Structure Determination of Novel Oxidation Products from Epicatechin: Thearubigin-Like Molecules

molecules

Article Structure Determination of Novel Oxidation Products from Epicatechin: Thearubigin-Like Molecules

Kazuhiro Uchida, Kazuki Ogawa and Emiko Yanase *

Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan; [email protected] (K.U.); [email protected] (K.O.) * Correspondence: [email protected]; Tel.: +80-58-234-2914

Academic Editor: Derek J. McPhee Received: 29 January 2016 ; Accepted: 23 February 2016 ; Published: 26 February 2016

Abstract: Following the oxidation of epicatechin (EC), three novel compounds and two known compounds were isolated. The chemical structures of these oxidation products were determined by mass spectrometry (MS) and various nuclear magnetic resonance (NMR) experiments, and the A-ring–B-ring linkage that is characteristic of catechin was found in each molecule. Three compounds showed similar ultraviolet–visible (UV-Vis) spectra to EC, whereas two compounds showed different spectral absorption in the region between 300 and 500 nm. A similar spectrum was obtained for the thearubigin fraction prepared from a black tea infusion. This result suggests that the condensation reaction between the A-ring and B-ring is more important than reaction between B-rings for thearubigin formation.

Keywords: epicatechin; oxidation; thearubigin; theaﬂavine; polymeric polyphenol; black tea

1. Introduction Black tea is a globally popular beverage that has attracted attention because of its health benefits, including antioxidative and anticarcinogenic activities. Tea can be classified by differences in processing methods, for example, black tea is a fermented tea. The term “fermented” refers to natural browning reactions induced by oxidative enzymes of the fresh tea leaves. Catechins are one of the components in tea that are drastically changed in this process, and dimers and polymers are generated. The polymer found in black tea is called thearubigin. The name thearubigin was given to an ill-defined group of substances by Roberts in 1962. Thearubigin has a rust-brown color and high water solubility. Many researchers have studied the chemical structure of thearubigin, and the average molecular weight was estimated to be approximately 5000 to 30,000. Thearubigin constitutes approximately 10%–20% of the dry weight of black tea leaves; however, detailed chemical studies have thus far been limited because the numerous components found in the extract leads to a complicated mixture, which makes preparative scale isolation difficult [1–4]. Theaflavins [5–8] are one of the pigments in black tea that have a familiar bright red tone and various bioactivities [9,10]. In the synthesis of theaflavin from epicatechin (1, EC) and epigallocatechin, the synthetic yield increased when an excessive amount of 1 was used for this reaction. However, the recovery of 1 was low after the reaction. This result suggested that another reaction could occur between catechol-type catechins. We consider reactions between catechol-type catechins likely to occur during the fermentation process of black tea and thus be involved in thearubigin generation. Therefore, it is important to clarify the chemical structures of the byproducts from the synthesis of theaflavin. In this study, to increase knowledge of the chemical structure of thearubigin, we investigated the oxidation reaction of 1. Structure determination of the three novel and two known compounds

Molecules 2016, 21, 273; doi:10.3390/molecules21030273 www.mdpi.com/journal/molecules Molecules 2016, 21, 273 2 of 9

MoleculesIn 2016this, 21study,, 273 to increase knowledge of the chemical structure of thearubigin, we investigated2 of 9 the oxidation reaction of 1. Structure determination of the three novel and two known compounds isolated showed the importance of condensation reactions between catechin A- and B-rings for isolated showed the importance of condensation reactions between catechin A- and B-rings for thearubigin formation. thearubigin formation. 2. Results and Discussion 2. Results and Discussion Oxidation of 1 was performed using copper chloride, and the reaction mixture was analyzed Oxidation of 1 was performed using copper chloride, and the reaction mixture was analyzed using using high-performance liquid chromatography with ultraviolet detection (HPLC-UV) (Figure 1). As high-performance liquid chromatography with ultraviolet detection (HPLC-UV) (Figure1). As a result, a result, 1 and five unknown peaks, which were determined to be two dimers (peak c and d) and 1 and ﬁve unknown peaks, which were determined to be two dimers (peak c and d) and three trimers three trimers (peak a, b and e), were observed. The yield of each peak depended on the reaction (peak a, b and e), were observed. The yield of each peak depended on the reaction conditions, oxidizing conditions, oxidizing agent, reagent equivalents, reaction time, etc. For isolation and structure agent, reagent equivalents, reaction time, etc. For isolation and structure determination of each peak, determination of each peak, the reaction was performed under optimum conditions for that peak. the reaction was performed under optimum conditions for that peak. The reaction solution was The reaction solution was extracted sequentially with ether and ethyl acetate. According to the HPLC extracted sequentially with ether and ethyl acetate. According to the HPLC analysis of each layer, analysis of each layer, the water layer contained peaks a and b, whereas the ethyl acetate layer the water layer contained peaks a and b, whereas the ethyl acetate layer contained peaks c, d, and e. contained peaks c, d, and e. Unreacted 1 was extracted into the ether layer. To collect peaks a and b, Unreacted 1 was extracted into the ether layer. To collect peaks a and b, the water layer was loaded into the water layer was loaded into a column packed with Diaion HP20SS particles and eluted with a column packed with Diaion HP20SS particles and eluted with MeOH. Each peak was separated using MeOH. Each peak was separated using preparative HPLC and characterized using spectroscopic preparative HPLC and characterized using spectroscopic techniques, including 1D and 2D nuclear techniques, including 1D and 2D nuclear magnetic resonance (NMR) and mass spectrometry. magnetic resonance (NMR) and mass spectrometry.

FigureFigure 1.1. HPLCHPLC chromatogramchromatogram ofofthe thereaction reactionmixture mixture obtained obtainedfollowing followingoxidation oxidation ofofepicatechin epicatechin (EC)(EC) 2 1 (monitored(monitored atat 280280 nm). Peaks Peaks were were shown shown as as a–d. a–d. a: a:dehydrotricatechin dehydrotricatechin B ( B5),2 b:(5 ),dehydrotricatechin b: dehydrotricatechin B (6), 1 1 Bc1: epicatechin-6(6), c: epicatechin-6′,6-dimer (,6-dimer2), d: epicatechin-6 (2), d: epicatechin-6′,8-dimer (3), e:,8-dimer dehydrotricatechin (3), e: dehydrotricatechin A (4), EC: epicatechin A ( (14).), EC: epicatechin (1). 2.1. Structure Determination for Peaks c and d 2.1. StructureNegative Determination mode electrospray for Peaks io cnization and d mass spectrometry (ESIMS) measurements of peaks c and Negatived both showed mode electrospray a pseudo-molecular ionization massion peak spectrometry at m/z 577 (ESIMS) ([M − measurements H]⁻), suggesting of peaks that cthese and 1 dcompounds both showed are a pseudo-moleculardimers of 1. The ionH-NMR peak atspectram/z 577 of peaks ([M ´ H]c and´), suggestingd were similar that theseand indicated compounds the arepresence dimers of of 1two. The EC1H-NMR units that spectra had oflost peaks onec andaromdaticwere proton similar from and indicatedboth the theA-ring presence and ofB-ring. two ECFurthermore, units that had the lost 1H- one and aromatic 13C-NMR proton spectral from signals both the were A-ring assigned and B-ring. using Furthermore, correlation spectroscopy the 1H- and 13(COSY),C-NMR spectralheteronuclear signals multiple were assigned quantum using cohere correlationnce (HMQC), spectroscopy and (COSY), heteronuclear heteronuclear multiple-bond multiple quantumcorrelation coherence (HMBC). (HMQC), As a result, and the heteronuclear chemical structure multiple-bond of peak correlation c was deduced (HMBC). to be As the a result, EC dimer the chemicalwith a connection structure of between peak c was the deduced6-position to beof the ECA-ring dimer in withone amolecule connection and between the 2′-position the 6-position of the ofB-ring the A-ring in the in other one molecule(compound and 2 the). On 21 -positionthe other of hand, the B-ring peak ind was the otherfound (compound to be the EC2). dimer On the with other a hand,connection peak d between was found the to 8-position be the EC of dimer the A-ring with a connectionand the 2′-position between theof the 8-position B-ring (compound of the A-ring 3 and), as

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Molecules 2016, 21, 273 3 of 9 the 21-position of the B-ring (compound 3), as shown in Figure2. Compounds 2 and 3 have with been previouslyshown in Figure reported 2. Compounds as the products 2 and of 3 radical have with oxidation been previously of 1, and the repo spectralrted as data the products were in agreement of radical publishedoxidation of data 1, and [11 ].the As spectral a result, data compounds were in agreement2 and 3 were published identiﬁed data as [11]. epicatechin-6 As a result,1,6-dimer compounds and epicatechin-62 and 3 were 1identified,8-dimer. as epicatechin-6′,6-dimer and epicatechin-6′,8-dimer.

Figure 2. Chemical structures of compounds 1–6.

2.2. Structure Determination for Peak e Negative mode mode ESIMS ESIMS measurements measurements of of peak peak e eshowed showed a pseudo-molecular a pseudo-molecular ion ion peak peak at m at/zm 863/z 863([M ([M− H]´⁻),H] suggesting´), suggesting that this that compound this compound is a trimer is a trimer of 1. The of 1 .mass The massnumber number was 2 was Da 2smaller Da smaller than thanthe expected the expected molecular molecular weight weight calculated calculated for a simple for a EC simple trimer EC with trimer connections with connections between betweenEC units ECsimilar units to similarthose in to dimers those 2 in and dimers 3. This2 and result3. Thisindicated result that indicated the EC units that the were EC connected units were via connected a double viabond a doubleor three bond linkages. or three The linkages. 1H-NMR The spectrum1H-NMR showed spectrum the showed presence the presenceof three pairs of three of pairssignals of signalscorresponding corresponding to the to2-positions the 2-positions and 3-positions and 3-positions of the of theC-rings, C-rings, indicating indicating that that these these rings rings were unmodiﬁed.unmodified. HMQCHMQC and and HMBC HMBC data data showed showed that that the linkagesthe linkages of the of three the catechin three catechin units were units between were thebetween A- and the B-rings. A- and In B-rings. the 13C-NMR In the spectrum,13C-NMR aspectrum, characteristic a characteristic signal at 190 signal ppm indicatedat 190 ppm the indicated presence ofthe a presence ketone. Thisof a ketone. signal wasThis correlated signal was with correla methineted with (3.1 methine ppm), which,(3.1 ppm), in HMBC, which, wasin HMBC, correlated was withcorrelated the 7- with and the 8-positions 7- and 8-positions of an A-ring of an and A-ring 11-position and 1′-position of a B-ring, of a asB-ring, shown as inshown Figure in3 Figure. These 3. resultsThese results indicated indicated the presence the presence of a ketone of a ketone in a B-ring in a B-ring that hadthat losthad aromaticitylost aromaticity and theand formation the formation of a ﬁve-memberedof a five-membered ring viaring an via ether an ether bond betweenbond between the A-ring the A-ring of one of EC one unit EC and unit the and B-ring the of B-ring another of unit.another Furthermore, unit. Furthermore, the 1H- andthe 113H-C-NMR and 13C-NMR spectral spectral signals weresignals assigned were assigned based on based COSY, on HMQC, COSY, andHMQC, HMBC and (Figure HMBC3). (Figure As a result, 3). As the a re structuresult, the of structure compound of compound4 was determined, 4 was determined, as shown in as Figure shown2. Wein Figure havenamed 2. We have4 dehydrotricatechin named 4 dehydrotricatechin A. A.

Molecules 2016, 21, 273 4 of 9 Molecules 2016, 21, 273 4 of 9

Figure 3. HMBCHMBC correlations for compounds 4 and 6.

2.3. Structure Structure Determination for Peaks a and b Peak a a was was analyzed analyzed by by negative negative mode mode ESIMS. ESIMS. The The appearance appearance of ofan an [M [M − H]´ ⁻H] ion´ peakion peak at m at/z 863,m/z which863, which was wasthe same the same as that as thatobserved observed for compound for compound 4, suggested4, suggested that that compound compound 5 is5 ais trimer a trimer of 1of with1 with three three linkages. linkages. In Incontrast contrast to to those those of of 4,4 ,the the 1H-1H- and and 1313C-NMRC-NMR spectra spectra of of 55 werewere simple simple and similar toto thosethose of of monomeric monomeric1, 1 except, except for for the the lack lack of one of one aromatic aromatic proton proton from eachfrom A-ringeach A-ring and B-ring. and B-ring.Although Although compound compound5 was clearly 5 was different clearly fromdifferent1, we from could 1, notwe obtaincould furthernot obtain spectral further evidence spectral to evidenceconfirm the to confirm complete the structure complete of structure peak a. of peak a. Peak b showed aa [M[M ´− H]´⁻ peakpeak at at mm//zz 861861 in in negative negative mode mode ESIM ESIMS,S, indicating indicating that this compound is another EC trimer. Three sets of sign signalsals arising from the 2-positions and 3-positions of the C-rings were observed in the 1H-NMRH-NMR spectrum, spectrum, indicating indicating the the pr presenceesence of of unmodified unmodified C-rings. C-rings. The HMBC correlations observed between A-ring pr protonsotons and B-ring carbons indicated that the EC units are connectedconnected betweenbetween the the A- A- and and B-rings. B-rings. The The¹³ C-NMR¹³C-NMR signals signals at 190at 190 and and 180 180 ppm ppm indicated indicated the thepresence presence of two of ketonetwo ketone groups. groups. Based Based on HMQC, on HMQC, the correlations the correlations between between these ketones these ketones and protons and protonswith signals with atsignals 4.47 and at 4.47 2.81 and ppm 2.81 indicated ppm indicated that the that ketones the ketones are geminal. are geminal. Furthermore, Furthermore, the HMBC the HMBCcorrelations correlations of these ketonesof these indicated ketones aindicated chemical a skeleton chemic thatal skeleton could be that attributed could tobe theattributed 1,4-conjugated to the 1,4-conjugatedaddition of the phenolicaddition OH of inthe the phenolic A-ring to OHO-quinone, in the A-ring resulting to in O a-quinone, loss of aromaticity. resulting Allin remaininga loss of aromaticity.signals in the All1H- remaining and 13C-NMR signals spectra in the were 1H- assignedand 13C-NMR based spectra on COSY, were HMQC, assigned and HMBCbased on (Figure COSY,3). HMQC,As a result, and the HMBC structure (Figure of compound3). As a result,6 was the determined, structure of ascompound shown in 6 Figure was determined,2. We have as named shown6 in Figure 2. We have named 6 dehydrotricatechin B1. dehydrotricatechin B1. Compound 6 was unstable in CD3OD and was converted to another compound with an NMR Compound 6 was unstable in CD3OD and was converted to another compound with an NMR spectrum consistent with that of peak a. On the other hand, the CD3OD solution of peak a did not spectrum consistent with that of peak a. On the other hand, the CD3OD solution of peak a did not show conversion toto compoundcompound6 6.. ThisThis result result indicated indicated that that compound compound6 6was was irreversibly irreversibly converted converted to tocompound compound5. Based5. Based on thison observationthis observation and the and NMR the andNMR ESIMS and dataESIMS described data described above, the above, chemical the chemical structure of compound 5 was proposed, as shown in Figure 2. We have named 5 structure of compound 5 was proposed, as shown in Figure2. We have named 5 dehydrotricatechin B2. dehydrotricatechin B2. 2.4. Formation Mechanisms of Compounds 2–6 2.4. Formation Mechanisms of Compounds 2–6 On the basis of the chemical structures of compounds 2–6, we proposed mechanisms for the formationOn the of basis these of compounds the chemical from structures1, as shown of compounds in Figure4 .2– When6, we proposed1 is treated mechanisms with an oxidizing for the formationagent, the catechol-typeof these compounds B-ring is from oxidized 1, as to shown form O in-quinone. Figure 4. The When nucleophilic 1 is treated addition with ofan the oxidizing B-rings agent,of epigallocatechin the catechol-type and epigallocatechin B-ring is oxidized gallate to form to O -quinoneO-quinone. occurs The becausenucleophilic of their addition high electron of the B-ringsdensity, of and epigallocatechin eventually leads and to theepigallocatechin formation of theaflavins gallate to O [11-quinone]. On the occurs other hand,because the of nucleophilic their high electronaddition density, of 1 to O and-quinone eventually occurs leads at the to A-ring the formation becauseof of its theaflav higherins electron [11]. On density the other compared hand, with the nucleophilicthat of the B-ring. addition Nucleophilic of 1 to O-quinone addition occurs at the C8at the or C6 A-ring position because results of inits thehigher formation electron of density dimers, comparedsuch as compounds with that 2ofand the3 ,B-ring. or trimers, Nucleophilic such as compound addition at5. the Furthermore, C8 or C6 theposition catechol results unit in of the formation of dimers, such as compounds 2 and 3, or trimers, such as compound 5. Furthermore, the catechol unit of the resulting product can be oxidized again to O-quinone and converted to

Molecules 2016, 21, 273 5 of 9 resultingMolecules product 2016, 21 can, 273 be oxidized again to O-quinone and converted to compound 4 by intramolecular5 of 9 cyclization of the C7-hydroxy group with the C51 position. Intramolecular cyclization can also occur compound 4 by intramolecular cyclization of the C7-hydroxy group with the C5′ position. with theIntramolecular C1 position cyclization to generate can compoundalso occur wi6th. Compoundthe C1 position4 is to converted generate compound into compound 6. Compound5 through a ring4 openingis converted reaction into compound caused by 5 through aromatization. a ring opening This formation reaction caused mechanism by aromatization. suggested This that the polymerizationformation mechanism of catechin suggested results from that itsthe oxidation, polymerization and further of catechin oxidation results offrom these its productsoxidation,results and in the formationsfurther oxidation of compounds of these products such as results4 and in6. the formations of compounds such as 4 and 6.

FigureFigure 4. Formation4. Formation mechanisms mechanisms of compounds compounds 2–26– from6 from 1. 1.

2.5. UV-Vis2.5. UV-Vis Spectra Spectra To study the relationship between chemical structure and color, the ultraviolet–visible (UV-Vis) To study the relationship between chemical structure and color, the ultraviolet–visible (UV-Vis) spectra of compounds 2–6 were obtained. Compounds 2, 3, and 5 showed similar spectra to that of 1, spectra of compounds 2–6 were obtained. Compounds 2, 3, and 5 showed similar spectra to that of which has a maximum absorption wavelength at 278 nm. On the other hand, compounds 4 and 6 1, whichshowed has spectral a maximum absorption absorption in the region wavelength between at 300 278 and nm. 500 On nm, the which other was hand, not compounds observed in the4 and 6 showedspectrumMolecules spectral 2016 of, 211 absorption ,(Figure 273 5). This in the result region indicated between that 300the formation and 500 nm,of the which new five-membered was not observed ring6 of in in9 the spectrumcompounds of 1 (Figure 4 and 65 ).allowed This result conjugation indicated between that an the A-ring formation and B-ring, of the resulting new ﬁve-membered in absorption in the ring in complex mixture that contains some products formed by conjugation between catechin and other compoundsvisible range.4 and 6 allowed conjugation between an A-ring and B-ring, resulting in absorption in the components in addition to polymers derived only from catechins. visible range.Oxidation of 1 was performed under strong oxidation conditions using K3Fe(CN)6/NaHCO3, and the reaction mixture was extracted using ether, EtOAc, and n-BuOH, in the listed order. When each extract was analyzed by HPLC, the n-BuOH extract produced broad overlapping peaks. In addition, line broadening was observed in the ¹H-NMR spectrum of the n-BuOH extract. These results suggested that the n-BuOH extract contained thearubigin-like polymers that were generated by the oxidation reaction. The UV-Vis spectrum of this n-BuOH fraction showed similar spectral features to those of compounds 4 and 6. This result indicated that the components in the n-BuOH fraction have chromophores with structures similar to those of compounds 4 and 6. Thearubigins are the most abundant pigments in black tea and are formed by enzymatic oxidization of catechins during the fermentation process. These compounds are polymeric polyphenols with unresolved structures [2–4]. To obtain the UV-Vis spectrum of these compounds, the hot water extract of black tea leaves was extracted using ether, EtOAc, and n-BuOH, in the listed order, and the n-BuOH extract was further fractionated into two fractions (Fr. 1 and 2) using a Toyopearl HW-40C column. The UV-Vis spectrum of Fr. 2, which had a reddish-brown color, showed a wide absorption band in the 300–500 nm region, similar to that of the n-BuOH extract of the oxidation mixture of 1 described above. The absorption band of the black tea extract was observed at 360 nm, which was somewhat blue-shifted in comparison to those of compounds 4 and 6 and the n-BuOH extract of the oxidation mixture of 1. It is assumed that this difference is because thearubigins from black tea are a

FigureFigure 5. UV-Vis 5. UV-Vis spectra spectra of of compounds compounds 11,, 4, and and 66, ,and and the the n-BuOHn-BuOH extracts. extracts.

3. Experimental Section

3.1. Materials EC was purchased from Sigma-Aldrich Co. LLC (St. Louis, MO, USA). Copper(II) chloride dihydrate and potassium hexacyanoferrate(III) were purchased from Nacalai Tesque Co. (Kyoto, Japan). Sodium hydrogen carbonate was purchased from Kishida Chemical Co., Ltd. (Osaka, Japan). CD3OD was purchased from Kanto Chemical Co. (Tokyo, Japan). Acetone-d6 was purchased from Acros Organics Co. (Morris Plains, NJ, USA).

3.2. Instrumentation

1H- and 13C-NMR, 1H-COSY, HMQC, and HMBC spectra were recorded on a JEOL ECA 600 spectrometer (JEOL, Tokyo, Japan) at 600 MHz for 1H and a Bruker Biospin AVANCE III 800 spectrometer (Bruker, Germany) at 200 MHz for 13C. ESIMS spectra were measured on a JMS-T100TD mass spectrometer (JEOL). Column chromatography was carried out using Diaion HP20SS (Mitsubishi Chemical Co., Tokyo, Japan) and Toyopearl HW-40C (Tosho Co., Tokyo, Japan). HPLC was performed with a JASCO PU-2089 or JASCO PU-887 system using a reverse-phase HPLC column (NB-ODS-9 4.6 mm I.D. × 250 mm, NX-ODS-9 10 mm I.D. × 250 mm, Nagara Science Co., Gifu, Japan) and JASCO MD-2010 or JASCO 875-UV detectors (JASCO Co., Tokyo, Japan). UV-Vis spectra were recorded on a Perkin Elmer Lambda 950 UV/VIS/NIR spectrometer (Kanagawa, Japan).

3.3. Synthesis of a and b

An aqueous solution of EC (0.2 mmol) and CuCl2·2H2O (1.0 mmol) in H2O was vigorously stirred at room temperature for 3 h. The reaction mixture was extracted with Et2O. The water layer was extracted with EtOAc. The water layer was concentrated until EtOAc had been removed and the resulting solution was subjected to column chromatography using a HP20SS resin. The column was eluted with MeOH after washing with water to remove CuCl2, and the resulting solution was separated by reverse-phase HPLC (25% MeOH, 0.5% HCOOH in H2O) to afford a and b.

Molecules 2016, 21, 273 6 of 9

Oxidation of 1 was performed under strong oxidation conditions using K3Fe(CN)6/NaHCO3, and the reaction mixture was extracted using ether, EtOAc, and n-BuOH, in the listed order. When each extract was analyzed by HPLC, the n-BuOH extract produced broad overlapping peaks. In addition, line broadening was observed in the ¹H-NMR spectrum of the n-BuOH extract. These results suggested that the n-BuOH extract contained thearubigin-like polymers that were generated by the oxidation reaction. The UV-Vis spectrum of this n-BuOH fraction showed similar spectral features to those of compounds 4 and 6. This result indicated that the components in the n-BuOH fraction have chromophores with structures similar to those of compounds 4 and 6. Thearubigins are the most abundant pigments in black tea and are formed by enzymatic oxidization of catechins during the fermentation process. These compounds are polymeric polyphenols with unresolved structures [2–4]. To obtain the UV-Vis spectrum of these compounds, the hot water extract of black tea leaves was extracted using ether, EtOAc, and n-BuOH, in the listed order, and the n-BuOH extract was further fractionated into two fractions (Fr. 1 and 2) using a Toyopearl HW-40C column. The UV-Vis spectrum of Fr. 2, which had a reddish-brown color, showed a wide absorption band in the 300–500 nm region, similar to that of the n-BuOH extract of the oxidation mixture of 1 described above. The absorption band of the black tea extract was observed at 360 nm, which was somewhat blue-shifted in comparison to those of compounds 4 and 6 and the n-BuOH extract of the oxidation mixture of 1. It is assumed that this difference is because thearubigins from black tea are a complex mixture that contains some products formed by conjugation between catechin and other components in addition to polymers derived only from catechins.

3. Experimental Section

3.2. Instrumentation 1H- and 13C-NMR, 1H-COSY, HMQC, and HMBC spectra were recorded on a JEOL ECA 600 spectrometer (JEOL, Tokyo, Japan) at 600 MHz for 1H and a Bruker Biospin AVANCE III 800 spectrometer (Bruker, Germany) at 200 MHz for 13C. ESIMS spectra were measured on a JMS-T100TD mass spectrometer (JEOL). Column chromatography was carried out using Diaion HP20SS (Mitsubishi Chemical Co., Tokyo, Japan) and Toyopearl HW-40C (Tosho Co., Tokyo, Japan). HPLC was performed with a JASCO PU-2089 or JASCO PU-887 system using a reverse-phase HPLC column (NB-ODS-9 4.6 mm I.D. ˆ 250 mm, NX-ODS-9 10 mm I.D. ˆ 250 mm, Nagara Science Co., Gifu, Japan) and JASCO MD-2010 or JASCO 875-UV detectors (JASCO Co., Tokyo, Japan). UV-Vis spectra were recorded on a Perkin Elmer Lambda 950 UV/VIS/NIR spectrometer (Kanagawa, Japan).

3.3. Synthesis of a and b

An aqueous solution of EC (0.2 mmol) and CuCl2¨ 2H2O (1.0 mmol) in H2O was vigorously stirred at room temperature for 3 h. The reaction mixture was extracted with Et2O. The water layer was extracted with EtOAc. The water layer was concentrated until EtOAc had been removed and the resulting solution was subjected to column chromatography using a HP20SS resin. The column was eluted with MeOH after washing with water to remove CuCl2, and the resulting solution was separated by reverse-phase HPLC (25% MeOH, 0.5% HCOOH in H2O) to afford a and b. ´ 1 Compound a (5): ESIMS, m/z: 863 ([M ´ H] ). H-NMR (600 MHz, CD3OD) δ: 6.74 (1H, s, H-b2), 6.64 (1H, s, H-b5), 6.01 (1H, s, H-6), 4.41 (1H, s, H-2), 3.90 (1H, m, H-3), 2.56 (1H, dd, J = 16.5 and 4.1 Hz, Molecules 2016, 21, 273 7 of 9

13 H-4). C-NMR (200 MHz, CD3OD) δ: 155.6 (C-5), 153.3 (C-7), 152.7 (C-8a), 143.7 (C-b3), 143.2 (C-b4), 130.7 (C-b1), 123.6 (C-b6), 117.8 (C-b5), 115.0 (C-b2), 106.5 (C-8), 98.2 (C-4a), 94.2 (C-6), 76.2 (C-2), 63.8 (C-3), 27.3 (C-4).

´ 1 Compound b (6): ESIMS, m/z: 861 ([M ´ H] ). H-NMR (600 MHz, acetone-d6) δ: 7.90 (1H, s, H-b211), 6.81 (1H, s, H-b511), 6.80 (1H, s, H-b5), 6.27 (1H, s, H-b2), 6.14 (1H, s, H-b51), 6.10 (1H, s, H-61), 6.07 (1H, s, H-611), 6.02 (1H, s, H-6), 4.73 (1H, s, H-211), 4.56 (1H, brs, H-31), 4.47 (1H, d, J = 14.4 Hz, H-b21), 4.45 (1H, brs, H-311), 4.36 (1H, s, H-2), 3.91 (1H, s, H-3), 3.52 (1H, s, H-21), 2.9 (1H, H-41; this 1 1 peak could not be observed in H-NMR due to overlap with the H2O signal), 2.84 (2H, m, H-b2 , 411), 2.68 (1H, dd, J = 12.6 and 3.0 Hz, H-411), 2.59 (1H, d, J = 12.6 Hz, H-4), 2.17 (1H, dd, J = 12.3 and 3.0 Hz, H-4), 2.1 (1H, H-41, this peak could not be observed in 1H-NMR due to overlap with 13 1 1 11 the acetone signal). C-NMR (200 MHz, acetone-d6) δ: 190.8 (C-b3 ), 177.4 (C-b4 ), 163.8 (C-7 ), 162.9 (C-8a11), 159.7 (C-b61), 156.1 (C-51), 156.0 (C-511), 155.4 (C-5), 154.2 (C-71), 153.9 (C-8a), 153.4 (C-7), 150.6 (C-8a1), 145.6 (C-b4), 144.0 (C-b411), 143.8 (C-b3), 131.4 (C-b1), 130.0 (C-b6), 127.3 (C-b111), 119.7 (C-b611), 119.5 (C-b2), 118.3 (C-b51), 117.9 (C-b511), 116.3 (C-b211), 114.7 (C-b5), 107.1 (C-8), 106.9 (C-81), 103.4 (C-811), 102.3 (C-4a11), 99.0 (C-4a1), 98.9 (C-4a), 95.7 (C-61), 94.0 (C-6), 90.6 (C-611), 90.0 (C-b11), 77.2 (C-21), 76.8 (C-211), 76.7 (C-2), 64.6 (C-311), 63.0 (C-3), 61.6 (C-31), 47.2 (C-21), 31.1 (C-411), 29.4 (C-41), 27.7 (C-4).

3.4. Synthesis of c and d

An aqueous solution of EC (0.2 mmol) and CuCl2¨ 2H2O (0.4 mmol) in H2O was vigorously stirred at room temperature for 1 h. The reaction mixture was extracted with Et2O. The water layer was extracted with EtOAc. The EtOAc layer was concentrated, and then separated by reverse-phase HPLC (25% MeOH, 0.5% HCOOH in H2O) to afford c and d.

3.5. Synthesis of e ˝ A solution of EC and K3Fe(CN)6 in NaHCO3 aq. was vigorously stirred at 0 C for 6 min. The mixture was sequentially extracted with Et2O, EtOAc, and n-BuOH, in the listed order. The EtOAc layer was concentrated, and then separated by reverse-phase HPLC (25% MeOH, 0.5% HCOOH in H2O) to afford e. The n-BuOH fraction was concentrated to afford a residue. ´ 1 Compound e (4): ESIMS, m/z: 863 ([M ´ H] ). H-NMR (600 MHz, CD3OD) δ: 7.04 (1H, d, J = 1.4 Hz, H-b211), 6.92 (1H, s, H-b2), 6.90 (1H, d, J = 6.0 Hz, H-b611), 6.81 (1H, d, J = 8.2 Hz, H-b511), 6.41 (1H, s, H-b5), 6.15 (1H, s, H-b21), 6.13 (1H, d, J = 2.1 Hz, H-8), 6.07 (1H, s, H-611), 6.03 (1H, s, H-61), 5.95 (1H, d, J = 2.0 Hz, H-6), 5.24 (1H, s, H-211), 5.00 (1H, d, J = 8.2 Hz, H-31), 4.38 (1H, s, H-2), 4.26 (1H, brs, H-311), 4.16 (1H, d, J = 2.7 Hz, H-21), 3.71 (1H, brs, H-3), 3.26 (1H, dd, J = 12.6 and 3.0 Hz, H-411), 3.1 (2H, m, H-b51, 41), 2.84 (1H, d, J = 12.0 Hz, H-411), 2.60 (1H, d, J = 13.8 Hz, H-41), 2.57 (1H, d, J = 12.6 Hz, H-4), 2.31 (1H, dd, J = 12.6 and 3.6 Hz, H-4). 13C-NMR (200 MHz, 1 1 11 11 CD3OD) δ: 191.3 (C-b4 ), 165.0 (C-b3 ), 164.1 (C-7 ), 163.5 (C-8a ), 156.7 (C-8a), 156.3 (C-5), 155.8 (C-7), 154.8 (C-51, 511), 153.6 (C-71), 151.2 (C-8a1), 144.4 (C-b411), 144.3 (C-b311), 143.2 (C-b4, C-b3), 130.8 (C-b111), 129.6 (C-b1), 122.1 (C-b6), 118.1 (C-b611), 117.7 (C-b5), 114.7 (C-b511), 114.1 (C-b2), 113.9 (C-b211), 108.5 (C-b21), 107.3 (C-81), 104.5 (C-811), 101.5 (C-4a11), 98.7 (C-4a1), 98.6 (C-4a), 95.6 (C-61), 95.2 (C-8), 94.6 (C-6), 93.8 (C-b61), 90.7 (C-611), 89.9 (C-b11), 78.7 (C-21), 78.6 (C-211), 76.1 (C-2), 68.1 (C-31), 65.4 (C-311), 64.1 (C-3), 39.0 (C-b51), 27.7 (C-4), 27.5 (C-411), 23.0 (C-41).

3.6. Extraction of Black Tea Black tea leaves (1.8 g) were reﬂuxed in water at 120 ˝C for 30 min. After cooling, the solution was extracted sequentially with Et2O, EtOAc, and n-BuOH, in the listed order. The n-BuOH extract was concentrated under reduced pressure to afford a residue. The n-BuOH extract was further fractionated using a Toyopearl HW-40C column (40% acetone, 8 M urea in 0.1 M HCl aq.) to afford Fr. 1, which had a light yellowish-brown color, and Fr. 2, which had a reddish-brown color. Molecules 2016, 21, 273 8 of 9

3.7. UV-Vis Spectra Each sample was dissolved in methanol at a concentration of 0.01 mg/mL. The spectrum of each sample was collected in the spectral range between 200 and 800 nm with a spectral resolution of 1 nm. The spectra were recorded against the corresponding solvent as a baseline.

4. Conclusions In conclusion, we investigated the oxidation reaction of 1 to increase knowledge of the chemical structure and formation mechanism of thearubigin. We determined the chemical structures of three novel trimers (compounds 4–6) and two known dimers (compounds 2 and 3). Furthermore, the UV-Vis spectrum of the black tea extract indicated that the chemical structures of thearubigins contain skeletons that are cyclized after the formation of linkages between A- and B-rings, as observed in compounds 4 and 6. Oxidative condensation between the B-rings of catechins has been considered the formation mechanism of thearubigins. However, this result suggests that the condensation reaction between A- and B-rings is another important mechanism for thearubigin formation. We consider this result important for resolving the chemical structures of thearubigins, and further studies are now in progress.

Acknowledgments: This work was supported by a JSPS KAKENHI Grant (Number 15K07427), by the Toyo Institute of Food Technology, and by Nagara Science Co., Ltd. Author Contributions: E.Y. conceived and designed the experiments; K.U. and K.O. performed the experiments and contributed to data collection. E.Y. and K.U. wrote the paper. Conﬂicts of Interest: The authors declare no conﬂict of interest.

Abbreviations

The following abbreviations are used in this manuscript:

EC Epicatechin HPLC High-performance liquid chromatography ESIMS Electrospray ionization mass spectrometry NMR Nuclear magnetic resonance COSY Correlation spectroscopy HMQC Heteronuclear multiple quantum coherence HMBC Heteronuclear multiple-bond correlation

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Sample Availability: Samples of the compounds are not available from the authors.

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