Sesquiterpenes from Ainsliaea fragrans and their inhibitory activities against cyclooxygenases-1 and 2

Article abstract of DOI:10.1248/cpb.57.597

One new guaiane-type sesquiterpene glycoside, 2′-O-E-caffeoyl- 8α-hydroxy-11α,13-dihydro-3β-O-β-D-glucozaluzanin C (1), was isolated from the whole herb of Ainsliaea fragrans (Compositae), together with five known sesquiterpene lactones: 8α-hydroxy-11α,13

Full text of DOI:10.1248/cpb.57.597

June 2009
Notes
Chem. Pharm. Bull. 57(6) 597—599 (2009)
597
Sesquiterpenes from Ainsliaea fragrans and Their Inhibitory Activities
against Cyclooxygenases-1 and 2
b
c
b
a
d
Hao WANG,a,b Ting WU, Ming YAN, Ge LIU, Ping LI, Xiao-Qi ZHANG, Wen-Cai YE, ,b,d and
*
,c
*
Lu-Yong ZHANG
a Key Laboratory of Modern Chinese Medicine, China Pharmceutical University; b Department of Natural Medicinal
Chemistry, China Pharmceutical University; c National Drug Screening Laboratory, China Pharmceutical University;
Nanjing 210009, P. R. China: and d Guangdong Province Key Laboratory of Pharmcodynamic Constituents of TCM and
New Drugs Research, Jinan University; Guangzhou 510632, P. R. China.
Received September 29, 2008; accepted February 10, 2009; published online March 31, 2009
One new guaiane-type sesquiterpene glycoside, 2ꢀ-O-E-caffeoyl-8a-hydroxy-11a,13-dihydro-3b-O-b-D-glu-
cozaluzanin C (1), was isolated from the whole herb of Ainsliaea fragrans (Compositae), together with five known
sesquiterpene lactones: 8a-hydroxy-11a,13-dihydro-3b-O-b-D-glucozaluzanin C (2), 8a-hydroxy-11a,13-dihy-
drozaluzanin C (3), 3a-hydroxy-11b,13-dihydro-8a-O-b-D-glucozaluzanin C (4), 3b-hydroxy-11b,13-dihydro-8a-
O-b-D-glucozaluzanin C (5), 3b-O-b-D-glucozaluzanin C (6). The structures of isolated compounds were estab-
lished by means of 1D and 2D NMR spectroscopy and chemical methods. All isolates obtained in the present
study were evaluated for their inhibitory effects against cyclooxygenases-1 and 2 in vitro, and the structure–activ-
ity relationships were also discussed.
Key words Ainslieae fragrans; Compositae; sesquiterpene; cyclooxygenase-1; cyclooxygenase-2
The plant Ainsliaea fragrans champ belongs to the Com- CHCl3, and n-BuOH. The n-BuOH portion was chroma-
positae family and grows in the southeast of P. R. China. The tographed on silica gel, Sephadex LH-20, and C18 repeatedly
whole herb of this plant is widely used in traditional Chinese to afford compounds 1—6. The other five known compounds
medicine for the treatment of coughing up blood, jaundice were subsequently identified as: 8a-hydroxy-11a,13-dihy-
with damp-heat pathogen, edema, and superficial infection.1) dro-3b-O-b-D-glucozaluzanin C (2),8) 8a-hydroxy-11a,13-
Nowadays, the aqueous extract of A. fragrans is widely ap- dihydrozaluzanin C (3),5) 3a-hydroxy-11b,13-dihydro-8a-O-
plied in treating cervicitis, endometritis, and pelvic inflam- b-D-glucozaluzanin C (4),10) 3b-hydroxy-11b,13-dihydro-
mation, and its clinical effects are significant in China.2—4) 8a-O-b-D-glucozaluzanin C (5),11) 3b-O-b-D-glucozaluzanin
From the aerial parts of A. fragrans, sesquiterpene lactones, C (6),12) by comparison of spectral data with literature data
flavonoids and lignans have been isolated.5—8) The ethanol values. The purity of these compounds was proved by TLC
extract of A. fragrans had been shown to possess significant and HPLC (purity ꢁ90% for all compounds).
anti-inflammatory activities on a rat model of cervicitis,
Compound 1 was obtained as white amorphous powder.
which was established by endovaginal injection of latex rub- The HR-electrospray ionization (ESI)-MS of 1 showed a
ber of oxybenzene in rats, decreasing the prostaglandin E2 quasimolecular ion [MꢂNa]ꢂ at m/z 611.2125, consistent
(PGE2) expressing in an earlier period and regulating the with a molecular formula C30H36O12. The IR spectrum of 1
subgroups of T lymphocyte in the rat cervix mucosa.9)
displayed absorption bands of hydroxyl group at 3425 cmꢃ1,
During our search for anti-inflammatory constituents from five-membered ring g-lactone carbonyl at 1753 cmꢃ1, and
Chinese medicinal plants, the n-BuOH portion from the a,b-unsaturated ester carbonyl group at 1706 cmꢃ1. Further-
ethanol extract of A. fragrans was found to inhibit the en-
zyme cyclooxygenases-1 and -2 (COX-1, and -2) in vitro,
with the concentration exhibiting 50% inhibition (IC50) being
20.28 and 1.18 mg/ml, respectively. Further bioassay-directed
fractionation of this extract led to the isolation of six
sesquiterpenes. In this paper, we describe the isolation and
structure elucidation of one new guaiane-type sesquiterpene
glycoside, 2ꢀ-O-E-caffeoyl-8a-hydroxy-11a,13-dihydro-3a-
O-b-D-glucozaluzanin
C (1), along with five known
sesquiterpenes obtained from this plant. All those isolated
were evaluated for their inhibitory effects against both COX-
1 and COX-2 in vitro, along with clinically used anti-inflam-
matory drug aspirin.
Results and Discussion
The air-dried and powdered herb of of A. fragrans was ex-
tracted thoroughly with EtOH at room temperature. The
ethanol extract of the plant was suspended in water and then
successively extracted with petroleum ether (60—90 °C),
∗ To whom correspondence should be addressed. e-mail: chywc@yahoo.com.cn; lyonzhang@sohu.com
© 2009 Pharmaceutical Society of Japan

598
Vol. 57, No. 6
Table 1. 1H- and 13C-NMR Spectra Data of Compounds 1 and 2a)
1
2
Position
1
dH
dC
dH
3.00 t (9.0)
1.97 ddd (5.1, 7.7, 14.5)
2.31 ddd (6.9, 8.8, 14.8)
4.62 dd (5.6, 6.8)
dC
2.99 t (9.1)
1.98 m
46.1
37.8
45.5
37.9
2.26 m
4.64 m
3
4
5
6
7
8
9
80.9
150.1
52.4
80.8
54.4
70.6
46.4
81.1
150.5
52.0
80.7
54.2
70.4
45.8
2.85 br d (9.4)
4.04 t (10.2)
2.34 dd (2.1, 8.1)
3.50 m
2.86 br d (9.2)
4.39 t (10.3)
2.38 dd (8.1, 10.2)
3.66 m
2.16 dd (8.3, 12.6)
2.72 dd (4.5, 12.5)
Fig. 1. H–H COSY and Key HMBC Correlations of 1
2.06 m
more, absorption bands of 1606 and 1524 cmꢃ1 revealed the
presence of an aromatic skeleton. The 13C and distortionless
enhancement by polarization transfer (DEPT) spectra of 1
displayed 30 signals, of which fifteen were assigned to the
guaiane-type sesquiterpene aglycone moiety, the remaining
fifteen signals corresponding to a hexose sugar residue and a
2.58 dd (4.7, 11.5)
10
11
12
13
14
144.8
39.9
181.9
11.5
145.4
40.0
182.0
11.3
2.72 t (7.8)
2.79 t (7.8)
1.09 3H d (7.7)
4.90 br s
5.11 br s
5.27 d (1.1)
5.32 d (1.1)
4.66 d (8.1)
4.85 m
3.58 t (8.6)
3.38 m
1.25 3H d (7.0)
4.99 br s
5.11 br s
5.33 d (1.4)
5.38 d (1.4)
4.45 d (7.8)
3.21 dd (7.9, 8.9)
3.36 t (8.9)
S. O.
116.6
115.9
15
116.2
115.2
1
1ꢀ
2ꢀ
3ꢀ
4ꢀ
5ꢀ
6ꢀ
99.4
75.3
76.4
72.1
78.1
62.8
102.1
75.3
78.3
71.9
77.9
62.9
caffeoyl moiety. The H-NMR spectrum of 1 showed the
presence of one methyl signal [d 1.09 (3H, d, Jꢄ7.7 Hz, H3-
13)], three hydroxylated methane signals [d 4.64 (1H, m, H-
3), 4.04 (1H, t, Jꢄ10.2 Hz, H-6), 3.50 (1H, m, H-8)], two
pairs of exocyclic olefinic protons [d 4.90 (1H, br s, H-14a),
5.11 (1H, br s, H-14b), 5.27 (1H, d, Jꢄ1.1 Hz, H-15a), 5.32
(1H, d, Jꢄ1.1 Hz, H-14b), and an anomeric proton [d 4.66
S. O.
3.70 dd (5.8, 12.0)
3.91 dd (2.1, 12.0)
3.26 m
3.56 m
3.88 dd (1.9, 12.1)
1ꢅ
2ꢅ
3ꢅ
4ꢅ
5ꢅ
127.8
115.4
149.7
146.8
116.5
123.0
115.3
147.3
168.3
7.02 d (2.0)
1
(1H, d, Jꢄ8.1 Hz, H-1ꢀ)]. The H-NMR spectrum of 1 also
6.78 d (8.1)
displayed an ABX system assigned to a 1,2,4-trisubstituted
aromatic ring [d 7.02 (1H, d, Jꢄ2.0 Hz, H-2ꢅ), 6.78 (1H, d,
Jꢄ8.1 Hz, H-5ꢅ), 6.94 (1H, dd, Jꢄ1.9, 8.2 Hz, H-6ꢅ)], and
two trans-olefinic protons [d 6.24 (1H, d, Jꢄ15.9 Hz, H-a),
7.55 (1H, d, Jꢄ15.9 Hz, H-b)], suggesting the presence of a
6ꢅ
6.94 dd (1.9, 8.2)
6.24 d (15.9)
7.55 d (15.9)
a
b
CꢄO
a) Measured in CD3OD. Assignments were established by DEPT, 1H–1H COSY,
HMQC and HMBC experiments. J values (in Hz) are in parentheses. S. O., signal ob-
scured by the solvent or other signals.
1
caffeoyl moiety. With the aid of H–1H correlation spec-
troscopy (COSY), heteronuclear multiple quantum correla-
tion (HMQC), and heteronuclear multiple bond connectivity
(HMBC) experiments (Fig. 1), all the 1H- and 13C-NMR sig-
Table 2. Inhibitory Activities of Compounds 1—6 against COX-1 and
COX-2 in Vitro
1
nals of 1 were assigned as shown in Table 1. Both the H-
and 13C-NMR signals of 1 were closely related to those of 2
(Table 1), except for the signals due to the caffeoyl moiety.
The location of E-caffeoyl group through an ester linkage to
the C-2ꢀ of the glucose was determined by the HMBC corre-
lations between H-2ꢀ (d 4.85) and the a,b-unsaturated ester
carbonyl carbon (d 168.3). In addition, the HMBC correla-
tions between H-1ꢀ (d 4.66) of glucose and C-3 (d 80.9), as
well as H-3 (d 4.64) and C-1ꢀ (d 99.4), indicated that the
glucose should be attached to C-3 of the aglycone. The rela-
tive stereochemistry of 1 was elucidated by means of a nu-
clear Overhauser effect spectroscopy (NOESY) spectrum,
which showed the significant correlations between the methyl
IC50ꢆS.D. (mM)
Compound
COX-1
COX-2
1
2
3
4
5
6
ꢁ200
ꢁ200
32.1ꢆ9.3
30.6ꢆ11.4
57.9ꢆ18.1
27.5ꢆ6.4
28.7ꢆ8.4
12.5ꢆ3.7
87.6ꢆ20.5
78.8ꢆ18.3
ꢁ200
ꢁ200
ꢁ200
Aspirin
77.2ꢆ17.6
group (d 1.08) and b-positioned H-8 (d 3.50), as well as be- uated against both COX-1 and COX-2 for the isolates ob-
tween H-6 (d 4.04) and H-8, indicating that the orientations tained in the present study and the results are summarized in
of the methyl group at C-11 and H-6 were both in b posi- Table 2. Compound 3, a guaianolide sesquiterpene lactone,
tions. The NOESY experiment also showed a strong cross showed moderate COX-1-inhibiting activity with IC50 value
peak between H-3 (d 4.64) and a-positioned H-5 (d 2.85), of 78.8 mM, comparable to that of representative anti-inflam-
suggesting that H-3 is a-oriented. Thus, compound 1 was matory drug aspirin with an IC50 value of 77.2 mM. However,
elucidated to a 2ꢀ-O-E-caffeoyl-8a-hydroxy-11a,13-dihydro- five sesquiterpene glycosides (1, 2, 4—6) exhibited no or lit-
3-b-O-b-D-glucozaluzanin C.
tle COX-1 inhibitory activity, suggesting that the insertion of
Compounds 1—6 were isolated from the active n-BuOH a glucose moiety at C-3 or C-8 of the guaianolide sesquiter-
fraction of the ethanol extract of A. fragrans. Compounds 2, pene aglycone severely reduced the COX-1 inhibitory po-
3, and 6 were the major guaiane-type sesquiterpene lactones tential. All tested compounds displayed potent COX-2
of this plant and the other compounds (1, 4, 5) were present inhibitory activities with IC50 values ranging from 12.5 to
in minor quantities. The in vitro inhibitory activity was eval- 57.9 mM, in comparison with that of aspirin with an IC50

June 2009
599
value of 87.6 mM. Among these compounds, the most potent
inhibition of the COX-2 enzyme was found for 6 (12.5 mM),
which differs from the other tested compounds by possessing
a conjugated exomethylene group in its lactone ring (a-
methylene-g-lactone), indicating the a-methylene-g-lactone
function in the molecule of sesquiterpene lactone increased
the COX-2 inhibiting activity. These results also postulate
that these sesquiterpene lactones may contribute partly to ex-
pression of anti-inflammatory activity of A. fragrans through
COX-2 inhibiting activity.
2ꢀ-O-E-Caffeoyl-8a-hydroxy-11a,13-dihydro-3b-O-b-D-glucozaluzanin
C (1): Colourless powder, mp 125—126 °C (MeOH). [a]D25 ꢂ35.8° (cꢄ
0.06, MeOH). UV lmax (MeOH) nm: 249. IR (KBr) cmꢃ1: 3425, 2923,
1753, 1706, 1606, 1524, 1268, 1071. ESI-MS m/z: 589 [MꢂH]ꢂ
(C30H36O12). HR-ESI-MS m/z: 611.2125 (Calcd for [C30H36O12ꢂNa]ꢂ:
611.2098). 1H- and 13C-NMR spectral data see Table 1.
8a-Hydroxy-11a,13-dihydro-3b-O-b-D-glucozaluzanin C (2): Colourless
powder, mp 118—120 °C (MeOH). [a]D25 ꢂ41.5° (cꢄ0.05, MeOH). UV
l
max (MeOH) nm: 193. IR (KBr) cmꢃ1: 3386, 2929, 2876, 1748, 1649, 1270,
1076. ESI-MS m/z: 444 [MꢂNH4]ꢂ (C211H30O9). HR-ESI-MS m/z: 449.3332
(Cald for [C21H30O9ꢂNa]ꢂ: 449.3259). H- and 13C-NMR spectral data see
Table 1.
Acid Hydrolysis of 1 A solution of compound 1 (5 mg) in 2 M
HCl–MeOH (1 : 1, 5 ml) was refluxed at 90 °C for 3 h, respectively. After
Experimental
General Experimental Procedures Melting points were determined on being neutralized with NaOH–H2O, the solution was extracted with EtOAc
an X-4 melting point microscopic apparatus (Beijing, China) and were un- (5 mlꢇ3). The H2O layer was concentrated and passed through an Alltech
corrected. Optical rotations were measured on a Jasco P-1030 polarimeter. C18 SPE cartridge and then separated repeatedly by HPLC [LichroCART
IR spectra were recorded on a Nicolet Impact 410 FT-IR instrument. UV NH2 column (5 mm, 4.6ꢇ250 mm); mobile phase: MeCN–H2O (75 : 25);
spectra were recorded using a Shimadzu UV-2501 spectrometer. NMR spec- column temperature: 30 °C; flow rate: 1.0 ml/min; detection: refractive index
tra were obtained on Bruker Avance 300 and Bruker Avance 500 spectrome-
ters with CD3OD as solvent. Electrospray ionization mass spectrometry
(ESI-MS) was performed on an Agilent 1100 HPLC/EST instrument. High
(RI)] to afford D-glucose [1: 1.2 mg, tR: 8.9 min, [a]D20: ꢂ47.2°].13)
Assay for Inhibition of COX Activity Inhibitory activity of the com-
pounds on COX-1 and COX-2 was assayed by the use of Colorimetric COX
resolution electrospray ionization mass spectrometry (HR-ESI-MS) was (ovine) Inhibitory Screening Assay Kit purchased from Cayman Chemical
conducted on a Micromass TOF spectrometer. Column chromatography was (Ann. Arbor, MI, U.S.A., Catalog No. 760111) according to the protocol
carried out using silica gel (200—300 mesh, 400 mesh, Qingdao Haiyang
Chemical Group Co., Ltd., China), octodecyl silica gel (ODS) (C18, 40—
63 mm, Merck), and Sephadex LH-20 (Pharmacia) as a stationary phase.
recommended by the supplier. Tested compounds were dissolved in DMSO
at a concentration of 1ꢇ10ꢃ2 M as stock solution, and then diluted into ap-
propriate concentrations (200, 100, 30, 10, 3, 1, 0.3 mM) with assay buffer.
TLC was performed on precoated silica gel 60 F254 or RP-18 F254 plates Results were expressed as meanꢆS.D. of three independent experiments,
(Merck) and visualized under UV light and by spraying with vanillin reagent each experiment including triplicate sets.
followed by heating.
Plant Material The whole herb of Ainsliaea fragrans was collected
from Wuyuan City, Jiangxi Province, P. R. China, in June 2005, and authen-
ticated by Prof. Ming-jian Qing (Dept. of Pharmacognosy, China Pharma-
ceutical University). A voucher specimen (No. 20050701) was deposited in
the herbarium of China Pharmaceutical University, Nanjing.
Acknowledgements This work was supported by a “211 Project” Uni-
versity Grant from China Pharmaceutical University awarded to Dr. Wang
Hao (No. 211015).
References
Extraction and Purification The air-dried and powdered herbs of A.
fragrans (5.5 kg) were extracted with 95% EtOH (20 lꢇ3) by maceration at
room temperature. The EtOH extract was suspended in water and then suc-
cessively extracted with petroleum ether (60—90 °C), chloroform, and n-
BuOH. The n-BuOH-soluble fraction (160 g) was subjected to silica gel
(200—300 mesh, 1000 g) column chromatography using CHCl3–MeOH
(95 : 5, 90 : 10, 85 : 15, 70 : 30, each 5 l) as eluents to yield 8 fractions (A—
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mesh, 50 g) column chromatography using CHCl3–MeOH (95 : 5, 500 ml) as
eluents to yield 25 fractions (Frs. B1—B25). Fractions B8—B12 were com-
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(54 mg, 1150—1500 ml). Fractions D21 and D22 were combined and sub-
jected to silica gel (400 mesh, 50 g) column chromatography using
CHCl3–MeOH (88 : 12, 500 ml) as eluent to yield 5 (21 mg, 80—140 ml),
and 6 (150 mg, 210—220 ml).
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