Prediction of the Adult T-Cell Leukemia Inhibitory Activity of Blueberry Leaves/Stems Using Direct-Injection Electron Ionization-Mass Spectrometry Metabolomics.

Although Vaccinium virgatum Aiton leaves and stems inhibit adult T-cell leukemia (ATL) cells, leaves and stems can differ between individual plants and by time and location. In this study, leaf and stem components were profiled in the same individual plant using direct-injection electron ionization-mass spectrometry (DI-EI-MS) metabolomics, with the aims of analyzing the anti-ATL activity, and quantifying proanthocyanidins (PACs). Leaves, stems, and leaf/stem mixtures showed distinct and characteristic spectra. Anti-ATL activity was stronger in stems than leaves, and the PAC content was higher in stems than leaves. These data were subjected to bivariate analysis to identify the factor (m/z) responsible for the inhibitory effect of ATL based on the highest coefficient of determination (R2). The results of this DI-EI-MS metabolomics analysis suggest that among PACs contained in V. virgatum stems and leaves, the fragment ion at m/z 149 contributes significantly to anti-ATL activity.

Effective Utilization of Vaccinium virgatum Aiton Stems as Functional Materials: Major Constituent Analysis and Bioactivity Evaluation.

We previously reported that rabbit-eye blueberry (Vaccinium virgatum Aiton) leaves exhibit multiple functions. In this study, we evaluated whether V. virgatum stems can also be used as functional materials similar to leaves and clarified the major constituents and their biological activity (antioxidant activity and anti-adult T cell leukemia (ATL) activity). Water extracts of V. virgatum stems were separated into 19 fractions using a Diaion HP-20 open column. Sugars and organic acids were detected in the highly water-soluble fractions. Polyphenols and proanthocyanidin were detected in the hydrous methanol-soluble fractions. In biological activity evaluations, a difference in antioxidant activity was observed in the water-containing methanol-eluted fractions, and fractions exhibiting anti-ATL activity differed depending on cell type. These results suggest that blueberry stems, like leaves, are rich in polyphenols and exhibit antioxidant activity and inhibit ATL cell growth. In the future, aerial parts of blueberries, including stems and leaves, could be used as functional materials and/or medicinal resources.

Vaccinium virgatum Aiton Leaves Extract Suppressed Lipid Accumulation and Uric Acid Production in 3T3-L1 Adipocytes.

Blueberry (Vaccinium virgatum Aiton; Kinisato 35 Gou) leaves have recently attracted increasing attention as a useful material for the prevention of lifestyle diseases. Here, we examined the effects of the hot water extract of blueberry leaves (BLEx) on lipogenesis and uric acid production in 3T3-L1 adipocytes. The results showed that BLEx suppressed lipid accumulation and the mRNA expression of differentiation markers in 3T3-L1 adipocytes. A fractionation study showed that the highly polymerized proanthocyanidin-rich fraction was responsible for this effect. Upon maturation to adipocytes, 3T3-L1 cells produced uric acid and tumor necrosis factor-α, and hypoxia stimulated the production of uric acid and xanthine oxidoreductase activity. BLEx suppressed the production of uric acid under these conditions. Although BLEx inhibited the enzymatic activity of xanthine oxidase, this activity was observed in several fractions containing catechin, epicatechin, chlorogenic acid, rutin, and low molecular weight proanthocyanidins. Taken together, these results indicate that BLEx contains various compounds with the ability to suppress lipid accumulation and uric acid production in adipocytes.

Brazilian propolis (AF-08) inhibits collagen-induced platelet aggregation without affecting blood coagulation.

Brazilian propolis (AF-08) is a dietary supplement containing a variety of flavonoids. It is used worldwide as a folk medicine. Flavonoids and a diet of fruits and vegetables containing them have been shown to reduce the risk of cardiovascular diseases (CVDs). Most of CVDs are caused by arterial thrombus formation. A thrombus is formed by the interaction between adhesion and aggregation of platelets to damaged blood vessels and blood coagulation consisting of extrisic and intrinsic pathways. Platelet aggregation and blood coagulation are closely linked to thrombosis. Therefore, we evaluated the effectiveness of AF-08 or its component flavonoids against thrombosis by examining their inhibition of platelet aggregation and blood coagulation. Human platelet-rich plasma was incubated with serial dilutions of AF-08 for 10 min to assess its inhibitory effect on platelet aggregation caused by collagen. The inhibitory effect of AF-08 on blood coagulation was evaluated by the prothrombin time (PT) and activated partial thromboplastin time (APTT), which reflect the coagulation function of extrinsic and intrinsic pathways, respectively. AF-08 significantly inhibited collagen-induced platelet aggregation but not PT and APTT, indicating that AF-08 inhibited platelet aggregation but not blood coagulation. Among three flavonoids contained in AF-08, apigenin and chrysin obviously inhibited platelet aggregation but the inhibitory effect of kaempferol was less effective. The three flavonoids did not affect PT and APTT. The inhibitory activity of AF-08 on human platelet aggregation without affecting blood coagulation was suggested to be partially due to apigenin and chrysin. AF-08 may be effective in suppressing platelet-based arterial thrombus formation and reducing the risk of CVDs.

[Predicting the Pharmacologic Activity of Natural Materials Based on Metabolomics].

Natural materials such as crude drugs and foods are mixtures composed of various metabolites. Metabolic profiling is often used to identify possible correlations between a compound's metabolic profile and pharmacologic activity. Direct-injection electron ionization-mass spectrometry (DI-EI-MS) is a novel metabolomics method useful for characterizing biological materials. This review demonstrates the establishment of a DI-EI-MS method for metabolic profiling using several closely related lichen species: Cladonia krempelhuberi, C. gracilis, C. pseudogymnopoda, and C. ramulosa. The qualitative DI-EI-MS method was used to profile major and/or minor constituents in extracts of lichen samples. Each lichen sample could be distinguished by altering the DI-EI-MS electron energy and examining the resulting data using one-way analysis of variance. We also attempted to predict pharmacologic activity using DI-EI-MS metabolomics. Blueberry leaf extracts inhibited the proliferation of adult T-cell leukemia (ATL) cells. Blueberry leaf extracts could be distinguished by principal component analysis based on the absolute intensity of characteristic fragment ions. Twenty cultivars were categorized into four species, and the most appropriate discriminative marker m/z value for identifying each cultivar was selected statistically. Components extracted based on DI-EI-MS analyses could be used to construct a model to predict ATL cell bioactivity. These data suggest that the novel DI-EI-MS metabolomics method is suitable for identifying species of natural materials and predicting their pharmacologic activity. This approach could enhance public health by facilitating evaluations of pharmacologic activity and functionality, leading to the elimination of counterfeit products.

Quantification of three triterpenic acids in dried rosemary using HPLC-fluorescence detection and 4-(4,5-diphenyl-1H-imidazole-2-yl)benzoyl chloride derivatization.

Functional triterpenic acids such as ursolic acid (UA), oleanolic acid (OA) and betulinic acid (BA) are representative ingredients in rosemary that may have health benefits. UA, OA and BA in rosemary extracts were derivatized with 4-(4,5-diphenyl-1H-imidazole-2-yl)benzoyl chloride (DIB-Cl) and detected using HPLC-fluorescence (FL). Dried rosemary (50 mg) was ground, added to 3 ml of ethanol, sonicated for 40 min, then the sample solution was added to a mixture of 1% trimethylamine and 1 mM DIB-Cl in acetonitrile. The mixture was settled for 5 min at room temperature, then the DIB-triterpenic acid derivatives were separated using a Wakopak Handy ODS column (250 × 4.6 mm, 6 µm) eluted with 25 mM acetate buffer (pH 4.5)/methanol/acetonitrile (= 8:10:82 v/v/v%). The fluorescence intensity of the eluent was monitored at 365 (λex ) and 490 nm (λem ) and the maximum retention time of the derivatives was 30 min. Calibration curves constructed using rosemary extract spiked with standards showed good linearity (r ≥ 0.997) in the range 2.5-100 ng/ml. The detection limits at 3σ for internal BA, UA and OA peaks in rosemary extract were 0.2, 0.4 and 0.5 ng/ml, respectively. This method was used to quantify BA, UA and OA in commercially available dried rosemary products.

Direct-Injection Electron Ionization-Mass Spectrometry Metabolomics Method for Analyzing Blueberry Leaf Metabolites That Inhibit Adult T-cell Leukemia Proliferation.

Metabolic profiling is often used to identify possible correlations between a compound's metabolic profile and biological activity. Direct-injection electron ionization-mass spectrometry "fingerprinting" is useful for characterizing biological materials. We demonstrate the utility of direct-injection electron ionization-mass spectrometry for metabolic profiling using 100 different extracts of leaves from 20 blueberry cultivars collected at 5 time points from April to December 2008. A qualitative direct-injection electron ionization-mass spectrometry method was used to profile the major and/or minor constituents in the blueberry leaf extracts. Blueberry leaf extracts could be distinguished by principal component analysis based on the absolute intensity of characteristic fragment ions. Twenty cultivars were categorized into four species, and the most appropriate discriminative marker m/z value for identifying each cultivar was selected statistically. Correlated m/z values indicating the collection month were determined in the same analysis, and air temperature variance factors were extracted from score plots by principal component analysis. We previously reported that blueberry extracts inhibit the proliferation of adult T-cell leukemia cells. Leaves of Vaccinium virgatum collected in December of 2008 exhibited significantly greater inhibition of adult T-cell leukemia cell proliferation than other species. Highly bioactive cultivars or species were identified by direct-injection electron ionization-mass spectrometry metabolomics analysis of blueberry leaf extracts. The components extracted based on our direct-injection electron ionization-mass spectrometry analyses could be used to construct a model to predict anti-adult T-cell leukemia bioactivity. This is the first study to report a relationship between seasonal variation and bioactivity of natural products using a direct-injection electron ionization-mass spectrometry metabolomics method.

Establishment of a Direct-Injection Electron Ionization-Mass Spectrometry Metabolomics Method and Its Application to Lichen Profiling.

Direct-injection electron ionization-mass spectrometry (DI-EI-MS) is a multivariate analysis method useful for characterizing biological materials. We demonstrated the use of DI-EI-MS for metabolic profiling using several closely related lichen species: Cladonia krempelhuberi, C. gracilis, C. pseudogymnopoda, and C. ramulosa. The methodology involves conversion of total ion chromatograms to integrated chromatograms and assessment of reproducibility. The qualitative DI-EI-MS method was used to profile the major and/or minor constituents in extracts of lichen samples. It was possible to distinguish each lichen sample by altering the electron energy in DI-EI-MS and examining the resulting data using one-way analysis of variance. Previously undetectable peaks, which are easy to fragment could be revealed by varying the electron energy. Our results suggest that metabolic profiling using DI-EI-MS would be useful for discriminating between subgroups within the same species. This is the first study to report the use of DI-EI-MS in a metabolomics application.

Application of Mixture Analysis to Crude Materials from Natural Resources (V) [1]: Discrimination of Glycyrrhiza uralensis and G. glabra by El mass spectrometry.

The roots and stolons of some Glycyrrhiza species are used worldwide for traditional folk medicines and commercial pharmaceuticals. Phenolic constituents such as flavonoids and coumarins are medicinal and vary according to species. Therefore, species identification is important for quality analysis. In order to identify Glycyrrhiza species by chemical fingerprinting, methanol extracts of the root bark of Glycyrrhiza uralensis Fischer and G. glabra Linn6 were analyzed using EI-MS. Differences in kinds and quantity of components are reflected in complex EI-MS data and determining characteristic peaks for each species is straightforward.. The chaiacteristic peaks were determined statistically by volcano plot, a multivariate analysis method. EI-MS data of G. uralensis and G. glabra showed differential patterns, and the notable peaks in each pattern were identified. Peaks at m/z 153 and 221 are signature peaks of G. uralensis, and at 11/z 173, 309, and 324 are those of G. glabra. In conclusion, we found species-specific patterns by EI-MS that distinguish G. uralensis and G. glabra. This method based on chemical constituent patterns can be applied to identify other Glycyrrhiza species and similar natural products.

Electron Ionization Mass Spectrometry-based Metabolomics Studies of Sophoraflavescens can Identify the Geographical Origin of Root Samples.

An electron ionization mass spectrometry (EI-MS)-based metabolomic approach was applied to Sophora flavescens to identify the geographical origin of each sample. The score plot from principal component analysis using the EI-MS data showed that Japanese S. flavescens samples tended to cluster away from Chinese S. flavescens samples. Statistical techniques showed that ions arising from kurarinol and kushenol H, which we previously identified as marker molecules for Japanese S. flavescens, were characteristic of Japanese S. flavescens. Therefore, metabolomics based on EI-MS data is a valuable tool for confirming the geographical origins of S. flavescens samples. The results suggest that EI-MS-based metabolomics is suitable for the quality control of traditional medicines containing many components.

Activation of Cellular Immunity in Herpes Simplex Virus Type 1-Infected Mice by the Oral Administration of Aqueous Extract of Moringa oleifera Lam. Leaves.

Moringa oleifera Lam. is used as a nutritive vegetable and spice. Its ethanol extract has been previously shown to be significantly effective in alleviating herpetic skin lesions in mice. In this study, we evaluated the alleviation by the aqueous extract (AqMOL) and assessed the mode of its anti-herpetic action in a murine cutaneous herpes simplex virus type 1 (HSV-1) infection model. AqMOL (300 mg/kg) was administered orally to HSV-1-infected mice three times daily on days 0 to 5 after infection. AqMOL significantly limited the development of herpetic skin lesions and reduced virus titers in the brain on day 4 without toxicity. Delayed-type hypersensitivity (DTH) reaction to inactivated HSV-1 antigen was significantly stronger in infected mice administered AqMOL and AqMOL augmented interferon (IFN)-γ production by HSV-1 antigen from splenocytes of HSV-1-infected mice at 4 days post-infection. AqMOL administration was effective in elevating the ratio of CD11b(+) and CD49b(+) subpopulations of splenocytes in infected mice. As DTH is a major host defense mechanism for intradermal HSV infection, augmentation of the DTH response by AqMOL may contribute to their efficacies against HSV-1 infection. These results provided an important insights into the mechanism by which AqMOL activates cellular immunity. Copyright © 2016 John Wiley & Sons, Ltd.

Comparison of Cultivars and Seasonal Variation in Blueberry (Vaccinium Species) Leaf Extract on Adult T-Cell Leukemia Cell Line Growth Suppression.

The inhibitory effects of blueberry leaves on the proliferation of adult T-cell leukemia (ATL) cell lines have previously been reported. A comparison of blueberry leaf extracts from different cultivars and seasonal variation were investigated regarding their effects on ATL cell line proliferation. The inhibitory effects of 80% ethanol leaf extracts from different blueberry cultivars collected from April to December in 2006 or 2008 were evaluated using two ATL cell lines. The bioactivities of leaf extracts of rabbit-eye blueberry (Vaccinium virgatum Aiton; RB species), southern highbush blueberry (V. spp.; SB species), northern highbush blueberry (V. corymbosum L.; NB species), and wild blueberry (V. bracteatum Thunb.; WB species) were compared. Of these, leaves of the RB species collected in December showed a significantly stronger inhibitory effect in both cell lines than the SB, NB, or WB species. These results suggest elevated biosynthesis of ATL-preventative bioactive compounds in the leaves of the RB species before the defoliation season.

Identification of a Bioactive Compound against Adult T-cell Leukaemia from Bitter Gourd Seeds.

In our previous report, an 80% ethanol bitter gourd seed extract (BGSE) was found to suppress proliferation of adult T-cell leukemia (ATL) cell lines. The present study aimed to identify the bioactive compounds from BGSE specific against ATL. From the result of an HPLC-MS analysis, α-eleostearic acid (α-ESA) was present in BGSE at 0.68% ± 0.0022% (±SD, n = 5). In the cell proliferation test, α-ESA potently suppressed proliferation of two ATL cell lines (ED and Su9T01; IC50 = 8.9 and 29.3 µM, respectively) more than several other octadecanoic acids. However, α-ESA moderately inhibited phytohemagglutinin-activated human peripheral blood mononuclear cells (PBMC; IC50 = 31.0 µM). These results suggest that BGSE-derived α-ESA has potential as a functional food constituent because of its activity against ATL, particularly against ED cells. Moreover, α-ESA might be effective for the prevention of moderate adverse effects of ATL on normal T cells.

Efficacy of Brazilian Propolis against Herpes Simplex Virus Type 1 Infection in Mice and Their Modes of Antiherpetic Efficacies.

Ethanol extracts (AF-06, 07, and 08, 10 mg/kg) of Brazilian propolis were administered orally to cutaneously herpes simplex virus type 1 (HSV-1)-infected mice three times daily on days 0 to 6 after infection to evaluate their efficacies against HSV-1 infection and significantly limited development of herpetic skin lesions. AF-07 and 08 significantly reduced virus titers in brain and/or skin on day 4 without toxicity, but AF-08 had no anti-HSV-1 activity in vitro. AF-06 and 08 significantly enhanced delayed-type hypersensitivity (DTH) to inactivated HSV-1 antigen in infected mice. Oral AF-08-administration significantly augmented interferon (IFN)-γ production by HSV-1 antigen from splenocytes of HSV-1-infected mice, while direct exposure of splenocytes of infected mice to AF-06 significantly elevated IFN-γ production in vitro. Thus, AF-08 might have components that are active in vivo even after oral administration and those of AF-06 might be active only in vitro. Because DTH is a major host defense for intradermal HSV-1 infection, augmentation of DTH response by AF-06 or 08, directly or indirectly, respectively, may contribute to their efficacies against HSV-1 infection. In addition, AF-06 and 07 possibly contain anti-HSV-1 components contributing to their efficacies. Such biological activities of Brazilian propolis may be useful to analyze its pharmacological actions.

Inhibition of proliferation by agricultural plant extracts in seven human adult T-cell leukaemia (ATL)-related cell lines.

Adult T-cell leukaemia (ATL) is caused by human T-cell leukaemia virus type I (HTLV-I) infection and is resistant to conventional chemotherapy. We evaluated the inhibitory effects of agricultural plants on the proliferation of seven ATL-related human leukaemia cells, using three ATL cell lines (ED, Su9T01 and S1T), two human T-cell lines transformed by HTLV-I infection (HUT-102 and MT-2) and two HTLV-I-negative human T-cell acute lymphoblastic leukaemia cell lines (Jurkat and MOLT-4). A total of 52 samples of 80% ethanol extracts obtained from 30 types of agricultural plants were examined. On the basis of IC(50) values, we selected samples with greater activity than genistein, which was used as a positive control. The highest inhibitory effect was observed with extracts from leaves of Vaccinium virgatum Aiton (blueberry) on four cell lines (ED, Su9T01, HUT-102 and Jurkat); seeds of Momordica charantia L. (bitter gourd) exhibited the second highest activity. The bitter gourd seeds suppressed the proliferation of three cell lines (Su9T01, HUT-102 and Jurkat). The extracts from edible parts of Ipomea batatas LAM. (sweet potato), edible parts of Colocasia esculenta (L.) Schott (taro), skin of taro and seeds of Prunus mume Sieb. et Zucc. (mume) showed markedly greater inhibitory effects on Su9T01 than genistein. These findings suggest that ATL-preventative bioactive compounds may exist in these agricultural plants, which are considered to be functional foods.

Placental transfer of melamine and its effects on rat dams and fetuses.

In 2008, an epidemic of cases of renal failure among Chinese infants, due to melamine contamination of milk, raised international concern. Thus, numerous studies on the metabolism of melamine were broadly undertaken. However, little is known about placental transfer of melamine. In this study, the possibility of placental transfer of melamine and its effects on fetuses and pregnant dams were determined. Melamine was respectively administered at 0, 40 and 400mg/kg body weight by daily gavage from gestation day (GD) 13 to GD 20 to control (C), low melamine (LM) and high melamine (HM) groups of pregnant female F344 rats. Rats were sacrificed 30min after the last gavage. Melamine was not detected in any of the control and placental samples, or in amniotic fluid from the LM group. Plasma and fetal melamine concentrations in the HM group were significantly higher than in the LM group (P<0.01). Liver enzyme determination revealed no differences among the three groups. However, plasma creatinine, plasma uric acid and blood urea nitrogen concentrations in dams were significantly increased by melamine (P<0.05). These results show that ingested melamine affects renal function in dams and dose-dependently passes the placental barrier to reach the fetus.

Proanthocyanidin derived from the leaves of Vaccinium virgatum suppresses platelet-derived growth factor-induced proliferation of the human hepatic stellate cell line LI90.

AIM: Hepatic stellate cell (HSC) proliferation plays a pivotal role in liver fibrogenesis, and agents that suppress HSC activation, including platelet-derived growth factor (PDGF)-induced HSC proliferation, are good candidates for antifibrogenic therapies. In this report, we use the LI90 HSC line to elucidate the antifibrogenic effects of proanthocyanidin derived from the leaves of Vaccinium virgatum. METHODS: Proanthocyanidin (PAC) was extracted from the leaves of blueberry V. virgatum (BB-PAC), grape seeds (GS-PAC) and Croton lechleri (CL-PAC). These extracts were examined for their effects on PDGF-BB-induced LI90 cell proliferation and DNA synthesis. Extracellular signal-regulated kinase (ERK) and Akt phosphorylation and PDGF receptor-beta (PDGFR-beta) expression were evaluated by western blot analysis. RESULTS: BB-PAC potently suppressed PDGF-BB-induced proliferation and DNA synthesis of LI90 cells. BB-PAC also suppressed PDGF-BB-induced DNA synthesis in primary cultured rat HSC. Moreover, GS-PAC and CL-PAC suppressed PDGF-BB-induced DNA synthesis in LI90 cells. In contrast, the monomeric PAC catechin and epicatechin and dimeric PAC procyanidin B2 only slightly suppressed PDGF-BB-induced DNA synthesis. Western blot analysis showed that BB-PAC completely or partially inhibited PDGF-BB-induced ERK and Akt phosphorylation, respectively. In addition, BB-PAC partially inhibited the PDGF-BB-induced degradation of PDGFR-beta. CONCLUSION: Our results suggest that BB-PAC suppresses activated HSC by inhibiting the PDGF signaling pathway. In addition, these results provide novel findings that may facilitate the development of antifibrogenic agents.

Inhibitory effect of Cucumis sativus on melanin production in melanoma B16 cells by downregulation of tyrosinase expression.

We compared the inhibitory effects on melanogenesis of six plant parts (leaves, stems, roots, whole fruits, calyxes, and fruits without calyxes) of Cucumis sativus. MeOH extracts of leaves and stems inhibited melanin production in B16 cells. These extracts did not affect the activity of mushroom tyrosinase or crude enzyme lysate from B16 cells. However, the extracts decreased tyrosinase expression at the protein level. These results suggest that the depigmenting mechanism of extracts from leaves and stems of C. SATIVUS involves the expression of tyrosinase. Of eight compounds isolated from the leaves, lutein ( 1) (IC (50) = 170.7 microM) and (+)-(1 R,2 S,5 R,6 S)-2,6-di-(4'-hydroxyphenyl)-3,7-dioxabicyclo[3.3.0]octane ( 2) (IC (50) = 270.8 microM) were found to suppress melanogenesis. Whereas 1 was found to markedly decrease the expression levels of tyrosinase, 2 only weakly reduced tyrosinase expression. This suggests that 1 is an active component in the leaves of C. sativus and is a potentially useful skin-whitening agent.

Two new megastigmanes from the leaves of Cucumis sativus.

Two new megastigmane, cucumegastigmanes I (1) and II (2), together with a known megastigmane, (+)-dehydrovomifoliol (3), and five other known compounds were isolated from the leaves of Cucumis sativus. The structures of the new compounds were elucidated from spectroscopic analysis and their absolute stereochemistries were determined in detail using the chemical conversion and a modified Mosher's method.