Steroidal constituents from the leaves of Hosta longipes and their inhibitory effects on nitric oxide production

Article abstract of DOI:10.1016/j.bmcl.2013.01.050

Hosta longipes (FR. et SAV.) MATSUMURA (Liliaceae) is an edible vegetable in Korea. This study was conducted with the aim of evaluating the potential of H. longipes as a functional food for the treatment of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. In this respect, the study resulted in the identification of three new steroidal compounds, longipenane (1), longipenane 26-O-β-d-glucopyranoside (2) and neogitogenin 3-O-α-l-rhamnopyranosyl-(1→2)-O-[β-d-glucopyranosyl-(1→4)] -β-d-galactopyranoside (3), along with two known steroidal saponins (4 and 5). The identification and structural elucidation of these compounds were based on 1D and 2D NMR measurements, high-resolution FAB mass spectroscopy (HR-FAB-MS), and chemical methods. A proinflammatory mediator, nitric oxide (NO), in murine microglial BV-2 cells was used to assess the anti-neuroinflammatory effect of the isolated compounds from H. longipes. Among them, compounds 4 and 5 showed strong inhibitory effects on NO production without high cell toxicity in lipopolysaccharide-activated BV-2 cells (IC ₅₀ = 17.66 and 13.16 μM, respectively).

Full text of DOI:10.1016/j.bmcl.2013.01.050

Bioorganic & Medicinal Chemistry Letters 23 (2013) 1771–1775
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Steroidal constituents from the leaves of Hosta longipes and their
inhibitory effects on nitric oxide production
Chung Sub Kim a, Sun Yeou Kim b, Eunjung Moon c, Mi Kyeong Lee d, Kang Ro Lee a,
⇑
a Natural Products Laboratory, School of Pharmacy, Sungkyunkwan University, 300 Chonchon-dong, Jangan-ku, Suwon, Gyeonggi-do 440-746, Republic of Korea
b College of Pharmacy, Gachon University, #191 Hambakmoe-ro, Yeonsu-gu, Incheon 406-799, Republic of Korea
c Graduate School of East-West Medical Science, Kyung Hee University Global Campus, #1732 Deogyeong-daero, Giheung-gu, Yongin, Gyeonggi-do 446-701, Republic of Korea
d College of Pharmacy, Chungbuk National University, #52 Naesudong-ro, Heungdeok-gu, Cheongju 361-763, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Hosta longipes (FR. et SAV.) MATSUMURA (Liliaceae) is an edible vegetable in Korea. This study was con-
ducted with the aim of evaluating the potential of H. longipes as a functional food for the treatment of
neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease. In this respect, the study
Received 22 October 2012
Revised 2 January 2013
Accepted 15 January 2013
Available online 26 January 2013
resulted in the identification of three new steroidal compounds, longipenane (1), longipenane 26-O-b-
glucopyranoside (2) and neogitogenin 3-O- -rhamnopyranosyl-(1?2)-O-[b- -glucopyranosyl-(1?4)]-
b- -galactopyranoside (3), along with two known steroidal saponins (4 and 5). The identification and
D-
a
-
L
D
D
Keywords:
Hosta longipes
Liliaceae
Steroid
Structural elucidation
Nitric oxide
structural elucidation of these compounds were based on 1D and 2D NMR measurements, high-resolu-
tion FAB mass spectroscopy (HR-FAB-MS), and chemical methods. A proinflammatory mediator, nitric
oxide (NO), in murine microglial BV-2 cells was used to assess the anti-neuroinflammatory effect of
the isolated compounds from H. longipes. Among them, compounds 4 and 5 showed strong inhibitory
effects on NO production without high cell toxicity in lipopolysaccharide-activated BV-2 cells
Neuroinflammation
(IC50 = 17.66 and 13.16 lM, respectively).
Ó 2013 Elsevier Ltd. All rights reserved.
Neuroinflammation is a defense mechanism in the central
nervous system against various harmful infections and injuries.
However, substantial evidence supports the idea that neuroinflam-
mation by activated microglia is strongly involved in the pathogen-
esis and progression of neurodegenerative diseases such as
multiple sclerosis, Alzheimer’s disease, and Parkinson’s disease.1
Microglia cells are the resident immune cells of the central nervous
system. Under normal conditions, these cells play a role in homeo-
stasis regulation and defense against injury.2 However, in response
to abnormal stimuli including environment toxins and neurotox-
ins, microglia are activated and release various proinflammatory
factors including nitric oxide (NO),3 leading to possible exacerba-
tion of neuronal cell death resulting in neurodegenerative
diseases.4
Hosta longipes (FR. et SAV.) MATSUMURA (Liliaceae) is an edible
vegetable in Korea and widely distributed throughout Korea, Chi-
na, and Japan.5 It is called ‘‘Bi-Bi-Chu’’ in Korea, and its young
leaves are consumed raw as a salad. This plant has been used as
a Korean traditional medicine for the treatment of swelling,
inflammation and snake bites.5,6 In previous phytochemical and
biological activity studies, its EtOH extract showed antioxidant
activity7 and steroidal saponins isolated from this plant showed
cytotoxic activity towards HeLa cells.8 However, little phytochem-
ical and biological investigations on H. longipes have been con-
ducted. In our screening procedures, the MeOH extract of leaves
of H. longipes showed an inhibitory effect on NO production in lipo-
polysaccharide (LPS)-activated BV-2 cells, a murine microglial cell
line. Therefore, as part of a continuing search for bioactive constit-
uents from Korean medicinal plant sources, we attempted to inves-
tigate the active constituents of this herb.
The MeOH extract from the leaves of H. longipes was suspended
in distilled water and then partitioned with n-hexane, CHCl3, EtOAc
and BuOH. The CHCl3 layer showed a considerable inhibitory effect
on nitric oxide (NO) production in LPS-activated BV-2 cells, and
EtOAc and BuOH layers displayed moderate inhibitory effects in
our screening procedures. Chemical investigation of these layers
using successive column chromatography over silica gel, Sephadex
LH-20, and preparative HPLC resulted in the isolation and identifi-
cation of three new steroidal compounds 1-3 and two known ste-
roidal saponins 4 and 5 (Fig. 1).
Compound 1 was obtained as a colorless gum. The molecular
formula was determined to be C27H44O5 from the molecular ion
peak [M+H]+ at m/z 449.3266 (calcd for 449.3267) in the posi-
tive-ion HR-FAB-MS. The IR spectrum displayed strong OH vibra-
tions at 3382 cmꢀ1 and a carbonyl band at 1660 cmꢀ1. The 1H
NMR spectrum showed the presence of four methyl groups [dH
1.08, 0.93 (each 3H, d, J = 7.0 Hz), 1.24 and 1.09 (each 3H, s)], three
⇑
Corresponding author. Tel.: +82 31 290 7710; fax: +82 31 290 7730.
E-mail address: krlee@skku.ac.kr (K.R. Lee).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.01.050

1772
C. S. Kim et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1771–1775
27
21
20
26
O
25
18
12
23
22
11
9
24
OH
26
21
OH18
HO
24
19
17
13
O
22
23
20
15
(R)
1
4
16
HO
O
2
3
OR
O 1'''
OH
HO
25
27
14
15
O
10
8
7
HO
OH
O
11
17
12
5
19
13
14
1
OH
16
HO
6
2
H
9
O1'
10
8
7
3
5
O
4
1''
6
O
HO
HO
OH
1: R = H
2: R = β -D-Glc
3
O
HO
HO
O
HO
O
O
O
O
O
OH
O
O
RO
HO
HO
OH HO
H
OH
O
HO
HO
OH
4: R = H
5: R =α -L-Rha
Figure 1. Structures of 1–5 from the leaves of H. longipes.
oxygenated methine [dH 4.34 (1H, dt, J = 7.0, 4.0 Hz), 4.04 (1H, t,
J = 6.0 Hz) and 3.35 (1H, m)], one oxygenated methylene [dH 3.44
(1H, dd, J = 5.5, 10.5 Hz), and 3.34 (1H, m)], and one olefinic proton
[dH 5.71 (1H, s)] signals (Table 1). The 13C NMR spectrum indicated
27 carbon resonances, which were classified by DEPT and HMQC
experiments as one carbonyl carbon (dC 201.0), one trisubstituted
double bond (dC 173.4 and 123.1), three oxygenated methines (dC
78.1, 72.9 and 70.9), one oxygenated methylene (dC 67.2), four
methyls, eight methylenes, six methines and two quaternary
carbons. These NMR data were very similar to those of (25S)-
16(S), 22(S), 26-trihydroxycholest-4-en-3-one,9 except for the ab-
sence of one methylene proton and carbon resonances and the
presence of one oxygenated methine [dH 3.35 (1H, m); dC 78.1].
The HMBC correlation of H-18/C-12 and NOE correlations of H-
12/H-9 and H-14 (Fig. 2) confirmed the presence of an OH group
at C-12. The relative stereochemistry was assumed to be the same
as that of (25S)-16(S),22(S),26-trihydroxycholest-4-en-3-one9
based on the J values and confirmed by the NOE correlations of
H-8/H-18 and H-19, H-9/H-12 and H-14, H-16/H-14, H-17 and H-
21, and H-18/H-20 (Fig. 2). The b-orientiation of the OH group at
C-12 was confirmed by the NOE correlations of H-12/H-9 and H-
14. The relative configuration of the OH group at C-22 was anti
to the Me group at C-20 by comparing the J value of H-22 (t,
J = 6.5 Hz) with that of (22S)-bethosides B and C [t, J = 6.9 Hz for
positive-ion HR-FAB-MS. The IR spectrum displayed strong OH
vibrations at 3382 cmꢀ1 and a carbonyl band at 1656 cmꢀ1. The
1H NMR spectrum of 2 showed an anomeric proton at dH 4.23
(1H, d, J = 8.0 Hz, H-10), which is a characteristic signal for a b-
glucopyranosyl unit, and six oxygenated protons attributed to a su-
gar at dH 3.87 (1H, dd, J = 12.0, 2.0 Hz, H-60a), 3.66 (1H, dd, J = 12.0,
10.5 Hz, H-60b), 3.34 (1H, m, H-30), 3.29 (1H, m, H-40), 3.27 (1H, m,
H-50) and 3.18 (1H, dd, J = 9.0, 8.0 Hz, H-20). The 13C NMR spectrum
of 2 showed signals for a b-glucopyranosyl unit (dC 103.4, 76.8,
76.6, 73.9, 70.5 and 61.5) and the other signals in the 1H and 13C
NMR spectra were fairly similar to those of 1. The chemical shifts
of C-26 in 2 [dH 3.74, 3.38 (each 1H, dd J = 10.5, 6.5 Hz); dC 74.9]
were downfield shifted compared with those of C-26 in 1, indicat-
ing the location of the b-glucopyranosyl unit at C-26. This was
confirmed by the HMBC correlation of H-10/C-26 (Fig. 3). The enzy-
matic hydrolysis of 2 with b-glucosidase (Almonds) yielded the
aglycone 2a, whose 1H NMR spectrum was in good agreement with
that of 1, and
pound 2 was characterized as (25R)-12b,16b,22b,26-(b-
D
-glucose {½a 2D5
ꢁ
+62.2 (c = 0.05, H2O)}. Thus, com-
-glucopyr-
D
anosyl)-tetrahydroxycholest-4-en-3-one and named longipenane
26-O-b- -glucopyranoside.
D
Compound 3 was obtained as a white powder. The molecular
formula was determined to be C45H74O18 from the molecular ion
peak [M+H]+ at m/z 903.4954 (calcd for 903.4953) in the posi-
tive-ion HR-FAB-MS. The 1H NMR spectrum indicated the presence
of two tertiary methyl groups [dH 0.93 and 0.81 (each 3H, s)], two
secondary methyl groups [dH 1.14 and 1.08 (each 3H, d, J = 7.0 Hz)],
typical steroid methyls, and three anomeric proton [dH 6.27 (1H, br
s), 5.20 and 4.97 (each 1H, d, J = 7.5 Hz)] signals showing HSQC cor-
relations with three anomeric carbon signals [dC 102.8, 107.6 and
101.4, respectively] (Table 2). Comparison of the signals from the
aglycone part of 3 in the 1H and 13C NMR spectra with those from
the aglycone part of cistocardin12 showed that the aglycone part of
3 was the same as that of cistocardin, and this was confirmed
H-22 (22b); ddd, J = 11.1, 3.3, 2.1 Hz for H-22 (22
configuration at C-25 was confirmed as R by examination of the
a
)].10 The absolute
Mosher esters of 1; R ester 1a showed a larger difference (
D
d
0.211) between two H-26 signals than that of S ester 1b (
D
d
0.081 ppm).11 Thus, compound 1 was characterized as (25R)-
12b,16b,22b,26-tetrahydroxycholest-4-en-3-one
and
named
longipenane.
Compound 2 was obtained as a colorless gum. The molecular
formula was determined to be C33H54O10 from the molecular ion
peak [M+H]+ at m/z 611.3795 (calcd for 611.3795) in the

C. S. Kim et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1771–1775
1773
Table 1
of the aglycone part were determined by NOE correlations (Fig. 4).
The 25S configuration was confirmed through the comparison
characteristic chemical shifts for C-23, C-24, C-25, C-26 and C-27
with those for C-23, C-24, C-26 and C-27 (27.3 0.3, 26.1 0.3,
65.1 0.1, and 16.2 0.2 ppm, respectively, 25S) and for C-23, C-
24, C-25 and C-26 (31.3 0.3, 28.8 0.3, 30.3 0.3 and 66.9 0.2,
respectively, 25R) in previously reported data.15–17 Thus, the struc-
1H (500 MHz) and 13C NMR (125 MHz) data of compounds 1 and 2 in CD3OD (d in
ppm, J values in parentheses)a
Position
1
2
dH
dC
dH
dC
1
2
3
4
5
6
7
8
2.07 m, 1.71 m
2.50 m, 2.30 m
35.5 t
33.4 t
201.0 s
123.1 d
173.4 s
32.7 t
31.4 t
33.9 d
53.0 d
38.6 s
30.1 t
78.1 d
47.3 s
52.5 d
35.5 t
70.9 d
62.9 d
9.5 q
2.08 m, 1.71 m
2.50 m, 2.30 m
35.5 t
33.5 t
201.1 s
123.1 d
172.8 s
32.7 t
30.7 t
33.8 d
53.0 d
38.6 s
30.1 t
78.1 d
47.5 s
52.5 d
35.8 t
70.8 d
63.0 d
9.5 q
ture of 3 was established as neogitogenin 3-O-a-L-rhamnopyrano-
5.71 s
5.72 s
syl-(1?2)-O-[b-D-glucopyranosyl-(1?4)]-b-D-galactopyranoside.
Compounds 4 and 5 were identified by comparing the 1H and
2.51 m, 2.33 m
1.89 m, 1.01 m
1.68 m
2.51 m, 2.33 m
1.89 m, 1.01 m
1.68 m
13C NMR, and MS spectra with the literature. They were
determined to be gitogenin 3-O-{O-b-
[b- -xylopyranosyl-(1?3)]-O-b- -glucopyranosyl-(1?4)-b-
topyranoside} (4) and gitogenin 3-O-{O-b- -glucopyranosyl-
(1?2)-O-[O- -rhamnopyranosyl-(1?4)-b- -xylopyranosyl-(1?3)]-
O-b- -glucopyranosyl-(1?4)-b-
-galactopyranoside} (5),18 which
D
-glucopyranosyl-(1?2)-O-
9
1.07 m
1.05 m
D
D
D-galac-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26a
26b
27
10
D
1.66 m, 1.46 m
3.35 m
1.66 m, 1.46 m
3.36 m
a-L
D
D
D
0.84 m
0.82 m
were suggested as the main constituents of H. longipes.
2.26 m, 1.35 m
4.32 dt, (7.0, 4.0)
1.44 m
2.27 m, 1.36 m
4.33 dt, (7.5, 4.0)
1.44 m
Neuroinflammation by overactivated microglia has been known
to contribute to progressive neuronal damage in many neurode-
generative diseases.1 In this study, we tested the anti-neuroinflam-
matory effects of compounds (1–5) isolated from H. longipes via
measurement of NO levels using a bacterial endotoxin, LPS, in mur-
ine microglia BV-2 cells.19,20 The inhibitory activities of the isolated
compounds on NO production were expressed as 50% inhibition
concentration (IC50). As shown in Table 3, compounds 2, 3, 4, and
5 decreased NO production. Among them, the IC50 values of com-
1.09 s
1.10 s
1.24 s
2.44 m
1.08 d (7.0)
4.04 t (6.0)
1.52 m
1.12 m
1.57 m
3.44 dd (10.5, 5.5)
3.34 m
0.93 d (6.5)
16.3 q
37.0 d
12.1 q
72.9 d
32.9 t
30.0 t
35.9 d
67.2 t
1.25 s
2.45 m
16.3 q
36.9 d
12.0 q
72.6 d
32.7 t
30.5 t
33.5 d
74.9 t
1.07 d (7.0)
4.05 t (6.0)
1.52 m
1.66 m, 1.15 m
1.77 m
3.74 dd (10.0, 6.5)
3.38 dd (10.0, 6.5)
0.95 d (6.5)
4.23 d (8.0)
3.18 dd (9.0, 8.0)
3.34 m
pounds 3, 4, and 5 were determined to be below than 20
exhibited inhibitory activity with an IC50 of 2.70, 17.66, and
13.16 M, respectively. It was shown that compound 3 strongly re-
duced the cell viability of BV-2 cells [38.9 2.2% at a concentration
of 20 M compared with LPS only treatment group, median lethal
dose (LD50) of 3.12 M]. We suggest that the inhibitory effect of
lM. They
15.9 q
16.1 q
103.4 d
73.9 d
76.8 d
70.5 d
76.6 d
61.5 t
l
20
30
l
40
3.29 m
3.27 m
3.87 dd (12.0, 2.0)
3.66 dd (12.0, 10.5)
l
50
compound 3 on NO production may be influenced by its high cyto-
toxic activities, although the percentage of inhibition of NO pro-
duction was more than that of cell cytotoxicity. Compounds 4
and 5 had no influence on cell viability at concentrations up to
60a
60b
a
The assignments were based on DEPT, 1H–1H COSY, HMQC and HMBC
experiments.
20
l
M. These compounds had shown the cell viability of BV-2 cells
M,
with 89.6 5.5% and 86.4 5.8% at a concentration of 20
l
through 1D and 2D NMR spectra (1H and 13C NMR, DEPT, 1H–1H
COSY, HSQC, HMBC and NOESY). The sugar parts were assumed
respectively. The results indicate that statistically significant dif-
ferences were not shown between LPS-only treatment group and
to
(1?4)]-b-
NMR spectra with those of gitogenin 3-O-{O-
syl-(1?2)-O-[b- -glucopyranosyl-(1?4)]-b-
-galactopyranoside}8
be
O-
-galactopyranosyl unit by comparing the 1H and 13C
-rhamnopyrano-
a
-
L
-rhamnopyranosyl-(1?2)-O-[b-
D
-glucopyranosyl-
each compound at 20 lM. Therefore, compounds 4 and 5 inhibited
D
LPS-induced NO production in BV-2 cells without the influence of
cytotoxicity. And these results indicate that steroidal saponins (4
and 5) may be the main active compounds of H. longipes, exhibiting
strong anti-neuroinflammatory properties. Moreover, these com-
pounds isolated from H. longipes can be candidates for treatment
of various neurodegenerative diseases associated with neuroin-
flammation via regulation of activated microglia.
a-L
D
D
and confirmed through HMBC correlation of H-10/C-3, H-100/C-20
and H-1000/C-40 (Fig. 4) and TOCSY (Supplementary data). Acid
hydrolysis of 3 gave neogitogenin (3a), which was identified
through 1H NMR and MS spectra with previously reported val-
ues,12,13 and three monosaccharides identified by GC/MS14 analysis
In conclusion, this study indicates that steroidal constituents
are the main components of the leaves of H. Longipes. Additionally,
were
L-rhamnose,
D
-glucose and D-galactose. The stereochemistries
OH
OH
H
OH
OH
OH
OH
H
OH
OH
H
H
H
O
H
H
O
1H-1H NOESY
1H-1H COSY
HMBC
Figure 2. HMBC, 1H–1H COSY and NOESY correlations of 1.

1774
C. S. Kim et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1771–1775
OH
H
OH
OH
OH
O
OH
H
OGlc
OH
HO
H
H
O
H
O
H
H
OH
O
HO
HO
1H-1H NOESY
1H-1H COSY
HMBC
Figure 3. HMBC, 1H–1H COSY and NOESY correlations of 2.
Table 3
Table 2
Effects of compounds 1–5 on NO production and cell viability in LPS-activated BV-2
cells
1H (500 MHz) and 13C NMR (125 MHz) data of compounds 3 in pyridine-d5 (d in ppm,
J values in parentheses)a
Compound
IC50
(l
M)
Cell viabilityb (%)
a
Position
Aglycone
Position
Sugar
1
2
3
4
5
>200
46.30
2.70
17.66
13.16
14.29
93.4 2.0
94.8 1.5
38.9 2.2⁄
89.6 5.5
86.4 5.8
102.3 3.7
dH
dC
dH
dC
1
2
3
4
5
6
7
8
2.27 m, 1.19 m 46.2 t
4.13 m
3.89 m
2.02 m, 1.83 m 33.9 t
1.03 m 45.1 d
1.24 m, 1.13 m 28.6 t
1.53 m, 0.82 m 32.6 t
1.42 m
0.62 br t (11.0) 54.8 d
37.3 s
1.50 m, 1.25 m 21.9 t
1.63 m, 1.03 m 40.5 t
41.2 s
1.02 m
2.03 m, 1.41 m 32.7 t
4.57 m
1.79 m
0.81 s
0.93 s
1.91 m
1.14 d (6.5)
10(Gal)
4.97 d (7.5)
4.58 m
4.37 m
4.59 m
4.14 m
4.60 m, 4.32 m 61.5 t
6.27 br s
4.81 m
4.60 m
4.33 m
101.4 d
77.2 d
76.9 d
81.0 d
76.1 d
71.0 d
85.4 d
20
30
40
L
-NMMAc
50
60
a
IC50 value of each compound was defined as the concentration (
50% inhibition of NO production in LPS-activated BV-2 cells.
Cell viability after treatment with 20 lM of each comound was expressed as a
percentage (%) of the LPS only treatment group. The results are averages of three
lM) causing
100(Rha)
200
102.8 d
72.8 d
73.2 d
74.5 d
69.9 d
19.0 q
107.6 d
76.0 d
79.4 d
72.6 d
79.1 d
b
35.1 d
9
300
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
400
independent experiments, and the data are expressed as mean SD. (Student’s t-
500
4.87 m
test, ⁄p-value <0.05)
600
1.63 d (6.5)
5.20 d (7.5)
4.15 m
4.23 m
4.11 m
c
L
-NMMA as a positive control.
1000(Glc)
2000
56.8 d
3000
81.8 d
63.3 d
17.0 q
14.0 q
42.9 d
15.3 q
110.2 s
4000
(3) were isolated from this plant source. Two main steroidal
saponins (4 and 5) of this plant, which significantly inhibited nitric
oxide (NO) production in LPS-activated BV-2 cells, were also
investigated. The present study thus indicates that these
compounds would be good candidates for further research as
anti-neuroinflammatory agents and show the potential of
H. longipes as a functional food for the treatment of neurodegener-
ative diseases such as Alzheimer’s disease and Parkinson’s disease.
5000
4.00 m
4.59 m, 4.22 m 63.5 t
6000
1.24 m, 1.12 m 28.6 t
1.90 m, 1.45 m 26.8 t
1.36 m
4.07 m, 3.38 m 65.5 t
1.08 d (7.0) 16.8 q
26.7 d
Acknowledgements
a
The assignments were based on DEPT, 1H–1H COSY, HSQC and HMBC
This research was supported by the Basic Science Research Pro-
gram through the National Research Foundation of Korea (NRF)
funded by the Ministry of Education, Science and Technology
(20100029358). We are thankful to the Korea Basic Science Insti-
tute (KBSI) for the measurements of NMR and MS spectra.
three new steroidal compounds, longipenane (1), longipenane
26-O-b- -glucopyranoside (2), and neogitogenin 3-O- -rhamnopyr-
anosyl-(1?2)-O-[b- -glucopyranosyl-(1?4)]-b- -galactopyranoside
D
a-L
D
D
O
HO
HO
O
H
O
HO
O
O
H
H
O
HO
OH
O
OH
HO
O
O
H
HO
Sugar
H
H
H
H
O
H
H
O
HO
HO
OH
1H-1H NOESY
HMBC
Figure 4. Key HMBC and NOESY correlations and of 3.

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