Monohydroxy-substituted polyunsaturated fatty acids from Swertia japonica

Article abstract of DOI:10.1002/hlca.200890198

Fourteen monohydroxy-substituted polyunsaturated fatty acids, including two new compounds, (9Z,12S,13E,15Z)-12-hydroxyoctadeca-9,13,15-trienoic acid (10) and (9Z,12Z,14E,16R)-16-hydroxyoctadeca-9,12,14-trienoic acid (13), and 12 known ones, i.e., 1-9, 11,

Full text of DOI:10.1002/hlca.200890198

Helvetica Chimica Acta – Vol. 91 (2008)
1857
Monohydroxy-Substituted Polyunsaturated Fatty Acids from
Swertia japonica
by Masafumi Kikuchi, Yasunori Yaoita, and Masao Kikuchi*
Department of Molecular Structural Analysis, Tohoku Pharmaceutical University, 4-4-1 Komatsushima,
Aoba-ku, Sendai, Miyagi 981-8558, Japan
(phone: þ 81-22-234-4181; fax: þ 81-22-275-2013; e-mail: mkikuchi@tohoku-pharm.ac.jp)
Fourteen monohydroxy-substituted polyunsaturated fatty acids, including two new compounds,
(9Z,12S,13E,15Z)-12-hydroxyoctadeca-9,13,15-trienoic acid (10) and (9Z,12Z,14E,16R)-16-hydroxyoc-
tadeca-9,12,14-trienoic acid (13), and 12 known ones, i.e., 1 – 9, 11, 12, and 14, were isolated from the
whole plants of Swertia japonica Makino, and characterized as the corresponding methyl esters 1a – 14a.
Their structures were elucidated by analysis of the corresponding spectroscopic data, and the absolute
configurations of 10a and 13a were determined by the Mosher-ester method. The CD spectra (Table) of
compounds 1a – 14a are briefly discussed. This is the first report on the isolation of monohydroxy-
substituted polyunsaturated fatty acids from the Swertia genus in Gentianaceae.
Introduction. – The whole plant of Swertia japonica Makino (Gentianaceae) is the
crude drug Swertia herb, used as a stomachic or stimulant of appetite in Japan [1]. In
previous articles, we reported the structure determination of fifteen new secoiridoid
glycosides [2 – 4], a new unsaturated alcohol glycoside [3], and a new lignan glycoside
[3], all isolated from the whole plants of S. japonica. Here, we describe the isolation and
structure elucidation of the monohydroxy-substituted polyunsaturated fatty acids
contained in the whole plants of S. japonica.
Results and Discussion. – The dried whole plants of S. japonica were extracted with
MeOH. The MeOH extract was partitioned between H2O and CHCl3, and the CHCl3-
soluble fraction was subjected to column chromatography (silica gel) to afford a
fraction containing the free acids 1 – 14. The latter was treated with (trimethylsilyl)-
diazomethane (¼(diazomethyl)trimethylsilane; TMSCHN2) to prepare the corre-
sponding methyl esters, which were purified by column chromatography (silica gel)
followed by prep. HPLC (chiral column) to furnish the fourteen monohydroxy-
substituted polyunsaturated fatty acid methyl esters 1a – 14a.
The known compounds were identified as (9R,10E,12Z)-9-hydroxyoctadeca-10,12-
dienoic acid (1) [5], (9S,10E,12Z)-9-hydroxyoctadeca-10,12-dienoic acid (2) [6],
(9R,10E,12Z, 15Z)-9-hydroxyoctadeca-10,12,15-trienoic acid (3) [5], (9S,10E,12Z,
15Z)-9-hydroxyoctadeca-10,12,15-trienoic acid (4) [7], (9Z,11E,13R)-13-hydroxyocta-
deca-9,11-dienoic acid (5) [8], (9Z,11E,13S)-13-hydroxyoctadeca-9,11-dienoic acid (6)
[6], (9Z,11E,13R,15Z)-13-hydroxyoctadeca-9,11,15-trienoic acid (7) [9], (9Z,11E,13S,
15Z)-13-hydroxyoctadeca-9,11,15-trienoic acid (8) [6], (9Z,12R,13E,15Z)-12-hydroxy-
octadeca-9,13,15-trienoic acid (9) [7], (9E,11E,13R)-13-hydroxyoctadeca-9,11-dienoic
ꢀ 2008 Verlag Helvetica Chimica Acta AG, Zürich

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acid (11) [10], (9E,11E,13S)-13-hydroxyoctadeca-9,11-dienoic acid (12) [10], and
(9Z,12Z,14E,16S)-16-hydroxyoctadeca-9,12,14-trienoic acid (14) [6] by comparison of
their physical and spectroscopic data with the published data.
.
Compound 10a had the molecular formula C19H32O3, based on the HR-EI-MS (Mþ
at m/z 308.2360). The IR spectrum showed the presence of an OH (3602 cmÀ1) and an
ester group (1731 cmÀ1). In the UV spectrum (Table), the typical absorption maximum
of a conjugated diene appeared at 232 nm [11]. Comparison of the H- and 13C-NMR
1

Helvetica Chimica Acta – Vol. 91 (2008)
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spectra (CDCl3), EI-MS, and circular-dichroism (CD) data (Table) of 10a with those of
ester 9a established their enantiomeric relationship.
Table. UV and CD Data ofCompounds 1a – 14a
Configuration of stereogenic center
UV lmax (log e)a)
CD De (l)a)
1a
2a
3a
4a
5a
6a
7a
8a
R
S
R
S
R
S
R
S
R
S
R
S
R
S
232 (4.2)
232 (4.2)
234 (4.3)
233 (4.0)
232 (4.4)
232 (4.4)
232 (4.4)
232 (4.4)
232 (4.2)
232 (4.3)
229 (4.0)
229 (4.0)
229 (4.0)
229 (4.0)
À 1.6 (232)
þ 1.8 (233)
À 1.9 (234)
þ 1.8 (235)
À 2.6 (231)
þ 2.7 (232)
À 0.4 (243)
þ 0.5 (239)
À 0.2 (243)
þ 0.3 (243)
À 0.8 (229)
þ 0.7 (228)
À 0.8 (229)
þ 0.7 (228)
9a
10a
11a
12a
13a
14a
a) Measurements were carried out in MeOH. l in nm.
1
The H- and 13C-NMR spectra of 10a exhibited signals due to one Me group (d(H) 1.00 (t, J ¼ 7.6
Hz); d(C) 14.2 (Me)), one MeO group (d(H) 3.67 (s); d(C) 51.5), one oxygenated CH (d(H) 4.19 – 4.23
(m); d(C) 72.2 (CH)), three CH¼CH moieties (d(H) 5.39 (dt, J ¼ 10.7, 7.6 Hz), 5.45 (dt, J ¼ 11.0, 7.6 Hz),
5.56 (dt, J ¼ 10.7, 7.3 Hz), 5.69 (dd, J ¼ 15.1, 6.6 Hz), 5.94 (t, J ¼ 11.0 Hz), and 6.53 (dd, J ¼ 15.1, 11.0 Hz);
d(C) 124.4 (CH), 125.8 (CH), 127.1 (CH), 133.6 (CH), 134.6 (CH), and 135.0 (CH)), and one C¼O
group (d(C) 174.3). The EI-MS of 10a with the fragment-ion peak at m/z 81 (cleavage of the C-chain on
either side of the OH group) indicated the position of the OH group at C(12) [12]. These spectral data
were identical to those of 9a, except for the CD data. The CD spectra of 9a and 10a exhibited mirror
images (9a: max. at 243 nm (De ¼ À0.2); 10a: max. at 243 nm (De ¼ þ0.3)), confirming their
enantiomeric relationship.
The absolute configuration at C(12) of 10a was determined by the Mosher-ester
method. While the Mosher-ester method is usually applied in a manner that the (R)-
and (S)-MTPA esters (¼(aR)- and (aS)-a-methoxy-a-(trifluoromethyl)benzeneace-
tates) of a given secondary alcohol are prepared for the determination of its absolute
configuration [13], in this case, the (R)-MTPA esters 9b and 10b of the two
enantiomeric compounds 9a and 10a were prepared to obtain the same information
[14]. Thus, the absolute configuration at C(12) of 10a should be (S). From these data,
the structure of 10a was determined to be (9Z,12S,13E,15Z)-12-hydroxyoctadeca-
9,13,15-trienoic acid methyl ester. Consequently, the structure of 10 is
(9Z,12S,13E,15Z)-12-hydroxyoctadeca-9,13,15-trienoic acid.
The subtraction of corresponding 1H-NMR signals of 10b from those of 9b resulted in positive Dd
values for CH2(8) (Dd ¼ 0.08), HÀC(10) (Dd ¼ 0.12), 1 H of CH2(11) (Dd ¼ 0.05), and 1 H of CH2(11)
(Dd ¼ 0.06), and in negative Dd values for HÀC(14) (Dd ¼ À0.09), HÀC(15) (Dd ¼ À0.05), CH2(17)
(Dd ¼ À0.03), and Me(18) (Dd ¼ À0.02).

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Helvetica Chimica Acta – Vol. 91 (2008)
.
Compound 13a had the molecular formula C19H32O3, based on the HR-EI-MS (Mþ
at m/z 308.2363). The IR spectrum showed the presence of an OH (3599 cmÀ1) and an
ester group (1731 cmÀ1). In the UV spectrum (Table), the typical absorption maximum
of a conjugated diene appeared at 229 nm [11]. Comparison of the H- and 13C-NMR
1
spectra (CDCl3), EI-MS, and CD data (Table) of 13a with those of ester 14a established
their enantiomeric relationship.
1
The H- and 13C-NMR spectra of 13a exhibited signals due to one Me group (d(H) 0.88 (t, J ¼
7.1 Hz ) ; d(C) 9.7 (Me)), one MeO group (d(H) 3.67 (s); d(C) 51.5), one oxygenated CH (d(H)
4.09 – 4.13 (m); d(C) 74.2 (CH)), three CH¼CH moieties (d(H) 5.34 – 5.38 (m), 5.39 – 5.43 (m, 2 H),
5.69 (dd, J ¼ 15.0, 7.0 Hz), 6.00 (t, J ¼ 11.0 Hz) and 6.52 (dd, J ¼ 15.0, 11.0 Hz); d(C) 125.7 (CH), 127.2
(CH), 127.8 (CH), 130.7 (CH), 130.8 (CH), and 136.1 (CH)), and one C¼O group (d(C) 174.4). The EI-
MS of 13a with the fragment ion peak at m/z 57 indicated the position of the OH group at C(16) [12].
These spectral data were identical to those of 14a except for the CD data. The CD spectra of 13a and 14a
exhibited mirror images (13a: max. at 229 nm (De ¼ À0.8); 14a: max. at 228 nm (De ¼ þ0.7)),
confirming their enantiomeric relationship.
The absolute configuration at C(16) of 13a was determined by the Mosher-ester
method in the above-described manner. Thus, the absolute configuration at C(16) of
13a should be (R). From these data, the structure of 13a was determined to be
(9Z,12Z,14E,16R)-16-hydroxyoctadeca-9,12,14-trienoic acid methyl ester. Therefore,
the structure of 13 is (9Z,12Z,14E,16R)-16-hydroxyoctadeca-9,12,14-trienoic acid.
The subtraction of corresponding 1H-NMR signals of 14b from those of 13b resulted in positive Dd
values for CH2(11) (Dd ¼ 0.04), HÀC(12) (Dd ¼ 0.04), HÀC(14) (Dd ¼ 0.07), and HÀC(15) (Dd ¼
0.10), and in a negative Dd value for Me(18) (Dd ¼ À0.09).
The CD data of esters 1a – 14a obtained in the present work are summarized in the
Table. In the (R) series, the OH group has a negative Cotton effect at ca. 230 nm,
whereas in the (S) series, it has a positive Cotton effect in this region. Thus, the absolute
configuration of acyclic hydroxylated diene moieties of the monohydroxy-substituted
polyunsaturated fatty acids can be deduced from a CD measurement.
This is the first report on the isolation of monohydroxy-substituted polyunsaturated
fatty acids from the Swertia genus in Gentianaceae.
We are grateful to Mr. S. Satoh and Mr. T. Matsuki for NMR and MS measurements.
Experimental Part
General. Column chromatography (CC): silica gel (SiO2, 230 – 400 mesh; Merck). Prep. HPLC:
CCPM pump (Tosoh); UV-8020 UV/VIS detector (Tosoh); if not stated otherwise, Chiralcel-OD column
(250 Â 4.6 mm; Daicel Chemical Industries, Ltd.), hexane/i-PrOH eluents, and detection at l 235 nm;
flow 1.0 ml/min; tR in min. UV Spectra: Beckman DU-64 spectrophotometer; lmax (log e) in nm. CD
Spectra: Jasco J-720 spectropolarimeter; l in nm, De in l molÀ1 cmÀ1. NMR Spectra: Jeol JNM-LA-600 (at
600 (1H) and 150 MHz (13C)) and JNM-LA-400 (at 400 (1H) and 100 MHz (13C)) spectrometers; d in ppm
rel. to Me4Si, J in Hz. EI- and HR-EI-MS: Jeol JMS-DX-303 mass spectrometer; in m/z.
Plant Material. The dried whole plants of Swertia japonica were purchased from Uchida Wakanyaku
Co., Ltd., Japan, in 2002. A voucher specimen (SJ-2002-01) was deposited with the Laboratory of
Molecular Structural Analysis, Tohoku Pharmaceutical University.

Helvetica Chimica Acta – Vol. 91 (2008)
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Extraction and Isolation. The dried whole plants of Swertia japonica (2.0 kg) were extracted three
times (14 d each time) with MeOH at r.t., and the extracts were filtered. The MeOH extracts were then
concentrated to give a residue (474.0 g), which was partitioned between H2O and CHCl3. The CHCl3-
soluble fraction was concentrated to afford a residue (308.0 g). A part of this residue (17.8 g) was
subjected to CC (SiO2, CHCl3/MeOH 19 :1 ! 2 :1): Fractions 1 – 20 (by TLC). Fr. 13 was methylated
with TMSCHN2 to give the corresponding methyl esters. The methyl ester mixture was subjected to CC
(SiO2, hexane/AcOEt 4 :1, CHCl3/MeOH 19 :1 ! 4 :1): Fr. 1’ – 20’ (by TLC). Fr. 5’, on prep. HPLC
(hexane/i-PrOH 300 :1) gave 0.4 mg of 11a (tR 105.0) and 0.4 mg of 12a (tR 126.3). Fr. 6’, on prep. HPLC
(hexane/i-PrOH 60 :1) gave 1.2 mg of 6a (tR 17.0), 0.2 mg of 1a (tR 19.3), 1.3 mg of 5a (tR 20.8), and
0.5 mg of 2a (tR 25.0). Fr. 7’, on prep. HPLC (hexane/i-PrOH 60 :1) gave 0.6 mg of 13a (tR 16.5), 1.0 mg of
7a (tR 18.1), a mixture 9a/10a/14a (tR 21.0), 0.3 mg of 4a (tR 21.6), 1.1 mg of 8a (tR 24.4), and 1.0 mg of 3a
(tR 34.4). The mixture 9a/10a/14a, on prep. HPLC (hexane/ i-PrOH 300 :1) gave 0.4 mg of 9a (tR 197.0),
0.4 mg of 10a (tR 266.0), and 0.3 mg of 14a (tR 338.0).
(9Z,12S,13E,15Z)-12-Hydroxyoctadeca-9,13,15-trienoic Acid Methyl Ester (10a): Colorless oil. UV
(MeOH): Table. CD (MeOH): Table. IR (CHCl3): 3602, 1731. 1H-NMR (600 MHz, CDCl3): 1.00 (t, J ¼
7.6, Me(18)); 2.05 (br. q, J ¼ 7.1, CH2(8)); 2.18 – 2.24 (m, CH2(17)); 2.30 (t, J ¼ 7.6, CH2(2)); 2.28 – 2.32 (m,
CH2(11)); 3.67 (s, MeO); 4.19 – 4.23 (m, HÀC(12)); 5.39 (dt, J ¼ 10.7, 7.6, HÀC(10)); 5.45 (dt, J ¼ 11.0,
7.6, HÀC(16)); 5.56 (dt, J ¼ 10.7, 7.3, HÀC(9)); 5.69 (dd, J ¼ 15.1, 6.6, HÀC(13)); 5.94 (t, J ¼ 11.0,
HÀC(15)); 6.53 (dd, J ¼ 15.1, 11.0, HÀC(14)). 13C-NMR (150 MHz, CDCl3): 14.2 (C(18)); 21.1 (C(17));
24.9 (C(3)); 27.4 (C(8)); 29.1 (C(5), C(6), C(7)); 29.5 (C(4)); 34.1 (C(2)); 35.4 (C(11)); 51.5 (MeO); 72.2
(C(12)); 124.4 (C(10)); 125.8 (C(16)); 127.1 (C(15)); 133.6 (C(13)); 134.6 (C(14)); 135.0 (C(9)); 174.3
.
.
(C(1)). HR-EI-MS: 308.2360 (Mþ , C19H32O3þ ; calc. 308.2351).
(9Z,12Z,14E,16R)-16-Hydroxyoctadeca-9,12,14-trienoic Acid Methyl Ester (13a): Colorless oil. UV
(MeOH): Table. CD (MeOH): Table. IR (CHCl3): 3599, 1731. 1H-NMR (600 MHz, CDCl3): 0.88 (t, J ¼
7.1, Me(18)); 2.06 (br. q, J ¼ 7.0, CH2(8)); 2.31 (t, J ¼ 7.7, CH2(2)); 2.93 (br. t, J ¼ 7.7, CH2(11)); 3.67 (s,
MeO); 4.09 – 4.13 (m, HÀC(16)); 5.34 – 5.38 (m, HÀC(10)); 5.39 – 5.43 (m, HÀC(9), HÀC(12)); 5.69
(dd, J ¼ 15.0, 7.0, HÀC(15)); 6.00 (t, J ¼ 11.0, HÀC(13)); 6.52 (dd, J ¼ 15.0, 11.0, HÀC(14)). 13C-NMR
(150 MHz, CDCl3): 9.7 (C(18)); 25.0 (C(3)); 26.1 (C(11)); 27.2 (C(8)); 29.1 (C(5), C(6), C(7)); 29.5
(C(4)); 30.2 (C(17)); 34.1 (C(2)); 51.5 (MeO); 74.2 (C(16)); 125.7 (C(14)); 127.2 (C(10)); 127.8 (C(13));
.
.
130.7 (C(12)); 130.8 (C(9)); 136.1 (C(15)); 174.4 (C(1)). HR-EI-MS: 308.2363 (Mþ , C19H32O3þ
;
calc. 308.2351).
MTPA Esterification of 9a, 10a, 13a, and 14a. To a soln. of each methyl ester 9a (0.4 mg), 10a
(0.4 mg), 13a (0.3 mg), and 14a (0.3 mg) in pyridine (20 ml), (S)-MTPA-Cl (1.7 ml) was added. Each
mixture was allowed to stand at r.t. for 24 h. Next, [(3-dimethylamino)propyl]amine (1.0 ml) was added,
and after 30 min, the mixture was concentrated and the residue purified by prep. HPLC (TSKgel-Silica-
60 column (300 Â 7.8 mm ; Tosoh), hexane/acetone 9 :1, l 230 nm) to give the (R)-MTPA esters 9b
(0.3 mg), 10b (0.3 mg), 13b (0.2 mg), and 14b (0.2 mg) from 9a, 10a, 13a, and 14a, resp.
Data of 9b: 1H-NMR (400 MHz, CDCl3): 0.98 (t, J ¼ 7.6, Me(18)); 2.01 (br. q, J ¼ 7.1, CH2(8)); 2.11 –
2.15 (m, CH2(17)); 2.43 – 2.47 (m, 1 H of CH2(11)); 2.49 – 2.53 (m, 1 H of CH2(11)); 5.35 (dt, J ¼ 10.7, 7.6,
HÀC(10)); 5.87 (t, J ¼ 11.0, HÀC(15)); 6.52 (dd, J ¼ 15.1, 11.0, HÀC(14)).
Data of 10b: 1H-NMR (400 MHz, CDCl3): 1.00 (t, J ¼ 7.6, Me(18)); 1.93 (br. q, J ¼ 7.1, CH2(8));
2.16 – 2.20 (m, CH2(17)); 2.38 – 2.42 (m, 1 H of CH2(11)); 2.43 – 2.47 (m, 1 H of CH2(11)); 5.23 (dt, J ¼
10.7, 7.6, HÀC(10)); 5.92 (t, J ¼ 11.0, HÀC(15)); 6.61 (dd, J ¼ 15.1, 11.0, HÀC(14)).
Data of 13b: 1H-NMR (400 MHz, CDCl3): 0.85 (t, J ¼ 7.1, Me(18)); 2.91 (br. t, J ¼ 7.7, CH2(11)); 5.63
(dd, J ¼ 15.0, 7.0, HÀC(15)); 5.97 (t, J ¼ 11.0, HÀC(13)); 6.64 (dd, J ¼ 15.0, 11.0, HÀC(14)).
Data of 14b: 1H-NMR (400 MHz, CDCl3): 0.94 (t, J ¼ 7.1, Me(18)); 2.87 (br. t, J ¼ 7.7, CH2(11)); 5.53
(dd, J ¼ 15.0, 7.0, HÀC(15)); 5.93 (t, J ¼ 11.0, HÀC(13)); 6.57 (dd, J ¼ 15.0, 11.0, HÀC(14)).

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Received March 25, 2008