Synthesis and Bioactivity Characterization of Scutellarein Sulfonated Derivative

Article abstract of DOI:10.3390/molecules22061028

Scutellarin (1) has been widely used to treat acute cerebral infarction in clinic, but poor aqueous solubility decreases its bioavailability. Interestingly, scutellarin (1) could be metabolized into scutellarein (2) in vivo. In this study, a sulfonic group was introduced at position C-8 of scutellarein (2) to enhance the aqueous solubility of the obtained derivative (3). DPPH (1,1-diphenyl-2-picrylhydrazyl)-radical scavenging ability and antithrombic activity were also conducted to determine its bioactivity. The result showed that scutellarein derivate (3) could be a better agent for ischemic cerebrovascular disease treatment.

Full text of DOI:10.3390/molecules22061028

molecules
Article
Synthesis and Bioactivity Characterization of
Scutellarein Sulfonated Derivative
Ting Gu 1,2, Yue Zhong 1,2, Yu-Ting Lu 1,2, Ying Sun 1,2, Ze-Xi Dong 1,2, Wen-Yu Wu 1,2
,
Zhi-Hao Shi 3, Nian-Guang Li 1,2,*, Xin Xue 1,2, Fang Fang 1,2, He-Min Li 1,2 and Yu-Ping Tang 1,
*
1
National and Local Collaborative Engineering Center of Chinese Medicinal Resources Industrialization and
Formulae Innovative Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, Jiangsu, China;
guting1992@163.com (T.G.); 18260028701@163.com (Y.Z.); lyt1996tian@outlook.com (Y.-T.L.);
18260028601@163.com (Y.S.); dongzexi1215@163.com (Z.-X.D.); 15150513147@163.com (W.-Y.W.);
xuexin0540121@163.com (X.X.); fylffh@163.com (F.F.); lihemin2006@sina.com (H.-M.L.)
Department of Medicinal Chemistry, Nanjing University of Chinese Medicine, Nanjing 210023,
Jiangsu, China
2
3
Department of Organic Chemistry, China Pharmaceutical University, Nanjing 211198, Jiangsu, China;
sszh163@163.com
*
Correspondence: linianguang@njutcm.edu.cn (N.-G.L.); yupingtang@njutcm.edu.cn (Y.-P.T.);
Tel./Fax: +86-25-8581-1916 (N.-G.L.); +86-25-8581-1916 (Y.-P.T.)
Received: 27 May 2017; Accepted: 20 June 2017; Published: 21 June 2017
Abstract: Scutellarin (
but poor aqueous solubility decreases its bioavailability. Interestingly, scutellarin (
metabolized into scutellarein ( in vivo. In this study, a sulfonic group was introduced at
position C-8 of scutellarein ( ) to enhance the aqueous solubility of the obtained derivative ( ).
DPPH (1,1-diphenyl-2-picrylhydrazyl)-radical scavenging ability and antithrombic activity were
1) has been widely used to treat acute cerebral infarction in clinic,
1
) could be
2)
2
3
also conducted to determine its bioactivity. The result showed that scutellarein derivate (
3) could be
a better agent for ischemic cerebrovascular disease treatment.
Keywords: scutellarin; scutellarein; sulfonated derivative; solubility; antioxidant; antithrombic
1. Introduction
Ischemic cerebrovascular disease is a common and high-incidence disease that threatens human
health seriously by severe neuronal injury and loss of brain function [
of ischemic cerebrovascular disease, and causes 9% of all deaths worldwide [
1
]. Stroke is a typical symptom
]. Thus, serious harm of
2
individual’s physical, mental, and social functioning can occur as a result of ischemic cerebrovascular
diseases [3,4].
Erigeron breviscapus (Vant.) Hand-Mazz is one of the well-known Chinese herbs, and has
an extensive application in traditional Chinese medicine treatment. The mechanism of E. breviscapus
has aroused increasing attention because of its significant curative effect. Liu studied the antioxidant
activity of the ethanol extract of E. breviscapus, and revealed it played an important role in
neuroprotective actions [
applications in neurological diseases due to the inhibition of
(GABA-T) and succinic semialdehyde dehydrogenase (SSADH) [6].
By modern analysis methods, scutellarin (40,5,6-trihydroxyflavone-7-glucuronide) (
could be confirmed as an important active ingredient in Erigeron breviscapus (Vant.) Hand-Mazz [7,8]
Ke found that scutellarin could inhibit hepatocellular carcinoma (HCC) cell metastasis in vivo
and migration and invasion in vitro by downregulating the STAT3/Girdin/Akt signaling [ ]. Further,
in Li’s research, scutellarin could attenuate vasospasm and neurological deficits by modulating the
5]. Tao also found that E. breviscapus ethanol extract had therapeutic
γ
-aminobutyric acid transaminase
1
) (Figure 1)
.
,
9
Molecules 2017, 22, 1028; doi:10.3390/molecules22061028
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Molecules 2017, 22, 1028
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Erk5-KLF2-eNOS pathway after subarachnoid hemorrhage (SAH) [10]. Furthermore, Zhao found that
scutellarin was a promising therapeutic agent for the prevention of wear particle-induced periprosthetic
osteolysis [11]. In addition, scutellarin (1) is effective in treating cerebral ischemia, angina pectoris,
myocardial infarction, stroke, cerebral thrombotic diseases and other kinds of cardiovascular diseases
and cerebrovascular injury [12].
Figure 1. Structures of scutellarin (1) and scutellarein (2).
However, because of the poor aqueous solubility (14.4
low bioavailability in Beagle dog (0.4%) [14], there is not a wide range of applications of scutellarin
) in clinic. Structure modification and dosage form transformation are two common methods used
±
0.17 µg/mL) [13], and the absolute oral
(1
to increase the bioavailability of this kind of natural product. By the approaches of being bound to
polyrotaxane (PR) [15], adopting different lipid excipients such as cremophor EL [16], or synthesizing
glucose-containing scutellarein derivatives [17], the bioavailability of scutellarin (1) can be improved
to some extent.
Scutellarein (
in vivo 18]. Our previous work indicated that scutellarein (
cerebral ischemia than scutellarin ( ) [19 20]. Nevertheless, the problem of poor aqueous solubility is
2) (Figure 1) is the hydrolysate of scutellarin (1), and is the main absorbing form
[
2
) displayed better protective effect in rat
1
,
still not resolved due to the flavonoid skeleton of scutellarein (2).
Sulfonation is an important reaction in increasing the solubility of flavonoids while retaining
the chemical properties of the parent compounds [21]; as a well-known type of in vivo metabolic
conversion, it also enhances the aqueous solubility of the ingested compounds [22]. Quercetin is
a typical flavonoid with an antiproliferative effect on a wide range of cancer cells [23
–25]. Cui, Woz´nicka,
and Zhang introduced sulfonic groups on quercetin [21 22 26], and all of these quercetin derivatives
,
,
showed good aqueous solubility on the basis of bioactivity preservation.
Therefore, in this research, we synthesized the sulfonated derivative of scutellarein (2),
and a series of biological activity assays including aqueous solubility, (1,1-diphenyl-2-picrylhydrazyl)
(DPPH)-radical scavenging ability, and antithrombic activity were also conducted to study its
biological activity.
2. Results and Discussion
2.1. Chemical Synthesis
In this study, one of our previous methods was applied to synthesize scutellarein (
hydrolysis of scutellarin ( ) [19]. In this method, 3.0 mol/L H2SO4 in 90% ethanol and heating
under a N2 atmosphere at 120 C for 48 h were selected as the best condition for the synthesis of
scutellarein ( ), and the yield could be up to 17.3%. Subsequently, concentrated sulfuric acid—as
2) by the
1
◦
2
reactant and solvent—was adopted to introduce a sulfonic group at the C-8 position of the scutellarein
◦
(2
). The reaction temperature should be controlled at about 25 C to avoid the side reaction in B
ring. According to the 1HNMR spectrum of
both 8.7, which indicated that the sulfonic group was not at C20,60 and C30,50 positions. Furthermore,
the hydrogen signal at 6.72 of C3-H was still present; this information confirmed that the sulfonic
group was introduced at the C-8 position. The synthesis route is shown in Scheme 1.
3
, the coupling constants of C20,60-H and C30,50-H were
δ

Molecules 2017, 22, 1028
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◦
Scheme 1. The route of the synthesis of scutellarein derivate (
(b) H2SO4, 25 C, 3 h, 18%.
3). (
a
) H2SO4, EtOH, 120 C, 48 h, 17.3%;
◦
2.2. Aqueous Solubility
The aqueous solubility of scutellarin (
1
), scutellarein (
2
), and
3
were determined by UV
spectrophotometry [27 ), scutellarein
), and were 7.62, 6.85, and 1949.64 µg/mL, respectively. The results showed that the introduction
–29]. As presented in Table 1, the aqueous solubility of scutellarin (1
(2
3
of the sulfonic group increased its aqueous solubility significantly compared to scutellarin (1) and
scutellarein (2).
Table 1. The solubility in water and antioxidant activity of scutellarin (1), scutellarein (2), and 3. DPPH:
(1,1-diphenyl-2-picrylhydrazyl).
Compd.
Solubility (µg/mL)
DPPH (µM)
Scutellarin (1)
Scutellarein (2)
(3)
7.62
6.85
1949.64
17.88
16.05
16.78
2.3. DPPH-Radical Scavenging Ability Assay
Antioxidants play important roles in removing free radicals, as antioxidants provide hydrogen
to free radicals and prohibit the adverse reactions from destroying normal tissues. This assay was
measured according to previous studies [30]. The results are displayed in Table 1.
As shown in the results, scutellarin (1), scutellarein (2), and 3 all exhibited good DPPH-radical
scavenging ability in this assay, as the values of IC50 were 17.88, 16.05, and 16.78 µM, respectively,
which indicated that compound 3 still had antioxidant activity.
2.4. Antithrombic Assay
According to our previous studies, the antithrombotic activity could be estimated by measuring
the prolongation of the plasma clotting time of thrombin time (TT), activated partial thromboplastin
time (APTT), international normalized ratio (INR) increase in prothrombin time (PT), and reduction in
fibrinogen (FIB) content [31]. The results are displayed in Table 2.
Table 2. The thrombin inhibition activity of scutellarin (1), scutellarein (2), and 3. APTT: activated
partial thromboplastin time; FIB: fibrinogen; PT: prothrombin time; TT: thrombin time.
Plasma Coagulation Parameters
Compd.
TT (s)
APTT (s)
PT (s)
FIB (g/L)
Blank plasma
Scutellarin (1)
Scutellarein (2)
3
19.85 ± 1.59
23.25 ± 1.55
24.48 ± 1.18
23.71 ± 1.82
29.83 ± 3.86
33.78 ± 2.32
36.12 ± 2.20
30.33 ± 1.06
5.03 ± 0.24
6.25 ± 0.08
5.93 ± 0.81
6.33 ± 1.87
7.23 ± 0.38
6.41 ± 0.15
6.91 ± 0.12
7.01 ± 0.96
Data represent mean ± SD n = 4.

Molecules 2017, 22, 1028
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As the results show, the antithrombotic activity was remained when the glucuronyl group in
scutellarin ( ) was hydrolyzed to produce scutellarein ( ); although the PT decreased and the FIB
content increased in scutellarein ( ), the TT and APTT in scutellarein ( ) increased compared with
those in scutellarin ( ); this result indicated that the glucuronyl group was not important for the
antithrombotic activity.
The results also indicated that scutellarein derivative with a sulfonic group at the C-8 position (
1
2
2
2
1
3
)
could retain the antithrombotic activity. On the one hand, the introduction of a sulfonic group at the
C-8 position increased TT and PT significantly compared with the control group. Specifically, the TT
prolongation of scutellarein derivative (
to scutellarein ( ) (24.48 s); nevertheless, the PT prolongation of scutellarein derivative (
superior to scutellarin ( ) (6.25 s) and scutellarein ( ) (5.93 s). On the other hand, the introduction of
a sulfonic group at the C-8 position of scutellarein ( ) increased APTT and decreased FIB to some degree
in comparison to the control group; however, the improvement of APTT (30.33 s) and FIB (7.01 g/L)
3) (23.71 s) was better than scutellarin (1) (23.25 s), but inferior
2
3) (6.33 s) was
1
2
3
was not as good as scutellarin (
FIB: 6.91 g/L).
1
) (APTT: 33.78 s, FIB: 6.41 g/L) and scutellarein (2) (APTT: 36.12 s,
Thus, the introduction of sulfonic group at C-8 position of the scutellarein (
antithrombotic activity of the parent compounds.
3) could retain the
3. Experimental Section
3.1. General Information
Scutellarin (1) was purchased from Sichuan Mianning Jiexiang Materials Co. Ltd. (Chengdu,
China). Reagents and solvents were purchased from commercial sources and used without further
purification unless otherwise specified. All non-aqueous reactions were carried out under nitrogen
production using flame-dried glassware; the anhydrous solvents were transferred via syringe or
◦
stainless steel cannula. Organic solvents were concentrated below 45 C by Büchi rotary evaporator at
approximately 20 mm Hg. Then, 0.15–0.20 mm silica gel plates (RSGF 254, Yantai, China), as thin-layer
chromatography (TLC), was used to monitored all of the reactions in 254 nm UV light. Chromatography
was carried out on silica gel (160–200 mesh, Qingdao, China) with mixtures of petroleum ether (60–90)
and ethyl acetate as eluant. The 1H NMR spectroscopy was carried out on a Bruker AV-300 (300 MHz)
(Bruker Corporation, Karlsruhe, Germany) in DMSO-d6. Abbreviations used are s (singlet), d (doublet),
t (triplet), q (quartet), b (broad), and m (multiplet). Mass spectrometry (MS) were performed on
a Waters Synapt HDMS spectrometer equipped with an electrospray ionization source (ESI).
3.2. Synthesis
3.2.1. 5,6,7-Trihydroxy-2-(4-hydroxyphenyl)-4H-chromen-4-one (2)
Concentrated H2SO4 (100 mL) was added into a stirring mixture of 1 (10 g, 21.65 mmol) and water
(10 mL) in ethanol (90 mL), and the reaction mixture was refluxed for 48 h under nitrogen and then was
allowed to cool to room temperature. Water (1000 mL) was added, and the solid that appeared was
filtered and then purified by column chromatography on silica gel with 50% ethyl acetate in petroleum
ether as eluent to afford
1 (1.05 g, 17% yield) as a yellow solid. IR (KBr): 3343.24, 1663.08, 1610.20,
1580.85, 1458.41, 1364.24, 1270.26, 1252.35, 1183.51, 1079.25, 1028.97, 1004.24, 830.86, 721.26, 598.71,
1
50740.65 cm−1. H-NMR (DMSO-d6) 6.73 (s, 1H, 8-H),
δ
6.78 (s, 1H, 3-H), 6.90–6.93 (d, 2H, J = 8.8 Hz
,
0
0
0
3 ,5 -H), 7.90–7.93 (d, 2H, J = 8.8 Hz, 2 ,6 -H), 8.71 (s, 1H, 6-OH), 10.30 (s, 1H, 4 -OH), 10.44 (s, 1H, 7-OH),
12.79 (s, 1H, 5-OH); ESI-MS: m/z 285 [M
C, 62.92; H, 3.49. The IR and H-NMR spectrums were included in the Supplementary Materials
available online.
−
H]−; Anal. Calcd. for C15H10O6: C, 62.94; H, 3.52; Found:
1

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3.2.2. 5,6,7-Trihydroxy-2-(4-hydroxyphenyl)-4-oxo-4H-chromene-8-sulfonic acid (3)
Concentrated sulfuric acid (15 mL) was added to
2 (1.0 g) in a 50 mL round-bottom flask,
the reaction mixture was allowed to continue stirring for 12 h at 25 ◦C. After that, 25 mL of saturated
sodium chloride solution was added into the mixture, and the mixture was kept for 4 h before filtering.
The orange–red precipitate was washed with saturated sodium chloride solution until the pH value of
the filtrate was up to 7, and was then recrystallized twice from the hot saturated aqueous solution;
3
(230 mg, 18% yield) was obtained as yellow solid. IR (KBr): 3272.90, 2918.66, 1659.91, 1592.18,
1558.24, 1472.13, 1417.77, 1373.40, 1321.34, 1259.45, 1229.59, 1163.55, 1105.26, 1025.93, 672.43, 579.93,
1
522.46 cm−1. H-NMR (DMSO-d6)
δ
6.72 (1H, s, 3-H), 6.97–6.99 (2H, d, J = 8.7 Hz, 30,50-H), 8.27–8.30
(2H, d, J = 8.7 Hz, 20,60-H), 8.55 (1H, s, 6-OH), 11.13 (1H, s, 4-OH), 12.52 (1H, s, 7-OH), 12.99 (1H,
s, 5-OH); ESI-MS: m/z 365 [M
−
H]−; Anal. Calcd. for C15H10O9S: C, 49.19; H, 2.75; S, 8.75; Found:
C, 49.21; H, 2.78; S. 8.77. The IR and 1H-NMR spectrums were included in the Supplementary Materials
available online.
3.3. Aqueous Solubility
The solubility of scutellarin (
method [27 29]. According to multicomponent exploitation method, 334 nm could be considered
as the wavelength of maximum absorbance of scutellarein ( ) by UV absorption spectrophotometry.
Scutellarin ( ) and scutellarein ( ) (300 g) were dissolved in 25 mL CH3OH, and (300 g) was
dissolved in 25 mL water. The solutions of these three compounds had concentrations ranging from
3 to 12 g/mL. Standard curves were determined on the basis of the absorbances of test samples
obtained by UV scanning, and all of them showed a good linear relationship. Each tested compound
(250
g) was ultrasound dissolved in 10 mL pure water for 1 h at 25 ◦C, and the solutions were stood
1), scutellarein (2), and 3 in water was determined using the known
–
2
1
2
µ
3
µ
µ
µ
for 30 min before centrifuging at the speed of 30,000 r/min. Absorbances of each compound were
obtained by UV scanning, and the aqueous solubility of all three compounds were obtained through
analysis of the standard curve.
3.4. DPPH-Radical Scavenging Ability Assay
The following method was adopted to evaluate the 1,1-diphenyl-2-picrylhydrazyl (DPPH)
scavenging property of the products: scutellarin (
to result in final concentrations ranging from 16 to 250
1
), scutellarein (
2
), and
3
were dissolved in DMSO
mol/L, 100 L)
µmol/L, and then DPPH (80
µ
µ
dissolved in DMSO was added in the testing samples. After incubating for 30 min in the dark,
the scavenging effect was calculated according to the following equation:
Scavenging effect (%) = [(1 − (A1 − A2)/A0] × 100%
(1)
where A0 is the absorbance of the control (without extract), A1 is the absorbance in the presence of the
extract, and A2 is the absorbance without DPPH.
3.5. Antithrombic Assay
Male New Zealand white rabbits weighing 2–2.5 kg were obtained from the experimental animal
center of Nanjing University of Chinese Medicine and were approved by Animal Ethics Committee
of Nanjing University of Chinese Medicine. They were kept in plastic cages at 22
±
2 ◦C with free
access to pellet food and water and on a 12 h light/dark cycle. Animal welfare and experimental
procedures were carried out in accordance with the guide for the care and use of laboratory animals
(National Research Council of USA, 1996) and related ethical regulations of Nanjing University of
Chinese Medicine. Rabbits were anesthetized with pentobarbital (50 mg/kg) and blood was drawn
from the common carotid artery. Blood was collected into plastic tubes with 3.8% sodium citrate
(citrate/blood: 1:9, v/v) for plasma anticoagulation. Platelet-poor plasma (PPP) was separated from
blood by centrifugation at 3000 rpm for 10 min.

Molecules 2017, 22, 1028
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TT, APTT, PT, and FIB were tested with commercial kits following the manufacturer’s instructions
by a coagulometer (Model LG-PABER-I, Steellex Co., Beijing, China). All the compounds were
dissolved in PBS with 20% DMSO, and the concentration was 100
µ
M. TT was determined by incubating
L thrombin solution and 20
L sample solution and 50 L PPP
L CaCl2.
40
µ
L PPP solution for 3 min at 37 ◦C, followed by addition of 40
µ
µL
◦
sample for 3 min at 37 C. APTT was determined by incubating 10
µ
µ
◦
solution with 50
PT was determined by incubating 40
40 L thromboplastin agent and 20
with 90
µ
L APTT-activating agent for 3 min at 37 C, followed by the addition of 50
µ
µ
L PPP solution for 3 min at 37 ◦C, followed by the addition of
µ
µ
L sample. FIB was determined by incubating 10 µL PPP solution
µ
L imidazole buffer for 3 min at 37 ◦C, followed by addition of 50
µL FIB agent and 10 µL
sample solution. By measuring the prolongation of the plasma clotting time of thrombin time (TT),
activated partial thromboplastin time (APTT), INR increase in prothrombin time (PT), and reduction
in fibrinogen (FIB) content, the antithrombotic activity was assessed.
4. Conclusions
Scutellarin (
nevertheless, clinical application is limited because of its poor solubility and low bioavailability. In this
study, we introduced a sulfonic group at the C-8 position of scutellarein ( ), which increased the
aqueous solubility obviously. Further, DPPH-radical scavenging ability assay and antithrombic assay
also proved that the sulfonated derivative ( ) still retained the bioactivity of parent compounds.
) could be an efficient agent for ischemic cerebrovascular
1) is effective in treating cardiovascular diseases and cerebrovascular injury [12];
2
3
This kind of scutellarein derivative (
3
disease treatment.
Supplementary Materials: The following are available online: IR and 1H-NMR spectrums of 2 and 3.
Acknowledgments: This work was supported by National Natural Science Foundation of China (No. 81274058,
21302225), China Scholarship Council (No. 201407060046), Natural Science Foundation of Jiangsu Province
(BK20151563), 333 High-level Talents Training Project Funded by Jiangsu Province, Six Talents Project Funded
by Jiangsu Province (2013-YY-010), Program for Excellent Talents in School of Pharmacy of Nanjing University
of Chinese Medicine (15ZYXET-1), Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources
Industrialization (ZDXMHT-1-13), Project Funded by the Priority Academic Program Development of Jiangsu
Higher Education Institutions and Project Funded by the Flagship Major Development of Jiangsu Higher Education
Institutions (PPZY2015A070), Key Laboratory of Therapeutic Material of Chinese Medicine, Jiangsu Province,
State Key Laboratory Cultivation Base for TCM Quality and Efficacy, Nanjing University of Chinese Medicine.
Author Contributions: T.G., Y.Z., Y.-T.L., Y.S., Z.-X.D., W.-Y.W., X.X., F.F. and H.-M.L. did the experiments, N.-G.L.
designed the synthetic route, Z.-H.S. wrote this paper and Y.-P.T. revised this paper.
Conflicts of Interest: All authors declare no financial/commercial conflict of interests.
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Sample Availability: Samples of the compounds 1, 2 and 3 are available from the authors.
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