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.

Antiviral Effect of Ephedrine Alkaloids-Free Ephedra Herb Extract against SARS-CoV-2 In Vitro.

We report for the first time that ephedrine alkaloids-free Ephedra Herb extract (EFE) directly inhibits the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in vitro and that the addition of EFE to the culture medium before viral infection reduces virus titers in the culture supernatant of SARS-CoV-2, including those of variant strains, by more than 99%, 24 h after infection. The addition of Ephedra Herb macromolecule condensed-tannin, which is the main active ingredient responsible for the anticancer, pain suppression, and anti-influenza effects of EFE, similarly suppressed virus production in the culture supernatant by 99% before infection and by more than 90% after infection. Since EFE does not have the side effects caused by ephedrine alkaloids, such as hypertension, palpitations, and insomnia, our results showed the potential of EFE as a safe therapeutic agent against coronavirus disease 2019.

Overlooked switch from transient sedation to sustained excitement in the Biphasic effects of Ephedra Herb extract administered orally to mice.

ETHNOPHARMACOLOGICAL RELEVANCE: In our previous study, we reported that Ephedra Herb extract (EHE) increased the locomotor activity of mice in the open-field test and reduced the immobility time in the forced swim test. Ephedrine alkaloids (EAs) are thought to be responsible for the adverse effects of Ephedra Herb. However, there are no reports to verify that the adverse effects of Ephedra Herb are caused by the amount of EAs in the herb. Therefore, we investigated whether these adverse effects of EHE are caused by the amounts of ephedrine (Eph) and pseudoephedrine (Pse) in the herbal extract. In a preliminary study of the time course analysis of the open field test, we newly observed that EHE evoked switching from transient sedation to sustained excitement. AIM OF THE STUDY: We aimed to confirm whether EHE evokes switching from transient sedation to sustained excitement, investigate whether these actions of EHE are caused by the amount of ephedrine (Eph) and pseudoephedrine (Pse) in the herbal extract, and clarify the molecular mechanism of the transient sedative effect. MATERIALS AND METHODS: The locomotor activity of mice was tested using the open-field test. The immobility times were measured using a forced swim test, and the motor dysfunction in mice was tested using the rotarod test. RESULTS: EHE, Eph, and Pse induced transient motionlessness between 0 and 15 min after oral administration, however, they did not induce depression-like behavior and motor dysfunction in mice, suggesting that the motionlessness induced by EHE, Eph, or Pse resulted from sedation. The α2a adrenoceptor inhibitor, atipamezole, decreased their sedative effects. Thus, immediately after EHE administration, the transient sedative effect is mediated through the activation of the α2a adrenoreceptor by Eph and Pse. EHE and Eph increased total locomotor activity for 15-120 min after oral administration; however, Pse had no effect. Therefore, the slow-onset and sustained excitatory effects of EHE are mediated by Eph. CONCLUSIONS: We discovered for the first time that EHE evokes diphasic action by switching from transient sedation to sustained excitement. The transient sedation was evoked by the Eph and Pse in the herbal extract via activation of the α2a adrenoceptor and the sustained excitement was caused by the Eph in the herbal extract.

N-Caffeoyltryptophan enhances adipogenic differentiation in preadipocytes and improves glucose tolerance in mice.

Coffee consumption has been shown to reduce the risk of developing type 2 diabetes mellitus (T2DM) in humans; however, the exact mechanism is not completely understood. Here, we demonstrate that N-caffeoyltryptophan (CTP), an ingredient of coffee, enhances adipogenic differentiation and promotes glucose uptake into adipocytes. CTP increased lipid accumulation and adipogenic markers (PPARγ, C/EBPα, and FABP4) expression in mouse 3T3-L1 preadipocyte cell lines and primary preadipocytes. In addition, CTP promoted glucose uptake in 3T3-L1 cells. In the oral glucose tolerance test, daily administration of CTP (30 mg/kg/day, i.p.) for a week reduced blood glucose levels in mice. In 3T3-L1 cells, adipogenic differentiation and increased adipogenic markers expression induced by CTP were inhibited by U0126, a selective MEK1/2 inhibitor. Furthermore, mRNA induction of Pparg by CTP was abrogated in SIRT1 siRNA-transfected 3T3-L1 cells. These results suggest the involvement of the MEK/ERK signaling and SIRT1 in the mechanism of adipogenic function of CTP. Taken together, CTP might contribute to the reduction in postprandial glycemia and a subsequent reduction in onset risk for T2DM.

Cynandione A causes a dynamic change in SIRT1 nuclear trafficking via PKA signaling and beige adipocyte differentiation in 3T3-L1 cells.

Inducible brown-like adipocytes, also known as beige adipocytes, dissipate energy through thermogenesis. Although recent reports suggest that silent information regulator 2 homolog 1 (SIRT1) promotes beige adipocyte differentiation (beiging), the activation mechanism of SIRT1 remains unknown. Here, we report that cynandione A (CA), a major component of Cynanchum wilfordii, causes dynamic changes in SIRT1 nuclear trafficking via protein kinase cAMP-dependent (PKA) signaling and induces the beiging process in adipocyte lineage cells. SIRT1 is located in both the cytoplasm and the nucleus of 3T3-L1 cells. Using cell fractionation and RNA interference experiments, we found that the translocation of SIRT1 from the cytoplasm to the nucleus was enhanced after CA treatment and was followed by upregulation of beige adipocyte-related gene expression. Moreover, we found that CA-induced SIRT1 nuclear trafficking is dependent on the PKA signaling pathway. These results suggest a novel mechanism of CA by which PKA signaling promotes SIRT1 nuclear trafficking, which permits the docking of SIRT1 to its nuclear substrates, leading to beiging in 3T3-L1 cells.

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.

Barricyclin D1-a dimeric ellagitannin with a macrocyclic structure-and accompanying tannins from Barringtonia racemosa.

Our examination of high molecular weight polyphenolic constituents in the leaves of Barringtonia racemosa of the family Lecythidaceae uncovered 5 previously undescribed ellagitannins. One, barringtin M1 (1), among them was a hydrolysable tannin monomer, while remaining 4, barringtins D1 (2), D2 (3), D3 (4), and barricyclin D1 (5), were all dimers. Barricyclin D1 had a first macrocyclic structure formed from casuarictin (6) and tellimagrandin I (7), and the other ellagitannins had structures related to 5. Two additional known phenolics, valoneic acid dilactone (8) and schimawalin A (9), were also isolated from the leaves. These results suggested that the leaves of B. racemosa are a natural resource rich in hydrolysable tannin oligomers.

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.

[The Adverse Effects of Ephedra Herb and the Safety of Ephedrine Alkaloids-free Ephedra Herb Extract (EFE)].

Ephedra Herb is defined in the 17th edition of the Japanese Pharmacopoeia (JP) as the terrestrial stem of Ephedra sinica Stapf., Ephedra intermedia Schrenk et C.A. Meyer, or Ephedra equisetina Bunge (Ephedraceae). The stems of Ephedra Herb contain greater than 0.7% ephedrine alkaloids (ephedrine and pseudoephedrine). Despite its high effectiveness, Ephedra Herb exert several adverse effects, including palpitation, excitation, insomnia, and dysuria. Both the primary and adverse effects of Ephedra Herb have been traditionally believed to be mediated by these ephedrine alkaloids. However, our study found that several pharmacological actions of Ephedra Herb were not associated with ephedrine alkaloids. We prepared an ephedrine alkaloid-free Ephedra Herb extract (EFE) by eliminating ephedrine alkaloids from Ephedra Herb extract (EHE) using ion-exchange column chromatography. EFE exerted analgesic, anti-influenza, and anticancer activities in the same manner as EHE. Moreover, EFE did not induce adverse effects due to ephedrine alkaloids, such as excitation, insomnia, and arrhythmias, and showed no toxicity. Furthermore, we evaluated the safety of EFE in healthy volunteers. The number of adverse event cases was higher in the EHE-treated group than in the EFE-treated group, although the difference was not significant. Our evidence suggested that EFE was safer than EHE.

Structures, NMR Spectroscopic Features, and Cytotoxic Properties of Oligomeric Hellinoyl (m-GO-m-GOG)-Type Ellagitannins from the Galls of Tamarix aphylla.

Ellagitannin oligomers are large molecules habitually showing complex NMR spectra that are sometimes misinterpreted and lead to incorrect structures. Understanding the NMR spectroscopic features of a group of ellagitannins would overcome these inadequacies. In this study, investigation of the galls of Tamarix aphylla led to the isolation of three new ellagitannin oligomers, phyllagallins T1 (1), T2 (2), and Q1 (3), a known monomer nilotinin M4 (4), four known dimers, nilotinins D7 (5) and D8 (6), hirtellin B (7), and tamarixinin A (8), and a simple phenolic, dehydrotrigallic acid (9). 1D and 2D NMR, HRESI-TOFMS, and ECD experiments show that compounds 1-8 are hellinoyl-type ellagitannins. The NMR spectroscopic features of this type of ellagitannins and the reasons for the abnormal upfield shifts of glucose anomeric proton and hellinoyl moiety proton signals are established considering the experimental results as well as quantum chemical calculation on a simple hellinoyl-type monomer, phyllagallin M2. Based on these results, the NMR assignments reported previously by a different research group for bracteatinin T1 and hirtellin T3 are revised. A cytotoxicity study against human oral squamous cell carcinoma cell lines (Ca9-22, HSC-2, and HSC-4) and human mesenchymal normal oral cells (HGF, HPC, and HPLF) showed cytotoxic effects with tumor-specificity higher than 5.2, 3.0, 1.6, and 2.0 for compounds 5, 2, 9, and 3, respectively.

Analgesic Effects of Ephedra Herb Extract, Ephedrine Alkaloids-Free Ephedra Herb Extract, Ephedrine, and Pseudoephedrine on Formalin-Induced Pain.

The analgesic effect of Ephedra Herb (EH) is believed to be derived from the anti-inflammatory action of pseudoephedrine (Pse). We recently reported that ephedrine alkaloids-free EH extract (EFE) attenuates formalin-induced pain to the same level as that achieved by EH extract (EHE), which suggests that the analgesic effect of EH may not be due to ephedrine alkaloids (EAs). To examine the contribution of EAs to the analgesic effect of EH, mice were injected with formalin to induce a biphasic pain reaction (first phase, 0-5 min; second phase, 10-45 min) at various time points after oral administration of the following test drugs: ephedrine (Eph), Pse, "authentic" EHE from Tsumura & Co. (EHE-Ts), EFE, and EHE that was used as the source of EFE (EHE-To). Biphasic pain was suppressed at 30 min after administration of Eph, EHE-Ts, and EHE-To. At 6 h after administration of EFE, EHE-To, and Pse-and at 4 to 6 h after administration of EHE-Ts-only second-phase pain was suppressed; however, the effect of Pse at 6 h was not significant. These results suggested that EHE has a biphasic analgesic effect against biphasic formalin-induced pain: in the first phase of analgesia (30 min after administration), biphasic pain is suppressed by Eph; in the second phase of analgesia (4-6 h after administration), second-phase pain is alleviated by constituents other than EAs, although Pse may partially contribute to the relief of second-phase pain.