Stability of Hydroxy-α-Sanshool in Medium-Chain Triglyceride Oil and Corresponding Oil/Water Emulsions.

The pungent component of sansho (Japanese pepper, Zanthoxylum pipritum) is sanshool, which is easily oxidized and decomposed. We have previously reported several sanshool stabilizers, such as α-tocopherol (α-Toc). Sansho pericarp powder treated with middle-chain triglycerides (MCTs) can be used to obtain extracts containing hydroxy-α-sanshool (HαS). Although HαS is stabilized when α-Toc is added to the MCT extracts, the loss of HαS is accelerated when it is mixed with a powder such as lactose. The separation of α-Toc from sanshools was thought to inevitably lead to their oxidation. Therefore, using sansho pericarp MCT extracts with or without α-Toc, oil/water (o/w) emulsions were prepared by adding a surfactant, glycerin, and water to these extracts. In both emulsions, HαS was stable in accelerated tests at 50 °C. However, when lactose powder was added to the emulsions and an accelerated test was performed, HαS in the emulsion containing α-Toc was stable, but HαS in the emulsion without α-Toc was unstable. These results highlight the importance of maintaining the close proximity of HαS and α-Toc in the emulsion. The stabilization of sanshools using emulsion technology can facilitate the production of various processed beverages, foods, cosmetics, and pharmaceuticals containing Japanese pepper.

Stabilization of Hydroxy-α-Sanshool by Antioxidants Present in the Genus Zanthoxylum.

Japanese pepper (sansho, Zanthoxylum piperitum) contains several types of sanshools belonging to N-alkylamides. Because of the long-chain unsaturated fatty acids present in their structure, sanshools are prone to oxidative deterioration, which poses problems in processing. In this paper, we evaluated the effects of antioxidants from the genus Zanthoxylum in preventing sanshool degradation using accelerated tests. An ethanolic extract of segment membranes of the sansho fruit pericarp was incubated at 70 °C for 7 days with different antioxidants to determine the residual amount of hydroxy-α-sanshool (HαS) in the extract. α-Tocopherol (α-Toc) showed excellent HαS-stabilizing activity at low concentrations. Among phenolic acids, we noted that the HαS-stabilizing activity increased with the number of hydroxy groups per molecule. For example, gallic acid and its derivatives exhibited excellent sanshool-stabilizing activity. Quercetin was found to be a superior HαS stabilizer compared with hesperetin and naringenin. However, the effective concentration was much higher for phenolic compounds than for α-Toc. These substances are believed to play a role in preventing the decomposition of sanshools in the pericarp of sansho. These sanshool stabilizers should be useful in the development of new beverages, foods, cosmetics, and pharmaceuticals that take advantage of the taste and flavor of sansho.

Potential hypotensive effects of Umezu polyphenols: a 14-week community-based, double-masked and placebo-controlled trial.

OBJECTIVE: To evaluate blood pressure (BP)-lowering effects of Umezu polyphenols, polyphenols contained in Japanese plums, in a community-based sample by double-masked and placebo-controlled design. METHODS: Seventy-two Japanese community-dwellers who were interested in prevention or control of their BP (preferably high-normal BP or grade I hypertension) but without antihypertensive medication were randomized into Umezu polyphenols or placebo groups. Each subject took 800 mg/day of Umezu polyphenols or placebo for 12 weeks, followed by a 2-week washout period. Their home and office BP were monitored for 14 weeks in a double-masked manner. We analyzed 56 subjects who met the inclusion criteria. RESULTS: Home BP increased gradually in both the groups during the intervention period, while diastolic office BP insignificantly decreased in the intervention group. During the washout period, home systolic BP in the morning elevated only in the intervention group. CONCLUSIONS: The study failed to collect consistent evidence of a clear persistent hypotensive effect of Umezu polyphenols.

Subcritical Methanol Extraction of the Stone of Japanese Apricot Prunus mume Sieb. et Zucc.

The pits of Japanese apricot, Prunus mume Sieb. et Zucc., which are composed of stones, husks, kernels, and seeds, are unused by-products of the processing industry in Japan. The processing of Japanese apricot fruits generates huge amounts of waste pits, which are disposed of in landfills or, to a lesser extent, burned to form charcoal. Mume stones mainly consist of cellulose, hemicellulose, and lignin. Herein, we attempted to solubilize the wood-like carapace (stone) encasing the pit by subcritical fluid extraction with the aim of extracting useful chemicals. The characteristics of the main phenolic constituents were elucidated by liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) analyses. The degrees of solubility for various treatments (190 °C; 3 h) were determined as follows: subcritical water (54.9%), subcritical 50% methanol (65.5%), subcritical 90% methanol (37.6%), subcritical methanol (23.6%), and subcritical isopropyl alcohol (14.4%). Syringaldehyde, sinapyl alcohol, coniferyl alcohol methyl ether, sinapyl alcohol methyl ether, 5-(hydroxymethyl)-2-furfural, and furfural were present in the subcritical 90% methanol extract. Coniferyl and sinapyl alcohols (monolignols) are source materials for the biosynthesis of lignin, and syringaldehyde occur in trace amounts in wood. Our current findings provide a solubilization method that allows the main phenolic constituents of the pits to be extracted under mild conditions. This technique for obtaining subcritical extracts shows great potential for further applications.

Antiviral and Virucidal Activities of Umesu Phenolics on Influenza Viruses.

In this study, umesu phenolics were purified from the salt extracts of Japanese apricot (Nanko-mume cultivar of Prunus mume Sieb. et Zucc.). Characterization of umesu phenolics revealed that, when added to the culture media of the infected cells, they inhibited the multiplication of influenza and many other RNA and DNA viruses. In addition to these antiviral activities, the phenolics significantly decreased the plating efficiency of influenza virus, if present in the virus inoculum. More drastic effects were observed in terms of virucidal activity; the infectivity of several strains of influenza viruses decreased less than 0.001 when they were incubated with 4 mg/ml phenolics at 30 ℃ for 5 min. The virucidal activity of phenolics was found to be more remarkable in acidic conditions; however, the activity was not merely a result of the acidity of the phenolics. These results clearly support the antiviral and virucidal activities of the umesu phenolics against influenza viruses and suggest their potential pharmacological usefulness as disinfectants or preventive medicine against superficial infections, such as the respiratory infections.

Antiviral and virucidal activities against herpes simplex viruses of umesu phenolics extracted from Japanese apricot.

Umesu phenolics were obtained from the salt extracts of Japanese apricot (Nanko-mume cultivar of Prunus mume Sieb. et Zucc.) as purified phenolics. The antiviral activities of umesu phenolics obtained were then examined against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2), enveloped DNA viruses. The phenolics inhibited the multiplication of these viruses when added to the culture media of the infected cells. This inhibition occurred at phenolic concentrations at which they showed no severe cytotoxicity. One-step growth experiments showed that the eclipse period in the HSV-1 multiplication process was extended in the presence of umesu phenolics and that the addition of phenolics after the completion of viral DNA replication did not affect their multiplication. More drastic effects were observed on virucidal activities against HSV-1 and HSV-2; the infectivity decreased to 0.0001 when infected cells were incubated with 3 mg/ml phenolics at 30°C for 5 min. These results demonstrate the antiviral and virucidal activities of umesu phenolics and suggest a potential pharmacological use for these phenolics as a sanitizing or preventive medicine against superficial HSV infections.

Process for the Purification of cis-p-Coumaric Acid by Cellulose Column Chromatography after the Treatment of the trans Isomer with Ultraviolet Irradiation.

A methanolic solution of trans-p-coumaric acid was exposed to ultraviolet radiation and a mixture solution of the trans and cis isomers was subjected to cellulose column chromatography, eluting with an aqueous 0.1% trifluoroacetic acid solution containing methanol (90:10, v/v). Separation of the trans and cis isomers was achieved. The identity of the cis isomer was confirmed by TLC, HPLC, and NMR. Since both the support and eluent are inexpensive, the cis isomers can be obtained economically on both the laboratory and industrial scales.

Antimicrobial Activity of the Phenolic Compounds of Prunus mume against Enterobacteria.

Mume fruit, the Japanese apricot (Prunus mume SIEB. et ZUCC.), is popular in Japan and is mostly consumed in the pickled form called umeboshi. This fruit is known to have anti-microbial properties, but the principal constituents responsible for the antimicrobial properties have not yet been elucidated. We investigated the antimicrobial activities of the phenolic compounds in P. mume against enterobacteria. In this study, growth inhibitory activities were measured as an index of the antibacterial activities. The phenolic compounds were prepared from a byproduct of umeboshi called umesu or umezu (often translated as "mume vinegar"). Umesu or umezu phenolics (UP) contain approximately 20% phenolic compounds with p-coumaric acid as a standard and do not contain citric acid. We observed the inhibitory effects of UP against the growth of some enterobacteria, at a relatively high concentration (1250-5000 µg/mL). Alkali hydrolysates of UP (AHUP) exhibited similar antibacterial activities, but at much lower concentrations of 37.5-300 µg/mL. Since AHUP comprises hydroxycinnamic acids such as caffeic acid, p-coumaric acid, and ferulic acid, the antibacterial activities of each of these acids were examined. Our study shows that the phenolic compounds in P. mume other than citric acid contribute to its antimicrobial activity against enterobacteria in the digestive tract.

Safety and adherence of Umezu polyphenols in the Japanese plum (Prunus mume) in a 12-week double-blind randomized placebo-controlled pilot trial to evaluate antihypertensive effects.

OBJECTIVES: Medications or lifestyle changes to prevent or improve hypertension often press considerable efforts on patients suffering from mild hypertension. Capsules including Umezu polyphenols (UP), polyphenols in Japanese plums, may help them to control their blood pressure (BP). The aim of this study is to evaluate the effectiveness of UP on BP and its safety. METHODS: A total of 15 healthy workers without antihypertensive medication who had some concerns about their BP, preferably normal-high BP or hypertension level 1, were randomized in a double-blind manner into UP ingesting and placebo groups. Each subject was instructed to take four capsules daily for 12 weeks (daily UP dose, 800 mg for the UP ingesting group; and 0 mg for the placebo group). These subjects were followed for 12 weeks, and their BP both at home and at the examination site, as well as self-perceived quality-of-life outcomes and possible side effects, was monitored during that period. Group × time interactions on BP changes were examined. RESULTS: All of the 15 subjects completed the 12-week intervention trial. The BP changes did not significantly differ between the UP ingesting and placebo groups, neither at the examination site nor at home. But during the study period, no adverse effects were observed. CONCLUSIONS: No remarkable effect of UP on BP was observed. However, a higher dose of UP was confirmed safe and high in adherence in this 12-week randomized controlled trial. Its effect on BP and other outcomes shall be confirmed in a larger sample.

Phenolics profile of mume, Japanese apricot (Prunus mume Sieb. et Zucc.) fruit.

The fruit of mume, Japanese apricot (Prunus mume Sieb. et Zucc.), was evaluated for its phenolics content, high performance liquid chromatography (HPLC) profile and antioxidative activities. The phenolics content of mume fruit was relatively high, the flesh of fully matured fruit containing up to 1% of phenolics on a dry weight basis. Reflecting such a high content of phenolics, the ORAC (oxygen radical absorbance capacity) value for mume fruit flesh showed high values, ranging from 150 to 320 µmol/g Trolox equivalent, depending upon the stage of maturation. 5-O-Caffeoylqunic acid (chlorogenic acid), 3-O-caffeoylquinic acid and tetra-O-acylated sucrose-related compounds were isolated from the flesh of mume fruit, although many unknown peaks were also apparent in the HPLC chromatogram. An alkali hydrolysate comprised four main phenolic acids, caffeic acid, cis/trans-p-coumaric acid and ferulic acid. No flavonoids were observed in the analysis. These results suggest that the majority of phenolics in mume fruit were hydroxycinnamic acid derivatives.

Caffeic acid inhibits vascular smooth muscle cell proliferation induced by angiotensin II in stroke-prone spontaneously hypertensive rats.

Epidemiological studies have linked the consumption of phenolic acids with reduced risk of cardiovascular diseases. In the present study, we sought to investigate whether caffeic acid, a phenolic acid which is abundant in normal diet, can antagonize angiotensin II (Ang II)-induced vascular smooth muscle cell (VSMC) proliferation in stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar-Kyoto (WKY) rats, and if so, to elucidate the underlying cell signaling mechanisms. We exposed VSMCs to Ang II and caffeic acid and found that caffeic acid significantly inhibited intracellular superoxide anion generation (decreased from 127 +/- 6.3% to 100.3 +/- 6.6% of the control cells) and the cell proliferation induced by Ang II. Furthermore, caffeic acid significantly abolished the tyrosine phosphorylation of JAK2 (decreased from 7.4 +/- 0.6-fold to 2.4 +/- 0.6-fold at 2 min) and STAT1 (decreased from 1.8 +/- 0.2-fold to 0.5 +/- 0.1-fold at 2 min) and the phosphorylation of ERK1/2 (decreased from 99.2 +/- 10.2-fold to 49.8 +/- 10.9-fold at 2 min) that were induced by Ang II. These effects of caffeic acid were consistent with the inhibition of the proliferation of VSMCs by DPI, an NADPH oxidase inhibitor, and by AG-490, a JAK2 inhibitor. In conclusion, our findings suggest that caffeic acid attenuates the proliferative reaction of VSMCs to Ang II stimulation in both SHRSP and WKY rats by inhibiting the generation of reactive oxygen species and then partially blocking the JAK/STAT signaling cascade and the Ras/Raf-1/ERK1/2 cascade.

Downregulation of Rac1 activation by caffeic acid in aortic smooth muscle cells.

Caffeic acid, a dietary phenol from coffee, fruits and vegetables, is an efficient antioxidant. However, little is known about its anti-oxidative mechanism in the modulation of fundamental cellular processes. In this study, we investigated whether caffeic acid regulates Rac1 GTPase activity, a partner of NADPH oxidase. Our results showed that caffeic acid decrease Rac1 protein level under basal conditions and incubation with angiotensin II (ANG II) in vascular smooth muscle cells. In a Rac-bound-to-PAK pull down assay, caffeic acid clearly inhibited Rac1 activity. We also observed that caffeic acid suppressed the generation of superoxide anion stimulated by ANG II that activates NADPH oxidase. On the other hand, co-incubation with caffei caid and cycloheximide significantly accelerated the Rac1 degradation. In addition, pretreatment with caffeic acid for 24 hours was able to prevent phosphorylation of MLC and HSP27, when cells were challenged with ANG II through the redox sensitive pathway. These results support the hypothesis that caffeic acid reduces Rac1 GTPase protein and activity level, followed by a down-regulation of NADPH oxidase activity.

A new bipyrrole and some phenolic constituents in prunes (Prunus domestica L.) and their oxygen radical absorbance capacity (ORAC).

Isolation and structural elucidation of prune constituents were performed and total 10 compounds were determined by NMR and MS analyses. A novel compound was identified to be 2-(5-hydroxymethyl-2',5'-dioxo-2',3',4',5'-tetrahydro-1'H-1,3'-bipyrrole)carbaldehyde, and 7 phenolic compounds were isolated from prunes for the first time. In addition, antioxidant activity of them was evaluated on the basis of the oxygen radical absorbance capacity (ORAC).

Abscisic acid related compounds and lignans in prunes (Prunus domestica L.) and their oxygen radical absorbance capacity (ORAC).

Four new abscisic acid related compounds (1-4), together with (+)-abscisic acid (5), (+)-beta-D-glucopyranosyl abscisate (6), (6S,9R)-roseoside (7), and two lignan glucosides ((+)-pinoresinol mono-beta-D-glucopyranoside (8) and 3-(beta-D-glucopyranosyloxymethyl)-2- (4-hydroxy-3-methoxyphenyl)-5-(3-hydroxypropyl)-7-methoxy-(2R,3S)-dihydrobenzofuran (9)) were isolated from the antioxidative ethanol extract of prunes (Prunus domestica L.). The structures of 1-4 were elucidated on the basis of NMR and MS spectrometric data to be rel-5-(3S,8S-dihydroxy-1R,5S-dimethyl-7-oxa-6-oxobicyclo[3,2,1]oct-8-yl)-3-methyl-2Z,4E-pentadienoic acid (1), rel-5-(3S,8S-dihydroxy-1R,5S-dimethyl-7-oxa-6-oxobicyclo[3,2,1]oct-8-yl)-3-methyl-2Z,4E-pentadienoic acid 3'-O-beta-d-glucopyranoside (2), rel-5-(1R,5S-dimethyl-3R,4R,8S-trihydroxy-7-oxa-6-oxobicyclo[3,2,1]oct-8-yl)-3-methyl-2Z,4E-pentadienoic acid (3), and rel-5-(1R,5S-dimethyl-3R,4R,8S-trihydroxy-7-oxabicyclo[3,2,1]- oct-8-yl)-3-methyl-2Z,4E-pentadienoic acid (4). The antioxidant activities of these isolated compounds were evaluated on the basis of oxygen radical absorbance capacity (ORAC). The ORAC values of abscisic acid related compounds (1-7) were very low. Two lignans (8 and 9) were more effective antioxidants whose ORAC values were 1.09 and 2.33 micromol of Trolox equiv/micromol, respectively.

Antioxidant properties of prunes (Prunus domestica L.) and their constituents.

Prunes contain large amounts of phenolics and show high antioxidant activity. The aim of this study is to clarify the contents of caffeoylquinic acid (CQA) isomers, and to estimate the contribution of these isomers to the antioxidant activity of prunes. Furthermore, structural elucidation and evaluation of antioxidant activity of prune components were also performed. CQA isomers in prunes were quantified by HPLC analysis, and it has become apparent that prunes contain relatively high amount of 4-O-caffeoylquinic acid. The contribution of CQA isomers to the antioxidant activity of prunes was revealed to be 28.4% on the basis of oxygen radical absorbance capacity (ORAC); hence, it was indicated that residual ORAC is dependent on unknown antioxidant components. Total 28 compounds were isolated and their structures were elucidated by NMR and MS analyses. Four abscisic acid related compounds, a chromanon, and a bipyrrole were novel. Each CQA isomer in prunes showed high antioxidant activities when measured by the oil stability index (OSI) method, O2- scavenging activity, and ORAC. Other isolated compounds such as hydroxycinnamic acids, benzoic acids, coumarins, lignans, and flavonoid showed high ORAC values. Furthermore, a novel chromanon indicated a remarkable synergistic effect on ORAC of CQA isomers.

Quantitative evaluation of antioxidant components in prunes (Prunus domestica L.).

Prunes are known to show high antioxidant activity on the basis of the oxygen radical absorbance capacity (ORAC), and their major antioxidant components are caffeoylquinic acid isomers. The aim of this study is to prove the contribution of caffeoylquinic acid isomers to the ORAC of prunes, and to investigate the existence of other antioxidant components. Caffeoylquinic acid isomers in ethanol (EtOH) extracts of prunes were quantified by HPLC analysis, and the degree of contribution of these isomers to the ORAC was found to be 28.4%; hence, it was speculated that the remaining ORAC is dependent on other antioxidant compounds. EtOH extract was partitioned between hexane and H(2)O. The H(2)O layer was further separated into H(2)O and 2-100% methanol (MeOH) eluates by Diaion HP-20 column chromatography. Both the H(2)O and 50% MeOH eluates showed high values of total phenolics and ORAC, although the contribution of caffeoylquinic acid isomers to the ORAC was low. Therefore, it was predicted that unknown antioxidants exist in these fractions, and several compounds were identified by HPLC analysis. Furthermore, hydrolysis of EtOH extract residue led to higher levels of total phenolics and ORAC, and these results suggested the existence of conjugated antioxidant components in prunes.

Antioxidant activity of prune (Prunus domestica L.) constituents and a new synergist.

Ethanol extract of prune was separated into hexane-soluble and H(2)O-soluble fractions, and the H(2)O-soluble fraction was further separated into a methanol (MeOH) eluate and an H(2)O eluate by Diaion HP-20 column chromatography. The MeOH eluate exhibited the strongest antioxidant activity among the separated fractions evaluated by oxygen radical absorbance capacity (ORAC). Further purification of the MeOH eluate led to isolation of a novel compound, 4-amino-4-carboxychroman-2-one, together with four known compounds (p-coumaric acid, vanillic acid beta-glucoside, protocatechuic acid, and caffeic acid), structures of which were identified by NMR and MS analyses. The ORAC values of these isolated compounds showed 0.15-1.43 units (micromol of Trolox equiv)/micromol, and the new compound showed a remarkable synergistic effect on caffeoylquinic acid isomers. The antioxidant activity of the MeOH eluate was highly dependent on the major prune components, caffeoylquinic acid isomers, with a contribution from the new synergist.