SUPPLEMENTARY MATERIAL
Isolation and identification of flavonoids from Coreopsis lanceolata L.
petals
Yoshiharu Okada,*† Mari Okita,† Yoshihiro Murai,‡ Yuka Okano† and
Masato Nomura†
†Graduate School of Systems Engineering, Kinki University, Umenobe 1, Takaya,
Higashi-Hiroshima 739-2116, Japan
‡Kinki University Joint Research Center, 3-4-1 Kowakae, Higashi-Osaka 577-8502,
Japan
Corresponding author. E-mail: okadasan@hiro.kindai.ac.jp.
Isolation and identification of flavonoids from Coreopsis lanceolata L.
petals
The methanol extraction of the Coreopsis lanceolata L. petals were acidhydrolyzed and 7,3’,4’-trihydroxy-8-methoxyflavanone (1) and 6,3’,4’trihydroxy-7-methoxyaurone (Leptosidin) (2) were successfully isolated. The
structure of compound 1 is designated as flavanone based on X-ray
crystallographic analysis and NMR spectroscopic analysis. The compound 1
showed high antioxidant effects based on DPPH radical scavenging assay
(94.3% scavenging rate) and SOD-like activity assay (23.9% inhibition rate).
Keywords: Coreopsis lanceolata L.; 7,3’,4’-Trihydroxy-8-methoxyflavanone;
Leptosidin
1. Experimental
1.1. General Procedures.
1H NMR and 13C NMR spectra were obtained on a JEOL JNM-EX400 spectrometer
in CDCl3 or DMSO-d6 operating at 400 Mz and 100 Mz respectively, with Me4Si as
internal standard. IR spectra were recorded with a Shimadzu FTIR-8100A
instrument. X-Ray analysis was made on a Rigaku VariMax with RAPID
diffractometer using multi-layer mirror monochromated Cu-K radiation (1.54187
Å). Melting points were measured in open capillary tubes and were uncorrected.
1.2. Extraction and Isolation.
Dried Coreopsis lanceolata L. petals (17.51 g) were extracted with methanol (300
mL) for 12 h using soxhlet apparatus. The methanol extract was concentrated under
reduced pressure to give dark red oil (9.77 g). An aqueous methanol solution was
heated with 2M hydrochloric acid at 60 °C for 3 hours. The resulting mixture was
extracted with ethyl acetate, and washed with water and brine. The mixture was dried
over anhydrous magnesium sulfate. The solvent was evaporated in vacuo, and the
residue was chromatographed on silica gel to give three fractions with methanolchloroform (1:9) as eluent. The three fractions were concentrated under reduced
pressure to give fraction 1 (1.58 g), fraction 2 (1.17 g), and fraction 3 (0.89 g)
respectively. Preparative thin layer chromatography of the fraction 2 with methanol-
chloroform (1:9) gave yellowish orange solid (Rf=0.41, 0.70 g) and orange solid
(Rf=0.28, 0.21 g). The yellowish orange solid was recrystallized from methanol to
give 7,3’,4’-trihydroxy-8-methoxyflavanone (1) as a citrine needle crystalline.
7,3’,4’-Trihydroxy-8-methoxyflavanone (1): m.p. 192-195 °C (195-197 °C (Geissman
& Heaton, 1943)); IR (KBr) 1647, 1597, 1194, 1013 cm-1 ; 1H NMR (DMSO-d6)
2.67 (dd, J=2.9 and 16.8 Hz, 1H, H-3eq), 3.07 (dd, J=12.7 and 16.8 Hz, 1H, H-3ax),
3.71 (s, 3H, OCH3), 5.43 (dd, J=2.9 and 12.5 Hz, 1H, H-2), 6.59 (d, J=8.8 Hz, 1H, H6), 6.78 (d, J=1.5 Hz, 1H, H-6’), 6.78 (s, 1H, H-5’), 6.94 (d, J=1.5 Hz, 1H, H-2’),
7.42 (d, J=8.8 Hz, 1H, H-5), 9.13 (s, 2H, OH-3’ and OH-4’), 10.38 (s, 1H, OH-7).;
13C NMR (DMSO-d ) 43.23 (C-3), 60.31 (OCH ), 79.31 (C-2), 110.38 (C-6),
6
3
114.28 (C-2’), 114.55 (C-10), 115.36 (C-5’), 117.88 (C-6’), 122.15 (C-5), 129.96 (C1’), 135.15 (C-8), 145.17 (C-3’), 145.60 (C-4’), 155.79 (C-9), 156.94 (C-7), 190.38
(C-4).; EIMS m/z 302 [M]+ (100).; Crystal system: Monoclinic, Space group: Pn,
a=4.1592(2) Å, b=7.8734(3) Å, c=20.446(2) Å, =91.096(7) °, V=669.41(6) Å3, Z=2,
R=0.0583, wR=0.0992.
6,3’,4’-Trihydroxy-7-methoxyaurone (Leptosidin) (2): 1H NMR (DMSO-d6) 
(s, 3H, OCH3), 6.63 (s, 1H, H-10), 6.76 (d, J=8.3 Hz, 1H, H-5), 6.84 (d, J=8.3 Hz,
1H, H-5’), 7.24 (dd, J=2.0 and 8.3 Hz, 1H, H-6’), 7.32 (d, J=8.3 Hz, 1H, H-4), 7.42
(d, J=2.0 Hz, 1H, H-2’).; 13C NMR (DMSO-d6) 60.78 (OCH3), 112.30 (C-10),
113.41 (C-5), 114.62 (C-9), 115.99 (C-5’), 117.83 (C-2’), 119.33 (C-4), 123.22 (C1’), 124.54 (C-6’), 132.12 (C-7), 145.36 (C-3’), 145.41 (C-4’), 148.03 (C-2), 157.73
(C-8), 157.83 (C-6), 181.15 (C-3).; EIMS m/z 300 [M]+ (100).
1.3. Preraration of (Z)-6,3’,4’-trihydroxy-7-methoxyaurone.
Aluminum oxide (basic, 5.00 g, 49.0 mmol) was added to a solution of 7-methoxy-6hydroxy-3(2H)-benzofuranone (0.27 g, 1.5 mmol) and 3,4di(methoxymethoxy)benzaldehyde (0.45 g, 2.0 mmol) in dichloromethane (5 mL)
(Bolek & Gutschow, 2005). The mixture was thoroughly stirred for 2 days at room
temperature. The suspension was filtered off, the residue was washed with
chloroform and the washed portions were combined with the filtrate. The solvent was
evaporated and the residue was chromatographed on preparative thin layer
chromatography (chloroform:diethyl ether=9:1) to give (Z)-6-hydroxy-7-methoxy-
3’,4’-di(methoxymethoxy)aurone (0.10 g, 0.26 mmol, 17% yield). A solution of (Z)6-hydroxy-7-methoxy-3’,4’-di(methoxymethoxy)aurone (0.10 g, 0.26 mmol) and
conc. HCl (0.2 mL) in aqueous THF (80%, 2 mL) was stirred overnight at room
temperature. The mixture was evaporated in vacuo and the residue was
chromatographed on preparative thin layer chromatography (chloroform:ethyl
acetate=1:1) to give (Z)-6,3’,4’-trihydroxy-7-methoxyaurone (0.07 g, 0.23 mmol,
91% yield). M.p. 235-239 °C (dec.) (236-240 °C (Shimokoriyama & Hattori, 1953));
IR (KBr) 1676.3, 1639.7, 1597.3, 1134.3, 1062.9 cm-1 ; 1H NMR (CD3OD) 
(s, 3H, OCH3), 4.98 (br s, 3H, OH), 6.66 (s, 1H, H-10), 6.70 (d, J=8.3 Hz, 1H, H-5),
6.83 (d, J=8.3 Hz, 1H, H-5’), 7.22 (d, J=8.1 Hz, 1H, H-6’), 7.30 (d, J=8.5 Hz, 1H, H4), 7.42 (s, 1H, H-2’).; 13C NMR (CD3OD) 61.45 (OCH3), 114.29 (C-10), 114.98
(C-5), 116.46 (C-9), 116.51 (C-5’), 118.87 (C-2’), 120.43 (C-4), 125.24 (C-1’),
126.31 (C-6’), 133.41 (C-7), 146.49 (C-3’), 147.23 (C-4’), 149.30 (C-2), 159.07 (C8), 159.34 (C-6), 184.21 (C-3).
Acknowledgements
We thank Ms. K. Hasegawa (Rigaku Co.) for X-ray analysis.
References
Bolek, D & Gutschow, M. (2005). Preparation of 4,6,3’,4’-tetrasubstituted aurones
via aluminium oxide-catalyzed condensation. Journal of Heterocyclic Chemistry, 42,
1399-1403.
Geissman, T. A. & Heaton, C. D. (1943). Anthochlor pigments. IV. The pigments of
Coreopsis grandiflora, Nutt. I. Journal of the American Chemical Society, 65, 677683.
Shimokoriyama, M. & Hattori, S. (1953). Anthochlor pigments of Cosmos sulphureus,
Coreopsis lanceolata and C. saxicola. Journal of the American Chemical Society, 75,
1900–1904.
5'
OMe
HO
OMe
9 O
4
6
5
OH
2
3
2'
OH
HO 6
5
O
OH
O
4
1
9
3 2 10
OH
O
2
Figure S1. Structures of the isolated compounds 1 and 2 from Coreopsis lanceolata L.
Figure S2. Ortep diagram of compound 1.
Figure S3. 1H NMR spectrum of 1 in DMSO-d6 (400 MHz).
Figure S4. 13C NMR spectrum of 1 in DMSO-d6 (100 MHz).
Figure S5. H-C COSY spectrum of 1.
Figure S6. 1H NMR spectrum of 2 in DMSO-d6 (400 MHz).
Figure S7. 13C NMR spectrum of 2 in DMSO-d6 (100 MHz).
Figure S8. H-C COSY spectrum of 2.
Figure S9. 1H NMR spectrum of the synthesized 6,3’,4’-trihydroxy-7-methoxyaurone
in CD3OD (400 MHz).
Figure S10. 13C NMR spectrum of the synthesized 6,3’,4’-trihydroxy-7methoxyaurone in CD3OD (100 MHz).