New transformation pathway and cytotoxic derivatives from the acid hydrolysis of timosaponin B III

Article abstract of DOI:10.1080/14786419.2018.1499640

Timosaponin B III is a major bioactive steroidal saponin isolated from Anemarrhena asphodeloides Bge. To potentially discover derivatives with better biological activity, timosaponin B III was structurally modified via acid hydrolysis to yield one new (2, timopregnane A I) C₂₁ steroidal glycoside and seven known compounds. Their structures were elucidated on the basis of NMR spectroscopy and mass spectrometry. All eight compounds were evaluated for cytotoxic activity against MCF7, SW480, HepG2, and SGC7901 cell lines in vitro. As a result, compounds 6 and 7 showed significant activity (IC₅₀ 2.94–12.2 μM) against all tested cell lines. Structure–activity relationships of these compounds were investigated and the preliminary conclusions were provided. Moreover, a new transformation pathway was discovered in the acid hydrolysis of timosaponin B III for the first time.

Full text of DOI:10.1080/14786419.2018.1499640

Natural Product Research
Formerly Natural Product Letters
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New transformation pathway and cytotoxic
derivatives from the acid hydrolysis of
timosaponin B III
Yun-Fang Zhao, Yu-Wei Zhang, Yinru Wang, Susan L. Morris-Natschke, Wei
Liu, Ting-Ting Shang, Hong Yin, Kuo-Hsiung Lee & Xue-Feng Huang
To cite this article: Yun-Fang Zhao, Yu-Wei Zhang, Yinru Wang, Susan L. Morris-Natschke, Wei
Liu, Ting-Ting Shang, Hong Yin, Kuo-Hsiung Lee & Xue-Feng Huang (2018): New transformation
pathway and cytotoxic derivatives from the acid hydrolysis of timosaponin B III, Natural Product
Research, DOI: 10.1080/14786419.2018.1499640
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2018.1499640
New transformation pathway and cytotoxic derivatives
from the acid hydrolysis of timosaponin B III
a
a
a
b
ꢀ
ꢀ
Yun-Fang Zhao , Yu-Wei Zhang , Yinru Wang , Susan L. Morris-Natschke ,
Wei Liub,c, Ting-Ting Shanga, Hong Yina, Kuo-Hsiung Leeb and
Xue-Feng Huanga,b
aState Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, School of
b
Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing, China; Natural Products
Research Laboratories, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill,
c
NC, USA; Department of Pharmacology, Nanjing Medical University, Nanjing, China
ABSTRACT
ARTICLE HISTORY
Received 22 May 2018
Accepted 9 July 2018
Timosaponin B III is a major bioactive steroidal saponin isolated
from Anemarrhena asphodeloides Bge. To potentially discover
derivatives with better biological activity, timosaponin B III was
structurally modified via acid hydrolysis to yield one new (2, timo-
pregnane A I) C21 steroidal glycoside and seven known com-
pounds. Their structures were elucidated on the basis of NMR
spectroscopy and mass spectrometry. All eight compounds were
evaluated for cytotoxic activity against MCF7, SW480, HepG2, and
SGC7901 cell lines in vitro. As a result, compounds 6 and 7
showed significant activity (IC50 2.94–12.2 lM) against all tested
cell lines. Structure–activity relationships of these compounds
were investigated and the preliminary conclusions were provided.
Moreover, a new transformation pathway was discovered in the
acid hydrolysis of timosaponin B III for the first time.
KEYWORDS
Timosaponin B III; Acid
hydrolysis; Steroidal
saponins; C21 steroidal
glycoside; Cytotoxic activity
CONTACT Kuo-Hsiung Lee
khlee@unc.edu; Xue-Feng Huang
hxf@cpu.edu.cn
†These authors contributed equally to this work.
Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2018.1499640.
ß 2018 Informa UK Limited, trading as Taylor & Francis Group

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Y.-F. ZHAO ET AL.
Figure 1. Structures of TB-III and compounds 1–8.
1. Introduction
Timosaponin B III (TB-III) is a major steroidal saponin originally isolated from Anemarrhena
asphodeloides Bge. In recent pharmacological studies, TB-III exhibited numerous bioactiv-
ities, including anti-inflammatory (Kim et al. 2009), anti-platelet aggregative (Zhang et al.
1999) and anti-depressive (Jiang et al. 2014) effects, but antitumor activity of TB-III has not
been reported. A study on the metabolism of TB-III reported several biotransformation
pathways, including deglycosylation, hydroxylation, oxidation, and ring cleavage (Jiang
et al. 2014). Because the metabolites were produced in an in vivo acidic or enzymatic
environment, in vitro acid hydrolysis of TB-III is a rational approach to study the mechan-
ism of drug action as well as to search for potential active anticancer compounds.
In the present studies, TB-III was subjected to structural modification by acid
hydrolysis to yield one new (timopregnane A I, 2) C21 steroidal glycoside and seven
known compounds (1, 4–9). Two C21 steroidal glycosides and their aglycone (1–3)
were obtained by acid hydrolysis of TB-III for the first time, and the transformation
pathways to all hydrolytic derivatives are discussed herein. Furthermore, these com-
pounds were evaluated for cytotoxic activity by the MTT method.
2. Results and discussion
The eight derivatives were obtained by acid hydrolysis (3% nitric acid) of TB-III at 100 ꢁC
for 3 h. One new C21 steroidal glycoside timopregnane A I (2), together with seven known
compounds, timopregnane A (1) (Zhu et al. 2012), 3b-hydroxy-5b-pregn-16(17)-ene-20-one
(3) (Blunt and Stothers 1977), timosaponin B III-d (4) (Fu et al. 2015), timosaponin B III-b
(5) (Liu et al. 2013), timosaponin A III (6) (Lu et al. 2016), timosaponin A I (7) (Zhao et al.
2015) and sarsapogenin (8) (Blunt and Stothers 1977) were obtained (Figure 1).
Compound 2 was obtained as a white amorphous powder. The molecular formula
was determined as C27H42O7 from a quasi-molecular ion peak at m/z 501.2821
1
[M þ Na]þ (calcd. for 501.2823) in the HR-ESI-MS spectrum. The H NMR spectrum of 2
(Table S1) showed three tertiary methyls at dH 0.93 (s, CH3-18), 0.86 (s, CH3-19) and

NATURAL PRODUCT RESEARCH
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Figure 2. The proposed pathways to the hydrolysis products (1–8) from TB-III.
2.25 (s, CH3-21) and an olefinic proton at dH 6.67 (m). One anomeric proton at dH 4.85
(d, J ¼ 7.5 Hz) confirmed the presence of one sugar moiety. The13C NMR spectrum
(Table S1) showed 27 signals, including those for one anomeric carbon (dC 104.3), two
olefinic carbons (dC 156.1, 145.1) and a carbonyl carbon (dC 196.7). These spectroscopic
features suggested that, like the known identified compounds, 2 is a C21 steroidal
glycoside containing with a carbonyl group. The spectroscopic data of 2 and 1 were
quite similar, particularly for the aglycone. The two compounds differed only in the
number of sugars attached at C-3 (one in 2, two in 1). In the HMBC spectrum of 2
(Figure S1), long-range corrections were observed between CH3-21 (dH 2.25) and C-20
(dc 196.7); CH3-18 (dH 0.93) and C-13 (dc 47.0), C-14 (dc 56.9), C-17 (dc 156.1); H-16 (dH
6.67) and C-14 (dc 56.9), C-15 (dc 32.7), C-17 (dc 156.1), C-20 (dc 196.7); CH3-19 (dH 0.86)
and C-1 (dc 31.2), C-5 (dc 37.5), C-10 (dc 35.9). In NOSEY spectrum, H-5 showed a correl-
ation with the b-oriented methyl protons of CH3-19, which confirmed the 5b configur-
ation. Based on the above information, the aglycone structure of compound 2 was
identified as 3b-hydroxy-5b-pregn-16(17)-ene-20-one.
The monosaccharide of 2 was confirmed as D-galactose following the acid hydroly-
sis of 2 and identification by HPLC analysis. The b-anomeric configuration of the sugar

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Y.-F. ZHAO ET AL.
was determined from its coupling constant. The sugar moiety was assigned at C-3
based on the observed HMBC correlation between Gal H-1' (dH 4.85) and C-3 (dC 74.8).
Thus, compound 2 (timopregnane AI) was fully identified as 3-O-b-D-galactopyranosyl-
(3b,5b)-pregn-16(17) -ene-20-one (Figure 1).
In this acid hydrolysis study, we found three obvious transformation pathways, I, II,
and III. Metabolite 1 was likely produced from TB-III by opening of the E-ring followed
by conversion to a C21 steroidal glycoside. In this transformation pathway (I), the
decisive factor is the ready oxidation of the 20, 22-double bond. Subsequently, oxida-
tive cleavage of this bond and loss of the ester on the C-17 OH, followed by dehydra-
tion would create the 16, 17-double bond in metabolites 1–3. Compounds 4 and 5
are products of successive hydrolysis of the C-3 sugar chain (II). In the final pathway
(III), the C-26 sugar chain is removed and the side chain at C-22 is cyclized to give spi-
rostanol-type steroidal glycosides (6–7) and an aglycone (8). Figure 2 illustrates the
proposed mechanisms for transformation of TB-III to 1–8.
TB-III and its eight hydrolytic derivatives were evaluated for cytotoxic effects against
MCF7, SW480, HepG2 and SGC7901 cell lines in vitro (Table S2), with 6 used as the
positive control (Kang et al. 2011). Compounds 1, 2, 4, and 5 as well as TB-III showed
no toxicity toward the four cell lines (IC50 > 100 lM). Compounds 6 and 7 exhibited
significant anticancer effects against the four cell lines (IC50 2.94–12.2 lM). Compounds
3 and 8 showed moderate cytotoxicity against three (MCF7, SW480, HepG2) and four
cell lines, respectively. The results demonstrated that the acid hydrolysis products 3
and 6–8 from TB-III exhibited greater cytotoxic activity than the parent compound,
which suggests that the inactive natural compound may incur certain bioactive effects
through metabolic transformation. Moreover, acid hydrolysis of timosaponins from A.
asphodeloides is a feasible method to obtain better biological activity.
The results of this study also provided preliminary information about the structure-
activity relationships of these compounds. These steroidal saponins have three differ-
ent aglycone skeletons, spirostanol, furostanol, and epipregnenolone saponins. In our
study, the six-ring derivatives (6–8) exhibited much greater activity than the five-ring
(TB-III, 4, 5) or four-ring (1–3) derivatives; thus, the steroidal saponins with an F-ring
showed obviously increased cytotoxic activity. To investigate the role of the number
of sugar moieties, we compared the activities of 1–3 and 6–8. Compounds 1 and 2
showed no activity against the four cell lines, but their aglycone structure 3 exhibited
moderate cytotoxicity against MCF7, SW480, and HepG2. However, the aglycone 8 was
less potent than the di- and mono-glycosides, 6 and 7, respectively. This result sug-
gested that the number of sugar moieties might affect the activity, but the aglycone
structure was more important in our study.
3. Experimental
3.1. General experimental procedures
ESI-MS and HR-ESI-MS experiments were performed on an Agilent 1100 Series LC/MSD
Trap SL mass spectrometer and Agilent 6520B Q-TOF spectrometer, respectively. NMR
spectra were recorded on Bruker ACF-300 and -500 spectrometers using pyridine-d5 as
solvent with tetramethylsilane (TMS) as the internal reference. The chemical shifts are

NATURAL PRODUCT RESEARCH
5
given in d (ppm) and coupling constants in Hz. Analytical HPLC was performed on a
Shimadzu LC-20AT with SPD-20A UV/VIS detector, using a YMC-Pack ODS-A column
(250 mm ꢂ4.6 mm, 5 lm). Semi-preparative HPLC was performed on a Shimadzu LC-
6AD with SPD-20A UV/VIS detector, using an YMC-Pack ODS-A column (250 mm
ꢂ20 mm, 5 lm). Column chromatography (CC) was carried out on silica gel (200–300
mesh, Qingdao, China), Silica gel 60 (230–400 mesh, Merck) and all chemical reagents
used in the studies were produced by Nanjing Chemical Reagent Co. Ltd.
(Nanjing, China).
3.2. Preparation of derivatives
The TB-III (1 g) was dissolved in 500 mL nitric acid aqueous solution (3% nitric acid).
The mixture was stirred at 100 ꢁC for 3 h. The reaction solution was neutralized with
bicarbonate, partitioned with ethyl acetate, and concentrated under reduced pressure
to dryness. The residue was separated by silica gel eluted with CHCl3:MeOH
(40:1 ꢃ 2:1, v/v) to give four fractions (Fr.1–4). Fr.1 was further separated by silica gel
CC to yield 3 (30 mg) and 8 (42 mg). Fr.2 was further separated by silica gel CC and
semi-preparative HPLC to give 2 (10 mg), 7 (15 mg), and 5 (25 mg). Fr.3 was further
separated by ODS CC and semi-preparative HPLC to yield 1 (20 mg), 4 (18 mg), and
6 (13 mg).
3.2.1. Timopregnane A I
25
White amorphous powder, ½aꢄD
ꢅ40.8 (c ¼ 0.12, MeOH), 1H-NMR (500 MHz, C5D5N) dH and 13C-NMR (125 MHz,
C5D5N) dC see Table S1. HR-ESI-MS m/z 501.2821 ([M þ Na]þ, calcd for
C27H42O7, 501.2823).
3.3. Acid hydrolysis of 2 and sugar analysis
Compound 2 (2 mg) was hydrolyzed with 2 M HCl-MeOH (5 ml) under reflux for 3 h.
The reaction mixture was diluted with H2O and extracted with CHCl3. The water layer
was neutralized with Na2CO3, concentrated, and subjected to TLC analysis together
with an authentic sample of D-galactose, and developed with CH2Cl2-MeOH-H2O (15: 6:
1). Detection was carried out with 5% vanillin-sulfuric acid spray.
3.4. Bioassay of cytotoxic activity
Human breast adenocarcinoma MCF7 cells, human colon adenocarcinoma SW480 cells,
human hepatocellular carcinoma HepG2 cells, and human gastric cancer SGC7901 cells
were obtained from the Cell Bank of Shanghai Institute of Biochemistry and Cell
Biology, Chinese Academy of Sciences. MCF7, SW480, and SGC7901 cells were cultured
in RPMI-1640 medium (Gibco, California) supplemented with 10% fetal bovine serum
(Gibco, Invitrogen Corporation, NY), 100 U/ml benzyl penicillin, and 100 U/ml strepto-
mycin in a humidified environment with 5% CO2 at 37 ꢁC. HepG2 cells were cultured
in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Invitrogen Corporation, NY)

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Y.-F. ZHAO ET AL.
supplemented with 10% fetal bovine serum (Gibco, Invitrogen Corporation, NY),
100 U/ml benzyl penicillin, and 100 U/ml streptomycin in a humidified environment
with 5% CO2 at 37 ꢁC.
Cell toxicity was measured by a colorimetric assay using MTT as described previ-
ously (Liu et al. 2016). Experiments were carried out in triplicate in a parallel manner.
Control cells were treated with culture media containing 0.1% DMSO. After incubation
for 24 h, absorbance (A) was measured at 570 nm.
Cell viability (%) was calculated using the following equation:
Cell viability (%) ¼ (Atreatment/Acontrol) ꢂ 100%.
IC50 (the concentration that caused 50% inhibition of cell proliferations) was calcu-
lated by the Logit method.
4. Conclusion
In summary, this study reports the isolation, structural elucidation, and cytotoxic activ-
ity of hydrolytic derivatives of TB-III. One new C21 steroidal glycoside was isolated
together with seven known compounds. Notably, a transformation pathway from TB-III
to epipregnenolone derivatives was demonstrated for the first time. This discovery
provides new thoughts for studying the metabolism of timosaponins in vitro and in
vivo. The cytotoxic activities of the nine compounds were evaluated against MCF7,
SW480, HepG2 and SGC7901 cell lines. Compounds 6 and 7 exhibited significant cyto-
toxic activity and may merit further research as potential anticancer leads.
Supplementary material
Supplementary material relating to this article is available online, alongside Tables
S1–S2 and Figures S1–S28.
Acknowledgments
The authors are grateful to the members of the analytical group of China Pharmaceutical
University for the spectral measurements.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was supported by the [National Natural Science Foundation of China] under Grant
[number 81573560]; and [National Natural Science Foundation of China] under Grant [num-
ber 81673513].
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