Analysis of Fluorescence Spectra of Citrus Polymethoxylated Flavones

Article pubs.acs.org/JAFC

Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Analysis of Fluorescence Spectra of Citrus Polymethoxylated Flavones and Their Incorporation into Mammalian Cells Danielle R. Gonçalves,† John A. Manthey,*,‡ Paulo I. da Costa,§ Marilia C. M. Rodrigues,† and Thais B. Cesar†

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Department of Food and Nutrition, Laboratory of Nutrition, Faculty of Pharmaceutical Sciences, São Paulo State University (UNESP), Araraquara 01049-010, Brazil ‡ U.S. Horticultural Research Laboratory, ARS, United States Department of Agriculture, 2001 South Rock Road, Fort Pierce, FL 34945, United States § Clinical Analysis Department, School of Pharmaceutical Sciences, São Paulo State University (UNESP), Araraquara 01049-010, Brazil ABSTRACT: Citrus polymethoxylated flavones (PMFs) influence biochemical cascades in human diseases, yet little is known about how these compounds interact with cells and how these associations influence the actions of these compounds. An innate attribute of PMFs is their ultraviolet-light-induced fluorescence, and the fluorescence spectra of 14 PMFs and 7 PMF metabolites were measured in methanol. These spectra were shown to be strongly influenced by the compounds’ hydroxy and methoxy substituents. For a subset of these compounds, the fluorescence spectra were measured when bound to human carcinoma Huh7.5 cells. Emission-wavelength maxima of PMF metabolites with free hydroxyl substituents exhibited 70−80 nm red shifts when bound to the Huh7.5 cells. Notable solvent effects of water were observed for nearly all these compounds, and these influences likely reflect the effects of localized microenvironments on the resonance structures of these compounds when bound to human cells. KEYWORDS: tangeretin, nobiletin, sinensetin, metabolites, citrus, Rutaceae, solvent effects, resonance, pyrylium, Huh7.5 cells

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tridesmethyl nobiletin. In our study, fluorescence spectra of the PMFs naturally occurring in orange juice as well as seven metabolites of these compounds were measured in methanol. The solvent effects of water on the fluorescence spectra of these PMFs and their metabolites were also investigated. Solvent effects of water are likely due to hydrogen bonding by water molecules with partial-charge-transfer centers within PMF resonance structures. An observation of the binding of PMFs to human Huh7.5 carcinoma cells was made by the detection of emissions from the 405 nm laser illumination of cells preincubated with a number of these compounds for 24 h. These spectra may possibly reflect features of the many chemical microenvironments involved in the binding of these compounds to human cells.

INTRODUCTION Polymethoxylated flavones (PMFs) are a group of flavonoids characteristically found in the genus Citrus (Rutaceae), particularly in sweet and sour oranges, grapefruits, and tangerines.1−3 They possess a typical flavonoid C6−C3−C6 carbon skeleton with methoxy groups (OCH3) distributed on the A- and B-rings and rarely at the 3-position of the middle pyrone ring (Figure 1). This polymethoxylation facilitates cellular absorption of these compounds4−6 and influences the levels of activities of these compounds against oxidative damage, cancer, inflammation, erythrocyte aggregation, and lipid biosynthesis.7−15 Recent studies have now shown that certain desmethylated and glucuronidated PMF metabolites, once thought to be less active than the original compounds, have in vitro actions that exceed those of the original compounds16−18 and thus are also likely to be active in vivo. The substitution patterns of the PMF A- and B-rings influence the molecular electronic structures of these compounds,19 and these influences are clearly observed in their UV−visible absorbance and fluorescence spectra. Likewise, large differences often occur in the in vitro biochemical effectiveness of individual PMFs and their metabolites. Such differences can arise from variations in membrane permeabilities as well as from differences in the binding of these compounds to catalytic enzymes, transport proteins, and nuclear receptors.20−23 A recent report described the detection within cells of a fluorescent conjugate of 5,3′,4′-tridesmethyl nobiletin and 2-aminoethyl diphenyl borate.24 Much weaker fluorescence was detected in cells incubated with only 5,3′,4′© XXXX American Chemical Society

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MATERIALS AND METHODS

Material. Distillation residues of cold-pressed orange oil with approximately 10% PMF content were obtained from a local Florida flavor company. The residues were mixed with methanol (1/1, v/v) and stirred overnight at room temperature, and the PMF-containing methanol solution was allowed to separate from the denser insolubleresidue mixture; it was then decanted and saved. The remaining residue was again mixed and stirred with another aliquot of methanol, and the first and second methanol solutions were combined and evaporated to an oil containing approximately 50% PMF. This Received: April 20, 2018 Revised: June 20, 2018 Accepted: June 21, 2018

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DOI: 10.1021/acs.jafc.8b02052 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

PMF administration, no rats were lost, but rather all showed good tolerance to the 200 mg kg−1 daily dose, maintained typical feed and water intake, and showed the weight gain normal for this species. Extraction of PMF Metabolites in Rat Urine. The pooled rat urine from each group was loaded onto a methanol- and waterprewashed SNAP RP-C18-HS (60 g) column (Biotage). The compounds retained on the column were eluted with methanol and dried under vacuum. This dried urine residue was dissolved in 200 mL of methanol and added to 800 mL of aqueous 0.5% formic acid. The metabolites were extracted three times with 500 mL of ethyl acetate. The combined ethyl acetate extracts were evaporated, and the recovered residue was kept at −20 °C. The urine of the control group contained no metabolites and was not processed further. Purification of PMF Metabolites. The initial step in the isolation of the PMF metabolites involved counter-current chromatography using fast-centrifugal-partition chromatography (FCPC). The FCPC conditions were optimized by choosing a biphasic-solvent system with a suitable partition coefficient.25 For this application, the biphasic-solvent system was composed of ethyl acetate/acetonitrile/ water with 0.5% formic acid (1/1/2, v/v/v), and the mobile phase consisted of the organic fraction (upper phase), whereas the stationary phase consisted of the aqueous fraction (lower phase). Prior to injection, the sample was diluted in the two-phase system (v/ v) and passed through a 0.45 μm filter. Chromatographic separations were achieved by using the Kromaton A-1000 rotor, operated at 1200 rpm, with the mobile phase delivered by a Waters Delta 600 HPLC pump at 20 mL min−1 with 20 mL collected fractions. Desmethyl aglycone metabolites were recovered in the early-eluting FCPC fractions, and the glucuronidated metabolites eluted later (data not shown). Detection of these compounds was achieved by HPLC-MS. Further purification of the metabolites was achieved by preparative thin-layer chromatography using silica-gel TLC plates (20 × 20 cm, Uniplate Analtech, Inc.) and toluene/acetone (3/1) as the developing solvent. Final purifications of the PMF glucuronide metabolites were accomplished with preparative HPLC using Varian ProStar 210/215 pumps (Varian) equipped with a Varian model 335 UV detector and the Star Chromatography Data System. Compound separations were achieved with either a Waters XBridge C8 (19 × 100 mm, 5 μm) or a Waters Atlantis dC18 (19 × 100 mm, 5 μm) reverse-phase semipreparative column, in which compound separations involved isocratic chromatographic runs with acetonitrile/0.5% formic acid in water with ratios of 20/80 to 30/70 (v/v), depending on the elution characteristics of each metabolite. All samples were diluted in DMSO prior to injection. HPLC-ESI-MS Analyses. Analyses of the PMFs and PMF metabolites were done with a Waters 2695 Alliance HPLC (Waters) connected in parallel with a Waters 996 PDA detector and a Waters/ Micromass ZQ single-quadrupole mass spectrometer equipped with an electrospray-ionization (ESI) source. Compound separations and MS detections were achieved as previously described.26 Fluorescence and UV Spectra. Fluorescence spectra were recorded with a PerkinElmer LS55 scanning fluorescence spectrometer. Spectra of the pure compounds were recorded in methanol at appropriate concentrations of either 0.01 or 0.001 mg mL−1. The exitand emission-slit widths were 5 nm with a scan speed of 200 nm/min. UV spectra were recorded with a UV-2401PC Shimadzu UV−vis recording spectrophotometer. Molecular-Structure Determination of PMF Metabolites. Structure investigations of the PMF metabolites involved UV−visible spectrophotometry,27 mass spectrometry,28,29 and nuclear magnetic resonance (NMR),30 as well as chemical modification using acid hydrolysis and alkaline degradation. PMF fragment ions formed during ESI-MS by retro-Diels−Alder fissions are reliable predictors of the substitution patterns of the flavone A- and B-rings.28,30−33 This information was used along with the results of the alkaline degradation to give preliminary structural analyses of these compounds. These details were further verified by 1H NMR and comparisons with known standards.

Figure 1. Sites of methoxylation and identities of the PMFs. material was dissolved in toluene and loaded onto 340 g HP-Sil silicagel columns (Biotage). Column chromatography was run using linear gradients of hexane and ethyl acetate to achieve partial separations of tangeretin (TAN), nobiletin (NOB), sinensetin (SIN), and 3′,4′,3,5,6,7,8-heptamethoxyflavone (HMF). The purifications of these compounds were completed by reverse-phase liquid chromatography using a 130 g RediSep C18 Reverse Phase column (Teledyne/ Isco) with linear gradients of methanol/aqueous 0.5% formic acid. Final purification was achieved by recrystallization in hexane, methanol, or mixtures of solvents at −20 °C and was followed by washes with 4 °C hexane. Production and Collection of PMF Metabolites from Rats. Forty male Wistar rats weighing 180 ± 5 g from the Animal Center of São Paulo State University (UNESP) were separated into 4 groups, with 10 animals in each group, to receive doses of (1) TAN, (2) NOB/(SIN) (70/30 w/w), (3) HMF, or (4) vehicle (the control group). They were placed in individual metabolic cages and maintained under controlled conditions: room temperature (23 ± 1 °C), 55 ± 5% relative humidity, and a 12−12 h day−night cycle. Initially, the rats were fed the normal lab chow diet (Purina Evialis do Brasil Nutriçaõ Animal Ltd.a.) and water ad libitum for 7 days to precondition the rats to their environment. Plain yogurt (Nestlé) was used as the vehicle for the PMF animal feeding via gavage. PMFs were separately incorporated into 1 mL of plain yogurt per day, using a homogenizer (Metabo, Marconi) operating at 27 000 rpm for 10 min at a controlled temperature of 25 °C. After the 1 week adaptation, the rats received 200 mg of a single PMF per kilogram of body weight by gavage once a day at 5:30 p.m. over 15 days. The rats were weighed every other day to calculate the correct daily doses. The control group received 1 mL of the vehicle per day. The animal care and study protocols were approved by the Ethics Committee for Animal Use at the School of Pharmaceutical Sciences, UNESP (No. 46/2015). Collection of urine was performed twice a day (8 a.m. and 5 p.m.). The entire daily volume of urine was homogenized and stored at −80 °C for 15 days. During the period of B


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Journal of Agricultural and Food Chemistry Alkaline Degradation. PMF metabolites were individually mixed (∼1.0 mg of dry weght) with 1 mL of 25% KOH in a 15 mL screwtop test tube and heated to 70 °C with a heating block for 6−24 h. Some compounds were initially dissolved in 200 μL of methanol. Upon completion of the heating, the KOH solution was combined with ∼1 mL of glacial acetic acid to adjust the pH to 3−6. The degradation products were analyzed by HPLC-ESI-MS. Alkalinedegradation products of SIN, TAN, NOB, and tetramethylscutellarein (TMS) were similarly analyzed for method validation. Acid Hydrolysis. PMF metabolites were mixed (∼1.0 mg) with 1 mL of 0.1 M HCl and heated to 100 °C for 2 h. Upon cooling, the hydrolysis solution was neutralized with 0.2 M KOH and then analyzed by HPLC-ESI-MS. 60 MHz NMR. PMF metabolites were also analyzed with a 60 MHz Nanalysis 60e permanent-magnet 1H NMR. Dried metabolite (1−5 mg) was dissolved in either 0.7 mL of deuterated dimethyl sulfoxide (DMSO-d6) or deuterated methanol. Spectra were recorded at 30 °C. Typically, spectra were collected as a sum of 100 to 4064 Fouriertransform scans depending on the amount of compound (5 to 1 mg) available for measurements. Some compounds were also analyzed by 400 MHz high-resolution NMR at 25 °C, and the compounds were dissolved in either DMSO-d6 or deuterated methanol. Cell Culture and Viability. The Huh7.5 human hepatoma cell line was kindly provided by Dr. Paula Rahal (Laboratory of Genomic Studies of the Biology Department of the Institute of Biosciences, Letters and Exact Sciences, IBILCE, UNESP, Sao Jose do Rio Preto, SP, Brazil). Huh7.5 cells are derived from the cell line Huh7, a lineage of hepatocytes from a carcinoma that were originally taken from a Japanese man, aged 57 years, in 1982. Cells were cultured in DMEM (Dulbecco’s modified Eagle’s medium, Sigma D1152) supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, and 1% nonessential amino acids (DMEM-C) in a 5% CO2-humidified incubator at 37 °C. The doses used to evaluate the incorporation of the PMF metabolites into mammalian cells were determined by cellviability assays using the MTT colorimetric assay.34 Confocal-Microscopy Analysis. The incorporation of pure PMFs and metabolites into the Huh7.5 cells was observed by confocal microscopy. The cells were plated at a density of 1 × 105 cells mL−1, prepared in DMEM-C, and distributed in 2 mL in a plate suitable for confocal microscopy (35 × 10 mm and adhered glass cover sheet, 22 × 22 mm). The plate was incubated at 37 °C in a humidified chamber with 5% CO2 for 12 h. Next, PMFs were added at 50 μM in DMEM-C, whereas the metabolites were added at 25 μM, and the cells were incubated over 24 h. These concentrations were selected from the cytotoxicity data to ensure high doses with cell viability greater than 80%. After this period, cells were washed twice with phosphate-buffer solution (PBS), and 1 mL of PBS was added to each plate to perform confocal-microscopy analysis. The images were obtained with a Confocal Zeiss 780 Microscope at the Physics Institute of São Carlos, University of São Paulo. The fluorescence spectra were obtained using a 405 nm laser, filters from 413 to 692 nm, a 98 μm pinhole, and a 40× objective. The images were obtained with a 405 nm laser, also with the 40× objective, and an emission-filter range between 415 and 550 nm. The negative control of the test consisted of cells treated with only DMEM-C medium.

Table 1. PMF Metabolites 1−7: ESI-MS of Protonated Molecular-Mass Ions, HPLC-Elution Times, and UVWavelength Maxima compound

[M + H]+1 ESI-MS+

RT (min)

UV spectrum (nm)

1

535/359

18.74

230, 271, 320

2

521/345

16.70

243, 264, 329

3 4 5 6 7

359 359 565/389 389 581/405

25.22 23.40 19.40 25.21 23.38

268, 325 242, 265, 333 249, 267, 336 249, 271, 332 254, 270, 346

proposed compound TAN-4′-Oglucuronide didesmethyl SINglucuronide 4′-desmethyl TAN monodesmethyl SIN NOB-glucuronide 4′-desmethyl NOB 3′,4′-didesmethyl HMF

Table 2. Fluorescence Parameters of PMF Standards in Orange-Peel-Oil-Distillation Residue compound

λEm max (nm)

FIUa

DMSO 5DMN 5DMHMF 5DMS 5DMTMS 5DMT ISOSIN SIN QHME TMS GTME ISOTMS TAN NOB HMF

none none none none none none 435 443 452 500 520/450(sh) 438 535 450 452

none none none none none none 45 190 343 96 25 62 trace 42 43

structure 5-desmethyl nobiletin 5-desmethyl heptamethoxyflavone 5-desmethyl sinensetin 5-desmethyl tetramethylscutellarein 5-desmethyl tangeretin 3′,4′,5,7,8-pentamethoxyflavone 3′,4′,5,6,7- pentamethoxyflavone 3′,4′,3,5,6,7-hexamethoxyflavone 4′,5,6,7-tetramethoxyflavone 3′,4′,3,5,7,8-hexamethoxyflavone 4′,5,7,8-tetramethoxyflavone 4′,5,6,7,8-pentamethoxyflavone 3′,4′,5,6,7,8-hexamethoxyflavone 3′,4′,3,5,6,7,8heptamethoxylflavone

a

Measurements recorded in methanol with excitation wavelengths (λ) at 285 and 330 nm. Compound concentrations were 0.01 mg mL−1 except ISOSIN, SIN, and QHME, which were 0.001 mg mL−1. For micromolar concentrations see Table 3. Fluorescence-intensity units (FIU) are measures against the maximum fluorescence intensity defined as 1000 units by the PerkinElmer LS-55 fluorescence spectrophotometer.

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RESULTS PMF Metabolites from Rat Urine. Seven metabolites, 1− 7, were isolated from PMF-dosed animals, and their main ESIMS fragment ions, HPLC elution times, and UV-absorbancewavelength maxima are listed in Table 1. The results of initial structural determinations of 1−7 are discussed below. MS fragmentation provided partial information about the substituents attached to the A- and B-rings,28,30,32 and alkaline hydrolysis provided further A- and B-ring-structure validation.35,36 There were not sufficient amounts of purified 2, 4, and 5 to achieve complete structure determinations.

Figure 2. Fluorescence spectra of QHME and SIN at 0.001 mg mL−1 and of NOB and HMF at 0.01 mg mL−1 in methanol. The Y-axis is presented in fluorescence-intensity units (FIU).

Metabolite 1. Tangeretin-4′-O-glucuronide. UV λmax 271, 320 nm. ESI+ 535 m/z [M + H]+1, 345, 211, 183, 121, 119. C


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and 153 m/z are evidence of a trimethoxy A-ring, and fragment ions 135 and 137 m/z are indicative of a dihydroxy B-ring. Acid hydrolysis yielded a flavone with ESI+ 345 m/z [M + H]+1 and fragment ions 195, 181, 153, 135, 137, and 125, which are identical to 2. It is suggested that hydrolyzed 2 is 3′,4′-didesmethyl-sinensetin. Sinensetin was the only PMF administered to the rats with a trimethoxy A-ring. Metabolite 3. 4′-Hydroxy-5,6,7,8-tetramethoxyflavone (4′desmethyl tangeretin). UV λmax 268, 325 nm. ESI+ 389 m/z [M + H]+1, 211, 183, 121, 119. Fragment ions 211 and 183 m/ z are evidence of a tetramethoxy A-ring, and fragment ions 119 and 121 m/z are evidence of a monohydroxy B-ring. Highresolution 1H NMR of 3 is identical to that for 4′-desmethyl tangeretin.37 Metabolite 4. 3′-Desmethyl sinensetin or 4′-desmethyl sinensetin. UV λmax 242, 265, 333 nm. ESI+ 359 m/z [M + H]+1, 181, 153, 151, 149. Fragment ions 181 and 153 m/z are evidence of a trimethoxy A-ring, and fragment ions 149 and 151 m/z are indicative of a monohydroxy-monomethoxy Bring. 1H NMR (60 MHz, CD3OD): H2′, H6′ (d, 2H, 7.48, 7.57), H8 (s, 7.17), H5′ (d, Jortho = 8.8 Hz, 1H, 6.80, 6.95), H3 (s, 1H, 6.68); four OCH3 at 3.92, 3.86, 3.77, 3.74 ppm. This metabolite was not isolated in sufficient amounts to allow complete structural elucidation relating to the position of the unsubstituted hydroxyl on the B-ring nor for accurate calculations of the coupling constants for H2′ and H6′ using 60 MHz NMR. Metabolite 5. Nobiletin-O-glucuronide. UV λmax 249, 267, 336 nm. ESI+ 565 m/z [M + H]+1, 389, 359, 197, 169, 165, 163. Fragment ions 197 and 169 m/z are evidence of a monohydroxy-trimethoxy A-ring, and fragment ions 165 and 163 m/z are indicative of a dimethoxy B-ring. Acid hydrolysis yielded a flavone aglycone with UV λmax 250, 279, and 336 nm and MS-fragmentation ions identical to those of 5, with the exception of the glucuronide-conjugate ion at 565 m/z. Alkaline hydrolysis of 5 produced further evidence of a monohydroxy-trimethoxy A-ring. Hydrolyzed 5 exhibited 1H NMR (60 MHz, CD3OD) as the following: H2′, H6′ (d, Jortho = 9.3 Hz; 2H, 7.52, 7.68), H5′ (d, Jortho = 8.3 Hz; 1H, 7.05, 7.19), H3 (s, 1H, 6.78). 1H NMR (60 MHz, CD3OD) of 5 exhibited nearly identical aromatic proton signals as those detected for hydrolyzed 5 as well as five OCH3 at 4.09, 4.01, 3.90 (×2), and 3.87 ppm. High-resolution 1H NMR (400 MHz, DMSO-d6) of hydrolyzed 5 exhibited the following: H2′, H6′ (d, 2H, 7.64, 7.65; 7.63, 7.62; 7.54, 7.53), H5′ (d, 1H, 7.17, 7.15), H3 (s, 1H, 6.78); five OCH3 at 3.96 (×2), 3.88, 3.85, 3.73. 13C NMR provides additional evidence of the occurrence of adjacent 3′- and 4′-methoxy substituents and no C7-methoxy signal (data not shown). Sufficient amounts were not available to achieve a complete structure identification of 5, but the results above support a flavone with a 3′,4′-dimethoxy B-ring and a monohydroxy-trimethoxy A-ring. Positive identifications of 7-desmethyl nobiletin and 6-desmethyl nobiletin have been reported in nobiletin metabolites produced by human liver microsomes,38 and we propose that 5 occurs as one of these metabolites. 7-Desmethyl nobiletin in citrus peels has also been previously reported.31,39 Metabolite 6. 3′-Methoxy-4′-hydroxy-5,6,7,8-tetramethoxyflavone (4′-desmethyl nobiletin). UV λmax 249, 271, 332 nm. ESI+ 389 m/z [M + H]+1, 211, 183, 149, 151. Fragment ions 211 and 183 m/z are evidence of a tetramethoxy A-ring, and fragment ions 149 and 151 m/z are indicative of a monohydroxy-monomethoxy B-ring. 1H NMR (60 MHz,

Figure 3. Fluorescence spectra of SIN at 0.001 mg mL−1 versus TMS and NOB at 0.01 mg mL−1 versus TAN at 0.01 mg mL−1 in methanol. The Y-axis is presented in fluorescence-intensity units (FIU).

Figure 4. (A) Fluorescence spectra of NOB(m) and HMF(m) at 0.01 mg mL−1 and SIN(m) at 0.001 mg mL−1 in methanol and of NOB(aq), HMF(aq), and SIN(aq) in 80/20 (v/v) water/methanol. The Y-axis is presented in fluorescence-intensity units (FIU). (B) Fluorescence spectra of TAN in methanol at 0.01 mg mL−1 with decreased percentages of methanol following dilution with water. The Y-axis is presented in fluorescence-intensity units (FIU).

Fragment ions 211 and 183 m/z are evidence of a tetramethoxy A-ring, and fragment ions 119 and 121 m/z are indicative of a monohydroxy B-ring. High-resolution 1H NMR of the aromatic protons of 1 is identical to that of 4′-hydroxy tangeretin.37 Acid hydrolysis of 1 yielded 3 (4′-desmethyl tangeretin). Metabolite 2. 3′,4′-Dihydroxy-5,6,7-trimethoxyflavone-4′glucuronide or 3′,4′-dihydroxy-5,6,7-trimethoxyflavone-3′-glucuronide. UV λmax 243, 264, 329 nm. ESI+ 521 m/z [M + H]+1, 345, 195, 181, 153, 135, 137, 125. Fragment ions 181 D


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Journal of Agricultural and Food Chemistry Table 3. Fluorescence Parameters of PMFs and PMF Metabolites Measured in Methanol and in Huh7.5 Cells compounda (μM)

λEm max (nm, cell bound)

λEm max (nm, methanol)

FUIb (cell bound)

FUIb (methanol)

solvent effectsc (ΔFIU/ΔλEm max)

1 (19) 2 (19) 3 (26) 4 (2.8) 5 (17) 6 (2.5) 7 (2.5) NOB (25) HMF (23) QHME (2.5) SIN (2.7) ISOSIN (2.7) TANd (27) TMSd (29) ISOTMS (29)

487 470 487 513 470 522 522 435 435    435  

425 508 412 450 446 456 494 450 452 450 443 430 535 496 434

14 27 12 22 9 48 48 23 74    25  

14 451 31 60 120 153 608 65 72 351 255 45 10 201 181

trace not measured +24/+28 −1036/+10 nm +640/+29 nm −125/+14 −539/−29 nm +475/+20 nm +211/+26 nm −69/+15 nm +573/+13 nm −2/+17 nm −7535 nm/+54419 nm −146496 nm/+170415 nm −39/+15 nm

a Structures include 1, tangeretin-4′-glucA; 2, dihydroxysinensetin-glucA; 3, tangeretin-4′-OH; 4, 3′- or 4′-hydroxysinensetin; 5, nobiletin-glucA; 6, 3′- or 4′-hydroxynobiletin; 7, 3′,4′-dihydroxyheptamethoxyflavone. For TAN, NOB, HMF, and SIN, see Table 2. bMeasurements recorded in methanol with excitation wavelengths (λ) at 330 nm and measurements recorded in aqueous solutions (cell bound) with excitation wavelengths (λ) at 405 nm using confocal microscopy. Concentrations are reported as micromolar (μM). Concentrations between 2.8 to 2.5 μM represent solutions of 0.001 mg mL−1, and higher values between 17 to 29 μM represent solutions of 0.01 mg mL−1. cWater-solvent effects are shown as ΔFIU/ΔλEm max. The value ΔFIU represents the gains or losses in fluorescence-intensity units (FIU) from the peak in methanol to the peak in water/methanol (80/20, v/v). The change in the wavelength of the emission maximum recorded in methanol versus that recorded in water/methanol (80/20, v/v) is represented as ΔλEm max. dTAN and TMS exhibited unusual shifts in their emission-wavelength maxima. The loss of FIU at the original λEm max is listed first, followed by the gain in FIU in the aqueous λEm max.

Fluorescence Spectra of PMFs and PMF Metabolites in Methanol. The fluorescence spectra of 14 PMFs were measured at room temperature in methanol using excitation wavelengths at 320 and 280 nm. These excitation wavelengths occur within typical PMF absorbance bands I and II, respectively.19 As shown in Table 2, no fluorescence occurred for any of the 5-desmethyl PMFs, including 5DMN (5desmethyl nobiletin), 5DMHMF (5-desmethyl heptamethoxflavone), 5DMTMS (5-desmethyl tetramethylscutellarein), 5DMT (5-desmethyl tangeretin), and 5-DMS (5-desmethyl sinensetin). The absence of fluorescence in 5-hydroxyflavones has been attributed to the formation of strong intramolecular hydrogen bonds between the 5-hydroxyl of the A-ring and the pyrone-ring carbonyl and the losses of planarity of the flavone molecules.40 Both attributes disrupt the fluorescence emissions arising from extended electron resonances in flavones. Among the PMFs exhibiting UV-induced fluorescence, most had similarly shaped emission spectra with wavelength maxima between 440 and 460 nm (Figure 2). No qualitative differences occurred in the spectra when either excitation wavelengths (320 and 280 nm) was used (data not shown). In contrast to the spectra in Figure 2, tangeretin (TAN) and tetramethylscutellarien (TMS) showed emission-wavelength maxima at 535 and 498 nm, respectively, with TAN exhibiting only trace fluorescence intensity compared with the other compounds (Figure 3). The influence of having adjacent methoxy substituents at the 3′- and 4′-positions of the B-ring is illustrated by the contrasting fluorescence spectra of TAN versus nobiletin (NOB) and TMS versus sinensetin (SIN), respectively, in Figure 3. The fluorescence spectra of many of the PMFs were notably affected by the addition of water to the PMF/methanol solutions. Although not all compounds in Table 1 were evaluated for this effect, the fluorescence spectra of NOB, SIN, quercetagetin hexamethyl ether (QHME), TMS, heptamethoxyflavone (HMF), and TAN all showed solvent effects with the

Figure 5. Fluorescence spectra of TMS(aq) in water/methanol (80/ 20, v/v) versus TMS(m) in methanol at 0.01 mg mL−1 and similarly for isoTMS(aq) and isoTMS(m). The Y-axis is presented in fluorescence-intensity units (FIU).

CD3OD): H2′, H6′ (d, 2H, 7.45, 7.17), H5′ (d, Jortho = 6.5 Hz, 1H, 7.01, 6.90), H3 (s, 1H, 6.70); four OCH3 at 3.98, 3.94, 3.81, 3.75. Alkaline hydrolysis of 6 produced vanillic acid, thus providing evidence of a 4′-hydroxy-3′-methoxy B-ring, consistent with 4′-desmethyl nobiletin. Metabolite 7. 3′,4′-Dihydroxy-3,5,6,7,8-pentamethoxyflavone (3′,4′-didesmethyl HMF). UV λmax 254, 270, 346 nm. ESI+ 405 m/z [M + H]+1, 211, 183, 137, 135. Fragment ions 211 and 183 m/z are evidence of a tetramethoxylated A-ring, and fragment ions 135 and 137 m/z are evidence of a dihydroxy B-ring. 1H NMR (60 MHz, CD3OD): 5 methoxy groups at 4.07, 3.99, 3.90, and 3.78 ppm; H2′, H6′ (d, Jortho = 5.9, 2H, 7.69/7.55, 7.52), H5′ (d, 1H, Jortho = 7.6 Hz, 6.97, 6.84). The signal for the C-3 proton was absent, as expected for an HMF metabolite. E


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and 3, originally from TAN, showed only trace fluorescence, whereas 2 and 4−7, derived from SIN (2 and 4), NOB (5 and 6), and HMF (7), exhibited moderate to strong fluorescence, with emission-wavelength maxima between 425 to 508 nm. These compounds showed a variety of different solvent effects with water. Because of the weak fluorescence-emission intensities of 1 and 3, these solvent effects were difficult to define, although there appeared to be decreased fluorescence intensity throughout the spectrum of 1 with increased water content, and for 3, the fluorescence intensities between 500 to 520 nm weakly increased, and there was no change at 420 nm (data not shown). Metabolites 2, 4, 6, and 7, with free hydroxyl substituents, showed dramatic losses in fluorescence intensities when measured in aqueous methanol (Table 3), whereas the fluorescence spectrum of 5 (lacking free hydroxyl substituents), showed a 7.5× increased intensity in 80% water compared with in 100% methanol. PMF and PMF Metabolite Binding to Intact Huh7.5 Cells. UV-induced fluorescence was detected by confocal microscopy in human carcinoma Huh7.5 cells preincubated 24 h with PMFs and PMF metabolites 1−7 using 405 nm laserinduced excitation. Untreated control cells exhibited trace background fluorescence, which contrasted with the intense fluorescence exhibited by the PMF- and metabolite-treated cells. Figure 6 shows the fluorescence images of illuminated Huh7.5 cells incubated separately with metabolites 1−7. Similar images were observed with the cells preincubated with the original PMFs: TAN, NOB, and HMF (data not shown). Despite the negligible levels of fluorescence of 1, 3, and TAN in methanol, intense fluorescence was observed for the Huh7.5 cells incubated with these compounds. Incubations of the Huh7.5 cells were performed separately with 50 μM PMFs and with 25 μM 1−7. These concentrations were determined to ensure cell viability of >80%, confirmed by MTT cell-viability measurements.34 MTT cytotoxicity assays showed IC50 values for 1−7 between 139 and 219 μM, with an average of 160 ± 30 μM (data not shown). Measurements of the emission spectra of the PMF-incubated Huh7.5 cells were made to more fully characterize the fluorescence of these compound-treated cells (Figure 7). Not all of the PMFs were analyzed in this manner. The fluorescence-emission spectra of the cells incubated with NOB and HMF showed wavelength maxima at 461 and 460 nm, respectively, and a second maximum between 435 and 440 nm. The longer-wavelength maxima at 454 and 461 nm for NOB and HMF are close to the wavelength maxima measured for these compounds in methanol. TAN, which exhibited an emission-wavelength maximum in methanol at 535 nm but at 434 nm in 80% water/methanol, exhibited an emissionwavelength maximum of 454 nm when bound to the cells. The spectrum obtained with cell-bound 2 shared some similarity with the spectra of cell-bound TAN, NOB, and HMF. The Huh7.5 cells treated with 4, 6, and 7 showed fluorescenceemission spectra with wavelength maxima near 525 nm, contrasting sharply with the spectra of Huh7.5-cell-bound NOB, HMF, and TAN. The weak emission spectra obtained from the Huh7.5 cells incubated with 1, 3, and 5 lacked sufficient intensities to make clear spectroscopic determinations, but in the cases of 1 and 5, the differences in the spectra versus the control spectrum revealed weak emission maxima between 440 and 450 nm and again at approximately 480 nm (data not shown).

Figure 6. Confocal-microscope image of Huh7.5 after 24 h of incubation with PMFs, metabolites, and a control. The scale is 10 μm.

addition of water. The intensities of the HMF, SIN, and NOB fluorescence emissions in water/methanol (80/20, v/v) solutions were sharply higher than the fluorescence intensities of these same compounds in methanol (Figure 4A). In water/ methanol (80/20, v/v) solutions, the emission-wavelength maxima of HMF, SIN, and NOB shifted to 480, 457, and 470 nm, respectively, compared with 452, 443, and 450 nm in methanol (Table 3), each representing a 15 to 30 nm red-shift to lower-energy transitions. Notably different solvent effects were observed with the addition of water to methanol solutions of TAN (Figure 4B). The new fluorescence-emission-wavelength maximum of TAN in water/methanol (80/20, v/v) occurred at 420 nm, compared with the previous value of 535 nm in methanol. Similar changes occurred in the spectrum of 4′,5,6,7-tetramethoxyflavone (TMS, Figure 5), yet the isomer iso-tetramethylscutellarein (ISOTMS, 4′,5,7,8-tetramethoxyflavone) did not show these same solvent effects. The emissionwavelength maximum of ISOTMS in methanol occurred at 438 nm, red-shifting to 450 nm in water/methanol (80/20, v/ v) and decreasing by 18% in emission intensity (Figure 5). The parameters of the fluorescence spectra of metabolites 1−7 measured in methanol are listed in Table 3. Metabolites 1 F


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Figure 7. Fluorescence spectra of PMFs and metabolites 1−7 in Huh7.5 cell culture. Metabolite 1, TAN-4′-glucA; metabolite 2, di-OH-SIN-glucA; metabolite 3, TAN-4′-OH; metabolite 4, 3′- or 4′-hydroxy-SIN; metabolite 5, NOB-glucA; metabolite 6, 4′-desmethyl NOB; metabolite 7, 3′,4′didesmethyl HMF. The Y-axis is presented in fluorescence-intensity units (FIU).

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DISCUSSION The PMFs obtained from citrus peels constitute an extremely small subset of the flavonoids in human diets, yet because of their potential health benefits to humans, these compounds have been extensively studied.16,41,42 Research has shown that these compounds, along with many other highly substituted flavones, exert beneficial effects by interacting with enzymes involved in the progression of human diseases. Such targeted enzymes include, in part, phosphodiesterases,43−45 xanthine oxidase, 46−48 adenosine receptors, 21,49,50 cell-signaling kinases,51−53 DNA topoisomerases,54−56 lipid-synthesis and glucose-metabolism enzymes,11,57 and transport proteins.23,58 To investigate interactions such as these in human cells, fluorescence spectroscopy may be useful because of the nonintrusive nature of fluorescence measurements and the sensitivity of these measurements to chemical microenvironments.59 The fluorescence spectra of flavonoids reflect to a large degree the extensive delocalization of electrons in the molecules’ ground and excited states, both of which are controlled by their chemical structures.60−62 A commonly observed structural influence is the long-wavelength greencolored fluorescence of flavonols (3-hydroxyflavones) produced by excited state keto−enol tautamers,62−64 and although flavonols often exhibit this long-wavelength fluorescence, most other flavones exhibit shorter-wavelength blue fluorescence. An important aspect for these electron delocalizations is the concurrent electron donation by the oxygen at the pyrone 1position (Figure 1) and the electron acceptance by the C4carbonyl oxygen to create charge-transfer zwitterionic resonance structures. These π-electron delocalizations are often

stabilized by hydrogen bonding with solvent molecules as well as by electron donation by the phenyl-ring substituents. As a result of this, most flavones exist as mixtures of pyrone and pyrylium resonance forms,65 both of which are differently influenced by chemical environments. In this study, the fluorescence spectra of 14 PMFs dissolved in methanol and 7 additional PMF metabolites, as well as the solvent effects of water on these spectra, were measured. An increase in solvent polarity generally produces red-shifts in the fluorescence-emission-wavelength maxima and lowers the emission intensities for many fluorescent compounds.60 However, our study showed that for most of the original unmodified PMF compounds (e.g., QHME, SIN, NOB, and HMF), the fluorescence-emission intensities sharply increased with the addition of water to the methanol solutions, but then the wavelength maxima of these original unmodified PMFs red-shifted by 15−30 nm, as seen in many other fluorescent compounds. These solvent effects with water appear to be selfcontradictory and seem not to fit the pattern normally observed with other fluorescent compounds. A structural feature common to the above group of compounds (QHME, SIN, NOB, and HMF) is the adjacent methoxyl substituents on the B-ring 3′- and 4′-positions. However, in contrast to these compounds, two other PMFs, tetramethylscutellarein (TMS, 4′-hydroxy-5,6,7-trimethoxyflavone) and tangeretin (TAN, 4′-hydroxy-5,6,7,8-trimethoxyflavone), lack the 3′-methoxyl substituent adjacent to the 4′position, and these compounds exhibited notably different water-solvent effects. The addition of water to methanol solutions of TMS and TAN created new intense fluorescence emissions near 425 nm excitation, and this occurred regardless G


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suggest that compounds with methoxylation at the C8 position (GHME, ISOTMS, TAN, NOB, and HMF) exhibit lower fluorescence intensities than those without a substituent (SIN, QHME, and TMS) at the C8 position. The research findings obtained with metabolites 1−7 further support this tendency, as well as the study of Wolfbeis,61 in which 5-methoxyflavone, 6-methoxyflavone, and 7-methoxyflavone were shown to exhibit substantially greater fluorescence intensities than 8methoxyflavone. The isolation and subsequent investigation of the fluorescence spectra of PMF metabolites 1−7 expanded the range of structures analyzed in this study and, in several cases, provided valuable comparisons between closely related compounds. The low fluorescence intensity of 1 is likely a result of the closely matching A- and B-ring substitutions shared with TAN, although the glucuronide substituent of 1 appears to cause differences in the solvent effects of water (nearly completely absent in 1, see Table 3, but prominent for TAN, see Figure 4), as well as differences in the fluorescencewavelength maxima of the two compounds (535 nm for TAN and 425 nm for 1) in methanol (Table 3). In contrast, the structurally similar NOB and 5 show strong similarities in their fluorescence spectra both in methanol and in 80% water/ methanol, but the fluorescence spectra of these compounds contrasted sharply when bound to the Huh7.5 cells. Compounds 2−4, 6, and 7, which possess free hydroxyl groups, showed dramatic drops in fluorescence emission in the presence of water, and as discussed above, these decreases are in sharp contrast to the changes of the original unmodified PMF compounds, which showed large emission increases with water. The solvent effect of water on the desmethylated metabolites (compounds 2−4, 6, and 7) is similar to that of quercetin (3′,4′,3,5,7-pentahydroxyflavone) and apigenin (4′,5,7-trihydroxyflavone) with which sharp decreases in fluorescence intensities occur in aqueous methanol and acetonitrile solutions.69 These sharp decreases in fluorescence emission can be readily attributed to intermolecular hydrogen bonding, which contorts the planarity of either the ground or excited state’s geometry or lowers the extent of π-electron delocalization in the excited electronic state. The images of the Huh7.5 cells preincubated with the PMFs and the PMF metabolites (Figure 6) show bright fluorescence associated with internal structures of the cells, presumably cellular organelles. Nearly circular structures putatively identified as oil droplets appear not to show any PMF sequestration, and as a consequence, it is proposed that most of the cellular associations of these compounds involve protein binding. Research into the fluorescence emission of flavones has led to earlier detections of these compounds in cultured cells,70−74 including direct detection of kaempferol and other flavonols in nuclei of Hepa-1c1c7 cells.73 An alternative method of detecting flavones involved the detection of the fluorescence of spontaneously formed flavonoid-2-aminoethoxydiphenyl borate (DPBA) conjugates in animal74 and plant cells.75−77 The nobiletin metabolite 5,3′,4′-tridesmethylnobiletin (TDN) was recently detected as the DPBA-TDN conjugate in mouse cells.74 Fluorescence emissions of many dietary plant flavonols are also strongly intensified by their binding to target proteins, typically producing intense greencolored fluorescence.25 This accentuated fluorescence can also be used with confocal microscopy to detect flavonol incorporation into cells, as previously shown for the Hepa1c1c7 cells.73

Figure 8. Resonance spectrum of PMFs with oxygen-containing substituents on the A-ring C6 positions. (A) Example of demethylation of the C6 methoxy of zapotin as discussed by Dryer.66 (B) Proposed resonance structure of ISOTMS with a free C6 phenyl A-ring position. The free C6 position allows additional resonance structures to further stabilize the ground and excited states of compounds like ISOTMS, thus differentiating ISTMS from TMS and TAN with methoxy substituents at the C6 position.

of the A-ring substituents at 5,6,7,8 in TAN or 5,6,7 in TMS. However, an isomer of TMS, isotetramethylscutellarein (ISOTMS, 4′-hydroxy-5,7,8-trimethoxyflavone) did not show this same solvent effect, although it has the same 4′-methoxy B-ring substitution. An analysis of these structures showed that the main difference between the combined structural features of TAN and TMS versus ISOTMS is the absence of an oxygencontaining substituent at the A-ring C6 position in ISOTMS. The manner by which the A-ring C6 position influences the stabilities and electronic resonance structures of PMF compounds is not certain, but it is known from other research that mass fragmentation involving demethylation of A-ring methoxyl substituents proceeds most readily at the C6 position relative to any of the other A-ring positions. As shown by Dreyer,66 a major factor contributing to this preference for demethylation at the C6 position is the formation of a highly stabilized pyrylium inner-C-ring resonance structure, such as that illustrated for the compound zapotin in Figure 8A. Without demethylation, the electron-donating properties of methoxyl substituents at the C6 position in TAN and TMS work against the formation of this type of pyrylium resonance structure. However, this is not the case for ISOTMS with no substituent at the C6 position (Figure 8B). Previous studies have shown examples of pyrulium-like resonance stabilization and enhanced fluorescence intensities accompanying the binding of flavonols (3-hydroxyflavones) to human and rat serum albumins.67,68 Further analysis of the fluorescence spectra of PMFs measured in this study suggests that intensities of fluorescence emissions are strongly influenced by the presence of a methoxy substituent at the C8 position. Results in Tables 2 and 3 H


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(6) Manach, C.; Morand, C.; Gil-Izquierdo, A.; BouteloupDemange, C.; Rémésy, C. Bioavailability in humans of the flavanones hesperidin and narirutin after the ingestion of two doses of orange juice. Eur. J. Clin. Nutr. 2003, 57, 235−242. (7) Robbins, R. C. Effect of methoxylated flavones on erythrocyte aggregation and sedimentation in blood or normal subjects: evidence of a dietary role for flavonoids. Int. J. Vitamin Nutr. Res. 1973, 43, 494−503. (8) Robbins, R. C. Action of flavonoids on blood cells: trimodal action of flavonoids elucidates their inconsistent physiological effects. Int. J. Vitamin Nutr. Res. 1974, 44, 203−216. (9) Manthey, J. A.; Guthrie, N. Antiproliferative activities of citrus flavonoids against six human cancer cell lines. J. Agric. Food Chem. 2002, 50, 5837−5843. (10) Manthey, J. A.; Bendele, P. Anti-inflammatory activity of an orange peel polymethoxylated flavone, 3′,4′,3,5,6,7,8-heptamethoxyflavone, in the rat carrageenan/paw edema and mouse lipopolysaccharide-challenge assays. J. Agric. Food Chem. 2008, 56, 9399−9403. (11) Kurowska, E. A.; Manthey, J. A.; Casaschi, A.; Theriault, A. G. Modulation of HepG2 cell net apolipoprotein B secretion by the citrus polymethoxyflavone, tangeretin. Lipids 2004, 39, 143−151. (12) Gosslau, A.; Chen, K. Y.; Ho, C. T.; Li, S. Anti-inflammatory effects of characterized orange peel extracts enriched with bioactive polymethoxyflavones. Food Sci. and Human Wellness 2014, 3, 26−35. (13) Huang, H.; Li, L.; Shi, W.; Liu, H.; Yang, J.; Yuan, X.; Wu, L. The multifunctional effects of nobiletin and its metabolites in vivo and in vitro. Evid. Based Complement. Alternat. Med. 2016, 2016, 2918798. (14) Kim, Y. J.; Choi, M. S.; Woo, J. T.; Jeong, M. J.; Kim, S. R.; Jung, U. J. Long-term dietary supplementation with low-dose nobiletin ameliorates hepatic steatosis, insulin resistance, and inflammation without altering fat mass in diet-induced obesity. Mol. Nutr. Food Res. 2017, 61, 1600889. (15) Wu, X.; Song, M.; Gao, Z.; Sun, Y.; Wang, M.; Li, F.; Zheng, J.; Xiao, H. Nobiletin and its colonic metabolites suppress colitisassociated colon carcinogenesis by down-regulating iNOS, inducing antioxidative enzymes and arresting cell cycle progression. J. Nutr. Biochem. 2017, 42, 17−25. (16) Li, S.; Wang, H.; Guo, L.; Zhao, H.; Ho, C. T. Chemistry and bioactivity of nobiletin and its metabolites. J. Funct. Foods 2014, 6, 2− 10. (17) Zheng, J.; Song, M.; Dong, P.; Qiu, P.; Guo, S.; Zhong, Z.; Li, S.; Ho, C. T.; Xiao, H. Identification of novel bioactive metabolites of 5-demethylnobiletin in mice. Mol. Nutr. Food Res. 2013, 57, 1999− 2007. (18) Xiao, H.; Yang, C. S.; Li, S.; Jin, H.; Ho, C. T.; Patel, T. Monodemethylated polymethoxyflavones from sweet orange (Citrus sinensis) peel inhibit growth of human lung cancer cells by apoptosis. Mol. Nutr. Food Res. 2009, 53, 398−406. (19) Jurd, L. Spectral properties of flavonoid compounds. In The Chemistry of Flavonoid Compounds; Geissman, T. A., Ed.; Pergamon Press: New York, 1962. (20) Conseil, G.; Baubichon-Cortay, H.; Dayan, G.; Jault, J.-M.; Barron, D.; Di Pietro, A. Biochemistry Flavonoids: A class of modulators with bifunctional interactions at vicinal ATP- and steroidbinding sites on mouse P-glycoprotein. Proc. Natl. Acad. Sci. U. S. A. 1998, 95, 9831−9836. (21) Jacobson, K. A.; Moro, S.; Manthey, J. A.; West, P. L.; Ji, X.-D. Interactions of flavones and other phytochemicals with adenosine receptors. Adv. Exp. Med. Biol. 2002, 505, 163−171. (22) Williams, R. J.; Spencer, J. P. E.; Rice-Evans, C. Flavonoids: antioxidants or signaling molecules? Free Radical Biol. Med. 2004, 36, 838−849. (23) Yue, Y.; Zhang, Y.; Li, Y.; Zhu, J.; Qin, J.; Chen, X. Interaction of nobiletin with human serum albumin studied using optical spectroscopy and molecular modeling methods. J. Lumin. 2008, 128, 513−520. (24) Chen, J.; Song, M.; Wu, X.; Zheng, J.; He, L.; McClements, D. J.; Decker, E.; Xiao, H. Direct fluorescent detection of a

The results in this study suggest that the interactions between cells and the citrus PMFs and their metabolites are complex, and at this time, details concerning solvent factors controlling their fluorescence remain poorly understood. Although the fully methylated PMFs and glucuronidated metabolites without free phenolic hydroxyls show small shifts in their emission-wavelength maxima when bound to the Huh7.5 cells, these differences are by no means similar to the large differences that occur in these spectra when measured in methanol versus in aqueous methanol solutions. However, this is not the case with metabolites with free hydroxyls, for which there are large red shifts in their emission-wavelength maxima when bound to the Huh7.5 cells. Curiously, these same compounds exhibit significant losses in their fluorescence intensities in aqueous solutions, but there are no obvious losses in the emission intensities of these compounds when bound to the Huh7.5 cells. These complexities are understandable in light of the many chemical microenvironments within cells, and the many factors yet to be considered in studying the fluorescence of PMFs, including pH at the binding site, the ionic interactions between the binding sites and resonance structures of the PMFs, and the viscosity effects of compound binding to proteins and membranes of intracellular organelles.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: (772) (462) 5930. E-mail: [email protected]. ORCID

Danielle R. Gonçalves: 0000-0003-4924-6696 John A. Manthey: 0000-0001-9122-0460 Notes

Disclaimer: This article is a U.S. Government work and is in the public domain in the United States. Mention of a trademark or proprietary product is for identification only and does not imply a guarantee or warranty of the product by the U.S. Department of Agriculture. The U.S. Department of Agriculture prohibits discrimination in all its programs and activities on the basis of race, color, national origin, gender, religion, age, disability, political beliefs, sexual orientation, and marital or family status. The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Veronica Cook for critical reading of the manuscript, assistance in figure and legend preparations, and expertise in conducting the fluorescence measurements and analyses by HPLC-MS.

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Analysis of Fluorescence Spectra of Citrus Polymethoxylated Flavones

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