Triterpenoids with Promoting Effects on the Differentiation of PC12 Cells from the Steamed Roots of Panax notoginseng

Article abstract of DOI:10.1021/acs.jnatprod.5b00027

(Chemical Equation Presented). The roots of Panax notoginseng, an important Chinese medicinal plant, have been used traditionally in both the raw and processed forms, due to the different chemical constituents and bioactivities found. Thirty-eight dammarane-type triterpenoid saponins were isolated from the steam-processed roots of P. notoginseng, including 18 new substances, namely, notoginsenosides SP1-SP18 (1-18). The structures of 1-18 were determined on the basis of spectroscopic analysis and acidic hydrolysis. The absolute configuration of the hydroxy group at C-24 in 1-4, 19, and 20 was determined in each case by Mo₂(AcO)₄-induced circular dichroism. The new compounds were found to feature a diversity of highly oxygenated side chains, formed by hydrolysis of the C-20 sugar moiety followed by dehydration, dehydrogenation, epoxidation, hydroxylation, or methoxylation of the main saponins in the raw roots. The new saponins 1, 2, 6-8, 14, and 17 and the known compounds 20-27 showed promoting effects on the differentiation of PC12 cells, at a concentration of 10 μM.

Full text of DOI:10.1021/acs.jnatprod.5b00027

Article
pubs.acs.org/jnp
Triterpenoids with Promoting Effects on the Differentiation of PC12
Cells from the Steamed Roots of Panax notoginseng
Cheng-Zhen Gu,†,‡ Jun-Jiang Lv,† Xiao-Xia Zhang,† Yi-Jun Qiao,† Hui Yan,† Yan Li,† Dong Wang,†
Hong-Tao Zhu,† Huai-Rong Luo,† Chong-Ren Yang,† Min Xu,*,† and Ying-Jun Zhang*,†
†State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of
Sciences, Kunming 650201, People’s Republic of China
‡University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China
S
* Supporting Information
ABSTRACT: The roots of Panax notoginseng, an important Chinese medicinal plant,
have been used traditionally in both the raw and processed forms, due to the different
chemical constituents and bioactivities found. Thirty-eight dammarane-type triterpenoid
saponins were isolated from the steam-processed roots of P. notoginseng, including 18
new substances, namely, notoginsenosides SP1−SP18 (1−18). The structures of 1−18
were determined on the basis of spectroscopic analysis and acidic hydrolysis. The
absolute configuration of the hydroxy group at C-24 in 1−4, 19, and 20 was determined
in each case by Mo2(AcO)4-induced circular dichroism. The new compounds were
found to feature a diversity of highly oxygenated side chains, formed by hydrolysis of the
C-20 sugar moiety followed by dehydration, dehydrogenation, epoxidation, hydrox-
ylation, or methoxylation of the main saponins in the raw roots. The new saponins 1, 2,
6−8, 14, and 17 and the known compounds 20−27 showed promoting effects on the
differentiation of PC12 cells, at a concentration of 10 μM.
ethods for the processing of traditional Chinese
notoginsenosides ST-1−ST-5.17 One of these substances,
Mmedicines (TCM) have been developed over thousands notoginsenoside ST-4, was found to be a promising agent for
herpes simplex virus infection, having EC50 values of 16.5 and
19.4 μM for HSV-1 and HSV-2, respectively.18 This preliminary
study also showed that the total saponins of the steamed roots
of P. notoginseng could enhance the neurite outgrowth of NGF-
mediated PC12 cells at a concentration of 50 μg/mL. In order
to determine the bioactive principles with neurite outgrowth-
promoting activities, a large-scale and detailed phytochemical
study on the processed roots of P. notoginseng was carried out,
which led to the isolation of 15 protopanaxadiol- and 23
protopanaxatriol-type saponins, including 18 new metabolites
(1−18). The structures of these new compounds were
determined by spectroscopic analysis and acidic hydrolysis,
using 13C−1H NMR spin-coupling constants (2,3JC,H) and
Mo2(AcO)4-induced circular dichroism (CD). A possible
transformation pathway of the new compounds in the
processed roots of P. notoginseng is also proposed. Moreover,
all the isolates were tested for their neurotrophic activities on
PC12 cells and cytotoxicity against five human cancer cell lines
(HL-60, SMMC-7712, A-549, MCF-7, and SW480), and the
results obtained are discussed herein.
of years. The processing methods have been used not only to
produce new medicinal or curative effects but also to reduce or
eliminate the toxicity and side effects of the raw medicinal
materials. They involve steaming, baking, cooking, frying, and
soaking with wine, vinegar, ginger juice, and/or other liquids.
Panax notoginseng (Burkill) F. H. Chen ex C. Y. Wu & K. M.
Feng (Araliaceae) is a significant medicinally used species, and
its roots have been used traditionally in both the raw and
processed forms. The raw form has been used to arrest various
internal or external hemorrhages, eliminate bruises, and treat
swelling and pain, as well as blood stasis and clots.1 In contrast,
the steamed root has been reported to generate a tonic that can
be used to nourish the blood and increase the production of
various blood cells in anemic conditions.2 To date, over 80
triterpenoid saponins have been isolated and reported from P.
notoginseng,3−5 among which notoginsenoside R1 and ginseno-
sides Rg1, Re, Rb1, and Rd are the main saponins of the raw
roots. The pharmacological activities of these saponins are
mainly associated with cardiovascular and cerebrovascular
diseases.6−11 Eight saponins that are absent in the raw form,
namely, ginsenosides 20(R/S)-Rh1, Rk3, Rh4, 20(S/R)-Rg3, Rk1,
and Rg5, have been found to be the main constituents in the
processed roots of P. notoginseng. Thus, the chemical
components are different in the raw and processed roots of
P. notoginseng.12−16
RESULTS AND DISCUSSION
The air-dried raw roots of P. notoginseng were crushed into
small grains and steamed at 120 °C with a pressure of 0.12 MPa
■
Our previous study on the steam-processed roots of P.
notoginseng led to the isolation of five new saponins,
Received: January 11, 2015
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.5b00027
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Chart 1
for 12 h.16 The 80% aqueous MeOH extract was subjected to
repeated column chromatography over D-101 resin, silica gel,
Diaion HP20SS, RP-18, and MCI-gel CHP20P, followed by
semipreparative HPLC, to afford 18 new dammarane-type
triterpenoid saponins (1−18), together with 20 known
compounds. The known substances were identified as
notoginsenosides T4 (19)19 and ST-1 (20),17 ginsenosides
20(R)-Rh1 (21),20 20(R)-Rg3 (22),21 Rk2 (23),22 20(S)- (24)
and 20(R)- (25) 6″-O-acetylginsenoside Rg3,23,24 20(R)-25-
hydroxyginsenoside Rh1 (26),20 25-hydroxyginsenoside Rk3
(27),25 ginsenosides 20(S)-Rh1,20 Rk3,22 Rh4,22 20(S)-Rg3,21
Rk1,22 Rg5,22 20(S)- and 20(R)-Rh2,26 and Rh3,22 20(R)-
dammarane-3β,6α,12β,20,25-pentol,27 and sanchinoside B1,28
respectively, using authentic samples and by comparison of
their spectroscopic data with literature values. Among them,
ginsenosides 20(S/R)-Rh1, 20(S/R)-Rg3, Rh4, Rk3, Rk1, and Rg5
are the main saponins found in the steamed processed roots of
P. notoginseng, and 25-hydroxyginsenoside Rk3 (27) was
isolated from P. notoginseng for the first time.
Compound 1 was obtained as a white, amorphous powder,
and its molecular formula was determined to be C42H72O15 by
the positive-mode HRESIMS (m/z 839.4766 [M + Na]+),
corresponding to seven degrees of unsaturation. The IR
spectrum indicated the presence of hydroxy groups (3423
cm−1) and an olefin moiety (1633 cm−1). The 13C NMR and
DEPT spectra showed 42 carbon resonances. Twelve of these
corresponded to two hexosyl units (δC 105.2, 83.4, 78.0, 71.6,
78.3, 62.7 and 106.1, 77.2, 78.2, 72.6, 78.3, 62.8) (Table 2),
which were determined to be D-glucosyl moieties on the basis
of acidic hydrolysis, followed by GC analysis of the
corresponding trimethylsilylated L-cysteine adduct. The other
30 carbons (Table 2) were assigned to nine methines with
three oxygen-bearing (δC 89.0, 71.0, and 80.1) and two olefinic
(δC 136.1 and 130.3) methines, eight methyls, seven
methylenes, and six quaternary carbons, of which two are
B
DOI: 10.1021/acs.jnatprod.5b00027
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Table 1. 1H (600 MHz) NMR Data of Compounds 1−3, 11,
and 17 in Pyridine-d5 (δ in ppm)
methylenes [δC 35.9 (C-22), and 23.0 (C-23)] in 20(S)-
ginsenoside Rg3, signals for a trans-disubstituted double bond
[δH 6.51 (1H, d, J = 15.6 Hz) and 6.44 (1H, dd, J = 6.0, 15.6
Hz)] occurred for 1, in addition to an oxymethine (δC 80.1, δH
4.47) and an oxygen-bearing quaternary carbon (δC 72.7). This
suggested that the side chain of compound 1 has two more
hydroxy groups than 20(S)-ginsenoside Rg3. The trans-
disubstituted double bond in 1 was located between C-22
no.
1
2
3
11
17
1
0.74 m
1.48 m
1.81 m
2.19 m
0.73 m
1.46 m
1.80 m
2.18 m
0.75 m
1.50 m
1.84 m
2.22 m
1.47 m
0.73 m
1.82 m
2.20 m
1.48 m
0.72 m
2.20 m
1.82 m
2
3
5
6
3.29 br d
(9.6)
3.28 dd (4.2, 3.31 dd (4.2, 3.29 dd (4.2, 3.29 m
1
and C-23, on the basis of the H−1H COSY correlations of an
11)
12.0)
11.4)
olefinic proton at δH 6.44 (H-23) with another olefinic proton
at δH 6.51 (H-22) and the oxymethine proton at δH 4.47 (d, J =
6.0 Hz, H-24), as well as the HMBC correlations of H-22 with
C-20 (δC 74.1), C-21 (δC 28.8), C-23 (δC 130.3), and C-24 (δC
80.1), H-23 (δH 6.44) with C-22 (δC 136.1) and C-24 (δC
80.1), H-24 (δH 4.47) with C-22, C-23, C-25 (δC 72.7) and C-
26 (δC 27.0), and H-26 (δH 1.60) and H-27 (δH 1.61) with
both C-24 and C-25. On the basis of the above HMBC
correlations, the oxymethine at δC 80.1 and the oxygen-bearing
quaternary carbon at δC 72.7 were assigned at C-24 and C-25,
respectively. Furthermore, the sugar location and sequence
could be confirmed by HMBC correlations from the glucosyl
H-1′ (δH 4.95) to the aglycone C-3 (δC 89.0) and from the
terminal glucosyl H-1″ (δH 5.41) to the inner glucosyl C-2′ (δC
83.4).
0.67 br d
(11.4)
0.66 br d
(12.0)
0.68 br d
(12.0)
0.66 br d
(11.4)
0.67 br d
(12.0)
1.35 m
1.47 m
1.21 m
1.47 m
1.39 m
1.40 m
2.02 m
3.95 m
2.04 m
1.03 m
1.59 m
1.60 m
1.91 m
2.41 m
1.01 s
1.34 m
1.47 m
1.21 m
1.43 m
1.39 m
1.48 m
2.00 m
3.94 m
1.99 m
1.03 m
1.59 m
1.57 m
1.92 m
2.40 m
1.01 s
1.37 m
1.49 m
1.22 m
1.44 m
1.41 m
1.41 m
2.07 m
3.95 m
2.02 m
1.01 m
1.55 m
1.44 m
1.87 m
2.41 m
1.03 s
1.34 m
1.47 m
1.20 m
1.43 m
1.37 m
1.42 m
1.92 m
3.90 m
2.08 m
1.06 m
1.65 m
1.55 m
1.97 m
2.77 m
0.97 s
1.37 m
1.49 m
1.23 m
1.47 m
1.38 m
1.44 m
1.91 m
3.89 m
2.02 m
1.11 m
1.69 m
1.54 m
2.01 m
2.88 m
1.03 s
7
9
11
12
13
15
16
17
18
19
21
22
The absolute configuration of the side chain in 1 was
1
determined from the H NMR coupling constants, the 13C
0.79 s
0.78 s
0.84 s
0.78 s
0.81 s
NMR chemical shifts, and analysis of the Mo2(AcO)4-induced
circular dichroism data. The large coupling constant of J22,23
(15.6 Hz) suggested the E-geometry of Δ22,23. The deshielded
chemical shifts of C-21 (δC 28.8) and C-17 (δC 53.7) indicated
the 20S configuration in 1, compared to a 20R configuration
[C-21 (δC 22.8) and C-17 (δC 50.7)].20 The absolute
configuration of C-24 was determined using Mo2(AcO)4-
induced circular dichroism. The aglycone 1a was mixed with
Mo2(AcO)4 in DMSO to give a metal complex, which displayed
a positive Cotton effect at 296 nm (Figure 1), suggesting the
24S configuration for 1a, according to the Snatzke rule.29,30
From the above evidence, the structure of notoginsenoside SP1
(1) was determined as (3β,12β,20S,22E,24S)-3,12,20,24,25-
pentahydroxydammar-22-ene-3-O-β-D-glucopyranosyl-(1→2)-
β-D-glucopyranoside.
The molecular formula of compound 2 was determined to be
C42H72O15, on the basis of the HRESIMS (m/z 839.4763 [M +
Na]+), which was the same value as that of 1. The NMR data of
2 (Tables 1 and 2) were identical to those of 1, except for a
difference for the upfield shift of H-22 to δH 6.42 ppm (vs δH
6.51 ppm in 1) and the downfield shift of H-23 to δH 6.55 ppm
(vs δH 6.44 ppm in 1). The chemical shifts of C-21 (δC 29.3)
and C-17 (δC 53.7) indicated the 20S configuration of 2,20 and
the large coupling constant of J22,23 (15.6 Hz) suggested the E-
geometry of Δ22,23, the same as those of 1. However, in a co-
HPLC analysis of 1 and 2, their retention times were different
[33.5 min (1) and 33.1 min (2)], revealing compound 2 to be a
stereoisomer of 1 at C-24, with the absolute configuration of C-
24 in 2 being R, opposite that of 1. Therefore, notoginsenoside
SP2 (2) was determined to be (3β,12β,20S,22E,24R)-
3,12,20,24,25-pentahydroxydammar-22-ene-3-O-β-D-glucopyra-
nosyl-(1→2)-β-D-glucopyranoside.
1.58 s
1.57 s
1.57 s
2.01 s
1.92 s
6.51 d
(15.6)
6.42 d
(15.6)
6.37 d
(15.6)
6.20d (8.4)
5.58 d (9.6)
23
6.44 dd
(15.6, 6.0)
6.55 dd
(15.6, 6.6)
6.47 dd
(15.6, 6.6)
5.19 dd (8.4, 4.11 dd
3.6) (9.6, 7.8)
24
26
27
28
29
30
1′
2′
3′
4′
5′
4.47 d (6.0) 4.50 d (6.6) 4.47 d (6.6) 3.79 d(3.6)
3.13 d (7.8)
1.24 s
1.60 s
1.61 s
1.29 s
1.10 s
0.97 s
1.62 s
1.63 s
1.29 s
1.10 s
0.97 s
1.57 s
1.58 s
1.31 s
1.13 s
0.93 s
1.63 s
1.63 s
1.29 s
1.11 s
0.93 s
1.47 s
1.31 s
1.13 s
0.97 s
4.95 d (7.8) 4.94 d (7.8) 4.96 d (7.8) 4.95d (7.8)
4.96d (7.8)
4.28 m
4.35 m
4.18 m
3.96 m
4.39 m
4.60 m
4.28 m
4.30 m
4.17 m
3.95 m
4.39 m
4.58 m
4.28 m
4.34 m
4.16 m
3.95 m
4.37 m
4.58 m
4.29 m
4.29 m
4.18 m
3.96 m
4.39 m
4.60 m
4.28 m
4.35 m
4.18 m
3.96 m
4.38 m
4.61 m
6′
1″
2″
3″
4″
5″
6″
5.41 d (7.2) 5.40 d (7.2) 5.42 d (7.8) 5.41 d (7.8) 5.41 d(7.2)
4.17 m
4.28 m
4.36 m
3.95 m
4.51 m
4.51 m
4.16 m
4.27 m
4.37 m
3.95 m
4.50 m
4.50 m
4.17 m
4.36 m
4.38 m
3.96 m
4.40 m
4.52 m
4.17 m
4.28 m
4.38 m
3.96 m
4.38 m
4.60 m
4.16 m
4.28 m
4.38 m
3.96 m
4.39 m
4.60 m
3.45 s
MeO
1
oxygen-bearing (δC 74.1 and 72.7). In the H NMR spectrum
(Table 1), two trans-coupled olefinic protons [δH 6.51 (1H, d, J
= 15.6 Hz) and 6.44 (1H, dd, J = 6.0, 15.6 Hz)] and eight
singlet methyls (δH 1.01, 0.79,1.58, 1.60, 1.61, 1.29, 1.10, and
0.97) were observed, in addition to two anomeric protons at δH
4.95 (1H, d, J = 7.8 Hz, H-1′) and δH 5.41 (1H, d, J = 7.2 Hz,
H-1″), suggesting the two glucosyl moieties as being β. The
above spectroscopic data were similar to those of 20(S)-
ginsenoside Rg3,21 a protopanaxadiol-type disaccharide saponin,
except for the C-20 side chain. Instead of a trisubstituted
double bond between C-24 and C-25 and two aliphatic
Notoginsenoside SP3 (3) gave the same molecular formula
as 1, namely, C42H72O15, on the basis of the HRESIMS [m/z
855.4494 [M + K]+]. The NMR data of 3 (Tables 1 and 2)
were similar to those of 1. These similarities, together with the
fact that 1 and 3 gave identical molecular formulas, indicated 3
C
DOI: 10.1021/acs.jnatprod.5b00027
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Table 2. 13C (150 MHz) NMR Data of Compounds 1−6, 11, 17, and 18 in Pyridine-d5 (δ in ppm)
no.
1
2
3
4
5
6
11
17
18
1
39.5 t
26.7 t
89.0 d
39.7 s
56.4 d
18.4 t
35.2 t
39.9 s
50.4 d
36.9 s
32.1 t
71.0 d
50.1 d
51.9 s
31.5 t
27.0 t
53.7 d
15.9 q
16.4 q
74.1 s
28.8 q
136.1 d
130.3 d
80.1 d
72.7 s
27.0 q
25.6 q
28.1 q
16.6 q
17.3 q
105.2 d
83.4 d
78.0 d
71.6 d
78.3 d
62.7 t
106.1 d
77.2 d
78.2 d
72.6 d
78.3 d
62.8 t
39.5 t
26.7 t
89.0 d
39.7 s
56.4 d
18.4 t
35.2 t
39.9 s
50.4 d
36.9 s
32.0 t
71.0 d
50.1 d
51.9 s
31.5 t
27.0 t
53.7 d
15.9 q
16.4 q
74.0 s
29.3 q
136.3 d
130.4 d
80.0 d
72.8 s
26.5 q
25.7 q
28.1 q
16.6 q
17.3 q
105.1 d
83.3 d
78.3 d
71.6 d
78.1 d
62.7 t
106.0 d
77.2 d
78.0 d
71.6 d
78.1 d
62.8 t
39.6 t
27.2 t
89.4 d
40.2 s
56.9 d
18.9 t
35.6 t
40.5 s
50.8 d
37.4 s
32.5 t
71.2 d
50.2 d
52.3 s
31.8 t
27.8 t
52.5 d
16.3 q
16.9 q
74.0 s
21.9 q
141.5 d
128.8 d
80.5 d
73.2 s
27.2 q
26.3 q
28.6 q
17.1 q
17.7 q
105.7 d
83.9 d
78.5 d
72.1 d
78.7 d
63.3 t
106.5 d
77.7 d
78.8 d
72.1 d
78.7 d
63.1 t
39.7 t
28.0 t
78.6 d
40.4 s
61.4 d
80.2 d
45.6 t
41.1 s
50.1 d
39.4 s
32.1 t
70.8 d
49.4 d
51.7 s
31.4 t
27.3 t
52.2 d
17.4 q
17.7 q
73.5 s
21.5 q
140.4 d
128.3 d
79.7 d
72.8 s
26.5 q
25.9 q
31.8 q
16.4 q
17.0 q
106.1 d
75.5 d
79.7 d
71.8 d
78.2 d
63.1 t
39.9 t
28.4 t
78.9 d
40.8 s
61.8 d
80.5d
39.7 t
27.9 t
78.5 d
40.3 s
61.4 d
80.0 d
45.3 t
41.2 s
50.4 d
39.4 s
32.5 t
72.0 d
50.7 d
50.7 s
32.3 t
27.9 t
50.9 d
17.3 q
17.7 q
140.1 s
13.1 q
127.9 d
69.2 d
80.7 d
72.9 s
28.9 q
25.5 q
31.7 q
16.3 q
16.6 q
106.0 d
75.4 d
79.6 d
71.7 d
78.1 d
63.0 t
39.2 t
26.8 t
88.9 d
39.7 s
56.4 d
18.4 t
35.3 t
40.2 s
50.8 d
37.0 s
32.6 t
72.2 d
51.6 d
51.0 s
32.5 t
29.1 t
50.0 d
15.8 q
16.5 q
144.7 s
15.0 q
127.3 d
68.3 d
80.2 d
73.2 s
28.1 q
26.7 q
28.1 q
16.6 q
17.0 q
105.2 d
83.4 d
78.3 d
71.6 d
78.3 d
62.8 t
106.0 d
77.1 d
78.2 d
71.6 d
78.3 d
62.7 t
39.2 t
26.7 t
89.0 d
39.7 s
56.5 d
18.4 t
35.4 t
40.2 s
50.8 d
37.0 s
32.7 t
72.1 d
51.1 d
51.0 s
32.8 t
29.3 t
50.8 d
15.8 q
16.5 q
146.5 s
14.0 q
120.4 d
78.1 d
66.8 d
57.3 s
25.0 q
20.0 q
28.2 q
16.6 q
16.9 q
105.3 d
83.5 d
78.4 d
71.6 d
78.4 d
62.7 t
106.2 d
77.2 d
78.0 d
71.6 d
78.4 d
62.8 t
55.6 q
40.1 t
28.4 t
78.8 d
40.8 s
61.7 d
80.5 d
45.8 t
41.5 s
51.1 d
39.8 s
33.6 t
72.0 d
52.8 d
51.7 s
33.4 t
30.0 t
52.3 d
17.8 q
18.2 q
167.9 s
17.7 q
120.6 d
196.4 s
67.0 d
61.1 s
25.2 q
19.1 q
32.2 q
16.8 q
17.2 q
106.5 d
75.8 d
80.1 d
72.2 d
78.6 d
63.4 t
2
3
4
5
6
7
45.6 t
41.7 s
51.0 d
40.1 s
33.1 t
72.8 d
51.7 d
51.3 s
32.8 t
29.8 t
50.4 d
17.8 q
18.2 q
142.0 s
15.4 q
127.7 d
69.0 d
80.8 d
73.6 s
28.6 q
27.1 q
32.2 q
16.8 q
17.2 q
106.5 d
75.9 d
80.1 d
72.2 d
78.6 d
63.4 t
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
1′
2′
3′
4′
5′
6′
1″
2″
3″
4″
5″
6″
MeO
to be an epimer of 1. The large coupling constant of J22,23 (15.6
Hz) indicated the E-geometry of Δ22,23, which was identical to
that of 1. However, the upfield shifts of C-17, C-21, and C-23
by 1.2, 6.9, and 1.5 ppm, respectively, and downfield shift of C-
22 by 5.4 ppm in 3 indicated that the configurations of C-20
and C-24 of 3 are different from those of 1. The 20R
configuration in 3 was confirmed by the chemical shift of C-21
(δC 21.9) and C-17 (δC 52.5).20 Moreover, the negative Cotton
effect at 310 nm (Figure 2) in the Mo2(AcO)4-induced CD
experiment of aglycone 3a confirmed C-24 in 3 as having the R
configuration. Thus, the structure of notoginsenoside SP3 (3)
was established as (3β,12β,20R,22E,24R)-3,12,20,24,25-penta-
hydroxydammar-22-ene-3-O-β-D-glucopyranosyl-(1→2)-β-D-
glucopyranoside.
693.4188 [M + Na]+). In the same manner as for 3, the
molecular formula and the NMR data (Tables 2 and 3) of 4
were closely comparable to those of notoginsenoside T4 (19), a
protopanaxatriol-type monodesmoside with an unknown
absolute configuration at C-24.19 In fact, the major differences
in the 13C NMR spectrum of 4 were the upfield shifts of C-17,
C-21, and C-23 and the downfield shift of C-22, which revealed
that 4 might be a geometrical isomer of 19 at C-20 and C-24.
The chemical shifts of C-17 [δC 52.2 (4) and 53.7 (19)] and C-
21 [δC 21.5 (4) and 29.2 (19)] indicated the 20R and 20S
configurations in 4 and 19, respectively.20 Moreover, the
absolute configurations of C-24 in 4 and 19 were determined,
respectively, to be S and R, according to the positive (4a) and
negative (19a) Cotton effects at 310 nm (Figures 1 and 2) in
the Mo2(AcO)4-induced CD experiments of 4a and 19a. The
absolute configuration of C-24 in notoginsenoside T4 (19) was
The molecular formula of notoginsenoside SP4 (4) was
elucidated as C36H62O11, on the basis of the HRESIMS (m/z
D
DOI: 10.1021/acs.jnatprod.5b00027
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
The relative configurations at C-23 and C-24 of compounds
5 and 6 were established using Murata’s method.31 In the
hetero half-filtered TOCSY (HETLOC) spectrum of 5, the
small coupling constant of 3JH‑23,H‑24 (3.0 Hz) indicated that H-
23 and H-24 are gauche, suggesting the presence of multiple
rotamers of A1, A2, the alternating A1/A2, B1, B2, and the
alternating B1/B2. The small coupling constants of 2JC‑23,H‑24 (0
Hz), 2JC‑24,H‑23 (−0.8 Hz), and 3JC‑22,H‑24 (0.8 Hz) indicated that
5 is mainly in the A1 configuration. In compound 6, the
3
medium coupling constant of JH‑23,H‑24 (4.8 Hz) suggested the
presence of two or more alternating rotamers (A2/A3, A1/A3,
3
B2/B3, or B1/B3). The small JC‑22,H‑24 (0.8 Hz) and medium
2JC‑24,H‑23 values (−2.4 Hz) indicated that the alternating
rotamer A1/A3 is predominant (Figure 3). Thus, Murata’s
method suggested that the relative configurations of C-23, C-24
in 5 and 6 are both threo. In the same manner as for 5 and 6,
the relative configurations of 7 (J23,24 = 4.8 Hz, threo) and 8
(J23,24 = 1.8 Hz, threo) were proposed.
Figure 1. Mo2(OAc)4-induced CD (ICD) spectra of 1a and 4a.
The absolute configurations of C-23 in compounds 5−8
were determined on the basis of the chemical shift of the C-21
methyl group.32 It has been reported that the absolute
configuration of C-23 can affect the chemical shift of the C-
21 methyl group. The deshielded chemical shift of C-21
showed an S configuration at C-23, and the comparatively
shielded chemical shift of C-21 indicated a 23R configuration.32
Therefore, the chemical shifts of C-21 [δC 15.4 (5), 13.1 (6),
20.3 (7), and 19.5 (8)] suggested the absolute configurations of
5 and 7 are 23S and 24R, while 6 and 8 are 23R and 24S,
respectively. Accordingly, the structures of notoginsenosides
SP5−SP8 were assigned as (3β,6α,12β,20E,23S,24R)-(5),
(3β,6α,12β,20E,23R,24S)-(6), (3β,6α,12β,20Z,23S,24R)-(7),
and (3β,6α,12β,20Z,23R,24S)-3,6,12,23,24,25- hexahydroxy-
dammar-20(22)-ene-6-O-β-D-glucopyranoside (8), respectively.
Both notoginsenosides SP9 (9) and SP10 (10) gave the
same molecular formula of C37H64O11, as deduced from the
HRESIMS. Their NMR data (Tables 3 and 5) were closely
related to those of 5 and 6, except that both 9 and 10 were
shown to possess one more methoxy group [δH 3.40 (9), 3.34
(10), δC 55.3 (9), 55.1 (10)] in the side chain. The methoxy
group was determined to be located at C-23, based on the
HMBC correlation of the methoxy protons with C-23.
Compounds 9 and 10 were also found to be C-23, C-24
diastereomers. In the same manner as for 5−7, the relative
configurations of 9 (J23,24 = 1.8 Hz, threo) were constructed.
Figure 2. Mo2(OAc)4-induced CD (ICD) spectra of 3a and 19a.
therefore determined as R for the first time, and the structure of
n o t o g i n s e n o s i d e S P 4 ( 4 ) w a s i d e n t i fi e d a s
(3β,6α,12β,20R,22E,24S)-3,6,12,20,24,25-hexahydroxydammar-
22-ene-6-O-β-D-glucopyranoside.
Compounds 5−8 were analyzed and found to have the same
molecular formula of C36H62O11, as established from their
HRESIMS data. The NMR data (Tables 2, 3, and 5) indicated
them all to have a protopanaxatriol skeleton and a
glucopyranosyl unit in their molecule, which were quite similar
to notoginsenoside ST-1 (20).17 Nevertheless, on comparing
with the side chain of 20 with two hydroxy groups at C-24 and
C-25, compounds 5−8 were found to have one more
oxymethine [δH 5.20 (5), 5.08 (6), 5.36 (7), and 5.42 (8),
δC 69.0 (5), 69.2 (6), 68.3 (7), and 67.2 (8)] in the side chain,
instead of a C-23 methylene group at 20. The additional
1
However, in compound 10, the H NMR coupling constant of
3JH‑23,H‑24 (6.6 Hz) was more than 5.0 Hz. The HETLOC
spectrum of 10 was therefore measured, from which the small
2
3JC‑22,H‑24 (0.8 Hz) and large JC‑24,H‑23 (−4.0 Hz) values
obtained suggested that the alternating rotamer B1/B3 is
predominant (Figure 3), indicating the erythro configuration of
C-23, C-24 in 10. Furthermore, the chemical shifts of C-21 [δC
15.2 (9) and 13.3 (10)] supported 9 as 23S, 24R and 10 as
23R, 24R.32 On the basis of the above evidence, notoginseno-
s i d e s S P 9 a n d S P 1 0 w e r e d e t e r m i n e d a s
(3β,6α,12β,20E,23S,24R)- (9) and (3β,6α,12β,20E,23R,24R)-
23-methoxy-3,6,12,24,25-pentadroxydammar-20(22)-ene-6-O-
β-D-glucopyranoside (10), respectively.
1
oxymethine was assigned as C-23, on the basis of the H−1H
COSY and HMBC spectra. The aforementioned data suggested
that compounds 5−8 are all 23-hydroxylated analogues of
notoginsenoside ST-1 (20), with different absolute config-
urations of the side chain. In the ROESY experiments,
compounds 7 and 8 showed correlations between H-21 and
H-22, indicating that the double bonds between C-20 and C-22
are in the Z configuration in both cases. However, the chemical
shifts of C-21 in 5 and 6 were shifted upfield, respectively, by
4.9 and 7.2 ppm, relative to 7, suggesting the double bonds
between C-20 and C-22 to have E-geometry in both 5 and 6.
Compound 11 gave a molecular formula of C42H72O15, as
established by the HRESIMS (m/z 839.4767 [M + Na]+). The
NMR data (Tables 1 and 2) of 11 were closely related to those
of notoginsenoside ST-2,17 a protopanaxadiol-type saponin
with a glucopyranosyl (1→2) glucopyranosyl unit linked to C-
1
The H and 13C NMR data revealed that compounds 5 and 6,
as well as 7 and 8, are C-23, C-24 diastereomers.
E
DOI: 10.1021/acs.jnatprod.5b00027
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
1
Table 3. H (600 MHz) NMR Data of Compounds 4−10 in Pyridine-d5 (δ in ppm)
no.
4
5
6
7
8
9
10
1.03 m
1
1.02 m
1.02 m
1.02 m
1.03 m
1.07 m
0.97 m
1.69 m
1.68 m
1.88 m
1.96 m
3.56 m
1.69 m
1.70 m
1.87 m
1.95 m
3.55 m
1.71 m
1.82 m
1.95 m
3.55 m
1.63 m
1.83 m
1.90 m
3.52 m
1.71 m
1.87 m
1.94 m
3.55 m
2
3
1.88 m
1.87 m
1.95 m
1.94 m
3.56 dd
(4.2, 11.4)
1.45 d (10.8)
4.46 m
3.55 dd
(4.8, 11.4)
1.44 d (10.8)
4.47 m
5
6
7
1.45 d (10.2)
4.46 m
1.45 d (10.2)
4.45 m
1.46 d (10.2)
4.47 m
1.95 m
2.56 dd
(3.0, 12.6)
1.61 m
1.50 m
2.09 m
3.96 m
2.07 m
1.18 m
1.70 m
1.52 m
1.70 m
3.61 m
1.25 s
1.41 d (12.6)
4.42 m
1.44 d (10.8)
4.46 m
1.97 m
2.55 br d
(12.6)
1.94 m
1.96 m
1.95 m
1.95 m
1.92 m
2.55 dd
(3, 12.6)
1.58 m
2.55 dd
(3.0, 12.6)
1.58 m
2.54 dd
(3.0, 12.6)
1.53 m
2.55 dd
(3.0, 12.6)
1.56 m
2.51 dd
(3.0, 15.0)
1.52 m
9
1.55 m
1.48 m
2.05 m
3.86 m
2.03 m
1.17 m
1.74 m
1.52 m
1.85 m
2.82 m
1.26 s
11
1.58 m
1.56 m
1.47 m
1.49 m
1.43 m
2.19 m
2.06 m
2.04 m
2.06 m
2.02 m
12
13
15
3.94 m
3.91 m
3.86 m
3.90 m
3.86 m
2.03 m
2.10 m
1.99 m
2.08 m
2.09 m
1.08 m
1.14 m
1.13 m
1.12 m
1.08 m
1.58 m
1.72 m
1.68 m
1.69 m
1.73 m
16
1.36 m
1.50 m
1.46 m
1.43 m
1.89 m
1.78 m
1.72 m
1.78 m
1.83 m
1.44 m
17
18
19
21
22
23
2.36 m
2.72 m
2.79 m
3.55 m
2.69 m
1.25 s
1.20 s
1.24 s
1.25 s
1.21 s
1.06 s
1.03 s
1.04 s
1.06 s
1.06 s
1.00 s
1.05 s
1.57 s
1.99 s
1.92 s
1.92 s
1.85 s
1.98 s
1.95 s
6.40 d (15.6)
6.47 dd
(15.6, 5.4)
4.49 d (5.4)
1.57 s
6.18 d (8.4)
5.20 dd
(8.4, 3.0)
3.78 d (3.0)
1.63 s
6.11 d (9.0)
5.08 dd
(9.0, 4.8)
3.88 d(4.8)
1.67 s
5.87 d(9.0)
5.36 dd
(9.0, 4.8)
4.00 d (4.8)
1.69 s
6.00 d (7.8)
5.42 dd
(1.8, 7.8)
3.72 d(1.8)
1.61 s
5.78 d (10.0)
4.49 dd
(10.0, 4.8)
3.75 d (4.8)
1.55 s
5.72 d(9.6)
4.35 dd
(9.6, 6.6)
3.90 d (6.6)
1.65 s
24
26
27
28
29
30
1′
2′
3′
4′
5′
1.59 s
1.64 s
1.59 s
1.68 s
1.66 s
1.52 s
1.52 s
2.10 s
2.11 s
2.10 s
2.11 s
2.10 s
2.07 s
2.10 s
1.64 s
1.63 s
1.64 s
1.64 s
1.65 s
1.60 s
1.64 s
0.79 s
0.79 s
0.78 s
0.82 s
0.80 s
0.80 s
0.80 s
5.06 d (7.8)
4.13 t (7.8)
4.31 t (9.0)
4.26 t (9.0)
3.99 m
5.06 d (7.8)
4.14 t (7.8)
4.31 m
5.06 d (7.8)
4.13 t (8.4)
4.30 t (8.4)
4.26 t (9.0)
3.98 m
5.07 d (7.8)
4.13 t (7.8)
4.30 t (8.4)
4.20 t (9.0)
3.90 m
5.07 d (7.8)
4.14 m
4.31 m
4.27 m
3.99 m
4.41, m
5.02 d (7.8)
4.10 t (9.0)
4.26 m
5.07 d (7.2)
4.13 t (7.8)
4.31 m
4.26 m
3.99 m
4.41 m
4.28 m
4.23 m
3.99 m
3.94 m
6′
4.40 dd
(11.4, 5.4)
4.57 dd
(11.4, 2.4)
4.41 m
4.41dd
4.41dd
4.37 m
(11.4, 5.4)
4.56 dd
(2.4, 11.4)
(5.4, 11.4)
4.60 dd
(2.4, 11.4)
4.57 br d
(10.8)
4.57 br d
(11.4)
4.53 br d
(13.8)
3.40
4.57 br d
(10.8)
3.34
MeO
3, and a methoxy group at C-23 of the side chain. However, C-
23 in notoginsenoside ST-2 was substituted by a hydroxy group
in 11. The side chain of 11 was identical to that of 5 with Δ20,22
and three hydroxy groups at C-23, C-24, and C-25. The relative
configurations of 11 (J23,24 = 3.6 Hz, threo) were established
Compounds 12 and 13 were determined also to be C-23, C-24
diastereomers. The coupling constants of J23,24 found for 12 and
13 were 8.4 and 7.8 Hz, respectively. Compared with those of
compounds 5−10, the relative configurations of C-23 and C-24
in 12 and 13 were proposed as erythro, due to their relatively
large coupling constants of J23,24 (>5.0 Hz). In order to confirm
this proposition, the carbon−proton spin-coupling constants of
1
due to similar H NMR coupling constants to those of 5. The
chemical shift of C-21 [δC 15.0 (11)] was also consistent with
that of 5 (δC 15.4), indicating the 23S and 24R absolute
configuration of 11. Therefore, the structure of notoginsenoside
SP11 (11) was elucidated as (3β,12β,20E,23S,24R)-
3,12,23,24,25-pentahydroxydmmar-20(22)-ene-3-O-β-D-gluco-
pyranosyl-(1→2)-β-D-glucopyranoside.
2,3
3
3
J
, as well as JC‑22,H‑24 and JC‑25,H‑23 of 13 were measured
C,H
from the HETLOC and phase-sensitive (ps)-HMBC NMR
3
3
spectra. The medium JH‑23,H‑24 (7.8 Hz), small JC‑22,H‑24 (0.8
3
Hz), and medium JC‑25,H‑23 (5.4 Hz) values suggested that the
alternating rotamer B1/B3 is predominant (Figure 3) in 13,
corresponding to the erythro isomer. Due to compound 12
sharing a similar coupling constant of J23,24 with that of 13, the
C-23, C-24 configurations of 12 were also established as erythro.
Furthermore, the chemical shifts of C-21 [δC 13.7 (12) and
The NMR data (Tables 4 and 5) of both 12 and 13 were
closely comparable to those of notoginsenoside T1,19
a
protopanaxatriol-type saponin with unknown configurations
of the C-23 hydroxy and the C-24, C-25 epoxy groups.
F
DOI: 10.1021/acs.jnatprod.5b00027
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
1
Table 4. H (600 MHz) NMR Data of Compounds 12−16 and 18 in Pyridine-d5 (δ in ppm)
no.
12
0.99 m
13
1.03 m
14
0.99 m
15
1.04 m
16
1.07 m
18
1.02 m
1
1.67 m
1.69 m
1.66 m
1.84 m
1.92 m
3.54 m
1.69 m
1.87 m
1.94 m
3.54 m
1.69 m
1.88 m
1.96 m
3.57 m
1.68 m
1.86 m
1.94 m
3.55 dd
2
3
1.86 m
1.89 m
1.94 m
1.95 m
3.55 dd
(4.2, 11.4)
1.43 d (10.2)
4.45 m
3.56 dd
(4.8, 12.0)
1.46 d (10.8)
4.46 m
(4.8, 11.4)
1.45 d (10.8)
4.46 m
1.97 m
2.57 dd
(3, 12.6)
1.57 m
1.46 m
2.06 m
3.88 m
2.16 m
1.21 m
1.76 m
1.68 m
1.88 m
2.82 m
1.25 s
5
6
7
1.43 d (10.2)
4.45 m
1.95 m
2.55 dd
(3, 12.6)
1.55 m
1.55 m
2.02 m
3.88 m
2.00 m
1.26 m
1.72 m
1.47 m
1.87 m
2.81 m
1.25 s
1.46 d (10.2)
4.47 m
1.48 d (10.8)
4.48 m
1.95 m
1.97 m
1.94 m
2.02 m
2.54 dd
(3, 12.6)
1.55 m
2.56 dd
(2.4, 12.6)
1.58 m
2.57 dd
(2.4, 12.6)
1.61 m
2.58 dd
(3, 13.2)
1.58 m
9
11
1.46 m
1.50 m
1.50 m
1.51 m
2.03 m
2.07 m
2.10 m
2.07 m
12
13
15
3.90 m
3.97 m
3.99 m
3.84 m
2.01 m
2.09 m
2.07 m
2.14 m
1.16 m
1.17 m
1.21 m
1.23 m
1.69 m
1.73 m
1.72 m
1.76 m
16
1.44 m
1.45 m
1.54 m
1.41 m
1.69 m
1.73 m
1.79 m
1.95 m
17
18
19
21
22
23
2.83 m
2.79 m
3.47 m
3.30 m
1.23 s
1.25 s
1.26 s
1.27 s
1.03 s
1.05 s
1.05 s
1.07 s
1.06 s
1.04 s
1.87 s
1.95 s
1.91 s
1.89 s
2.04 s
2.54 s
5.93 d (9.6)
4.72 dd
(9.6, 8.4)
3.27 d (8.4)
1.30 s
5.88 d (9.0)
4.68 dd
(9.0, 7.8)
3.27 d (7.8)
1.31 s
5.55 d (9.6)
4.09 dd
(9.6, 7.8)
3.11 d(7.8)
1.24 s
5.61 d (8.4)
4.81 dd
(8.4, 7.8)
3.23 d (7.8)
1.32 s
5.33 d (9.0)
4.26 dd
(9.0, 7.8)
3.16 d (7.8)
1.25 s
6.78 s
24
26
27
28
29
30
1′
2′
3′
4′
5′
3.58 s
1.29 s
1.51 s
1.38 s
1.47 s
1.35 s
1.63 s
1.34 s
2.10 s
2.11 s
2.11 s
2.11 s
2.13 s
2.12 s
1.63 s
1.65 s
1.65 s
1.65 s
1.66 s
1.64 s
0.82 s
0.84 s
0.84 s
0.84 s
0.90 s
0.84 s
5.05 d (7.8)
4.13 t (8.4)
4.31 t (8.4)
4.26 t (7.8)
3.99 m
5.00 d (7.8)
4.14 t (8.4)
4.31 t (8.4)
4.26 t (9.0)
3.99 m
5.06 d (7.8)
4.13 t (7.8)
4.30 m
4.26 m
3.99 m
4.40 m
5.07 d (7.8)
4.14 t (7.8)
4.31 t (8.4)
4.26 t (9.0)
4.00 m
5.09 d (7.8)
4.16 t (7.8)
4.32 m
5.08 d (7.8)
4.14 t (7.8)
4.30 t (7.8)
4.27 t (9.0)
3.99 m
4.26 m
4.01 m
6′
4.41 dd
(5.4, 11.4)
4.57 dd
(2.4, 11.4)
4.40 dd
(5.4, 12.0)
4.57 dd
(2.4, 11.4)
4.41 m
4.41 m
4.41 dd
(5.4, 11.4)
4.57 dd
4.56 br d
(11.4)
4.58 br d
(11.4)
4.59 br d
(10.8)
(2.4, 11.4)
MeO
3.43 s
3.43 s
14.1 (13)] indicated the absolute configurations of 12
(23R,24R) and 13 (23S,24S), respectively. Therefore, com-
pounds 12 and 13 (notoginsenosides SP12 and SP13) were
determined as (3β, 6α, 12β,20E, 23R, 24R)- and
(3β,6α,12β,20E,23S,24S)-24,25-epoxy-3,6,12,23-tetrahydroxy-
dammar-20(22)-ene-6-O-β-D-glucopyranoside, respectively.
On the basis of detailed analysis of the NMR data (Tables 4
and 5), compound 14 was determined to have the same planar
structure as notoginsenoside T2,19 a protopanaxatriol-type
saponin with unknown configurations of the C-23 methoxy
and the C-24/C-25 epoxy groups. The relative configuration of
C-23, C-24 of 14 was established as erythro due to the same
coupling constant of J23,24 [7.8 Hz (14)] as those of 12 (8.4
Hz) and 13 (7.8 Hz). Moreover, the chemical shift of C-21 (δC
14.4) was similar to that of 13 [C-21 (δC 14.1)] and indicated
that the absolute configurations of 14 are also 23S and 24S.
Accordingly, notoginsenoside SP14 (14) was identified as
(3β,6α,12β,20E,23S,24S)-24,25-epoxy-23-methoxy-3,6,12-trihy-
droxydammar-20(22)-ene-6-O-β-D-glucopyranoside.
Compound 15 was shown to have the same planar structure
as 12 and 13, by analysis of its NMR data (Tables 4 and 5).
The ROESY correlation between H-21 and H-22 revealed the
Z-geometry of Δ20,22 in 15. The large coupling constant of J23,24
[7.8 Hz (15)] suggested that the relative configuration of C-23,
C-24 in 15 is erythro, being the same as compounds 12−14.
Moreover, the same chemical shift of C-21 (δC 19.5) as that in
8 indicated the 23R and 24R configurations in 15.
Notoginsenoside SP15 (15) was therefore assigned as
(3β,6α,12β,20Z,23R,24R)-24,25-epoxy- 3,6,12,23-tetrahydroxy-
dammar-20(22)-ene-6-O-β-D-glucopyranoside.
The molecular formula of 16 was determined to be
C37H62O10, on the basis of the HRESIMS (m/z 689.4241 [M
G
DOI: 10.1021/acs.jnatprod.5b00027
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Journal of Natural Products
Article
Table 5. 13C (150 MHz) NMR Data of Compounds 7−10 and 12−16 in Pyridine-d5 (δ in ppm)
no.
7
8
9
10
12
13
14
15
16
1
39.9 t
28.4 t
79.0 d
40.8 s
61.9 d
80.6 d
45.8 t
41.1 s
51.1 d
40.1 s
33.0 t
72.4 d
51.1 d
51.5 s
33.1 t
28.8 t
41.7 d
17.8 q
18.2 q
140.6 s
20.3 q
128.8 d
68.3 d
81.0 d
73.6 s
29.6 q
26.4 q
32.1 q
16.8 q
17.1 q
106.6 d
75.6 d
80.1 d
72.3 d
78.7 d
63.4 t
39.9 t
28.0 t
78.9 d
40.8 s
61.8 d
80.5 d
45.7 t
41.7 s
51.2 d
40.1 s
32.7 t
73.1 d
51.7 d
51.7 s
33.6 t
28.4 t
41.5 d
17.6 q
18.2 q
144.2 s
19.5 q
127.7 d
67.2 d
80.3 d
73.5 s
28.4 q
27.5 q
32.2 q
16.8 q
17.1 q
106.5 d
75.9 d
80.2 d
72.2 d
78.7 d
63.4 t
39.5 t
27.9 t
78.5 d
40.3 s
61.4 d
80.0 d
45.1 t
41.3 s
50.7 d
39.7 s
33.1 t
72.1 d
51.5 d
50.9 s
32.3 t
29.5 t
50.3 d
17.3 q
17.7 q
145.1 s
15.2 q
123.1 d
78.2 d
80.5 d
72.4 s
28.3 q
26.0 q
31.7 q
16.3 q
16.8 q
106.0 d
75.4 d
79.6 d
71.8 d
78.1 d
63.1 t
55.3 q
39.5 t
27.9 t
78.5 d
40.3 s
61.4 d
80.1 d
45.4 t
41.3 s
50.5 d
39.7 s
32.2 t
72.2 d
50.7 d
50.7 s
32.5 t
28.4 t
51.2 d
17.4 q
17.7 q
143.4 s
13.3 q
123.0 d
79.4 d
80.6 d
72.5 s
29.2 q
24.8 q
31.7 q
16.4 q
16.7 q
106.0 d
75.4 d
79.6 d
71.8 d
78.1d
39.5 t
27.9 t
78.6 d
40.4 s
61.4 d
80.1 d
45.2 t
41.7 s
50.7 d
39.7 s
32.5 t
72.3 d
50.9 d
50.7 s
32.7 t
28.6 t
50.6 d
17.3 q
17.7 q
143.0 s
13.7 q
124.7 d
68.6 d
68.7 d
58.4 s
25.2 q
20.1 q
31.7 q
16.3 q
16.7 q
106.0 d
75.5 d
79.7 d
71.9 d
78.2 d
63.1 t
39.5 t
27.9 t
78.6 d
40.4 s
61.4 d
80.1 d
45.3 t
41.3 s
50.6 d
39.7 s
32.5 t
72.3 d
50.6 d
51.0 s
32.4 t
29.2 t
50.4 d
17.4 q
17.7 q
143.6 s
14.1 q
124.3 d
68.4 d
68.7 d
58.7 s
25.1 q
19.9 q
31.7 q
16.3 q
16.8 q
106.0 d
75.5 d
79.7 d
71.7 d
78.2 d
63.1 t
39.8 t
28.4 t
78.9 d
40.8 s
61.8 d
80.5 d
45.7 t
41.7 s
51.0 d
40.1 s
33.1 t
72.5 d
51.1 d
51.3 s
33.2 t
29.6 t
51.1 d
17.7 q
18.2 q
146.9 s
14.4 q
120.7 d
78.4 d
67.1 d
57.8 s
25.4 q
20.4 q
32.2 q
16.8 q
17.1 q
106.5 d
75.9 d
80.1 d
72.2 d
78.7 d
63.4 t
56.1 q
39.9 t
28.4 t
78.7 d
41.6 s
61.8 d
80.5 d
45.8 t
41.8 s
51.1 d
40.1 s
32.6 t
73.1 d
51.7 d
51.8 s
33.6 t
27.9 t
41.6 d
17.6 q
18.2 q
146.0 s
19.5 q
123.6 d
67.8 d
68.7 d
58.2 s
25.6 q
19.8 q
32.1 q
16.8 q
17.1 q
106.5 d
75.9 d
80.1 d
72.2 d
78.9 d
63.5 t
39.9 t
28.4 t
79.0 d
40.9 s
61.8 d
80.4 d
45.7 t
41.8 s
51.0 d
40.1 s
33.5 t
72.3 d
50.4 d
51.6 s
32.9 t
30.0 t
42.1 d
17.8 q
18.2 q
147.4 s
21.3 q
121.1 d
77.7 d
67.1 d
58.0 s
25.5 q
20.4 q
32.1 q
16.8 q
17.2 q
106.4 d
75.9 d
80.2 d
72.2 d
78.7 d
63.5t
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
1′
2′
3′
4′
5′
6′
MeO
63.1 t
55.1 q
56.3 q
similar to that in 7 [C-21 (δC 20.3)] indicated the absolute
configuration of 16 to be 23S and 24S. Thus, notoginsenoside
SP16 (16) was elucidated as (3β,6α,12β,20Z,23S,24S)-24,25-
epoxy-23-methoxy-3,6,12-trihydroxydammar-20(22)-ene-6-O-
β-D-glucopyranoside.
Compound 17 gave a molecular formula of C43H72O14, on
the basis of the HRESIMS (m/z 835.4820 [M + Na]+). The 1H
and 13CNMR data (Tables 1 and 2) of 17 were similar to those
of notoginsenoside ST-2,17 a protopanaxatriol-type saponin,
except for the signals due to the side chain. Moreover,
compound 17 had one more degree of unsaturation than
notoginsenoside ST-2. Since the number of double bonds in 17
was the same as that in notoginsenoside ST-2, it was concluded
there must be one more ring on the side chain of 17. The
oxygen bridge between C-24 and C-25 and a methoxy group at
Figure 3. Newman projections of C-22−C-25 in compounds 5, 6, 10,
and 13.
1
C-23 were determined according to the H−1H COSY and
+ Na]+). Compared to 15, compound 16 had one more
methoxy group (δC 56.3, δH 3.43) located in the side chain. The
HMBC correlation of the methoxy proton with C-23 (δC 77.7)
indicated its location at C-23. The ROESY correlation between
H-21 and H-22 revealed the Z-geometry of Δ20,22. Moreover,
the J23,24 value (7.8 Hz) supported the erythro C-23, C-24
configuration, and the chemical shift of C-21 (δC 21.3) in being
HMBC correlations. The J23,24 value of 17 (7.8 Hz) indicated
that C-23, C-24 in 17 are erythro configured, since the chemical
shift of C-21 (δC 14.0) was identical to that of C-21 of 13 (δC
14.1). This suggested that the absolute configuration of 17 is
23S, 24S. Therefore, the structure of notoginsenoside SP17
(17) was elucidated as (3β,12β,20E,23S,24S)-24,25-epoxy-23-
H
DOI: 10.1021/acs.jnatprod.5b00027
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Journal of Natural Products
Article
methoxy-3,12-dihydroxydammar-20(22)-ene-3-O-β-D-glucopyr-
anosyl-(1→2)-β-D-glucopyranoside.
roots of P. notoginseng, were also tested, in order to compare
their bioactivities. As shown in Table 6, the new compounds 1,
Compound 18 gave a molecular formula of C36H58O10, as
deduced from the HRESIMS (m/z 673.3927 [M + Na]+),
representing eight degrees of unsaturation. The NMR data of
18 were similar to those of ginsenoside Rh4,22 except for signals
arising from the side chain. The 13C NMR data (Tables 2 and
4) at δC 196.4 indicated the presence of a ketone group in 18.
Besides the five degrees of unsaturation due to the glucosyl and
the four rings from the aglycon skeleton, three degrees of
unsaturation were represented by the side chain of 18.
However, the NMR data suggested that only one double
bond (δC 167.9, 120.6) and one carbonyl group (δC 196.4) are
present in 18. Thus, compound 18 was found to have an
additional ring in the side chain. On the basis of the HMBC
correlations, a double bond between C-20 and C-22, a carbonyl
group at C-23, and an oxygen bridge between C-24 and C-25
were determined. The configuration of the double bond was
identified as having E-geometry due to the chemical shift of C-
21 (δC 17.7), being similar to this carbon (δC 16.7) of cordialia
A,33 while the Z-geometry corresponded to the relative
deshielded chemical shift of C-21 (δC 20.2).34 Therefore,
notoginsenoside SP18 (18) was determined as
(3β,6α,12β,20E,24)-24,25-epoxy-23-one-3,6,12-trihydroxydam-
mar-20(22)-ene-6-O-β-D-glucopyranoside.
In the present study, 18 new dammarane-type saponins
featuring a highly oxygenated side chain were obtained from the
steam-processed roots of P. notoginseng. Most of these exhibit a
di- or trioxygenated side chain occurring as stereoisomers (1
and 2, 5 and 6, 7 and 8, 9 and 10, along with 12 and 13). These
were obtained in pure form, and their absolute configurations at
the side chains were determined. The relative configurations at
C-23 and C-24 were determined using Murata’s method. The
results suggested that a small coupling constant J23,24 (<5.0 Hz)
corresponded to the threo configuration of C-23, C-24, while a
large J23,24 value (>5.0 Hz) corresponded to the erythro
configuration. The chemical shifts of C-21 revealed the absolute
configurations of C-23 and C-24.32 Moreover, the absolute
configurations of C-24 in 1, 3, 4, 19, and 20 were determined
according to the positive (S configuration) or negative (R
configuration) Cotton effects in the Mo2(AcO)4-induced CD
experiments of their corresponding aglycons.29,30 It is noted
that the R and S configurations of C-20 affected significantly the
chemical shifts of C-17 and C-21, while the R and S
configurations of C-24 (1−4 and 19) have no such influence
on the chemical shifts of C-24 and C-25.
Table 6. Differentiation Rates of Compounds from the
Processed Roots of P. notoginseng on PC12 Cells
compound
differentiation rate (%)
a
blank
0
b
NGF
3.6
c
NGF
21.7
8.2
1
2
10.8
9.9
6
7
11.4
10.9
11.6
11.4
9.3
8
14
17
20
21
22
23
24
25
26
27
8.5
9.5
8.9
9.3
13.4
8.6
8.3
a
b
Blank with no added NGF. Negative control: 5 ng/mL of NGF.
c
Positive control: 50 ng/mL of NGF.
2, 6−8, 14, and 17 and the known metabolites 20−27 were
found to promote the differentiation of PC12 cells after 72 h at
a concentration of 10 μM, while the major compounds in the
raw roots showed no promoting properties. Although the total
saponins of the raw roots of P. notoginseng, ginsenosides Rg1,
Rb1, and Rd and notoginsenoside R1, were reported to have
some cytotoxicity for cancer cells,35−38 all the compounds from
the steam-processed roots of P. notoginseng showed no
cytotoxicity against the HL-60, SMMC-7712, A-549, MCF-7,
and SW480 cell lines (IC50 values >10 μM). The present results
may provide a scientific basis for ethnomedical usage of the
processed P. notoginseng roots.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
performed on a JASCO P-1020 polarimeter. UV spectra were
recorded on a Shimadzu UV-2401A spectrophotometer. IR spectra
were measured on a Bruker Tensor 27 spectrometer with KBr pellets.
1D and 2D NMR spectra were run on Bruker DRX-400, -500, and
AVANCE III-600 NMR spectrometers operating at 400, 500, and 600
The protopanaxatriol saponins, ginsenosides Rg1, Re, and
notoginsenoside R1, and the protopanaxadiol saponins,
ginsenosides Rb1 and Rd, are the five main saponins of the
raw roots of P. notoginseng. After a steaming process conducted
for 12 h at 0.12 MPa, these major saponins with one or two
sugars at C-20 decreased in abundance, while other new
saponins were formed, including the major products ginseno-
sides 20(R)-(21) and 20(S)-Rh1, Rk3, Rh4, 20(S)- and 20(R)-
(22) Rg3, Rk1, and Rg5. A proposed transformation pathway of
these metabolites is shown in the Supporting Information
(Figure S177).
Since the processed roots of P. notoginseng have been used as
a tonic by local people in its growing area in southwest China,
all the compounds were evaluated for their ability to stimulate
nerve growth factor (NGF)-mediated neurite outgrowth on
PC12 cells. The ginsenosides Rg1, Re, Rb1, and Rd and
notoginsenoside R1, which are main components in the raw
1
MHz for H and 100, 125, and 150 MHz for 13C, respectively. The
carbon−proton spin-coupling constants (2,3JC,H) were run on a Bruker
AVANCE 800 MHz NMR spectrometer for 1H and 200 MHz for 13C.
Coupling constants are expressed in hertz, and chemical shifts are
given on the ppm scale with solvents as internal standard. ESIMS and
HRESIMS were measured on a Bruker HCT/Esquire and an Agilent
G6230. ICD was detected with a Chirascan circular dichroism
spectrograph (Applied Photophysis, England). Preparative HPLC
was carried out on an Agilent 1260 apparatus with a DAD detector.
Semipreparative HPLC was performed on an Agilent 1260 liquid
chromatograph with a 5 μm Thermo BDS Hypersil-C18 column (10 ×
250 mm). Column chromatography (CC) was performed with Diaion
101 resin (Shandong Lukang Pharmaceutical Co., Ltd., People’s
Republic of China), Diaion HP20SS (Mitsubishi Chemical Co.,
Tokyo, Japan), silica gel (200−300 mesh) (Qingdao Marine Chemical
and Industrial Factory, Qingdao People’s Republic of China), MCI-gel
CHP20P (75−100 μm) (Mitsubishi Chemical Co., Ltd.), and RP-8 or
I
DOI: 10.1021/acs.jnatprod.5b00027
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Journal of Natural Products
Article
RP-18 gel (40−60 μm) (Merck, Darmstadt, Germany). Fractions were
monitored by TLC, and spots were visualized by heating the silica gel
plates sprayed with 10% sulfuric ethanol solution.
Notoginsenoside SP2 (2): amorphous powder; [α]2D1 +5.6 (c 1.4,
MeOH); IR (KBr) νmax 3418, 2965, 2944, 2877, 1632, 1452, 1371,
1165, 1077, 1028, 625, 576 cm−1; 1H and 13C NMR data, see Tables 1
and 2; HRESIMS (positive) m/z 839.4763 [M + Na]+ (calcd for
C42H72O15Na, 839.4763).
Plant Material. Air-dried roots of P. notoginseng were collected
from Wenshan County, Yunnan Province, People’s Republic of China,
in April 2011 and identified by one of the authors (C.-R.Y.). A voucher
specimen (KIB-Z-00336) has been deposited in the State Key
Laboratory of Phytochemistry and Plant Resources in West China,
Kunming Institute of Botany, Chinese Academy of Science. The raw
plant sample was crushed into small pieces and then steamed at high
temperature (120 °C) and pressure (0.12 MPa) for 12 h, yielding a
steamed P. notoginseng preparation.
Extraction and Isolation. The steamed P. notoginseng (15.0 kg)
was extracted with 80% aqueous MeOH (3 × 20 L, 2 days each) at
room temperature. After removal of the organic solvent, the extract
(3.0 kg) was subjected to Diaion 101 column chromatography (250 ×
30 cm), eluting with H2O to remove saccharides and then with MeOH
to give a total saponin fraction (2.12 kg), which were further
fractionated on a silica gel column (250 × 30 cm), eluting with
CHCl3−MeOH−H2O (85:15:1−75:25:2), to afford eight fractions
(Frs. A−H).
Fr. A (10 g) was chromatographed over an RP-18 column, eluting
with MeOH−H2O (7:3 to 9:1), to yield three subfractions (Fr. A1−
A3). Fr. A1 (45 mg) was purified by semipreparative HPLC
(CH3CN−H2O, 7:3) to give 23 (9 mg) and ginsenoside Rh3 (16
mg). Fr. B (200 g) was applied to RP-18 CC (MeOH−H2O, 1:1 to
9:1), to give five subfractions (Fr. B1−B5). Fr. B1 (120 g) was purified
by RP-18 CC (MeOH−H2O, 7:3), followed with recrystallization in
MeOH−H2O (75:25), to yield ginsenoside Rk1 (40 g) and
ginsenoside Rg5 (43 g). Fr. B2 (8 g) was applied to MCI-gel
CHP20P (MeOH−H2O, 6:4 to 9:1) and RP-18 (MeOH−H2O,
75:25) CC, followed by recrystallization, to give ginsenoside 20(S)-
Rh2 (800 mg) and 20(R)-Rh2 (1 g). Fr. B3 (200 mg) was purified by
RP-18 CC (MeOH−H2O, 78:22) to give 20(R)-dammarane-
3β,6α,12β,20,25-pentol (30 mg).
Fr. C (250 g) was subjected to a Diaion HP20SS CC (MeOH−
H2O, 4:6 to 9:1) to afford five subfractions (Frs. C1−C5). Frs. C1 (90
g) and C2 (102 g) were applied separately to RP-18 CC (MeOH−
H2O, 64:36), followed by recrystallization in MeOH−H2O (60:40,
55:45), to give ginsenoside Rk3 (30 g), Rh4 (34 g), and 20(S)-Rg3 (38
g) and 22 (36 g), respectively. Fr. C4 (63 mg) was purified by
semipreparative HPLC (CH3CN−H2O, 1:1) to give 24 (6 mg) and 25
(13 mg).
Fr. D (150 g), Fr. E (50 g), Fr. F (40 g), Fr. G (30 g), and Fr. H (10
g) were separately applied to an RP-18 column, eluting with MeOH−
H2O (4:6 to 9:1), to yield subfractions D1−D5, E1−E5, F1−F5, G1−
G4, and H1−H4, respectively. Fr. D2 (96 g) was further recrystallized
in MeOH−H2O (40:60) to yield 21 (33 g) and 20(S)-Rh1 (30 g). Fr.
E1 (173 mg) was purified by semipreparative HPLC (CH3CN−H2O,
23:77 to 30:70) to give 1 (13 mg), 2 (12 mg), and 3 (11 mg). Fr. E2
(27 g) was subjected to RP-18 CC (MeOH−H2O, 43:57), followed
with recrystallization in MeOH−H2O (35:65), to yield sanchinoside
B1 (9 g) and 26 (4 g). Fr. E3 (196 mg) was purified by
semipreparative HPLC (CH3CN−H2O, 33:67 to 40:60) to yield 18
(3 mg), 11 (15 mg), 17 (16 mg), and 27 (6 mg).
Fr. F2 (182 mg) was purified by semipreparative HPLC (CH3CN−
H2O, 23:77 to 28:72) to afford 4 (8 mg), 15 (15 mg), and 20 (7 mg).
Fr. F3 (121 mg) was purified by semipreparative HPLC (CH3CN−
H2O, 25:75 to 30:70) to yield 14 (12 mg) and 16 (14 mg). Fr. G3
(172 mg) was purified by semipreparative HPLC (CH3CN−H2O,
21:79 to 24:76) to yield 9 (6 mg), 10 (13 mg), 12 (15 mg), and 13
(17 mg). Fr. H1 (121 mg) was purified by semipreparative HPLC
(CH3CN−H2O, 15:85 to 18:82) to afford 6 (21 mg), 7 (7 mg), and
19 (35 mg). Fr. H2 (133 mg) was purified by semipreparative HPLC
(CH3CN−H2O, 19:81 to 22:78) to yield 5 (13 mg) and 8 (9 mg).
Notoginsenoside SP1 (1): amorphous powder; [α]2D1 −6.6 (c 0.86,
MeOH); IR (KBr) νmax 3423, 2964, 2935, 2876, 1633, 1463, 1368,
1154, 1076, 1043, 659, 571 cm−1; 1H and 13C NMR data, see Tables 1
and 2; HRESIMS (positive) m/z 839.4766 [M + Na]+ (calcd for
C42H72O15Na, 839.4763).
Notoginsenoside SP3 (3): amorphous powder; [α]2D1 −5.5 (c 1.7,
MeOH); IR (KBr) νmax 3424, 2941, 2878, 1639, 1464, 1454, 1414,
1
1384, 1198, 1170, 1077, 1038, 979, 646, 602, 579 cm−1; H and 13C
NMR data, see Tables 1 and 2; HRESIMS (positive) m/z 855.4494
[M + K]+ (calcd for C42H72O15K, 855.4503).
Notoginsenoside SP4 (4): amorphous powder; [α]2D1 −12.9 (c 0.55,
MeOH); IR (KBr) νmax 3428, 2962, 2930, 2876, 2856, 1636, 1464,
1414, 1384, 1154, 1077, 1033, 641, 534 cm−1; 1H and 13C NMR data,
see Tables 2 and 3; HRESIMS (positive) m/z 693.4188 [M + Na]+
(calcd for C36H62O11Na, 693.4184).
Notoginsenoside SP5 (5): amorphous powder; [α]2D1 +10.2 (c 0.54,
MeOH); IR (KBr) νmax 3424, 2961, 2933, 2876, 1631, 1462, 1384,
1
1156, 1075, 1029, 928, 574, 532 cm−1; H and 13C NMR data, see
Tables 2 and 3; HRESIMS (positive) m/z 693.4188 [M + Na]+ (calcd
for C36H62O11Na, 693.4184).
NotoginsenosideSP6 (6): amorphous powder; [α]2D1 +19.9 (c 1.4,
MeOH); IR (KBr) νmax 3425, 2964, 2934, 2878, 1632, 1464, 1384,
1
1154, 1074, 1027, 928, 580, 530 cm−1; H and 13C NMR data, see
Tables 2 and 3; HRESIMS (positive) m/z 693.4187 [M + Na]+ (calcd
for C36H62O11Na, 693.4184).
Notoginsenoside SP7 (7): amorphous powder; [α]2D1 −5.7 (c 0.77,
MeOH); IR (KBr) νmax 3421, 2964, 2935, 2878, 1629, 1462, 1384,
1155, 1074, 1031, 928, 891, 588, 533 cm−1; 1H and 13C NMR data, see
Tables 3 and 5; HRESIMS (positive) m/z 693.4187 [M + Na]+ (calcd
for C36H62O11Na, 693.4184).
Notoginsenoside SP8 (8): amorphous powder; [α]2D1 −4.7 (c 1.0,
MeOH); IR (KBr) νmax 3423, 2962, 2933, 2876, 1631, 1462, 1383,
1155, 1076, 1031, 612, 552 cm−1; 1H and 13C NMR data, see Tables 3
and 5; HRESIMS (positive) m/z 693.4184 [M + Na]+ (calcd for
C36H62O11Na, 693.4184).
Notoginsenoside SP9 (9): amorphous powder; [α]2D4 +22.2 (c 0.19,
MeOH); IR (KBr) νmax 3426, 2960, 2933, 2877, 1631, 1457, 1384,
1
1155, 1074, 1030, 893, 573, 529 cm−1; H and 13C NMR data, see
Tables 3 and 5; HRESIMS (positive) m/z 707.4326 [M + Na]+ (calcd
for C37H64O11Na, 707.4341).
Notoginsenoside SP10 (10): amorphous powder; [α]2D1 −23.7 (c
0.43, MeOH); IR (KBr) νmax 3431, 2960, 2932, 2877, 1631, 1452,
1
1384, 1155, 1073, 1030, 928, 579, 530 cm−1; H and 13C NMR data,
see Tables 3 and 5; HRESIMS (positive) m/z 707.4343 [M + Na]+
(calcd for C37H64O11Na, 707.4341).
Notoginsenoside SP11 (11): amorphous powder; [α]2D1 −9.4 (c
0.89, MeOH); IR (KBr) νmax 3424, 2944, 2877, 1632, 1552, 1463,
1
1453, 1385, 1161, 1077, 1030, 895, 593, 579 cm−1; H and 13C NMR
data, see Tables 1 and 2; HRESIMS (positive) m/z 839.4767 [M +
Na]+ (calcd for C42H72O15Na, 839.4763).
Notoginsenoside SP12 (12): amorphous powder; [α]2D1 +19.0 (c
0.92, MeOH); IR (KBr) νmax 3425, 2961, 2929, 2875, 1631, 1583,
1
1383, 1153, 1075, 1026, 972, 578 cm−1; H and 13C NMR data, see
Tables 4 and 5; HRESIMS (positive) m/z 675.4082 [M + Na]+ (calcd
for C36H60O10Na, 675.4079).
Notoginsenoside SP13 (13): amorphous powder; [α]2D1 +17.0 (c
0.64, MeOH); IR (KBr) νmax 3421, 2959, 2931, 2875, 1631, 1460,
1
1382, 1154, 1073, 1029, 892, 574 cm−1; H and 13C NMR data, see
Tables 4 and 5; HRESIMS (positive) m/z 675.4082 [M + Na]+ (calcd
for C36H60O10Na, 675.4079).
Notoginsenoside SP14 (14): amorphous powder; [α]2D1 +3.9 (c 1.0,
MeOH); IR (KBr) νmax 3431, 3426, 2960, 2932, 2878, 1633, 1452,
1
1380, 1152, 1071, 1027, 645, 561 cm−1; H and 13C NMR data, see
Tables 4 and 5; HRESIMS (positive) m/z 689.4238 [M + Na]+ (calcd
for C37H62O10Na, 689.4235).
Notoginsenoside SP15 (15): amorphous powder; [α]2D1 −24.2 (c
0.83, MeOH); IR (KBr) νmax 3424, 2960, 2935, 2876, 1631, 1453,
1
1383, 1074, 1034, 973, 596, 531 cm−1; H and 13C NMR data, see
Tables 4 and 5; HRESIMS (positive) m/z 675.4084 [M + Na]+ (calcd
for C36H60O10Na, 675.4079).
J
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Journal of Natural Products
Article
Notoginsenoside SP16 (16): amorphous powder; [α]2D1 −13.7 (c
1.0, MeOH); IR (KBr) νmax 3433, 2961, 2934, 2877, 1631, 1553, 1460,
1384, 1073, 1033, 591, 538 cm−1; 1H and 13C NMR data, see Tables 4
and 5; HRESIMS (positive) m/z 689.4241 [M + Na]+ (calcd for
C37H62O10Na, 689.4235).
Cytotoxicity Assay. As previously reported.40
ASSOCIATED CONTENT
■
S
* Supporting Information
The 1H and 13C NMR, HSQC, HMBC, 1H−1H COSY,
Notoginsenoside SP17 (17): amorphous powder; [α]2D1 −9.0 (c 0.9,
MeOH); IR (KBr) νmax 3423, 2944, 2876, 1632, 1564, 1453, 1414,
1
ROESY, and mass spectra for compounds 1−18, the H NMR
1
1384, 1160, 1076, 1038, 980, 627 cm−1; H and 13C NMR data, see
and ESIMS data of the aglycon (1a, 3a, 4a, 19a, 20a), the
HETLOC spectra of 5, 6, 10, and 13, and the ICD spectrum of
the aglycon (20a). The Supporting Information is available free
of charge on the ACS Publications website at DOI: 10.1021/
acs.jnatprod.5b00027.
Tables 1 and 2; HRESIMS (positive) m/z 835.4820 [M + Na]+ (calcd
for C43H72O14Na, 835.4814).
Notoginsenoside SP18 (18): amorphous powder; [α]2D1 +18.7 (c
0.57, MeOH); UV (MeOH) λmax (log ε) 257 (3.26) nm; IR (KBr)
νmax 3432, 2960, 2932, 2875, 1630, 1614, 1384, 1155, 1075, 1033, 576
cm−1; 1H and 13C NMR data, see Tables 2 and 4; HRESIMS
(positive) m/z 673.3927 [M + Na]+ (calcd for C36H58O10Na,
673.3922).
AUTHOR INFORMATION
Corresponding Authors
*Tel/Fax: +86-871-6522-3235. E-mail: zhangyj@mail.kib.ac.cn
(Y.-J. Zhang).
■
Acid Hydrolysis of Compounds 1 and 6. Compounds 1 and 6
(each 5 mg) were hydrolyzed in 2 M HCl (5 mL) at 65 °C for 6 h,
respectively. The reaction mixture was extracted with CHCl3 three
times (3 × 5 mL). The aqueous layer was neutralized with 2 M NaOH
and dried to produce a monosaccharide mixture. Next, a solution of
the sugar mixture in pyridine (2 mL) was added to L-cysteine methyl
ester hydrochloride (1.5 mg) and kept at 60 °C for 1 h. After this,
trimethylsilylimidazole (1.5 mL) was added to the reaction mixture in
an ice−water bath and kept at 60 °C for 30 min. The mixture was
subjected to GC analysis, run on a Shimadzu GC-14C gas
chromatograph, equipped with a 30 m × 0.32 mm i.d. 30QC2/AC-5
quartz capillary column and a H2 flame ionization detector with the
following conditions: column temperature, 180−280 °C; programmed
increase, 3 °C/min; carrier gas, N2 (1 mL/min); injector and detector
temperature, 250 °C; injection volume, 4 μL; and split ratio 1/50. The
configuration of the sugar moiety was determined by comparing the
retention time with the derivatives of the authentic samples. The
retention times of D-/L-glucose were 21.135/21.580 min, and the
configuration of the respective sugar moiety from compounds 1 and 6
was determined as D-glucose.
Enzymatic Hydrolysis of Compounds 1, 3, 4, 19, and 20.
Compounds 1, 3, 4, 19, and 20 (each 6 mg) in MeOH−H2O (1:100,
20 mL) were hydrolyzed with cellulase (300 mg) at 37 °C for 4 days.
The reaction mixture was subjected to an MCI-gel CHP20P column,
eluted initially with 10% MeOH and then with 70% MeOH. The 70%
MeOH fraction was evaporated to dryness and yielded the aglycones
1a, 3a, 4a, 19a, and 20a (1.5 mg, respectively) from 1, 3, 4, 19, and 20,
respectively.
*E-mail: minxu@mail.kib.ac.cn (M. Xu).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors are grateful to the members of the analytical group
at the State Key Laboratory of Phytochemistry and Plant
Resources in West China, Kunming Institute of Botany,
Chinese Academy of Science, for measuring the spectroscopic
data. This work was supported by the Science and Technology
Planning Project of Yunnan Province, China (2013FC008), the
973 Program of Ministry of Science and Technology of China
(2011CB915503), the National Natural Science Foundation of
China (No. 81173477), and the 12th Five-Year National
Science and Technology Supporting Program of China
(2012BAI29B06).
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