Isolation, synthesis and structures of ginsenoside derivatives and their anti-tumor bioactivity

Article abstract of DOI:10.3390/molecules15010399

Protopanaxatriol saponins obtained with AB-8 macroporous resin mainly consisted of ginsenosides Rgi and Re. A novel mono-ester of ginsenoside-Rh ₁ (ginsenoside-ORh₁) was synthesized through further enzymatic hydrolysis and octanoyl chloride modifications. A 53% yield was obtained by a facile synthetic method. The structures were identified on the basis of ID-NMR and 2D-NMR, as well as ESI-TOF-MS mass spectroscopic analyses. The isolated and synthetic compounds were applied in an anti-tumor bioassay, in which ginsenoside ORhi showed moderate effects on Murine H22 Hepatoma Cells.

Full text of DOI:10.3390/molecules15010399

Molecules 2010, 15, 399-406; doi:10.3390/molecules15010399
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Isolation, Synthesis and Structures of Ginsenoside Derivatives
and Their Anti-Tumor Bioactivity
Mei Han 1,†, Jin-Gang Hou 1,†, Cheng-Ming Dong 2,*, Wei Li 1,3, Hao-Lun Yu 1, Yi-Nan Zheng 1,*
and Li Chen 3
1
College of Chinese Medicinal Materials, Jilin Agricultural University, Changchun 130118, China;
E-Mail: houjingang1225@126.com (J.-G.H.)
2
Department of Pharmaceutical Science, Henan College of Traditional Chinese Medicine,
Zhengzhou 450008, China
3
Norman Bethune College of Medicine, Jilin University, Changchun, 130000, China
†
These authors contributed equally to this work.
* Authors to whom correspondence should be addressed; E-Mails: dcm371@hactcm.edu.cn (C.-M.D.);
zhenyinan@tom.com (Y.-N.Z.); Tel.: +86-37-65680041, Fax: +86-37-65680011.
Received: 4 December 2009; in revised form: 8 January 2010 / Accepted: 11 January 2010 /
Published: 19 January 2010
Abstract: Protopanaxatriol saponins obtained with AB-8 macroporous resin mainly
consisted of ginsenosides Rg1 and Re. A novel mono-ester of ginsenoside-Rh1
(ginsenoside-ORh1) was synthesized through further enzymatic hydrolysis and octanoyl
chloride modifications. A 53% yield was obtained by a facile synthetic method. The
structures were identified on the basis of 1D-NMR and 2D-NMR, as well as ESI-TOF-MS
mass spectroscopic analyses. The isolated and synthetic compounds were applied in an
anti-tumor bioassay, in which ginsenoside ORh1 showed moderate effects on Murine H22
Hepatoma Cells.
Keywords: isolation; synthesis; ginsenoside-ORh1

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400
Introduction
Panax ginseng, which belongs to the Araliaceae family, has been used as a traditional medicine for
thousands of years and is now a popular natural medicine used worldwide [1,2]. Ginsenosides [3,4]
have been regarded as the principal components responsible for the pharmacological activities of
ginseng including anti-inflammatory activity, increasing the free radical scavenging activities and
reducing weight [5–9]. Recent studies pointed that some rare ginsenosides such as Rh1, M1 had strong
anticancer activity both in vivo and in vitro [10,11]. Pharmaceutical studies [12] have shown that
ginseng saponins were ingested mainly by the bacteria of the small intestine and thus converted into
their final forms: Re→Rg1→F1 or Rh1→M4; Rb1→Rd→F2→M1; Rb2→M6→M2→M1;
Rc→M7→M3→M1. Previous studies also showed that ginsenoside-M1 was further esterified with
fatty acids and thus could be sustained longer in the body, a result that suggested that the fatty acid
ester of the M1 might be the real anti-tumor active species in vivo [13]. Our laboratory previously
reported the synthesis of three novel mono-esters of ginsenoside-M1 (DM1, SM1, PM1) and their
bioactivity [14]. According to the previous study, we established a method for synthesizing ORh1 and
evaluated its bioactivity. Ginsenoside Rh1 was obtained through enzymatic hydrolysis and the fatty
ester was modified by octanoyl chloride. The structure of ginsenoside-ORh1 was identified through 1H-
13
and C-NMR and ESI-TOF-MS spectroscopic analyses. The pure ginsenoside-ORh1 was used to
determine the anticancer bioactivity against Murine H22 Hepatoma Cells; the results showed that
ginsenoside-ORh1 had moderate effects on H22 cells. This is the first time ginsenoside-ORh1 had been
synthesized and reported. In this paper, we present the synthesis method and the bioactivity evaluation
of the new ginsenoside fatty acid ester ORh1.
Results and Discussion
Characterizations of compounds 1-4
Compound 1 and compound 2 have been characterized as ginsenosides Rg1 and Re, whose
structures were identified by MS and NMR data analysis (not shown). After an enzymatic hydrolysis
process, we obtained ginsenoside Rh1 (Compound 3). Then, ginsenoside Rh1 was modified by octanoyl
chloride and formed ginsenoside fatty acid ester (compound 4). The structures of compounds 1-4 are
shown in Figure 1. The HPLC analysis of compounds 3 and 4 is shown in Figure 2. From the HPLC
13
analysis, the polarity of compound 4 was lower than that of Rh1 (compound 3). From the C-NMR
data analysis and comparison, we found no significant chemical shifts changes for the main skeleton
but an upfield shift of C’-6 of the 6-O-Glu (δ 63.5 to δ 64.8, see Table 1), indicating that the fatty acid
ester subsituent was connected to that position of 6-O-Glu was observed. This assumption had been
verified by HMBC (see Figure 3) which showed a cross-peak between H-6’ to the carboxyl carbon.

Molecules 2010, 15
Table 1. 13C-NMR Chemical Shifts(δ) of protopanaxatriol and compounds 3 and 4. a
401
No.(C)
1
Rh1
39.4
27.9
78.6
40.3
61.4
78.0
45.2
41.1
50.2
39.6
32.0
71.0
48.2
51.6
31.1
27.2
54.7
17.4
17.6
78.0
26.8
25.8
28.0
126.0
130.6
25.8
17.6
31.7
16.4
16.8
Compound 3
39.2
27.2
78.7
40.2
61.4
77.7
45.4
41.3
50.4
39.9
32.2
71.2
48.4
51.8
31.4
27.2
54.9
17.2
17.8
78.3
27.0
26.0
28.1
126.5
130.9
26.0
17.5
31.8
16.5
17.0
Compound 4
39.8
27.2
78.7
39.4
60.0
80.2
44.6
41.1
50.0
39.0
31.6
70.0
49.7
50.8
29.6
26.7
50.3
17.2
17.8
73.9
27.0
34.1
22.6
125.7
130.9
25.6
17.7
31.6
15.8
16.6
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
a Compounds 3 and 4 were measured in CDCl3 and chemical shifts are expressed in ppm.
Figure 1. Isolated, enzyme produced and synthetic ginsenosides.
R3
OH
R1
R2
Rg1: R1=-OH, R2=-Oglc, R3=-Oglc; Re: R1=-OH , R2= -Oglc (2-1) rha; R3=-Oglc; Rh1:R1=-OH,
R2=-Oglc, R3=OH; ORh1:R1=-OH,R2=-Oglc(6-1)-octanoyl ester;R3=OH.

Molecules 2010, 15
402
Figure 2. HPLC spectra of Rh1 (compound 3) and the reaction product of ORh1
(compound 4) HPLC conditions: isocratic elution with 100% MeOH for 60 min.
Figure 3. Partial HMBC and COSY correlation of the synthetic compound.
HO
OH
O
C
O
6'
O
HO
OH
COSY
O
HO
HMBC
OH
Table 2. 13C-NMR data of sugar and fatty acid ester moieties of compound 3 and 4.
No.(C)
1′
Rh1
105.7
75.4
80.0
71.8
79.5
63.5
Compound 3 Compound 4
106.2
75.6
80.2
72.0
79.8
63.3
104.4
74.0
80.1
72.0
79.2
64.8
174.3
34.1
24.9
30.3
29.1
30.9
22.6
14.1
2′
3 ′
4′
5′
6′
1′′
2′′
3′′
4′′
5′′
6′′
7′′
8′′
a Compounds 3 and 4 were measured in CDCl3 and chemical shifts are expressed in ppm.

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403
Bioactivity evaluation
The purified and synthetic compounds were tested in an anti-tumor bioassay. Compounds 3 and 4
showed moderate cytotoxicity effects against the Murine H22 Hepatoma Cells. The results are shown
in Table 3 and Figure 4. We tested the sample for four concentrations (10 μM, 20 μM, 40 μM, 80 μM).
The highest inhibitory rate of ORh1 was 87.16% at the concentration of 80 μM. The IC50 of ORh1 was
obtained at the concentration of 42.44 μM.
Table 3. Anti-tumor activities of compound 3 and 4 (IC50 values in μM).
Inhibitory rate (%)
Ginsenoside Rh1 ORh1
IC50(μM)
Concentration
(μM)
Ginsenoside Rh1
ORh1
0 μM
0
0
10 μM
8.97
9.28
11.44
11.09
62.34
87.16
20 μM
48.63
42.44
40 μM
54.21
76.54
80 μM
Figure 4. The rate of inhibition of ORh1 on H22 cell lines after 24 h.
Conclusions
The anti-tumor effects of some ginsenosides were known. For instance, Rg3 has been shown to
possess anti-tumor properties and have an effect on drug-resistant cultured cancer cells [15,16]. Rh2
can reduce the proliferation of a variety of cultured cancer cells and can influence apoptosis [17–19].
Most of the ginsenosides with significant anti-tumor activities belongs to protopanaxdiol-type saponins.
In our study, we investigated the protopanaxtriol-type ginsenoside Rh1. The protopanaxtriol-type
saponins, including Rg1 and Re, were finally transformed into ginsenoside Rh1 after intestinal
metabolism. We obtained the ginsenoside Rh1 by means of enzymolysis and further chemical
modification led to the synthesis of Rh1 mono-fatty acid ester. The bioactivity evaluation of fatty acid

Molecules 2010, 15
404
ester (ORh1) showed that this kind of compound was more active against human tumor cells. We
concluded that the membrane transportation of small molecules depends on their lipophilic abilities
and the fatty acid ester (ORh1) with lower polarity met this requirement. In this study, we synthesized
ORh1 with fatty acid acyl-chlorides and the synthetic product had the desired lower polarity. In this
study, ORh1 showed more activity and higher anti-tumor efficiency than ginseonside Rh1 on Murine
H22 Hepatoma Cells, and as time increased (more than 24 hr), the anti-tumor action of Rh1 was almost
eliminated, whereas ORh1 still displayed considerable anti-tumor action.
Experimental
General
1
13
The H- and C-NMR spectra were measured on a Bruker AV 400 NMR spectrometer in CDCl3,
using TMS as an internal standard. Chemical shifts (δ) are expressed in parts per million (ppm). The
HR-ESI-TOF mass spectra were obtained from a MDS SCIEX API QSTAR-MS instrument. Column
chromatographies were carried out with silica gel 60M (200-300 mesh) and AB-8 macroporous resin
and HPLC were carried out on an LC-2010 system (Shimadzu).
Chemicals and reagents
Octanoyl chloride was purchased from ABCR GmbH& Co. KG. Enzyme (snailase) was purchased
from BioDee BioTech Corporation Ltd. (code: S0100, Beijing, China). Other chemicals and reagents
were purchased from the Chinese Chemical Group (Beijing, China).
Extraction and isolation
Extracts of leaves from Panax ginseng (100 g) was dissolved with sufficient amount of distilled
water and filtered, then the filtrate was absorbed on a AB-8 macroporous resin column (100 × 10 cm)
for 8 h. Gradient elution with 25%, 30%, 80% ethanol was used to elute the column. The dry eluate
was obtained from 30% ethanol fraction and weighted (45 g). The fraction consisted of the
o
protopananxtriol-type saponins Ginsenoside Rg1 (1), a white amorphous powder, mp 194–196 C;
o
ESI-MS[+]: m/z=801 [M+H]+ and Ginsenoside Re (2), a white amorphous powder, mp 201-203 C;
ESI-MS[+]: m/z=969.3 [M+Na]+.
Enzymatic reaction
A saponin fraction (1 g) obtained from the former procedure was weighed out and dissolved in
distilled water (400 mL), enzyme (110 mg) was added and the mixture was cultured at 37 °C for 24 h
while the pH was maintained at a value of 4.5. Then the enzyme reaction mixture was subjected to
silica gel column chromatography eluting with CHCl3-MeOH (9:1) to afford purified Ginsenoside Rh1
(3, 320 mg) as a white amorphous powder, mp 190–192 °C; ESI-MS[+]: m/z=639 [M+H]+; 13C-NMR
data, see Table 1 and Table 2.

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Synthesis of Ginsenoside ORh1 (4)
Octanoyl chloride (125 mg) and K2CO3 (80 mg) were dissolved in CHCl2 (80 mL) under
ice-cooling and stirred for 20min, then ginsenoside Rh1 (100 mg) dissolved in CHCl2 (1 mL) was
added slowly. The mixture was reacted under stirring and with ice-cooling state for 24 h. The reacted
solution was filtered through a 0.22 μm membrane, then the filtrate was condensed and subjected to
silica gel column chromatography, eluted with CHCl3-MeOH=10:1 to give pure 4 (66 mg) as a
colorless oil; HR-ESI-TOF-MS [+]+: m/z=764.2533 [M+H]+; 13C-NMR data, see Table 1 and Table 2.
The purity was analyzed by HPLC (100%MeOH, Hypersil C18, 35 °C, 0.5 mL/min). The synthesis
procedure is shown in Scheme 1.
Scheme 1. Preparation of ginsenoside ORh1 (4).
HO
HO
OH
OH
O
ice-cooling
O
HO
HO
CH3(CH2)6
Cl
H2C
HO
O C(CH2)6CH3
O
stirring
CH2OH
O
O
O
OH
OH
OH
OH
OH
In vitro anti-tumor assays
Mono-nuclear cell direct cytotoxicity assay against human tumors cell lines were carried out at the
Cell Culture Laboratory, Pharmaceutical College, Jilin University, using Murine H22 Hepatoma Cells.
A blank control was used in this study. Generally, 5 × 105/mL cells (190 μL) were placed in a 96-well
plate and treated with obtained compound (10 μL). The normal cell was added culture medium. The
plate was incubated at 37 °C for 24 h.Then, each well was added MTT (20 μL) with the concentration
of 5 mg/mL and incubation at 37 °C was continued for 4 h. After that, the supernatant was removed
and DMSO (150 μL) was added, the samples were agitated and the absorbance read at 490 nm.
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