Article

pubs.acs.org/JAFC

Polymethoxyflavones Isolated from the Peel of Miaray Mandarin

(Citrus miaray) Have Biofilm Inhibitory Activity in Vibrio harveyi
Ram M. Uckoo, G. K. Jayaprakasha,* Amit Vikram, and Bhimanagouda S. Patil*
Vegetable and Fruit Improvement Center, Department of Horticultural Sciences, Texas A&M University, 1500 Research Parkway
Suite A120, College Station, Texas 77845, United States
*
S Supporting Information

ABSTRACT: Citrus fruits are a good source of bioactive compounds with numerous beneficial biological activities. In the
present study, fruits of the unexplored Miaray mandarin were used for the isolation of 10 bioactive compounds. Dried peels were
sequentially extracted with hexane and chloroform in a Soxhlet-type apparatus for 8 h. The extracts were concentrated under
vacuum and separated by flash chromatography to obtain nine polymethoxyflavones and a limonoid. The purity of each
compound was analyzed by high-performance liquid chromatography (HPLC), and the compounds were identified by spectral
analysis using MALDI-TOF-MS and NMR. The isolated compounds were identified as 5-hydroxy-3,7,3′,4′-tetramethoxyflavone,
5,6,7,8,4′-pentamethoxyflavone (tangeretin), 3,5,6,7,8,3′,4′-heptamethoxyflavone, 5,6,7,8,3′,4′-hexamethoxyflavone (nobiletin),
3,5,7,8,3′,4′-hexamethoxyflavone, 3,5,7,3′,4′-pentamethoxyflavone (pentamethylquercetin), 5,7,4′-trimethoxyflavone, 5,7,8,4′-
tetramethoxyflavone, 5,7,8,3′,4′-pentamethoxyflavone, and limonin. These compounds were further tested for their ability to
inhibit cell−cell signaling and biofilm formation in Vibrio harveyi. Among the evaluated polymethoxyflavones, 3,5,6,7,8,3′,4′-
heptamethoxyflavone and 3,5,7,8,3′,4′-hexamethoxyflavone inhibited autoinducer-mediated cell−cell signaling and biofilm
formation. These results suggest that Miaray mandarin fruits are a good source of polymethoxyflavones. This is the first report on
the isolation of bioactive compounds from Miaray mandarin and evaluation of their biofilm inhibitory activity as well as isolation
of pentamethylquercetin from the Citrus genus.
KEYWORDS: citrus, flash chromatography, identification, pentamethylquercetin, polymethoxyflavones, biofilm

■ INTRODUCTION
Citrus fruits including oranges, mandarins, grapefruits, limes,
and lemons are some of the most-consumed fruits in the world.
These fruits are excellent sources of phytochemicals such as
flavonoids, limonoids, organic acids, and carotenoids.1−4 The
mandarin (Citrus reticulata) citrus cultivars have a loose peel,
bright orange-red color, and juicy, succulent segment
membranes. They are among the most rapidly increasing citrus
varieties in terms of worldwide production and consumption.5
Native to Southeast Asia, mandarins are commercially
cultivated in the temperate regions of the world,6 with annual
production estimated at 20.3 million metric tons.7 Due to the
economic benefits of cultivation of mandarins, these fruits have
been extensively studied to enhance their yield and quality.
Mandarins typically show high diversity due to hybridization
and somatic mutations.8−10 These factors have led to significant
phenotypic and genetic variation, resulting in numerous species.
Miaray mandarin (Citrus miaray Tan.) is used as a rootstock for
citrus propagation.11 Miaray mandarin fruits are round, bright
yellow in color, and 5−7 cm in diameter, with sour, juicy
segment membranes. Only a few scientific studies have
examined this species, and most of these studies have focused
on evaluation of genetic heritability. These studies suggest that
Miaray mandarins are unique, with distinct genotypic character-
istics in comparison to other mandarin species.10
Polymethoxyflavones are a group of flavonoids that have
three or more methoxyl moieties attached to their basic flavone
structure. Polymethoxyflavones have been extensively studied
for their biological properties such as anticancer, antilipogenic,
antitumor, antiviral, and anti-inflammatory activities.12−14
Nobiletin, a polymethoxyflavone present in the majority of
citrus fruits, is reported to have potential antidementia activity
in both in vitro and in vivo studies.15 However, a major
bottleneck in investigating the potential biological activity of
polymethoxyflavones is their unavailability in large-scale
quantity and the relative impurity of most commercial
preparations. Prior research from our laboratory reported the
development of chromatographic methods for the analysis of
amines in different mandarin species and the isolation of
polymethoxyflavones from mandarin, grapefruit, and or-
ange.16,17 Our results indicate that mandarins are a rich source
of polymethoxyflavones and could be exploited for large-scale
isolation. Polymethoxyflavones primarily occur in the fruit peels
and to a lesser extent in the juice. These compounds are also
reported to be present in high levels in the fruits of Shiikuwasha
(Citrus depressa) and Calamondin (Citrus madurensis).18,19
In recent years, advances in chromatographic techniques
have led to improved methods for the rapid separation and
isolation of bioactive compounds. Simultaneously separation
and spectroscopic detection methods, termed hyphenated
chromatography, can be used to elucidate the tentative identity
of the compounds of interest.20−22 Among separation
techniques, flash chromatography provides several advantages,
Received: January 2, 2015
Revised: June 27, 2015
Accepted: July 3, 2015

© XXXX American Chemical Society A DOI: 10.1021/acs.jafc.5b02445

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Journal of Agricultural and Food Chemistry
Figure 1. Flash chromatograms obtained by the separation of extracts of Miaray mandarin peel using silica gel columns. The individual peaks A−G
yielded compounds 1−7, and H−N yielded compounds 1, 3, 4, and 7−10.
including large sample volumes, a wide range of solvents,
automated detection, and inline ultraviolet−visible spectral
scanning (UV−vis). This improved technique enables medium-
pressure liquid chromatographic separations as well as real-time
monitoring of the compounds by their unique UV−vis spectral
characteristics. Flash chromatography has been successfully
applied for the separation and isolation of several bioactive
compounds such as curcuminoids, flavonoids, and coumarins
from a wide range of plant extracts and industrial by-
products.17,23,24
The antimicrobial activity of citrus peel extracts has been
intensively investigated,25,26 but only a few studies have
evaluated the effect of pure polymethoxyflavones, primarily
due to commercial unavailability. Furthermore, polymethoxy-
flavones do not demonstrate potent bactericidal or bacterio-
static effects. For example, a recent study showed that
polymethoxyflavones nobiletin and tangeretin exhibit low
antimicrobial activities against six strains of microorganisms,
including Escherichia coli, Staphylococcus sp., and Salmonella
typhi.26 An alternative mechanism to attenuate bacterial
pathogenicity is interference with bacterial cell−cell communi-
cation pathways. We have previously demonstrated that several
citrus limonoids and flavonoids interfere with bacterial signaling
cascades, leading to attenuation of various virulence mecha-
nisms including biofilm formation.27−29 Our prior study
showed that 3′,4′,5,6,7-pentamethoxyflavone (sinensitin) exerts
B DOI: 10.1021/acs.jafc.5b02445
Article
biofilm inhibitory activity in a dose-dependent manner. Citrus
has approximately 28 structurally distinct reported polymethox-
yflavones, with variation in the number and position of the
methoxyls attached to the flavone backbone.30 These structural
variants may potentially demonstrate a wide range of inhibitory
activities on bacterial biofilms and help in the identification of
novel antimicrobial agents.
In the present study, nine polymethoxyflavones and one
limonoid were isolated for the first time from Miaray mandarins
using rapid flash chromatography along with 3,5,7,3′,4′-
pentamethoxyflavone (pentamethylquercetin). These com-
pounds were characterized by spectral analysis using high-
performance liquid chromatography (HPLC), matrix-assisted
laser desorption/ionization-time-of-flight-mass spectrometry
(MALDI-TOF), and 1D and 2D nuclear magnetic spectroscopy
(NMR). All identified compounds were evaluated for their
biofilm inhibitory activity using Vibrio harveyi.
■
MATERIALS AND METHODS
Plant Material. Mature Citrus miaray mandarin fruits (Figure S1)
were harvested in December 2010 from the Texas A&M University−
Kingsville Citrus Center orchard (Weslaco, TX, USA). A voucher
specimen (249960) has been submitted to the S. M. Tracy Herbarium,
Texas A&M University (College Station, TX, USA). The peels were
separated and dried to ≤5% moisture. The peels were blended to
obtain 40−60 mesh size powder in a Vita-prep blender (Vita-Mix
Corp., Cleveland, OH, USA) and used for extraction.
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Journal of Agricultural and Food Chemistry
Reagents and Instrumentation. Solvents used for analysis were
of HPLC grade and obtained from Fisher Scientific (Pittsburgh, PA,
USA). Nanopure water (NANOpure, Barnstead/Thermolyne, Dubu-
que, IA, USA) was used for HPLC analysis. The solvents used for flash
chromatography were of analytical grade and purchased from Fisher
Scientific. The separation of polymethoxyflavones was carried out on a
flash chromatography system (Combiflash Rf, Teledyne ISCO,
Lincoln, NE, USA). Silica gel (particle size 35−60 μm) columns
(220 g) were purchased from ISCO Inc. (RediSep Rf ISCO Inc.).
Soxhlet Extraction of Peels. Eight hundred grams of dried peel
powder was loaded into a Soxhlet-type apparatus and extracted with
hexane and chloroform sequentially for 16 h each. The extracts were
concentrated to obtain 31 and 23 g of hexane and chloroform extracts,
respectively.
Sample Preparation and Chromatographic Separation. The
hexane extract (31 g) was dissolved in 50 mL of hexane, impregnated
with 20 g of silica gel, and dried at room temperature in a hood. A
similar procedure was followed for the chloroform extract and used for
purification. The chromatography system Teledyne ISCO CombiFlash
Rf 4x system equipped with a fiber optic spectrometer, a UV−vis
(λ200 nm−λ700 nm) detector, and a 220 g silica gel flash column was used
for the separation. This detector can detect and monitor UV spectra of
the eluent after separation from the flash column using PeakTrak 2.0.0
software.
Hexane Extract. The silica gel impregnated hexane extract of
Miaray mandarin was subjected to flash chromatography on a silica gel
column. The column was equilibrated with hexane for 3 column
volumes prior to separations. Polymethoxyflavones were separated
with a 65 min gradient program of solvent A (hexane) and solvent B
(chloroform): 100% A held for 4 min, linearly increased to 10% B over
1 min, held for 12 min, linearly increased to 40% B over 6 min, held
for 14 min, linearly increased to 100% B over 6 min, held for 12 min,
and then finally returned to the initial conditions and held for 10 min.
The flow rate was maintained at 150 mL/min, and individual fractions
were collected by monitoring the eluting analytes at λ210 nm and λ340 nm.
The eluent from the column was also monitored using an inline UV−
vis detector for monitoring the absorption spectra of the separating
compounds. Individual peaks were tentatively identified on the basis of
the distinct absorption spectrum of each fraction. Seven major peaks
(A−G) were observed and collected in individual fractions (Figure
1A). The retention times of the separated peaks were as follows: peak
A, 22.5−25 min; peak B, 25−29.5 min; peak C, 29.5−34 min; peak D,
34−37.5 min; peak E, 39−42 min; peak F, 42−45 min; peak G, 45−50
min. The individual peak fractions were further analyzed by HPLC and
pooled on the basis of their similarity in retention time and matching
UV spectral data. Evaporation of the solvent from the pooled fractions
of peaks A−G yielded crystallization of compounds 1−5, 8, and 9,
respectively.
Chloroform Extract. The silica gel impregnated chloroform extract
was subjected to flash chromatography on a silica gel column (220 g).
The column was equilibrated with hexane for 3 column volumes prior
to separations. Polymethoxyflavones were separated with a 75 min
gradient program of solvent A and solvent B: 100% A held for 6 min,
linearly increased to 10% B over 4 min, held for 15 min, linearly
increased to 40% B over 5 min, held for 15.5 min, linearly increased to
60% B over 5 min, held for 4.5 min, linearly increased to 100% B over
6 min, held for 12 min, and then finally returned to the initial
conditions and held for 2 min. The flow rate was maintained at 150
mL/min, and individual fractions were collected by monitoring the
eluting analytes at λ210 nm and λ340 nm. The eluent from the column was
also monitored using an inline UV−vis detector for monitoring the
absorption spectra of the separating compounds. Individual peaks were
tentatively identified on the basis of the distinct absorption spectrum
of each fraction. Seven major peaks (H−N) were observed and
collected (Figure 1B). The retention times of the separated peaks were
as follows: peak H, 30−32.5 min; peak I, 32.5−35 min; peak J, 35−39
min; peak K, 39−44 min; peak L, 51−55 min; peak M, 55−59 min;
peak N, 62−70 min. Twenty microliters of each fraction was diluted
with 500 μL of acetone and analyzed by HPLC for detection of
polymethoxyflavones and further pooling of fractions with similar
C DOI: 10.1021/acs.jafc.5b02445
Article
compounds. Evaporation of the solvent from the pooled fractions of
peaks H−N yielded crystallized compounds 1, 3 4, 5, 6, 7, and 10.
Liquid Chromatography. The HPLC system consisted of a
Waters 1525 HPLC series (Milford, MA, USA) connected to a
photodiode array detector. A Gemini C18 column (3 μm, 250 × 4.6
mm) (Phenomenex, Torrance, CA, USA) was used for the separations.
A gradient mobile phase of 3 mM phosphoric acid (A) and acetonitrile
(B) was used for the separations at a flow rate of 1 mL/min. Initially,
elution was started with a gradient of 5% B followed by a linear
increase to 50% in 20 min and then returned to 5% in 5 min. The
injection volume was set at 10 μL, and the polymethoxyflavones were
detected at λ280 nm and λ340 nm. Chromatographic data were collected
and processed using Empower2 software (Waters).
Matrix-Assisted Laser Desorption/Ionization-Time of Flight-
Mass Spectrometry (MALDI-TOF-MS) Analysis. The samples for
MS analysis were prepared by dissolving the isolated compounds (1−
10) in acetonitrile and mixed with 2′,4′,6′-trihydroxyacetophenone
(THAP) matrix. Half a microliter of the matrix mixture was spotted on
a MALDI sample plate and air-dried. MALDI-TOF mass spectra were
acquired using a Voyager DE-Pro (Applied Biosystems, Carlsbad, CA,
USA) mass spectrometer in positive reflector ion mode. After time-
delayed extraction of 275 ns, the ions were accelerated to 20 kV for
TOF mass spectrometric analysis. A total of 100 laser shots were
acquired with the signal averaged per mass spectrum.
NMR Analysis. 1H and 13C NMR attached proton test (APT)
spectra were recorded at 400 and 100 MHz, respectively, by FT NMR
(JEOL USA, Inc., Peabody, MA, USA). The isolated compounds were
dissolved in chloroform (CDCl3) except compound 8, which was
dissolved in deuterated dimethyl sufoxide (DMSO-d6). Compounds 1,
5, 6, 8, and 10) were identified by using 1D and 2D NMR data
including heteronuclear single-quantum correlation spectroscopy
(HSQC); heteronuclear multiple-bond correlation spectroscopy
(HMBC), double quantum filter correlation spectroscopy (DQF-
COSY), and nuclear Overhauser effect spectroscopy (NOSEY).
Bacterial Strains and Media. V. harveyi strains BB170
(luxN::Tn5), BB886 (luxPQ::Tn5), BB120 (wild-type), JAF483
(luxO D47A), and BNL258 (hfq::Tn5lacZ) were kindly provided by
B. L. Bassler (Princeton University, Princeton, NJ, USA).31−34 E. coli 5,
an environmental isolate, was used as a positive control for
autoinducer-2 (AI-2) activity.35 Autoinducer bioassay (AB) or Luria
Marine (LM) media were used to culture the V. harveyi strains.36
Growth Assay. Overnight cultures of V. harveyi BB120 were
diluted 100-fold in AB media and treated with polymethoxyflavones
(50 μM) or an equivalent volume of DMSO. The cultures were grown
for 16 h, and OD570 was measured every 15 min by using a Synergy
HT multimode microplate reader (BioTek Instruments, Winooski,
VT, USA). The instrument was set to maintain a temperature of 30
°C, and plates were constantly shaken at medium speed between
readings. The data are presented as the mean of three biological
replicates.
Bioluminescence Assay. The bioluminescence was measured
using the method described previously from our laboratory.27 In brief,
E. coli 5 and V. harveyi BB120 were cultured overnight in Luria−
Bertani (LB) and LM media, respectively, to obtain high
concentrations of autoinducer activity. The overnight cultures were
centrifuged at 10000 rpm for 10 min in a microcentrifuge and filtered
using a 0.2 μm cellulose acetate membrane filter to obtain clear cell-
free supernatant (CFS). The CFSs were stored at −20 °C until use.
Inhibition of autoinducer [harveyi autoinducer (HAI) and AI-2]-
mediated bioluminescence was measured in a 96-well plate assay.27
The final concentrations of polymethoxyflavones tested were 12.5, 25,
and 50 μM. Diluted (2500-fold) overnight cultures (900 μL) of
reporter strains BB886 (for HAI) and BB170 (for AI-2) were
incubated with 5 μL of CFS, 0.5 μL of polymethoxyflavones or
DMSO, and 4.5 μL of sterile Autoinducer Bioassay (AB) medium at
30 °C with shaking at 100 rpm. Light production was measured by a
Victor2 1420 multilabel counter (Beckman Coulter) in luminescence
mode. The values were recorded as relative light units and used for
calculations. The relative activity was calculated as the ratio of
luminescence of the test sample to the control (DMSO) sample.36
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Journal of Agricultural and Food Chemistry Article

Biofilm Assay. The biofilm assay was conducted using the method The chromatograms of the HPLC analysis of the isolated
described previously from our laboratory.29 Briefly, an overnight compounds are presented in Figure 2. The absence of other
culture of V. harveyi BB120 was diluted 1:50 in LM medium, and 190
μL of this fresh culture was incubated with 7 μL of sterile medium and peaks demonstrates the purity of the isolated compounds. The
0.5 μL of DMSO or polymethoxyflavones (12.5, 25, 50 μM) dissolved purity of the isolated compounds ranged between ≥95 and
in DMSO. The biofilm mass was quantified by washing with ≤98%. The absorption spectra of the compounds were
phosphate buffer (0.1 M, pH 7.4), followed by staining with 0.3%
Crystal Violet (Fisher) for 20 min. The dye associated with biofilm obtained using a photodiode array detector. All of the isolated
was dissolved with 200 μL of 33% acetic acid, and A570 was measured.
The mean ± standard deviation of three biological replicates is
presented.

■ RESULTS AND DISCUSSION

Extraction. Successive extractions of mandarin peels using
nonpolar hexane and medium-polar chloroform in a Soxhlet
type apparatus yielded 3.9 and 2.9% of concentrated crude
extract, respectively. HPLC analysis of the extracts (Figure S2)
suggested that the extracts contain high levels of polymethoxy-
flavones. Additionally, the chromatograms suggested that the
successive extractions with hexane and chloroform enabled
fractionation of low-polar and medium-polar molecules,
respectively. The individual polymethoxyflavones were purified
by flash chromatography.
Purification of Polymethoxyflavones. Hexane Extract.
The step gradient elution using hexane and acetone enabled
clear separation of individual polymethoxyflavones (Figure 1a).
The eluent from the column was monitored using an inline
UV−vis detector to show the absorption spectra of the
compounds, which were subsequently collected in fractions.
The initial isocratic elution using 10% acetone separated the oil
from the crude extract. After separation of the oils, the linear
change in gradient elution to 60:40 hexane/acetone resulted in
good separation of the low-polarity polymethoxyflavones,
whereas medium-polar polymethoxyflavones were separated
by the gradual increase in the solvent polarity to 100% acetone.
On the basis of HPLC analysis, fractions showing similar peak
profiles were pooled and concentrated under vacuum. The
concentrated peak (A−G) fractions yielded crystallized
compounds, which were collected, their purity was analyzed
by HPLC, and they were identified by spectroscopic data.
Chloroform Extract. The impregnated chloroform extract
was subjected to flash chromatographic separation using a step
gradient of hexane and acetone solvents with a total run time of
75 min. To enable good separation of medium- and low-polar
polymethoxyflavones, an additional step gradient of 40:60
hexane/acetone was used, followed by a linear increase to 100%
acetone (Figure 1b). The step gradient elution resulted in
separation of peaks H−N. Similar to the chromatographic
separation of the hexane extract, the eluent from the column
was monitored using an inline UV−vis detector. Individual
peaks with distinct absorption spectra were collected in
individual fractions. The fractions collected for individual
peaks were analyzed by HPLC and concentrated to obtain
crystallized compounds. These were further subjected to
spectral analysis using MS and NMR to identify the
compounds.
Altogether, 10 purified compounds were isolated from
hexane and chloroform extracts. The yields of the isolated
compounds were as follows: 1, 18 mg; 2, 76 mg; 3, 8628 mg; 4,
2012 mg; 5, 210 mg; 6, 121 mg; 7, 1077 mg; 8, 79 mg; 9, 810
mg; and 10, 820 mg.
Identification. The identification and structural elucidation Figure 2. HPLC chromatograms of isolated compounds (1−10) along
of the isolated compounds from Miaray mandarin were with their UV spectra. The separation was conducted on a Gemini C18
conducted using spectral analysis by HPLC, MS, and NMR. column using gradient mobile phase.

D DOI: 10.1021/acs.jafc.5b02445
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Figure 3. 1 H NMR spectra of purified compounds (1, 5, 6, 8, and 10) recorded on a JEOL ECS NMR spectrometer at 400 MHz. All compounds
were dissolved in CDCl3 except 8, which was dissolved in DMSO-d6. The solvent peak is denoted by an asterisk (*).

compounds had distinct UV maxima in the range of
λ325 nm−λ353 nm, which is characteristic of polymethoxyflavones.
The results are consistent with earlier studies.16,37,38 The
MALDI-TOF mass spectral data for compounds 2, 3, 4, 7, and
9 were found to be 373.04, 433.16, 403.31, 470.21, and 343.26,
respectively (Figure S3). The NMR chemical shifts (not
shown) of these compounds were matched to reported
values.16,39 Using these data, the structures of compounds 2,
3, 4, 7, and 9 were confirmed and identified as 5,6,7,8,4′-
pentamethoxyflavone (tangeretin), 3,5,6,7,8,3′,4′-heptamethox-
yflavone, 5,6,7,8,3′,4′-hexamethoxyflavone (nobiletin), limonin,
and 5,7,8,4′-tetramethoxyflavone, respectively. The structure of
tangeretin was also confirmed by 2D NMR spectral assign-
E DOI: 10.1021/acs.jafc.5b02445
ments (Figure S4). These data were helpful when assigning the
chemical shifts of another three pentamethoxyflavones of
compounds 6 and 10.
Compounds 4 and 5 are hexamethoxyflavones, whereas
compound 2, 6, and 10 are pentamethoxyflavones with [M +
1]+ of 403 and 373, respectively. The structural confirmation is
not very accurate using only mass spectral data. Therefore, we
have used 1D and 2D NMR spectral data to validate the
findings (Figures 3 and 4 and Figures S4−S9). 1H NMR signals
(Figure 3) of compounds 1, 5, 6, 8, and 10 showed at δ 3.5−4.0
and 6.3−8.0, indicating that all compounds had structures
typical of polymethoxyflavones. Their characteristics and
assignments were made using 13C APT NMR (Figure 4) and
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Figure 4. 13C NMR attached proton test (APT) spectra of the isolated compounds (1, 5, 6, 8, and 10) recorded on a JEOL ECS spectrometer at 100
MHz. All compounds were dissolved in CDCl3 except 8, which was dissolved in DMSO-d6.

2D experiments including HMBC, HMQC, DQFCOSY, and
NOSEY.
Compound 1: The presence of a downfield proton resonance at
12.5 ppm on 1H NMR is a characteristic indicator of a chelated
hydroxyl group at the fifth position of the flavone skeleton. Due
to the presence of the hydroxyl group at C-5, the carbonyl
signal at the fourth position in 13C NMR showed at δ 178.5.
Moreover, the lack of a resonance at δ 6.6 for the olefinic
proton in 1H NMR indicated that there is a substituent at the 3-
position. The 1H NMR spectrum showed the ABX-type
aromatic proton signals indicating the existence of 1′,3′,4′-
trisubstituted flavones. 13C NMR showed the presence of three
methyl groups at around δ 56 and one distinct signal at δ 60.2
for the 7-methoxyl group. The MALDI-TOF-MS spectrum
showed the [M + 1]+ at m/z 359.3011. Using these spectral
data identified compound 1 as 5-hydroxyl-3,7,3′,4′-tetrame-
thoxyflavone. The chemical shifts show good agreement with
the earlier papers by Li et al.40
Compound 5: The 1H NMR spectrum showed the presence of
six methoxyl groups at δ 3.88−3.98, and those were tentatively
assigned to the 3-, 5-, 7-, 8-, 3′-, and 4′-positions. These
assignments were further confirmed from 13C NMR APT
chemical shifts with two signals observed at low field (δ 59.9
and 61.5) for two typical methoxyl groups at 3- and 8-
F DOI: 10.1021/acs.jafc.5b02445
substitution. A sharp singlet at δ 6.4 was assigned to the sixth
position, which was evidenced from the δ 92.32 signal. The B-
ring proton signals showed a pattern similar to ABX coupling
for three protons at H-2′, H-5′, and H-6′ (Figure S5).
However, the H-2′ displayed a broad singlet due to NOE effect
caused by methoxyl substitution at the C-3 position. Using
these spectral data and MALDI-TOF-MS (m/z at 403.3491),
compound 5 was identified as 3,5,7,8,3′,4′-hexamethoxyflavone
and signals were matched to reported values.45
Compound 6: The 1H NMR spectrum showed the presence of
five methoxyl groups, and those were shown from the 13CNMR
APT spectrum to be similar to compound 5, with substitutions
at 3-, 5-, 7-, 3′-, and 4′-positions. These data suggested that
compound 6 is a pentamethoxylated flavonoid. In 1H NMR two
aromatic broad singlets at δ 6.34 and 6.48 well correlated in
DQFCOSY to two different neighboring methoxy groups at δ
3.88 and 3.93, which indicates meta substitution in the A-ring
(Figure S6). On the other hand, in the proton NMR spectrum,
the δ 6.34 proton had a meta coupling to δ 6.48 (J = 1.8 Hz),
which was assigned to H-6 and H-8. Spectra in the B-ring had
ABX type aromatic proton signals, which confirms the presence
of three protons, and it was confirmed by integrated area of
three protons. The size of the coupling constant (J = 1.1 and
8.4 Hz) is characteristic of meta and ortho coupling as found in
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3′,4′-methoxylated flavonoids, whereas such correction were

not found in tangeretin due to the absence of ABX coupling
(Figure S7). Moreover, proton signals for H-2′ displayed
downfield at δ 7.9 as compared to PMF (10) due to the
presence of methoxyl substitution at the C-3-position, which
will cause NOE effect. A similar trend was observed for
hexamethoxyflavone (5). Using this NMR and mass spectral
data ([M + 1]+ − 373.3003), we confirmed compound 6 as
3,5,7,3′,4′-pentamethoxyflavone (pentamethylquercetin).
These chemical shifts were identical to those from the scientific
literature.42 This compound was previously identified in the
bark extract of Melicope subunifoliolata.43
Compound 8: The TOF-MS spectrum showed a molecular ion
peak at m/z 313.2550. The 1H NMR (DMSO-d6) spectral
analysis showed the presence of three methoxyl signals at δ
3.78, 3.8, and 3.86, which were confirmed in the 13C NMR APT
spectrum at δ 55.5, 55.9, and 56.5. Two broad singlets for one
proton in the aromatic region showed the presence of two
meta-coupled doublets at δ 6.38 (J = 2 Hz) and δ 6.55 (J = 2
Hz) in the A-ring for H-6 and H-8 protons. Another two ortho-
coupled doublets at δ 7.05 (J = 9 Hz, 2H) and δ 7.92 (J = 9 Hz,
2H) were assigned to the B-ring (H-3′, H-5′, H-2′, H-6′)
(Figure 3). This compound had one proton singlet at δ 6.61,
which was assigned to the olefinic proton at H-3.44 Thus,
compound 8 was determined to be 5,7,4′-trimethoxyflavone,
and the spectral data were consistent with data from the
literature.30,41
Compound 10 had a TOF-MS m/z 373.2725 [M + 1]+,
which indicates the compound is a pentamethoxyflavone. The
1
H NMR spectrum showed the presence of five methoxyls at δ
3.91−3.99 and two proton singlets at δ 6.4 and 6.6 (Figure 3).
In the APT spectrum (Figure 4), the A-ring carbon signals
coincided well with those of compound 9 (tetramethoxy- Figure 5. Structures of isolated compounds (1−10) from Miaray
flavone).17 The 1H NMR spectrum revealed the presence of an mandarins. Compounds 1, 5, 6, 8, and 10 were identified by NMR and
ABX system for the three aromatic protons in the B-ring at H- mass spectral analysis as 5-hydroxy-3,7,3′,4′-tetramethoxyflavone,
2′, H-5′, and H-6′ as demonstrated by coupling constant signals 3,5,7,8,3′,4′-hexamethoxyflavone, 3,5,7,3′,4′-pentamethoxyflavone
at δ 7.39 (d, 1H, J = 1.8 Hz), δ 6.95 (d, 1H, J = 8.2 Hz), and δ (pentamethylquercetin), 5,7,4′-trimethoxyflavone, and 5,7,8,3′,4′-pen-
7.6 (d, 1H, J = 8.2 Hz), respectively (Figure 3 and Figure S8). tamethoxyflavone, respectively. Compounds 2, 3, 4, 7, and 9 were
identified by mass spectral analysis as tangeretin, heptamethoxyflavone,
These results indicate the presence of methoxyls at the 3′- and nobiletin, limonin, and tetramethoxyflavone, respectively.
4′-positions in the B-ring. The singlets at δ 6.58 and 6.4 were
assigned to the H-3 and H-6 protons, respectively, using review describing the various compounds present in citrus
DQFCOSY data in Figure S8. 1HNMR spectra of four species by Manthey et al.47
pentamethoxyflavones for understanding the chemical shifts Antimicrobial Activity of Polymethoxyflavones. We
and structural assignments of all signals are presented in Figure next examined whether these compounds affect bacterial
S9. Although sinensetin is not isolated from Miaray mandarins, growth by treating V. harveyi with polymethoxyflavones. The
the spectrum was given for comparison purposes from our kinetic growth curve was calculated by recording the OD570 for
earlier study. Using these spectral data, compound 10 was 16 h of growth at optimal temperature. Figure 6 illustrates the
determined to be 5,7,8,3′,4′-pentamethoxyflavone, and the growth kinetics of V. harveyi BB120 after treatment with 50 μM
chemical shifts were compared to reported data.33,45 of the eight polymethoxyflavones. The sigmoid bacterial growth
In summary, results from the spectral analysis of the isolated curve observed indicates the polymethoxyflavones do not
compounds confirm the identity of the isolated compounds as inhibit the bacterial growth at 50 μM concentration. Similar
5-hydroxy-3,7,3′,4′-tetramethoxyflavone (1), 5,6,7,8,4′-pentam- results were also observed in other studies evaluating the
ethoxyflavone (tangeretin) (2), 3,5,6,7,8,3′,4′-heptamethoxy- antimicrobial activity of polymethoxyflavones (nobiletin and
flavone (3), 5,6,7,8,3′,4′-hexamethoxyflavone (nobiletin) (4), tangeretin), which had minimum inhibitory concentration
3,5,7,8,3′,4′-hexamethoxyflavone (5), 3,5,7,3′,4′-pentamethoxy- (MIC) of ≥1600 μg/mL,26 suggesting that these compounds
flavone (pentamethylquercetin) (6), limonin (7), 5,7,4′- are not bactericidal.
trimethoxyflavone (8), 5,7,8,4′-tetramethoxyflavone (9), and Inhibition of Biofilm Formation and Bioluminescence.
5,7,8,3′,4′-pentamethoxyflavone (10), respectively (Figure 5). Biofilms are surface adherent colonies of bacteria that form an
The results are consistent with the reported values.38,45,46 To extracellular polymeric matrix to protect themselves. Control of
the best of our knowledge, this is the first report of the isolation biofilm formation remains a concern among medical personnel,
of 3,5,7,3′,4′-pentamethylquercetin from the genus Citrus. It is primarily related to medical devices and implants. The bacterial
worth noting that this compound was previously reported in a biofilms in medical devices and implants are difficult to control
G DOI: 10.1021/acs.jafc.5b02445
J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry Article

Figure 6. Growth curve of V. harveyi BB120 in the presence of

polymethoxyflavones. The evaluated polymethoxyflavones were
tangeretin, 3,5,6,7,8,3′,4′-heptamethoxyflavone (Hepta-MF), nobiletin,
5,7,4′-trimethoxyflavone (Tri-MF), 3,5,7,8,3′,4′-hexamethoxyflavone
(Hexa-MF), 5,7,8,4′-tetramethoxyflavone (Tet-MF), 3,5,7,3′,4′-pen-
tamethoxyflavone (Penta-MF1), and 5,7,8,3′,4′-pentamethoxyflavone
(Penta-MF2).

due to increased antimicrobial resistance of biofilms.48 As

biofilm formation is regulated by several factors including
quorum sensing, interference with the quorum sensing pathway
is currently thought to be a viable strategy to control biofilms.
Quorum sensing is a population-dependent phenomenon
that is mediated through the concentration of autoinducers, Figure 7. HAI-1 and AI-2 induced bioluminescence inhibition in V.
signaling molecules synthesized by bacteria.49 Quorum sensing harveyi BB886 (A) and BB170 in the presence of polymethoxyflavones
regulated bioluminescence production in V. harveyi and its (B). The evaluated polymethoxyflavones were tangeretin (TANG),
signal transduction pathway has been well studied. The 3,5,6,7,8,3′,4′-heptamethoxyflavone (Hep-MF), nobiletin (NOB),
autoinducers are detected by three coincidence detectors, 5,7,4′-trimethoxyflavone (Tri-MF), 3,5,7,8,3′,4′-hexamethoxyflavone
LuxN, LuxPQ, and CqsS, that phosphorylate LuxU, a (Hex-MF), 5,7,8,4′-tetramethoxyflavone (Tet-MF), 3,5,7,3′,4′-pen-
phosphorelay protein. LuxU then phosphorylates the response tamethoxyflavone (Penta-MF-1), and 5,7,8,3′,4′-pentamethoxyflavone
(Penta-MF-2).
regulator LuxO that in turn regulates the expression of
downstream genes including bioluminescence genes.31−34
Furthermore, interference with V. harveyi bioluminescence
has been widely used as a model to study and identify quorum
sensing inhibitors.27−29 Panels A and B of Figure 7 illustrate the
results obtained from the analysis of bioluminescence in BB886
and BB170 strains of V. harveyi, respectively. Among the
evaluated polymethoxyflavones, only 3,5,6,7,8,3′,4′-heptame-
thoxyflavone and 3,5,7,8,3′,4′-hexamethoxyflavone seem to
interfere with AI-2-mediated bioluminescence in BB170. The
treatment of BB886 strain with polymethoxyflavones demon-
strated <40% inhibition over control (DMSO).
The inhibition of bioluminescence suggests that certain
polymethoxyflavones may interfere with biofilm formation. Figure 8. Inhibition of V. harveyi BB120 biofilm after treatment with
Consistent with the bioluminescence interference results, only polymethoxyflavones. The evaluated polymethoxyflavones were
3,5,6,7,8,3′,4′-heptamethoxyflavone and 3,5,7,8,3′,4′-hexame- tangeretin, 3,5,6,7,8,3′,4′-heptamethoxyflavone (Hepta-MF), nobiletin,
5,7,4′-trimethoxyflavone (Tri-MF), 3,5,7,8,3′,4′-hexamethoxyflavone
thoxyflavone inhibited biofilm formation in a dose-dependent
(Hexa-MF), 5,7,8,4′-tetramethoxyflavone (Tet-MF), 3,5,7,3′,4′-pen-
manner (Figure 8). Approximately 61% (with respect to tamethoxyflavone (Penta-MF1), and 5,7,8,3′,4′-pentamethoxyflavone
control−DMSO treatment) of biofilm formation was inhibited (Penta-MF2).
by 3,5,7,8,3′,4′-hexamethoxyflavone at a concentration of 50
μM. The IC50 value of 3,5,7,8,3′,4′-hexamethoxyflavone was
calculated to be 37.38 μM. Although 3,5,6,7,8,3′,4′-heptame- compounds might inhibit biofilm formation. Therefore,
thoxyflavone inhibited biofilm, the percent inhibition was constitutively luminescent V. harveyi mutants were investigated
approximately 35% at the maximum evaluated concentration of after treatment with 50 μM 3,5,7,8,3′,4′-hexamethoxyflavone
50 μM. Other polymethoxyflavones did not have >35% (Figure 9). The results demonstrated an increase in bio-
inhibition of biofilm with respect to control. luminescence in the luxO mutant strain (JAF483), but showed
Analyzing the bioluminescence in specific mutant strains no change in the hfq mutant (BNL258), suggesting an effect on
after treatment with polymethoxyflavones may enable us to LuxO. However, further studies are needed to validate the effect
understand the possible pathway through which these on LuxO. The results indicate that 3,5,7,8,3′,4′-hexamethoxy-
H DOI: 10.1021/acs.jafc.5b02445
J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry
Figure 9. Bioluminescence inhibition in V. harveyi mutants JAF483
(luxO) and BNL258 (hfq) by 3,5,7,8,3′,4′-hexamethoxyflavone (50
μm).
flavone is a potent inhibitor of V. harveyi quorum sensing and
appears to have larger effect on the AI-2 system. The effect on
luxO mutants seems to suggest that 3,5,7,8,3′,4′-hexamethoxy-
flavone may be a nonspecific inhibitor. Further studies are
required to evaluate the ability of polymethoxyflavones to
interfere with quorum sensing-mediated biofilm formation in
human pathogens.
In summary, 10 bioactive compounds were successfully
isolated from Miaray mandarin using flash chromatography,
and their structural identification was confirmed by spectral
analysis using MALDI-TOF and NMR. Among the isolated
compounds, 3,5,7,3′,4′-pentamethoxyflavone is reported here
for the first time from Citrus spp.. Among the evaluated
polymethoxyflavones, 3,5,7,8,3′,4′-hexamethoxyflavone demon-
strated inhibitory activity on autoinducer-mediated cell−cell
signaling and biofilm formation in V. harveyi. Further research is
required to understand the role of polymethoxyflavones in the
inhibition of biofilm in pathogenic bacteria. Results from this
study could enable the development of strategies using
polymethoxyflavones for preventing bacterial pathogenicity.
Moreover, identification of potent antimicrobials from citrus
species will provide added economic benefits to both producers
and the citrus-processing industry.
■
ASSOCIATED CONTENT
*
S Supporting Information
Additional figures as cited in the text. The Supporting
Information is available free of charge on the ACS Publications
website at DOI: 10.1021/acs.jafc.5b02445.
■
AUTHOR INFORMATION
Corresponding Authors
*(B.S.P.) Phone: (979) 862-4521. Fax: (979) 862-4522. E-
mail: b-patil@tamu.edu.
*(G.K.J.) E-mail: gkjp@tamu.edu.
Funding
This project is based upon work supported by the USDA-NIFA
2010-34402-20875, “Designing Foods for Health”, through the
Vegetable & Fruit Improvement Center.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
We acknowledge Chandra Mohan, Shiva Rani, and Kalpana for
assisting in harvesting and peeling the fruits.
■
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J DOI: 10.1021/acs.jafc.5b02445
J. Agric. Food Chem. XXXX, XXX, XXX−XXX