Microbial transformation of pseudoprotodioscin by Gibberella fujikuroi

Article abstract of DOI:10.1080/10286020.2018.1468438

Three new (6, 9, and 12) and nine known steroidal saponins were obtained from the fermentation broth of pseudoprotodioscin (PPD) incubated with a fungus Gibberella fujikuroi CGMCC 3.4663. Structures of the metabolites were elucidated by 1-D (¹H, ¹³C), 2-D (HMBC, HSQC, NOESY) NMR, and HR-MS analyses. The biotransformation pathway of pseudoprotodioscin by Gibberella fujikuroi CGMCC 3.4663 was proposed. Compounds 1–11 were tested in vitro for their cytotoxic activities against two human cancer cell lines (HepG2 and Hela). Compounds 1, 6, 9, and 10 exhibited cytotoxic activity against HepG2 cells. Compound 10 exhibited cytotoxicity to Hela cells.

Full text of DOI:10.1080/10286020.2018.1468438

Journal of Asian Natural Products Research
ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: http://www.tandfonline.com/loi/ganp20
Microbial transformation of pseudoprotodioscin
by Gibberella fujikuroi
Hong-Xiu Hu, Ran-Ran Gao, Zhao-Hui Gao, Yue Qiao, Xin-Ran Dong, Gang
Ding & Di-An Sun
To cite this article: Hong-Xiu Hu, Ran-Ran Gao, Zhao-Hui Gao, Yue Qiao, Xin-Ran Dong, Gang
Ding & Di-An Sun (2018): Microbial transformation of pseudoprotodioscin by Gibberella fujikuroi,
Journal of Asian Natural Products Research
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Journal of asian natural Products research, 2018
https://doi.org/10.1080/10286020.2018.1468438
Microbial transformation of pseudoprotodioscin by Gibberella
fujikuroi
Hong-Xiu Hu#, Ran-Ran Gao#, Zhao-Hui Gao, Yue Qiao, Xin-Ran Dong, Gang Ding and
Di-An Sun
natural Medicine chemistry research center, institute of Medicinal Plant development, chinese academy of
Medical sciences & Peking union Medical college, Beijing 100193, china
ABSTRACT
ARTICLE HISTORY
received 29 January 2018
accepted 19 april 2018
Three new (6, 9, and 12) and nine known steroidal saponins were
obtained from the fermentation broth of pseudoprotodioscin (PPD)
incubated with a fungus Gibberella fujikuroi CGMCC 3.4663. Structures
of the metabolites were elucidated by 1-D (1H, 13C), 2-D (HMBC, HSQC,
NOESY) NMR, and HR-MS analyses. The biotransformation pathway
of pseudoprotodioscin by Gibberella fujikuroi CGMCC 3.4663 was
proposed. Compounds 1–11 were tested in vitro for their cytotoxic
activities against two human cancer cell lines (HepG2 and Hela).
Compounds 1, 6, 9, and 10 exhibited cytotoxic activity against HepG2
cells. Compound 10 exhibited cytotoxicity to Hela cells.
KEYWORDS
Biotransformation;
pseudoprotodioscin;
Gibberella fujikuroi; steroidal
saponins; cytotoxic activity;
micro-organism
1. Introduction
Herbs and natural products have been used to treat disease since ancient time. Plant-
derived steroidal saponins are mostly found in the plant families Dioscoreaceae, Liliaceae,
Agavaceae, Allaceae, and so on, which had remained attractive to scientists and doctors
owing to their multiple therapeutic effects [1]. Previous studies showed steroidal sapon-
ins exhibited a wide range of pharmacological activities such as antiplatelet aggregation,
antidiabetes, cytotoxicity, and anticancer [2–5]. Pseudoprotodioscin (PPD) was a steroidal
saponin mainly isolated from traditional Chinese herbal Dioscorea panthaica Prain et Burkill
or Dioscorea nipponica Makino, which has been put into clinic for remedying patients with
symptom of myocardial ischemia in China for many years. Microbial biotransformation
CONTACT di-an sun
diansun@sina.com
#these authors contributed equally to this work.
the supplemental data for this article is available online at https://doi.org/10.1080/10286020.2018.1468438.
© 2018 informa uK limited, trading as taylor & francis Group

2
H.-X. HU ET AL.
has attracted extensive attention in the field of pharmaceutical research [6,7]. rough their
unique and complex multi-enzyme catalytic systems including hydroxylation, oxidation,
reduction, and introduction of functional groups, microbial transformation can generate
some compounds that were impossible to be obtained by traditional chemical modification
[8–10]. In this aspect, the microbial biotransformation shows obvious advantages over
conventional chemical synthesis in novel candidate screening and bulk amount yield of
complicate structural compounds [11–13].
To obtain derivatives with higher activity and less toxicity, microbial biotransformation
of pseudoprotodioscin was carried out. Twelve steroidal saponins (1–12) were isolated and
identified from the fermentation broth (Figure 1) of pseudoprotodioscin incubated with
Gibberella fujikuroi CGMCC 3.4663. We reported in this article the isolation, structural
elucidation, and cytotoxic activities of metabolites. e biotransformation pathway of pseu-
doprotodioscin by Gibberella fujikuroi CGMCC 3.4663 was also proposed.
2. Result and discussion
2.1. The structure elucidation of new products
e fermented broth was extracted with an equal volume of n-butyl alcohol for three times.
e crude extract was subjected to column chromatography on silica gel, Sephadex LH-20,
and semi-preparative HPLC to afford compounds 1–12 (Figure 1), including three new
compounds (6, 9, and 12) and nine known compounds. Compounds 1, 2, and 8 were
reported before in our group as microbial transformation products of pseudoprotodioscin
by Chaetomium olivaceum [14]. Compounds 3, 4, 5, 7, 10, and 11 were known compounds.
e data of them can be found in the reported literature [15–20].
Compound 6 was isolated as a white amorphous powder. e molecular formula of
C39H62O14 was deduced from the [M + Na]+ ion at m/z 777.4059. e 13C NMR spectrum
of 6 showed 39 signals. Since there were two anomeric proton signals appearing at δ 4.98
(1H, o), δ 5.93 (1H, s) in the 1H-NMR spectrum and two anomeric carbons observed at
δ 102.9, 103.2 in the 13C-NMR spectrum, 12 carbon signals were attributed to two sugar
moieties. e 26-glucose signals disappeared. e other 27 carbon signals were assigned
to the aglycone moiety. e NMR data of aglycone moiety were obviously different from
that of the substrate. e signals of double bond between C-20 and C-22 in the substrate
were replaced by the carbonyl groups (δ 205.9, C-20; δ 173.8, C-22) in compound 6. It was
confirmed by the long-range correlations of H-21 (δ 2.15) with C-17 (δ 67.2) and C-20 (δ
205.9) and correlations of C-22 (δ 173.8) with H-16 (δ 5.71–5.73), H-23 (δ 2.51, δ 2.55), and
H-24 (δ 2.12) in the HMBC spectrum (Figure 2). A cross-peak between H-16 and H-17 and
the cross-peak between H-14 and H-17 could be found in the NOESY spectrum (Figure 3).
us, C-22 was on the β-position. According to the chemical shifs of the two proton
signals H-26 a (δ 3.62) and H-26 b (δ 3.70) (δ ab = 0.02 < 0.48), the C-25 configuration
was deduced to be R [21,22]. e structure of compound 6 was elucidated as 16β-(4′R-
methyl-5′-ol-pentanoxyl)-pregn-5-ene-3β-ol-20-one-3-O-α-L-rhamnopyranosyl-(1→4)-
β-D-glucopyranoside.
Compound 9 was isolated as a white amorphous powder. Its molecular formula of
C39H60O12 was deduced from the [M + Na]+ ion at m/z 743.3973. It had two mass units less
than that of compound 11. When comparing its NMR data with that of compound 11, it lost

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
3

4
H.-X. HU ET AL.
Figure 2. Key hMBc correlations in compounds 6, 9, 12.
Figure 3. Key noesY correlations in compounds 6 and 12.
one methyl group. In the meantime, it has one more double bond. Since the double between
C5–C6 was unchanged, the other double bond was assigned to C20–C21. is assignment
was confirmed by the long-range correlation of H-21 (δ 5.32, δ 5.09) with C-20 (δ 153.2),
C-22 (δ 107.7), and C-17 (δ 60.6) in the HMBC spectrum. us, compound 9 was elucidated
as 25R-spirost-5, 20-diene-3β-ol-3-O-α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranoside.
Compound 12 was isolated as a white amorphous powder. e molecular formula
was assigned as C39H62O13 based on its HR-ESI-MS data that displayed an [M + Na]+
ion at m/z 761.4096. e 13C NMR spectrum of compound 12 showed 39 signals and
two anomeric carbons were observed at δ 102.7, 102.5. is compound has the same
molecular formula as that of compound 8. e 13C NMR data of compounds 8 and 12
were very similar. e proton NMR data of these two compounds were also very simi-
lar except for the chemical shifs of the Me groups (δ 1.75 and δ 1.53). A singlet methyl
signal was observed at δ 1.75 (21-CH3) which had long-range correlations with C-20 (δ
82.7) and C-22 (δ 110.7). It was deduced that a hydroxyl group was attached at C-20 posi-
tion. In the NOESY spectrum, a cross peak between H-21 (δ 1.75) and H-18 (δ 1.21)
indicated that the–OH was on the α-position. According to the chemical shifs of H-26
a (δ 3.46) and H-26 b (δ 4.31–4.33) (δ ab = 0.86 > 0.48), the C-25 configuration was
deduced to be S. Based on the above data, the structure of compound 12 was established
as 25S-spirost-5-ene-3β,20α-diol-3-O-α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranoside.
2.2. The possible transformation pathway
According to the structures of the obtained compounds and the literature [14,17,23,24], we
proposed the possible transformation pathway of pseudoprotodioscin by Gibberella fujikuroi

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
5
CGMCC 3.4663 as shown in Figure 1. e 1–2 linked rhamnose of the substrate was first
hydrolyzed to form the possible intermediate. is intermediate was then transformed to
other metabolites through four directions.
e first direction was the epoxidation of intermediate I to form intermediate II.
Intermediate II was then transformed to other products through four directions. Intermediate
III was formed through the addition of a methoxy group at C-20 position of II. Product 1
could be formed through III by loss of one molecular water. A hydroxyl group was added
on II to form intermediates IV and V. ey were unstable and were transformed to com-
pounds 3, 8, 9, 12 through the cleavage of the 26-glucose, cyclization, dehyroxylation, and
isomerization as shown in Figure 1. Compound 2 could be obtained with the addition of
a methoxy group at C-22 of II.
e second direction was the cleavage of 26-glucose and the oxidative cleavage of C20
(22) double bond to afford compound 6. e ester bond of compound 6 was hydrolyzed
to form intermediate VI. is intermediate was then transformed to compound 7 by loss
of one molecular water.
e third direction was the direct hydroxylation at C-22 to afford compound 4.
Compound 10 could be formed through the cleavage of 26-glucose and cyclization reaction
of compound 4. Intermediate VII was transformed to compound 11 through the cleavage
of 26-glucose and cyclization.
e fourth direction was the methoxylation at C-22 to afford compound 5.
e biotransformation process reported here was very complicated with oxidation,
methoxylation, hydroxylation, and other reactions involved. It enriched steroidal saponins
structural types and provided a possible new way for steroid hormone producing. It also
provided new clues for the investigation of new enzymes of this fungus.
2.3. Cytotoxic activities
Compounds 1–11 were evaluated for cytotoxic activities against two kinds of human can-
cer cell lines (HepG2 and Hela), with polyphyllin I as a positive control. e IC50 values of
polyphyllin I was 3.2 0.3 μM against HepG2 cells and was 4.3 0.5 μM against Hela cells,
respectively. Compound 10 exhibited cytotoxicity (IC50<100 μM) to Hela cells with IC50
value of 30.97 0.5 μM. Compounds 1, 6, 9, 10 exhibited cytotoxic activity against HepG2
cells with IC50 values of 68.6 0.6, 94.3 1.1, 46.3 0.8, 22.1 0.3 μM, respectively. By
comparing the structures of the compounds 10 and 11, it was deduced that the orientation
of Me-21 in the structure could influence the toxic effect on the HepG2 cells. e saponin
10 with α-orientated Me-21 (20R) showed strong cytotoxicity while no cytotoxicity was
observed in compound 11 with β-orientated Me-21 (20S).
3. Experimental
3.1. General experimental procedures
e optical rotations were measured in methanol in a Perkin-Elmer 341 polarimeter (Perkin-
Elmer, Massachusetts, U.S.A.). Infrared (IR) spectra were recorded with a SHIMADZU
FTIR-8400S spectrophotometer (Tokyo, Japan). High-resolution electrospray ionization
mass spectra (HR–ESI–MS) were measured on ermo LTQ Orbitrap XL mass spectrometer

6
H.-X. HU ET AL.
(ermo Fisher Scientific, Bremen, Germany). Nuclear magnetic resonance (NMR) spec-
tra were performed on Bruker DRX-600 spectrometer (Bruker BioSpin, Rheinstetten,
1
Germany), 600 MHz for H NMR and 150 MHz for 13C NMR in pyridine-d5 (Sigma–
Aldrich, U.S.A.), the chemical shifs were given in δ (ppm). Pre-coated silica gel GF254
plates (Qingdao Oceanic Chemicals, Qingdao, China) were used to perform thin layer
chromatography (TLC). Silica gel (200–300 mesh, Qingdao Oceanic Chemicals, Qingdao,
China) was used for column chromatography. Semi-preparative HPLC was carried out on
a SHIMADZU system (Tokyo, Japan) with a LC-6AD pump, a SPD-20A UV detector, and
a Dr. Maisch GmbH Reprosil 100 C18 column (5 μm, 10 × 250 mm) with H2O–MeOH as
mobile phase and UV detector at 210 nm.
Dulbecco’s modified eagle medium (DMEM) and antibiotics (penicillin and streptomy-
cin) were purchased from Gibco (Grand Island, NY, U.S.A.). Phosphate buffered saline (PBS)
and trypsin were obtained from Solarbio (Beijing, China). Fetal bovine serum (FBS) was
purchased from Zhejiang Tianhang Biotechnology Co., Ltd. (Hangzhou, China). 96-Well
plates were obtained from Corning Costar (CorningCostar, Cambridge, MA, U.S.A.).
Human hepatocarcinoma cells (HepG2) and Human cervical cancer cell line (Hela) were
originally obtained from American Type Culture Collection (ATCC, Manassas, VA, U.S.A.).
e cells were maintained in a CO2 incubator (RevcoAshiville, NC, U.S.A.). Absorbance
was read on Bio-rad Model 450 (Bio-Rad Laboratories, CA, U.S.A.) microplate reader. All
other chemicals used were of biochemical regent grade.
3.2. Micro-organism
e fungal strain Gibberella fujikuroi CGMCC 3.4663 used in the experiment was purchased
from the China General Microbiological Culture Collection Center (CGMCC, Beijing,
China) and was cultured in the potato broth medium.
3.3. The substrate and culture medium
Pseudoprotodioscin was isolated and purified from Di-ao-xin-xue-kang capsules in our
lab. e biotransformation was performed in potato medium. e culture medium was
prepared by the following procedure: 1000 ml distilled water was added to 200 g mincing
husked potato and was boiled for half an hour before it was filtered. en 20 g glucose
and distilled water were added to the filtrate to the volume of 1000 ml. e medium was
sterilized in an autoclave at 121 °C for half an hour before use.
3.4. Biotransformation procedure
e biotransformation of pseudoprotodioscin was carried in 1000 ml Erlenmeyer flasks,
each containing 400 ml culture medium. Micro-organism was added to the flasks and
incubated on a rotary shaker at 160 rpm at 27 °C for 48 h, and to each flask 60 mg substrate
(dissolved in 0.1 ml methanol) was added. e biotransformation was carried out in 20
flasks (1000 ml). A total of 1200 mg substrate was equally added to flasks. e reaction was
maintained for another 5 days. e cultures were ultrasonically extracted for three times
(30 min each) with an equal volume of n-butyl alcohol. e extracts were evaporated (52 °C)
under decreased pressure on a rotary evaporator and were analyzed by TLC. Two controls

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
7
were set under the same conditions: substrate control that had the substrate but no fungus
and culture control that had the fungus but no substrate.
3.5. Isolation and purification of transformed products
A total of 5.76 g crude extract was obtained from the fermented broth. It was first purified
by silica gel (200–300 mesh 140 g) column and was eluted with CHCl3–MeOH–H2O from
12:1:0.05 (v/v/v) to 1:1:0.1 (v/v/v). irteen fractions (1–13) were obtained by TLC analysis.
Fraction 6 (0.908 g) was further purified on a silica gel column (300–400 mesh, 30 g) with
a gradient elution of CHCl3–MeOH–H2O (v/v/v, 6:1:0.1, 5:1:0.1, 4:1:0.1, 3:1:0.1, 2:1:0.1
and 1:1:0.1). Eight fractions (6A–6H) were obtained. Fraction 6C (381 mg) was purified by
semi-preparative HPLC and was eluted with MeOH–H2O (v/v, 88:12, 2 ml·min−1) to yield
pure compound 11 (155.5 mg, tR = 19.5 min), compound 10 (123.9 mg, tR = 39.9 min), and
compound 8 (56.2 mg, tR = 43.5 min). Fraction 6D was also purified using semi-prepara-
tive HPLC with MeOH–H2O (v/v, 85:15, 2 ml·min−1) to yield compound 12 (1.1 mg, tR =
22.5 min) and compound 9 (6.8 mg, tR = 43.0 min). Fraction 7 (459 mg) was purified by silica
gel column chromatography (300–400 mesh, 20 g) that was eluted with CHCl3–MeOH–
H2O in the ratios of 6:1:0.1, 4:1:0.1, 3:1:0.1, 1:1:0.1. A total of seven fractions (7A–7G)
were collected. Fraction 7D were purified using semi-preparative HPLC and eluted with
MeOH–H2O (v/v, 75:25, 2 ml·min−1) to yield pure compound 6 (5.7 mg, tR = 23.1 min) and
compound 7 (11.2 mg, tR = 33.2 min).
Fraction 11 (1.03 g) was purified on Sephadex gel column chromatography and eluted
with MeOH. Eight fractions (11A–11H) were obtained. Fraction 11D was subjected to
semi-preparative HPLC and eluted with MeOH–H2O (v/v, 70:30, 2 ml·min−1) to afford pure
compounds 3 (8.1 mg, tR = 11.5 min), 4 (1.8 mg, tR = 13 min) and 5 (5.6 mg, tR = 33.4 min).
Fraction 11E was subjected to semi-preparative HPLC and eluted with MeOH–H2O (v/v,
70:30, 2 ml·min−1) to afford pure compound 2 (2.1 mg, tR = 13 min) and compound 1
(5.6 mg, tR = 27.6 min). e amount and yield of each compound were shown in Table 1.
3.5.1. Compound 6
White amorphous powder;[ꢀ]D20 –58.7 (c 0.162, MeOH); IR (Neat) vmax (cm−1): 3411, 2936,
1
2848, 1708, 1645, 1435, 1130, 1380, 1064, 1032, 811; H and 13C NMR spectral data, see
Table 2; HR-ESI-MS m/z: 777.4059 [M + Na]+ (calcd for C39H62O14Na, 777.4038).
3.5.2. Compound 9
White amorphous powder; [ꢀ]2D0 –190 (c 0.4, MeOH); IR (Neat) v (cm−1): 3420, 2949,
max
1632, 1032, 1066, 1032, 989, 861; 1H and 13C NMR spectral data, see Table 2; HR-ESI-MS
m/z: 743.3973 [M + Na]+ (calcd for C39H60O12Na, 743.3983).
3.5.3. Compound 12
White amorphous powder;[ꢀ]D20 –49.56 (c 0.081, MeOH); IR (Neat) vmax (cm−1):3372, 2924,
1658, 1450, 1067, 1032, 913, 812; 1H and 13C NMR spectral data, see Table 2; HR-ESI-MS
m/z: 761.4096 [M + Na]+ (calcd for C39H62O13Na, 761.4088).

8
H.-X. HU ET AL.
Table 1. the amount and yield of each compound.
Com-
pounds
PPD
1
2
3
4
5
6
7
8
9
10
11
12
amounts 1200.0
5.6
2.1
8.1
1.8
5.6
5.7
11.2
56.2
6.8
123.9 155.5
1.1
(mg)
Yield (%)
-
0.47
0.18
0.68
0.15
0.47
0.48
0.93
4.7
0.57
10.3
13.0
0.09
1
Table 2. h nMr (600 Mhz) and 13c nMr (150 Mhz) spectral data of new compounds 6, 9, and 12 in
pyridine-d5 (δ in ppm, J in hz).
6
9
12
position
δC
δH
δC
δH
δC
δH
1
2
3
4
37.9
30.6
78.6
39.8
0.97, o; 1.70, o
1.70, o; 2.10, o
3.88–3.10 (m)
2.72, dd, J = 3.6, 15;
2.45–2.47 (m)
-
37.4
30.6
78.6
39.2
0.97, o; 1.70–1.72 (m)
1.87, o; 2.08–2.10 (m)
3.73–3.75 (m)
2.72, d, J = 7.2;
2.45–2.47 (m)
-
37.4
30.2
78.2
39.4
1.66, o; 0.93, o
1.21, o; 1.64, o
3.88–3.90 (m)
2.72–2.74 (m);
2.45–2.47 (m)
-
5
141.5
122.0
32.3
31.4
50.8
37.5
21.0
38.6
42.7
54.5
33.1
75.2
67.2
14.2
19.9
205.9
30.8
173.8
36.4
29.5
36.0
67.6
141.3
122.2
33.4
32.4
50.6
37.9
21.2
39.2
43.2
57.2
32.6
81.8
60.6
15.3
19.8
153.2
108.2
107.7
32.1
29.5
31.1
68.1
141.0
122.0
34.2
30.8
50.3
37.1
20.9
41.5
41.6
56.3
32.3
82.8
70.1
15.9
16.6
82.7
18.6
110.7
26.5
24.7
27.7
65.6
6
7
8
9
5.32, br,d, J = 3.6
1.87, o; 1.48, o
1.50, o
0.90, o
-
1.44–1.46 (m)
1.11–1.13 (m); 2.21, o
-
5.32, d, J = 1.8
2.12, o; 1.51, o
1.51, o
0.88, o
-
1.44–1.46 (m)
1.81, o; 1.11, o
-
5.34, br,d, J = 4.8
1.99, o; 1.43, o
1.51, o
0.83–0.85 (m)
-
1.44–1.46 (m)
2.01, o; 1.23, o
-
0.89–0.91 (m)
1.87, o; 1.27, o
5.06, o
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
0.83, o
1.07, o
2.49, o; 2.56, o
5.71–5.73 (m)
2.48, o
2.11–2.13 (m); 1.53, o
4.74–4.76 (m)
2.72, d, o
2.45, d, J = 8.4
1.21, s
1.24, s
0.92, s
-
2.15, s
-
0.78, s
0.91, s
-
1.11, s
-
1.75, s
-
5.32,o; 5.09,o
-
1.93–1.95 (m); 1.78, o
1.64, o
2.51, o; 2.55, o
2.12, o
1.56–1.58 (m); 1.48, o
2.00, o; 2.10–2.12 (m)
1.62, o
4.31–3.33 (m); 3.46,
d，J = 11.4
1.14, d, J = 6.6
4.98, o
1.31, o
3.62, o;
3.70, o
1.70–1.72 (m)
3.54, o;
3.60,o
0.72, d, J = 6.6
4.98, o
27
3-Glc-1
2
3
4
5
6
17.3
102.9
76.0
77.2
78.6
77.6
61.9
103.2
73.1
73.3
74.5
70.8
19.1
1.04, d, J = 6.6
4.98, o
17.8
102.9
75.2
77.2
78.6
77.7
62.0
103.18
73.1
73.3
74.5
70.9
19.0
16.6
102.5
75.6
76.7
78.2
77.2
61.5
102.7
72.7
72.9
74.1
70.4
18.7
4.02, t
4.26, o
4.50, t
4.02, t
4.26, o
4.50, t
4.01, t
4.26, o
4.50, t
3.74–3.76 (m)
4.28, o; 4.17, o
5.93, s
4.74, br, s
4.62, dd, J = 2.4, 9.0
4.38, t, J = 9.6
5.05–5.07 (m)
1.75, d, J = 6.0
3.74–3.76 (m)
4.28, o; 4.17, o
5.93, s
3.74–3.76 (m)
4.25, o; 4.15, o
5.96, s
4.73, br, s
4.62, dd, J = 3.0, 9.0
4.38, t
rha(1–4)-1
2
3
4
5
6
4.74, br, s
4.62, dd, J = 2.4, 9.0
4.38, t, J = 9.6
5.06–5.08 (m)
1.75, o
5.03–5.05 (m)
1.75, d, J = 6.0
s – singlet; d – doublet; t – triplet; br – broad; o – overlapped; m – multiple.
3.6. Cytotoxic activities
Human hepatocarcinoma (HepG2) and Human cervical cancer (Hela) cell lines were
maintained in RPMI-1640 medium containing 10% fetal bovine serum and 100 units·ml
−1
−1
penicillin and 100 μg·ml streptomycin. Afer they were digested with 0.25% trypsin

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
9
solution, the cells were resuspended in the medium to about 5 × 104 cells·ml −1 then seeded
the cell into 96-well flat microtiter plates. Afer incubation with different concentrations
of compounds 1–11 (1.56–100 μM) for 24 h, the cell growth was added 20 μl of 5 mg·ml
−1 MTT dissolved in phosphate buffer and the plate was reincubated at 37 °C in 4% CO2
for 4 h, then the supernatant was removed from every well, and 150 μl DMSO was added.
e plate was shaken on a micro-shaker for 10 min, and then read on a microplate reader
at 540 nm. Polyphyllin I was used as a positive control.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
is work was financially supported by the National Natural Science Foundation of China [grant num-
ber 21172267] and the Program for Innovative Research Team in IMPLAD [grant number IT1305].
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