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Article

Characterization and Identification of Bioactive Polyphenols in the Trapabispinosa Roxb. Pericarp Extract

1
Department of Nutritional Science, Faculty of Health and Welfare Sciences, Okayama Prefectural University, Okayama 719-1197, Japan
2
Hayashikane Sangyo Co., Ltd., 2-4-8 Yamatomachi, Shimonoseki 750-8608, Japan
*
Author to whom correspondence should be addressed.
Submission received: 13 August 2021 / Revised: 18 September 2021 / Accepted: 22 September 2021 / Published: 24 September 2021
(This article belongs to the Special Issue Bioactives and Functional Ingredients in Foods)

Abstract

:
In this study, we present the isolation and characterization of the structure of six gallotannins (16), three ellagitannins (79), a neolignan glucoside (10), and three related polyphenolic compounds (gallic acid, 11 and 12) from Trapa bispinosa Roxb. pericarp extract (TBE). Among the isolates, the structure of compound 10 possessing a previously unclear absolute configuration was unambiguously determined through nuclear magnetic resonance and circular dichroism analyses. The α-glucosidase activity and glycation inhibitory effects of the isolates were evaluated. Decarboxylated rugosin A (8) showed an α-glucosidase inhibitory activity, while hydrolyzable tannins revealed stronger antiglycation activity than that of the positive control. Furthermore, the identification and quantification of the TBE polyphenols were investigated by high-performance liquid chromatography coupled to ultraviolet detection and electrospray ionization mass spectrometry analysis, indicating the predominance of gallic acid, ellagic acid, and galloyl glucoses showing marked antiglycation properties. These findings suggest that there is a potential food industry application of polyphenols in TBE as a functional food with antidiabetic and antiglycation activities.

Graphical Abstract

1. Introduction

Water chestnut (Trapa bispinosa Roxb.)—belonging to the family Lythraceae—is a floating annual aquatic plant originally distributed in Southeast Asia, and its cultivation is widely extended to Southern Europe, Africa, and Asia. The dried pericarp has been popular as a tea in the Fukuoka and Saga prefectures in Japan [1], and it has been used as a traditional folk medicine in China, such as an antidiarrheal and antipyretic agent [2]. The fruit and leaf extracts reportedly exhibit diverse biological, such as antioxidant [3] and anticancer [4] activities. The removal effect of industrial pollutant by this plant was reported as a sorbent [5]. The phytochemical studies on this plant revealed the presence of tannins, flavonoids, and saponins, while its detailed components remain elusive. The phytochemical and biological studies of Trapa japonica Flerov.—a species closely related to Trapa bispinosa Roxb.—described the isolation of ellagitannins (including trapanin, tellimagrandin II, trapanins A and B, rugosin D, and cornusiin G) and gallotannins (including 1,2,3- and 1,2,6-tri-O-galloyl-β-d-glucoses) from the leaves and pericarps, and the biological properties of the polyphenols [6,7,8,9]. Based on these studies, Trapa bispinosa Roxb. polyphenols are believed to contribute to various biological effects.
The accumulation of advanced glycation end products (AGEs) in the various tissues of our body is a cause of Alzheimer’s disease [10] and diabetes [11], suggesting that the inhibition of glycation reaction delays the chronic glycative stress-associated diseases. From a disease prevention perspective, food components and pharmaceutical ingredients that inhibit the α-glucosidase activity and glycation reaction are beneficial for our health. The Trapa bispinosa Roxb. pericarp extract (TBE) reportedly inhibits the α-glucosidase and glycation reactions [1,12]. However, the TBE components that contribute to these inhibitory effects remain largely unknown. In this study, with the aim of developing functional food materials for TBE that is popular as tea in Japan and effectively utilizing the pericarp which is often wasted, we isolated and characterized TBE polyphenols and evaluated the α-glucosidase and AGE-formation inhibitory effects of the isolated compounds. In addition, the TBE polyphenols showing α-glucosidase and AGE-formation inhibitory effects were identified and quantified by high-performance liquid chromatography coupled to ultraviolet detection and electrospray ionization mass spectrometry (LC/UV/ESIMS) analysis. As the results, we believe that this study might be attract the interest of the food industry due to the development and evaluation of potential application of TBE ingredients.

2. Results and Discussion

2.1. Isolation and Structural Elucidation of TBE-Derived Polyphenols and Related Compounds

A 70% aqueous acetone extract of TBE was subsequently extracted with Et2O, EtOAc, and water-saturated n-BuOH. The EtOAc extract was repeatedly purified by Toyopearl HW-40 (coarse grade), MCI gel CHP20/P120, Sephadex LH-20, Mega Bond Elute C18 and preparative HPLC to obtain eight known compounds including gallic acid; six gallotannins as 2,6-di-O-galloyl-β-d-glucose (1) [13], 1,2,3-tri-O-galloyl-β-d-glucose (2) [14], 1,2,6-tri-O-galloyl-β-d-glucose (3) [14], 2,3,6-tri-O-galloyl-β-d-glucose (4) [15], 1,2,3,6-tetra-O-galloyl-β-d-glucose (5) [16], and 1,2,4,6-tetra-O-galloyl-β-d-glucose (6) [16]; and an ellagitannin as tellimagrandin II (7) [17]. The n-BuOH extract was subsequently subjected to column chromatography over Diaion HP-20 and Toyopearl HW-40 (coarse grade) to give compounds 5 and 7. In a separate experiment, TBE aqueous solution was chromatographed over Diaion HP-20, Toyopearl HW-40 (coarse grade), MCI gel CHP20/P120, Bond Elute Plexa, and Mega Bond Elute C18 to give five known compounds including two ellagitannins as decarboxylated rugosin A (8) [18] and camptothin B (9) [7], a neolignan as (7′S,8′R)-dihydrodehydrodiconiferyl alcohol-9′-O-β-d-glucoside (10) [19], and two ellagic acid derivatives as rubuphenol (11) [20] and eschweilenol A (12) [21]. The known compounds were identified by direct comparison with authentic specimens or by comparison of spectroscopic data with those reported in the literature (Figure 1).
The structure of neolignan glucoside (10) was characterized as a known dihydrodehydrodiconiferyl alcohol-9′-O-glucoside [19] based on the 1D and 2D-nuclear magnetic resonance (NMR) analyses including 1H-1H correlation spectroscopy (COSY), heteronuclear single quantum correlation (HSQC), heteronuclear multiple bond correlation (HMBC), and nuclear Overhauser effect spectroscopy (NOESY) experiments (Figure S1) and electrospray ionization-mass spectrometry (ESI-MS) analysis. However, the absolute configuration in 10 was still unknown. The stereochemistry of glucose in 10 was confirmed to D-series since the released glucose by acid hydrolysis of 10 was a positive response to the reaction with glucose oxidase [22]. The coupling constant (J7′,8 = 6.6 Hz) between the H-7′ and H-8′ protons suggested that the relative configuration of 10 at C-7′ and C-8′ was threo [23,24]. This relative configuration was further supported by the NOE correlations between the H-7′ and H-9′ protons, as well as those between the H-8′ and H-2′ and H-6′ protons. The absolute configuration of neolignan with dihydrobenzo[b]furan skeleton has been determined in agreement with the aromatic quadrant and P/M helicity rules [25,26].
The structure of compound 13, which was obtained by the hydrolysis of 10, was confirmed based on 1H-NMR (Figure S2), 13C-NMR and atmospheric pressure ionization (APCI) MS analyses. The circular dichroism (CD) spectrum of 13 showed negative cotton at the 1La (around 230 nm), indicating that the absolute configuration of C-8′ was R series based on the aromatic quadrant rule. Furthermore, the P/M helicity rule provided evidence that the 7′S, 8′R configurations of 13 by showing positive cotton at 1Lb (around 290 nm) band in the CD spectrum. Based on these findings, the stereochemistry of 10 was determined as shown by the formula in Figure 2.
Compound 14 was also obtained as a side product from the hydrolysis of 10 (Figure 2 and Figure S2). The product has been reported to be an intermediate of adenosine A1 receptor ligand [27]. The production of 14 by acid hydrolysis of 10 was further supported the characterization of aglycone moiety of 10.
Rubuphenol (11) and eschweilenol A (12) were identified by their 1H- and 13C-NMR, 1H-1H COSY, HSQC, HMBC, and NOESY experiments (Figure S3). The NOESY spectrum of 12 showed the correlation between H-5 and H-6″ protons, while that correlation was not observed in 11. Compounds 11 and 12 were methylated to confirm the structures as the corresponding methylated derivatives (compounds 1518) based on spectral analyses (Figure S4). The 1H-1H COSY and NOESY spectra of compounds 15 and 16 showed the correlations between H-5 and 4-OCH3 protons, while those correlations were not observed in 17 and 18. The observations of 1H-1H COSY and NOE correlations between H-5′ and 4′-OCH3 and, H-5″ and 4″-OCH3 protons in 16 and 18 supported the positions of hydroxyl group and ether linkage of 11 and 12. Based on these findings, the structures of 11 and 12 were established as shown by the formulas in Figure 1. In this study, compounds 1, 4, 6, 8, 11, and 12 were firstly isolated from Trapa species and the absolute configuration of 10 was confirmed based on NMR and CD analyses.

2.2. α-Glucosidase Inhibitory Activity of the TBE-Derived Compounds

The α-glucosidase inhibitory activities of the TBE-derived and the related polyphenols are shown in Table 1 as IC50 (μM) values. The inhibitory activities of gallic acid and ellagic acid, which are well-known metabolites of gallotannins and ellagitannins, respectively [28], showed little effect similar to that of gallotannins. (7′S,8′R)-Dihydrodehydrodiconiferyl alcohol-9′-O-β-d-glucoside (10) and its hydrolysates 13 and 14, and rubuphenol (11) and eschweilenol A (12) also showed no inhibitory activity. Among the tested compounds, 1,2,3,4,6-penta-O-galloyl-β-d-glucose and decarboxylated rugosin A (8) showed inhibitory activity with IC50 values at 59.0 ± 0.4 μM and 20.7 ± 0.1 μM, respectively. However, the inhibitory activities of all tested compounds were not reached to that of acarbose as a positive inhibitor [29]. Tannin-containing plant extracts reportedly exert a strong α-glucosidase inhibitory effect [30], but little is known about the tannin contributors themselves. It has been reported that the extract of Trapa japonica belonging to the same genus as T. bispinosa and the isolated ellagitannin dimers cornusiin G and rugosin D from T. japonica showed the inhibitory activity on α-glucosidase comparable to that of acarbose [8]. The isolated polyphenols from T. bispinosa showed no effect, but the presence of cornusiin G was confirmed by HPLC analysis, which was described in Section 2.4, indicating cornusiin G and unidentified ellagitannin dimers might be contributed to inhibition on α-glucosidase. Further study is necessary to investigate the possibility that the other components besides ellagitannin dimers contribute to the activity.

2.3. Antiglycation Effects of the TBE-Derived Compounds

The inhibitory effect of the TBE-derived polyphenols and the related compounds on AGEs, generated by the glycation reaction between human serum albumin (HSA) and glucose or fructose was evaluated. All tested compounds exhibited significantly stronger AGE-formation inhibitory activity than the positive control aminoguanidine [31] with IC50 values in the range of 0.1 ± 0.0–14.7 ± 2.0 μM with glucose and of 0.2 ± 0.0–27.0 ± 2.6 μM with fructose (Table 2), except for (7′S, 8′R)-dihydrodehydrodiconiferyl alcohol-9′-O-β-d-glucoside (10) and its hydrolysates 13 and 14. It is noteworthy that the gallotannin (26) and 1,2,3,4,6-penta-O-galloyl-β-d-glucose and ellagitannin (79) potencies were incomparably stronger than that of aminoguanidine.
AGEs are known to be generated via multiple pathways in glycation reactions [32]. Therefore, we also evaluated the AGE cross-link cleaving effects of the TBE polyphenols and the related compounds. Almost all tested compounds exhibited a stronger activity than the positive control N-phenacylthiazolium bromide (PTB) [33], except for ellagic acid, 2,6-di-O-galloyl-β-d-glucose (1), 1,2,3,4,6-penta-O-galloyl-β-d-glucose, 10, 13, and 14. In particular, gallic acid, rubuphenol (11), and eschweilenol A (12) showed remarkable activities in this assay. Some of isolates have not been evaluated for antiglycation effects, since the isolated amount of the compounds were insufficient to test. However, these results indicated that the TBE-derived polyphenols exert AGE-formation inhibitory activity and might contribute to the antiglycative effect of TBE.

2.4. LC/UV/ESIMS Analysis of TBE

The presence of phenolic compounds, such as the gallotannins and ellagitannins, has already been previously reported in the pericarp of Trapa species [8,34]. However, the detailed polyphenol content in the pericarp of the Trapa species has not yet been revealed. There are few reports on the qualitative and quantification of hydrolyzable tannins containing both gallotannins and ellagitannins by LC-MS method. Here, we could identify and quantify a total of 30 polyphenols including gallotannins and ellagitannins in TBE by LC/UV/ESIMS method with each polyphenol specimen (Figure 3). The total ion and UV at 280 nm chromatograms of TBE displayed with good separation in Figure 3A,B, respectively. Among the candidates, compounds having lactones were clearly detected at UV at 360 nm (Figure 3C) for more separation and accurate quantification. In Table 3, gallic acid (32.2 ± 0.1 mg/g) exhibiting the most potent AGE cross-link cleaving activity among the isolated polyphenols is shown as a main TBE component, suggesting that it was produced from gallotannins or ellagitannins during TBE manufacturing, as well as ellagic acid (6.9 ± 0.1 mg/g). The various gallotannins possessing significant AGE-formation inhibitory activity were contained in the range of 0.2 ± 0.1–16.8 ± 1.2 mg/g. Valoneic acid dilactone (1.8 ± 0.2 mg/g), rubuphenol (11) (4.3 ± 0.1 mg/g), and eschweilenol A (12) (0.9 ± 0.2 mg/g) were minor TBE components, implying that these polyphenols were also ascribable to TBE ellagitannins. Urolithin M5 (1.4 ± 0.4 mg/g), a well-known ellagitannin metabolite, was also found in TBE. Urolithin M5 might be produced by biosynthesis in Trapa bispinosa, since urolithins A and B and isourolithin A reportedly contained in the plant of the same genus, Trapa natans [35]. Urolithin M5 has also been isolated from Tamarix nilotica [36]. The presence of ellagitannin dimers, camptothin B (9) and cornusiin G, and an ellagitannin monomer, 1,2-Di-O-galloyl-4,6-hexahydroxydiphenoyl-d-glucose in TBE could also be identified by LC/UV/ESIMS analysis. Gallotannins (26), tellimagrandin II (7), and decarboxylated rugosin A (8), which showed strong effects of both AGE-formation inhibition and AGE-derived crosslink cleaving, were contained in TBE at high levels. These results clearly provided the basic confirmation to the potential contribution of TBE polyphenols to antidiabetic and antiglycative effects.

3. Materials and Methods

3.1. Chemicals

The TBE was prepared as follows: the Trapa bispinosa pericarp cultivated in Thailand was dried, sterilized, and crushed at ambient conditions, followed by extraction with hot water (approximately six times the weight of the water chestnut pericarp). Dextrin was added to the extracted liquid so that the ratio of chestnut pericarp water extract to dextrin would be 67:33 using the dry weight. TBE was obtained after spray drying the extract and its moisture content was less than 10%. Ellagic acid, N-phenacylthiazolium bromide (PTB), and aminoguanidine hydrochloride were obtained from Wako Pure Chemical Industries (Osaka, Japan). 1-Phenyl-1,2-propanedione (PPD) and rat intestinal acetone powder were purchased from Sigma Aldrich (St Louis, MO, USA). Trimethylsilyldiazomethane (TMS-CHN2) was purchased from Tokyo Chemical Industry (Tokyo, Japan). Each polyphenol specimen was used compounds isolated from natural sources held in our library: gallotannins [37,38,39,40,41,42,43,44], 1,2-di-O-galloyl-4,6-hexahydroxydiphenoyl-d-glucose [45], cornusiin G [8], brevifolincarboxylic acid [39], urolithin M5 [46], and valoneic acid dilactone [41].

3.2. General Experimental Procedure

Optical rotations were recorded using a Jasco DIP-1000 polarimeter (Jasco, Tokyo, Japan). UV and CD spectra were measured by using Jasco V-530 spectrophotometer (Jasco, Tokyo, Japan) and Jasco J-710 spectropolarimeter (Jasco, Tokyo, Japan), respectively. 1H-NMR (600 MHz) and 13C-NMR (151 MHz) spectra including 1H-1H COSY, NOESY, HSQC, and HMBC were recorded on a Varian NMR system (Varian, Palo Alto, CA, USA) and chemical shifts are given in ppm (ppm) values relative to acetone-d6 (2.04 ppm for 1H and 29.8 ppm for 13C), CD3OD (3.35 ppm for 1H and 49.0 ppm for 13C), and CDCl3 (7.26 ppm for 1H and 77.0 ppm for 13C). ESI or APCI mass spectra were performed on a Bruker MicrOTOF II instrument (Bruker, Billerica, MA, USA) using direct sample injection. Reversed-phase HPLC was conducted on InertSustain C18 column (150 mm × 4.6 mm i.d., 5 μm, GL Sciences, Tokyo, Japan) at 40 °C with the mobile phase consisted of CH3CN:H2O:HCOOH (5:90:5) (solvent A) and CH3CN:H2O:HCOOH (45:50:5) (solvent B). The flow rate was 1.0 mL/min, and a linear gradient was programmed as follows: 0–15 min (solvent B: 10–30%), 15–20 min (solvent B: 30–50%), 20–30 min (solvent B: 10%) and the absorbance was monitored at 280 and 360 nm. Normal-phase HPLC was conducted on YMC-Pack SIL column (250 mm × 4.6 mm i.d., 5 μm, YMC, Kyoto, Japan) with n-hexane:MeOH:THF:HCOOH (55:33:11:1) containing oxalic acid (450 mg/L) by isocratic elution. The flow rate was 1.5 mL/min and the absorbance was monitored at 280 nm. Preparative HPLC was carried out under the same conditions as reversed-phase HPLC condition by using the mobile phase consisted of CH3CN:H2O:HCOOH (10:85:5) (condition 1) or MeOH:H2O:HCOOH (10:85:5) (condition 2). Column chromatography was carried out by Diaion HP-20 (Mitsubishi Chemical, Tokyo, Japan), Toyopearl HW-40 (coarse grade) (Tosoh, Tokyo, Japan), MCI gel CHP20/P120 (Mitsubishi Chemical, Tokyo, Japan), Sephadex LH-20 (GE Healthcare, Chicago, IL, USA), Mega Bond Elut C18 (Agilent technologies, Santa Clara, CA, USA), Bond Elut Plexa (Agilent technologies, Santa Clara, CA, USA), and YMC Gel ODS-AQ-HG (YMC, Kyoto, Japan).

3.3. Extraction and Isolation

TBE (450 g) was dissolved in H2O (700 mL) and the solution was extracted subsequently extracted with Et2O (3 × 700 mL), EtOAc (3 × 700 mL), and water-saturated n-BuOH (3 × 700 mL), to give Et2O (5.2 g), EtOAc (16.7 g), n-BuOH extracts (14.3 g), and H2O soluble portion (163.5 g). A part of EtOAc extract (5.0 g) was chromatographed over Toyopearl HW-40 (coarse grade) (40 cm × 2.2 i.d. cm) with 40%, 50%, 60%, and 70% aqueous MeOH—MeOH:H2O:acetone (7:2:1)—MeOH:H2O:acetone (7:1:2)—70% aqueous acetone in a stepwise elution mode. The 40% aqueous MeOH fraction gave gallic acid (779.9 mg), the 50% aqueous MeOH fraction gave 1,2,3-tri-O-galloyl-β-d-glucose (2) (148.1 mg), the 60% aqueous MeOH fraction gave 1,2,3,6-tetra-O-galloyl-β-d-glucose (5) (301.1 mg), and the 70% aqueous MeOH fraction gave 1,2,4,6-tetra-O-galloyl-β-d-glucose (6) (67.7 mg) and tellimagrandin II (7) (189.2 mg), by column chromatographic purification on each MCI gel CHP20/P120 (40 cm × 1.1 i.d. cm). The 40% aqueous MeOH fraction (200 mg) was purified by column chromatographies over MCI gel CHP20/P120 (40 cm × 1.1 i.d. cm) with aqueous MeOH, Sephadex LH-20 (40 cm × 1.1 i.d. cm) with EtOH-MeOH solvent system, Mega Bond Elut C18 cartridge column with aqueous MeOH, and preparative HPLC under the condition 1, to give 2,6-Di-O-galloyl-β-d-glucose (1) (3.0 mg). The 50% aqueous MeOH fraction (300 mg) was further purified by Mega Bond Elut C18 with 10%, 20%, 30%, 40%, 50%, 60%, and 70% aqueous MeOH–100% MeOH–70% aqueous acetone and preparative HPLC under the condition 2 to give 1,2,6-tri-O-galloyl-β-d-glucose (3) (0.8 mg) and 2,3,6-tri-O-galloyl-β-d-glucose (4) (0.9 mg). A part of n-BuOH extract (10 g) was chromatographed over Diaion HP-20 (40 cm × 5.0 i.d. cm) with H2O–10, 30, and 50% aqueous MeOH–100% MeOH–70% aqueous acetone in a stepwise elution mode. The 50% aqueous MeOH eluate (1.5 g) was further chromatographed over Toyopearl HW-40 (coarse grade) (40 cm × 2.2 i.d. cm) with 50%, 60%, and 70% aqueous MeOH–MeOH:H2O:acetone (7:2:1)–MeOH:H2O:acetone (7:1:2)–70% aqueous acetone in stepwise elution mode. Compounds 5 (40.3 mg) and 7 (43.8 mg) were obtained from the 60% aqueous MeOH and MeOH:H2O:acetone (7:2:1) fractions, respectively. TBE (500 g) was dissolved in H2O (10 L) and, the solution was subjected to Diaion HP-20 (80 cm × 5.0 i.d. cm) and eluted with H2O increasing amounts of MeOH (0–10–30–50–100% MeOH) and 70% aqueous acetone. A part (7.75 g) of the 50% aqueous MeOH eluate (23.0 g) was chromatographed over Toyopearl HW-40 (coarse grade) (40 cm × 2.2 i.d. cm) with 50, 60, and 70% aqueous MeOH–MeOH:H2O:acetone (7:2:1)–MeOH:H2O:acetone (7:1:2)–70% aqueous acetone as eluent. The combined fraction (400 mg) consisted of 70% aqueous MeOH and MeOH:H2O:acetone (7:1:2) fractions was purified by column chromatography over MCI gel CHP20/P120 (40 cm × 1.1 i.d. cm) with 30%, 40%, and 50% aqueous MeOH–100% MeOH–70% aqueous acetone. Subsequently, the 40% MeOH eluate (23.2 mg) was purified by Bond Elut Plexa with 30%, 40%, 50%, 60% aqueous MeOH and MeOH to give decarboxylated rugosin A (8) (8.4 mg) from the 60% aqueous MeOH eluate. The MeOH:H2O:acetone (7:2:1) eluate (120 mg) was purified by Mega Bond Elut C18 and YMC Gel ODS-AQ-HG (21 cm × 1.1 i.d. cm) with H2O-MeOH solvent system to give camptothin B (9) (7.3 mg). The 100% MeOH eluate (8.0 g) obtained by Diaion HP-20 separation was chromatographed over Toyopearl HW-40 (coarse grade) (43 cm × 2.2 i.d. cm) with H2O increasing amounts of MeOH (50–60–100% MeOH) followed by 70% aqueous acetone. (7′S, 8′R)-Dihydrodehydrodiconiferyl alcohol-9′-O-β-d-glucoside (10) (347.2 mg) was obtained from the 50% aqueous MeOH eluate. The fraction eluted with 100% MeOH and 70% aqueous acetone was dissolved in hot MeOH and the resulting solution was stored at 4 °C to remove ellagic acid as a precipitate. The collected supernatant (2.6 g) was chromatographed over Toyopearl HW-40 (coarse grade) (44 cm × 2.2 i.d. cm) with 70% aqueous MeOH–MeOH:H2O:acetone (7:2:1)–MeOH:H2O:acetone (7:1:2)–70% aqueous acetone. The MeOH:H2O:acetone (7:2:1)-MeOH:H2O:acetone (7:1:2) eluate was purified by MCI gel CHP20/P120 (24 cm × 1.1 i.d. cm) and YMC Gel ODS-AQ-HG (27 cm × 1.1 i.d. cm) with MeOH-H2O solvent system to afford rubuphenol (11) (33.1 mg) and eschweilenol A (12) (5.5 mg).
2,6-Di-O-galloyl-β-d-glucose (1): pale yellow amorphous powder; 1H-NMR [600 MHz, acetone-d6-D2O (9:1)] δ 7.11–7.14 (4H in total each, s, galloyl-H), 5.35 (1H, d, J = 3.6 Hz, Glc α-1), 4.88 (1H, t, J = 8.4 Hz, Glc β-2), 4.83 (1H, d, J = 7.8 Hz, Glc β-1), 4.74 (1H, dd, J = 3.6, 10.2 Hz, Glc α-2), 4.56 (1H, dd, J = 1.8, 11.4 Hz, Glc β-6), 4.51 (1H, dd, J = 1.8, 11.4 Hz, Glc α-6), 4.36 (2H, m, Glc α-6, β-6), 4.13 (1H, m, Glc α-5), 4.09 (1H, t, J = 9.6 Hz, Glc α-3), 3.74 (1H, t, J = 9 Hz, Glc β-3), 3.68 (1H, m, Glc β-5), 3.5–3.7 (Glc- and β-4, overlapped with DOH). HR-ESI-MS m/z 483.0787 [M − H] (calcd for C20H20O14-H, 483.0780).
1,2,3-Tri-O-galloyl-β-D-glucose (2): pale yellow amorphous powder; 1H-NMR [600 MHz, acetone-d6-D2O (9:1)] δ 7.09, 7.08, 7.00 (2H, each, s, galloyl-H), 6.06 (1H, d, J = 8.4 Hz, Glc H-1), 5.61 (1H, t, J = 9.6 Hz, Glc H-3), 5.40 (1H, dd, J = 8.4, 9.6 Hz, Glc H-2), 3.97 (1H, t, J = 9.6 Hz, Glc H-4), 3.93 (1H, d, J = 10.2 Hz, Glc H-6), 3.81 (2H, m, Glc H-5, 6). HR-ESI-MS m/z 635.0890 [M − H] (calcd for C27H24O18-H, 635.0890)
1,2,6-Tri-O-galloyl-β-d-glucose (3): pale yellow amorphous powder; 1H-NMR [600 MHz, acetone-d6-D2O (9:1)] δ 7.11, 7.07, 7.03 (2H, each, s, galloyl-H), 5.90 (1H, d, J = 9.0 Hz, Glc H-1), 5.22 (1H, t, J = 9.0 Hz, Glc H-2), 4.59 (1H, dd, J = 1.8, 12.0 Hz, Glc H-6), 4.37 (1H, dd, J = 5.4, 12.0 Hz, Glc H-6), 3.96 (1H, t, J = 9.0 Hz, Glc H-3), 3.91 (1H, m, Glc H-5), 3.71 (1H, t, J = 9.0 Hz, Glc H-4). HR-ESI-MS m/z 635.0898 [M − H] (calcd for C27H24O18-H, 635.0890).
2,3,6-Tri-O-galloyl-β-d-glucose (4): pale yellow amorphous powder; 1H-NMR [600 MHz, acetone-d6-D2O (9:1)] δ 7.00–7.14 (6H in total each, s, galloyl-H), 5.77 (1H, t, J = 9.6 Hz, Glc α-3), 5.47 (1H, d, J = 3.6 Hz, Glc α-1), 5.43 (1H, m, Glc β-3), 5.10 (1H, dd, J = 7.8, 9.6 Hz, Glc β-2), 5.01 (1H, d, J = 7.8 Hz, Glc β-1), 4.94 (1H, dd, J = 3.6, 9.6 Hz, Glc α-2), 4.59 (1H, d, J = 11.4 Hz, Glc β-6), 4.55 (1H, dd, J = 1.8, 12 Hz Glc α-6), 4.47–4.40 (3H, m, Glc α-6, β-6, β-5), 4.29 (1H, m, Glc α-5), 3.93 (1H, t, J = 9.6 Hz, Glc α-4), 3.88 (1H, d, J = 6 Hz, Glc β-4). HR-ESI-MS m/z 635.0892 [M − H] (calcd for C27H24O18-H, 635.0890).
1,2,3,6-Tetra-O-galloyl-β-d-glucose (5): pale yellow amorphous powder; 1H-NMR [600 MHz, acetone-d6-D2O (9:1)] δ 7.16, 7.09, 7.08, 7.01 (2H, each, s, galloyl-H), 6.14 (1H, d, J = 8.4 Hz, Glc H-1), 5.69 (1H, t, J = 9.6 Hz, Glc H-3), 5.49 (1H, dd, J = 8.4, 9.6 Hz, Glc H-2), 4.66 (1H, dd, J = 1.8, 12.6 Hz, Glc H-6), 4.50 (1H, dd, J = 5.4, 12.6 Hz, Glc H-6), 4.17 (1H, m, Glc H-5), 4.08 (1H, t, J = 9.6 Hz, Glc H-4). HR-ESI-MS m/z 787.1034 [M − H] (calcd for C34H28O22-H, 787.0999).
1,2,4,6-Tetra-O-galloyl-β-d-glucose (6): pale yellow amorphous powder; 1H-NMR [600 MHz, acetone-d6-D2O (9:1)] δ 7.14, 7.13, 7.09, 7.06 (2H, each, s, galloyl-H), 6.04 (1H, d, J = 8.4 Hz, Glc H-1), 5.40 (1H, t, J = 9.6 Hz, Glc H-4), 5.38 (1H, dd, J = 8.4, 9.6 Hz, Glc H-2), 4.53 (1H, dd, J = 1.2, 12.6 Hz, Glc H-6), 4.36 (1H, t, J = 9.6 Hz, Glc H-3), 4.30 (1H, m, Glc H-5), 4.20 (1H, dd, J = 5.4, 12.6 Hz, Glc H-6). HR-ESI-MS m/z 787.1008 [M − H] (calcd for C34H28O22-H, 787.0999).
Tellimagrandin II (7): pale yellow amorphous powder; 1H-NMR [600 MHz, acetone-d6-D2O (9:1)] δ 7.09, 6.99, 6.95 (2H, each, s, galloyl-H), 6.63, 6.47 [1H each, HHDP-H], 6.17 (1H, d, J = 7.8 Hz, Glc H-1), 5.81 (1H, t, J = 9.6 Hz, Glc H-3), 5.59 (1H, dd, J = 7.8, 9.6 Hz, Glc H-2), 5.32 (1H, dd, J = 6.6, 13.2 Hz, Glc H-6), 5.20 (1H, t, J = 9.6 Hz, Glc H-4), 4.53 (1H, dd, J = 6.6, 9.6 Hz, Glc H-5), 3.87 (1H, d, J = 13.2 Hz, Glc H-6). HR-ESI-MS m/z 937.0956 [M − H] (calcd for C41H30O26-H, 937.0953).
Decarboxylated rugosin A (8): dark brown amorphous powder; 1H-NMR [600 MHz, acetone-d6-D2O (9:1)] δ 7.08, 7.00, 6.98 (2H, each, s, galloyl-H), 6.49, 6.39 (1H each, s, decarboxylated valoneoyl group -Ha, Hb), 6.43, 6.38 (1H each, d, J = 9.0 Hz, decarboxylated valoneoyl group -Hc, Hd), 6.15 (1H, d, J = 7.8 Hz, Glc H-1), 5.81 (1H, t, J = 9.6 Hz, Glc H-3), 5.59 (1H, dd, J = 7.8, 9.6 Hz, Glc H-2), 5.27 (1H, dd, J = 6.6, 13.2 Hz, Glc H-6), 5.17 (1H, t, J = 9.6 Hz, Glc H-4), 4.13 (1H, dd, J = 6.6, 9.6 Hz, Glc H-5), 3.82 (1H, d, J = 13.2 Hz, Glc H-6). HR-ESI-MS m/z 1061.1114 [M − H] (calcd for C47H34O29-H, 1061.1113).
Camptothin B (9): dark brown amorphous powder; 1H-NMR [600 MHz, acetone-d6-D2O (9:1)] δ 7.09, 6.99, 6.97, 6.84 (4/3 H, each, s, galloyl-H), 7.08, 7.02, 6.98, 6.91 (2/3 H, each, s, galloyl-H), 7.11 (1/3H, s, valoneoyl-Hc), 7.00 (2/3H, s, valoneoyl-Hc), 6.65 (2/3H, s, HHDP-Hd), 6.64 (1/3H, s, HHDP-Hd), 6.498 (1/3H, s, HHDP-He), 6.496 (2/3H, s, HHDP-He), 6.62 (1/3H, s, valoneoyl-Ha), 6.59 (2/3H, s, valoneoyl-Ha), 6.18 (1/3H, s, valoneoyl-Hb), 6.16 (2/3H, s, valoneoyl-Hb), 6.18 (2/3H, d, J = 7.8 Hz, Glc HR-1), 6.16 (1/3H, d, J = 7.8 Hz, Glc HR-1), 5.80 (1/3H, t, J = 9.6 Hz, Glc HL-3), 5.60 (1/3H, t, J = 9.6 Hz, Glc HR-3), 5.59 (2/3H, t, J = 9.6 Hz, Glc HR-3), 5.55 (1/3H, dd, J = 7.8, 9.6 Hz, Glc HR-2), 5.54 (2/3H, dd, J = 7.8, 9.6 Hz, Glc HR-2), 5.46 (2/3H, t, J = 9.6 Hz, Glc HL-3), 5.44 (1/3H, dd, J = 6.6, 13.2 Hz, Glc HL-6), 5.35 (1/3H, dd, J = 4.2 Hz, Glc HL-1α), 5.27 (1/3H, dd, J = 6.6, 13.2 Hz, Glc HR-6), 5.21 (1/3H, d, J = 6.6, 13.2 Hz, Glc HR-6′), 5.17 (2/3H, dd, J = 6.6, 13.2 Hz, Glc HL-6), 5.12 (2/3H, dd, J = 8.4, 9.6 Hz, Glc HL-2), 5.09 (1/3H, t, J = 9.6 Hz, Glc HL-4), 5.07 (1/3H,2/3H t, J = 9.6 Hz, Glc HR-4), 5.06 (1/3H, t, J = 4.2, 9.6 Hz, Glc HL-2), 5.00 (2/3H, t, J = 9.6 Hz, Glc HL-4), 5.00 (2/3H, t, J = 9.6 Hz, Glc HL-4), 4.61 (1/3H, dd, J = 6.6, 9.6 Hz, Glc HR-5), 4.57 (1/3H, dd, J = 6.6, 9.6 Hz, Glc HL-5), 4.45 (2/3H, dd, J = 6.6, 9.6 Hz, Glc HL-4), 4.49 (2/3H, t, J = 8.4 Hz, Glc HL-1β), 3.93 (1/3H, d, J = 13.2 Hz, Glc HL-6), 3.87 (2/3H, d, J = 13.2 Hz, Glc HR-6′), 3.80 (2/3H, d, J = 13.2 Hz, Glc HL-6), 3.74 (1/3H, d, J = 13.2 Hz, Glc HR-6). HR-ESI-MS m/z 1721.1710 [M − H] (calcd for C75H54O48-H, 1721.1712).
(7′S,8′R)-Dihydrodehydrodiconiferyl alcohol-9′-O-β-d-glucoside (10): pale brown amorphous powder; [ α ] D 25 + 12.8° (c 0.5, MeOH); UV (MeOH) λmax (log ε) 225 (4.30), 282 (3.92) nm; CD (MeOH) [α] (nm) − 4.3 × 103 (223), +1.7 × 104 (240), +1.0 × 104 (291); 1H-NMR [600 MHz, CD3OD] δ 6.99 (1H, d, J = 1.8 Hz, H-2′), 6.84 (1H, dd, J = 1.8, 7.8 Hz, H-6′), 6.79 (1H, brs, H-6), 6.75 (1H, d, J = 7.8 Hz, H-5′), 6.71 (1H, brd, J = 1.2 Hz, H-2), 5.57 (1H, d, J = 6.6 Hz, H-7′), 4.35 (1H, d, J = 7.8 Hz, H-1″), 4.10 (1H, dd, J = 8.4, 9.6 Hz, H-9′a), 3.86 (1H, dd, J = 2.4, 12.0 Hz, H-6”a), 3.84 (3H, s, OCH3-3), 3.83 (1H, m, H-9′b), 3.81 (3H, s, OCH3-3′), 3.68 (1H, dd, J = 6.0, 12.0 Hz, H-6″a), 3.64 (1H, brdd, J = 6.6, 13.2 Hz, H-8′), 3.58 (2H, t, J = 6.6 Hz, H-9), 3.37 (1H, t, J = 9.0 Hz, H-3″), 3.31 (1H, t, J = 9.0 Hz, H-4″), 3.27 (1H, ddd, J = 2.4, 6.0, 9.0 Hz, H-5″), 3.23 (1H, dd, J = 7.8, 9.0 Hz, H-2″), 2.61 (2H, brt, J = 7.8 Hz, H-7), 1.80 (2H, m, H-8); 13C-NMR [151 MHz, CD3OD] δ 147.6 (C-3′), 146.00 (C-4′), 145.97 (C-4), 143.8 (C-3), 135.5 (C-1), 133.2 (C-1′), 128.3 (C-5), 118.4 (C-6′), 116.8 (C-6), 114.6 (C-5′), 112.6 (C-2), 109.4 (C-2′), 102.8 (C-1″), 87.8 (C-7′), 76.7 (C-3″), 76.6 (C-5″), 73.7 (C-2″), 70.9 (C-9′), 70.2 (C-4″), 61.3 (C-6″), 60.8 (C-9), 55.3 (C-3OCH3), 55.0 (C-3′OCH3), 51.5 (C-8′), 34.4 (C-8), 31.5 (C-7); HR-ESI-MS m/z 521.2041 [M − H] (calcd for C26H34O11-H, 521.2028).
Acid hydrolysis of 10: A solution of 10 (50 mg) in 1 M HCl was heated in boiled water for 1 h. The reaction mixture was purified by Mega Bond Elut C18 with MeOH-H2O (0:100–10:90–20:80–30:70–40:60–50:50–60:40) in stepwise gradient. Compounds 13 (5.4 mg) and 14 (4.3 mg) were obtained from 40% and 60% MeOH fractions, respectively. The obtained H2O fraction was tested by Glucose CII Test Wako kit (Wako Pure Chemical Industries, Osaka, Japan) to determine D-series of glucose in 10 [22].
Compound 13 (aglycone of 10): off-white amorphous powder; [ α ] D 25 + 14.1° (c 0.5, MeOH); UV (MeOH) λmax (log ε) 227 (4.30), 286 (3.87) nm; CD (MeOH) [α] (nm) − 3.4 × 103 (225), + 1.5 × 104 (240), + 6.7 × 103 (292); 1H-NMR [600 MHz, acetone-d6-D2O (9:1)] δ 6.99 (1H, d, J = 1.8 Hz, H-2′), 6.83 (1H, dd, J = 1.8, 7.8 Hz, H-6′), 6.78 (1H, d, J = 7.8 Hz, H-5′), 6.72 (1H, brs, H-6), 6.71 (1H, brs, H-2), 5.50 (1H, d, J = 6.6 Hz, H-7′), 3.82 (1H, m, H-9′a), 3.80 (3H, s, OCH3-3′), 3.78 (3H, s, OCH3-3), 3.73 (1H, dd, J = 7.2, 10.8 Hz, H-9′b), 3.52 (2H, t, J = 6.6 Hz, H-9), 3.47 (1H, brdd, J = 6.6, 13.2 Hz, H-8′), 2.58 (2H, brt, J = 7.8 Hz, H-7), 1.76 (2H, m, H-8); 13C-NMR [151 MHz, acetone-d6-D2O (9:1)] δ 147.6 (C-3′), 146.2 (C-4, 4′), 143.9 (C-3), 135.5 (C-1), 133.6 (C-1′), 128.9 (C-5), 118.4 (C-6′), 116.7 (C-6), 114.9 (C-5′), 112.8 (C-2), 109.7 (C-2′), 87.2 (C-7′), 63.6 (C-9′), 60.7 (C-9), 55.5 (C-3OCH3), 55.4 (C-3′OCH3), 54.0 (C-8′), 34.7 (C-8), 31.7 (C-7); HR-APCI-MS m/z 359.1499 [M − H] (calcd for C20H24O6-H, 359.1500).
Compound 14: off-white amorphous powder; 1H-NMR [600 MHz, acetone-d6-D2O (9:1)] δ 7.32 (1H, d, J = 1.8 Hz, H-2′), 7.25 (1H, dd, J = 1.8, 8.4 Hz, H-6′), 6.97 (1H, d, J = 1.2 Hz, H-6), 6.95 (1H, d, J = 8.4 Hz, H-5′), 6.77 (1H, brd, J = 1.2 Hz, H-2), 3.97 (3H, s, OCH3-3), 3.91 (3H, s, OCH3-3′), 3.56 (2H, t, J = 6.6 Hz, H-9), 2.74 (2H, brt, J = 7.2 Hz, H-7), 2.39 (3H, s, H-9′), 1.86 (2H, m, H-8); 13C-NMR [151 MHz, acetone-d6-D2O (9:1)] δ 151.1 (C-7′), 147.8 (C-3′), 147.0 (C-4′), 144.7 (C-3), 141.0 (C-4), 137.9 (C-1), 132.9 (C-8′), 123.0 (C-5), 120.0 (C-6′), 115.4 (C-5′), 110.6 (C-6), 110.2 (C-2′), 109.5 (C-1′), 107.6 (C-2), 60.8 (C-9), 55.50 (C-3′OCH3), 55.46 (C-3OCH3), 34.9 (C-8), 32.3 (C-7), 8.8 (C-9′); HR-APCI-MS m/z 341.1394 [M − H] (calcd for C20H22O5-H, 341.1394).
Rubuphenol (11): off-white amorphous powder; 1H-NMR [600 MHz, CD3OD]: δ 7.53 (1H, s, H-5′), 7.33 (1H, s, H-5), 6.19 (1H, d, J = 9.0 Hz, H-6″), 6.16 (1H, d, J = 9.0 Hz, H-5′); 13C-NMR [151 MHz, CD3OD]: δ 160.7 (C-7), 160.6 (C-7′), 153.8 (C-4′), 149.5 (C-4), 143.4 (C-2 or 2′), 143.1 (C-4″), 141.2 (C-1″), 141.0 (C-3), 137.9 (C-3′), 137.15 (C-2”), 137.09 (C-2 or 2′), 135.5 (C-3″), 115.2 (C-6′), 113.4 (C-1), 113.3 (C-1′), 113.1 (C-5′), 111.8 (C-5), 108.9 (C-6), 107.4 (C-6″), 106.3 (C-5″); HR-ESI-MS m/z 425.0144 [M − H] (calcd for C20H10O11-H, 425.0150).
Eschweilenol A (12): off-white amorphous powder; 1H-NMR [600 MHz, CD3OD]: δ 7.50 (1H, s, H-5′), 7.30 (1H, s, H-5), 6.51 (1H, d, J = 9.0 Hz, H-6″), 6.40 (1H, d, J = 9.0 Hz, H-5″); 13C-NMR [151 MHz, CD3OD]: δ 161.34 (C-7′), 161.27 (C-7), 150.7 (C-4), 149.9 (C-4′), 145.0 (C-4″), 142.8 (C-3), 141.0 (C-3′), 139.7 (C-2″), 137.9 (2C, C-2, 2′) (2C), 137.4 (C-1″), 136.2 (C-3″), 115.9 (C-1), 113.6 (C-1′), 112.8 (C-6″), 111.9 (C-5), 111.8 (C-5′), 109.9 (C-6′), 108.7 (C-6), 107.3 (C-5″); HR-ESI-MS m/z 425.0134 [M − H] (calcd for C20H10O11-H, 425.0150).
Methylation of compounds 11 and 12: Each solution of 11 (10 mg) or 12 (10 mg) in acetone was treated with an excess of TMS-CHN2 in hexane at room temperature overnight. Each reaction mixture was evaporated in vacuo and purified by preparative TLC (Merck, North Wales, PA, USA) with toluene:acetone (3:1) to obtain compounds 15 (Rf 0.55, 1.8 mg) and 16 (Rf 0.70, 2.1 mg) from 11, and 17 (Rf 0.60, 0.7 mg) and 18 (Rf 0.74, 1.2 mg) from 12, respectively.
Compound 15: off-white amorphous powder; 1H-NMR [600 MHz, CDCl3]: δ 7.76 (1H, s, H-5′), 7.67 (1H, s, H-5), 6.52 (1H, d, J = 9.0 Hz, H-5″), 6.23 (1H, d, J = 9.0 Hz, H-6″), 4.24, 4.032, 4.026, 4.02, 3.95 (each 3H, s, OCH3-H); HR-APCI-MS m/z 497.1102 [M + H]+ (calcd for C25H20O11 + H, 497.1078).
Compound 16: off-white amorphous powder; 1H-NMR [600 MHz, CDCl3]: δ 7.76 (1H, s, H-5′), 7.67 (1H, s, H-5), 6.47 (1H, d, J = 9.0 Hz, H-5″), 6.41 (1H, d, J = 9.0 Hz, H-6″), 4.24, 4.03, 4.00 (each 3H, s, OCH3-H), 3.94 (6H, s, OCH3-H), 3.82 (3H, s, OCH3-H); HR-APCI-MS m/z 511.1258 [M + H]+ (calcd for C26H22O11 + H, 511.1235).
Compound 17: off-white amorphous powder; 1H-NMR [600 MHz, CDCl3] δ 7.71 (1H, s, H-5′), 7.49 (1H, s, H-5), 6.79 (1H, d, J = 9.0 Hz, H-6″), 6.75 (1H, d, J = 9.0 Hz, H-5″), 4.32, 4.21, 4.04, 3.98, 3.83 (each 3H, s, OCH3-H); HR-APCI-MS m/z 497.1065 [M + H]+ (calcd for C25H20O11 + H, 497.1078).
Compound 18: off-white amorphous powder; 1H-NMR [600 MHz, CDCl3] δ 7.71 (1H, s, H-5′), 7.47 (1H, s, H-5), 6.85 (1H, d, J = 9.0 Hz, H-6″), 6.69 (1H, d, J = 9.0 Hz, H-5″), 4.32, 4.20, 4.04, 3.92, 3.91, 3.82 (each 3H, s, OCH3-H); HR-APCI-MS m/z 511.1245 [M + H]+ (calcd for C26H22O11 + H, 511.1235).

3.4. α-Glucosidase Inhibitory Activity

The α-glucosidase inhibitory activity was tested according to the previously described method by Kirino et al. [47] with a slight modification. Rat intestinal acetone powder was mixed with 0.1 M phosphate buffer (pH 7) and centrifuged at 18,500× g and 4 °C for 20 min. The resulting supernatant was collected and used as a glucosidase solution for enzymatic assay. Each sample solution (160 μL) was mixed with 250 mM maltose solution (20 μL) in 0.2 M phosphate buffer (pH 7) and then incubated at 37 °C for 3 min. After incubation, the enzymatic reaction was started by adding glucosidase solution from the rat intestine (20 μL) and the resulting reaction mixtures were further incubated at 37 °C for 15 min. After 15 min, the reaction mixtures were immediately heated at 100 °C for 5 min to stop the reaction followed by cooling on ice for 5 min. The amount of glucose in the reaction mixtures was determined using the F-Kit glucose (Roche diagnostics, Co., Tokyo, Japan) by measuring the absorbance at 340 nm. A control was carried out with 0.1 M phosphate buffer (pH 7) instead of sample solution. For the blank, the glucosidase solution was replaced with distilled water. The glucosidase inhibitory rates of tested samples were calculated as;
Inhibitory rate (%) = 100 − [(AsampleAblank)/(AcontrolAblank)] × 100
where Asample, Acontrol, and Ablank is the absorbance of the tested sample, control, and blank, respectively. The experimental data are represented as IC50 (μM) values.

3.5. Inhibitory Effect on AGE-Formation

The antiglycation effects of compounds isolated from TBE and several other related compounds were evaluated based on their AGE inhibitory activities as described previously [25], with slight modifications. Briefly, the sample solution was added to a reaction mixture containing 83.3 mM phosphate buffer (pH 7.2), 2.0 M glucose, 2.0 M fructose, 8.0 mg/mL human serum albumin (HSA), and distilled water (6:1:1:1:1, v/v). As a control, the vehicle was supplemented instead of the sample solution. For each blank, glucose or fructose was replaced with distilled water, and the total volume was set to 1000 μL. After incubation of the sample mixture at 60 °C for 40 h, the solutions were diluted 8-fold with distilled water, dispensed into a black microplate in 200 µL portions, and their fluorescence intensities were measured at excitation and emission wavelengths of 370 and 465 nm, respectively, using a Power Scan HT (DS Pharma Biomedical Co. Ltd., Osaka, Japan). The inhibitory rate was calculated as;
Inhibitory rate (%) = 100 − [(SSB)/(CCB)] × 100
where S is the relative intensity of the sample solution, C is the relative intensity of the control solution, and SB and CB are the intensities of the glucose or fructose-omitted blank solutions. The experimental data are represented as IC50 (μM) values.

3.6. AGE-Derived Crosslink-Cleaving Effect

The AGE crosslink-cleaving activity of the same samples was evaluated according to the previously described method by Kato et al. [28] with a slight modification. Briefly, the 1 mg/mL of tested samples prepared with H2O were mixed with 1.13 mM PPD solution in MeOH:50 mM phosphate buffer (pH 7.4) (1:1) and incubated at 37 °C for 4 h. After 4 h, the reaction was stopped with 200 μL of 2 M HCl, then the stopped reaction mixtures were centrifuged at 8200× g for 5 min. The amount of benzoic acid in the supernatant was measured by reversed-phase HPLC. The following conditions were applied: column, InertSustain C18 column (150 mm × 4.6 i.d mm., 5 μm); mobile phase, 50 mM phosphate buffer (pH 2.2):CH3CN (80:20) (solvent A) and 50 mM phosphate buffer (pH 2.2):CH3CN (50:50) (solvent B), the gradient program, 0–20 min (solvent B: 0–25%), 20–25 min (solvent B: 25–100%), 25–36 min (solvent B: 100–25%); column temperature, 40 °C; detection, UV at 230 nm; flow rate, 1.0 mL/min. These cleaving effects were calculated as equivalents to benzoic acid.

3.7. TBE Polyphenol Identification and Quantification by LC/UV/ESIMS Analysis

TBE (1.0 g) was sonicated with 70% aqueous acetone (3 × 10 mL) and the resulting suspension was centrifuged at 2200× g for 5 min. The supernatant was collected and dried. The obtained acetone extract from TBE (0.68 g) was dissolved in 50% aqueous MeOH to a concentration of 5 mg/mL and the solution of TBE extract was subjected to LC/UV/ESIMS. This analysis was performed on Waters 2695 separation module (Waters, Milford, MA, USA) coupled to Shimadzu SPD-6AV UV–vis spectrophotometric detector (Shimadzu, Kyoto, Japan) and Bruker MicrOTOF II instrument equipped with ESI source. The analyte was separated by InertSustain C18 column (150 mm × 4.6 mm i.d., 5 μm) at 40 °C with the mobile phase consisted of CH3CN:H2O:HCOOH (94.5:5:0.1) (solvent A) and CH3CN:H2O:HCOOH (54.9:45:0.1) (solvent B). The flow rate was 1.0 mL/min (splitting flow rate at 0.2 mL/min to MS unit), and a linear gradient was programmed as follows: 0–15 min (solvent B: 10–30%), 15–20 min (solvent B: 30–50%), 20–30 min (solvent B: 10%) and UV detection was monitored at 280 nm and 360 nm. MS parameters in negative ion mode were as follows: capillary voltage, 3.5 kV; nebulizer, 0.4 bar; dry gas, 4.0 L/min; dry temperature, 180 °C. The MS spectra were recorded in the range of m/z 50–3000. The polyphenol contents were expressed as mg/g (dry weight) by the absolute calibration curve method based on UV chromatogram.

4. Conclusions

In summary, we isolated the 13 known polyphenolic compounds including gallic acid, six galloyl glucoses (16), three ellagitannins (79), one neolignan (10), and ellagic acid derivatives 11 and 12 from TBE. The absolute configuration of 10 was confirmed by the aromatic quadrant and P/M helicity rules based on our CD analysis. Among the isolated polyphenols, decarboxylated rugosin A (8) and 1,2,3,4,6-penta-O-galloyl-β-d-glucose showed α-glucosidase inhibitory activities. Gallotannins and ellagitannins showed more significant inhibitory effect on AGE formation than that of gallic acid. Furthermore, gallic acid showed most potent AGE-derived crosslink cleaving activity among the tested polyphenols. A total of 30 TBE polyphenols were comprehensively identified by LC/UV/ESIMS analysis. The contents of tannins and the related polyphenols were also analyzed using LC/UV/ESIMS, indicating that gallic acid and gallotannins showing antiglycation effects were contained the major level in TBE. Further investigations are required to develop a deeper understanding of the TBE antidiabetic and antiglycation effects as well as the safety by in vivo experiments or clinical trial. Since the pericarp of this plant has experience in food as tea, it is considered that safety is guaranteed to some extent. The results of this study suggested the TBE polyphenols are a good source for antidiabetic and antiglycation effects, which could be applied as functional foods or nutritional supplements to improve human health benefits.

Supplementary Materials

The following are available online, Figure S1: 1D and 2D-NMR spectra of (7′S, 8′R)-dihydrodehydrodiconiferyl alcohol-9′-O-β-d-glucoside (10); Figure S2: 1H-NMR spectrum of compounds 13 and 14; Figure S3: 1D and 2D-NMR spectra of rubuphenol (11) and eschweilenol A (12); Figure S4: 1D and 2D-NMR spectra of compounds 1518.

Author Contributions

Conceptualization, H.I.; Methodology, Y.I., S.K. and N.G.; Validation, Y.I., S.S. and N.K.; Formal analysis, S.S. and N.K.; Investigation, Y.I., S.S., N.K., C.H. and S.K.; Data curation, Y.I., S.S., N.K., C.H., S.K. and N.G.; Visualization, Y.I.; Resources, T.U.; Funding acquisition, T.U. and H.I.; Writing—original draft preparation, Y.I.; Writing—review and editing, T.U. and H.I.; Supervision, H.I.; Project administration, H.I. All authors have read and agreed to the published version of the manuscript.

Funding

This study was funded by Okayama Prefectural University (Grant number: 2018D15 and 2019D11 to H.I.).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data is contained within the article and supplementary materials.

Acknowledgments

The authors grateful to the Faculty of Pharmaceutical Sciences of the Okayama University for the UV and CD spectral measurements, just as well as for the optical rotation. The authors thank to the SC-NMR Laboratory of the Okayama University for the NMR measurements.

Conflicts of Interest

The authors declare no conflict of interest.

Sample Availability

Not available.

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Figure 1. Chemical structures of the polyphenols isolated from TBE and the methylated compounds of 11 and 12.
Figure 1. Chemical structures of the polyphenols isolated from TBE and the methylated compounds of 11 and 12.
Molecules 26 05802 g001
Figure 2. The key HMBC and NOESY correlations for compound 10, compound 13 and 14 chemical structures, and projection for 1La (a) projection in the direction of the arrow, the wedge indicates the plane of the A-ring); and 1Lb (b) transition for 13.
Figure 2. The key HMBC and NOESY correlations for compound 10, compound 13 and 14 chemical structures, and projection for 1La (a) projection in the direction of the arrow, the wedge indicates the plane of the A-ring); and 1Lb (b) transition for 13.
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Figure 3. LC/UV/ESIMS analysis of TBE. Total ion chromatogram (A) and UV chromatogram at 280 nm (B) and 360 nm (C).
Figure 3. LC/UV/ESIMS analysis of TBE. Total ion chromatogram (A) and UV chromatogram at 280 nm (B) and 360 nm (C).
Molecules 26 05802 g003
Table 1. α-Glucosidase inhibitory activity of polyphenols isolated from TBE and related compounds.
Table 1. α-Glucosidase inhibitory activity of polyphenols isolated from TBE and related compounds.
CompoundInhibitory Effects on
α-Glucosidase Activity
IC50 (μM)
Gallic acid [8]>100
Ellagic acid >100
1,2,3-Tri-O-galloyl-β-d-glucose (2) [8]>100
1,2,6-Tri-O-galloyl-β-d-glucose (3) [8]>100
1,2,3,6-Tetra-O-galloyl-β-d-glucose (5) [8]>100
1,2,4,6-Tetra-O-galloyl-β-d-glucose (6)>100
1,2,3,4,6-Penta-O-galloyl-β-d-glucose 59.0 ± 0.4
Tellimagrandin II (7) [8]>100
Decarboxylated rugosin A (8)20.7 ± 0.1
Camptothin B (9)>100
Compound 10>100
Compound 13>100
Compound 14>100
Rubuphenol (11)>100
Eschweilenol A (12)>100
Cornusiin G [8]6.3 ± 0.1
Acarbose4.0 ± 0.1
Data are expressed as the means ± SE (n = 3).
Table 2. AGE-formation inhibitory effects in HSA/glucose or fructose and AGE-derived crosslink-cleaving activities of polyphenols isolated from TBE and related compounds.
Table 2. AGE-formation inhibitory effects in HSA/glucose or fructose and AGE-derived crosslink-cleaving activities of polyphenols isolated from TBE and related compounds.
CompoundInhibitory Effects on AGE FormationCrosslink-Cleaving Activities
IC50 (μM)
GlucoseFructoseRelative Ratio *
Gallic acid 14.7 ± 2.027.0 ± 2.6720.1 ± 54.1
Ellagic acid 1.8 ± 0.12.5 ± 0.111.4 ± 0.1
2,6-Di-O-galloyl-β-d-glucose (1)1.5 ± 0.13.3 ± 0.077.4 ± 4.0
1,2,3-Tri-O-galloyl-β-d-glucose (2)0.4 ± 0.00.4 ± 0.0190.5 ± 2.5
1,2,6-Tri-O-galloyl-β-d-glucose (3)0.3 ± 0.00.4 ± 0.0146.6 ± 12.6
2,3,6-Tri-O-galloyl-β-d-glucose (4)0.3 ± 0.01.0 ± 0.0N.T.
1,2,3,6-Tetra-O-galloyl-β-d-glucose (5)0.3 ± 0.00.3 ± 0.0209.0 ± 33.7
1,2,4,6-Tetra-O-galloyl-β-d-glucose (6)0.3 ± 0.00.3 ± 0.0159.6 ± 12.7
1,2,3,4,6-Penta-O-galloyl-β-d-glucose0.2 ± 0.00.2 ± 0.097.9 ± 2.1
Tellimagrandin II (7)0.2 ± 0.00.3 ± 0.0230.8 ± 12.2
Decarboxylated rugosin A (8)0.3 ± 0.00.3 ± 0.0233.0 ± 5.8
Camptothin B (9)0.1 ± 0.00.2 ± 0.0180.6 ± 4.2
Compound 10>500>100016.7 ± 1.4
Compound 13>500>100017.2 ± 1.1
Compound 14>500>10000.5 ± 1.1
Rubuphenol (11)2.4 ± 0.04.7 ± 0.3514.8 ± 11.2
Eschweilenol A (12)2.4 ± 0.02.9 ± 0.1484.5 ± 12.5
Aminoguanidine258.9 ± 6.8801.0 ± 17.7N.T.
N-Phenacylthiazolium bromide (PTB)N.T.N.T.100
Data are expressed as the means ± SE (n = 3), N.T. means not tested, * The concentrations of the tested samples are 100 μg/mL.
Table 3. TBE polyphenol content.
Table 3. TBE polyphenol content.
Peak No. CompoundtR (min)MS (m/z)Content (mg/g of Dry Weight)
12,3-Di-O-galloyl-β-d-glucose3.18, 3.94483 [M − H]15.5 ± 0.2
2Gallic acid3.23339 [2M − H]32.2 ± 0.1
32,6-Di-O-galloyl-β-d-glucose (1)4.90, 7.52483 [M − H]N.T.
43,6-Di-O-galloyl-β-d-glucose5.55, 6.58483 [M − H]3.9 ± 0.0
51,6-Di-O-galloyl-β-d-glucose7.53483 [M − H]16.8 ± 1.2
6Digalloyl glucose8.07483 [M − H]N.T.
71,2,3-Tri-O-galloyl-β-d-glucose (2)8.75635 [M − H]4.8 ± 0.0
83,4,6-Tri-O-galloyl-β-d-glucose8.75, 11.6635 [M − H]3.4 ± 0.1
9Brevifolincarboxylic acid9.22291 [M − H]1.7 ± 0.1
102,3,6-Tri-O-galloyl-β-d-glucose (4)9.86, 12.6635 [M − H]N.T.
112,4,6-Tri-O-galloyl-β-d-glucose10.2, 13.5635 [M − H]0.2 ± 0.1
12Trigalloyl glucose11.3635 [M − H]N.T.
131,2,6-Tri-O-galloyl-β-d-glucose (3)12.5635 [M − H]4.1 ± 0.5
141,3,6-Tri-O-galloyl-β-d-glucose13.5635 [M − H]1.8 ± 0.7
151,2-Di-O-galloyl-4,6-hexahydroxydiphenoyl-β-d-glucose13.8785 [M − H]0.9 ± 0.0
16Valoneic acid dilactone13.9469 [M − H]1.8 ± 0.2
17Trigalloyl glucose14.5635 [M − H]N.T.
18Urolithin M515.1275 [M − H]1.4 ± 0.4
191,4,6-Tri-O-galloyl-β-d-glucose15.1635 [M − H]0.6 ± 0.0
20Camptothin B (9)16.2860 [M − 2H]2−N.T.
21Tellimagrandin II (7)17.7937 [M − H]5.7 ± 0.0
221,2,3,6-Tetra-O-galloyl-β-d-glucose (5)18.8787 [M − H]13.3 ± 0.0
231,2,4,6-Tetra-O-galloyl-β-d-glucose (6)19.7787 [M − H]1.5 ± 0.0
24Ellagic acid19.9301 [M − H]6.9 ± 0.1
25Decarboxylated rugosin A (8)20.31061 [M − H]2.4 ± 0.0
261,2,3,4,6-Penta-O-galloyl-β-d-glucose21.5939 [M − H]0.7 ± 0.0
27Cornusiin G21.7861 [M − 2H]2−0.3 ± 0.0
28Rubuphenol (11)22.2425 [M − H]4.3 ± 0.1
29Compound 1022.1521 [M − H]1.3 ± 0.1
30Eschweilenol A (12)24.0425 [M − H]0.9 ± 0.2
Data are expressed as the means ± SE (n = 3), N.T. means not tested, tR: retention time.
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Iwaoka, Y.; Suzuki, S.; Kato, N.; Hayakawa, C.; Kawabe, S.; Ganeko, N.; Uemura, T.; Ito, H. Characterization and Identification of Bioactive Polyphenols in the Trapabispinosa Roxb. Pericarp Extract. Molecules 2021, 26, 5802. https://0-doi-org.brum.beds.ac.uk/10.3390/molecules26195802

AMA Style

Iwaoka Y, Suzuki S, Kato N, Hayakawa C, Kawabe S, Ganeko N, Uemura T, Ito H. Characterization and Identification of Bioactive Polyphenols in the Trapabispinosa Roxb. Pericarp Extract. Molecules. 2021; 26(19):5802. https://0-doi-org.brum.beds.ac.uk/10.3390/molecules26195802

Chicago/Turabian Style

Iwaoka, Yuji, Shoichi Suzuki, Nana Kato, Chisa Hayakawa, Satoko Kawabe, Natsuki Ganeko, Tomohiro Uemura, and Hideyuki Ito. 2021. "Characterization and Identification of Bioactive Polyphenols in the Trapabispinosa Roxb. Pericarp Extract" Molecules 26, no. 19: 5802. https://0-doi-org.brum.beds.ac.uk/10.3390/molecules26195802

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