The bioassay-guided isolation of antifungal saponins from Hosta plantaginea leaves

Article abstract of DOI:10.1080/10286020.2017.1329304

Four new steroidal saponins hostaside Ⅰ (1), hostaside Ⅱ (2), hostaside Ⅲ (3), and hostaside Ⅳ (4), together with five known steroidal saponins (5–9), were isolated by the bioassay-guided fractionation from the leaves of Hosta plantaginea (Lam.) Aschers, a worldwide well-known ornamental plant. Hostasides Ⅰ and Ⅱ showed significant antifungal activities, and they could inhibit the growth of Candida albicans and Fusarium oxysporium with MIC values as low as 4?μg/ml.

Full text of DOI:10.1080/10286020.2017.1329304

Journal of Asian Natural Products Research
ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: http://www.tandfonline.com/loi/ganp20
The bioassay-guided isolation of antifungal
saponins from Hosta plantaginea leaves
Meng-Yue Wang, Ying Peng, Chong-Sheng Peng, Jiang-Yuan Qu & Xiao-Bo Li
To cite this article: Meng-Yue Wang, Ying Peng, Chong-Sheng Peng, Jiang-Yuan Qu & Xiao-Bo
Li (2017): The bioassay-guided isolation of antifungal saponins from Hosta plantaginea leaves,
Journal of Asian Natural Products Research, DOI: 10.1080/10286020.2017.1329304
To link to this article: http://dx.doi.org/10.1080/10286020.2017.1329304
Published online: 23 May 2017.
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Date: 24 May 2017, At: 08:30

Journal of asian natural Products research, 2017
https://doi.org/10.1080/10286020.2017.1329304
The bioassay-guided isolation of antifungal saponins from
Hosta plantaginea leaves
Meng-Yue Wang, Ying Peng, Chong-Sheng Peng, Jiang-Yuan Qu and Xiao-Bo Li
school of Pharmacy, shanghai Jiao tong university, shanghai 200240, china
ABSTRACT
ARTICLE HISTORY
received 21 february 2017
accepted 7 May 2017
Four new steroidal saponins hostaside Ⅰ (1), hostaside Ⅱ (2), hostaside
Ⅲ (3), and hostaside Ⅳ (4), together with five known steroidal
saponins (5–9), were isolated by the bioassay-guided fractionation
from the leaves of Hosta plantaginea (Lam.) Aschers, a worldwide
well-known ornamental plant. Hostasides Ⅰ and Ⅱ showed significant
antifungal activities, and they could inhibit the growth of Candida
albicans and Fusarium oxysporium with MIC values as low as 4 μg/ml.
KEYWORDS
liliaceae; Hosta plantaginea;
steroidal saponin; antifungal;
Candida albicans; Fusarium
oxysporium
1. Introduction
Hosta plantaginea (Lam.) Aschers, belonging to genus Hosta (Liliaceae), is a perennial herb
widely cultivated in Europe, Asia, and America as garden plant. Its young leaves and buds
are edible, and the leaves, flowers, and rhizomes are commonly used as important folk med-
icine in China for the treatment of fungal infection, sore throat, mastitis, otitis media, and
so on [1,2]. Recently, some steroidal saponins [3–6] and benzylphenethylamine alkaloids
[7,8] with the antitumor and antivirus activities were found.
In our continuing efforts to find the bioactive constituents from genus Hosta [9–11], some ste-
roidal saponins were isolated from H. plantaginea rhizome. In the present study, the antifungal
constituents in H. plantaginea leaves were investigated for the first time. Using bioassay-guided
fractionation, four new saponins hostasides Ⅰ (1), Ⅱ (2), Ⅲ (3), and Ⅳ (4), together with five
known saponins, were isolated, which were (25R)-2α,3β-dihydroxy-5α-spirostane 3-O-α-L-
rhamnopyranosyl-(1→2)-[β-D-glucopyranosyl-(1→4)]-β-D-galactopyranoside (5) [12], (25R)-
2α,3β,12β-trihydroxy-5α-spirostane 3-O-α-L-rhamnopyranosyl-(1→2)-β-D-galactopyranoside
CONTACT Xiao-Bo li
xbli@sjtu.edu.cn
© 2017 informa uK limited, trading as taylor & francis Group

2
M.-Y. WANG ET AL.
21
R3
26
O
21
25
18 H
O
20
23
18
20
17
22
24
12
17
16
15
11
9
13
19
12
O
H
13
14
11
9
19
14
16
H
HO
O
1
4
OR
H
15
2 OH
HO
O
2
10
1
4
8
7
OR
HO
H
6
H
2
3
10
8
3
5
OH
O
H
6
H
5
7
HO
H
OR1
H
OH
R2
R3
H
R
R1
1
Glu-(1 2) -Glu
Xyl-(1 2) -Glu
H
4 (25S)
2
3
Xyl-(1 2) -Glu
H
O
O
H
H
Glu
Rha
Rha
H
5 (25R)
6 (25R)
O
OH
H
OH
HO
O
HO
H
O
HO
HO
O
O
OH
H
H
H
H
Glu
H
7 (25R)
8 (25R)
OH
O
O
O
OH
HO
HO
O
HO
H
OH
Rha
OH
9
Figure 1. chemical structures of steroidal saponins 1–9.
(6) [13], (25R)-2α,3β-dihydroxy-5α-spirostane 3-O-β-D-glucopyranosyl-(1→4)-β-D-galacto-
pyranoside (7) [12], (25R)-2α,3β-dihydroxy-5α-spirostane 3-O-α-L-rhamnopyranosyl-
(1→2)-β-D-galactopyranoside (8) [4], and (25R)-2α,3β-dihydroxy-5α-spirost-9-en-12-one
3-O-β-D-glucopyranosyl-(1→2)-[β-D-xylopyranosyl-(1→3)]-β-D-glucopyranosyl-(1→4)-β-
D-galactopyranoside (9) [14] (Figure 1).
2. Results and discussion
Compound 1 was obtained as white amorphous powder with the molecular formula
C39H62O18 which was deduced from HR-ESI-MS at m/z 841.3832 [M + Na]+. e 1H NMR
spectrum (Tables 1 and 2) indicated the presence of three methyls (singlets at δH 0.91,
0.87, and 2.23, each 3H), three anomeric protons [δH 4.93 (1H, d, J = 7.7 Hz), 5.15 (1H, d,
J = 7.8 Hz), and 5.22 (1H, d, J = 7.5 Hz)], and one vinylic proton (δH 6.59 (1H, dd, J = 3.1,
1.7 Hz). e 13C NMR spectrum indicated the presence of one carbonyl carbon (δC 196.5)
and two vinylic carbon signals (δC 144.9 and 155.5, respectively). e above data primarily
suggested that 1 was a progesterone triglycoside and its aglycone was further determined
to be 2α,3β-dihydroxy-5α-pregna-16-en-20-one. e α-orientation of C-5 is deduced by
its chemical shif at 44.7. For 5α compounds, the chemical shifs of C-5 appear at 43–46;
while for 5β compounds, the chemical shifs are observed at 35–36.5 [15]. NOESY corre-
lations between H-3 and H-5, H-2 and H-19 indicated the α-equatorial configuration of
H-3 and β-equatorial configuration of H-2. Acid hydrolysis of 1 gave a sapogenin 2α,3β-
dihydroxy-5α-pregna-16-en-20-one (1a) [16] and a sugar portion. Two monosaccharides
identified from the sugar portion by GC analysis were D-glucose and D-galactose in a ratio
of 2:1. e β-anomeric configurations for both glucose and galactose were judged from
their coupling constants (J > 7.0 Hz). e sugar portion was further confirmed to be O-β-
D-glucopyranosyl-(1→2)-β-D-glucopyranosyl-(1→4)-β-D-galactopyranosyl unit through
HMBC correlations of H-1′/C-3, H- 1′′/C-4′, and H-1′′′/C-2′′. us, the structure of 1 was
established as 2α,3β-dihydroxy-5α-pregna-16-en-20-one 3-O-β-D-glucopyranosyl-(1→2)-
β-D-glucopyranosyl-(1→4)-β-D-galactopyranoside, and named hostaside Ⅰ.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
3
Table 1. 1h (600 Mhz) and 13c (150 Mhz) nMr spectral data of the aglycone moieties of compounds 1–4
(δ in ppm, in c5d5n).
1
2
3
4
Position
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
1.14 dd (12.3, 45.5
1.14 dd (12.3,
10.2)
45.5
1.13 dd (12.2,
10.2)
2.14 dd (12.2,
4.6)
3.92–3.93 m
3.88–3.89 m
1.44a
45.6
1.10 dd (12.6,
10.2)
2.18 dd (12.6,
4.8)
3.89−3.90 m
3.82−3.83 m
1.43 dd (10.8,
5.6)
45.6
10.2)
2.15 dd (12.3,
4.8)
2.15 dd (12.3,
4.8)
2
3
4
3.94a
70.4
84.4
3.95a
70.4
84.3
34.1
70.6
84.2
34.2
70.5
84.6
34.1
3.89–3.90 m
3.88–3.89 m
1.45 dd (12.5,
9.0)
1.45 dd (12.5, 34.1
9.0)
1.83 dd (12.5,
1.6)
1.88 dd (12.5,
1.6)
1.86 dd (12.3,
1.6)
1.83 dd (10.8,
4.2)
5
6
0.99a
1.00a
44.7
27.9
0.99a
44.7
27.9
0.98a
44.9
28.0
1.00a
44.7
27.6
1.00a
0.99a
1.01a
1.11–1.14 m
0.72 dd (15.0, 32.0
4.7)
1.11–1.13 m
0.72 dd (15.0,
4.7)
1.10–1.12 m
0.72 dd (15.0,
4.7)
1.08a
7
32.0
32.1
0.62 dd (12.0,
4.2)
32.1
1.51–1.53 m
1.61–1.63 m
0.86–0.88 m
1.51–1.53 m
1.61–1.63 m
0.86–0.88 m
1.51–1.51 m
1.62–1.63 m
0.85–0.87 m
1.57–1.59 m
1.63–1.62 m
0.58 dd (11.2,
4.8)
8
9
33.3
55
33.3
55.0
33.3
55.0
34.5
54.4
10
11
37.1
21.5
37.1
21.5
37.1
21.5
36.9
21.4
1.21–1.23 m
1.38–1.39 m
1.21–1.22 m
1.38–1.39 m
1.43 br.d (14.5)
2.56 dd (14.5,
3.0)
1.21–1.22 m
1.38−1.39 m
1.44a
2.56 dd (14.5,
3.0)
1.08a
1.31–1.32 m
0.94–0.95 m
1.58 d (11.5)
12
1.43 br.d (14.5) 35.3
35.3
35.3
39.9
2.56 dd (14.5,
3.0)
13
14
15
46.6
56.3
46.6
56.3
32.2
46.6
56.3
32.2
40.7
56.2
32.1
1.40–1.41 m
1.40–1.41 m
1.91 dd (12.0,
1.7)
1.40–1.42 m
1.91 dd (12.0,
1.7)
0.80–0.81 m
0.91–0.92 m
1.91 dd (12.0, 32.2
1.7)
2.08–2.11 m
6.59 dd (3.1,
2.08–2.11 m
6.58 dd (3.1,
1.7)
2.08–2.10 m
6.59 dd (3.1,
1.7)
1.41–1.43 m
4.48a
16
144.9
144.9
144.9
81.2
1.7)
17
18
19
20
21
22
23
155.5
16.3
13.3
196.5
27.1
155.5
16.3
13.3
196.5
27.1
155.5
16.3
13.3
196.4
27.0
1.80–1.82 m
0.79 s
62.8
16.6
13.2
42.2
14.7
109.6
26.2
0.91 s
0.87 s
0.92 s
0.87 s
0.91 s
0.86 s
0.68 s
1.83a
2.23 s
2.23 s
2.23 s
1.85 d (6.6)
1.35−1.36 m
1.83a
24
1.29–1.30 m
2.05–2.07 m
26.3
25
26
27.5
65.0
3.98 dd (10.5,
3.4)
3.32 dd (10.5,
10.5)
1.01 d (7.2)
27
16.2
aoverlapped.
Compound 2 was obtained as a white amorphous powder. Its molecular formula was
deduced to be C38H60O17 by HR-ESI-MS at m/z 811.3732 [M + Na]+. e 1H and 13C NMR
spectra indicated that the aglycone of 2 was essentially identical to that of 1. However,
the sugar portion was different from 1 in the lack of a glucopyranosyl unit but the pres-
ence of an xylopyranosyl unit. Acid hydrolysis of 2 afforded D-xylose, D-glucose, and D-
galactose in a ratio of 1:1:1 on the basis of GC analysis. e HMBC correlations of H-1′/C-3,

4
M.-Y. WANG ET AL.
Table 2. 1h (600 Mhz) and 13c (150 Mhz) nMr spectral data of the sugar portions of compounds 1–4 (δ
in ppm, in c5d5n).
1
2
3
4
Position
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
β-D-Gal
4.93 d (7.7)
4.47 dd (9.0,
7.7)
4.18 dd (9.0,
2.7)
β-D-Gal
4.93 d (7.7)
4.47a
β-D-Gal
4.96 d (7.8)
4.52 dd (9.3,
7.8)
4.36 dd (9.3,
3.0)
β-D-Gal
4.94 d (7.7)
4.49a
1′
2′
103.8
72.8
103.8
72.8
103.9
72.2
103.9
72.7
3′
75.5
4.18 dd (9.0,
2.7)
75.5
75.1
4.17 dd (9.6,
2.4)
75.6
4′
5′
6′
4.57 d (2.7)
4.07–4.09 m
4.74 dd (9.6,
9.6)
80.8
75.7
60.4
4.57 (d, 2.7)
4.07–4.08 m
4.74 dd (9.6,
9.6)
4.22 dd (9.6,
4.8)
80.4
75.8
60.4
4.58 d (3.0)
4.12–4.13 m
4.59 dd (9.3,
9.3)
4.32 dd (9.3,
4.2)
70.0
77.2
61.1
4.58 d (2.4)
4.07–4.08 m
4.75 dd (9.6,
9.6)
4.24 dd (9.6,
4.8)
80.4
75.8
60.4
4.22a
β-D-Glu
β-D-Glu
β-D-Glu
1′′
2′′
5. 5.15 d (7.8) 105.2
5.18 d (7.8)
4.13 dd (8.4,
7.8)
105.2
86.5
5.19 d (7.8)
4.14 dd (8.4,
7.8)
105.2
86.5
4.15 dd (8.6,
7.8)
86.2
3′′
4.27 dd (8.6,
8.6)
78.3
4.27 dd (8.4,
8.4)
78
4.27 dd (8.4,
8.4)
78.1
4′′
5′′
6′′
3.96a
71.9
78.1
63.1
3.96a
72.3
78.1
63.3
3.96a
72.3
78.1
63.4
3.95a
4.01a
4.63 d (10.2)
4.11–4.12 m
β-D-xyl
5.08 d (6.0)
4.03–4.05 m
4.02a
4.01a
4.64 d (10.0)
4.11−4.12 m
β-D-xyl
5.09 d (5.8)
4.03–4.04 m
4.02a
4.63 d (10.2)
4.12–4.13 m
β-D-Glu
5.22 d (7.5)
1′′′
2′′′
3′′′
107.0
104.8
76.5
77.9
104.7
76.5
77.9
4.06 d (9.0, 7.2) 76.6
4.12 dd (9.0,
9.0)
77.5
4′′′
5′′′
4.22a
70.3
78.8
3.95a
4.48a
70.4
67.4
3.96a
4.49a
70.4
67.4
3.80–3.81 m
3.67 d (10.6)
3.68 d (11.0)
6′′′
4.60 dd (12.4,
3.2)
61.5
4.37 dd (12.4,
3.6)
aoverlapped.
Table 3. the Mic values (μg/ml) of steroidal saponins against Candida albicans and Fusarium oxysporium.
Sample
Candida albicans
Fusarium oxysporium
1
2
4
4
8
8
3
9
32
16
4
64
>64
16
nystatin
H-1′′/C-4′, and H-1′′′/C-2′′ indicated the sugar portion to be O-β-D-xylopyranosyl-
(1→2)-β-D-glucopyranosyl-(1→4)-β-D-galactopyranosyl unit. us, the structure of 2 was
established as 2α,3β-dihydroxy-5α-pregna-16-en-20-one 3-O-β-D-xylopyranosyl-(1→2)-
β-D-glucopyranosyl-(1→4)-β-D-galactopyranoside, and named hostaside Ⅱ.
Compound 3 was obtained as a white needle solid. Its HR-ESI-MS gave a quasi-molecular
ion at m/z 517.2774 [M + Na]+, corresponding to the molecular formula C27H42O8. e 1H
and 13C NMR spectra revealed that the aglycone of 3 was essentially identical to that of 1,
and 3 only possessed a galactopyranosyl (Tables 1 and 2). Acid hydrolysis of 3 afforded 1a

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
5
and D-galactose. e HMBC experiment showed correlation between the proton signal
at δH 4.96 (H-1′, galactosyl group) and the carbon signal at δC 84.2 (C-3, aglycone). us,
the structure of 3 was elucidated as 2α,3β-dihydroxy-5α-pregna-16-en-20-one 3-O-β-D-
galactopyranoside, and named hostaside Ⅲ.
Compound 4 was obtained as white amorphous powder with the molecular formula
C44H70O18, deduced from the HR-ESI-MS at m/z 909.4458 [M + Na]+. e 1H NMR spec-
trum indicated the presence of two tertiary methyl groups [δH 0.79 and 0.68 (each 3H, s)],
two secondary methyl groups [δH 1.85 (d) and 1.01 (d)], and three anomeric proton [δH
4.94, 5.19, and 5.09 (each 1H, d)] signals. So, 4 was determined to be a steroidal triglycoside
with (25S)-2α,3β-dihydroxy-5α-spirostane (neogitogenin) as aglycone. e 25S configura-
tion was deduced through the comparison of characteristic chemical shifs for C-23, C-24,
C-25, C-26, and C-27 with those data reported [17]. However, acid hydrolysis of 4 gave both
neogitogenin with 25 S-configuration and gitogenin with 25 R-configuration in a ratio of
1:1. Since 25 S steroidal aglycones were known to easily change to 25 R aglycones with acid
treatment [18], the configuration at the 25-position of 4 was primarily deduced to be S.
e further enzymatic hydrolysis of 4 only gave neogitogenin (4a) [19], confirming that the
aglycone of 4 was 25 S neogitogenin. Acid hydrolysis of 4 also gave D-xylose, D-glucose, and
D-galactose in a ratio of 1:1:1 on the basis of GC analysis. e β-anomeric configurations
for xylose, glucose, and galactose were judged from their coupling constants. e sugar
portion was further confirmed to be O-β-D-xylopyranosyl-(1→2)-β-D-glucopyranosyl-
1
(1→4)-β-D-galactopyranosyl unit by comparing the H and 13C NMR spectra with those
of 2 and confirmed through HMBC correlations of H- 1′/C-3, H-1′′/C-4′, and H-1′′′/C-2′′.
us, the structure of 4 was established as (25S)-2α,3β-dihydroxy-5α-spirostane 3-O-β-
D-xylopyranosyl-(1→2)-β-D-glucopyranosyl-(1→4)-β-D-galactopyranoside, and named
hostaside Ⅳ.
Saponins 1–3 with progesterone as aglycone and 9 with manogenin as aglycone were all
observed to significantly inhibit the growth of fungi Candida albicans and Fusarium oxyspo-
rium (Table 3). However, their aglycones (1a and 4a) and the saponins with spirostanol
and isospirostanol as aglycones (4–8) didn’t show the obvious activity. e results revealed
that the antifungal activity of saponins was related to both the structures of aglycones and
the sugar chains.
3. Experimental
3.1. General experimental procedures
Melting point was determined on an XT4A micro melting point apparatus (unchecked,
Shanghai Iwise Scientific Instrument Co., Ltd., Shanghai, China). Optical rotations were
measured on a JASCO P-2000 polarimeter (JASCO, Tokyo, Japan). UV spectra were meas-
ured on a UV1102 spectrophotometer (Shanghai Tianmei Scientific Instrument Co., Ltd.,
Shanghai, China). IR spectra were recorded on an Equinox 55 FTIR spectrophotometer
(Bruker, Karlsruhe, Germany). ESI-MS analyses were achieved on an Agilent 6410 tri-
ple-quad mass spectrometer (Agilent, Santa Clara, USA). GC analysis was performed on
a Shimadzu GC-2010 gas chromatograph (Shimadzu, Tokyo, Japan) equipped with an H2
flame ionization detector and a DB-5 quartz capillary column (30 m × 0.25 mm × 0.25 μm).
1H and 13C NMR spectra were recorded on Bruker Avance DRX-600 spectrometers with a

6
M.-Y. WANG ET AL.
5 mm 13C/1H/15N TCI CryoProbe (Bruker, Karlsruhe, Germany). HPD 100 macroporous
resin (16–60 mesh, Tianjin Haiguang Chemical Group Co., Ltd., Tianjin, China), silica
gel (200–300 mesh, Qingdao Haiyang Chemical Group Co., Ltd., Qingdao, China), and
ODS (40–63 μm, Merck, Darmstadt, Germany) were used for column chromatography
(CC). Nystatin and naringinase were purchased from China National Medicines CO. Ltd
(Shanghai, China). L-cysteine methyl ester and hydrochloride and 1-trimethylsilylimida-
zole were purchased from Sigma-Aldrich (St. Louis, USA). Potato dextrose agar media
C. albicans and F. oxysporium were provided by Shanghai Institute of Pharmaceutical
Industry. Preparative HPLC chromatography was performed on a Water 2535 preparative
liquid chromatography system (Waters, Milford, USA), equipped with an auto-sampler,
an Xbridge Prep C18 OBD column (19.0 × 150 mm, 5 μm), and a WFC III collector. Semi-
preparative HPLC chromatography was performed on a Shimadzu LC 2010 AHT liquid
chromatography system (Shimadzu, Tokyo, Japan), equipped with a Nova-Pak HR C18
column (7.9 × 300 mm, 6 μm).
3.2. Plant material
e leaves of Hosta plantaginea were collected in July 2013 from Chongqing, China, and
authenticated by one of the authors (Mengyue Wang). A voucher specimen (CQ20130722)
has been deposited at the herbarium at the School of Pharmacy, Shanghai Jiao Tong
University, Shanghai, China.
3.3. Bioassay-guided isolation
e air-dried leaves of H. plantaginea (1.2 kg) were powdered and extracted by refluxing with
10 L 95% alcohol for three times (2 h each). e alcohol extract was completely evaporated
under reduced pressure to afford a residue (A, 260 g). Part of A (255 g) was suspended in
water (750 ml), and then successively partitioned with CH2Cl2, EtOAc, and n-BuOH (each
500 ml × 3). e CH2Cl2, EtOAc，and n-BuOH layers were dried under vacuum to afford
CH2Cl2 fraction (CA, 54 g), EtOAc fraction (EA, 31 g), and n-BuOH fraction (BA, 63 g),
respectively. e water layer remained was dried under vacuum to afford water layer fraction
(WA, 104 g). Fractions A, CA, EA, BA, and WA were subjected to the antifungal (C. albicans
and F. oxysporium) evaluation by agar diffusion method at the same concentration of 100 μg/
ml in methanol [20]. e most active fraction BA was subjected to the further fractionation.
Part of BA (60 g) was fractionated by macroporous resin CC (8.0 × 90 cm) successively
eluted with water, 30, 60, and 90% ethanol (2000 ml each). e eluents were completely
evaporated under vacuum to afford water portion (BA0, 6.9 g), 30% ethanol portion (BA30,
27.4 g), 60% ethanol portion (BA60, 17.3 g), and 90% ethanol portion (BA90, 7.4 g), respec-
tively. Portions BA0, BA30, BA60, and BA90 were subjected to the antifungal evaluation at
the same concentrations of 100 μg/ml again. e most active portion BA60 was subjected
to the further chemical investigation.
Part of BA60 (15.4 g) was fractionated by ODS CC (5.5 × 60 cm; MeOH/H2O; 20:80 to
75:25) to give Frs.1–21 (200 ml each). Fr.3 was further purified by silica gel CC (CH2Cl2/
MeOH/H2O; 60:35:2) to obtain 9 (146 mg). Fr.9 and Fr.10 were combined and further puri-
fied by prep. HPLC (MeCN/H2O 20:80; 8.0 ml/min; 210 nm) to obtain fraction Frs.9.1–9.4
(collected by peaks). Fr.9.3 was further purified by semi-prep. HPLC (MeCN/H2O 20:80;

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
7
3.0 ml/min; 210 nm) to afford 1 (tR 10.3 min, 31 mg) and 2 (tR 11.1 min, 40 mg). Fr.14
was further purified by ODS CC (3.5 × 60 cm; MeCN/H2O 35:65 to 55:45) to afford 4
(23 mg) and 5 (78 mg). Fr.17 and Fr.18 were combined and further purified on silica gel
CC (CH2Cl2/MeOH 9:1 to 8:2) to obtain 6 (31 mg), 7 (20 mg), and 8 (19 mg). Fr.20 was
further purified by semi-preparative HPLC (MeCN/H2O 30:70; 2.0 ml/min; 210 nm) to
afford 3 (tR 18.7 min, 32 mg).
3.3.1. Hostaside Ⅰ (1)
White amorphous powder (MeOH); mp 271–273 °C; [ꢀ]2D0 −18.8 (c 0.10, MeOH); UV
(MeOH) λmax (log ε): 240 (3.74) nm; IR (KBr) νmax: 3407 (OH), 2932 (CH), 1660 (C = O),
1
1585 (C = C), 1380, 1232, 1164, 1074, 924, 892 cm−1; H and 13C NMR spectral data see
Tables 1 and 2. HR-ESI-MS: m/z 841.3832 [M + Na]+ (calcd for C39H62O18Na, 841.3834).
3.3.2. Hostaside Ⅱ (2)
White power (MeOH); mp 266–267 °C; [ꢀ]2D0 −15.6 (c 0.10, MeOH); UV (MeOH) λmax
(log ε): 239 (3.77) nm; IR (KBr) νmax: 3405 (OH), 2931 (CH), 1660 (C=O), 1585 (C=C),
1379, 1231, 1162, 1073, 924, 892 cm−1; 1H and 13C NMR spectral data see Tables 1 and 2.
HR-ESI-MS: m/z 811.3732 [M + Na]+ (calcd for C38H60O17Na, 811.3728).
3.3.3. Hostaside Ⅲ (3)
White amorphous powder; mp 245–247 °C; [ꢀ]2D0 −7.0 (c 0.10, MeOH); UV (MeOH) λmax
(log ε): 239 (3.98) nm; IR (KBr) νmax: 3402 (OH), 2931(CH), 1659 (C=O), 1584 (C=C),
1375, 1230, 1163, 1074, 924, 892 cm−1; 1H and 13C NMR spectral data see Tables 1 and 2.
HR-ESI-MS: m/z 517.2774 [M + Na]+ (calcd for C27H42O8Na, 517.2769).
3.3.4. Hostaside Ⅳ (4)
White powder; mp 275–277 °C; [ꢀ]D20 −11.3 (c 0.10, MeOH); IR (KBr) νmax: 3410 (OH),
2935 (CH), 1452, 1378, 1240, 1057, 980, 921, 898, 865 cm−1 (intensity: 898 > 921); 1H and
13C NMR spectral data see Tables 1 and 2. HR-ESI-MS: m/z 909.4458 [M + Na]+ (calcd for
C44H70O18Na, 909.4462).
3.4. Acid hydrolysis of 1, 2, 3, and 4
A solution of sample (9 mg) in HCl/MeOH (4:1, 10 ml) was refluxed at 90 °C for 2 h.
en, the reaction mixture was diluted with H2O and extracted with CH2Cl2 (15 ml × 2).
e CH2Cl2 layer was washed with water to neutrality, concentrated under vacuum and
then subjected to silica gel CC (CH2Cl2/MeOH 95:5) to give aglycone. e water layer was
neutralized with 10% NaOH solution and concentrated in vacuo to dryness to give sugar
fraction. e sugar fraction was dissolved in anhydrous pyridine (2.0 ml) followed by adding
of L-cysteine methyl ester hydrochloride (5 mg). e mixture was stirred at 60 °C for 1.5 h
and trimethylsilylated with 1-trimethylsilylimidazole (0.3 ml) for 2 h. e mixture was par-
titioned between n-hexane and H2O (2 ml each), and the organic layer was analyzed by GC
(column temperature, 100/280 °C, programmed increase, 10 °C/min; carrier gas, N2, 1.5 ml/
min; injector and detector temperature, 280 °C; injection volume, 1 μl; split ratio, 10:1) [3].
Identification of D-glucose, D-galactose, and D-xylose was detected by co-injection of the
hydrolysate with standard silylated samples.

8
M.-Y. WANG ET AL.
3.5. Enzymatic hydrolysis of 4
Compound 4 (7.5 mg) was treated with naringinase (90.0 mg) in HCOOH/KOAc buffer
(pH 4.5, 3.0 ml) at room temperature for 5 days. e reaction mixture was purified by CC
on silica gel (2 × 15 cm) eluted with CH2Cl2/MeOH (95:5) to yield aglycone (1.4 mg).
3.6. Antifungal activity assay of saponins
e primary antifungal screening was carried out using agar disk-diffusion, at an initial
concentration of 64 μg/ml in methanol [20]. e active compounds with inhibition zones
more than 10 mm were subjected to the further MIC investigation by the microdilution
method [21].
Acknowledgments
e authors are really grateful to Dr. Bona Dai of the Instrumental Analysis Center of Shanghai Jiao
Tong University, for the measurements of NMR.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
is study was financially supported by a grant of National Natural Science Fund of China [grant
number 81374067].
References
[1] J.W. He, L. Yang, J.X. Zhu, X.M. Wang, Z.R. Zou, W.W. He, and G.Y. Zhong, J. Jiangxi Norm.
Univ. (Nat. Sci.). 40, 183 (2016).
[2] Y. Xin and Y.H. Bai, Chin. Tradit. Patent Med. 37, 653 (2015).
[3] X.J. Li, L. Wang, P.F. Xue, H.X. Xie, H. Wei, and J. Wang, J. Asian Nat. Prod. Res. 17, 224 (2015).
[4] J.Q. Liu, C.F. Wang, M.H. Qiu, and W.X. Hu, Chin. Tradit. Herb. Drugs. 41, 520 (2010).
[5] J.H. Zhang, H.X. Xie, P.F. Xue, H.G. Zhang, X.Y. Liu, and J.M.S. Bai, Chin. Pharm. J. 45, 335
(2010).
[6] H.X. Xie, J.H. Zhang, H.G. Zhang, and P.F. Xue, Chin. Pharm. J. 44, 733 (2009).
[7] Y.H. Wang, Z.K. Zhang, F.M. Yang, Q.Y. Sun, H.P. He, Y.T. Di, S.Z. Mu, Y. Lu, Y. Chang, Q.T.
Zheng, M. Ding, J.H. Dong, and X.J. Hao, J. Nat. Prod. 70, 1458 (2007).
[8] Y.H. Wang, S. Gao, F.M. Yang, Q.Y. Sun, J.S. Wang, H.Y. Liu, C.S. Li, Y.T. Di, S.L. Li, H.P. He,
and X.J. Hao, Org. Lett. 9, 5279 (2007).
[9] R. Li, M.Y. Wang, and X.B. Li, J. Med. Plants Res. 6, 2704 (2012).
[10] J.Y. Qu, M.Y. Wang, C.M. Wang, G.Y. Zhong, and X.B. Li, Chin. Tradit. Herb. Drugs. 42, 217
(2011).
[11] M.Y. Wang, Z.H. Xu, Y. Peng, G.Y. Zhong, and X.B. Li, Chem. Nat. Compd. 52, 1047 (2016).
[12] Y. Mimaki, T. Kanmoto, M. Kuroda, Y. Sashida, Y. Satomi, A. Nishino, and H. Nishino,
Phytochemistry 42, 1065 (1996).
[13] Y. Mimaki, M. Kuroda, A. Kameyama, A. Yokosuka, and Y. Sashida, Phytochemistry 48, 1361
(1998).

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
9
[14] Y. Mimaki, T. Kanmoto, M. Kuroda, Y. Sashida, A. Nishino, Y. Satomi, and H. Nishino, Chem.
Pharm. Bull. 43, 1190 (1995).
[15] I. Kostova and D. Dinchev, Phytochem. Rev. 4, 111 (2005).
[16] M.Y. Wang, Z.H. Xu, Y. Peng, G.Y. Zhong, and X.B. Li, Chem. Nat. Compd. 45, 1047 (2016).
[17] P.K. Agrawal, D.C. Jain, and A.K. Pathak, Magn. Reson. Chem. 33, 923 (1995).
[18] M. Yoshikawa, T. Murakami, H. Komatsu, J. Yamahara, and H. Matsuda, Heterocycles 47, 397
(1998).
[19] A. Temraz, O.D. EI Gindi, H.A. Kadry, N.D. Tommasi, and A. Braca, Phytochemistry 67, 1011
(2006).
[20] X.L. Xu, L.Y. Yin, J.H. Gao, L.J. Gao, and F.H. Song, Chem. Biodivers. 11, 807 (2014).
[21] R.S. Cong, W.P. Yuan, T.J. Fan, R.C. Guo, Z. Xuan, C.Y. Zhang, and H.K. Cai, Period. Ocean
Univ. Chin. 36, 959 (2006).