Characterization of Proanthocyanidin Oligomers of Ephedra sinica

Ephedra sinica, an important plant in Chinese traditional medicine, contains a complex mixture of proanthocyanidin oligomers as major constituents; however, only the minor components have been chemically characterized. In this study, oligomers with relatively large molecular weights, which form the main body of the proanthocyanidin fractions, were separated by adsorption and size-exclusion chromatography. Acid-catalyzed degradation in the presence of mercaptoethanol or phloroglucinol led to the isolation of 18 fragments, the structures of which were elucidated from their experimental and TDDFT-calculated ECD spectra. The results indicated that (−)-epigallocatechin was the main extension unit, while catechin, the A-type epigallocatechin–gallocatechin dimer, and the A-type epigallocatechin homodimer, were identified as the terminal units. Among the degradation products, thioethers of gallocatechin with 3,4-cis configurations, a B-type prodelphinidin dimer, a prodelphinidin trimer with both A- and B-type linkages, and a prodelphinidin dimer with an α-substituted A-type linkage were new compounds. In addition, a phloroglucinol adduct of an A-type prodelphinidin dimer, a doubly-linked phloroglucinol adduct of epigallocatechin, and a unique product with a flavan-3-ol skeleton generated by the rearrangement of the aromatic rings were also isolated.


Introduction
Ephedra sinica Stapf (Fam. Ephedraceae) is one of the most important plants in traditional medicine, and is used as a diuretic, antipyretic, diaphoretic, and for relieving a cough and asthma [1]. As the crude drug, it has an official monograph in both the Chinese and Japanese Pharmacopoeias, where it is standardized against the major alkaloids, ephedrine and pseudoephedrine [2]. Thus, the main emphasis is conventionally given to its alkaloidal content, despite the fact that this only constitutes about 0.7-0.8% of the whole plant [3,4]. Clearly, the motivation for this is the proven clinical effects of these alkaloids on the respiratory, central nervous, and cardiovascular systems [5]. However, many species of Ephedra have also been shown to contain significant amounts of proanthocyanidins [6]. Recently, many health benefits of foods and medicinal plants have been attributed to proanthocyanidins [7], and some of their biological activities, including hypotensive and vasorelaxant effects [8,9], improvement of the airway microenvironment in asthma [10], and the inhibition of inflammation and remodeling in murine models of chronic asthma [11], are responsible for the aforementioned activities of E. sinica, especially its respiratory and cardiovascular effects. A number of studies have shown that Ephedra spp. also display other biological activities that are not attributed to alkaloids, including antimicrobial [12,13], antioxidant [14], anti-inflammatory [15,16], immunosuppressive [17], antiviral [18], anti-invasive, antiangiogenic, antitumor [19], and cytotoxic [20] cell lines SGC-7901, HepG2, and HeLa [21]. In addition, a decrease in the uremic toxin parameters of rats was reportedly induced by the administration of proanthocyanidin oligomers of E. sinica [22,23].
As for the composition of the proanthocyanidins of E. sinica, monomeric flavan-3-ols [12,24] and dimeric proanthocyanidins with A-type linkages have been isolated [12,21,[25][26][27][28]. The presence of prodelphinidin trimers and tetramers with A-and B-type linkages has also been shown [12]. However, these flavan-3-ols and proanthocyanidins are minor components of the total polyphenol content, and our preliminary HPLC and TLC analysis of the extract suggested that the main body of the polyphenols was a complex mixture of oligomers, detected as a broad hump on the HPLC baseline and at the origin of the TLC plate ( Figure 1a). Thus, the present study aimed at characterizing these proanthocyanidin oligomers by acid-catalyzed degradation in the presence of nucleophilic agents, that is, 2-mercaptoethanol or phloroglucinol. The degradation involved the cleavage of the interflavan bonds under acidic conditions, generating flavan-3-ols from the terminal units and flavanyl-4 cations from the extension units, which were trapped by nucleophilic agents (Scheme 1) [29].

Composition of the Intact Proanthocyanidin Oligomer
The dried aerial parts of E. sinica were extracted with aqueous acetone and fractionated by a series of chromatographic separation methods, including size-exclusion chromatography [30]. The fractions containing only oligomeric proanthocyanidins accounted for 2.7% of the dried plant material, and the HPLC profile showed a broad hump on the baseline (Figure 1b). The 13 C-NMR spectrum of the oligomer fraction in DMSO-d6 ( Figure 2) showed signals characteristic of proanthocyanidins [31]. Based on a comparison with the literature data [11], the signals at δC 77 and cell lines SGC-7901, HepG2, and HeLa [21]. In addition, a decrease in the uremic toxin parameters of rats was reportedly induced by the administration of proanthocyanidin oligomers of E. sinica [22,23].
As for the composition of the proanthocyanidins of E. sinica, monomeric flavan-3-ols [12,24] and dimeric proanthocyanidins with A-type linkages have been isolated [12,21,[25][26][27][28]. The presence of prodelphinidin trimers and tetramers with A-and B-type linkages has also been shown [12]. However, these flavan-3-ols and proanthocyanidins are minor components of the total polyphenol content, and our preliminary HPLC and TLC analysis of the extract suggested that the main body of the polyphenols was a complex mixture of oligomers, detected as a broad hump on the HPLC baseline and at the origin of the TLC plate ( Figure 1a). Thus, the present study aimed at characterizing these proanthocyanidin oligomers by acid-catalyzed degradation in the presence of nucleophilic agents, that is, 2-mercaptoethanol or phloroglucinol. The degradation involved the cleavage of the interflavan bonds under acidic conditions, generating flavan-3-ols from the terminal units and flavanyl-4 cations from the extension units, which were trapped by nucleophilic agents (Scheme 1) [29].

Scheme 1.
Reaction mechanism of the acid-catalyzed cleavage of the interflavan bond in the presence of nucleophiles (Nu).

Composition of the Intact Proanthocyanidin Oligomer
The dried aerial parts of E. sinica were extracted with aqueous acetone and fractionated by a series of chromatographic separation methods, including size-exclusion chromatography [30]. The fractions containing only oligomeric proanthocyanidins accounted for 2.7% of the dried plant material, and the HPLC profile showed a broad hump on the baseline (Figure 1b). The 13 C-NMR spectrum of the oligomer fraction in DMSO-d6 ( Figure 2) showed signals characteristic of proanthocyanidins [31]. Based on a comparison with the literature data [11], the signals at δC 77 and

Composition of the Intact Proanthocyanidin Oligomer
The dried aerial parts of E. sinica were extracted with aqueous acetone and fractionated by a series of chromatographic separation methods, including size-exclusion chromatography [30]. The fractions containing only oligomeric proanthocyanidins accounted for 2.7% of the dried plant material, and the HPLC profile showed a broad hump on the baseline (Figure 1b). The 13 C-NMR spectrum of the oligomer fraction in DMSO-d 6 ( Figure 2) showed signals characteristic of proanthocyanidins [31]. Based on a comparison with the literature data [11], the signals at δ C 77 and δ C 70-73, which were attributable to flavan C-ring C-2 and C-3 methine carbons, respectively, suggested the occurrence of B-type linkages. The chemical shifts also indicated that the 2,3-cis configuration was more abundant than 2,3-trans [32,33]. The signals in the range of δ C 27-31 were attributable to the C-4 carbons of A-type proanthocyanidin extension units [12] and of terminal units [34,35]. The prominent aromatic signals observed at δ C 106, 130, 132, and 145 suggested the predominance of pyrogallol-type B-rings over catechol-type B-rings (δ C 115-120).

Identification of Known Products
Thiol degradation was performed according to the previously described method [35] with modifications of the reaction time and temperature, and 10 compounds (1-10) were isolated and characterized ( Figure 3a). Acid-catalyzed degradation with phloroglucinol [29,36] yielded a different set of 10 products (5, 8, 11-18), among which two products (5 and 8) were identical to those obtained by thiol degradation (Figure 3b).

Identification of Known Products
Thiol degradation was performed according to the previously described method [35] with modifications of the reaction time and temperature, and 10 compounds (1-10) were isolated and characterized ( Figure 3a). Acid-catalyzed degradation with phloroglucinol [29,36] yielded a different set of 10 products (5, 8, 11-18), among which two products (5 and 8) were identical to those obtained by thiol degradation (Figure 3b).

Structure Elucidation of New Degradation Products
Among the 18 isolated products, 1, 2, 3, 7, 13, 14, and 16 are reported here for the first time. Their structures are shown in Figure 5 and the 1 H-and 13 C-NMR spectroscopic data are summarized in Tables 1 and 2.
The molecular formula of 1 was shown to be C32H30O15S based on the [M + H] + peak at m/z 687.1382 in HRFABMS, indicating that 1 was a mercaptoethanol adduct of a prodelphinidin dimer with a B-type linkage. This was confirmed by the appearance of two intense signals at δH 6.57 and δH 6.67 (each 2H) arising from two pyrogallol-type B-rings and two methine proton signals attributable to C-ring H-2 in the 1 H-NMR signals (Table 1). In the HSQC spectrum, the C-ring H-2 signal at δH 4.34 (J = 9.4 Hz) was correlated to a carbon signal at δC 83.2, while the other F-ring H-2 ( Figure 6) at δH 5.31 (br s) was found to be connected to the carbon that resonated at δC 75.05. The former indicated a 2,3-trans configuration and the latter, a 2,3-cis configuration [33]; thus, the dimer was composed of gallocatechin and epigallocatechin. The 1 H-1 H COSY and HMBC correlations ( Figure 6) allowed the determination of the connectivity of the two catechin units and hydroxyethylthiol group. The strong NOESY correlations between C-ring H-2 and H-4 ( Figure 6), and between -SCH2-and F-ring H-3, confirmed the configuration of the C-and F-rings [39,40]. A linkage between C-ring C-4 and D-ring C-8 was deduced from the NOESY correlation between aromatic E-ring H-2, 6 and C-ring H-4 [41,42].

Structure Elucidation of New Degradation Products
Among the 18 isolated products, 1, 2, 3, 7, 13, 14, and 16 are reported here for the first time. Their structures are shown in Figure 5 and the 1 H-and 13 C-NMR spectroscopic data are summarized in Tables 1 and 2.
The molecular formula of 1 was shown to be C 32 H 30 O 15 S based on the [M + H] + peak at m/z 687.1382 in HRFABMS, indicating that 1 was a mercaptoethanol adduct of a prodelphinidin dimer with a B-type linkage. This was confirmed by the appearance of two intense signals at δ H 6.57 and δ H 6.67 (each 2H) arising from two pyrogallol-type B-rings and two methine proton signals attributable to C-ring H-2 in the 1 H-NMR signals (Table 1). In the HSQC spectrum, the C-ring H-2 signal at δ H 4.34 (J = 9.4 Hz) was correlated to a carbon signal at δ C 83.2, while the other F-ring H-2 ( Figure 6) at δ H 5.31 (br s) was found to be connected to the carbon that resonated at δ C 75.05. The former indicated a 2,3-trans configuration and the latter, a 2,3-cis configuration [33]; thus, the dimer was composed of gallocatechin and epigallocatechin. The 1 H-1 H COSY and HMBC correlations ( Figure 6) allowed the determination of the connectivity of the two catechin units and hydroxyethylthiol group. The strong NOESY correlations between C-ring H-2 and H-4 ( Figure 6), and between -SCH 2 -and F-ring H-3, confirmed the configuration of the C-and F-rings [39,40]. A linkage between C-ring C-4 and D-ring C-8 was deduced from the NOESY correlation between aromatic E-ring H-2, 6 and C-ring H-4 [41,42].    the 1 Lb band (280 nm) rather than the 1 La band (220-240 nm) [40]. Here, the negative Cotton effect at 288 nm, in addition to the predominance of E-conformers, both led to the conclusion that the pyrogallol E-ring had an α-orientation relative to the F-2 carbon. The terminal unit was thereby designated as (−)-epigallocatechin. Furthermore, the ECD spectrum for 1 showed a close resemblance to those of procyanidins B-4 previously observed by Barrett and colleagues [43]. Accordingly, 1 was concluded to be (+)-gallocatechin-(4→8)-(−)-epigallocatechin-4-(2-hydroxyethyl)thioether.  (Table 1) showed a doublet signal at δH 4.77 (J = 9.6 Hz), indicating the 2,3-trans configuration characteristic of gallocatechin. The C-ring H-4 resonated as a doublet at δH 4.36 (J = 4.3 Hz), which indicated the 3,4-cis configuration [31]. This was further confirmed by the appearance of a strong NOESY correlation between H-3 and H-4, and the absence of NOE between H-2 and H-4 ( Figure 7). As for the absolute configuration, a negative Cotton effect at 284 nm in the ECD spectrum, which was similar to that of (+)-catechin [43], suggested P-helicity for the flavan A-and B-rings. Based on these results, 2 was concluded to be (+)-gallocatechin-4-(2hydroxyethyl)thioether. Compound 3 was found to have the molecular formula C47H40O22S based on the [M + Na] + peak at m/z 1011.1639 in HRFABMS. This implied that 3 was a thioether of a prodelphinidin trimer involving both A-type and B-type linkages. In the 1 H-NMR spectrum (Table 1), three intense aromatic singlets at δH 6.77, δH 6.70, and δH 6.56 (each 2H) indicated the presence of three pyrogallol-type Brings. The presence of two B-type linkages was apparent from the two C-ring H-2 signals resonating at δH 5.31 and δH 5.29 with small J2,3 values (<2 Hz). This again indicated that the two units were epigallocatechin. These spectroscopic features suggested a close relationship between 3 and the epigallocatechin trimer isolated from E. sinica with A-and B-type linkages [12]. In the HMBC spectrum of 3 (Figure 8), the ketal carbon C-2 (δC 99.91) of the A-type linkage was correlated to Cring H-4 (δH 4.38, J = 3.4 Hz), which was in turn correlated to a D-ring C-9 (δC 151.31) of the middle unit. Another benzylic methine H-4 of the middle unit F-ring (δH 4.79, J = 2.2 Hz) showed an HMBC correlation to the D-ring C-9 and terminal unit G-ring C-9 (δC 153.90). This indicated that an A-type linkage was involved between the top and middle units. The 13 C-NMR chemical shift for F-ring C-4 at δC 36.21 was consistent with its involvement in a B-type linkage at this position [12,33,44], and the I-ring C-4 at a lower field (δC 43.83) was indicative of a thioether at this position [38]. The F-ring H-4 and I-ring H-4 were observed as doublet signals with coupling constants of J = 2.3 Hz and J = 2.4 Hz, respectively, indicating that both flavan rings adopted a 3,4-trans configuration [31]. This was further supported by the absence of a NOESY correlation between H-2 and H-4 in both the F-ring and the Iring (Figure 8). The linkage between rings C and D was established as 4→8, 2→O→7 by the presence of a NOESY correlation between E-ring H-2,6 and C-ring H-4 [41]. The connection between the F-ring and the G-ring was also determined to be from C-4 to C-8 based on the NOESY cross peaks of H-ring ECD spectroscopy allowed the determination of the absolute configuration at C-4. The 1 H coupling constants of the C-ring indicated that the B-ring was in an equatorial position (E-conformer). Taking this observation into account, the negative Cotton effect at 218 nm implied an α-orientation of the terminal unit at C-4 [40]. Thus, the extension unit was concluded to be (+)-gallocatechin. The establishment of the absolute configuration of the epigallocatechin unit relied on the Cotton effect at the 1 L b band (280 nm) rather than the 1 L a band (220-240 nm) [40]. Here, the negative Cotton effect at 288 nm, in addition to the predominance of E-conformers, both led to the conclusion that the pyrogallol E-ring had an α-orientation relative to the F-2 carbon. The terminal unit was thereby designated as (−)-epigallocatechin. Furthermore, the ECD spectrum for 1 showed a close resemblance to those of procyanidins B-4 previously observed by Barrett and colleagues [43]. Accordingly  [31]. This was further confirmed by the appearance of a strong NOESY correlation between H-3 and H-4, and the absence of NOE between H-2 and H-4 ( Figure 7). As for the absolute configuration, a negative Cotton effect at 284 nm in the ECD spectrum, which was similar to that of (+)-catechin [43], suggested P-helicity for the flavan A-and B-rings. Based on these results, 2 was concluded to be (+)-gallocatechin-4-(2-hydroxyethyl)thioether.  (Table 1) showed a doublet signal at δH 4.77 (J = 9.6 Hz), indicating the 2,3-trans configuration characteristic of gallocatechin. The C-ring H-4 resonated as a doublet at δH 4.36 (J = 4.3 Hz), which indicated the 3,4-cis configuration [31]. This was further confirmed by the appearance of a strong NOESY correlation between H-3 and H-4, and the absence of NOE between H-2 and H-4 ( Figure 7). As for the absolute configuration, a negative Cotton effect at 284 nm in the ECD spectrum, which was similar to that of (+)-catechin [43], suggested P-helicity for the flavan A-and B-rings. Based on these results, 2 was concluded to be (+)-gallocatechin-4-(2hydroxyethyl)thioether. Compound 3 was found to have the molecular formula C47H40O22S based on the [M + Na] + peak at m/z 1011.1639 in HRFABMS. This implied that 3 was a thioether of a prodelphinidin trimer involving both A-type and B-type linkages. In the 1 H-NMR spectrum (Table 1), three intense aromatic singlets at δH 6.77, δH 6.70, and δH 6.56 (each 2H) indicated the presence of three pyrogallol-type Brings. The presence of two B-type linkages was apparent from the two C-ring H-2 signals resonating at δH 5.31 and δH 5.29 with small J2,3 values (<2 Hz). This again indicated that the two units were epigallocatechin. These spectroscopic features suggested a close relationship between 3 and the epigallocatechin trimer isolated from E. sinica with A-and B-type linkages [12]. In the HMBC spectrum of 3 (Figure 8), the ketal carbon C-2 (δC 99.91) of the A-type linkage was correlated to Cring H-4 (δH 4.38, J = 3.4 Hz), which was in turn correlated to a D-ring C-9 (δC 151.31) of the middle Compound 3 was found to have the molecular formula C 47 H 40 O 22 S based on the [M + Na] + peak at m/z 1011.1639 in HRFABMS. This implied that 3 was a thioether of a prodelphinidin trimer involving both A-type and B-type linkages. In the 1 H-NMR spectrum (Table 1), three intense aromatic singlets at δ H 6.77, δ H 6.70, and δ H 6.56 (each 2H) indicated the presence of three pyrogallol-type B-rings. The presence of two B-type linkages was apparent from the two C-ring H-2 signals resonating at δ H 5.31 and δ H 5.29 with small J 2,3 values (<2 Hz). This again indicated that the two units were epigallocatechin. These spectroscopic features suggested a close relationship between 3 and the epigallocatechin trimer isolated from E. sinica with A-and B-type linkages [12]. In the HMBC spectrum of 3 (Figure 8), the ketal carbon C-2 (δ C 99.91) of the A-type linkage was correlated to C-ring H-4 (δ H 4.38, J = 3.4 Hz), which was in turn correlated to a D-ring C-9 (δ C 151.31) of the middle unit. Another benzylic methine H-4 of the middle unit F-ring (δ H 4.79, J = 2.2 Hz) showed an HMBC correlation to the D-ring C-9 and terminal unit G-ring C-9 (δ C 153.90). This indicated that an A-type linkage was involved between the top and middle units. The 13 C-NMR chemical shift for F-ring C-4 at δ C 36.21 was consistent with its involvement in a B-type linkage at this position [12,33,44], and the I-ring C-4 at a lower field (δ C 43.83) was indicative of a thioether at this position [38]. The F-ring H-4 and I-ring H-4 were observed as doublet signals with coupling constants of J = 2.3 Hz and J = 2.4 Hz, respectively, indicating that both flavan rings adopted a 3,4-trans configuration [31]. This was further supported by the absence of a NOESY correlation between H-2 and H-4 in both the F-ring and the I-ring (Figure 8). The linkage between rings C and D was established as 4→8, 2→O→7 by the presence of a NOESY correlation between E-ring H-2,6 and C-ring H-4 [41]. The connection between the F-ring and the G-ring was also determined to be from C-4 to C-8 based on the NOESY cross peaks of H-ring H-2,6 with F-ring H-4 and H-3. The ECD spectrum of 3 showed a strong positive Cotton effect at 233 nm, reflecting the configuration at C-ring C-4, thereby establishing the top extension unit as (−)-epigallocatechin; however, the configuration of the middle and bottom epigallocatechin units could not be determined from the ECD data. Prodelphinidin oligomers with (+)-epigallocatechin units were previously isolated from the same plant source [12]; therefore, the absolute configuration Compound 7 was characterized as an A-type prodelphinidin dimer with a mercaptoethanol substituent, and its molecular formula was determined as C32H28O15S from the [M + Na] + peak at m/z 707.1043 in HRFABMS. The presence of an A-type linkage was apparent from the signal at δC 100.07, The ECD spectrum of 3 showed a strong positive Cotton effect at 233 nm, reflecting the configuration at C-ring C-4, thereby establishing the top extension unit as (−)-epigallocatechin; however, the configuration of the middle and bottom epigallocatechin units could not be determined from the ECD data. Prodelphinidin oligomers with (+)-epigallocatechin units were previously isolated from the same plant source [12]; therefore, the absolute configuration  Compound 13 was obtained as a product of phloroglucinolysis, and the HRFABMS peak (m/z 431.0979 [M + H] + ) confirmed the molecular formula as C21H18O10, the same as that of 11 and 12.
Because of overlapping C-ring proton signals in the 1 H-NMR spectrum measured in acetone-d6, the 2D NMR spectra were measured in methanol-d4 ( Table 2). The resulting 1 H-NMR spectrum showed signals attributable to pyrogallol (δH 6.19, 2H) and phloroglucinol (δH 5.79, 2H) rings, as well as mutually meta-coupled A-ring H-6 and H-8 (δH 5.94 and 5.95, J = 2.4 Hz), which were related to those observed in the spectra of 11 and 12 [12,37,42]. In the 1 H-1 H COSY spectrum (Figure 11), a broad aliphatic singlet signal at δH 5.53 was correlated to a methine signal at δH 4.05 (J = 2.4, 1.0 Hz), and these signals were attributed to C-ring H-2 and H-3, respectively. The small coupling constant suggested the 2,3-cis configuration [33]. Another aliphatic methine signal at δH 4.06 was assigned to C-ring H-4 based on its HMBC correlations to A-ring C-5, 9 and 10 ( Figure 11). H-4 also showed HMBC correlations with pyrogallol H-2,6 (δH 6.19), indicating that the pyrogallol ring was attached to C-4. This was further supported by the long-range 1 H-1 H coupling between C-ring H-4 and pyrogallol B-ring H-2,6 in the 1 H-1 H COSY spectrum and the HMBC cross peak between C-ring H-3 and pyrogallol C-1 [45]. The remaining moiety, i.e., the phloroglucinol ring with a symmetrical structure, was shown to be located at C-ring C-2 by the HMBC correlation of C-ring H-2 to the phloroglucinol C-1, 2, and 6. The 2,3-cis-3,4-trans configuration was inferred by a comparison of the coupling constants with those in the literature [31], and this was further supported by the absence of NOE between H-2 and H-4 and occurrence of the strong NOE between C-ring H-2 and pyrogallol Bring H-2,6 ( Figure 11). The weak correlation observed between the C-ring H-2 and A-ring H-8 protons suggested that H-2 was at the axial position, thereby implying that the C-ring adopted the Econformation. From the positive Cotton effect at 227 nm, the absolute configuration at C-4 was determined to be S [31]. Accordingly, 13 was concluded to be 2-(2,4,6-trihydroxyphenyl)-4-(3,4,5-  Compound 7 was characterized as an A-type prodelphinidin dimer with a mercaptoethanol substituent, and its molecular formula was determined as C 32 H 28 O 15 S from the [M + Na] + peak at m/z 707.1043 in HRFABMS. The presence of an A-type linkage was apparent from the signal at δ C 100.07, attributable to the C-ring C-2 ketal carbon [12,42]. The large coupling constant (J = 9.8 Hz) of the H-2 at δ H 4.99 indicated the 2,3-trans configuration of the lower unit F-ring. The coupling constants of the C-ring H-4 at δ H 4.17 (J = 3.6 Hz) and F-ring H-4 at δ H 4.39 (J = 4.3 Hz), which were similar to the values observed in 2, suggested the 3,4-cis configuration of these rings. A comparison of the 1 H-and 13 C-NMR data with those in the literature suggested that the dimer was composed of epigallocatechin and gallocatechin [12,37]. This was supported by a strong NOE between F-3 and F-4, and weak NOE between F-2 and F-3 ( Figure 10). The linkage between the C-and D-rings was established to be 4→8 by the observation of a NOESY correlation between C-ring H-4 and E-ring H-2,6. The absolute configuration at the C-ring C-4 was established by ECD spectroscopy, where the strong negative Cotton effect at 228 nm indicated that the extension unit was (+)-epigallocatechin. The terminal unit was designated as (+)-gallocatechin based on a comparison of the ECD spectrum with that of compound 2, which also had a negative Cotton effect, of a lesser amplitude, at 284 nm. Compound 7 was thereby established as (−)-epigallocatechin-(4α→8,2→O→7)-(+)-gallocatechin-4-(2-hydroxyethyl)thioether. Compound 13 was obtained as a product of phloroglucinolysis, and the HRFABMS peak (m/z 431.0979 [M + H] + ) confirmed the molecular formula as C21H18O10, the same as that of 11 and 12.
Because of overlapping C-ring proton signals in the 1 H-NMR spectrum measured in acetone-d6, the 2D NMR spectra were measured in methanol-d4 ( Table 2). The resulting 1 H-NMR spectrum showed signals attributable to pyrogallol (δH 6.19, 2H) and phloroglucinol (δH 5.79, 2H) rings, as well as mutually meta-coupled A-ring H-6 and H-8 (δH 5.94 and 5.95, J = 2.4 Hz), which were related to those Compound 13 was obtained as a product of phloroglucinolysis, and the HRFABMS peak (m/z 431.0979 [M + H] + ) confirmed the molecular formula as C 21 H 18 O 10 , the same as that of 11 and 12.
Because of overlapping C-ring proton signals in the 1 H-NMR spectrum measured in acetone-d 6 , the 2D NMR spectra were measured in methanol-d 4 ( Table 2). The resulting 1 H-NMR spectrum showed signals attributable to pyrogallol (δ H 6.19, 2H) and phloroglucinol (δ H 5.79, 2H) rings, as well as mutually meta-coupled A-ring H-6 and H-8 (δ H 5.94 and 5.95, J = 2.4 Hz), which were related to those observed in the spectra of 11 and 12 [12,37,42]. In the 1 H-1 H COSY spectrum (Figure 11), a broad aliphatic singlet signal at δ H 5.53 was correlated to a methine signal at δ H 4.05 (J = 2.4, 1.0 Hz), and these signals were attributed to C-ring H-2 and H-3, respectively. The small coupling constant suggested the 2,3-cis configuration [33]. Another aliphatic methine signal at δ H 4.06 was assigned to C-ring H-4 based on its HMBC correlations to A-ring C-5, 9 and 10 ( Figure 11). H-4 also showed HMBC correlations with pyrogallol H-2,6 (δ H 6.19), indicating that the pyrogallol ring was attached to C-4. This was further supported by the long-range 1 H-1 H coupling between C-ring H-4 and pyrogallol B-ring H-2,6 in the 1 H-1 H COSY spectrum and the HMBC cross peak between C-ring H-3 and pyrogallol C-1 [45]. The remaining moiety, i.e., the phloroglucinol ring with a symmetrical structure, was shown to be located at C-ring C-2 by the HMBC correlation of C-ring H-2 to the phloroglucinol C-1, 2, and 6. The 2,3-cis-3,4-trans configuration was inferred by a comparison of the coupling constants with those in the literature [31], and this was further supported by the absence of NOE between H-2 and H-4 and occurrence of the strong NOE between C-ring H-2 and pyrogallol B-ring H-2,6 ( Figure 11). The weak correlation observed between the C-ring H-2 and A-ring H-8 protons suggested that H-2 was at the axial position, thereby implying that the C-ring adopted the E-conformation. From the positive Cotton effect at 227 nm, the absolute configuration at C-4 was determined to be S [31]. Accordingly, 13 was concluded to be 2-(2,4,6-trihydroxyphenyl)-4-(3,4,5-trihydroxyphenyl)-3,4-dihydro-2H-1-benzopyran-3,5,7-triol (2R,3R,4S). This compound was a byproduct of phloroglucinolysis, and a plausible production mechanism is proposed in Scheme 2.
Molecules 2017, 22, 1308 11 of 18 trihydroxyphenyl)-3,4-dihydro-2H-1-benzopyran-3,5,7-triol (2R,3R,4S). This compound was a byproduct of phloroglucinolysis, and a plausible production mechanism is proposed in Scheme 2.  Compound 14 was determined to have the molecular formula C36H28O17 (m/z 733.1406, [M + H] + ), identifying it as a phloroglucinol adduct of a prodelphinidin dimer involving an A-type linkage. The signals in the 1 H-and 13 C-NMR spectra were related to those of the epigallocatechin-epigallocatechin dimer [12] and the procyanidin A2-phloroglucinol adduct [46], and their assignments ( Table 2) were based on a comparison with the reported data. The signal of F-ring H-2 (δH 5.32) was observed as a singlet, indicating the 2,3-cis configuration of the F-ring [33]. In addition, the coupling constant of the   Compound 14 was determined to have the molecular formula C36H28O17 (m/z 733.1406, [M + H] + ), identifying it as a phloroglucinol adduct of a prodelphinidin dimer involving an A-type linkage. The signals in the 1 H-and 13 C-NMR spectra were related to those of the epigallocatechin-epigallocatechin dimer [12] and the procyanidin A2-phloroglucinol adduct [46], and their assignments ( Table 2) were based on a comparison with the reported data. The signal of F-ring H-2 (δH 5.32) was observed as a singlet, indicating the 2,3-cis configuration of the F-ring [33]. In addition, the coupling constant of the Scheme 2. A possible production mechanism of 13.
Compound 14 was determined to have the molecular formula C 36 H 28 O 17 (m/z 733.1406, [M + H] + ), identifying it as a phloroglucinol adduct of a prodelphinidin dimer involving an A-type linkage. The signals in the 1 H-and 13 C-NMR spectra were related to those of the epigallocatechin-epigallocatechin dimer [12] and the procyanidin A2-phloroglucinol adduct [46], and their assignments ( Table 2) were based on a comparison with the reported data. The signal of F-ring H-2 (δ H 5.32) was observed as a singlet, indicating the 2,3-cis configuration of the F-ring [33]. In addition, the coupling constant of the F-ring H-4 (δ H 4.63, J = 2.3 Hz) was consistent with the 3,4-trans configuration [31]. This was confirmed by the NOESY spectrum, which displayed a strong correlation between F-ring H-3 and H-4, but no NOE between H-2 and H-4 ( Figure 12). The 4→8 linkage between the C-and D-rings was established by the NOE between E-ring H-2,6 and C-ring H-4. Furthermore, the strong positive Cotton effect at 232 nm established that the upper unit was (−)-epigallocatechin [12]. On the basis of previous studies of compound 7, by Nam et al. [42] and Barrett et al. [43], and considering the weak positive Cotton effect at 220-240 nm of 12, the lower unit was deduced to be (−)-epigallocatechin. It was therefore concluded that compound 14 was (−)-epigallocatechin-(4β→8,2→O→7)-(−)-epigallocatechin-4-phloroglucinol. Compound 14 was determined to have the molecular formula C36H28O17 (m/z 733.1406, [M + H] + ), identifying it as a phloroglucinol adduct of a prodelphinidin dimer involving an A-type linkage. The signals in the 1 H-and 13 C-NMR spectra were related to those of the epigallocatechin-epigallocatechin dimer [12] and the procyanidin A2-phloroglucinol adduct [46], and their assignments ( Table 2) were based on a comparison with the reported data. The signal of F-ring H-2 (δH 5.32) was observed as a singlet, indicating the 2,3-cis configuration of the F-ring [33]. In addition, the coupling constant of the F-ring H-4 (δH 4.63, J = 2.3 Hz) was consistent with the 3,4-trans configuration [31]. This was confirmed by the NOESY spectrum, which displayed a strong correlation between F-ring H-3 and H-4, but no NOE between H-2 and H-4 ( Figure 12). The 4→8 linkage between the C-and D-rings was established by the NOE between E-ring H-2,6 and C-ring H-4. Furthermore, the strong positive Cotton effect at 232 nm established that the upper unit was (−)-epigallocatechin [12]. On the basis of previous studies of compound 7, by Nam et al. [42] and Barrett et al. [43], and considering the weak positive Cotton effect at 220-240 nm of 12, the lower unit was deduced to be (−)-epigallocatechin. It was therefore concluded that compound 14 was (−)-epigallocatechin-(4β→8,2→O→7)-(−)-epigallocatechin-4phloroglucinol. Product 16 showed the [M + H] + peak at m/z 429.0818 in HRFABMS, indicating the molecular formula C21H16O10. An unambiguous assignment of the 1 H-and 13 C-NMR signals was achieved by 1 H-1 H COSY, HSQC, HMBC, and NOESY spectroscopy. The absence of a C-2 proton signal and appearance of a C-2 carbon signal at δC 100.21 confirmed the presence of an A-type linkage [12,44,46]. The HMBC cross peaks ( Figure 13) between C-ring H-4 and D-ring C-1,2,6 indicated the linkage of the phloroglucinol moiety to C-ring C-4. The NOE cross peaks between H-3 and B-ring H-2,6 indicated the 3,4-trans configuration [47]. Furthermore, the ECD spectrum showed a positive Cotton effect at 220-240 nm, indicating a β-configuration at C-4. Accordingly, 16 was established to be (−)epigallocatechin-(4β→1,2→O→2)-phloroglucinol. Compound 16 was regarded as a byproduct of phloroglucinolysis involving the oxidation of the pyrogallol-type B-ring (Scheme 3) [45]. Product 16 showed the [M + H] + peak at m/z 429.0818 in HRFABMS, indicating the molecular formula C 21 H 16 O 10 . An unambiguous assignment of the 1 H-and 13 C-NMR signals was achieved by 1 H-1 H COSY, HSQC, HMBC, and NOESY spectroscopy. The absence of a C-2 proton signal and appearance of a C-2 carbon signal at δ C 100.21 confirmed the presence of an A-type linkage [12,44,46]. The HMBC cross peaks ( Figure 13) between C-ring H-4 and D-ring C-1,2,6 indicated the linkage of the phloroglucinol moiety to C-ring C-4. The NOE cross peaks between H-3 and B-ring H-2,6 indicated the 3,4-trans configuration [47]. Furthermore, the ECD spectrum showed a positive Cotton effect at 220-240 nm, indicating a βconfiguration at C-4. Accordingly, 16 was established to be (−)-epigallocatechin-(4β→1,2→O→2)-phloroglucinol. Compound 16 was regarded as a byproduct of phloroglucinolysis involving the oxidation of the pyrogallol-type B-ring (Scheme 3) [45].

General
NMR spectra were recorded in acetone-d6 (Wako Pure Chem. Ind. Ltd., Osaka, Japan), methanol-d4 (Kanto Chem. Co., Inc., Tokyo, Japan), and DMSO-d6 (Kanto Chemical Co., Inc., Tokyo, Japan) with a Varian Unity Plus 500 spectrometer (Palo Alto, CA, USA) operating at 500 MHz for 1 H and 125 MHz for 13 C, and with a JEOL JNM-AL 400 spectrometer (JEOL Ltd, Tokyo, Japan) at 400 MHz for 1 H and 100 MHz for 13 C. HRFABMS spectra were recorded on a JMS 700N spectrometer (JEOL Ltd., Tokyo, Japan) in positive ion mode, with glycerol or m-nitrobenzyl alcohol, with or without NaCl, as the matrix. UV spectra were recorded in MeOH with a Jasco V-560 UV/Vis spectrometer (Jasco Co. Ltd.,

Plant Material
Dried aerial parts of Ephedra sinica were purchased from Uchida Wakanyaku Ltd., Tokyo, Japan.

Thiolysis
Thiol degradation was performed according to the method of Kusano et al. [35] with modifications. Proanthocyanidin oligomers (Fr. 1-1, 1.0 g) were dissolved in 60% EtOH (200 mL) containing mercaptoethanol (10 mL) (Kanto Chemical Co. Inc., Tokyo, Japan) and concentrated HCl (0.5 mL) (Kishida Chemical Co., Osaka, Japan). The reaction mixture was then heated at 70 • C for 22 h. The reaction mixture was then analyzed by HPLC and was further fractionated. Fr. 2-2-2 (1.0 g) was also subjected to thiolysis in the same manner. The reaction mixture of Fr. 1-1 was first concentrated to remove EtOH. The resulting aqueous solution was subjected to Sephadex LH-20 chromatography