Triterpenoid glycosides from the rhizomes of Allium ascalonicum and their anoctamin-1 inhibitory activity

Article abstract of DOI:10.1080/14786419.2020.1713122

Ten triterpenoid glycosides including two undescribed compounds (1 and 2) were isolated from the methanol extract of Allium ascalonicum rhizomes. These compounds were structurally elucidated to be 3β-O-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl-19α-hydroxyolean-12-ene-28-oic acid 28-O-[α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl] ester (1), 3-O-β-D-glucopyranosyl-(1→3)-[α-L-rhamnopyranosyl-(1→2)]-α-L-arabinopyranosyl-3β,19α-dihydroxyoleanane-12-en-28-oic acid (2), lactifoloside C (3), lactifoloside H (4), randiasaponin IV (5), kudinoside G (6), ilexkudinoside W (7), lactifoloside G (8), kudinoside D (9), and ilexkudinoside T (10) by analyzing their HR-ESI-MS, NMR spectral data and by comparison with those reported in the literature. Compounds 1–10 were evaluated for anoctamin-1 (ANO1) inhibitory activity using yellow fluorescent protein reduction assays. At the concentration of 30 μM, compounds 2 and 9 displayed moderate ANO1 inhibitory percentages of 28.9 ± 0.85% and 26.2 ± 0.65%, respectively.

Full text of DOI:10.1080/14786419.2020.1713122

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20
Triterpenoid glycosides from the rhizomes
of Allium ascalonicum and their anoctamin-1
inhibitory activity
Nguyen Thi Mai, Duong Thi Dung, Tran Thuy Nga, Vu Thi Xuan, Vu Van Doan,
Bui Huu Tai, Nguyen Xuan Nhiem, Pham Hai Yen, Phan Van Kiem, Yohan Seo,
Wan Namkung, SeonJu Park & Seung Hyun Kim
To cite this article: Nguyen Thi Mai, Duong Thi Dung, Tran Thuy Nga, Vu Thi Xuan, Vu Van
Doan, Bui Huu Tai, Nguyen Xuan Nhiem, Pham Hai Yen, Phan Van Kiem, Yohan Seo, Wan
Namkung, SeonJu Park & Seung Hyun Kim (2020): Triterpenoid glycosides from the rhizomes
of Alliumꢀascalonicum and their anoctamin-1 inhibitory activity, Natural Product Research, DOI:
10.1080/14786419.2020.1713122
To link to this article: https://doi.org/10.1080/14786419.2020.1713122
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2020.1713122
Triterpenoid glycosides from the rhizomes of Allium
ascalonicum and their anoctamin-1 inhibitory activity
Nguyen Thi Maia, Duong Thi Dungb, Tran Thuy Ngaa, Vu Thi Xuana, Vu Van
Doanb, Bui Huu Taib,c, Nguyen Xuan Nhiemb,c, Pham Hai Yenb, Phan Van
Kiemb,c, Yohan Seod, Wan Namkungd, SeonJu Parke
and Seung Hyun Kimd
b
aUniversity of Transport and Communications, Hanoi, Viet Nam; Institute of Marine Biochemistry,
c
Vietnam Academy of Science and Technology (VAST), Hanoi, Viet Nam; Graduate University of
d
Science and Technology, VAST, Hanoi, Viet Nam; College of Pharmacy, Yonsei Institute of
e
Pharmaceutical Science, Yonsei University, Incheon, Korea; Chuncheon Center, Korea Basic Science
Institute (KBSI), Chuncheon, Republic of Korea
ABSTRACT
ARTICLE HISTORY
Received 18 October 2019
Accepted 30 December 2019
Ten triterpenoid glycosides including two undescribed com-
pounds (1 and 2) were isolated from the methanol extract of
Allium ascalonicum rhizomes. These compounds were structurally
elucidated to be 3b-O-a-L-rhamnopyranosyl-(1!2)-a-L-arabinopyr-
anosyl-19a-hydroxyolean-12-ene-28-oic acid 28-O-[a-L-rhamnopyr-
KEYWORDS
Allium ascalonicum;
Alliaceae; alliumascaside A;
alliumascaside B;
ANO1 inhibitor
anosyl-(1!2)-b-D-glucopyranosyl]
ester
(1),
3-O-b-D-
glucopyranosyl-(1!3)-[a-L-rhamnopyranosyl-(1!2)]-a-L-arabino-
pyranosyl-3b,19a-dihydroxyoleanane-12-en-28-oic acid (2), lactifo-
loside C (3), lactifoloside H (4), randiasaponin IV (5), kudinoside G
(6), ilexkudinoside W (7), lactifoloside G (8), kudinoside D (9), and
ilexkudinoside T (10) by analyzing their HR-ESI-MS, NMR spectral
data and by comparison with those reported in the literature.
Compounds 1–10 were evaluated for anoctamin-1 (ANO1) inhibi-
tory activity using yellow fluorescent protein reduction assays. At
the concentration of 30 mM, compounds 2 and 9 displayed
moderate ANO1 inhibitory percentages of 28.9 0.85% and
26.2 0.65%, respectively.
CONTACT Seung Hyun Kim
kimsh11@yonsei.ac.kr
College of Pharmacy, Yonsei Institute of Pharmaceutical
Science, Yonsei University, Incheon 406-840, Korea
Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2020.1713122.
ß 2020 Informa UK Limited, trading as Taylor & Francis Group

2
N. T. MAI ET AL.
1. Introduction
Allium is a large genus of the Alliaceae family, comprising approximately 500 species.
Some of them such as A. cepa (onion), A. sativum (garlic), A. porrum (leek), A. schoeno-
prasum (chive), A. fistulosum (scallion), and A. ascalonicum (shallot) have been com-
monly used all over the world as vegetables or culinary supplements (Lanzotti et al.
2014; Singh et al. 2018). Allium vegetables as well as garlic were well known for thou-
sands years in traditional medicines to treat inflammation and cardiovascular diseases.
They have also been known as potent antitumor, antimicrobial, antiarthritic, hypolipi-
daemic, and hypoglycemic agents (Sengupta et al. 2004). A. ascalonicum is cultivated
as a common vegetable in Viet Nam and many countries in Asia, Europe, America, and
Africa (Loi 2012). In Vietnamese folk medicine, this plant is used to treat typhoid, fever,
colds, headaches, bloating and poisoning, blurred vision, deaf ears, and breast swelling
(Loi 2012). Previous phytochemical studies on A. ascalonicum indicated that it contains
sulphur-containing compounds (Adeniyi and Anyiam 2004), flavonoids and furostanol
saponins (Fattorusso et al. 2002). Herein, we report the isolation and structural elucida-
tion of two new and eight known triterpenoid glycosides from the methanol extract
of A. ascalonicum rhizomes. Additionally, the anoctamin-1 (ANO1) inhibitory activity of
the isolated compounds was also investigated. ANO1, known as transmembrane pro-
tein 16A (TMEM16A), has been identified as a calcium-activated chloride channel
expressed in various cell types (Yang et al. 2008; Seo et al. 2017). Since ANO1 plays
pivotal roles in the regulation of various biological process, such as epithelia fluid
secretion, smooth muscle contraction, cell proliferation and sensory signal transduc-
tion, the finding of ANO1 inhibitors would be considered as a new strategy for the
treatment of several diseases such as pain, hypertension, asthma, diarrhea, and cancer
(Bill and Gaither 2017). To date, only a few ANO1 inhibitors have been described, but
several flavonoids and cycloartane triterpenoids have been reported to inhibit ANO1
(Seo et al. 2017; Thu et al. 2019). As mentioned above, inhibition and modulation of
ANO1 function could be beneficial to therapeutics, so the chemical constituents of A.
ascalnoicum were investigated for ANO1 inhibitory activity.
2. Results and discussion
Compound 1 was obtained as a white amorphous powder. Its molecular formula of
35
C53H86O21 was deduced from the quasi molecular ion peaks [Mþ Cl]ꢀ at m/z
37
1093.5370 and [Mþ Cl]ꢀ at m/z 1095.5386 (calcd. for C53H86O2135Cl: 1093.5350 and
1
calcd. for C53H86O2137Cl: 1095.5321) in the negative ion mode HR-ESI-MS. The H NMR
spectrum of 1 exhibited one olefin proton signal [dH 5.35 (br s)], seven methyl singlets
[dH 0.78, 0.87, 0.95, 0.96, 0.98, 1.03, and 1.31 (each, 3H)], and two methyl doublets [dH
1.25 and 1.26 (each, 3H, d, J ¼ 6.0 Hz)]. Four anomeric protons were identified at dH
4.58 (d, J ¼ 5.0 Hz), 5.11 (d, J ¼ 1.5 Hz), 5.39 (d, J ¼ 1.5 Hz), and 5.44 (d, J ¼ 7.5 Hz) sug-
gesting the presence of four sugar units. The 13C NMR and DEPT spectra revealed the
signals of 53 carbons, including 8 non-protonated carbons, 25 methines, 11 methyl-
enes, and 9 methyl groups. A double bond (C¼C) was assigned by a pair of
deshielded carbon signals at dC 124.8 and 144.3. The most deshielded carbon signal at
dC 178.5 suggested the presence of one carboxyl group. Four carbon signals at dC

NATURAL PRODUCT RESEARCH
3
104.7, 102.1, 101.6, and 95.3 demonstrated the anomeric carbons of four sugar units.
The aforementioned data suggested that compound 1 to be an oleanane-type triter-
pene glycoside having four sugar units. The HMBC correlations between H3-23 (dH
1.03)/H3-24 (dH 0.87) and C-3 (dC 90.8)/C-4 (dC 40.3)/C-5 (dC 57.2) suggested the pos-
ition of the oxygenated group at C-3 and assignment of two methyl groups C-23/C-24
of the oleanane skeleton (Supplementary material, Figure S1). The HMBC correlations
between H3-25 (dH 0.95) and C-5 (dC 57.2)/C-9 (dC 49.1) and between H3-26 (dH 0.78)
and C-9 indicated assignment of two methyl groups C-25 and C-26. The HMBC correla-
tions from H3-26/H3-27 (dH 1.31) to C-8 (dC 40.9)/C-9 (dC 49.1) and from H3-27 to C-13
(dC 144.3) confirmed the presence of a methyl group at C-27 and a double bond at C-
12/C-13, respectively. Moreover, COSY cross-peaks of H-9 (dH 1.75)/H2-11 (dH 1.96 and
1.98)/H-12 (dH 5.35) further confirmed the position of double bond C-12/C-13. An add-
itional hydroxy group at C-19 was deduced from the COSY cross peak of H-18 (dH
3.02)/H-19 (dH 3.28) and further reconfirmed by HMBC correlations between H3-29
(dH 0.96)/H3-30 (dH 0.98) and C-19 (dC 82.7). The carboxyl group was characterized by
deshielded carbon signal at dC 178.5. Next, the orientation of two substituted groups
(at C-3 and C-19) in the oleanane backbone was examined by J coupling constant of
related carbinol protons as well as by NOESY analysis. The double doublet signals
(J ¼ 11.5, 4.5 Hz) of H-3 (dH 3.14) and the NOESY correlation of H-3/H-5 (dH 0.82) indi-
cated axial-alpha orientation of H-3. NOESY correlations between H-18 (dH 3.02) and
H3-29 (dH 0.96)/H-19 (dH 3.28) and between H-19 and H3-29 (dH 0.96) suggested
axial-alpha orientation of OH-19 (Supplementary material, Figure S2). Therefore, the
aglycone moiety of 1 was identified to be 3b,19a-dihydroxyolean-12-ene-28-oic acid.
Except 30 carbon signals belonging aglycone moiety, remaining 23 carbons sug-
gested the presence of three hexose and one pentose sugar units. Two broad sin-
glets of anomeric protons (dH 5.11 and 5.39) implied a-linkages of two rhamnosyl
sugars. The glucopyranosyl linkage must be in the b-form as judged from the large J
coupling constant (J ¼ 7.5 Hz) of the anomeric proton at dH 5.44. The pentose was
assigned to be a-arabinopyranose indicated by JH1 - H2 coupling constant (J ¼ 5.0 Hz)
and a broad singlet of proton Ara H-40 (dH 3.80). The presence of D-glucose, L-arabin-
ose, and L-rhamnose in compound 1 was confirmed by acid hydrolysis. The sugars
were converted into their TMS derivatives, and analyzed by GC in comparison with
authentic sugar (Kiem et al. 2009). Continuously, the HMBC correlations between
anomeric protons Ara H-10 (dH 4.58) and C-3 (dC 90.8) and between Glc H-1000 (dH
5.44) and C-28 (dC 178.5) suggested arabinopyranosyl group binding at C-3, and a
glucopyranosyl group binding at C-28 of the aglycone. The COSY cross peak of Ara
H-10 (dH 4.58)/Ara H-20 (dH 3.79) and HMBC correlation from RhaI H-1ʺ (dH 5.11) to
Ara C-20 (dC 76.9) indicated the first rhamnopyranosyl group link to C-2 of arabino-
pyranosyl group. Similarly, the COSY cross peak of Glc H-1000 (dH 5.44)/Glc H-2000 (dH
3.62) and the HMBC correlation from RhaII H-10000 (dH 5.39) to Glc C-2000 (dC 77.0) indi-
cated the second rhamnopyranosyl group link to C-2 of glucopyranosyl group.
Consequently, the structure of compound 1 was determined to be 3b-O-a-L-rhamno-
pyranosyl-(1!2)-a-L-arabinopyranosyl-19a-hydroxyolean-12-ene-28-oic acid 28-O-
[a-L-rhamnopyranosyl-(1!2)-b-D-glucopyranosyl] ester, a new compound named as
alliumascaside A (Figure 1).

4
N. T. MAI ET AL.
Figure 1. Chemical structures of compounds 1–10.
The HR-ESI-MS analysis of 2 revealed quasi molecular ion peaks at m/z 947.4739
35
37
[Mþ Cl]ꢀ and m/z 949.4755 [Mþ Cl]ꢀ (calcd. for C47H76O1735Cl: 947.4771 and
C47H76O1737Cl: 949.4742, respectively), indicating a molecular formula of C47H76O17.
1
The H NMR spectrum of 2 exhibited one olefin proton signal [dH 5.33 (br s)], seven
methyl singlets [dH 0.77, 0.88, 0.98, 1.05, 1.31 (each, 3H) and 0.96 (6H)], a methyl doub-
let [dH 1.25 (3H, d, J ¼ 6.0 Hz)], and three anomeric protons [dH 4.51 (d, J ¼ 7.5 Hz), 4.53
(d, J ¼ 5.0 Hz), and 5.23 (br s)]. The 13C NMR and DEPT spectra showed signals for 47
carbons, including 8 non-protonated carbons, 20 methines, 11 methylenes, and 8
methyl groups. A pair of deshielded carbon signals at dC 124.8 and 144.7 suggested
the presence of a C¼C double bond. Three anomeric carbons at dC 105.1, 102.0, and
1
104.3 further confirmed the presence of three sugar units. The H and 13C NMR spec-
tral data of 2 were recognized to have close similarity with those of 1, except signals
corresponding to the sugar moieties (Supplementary material, Table S1). In particular,
three pairs of anomeric protons/carbons containing a broad singlet of an anomeric
proton (dH 5.23) and doublet methyl group (dH 1.25) indicated one rhamnose unit in
compound 2, which was different from the two rhamnose units in the structure of
compound 1. The presence of D-glucose, L-arabinose, and L-rhamnose sugars in com-
pound 2 were also confirmed by GC analysis and comparison with authentic sugar
standards after acid hydrolysis and TMS derivatization (Kiem et al. 2009). Furthermore,
the multiplicity of ara H-10 [4.58 (d, J ¼ 5.0 Hz)], rha H-100 [5.23 (br s)], and glc H-1000
1
[4.51 (d, J ¼ 7.5 Hz)] in the H-NMR spectrum of compound 2 indicated a-form of the
arabinopyranosyl, a-form of the rhamnopyranosyl, and b-form of the glucopyranosyl
linkages, respectively. Additionally, the HMBC correlation from Rha H-1ʺ (dH 5.23) to
Ara C-20 (dC 75.3) and from Glc H-1000 (dH 4.51) to Ara C-30 (dC 81.9) indicated the struc-
ture of a trisaccharide in compound 2 as b-D-glucopyranosyl (1!3)-[a-L-rhamnopyra-
nosyl-(1!2)]-a-L-arabinopyranosyl group. The HMBC correlation between Ara H-10 (dH
4.58) and C-3 (dC 89.7) indicated the O-glycosidic linkage of that trisaccharide at C-3 of
the aglycone moiety. Consequently, structure 2 was established to be 3-O-b-D-gluco-
pyranosyl-(1!3)-[a-L-rhamnopyranosyl-(1!2)]-a-L-arabinopyranosyl-3b,19a-dihydroxyo-
leanane-12-en-28-oic acid, a new compound named as alliumascaside B.

NATURAL PRODUCT RESEARCH
5
Compounds 3–10 were identified as lactifoloside C (Ming An et al. 1997), lactifolo-
side H (Ouyang et al. 1998), randiasaponin IV (Sahpaz et al. 2000), kudinoside G (Ming
An et al. 1997), ilexkudinoside W (Che et al. 2011), lactifoloside G (Ouyang et al. 1998),
kudinoside D (Ouyang et al. 1996), and ilexkudinoside T (Tang et al. 2009), respect-
ively, by analyses of their MS, 1D and 2D NMR spectroscopic data and by comparison
with those reported in the literature (Figure 1). Compounds 1–10 were then evaluated
for their ANO1 inhibitory effect using yellow fluorescent protein reduction assay (Seo
et al. 2016). Experiments were performed in triplicate and the biological results were
described in the percentage of inhibition (mean SD). Ani9 was purchased from
ChemDiv (San Diego, CA) and the 3 lM Ani9 solution was used as positive control
(97.5 0.34% ANO1 inhibition). At the concentration of 30 mM, compounds 2 and 9
exhibited 28.9 0.85% and 26.2 0.65% ANO1 inhibitory activity. Other compounds (1,
3–8, 10) were inactive. For the structure-activity relationship of ten isolated triterpene
saponins, it is possible to suggest that compounds possessing free acid functional
group at C-28 or having cyclic carboxylic ester between C-28 and C-20 showed moder-
ate inhibitory activity against ANO1 while the attachment of sugar units at C-28 dimin-
ish the inhibitory activity against ANO1. Since modulation of ANO1 can play a critical
role in the treatment of various diseases, screens of various compounds including nat-
ural compounds have been conducted to identify more potent and specific inhibitors
of ANO1. However, many reported ANO1 inhibitors from natural products have been
focused on small-sized phenolic compounds such as flavonoids and stilbenes (Chai
et al. 2017; Seo et al. 2017). There have been few reports suggesting triterpene sapo-
nins as ANO1 inhibitors. In this study, ten triterpene saponins including two new com-
pounds were identified from A. ascalonicum and two of them showed ANO1 inhibitory
activity, suggesting the possibility of finding ANO1 inhibitors of various chemical struc-
tures from natural products.
3. Experimental
3.1. General
HR-ESI-MS were recorded on an Agilent 6530 accurate mass QTOF LC/MS systems.
NMR spectra were recorded on a Bruker AM500 FT-NMR spectrometer using TMS as
an internal Standard. Semi-preparative HPLC was performed on an Agilent 1100
HPLC system including degasser, quaternary pump, autosampler, DAD detector,
J’sphere ODS M-80 column (150 ꢁ 20 mm), and flow rate of 3 mL/min. Silica gel,
reverse phase C-18, and diaion HP-20 were used as adsorbent in flash column
chromatography.
3.2. Plant material
The rhizomes of A. ascalonicum L. were collected in Hanoi City in December 2018, and
identified by Dr. Nguyen The Cuong, Institute of Ecology and Biological Resources. A
voucher specimen (No: NCCT.2019.78) was deposited at the Institute of Marine
Biochemistry, VAST.

6
N. T. MAI ET AL.
3.3. Extraction and isolation
The dried powder of the rhizomes of A. ascalonicum L. (5.0 kg) was sonicated 3 times
with methanol (10 L, each). After removal of solvent, the methanol extract (340 g) was
suspended with water (4 L) and successively separated with dichloromethane to give
dichloromethane residue (AA1, 150 g) and water layer (AA2). The water layer was chro-
matographed on a Diaion HP-20 column, eluting with water, and then methanol/water
(1/3, 1/1, 3/1, and 1/0, v/v) to obtain four fractions W1-W4, respectively. The W2 frac-
tion (38.0 g) was roughly separated on a silica gel column, eluting with CH2Cl2/MeOH
(20/1, 10/1, 5/1, 2.5/1, 1/1, v/v) to give five sub-fractions W2A-W2E, respectively.
Fraction W2C (16.0 g) was chromatographed on a RP-18 column, eluting with acetone/
water (1/1.2, v/v) to give four fractions W2C1-W2C4. Fraction W2C2 (3.5 g) was chroma-
tographed on a silica gel column eluting with dichloromethane/acetone/water (1/4/
0.2, v/v/v) to give two fractions W2C2A and W2C2B. Compounds 2 (3.2 mg) and 5
(3.6 mg) were obtained from W2C2A by HPLC running with ACN in H2O (33%, v/v).
Compounds 9 (3.6 mg) and 10 (9.1 mg) were obtained from fraction W2C2B by HPLC
running with ACN in H2O (40%, v/v). Fraction W2C3 was chromatographed on a silica
gel column eluting with dichloromethane/acetone/water (1/4/0.3, v/v/v) to give three
fractions W2C3A-W2C3C. Fraction W2C3A was chromatographed on HPLC using ACN
in H2O (30%, v/v) to obtained compounds 1 (3.6 mg) and 7 (3.9 mg). Fraction W3
(48 g) was separated on a silica gel column eluting with CH2Cl2/MeOH (20/1, 5/1, 1/1,
v/v) to give three sub-fractions W3A-W3C. Fraction W3A (14.0 g) was chromatographed
on a RP-18 column eluting with acetone/water (1/1.2, v/v) to give four fractions W3A1-
W3A4. Fraction W3A2 was first chromatographed on a RP-18 column chromatography
eluting with acetone/water (4/1, v/v) and then purified on HPLC running with 30%
acetonitrile to yield compounds 3 (4.1 mg) and 6 (6.4 mg). The W3B fraction (15.0 g)
was chromatographed on a RP-18 column eluting with acetone/water (1/1.2, v/v) to
give five fractions W3B2A-W3B2E. Fraction W3B2B (3.5 g) was first chromatographed
on a RP-18 column chromatography eluting with acetone/water (4/1, v/v) and then
purified on HPLC running with 35% acetonitrile to yield compounds 8 (16.6 mg) and
4 (5.0 mg).
3.3.1. Alliumascaside A (1)
25
35
Amorphous powder, ½aꢂD : ꢀ95.0ꢃ (c 0.1, MeOH); HR-ESI-MS: m/z 1093.5370 [Mþ Cl]ꢀ
(Calcd for C53H86O2135Cl, 1093.5350) and 1095.5386 [Mþ Cl]ꢀ (Calcd. for
37
1
C53H86O2137Cl, 1095.5321); H NMR (CD3OD, 500 MHz) dH (ppm): 1.00 (m, Ha-1), 1.63
(m, Hb-1), 1.75 (m, Ha-2), 1.85 (m, Hb-2), 3.14 (dd, J ¼ 4.5, 11.5 Hz, H-3), 0.82 (br d,
11.5 Hz, H-5), 1.45 (m, Ha-6), 1.58 (m, Hb-6), 1.43 (m, Ha-7), 1.51 (m, Hb-7), 1.75 (m, H-9),
1.96 (m, Ha-11), 1.98 (m, Hb-11), 5.35 (br s, H-12), 1.05 (m, Ha-15), 1.80 (m, Hb-15), 1.70
(m, Ha-16), 2.35 (m, Hb-16), 3.02 (br s, H-18), 3.28 (br d, J ¼ 3.5 Hz, H-19), 1.16 (m, Ha-
21), 1.47 (m, Hb-21), 1.66 (m, Ha-22), 1.84 (m, Hb-22), 1.03 (s, H-23), 0.87 (s, H-24), 0.95
(s, H-25), 0.78 (s, H-26), 1.31 (s, H-27), 0.96 (s, H-29), 0.98 (s, H-30), Ara: 4.58 (d, 5.0 Hz,
H-10), 3.79 (dd, J ¼ 5.0, 8.5 Hz, H-20), 3.76 (dd, J ¼ 3.0, 8.5 Hz, H-30), 3.80 (br s, H-40), 3.87
(br d, J ¼ 11.5 Hz, Ha-50), 3.50 (dd, J ¼ 2.5, 11.5 Hz, Hb-50), RhaI: 5.11 (br s, H-100), 3.96 (br
d, J ¼ 3.0 Hz, H-200), 3.70 (dd, J ¼ 3.0, 9.0) Hz, H-300), 3.40 (t, J ¼ 9.0 Hz, H-400), 3.82 (m, H-
500), 1.25 (d, J ¼ 6.0 Hz, H-600), Glc: 5.44 (d, J ¼ 7.5 Hz, H-1000), 3.62 (dd, J ¼ 7.5, 9.0 Hz, H-

NATURAL PRODUCT RESEARCH
7
2000), 3.57 (t, J ¼ 9.0 Hz, H-3000), 3.43 (t, J ¼ 9.0 Hz, H-4000), 3.35 (m, H-5000), 3.78 (dd, J ¼ 2.0,
11.5 Hz, Ha-6000), 3.70 (dd, J ¼ 5.5, 11.5 Hz, Hb-6000), RhaII: 5.39 (br s, H-10000), 3.91 (br d,
J ¼ 3.0 Hz, H-20000), 3.70 (dd, J ¼ 3.0, 9.0 Hz, H-30000), 3.40 (t, J ¼ 9.0 Hz, H-40000), 3.83 (m, H-
5
0000), 1.26 (d, J ¼ 6.0 Hz, H-60000). 13C NMR (CD3OD, 125 MHz) dC (ppm): 39.8 (C-1), 27.2
(C-2), 90.8 (C-3), 40.3 (C-4), 57.2 (C-5), 19.5 (C-6), 33.1 (C-7), 40.9 (C-8), 49.1 (C-9), 38.1
(C-10), 24.7 (C-11), 124.8 (C-12), 144.3 (C-13), 42.8 (C-14), 29.6 (C-15), 28.5 (C-16), 47.3
(C-17), 45.3 (C-18), 82.7 (C-19), 35.9 (C-20), 29.6 (C-21), 34.1 (C-22), 28.5 (C-23), 17.0 (C-
24), 16.0 (C-25), 17.8 (C-26), 24.7 (C-27), 178.5 (C-28), 28.6 (C-29), 25.3 (C-30), Ara: 104.7
(C-10), 76.9 (C-20), 73.0 (C-30), 68.3 (C-40), 64.2 (C-50), RhaI: 102.1 (C-100), 71.9 (C-200), 72.2
(C-300), 73.8 (C-400), 70.2 (C-500), 18.0 (C-600), Glc: 95.3 (C-1000), 77.0 (C-2000), 79.2 (C-3000),
71.4 (C-4000), 78.4 (C-5000), 62.4 (C-6000), RhaII: 101.6 (C-10000), 72.2 (C-20000), 72.2 (C-30000),
73.9 (C-40000), 70.3 (C-50000), 18.2 (C-60000).
3.3.2. Alliumascaside B (2)
25
35
Amorphous powder, ½aꢂD : - 76.0ꢃ (c 0.1, MeOH); HR-ESI-MS: m/z 947.4739 [Mþ Cl]ꢀ
(Calcd. for C47H76O1735Cl, 947.4771) and 949.4755 [Mþ Cl]ꢀ (Calcd. for C47H76O1737Cl,
37
1
949.4742); H NMR (CD3OD, 500 MHz) dH (ppm): 1.01 (m, Ha-1), 1.63 (m, Hb-1), 1.75 (m,
Ha-2)/1.85 (m, Hb-2), 3.13 (dd, J ¼ 4.5, 11.5 Hz, H-3), 0.82 (br d, J ¼ 11.5 Hz, H-5), 1.45
(m, Ha-6), 1.58 (m, Hb-6), 1.32 (m, Ha-7), 1.54 (m, Hb-7), 1.75 (m, H-9), 1.92 (m, Ha-11),
1.97 (m, Hb-11), 5.33 (br s, H-12), 1.06 (m, Ha-15), 1.80 (m, Hb-15), 2.28 (m, Ha-16), 1.64
(m, Hb-16), 3.09 (br s, H-18), 3.28 (br d, J ¼ 3.0 Hz, H-19), 1.66 (m, Ha-21), 1.78 (m, Hb-
21), 1.31 (m, Ha-22), 1.63 (m, Hb-22), 1.05 (s, H-23), 0.88 (s, H-24), 0.96 (s, H-25), 0.77 (s,
H-26), 1.31 (s, H-27), 0.96 (s, H-29), 0.98 (s, H-30), Ara: 4.58 (d, J ¼ 5.0 Hz, H-10), 3.90 (dd,
J ¼ 5.0, 8.5 Hz, H-20), 3.88 (dd, J ¼ 3.0, 8.5 Hz, H-30), 4.04 (br s, H-40), 3.91 (br d,
J ¼ 11.5 Hz, Ha-50), 3.52 (dd, J ¼ 2.5, 11.5 Hz, Hb-50), RhaI: 5.23 (br s, H-100), 3.93 (br d,
J ¼ 3.0 Hz, H-200), 3.73 (dd, J ¼ 3.0, 9.0 Hz, H-300), 3.40 (t, J ¼ 9.0 Hz, H-400), 3.86 (m, H-500),
1.25 (d, J ¼ 6.0 Hz, H-600), Glc: 4.51 (d, J ¼ 7.5 Hz, H-1000), 3.30 (dd, J ¼ 7.5, 9.0 Hz, H-2000),
3.32 (t, J ¼ 9.0 Hz, H-3000), 3.35 (t, J ¼ 9.0 Hz, H-4000), 3.38 (m, H-5000), 3.83 (dd, J ¼ 2.5,
11.5 Hz, Ha-6000), 3.70 (dd, J ¼ 5.5, 11.5 Hz, Hb-6000). 13C NMR (CD3OD, 125 MHz) dC (ppm):
39.9 (C-1), 27.2 (C-2), 89.7 (C-3), 40.4 (C-4), 57.3 (C-5), 19.5 (C-6), 33.1 (C-7), 40.9 (C-8),
49.1 (C-9), 38.1 (C-10), 24.8 (C-11), 124.8 (C-12), 144.7 (C-13), 42.6 (C-14), 29.7 (C-15),
28.6 (C-16), 47.0 (C-17), 45.4 (C-18), 82.6 (C-19), 36.0 (C-20), 29.5 (C-21), 34.0 (C-22),
28.6 (C-23), 17.2 (C-24), 15.9 (C-25), 17.8 (C-26), 25.0 (C-27), (C-28, not detected), 28.7
(C-29), 25.2 (C-30), Ara: 105.1 (C-10), 75.3 (C-20), 81.9 (C-30), 68.5 (C-40), 64.6 (C-50), RhaI:
102.0 (C-100), 72.1 (C-200), 72.1 (C-300), 73.8 (C-400), 70.3 (C-500), 18.0 (C-600), Glc: 104.3 (C-
1000), 75.1 (C-2000), 78.0 (C-3000), 71.2 (C-4000), 78.0 (C-5000), 62.4 (C-6000).
4. Conclusions
Ten triterpene glycosides including alliumascaside A (1) and alliumascaside B (2), lacti-
foloside C (3), lactifoloside H (4), randiasaponin IV (5), kudinoside G (6), ilexkudinoside
W (7), lactifoloside G (8), kudinoside D (9), and ilexkudinoside T (10) were isolated
from the rhizomes of A. ascalonicum. Compounds 1 and 2 were elucidated to be new
compounds. All of the isolated compounds were evaluated for ANO1 inhibitory activity

8
N. T. MAI ET AL.
at a concentration of 30 mM. Compounds 2 and 9 moderately exhibited activity with
inhibition rates of 28.9 0.85% and 26.2 0.65%, respectively.
Disclosure statement
The authors declare no conflict of interest.
Funding
This research is funded by Vietnam National Foundation for Science and Technology
Development (NAFOSTED) under Grant number 104.01-2018.332 and by the National Research
Foundation of Korea (NRF) grant [NRF-2016R1A2B4006742] funded by the Ministry of Education,
Science and Technology, Republic of Korea.
ORCID
SeonJu Park
Seung Hyun Kim
http://orcid.org/0000-0002-0532-5977
http://orcid.org/0000-0003-2613-0674
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