2.1. Isolation and LC-MS Analysis of RGWRT
In order to obtain the diverse structures of tea polyphenols, the chemical components of RGWRT were systematically studied. As a result, fifty-eight polyphenolic compounds were afforded and could be classified into two main categories: flavan-3-ols (1–21) and flavones (22–58). These isolated substances were used as standards in the UPLC-Q-TOF-MS analysis of RGWRT extract. The chromatographic peaks were identified by the comparison with retention time, accurate parent mass, and fragment ion spectrogram of standards, and the results confirmed that the main tea polyphenols have been obtained in the present study (Figure 1). Furthermore, the flavan-3-ols in RGWRT could be further subdivided into several subgroups, including catechins, flavoalkaloids, procyanidins, theasinensins and their corresponding derivatives. The flavones could be further categorized into different classes based on the number of hydroxyl groups, encompassing kaempferol, quercetin, and myricetin, along with their respective O-glycosides. The C-glycosides of flavone are presented as a distinct subclass. The specific structures of various types of tea polyphenols are illustrated in Figure 2. This is the first time that a wide variety of tea polyphenols with diverse structural types were obtained for sequential studies.
The structures of all polyphenols were elucidated on the basis of comprehensive analysis of 1D and 2D NMR and HRMS spectroscopic data, including eleven undescribed compounds (11, 12, 20, 30, 32, 34, 36, 43, 44, 46, and 48) and forty-seven known tea polyphenols. The known compounds were identified by comparing their spectral data with those reported in the literature as (−)-epicatechin (1) [34], (−)-epicatechin-3-O-gallate (2) [35], (−)-epicatechin-3-(3′-O-methyl)-gallate (3) [36], (−)-epigallocatechin (4) [37], (−)-epigallocatechin-3-O-gallate (5) [38], (−)-epigallocatechin-3-(3′-O-methyl)-gallate (6) [39], (−)-epiafzelechin-3-O-gallate (7) [40], (+)-catechin (8) [41], (−)-gallocatechin-3-O-gallate (9) [38], etc-pyrrolidinone F (10) [42], puerin IV (13) [19], puerin V (14) [19], puerin VI (15) [19], procyanidin B2 (16) [43], procyanidin B2-3′-O-gallate (17) [44], procyanidin B4-3′-O-gallate (18) [45], catechin-(4α→8)-epigallocatechin-3-O-gallate (19) [46], theasinensin A (21) [47], kaempferol (22) [48], astragalin (23) [49], kaempferol-3-O–β-D-galactopyranoside (24) [50], kaempferol-3-O-rutinoside (25) [51], kaempferol-3-O-robinobioside (26) [52], kaempferol-3-O-[β-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→6)]-β-D-glucopyranoside (27) [53], kaempferol-3-O-[β-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→6)]-β-D-galactopyranoside (28) [54], kaempferol-3-O-[2″-O-(E)-p-coumaroyl] [β-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→6)]-β-D-glucopyranoside (29) [55], kamoreokchaside I (31) [56], camellikaempferoside A (33) [57], camellikaempferoside C (35) [58], quercetin-3-O–β-D-glucoside (37) [59], quercetin-3-O–β-D-galactoside (38) [59], rutin (39) [60], quercetin-3-O-[β-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→6)]-β-D-glucopyranoside (40) [53], quercetin-3-O-[β-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→6)]-β-D-galcopyranoside (41) [53], camelliquercetiside B (42) [61], camelliquercetiside C (45) [61], camelliquercetiside A (47) [61], myricetin-3-O–β-D-glucopyranoside (49) [62], myricetin-3-O–β-D-rutinoside (50) [63], isovitexin (51) [64], isovitexin-2″-O-glucoside (52) [65], apigenin-6-C–α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranoside (53) [66], apigenin-6-C–α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranoside (54) [67], schaftoside (55) [68], isoschaftoside (56) [53], vitexin (57) [69], vitexin-2″-O-glucoside (58) [65], respectively.
2.2. Structure Elucidation of New Tea Polyphenols from RGWRT
Compound 11 was obtained as white amorphous powder. The molecular formula was deduced as C21H23NO8 from a molecular ion peak at m/z 416.1331 [M − H]− (calcd for C21H22NO8, 416.1345) in the HR-ESI-MS. The presence of a flavan-3-ol skeleton was inferred obviously from the 1H and 13C NMR spectra (recorded in methanol-d4) of 11 (Table 1, Figure S2) [37]. More specifically, the typical proton signals at δH 6.01 (1H, s) for ring A, at δH 6.51 (2H, s, H-2′, H-6′) for ring B, and at δH 4.76 (1H, s, H-2), δH 4.20 (1H, s, H-3), δH 2.86 (1H, m, H-4β), δH 2.76 (1H, m, H-4α) for ring C, were similar to those of (−)-epigallocatechin (EGC) (Table 1). A single proton signal on the A ring indicated a replacement at C-6 or C-8. Besides the similar signals with EGC skeleton, the 1H NMR spectrum of 11 indicated the presence of three sets of methylene signals at δH 2.66, 2.42 (each 1H, m) for H-3″, δH 2.17 and 2.34 (each 1H, m) for H-4″, δH 2.66 and 3.50 (each 1H, m) for H-6″, a methine proton at δH 5.45 (1H, s) for H-5″, and one methyl group at δH 1.01 (3H, t, J = 7.2 Hz) for H-7″, and 13C NMR spectrum showed the corresponding signals attributable to one carbonyl (δC 177.6, C-2″), two methylenes (δC 32.6, 24.5, C-3″ and 4″), a methine (δC 54.1, C-5″), and an ethyl group (δC 36.3, 12.6, C-6″ and 7″), which was further supported by HSQC correlations. The HMBC correlations between H-3″ with C-5″, C-4″, and C-2″ suggested the presence of a N-ethyl-2-pyrrolidinone ring [70] (Figure 3). The existence of a partial structure of –C(3″)H2−C(4″)H2−C(5″)H− could be further inferred through the 1H−1H COSY correlations of H-4″ with H-3″/H-5″ [42]. The combination of the HMBC correlations between H-4″ to C-6/C-2″/C-5″/C-3″ and H-8 to C-6/C-7/C-9/C-10, as well as the ROESY (recorded in DMSO-d6) correlation of the signal (δH 5.89, H-8) with the 7-OH proton (δH 9.23) (Figure S8) showed that the N-ethyl-2-pyrrolidinone moiety was connected to the C-6 position [71]. Thus, the planar structure of compound 11 was determined as 6-N-ethyl-2-pyrrolidinone-epigallocatechin.
Compound 12 was found to have the same molecular formula as 11 (C21H23NO8) based on a molecular ion peak at m/z 416.1336 [M − H]− (calcd for C21H22NO8, 416.1345) in the HR-ESI-MS. The 1H NMR and 13C NMR spectra of 12 were similar to those of 11, indicating they possess the same structural scaffold. The position of N-ethyl-2-pyrrolidinone in compound 12 was also determined to be at C-6 via the similar key correlations of ROESY (recorded in DMSO-d6, Figure S17) and HMBC spectra (recorded in methanol-d4, Figure S16).
The difference between 12 and 11 is the configuration of C-5″, and previous studies have shown that the absolute configuration at C-5″ can be distinguished by subtracting one CD spectrum from its stereoisomer with the same configurations at C-2/C-3 [19,42]. To identify the absolute configuration at C-5″ of compounds 11 and 12, a pair of stereoisomers at C-5″, the CD spectra of two compounds were compared after subtracting the CD spectrum from each other (Figure 4) [42]. For compound 11, the arithmetically independent CD curves of C-5″ exhibited a strong negative cotton effect at 211 nm (Δε − 14.0) and was confirmed to be of the C-5″ R configuration. The configuration of 12 was deduced as S configuration due to a positive cotton effect at 211 nm (Δε + 14.0) in the CD spectrum (Figure 4). Therefore, the structures of compound 11 and 12 were determined to be 6-(5″R)-N-ethyl-2-pyrrolidinone-epigallocatechin and 6-(5″S)-N-ethyl-2-pyrrolidinone-epigallocatechin.
Compound 20 was determined to have a molecular formula of C38H32O18 based on HR-ESI-MS at m/z 775.1505 [M − H]− (calcd for C38H31O18, 775.1510). The 1H and 13C NMR data of 20 were similar to theasinensin B except for the addition of a methoxy group [δH 3.81, (3H, s)], indicating that 20 was a methylated theasinensin B [45]. The HMBC correlations from the methoxy proton to C-3″, and from H-2″ (δH 6.99) to C-1″/C-3″/C-4″/C-6″/C-7″ (Figure 3) confirmed that the methoxy group was linked to C-3″. Therefore, the structure of compound 20 was assigned as epigallocatechin-(2′→2′)-epigallocatechin-3-(3″-O-methyl)-gallate.
Compound 30 was presented as yellow amorphous powder with a molecular formula of C42H46O22 by HR-ESI-MS ion at m/z 901.2394 [M − H]− (calcd for C42H45O22, 901.2402). The 1H NMR and 1H−1H COSY spectrum of 30 suggested the presence of characteristic kaempferol aglycone [δH 6.19, (1H, d, J = 2.0 Hz) and 6.38 (1H, d, J = 2.0 Hz) for A ring; δH 6.91 (2H, d, J = 8.7 Hz) and 8.00 (2H, d, J = 8.7 Hz) for B ring], Z–p-coumaroyl group [δH 6.70 (2H, d, J = 8.5 Hz), 7.64 (2H, d, J = 8.6 Hz), 5.89 (1H, d, J = 12.7 Hz) and 6.88 (1H, d, J = 12.7 Hz)] [72], and three monocarbohydrate moieties [δH 4.41 (1H, d, J = 7.7 Hz), 4.55 (1H, br s) and 5.51 (1H, d, J = 8.0 Hz) for three anomeric protons] (Table 2). In addition, the corresponding anomeric carbons of the sugars were observed at δC 105.7, 102.1 and 100.7 in the 13C NMR spectrum, with the help of the HSQC experiment. Acid hydrolysis of 30 followed by UPLC-CAD analysis identified two D-glucose, and a L-rhamnose. The key HMBC correlations of H-1″ (δH 5.51, Glc) to C-3 (δC 134.6), H-1‴ (δH 4.55, Rha) to C-6″ (δC 68.2, Glc), and H-1′′′′ (δH 4.41, Glc) to C-3‴ (δC 83.2, Rha) determined the linkage of sugar chain [55,56]. The p-coumaroyl group was attached at C-2″ by the correlation between δH 5.00 (H-2″) to δC 167.6 (C=O of Z–p-coumaroyl group). Therefore, the chemical structure of 30 was determined to be kaempferol-3-O-[2″-O-(Z)–p-coumaroyl] [β-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→6)]-β-D-glucopyranoside.
Compound 32, a yellow amorphous powder, showed an anion peak at m/z 901.2384 [M − H]− (C42H45O22, 901.2402) in HR-ESI-MS spectrum, exhibited similar 1H and 13C NMR spectra compared to 30. The main difference was that the glucopyranoside attached at C-3 in 30 was replaced by a galactopyranoside moiety, which was confirmed by the acid hydrolysis and the anomeric proton [δH 5.41 (1H, d, J = 7.9 Hz)]. Thus, compound 32 was elucidated as kaempferol-3-O-[2″-O-(Z)-p-coumaroyl] [β-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→6)]-β-D-galactopyranoside.
Compound 34, a yellow amorphous powder, was determined as C38H48O24 by HR-ESI-MS at m/z 887.2448 [M − H]− (calcd for C38H47O24, 887.2457). The 1H and 13C NMR data of 34 were similar to those of 30 except for the addition of a sugar unit at C-3″ and the absence of p-coumaroyl group, indicating that 34 was a kaempferol tetraglycoside. The presence of two β-D-glucopyranosyl, one α-L-arabinopyranosyl, and one α-L-rhamnopyranosyl units were confirmed by the acid hydrolysis and coupling constants. The glycosidic linkage of oligosaccharide chain was established via the key HMBC correlations from δH 5.17 (H-1″, Glc) to δC 135.5 (C-3), δH 4.56 (H-1‴, Rha) to δC 68.8 (C-6″, Glc), δH 4.40 (H-1′′′′, Glc) to δC 83.2 (C-3‴, Rha), and δH 4.57 (H-1′′′′′, Ara) to δC 86.9 (C-3″, Glc) (Figure 3). Thus, the structure of 34 was determined to be kaempferol-3-O-[α-L-arabinopyranosyl-(1→3)] [β-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→6)]-β-D-glucopyranoside.
Compound 36 was purified as a yellow amorphous powder, and its molecular formula was deduced as C47H54O26 by HR-ESI-MS at m/z 1033.2805 [M − H]− (calcd for C47H53O26, 1033.2825). The NMR data obtained for 36 resembled those of 30, except for the additional arabinopyranosyl unit attached at C-3″, which was supported by the acid hydrolysis and HMBC correlation of H-1′′′′′ (δH 4.30) to C-3″ (δC 80.5). The α-configuration of the arabinopyranosyl group was determined by the coupling constant (J = 5.9 Hz) of the anomeric proton. Consequently, compound 36 was elucidated as kaempferol-3-O-[2″-O-(Z)-p-coumaroyl] [α-L-arabinopyranosyl-(1→3)] [β-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→6)]-β-D-glucopyranoside.
Compound 43 was presented as yellow amorphous powder with a molecular formula of C42H46O23 by HR-ESI-MS ion at m/z 917.2331 [M − H]− (calcd for C42H45O23, 917.2352). The 1H NMR spectrum indicated the typical quercetin unit signals at δH 7.46 (1H, d, J = 2.2 Hz, H-2′), δH 7.63 (1H, dd, J = 8.5, 2.3 Hz, H-6′), δH 6.79 (1H, m, H-5′), δH 6.29 (1H, br s, H-8) and δH 6.11 (1H, br s, H-6), and E–p-coumaroyl group with protons at δH 7.56 (1H, d, J = 15.9 Hz, H-Cou-7), δH 7.52 (2H, d, J = 8.6 Hz, H-Cou-2, H-Cou-6), δH 6.79 (2H, d, J = 8.4 Hz, H-Cou-3, H-Cou-5), and δH 6.38 (1H, d, J = 15.9 Hz, H-Cou-8) [72]. Furthermore, three anomeric proton signals of sugar moieties were observed at δH 5.59 (1H, d, J = 8.0 Hz, H-1″), δH 4.49 (1H, br s, H-1‴), and δH 4.32 (1H, d, J = 7.7 Hz, H-1′′′′) (Table 2). 2D NMR and acid hydrolysis experiments were performed to determine the sugar unit and the glycosidic linkage of 43, which suggested the existence of a β-D-galactopyranosyl, an α-L-rhamnopyranosyl, and β-D-glucopyranosyl moieties. The attachments of sugar chains and E–p-coumaroyl group were determined by the HMBC correlations of δH 5.59 (H-1″, Gal) to δC 132.8 (C-3), δH 4.49 (H-1‴, Rha) to δC 64.9 (C-6″, Gal), δH 4.32 (H-1′′′′, Glc) to δC 81.7 (C-3‴, Rha) and δH 5.22 (H-2″, Gal) to δC 166.0 (C=O of E–p-coumaroyl group) [61]. Accordingly, compound 43 was identified as quercetin-3-O-[2″-O-(E)-p-coumaroyl] [β-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→6)]-β-D-galcopyranoside.
Compound 44 was obtained as a yellow amorphous powder, and its molecular formula was deduced as C42H46O23 by HR-ESI-MS at m/z 917.2332 [M − H]− (calcd forC42H45O23, 917.2352). The 1H and 13C NMR data of 44 exhibited high similarity to that of 43, with the main distinction being a configuration of coumaroyl moiety shift from E– in 43 to Z– in 44 (Table 3), which was supported by the two olefinic protons at δH 6.82 (1H, d, J = 12.0 Hz) and δH 5.81 (1H, d, J = 12.9 Hz). Hence, the structure of compound 44 was established as quercetin-3-O-[2″-O-(Z)-p-coumaroyl] [β-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→6)]-β-D-galcopyranoside.
Compound 46 was isolated as a yellow amorphous powder with a molecular formula of C38H48O25 by HR-ESI-MS ion at m/z 903.2400 [M − H]− (calcd for C38H47O25, 903.2406). The 1H and 13C NMR spectroscopic data of 46 were similar to those of 44 except for the addition of an arabinopyranosyl unit at C-3″ and the absence of Z–p-coumaroyl group, indicating that 46 was a quercetin tetraglycoside, which was further confirmed by the acid hydrolysis. The attachment of an arabinopyranosyl group to C-3″ was established from the HMBC correlation between H-3″ (δH 3.60, Glc) with δC 105.6 (H-1′′′′′, Ara). Consequently, compound 46 was elucidated as quercetin-3-O-[α-L-arabinopyranosyl-(1→3)] [β-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→6)]-β-D-glucopyranoside.
Compound 48, a yellow amorphous powder, was determined as C47H54O27 by HR-ESI-MS at m/z 1049.2751 [M − H]− (calcd for C47H53O27, 1049.2774). Compound 48 showed similar NMR data to that of camelliquercetiside A [62], except for slight differences of the two olefinic proton signals at δH 6.82 (1H, d, J = 13.3 Hz) and δH 5.83 (1H, d, J = 12.8 Hz), indicating the different configuration of the double bond in 48. Moreover, the linkage of the Z–p-coumaroyl unit was assigned at C-2″, which was confirmed through the HMBC correlation from H-2″ (δH 5.04, m) to C-Cou 9 (the carbonyl carbon of the Z-coumaroyl moiety, δC 164.9). Therefore, the structure of compound 48 was characterized as quercetin-3-O-[2″-O-(Z)-p-coumaroyl] [α-L-arabinopyranosyl-(1→3)] [β-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→6)]-β-D-glucoside.
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