Phenolics Profile of Mume, Japanese Apricot (Prunus Mume Sieb. Et Zucc

Biosci. Biotechnol. Biochem., 77 (8), 1623–1627, 2013

Phenolics Profile of Mume, Japanese Apricot (Prunus mume Sieb. et Zucc.) Fruit

y Takahiko MITANI,1; Asako HORINISHI,1 Kunihiro KISHIDA,1 Tomoaki KAWABATA,1 Fumiko YANO,1 Hisa MIMURA,1 Nobuya INABA,2 Hisako YAMANISHI,3 Takaaki OE,4 Keiichi NEGORO,4 Hajime MORI,3 Yasuhito MIYAKE,3 Asao HOSODA,3 Yoshie TANAKA,3 Megumi MORI,3 and Yoshihiko OZAKI1

1Faculty of Biology-oriented Science and Technology, Kinki University, Kinokawa, Wakayama 649-6493, Japan 2Wakayama Agricultural Processing Research Corporation, Wakayama, Wakayama 649-6212, Japan 3Industrial Technology Center of Wakayama Prefecture, Ogura, Wakayama, Wakayama 649-6261, Japan 4Fruit Tree Experimental Station, Wakayama Research Center of Agriculture and Fisheries, Minabe, Hidaka, Wakayama 645-0021, Japan

Received February 1, 2013; Accepted April 23, 2013; Online Publication, August 7, 2013 [doi:10.1271/bbb.130077]

The fruit of mume, Japanese apricot (Prunus mume been used as a folk remedy in Japan with anticipated Sieb. et Zucc.), was evaluated for its phenolics content, medicinal effects on symptoms including fatigue, diar- high performance liquid chromatography (HPLC) pro- rhea, and fever. In other Asian countries, such other file and antioxidative activities. The phenolics content of types of processed mume fruit as fructus mume, which is mume fruit was relatively high, the flesh of fully dried and smoked, have been traditionally used as a matured fruit containing up to 1% of phenolics on a medical alternative that is effective for its antitussive, dry weight basis. Reflecting such a high content of expectoration, antiemetic, antidiarrheal, anthelmintic phenolics, the ORAC (oxygen radical absorbance and antipyretic actions.3) capacity) value for mume fruit flesh showed high values, Epidemiological studies suggest that the intake of ranging from 150 to 320 mol/g Trolox equivalent, fruits and vegetables that contain plant phenolics is depending upon the stage of maturation. 5-O-Caffeoyl- associated with a reduction in the risk of such chronic qunic acid (chlorogenic acid), 3-O-caffeoylquinic acid ailments as cardiovascular disease, common cancers, and tetra-O-acylated sucrose-related compounds were and osteoporosis.4) Mume fruit could also have the isolated from the flesh of mume fruit, although many potential to reduce the risks of these diseases. It has been unknown peaks were also apparent in the HPLC reported that prunate, one of the coumarin derivatives chromatogram. An alkali hydrolysate comprised four isolated from mume fruit, specifically inhibited the main phenolic acids, caffeic acid, cis/trans-p-coumaric growth of several cultured cancer cells.5) Aryltetralin acid and ferulic acid. No flavonoids were observed in the lignan, a lyoniresinol in umeshu (mume liqueur) and analysis. These results suggest that the majority of umezu (a by-product of the pickling process for mume phenolics in mume fruit were hydroxycinnamic acid fruit containing a high concentration of salt and citric derivatives. acid) has been reported to possess antioxidative activ- ity6,7) or to show inhibiting activity against the mobility Key words: Prunus mume; phenolics; oxygen radical of Helicobacter pylori.8) Ina et al. have found that absorbance capacity (ORAC); hydroxycin- benzyl -D-glucoside and chlorogenic acids contained in namic acid; chlorogenic acid mume fruit9) had several physiological activities in rats, including an inhibitory effect on acetic acid-induced Mume, Japanese apricot (Prunus mume Sieb. et writhing behavior. The inhibition of the production of Zucc.), belongs to the Rosaceae family and is one of bradykinin and prostaglandin is thought to be respon- the most popular fruit trees in Japan. Unlike many other sible for this anti-nociceptive action.10) It has also been fruits, most of the harvested fruits are first processed and observed that orally administered benzyl -D-glucoside then consumed, due to the presence of cyanoglycoside, decreased aldosterone and increased corticosterone in prunasin and amygdalin1,2) and the extreme sourness of the plasma.11) New flavonol oligoglycosides, prunoses I, the fruit. The majority of the accumulated acids in the II and III,12,13) have been isolated from a methanolic fruit are citric acid, and the acid content of the mature extract of the mume flower. Prunoses I and II inhibited fruit may be up to 6–7% on a fresh weight basis. Due to platelet aggregation induced by thrombin.12) However, these properties, the pickled mume fruit (umeboshi) is little research has been conducted on a systemic analysis popular in Japan and used often as a preservative to of the mume fruit constituents. extend the life of other food items. A few of these We focused this present study on the phenolic processed products of mume fruit, such as umeboshi and compounds contained in mume fruit. We first analyzed bainiku ekisu (concentrated juice of mume fruit) have the phenolic profile changes in the mume nanko cultivar

y To whom correspondence should be addressed. Present address: Wakayama Industry Promotion Foundation, 2-1 Honmachi, Wakayama, Wakayama 640-8033, Japan; Tel: +81-73-432-5155; Fax: +81-73-432-3314; E-mail: [email protected] Abbreviations: ORAC, oxygen radical absorbance capacity; DMSO, dimethyl sulfoxide; HPLC, high performance liquid chromatography; LC/MS, liquid chromatography/mass spectrometry; NMR, nuclear magnetic resonance 1624 T. MITANI et al. during fruit maturation, this being the most popular Separation was performed at a flow rate of 1.5 mL/min and 4 C cultivar grown in Wakayama Japan where over 50% of with C:B gradient steps of 75:15 to 45:35 in 220 min, and then 0:100 in national production is originated. We also evaluated the 40 min. Aliquots of the eluate corresponding to each peak were collected, evaporated to dryness and dissolved in 1 mL of methanol. antioxidative properties of the mume fruit phenolics by the oxygen radical absorbance capacity (ORAC) method Determination of total phenolics. The total phenolic content was and elucidated the chemical structures of some of the determined by the Folin-Ciocalteu method,14) using gallic acid as a phenolics. standard. The value is expressed as gallic acid (mg) per gram dry weight. Materials and Methods ORAC assay. The antioxidative activity was determined by an Fruit samples. Fruit samples of P. mume cv. nanko were randomly oxygen radical absorbance capacity (ORAC) assay as described by Ou collected from a single fixed tree, which was grown in the experimental et al.15) Each sample was diluted to the appropriate concentration, and orchard of the Laboratory of the Japanese Apricot at the Fruit Tree the fluorescence loss was monitored by a 96-well fluorescent micro- Experiment Station of Wakayama prefectural government, at three plate reader (TEKAN Spectrafluor Plus, Austria). Each ORAC value is different growth stages from 2004–2009. The harvesting of the samples expressed as micromole Trolox equivalent per gram dry weight. was at the stone-hardening stage (i), premature stage with a deep green peel color approximately 105–110 d after flowering (ii), and mature Alkaline hydrolysis. An ester or ether linkage was hydrolyzed by stage with a yellow peel color approximately 130 d after flowering (iii). following the procedure described by Krygier.16) Two hundred and The samples were washed in tap water, dried, and then manually fifty mg of freeze-dried flesh powder was suspended in 10 mL of 1 N separated into the flesh and stone parts. The stone part was further NaOH that had been deoxidized in advance by bubbling nitrogen gas. divided into the endocarp, internal seed coat and kernel. The freeze- After 4 h of incubation at 37 C, the suspension was acidified by adding dried and powdered flesh was sieved through a wire screen (425 mm phosphoric acid to pH 4.5 and then extracted with ethyl acetate. The aperture) and was stored in light-shielded bottles at 20 C. Any flesh ethyl acetate layer was evaporated to dryness, and the residue was that remained on the surface of the endocarp was removed by brushing, dissolved in a minimal volume of methanol for analysis under the the endocarp was dried in a vacuum desiccator for two days, and the above-mentioned HPLC conditions. milled powder was stored in a light-shielded bottle at 20 C until needed for further extraction. The kernel was cut and dried in the Determination of NMR spectra. The isolated sample was dissolved 1 13 vacuum desiccator to constant weight. The dried kernel was further in methanol-d6 or DMSO-d6. The H-NMR and C-NMR spectra shredded prior to subsequent extraction. were recorded by a Bruker-Avance 400 MHz spectrometer (100 MHz for 13C). Chemical shifts are reported as (ppm) and coupling Preparation of the phenolic fractions. Five hundred mg of freeze- constants (J) are given in Hertz. dried powder of the flesh and of vacuum-dried powder of the kernel and endocarp were sonicated in 25 mL of 90% ethanol for 2 min by a Results and Discussion Branson 450 sonifier (20 kHz, 200 W). Each sonicated suspension was then centrifuged at 2000 g for 10 min at 4 C, and the supernatant was collected. Whenever centrifugal separation was insufficient, the super- Phenolic content of mume fruit natant was further filtered through Millex-LG (0.2 mm pore size, Changes in the phenolic concentrations of the mume Millipore Corporation). A 1 g amount of the shredded sample was fruit are summarized in Table 1. The flesh demonstrated defatted with 20 mL of n-hexane for albumen, prior to being extracted the highest concentrations throughout the three stages of with 90% ethanol. maturation. At the stone-hardening stage, the flesh HPLC and LC/MS analyses. An Agilent 1100 system was used for contained over 1% of phenolics on a dry weight basis, the HPLC analyses. A portion of each phenolic fraction collected from after which the phenolics in the flesh decreased with the flesh, endocarp and kernel of the fruit was evaporated to dryness, increasing maturation. The weight of the fruit increased and the residue was dissolved in a minimal volume of dimethyl as it matured. The mean wet weight of one fruit was sulfoxide (DMSO) before being injected into a Hydrosphere C18 19.5 g at the stone-hardening stage, 33.9 g at the column (’4:6mm 250 mm; YMC, Kyoto, Japan). The column was premature stage and 46.6 g at the fully mature stage. eluted with mixed solvent A (0.1% TFA):B (methanol) at a flow rate of 1.0 mL/min. The eluent was A:B ¼ 80:20 for first 10 min, and then a The mean wet flesh weight of one fruit was 16.0 g, linear gradient ending with 25:75 after 80 min. All analyses were 22.9 g and 42.3 g at each stage. It has been reported that carried out at 30 C and monitored by UV absorption at 280 nm. The organic acids and sorbitol were accumulated, but that the LC/MS analyses of compounds 1 and 2 replaced solvent A with 0.5% phenolic content on a wet weight basis was almost formic acid (solvent C). The column was eluted with C and B constant during the maturation process of mume fruit.17) according to the same elution program as that for A and B. An aliquot of the eluate was introduced into the ESI-MS interface of a Marinar Biospectrometry Workstation (Applied Biosystems) set in the positive Table 1. Changes in Phenolics Content and Antioxidative Activity of mode with a spray tip potential of 3500 V, nozzle potential of 80 V, and Mume Fruit through Different Stages of Growth nozzle temperature of 140 C. LC/MS analyses of compounds A, C, D and 3 were performed by Stone-hardening Premature Full mature ESI-MS with an Exactive mass spectrometer (Thermo Scientific) stagea stageb stagec interfaced with an Accela 600 HPLC system (Thermo Scientific). The same method as that already described was used for LC separation, Phenolics content (mg/g gallaic acid equivalent, dry weight basis) except that solvent A was replaced by 0.2% formic acid and solvent B Flesh 11:7 0:29:5 0:18:1 0:2 was replaced by 0.2% formic acid in methanol. The MS detector was Stone 2:6 0:26:4 0:27:7 0:2 set to the negative mode for the analyses of compounds A, C and D, Kernel 5:9 0:12:2 0:11:9 0:0 and the positive mode was used for detecting compound 3. The detector conditions for the both modes were a spray voltage of 4000 V ORAC value (micromole/g, Trolox equivalent, dry weight basis) and a capillary temperature of 300 C. Flesh 320 14 239 14 158 11 Stone 55 696 9 109 10 Preparative HPLC. The AKTA Explorer 10S System (GE Kernel 49 218 117 2 Healthcare Bioscience) was used for preparative HPLC. A portion of the phenolic fraction was injected into the Hydrosphere C18 column a, 75–80 d after flowering, peel color was deep green; b, 105–110 d after (10 250 mm, 5 mm; YMC). flowering, peel color was green; 130 d after flowering, peel color was yellow. Phenolics Profile of Mume Fruit 1625

The reason for the decrease in phenolic content may be 1 2 3 related to the change in weight of each of the fruit A components. The increased phenolic concentration in the stone may be explained by the deposition of a phenolic compound such as a lignin precursor or monomer. In contrast, the weight of the kernel remained B

almost constant, being 2.3 g at the stone-hardening Absorbance stage, 2.2 g at the premature stage and 2.4 g at the fully mature stage. The concentration of phenolics remained fairly steady throughout the harvesting season. C The ORAC values at each stage are shown in Table 1. Flesh harvested at the stone-hardening stage showed the highest ORAC value among the analyzed samples. The ORAC value of the flesh decreased with maturation as Retention time the change in concentration of the phenolics. In contrast, Fig. 1. HPLC Analysis of the Phenolic Fraction of Mume Flesh at the ORAC values of the endocarp reached twice the Three Different Growth Stages. level of that of the immature fruits in the last stage of A, stone-hardening stage; B, stage with a deep green peel color; fruit maturation. The overall ORAC values for the and C, mature stage. A portion of the phenolic fractions collected from the fruit’s flesh was evaporated to dryness, and the residue was kernel were not very high and tended to decrease during dissolved in a minimal volume of dimethyl sulfoxide (DMSO) and fruit maturation. It was noted that the change in the injected into a Hydrosphere C18 column (’4:6mm 250 mm; phenolic concentrations of the endocarp throughout the YMC, Kyoto, Japan). The column was eluted with mixed solvent fruit maturation process was substantially greater than A (0.1% TFA):B (methanol) at a flow rate of 1.0 mL/min. Elution that of the flesh. The ORAC values of various fruits have was conducted at A:B ¼ 80:20 for the first 10 min, and then with a 18) linear gradient to 25:75 up to 80 min. All analyses were carried out been reported to vary widely among fruit species. The at 30 C and monitored by UV absorption at 280 nm. Compound 1, ORAC values presented in Table 1 were obtained from 3-O-caffeoilquinic acid (neochlorogenic acid); compound 2, 5-O- the samples extracted with a 90% ethanol solution, so caffeoilquinic acid (chlorogenic acid); compound 3, 1,30,40,60-tetra- the flesh of mume fruit was extracted with water and O-acetyl-6-O-p-coumaroyl-sucrose (prunose II) or 4,30,40,60-tetra-O- then with acetone to compare with other fruits.18) The acetyl-6-O-p-coumaroyl-sucrose (prunose III) or their isomers. total respective ORAC values of the water extract and acetone extract of the flesh were 315, 171 and 139 (neochlorogenic acid) and 5-O-caffeoilquinic acid (Trolox equivalent micromole/g on a dry weight basis) (chlorogenic acid) by comparing their MS and 1H- at the stone hardening stage, premature stage, and fully NMR spectral data (Fig. 2). mature stage. The value for the mume flesh was Compound 1 and 3-O-caffeoylquinic acid (neochloro- relatively higher than those of the other fruits.18) On genic acid) 1 the basis of the dry weight of fruit, strawberry had the H-NMR (400 MHz, methanol-d4) : 1.95 (1H, dd, highest ORAC activity (153.6) followed by plum (79.1), J ¼ 7, 11 Hz, H-6ax), 2.13 (2H, m, H-2eq and 6eq), 2.20 orange (51.7). Mume fruit may therefore be regarded as (1H, dd, J ¼ 3, 12 Hz, H-2ax), 3.63 (1H, dd, J ¼ 1, a fruit with high antioxidative activity. 7 Hz, H-4), 4.14 (1H, ddd, J ¼ 2, 7, 7 Hz, H-5), 5.34 (1H, ddd, J ¼ 2, 2, 3 Hz, H-3), 6.30 (1H, d, J ¼ 13 Hz, HPLC profiles H-80), 6.76 (1H, d, J ¼ 6 Hz, H-50), 6.93 (1H, dd, J ¼ 2, Figure 1 shows that the analysis of the flesh presented 6 Hz, H-60), 7.04 (1H, d, J ¼ 2 Hz, H-20), 7.58 (1H, d, a unique HPLC profile at each growth stage. Certain J ¼ 13 Hz, H-70). differences were apparent between the stone-hardening Compound 2 and 5-O-caffeoylquinic acid (chloro- stage (not edible) and other stages (edible), although genic acid) 1 there were common peaks across all the stages. The H-NMR (400 MHz, methanol-d4) : 2.02 (1H, dd, chromatograms of the premature and fully mature J ¼ 7, 11 Hz, H-6ax), 2.16 (2H, m, H-2eq and 6eq), 2.22 samples demonstrated a similar peak profile. (1H, dd, J ¼ 3, 12 Hz, H-2ax), 3.72 (1H, dd, J ¼ 1, 7 Hz, H-4), 4.17 (1H, ddd, J ¼ 2, 7, 7 Hz, H-5), 5.34 Identification of several compounds (1H, ddd, J ¼ 2, 2, 3 Hz, H-3), 6.26 (1H, d, J ¼ 16 Hz, The HPLC analysis of the phenolic fraction of the H-80), 6.76 (1H, d, J ¼ 8 Hz, H-50), 6.94 (1H, dd, J ¼ 2, fruit flesh revealed several common peaks at all stages 6 Hz, H-60), 7.04 (1H, d, J ¼ 4 Hz, H-20), 7.56 (1H, d, of the fruit growth (Fig. 1). We isolated three common J ¼ 13 Hz, H-70). main peaks from among the various peaks shown. Compound 3 was obtained as a white powder with the Compounds 1 and 2 were isolated from the phenolic elemental composition of C29H36O17 established by the fraction of the flesh of mume fruit at the premature high-resolution LC-MS/MS (ESI, positive) data. The stage, while compound 3 was isolated from the sample LC-MS data of compound 3 showed a molecular ion at the stone-hardening stage. (M þ Naþ) peak at m=z 679, in addition to a fragment More than 10 mg of each compound was obtained, ion (Mþ) peak by LC-MS/MS at m=z 147 derived from and the final structural evidence was achieved by 1H- the p-coumarate moiety. The 1H-NMR (400 MHz, þ NMR. Compounds 1 and 2 gave the same [M þ 1] ion methanol-d4) spectrum suggested that compound 3 at m=z 355 in the positive ion mode of their ESI-TOF- contained a p-coumaroyl group [: 6.42 (1H, d, MS data. Their 1H-NMR spectra were compared to the J ¼ 15:9 Hz), 7.74 (1H, d, J ¼ 15:9 Hz); 2000 and 3000-H, spectra previously reported,19,20) compounds 1 and 2 6.81 (2H, d, J ¼ 8:6 Hz), 7.54 (2H, d, J ¼ 8:6 Hz); 30000, being respectively identified as 3-O-caffeoilquinic acid 50000 and 20000,60000-H] and acetyl groups [: 1.77, 1.99, 1626 T. MITANI et al.

1 2

3-1 3-2

Fig. 2. Chemical Structures of 3-O-Caffeoylquinic Acid (neochlorogenic acid; 1), 5-O-Caffeoylquinic Acid (chlorogenic acid; 2), 1,30,40,60-Tetra-O- acetyl-6-O-p-coumaroyl-sucrose (prunose II; 3-1) and 4,30,40,60-Tetra-O-acetyl-6-O-p-coumaroyl-sucrose (prunose III; 3-2).

C A

B D

Fig. 3. Typical HPL Chromatogram for the Alkali Hydrolysate at the Premature Stage in 2008. The freeze-dried flesh powder (250 mg) was suspended in 10 mL of 1 N NaOH that had previously been deoxidized by bubbling nitrogen gas. After 4 h of incubation at 37 C, the suspension was acidified by adding phosphoric acid to pH 4.5 and then extracted with ethyl acetate. The ethyl acetate layer was evaporated to dryness, and the resulting residue was dissolved in a minimal volume of methanol and analyzed under the HPLC conditions described in Fig. 1. Compound A, caffeic acid; compound B, cis-p-coumaric acid; compound C, trans-p-coumaric acid; compound D, ferulic acid.

13 Table 2. C-NMR (100 MHz) Data for Compound 3 (DMSO-d6) tion. Typical HPLC data for the alkali hydrolysate at the premature stage in 2008 are shown in Fig. 3. The Chemical shift (ppm) chromatograms of the alkali hydrolysate showed similar 170.2 115.7 68.5 patterns regardless of the maturity stage or the produc- 170.1 113.5 67.5 tion year of the fruit. These chromatograms had four 169.6 103.6 64.5 common main peaks (A, B, C and D). Compounds A, C 169.4 90.7 62.7 and D were tentatively identified from their spectral 165.3 79.4 62.5 160.0 76.3 20.6 characteristics and retention times by comparing with 145.4 72.5 20.5 the respective commercially available pure compounds, 130.3 72.4 20.4 caffeic acid, trans-p-coumaric acid, and ferulic acid. 124.9 68.6 20.2 They were respectively identified from an HPLC negative-ion ESI-MS analysis as the ions of caffeic acid, p- coumaric acid, and ferulic acid. 2.07, 2.09 (3H, s)]. Compound 3 was thus assigned to Compound A: m=z 179.0343 (179.0350 calcd. for 0 0 0 the group of polyacylated sucrose, 1,3 ,4 ,6 -tetra-O- C9H7O4 ½M H ) acetyl-6-O-p-coumaroyl-sucrose (prunose II) or Compound C: m=z 163.0391 (163.0401 calcd. for 0 0 0 4,3 ,4 ,6 -tetra-O-acetyl-6-O-p-coumaroyl-sucrose (pru- C9H7O3 ½M H ) nose III), or to their isomer which has previously been Compound D: m=z 193.0501 (193.0506 calcd. for 12,13) reported in the fresh flowers of P. mume. The C10H9O4 ½M H ) amount of the isolated sample of compound 3 was Compounds A to D were isolated and purified by insufficient to determine the structure of its sugar moiety using preparative HPLC. The 1H-NMR spectrum of and the absolute configuration of the acetylated position compound B was consistent with the spectrum of cis-p- from the 13C-NMR spectrum. However, the spectrum of coumaric acid previously reported, and compounds A, C compound 3 (Table 2) was almost identical to that of and D were also confirmed.21–23) prunose II. Compound 3 could therefore be prunose II or Alkali-hydrolysis experiments revealed that agly- a related compound. cones of the hydroxycinnamic acid derivatives were largely made up of trans-p-coumaric acid, caffeic acid, Alkaline hydrolysis ferulic acid and cis-p-coumaric acid in the order of peak The freeze-dried flesh powder was hydrolyzed under area. The experiments also showed the relative propor- alkaline conditions to elucidate the phenolic composi- tions of the aglycones to be constant during the Phenolics Profile of Mume Fruit 1627 maturation of mume fruit, although there were some Acknowledgment differences in the HPLC profiles between the samples from the stone-hardening stage and other stages. It This study was supported by research grants funded appears that these hydroxycinnamic acids were present by the Regional Innovation Strategy Support Program of in such forms as ester bound or glycosidic. the Ministry of Education, Culture, Sports, Science and Compound A and caffeic acid Technology of Japan. 1H-NMR (400 MHz, MeOD, TMS) : 7.52 (1H, d, J ¼ 16 Hz, 7.03 (1H, d, J ¼ 2:0 Hz), 6.93 (1H, dd, References J ¼ 8:0, 2.0 Hz), 6.77 (1H, d, J ¼ 8:0 Hz), 6.21 (1H, d, J ¼ 16 Hz). 1) Ohtsubo T and Ikeda F, J. Engeigaku Zasshi (in Japanese), 62, Compound B and cis-p-coumaric acid 695–700 (1994). 1H-NMR (400 MHz, MeOD, TMS) : 7.59 (2H, d, 2) Terada H and Sakabe Y, Eisei Kagaku (in Japanese), 34, 36–40 J ¼ 8:0 Hz), 6.77 (1H, d, J ¼ 12 Hz), 6.73 (2H, d, (1988). J ¼ 8:0 Hz), 5.77 (1H, d, J ¼ 16 Hz). 3) Jee HJ, ‘‘Health Foods from Herbs,’’ Seoul National University Press, Seoul, p. 52 (1999). Compound C and trans-p-coumaric acid 4) World Health Organization, ‘‘Diet, Nutrition and Prevention of 1 H-NMR (400 MHz, MeOD, TMS) : 7.59 (1H, d, Chronic Diseases,’’ WHO technical report series 916, Report of J ¼ 16 Hz), 7.45 (2H, d, J ¼ 12 Hz), 6.80 (2H, d, a joint WHO/FAO expert consultation (2003). J ¼ 8 Hz), 6.28 (1H, d, J ¼ 16 Hz). 5) Jeong JT, Moon JH, Park KH, and Shin CS, J. Agric. Food Compound D and ferulic acid Chem., 54, 2123–2128 (2006). 1H-NMR (400 MHz, MeOD, TMS) : 7.59 (1H, 22d, 6) Shirasaka N, Kurematsu A, Kondo N, Kondo S, Iida M, Nippon Shokuhin J ¼ 16 Hz), 7.18 (1H, d, J ¼ 2:0 Hz), 7.06 (1H, dd, Hasegawa T, Murakami T, and Yoshizumi H, Kagaku Kogaku Kaishi (in Japanese), 46, 792–798 (1999). J ¼ 8:0, 2.0 Hz), 6.81 (1H, d, J ¼ 8:0 Hz), 6.31 (1H, d, 7) Shirasaka N, Nomura T, Murakami T, and Yoshizumi H, J ¼ 16 Hz), 3.89 (3H, s). Nippon Shokuhin Kagaku Kogaku Kaishi (in Japanese), 50, It is generally understood that the beneficial effects of 203–206 (2003). phenolic compounds would reduce the risk of major 8) Miyazawa M, Utsunomiya H, Inada K, Yamada T, Okuno Y, chronic diseases. However, information on their efficacy Tanaka H, and Tatematsu M, Biol. Pharm. Bull., 29, 172–173 in humans is limited, although the health benefits of (2006). 9) Ina H, Yamada K, and Miyazaki T, Nat. Med., 53, 109 phenolic compounds have been shown in in vitro and (1999). animal studies. The main obstacles to enhancing their 10) Ina H, Yamada K, Matsumoto K, and Miyazaki T, Nat. Med., efficacy are the poor absorption of phenolic compounds 56, 184–186 (2002). in the alimentary canal and extensive metabolism in the 11) Ina H, Yamada K, Matsumoto K, and Miyazaki T, Nat. Med., intestines and liver. Phenolic acids are considered to be 57, 178–180 (2003). anti-inflammatory, anticarcinogenic, and antimicrobial 12) Yoshikawa M, Murakami T, Ishiwada T, Morikawa T, Kagawa agents, as well as antioxidants.24–26) The benefits pro- M, Higashi Y, and Matsuda H, J. Nat. Prod., 65, 1151–1155 (2002). vided by phenolic acids may lie in their antioxidative 13) Matsuda H, Morikawa T, Ishiwada T, Managi H, Kagawa M, properties and their bioavailability. Konishi et al. have Higashi Y, and Yoshikawa M, Chem. Pharm. Bull., 51, 440–443 reported the transport rate of p-coumaric acid being 100 (2003). times higher than that of gallic acid across Caco-2 cell 14) Singleton VL and Rossi J, Am. J. Enol. Viticult., 16, 144–158 monolayers and the relative bioavailability of p-couma- (1965). ric acid against gallic acid being about 70 based on 15) Ou B, Huang D, Hampsch-Woodill M, Flanagan JA, and calculations from the serum concentration profile in the Deemer EK, J. Agric. Food Chem., 50, 3122–3128 (2002). 26,27) 16) Krygier K, Sosulski F, and Hogge L, J. Agric. Food Chem., 30, portal vein of rats. We also confirmed that the 330–334 (1982). bioavailability of p-coumaric acid was significantly 17) Oe T, Kuwabara A, Negoro K, Yamada S, and Sugai H, higher than that of caffeic or ferulic acid in mice and Engeigaku Kenkyu (in Japanese), 5, 141–148 (2006). rats (unpublished data). Mume fruit phenolics may have 18) Wang H, Guohua C, and Prior RL, J. Agric. Food Chem., 44, the capacity to reduce the risk of certain chronic diseases. 701–705 (1996). We clarified in this study that the total content of 19) Morishita H, Iwahashi H, Osaka N, and Kido R, J. Chromatogr., 315, 253–260 (1984). extractable phenolics in mume fruit flesh was about 1% 20) Tatefuji T, Izumi N, Ohta T, Arai S, Ikeda M, and Kurimoto M, on a dry weight basis and that the phenolics mostly Biol. Pharm. Bull., 19, 966–970 (1996). consisted of hydroxycinnamic acid derivatives. It is well 21) Lim EK, Higgins GS, Li Y, and Bowles DJ, Biochem. J., 373, known that there are many economically valuable fruits 987–992 (2003). throughout the world in the group of genus Prunus; for 22) Kor R, Vonk H, Xu X, Hoff WD, Crielaard W, and Hellingwerf example, dried plums (P. domesticus) contain a large KJ, FEBS Lett., 382, 73–78 (1996). amount of chlorogenic acids which constitute as much 23) Huang Z, Dostal L, and Rosazza JP, J. Biol. Chem., 268, 23954– 24) 23958 (1993). as 90% of the entire phenolic content. Nectarines, 24) Donovan JL, Meyer AS, and Waterhouse A, J. Agric. Food peaches, and plums contain such phenolics as hydro- Chem., 46, 1247–1252 (1998). xycinnamic acids, flavan-3-ols, and flavonols. Chloro- 25) Shahadi F and Naczk M, ‘‘Phenolics in Food and Nutraceut- genic acids were the predominant hydroxycinnamic icals,’’ CRC Press, Boca Raton, p. 158 (2004). acids, this being different from the present finding for 26) Konishi Y, Kobayashi S, and Shimizu M, Biosci. Biotechnol. mume fruit.28) The contents and proportion of phenolics Biochem., 67, 2317–2324 (2003). in Prunus fruits vary with the species within the genus. 27) Konishi Y, Hitomi Y, and Yoshioka E, J. Agric. Food Chem., 52, 2527–2532 (2004). The characteristic health benefits that stone fruits 28) Toma´s-Barbera´n FA, Gil MI, Cremin P, Waterhouse AL, provide would consequently also vary to reflect these Hess-Pierce B, and Kader AA, J. Agric. Food Chem., 49, 4748– differences in the fruit constituents. 4760 (2001).