Two new flavones from Tridax procumbens Linn

Article abstract of DOI:10.3390/molecules15096357

Two new flavones, 8,3′-dihydroxy-3,7,4′-trimethoxy-6-O-β- D-glucopyranosyl flavone (1) and 6,8,3′-trihydroxy-3,7,4′- trimethoxyflavone (2) were isolated from Tridax procumbens Linn., together with the four known compounds puerarin (3), esculetin (4), olea

Full text of DOI:10.3390/molecules15096357

Molecules 2010, 15, 6357-6364; doi:10.3390/molecules15096357
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Two New Flavones from Tridax procumbens Linn.
Runsheng Xu 1,2, Jing Zhang 3 and Ke Yuan 1,*
1
Nurturing Station for the State Key Laboratory of Subtropical Silviculture, Zhejiang Agriculture
and Forestry University, Linan 311300, China
2
College of Chemistry and Life Science, Zhejiang Normal University, Jinhua 321004, China
3
College of Pharmacy, Henan University of Traditional Chinese Medicine, Zhengzhou 450008,
China
* Author to whom correspondence should be addressed; E-Mail: yuan_ke001@yahoo.com.cn;
Tel.: +86-571-63743607; Fax: +86-571-63740809.
Received: 10 July 2010; in revised form: 11 August 2010; Accepted: 13 August 2010 /
Published: 9 September 2010
Abstract: Two new flavones, 8,3′-dihydroxy-3,7,4′-trimethoxy-6-O-β-D-glucopyranosyl
flavone (1) and 6,8,3′-trihydroxy-3,7,4′-trimethoxyflavone (2) were isolated from Tridax
procumbens Linn., together with the four known compounds puerarin (3), esculetin (4),
oleanolic acid (5) and betulinic acid (6). The structures of the two new flavones were
elucidated based on chemical analysis and spectral methods (IR, 1D and 2D NMR, ESI-
MS, HR-ESI-MS). The antioxidant activity of the two new flavones were evaluated by two
methods, the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity and ferric
reducing antioxidant power (FRAP) assays, and the data showed that compounds 1 and 2
have certain antioxidant activity, with the antioxidant activity of compound 2 being
stronger than that of compound 1.
Keywords: Tridax procumbens Linn; 8,3′-dihydroxy-3,7,4′-trimethoxy-6-O-β-D-
glucopyranosyl flavone; 6,8,3′-trihydroxy-3,7,4′-trimethoxyflavone; antioxidant activity;
DPPH; FRAP
1. Introduction
Tridax procumbens Linn., growing in the tropical areas of the Americas, belongs to the Compositae
family [1]. It was introduced into China in the 1940s [2], and has been used in traditional medicine by

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the native populations to treat bronchitis, dysentery, diarrhoea and to prevent hair loss. Recently, the
chemical components of its essential oils have been investigated [3,4]. In this article, we report the
isolation and structural identification of six compounds, including three flavones: 8,3′-dihydroxy-
3,7,4′-trimethoxy-6-O-β-D-glucopyranosyl flavone (1), 6,8,3′-trihydroxy-3,7,4′-trimethoxyflavone (2),
and puerarin (3) [5], one coumarin, esculetin (4) [6], and two triterpenoids: oleanolic acid (5) [7] and
betulinic acid (6) [8] (Figure 1). Among them, compounds 1 and 2 are two new flavones and
compounds 3~6 were isolated from this species for the first time. This paper presents the isolation and
structural elucidation of compounds 1 and 2, and their antioxidant activity, evaluated using the well
known DPPH and FRAP assays.
Figure 1. Structures of compounds 1-6.
OH
OH
OCH3
O
OH
OH
HO
HO
OH
H3CO
RO
O
O
OH
O
O
OCH3
:R=Glu
:R=H
1
2
3
COOH
COOH
HO
HO
O
O
HO
HO
4
5
6
2. Results and Discussion
Compound 1 was obtained as a yellow amorphous powder. The HR-ESI-MS showed an ion at m/z
1
545.1274 [M+Na]+ (calcd 545.1271), corresponding to the molecular formula, C24H26O13. The H-
NMR spectrum displayed three aromatic protons at δ 7.65 (1H, dd, J = 8.0, 2.0 Hz), 7.03 (1H, d,
J = 8.0 Hz) and 7.60 (1H, d, J = 2.0 Hz), suggesting an ABX spin system in the B-ring [9], a signal of
an isolated aromatic proton at δ 6.87 (s, 1H) was also seen. Three methoxy groups appeared at δ 3.80
(1H, s), 3.88 (s, 1H), 3.93 (1H, s). The sugar protons appeared between δ 3.41~5.10. The doublet peak
of one single proton at δ 5.10 (1H, d, J = 7.4 Hz) was attributed to the H-1′′, and suggested that the D-
13
glucose moiety was in a β-orientation. The C-NMR spectrum (Table 1) indicated that compound 1
was a flavonol glycoside with twenty-four carbons, including fifteen flavonoid carbons and six sugar
carbons, with δ 102.0 being a characteristic signal for the C-1′′of the sugar. Acid hydrolysis of
compound 1 gave glucose, identified by paper chromatography and comparison with an authentic
sample. A HSQC experiment showed a correlation between the proton signal at H-1′′ (δ 5.10) and the
carbon signal at C-1′′ (δ 102.0). The location of the glucose was established on the basis of HMBC

Molecules 2010, 15
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correlations from H-1′′ (δ 5.10) to C-6 (δ 157.9) (Figure 2). The β-D-Glycosyl was attached to the C-6
1
of A-ring. The H-NMR and 13C-NMR data of the D-Glycosyl was in agreement with those of a
reference. The HMBC spectrum showed the three methoxy groups were linked to C-3, C-7 and C-4′ of
the flavonoid skeleton. On the basis of the above evidence, The chemical structure of compound 1 was
formulated as: 8,3′-dihydroxy-3,7,4′-trimethoxy-6-O-β-D-glucopyranosyl flavone.
Table 1. 1H-NMR and 13C-NMR data for compounds 1 and 2 in CD3OD, δ (ppm), J (Hz).
Compound 1
δ C
Compound 2
δ C
Position
δ H
HMBC
δ H
HMBC
2
158.3
115.9
138.8
179.4
93.2
3
139.9
4
180.4
5
6.87 s
95.6 C10, C7, C9, C6, C4
6.54 s
C10, C7, C9
6
157.9
133.8
153.8
153.4
108.1
124.1
155.1
130.1
152.4
151.9
106.4
123.7
7
8
9
10
1′
2′
7.60 d (2.0)
7.03 d (8.0)
116.3
147.7
151.9
112.3
C6′, C3′, C4′, C2
7.73 d (2.0)
6.97 d (8.0)
114.6 C6′, C3′, C4′, C2
3′
145.6
148.9
4′
5′
6′
C1′, C3′, C4′
C2′, C4′
C3
110.5
C1′, C3′, C4′
C2′, C4′
C3
7.65 dd (8.0, 2.0) 122.4
7.71 dd (8.0, 2.0) 121.7
3-OCH3
7-OCH3
4′-OCH3
1′′
3.80 s
3.88 s
60.6
61.5
56.4
102.0
74.8
78.0
71.3
78.5
62.6
3.86 s
4.04 s
3.99 s
60.3
61.1
56.2
C7
C7
3.93 s
C4′
C4′
5.10 d (7.4)
3.49 m
C6
2′′
C1′′, C3′′
C2′′, C4′′
C3′′, C5′′
C4′′
3′′
3.53 dd
4′′
3.41 m
5′′
3.42 m
6′′
3.69 dd, 3.95 m
C5′′
Figure 2. The key HMBC correlations of compound 1.
OH
OH
OCH3
H
OCH3
H
OH
OH
H3CO
OH
O
O
H3CO
OH
O
O
H
OCH3
O
O
OCH3
O
O
H
OH
OH
HO
H
HO
OH
OH

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Compound 2 was obtained as a yellow amorphous powder. The HR-ESI-MS showed an [M-H]- ion
1
at m/z 359.0709 (calcd 359.0748), corresponding to the molecular formula, C18H16O8. The H-NMR
spectrum showed three aromatic protons at δ 7.71 (1H, dd, J = 8.0, 2.0 Hz), 6.97 (1H, d, J = 8.0 Hz)
and 7.73 (1H, d, J = 2.0 Hz) suggesting an ABX spin system in the B-ring, and an isolated aromatic
proton signal at δ 6.87 (s, 1H) was also seen. Three methoxy groups were noted at δ 3.80 (s, 3H), 3.88
(3H, s), 3.93 (3H, s). The 1H-1H COSY spectra of compound 2 disclosed that the signal at δ 6.97 (1H,
d, J = 8.0 Hz) was correlated to the one at δ 7.71 (1H, d. J = 8.0, 2.0 Hz). In the HMBC, H-1′′ signal at
δ 5.10 correlated to C-6 (δ 157.9, Figure 3). The HMBC spectrum showed the three methoxy groups
were linked to C-3, C-7 and C-4′ of the flavonoid skeleton. On the basis of the above evidence, the
chemical structure of compound 2 was formulated as: 6,8,3′-trihydroxy-3,7,4′-trimethoxyflavone.
Figure 3. The key HMBC correlations of compound 2.
OH
OH
OCH3
H
OCH3
H
OH
OH
H3CO
HO
O
O
H3CO
HO
O
O
H
OCH3
OCH3
H
Antioxidant activity
The antioxidant activities of compounds 1 and 2 were evaluated using the DPPH and FRAP assays.
The DPPH assay has been widely used to test the free radical scavenging ability of plant components.
In the present study, it was found that both the two new compounds had DPPH free-radical scavenging
activity. The DPPH scavenging rates are shown in Figure 4(A), and the IC50 values are shown in
Table 2. From these data, it can be concluded that the sequence of antioxidant activity of these
compounds was as follows: Trolox (reference) > compound 1 > compound 2. The FRAP assay can
reflect the antioxidant activity through the variation of absorbance at a certain wavelength. The
absorbances of the two new compounds are shown in Figure 4(B), and TEAC values are shown in
Table 2. These TEAC values demonstrated that the sequence of reducing power of these compounds
was as follows: Trolox > compound 1 > compound 2.
Table 2. IC50 values and TEAC values of compounds 1, 2 and Trolox with different
concentration.
Compound 1
0.03180
Compound 2
0.04618
Trolox
0.00948
1.00000
IC50 value (mg/mL)
TEAC value (mg/g)
0.29784
0.07017

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Figure 4. The antioxidant activity of compounds 1, 2 and Trolox with different
concentrations (A: DPPH assay, B: FRAP assay).
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
100
A
B
90
80
70
60
50
40
30
20
1
2
Trolox
1
2
Trolox
0.00
0.02
0.04
0.06
0.08
0.10
0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40
Concentration(mg/mL)
Concentration/(mg/mL)
3. Experimental
3.1. General
1D and 2D NMR spectra (chemical shifts in ppm, coupling constants in Hz) were recorded on a
1
Bruker DPX 400 instrument. The H NMR spectra were reported in delta (δ) units, parts per million
(ppm) downfield from the internal standard. Coupling constants are reported in Hertz (Hz). ESI mass
spectra were obtained on a Waters Q-Tof Micro TM mass spectrometer coupled to a Waters 2795
system that included an online photodiode array detector. HR-ESI-MS spectra were determined on a
Waters Q-Tof MicroTM ESI-MS. The compounds were detected from their UV absorbance and by
spraying on TLC plates with FeCl3-K3[Fe(SCN)6] reagent.
3.2. Plant aaterial
The whole plants of Tridax procumbens was collected in Sanya city, Hainan Province, China, in
September 2007. The plant was identified by Prof. Shi-man Huang of Hainan University. A voucher
specimen of this plant (ZJFU-20070910-10) has been deposited in our laboratory.
3.3. Extraction and isolation [10]
Air-dried and powdered whole plants of Tridax procumbens (5 kg) was extracted with 70% EtOH
(20 L × 3) at room temperature for 24 h. The combined EtOH extracts was concentrated by vacuum
thin film evaporation below 50 °C. After concentration the extracts were dissolved in H2O (5 L) and
extracted successively with petroleum ether (15 L × 3) and EtOAc (15 L × 3). The EtOAc layer was
separated by repeated column chromatography on Sephadex LH-20, RP-18 using MeOH/H2O as eluant
to produce six fractions. Fr. 4 (15.5 g) was eluted with MeOH-H2O (40:60) and was then repeatedly
subjected to column chromatography (CC) to yield 8,3′-dihydroxy-3,7,4′-trimethoxy-6-O-β- D-
glucopyranosyl flavone (1, 85.6 mg) and 6,8,3′-trihydroxy-3,7,4′-trimethoxyflavone (2, 51.0 mg).
Puerarin (3, 68.2 mg), esculetin (4, 27.4 mg), oleanolic acid (5, 58.5 mg) and betulinic acid (6, 35.5 mg).

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3.4. Spectral data
Compound 1: 35.6 mg, yellow amorphous powder, mp: 206–208 °C (MeOH); [α]D20 = -45.6(c 0.30,
MeOH); UV λmax nm (logε): 231 (3.80), 291 (3.65); IR υmax cm-1 (KBr): 3432 (OH), 1600, 1545, 1456,
1
13
1260, 1223, 829 cm-1; HR-EI-MS m/z 545.1274 [M+Na]+ (Calcd 545.1254); H-NMR, C-NMR and
2D experiments see Table 1.
Compound 2: 51.0 mg, yellow amorphous powder, mp: 195–197 °C (MeOH); [α]D20 = -49.5(c 0.30,
MeOH); UV λmax nm (logε): 254 (3.82), 275 (3.66), 277 (1.98); IR υmax cm-1 (KBr): 3400 (OH), 1655,
13
1600, 1545, 1456, 829 cm-1; HR-EI-MS m/z 359.0709 (Calcd 359.0748); 1H-NMR, C-NMR and 2D
experiments see Table 1.
3.5. Process of acid hydrolysis
The process of acid hydrolysis was: compound 1 (30 mg) was dissolved in 2% sulfuric acid aqueous
solution (30 mL) in a 100 mL besker by heating of electric furnace. Solution was heated for one hour
in boiling condition and then filtered. the filter residue was aglycone. The concentrated hydrolysate
was identified by paper chromatography by D-glucose as authentic sample and B:A:W (4:1:5) as the
solvent system. The sugars was identified as D-glucose. Aglycone was yellow powder,
mp 235–236 °C.
3.6. DPPH and FRAP assays
3.6.1. DPPH assay
The DPPH radical scavenging activity was evaluated according to the literature [11] with the slight
modification. Briefly, each sample (100 µL of various concentrations) was added to ethanolic DPPH
solution (100 µL, 2.5 × 10-2 mg/mL). After mixing gently and standing at 24 °C for 30 min, the
absorbances were measured at 530 nm using a VERSAmax microplate reader spectrophotometer. The
percentage of DPPH which was scavenged was calculated using the following formula:
Scavenging %= 1- (Ap-Ac) /Amax × 100%.
where Ap was the stable absorbance of DPPH ethanol solution (100 μL) plus sample solution (100 μL),
Ac was the stable absorbance of 70% ethanol solution (100 μL) plus sample solution (100 μL) and Amax
was the stable absorbance of DPPH ethanol solution (100 μL) plus 70% ethanol solution (100 μL).
3.6.2. FRAP assay [12]
FRAP assay was carried out in a microtiter plate. Each sample (80 μL) with appropriate dilution if
necessary was added to FRAP reagent [13] (150 μL, 10 parts of 0.3 mol/L sodium acetate buffer at pH
.
3.7, 1 part of 0.01 mol/L TPTZ solution, and 1 part of 0.02 mol/L FeCl3 6H2O solution), and 70%
ethanol (80 μL) plus FRAP reagent (150 μL) was used as a blank, then the wavelength at 593 nm was
read after 10 min. Fresh working solutions of Trolox were used for calibration, and the total
antioxidant capacity were expressed as TEAC (Trolox Equivalent Antioxidant Capacity) values, it was
calculated when the absorbency at 0.4 TEAC value means mg TE/mg flavone.

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4. Conclusions
In our present study, two new flavones 1-2, together with four known compounds 3~6 were
separated from the aerial parts of Tridax procumbens, and their structures were identified by chemical
analysis and extensive spectral evidence, including IR, 1D and 2D NMR, ESI-MS, HR-ESI-MS data.
The antioxidant activity of the two new flavones were evaluated by the DPPH and FRAP assays, and
the data showed that compounds 1 and 2 have certain antioxidant activity. The antioxidant activity of
compound 2 was weaker than that of Trolox, the positive control, but stronger than that of compound 1.
Supplementary Materials
Supplementary materials can be accessed at http://www.mdpi.com/1420-3049/15/9/6357/s1
Acknowledgments
The authors are grateful to J. X. Kang, W. G. Zhu, S. M. Wang in the Analysis and Testing Center,
Zhengzhou University, for measuring NMR and MS data.
References
1. An, F.; Kan L.Y.; Xie, G.S.; Chen, Z.Q. Analyses of the Germination in the Invasive Plant
Eupatorium Catarium Seeds in Hainan Province. J. Northwest Forest. Univ. 2007, 22, 198-206.
2. Saxena, V.K.; Albert, S. β-Sitosterol-3-O-β-D-xylopyranoside from the flowers of Tridax
procumbens Linn. J .Chem. Sci. 2005, 117, 263-266.
3. Martin-Quintal, Z.; Moo-Puc, R., Gonzalez-Salazar, F.; Chan-Bacab, M.J.; Torres-Tapia, L.W.;
Peraza-Sánchez, S.R. In vitro activity of Tridax procumbens against promastigotes of Leishmania
mexicana. J. Ethnopharmacol. 2009, 122, 463-467.
4. Gautam, R.; Saklani, A.; Jachak, S.M. Indian medicinal plants as a source of antimycobacterial
agents. J. Ethnopharmacol. 2007, 110, 200-234.
5. Cheng, G.; Bai, Y.J.; Zhao, Y.Y.; Tao, J.; Liu, Y.; Tu, G.Z.; Ma, L.B.; Liao, N.; Xu, X.J.
Flavonoids from Ziziphus jujuba Mill var. spinosa. Tetrahedron 2000, 56, 8915-8920.
6. Sharma, R.K.; Negi, D.S.; Gibbons, S.; Otsuka, H. Chemical and antibacterial constituents of
Skimmia anquetelia. Planta Med. 2008, 74, 175-177.
7. Wang, Y.L.; Song, D.D.; Li, Z.L.; Yuan, T.; Zhang, H.L.; Pei, Y.H.; Jing, Y.K.; Hua, H.M.
Triterpenoids isolated from the aerial parts of Salvia chinensis. Phytochem. Lett. 2009, 2, 81-84.
8. Dimitroula, T.; Nektarios, A.; Konstantia, G.; Caroline, S.; Ioanna, C. Triterpenoids from
Eucalyptus camaldulensis Dehnh. Tissue Cultures. Helv. Chim. Acta 2008, 91, 2110-2114.
9. Fang, J.M.; Liu, M.Y.; Cheng, Y.S. Lignans from wood of Calocedrus formosana. Phytochemistry
1990, 29, 3048-3049.
10. Sayed, A.A.; Hossain, M.B.; Dick,V.H.; Francis, J.S. Alkaloids from the Tunicate Polycarpa
aurata from Chuuk Atoll. J. Org. Chem. 1996, 61, 2709-2712.

Molecules 2010, 15
6364
11. Liu, J.K.; Hu, L.; Dong, Z.J.; Hu, Q. DPPH radical scavenging activity of ten natural p-terphenyl
derivatives obtained from three edible mushrooms indigenous to China. Chem. Biodiversi. 2004, 1,
601-605.
12. Gao, Y.H.; Zheng, J.P.; Zhu, C.Y.; Li, Y.J.; Ke, Y.; Bian, K. Evaluation and Miniaturization for
Antioxidant Capacity Measurement. Chin. Pham. J. 2008, 24, 1863-1867.
13. Thaipong K.; Boonprakob U.; Crosby K.; Cisneros-Zevallos L.; Byrne D.H. Comparison of
ABTS, DPPH, FRAP and ORAC assays for estimating antioxidant activity from guava fruit
extracts. J. Food Compos. Anal. 2006, 19, 669-675.
Sample Availability: Samples of ccompound 1 and compound 2 are available from the authors (contact
yuan_ke001@yahoo.com.cn).
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