Amazake made from sake cake and rice koji suppresses sebum content in differentiated hamster sebocytes and improves skin properties in humans.

Amazake is a traditional Japanese health drink. Here, we examined the effects of amazake on skin in cells and humans. Treatment with sake cake or rice koji suppressed intracellular lipid accumulation in differentiated hamster sebocytes, likely through the reduced expression of peroxisome proliferator-activated receptor-gamma (PPARγ) mRNA. In double-blind, placebo-controlled trial, seventeen Japanese women ingested either amazake or placebo for 4 weeks. Ingestion of the amazake decreased the sebum content compared to the placebo. The questionnaires showed improvements in "face color," "dark circles under the eyes," "glossy hair," and "waking up well", only in the amazake. In accordance with the questionnaires, additional analysis revealed the change in the L* values under the eyes was statistically increased in the amazake compared to the placebo. These results indicate that amazake may decrease sebum content in cells and humans and increase the L* values under the eyes, with some additional beneficial effects in humans.

Intake of a Mixture of Sake Cake and Rice Malt Increases Mucin Levels and Changes in Intestinal Microbiota in Mice.

Amazake is a traditional Japanese beverage. Its main ingredients are sake cake and rice malt. In this study, we examined the effect of sake cake and rice malt on the intestinal barrier function and gut microbiota. BALB/c mice were fed a control diet or a diet containing a mixture of sake cake and rice malt powder (SRP) for four weeks. Fecal IgA values did not change between groups, but the fecal mucin level was significantly greater in the SRP-fed group. Gene expression analysis in the ileum by real-time PCR demonstrated Muc2 expression did not change, while the Muc3 expression was upregulated in the SRP-fed group. Furthermore, microbiota analysis demonstrated a change by SRP intake at the family level, and the proportion of Lactobacillaceae significantly increased in the SRP-fed group. At the genus level, the proportion of Lactobacillus also significantly increased in the SRP-fed group. These results suggest that the intake of a mixture of sake cake and rice malt improves intestinal barrier function by increasing mucin levels and inducing changes in intestinal microbiota.

Effect of piceatannol-rich passion fruit seed extract on human glyoxalase I-mediated cancer cell growth.

Passion fruit seed extract (PFSE), a product rich in stilbenes such as piceatannol and scirpusin B, has various physiological effects. It is unclear whether PFSE and its stilbene derivatives inhibit cancer cell proliferation via human glyoxalase I (GLO I), the rate-limiting enzyme for detoxification of methylglyoxal. We examined the anticancer effects of PFSE in two types of human cancer cell lines with different GLO I expression levels, NCI-H522 cells (highly-expressed GLO I) and HCT116 cells (lowly-expressed GLO I). PFSE and its stilbenes inhibited GLO I activity. In addition, PFSE and its stilbenes supressed the cancer cell proliferation of NCI-H522 cells more than HCT116 cells. These observations suggest that PFSE can provide a novel anticancer strategy for prevention and treatment.

Isolation and characterization of Gram-negative and Gram-positive bacteria capable of producing piceatannol from resveratrol.

Piceatannol is a valuable natural polyphenol with therapeutic potential in cardiovascular and metabolic disease treatment. In this study, we screened for microorganisms capable of producing piceatannol from resveratrol via regioselective hydroxylation. In the first screening, we isolated microorganisms utilizing resveratrol, phenol, or 4-hydroxyphenylacetic acid as a carbon source for growth. In the second screening, we assayed the isolated microorganisms for hydroxylation of resveratrol. Using this screening procedure, a variety of resveratrol-converting microorganisms were obtained. One Gram-negative bacterium, Ensifer sp. KSH1, and one Gram-positive bacterium, Arthrobacter sp. KSH3, utilized 4-hydroxyphenylacetic acid as a carbon source for growth and efficiently hydroxylated resveratrol to piceatannol without producing any detectable by-products. The hydroxylation activity of strains KSH1 and KSH3 was strongly induced by cultivation with 4-hydroxyphenylacetic acid as a carbon source during stationary growth phase. Using the 4-hydroxyphenylacetic acid-induced cells as a biocatalyst under optimal conditions, production of piceatannol by strains KSH1 and KSH3 reached 3.6 mM (0.88 g/L) and 2.6 mM (0.64 g/L), respectively. We also cloned genes homologous to the monooxygenase gene hpaBC from strains KSH1 and KSH3. Introduction of either hpaBC homolog into Escherichia coli endowed the host with resveratrol-hydroxylating activity.

Efficient monooxygenase-catalyzed piceatannol production: Application of cyclodextrins for reducing product inhibition.

Piceatannol is a rare, costly plant-based stilbene derivative and exhibits various health-enhancing properties. Recently, we demonstrated that piceatannol could be produced from resveratrol through site-selective hydroxylation using Escherichia coli cells expressing the monooxygenase HpaBC. However, piceatannol production ceased at approximately 25 mM, even when sufficient levels of the substrate resveratrol remained in the reaction mixture. In this study, we found that high concentrations (>20-25 mM) of piceatannol significantly inhibited the HpaBC-catalyzed reaction. Cyclodextrins (CDs) reportedly encapsulate various hydrophobic compounds. We found that the addition of ß-CD or γ-CD to the reaction mixture reduced the inhibition caused by the product piceatannol. The effects of ß-CD on piceatannol production were more pronounced than those of γ-CD at high concentrations of the substrate resveratrol and CDs. The production of piceatannol reached 49 mM (12 g L-1) in the presence of ß-CD, a level twice that achieved in the absence of ß-CD. The technique described here might be applicable to the bioproduction of other stilbenes and structurally related compounds.

Effect of Passion Fruit Seed Extract Rich in Piceatannol on the Skin of Women: A Randomized, Placebo-Controlled, Double-Blind Trial.

Piceatannol has been reported to have a wide variety of effects on the skin, including promoting collagen production, inhibiting melanin synthesis, inducing the antioxidant glutathione, and eliminating reactive oxygen species. In this study, a randomized, placebo-controlled, double-blind trial was conducted to clinically evaluate the effects of piceatannol-rich passion fruit seed extract on the skin of healthy Japanese women (age, 35-54 y). Thirty-two women with dry skin received either passion fruit seed extract (5 mg piceatannol) or a placebo (dextrin) for 8 wk. Skin hydration and other parameters on the face were assessed at 0, 4, and 8 wk by using specialized equipment. Furthermore, questionnaire interviews were conducted regarding the physical condition of subjects at 0, 4, and 8 wk. The results showed that consumption of passion fruit seed extract led to significant increases in the moisture content of human skin after 4 and 8 wk compared with that before the trial. The amount of transepidermal water loss decreased over time, although the differences were not significant. Moreover, a stratified analysis of subjects with moisture values of ≤200 µS revealed increased moisture content in the passion fruit seed extract group as compared with the placebo group. Furthermore, the results of questionnaires showed significant reductions in "perspiration" and "fatigue" in the passion fruit seed extract group as compared with the placebo group. These results indicate that oral intake of passion fruit seed extract that is rich in piceatannol could improve the moisture of dry skin and reduce fatigue.

Investigation of epigallocatechin-3-O-caffeoate and epigallocatechin-3-O-p-coumaroate in tea leaves by LC/MS-MS analysis.

(-)-Epigallocatechin-3-O-gallate (EGCG), the major catechin present in green tea, exhibits potent antioxidant activity. We thereby investigated the presence of unknown components bearing the (-)-epigallocatechin (EGC) moiety in fresh tea leaf samples. Initially, liquid chromatography tandem mass spectrometry (LC-MS/MS) was employed to examine fresh tea leaves of the Yabukita, the most popular tea cultivar in Japan, which suggested the presence of the EGC phenylpropanoid derivatives, (-)-epigallocatechin-3-O-p-coumaroate (EGCpCA) and (-)-epigallocatechin-3-O-caffeoate (EGCCA). The structures of the detected EGCpCA and EGCCA were then confirmed by LC-MS/MS using synthesized EGCpCA and EGCCA as standards. In addition, EGCpCA and EGCCA were evaluated for their antioxidant activity in the ORAC (oxygen radical antioxidant capacity) and DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging assays, where EGCCA (8.60µmolTE/µmol, TE=Trolox equivalents) exhibited a stronger antioxidant activity than EGCG (5.52µmolTE/µmol) in the ORAC assay. Finally, EGCpCA and EGCCA were quantitated in several tea leaf samples using LC-MS/MS, and it was found that these compounds were present in lower quantities (EGCpCA, 16.8-345.8µg/g, EGCCA, 4.3-75.1µg/g in the dry tea leaves) than the major catechins. In this study, we found the potent antioxidant EGCCA using LC-MS/MS and revealed its wide existence in various tea leaves.

The Effect of Piceatannol from Passion Fruit (Passiflora edulis) Seeds on Metabolic Health in Humans.

Animal studies have shown the beneficial effects of piceatannol on metabolic health; however, there is a lack of human studies designed to examine these effects. The objective of this study was to investigate the effects of piceatannol on metabolic health in humans. This randomized, placebo-controlled study was conducted on 39 subjects, including 10 overweight men and 9 overweight women (BMI ≥ 25), as well as 10 non-overweight men and 10 non-overweight women (BMI < 25). Subjects received piceatannol (20 mg/day) or placebo capsules for eight weeks in a random order. The primary outcome was the effect of piceatannol on glucose-metabolism, including insulin sensitivity. The secondary outcomes were the effects on other parameters, including blood pressure (BP), heart rate (HR), endothelial function, lipids, inflammation, oxidative stress, mood status, and Sirt1 and phospho-AMP-activated kinase (p-AMPK) expression in isolated peripheral blood mononuclear cells (PBMNCs). Supplementation with piceatannol in overweight men reduced serum insulin levels, HOMA-IR, BP and HR. Other groups, including non-overweight men, as well as overweight and non-overweight women, showed no beneficial effects on insulin sensitivity, BP and HR. Furthermore, piceatannol is not associated with other data, including body weight (BW), body composition, endothelial function, lipids, inflammation, oxidative stress, mood status, and Sirt1/p-AMPK expression in PBMNCs. In conclusion, supplementation with piceatannol can improve metabolic health, including insulin sensitivity, BP and HR, in overweight men.

Evaluation of the innate immune-stimulating activity of amazake using a silkworm muscle contraction assay.

We evaluated the innate immune-stimulating activity of amazake using a silkworm muscle contraction assay. Sake cake, a raw material used to make amazake, had high innate immunity-stimulating activity, whereas rice malt, another raw material used to make amazake, did not, even after fermentation. These results suggest that the silkworm muscle contraction assay is a useful tool to screen foods with high innate immune-stimulating activity and that amazake made from sake cake has immunomodulatory potential.

Piceatannol, a natural trans-stilbene compound, inhibits human glyoxalase I.

Human glyoxalase I (GLO I), a rate-limiting enzyme for detoxification of methylglyoxal (MG), a by-product of glycolysis, is known to be a potential therapeutic target for cancer. Here, we searched new scaffolds from natural compounds for designing novel GLO I inhibitors and found trans-stilbene scaffold. We examined the inhibitory abilities to human GLO I of commercially available trans-stilbene compounds. Among them, piceatannol was found to have the most potent inhibitory activity against human GLO I. Piceatannol could inhibit the proliferation of human lung cancer NCI-H522 cells, which are dependent on GLO I for survival, in a dose- and time-dependent manner. In addition, piceatannol more significantly inhibited the proliferation of NCI-H522 cells than that of NCI-H460 cells, which are less dependent on GLO I. Importantly, overexpression of GLO I in NCI-H522 cells resulted in less sensitive to the antiproliferative activity of piceatannol. Taken together, this is the first report demonstrating that piceatannol inhibits GLO I activity and the GLO I-dependent proliferation of cancer cells. Furthermore, we determined a pharmacophore for novel inhibitors of human GLO I by computational simulation analyses of the binding mode of piceatannol to the enzyme hot spot in the active site. We suggest that piceatannol is a possible lead compound for the development of novel GLO I inhibitory anticancer drugs.

Piceatannol exhibits anti-inflammatory effects on macrophages interacting with adipocytes.

Piceatannol (PIC), a natural analog of resveratrol (RES), is a phytochemical found in passion fruit seeds. To clarify the effects of PIC on obesity-induced inflammation in adipose tissue, we investigated the anti-inflammatory activity of PIC-related compounds (PIC, RES, and metabolites from PIC) in culture models of obese adipose tissue. Lipopolysaccharide (LPS) and conditioned medium from 3T3-L1 adipocytes (3T3-L1-CM) enhanced proinflammatory gene expression and synthesis of nitric oxide (NO), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) in RAW264.7 macrophages. Although each compound inhibited the mRNA expression of iNOS (inducible NO synthase), TNF-α, and IL-6, PIC potently inhibited them, and 30 µmol/L PIC suppressed the LPS- and 3T3-L1-CM-induced mRNA expression of iNOS (70.4% and 69.2% suppression, respectively), TNF-α (42.6% and 47.0% suppression), and IL-6 (27.3% and 42.1% suppression). PIC also significantly suppressed production of NO (80.3% suppression) and inflammatory cytokines (TNF-α; 33.7% suppression, IL-6; 66.5% suppression). Furthermore, PIC was found to rescue the uncoupling protein 1 mRNA expression induced by isoproterenol in 10T1/2 adipocytes, which was suppressed by LPS-activated macrophages. These results suggest that PIC may attenuate the pathologic inflammation triggered by adipose tissues.

Piceatannol is superior to resveratrol in promoting neural stem cell differentiation into astrocytes.

Piceatannol (3,3',4',5-trans-tetrahydroxystilbene) is a polyphenolic compound abundant in the seeds of passion fruit (Passiflora edulis). Piceatannol is an analogue of resveratrol (3,4',5-trans-trihydroxystilbene) and shares the structural motif and biological activities such as activation of SIRT1. Several studies have shown that piceatannol is more potent than resveratrol. In this study, we examined the effects of piceatannol on neural stem cell differentiation into astrocytes compared with those of resveratrol. At a concentration of 2.5 µM, piceatannol promoted astrocyte differentiation, while resveratrol had no effect at this concentration. Furthermore, we found that oral administration of piceatannol increased the number of astrocytes in the brains of adult mice, while resveratrol administration showed no effects. These results suggest that piceatannol has a superior effect to resveratrol in promoting astrocyte differentiation.

Vascular- and hepato-protective effects of passion fruit seed extract containing piceatannol in chronic high-fat diet-fed rats.

The effects of chronic administration of piceatannol-enriched (9.5% w/w) passion fruit seed extract (PFSE) on the cardiovascular damage induced in a high-fat (HF) diet-fed model of Fischer 344 rats were evaluated. Rats were fed the control, HF, or HF diets containing PFSE (0.5% w/w) for 16 weeks, and the effects of the various diets on the tissue weight, serum lipid profile, hepatic fibrosis, hepatic ductular reaction, cardiac function and aortic ring reactivity were examined. HF diet-fed rats developed signs of cardiovascular disease with abnormal serum profiles compared to control diet-fed rats. PFSE supplementation improved the liver hypertrophy and hepatic histology of the HF diet-fed rats. In addition, the triglyceride and cholesterol levels, platelet aggregation, cardiac function, and acetylcholine-mediated relaxation of the aortic ring were improved. These results suggest that the chronic intake of PFSE containing piceatannol prevents HF diet-induced cardiovascular disease in rats.

Enhanced glucose tolerance by intravascularly administered piceatannol in freely moving healthy rats.

Piceatannol is a phytochemical in the seeds of passion fruit that has a hypoglycemic effect when orally administered. To elucidate the contribution of intact and metabolites of piceatannol after gastro-intestinal absorption to hypoglycemic effect, we examined the influence of piceatannol and isorhapontigenin on blood glucose concentrations during fasting and glucose tolerance tests by administering them intravascularly to freely moving healthy rats. We found that intravascularly administered piceatannol reduced the blood glucose concentrations during both fasting and glucose tolerance tests, but isorhapontigenin did not during either of them. Furthermore, we found that piceatannol increased the insulinogenic index during glucose tolerance tests and that piceatannol had no influence on insulin sensitivity by performing hyperinsulinemic euglycemic clamping tests. These results suggest that piceatannol orally intaken may enhance glucose tolerance by the effect of intact piceatannol through enhanced early-phase secretion of insulin. Therefore, oral intake of piceatannol might contribute to proper control of postprandial glycemic excursions in healthy subjects.

Biocatalytic synthesis of 3,4,5,3',5'-pentahydroxy-trans-stilbene from piceatannol by two-component flavin-dependent monooxygenase HpaBC.

HpaBC monooxygenase was previously reported to hydroxylate resveratrol to piceatannol. In this article, we report a novel catalytic activity of HpaBC for the synthesis of a pentahydroxylated stilbene. When Escherichia coli cells expressing HpaBC were incubated with resveratrol, the resulting piceatannol was further converted to a new product. This product was identified by mass spectrometry and NMR spectroscopy as a 5-hydroxylated piceatannol, 3,4,5,3',5'-pentahydroxy-trans-stilbene (PHS), which is a reportedly valuable biologically active stilbene derivative. We attempted to produce PHS from piceatannol on a flask scale. After examining the effects of detergents and buffers on PHS production, E. coli cells expressing HpaBC efficiently hydroxylated piceatannol to PHS in a reaction mixture containing 1.5% (v/v) Tween 80 and 100 mM 3-morpholinopropanesulfonic acid-NaOH buffer at pH 7.5. Under the optimized conditions, the whole cells regioselectively hydroxylated piceatannol, and the production of PHS reached 6.9 mM (1.8 g L(-1)) in 48 h.

Piceatannol lowers the blood glucose level in diabetic mice.

We previously found that passion fruit (Passiflora edulis) seeds contained a high amount of piceatannol (3,5,3',4'-trans-tetrahydroxystilbene), a natural analog of resveratrol (3,5,4'-trans-trihydroxystilbene). Resveratrol has been proposed as a potential anti-metabolic disorder compound, by its activation of sirtuin and AMP-activated protein kinase. Many reports show that resveratrol ameliorates diet-induced obesity and insulin resistance. However, it is not known whether piceatannol also affects diet-induced obesity. We explored the effect of piceatannol on high fat diet-fed mice. The results showed that piceatannol did not affect high fat diet-induced body weight gain or visceral fat gain in mice. However, piceatannol did reduce fasting blood glucose levels. Furthermore, to explore the potential of passion fruit seed extract containing piceatannol as a functional food, passion fruit seed extract was administered in a genetic diabetic mouse model (db/db mice). Single administration of passion fruit seed extract, as well as piceatannol reduced the blood glucose levels of these db/db mice. These results suggest that piceatannol and passion fruit seed extract may have potential application in the prevention of diabetes.

Piceatannol and its metabolite, isorhapontigenin, induce SIRT1 expression in THP-1 human monocytic cell line.

Piceatannol is a phytochemical that is present in large amounts in passion fruit (Passiflora edulis) seeds, and is an analog of resveratrol. Recently, the absorption and metabolism of piceatannol were investigated in rats, and isorhapontigenin, O-methyl piceatannol, was detected as a piceatannol metabolite in rat plasma. To elucidate the function of piceatannol and its metabolites, we investigated the expression of sirtuin 1 (SIRT1) in THP-1 monocytic cells after treatment with piceatannol and its metabolites, and compared their effects with those of resveratrol and its metabolites. Piceatannol and resveratrol upregulated the expression levels of SIRT1 mRNA and SIRT1 protein. An extract of passion fruit seeds, which contained high levels of piceatannol, also upregulated SIRT1 mRNA expression. As for the metabolites, isorhapontigenin upregulated SIRT1 mRNA expression, whereas resveratrol glucuronides and sulfate did not affect SIRT1 expression. These findings indicate that after intake of piceatannol, not only piceatannol itself, but also its metabolite, isorhapontigenin, contributed to the upregulation of SIRT1 expression.

The protective effects of piceatannol from passion fruit (Passiflora edulis) seeds in UVB-irradiated keratinocytes.

The use of naturally occurring botanicals with substantial antioxidant activity to prevent photoageing is receiving increasing attention. We have previously identified piceatannol and scirpusin B, which is a dimer of piceatannol, as strong antioxidants that are present in passion fruit (Passiflora edulis) seeds. In the present study, the effects of passion fruit seed extract, piceatannol, and scirpusin B on human keratinocytes were investigated. The passion fruit seed extract and piceatannol upregulated the glutathione (GSH) levels in keratinocytes in a dose-dependent manner, indicating that piceatannol is an active component of the passion fruit seed extract in keratinocytes. The pretreatment with piceatannol also suppressed the UVB-induced generation of reactive oxygen species (ROS) in the keratinocytes. In addition, the transfer of the medium from the UVB-irradiated keratinocytes to non-irradiated fibroblasts enhanced matrix-metalloproteinase (MMP)-1 activity, and this MMP-1 induction was reduced when the keratinocytes were pretreated with piceatannol. These results suggest that piceatannol attenuates the UVB-induced activity of MMP-1 along with a reduction of ROS generation in keratinocytes. Thus, piceatannol and passion fruit seed extract containing high amounts of piceatannol are potential anti-photoageing cosmetic ingredients.