Identification of Antioxidative Hydrolyzable Tannins in Water Chestnut

Despite the various biological activities exhibited by water chestnut (the fruit of the Trapa genus), the phenolic compounds present in its extract require comprehensive characterization. Accordingly, we analyzed a 80% methanol extract of commercially available water chestnut and identified a new hydrolyzable tannin dimer termed trapadin A. Additionally, 22 known compounds, including 10 hydrolyzable tannin monomers and 2 dimers, were also detected in the extract. Spectroscopic and chemical methods were used to elucidate the structure of trapadin A, revealing it to be a hydrolyzable tannin dimer formed from units of tellimagrandin II and 1,2,3,6-tetra-O-galloyl-β-d-glucose. Moreover, the 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity assay used to determine the half-maximal effective concentration values for the 23 compounds isolated from water chestnut indicated significant radical scavenging activity associated with hydrolyzable tannins. Notably, trapadin A, the new hydrolyzable tannin dimer, exhibited the highest activity value among the tested compounds.


Introduction
The water chestnut is the fruit of the aquatic plant Trapa genus, which includes several species such as T. japonica, T. incisa, and T. bispinosa.It thrives in swamps and lakes across Asia and features a hard walnut-like shell.In Japan, particularly in the Kyushu region, the edible starchy portion of the water chestnut is boiled and consumed along with the husks, yielding a tea that is considered to have stomachic and nourishing effects [1].Notably, water chestnut primarily consists of carbohydrates, with lipids, proteins, and carbohydrates constituting 0.5, 5.8, and 40.6 g per 100 g, respectively.Additionally, this fruit is rich in vitamin B1 and folic acid [2].Water chestnuts from T. japonica and T. bispinosa also reportedly contain hydrolyzable tannins [1,[3][4][5].Biological studies have indicated diverse health benefits associated with water chestnut, including antioxidant [1,6], antidiabetic [5,7,8], antiobesity [9,10], anti-glycation [5], anti-inflammatory [11,12], antiadipogenic [6], and hepatic protective effects [13].Furthermore, it has demonstrated inhibitory activity against αamylase [14] and α-glucosidase [3,5,14].Recently, water chestnut fruit extract has garnered attention for its potential in skin improvement [15] and as a treatment for alopecia [16], positioning it as a promising cosmeceutical and beauty food.
Considering the presence of diverse polyphenolic compounds in water chestnuts that exert various biological activities, a comprehensive investigation to identify its characteristic phenolic compounds is warranted.Moreover, antioxidant function is a typical biological activity of polyphenols [17].Antioxidants in foods contribute to the maintenance of health and reduce the risk of various age-related diseases [18].The contribution of their activity has been attributed to their chemical structure [17].Therefore, detailed structural elucidation of the polyphenol activity in foods is important.
cornusiin A (21) [30], rugosin D ( 22) [31], and (7′S,8′R)-d hol-9′-O-β-D-glucose ( 23) [5] (Figure 1).The known com direct HPLC comparison with authentic standards and data with those reported in the literature.To the best of o brevifolincarboxylic acid (5), urolithin A ( 7), urolithin M p-coumaroyl-β-D-glucose (17) were isolated for the first t study.Two carbonyls of the hexahydroxydiphenoyl (HHDP) part of the Val group were located at O-4 and O-6 of the glucose I core in 1, which indicated the existence of two regioisomers.Orientation of the Val group in 1 was found to be of the isorugosin type, similar to that in cornusiin G, based on a comparison of the 1 H NMR signal (δ 6.60) for Val H-3 and the 13 C NMR signal (δ 147.9) for Val C-4″ of 1 with those of the rugosin and isorugosin types [31,33,36,37].The sugar unit obtained following acid hydrolysis of 1 was identified as D-glucose based on HPLC analysis of the derivatives prepared by the reaction with L-cysteine methyl ester and o-tolyl isothiocyanate according to a previously reported method [35].Based on these data, the structure of trapadin A was established as 1.

DPPH Radical Scavenging Activity
Several methods for evaluating antioxidant activity based on their principles and simplicity have been reported.Among these, the DPPH assay showed relatively high measurement accuracy for single compounds [38].The DPPH method has extremely high reproducibility and is effective as a standard test method [39].Therefore, in this study, the antioxidant activity was evaluated using the DPPH method.
DPPH radical scavenging activity assays were performed for compounds 1-23 (Table 2).The degree of activity was indicated by the half-maximal effective concentration Two carbonyls of the hexahydroxydiphenoyl (HHDP) part of the Val group were located at O-4 and O-6 of the glucose I core in 1, which indicated the existence of two regioisomers.Orientation of the Val group in 1 was found to be of the isorugosin type, similar to that in cornusiin G, based on a comparison of the 1 H NMR signal (δ 6.60) for Val H-3 and the 13 C NMR signal (δ 147.9) for Val C-4 of 1 with those of the rugosin and isorugosin types [31,33,36,37].The sugar unit obtained following acid hydrolysis of 1 was identified as D-glucose based on HPLC analysis of the derivatives prepared by the reaction with L-cysteine methyl ester and o-tolyl isothiocyanate according to a previously reported method [35].Based on these data, the structure of trapadin A was established as 1.

DPPH Radical Scavenging Activity
Several methods for evaluating antioxidant activity based on their principles and simplicity have been reported.Among these, the DPPH assay showed relatively high measurement accuracy for single compounds [38].The DPPH method has extremely high reproducibility and is effective as a standard test method [39].Therefore, in this study, the antioxidant activity was evaluated using the DPPH method.
DPPH radical scavenging activity assays were performed for compounds 1-23 (Table 2).The degree of activity was indicated by the half-maximal effective concentration (EC 50 ) values.Compounds 4 and 7-9 were inactive (EC 50 : >100 µM), as evidenced by their antioxidant activity, and none of them had adjacent phenolic hydroxyl groups in its molecules.All the other compounds had adjacent phenolic hydroxyl groups in their molecules, and the greater the number, the stronger the activity.The EC 50 values showed notable activity for hydrolyzable tannins.The EC 50 values of hydrolyzable tannin monomers and dimers were between 5.06 and 12.5 µM and 3.48 and 3.91 µM, respectively, and the dimer with a higher number of phenolic hydroxyl groups in the molecule exhibited enhanced activity.Among these values, the EC 50 value of trapadin A (1), the new compound, was 3.14 µM, demonstrating the highest activity.Therefore, we concluded that hydrolyzable tannins, especially dimers, contribute enormously to the antioxidant activity of water chestnuts.Therefore, further investigation of hydrolyzable tannin oligomers in water chestnuts is necessary.

Materials
The water chestnuts (lot.nos.06047F209 and 06047D280, the fruit of Trapa bispinosa) used for the phytochemical investigation were purchased from Nakajima Shoyaku Ltd. (Kyoto, Japan).All other reagents used were of special or analytical grade.

Methylation of Compound 1 Followed by Methanolysis
A solution of compound 1 (4 mg) in EtOH (2 mL) was treated with (trimethylsilyl)diazomethane in hexane solution (2 mL) at room temperature overnight.After solvent removal, the residue was directly methanolyzed without further purification using 0.2% sodium methoxide in MeOH (2 mL) at room temperature for 12 h.After acidification with a few drops of 10% hydrochloric acid, the reaction mixture was evaporated, and the residue was purified using preparative TLC (n-hexane-acetone (1:1)) to yield methyl tri-Omethylgallate (24, 1.7

Determination of the Sugar Configuration of Compound 1
The sugar configuration was determined using a previously described method [35].Compound 1 (1.0 mg) was hydrolyzed by heating in 1 mol/L hydrochloric acid (0.2 mL) and neutralized using Amberlite IRA400 (Organo Corporation, Tokyo, Japan).After evaporation, the residue was dissolved in pyridine (0.2 mL) containing L-cysteine methyl ester hydrochloride (1.0 mg) and heated at 60 • C for 1 h.o-Tolyl isothiocyanate (1.0 mg) in pyridine (0.2 mL) was then added to each mixture, followed by direct analysis using RP-HPLC (Condition 3).The peak from compound 1 coincided with that of the derivative similarly prepared from an authentic D-glucose sample.

DPPH Radical Scavenging Activities of Compounds 1-23
The DPPH radical scavenging activity of each compound was determined using the DPPH Antioxidant Assay Kit (Dojin Laboratories, Kumamoto, Japan) following the manufacturer's instructions [39,40].The sample solution, assay buffer, and DPPH working solution were mixed in 96-well plates and incubated in the dark at 25 • C for 30 min.The absorbance was measured at 517 nm using an Infinite F200 microplate reader (Tecan Group Ltd., Mannedorf, Switzerland).The EC 50 was determined via regression line analysis, and Trolox was used as the positive control.All experiments were performed in triplicate.

Conclusions
In conclusion, we successfully isolated a new hydrolyzable tannin dimer, trapadin A (1), from water chestnut, along with 22 known compounds.Through the application of spectroscopic and chemical methods, the structure of trapadin A (1) was elucidated, revealing a hydrolyzable tannin dimer formed from units of tellimagrandin II and 1,2,3,6tetra-O-galloyl-β-D-glucose.Furthermore, the determined EC 50 values in the DPPH radical scavenging assay revealed substantial activity for hydrolyzable tannins, with trapadin A (1), the new hydrolyzable tannin dimer, exhibiting the highest activity value.The findings of

Table 1 .
1H and13C NMR data for glucose residues of compound 1 measured in MeOH-d 4 .