Terpenoids and phenethyl glucosides from Hyssopus cuspidatus (Labiatae)

Article abstract of DOI:10.1016/j.phytochem.2011.07.008

Monoterpenoids (3 and 4), sesquiterpenoid (2), diterpenoid (1) and four phenethyl glucosides (5-8), together with fourteen known compounds, were isolated from the whole herb of Hyssopus cuspidatus. Their structures were determined by spectroscopic means.

Full text of DOI:10.1016/j.phytochem.2011.07.008

Phytochemistry 72 (2011) 2244–2252
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Phytochemistry
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Terpenoids and phenethyl glucosides from Hyssopus cuspidatus (Labiatae)
Megumi Furukawa a, Mitsuko Makino b, Emika Ohkoshi a,1, Taketo Uchiyama a, Yasuo Fujimoto a,
⇑
a College of Pharmacy, Nihon University, 7-7-1 Narashinodai, Funabashi, Chiba 274-8555, Japan
b Department of General Education, College of Humanities and Sciences, Nihon University, 3-25-40 Sakurajousui, Setagaya, Tokyo 156-8550, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Monoterpenoids (3 and 4), sesquiterpenoid (2), diterpenoid (1) and four phenethyl glucosides (5–8),
together with fourteen known compounds, were isolated from the whole herb of Hyssopus cuspidatus.
Their structures were determined by spectroscopic means. The abietane-type diterpenoids (1, 9, 10), ros-
marinic acid (15) and salvigenin (17) inhibited leukotriene (LT) C4 secretion from primary alveolar cells of
Wistar rats.
Received 14 March 2011
Received in revised form 15 June 2011
Accepted 8 July 2011
Available online 3 September 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Hyssopus cuspidatus
Labiatae
Abietane-type diterpenoids
Eremophilane-type sesquiterpenoid
Phenethyl glycosides
Leukotriene
1. Introduction
et al., 1998), ursolic acid (19) (Tundisa et al., 2002), 2a-hydroxyur-
solic acid (20) (Murakami et al., 1993), 2 -hydroxyoleanolic acid
a
Hyssopus cuspidatus (Labiatae) is a folk medicine for treatment
of fever and bronchus asthma in Xinjiang province, China.
Although there are some reports on the constituents of this plant
(Ablizl et al., 2009; Xue et al., 1990), those studies mainly focused
on the essential oil of H. cuspidatus. In our biological studies on an
alcohol extract of the whole herb of this plant, the extract was
found to inhibit leukotriene (LT) C4 secretion from primary alveolar
cells of Wistar rats. In this paper, the isolation and structure eluci-
dation are described of a new abietane-type diterpenoid (1), an
eremophilane-type sesquiterpenoid (2), a pinane-type monoterpe-
noid (3), a menthane-type monoterpenoid (4) and four phenyleth-
anoids (5–8) along with fourteen known compounds, 19, 20-
epoxy-12-methoxy-11, 14, 19-trihydroxy-7-oxo-8, 11, 13-abiet-
atriene (9) (Araújo et al., 2005), 11, 14-dihydroxy-12-methoxy-7-
oxo-8, 11, 13-abietatrien-19–20b-olide (10) (Araújo et al., 2005),
10-hydroxycarvone (11) (Hamada et al., 1997), pinonic acid (12)
(Fernández et al., 1993), loliolide (13) (Fernández et al., 1993), des-
acetylmatricarin (14) (Wong and Brown, 2002), rosmarinic acid
(15) (Dapkevicius et al., 2002), coulterone (16) (Frontana et al.,
1994), salvigenin (17) (Hòrie et al., 1998), genkwanin (18) (Hòrie
(21) (Murakami et al., 1993), hyptadienic acid (22) (Taniguchi
et al., 2002). The effect of the isolated compounds on the secretion
of LTC4 from primary alveolar cells of Wistar rats is also described.
2. Results and discussion
2.1. Structure elucidation
Compound 1 was obtained as a yellow amorphous powder. In
the positive-ion HR EI-MS, a molecular ion peak at m/z 390.2035
(calcd 390.2042) [M]+, was observed which is consistent with a
molecular formula C22H30O6. The 1H NMR and 1H–1H correlation
spectroscopy (COSY) spectra exhibited the presence of a tertiary
methyl group [d 0.97 (3H, s)], an isopropyl group [d 1.38 (6H, d,
J = 6.9 Hz), d 3.31 (1H, sep, J = 6.9 Hz)], two methoxyl groups [d
3.30, 3.78 (3H each, s)], a trimethylene (C-1–C-3) and a methine-
methylene (C-5–C-6) sequences, and two hydroxyl proton signals
[d 5.76, 12.0 (1H each, s)] which were exchanged by adding D2O.
(Fig. 2 and Table 1). Analysis of the 13C and 13C-1H COSY spectra
indicates the presence of carbonyl (d 205.4), acetal [dH 4.28 (1H,
s), dC 105.4], and oxymethylene [d 3.94 (1H, dd, J = 11.7, 2.1 Hz),
d 4.36 (1H, d, J = 11.7 Hz), dC 59.6] groups as well as benzene ring
(d 113.2, 127.0, 128.3, 139.8, 152.0, 157.0). The substitution pat-
tern of the benzene ring was investigated by analysis of the heter-
onuclear multiple bond correlation (HMBC) spectrum of 1. The
methine proton signal (d 3.31) of the isopropyl group exhibited
the long-range correlations with the carbon resonances at d
⇑
Corresponding author. Present address: Department of General Education,
College of Humanities and Sciences, Nihon University, 3-25-40 Sakurajousui,
Setagaya, Tokyo 156-8550, Japan. Tel.: +81 3 5317 9365; fax: +81 3 5317 9433.
E-mail address: fujim-y@chs.nihon-u.ac.jp (Y. Fujimoto).
1
Present address: Natural Products Research Laboratories, Eshelman School of
Pharmacy, University of North Carolina, Chapel Hill, NC 27599, United States.
0031-9422/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2011.07.008

M. Furukawa et al. / Phytochemistry 72 (2011) 2244–2252
2245
Fig. 1. Structures of Compounds 1–22.
127.0 (C-13), d 157.0 (C-14) and d 152.0 (C-12) which have corre-
lations with a methoxyl proton (d 3.30) and as a hydroxyl proton (d
5.76), signals suggesting the presence of a methoxyl group at C-12
and a hydroxyl group at C-11. The chemical shift value of the C-14
resonance (d 157.0) and the low field shift of a hydroxyl proton sig-
nal (d 12.0) indicated the presence of a hydrogen bonded-hydroxyl
group at C-14 and the C-7 should be a carbonyl carbon (d 205.4).
This was further confirmed by the observation of long-range corre-
lations between the hydroxyl proton (d 12.0) and C-14 (d 157.0)
resonances besides the C-8 signal (d 113.2). The connectivity of
the other functional groups was also deduced by analysis of the
HMBC spectrum. As shown in Fig. 2, long-range correlations were

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M. Furukawa et al. / Phytochemistry 72 (2011) 2244–2252
Fig. 3. NOESY Correlations for Compound 1.
Fig. 2. Key HMBC Correlations for Compound 1.
long-range coupling were observed between the following proton
and carbon signals: H-1 and C-5, C-9, C-10; H-3 and C-4, C-5; H-
15 and C-4, C-5; H-14 and C-4, C-5, C-6, C-10; H-12 and C-7, C-
13, H-13 and C-7; H-7 and C-9; H-8 and C-9, suggesting the struc-
ture of 2 was as represented. Its relative stereochemistry was
determined as shown in Fig. 4 by observation of the following
observed between the following proton and carbon signals: H-18
and C-3, C-4, C-5; H-19 and C-18, C-20; H-20 and C-1, C-10; H-5
and C-4, C-10; H-6 and C-7, C-8; HO-11 and C-9, C-11, C-12; H-
15 and C-12, C-13, C-14; HO-14 and C-13, C-14, C-8. Thus, the
structure of 1 was confirmed as shown in Fig. 2. The relative ste-
reochemistry of 1 was assigned by analysis of the NOESY spectrum.
NOESY correlations: H-15 and H-3, H-14; H-14 and H-7, H-6
a
and H-4, H-12. The absolute configuration of 2 was determined
by application of new Mosher’s method (Ohtani et al.,1991). The
signals due to H-4, 14, and 15 of the (S)- a-methoxy-a-(trifluoro-
methyl) phenylacetyl (MTPA) ester of 2 appeared at higher fields
than those of (R)-MTPA ester, while the H-1 signal of (S)-MTPA es-
ter appeared at a lower field than that of (R)-MTPA ester, thereby
indicating the stereochemistry at C-3 to be R-configuration. Thus,
the absolute configuration of 2 could be assigned as 3R, 4R, 5R,
7R (Fig. 5).
The H-5 signal showed correlations with the H-1a, H-3a and H-18
signals, and the H-6b signal exhibited the correlations with H-19
and H-20 signals. Thus, the relative stereostructure of 1 was con-
firmed as shown in Fig. 3.
Compound 2 was obtained as a colorless oil. In the positive-ion
HR EI-MS, a molecular ion peak at m/z 234.1616 (calcd 234.1620)
[M]+, consistent with a molecular formula C15H22O2 was observed.
The 1H NMR spectrum of 2 showed the presence of a tertiary
methyl [d 0.98 (3H, s)], secondary methyl [d 1.08 (3H, d,
J = 6.5 Hz)], vinyl methyl [d 1.76 (3H, brs)], oxymethine [d 3.61
(1H, m)], and exomethylene [d 4.76, 4.78 (1H each, brs)] groups
and a vinyl proton [d 6.45 (1H, s)] (Table 2). The 13C NMR spectrum
exhibited fifteen signals due to three methyl carbons (d 11.5, 20.8,
26.1), an oxymethine carbon (d 69.2), four sp2 carbons (d 110.7,
134.0, 145.3, 148.9), and a carbonyl carbon (d 205.4) (Table 2).
These spectroscopic data and the unsaturation degrees indicated
that compound 2 should be a bicyclic sesquiterpene. Analysis of
the 1H-1H COSY spectrum of 2 indicates the sequences of the C-
1–C-15 through C-4 and the C-6–C-8 besides the presence of an
isopropenyl group. The connectivity of these partial structures
and the functional groups were investigated by analysis of HMBC
spectrum. As shown in Fig. 4, the 13C–1H correlations due to the
Compound 3 was obtained as a pale yellow oil. In the positive-
ion HR EI-MS, a molecular ion peak at m/z 184.1099 (calcd.
184.1099) [M]+, which is consistent with a molecular formula
C
10H16O3, was observed. Analysis of the 1H and 1H–1H COSY spec-
tra indicates the presence of a geminal dimethyl [d 0.90, 1.38 (3H
each, s)] and an oxymethylene [d 3.37, 4.07 (1H each, d,
J = 12.0 Hz)] group, and a sequence of CH-1–CH2-7–CH-5–CH2-4
(Table 2). The 13C NMR spectra including DEPT showed 10 signals
due to geminal dimethyl group [d 22.6 (CH3), 26.8 (CH3), 39.3 (C),
two methylene groups (d 27.8 and 43.5), two methine groups (d
38.1 and 46.7), an oxymethylene group (d 67.7) and an oxygenated
quaternary carbon (d 77.1) and a carbonyl carbon (d 216.2). These
spectroscopic data suggested that compound 3 should be a bicyclic
monoterpenoid. The connectivity of these partial structures and
Table 1
1H and 13C NMR Spectroscopic Data for Compound 1 (600 MHz, in CDCl3).
Position
1H (J, Hz)
13C
Position
1H (J, Hz)
13C
1
1b
a
1.33 (1H, ddd, 13.8, 3.4, 2.1)
3.42 (1H, dd, 13.8, 6.2)
1.62a
34.8
11
12
13
14
15
16
17
18
19
20
139.8
152.0
127.0
157.0
26.1
20.3
20.3
23.3
105.4
59.6
2a
21.7
39.5
2b
2.50 (1H, m)
3a
1.40 (1H, m)
3.31b(1H, sep, 6.9)
1.38 (3H, d, 6.9)
1.38 (3H, d, 6.9)
0.97 (3H, s)
3b
4
5
1.62a
36.4
44.3
37.4
1.88 (1H, dd, 15.8, 2.7)
3.27b
2.67 (1H, s, 15.8, 2.7)
6a
4.28 (1H, s)
6b
7
8
9
3.94 (1H, dd, 11.7, 2.1)
4.36 (1H, d, 11.7)
3.30 (3H, s)
3.78 (3H, s)
5.76 (1H, s)
205.4
113.2
128.3
38.8
OCH3
OCH3
OH
54.8
62.1
10
OH
12.0 (1H, s)
The values in parentheses represent the coupling constants in Hz. The d values are in ppm downfield from TMS.
a
show overlapping of the signals.
show overlapping of the signals.
b

M. Furukawa et al. / Phytochemistry 72 (2011) 2244–2252
2247
Table 2
1H- and 13C-NMR Spectroscopic Data for Compound 2–4 (in CDCl3).
Compound 2
Compound 3
1H (J, Hz)
Compound 4
1H (J, Hz)
Position
1H (J, Hz)
13C
13C
13C
1
2
6.45 (1H, s)
2.59 (1H, m)
2.14 (1H, m)
3.61 (1H, m)
134.0
36.3
2.07(1H, t, 6.1)
46.7
77.1
134.4
68.5
4.03 (1H, m)
3
69.2
216.2
1.63 (1H, m)
1.96 (1H, m)
2.43 (1H, m)
2.18 (1H, m)
1.89 (1H, m)
5.59 (1H, m)
31.5
4
5
a1.57 (1H, m)
47.5
39.4
2.60 (2H, s)
2.08 (1H, m)
43.5
38.1
31.2
37.0
6
2.00 (1H, m)
a1.57 (1H, m)
2.36 (1H, m)
43.0
2.60 (2H, s)
39.3
27.8
22.6
26.8
67.7
125.2
20.8
7
40.4
1.82 (1H,d, 11.0)
2.49 (1H, m)
0.90 (3H, s)
1.81 (3H, br s)
8
2.34 (1H, m)
2.43 (1H, m)
44.1
152.7
108.7
65.2
9
205.4
145.3
1.38 (3H, s)
4.92 (1H, br s)
5.09 (1H, br s)
4.16 (2H, m)
10
3.37 (1H, d, 12.0)
4.07 (1H, d, 12.0)
11
12
13
148.9
20.8
110.7
1.76 (3H, br s)
4.76 (s)
4.78 (s)
14
15
0.98 (3H, s)
1.08 (3H, d, 6.5)
26.1
11.5
The values in parentheses represent the coupling constants in Hz. The d values are in ppm downfield from TMS.
a
shows overlapping of signals.
functional groups were investigated by analysis of the 13C–1H long-
range correlations. The HMBC spectrum of 3 exhibited cross-peaks
between the following proton and carbon signals: H-10 and C-2, C-
3; H-4 and C-3; H-8 and C-1, C-6; H-9 and C-5, C-6. In addition, the
NOESY spectrum showed a correlation between H-8 and H-10 sig-
nals. Thus, the structure of 3 was defined as shown in Fig. 6.
Compound 4 was obtained as a colorless oil. In the positive-ion
HR EI-MS, a molecular ion peak at m/z 168.1157 (calcd. 168.1150)
[M]+ was observed indicator of molecular formula C10H16O2. The
1H NMR spectrum showed the presence of vinyl methyl [d 1.81
(3H, br s)], oxymethylene [d 4.16 (2H, m)], oxymethine [d 4.03
(1H, m)], and exomethylene [d 4.92, 5.09 (1H each, br s)] groups,
as well as a vinyl proton [d 5.59 (1H, m)] (Table 2). Detailed anal-
ysis of 1H–1H COSY spectra suggested connectivity from C-2 to C-6
and C-9 to C-10 through C-8. Furthermore, the linkages between C-
1 and C-2, and C-4 and C-8 were deduced by observation of the
long-range correlations between following proton and carbon sig-
nals: H-7 and C-1, C-2, C-6; H-6 and C-1, C-2; H-2 and C-1; H-4 and
Fig. 4. Key HMBC and Difference NOE Correlations for Compound 2.
Fig. 5. Absolute Configuration for Compound 2.

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M. Furukawa et al. / Phytochemistry 72 (2011) 2244–2252
C-8; H-9 and C-4, C-10. The relative stereochemistry of 4 was
determined by difference NOE experiments. Irradiation of the H-
10 resonances at d 4.16 produced a significant enhancement of
the H-2 signal at d 4.03. Thus, the relative stereostructure of 4
was confirmed as shown in Fig. 7. The absolute configuration of
4 was confirmed by application of the new Mosher’s method. The
resonance due to H-7 of the 4-(R)-MTPA ester appeared at a higher
field than that of 4-(S)-MTPA ester, and H-3 and H-4 signals of 4-
(R)-MTPA ester appeared at lower fields than those of 4-(S)-MTPA
ester. Thus, the absolute configuration of 4 could be assigned as 2S,
4R (Fig. 8).
Compound 5 was obtained as a pale yellow oil. In the negative-
ion HR FAB-MS, a quasi-molecular ion peak at m/z 479.1552 (calcd.
479.1553) [M–H]ꢀ, consistent with a molecular formula C23H27O11
was observed. The 1H–1H and 13C–1H COSY spectra indicated pres-
ence of a p-hydroxyphenethyl group [A] [d 3.69 and 3.91 (1H each,
m, H-1), d 2.80 (2H, m, H-2), d 6.65 (2H, d, J = 8.5 Hz, H-30and H-50),
d 6.95 (2H, d, J = 8.5 Hz, H-20and H-60)] and 30,50-O-dimethylgalloyl
group [B] [d 3.78 (6H, s, OCH3 ꢁ 2), d 7.32 (2H, br s, H-2000and H-6000),
dc 168.0 (COO) and a sugar moiety (see Tables 3 and 4)]. The con-
nectivity of these partial structures was investigated by analysis of
the HMBC spectrum. The H-100 (anomeric proton) and the H-600 sig-
nals of the sugar moiety exhibited cross-peaks with the carbon sig-
nals at d 72.4 (C-1) and at d 168.0 (COO), respectively, as well as the
long-range correlations shown in Fig. 9. The sugar was identified as
Fig. 7. Key HMBC and Difference NOE Correlations for Compound 4.
D-glucose by both TLC and gas chromatographic analysis of its tri-
methylsilyl thiazolidine derivative after alkaline and acid hydroly-
sis of 5 (see Experimental). Thus, structure 5 was confirmed as
shown in Fig. 5.
Compound 6 was obtained as a pale yellow oil. In the negative-
ion HR FAB-MS, a quasi-molecular ion peak at m/z 495.1500 (calcd.
495.1502) [MꢀH]ꢀ, which is consistent with a molecular formula
C
23H27O12 was observed. The 1H and 13C NMR spectra were very
similar to those of 5 except for the presence of a 3,4-dihydroxy-
phenethyl group [d 3.53 (2H, m, H-1), d 2.44 (2H, m, H-2), d 6.92
(1H, d, J = 2.0 Hz, H-20), d 6.74 (1H, d, J = 8.0 Hz, H-50), and d 6.75
(1H, dd, J = 2.0, 8.0 Hz, H-60)] in place of the p-hydroxyphenethyl
group. Thus, structure 6 was as shown in Fig. 1.
Fig. 8. Absolute Configuration for Compound 4.
495.1502) [MꢀH]ꢀ, indicative of molecular formula C23H27O12. The
1H and 13C NMR spectra were similar to those of 7 except for the
presence of 1,2,4-trisubstituted benzene ring [d 6.91 (1H, d,
J = 2.0 Hz, H-30), d 6.74 (1H, dd, J = 2.0, 8.0 Hz, H-60), d 6.74 (1H, d,
J = 8.0 Hz, H-50)] in place of 1,4-disubstituted benzene ring. Thus,
structure 8 was as shown in Fig. 1.
Compound 7 was obtained as a pale yellow oil. In negative-ion
HR FAB-MS, a quasi-molecular ion peak at m/z 479.1554 (calcd
479.1553) [MꢀH]ꢀ, which is consistent with a molecular formula
C
23H27O11 was observed. The 1H and 13C NMR spectra of 7 showed
close similarity to those of 5. The HMBC spectrum of 7 exhibited a
cross-peak between the anomeric proton signal (d 4.84) and an
oxygenated aromatic carbon resonance (d 157.4, C-40), suggesting
the glycosidic linkage with a phenolic hydroxy group. Thus, the
structure of 7 was represented as shown in Fig. 1.
2.2. Assay
Compound 8 was obtained as a pale yellow oil. In the negative-
ion HR FAB-MS, a quasi-molecular ion peak at m/z 495.1496 (calcd.
Metabolites of arachidonate such as LTC4, LTD4 and LTB4, that
are released from mast cells by an allergic reaction and from
Fig. 6. Key HMBC and NOESY Correlations for Compound 3.

M. Furukawa et al. / Phytochemistry 72 (2011) 2244–2252
2249
Table 3
1H-NMR Spectroscopic Data for Compound 5–8 (in MeOH-d4).
1H
Compound 5
Compound 6
Compound 7
Compound 8
Aglycone
1
2
20
30
50
60
3.69–3.91 (2H, m)
2.80 (2H, m)
6.95 (d, 8.5)
6.65 (d, 8.5)
6.65 (d, 8.5)
6.95 (d, 8.5)
3.53 (2H, m)
2.44 (2H, m)
6.92 (d, 2.0)
3.65 (2H, t, 7.1)
2.69 (2H, t, 7.1)
6.93 (s)
6.93 (s)
6.93 (s)
3.51 (2H, m)
2.74 (2H, m)
6.91 (d, 2.0)
6.74 (dd, 8.0, 2.0)
6.74 (d, 8.0)
6.74 (d, 8.0)
6.75 (dd, 8.0, 2.0)
6.93 (s)
Glucose
100
4.35 (d, 8.0)
3.22 (t, 8.0)
3.34–3.42 (2H, m)
4.77 (d, 7.8)
3.34–3.70 (4H, m)
4.84 (d, 7.3)
3.35–3.56 (4H, m)
4.77 (d, 6.1)
3.45–3.56 (4H, m)
200
300
400
500
3.62 (m)
4.38 (dd, 11.7, 6.8)
4.67 (dd, 11.7, 2.2)
600
4.43 (dd, 12.0, 6.0)
4.72 (dd, 12.0, 2.2)
4.42 (dd, 12.0, 7.8)
4.71 (dd, 12.0, 2.2)
4.48 (dd, 11.9, 6.7)
4.72 (dd, 11.9, 2.1)
Galloyl
2000, 6’’’
OCH3
7.32 (2H, br s)
3.78 (6H, s)
7.34 (2H, br s)
3.86 (6H, s)
7.34 (2H, br s)
3.85 (6H, s)
7.34 (2H, br s)
3.86 (6H, s)
The Spectroscopic data for 5–7 were measured at 400 MHz and those for 8 were taken at 500 MHz.
Table 4
13C NMR Spectroscopic Data for Compound 5–8 (in MeOH-d4).
13C
5
6
7
8
13C
5
6
7
8
Aglycone
Galloyl
1
2
72.4
36.5
70.1
36.3
64.2
39.4
64.1
39.2
1000
121.2
108.3
150.0
142.3
56.8
121.2
108.5
148.9
142.2
57.0
121.3
108.6
149.0
142.3
57.0
121.2
108.5
148.9
142.2
57.0
2000, 6000
3000, 5000
4000
1’
2’
3’
4’
5’
6’
130.5
130.8
116.1
156.8
116.1
130.8
132.2
119.9
146.4
146.9
118.0
125.8
157.4
117.8
130.7
157.4
130.7
117.8
132.1
119.9
146.3
146.8
117.0
125.5
OCH3
COO
168.0
167.9
167.8
167.9
Glucose
1’’
2’’
3’’
4’’
104.6
75.1
78.0
72.2
75.4
65.3
104.5
75.8
77.4
71.9
74.8
65.1
102.5
75.5
77.9
72.2
74.9
65.2
104.5
75.8
77.4
71.9
74.8
65.1
5’’
6’’
The Spectroscopic data for 5–7 were measured at 100 MHz and those for 8 were taken at 125 MHz.
Fig. 9. Key HMBC and NOESY Correlations for Compound 5.
eosinophils by inflammatory stimulations, are important in the
pathogenesis of bronchial asthma. LTC4 and LTD4 constrict bron-
chial smooth muscle, stimulate mucus secretion from bronchial
glands, and increase the permeability of airway vasculature. So the
inhibition of LTC4 secretion from primary alveolar cells of Wistar
rats was next investigated. As shown in Table 5, among the isolated
compounds, the abietane-type diterpenoids (1, 9, 10), rosmarinic
acid (15) and salvigenin (17) showed inhibition of LTC4 secretion
to a similar extent or more potently than the ketotifen used as a
positive control (Table 5). These results suggest that the phenolic

2250
M. Furukawa et al. / Phytochemistry 72 (2011) 2244–2252
Table 5
30 °C. The combine ethanol extract was concentrated under re-
Inhibitory Effects of Isolated Compounds on Release of LTC4.
duced pressure to give an oily material (81.6 g) which was applied
to a Diaion HP-20 resin column, and then eluted successively with
MeOH–H2O (4:6, Fr. A), (7:3, Fr. B), MeOH (Fr. C) and acetone (Fr.
D). Each fraction was concentrated in vacuo to give Fr.A (22.5 g),
Fr.B (5.4 g), Fr.C (28.9 g), and Fr.D (18.9 g). Only Fr.B and Fr.C
showed inhibitory activity on secretion of LTC4 at a concentration
Compounds
IC50 (
l
M)
Compounds
IC50 (lM)
1
2
3
4
5
6
7
8
42.9
12
13
14
15
16
17
18
19
>100
>100
92.5
13.6
>100
>100
>100
>100
>100
>100
>100
39.9
a
-
of 100 lg/ml, in the bio-assay guided fractionation Fr. C was tritu-
39.9
76.0
rated with MeOH and the insoluble material was collected and
recrystallized from EtOAc to give 19 (6100 mg). The mother liquor
was concentrated to leave an oily material which was subjected to
silica gel CC, this being eluted successively with hexane–EtOAc
(5:1, 3.6 l, Fr. C-1–C-3), (3:1, 2.0 l, Fr.C-4), (2:1, 1.0 l, Fr.C-5), (1:1,
2.4 l, Fr.C-6), EtOAc (2.0 l, Fr.C-7) and MeOH (2.0 l, Fr.C-8) to give
eight fractions. Fr. C-3 was fractionated by reversed phase HPLC
{Kaseisorb LC PH SUPER, 20 ꢁ 250 mm (column A), MeOH–H2O
(75:25), flow rate 8 ml minꢀ1} to afford four fractions (Fr.C-3–1-
C-3–4). Fr.C-3–3 was purified by reversed phase HPLC {Kaseisorb
LC ODS PH SUPER, 10 ꢁ 250 mm (column B), MeOH–H2O (9:1),
flow rate 3 ml minꢀ1} to yield 16 (38 mg). Fr.C-4 was fractionated
by reversed phase HPLC {column A, MeOH-H2O (8:2), flow rate
8 ml minꢀ1} to afford four fractions (Fr. C-4–1–C-4–4). Fr.C-4–1
was further separated by reversed phase HPLC {column B,
MeOH–H2O (85:15), flow rate 3 ml minꢀ1} to give 1 (12 mg) and
10 (12 mg). Purification of Fr.C-4–2 using reversed phase HPLC
{column B, MeOH–H2O (85:15), flow rate 3 ml minꢀ1} to give 9
(75 mg). Separation of Fr. C-5 by reversed phase HPLC {column A,
MeOH–H2O (8:2), flow rate 8 ml minꢀ1} gave fourteen fractions
(Fr.C-5–1–C-5–14). Purification of Fr. C-5–3 by reversed phase
b
-
b
9
10
11
20
21
Ketotifen
-
b
23.3
>100
-
46.8
a
not done.
b
Compounds (19, 20 and 21) exhibit cytotoxicity at the concentration of >10 lM.
compounds (1, 9, 10 and 15) having 1,4- or 1,2-dihydroxybenzene
moieties and flavones (17 and 18) exhibit potent inhibition of re-
lease of LTC4.
2.3. Conclusions
The constituents of H. cuspidatus have been investigated by sev-
eral Chinese research groups (Ablizl et al., 2009; Xue et al., 1990).
However, those studies have mainly focused on the essential oil of
this plant. In the present studies, two monoterpenoids (3 and 4), a
sesquiterpenoid (2), a diterpenoid (1) and four phenethyl gluco-
sides (5–8), together with fourteen known compounds, were iso-
lated from the whole herb of H. cuspidatus. The effect of the
isolated compounds on the secretion of LTC4 from primary alveolar
cells of Wistar ratss is also investigated. The phenolic compounds
(1, 9, 10 and 15) having 1,4- or 1,2-dihydroxybenzene moieties
and flavones (17 and 18) potently inhibited release of LTC4 from
primary alveolar cells of Wistar rats.
HPLC {column B, MeOH–H2O (75:25), flow rate 3 ml minꢀ1
}
yielded 2 (15 mg). Fr. C-5–4 was purified by reversed phase HPLC
{column B, MeOH–H2O (75:25), flow rate 3 ml minꢀ1} to give 18
(4 mg). Compound 17 (7 mg) was obtained from Fr.C-5–6 by puri-
fication using reversed phase HPLC {column B (MeOH–H2O (8:2),
flow rate 3 ml minꢀ1}. Fr.C-5–10 was purified by reversed phase
HPLC {Senshu Pak PEGASIL ODS, 10
u
ꢁ 250 mm (column C),
MeOH–H2O (9:1), flow rate 3 ml minꢀ1} to give 22 (16 mg). Com-
pounds 20 (47 mg) and 21 (29 mg) were obtained from Fr. C-5–
12 and Fr. C-5–11, respectively, by purification using reversed
phase HPLC {column C, MeOH-H2O (9:1), flow rate 3 ml minꢀ1}.
Fr. B (5.4 g) was subjected to silica gel CC, this being eluted, succes-
sively with CHCl3–MeOH–H2O {100:1:0 (300 ml, Fr. B-1), 10:1:0.05
(1650 ml, Fr. B-2), 8:2:0.2 (2500 ml, Fr. B-3), 7:3:0.3 (1000 ml, Fr.
B-4, B-5), 6:4:0.5 (1000 ml, Fr. B-6), 6:4:1 (2000 ml, Fr. B-7) and
acetone (Fr. B-8)} to give eight fractions. Fr.B-1 was further frac-
tionated by silica gel CC {toluene–acetone 20:1 (500 ml, Fr.B-1–
1), 10:1 (550 ml, Fr.B-1–2), 5:1 (1020 ml, Fr.B-1–3), 3:1 (600 ml,
Fr.B-1–4), 2:1 (600 ml), 1:1 (600 ml), acetone (800 ml) Fr.B-1–5,
MeOH (1000 ml, Fr.B-1–6)} to yield six fractions. Separation of
Fr.B-1–4 by reversed phase HPLC {column A, MeOH–H2O (4:6),
flow rate 3 ml minꢀ1} afforded twelve fractions (Fr.B-1–4-1-B-1–
4-12). Concentration of Fr.B-1–4-5 gave 3 (37 mg). Further separa-
tion of Fr.B-1–4-6 by reversed phase HPLC {column B, CH3CN–H2O
(3:7), flow rate 3 ml minꢀ1} gave 13 (7 mg). Fr.B-1–4-7 was further
purified by reversed phase HPLC (column B, CH3CN–H2O (32:68),
flow rate 3 ml minꢀ1) to give 11 (5 mg). Compound 12 (17 mg)
was obtained from Fr. B-1–4-8 by purification using reversed phase
HPLC {column B, CH3CN–H2O (3:7), flow rate 3 ml minꢀ1}. Separa-
tion of Fr.B-1–4-9 by reversed phase HPLC {column B, CH3CN–H2O
(25:75), flow rate 3 ml minꢀ1} yielded 14 (9 mg). Fr.B-1–4-11 was
further purified by reversed phase HPLC {column B, CH3CN–H2O
(3:7), flow rate 3 ml minꢀ1} to give 4 (6 mg). Fr.B-3 was fraction-
3. Experimental
3.1. General
1H and 13C NMR spectra were measured on a JEOL JNM lambda-
400, 500 spectrometer or JEOL ECA-600 in CDCl3 or MeOH-d4
containing TMS as an internal standard, with MS spectra being
recorded on a HITACHI M-2000 instrument. IR spectra were
acquired on a JASCO IR A-2. Column chromatography (cc) was car-
ried out on silica gel (Wakogel C-200) and Diaion HP-20 (Nippon
Rensui). HPLC was conducted with a Spectra Physics SP 8800 and
Sensyu SSC-3160 pump equipped with either ERC-7520 (ERMA)-
RI or HITACHI L-400-UV detector. Silica gel 60 F254 (Merck) pre-
coated TLC plates were used and the detection was carried out
by spraying 5% H2SO4 solution followed by heating.
3.2. Plant material
H. cuspidatus was collected in Xinjiang province, China and
identified by Mr. Shen Jin Gui, Institute of Materia Medica, Shang-
hai Institutes for Biological Sciences, Chinese Academy of Sciences.
A voucher specimen has been deposited in the above Institute and
the Laboratory of Medicinal Chemistry, College of Pharmacy, Nihon
University. (voucher No. HC-001, Laboratory of Medicinal
Chemistry).
3.3. Extraction and isolation
ated by reversed phase HPLC {CAPCELL PAK UG, 20
u
ꢁ 250 mm
(column D), MeOH–H2O (45:55), flow rate 8 ml minꢀ1} to afford
four fractions (Fr.B-3–1–B-3–4). Separation of Fr.B-3–2 by reversed
Dried whole herb of H. cuspidatus (1.5 kg) was extracted with
EtOH of (6.0 l ꢁ 4) under ultrasonication for each 30 min at 20–
phase HPLC {column A, CH3CN–H2O (15:85), flow rate 3 ml minꢀ1
}

M. Furukawa et al. / Phytochemistry 72 (2011) 2244–2252
2251
gave 7 (10 mg) and 8 (3 mg). Compounds 5 (50 mg) and 6 (30 mg)
were obtained by separation of Fr. B-3–3 using reversed phase
HPLC {column B, MeOH–H2O (1:1), flow rate 3 ml minꢀ1}. Fr.B-5
was further fractionated by reversed phase HPLC {column A,
MeOH–H2O (4:6), flow rate 8 ml minꢀ1} to afford five fractions
(Fr.B-5–1–B-5–5). Compound 15 (15 mg) was isolated from Fr.B-
5–1 by purification using reversed phase HPLC {column B,
CH3CN–H2O (1:9), flow rate 3 ml minꢀ1}.
tinued overnight at room temperature. The mixture was diluted
with EtOAc (1 ml) and then washed successively with brine and
H2O, dried (Na2SO4) and then concentrated in vacuo. 1H NMR
(500 MHz): Hz 410.55 (H-15), 476.40 (H-14), 756.70 (H-4),
857.75 (H-12), 2417.20 (H-13), 3283.25 (H-1).
3.13. (R)-(+)-MTPA Ester of 2
The reaction was carried out in the similar manner as above. 1H
NMR (500 MHz): Hz 420.40 (H-15), 483.40 (H-14), 759.60 (H-4),
866.10 (H-12), 2424.40 (H-13), 3264.70 (H-1).
3.4. Compound 1
Yellow amorphous powder; HR-EI-MS [M]+ m/z 390.2035
(calcd. 390.2042 for C22H30O6); [
a]
D +95.2 (MeOH, c 0.25); UV kmax
3.14. (S)-(-)-MTPA Ester of 4
(MeOH) nm (e): 216 (4900), 240 (3800), 277 (3600), 368 (2200);
For 1H and 13C NMR spectroscopic data, see Table 1.
3.5. Compound 2
The reaction was carried out in a similar manner as described in
the preparation of MTPA ester of 2. 1H NMR (500 MHz): Hz 855.45
(H-7), 902.75 (H-3), 961.60 (H-5) 1021.70 (H-3), 1029.35 (H-5),
1160.20 (H-4), 2818.30 (H-6).
Colorless oil; HR-EI-MS [M]+ m/z 234.1616 (calcd. 234.1620 for
C15H22O2); [
a]
D +21.5 (MeOH, c 1.0); UV kmax (MeOH) nm (
e
): 241
3.15. (R)-(+)-MTPA Ester of 4
(3800): For 1H and 13C NMR spectroscopic data, see Table 2.
The reaction was carried out in the similar manner as above. 1H
NMR (500 MHz): Hz 854.85 (H-7), 911.90 (H-3), 959.15 (H-5)
1024.45 (H-3), 1029.05 (H-5), 1160.50 (H-4), 2818.30 (H-6).
3.6. Compound 3
Pale yellow oil; HR-EI-MS [M]+ m/z 184.1099 (calcd. 184.1099
for C10H16O3); [
D +21.5 (MeOH, c 2.0); For 1H and 13C NMR spec-
a
]
3.16. Absolute configuration of sugar in compound 5, 6, 7 and 8 (Hara
troscopic data, see Table 2.
et al., 1987)
3.7. Compound 4
Each glycoside (1 mg) was dissolved in 2% NaOMe/MeOH (1 ml)
at room temperature. After 1 h, the reaction mixture was neutral-
ized by passing each through an ion-exchange resion (Amberlite
IRA 400, H+ form) column. These were then heated in 1 M HCl
(2 ml) at 90 °C for 3 h. After cooling, each reaction mixture was
neutralized by individually passing each through an ion-exchange
resin (Amberlite IRA 400, OHꢀ form) column. Each filtrate was then
transferred to a Sep-Pak C18 cartridge and eluted with H2O and
MeOH, and the H2O eluated were individuals concentrated and
Colorless oil; HR-EI-MS [M]+ m/z 168.1157 (calcd. 168.1150 for
C
10H16O2); [
a
]
D ꢀ7.7 (MeOH, c 0.5); For 1H and 13C NMR spectro-
scopic data, see Table 2.
3.8. Compound 5
Pale yellow oil; HR-FAB-MS [MꢀH]ꢀ m/z 479.1552 (calcd.
479.1553 for C23H27O11); [
a
]
D ꢀ9.3 (MeOH, c 1.0); UV kmax (MeOH)
the residued were treated with
ride (2 mg) in pyridine (0.25 ml) at 60 °C for 1 h. After the reaction,
the solution was treated with TMS-HT (100 l, hexamethyldisilaz-
L-cysteine methyl ester hydrochlo-
nm (e
): 220 (11100), 278 (7 1 0 0); For 1H and 13C NMR spectro-
scopic data, see Tables 3 and 4.
l
ane and trimethylchlorosilamine in pyridine, TOKYO KASEI Co., To-
3.9. Compound 6
kyo, Japan) at 40 °C for 10 min. The reaction mixture was then
subjected to GLC analysis to identify the derivatives of
from 5, 6, 7 and 8. GLC conditions: column, ULBON HR-1,
m; detector, FID; injector tempera-
ture, 250 °C; detector temperature, 280 °C; column temperature,
250 °C for 0.5 min and then 1.5 °C/min up to 270 °C; He carrier,
D-glucose
Pale yellow oil; HR-FAB-MS [MꢀH]ꢀ m/z 495.1500 (calcd.
495.1502 for C23H27O12); [
a]
D ꢀ56.2 (MeOH, c 1.0); UV kmax
25 m ꢁ 0.25 mm (i.d.), 0.25
l
(MeOH) nm (
e
): 278 (8000), 379 (7700); For 1H and 13C NMR spec-
troscopic data, see Tables 3 and 4.
24 cm/s; D-glucose tR, 6.83 min, respectively.
3.10. Compound 7
3.17. Measurement of LTC4 secretion (Suzuki et al., 2001)
Pale yellow oil; HR-FAB-MS [MꢀH]ꢀ m/z 479.1554 (calcd.
479.1553 for C23H27O11); [
a
]
D ꢀ38.1 (MeOH, c 1.4); UV kmax
Wistar rats (6–10 weeks, female) were euthanized by decapita-
tion and the pulmonary cavities were opened. After severing the
descending aorta, the blood in the lungs was cleared by perfusion
through the right heart with PBS (5 ml) containing of heparin 50 U/
ml until the lungs became whitish. Using an 18-gauge needle, the
trachea was cannulated, and of heparin/PBS (1 ml) was slowly in-
jected into the lungs and then withdrawn. This procedure was re-
peated 7–10 times, and a total of 5–8 ml of lavage fluid was
collected.
(MeOH) nm (e
): 278 (8000), 379 (7700); For 1H and 13C NMR spec-
troscopic data, see Tables 3 and 4.
3.11. Compound 8
Pale yellow oil; HR-FAB-MS [MꢀH]ꢀ m/z 495.1496 (calcd.
495.1502 for C23H27O12); [
a
]
D ꢀ33.8 (MeOH, c 0.12); UV kmax
(MeOH) nm (e
): 278 (8000), 379 (7700); For 1H and 13C NMR spec-
troscopic data, see Tables 3 and 4.
To obtain alveolar cell populations, lungs were perfused with
heparin solution as above. Thereafter, they were aseptically re-
moved and cut into small pieces. The dissected tissue was incu-
bated in Dullbecco’s modified Eagle’s MEM (D-MEM) containing
1% penicillin–streptomycin (Cellgro) and 10% FBS, and digested
lungs were further disrupted by gently pushing the tissue through
3.12. (S)-(ꢀ)-MTPA Ester of 2 (I. Ohtani et al., 1991)
To a stirred solution of 2 (1 mg) in pyridine (2 drops) was added
one drop (large excess) of (R)-(ꢀ)-MTPA–Cl and stirring was con-

2252
M. Furukawa et al. / Phytochemistry 72 (2011) 2244–2252
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centrifuged at 500g. Cells were washed and resuspended
2 ꢁ 105 cells/ml of D-MEM, and plated on a 24-well plate for
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500 ll/well incubated at 37 °C for 2 h. These cells were washed
with PBS, and incubated in D-MEM containing 20 mM Hepes,
pH 7.4 (Hepes-D-MEM) and test sample at 37 °C for 30 min and
the cells were stimulated with calcium ionophore A23187 in
Hepes-D-MEM, and incubated at 37 °C for 30 min.
LTC4 content in supernatants was determined by LTC4 Enzyme
immunoassay (EIA) kit (Cayman Chemical, Ann Arbor, MI) accord-
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