Synthesis and antitumor activity of sacroflavonoside

Article abstract of DOI:10.1080/14786419.2019.1645660

Sacroflavonoside, a new derivative of diphenylethene, was isolated from Artemisia sacrorum, which have been found to possess the inhibitory effect on the proliferation of gastric carcinoma cells (MKN-45) in vitro in our previous studies. With anisaldehyde (SM-A) as starting material, the sacroflavonoside was synthesized by nucleophilic addition, electrophilic substitution and dehydration cyclization. The structure of sacroflavonoside was established by 1 D (¹H NMR and ¹³C NMR) and 2 D-NMR (HSQC and HMBC) spectral analysis. The antitumor activity and potential mechanism against MKN-45 cells of sacroflavonoside were evaluated in vitro. The results showed that sacroflavonoside could significantly induce MKN-45 cells apoptosis and autophagy by increasing the expression of Bax, Caspase-3, Beclin1 and LC3-II proteins and decreasing the expression of Bcl-2 protein at low micromole level. This investigation provided a valuable lead structure for the development of antitumor drugs.

Full text of DOI:10.1080/14786419.2019.1645660

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20
Synthesis and antitumor activity of
sacroflavonoside
Qinghu Wang, Ying Xin, Biligetu Pa, Xiang He & Wenqiang Bao
To cite this article: Qinghu Wang, Ying Xin, Biligetu Pa, Xiang He & Wenqiang Bao (2019):
Synthesis and antitumor activity of sacroflavonoside, Natural Product Research, DOI:
10.1080/14786419.2019.1645660
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2019.1645660
SHORT COMMUNICATION
Synthesis and antitumor activity of sacroflavonoside
ꢀ
Qinghu Wang, Ying Xin , Biligetu Pa, Xiang He and Wenqiang Bao
College of Traditional Mongolian Medicine, Inner Mongolia University for Nationalities, Tongliao,
Inner Mongolia, China
ABSTRACT
ARTICLE HISTORY
Received 24 May 2019
Accepted 14 July 2019
Sacroflavonoside, a new derivative of diphenylethene, was iso-
lated from Artemisia sacrorum, which have been found to possess
the inhibitory effect on the proliferation of gastric carcinoma cells
(MKN-45) in vitro in our previous studies. With anisaldehyde (SM-
A) as starting material, the sacroflavonoside was synthesized by
nucleophilic addition, electrophilic substitution and dehydration
cyclization. The structure of sacroflavonoside was established by
1 D (1H NMR and 13C NMR) and 2 D-NMR (HSQC and HMBC) spec-
tral analysis. The antitumor activity and potential mechanism
against MKN-45 cells of sacroflavonoside were evaluated in vitro.
The results showed that sacroflavonoside could significantly
induce MKN-45 cells apoptosis and autophagy by increasing the
expression of Bax, Caspase-3, Beclin1 and LC3-II proteins and
decreasing the expression of Bcl-2 protein at low micromole level.
This investigation provided a valuable lead structure for the
development of antitumor drugs.
KEYWORDS
Sacroflavonoside;
Diphenylethene; Synthesis;
Antitumor activity
1. Introduction
Although the treatment of cancer had made significant progress, it remains the lead-
ing cause of death worldwide. It was reported that annual cancer cases will rise to 22
million in the next two decades (Song 2015). The treatments of cancer mainly include
surgery, chemotherapy and radiotherapy. However, surgery is usually the treatments
of choice for early stage cancers, for patients with unrespectable disease, local control
CONTACT Qinghu Wang
wqh196812@163.com
College of Traditional Mongolian Medicine, Inner Mongolia
University for Nationalities, Tongliao 028000, Inner Mongolia, China
ꢀ
The author contributed equally to this work
Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2019.1645660.
ß 2019 Informa UK Limited, trading as Taylor & Francis Group

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Q. WANG ET AL.
Figure 1. Synthesis route of sacroflavonoside.
can be achieved with chemotherapy combined with radiotherapy or some combin-
ation (Torre et al. 2015; Li et al. 2015). At present, chemotherapy is still the main type
of cancer treatment, and phytochemicals have been a crucial part of anti-cancer drugs.
Indeed, more than half of the approved anticancer drugs are either natural products
or developed on the basis of knowledge gained from natural products. In addition,
about 75% of plant-derived drugs used today in the clinic come from traditional medi-
cines (Li-Weber 2009).
The aerial parts of Artemisia sacrorum Ledeb. (family Composite), also named
Hareshabake in Mongolian, is one of the most popular and multi-purpose herbs used
in China. In the traditional Mongolian medicines, A. sacrorum are widely used for clin-
ical treatment of insecticidal, analgesic, convergence of pus and yellow water, detu-
mescence, breaking the lump and so on (Wang et al. 2015). Breaking the lump is a
classification type of Mongolian medicine efficacy, equivalent to the antitumor effect
of modern medicine (Wang et al. 2015). It was found that the extract of dichlorome-
thane and ethyl acetate of A. sacrorum had certain toxicity on breast cancer cells
(MCF-7) and gastric cancer cells (MKN45) in vitro in our previous studies (Yu et al.
2016). The chemical constituents of A. sacrorum reported mainly contained terpenoids

NATURAL PRODUCT RESEARCH
3
(Wu et al. 1994; Zhang 1998), coumarins (Zhang and Li 1993) and flavonoids (Zhang
et al. 1990).
Sacroflavonoside, a new derivative of diphenylethene, is isolated from A. sacrorum.
Diphenylethens are the secondary metabolites with a C6-C2-C6 skeleton and possesses
a variety of biological activities including antitumor, antihypertensive, antioxidant, and
so on (Busquets et al. 2007; Csiszar et al. 2009). It has been showed that sacroflavono-
side had inhibitory effect on the proliferation of MNK45 cells in vitro in our previous
studies, with relatively low IC50 value of 5.03 lM (Yu et al. 2016). However, it is difficult
to get enough sacroflavonoside by isolation and purification due to the low content
in A. sacrorum.
In the present study, we carried out the synthesis of sacroflavonoside by using
nucleophilic addition, electrophilic substitution and dehydration cyclization. The struc-
ture of sacroflavonoside was established by 1 D (1H NMR and 13C NMR) and 2 D-NMR
(HSQC, HMBC and HR-MS) spectral analysis. To further investigate the potential use of
sacroflavonoside as antitumor compound, the mechanism of antitumor effects of
sacroflavonoside was studied.
2. Results and discussion
2.1. Synthesis of sacroflavonoside
Scroflavonoside was synthesized by nucleophilic addition, electrophilic substitution
and dehydration cyclization. A synthetic route to the compound is illustrated in
Figure 1. When intermediate-01 (Int-01) was synthesized, trimethylcyanosilane was
used as reactant instead of potassium cyanide in this experiment. It was first carried
out in dichloromethane, and the yield was only 35%. When the solvent was changed
to acetonitrile, the yield increased to 59%. During the synthesis of intermediate-02
(Int-02), yellow viscous liquid was easily obtained at room temperature, which reduced
the yield. The reaction was controlled in ice bath and satisfactory results are obtained.
Under the experimental conditions, the yield of scroflavonoside was 55%. The purity
of scroflavonoside was 97.0% after separation and purification by silica gel column
chromatography. In conclusion, a facile synthetic route of scroflavonoside was found
by studying the reaction temperature and solvent conditions.
2.2. Structure elucidation of sacroflavonoside
The structure of sacroflavonoside was lucidated on the basis of spectroscopic methods
1
including IR, 1 D NMR, 2 D NMR and HR-ESI-MS. In the H NMR spectrum, a signal was
observed as a singlet at 5.10 ppm. This proton signal provided correlation with the car-
bon at 87.9 ppm in 2 D HSQC, leading to its assignment as one oxidated methylene
proton (Figure 2a). The proton showed strong HMBC correlations with C-4 (dC
104.1 ppm), C-3 (dC 183.7 ppm) and C-2’,6’ (dC 122.4 ppm) attached to C-2 at 87.9 ppm
(Figure 2b), indicating the formation of furanone ring. Moreover, the anomeric proton
at C-1’’ position (5.05 ppm, J ¼ 7.5 Hz) appeared as a doublet (Figure S1a) indicating it
was a b-D-glucopyranose based on the spectral data with those reported in the litera-
ture (Tanaka et al. 2007) and the coupling constant of the anomeric proton. The

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Q. WANG ET AL.
HMBC correlations (Figure S1b) from this proton to C-7 (dC 163.2 ppm) indicated
that the b-D-glucopyranose was attached to C-7. All the proton carbon correlations
were studied to deduce the structure of this compound and it was named as sacro-
flavonoside. The IR analysis ascertained the presence of carbonyl (1677 cmꢁ1).
Further, it was confirmed by high resolution mass which was in close agreement
with the calculated mass ([M-H]- for C21H22O10, calculated: m/z 433.1135, found: m/
z 433.1144).
2.3. Influence of sacroflavonoside on apoptosis and autophagy
In our previous study, we have demonstrated that sacroflavonoside can inhibit the
growth of different digestive tumor cells, and MKN45 was the most sensitive one
among the gastric cancer cell lines tested with relatively low IC50 value of 5.03lM (Yu
et al. 2016). With a view to determine whether sacroflavonoside inhibit the prolifer-
ation of MKN45 cells by activating apoptotic pathway and another autophagic path-
way, we observed the expression of Bax, Bcl-2, Caspase-3, Beclin1 and LC3B by
Western blot analysis. The results showed that the expression of Bax, Caspase-3,
Beclin1 and LC3-II proteins increased and the expression of Bcl-2 protein decreased in
MKN45 cells treated with sacroflavonoside (Figure S2).
Caspase-3, as an important member of Caspases family that initiates and executes
endogenous and exogenous apoptosis induced by various inducing factors, is a key
factor in the interaction of endogenous and exogenous pathways of apoptosis (Islam
et al. 2000). Activation of Caspase-3 can cause DNA repair and apoptosis by hydrolyz-
ing PARP in the nucleus (Li et al. 2004). The activity of caspase-3 in MKN45 cells was
increased 24 h after sacroflavonoside was added. Bcl-2 family proteins (including Bad,
Bax, Bid, Bcl-2, and Bcl-xL) play a key role in mitochondrial-mediated apoptosis. Bad
and Bax induce apoptosis by promoting mitochondrial release of cytochrome c and
Smac. Bcl-2 and BclxL are known as anti-apoptotic proteins because they inhibit mito-
chondrial-mediated apoptosis by blocking mitochondrial release of cytochrome c or
Smac. A shift in the balance between anti-apoptotic and pro-apoptotic proteins in the
Bcl-2 family could trigger mitochondrial-dependent caspase activation and further
induce cell apoptosis (Danial and Korsmeyer 2004; Green and Kroemer 2004).
Sacroflavonoside can affect the expression of the Bcl-2 family of proteins by up-regu-
lating Bax and down-regulating Bcl-2. Thus, it is possible that sacroflavonoside induces
mitochondrial release of cytochrome-C and Smac by affecting the expression of the
bcl-2 family of proteins.
On the other hand, there was increase in Beclin-1, which was identified as regula-
tory factors of autophagy by its existence in pro-autophagic complexes (Kang et al.
2011). LC3 is a recognized autophagic marker that can be detected in the initiation of
autophagy. LC3-II is a truncated form of LC-3, and is necessary to combine with the
seclusion membrane for wrapping around cytoplasm components fundamentally at
random in the primary stage of autophagy (Takeuchi et al. 2005). These results sug-
gest that treatment with sacroflavonoside induces activation of two types of cell death
pathway, not only apoptosis but also autophagy.

NATURAL PRODUCT RESEARCH
5
3. Experimental
3.1. General experimental procedures
Anisaldehyde, trimethylsilyl cyanide, phloroglucin and tetraacetyl-a-D- bromide glucose
was purchased from Aladdin Company, China. All the other reagents and chemicals
were purchased from commercial suppliers and used as received unless otherwise
stated. When needed, the reactions were carried out in oven dried glassware under a
positive pressure of dry nitrogen. Melting points were determined on a STUART-SMP3
melting point apparatus without correction. IR spectra were recorded on PE-2000
spectrometer in KBr pellets and were reported in cmꢁ1. All NMR spectra were recorded
on a Bruker AV-II 500 MHz NMR spectrometer with CDCl3 or DMSO-d6 as solvent and
tetramethylsilane (TMS) as the internal standard, operating at 500 MHz for 1H, and
125 MHz for 13C, respectively. All chemical shift values were reported in units of d
(ppm). The following abbreviations were used to indicate the peak multiplicity:
s ¼ singlet; d ¼ doublet; t ¼ triplet; m ¼ multiplet; br ¼ broad. MS spectra were recorded
on a Waters Xevo Q-TOF HRMS instrument. Reactions and chromatography fractions
were performed on silica gel (200-300mesh) using the indicated eluents and thin-layer
chromatography was carried out on silica gel plates with a layer thickness of 0.25 mm.
3.2. Synthesis of sacroflavonoside
To a solution of anisaldehyde (1.02 g, 7.5 mmol) in acetonitrile (36 mL) was added tri-
methylsilyl cyanide 1.18 mL (0.878 g, 8.8 mmol) at room temperature and zinc iodide
(0.0306 g) in the environment of nitrogen protection successively. The reaction mixture
was refluxed for 1.5 h and then filtered and concentrated. The residue of p-methyl
benzol cyanohydrin (1.63 g, 1.0 mmol) was dissolved in absolute ether (90 mL).
Phloroglucin (1.38 g, 10.9 mmol) dried by vacuum in 110 ꢂC for 3 hours is added to the
above solution. The reaction mixture was cooled immediately in ice bath and then
poured into dry hydrochloric acid gas for 1 h, and was placed in the refrigerator for
3 days and then filtered, washed with ether and removed aether. The product was
added to 150 mL aqueous solution in hydrochloric acid (0.1 mmol/L), refluxed for 3 h,
filtered and washed with water, dried by vacuum, and got coarse powder called
Int-02.
To a solution of Int-02 (136 mg, 0.5 mmol) in acetonitrile (10 mL), was added tetraa-
cetyl-a-D- bromide glucose (369 mg, 0.9 mmol) and then was stirred evenly. To the
reaction mixture was added silver oxide (173 mg, 0.75 mmol) and then stirred evenly
for 5 h in the environment of nitrogen protection successively. The reaction liquid was
filtered, concentrated and purified by silica gel chromatography with petroleum ether/
acetone (4/1, v/v) to afford the pure product as a yellow needle.
Sacroflavonoside: Yellow needle. UV (MeOH) kmax nm (log e): 281 (3.92); IR (neat) ꢀ
1
(cmꢁ1): 3452, 1668, 1517; H-NMR(500 MHz, DMSO-d6) d: 5.10 (1H, s, H-2), 6. 47 (1H, d,
J ¼ 2.0 Hz, H-5), 6.72 (1H, d, J ¼ 2.0 Hz, H-7), 7.36 (2H, d, J ¼ 9.0 Hz, H-2’, 6’), 7.08 (2H, d,
J ¼ 9.0 Hz, H-3’, 5’), 3.89 (3H, s, OCH3), 5.05 (1H, d, J ¼ 7.5 Hz, H-1’’). 13C-NMR (125 MHz,
DMSO-d6) d: 87.9 (C-2), 183.7 (C-3), 161.5 (C-4), 100.5 (C-5) , 163.2 (C-6), 95.2 (C-7),
154.9 (C-8), 104.1 (C-9), 144.8 (C-1’), 122.4 (C-2’), 115. 9 (C-3’), 158.3 (C-4’), 115.9 (C-5’),

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Q. WANG ET AL.
122.4 (C-6’), 56.5 (OCH3), 100.3 (C-1’’), 76.9 (C-2’’), 73.5 (C-3’’), 70.0 (C-4’’), 77.6 (C-5’’),
61.0 (C-6’’). HR-ESI-MS: m/z 433.1144 [M-H]- (calcd for C21H22O10, 433.1135).
3.3. Cell lines and culture conditions
Human gastric cancer cell line MKN-45 was purchased from American Type Culture
Collection (ATCC), and was cultivated in RPMI 1640 medium according to the man-
ufacturer’s instructions. Medium was supplemented with 10% FBS, and all tumor cells
were incubated at 37 ꢂC and 5% CO2 incubator.
3.4. Western blot analysis
Cells (1 ꢃ 106cells/mL) grown in six-well plates were incubated at 37 ꢂC for 24 h with
furanoside of Artemisia sacrorum treatment at various concentrations (2.5 lmol,
5 lmol). Proteins from cell lysates were extracted using a Tissue or Cell Total Protein
Extraction Kit containing protease and phosphatase inhibitor cocktails. Cell lysates
were centrifuged at 20,000 g for 10 min at 4ꢂC, and protein concentration in super-
natant was measured by using MicroBCA Protein Assay Reagent Kit. Protein lysates
were heated in 99 ꢂC for 10 min before being mixed evenly and centrifuged slightly.
The proteins were separated by SDS-PAGE electrophoresis and transferred to nitrocel-
lulose membranes followed by blocking for 2 h with 5% nonfat milk dissolved in
water. The membranes were incubated with primary antibodies (cleaved GAPDH, Bax,
Bcl-2, Caspase-3, Beclin1 and LC3B) overnight at 4 ꢂC. All antibodies for Western blot
were purchased from Santa Cruz Biotechnology, Santa Cruz, CA, USA. GAPDH was
tested in the same gel as internal control for the loading sample. The membranes
were incubated with fuorescent antibodies at room temperature for 2 h. After being
washed, the bound antibodies were detected by the ECL Western blot detection
system. Western blot analyses were repeated 3 times.
4. Conclusion
In conclusion, the present study demonstrated that sacroflavonoside could induce
apoptosis and autophagy of the human gastric cancer cell line MKN45. The apoptotic
pathway triggered by sacroflavonoside in MKN45 cells was dependent on mitochon-
drial and caspase-3 pathways, also controlled by the ratio of Bcl-2/Bax. The autophagy
pathway activated by sacroflavonoside in MKN45 cells was dependent on Beclin-1.
Sacroflavonoside may be useful as a novel effective reagent in treating gastric cancers,
which generally have poor outcomes. We plan to investigate the induction mechanism
of tumour cell death in detail, especially how sacroflavonoside turns the cell death
programme on, and continue research for in vivo models of antitumour therapy.
Disclosure statement
No potential conflict of interest was reported by the authors.

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Funding
This work was supported by the National Natural Science Foundation of China (No. 81660698).
Jazan University for providing grant to conduct this study through Ethnopharmacology
Research Group.
References
ꢀ
ꢀ
Busquets S, Ametller E, Fuster G, Olivan M, Raab V, Argiles JM, Lopez-Soriano FJ. 2007.
Resveratrol, a natural diphenol, reduces metastatic growth in an experimental cancer model.
Cancer Lett. 245(1–2):144–148.
Csiszar A, Labinskyy N, Olson S, Pinto JT, Gupte S, Wu JM, Hu F, Ballabh P, Podlutsky A,
Losonczy G, et al. 2009. Resveratrol prevents monocrotaline-induced pulmonary hypertension
in rats. Hypertension. 54(3):668–675.
Danial NN, Korsmeyer SJ. 2004. Cell death: critical control points. Cell. 116(2):205–219.
Green DR, Kroemer G. 2004. The pathophysiology of mitochondrial cell death. Science.
305(5684):626–629.
Islam S, Islam N, Kermode T, Johnstone B, Mukhtar H, Moskowitz RW, Goldberg VM, Malemud
CJ, Haqqi TM. 2000. Involvement of caspase-3 in epigallocatechin-3-gallatemediated apoptosis
of human chondrosarcoma cells. Biochem Biophys Res Commun. 270(3):793–797.
Kang R, Zeh HJ, Lotze MT, Tang D. 2011. The Beclin 1 network regulates autophagy and apop-
tosis. Cell Death Differ. 18(4):571–580.
Li-Weber M. 2009. New therapeutic aspects of flavones: the anticancer properties of Scutellaria
and its main active constituents Wogonin, Baicalein and Baicalin. Cancer Treat Rev. 35(1):
57–68.
Li YH, Wang C, Meng K, Chen LB, Zhou XJ. 2004. Influence of survivin and caspase-3 on cell
apoptosis and prognosis in gastric carcinoma. World J Gastroenterol. 10(13):1984–1988.
Li Z, Zhang Y, Jin T, Men J, Lin Z, Qi P, Piao Y, Yan G. 2015. NQO1 protein expression predicts
poor prognosis of non-small cell lung cancers. BMC Cancer. 15:207–209.
Song M. 2015. Progress in discovery of KIF5B-RET kinase inhibitors for the treatment of non-
small-cell lung cancer. J Med Chem. 58(9):3672–3681.
Takeuchi H, Kondo Y, Fujiwara K, Kanzawa T, Aoki H, Mills GB, Kondo S. 2005. Synergistic aug-
mentation of rapamycin-induced autophagy in malignant glioma cells by phosphatidylinositol
3-kinase/protein kinase B inhibitors. Cancer Res. 65(8):3336–3346.
Tanaka T, Nakashima T, Ueda T, Tomii K, Kouno I. 2007. Facile discrimination of aldose enan-
tiomers by reversed-phase HPLC. Chem Pharm Bull. 55(6):899–905.
Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, Jemal A. 2015. Global cancer statistics,
2012. CA Cancer J Clin. 65(2):87–108.
Wang QH, Wu RJ, Qi GQ, Wu JS, Dai NYT, Na RCGT. 2015. Chemical Constituents of Artemisa sac-
rorum Ledeb. Chin Pharm J. 50:1380–1384.
Wu LJ, Ban XD, Wang CX. 1994. Study on the chemical constituents of Artemisa sacrorum
Ledeb. J Shenyang Pharm Univ. 11:54–57.
Yu H, Wang QH, Han N, Tu BX, Bu R. 2016. Isolation and identification of six terpenes from the
petroleum ether extracts from Artemisa sacrorum Ledeb. Chin J Med Chem. 26:142–145.
Zhang DZ. 1998. Isolition and identification of two new ent-kauranoid diterpenes from Artemisa
sacrorum Ledeb. Nat Prod Res Dev. 10:34–37.
Zhang DZ, Li X. 1993. Studies on the aromatic constituents from Artemisa sacrorum Ledeb. Chin
Trad Herb Drug. 24:283–286.
Zhang DZ, Li X, Zhu YR. 1990. Study on the chemical constituents of Artemisa sacrorum Ledeb.
J Shenyang Pharm Univ. 6:146–149.