Influenza A (H1N1) antiviral and cytotoxic agents from Ferula assa-foetida

Article abstract of DOI:10.1021/np900158f

Two new sesquiterpene coumarins, designated 5′-acetoxy-8′- hydroxyumbelliprenin (1) and 10′R-acetoxy-11′-hydroxyumbelliprenin (2), and a new diterpene, 15-hydroxy-6-en-dehydroabietic acid (3), along with 27 known compounds, were isolated from a CHCl₃

Full text of DOI:10.1021/np900158f

1568
J. Nat. Prod. 2009, 72, 1568–1572
Influenza A (H1N1) Antiviral and Cytotoxic Agents from Ferula assa-foetida
Chia-Lin Lee,† Lien-Chai Chiang,‡ Li-Hung Cheng,† Chih-Chuang Liaw,§ Mohamed H. Abd El-Razek,⊥ Fang-Rong Chang,*,†
and Yang-Chang Wu*,†
Graduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical UniVersity, Kaohsiung 807, Taiwan, Republic of China,
Department of Microbiology, College of Medicine, Kaohsiung Medical UniVersity, Kaohsiung 807, Taiwan, Republic of China, Graduate
Institute of Pharmaceutical Chemistry, China Medical UniVersity, Taichung 404, Taiwan, Republic of China, and Chemistry of Natural
Products Department, National Research Center, Cairo, 12622, Egypt
ReceiVed March 9, 2009
Two new sesquiterpene coumarins, designated 5′-acetoxy-8′-hydroxyumbelliprenin (1) and 10′R-acetoxy-11′-hydroxy-
umbelliprenin (2), and a new diterpene, 15-hydroxy-6-en-dehydroabietic acid (3), along with 27 known compounds,
were isolated from a CHCl3-soluble extract of Ferula assa-foetida through bioassay-guided fractionation. The structures
of the new metabolites 1-3 were identified by spectroscopic data interpretation and by the Mosher ester method.
Compounds 4 and 6-13 showed greater potency against influenza A virus (H1N1) (IC50 0.26-0.86 µg/mL) than
amantadine (IC50 0.92 µg/mL), and 11 exhibited the best potency (IC50 0.51, 2.6, and 3.4 µg/mL) of these compounds
against the HepG2, Hep3B, and MCF-7 cancer cell lines, respectively.
Influenza occurs with seasonal variations and reaches peak
prevalence in winter, with many people killed worldwide every
year. Until now, only a few organic compounds including aman-
tadine, rimantadine, and ribavirin have been used for influenza
therapy. However, drug-resistant influenza viruses are generated
quickly. During the course of an anti-influenza and cytotoxicity
screening program on natural products, it was found that a CHCl3
extract of Ferula assa-foetida was active against H1N1 influenza
virus (IC50 < 3.4 µg/mL) and various human cancer cell lines (IC50
< 20 µg/mL). Therefore, the plant was selected as a lead to be
investigated.
F. assa-foetida L. (Apiaceae) is distributed mainly in Iran,
Afghanistan, and mainland China. Nearly a hundred years ago, the
roots of this plant were an important remedy for the well-known
“Spanish Flu” (type A influenza, H1N1 subtype), which led to
20-100 million people dying at that time.1 The roots of F. assa-
foetida have been used for their perceived anthelmintic, anticar-
cinogenesis, anti-HIV, antimicrobial, antirheumatic, antispasmodic,
diuretic, and emmenagogue actions in folk medicine.2-7 There are
236 natural products, including 129 sesquiterpene coumarins, seven
monoterpene coumarins, 49 daucane sesquiterpenes, nine polysul-
fide derivatives, 10 sesquiterpene phenylpropanoids, 21 simple
sesquiterpenes, six simple monoterpenes, and five oleanane triter-
penes, reported from the genus Ferula to date. While F. assa-foetida
has had extensive use in folk medicine for the treatment of influenza,
it is interesting that the antiviral activities of the isolated compounds
from this plant have not been evaluated in a systematic manner.
Bioassay-directed chromatographic fractionation of F. assa-foetida
resin led to the isolation of 30 compounds, including 20 sesquit-
erpene coumarins, 5′-acetoxy-8′-hydroxyumbelliprenin (1), 10′R-
acetoxy-11′-hydroxyumbelliprenin (2), 5′S-hydroxyumbelliprenin
(4),8 10′R-karatavicinol (5),9 8′-acetoxy-5′S-hydroxyumbelliprenin
(6),8,9 methyl galbanate (7),8,10,11 galbanic acid (8),10,11 farnesiferol
C (9),10,12,13 farnesiferol A (10),12,13 farnesiferol B,12,13 assafoe-
tidin,14 lehmferin,15 conferol (11),16 feselol,17 ligupersin A (12),18
epi-conferdione (13),9 microlobin,11 lehmannolone,19 polyanthi-
nin,20 and kamolonol;21 two polysulfide derivatives, foetisulfide A
and foetisulfide C;22 a simple coumarin, umbelliferone; a benzal-
dehyde, vanillin; a drimane sesquiterpenoid, fetidone B;23 three
diterpenes, 7-oxocallitrisic acid, picealactone C, and 15-hydroxy-
6-en-dehydroabietic acid (3); a sterol, ꢀ-sitosterol; and a fatty acid,
oleic acid. Among them, two new sesquiterpene coumarins (1 and
2) and a new diterpene (3) were identified. Most of these compounds
were evaluated in influenza A (H1N1) antiviral and cytotoxicity
assays, and the results are described herein.
Results and Discussion
A MeOH extract of the resin of F. assa-foetida was partitioned
between n-hexane-MeOH (1:1), and then the MeOH layer was
partitioned between CHCl3-H2O (1:1) to obtain a CHCl3 extract,
which showed significant anti-H1N1 activity (IC50 < 3.4 µg/mL)
and cytotoxicity for three cancer cell lines (IC50 < 20 µg/mL). Initial
fractionation of the CHCl3 extract was carried out by open liquid
chromatography on silica gel to give 11 fractions. Chromatographic
fractionation of these active subfractions provided two new ses-
quiterpene coumarins (1 and 2), a new diterpene (3), and 27 known
compounds.
HRESIMS of 1 exhibited a [M + Na]+ ion at m/z 463.2099
(C26H32O6Na). The IR spectrum showed absorptions for hydroxy
(3440 cm-1), acetoxy (1729 cm-1), and aromatic (1613 and 1555
cm-1) functional groups. UV absorptions at 211 and 318 nm also
indicated a coumarin nucleus oxygenated at the C-7 position.12 In
the 13C NMR spectrum, compound 1 displayed 26 carbon signals,
with nine being typical for an umbelliferone skeleton [δ 101.5 (C-
8), 112.5 (C-10), 113.1 (C-3), 114.2 (C-6), 128.7 (C-5), 143.4 (C-
4), 155.8 (C-9), 161.2 (C-2), and 161.9 (C-7)] and the remaining
17 signals ascribable to a sesquiterpene moiety [δ 18.0 (C-13′),
13.1 (C-14′), 17.1 (C-15′), 25.9 (C-12′), 34.2 (C-9′), 44.9 (C-4′),
65.2 (C-1′), 68.9 (C-5′), 75.9 (C-8′), 119.5 (C-10′), 122.1 (C-2′),
123.3 (C-6′), 135.5 (C-11′), 137.6 (C-3′), 141.9 (C-7′)] with an
acetoxy group (δ 21.2 and 170.3). In the 1H NMR spectrum, signals
for two main moieties, a coumarin and a sesquiterpene, were
revealed. The coumarin moiety appeared as five signals [δΗ 6.24
and 7.63 (each 1H, d, J ) 9.4 Hz), 7.35 (1H, d, J ) 8.4 Hz), 6.82
(1H, dd, J ) 2.4, 8.4 Hz), 6.79 (1H, d, J ) 2.4 Hz)]. In turn, the
sesquiterpene moiety displayed signals for four methyls [δH 1.62,
(3H, brs), 1.71 (3H, brs), 1.72 (3H, brs), 1.81, (3H, s)], two
methylenes [δH 2.23 (2H, m), 2.28 (1H, d, J ) 6.0 Hz), 2.45 (1H,
dd, J ) 7.6, 13.6 Hz)], an oxygenated methylene [δH 4.57 (2H, d,
J ) 6.2 Hz)], three olefinic methines [δH 5.08 (1H, brt, J ) 6.4
Hz), 5.41 (1H, d, J ) 8.8 Hz), 5.50 (1H, brt, J ) 6.2 Hz)], and
two oxygenated methines [δH 3.99 (1H, brt, J ) 6.4 Hz), 5.70 (1H,
* To whom correspondence should be addressed. Tel.: +886-7-312-1101,
ext. 2197. Fax: +886-7-311-4773. E-mail: yachwu@kmu.edu.tw or
aaronfrc@kmu.edu.tw.
† Graduate Institute of Natural Products, Kaohsiung Medical University.
‡ Department of Microbiology, Kaohsiung Medical University.
§ China Medical University.
⊥ National Research Center, Cairo.
10.1021/np900158f CCC: $40.75  2009 American Chemical Society and American Society of Pharmacognosy
Published on Web 08/19/2009

AntiViral and Cytotoxic Agents from Ferula assa-foetida
Journal of Natural Products, 2009, Vol. 72, No. 9 1569
Figure 1. Important HMBC correlations for compounds 1 and 2.
Table 1. 1H and 13C NMR Data of Compounds 1 and 2 (400
and 100 MHz in CDCl3, δ in ppm, J in Hz)
1
2
position
δH (J in Hz)
δC
δH (J in Hz)
δC
2
3
4
5
6
7
8
9
10
1′
2′
3′
4′
161.2 (s)
161.3 (s)
112.9 (d)
143.5 (d)
128.7 (d)
113.2 (d)
162.1 (s)
101.5 (d)
155.8 (s)
112.4 (s)
65.5 (t)
6.24 (d, 9.4)
7.63 (d, 9.4)
7.35 (d, 8.4)
6.82 (dd, 8.4, 2.4)
113.1 (d) 6.23 (d, 9.4)
143.4 (d) 7.63 (d, 9.4)
128.7 (d) 7.35 (d, 8.4),
114.2 (d) 6.82 (dd, 8.4, 2.4)
161.9 (s)
101.5 (d) 6.79 (d, 2.4)
155.8 (s)
112.5 (s)
6.79 (d, 2.4)
4.57 (d, 6.2)
5.50 (brt, 6.2)
65.2 (t)
122.1 (d) 5.45 (brt, 6.4)
137.6 (s)
4.59 (d, 6.4)
118.4 (d)
142.2 (s)
2.45 (brdd, 13.6, 7.6) 44.9 (t)
2.28 (d, 6.0)
2.14-2.06 (2H, m) 39.3 (t)
5′
5.70 (m)
5.41 (d, 8.8)
68.9 (d)
123.3 (d) 5.09 (brt, 6.2)
141.9 (s)
75.9 (d)
34.2 (t)
2.14-2.06 (2H, m) 26.1 (t)
6′
124.2 (d)
134.5 (s)
36.0 (t)
7′
8′
3.99 (brt, 6.4)
2.23 (m)
5.08 (brt, 6.4)
1.93 (2H, m)
1.68 (2H, m)
9′
27.8 (t)
10′
11′
12′
13′
14′
15′
OAc
119.5 (d) 4.77 (dd, 10.0, 2.8) 79.5 (d)
135.5 (s)
25.9 (q)
18.0 (q)
13.1 (q)
17.1 (q)
21.2 (q)
170.3 (s)
72.4 (s)
24.8 (q)
26.7 (q)
16.0 (q)
16.7 (q)
21.1 (q)
171.2 (s)
1.62 (brs)
1.71 (brs)
1.72 (brs)
1.81 (s)
1.17 (brs)
1.18 (brs)
1.58 (brs)
1.74 (brs)
1.99 (s) (C-5′)
2.10 (s) (C-10′)
of the coumarin moiety via an ether linkage, and the acetoxy and
hydroxy groups were positioned at C-10′ (δ 79.5) and C-11′ (δ
72.4), respectively. Compound 2 was elucidated as 10′-acetoxy-
11′-hydroxyumbelliprenin.24
m)]. Compound 1 gave the same molecular formula, C26H32O6, and
similar 1D NMR data to compound 6, 8′-acetoxy-5′-hydroxyum-
belliprenin,12 with the only difference being due to the placement
of an acetoxy group and a hydroxy group. The key HMBC
correlations suggested that the acetoxy and hydroxy groups could
be positioned at C-5′ and C-8′, respectively (Figure 1). The HMBC
correlation of an oxygenated methylene at δH 4.57 (H-1′) with a
carbon signal at δC 161.9 (C-7) indicated that the sesquiterpene
unit is attached to C-7 of the coumarin moiety via an ether linkage.
The absolute configuration at C-8′ was not determined due to the
small amount of 1 available and also because no previous literature
values could be used for comparison. The new compound 1 was
therefore assigned as 5′-acetoxy-8′-hydroxyumbelliprenin.
To determine the only chiral center at C-10′, we speculated that
2 and 10′-karatavicinol (5) possess the same biogenetic origin.
Consequently, 5 was treated separately with (R)- and (S)-R-
methoxy-R-(trifluoromethyl)phenylacetyl chloride [(R)- and (S)-
MTPA-Cl] in the presence of C5D5N, to yield the (S)- and (R)-
MTPA esters (5a and 5b), respectively.25 The MTPA esters were
1
generated successfully at C-10′ as elucidated from the H NMR
spectra (5a, H-8′ δ 2.15, H-9′ δ 2.15, H-10′ δ 5.49, H-12′ δ 1.37,
H-13′ δ 1.70; 5b, H-8′ δ 2.13, H-9′ δ 2.13, H-10′ δ 5.50, H-12′ δ
1.43, H-13′ δ 1.71). The differences between the 1H NMR chemical
shifts for 5a and 5b (∆ values shown in Figure 3) led to the
assignment of the R-configuration at C-10′ of 5. From a comparison
of the specific rotation data of 5 ([R]25 +16.2) and 2 ([R]25
A [M + H]+ ion at m/z 443.2433 (C26H35O6) was present in the
HRFABMS of 2. IR absorptions at 3480, 1731, and (1555 and 1613)
cm-1 supported the presence of hydroxy, acetoxy, and aromatic
ring functions, respectively. UV absorptions at 213 and 321 nm
indicated a coumarin nucleus oxygenated at the C-7 position.12 In
the 1D NMR spectrum (Table 1), the signals indicated a carbon
skeleton with two main moieties, a coumarin and a sesquiterpene.
On the basis of HMBC correlations [δH 4.59 (H-1′)/δC 162.1 (C-
7), 118.4 (C-2′), and 142.2 (C-3′); δH 4.77 (H-10′)/δC 171.2
(OCOCH3); δH 1.17 (H-12′)/δC 79.5 (C-10′), 72.4 (C-11′), and 26.7
(C-13′); δH 1.18 (H-13′)/δC 79.5 (C-10′), 72.4 (C-11′), and 24.8
(C-12′)], the sesquiterpene unit could be attached to C-7 (δ 162.1)
D
D
+11.2), the absolute configuration of 2 at C-10′ could therefore be
assigned with an R-configuration. Compound 2 was established as
10′R-acetoxy-11′-hydroxyumbelliprenin.
Compound 3 gave the molecular formula C20H26O3, as deter-
mined by HRESIMS (m/z 337.1784 [M + Na]+), indicating eight
degrees of unsaturation. Its IR spectrum showed absorptions
attributable to hydroxy (3417 cm-1), carboxylic acid (1696 cm-1),
and aromatic ring (1613 and 1514 cm-1) functions. On the basis
of its 13C NMR and DEPT data, 3 showed 20 carbon signals,

1570 Journal of Natural Products, 2009, Vol. 72, No. 9
Lee et al.
Table 3. Influenza A (H1N1) Antiviral Activity for Compounds
2 and 4-13
compound
IC50 (µg/mL)
IC90 (µg/mL)
2
4
5
6
7
8
9
10
0.94 ( 0.07
0.36 ( 0.03
0.99 ( 0.07
0.81 ( 0.06
0.26 ( 0.03
0.45 ( 0.04
0.29 ( 0.02
0.51 ( 0.04
0.47 ( 0.05
0.86 ( 0.08
0.32 ( 0.02
0.92 ( 0.04
1.98 ( 0.12
0.62 ( 0.08
2.05 ( 0.14
1.56 ( 0.11
0.44 ( 0.05
0.88 ( 0.07
0.50 ( 0.07
0.64 ( 0.07
0.83 ( 0.09
1.75 ( 0.12
0.37 ( 0.03
1.73 ( 0.11
11
12
13
Figure 2. Important HMBC correlations for compound 3.
amantadine
H-5/Me-19 and H-5/Me-20 indicated ꢀ- and R-orientations, re-
spectively, for Me-20 and H-5 (trans A/B ring junction). Compound
3 was therefore elucidated as 15-hydroxy-6-en-dehydroabietic acid.
To the best of our knowledge, this is the first report of a diterpene
with an abietane skeleton from the genus Ferula.
On the basis of the work of Nittala et al.,26 the chiral center of
the known 5′-hydroxyumbelliprenin (4) is similar to that of 12,13-
didehydrofurospongin-1. The absolute configuration, the S form,
of 12,13-didehydrofurospongin-1 was determined by the Mosher
method. From a comparison of their specific rotation data {12,13-
didehydrofurospongin-1 ([R]28D -8); 4 ([R]25D -3.8)}, the absolute
configuration of 4 at C-5′ was assigned the S form. Compound 4 is
accordingly 5′S- hydroxyumbelliprenin.
1
Figure 3. H NMR chemical shift differences [∆δ ) δ H(S)-MTPA
- δ H(R)-MTPA (ppm)] of the MTPA esters of 5 and 6.
The absolute configuration of the known 8′-acetoxy-5′-hydroxy-
umbelliprenin (6)12 at C-5′ was still unknown at the time of the
present study. Compound 6 was treated separately with (R)- and
(S)-MTPA-Cl in C5D5N to yield the (S)- and (R)-MTPA esters (6a
and 6b), respectively. The MTPA esters were generated successfully
Table 2. 1H and 13C NMR Data of Compound 3 (400 and 100
MHz in CDCl3, δ in ppm, J in Hz)
position
δH (J in Hz)
δC
1
2
3
2.21 (brd, 11.2)
1.81 (m)
1.81 (m)
35.2 (t)
18.3 (t)
35.6 (t)
1
at C-5′, as elucidated from the H NMR spectra (6a, H-2′ δ 5.58,
H-4′a δ 2.37, H-4′b δ 2.31, H-5′ δ 6.20, H-6′ δ 5.70, H-8′ δ 5.35,
H-14′ δ 1.93, H-15′ δ 1.72; 6b, H-2′ δ 5.69, H-4′a δ 2.41, H-4′b
δ 2.35, H-5′ δ 6.13, H-6′ δ 5.49, H-8′ δ 5.30, H-14′ δ 1.79, H-15′
δ 1.94). The differences between the 1H NMR chemical shifts for
6a and 6b (∆ values shown in Figure 3) led to the assignment of
the S-configuration at C-5′, and 6 was determined as 8′-acetoxy-
5′S-hydroxyumbelliprenin.
1.76 (m)
4
46.0 (s)
5
6
7
8
2.91 (t, 2.8)
5.81 (dd, 9.6, 2.7)
6.55 (dd, 9.6, 2.9)
46.3 (d)
130.0 (d)
128.3 (d)
132.4 (s)
145.9 (s)
37.1 (s)
121.6 (d)
123.7 (d)
146.6 (s)
122.8 (d)
72.4 (s)
31.6 (q)
31.6 (q)
183.5 (s)
17.7 (q)
20.7 (q)
9
10
11
12
13
14
15
16
17
18
19
20
Sesquiterpene coumarin ethers (7-hydroxycoumarin analogues)
have been reported as possessing various biological activies,
including potent NF-κB inhibition [8′-acetoxy-5′S-hydroxyumbel-
liprenin (6)],22 antibacterial activity [galbanic acid (8)],27 a potential
cancer chemopreventive effect [farnesiferol C (9)],28 squalene-
hopene cyclase inhibition [karatavicinol (5) and farnesiferol C (9)],29
and human rhinovirus coat protein inhibition [karatavicinol (5) and
farnesiferol B].30 Since 7-hydroxycoumarin analogues showed good
anti-HIV activity in previous studies,3,22 the pure 7-O-sesquiterpene
coumarins, compounds 2 and 4-6 (acyclic), 7-9 (monocyclic),
and 10-13 (bicyclic), were screened in an in vitro anti-influenza
A viral (H1N1) assay, with amantadine as a positive control (Table
3). Most of these compounds exhibited a higher antiviral potency
than amantadine, except for 2, 5, and 12. These results are of interest
because the structures of many of these compounds are quite similar.
For example, 6 (OH-5′, OAc-8′) has an additional acetoxy group
in comparison with 4 (OH-5′), but 6 was much less potent than 4.
Compound 2 (OAc-10′) has the same skeleton as 5 (OH-10′), and
these two compounds showed a similar potency to the positive
control, amantadine. Between compounds 7 (COOCH3-3′) and 8
(COOH-3′), 7 showed better potency than 8 and, therefore, indicated
that methyl esterification of C-3′ enhanced the activity in the
bioassay used. For the bicyclic-sesquiterpene coumarins, 12 and
13, a C-3′-carbonyl afforded greater potency than a C-3′-OH in
this kind of skeleton. Overall, the present study has determined
that sesquiterpene coumarins from F. assa-foetida may serve as
7.13 (d, 8.4)
7.30 (dd, 8.4, 2.0)
7.17 (d, 2.0)
1.57 (brs)
1.57 (brs)
1.40 (s)
1.08 (s)
including four methyls (δC 17.7, 20.7, 31.6, 31.6), three methylenes
(δC 18.3, 35.2, 35.6), six methines (δC 46.3, 121.6, 122.8, 123.7,
128.3, 130.0), and seven quaternary (δC 37.1, 46.0, 72.4, 132.4,
145.9, 146.6, 183.5) carbons (Table 2). Among the seven quaternary
carbons, one was assigned as a carbonyl carbon at δ 183.5.
Therefore, the data supported the presence of one carbonyl, four
olefins, and three ring moieties to fulfill the eight degrees of apparent
unsaturation, and 3 was postulated to be an abietane-type diter-
pene.26 The HMBC correlations [δH 1.57 (H-16)/δC 72.4 (C-15),
31.6 (C-17), and 146.6 (C-13); δH 1.57 (H-17)/δC 72.4 (C-15), 31.6
(C-16), and 146.6 (C-13); δH 1.40 (H-19)/δC 35.6 (C-3), 46.0 (C-
4), 46.3 (C-5), and 183.5 (C-18)] suggested the hydroxy and
carboxylic groups to be located at C-15 (δ 72.4) and C-4 (δ 46.0),
respectively (Figure 2). The NOESY correlation between Me-19
and Me-20 as well as the absence of NOESY correlations between

AntiViral and Cytotoxic Agents from Ferula assa-foetida
Journal of Natural Products, 2009, Vol. 72, No. 9 1571
was purified by preparative TLC (CHCl3) to give 4 (5.7 mg) and
lehmferin (2.8 mg). Subfraction 4-6-5 (400 mg) was chromatographed
on a silica gel column (70-230 mesh, column: 2.5 × 28 cm), using
n-hexane-CH2Cl2 (2:7) as eluent, and subfraction 4-6-5-1 (253.30 mg)
was purified subsequently with n-hexane-EtOAc (7:2), to obtain seven
subfractions. Subfractions 4-6-5-1-2 (9.5 mg), 4-6-5-1-3 (68.7 mg), and
4-6-5-1-6 (21.9 mg) were purified by RP-HPLC (flow rate: 3.0 mL/
min) to give fetidone B (2.2 mg, MeOH-H2O, 70:30, tR ) 14 min), a
farnesiferol B and assafoetidin mixture (16.4 mg, MeOH-H2O, 63:
37, tR ) 123 min), feselol (3.3 mg, MeOH-H2O, 63:37, tR ) 136
min), and 1 (2.3 mg, MeOH-H2O 70:30, tR ) 34 min), respectively.
Subfractions 4-6-5-2 (43.4 mg) and 4-6-7 (354.7 mg) were purified by
RP-HPLC (MeOH-H2O, 76:24; flow rate: 3.0 mL/min) to give 10 (8.9
mg, tR ) 38 min) and 11 (6.2 mg, tR ) 41 min), and 7 (58.9 mg, tR )
27 min), 8 (15.7 mg, tR ) 31 min), and 7-oxocallitrisic acid (1.5 mg,
tR ) 53 min), respectively. Subfraction 4-7 (12.40 g) was separated by
silica gel chromatography (70-230 mesh, column: 5 × 25 cm;
CHCl3-MeOH, 60:1) into six subfractions. Subfraction 4-7-4 (746.80
mg) was subjected to silica gel chromatography (70-230 mesh, column:
3 × 20 cm; n-hexane-EtOAc, 5:2) and purified by RP-HPLC
(MeOH-H2O, 75:25; flow rate: 3.0 mL/min) to give 2 (19.7 mg, tR )
24 min), 6 (57.0 mg, tR ) 34 min), and 13 (11.6 mg, tR ) 12 min).
The CHCl3/MeOH-insoluble umbelliferone (3.4 mg) was obtained from
subfraction 4-8-7 (58.2 mg).
Fraction 5 (11.77 g) was chromatographed over silica gel (70-230
mesh, column: 5 × 30 cm; CHCl3-MeOH, 50:1), with subfraction
5-2 (334.30 mg) then subjected to RP-MPLC (MeOH-H2O, 65:35),
to give five subfractions. Subfraction 5-2-1 was purified by RP-HPLC
(MeOH-H2O, 61:39; flow rate: 3.0 mL/min) to give foetisulfide A
(1.5 mg, tR ) 43 min). Subfraction 5-3 (255.2 mg) was subjected to
RP-MPLC (MeOH-H2O, 70:30) and subfraction 5-3-3 (112.3 mg) was
purified by silica gel chromatography (70-230 mesh, column: 2.5 ×
30 cm; CH2Cl2-MeOH, 40:1) to obtain kamolonol (14.3 mg). Sub-
fraction 5-3-4 (351.4 mg) was chromatographed by RP-HPLC
(MeOH-H2O, 65:35; flow rate: 3.0 mL/min) to give microlobin (30.4
mg, tR ) 73 min), and then the mother liquor was purified by RP-
HPLC (MeOH-H2O, 53:47; flow rate: 3.0 mL/min) to give 12 (21.3
mg, tR ) 64 min) and foetisulfide C (3.6 mg, tR ) 50 min). Subfractions
5-3-5 and 5-3-6 were chromatographed over silica gel (70-230 mesh,
column: 1.5 × 25 cm; CH2Cl2-MeOH, 60:1 and 50:1) to give
picealactone C (0.7 mg) and 5 (241.7 mg). Subfraction 5-5 (352.3 mg)
was chromatographed by RP-HPLC (MeOH-H2O, 72:28; flow rate:
3.0 mL/min) to give 3 (7.0 mg, tR ) 16 min).
promising lead compounds for new drug development against
influenza A (H1N1) viral infection. A standardized plant extract of
F. assa-foetida, a species that has been used to treat influenza for
many years, may also be worthy of being further investigated as a
new phytomedicine.
Furthermore, the pure compounds were screened in a cytotoxicity
assay with doxorubicin as the positive control. The minor compound
11 exhibited the best potency (IC50 0.51, 2.6, and 3.4 µg/mL) against
HepG2, Hep3B, and MCF-7 tested cancer cell lines, respectively.
Interestingly, compound 11 also showed high potency influenza
activity (IC50 0.47 ( 0.05 µg/mL). The remaining compounds were
all inactive for all cancer cell lines (IC50 > 4 µg/mL; Table S1,
Supporting Information).
Experimental Section
General Experimental Procedures. Optical rotations were taken
on a JASCO-P-1020 polarimeter (cell length 10 mm). UV spectra were
measured on a JASCO V-530 UV/vis spectrophotometer. IR spectra
were recorded on a Mattson Genesis II FT-IR spectrophotometer. NMR
spectra were recorded on Varian Gemini-20000 (200 MHz), Varian
Unity-plus (400 MHz), and Varian Unity-plus (600 MHz) FT-NMR
NMR spectrometers. Chemical shift (δ) values are in ppm (parts per
million), with CDCl3 as internal standard, and coupling constants (J)
are in Hz. HRFABMS, HRESIMS, and ESIMS measurements were
performed on JEOL JMS-700, Bruker APEX II, and Finnigan PO-
LARISQ mass spectrometers. TLC was performed on Kieselgel 60,
F
254 (0.20 mm, Merck), and spots were viewed under UV light at 254
and 356 nm and/or stained by spraying with 50% H2SO4 and heating
on a hot plate. For column chromatography, silica gel (Kieselgel 60,
70-230 and 230-400 mesh, Merck) and Sephadex LH-20 were used.
The instrumentation for the RP-MPLC experiment was composed of a
Supelco VersaFlash flash chromatography apparatus and VersaFlash
C-18 cartridges (40 × 150 mm). Further purification of some
compounds obtained was achieved by preparative HPLC, using a
Shimadzu LC-10ATVp/Shimadzu SCL-10AVp UV-vis detector, and
Thermo columns (analytical: 5 µm, 250 × 4.6 mm; preparative: 8 µm,
250 × 10 mm; C18) were used. For the preparation of Mosher ester
derivatives, (S)-(+)- and (R)-(-)-R-methoxy-R-(trifluoromethyl)phe-
nylacetyl chloride were used as the reagents.
Plant Material. Ferula assa-foetida resin (3.23 kg) was purchased
from a Chinese herb shop in Taipei, Taiwan, in November 2006 and
identified by one of the authors (M.H.A.R.). A voucher specimen
(FA200611) was deposited at the Graduate Institute of Natural Products,
Kaohsiung Medical University, Kaohsiung, Taiwan.
Extraction and Isolation. F. assa-foetida resin (3.23 kg) was
extracted four times with MeOH (5 L each) at room temperature to
obtain a crude extract (313.8 g). The crude extract was partitioned
between n-hexane-MeOH (1:1), and then the MeOH extract (120.1
g) was partitioned between CHCl3-H2O (1:1), to obtain a CHCl3 extract
(65.2 g), which showed significant anti-H1N1 antiviral and cytotoxic
activities. Initial fractionation of the CHCl3 extract (65.2 g) was carried
out by open column chromatography on silica gel (230-400 mesh,
column: 7 × 30 cm), using gradients of n-hexane-CHCl3-MeOH (100:
0:0 to 0:80:20), and gave 11 fractions. A precipitation was obtained
from the first chromatographic step, using n-hexane as the eluent, and
was washed with a trace amount of n-hexane to afford oleic acid
(2.0 g).
Fraction 3 (6.66 g) was fractionated into 15 fractions by silica gel
chromatography (70-230 mesh, column: 5 × 23 cm; CHCl3). Sub-
fraction 3-6 (602.06 mg) was subjected to silica chromatography
(70-230 mesh, column: 3 × 20 cm; n-hexane-EtOAc, 1:1) and
purified by preparative TLC (CHCl3) to give 9 (1.9 mg). Subfraction
3-7 (619.90 mg) was purified by solid-phase extraction (SPE) (DSC-
18, 60 mL, 10GMS, No. 52609-U; MeOH-H2O, 80:20) to obtain
vanillin (23.5 mg).
Fraction 6 (4.33 g) was chromatographed over silica gel (70-230
mesh, column: 7 × 35 cm; CH2Cl2-MeOH, 25:1) to give five
subfractions. The third subfraction was purified with RP-MPLC
(MeOH-H2O, 55:45) to give polyanthinin (23.4 mg).
5′-Acetoxy-8′-hydroxyumbelliprenin (1): yellow oil; [R]25 +8.6
D
(c 0.12, CHCl3); UV (MeOH) λmax (log ε) 211 (4.54), 318 (3.95) nm;
1
IR (neat) νmax 3440, 1729, 1613, 1555, 1233, 836 cm-1; H and 13C
NMR (CDCl3, 400 MHz), see Table 1; ESIMS m/z 463 [M + Na]+;
HRESIMS m/z 463.2099 [M + Na]+(calcd for C26H32O6Na, 463.2096).
10′R-Acetoxy-11′-hydroxyumbelliprenin (2): yellow oil; [R]25
D
+11.2 (c 0.5, CHCl3); UV (MeOH) λmax (log ε) 213 (4.77), 321 (4.39)
1
nm; IR (neat) νmax 3480, 1731, 1613, 1555, 1234, 836 cm-1; H and
13C NMR (CDCl3, 400 MHz), see Table 1; ESIMS m/z 465 [M + Na]+;
HRFABMS m/z 443.2433 [M + H]+ (calcd for C26H34O6 + H,
443.2434).
15-Hydroxy-6-en-dehydroabietic acid (3): yellow oil; [R]25D -14.6
(c 0.16, CHCl3); UV (MeOH) λmax (log ε) 243 (3.95), 271 (3.74) nm;
IR (neat) νmax 3417, 1696, 1613, 1514 cm-1; 1H and 13C NMR (CDCl3,
400 MHz), see Table 2; ESIMS m/z 337 [M + Na]+; HRESIMS m/z
337.1784 [M + Na]+ (calcd for C20H26O3Na, 337.1780).
Anti-influenza A Virus (H1N1) Bioassay.31,32 Madin-Darby canine
kidney (MDCK) cells (ATCC CCL34) were used as target cells for
viral infection in the XTT (tetrazolium hydroxide salt) assay. They were
grown as adherent cells in MEM medium supplemented with 10% fetal
calf serum (FCS), 100 U/mL penicillin G, 100 µg/mL streptomycin,
and 0.25 µg/mL amphotericin B. In the antiviral assay, the medium
was supplemented with 2% FCS and the above-mentioned antibiotics.
Virus titers were determined by the cytopathic effect in MDCK cells
and expressed as 50% tissue culture infective dose (TCID50) values
per mL. All viruses were stored at -70 °C until use. The antiviral
activity against influenza A virus (H1N1) was evaluated by the XTT
method. MDCK cells, treated by trypsin, were seeded onto 96-well
Fraction 4 (11.78 g) was separated into eight subfractions by column
chromatography on silica gel (70-230 mesh, column: 7 × 24 cm;
n-hexane-EtOAc, 1:1), and crystalline ꢀ-sitosterol (0.30 g) was
obtained with MeOH. Subfraction 4-6 was subjected to silica gel
chromatography (70-230 mesh, column: 5 × 17 cm; CHCl3-MeOH,
60:1) to give six subfrations. Subfraction 4-6-2 was purified with HPLC
(ODS Thermo, 10 × 250 mm; MeOH-H2O, 78:22; flow rate: 3.0 mL/
min) to give lehmannolone (1.7 mg, tR ) 31 min). Subfraction 4-6-4

1572 Journal of Natural Products, 2009, Vol. 72, No. 9
Lee et al.
plates with a concentration of 1.0 × 105 cells per mL and a volume of
70 µL per well. After incubation at 35 °C with 5% CO2 for 24 h, 20
µL of test virus solution was added and incubated for another 1 h.
Different concentrations of test substances were then added to culture
wells in triplicate. Amantadine was used as a positive control. After
incubation at 35 °C with 5% CO2 for 3 days, XTT reagent was added
and incubated for 3 h. The viral inhibition rate (%) was calculated as
[100 - (OD492/OD690) × 100]%. The antiviral concentration of 50%
inhibition (IC50) was defined as the concentration achieving 50%
cytoprotection against virus infection.
Cytotoxicity Bioassays. Fractions and isolates were tested against
lung (A549), breast (MEA-MB-231 and MCF7), liver (HepG2 and
Hep3B), and oral (Ca9-22) human cancer cell lines using an established
colorimetric MTT (diphenyltetrazolium bromide) assay protocol.33
Doxorubicin was used as a positive control. In brief, freshly trypsinized
cell suspensions were seeded in 96-well microtiter plates at densities
of 5000-10 000 cells per well with test compounds added from a
DMSO stock solution. After 3 days in culture, the attached cells were
incubated with MTT (0.5 mg/mL, 1 h) and subsequently solubilized in
DMSO. The absorbance was measured at 550 nm using an ELISA
reader. The IC50 is the concentration of agent that reduced cell growth
by 50%, under the experimental conditions used.
(9) Nabiev, A. A.; Khasanov, T. K.; Malikov, V. M. Khim. Prir. Soedin.
1982, 578–581; Chem. Abstr. 1983, 98, 86223.
(10) Yang, J. R.; An, Z.; Jing, S.; Qin, H. L. Chem. Pharm. Bull. 2006,
54, 1595–1598.
(11) Bizhanova, K.; Saidkhodzhaev, A. I. Khim. Prir. Soedin. 1978, 265–
267; Chem. Abstr. 1978, 89, 56443.
(12) Kuliev, Z. A.; Khasanov, T. K.; Malikov, V. M. Khim. Prir. Soedin.
1982, 120–121; Chem. Abstr. 1982, 96, 177967.
(13) Abd E1-Razek, M. H.; Wu, Y. C.; Chang, F. R. C. J. Chin. Chem.
Soc. 2007, 54, 235–238.
(14) Caglioti, L.; Naef, H.; Arigoni, D.; Jeper, O. HelV. Chem. Acta 1959,
42, 2557–2570.
(15) Sagitdinova, G. V.; Saidkhodzhaev, A. I.; Malikov, V. M. Khim. Prir.
Soedin. 1983, 709–712; Chem. Abstr. 1984, 100, 171545.
(16) Bagirov, V. Yu.; Gasanova, R. Yu.; Burma, O. I.; Ban’kovskii, A. I.
Khim. Prir. Soedin. 1977, 279–280; Chem. Abstr. 1977, 87, 81275.
(17) Nabiev, A. A.; Khasanov, T. Kh.; Melibaev, S. Khim. Prir. Soedin.
1978, 517–518; Chem. Abstr. 1978, 89, 211931.
(18) Nabiev, A. A.; Malikov, V. M. Khim. Prir. Soedin. 1983, 700–704;
Chem. Abstr. 1984, 100, 171543.
(19) Appendino, G.; Maxia, L.; Bascope, M.; Houghton, P. J.; Sanchez-
Duffhues, G.; Munoz, E.; Sterner, O. J. Nat. Prod 2006, 69, 1101–
1104.
(20) Vandyshev, V. V.; Sklyar, Yu. E.; Perel’son, M. E.; Moroz, M. D.;
Pimenov, M. G. Khim. Prir. Soedin. 1972, 670–671; Chem. Abstr.
1973, 78, 108265.
(21) Duan, H.; Takaishi, Y.; Tori, M.; Takaoka, S.; Honda, G.; Ito, M.;
Takeda, Y.; Kodzhimatov, O. K.; Kodzhimatov, K.; Ashurmetov, O.
J. Nat. Prod. 2002, 65, 1667–1669.
Acknowledgment. This work was supported by grants from the
National Science Council and the Committee on Chinese Medicine and
Pharmacy, Department of Health, Executive Yuan, Taiwan, awarded
to Y.-C.W. and F.-R.C.
(22) Banerji, A.; Mallick, B.; Chatterjee, A.; Budzikiewicz, H.; Breuer,
M. Tetrahedron Lett. 1988, 29, 1557–1560.
(23) Caglioti, L.; Naef, H.; Arigoni, D.; Jeper, O. HelV. Chem. Acta 1958,
41, 2278–2292.
(24) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am. Chem.
Soc. 1991, 113, 4092–4096.
(25) Kobayashi, M.; Chavakula, R.; Murata, O.; Sarma, N. S. Chem. Pharm.
Bull. 1992, 40, 599–601.
Supporting Information Available: 1H, 13C, HSQC, HMBC,
COSY, and NOESY spectra of compounds 1-3 along with cytotox-
icity data. This material is available free of charge via the Internet
at http://pubs.acs.org.
(26) Gigante, B.; Silva, A. M.; Marcelo-Curto, M. J.; Feio, S. S.; Roseiro,
References and Notes
J.; Reis, L. V. Planta Med. 2002, 68, 680–684.
(27) Shahverdi, A. R.; Fakhimi, A.; Zarrini, G.; Dehghan, G.; Iranshahi,
M. Biol. Pharm. Bull. 2007, 30, 1805–1807.
(1) Krebs, T. E. Bulletin of the NeVada State Board of Health; Carson
City, NV, 1920; No. 1, pp 7-9.
(2) Zhou, P.; Takaishi, Y.; Duan, H.; Chen, B.; Honda, G.; Itoh, M.;
Takeda, Y.; Kodzhimatov, O. K.; Lee, K. Phytochemistry 2000, 53,
689–697.
(3) Lee, T. T. Y.; Kashiwada, Y.; Huang, L.; Snider, J.; Cosentino, M.;
Lee, K. H. Bioorg. Med. Chem. 1994, 2, 1051–1056.
(4) Appendino, G.; Cravotto, G.; Sterner, O.; Ballero, M. J. Nat. Prod.
2001, 64, 393–395.
(5) Iranshahi, M.; Kalategi, F.; Rezaee, R.; Shahverdi, A. R.; Ito, C.;
Furukawa, H.; Tokuda, H.; Itoigawa, M. Planta Med. 2008, 74, 147–
150.
(6) Eigner, D.; Scholz, D. J. Ethnopharmacol. 1999, 67, 1–6.
(7) Banerji, A.; Malliok, B.; Chatterjee, A. Tetrahedron Lett. 1988, 29,
1557–1560.
(8) Appendino, G.; Tagliapietra, S.; Nano, G. M.; Jakupovic, J. Phy-
tochemistry 1994, 35, 183–186.
(28) Pillai, S. P.; Menon, S. R.; Mitscher, L. A.; Pillai, C. A.; Shankel,
D. M. J. Nat. Prod. 1999, 62, 1358–1362.
(29) Gravotto, G.; Balliano, G.; Robaldo, B.; Oliaro-Bosso, S.; Chimichi,
S.; Boccalini, M. Bioorg. Med. Chem. Lett. 2004, 14, 1931–1934.
(30) Rollinger, J. M.; Steindl, T. M.; Schuster, D.; Kirchmair, J.; Anrain,
K.; Ellmerer, E. P.; Langer, T.; Stuppner, H.; Schmidtke, M. J. Med.
Chem. 2008, 51, 842–851.
(31) Chiang, L. C.; Chiang, W.; Chang, M. Y.; Ng, L. T.; Lin, C. C.
AntiViral Res. 2002, 55, 53–62.
(32) Kodama, E.; Shigeta, S.; Suzuki, T.; De Clercq, E. AntiViral Res. 1996,
31, 159–164.
(33) Chan, M. L.; Chen, W. Y.; Tsai, C. Y.; Chang, F. R.; Wu, Y. C. Mol.
Cancer Ther. 2005, 4, 1277–1285.
NP900158F