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

In this study, we present the isolation and characterization of the structure of six gallotannins (1–6), three ellagitannins (7–9), 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.


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 1,2,6tri-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  The structure of compound 13, which was obtained by the hydrolysis of 10, was confirmed based on 1 H-NMR ( Figure S2), 13 C-NMR and atmospheric pressure ionization (APCI) MS analyses. The circular dichroism (CD) spectrum of 13 showed negative cotton at the 1 L a (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 1 L b (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 (Figures 2 and S2). The product has been reported to be an intermediate of adenosine A 1 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 1 H-and 13 C-NMR, 1 H-1 H 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 15-18) based on spectral analyses ( Figure S4). The 1 H-1 H COSY and NOESY spectra of compounds 15 and 16 showed the correlations between H-5 and 4-OCH 3 protons, while those correlations were not observed in 17 and 18. The observations of 1 H-1 H COSY and NOE correlations between H-5 and

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

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 IC 50 (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 IC 50 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. Table 1. α-Glucosidase inhibitory activity of polyphenols isolated from TBE and related compounds.
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.   (7) 0.
Data are expressed as the means ± SE (n = 3), N.T. means not tested, * The concentrations of the tested samples are 100 µg/mL.

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 (2-6), 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.

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; (1) where A sample , A control , and A blank is the absorbance of the tested sample, control, and blank, respectively. The experimental data are represented as IC 50 (µM) values.

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; 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 IC 50 (µM) values.

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 H 2 O 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. 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.

Conclusions
In summary, we isolated the 13 known polyphenolic compounds including gallic acid, six galloyl glucoses (1-6), three ellagitannins (7-9), 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-Dglucose 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.