Saponins from the processed rhizomes of Polygonatum kingianum

Article abstract of DOI:10.1248/cpb.57.1011

Two new spirostanol saponins, named kingianoside H (1) and kingianoside I (2), were isolated from the processed rhizomes of Polygonatum kingianum, along with a known triterpenoid saponin ginsenoside-Rc (3), four known spirostanol saponins Tg (4), (5), pol

Full text of DOI:10.1248/cpb.57.1011

September 2009
Notes
Chem. Pharm. Bull. 57(9) 1011—1014 (2009)
1011
Saponins from the Processed Rhizomes of Polygonatum kingianum
He-Shui YU,a,b Bai-Ping MA, ,a Li-Ping KANG,a Tao ZHANG,a Feng-Juan JIANG,a Jie ZHANG,a
*
c
Peng ZOU,a Yang ZHAO,a Cheng-Qi XIONG,a Da-Wei TAN,a Xin-bo SONG,b and Kate YU
b
a Beijing Institute of Radiation Medicine; Beijing 100850, People’s Republic of China: Tianjin University of Traditional
Chinese Medicine; Tianjin 300193, People’s Republic of China: and c Waters Corporation; Milford, MA 01757, U.S.A.
Received April 30, 2009; accepted June 19, 2009; published online June 30, 2009
Two new spirostanol saponins, named kingianoside H (1) and kingianoside I (2), were isolated from the
processed rhizomes of Polygonatum kingianum, along with a known triterpenoid saponin ginsenoside-Rc (3), four
known spirostanol saponins Tg (4), (5), polygonatoside C1 (6) and ophiopogonin Cꢀ (7). The structures of the new
compounds were elucidated by detailed spectroscopic analyses, including 1D and 2D NMR techniques and chem-
ical methods. Compounds 3 and 5 were first reported from the genus Polygonatum. Compounds 4, 6 and 7 are re-
ported for the first time from the processed Polygonatum kingianum.
Key words Polygonatum kingianum; processing; steroidal saponin; triterpenoid saponin; identification
The rhizomes of Polygonatum kingianum COLL. et HEMSL. their structures elucidated by 1D and 2D NMR in combi-
(Liliaceae), one of the original plants known as Huang-jing nation with MS studies. Compounds 3—7 were known
in traditional Chinese medicine, were used as a tonic remedy saponins and identified by comparison of their NMR data
to treat lung troubles and ringworm.1) Clinically, the with those reported in the literature. Compound 3 is a known
processed rhizomes products (processed with yellow rice triterpenoid saponin and its structure was identified as (20S)-
wine) were routinely used, for it is widely believed that this
process would enhance the effect of tonic remedy.
Previous phytochemical investigations on the fresh rhi-
zomes of several Polygonatum species have resulted in the
isolation of steroidal saponins and triterpenoid saponins,2—8)
but no systematic study on the chemical constituents of the
processed products has been reported so far. We had previ-
ously reported some steroidal saponins from the fresh rhi-
zomes of P. kingianum.9,10) As a part of our ongoing research,
we focused on investigating the variation chemical con-
stituents of the fresh and the processed rhizomes. Our pre-
liminary phytochemical studies on processed P. kingianum
had indicated that the amounts of certain chemical con-
stituents are higher in processed P. kingianum than those in
fresh P. kingianum. Meanwhile, some other chemical con-
stituents are lower and a few are new. Furthermore, the ultra
performance liquid chromatograph (UPLC)-MS profile of
the processed products revealed that a few constituents that
were not reported previously. Therefore, our detailed chemi-
cal investigation on the processed P. kingianum led to the iso-
lation of two new spirostanol saponins, along with a known
triterpenoid saponin and four known spirostanol saponins.
Compounds 3 and 5 were reported for the first time from the
genus Polygonatum. These compounds are reported from the
processed P. kingianum for the first time. In this paper, we
describe the isolation and structure elucidation of the two
new saponins on the basis of extensive spectral analyses, in-
cluding 1D (dimensional) and 2D NMR spectral data and
chemical evidences.
Results and Discussion
The crude extract of the processed P. kingianum was frac-
tionated by using a combination of macroporous resin, silica-
gel and octadecyl silica (ODS) silica-gel column chromatog-
raphy and semi-preparative HPLC to afford compounds 1—
7.
Compounds 1 and 2 were found to be new saponins and
© 2009 Pharmaceutical Society of Japan
∗ To whom correspondence should be addressed. e-mail: ma_bp@sohu.com

1012
Vol. 57, No. 9
1
protopanaxadiol-3-O-b-D-glucopyranosyl-(1→2)-b-D-glu- evidence for the 24S and 25R configurations. The H- and
copyranoside-20-O-a-L-arabinofuranosyl-(1→6)-b-D-glu- 13C-NMR data for the F-ring of 1 was identical with those of
copyranoside (ginsenoside-Rc).11) Compounds 4—7 are the compound 216) in the literature, also provided evidence
known spirostanol saponins and their structures were identi- for the 24S and 25R configurations. In the heteronulcear mul-
fied as (25R)-spirost-5-en-3b,17a-diol-3-O-a-L-rhamnopyra- tiple bond coherence (HMBC) spectrum of 1 (Fig. 1), the
nosyl-(1→4)-a-L-rhamnopyranosyl-(1→4)-[a-L-rhamnopy- long range correlations between the carbon signal d 66.4 (C-
ranosyl-(1→2)]-b-D-glucopyranoside (Tg),12) (25R)-spirost- 24) and d 2.05 (H-23) and 3.58 (H-26) were confirmed the
5-en-3b,17a-diol-3-O-b-D-glucopyranosyl-(1→3)-[a-L- location of the one hydroxyl group at C-24. Furthermore, the
rhamnopyranosyl-(1→2)]-b-D-glucopyranoside,13)
(25R)-
Table 1. 1H- and 13C-NMR Data of Compounds 1 and 2 (d in Pyridine-
d5)a)
spirost-5-en-3b,17a-diol-3-O-a-L-arabinofuranosyl-(1→4)-
[a-L-rhamnopyranosyl-(1→2)]-b-D-glucopyranoside (poly-
gonatoside C1)14) and (25R)-spirost-5-en-3b-ol-3-O-a-L-
rhamnopyranosyl-(1→4)-b-D-glucopyranoside (ophiopogo-
nin Cꢀ),15) respectively.
1
2
Position
dC
d
H J (Hz)
dC
dH J (Hz)
Compound 1 was obtained as a white amorphous powder.
It gave positive Liebermann–Burchard and negative Ehrlich
reagent tests, which suggested that 1 was a spirostanol
saponin. The molecular formula was determined as
C39H60O15 by the negative-ion HR-electrospray ionizatoin
(ESI)-MS (m/z 767.3854 [MꢁH]ꢁ). The positive ion FAB-
MS also showed the characteristic fragment ion peaks at m/z:
769.4 [MꢂH]ꢂ, 607.3 [MꢂHꢁ162]ꢂ, 445.2 [MꢂHꢁ
162ꢁ162]ꢂ, suggesting the existence of two hexose units in
the molecule. Compound 1 was hydrolyzed with acid to af-
Aglycone
1
2
3
37.0 1.47 m, 0.87 m
30.0 2.06 m, 1.63 m
77. 7 3.83 m
37.0 1.47 m, 0.86 m
30.0 2.06 m, 1.65 m
77.7 3.85 m
4
39.1 2.67 m, 2.35 m
39.1 2.67 m, 2.40 m
5
140.8
—
140.8
—
6
121.4 5.27 br s
121.4 5.27 br s
31.7 1.46 m, 1.84 m
30.9 1.80 m
7
8
9
10
11
31.7 1.46 m, 1.84 m
30.9 1.82 m
52.3 1.29 m
52.3 1.29 m
37.6
—
37.6
—
1
37.5 2.50 m, 2.28 dd (6.0, 14.4) 37.5 2.49 m, 2.27 br d
(9.0)
ford D-galactose and D-glucose. The H-NMR spectrum of
1 revealed the presence of two singlet methyl signals at d
0.89 (3H, s, 19-CH3) and 1.07 (3H, s, 18-CH3), and two dou-
blet methyl signals at d 1.29 (3H, d, Jꢃ6.6 Hz, 27-CH3) and
1.38 (3H, d, Jꢃ6.6 Hz, 21-CH3), which were characteristic of
spirostanol saponin methyls. Furthermore, an olefinic proton
at d 5.27 (H, br s, H-6) was assigned, same for two anomeric
protons at d 4.86 (1H, d, Jꢃ7.2 Hz) and 5.28 (1H, d,
Jꢃ8.4 Hz). The 13C-NMR spectrum of 1 showed two
anomeric carbon signals at d 102.9 and 107.2, as well as two
olefinic carbon signals at d 140.8 and 121.4. Comparing the
13C-NMR data of 1 with that of pratioside D1,4) significant
differences of chemical shifts in F-ring (d 109.3, 31.7, 29.2,
30.5, 67.0, 17.3) indicated that 1 had one hydroxyl group at-
12 212.5
—
—
212.5
54.9
—
—
13
14
15
16
17
18
19
20
21
54.9
56.0 1.41 m
31.5 2.06 m, 1.58 m
80.1 4.45 m
53.6 2.78 dd (6.6, 8.4)
15.8 1.07 s
18.8 0.89 s
43.2 1.97 m
13.7 1.38 d (6.6)
56.0 1.41 m
31.5 2.06 m, 1.58 m
80.1 4.45 m
53.6 2.78 dd (7.2, 8.4)
15.8 1.07 s
18.8 0.89 s
43.2 1.97 m
13.7 1.38 d (6.6)
111.5
22 111.5
—
—
23
36.0 2.05 m, 2.15 dd (12.6, 12.0) 36.0 2.05 m, 2.15 dd
(12.6, 12.0)
24
25
26
66.4 4.62 m
66.4 4.62 m
35.9 2.03 m
64.6 4.04 br d (10.2),
3.58 br d (10.2)
9.7 1.28 d (7.8)
35.9 2.03 m
64.6 4.03b) o, 3.56 br d (9.6)
1
tached at F-ring of the spirostanol skeleton. The H–1H cor-
relation spectroscopy (COSY) spectrum was carefully in-
spected to assign the structure of the F-ring part, with the
three-proton doublet signal at d 1.29 (3H, d, Jꢃ6.6 Hz) at-
tributable to 27-CH3, being used as the starting point of
analysis. As a result, the structural fragment of the F-ring,
–C(23)H2–C(24)H(–O–)–C(25)H (–C(26)H2–O–)–CH3(27), was re-
vealed and the location of one hydroxyl group at C-24 was
evident. The proton signals of H-23ax at d 2.15 dd (Jꢃ12.0,
12.6 Hz) and signal of H-26ax at d 2.15 br d (Jꢃ9.6 Hz) gave
27
9.7 1.29 d (6.6)
Glycone
Gal
1
2
102.9 4.86 d (7.2)
73.5 4.38 dd (7.2, 9.0)
75.4 4.23 m
102.7 4.88 d (7.2)
73.3 4.48 m
75.6 4.09 m
81.0 4.57 m
76.8 4.06 m
3
4
80.0 4.69 br s
5
76.0 4.04 m
6
61.0 4.23 m, 4.63 m
60.4 4.73 m, 4.19 m
Glc
1
2
107.2 5.28 d (8.4)
75.2 4.13 dd (8.4, 9.0)
78.7 4.22 m
105.2 5.14 d (7.8)
86.2 4.15 m
78.5 4.27 m
3
4
72.3 4.06 m
71.9 3.96 m
5
78.5 4.01 m
78.2 3.97 m
6
63.2 4.20 m, 4.60 m
63.2 4.09 m, 4.62 m
Glcꢀ
1
2
107.0 5.23 d (7.8)
75.2 3.98 m
3
77.8 4.12 m
4
70.3 4.22 m
5
79.0 3.81 m
6
61.6 4.57 m, 4.37 m
a) The assignments were based on the 1H-NMR, 13C-NMR, 1H–1H COSY, HSQC
and HMBC experiments. b) o: overlapping, indicates overlapping signals.
Fig. 1. The Key HMBC Correlations of Compound 1

September 2009
1013
raphy.
anomeric proton signals at d 4.86 (H-1 of the galactose) and
5.28 (H-1 of the glucose) showed correlations with the car-
bon signals at d 77.7 (C-3) and 80.0 (C-4 of the galactose),
respectively. The full assignments of these sugar signals were
Plant Material The material was collected from Fenggang county of
Guizhou province, People’s Republic of China in December 2006, and was
identified as rhizomes of Polygonatum kingianum COLL. et. HEMSL. by Prof.
Li-juan Zhang of the Tianjing University of Traditional Chinese Medicine.
The processed products were processed according to the procedures from
the Chinese pharmacopoeia: the dried fresh rhizomes of P. kingianum were
mixed thoroughly with yellow rice wine with 5 : 1 ratio and kept in a con-
tainer with cap tightly closed till the wine was all absorbed. The wine
soaked rhizomes well and steamed thoroughly according to the specification
of the procedures, afterwards samples were cooled to room temperature and
cut to thin slices. Finally dried for 48 h at 50 °C and cooled to room tempera-
ture to become the processed samples. A voucher specimen (No. 061201)
was deposited in the herbarium of Beijing Institute of Radiation Medicine,
Beijing.
1
confirmed by H–1H COSY, heteronuclear single quantum
coherence (HSQC) and HMBC experiments. Therefore, the
structure of 1 was determined to be (24S, 25R)-3b,24-di-
hydroxy-spirostan-5-en-12-one-3-O-b-D-glucopyranosyl-
(1→4)-b-D-galactopyranoside, and named kingianoside H.
Compound 2 was obtained as a white amorphous powder.
It gave positive Liebermann–Burchard and negative Ehrlich
reagent tests, which suggested that 2 was a spirostanol
saponin. The molecular formula was determined as
C45H70O20 by the negative-ion HR-ESI-MS (m/z 929.4382
Extraction and Isolation The decoction pieces of processed P.
kingianum (5.5 kg) were extracted for three times with 45% Me2CO. The
[MꢁH]ꢁ). The positive ion FAB-MS also showed the charac- combined extract was concentrated under reduced pressure to give 72.0 g of
teristic fragment ion peaks at m/z: 931.4 [MꢂH]ꢂ, 769.4
residue. The extract was fractionated by macroporous resin SP825 and
eluted with a gradient mixture of Me2CO–H2O (10 : 90, 40 : 60, 80 : 20), to
give three fractions (Fr. A—C). Fraction A was further purified on a macrop-
orous resin SP825 column and eluted with gradient mixtures of
[MꢂHꢁ162]ꢂ, 607.3 [MꢂHꢁ162ꢁ162]ꢂ, 589.3 [MꢂHꢁ
162ꢁ162ꢁ18]ꢂ, 445.3 [MꢂHꢁ162ꢁ162ꢁ162]ꢂ, 427.3
[MꢂHꢁ162ꢁ162ꢁ162ꢁ18]ꢂ, suggesting the existence of
Me2CO–H2O (25 : 75, 35 : 65, 60 : 40), to give three fractions, A1 (1.8 g), A2
(0.9 g) and A3 (4.0 g). A part of fraction A3 (3.6 g) was chromatographed on
three hexose units in the molecule. Compound 2 was hy-
silica-gel with
a CHCl3–MeOH–H2O solvent system (15 : 1 : 0.01→
drolyzed with acid to afford D-galactose and D-glucose. The
1H-NMR spectrum of 2 revealed the presence of two singlet
methyl signals at d 0.89 (3H, s, 19-CH3) and 1.07 (3H, s, 18-
CH3), and two doublet methyl signals at d 1.28 (3H, d,
Jꢃ7.8 Hz, 27-CH3) and 1.39 (3H, d, Jꢃ7.2 Hz, 21-CH3),
which were recognized as typical spirostanol saponin
methyls. Furthermore, an olefinic proton at d 5.26 (H, br s,
H-6) was readily assigned, as well as signals for three
anomeric protons at d 4.88 (1H, d, Jꢃ7.2 Hz), 5.14 (1H, d,
Jꢃ7.8 Hz) and 5.25 (1H, d, Jꢃ9.6 Hz). The 13C-NMR spec-
trum of 2 showed three anomeric carbon signals at d 102.7,
105.2 and 107.0, in addition, two olefinic carbon signals at d
2 : 1 : 0.01), and fractions A3-96—106 was separated by semi-preparative
HPLC with MeOH–H2O (62 : 38), to yield compound 1 (8.9 mg), fractions
A3-107—156 was separated by semi-preparative HPLC with MeOH–H2O
(60 : 40), to yield compound 2 (9.6 mg), fractions A3-205—220 were further
separated by semi-preparative HPLC with MeCN–H2O (22 : 78) to yield
compounds 3 (7.3 mg). Fraction C (2.4 g) was subjected to column chro-
matography on ODS silica-gel with a MeCN–H2O solvent system (45 : 55,
48 : 52, 52 : 48), to yield compound 4 (fractions C-71—73) (36.3 mg). Fi-
nally, fractions C-108—142 were further separated by semi-preparative
HPLC with MeCN–H2O (49 : 51) to yield compounds 5 (8.4 mg), 6 (9.7 mg)
and 7 (18.7 mg).
Compound 1: White amorphous power, [a]D20 ꢁ34.3° (cꢃ0.046, py-
ridine); 1H- and 13C-NMR: see Table 1. HR-ESI-MS (negative) m/z:
767.3854 [MꢁH]ꢁ (Calcd for C39H59O15: 767.3867). FAB-MS m/z: 769.4
[MꢂH]ꢂ, 607.5 [MꢂHꢁ162]ꢂ, 445.2 [MꢂHꢁ162ꢁ162]ꢂ
1
140.8 and 121.4. Analysis of the H- and 13C-NMR spectral
data of 2 in comparison with those of 1 implied that the agly-
cone of 2 was the same as 1. In the HMBC spectrum of 2, a
cross peak between the 1H-NMR signal at d 4.88 (H-1 of the
galactose) and the carbon signal at d 77.7 (C-3, aglycone) in-
dicated glycosylation of the aglycone at C-3. Furthermore,
the anomeric proton signals at d 5.14 (H-1of the glucose)
and 5.25 (H-1ꢀ of the glucose) showed correlations with the
carbon signals at d 81.0 (C-4 of the galactose) and 86.2 (C-2
of the glucose), respectively. The full assignments of these
Compound 2: White amorphous power, [a]D20 ꢁ43.2° (cꢃ0.038, py-
ridine); 1H- and 13C-NMR: see Table 1. HR-ESI-MS (negative) m/z:
929.4382 [MꢁH]ꢁ (Calcd for C45H69O20: 929.4396). FAB-MS m/z: 931.4
[MꢂH]ꢂ, 769.4 [MꢂHꢁ162]ꢂ, 607.3 [MꢂHꢁ162ꢁ162]ꢂ, 589.3
[MꢂHꢁ162ꢁ162ꢁ18]ꢂ,
[MꢂHꢁ162ꢁ162ꢁ162ꢁ18]ꢂ.
445.3
[MꢂHꢁ162ꢁ162ꢁ162]ꢂ,
427.3
Acid Hydrolysis of Compound 1 Compound 1 (about 2.0 mg) was
treated in 1 M HCl (dioxane–H2O, 1 : 1, 2 ml) at 100 °C for 1.5 h. The reac-
tion mixture was neutralized with silver carbonate and evaporated to dryness
under N2 gas overnight. The residue was extracted with CHCl3 and H2O.
Then, in monosaccharide mixture, glucose and galactose were detected by
TLC analysis on a cellulose plate using n-BuOH–EtOAc–C5H5N–H2O
(6 : 1 : 5 : 4) as development and aniline-o-phthalic acid as detection, compar-
ing with the authentic samples: glucose (Rf 0.46) and galactose (Rf 0.39).
The determination of the configuration of sugar moieties followed the proce-
dure was described in our previous paper.10) The retention times of the deriv-
atives for the standards were: tR: 20.31 min (D-glucose derivative), tR:
20.82 min (L-glucose derivative), tR: 22.08 min (D-galactose derivative) and
tR: 22.65 min (L-galactose derivative). The retention times of the derivatives
of D-glucose (D-glucose derivative) and D-galactose (D-galactose derivative)
for compound 1 were 20.29 min and 22.03 min, respectively.
1
sugar signals were confirmed by HSQC and H–1H COSY
experiments. Thus, the structure of 2 was determined to be
(24S,25R)-3b,24-dihydroxy-spirostan-5-en-12-one-3-O-b-D-
glucopyranosyl-(1→2)-b-D-glucopyranosyl-(1→4)-b-D-
galactopyranoside, and named Kingianoside I.
Experimental
General Methods The HR-ESI-MS was recorded on 9.4 T Q-FT-MS
Apex Qe (Bruker Co.). FAB-MS: Micromass Zabspec. Optical rotations
were measured with Perkin-Elmer 343 polarimeter. The NMR spectra were
recorded with Varian UNITYINOVA 600 (599.8 MHz for 1H-NMR and
150.8 MHz for 13C-NMR), and the chemical shifts were given on d (ppm)
scale with tetramethylsilane as an internal standard. The HPLC analysis was
performed using Agilent 1100 system (pump, quaternary pump. Detector,
RID and DAD, U.S.A.), Apollo C18 (Alltech, 8.0 mm i.d.ꢄ250 mm, ODS,
10 mm, U.S.A.) and YMC-Pack ODS-A C18 (YMC, 4.6 mm i.d.ꢄ250 mm,
Compound 2 (about 2.0 mg) was subjected to acid hydrolysis as described
for 1. The derivatives of D-glucose (D-glucose derivative) and D-galactose (D-
galactose derivative) were observed at 20.29 min and 22.03 min, respec-
tively.
Acknowledgment We are grateful to Mrs. Yan Xue and Mr. He-bing
Chen of the National Center of Biomedical Analysis for the measurements
ODS, 5 mm, Japan). The Gas chromatographic analysis was performed with of the MS and NMR spectra. This work was supported by the National Nat-
an Agilent 6890 Series, gas chromatograph equipped with an H2 flameion- ural Science Foundation of China (30600822).
ization detector. The column was an HP-5 capillary column (30 mꢄ
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