Three new flavonoids, proanthocyanidin, and accompanying phenolic constituents from Feijoa sellowiana

Article abstract of DOI:10.1080/09168451.2017.1412246

Our investigation of phenolic constituents of fruits, flower buds, and leaves of Feijoa sellowiana led to the isolation of twenty-one phenolics including three new gossypetin glycosides 1-3, and also the purification of a proanthocyanidin fraction. A high-performance liquid chromatography method for simultaneous analysis of phenolic constituents was established and then used to investigate the phenolic profiles of the parts of the plant species, to show the presence of characteristic flavonoids and ellagic acid derivatives or ellagitannins in the extracts from fruits, flower buds, and leaves. The branch extract profile also suggested the presence of alkylated ellagic acids as characteristic constituents. Inhibitory effects of feijoa flavonoids on mushroom tyrosinase were seen, although in some cases this may have resulted from direct interaction with the enzyme. Cytotoxic effect of the proanthocyanidin fraction was also shown.

Full text of DOI:10.1080/09168451.2017.1412246

Bioscience, Biotechnology, and Biochemistry
ISSN: 0916-8451 (Print) 1347-6947 (Online) Journal homepage: http://www.tandfonline.com/loi/tbbb20
Three new flavonoids, proanthocyanidin, and
accompanying phenolic constituents from Feijoa
sellowiana
Hiroe Aoyama, Hiroshi Sakagami & Tsutomu Hatano
To cite this article: Hiroe Aoyama, Hiroshi Sakagami & Tsutomu Hatano (2018): Three new
flavonoids, proanthocyanidin, and accompanying phenolic constituents from Feijoa sellowiana,
Bioscience, Biotechnology, and Biochemistry, DOI: 10.1080/09168451.2017.1412246
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Date: 05 January 2018, At: 02:26

Bioscience, Biotechnology, and Biochemistry, 2017
https://doi.org/10.1080/09168451.2017.1412246
Three new flavonoids, proanthocyanidin, and accompanying phenolic
constituents from Feijoa sellowiana
Hiroe Aoyamaa, Hiroshi Sakagamib and Tsutomu Hatanoa
b
agraduate school of medicine, dentistry and Pharmaceutical sciences, okayama University, okayama, Japan; meikai University research
institute of odontology (m-rio), saitama, Japan
ABSTRACT
ARTICLE HISTORY
received 24 august 2017
accepted 20 november 2017
Our investigation of phenolic constituents of fruits, flower buds, and leaves of Feijoa sellowiana
led to the isolation of twenty-one phenolics including three new gossypetin glycosides 1–3, and
also the purification of a proanthocyanidin fraction. A high-performance liquid chromatography
method for simultaneous analysis of phenolic constituents was established and then used
to investigate the phenolic profiles of the parts of the plant species, to show the presence of
characteristic flavonoids and ellagic acid derivatives or ellagitannins in the extracts from fruits,
flower buds, and leaves. The branch extract profile also suggested the presence of alkylated
ellagic acids as characteristic constituents. Inhibitory effects of feijoa flavonoids on mushroom
tyrosinase were seen, although in some cases this may have resulted from direct interaction with
the enzyme. Cytotoxic effect of the proanthocyanidin fraction was also shown.
KEYWORDS
Feijoa sellowiana; myrtaceae;
flavonoid; tyrosinase
inhibitor; cytotoxicity
Use of polyphenol-rich plants in medicine, cosmetics,
and health foods is expected [1,2] based on recent find-
ings regarding the health effects of their polyphenolic
constituents, including antioxidant, antibacterial, and
antitumor effects. Several distinctive plants in the fam-
ily Myrtaceae are rich in polyphenolics, including fla-
vonoids and ellagic acids [3,4], as exemplified by guava
(Psidium guajava) [5], clove (Syzygium aromaticum)
[6,7], and allspice (Pimenta dioica) [8].
Feijoa sellowiana Berg, also in the family Myrtaceae,
is known by its edible fruits [9], and this evergreen tree
is widely cultivated in Oceania and the Americas. Our
preliminary examination of the constituents of this plant
suggested the presence of unidentified phenolics in addi-
tion to known flavonoids and related phenolics [10–17].
e objective of this paper is therefore to characterize
the polyphenolic constituents, especially flavonoids and
ellagic acids, in this species to provide the basis for using
this plant as a polyphenolic resource.
performed on an Agilent 6520 + 4240 Series Accurate-
Mass Quadrupole Time-of-Flight (Q-TOF) LC/MS
instrument (Agilent, Santa Clara, CA, USA) with 50%
aq. acetonitrile containing 0.1% formic acid. 1H and 13C
NMR spectra were recorded on Varian INOVA 600AS
1
(600 MHz for H, and 151 MHz for 13C) and Varian
INOVA 400MR (400 MHz for 1H, and 100 MHz for 13C)
instruments (Varian, Palo Alto, CA, USA). Chemical
shifs were given in δ (ppm) downfield from tetramethyl-
silane, based on those of the solvent signals [δH 2.04 and
δC 29.8 for acetone-d6, and δH 3.30 and δC 49.8 for meth-
anol-d4]. Optical rotations were measured on a JASCO
DIP-1000 digital polarimeter (JASCO, Tokyo, Japan).
Electronic circular dichroism (ECD) spectra were
measured on a JASCO J-720 W spectrometer. e flow
rate was set to 1.0 mL/min. A YMC Pack ODS Pro C8
(4.6 mm i.d. × 150 mm) column was also used as men-
tioned below. Preparative HPLC was performed on a
YMC ODS-A324 (10 mm i.d. × 300 mm) column (YMC,
Kyoto, Japan) with H2O-CH3CN-HCOOH (85:15:5, by
volume) as the eluent. A Hitachi L-2455 diode-array
detector (DAD) (Hitachi, Tokyo, Japan) was used as a
UV detector for HPLC. Analytical normal-phase (NP)
HPLC was performed on a YMC-Pack SIL A-003 column
(4.6 mm i.d. × 250 mm) with n-hexane−MeOH−tetrahy-
drofuran−formic acid (55:33:11:1) containing oxalic acid
(450 mg/L) at room temperature. e flow rate was set at
1.5 mL/min, and UV detection at 280 nm was effected.
Materials and methods
General
Electrospray ionization mass spectrometry (ESI-MS)
measurements were performed on a Bruker amaZon
X and ETD instrument (Bruker, Billerica, MA, USA)
with 50% aq. acetonitrile containing 0.1% formic acid.
High-resolution (HR)-ESI-MS measurements were
CONTACT tsutomu hatano
hatano-t@cc.okayama-u.ac.jp
supplementary material for this paper is available online at https://doi.org/10.1080/09168451.2017.1412246.
© 2017 Japan society for Bioscience, Biotechnology, and agrochemistry

2
H. AOYAMA ET AL.
Silica gel (YMC), Toyopearl HW-40C (TOSOH, Tokyo,
Japan), YMC-gel ODS-A (75 μm) (YMC), and MCI-gel
CHP-20P (Mitsubishi Chemical, Tokyo, Japan) were
used for column chromatography. Apigenin, flavone,
kaempferol, luteolin, morin, myricetin, naringenin,
quercetagenin, quercetin, hyperoside, isoquercitrin and
quercitrin were purchased from Extrasynthese (Lyon,
France), cyanidin-3-O-β-glucoside were purchased
from Funakoshi Co., Ltd. (Tokyo, Japan) and gossypetin
was from Indofine Chemical (Hillsborough, NJ, USA).
Phloroglucinol was from Ishizu Pharmaceutical (Osaka,
Japan). Ellagic acid, L-tyrosine, and mushroom tyrosi-
nase were from Sigma (USA), and kojic acid was from
TCI (Tokyo, Japan).
chromatographed on a Sephadex LH-20 column (1.1 cm
i.d. × 30 cm) with the mixtures of 70% EtOH and 70%
acetone, to yield PAOF-1 (22) (164.9 mg) from the eluate
with a mixture of 70% EtOH–70% acetone [4:6 (v/v)].
Fresh flower buds (105 g) were extracted with
MeOH and the concentrated filtrate was then treated
with n-hexane to remove the lipophilic materials. e
remaining MeOH soluble part (12.2 g) was subjected
to column chromatography on Diaion HP-20 (4.0 cm
i.d. × 40 cm) with increasing concentrations of MeOH
in H2O. e H2O eluate (8.3 g) from the column was
chromatographed on a Sephadex LH-20 column (2.2 cm
i.d. × 50 cm) with 70% EtOH, to yield pedunculagin (21)
(254.1 mg). e eluate with 20% MeOH (1.3 g) from the
Diaion column was fractionated with column chroma-
tography on YMC-gel ODS-A (75 μm) (2.2 cm i.d. ×
50 cm) with increasing concentrations of MeOH in H2O
containing 5% acetic acid, to yield cyanidin-3-O-β-glu-
coside (20) (12.7 mg) and gossypetin-3-O-α-l-arabino-
furanoside (1) (6.4 mg).
Fresh leaves (111 g) were homogenized in 70% ace-
tone and the concentrated filtrate from the homogenate
was extracted with ethyl acetate and then with n-butanol.
e ethyl acetate extract (1.8 g) was subjected to col-
umn chromatography on Toyopearl HW-40C (2.2 cm
i.d. × 40 cm) with 70% EtOH and then 70% acetone as
the eluents. Combined fractions 21–25 (257 mg) from
the 70% EtOH eluate were fractionated by column
chromatography on MCI gel CHP-20P (1.1 cm i.d. ×
30 cm) with increasing concentrations of MeOH in
H2O, to yield hyperoside (5) (30.0 mg) from combined
fractions 1–20 of the 35% MeOH eluate. Combined
fractions 31–150 of the 35% MeOH eluate were fur-
ther purified by preparative HPLC to yield hyperoside
(5) (6.6 mg), isoquercitrin (6) (1.2 mg), reynoutrin
(7) (11.6 mg), avicularin (9) (31.0 mg), and quercitrin
(10) (19.6 mg). e 40% MeOH eluate from the MCI
column, and the 100% MeOH eluate were respectively
identified as guaijaverin (8) (19.0 mg) and flavone (11)
(7.3 mg). In a separate experiment, fresh leaves (531 g)
were homogenized in MeOH and, afer removal of the
lipophilic material from the concentrated filtrate with
n-hexane, the remaining MeOH solution was evaporated
to yield an extract (38.7 g). is extract was subjected to
column chromatography on Diaion HP-20 (4.0 cm i.d.
× 40 cm) with increasing concentrations of MeOH in
H2O. e 40% MeOH eluate (3.4 g) was fractionated by
column chromatography on Toyopearl HW-40C (2.2 cm
i.d. × 40 cm) with 70% EtOH, and combined fractions
16–20 (274.2 mg) were purified by column chromatog-
raphy on MCI gel CHP-20P (1.1 cm i.d. × 40 cm) with
increasing concentrations of MeOH in H2O, to yield
4-O-α-arabinofuranosylellagic acid (13) (8.8 mg) from
fractions 21–30 of the 50% MeOH eluate, and trifolin
(12) (1.4 mg) from fractions 41–50 of the 50% MeOH
eluate. e 80% MeOH eluate (341.5 mg) from the
Plant material
Fresh fruits, flower buds, leaves, and branches of a single
tree of F. sellowiana (cv. “Coolidge”) were collected at the
Medicinal Plant Garden, Okayama University Graduate
School of Medicine, Dentistry and Pharmaceutical
Sciences. Leaves and buds were collected in June,
and fruits were collected in October and November.
e voucher specimen No. FS-001HS was kept at the
Medicinal Plant Garden.
Extraction and isolation
Fresh fruits (309 g) were homogenized in 70% acetone
and the concentrated filtrate from the homogenate was
extracted successively with ethyl acetate and n-butanol.
e ethyl acetate extract (2.8 g) was subjected to column
chromatography on Toyopearl HW-40C (2.2 cm i.d. ×
40 cm) with 70% EtOH and then with 70% acetone.
Combined fractions 21–25 (74.7 mg) of the 70% EtOH
eluate were fractionated by column chromatography on
MCI gel CHP-20P (1.1 cm i.d. × 30 cm) with increas-
ing concentrations of MeOH in H2O, to yield phyllan-
thusiin E (17) (9.2 mg) from the 35% MeOH eluate,
and gossypetin-3-O-l-α-arabinofuranoside (3.3 mg)
(1) from the 40 and 45% MeOH eluates. e remain-
ing part of the combined eluates with 40% MeOH and
45% MeOH, as well as the eluate with 60% MeOH from
the MCI gel column were purified by preparative HPLC
to yield aromadendrin-7-O-β-glucoside (1.2 mg) (19),
4-O-α-arabinofuranosylellagic acid (13) (0.4 mg), and
naringenin-7-O-β-glucoside (0.8 mg) (18). Combined
fractions 26–30 (81.7 mg) of the 70% EtOH eluate from
the Toyopearl column were fractionated by column
chromatography on MCI gel CHP-20P (1.1 cm i.d. ×
20 cm) with increasing concentrations of MeOH in H2O,
to yield ellagic acid (16) (3.5 mg) from the 65% MeOH
eluate. e water-soluble portion (27.9 g) obtained afer
the n-butanol extraction was subjected to column chro-
matography on Diaion HP-20 (2.2 cm i.d. × 35 cm) with
increasing concentrations of MeOH in H2O. e 60%
MeOH eluate (396.6 mg) from the Diaion column was

BIOSCIENCE, BIOTECHNOLOGY, AND BIOCHEMISTRY
3
1
Diaion column was further purified by column chro-
matography on MCI gel CHP-20P (2.2 cm i.d. × 30 cm)
with increasing concentrations of MeOH in H2O, and
further purified by preparative HPLC to yield quercetin
(14) (2.0 mg). Separately, dried leaves (1.78 kg) were
pulverized, and extracted successively with n-hexane,
ethyl acetate, MeOH, and 70% acetone. e MeOH
extract (265.1 g) and the 70% acetone extract (230.7 g)
were combined, and the mixture dissolved in water was
extracted with n-hexane, ethyl acetate, and n-butanol,
to yield the following extracts: n-hexane (14.8 g), ethyl
acetate (63.0 g), n-butanol (139.7 g), and aq. extracts
(265.7 g). e n-butanol extract was subjected to column
chromatography on a Diaion HP-20 column (10.0 cm
i.d. × 70 cm) with increasing concentrations of MeOH in
H2O, and the 70% MeOH eluate was subjected to column
chromatography on a YMC gel ODS-A column (75 μm)
(5.0 cm i.d. × 35 cm) with increasing concentrations of
MeOH in H2O. e second fraction of the 25% MeOH
eluate (2.47 g) was further chromatographed on a col-
umn of MCI gel CHP-20P and preparative HPLC to give
gossypetin (3.9 mg) (15). e second fraction of the 30%
MeOH eluate (1.80 g) from the MCI gel column was
chromatographed again on a column of MCI gel CHP-
20P (2.2 cm i.d. × 35 cm) with increasing concentrations
of MeOH in H2O. e 40% MeOH eluate (65.8 mg) and
the first part of the 45% MeOH eluate (109.1 mg) from
the MCI gel column were respectively purified by pre-
parative HPLC to yield gossypetin-3-O-β-galactopyra-
noside (4) (3.4 mg), gossypetin-3-O-β-xylopyranoside
(3) (4.0 mg), gossypetin-3-O-α-rhamnopyranoside (2)
(7.5 mg), and gossypetin-3-O-α-l-arabinofuranoside
(4.5 mg) (1).
([M − pentose − H]−) (negative-ion mode). H NMR,
see Table 1. 13C NMR [acetone-d6–D2O (13:2, v/v),
151 MHz] δC 158.2 (C-2), 133.9 (C-3), 179.5 (C-4), 153.9
(C-5), 99.2 (C-6), 153.9 (C-7), 125.7 (C-8), 145.6 (C-9),
104.5 (C-10), 122.5 (C-1′), 116.6 (C-2′), 145.5 (C-3′),
149.1 (C-4′), 116.1 (C-5′), 122.5 (C-6′), 108.9 (C-1″),
1
82.3 (C-2″), 77.9 (C-3″), 87.7 (C-4″), 61.6 (C-5″). H
NMR [methanol-d4, 600 MHz] δH 7.63 (1H d J = 2.4 Hz)
(H-2′), 7.59 (1H dd J = 8.4, 2.4 Hz) (H-6′), 6.90 (1H d
J = 8.4 Hz) (H-5′), 6.28 (1H s) (H-6), 5.46 (1H br s)
(H-1″), 4.33 (1H dd J = 3.0, 0.6 Hz) (H-2″), 3.91 (1H
dd J = 5.4, 3.0 Hz) (H-3″), 3.91 (1H dd J = 8.4, 3.6 Hz)
(H-4″), 3.51 (1H dd J = 12.0, 3.6 Hz) (H-5″a), 3.48 (1H
dd J = 12.0, 8.4 Hz) (H-5″b). 13C NMR [methanol-d4,
151 MHz] δC 160.1 (C-2), 135.4 (C-3), 181.2 (C-4), 155.7
(C-5), 100.4 (C-6), 155.8 (C-7), 127.0 (C-8), 147.5 (C-9),
106.1 (C-10), 124.0 (C-1′), 117.9 (C-2′), 147.1 (C-3′),
150.7 (C-4′), 114.2 (C-5′), 124.1 (C-6′), 110.3 (C-1″),
84.1 (C-2″), 79.5 (C-3″), 88.9 (C-4″), 63.4 (C-5″).
Gossypetin-3-O-α-rhamnopyranoside (2)
An amorphous yellow powder, [α]D −98.3° (c 1, MeOH).
ECD (c 0.01, MeOH) [θ] (nm) −7.2 × 103 (231),+8.0 × 103
(255), −7.2 × 103 (322). HR-ESI-MS Found, m/z 463.0889
([M−H]−); Calcd for C21H20O12−H, 463.0871. ESI-MS
m/z 465 ([M + H]+), 319 (MS/MS) ([M − deoxyhex-
ose + H]+) (positive-ion mode); m/z 463 ([M−H]−),
317 (MS/MS) ([M − deoxyhexose − H]−) (negative-ion
mode). 1H NMR, see Table 1. 13C NMR [acetone-d6–D2O
(13:2, v/v), 151 MHz] δC 158.4 (C-2), 135.3 (C-3), 179.3
(C-4), 153.5 (C-5), 99.1 (C-6), 154.1 (C-7), 125.7 (C-8),
145.6 (C-9), 104.9 (C-10), 122.4 (C-1′), 116.8 (C-2′),
145.5 (C-3′), 149.0 (C-4′), 115.9 (C-5′), 122.5 (C-6′),
102.7 (C-1″), 71.3 (C-2″), 72.4 (C-3″), 71.5 (C-4″), 71.1
(C-5″), 17.5 (C-6″).
Gossypetin-3-O-α-L-arabinofuranoside (1)
An amorphous yellow powder, [α]D − 72.8° (c 1, MeOH).
ECD (c 0.01, MeOH) [θ] (nm) −4.9 × 103 (233), +5.4 × 103
(258), −1.1 × 104 (304). HR-ESI-MS found, m/z 451.0864
([M + H]+); Calcd for C20H18O12 + H, 451.0877. ESI-MS
m/z 451 ([M + H]+), 319 (MS/MS) ([M − pentose + H]+)
(positive-ion mode); m/z 449 ([M−H]−), 317 (MS/MS)
Gossypetin-3-O-β-xylopyranoside (3)
An amorphous yellow powder, [α]D −139.0° (c 1,
MeOH). ECD (c 0.01, MeOH) [θ] (nm) −7.8 × 103
(207), +1.1 × 104 (221), +9.1 × 103 (239). HR-ESI-MS
Table 1. 1h-nmr assignment of compound 1–3 and correspond known compounds 7, 9, and 10 [1h-nmr 600 mhz, acetone-d6–
d2o (13:2), 27 °c] (J in hz).
Position
1
9
2
10
3
7
2′
5′
6′
6
7.70 (d 2.4)
6.95 (d 8.4)
7.59 (dd 8.4, 2.4)
6.33 (s)
7.62 (d 1.8)
6.95 (d 8.4)
7.50 (dd 8.4, 1.8)
6.23 (d 1.8)
6.47 (d 1.8)
7.49 (d 1.8)
6.97 (d 8.4)
7.40 (dd 8.4, 1.8)
6.32 (s)
7.42 (d 2.4)
6.96 (d 8.4)
7.32 (dd 8.4, 2.4)
6.22 (d 1.8)
6.43 (d 1.8)
7.79 (d 2.4)
6.92 (d 8.4)
7.68 (dd 8.4, 2.4)
6.31 (s)
7.70 (d 2.4)
6.91 (d 8.4)
7.58 (dd 8.4, 2.4)
6.22 (d 1.8)
6.47 (d 1.8)
8
1″
2″
3″
4″
5″
5.43 (br s)
5.49 (br s)
5.37 (d 1.8)
5.37 (d 1.8)
5.22 (d 7.2)
3.47 (d 7.2)
3.54 (dd 9.0, 7.2)
3.50 (dd 9.0, 5.4)
3.10 (dd 12.0, 9.0)
3.72 (dd 12.0, 5.4)
5.24 (d 7.2)
3.46 (d 7.2)
3.52 (dd 9.0, 7.2)
3.51 (dd 9.0, 5.4)
3.09 (dd 12.0, 9.0)
3.71 (dd 12.0, 5.4)
a
4.33 (d 1.8)
4.22 (dd 3.6, 1.8)
3.74 (dd 6.0, 3.6),
3.34–3.36 (m)
4.21 (dd 3.6, 1.8)
3.74 (dd 6.0, 3.6)
3.34–3.35 (m)
a
3.97 (dd 4.8, 2.4)
3.90 (dd 8.4, 4.8)
3.48 (dd 12.0, 4.8)
3.45 (dd 12.0, 8.4)
3.88 (dd 9.0, 4.2)
3.48 (dd 12.0, 4.2)
3.45 (dd 12.0, 9.0)
6″
0.85 (d 6.0)
0.84 (d 5.4)
aoverlapped with the hdo signals.

4
H. AOYAMA ET AL.
Found, m/z 449.0723 ([M−H]−); Calcd for C20H18O12−H,
449.0715. ESI-MS m/z 451 ([M + H]+), 319 (MS/MS)
([M − pentose + H]+) (positive-ion mode); m/z 449
([M−H]－), 317 (MS/MS) ([M − pentose − H]−) (neg-
ative-ion mode). 1H NMR, see Table 1. 13C NMR [ace-
tone-d6–D2O (13:2, v/v), 151 MHz] δC 157.9 (C-2), 134.5
(C-3), 179.0 (C-4), 153.7 (C-5), 99.1 (C-6), 153.9 (C-7),
125.7 (C-8), 145.5 (C-9), 104.7 (C-10), 122.5 (C-1′),
117.1 (C-2′), 145.3 (C-3′), 149.2 (C-4′), 115.7 (C-5′),
122.8 (C-6′), 103.7 (C-1″), 74.3 (C-2″), 76.5 (C-3″), 70.0
(C-4″), 66.4 (C-5″).
weight distribution was prepared using (−)-epicatechin
(monomer), procyanidin B2 (dimer), procyanidin C1
(trimer) and cinnamtannin A2 (tetramer), isolated from
bark of Cinnamomum cassia following a method applied
to Cynomorium songaricum [20].
The HPLC-DAD (diode array detector) analyses
of the extracts and quantitation of the major
constituents
Extracts for the quantitative HPLC analyses (other than
the MeOH extracts of fresh leaves and flower buds) were
prepared as follows. Fresh fruits (127.7 g) were homoge-
nized in MeOH (500 mL), and the concentrated filtrate
(100 mL) was extracted with n-hexane (100 mL × 3) to
remove the lipophilic material. e MeOH-soluble por-
tion was evaporated to yield the extract (13.8 g) used
for the HPLC analysis. Fresh branches (570.0 g) were
homogenized in MeOH (300 mL), and the concentrated
filtrate (120 mL) was treated with n-hexane (120 mL × 3)
to remove the lipophilic material. e remaining MeOH
solution was evaporated to yield the extract (7.5 g) for
the HPLC analysis.
Hydrolysis of compounds 1–3
Compound 1 (100 μg) in 1 M HCl (200 μL) was heated
for 4 h at 100 °C in water bath. e solution was then
concentrated to dryness, and analyzed by RP-HPLC, to
show the presence of gossypetin in the reaction mix-
ture based on the comparison of the authentic one.
Compounds 2 and 3 were treated analogously, to show
the formation of gossypetin.
Proanthocyanidin PAOF-1 (22)
Each of the extracts (5 mg) thus obtained was dis-
solved in MeOH (100 μL), and 3 μL each of the solutions
was injected into a YMC Pack ODS Pro C8 (4.6 mm i.d. ×
150 mm) column (YMC, Kyoto, Japan) with the follow-
ing gradient profile of solvents A [H2O–CH3CN–AcOH
(95:5:1, v/v/v)] and B [H2O–CH3CN–AcOH (60:40:1,
v/v/v)] using a Hitachi L-2130 gradient system (Hitachi,
Tokyo, Japan): 0–40 min, 0% → 30%, linearly; 40–60 min,
30% → 100% (%B in A + B) for the simultaneous detec-
tion of the phenolics. e column temperature was set to
40 °C in a column oven. Flow rate was set to 1.0 mL/min.
Detection was accomplished using UV absorption in
the 200–400 nm range on the L-2155 photodiode-array
instrument (Hitachi), and monitored at 280 and 320 nm
for flavonoids and ellagic acids, respectively.
e following isocratic conditions were used for
quantitation purposes. Each solution (2 μL) of the
extracts (5 mg) in MeOH (100 μL) was injected into a
YMC Pack ODS Pro C8 (4.6 mm i.d. × 150 mm) column
(YMC, Kyoto, Japan) with a mixture of H2O and CH3CN
(85:15, by volume) containing 1% acetic acid for flavo-
noid glycosides and ellagic acids and a mixture of H2O
and CH3CN (7:3, by volume) containing 1% acetic acid
for flavone (11) as the eluents. Solutions of the authentic
samples with concentrations of 100, 50, and 25 μg/100
μL were prepared and injected onto the column, and the
quantitation of the constituents was based on their peak
areas, and on triplicate experiments.
13C NMR [acetone-d6–D2O (13:2, v/v), 151 MHz] δC
169.3 (galloyl), 156.0, 154.3 (C-5, 7, 8a), 145.8, 145.1
(C-3′, 4′), 131.9 (C-1′), 118.7 (C-6′), 115.6 (C-5′), 114.5
(C-2′), 110.1 (C-6, 8), 107.1 (C-4a), 101.6 (C-4a), 97.1
(C-6, 8), 76.4 (C-2), 72.1 (C-3 extension), 67.8 (C-3
terminal), 36.9 (C-4). ECD (c 0.01, MeOH) [θ] (nm)
−5.3 × 105 (206), +1.1 × 106 (238), −3.5 × 104 (277).
Constituent analysis of PAOF-1 (22)
e constituent units of PAOF-1 (22) were identified by
phloroglucinolysis as follows [18]: PAOF-1 (30.1 mg) was
dissolved in 1% HCl-EtOH containing 0.5% (w/v) phloro-
glucinol, and lef to stand for 3 days at room temperature,
and the reaction mixture (38.6 mg) was subjected to col-
umn chromatography on MCI gel CHP-20P (1.1 cm i.d. ×
20 cm), to give (−)- epicatechin-(4β-2)-phloroglucinol (22a)
(2.1 mg) (from the eluate with 25% + 30% MeOH) and
(−)-3-O-galloylepicatechin-(4β-2)-phloroglucinol (22b)
(2.8 mg) (from the eluate with 40% MeOH eluate. e pres-
ence of catechin from the terminal unit in the reaction mix-
ture was also observed in the NP- and RP-HPLC analyses.
Molecular weight distribution analysis of
proanthocyanidins in PAOF-1 (22) [19]
e molecular weight distribution was estimated by
size exclusion chromatography (SEC) on a TSK-gel
SuperAW3000 column (6.0 mm i.d. × 150 mm) (TOSOH,
Tokyo, Japan) using N,N-dimethylformamide (DMF)
containing 3 M aq. ammonium formate [0.5% (v/v)] as
the eluent at 40 °C in a column oven. e flow rate was
set at 0.5 mL/min. e calibration line for the molecular
Tyrosinase inhibitory assay
A tyrosinase inhibitory assay was performed based on a
method reported previously [21] with slight modifica-
tion using 96-well microplates. A mixture of phosphate

BIOSCIENCE, BIOTECHNOLOGY, AND BIOCHEMISTRY
5
buffer (pH 6.8) (160 μL) and MeOH (20 μL) contain-
ing the followings additives were prepared: mushroom
tyrosinase (62.5 mU) (A), without any additives (B),
mushroom tyrosinase and the tested compound (C),
and the tested compound without the enzyme (D).
e mushroom tyrosinase was added as the buffered
solution and the tested compounds were added as the
MeOH solution. Afer pre-incubation of these solutions
for 10 min at room temperature, L-tyrosine in 20 μM
phosphate buffer (1 μM, 20 μL) was added to each solu-
tion and the mixtures were incubated for 20 min at room
temperature. Based on the absorbance at 475 nm before
(λbefore) and afer (λafer) the incubation for each solution,
the inhibition (%) for the tested compound was calcu-
lated using the equation
Structures of new flavonoid glycosides
Compound 1 was obtained as an amorphous yellow
powder. e molecular formula of this compound was
determined to be C20H18O12 based on the [M + H]+ ion
peak from positive-ion mode of the HR-ESI-MS. e
low-resolution ESI-MS also showed the ions at m/z 451
in the positive-ion mode and m/z 449 in the negative-ion
mode, corresponding to the [M + H]+ and the [M − H]−
ions, respectively, and the MS/MS of these ions showed
the ions at m/z 319 [M − pentose + H]+ (positive-ion
mode) and m/z 317 [M − pentose–H]− (negative-ion
mode), corresponding to the aglycone of mass 318. e
1H NMR spectrum (Table 1) in acetone-d6 containing
D2O showed a singlet at δH 6.33, and signals of three
protons forming an ABX system at δH 7.70, 7.59, and
6.95. e former singlet was assigned to an A-ring pro-
ton of its flavonol skeleton, and the latter ABX signals
were attributed to the B-ring protons of the skeleton. e
spectrum also showed proton signals corresponding to
the pentose moiety. e chemical shifs and the coupling
constants of the 1H-NMR signals of the pentose are sim-
ilar to those of avicularin (9) (= quercetin-3-O-α-l-ara-
binofuranoside) (see Table 1), suggesting the presence of
arabinose as the pentose in 1. Combined with the molec-
ular formula of 1 shown above, the structure where an
A-ring proton in 9 is replaced with a hydroxyl group
was assigned to compound 1. e 13C NMR spectrum
of 1 substantiated this structural assignment as shown
in the “Materials and methods” section). An A-ring
carbon signal in the spectrum of 1 shifed downfield
noticeably (δC 125.7, C-8), relative to the corresponding
carbon signal (δC 94.6) of 9, and three A-ring carbon
signals shifed upfield [δC 153.9 (C-5), 153.9 (C-7), 145.6
(C-9)], relative to the corresponding carbon signals of 9
[δC 162.2 (C-5), 165.4 (C-7), 158.3 (C-9)]. ese shifs
in the A-ring carbons indicated that the flavonol agly-
cone in 1 is gossypetin. e assignments of the A-ring
carbon signals were further substantiated by the cor-
relations of H-6 to C-5, 7, 8, and 10 in the heteronu-
clear multiple-bond correlation (HMBC) spectrum of 1
(Figure S4 in the Supplementary data) in methanol-d4.
e HMBC spectrum also showed a correlation of H-1″
to C-3 (Figure S3 in the Supplementary data) in ace-
tone-d6–D2O, substantiating the location O-3 of the
arabinofuranose residue on the flavonol structure in 1.
e electronic circular dichroism (ECD) spectrum of
compound 1 showed negative and positive maxima at
304 and 258 nm ([θ]304 −1.1 × 104, [θ]258 + 0.5 × 104),
forming a pattern similar to that of 9 ([θ]295 − 0.8 × 104,
[θ]254 + 1.1 × 104). Because the l-configuration of the
arabinofuranose moiety in 9 found in the present study
was shown by its specific rotation ([α]D −152°; lit. [25],
[α]D −152°), the arabinofuranose residue in compound 1
was determined to have the l-configuration. e struc-
ture of compound 1 was thus considered to be gossype-
tin- 3-O-α-l-arabinofuranoside (Figure 1).
% = 100 × [(A − B) − (C − D)] ∕ (A − B)
where A, B, C, and D are the differences between λafer
and λbefore for the respective solutions mentioned above.
e inhibition % was expressed as the average from trip-
licate experiments. Kojic acid and quercetin were used
as the positive controls.
Antitumor assay
e antitumor assay was performed according to the
method reported previously [22], using human oral
squamous cell carcinoma (OSCC) cell lines (gingi-
val tissue-derived Ca9-22, and tongue tissue-derived
HSC-2, HSC-3 and HSC-4) and human normal oral cells
[gingival fibroblast (HGF), pulp cell (HPC), periodontal
ligament fibroblast (HPLF)]. e tumor selectivity index
(TS) was calculated by dividing the mean CC50 for four
OSCC cell lines to mean CC50 for three normal oral cells
[(D/B) in Table 4]. Since both Ca9-22 and HGF cells
were derived from the gingival tissue, the relative sensi-
tivity of these cells was also compared [(C/A) in Table 4].
Results and discussion
Twenty-one phenolic compounds (1–21), including
three new ones (1, 2, and 3; Figure 1), and a proantho-
cyanidin oligomer fraction named PAOF-1 (22), were
obtained from the fruits, flower buds, and leaves of an
F. sellowiana tree. Among them, seven compounds (1,
13, 16–19) and PAOF-1 (22) were from the fruits, three
(1, 20, and 21) were from the flower buds, and 1–15
were from the leaves. e known compounds among
those isolated were identified based on the comparisons
of the NMR and MS data with those in the literature (4
[23], 7 [24], 8 [25], 9 [25], 12 [26], 13 [27], 17 [28], 18
[29], 19 [30]), and direct comparisons with the authen-
tic samples on HPLC (5, 6, 10, 11, 14–16, 20, 21, 23).
Although compounds 5, 9, 10, 13, 14, 16 and 20, 21, 23
were reported as the constituents of this plant species,
the isolation of compounds 4, 6–8 12, 15, 17–19, 22 was
not reported previously.

6
H. AOYAMA ET AL.
Figure 1. Phenolic constituents of Feijoa sellowiana. arrows in structure 1 indicate key hmBc correlations.
Compound 2 was obtained as an amorphous yel-
low powder. e molecular formula C21H20O12, was
indicated by the [M − H]− ion peak in the HR-ESI-MS.
e low-resolution ESI-MS also showed the [M + H]+
ion peak at m/z 465 and the [M − deoxyhexose + H]+
ion peak at m/z 319 (MS/MS of the ion) (positive-ion
mode), and the [M − H]− ion peak at m/z 463 and the
[M–deoxyhexose–H]− ion peak at m/z 317 (MS/MS of
the ion) (negative-ion mode). us, the aglycone with
mass 318 was consistent with the gossypetin structure.
structure of gossypetin-3-O-α-rhamnopyranoside was
thus assigned to compound 2.
Compound 3 was also obtained as an amorphous
yellow powder. Its molecular formula C20H18O12 was
indicated by the [M − H]− ion peak in the HR-ESI-MS.
e low-resolution ESI-MS showed the [M + H]+ ion
peak at m/z 451 and the ion peak at [M − pentose + H]+
ion peak at m/z 319 (MS/MS) (positive-ion mode), and
the [M − H]− ion peaks at m/z 449 and the [M − pen-
tose − H]− ion peak at m/z 317 (MS/MS) (negative-ion
mode). e ions for the mass-318 aglycone are also
consistent with the gossypetin structure. e 1H NMR
spectrum (Table 1) showed the signals of the aglycone
gossypetin, too. e sugar proton signals showed a pat-
tern similar to the corresponding one of reynoutrin
(= quercetin-3-O-β-xylopyranoside) (7), indicating that
the pentose in 3 is β-xylopyranose. e 13C NMR spec-
trum also showed the signals assignable to structure 3
1
e H NMR spectrum of compound 2 also showed
the presence of gossypetin as the aglycone moiety
(Table 1). e sugar proton signals corresponding to
the deoxyhexose moiety in 2 forms a pattern similar to
that of the rhamnose moiety of quercitrin (= querce-
tin-3-O-α-rhamnopyranoside) (10). e 13C NMR spec-
trum also showed the signals attributed to structure 2
as shown in the “Materials and methods” section. e

BIOSCIENCE, BIOTECHNOLOGY, AND BIOCHEMISTRY
7
(See the “Materials and methods” section). e structure
of gossypetin-3-O-β-xylopyranoside was thus assigned
to compound 3.
e aglycone structure in 1–3 was further con-
firmed by acid hydrolysis of these compounds, to give
gossypetin.
the remaining four prominent peaks were assigned to
those of isoquercitrin (6), 4-O-α-arabinofuranosylellagic
acid (13), quercetin (14), and flavone (11). Compound
11 was reported as one of the characteristic constituents
with anticancer activity in a feijoa fruit extract [32].
Furthermore, the peaks corresponding to those of gallic
acid (23) and pedunculagin (21) were also assigned based
on their DAD patterns in addition to their retention times
(data not shown). Although some other phenolic constit-
uents (16–19) were also isolated from the fruit extract in
the present study, their HPLC peaks were much smaller.
e flower bud extract showed the profile of Figure
2(b). e peaks of pedunculagin (21) and gallic acid (23)
were also seen there, as described above. e HPLC peak
of an anthocyanin constituent, cyanidin glucoside (20),
which is ascribed to the red color of feijoa flowers, also
appeared. ere were also HPLC peaks due to flavone
(11), ellagic acid (16), and the new flavonoid, gossypetin
arabinofuranoside (1).
e leaf extract showed the HPLC peaks of the phe-
nolic compounds 5–14 isolated from the leaf extract,
as shown by Figure 2(c). Although the peaks due to the
quercetin glycosides (5–10) were distinctive, the HPLC
peak of their aglycone, quercetin (14), was much smaller
than those of the corresponding glycosides. Flavone (11)
was one of the major constituents in the leaf extract.
Peaks due to ellagic acid (16), pedunculagin (21), and
gallic acid (23) were also observed.
e branch extract showed remarkable HPLC peaks
in the region of retention time 60–72 min, as shown by
peak group A in Figure 2(d). e UV (DAD) spectra
of the HPLC peaks of group A (except for the peak at
retention time 70.5 min) had characteristic absorption
maxima in the UV region 350–365 nm (Figure S15 in
the Supplementary data), which are similar to that of
ellagic acid (16), shown in Figure 2(d). e similarity
of the DAD spectra suggested the presence of alkylated
derivatives of ellagic acids [27] such as O-methyl ethers.
Although the presence of the alkylated derivatives was
reported for some myrtaceous plants [3], further con-
firmation of the presence of those types of compounds
in this source plant is still awaited.
e amounts of the flavonoids (5–10 and 11) and
ellagic acids (13 and 16) in the extracts were quantitated
and the results are summarized in Table 2. Isoquercitrin
(6) was the most abundant flavonoid in the fruit extract,
while hyperoside (5) was the most abundant flavonoid
in the leaf extract (Table 2). It should be noted that con-
siderable contents of flavone (11) were seen in all the
extracts from the tree. e content of 16 in the fruit
extract, in addition to that of arabinofuranosylellagic
acid (13), was also noteworthy. Ellagic acid (16) was one
of the most abundant phenolics in the extracts from the
leaves, flower buds, and branches of the tree (Table 2).
Ellagic acid was found in edible fruits of some plants
such as strawberry, and the plants containing ellagic acid
Characterization of PAOF-1
PAOF-1 (22) was obtained from the water-soluble res-
idue of the feijoa fruit extract as a brown amorphous
powder. e 13C NMR spectrum indicated the signals
assignable to the galloyl moieties in addition to those of
the procyanidin skeleton (see the Materials and Method
section), and the galloylated procyanidin structure was
further substantiated by acid degradation in the presence
of phloroglucinol, to show that it is composed of (−)-epi-
catechin and (−)-3-O-galloylepicatechin as the extension
units and (+)-catechin as the terminal unit. ese constit-
uent units were identified based on their comparisons on
RP-/NP-HPLC with the authentic samples. e relative
molar ratio of (−)-epicatechin-(4β-2)-phloroglucinol
and (−)-3-O-galloylepicatechin-(4β-2)-phloroglucinol
in the reaction mixture was estimated to be 5:7. e
2R stereochemistry [31] of the respective flavan units
were indicated by the negative Cotton effect at 273 nm
in the ECD spectrum. e number-average (Mn) and
weight-average (Mw) molecular weights of the procya-
nidin were estimated to be 6400 and 7600, respectively,
based on the SEC analysis [19]. e polymerization
degree (PD) 17 was also calculated based on the Mn data.
Although these values are based on the extrapolation of
the standard procyanidins and epicatechin, this value is
not so far from that calculated from the phloroglucinol
treatment (PD, 14).
Comparison of composition profiles of the extracts
from various parts of F. sellowiana
Because differences were seen among the phenolic con-
stituents isolated from the parts obtained from the single
tree, we compared the composition profiles of the extracts
from the fruits, flower buds, leaves, and branches using an
HPLC system equipped with a photodiode-array detector.
An HPLC condition with a gradient elution system using
(A) H2O–CH3CN (95:5) containing 1% acetic acid and
(B) H2O–CH3CN (60:40) containing 1% acetic acid was
established for simultaneous analyses of the phenolics iso-
lated from three of the four parts. e chromatographic
profiles of the extracts from the four parts, monitored at
280 and 320 nm, are shown in Figure 2; this wavelength
combination was adjusted to detect the phenolics, includ-
ing flavonoids and ellagic acids, in the extracts.
e fruit extract exhibited the HPLC profile of Figure
2(a). A pair of characteristic peaks of pedunculagin (21)
forming an anomer mixture was observed there, and

8
H. AOYAMA ET AL.
Figure 2. rP-hPlc profiles of the crude methanol extracts from (a) fruits, (b) buds, (c) leaves, and (d) branches of Feijoa sellowiana.
red and blue lines indicate profiles monitored at 280 and 320 nm, respectively.
or its derivatives have been expected to be beneficial for
health [33], since various pharmacological effects, such
as anticancer, anti-inflammatory, anti-mutagenic, anti-
oxidant, and antimicrobial effects, have been reported for
ellagic acid. e presence of ellagic acid also can be cor-
related with the presence of ellagitannins because ellagic
acid is known as the product formed upon hydrolysis
of ellagitannins. Ellagitannins have been found in plant
species of the family Myrtaceae, and an ellagitannin
pedunculagin (21) was obtained from flower buds in the
present study. Additional studies to identify hydrolysable
tannins of this plant species are in progress.
e accumulation of these antioxidant flavonoids and
phenolics in plants can be explained by the roles in the
plant survival in tropical and subtropical regions [34].
e present study indicated that all four parts of feijoa
trees, including leaves, can be used as resources for anti-
oxidant phenolics.
Tyrosinase inhibitory assay
Tyrosinase inhibitors are expected to have suppress-
ing effects on diet browning via affecting production
of melanin or melanin-like substances [35], and the

BIOSCIENCE, BIOTECHNOLOGY, AND BIOCHEMISTRY
9
inhibitory effects of various flavonoids on this enzyme
including the structure-activity relationship have been
revealed [35]. erefore, we examined the effects of
flavonoids found in F. sellowiana and related phenolics
on the tyrosinase activity. e results are summarized
in Table 3. e flavonoid glycosides examined did not
affect the tyrosinase activity at the concentration used,
as observed in previous reports [36–38]. However, gos-
sypetin (15), the aglycone of the new compounds, 1–3,
had an inhibitory effect, along with the other unglyco-
sylated flavonoids examined in the present study. e
inhibitory effect of gossypetin on tyrosinase has not yet
been reported, and the strength of the inhibition was
almost the same as that observed for one of the positive
controls, kojic acid, and somewhat weaker than that of
quercetin (14) (Table 3). A hydrolysable tannin pedun-
culagin (21), and the proanthocyanidin (condensed tan-
nin) PAOF-1 (22) did not show the inhibitory activity
(< 10% inhibition). e inhibitory effects seen in the
present study could be attributable to their antioxidant
effects, including those via trapping copper ions in the
enzyme. Because the tyrosinase used in this study was
from mushrooms, the effects may be different from the
findings using the human enzyme; further experiments
using that type are expected.
papers suggested that polyphenols might work as sub-
strates and/or cofactors of tyrosinase, or as an enzyme
binding agent [38]. Although these explanations might
rationalize the unusual effects of these two compounds
on tyrosinase activity, further studies are needed to clar-
ify them.
Antitumor assay
Although various antitumor drugs have been developed,
most of them had potent cytotoxicity on the normal
cells [31]. On the other hand, various tannins with large
molecular weights, especially oligomeric hydrolysable
tannins, showed remarkable tumor selectivity (TS) in
the cytotoxic experiments [39]. erefore, we exam-
ined cytotoxicity of the galloylated proanthocyanidin
oligomer PAOF-1 (22) using OSCC lines (Ca9–22, HSC-
2, HSC-3, HSC-4) and normal oral cells (HGF, HPC,
HPLF) in the present study. As a result, CC50 of PAOF-1
against human oral squamous cell carcinoma cell lines
were 151 μM, while CC50 for normal human oral cells
showed over 477 μM (Table 4). e TS index was over
3.2 based on these values [(D)/(B) in Table 4]. When
tumor selectivity was calculated using cells both derived
from gingival tissue (Ca9-22 vs. HGF), PAOF-1 had
slightly higher TS (>4.6), comparable with that of 5-FU
On the other hand, ellagic acid (16) and myri-
cetin (26) showed unanticipated inhibitory values
(−47.0 7.5%, and 139.8 6.9% inhibition of tyrosinase,
respectively). ese results suggest that ellagic acid acts
as a substrate or cofactor of tyrosinase, and myricetin
binds to the products of the enzyme reaction. Previous
Table 3. tyrosinase inhibitory activity of flavonoids.
% Inhibition of
Compound type
Compound
tyrosinase a
glycosides
hyperoside (5)
isoquercitrin (6)
reynoutrin (7)
guaijaverin (8)
avicularin (9)
Quercitrin (10)
Flavone (11)
29.4 3.8
1.1 4.4
16.2 6.8
5.4 2.0
18.7 8.8
4.8 6.4
15.4 5.6
97.6 8.1
91.6 1.7
68.2 3.0
139.8 6.9
81.5 10.2
65.4 5.4
32.7 4.6
30.3 5.0
−47.0 7.5
−1.4 10.4
7.2 6.5
Table 2. contents of flavonoids and ellagic acids in the leaves,
flower buds, branches, and fruits of Feijoa sellowianaa.
Unglycosylated com-
pounds
Flower
buds
Kaempferol (24)
Quercetagetin (25)
gossypetin (15)
myricetin (26)
morin (27)
Leaves
Branches
Fruits
hyperoside (5)
isoquercitrin (6)
reynoutrin (7)
guaijaverin (8)
avicularin (9)
Quercitrin (10)
Flavone (11)
arabinofuranosylel-
lagic acid (13)
ellagic acid (16)
94.7 5.1 30.2 0.2 28.6 0.3
tr b
57.2 4.2
tr
tr
tr
tr
9.5 0.5
57.8 1.8
90.9 5.4
88.0 5.3
46.2 1.0
tr
tr
tr
tr
tr
tr
tr
tr
tr
tr
luteolin (28)
apigenin (29)
naringenin (30)
ellagic acid (16)
Pedunculagin (21)
PaoF-1 (22)
85.2 5.3 56.3 2.8 26.0 0.1 57.2 4.2
tr tr tr 8.3 0.5
tannins
120.4 7.7 76.4 1.7 44.7 1.6 45.3 3.0
Positive controls
Quercetin (14)
Kojic acid
90.8 0.7
73.1 0.8
adata are expressed as the mean μg sd/10 mg of the extract based on
the triplicate experiments.
adata are expressed as the mean % inhibition sd for 0.1 mm of the
sample based on triplicate experiments.
btr, trace. it means lower than the loQ of each of the flavonoids (ca.
5 μg/10 mg of the extracts), and 13, 16 (ca. 5 μg/10 mg of the extracts).
Table 4. antitumor activity of PaoF-1 and antitumor agents.
CC50 (μM)
Human oral squamous cell carcinoma cell lines
Ca9-22
Human normal oral cells
Mean
HGF
Mean
TS
(A)
HSC-2
HSC-3
HSC-4
(B)
(C)
HPLF
HPC
(D)
(D)/(B)
(C)/(A)
PaoF-1
109 3.5
83 4.5
323 6.7
88 3.0
151
>500
430 61
>500
>477
>3.2
>4.6
Positive controls
5-FU
doxorubicin
docetaxel
389 206
34 10
256 133
54 20
183
0.49
<0.12
>2000
>5
>1
>2000
>5
>1
>2000
3.7 0.47
>1
>2000
>4.6
>1
>10.9
>9.4
>8.3
>5.1
>5
>128
1.00 0.45 0.22 0.06 0.45 0.06 0.29 0.01
<0.0078 <0.0078 0.18 0.17 0.29 0.09

10
H. AOYAMA ET AL.
(TS > 5.1) and doxorubicin (>5), except for docetaxel
(TS > 128) [(C)/(A) in Table 4].
Since the cytotoxic property of an acetonic extract
from feijoa fruit peels was reported previously [40], this
result suggested that one of the active constituents may
be regarded to be the oligomeric proanthocyanidin in
this plant.
Funding
is work was supported by a Grant-in-Aid from JSPS [grant
number 25450170].
References
[1] Manach C, Williamson G, Morand C, et al.
Bioavailability and bioefficacy of polyphenols in
humans. I. Review of 97 bioavailability studies. Am J
Clin Nutr. 2005;81:230S–242S.
[2] Gharras HE. Polyphenols: food sources, properties
and applications – a review. Int J Food Sci Technol.
2009;44:2512–2518.
[3] Lowry JB. e distribution and potential taxonomic
value of alkylated ellagic acid. Phytochemistry.
1968;7:1803–1813.
[4] Reynertson KA, Yang H, Jiang B, et al. Quantitative
analysis of antiradical phenolic constituents from
fourteen edible Myrtaceae fruits. Food Chem.
2008;109:883–890.
[5] Gutiérrez RMP, Mitchell S, Solis RV. Psidium guajava:
a review of its traditional uses, phytochemistry and
pharmacology. J Ethnopharmacol. 2008;117:1–27.
[6] Cai L, Wu CD. Compounds from Syzygium aromaticum
possessing growth inhibitory activity against oral
pathogens. J Nat Prod. 1996;59:987–990.
Conclusions
In the present study, we isolated new gossypetin glycosides
(1–3), along with eighteen known compounds, including
another gossypetin glycoside 4 and its aglycone gossy-
petin (15), quercetin (14) and its glycosides (5–10), a
kaempferol glycoside (12), a naringenin glycoside (18), an
aromadendrin glycoside (19), a cyanidin glycoside (20),
flavone (11), ellagic acid (16) and its derivatives (13 and
17), pedunculagin (21) and proanthocyanidin oligomer
PAOF-1 (22) as the phenolic constituents of the leaves,
fruits, and flower buds of F. sellowiana. is is the first
report of the isolation of gossypetin-3-O-β-galactopyra-
noside (4), isoquercitrin (6), reynoutrin (7), guaijaverin
(8) trifolin (12), phyllanthusiin E (17), naringenin-7-
O-β-glucoside (18), and aromdendrin-7-O-β-glucoside
(19) from feijoa. e presence of the alkylated ellagic acid
derivatives was also suggested in the branch extract.
e contents of nine of the phenolic compounds in
the extracts from various parts were quantitated (Table
2), to reveal that feijoa can be regarded as a resource for
those polyphenolic compounds. Unglycosylated flavo-
noids examined in this study showed inhibitory effects
on tyrosinase, comparable to that of kojic acid (Table 3).
e proanthocyanidin oligomer fraction (PAOF-1) (22)
derived from F. sellowiana fruits showed selective cyto-
toxity against OSCC lines, comparable to that of 5-FU
(Table 4). Previous studies on feijoa constituents generally
focused on its edible fruits; however, the findings in the
present study suggested not only fruits, but also the other
parts, especially leaves, of this plant species might be worth
studying to find bioactive polyphenolics or flavonoids.
[7] Nassa MI. Flavonoid triglycosides from the seeds of
Syzygium aromaticum. Carbohydr Res. 2006;341:160–163.
[8] Kikuzaki H, Miyajima Y, Nakatani N. Phenolic
glycosides from berries of pimenta dioica. J Nat Prod.
2008;71:861–865.
[9] Weston RJ. Bioactive products from fruit of the feijoa
(Feijoa sellowiana, Myrtaceae): a review. Food Chem.
2010;121:923–926.
[10] Okuda T, Yoshida T, Hatano T, et al. Ellagitannins
of the casuarinaceae, Stachyuraceae and Myrtaceae.
Phytochemistry. 1982;21:2871–2874.
[11] Lowry JB. Anthocyanins of the melastomataceae,
Myrtaceae and some allied families. Phytochemistry.
1976;15:513–516.
[12] Hardy PJ, Michael BJ.Volatile components of feijoa
fruits. Phytochemisty. 1970;9:1355–1357.
[13] Shaw GJ, Allen JM, Yates MK. Volatile flavour
constituents of the skin oil from Feijoa sellowiana.
Phytochemistry. 1989;28:1529–1530.
[14] Ruberto G, Tringali C. Secondary metabolites from
the leaves of Feijoa sellowiana berg. Phytochemistry.
2004;65:2947–2951.
Author contributions
[15] Vanidze MR, Shalashvili AG, Chkhikvishvili ID, et al.
Catechins of feijoa leaves. Subtropicheskie Kul’tury.
1991;6:87–90.
H. A. performed isolation, identification, structure
elucidation, tyrosinase inhibitory assay of the constit-
uents, and prepared the manuscript; H. S. contributed
to antitumor assay and improved the manuscript; T. H.
designed the study and reviewed manuscript.
[16] ElShenawy SM, Marzouk MS, Dib RAE, Elyazed HEA,
et al. A polyphenols and biological activities of Feijoa
sellowiana leaves and twigs. Rev Latinoamer Quím.
2008;36:104–120.
[17] Oldrich L, Borivoj K, Ladislav K, et al. Identification of
isoflavones in Acca sellowiana and two Psidium spesies
(Myrtaceae). Biochem Syst Ecol. 2005;33:983–992.
[18] Foo LY, Karchesy JJ. Procyanidin tetramers and
pentamers from Douglas fir bark. Phytochemistry.
1991;30:667–670.
[19] Kusuda M, Inada K, Ogawa T, et al. Polyphenolic
constituent structures of Zanthoxylum piperitum fruit
and the antibacterial effects of its polymeric procyanidin
on methicillin-resistant Staphylococcus aureus. Biosci
Biotechnol Biochem. 2006;70:1423–1431.
Acknowledgements
e Varian INOVA 600AS NMR and Varian INOVA 400-MR
NMR instruments used in this study are the property of the
SC-NMR laboratory of Okayama University.
Disclosure statement
No potential conflict of interest was reported by the authors.

BIOSCIENCE, BIOTECHNOLOGY, AND BIOCHEMISTRY
11
[20] Jin S, Eerdunbayaer, Doi A, et al. Polyphenolic
constituents of Cynomorium songaricum Rupr. and
antibacterial effect of polymeric proanthocyanidin on
methicillin-resistant Staphylococcus aureus. J Agric
Food Chem. 2012;60:7297–7305.
[29] Moco S, Tseng LH, Spraul M, et al. Building-up
a comprehensive database of flavonoids based on
nuclear magnetic resonance data. Chromatographia.
2006;64:503–508.
[30] Slimestad R, Andersen OM, Francis GW. Ampelopsin
7-glucoside and other dihydroflavonol 7-glucosides from
needles of Picea Abies. Phytochemistry. 1984;35:550–552.
[31] Korver O, Wilkins CK. Circular dichroism spectra of
flavonols. Tetarhedron. 1971;27:5459–5465.
[32] Bontempo P, Mita L, Miceli M, et al. Feijoa sellowiana
derived natural flavone exerts anti-cancer action
displaying HDAC inhibitory activities. Int J Biochem
Cell Biol. 2007;39:1902–1914.
[33] Vattem DA, Shetty K. Biological functionality of ellagic
acid: review. J Food Biochem. 2005;29:234–266.
[34] CasatiP, WalbotV. Geneexpressionprofilinginresponse
to ultraviolet radiation in maize genotypes with varying
flavonoid content. Plant Physiol. 2003;132:1739–1754.
[35] Chang T. An updated review of tyrosinase inhibitor. Int
J Mol Sci. 2009;10:2440–2475.
[36] Nugroho A, Choi J, Park J, et al. two new flavonol
glycosides from Lamium amplexicaule L. and their in
vitro free radical scavenging and tyrosinase inhibitory
activities. Planta Med. 2009;75:364–366.
[37] Kubo I, Kinst-Hori I. Flavonols from saffron flower:
tyrosinaseinhibitoryactivityandinhibitionmechanism.
J Agric Food Chem. 1999;47:4121–4125.
[38] Kim Y, Uyama H. Tyrosinase inhibitors from natural
and synthetic sources: structure, inhibition mechanism
and perspective for the future. Cell Mol Life Sci.
2005;62:1707–1723.
[21] Likhitwitayawuid K, Srutularak B. A new dimeric
stilbene with tyrosinase inhibitiory activity from
Artocarpus gomezianus. J Nat Prod. 2001;64:1457–1459.
[22] Orabi MAA, Taniguchi S, Sakagami H, et al.
Hydrolyzable tannins of tamaricaceous plants. v.
structures of monomeric–trimeric tannins and
cytotoxicity of macrocyclic-type tannins isolated from
Tamarix nilotica. J Nat Prod. 2013;76:947–956.
[23] Suzuki H, Sasaki R, Ogata Y, et al. Metabolic
profiling of flavonoids in Lotus japonicus using liquid
chromatography Fourier transform ion cyclotron
resonance mass spectrometry. Phytochemistry.
2008;69:99–111.
[24] Olszawska M, Wolbiś M. Further flavonoids from the
flowers of Prunus spinosa L. Drug Res. 2007;64:241–
245.
[25] Andres S, Petra S, Jurgen C, et al. Elution order of
quercetin glycosides from apple pomace extracts
on a new HPLC stationary phase with hydrophilic
endcapping. J Sep Sci. 2002;23:361–364.
[26] Wolbiś M, Nowak S, Kicel A. Polyphenolic compounds
in Scopolia caucasica Kolesn. ex Kreyer (Solanaceae).
Acta Pol Pharm. 2007;64:241–246.
[27] Zafrilla P, Ferreres F, Tomás-Barberán FA. Effect of
processing and storage on the antioxidant ellagic acid
derivatives and flavonoids of red raspberry (Rubus
idaeus) jams. J Agric Food Chem. 2001;49:3651–3655.
[28] Yoshida T, Ito H, Matsunaga S, et al. Tannins and
related polyphenols of euphorbiaceous plants. IX.
Hydrolyzable tannins with 1C4 glucose core form
Phyllanthus flexuosus Muell. Arg Chem Pharm Bull.
1992;40:53–60.
[39] Sakagami H. Biological activities and possible dental
application of three major groups of polyphenols. J
Pharmacol Sci. 2014;126:92–106.
[40] Motohashi N, Kawase M, Shirataki Y, et al. Biological
activity of feijoa peel extracts. Anticancer Res.
2000;20:4323–4329.