New polyacetylenes, DGAT inhibitors from the roots of Panax ginseng

Article abstract of DOI:10.1055/s-2004-815534

The petroleum ether extract of Panax ginseng showed a significant inhibition of the diacylglycerol acyltransferase (DGAT) enzyme from rat liver microsomes. Bioactivity-guided fractionation led to the isolation of two new polyacetylenic compounds, (9R,10S)-epoxyheptadecan-4,6-diyn-3-one (1) and 1-methoxy-(9R,10S)-epoxyheptadecan-4,6-diyn-3-one (2). Their chemical structures were elucidated on the basis of spectroscopic evidence and asymmetric synthesis. IC₅₀ values of 9 μg/mL (1) and 32 μg/ml (2) were obtained.

Full text of DOI:10.1055/s-2004-815534

Seung Woong Lee1
Koanhoi Kim1
Mun-Chual Rho1
Mi Yeon Chung1
Young Ho Kim2
Sangku Lee1
Hyun Sun Lee1
Young Kook Kim1
New Polyacetylenes, DGAT Inhibitors from the Roots
of Panax ginseng
Abstract
structures were elucidated on the basis of spectroscopic evi-
dence and asymmetric synthesis. IC50 values of 9 mg/mL #1) and
The petroleum ether extract of Panax ginseng showed a signifi- 32 mg/mL #2) were obtained.
cant inhibition of the diacylglycerol acyltransferase #DGAT) en-
zyme from rat liver microsomes. Bioactivity-guided fractiona- Key words
tion led to the isolation of two new polyacetylenic compounds, Panax ginseng ´ Araliaceae ´ diacylglycerol acyltransferase #DGAT) ´
#9R,10S)-epoxyheptadecan-4,6-diyn-3-one #1) and 1-methoxy- polyacetylenes ´ structure elucidation
#9R,10S)-epoxyheptadecan-4,6-diyn-3-one #2). Their chemical
Introduction
analeptic, stomachic, and erythropoietic agent in Korea, Japan
and China [4]. A number of polyacetylenic compounds, such as
Triglycerides represent the major form of stored energy in eukar- ginsenoynes A ± E [5], panaxydol [6], panaxytriol [7], and pa-
yotes. But, a high triglyceride level is known to be a major risk naxacol [8] were isolated from the roots of Panax ginseng.
factor for coronary heart disease, obesity and hypertriglyceride- These compounds have shown various biological activities in-
mia [1]. Triglyceride synthesis has been assumed to occur prima- cluding cytotoxicity [9], inhibition of iNOS production [10]
rily through acyl CoA; diacylglycerol acyltransferase #DGAT), a and inhibition of CETP #cholesteryl ester transfer protein) en-
microsomal enzyme that catalyses the final step in the glycerol zyme [11].
197
phosphate pathway, has been cloned [2]. Recently, the genera-
tion of DGAT-deficient mice has provided a better understanding In our screening for DGAT enzyme inhibitors from medicinal
of the relationship between triacylglycerol synthesis and obesity plants, a petroleum ether extract of Panax ginseng significantly
[3]. Therefore, DGAT is considered as a potential target of inhibi- inhibited activity of the DGAT enzyme that was prepared from
tion for the specific control of high triglyceride-induced disor- rat liver. Bioactivity-guided fractionation of the petroleum ether
ders, such as obesity or hypertriglyceridemia.
extract led to the isolation of two new polyacetylenic com-
pounds 1 and 2 #Fig.1).
Panax ginseng C.A. Meyer #Araliaceae) is one of the most impor-
tant oriental medicinal plants and has been employed as an
Affiliation
1 Laboratory of Lipid Metabolism, Korea Research Institute of Bioscience and Biotechnology, Taejon, Korea
2 College of Pharmacy, Chungnam National University, Taejon, Korea
Correspondence
Hyun Sun Lee, Ph.D. ´ Laboratory of Lipid Metabolism ´ Korea Research Institute of Bioscience
and Biotechnology ´ P.O. Box 115 ´ Yusong ´ Taejon 305±333 ´ Korea ´ Phone: +82-42-860-4314 ´
Fax: +82-42-861-2675 ´ E-mail: leehs@kribb.re.kr
Funding
This research was supported by grants from Plant Diversity Research Center of 21st Century Frontier Research
Program and KRIBB Research Initiative Program funded by the Ministry of Science and Technology of the
Korean government
Received August 15, 2003 ´ Accepted November 22, 2003
Bibliography
Planta Med 2004; 70: 197±200 ´ ꢀ Georg Thieme Verlag Stuttgart ´ New York ´ ISSN 0032-0943 ´
DOI 10.1055/s-2004-815534

Materials and Methods
showed the highest inhibitory effect and were further separat-
ed by low pressure liquid chromatography [Lichroprep CN Lo-
bar column, 40±63 mm, size B #310±25); flow rate, 5 mL/min;
General experimental procedures
Optical rotations were determined on a JASCO DIP-370 polarime- UV, 254 nm] with elution by hexane-CHCl3 #90:10, v/v) to give
ter. IR spectra were recorded on a JASCO A-102 spectrophoto- three sub-fractions. The second fraction #2.4 g) with the highest
meter. UV spectra were obtained on a Hewlett Packard model inhibitory effect was successively separated by semi-prepara-
8453 spectrophotometer. 1H-NMR #300 MHz), 13C-NMR #75 tive HPLC[YMCJ'sphere ODS H-80 column, 4 mm, 20”250 mm;
MHz), 1H-1H COSY, HMQC and HMBC spectra were obtained on flow rate, 7 mL/min; UV, 254 nm] with elution of 88% aqueous
a Varian Unity 300 spectrometer using benzene-d6 as solvent. MeOH to give 1 #250 mg) and 2 #50 mg).
HRFAB-MS was measured on a JEOL HX 110A/HX 100A spectro-
meter. Silica gel #230 ± 400 mesh, 0.040 ± 0.063 mm, Merck) Compound 1: C17H24O2; light yellow oil; [a]D25: ±70.08 #c 1.0,
was used for column chromatography and silica gel 60 F254 CHCl3); IR #neat/CHCl3): nmax = 1680 #C = O), 2240 #C = C)
#Merck) for TLC. Bovine serum albumin and sn-1,2-dioleoylgly- cm±1; UV #MeOH): lmax = 210, 228 #sh), 240, 253, 267, 283 nm;
cerol were obtained from Sigma Chemical Co #St. Louis. Mo). HRFAB-MS: m/z = 261.1852 #calcd. for C17H24O2 [M + H]+:
[1±14C]Palmitoyl CoA was purchased from Amersham Pharma- 261.1855); 1H-NMR #300 MHz, C6D6), 13C-NMR #75 MHz, C6D6),
cia Biotech Inc. The HPLCsystem consisted of a Shimadzu Model
LC-6AD pump and SPD-10A detector.
HMBCspectra: see Table 1.
Compound 2: C18H26O3; light yellow oil; [a]D25: ±75.58 #c 0.5,
CHCl3); IR #neat/CHCl3): nmax = 1220, 1700 #C= O), 2250
Extraction and isolation
The roots of 4-year-old Panax ginseng were purchased at Keum- #C= C) cm ±1; UV #MeOH): lmax = 208, 228 #sh), 243, 253, 269,
san Ginseng Market, Korea, in May 2001. The authenticity of the 285 nm; HRFAB-MS: m/z = 291.1655 #calcd. for C18H26O3 [M +
plant was confirmed by Prof. K. H. Bae, College of Pharmacy, H]+: 291.1660); 1H-NMR #300 MHz, C6D6), 13C-NMR #75 MHz,
Chungnam National University. A voucher specimen #CA-03± C6D6), HMBCspectra: see Table 1.
041) was deposited in the Korea Plant Extract Bank, Korea Re-
search Institute of Bioscience and Biotechnology. Fresh roots of Synthesis of 1
Panax ginseng #7.5 kg) were extracted twice with petroleum Sharpless asymmetric epoxidation of cis-2-decen-1-ol using di-
ether #15 L) at room temperature for 3 days. The petroleum ether ethyl D-tartrate afforded #2R,3S)-3-heptyloxiranemethanol in
extract #22 g) was subjected to silica gel #400 g) column chroma- > 99% ee after recrystallization from petroleum ether. #2R,3S)-3-
tography and eluted with a step gradient of hexane-EtOAc Heptyloxiranemethanol was converted to the corresponding tri-
#100:1, 70:1, 50:1, 30:1, 10:1, each of 2 L, v/v). Based on the flate utilizing trifluoromethanesulfonic anhydride. Then, the tri-
TLCprofile, eighteen fractions were collected and each fraction
flate was treated with the dilithio complex of hepta-4,6-diyn-3-
was monitored by the in vitro DGAT enzyme assay. Fractions ol formed by addition of propionaldehyde to lithium complex of
#F-8 to F-10, hexane-EtOAc, 50:1, 30:1 fractions, 5.72 g) 1,4-dichloro-2-butyne, to yield #9R,10S)-heptadeca-4,6-diyn-
198
Table 1 NMR data of 1 and 2 ꢀ75 MHz for 13C, 300 MHz for 1H, benzene-d6).
No.
1
2
13C
1H
MHBC ꢀH to C)
13C
1H
HMBC ꢀH to C)
1
2
3
8.09 ꢀq)
0.79 t ꢀ7.5)
2.06 q ꢀ7.5)
±
186.40, 39.11
186.40, 8.09
±
67.42 ꢀt)
3.33 t ꢀ6.0)
2.38 t ꢀ6.0)
±
58.70, 45.96, 184.20
39.11 ꢀt)
186.40 ꢀs)
±
45.96 ꢀt)
184.20 ꢀs)
±
67.42, 184.2
±
485.37 ꢀs)
±
85.67 ꢀs)
±
5
6
7
74.00 ꢀs)
66.41 ꢀs)
74.81 ꢀs)
±
±
±
±
74.02 ꢀs)
66.42 ꢀs)
75.20 ꢀs)
±
±
±
±
±
±
±
±
2.17 dd ꢀ5.7, 18.0)
85.37, 74.81, 74.00
2.14 dd ꢀ5.7, 18.0)
85.67, 75.20, 74.02
8
20.15 ꢀt)
20.13 ꢀt)
1.92 dd ꢀ6.6, 18.0)
66.41, 56.69, 53.91
20.15
1.87 dd ꢀ6.9, 18.0)
66.42, 56.67, 53.85
9
53.91 ꢀd)
56.69 ꢀd)
30.02 ꢀt)
29.91 ꢀt)
28.11 ꢀt)
2.70 ddd ꢀ5.7, 6.6, 4.0)
2.55 m
53.85 ꢀd)
56.67 ꢀd)
2.68 ddd ꢀ5.7, 6.6, 4.0)
2.53 m
20.13, 56.67
10
30.02, 29.91
28.08
11
1.13±1.29 m
1.13±1.29 m
1.13±1.29 m
±
28.08 ꢀt)
1.13±1.29 m
1.13±1.29 m
1.13±1.29 m
±
±
±
±
12
±
±
29.90 ꢀt)
13
20.01 ꢀt)
1427.16 ꢀt)
1.13±1.29 m
±
ꢀt) 27.141.13±1.29 m
32.47 ꢀt)
15
32.48 ꢀt)
23.39 ꢀt)
14.68 ꢀq)
±
1.13±1.29 m
1.13±1.29 m
0.90 t ꢀ6.9)
±
±
±
1.13±1.29 m
1.13±1.29 m
0.91 t ꢀ6.9)
2.96 s
±
16
23.39 ꢀt)
±
17
23.39, 32.48
14.63 ꢀq)
58.70 ꢀq)
32.47, 23.39
±
1-OCH3
±
Lee SW et al. New Polyacetylenes, DGAT¼ Planta Med 2004; 70: 197±200

9,10-epoxy-3-ol. Finally, the polyacetylenic alcohol, #9R,10S)- Compound 1 was obtained as a light yellow oil which showed
heptadeca-4,6-diyn-9,10-epoxy-3-ol, was oxidized to 1 by treat- [a]D25: ±70.08. The UV spectrum of 1 showed a typical absorption
ment with tetrapropylammonium perruthenate and 4-methyl- band for a diyne chromophore [14]. The IR spectrum of 1 showed
morpholine N-oxide. The details of total synthesis of 1 will be re- the presence of conjugated triple bonds #2240 cm-1) and a carbo-
ported elsewhere.
nyl group #1678 cm±1). In the HRFAB-MS, a peak corresponding to
[M + H]+ was observed at m/z = 261.18 which suggested the mo-
lecular formula of 1 to be C17H24O2. The 13C-NMR spectrum #Ta-
DGAT assay using rat liver microsomes
The DGAT assay was measured as reported previously with some ble 1) suggested the presence of 17 carbons: one carbonyl carbon
modifications [12]. In brief, the reaction mixture, containing #dC = 186.4), two methyl #dC = 8.09 and 14.68) and two oxygen-
175 mM Tris-HCl #pH 8.0), 8.0 mM MgCl2, 0.2 mM sn-1,2-diacyl- bearing methine carbons #dC = 53.91 and 56.69), four conjugated
glycerol, 0.25 mg of fatty acid-free bovine serum albumin, and acetylene carbons #dC = 66.41, 74.0, 74.81 and 85.37), and eight
30 mM [1±14C]palmitoyl-CoA #0.02 mCi) in a total volume of methylene carbons. The 1H-NMR spectrum of 1 #Table 1) showed
200 mL, was initiated by the addition of the rat liver microsomal the presence of terminal methyl protons, with the signals at
fraction, followed by gentle and brief vortexing. After incubation dH = 0.79 #3H, t, J = 7.5 Hz), 0.9 #3H, t, J = 6.9 Hz), methylene
for 10 min at 258C, the reaction was stopped by the addition of protons, with the signals at dH = 1.13 ± 1.29 #12H, m), and a me-
1.5 mL 2-propanol-heptane-water #80:20:2, v/v) and 1.0 mL hep- thylene proton, signals at dH = 2.06 #2H, q, J = 7.5 Hz). The pro-
tane and 0.5 mL water to extract lipids. After vortexing, 1.2 mL of tons of the epoxy group in 1 were determined by 1H-NMR as
the organic phase was transferred to a glass tube and washed once dH = 2.55 #1H, m), 2.70 #1H, ddd, J = 5.7, 6.6, 4.0 Hz), respective-
with 2.0 mL of an alkaline ethanol solution [ethanol-0.5 N NaOH- ly. The signals of methylene protons between a triple bond and
water #50:10:40, v/v)]. The amount of radioactivity was deter- an epoxide were observed at dH = 1.92 #1H, dd, J = 6.6, 18.0 Hz)
1
mined in a liquid scintillation counter #1450 micro beta TRIUX). and 2.17 #1H, dd, J = 5.7, 18.0 Hz) in the H-NMR spectrum of 1.
The samples were tested for DGAT inhibitory activity in three in- Also, we were able to observe that the methylene protons located
dependent experiments and evocarpine was used as a positive between a triple bond and the epoxide showed the long-range cor-
control [13]. All inhibitors were added as solutions in DMSO.
relations between H-8 and C-4, -5, -6, -7, -9, -10 in the HMBC ex-
periment #Table 1). The 1H-1H COSY spectrum of 1 showed the sig-
nal of correlated protons, dH = 0.79 and 2.06, and a germinal cou-
Data analysis
All values are expressed as means  standard error #SE). Statisti- pling between dH = 2.17 and 1.92. The synthetic strategy of 1 was
cal significance was set at p < 0.05 as determined by Student's based on establishment of the absolute configuration of a chiral
paired t test.
epoxy-alcohol and manipulation of the epoxy-alcohol to 1 without
loss of chirality. The synthetic compound was in good accord with
an authentic sample 1 in all aspects including 1H-, 13C-NMR, optical
rotation, and TLCin three different solvent systems. Therefore, the
absolute configuration of 1 was determined to be 9R and 10S by its
Results and Discussion
199
Extracts of herbal medicines were screened with an in vitro assay asymmetric synthesis. On the basis of these data the structure of 1
using rat liver microsomes as DGAT enzyme sources. We found was elucidated as #9R,10S)-epoxyheptadecan-4,6-diyn-3-one.
that the petroleum ether extract of the fresh roots of Panax
ginseng caused a relatively strong inhibition of DGAT enzyme ac- Compound 2 was obtained as a light yellow oil which showed
tivity. The petroleum ether extract of Panax ginseng roots was [a]D25: ±75.58. It displayed a peak at m/z = 291.1655 in the
fractionated by open column chromatography on silica gel, HRFAB-MS, corresponding to [M + H]+, indicating a molecular
MPLC, and semi-preparative HPLC to afford 1 and 2 #Fig.1).
formula of C18H26O3. The UV spectrum of 2 was similar to that of
1, thus, we deduced its structure as a polyacetylene with the
same chromophore. The IR spectrum of 2 showed the presence
of conjugated triple bonds #2250 cm±1), a carbonyl group #1700
cm-1), and C-O stretching band #1220 cm±1). The presence of a
1
methoxy group was noted in the H-NMR and 13C-NMR spectra
of 2 #Table 1), dH = 2.96 #3H, s) and dC = 58.70, respectively.
Also, HMBCcorrelations #Table 1) clearly revealed that the me-
thylene proton #dH = 3.33, H-1) is long-range coupled to the
methoxy group #dC = 58.70, C-1-OCH3), methylene carbon
#dC = 45.96, C-2), and carbonyl group #dC = 184.2, C-3). There-
fore, the methoxy group is attached at C-1 in 2. Optical rotation
and 1H-NMR experiments were used to determine the stereo-
chemistry of the epoxy group in 2. The sign of the optical rotation
of 2 was shown to be negative and the 1H-NMR spectrum of 2
displayed H-8 as a dd and dd at dH = 1.87 and 2.14 with J values
of 6.9, 18.0 and 5.7, 18.0 Hz, respectively. This splitting pattern of
the peaks in the 1H-NMR spectrum of 2 coincided with those of 1.
Therefore, absolute configuration of the epoxy group in 2 is 9R
and 10S and its structure was elucidated as 1-methoxy-#9R,10S)-
epoxyheptadecan-4,6-diyn-3-one.
Fig. 1 Structures of polyacetylenic compounds 1 and 2 isolated from
the root of Panax ginseng.
Lee SW et al. New Polyacetylenes, DGAT¼ Planta Med 2004; 70: 197±200

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The inhibitory effects of the compounds 1 and 2 on the DGAT ac-
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liver. They inhibited the DGAT activity dose-dependently with
IC50 values of 9 mg/mL #1) and 32 mg/mL #2), respectively #Fig. 2).
The inhibitory activities of 1 and 2 were confirmed by the posi-
tive control with evocarpine #IC50 value of 8.1 mg/mL) isolated
from fruits of Evodia rutaecarpa [13].
11
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13
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