Relationship between flavonoid structure and reactive oxygen species generation upon ultraviolet and X-ray irradiation

Journal of Photochemistry & Photobiology A: Chemistry 384 (2019) 112044

Contents lists available at ScienceDirect

Journal of Photochemistry & Photobiology A: Chemistry journal homepage: www.elsevier.com/locate/jphotochem

Relationship between flavonoid structure and reactive oxygen species generation upon ultraviolet and X-ray irradiation

T

Lei Jianga, , Emiko Yanaseb, Takashi Moric, Kanae Kuratad, Michiru Toyamae, Ayaka Tsuchiyaf, Kosei Yamauchif, Tohru Mitsunagaf, Hitoshi Iwahashig, Junko Takahashih ⁎

a

United Graduate School of Agricultural Science, Gifu University, Gifu 501-1193, Japan Laboratory of Bio-organic Chemistry, Faculty of Applied Biological Sciences, Gifu University, Gifu 501-1193, Japan c Animal Medical Center, Gifu University, Gifu 501-1193, Japan d Graduate School of Applied Biological Sciences, Gifu University, Gifu 501-1193, Japan e Institute of Radioisotope, Asahi University, Gifu 501-0296, Japan f Laboratory of Natural Products Chemistry, Faculty of Applied Biological Sciences, Gifu University, Gifu 501-1193, Japan g Laboratory of Applied Microbiology, Faculty of Applied Biological Sciences, Gifu University, Gifu 501-1193, Japan h Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba 305-8566, Japan b

ARTICLE INFO

ABSTRACT

Keywords: Free radicals ORAC Non-ionizing and ionizing radiation Radiosensitizer Cancer radiotherapy

In this research, we aimed to reveal structures related to reactive oxygen species (ROS) generation of flavonoids upon ultraviolet (UV) or X-ray irradiation. ROS production of selected flavonoids was studied by using ROS fluorescence probes. We discuss the relationship between the structures of the flavonoids and the generation of three ROSs, superoxide anion radical (O2•−), hydroxyl radical (•OH), and singlet oxygen (1O2). Results showed that 2,3-double bond was an key structure for natural flavonoids to generate O2•−. We also discuss the effects of the ROS scavenging capacities on ROS generation through oxygen radical absorbance capacity (ORAC) tests. Derivatives of quercetin could produce •OH due to differences of ROS scavenging ability. Our results indicated that flavonoids and their derivatives, which were used as antioxidant reagents, can serve as ROS generators upon ultraviolet (UV) or X-ray irradiation.

1. Introduction Polyphenols (or polyhydroxyphenols) are a large class of natural chemicals characterized by the presence of numerous phenol structural units. Polyphenols are divided into several groups, one of which is represented by flavonoids. Polyphenols are widely known as efficient radical scavengers [1–3], which protect cellular structures from radical attack as well as bring about fast chemical repair of free radical damage on biomolecules through H-atom donation or electron transfer. In the case of flavonoids, the number of hydroxy groups in the aromatic B-ring (numbering system, see Scheme 1) directly determines the scavenging activity [3]. On the other hand, polyphenols can serve as reactive oxygen species (ROS) generators in certain situations. When auto-oxidation occurs on polyphenols, they can react with molecular oxygen and produce superoxide anion radicals (O2•−) through a one- or two-step electron

transfer, which produces a pro-oxidant effect [4,5]. Flavonoids can further donate a hydrogen atom from their pyrogallol or catechol structure to O2•− generated during auto-oxidation to produce hydrogen peroxide (H2O2) [6]. It has been reported that essential OH groups, a 2,3-double bond, and a carbonyl group at the 4-position in the C-ring are structural determinants of the pro-oxidant capacity of flavonoids [7,8]. Polyphenols have also been reported to enhance hydroxyl radical (•OH) formation in the presence of H2O2 and Fe3+ and accelerate deoxyribonucleic acid (DNA) damage [9]. Latest research also showed that metal (Fe3+)-phenolic networks could degradate H2O2 into reactive species, which is useful for water clean-up [10]. In 2018, Toyama et al. found that 3′,4′,5,7-tetrahydroxyflavone (luteolin) can serve as a ROS generator (only O2•−) under X-ray and ultraviolet (UV) irradiation [11]. Recent reports showed that ROS generation of natural chemical has a possibility to improve traditional radiotherapy. 5-Aminolevulinic acid

Corresponding author. E-mail addresses: [email protected] (L. Jiang), [email protected] (E. Yanase), [email protected] (T. Mori), [email protected] (K. Kurata), [email protected] (M. Toyama), [email protected] (A. Tsuchiya), [email protected] (K. Yamauchi), [email protected] (T. Mitsunaga), [email protected] (H. Iwahashi), [email protected] (J. Takahashi). ⁎

https://doi.org/10.1016/j.jphotochem.2019.112044 Received 12 July 2019; Received in revised form 18 August 2019; Accepted 20 August 2019 Available online 20 August 2019 1010-6030/ © 2019 Elsevier B.V. All rights reserved.

Journal of Photochemistry & Photobiology A: Chemistry 384 (2019) 112044

L. Jiang, et al.

powerful than UV light [16], and it can break chemical bonds so called ionization capacity [17]. We found significant relationships between flavonoid structure and ROS generation ability. This paper reports and discusses these relationships. We limited our examination to three dominant species, •OH, O2•−, and singlet oxygen (1O2), which are the most likely to affect cell viability [18]. For a long time, flavonoids are used as antioxidants in various fields, such as food industry and cosmetic industry. This work aimed to reveal the direct oxidant ability (ROS generation) of flavonoids and it will be beneficial to develop novel function of flavonoids as radiosensitizers. In addition, studies on structure relationships were also useful for deeply understanding of ROS generation of polyphenols. 2. Materials and methods Scheme 1. Flavonoid numbering system.

2.1. Materials and instruments

(ALA) is a natural chemical that can serve as a photosensitizer in photodynamic therapy (PDT) because it causes preferential accumulation of protoporphyrin IX (PpIX) in tumor cells. ROS production from PpIX under X-ray irradiation was well confirmed in previous studies and ALA has been reported to be a potential radiosensitizer for radiotherapy [12,13]. Recent research had confirmed the effect of ALA on developing traditional radiotherapy by using animal experiments [14]. As increasing oxidative stresses caused by ROSs induces cancer cell death during radiotherapy [15], it would be meaningful to reveal the ROS generation capacities of flavonoids and their derivatives because they are also potential radiosensitizers. In addition to luteolin, there are many flavonoids; they are widely distributed in nature, and some flavonoids have similar structures to luteolin. These species were considered to produce ROSs. We focused on various flavonoids (Scheme 1, Table 1, see supporting information, Fig. S1) and attempted to determine their ROS generation ability. In this research, 14 basic flavonoids (mainly flavonol, dihydroflavonol, flavone, and flavanone types, compounds 1–14) were selected and their structures and ROS producing activities were evaluated. The ROS productions of several analogues or derivatives was also investigated (compounds 15–19). In addition, ROS production under long-wavelength UV (365 nm) radiation and short-wavelength X-ray (< 0.005 nm) were detected. Both UV light and X-ray are electromagnetic waves; however, X-ray is ionizing radiation while UV light is non-ionizing radiation. X-ray has a higher frequency and are more

Chemicals: Protoporphyrin IX (PpIX, purity > 95% by HPLC), galangin (purity > 95% by HPLC) and (+)-Catechin (purity > 98% by HPLC) were purchased from Sigma Aldrich (USA). Quercetin (purity > 95% by HPLC), kaempferol (purity > 95% by HPLC), apigenin (purity > 95% by HPLC), rutin (purity > 95% by HPLC), and hematoporphyrin (HP, purity > 45% by HPLC, a kind of photosensitizer which can generate 1O2 under UV [19]) were purchased from Wako Pure Chemical Industries Ltd. (Japan). Fisetin (purity > 96% by HPLC), 3-hydroxyflavone (purity > 98% by HPLC), 3′,4′,5,7-tetrahydroxyflavone (luteolin, purity > 98%) and morin hydrate (purity > 90% by HPLC) were purchased from Tokyo Chemical Industry Co., Ltd. (Japan). 3,5-Dihydroxyflavone (purity > 99% by HPLC) was purchased from Indofine Chemical Company, Inc. (USA). (-)-epigallocatechin-3gallate (purity > 94% by HPLC) was purchased from Taiyo Kagaku Co., Ltd. (Japan). Robinetin (purity > 99% by HPLC) and fustin (purity > 99% by HPLC) were purchased from Extrasynthese (France). Naringenin (purity > 95% by HPLC) was purchased from Nacalai Tesque, Inc. (Japan). Myricetin (purity > 99% by HPLC) was prepared from myricitrin via an acid hydrolysis reaction and myricitrin was isolated and purified from the bark of Myrica rubra Siebold et Zucc [20]. (+)-Taxifolin (purity > 99% by HPLC) was isolated and purified from larch heartwood [21]. The synthesis method for 3′,4′,5,7-tetra-O-benzylquercetin (purity > 99% by HPLC) was as described in a previous report. After the benzylation of rutin, the resulting product was hydrolyzed using HCl [22]. Using a similar method, rutin was methylated

Table 1 Structural differences of natural chemical samples. Number

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Ring Carbon

Myricetin Quercetin Morin Kaempferol Galangin Robinetin Fisetin 3,5-Dihydroxyflavone 3-Hydroxyflavone Luteolin Apigenin Taxifolin Fustin Naringenin Catechin 3’,4’,5,7-Tetra-O-methylquercetin 3’,4’,5,7-Tetra-O-benzylquercetin Rutin Epigallocatechin-3-gallate

A

C

B

C5

C7

C2-C3

C3

C4

C2’

C3’

C4’

C5’

OH OH OH OH OH H H OH H OH OH OH H OH OH OMe OBn OH OH

OH OH OH OH OH OH OH H H OH OH OH OH OH OH OMe OBn OH OH

= = = = = = = = = = = – – – – = = = –

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

C=O C=O C=O C=O C=O C=O C=O C=O C=O C=O C=O C=O C=O C=O CH2 C=O C=O C=O CH2

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

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

OH OH OH OH H OH OH H H OH OH OH OH OH OH OMe OBn OH OH

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

2

OH OH OH ORut O-gallate

OH OMe OBn OH OH

Journal of Photochemistry & Photobiology A: Chemistry 384 (2019) 112044

L. Jiang, et al.

using excess dimethyl sulfate and then hydrolyzed to obtain 3’,4’,5,7tetra-O-methylquercetin (purity > 99% by HPLC) [23]. Reagents: Dihydroethidium (DHE) was purchased from Wako Pure Chemical Industries Ltd. (Japan). Aminophenyl fluorescein (APF) was purchased from Goryo Chemical, Inc. (Japan). Singlet oxygen sensor green (SOSG) was purchased from Thermo Fisher Scientific (USA). Superoxide dismutase (SOD), N,N-dimethylformamide (DMF), 99.5% EtOH, hydrogen peroxide (30% H2O2), and methanol were purchased from Wako Pure Chemical Industries Ltd. (Japan). Sodium azide (NaN3) was purchased from Kishida Chemical Co., Ltd. (Japan). Sodium hypochlorite pentahydrate (NaClO·5H2O), cobalt(II) chloride hexahydrate (CoCl2·6H2O), and xanthine were purchased from Tokyo Chemical Industry Co., Ltd. (Japan). Xanthine oxidase (XOD) from bovine milk was ordered from Sigma Aldrich (USA). Equipment: The UV lamp (SLUV-6, AS ONE, Japan) had a wavelength of 365 nm. The UV lamp intensity, as measured using a UV light meter (UV-340A, Lutron Electronic Enterprise), was 0.50 mW/m2 0.2 m below the lamp. In this research, we put plates 13 cm below the UV lamp. Exposure time of 60 min for UV corresponded to total 0.14 mJ per well. X-rays were generated from a linear accelerator (Primus MidEnergy, Toshiba). The X-ray absorption dose rate was measured using an X-ray dosimeter (RAMTEC Smart, Toyo Medic) to be 240.5 cGy/min 1.0 m from the source (exposure time of 3.7 min for X-ray dose of 9 Gy, total 1.8 mJ per well).

Table 3 Evaluation methods of specific ROS scavenging capacity.

All chemicals were dissolved in DMF as 8 mM stock solutions and diluted by 400 times with phosphate-buffered saline (PBS) before use. DHE, APF, and SOSG was used to detect superoxide radicals (O2•−), hydroxyl radicals (•OH), and singlet oxygen (1O2), respectively. For specific detection of those radicals, SOD (30 U/mL), EtOH (40%), and NaN3 (4 mM) were used as quenchers (Table 2) [11]. Fluorescence quenching by quenchers were used for confirming the generation of specific ROSs. DHE was dissolved in DMF as a 25 mM stock solution and diluted to a final concentration of 20 μM using PBS. APF was dissolved in DMF as a 5 mM stock solution and diluted to a final concentration of 5 μM. SOSG was dissolved in methanol as a 5 mM stock solution and diluted to a final concentration of 5 μM. In a 96-well black plate, 100 μL of chemical and probe were mixed, and the fluorescence intensity after UV or X-ray irradiation was determined. Non-irradiated chemicals served as controls. Wavelengths of 485/610, 490/514, and 500/530 nm were used for DHE, APF, and SOSG, respectively. The fluorescence intensities were expressed in arbitrary units (arb. unit).

DHE APF SOSG

SOD EtOH NaN3

1). Superoxide anion 2). Hydroxyl radical 3). Singlet oxygen

SOD EtOH NaN3

DHE APF SOSG

[24,25] [3,26] [27,28]

Xanthine + XOD H2O2 + CoCl2 H2O2 + NaClO

3. Results 3.1. Production of individual ROSs by each chemical 3.1.1. O2•− production To understand structural contribution to O2•− production of flavonoids under UV and X-ray, we estimated the production using DHE as a fluorescence probe. DHE is known as a fluorescence probe that is mainly sensitive to O2•−, however it also has little sensitivities to •OH and hydroperoxyl radicals (protonated form of O2•− [29]), O2•− generation could be characterized with or without its quencher, SOD [11,12]. We obtained O2•− production by comparing with non-irradiated group, so we could compare relationships between O2•− production and structure of flavonoids. Fig. 1(a) shows the DHE fluorescence intensities of chemicals under UV. Quercetin (2), robinetin (6) and 3’,4’,5,7-tetra-O-methylquercetin (16) had relatively high fluorescence intensities among all chemicals.

Table 2 Detection methods of specific ROSs.

Superoxide anion Hydroxyl radical Singlet oxygen

Ref.

Mean values of the fluorescence intensities of probes corresponding to the amounts of generated ROS were statistically evaluated using Student’s t-test.

The scavenging capacities for individual ROSs were determined using modifications of the ORAC, which was published in previous papers [3,24–28]. In our study, we evaluated scavenging capacities for individual ROSs by comparison with known scavengers (Table 3). Reagent preparation: Solutions of all chemicals and fluorescent probes were prepared at twice the concentration of those shown in section 2.2 and 50 μL was added to each well, so that the final concentration would be the same.

Quncher

Probe

2.4. Statistics

2.3. ROS scavenging capacities of chemicals for individual ROSs

Probes

Scavenger

1) A xanthine solution was prepared by dissolving 0.015 g of xanthine in 5 mL of 0.1 N sodium hydroxide and 95 mL of the buffer. Ten milliliters of a 0.125 unit/mL XOD solution was prepared using the buffer solution and kept in an ice bath. SOD powder was dissolved in PBS to prepare a 30 U/mL stock solution. First, we added 50 μL of the xanthine solution, each chemical (or the SOD solution), and the DHE solution to a 96-well black plate, and then we added the XOD solution to initiate the reaction. The total liquid volume in each well was 200 μL. The chemicals were analyzed using a plate reader after a reaction time of 15 min (dark environment, room temperature). 2) A 1.1 M hydrogen peroxide solution was prepared by diluting a 30% H2O2 solution (8.8 M) with distilled water. A cobalt solution was prepared by dissolving 23.8 mg (10 mM) of cobalt(II) chloride hexahydrate and 20 mg of picolinic acid (promoting the dissolution of cobalt(II) salts) in 10 mL of distilled water. A 10% EtOH solution was prepared from 99.5% EtOH by diluting with PBS. First, we added 50 μL of the H2O2 solution, each chemical (or the EtOH solution), and the APF solution to a 96-well black plate, and then added the cobalt solution to initiate the reaction. The total liquid volume in each well was 200 μL. The chemicals were analyzed using a plate reader after a reaction time of 30 min (dark environment, room temperature). 3) We chose to use a 100 mM H2O2 solution, so we prepared a 400 mM stock solution in PBS and added 50 μL to the 200 μL reaction system. We chose to use a 1 mM NaClO solution, so we prepared a 4 mM stock solution in PBS and added 50 μL to the 200 μL reaction system. A 4 mM stock solution of NaN3 was prepared by dissolving it in distilled water. First, we added 50 μL of the H2O2 solution, each chemical (or the NaN3 solution), and the SOSG solution to a 96-well black plate, and then added the NaClO solution to initiate the reaction. The total liquid volume in each well was 200 μL. The chemicals were analyzed using a plate reader after a reaction time of 15 min (dark environment, room temperature).

2.2. Detection method for ROSs after exposure of chemicals to UV and Xray radiation

ROS species

Free Radical Generation System

3

Journal of Photochemistry & Photobiology A: Chemistry 384 (2019) 112044

L. Jiang, et al.

Fig. 1. O2•− production of chemicals 1–19. Fluorescence intensities after mixing 100 μL of each 20 μM chemical and 100 μL of 50 μM DHE. (a) Fluorescence intensity after exposure to UV radiation for 20, 40, and 60 min. Red numbers in parentheses indicate number of additional aromatic hydroxy groups besides 3-OH group. Blue lane shows the difference of O2•− production as the structure changes. (b) Fluorescence intensity after exposure to X-rays (9 Gy). C represents control and was a 0.25% DMF solution with PBS buffer (blank). PpIX was used as a positive control. The number indicated in the figure corresponding to chemicals shown in text and Table 1. Error bars show the standard deviation (s.d.). * 0.01 < p < 0.05, ** p < 0.01.

3-hydroxyflavone (9) and apigenin (11) ˜ catechin (15) did not increased significantly compared with that of control. When exposed to UV, several trends regarding the relationships between the structures of basic flavonoids (compounds 1–14) and O2•− production were discovered (Fig. 1(a)):

Their fluorescence intensities were 4, 5.3 and 4.6 times that of control (60 min) respectively, meanwhile they were lower than that of PpIX. Fluorescence intensity of fisetin (7) followed those three chemicals, 3.3 times that of control (60 min). Myricetin (1), morin (3) ˜ galangin (5), 3,5-dihydroxyflavone (8), luteolin (10) and 3’,4’,5,7-tetra-O-benzylquercetin (17) ˜ epigallocatechin-3-gallate (19) had relatively lower fluorescence intensities. On the other hand, fluorescence intensities of 3-hydroxyflavone (9) and apigenin (11) ˜ catechin (15) did not show a significant increasing compared with that of control. Fig. 1(b) showed the DHE fluorescence intensities of chemicals under X-ray. Quercetin (2), robinetin (6), fisetin (7) and 3’,4’,5,7-tetraO-methylquercetin (16) had higher fluorescence intensities than that of PpIX. Their fluorescence intensities were 3.1, 2.6, 2.6 and 2.8 times that of control, respectively. Myricetin (1), morin (3) ˜ galangin (5), 3,5dihydroxyflavone (8), luteolin (10) and 3’,4’,5,7-tetra-O-benzylquercetin (17) ˜ epigallocatechin-3-gallate (19) had relatively lower fluorescence intensities under X-ray. In addition, fluorescence intensities of

1) When the DHE fluorescence of quercetin (2) and taxifolin (12) and of fisetin (7) and fustin (13) were compared, it was found that the lack of a 2,3-double bond led to inefficient O2•− generation for taxifolin (12) and fustin (13). In addition, naringenin (14) also yielded no O2•−. Thus, 2,3-double bonds are essential for O2•− generation by flavonoids. 2) Flavonoids could produce more O2•− when they had four additional aromatic hydroxy groups (in addition to the 3-OH group). The red numbers in parentheses in Fig. 1(a) show the number of additional aromatic hydroxy groups besides the 3-OH group, and blue lane in Fig. 1(a) showed the difference of O2•− production as the structure 4

Journal of Photochemistry & Photobiology A: Chemistry 384 (2019) 112044

L. Jiang, et al.

fluorescence probe. SOSG was used for specific detection of 1O2, NaN3 was known as a 1O2 quencher. SOSG with or without NaN3 were used for characterizing 1O2 [11,12]. We obtained 1O2 production by comparing with non-irradiated group. The fluorescence intensity in Fig. 3 indicated the 1O2 production of each chemical. Fig. 3(a) showed the SOSG fluorescence intensities of chemicals under UV. No chemical showed significant fluorescence intensity in this condition. SOSG fluorescence intensities of chemicals under X-ray were showed in Fig. 3(b). 3’,4’,5,7-tetra-O-methylquercetin (16) and 3′,4′,5,7-tetra-O-benzylquercetin (17) showed higher fluorescence intensities than that of hematoporphyrin (HP). Fluorescence intensities of galangin (5), 3,5-dihydroxyflavone (8) and 3-hydroxyflavone (9) were slightly higher than that of control. On the other hand, other chemicals showed lower fluorescence intensities than that of control. All chemicals were inefficient in 1O2 production under UV irradiation (Fig. 3(a)). Thus, UV exposure was not powerful enough to promote 1O2 generation by each chemical. In the case of 9 Gy X-rays, galangin (5), 3,5-dihydroxyflavone (8) and 3-hydroxyflavone (9) displayed low 1O2 production (Fig. 3(b)). Meanwhile, 3’,4’,5,7-tetra-O-methylquercetin (16) and 3′,4′,5,7-tetra-O-benzylquercetin (17) were efficient in 1O2 generation. 1 O2 can hardly be directly prepared from ground-state molecular oxygen by light irradiation because of spin-forbidden. In the case of UV irradiation, photosensitizers are needed [31]. High energy of X-ray makes the transition of ground-state molecular oxygen to 1O2 possible. Low 1O2 production of galangin (5), 3,5-dihydroxyflavone (8) and 3hydroxyflavone (9) under X-ray were considered resulting from diffusion controlled limit once 1O2 generated by radiolysis of ambient oxygen molecular. High 1O2 productions of 3’,4’,5,7-tetra-O-methylquercetin (16) and 3′,4′,5,7-tetra-O-benzylquercetin (17) indicate that they can serve as sensitizers to transfer energy from X-ray to groundstate molecular oxygen to form 1O2.

changes. For example, quercetin (2), robinetin (6), and luteolin (10) displayed better O2•− production compared to other compounds with similar structures but different additional aromatic OH groups. 3) Comparison between quercetin (2), luteolin (10), and rutin (18) and between kaempferol (4) and apigenin (11) revealed that the existence of the 3-OH group made quercetin (2) and kaempferol (4) much more efficient in O2•− generation. However, luteolin (10) showed significant O2•− generation without a 3-OH group. Thus, the 3-OH group is not essential but serves as a booster. 4) Catechin-type catechin (15) was inefficient in O2•− generation as expected because of its lack of a 2,3-double bond, 3-OH group, and even 4-carbonyl. Meanwhile, its analogue, (-)-epigallocatechin-3gallate (19), showed poor O2•− generation under UV irradiation. 5) Comparison of quercetin (2), 3’,4’,5,7-tetra-O-methylquercetin (16), and 3′,4′,5,7-tetra-O-benzylquercetin (17) revealed that methoxy groups have the same function as aromatic OH groups. However, the benzylation of aromatic OH groups decreased the O2•− production. On the other hand, similar conclusions could be drawn following exposure to 9 Gy X-ray radiation (Fig. 1(b)). Based on the control, the O2•− generation abilities of the chemicals due to X-ray irradiation and UV irradiation showed similar tendencies, except for those of fisetin (7). Different ionization capacities of UV light and X-rays were thought to lead to those discrepancies. To form O2•− with oxygen molecules, energy is needed for removing electrons from the neutral flavonoids [30]. These trends presented in Fig. 1 could indicate that quercetin (2) and robinetin (6) which have 2,3-double bond, a 3-OH group and four additional aromatic hydroxy groups needed much lower energy than other chemicals to generate O2•− upon UV and X-ray irradiation. The deficiency of 2,3-double bond or 3-OH group and change of aromatic hydroxy groups, may significantly increase such energy demand, and 2,3-double bond affected the most. Energy demand of fisetin (7) was thought to close to the energy of X-ray irradiation. In addition, quercetin (2), morin (3) and robenetin (6) had the same amount of aromatic OHs, however, they had different O2•− production. Thus, relocation of aromatic OHs could also affect such energy demand.

3.2. Scavenging capacity of individual ROSs of chemicals Polyphenols were known as efficient radical scavengers for many years. However, quercetin (2) and robinetin (6) significantly produced O2•− upon UV or X-ray. To evaluate ROS production of flavonoids more correctly, we have to also characterize scavenging activity under the same concentration that of ROS production. Thus, we attempted to determine the scavenging capacities for individual ROSs of the same concentration as section 3.1 of each chemical by using modifications of the oxygen radical absorbance capacity (ORAC) [3,24–28].

3.1.2. •OH production To understand structural contribution to •OH production of flavonoids under UV and X-ray, we estimated the production using APF as a fluorescence probe. APF is a hydroxyl-radical-sensitive fluorescence probe, EtOH can serve as a •OH quencher. APF with or without EtOH was used for characterizing •OH generation [11,12]. We obtained •OH production by comparing with non-irradiated group. Fig. 2(a) showed the APF fluorescence intensities of chemicals under UV. 3’,4’,5,7-tetra-O-methylquercetin (16) showed a relatively higher fluorescence intensity among all chemicals. APF fluorescence intensities of chemicals under X-ray were showed in Fig. 2(b). 3’,4’,5,7-tetra-O-methylquercetin (16) and 3′,4′,5,7-tetra-Obenzylquercetin (17) showed high fluorescence intensities which 3.4 and 2.5 times that of control. Except for 3’,4’,5,7-tetra-O-methylquercetin (16), all chemicals were inefficient in •OH generation under UV irradiation (Fig. 2(a)). The results were similar in the case of 9 Gy X-ray irradiation (Fig. 2(b)); however, high •OH production by 3′,4′,5,7-tetraO-benzylquercetin (17) could be observed under the X-ray conditions. 3′,4′,5,7-tetra-O-benzylquercetin (17) showed a trend that it was more active in •OH generation under X-ray. It was known that ionizing radiation was more powerful than non-ionizing radiation [16]. Thus, this chemical needed high energy to generate •OH.

3.2.1. O2•− scavenging capacity of flavonoid To determine O2•− scavenging capacity, we used xanthine and XOD system to generate O2•− artificially and SOD as a O2•− scavenger [24,25]. Comparing with data of control and SOD, we could evaluate O2•− scavenging capacity of each chemical. Fig. 4 showed that chemical quercetin (2) ˜ galangin (5), fisetin (7), 3,5-dihydroxyflavone (8), and 3’,4’,5,7-tetra-O-methylquercetin (16) enhanced the DHE fluorescence intensities when compared to the control. Fluorescence intensities of other chemicals didn’t significantly decrease compared with that of control. Thus, every chemical at the final concentration of 10 μM showed almost no O2•− scavenging capacity. To reveal the reason of inefficiency of those chemicals in O2•− scavenging, we also attempted to evaluate spontaneous O2•− generation by the chemicals, xanthine, and XOD (see supporting information, Fig. S2). Myricetin (1) and robinetin (6) generated small amounts of O2•− spontaneously without any irradiation. Xanthine had no significant effect while XOD enhanced the fluorescence intensities of myricetin (1) and robinetin (6). In Fig. 1, myricetin (1) ˜ 3,5-dihydroxyflavone (8) and 3’,4’,5,7tetra-O-methylquercetin (16) had apparent O2•− production both under

3.1.3. 1O2 production To understand structural contribution to 1O2 production of flavonoids under UV and X-ray, we estimated the production using SOSG as a

5

Journal of Photochemistry & Photobiology A: Chemistry 384 (2019) 112044

L. Jiang, et al.

Fig. 2. •OH production of chemicals 1–19. Fluorescence intensities after mixing 100 μL of each 20 μM chemical and 100 μL of 10 μM APF. (a) Fluorescence intensity after exposure to UV radiation for 20, 40, and 60 min. (b) Fluorescence intensity after exposure to X-rays (9 Gy). C represents control and was a 0.25% DMF solution with PBS buffer (blank). PpIX was used as a positive control. Error bars show the standard deviation (s.d.). * 0.01 < p < 0.05, ** p < 0.01.

UV and X-ray. According to the results in Fig. 1, Fig. 4 and Fig. S2, simple comparison is difficult, O2•− generation is enhanced by some chemicals under irradiation. Furthermore, for some chemicals, the degree of radical generation does not change under irradiation and dark conditions.

scavenging capacity, even better than 10% EtOH. Although myricetin (1) had the strongest and 3-hydroxyflavone (9) had the weakest •OH scavenging ability, there were no obvious connection between •OH scavenging capacity and number of OH groups at concentration of 10 μM. Both methylation and benzylation of aromatic OH groups significantly decreased •OH scavenging ability (3’,4’,5,7tetra-O-methylquercetin (16) and 3′,4′,5,7-tetra-O-benzylquercetin (17)). In addition, we found deficiency of 2,3-double bond of flavonoids made them lower active in •OH scavenging no matter how many OH groups they have (taxifolin (12), fustin (13) and naringenin (14)). According to results in section 3.1.1, taxifolin (12), fustin (13) and naringenin (14) also had no activity in O2•− production. Thus, we considered 2,3-double bond is an important structure which takes part in both production and scavenging reactions of flavonoids. Quercetin (2) had a higher •OH scavenging capacity than 3’,4’,5,7tetra-O-methylquercetin (16) and 3′,4′,5,7-tetra-O-benzylquercetin (17). It is known that phenolic OH groups are major •OH scavengers [3]. 3’,4’,5,7-tetra-O-methylquercetin (16) and 3′,4′,5,7-tetra-O-

3.2.2. •OH scavenging capacity of flavonoid To determine •OH scavenging capacity, we used H2O2 and CoCl2·6H2O (instead of cobalt(II) fluoride tetrahydrate) reaction system to artificially produce •OH and 10% ethanol (EtOH) as a •OH scavenger [3,26]. We evaluated •OH scavenging capacity of each chemical by comparing their data with those of the control and EtOH. Fig. 5 showed all chemicals lead the decreasing of APF fluorescence intensities. Fluorescence intensities decreasing of the chemicals were in the order 1 > EtOH > 3 > 2 > 7 > 10 > 6 > 19 > 15 > 8 > 4 = 5 > 13 > 14 > 18 > 17 > 11 > 16 > 12 > 9. All chemicals were active in •OH scavenging. Myricetin (1) decreased the most fluorescence intensity. Thus, it had the strongest •OH 6

Journal of Photochemistry & Photobiology A: Chemistry 384 (2019) 112044

L. Jiang, et al.

Fig. 3. 1O2 production of chemicals 1–19. Fluorescence intensities after mixing 100 μL of each 20 μM chemical and 100 μL of 10 μM SOSG. (a) Fluorescence intensity after exposure to UV radiation for 20, 40, and 60 min. (b) Fluorescence intensity after exposure to X-rays (9 Gy). C represents control and was a 0.25% DMF solution with PBS buffer (blank). Hematoporphyrin (HP) was used as a positive control. Error bars show the standard deviation (s.d.). * 0.01 < p < 0.05, ** p < 0.01.

Fig. 4. O2•− scavenging capacity of each chemical. Fluorescence intensities after reaction time of 15 min. C represents control and was a 0.25% DMF solution with PBS buffer (blank) containing the xanthine and XOD reaction system. SOD was used as a negative control. Error bars show the standard deviation (s.d.). * 0.01 < p < 0.05, ** p < 0.01.

7

Journal of Photochemistry & Photobiology A: Chemistry 384 (2019) 112044

L. Jiang, et al.

Fig. 5. •OH scavenging capacity of each chemical. Fluorescence intensities after reaction time of 30 min. C represents control and was a 0.25% DMF solution with PBS buffer (blank) containing a H2O2 and CoCl2·6H2O reaction system. EtOH was used as a negative control. Error bars show the standard deviation (s.d.). * 0.01 < p < 0.05, ** p < 0.01.

Lower 1O2 productions than control of chemicals are assumed to be due to differences in the radical scavenging abilities (Fig. 3(b)).

benzylquercetin (17) are derived from quercetin (2) through etherification and have no free phenolic OH groups; therefore, •OH scavenging ability was assumed to be lacking. Thus, 3’,4’,5,7-tetra-O-methylquercetin (16) and 3′,4′,5,7-tetra-O-benzylquercetin (17) could generate •OH under UV or X-ray irradiation, but quercetin (2) could not (Fig. 2). Quercetin (2) was thought to generate •OH as well as 3’,4’,5,7tetra-O-methylquercetin (16) and 3′,4′,5,7-tetra-O-benzylquercetin (17) do, but the generated •OH were not observed because they are scavenged immediately.

4. Discussion Natural flavonoids didn’t show activities in •OH and 1O2 generation. However, 3’,4’,5,7-tetra-O-methylquercetin (16) which was derived from quercetin (2) could serve as an effective •OH and 1O2 generator under both UV and X-ray irradiation. 2,3-double bond, 3-OH group, and number of aromatic hydroxy groups were closely related to the O2•− generation capacities of flavonoids (Table 4). Natural flavonoids with 2,3-double bond, 3-OH and a certain number of aromatic OH groups showed O2•− generation activities. Four aromatic OH groups made flavonoids relatively easier to produce O2•−. Based on our data, we proposed our own hypothesis regarding how flavonoids generate three types of ROSs. Flavonoids with a 2,3-double bond and 3-OH group contribute on the generation of O2•−. This can be explained through the speculation that when exposed to UV light or Xrays, the 2,3-double bond and 3-OH group have the functions of absorbing energy. Energy from UV or X-ray can activate the 2,3-double bond to donate one electron to O2 to produce O2•− while the 3-OH group is separated from the flavonoid molecule to form •OH at the same time. Compound luteolin (10) produced low amounts of O2•− without the help of a 3-OH group; perhaps aromatic OH groups undergo similar reactions, but with much more difficulty than the 3-OH group does. Aromatic OH groups have strong •OH scavenging capacity. Thus, we could only detect •OH generated by the derivatives of quercetin (2), 3’,4’,5,7-tetra-O-methylquercetin (16) and 3′,4′,5,7-tetra-O-

3.2.3. 1O2 scavenging capacity To determine 1O2 scavenging capacity, a H2O2 and NaClO·5H2O reaction system was used to artificially produce 1O2. NaN3 was used as a 1 O2 scavenger [27,28]. We evaluated 1O2 scavenging capacity of each chemical by comparing their data with those of the control and NaN3. Fig. 6 showed that all chemicals decreased SOSG fluorescence intensities very slightly. The fluorescence intensities decreasing of the chemicals were in the order NaN3 > 13 > 1 > 10 > 3 > 16 > 8 > 18 > 19 > 5 > 4 > 6 > 2 > 17 > 7 > 9 > 11 > 12 > 15 > 14. We found that all chemicals only showed a slight 1O2 scavenging capacity, which was much lower than NaN3. Results indicated that flavonoids were not efficient 1O2 scavengers at concentration of 10 μM. There were also no significant relationships between 1O2 scavenging ability and structure. Interestingly, although 3’,4’,5,7-tetra-O-methylquercetin (16) and 3′,4′,5,7-tetra-O-benzylquercetin (17) could generate 1O2 under X-rays (Fig. 3(b)), 3′,4′,5,7-tetra-O-benzylquercetin (17) had a similar 1O2 scavenging capacity to that of quercetin (2), while that of 3’,4’,5,7-tetra-O-methylquercetin (16) was much higher.

Fig. 6. 1O2 scavenging capacity of chemicals 1–19. Fluorescence intensities after reaction time of 15 min. C represents control and was a 0.25% DMF solution with PBS buffer (blank) containing a H2O2 and NaClO·5H2O reaction system. NaN3 was used as a negative control. Error bars show the standard deviation (s.d.). * 0.01 < p < 0.05, ** p < 0.01.

8

Journal of Photochemistry & Photobiology A: Chemistry 384 (2019) 112044

L. Jiang, et al.

Table 4 Summary of results (++: relatively high production; +: relatively low production; -: almost no effect). Number

Structure

ROS generating capacity UV

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

ROS scavenging rate / % X-ray

2,3-double bond

3-OH

Aromatic OHs

O2•−

•

Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No No No No Yes Yes Yes No

Yes Yes Yes Yes Yes Yes Yes Yes Yes No No Yes Yes No Yes Yes Yes No No

5 4 4 3 2 4 3 1 0 4 3 4 3 3 4 0 (OMe×4) 0 (OBn×4) 4 5

+ ++ + + + ++ + + – + – – – – – ++ + + +

– – – – – – – – – – – – – – – + – – –

OH

1

O2•−

•

1

– – – – – – – – – – – – – – – – – – –

+ ++ + + + ++ ++ + – + – – – – – ++ + + +

– – – – – – – – – – – – – – – ++ ++ – –

– – – – + – – + + – + – – – – ++ ++ – –

O2

benzylquercetin (17). In addition, 3’,4’,5,7-tetra-O-methylquercetin (16) and 3′,4′,5,7-tetra-O-benzylquercetin (17) are thought to generate 1 O2 by energy transfer from X-ray to oxygen molecules.

[5] [6]

5. Conclusion

[7]

Polyphenols are important natural products for humans, and they are recognized by the public because of their antioxidant capacities and other health benefits. Several papers have mentioned anticancer effects resulting from the pro-oxidant effects of polyphenols [9,32]. Our research revealed a direct oxidation effect that comes from the ROS generation capacities of polyphenols. In this work, quercetin (2), robinetin (6), 3’,4’,5,7-tetra-O-methylquercetin (16) and 3′,4′,5,7-tetra-Obenzylquercetin (17) showed high ROS production under X-ray. Our next work will verify their potentials as novel radiosensitizers.

[8] [9]

[10] [11]

Acknowledgments

[12]

X-ray equipment was provided by the Animal Medical Center, Gifu University. This work was partially supported by the Canon Foundation. We would like to thank Editage (www.editage.jp) for English language editing. We would also like to thank Takema Hasegawa and Akihiro Moriyama (United Graduate School of Agricultural Science, Gifu University) for proofreading.

[13] [14] [15] [16]

Appendix A. Supplementary data

[17]

Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.jphotochem.2019. 112044.

[18] [19]

References [20]

[1] J. Baumann, G. Wurm, F.V. von Bruchhausen, Prostaglandin synthetase inhibition by flavonoids and phenolic compounds in relation to their O2•− scavenging properties (author’s translation), Arch Pharm (Weinheim) 313 (1980) 330–337. [2] Y. Sorata, U. Takahama, M. Kimura, Protective effect of quercetin and rutin on photosensitized lysis of human erythrocytes in the presence of hematoporphyrin, Biochim. Biophys. Acta 799 (1984) 313–317. [3] S.R. Husain, J. Cillard, P. Cillard, Hydroxyl radical scavenging activity of flavonoids, Phytochemistry 26 (1987) 2489–2491. [4] K. Brudzynski, L. Maldonado-Alvarez, Polyphenol-protein complexes and their

[21] [22]

9

OH

O2

O2•−

•

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 SOD (70)

51 41 45 31 31 37 39 32 20 38 27 25 31 30 32 27 29 29 36 EtOH (47)

OH

1

O2

26 17 26 18 19 18 15 21 10 26 9 7 31 3 4 22 17 20 19 NaN3 (74)

consequences for the redox activity, structure and function of honey. a current view and new hypothesis – a review, Pol. J. Food Nutr. Sci. 65 (2015) 71–80. D. Metodiewa, A.K. Jaiswal, N. Cenas, E. Dickancaité, J. Segura-Aguilar, Quercetin may act as a cytotoxic prooxidant after its metabolic activation to semiquinone and quinoidal product, Free Radic. Biol. Med. 26 (1999) 107–116. Y.H. Miura, I. Tomita, T. Watanabe, T. Hirayama, S. Fukui, Active oxygens generation by flavonoids, Biol. Pharm. Bull. 21 (1998) 93–96. W. Bors, W. Heller, C. Michel, M. Saran, Flavonoids as antioxidants: determination of radical scavenging efficiencies, Meth. Enzymol. 186 (1990) 343–355. C.A. Rice-Evans, J. Miller, G. Paganga, Antioxidant properties of phenolic compounds, Trends Plant Sci. 2 (1997) 152–159. M.J. Laughton, B. Halliwell, P.J. Evans, J.R.S. Hoult, Antioxidant and pro-oxidant actions of the plant phenolics quercetin, gossypol and myricetin: effects on lipid peroxidation, hydroxyl radical generation and bleomycin-dependent damage to DNA, Biochem. Pharmacol. 38 (1989) 2859–2865. B.L. Tardy, J.J. Richardson, J. Guo, J. Lehtonen, M. Ago, O.J. Rojas, Lignin nanoand microparticles as template for nanostructured materials: formation of hollow metal-phenolic capsules, Green Chem. 20 (2018) 1335–1344. M. Toyama, T. Mori, J. Takahashi, H. Iwahashi, Luteolin as reactive oxygen generator by X-ray and UV irradiation, Radiat. Phys. Chem. Oxf. Engl. 146 (2018) (1993) 11–18. J. Takahashi, M. Misawa, Characterization of reactive oxygen species generated by protoporphyrin IX under X-ray irradiation, Radiat. Phys. Chem. Oxf. Engl. 78 (2009) (1993) 889–898. J. Takahashi, M. Misawa, M. Murakami, T. Mori, K. Nomura, H. Iwahashi, 5Aminolevulinic acid enhances cancer radiotherapy in a mouse tumor model, Springerplus 2 (2013) 602. J. Takahashi, M. Murakami, T. Mori, H. Iwahashi, Verification of radiodynamic therapy by medical linear accelerator using a mouse melanoma tumor model, Sci. Rep. 8 (2018) 2728. A. Glasauer, N.S. Chandel, Targeting antioxidants for cancer therapy, Biochem. Pharmacol. 92 (2014) 90–101. R. Sommer, W. Pribil, S. Appelt, P. Gehringer, H. Eschweiler, H. Leth, Inactivation of bacteriophages in water by means of non-ionizing (UV-253.7 nm) and ionizing (gamma) radiation: a comparative approach, Water Res. 35 (2001) 3109–3116. L. Jiang, H. Iwahashi, The roles of radio-functional natural chemicals for the development of cancer radiation therapy, Rev. Environ. Health 34 (2018) 5–12. J. Cadet, J.R. Wagner, DNA base damage by reactive oxygen species, oxidizing agents, and UV radiation, Cold Spring Harb. Perspect. Biol. 5 (2013) a012559. T. Uchino, H. Tokunaga, M. Ando, ProstaglandinE(2) Release, Squalene Monohydroperoxide Production and Cell Toxicity of Skin((2TM)) ZK1301 as a Human Skin Model in the Presence of Haematoporphylin and Ultraviolet-A Irradiation, Toxicol. In Vitro 13 (1999) 483–489. H. Matsuda, M. Yamazaki, K. Matsuo, Y. Asanuma, M. Kubo, Anti-androgenic activity of Myricae Cortex–isolation of active constituents from bark of Myrica rubra, Biol. Pharm. Bull. (2001) 259–263. T. Mitsunaga, I. Abe, M. Kontani, H. Ono, T. Tanaka, Inhibitory effects of bark proanthocyanidins on the activities of glucosyltransferases of Streptococcus sobrinus, J. Wood Chem. Technol. 17 (1997) 327–340. K. Yamauchi, T. Mitsunaga, I. Batubara, Synthesis of quercetin glycosides and their melanogenesis stimulatory activity in B16 melanoma cells, Bioorg. Med. Chem. 22 (2014) 937–944.

Journal of Photochemistry & Photobiology A: Chemistry 384 (2019) 112044

L. Jiang, et al. [23] K. Yamauchi, T. Mitsunaga, M. Inagaki, T. Suzuki, Synthesized quercetin derivatives stimulate melanogenesis in B16 melanoma cells by influencing the expression of melanin biosynthesis proteins MITF and p38 MAPK, Bioorg. Med. Chem. 22 (2014) 3331–3340. [24] R.L. Prior, M. Sintara, T. Chang, Multi-radical (ORACMR5) antioxidant capacity of selected berries and effects of food processing, J. Berry Res. 6 (2016) 159–173. [25] L. Zhang, D. Huang, M. Kondo, E. Fan, H. Ji, Y. Kou, B. Ou, Novel high-throughput assay for antioxidant capacity against superoxide anion, J. Agric. Food Chem. 57 (2009) 2661–2667. [26] B. Ou, M. Hampsch-Woodill, J. Flanagan, E.K. Deemer, R.L. Prior, D. Huang, Novel fluorometric assay for hydroxyl radical prevention capacity using fluorescein as the probe, J. Agric. Food Chem. 50 (2002) 2772–2777. [27] A. Ryu, E. Naru, K. Arakane, T. Masunaga, K. Shinmoto, Cross-linking of collagen by singlet oxygen generated with UV-A, Chem. Pharm. Bull. (Tokyo) 45 (1997)

1243–1247. [28] S. Luqman, K. Fatima, N. Masood, A high throughput spectrophotometric method for singlet oxygen quenching, Protoc. Exch. (2016), https://doi.org/10.1038/ protex.2016.1054. [29] R. Luc, C. Vergely, Forgotten radicals in biology, Int. J. Biomed. Sci. 4 (2008) 255–259. [30] G.C. Justino, A.J. Vieira, Antioxidant mechanisms of Quercetin and Myricetin in the gas phase and in solution–a comparison and validation of semi-empirical methods, J. Mol. Model. 16 (2010) 863–876. [31] B. Marchetti, T.N. Karsili, An exploration of the reactivity of singlet oxygen with biomolecular constituents, Chem. Commun. (Camb.) 52 (2016) 10996–10999. [32] H.Y. Khan, H. Zubair, M.F. Ullah, A. Ahmad, A prooxidant mechanism for the anticancer and chemopreventive properties of plant polyphenols, Curr. Drug Targets 13 (2012) 1738–1749.

10