Comparison of colorimetric methods for the analysis of total polyphenols in green tea extracts.

Two colorimetric methods are used to determine the total polyphenol contents of tea, namely, "the Folin-Ciocalteu method" defined by the International Organization for Standardization (ISO) and the "iron tartrate method" specified in the Standard Tables of Food Composition in Japan. In this study, we compared the Folin-Ciocalteu and iron tartrate methods using green tea extracts. When comparing the two methods, the sum of the four major catechins measured using high-performance liquid chromatography (HPLC) was regarded as the standard value. The total polyphenol contents obtained using the Folin-Ciocalteu method were closer to the HPLC value than those obtained using iron tartrate method. However, iron tartrate method is adequate if the current official method is improved, that is, our results suggest that the coefficients appropriate for common green tea varieties, as well as the degree and duration of cover cultivation, in the official iron tartrate method must be considered.

Catechol-O-methyltransferase and monoamine oxidase B inhibitory activities of Australian bee pollen.

Bee pollen is an apicultural product collected by honeybees from flower stamens and used as a functional food worldwide. In the present study, we aim to elucidate the functions of Australian bee pollen. Australian bee pollen extracts and their main components were tested for catechol-O-methyltransferase (COMT) and monoamine oxidase B (MAOB) inhibitory activities. These enzymes are key neurotransmitters involved in Parkinson's disease and depression. Myricetin (5), tricetin (6), and luteolin (7) exhibited high COMT inhibitory activities (half maximal inhibitory concentration [IC50] = 23.3, 13.8, and 47.4 µM, respectively). In contrast, 5, 7, and annulatin (8) exhibited MAOB inhibitory activities (IC50 = 89.7, 32.8, and 153 µM, respectively). Quantitative analysis via high-performance liquid chromatography revealed that 5 was abundant in Australian bee pollen extracts. Our findings suggest that 5 contributes to the COMT and MAOB inhibitory activities of Australian bee pollen.

Prenylated acetophenones from the roots of Calendula officinalis and their anti-inflammatory activity.

Calendula officinalis is a medicinal plant in the Asteraceae family, and it has a broad range of biological activities. In this study, we focused on the roots of C. officinalis, which have remarkable anti-inflammatory properties. By using a bioassay-guided fractionation approach, prenylated acetophenones 1 and 2-of which 1 was previously unknown-were isolated, and their structures were determined by spectroscopic analysis. Both compounds decreased lipopolysaccharide-stimulated NO production in J774.1 cells. This study could lead to the use of the Calendula roots as a natural source of inflammatory mediators.

Catechol-O-methyltransferase Inhibitors from Calendula officinalis Leaf.

Calendula officinalis is commonly known as marigold and its flowers are used in herbal medicines, cosmetics, perfumes, dyes, pharmaceutical preparations, and food products. However, the utility of its leaves has not been studied in depth. The purpose of the present study was to identify the major compounds in C. officinalis leaves and to determine the inhibitory properties of the isolated compounds toward human catechol-O-methyltransferase (COMT), a key neurotransmitter involved in Parkinson's disease and depression. We isolated and identified ten compounds, including two phenylpropanoids and seven flavonoids, from C. officinalis leaf extracts, of which four flavonoids were identified from C. officinalis leaves for the first time. Eight compounds exhibited COMT inhibitory activities with IC50 values of less than 100 µM. Our results indicate that compounds in C. officinalis leaves are potentially effective for preventing Parkinson's disease and depression. Thus, C. officinalis leaves may hold promise as dietary supplements.

Thermostability and catechol-O-methyltransferase inhibitory activity of acylated anthocyanins from purple yam.

The thermostability of purple yam was investigated to be used as natural colorants. In addition, the inhibitory properties of purple yam and its isolated anthocyanins toward human catechol-O-methyltransferase (COMT), a key neurotransmitter involved in Parkinson's disease and depression, were also investigated. The thermostability of purple yam was higher than that of the reference samples (purple sweet potato and purple potato). Quantitative HPLC analysis revealed that alatanin A (2) contributed to the thermostability of purple yam. Methanol extracts of purple yam exhibited the highest COMT inhibitory activity of the tested samples. Alatanin D (1) showed the highest inhibitory activity of the anthocyanins in purple yam (IC50 19 µM). This study revealed the thermostability and COMT inhibitory activity of purple yam and may lead to its use not only as a thermostable natural source of colorants, but also for the prevention and treatment of Parkinson's disease and depression.

Chemical Profiles of Korean Bee Pollens and Their Catechol-O-methyltransferase Inhibitory Activities.

Bee pollen is an apicultural product collected by honeybees from flower stamens and is consumed to help maintain a healthy diet. In this study, the chemical profiles of 11 Korean bee pollens were investigated using molecular networking analysis. This analysis elucidated the presence of two major clusters, hydroxycinnamoyl acid amides (HCAAs, molecular network 1 (MN1)) and flavonoid glycosides (MN2), in the bee pollen samples. The inhibitory properties of the bee pollens and the isolated HCAAs toward human catechol-O-methyltransferase (COMT), a key neurotransmitter involved in Parkinson's disease and depression, were determined. N1,N5,N10-(E)-tricaffeoylspermidine ((E,E,E)-1) exhibited the highest activity of the four compounds isolated, with an IC50 value 16 µM, and inhibited COMT competitively. Quantitative analysis of HCAAs showed that the amounts of N1,N10-dicaffeoyl-N5-p-coumaroylspermidine (2) and N10-caffeoyl-N1,N5-di-p-coumaroylspermidine (3) contributed to the observed differences in the COMT inhibitory activities of Korean bee pollens. This study may lead to the prevention and treatment of Parkinson's disease and depression using bee pollens.

Propolis: Chemical Constituents, Plant Origin, and Possible Role in the Prevention and Treatment of Obesity and Diabetes.

Honeybee products are not only beneficial to human health but also important to the food industry. One such product is propolis, a resinous substance that honeybees collect from certain trees and plants and store inside their hives. Although various health benefits of propolis have been reported, the chemical composition of propolis varies greatly depending on the growing region and plant origin. These differences have led to many misconceptions and conflicting research results. In this paper, we review research findings on how the growing region and plant origin of propolis affects its composition. We also discuss trends in research on the antiobesity and antidiabetes effects of propolis as well as recent findings that a major component of Brazilian green propolis modulates adipocyte function. Finally, we discuss challenges to be tackled in future research on the health benefits of propolis and share our perspective on the future of this field.

Composition of Algerian Propolis, Plant Origin, and Its Antiangiogenic Activity In Vitro.

The antiangiogenic activity of the ethanol extract of propolis collected from different regions in western Algeria was investigated using in vitro human umbilical vein endothelial cells (HUVECs). The ethanol extract with the strongest activity, i.e., Algerian propolis 1 (EEPA1), inhibited the formation of capillary networks in a dose-dependent manner (6.25-50 µg/mL) within 12 h and induced cell fragmentation of HUVECs at 50 µg/mL after treatment for 24 h. To identify the active compounds in EEAP1, a high-performance liquid chromatography (HPLC) analysis was performed, revealing that EEAP1 contains two major compounds. Both compounds were isolated by repeated column chromatography and identified as ω-hydroxyferulenol (1) and ferulenol (2), which have a coumarin structure conjugated with a farnesyl group according to NMR, high-resolution electrospray ionization mass spectroscopy, and chemical modification. Compounds 1 and 2 inhibited the tube-forming activity of HUVECs, especially 2, which exhibited a stronger antiangiogenic effect even at a low concentration of 3.31 µg/mL. Moreover, 2 suppressed the elongation and induced cell fragmentation at the same dose. The molecular changes in tube-forming HUVECs induced by 2 were found to be related to the activation of the caspase signals. To confirm the plant origin of propolis, an HPLC comparative analysis of the ethanol extracts of some plants near beekeeping areas and that of Algerian propolis (EEAP1) was performed, and similar chromatographic patterns were observed. This result suggests that the plant origin of this Algerian propolis is the resin of Ferula communis.

Promotion effect of the propolis from Jeju Island, Korea, on NGF secretion in human glioblastoma cells.

Propolis is a resinous mixture of substances collected and processed from various botanical sources by honeybees (Apis mellifera). We previously found that propolis collected on Jeju Island, located off the southern coast of Korea, originates from a single plant, Angelica keiskei KOIDZUMI (Ashitaba). A. keiskei has been well-studied as a health food and has been reported to promote nerve growth factor (NGF) production. Propolis formed from the resin of A. keiskei is expected to have a similar promotional effect on NGF production. NGF is a potential pharmacological agent for Alzheimer's disease. In this study, the effects of an ethanolic extract of propolis from Jeju Island (EEPJ) on NGF secretion and cell viability in T98G human glioblastoma cells were evaluated. Ethanolic extracts of propolis from Brazil (Baccharis type) and from Uruguay (Populus type) were also studied for comparison. We found that EEPJ significantly increased NGF secretion in the cells in a concentration-dependent manner. Furthermore, the effects of 27 compounds previously isolated from EEPJ were also evaluated. Several compounds were found to have a promotion effect on NGF secretion, and the structure-activity relationships of the compounds were considered relative to their promotional effect on NGF biosynthesis. The promotional effect of EEPJ is a characteristic biological activity that is not present with other propolis types, so the propolis from Jeju Island may have potential applications as a therapeutic candidate for Alzheimer's disease.

Carob pod polyphenols suppress the differentiation of adipocytes through posttranscriptional regulation of C/EBPß.

Obesity is a major risk factor for various chronic diseases such as diabetes, cardiovascular disease, and cancer; hence, there is an urgent need for an effective strategy to prevent this disorder. Currently, the anti-obesity effects of food ingredients are drawing attention. Therefore, we focused on carob, which has high antioxidant capacity and various physiological effects, and examined its anti-obesity effect. Carob is cultivated in the Mediterranean region, and its roasted powder is used as a substitute for cocoa powder. We investigated the effect of carob pod polyphenols (CPPs) on suppressing increases in adipose tissue weight and adipocyte hypertrophy in high fat diet-induced obesity model mice, and the mechanism by which CPPs inhibit the differentiation of 3T3-L1 preadipocytes into adipocytes in vitro. In an in vivo experimental system, we revealed that CPPs significantly suppressed the increase in adipose tissue weight and adipocyte hypertrophy. Moreover, in an in vitro experimental system, CPPs acted at the early stage of differentiation of 3T3-L1 preadipocytes and suppressed cell proliferation because of differentiation induction. They also suppressed the expression of transcription factors involved in adipocyte differentiation, thereby reducing triacylglycerol synthesis ability and triglycerol (TG) accumulation. Notably, CPPs regulated CCAAT/enhancer binding protein (C/EBP)ß, which is expressed at the early stage of differentiation, at the posttranscriptional level. These results demonstrate that CPPs suppress the differentiation of adipocytes through the posttranscriptional regulation of C/EBPß and may serve as an effective anti-obesity compound.

Phytochemical and anti-inflammatory properties of Senegalese propolis and isolated compounds.

Propolis is a chemically complex resinous product collected from various plant sources by honeybees that has been used historically a traditional folk medicine in many parts of the world. The main constituents of propolis are beeswax and plant resins. We recently obtained Senegalese propolis, which, to our knowledge, has not been previously reported. The purpose of this study was to analyze the composition of Senegalese propolis and evaluate its anti-inflammatory activity. Ten known phenolic compounds with phenanthrene or stilbene skeletons were isolated. Nitric oxide (NO) production assay revealed that Senegalese propolis suppresses lipopolysaccharide (LPS)-stimulated production of NO in J774.1 cells in a dose-dependent manner. The anti-inflammatory potency of Senegalese propolis was higher than that of other previously reported propolis. Furthermore, the eight compounds isolated from Senegalese propolis showed high anti-inflammatory activity by inhibiting the LPS-induced expression of inducible NO synthase (iNOS). These results suggest that Senegalese propolis and its components have potential applications as anti-inflammatory agents.

Chemoenzymatic synthesis of 3-ethyl-2,5-dimethylpyrazine by L-threonine 3-dehydrogenase and 2-amino-3-ketobutyrate CoA ligase/L-threonine aldolase.

Pyrazines are typically formed from amino acids and sugars in chemical reactions such as the Maillard reaction. In this study, we demonstrate that 3-ethyl-2,5-dimethylpyrazine can be produced from L-Thr by a simple bacterial operon. We conclude that EDMP is synthesized chemoenzymatically from L-Thr via the condensation reaction of two molecules of aminoacetone and one molecule of acetaldehyde. Aminoacetone is supplied by L-threonine 3-dehydrogenase using L-Thr as a substrate via 2-amino-3-ketobutyrate. Acetaldehyde is supplied by 2-amino-3-ketobutyrate CoA ligase bearing threonine aldolase activity from L-Thr when CoA was at low concentrations. Considering the rate of EDMP production, the reaction intermediate is stable for a certain time, and moderate reaction temperature is important for the synthesis of EDMP. When the precursor was supplied from L-Thr by these enzymes, the yield of EDMP was increased up to 20.2%. Furthermore, we demonstrate that this reaction is useful for synthesizing various alkylpyrazines.

Artepillin C, a Key Component of Brazilian Propolis, Induces Thermogenesis in Inguinal White Adipose Tissue of Mice through a Creatine-Metabolism-Related Thermogenic Pathway.

Induction of beige adipocytes in white adipose tissue (WAT) is a potential therapeutic target for the treatment of obesity because beige adipocytes release excess energy via uncoupling-protein-1-associated thermogenesis. In this study, we investigated how artepillin C (ArtC) promotes thermogenesis in vivo. We demonstrated that 28 day administration of ArtC (10 mg/kg of body weight) to mice significantly induced thermogenesis in beige adipocytes in inguinal WAT (iWAT) and suppressed reductions in core body temperature induced by cold exposure at 4 °C. Moreover, ArtC-induced thermogenesis in iWAT was significantly inhibited by treatment with a creatine metabolism inhibitor, and ArtC significantly upregulated the expression of creatine-metabolism-related enzymes in the thermogenic pathway. These results indicate that ArtC induces thermogenesis in beige adipocytes in iWAT, and the observed ArtC-induced thermogenesis is associated with the creatine-metabolism-related thermogenic pathway, which is characteristically observed in beige adipocytes.

Co-Administration of Curcumin and Artepillin C Induces Development of Brown-Like Adipocytes in Association with Local Norepinephrine Production by Alternatively Activated Macrophages in Mice.

Classical brown adipocytes, characterized by interscapular depots, have multilocular fat depots and are known to release excess energy. Recent studies have shown that induction of brown-like adipocytes, also referred to as beige or brite cells, in white adipose tissue (WAT) results in the release of excess energy through mitochondrial heat production via uncoupling protein 1. This has potential a therapeutic strategy for obesity and related diseases as well as classical brown adipocytes. In our previous studies, we found that artepillin C (ArtC, 10 mg/kg body weight), a characteristic constituent of Brazilian propolis, significantly induced the development of brown-like adipocytes in inguinal WAT (iWAT) of mice. Furthermore, we recently demonstrated that curcumin (Cur, 4.5 mg/kg) also significantly induced the development of brown-like adipocytes in mice. The combined administration of several food-derived factors can enhance their bioactivity and reduce their required functional doses. In this study, we showed that co-administration of Cur and ArtC at lower doses (Cur, 1.5 mg/kg; ArtC, 5 mg/kg) additively induce brown-like adipocyte development in mouse iWAT. Moreover, this induction is associated with the localized production of norepinephrine following accumulation of alternatively activated macrophages in iWAT. These findings suggest that co-administration of Cur and ArtC is significantly effective to reduce the dose and enhance the formation of brown-like adipocyte via a unique molecular mechanism.

Propolis Components from Stingless Bees Collected on South Sulawesi, Indonesia, and Their Xanthine Oxidase Inhibitory Activity.

Three new compounds, namely, 4-(4'-hydroxy-3'-methoxyphenyl)-3,5,7-trihydroxycoumarin (1) and sulawesins A (2) and B (3), were isolated from the propolis of stingless bees ( Tetragonula aff. biroi) collected on South Sulawesi, Indonesia. In addition, five known compounds, glyasperin A, broussoflavonol F, (2 S)-5,7-dihydroxy-4'-methoxy-8-prenylflavanone, (1' S)-2- trans,4- trans-abscisic acid, and (1' S)-2- cis,4- trans-abscisic acid, were identified. The structures of the new compounds were determined by a combination of methods that included mass spectrometry and NMR spectroscopy. The absolute configuration of sulawesin A (2), a new podophyllotoxin derivative, was determined by X-ray crystallography. The absolute configuration of sulawesin B (3) was also determined by the ECD calculation. 4-(4'-Hydroxy-3'-methoxyphenyl)-3,5,7-trihydroxycoumarin (1) and sulawesin A (2) were examined for xanthine oxidase (XO) inhibitory activity; 1 exhibited XO inhibitory activity, with an IC50 value of 3.9 µM.

5-Hydroxyferulic acid methyl ester isolated from wasabi leaves inhibits 3T3-L1 adipocyte differentiation.

To investigate the compounds present in wasabi leaves (Wasabia japonica Matsumura) that inhibit the adipocyte differentiation, activity-guided fractionation was performed on these leaves. 5-Hydroxyferulic acid methyl ester (1: 5-HFA ester), one of the phenylpropanoids, was isolated from wasabi leaves as a compound that inhibits the adipocyte differentiation. Compound 1 suppressed the intracellular lipid accumulation of 3T3-L1 cells without significant cytotoxicity. Gene expression analysis revealed that 1 suppressed the mRNA expression of 2 master regulators of adipocyte differentiation, PPARγ and C/EBPα. Furthermore, 1 downregulated the expression of adipogenesis-related genes, GLUT4, LPL, SREBP-1c, ACC, and FAS. Protein expression analysis revealed that 1 suppressed PPARγ protein expression. Moreover, to investigate the relationship between the structure and activity of inhibiting the adipocyte differentiation, we synthesized 12 kinds of phenylpropanoid analog. Comparison of the activity among 1 and its analogs suggested that the compound containing the substructure that possess a common functional group at the ortho position such as a catechol group exhibits the activity of inhibiting the adipocyte differentiation. Taken together, our findings suggest that 1 from wasabi leaves inhibits adipocyte differentiation via the downregulation of PPARγ.

Nymphaeol-C, a prenylflavonoid from Macaranga tanarius, suppresses the expression of fibroblast growth factor 18.

BACKGROUND: Fibroblast growth factor 18 (FGF18) is one of the key factors in human signaling pathways and has been reported to be associated with the formation of various tissues. Additionally, FGF18 has been reported to maintain the telogen stage of the hair cycle, and its over-expression has also been observed in cancer cells. HYPOTHESIS/PURPOSE: We searched for natural compounds that inhibit the expression of FGF18 expression in vitro and evaluated their inhibitory mechanisms. STUDY DESIGN: Various plant samples were screened using a luciferase assay targeting FGF18. One active compound was selected by the screening, isolated and identified. METHODS: The active compound was isolated using chromatographic techniques and identified by specific rotation measurements, LC-MS and NMR. Additionally, its inhibitory mechanism was evaluated using real-time RT-PCR and Western blotting. RESULTS: As a result of screening various plant leaf samples, Macaranga tanarius was identified as the most active plant and a prenylflavonoid nymphaeol-C was isolated as the active compound. Using real-time RT-PCR and Western blotting analysis, this compound was confirmed to strongly suppress the expression of FGF18. The compound lowered the ß-catenin level in the Wnt/ß-catenin pathway. Thus, it was suggested that nymphaeol-C suppresses the expression of FGF18 by suppressing ß-catenin expression. Additionally, the compound lowered the extracellular signal-regulated kinase1/2 (ERK1/2) phosphorylation level in the mitogen-activated protein kinase cascade (MAPK cascade). Therefore, nymphaeol-C suppressed downstream signals of FGF18 by suppressing the expression of FGF18. CONCLUSION: We isolated and identified prenylflavonoid nymphaeol-C from M. tanarius. The compound suppresses the expression of FGF18 and affects FGF18 related signals.

Frondoside A from sea cucumber and nymphaeols from Okinawa propolis: Natural anti-cancer agents that selectively inhibit PAK1 in vitro.

A sulfated saponin called "Frondoside A" (FRA) from sea cucumber and ingredients from Okinawa propolis (OP) have been previously shown to suppress the PAK1-dependent growth of A549 lung cancer as well as pancreatic cancer cells. However, the precise molecular mechanism underlying their anti-cancer action still remains to be clarified. In this study, for the first time, we found that both FRA and OP directly inhibit PAK1 in vitro in a selective manner (far more effectively than two other oncogenic kinases, LIMK and AKT). Furthermore, at least two major anti-cancer ingredients of OP, nymphaeols A and C, also directly inhibit PAK1 in vitro in a selective manner. To the best of our knowledge, FRA is the first marine compound that selectively inhibits PAK1. Likewise, these nymphaeols are the first propolis ingredients that selectively inhibit PAK1.