Reaction coupling separation for isosteviol production from stevioside catalyzed by acidic ion-exchange resin

Article abstract of DOI:10.1007/s00449-020-02431-4

Abstract: Isosteviol, a prodrug used to be obtained via Wagner–Meerwein rearrangement from steviol with low yield and long reaction?time. Herein, an in-situ separation-coupling-reaction is presented to prepare isosteviol from the natural sweetener stevioside. Simply with in-situ water-washing, the product containing 92.98% purity of isosteviol was obtained with a stevioside conversion of 97.23% from a packet bed reactor without further separation. Within the assayed inorganic acid, organic acids and acidic ionic liquids, the acidic ion-exchange resins provided higher product specificity towards isosteviol. Furthermore, comparing to 5-Fluorouracil, the product presented similar and even stronger inhibition on proliferation of the assayed human cancer cells in a time and dose-dependence by causing cell phase arrest. Isosteviol treatment caused G1 arrest on SGC-7901, HCT-8 and HCT-116 cells, S arrest on HepG2, Huh-7 and HepG3B cells, and G2 arrest on MGC-803 cells, respectively. Graphic abstract: Reaction coupling separation for isosteviol production catalyzed by acidic ion-exchange resin.[Figure not available: see fulltext.]

Full text of DOI:10.1007/s00449-020-02431-4

Bioprocess and Biosystems Engineering
https://doi.org/10.1007/s00449-020-02431-4
RESEARCH PAPER
Reaction coupling separation for isosteviol production from stevioside
catalyzed by acidic ion‑exchange resin
Xueyi Hu1,2 · Zhuoyu Zhou1,2 · Zongying Zhang1,2 · Xiaoxia Wang1,2 · Xiaochen Sui1,2 · Junming Chen1,2
Yongmei Xia1,2 · Jue Zhang3 · Jianguo Lin3
·
Received: 17 December 2019 / Accepted: 17 August 2020
© Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract
Isosteviol, a prodrug used to be obtained via Wagner–Meerwein rearrangement from steviol with low yield and long reac-
tion time. Herein, an in-situ separation-coupling-reaction is presented to prepare isosteviol from the natural sweetener stevio-
side. Simply with in-situ water-washing, the product containing 92.98% purity of isosteviol was obtained with a stevioside
conversion of 97.23% from a packet bed reactor without further separation. Within the assayed inorganic acid, organic acids
and acidic ionic liquids, the acidic ion-exchange resins provided higher product specifcity towards isosteviol. Furthermore,
comparing to 5-Fluorouracil, the product presented similar and even stronger inhibition on proliferation of the assayed human
cancer cells in a time and dose-dependence by causing cell phase arrest. Isosteviol treatment caused G1 arrest on SGC-7901,
HCT-8 and HCT-116 cells, S arrest on HepG2, Huh-7 and HepG3B cells, and G2 arrest on MGC-803 cells, respectively.
Graphic abstract
Reaction coupling separation for isosteviol production catalyzed by acidic ion-exchange resin.
Keywords Isosteviol · Stevioside · Hydrolysis · Cation-exchange resin · Anticancer · Ionic liquid
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s00449-020-02431-4) contains
Introduction
supplementary material, which is available to authorized users.
Isosteviol, (4α, 8β, 13β)-13-Methyl-16-oxo-17-norkauran-
18-oic acid, was reported on its structure, stereochemistry
[1] and absolute confguration in 1960s [2]. To date, isoste-
viol has been mostly recognizing as a popular starting mate-
rial for drug synthesis [3–7]; for instance, as precursors of
synthetic drugs for the treatment of cancer and infammation
[8, 9].
* Yongmei Xia
ymxia@jiangnan.edu.cn
1
State Key Laboratory of Food Science and Technology,
School of Chemical and Materials Engineering, Jiangnan
University, 1800 Lihu Avenue, Wuxi 214122, Jiangsu, China
2
Key Laboratory of Synthetic and Biological Colloids
(Ministry of Education), School of Chemical and Materials
Engineering, Jiangnan University, 1800 Lihu Avenue,
Wuxi 214122, Jiangsu, China
Recently, isosteviol itself has gained intensive inter-
ests as it possesses various biological activities [10], such
as reducing vasoconstriction [11], improving glucose and
insulin sensitivity, lowering plasma triglycerides and weight,
3
Key Laboratory of Nuclear Medicine of Ministry of Health,
Jiangsu Institute of Nuclear Medicine, Wuxi 214063, Jiangsu,
China
Vol.:(0123456789)
1 3

Bioprocess and Biosystems Engineering
and regulating the gene expression profle of key insulin
regulatory genes for the treatment of type II diabetes [12,
13], relieving I/R injury in rat brains [14] and so on [6].
Therefore, efcient massive production of isosteviol with
high purity is expected and beneft to its application.
conducted. Meanwhile, to elucidate isosteviol’s cytotoxic-
ity and anticancer activity, the cytotoxicity of isosteviol on
several human normal cells and carcinoma cell lines was
investigated as well.
Isosteviol can be obtained by Wagner–Meerwein rear-
rangement from steviol [15], the latter is a product hydro-
lyzed from natural glycosyl steviosides such as stevioside
or rebaudiosides catalyzed by acids (Scheme 1), such as
hydrochloric acid, sulfuric acid and hydrobromic [16–18].
However, the acidic hydrolysis of glycosyl steviosides would
simultaneously yield steviol and endocyclic isomers of ste-
viol [19, 20]. For example, an analogous mixture of isoste-
viol, steviol, and Δ15-steviol was obtained using 0.7% of
hydrochloric acid, and the yield of isosteviol was only 36%
[16]. Cherney et al. synthesized steviol by multistep reac-
tions, which was then converted to isosteviol by the acid-
induced rearrangement [21]. In another case, an isosteviol
yield of 83% along with steviol (17% yield) was obtained,
assisted with FeCl3 from hydrolyzing stevioside at a refex-
ing temperature [20]. Enzymatic hydrolysis may provide
higher product specifcity on isosteviol. Milagre disclosed
that stevioside was hydrolyzed with the catalysis of pancrea-
tin and ofered isosteviol in a yield of 93.9% in 7 days [22].
Therefore, quick and afordable preparation of isosteviol
might be still expected on acidic hydrolysis of stevioside.
In this experiment, aiming at fnding a practicable process
to produce isosteviol in high yields, various acidic catalysts
including acids, acidic ionic liquids and cation-exchange res-
ins were investigated on their catalysis activity and selec-
tivity to fnd a suitable catalyst for isosteviol production;
subsequently, an in-situ separation coupling production of
isosteviol from hydrolyzing stevioside was designed and
Materials and methods
Chemicals
Stevioside (97% of HPLC purity) was from Qing-
dao Runde Biotechnology Co., Ltd (Qingdao, China).
Na2CO3, CH3COONa, CH3COOH, Na2HPO4⋅12H2O,
NaH2PO4⋅2H2O, NaCl, KCl, dimethyl sulfoxide (DMSO,
BR) and 5-Fluorouracil (5-FU, BR) were purchased from
Sinopharm Chemical Reagent Co., Ltd (Shanghai, China).
Isosteviol (97% of HPLC purity) was from Nanjing Spring &
Autumn Biological Engineering Co., Ltd (Shanghai, China).
Acidic ionic liquids [MIMPS]3PWO40, [C3IMPS]3PWO40,
[C5IMPS]3PWO40, [C7IMPS]3PWO40, H3PWO40 were pro-
vided by Dr Wei Li from School of Chemical and Materials
Engineering at Jiangnan University. Resin 732 (Na+ form,
styrene–divinyl benzene copolymer, total exchange capac-
ity of 4.6 mmol/g (Na+ form), resin 0014 (Na+ form, sty-
rene–divinyl benzene copolymer, total exchange capacity of
4.5 mmol/g), resin 00112 (Na+ form, matrix styrene–divinyl
benzene copolymer, total exchange capacity of 4.0 mmol/g)
and resin D113 (H+ form, acrylic copolymer, total exchange
capacity of 10.8 mmol/g) cation-exchange resins were pur-
chased from Sinopharm Chemical Reagent Co., Ltd (Shang-
hai, China). Trypsin–EDTA solution, propidium iodide (PI),
Triton X-100, endonuclease (RNase A), 3-(4,5-dimethylth-
iazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and
Scheme 1 Acid-catalyzed preparation of isosteviol from hydrolysis of stevioside
1 3

Bioprocess and Biosystems Engineering
penicillin–streptomycin solution (100×) were purchased
from Beyotime Institute of Biotechnology Co., Ltd (Shang-
hai, China). All other reagents were of analytical grade and
used as received unless otherwise stated.
The yield of isosteviol or steviol was production yield,
calculated based on the whole analysis of precipitated solid
with mass measurement. But the yields of other byprod-
ucts, if needed, were calculated based on their corresponding
HPLC chromatograph peak area (Ax) with chromatograph
peak area of stevioside (ASt) as the reference, calculated as
follows:
Cell culture
The human moderately diferentiated gastric cancer cell
SGC-7901, human colon adenocarcinoma cell Caco-2,
human ileocecal adenocarcinoma epithelial cell HCT-8
and human colorectal cell HCT 116 were purchased from
Shanghai Institute of Biochemistry and Cell Biology, Chi-
nese Academy of Science. The human gastric mucosal epi-
thelial cell GES-1, human poorly diferentiated gastric can-
cer cell MGC-803, three hepatoma cells, Huh-7 (mutated
p53), HepG2 (wild-type p53) and Hep3B (p53 deleted)
were purchased from the American Type Culture Collec-
tion. SGC-7901 was cultured in RPMI 1640 medium, and
other cells were cultured in DMEM medium containing 10%
fetal bovine serum, 1% glutamine (200 mmol/L), penicillin
(100 IU/mL), and streptomycin (100 mg/L) in a humidifed
5% CO2 atmosphere at 37 °C before use.
Ct × Ax∕Ast
Yield =
× 100%
C0
All tests were performed in triplicate at least; all data
presented were with standard deviations less than 5%.
MTT assay on cell proliferation
The efect of isosteviol on the proliferation of human carci-
noma or normal cell was evaluated with MTT assay [25–27].
The cells in the logarithmic growth phase were digested with
0.25% trypsin and adjusted to 5,000 cells/well using DMEM
complete medium. Prior to the isosteviol treatment, 100 μL
cell suspension was pipetted into each well in 96-well plates
and cultured for 24 h at 37˚C in 5%CO2. Subsequently, cells
were incubated with isosteviol at 37 °C in 5% CO2 for 48 h.
The culture medium was then removed and 100 μL MTT
reagent (0.5 mg/mL in culture medium) was added. Follow-
ing an additional 4 h of incubation, the MTT/medium was
removed and 150 μL of DMSO was added to dissolve the
formazan crystals. Absorbance of the solution at 570 nm
was recorded to calculate the inhibition rate on cell growth.
Chemotherapy agent 5-FU was employed as a positive con-
trast. All measurements were performed in triplicate and
recorded as means. The inhibition rate was calculated as
follows:
Hydrolysis of stevioside in a batch reactor
The prepared selected cation exchange resins in Na+ form
was converted to H+: cation exchange resins were condi-
tioned in hydrochloric acid solution (1 mol/L HCl) and then
several washings with deionized water to remove excess acid
[23]. In a typical reaction, 20 mL stevioside aqueous solu-
tion (20 g/L) in a 50 mL Erlenmeyer fask was kept at 95 °C
for 30 min, then mixed with the cation-exchange resin and
shaken in a water bath at 95 °C for 24 h. The precipitated
white powder was fltered and washed with warm DI water
until no stevioside was detectable from the eluting solution.
The powder was then recrystallized from 95% aqueous
methanol, and white needles were obtained as the fnal prod-
uct. The product was characterized with NMR (AVANCE III
400 MHz Digital NMR Spectrometer (Bruker, USA)) and
LC–MS (BEH HILIC C18 column; mobile phase: acetoni-
trile and water (75:25, v/v), 0.3 mL/min; column tempera-
ture: 30 °C; collision energy: 20–55 eV; polarity: ES−)[24,
25]. The conversion of stevioside (St) was calculated based
on HPLC analysis with the calibration of a standard solution
of stevioside, described as following:
Cell growth inhibition rate (%)
A570 of control − A570 of sample
=
× 100
A570 of control
Cell cycle analysis
The human carcinoma cells were plated at 2 × 105/well in
6-well plates and treated with isosteviol. After 48 h, the cells
were then harvested with trypsin, washed three times for
1 min each, resuspended in cold PBS and fxed in cold (4
°C) 70% ethanol for 4 h, and kept storage at − 20 °C over-
night. Next, the cells were washed three times for 1 min each
again and resuspended in PBS containing 40 μg/mL PI and
0.1 mg/mL RNase (#C1052, Beyotime Institute of Biotech-
nology Co., Ltd, Shanghai, China), and then incubated for
30 min at room temperature. PI-stained cells were analyzed
using a fow cytometer and ModFit LT 5.0 software (Verity
Software House, USA) [26].
C0 − Ct
St conversion =
× 100%
C0
where C0 and Ct is the initial and real-time concentration
(g/L) of stevioside in the reaction mixture, respectively. The
stevioside concentration was determined with a standard
calibration curve.
1 3

Bioprocess and Biosystems Engineering
2:1 (Fig. 1b). Similarly, weak acidic cation-exchange resins
(D113) did not accelerate the hydrolysis of stevioside; but
the stronger acidic resins reduced more isosteviol, especially
the 732 cation-exchange resin, the yield of isosteviol and
steviol is about 6:1 (Fig. 1c, Run 2). In summary, compared
to the assayed inorganic acid, organic acids and acidic ionic
liquids, the acidic ion-exchange resins provided high product
selectivity towards isosteviol.
Results and discussion
Comparative study with inorganic acids and acidic
cation‑exchange resins
Four inorganic acids and four organic acids (Fig. 1a), fve
acidic ionic liquids (Fig. 1b), and fve cation-exchange resins
(Fig. 1c) were tested for their product specifcity of hydro-
lyzing stevioside(Fig. 1).
Therefore, 732 cation-exchange resin was then chosen
for the subsequent experiments due to its higher product
specifc activity, in which isosteviol was the main product
as the IR, LC–MS and NMR profle indicated [16, 28] (see
also Supporting material, Figs. 1s, 2s, 3s). The product was
recrystallized with 95% methanol twice, giving white crystal
needles with a purity of 99% (HPLC). NMR profles of the
main product from hydrolysis of stevioside with 732 resin
As presented in Fig. 1, strong acids (Fig. 1a) mainly
induced steviol in the hydrolysis products, in accordance
with that reported in the literature [16] where the yield of
isosteviol is only 36%; while the middle strong and weak
acids did not promote hydrolysis of stevioside (Fig. 1a). The
main products of the hydrolysis catalyzed by acidic ionic
liquid were isosteviol and steviol, the yield ratio is about
Fig. 1 Acidic hydrolysis of stevioside. 95 oC, St 50 g/L, H+ 10 mol%/
St, 3 h; catalyzed by A (acids): 1- HCl, 2- H2SO4, 3- HNO3, 4
-H3PO4, 5- CH3COOH, 6- NH2SO3H, 7- C2H2O4, 8- C6H8O7; B
(acidic ionic liquids): 1- [MIMPS]3PWO40, 2- [C3IMPS]3PWO40,
3- [C5IMPS]3PWO40, 4- [C7IMPS]3PWO40, 5- H3PWO40; C (cat-
ion-exchange resins): 1- 001×4, 2- 732, 3- 001×12, 4- D113, 5-
001×7.5
Fig. 2 Single factor test of hydrolyzing stevioside catalyzed by 732 cation-exchange resin. A: St 100 g/L, H+ 15 mol%/St, 10 h; B: 95 °C, H+
15 mol%/St, 10 h; C: 95 °C, St 200 g/L, 10 h; D: 95 °C, 200 g/L, H+ 15 mol%/St
1 3

Bioprocess and Biosystems Engineering
are as follows: 1H NMR: (400 MHz, Pyr) δ 14.66 (s, 1H),
2.66 (dd, J=18.4, 3.5 Hz, 1H), 2.45 (d, J=13.0 Hz, 1H),
2.25–2.11 (m, 1H), 2.10 – 2.01 (m, 2H), 1.78 (d, J=18.4 Hz,
1H), 1.66–1.52 (m, 4H), 1.45 (dt, J=11.4, 4.2 Hz, 3H), 1.35
(s, 3H), 1.34–1.24 (m, 2H), 1.24–1.03 (m, 4H), 1.02 (s,
3H), 0.96 (s, 3H), 0.87 (td, J=13.3, 3.9 Hz, 1H).. 13CNMR
(101 MHz, Pyr) δ 39.34 (C-1), 19.41 (C-2), 37.21 (C-3),
43.62(C-4), 56.73 (C-5), 22.17 (C-6), 41.43 (C-7), 48.34
(C-8), 54.49 (C-9), 38.34 (C-10), 20.35 (C-11), 38.10(C-
12), 39.85 (C-13), 53.98 (C-14), 48.34 (C-15), 224.5 (C-16),
20.02 (C-17), 29.15 (C-18), 180.04 (C-19), 39.34(C-20).
Reaction coupling separation for isosteviol
production from stevioside with 732 acidic
ion‑exchange resin
First of all, using 732 acidic ion-exchange resin as the cata-
lyst, a single factor test was applied to optimize the reaction
parameters as shown in Fig. 2; where St completely con-
verted with 91.9% of isosteviol yield and 4.8% of steviol
yield in 24 h (Fig. 2d).
Subsequently, a packed bed reactor was used to utilize
the resin (Fig. 3), where the reaction coupling separation
Fig. 3 Reaction coupling
separation of isosteviol from
stevioside with 732 acidic ion-
exchange resin
Fig. 4 Hydrolysis of stevioside with the acidic cation exchange resin in the packed bed reactor at 95 °C. 6 g ion exchange resins, a St 20 g/L; b
fow rate: 5.0 mL/h; c St 20 g/L, fow rate: 5.0 mL/h
1 3

Bioprocess and Biosystems Engineering
of isosteviol production from stevioside was realized. With
an in-situ separation, the product with 92.98% purity of
isosteviol was obtained with a stevioside conversion of
97.23%, simply after water washing. Considering the pro-
duction strength and production yield, the suitable param-
eters would be obtained as (Fig. 4): 95 °C, 20 g/L of St,
Fig. 5 Inhibition on human cancer cells proliferation with isosteviol (48 h)
1 3

Bioprocess and Biosystems Engineering
5.0 mL/h of fow rate and 6 g acidic ion-exchange resin;
the isosteviol production intensity was up to 1.54 g/L/h
with 97.23% of St conversion. Except greatly expediting
the reaction with the pure product, this process can erase
the need for recrystallizing.
Furthermore, fow cytometry analysis was performed
to determine the cell cycle distribution and population of
dead cell in isosteviol-treated cells. As it was reported, 5-FU
(1000 ng/mL) inhibited SW480 and COLO320DM by a tran-
sient G1-S phase arrest [34]. Similarly, as Fig. 6 and Table 1
indicated, isosteviol also caused cell phase arrest and apop-
tosis. Specifcally, isosteviol treatment caused G1 arrest on
SGC-7901, HCT-8 and HCT-116, S arrest on HepG2, Huh-7
and HepG3B, G2 arrest on MGC-803, respectively.
Anticancer potential of isosteviol
As aforementioned, there are many reports on the antican-
cer activity of isosteviol derivative [4, 9, 29]. Isosteviol
was shown to prevent the growth of human cancer cells
(human B cell acute lymphoblastoid leukemia cell BALL-
1, human T cell acute lymphoblastic leukemia cell MOLT-4,
and human gastric cancer cell NUGC-3) with LD50 values
of 84–167 μM, and IC50 value for DNA polymerases was
64.0 μM [30, 31]. Isosteviol showed exhibited strong inhibi-
tory efects in a two-stage carcinogenesis test using mouse
skin induced by DMBA and 12-O-tetradecanoylphorbol-
13-acetate (TPA), and the inhibitory was greater than that
of glycyrrhizin [32].
Conclusions
In this work, a reaction coupled separation for preparing
isosteviol was achieved, and for the frst time, anticancer
activity of isosteviol on human cancer cells was preliminar-
ily explored.
With an in-situ separation, simply with water-washing,
the product with 92.98% purity of isosteviol was obtained
with a stevioside conversion of 97.23% in a packet bed
reactor. Compared to the assayed inorganic acid, organic
acids and acidic ionic liquids, the acidic ion-exchange res-
ins provided high product selectivity towards isosteviol.
Isosteviol presented similar inhibition on several human
cancer cell lines as 5-Fluorouracil (5-FU) did, in a time and
dose-dependent manner through inducing cell phase arrest;
and it even performed stronger inhibition on Huh-7 and
MGC-803 cells. Isosteviol treatment caused G1 arrest on
SGC-7901, HCT-8 and HCT-116 cells, S arrest on HepG2,
Huh-7 and HepG3B cells, and G2 arrest on MGC-803 cells,
In this study, nine human cancer cell and normal lines
were tested with isosteviol treatment (Fig. 5). For compari-
son, 5FU (LD50 is 115 mg/kg, oral, mouse) was used as the
positive control in this experiment. And the LD50 of isoste-
viol is 0.5 g/kg body weight (dogs, rats and mice, oral) [33],
which is 3 folders of that of 5-FU.
As shown in Fig. 5, isosteviol presented similar inhibition
as 5-FU, and inhibited the cells viability in a time and dose-
dependent manner (see also Supporting material Fig. 4S).
Moreover, isosteviol even performed stronger inhibition on
some cell lines, such as Huh-7 and MGC-803.
Fig. 6 Efect of isosteviol on cell cycle distribution in the human gastrointestinal and liver cancer cells
1 3

Bioprocess and Biosystems Engineering
Table 1 Efect of isosteviol on
cell cycle progression of the
gastrointestinal and liver cancer
cells
Cell
Dosage (μg/mL)
G1 (%)
S (%)
G2 (%)
HepG2
Control
50
150
Control
50
150
Control
50
100
69.91
65.69
65.96
70.89
65.69
62.55
70.81
65.68
59.20
67.66
64.99
49.63
45.05
60.41
66.86
73.05
73.52
86.05
57.43
66.77
69.95
22.09
29.39
30.47
21.22
29.39
32.69
5.02
33.55
39.50
24.40
19.57
30.31
43.81
39.29
33.14
24.41
23.00
5.95
8.00
4.91
3.57
8.00
4.91
4.77
24.17
0.77
1.30
Huh-7
HepG3B
MGC-803
SGC-7901
HCT-8
Control
50
150
Control
50
200
Control
50
150
Control
50
200
7.95
15.44
20.06
11.15
0.30
0.00
2.55
3.48
8.00
8.00
8.00
6.04
HCT-116
34.57
25.23
24.01
5. Luan T, Cao LH, Deng H, Shen QK, Tian YS, Quan ZS (2019)
Design and synthesis of C-19 isosteviol derivatives as potent and
highly selective antiproliferative agents. Molecules 24:121
6. Ullah A, Munir S, Mabkhot Y, Badshah SL (2019) Bioactivity
profle of the diterpene isosteviol and its derivatives. Molecules
24:678
respectively. This implies isosteviol a hopeful potential on
anti-cancer activity.
Acknowledgements Financial support from National Natural Science
Foundation of China (31772017, 31371837), the national frst-class
discipline program of Light Industry Technology and Engineering
(LITE2018-03), and the project of outstanding scientifc & techno-
logical innovation group of Jiangsu Province are gratefully appreciated.
7. Garifullin BF, Strobykina IY, Khabibulina LR, Sapunova AS,
Voloshina AD, Sharipova RR, Khairutdinov BI, Zuev YF, Kataev
VE (2019) Synthesis and cytotoxicity of the conjugates of diterpe-
noid isosteviol and N-acetyl-D-glucosamine. Nat Prod Res. https
://doi.org/10.1080/14786419.2019.1650355
Compliance with ethical standards
8. Liu CJ, Zhang T, Yu SL, Dai XJ, Wu Y, Tao JC (2017) Synthesis,
cytotoxic activity, and 2D-and 3D-QSAR studies of 19-carboxyl-
modified novel isosteviol derivatives as potential anticancer
agents. Chem Biol Drug Des 89:870–887
Conflict of interest There are no conficts to declare.
9. Malki A, El-Sharkawy A, El Syaed M, Bergmeier S (2017) Anti-
tumor activities of the novel isosteviol derivative 10C against liver
cancer. Anticancer Res 37:1591–1601
References
10. Meninno S (2019) Valorization of waste: sustainable organo-
catalysts from renewable resources. Chemsuschem. https://doi.
org/10.1002/cssc.201902500
1. Dolder F, Lichti H, Mosettig E, Quitt P (1960) The Structure
and Stereochemistry of Steviol and Isosteviol. J Am Chem Soc
82:246–247
11. Mathur S, Bulchandani N, Parihar S, Shekhawat GS (2017) Criti-
cal review on steviol glycosides: Pharmacological, toxicological
and therapeutic aspects of high potency zero caloric sweetener.
Int J Pharmacol 13:916–928
2. Mosettig E, Beglinger U, Dolder F, Lichti H, Quitt P, Waters JA
(1963) The absolute confguration of steviol and isosteviol. J Am
Chem Soc 85:2305–2309
12. Ruiz-Ruiz JC, Moguel-Ordonez YB, Segura-Campos MR (2017)
Biological activity of Stevia rebaudiana Bertoni and their relation-
ship to health. Crit Rev Food Sci 57:2680–2690
3. Sharipova RR, Andreeva OV, Garifullin BF, Strobykina IY, Stro-
bykina AS, Voloshina AD, Kravchenko MA, Kataev VE (2018)
Synthesis and antimicrobial and antituberculosis activity of the
frst conjugates of the diterpenoid isosteviol and D-arabinofura-
nose. Chem Nat Compd 54:92–97
13. Carrera-Lanestosa A, Moguel-Ordonez Y, Segura-Campos M
(2017) Stevia rebaudiana Bertoni: a natural alternative for treat-
ing diseases associated with metabolic syndrome. J Med Food
20:933–943
4. Liu Y, Wang TT, Ling Y, Bao N, Shi W, Chen L, Sun JB (2017)
Design, synthesis and cytotoxic evaluation of nitric oxide-releas-
ing derivatives of isosteviol. Chem Biol Drug Des 90:473–477
1 3

Bioprocess and Biosystems Engineering
27. Ye J, Pei X, Cui H, Yu Z, Lee H, Wang J, Wang X, Sun L, He
H, Yang VC (2018) Cellular uptake mechanism and comparative
in vitro cytotoxicity studies of monomeric LMWP-siRNA conju-
gate. J Ind Eng Chem 63:103–111
14. Zhang H, Sun X, Xie Y, Tian F, Hu H, Tan W (2018) Isosteviol
sodium inhibits astrogliosis after cerebral ischemia/ reperfusion
injury in rats. Biol Pharm Bull 41:575–584
15. Avent AG, Hanson JR, Hitchcock PB, De Oliveira BH (1990)
The infuence of a 15-hydroxy group on the rearrangement reac-
tions of steviol and its 16,17-epoxide. J Chem Soc Perkin Trans
1:2661–2665
28. Oliveira BH, Strapasson RA (1996) Biotransformation of isoste-
viol by Fusarium verticilloides. Phytochemistry 43:393–395
29. Strobykina IY, Belenok MG, Semenova MN, Semenov VV,
Babaev VM, Rizvanov IK, Mironov VF, Kataev VE (2015) Triph-
enylphosphonium cations of the diterpenoid isosteviol: synthesis
and antimitotic activity in a sea urchin embryo model. J Nat Prod
78:1300–1308
16. Khaibullin RN, Strobykina IY, Kataev VE, Lodochnikova OA,
Gubaidullin AT, Musin RZ (2009) New synthesis of diterpenoid
(16S)-dihydrosteviol. Russ J Gen Chem 79:967–971
17. Li HP, Dong CM, Hou YM, Liu HM (2009) Synthesis of 17-OH-
Isosteviol. Chem Res Chin Univ 25:116–117
30. Mizushina Y, Akihisa T, Ukiya M, Hamasaki Y, Murakami-
Nakai C, Kuriyama I, Takeuchi T, Sugawara F, Yoshida H
(2005) Structural analysis of isosteviol and related compounds
as DNA polymerase and DNA topoisomerase inhibitors. Life Sci
77:2127–2140
18. Lohoelter C, Weckbecker M, Waldvogel SR (2013) (–)-Isosteviol
as a versatile ex-chiral-pool building block for organic chemistry.
Eur J. Org Chem 2013:5539–5554
19. Perera WH, Docampo ML, Wiggers FT, Huford CD, Fronczek
FR, Avula B, Khan IA, McChesney JD (2018) Endocyclic double
bond isomers and by-products from rebaudioside A and stevioside
formed under acid conditions. Phytochem Lett 25:163–170
20. Wan HD, He GZ, Zhang HJ (2019) Isosteviol preparation and
inclusion complexation of it with -cyclodextrin. J Incl Phenom
Macro 94:65–73
31. Momtazi-Borojeni AA, Esmaeili SA, Abdollahi E, Sahebkar A
(2017) A review on the pharmacology and toxicology of steviol
glycosides extracted from stevia rebaudiana. Curr Pharm Design
23:1616–1622
32. Takasaki M, Konoshima T, Kozuka M, Tokuda H, Takayasu J,
Nishino H, Miyakoshi M, Mizutani K, Lee KH (2009) Cancer
preventive agents. Part 8: chemopreventive efects of stevioside
and related compounds. Bioorgan Med Chem 17:600–605
33. Bazotte RB, Lonardoni MTC, Alvarez M, Gaeti WP, Amado
CAB (1986) Determination of the lethal dose (LD50) for the
lethal isosteviol in laboratory animals. Dev Med Child Neurol
18:103–116
21. Cherney EC, Green JC, Baran PS (2013) Synthesis of ent-kaurane
and beyerane diterpenoids by controlled fragmentations of over-
bred intermediates. Angew Chem Int Ed 52:9019–9022
22. Milagre HMS, Martins LR, Takahashi JA (2009) Novel agents for
enzymatic and fungal hydrolysis of stevioside. Brazilian Journal
of Microbiology 40:367–372
34. Yoshikawa R, Kusunoki M, Yanagi H, Noda M, Furuyama J-i,
Yamamura T, Hashimoto-Tamaoki T (2001) Dual antitumor
efects of 5-fuorouracil on the cell cycle in colorectal carcinoma
cells: a novel target mechanism concept for pharmacokinetic
modulating chemotherapy. Can Res 61:1029–1037
23. Víctor-Ortega MD, Ochando-Pulido JM, Martínez-Ferez A (2016)
Iron removal and reuse from Fenton-like pretreated olive mill
wastewater with novel strong-acid cation exchange resin fxed-
bed column. J Ind Eng Chem 36:298–305
24. Chen JM, Xia YM, Wan HD, Wang HJ, Liu X (2014) A complete
specifc cleavage of glucosyl and ester linkages of stevioside for
preparing steviol with a β-galactosidase from Sulfolobus solfa-
taricus. J Mol Catal B Enzym 105:126–131
Publisher’s Note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional afliations.
25. Chen JM, Ding L, Sui XC, Xia YM, Wan HD, Lu T (2016) Pro-
duction of a bioactive sweetener steviolbioside via specifc hydro-
lyzing ester linkage of stevioside with a beta-galactosidase. Food
Chem 196:155–160
26. Chen J-M, Zhang J, Xia Y-M, Wang X-X, Li J (2018) The natural
sweetener metabolite steviol inhibits the proliferation of human
osteosarcoma U2OS cell line. Oncol Lett 15:5250–5256
1 3