Resin glycosides from the aerial parts of Operculina turpethum

Article abstract of DOI:10.1016/j.phytochem.2012.05.010

Three glycosidic acids, turpethic acids A-C, and two intact resin glycosides, turpethosides A and B, all having a common pentasaccharide moiety and 12-hydroxy fatty acid aglycones of different chain lengths, were obtained from the aerial parts of Operculina turpethum. Their structures were elucidated by spectroscopic analyses and chemical correlations. The aglycones were characterized as 12-hydroxypentadecanoic acid in two compounds, 12-hydroxyhexadecanoic acid in two other components, and 12-hydroxyheptadecanoic acid in the fifth compound, which were all confirmed by synthesis. The absolute configurations of these aglycones were all established as S by Mosher's method. These compounds represent the first examples of resin glycosides with a monohydroxylated 12-hydroxy fatty acid as an aglycone, and one compound is the first described resin glycoside having a hydroxylated C₁₇ fatty acid as its aglycone.

Full text of DOI:10.1016/j.phytochem.2012.05.010

Phytochemistry 81 (2012) 165–174
Contents lists available at SciVerse ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Resin glycosides from the aerial parts of Operculina turpethum
a
a
a,
Wenbing Ding a,c, Zi-Hua Jiang b, , Ping Wu , Liangxiong Xu , Xiaoyi Wei
⇑
⇑
a Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Xingke Road 723, Tianhe District,
Guangzhou 510650, China
b Department of Chemistry, Lakehead University, 955 Oliver Road, Thunder Bay, ON, Canada P7B 5E1
c Graduate School of Chinese Academy of Sciences, Yuquanlu 19A, Beijing 100049, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Three glycosidic acids, turpethic acids AꢀC, and two intact resin glycosides, turpethosides A and B, all
having a common pentasaccharide moiety and 12-hydroxy fatty acid aglycones of different chain lengths,
were obtained from the aerial parts of Operculina turpethum. Their structures were elucidated by
spectroscopic analyses and chemical correlations. The aglycones were characterized as 12-hydroxypen-
tadecanoic acid in two compounds, 12-hydroxyhexadecanoic acid in two other components, and 12-
hydroxyheptadecanoic acid in the fifth compound, which were all confirmed by synthesis. The absolute
configurations of these aglycones were all established as S by Mosher’s method. These compounds
represent the first examples of resin glycosides with a monohydroxylated 12-hydroxy fatty acid as an
aglycone, and one compound is the first described resin glycoside having a hydroxylated C17 fatty acid
as its aglycone.
Received 4 November 2011
Received in revised form 2 February 2012
Available online 18 June 2012
Keywords:
Operculina turpethum
Convolvulaceae
Resin glycosides
Glycosidic acids
12-Hydroxy fatty acids
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
of resin glycosides, certain positions of the sugar units are further
acylated with residues of small organic acids (C2–C5, volatile),
Resin glycosides are unique secondary metabolites in the plant
kingdom confined to the morning glory family (Convolvulaceae)
(Pereda-Miranda et al., 2010; Pereda-Miranda and Bah, 2003). They
are the principles responsible for the purgative action of all the
important Convolvulaceous species used in traditional medicine
throughout the world. They have also been reported to have other
various biological activities including being haemolytic, antibacte-
rial, antifungal, plant growth regulatory, and cytotoxic (Dembitsky,
2004; Figueroa-González et al., 2012; Fürstner, 2004; Pereda-
Miranda et al., 2006a; Pereda-Miranda and Bah, 2003). To date,
hundreds of resin glycosides have been isolated from the Convol-
vulaceae family. They are composed of differently acylated oligo-
saccharides glycosidically linked to hydroxylated fatty acids
which are usually linked back to the sugar chain to form macrolac-
tone rings of various sizes (Eich, 2008; Pereda-Miranda et al.,
2010). The hydroxylated fatty acids are structurally quite con-
served among the large number of resin glycosides reported so
far. Jalapinolic acid [(S)-11-hydroxyhexadecanoic acid] and con-
volvulinic acid [(S)-11-hydroxytetradecanoic acid] are the most
frequently found aglycones for the macrocyclic lipooligosaccharide
core, although other different hydroxylated fatty acids with chain
length of C10, C12, C14, C15, C16, and C18 also being reported (Eich,
2008; Pereda-Miranda et al., 2010, 2006b). For the vast majority
straight-chain saturated fatty acids (non-volatile), and arylalkyl
acids, respectively (Eich, 2008).
The genus Operculina (Fam. Convolvulaceae) consists of about
25 species occurring in tropical areas worldwide. Operculina turpet-
hum (L.) S. Manso (syn. Ipomoea turpethum), a perennial herba-
ceous vine, is distributed in southern China (Guangdong,
Guangxi, Hainan, Taiwan, and Yunnan), Southeast and South Asia
(Bangladesh, Cambodia, India, Indonesia, Ryukyu Islands, Laos,
Malaysia, Burma, Nepal, New Guinea, Pakistan, Philippines, Sri
Lanka, Thailand, and Vietnam), the Pacific Islands, and Australia
(Staples, 2007; Wu, 1979). This plant has significant ethnomedici-
nal values, and its roots, known as ‘‘Indian jalap’’, are commercially
available as mild but very effective laxatives. Partly due to the
introduction to the world market of jalap and other plants such
as Brazilian-grown jalap (I. operculata, syn. O. macrocarpa), the
demand for Mexican jalap root (I. purga) has declined (Pereda-
Miranda et al., 2010). The roots and stems of O. turpethum have
traditionally been used in Indian medicine to treat a wide range
of ailments, such as tumors, burning diseases, jaundice, paralysis,
and pain in the joints and muscles (Ahbuselvam et al., 2007). Fur-
thermore, an O. turpethum extract has been used to relieve fevers
and to treat anaemia, splenomegaly, raised lipid levels, obesity,
gastric ulcer, and related gastrointestinal disturbances (Ahmad
et al., 2009; Austin, 1982; Laakshmayya et al., 2006). Recent
studies have shown that the methanolic extract of O. turpethum
stems also has antioxidant activity and a protective effect against
⇑
Corresponding authors. Tel.: +86 20 37252538.
E-mail addresses: zjiang@lakeheadu.ca (Z.-H. Jiang), wxy@scbg.ac.cn (X. Wei).
0031-9422/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.phytochem.2012.05.010

166
W. Ding et al. / Phytochemistry 81 (2012) 165–174
7,12-dimethylbenz(a)anthracene (DMBA)-induced breast cancer in
female rats (Ahbuselvam et al., 2007); moreover, the aqueous ex-
tract of O. trupethum roots provides hepatoprotective and anti-
clastogenic effects against hepatic fibrosis (Ahmad et al., 2009).
Previously the isolation and hepato-protective activity of four
dammarane-type triterpenoid saponins was reported from the aer-
ial parts of this plant (Ding et al., 2011). Wagner et al. (1978) also
reported the isolation from the roots of this plant of several resin
glycosides which carried a number of dihydroxylated fatty acids,
in addition to the common jalapinolic acid, as the aglycones. These
dihydroxylated fatty acids include 3,12-dihydroxypentadecanoic
acid, 3,12-dihydroxypalmitic acid, 4,12-dihydroxypentadecanoic
acid and 4,12-dihydroxypalmitic acid. In continuation of our phy-
tochemical studies on this plant, a group of new resin glycosides
(4 and 5) and glycosidic acids (1–3) have been isolated. They are
structurally interesting because they are the first examples of resin
glycosides having a monohydroxylated 12-hydroxy fatty acid as
aglycone and 3 is the first resin glycoside with a hydroxy C17 fatty
acid as its aglycone.
similar. Careful comparison of their spectra (Table 1) showed that
1–3 had a common oligosaccharide moiety comprised of three 6-
deoxyhexose and two hexose residues. Taking turpethic acid B
(2) as an example, its 1H NMR spectrum exhibited five anomeric
proton signals at d 4.97 (1H, d, J = 7.4, Hz), 5.22 (1H, d, J = 7.8,
Hz), 5.89 (1H, br s), 6.20 (1H, br s) and 6.33 (1H, br s) and three sec-
ondary methyl resonances at d 1.56 (3H, d, J = 6.2 Hz), 1.59 (3H, d,
J = 6.1 Hz) and 1.69 (3H, d, J = 6.2 Hz), assignable to the CH3-6 of 6-
deoxyhexoses. These structural features were also evident in the
13C NMR spectrum of 2, which displayed five anomeric carbon sig-
nals in the range d 100.5–105.1. Analysis of COSY and HSQC spectra
led to assignments of proton and carbon resonances for each
monosaccharide residue in 1–3 (Table 1). The oligosaccharide moi-
eties in 1–3 were found to be identical with those of operculinic
acid B, a known glycosidic acid previously obtained from Ipomoea
operculata (Ono et al., 1989). The connectivities between the
hydroxyfatty acid aglycones (Ag) and sugar chains, and among
monosaccharides in 1–3 were supported by their HMBC spectra,
in which, also using 2 as an example, long range correlations were
observed for dH 4.97 (H-1, Glc) with dC 77.7 (C-12, Ag), dH 4.20 (H-2,
Glc) with dC 101.1 (C-1, Rha), dH 6.33 (H-1, Rha) with dC 76.9 (C-2,
Glc), dH 4.22 (H-4, Rha) with dC 103.1 (C-1, Rha0), dH 5.89 (H-1, Rha0)
with dC 82.0 (C-4, Rha), dH 4.49 (H-4, Rha0) with dC 102.7 (C-1,
Rha00), dH 6.20 (H-1, Rha00) with dC 78.3 (C-4, Rha0) and dH 5.22
(H-1, Glc0) with dC 82.4 (C-3, Rha0). In order to confirm the mono-
saccharide compositions and absolute configurations, 1–3 were
subjected to acid hydrolysis. Only two monosaccarides were de-
2. Results and discussion
The EtOH extract of the powdered dry aerial parts of O. turpet-
hum was suspended in water and partitioned by successive extrac-
tions with petroleum ether, EtOAc, and n-BuOH. The petroleum
ether-soluble fraction was subjected to silica gel column chroma-
tography (CC) and eluted with petroleum ether–acetone of increas-
ing polarity to afford four fractions (DꢀG) of resin glycosides.
Fraction G was further separated by ODS and Sephadex LH-20 CC
followed by preparative HPLC to give two intact resin glycosides,
trivially named turpethosides A (4) and B (5). Fractions DꢀF were
investigated by the same methods, but no pure resin glycosides
were obtained.
tected and isolated from the hydrolysates and identified
and -rhamnose, respectively, on the basis of their specific optical
rotation and TLC comparison with authentic samples. Thus, the oli-
gosaccharide moieties in 1–3 were all established to be b- -Glc-
(1?3)-[ -Rha-(1?4)]- -Rha-(1?4)- -Rha-(1?2)-b- -Glc.
D-glucose
L
D
a
-L
a-
L
a
-L
D
Obviously, the difference between turpethic acids AꢀC (1–3)
lie in their aglycones, the hydroxylated fatty acids. On mild acid
hydrolysis, turpethic acids AꢀC (1–3) afforded hydroxylated fatty
acids 6–8, respectively. ESIMS of 6 gave quasi-molecular ions at
m/z 281 [M+Na]+ and 259 [M+H]+ in positive ion mode and m/z
257 [M–H]– in negative ion mode. Its 13C NMR spectrum (Ta-
ble 2) exhibited signals indicating the presence of 15 carbons
including a carboxyl and an oxygenated methine, indicating a
monohydroxylated pentadecanoic acid. In the HMBC spectrum
of 6, a long range correlation was observed from the terminal
methyl proton signal at dH 0.95 (H-15) to the carbon resonance
at dC 40.2, which was assignable to the methylene carbon adja-
cent to the oxygenated methine group, suggesting that the hy-
droxy group was located at C-12 of the fatty acid. Thus,
aglycone 6 was identified as 12-hydroxypentadecanoic acid. In
order to confirm this structure, compound 6 was converted to
its methyl ester 9-(S) and 12-hydroxypentadecanoic acid methyl
ester in its racemic form (9) was synthesized starting from 12-
hydroxydodecanoic acid (12) (Scheme 1). Indistinguishable 1H
and 13C NMR spectra between 9-(S) and 9 were observed and
unambiguously confirmed that the hydroxy substituent in 6
was at C-12.
The 1D NMR spectroscopic and ESIMS data indicated that 7 and
8 were monohydroxylated hexadecanoic acid and monohydroxy-
lated heptadecanoic acid, respectively, in accordance with deduc-
tions from the ESIMS data of 2 and 3. By comparison of the 13C
NMR spectroscopic data of 7 and 8 (Table 2) with those reported
for the commonly encountered jalapinolic acid (Pereda-Miranda
et al., 2006b), small but evident differences in the chemical shift
values of two methylene carbons adjacent to the oxymethine car-
bon were found between 7 (dC 37.4 and 37.1) and jalapinolic acid
(dC 37.5 and 37.4), whereas the values of these two carbons (both
at dC 37.5) in 8 were almost identical with those of jalapinolic acid.
Careful analysis of the HSQC and HMBC spectra led to assignment
Since purification of intact resin glycosides is problematic
(Pereda-Miranda et al., 2010, 2006b), our research then focused
on the glycosidic acids of the resin glycosides in this plant. The
unseparated resin glycoside mixtures were combined and treated
with 5% KOH. The reaction mixture afforded three glycosidic acids
1–3 which were trivially named turpethic acids AꢀC, respectively.
By analysis of the HRESIMS and NMR spectroscopic data, the
molecular formulae of turpethic acids AꢀC (1–3) were determined
to be C45H80O25, C46H82O25, and C47H84O25, respectively, each dif-
fering from the others by one or two CH2 units. The negative ion
ESIMS of 1 exhibited an [M–H]ꢀ ion peak at m/z 1019 as well as
a series of fragment ions at m/z 873 [1019 – C6H10O4]ꢀ, 857
[1019 – C6H10O5]ꢀ, 711 [1019 – C6H10O4 – C6H10O5]ꢀ, 565 [711 –
C6H10O4]ꢀ, 419 [565 – C6H10O4]ꢀ, and 257 [419 – C6H10O5]ꢀ, obvi-
ously generated from the quasi-molecular [M–H]ꢀ ion precursor by
sequential loss of five monosaccharide units including three deox-
yhexose (C6H10O4) units and two hexose (C6H10O5) units to yield
the aglycone ion (m/z 257) in accordance with a hydroxypenta-
decanoic acid anion. The negative ESIMS of 2 exhibited, besides
an [M–H]ꢀ ion peak at m/z 1033, characteristic fragment ion peaks
at m/z 887 [1033 – C6H10O4]ꢀ, 871 [1033 – C6H10O5]ꢀ, 725 [1033 –
C6H10O4 – C6H10O5]ꢀ, 579 [725 – C6H10O4]ꢀ, 433 [579 – C6H10O4]ꢀ,
and 271 [433 – C6H10O5], which all were 14 mass units (CH2) more
than those of 1. For turpethic acid C (3), its negative ESIMS dis-
played an [M–H]ꢀ peak at m/z 1047 and characteristic fragment
ions at m/z 901 [1047 – C6H10O4]ꢀ, 885 [1047 – C6H10O5]ꢀ, 739
[1047 – C6H10O4 – C6H10O5]ꢀ, 593 [739 – C6H10O4]ꢀ, 447 [593 –
C6H10O4]ꢀ and 285 [447 – C6H10O5]ꢀ, all having 28 mass units
(2 ꢁ CH2) more than those of 1. These data suggested that 1–3
are pentasaccharides of monohydroxy fatty acids with chain
lengths of C15, C16, and C17, respectively.
The 1H NMR spectra of turpethic acids AꢀC (1–3) were almost
identical with each other and their 13C NMR spectra were also very

W. Ding et al. / Phytochemistry 81 (2012) 165–174
167
Table 1
1H (600 MHz) and 13C NMR (150 MHz) data of 1–3 in C5D5N.
Position
1
2
3
dH (J in Hz)
dC
dH (J in Hz)
dC
dH (J in Hz)
dC
Glc-1
4.96 d (7.5)
4.20 m
4.25 m
4.14 t (9.1)
3.88 ddd (9.1, 5.0, 2.5)
4.34 dd (11.7, 5.0)
4.52 br d (11.7)
100.5
77.0
79.2
71.6
77.7
62.5
4.97 d (7.4)
4.20 dd (9.1, 7.4)
4.23 m
100.5
76.9
79.2
71.4
77.7
62.5
4.98 d (7.3)
4.22 dd (9.2, 7.3)
4.24 m
100.6
77.0
79.2
71.4
77.7
62.5
2
3
4
5
6
4.14 t (9.0)
4.14 t (9.2)
3.89 ddd (9.0, 5.0, 2.5)
4.34 dd (11.7, 5.0)
4.52 br d (11.7)
3.89 ddd (9.2, 5.0, 2.6)
4.34 dd (11.7, 5.0)
4.53 br d (11.7)
Glc0-1
5.21 d (7.7)
105.2
74.7
78.0
71.4
78.1
62.6
5.22 d (7.8)
3.96 dd (9.0, 7.8)
4.17 m
4.10 t (9.1)
3.94 ddd (9.1, 6.0, 2.7)
4.26 m
105.1
74.7
78.1
71.6
78.1
62.6
5.22 d (7.8)
105.1
74.9
78.1
71.6
78.1
62.6
2
3
4
5
6
3.96 dd (9.1, 7.7)
4.18 dd (9.1, 9.1)
4.09 t (9.1)
3.94 ddd (9.1, 5.1, 2.2)
4.25 m
3.96 dd (9.0, 7.8)
4.18 dd (9.0, 9.0)
4.11 t (9.0)
3.94 ddd (9.0, 5.0, 2.3)
4.27 m
4.49 m
4.49 m
4.50 m
Rha-1
6.32 br s
101.2
72.3
72.2
81.4
67.4
18.4
6.33 br s
101.1
72.2
72.3
82.0
67.3
18.5
6.34 br s
101.2
72.3
72.2
82.0
67.4
18.5
2
3
4
5
6
4.65 dd (3.1, 1.1)
4.60 dd (9.3, 3.1)
4.24 m
4.89 m
1.68 d (6.2)
4.66 br d (3.4)
4.61 dd (9.2, 3.4)
4.22 m
4.90 m
1.69 d (6.2)
4.66 br d (3.5)
4.61 dd (9.3, 3.4)
4.26 m
4.91 m
1.70 d (6.2)
Rha0-1
5.85 br s
5.18 br s
4.72 dd (8.9, 2.8)
4.49 m
4.40 m
103.2
71.8
82.5
78.3
68.2
18.4
5.89 br s
5.18 br s
4.72 dd (8.9, 2.8)
4.49 m
4.40 m
103.1
71.8
82.4
78.3
68.2
18.6
5.89 br s
5.18 br s
4.72 dd (8.8, 2.8)
4.49 m
4.40 m
103.1
71.8
82.4
78.3
68.2
18.6
2
3
4
5
6
1.59 d (6.1)
1.59 d (6.1)
1.60 d (6.2)
Rha00-1
6.20 br s
4.88 br s
4.42 dd (9.0, 3.3)
4.22 m
4.29 m
102.7
72.2
72.4
73.6
70.0
18.0
6.20 br s
4.87 br s
4.42 dd (8.8,3.3)
4.20 m
4.30 m
102.7
72.2
72.4
73.6
70.1
18.0
6.20 br s
102.7
72.3
72.4
73.6
70.1
18.0
2
3
4
5
6
4.87 br d (3.4)
4.42 dd (9.1, 3.5)
4.21 m
4.29 m
1.56 d (6.2)
1.56 d (6.2)
1.56 d (6.2)
Ag-1
2
3
176.6
35.0
25.5
25.2
33.7
77.4
37.3
18.6
14.2
175.9
34.8
25.4
25.3
33.6
77.7
34.7
27.3
22.9
14.0
175.9
34.7
25.4
25.3
33.7
77.7
35.1
24.9
32.1
22.7
14.0
2.49 t (6.7)
1.77 m
1.59 m
1.83 m
4.03 m
1.72 m, 1.68 m
1.58 m
0.93 t (7.2)
2.49 t (7.4)
1.77 m
1.61 m
1.83 m
4.04 m
1.73 m
1.57 m, 1.48 m
1.34 m
2.49 t (7.4)
1.77 m
1.61 m
1.85 m
4.05 m
1.75 m
1.61 m, 1.51 m
1.29 m
1.31 m
0.89 t (6.9)
10
11
12
13
14
15
16
17
0.92 t (7.3)
Table 2
13C NMR spectroscopic data of 6–8 in C5D5N and 9-(S)ꢀ11-(S) in CDCl3.
Position
6
7
8
9-(S)
10-(S)
11-(S)
1
2
175.8
34.6
175.7
34.6
175.7
34.6
174.3
34.1
174.4
34.1
174.4
34.1
3
25.3
25.3
25.3
24.9
24.9
24.9
4–9
10
11
12
13
14
15
16
17
OMe
29.8–29.2
25.9
38.1
70.3
40.2
29.9–29.3
26.1
29.9–29.3
26.1
29.6–29.1
25.6
37.4
71.7
39.6
29.6–29.1
25.6
29.6–29.1
25.6
37.4
72.0
37.4
25.3
31.9
22.6
14.0
38.2a
70.6
38.1a
70.3
37.4a
72.0
37.8a
28.2
22.9
38.2a
25.7
32.1
22.7
13.9
37.1a
27.8
22.7
19.1
14.1
18.8
14.1
14.0
14.0
51.4
51.4
51.4
a
Assignments interchangeable in the same column.
of the 13C NMR spectroscopic data of 6–8 (Table 2) and indicated
that the chemical shifts of C-1–C-11 for 7 and 8 were very similar
to those for 6. These findings suggested that the hydroxy substitu-
ents in both 7 and 8 were also located at the C-12 position.
Similarly, the structures of 7 and 8 were finally confirmed by direct
comparison of the spectroscopic data of their methyl esters 10-(S)
and 11-(S) with those of their synthetic counterparts (10 and 11)
which were synthesized as racemic forms (Scheme 1).

168
W. Ding et al. / Phytochemistry 81 (2012) 165–174
osyl-(1?3)-O-[
syl-(1?4)-O-
(S)-12-hydroxyhexadecanoic acid 12-O-b-
O-[ -rhamnopyranosyl-(1?4)]-O- -rhamnopyranosyl-(1?4)-
O- -rhamnopyranosyl-(1?2)-b- -glucopyranoside (2) and (S)-12-
hydroxyheptadecanoic acid 12-O-b- -glucopyranosyl-(1?3)-O-
-rhamnopyranosyl-(1?4)]-O- -rhamnopyranosyl-(1?4)-O-
-rhamnopyranosyl-(1?2)-b- -glucopyranoside (3), respectively
(Fig. 1).
a
-
L
-rhamnopyranosyl-(1?4)]-O-
-rhamnopyranosyl-(1?2)-b- -glucopyranoside (1),
-glucopyranosyl-(1?3)-
a-L-rhamnopyrano-
a
-L
D
D
a
-
L
a-L
a-L
D
D
[
a
-
L
a-L
a-L
D
Assignments of the NMR spectroscopic data for hydroxylated
long chain fatty acids are quite challenging due to signal overlap,
which makes it difficult to determine their structures based on
Scheme 1. Synthesis of 12-hydroxy fatty acid methyl esters 9–11. Reagents and
conditions: (a) CH3OH, H2SO4; (b) IBX (5.0 equiv), DMSO, RT; (c) n-C3H7MgBr, THF,
RT; (d) n-C4H9MgBr, THF, RT; (e) n-C5H11MgBr, THF, RT.
The configurations at C-12 of 6–8 were determined by Mosher’s
method (Dale and Mosher, 1973). Hydroxyfatty acid methyl esters
9-(S)ꢀ11-(S) were treated with (R)-
a-methoxy-a-phenylacetic acid
(R-MPA) and S-MPA to afford three pairs of Mosher esters 16-(S,R)/
16-(S,S), 17-(S,R)/17-(S,S), and 18-(S,R)/18-(S,S), respectively. The
1H NMR spectroscopic data of each ester were recorded and as-
signed (see Experimental) with the aid of their COSY spectra The
chemical shift differences (
the stereogenic center of each pair of Mosher esters were then
analyzed (Table 3). A positive d value for H-10 and H-11 and a
negative d for H-13, H-14, H-15, H-16, and/or H-17 denoted an
S-configuration at C-12 (Cao et al., 2005; Yin et al., 2008). In partic-
ular, significant chemical shift differences ( d) were observed
between the corresponding terminal methyl groups which were
readily identifiable. A d value of –0.16 ppm between the terminal
D
d = dR ꢀ dS) of those protons around
D
D
D
D
methyl groups in both 16 and 17, and –0.09 ppm in 18, confirmed a
12S-absolute configuration according to the configurational model
proposed by Kakisawa and coworkers (Ohtani et al., 1991). The
of –0.09 ppm between the terminal methyl signals in 18 was also
consistent with previously reported values (–0.07 to
Dd
D
d
–
0.035 ppm) for the corresponding methyl signals which are sepa-
rated by as many as four methylene groups (Ono et al., 1993; Yin
et al., 2008). Furthermore, the specific optical rotations for all free
acids 6–8 and their methyl esters 9-(S)–11-(S) had positive values,
which is in accordance with the observation that monohydroxylat-
ed fatty acids with a dextrorotatory optical activity have an (S)-
absolute configuration while their laevoisomers have the opposite
(R)-configuration (Chérigo et al., 2008). Thus, the absolute configu-
rations of 6–8 were determined to be S (Fig. 1).
Therefore, the structures of turpethic acids AꢀC were estab-
lished as (S)-12-hydroxypentadecanoic acid 12-O-b-D-glucopyran-
Table 3
1H NMR
Dd values of R/S Mosher’s esters 16–18.
Compound
D
d (dR ꢀ dS, in ppm)
H-10
H-11
H-13
H-14
H-15
H-16
H-17
16
17
18
0.32
0.32
0.32
0.13
0.13
0.12
ꢀ0.11
ꢀ0.13
ꢀ0.12
ꢀ0.28
ꢀ0.39
ꢀ0.30
ꢀ0.16
ꢀ0.18
ꢀ0.24
ꢀ0.16
ꢀ0.15
ꢀ0.09
Fig. 1. Structural formulae of compounds 1–18.

W. Ding et al. / Phytochemistry 81 (2012) 165–174
169
spectroscopic data alone. During the course of this study, with syn-
thetic authentic samples, it was possible to make a full assignment
of all carbon signals, except C-4–C-9, for the 12-hydroxy fatty acids
6–8 and their corresponding methyl esters 9–11, based on their
HSQC and HMBC data (Table 2). It was found that the chemical
shifts of C-11 and C-13, adjacent to the hydroxylated methine car-
bon (C-12), were quite distinct in 12-hydroxypentadecanoic acid
derivatives [dC 38.1 (C-11) and 40.2 (C-13) in 6; dC 37.4 (C-11)
and 39.6 (C-13) in 9]. As the chain length increased, the absolute
value of the chemical shift difference between C-11 and C-13
Turpethoside A (4) gave a quasi-molecular ion peak at m/z
1287.6097 [M+Cl]– in its HRESIMS, corresponding to a molecular
formula of C60H100O27. Its 1H and 13C NMR spectra (Table 4) dis-
played characteristic signals for a glycosidic acid core closely sim-
ilar to that turpethic acids 1–3, as well as additional resonances
indicating the presence of three short organic acid moieties. The
signals readily distinguished for an olefinic methine at dH 6.95
(m), an olefinic tertiary methyl group at dH 1.80 (br s) and an ole-
finic secondary methyl group at dH 1.57 (dd, J = 7.1, 1.1 Hz) in the
1H NMR spectrum, and resonances for a conjugated ester carbonyl
at dC 167.6, an olefinic methine at dC 137.3, an olefinic quaternary
(D
d = |dC-13 ꢀ dC-11|) became smaller [dC 38.2 and 37.8 (C-11, C-
13) in 7; dC 37.4 and 37.1 (C-11, C-13) in 10; dC 38.1 and 38.2 (C-
11, C-13) in 8; dC 37.4 and 37.4 (C-11, C-13) in 11] (Table 2 and
Fig. 2). In fact, C-11 and C-13 collapsed into a single signal in 12-
hydroxyheptadecanoic acid methyl ester 11 (Fig. 2). In order to
demonstrate that such spectroscopic features also apply to 11-
hydroxylated fatty acids, 11-hydroxyheptadecanoic acid methyl
ester (15) were synthesized according to a known procedure (Jiang
et al., 1995). Indeed, the two carbon atoms (C-10 and C-12) adja-
cent to the hydroxylated methine gave rise to only one signal in
its 13C NMR spectrum (Fig. 2). This trend was found to be true also
for the carbon resonances reported for (S)-11-hydroxyhexadeca-
noic acid methyl ester [dC 37.5 (C-10 or C-12)/37.4 (C-12 or C-
Table 4
1H (600 MHz) and 13C NMR (150 MHz) spectroscopic data of 4 and 5 in C5D5N.
Position
4
5
dH (J in Hz)
dC
dH (J in Hz)
dC
Glc-1
4.81 d (7.6)
3.91 dd (9.1, 7.6)
4.10 m
4.12 m
3.83 m
104.2
81.4
76.7
71.8
77.8
62.7
4.82 d (7.5)
3.91 dd (9.1, 7.5)
4.11 m
4.12 m
3.83 m
104.3
81.4
76.7
71.8
77.8
62.7
2
3
4
5
6
4.30 dd (11.7, 6.4)
4.45 br d (11.7)
4.30 dd (11.3, 6.2)
4.45 br d (11.3)
10), with a
D
d = 0.1 ppm] and (S)-11-hydroxytetradecanoic acid
d = 2.1 ppm]
Glc0-1
5.07 d (7.7)
3.94 dd (9.0, 7.7)
4.03 t (9.0)
3.96 m
3.71 m
105.1
75.0
78.3
70.7
75.1
63.6
5.07 d (7.7)
3.94 dd (9.0, 7.7)
4.03 t (9.0)
3.96 m
3.71 m
105.1
75.0
78.3
70.7
75.1
63.6
methyl ester [dC 37.5 (C-10)/39.6 (C-12), with a
D
2
3
4
5
6
(Pereda-Miranda et al., 2006b). It seems that for both 11-hydroxyl-
ated and 12-hydroxylated fatty acids and their derivatives, the
chemical shifts of the carbon atoms adjacent to the hydroxylated
methine tend to concur as the chain length grows, and eventually
4.50 dd (11.6, 4.9)
4.94 dd (11.6, 1.8)
4.49 dd (11.5, 4.9)
4.93 br d (11.5)
become very close (Dd < 0.1 ppm) or identical when the hydroxyl-
Rha-1
5.57 br s
6.11 br s
5.19 br d (9.6)
4.26 t (9.6)
4.43 m
98.5
73.8
69.8
79.0
68.7
19.6
5.57 br s
6.11 br s
5.19 br d (9.6)
4.26 t (9.6)
4.43 m
98.6
73.8
69.8
79.1
68.7
19.6
ated methine is separated by at least four CH2 units from the ter-
minal methyl group. Such 13C NMR spectroscopic characteristics
may apply also to other monohydroxylated long chain fatty acids
and their derivatives.
2
3
4
5
6
1.62 d (6.2)
1.63 d (6.2)
Rha0-1
6.19 br s
98.8
73.0
79.5
79.1
68.0
18.6
6.19 br s
5.98 br s
4.72 dd (9.1, 3.3)
4.33 t (9.3)
4.41 m
98.8
73.0
79.5
79.1
68.0
18.7
2
3
4
5
6
5.98 dd (3.3, 1.8)
4.72 dd (9.1, 3.3)
4.33 t (9.3)
4.40 m
1.71 d (6.1)
1.71 d (6.1)
Rha00-1
6.22 br s
4.97 br s
4.60 dd (8.9, 3.4)
5.84 t (9.2)
4.42 m
103.2
72.3
70.3
75.6
68.7
17.9
6.21 br s
4.97 br s
4.60 dd (9.0, 3.3)
5.84 t (9.0)
4.43 m
103.2
72.3
70.3
75.6
68.2
17.9
2
3
4
5
6
1.41 d (6.3)
1.41 d (6.3)
Ag-1
2
173.1
34.6
34.3
82.0
37.3
18.6
14.3
173.1
34.6
34.3
82.4
34.9
27.7
23.1
14.1
2.22 m, 2.36 m
1.58 m, 1.64 m
3.82 m
1.61 m, 1.88 m
1.73 m
2.22 m, 2.37 m
1.62 m, 1.66 m
3.82 m
1.66 m, 1.94 m
1.30 m
11
12
13
14
15
16
0.82 t (7.5)
1.22 m
0.82 t (7.4)
Mba-1
2
3
4
175.1
41.0
26.8
11.3
16.4
175.1
41.1
26.8
11.4
16.4
2.38 m
2.38 m
1.41 m, 1.64 m
0.79 t (7.5)
1.03 d (7.0)
1.41 m, 1.64 m
0.80 t (7.5)
1.03 d (7.0)
2-Me
Mba0-1
2
3
4
176.5
41.1
26.8
11.7
16.6
176.5
41.1
26.8
11.7
16.6
2.50 m
2.50 m
1.50 m, 1.73 m
0.88 t (7.5)
1.24 d (7.0)
1.50 m, 1.73 m
0.88 t (7.4)
1.24 d (7.0)
2-Me
Tga-1
2
3
4
167.6
128.9
137.3
14.0
167.6
129.0
137.3
14.0
6.95 m
1.57 dd (7.1, 1.1)
1.81 br s
6.95 m
1.57 br d (7.1)
1.80 br s
Fig. 2. 13C NMR spectra of 6–8 in C5D5N and 9–11 and 15 in CDCl3 showing
chemical shifts of the carbons adjacent to the hydroxylated methine carbon.
2-Me
12.1
12.2

170
W. Ding et al. / Phytochemistry 81 (2012) 165–174
carbon at dC 128.9 and two methyl carbons at dC 12.1 and 14.0 in
the 13C NMR spectrum which were characteristic of a tigloyl group
(Mills et al., 2005). Additional 1H NMR signals due to short organic
acid moieties were also observed for four methyl groups including
two primary methyl at dH 0.88 and 0.79 (each t, J = 7.5 Hz) and two
secondary methyls at dH 1.24 and 1.03 (each d, J = 7.0 Hz). Further
analysis of these methyl signals in the COSY spectrum indicated
the presence of two 2-butyl groups. This, in combination with
the presence of four ester carbonyl carbon resonances at dC
176.5–167.6 in the 13C NMR spectrum, including those for tigloyl
group and the hydroxylated fatty acid aglycone, indicated the pres-
ence of two 2-methylbutyric acid (Mba) moieties. Compound 4
was then subjected to saponification with 5% KOH and the prod-
ucts fractionated into organic and glycosidic acid fractions. The for-
mer examined by GC–MS gave two peaks corresponding to tiglic
acid (Tga) and 2-methylbutyric acid by direct comparison with
authentic samples. The absolute configuration of Mba was con-
firmed to be S by GC analysis using a chiral capillary column
according to the previously reported method (Noda and Horiuchi,
2008; Gaspar and Barroso, 2006). The glycosidic acid fraction fur-
nished turpethic acid A (1), thus confirming the structure of the
glycosidic acid core of 4.
ment ions (see Experimental) all having 14 mass units (CH2)
more than those of 4. Alkaline hydrolysis of 5 afforded turpethic
acid B (2), indicating that 5 has a glycosidic acid core identical to
that of 2. The presence of two S-Mba and a Tga moieties in 5 was
also supported by GC–MS and chiral GC analyses. Therefore,
the structure of turpethoside B was elucidated as (S)-12-hydroxy-
hexadecanoic acid 12-O-(6-O-(S)-2-methylbutyryl)-b-
anosyl-(1?3)-O-[4-O-tigloyl- -rhamnopyranosyl-(1?4)]-O-
[2-O-(S)-2- methylbuty-ryl]- -rhamnopyranosyl-(1?4)-O-
-glucopyranoside, intramolecular 1,
D-glucopyr-
a
-L
a
-
L
a-L-
rhamnopyranosyl-(1?2)-b-
200-ester (5).
D
3. Conclusions
A group of resin glycosides with 12-hydroxy C15ꢀC17 fatty acid
as aglycones, turpethic acids AꢀC (1–3) and turpethosides A and B
(4 and 5), were obtained from the aerial parts of O. turpethum
growing in southern China. Their structures were unambiguously
assigned by spectroscopic analysis and chemical correlations. 12-
Hydroxylated fatty acids are rarely found as aglycones of resin gly-
cosides. Ricinoleic acid (12-hydroxy-9-cis-octadecenoic acid) was
once reported to be an aglycone for ipopurpuroside, a resin glyco-
side from Ipomoea purpurea (Nikolin et al., 1978), but the structure
of this resin glycoside was not established. 3,12- and 4,12-dihy-
droxylated fatty acids were also reported as aglycones of resin gly-
cosides from the same plant (Wagner et al., 1978) and other
species (Çalißs et al., 2007; Ono et al., 2009). However, monohydr-
oxylated 12-hydroxy fatty acids are being reported for the first
time in the present study as aglycones of resin glycosides. In addi-
tion, the aglycone of 3 is a hydroxylated C17 fatty acid, which is
unprecedented for resin glycosides. These findings represent an
important contribution to the structural diversity of these intrigu-
ing glycolipid conjugates. Furthermore, by analysis of 13C NMR
spectroscopic data of 12-hydroxy C15ꢀC17 fatty acids and their
The negative ESIMS of 4 exhibited, besides the [M–H]– ion at
m/z 1251, fragment ions at m/z 1151 [1251 – C5H8O2 (Tga)]–,
1149 [1251
–
C5H10O2 (Mba)]–, 1049 [1251
–
C5H8O2
–
–
C5H10O2]–, 1047 [1251 – 2 ꢁ C5H10O2]– and 947 [1251 – C5H8O2
2 ꢁ C5H10O2]– due to loss of the short organic acids. Furthermore,
the negative ion ESIMS displayed a series of diagnostic fragment
ions (Noda et al., 1988) at m/z 1023 [1251 – C11H16O5 (O-tigloylr-
hamnose unit)]ꢀ, 1005 [1251 – C11H18O6 (O-methylbutyrylglucose
unit)]–, 547 [C27H48O11 (hydroxypentadecanoic acid O-rhamno-
sylglucoside, intramolecular ester)
–
H]– and 419 [C21H40O8
(hydroxypentadecanoic acid O-glucoside) – H]–, arising from glyco-
sidic cleavages, which suggested ester linkages of Tga to Rha00, Mba
to Rha0, Mba0 to Glc0, and 12-hydroxypentadecanoic acid to Rha.
The exact O-acylated positions in the monosaccharide moieties
were established by detailed analysis of the 2D NMR spectra of 4,
in particular the HMBC spectrum. By analysis of the COSY and
HSQC spectra, the proton signals and corresponding carbon signals
for the five individual monosaccharides were assigned (Table 4). It
was found that the proton resonances for H-2 of Rha (dH 6.11), H-2
of Rha0 (dH 5.98), H-4 of Rha00 (dH 5.84), and H2-6 of Glc0 (dH 4.94 and
4.50) were shifted downfield relative to those in 1 (Table 1), indi-
cating esterification of these positions. Subsequently, long range
correlation between these protons and the ester carbonyl carbons
were examined in the HMBC spectrum. As a result, correlations
were observed from H-2 of Rha, H-2 of Rha0, H-4 of Rha00, and H2-
6 of Glc0 to carbonyls (C-1) of the aglycone (Ag) (dC 173.1), Mba
(dC 175.1), Tga (dC 167.6) and Mba0 (dC 176.5), respectively. These
observations indicated that the carboxyl group of (S)-12-hydroxy-
pentadecanoic acid was linked to C-2 of Rha to form a macrolac-
tone ring, Mba and Mba0 moieties were attached at C-2 of Rha0
and C-6 of Glc0, respectively, and a Tga moiety was located at C-4
of Rha00. Therefore, the structure of turpethoside A (4) was
elucidated as (S)-12-hydroxypentadecanoic acid 12-O-(6-O-(S)-
methyl esters, it has been shown that the difference (D
d) of 13C
NMR chemical shifts between two methylene carbons adjacent to
the hydroxylated methine carbon, which were readily distin-
guished from other methylene carbons by their downfield shifts
(dC > 37 ppm), was correlated with the position of the hydroxy
group in the fatty acid chain. This finding provides an empirical
13C NMR spectroscopic method for the facile location of hydroxy
groups in hydroxy fatty acids.
On the other hand, a previous investigation on Indian jalap res-
ins demonstrated that the aglycones of resin glycosides in this spe-
cies were rare 3,12- and 4,12-dihydroxylated C15 and C16 fatty
acids, in addition to the common jalapinolic acid (Wagner et al.,
1978). However, resin glycosides with these fatty acid aglycones,
including the jalapinolic acid aglycone, were not obtained in the
present study. Instead, the aglycones of resin glycosides in this
plant growing in southern China were found to be another group
of rare hydroxy fatty acids, monohydroxylated 12-hydroxy C15
–
C17 fatty acids. These suggest that the fatty acid aglycones of resin
glycosides in this taxon are peculiar, diverse, and possibly habitat-
specific.
2-methylbutyryl)-b-
rhamnopyranosyl-(1?4)]-O-[2-O-(S)-2-methylbutyryl]-
pyranosyl-(1?4)-O- -rhamnopyranosyl-(1?2)-b- -glucopyran-
oside, intramolecular 1,200-ester.
D
-glucopyranosyl-(1?3)-O-[4-O-tigloyl-
a-L-
a-L-rhamno-
4. Experimental
a
-
L
D
4.1. General experimental procedures
Turpethoside B (5) had a molecular formula of C61H102O27, dif-
fering from 4 by an additional CH2 unit, as determined from a qua-
si-molecular ion at m/z 1325.6747 [M+AcOH–H]– in the negative
ion HRESIMS. Its 1H and 13C NMR spectra (Table 4) were very sim-
ilar to those of 4, except for signals due to the hydroxylated fatty
acid moiety. The negative ion ESIMS of 5 showed the same frag-
mentation pattern as that of 4 and provided characteristic frag-
Optical rotations were obtained on a Perkin-Elmer 341 polarim-
eter with MeOH as solvent. The 1H, 13C, and 2D NMR spectra were
recorded on a Bruker Avance-600 or a Bruker DRX-400 instrument
using TMS as an internal standard. HRESIMS data were obtained on
an API QSTAR mass spectrometer. ESIMS were collected on an MDS
SCIEX API 2000 LC/MS instrument. EIMS were aquired on a

W. Ding et al. / Phytochemistry 81 (2012) 165–174
171
Shimadzu GCMS-QP2000Plus apparatus with ionization energy of
70 eV. Preparative HPLC was performed with a Shimadzu LC-6A
pump and a Shimadzu RID-10A refractive index detector using a
m/z: 1069 [M+Cl]–, 1033 [M–H]–, 887, 871, 725, 579, 433, and 271;
HRESIMS m/z: 1033.50537 [M–H]– (calcd for C46H81O25, 1033.
5072).
YMC-Pack ODS-A column (5
matography (CC), silica gel 60 (100–200 mesh, Qingdao Marine
Chemical Ltd., Qingdao, China), Develosil ODS (75 m, Nomura
l
m, 250 ꢁ 20 mm). For column chro-
4.6. Turpethic acid C (3)
l
Chemical Co. Ltd., Osaka, Japan), and Sephadex LH-20 (GE Health-
care, Uppsala, Sweden) were used.
20
White powder, [
a
]
–69.4 (c 1.9, MeOH); for 1H (600 MHz,
D
C5D5N) and 13C (150 MHz, C5D5N) NMR spectroscopic data, see
Table 1; positive ion ESIMS m/z: 1071 [M+Na]+; negative ion ESIMS
m/z: 1083 [M+Cl]–, 1047 [M–H]–, 901, 885, 739, 593, 447, and 285;
4.2. Plant material
HRESIMS m/z: 1047.52173 [M–H]– (calcd for C47H83O25
,
Aerial parts of O. turpethum were collected from Hengqin island,
Zhuhai, Guangdong, China, in October 2008, and authenticated by
Prof. Fuwu Xing, South China Botanical Garden, Chinese Academy
of Sciences. A voucher specimen (743902) has been deposited at
the Herbarium of South China Botanical Garden, Chinese Academy
of Sciences.
1047.5229).
4.7. Turpethoside A (4)
20
Colorless syrup, [
a
]
–0.28 (c 0.19, MeOH); for 1H (600 MHz,
D
C5D5N) and 13C (150 MHz, C5D5N) NMR spectroscopic data, see
Table 4; positive ion ESIMS m/z: 1275 [M+Na]+; negative ion ESIMS
m/z: 1287 [M+Cl]–, 1251 [M–H]–, 1151 [1251 – C5H8O2 (Tga)]–,
4.3. Extraction and isolation
1149 [1251 – C5H10O2 (Mba)]–, 1067 [1251 + H2O – C5H8O2
–
–
Powdered, air-dried, aerial parts of O. turpethum (8.0 kg) were
extracted with EtOH–H2O (3 ꢁ 20 L, 95:5, v/v, 48 h each) at room
temperature and the EtOH solution was concentrated under
reduced pressure to give a residue (1.1 kg). This latter was sus-
pended in H2O and sequentially extracted with petroleum ether
(60–90 °C), EtOAc, and n-BuOH. The petroleum ether layer was
concentrated in vacuo to yield a petroleum ether-soluble fraction
(148.0 g). This was subjected to silica gel CC eluted with petroleum
ether (60–90 °C)–acetone mixtures of increasing polarity (10:0 to
5:5), to yield ten fractions (A–J). Fraction G (6.3 g), obtained on elu-
tion with petroleum ether–acetone (6:4), was further subjected to
ODS CC, using MeOH–H2O mixtures of decreasing polarities (80:20
to 95:5) to give four subfractions (G1–G4). Subfraction G3
(276 mg) was further separated by Sephadex LH-20 CC using
MeOH followed by preparative HPLC using MeOH–H2O (85:15,
v/v) with a flow-rate of 5 mL/min to afford turpethoside A (4)
(7 mg, tR = 50 min) and turpethoside B (5) (16 mg, tR = 70 min).
Compound 4 was obtained pure after a single HPLC separation
while compound 5 was purified using peak shaving (Pereda-Miran-
da and Hernández-Carlos, 2002) by manually recycling the peak
three times.
C5H10O2]–, 1065 [1251 + H2O
–
2 ꢁ C5H10O2]–, 1049 [1251
C5H8O2 – C5H10O2]–, 1047 [1251 – 2 ꢁ C5H10O2]–, 1023, 1005,
965, 947, 921, 565, 547, 419; HRESIMS m/z: 1287.6097 [M+Cl]–
(calcd for C60H100O27Cl, 1287.6141).
4.8. Turpethoside B (5)
20
Colorless syrup, [
a
]
–0.27 (c 0.39, MeOH); for 1H (600 MHz,
D
C5D5N) and 13C (150 MHz, C5D5N) NMR spectroscopic data, see
Table 4; positive ion ESIMS m/z: 1289 [M+Na]+; negative ion ESIMS
m/z: 1301 [M+Cl]–, 1265 [M–H]–, 1165 [1265 – C5H8O2]–, 1163
[1265 – C5H10O2]–, 1081 [1265 + H2O – C5H8O2 – C5H10O2]–, 1079
[1265 + H2O – 2 ꢁ C5H10O2]–, 1063 [1265 – C5H8O2 – C5H10O2]–,
1061 [1265 – 2 ꢁ C5H10O2]–, 1037, 1019, 979, 961, 935, 917, 579,
561, 433; HRESIMS m/z 1325.6711 [M+AcOH – H]– (calcd for
C
63H105O29, 1325.6747).
4.9. Acid hydrolysis of 1–3 and preparation of (S)-12-hydroxy fatty
acids 6–8
Fractions D–F were then combined and a portion (800 mg) of
the combined mass (41.6 g) was treated with 5% KOH at 85 °C for
4 h. The reaction mixture was acidified to pH 4.0 and extracted
with Et2O (50 mL ꢁ 3). The aqueous layer was further extracted
with n-BuOH (3 ꢁ 100 mL) and the n-BuOH layer was concentrated
in vacuo to yield a residue (540 mg) which was subjected to Sepha-
dex LH-20 CC using MeOH to afford a glycosidic acid fraction
(400 mg). A portion (100 mg) of this fraction was subjected to
preparative HPLC using CH3OH–H2O (6:1) with a flow-rate of
5 mL/min to obtain turpethic acids A (1) (28 mg, tR = 25 min), B
(2) (36 mg, tR = 35 min), and C (3) (22 mg, tR = 53 min).
The remaining portion (300 mg) of the glycosidic acid fraction
was hydrolyzed by 10% H2SO4 at 80 °C for 4 h. The hydrolysate
was cooled to room temperature and extracted with Et2O
(30 mL ꢁ 3) to afford a mixture of hydroxy fatty acids (50 mg).
The mixture was separated by HPLC using MeOH–H2O (75:25,
v/v) with a flow-rate of 5 mL/min to give 6 (15.0 mg, tR = 32 min),
7 (18 mg, tR = 41 min), and 8 (14 mg, tR = 56 min). The aqueous
layer was neutralized with 5% NaOH and desalted by passage
through a Sephadex LH-20 column using MeOH to afford a mix-
ture of sugars (90 mg), from which glucose (Rf = 0.3) and rhamnose
(Rf = 0.52) were detected by TLC (CHCl3–MeOH–H2O, 6:4:1). The
4.4. Turpethic acid A (1)
mixture was subjected to silica gel CC eluted with CHCl3–MeOH–
20
H2O (10:3:1) to obtain
L
-rhamnose (29 mg): [
a
]
D
+8.2 (c 0.97,
20
White powder, [
a
]
D
–65.0 (c 1.5, MeOH); for 1H (600 MHz,
H2O), and further eluted with CHCl3–MeOH–H2O (7:3:1) to give
C5D5N) and 13C (150 MHz, C5D5N) NMR spectroscopic data, see
Table 1; positive ion ESIMS m/z: 1,043 [M+Na]+; negative ESIMS
m/z: 1019 [M–H]–, 873, 857, 711, 565, 419, and 257; negative ion
HRESIMS m/z: 1019.4912 [M–H]– (calcd for C45H79O25, 1019.4916).
20
D-glucose (10.9 mg): [
a]
D
+69.1° (c 0.55, H2O).
4.9.1. (S)-12-Hydroxypentadecanoic acid (6)
20
Colorless oil; [
a]
+2.2 (c 1.35, CHCl3); 1H NMR (600 MHz,
D
4.5. Turpethic acid B (2)
C5D5N) d: 3.84 (1H, m, H-12), 2.51 (2H, t, J = 7.4 Hz, H-2), 1.78
(2H, m, H-3), 1.47–1.74 (8H, m), 1.18–1.41 (12H, m), and 0.95
(3H, t, J = 7.1 Hz, H-15); for 13C NMR (150 MHz, C5D5N) see Table 3;
positive ion ESIMS m/z: 281 [M+Na]+; negative ion ESIMS m/z: 257
[M–H]–.
–72.9 (c 1.7, MeOH); for 1H (600 MHz,
20
White powder, [
a]
D
C5D5N) and 13C (150 MHz, C5D5N) NMR spectroscopic data, see
Table 1; positive ion ESIMS m/z: 1057 [M+Na]+; negative ion ESIMS

172
W. Ding et al. / Phytochemistry 81 (2012) 165–174
4.9.2. (S)-12-Hydroxyhexadecanoic acid (7)
added dropwise to the resulting mixture under N2 and vigorous
20
Colorless oil; [
a
]
D
+0.6 (c 1.42, CHCl3); 1H NMR (600 MHz,
stirring. After the addition was completed, the reaction mixture
was stirred for 1 h to provide a Grignard reagent of n-C3H7MgBr
(1.6 mol/L, in THF). By the same method, n-C4H9MgBr (1.6 mol/L,
in THF) and n-C5H11MgBr (1.6 mol/L, in THF) were obtained.
C5D5N) d: 3.82 (1H, m, H-12), 2.51 (2H, t, J = 7.4 Hz, H-2), 1.78
(2H, m, H-3), 1.57–1.73 (6H, m), 1.44–1.57 (2H, m), 1.20–1.42
(14H, m), and 0.89 (3H, t, J = 7.3 Hz, H-16); for 13C NMR
(150 MHz, C5D5N) spectroscopic data, see Table 3; positive ion
ESIMS m/z: 295 [M+Na]+; negative ion ESIMS m/z: 271 [M–H]–.
4.11.2. 12-Hydroxydodecanoic acid methyl ester (13)
12-Hydroxydodecanoic acid (1.10 g, 5.09 mmol) was stirred
with CH3OH (30 mL, 0.74 mol) in the presence of a catalytic
amount of concentrated H2SO4 (20 drops) under reflux for 8 h. Vol-
atile materials were removed via rotary evaporation, the residue
was dissolved in Et2O (200 mL), and successively washed with
10% NaHCO3 (3 ꢁ 100 mL) and H2O (3 ꢁ 100 mL). The organic layer
was then dried (anhydr. Na2SO4) and concentrated to yield 12-
hydroxydodecanoic acid methyl ester (13, 0.81 g, 80%): a white so-
lid; 1H NMR (400 MHz, CDCl3) d: 3.62 (3H, s, OCH3), 3.58 (2H, t,
J = 6.7 Hz, H-12), 2.25 (2H, t, J = 7.6 Hz, H-2), 1.47–1.58 (4H, m),
1.23–1.30 (14H, m).
4.9.3. (S)-12-Hydroxyheptadecanoic acid (8)
+0.5 (c 0.77, CHCl3); 1H NMR (600 MHz,
20
Colorless oil; [
a]
D
C5D5N) d: 3.83 (1H, m, H-12), 2.51 (2H, t, J = 7.4 Hz, H-2), 1.78
(2H, m, H-3), 1.46–1.74 (8H, m), 1.20–1.42 (16H, m), and 0.85
(3H, t, J = 7.1 Hz, H-17); for 13C NMR (150 MHz, C5D5N) spectro-
scopic data, see Table 3; negative ion ESIMS m/z: 285 [M–H]–.
4.10. Preparation of (S)-12-hydroxy fatty acid methyl esters 9-(S)–11-
(S) from (S)-12-hydroxy fatty acids 6–8
In a typical experiment (Hecht and Kozarich, 1973), hydroxy
fatty acid 6 (13 mg, 0.05 mmol) and N-nitrosomethylurea (MUN,
0.21 g, 0.2 mmol) were dissolved in 1,2-dimethoxyethane–H2O
(5:1, 10 mL). The solution was maintained at ice-bath temperature
and 0.6 N KOH (10 mL added dropwise). The reaction mixture was
concentrated under reduced pressure and the aqueous solution ex-
tracted with Et2O (10 mLꢁ3). The Et2O layer was dried (anhydrous
MgSO4) and evaporated to afford the methyl ester 9-(S) (11.4 mg,
83%). By the same method, hydroxy fatty acids 7 and 8 (each
13 mg) yielded 10-(S) (12.0 mg, 88%) and 11-(S) (11.1 mg, 81%),
respectively.
4.11.3. Methyl 12-oxododecanoate (14)
1-Hydroxy-1,2-benziodoxol-3(1H)-one-1-oxide (IBX, 4.2 g,
15 mmol) was added to a solution of methyl ester 13 (0.8 g,
3.4 mmol) in DMSO (10 mL), and the mixture was stirred at rt for
4 h. The pale-yellow colored solution was diluted with Et2O and
successively washed with saturated NaCl and H2O. The organic
layer was dried (anhydr. Na2SO4) and concentrated in vacuo. The
resulting residue was purified by silica gel CC to give methyl 12-
oxododecanoate (14, 0.68 g, 85%): white waxy solid; 1H NMR
(400 MHz, CDCl3) d: 9.77 (1H, t, J = 1.8 Hz, H-12), 3.68 (3H, s,
OCH3), 2.42 (1H, td, J = 7.4, 1.8 Hz, H-11), 2.30 (2H, t, J = 7.5 Hz,
H-2), 1.62 (4H, m), 1.24–1.29 (12H, m).
4.10.1. (S)-12-Hydroxypentadcanoic acid methyl ester [9-(S)]
20
Colorless oil, [
a]
+0.8 (c 1.1, CHCl3); 1H NMR (400 MHz,
D
CDCl3) d: 3.67 (3H, s, OCH3), 3.59 (1H, m, H-12), 2.30 (2H, t,
J = 7.6 Hz, H-2), 1.60 (2H, m, H-3), 1.23–1.50 (20H, m), 0.93 (3H,
t, J = 6.9 Hz, H-15); for 13C NMR (100 MHz, CDCl3) spectroscopic
data, see Table 3; EIMS m/z (rel. int.): 255 [M ꢀ OH]+ (1), 229 [M
ꢀ C3H7]+ (9), 200 [M ꢀ C4H8O]+ (20), 197 [M ꢀ C3H7 ꢀ MeOH]+
(55), 179 [M ꢀ C3H7 ꢀ MeOH – H2O]+ (12), 157 (30), 143 (44), 95
(55), 87 (100).
4.11.4. Racemic12-hydroxy fatty acid methyl esters 9–11
Compound 14 (200 mg, 0.88 mmol) was dissolved in anhydrous
THF (3 mL). The fresh n-C5H11MgBr in THF (1.6 mol/L, 0.9 mmol)
was added under N2 and the reaction mixture stirred for 4 h. The
reaction was quenched with saturated NH4Cl (30 mL). The result-
ing mixture was then extracted with Et2O (3 ꢁ 20 mL), dried
(Na2SO4), filtered, and evaporated under reduced pressure. The res-
idue was purified by flash chromatography over silica gel (n-hex-
ane–EtOAc, 9:1) to afford 9 (60 mg, 25%). By the same method,
10 (100 mg, 40%) and 11 (90 mg, 34%) were obtained. The 1H and
13C NMR spectroscopic data of 9–11 were identical with those of
9-(S)–11-(S).
4.10.2. (S)-12-Hydroxyhexadecanoic acid methyl ester [10-(S)]
20
Colorless oil, [
a]
+1.1 (c 0.9, CHCl3); 1H NMR (400 MHz,
D
CDCl3) d: 3.67 (3H, s, OCH3), 3.59 (1H, m, H-12), 2.30 (2H, t,
J = 7.6 Hz, H-2), 1.62 (2H, m, H-3), 1.20–1.52 (22H, m), 0.91 (3H,
t, J = 7.0 Hz, H-16); for 13C NMR (100 MHz, CDCl3) spectroscopic
data, see Table 3; EIMS m/z (rel. int.): 269 [M ꢀ OH]+ (1), 229 [M
ꢀ C4H9]+ (12), 200 [M ꢀ C4H8O]+ (20), 197 [M ꢀ C4H9 ꢀ MeOH]+
(60), 179 [M ꢀ C4H9 ꢀ MeOH – H2O]+ (14), 157 (31), 143 (42), 95
(47), 87 (100).
4.12. Synthesis of methyl 11-hydroxyheptadecanoate (15)
By a reported procedure (Jiang et al., 1995) 10-undecenoic acid
(1.0 g) was first converted to methyl 11-oxoundecanoate (300 mg),
which was subsequently treated with n-C6H13MgBr to give 11-hy-
droxy-heptadecanoic acid methyl ester (15, 140 mg): colorless oil;
1H NMR (400 MHz, CDCl3) d: 3.67 (3H, s, OCH3), 3.58 (1H, m, H-11),
2.30 (2H, t, J = 7.6 Hz, H-2), 1.61–1.28 (26 H, m), 0.88 (3H, t,
J = 6.3 Hz, H-17); 13C NMR (100 MHz, CDCl3) d: 174.3 (C-1), 72.0
(C-11), 51.4 (OCH3), 37.4 (C-10 and C-12), 34.1 (C-2), 31.8, 29.6,
29.5, 29.3, 29.3, 29.2, 29.1, 25.6 (C-9 and C-13), 24.9 (C-3), 22.6
(C-16), 14.0 (C-17); positive ion ESIMS m/z 301 [M+H]+.
4.10.3. (S)-12-Hydroxyheptadecanoic acid methyl ester [11-(S)]
20
Colorless oil, [
a
]
D
+0.6 (c 0.8, CHCl3); 1H NMR (400 MHz,
CDCl3) d: 3.67 (3H, s, OCH3), 3.59 (1H, m, H-12), 2.30 (2H, t,
J = 7.6 Hz, H-2), 1.62 (2H, m, H-3), 1.23–1.50 (24H, m), 0.93 (3H,
t, J = 6.8 Hz, H-17); for 13C NMR (100 MHz, CDCl3) spectroscopic
data, see Table 3; EIMS m/z (rel. int.): 283 [M ꢀ OH]+ (1), 229 [M
ꢀ C5H11]+ (17), 200 [M ꢀ C4H8O]+ (28), 197 [M ꢀ C5H11 ꢀ MeOH]+
(84), 179 [M ꢀ C5H11 ꢀ MeOH – H2O]+ (19), 157 (41), 143 (51), 95
(58), 87 (100).
4.13. Preparation of Mosher’s esters of 9-(S)–11-(S)
4.11. Synthesis of racemic 12-hydroxy fatty acid methyl esters 9–11
A
solution of (R)-a-methoxy-a-phenylacetic acid (MPA,
12.0 mg), 4-dimethylaminopyridine (DMAP, 10.0 mg), and N,N-
dicyclohexylcarbodiimide (DCC, 10.0 mg) in CH2Cl2 (1.0 mL) was
added to compound 9-(S) (5.0 mg) in CH2Cl2 (1.5 mL). The solution
was stirred for 20 h at 25 °C, treated with EtOAc (30.0 mL) and
filtered. The filtrate was concentrated and the residue purified by
4.11.1. Preparation of Grignard reagents
To a three-necked flask were added anhydrous THF (3 mL), Mg
(1.0 g, 41.7 mmol), and an iodine crystal at room temperature. A
solution of 1-bromopropane (2.0 mol/L in THF, 30 mmol) was then

W. Ding et al. / Phytochemistry 81 (2012) 165–174
173
silica gel CC eluted with cyclohexane–EtOAc (95:5) to give 16-(S,R)
(5.5 mg, 71%). Similarly, treatment of 9-(S) (5.0 mg) with (S)-MPA
yielded 16-(S,S) (6.0 mg, 78%). Using the same method, esterifica-
tion of 10-(S) and 11-(S) with (R)-MPA and (S)-MPA gave two pairs
of Mosher’s esters 17-(S,R)/17-(S,R) and 18-(S,R)/18-(S,S),
respectively.
4.14. Alkaline hydrolysis of 4 and 5 and analysis of short organic acids
Compound 4 (5 mg) was treated with 5% KOH (3 mL) at 85 °C
for 4 h. The reaction mixture was acidified to pH 4.0 and extracted
with Et2O (3 ꢁ 1 mL). The Et2O layer was dried (anhydr. MgSO4)
and concentrated to a small volume (about 0.2 mL) to afford a short
chain organic acid fraction. The aqueous layer was further ex-
tracted with n-BuOH (3 ꢁ 2 mL). The n-BuOH solution was concen-
trated in vacuo to yield a glycosidic acid (3 mg) which was
identified as turpethic acid A (1) on the basis of the 1H NMR spec-
troscopic data and HPLC analysis. By the same method, compound
5 (5 mg) afforded another short chain organic acid fraction and tur-
pethic acid B (3 mg, 2).
4.13.1. (S)-12-[(R)-a-Methoxy-a-phenylacetyloxy]pentadecanoic acid
methyl ester [16-(S,R)]
20
Colorless oil, [a]
D
–3.5 (c 0.25, CHCl3); 1H NMR (400 MHz,
CDCl3) d: 7.44 (2H, m, Ar-H), 7.33 (3H, m, Ar-H), 4.91 (m, H-12),
4.74 (1H, s, MPA-H- ), 3.67 (3H, s, 1-OCH3), 3.42 (3H, s, MPA-a-
a
OCH3), 2.30 (2H, t, J = 7.6 Hz, H-2), 1.62 (2H, m, H-3), 1.50 (2H,
m, H-11), 1.38 (2H, m, H-13), 1.22 (2H, m, H-10), 0.99 (2H, m, H-
14), 0.71 (t, J = 7.3 Hz, H-15); ESIMS m/z: 421 [M+H]+, 443 [M+Na]+.
Both short chain organic acid fractions obtained from 4 and 5
were analyzed by GC–MS using a Shimadzu GCMS-QP2000Plus
apparatus, equipped with a RxiÒ-5 ms fused silica capillary column
(30 m ꢁ 0.25 mm, 0.25
lm). The carrier gas was helium. Column
4.13.2. (S)-12-[(S)-
a
-Methoxy-
a
-phenylacetyloxy]pentadecanoic acid
temperature was initially 50 °C, increased to 160 °C at 20 °C/min,
then increased to 220 °C at 5 °C/min. For GC–MS detection, an elec-
tron ionization system was used with ionization energy of 70 eV.
Both fractions gave two predominant peaks which were identified
to be 2-methylbutyric acid (tR 3.14 min): EIMS m/z 102 [M]+ (0.8),
87 (24), 74 (100), 57 (64), 45 (16), and 41 (52), and tiglic acid (tR
3.70 min): EIMS m/z 100 [M]+ (100), 85 (29), 72 (0.8), 55 (86), 53
(16), and 39 (25), by comparison of their retention times (tR) with
those of authentic ( )-2-methylbutyric acid and tiglic acid (both
98%, Acros Organics, New Jersey).
methyl ester [16-(S,S)]
20
Colorless oil, [a]
D
+4.0 (c 0.25, CHCl3); 1H NMR (400 MHz,
CDCl3) d: 7.44 (2H, m, Ar-H), 7.33 (3H, m, Ar-H), 4.92 (1H, m, H-
12), 4.73 (1H, s, MPA-H- ), 3.67 (3H, s, 1-OCH3), 3.42 (3H, s,
MPA- -OCH3), 2.31 (2H, t, J = 7.5 Hz, H-2), 1.61 (2H, m, H-3), 1.49
a
a
(2H, m, H-13), 1.38 (2H, m, H-11), 1.27 (2H, m, H-14), 0.90 (2H,
m, H-10), 0.88 (3H, t, J = 7.3 Hz, H-15); ESIMS m/z: 421 [M+H]+,
443 [M+Na]+.
4.13.3. (S)-12-[(R)-a-Methoxy-
a-phenylacetyloxy]hexadecanoic acid
Subsequently, 2-methylbutyric acid in fractions obtained from
4 and 5 was further analyzed by GC with an Agilent 7890A GC sys-
methyl ester [17-(S,R)]
Colorless oil, [a]
D
–3.6 (c 0.25, CHCl3); 1H NMR (400 MHz,
20
tem using a Chir-
siloxane) chiral capillary column (Varian WCOT Fused Silica
CP7495, 25 m ꢁ 0.25 mm, 0.12 m), column temp.: 50 °C (hold
L-Val (N-propionyl-L-valine tert-butylamide poly-
CDCl3) d: 7.44 (2H, m, Ar-H), 7.33 (3H, m, Ar-H), 4.90 (1H, m, H-
12), 4.73 (1H, s, MPA-H- ), 3.67 (3H, s, 1-OCH3), 3.42 (3H, s,
MPA- -OCH3), 2.30 (2H, t, J = 7.6 Hz, H-2), 1.61 (2H, m, H-3), 1.50
a
l
a
2 min)?180 °C at 3 °C/min, carrier gas H2. The authentic sample
( )-2-methylbutyric acid gave two peaks at tR 17.05 min and tR
17.19 min. Each of the fractions from 4 and 5 and the authentic
(S)-(+)-2-methylbutyric acid (98%, Acros Organics, New Jersey)
gave a single peak at tR ꢂ17 min, and a mixture sample containing
two fractions and authentic (S)-(+)-2-methylbutyric acid provided
only one peak at tR 17.22 min. Thus, the absolute configuration of
2-methylbutyric acid in both fractions from 4 and 5 was deter-
mined to be S.
(2H, m, H-11), 1.38 (2H, m, H-13), 1.22 (2H, m, H-10), 1.07 (2H,
m, H-15), 0.90 (2H, m, H-14), 0.70 (3H, t, J = 7.3 Hz, H-16); ESIMS
m/z: 435 [M+H]+, 457 [M+Na]+.
4.13.4. (S)-12-[(S)-
a
-Methoxy-
a
-phenylacetyloxy]hexadecanoic acid
methyl ester [17-(S,S)]
Colorless oil, [a]
D
+3.6 (c 0.25, CHCl3); 1H NMR (400 MHz,
20
CDCl3) d: 7.44 (2H, m, Ar-H), 7.33 (3H, m, Ar-H), 4.90 (1H, m, H-
12), 4.74 (1H, s, MPA-H- ), 3.67 (3H, s, 1-OCH3), 3.42 (3H, s,
MPA- -OCH3), 2.31 (2H, t, J = 7.6 Hz, H-2), 1.62 (2H, m, H-3), 1.51
a
a
Acknowledgments
(2H, m, H-13), 1.37 (2H, m, H-11), 1.29 (2H, m, H-14), 1.25 (2H,
m, H-15), 0.90 (2H, m, H-10), 0.86 (3H, t, J = 7.0 Hz, H-16); ESIMS
m/z: 435 [M+H]+, 457 [M+Na]+.
We thank Mr. Ming Zhu, Zhuhai Action Pharmaceutical Science
and Technology Co. Ltd., Zhuhai, Guangdong, China, for the collec-
tion of plant material and Prof. Fuwu Xing, South China Botanical
Garden, Chinese Academy of Sciences for the authentication of
plant material. The author, Dr. Jiang, gratefully acknowledges the
support of K.C. Wong Education Foundation. This work was sup-
ported by the Knowledge Innovation Programs of the Chinese
Academy of Sciences (Grant Nos. KSCX2-EW-R-15 and KSCX2-
YW-R-218).
4.13.5. (S)-12-[(R)-a-Methoxy-
a-phenylacetyloxy]heptadecanoic acid
methyl ester [18-(S,R)]
Colorless oil, [a]
D
–3.6 (c 0.25, CHCl3); 1H NMR (400 MHz,
20
CDCl3) d: 7.44 (2H, m, Ar-H), 7.34 (3H, m, Ar-H), 4.90 (1H, m, H-
12), 4.73 (1H, s, MPA-H- ), 3.67 (3H, s, 1-OCH3), 3.42 (3H, s,
MPA- -OCH3), 2.30 (2H, t, J = 7.6 Hz, H-2), 1.62 (2H, m, H-3), 1.50
a
a
(2H, m, H-11), 1.38 (2H, m, H-13), 1.22 (2H, m, H-10), 1.10 (2H,
m, H-16), 1.05 (2H, m, H-15), 0.93 (2H, m, H-14), 0.77 (3H, t,
J = 6.9 Hz, H-17); ESIMS m/z: 449 [M+H]+, 471 [M+Na]+.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.phytochem.2012.
05.010.
4.13.6. (S)-12-[(S)-
methyl ester [18-(S,S)]
a
-Methoxy-
a
-phenylacetyloxy]heptadecanoic acid
+3.9 (c 0.25, CHCl3); 1H NMR (400 MHz,
20
Colorless oil, [a]
D
CDCl3) d: 7.44 (2H, m, Ar-H), 7.33 (3H, m, Ar-H), 4.90 (1H, m, H-
12), 4.73 (1H, s, MPA-H- ), 3.67 (3H, s, 1-OCH3), 3.42 (3H, s,
MPA- -OCH3), 2.31 (2H, t, J = 7.6 Hz, H-2), 1.62 (2H, m, H-3), 1.50
(2H, m, H-13), 1.38 (2H, m, H-11), 1.29 (2H, m, H-15), 1.25 (2H,
m, H-16), 1.23 (2H, m, H-14), 0.90 (2H, m, H-10), 0.86 (3H, t,
J = 6.8 Hz, H-17); ESIMS m/z: 449 [M+H]+, 471 [M+Na]+.
a
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