Ultraviolet B (UVB) Photosensitivities of Tea Catechins and the Relevant Chemical Conversions

molecules

Article Ultraviolet B (UVB) Photosensitivities of Tea Catechins and the Relevant Chemical Conversions

Meng Shi, Ying Nie, Xin-Qiang Zheng, Jian-Liang Lu, Yue-Rong Liang and Jian-Hui Ye *

Tea Research Institute, Zhejiang University, Hangzhou 310058, China; [email protected] (M.S.); [email protected] (Y.N.); [email protected] (X.-Q.Z.); [email protected] (J.-L.L.); [email protected] (Y.-R.L.) * Correspondence: [email protected]; Tel.: +86-571-8898-2704

Academic Editors: Declan P. Naughton and Derek J. McPhee Received: 13 August 2016; Accepted: 6 October 2016; Published: 10 October 2016

Abstract: Ultraviolet B (UVB) photosensitivities of eight catechins were screened. In both water and ethanol, epicatechin (EC, 575 µM) and catechin (C, 575 µM) exhibited low photostabilities under 6 h UVB radiation with the generation of yellow photoproducts, while other catechins (epigallocatechin gallate, epigallocatechin, epicatechin gallate, gallocatechingallate, gallocatechin, catechin gallate) were relatively UVB-insensitive. Photoisomerization and photolysis were two important UVB-induced reactions to EC whereas photolysis was the dominant reaction for C. The inﬂuencing factors of time (2–10 h), solvent (water, ethanol) and substrate concentration (71.875–1150 µM) on UVB-induced chemical conversions of EC and C were investigated, and eight photoproducts were identiﬁed through ultra performance liquid chromatography-diode array detection-tandem mass spectrometry (UPLC-DAD-MS/MS) and 1H nuclear magnetic resonance (1H-NMR analysis). Photolysis reaction involved two pathways, including radical reaction and photo-induced electron transfer reaction. The 2,2-diphenylpicrylhydrazyl (DPPH) scavenging abilities of eight catechins did not change upon 6 h UVB irradiation. EC and C are photosensitive catechins among eight catechins causing deep color.

Keywords: photostability; catechins; photoisomerization; photolysis; photoproducts

1. Introduction Tea catechins are the major components of the tea polyphenols present in tea leaves, which have been associated with many health beneﬁts and functionalities such as anti-radical, anti-bacterial, anticarcinogenic and anti-aging activities [1,2]. They are mainly composed of (−)-epigallocatechin gallate (EGCg), (−)-epigallocatechin (EGC), (−)-epicatechin gallate (ECg), (−)-epicatechin(EC), and their geometric epimers (−)-gallocatechingallate (GCg), (−)-gallocatechin (GC), (−)-catechin gallate (Cg), and (−)-catechin (C). In the past decades, the protective effects of green tea or tea extract against Ultraviolet (UV)-induced skin damage and skin aging have been reported [3–5], leading to the growing interest in utilization of green tea or tea extract for cosmetics and skin creams [6–8]. However, tea catechins or tea extracts inevitably turn brown during storage, which limits their application not only in the cosmetic industry but also in the food industry due to the undesirable dark color. The bioactivity changes of catechins upon browning draw great interest [9,10]. Illumination, temperature and pH value are important influential factors for non-enzymatic browning of catechin-containing products [11]. The stabilities of catechins at different temperatures and pH values have been excessively studied [12–15]. Epimerization, hydrolysis and oxidation/condensation reactions are three important reactions accounting for instability or degradation of catechins with the generation of epimer, gallic acid (GA) and non-gallated catechin moiety, as well as dark polymeric compounds [12–15]. However, these may not represent the photo-induced chemical reactions of tea catechins that are driven by an entirely different energy. Illumination is a nonnegligible environmental

Molecules 2016, 21, 1345; doi:10.3390/molecules21101345 www.mdpi.com/journal/molecules Molecules 2016, 21, 1345 2 of 13 condition for storage, thus the photostabilities of tea catechins deserve a systematic investigation in order to resolve the browning problem of tea catechins caused by illumination. Ultraviolet B (UVB) is electromagnetic radiation with a wavelength ranging from 320 nm to 290 nm, and is regarded as the most destructive natural radiation on account of its energy, which is high enough to cause many photochemical reactions [16]. UVB radiation has been used to investigate the photosensitivity of natural polyphenols due to the advantages of its constant and adjustable intensity, easy control and simplicity compared with solar radiation [17–19]. Various phenolic compounds, such as apigenin, quercetin, resveratrol, EGCg, C and EC have been reported to be susceptible to photo-oxidation in the presence or absence of exogenous photosensitizers/free radical initiators [17–22]. Severe photodegradation of EGCg in topical cream was reported with the degradation percentage being ~70% [9,23], and 83% of C (950 µM, methanol) was lost after 3 h of ultraviolet C (UVC) exposure [24]. Phenoxyl radicals were produced from the catechol or resorcinol rings of C by photo-oxidation, leading to further chemical evolution [25]. For the eight catechins, substituent groups may impact their chemical properties, resulting in various responses to UVB radiation and different contributions to browning process. However, relevant information on the photosensitivities of the eight catechins is insufﬁcient. In the present work, the photostabilities of eight catechins under UVB radiation were investigated, and the UVB-susceptible catechins were selected for further studies in terms of the effects of solvent, radiation time and substrate concentration. Photoproducts were identiﬁed by ultra performance liquid chromatography-diode array detection-tandem mass spectrometry (UPLC-DAD-MS/MS) and 1H nuclear magnetic resonance (NMR), and the reaction mechanisms of UVB-induced transformations were discussed. The impact of UVB radiation on the antioxidant activities of the eight catechins was evaluated by 2,2-diphenylpicrylhydrazyl (DPPH) assay.

2. Results and Discussion

2.1. The Photostabilities of Catechins under UVB Radiation One molecule of catechin converts to one molecule of its epimer through an epimerization reaction [26], and one molecule of gallated catechins produces one molecule of GA via hydrolysis of the ester bond [27]. The percentage of conversion of catechins due to epimerization and hydrolysis reactions was calculated by the equation in Table1. The percentage of catechin loss due to other non-identified reactions was calculated by subtracting the percentage of epimerization and the percentage of hydrolysis from 100%, and was termed other percentage degradation for representation. Table1 shows the photostabilities of individual catechins in both water and ethanol. After 6 h of UVB radiation, no GA was detected in any of the catechin samples, indicating that no hydrolysis reaction occurred under UVB radiation. Therefore the GA data was not presented in the following studies. In Table1, GC, EGC, GCg, EGCg, Cg and ECg were insensitive to UVB irradiation with percentage maintained of 90.7%–99.6%, whereas only 58.0%–80.3% of EC and C were preserved after 6 h UVB radiation. A decrease below 10% is considered as insensitivity to UVB. Hence, EC and C both in water and ethanol showed high photosensitivities to UVB, which was also reflected by their color change from colorless to yellow under UVB radiation (Table1). Epimers were only detected in the aqueous and ethanol solutions of EC and C but not the other six catechins. For EC, 31.1% and 20.0% converted to its epimer C in water and ethanol respectively, compared with 2.6% and 5.2% of C converting to EC accordingly, which suggests that EC and C undergo isomerization under UVB radiation and that the epi-type catechin is favorable for photoisomerization. Forest et al. [28] also reported that photoisomerization occurred to C with the generation of unknown yellow compounds as side products. 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(58.0% ± 1.11 ± 0.2%) catechinscatechins and Tableandwere were therefore1. The therefore photostabilities3' selected1' Bselected for of further foreight further catechin study. study.s under ultraviolet B (UVB)341.0 radiation ± 6.3 (μM) 333.7. ± 1.1 unknown UVB-induced2 reactions.O OH 3'OH EC OHand C showed the highest3 EC photosensitivities among4 the eight 4 Catechins HO Molecular2' StructuresOH 2' OH After UV Radiation CompositionsEC Water Ethanol catechinscatechinsEC and wereand were therefore1 therefore selected4' selected5' for further for further study. study. (59.3%341.0 ±± 3.1%)6.3 (58.0%333.71 ±± 0.2%)1.11 Table2 Table 1. The 1. photostabilities The photostabilities13'B2 OH6' of4' eight of eightcatechin catechins under3s under ultraviolet ultraviolet B (UVB) B (UVB)341.0 radiation(59.3% ±341.0 4 radiation6.3 ± ± (3.1%) μ6.3M) 333.7(μ. M)(58.0% ± 333.7 4.1.1 ± ± 0.2%) 1.1 Catechins MolecularA Structures1'C3' 1' 3'OHB After UV Radiation CompositionsEC Water Ethanol catechins and 2were thereforeO 2' O selectedOH forOH further study. 3 EC EC 4 4 1 ECCatechin s HO HO Molecular2' Structures2'5'4' After UV Radiation Compositions(59.3% ± 3.1%)178.5 Water ± 3.6 (58.0% 1 ± 0.2%)115.0Ethanol ± 0.7 TableEC 1. The photostabilities1 OH4' 5' of eight catechins under ultraviolet(59.3% ±341.0 3.1%) B (UVB) ± 6.3 (58.0% radiation ±333.71 0.2%) ± 1.1 (µM) . TableTable 1. The 1. photostabilitiesThe1 photostabilities21' 1 BOH6'3 3' of4' eight of eight catechin catechins unders under ultraviolet ultravioletEpimer B (UVB) B (UVB) radiation(59.3% radiation ± (3.1%)μM) ( μ. M)(58.0% . ± 0.2%) 2 2 A OC 1'4 B 21' B6' OH 3 3 4 4 4 4 CatechinCatechins s HO Molecular MolecularOA StructuresCO Structures2' OH After AfterUV Radiation UV RadiationCompositionsCompositionsEC Water Water Ethanol1 Ethanol1 EC TableHO 1. TheHO photostabilitiesOH 3' 5' of eight catechins under ultraviolet B (UVB)341.0178.5 radiation(31.1% ± 6.33.6 ± 1.2%)(μM)333.7115.0 . (20.0%± 1.10.7 ± 0.1%) EC EC Table 1. The3 photostabilities2 OH1 6' 5' OH 5'4' of eight catechins under ultraviolet B (UVB)(59.3%341.0178.5 radiation ± ± 3.1%) 6.33.6 (μM)(58.0%333.7 115.0. ± ± 0.2%) 1.10.7 2 A C 22' 6'3 21'OH3'B 3 EpimerEC 4 1 4 2 A C4 O OH 6' OH 3 Epimer 4 4 CatechinCatechins Table s 1. Molecular TheHO Molecular photostabilities1A StructuresC4 Structures2' 4' OH of eight After AftercatechinUV Radiation UV sRadiation under ultravioletCompositionsCompositions ECB (UVB) (59.3%(31.1%178.5 Waterradiation ±±± 3.1%)1.2%)3.6 Water ( μ(58.0% (20.0%M)115.0 Ethanol. ±±± 0.2%)0.1%)0.7Ethanol EC OH 3 1'OH B 5' 341.0178.5(59.3%(31.1% ± 6.33.6 ±± 3.1%)1.2%)333.7 115.0 (58.0%(20.0% ± 1.10.7 ±± 0.2%)0.1%) 2 2 2 OOH 3 OH13' 2 OH 4' 3 3 Epimer3 341.0178.54 ±± 6.33.64 333.7115.04 ±± 1.10.74 4 CatechinCatechins s HO Molecular Molecular4A Structures Structures3 1'OH3' OHB6' OH After AfterUV Radiation UV RadiationCompositionsEpimerCompositions Water Water EthanolEthanol CatechinsEC Molecular4 Structures2'CO4 OH 5' OH After UV Radiation EC EpimerECCompositions (31.1% ± 1.2%)461.5 ± 4.5(20.0% Water ± 0.1%)339.2 ± 3.4 Ethanol 2 HOOH 2 6' 2' 3 (59.3%(31.1%(31.1% ±±178.5 3.1%)1.2%)4 ± 1.2%)3.6(58.0% (20.0% (20.0% ±±115.0 0.2%)0.1%)4 ± 0.1%)0.7 Catechins EC MolecularAOH C1OH StructuresOH OH4' 5'4'OH After UV Radiation CompositionsC 341.0 Water(59.3% 341.0± 6.3 ±± 3.1%)6.3 333.7Ethanol(58.0% 333.7± 1.1 ±± 0.2%)1.1 1' 13'BOH3 2 3'OHOH OA 4 OH 1' BOH6' OH EC EpimerEC 178.5 ± 3.6 115.0 ± 0.7 HO 2'OHCO 2' 461.5(80.3% ± 4.5 ± 0.0%)339.2 (59.0% ± 3.4 ± 0.6%) EC HO OH 3 5' OH (59.3%341.0(31.1% ± 341.0±461.5 3.1%) 6.3 ± ±± 1.2%) 6.34.5 (58.0% 333.7(20.0% ± 333.7±339.2 0.2%) 1.1 ± ±± 0.1%) 1.13.4 ± EC 41 13'OH 4'3'OH 5'4' OH EpimerC 341.0(59.3% 178.5± 6.3 ± ± 3.1%) 3.6 341.0 333.7(58.0% 115.0± 1.16.3 ± ± 0.2%) 0.7 333.7 1.1 21'O3'OHB6'3 2 OH BOH6' OH EC ECC AHOOH COA 2' 1'2' OH EC Epimer (31.1%(80.3%461.5EC ±±± 1.2%)0.0%)4.5 (20.0%(59.0%339.2 ±±± 0.1%)0.6%)3.4 C HO 2'OC4 (59.3%(59.3% ± 3.1%) ± 3.1%)(58.0% (58.0% ± 0.2%) ± 0.2%) EC HO 1 1 5'4'OH 5'4' C (59.3%178.5461.5(31.1%(80.3% ±±178.5461.5 3.1%)3.64.5 ±± ±± 1.2%)0.0%) 3.64.5 (58.0% 115.0 339.2 (20.0%(59.0%± ±±115.0339.2 0.2%)0.73.4 ± ± ±± 0.1%)0.6%) 0.73.4 ± EC EC 1OOH 3 21'OH B 1' OHB (59.3 3.1%) (58.0 0.2%) HO O 1'OO B6'32 OH OH6' OH EpimerC C C HO HOAHO C4AO C4 Epimer (80.3% ± 0.0%)14.9 ± 0.1(59.0% ± 0.6%)30.0 ± 0.5 EC ECC HO OH OH5' 5' (31.1%(80.3%(80.3% ±±461.5 1.2%)0.0%) ± 0.0%)4.5(20.0% (59.0% (59.0% ±±339.2 0.1%)0.6%) ± 0.6%)3.4 EC OH OOH 2 6' 2 5' Epimer 178.5(31.1% 178.5± 3.6 ±± 1.2%)3.6 115.0(20.0% 115.0± 0.7 ±± 0.1%)0.7 HO A CA 3 2 OH 6'3 OH 6' OH C C A C4O CO OH EpimerEpimer 461.514.9178.5 ±(2.6%± 0.1±4.5 3.6 ± 0.0%)339.2178.530.0 115.0 ±±(5.2% 0.53.4± 0.7 3.6 ± 0.1%) 115.0 ± 0.7 HO HO OH OH4 OH 178.5(80.3% 178.5± 3.6 ± ± 0.0%)3.6 115.0(59.0% 115.0± 0.7 ± ± 0.6%)0.7 C C OH OH EpimerEpimerC (31.1%(31.1% ±461.5 14.91.2%) ± ±± 1.2%) 0.14.5 (20.0% (20.0% ±339.2 30.00.1%) ± ±± 0.1%) 0.53.4 OH 3 OH 3 OH Epimer Epimer 4 4O OH OH OH EpimerEpimerC (80.3% (2.6%(31.1%14.9 ±± 0.0%)±0.10.0%) 1.2%) (59.0%(5.2%(20.0%30.0 ±± 0.1%) 0.50.6%)± 0.1%) HOOH 4 OH (31.1 ± 1.2%) (20.0 ± 0.1%) C OH OH (31.1%14.9(31.1%(2.6% ± 1.2%)0.1 ±± 0.0%) 1.2%)(20.0% 30.0(20.0%(5.2% ± 0.1%)0.5 ±± 0.1%) 0.1%) OH OOH OH Epimer 461.5(80.3% ±461.514.9 4.5 ± ±± 0.0%)0.14.5 339.2 (59.0% ±339.230.0 3.4 ± ±± 0.6%) 0.53.4 HO OHOHOH OH Epimer C OH O OH OH C EpimerC (2.6% ± 0.0%) (5.2% ± 0.1%) HO (80.3%(2.6% ±±531.6 14.90.0%)0.0%) ±± 0.19.4 (59.0% (5.2% ±±521.6 30.00.1%)0.6%) ±± 0.57.1 C OH OH OH OH EGC 461.5(80.3%(2.6% 461.5± 4.5 ± ±± 0.0%) 0.0%)4.5 339.2(59.0%(5.2% 339.2± 3.4 ± ±± 0.1%) 0.6%)3.4 ± OH OHOHOH 461.5 4.5 339.2 3.4 O OH OH C Epimer 531.614.9 ± ± 0.1 9.4 521.630.0 ± ± 0.5 7.1 HO OHO C C (92.5% ± 1.6%) (90.7% ± 1.2%) C HO OH OH EpimerEGC (80.3%461.5(2.6% ± 461.5±531.6 0.0%) 4.5 ± ±±0.0%) 4.59.4 (59.0% 339.2(5.2% ± 339.2±521.6 0.6%) 3.4 ± ±±0.1%) 3.47.1 C OH OH OH (80.3%14.9 ± 0.0%)0.1 (80.3 (59.0%±30.00.0%) ± 0.6%)0.5 (59.0 ± 0.6%) C O OH OH OH C EGCC (92.5%(2.6% ± ± 0.0%) 1.6%) (90.7%(5.2% ± ± 0.1%) 1.2%) HOOH O O OH Epimer 531.6 ± 9.4 521.6 ± 7.1 C EGC HO HO OH OH EGC (80.3%531.614.9(80.3%(92.5%(2.6% ± ±531.6 0.10.0%) 9.4 ± ±± ± 0.0%) 0.0%)1.6%) 9.4(59.0% 521.6 30.0(59.0%(90.7%(5.2% ± ±521.6 0.50.6%) 7.1 ± ±± ± 0.1%) 0.6%)1.2%) 7.1 C OOH OH 14.9 ± 0.1 30.0 ± 0.5 HO O O OHOHOH OH EpimerEGC EGC (92.5%14.9 ± ±1.6%) 0.1 (90.7%30.0 ± 1.2%)± 0.5 EGC HO HO O OH Epimer 14.9 ± 0.1 30.0 ± 0.5 C C HO OH Epimer (92.5%(2.6% (92.5%± ± 531.60.0%) 1.6%) ± 1.6%)9.4 (90.7%(5.2% (90.7%± ± 521.60.1%) 1.2%) ± 1.2%)7.1 EGC OH OOH OH OH 14.9(2.6% ±14.9 0.1 ±± 0.0%)0.1 30.0(5.2% ±30.0 0.5 ±± 0.1%)0.5 HO OH OH OH EpimerEGC Epimer (2.6%531.6 ±± 9.40.0%) UD 521.6(5.2% ± ± 7.1 0.1%) UD EGC O O OHOH EpimerEpimer HO HO OH OH OH EGC 14.9(92.5% ±14.9 0.1 ± 0.11.6%) (2.6 30.0±(90.7% ±30.0 0.0%)0.5 ± 0.51.2%) (5.2 ± 0.1%) EGC EGC OH OH OH OH Epimer (2.6%UD(2.6% ± 531.60.0%) ± ±0.0%) 9.4 (5.2% UD(5.2% ± 521.6 0.1%) ± ±0.1%) 7.1 OH OHOH EpimerEpimer (92.5% ± 1.6%) (90.7% ± 1.2%) O OH OH EpimerEGC UD UD HOOH OH OH (2.6% ± 0.0%) (5.2% ± 0.1%) EGC OH OOH OH Epimer 531.6UD(92.5%(2.6% ± 9.4 ±± 0.0%) 1.6%) 521.6 UD(90.7%(5.2% ± 7.1 ±± 0.1%) 1.2%) HO OH OH 531.6 ± 9.4 521.6± ± 7.1 ± EGC OHOHOHOH OH EpimerEGC UD 531.6 UD 9.4 521.6 7.1 OH O OH OH EpimerEGC UD UD HO EGC(92.5%(92.5%531.6(92.5% ± ±557.5± 1.6%) 1.6%)9.4 ± ± 1.6%)3.3 (90.7%(90.7% 521.6(90.7% ± ±546.7± 1.2%) 1.2%)7.1 ± ± 1.2%)1.8 EGC OH OH OH OH OHOH 531.6 ± 9.4 521.6 ± 7.1 EGC OH OHOH GC (92.5 ± 1.6%) (90.7 ± 1.2%) O OH OH OH EGCEpimer EGC 557.5 ± 3.3 UD 546.7 ± 1.8 UD HO O Epimer 531.6UD(97.0% ± 9.4 ± 3.2%) 521.6 UD(95.1% ± 7.1 ± 0.3%) EGC HO OH OHOH GC (92.5%(92.5% ±531.6 1.6%) ± ± 1.6%) 9.4 (90.7% (90.7% ±521.6 1.2%) ± ± 1.2%) 7.1 EGC OH OH OH 557.5 ± 3.3 546.7 ± 1.8 OH O OH OH EGC EGC (97.0%557.5 ±± 3.2%)3.3 (95.1%546.7 ±± 0.3%)1.8 HO O O OH OH EpimerGC UD UD GC HO HO OH OH GC (92.5%(92.5% ± 1.6%) ± 1.6%)(90.7% (90.7% ± 1.2%) ± 1.2%) EGC O OH OH Epimer 557.5UD(97.0% ± 3.3 ± 3.2%)546.7 UD(95.1% ± 1.8 ± 0.3%) EGC HO OH OH 557.5 ± 3.3 546.7 ± 1.8 O O OHOH Epimer Epimer(97.0% UD± 3.2%) (95.1% ± UD 0.3%) UD GC HO HO O OH OH GC EpimerGC UD UD EGC EGC HO OH OH OH GC OH O OH OH (97.0%(97.0% ±557.5 3.2%) ± 3.2%)3.3 (95.1% (95.1% ±546.7 0.3%) ± 0.3%)1.8 HO OH OH OH Epimer 557.5 ± 3.3 UD 546.7 ± 1.8 UD GC OH OH OH Epimer GC UD UD O OHO OHOH GC Epimer UD UD HO HO OH Epimer 557.5UD(97.0% ± 3.3 ± 3.2%) 546.7 UD(95.1% ± 1.8 ± 0.3%) GC GC OH OH OH OHOH OH (97.0% ± 3.2%)557.5 ± 3.3(95.1% ± 0.3%)546.7± ± 1.8 ± OHOH OH EpimerGC Epimer UD UD 557.5 UD UD3.3 546.7 1.8 OH OHO OH OH EpimerGC UD UD HO O Epimer (97.0%GCUD ± 3.2%) (95.1% UD ± 0.3%) GC HO OH OH OH 557.5(97.0% ±557.5 3.3 ± ± 3.2%)3.3 546.7 (95.1% ±546.7 1.8 ± ± 0.3%) 1.8 GC OH OH OH OH GC OH OHOH OH (97.0 ± 3.2%) (95.1 ± 0.3%) O OH OH EpimerGC Epimer GC UD UD UD UD HO OH O 567.7 ± 1.2 544.1 ± 4.1 GC HO OHOH (97.0%557.5(97.0% ± ± 3.2%) 3.3 ± 3.2%)(95.1% 546.7(95.1% ± ± 0.3%) 1.8 ± 0.3%) GC OH OH OH OH ECg 557.5 ± 3.3 546.7 ± 1.8 OH OHOH OHOH OH GC Epimer 567.7 ± 1.2 UD 544.1 ± 4.1 UD O O OH Epimer GC UD UD HO HO OH EpimerECg (97.0%557.5UD(98.7% ± 557.5± 3.2%) 3.3 ± ± 0.2%)3.3 (95.1% 546.7 UD(94.6% ± 546.7± 0.3%) 1.8 ± ± 0.7%)1.8 GC GC OHO OH OH (97.0%567.7 ± ± 3.2%) 1.2 (95.1%544.1 ± ± 0.3%) 4.1 HO OH OH OHOH OH GC (98.7% ± 0.2%) (94.6% ± 0.7%) OH OH OH GC Epimer567.7 ± 1.2 544.1 UD± 4.1 UD O O OH EpimerECg UD UD HOHO O OH ECg (97.0%(97.0% ± 3.2%) ± 3.2%)(95.1% (95.1% ± 0.3%) ± 0.3%) GC ECg HOOH OH 567.7(98.7% ± 1.2 ± 0.2%)544.1 (94.6% ± 4.1 ± 0.7%) GC OH OH OH OH OH (98.7% ± 0.2%)567.7 ± 1.2(94.6% ± 0.7%)544.1 ± 4.1 ECg O OOH O OH OH OH Epimer UD UD HO HOHO O OH ECgEpimer ECg UD UD GC GC HO OCOH OHOHOH (98.7%567.7 ±567.7± 0.2%)1.2 ± 1.2 (94.6% 544.1 ±544.1± 0.7%)4.1 ± 4.1 ECg OH OH OH OHOH 567.7 (98.7%± 1.2 ± 0.2%)544.1 ±(94.6% 4.1 ± 0.7%) ECg O OH OCO OH OH OH OH ECgEpimer UD 567.7 ± UD1.2 544.1 ± 4.1 HO HO OHO EpimerECg EpimerECg UD UD UD UD OH OH Epimer ECg(98.7%UD ± 0.2%) (94.6% UD ± 0.7%) ECg OH OOH OC OHOH OH (98.7% (98.7%± 0.2%) ± 0.2%)(94.6% ±(94.6% 0.7%) ± 0.7%) ECg O OHOC OH OH 567.7 ± 1.2 544.1 ± 4.1 ECg O O OH EpimerEpimer UD UD (98.7 UD± 0.2%) UD (94.6 ± 0.7%) HOHO HO OH OH OH OH EpimerECg UD UD OH O OH OH Epimer UD UD ECg ECg OH OHOCO OH OH 567.7(98.7% ± 1.2 ± 0.2%)544.1 (94.6% ± 4.1 ± 0.7%) ECg OHOC OH OH 567.7 ± 1.2 544.1 ± 4.1 O OH OHOH OHHO OH OH OH EpimerECg EpimerECg UD UD UD UD OHO OC OCO OH OH OH Epimer (98.7%567.7UD(98.7% ± 569.8± 0.2%) 1.2 ± ± 0.2%)3.0 (94.6% 544.1 UD(94.6% ± 536.9± 0.7%) 4.1 ± ± 0.7%)3.8 ECg OC OH OHOHOH 567.7 ± 1.2 544.1 ± 4.1 O OOH OH OH OH Cg Epimer569.8 ± 3.0 536.9 UD± 3.8 UD HO OHOHHO OH OHOH EpimerECgEpimer ECg UD UD UD UD OHO O OH Cg 567.7(99.1% 567.7± 1.2 ± ± 0.5%) 1.2 544.1(93.4% 544.1± 4.1 ± ± 0.7%) 4.1 ECg OHOC OH OH OH (98.7%(98.7% ±569.8 0.2%) ± ± 0.2%) 3.0(94.6% (94.6% ±536.9 0.7%) ± ± 0.7%) 3.8 ECg O O OHOOHH OH ECg (99.1%569.8 ±± 0.5%)3.0 (93.4%536.9 ±± 0.7%)3.8 HO HO O OH ECg HO O OH OH OH EpimerCg UD UD HO O OH Cg (98.7%(98.7% ± 0.2%) ± 0.2%)(94.6% (94.6% ± 0.7%) ± 0.7%) Cg OC OH OH OH 569.8(99.1% ± 3.0 ± 0.5%)536.9 (93.4% ± 3.8 ± 0.7%) ECg ECg O O OHOCOH OH (99.1%569.8 ± 0.5%)±569.8 3.0 ± 3.0(93.4% 536.9 ± 0.7%) ±536.9 3.8 ± 3.8 Cg HO HO O OH OH OH 569.8 ± 3.0 536.9 ± 3.8 OHHO O OH OH EpimerCg UD UD HO OH O OH OH EpimerCg UD UD ECg OCO OHOH Cg ECg OC OCOH OH OH (99.1%569.8 (99.1%± ± 569.83.0 0.5%) ± 0.5%)3.0 (93.4%536.9 ±(93.4% ±3.8536.9 0.7%) ± 0.7%)3.8 Cg Cg O OH OC OH OHOHOH OH Cg HO O O Epimer UD (99.1 ± 0.5%) UD (93.4 ± 0.7%) HO OHOHHO OH OHO EpimerCg Epimer Cg UD UD UD UD OCOO OCO OH OH OH Epimer UD UD Cg OCOH OHOH OH (99.1% (99.1%± 0.5%) ± 0.5%)(93.4% ±(93.4% 0.7%) ± 0.7%) Cg Cg OC OHOH 569.8 ± 3.0 536.9 ± 3.8 OH O OH O OHOH OH EpimerEpimer UD UD UD UD HO OHHO OH OHO OHO EpimerEpimer Cg UD UD UD UD O O OH OH Cg Cg OC OCOH OH OH OH Epimer 569.8UD(99.1% ± 3.0 ± 0.5%)536.9 UD(93.4% ± 3.8 ± 0.7%) OH OH OH 569.8 ± 3.0 536.9 ± 3.8 OH O OH OH Epimer UD UD OHHO OH OHOH OH EpimerCg Cg UD UD O O Epimer UD UD OC OC OH OH OH (99.1% ±572.4 0.5%) ± 2.6 (93.4% ±525.3 0.7%) ± 0.7 OHOH 572.4569.8(99.1% ± 569.8 ±2.6 3.0 ±± 0.5%)3.0 525.3 536.9 (93.4%± 536.90.7± 3.8 ±± 0.7%)3.8 Cg O OHOH OH OH HO OH OH OOH OH EpimerEGCg UD UD HO OHO OHO EGCgCg Epimer Cg UD UD OH OH 569.8(99.6% 569.8± 3.0 ± ± 0.5%) 3.0 536.9(91.4% 536.9± 3.8 ± ± 0.1%) 3.8 Cg OHOCOHOH OH (99.6%(99.1%572.4572.4 (99.1% ±± ± 0.5%)±2.6572.4 0.5%) 2.6 ± ± 0.5%) 2.6(91.4%(93.4% 525.3 525.3 (93.4%±± ±0.1%) 0.7±525.3 0.7%) 0.7 ± ± 0.7%) 0.7 Cg O OH OH OH ± ± HO OO O Cg Cg 572.4 2.6 525.3 0.7 HOHO HO OH OH OH OH EGCgEGCg EpimerEGCg UD UD OH O OH (99.1% ± 0.5%) (93.4% ± 0.7%) EGCg OC OH OH OH EGCg(99.6%(99.1% ± 0.5%) ± 0.5%)(91.4% (93.4% ± 0.1%) ± 0.7%) Cg O OC OH (99.6%572.4 ±(99.6% 0.5%)± 2.6 ± 0.5%)(91.4%525.3 ±(91.4% 0.1%) ± 0.7 ± 0.1%) EGCg Cg HO O O OH OH 572.4 ± 2.6 ±525.3 ± 0.7 ± EGCg HO OHHO O OOH OH OH (99.6 0.5%) (91.4 0.1%) HO HO OH OH OH EpimerEGCgEpimerEGCg UD UD UD UD O OH O OHOH OH Cg Cg OC OCOH OH OHOHOH (99.6%572.4 ± ± 572.42.6 0.5%) ± 2.6 (91.4%525.3 ± ±0.7525.3 0.1%) ± 0.7 EGCgEGCgEGCg OC OH OHOHOH (99.6% ± 0.5%) (91.4% ± 0.1%) O OH EGCg HO OH OH O EpimerEpimerEGCg UD UD UD UD OHHO O OHOHO EpimerEpimer UD UD UD UD OCO OCO OHOHOH OH (99.6% (99.6%± 0.5%) ± 0.5%)(91.4% ±(91.4% 0.1%) ± 0.1%) EGCg OC OCOHOHOH OHOH OH Epimer UD572.4 ± 2.6 UD525.3 ± 0.7 EGCg OH O OH O OHOH OH EpimerEpimer Epimer UD UD UD UD UD HO HO OHO O OHOH OH OHOH OH EpimerEpimer EGCg UD UD UD UD O O OH OH EGCg OC OH OH OH 572.4(99.6% ± 2.6 ± 0.5%)525.3 (91.4% ± 0.7 ± 0.1%) EGCg OHOCOHOH OH 572.4 ± 2.6 525.3 ± 0.7 O OH OH OH OHHO OHOH OH EpimerEGCgEGCg UD UD OH OH Epimer UD UD OCO O OH OH (99.6% ± 0.5%) (91.4% ± 0.1%) OC OH OH 566.2572.4566.2(99.6% ± 572.4 566.2±0.0 2.60.0 ±± 0.5%)2.60.0 524.0 525.3524.0 (91.4%± 525.3524.02.8± 0.72.8 ±± 0.1%)0.72.8 EGCg O OH OHOH OH Epimer UD 566.2 UD ± 0.0 524.0 ± 2.8 HO OH OOH OH EGCg GCg HO OH O OHOH GCgGCg Epimer EGCg UD UD O OHOH GCg572.4(98.5% 572.4± 2.6 ± ± 0.0%) 2.6 525.3(91.1% 525.3± 0.7 ± ± 0.5%) 0.7 OH OHOH OH (98.5%(99.6%(98.5%566.2 (99.6% ±± ± 0.0%)0.0 0.5%)0.0%) ± 0.5%) (91.1%(91.4%(91.1%524.0 (91.4%±± ±0.5%)2.8 0.1%)0.5%) ± 0.1%) EGCgEGCg O OHOC OH OH 566.2 ± 0.0 524.0 ± 2.8 GCg HO OO O OH EGCgEGCg (98.5 ± 0.0%) (91.1 ± 0.5%) HOHOHO HO GCg OH OH OHOH OH EpimerGCg (99.6%(99.6% ± 0.5%)UD ± 0.5%)(91.4% (91.4% ± 0.1%) UD ± 0.1%) O OH OH (98.5% ± 0.0%) (91.1% ± 0.5%) EGCgGCgGCg EGCg GCg O OCO OC OH OH 566.2(98.5% ± 0.0 ± 0.0%)524.0 (91.1% ± 2.8 ± 0.5%) HO HO O OH OH OH 566.2 ± 0.0 524.0 ± 2.8 HO O OH OHOH Epimer UD UD OHHO OH OHOH OH OH GCgEpimer GCg UD UD EGCg O OCO OHOH OH 566.2 ± 0.0 524.0 ± 2.8 GCgEGCg OCOC OHOC OHOH OH OH Epimer (98.5%UD ± 0.0%) (91.1% UD ± 0.5%) GCg OH (98.5%566.2 ± 0.0%)0.0 (91.1%524.0 ± 0.5%)2.8 O OH OH OH GCg Epimer UD UD HO OHOHOH O EpimerEpimerEpimer GCg UDUD UD UD UD UD HO OH OO OHOHO (98.5% ± 0.0%) (91.1% ± 0.5%) OC OCO OHOHOHOH OH (98.5% ± 0.0%) (91.1% ± 0.5%) GCg O OCOH OHOH OH 566.2 ± 0.0 524.0 ± 2.8 GCg HO OH OH O OOHOHH OH EpimerEpimer UD UD UD UD OHHO OH OH Epimer UD UD OH O OHO OHOH EpimerGCg UD UD GCg O OH 1 1 OC OHOHOH OH GCg OHOC OH µ 566.2(98.5% ±566.2 0.0 ± ± 0.0%)0.0 524.0 (91.1% ±524.0 2.8 ± ± 0.5%) 2.8 The solutions1 The1 solutions of of individual individualO catechins catechinsOH OH were were prepared prepared at 575 μM at with 575 waterM and with ethanol water respectively; and ethanol respectively; OHHO OH OH EpimerGCg GCg UD UD The solutionsThe solutions of individualOH ofOC individualO catechinsOHOH catechins were wereprepared prepared at 575 at μ M575 with μM Epimerwater with water and ethanol and ethanolUD respectively; respectively; UD 2 2 OCO OH OH 3 3 (98.5%566.2(98.5% ± 566.2± 0.0%) 0.0 ±± 0.0%)0.0 (91.1% 524.0(91.1% ± 524.0± 0.5%) 2.8 ±± 0.5%)2.8 1 GCg OH O OH OHOH OHOH OH Epimer UD UD EC-C,2 EC-C, EGC-GC,2 EGC-GC,HO ECg-Cg, OHECg-Cg,O O EGCg-GCg EGCg-GCg areare geometric isomers; isomers; 3 Pictures3 Pictures of the ofwater the and water ethanol and ethanol solutions The1 solutionsHO of individualOH catechins were prepared at 575 μM withGCgEpimer waterGCg and ethanolUD respectively; UD EC-C, TheEC-C, EGC-GC, solutions EGC-GC, ECg-Cg, of individualECg-Cg, EGCg-GCgO OHOH EGCg-GCg catechins are weregeometric are geometricprepared isomers; at isomers; 575 PicturesμM with Pictures of water the of waterand the ethanol waterand ethanol respectively;and ethanol OH OH (98.5%566.2 ± 566.2± 0.0%) 0.0 ± 0.0 (91.1% 524.0 ± 524.0± 0.5%) 2.8 ± 2.8 GCg GCg OC OH OH OH 4 4(98.5% ± 0.0%) (91.1% ± 0.5%) 2 O O 3GCg GCg of individualsolutions1 HO catechinsof individualHO after catechins 6 h after UVB 6 h radiation: UVB radiation: W-water; W-water; E-ethanol;4 Data4Data in round in blankets round blankets were the EC-C,21 EGC-GC, OHECg-Cg,OH EGCg-GCgOH are geometric isomers; PicturesEpimer3 of the waterUD and ethanol UD solutions Thesolutions solutions TheEC-C, of solutions individual EGC-GC, of of individual individual of individualcatechinsECg-Cg, catechins catechinsO OH afterEGCg-GCg catechinsOH 6after were h UVB 6 werepreparedh are radiation:UVB preparedgeometric radiation: at W-water;575 at μisomers; W-water;575M with μE-ethanol;M waterwith E-ethanol;Pictures water (98.5%and Data ethanolof (98.5%and ±in Datathe 0.0%) round ethanol ±water respectively;in 0.0%) roundblankets(91.1% respectively;and(91.1% blankets ±ethanol 0.5%) ± 0.5%) GCg1 GCg O OCO OC OH OH The solutionsHO HO of individual catechinsOH were prepared at 575 μM with water and4 ethanol respectively; maintainedwere2 1 the percentage maintainedOH OH ofpercentage catechins.OH OH of catechins. Data in Da squareta in square blanketsEpimer3 blanketsEpimer were were the UDthe percentages UDpercentages UD of UD epimerization of GCg solutions EC-C, GCg2 The EGC-GC, of solutions individual ECg-Cg, of individualcatechinsO EGCg-GCgO OH after catechinsOH 6 h areUVB weregeometric radiation: prepared isomers; W-water; at 575 μ E-ethanol;PicturesM with3 waterof Data the and inwater4 round ethanol and blankets respectively;ethanol solutions EC-C, EGC-GC, of individualOC ECg-Cg,OC catechins OHEGCg-GCg OHafter 6 h are UVB geometric radiation: isomers; W-water; PicturesE-ethanol; of theData water in round and blankets ethanol 2 EC-C, EGC-GC, ECg-Cg, EGCg-GCgOH OH are geometric isomers; 3 Pictures of the water and ethanol individual epimerization21catechinsOH of =individualOH concentration catechinsof epimer= concentration (µM)/575 of epimerµEpimerM ×Epimer(μ 3 100%.M)/575 4 EC,μMUD × ( −UD100%.)-epicatechin; EC, UD(−)- UD C, (−)-catechin; solutions TheEC-C, of solutions individual EGC-GC, ofOC individualcatechinsECg-Cg,O OCO afterOHEGCg-GCg catechins OH6 h UVB were are radiation: preparedgeometric W-water; at isomers; 575 μ E-ethanol;M with Pictures water Data of and 4the in ethanol round water blankets respectively;and ethanol solutions of individual catechinsOH after 6 h UVB radiation: W-water; E-ethanol;4 Data in round blankets 1solutions of individualOH OH catechins after OH 6 h UVB radiation: W-water;Epimer E-ethanol;Epimer DataUD in roundUD blankets UD UD EGC, (epicatechin;− The)-epigallocatechin; 12solutions The solutions C, of (− individual)-catechin; of individualO GC, catechinsOEGC, (− catechins )-gallocatechin;(−)-epigallocatechin; were wereprepared prepared at ECg, 575GC, at μ 575M(− ( −)-gallocatechin;with μ)-epicatechinM waterwith3 water and ethanolECg, and4 gallate; ethanol(− )-epicatechinrespectively; Cg,respectively; (− )-catechin gallate; solutions EC-C, EGC-GC, of individual ECg-Cg, catechinsOH EGCg-GCg OH after 6 h are UVB geometric radiation: isomers; W-water; PicturesE-ethanol; of theData water in round and blankets ethanol 21 − 12 − 3 3 EGCg, gallate;( TheEC-C,)-epigallocatechin solutionssolutions TheEC-C, Cg,EGC-GC, solutions ( −EGC-GC,)-catechin of of individual individualECg-Cg, of individual ECg-Cg,gallate; gallate; EGCg-GCgcatechins catechins EGCg, EGCg-GCg catechins GCg, after were( −are)-epigallocatechin ( 6 were preparedgeometrich )-gallocatechinare UVB preparedgeometric radiation: at isomers; 575 atgallate; μisomers; 575 W-water;M gallate; with μ PicturesGCg,M withwater PicturesE-ethanol; ( UD,− )-gallocatechinwaterof and the undetectable. of ethanoland water4 theData ethanol water respectively; inandgallate; round respectively;ethanoland UD, blankets ethanol 1 1 2 The solutions The solutions of individual of individual catechins catechins were were prepared prepared at 575 at μ 575M with3 μM withwater water and ethanol and ethanol respectively; respectively; solutionsundetectable. EC-C,2solutions EC-C, EGC-GC, of individual EGC-GC, of individual ECg-Cg, catechinsECg-Cg, EGCg-GCgcatechins afterEGCg-GCg 6after h are UVB 6 geometrichare UVBradiation: geometric radiation: isomers; W-water; isomers; W-water; PicturesE-ethanol; 3 PicturesE-ethanol; of 4 theData of water4 theDatain round water inand round blankets ethanoland blanketsethanol 2 2 3 3 solutions EC-C,solutions EC-C, EGC-GC, of individual EGC-GC, of individual ECg-Cg, catechinsECg-Cg, catechinsEGCg-GCg afterEGCg-GCg after6 h are UVB 6 hgeometric are UVBradiation: geometric radiation: isomers; W-water; isomers; W-water; PicturesE-ethanol; PicturesE-ethanol; of 4 theData of 4 water theDatain round water inand round blankets ethanoland blankets ethanol In our solutions study,solutions of EC individual of andindividual catechins C, catechins the after basic after6 h UVB 6 unith UVBradiation: radiation: of flavan-3-ols, W-water; W-water; E-ethanol; E-ethanol; exhibited 4 Data 4 Datain round in round high blankets blankets susceptibility to UVB radiation whereas EGC, GC, EGCg, GCg, ECg and Cg were UVB-insensitive, indicating that the presence of gallated and pyrogallol moieties hindered the UVB-induced chemical transformations. This is in accordance with the previous report showing that epimerization reaction was closely related with chemical structure of flavan-3-ols [29]. Dobashi et al. [17] demonstrated that EC and C showed higher photo-antioxidant activity than EGC whereas no photo-antioxidant activity of gallated catechins was observed. Hence, a substituent group has a decisive influence on the photosensitivity of flavan-3-ols. In addition, a clear solvation effect on the photoisomerization of EC and C was observed (Table1). An aqueous solution was favorable to photoisomerization. It was reported that UVB-induced epimerization of EC to C was via an intermediate quinone methide through ionization [20]. Thus, higher polarity of water facilitated the ionization process, increasing the generation of intermediate quinone methide compared with ethanol medium. This explained why more EC converted to C in Molecules 2016, 21, 1345 4 of 14

In our study, EC and C, the basic unit of flavan-3-ols, exhibited high susceptibility to UVB radiation whereas EGC, GC, EGCg, GCg, ECg and Cg were UVB-insensitive, indicating that the presence of gallated and pyrogallol moieties hindered the UVB-induced chemical transformations. This is in accordance with the previous report showing that epimerization reaction was closely related with chemical structure of flavan-3-ols [29]. Dobashi et al. [17] demonstrated that EC and C showed higher photo-antioxidant activity than EGC whereas no photo-antioxidant activity of gallated catechins was observed. Hence, a substituent group has a decisive influence on the photosensitivity of flavan-3-ols. In addition, a clear solvation effect on the photoisomerization of EC and C was observed (Table 1). An aqueous solution was favorable to photoisomerization. It was reported that UVB-induced epimerization of EC to C was via an intermediate quinone methide through ionization [20]. Thus, higher polarity of water facilitated the ionization process, increasing Moleculesthe generation2016, 21, of 1345 intermediate quinone methide compared with ethanol medium. This explained4 why of 13 more EC converted to C in water than ethanol. Solvation effect on epimerization reaction of catechins waterwas also than reported ethanol. in Solvation other studies effect [29,30]. on epimerization Solvation effect reaction on the of catechinsunidentified was reaction also reported was discussed in other studiesin a later [29 section.,30]. Solvation effect on the unidentiﬁed reaction was discussed in a later section.

2.2.2.2. The Chemical TransformationsTransformations ofof ECEC andand CC withwith UVUV BB RadiationRadiation TimeTime ThereThere areare differentdifferent typestypes ofof reactionsreactions occurringoccurring toto ECEC andand CC underunder UVBUVB radiation,radiation, whichwhich mightmight playplay differentlydifferently withwith radiationradiation time.time. FigureFigure1 1 shows shows the the changes changes in in percentages percentages of of epimerization epimerization and and percentagespercentages maintainedmaintained of of EC EC and and C C over over time. time. The The corresponding corresponding percentages percentages of of degradation degradation due due to otherto other reactions reactions can can be be obtained obtained by by subtracting subtracting the the percentage percentage of of epimerization epimerizationand and thethe percentagepercentage maintainedmaintained fromfrom 100%.100%. Upon 2 h of UVB irradiation, 15.4% and 0.5% of ECEC inin waterwater underwentunderwent epimerizationepimerization andand degradation degradation reactions reactions respectively, respectively, compared compared with with 17.2% 17.2% and 15.7% and of15.7% EC in of ethanol EC in (Figureethanol1 ).(Figure Upon 21). h Upon of UVB 2 radiation,h of UVB theradiation, percentage the ofpercentage epimerization of epimerization and percentage and of percentage degradation of ofdegradation C were 1.7% of andC were 8.8% 1.7% in water, and 8.8% and in 1.7% water, and and 22.7% 1.7% in and ethanol 22.7% (Figure in ethanol1). These (Figure results 1). indicateThese results that epimerizationindicate that epimerization was the dominant was the reaction dominant for ECreaction in both for water EC in and both ethanol water and for theethanol first 2for h ofthe UVB first radiation,2 h of UVB and radiation, other degradation and other reactions degradation were reactions the principal were reactions the principal to C under reactions the same to C conditions.under the Assame radiation conditions. time increased,As radiation the percentagetime increased, of epimerization the percentage of EC inof waterepimerization increased rapidlyof EC in after water the firstincreased 8 h and rapidly then maintained after the first stable 8 h atand ~35.4% then maintained from 8 h to 10stable h, while at ~35.4% the percentage from 8 h to of degradation10 h, while the of ECpercentage slowly increased of degradation from 0.5% of toEC 9.6% slowly after increased the first 6 from h and 0.5% then rapidlyto 9.6% increasedafter the fromfirst 9.6%6 h and to 29.9% then inrapidly the subsequent increased from 4 h (Figure 9.6% to1A). 29.9% This in suggests the subsequent an antagonistic 4 h (Figure relationship 1A). This suggests between an percentage antagonistic of epimerizationrelationship between and percentage percentage of degradation of epimerization of EC inand water, percentage which is of possibly degradation due to of the EC competition in water, forwhich UVB is energypossibly between due to epimerizationthe competition and for degradation UVB energy reactions. between Theepimerization percentages and of degradationdegradation ofreactions. EC and The C in percentages ethanol gradually of degradation increased of withEC and radiation C in ethanol time, andgradually were muchincreased higher with than radiation those intime, water. and Thus, were ethanol much higher medium than exacerbated those in wate the photo-degradationr. Thus, ethanol medium of EC and exacerbated C. The 6 h the point photo- was selecteddegradation for the of followingEC and C. studies The 6 h on point account was of selected the high for percentage the following of conversion studies on in account both epimerization of the high andpercentage degradation of conversion reactions. in both epimerization and degradation reactions.

Percentage maintained of EC Percentage maintained of EC Percentage maintained of C Percentage maintained of C Percentage of epimerization B 100 A 100 Percentage of epimerization 80 80 60 60 % % 40 40

20 20

0 0 12345 12345 2 4 6 8 10 2 4 6 8 10 Time (h) Time (h)

FigureFigure 1.1. TheThechanges changes in in percentage percentage of of epimerization epimerization and and the the percentage percentage maintained maintained of EC of and EC Cand with C UVBwith radiationUVB radiation time: time: (A) water, (A) water, 575 µ M;575 ( Bμ)M; ethanol, (B) ethanol, 575 µ M.575 μM.

2.3. The Effect of Substrate Concentrations of EC and C Figure 2 shows the effect of substrate concentration on percentage of epimerization and percentage maintained of EC and C in water and ethanol. As substrate concentration increased from 71.875 to 1150 μM, the percentage of epimerization and percentage of degradation of EC and C in both water and ethanol decreased and resulted in an elevated percentage of maintained EC and C at high substrate concentration. This indicates that high substrate concentration suppressed the UVB- induced chemical transformations of EC and C. From Figure 2, we can see that the solvent exerted a great influence on the chemical conversions of EC and C under UVB radiation. At the substrate concentration of 71.875–1150 μM, the percentages of epimerization of EC were 29.0%–52.7% for water Molecules 2016, 21, 1345 5 of 13 and 5.8%–33.2% for ethanol, while the percentages of degradation of EC in water and ethanol were 7%–20.2% and 1.5%–34.5% respectively. Hence, in contrast to water, ethanol medium suppressed the photoisomerizationthe photoisomerization of EC of but EC butincreased increased the thepercentages percentages of ofdegradation degradation of ofEC EC at atlow low substrate substrate concentrationconcentration (71.875 (71.875 μµM to 575575 µμM).M). The The percentages percentages of of epimerization epimerization of Cof inC waterin water and and ethanol ethanol were wereless thanless 10.4%,than 10.4%, while thewhile percentages the percentages of degradation of degradation of C were of 8.4%–20.4% C were 8.4%–20.4% for water and for 12.4%–55.3%water and 12.4%–55.3%for ethanol, for hence ethanol, other hence reaction other was reaction the dominating was the dominating reaction to reaction C upon to UVB C upon irradiation UVB irradiation (Figure2). (FigureThis was 2). consistent This was with consistent previous with result previous showing result that epi-structure showing that is favorableepi-structure for photoisomerization is favorable for photoisomerizationof catechin. of catechin.

Percentage maintained of EC Percentage maintained of EC Percentage maintained of C Percentage maintained of C Percentage of epimerization Percentage of epimerization A 100 B 100 80 80 60 60 % 40 % 40

20 20

0 12345 0 71.875 143.75 287.5 575 1150 71.87512345143.75 287.5 575 1150 Concentration (µM) Concentration (µM)

FigureFigure 2. 2. TheThe effect effect of of substrate substrate concentration concentration on on th thee percentage percentage of of epimerization epimerization and and percentage percentage maintainedmaintained of of EC EC and and C C under under 6 6h hUVB UVB radiation: radiation: (A (A) )water water and and (B (B) )ethanol. ethanol.

2.4.2.4. Structural Structural Characterization Characterization of of Photoproducts Photoproducts BasedBased on on UPLC-DAD-MS UPLC-DAD-MS analysis, analysis, the the UVB UVB irradiat irradiateded aqueous aqueous and andethanol ethanol solutions solutions of EC of and EC Cand had C the had same the same types types of photoproducts. of photoproducts. The The UPLC UPLC chromatogram chromatogram and and MS MS information information of of concentratedconcentrated EC EC sample sample (287.5 (287.5 μµMM in ethanol, 6 hh UVBUVB radiation)radiation) hashas been been presented presented in in Figure Figure3 as3 as an −1 anexample. example. Except Except forfor the typical peaks peaks corresponding corresponding to toEC EC (P4, (P4, [M [M− H]− H] m/−z 1289)m/ andz 289) its andepimer its epimerC (P3, −1 −1 [MC( −P3, H] [M m−/z 289),H]−1 sevenm/z 289), new sevenpeaks newfor P1, peaks P2, P5–9 for P1, were P2, detected P5–9 were with detected [M − H] with m/ [Mz at− 137,H] −287,1 m 335,/z at 427,137, 427, 287, 427, 335, 427 427, (in 427, order), 427, suggesting 427 (in order), that suggestingnew photoproducts that new were photoproducts generated wereunder generated UVB radiation under dueUVB to radiationthe unidentified due to reactions. the unidentified The photolytic reactions. clea Thevage photolytic of flavan-3-ols cleavage (fisetinidol of flavan-3-ols and C) in (fisetinidolmethanol occurredand C) inat methanolthe ether occurredlinkage C-O at theof the ether heterocyclic linkage C-O ring of with the heterocyclicproduction of ring the with corresponding production diradicals.of the corresponding These diradicals diradicals. underwent These two diradicals pathways: underwent radical reaction two pathways: and photo-induced radical reaction electron and transferphoto-induced reaction, electron which transferrespectively reaction, led whichto the respectivelygeneration ledof ortho- to thequinone generation methides of ortho and-quinone 1,3- diarylpropan-2-olsmethides and 1,3-diarylpropan-2-ols with an addition withreaction an addition of methanol reaction to ofthe methanol diradical to [18]. the diradicalIn the case [18 ].of In EC the (molecular weight abbreviated Mr, 290 Da) as reactant and ethanol as medium (Mr 46 Da), case of EC (molecular weight abbreviated Mr, 290 Da) as reactant and ethanol as medium (Mr 46 Da), theoreticallytheoretically the the molecular molecular weights weights of of ortho-orthoquinone-quinone methide-like methide-like and and 1,3-diarylpropan-2-ols-like 1,3-diarylpropan-2-ols-like productsproducts should should be be 138 138 Da Da and and 336 336 Da, Da, which which was was observed observed in Figure in Figure 3 corresponding3 corresponding to P1 to and P1 and P5 −1 −1 withP5 with [M −[M H] − mH]/z −1371 m and/z 137 m/z and 335 mrespectively./z 335 respectively. Compound Compound P2 at [M − P2 H] at [Mm/z− 287H] was−1 m considered/z 287 was asconsidered quinone on as account quinone of on the account typical of transformation the typical transformation from phenol from to quinone phenol to with quinone a loss with of 2H aloss [31], of and2H this [31], was and affirmed this was affirmedlater by its later UV by adsorption its UV adsorption spectrum. spectrum. New types New of types photoproducts of photoproducts P6-9 were P6-9 found with [M − H]−1 m/z 427−1 in Figure 3. Coincidently, the Mr of new compounds (428 Da) was the were found with [M − H] m/z 427 in Figure3. Coincidently, the M r of new compounds (428 Da) was the sum of 138 and 290 Da, suggesting that these four compounds are possibly the coupling products of the biradical of EC (Mr 290 Da) and the radical precursor of ortho-quinone methide (Mr 138 Da). Hence, a daughter ion scan MS experiment with a mass of parent ion set at 427 m/z was carried out to further investigate the molecular structures of Compounds P6-9, and their MS/MS spectra are shown in the inset of Figure3. Obviously, Compounds P6-9 were isomers on account of the same [M − H]−1 m/z 427 and similar MS/MS spectra. Based on our speculation that Compounds P6-9 might be formed from addition of ortho-quinone methide-like compound to the biradical of EC, the proposed fragmentation pathway of isomers P6-9 is shown in Figure4, which was in an agreement with the MS/MS spectrum. The presence of one double bond and one chiral carbon in the molecule is the reason for the existence of four isomers. MoleculesMolecules 2016 2016, ,21 21, ,1345 1345 66 of of 14 14 sumsum of of 138 138 and and 290 290 Da, Da, suggesting suggesting that that these these four four compounds compounds are are possibly possibly the the coupling coupling products products of of r r thethe biradicalbiradical ofof ECEC (M(Mr 290290 Da)Da) andand thethe radicalradical precursorprecursor ofof ortho-ortho-quinonequinone methidemethide (M(Mr 138138 Da).Da). Hence,Hence, a a daughter daughter ion ion scan scan MS MS experiment experiment with with a a mass mass of of parent parent ion ion set set at at 427 427 m m//zz was was carried carried out out toto furtherfurther investigateinvestigate thethe molecularmolecular structuresstructures ofof CompoundsCompounds P6-9,P6-9, andand theirtheir MS/MSMS/MS spectraspectra areare shownshown inin thethe insetinset ofof FigureFigure 3.3. Obviously,Obviously, CompouCompoundsnds P6-9P6-9 werewere isomersisomers onon accountaccount ofof thethe samesame −1 [M[M − − H] H]−1 m m//zz 427 427 and and similar similar MS/MS MS/MS spectra. spectra. Based Based on on our our speculation speculation that that Compounds Compounds P6-9 P6-9 might might bebe formedformed fromfrom additionaddition ofof ortho-ortho-quinonequinone methide-likemethide-like compoundcompound toto thethe biradicalbiradical ofof EC,EC, thethe proposedproposed fragmentation fragmentation pathwaypathway ofof isomers isomers P6-9 P6-9 is is shownshown inin FigureFigure 4, 4, whichwhich waswas inin an an agreementagreement with the MS/MS spectrum. The presence of one double bond and one chiral carbon in the molecule Moleculeswith the2016 MS/MS, 21, 1345 spectrum. The presence of one double bond and one chiral carbon in the molecule6 of 13 isis the the reason reason for for the the existence existence of of four four isomers. isomers.

FigureFigure 3. 3. The TheThe ultra ultra ultra performance performance performance liquid liquid liquid chromatography chromatography chromatography (UPLC) (UPLC) and(UPLC) and mass massand spectrometry spectrometry mass spectrometry (MS) (MS) information information (MS) informationofof UVB UVB irradiated irradiated of UVB EC EC irradiatedin in ethanol ethanol ECafter after in concentratio concentratio ethanol aftern.n. concentration.InsetInset P6-9: P6-9: the the MS/MS MS/MS Inset P6-9: spectra spectra the of of MS/MS photoproducts photoproducts spectra ofassignedassigned photoproducts to to P6-9. P6-9. assigned to P6-9.

OH OH OH OH - OH OH OH - OH - OH - OH HO - HO - HO CH CH HO HO - H CH CH HO - H - H - H OH - H OH - H OH OH OH OH HO HO m/zm/z 125 125 HO HO OH OH - OH OH OH OH - OH OH HO OH OH HO OH m/z 301 m/z 275 OH m/z 301 m/z 275

OH OH OH OH - - H H HO HO

OH OH m/zm/z 427 427

OH - OH - OH - OH - HO - H HO OH OH HO - H HO OH OH - H O - H O OH OH OH OH m/zm/z 137 137 m/zm/z 289 289 −1 FigureFigureFigure 4. 4. 4.Proposed Proposed Proposed fragmentation fragmentation fragmentation pathway pathwa pathway ofy of of isomeric isomeric isomeric ions ions ions at at at [M [M [M− − −H] H] H]−−11 m m//zz z427. 427.427.

TheThe UV-visibleUV-visible spectraspectra (190–400(190–400 nm)nm) ofof allall compcompoundsounds areare shownshown inin FigureFigure 5.5. TheThe UV-visibleUV-visible The UV-visible spectra (190–400 nm) of all compounds are shown in Figure5. The UV-visible spectraspectra of of Compounds Compounds P1 P1 and and P2 P2 exhibited exhibited two two strong strong absorption absorption peaks peaks at at 280 280 nm/307 nm/307 nm nm and and 250 250 spectra of Compounds P1 and P2 exhibited two strong absorption peaks at 280 nm/307 nm and nm/283nm/283 nm nm respectively, respectively, which which was was distinctly distinctly differe differentnt from from the the maximum maximum absorbance absorbance at at ~280 ~280 nm nm of of 250 nm/283 nm respectively, which was distinctly different from the maximum absorbance at ~280 nm CompoundsCompounds P3-9P3-9 duedue toto aromaticaromatic ring.ring. TheThe parapara-Quinone-Quinone methidesmethides havehave typicaltypical UVUV absorbanceabsorbance atat of Compounds P3-9 due to aromatic ring. The para-Quinone methides have typical UV absorbance at 300300 nm nm and and benzoquinone benzoquinone has has strong strong absorbance absorbance at at 244 244 nm nm [32,33]. [32,33]. The The UV UV absorption absorption spectra spectra also also 300 nm and benzoquinone has strong absorbance at 244 nm [32,33]. The UV absorption spectra also confirmedconfirmed that that Compounds Compounds P1 P1 and and P2 P2 belonged belonged to to quinone quinone methides methides and and quinone quinone respectively, respectively, due due conﬁrmed that Compounds P1 and P2 belonged to quinone methides and quinone respectively, due toto thethe possessionpossession ofof characteristiccharacteristic absorptionabsorption bandsbands nearnear 300300 nmnm andand 244244 nm.nm. TheThe lessless thanthan 1010 nmnm to the possession of characteristic absorption bands near 300 nm and 244 nm. The less than 10 nm differencedifference might might be be attributable attributable to to the the substitution substitution of of hydroxyl hydroxyl groups groups on on aromatic aromatic ring ring leading leading to to a a difference might be attributable to the substitution of hydroxyl groups on aromatic ring leading to a shift of λmax value to longer wavelength (bathochromic effect) because of electron donating effect of hydroxyls. In addition, ortho-Quinone methides and quinone are responsible for the yellow color of UVB irradiated EC sample, since quinonoid compounds are often yellow to red in color [34]. Molecules 2016, 21, 1345 7 of 13 Molecules 2016, 21, 1345 8 of 15

FigureFigure 5. 5. TheThe ultraviolet ultraviolet (UV) (UV) spectra spectra of of Compounds Compounds P1-9. P1-9.

1 TheThe 1H-NMRH-NMR spectra spectra of of EC EC and and its its UV UVBB irradiated irradiated reaction reaction mixture mixture with with higher higher percentages percentages of of 1 conversionconversion were were studied studied to resolve resolve the characteristic characteristic 1HH frequency frequency bands bands of of new new photoproducts photoproducts from from EC for further testifying the chemical structures of photoproducts. Since nine compounds are present EC for further testifying the chemical structures of photoproducts. Since nine compounds are present in the UVB irradiated reaction mixtures including four isomeric products and several products at a in the UVB irradiated reaction mixtures including four isomeric products and several products at a low level, it is not practical to isolate each photoproduct. The NMR spectra data of EC is as follows: low level, it is not practical to isolate each photoproduct. The NMR spectra data of EC is as follows: 1H-NMR of EC (600 MHz, Acetone-d6) δ ppm: 8.10 (s, H, H-3’), 7.94 (s, H, OH-5), 7.79 (s, H, OH-4’), 7.73 (s, H, OH-7), 7.05 (d, 1H, H-2’, J2’,6’ = 1.7 Hz), 6.83 (dd, 1H, H-6’, J6’,2’ = 1.7 Hz, J6’,5’ = 8.3 Hz), 6.78

Molecules 2016, 21, 1345 8 of 13

1H-NMR of EC (600 MHz, Acetone-d6) δ ppm: 8.10 (s, H, H-3’), 7.94 (s, H, OH-5), 7.79 (s, H, OH-4’), 7.73 (s, H, OH-7), 7.05 (d, 1H, H-2’, J2’,6’ = 1.7 Hz), 6.83 (dd, 1H, H-6’, J6’,2’ = 1.7 Hz, J6’,5’ = 8.3 Hz), 6.78 (d, 1H, H-5’, J5’,6’ = 8.3 Hz), 6.02 (d, 1H, H-6, J6,8 = 2.3 Hz), 5.91 (d, 1H, H-8, J8,6 = 2.3 H), 4.88 (br, s, 1H, H-2), 4.21 (m, 1H, H-3), 3.58 (d, 1H, OH-3, JOH-3,3 = 5.5 Hz), 2.86 (dd, 1H, H-4a, J4a,4b = 16.6 Hz, 1 J4a,3 = 4.7 Hz), 2.74 (dd, 1H, H-4b, J4b,4a = 16.6 Hz, J4b,3 = 3.2 Hz). The H-NMR data of EC was consistent with reported results [15,35]. After UVB radiation, new 1H frequency bands were found in the 1H-NMR spectra of reaction mixture from EC in addition to the original bands of EC, including 1.12 ppm (t, J = 6.8 Hz) corresponding to methyl proton of characteristic branch -O-CH2-CH3 in new Compound 5, 2.91 ppm (dd, J = 5.4 Hz, J = 16.1 Hz) corresponding to methylene proton of characteristic branch -O-CH2-CH3 in new Compound P5, 6.89 ppm (d) and 6.85 ppm (d) corresponding to protons on the carbon-carbon double bond in new Compounds P6-9. This indicated the presence of speculated functional group or structure in the UVB irradiated reaction mixture of EC.

2.5. Proposed Reaction Mechanisms The photolytic reaction mechanism of EC/C is given in Figure6, referring to the reported two photolysis pathways of ﬂavan-3-ols [18]. Upon UVB irradiation, EC/C molecule was excited at two positions: -OH bond and heterocyclic ring. The excitation of -OH bond led to the generation of quinone compound P2 at [M − H]−1 m/z 287, while the heterocyclic ring of EC/C was preferentially opened via photolytic cleavage at the ether linkage C-O with low bond dissociation energies, which resulted in the generation of free radicals A and B at m/z 137 and 289. Free radical reaction led to the generation of quinone methides such as Compound P1 at m/z 137 and four isomers P6-9 at m/z 427 through grafting reaction of free radical A m/z 137 onto free radical B m/z 289 after intramolecular rearrangement of H and -OH (Figure6). The phloroglucinol grafted derivatives of EC and C were also photosynthesized by Wilhelm-Mouton et al. [36] via photolytic cleavage of the ether bond on the heterocyclic ring of ﬂavan-3-ols. Besides, neutral radicals can also be ionized in a polar solvent due to photo-induced electron transfer reaction [37], which is the other pathway of photolysis in competition with free radical reaction. The photo-induced electron transfer reaction product at m/z 335 was obtained in our study with an addition of -OCH2CH3 from ethanol, indicating the occurrence of electron transfer reaction under UVB radiation. Thus, photolysis reaction was responsible for the percentages of EC and C loss previously termed percentages of degradation. In the present study, two reaction pathways of radical and photo-induced electron transfer reactions synchronously occurred to UVB irradiated EC and C with the generation of three types of photoproducts, which complemented the reported conclusion [18].

2.6. Antioxidant Activities The antioxidant activities of individual catechins (575 µM, ethanol) determined by DPPH assay were shown in Table2. EGCg had the highest DPPH scavenging abilities; 2064 ± 29 µM trolox, followed by GCg, ECg, Cg, EGC, GC, EC and C in that order, which indicates that gallated catechins possessed higher antioxidant capacities than non-gallated catechins and epi-type catechin was more antioxidant active than the corresponding non-epi type at the same molality. This result was consistent with the antioxidant activity rank of different catechins reported by Lee et al. [38]. Upon 6 h of UVB irradiation, no significant difference in DPPH scavenging ability was observed for the eight catechins. This was in line with previous result that no obvious photo-degradation was observed for catechins except EC and C under 6 h UVB radiation. For EC and C, photoisomerization and photolysis reaction may not significantly influence their antioxidant activities in the short-term but could be an important cause of browning deterioration of catechins-containing products in color. Besides, a yellowish color change was also observed in the aqueous solutions of EC and C after 15-day-storage under laboratory illumination (Supplementary Materials Figure S1), indicating that UVB radiation is not a necessary condition for photo-degradation of EC and C. Nevertheless, the photoproducts of EC and C under laboratory illumination need identification. The photosensitivities of EC and C provide a new aspect Molecules 2016,, 21,, 13451345 9 of 1314 and C under laboratory illumination need identification. The photosensitivities of EC and C provide fora new solving aspect the for browning solving the problem browning of catechin-containing problem of catechin-containing products via diminishingproducts via color diminishing deterioration color originatingdeterioration from originating EC and C.from EC and C.

O OH O O OH O

O HO O HO hv hv HO O OH OH OH OH OH C/EC OH m/z 289 Compound P2 m/z 287

Photolysis hv OH OH OH OH OH O OH HO OH HO O OH HO OH OH hv CH3CH2OH Photo-induced

electron transfer OH OCH2CH3 OH OH OH Compound P5 O OH m/z 335 HO OH hv H OH

OH OH OH OH O OH OH HO HO HO OH OH + CH2 H OH OH OH O

Free radical A Free radical B CH2 m/z 137 m/z 289 HO OH

Rearrangement of -OH hv OH

HO OH OH OH HO O OH OH CH2 OH HO Compound P1 m/z 137 OH Isomers P6-9 m/z 427

FigureFigure 6. Proposed 6. Proposed photolytic photolytic reac reactiontion mechanism mechanism of ofEC/C. EC/C.

Table 2. The 2,2-diphenylpicrylhydrazyl (DPPH) scavenging abilities of individual catechins before Table 2. The 2,2-diphenylpicrylhydrazyl (DPPH) scavenging abilities of individual catechins before 1 and after UVB radiation (µμM trolox) 1. Treatment EC C EGC GC ECg Cg EGCg GCg BeforeTreatment UVB radiation 1219 EC ± 49 967 C ± 48 EGC1376 ± 27 1285 GC ± 7 1836 ECg ± 28 1736 Cg ± 43 2064 EGCg ± 29 1996 GCg ± 53 BeforeAfter UVB radiationradiation 1219 1136± 49 ± 33 967 921± 48 ± 30 1376 1363± 27 ± 12 1285 1274± ±7 30 1836 1808± 28± 31 1736 1745± ±43 23 2064 2064± ±29 22 1996 2026± ±53 21 After UVB radiation 1136 ± 33 921 ± 30 1363 ± 12 1274 ± 30 1808 ± 31 1745 ± 23 2064 ± 22 2026 ± 21 1 The ethanol solutions of individual catechins (575 μM) were UVB irradiated for 6 h. 1 The ethanol solutions of individual catechins (575 µM) were UVB irradiated for 6 h.

Molecules 2016, 21, 1345 10 of 13

3. Materials and Methods

3.1. Chemicals The individual catechins (−)-epigallocatechin gallate (EGCg, ≥98%), (−)-epigallocatechin (EGC, ≥98%), (−)-epicatechin gallate (ECg, ≥98%), (−)-epicatechin (EC, ≥98%), (−)-gallocatechingallate (GCg, ≥98%), (−)-gallocatechin (GC, ≥98%), (−)-catechin gallate (Cg, ≥98%), (−)-catechin (C, ≥98%), and gallic acid (GA, ≥98%) were purchased from Aladdin Industrial Corporation (Shanghai, China). The DPPH was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). The (±)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) was purchased from Sigma-Aldrich (Shanghai, China). Chemical purity grade ethanol was purchased from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). Acetic acid glacial (TEDIA company, Fairﬁeld, OH, USA), methanol and acetonitrile (Avantor performance materials, Inc., Center Valley, PA, USA) were of HPLC (High performance liquid chromatography) grade. The Milli-Q water was prepared by an EASYPure II UV UltraPure Water System (Barnstead International, Dubuque, IA, USA).

3.2. Photosensitivities of Eight Catechins to UVB The solutions of individual catechins were freshly prepared at 575 µM with water and ethanol respectively. Two milliliters of each catechin solution was placed in a 2 mL polypropylene centrifuge tube which was exposed to UVB for 6 h with a fully opening lid, and was then stored at −20 ◦C to terminate reactions. The intensity of UVB was achieved at 100 µW·cm−2 by placing samples under six UV lamps (SPECTRONICS BLE-1T158 Tube 15 watt, main output at 312 nm) at a distance of 45 cm, which was close to the UVB intensity in the sunlight. All samples were turned back to room temperature, and made up to 2 mL prior to HPLC analysis. In a pretest, the potential UVB absorbing effect of polypropylene tube wall on the UVB-induced chemical conversion of catechins (575 µM, ethanol) was evaluated by comparing with UVB-transmissive Q-cuvette. The result showed that the photo-induced conversion rates of EC and C in polypropylene tubes slightly decreased by ~8% compared with Q-cuvette, while no signiﬁcant difference was observed for other catechins. The polypropylene centrifuge tubes were used in the following studies considering operational convenience and practical use.

3.3. Effect of UVB Radiation Time on Conversions of EC and C According to the recovery of catechins in Section 3.2, EC and C were UVB-susceptible and selected for further investigation. The solutions of EC and C were prepared at 575 µM with water and ethanol respectively. Two milliliters of EC and C solutions were exposed to UVB for 2 h, 4 h, 6 h, 8 h and 10 h, respectively. All samples were treated as above and analyzed by HPLC.

3.4. Effect of Substrate Concentration on Conversions of EC and C The solutions of EC and C were respectively prepared at 71.875, 143.75, 287.5, 575 and 1150 µM by water and ethanol. Two milliliters of C and EC solutions at various concentrations were exposed to UVB for 6 h. All samples were treated as described in Section 3.2 and analyzed by HPLC.

3.5. HPLC Analysis All samples were centrifuged at 12,000 rpm for 10 min before HPLC analysis. The concentrations of catechins and GA were analyzed by HPLC according to a previous paper [39]. The HPLC conditions were: injection volume 10 µL, Agilent TC-C18 column (4.6 mm × 250 mm, Agilent Technologies, Santa Clara, CA, USA), column temperature 32 ◦C, mobile phase A = acetonitrile/acetic acid/water (6:1:193, v), mobile phase B = acetonitrile/acetic acid/water (60:1:139, v), linear gradient elution: from 80% (v) A and 20% (v) B to 35% (v) A and 65% (v) B for the first 35 min and then 80% (v) A and 20% (v) B until 40 min, flow rate 1 mL·min−1, Shimadzu SPD ultraviolet detector at 280 nm. Catechins were quantified according to external standard calibration by comparing with the corresponding authentic standards. Molecules 2016, 21, 1345 11 of 13

3.6. Identiﬁcation of the Photoproducts of EC and C Ninety milliliters of the aqueous and ethanol solutions of EC and C (287.5 µM) were respectively exposed to UVB for 6 h as described in Section 3.2. The reaction mixtures of EC and C were submitted to rotary evaporator, concentrated to 10 mL at 40 ◦C under vacuum and sampled for UPLC-DAD-MS/MS analysis and the left concentrate was freeze dried for 1H-NMR measurement.

3.6.1. UPLC-DAD-MS/MS Analysis UPLC-DAD-MS/MS (Waters Corporation, Milford, DE, USA) was employed to provide the MS information of relevant photoproducts. The UPLC conditions were: Acquity UPLC HSST3 column (2.1 mm × 150 mm, 1.8 µm), column temperature 35 ◦C, injection volume 5 µL, mobile phase A = 0.1% formic acid + 99.9% water (v/v), mobile phase B = 0.1% formic acid + 99.9% acetonitrile (v/v), linear gradient elution: from 99.9% (v) A/0.1% (v) B to 10% (v) A/90% (v) B for 38 min, ﬂow rate 0.3 mL·min−1. An electrospray ionization (ESI) technique in a negative ion mode was employed for MS analysis. The ion source conditions were set as follows: capillary voltage 3000 V, cone voltage 30 V, extractor 3.0 V and RF lens 0.2 V, ion source temperature 150 ◦C, desolvation gas nitrogen at a ﬂow rate of 800 L·h−1 and temperature at 300 ◦C. Full scan ranging from 100 to 1000 atomic mass unit (amu) were recorded. Argon was used as the collision gas. The collision energy was 20 eV. Triple quadrupole was set-up to daughter ion scan experiment, and the mass of parent ion was set at 427 m/z. UV-visible spectrum of reaction products was recorded in the 190–400 nm range by a photodiode detector.

3.6.2. 1H Nuclear Magnetic Resonance (NMR) Measurement 1H-NMR spectra were obtained from ~5 mg of EC and the corresponding reaction mixture suspended in 0.8 mL acetone-d 6. The spectra were recorded on an Agilent 600 MHz DD 2 with a 5 mm One NMR probe (Agilent Technologies, Santa Clara, CA, USA). Integration of the spectra was performed with Mnova NMR software (version 6.1.0, Mestrelab Research S. L., Santiago de Compostela, Spain).

3.7. Antioxidant Activity Measurement Antioxidant activity was evaluated by scavenging activity of DPPH free radical according to the modiﬁed method [40]. One hundred and eighty microliters of DPPH solution (0.4 mM, ethanol) and 20 µL of individual catechin solution (575 µM, ethanol) with and without 6 h UVB radiation were loaded onto a microplate, and then the mixture was incubated in dark at 30 ◦C for 30 min and measured by Epoch Microplate Spectrophotometer (BioTek Instruments Inc., Winooski, VT, USA) at 517 nm. Antioxidant capacity was expressed as µM trolox equivalents, using the linear calibration curve of trolox (100–1250 µM).

3.8. Data Analysis All analyses were carried out in triplicate. The mean values of the triplicate analysis ±SD are presented.

4. Conclusions Eight catechins showed different photosensitivities to UVB. EC and C were susceptible under UVB radiation while other six catechins were insensitive. Photoisomerization and photolysis were the main reactions in the photo-induced chemical transformations of EC and C, which were inﬂuenced by radiation time, solvent and substrate concentration. Two reaction pathways including radical reaction and photo-induced electron transfer reaction were involved in the photolysis reaction of EC and C. No signiﬁcant change was observed in the antioxidant activities of eight catechins upon 6 h UVB irradiation.

Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/21/ 10/1345/s1. Molecules 2016, 21, 1345 12 of 13

Acknowledgments: This work was supported by the Zhejiang Provincial Natural Science Foundation of China under Grant (LQ16C160001) awarded to Jian-Hui Ye. Author Contributions: Y.-R.L. and J.-H.Y. conceived and designed the experiments; M.S. performed the experiments; Y.N. and J.-L.L. analyzed the data; X.-Q.Z. contributed reagents/materials/analysis tools; J.-H.Y. wrote the paper. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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Sample Availability: Samples of UV B irradiated products of EC and C are available from the authors.

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