Steamed ginseng-leaf components enhance cytotoxic effects on human leukemia HL-60 cells

Article abstract of DOI:10.1248/cpb.58.1111

Three new dammarane-type glycosides, named ginsenosides SL ₁-SL₃ (1-3), and eleven known compounds (4-14) were isolated from the heat-processed leaves of Panax ginseng. Their structures were elucidated on the basis of extensive chemi

Full text of DOI:10.1248/cpb.58.1111

August 2010
Note
Chem. Pharm. Bull. 58(8) 1111—1115 (2010)
1111
Steamed Ginseng-Leaf Components Enhance Cytotoxic Effects on Human
Leukemia HL-60 Cells
Nguyen Huu TUNG,a,b Gyu Yong SONG,a Chau Van MINH,b Phan Van KIEM,b Long Guo JIN,a
c
c
,a
*
Hye-Jin BOO, Hee-Kyoung KANG, and Young Ho KIM
a College of Pharmacy, Chungnam National University; Daejeon 305–764, Korea: b Institute of Natural Products
Chemistry, Vietnam Academy of Science and Technology; 18 Hoang Quoc Viet, Nghiado, Caugiay, Hanoi 10000, Vietnam:
and c Department of Pharmacology, School of Medicine, Institute of Medical Sciences, Jeju National University; 66
Jejudaehakno, Jeju 690–756, Korea. Received April 28, 2010; accepted May 20, 2010; published online May 21, 2010
Three new dammarane-type glycosides, named ginsenosides SL1—SL3 (1—3), and eleven known compounds
(4—14) were isolated from the heat-processed leaves of Panax ginseng. Their structures were elucidated on the
basis of extensive chemical and spectroscopic methods. Cytotoxic-activity testing of compounds 1—14 against
human leukemia HL-60 cells showed that ginsenosides Rh3 (11) and Rk2 (12) exhibited potent effects with IC50
values of 0.8 and 0.9 mM. In addition, ginsenosides SL3 (3), 20S-Rg2 (7), F4 (10), 20S-Rh2 (13) displayed strong ac-
tivity with IC50 values of 9.0, 9.0, 7.5, and 8.2 mM, respectively. This is the first report on chemical components of
the steamed ginseng leaves.
Key words Panax ginseng; Araliaceae; ginsenoside SL1; ginsenoside SL2; ginsenoside SL3; cytotoxicity
Panax ginseng (C.A. MEYER, Araliaceae), an ancient and (20R)-ginsenoside Rg2 (8),10) ginsenoside Rg6 (9),11) ginseno-
famous herbal drug in oriental traditional medicine, has been side F4 (10),11) ginsenoside Rh3 (11),8) ginsenoside Rk2
used as a tonic and for the treatment of various diseases.1,2) (12),8) (20S)-ginsenoside Rh2 (13),12) and (20R)-ginsenoside
Biologically active constituents of whole parts of P. ginseng Rh2 (14),12) respectively (Fig. 2).
have been pursued extensively and many dammarane-type
triterpene oligoglycosides have been characterized as the lecular formula C36H62O11 as deduced by a high-resolution
principal components.1—4)
Fourier-transform ion-cyclotron-resonance mass spectrome-
Ginsenoside SL1 (1), an amorphous powder, has the mo-
Traditionally, the root of P. ginseng (ginseng), the most try (HR-FT-ICR-MS) experiment (Found at m/z [MϩNa]ϩ
used and valuable part, has been processed to make white 693.4141, Calcd for C36H62O11Na 693.4190). Acid hydroly-
ginseng (WG, roots air-dried after peeling) and red ginseng sis of 1 liberated D-glucose, confirmed by gas chromatogra-
(RG, roots steamed at 98—100 °C without peeling) to en- phy (GC) analysis. It was proposed to possess a hydroperoxyl
hance its preservation and efficacy. In which, RG is more group due to positive response to N,N-dimethyl-p-phenylene-
common as an herbal medicine than WG, because steaming diammonium dichloride reagent. The 1H-NMR spectrum of 1
induces changes in the chemical constituents and enhances showed signals due to the aglycone part [d 0.88, 1.07, 1.25,
the biological activities of ginseng.
1.43, 1.65, 1.92, 2.09 (3H each, s, H3-30, 19, 18, 21, 29, 27,
Extracts from roots and leaves have similar multifaceted
pharmacological activities (e.g. central nervous and cardio-
vascular systems). Moreover, in terms of costs and source
availability, ginseng leaf has advantages over its root.5) Nev-
ertheless, there has been no study concerning processed
leaves of this plant. In our ongoing research on P. ginseng,6,7)
it was found that a saponin extract of steamed leaves showed
potent cytotoxic effect on HL-60 cells. Subsequently, the cur-
rent study on chemical components of the steamed leaves led
to the isolation of three new damarane-type saponins, gin-
senosides SL1—SL3 (1—3), and eleven known ones (4—14).
Here, this paper deals with the structure elucidation of the
new ginsenosides SL1—SL3 (1—3) and evaluation of cyto-
toxic activity against HL-60 cells of all compounds.
Results and Discussion
The methanolic extract of the steamed leaves of P. ginseng
was suspended in H2O and partitioned with CH2Cl2. Then,
the H2O layer was subjected to a Diaion HP-20 column, fol-
lowed by various silica gel and YMC reversed-phase
columns to yield three new dammarane-type saponins, gin-
senoside SL1—SL3 (1—3) (Fig. 1), and eleven known ones
(4—14), including (20S)-ginsenoside Rh1 (4),8) ginsenoside
F1 (5),9) ginsenoside Rh4 (6),8) (20S)-ginsenoside Rg2 (7),10)
Fig. 1. Structures of Ginsenosides SL1—SL3 (1—3)
∗ To whom correspondence should be addressed. e-mail: yhk@cnu.ac.kr
© 2010 Pharmaceutical Society of Japan

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Vol. 58, No. 8
28), 3.54 (1H, dd, Jϭ11.6, 4.8 Hz, H-3), 3.98 (1H, m, H-12), 13C-NMR (Table 1) spectra of 2 due to the dammarane-type
4.42 (1H, m, H-6), 5.11 and 5.27 (1H each, br s, H-26)] and triterpene part and 6-O-b-D-[a-L-rhamnopyranosyl-(1→2)-b-
an anomeric proton at d 5.07 (d, Jϭ7.2 Hz, H-1Ј), which was D-glucopyranosyl] moiety were superimposable on those of
11)
assignable to a b-glucopyranosyl unit. The 13C-NMR spec- (20E)-ginsenoside F4 except for the signals of the side-
trum of 1 exhibited 36 signals including a set of six signals chain part (C-24—C-27), which were similar to that of 1.
(d 106.0, 75.4, 79.6, 71.8, 78.2, 63.0) revealing a b-D-glu- Moreover, comprehensive analyses of the 1H–1H COSY,
copyranosyl unit and 30 remaining ones of a sapogenol moi- heteronuclear multiple quantum coherence (HMQC), HMBC
ety. The signal of C-5 at d 61.4 is a characteristic of a pro- (Fig. 3), and ROESY spectra of 2 (Fig. 4) permitted complete
topanaxatriol-type aglycone common among dammarane- assignments of its NMR data as well as partial structures. E-
type saponins in P. ginseng with variations in its side-chain. Geometry of the double bond at C-20(22) of 2 was con-
Furthermore, the 1H- and 13C-NMR data of 1 were similar to cluded on the basis of the methyl carbon signal C-21 at d
those of (20R)-ginsenoside Rh113,14) except for the signals be- 13.2; whereas in case of Z-form, the chemical shift of C-21 is
longing to the side-chain part (C-24—C-27) of the aglycone, expected at ca. d 30.0,15) respectively. Hence, the structure of
which was identical to that of floralginsenosides A and C.4) ginsenoside SL2 (2) was identified as (20E)-24-hydroper-
20R-Configuration was suggested based on the 13C-NMR ev- oxyl-3b,6a,12b-trihydroxydammar-20(22),25-diene 6-O-a-
idence of C-17 at d 51.1 and C-21 at d 22.5, which were L-rhamnopyranosyl-(1→2)-b-D-glucopyranoside.
compatible with those of related structures.13,14) The pro-
Ginsenoside SL3 (3), an amorphous powder, has the mo-
posed structure of 1 was further confirmed by the 1H–1H cor- lecular formula C42H70O14 from a HR-FT-ICR-MS experi-
relation spectroscopy (COSY), heteronuclear multiple bond ment. The molecule of 3 was also proposed to have a hy-
correlation (HMBC) (Fig. 3), and rotating frame Overhauser droperoxyl group according to positive response to N,N-di-
effect spectroscopy (ROESY) spectra, respectively. As shown methyl-p-phenylenediammonium dichloride reagent. Like
1
in Fig. 2, the H–1H COSY experiment on 1 indicated the compound 2, the acid hydrolysis of 3 gave D-glucose and L-
presence of partial structures written in bold lines; and in the rhamnose. The H- and 13C-NMR (Table 1) spectra of 3 re-
1
HMBC spectrum, the long-range correlations were observed sembled those of ginsenoside Rg6 (9)11) except for the signals
between the following protons and carbons: H-6 and C-8; H- of the side-chain part (C-22—C-27). Furthermore, the struc-
1
12 and C-9,17; H-18 and C-7,9,14; H-19 and C-1,5,9; H-21 ture of 3, especially the side chain, was assigned by H–1H
and C-17; H-23 and C-20,25; H-24 and C-26; H-26 and C- COSY, HMQC, HMBC (Fig. 3), and ROESY spectra (Fig.
24; H-27 and C-24; H-1Ј and C-6. Consequently, the struc- 4). Accordingly, the location of hydroperoxyl group at C-23
ture of ginsenoside SL1 (1) was characterized as (20R)-24- and double bond at C-24 were assured from following
hydroperoxyl-3b,6a,12b,20a-tetrahydroxy-dammar-25-ene HMBC correlations: H-21 with C-22, H-22 with C-24, H-24
6-O-b-D-glucopyranoside.
with C-22,26, respectively. Thus, the structure of ginsenoside
Ginsenoside SL2 (2), also an amorphous powder, has the SL3 (3) was identified as 23-hydroperoxyl-3b,6a,12b-tri-
molecular formula C42H70O14 on the basis of a HR-FT-ICR- hydroxydammar-20(21),24-diene 6-O-a-L-rhamnopyranosyl-
MS experiment. Like compound 1, the molecule of 2 was (1→2)-b-D-glucopyranoside.
proposed to have a hydroperoxyl group due to positive re-
On the basis of traditionally-oriental medical theories, the
sponse to N,N-dimethyl-p-phenylenediammonium dichloride herbs need processing for different purposes. Like roots of
reagent. On the acid hydrolysis, it yielded D-glucose and L- P. ginseng, chemical compositions of the steamed leaves
1
rhamnose as identified by the GC procedure. The H- and were significantly different from those in the raw materials
(Fig. 5). Because steaming was carried out under high tem-
perature, the new monodesmosides should be formed by
chemical degradation of the C-20 glycosyl moiety of the
dammarane skeleton during the processing.
To evaluate the potential of the steamed-leaf components
for leukemia treatment, their cytotoxic activity was tested
against the HL-60 cell line, a type of human leukemia, using
the 3-(dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) assay.16) As the results, ginsenosides Rh3 (11)
and Rk2 (12) exhibited potent activity with IC50 values of 0.8
and 0.9 mM. In addition, new ginsenoside SL3 (3), ginseno-
sides 20S-Rg2 (7), F4 (10), and 20S-Rh2 (13) displayed strong
activity with IC50 values of 9.0, 9.0, 7.5, and 8.2 mM, respec-
tively. Besides, the activity of ginsenosides SL2 (2) and Rg6
(9) was relatively weak with IC50 values of 78.6 and 35.7 mM
as compared with mitoxantrone (MX) used as the positive
control with the IC50 value of 7.9 mM (Table 2). It is notewor-
thy that these components are unique in steamed leaves and
not found in non-processed samples as reported previously.6)
Structurally, it is suggested that variations in structures of
ginsenosides, especially the side-chain, influenced the cyto-
toxic activity against HL-60 cells. In particular, S stereo-
Fig. 2. Structures of Known Ginsenosides 4—14
specificity at C-20 in cytotoxic action against HL-60 cells

August 2010
1113
Table 1. 1H- and 13C-NMR Data for Ginsenosides SL1—SL3 (1—3) in Pyridine-d5
1
2
3
Position
dC
d
H (J in Hz)
dC
d
H (J in Hz)
dC
dH (J in Hz)
1
2
39.3
27.9
1.03 m
1.73 m
1.87 m
39.6
27.8
1.00 m
1.72 m
1.44 m
39.6
27.7
1.01 m
1.73 m
1.44 m
1.95 m
1.83 m
1.84 m
3
4
5
6
7
78.5
40.4
61.4
80.1
45.1
3.54 dd (11.6, 4.8)
78.4
40.0
60.8
74.5
46.0
3.50 dd (11.6, 5.2)
78.4
40.0
60.8
74.3
46.2
3.51 dd (11.6, 5.2)
1.42 d (9.2)
4.42 m
1.97 m
1.41 d (10.0)
4.70 m
2.00 m
1.42 d (10.0)
4.70 m
2.02 m
2.54 m
2.32 m
2.32 m
8
9
10
11
41.0
50.2
39.6
31.2
41.4
49.7
39.5
32.2
41.3
50.0
39.4
32.1
1.61 m
1.60 m
1.62 m
1.46 m
2.13 m
3.98 m
2.02 m
1.44 m
2.04 m
3.92 m
1.98 m
1.44 m
2.04 m
3.91 m
2.00 m
12
13
14
15
70.9
49.6
51.6
31.7
72.1
50.6
50.9
32.4
72.3
52.0
51.2
32.6
1.10 m
1.61 m
1.29 m
1.85 m
2.38 m
1.25 s
0.98 m
1.58 m
1.74 m
1.85 m
2.81 m
1.23 s
0.98 m
1.60 m
1.76 m
1.88 m
3.03 m
1.23 s
16
26.3
27.6
25.2
17
18
19
20
21
51.1
17.7
17.4
73.7
22.5
50.5
17.7
17.8
142.6
13.2
50.2
17.7
17.6
154.4
114.0
1.07 s
0.97 s
0.98 s
1.43 s
1.80 s
4.82 br s
5.12 br s
2.50 m
2.78 m
4.71 m
22
23
40.4
25.3
2.16 m
2.54 m
1.83 m
2.23 m
4.73 m
121.9
30.7
5.80 m
37.1
89.0
2.25 m
2.80 m
4.71 m
24
25
26
90.2
146.1
113.5
90.1
145.7
113.9
128.8
137.2
25.1
5.27 d (8.0)
1.38 s
5.11 br s
5.27 br s
1.92 s
2.09 s
1.65 s
5.08 br s
5.25 br s
1.88 s
2.13 s
1.34 s
0.97 s
5.28 d (7.2)
4.37 m
4.35 m
4.20 m
27
28
29
30
Glc-1Ј
2Ј
3Ј
4Ј
5Ј
6Ј
17.7
31.7
16.8
17.3
106.0
75.4
79.6
71.8
78.2
63.0
18.1
32.2
16.8
17.3
101.9
79.5
78.4
72.4
78.2
63.0
17.6
32.1
16.8
17.1
101.8
79.3
78.4
72.3
78.3
62.9
1.56 s
2.15 s
1.38 s
0.98 s
5.29 d (7.2)
4.38 m
4.34 m
4.22 m
3.98 m
0.88 s
5.07 d (7.2)
4.13 t (8.0)
4.28 t (8.4)
4.22 m
3.97 m
4.40 m
3.98 m
4.38 m
4.38 m
4.57 br d (11.2)
4.53 br d (11.2)
6.52 br s
4.81 br s
4.70 m
4.37 m
4.98 m
4.54 br d (11.2)
6.53 br s
4.82 br s
4.70 m
4.37 m
4.98 m
Rha-1Љ
2Љ
3Љ
4Љ
5Љ
102.0
71.9
72.2
74.0
69.4
18.8
101.9
72.1
72.4
74.0
69.4
18.7
6Љ
1.84 d (6.4)
1.82 d (6.4)
Assignments were confirmed by COSY, HMQC, HMBC, and ROESY spectra.
was observed since of two isomeric pairs at C-20 of ginseno- and Rh2 has been reported to show anti-cancer activity.17—19)
side Rg2 (7, 8) and ginsenoside Rh2 (13, 14), only the 20S-
isomers (7, 13) were strongly active.
Taken together, it is apparent that rich dammarane-type
monodesmosides presented are not only chemically charac-
After oral administration of ginseng, ginsenosides are well teristic of the steamed leaves but also give special biological
known to be metabolized by intestinal microflora, and thus activities to this processed herb. These results warrant further
intestinal bacterial metabolites of saponin extracts of studies concerning potential of saponin extracts of steamed
steamed ginseng-leaves as well as their activities should be ginseng-leaves for leukemia treatments.
investigated in further study. On the other hand, it is notice-
able that oral administration of ginsenosides such as Rg1, Rg3

1114
Vol. 58, No. 8
Fig. 3. COSY, Selected HMBC Correlations of Ginsenosides SL1—SL3 (1—3)
Fig. 5. HPLC/ELSD Profiles of Raw Leaves (A) and Steamed Leaves (B)
of P. ginseng
Key to peak identity: 1, Rg1; 2, Re; 3, F5; 4, F3; 5, Rb1; 6, Rc; 7, Rb2; 8, Rd; 9, F1; 10,
Rh1; 11, Rh2(20S); 12, Rh2(20R); 13, SL1; 14, Rh4; 15, Rg6; 16, F4; 17, SL2; 18, SL3;
19, Rg2(20S); 20, Rg2(20R); 21, Rk2; 22, Rh3.
Table 2. Cytotoxic Effects of Ginsenosides 1—14 on HL-60 Human
Leukemia Cells
Ginsenoside
IC50 (mM)a)
Fig. 4. Selected ROESY Correlations of 2 and 3
1
2
3
4
Ͼ100
78.6Ϯ1.6
9.0Ϯ0.4
Ͼ100
Experimental
General Procedures Optical rotations were obtained using a DIP-360
digital polarimeter (Jasco, Easton, MD, U.S.A.). IR spectra were measured
using a Perkin-Elmer 577 spectrometer (Perkin Elmer, Waltham, MA,
U.S.A.). NMR spectra were recorded on Bruker DRX 400 and 500 NMR
spectrometers (Bruker, Billerica, MA, U.S.A.). HR-FT-ICR-MS measure-
ments utilized a Variant 910 FT-ICR mass spectrometer (Varian, CA,
U.S.A.). GC (Shimadzu-2010, Kyoto, Japan) using a DB-05 capillary col-
umn (0.5 mm i.d.ϫ30 m) [column temperature: 210 °C; detector tempera-
ture: 300 °C; injector temperature: 270 °C; He gas flow rate: 30 ml/min
(splitting ratio: 1/20)] was used for sugar determination. Column chromatog-
raphy was performed on silica gel (70—230 and 230—400 mesh, Merck,
Darmstadt, Germany), YMC RP-18 resins (30—50 mm, Fuji Silysia Chemi-
cal Ltd., Aichi, Japan), and HP-20 Diaion (Mitsubishi Chemical, Tokyo,
Japan). TLC was performed on Kieselgel 60 F254 (1.05715; Merck, Darm-
stadt, Germany) or RP-18 F254s (Merck) plates. Spots were visualized by
5
6
7
8
9
10
11
12
Ͼ100
Ͼ100
9.0Ϯ0.5
Ͼ100
35.7Ϯ1.1
7.5Ϯ1.5
0.8Ϯ0.1
0.9Ϯ0.1
8.2Ϯ0.1
Ͼ100
13
14
Mitoxantroneb)
7.9Ϯ0.5
a) Results are the meansϮS.D. of three independent experiment in triplicate, and
values Ͻ100 mM are considered to be active. b) Positive control.

August 2010
1115
spraying with 10% aqueous H2SO4 solution, followed by heating.
analysis was run on Shimadzu LC-6AD (Kyoto, Japan) equipped with a Var-
Plant Material The leaves of P. ginseng were collected in Geumsan ian column (Polaris XRs5 C18, 250ϫ4.6 mm) and an ELSD-LT evaporative
province, which is well-known for P. ginseng cultivation in Korea, in August light scattering detector (Shimadzu, Kyoto, Japan). The separation was ef-
2008, and were taxonomically identified by one of the authors (Y. H. Kim). fected by gradient elution, using eluents (A) CH3CN : H2O : 5% acetic acid
Voucher specimens (CNU 08201) have been deposited at the College of (15 : 80 : 5) and (B) CH3CN : H2O (80 : 20) according to the following pro-
Pharmacy, Chungnam National University. The air-dried sample (1.0 kg) file: 0—10 min, 30% B (70% A); 10—25 min, 50% B; 25—40 min, 80% B;
was crushed finely and then steamed at 120 °C for 4 h under 0.15 MPa pres- 40—60 min, 100% B. The solvent flow rate was held constant at 1.0 ml/min
sure, without mixing with water, to give the steamed-leaf sample, which was at ambient temperature throughout the analysis.
used for extraction and isolation in this study.
Extraction and Isolation The steamed-leaf sample of P. ginseng was
Cytotoxic Assay Cell growth inhibition by different samples was ana-
lyzed using colorimetric MTT assay in HL-60 cell lilne. HL-60 cells were
extracted in MeOH (4.0 lϫ3, 50 °C) and the combined extracts were concen- obtained from the Korea Cell Ling Bank (KCLB, Seoul, Korea) and cultured
trated in vacuo to dryness. The MeOH residue (160 g) was suspended in in RPMI 1640 supplemented with 10% fetal bovine serum (GIBCO Inc.,
H2O (2.0 l), then partitioned with CH2Cl2 (2.0 lϫ3), and the water layer was Grand Island, NY, U.S.A.) and 100 U/ml penicillin and 100 mg/ml strepto-
subjected to a Diaion HP-20 column eluted with a gradient of MeOH in H2O mycin (GIBCO Inc., Grand Island, NY, U.S.A.) at 37 °C in a humidified 5%
(25, 50, 75, 100% MeOH; v/v) to give eight fractions (fr. 1.1—fr. 1.8). Next, CO2. Briefly, HL-60 cells were seeded into 96-well plates at a density of
fr. 1.6 (4.5 g) was chromatographed on a silica gel column using CHCl3– 3ϫ105 cells/well. The cells were then treated with the samples at concentra-
MeOH–H2O (10 : 3 : 0.4, v/v/v) to afford eleven subfractions (fr. 2.1—fr.
2.11). Fr. 2.1 (300 mg) was further chromatographed on a reversed-phase U.S.A.) was used as the positive control. After 3 d, the cells were treated
column with MeOH–H2O (4 : 1) to obtain ginsenoside Rh4 (6, 80 mg). Simi- with 50 ml MTT (2 mg/ml, Sigma Chemical Co., MO, U.S.A.). Plates were
tions ranging from 0.1 to 100 mM. Mitoxantrone (MX) (Sigma-Aldrich, MO,
larly, fr. 2.3 (350 mg) was subjected to a reversed-phase column with incubated at 37 °C for 4 h, the media was carefully aspirated. 150 ml Di-
MeOH–H2O (5 : 3) to afford ginsenoside Rh1 (4, 23 mg) and ginsenoside F1 methylsulfoxide (DMSO, Amresco, OH, U.S.A.) was then added to each
(5, 45 mg). Fr. 2.4 (330 mg) was repeatedly chromatographed on a reversed- well to dissolve the formazan crystals. The plates were read immediately at
phase column with MeOH–H2O (5 : 2) to furnish ginsenoside SL1 (1, 540 nm on a microplate reader (Amersham Pharmacia Biotech., NY,
13 mg), ginsenoside Rg6 (9, 15 mg), and ginsenoside F4 (10, 12 mg). Again, U.S.A.). All the experiments were performed at least three times in triplicate
fr. 2.7 (550 mg) was chromatographed on a reversed-phase column with and the mean absorbance values were calculated.
MeOH–H2O (5 : 2) to give ginsenoside SL2 (2, 15 mg), ginsenoside SL3 (3,
12 mg), (20S)-ginsenoside Rg2 (7, 14 mg), and (20R)-ginsenoside Rg2 (8,
20 mg).
Fr. 1.8 (10.0 g) was subjected to a silica gel column with CHCl3–MeOH–
Acknowledgements This study was supported by the Technology De-
velopment Program for Agriculture and Forestry (No. 108079-3), the Min-
istry for Agriculture, Forestry and Fisheries; and the Priority Research Cen-
H2O (7 : 1 : 0.1) to furnish five subfractions (fr. 3.1—fr. 3.5). Then, fr. 3.1 ter Program through the National Research Foundation of Korea (NRF)
(130 mg) was repeatedly chromatographed on a reversed-phase column with funded by the Ministry of Education, Science and Technology (2009-
MeOH–H2O (8 : 1) to give ginsenoside Rh3 (11, 18 mg) and ginsenoside Rk2 0093815), Republic of Korea. The authors would like to thank the Korean
(12, 20 mg). Finally, fr. 3.4 (350 mg) was chromatographed on a reversed- Basic Science Institute (KBSI) for taking NMR and MS experiments.
phase column with MeOH–H2O (6 : 1) to obtain (20S)-ginsenoside Rh2 (13,
25 mg) and (20R)-ginsenoside Rh2 (14, 15 mg).
References
Ginsenoside SL1 (1): White amorphous powder; [a]D20 ϩ12° (cϭ0.22,
MeOH); IR (KBr) nmax: 3448, 2922, 1637, 1262, 1054 cmϪ1; 1H-NMR (pyri-
dine-d5, 400 MHz) and 13C-NMR (pyridine-d5, 100 MHz): see Table 1; HR-
FT-ICR-MS m/z: 693.4141 [MϩNa]ϩ (Calcd for C36H62O11Na: 693.4190).
Ginsenoside SL2 (2): White amorphous powder; [a]D20 Ϫ9° (cϭ0.25,
MeOH); IR (KBr) nmax: 3436, 2931, 1634, 1260, 1068 cmϪ1; 1H-NMR (pyri-
dine-d5, 400 MHz) and 13C-NMR (pyridine-d5, 100 MHz): see Table 1; HR-
FT-ICR-MS m/z: 821.4596 [MϩNa]ϩ (Calcd for C42H70O14Na: 821.4663).
Ginsenoside SL3 (3): White amorphous powder; [a]D20 Ϫ11° (cϭ0.18,
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MeOH); IR (KBr) nmax: 3427, 2942, 1637, 1256, 1072 cmϪ1 1H-NMR
;
(pyridine-d5, 400 MHz) and 13C-NMR (pyridine-d5, 100 MHz): see Table 1;
HR-FT-ICR-MS m/z: 821.4660 [MϩNa]ϩ (Calcd for C42H70O14Na:
821.4663).
Acid Hydrolysis and Sugar Determination of Ginsenosides SL1—SL3
(1—3) A solution of each (2.0 mg) in 1.0 M HCl (3.0 ml) was heated under
reflux for 2 h. Then, the reaction mixture was concentrated in vacuo to dry-
ness. The residue was extracted with EtOAc and H2O (5 ml each, 3 times).
Next, the sugar residue, obtained by concentration of the water layer, was
dissolved in dry pyridine (0.1 ml). Then L-cysteine methyl ester hydrochlo-
ride in pyridine (0.06 M, 0.1 ml) was added to the solution. After heating the
reaction mixture at 60 °C for 2 h, 0.1 ml of trimethylsilylimidazole was 12) Baek N. I., Kim D. S., Lee Y. H., Park J. D., Jeong S. Y., Lee C. B.,
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evaporated in vacuo to give a dried product, which was partitioned between 13) Zhao P., Liu Y. Q., Yang C. R., Phytochemistry, 41, 1419—1422
hexane and H2O.6) The hexane layer was analyzed by the GC procedure
(1996).
(General Procedures). The peaks of the hydrolysate of the ginsenoside were 14) Teng R. W., Li H. Z., Chen J. T., Wang D., He Y., Yang C. R., Magn.
detected at tR 14.12 min for D-glucose in 1 and at tR 14.12 and 4.50 min for
D-glucose and L-rhamnose in 2 and 3, respectively. The retention times for
the authentic samples (Sigma), after being treated in the similar manner,
were 14.12 min (D-glucose), 14.25 min (L-glucose), and 4.50 min (L-rham-
nose). Co-injection of the hydrolysates of the ginsenoside with standard D-
glucose/L-rhamnose gave single peaks.
Reson. Chem., 40, 483—488 (2002).
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HPLC Analysis of Ginsenosides The standard ginsenosides were iso-
lated from leaves and steamed leaves as identified in this study and previous 19) Nakata H., Kikuchi Y., Tode T., Hirata J., Kita T., Ishii K., Kudoh K.,
report.6) The saponin fractions (20 mg) from the leaves and steamed-leaves
Nagata I., Shinomiya N., Jpn. J. Cancer Res., 89, 733—740 (1998).
were dissolved in MeOH (1 ml) and filtered by 0.45 mm syringe filter. HPLC