Megastigmane glycosides from leaves of Eucommia ulmoides Oliver with ACE inhibitory activity

Article abstract of DOI:10.1016/j.fitote.2016.12.001

Four new megastigmane glycosides, eucomegastigsides A-D (2, 3, 5 and 7), together with three known megastigmane glycosides, (6R, 7E, 9R)-9-hydroxy-4, 7-megastigmadien-3-one-9-O-[α-L-arabinopyranosyl-(l → 6)-β-D-glucopyranoside (1), foliasalacioside B₁ (4) and eleganoside A (6), were isolated from the leaves of Eucommia ulmoides Oliver. Their anti-hypertensive effect was investigated in vitro based on the inhibition of Angiotensin Converting Enzyme (ACE) using HPLC. The results showed that the isolates (2, 3, 4, 5, 7) had moderate inhibitory effects on ACE in vitro compared with captopril.

Full text of DOI:10.1016/j.fitote.2016.12.001

Fitoterapia 116 (2017) 121–125
Contents lists available at ScienceDirect
Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
Megastigmane glycosides from leaves of Eucommia ulmoides Oliver with
ACE inhibitory activity
Jian-Kun Yan a,c, Li-Qin Ding c, Xu-Liu Shi b,c, Paul Owusu Donkor b,c, Li-Xia Chen a, , Feng Qiu
⁎
a,b,c,⁎⁎
a
Department of Natural Products Chemistry, School of Traditional Chinese Materia Medica, Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, Shenyang Phar-
maceutical University, Shenyang 110016, PR China
b
School of Chinese Materia Medica, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, PR China
Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, PR China
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Four new megastigmane glycosides, eucomegastigsides A-D (2, 3, 5 and 7), together with three known
megastigmane glycosides, (6R, 7E, 9R)-9-hydroxy-4, 7-megastigmadien-3-one-9-O-[α-L-arabinopyranosyl-
(l → 6)-β-D-glucopyranoside (1), foliasalacioside B1 (4) and eleganoside A (6), were isolated from the leaves of
Eucommia ulmoides Oliver. Their anti-hypertensive effect was investigated in vitro based on the inhibition of An-
giotensin Converting Enzyme (ACE) using HPLC. The results showed that the isolates (2, 3, 4, 5, 7) had moderate
inhibitory effects on ACE in vitro compared with captopril.
Received 2 November 2016
Received in revised form 26 November 2016
Accepted 1 December 2016
Available online 3 December 2016
Keywords:
© 2016 Elsevier B.V. All rights reserved.
Leaves of Eucommia ulmoides Oliver
Megastigmane glycosides
Eucomegastigsides
Angiotensin converting enzyme (ACE)
1. Introduction
megastigmadien-3-one-9-O-[α-L-arabinopyranosyl-(l
→
6)-β-D-
glucopyranoside (1) [11], foliasalacioside B1 (5) [12] and eleganoside
A (6) [13] (Fig. 1), from the leaves of E. ulmoides. In addition, the in
vitro ACE inhibitory activities of these compounds were measured by
HPLC assay.
Eucommia ulmoides Oliver is one of the oldest known and used tonic
herbs in Asia. The medicinal parts of E. ulmoides are the bark and leaf as
mentioned in the Pharmacopoeia of the People's Republic of China
(2015) [1]. Leaves of E. ulmoides have been reported to possess anti-hy-
pertensive [2–5] property in vitro and in vivo. In particular, the glycosidic
ingredients play an important role in the anti-hypertensive effect. Previ-
ous studies have found many bioactive components in the leaves of E.
ulmoides, including iridoids [6], phenylpropanoids [7] and flavonoids
[8]. However, only few megastigmanes have been obtained from the
leaves of E. ulmoides [7,9].
Angiotensin Converting Enzyme (ACE) is the key enzyme that regu-
lates the renin-angiotensin system (RAAS) which is a hormone system
that is involved in the regulation of plasma sodium concentration and
arterial blood pressure [10]. Inhibitors of ACE are the first line of therapy
for hypertension. However, although all these drugs produce side ef-
fects such as digestive tract symptoms, dry cough or allergic phenome-
na, no relevant adverse effects were observed in the experimental group
who took the extract of the leaves of E. ulmoides [5].
2. Experimental
2.1. General
Optical rotations were recorded on a Rudolph (Hackettstown, NJ)
Autopol V automatic polarimeter, and CD spectra were measured on a
JASCO J-815 spectrophotometer using MeOH as solvent. UV spectra
were recorded on a UNICO UV-2102PCS spectrophotometer. IR spectra
were obtained on a Perkin Elmer (Boston, MA) Spectrum 65 FT-IR Spec-
trometer. NMR spectra were carried out on a Bruker (Billerica, MA) AM-
400/600 spectrometer at 25 °C. High-Resolution ESIMS (HRESIMS) was
performed on a Waters (Milford, MA) Xevo G2-S UPLC-Q/TOF. A Waters
e2695 equipped with a 2998 PDA detector and RP C18 reversed-phase
silica gel (4.6 × 250 mm, ODS-3 5 μm, Japan) was used for High Perfor-
mance Liquid Chromatography (HPLC). A Waters 2535 preparative High
Performance Liquid Chromatography (pHPLC) equipped with RP C18 re-
versed-phase silica gel (20 × 250 mm, ODS-3 5 μm, Japan) and semi-
preparative High Performance Liquid Chromatography (semi-pHPLC)
equipped with A chiral column (Daicel Chiralpak IE, 4.6 × 250 mm,
Japan) were used for separation. Jasco LC Net II/ADC with a Jasco OR-
4090 detector and Shodex Asahipak NH2P-50 4E column was used for
the analysis of the sugars. Silica gel (200–400 mesh, Qingdao Haiyang
In this study, we explored the isolation and structure elucidation of
four new megastigmane glycosides 2, 3, 5 and 7, together with three
known megastigmane glycosides, (6R, 7E, 9R)-9-hydroxy-4, 7-
⁎
*Corresponding author.
⁎⁎ Correspondence to: F. Qiu, School of Chinese Materia Medica, Tianjin University of
Traditional Chinese Medicine, Tianjin 300193, PR China.
E-mail addresses: syzyclx@163.com (L.-X. Chen), fengqiu20070118@163.com (F. Qiu).
http://dx.doi.org/10.1016/j.fitote.2016.12.001
0367-326X/© 2016 Elsevier B.V. All rights reserved.

122
J.-K. Yan et al. / Fitoterapia 116 (2017) 121–125
Fig. 1. The structures of compounds 1-7.
Chemical, China), C18 reversed-phase silica gel (12 nm, S-50 μm, YMC,
Wilmington, NC), Sephadex LH-20 gel (GE Healthcare (USA) Co. Ltd),
were used for column chromatography.
ACE from rabbit lung, hippuryl-histidyl-leucine (HHL), hippuric acid
(HA), captopril, orthoboric acid (H3BO3), were purchased from Sigma-
Aldrich (Bornem, Belgium; St. Louis, MO). NaOH, NaCl, trifluoroacetic
acid (FA) and other solvents used were of analytical grade (Concord
Technology (Tianjin) Co. Ltd.).
preparative column (MeOH-H2O, 38:62, 6 mL/min) to obtain three frac-
tions (B83211–B83213), with B83211 and B83213 as compounds 1
(360.0 mg) and 2 (120.0 mg), respectively. B83212 was further subject-
ed to HPLC (MeCN-H2O, 18:82, 6 mL/min) to give compound 3 (5.4 mg).
B8331 was separated by preparative HPLC (MeOH-H2O, 38:62,
6 mL/min) to give five fractions (B83311-B83315). B83315 was further
separated by preparative HPLC (MeCN-H2O, 18:82, 6 mL/min) to obtain
compounds 4 (120.0 mg) and 5 (41.0 mg). B834 was separated by
Sephadex LH-20 (MeOH) to give six fractions (B8341–B8346). B8341
was separated by preparative HPLC (MeOH-H2O, 35:65, 6 mL/min) to
give eight fractions (B83411–B83418). B83415 was compound 6
(20.0 mg). B83416 (23.0 mg) was further separated by preparative
HPLC (MeCN-H2O, 18:82, 6 mL/min) to obtain compound 7 (7.1 mg).
Compounds 2, 3, 5 and 7 were identified as new compounds. The
known compounds, by comparing with the published literature, were
identified as (6R, 7E, 9R)-9-hydroxy-4, 7-megastigmadien-3-one 9-O-
2.2. Plant materials
Leaves of Eucommia ulmoides Oliver were purchased from Anguo in
Hebei Province, P.R. China in August 2014, and were authenticated by
Prof. Lijuan Zhang of Tianjin University of Traditional Chinese Medicine.
A voucher sample was deposited at the School of Chinese Materia
Medica, Tianjin University of Traditional Chinese Medicine.
[α-L-arabinopyranosyl-(l
→
6)-β-D-glucopyranoside
(1),
2.3. Extraction and isolation
foliasalacioside B1 (4) and eleganoside A (6). The structures of com-
pounds 1–7 were shown in Fig. 1, and the 1H NMR and 13C NMR data
of 2, 3, 5 and 7 were listed in Table 1.
The air-dried leaves of Eucommia ulmoides Oliver (9.5 kg) were ex-
tracted with 80% EtOH/H2O (3 × 100 L) after 2-hour reflux to obtain
700 g of concentrated extract. The extract was suspended in water
(5 L) and partitioned successively with CHCl3 (4 × 1 L), EtOAc
(4 × 1 L), and n-BuOH (4 × 1 L) to give the corresponding extracts.
The n-BuOH extract (180 g) was subjected to silica gel column
(CH2Cl2-MeOH, 10:0 → 0:10) to yield eight fractions, B1–B8. Fraction
2.3.1. Eucomegastigsides A (2)
Pale yellow gum; [α]25D + 37 (c 0.19, MeOH); UV (MeOH) λmax (log
ε) 271 (0.122) nm, 236 (0.463) nm; ECD (c 3.18 × 10−3 M, MeOH) λmax
(Δε) 261 (+74.52), 317 (−12.08); IR (KBr) νmax 2938, 1647, 1384,
1259 and 1053 cm−1 1H and 13C NMR data, see Table 1; positive
;
B8 (24.0 g) was chromatographed over reversed-phase C18 (RP-C18
)
HRESIMS m/z 503.2486 [M + H]+ (calcd. for C24H38O11, 503.2492).
silica gel CC eluted with MeOH-H2O (0:10 → 10:0) to afford nineteen
subfractions (B811–B815, B821–B825, B831–B835, B841–B844). B832
and B833 were separated by Sephadex LH-20 (MeOH) to give seven
fractions (B8321–B8327) and nine fractions (B8331–B8339) respective-
ly. B8321 was separated by preparative HPLC equipped with a semi-
2.3.2. Eucomegastigsides B (3)
Pale yellow gum; [α]25D - 90 (c 0.04, MeOH); UV (MeOH) λmax (log
ε) 267 (0.116) nm, 234 (0.163) nm; ECD (c 1.47 × 10−2 M, MeOH) λmax
(Δε) 251 (−42.40), 311 (+3.56); IR (KBr) νmax 2981, 2966, 1655, 1384,

J.-K. Yan et al. / Fitoterapia 116 (2017) 121–125
123
Table 1
1H NMR and 13C NMR data for compounds 2 (MeOH-d4, 600/150 MHz) and 3, 5 and 7 (MeOH-d4, 400/100 MHz).
2
δC
3
δC
5
δC
7
δC
Position
δH (mult, J in Hz)
δH (mult, J in Hz)
δH (mult, J in Hz)
δH (mult, J in Hz)
1
2
37.2
48.5
202.1
126.3 5.90 (1H, s)
165.7
37.3
48.6
202.2
126.2 5.87 (1H, s)
166.2
37.5
48.2
202.4
125.5 5.74 (1H, s)
170.0
37.8
38.5
2.50 (1H, d, 16.8); 2.06 (1H, d,
16.7)
2.41 (1H, d, 16.8); 2.05 (1H, d,
16.6)
2.42 (1H, d, 17.4); 1.92 (1H, d,
17.4)
1.74 (2H, dd, 11.8, 5.1)
2.37 (2H, t, 6.8)
3
4
5
6
35.2
201.6
131.8
168.6
57.0
2.69 (1H, d, 9.4)
56.9
2.66 (1H, d, 9.2)
52.6
1.91 (1H, m)
1.66–1.54 (1H, m); 1.75–1.71
(1H, m)
1.66–1.54 (2H, m)
3.81–3.74 (1H, m)
1.18 (3H, d, 6.2)
1.03 (3H, s)
2.29–2.17 (1H, m); 2.54–2.43
(1H, m)
1.64–1.55 (2H, m)
3.75–3.71 (1H, m)
1.22 (3H, d, 6.1)
1.13 (3H, s)
7
131.1 5.77 (1H, dd, 15.3, 9.4)
137.1 5.60 (1H, dd, 15.3, 7.3)
129.1 5.73 (1H, dd, 15.3, 6.4)
138.3 5.62 (1H, dd, 15.3, 9.3)
26.8
27.7
8
9
37.6
77.9
22.2
27.6
29.1
25.1
36.6
77.9
22.1
27.3
27.3
11.9
74.9
22.4
27.6
28.1
24.0
4.45 (1H, m)
1.29 (3H, d, 6.4)
0.99 (3H, s)
1.03 (3H, s)
1.98 (3H, d, 1.1)
77.1
21.2
27.6
28.1
24.1
4.43–4.36 (1H, m)
1.28 (3H, d, 6.3)
0.96 (3H, s)
1.01 (3H, s)
1.95 (3H, s)
10
11
12
13
1′
2′
3′
4′
5′
0.95 (3H, s)
1.99 (3H, s)
1.13 (3H, s)
1.70 (3H, s)
101.5 4.29 (1H, d, 7.8)
102.8 4.33 (1H, d, 7.9)
104.2 4.26 (1H, d, 7.3)
104.4 4.30 (1H, d, 7.8)
75.1
78.2
71.7
77.0
3.23–3.18 (1H, m)
3.28 (1H, t, 8.9)
3.33 (1H, t, 3.4)
75.3
78.0
71.6
76.9
3.16 (1H, t, 8.0)
75.3
78.1
72.4
76.8
3.11–3.07 (1H, m)
3.28 (1H, m)
3.55–3.51 (1H, m)
3.36 (1H, br s)
4.01 (1H, d, 11.0), 3.66 (1H, dd,
11.0, 5.3)
75.4
78.3
71.7
76.9
3.15–3.09 (1H, m)
3.29–3.26 (1H, m)
3.29–3.26 (1H, m)
3.36 (1H, d, 5.8)
4.01 (1H, d, 9.6); 3.66 (1H, dd,
11.4, 5.5)
3.37–3.32 (1H, m)
3.37–3.32 (1H, m)
3.37–3.32 (1H, m)
4.02 (1H, d, 10.8); 3.68 (1H, d,
11.3)
3.33 (1H, t, 3.4)
4.07 (1H, dd, 11.4, 1.6); 3.72 (1H,
dd, 11.4, 5.3)
6′
69.6
69.6
69.6
69.6
1″
2″
3″
4″
105.3 4.32 (1H, d, 6.7)
105.3 4.25 (1H, d, 6.8)
105.2 4.26 (1H, d, 7.3)
105.2 4.26 (1H, d, 6.6)
74.3
72.4
69.5
3.52–3.51 (1H, m)
3.58 (1H, dd, 8.7, 6.8)
3.81 (1H, dd, 4.9, 3.2)
3.85 (1H, dd, 12.4, 3.3); 3.54 (1H,
m)
74.3
72.5
69.5
3.53–3.47 (1H, m)
3.59–3.54 (1H, m)
3.78 (1H, m)
71.7
74.2
69.5
3.28 (1H, m)
3.48–3.45 (1H, m)
3.76 (1H, s)
72.4
74.3
69.5
3.52 (1H, t, 7.6)
3.46 (1H, m)
3.75–3.71 (1H, m)
3.46 (1H, m); 3.87–3.75 (1H,
m)
3.84 (1H, dd, 12.3, 3.1);
3.53–3.47 (1H, m)
3.81–3.74 (1H, m); 3.48–3.45
(1H, m)
5″
66.8
66.8
66.7
66.7
1052 cm−1
;
1H and 13C NMR data, see Table 1; positive HRESIMS m/z
The experimental procedure is described in Table 2. Captopril was used
as a positive control. Triplicate determinations were carried out for each
compound. Samples (20 μL) were analyzed on a Waters e2695 equipped
with a 2998 PDA detector and RP C18 reversed-phase silica gel
(4.6 × 250 mm, ODS-3 5 μm, Japan), and HA was detected on 227 nm.
Acetonitrile and water (containing 0.01% TFA) (25:75, 1 mL/min)
were used as the mobile phases A and B, respectively. The inhibitory ac-
tivities of compounds were evaluated by determining ACE inhibition ra-
tios. ACE inhibition ratio was obtained using the equation:
503.2492 [M + H]+ (calcd. for C24H38O11, 503.2492).
2.3.3. Eucomegastigsides C (5)
Pale yellow gum; [α]25D + 6 (c 0.10, MeOH); UV (MeOH) λmax (log
ε) 272 (0.100) nm, 238 (0.626) nm; ECD (c 8.09 × 10−3 M, MeOH) λmax
(Δε) 213 (+5.85), 233 (−10.10), 331 (+4.88); IR (KBr) νmax 2931,
1651, 1415, 1384, 1258 cm−1; 1H and 13C NMR data, see Table 1; posi-
tive HRESIMS m/z 527.2463 [M + Na]+ (calcd. for C24H40O11Na,
527.2468).
IR ¼ ðS1–S2Þ=ðS1–SÞ ꢀ 100%
2.3.4. Eucomegastigsides D (7)
Pale yellow gum; [α]25D - 9 (c 0.05, MeOH); UV (MeOH) λmax (log ε)
273 (0.078) nm, 239 (0.226) nm; IR (KBr) νmax 2966, 1641, 1384,
where S1 is the mean peak area of HA in control group, S is the mean
peak area of HA in blank group, and S2 is the mean peak area of HA in
the sample.
1033 cm−1 1H and 13C NMR data, see Table 1; positive HRESIMS m/z
;
527.2466 [M + Na]+ (calcd. for C24H40O11Na, 527.2468).
3. Results and discussion
2.4. Acid hydrolysis of compound 2,3, 5, and 7
3.1. Chemistry
A solution of 2, 3, 5, 7 (1.0 mg, respectively) in 2 M HCl (5.0 mL) was
heated for 5 h. The reaction mixture was neutralized with Amberlite IR-
120, then the mixture was extracted with CH2Cl2. The aqueous layer
was subjected to HPLC analysis with OR detector using the following
conditions: Mobile phase: MeCN-H2O (75: 25), flow rate:
Compound 2 was obtained as a pale yellow gum. The molecular for-
mula C24H38O11 was determined by HRESIMS with the [M + H]+ peak
at m/z 503.2486 (calcd. for C24H39O11, 503.2492). Its IR absorption
1.0 mL∙min−1, tR = 6.43 min. (L-arabinose, [α]20 + 29), tR
=
D
8.61 min (D-glucose, [α]20D + 27). The sugars showed the retention
Table 2
The experimental procedure of ACE inhibitory activity.
times to be identical with those of authentic L-arabinose and D-glucose.
Sample (μL)
Control (μL)
Blank (μL)
2.5. ACE inhibitory activity assay
SBB
ACE
ACEI
350-(X + 10)
10
X
(37 °C water-bath for 10 min)
50
(37 °C water-bath for 40 min)
600
340
10
0
350
0
0
ACE activity was determined by HPLC assay according to the method
described [14,15]. In general, H3BO3 (6.18 g) and NaCl (17.53 g) were
dissolved in Ultra Pure Water (which was adjusted by NaOH to
pH 8.3) to 1000 mL (SBB). HHL (21.5 mg), HA (17.9 mg), captopril
(50 mg) and ACE (1 U) were dissolved in a 10 mL volumetric flask of
the above-mentioned borate solution, respectively. All compounds
were prepared by dissolving each of them in 2 mg/mL SBB, respectively.
HHL
50
50
1 M HCl
600
600
(10,000 g/min centrifugation for
10 min)
X = 30, 60, 120 μL.

124
J.-K. Yan et al. / Fitoterapia 116 (2017) 121–125
bands at 2938, 1647, 1647 and 1260 cm−1 indicated the presence of α,
β-unsaturated carbonyl and olefin groups. The 1H NMR and 13C NMR
data of 2 (Table 1) indicated a glycosylated megastigmane skeleton.
The 1H NMR spectrum displayed two singlet methyl proton signals at
spectrum, which showed negative Cotton effects at 251 nm (Δε −
42.40) and positive Cotton effects at 311 nm (Δε + 3.56) [12]. Com-
pound
3
was elucidated as (6S, 7E, 9R)-9-hydroxy-4,7-
6)]-β-D-
new compound and named as
megastigmadien-3-one-9-O-[α-L-arabinopyranosyl-(l
glucopyranoside. It was
eucomegastigsides B.
→
δH 1.03 (3H, s) and 0.99 (3H, s), a doublet methyl proton signal at δH
a
1.29 (3H, d, J = 6.4 Hz), a vinyl methyl proton signal at δH 1.98 (3H, d,
J = 1.1 Hz), and three olefinic proton signals at δH 5.74 (1H, s), 5.77
(1H, dd, J = 15.3, 9.4 Hz), 5.60 (1H, dd, J = 15.3, 7.3 Hz) and two doublet
signals at δH 4.29 (1H, d, J = 7.8 Hz) and 4.32 (1H, d, J = 6.7 Hz) which
were assigned as two anomeric protons of sugar moieties. Comparing
the 13C NMR data of 2 with the known compound 1 showed that they
are similar to each other. The main differences were that the 13C NMR
signals at δC 138.2 (C-8), 77.1 (C-9), 21.2 (C-10) and 102.6 (C-1′) in 1
shifted to 137.1 (C-8), 74.9 (C-9), 22.4 (C-10) and 101.5 (C-1′) in 2, sug-
gesting that the absolute configuration at C-9 in 2 was different from
that in 1. The chemical shift value of C-9 was important for deducing
the configuration of C-9 in Δ7,8-type of 9-hydroxymegastigmane 9-O-
β-D-glucopyranoside in Methanol-d4. For the 9S, the chemical shift ap-
pears at around δC (74–76), and in the 9R, the carbon resonates near
Compound 5 was obtained as a pale yellow gum. The molecular for-
mula C24H40O11 was determined by HRESIMS with the [M + Na]+ peak
at m/z 527.2463 (calcd. for C24H40O11Na, 527.2468). Its IR spectrum
showed absorption bands at 2930, 1651, 1415 and 1259 cm−1 assign-
able to α, β-unsaturated carbonyl and olefin groups. The 1H and 13C
NMR data of 5 (Table 1) indicated a glycosylated megastigmane skele-
ton. The 1H NMR spectrum displayed two singlet methyl proton signals
at δH 1.03 (3H, s) and 0.95 (3H, s), a doublet methyl proton signal at δH
1.18 (3H, d, J = 6.2 Hz), a vinyl methyl proton signal at δH 1.99 (3H, s),
and an olefinic proton signal at δH 5.74 (1H, s). The 1H and 13C NMR data
of 5 were very similar to those of 4, with the differences being raised
from the chiral centers C-9, indicating the absolute configuration at C-
9 in 5 different from that in 4. Based on the empirical rule and CD spec-
trum of 5, the absolute configurations of C-6 and C-9 were confirmed as
6R,9S. On the basis of 2D-NMR spectra and above-mentioned evidence,
the structure of 5 was characterized as shown (Fig. 1) and named
eucomegastigsides C.
Compound 7 was obtained as a pale yellow gum. The molecular for-
mula C24H40O11 was determined by HRESIMS with the [M + Na]+ peak
at m/z 527.2466 (calcd. for C24H40O11Na, 527.2468). Its IR absorption
bands at 2966, 1641 and 1384 cm−1 indicated the presence of α, β-un-
saturated carbonyl. The 1H and 13C NMR data of 5 (Table 1) indicated a
glycosylated megastigmane skeleton. The 1H NMR spectrum displayed
two singlet methyl proton signals at δH 1.13 (6H, s), a doublet methyl
proton signal at δH 1.22 (3H, d, J = 6.1 Hz), a vinyl methyl proton signal
at δH 1.70 (3H, s), and two doublet signals at δH 4.30 (1H, d, J = 7.8 Hz)
and 4.26 (1H, d, J = 6.6 Hz) which were assigned as two anomeric pro-
tons of sugar moieties. Comparing the 13C NMR data of 7 with 5 showed
that 7 was consistent with 5 in the saccharide part. The 13C NMR data of
the aglycone part of 7 were similar with those of Foliasalaciosides D
[12]. The main differences were that the carbon signals at δC 37.3 (C-
8), 76.0 (C-9) and 20.0 (C-10) in Foliasalaciosides D shifted to δC 36.6
(C-8), 77.9 (C-9) and 22.1 (C-10) in 7, suggesting that the absolute con-
figuration at C-9 in 7 was different from that in Foliasalaciosides D. It is
reported that the chemical shift at C-9 is indicative for 9S δC (77.7–78.1)
and 9R δC (75.7–76.8) configuration for 9-hydroxymegastigmane 9-O-
β-D-glucopyranoside in Methanol-d4 [16]. The absolute configuration
at C-9 in 7 was also confirmed to be (S) based on chemical shift at C-9
δC (77–79) [16]. The absolute configuration at C-9 in 2 was confirmed
to be (S) based on chemical shift at C-9 δC (74.9). The absolute configu-
ration at C-6 in 2 was further confirmed as (R) by CD spectrum, which
showed positive Cotton effects at 261 nm (Δε + 74.52) and negative
Cotton effects at 317 nm (Δε − 12.08) [12]. As shown in (Fig. 2), in
the HMBC spectrum, some key long-range correlations were observed
between 0.99 (11-CH3) and C-2, C-3, C-6 and C-12, as well as 4.29 (1′-
CH) and C-9, and between 4.32 (1″-CH) and C-6′. The positions of the
glycoside linkages in 2 were determined unambiguously by HMBC ex-
periment. The acid hydrolysis of 2 liberated D-glucose and L-arabinose,
which were identified by HPLC analysis using an optical rotation detec-
tor by comparison of the retention times with the authentic samples
[12]. The glucose and the arabinose moieties were deduced to be β
and α configuration respectively by comparing coupling constants and
chemical shift of signals of sugar moieties with those reported [11–
13]. On the basis of the above analysis, compound 2 was elucidated as
(6R,
7E,
9S)-9-hydroxy-4,7-megastigmadien-3-one-9-O-[α-L-
arabinopyranosyl-(l → 6)]-β-D-glucopyranoside. It was a new com-
pound and named as eucomegastigsides A.
Compound 3 was obtained as a pale yellow gum. Its molecular for-
mula C24H38O11 was determined by HRESIMS with the [M + H]+ peak
at m/z 503.24892 (calcd. for C24H39O11, 503.2492). Comparing the pro-
ton and carbon signals of 3 with compound 1 showed that they are al-
most the same with little differences. The CD spectrum suggested that
the absolute configuration at C-6 in 3 was different from that in 1. The
absolute configuration at C-6 in 3 was further confirmed as (S) by CD
δC (77.9). The connection of glucosyl to C-9 and arabinosyl to glucosyl
were determined unambiguously from key long-range correlations in
HMBC experiment in (Fig. 2). Finally, the structure of 7 was character-
ized as shown (Fig. 1) and named eucomegastigsides D.
3.2. ACE inhibitory activity
All the compounds (1–7) and control drug coptopril were evaluated
for their ACE inhibitory activities using HPLC method. The inhibitory ef-
fects of 1–7 were computed using percentage inhibition ratio (%). All
isolates showed either moderate or weak inhibition ratios. Compounds
2, 3, 4, 5, 7 with inhibition ratios of 24.6
0.5%, 29.1
0.6%, 31.6
0.6%, 31.2 0.2% and 29.7 0.4% respectively at the concentration of
240 μg/mL showed moderate activities compared with captopril
(98.0 0.1% at 240 μg/mL).
4. Conclusion
Four new megastigmane glycosides, eucomegastigsides A–D (2, 3, 5
and 7), together with three known megastigmane glycosides, (6R, 7E,
9R)-9-hydroxy-4,7-megastigmadien-3-one-9-O-[α-L-
Fig. 2. Key HMBC correlations of compounds 2, 3, 5 and 7.
arabinopyranosyl-(l → 6)]-β-D-glucopyranoside (1), foliasalacioside B1

J.-K. Yan et al. / Fitoterapia 116 (2017) 121–125
125
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This work was supported by grants from the National Science Foun-
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