3
15.6
16.8
25.0
23.6
20.6
180.2
38.9
26.5
90.3
44.3
56.3
18.9
33.5
42.8
47.6
36.6
23.9
125.0
140.6
39.7
25.1
26.6
36.0
45.2
42.6
42.2
38.4
84.3
23.3
63.1
15.5
16.8
24.9
23.5
20.5
179.9
C-1′
C-2′
C-3′
C-4′
C-5′
C-6′
104.9
78.2
77.5
73.7
78.2
172.4
104.9
78.3
77.6
73.7
78.3
172.3
105.2
78.4
78.5
73.7
77.4
172.2
105.2
78.4
78.4
73.7
77.4
172.3
104.9
78.0
78.3
73.7
77.7
172.2
104.7
78.2
76.5
73.5
78.3
169.6
51.9
102.1
78.6
76.5
72.6
78.4
183.5
102.3
72.2
72.6
74.2
69.5
18.9
COOCH3
C-1′′
C-2′′
C-3′′
C-4′′
C-5′′
C-6′′
C-1′′
C-2′′
C-3′′
C-4′′
C-5′′
C-6′′
C-1′′′
C-2′′′
C-3′′′
C-4′′′
C-5′′′
C-6′′′
102.1
78.6
77.1
73.0
78.4
171.8
102.2
72.3
72.6
74.4
69.5
18.9
102.1
78.7
77.2
73.1
78.3
171.5
102.2
72.2
72.6
74.4
69.5
18.9
101.8
77.3
88.9
67.9
77.6
61.3
102.3
72.3
72.8
74.4
69.5
19.0
104.5
75.0
78.6
71.6
78.6
62.5
101.8
77.6
87.6
70.7
77.7
61.3
102.4
72.2
72.7
74.3
69.5
19.0
105.2
74.7
78.4
67.8
67.1
101.9
78.6
85.9
71.0
77.1
170.9
100.6
72.3
72.8
74.4
69.7
18.9
105.1
72.3
74.3
69.3
67.5
C-9
C-10
C-11
C-12
C-13
C-14
C-15
C-16
C-17
C-18
C-19
C-20
C-21
C-22
C-23
C-24
C-25
C-26
C-27
C-28
C-29
C-30
a
Measured in pyridine-d5 at 125 MHz.
[M - C5H9O4]- and 953 [M - C6H11O4]- were observed for
5. HRFABMS analysis revealed the molecular formulas of
4 and 5 to be C53H82O23 and C53H80O24, respectively.
Enzymatic hydrolysis of 4 and 5 with glycyrrhizic acid
hydrolase furnished 6 as their common aglycon. Acid
hydrolysis of 4 with 5% aqueous H2SO4-1,4-dioxane (1:1)
liberated D-glucuronic acid, D-glucose, L-rhamnose, and
D-xylose, while D-glucuronic acid, L-rhamnose, and D-xylose
were obtained by acid hydrolysis of 5.7 The 1H and 13C NMR
(pyridine-d5, Table 1) spectra of 4 showed signals due to a
xylopyranosyl moiety [δ 4.86 (d, J ) 7.3 Hz, H-1′′′′)], a
glucuronopyranosyl moiety [δ 4.97 (d, J ) 8.0 Hz, H-1′)], a
glucopyranosyl moiety [δ 5.74 (d, J ) 7.6 Hz, H-1′′)], and
a rhamnopyranosyl moiety [δ 1.74 (d, J ) 6.1 Hz, H-6′′′)
and 6.24 (br s, H-1′′′)], which were similar to those of 3,
group at the C-30 position seems to be essential for the
potent sweetness with this compound class. Compound 2
was also the most abundant sweet-tasting constituent of
this plant.
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. The following in-
struments were used to obtain physical data: specific rota-
tions, Horiba SEPA-300 digital polarimeter (l ) 5 cm); IR
spectra, Shimadzu FTIR-8100 spectrometer; 1H NMR spectra,
J EOL LNM-500 (500 MHz) spectrometer; 13C NMR spectra,
J EOL LNM-500 (125 MHz) spectrometer with tetramethyl-
silane as an internal standard; FABMS and HRFABMS, J EOL
J MS-SX 102A mass spectrometer; HPLC detector, Shimadzu
RID-6A refractive index detector.
The following materials were used for chromatography:
normal-phase silica gel column chromatography, silica gel BW-
200 (Fuji Silysia Chemical, Ltd., 150-350 mesh); reversed-
phase silica gel column chromatography, Chromatorex ODS
DM1020T (Fuji Silysia Chemical, Ltd., 100-200 mesh); TLC,
precoated TLC plates, silica gel 60F254 (Merck, 0.25 mm)
(normal-phase) and silica gel RP-18 F254S (Merck, 0.25 mm)
(reversed-phase); reversed-phase HPTLC, precoated TLC plates,
silica gel RP-18 WF254S (Merck, 0.25 mm); detection was
performed by spraying with 1% Ce(SO4)2-10% aqueous H2-
SO4 followed by heating.
P la n t Ma ter ia l. The stems of A. myriophylla were collected
in Muang District, Kanchanaburi Province, Thailand, in J uly
2001. It was identified by one of the authors (Y.P.). A voucher
of the plant is on file in our laboratory (T-02).
Extr a ction a n d Isola tion . The dried stems of A. myrio-
phylla (1.0 kg) were cut and extracted three times with MeOH
under reflux. Evaporation of the solvent under reduced pres-
sure provided the MeOH extract (151.5 g, 15.2%), and a part
of it (121.2 g) was partitioned with EtOAc-H2O (1:1). Removal
of the solvents under reduced pressure yielded an EtOAc-
soluble fraction (24.6 g, 3.1%) and a H2O-soluble fraction (96.6
g, 12.1%). The H2O-soluble fraction (83.0 g) was separated by
reversed-phase silica gel column chromatography [2.0 kg, H2O
1
except for the C-3′′ xylopyranosyl moiety. In turn, the H
and 13C NMR (pyridine-d5, Table 1) spectra of 5 showed
signals assignable to a xylopyranosyl moiety [δ 4.89 (d, J
) 7.3 Hz, H-1′′′′)], two glucuronopyranosyl moieties [δ 5.03
(d, J ) 7.6 Hz, H-1′) and 5.89 (d, J ) 7.6 Hz, H-1′′)], and a
rhamnopyranosyl moiety [δ 1.75 (d, J ) 6.4 Hz, H-6′′′) and
6.15 (br s, H-1′′′)], which were similar to those of 1, except
for the C-3′′ xylopyranosyl moiety. Finally, the oligoglyco-
side structures of 4 and 5 were characterized by HMBC
experiments on 4 and 5, in which long-range correlations
were observed between the following proton and carbon
signals: H-1′ and C-3, H-1′′ and C-2′, H-1′′′ and C-2′′, and
H-1′′′′ and C-3′′, respectively. Consequently, the structures
of albiziasaponins D (4) and E (5) were elucidated as shown.
Since the stems of A. myriophylla have been used as a
substitute for licorice and the methanolic extract showed
sweetness, we examined the sweetness of the principal
saponins (1 and 2). Compound 2, with a carboxyl group at
the C-30 position, showed potent sweetness, which was 600
times sweeter than that of sucrose, while the sweetness of
1, which has a lactone group at the C-30 position, was only
5 times sweeter than sucrose. Accordingly, a free carboxyl

New Sweet Triterpene Saponins from Albizia
J ournal of Natural Products, 2002, Vol. 65, No. 11 1641
f MeOH-H2O (60:40) f MeOH] to give six fractions [Fr. 1
(43.1 g), Fr. 2 (0.9 g), Fr. 3 (9.1 g), Fr. 4 (12.1 g), Fr. 5 (16.0 g),
and Fr. 6 (1.8 g)]. Fraction 3 (9.1 g) was subjected to normal-
phase silica gel column chromatography [30 g, CHCl3-MeOH-
H2O (15:3:1, lower layer f 6:4:1) f MeOH] to give albiziasa-
ponin A (1, 850 mg, 0.12%). Fraction 4 (200 mg) was purified
by HPLC [YMC-Pack Ph, 250 × 20 mm i.d., MeOH-1%
aqueous AcOH (70:30)] to furnish 1 (97 mg, 0.85%). Fraction
5 (16.0 g) was further separated by reversed-phase silica gel
column chromatography [480 g, MeOH-H2O (70:30) f MeOH]
to afford four fractions [Fr. 5-1 (2.6 g), Fr. 5-2 (6.0 g), Fr. 5-3
(2.2 g), and Fr. 5-4 (5.2 g)]. Fraction 5-2 (3.0 g) was purified
by HPLC [YMC-Pack ODS-A, 250 × 20 mm i.d., MeOH-1%
aqueous AcOH (70:30)] to give eight further fractions [Fr. 5-2-1
(213 mg), Fr. 5-2-2 (102 mg), Fr. 5-2-3 (24 mg), Fr. 5-2-4 (89
mg), Fr. 5-2-5 ()1, 1800 mg, 0.52%), Fr. 5-2-6 (339 mg), Fr.
5-2-7 (324 mg), and Fr. 5-2-8 (109 mg)]. Fraction 5-2-4 (89 mg)
was purified by HPLC [YMC-Pack ODS-A, 250 × 20 mm i.d.,
CH3CN-1% aqueous AcOH (35:75)] to furnish albiziasaponin
E (5, 56 mg, 0.016%). Fraction 5-2-6 (339 mg) was purified by
HPLC [YMC-Pack ODS-A, 250 × 20 mm i.d., CH3CN-1%
aqueous AcOH (35:75)] to afford albiziasaponins C (3, 24 mg,
0.007%) and D (4, 15 mg, 0.004%). Fraction 5-2-7 (324 mg)
was purified by HPLC [YMC-Pack ODS-A, 250 × 20 mm i.d.,
CH3CN-1% aqueous AcOH (40:60)] to give albiziasaponin A
monomethyl ester (1a , 40 mg, 0.012%) and 4 (59 mg, 0.017%).
Fraction 5-2-8 (109 mg) was purified by HPLC [YMC-Pack
ODS-A, 250 × 20 mm i.d., CH3CN-1% aqueous AcOH (40:
60)] to give licorice-saponin F3 (29 mg, 0.008%). Fraction 5-3
(1.8 g) was purified by HPLC [YMC-Pack ODS-A, 250 × 20
mm i.d., MeOH-1% aqueous AcOH (80:20)] to give Fr. 5-3-1
(134 mg), Fr. 5-3-2 (30 mg), Fr. 5-3-3 (79 mg), Fr. 5-3-4
[albiziasaponin B (2, 800 mg, 0.13%)], and Fr. 5-3-5 (757 mg).
Finally, fraction 5-3-1 (134 mg) was purified by HPLC [YMC-
Pack ODS-A, 250 × 20 mm i.d., MeOH-1% aqueous AcOH
(75:35)] to give yunganoside B1 (19 mg, 0.003%).
see Table 1; positive-ion FABMS m/z 993 [M + Na]+; negative-
ion FABMS m/z 969 [M - H]-, 823 [M - C6H11O4]-, 647 [M -
C12H19O10]-, 471 [M - C18H27O16]-; HRFABMS m/z 993.4696
(calcd for C48H74O20Na [M + Na]+, 993.4671).
Albizia sa p on in C (3): colorless fine crystals from CHCl3-
MeOH, mp 269-272 °C; [R]D24 -12.6° (c 0.10, MeOH); IR (KBr)
νmax 3453, 2926, 1765, 1632, 1078, 1048 cm-1; 1H NMR
(pyridine-d5, 500 MHz) δ 0.68, 0.82, 0.98, 1.18, 1.19, 1.46 (3H
each, all s, H3-25, 26, 28, 29, 27, and 23), 1.75 (3H, d, J ) 6.4
Hz, H-6′′′), 2.13 (1H, dd, J ) 6.7, 12.8 Hz, H-18), 3.42 (1H, dd,
J ) 4.6, 11.9 Hz, H-3), 3.33, 4.28 (1H each, both d-like, H2-
24), 4.15 (1H, br d, J ) ca. 6 Hz, H-22), 4.97 (1H, d, J ) 7.6
Hz, H-1′), 5.01 (1H, d, J ) 7.6 Hz, H-1′′′′), 5.12 (1H, br s, H-12),
5.75 (1H, d, J ) 7.6 Hz, H-1′′), 6.33 (1H, br s, H-1′′′); 13C NMR
data, see Table 1; positive-ion FABMS m/z 1139 [M + Na]+;
negative-ion FABMS m/z 1115 [M - H]-, 969 [M - C6H11O4]-,
953 [M - C6H11O5]-, 645 [M - C18H31O14]-; HRFABMS m/z
1139.5261 (calcd for C54H84O24Na [M + Na]+, 1139.5250).
Albizia sa p on in D (4): colorless fine crystals from CHCl3-
MeOH, mp 238-240 °C; [R]D24 -13.3° (c 0.10, MeOH); IR (KBr)
νmax 3445, 2930, 1765, 1655, 1078, 1043 cm-1 1H NMR
;
(pyridine-d5, 500 MHz) δ 0.68, 0.82, 0.98, 1.19, 1.24, 1.49 (3H
each, all s, H3-25, 26, 28, 29, 27, and 23), 1.74 (3H, d, J ) 6.1
Hz, H-6′′′), 2.13 (1H, dd, J ) 6.4, 13.1 Hz, H-18), 3.42 (1H, dd,
J ) 4.3, 11.6 Hz, H-3), 3.33, 4.28 (1H each, both d-like, H2-
24), 4.16 (1H, br d, J ) ca. 6 Hz, H-22), 4.86 (1H, d, J ) 7.3
Hz, H-1′′′′), 4.97 (1H, d, J ) 8.0 Hz, H-1′), 5.12 (1H, br s, H-12),
5.74 (1H, d, J ) 7.6 Hz, H-1′′), 6.24 (1H, br s, H-1′′′); 13C NMR
data, see Table 1; positive-ion FABMS m/z 1087 [M + H]+,
1109 [M + Na]+; negative-ion FABMS m/z 1085 [M - H]-, 953
[M - C5H9O4]-, 939 [M - C6H11O4]-, 645 [M - C17H29O13]-;
HRFABMS m/z 1109.5157 (calcd for C53H82O23Na [M + Na]+,
1109.5145).
Albizia sa p on in E (5): colorless fine crystals from CHCl3-
MeOH, mp 240-242 °C; [R]D24 -11.4° (c 0.10, MeOH); IR (KBr)
νmax 3445, 2933, 1752, 1655, 1078, 1047 cm-1; 1H NMR
(pyridine-d5, 500 MHz) δ 0.73, 0.83, 0.97, 1.18, 1.21, 1.49 (3H
each, all s, H3-25, 26, 28, 29, 27, and 23), 1.75 (3H, d, J ) 6.4
Hz, H-6′′′), 2.13 (1H, dd, J ) 6.4, 13.1 Hz, H-18), 3.43 (1H, dd,
J ) 4.3, 11.6 Hz, H-3), 3.53, 4.40 (1H each, both d-like, H2-
24), 4.15 (1H, br d, J ) ca. 7 Hz, H-22), 4.89 (1H, d, J ) 7.3
Hz, H-1′′′′), 5.03 (1H, d, J ) 7.6 Hz, H-1′), 5.12 (1H, br s, H-12),
5.89 (1H, d, J ) 7.6 Hz, H-1′′), 6.15 (1H, br s, H-1′′′); 13C NMR
data, see Table 1; positive-ion FABMS m/z 1101 [M + H]+,
1123 [M + Na]+; negative-ion FABMS m/z 1099 [M - H]-, 967
[M - C5H9O4]-, 953 [M - C6H11O4]-; HRFABMS m/z 1123.4954
(calcd for C53H80O24Na [M + Na]+, 1123.4937).
Acid Hyd r olysis of Albizia sa p on in s A (1), B (2), C (3),
D (4), a n d E (5). A solution of 1-5 (5 mg each) in 5% aqueous
H2SO4-1,4-dioxane (1:1, 2 mL) was heated under reflux for 1
h. After cooling, the reaction mixture was neutralized with
Amberlite IRA-400 (OH- form) and the resin was removed by
filtration. After removal of the solvent from the filtrate under
reduced pressure, the residue was transferred to a Sep-Pak
C18 cartridge with H2O and MeOH. The H2O eluate was
concentrated in vacuo to give a residue, which was treated with
L-cysteine methyl ester hydrochloride (4 mg) in pyridine (0.5
mL) at 60 °C for 1 h. After the reaction, the solution was
treated with N,O-bis(trimethylsilyl)trifluoroacetamide (0.2 mL)
at 60 °C for 1 h. The supernatant of the reaction mixture was
then subjected to GLC analysis to identify the derivatives of
D-glucuronic acid (i) and L-rhamnose (ii) from 1-5; D-glucose
(iii) from 3 and 4; and D-xylose (iv) from 4 and 5. GLC
conditions: Supelco STB-1, 30 m × 0.25 mm (i.d.) capillary
column; injector temperature, 230 °C; detector temperature,
230 °C; column temperature, 230 °C; He flow rate, 15 mL/
min; tR: (i) 26.4 min, (ii) 15.4 min, (iii) 24.2 min, and (iv) 13.8
min.
Licorice-saponin F34 and yunganoside B1 were identified
5
by comparison of their physical data ([R]D, IR, 1H NMR, 13C
NMR, MS) with reported values.
Albizia sa p on in A (1): colorless fine crystals from CHCl3-
MeOH, mp 226-229 °C; [R]D24 -28.2° (c 0.10, MeOH); IR (KBr)
1
νmax 3453, 2940, 1760, 1640, 1047 cm-1; H NMR (pyridine-
d5, 500 MHz) δ 0.71, 0.82, 0.98, 1.20, 1.22, 1.51 (3H each, all
s, H3-25, 26, 28, 29, 27, and 23), 1.81 (3H, d, J ) 6.4 Hz, H-6′′′),
2.13 (1H, dd, J ) 6.4, 13.3 Hz, H-18), 3.45 (1H, dd, J ) 4.3,
11.6 Hz, H-3), 3.48, 4.40 (1H each, both d, J ) 11.6 Hz, H2-
24), 4.20 (1H, br d, J ) ca. 6 Hz, H-22), 5.06 (1H, d, J ) 7.6
Hz, H-1′), 5.13 (1H, br s, H-12) 5.87 (1H, d, J ) 7.6 Hz, H-1′′),
6.27 (1H, br s, H-1′′′); 13C NMR data, see Table 1; positive-ion
FABMS m/z 969 [M + H]+, 991 [M + Na]+; negative-ion
FABMS m/z 967 [M - H]-, 821 [M - C6H11O4]-, 645 [M -
C
12H19O10]-; HRFABMS m/z 969.4724 (calcd for C48H73O20 [M
+ H]+, 969.4695), 991.4543 (calcd for C48H72O20Na [M + Na]+,
991.4515).
Albizia sa p on in A m on om eth yl ester (1a ): colorless fine
crystals from CHCl3-MeOH, mp 209-212 °C; [R]D25 -16.6° (c
0.86, MeOH); 1H NMR (pyridine-d5, 500 MHz) δ 0.79, 0.84,
0.97, 1.19, 1.20, 1.54 (3H each, all s, H3-25, 26, 28, 29, 27, and
23), 1.78 (3H, d, J ) 6.1 Hz, H-6′′′), 2.13 (1H, dd like, H-18),
3.45 (1H, dd, J ) 4.8, 11.9 Hz, H-3), 3.54, 4.43 (1H each, both
d like, H2-24), 3.77 (3H, s, -OCH3), 4.15 (1H, br d, J ) ca. 6
Hz, H-22), 5.03 (1H, d, J ) 7.4 Hz, H-1′), 5.13 (1H, br s, H-12)
5.87 (1H, d, J ) 7.7 Hz, H-1′′), 6.29 (1H, br s, H-1′′′); 13C NMR
data, see Table 1; positive-ion FABMS m/z 983 [M + H]+, 1005
[M + Na]+; negative-ion FABMS m/z 981 [M - H]-, 835 [M -
C6H11O4]-.
Albizia sa p on in B (2): colorless fine crystals from CHCl3-
MeOH, mp 220-223 °C; [R]D24 +4.6° (c 0.10, MeOH); IR (KBr)
1
νmax 3453, 2930, 1730, 1645, 1047 cm-1; H NMR (pyridine-
En zym a tic Hyd r olysis of Albizia sa p on in s A (1) a n d
C-E (3-5) To Affor d 6. A solution of 1 (20.0 mg) in 0.1 M
acetate buffer (pH 4.4, 2.0 mL) was treated with glycyrrhizinic
acid hydrolase (Maruzen Pharmaceutical Co., Ltd., Hiroshima,
J apan, 1.0 mL), and the mixture was stirred at 44 °C for 3 h.
After treatment of the reaction mixture with EtOH, the solvent
was evaporated to dryness under reduced pressure and the
d5, 500 MHz) δ 0.72, 0.88, 0.90, 1.31, 1.37, 1.52, (3H each, all
s, H3-25, 28, 26, 27, 29, and 23), 1.80 (3H, d, J ) 6.1 Hz, H-6′′′),
2.39 (1H, dd, J ) 3.1, 12.8 Hz, H-18), 3.45 (1H, dd, J ) 4.0,
12.8 Hz, H-3), 3.49, 4.39 (1H each, both d, J ) 11.6 Hz, H2-
24), 5.07 (1H, d, J ) 7.6 Hz, H-1′), 5.44 (1H, br s, H-12), 5.90
(1H, d, J ) 7.9 Hz, H-1′′), 6.31 (1H, br s, H-1′′′); 13C NMR data,

1642 J ournal of Natural Products, 2002, Vol. 65, No. 11
Yoshikawa et al.
residue was purified by silica gel column chromatography [1.0
g, n-hexanes-EtOAc (1:1)] to give 6 (7.0 mg, 72%). Using a
similar procedure, a solution of 3 (10.0 mg), 4 (8.5 mg), or 5
(10.0 mg) in 0.1 M acetate buffer (pH 4.4, 1.0 mL) was treated
with glycyrrhizinic acid hydrolase (0.5 mL each), and the
mixture was stirred at 44 °C for 3 h. After EtOH was added
to the reaction mixture, the solvent was removed under
reduced pressure and the residue was purified by silica gel
column chromatography [1.0 g, n-hexane-EtOAc (1:1)] to
furnish 6 (from 3: 3.0 mg, 71%; from 4: 2.1 mg, 57%; from
5: 3.0 mg, 70%), which was identified by comparison of
physical data ([R]D, IR, 1H NMR, 13C NMR) with reported
values.6
Ack n ow led gm en t. We are grateful to Dr. K. Mizutani and
Ms. M. Yamana, Research Laboratories (Maruzen Pharma-
ceutical Co., Ltd., Hiroshima, J apan), for the sensory testing.
Refer en ces a n d Notes
(1) This paper is number 20 in our series Bioactive Saponins and
Glycosides. For paper number 19, see: Yoshikawa, M.; Morikawa,
T.; Yashiro, K.; Murakami, T.; Matsuda, H. Chem. Pharm. Bull. 2001,
49, 1452-1456.
(2) Ito, A.; Kasai, R.; Yamasaki, K.; Duc, N. M.; Nham, N. T. Phytochem-
istory 1994, 37, 1455-1458. (b) Ito, A.; Kasai, R.; Duc, N. M.; Ohtani,
K.; Nham, N. T.; Yamasaki, K. Chem. Pharm. Bull. 1994, 42 (2),
1966-1967.
(3) Yoshikawa, M.; Murakami, T.; Oominami, H.; Matsuda, H. Chem.
Pharm. Bull. 2000, 48, 1045-1050. (b) Murakami, T.; Nakamura,
J .; Kageura, T.; Matsuda, H.; Yoshikawa, M. Chem. Pharm. Bull.
2000, 48, 1720-1725. (c) Murakami, T.; Oominami, H.; Matsuda, H.;
Yoshikawa, M. Chem. Pharm. Bull. 2001, 49, 741-746. (d) Yoshika-
wa, M.; Morikawa, T.; Yashiro, K.; Murakami, T.; Matsuda, H. Chem.
Pharm. Bull. 2001, 49, 1452-1456.
En zym a tic Hyd r olysis of Albizia sa p on in B (2) Givin g
Azu k isa p ogen ol (7). A solution of 2 (20.0 mg) in 0.1 M
acetate buffer (pH 4.4, 0.5 mL) was treated with glycyrrhizinic
acid hydrolase (1.0 mL), and the mixture was stirred at 44 °C
for 3 h. After the addition of EtOH to the reaction mixture,
the solvent was removed under reduced pressure. The crude
product was purified by silica gel column chromatography [1.0
g, n-hexane-EtOAc (1:1)] to give azukisapogenol (7, 9.2 mg,
97%), which was identified by a comparison of physical data
(4) Kitagawa, I.; Hori, K.; Sakagami, M.; Zhou, J .-L.; Yoshikawa, M.
Chem. Pharm. Bull. 1993, 41, 1337-1345.
(5) Ohtani, K.; Ogawa, K.; Kasai, R.; Yang, C.-R.; Yamasaki, K.; Zhou,
J .; Tanaka, O. Phytochemistry 1992, 31, 1747-1752.
1
([R]D, IR, H NMR, 13C NMR) with reported values.9
Bioa ssa y. Sen sor y Testin g. Sweetness relative to sucrose
was evaluated by a human sensory panel. Albiziasaponin A
(1) was dissolved in water-ethanol (100:1) to make a 0.1%
(w/v) solution, and sucrose solutions were prepared at gradu-
ated concentrations from 0.2 to 0.8% (w/v). Albiziasaponin B
(2) was dissolved in water-ethanol (400:1) to make a 0.006%
(w/v) solution, and sucrose solutions were prepared at gradu-
ated concentrations from 3.2 to 4.0% (w/v). Both samples were
evaluated by a panel of five trained tasters in the manner
described previously.10 The panelists were asked to taste a
sample solution and to estimate its sweetness intensity relative
to that of the sucrose solution of appropriate concentration.
Panelists were asked to taste samples in this way four times.
(6) Kitagawa, I.; Hori, K.; Uchida, E.; Chen W.-Z.; Yoshikawa, M.; Ren,
J . Chem. Pharm. Bull. 1993, 41, 1567-1572.
(7) Hara, S.; Okabe H.; Mihashi, K. Chem. Pharm. Bull. 1986, 34, 1843-
1845.
(8) The 1H and 13C NMR spectra of 1-5 and 1a were assigned with the
aid of distortionless enhancement by polarization transfer (DEPT),
homo- and heterocorrelation spectroscopy (1H-1H, 13C-1H COSY),
homo- and heteronuclear Hartmann-Hahn spectroscopy (1H-1H,
13C-1H HOHAHA), and HMBC experiments.
(9) Kitagawa, I.; Wang, H. K.; Saito, M.; Yoshikawa, M. Chem. Pharm.
Bull. 1983, 31, 664-673.
(10) Mizutani, K.; Kuramoto, T.; Tamura, Y.; Ohtake, N.; Doi, S.; Nakaura,
M.; Tanaka, O. Biosci. Biotech. Biochem. 1994, 58, 554-555.
NP020220L