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Biosci. Biotechnol. Biochem., 77 (6), 121023-1–4, 2013 Note Peanut-Skin Polyphenols, A1 and Epicatechin-(4 !6)-epicatechin-(2 !O!7, 4 !8)-, Exert Cholesterol Micelle-Degrading Activity in Vitro

Tomoko TAMURA,1 Naoko INOUE,1 Megumi OZAWA,1 Akiko SHIMIZU-IBUKA,2 Soichi ARAI,1 y Naoki ABE,1 Hiroyuki KOSHINO,3 and Kiyoshi MURA1;

1Department of Nutritional Science, Faculty of Applied Bioscience, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan 2Department of Material and Biological Chemistry, Faculty of Science, Yamagata University, 1-4-12 Kojirakawa-machi, Yamagata 990-8560, Japan 3RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan

Received January 4, 2013; Accepted February 27, 2013; Online Publication, June 7, 2013 [doi:10.1271/bbb.121023]

We identified epicatechin-(4 !6)-epicatechin-(2 ! Here we report that a major peanut-skin polyphenol O!7, 4 !8)-catechin (EEC) in the skin of the peanut having a cholesterol micelle-degrading effect is epica- (Arachis hypogaea L.). EEC (a trimer) showed more techin-(4!6)-epicatechin-(2!O!7, 4!8) cate- potent cholesterol micelle-degrading activity than pro- chin (EEC), in addition to the previously identified cyanidinAdvance A1 (a dimer) did in vitro. The hypercholester- View 3) . olemia suppressing effect of a peanut skin polyphenol on Roasted peanut skin (40 g) was boiled in purified rats fed high-cholesterol diet in our preceding experi- water (1,000 mL) for 30 min, and the supernatant was ments might thus have been due primarily to a micelle taken by decantation and filtration. This treatment was degrading effect in the intestine. done in triplicate, and then the three supernatants were pooled prior to vacuum concentration to 1,000 mL. The Key words: catechin; cholesterol; micellar solubility; total polyphenol in this concentrate was quantified by peanut skin; procyanidin Folin-Ciocalteu assay.6) As catechin equivalent, 4.65 g of polyphenol was obtained from 40 g of peanut skin. A great many polyphenols of food origin are known Then a 50-mL aliquot of the 1,000-mL polyphenol to have physiologic effects, including anti-oxidative, solution was subjectedProofs to filtration with an ultrafilter anti-inflammation, and antimicrobial one.1) These are (MWCO 3000; Advantec, Tokyo). Seventy-five point closely related to stereochemistry. Tatsuno et al.2) five mg of polyphenol was obtained in a lower-molecu- indicated that within the group of , the lar-weight (Mr < 3000) fraction, and its filtrate dimers and trimers showed similar suppressive activ- (Mr < 3000) was chromatographed on a TSK-gel ities to TNF- and IL-6, whereas the tetramers showed Toyopearl HW-40 F column (26 800 mm) (Tosoh no such activity. We have found that polyphenols Bioscience, Tokyo) using methanol as the mobile phase. occurring in peanut skin are anti-allergenic by sup- Procyanidin A1 was obtained from the fourth of gel pression of degranulation in immunocytics.3) In this filtration peak as described previously.3) The remaining case, showed inhibitory activity like polyphenols in the TSK-gel Toyopearl HW-40 F column procyanidin A1, while (þ)-catechin was not involved were eluted with 80% acetone. The resulting eluate in inhibiting -hexosaminidase release.3) Almost all contained 60.8% polyphenol. Then, the eluate was studies of the mechanism of the cholesterol micelle- purified by chromatography on a YMC gel ODS-AQ degrading effect of the polyphenols have been con- 12S50 column (YMC, Kyoto, Japan) pre-equilibrated ducted with extract solutions containing polyphenol or with solvent A (0.1% formic acid v/v). Elution was with monomeric polyphenols: catechin, epicatechin, and conducted by linear gradient (20%!40% methanol as EGCg. solvent B) at a flow rate of 5 mL/min for 270 min, and We have reported that a peanut-skin polyphenol effect was separated into peaks. A major component of the occurred in the skin at 5% on a dry-matter basis and had main peak of the chromatography was isolated. a serum cholesterol-reducing effect when administrated The primary nuclear magnetic resonance (NMR) to rats fed a high-cholesterol diet, and that the effect was spectra of a major component were recorded by JNM- predominant at lower-molecular-weights.4,5) In addition, ECX 400 (JEOL, Tokyo) at 400 MHz (1H) and at 13 a major portion of dietary cholesterol was found to reach 100 MHz ( C), with methanol-d4 as solvent and the feces after degrading the intestinal cholesterol tetramethylsilane as internal standard at ambient tem- micelle without entry into the blood.5) perature. At that temperature, the 1H-NMR (400 MHz)

y To whom correspondence should be addressed. Tel: +81-3-5477-2459; Fax: +81-3-5477-2392; E-mail: [email protected] Abbreviations: DQF-COSY, double-quantum filtered correlation spectroscopy; EEC, epicatechin-(4!6)-epicatechin-(2!O!7, 4!8)- catechin; EGCg, epigallocatechingallate; HMBC, heteronuclear multiple-bond correlation; HPLC, high performance liquid chromatography; HSQC, heteronuclear single-quantum coherence; NMR, nuclear magnetic resonance; NOESY, nuclear overhauser effect correlation spectroscopy; 2D, two- dimensional 121023-2 T. TAMURA et al. spectrum of major component gave a number of broad assignments were achieved by careful spectral analysis. signals, and hence full assignments for structure iden- There are summarized in Table 1. The connectivity of tification were not completed. It has been reported that the inter-unit was confirmed by HMBC correlations trimeric procyanidin with an interflavonoid linkage from H-4 to C-50, C-60, and C70, and also from H-40 to between 4 and 6 or between 4 and 8 has atropisomers C-700, C-800, and C-800a. In the NOESY experiment with owing to slow rotation of the linkage bond at ambient two mixing times (100 ms and 500 ms), several chemical temperature.7) The behavior of the 1H-NMR spectrum in exchange cross peaks were observed for the two our experiment suggested that is component possessed atropisomers. Based on the NMR spectral data, the the 4- > 6- or the 4- > 8-interflavonoid linkage. structure of the major component based on chromatog- Further NMR spectral analysis of 1H-(600 MHz), 13C- raphy on the YMC gel ODS-AQ 12S50 column was (150 MHz), and several sets of two-dimensional (2D) determined to be in agreement with the data for an NMR data, including DQF-COSY, HSQC, HMBC, and abundance ratio of a 1:1 mixture of the two atropisomers NOESY, were recorded under low temperature condi- (conformers-1 and -2) of a epicatechin-(4!6)-epica- tions (40 C). Under these conditions, two sets of techin-(2!O!7, 4!8) catechin, ECC (Fig. 1). 1H NMR signals were observed for the major compo- The conformations of the atropisomers were eluci- nent from peak A, at ratio of abundance of 1:1 of two dated by a NOESY experiment. NOEZY correlations atropisomers (conformers-1 and -2). Complete NMR between H-2 and H-1200 and H-1600 and between H-8 and

1 13 Table 1. H and C NMR Spectral Data for EEC in CD3OD at 40 C

13 1 C-NMR (150 MHz) H-NMR (600 MHz) in CD3OD at 40 C No. conformer-1 conformer-2 conformer-1 conformer-2 2 77.75 76.91 5.48 s 5.03 s Advance3 73.46 View72.85 3.79 br.s 3.85 br.s 4 36.65 37.41 4.62 br.s 4.64 br.s 4a 103.25 102.28 — — 5 157.96 158.19 — — 6 95.42 95.71 5.83 d (2.3) 5.89 d (2.3) 7 156.79 157.76 — — 8 95.71 95.42 6.16 d (2.3) 5.93 d (2.3) 8a 157.76 157.84 — — 9 132.90 132.59 — — 10 115.08 114.97 6.91 d (1.8) 6.83 d (1.3) 11 145.90 145.75 — — 12 145.54a 145.42 — — 13 115.67 115.49 6.70 d (8.2)Proofs 6.66 d (8.3) 14 119.32 119.01 6.59 dd (8.2, 1.8) 6.61 dd (8.3, 1.3) 20 99.78 100.05 — — 30 67.54 67.65 4.03 s 4.07 d (3.5) 40 29.03 29.48 4.03 s 4.16 d (3.5) 40a 104.06 103.67 — — 50 154.86 154.05 — — 60 111.33 110.58 — — 70 156.31 157.19 — — 80 96.27 97.37 6.19 s 6.01 s 80a 151.92 151.92 — — 110 131.97b 131.99b —— 120 115.40c 115.42c 7.07 d (1.9) 7.09 d (1.9) 130 145.54a 145.58a —— 140 146.67d 146.72d —— 150 115.30e 115.35e 6.78 d (7.8) 6.79 d (8.3) 160 119.69f 119.72f 6.97 dd (7.8, 1.9) 6.99 dd (8.3, 1.9) 200 85.77 85.14 4.35 d (9.2) 4.73 d (8.3) 300 68.91 68.14 3.87 ddd (10.1, 9.2, 5.9) 4.19 ddd (9.2, 8.3, 5.5) 400 31.18 29.24 3.06 dd (16.1, 5.9) 2.99 dd (16.1, 5.5) 2.46 dd (16.1, 10.1) 2.55 dd (16.1, 9.2) 400a 103.67 103.00 — — 500 156.22 156.31 — — 600 96.64 96.39 6.098 s 6.096 s 700 152.20 152.20 — — 800 106.73 106.26 — — 800a 151.76 151.22 — — 1100 129.50 129.91 — — 1200 117.88 115.75 6.72 d (1.4) 6.96 d (1.4) 1300 145.37 146.17 — — 1400 146.45 148.86 — — 1500 118.20 116.54 7.03 d (7.8) 6.85 d (8.2) 1600 119.45 121.11 6.77 dd (7.8, 1.4) 6.89 dd (8.2, 1.4)

a–f, These assignments are exchangeable. Cholesterol Micelle-Degradation by Peanut-Skin Polyphenols 121023-3

13 12 A 14 1 8 9 11 7 8a 2 10 3 Catechin : R =H, R =OH 6 4a 1 2 5 4 R1 R2 Epicatechin : R1=OH, R2=H

B 4a 5’ 7’

15’ 4a 16’ 14’ 8’ 11’ 13’ 13 8’a 7’ 2’ 12’ 12 14 3’ 9 3 6’ 4’a 11 5’ 4’ 10 2 4 8’’ 7’’ 4a 6’’ 8a 5 8’’a 5’’ 8 4’’a 6 12’’ 7 13’’ 2’’ 4’’ 11’’ 3’’ 14’’ 16’’ 15’’

Conformer-1 Conformer-2

Fig. 1. Structures of Epicatechin-(4!6)-epicatechin-(2!O!7, 4!8)-catechin (EEC) and Correlations. Structures of building blocks of the construction unit of EEC (A), and its two conformers, conformer-1 and -2 (B).

00 H-15 on conformer-1 were observed, which suggests ) M A ) µ the isomers had the conformations shown in Fig. 1. EEC 500 6.0 M is known to occur in Vacciniumvitis-idaea L. and to Advance View400 5.0 have the same complicated NMR spectrum predicting 8) 4.0 the presence of the two conformers. This is the first 300 report that EEC was detected in peanut skin. The amount 3.0 200 of EEC in peanut skin was 2.96 mg/g on a dry-matter Cholesterol 2.0 basis as measured by high-performance liquid chroma- Bile acie 100 tography (HPLC). EEC has potent anti-superoxidation 1.0 9) activity, but no information is available regarding other 0 0.0 Bile acid in micelle solution (m

physiologic functionalities. Cholesterol remaining in micelle ( 0 10203040506070 Micellar solubility was assayed as described previ- Polyphenol added (µg/mL) ously.5) In brief, a cholesterol micellar solution in a mixture of 6.6 mM sodium taurocholate, 0.6 mM phos- B

) Proofs 500 M phatidylcholine, 0.5 mM cholesterol, 132 mM NaCl, and µ 15 mM sodium phosphate at pH 7.4 was prepared by the 400 method of Ikeda et al.10) and kept at 37 C for 24 h. A 100-mL aliquot of water as negative control and a similar 300 volume of a water-soluble polyphenol fraction were (+)-Catechin adding to 5 mL of a micellar solution. One h after adding 200 Procyanidin A1 each of the samples, the resulting solution was centri- EEC fuged at 1,000 rpm for 10 min, and then passed through a 100 220-nm Milex-GV filter (Japan Millipore, Tokyo) to 0 remove turbidity. The cholesterol concentration in the Colesterol remaining in micelle ( 0 250 500 750 1,000 1,250 1,500 supernatant was assayed by GC-1700 gas chromatograph Polyphenol added (µg/mL) (Shimadzu, Kyoto, Japan). The bile acid in the micelle was quantified by an enzyme-colorimetric method by Fig. 2. Polyphenol Concentration-Dependent Decreases in the Cho- Total bile acid-Testwako (Wako, Osaka, Japan). lesterol Remaining in the Micelles. The addition of water-soluble peanut-skin polyphe- Micellar cholesterol and bile acid levels in the presence of added polyphenol (A), and effects of the three polyphenols on the nols degraded the micelle in proportion to the amount of solubility of cholesterol in the micelles (B). Each point with an polyphenol added, but the addition of polyphenol did not error bar represents mean standard error for three samples. EEC, affect the bile acid in the micelle (Fig. 2A), suggesting epicatechin-(4!6)-epicatechin-(2!O!7, 4!8)-catechin. that the water-soluble polyphenol-aided release of cholesterol was not due to a taurocholic acid-related 1,000 mM procyanidin A1 were added, the remaining decrease in surfectancy. Yasuda et al.11) found that cholesterol was reduced to 69% (355:5 9:1 mg/mL) dimeric, trimeric, and tetrameric polyphenols can de- and to 38% (194:3 19:3 mg/mL) respectively, whereas grade the cholesterol micelle at up to 1,000 mg/mL. As the addition of 250 mM and 500 mM EEC resulted in shown in Fig. 2B, the catechin (a monomer) did not reductions of the remaining cholesterol to 11% degrade the cholesterol micelle, while a procyanidin A1 (54:6 0:3 mg/mL) and 5% (24:2 2:7 mg/mL) respec- (a dimer) and EEC (a trimer) induced degradation in a tively. Thus almost all the micelle-forming cholesterol dose-dependent manner. When 500 mM procyanidin A1 was released to degrade the micelle at concentrations was added, an 87%-portion of cholesterol (445:2 of higher than 1,250 mM procyanidin A1 and at a much 4:7 mg/mL) remained in the micelle. When 750 mM and lower concentration of EEC. 121023-4 T. TAMURA et al. As for the structure-function relationship of polyphe- stereochemically different oligomer procyanidins show nol, investigation has been conducted with tea epicate- different ways of interaction with cholesterol micelles. chin and EGCg, which have some bioactive effect by insertion of their hydrophobic galloyl moieties into the Acknowledgment lipid bilayers.12) It has been stated that energetic stabilization by in a phospholipid is a This work was supported by a Grant-in-Aid for significant factor in interaction with the lipid bilayer.13) Young Scientists (B) no. 24700848 from the Japan , a single-linked procyanidin, displayed Society for the Promotion of Science (JSPS). stronger superoxide scavenging activity than procyani- din A1, a double-linked procyanidin.9) On the other References hand, the epicatechin-(4!8)-epicatechin-(2!O! 7, 4!8) catechin contains a 4!8 linkage has a 1) Scalbert A, Manach C, Morand C, Remesy C, and Jimenez L, Crit. Rev. Food Sci. Nutr. 45 4-times stronger antimicrobial activity than EEC, with a , , 287–306 (2005). 14) 2) Tatsuno T, Jinno M, Arima Y, Kawabata T, Hasegawa T, 4!6 linkage. As to the cholesterol micelle-degrad- Yahagi N, Takano F, and Ohta T, Biol. Pharm. Bull., 35, 909– ing activity of polyphenols, investigation of cacao 916 (2012). oligomeric polyphenols for micelle-degrading effects 3) Tomochika K, Shimizu-Ibuka A, Tamura T, Mura K, Abe N, has given the result that , epicatechin- Onose J, and Arai S, Biosci. Biotechnol. Biochem., 75, 1164– (4!8)-epicatechin is stronger than , 1168 (2011). epicatechin-(4!6)-epicatechin in terms of micelle- 4) Shimizu-Ibuka A, Udagawa H, Kobayashi-Hattori K, Mura K, Tokue C, Takita T, and Arai S, Biosci. Biotechnol. Biochem., degrading activity, a result suggesting a between-dimer 73, 205–208 (2009). 11) difference in stereo chemistry. In view of these 5) Tamura T, Inoue N, Shimizu-Ibuka A, Tadaishi M, Takita T, published data, we think that the steric difference Arai S, and Mura K, Biosci. Biotechnol. Biochem., 76, 834–837 between procyanidin A1 (a dimer) and EEC (a trimer) (2012). as peanut-skin polyphenols is involved in the different 6) Singleton VL and Rossi JAJr, Am. J. Enol. Viticult., 16, 144– Advance View158 (1965). cholesterol micelle-degrading effects, although it re- mains to be determined how different this effect is as 7) Shoji T, Mutsuga M, Nakamura T, Kanda T, Akiyama H, and Goda Y, J. Agric. Food Chem., 51, 3806–3813 (2003). between the two conformers we found (Fig. 1). 8) Satoshi M, Nonaka G, and Nishioka I, Pharm. Bull., 36, 33–38 Cholesterol micelle degradation by polyphenol re- (1988). flects the in vivo phenomenon.5) The most effective 9) Ho KY, Huang JS, Tsai CC, Lin TC, Hsu YF, and Lin CC, factor we found, EEC, occurred in dried peanut skin at J. Pharm. Pharmacol., 51, 1075–1078 (1999). 0.206%, and the other polyphenols, procyanidin A1, and 10) Ikeda I, Imasato Y, Sasaki E, Nakayama M, Nagao H, Takeo T, (þ)-catechin, were present at 0.755% and 0.032% Yayabe E, and Sugano M, Biochim. Biophys. Acta, 1127, 141– respectively. Administration of a water-soluble peanut- 146 (1992). 11) Yasuda A, Natsume M, Sasaki K, Baba S, Nakamura Y, skin polyphenol fraction to rats fed a high-cholesterol Kanegae M, and Nagaoka S, BioFactors, 33, 211–223 (2008). diet improved hypercholesterolemia as a result of an 12) Kajiya K, KumazawaProofs S, and Nakayama T, Biosci. Biotechnol. increase in fecal cholesterol amount. Biochem., 65, 2638–2643 (2001). The significance of hyperlipidemia suppression due to 13) Uekusa Y, Kamihira-Ishijima M, Sugimoto O, Ishii T, degraded cholesterol micelle in the lumen has been Kumazawa S, Nakamura K, Tanji K, Naito A, and Nakayama emphasized in recent years.15) Future investigation of T, Biochi. Biophy. Acta, 1808, 1654–1660 (2011). 14) Ho KY, Tsai CC, Huang JS, Chen CP, Lin TC, and Lin CC, oligomeric procyanidins occurring in peanut skin as a J. Pharm. Pharmacol., 53, 187–191 (2001). food waste is important in the industrial production of a 15) Chandra K, Paul FR, Louise EB, Mahinda YA, and Glen SP, safe medicine with a hypercholesterolemia suppressing J. Agric. Food Chem., 53, 4623–4627 (2005). effect as well as in the scientific elucidation of the ways