The structure-antifungal activity relationship of 5,7-dihydroxyflavonoids against Penicillium italicum

Food Chemistry 224 (2017) 26–31

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The structure-antifungal activity relationship of 5,7-dihydroxyflavonoids against Penicillium italicum Shuzhen Yang a, Jie Zhou a, Dongmei Li b, Chunyu Shang a, Litao Peng a,⇑, Siyi Pan a a b

Key Laboratory of Environment Correlative Dietology, Ministry of Education, College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, PR China Georgetown University Medical Center, Washington, DC, USA

a r t i c l e

i n f o

Article history: Received 9 August 2016 Received in revised form 27 October 2016 Accepted 1 December 2016 Available online 9 December 2016 Keywords: 5,7-Dihydroxyflavonoids P. italicum Spore germination Respiration Structure-activity relationship

a b s t r a c t To evaluate the structure-activity relationship of 5,7-dihydroxyflavonoids against P. italicum, we tested the antifungal activity of 23 selected 5,7-dihydroxyflavonoids against spore germination of P. italicum, and the effects of hydroxyl group, hydrogenation, methylation and glycosylation on the antifungal activity are explored. C-40 -OH and C-3-OH are active groups for the 5,7-dihydroxyflavonoids against P. italicum. We find that hydrogenation of the C2/C3 bond decreases the antifungal activity of 5,7dihydroxyflavonoids. Antifungal activity of 5,7-dihydroxyflavonoids against P. italicum was affected by the conjugation site of glycosylation and the class of sugar moiety. The correlation between antifungal activity and the inhibition of respiration of 5,7-dihydroxyflavonoids was further evaluated. We find no significant relationship among the IC50 of 5,7-dihydroxyflavonoids on spore germination and on respiration. Some 5,7-dihydroxyflavonoids even enhance the respiration of P. italicum. This indicate respiration is not the only target for 5,7-dihydroxyflavonoids against P. italicum. Ó 2016 Published by Elsevier Ltd.

1. Introduction Blue mold, caused by Penicillium italicum, is one of the most common postharvest diseases of citrus fruits, which is responsible for the loss of up to 25% of total citrus production worldwide (Li et al., 2009; Tayel, Moussa, Salem, Mazrou, & El-Tras, 2016). Synthetic fungicides such as sodium o-phenylphenate (SOPP), thiabendazole (TBZ), and imazalil (IMZ) have been applied for postharvest control of the main pathogens of citrus fruits in various citrusproducing countries for many years. However, this continuous use of synthetic fungicides has not only increased the potential to develop drug resistance and to pollute the environment, but is also undesirable due to a long degradation period that may leave unpleasant residues on the fruit (Talibi, Boubaker, Boudyach, & Ait Ben Aoumar, 2014; Yang et al., 2016; Zhang, 2007). Lately many attempts have been undertaken in order to develop safer and more eco-friendly alternatives for controlling citrus postharvest disease, among which, the use of naturally derived bioactive compounds seems to be one of the most promising. Flavonoids, a large group of phenolic compounds produced by a broad spectrum of fruits and vegetables during their secondary ⇑ Corresponding author. E-mail addresses: [email protected] (S. Yang), [email protected] (J. Zhou), [email protected] (D. Li), [email protected] (C. Shang), [email protected] (L. Peng), [email protected] (S. Pan). http://dx.doi.org/10.1016/j.foodchem.2016.12.001 0308-8146/Ó 2016 Published by Elsevier Ltd.

metabolism, have long been associated with a wide range of biological activities, including antioxidant, antimicrobial, antimutagenic, cytotoxic and anticarcinogenic activities (Orhan, Özçelik, Özgen, & Ergun, 2010). The inherent antimicrobial properties of flavonoids in fact are determined by their chemical structure (Filho et al., 2015; Orhan et al., 2010; Romano et al., 2013). The core structure of flavonoid compounds is the 2-phenyl-benz[a]pyrane or flavan nucleus, which consists of two benzene rings (A and B) that are linked through a heterocyclic pyran ring (C) (Cushnie & Lamb, 2005). In the A ring, the hydroxylations at positions 5 and 7 are the most critical for the antimicrobial activity of flavonoids according to quantum chemistry calculations. For example, hydroxyl groups at positions 5 and 7 in flavones and flavonols used in Brunskole et al. (2009) study can effectively inhibit trihydroxynaphthalene reductase, which is a target for development of new fungicides and selective antimycotics (Brunskole et al., 2009). In our previous study, we found that some active components from poplar bud and propolis including pinocembrin, chrysin and galangin, could inhibit Penicillium italicum growth (Yang et al., 2011, 2016). These compounds all belong to 5,7-dihydroxyflavonoids. Cushnie and Lamb (2011) reported that more than 50% of antimicrobial flavonoids are 5,7-dihydroxyflavonoids (Cushnie & Lamb, 2011). These results indicate that bioactive 5,7dihydroxyflavonoids have a good potential to be a fungicidal alternative.

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Like most microbes, fungi rely on respiration to meet their major energy demands for germination and growth. Therefore, the energy pathway can be an ideal target for fungicide development (Haraguchi, Tanimoto, Tamura, Mizutani, & Kinoshita, 1998; Martins, Dinamarco, Curti, & Uyemura, 2011; Raafat, Von Bargen, Haas, & Sahl, 2008). In fact, flavonoids are one such example. In our previous reports, we also found that the antimicrobial actions of propolis and its active compound pinocembrin could inhibit the mycelial growth of P. italicum by interfering with energy homeostasis and cell membrane integrity (Peng et al., 2012;Yang et al., 2016). To explore the antifungal activity and their action modes of 5,7-dihydroxyflavonoids against P. italicum, 23 kinds of 5,7-dihydroxyflavonoids with different structure characteristics (Table 1) were selected for this study. The inhibitory effects of each 5,7-dihydroxyflavonoid on P. italicum respiration are analyzed and correlated with their structure.

2.2. Chemicals All the tested flavonoids, including chrysin, pinocembrin, biochanin A, baicalein, galangin, apigenin, genistein, naringenin, luteolin, diosmetin, kaempferide, kaempferol, hesperetin, morin, quercetin, dihydroquercetin, myricetin, dihydromyricetin, rutin, hyperoside, isoquercitrin, myticetrin, genistin (Table 1) were purchased from Aladdin (Shanghai) Reagent Co. (Shanghai, China) and the purity is up to 98% by HPLC. All of the chemicals were dissolved in 80% ethanol to prepare stock solution and the final ethanol concentrations were <1%. 2.3. Antifungal activity assay

2. Materials and methods

To evaluate the antifungal activity of 5,7-dihydroxy flavonoids, inhibitory activity of the tested 5,7-dihydroxy flavonoids on spore germination of P. italicum was determined according to Yang et al. (2011) method (Yang et al., 2011). The inhibitory concentration (IC50) represents as inhibiting spore germination by 50% relative to control.

2.1. Testing strains and spore suspension preparation

2.4. Inhibition of P. italicum respiration by 5,7-dihydroxy flavonoids

P. italicum was isolated from infected citrus fruit with typical blue mold symptoms, and confirmed by morphological characteristics of conidia and their growth feathers on healthy fruits. Spores were from 1-week-old fungal cultures that had been incubated at 26 °C, resuspended in sterile water containing 0.05% (v/v) Tween80 and adjusted to 1  105 spores/ml.

24-h-cultured mycelia from spores at 26 °C in PDB were used to test the inhibition of 5,7-dihydroxy flavonoids on respiration of P. italicum. The mycelia were washed with physiological saline thrice and suspended in 20 mmol/l HEPES-TRIS buffer (containing 250 mmol/l sucrose, 10 mmol/l Tris-HCl, 5 mmol/l KH2PO4, 1 mmol/l MgCl2, pH 7.2), and then each 5,7-dihydroxyflavonoid

Table 1 Structure of 23 selected 5,7-dihygroxy flavonoids.

Compound Chrysin Pinocembrin Biochanin A Baicalein Galangin Apigenin Genistein Naringenin Luteolin Diosmetin Kaempferide Kaempferol Hesperetin Morin Quercetin Dihydroquercetin Myricetin Dihydromyricetin Rutin Hyperoside Isoquercitrin Myticetrin Genistin

2 H

H

H

H H

3

5

6

7

20

30

40

50

H H H H OH H H H H H OH OH H OH OH OH OH OH O-R1 O-R2 O-R3 O-R4 H

OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH

H H H OH H H H H H H H H H H H H H H H H H H H

OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH O-R4

H H H H H H H H H H H H H OH H H H H H H H H H

H H H H H H H H OH OH H H OH H OH OH OH OH OH OH OH OH H

H H OCH3 H H OH OH OH OH OCH3 OCH3 OH OCH3 OH OH OH OH OH OH OH OH OH OH

H H H H H H H H H H H H H H H H OH OH H H H OH H

Note: R1: rhamnose-glucoside; R2-galactoside; R3-glucoside; R4-rhamnoside.

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in a series of concentration was added into the mycelial suspensions and oxygen consumption assay was carried out at 26 °C with a Clark-type oxygen electrode immediately. Respiration state is expressed as the oxygen consumption rate as described previously (Peng et al., 2012). The inhibitory concentration (IC50) represents an inhibiting respiration rate by 50% relative to control.

2.5. Statistical analysis Statistical analysis was performed using SPSS version 9 for Windows (SPSS Inc., Chicago, IL, USA). All assays were carried out in triplicates. The IC50 value were calculated from the graph of inhibition rate (expressed as means ± SD) against concentration of testing compound using Microsoft Excel software. Correlations between IC50 of antifungal activity and respiration rate are determined by linear regression analysis employing Microsoft excel software.

3.2. Effect of hydrogenation of the C2–C3 double bond on the antifungal activity of 5,7-dihydroxyflavonoids The C2@C3 double bond in conjugation with a carbonyl group has been considered as the function group that is attributed to the bioactivity of this group of compounds (Modak, Leonor Contreras, González-Nilo, & Torres, 2005). However, Bylka, Matlawska, and Pilewski (2004) reported that flavonones such as naringenin, taxifolin, and dihy-drokaempferide have no inhibitory activities to Escherichia coli, Staphylococcus aureus and Enterococcus faecalis (Bylka et al., 2004). The molecular structure-activity study found that the hydrogenation of the C2@C3 bond in some flavonoids would decrease the binding affinity for the target proteins (Xiao et al., 2011). In our case, that the IC50 values of 5,7dihydroxyflavonones and 5,7-dihydroxyflavononols on P. italicum are lower than those of 5,7-dihydroxyflavones and 5,7dihydroxyflavonols with C2@C3 double bond, as shown in Table 3 (apignin vs. naringenin, quercetin vs. dihydroquercetin, myricetin vs. dihydromyricetin). Our results supported that the hydrogenation of C2@C3 bond decrease the antifungal activity of 5,7dihydroxyflavonoids.

3. Results and discussion 3.1. Effect of hydroxyl groups of 5,7-dihydroxyflavones on the antifungal activity

3.3. Effect of methylation on the antifungal activity of 5,7dihydroxyflavones

The hydroxyl groups of flavonoids are important for their antimicrobial activities according to Smejkal et al. (2008). Others also noted that the presence of hydroxyl groups 30 ,40 ,50 in ring B of flavonoids is responsible for antimicrobial activities against P. vulgaris and S. aureus (Mori, Nishino, Enoki, & Tawata, 1987), and two flavonoids with 40 -OH from Retama raetam flowers had strong antifungal activity against Candida species (Edziri et al., 2012). As shown in the Table 2, we find that C-40 -OH may improve the antifungal activity of 5,7-dihygroxy flavone against P. italicum according to comparing the activities between chrysin and apigenin or luteolin; baicalin and apigenin. Conversely, the introduction of C-40 -OH decreased the inhibitory activities of 5,7dihygroxyflavonones and 5,7-dihygroxyflavanonol by comparing the IC50 of Naringenin and pinocembrin; dihydroquercetin and dihydromyricetin. From Table 2, we also find that the antifungal activities of 5,7-dihydroxy flavonol with C-3-OH are lower than those of 5,7-dihydroxy flavone without C-3-OH (galangin vs. chysin; kaempferol vs. apigenin; quercetin vs. luteolin), which indicates that the introduction of C-3-OH decreases the activity of 5,7-dihygroxy flavonol against P. italicum. Therefore, C-40 -OH and C-3-OH are active groups for 5,7-dihygroxy flavonoids against P. italicum.

A recent survey on current available chemicals suggested that the main classes of antimicrobial and antiviral flavonoids found in medicinal plants are methylated flavones and flavonols. For example, 5,6,7,8-tetramethoxyflavone, 5,6,7-trimethoxyflavone, 40 -hydroxy-5,6,7,8-tetramethoxyflavone and 40 -hydroxy-5,6,7-trime thoxyflavone, extracted from Zeyheria tuberculosan, have antimicrobial activities against Staphylococcus aureus and Candida albicans (Bastos et al., 2009). Also, 40 -methoxyl group in chalcone seems to be the second requirement for its antibacterial activity. In our study, we find that the methylated 5,7dihydroxyflavonoids did not increase antifungal activity against P. italicum when compared with corresponding 5,7dihydroxyflavonoids without methoxyl group (Table 4). Biochanin A, kaempferide with methoxyl groups at 40 position even showed higher IC50 values than those of naringenin and pinocemcrine with other groups at 40 position. Our results are in agreement with the previous report that the flavonoids in which the hydroxyl was replaced with a methoxy group (kaempferide, tamarixetin) would diminish their antibacterial effectiveness (Xu & Lee, 2001). Those results suggested that methoxylation at 40 position of flavonoids may have different effects upon antifungal activity against P. italicum.

Table 2 Effect of hydroxyl group on IC50 of 5,7-dihygroxy flavonoids against spore germination. Kind

5,7-Dihygroxy flavonoids

Number

Position

IC50

Flavone

Chrysin Baicalein Apigenin Luteolin

2 3 3 4

5,7 5,6,7 5,7,40 5,7,30 ,40

0.4188 0.4664 0.2252 0.3037

Flavonol

Galangin Kaempferol Morin Quercetin Myricetin

4 4 5 5 6

3,5,7 3,5,7,40 3,5,7,20 ,40 3,5,7,30 ,40 3,5,7,30 ,40 ,50

0.4338 0.6754 0.4223 0.3408 0.377

Flavonone

Pinocembrin Naringenin

2 3

5,7 5,7,40

0.1041 0.3159

Flavanonol

Dihydroquercetin Dihydromyricetin

5 6

3,5,7,30 ,40 3,5,7, 30 ,40 ,50

0.4344 0.644

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S. Yang et al. / Food Chemistry 224 (2017) 26–31 Table 3 Effect of hydrogenated C@C bond on IC50 of 5,7-dihydroxyflavonoids against spore germination. 5,7-Dihydroxyflavonoids Apigenin Naringenin Quercetin Dihydroquercetin Myricetin Dihydromyricetin

2 H H H

3

5

6

7

20

30

40

50

IC50

H H OH OH OH OH

OH OH OH OH OH OH

H H H H H H

OH OH OH OH OH OH

H H H H H H

H H OH OH OH OH

OH OH OH OH OH OH

H H H H OH OH

0.2252 0.3159 0.3408 0.4344 0.3770 0.6440

Table 4 Effect of methoxyl group on IC50 of 5,7-dihydroxyflavonoids against spore germination. 5,7-Dihydroxyflavonoids

2

3

5

6

7

20

30

40

50

IC50

Biochanin A Naringenin Pinocembrin Kaempferide Kaempferol Diosmetin Luteolin Hesperetin

H H H

H H H OH OH H H H

OH OH OH OH OH OH OH OH

H H H H H H H H

OH OH OH OH OH OH OH OH

H H H H H H H H

H H H H H OH OH H

OCH3 OH H OCH3 OH OCH3 OH OCH3

H H H H H H H OH

0.424 0.3159 0.1041 0.7619 0.6754 0.2903 0.3037 0.5354

H

3.4. Effect of glycosylation on the antifungal activity of 5,7dihydroxyflavonoids

3.5. Inhibitory effect of 5,7-dihydroxy flavonoids on respiration of P. italicum

The glycosylation of natural products through carbon–carbon bonds is a commonly biochemical reaction that can stabilize the bond between the secondary metabolite and sugar acceptors (Brazier-Hicks et al., 2009). Many flavonoid glycosides with antimicrobial activities have been recently identified from plants such as Galium fissurense Ehrend, Viscum album, Cirsium hypoleucum, the roots of L. alata, Calotropis procera, Pavetta crassipes leaves, Marrubium globosum, leaves of mango (Bello, Ndukwe, Audu, & Habila, 2011; Kanwal, Hussain, Siddiqui, & Javaid, 2009; Nenaah, 2013; Okoth, Chenia, & Koorbanally, 2013; Orhan et al., 2010). In Table 5, the antifungal activities of monoglycosylated 5,7dihydroxyflavonoids are affected by their conjugation sites. A slight decrease of the antifungal activity against P. italicum is seen in monoglycoslation of flavonoid at 3-OH (isoquercitrin vs. quercetin; myticetrin vs. myricetin). On the other hand, the antifungal activity decreased greatly when 7-OH is glycosylated (genistein vs. genistin). From Table 5, class of sugar moiety also has effect on the activity of glycoslated 5,7-dihydroxyflavonoids against the pathogen. Glycosylated quercetin with O-rha-glu shows the strongest inhibitory activity when compared with the glycosylated quercetin with O-gal and O-glu (Rutin vs. hyperoside and isoquercitrin). Waage and Hedin (1985) also reported that quercetin-3-O-rhamnoside exhibited the strongest activity against Pseudomonas maltophilia and Enterobacter cloacae among the quercetin glycosides, while the other quercetin-O-glycosides (3-O-glucoside,3-O-galactoside, 30 -O-galactoside, 3-O-rutinoside, 3-O-galactoglucoside, and 7-O-glucoside) exhibited weak or no activities to these two bacterial species (Waage & Hedin, 1985).

The respiration chain of mitochondria is a major source of Reactive Oxygen Species production in most eukaryotic cells. The balance between Reactive Oxygen Species and antioxidants is very important for cellular activities, as both extremes, oxidative and antioxidative stress, are damaging. Flavonoids are weak acids that possess well recognized antioxidant and prooxidant properties (Poljsak, Šuput, & Milisav, 2013). Interfering respiration is one of the important antimicrobial mechanisms of flavonoids. Some flavonoids such as quercetin and its glycosides and dihydromycetin at high concentrations can decrease respiration rate of mitochondria and ATP synthesis in fungal cells, leading to cell death (Trumbeckaite et al., 2006). However, some flavonoids such as biochanin A, galangin could increase the respiration rate and ATP level of renal proximal tubules cells (Rasbach & Schnellmann, 2008). In order to evaluate the action mode of 5,7-dihydroxyflavonoids against P. italicum, we also measure the respiration rate of P. italicum after treated with 5,7-dihydroxyflavonoids. The results show that most of the 5,7-dihydroxyflavonoids have inhibitory effects on the respiration of P. italicum with IC50 at ranges from 0.3769 mg/ml to 2.8505 mg/ml, which are slightly higher than the corresponding IC50 against spore germination (Table 6). However, no significant positive relationship between IC50 of 5,7dihydroxyflavonoids on spore germination and on respiration rate of P. italicum (y = 1.2929x + 0.6813, R2 = 0.1297). We also find that dihydromyricetin, biochanin A, hyperoside, and apigenin with antifungal activity have no inhibitory or even enhanced effects on the respiration of P. italicum. The diversity of mitochondrial responses for flavonoids was also observed by Poór et al. (2014),

Table 5 Effect of glycosylation on IC50 of 5,7-dihydroxyflavonoids against spore germination. 5,7-Dihydroxyflavonoids Quercetin Hyperoside Isoquercitrin Rutin Myricetin Myticetrin Genistein Genistin

2

3

5

6

7

20

30

40

50

IC50

OH O-gal O-glu O-rha-glu OH O-Rha H H

OH OH OH OH OH OH OH OH

H H H H H H H H

OH OH OH OH OH OH OH O-Rha

H H H H H H H H

OH OH OH OH OH OH H H

OH OH OH OH OH OH OH OH

H H H H OH OH H H

0.3408 0.3383 0.3736 0.1808 0.377 0.4046 0.3632 0.8118

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Table 6 Antifungal activity of 5,7-dihydroxy flavonoids against spore germination and respiration of P. italicum (lmol/ml). Flavonoids

Chrysin Pinocembrin Biochanin A Baicalein Galangin Apigenin Genistein Naringenin Luteolin Diosmetin Kaempferide

IC50

Flavonoids

Spore germination

Respiration

0.4188 0.1041 0.4224 0.4664 0.4338 0.2252 0.3632 0.3159 0.3037 0.2903 0.7619

2.1679 0.6444 – 2.8505 0.3769 – 1.2683 1.2807 1.3030 0.7559 2.3207

in which most of the flavonoids (17 flavonoids) had no effects on ATP synthesis in kidney cells, but diosmetin, apigenin, luteolin increased the intracellular ATP levels (Poór et al., 2014). These results suggest that the action modes of 5,7-dihydroxyflavonoids against P. italicum were diversity and could not be explained simply by an interference in the pathogen’s energy metabolism. 4. Conclusions The results obtained in this study show that all of the 23 selected 5,7-dihydroxyflavonoids have antifungal activities against P. italicum. The sites of hydroxyl group at positions of C-40 -OH and C-3-OH are crucial for antifungal activities of 5,7dihydroxyflavonoids against P. italicum. Hydrogenation of the C2@C3 bond decrease the antifungal activity of 5,7dihydroxyflavonoids. Methoxylation at 40 -postion has no positive effects on the activity of 5,7-dihydroxyflavonoids. However, the conjugation site of glycosylation and the class sugar moiety affect the antifungal activity of 5,7-dihydroxyflavonoids. Most of tested 5,7-dihydroxyflavonoids can suppress the respiration of P. italicum except a few 5,7-dihydroxyflavonoids such as dihydromyricetin, biochanin A, hyperoside, and apigenin that stimulated P. italicum respiration. In summary, we demonstrate that 5,7-dihydroxyflavonoids can serve as potent alternatives against P. italicum and we find further that the antifungal activity is sensitive to the molecular structure. Although some have speculated that the antifungal activity of these compounds is a consequence of the inhibition of respiration, we find that the mechanism of these compounds is more complex. Conflicts of interest The authors declare no conflict of interest. Acknowledgement This research was supported by the National Natural Science Foundation of China (NSFC No. 31271969 and No. 31471633). References Bastos, M. L. A., Lima, M. R. F., Conserva, L. M., Andrade, V. S., Rocha, E. M. M., & Lemos, R. P. L. (2009). Studies on the antimicrobial activity and brine shrimp toxicity of Zeyheria tuberculosa (Vell.) Bur. (Bignoniaceae) extracts and their main constituents. Annals of Clinical Microbiology and Antimicrobials, 8, 16. Bello, I. A., Ndukwe, G. I., Audu, O. T., & Habila, J. D. (2011). A bioactive flavonoid from Pavetta crassipes K. Schum. Organic and Medicinal Chemistry Letters, 1(1), 14. Brazier-Hicks, M., Evans, K. M., Gershater, M. C., Puschmann, H., Steel, P. G., & Edwards, R. (2009). The C-glycosylation of flavonoids in cereals. Journal of Biological Chemistry, 284(27), 17926–17934.

Hesperetin Morin Quercetin Dihydroquercetin Myricetin Dihydromyricetin Rutin Hyperoside Isoquercitrin Myticetrin Genistin

IC50 Spore germination

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0.5354 0.4223 0.3408 0.4344 0.377 0.644 0.1808 0.3383 0.3736 0.4046 0.8118

1.2592 1.2056 0.6913 1.5592 1.2459 – 0.5195 – 1.0533 0.8144 0.8504

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