In vitro study on α-amylase and α-glucosidase inhibitory activities of a new stigmastane-type steroid saponin from the leaves of Vernonia amygdalina

Article abstract of DOI:10.1080/14786419.2019.1607853

Using various chromatographic separations, four compounds, including one new steroid saponin named vernoamyoside E (1), were isolated from the leaves of the Vietnamese medicinal plant Vernonia amygdalina Delile (Asteraceae). Their structures were established by spectroscopic methods such as 1D- and 2D-NMR, HR-ESI-MS, and HPLC analysis. The inhibitory activities against α-glucosidase and α-amylase of the isolated compounds from V. amygdalina were reported for the first time. The results indicated that compound 1 significantly inhibited both against α-amylase and α-glucosidase activity.

Full text of DOI:10.1080/14786419.2019.1607853

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20
In vitro study on α-amylase inhibitory and α-
glucosidase of a new stigmastane-type steroid
saponin from the leaves of Vernonia amygdalina
Hoang Le Tuan Anh, Le Ba Vinh, Le Thi Lien, Pham Viet Cuong, Masayoshi
Arai, Tran Phuong Ha, Hoang Ngoc Lin, Ton That Huu Dat, Le Canh Viet
Cuong & Young Ho Kim
To cite this article: Hoang Le Tuan Anh, Le Ba Vinh, Le Thi Lien, Pham Viet Cuong, Masayoshi
Arai, Tran Phuong Ha, Hoang Ngoc Lin, Ton That Huu Dat, Le Canh Viet Cuong & Young Ho
Kim (2019): Inꢀvitro study on α-amylase inhibitory and α-glucosidase of a new stigmastane-
type steroid saponin from the leaves of Vernoniaꢀamygdalina, Natural Product Research, DOI:
10.1080/14786419.2019.1607853
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2019.1607853
In vitro study on a-amylase inhibitory and a-glucosidase
of a new stigmastane-type steroid saponin from the
leaves of Vernonia amygdalina
a,b
ꢀ
d,e
ꢀ
a,b
a
Hoang Le Tuan Anh , Le Ba Vinh , Le Thi Lien , Pham Viet Cuong ,
c
a
a
a
Masayoshi Arai , Tran Phuong Ha , Hoang Ngoc Lin , Ton That Huu Dat ,
a
d
Le Canh Viet Cuong and Young Ho Kim
a
Mientrung Institute for Scientific Research Vietnam Academy of Science and Technology, Hue city,
b
Vietnam; Graduate University of Science and Technology Vietnam Academy of Science and
Technology (VAST), Hanoi, Vietnam; Research Center for Drug Discovery, Graduate School of
Pharmaceutical Sciences, Osaka University, Osaka, Japan; College of Pharmacy, Chungnam National
University, Daejeon, Republic of Korea; Institute of Marine Biochemistry Vietnam Academy of
c
d
e
Science and Technology, Ha Noi, Vietnam
ABSTRACT
ARTICLE HISTORY
Using various chromatographic separations, four compounds,
including one new steroid saponin named vernoamyoside E (1),
were isolated from the leaves of the Vietnamese medicinal plant
Vernonia amygdalina Delile (Asteraceae). Their structures were
established by spectroscopic methods such as 1D- and 2D-NMR,
HR-ESI-MS, and HPLC analysis. The inhibitory activities against
a-glucosidase and a-amylase of the isolated compounds from V.
amygdalina were reported for the first time. The results indicated
that compound 1 significantly inhibited both against a-amylase
and a-glucosidase activity.
Received 27 December 2018
Accepted 31 March 2019
KEYWORDS
Vernonia amygdalina;
Asteraceae; vernomyoside E;
a-amylase inhibitory;
a-glucosidase inhibitory
CONTACT Young Ho Kim
yhk@cnu.ac.kr
College of Pharmacy, Chungnam National University, Daejeon,
cuongwg@yahoo.com Mientrung Institute for Scientific Research
3
4134, Republic of Korea; Pham Viet Cuong
Vietnam Academy of Science and Technology, 321 Huynh Thuc Khang, Hue city, Thua Thien Hue, Vietnam
ꢀ
These authors are contributed equally to this work.
Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2019.1607853.
ß 2019 Informa UK Limited, trading as Taylor & Francis Group

2
H. LETUANANH ET AL.
1. Introduction
Vernonia amygdalina Del. is a shrub or small tree that is mainly grown in tropical areas
of Africa (Atangwho et al. 2012). In Africa, it can be used as a traditional treatment for
diabetes, nausea, dermatitis, arthritis, ascariasis, stomached, anaemia, jaundice, pneu-
monia, fever, tonsillitis, schistosomiasis, malaria, amoeba, and intestinal upsets
(Farombi and Owoeye 2011). The principal chemical components have shown that V.
amygdalina contains steroids, polyphenolics, alkaloids, anthraquinones, lignans, flavo-
noids, and coumarins (Liu et al. 2019). Biological activities of extracts and isolated
compounds from V. amygdalina have been reported, such as antibacterial, antifungal,
antiparasite, antiviral, anticancer, anti-obesity, antioxidant, antihypertensive, and liver
protective activity (Atangwho et al. 2012; Atangwho et al. 2013; Igile et al. 1994;
Kupchan et al. 1969; Taiwo et al. 2009; Yeap et al. 2010). In continuation to study sec-
ondary metabolites and evaluation anti-diabetic compounds of Vietnamese medicinal
plants, herein, we reported the isolation and structural elucidation of four steroid sap-
onins including one new compound (1) from the leaves of V. amygdalina.
2. Results and discussion
In the present study, the methanol (MeOH) extract from the leaves of V. amygdalina
were evaluated to against a-amylase and a-glucosidase activity. In this regard, the
MeOH extract significantly inhibited both against a-amylase and a-glucosidase activity
with IC50 0.64 ± 0.12 mg/mL and 1.78 ± 0.31 mg/mL, respectively. Anti-diabetic guide
fraction and purification led to isolation of four compounds including one new com-
pound (1), and three known compounds (2–4) (Figure 1). The known compounds
were identified vernoniacums B (2) (Ma et al. 2016), vernonioside B (3) (Jisaka et al.
1
1
992), and vernonioside B (4) (Jisaka et al. 1993) by comparing their spectroscopic
2
2
0
data (½aꢁ :1D, 2D NMR, MS) with reported values.
D
20
Vernoamyoside E (1), ½aꢁ :þ24.7 (c 0.15, MeOH), was obtained as a white amorphous
D
powder. The molecular formula (C H O ) of 1 was determined by HR-ESI-MS with a
36 56 11
þ
þ
,
11
sodium molecular ion peak at m/z 687.3708 [Mþ Na] (calcd for C H NaO
36
56
687.3720). The NMR spectra (Supplementary material) are indicative of a steroid saponin
with three tert-methyl proton signals at d 0.57, 0.93, and 1.42 (each, 3H, s), two doublet
H
methyl signals at dH 0.94 (3H, d, J ¼ 7.0 Hz, H-26), 0.95 (3H, d, J ¼ 7.0Hz, H-27), a
methoxy signal at d 3.21 (3H, s, 28-OCH ), and two doublet olefinic protons at d 5.43
H
3
H
(
1H, d, J ¼ 5.0Hz, H-7), 5.53 (1H, d, J ¼ 5.5 Hz, H-11), as well as one anomeric proton sig-
0
13
nals at d 4.42 (1H, d, J ¼ 8.0 Hz, H-1 ). The C-NMR spectrum of 1 exhibited the signals
H
of 36, of which 6 carbon signals were assigned to a sugar unit and the remaining 29
carbon signals were assigned to a steroid aglycon (one of the main constituents of V.
13
amygdalina) on the basis 1D-2D-NMR. Analysis C-NMR signals were indicated an iso-
propyl unit at d 32.7 (C-25), 17.4 (C-26), 18.1 (C-27), three hydroxylated carbons at dC
C
8
2
1.0 (C-22), 91.3 (C-23), 83.2 (C-24), two hemiacetal carbons at d 100.0 (C-21), 112.9 (C-
C
8), a methoxy group at d 48.3 (28-OCH ), and a methyl group at d 17.4 (C-29) by
C
3
C
7,9(11)
combined HSQC signals. The aglycon of 1 was identified as D
glycoside previously reported from V. amygdalina by clear agreement of its H and
NMR data (Quasie et al. 2016). Comparison of the H, and C-NMR data of 1 with those
dienstigmastane
1
13
C
1
13

NATURAL PRODUCT RESEARCH
3
of vernoniosides B (3) demonstrated that two compounds were almost identical in the
1
same NMR solvent. The only difference between these two compounds was the replace-
ment of the hydroxyl group at C-16 (d 28.0 ppm). In addition, the NOESY spectrum
C
revealed significant cross-peaks between H-20 and each proton at C-21, C-22, and C-23.
H-22 and each proton at C-21, and C-23, H-23 and one of methyl (H-27) of the isopropyl
group, and H-22 and the methoxy group showing that all of the hydrogens at C-20-C-
23, and the isopropyl and the methoxy groups are b-orientated. Furthermore, absolute
configurations of the sugar moieties were identified as b-D-glucopyranosyl by coupling
0
constants of anomeric position d 4.42 (J ¼ 8.0, H-1 ), and acid hydrolysis of 1 followed
H
by TLC and HPLC analysis in comparison with the authentic samples (Supplementary
data). Attachment of the monosaccharide chain at C-3 of the aglycon was confirmed by
HMBC correlation of the anomeric proton H-3 (d 3.71) with C-1 (d 102.6). Therefore,
0
H
C
the structure of 1 was established as (22R,23S,24R,28S)-3b-glucosyl-28-methoxy-7,8,9,11-
tetradehydro-21,24-dihydroxy-21,23:22,28-diepoxy-5a-stigmastane.
Diabetes mellitus is a chronic disease that affects millions of people worldwide. To
date, much effort has been studied to identify effective with a-amylase, and a-glucosi-
dase inhibitors from natural sources by isolation compounds develop functional food
for use against diabetes (Fatmawati et al. 2011). Previously, we were investigated that
isolated compounds which were exhibited potential treatment of diabetes from Rosa
rugose, and Ecklonia cava (Park et al. 2018; Thao et al. 2014). Bitter leaf (V. amygdalina)
is a popular vegetable commonly consumed and which has been employed in trad-
itional medicine for the prevention and management of diabetes and hypertension
(Saliu et al. 2012). In this study, the inhibitory effects of the MeOH extract, and isolated
compounds against a-amylase and a-glucosidase were evaluated. As the obtained
results, compound 1 exhibited potent inhibitory activity against a-glucosidase enzyme,
more than positive control on both 100 and 500 lg/mL concentrations with values
5
0.17 ± 1,03 mg/mL and 60,03 ± 3,85 mg/mL, respectively. While, compounds 1, 2, and 3
showed significant inhibition against a-amylase enzyme with IC50 values of 102.23,
3.17, and 87.09 mg/mL, respectively when compared with that of a positive control,
8
acarbose, 6.3 mg/mL.
All compounds (1–3) have shown significantly against both a-amylase and a-gluco-
sidase. The most active compound 1, not only indicated a more potent a-glucosidase
inhibitory activity than acarbose (positive control) but also showed significant activity
against a-amylase.
Given the potential against both a-amylase and a-glucosidase of V. amygdalina
extracts, and isolated compounds, compound 1 might be potential candidate for fur-
ther investigation on the molecular mechanisms of action on specific anti-dia-
betic targets.
3. Experimental
3.1. General experimental procedures
Optical rotations were determined on a JASCO P-2000 polarimeter (Hachioji, Tokyo,
Japan). The high-resolution electrospray ionisation mass spectra (HR-ESI-MS) were
obtained from a MicroQ-TOF III mass spectrometer (Bruker Daltonics, 255748

4
H. LETUANANH ET AL.
Germany). IR spectra were obtained on a Bruker TENSOR 37 FT-IR spectrometer. The
NMR spectra were recorded on a Bruker Advance III HD 500 FT NMR spectrometers
with TMS as an internal standard. HPLC was used with Kinetex C18 column
(
250 ꢂ 20.0 mm, 5 mm; Phenomenex, Torrance, CA). Column chromatography (CC) was
performed on silica gel (Kiesel gel 60, 70–230 mesh and 230–400 mesh, Merck,
ꢀ
Darmstadt, Germany), YMC GEL (ODS-A, 12 nm S-150 lm, YMC Co., Ltd.) and Sephadex
LH-20 (Sigma-Aldrich, USA) resins. TLC used pre-coated silica gel 60 F254 (1.05554.0001,
Merck) and RP-18 F254S plated (1.15685.0001, Merck), and compounds were visualized
by spraying aqueous 10% H SO and heating for 3–5 min.
2
4
3.2. Biological material
The leaves of V. amygdalina were collected at Phong Dien, Thua Thien Hue Province,
Vietnam in August 2017, and were identified by Dr. Nguyen Quynh Nga (Institute of
Medicinal Material, Vietnam). A voucher plant (MISR-2017-14) has been deposited at
Mientrung Institute for Scientific Research, VAST, Vietnam.
3.3. Assay for a-glucosidase inhibition
The a-glucosidase enzyme inhibition assay was carry out according to similar the pre-
viously method (Luyen et al. 2019) and provide in supplementary data.
3
.4. Assay for a-amylase inhibition
The a-amylase enzyme inhibitory activity was performed using the modification
Luyen et al. 2019) (Supplementary data).
(
3.5. Extraction and isolation
Air-dried leaves of V. amygdalina (1.2 kg) were extracted with 100% methanol (5 L ꢂ 3
ꢃ
times) under sonication at 50 C for 4 h to yield a dark solid extract (200 g). This extract
was suspended in water and successively partitioned with n-hexane, dichloromethane
and ethyl acetate to give corresponding n-hexane (VAH, 52 g), dichloromethane (VAD,
42 g), ethyl acetate (VAE, 31 g) and water layer (VAW, 75 g). The VAE fraction (31 g)
was subsequently chromatographed on a silica gel column eluting with dichlorome-
thane/methanol (gradient from 100/1 – 0/1, v/v) to give five fractions (VAE1-VAE5).
The VAE1 (1.2 g) fraction was further separated on a silica gel column eluting with
dichloromethane/methanol/water (25/1/0.05, v/v/v) to give two smaller fractions
(VAE1.1 and VAE1.2). Compound 1 (5 mg) was yielded from VAE1.1 (0.3 g) fraction by
purify on a RP-18 column eluting with methanol/water (4/1, v/v). The VAE1.2 (0.2 g)
fraction was further separated on a Sephadex LH-20 column eluting with methanol/
water (1/1, v/v) to afford compound 3 (15 mg). The VAE2 (0.9 g) fraction was continu-
ously chromatographed on a RP-18 column eluting with methanol/water (3/1, v/v) to
yield compounds 2 (7 mg) and 4 (6 mg).

NATURAL PRODUCT RESEARCH
5
Figure 1. The structures of compounds 1–4.
3
.5.1. Vernoamyoside E (1) 2
0
White amorphous powder, ½aꢁ : þ24.7 (c 0.15, MeOH); IR (KBr) m
3424, 2940, 1598,
D
max
1
1448, 1380; H-NMR (MeOD-d , 500 MHz): d 1.34 (1H, m, H -1), 1.99 (1H, m, H -1), 1.93
4
H
a
b
(
1H, m, H -2), 3.71 (1H, m, H -2), 3.71 (1H, m, H-3), 1.32 (1H, m, H -4), 1.88 (1H, m, H -
a b a b
4
5
1
2
), 1.41 (1H, m, H-5), 1.61 (1H, m, H -6), 1.97 (1H, m, H -6), 5.43 (1H, d, J ¼ 5.0 Hz, H-7),
a
b
.53 (1H, d, J ¼ 5.5 Hz, H-11), 2.04 (1H, m, H -12), 2.22 (1H, m, H -12), 2.26 (1H, m, H-
a
b
4), 1.49 (1H, m, H -15), 1.86 (1H, m, H -15), 1.53 (1H, m, H -16), 2.18 (1H, m, H -16),
a
b
a
b
.08 (1H, m, H-17), 0.57 (3H, s, H-18), 0.93 (3H, s, H-19), 1.89 (1H, m, H-20), 5.43 (1H, d,
J ¼ 5.0 Hz, H-21), 4.26 (1H, t, J ¼ 5.5 Hz, H-22), 4.45 (1H, d, J ¼ 5.5 Hz, H-23), 2.20 (1H, m,
H-25), 0.94 (3H, d, J ¼ 7.0 Hz, H-26), 0.95 (3H, d, J ¼ 7.0 Hz, H-27), 1.42 (3H, s, H-29), 4.42
0
0
0
(
(
1H, d, J ¼ 8.0, H-1 ), 3.18 (1H, m, H-2 ), 3.29 (1H, ddd, J ¼ 2.5, 5.0, 9.5 Hz, H-3 ), 3.32
0
0
0
1H, m, H-4 ), 3.39 (1H, t, J ¼ 9.0 Hz, H-5 ), 3.70 (1H, dd, J ¼ 5.5, 12.0 Hz, H -6 ), 3.87 (1H,
a
0
13
J ¼ 2.5, 12.0 Hz, H -6 ) 3.21 (3H, s, OCH -28); C-NMR (MeOD-d , 125 MHz): d 35.8 (C-
b
3
4
C
1
), 30.8 (C-2), 78.9 (C-3), 34.8 (C-4), 40.1 (C-5), 30.3 (C-6), 121.4 (C-7), 137.2 (C-8), 144.8

6
H. LETUANANH ET AL.
(C-9), 36.8 (C-10), 119.5 (C-11), 41.9 (C-12), 42.7 (C-13), 51.9 (C-14), 24.2 (C-15), 28.0 (C-
1
2
1
6), 45.4 (C-17), 12.9 (C-18), 19.8 (C-19), 50.2 (C-20), 100.0 (C-21), 81.0 (C-22), 91.3 (C-
3), 83.2 (C-24), 32.7 (C-25), 17.4 (C-26), 18.1 (C-27), 112.9 (C-28), 17.4 (C-29), 102.6 (C-
0
0
0
0
0
0
), 74.8 (C-2 ), 77.4 (C-3 ), 71.4 (C-4 ), 77.7 (C-5 ), 62.6 (C-6 ), 48.3 (OCH -28); HR-ESI-MS
3
þ
þ
m/z 687.3708 [M þ Na] (calcd for C H NaO , 687.3720).
36
56
11
Conflict of interest
The authors have declared that there is no conflict of interest.
Funding
This work was supported by Vietnam Academy of Science and Technology under grant number:
VAST.N D- P.03/17-18).
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