Food

Chemistry
Food Chemistry 105 (2007) 48–56
www.elsevier.com/locate/foodchem

Identification of phenolic antioxidants from Sword Brake

fern (Pteris ensiformis Burm.)
Yung-Husan Chen a, Fang-Rong Chang a, Yih-Jer Lin b, Lisu Wang c, Jinn-Fen Chen d,
Yang-Chang Wu a,*, Ming-Jiuan Wu b,*
a
Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung 807, Taiwan
b
Department of Biotechnology, Chia-Nan University of Pharmacy and Science, Tainan 717, Taiwan
c
Department of Environmental and Occupational Health, Medical College, Cheng-Kung University, Tainan 701, Taiwan
d
Taitung District Agricultural Research and Extension Station, Taitung 950, Taiwan

Received 11 October 2006; received in revised form 24 January 2007; accepted 13 March 2007

Abstract

Sword Brake fern (Pteris ensiformis Burm.) is one of the most common ingredients of traditional herbal drinks in Taiwan. In an effort
to identify antioxidants from the aqueous extract of Sword Brake fern (SBF), the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scaveng-
ing activity-guided isolation was employed. Three new compounds, kaempferol 3-O-a-L-rhamnopyranoside-7-O-[a-D-apiofuranosyl-
(1-2)-b-D-glucopyranoside] (1), 7-O-caffeoylhydroxymaltol 3-O-b-D-glucopyranoside (3) and hispidin 4-O-b-D-glucopyranoside (4),
together with five known compounds, kaempferol 3-O-a-L-rhamnopyranosid-7-O-b-D-glucopyranoside (2), caffeic acid (5), 5-O-caffeoyl-
quinic acid (6), 3,5-di-O-caffeoylquinic acid (7) and 4,5-di-O-caffeoylquinic acid (8) were isolated and determined on the basis of spec-
troscopic analyses. HPLC with UV detector was further employed to analyze the content of each compound in SBF based on the
retention time by comparison with isolated pure compounds. It was found that the most abundant phenolic compound was compound
3, followed by compounds 7 and 4. The di-O-caffeoylquinic acids (7 and 8) have the strongest DPPH scavenging potential with IC50
around 10 lM and the highest Trolox equivalent antioxidant capacity (TEAC) about 2 mM. This data indicates that SBF is rich in phe-
nolic antioxidants.
Ó 2007 Elsevier Ltd. All rights reserved.

Keywords: Pteris ensiformis; DPPH; TEAC; Caffeic acid; Hispidin

1. Introduction beverages in Taiwan. It has been previously demonstrated

that the aqueous extract of Sword Brake fern (SBF) exerts
Botanicals have been used for treatment or prevention immunomodulatory effect by inhibiting the release of
of various human diseases throughout history. Many indig- tumor necrosis factor (TNF)-a, interleukin (IL)-1b, IL-6,
enous herbal plants of regional interest have been used nitric oxide (NO), and prostaglandin E2 (PGE2) in
popularly as folk medicines in Taiwan or other Asian coun- lipopolysaccharide (LPS)-activated RAW264.7 cells (Wu,
tries; however, their active phytochemicals or biological Wang, Weng, & Lian, 2005). In view that a variety of
effects remained to be elucidated. Sword Brake fern (Pteris anti-inflammatory natural components also possess antiox-
ensiformis Burm.) is one of the most popular herbs used in idant activity (Chamundeeswari, Vasantha, Gopalakrish-
nan, & Sukumar, 2003; Lo, Liang, Lin-Shiau, Ho, & Lin,
2002; Wu, Yen, Wang, & Weng, 2004; Wu, Huang, Lian,
*
Corresponding authors. Tel.: +886 7 312 1101x2197; fax: +886 7 311 Kou, & Wang, 2005) the possibility that SBF has a free
4773 (Y.-C. Wu); Tel: +886 6 266 4911x525; fax: +886 6 266 6411 (M.-J.
radical scavenging activity arose.
Wu).
E-mail addresses: yachwu@kmu.edu.tw (Y.-C. Wu), imwu@mail. Phytochemical investigations on the Pteris genus have
chna.edu.tw (M.-J. Wu). yielded various phenolic compounds (Imperato, 1994; Lu,

0308-8146/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2007.03.055
 Y.-H. Chen et al. / Food Chemistry 105 (2007) 48–56 49

Hu, Zhang, & Tan, 1999) and diterpenes (Deng & Liang, Institute of Natural Products, Kaohsiung Medical Univer-
2005; Woerdenbag, Lutke, Bos, & Stevens, 1996). Phenolic sity, Kaohsiung, Taiwan.
compounds are potent antioxidants that play an important
role in human nutrition as preventive agents against several 2.3. Extraction and isolation
diseases and protecting the body tissues from oxidative
stress. Epidemiological evidences indicate an inverse rela- Fresh whole plants (8.1 kg wet weight) of Sword Brake
tionship between the intake of polyphenol-rich foods and fern were cut into small pieces and extracted with boiling
the risk of coronary heart disease as well as some types water (3  20 l). A small portion of the combined aqueous
of cancer (Scalbert, Johnson, & Saltmarsh, 2005). The extract was lyophilized to yield a dark-brown powder,
diterpene isolated from Pteris has been shown to be a which was redissolved in deionized water prior to use. It
potent antitumor agent by inducing apoptosis (Chen was denoted as the crude aqueous extract, SBF.
et al., 2004; Liu, Chen, et al., 2005; Liu, Ng, et al., 2005). The rest of the aqueous extract was concentrated to a
The aim of this study is to search for the antioxidant small volume and partitioned with chloroform to yield the
principals using 1,1-diphenyl-2-picrylhydrazyl (DPPH) rad- chloroform and aqueous layers. The resulting aqueous layer
ical scavenging activity as an index. Our result demon- was further partitioned with n-butanol to give n-butanol
strated that the aqueous extract of Sword Brake fern and aqueous layers. The n-butanol layer (30.0 g) was then
(SBF) had strong antioxidant activity. Eight phenolic passed through Diaion HP20SS chromatography and was
compounds were identified (1–8) (Fig. 1) from the n-butanol eluted with water and methanol (1:0–0:1) to give five frac-
layer and kaempferol 3-O-a-L-rhamnopyranoside-7-O-[a- tions (A1–A5).
D-apiofuranosyl-(1-2)-b-D-glucopyranoside] (1), 7-O-caf-
Fr. A2 (7.6 g) was passed through silica gel column chro-
feoylhydroxymaltol 3-O-b-D-glucopyranoside (3), and matography and eluted with chloroform–methanol (4:1)
hispidin 4-O-b-D-glucopyranoside (4) are novel compounds. followed by preparative reverse-phase HPLC (Purospner,
The Trolox equivalent antioxidant capacity (TEAC) of each 20 250 mm, methanol: water = 1:3, flow rate 3 ml/min)
compound was further analyzed using the (ABTS/H2O2/ to yield compounds 1 (37.5 mg) and 2 (30.0 mg).
HRP) methods. Compound 3 (ca. 100.0 mg) was obtained by re-crystalli-
zation of the marc of the Fr. A3 (3.6 g) with methanol and
2. Materials and methods water (1:1). The rest of Fr. A3 was then passed through pre-
parative reverse-phase HPLC (Purospner, 20  250 mm,
2.1. General procedures methanol: water = 1:1, flow rate 3 ml/min) to yield com-
pound 4 (ca. 100.0 mg).
Optical rotations were measured on a JASCO DIP-370 Fr. A4 (6.2 g) was chromatographed on Sephadex LH-
digital polarimeter. UV spectra were obtained on a 20 (eluted with 100% methanol) followed by preparative
Hitachi 220-20 spectrophotometer. IR spectra were mea- reverse-phase HPLC (Purospner, 20  250 mm, methanol:
sured on a Hitachi 260-30 spectrophotometer. 1H NMR water = 1:1, flow rate 3 ml/min) to yield compounds 5
and 13C NMR spectra were recorded on a Varian Inova (30.1 mg), 6 (20.2 mg), 7 (19.8 mg), and 8 (18.0 mg).
500, Varian Unity Plus 400 MHz, or Varian Gemini
200 MHz spectrometer using TMS as an internal stan-
dard. Chemical shifts were reported in parts per million 2.4. Acid hydrolysis of compounds 1 and 2
(d) and coupling constants (J) were expressed in Hertz.
LR-EI-MS were collected on a Bruker APEX II mass A solution of each compound (3.0 mg) in 6% aqueous
or a Quattro GCMS spectrometer having a direct inlet HCl (3.5 ml) was refluxed for 2 h. The reaction mixture
system. LR-ESI-MS and HR-ESI-MS were measured on was diluted with water and then extracted with ethyl ace-
a Bruker APEX II mass spectrometer. Purospner STAR tate. The resulting aglycones were identified by their 1H
RP-18e (Merck KGaA, Darmstadt, Germany), Silica gel NMR spectra.
60 (230–400 mesh, Merck), Sephadex LH-20 (GE Health-
care UK Ltd., Buckinghamshire, England), and Diaion 2.5. Acetylation of compounds 1 and 2
HP20SS (Mitsubishi Chemical Co., Japan) were used
for column chromatography. Spots were detected by Compounds 1 or 2 (2.0 mg of each) was heated in a
spraying TLC with 50% H2SO4 followed by heating on sealed vial for 24 h at 80 °C in 2% methanol–HCl (2
a hot plate. ml). The mixture was extracted with ethyl acetate. The
aqueous hydrolysate was neutralized with Na2CO3 and fil-
2.2. Plant tered. L-Rhamnose, D-glucose, and D-apiose in the filtrate
were acetylated with pyridine/Ac2O. The acetylated sugar
Sword Brake fern (Pteris ensiformis Burm.) was residues were compared with the acetylated references,
obtained from the Taitung District Agricultural Research L-rhamnose, D-glucose, and D-apiose with GC–MS. The
and Extension Station, Taitung, Taiwan in Dec. 2004. A results showed that peracetylrhamnose, peracetylglucose,
voucher specimen (PE001) was deposited in the Graduate and peracetylapiose were derived from 1 and 2.
50 Y.-H. Chen et al. / Food Chemistry 105 (2007) 48–56

O
OH
O
O
R2O O

HO Glucose_O

OR1 OH O

OH O

1 R1 = rha R2 = glc (2-1) api 3 7-O-Caffeoylhydroxymaltol O-β -D-glucopyranoside

2 R1 = rha R2 = glc

O_ Glucose
O

OH
HO

O O
OH
HO

OH
5 Caffeic acid
4 Hispidin 4-O-β -D-glucopyranoside

O OH

R4O

R3O OR1

OR2

6 R1 = Caffeoyl R2 = H R3 = H R4 = H
7 R1 = Caffeoyl R2 = H R3 = Caffeoyl R4 = H
8 R1 = Caffeoyl R2 = Caffeoyl R3 = H R4 = H

Fig. 1. Structures of phenolic compounds 1–8 isolated from the aqueous extract of Sword Brake fern (Pteris ensiformis Burm.).

2.6. Quantification of phenolic compounds with HPLC were as follows: initial methanol composition, 20%,
increased to 40% in 20 min, and increased to 60% in
The contents of phenolic compounds in the aqueous 5 min.
extract of Sword Brake fern (SBF) were analyzed by
HPLC. Dried whole plants of SBF (1 g) were dispersed 2.7. DPPH scavenging capacities
and ground in boiling water (10 ml  3). A total of
30 ml filtrate was transferred into a volumetric flask and The crude aqueous extract of Sword Brake fern (SBF),
then passed through a C18-E SPE cartridge (500 mg/ different layers and isolated compounds were evaluated
3 ml) (Merck). The cartridge was eluted successively with for activities to scavenge the stable DPPH radical
3 ml of 10% methanol/water (v/v), and 3 ml of 60% meth- (0.1 mM, Sigma Chemical, St. Louis, MO, USA) according
anol/water (v/v).The 60% methanol fraction was collected to the method (Dinis, Maderia, & Almeida, 1994). The
and subjected to HPLC analysis. HPLC analysis was per- affinity of the test material to quench the DPPH free radical
formed using Shimadzu LC-10AT pumps, SPD-10A UV– was evaluated according to the equation: scavenging % =
Vis detector, and TSK-GEL ODS-80TM (250  4.6 mm (Ac  As)/Ac  100%. As and Ac are absorbance at
i.d.). The wavelength of the UV detector was set to be 517 nm of the reaction mixture with sample and control,
297 nm and the flow rate was 1 ml/min. The stepwise respectively. The IC50 values were obtained through extra-
HPLC conditions with methanol/water as mobile phase polation from linear regression analysis and denoted the
 Y.-H. Chen et al. / Food Chemistry 105 (2007) 48–56 51

concentration of sample required to scavenge 50% of DPPH Kaempferol 3-O-a-L-rhamnopyranoside-7-O-[a-D-apio-

radicals. All experiments were repeated at least three times. furanosyl-(1-2)-b-D-glucopyranoside] (1): C32H38O19, yel-
low amorphous solid, LR-ESI-MS m/z: 749.0 [100%,
2.8. Assay of Trolox equivalent antioxidant capacity M+Na]+; HRESIMS m/z: 749.1906 (calcd. for C32H38
25
(TEAC) O19Na+, 749.1899), ½aD : 60° (c 0.35, MeOH), UV
kMeOH
max (log e): 337 (3.8), 227 (3.9) nm, IR mmax (neat):
The antioxidant activity of each compound (1–8) was 3410, 1650 cm1, 1H NMR (CD3OD, 400 MHz) and 13C
further measured using the TEAC assay as described by NMR (CD3OD, 100 MHz) are given in Table 1.
Miller, Rice-Evans, Davies, Gopinathan, and Milner Kaempferol 3-O-a-L-rhamnopyranosid-7-O-b-D-gluco-
(1993) with minor modification. The TEAC value is based pyranoside (2): C27H30O15, yellow amorphous solid, LR-
on the ability of samples to scavenge the blue-green ABTS+ ESI-MS m/z: 617.0 [100%, M+Na]+, HR-ESI-MS m/z:
25
radical cation relative to the ability of the water-soluble 617.1486 (calcd for C27H30O15Na+, 617.1482), ½aD : 72°
MeOH
vitamin E analogue 6-hydroxy-2,5,7,8-tetramethylchro- (c 0.42, MeOH), UV kmax (log e): 349 (3.9), 263 (4.1)
man-2-carboxylic acid (Trolox). ABTS+ is generated by nm, IR mmax (Neat): 3400, 1654 cm1, 1H NMR (CD3OD,
the interaction of ABTS (2,20 -azino-bis(3-ethylbenzthiazo- 400 MHz) and 13C NMR (CD3OD, 100 MHz) are given in
line-6-sulfonic acid, 100 lM), H2O2 (50 lM), and horse rad- Table 1, NMR data are consistent with (El-Sayeda, Awa-
ish peroxidase (Sigma, 4.4 U/mL). After 1 ml of ABTS+ adb, Hifnawyb, & Mabryc, 1999).
was added to samples or Trolox, the absorbance at Acid hydrolysis of compounds 1 and 2 released kaempf-
734 nm was recorded after 10 min of incubation. TEAC is erol, identified by 1H and 13C NMR spectroscopy. The gas
defined as the concentration (mM) of Trolox having the chromatographic analysis of the acetylation products of
antioxidant equivalent to a 1.0 mM of the compound under both compounds shows the presence of glucose, rhamnose,
investigation. To calculate the TEAC, the gradient of the and apiose in the ratio 1:1:1. The molecular formula of 1
plot of the percentage inhibition of absorbance vs. concen- and 2, C32H38O19 and C27H30O15, respectively, were estab-
tration plot for the antioxidant in question is divided by the lished by HR-ESI-MS (1: [M+Na]+ m/z 749.1906, calcd.
gradient of the plot for Trolox (Re et al., 1999). 749.1899, 2: [M+Na]+ m/z 617.1486, calcd. 617.1477).
The complete structures of 1 and 2 were elucidated by 1D
3. Results and discussion and 2D NMR experiments. The 1H NMR spectrum of com-
pound 1 showed the presence of two meta-coupled aromatic
3.1. Identification of compounds 1–8 protons at d 6.47 and 6.72 (both d, J = 2 Hz), which were
assigned to the protons at H-6 and H-8 respectively; as well
The aqueous extract of Sword Brake fern (Pteris ensifor- as ortho-coupled aromatic protons at d 8.12 (d, J = 8.0 Hz,
mis Burm.) was partitioned successively with chloroform H-20 and H-60 ) and 6.90 (d, J = 8.0 Hz, H-30 and H-50 ). The
13
and n-butanol. The IC50 values for DPPH scavenging of C NMR shifts of the aglycone moiety of 1 corresponded
crude extract (SBF), chloroform, n-butanol, and aqueous to the shifts for kaempferol with significant differences on
layers were 50.3, >200, 15.7, and 61.5 lg/ml, respectively. C-3 and C-7. These shifts were analogous to those reported
This result indicates that SBF is a strong antioxidant and for the glycosylation of hydroxy group in a flavonol glyco-
most of the active components may exist in the n-butanol side (Agrawal, 1989). Three anomeric protons were easily
layer. identified in the spectra of 1 at dH 5.12 (d, J = 7.5 Hz),
Further isolation of compounds from the n-butanol layer 5.39 (d, J = 1.5 Hz), 5.46 (d, J = 1.5 Hz) and correlated
was carried out with Diaion HP20SS, Sephadex LH-20, with carbons at dC 100.1, 103.5, and 110.8, respectively.
silica gel column chromatography, and preparative From the assigned aglycone and sugar values, it was appar-
reverse-phase HPLC. This led to the isolation of eight ent that one saccharide and one disaccharide unit were
phenolic compounds, whose structures were determined to attached to C-3 and C-7 of the aglycone. The structure of
be kaempferol 3-O-a-L-rhamnopyranoside-7-O-[a-D-apio- the sugar chain was assigned by a combination of COSY,
furanosyl-(1-2)-b-D-glucopyranoside] (1), kaempferol HMQC, and HMBC experiments. Starting from the ano-
3-O-a-L-rhamnopyranoside-7-O-b-D-glucopyranoside (2), meric protons of each sugar unit, all of the hydrogens could
7-O-caffeoylhydroxymaltol 3-O-b-D-glucopyranoside (3), be identified using a combination of COSY experiments
hispidin 4-O-b-D-glucopyranoside (4), caffeic acid (5), 5-caf- (Table 1). The assignments of all proton resonances for
feoylquinic acid (6), 3,5-di-caffeoylquinic acid (7), and the sugar moieties allowed us to assign the resonances of
4,5-di-caffeoylquinic acid (8), respectively, by detailed spec- the linked carbon atoms by HMQC experiment (Table 1).
troscopic analysis and comparing with literature data. Many Information about the sequence of the oligosaccharide
phenolic compounds have been isolated from Pteris genus chain was deduced from HMBC experiments. Key correla-
(Imperato, 1994; Lu et al., 1999); however, compounds 1–8 tion peaks were observed between the anomeric proton of
were identified from the genus Pteris for the first time the rhamnose (d 5.39) and the C-3 of the kaempferol
possibly due to the high biodiversity of Pteris genus in terms (d 136.4), between the anomeric proton signal of the glucose
of phenolic composition. Compounds 2, 3, and 4 are new (d 5.12) and C-7 of the kaempferol (d 164.4) as well as
compounds. Their structures are shown in Fig. 1. between the anomeric proton of the apiose (d 5.46) and
52 Y.-H. Chen et al. / Food Chemistry 105 (2007) 48–56

Table 1
1 13
H NMR (400 MHz) and C NMR (100 MHz) spectral data of compound 1 and 2 in CD3OD (d in ppm, J in Hz)
1 13
Position H NMR C NMRa HMBC
1 2 1 2 1
2 159.8 159.7 H-20 , H-60
3 136.4 136.4 H-100
4 179.7 179.7
5 162.8 162.7 H-6
6 6.47 (1H, d, 2) 6.47 (1H, d, 2) 100.6 100.8 H-8
7 164.4 164.6 H-6, H-8, H-1000
8 6.72 (1H, d, 2) 6.72 (1H, d, 2) 95.7 95.7 H-6
9 158.0 158.0 H-8
10 107.6 107.6 H-6, H-8
10 122.3 122.3 H-30 , H-50
20 7.79 (1H, d, 8.0) 7.78 (1H, d, 8.0) 132.1 132.0 H-60
30 6.93 (1H, d, 8.0) 6.94 (1H, d, 8.0) 116.5 116.5 H-50
40 161.7 161.7 H-20 , H-60
50 6.93 (1H, d, 8.0) 6.94 (1H, d, 8.0) 116.5 116.5 H-30
60 7.79 (1H, d, 8.0) 7.78 (1H, d, 8.0) 132.1 132.0 H-20
Rhamnoside
100 5.39 (1H, s) 5.39 (1H, s) 103.5 103.4
200 4.23 (1H, br s) 4.23 (1H, br s) 71.2 71.2 H-100
300 3.94 (1H, m) 3.94 (1H, m) 72.1 72.1 H-400 , H-500
400 3.43 (1H, m) 3.45 (1H, m) 73.1 73.1 H-500 , H-600
500 3.64 (1H, m) 3.50 (1H, m) 71.8 71.8 H-400 , H-600
600 0.92 (3H, d, 6.2) 0.92 (3H, d, 6.2) 17.6 17.6
Glucoside
1000 5.12 (1H, d, 7.2) 5.06 (1H, d, 7.2) 100.1 101.5 H-2000 , H-3000
2000 3.66 (1H, m) 3.42 (1H, m) 78.2 75.1 H-3000 , 10000
3000 3.43 (1H, m) 3.40 (1H, m) 78.6 78.0 H-2000
4000 3.31 (1H, m) 3.31 (1H, m) 72.1 72.1 H-5000 , H-6000
5000 3.19 (1H, m) 3.19 (1H, m) 78.4 78.3 H-3000 , H-6000
6000 3.51 (2H, m) 3.51 (2H, m) 62.4 62.4
Apioyl
10000 5.46 (1H, s) 110.8 H-4a0000
20000 3.96 (1H, s) 78.1 H-10000 , H-4a0000
30000 80.7 H-10000 , H-20000 , H-4a0000
4a0000 3.82 (1H, d, 10.0) 75.4 H-10000 , H-20000
4b0000 4.04 (1H, d, 10.0)
50000 3.55 (2H, br s) 65.4 H-20000 , H-4b0000
a 1 1
Assignments confirmed by decoupling, H– H COSY, NOESY, HMQC and HMBC.

the C-2 of the glucose (d 78.2). From these considerations, 5.62 (1H, d, J = 14.0, H-7b), 7.46 (1H, s, H-20 ), 7.10
the structure of kaempferol 3-O-a-L-rhamnopyranoside-7- (2H, s, H-50 , H-60 ), 6.49 (1H, d, J = 15.6, H-70 ) 7.86
O-[a-D-apiofuranosyl-(1-2)-b-D-glucopyranoside] (Fig. 1) (1H, d, J = 15.6, H-80 ), sugar signals: d 5.52 (1H, d,
was assigned to 1. The 1H NMR spectrum of 2 also showed J = 7.6, H-100 ), 3.39 (1H, m, H-200 ), 4.15 (1H, m, H-300 ),
a kaempferol derivative. The signals in the 1H, 13C, and 2D 4.06 (1H, m, H-400 ), 4.02 (1H, m, H-500 ), 4.32 (1H, m,
NMR spectra were superimposable to those of 1, as a result, H-600 ), and 13C NMR (C5D5N, 100 MHz): ppm 157.6
the structure of 2 was kaempferol 3-O-a-L-rhamnopyrano- (C-2), 143.2 (C-3), 175.4 (C-4), 117.2 (C-5), 155.7 (C-6)
side-7-O-b-D-glucopyranoside (Fig. 1), which was reported 58.1 (C-7), 126.4 (C-10 ) 115.3 (C-20 ), 147.8 (C-30 ) 146.9
previously (El-Sayeda et al., 1999; Sharaf, El-Ansari, & (C-40 ), 122.2 (C-50 ), 116.3 (C-60 ), 113.3 (C-70 ), 166.6
Saleh, 1997) (Table 2). (C-80 ), 104.4 (C-100 ), 75.0 (C-200 ), 77.6 (C-300 ), 70.6 (C-400 ),
7-O-Caffeoylhydroxymaltol 3-O-b-D-glucopyranoside 78.6 ( C-600 ), 62.0 (C-600 ), key HMBC correlations: H-100 /
(3): C21H22O12, colorless amorphous solid, LR-ESI-MS C-3, H-7/C-90 .
m/z: 489.0 [M+Na]+; HR-ESI-MS m/z: 489.1006 (calcd. Hispidin 4-O-b-D-glucopyranoside (4): C19H20O10, yel-
25
for C21H22O12Na+, 489.1003, ½aD : 20.5° (c 0.62, low powder, LR-ESI-MS m/z: 431.1 [M+Na]+; HR-ESI-
MeOH
MeOH), UV kmax (log e): 324 (3.57), 270 (3.71) nm, IR MS m/z: 431.0952 (calcd for C19H20O10Na+, 431.0949),
25
mmax (Neat): 3370, 1710, 1650 cm1, 1H NMR (C5D5N, ½aD :-65.1° (c 0.52, MeOH), UV kMeOH max (log e): 263 (4.53),
400 MHz), aglycone signals: d 6.48 (1H, d, J = 5.0, H-5), 339 (3.90) nm, IR mmax (Neat): 3400, 2942, 1702 cm1, 1H
7.94 (1H, d J = 5.0, H-6), 5.56 (1H, d, J = 14.0, H-7a), NMR (CD3OD, 400 MHz), aglycone signals: d 5.73 (1H,
 Y.-H. Chen et al. / Food Chemistry 105 (2007) 48–56 53

Table 2 the proposed structure of hispidin 4-O-b-D-glucopyrano-

13
C NMR (100 MHz) spectral data of compound 5, 6, 7, and 8 in CD3OD side (4) was determined unambiguously.
(d in ppm)
Caffeic acid (5): C9H8O4, yellow powder, EI-MS: m/z
13
Position C NMRa 181.1 [M+H]+, 1H NMR (400 MHz, CD3OD): d 6.31
5 6 7 8 (1H, d, J = 16.0 Hz, H-8), 6.75 (1H, d, J = 8.0 Hz, H-5),
1 78.0 74.7 76.3 6.93 (1H, dd, J = 2.0, 8.0 Hz, H-6), 7.04 (1H, d,
2 38.9 36.1 38.4 J = 2.0 Hz, H-2), 7.57 (1H, d, J = 16.0 Hz, H-7); 13C
3 72.6 72.5 69.5 NMR (CD3OD, 100 MHz) were given in Table 1, identical
4 75.0 69.9 75.8
5 73.1 72.8 69.0
to data in the literature (Kumaran & Karunakaran, 2007).
6 40.6 37.9 39.5 5-O-Caffeoylquinic acid (6): C16H18O8, yellow powder,
7 180.9 177.0 177.2 ESI-MS: m/z 355.5 [M+H]+.1H NMR (400 MHz,
Coffeoyl CD3OD): d 2.00 (2H, m, H-2, H-6), 2.13 (2H, m, H-2,
10 127.7 127.8 127.7 127.8 127.7 127.6 H-6), 3.63 (1H, dd, J = 3.0, 9.0 Hz, H-4), 4.14 (1H, s,
20 115.1 115.5 114.7 114.7 114.7 114.7 H-3), 5.34 (1H, m, H-5), 6.30 (1H, d, J = 16.0 Hz, H-80 ),
30 146.8 146.8 146.7 146.7 146.7 146.7 6.76 (1H, d, J = 8 Hz, H-50 ), 6.93 (1H, dd, J = 2.0,
40 149.4 149.5 149.6 149.6 149.6 149.6
8.0 Hz, H-60 ), 7.04 (1H, d, J = 2.0 Hz, H-20 ), 7.58 (1H, d,
50 116.5 116.5 116.5 116.5 116.5 116.5
60 122.8 122.9 123.2 123.1 123.2 123.1 J = 16 Hz, H-70 ); 13C NMR (CD3OD, 100 MHz) were
70 147.1 147.0 147.7 147.5 147.7 147.5 given in Table 1, identical to NMR data in the literature
80 115.5 115.1 114.6 114.6 114.6 114.6 (Iwai, Kishimoto, Kakino, Mochida, & Fujita, 2004).
90 171.1 169.1 168.5 168.2 168.5 168.2 3,5-Di-O-caffeoylquinic acid (7): C25H24O12, yellow
a
Assignments confirmed by decoupling, 1
H–1H COSY, NOESY, power, ESI-MS: m/z 539.0 [M+Na]+, 1H NMR (CD3OD,
HMQC and HMBC. 400 MHz): d 2.13–2.34 (4H, m, H-2, -6), 4.16 (1H, m, H-4),
5.12 (1H, m, H-5), 5.63 m (1H, m, H-3), 6.26 and 6.31 (1H
each, d, J = 16.0 Hz, H-80 ,-800 ), 6.71 and 6.75 (1H each, d,
s, H-3), 6.25 (1H, s, H-5), 6.61 (1H, d, J = 15.6, H-7) 7.31 J = 8.0 Hz, H-50 , -500 ), 6.96 and 6.97 (1H each, dd, J = 2.0,
(1H, d, J = 15.6, H-8), 7.03 (1H, d, J = 1.2, H-2), 6.95 (1H, 8.2 Hz, H-60 , -600 ), 7.06 and 7.07 (1H each, d, J = 2.0 Hz,
dd, J = 8.0, 1.2, H-5), 6.77 (1H, d, J = 8.0, H-6), sugar sig- H-20 , -200 ), 7.57 and 7.61 (1H each, d, J = 16.0 Hz, H-70 ,
nals: d 4.66 d (1H, d, J = 7.6, H-100 ), 3.38 (1H, m, H-200 ), -700 ); 13C NMR (CD3OD, 100 MHz) were given in Table
3.40 (1H, m, H-300 ) 3.31 (1H, m, H-400 ), 3.22 (1H, m, H- 1. The NMR data were consistent with the literature (Iwai
500 ), 3.75 (1H, m, H-600 ), and 13C NMR (C5D5N, et al., 2004; Zhu, Zhang, & Lo, 2004).
100 MHz): ppm 166.6 (C-2), 92.3 (C-3), 171.6 (C-4), 4,5-Di-O-caffeoylquinic acid (8): C25H24O12, yellow
101.1 (C-5), 161.7 (C-6), 116.7 (C-7), 137.5 (C-8), 128.7 powder, ESI-MS: m/z 539.1 [M+Na]+. 1H NMR (CD3OD,
(C-9), 114.8(C-10), 148.7 (C-11), 146.8 (C-12),116.5 400 MHz): d 1.94–2.34 (4H, m, H-2, -6), 4.36 (1H, s, H-3),
(C-13), 122.0 (C-14), 100.8 (C-10 ), 74.4 (C-20 ), 77.6 (C-30 ), 5.11 (1H, m, H-4), 5.55 (1H, m, H-5), 6.19 and 6.29 (1H
70.9 (C-40 ), 78.5 (C-50 ), 62.0 (C-60 ), key HMBC correlation: each, d, J = 16.0 Hz, H-80 , -800 ), 6.74 and 6.76 (1H each,
H-10 /C-4. d, J = 8.0 Hz, H-50 , -500 ), 6.90 and 6.92 (1H each, dd,
The HMBC spectrum of compound 3 showed the corre- J = 2.0, 8.0 Hz, H-60 , -600 ), 7.01 and 7.03 (1H each, d,
lations of H-5/C-4, H-5/C-3, H-6/C-4, H-6/C-2, H-7/C-2, J = 2.0 Hz, H-20 , -200 ), 7.52 and 7.60 (1H each, d,
and H-7/C-3, which confirmed the presence of the J = 16.0 Hz, H-70 , -700 ); 13C NMR (CD3OD, 100 MHz)
hydroxymaltol moiety. The HMBC correlation of H-7ab/ were given in Table 1. The NMR data were consistent with
C-90 was observed corresponding to the linkage between the literature (Iwai et al., 2004; Zhu et al., 2004).
the hydroxymaltol and the caffeoyl moiety (Guo, Koike,
Li, Guo, & Nikaido, 2004). Furthermore, the b-glucopyra- 3.2. The contents of compounds 1–8
nose group was identified by direct comparison with spec-
troscopic data (Guo et al., 2004). An obvious correlation The contents of phenolic compounds 1–8 in the aqueous
between dH 5.52 (anomeric proton) and dC 143.2 (C-3) extract of Sword Brake fern (SBF) was analyzed by HPLC
has been observed in the HMBC experiment. This evidence as described in Section 2. A representative HPLC chro-
confirmed that the glucose was linked at C-3 of hyroxymal- matogram is shown in Fig. 2. The purified compounds
tol. Thus, the proposed structure 3 was determined as 1–8 were used in parallel to obtain the retention time for
hydroxymaltol 7-O-caffeoyl-3-O-b-D-glucopyranoside. The quantification analysis. Table 3 shows the contents of com-
aglycone part of 4 was a known compound, hispidin, iso- pounds 1–8 in SBF. It was found that caffeic acid (5) and
lated from Inonotus hispidus. The structure was identified its derivatives (3, 7, and 8) were the major class of phenolic
by the NMR data, which were reported in the literature acids in SBF, with levels of 2.64 ± 0.23, 9.99 ± 0.17,
(Ali, Jansen, Horst, Liberra, & Lindequist, 1996). The 4.47 ± 0.15 and 3.68 ± 0.22 mg/g dry weight, respectively.
HMBC spectrum correlations of dH 5.52 (anomeric proton) Hispidin 4-O-b-D-glucopyranoside (4) is another impor-
and dC 171.6 (C-4) has been observed. This evidence con- tant composition with content of 4.11 ± 0.08 mg/g dry
firmed that the glucose was linked at C-4 of hispidin. Thus, weight. Kaempferol glycosides (1 and 2) account for the
54 Y.-H. Chen et al. / Food Chemistry 105 (2007) 48–56

Fig. 2. A representative HPLC chromatograms of phenolic compounds 1–8 from the aqueous extract of Sword Brake fern (Pteris ensiformis Burm.).

Table 3 the antioxidant activity increased with the number of caf-

Contents of the isolated phenolic compounds in Sword Brake fern feoyl moeity in the molecule and this agreed with a previ-
Samples Amounts (mg/g dry ous report (Iwai et al., 2004).
weight)a Phenolic acids, known as a kind of multipurpose bioac-
Kaempferol 3-O-a-L-rhamnopyranoside-7-O-[a-D- 2.41 ± 0.04 tive agent, frequently occur in herbal plants (He, 2000).
apiofuranosyl-(1-2)-b-D-glucopyranoside] (1) Besides the antibacterial and antifungal activity, phenolic
Kaempferol 3-O-a-L-rhamnopyranosid-7-O-b-D- 2.62 ± 0.02
glucopyranoside (2)
acids were considered in recent years as potentially protec-
7-O-caffeoylhydroxymaltol 3-O-b-D- 9.99 ± 0.17 tive compounds against cancer and heart disease, in part
glucopyranoside (3) because of their antioxidant properties (Morton, Caccetta,
Hispidin 4-O-b-D-glucopyranoside (4) 4.11 ± 0.08 Puddey, & Croft, 2000). Caffeic acid (3,4-dihydroxycin-
Caffeic acid (5) 2.64 ± 0.23 namic acid) is among the major hydroxycinnamic acids
5-O-Caffeoylquinic acid (6) 0.86 ± 0.09
3,5-Di-O-caffeoylquinic acid (7) 4.47 ± 0.15
present in nature and is a potent antioxidant (Gulcin,
4,5-Di-O-caffeoylquinic acid (8) 3.68 ± 0.22 2006). In this research, it was found that caffeic acid deriv-
a
Values were determined from integration of HPLC signals and
atives (compounds 3, 5, 6, 7 and 8) were the major antiox-
response factors calculated from isolated pure compounds. Values repre- idant phenolics in SBF.
sent means ± SEM (n = 3). Hispidin inhibits protein kinase C (PKC) b-isoform
(IC50 = 2 lM) and is preferentially cytotoxic to cancer
major flavonols in SBF with 2.41 ± 0.04 and 2.62 ± cells (Gonindard et al., 1997). The derivatives and analogs
0.02 mg/g dry weight, respectively.
Table 4
3.3. DPPH radical scavenging assay and TEAC analysis The antioxidant activities of phenolic compounds isolated from P.
ensiformis
DPPH (1,1-diphenyl-2-picrylhydrazyl) scavenging assay Samples IC50 (lM)a TEAC
and Trolox equivalent antioxidant capacity (TEAC) are (mM)b
two widely used methods to evaluate antioxidant capacity Kaempferol 3-O-a-L-rhamnopyranoside-7- 104.27 ± 2.36 0.58 ± 0.01
in a short time (Blois, 1958; Re et al., 1999). Table 4 shows O-[a-D-apiofuranosyl-(1-2)-b-D-
glucopyranoside] (1)
the DPPH scavenging activities and TEAC values of com-
Kaempferol 3-O-a-L-rhamnopyranosid-7- 128.78 ± 2.52 0.52 ± 0.02
pounds 1–8 isolated from SBF. All of these compounds O-b-D-glucopyranoside (2)
exhibited considerable DPPH and ABTS.+ radical cation 7-O-caffeoylhydroxymaltol 3-O-b-D- 21.06 ± 0.81 0.89 ± 0.02
scavenging activities. Among them, caffeic acid (5) and its glucopyranoside (3)
derivatives (3, 6, 7, and 8) as well as hispidin glucoside Hispidin 4-O-b-D-glucopyranoside (4) 28.21 ± 1.51 0.85 ± 0.04
Caffeic acid (5) 17.48 ± 0.33 0.92 ± 0.04
(4) displayed stronger or equal DPPH scavenging activities
5-O-Caffeoylquinic acid (6) 21.63 ± 1.37 1.27 ± 0.03
as compared with the common antioxidant supplement, 3,5-Di-O-caffeoylquinic acid (7) 10.71± 1.13 1.99 ± 0.02
a-tocopherol. This result indicates that the structure prere- 4,5-Di-O-caffeoylquinic acid (8) 10.30 ± 0.72 2.19 ± 0.03
quisite to reinforce DPPH scavenging is catechol moiety. In Kaempferol (reference) 25.70 ± 1.02 1.19 ± 0.02
addition to OH moieties in the structural arrangements, the Hispidin (reference) 29.97 ± 2.84 0.91 ± 0.02
a-Tocopherol (reference) 28.08 ± 2.69
resonance of electrons between rings may also be impor-
Trolox (reference) 1.00 ± 0.01
tant for their DPPH scavenging activities. a
IC50: concentration (in lM) necessary for reduction 50% DPPH
Among the isolated compounds, di-O-caffeoylquinic
radical. Values represent means ± SEM (n = 3).
acids (7, 8) were both the strongest DPPH and ABTS.+ b
TEAC: concentration (in mM) of Trolox having the antioxidant
radical cation scavengers with IC50 and TEAC value about equivalent to 1.0 mM of the tested compound. Values represent means
10 lM and 2 mM, respectively. It is worthy of noting that ± SEM (n = 3).
 Y.-H. Chen et al. / Food Chemistry 105 (2007) 48–56 55

of hispidin have also been shown to be potent free radical Deng, Y., & Liang, N. (2005). Investigation on the chromatogram of
scavengers (Lee, Seok, Kim, & Yun, 2006; Lee & Yun, diterpenoids in Pteris semipinnata by HPLC-APCI-MS. Zhong Yao
Cai, 28, 278–280.
2006). In this research, we identified a new compound, Dinis, T. C., Maderia, V. M., & Almeida, L. M. (1994). Action of phenolic
hispidin 4-O-b-D-glucopyranoside (4), exhibited compatible derivatives (acetaminophen, salicylate, and 5-aminosalicylate) as
DPPH scavenging activity and TEAC value as compared inhibitors of membrane lipid peroxidation and as peroxyl radical
with its aglycone, hispidin. scavengers. Archives of Biochemistry and Biophysics, 315, 161–169.
It has been suggested that flavonols with a free 3-OH El-Sayeda, N. H., Awaadb, A. S., Hifnawyb, M. S., & Mabryc, T. J.
(1999). A favonol triglycoside from Chenopodium murale. Phytochem-
group, are the strongest antioxidants among flavonoids istry, 51, 591–593.
(Burda & Oleszek, 2001; Hollman & Katan, 1997). The iso- Gonindard, C., Bergonzi, C., Denier, C., Sergheraert, C., Klaebe, A.,
lated flavonol glycosides (1–2) in SBF displayed much Chavant, L., et al. (1997). Synthetic hispidin, a PKC inhibitor, is more
weaker DPPH radical scavenging activity (IC50 > cytotoxic toward cancer cells than normal cells in vitro. Cell Biology
 100 lM) and TEAC values (0.5 mM) than the aglycone, and Toxicology, 13, 141–153.
Gulcin, I. (2006). Antioxidant activity of caffeic acid (3,4-dihydroxycin-
kaempferol (IC50 = 25.70 ± 1.02 lM; TEAC  1 mM). namic acid). Toxicology, 217, 213–220.
Current result supported previous notion that O-glycosyla- Guo, H., Koike, K., Li, W., Guo, D., & Nikaido, T. (2004). Maltol
tion had a tremendous negative effect on free radical scav- glucosides from the tuber of Smilax bockii. Phytochemistry, 65,
enging of kaempferol (Cardoso, Silva, Castro-Gamboa, & 481–484.
Bolzani, 2005). He, X. G. (2000). On-line identification of phytochemical constituents in
botanical extracts by combined high-performance liquid chromato-
Previously, it has been demonstrated that SBF inhibited graphic-diode array detection–mass spectrometric techniques. Journal
inflammatory mediator production in LPS-activated of Chromatography A, 880, 203–232.
RAW264.7 cells. From current research, it suggests that Hollman, P. C., & Katan, M. B. (1997). Absorption, metabolism and
the health promotion effect of SBF may be the result of health effects of dietary flavonoids in man. Biomedical Pharmacother-
combinatory activities, including antioxidant and antiin- apy, 51, 305–310.
Imperato, F. (1994). A new flavone glycoside from the fern Pteris cretica.
flammatory. This result supports the hypothesis that the Experientia, 50, 1115–1116.
additive and synergistic effects of phytochemicals in fruits Iwai, K., Kishimoto, N., Kakino, Y., Mochida, K., & Fujita, T. (2004). In
and vegetables are responsible for their health benefits vitro antioxidative effects and tyrosinase inhibitory activities of seven
(Liu, 2003). In conclusion, we demonstrated for the first hydroxycinnamoyl derivatives in green coffee beans. Journal of
time that SBF exhibited strong antioxidant activity, attrib- Agricultural and Food Chemistry, 52, 4893–4898.
Kumaran, A., & Karunakaran, R. J. (2007). Activity-guided isolation and
uted to the phenolic compounds, especially derivatives of identification of free radical-scavenging components from an aqueous
caffeic acid, hispidin and kaempferol. extract of Coleus aromaticus. Food Chemistry, 100, 356–361.
Lee, I. K., Seok, S. J., Kim, W. K., & Yun, B. S. (2006). Hispidin
Acknowledgements derivatives from the mushroom Inonotus xeranticus and their antiox-
idant activity. Journal of Natural Products, 69, 299–301.
Lee, I. K., & Yun, B. S. (2006). Hispidin analogs from the mushroom
This research was supported partially by the research Inonotus xeranticus and their free radical scavenging activity. Bioor-
Grants NSC-93-2323-B-037-002 (to Y.-C. Wu) and NSC- ganic & Medicinal Chemistry Letters, 16, 2376–2379.
93-2320-B-041-003 (to M.-J. Wu) from the National Sci- Liu, R. H., Health benefits of fruit and vegetables are from additive and
ence Council, Taiwan. synergistic combinations of phytochemicals. American Journal of
Chinese Medicine 78, 517–520.
Liu, Z. M., Chen, G. G., Vlantis, A. C., Liang, N. C., Deng, Y. F.,
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