Antifungal action and inhibitory mechanism of polymethoxylated flavones from Citrus reticulata Blanco peel against Aspergillus niger

Accepted Manuscript Antifungal action and inhibitory mechanism of polymethoxylated flavones from Citrus reticulata Blanco peel against Aspergillus niger Ting Wu, Dan Cheng, Mengying He, Siyi Pan, Xiaolin Yao, Xiaoyun Xu PII:

S0956-7135(13)00375-7

DOI:

10.1016/j.foodcont.2013.07.027

Reference:

JFCO 3382

To appear in:

Food Control

Received Date: 18 April 2013 Revised Date:

11 July 2013

Accepted Date: 22 July 2013

Please cite this article as: WuT., ChengD., HeM., PanS., YaoX. & XuX., Antifungal action and inhibitory mechanism of polymethoxylated flavones from Citrus reticulata Blanco peel against Aspergillus niger, Food Control (2013), doi: 10.1016/j.foodcont.2013.07.027. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Antifungal action and inhibitory mechanism of polymethoxylated flavones from Citrus reticulata Blanco peel against Aspergillus niger

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Ting Wu a, Dan Cheng a, Mengying He a, Siyi Pan a, Xiaolin Yao b, Xiaoyun Xu *,a

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University), Ministry of Education, Wuhan, 430070, PR China

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College of Biological Engineering, Hubei University of Technology, Wuhan, 430068,

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Key Laboratory of Environment Correlative Dietology (Huazhong Agricultural

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PR China

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* Corresponding author. Tel.: +86 27 87671056; fax: +86 27 87288373.

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E-mail address: [email protected] (Xiaoyun Xu).

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Choice of journal/section： ：Microbial food safety and antimicrobial systems

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ABSTRACT

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Polymethoxylated flavones (PMFs), isolated from the peels of Citrus reticulata Blanco, were

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identified and quantitated as tangeretin (TAN) (33.87%), nobiletin (NOB) (20.98%),

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5-demethylnobiletin (3.52%), tetramethyl-o-scutellarein (1.61%), tetramethyl-o-isoscutellarein

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(1.23%), pentamethoxyflavone (1.08%) and sinensetin (0.35 %). PMFs are promising natural

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antimicrobial compounds with potential applications in the food industry. The antifungal effects

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of PMFs on Aspergillus niger (A. niger)were evaluated by microbroth dilution assay and

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growth curve determination. The minimum inhibition concentration (MIC) of PMFs extract and

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of TAN against A.niger was determined to be 0.12mg/mL and 1.5mg/mL respectively. PMFs

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affected the permeability of cytomembrane, resulting in instant increased flux of K+ and

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increased relative electrical conductivity. PMFs also dose-dependently reduced the chitin

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production. These results suggest that the antifungal effects of PMFs could be explained by

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the permeability change of cytomembrane and the fragility of cell walls caused by chitin

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inhibition.

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Keywords: Polymethoxylated flavones; Aspergillus niger; Citrus reticulata Blanco; Antifungal;

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Permeability; Chitin

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44 45 1. Introduction

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Fungi are the primary spoilage agents of freshly harvested fruits, vegetables (Mehyar,

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Al-Qadiri, Abu-Blan, & Swanson, 2011) and bakery products (Marín, Guynot, Sanchis,

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Arbonés, & Ramos, 2002). A. niger, the most important member of the genus Aspergillus, is

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capable of growing upon a wide range of organic substrates, and often causes deterioration of

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stored food material (Barrios, Medina, Cordoba, & Jordano, 1997; Mishra & Dubey, 1994;

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Paster, et al., 1990). A.niger also plays a major role in otomycosis and mycotoxin production

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(Baker, 2006; Schuster, Dunn-Coleman, Frisvad, & van Dijck, 2002). Although synthetic

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antimicrobial compounds are used to combat fungi, negative consumer perception of chemical

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preservatives is driving attention towards natural alternatives.

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Citrus peel is a rich source of polymethoxylated flavones (PMFs), which are rarely found

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in other plants. PMFs are of particular interest for their broad spectrum of biological activities,

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including anticarcinogenic (J. Chen, Montanari, & Widmer, 1997), antiproliferative (Yanez, et

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al., 2004), anti-inflammatory (Benavente-Garcia & Castillo, 2008), and effects on mammalian

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metabolism (Middleton, Kandaswami, & Theoharides, 2000). Studies have showed that PMFs

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also have the ability to inhibit microbial growth, including antibacterial, antifungal and antiviral

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activities (Jayaprakasha, Selvi, & Sakariah, 2003; Li, et al., 2007; Ortuño, et al., 2006). Thus,

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PMFs are promising natural antimicrobial compounds with potential applications in food,

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contributing to safety and preservation. However, compared to antibacterial capacity, the

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antifungal activity of PMFs including mechanism has received little attention. Several known

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mechanisms of action for commercial antifungal agents have been investigated from different

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approaches including, inhibition of the biosynthesis of macromolecules such as DNA, RNA

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and proteins, inhibition of squalene epoxide, ergosterol, folic acid biosynthesis and inhibition

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of cytoplasmic membrane function (Carmona, Gandía, López-García, & Marcos, 2012; Kocsis,

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et al., 2009). Unlike glycosidic flavonoids, PMFs are considerably less polar and assume a

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planar structure, which could interfere with their permeability and hence biological properties.

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In this study, the antifungal activity of PMFs extract from the peels of Citrus reticulata

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Blanco was studied against A.niger. The permeability of cytoderm and cytomembrane was the

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main objective in this research to elucidate the probable mechanism of PMFs against A. niger.

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2.1. Plant material

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Heyang II, a citrus variety of Citrus reticulata Blanco, was obtained from Citrus reticulata Blanco Comprehensive Experiment Station (Quzhou, China).

2.2. Fungal strain and culture media

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A.niger (CICC 2273), obtained from China Center of Industrial Culture Collection (CICC),

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was cultured on potato dextrose agar (PDA). Spore suspensions were prepared and diluted in

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sterile water to a concentration of approximately 105 spores/ml. Spore population was counted

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using a haemocytometer.

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2.3 Chemicals 4

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Standards of NOB and TAN (purity ≥98%) were purchased from Shanghai Yuanye

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Biotechnology Co., Ltd. (Shanghai, China). Baicalein (5,6,7- trihydroxyflavone) standard was

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purchased from the National Institutes for Food and Drug Control (Beijing, China) (purity＞

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98.5%). Roswell Park Memorial Institute (RPMI) Medium 1640 was purchased from Invitrogen

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Corporation (San Diego, CA, USA). All reagents used in the study were of analytical grade,

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except for the chromatographic grade solvents used in HPLC and LC-MS/MS.

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94 2.4 Extraction of PMFs

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PMFs were extracted from the peels of Citrus reticulata Blanco using the method of Yao,

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Xu, Fan, Qiao, Cao, and Pan (2009) with minor modification. The citrus peels were dried at

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40°C to constant weight and milled into powder (siz e 0.45mm). The powder (200g) was

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extracted with 2000mL of 95% ethanol (40°C, 12h). T he suspension was concentrated to

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about 50mL using a rotary evaporator at 40°C and tr eated with 400mL of petroleum ether (×3,

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30min) in a separatory funnel. The combined petroleum ether extracts were washed with 0.4%

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sodium hydroxide solution until the aqueous fraction was colorless. The petroleum ether layer

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was collected, concentrated, freeze-dried (Chirst Beta 2-8 LD plus, Osterode, Germany) with

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a yield of crude PMFs about 960mg.

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2.5 HPLC and LC-MS/MS Analysis

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The identification and quantitative analysis of the PMFs extract were performed by HPLC

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and LC-MS/MS. Identification of NOB and TAN was accomplished by matching the spectra,

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mass spectrum and the retention time of peaks in the sample with the authentic standards. 5

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Other PMFs were quantified as baicalein equivalents. For HPLC analysis, an Agilent 1100 series LC/MSD Trap (Agilent, USA), coupled with a

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photodiode-array detector (PDA) set at 330nm (Pupin et al., 1998) was used. UV spectra were

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taken in the region of 200-800nm. According to the method described by Mouly, Gaydou and

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Auffray (1998), chromatographic conditions were as follows: column, Amethyst C18-H

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(4.6mm×250mm, 5µm) (Delaware Technology Park, Delaware, USA ); Eluent: (A) 1.5% glacial

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acetic acid dissolved in water, (B) methyl alcohol. The gradient elution had the following profile:

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0-45min, 50-72% B, 45-50min, 72%-50% B. The flow rate was 1.00 mL/min. 10µL of sample

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was injected for analysis.

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For LC-MS/MS analysis, the Agilent 1100 series LC/MSD Trap (Agilent, USA) was

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equipped with an electrospray ionization ion source (ESI). The method with minor

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modifications was as reported by Dugo, Mondello, Dugo, Stancanelli and Dugo (2000). The

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mass spectrometer was operated in positive ion mode with a capillary voltage of 4.5kV,

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pressure of nebulizing nitrogen of 40psi, capillary temperature of 250°C, dry gas flow at

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10L/min, a scan scope ranged from 100 to 1000m/z.

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2.6 Antifungal activity

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2.6.1 Minimal inhibitory concentration (MIC) determination

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The MIC of PMFs extract was evaluated according to the microdilution method described

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by Tang et al. (2010) with minor modifications. Stock solutions of PMFs were prepared in

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dimethyl sulfoxide (DMSO) into RPMI 1640 medium. Then, 180 µL of sample solutions were

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diluted two fold into 96-well plates with concentrations ranging from 6.5 to 0.013 mg/mL. 20µL 6

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aliquot of suspension of 105spores/mL of A.niger was inoculated onto microplates, and the

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test was performed in a final volume of 200 µL. The positive control (TAN + fungi), growth

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control (RPMI + fungi) and sterility control (RPMI) were evaluated similarly. The plates were

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incubated in a shaker at 35 °C for 48 h. The MIC va lues were read at 560 nm using a

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microplate reader. The MIC of the PMFs extract was defined as the lowest concentration

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preventing any discernible growth.

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2.6.2 Growth curve determination

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The growth curve was constructed by measuring the dry mycelial weight of A.niger, using

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the method reported by Rasooli, Rezaei and Allameh (2006). Briefly, A.niger inoculum was

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cultivated on potato dextrose broth (PDB) and incubated for 48h in an incubator shaker (28°C,

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150rpm). In order to observe a significant inhibition, the inoculum was then added to either

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PDB alone (control) or a solution of PDB plus an appropriate amount of PMFs extract stock

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solution with a final concentration 0.16mg/mL, slightly higher than the MIC value determined,

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then incubated at 28°C, 150rpm. At determined times , tubes of samples (with or without PMFs)

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were removed and stored at 4°C. Following incubatio n, all samples were centrifuged at 8000

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× g for 25min, supernatants were discarded and the sediments were dried at 105°C in a

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circulation oven for 4h. The mycelial dry weight was then determined.

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2.7 Extracellular Na+ and K+ determination

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Concentrations of extracellular Na+ and K+ were determined by flame atomic absorption

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spectrophotometer as outlined by Rabaste, Jeminet, Dauphin and Delort (1992) with slight

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modifications. The A.niger suspension of 105 spores/mL was incubated (28°C, 150rpm, 48h), 7

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then centrifuged (4°C, 10000 × g, 10min) (Avanti J- E, Beckman Coulter, Fullerton, USA). The

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fungal mass was washed (×2) in 3-(N-morpholino)propanesulfonic acid (MOPS) buffer (50mM,

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pH 6.0) , suspended in 4mL of MOPS buffer (50mM, pH 6.0) with 1% glucose, and incubated

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with 2.4mg/mL PMFs extract for varying times (0-40min, 10min intervals). Distilled water

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replaced the PMFs extract as the control group. The samples were placed on an incubator

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shaker (28°C, 150rpm), centrifugated (10000×g, 10mi n, 4°C), 3mL of supernatant was

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digested in acid mixture (nitric acid: perchloric acid = 4: 1, v/v) and diluted to appropriate

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concentration for analyses using an AA-6300C series atomic absorption spectrophotometer

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(Shimazu, Kyoto, Japan).

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2.8 Electrical conductivity determination

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The electrical conductivity of the fungal mass was determined by the method reported by

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Kong, et al. (2008). The A. niger inoculum after incubation (28°C, 48h) was centrifu ged at

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3000 × g for 20min, then washed with 5% glucose until the electrical conductivity was close to

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that of 5% glucose. The isotonic cell was obtained with electrical conductivity marked as L0.

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Different concentrations of PMFs solution (0.6, 1.2, 2.4mg/mL respectively) were added

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to 5% glucose with the electrical conductivities of these mixtures defined as L1, L2, L3. The

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same concentrations of PMFs solution were added to the isotonic cell. The samples were

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mixed and incubated at 28°C for 2h, then the electr ical conductivities were measured and

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marked as L1’, L2’, L3’. The value of L-L’ was expressed as △L. The ratio of △L/L0 indicates

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the relative electrical conductivity of PMFs extracts.

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2.9 Chitin content estimation Chitin content was estimated according to the method described by Fang, Hassanien,

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Wong, Bah, Soliman and Ng (2010) with minor modifications. After incubation with different

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concentrations of PMFs extract for 12 h, hyphae of A. niger were stained with 0.025% Congo

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red with agitation in the dark for 5 min. After rinsing with sterile water (×3), hyphae from

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different treatments were examined using a spectrofluorometer. Excitation wavelength of

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540nm and an emission wavelength of 560-900nm were used for analyses.

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All determinations were carried out in triplicate. Data analyses were performed by

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analysis of variance. One-way ANOVA was applied to determine differences (P<0.05) using

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SAS version 8.1.

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3. Results

The main constituents of PMFs extracts from Citrus reticulata Blanco (Table 1) were

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identified based on their UV spectra, molecular ions, fragment ions, and elution order

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comparison to the reported data (Dugo, et al., 2000; Wang, Wang, Huang, Tu, & Ni, 2007; Yao,

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et al., 2009 ). The fragments of [M+H–n×15]+ produced by loss of one or more methyl groups

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from the protonated molecule, as well as [M+H–28]+, [M + H − 33]+ , [M + H − 43]+ , [M+H–46]+,

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and [M+H–61]+ fragment ions were diagnostic for the polymethoxylated species (Morin et

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al.,1991). In addition, characteristic “double-peaks” and the absorption maxima near 330 nm

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in the UV spectra could be another indicators for PMFs (Mouly, P., Gaydou, E. M., & Auffray,

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A., 1998). Fig. 1 shows the chromatogram profiles of PMFs. Compounds 1, 2, 3, 4, 5, 7 were

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identified as tetra-, penta-, and hexa-substituted flavones due to the intense molecular ions

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[M+H]+ at m/z 343, 373 and 403 in the positive mode. Compound 6 could be assigned as

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being monohydroxy-pentamethoxyflavone due to the intense protonated molecular ions

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[M+H]+ at m/z 389. Seven compounds were quantified out of which, TAN (33.87%) was the

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principal

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tetramethyl-o-scutellarein

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pentamethoxyflavone (1.08%) and sinensetin (0.35 %).

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5-demethylnobiletin

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tetramethyl-o-isoscutellarein

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The MIC values of PMFs extract and TAN for the growth inhibition of A. niger were found

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to be 0.12 mg/mL and1.5mg/mL respectively, which indicated that PMFs extract had better

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inhibitory activity than TAN. Results from exposure of A.niger to PMFs for a time period up to

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153 h at 28 °C in PDB are shown in Fig. 2. Growth p hases of A. niger could be distinguished

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as the lag phase (0-39h), log phase (40-69h) and stationary phase (after 69h). The mycelial

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dry weight of the sample treated with PMFs followed the same trend of the control until 50h,

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then decreased sharply to constant level after a shorter log phase. Fungal growth inhibition

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was significant compared with the non-treated control (p < 0.05).

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As indicated in Fig. 3, the concentration of extracellular K+ was much higher than the

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control as shown in Fig.3-A. However, the concentration of Na+ in medium with PMFs extract

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was lower than the non-treated control, although no significant difference was shown (Fig.

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3-B). The variation curve declined sharply and demonstrated a significant difference (P<0.05).

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As illustrated in Fig. 4, the application of up to approximately 0.6 mg/mL PMFs extract caused

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the relative electrical conductivity of the growth matrix to increase dramatically. However, this 10

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change was not dose-dependent, as the relative electrical conductivity remained around 0.8

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even when the concentration of PMFs reached 2.4 mg/mL. The effect of PMFs extract on the chitin content in the plasma membrane of A. niger is

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shown in Fig. 5. A dose-dependent decrease in chitin production was observed when cells

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were grown in the presence of PMFs. The chitin content decreased by 31.9%, 48.9%, 59.6%,

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63.8% and 70.2%, respectively, when A. niger cells were exposed to MIC, 2×MIC, 3×MIC, 4

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×MIC and 5×MIC values of PMFs. The results demonstrate that the chitin content (at 608

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nm) of A. niger was significantly inhibited by the different concentrations of PMFs .

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4. Discussion

This study investigated the antifungal effects and mechanism of attack of a mixture of

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PMFs isolated from Citrus reticulata Blanco peel on A. niger. Although results were obtained

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using PMFs extract and the individual major PMF component TAN, in commercial practice, a

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mixture of PMFs would be more applicable because the cost of isolating pure PMF

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components would reduce their industrial feasibility unless warranted.

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The composition and content of PMFs vary with citrus variety, the production origin as

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well as the genuine production place (H.-F. Chen, et al., 2012). Such variation in composition

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of PMFs would alter their biological activity. Hence, the chemical profile of PMFs extract

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needs to be standardized for optimizing their potential as antifungal agents. The most

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abundant PMFs in citrus are NOB ,TAN, sinensetin and heptamethoxyflavone (Mak, et al.,

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1996; Nogata, et al., 2006). In the present study, TAN followed by NOB were the major

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components, and their concentrations were much higher than 5-demethylnobiletin,

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tetramethyl-o-scutellarein,

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sinensetin .

tetramethyl-o-isoscutellarein,

pentamethoxyflavone

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Some research studies have identified flavonoids (including from citrus) and assessed

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their antimicrobial activity (Arima, Ashida, & Danno, 2002; Mandalari, et al., 2007; Rauha, et

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al., 2000), but there are few reported studies about the possible antifungal activity of

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flavonoids, especially PMFs. Ortuno et al. (2006) reported that PMFs are more active

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antifungal agents than flavanones against Penicillium digitatum. Our previous studies showed

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that the inhibiting effect of PMFs against A.niger is more effective than against Penicillium

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corylophilum (data not shown). In this research, A.niger was chosen as representative of

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filamentous fungal species.

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The inhibition capacity of PMFs varies when acting on different microorganisms. As

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reported by Yi, Yu, Liang and Zeng (2008), the MIC of NOB and TAN against Staphylococcus

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aureus was 1.6mg/mL while the MIC against Salmonella typhi/ and Enterobacter cloacae was

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over 2mg/mL. In this research, the MIC of TAN against A.niger was 1.5mg/mL, similar to the

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value which has been reported against Staphylococcus aureus. The MIC of PMFs extract in

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this study was 0.12mg/mL, which is significantly lower than that of TAN. Synergistic action of

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different PMFs could be an explanation, as it has been reported by Silva, Weidenbörner and

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Cavaleiro (1998), that flavonoid mixtures at different concentrations caused significantly

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higher inhibition than each component alone. Akao, itoh, Ohguchi, Iinuma and Nozawa (2008)

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also showed that TAN, NOB, and 5-DN exhibited a synergistic effect on cell growth of human

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neuroblastoma SH-SY5Y. In this study, the sharp decrease of mycelial dry weight after 50h

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indicated that the fungus was mainly inhibited from the log phase to stationary phase and

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finally disintegrated by PMFs (Fig. 2). Determination of extracellular Na+ and K+ as well as the electrical conductivity was carried

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out to investigate the change in permeability of cytomembrane. It has been reported that the

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intracellular K+ concentration decreases and the Na+ concentration increases as the culture is

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aging (Bot & Prodan, 2010). The Na+ and K+ curves in Fig. 3 showed that Na+ inflowed from

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extracellular to intracellular when stimulated by PMFs, while the K+ flowed from intracellular to

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extracellular, which implied that the A.niger was rapidly aging when in contact with PMFs.

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With time, the reduction of extracellular K + also corresponded with its function to maintain the

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intracellular osmotic pressure: a high concentration of extracellular K+ would activate the

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Na+-K+ pump, which could enhance the influx of K+ from extracellular to intracellular. The

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relative electrical conductivity of the growth matrix increased immediately when PMFs extract

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was added, then remained constant even with a further increase of PMFs extract, indicating

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that the permeability of cytomembrane was affected (Fig. 4). Recent studies have shown that

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some essential oils primarily affect fungal cell permeability through direct interaction with the

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cytomembrane (Sharma & Tripathi, 2008; Tolouee, et al., 2010). This study led to a similar

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conclusion that the membrane structure of the fungus is significantly damaged by PMFs. The

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fluxion of Na+ and K+ and the increased relative electrical conductivity are indicators of

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membrane damage.

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In order to predict target components in the plasma membrane, the effect of PMFs extract

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on the chitin content was assessed. Chitin, the main constituent of hyphal walls in

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Zygomycetes, contributes significantly to the mechanical strength of the cell wall. When chitin

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synthesis is affected, growing hyphae tend to lyse and form pronounced bulges unless the 13

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osmolarity of the medium is increased (Datema, Van Den Ende, & Wessels, 1977; Ram, et al.,

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2004). In this study, by using the dye Congo red, a dose-dependent decrease of chitin was

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observed (Fig. 5). Similarly, Luo et al. (2012) showed that inactivation of Bbslt2 interfered with

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chitin synthesis. Mizuhara, et al. (2011) concluded that the growth inhibition of several

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filamentous fungi caused by CTB1 (an antifungal cyclic thiopeptide) could be explained by cell

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wall fragility induced via CTB1 binding to chitin. Yutani, et al. (2011) stated that anethole

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induces morphological changes in M. mucedo hyphae by inhibiting chitin synthase and

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thereby restricting hyphal growth. The presence of uncompetitive inhibitors indicates the

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possibility of regulation sites for the activity of chitin synthase itself. Therefore, we suggest that

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PMFs induce a considerable impairment to the chitin biosynthesis for A.niger, indicating

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cytomembrane is the antifungal target. Other mechanisms, including energy metabolism

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system disruption and the inhibition of nucleic acids formation may also contribute.

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In conclusion, PMFs extract from Citrus reticulata Blanco peel possesses antifungal

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activity inhibiting the growth of A. niger .The MIC value (0.12mg/mL) demonstrates the

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feasibility of commercial application of PMFs against fungi. The inhibition could be explained

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by permeability change of cytomembrane and cell wall fragility induced via PMFs inhibiting

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chitin synthase. The findings demonstrate that PMFs may be considered as potential natural

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antifungal agents for use in the food industry .

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Acknowledgements

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This research was supported by National Natural Science Foundation of China

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(No.31171756) and the Fundamental Research Funds for the Central Universities 14

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(No.2009JC012). Project support was also by the Scientific Research Foundation for the

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Returned Overseas Chinese Scholars, State Education Ministry. Special thanks to the Citrus

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reticulata Blanco Comprehensive Experiment Station (Quzhou) for kindly providing the Citrus

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reticulata Blanco fruits.

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Figure captions Fig. 1

HPLC profile of PMFs extract from Creticulata Blanco orange peel.

Fig. 2

The growth curve of Aspergillus niger: the sample line represents added PMFs with a final

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concentration of 0.16mg/mL, the control line without PMFs.

The concentrations of extracellular Na+ and K+ with time: figure A- K+, B- Na+.

Fig. 4

The relative electrical conductivity of growth matrix with different concentration of PMFs.

Fig. 5

Spectrophotometric chitin profiles of A. niger .(A)Control experiment(untreated).(B) A.

niger treated with

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Fig. 3

MIC of PMFs.(C) A. niger treated with 2× MIC of PMFs. (D) A. niger treated

with 3×MIC of PMFs.

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(E) A. niger treated with 4×MIC of PMFs. (F) A. niger treated with 5×MIC of PMFs.

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Highlights: The identification and quantitative analysis of the PMFs extracts was performed




Antifungal activity of PMFs was studied




Inhibitory mechanism of PMFs was explored

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Table 1 Peak assignment for analysis of PMFs Peak no.

tR(min)

[M+H]+

MS/MS(m/z)

(m/z)

UV Peak

Identification

(λ/nm)

1

22.0

373

358, 343, 329, 312

239, 269, 331

2

24.2

373

358, 343, 328, 312, 288

251, 268, 334

3

28.9

343

328, 313, 283

268, 324

tetramethyl-o-isoscutellarein

4

31.3

343

328, 313, 299, 282

248, 268, 346

tetramethyl-o-scutellarein

5

31.9

403

388, 373, 355, 342

248, 269, 335

nobiletin

6

38.5

389

374, 359, 356, 341, 328

270, 344

5-demethylnobiletin

7

39.3

373

358, 343, 312

232, 270, 326

tangeretin

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pentamethoxyflavone

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tR, retention time

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sinensetin

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A

B

1

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