Synthesis and antioxidant activities of flavonoids derivatives, troxerutin and 3', 4', 7-triacetoxyethoxyquercetin

Article abstract of DOI:10.1016/j.cclet.2013.01.016

Syntheses of two very important derivatives of quercetin, troxerutin and 3', 4', 7-triacetoxyethoxy-quercetin were described. The latter was synthesized by highly selective esterification reaction in first time. The compounds were characterized by NMR, IR and Mass spectroscopy. Additionally, the antioxidant activities of the compounds were tested by means of improved pyrogallol autoxidation method. This was the first in using this method to test the antioxidant activities of these two compounds in vitro. The optimum system of pyorgallol autoxidation spectrophotometry was investigated and established according to the reaction rules. The assay indicated that these compounds showed noticeable antioxidant activities, and compound 2 was much more effective as a free radical scavenger than the compound 1 vitamin C was used as a reference material.

Full text of DOI:10.1016/j.cclet.2013.01.016

Chinese Chemical Letters 24 (2013) 223–226
Contents lists available at SciVerse ScienceDirect
Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
Original Article
Synthesis and antioxidant activities of flavonoids derivatives, troxerutin
and 3’, 4’, 7-triacetoxyethoxyquercetin
*
Jian-Dong Xu, Li-Wei Zhang, Yu-Fa Liu
College of Chemistry, Chemical Engineering and Materials Science, Engineering Research Center of Pesticide and Medicine Intermediate Clean Production, Ministry of Education,
Shandong Normal University, Jinan 250014, China
A R T I C L E I N F O
A B S T R A C T
Article history:
Syntheses of two very important derivatives of quercetin, troxerutin and 3’, 4’, 7-triacetoxyethoxy-
quercetin were described. The latter was synthesized by highly selective esterification reaction in first
time. The compounds were characterized by NMR, IR and Mass spectroscopy. Additionally, the
antioxidant activities of the compounds were tested by means of improved pyrogallol autoxidation
method. This was the first in using this method to test the antioxidant activities of these two compounds
in vitro. The optimum system of pyorgallol autoxidation spectrophotometry was investigated and
established according to the reaction rules. The assay indicated that these compounds showed
noticeable antioxidant activities, and compound 2 was much more effective as a free radical scavenger
than the compound 1 vitamin C was used as a reference material.
Received 12 November 2012
Received in revised form 9 December 2012
Accepted 21 December 2012
Available online 5 February 2013
Keywords:
Quercetin
Troxerutin
3’,4’, 7-Triacetoxyethoxyquercetin
Antioxidant
ß 2013 Yu-Fa Liu. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
1. Introduction
the inhibition of platelet aggregation and the prevention of
thrombosis [16]. Therefore, it is used as a treatment against the 5-
Flavonoids, such as flavones and flavonols, are plant polyphe-
nolic compounds, which are found in many foods, especially in
fruits and vegetables [1]. As the most common natural flavonoid,
quercetin and its derivatives are widely present in nuts, beverages
and Chinese herbal medicine. Given their broad range of
pharmacological effects and biological activities, such as efficient
anti-oxidant and scavenging oxygen free radicals injury [2,3],
quercetin and its derivatives not only are very efficient drugs for
reducing the blood pressure [4], but also appear to be active in
many diseases related to aging such as vasodilatation [5,6],
cardiovascular [7], neurodegenerative [8] diseases and cancer [9],
especially ovarian cancer, breast cancer and leukemia [10,11].
Furthermore, an enormous number of scientific studies indicate
that they possess antimicrobial activity [12], anti-inflammatory
and analgesic properties [13]. Nevertheless, only a small percent-
age of the ingested quercetin can be absorbed by the human body,
as previously reported [14]. Thus, arduous efforts should be made
to increase their solubility and slow down their metabolism by
chemical modification of the natural compound on the premise of
maintaining the ability to regenerate the original molecules [15].
Troxerutin (trihydroxy-ethylrutoside) possesses extensive
pharmacological activities. Its mechanism is probably related to
hydroxytryptamine, bradykinin-induced vascular injury and
increases the healing of capillary endothelial defects. Moreover,
it can also reduce capillary permeability and prevent edemas
caused by increasing vascular permeability [17,18]. Thus, two
quercetin derivatives were designed and synthesized by using
rutin as a substrate, expecting to obtain more active derivatives. In
addition, there are several methods for determining the superoxide
scavenging activities of foods, including cytochrome c reduction,
electron spin resonance (ESR), chemiluminescence [19], and so on.
All of these methods require special and expensive instruments or
biological agents. Yet, pyrogallol (1,2,3-trihydroxybenzene) can
autoxidize in alkaline solutions to produce ꢀO2ꢁ anion radicals and
colored intermediate products, quinones, which are easily
detected by a spectrophotometer. The antioxidant can inhibit
the autoxidation of pyrogallol. Thus, it can inhibit the colored
intermediate products. So the absorbance reflects the generation of
both oxidation products and superoxide radicals (ꢀO2ꢁ). The
improved pyrogallol method is a reliable and cheap superoxide-
scavenging assay suitable for many types of flavonoids. So the
scavenging activities of the compounds were evaluated by means
of an improved pyrogallol autoxidation method. And what is more,
in previous reports, flavonoids played a remarkable role in
antioxidant activities mainly by means of chelating with variable
valence metal ion or scavenging oxygen free radicals [20]. The first
site involved the complex formation process was the 3-hydroxy-4-
carbonyl in the original structure [21].
* Corresponding author.
E-mail address: yufaliu@sdnu.edu.cn (Y.-F. Liu).
1001-8417/$ – see front matter ß 2013 Yu-Fa Liu. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
http://dx.doi.org/10.1016/j.cclet.2013.01.016

224
J.-D. Xu et al. / Chinese Chemical Letters 24 (2013) 223–226
OH
OH
OH
O
O
HO
O
O
O
O
O
OH
HO
a
O
+
b
O
O
OH
rutinose
OH
rutinose
1
O
O
O
+
O
O
c
O
O
O
O
O
O
O
O
OH
OH
O
2
Scheme 1. Condition and regent: (a) NaOH; (b) H2O; (c) H2SO4.
2. Experimental
The pyrogallol autoxidation system was established according
to previous studies [22,23]. A pyrogallol solution (60 mmol/L, in
1 mol/L HCl, 0.10 mL) was thoroughly mixed with Tris–HCl
(physiological pH = 7.4, 5.00 mL) buffer containing 1 mmol/L
Na2EDTA. Additionally, ultrapure water (4.90 mL) was added
slowly into the system. After the mixture was vigorously shaken,
the absorbance of the mixture was measured by UV–vis at RT
(Fig. 1). The procedure was repeated, using different concentration
solutions of three drugs, 4 ꢂ 10ꢁ6 mol/L, 8 ꢂ 10ꢁ6 mol/L,
12 ꢂ 10ꢁ6 mol/L, 16 ꢂ 10ꢁ6 mol/L, 20 ꢂ 10ꢁ6 mol/L. The protocol
of velocity determination of pyrogallol autoxidation was shown in
Table 1.
Ethylene oxide (6.60 g, 150 mmol) and NaOH (0.28 g, 7 mmol)
were added to a solution of rutin (12.21 g, 20 mmol) in H2O
(100 mL) at room temperature. After stirring at 75 8C for 6 h, the pH
value of the mixture was adjusted to 4.0 by concentrated
hydrochloric acid. Then, the yellow solid was formed. The crude
product was collected by filtration and washed with cold water. It
was repeatedly recrystallized by methyl alcohol and the yellow
powder, compound 1, was obtained. The purity was 99.84% tested
by HPLC (CH3OH/H2O = 8:1; v/v) equipment (Shimadzu).
Troxerutin (1.00 g, 1.35 mmol) was dissolved in acetic anhy-
dride (20.00 mL) in a 250 mL round-bottomed flask and the
solution was stirred for half an hour at room temperature. H2SO4
(0.40 mL, 50%) was added dropwise into the reaction system on the
stir condition. After completion of the addition, the resulting
solution was stirred at RT for 2 h followed by keeping it at RT for
5 h. An additional 150 mL of ultrapure water was added and the
resulting mixture was stirred at RT for 2 h until the faint yellow
deposition separated out. Extract the deposited material and wash
them with ultrapure water several times to obtain the faint yellow
crystals. The filter cake was dried in a vacuum oven to give 0.57 g
(57%, m) of the desired compound 2 (Scheme 1).
3. Results and discussion
Compound 1. 1H NMR (300 MHz, DMSO-d6):
d 12.50 (s, 1 H, 5-
OH), 7.83 (s, 1H, 20-ArH), 7.70 (d, 1H, J = 8.46 Hz, 60-ArH), 7.13–7.10
(d, 1H, J = 8.73 Hz, 50-ArH), 6.72 (s, 1H, 8-ArH), 6.36 (s, 1H, 6-ArH),
4.95–4.89 (m, 3H, CH2CH2O*H), 4.10–4.04 (m, 6H, OC*H2CH2OH),
3.73–3.28 (m, 6H, OCH2C*H2OH), 0.96–0.93 (m, 3H, CH3). 13C NMR
(75 MHz, DMSO-d6):
d
60.0 (30,40,7-O-CH2C*H2OH), 70.63 (30,40,7 -
O-*CH2CH2OH), 98.8 (6-ArH), 105.5 (10-ArH), 113.3 (20-ArH), 114.9
(50-ArH), 122.8 (60-ArH), 123.0 (10-ArH), 134.1 (3-C), 148.0 (30-
ArH), 151.4 (40-ArH), 156.9 (2-C), 157.0 (5-ArH), 161.3 (9-C), 165.1
(7-ArH), 177.9 (4-C). mp 185.2–187.0 8C. The 13C NMR data agrees
well with previous studies [24].
2.00
Compound 2. 1H NMR (300 MHz, DMSO-d6):
d 12.38 (s, 1H, 5-
1.75
(321,1.681)
.
OH), 9.71 (s, 1H, 3-OH), 7.86–7.88 (d, 1H, J = 8.52 Hz, 20-ArH), 7.81
(d, 1H, J = 7.20 Hz, 60-ArH), 7.17–7.20 (d, 1H, J = 8.70 Hz, 50-ArH),
6.80 (s, 1H, 8-ArH), 6.37 (s, 1H, 6-ArH), 4.02–4.34 (m, 12H,
OC2*H4O ꢂ 3), 2.04 (s, 9H, COC*H3). 13C NMR (75 MHz, DMSO-d6):
1.50
1.25
1.00
0.75
d
21.05(*CH3CO), 62.58–63.04 (30, 40, 7-OCH2*CH2- ꢂ 3), 67.10–
68.02 (30, 40, 7-O*CH2CH2- ꢂ 3), 93.22 (C-8), 98.25 (C-6), 104.71 (C-
10), 114.51 (C-20), 115.10 (C-50), 122.97 (C-60), 124.45 (C-10),
Table 1
(436,0.459)
0.50
0.25
0.00
.
ꢁ
Sample preparation for the elimination experiment of ꢀO2 of compounds.
No.
Tris–HCla
(mL)
Ultrapure
water/mL
Samples (mL)
Pyrogallol
(mL)
1
2
VC
225 250 275 300 325 350 375 400 425 450 475 500
W (nm)
1
2
3
4
5
5
5
5
4.9
4.8
4.8
4.8
0
0
0
0.1
0.1
0.1
0.1
0.1
0
0
0
0.1
0
0
Fig. 1. Absorption spectrum of pyrogallol autoxidation at different wavelengths.
(For interpretation of the references to color in this figure legend, the reader is
referred to the web version of this article.)
0
0.1
a
The concentration of Tris–HCl is 50 mmol/L

J.-D. Xu et al. / Chinese Chemical Letters 24 (2013) 223–226
225
0 mol/L
1.5
1.0
0.5
(A)
0 mol/L
1.5
1.0
0.5
(B)
-6
4 × 10 mol/L
-6
4 ×10 mol/L
-6
12 × 10 mol/L
-6
12 ×10 mol/L
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
T
T
Fig. 2. Effect of compound 1(A) and 2(B) concentration on their ꢀO2 elimination activity.
ꢁ
Table 2
selective esterification reaction from the reaction mechanism of
synthesis of compounds. The glycosyl in 3-O-rutinose of com-
pound 1 was substituted by 3-OH in compound 2. Only three
acetoxyethoxy groups in 7, 3’, 4’ were prepared by acylation on the
strict controlled reaction condition. The results indicated that
compound 1 and 2 were all showed antioxidant activity. However,
the compound 2 was much more effective in inhibiting the
antioxidant of pyrogallol than the compound 1. A possible reason
was that chemical structure modification enhanced its complex
ability.
ꢀO2 elimination rate of compounds (%).
ꢁ
Compounds
Concentration (ꢂ10ꢁ6 mol/L)
4
8
12
16
20
1
2
14.28
16.32
15.07
15.79
18.18
21.66
18.35
24.63
29.81
25.47
31.07
38.11
32.84
40.16
55.37
VC
137.18 (C-3), 146.66 (C-30), 148.13 (C-2), 150.54 (C-40), 156.52 (C-
9), 160.90 (C-5), 164.30 (C-7), 170.73–170.77 (CH3*CO ꢂ 3), 176.59
(C = O). m.p.139.2–140.5 8C. IR (Bruker Tensor 27, KBr, cmꢁ1):
Acknowledgment
v
3299, 1737, 1650, 1379, 1259. ESI-MS: m/z 561.5 (M + 1).
Elemental Anal. (Perkin Elmer PE 2400 II CHNS/O): calcd. for
27H28O13 (560.50): C, 57.85; H, 5.03; O, 37.11. Found: C, 57.90; H,
5.0; O, 37.1.
As shown in Fig. 1, two bands, 321 nm and 436 nm, were
obtained in our experimental conditions. In earlier studies [25],
We sincerely appreciate the determinations of NMR, IR, MS and
elemental analysis conducted by Zhi-Xian Liu, Li-Xin Yu and Xin-
Jian Qiu.
C
Appendix A. Supplementary data
ꢁ
420 nm was selected as the monitoring wavelength for ꢀO2
generation. However, the UV spectra showed that 321 nm was
much more sensitive than 436 nm. In this study, 321 nm was
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.cclet.2013.01.016.
therefore regarded as the best monitoring wavelength for
References
ꢀ
detecting concentrations of O2ꢁ. The ratio of
DA/min was in the
specified range. The absorbance of the solutions at 321 nm was
measured up to 5 min. The oxidation rate of pyrogallol for samples
[1] P.C.H. Hollman, I.C.W. Arts, Flavonols, flavones and flavanols-nature, occurrence
and dietary burden, J. Sci. Food Agric. 80 (2000) 1081–1093.
[2] Y.L. Mi, C.Q. Zhang, C.M. Li, et al., Quercetin attenuates oxidative damage induced
by treatment of embryonic chicken spermatogonial cells with 4-nitro-3-phenyl-
phenol in diesel exhaust particles, Biosci. Biotechnol. Biochem. 5 (2010) 934–938.
[3] B. da cruz. Pa´dua, L.D. Silva, J.V. Rossoni, et al., Antioxidant properties of Baccharis
trimera in the neutrophils of Fisher rats, J. Ethnopharmacol. 3 (2010) 381–386.
[4] J. Duarte, R. Jimenez, F. OValle, et al., Protective effects of the flavonoid quercetin
in chronic nitric oxide deficient rats, J. Hypertens. 20 (9) (2002) 1843–1854.
[5] G. Bobe, P.S. Albert, L.B. Sansbury, et al., Interleukin-6 as a potential indicator for
prevention of high-risk adenoma recurrence by dietary flavonols in the polyp
prevention trial, Cancer Prev. Res. (Phila Pa) 6 (2010) 764–775.
[6] N.K. Khoo, C.R. White, R.P. Patel, et al., Dietary flavonoid quercetin stimulates
vasorelaxation in aortic vessels, Free Radic. Biol. Med. 3 (2010) 339–347.
[7] M. Yoshizumi, K. Tsuchiya, K. Kirima, et al., Quercetin inhibits shc-and phospha-
tidylinositol 3-kinase-mediated c-Jun N-terminal kinase activation by angioten-
sin II in cultured rat aortic smooth muscle cells, Mol. Pharmacol. 60 (2001)
656–665.
was calculated as the slope of the absorbance line (
DA1). The value
of A321nm was measured against the Tris–HCl buffer every 30 s for
ꢀ
ꢁ
5 min. The O2 scavenging ability was calculated as:
Scavenging (%) = [(A0 ꢁ A) ꢃ A0] ꢂ 100%
Here, A0 was the absorbance of the reaction mixture without
trolox, and A was that with trolox.
The results of effect of compound 1 (A) and 2 (B) concentration
ꢀ
ꢁ
on its O2 elimination activity were shown in Fig. 2. The results
suggested that the antioxidant activity of the compound 2 was
more sensitive in the same concentration than theꢁcompound 1.
Effect of compounds concentration on their ꢀO2 elimination
activities was investigated and the results showed in Table 2. The
scavenging rate was easily figured out. The antioxidant activity of
compound 2 made up 81.46% of the activity of VC, and compound 1
was 66.69% in our experimental conditions.
[8] N. Suematsu, M. Hosoda, K. Fujimori, Protective effects of quercetin against
hydrogen peroxide-induced apoptosis in human neuronal SH-SY5Y cells,
Neurosci. Lett. 504 (2011) 223–227.
[9] G.J. Du, H.H. Lin, Y.M. Yang, et al., Dietary quercetin combining intratumoral
doxorubicin injection synergistically induces rejection of established breast
cancer in mice, Int. Immunopharmacol. 10 (2010) 819–826.
4. Conclusion
[10] S. Kwaii, Y. Tomono, E. Katase, K. Ogawa, M. Yano, Effect of citrus flavonoids on HL-
60 cell differentiation, Anticancer Res. 19 (2A) (1999) 1261–1269.
[11] B. Csokay, N. Prajda, G. Weber, E. Olah, Molecular mechanisms in the antipro-
liferative action of quercetin, Life Sci. 64 (24) (1997) 2157–2163.
[12] A. Chatzopoulou, A. Karioti, C. Gousiadou, et al., Depsides and other polar con-
stituents from Origanum dictamnus L. and their in vitro antimicrobial activity in
clinical strains, J. Agric. Food Chem. 58 (2010) 6064–6068.
In recent years, the studies focusing on the flavonoids and their
bioactivities have been increasing gradually [26]. Our chemical
structure modification and activity test of flavonoids are of great
importance and necessity. The structure was modified via highly

226
J.-D. Xu et al. / Chinese Chemical Letters 24 (2013) 223–226
[13] C. Delporte, N. Backhouse, et al., Analgesic-antiinflammatory properties of Prous-
tia pyrifolia, J. Ethnopharmacol. 99 (2005) 119–124.
fluorescence and lucigenin-enhanced chemiluminescence techniques, Free Radic.
Biol. Med. 29 (2000) 388–396.
[14] J. She, L.E. Mo, T.B. Kang, Z.J. Song, Liang N.C, Quercetin water-soluble derivatives
preparation and biological activity, Chin. J. Med. Chem. 8 (4) (1998) 287–289.
[15] K.H. Wong, G.Q. Li, K.M. Li, V.R. Naumovski, K. Chan, Kudzu root: traditional uses
and potential medicinal benefits in diabetes and cardiovascular diseases, J.
Ethnopharmacol. 134 (2011) 584–607.
[16] S.H. Fan, Z.F. Zhang, Y.L. Zheng, et al., Troxerutin protects the mouse kidney from
D-galactose-caused injury through anti-inflammation and anti-oxidation, Int.
Immunopharmacol. 9 (2009) 91–96.
[17] C.M. Liu, J.Q. Ma, Y. Lou, Chronic administration of troxerutin protects mouse
kidney against D-galactose-induced oxidative DNA damage, Food Chem. Toxicol.
48 (2010) 2809–2817.
[18] A. Celik, O. Ersoy, N. Ozkan, et al., Comparison of the effects of troxerutin
and heparinoid on flap necrosis, J Plast. Reconstr. Aesthetic Surg. 63 (2010)
875–883.
[20] J.M. You, J.T. Lan, Extraction technology of naringin from orange, Modern Food Sci.
Technol. 2 (2008) 160–166.
[21] T.L. Lin, B.Z. Yan, G.F. Hu, W. Mei, Spectral analysis of AI (11I)-quercetin com-
plexes, Chin. J. Anal. Chem. 8 (2006) 1125–1128.
[22] X.C. Li, Improved pyrogallol autoxidation method: a reliable and cheap superox-
ide-scavenging assay suitable for all antioxidants, J. Agric. Food Chem. 60 (2012)
6418–6424.
[23] S.H. Han, J.B. Zhu, Y.Y. Wang, Measurement of the antioxidant activity by
pyrogallol autoxidation, China Brewing 207 (6) (2009) 115–157.
[24] Y.M. Xiao, P. Mao, Z.H. Zhao, L.R. Yang, X.F. Lin, Regioselective enzymatic acylation
of troxerutin in nonaqueous medium, Chin. Chem. Lett. 21 (2010) 59–62.
[25] J.L. Li, M. Zhang, T.S. Zheng, The in vitro antioxidant activity of lotus germ oil from
supercritical fluid carbon dioxide extraction, Food Chem. 115 (2009) 939–944.
[26] J.G. Cao, X. Xia, X.F. Chen, J.B. Xiao, Q.X. Wang, Characterization of flavonoids from
dryopteris erythrosora and evaluation of their antioxidant, anticancer and acetyl-
cholinesterase inhibition activities, Food Chem. Toxicol. 51 (2013) 242–250.
[19] M.A. Barbacanne, J.P. Souchard, B. Darblade, et al., Detection of superoxide anion
released extracellularly by endothelial cells using cytochrome c reduction, ESR,