Cytotoxic Cardiac Glycoside Constituents of Vallaris glabra Leaves

Article abstract of DOI:10.1021/acs.jnatprod.7b00554

Thirteen cardenolide glycosides (1-13) were isolated from the CH₂Cl₂ and MeOH extracts of Vallaris glabra leaves. The structures of the new compounds (2-13) were identified by spectroscopic methods, with the absolute configurations o

Full text of DOI:10.1021/acs.jnatprod.7b00554

Article
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Cite This: J. Nat. Prod. XXXX, XXX, XXX-XXX
Cytotoxic Cardiac Glycoside Constituents of Vallaris glabra Leaves
Sudarat Kruakaew,† Chonticha Seeka,† Thitima Lhinhatrakool,‡ Sanit Thongnest,§ Jantana Yahuafai,⊥
Suratsawadee Piyaviriyakul,⊥ Pongpun Siripong,⊥ and Somyote Sutthivaiyakit*,†
†Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Ramkhamhaeng University,
Hua Mark, Bangkapi, Bangkok 10240, Thailand
‡College of Oriental Medicine, Rangsit University, Muang Ake, Pathumthani 12000, Thailand
§Chulabhorn Research Institute, Kamphang Phet 6 Road, Bangkok 10210, Thailand
⊥Natural Products and Integrative Medicine Research Section, Research Division, National Cancer Institute, Rama VI Road, Bangkok
10400, Thailand
S
* Supporting Information
ABSTRACT: Thirteen cardenolide glycosides (1−13) were isolated from
the CH2Cl2 and MeOH extracts of Vallaris glabra leaves. The structures of
the new compounds (2−13) were identified by spectroscopic methods,
with the absolute configurations of the sugar moieties determined by acid
hydrolysis. All compounds were evaluated for their cytotoxic activity
against human cervix adenocarcinoma, lung carcinoma, and colorectal
adenocarcinoma cell lines. The two most potent compounds [2′-O-
acetylacoschimperoside P (1) and oleandrigenin-3-O-α-L-2′-O-acetylvallar-
opyranoside (2)] exhibited IC50 values in the range of 0.03−0.07 μM.
Vallaris glabra (L.) Kuntze (Apocynaceae), commonly known
as bread flower and named “com-ma-nat” in Thailand, is a
shrub growing up to 8 m in height.1 Its latex was reported to
possess wound-healing and strong laxative properties and can
cause vomiting, stimulate striated muscle, increase blood
pressure, and induce uterine contractions.2 A previous study
on this species demonstrated the presence of two cardenolide
glycosides, 2′-O-acetylacoschimperoside P (1) and 1-acetox-
ydigitoxigenin-3-O-α-D-2′-O-acetylacofrioside, with hedgehog
signaling inhibitory and cytotoxic activities.3 Subsequently, the
isolation of ursolic acid, stearic acid, and caffeoyl esters of
quinic acid from the leaves was reported.4 Several mono-
terpenoids and 2-acetyl-1-pyrroline were also found to occur in
the flowers.5 In the present work, a TLC investigation, using
Kedde’s reagent as a staining reagent, revealed the presence of
several cardenolides in the CH2Cl2 and MeOH extracts of the
fresh leaves of V. glabra. The CH2Cl2 extract showed cytotoxic
activity against human cervix adenocarcinoma (HeLaS3),
human lung carcinoma (A549), and human colorectal
adenocarcinoma (HT-29) cells with IC50 values of 1.3 0.1,
schimperoside P (1),3 together with three cardenolide
glycosides (2−4), while nine cardenolide glycosides (5−13)
were isolated from the MeOH extract. We report herein the
spectroscopic identification of these cardenolides (2−13)
1
together with the H and 13C NMR spectroscopic data of 1
in MeOH-d4 and their cytotoxic activities against three cancer
cell lines.
RESULTS AND DISCUSSION
■
Compound 2 was obtained as a colorless solid, mp 172−173
°C, with a molecular formula of C34H50O11 based on its
HRESIMS [M + Na]+ ion at m/z 657.3263 (calcd for
C34H50O11Na, 657.3353). The IR absorption bands at νmax
1732 (CO stretch) and 1650 cm−1(CC stretch), the H
1
NMR resonances at δH 4.99 and 4.96 (both as dd, J = 18.5 and
1.7 Hz, H2-21) and δH 5.97 (t, J = 1.7 Hz, H-22), and 13C NMR
resonances at δC 175.4, 170.9, 120.3, and 76.7 indicated the
presence of an α,β-unsaturated-γ-lactone moiety in 2 (Tables 3
and 4). The cardenolide aglycone was characterized as
1
oleandrigenin based on the H and 13C NMR spectroscopic
1.6
0.1, and 1.9
0.1 μg/mL, respectively. The MeOH
data, particularly the 1H NMR resonances at δH 3.90 (brs, W1/2
= 8.0 Hz, H-3α),10 2.07 (s, OCOCH3-2′), 1.93 (s, OCOCH3-
16), 5.46 (ddd, J = 9.5, 8.7, and 2.0 Hz, H-16), and 3.25 (d, J =
8.7 Hz, H-17) as found in 2′-O-acetylacoschimperoside P
extract was inactive against these three cell lines (IC50 > 20 μg/
mL). Thorough chromatographic purification of the CH2Cl2
extract led to the isolation of 10 known compounds, 3β,28-
dihydroxyurs-12-ene (uvaol),6 3β-hydroxyolean-12-en-28-oic
acid,7 3β-hydroxyurs-12-en-28-oic acid,7 3β,27-dihydroxyurs-
12-en-28-oic acid,8 cycloeucalenol,9 lupeol,7 stigmast-4-en-3-
one, β-sitosterol, β-sitosteryl glucoside, and 2′-O-acetylaco-
Received: June 29, 2017
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.7b00554
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
3′′), 71.7 (C-4′′), 77.8 (C-5′′), and 62.9 (C-6′′)]. The key
HMBC cross-peak between H-1′′/C-4′ revealed the con-
nectivity of C-1′′ to C-4′-O. Upon acid hydrolysis of 3 with 5%
HCl at 70 °C for 6 h and subsequent chromatographic
purification, the aglycone 14,16-dianhydrogitoxigenin,11 togeth-
er with L-acofriose (3-O-methyl-L-rhamnose)12 and D-glucose,13
was obtained. On the basis of its NMR spectroscopic data and
the acid hydrolysis procedure, compound 3 was thus elucidated
as oleandrigenin-3-O-β-D-glucopyranosyl-(1→4)-α-L-2′-O-ace-
tylacofriopyranoside.
Compound 4 was isolated as a colorless solid with mp 183−
184 °C. The high-resolution ESIMS suggested a molecular
formula of C40H60O16, as indicated from the [M + Na]+ ion at
m/z 819.3793 (calcd for C40H60O16Na, 819.3773). Most of the
1H and 13C resonances were similar to those of compound 3
(Tables 1−4), except for the data of H-3′ (δH 3.73, dd, J = 5.6
and 3.3 Hz) and H-4′ (δH 3.90, dd, J = 6.7 and 3.3 Hz) and a
deshielded quintet at δH 4.28 (J = 6.7 Hz) of H-5′. This
indicated a β-oriented OCH3-3′, thus establishing the presence
of a vallaropyranosyl moiety. A glucopyranosyl unit was
1
detected from H and 13C NMR resonances particularly at δH
4.39 (d, J = 7.7 Hz, H-1″) and δC 103.0 (C-1″) and of an
oxymethylene group at δH 3.86 and 3.65 and δC 62.8. The key
HMBC cross-peak between H-1″/C-4′ indicated a connectivity
of C-1′′ to C-4′-O. Based on the acid hydrolysis of 2 and 3,
which furnished L-vallarose, and L-acofriose and D-glucose,
respectively, compound 4 was therefore assigned as olean-
drigenin-3-O-β-D-glucopyranosyl-(1→4)-α-L-2′-O-acetylvallaro-
pyranoside.
(oleandrigenin-3-O-α-L-2′-O-acetylacofriopyranoside, 1),3 pre-
Compound 5 was isolated as a colorless, amorphous solid
with a molecular formula of C38H56O14, as indicated from the
HRESIMS {m/z 759.3580 [M + Na]+ (calcd for C38H56O14Na
viously isolated from this plant and also in the present study.
1
The H NMR resonances of the deoxy sugar unit and its
coupling constants [δH 4.70 (brs, H-1′), 5.08 (dd, J1′,2′ = 1.8
1
759.3562)}. Patterns of the H and 13C NMR resonances of 5
Hz, J2′,3′ = 3.5 Hz, H-2′), 3.49 (t, J2′,3′ = J3′,4′ = 3.5 Hz, H-3′),
1
resembled those of 3 (Tables 1, 2, and 5) except that the H
3.45 (dd, J3′,4′ = 3.5 Hz, J4′,5′ = 8.7 Hz, H-4′), 4.08 (dq, J4′,5′
=
NMR resonances of H-16 at ca. δH 5.46 and a singlet at ca. δH
1.93 (CH3CO-16) as encountered in 3 were replaced by
resonances at δH 6.25 (brs, H-16), together with a doublet at δH
2.77 and a doublet of doublets at δH 2.31 of H2-15, indicating
the cardenolide aglycone as 16-anhydrogitoxigenin.14 Based on
8.7 Hz, J5′,6′ = 6.4 Hz, H-5′), and 1.20 (d, J5′,6′ = 6.4 Hz, H3-6′)]
were slightly different from those of the 2′-O-acetylacofriopyr-
anosyl group [δH 3.45 (dd, J2′,3′ = 3.4 Hz, J3′,4′ = 9.5 Hz, H-3′),
3.35 (t, J3′,4′ = J4′,5′ = 9.5 Hz, H-4′)] in 1 (Tables 1 and 3),
revealed the orientation of the OCH3-3′ as β, and thus
established the sugar moiety as a 2′-O-acetylvallaropyranosyl
group. In addition, the ROESY spectrum exhibited cross-peaks
between OCH3-3′/H-5′, H-3/H-1′, H-16/H-17, and H3-18/H-
22. The absolute configuration of the sugar was assigned after
acid hydrolysis of 2 with 5% HCl at 70 °C for 6 h to give the
aglycone 14,16-dianhydrogitoxigenin [3-hydroxycarda-
14,16,20(22)-trienolide]11 and L-vallarose (3-O-methyl-6-des-
oxy-L-altrose) showing [α]D26 −38.9 (c 0.04, H2O), lit.12 [α]2D3
−17.2 (c 0.90, H2O). The structure of compound 2 was thus
elucidated as oleandrigenin-3-O-α-L-2′-O-acetylvallaropyrano-
side. This compound was obtained from vallarosolanoside,
previously reported in V. solanacea,12 after acetylation using
acetic anhydride in pyridine; however, there were no NMR
spectroscopic data reported for this derivative.
1
the H−1H COSY, HSQC, and HMBC data and the acid
hydrolysis of 3, which provided L-acofriose and D-glucose, the
structure of compound 5 was thus proposed as 16-
anhydrogitoxigenin-3-O-β-D-glucopyranosyl-(1→4)-α-L-2′-O-
acetylacofriopyranoside.
Compound 6, a colorless, amorphous solid with a molecular
formula of C36H54O13, exhibited a molecular mass 42 amu
lower than that of 5 {HRESIMS [M + Na]+ ion at m/z
1
717.3462 (calcd for C36H54NaO13, 717.3447)}. The H NMR
and 1H−1H COSY spectra revealed the absence a singlet of an
O-acetyl group at ca. δH 2.09, and a resonance of H-2′ at ca. δH
5.19 (dd, J = 3.3 and 1.6 Hz), as found in 5, was replaced by a
shielded resonance of H-2′ at δH 4.00 (dd, J = 3.1 and 1.8 Hz),
thus indicating the deoxy sugar as an acofriose instead of 2′-O-
acetylacofriose. The remainder of the 1H and 13C NMR
resonances, particularly of the aglycone, resembled those of 5
(Table 5). On the basis of acid hydrolysis of 3, which gave D-
glucose and L-acofriose, the structure of 6 was proposed as 16-
anhydrogitoxigenin-3-O-β-D-glucopyranosyl-(1→4)-α-L-aco-
friopyranoside.
Compound 3, a colorless solid with mp 179−181 °C, was
assigned a molecular formula of C40H60O16 as deduced from
the HRESIMS, which gave a [M + Na]+ ion at m/z 819.3796
1
(calcd for C40H60O16Na, 819.3773). The H and 13C NMR
spectra showed similar resonance patterns to those found for 1
(Tables 1 and 2), with additional resonances of a glucopyr-
anosyl ring [(δH 4.57, d, J = 7.8 Hz, H-1″), 3.15 (dd, J = 9.1 and
7.8 Hz, H-2″), 3.35 (H-3′′), 3.29 (H-4′′), 3.24 (H-5′′), and
3.86 (dd, J = 11.9 and 2.1 Hz, H-6a″), and 3.67 (dd, J = 11.9
and 5.3 Hz, H-6b″), δC 104.9 (C-1″), 75.6 (C-2′′), 77.8 (C-
Compound 7 was obtained as an amophous solid showing
1
the same molecular mass as 6. The H and 13C NMR spectra
exhibited similar resonances to those of 6 (Table 5), except that
the resonances of an acofriopyranosyl moiety were replaced by
B
DOI: 10.1021/acs.jnatprod.7b00554
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
1
Table 1. H NMR Spectroscopic Data (400 MHz) of 1, 3, 8, 12, and 13 (in MeOH-d4)
1
3
8
12
13
δH (J in Hz)
1.59, 1.25
position
δH (J in Hz)
δH (J in Hz)
δH (J in Hz)
δH (J in Hz)
1
1.91, 1.43
1.88, 1.32
1.44, 1.56
1.79, 1.48
2
1.73, 1.59
3.96 (w1/2 = 9.0)
1.64, 1.48
1.69
1.62a
191, 1.31
3.97 (w1/2 = 8.0)
1.89, 1.50
1.70
1.91, 1.27
3.97d
1.59, 1.25
3.95 (w1/2 = 9.0)
1.88
3
3.95 brs (w1/2 = 9.0)
4
1.66, 1.46
1.67
1.87, 1.28
1.67
5
1.65
6
1.88, 1.29
1.82, 1.25
1.62
1.29
1.65, 1.60
1.86, 1.24
1.68
1.57
1.56
7
1.23
1.62a
1.81
1.87, 1.23
1.62
8
1.62
9
1.68
1.69
1.75
1.73
1.69
10
11
12
13
14
15
1.44
1.44
1.48, 1.29
1.57, 1.46
1.41, 1.23
1.44
1.79, 1.44
1.54, 1.41
1.55, 1.41
1.54, 1.43
2.78 dd (15.6, 9.6), 1.79 dd 2.78 brd (15.6, 9.6), 1.79
2.81 dd (15.5, 9.7), 1.83 dd 1.82 dd (15.4, 2.3), 2.81 dd 1.79 dd (15.6, 2.2), 2.75 dd
(15.6, 2.4)
5.46 ddd (9.6, 8.7, 2.4)
3.25 d (8.7)
0.93 s
dd (15.6, 2.4)
(15.5, 2.4)
5.49 dt (9.7, 2.4)
3.30 d (9.7)i
0.96 s
(15.4, 9.6)
(15.6, 9.5)
5.47 dt (9.5, 2.2)
3.26 d (9.5)
0.93 s
16
17
18
19
20
21
5.46 ddd (11.2, 9.4, 2.3)
3.26b
5.50 ddd (9.6, 7.3, 2.3)
3.30i
0.93 s
0.96 s
0.96 s
0.98 s
0.97 s
0.98 s
0.96 s
j
4.99 dd (18.2, 1.8), 4.96 dd 4.97 dd (18.2, 1.8), 4.9
(18.2, 1.8)
5.04 dd (18.5, 1.7), 5.00 dd 5.04 dd (18.6, 1.8), 4.9 dd 5.01 dd (18.6, 1.7), 4.94 dd
(18.5, 1.7)
(18.6, 1.8)
(18.6, 1.7)
22
23
1′
2′
3′
4′
5′
6′
5.97
5.97 dd (1.8, 1.48)
6.00 t (1.7)
6.00 s
5.97 s
4.80 d (1.7)
4.81 d (1.7)
5.19 dd (3.3, 1.7)
3.73 dd (9.4, 3.3)
3.61 t (9.4)
3.76 dq (9.4, 6.2)
1.30 d (6.2)
1.93 s
4.83 d (1.7)
4.83 d (1.8)
3.99 dd (3.0, 1.8)d
3.65 dd (8.9, 3.0)
3.75e
3.76 quint (5.6)e
1.28 d (5.6)
1.96 s
4.80 d (1.7)
5.18 dd (3.8, 1.7)
3.73 dd (9.3, 3.8)g
3.62 t (9.3)h
3.76 dq (6.1, 9.2)g
1.30 d (6.1)
1.93 s
5.17 dd (3.4, 1.7)
3.45 dd (9.5, 3.4)
3.35 t (9.5)
3.99 dd (3.1, 1.7)
3.62 dd (8.8, 3.1)
3.72 t (9.6, 8.8)c
3.74 dq (9.6, 5.5)c
1.31 d (5.5)
3.70 dq (9.5, 6.2)
1.24 d (6.2)
OCOCH3-16 1.93 s
1.96 s
OCOCH3-2′ 2.07 s
2.08 s
2.11 s
OCH3-3′
3.39 s
3.38 s
3.46 s
3.46 s
3.39 s
1″
2″
3″
4″
5″
6″
4.57 d (7.8)
3.15 dd (9.1, 7.8)
3.35
4.61 d (7.7)
4.62 d (7.7)
4.58 d (7.8)
3.16 dd (8.4, 7.8)
3.36i
3.19 dd (8.7, 7.7)
3.39 t (8.7)
3.16 dd (8.7, 7.7)
3.36 t (8.7)
3.29
3.31 t (8.7)
3.30 t (8.7)
3.32 dd (9.3, 7.1)i
3.24b
3.25 ddd (8.7, 5.5, 2.2)
3.42 ddd (8.7, 5.9, 2.1)
3.42 ddd (9.3, 7.1, 2.0)
3.86 dd (11.9, 2.1), 3.67 dd 3.87 dd (12.1, 2.2), 3.69 dd 4.16 dd (11.7, 2.1), 3.78 dd 4.14 dd (11.7, 2.0), 3.78f
(11.9, 5.3)
(12.1, 5.5)
(11.7, 5.9)
1‴
2‴
3‴
4‴
5‴
6‴
4.40 d (7.8)
4.37 d (7.8)
3.19 dd (8.6, 7.8)
3.36 t (8.6)
3.28 dd (9.4, 7.8)
3.36 t (9.4)
3.27 t (8.6)f
3.28 t (9.4)
3.26 ddd (10, 5.4, 2.2)f
3.27 dt (9.4, 4.0)
3.88 brd (11.9), 3.69 dd
(11.9, 5.4)
3.86 dd (12.0, 1.9), 3.66 dd
(12.0, 4.0)h
a−i
j
Overlapped signals. Obscured by solvent signal.
those of a vallaropyranosyl moiety [δH 4.65 (d, J = 3.1 Hz, H-
1′), 3.92 (dd, J = 6.9 and 3.1 Hz, H-2′), 3.63 (dd, J = 6.9 and
3.1 Hz, H-3′), 3.95 (dd, J = 6.9 and 3.1 Hz, H-4′), 4.21 (dq, J =
6.9 and 6.6 Hz, H-5′), and 1.23 (d, J = 6.6 Hz, H-6′)]. A
glucopyranosyl moiety was deduced from the 1H and 13C NMR
resonances at δH 4.40 (d, J = 7.7 Hz, H-1″) and δC 102.6 (C-
1″) and an oxymethylene group at δH 3.85 and 3.66 and δC
62.8. The key HMBC cross-peak between H-1″/C-4′ indicated
the connectivity of C-1′′ to C-4′-O. Based on the acid
hydrolyses of 2 and 3, which provided L-vallarose, and L-
acofriose and D-glucose, respectively, the structure of 7 was thus
assigned as 16-anhydrogitoxigenin-3-O-β-D-glucopyranosyl-
(1→4)-α-L-vallaropyranoside.
Compound 8 was proposed with a molecular formula of
C38H58O15, based on its HRESIMS. Comparison of its NMR
spectroscopic data with those of 6 (Tables 1, 2, and 5) revealed
1
the H and 13C resonances of sugar moieties to have almost
identical patterns, while the resonances of the aglycone showed
the corresponding resonances of OCOCH3-16 (δH 1.96), H-16
(δH 5.49, dt, J = 9.7 and 2.4 Hz), and H-17 (δH 3.30, d, J = 9.7
C
DOI: 10.1021/acs.jnatprod.7b00554
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Table 2. 13C NMR Spectroscopic Data (100 MHz) of 1, 3, 8, 12, and 13 (in MeOH-d4)
1
3
8
12
13
position
δC, type
δC, type
δC, type
δC, type
δC, type
1
29.4, CH2
31.6, CH2
31.6, CH2
31.6, CH2
30.9, CH2
2
25.9, CH2
73.1, CH
30.2, CH2
36.8, CH
26.3, CH2
20.8, CH2
41.4, CH
35.3, CH
34.9, C
27.3, CH2
74.6, CH
30.9, CH2
38.6, CH
27.7, CH2
22.2, CH2
42.7, CH
36.6, CH
36.3, C
27.7, CH2
74.0, CH
30.9, CH2
38.1, CH
27.4, CH2
22.2, CH2
42.7, CH
36.6, CH
36.3, C
27.7, CH2
74.0, CH
30.9,CH2
38. 2, CH
27.4, CH2
22.2, CH2
42.7, CH
39.7, CH
36.3, C
27.3, CH2
74.7, CH
31.6, CH2
38.2, CH
27.6, CH2
22.2, CH2
42.7, CH
36.7, CH
36.3, C
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
1′
2′
3′
4′
5′
6′
20.6, CH2
38.6, CH2
50.0, C
22.0, CH2
39.9, CH2
51.4, C
22.0, CH2
39.9, CH2
51.4, C
22.0, CH2
39.9 CH2
51.4, C
22.0, CH2
40.0, CH2
51.4, C
83.6, C
85.0, C
84.9, C
85.0, C
84.9, C
39.9 CH2
74.6 CH
41.3, CH2
76.0, CH
57.4, CH
16.4, CH3
24.4, CH3
171.6, C
41.3, CH2
76.0, CH
57.4, CH
16.4, CH3
24.3, CH3
171.6, C
41.4, CH2
76.0, CH
57.4, CH
16.4, CH3
24.3, CH3
171.6, C
41.4, CH2
76.0, CH
57.5, CH
16.4, CH3
24.3, CH3
171.6, C
77.6, CH2
121.8, CH
176.8, C
97.3, CH
70.2, CH
81.0, CH
79.3, CH
68.8, CH
18.4, CH3
56.1, CH
15.0, CH3
22.9, CH3
170.8, C
76.2, CH2
120.4, CH
175.4, C
77.6, CH2
121.7, CH
176.8, C
77.5, CH2
121.7, CH
176.7, C
77.6, CH2
121.7, CH
176.8, C
96.0, CH
69.0, CH
79.3, CH
71.8, CH
68.7, CH
16.6, CH3
170.6, C; 19.5, CH3
170.3, C; 19.4, CH3
56.7, CH3
97.3, CH
70.1, CH
81.0, CH
79.2 CH
99.8, CH
68.5, CH
82.6, CH
79.3, CH
68.8, CH
18.2, CH3
21.0, CH3, 172.2, C
99.8, CH
68.6, CH
82.7, CH
79.0, CH
68.8, CH
18.4, CH3
172.2, C, 20.9, CH3
68.8, CH
18.2, CH3
172.1,a C; 20.9, CH3
172.2,a C; 20.8, CH3
57.7, CH3
104.9, CH
75.6, CH
77.8, CHb
71.7, CH
77.8, CHb
62.9, CH2
OCOCH3-16
172.3, C; 20.9, CH3
172.2, C; 20.9, CH3
57.7, CH3
104.9, CH
75.6, CH
OCOCH3-2′
OCH3-3′
1″
2″
3″
4″
5″
6″
1‴
2‴
3‴
4‴
5‴
56.9, CH3
105.0, CH
75.8, CH
77.8, CH
71.7, CH
77.9, CH
62.9, CH2
56.9, CH3
104.9, CH
75.7, CH
77.9, CH
71.6, CH
77.0, CH
70.5, CH2
105.0, CH
75.2, CH
78.0, CH
71.9, CH
77.9, CH
62.8, CH2
78.0, CH
71.8, CH
77.0, CH
70.5, CH2
105.1, CH
75.2, CH
78.0, CH
71.6, CH
77.8, CH
6‴
62.2, CH2
a,b
Overlapped signals.
Hz), as in the oleandrigenin unit of 1−3. On the basis of acid
hydrolysis of 3, which provided L-acofriose and D-glucose, and
field than H-5′ of 8. Considering the 1H NMR resonances and J
values of H-1′, H-2′, H-3′, H-4′, and H-6′, which were detected
at δH 4.68 (d, J = 3.1 Hz), 3.95 (dd, J = 5.4 and 3.1 Hz), 3.65
(dd, J = 5.4 and 3.3 Hz), 3.98 (dd, J = 6.7 and 3.3 Hz), and 1.29
(d, J = 6.7 Hz), respectively, it could be deduced that one of the
sugar moieties is a α-vallaropyranosyl unit. Characteristic
resonances of a β-glucopyranosyl group were also present.
The HMBC cross-peaks between H-3/C-1′ and H-1″/C-4′
indicated connectivities between the oxygen atom at C-3 to C-
1′, and C-4′-O to C-1″. On the basis of acid hydrolyses of 2 and
3, which provided L-vallarose, and L-acofriose and D-glucose,
respectively, compound 9 could thus be proposed as
1
further perusal of the H,1H−COSY, HSQC, HMBC, and 1D-
TOCSY data, the structure of compound 8 was elucidated as
oleandrigenin-3-O-β-D-glucopyranosyl-(1→4)-α-L-O-acofrio-
pyranoside.
Compound 9 was isolated as a colorless, amorphous solid,
having the same molecular formula, C38H58O15, as that of 8
based on the HRESIMS. The 1H and 13C NMR spectra (Tables
3 and 4) showed characteristic resonances of an oleandrigenin
aglycone, with two sugar moieties quite similar to those of 8.
The 1H NMR resonance of H-5′ was notably detected as a well-
separated quintet at δH 4.24 (J = 6.7 Hz), at somewhat lower
D
DOI: 10.1021/acs.jnatprod.7b00554
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
1
Table 3. H NMR Spectroscopic Data (400 MHz) of 2, 4, 9, 10, and 11 (in MeOH-d4)
2
4
9
10
11
δH (J in Hz)
1.86, 1.57
position
δH (J in Hz)
δH (J in Hz)
δH (J in Hz)
δH (J in Hz)
1
1.73, 1.27
1.81
1.57
3.91a
1.49
1.65
1.89
1.31
2
1.58
1.93, 1.29
4.00b
1.94, 1.29
4.00c
1.90, 1.27
3.92e
1.51
3
3.90 (w1/2 = 8.0)
1.89. 1.49
1.63
4
1.80, 1.53
1.75
1.86, 1.53
1.79
5
1.75
6
1.87, 1.25
1.82, 1.18
1.51
1.87, 1.24
1.44
1.64
1.63
1.60
7
1.85, 1.26
1.67
1.82
1.80
8
1.61
1.56
1.63
9
1.59
1.67
1.72
1.66
1.69
10
11
12
13
14
15
1.51
1.77, 1.18
1.52, 1.42
1.49, 1.27
1.60, 1.46
1.46,
1.48, 1.24
1.54, 1.44
1.45, 1.33
1.59, 1.39
2.77 dd (15.4, 9.5), 1.79 dd 2.77 dd (15.7, 9.6), 1.78 dd 2.88 dd (15.6, 9.6), 1.82 dd 2.64 dd (14.9, 8.4), 1.72 dd 1.79 dd (15.6, 2.4), 2.78 dd
(15.4, 2.0)
5.46 ddd (9.5, 8.7, 2.0)
3.25 d (8.7)
0.93 s
(15.7, 2.1)
5.46 ddd (9.6, 8.2, 2.1)
3.26 d (8.2)
0.93 s
(15.6, 2.2)
5.49 dt (9.6, 2.2)
3.30 d (9.6)e
0.96 s
(14.9, 2.2),
4.65 dt (8.2, 2.1)
3.15 d (8.2)e
0.94 s
(15.6, 9.4)
5.46 dt (9.4, 2.4)
3.26 d (9.4)
0.91 s
16
17
18
19
20
21
0.95 s
0.94 s
0.98 s
0.97 s
0.93 s
4.99 dd (18.5, 1.7), 4.96 dd 5.01 dd (18.5, 1.7), 4.94 dd 5.09 dd (18.5, 1.7), 4.97 dd 5.19 dd (1.76, 18.6), 5.13
(18.5, 1.7)
5.01 dd (18.5, 1.7), 4.94 dd
(18.5, 1.7)
(18.5, 1.7)
(18.5, 1.7)
dd (1.6, 18.5)
22
23
1′
2′
3′
4′
5′
6′
5.97 t (1.7)
5.97 brs
6.00 s
5.96 brs
5.97 s
4.70 brs
4.73 d (3.3)
5.11 dd (5.6, 3.3)
3.73 dd (5.6, 3.3)
3.90 dd (6.7, 3.3)a
4.28 quint (6.7)
1.25 d (6.7)
1.93 s
4.68 d (3.1)
4.68 d (3.1)
4.66 d (2.9)
5.08 dd (3.5, 1.8)
3.49 t (3.5)
3.95 dd (5.4, 3.1)
3.65 dd (5.4, 3.3)
3.98 dd (6.7, 3.3)b
4.24 quint (6.7)
1.29 d (6.7)
3.95 dd (5.4, 3.1)
3.65 dd (5.4, 3.2)
3.97 dd (6.7, 3.2)c
4.24 quint (6.7)
1.26 d (6.7)
3.93 dd (5.2, 2.9)e
3.62 dd (5.2, 3.3)
3.81 dd (7.0, 3.3)
4.20 dq (7.0, 6.6)
1.24 d (6.6)
3.45 dd (8.7, 3.5)
4.08 dq (8.7, 6.4)
1.20 d (6.4)
OCOCH3-16 1.93 s
1.96 s
1.93 s
OCOCH3-2′ 2.07 s
2.07 s
OCH3-3′
3.48 s
3.47 s
3.51 s
3.51 s
3.47 s
1″
2″
3″
4″
5″
6″
4.39 d (7.7)
3.21 dd (9.0, 7.7)
3.36
4.43 d (7.7)
4.43 d (7.7)
4.39 d (7.4)
3.24 dd (9.4, 7.7)
3.39 t (9.4)
3.21 dd (9.2, 7.7)
3.22 dd (8.8, 7.4)
3.36 t (8.8)
3.37 t (9.2)
3.27b
3.32 t (9.4)
3.32 dd (9.6, 9.2)d,g
3.28 ddd (9.6, 4.2, 1.2)d
3.31 t (8.8)
3.27b
3.29 ddd (9.4, 5.4, 1.9)
3.46.ddd (8.8, 6.1, 2.1)
3.86 d (11.8), 3.65 dd
(11.8, 5.4)
3.88 dd (12.1, 1.9), 3.69 dd 3.88 dd (12.0, 1.2), 3.69 dd 4.14 dd (11.7, 2.1), 3.77 dd
(12.1, 5.4)
(12.0, 4.2)
(11.7, 6.1)
1‴
2‴
3‴
4‴
5‴
6‴
4.39 d (7.6)
3.19 dd (8.7, 7.6)
3.37 t (8.7)
3.28 obs dd (8.7, 7.3)f
3.24f
3.86 dd (11.6, 1.9) 3.66 dd
(11.6, 5.2)
a−f
g
Overlapped signals. Obscured by solvent signal.
oleandrigenin-3-O-β-D-glucopyranosyl-(1→4)-α-L-vallaropyra-
noside.
(gitoxigenin). The structure of compound 10 was thus
elucidated to be gitoxigenin-3-O-β-D-glucopyranosyl-(1→4)-α-
L-vallaropyranoside.
Compound 11 was isolated as an amorphous solid with a
molecular formula of C44H68O20 based on the HRESIMS. The
1H and 13C NMR spectra showed some complex sets of
resonances, however, with recognizable resonances of the
aglycone oleandrigenin and three dioxygenated methine groups
being evident at δH 4.66 (d, J = 2.9 Hz), 4.39 (d, J = 7.4 Hz),
and 4.39 (d, J = 7.6 Hz) and δC 100.3, 102.4, and 105.0 (Tables
Compound 10 was isolated as an amorphous solid with a
molecular formula of C36H56O14 as detected by HRESIMS, a
molecular mass of 42 amu lower than that of 9. The 1H and 13C
NMR spectra of 10 were similar to those of 9 (Tables 3 and 4),
but a singlet of OCOCH3-16 at ca. δH 1.96 was absent, and
double triplets at δH 5.49 (assigned for H-16 in 9) were
replaced by shielded double triplets of H-16 at δH 4.65, thus
indicating the aglycone of 10 to be 16-desacetyloleandrigenin
E
DOI: 10.1021/acs.jnatprod.7b00554
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Table 4. 13C NMR Spectroscopic Data (100 MHz) of 2, 4, 9, 10, and 11 (in MeOH-d4)
2
4
9
10
11
position
δC, type
δC, type
δC, type
δC, type
δC, type
1
29.9, CH2
31.3, CH2
31.5, CH2
31.5, CH2
27.9, CH2
75.4, CH
31.5, CH2
38. 0, CH
27.6, CH2
22.5, CH2
42.9, CH
36.8, CH
36.3, C
31.5, CH2
2
26.2, CH2
73.2, CH
30.1, CH2
36.7, CH
26.4, CH2
20.8, CH2
41.3, CH
35.3, CH
34.9, C
27.5, CH2
75.0, CH
31.5, CH2
38.1, CH
27.8, CH2
22.2, CH2
42.7, CH
36.7, CH
36.4, C
27.8, CH2
74.9, CH
31.4, CH2
37.6, CH
27.5, CH2
22.2, CH2
42.7, CH
36.7, CH
36.3, C
27.8, CH2
74.8, CH
31.5, CH2
38.0, CH
27.6, CH2
22.2, CH2
42.7, CH
36.7, CH
36.3, C
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
1′
2′
3′
4′
5′
6′
20.6, CH2
38.6, CH2
50.0, C
22.0, CH2
40.0, CH2
51.4, C
22.0, CH2
40.0, CH2
51.4, C
22.1, CH2
41.0, CH2
51.3, C
22.0, CH2
40.0, CH2
51.4, C
83.6, C
85.0, C
85.0, C
85.7, C
85.0, C
40.0 CH2
74.6 CH
41.4, CH2
76.0, CH
57.5, CH
16.4, CH3
24.4, CH3
171.6, C
41.3, CH2
76.0, CH
57.4, CH
16.4, CH3
24.3, CH3
171.6, C
43.8, CH2
73.2, CH
59.7, CH
17.1, CH3
24.4, CH3
173.7, C
41.4, CH2
76.0, CH
57.4, CH
16.4, CH3
24.3, CH3
171.6, C
56.0, CH
15.0, CH3
23.0, CH3
170.9, C
76.7, CH2
120.3, CH
175.4, C
77.6, CH2
121.7, CH
176.8, C
77.5, CH2
121.7, CH
176.7, C
77.8, CH2
120.6, CH
178.0 C
77.5, CH2
121.7, CH
176.8, C
96.0, CH
69.1, CH
77.4, CH
69.7, CH
65.2, CH
16.4, CH3
170.2,a C; 19.6, CH3
170.2,a C; 19.5, CH3
56.4, CH3
97.2, CH
71.0, CH
76.8, CH
77.2 CH
100.2, CH
69.3, CH
79.0, CH
76.7, CH
67.4, CH
17.6, CH3
172.1, C; 20.9, CH3
100.3,CH
69.3, CH
79.1, CH
76.9, CH
67.5, CH
17.7, CH3
100.3, CH
69.2, CH
78.9, CH
76.7, CH
67.2, CH
17.8, CH3
67.7, CH
17.4, CH3
172.2, C; 24.4,b CH3
171.7, C; 24.4,b CH3
57.8, CH3
103.0, CH
75.3, CH
77.8, CH
71.6, CH
78.0, CH
62.8, CH2
OCOCH3-16
172.2, C; 20.9, CH3
OCOCH3-2′
OCH3-3′
1″
2″
3″
4″
5″
6″
1‴
2‴
3‴
4‴
5‴
56.9, CH3
105.0, CH
75.8, CH
77.8, CH
71.7, CH
77.9, CH
62.9, CH2
57.9, CH3
102.6, CH
74.9, CH
71.8, CH
71.7, CH
78.1, CH
62.9, CH2
57.8, CH3
102.4, CH
75.2, CH
77.9, CH
71.7, CH
77.3, CH
62.7, CH2
105.0 CH
75.3, CH
77.7, CH
71.6, CH
78.0, CH
70.2, CH2
6‴
a,b
Overlapped signals.
3 and 4). The 1H−1H COSY cross-peaks helped identify the 1H
NMR resonances of a vallaropyranosyl unit at δH 4.66 (d, J =
2.9 Hz, H-1′), 3.93 (dd, J = 5.2 and 2.9 Hz, H-2′), 3.62 (dd, J =
5.2 and 3.3 Hz, H-3′), 3.81 (dd, J = 7.0 and 3.3 Hz, H-4′), 4.20
(dq, J = 7.0 and 6.6 Hz, H-5′), and 1.24 (d, J = 6.6 Hz, H-6′).
The presence of two glucopyranosyl moieties was shown from
the resonances of two anomeric protons and two oxymethylene
groups. The HMBC cross-peaks between H-1′/C-3, H-1″/C-
4′, and H-1‴/C-6′′ indicated connectivities of C-1′-O to C-3,
C-1″-O to C-4′, and C-1‴-O to C-6″. On the basis of the
1H−1H COSY, HSQC, HMBC, and 1D-TOCSY spectra and
the acid hydrolyses of 2 and 3, which gave L-vallarose, and L-
acrofriose and D-glucose, respectively, the structure of
compound 11 was therefore elucidated as oleandrigenin-3-O-
β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl-(1→4)-α-L-val-
laropyranoside.
Compound 12 exhibited the same molecular formula as 11
based on the HRESIMS. The 1H and 13C NMR spectra
1
resembled those of 11 (Tables 1−4). However, the H,1H-
COSY spectrum showed H-5′ resonating at δH 3.76 (as quint, J
= 5.6 Hz), at higher field than that in 11, and occurred together
with resonances at δH 4.83 (d, J = 1.8 Hz, H-1′), 3.99 (dd, J =
3.0 and 1.8 Hz, H-2′), 3.65 (dd, J = 8.9 and 3.0 Hz, H-3′), 3.75
(H-4′), and 1.28 (d, J = 5.6 Hz, H-6′), similar to those of an
1
acofriopyranosyl moiety as displayed in 8. The H and 13C
NMR resonances of two glucosyl units were also encountered
F
DOI: 10.1021/acs.jnatprod.7b00554
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
1
Table 5. H (400 MHz) and 13C (100 MHz) NMR Spectroscopic Data of 5−7 (in MeOH-d4)
5
6
7
position
δH (J in Hz)
δC, type
31.7, CH2
δH (J in Hz)
δC, type
δH (J in Hz)
δC, type
1
1.59, 1.45
1.95, 1.29
1.57
31.7, CH2 1.50, 1.55
27.7, CH2 1.93, 1.27
73.9, CH 3.94e
30.9, CH2 1.87, 1.80
37.6, CH 1.77
27.5, CH2 1.60
22.2, CH2 1.83, 1.28
42.1, CH 1.74
38.1, CH 1.67
36.3, C
31.6, CH2
27.7, CH2
74.9, CH
31.5, CH2
38.0, CH
27.6, CH2
22.2, CH2
42.2, CH
37.6, CH
36.3, C
2
27.6, CH2
74.6, CH
30.9, CH2
37.6, CH
27.4, CH2
22.2, CH2
42.1, CH
38.2, CH
36.3, C
1.94
3.97c
3
3.95 brs (w1/2 = 9)
4
1.90, 1.49
1.68
1.89, 1.50
1.65
5
6
1.60
1.59
7
1.82, 1.29
1.75
1.80, 1.29
1.75
8
9
1.67
1.66
10
11
12
13
14
15
1.45, 1.18
2.05, 1.18
21.1, CH2
39.4, CH2
53.4, C
1.84, 1.12
21.0, CH2 1.46, 1.14
21.1, CH2
39.5, CH2
53.4, C
2.06 dt (9.4, 2.1), 1.17 t (9.4) 39.4, CH2 2.05, 1.17
53.4, C
86.7, C
86.7, C
86.8, C
2.77 brd (17.8), 2.31 dd (17.8, 41.1, CH2
3.2)
2.80 d (17.5), 2.34 dd (17.5,
2.9)
41.1, CH2 2.77 brd (18.2), 2.31 dd (18.2, 41.1, CH2
3.4)
16
17
18
19
20
21
6.25 brs
134.9, CH
145.1, C
6.28 t (2.9)
134.9, CH 6.25 brs
145.0, C
134.9, CH
145.1, C
1.27 s
0.99 s
17.0, CH3
24.6, CH3
162.0, C
1.29 s
1.01 s
17.0, CH3 1.27 s
24.6, CH3 0.98 s
162.0, C
16.9, CH3
24.6, CH3
162.0, C
5.12 dd (16.8, 1.5), 5.02 dd
(16.8, 1.5)
73.6, CH2
5.17 dd (16.7, 1.6), 5.03 dd
(16.7, 1.6)
73.5, CH2 5.14 dd (16.7, 1.4), 5.00 dd
(16.7, 1.4)
73.6, CH2
22
23
1′
2′
3′
4′
5′
6′
5.99 brs
112.0 CH
177.4, C
97.3, CH
68.8, CH
81.0, CH
79.3, CH
70.1, CH
18.2, CH3
6.01 s
111.9, CH 5.99 brs
111.9, CH
177.4, C
177.3, C
4.80 d (1.6)
4.83 d (1.8)
99.8, CH 4.65 d (3.1)
68.5, CH 3.92 dd (6.9, 3.1)e
82.6, CH 3.63 dd (6.9, 3.1)
79.3, CH 3.95 dd (6.9, 3.1)
68.8, CH 4.21 dq (6.9, 6.6)
18.2, CH3 1.23 d (6.6)
100.2, CH
69.3, CH
79.0, CH
76.8, CH
67.4, CH
17.6, CH3
5.19 dd (3.3, 1.6)
3.73 dd (9.4, 3.3)
3.61 t (9.4)
4.00 dd (3.1, 1.8)c
3.62 dd (9.2, 3.1)
3.73 t (9.2)d
3.76 dq (9.4, 6.3)
1.29 d (6.3)
3.74 dq (9.2, 5.7)d
1.31 d (5.7)
OCOCH3-2′ 2.09 s
172.1, C; 20.9,
CH3
OCH3-3′
3.39 s
57.7, CH3
105.0, CH
75.6, CH
77.9, CHb
71.8, CH
77.9, CHb
62.9, CH2
3.46 s
56.9, CH3 3.48 s
57.8, CH3
102.6, CH
75.3, CH
77.8, CH
71.6, CH
78.1, CH
62.8, CH2
1″
2″
3″
4″
5″
6″
4.57 d (8.0)
3.15 dd (8.8, 8.0)
3.39 dd (9.4, 8.8)
3.31 dd (9.4, 8.2)a
3.27a
4.62 d (7.8)
105.0, CH 4.40 d (7.7)
75.8, CH 3.21 dd (9.1, 7.7)
77.9, CH 3.36 t (9.1)
71.7, CH 3.29 t (9.1)f
77.9, CH 3.27f
3.19 dd (9.1, 7.8)
3.39 dd (9.1, 8.8)
3.31 t (8.8)
3.25 ddd (8.8, 5.4, 2.4)
3.86 dd (11.8, 2.1), 3.73 dd
(11.8, 5.2)
3.87 dd (11.8, 2.4), 3.69 dd
(11.8, 5.4)
62.9, CH2 3.85 dd (11.9, 1.8), 3.66 dd
(11.9, 5.3)
a−f
Overlapped signals.
(Tables 1 and 2). The HMBC cross-peaks between H-1′/C-3,
H-1″/C-4′, and H-1‴/C-6′′ indicated connectivities between
C-1′-O and C-3, C-1″-O and C-4′, and C-1‴-O and C-6″. On
the basis of acid hydrolysis of 3, which provided L-acofriose, and
D-glucose, the structure of compound 12 could be deduced as
oleandrigenin-3-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyra-
nosyl-(1→4)-α-L-acofriopyranoside.
3-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl-(1→4)-α-
L-2′-O-acetylacofriopyranoside.
Compounds 1−13 were evaluated for their cytotoxicity
against HeLaS3 [human cervix adenocarcinoma (ATCC; CCL-
2.2)], A549 [human lung carcinoma (ATCC: CCL-185)], HT-
29 [human colorectal adenocarcinoma (ATCC; HTB-38)], and
Vero (normal African green monkey kidney, ATCC; CCL-81)
cells (Table 6). Among the test compounds, 1 (the
monoglycoside of oleandrigenin with a 2′-O-acetylacofriosyl
moiety) was the most potent against HeLaS3 and HT-29 cells,
Compound 13 was obtained as a colorless solid with a
molecular formula of C46H70O21, based on HRESIMS, of
molecular mass 42 amu higher than that of 12. The 1H and 13
C
NMR spectra exhibited 1H and 13C NMR resonances similar to
those of 12, but the resonances due to an acofriopyranosyl
moiety were replaced by those of a 2′-O-acetylacofriopyranosyl
unit (Tables 1 and 2). After further investigation of the 1H,1H-
COSY, HSQC, HMBC, and 1D-TOCSY data, and on the basis
of acid hydrolysis of 3 providing L-acofriose and D-glucose, the
structure of compound 13 could be assigned as oleandrigenin-
showing IC50 values of 0.04
0.00 and 0.07
0.01 μM,
respectively, and 2 (the monoglycoside of oleandrigenin with a
2′-O-acetylvallarosyl moiety) was the most potent against A549
cells, exhibiting an IC50 value of 0.03
comparing the diglycosides of oleandrigenin (3 and 4), 4 (with
a 2′-O-acetylvallarosyl moiety) was more active than 3 (with a
2′-O-acetylacofriosyl moiety) against all cell lines. Among the
0.02 μM. When
G
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Journal of Natural Products
Article
0:100)) to give four subfractions (5.1−5.4). Also obtained was
subfraction 5.2 (360 mg) after CC (silica gel, hexanes−EtOAc, 97:3)
and passage over Sephadex LH-20 (CH2Cl2−MeOH, 5:95) to afford
lupeol (70 mg). Subfraction 5.3 (1.93 g) was further purified by CC
(silica gel, hexanes−EtOAc, 96:4, then Sephadex LH-20, MeOH) to
give 3β,27-dihydroxyurs-12-en-28-oic acid (13 mg). Fraction 6 (3.98
g) was fractionated by CC (silica gel, hexanes−EtOAc, 92:8 to 80:20)
to give seven subfractions (6.1−6.7), and subfraction 6.2 (380 mg)
after CC (silica gel, hexanes−EtOAc, 95:5) furnished stigmast-4-en-3-
one (4 mg) and an additional quantity of lupeol (69 mg). Subfraction
6.3 (421 mg) was purified (CC, RP-18, MeOH) to give five
subfractions (6.3.1−6.3.5), and subfraction 6.3.4 after CC (RP-18,
MeOH, then silica gel, hexanes−CH2Cl2, 35:65 to 25:75) gave
cycloeucalenol (161 mg). Subfraction 6.4 (349 mg) was purified by
CC (silica gel, hexanes−EtOAc, 97:3 to 96:4) to give three
subfractions (6.4.1−6.4.3), and subfraction 6.4.2 (168 mg) after
further CC (silica gel, hexanes−CH2Cl2, 45:55) gave β-sitosterol (367
mg). Fraction 7 (3.94 g) after CC (silica gel, CH2Cl2−MeOH, 99.6:0.4
to 97:3) gave seven subfractions (7.1−7.7), and subfraction 7.2 (1.67
g) was further purified (CC, silica gel, hexanes−EtOAc, 92:8 to 80:20,
then hexanes−CH2Cl2, 20:80) to give uvaol (urs-12-ene-3β,28-diol)
(19 mg). Subfraction 7.5 (80 mg) after CC (silica gel, CH2Cl2−
MeOH, 99.2:0.8) gave oleanolic acid (31 mg) and ursolic acid (20
mg). Fraction 8 (4.2 g) was fractionated (CC, CH2Cl2−MeOH,
99.6:0.4 to 97:3) to give 10 subfractions (8.1−8.10). Subfraction 8.5
(143 mg) was purified by reversed-phase CC (RP-18, MeOH−H2O,
65:35−100:0) to give five subfractions (8.5.1−8.5.5), and subfraction
8.5.2 gave 2 (37 mg), while subfraction 8.5.4 (37 mg) after CC
(Sephadex LH-20, MeOH) gave an additional quantity of 3β,27-
dihydroxyurs-12-en-28-oic acid (7 mg). Subfraction 8.6 (131 mg) after
CC (RP-18, MeOH−H2O, 70:30) gave three subfractions (8.6.1−
8.6.3), and the polar fraction of those (41 mg) after CC (Sephadex
LH-20, MeOH) furnished 2 (9 mg). Subfraction 8.7 (210 mg) after
CC (RP-18, MeOH−H2O, 60:40 to 100:0) gave four subfractions
(8.7.1−8.7.4), and subfraction 8.7.2 (86 mg) was further purified by
CC (Sephadex LH-20, MeOH) to give additional quantities of 2 (3
mg) and 1 (16 mg). Fraction 11 (10.3 g) was fractionated by CC
(silica gel, CH2Cl2−MeOH, 98.5:1.5−86:14) to give six subfractions
(11.1−11.6), and subfraction 11.2 gave an additional quantity of
ursolic acid (700 mg). Subfraction 11.6 (922 mg) after CC (RP-18,
MeOH−H2O, 55:45 to 100:0) gave β-sitosteryl glucoside (64 mg).
Fraction 12 (1.70 g) was fractionated by CC (silica gel, CH2Cl2−
MeOH, 99:1−93:7) to give three subfractions (12.1−12.3), and
subfraction 12.3 (700 mg) was further purified by CC (Sephadex LH-
20, MeOH) to give four subfractions, (12.3.1−12.3.4). Subfraction
12.3.3 (176 mg), after reversed-phase CC (RP-18, MeOH−H2O,
60:40 to 100:0), gave four additional subfractions (12.3.3.1−12.3.3.4).
Compounds 4 (9 mg) and 3 (19 mg) were obtained after subfraction
12.3.3.2 (70 mg) was purified by reversed-phase CC (RP-18, MeOH−
H2O, 50:50 to 55:45). Fraction 13 (13.4 g) was fractionated by CC
(silica gel, CH2Cl2−MeOH, 96:4 to 50:50) to give three subfractions
(13.1−13.3), and subfraction 13.2 (1.34 g) after further purification by
reversed-phase CC (RP-18, MeOH−H2O, 80:20−100:0) gave four
subfractions (13.2.1−13.2.4). Subfraction 13.2.3 (424 mg) after CC
(CH2Cl2−MeOH, 96:4 to 92:8) gave four subfractions (13.2.3.1−
13.2.3.4), and subfraction 13.2.3.2 (34 mg) after further CC (Sephadex
LH-20, MeOH) furnished an additional quantity of 3 (13 mg).
Subfraction 13.2.3.3 (156 mg) after purification by CC (silica gel,
CH2Cl2−MeOH, 90:10, then reversed-phase RP-18 CC (MeOH−
H2O, 70:30−100:0), gave 5 (7 mg).
Table 6. Cell Growth Inhibitory Activities of Crude Extracts
and Cardenolides 1−13 against Cancer and Noncancerous
(Vero) Cellsa
HeLaS3
(IC50)
HT-29
(IC50)
compound
A 549 (IC50)
Vero (IC50)
Crude Extract (μg/mL)
VG-L-CH2Cl2
1.2 0.1
1.6 0.1
>20
1.9 0.1
>20
5.1 1.3
>20
VG-L-MeOH
>20
Compound (μM)
1
0.04 0.00
0.1 0.0
1.4 0.1
0.4 0.0
>10
0.5 0.1
0.03 0.01
1.5 0.2
0.6 0.1
>10
0.1 0.0
0.1 0.0
1.9 0.2
0.6 0.0
>10
0.2 0.0
0.3 0.0
5.8 0.4
2.2 0.2
>10
2
3
4
5
6
0.7 0.1
0.7 0.1
0.2 0.0
0.1 0.0
6.8 0.9
0.5 0.1
0.4 0.0
1.6 0.1
<0.1
0.3 0.1
1.6 0.1
0.1 0.0
0.1 0.0
3.8 0.3
1.2 0.1
0.6 0.1
2.1 0.2
5.0 1.7
>10
0.8 0.1
1.9 0.1
0.2 0.0
0.2 0.0
6.4 0.9
1.6 0.1
1.4 0.0
5.0 1.0
0.4 0.0
7.6 0.7
2.5 0.2
6.2 0.4
0.7 0.1
0.6 0.1
>10
7
8
9
10
11
6.9 1.0
4.8 1.4
>10
12
13
doxorubicin
etoposide
6.6 0.2
>10
0.5 0.1
a
Data are represented as mean SD (n = 6); IC50 = 50% inhibition
concentration.
diglycosides possessing similar glycosyl units (with an L-
vallarosyl moiety) of oleandrigenin (9), 16-anhydrogitoxigenin
(7), and gitoxigenin (10), the order of cytotoxic activity was
found to be 9 > 7 > 10, and furthermore among the
diglycosides possessing similar glycosyl units (with an L-
acofriosyl moiety) of oleandrigenin (8) and 16-anhydrogitox-
igenin (6), 8 was more potent. Based on these results, it could
be concluded that the aglycone oleandrigenin moiety was the
most crucial for higher cytotoxic potency and the gitoxigenin
moiety less effective.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Melting points were
measured using an Electrothermal melting point apparatus and are
uncorrected. Optical rotations were recorded on a JASCO DIP 1020
polarimeter. The IR spectra were obtained on a PerkinElmer 1760x
FT-IR spectrophotometer. The 1H and 13C NMR spectra were
recorded with Bruker AVANCE 400 MHz and Bruker AVANCE III
HD 400 MHz NMR spectrometers. Chemical shifts are referenced to
the residual solvent signals (MeOH-d4: δH 3.30 and δC 49.0 ppm).
HRESIMS were recorded on a Bruker Daltonics microTOF mass
spectrometer.
Plant Material. The plant Vallaris glabra was collected from a
garden in Lat Phrao District, Bangkok, in March 2012. The plant was
identified by Assoc. Prof. Dr. Nijsiri Ruangrungsi, Department of
Pharmacognosy, Faculty of Pharmaceutical Science, Chulalongkorn
University, Bangkok, Thailand. A voucher specimen (SSVG-1/2012) is
maintained at the Department of Chemistry, Ramkhamhaeng
University.
Extraction and Isolation. Fresh V. glabra leaves (6.2 kg) were
ground and soaked in MeOH (36 L) at room temperature for 3 weeks.
The filtrate was concentrated and partitioned successively with
hexanes and CH2Cl2 (8 L) to obtain hexanes (dark green solid, 82.6
g), CH2Cl2 (dark green solid, 124.8 g), and MeOH (reddish-brown,
212.4 g) extracts. The CH2Cl2 extract was fractionated by column
chromatography (CC, silica gel, hexanes−CH2Cl2, 80:20 to CH2Cl2−
MeOH, 40:60) to obtain 14 fractions. The moderately polar fraction 5
(2.7 g) was purified by CC (silica gel, hexanes−CH2Cl2 (40:60 to
The crude MeOH extract (100 g) was fractionated by CC (Diaion
HP20, H2O−MeOH, 100:0 to 0:100) to obtain six fractions (1−6).
An aliquot of the less polar fraction 5 (1.4 g) was further fractionated
(CC, Sephadex LH-20, MeOH) to obtain six subfractions (5.1−5.6).
Subfraction 5.2 (135 mg) was purified by reversed-phase CC (H2O−
MeOH, 55:45 to 0:100) to give 12 (7 mg) and 13 (7 mg). Subfraction
5.3 (220 mg) after CC (RP-18, H2O−MeOH, 65:35 to 0:100) gave
seven subfractions (5.3.1−5.3.7), and further purification of sub-
fraction 5.3.3 (13 mg) by reversed-phase CC (RP-18, H2O−MeOH,
45:65 to 0:100) gave 11 (7 mg). Subfraction 5.3.6 (59 mg) was
H
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Article
fractionated by CC (silica gel, EtOAc−MeOH, 93:7) to give four
subfractions (5.3.6.1−5.3.6.4), and subfraction 5.3.6.3 gave an
additional quantity of 13 (17 mg). Subfraction 5.4 (336 mg) was
purified by reversed-phase CC (RP-18, H2O−MeOH, 75:5 to 0:100)
to give seven subfractions (5.4.1−5.4.7). Subfraction 5.4.4 gave 10 (5
mg), and subfraction 5.4.5 after further purification by CC (Sephadex
LH-20, MeOH) gave five subfractions (5.4.5.1−5.4.5.5). Subfraction
5.4.5.2 gave 9 (8 mg), and subfraction 5.4.5.3 (82 mg) after further
purification by CC (RP-18, H2O−MeOH, 75:5 to 0:100, then silica
gel, CH2Cl2−MeOH, 92:8) gave 7 (5 mg). Subfraction 5.4.6 (17 mg)
after CC (silica gel, CH2Cl2−MeOH, 92:8) gave 6 (9 mg) and 8 (5
mg).
Oleandrigenin-3-O-α-L-2′-O-acetylvallaropyranoside (2′-O-ace-
tylvallarosolanoside, 2): colorless solid, mp 172−173 °C (MeOH/
EtOAc); [α]2D5 −55.6 (c 0.30, MeOH); FT-IR (ATR) νmax 3451, 2932,
1732, 1650, 1371, 1234, 1096, 1034, 1012 cm−1; 1H NMR (MeOH-d4,
400 MHz) and 13C NMR ((MeOH-d4, 100 MHz) data see Tables 3
and 4; HRESIMS m/z 657.3263 [M + Na]+ (calcd for C34H50NaO11,
657.3353).
NMR (MeOH-d4, 100 MHz) data see Tables 3 and 4; HRESIMS m/z
735.3564 [M + Na]+ (calcd for C36H56NaO14, 735.3552).
Oleandrigenin-3-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyrano-
syl-(1→4)-α-L-vallaropyranoside (11): [α]3D2 −45.8 (c 0.33, MeOH);
FT-IR (ATR) νmax 3350, 2924, 2878, 2855, 1733, 1628, 1449, 1378,
1257, 1165, 1102, 1068, 1033, 1014, 905, 801 cm−1; 1H NMR
(MeOH-d4, 400 MHz), 13C NMR (MeOH-d4, 100 MHz) data see
Tables 3 and 4; HRESIMS m/z 939.4209 [M + Na]+ (calcd for
C44H68NaO20, 939.4182).
Oleandrigenin-3-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyrano-
syl-(1→4)-α-L-acofriopyranoside (12): [α]3D2 −40.6 (c 0.35, MeOH);
FT-IR (ATR) νmax 3369, 2932, 2876, 1733, 1618, 1447, 1378, 1248,
1162, 1101, 1066, 1033, 1018, 986, 907, 889 cm−1; 1H NMR (MeOH-
d4, 400 MHz), 13C NMR (MeOH-d4, 100 MHz) data see Tables 1 and
2; HRESIMS m/z 939.4208 [M + Na]+ (calcd for C44H68NaO20,
939.4182).
Oleandrigenin-3-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyrano-
syl-(1→4)-α-L-2′-O-acetylacofriopyranoside (13): colorless solid, mp
182−184 °C (MeOH/EtOAc); [α]3D2 −37.9 (c 0.33, MeOH); FT-IR
(ATR) νmax 3381, 2931, 2894, 2868, 1735, 1728, 1609, 1445, 1370,
1235, 1168, 1130, 1073, 1051, 1022, 985, 912 cm−1; 1H NMR
(MeOH-d4, 400 MHz), 13C NMR (MeOH-d4, 100 MHz) data see
Tables 1 and 2; HRESIMS m/z 981.4306 [M + Na]+ (calcd for
C46H70NaO21, 981.4287).
Oleandrigenin-3-O-β-D-glucopyranosyl-(1→4)-α-L-2′-O-acetyla-
cofriopyranoside (3): colorless solid, mp 179−181 °C (MeOH/
EtOAc); [α]2D5 −24.1 (c 0.35, MeOH); FT-IR (ATR) νmax 3369, 2929,
1731, 1620, 1371, 1234, 1071, 1053, 1033, 1025 cm−1; 1H NMR
(MeOH-d4, 400 MHz), 13C NMR (MeOH-d4, 100 MHz) data see
Tables 1 and 2; HRESIMS m/z 819.3796 [M + Na]+ (calcd for
C40H60NaO16, 819.3773).
Acid Hydrolysis of 2. A mixture of compound 2 (4.2 mg) and 5%
HCl (5 mL) was heated at 70 °C for 5 h. The cold reaction mixture
was extracted with EtOAc (3 × 10 mL). The combined EtOAc extract,
after washing with water, was concentrated to give the agylcone, 14,16-
dianhydrogitoxigenin, Rf 0.5 (CH2Cl2−MeOH, 99:1). The aqueous
extract, after removal of water, gave L-vallarose (0.7 mg), Rf 0.7 (silica
gel TLC, thickness 0.2 mm, CH2Cl2−MeOH, 92:8). The optical
rotation value was measured after 24 h of dissolution in H2O: L-
vallarose [α]2D6 −38.9 (c 0.04, H2O) [lit.12 [α]D23 −17.2 (c 0.90, H2O)].
Acid Hydrolysis of 3. A mixture of compound 3 (4.2 mg) and 5%
HCl (5 mL) was heated at 70 °C for 5 h, then left to cool to room
temperature. The reaction mixture was then partitioned with EtOAc to
give an EtOAc extract, from which the aglycone, 14,16-dianhydrogi-
toxigenin (1.0 mg), Rf 0.72 (CH2Cl2−MeOH, 98:2), was obtained
after concentration. The aqueous phase was neutralized with saturated
NaHCO3 and concentrated to give an aqueous extract (3.1 mg).
Reversed-phase column chromatography (RP-18, MeOH−H2O,
15:85) gave D-glucose (1.0 mg, Rf 0.1 (CH2Cl2−MeOH, 90:10) and
L-acofriose, 0.5 mg) Rf 0.8 [silica gel TLC, thickness 0.2 mm, CH2Cl2−
MeOH (86:14)]. The optical rotation values were measured after 24 h
of dissolution in H2O: D-glucose [α]2D9 +40.2 (c 0.1, H2O) [lit.13 +53.2
(c 0.1, H2O)] and L-acofriose, [α]2D9 +30.0 (c 0.05) [lit.12 [α]2D1 +37.3 (c
0.95, H2O)].
Oleandrigenin-3-O-β-D-glucopyranosyl-(1→4)-α-L-2′-O-acetyl-
vallaropyranoside (4): colorless solid, mp 183−184 °C (MeOH/
EtOAc); [α]2D5 −15.2 (c 0.37, MeOH); FT-IR (ATR) νmax 3396, 2929,
1
1732, 1630, 1238, 1033, 1026, 1015 cm−1; H NMR (MeOH-d4, 400
MHz), 13C NMR (MeOH-d4, 100 MHz) data see Tables 3 and 4;
HRESIMS m/z 819.3793 [M + Na]+ (calcd for C40H60NaO16,
819.3773).
16-Anhydrogitoxigenin-3-O-β-D-glucopyranosyl-(1→4)-α-L-2′-O-
acetylacofriopyranoside (5): [α]2D5 −9.6 (c 0.15, MeOH); FT-IR
(ATR) νmax 3389, 2927, 2881, 2852, 1728, 1619, 1449, 1374, 1235,
1
1161, 1105, 1051, 1029, 981, 902, 888 cm−1; H NMR (MeOH-d4,
400 MHz), 13C NMR (MeOH-d4, 100 MHz) data see Table 5;
HRESIMS m/z 759.3580 [M + Na]+ (calcd for C38H56O14Na
759.3562).
16-Anhydrogitoxigenin-3-O-β-D-glucopyranosyl-(1→4)-α-L-aco-
friopyranoside (6): colorless solid, mp 198−200 °C (MeOH/EtOAc);
[α]3D1 −18.6 (c 0.45, MeOH); FT-IR (ATR) νmax 3528, 3371, 2930,
2880,1724, 1695, 1623, 1449, 1379, 1260, 1108, 1072, 1047, 1030,
1
982, 887 cm−1; H NMR (MeOH-d4, 400 MHz), 13C NMR (MeOH-
d4, 100 MHz) data see Table 5; HRESIMS m/z 717.3462 [M + Na]+
(calcd for C36H54NaO13, 717.3447).
16-Anhydrogitoxigenin-3-O-β-D-glucopyranosyl-(1→4)-α-L-val-
laropyranoside (7): colorless solid, mp 204−206 °C (MeOH/
EtOAc); [α]3D1 −18.3 (c 0.25, MeOH); FT-IR (ATR) νmax 3476,
3383, 2933, 2885, 2851, 1727, 1621, 1447, 1378, 1255, 1163, 1076,
Bioassay. The antiproliferative effects of the isolated compounds
on HeLaS3 [human cervix adenocarcinoma (ATCC; CCL-2.2)], A549
[human lung carcinoma (ATCC; CCL 185)], HT-29 [human
colorectal adenocarcinoma (ATCC HTB-38)], and nontumorigenic
(Vero, normal African green monkey kidney, ATCC; CCL-81) cells
using a microculture tetrazolium (MTT) assay were determined
according to a previously reported protocol.15
1030, 1015, 984, 891, 829 cm−1; 1H NMR (MeOH-d4, 400 MHz), 13
C
NMR (MeOH-d4, 100 MHz) data see Table 5; HRESIMS m/z
717.3455 [M + Na]+ (calcd for C36H54NaO13, 717.3447).
Oleandrigenin-3-O-β-D-glucopyranosyl-(1→4)-α-L-O-acofriopyr-
anoside (8): [α]3D1 −49.1 (c 0.26, MeOH); FT-IR (ATR) νmax 3376,
2928, 2874, 1732, 1631, 1447, 1377, 1246, 1163, 1107, 1070, 1031,
1
983, 892 cm−1; H NMR (MeOH-d4, 400 MHz), 13C NMR (MeOH-
d4, 100 MHz) data see Tables 1 and 2; HRESIMS m/z 777. 3667 [M +
ASSOCIATED CONTENT
■
Na]+ (calcd for C38H58NaO15, 777.3657.
S
* Supporting Information
Oleandrigenin-3-O-β-D-glucopyranosyl-(1→4)-α-L-vallaropyra-
noside (9): [α]3D1 −39.5 (c 0.40, MeOH); FT-IR (ATR) νmax 3377,
2926, 2873, 2856, 1732, 1630, 1447, 1375, 1246, 1162, 1071, 1032,
1013, 896 cm−1; 1H NMR (MeOH-d4, 400 MHz), 13C NMR (MeOH-
d4, 100 MHz) data see Tables 3 and 4; HRESIMS m/z 777. 3661 [M +
Na]+ (calcd for C38H58NaO15, 777.3657).
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.7b00554.
1H and 13C NMR spectra of compounds 1−13, 2D NMR
spectra of 2, and 1D TOCSY spectra of 5, 7, 11, and 12
(PDF)
Gitoxigenin-3-O-β-D-glucopyranosyl-(1→4)-α-L-vallaropyrano-
side (10): [α]3D1 −21.4 (c 0.38, MeOH); FT-IR (ATR) νmax 3351,
2925, 2854, 1759, 1733, 1627, 1597, 1451, 1376, 1261, 1160, 1072,
1
1028, 1008, 961, 889 cm−1; H NMR (MeOH-d4, 400 MHz), 13C
HMBC data of compounds 1−13 (PDF)
I
DOI: 10.1021/acs.jnatprod.7b00554
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
AUTHOR INFORMATION
Corresponding Author
*Tel: (662) 319-0931. Fax: (662) 319-1900. E-mail: somyote_
s@yahoo.com; s_somyote@ru.ac.th.
■
ORCID
Somyote Sutthivaiyakit: 0000-0002-3249-1380
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are grateful to Ramkhamhaeng University, the Thailand
Research Fund, and the Center of Excellence for Innovation in
Chemistry (PERCH−CIC), Commission on Higher Education,
Ministry of Education, for financial support. We acknowledge
Miss W. Hadtugvong, Miss P. Pongpinit, Miss S. Karoonbor-
irak, Miss N. Polteera, Miss N. Tepnuan, Miss J. Khambudda,
Miss T. Srithong, and the late Mr. W. Komkhunthot for
technical assistance.
REFERENCES
■
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