Chromatographic Evaluation and Characterization of Constituents of Sunflower Seed Extract Used as Food Additives.

Sunflower seed extract, an antioxidant agent registered on the List of Existing Food Additives in Japan, was evaluated using HPLC, and three common constituents were detected. These peaks were identified as monocaffeoylquinic acids (3-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, and 5-O-caffeoylquinic acid [chlorogenic acid]). Upon scrutinizing other components, dicaffeoylquinic acids (isochlorogenic acids; 3,4-di-O-caffeoylquinic, 3,5-di-O-caffeoylquinic, and 4,5-di-O-caffeoylquinic acids) were also identified. Structures of two newly isolated compounds were determined to be 3-O-(3S-2-oxo-3-hydroxy-indole-3-acetyl)-5-O-caffeoylquinic and 4-O-(3S-2-oxo-3-hydroxy-indole-3-acetyl)-5-O-caffeoylquinic acids. To identify the components that contribute to the antioxidant activity of sunflower seed extract, we fractionated the food additive sample solution and examined the active fractions for 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity. Monocaffeoylquinic and dicaffeoylquinic acids showed high DPPH activity, including their contribution to the antioxidant activity of this food additive. DPPH radical scavenging activity of the new compounds showed almost the same value as that of the positive control, Trolox. Therefore, the contribution of these compounds was also considered.

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-ß-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.

Characterization of phenolic constituents in hazelnut kernels.

Hazelnuts contain biologically active phenolic compounds and are widely used for their nutritional value. In this study, the phenolic compounds contained in hazelnuts were isolated from the kernels of Corylus avellana L. and investigated. Spectral analyses revealed 2 new acetophenone glycosides, characterized as 2',4',6'-trihydroxyacetophenone-4'-O-(2-O-ß-d-apiosyl)-ß-d-glucoside and 2',4',6'-trihydroxyacetophenone-4'-O-(2-O-ß-d-apiosyl-6-O-α-l-arabinosyl)-ß-d-glucoside, and 4 known compounds. Four high-molecular-mass condensed tannin fractions were detected in the water-soluble fraction of the extract, characterized as B-type procyanidin consisting of extension and terminal units. Gel permeation chromatography analyses revealed that the average molecular mass, based on the polystyrene standard, was approximately 15 000-113 000. These high-molecular-mass condensed tannin fractions were chemically characterized and exhibited different molecular weights. The fractions of high-molecular-mass condensed tannins were obtained from hazelnuts and tested for 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity. The EC50 values indicated significant activity for all the fractions.

Oenothein B, a Bioactive Ellagitannin, Activates the Extracellular Signal-Regulated Kinase 2 Signaling Pathway in the Mouse Brain.

(1) Background: Oenothein B, a cyclic dimeric ellagitannin present in various medicinal plants, has been reported to exert diverse effects that are beneficial for the treatment and prevention of diseases, including cancer and infections. We recently showed that oenothein B also functions in the brain because its oral administration to systemic inflammatory model mice reduced inflammatory responses in the brain and suppressed abnormal behavior. (2) Results: The present in vivo results demonstrated that oenothein B activated extracellular signal-regulated kinase 2 and cAMP response element-binding protein in the brain, both of which play important roles in synaptic transmission and learning/memory in the central nervous system (CNS). (3) Conclusions: These results suggest that oenothein B exerts neuroprotective effects on the CNS by not only its anti-inflammatory activity but also by enhancing neuronal signaling pathways.

Characterization of Components in Natural Products for the Evaluation of Existing Food Additives in Japan.

In Japan, existing food additives are those included in the List of Existing Food Additives specified in the Supplementary Provisions to the Law Concerning Amendments to the Food Sanitation Law and Nutrition Improvement Law. Most of the currently available food additives are natural extracts containing various ingredients. However, the characteristic and active components of existing food additives are not always properly defined due to poor characterization of the constituents of the respective raw materials. For that reason, the characteristic components of existing food additives from natural extracts have been evaluated using various methods and reported. Here we review examples of our research on the characterization of marker constituents of existing food additives from natural products.

Isolation and Characterization of Neuroprotective Components from Citrus Peel and Their Application as Functional Food.

The elderly experience numerous physiological alterations. In the brain, aging causes degeneration or loss of distinct populations of neurons, resulting in declining cognitive function, locomotor capability, etc. The pathogenic factors of such neurodegeneration are oxidative stress, mitochondrial dysfunction, inflammation, reduced energy homeostatis, decreased levels of neurotrophic factor, etc. On the other hand, numerous studies have investigated various biologically active substances in fruit and vegetables. We focused on the peel of citrus fruit to search for neuroprotective components and found that: 1) 3,5,6,7,8,3',4'-heptamethoxyflavone (HMF) and auraptene (AUR) in the peel of Kawachi Bankan (Citrus kawachiensis) exert neuroprotective effects; 2) both HMF and AUR can pass through the blood-brain barrier, suggesting that they act directly in the brain; 3) the content of AUR in the peel of K. Bankan was exceptionally high, and consequently the oral administration of the dried peel powder of K. Bankan exerts neuroprotective effects; and 4) intake of K. Bankan juice, which was enriched in AUR by adding peel paste to the raw juice, contributed to the prevention of cognitive dysfunction in aged healthy volunteers. This review summarizes our studies in terms of the isolation/characterization of HMF and AUR in K. Bankan peel, analysis of their actions in the brain, mechanisms of their actions, and trials to develop food that retains their functions.

Identification of Phenolic Constituents and Inhibitory Activity of Persimmon Calyx and Shiteito against Tumor Cell Proliferation.

The persistent calyx on the fruit of Diospyros kaki, called "Shitei" in Japanese, is reported to contain phenolic compounds including condensed tannins. In this study, we isolated and characterized a new compound, together with 26 phenolic components, from the 70% acetone extract of Shitei, with structural elucidation based on spectroscopic analyses. In addition, we confirmed the presence of condensed tannins by 13C-NMR spectra, and the weight-average molecular weight was estimated by gel permeation chromatography (GPC) analysis. Next, Shiteito, a Kampo medicine consisting of Shitei, ginger, and clove clinically used to treat chronic hiccoughs occurring in association with anticancer drug treatments, and hot-water extracts of each of its components, were analyzed by HPLC, which determined that the main ingredient in Shiteito was derived from clove. We therefore isolated the ingredients and investigated their anti-tumor cell proliferative activity, together with Shiteito and Shitei extracts. As a result, Shiteito showed weak inhibition of hepatocellular carcinoma (Hep3B) cell proliferation at a high concentration. In contrast, ellagic acid, one of the main constituents of Shiteito, showed significant cytotoxicity against Hep3B cells, and significant inhibition of gastric adenocarcinoma (AGS) cell proliferation in a concentration-dependent manner. The ethyl acetate (EtOAc) fraction of the 70% acetone extract of Shitei significantly inhibited the proliferation of colon adenocarcinoma (Caco-2) and AGS cells at low to middle concentration, while showing strong cytotoxicity against Hep3B. These data indicate that Shiteito and Shitei extracts could enhance cancer drug treatment by preventing the associated chronic hiccups, and have the potential to be adjuvant treatments as well.

Oenothein B in Eucalyptus Leaf Extract Suppresses Fructose Absorption in Caco-2 Cells.

Inhibition of fructose absorption may suppress adiposity and adiposity-related diseases caused by fructose ingestion. Eucalyptus leaf extract (ELE) inhibits intestinal fructose absorption (but not glucose absorption); however, its active compound has not yet been identified. Therefore, we evaluated the inhibitory activity of ELE obtained from Eucalyptus globulus using an intestinal fructose permeation assay with the human intestinal epithelial cell line Caco-2. The luminal sides of a cell monolayer model cultured on membrane filters were exposed to fructose with or without the ELE. Cellular fructose permeation was evaluated by measuring the fructose concentration in the medium on the basolateral side. ELE inhibited 65% of fructose absorption at a final concentration of 1 mg/mL. Oenothein B isolated from the ELE strongly inhibited fructose absorption; the inhibition rate was 63% at a final concentration of 5 µg/mL. Oenothein B did not affect glucose absorption. In contrast, the other major constituents (i.e., gallic acid and ellagic acid) showed little fructose-inhibitory activity. To our knowledge, this is the first report that oenothein B in ELE strongly inhibits fructose absorption in vitro. ELE containing oenothein B can prevent and ameliorate obesity and other diseases caused by dietary fructose consumption.

Quality Evaluation and Characterization of Fractions with Biological Activity from Ephedra Herb Extract and Ephedrine Alkaloids-Free Ephedra Herb Extract.

Previously, we reported that the c-Met inhibitory effect of Ephedra Herb extract (EHE) is derived from ingredients besides ephedrine alkaloids. Moreover, analgesic and anti-influenza activities of EHE and ephedrine alkaloids-free Ephedra Herb extract (EFE) have been reported recently. In this study, we examined the fractions containing c-Met kinase inhibitory activity from EHE and the fractions with analgesic and anti-influenza activities from EFE, and elucidated the structural characteristics of the active fractions. Significant c-Met kinase activity was observed in 30, 40, and 50% methanol (MeOH) eluate fractions obtained from water extract of EHE using Diaion HP-20 column chromatography. Similarly, 20 and 40% MeOH, and MeOH eluate fractions obtained from water extract of EFE were found to display analgesic and anti-influenza activities. Reversed phase-HPLC analysis of the active fractions commonly showed broad peaks characteristic of high-molecular mass condensed tannin. The active fractions were analyzed using 13C-NMR and decomposition reactions; the deduced structures of active components were high-molecular mass condensed tannins, which were mainly procyanidin B-type and partly procyanidin A-type, including pyrogallol- and catechol-type flavan 3-ols as extension and terminal units. HPLC and gel permeation chromatography (GPC) analyses estimated that the ratio of pyrogallol- and catechol-type was approximately 9 : 2, and the weight-average molecular weight based on the polystyrene standard was >45000. Furthermore, GPC-based analysis was proposed as the quality evaluation method for high-molecular mass condensed tannin in EHE and EFE.

Identification of the characteristic components in walnut and anti-inflammatory effect of glansreginin A as an indicator for quality evaluation.

Walnut is a nutritious food material, but only a few studies have been conducted on the mechanisms of its functions and the technique for quality evaluation. Therefore, we analyzed the components in aqueous methanol extract of walnut, and characterized 30 components, including three new compounds, glansreginin C, ellagic acid 4-O-(3'-O-galloyl)-ß-D-xyloside, and platycaryanin A methyl ester. We analyzed the extracts of other nuts using HPLC and clarified that a characteristic peak corresponding to glansreginin A was mainly observed in walnut. These results suggested that glansreginin A might be an indicator component of the quality of walnut. We then examined whether glansreginin A has neuroprotective effect, using lipopolysaccharide (LPS)-induced inflammatory model mice. The results revealed that oral administration of glansreginin A prevented LPS-induced abnormal behavior and LPS-induced hyper-activation of microglia in the hippocampus. These results suggested that glansreginin A has the ability to exert neuroprotective effect via anti-inflammation in the brain.

T-cell activation-inhibitory assay: a proposed novel method for screening caloric restriction mimetics.

Caloric restriction (CR) is a major contributor to good health and longevity. CR mimetics (CRMs) are a group of plant-derived compounds capable of inducing the benefits of CR. Since a longevity gene, SIRT1, inhibits T-cell activation and SIRT1 loss results in increased T-cell activation, we hypothesized that compounds capable of activating SIRT1 signaling can inhibit T-cell activation and function as CRMs. Thus we propose, in the present study, the application of a T-cell activation-inhibitory assay to screen candidate CRMs. Well-known CRMs, such as resveratrol, butein, and fisetin, suppressed the anti-CD3/CD28 antibody-induced activation of mouse spleen T-cells. We next randomly assessed 68 plant-derived compounds for screening novel candidate CRMs using this bioassay and found that all four compounds showing IC50 values <5 µM, such as curcumin, α-mangostin, nobiletin, and heptamethoxyflavone, have beneficial functions for health such as anti-inflammatory effect. These results suggest that the T-cell activation-inhibitory assay can be used to screen candidate CRMs.

Auraptene/Naringin-Rich Fruit Juice of Citrus kawachiensis (Kawachi Bankan) Prevents Ischemia-Induced Neuronal Cell Death in Mouse Brain through Anti-Inflammatory Responses.

Cerebral ischemia/reperfusion leads to delayed neuronal cell death, resulting in brain damage. Auraptene (AUR) and naringin (NGIN), which exert neuroprotective effects in ischemic brain, are abundant in the peel of Citrus kawachiensis. Although parts of AUR/NGIN are transited from the peel to the juice during the squeezing of this fruit, these amounts in juice might be too low to exert effects. We thus prepared the AUR/NGIN-rich fruit juice of C. kawachiensis by addition of peel paste to the raw juice. The present study revealed that orally administration of the dried powder of this AUR/NGIN-rich fruit juice (2.5 g/kg/d) for 7 d to ischemic mice significantly suppressed the ischemia-induced neuronal cell death in the hippocampus, which was coincidently with the reduction of hyperactivation of microglia and astrocytes. These results suggest that AUR/NGIN-rich juice of C. kawachiensis may possess therapeutic potential for the prevention of neurodegenerative diseases via inhibition of inflammatory processes.

Characterization of UV-Sensitive Marker Constituents of Polygala Root for TLC: Applications in Quality Control of Single Crude Drug Extract Preparations.

Polygala Root (the root of Polygala tenuifolia WILLDENOW; Japanese name "Onji"), a well-known crude drug, traditionally used as an expectorant and sedative, has been attracting increased interest in recent years owing to its newly found pharmacological effect related to neuroprotection. However, there is no specific method for identifying and estimating the quality of this crude drug in the Japanese Pharmacopoeia, 17th edition. Therefore, in order to develop a TLC-based simple and convenient identification method using characteristic chemical marker(s) for the drug and its extract products, UV-sensitive constituents of Polygala Root were first investigated. A total of 23 aromatic compounds were isolated and characterized. Two new compounds, namely, polygalaonjisides A (1) and B (2), were characterized as syringic acid 4-O-(2'-O-ß-D-apiosyl)-ß-D-glucoside and 2-O-(ß-D-glucosyl)-3'-O-benzoylsucrose, respectively. Based on these phytochemical results, a TLC method focusing on three marker spots with Rf value of approximately 0.4-0.5 due to tenuifolisides A and B and 3,6'-di-O-sinapoylsucrose was proposed as a simple and convenient test to identify Polygala Root or its single-extract products on the market. The data presented in this paper could be useful in stipulating a confirmation test to identify Polygala Root.

Neuroprotective effect of Citrus kawachiensis (Kawachi Bankan) peels, a rich source of naringin, against global cerebral ischemia/reperfusion injury in mice.

Cerebral ischemia/reperfusion is known to induce the generation of reactive oxygen species and inflammatory responses. Numerous studies have demonstrated that naringin (NGIN) has anti-oxidant and anti-inflammatory properties. We previously reported that Citrus kawachiensis contains a large quantity of NGIN in its peel. In the present study, we orally (p.o.) administered dried peel powder of C. kawachiensis to mice of a transient global ischemia model and found in the hippocampus region that it 1) suppressed neuronal cell death, 2) reversed the reduction in the level of phosphorylated calcium-calmodulin-dependent protein kinase II, 3) had the tendency to reverse the reduction in the level of glutathione, and 4) blocked excessive activation of microglia and astrocytes. These results suggested that the dried peel powder of C. kawachiensis had a neuroprotective effect against ischemic brain via anti-oxidative and anti-inflammatory effects. We also showed that these effects of the dried peel powder were more powerful than those obtained with a comparable amount of NGIN alone.

Chemical and Biological Significance of Oenothein B and Related Ellagitannin Oligomers with Macrocyclic Structure.

In 1990, Okuda et al. reported the first isolation and characterization of oenothein B, a unique ellagitannin dimer with a macrocyclic structure, from the Oenothera erythrosepala leaves. Since then, a variety of macrocyclic analogs, including trimeric-heptameric oligomers have been isolated from various medicinal plants belonging to Onagraceae, Lythraceae, and Myrtaceae. Among notable in vitro and in vivo biological activities reported for oenothein B are antioxidant, anti-inflammatory, enzyme inhibitory, antitumor, antimicrobial, and immunomodulatory activities. Oenothein B and related oligomers, and/or plant extracts containing them have thus attracted increasing interest as promising targets for the development of chemopreventive agents of life-related diseases associated with oxygen stress in human health. In order to better understand the significance of this type of ellagitannin in medicinal plants, this review summarizes (1) the structural characteristics of oenothein B and related dimers; (2) the oxidative metabolites of oenothein B up to heptameric oligomers; (3) the distribution of oenotheins and other macrocyclic analogs in the plant kingdom; and (4) the pharmacological activities hitherto documented for oenothein B, including those recently found by our laboratory.

Sansoninto as evidence-based remedial medicine for depression-like behavior.

In vitro screening methods using cultured Neuro2a cells to examine the activation (phosphorylation) of extracellular signal-regulated kinase (ERK) 1/2 and promotion of neurite outgrowth revealed that the extracts of 5 Kampo (Japanese traditional) formulations have potential as medicines for the treatment of behavioral abnormalities. Since sansoninto (SAT) extract exerted stronger effects than the other candidates tested, we investigated whether its oral administration ameliorates the pathologies of some mouse models of behavioral impairments. The results obtained suggested that SAT extract exerted anti-depression-like effects in the forced swim test, which may be mediated by the up-regulated expression of brain-derived neurotrophic factor (BDNF) in the hippocampus. They may also be mediated by the enhanced phosphorylation of the cAMP response element-binding protein (CREB) via the mitogen-activated protein kinase (MAPK) cascade and Ca2+/calmodulin-dependent protein kinase II (CaMK II) cascade, a downstream signaling cascade of the N-methyl-D-aspartate (NMDA) receptor. These results indicate that the extract of SAT has potential as a new remedial medicine in the treatment of depression-like behavior.

Survey on the Original Plant Species of Crude Drugs Distributed as Cynanchi Wilfordii Radix and Its Related Crude Drugs in the Korean and Chinese Markets.

Cynanchi Wilfordii Radix (CWR) is used in Korea as a substitute for Polygoni Multiflori Radix (PMR), which is a crude drug traditionally used in East Asian countries. Recently, the use of Cynanchi Auriculati Radix (CAR) in place of PMR and CWR has emerged a major concern in the Korean market. In Japan, PMR is permitted to be distributed as a pharmaceutical regulated by the Japanese Pharmacopoeia 17th edition (JP17). Although CWR and CAR have not traditionally been used as medicines, CWR was recently introduced as a health food. The distribution of unfamiliar CWR-containing products could lead to the misuse of original species for PMR and CWR like in Korea. To prevent this situation, the original species of plant products distributed as PMR, CWR, and CAR in the Korean and Chinese markets were surveyed and identified by their genes and components. The results revealed that all two PMR in the Korean market were misapplied as CAR, and that CAR was incorrectly used in eight of thirteen products distributed as CWR in both markets. As PMR is strictly controlled by JP17, the risk of mistaking PMR for CWR and CAR would be low in Japan. In contrast, the less stringent regulation of health food products and the present situation of misidentification of CWR in the Korean and Chinese markets could lead to unexpected health hazards. To ensure the quality and safety of crude drugs, it is important to use the information about the genes and components of these crude drugs.

Identification of Characteristic Phenolic Constituents in Mousouchiku Extract Used as Food Additives.

Mousouchiku extract is prepared from the bamboo-sheath of Phyllostachys heterocycla MITF. (Poaceae), and is registered as a food manufacturing agent in the List of Existing Food Additives in Japan. This study describes the chromatographic evaluation of characteristic components of this extract to obtain the chemical data needed for standardized specifications. We isolated 12 known compounds from this extract: 5-hydroxymethyl-2-furfural, 4-hydroxybenzoic acid, trans-p-coumaric acid, trans-ferulic acid, N,N'-diferuloylputrescine, 4'-hydroxypropiophenone, ß-arbutin, tachioside, isotachioside, 3,4'-dihydroxypropiophenone 3-O-glucoside, koaburaside, and (+)-lyoniresinol 9'-O-glucoside. Moreover, a new propiophenone glycoside, propiophenone 4'-O-(6-ß-D-xylosyl)-ß-D-glucoside (propiophenone 4'-O-primeveroside), was isolated. The structure of each isolated compound was elucidated based on NMR and MS data or direct HPLC comparisons with authentic samples. Among the isolates, (+)-lyoniresinol 9'-O-glucoside was found to be the major ingredients of the extract as observed using HPLC analysis. However, 2,6-dimethoxy-1,4-benzoquinone, which is considered the main constituent of mousouchiku extract, was only detected as a trace constituent and not isolated in this study.

Polyphenols from flowers of Magnolia coco and their anti-glycation effects.

We investigated the inhibitory effects of several plant extracts on advanced glycation end-products (AGEs) formation. Among tested samples, the flower extract of Magnolia coco showed significant inhibition of AGE formation. We isolated and characterized procyanidin oligomer and four other compounds from the flowers, and evaluated their inhibitory effects on AGE formation and the AGE-derived crosslink-cleaving activity of the isolated compounds.

Permeation of Polymethoxyflavones into the Mouse Brain and Their Effect on MK-801-Induced Locomotive Hyperactivity.

Accumulating data have indicated that citrus polymethoxyflavones (PMFs) have the ability to affect brain function. In the present study, we showed that 3,5,6,7,8,3',4'-heptamethoxy- flavone (HMF) given intraperitoneally to mice was immediately detected in the brain and that the permeability of the brain tissues to it was significantly higher than that of other citrus PMFs (nobiletin, tangeretin, and natsudaidain). The permeation of these PMFs into the brain well correlated with their abilities to suppress MK-801-induced locomotive hyperactivity, suggesting that HMF had the ability to act directly in the brain. We also obtained data suggesting that the suppressive effect of HMF on MK-801-induced locomotive hyperactivity was mediated by phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2) in the hippocampus.