Polymethoxyflavones Isolated from the Peel of Miaray Mandarin

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

■

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 orange.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,

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 characteristics 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, © XXXX American Chemical Society

Received: January 2, 2015 Revised: June 27, 2015 Accepted: July 3, 2015

A

DOI: 10.1021/acs.jafc.5b02445 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

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.

biofilm inhibitory activity in a dose-dependent manner. Citrus has approximately 28 structurally distinct reported polymethoxyflavones, 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 compounds were characterized by spectral analysis using highperformance 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.

including large sample volumes, a wide range of solvents, automated detection, and inline ultraviolet−visible spectral scanning (UV−vis). This improved technique enables mediumpressure 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 byproducts.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, polymethoxyflavones do not demonstrate potent bactericidal or bacteriostatic 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 communication pathways. We have previously demonstrated that several citrus limonoids and flavonoids interfere with bacterial signaling cascades, leading to attenuation of various virulence mechanisms including biofilm formation.27−29 Our prior study showed that 3′,4′,5,6,7-pentamethoxyflavone (sinensitin) exerts

■

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. B


Article

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, Dubuque, 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

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 FlightMass 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 timedelayed 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 (DQFCOSY), 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 cellfree 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 C


Article

Journal of Agricultural and Food Chemistry Biofilm Assay. The biofilm assay was conducted using the method described previously from our laboratory.29 Briefly, an overnight 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 0.5 μL of DMSO or polymethoxyflavones (12.5, 25, 50 μM) dissolved in DMSO. The biofilm mass was quantified by washing with 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 was dissolved with 200 μL of 33% acetic acid, and A570 was measured. The mean ± standard deviation of three biological replicates is presented.

The chromatograms of the HPLC analysis of the isolated compounds are presented in Figure 2. The absence of other peaks demonstrates the purity of the isolated compounds. The purity of the isolated compounds ranged between ≥95 and ≤98%. The absorption spectra of the compounds were obtained using a photodiode array detector. All of the isolated

■

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 polymethoxyflavones. 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 of the isolated compounds from Miaray mandarin were conducted using spectral analysis by HPLC, MS, and NMR.

Figure 2. HPLC chromatograms of isolated compounds (1−10) along with their UV spectra. The separation was conducted on a Gemini C18 column using gradient mobile phase. D


Article


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′-heptamethoxyflavone, 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-

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 E


Article


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.

substitution. A sharp singlet at δ 6.4 was assigned to the sixth position, which was evidenced from the δ 92.32 signal. The Bring 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

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 3position. 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′-tetramethoxyflavone. 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 8F


Article

Journal of Agricultural and Food Chemistry 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 orthocoupled 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 (tetramethoxyflavone).17 The 1H NMR spectrum revealed the presence of an ABX system for the three aromatic protons in the B-ring at H2′, H-5′, and H-6′ as demonstrated by coupling constant signals at δ 7.39 (d, 1H, J = 1.8 Hz), δ 6.95 (d, 1H, J = 8.2 Hz), and δ 7.6 (d, 1H, J = 8.2 Hz), respectively (Figure 3 and Figure S8). These results indicate the presence of methoxyls at the 3′- and 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 DQFCOSY data in Figure S8. 1HNMR spectra of four pentamethoxyflavones for understanding the chemical shifts and structural assignments of all signals are presented in Figure S9. Although sinensetin is not isolated from Miaray mandarins, the spectrum was given for comparison purposes from our earlier study. Using these spectral data, compound 10 was determined to be 5,7,8,3′,4′-pentamethoxyflavone, and the chemical shifts were compared to reported data.33,45 In summary, results from the spectral analysis of the isolated compounds confirm the identity of the isolated compounds as 5-hydroxy-3,7,3′,4′-tetramethoxyflavone (1), 5,6,7,8,4′-pentamethoxyflavone (tangeretin) (2), 3,5,6,7,8,3′,4′-heptamethoxyflavone (3), 5,6,7,8,3′,4′-hexamethoxyflavone (nobiletin) (4), 3,5,7,8,3′,4′-hexamethoxyflavone (5), 3,5,7,3′,4′-pentamethoxyflavone (pentamethylquercetin) (6), limonin (7), 5,7,4′trimethoxyflavone (8), 5,7,8,4′-tetramethoxyflavone (9), and 5,7,8,3′,4′-pentamethoxyflavone (10), respectively (Figure 5). The results are consistent with the reported values.38,45,46 To the best of our knowledge, this is the first report of the isolation of 3,5,7,3′,4′-pentamethylquercetin from the genus Citrus. It is worth noting that this compound was previously reported in a

Figure 5. Structures of isolated compounds (1−10) from Miaray mandarins. Compounds 1, 5, 6, 8, and 10 were identified by NMR and mass spectral analysis as 5-hydroxy-3,7,3′,4′-tetramethoxyflavone, 3,5,7,8,3′,4′-hexamethoxyflavone, 3,5,7,3′,4′-pentamethoxyflavone (pentamethylquercetin), 5,7,4′-trimethoxyflavone, and 5,7,8,3′,4′-pentamethoxyflavone, respectively. Compounds 2, 3, 4, 7, and 9 were identified by mass spectral analysis as tangeretin, heptamethoxyflavone, nobiletin, limonin, and tetramethoxyflavone, respectively.

review describing the various compounds present in citrus species by Manthey et al.47 Antimicrobial Activity of Polymethoxyflavones. We next examined whether these compounds affect bacterial growth by treating V. harveyi with polymethoxyflavones. The kinetic growth curve was calculated by recording the OD570 for 16 h of growth at optimal temperature. Figure 6 illustrates the growth kinetics of V. harveyi BB120 after treatment with 50 μM of the eight polymethoxyflavones. The sigmoid bacterial growth curve observed indicates the polymethoxyflavones do not inhibit the bacterial growth at 50 μM concentration. Similar results were also observed in other studies evaluating the antimicrobial activity of polymethoxyflavones (nobiletin and tangeretin), which had minimum inhibitory concentration (MIC) of ≥1600 μg/mL,26 suggesting that these compounds are not bactericidal. Inhibition of Biofilm Formation and Bioluminescence. Biofilms are surface adherent colonies of bacteria that form an extracellular polymeric matrix to protect themselves. Control of biofilm formation remains a concern among medical personnel, primarily related to medical devices and implants. The bacterial biofilms in medical devices and implants are difficult to control G


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′-pentamethoxyflavone (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, signaling molecules synthesized by bacteria.49 Quorum sensing regulated bioluminescence production in V. harveyi and its signal transduction pathway has been well studied. The autoinducers are detected by three coincidence detectors, LuxN, LuxPQ, and CqsS, that phosphorylate LuxU, a phosphorelay protein. LuxU then phosphorylates the response 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′-heptamethoxyflavone 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 demonstrated 35% inhibition of biofilm with respect to control. Analyzing the bioluminescence in specific mutant strains after treatment with polymethoxyflavones may enable us to understand the possible pathway through which these

Figure 7. HAI-1 and AI-2 induced bioluminescence inhibition in V. harveyi BB886 (A) and BB170 in the presence of polymethoxyflavones (B). The evaluated polymethoxyflavones were tangeretin (TANG), 3,5,6,7,8,3′,4′-heptamethoxyflavone (Hep-MF), nobiletin (NOB), 5,7,4′-trimethoxyflavone (Tri-MF), 3,5,7,8,3′,4′-hexamethoxyflavone (Hex-MF), 5,7,8,4′-tetramethoxyflavone (Tet-MF), 3,5,7,3′,4′-pentamethoxyflavone (Penta-MF-1), and 5,7,8,3′,4′-pentamethoxyflavone (Penta-MF-2).

Figure 8. Inhibition of V. harveyi BB120 biofilm after treatment with 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′-pentamethoxyflavone (Penta-MF1), and 5,7,8,3′,4′-pentamethoxyflavone (Penta-MF2).

compounds might inhibit biofilm formation. Therefore, constitutively luminescent V. harveyi mutants were investigated after treatment with 50 μM 3,5,7,8,3′,4′-hexamethoxyflavone (Figure 9). The results demonstrated an increase in bioluminescence in the luxO mutant strain (JAF483), but showed no change in the hfq mutant (BNL258), suggesting an effect on LuxO. However, further studies are needed to validate the effect on LuxO. The results indicate that 3,5,7,8,3′,4′-hexamethoxyH


Article


composition is modulated by household processing techniques. J. Food Sci. 2012, 77 (9), C921−C926. (2) Uckoo, R. M.; Jayaprakasha, G. K.; Somerville, J. A.; Balasubramaniam, V. M.; Pinarte, M.; Patil, B. S. High pressure processing controls microbial growth and minimally alters the levels of health promoting compounds in grapefruit (Citrus paradisi Macfad) juice. Innovative Food Sci. Emerging Technol. 2013, 18, 7−14. (3) Kim, J.; Jayaprakasha, G. K.; Uckoo, R. M.; Patil, B. S. Evaluation of chemopreventive and cytotoxic effect of lemon seed extracts on human breast cancer (MCF-7) cells. Food Chem. Toxicol. 2012, 50 (2), 423−430. (4) Uckoo, R. M.; Jayaprakasha, G. K.; Patil, B. S. Phytochemical analysis of organic and conventionally cultivated Meyer lemons (Citrus meyeri Tan.) during refrigerated storage. J. Food Compos. Anal. 2015, 42, 63−70. (5) Androula, G. Evaluation of rootstocks for ‘Clementine’ mandarin in Cyprus. Sci. Hortic. 2002, 93 (1), 29−38. (6) Gmitter, F.; Hu, X. The possible role of Yunnan, China, in the origin of contemporary citrus species (rutaceae). Econ. Bot. 1990, 44 (2), 267−277. (7) USDA. Citrus: World Markets and Trade; U.S. Department of Agriculture, Foreign Agricultural Service, Government Printing Office: Washington, DC, USA, 2011. (8) Wu, G. A.; Prochnik, S.; Jenkins, J.; Salse, J.; Hellsten, U.; Murat, F.; Perrier, X.; Ruiz, M.; Scalabrin, S.; Terol, J. Sequencing of diverse mandarin, pummelo and orange genomes reveals complex history of admixture during citrus domestication. Nat. Biotechnol. 2014, 32 (7), 656−662. (9) Luro, F.; Gatto, J.; Costantino, G.; Pailly, O. Analysis of genetic diversity in Citrus. Plant Genet. Resour. 2011, 9 (2), 218−221. (10) Coletta Filho, H. D.; Machado, M. A.; Targon, M. L. P. N.; Moreira, M. C. P. Q. D. G.; Pompeu, J. Analysis of the genetic diversity among mandarins (Citrus spp.) using RAPD markers. Euphytica 1998, 102 (1), 133−139. (11) Hutchison, D. J.; Hearne, C. J.; Bistline, F. W. The performance of ‘Valencia’ orange trees on 21 rootstocks in the Florida flatwoods. Proc. Fla. State Hortic. Soc. 1992, 105, 60−63. (12) Kurowska, E.; Manthey, J.; Casaschi, A.; Theriault, A. Modulation of hepG2 cell net apolipoprotein B secretion by the citrus polymethoxyflavone, tangeretin. Lipids 2004, 39 (2), 143−151. (13) Li, S.; Pan, M.-H.; Lai, C.-S.; Lo, C.-Y.; Dushenkov, S.; Ho, C.T. Isolation and syntheses of polymethoxyflavones and hydroxylated polymethoxyflavones as inhibitors of HL-60 cell lines. Bioorg. Med. Chem. 2007, 15 (10), 3381−3389. (14) Sergeev, I. N.; Li, S.; Ho, C.-T.; Rawson, N. E.; Dushenkov, S. Polymethoxyflavones cctivate Ca2+-dependent apoptotic targets in adipocytes. J. Agric. Food Chem. 2009, 57 (13), 5771−5776. (15) Nakajima, A.; Ohizumi, Y.; Yamada, K. Anti-dementia activity of nobiletin, a citrus flavonoid: a review of animal studies. Clin. Psychopharmacol. Neurosci. 2014, 12 (2), 75−82. (16) Uckoo, R. M.; Jayaprakasha, G. K.; Patil, B. S. Rapid separation method of polymethoxyflavones from citrus using flash chromatography. Sep. Purif. Technol. 2011, 81 (2), 151−158. (17) Uckoo, R. M.; Jayaprakasha, G. K.; Patil, B. S. Hyphenated flash chromatographic separation and isolation of coumarins and polymethoxyflavones from byproduct of citrus juice processing industry. Sep. Sci. Technol. 2013, 48 (10), 1467−1472. (18) Yamamoto, K.; Yahada, A.; Sasaki, K.; Ogawa, K.; Koga, N.; Ohta, H. Chemical markers of shiikuwasha juice adulterated with calamondin juice. J. Agric. Food Chem. 2012, 60 (44), 11182−11187. (19) Lou, S.-N.; Hsu, Y.-S.; Ho, C.-T. Flavonoid compositions and antioxidant activity of calamondin extracts prepared using different solvents. J. Food Drug Anal. 2014, 22 (3), 290−295. (20) Wilson, I. D.; Brinkman, U. A. T. Hyphenation and hypernation: the practice and prospects of multiple hyphenation. J. Chromatogr., A 2003, 1000 (1−2), 325−356. (21) Griffiths, P. R.; Pentoney, S. L.; Giorgetti, A.; Shafer, K. H. The hyphenation of chromatography and FT-IR. Anal. Chem. 1986, 58 (13), 1349A−1366A.

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′-hexamethoxyflavone 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 demonstrated 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

■

AUTHOR INFORMATION

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. Corresponding Authors

*(B.S.P.) Phone: (979) 862-4521. Fax: (979) 862-4522. Email: [email protected]. *(G.K.J.) E-mail: [email protected]. 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. REFERENCES

(1) Uckoo, R. M.; Jayaprakasha, G. K.; Balasubramaniam, V. M.; Patil, B. S. Grapefruit (Citrus paradisi Macfad) phytochemicals I


Article

Journal of Agricultural and Food Chemistry (22) Marston, A.; Hostettmann, K. Natural product analysis over the last decades. Planta Med. 2009, 75, 672−682. (23) Uckoo, R. M.; Jayaprakasha, G. K.; Patil, B. S. Rapid separation method of polymethoxyflavones from citrus using flash chromatography. Sep. Purif. Technol. 2011, 81 (2), 151−158. (24) Jayaprakasha, G. K.; Negi, P. S.; Sikder, S.; Mohanrao, L. J.; Sakariah, K. K. Antibacterial activity of Citrus reticulata peel extracts. Z. Naturforsch., C: J. Biosci. 2000, 55, 1030. (25) Nannapaneni, R.; Muthaiyan, A.; Crandall, P. G.; Johnson, M. G.; O’Bryan, C. A.; Chalova, V. I.; Callaway, T. R.; Carroll, J. A.; Arthington, J. D.; Nisbet, D. J.; Ricke, S. C. Antimicrobial activity of commercial citrus-based natural extracts against Escherichia coli O157:H7 isolates and mutant strains. Foodborne Pathog. Dis. 2008, 5 (5), 695−699. (26) Yi, Z.; Yu, Y.; Liang, Y.; Zeng, B. In vitro antioxidant and antimicrobial activities of the extract of Pericarpium Citri Reticulatae of a new Citrus cultivar and its main flavonoids. LWT−Food Sci. Technol. 2008, 41 (4), 597−603. (27) Vikram, A.; Jesudhasan, P. R.; Jayaprakasha, G. K.; Pillai, S. D.; Patil, B. S. Citrus limonoids interfere with Vibrio harveyi cell−cell signalling and biofilm formation by modulating the response regulator LuxO. Microbiology 2011, 157 (1), 99−110. (28) Vikram, A.; Jesudhasan, P. R.; Jayaprakasha, G. K.; Pillai, S. D.; Jayaraman, A.; Patil, B. S. Citrus flavonoid represses Salmonella pathogenicity island 1 and motility in S. Typhimurium LT2. Int. J. Food Microbiol. 2011, 145 (1), 28−36. (29) Vikram, A.; Jayaprakasha, G. K.; Jesudhasan, P. R.; Pillai, S. D.; Patil, B. S. Suppression of bacterial cell−cell signalling, biofilm formation and type III secretion system by citrus flavonoids. J. Appl. Microbiol. 2010, 109 (2), 515−527. (30) Uckoo, R. M.; Jayaprakasha, G. K.; Patil, B. S. Chromatographic techniques for the separation of polymethoxyflavones from citrus. In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B. S., Jayaprakasha, G. K., Murthy, K. N. C., Seeram, N., Eds.; ACS Symposium Series 1093; American Chemical Society: Washington, DC, USA, 2012. (31) Surette, M. G.; Bassler, B. L. Quorum sensing in Escherichia coli and Salmonella typhimurium. Proc. Natl. Acad. Sci. U. S. A. 1998, 95 (12), 7046−7050. (32) Freeman, J. A.; Bassler, B. L. A genetic analysis of the function of LuxO, a two-component response regulator involved in quorum sensing in Vibrio harveyi. Mol. Microbiol. 1999, 31 (2), 665−677. (33) Freeman, J. A.; Bassler, B. L. Sequence and function of LuxU: a two-component phosphorelay protein that regulates quorum sensing in Vibrio harveyi. J. Bacteriol. 1999, 181, 899−906. (34) Lenz, D. H.; Mok, K. C.; Lilley, B. N.; Kulkarni, R. V.; Wingreen, N. S.; Bassler, B. L. The small RNA chaperone Hfq and multiple small RNAs control quorum sensing in Vibrio harveyi and Vibrio cholerae. Cell 2004, 118 (1), 69−82. (35) Qin, Y.; Luo, Z.-Q.; Farrand, S. K. Domains formed within the N-terminal region of the quorumsensing activator TraR are required for transcriptional activation and direct interaction with RpoA from Agrobacterium. J. Biol. Chem. 2004, 279 (39), 40844−40851. (36) Lu, L.; Hume, M. E.; Pillai, S. D. Autoinducer-2-like activity associated with foods and its interaction with food additives. J. Food Prot. 2004, 67 (7), 1457−1462. (37) Yao, X.; Xu, X.; Fan, G.; Qiao, Y.; Cao, S.; Pan, S. Determination of synergistic effects of polymethoxylated flavone extracts of Jinchen orange peels (Citrus sinensis Osberk) with amino acids and organic acids using chemiluminescence. Eur. Food Res. Technol. 2009, 229 (5), 743−750. (38) Wang, D.; Wang, J.; Huang, X.; Tu, Y.; Ni, K. Identification of polymethoxylated flavones from green tangerine peel (Pericarpium Citri Reticulatae Viride) by chromatographic and spectroscopic techniques. J. Pharm. Biomed. Anal. 2007, 44 (1), 63−69. (39) Perez, J. L.; Jayaprakasha, G. K.; Valdivia, V.; Munoz, D.; Dandekar, D. V.; Ahmad, H.; Patil, B. S. Limonin methoxylation influences the induction of glutathione S-transferase and quinone reductase. J. Agric. Food Chem. 2009, 57 (12), 5279−5286.

(40) Li, S.; Yu, H.; Ho, C.-T. Nobiletin: efficient and large quantity isolation from orange peel extract. Biomed. Chromatogr. 2006, 20 (1), 133−138. (41) Machida, K.; Osawa, K. On the flavonoid constituents from the peels of Citrus hassaku Hort. ex Tanaka. Chem. Pharm. Bull. 2012, 37 (4), 1092−1094. (42) Silva, T.; Carvalho, M.; Braz-Filho, R. Estudo espectroscópico em elucidaçaõ estrutural de flavonóides de Solanum jabrense Agra & Nee e S. paludosum Moric. Quim. Nova 2009, 32, 1119−1128. (43) Chung, L. Y.; Yap, K. F.; Goh, S. H.; Mustafa, M. R.; Imiyabir, Z. Muscarinic receptor binding activity of polyoxygenated flavones from Melicope subunifoliolata. Phytochemistry 2008, 69 (7), 1548−1554. (44) Sugiyama, S.; Umehara, K.; Kuroyanagi, M.; Ueno, A.; Taki, T. Studies on the differentiation inducers of myeloid leukemic cells from Citrus species. Chem. Pharm. Bull. 1993, 41 (4), 714−719. (45) Koichi, M.; Keisuke, O. On the flavonoid constituents from the peels of Citrus hassaku Hort. ex Tanaka. Chem. Pharm. Bull. 1989, 37 (4), 1092−1094. (46) Raman, G.; Cho, M.; Brodbelt, J. S.; Patil, B. S. Isolation and purification of closely related Citrus limonoid glucosides by flash chromatography. Phytochem. Anal. 2005, 16 (3), 155−160. (47) Manthey, J. A.; Guthrie, N.; Grohmann, K. Biological properties of citrus flavonoids pertaining to cancer and inflammation. Curr. Med. Chem. 2001, 8 (2), 135−153. (48) Djeribi, R.; Bouchloukh, W.; Jouenne, T.; Menaa, B. Characterization of bacterial biofilms formed on urinary catheters. Am. J. Infect. Control 2012, 40 (9), 854−859. (49) Reading, N. C.; Sperandio, V. Quorum sensing: the many languages of bacteria. FEMS Microbiol. Lett. 2006, 254 (1), 1−11.

J


Polymethoxyflavones Isolated from the Peel of Miaray Mandarin

Recommend Documents