Umbelliferose Isolated from Cuminum cyminum L. Seeds Inhibits Antigen-Induced Degranulation in Rat Basophilic Leukemia RBL-2H3 Cells.

Cuminum cyminum L. (cumin) is an annual plant of the Umbelliferae family native to Egypt. We previously showed that the aqueous extract of cumin seeds suppresses degranulation by downregulating the activation of antigen-induced intracellular signaling molecules in rat basophilic leukemia RBL-2H3 cells. However, the active substances in the extract have not yet been identified. Accordingly, herein, we aimed to ascertain the water-soluble substances present in cumin seeds that inhibit degranulation, which led to the identification of umbelliferose, a characteristic trisaccharide present in plants of the Umbelliferae family. Our study is the first to reveal the degranulation-suppressing activity of umbelliferose, and quantification studies suggest that cumin seed powder contains 1.6% umbelliferose. Raffinose, an isomer of umbelliferose, was also found to significantly suppress antigen-induced degranulation, but less so than umbelliferose. Both umbelliferose and raffinose contain sucrose subunits in their structures, with galactose moieties bound at different sites. These differences in structure suggest that the binding of galactose to the sucrose subunit at the α1-2 bond contributes to its strong degranulation-inhibiting properties.

Discovery of stereospecific cytotoxicity of (8R,8'R)-trans-arctigenin against insect cells and structure-activity relationship on aromatic ring.

One of the arctigenin stereoisomers, (8R,8'R)-trans-form 1, showed stereospecific cytotoxicity against insect cells, Sf9 and NIAS-AeAl-2 cells. By the comparison with other stereoisomers, the most importance of the 8'R stereochemistry for the higher activities was clarified. On the other hand, the wider range of activity level among stereoisomers against cancer cells, HL-60, was not observed. The structure-activity relationship research using derivatives bearing (8R,8'R)-trans-form was performed to show the same level of activities of 3-iodo, 4-iodo, and 3,4-methylenedioxy derivatives 28, 29, and 36 as (8R,8'R)-trans-arctigenin 1. In the examination of thiono derivatives, 4-iodo thiono and 3,4-methylenedioxy thiono derivatives 66, 67 showed similar level of activities to that of (8R,8'R)-trans-arctigenin 1. The expression of ribosomal 28S rRNA gene of Sf9 cells was increased by (8R,8'R)-trans-arctigenin 1, whereas a degradation of DNA was not observed.

A simple and versatile authenticity assay of coffee products by single-nucleotide polymorphism genotyping.

Interspecific single-nucleotide polymorphisms (SNPs) in the rbcL DNA barcode have been strictly validated and adopted as a designed SNP genotyping maker to discriminate between two major coffee species, Coffea arabica and C. canephora, and to estimate the mixing ratio of DNA from C. arabica/C. canephora in this study. The SNP genotyping is applicable to not only green (unroasted) coffee beans, but also processed coffee products (roasted coffee beans and instant coffee powder), in which genomic DNA is degraded, because the genotyping developed in this study requires only 10 copies of 63-bp-long DNA fragments of rbcL gene. The authenticity assay established in this study has several advantages: a high versatility to DNA sample conditions; simple and rapid procedures (only two steps; DNA extraction and SNP genotyping); the feasibility in coffee business for practical use to prevent false advertising and provide quality control. Abbreviations: SNP: single-nucleotide polymorphism; SBS: single base substitution; ISR: intergenic spacer region; INDEL: insertion-deletion.

Cytotoxicity against HL60 Cells of Ficifolidione Derivatives with Methyl, n-Pentyl, and n-Heptyl Groups.

Ficifolidione, a natural insecticidal compound isolated from the essential oils of Myetaceae species, is a spiro phloroglucinol with an isobutyl group at the C-4 position. We found that ficifolidione showed cytotoxicity against cancer cells via apoptosis. Replacement of the isobutyl group by n-propyl group did not influence the potency, but the effect of the replacement of this group by a shorter or longer alkyl group on the biological activity remains unknown. In this study, ficifolidione derivatives with alkyl groups such as methyl, n-pentyl, and n-heptyl group-instead of the isobutyl group at the C-4 position-were synthesized to evaluate their cytotoxicity against the human promyelocytic leukaemia cell line HL60 and their insecticidal activity against mosquito larvae. The biological activities of their corresponding 4-epimers were also evaluated. As a result, the conversion of the isobutyl group to another alkyl group did not significantly influence the cytotoxicity or insecticidal activity. In HL60 cells treated with the n-heptyl-ficifolidione derivative, the activation of caspase 3/7 and the early stages of apoptosis were detected by using immunofluorescence and flow cytometric techniques, respectively, suggesting that the cytotoxicity should be induced by apoptosis even though the alkyl group was changed.

Suppressive effect of ethanol extract from mango (Mangifera indica L.) peel on IgE production in vitro and in vivo.

Immunoglobulin E (IgE) is involved in the onset of allergic reaction, and the suppression of IgE production leads to alleviation of allergic symptoms. We found that mango peel ethanol extract (MPE) significantly suppresses IgE production by human myeloma cell line U266 cells, suggesting that MPE has an anti-allergic effect by inhibiting the production of IgE. Although mangiferin is contained in mango, which suppresses IgE production by U266 cells, it was not contained in MPE. We investigated the suppressive effect of MPE in 2,4-dinitrofluorobenzene (DNFB)-induced allergic contact dermatitis model mice. The elevation of serum IgE level was significantly suppressed by oral administration of MPE. Intake of MPE also suppressed the expression level of IL-4 in the DNFB-challenged ears, suggesting that MPE suppresses the IL-4-mediated maturation into IgE-producing cells. Our findings indicate that MPE has a potential to alleviate the increase in serum IgE level that is feature of type I allergy.

Syntheses of cytotoxic novel arctigenin derivatives bearing halogen and alkyl groups on aromatic rings.

The new lignano-9,9'-lactones (α,ß-dibenzyl-γ-butyrolactone lignans), which showed the higher cytotoxicity than arctigenin, were synthesized. The well-known cytotoxic arctigenin showed activity against HL-60 cells (EC50=12µM), however, it was inactive against HeLa cells (EC50>100µM). The synthesized (3,4-dichloro, 2'-butoxy)-derivative 55 and (3,4-dichloro, 4'-butyl)-derivative 66 bearing the lignano-9,9'-lactone structures showed the EC50 values of 10µM and 9.4µM against HL-60 cells, respectively. Against HeLa cells, the EC50 value of the derivative 66 was 27µM. By comparing the activities with the corresponding 9,9'-epoxy structure (tetrahydrofuran compounds), the importance of the lactone structure of 55 and 66 for the higher activities was shown. The substituents on the aromatic ring of the lignano-9,9'-lactones affected the cytotoxicity level, observing more than 10-fold difference.

Immunomodulatory Activity of Octenyl Succinic Anhydride Modified Porang (Amorphophallus oncophyllus) Glucomannan on Mouse Macrophage-Like J774.1 Cells and Mouse Primary Peritoneal Macrophages.

Porang is a local plant of Indonesia, which has a high content of glucomannan. In this study, porang glucomannan (PG) was esterified with octenyl succinic anhydride (OSA) to enhance emulsion properties to be widely used in food industry. OSA-modified PG (OSA-PG) enhanced the phagocytosis activity of macrophage-like J774.1 cells and mouse peritoneal macrophages. In addition, OSA-PG increased the production of IL-6 and TNF-α by enhancing their gene expression. Immunoblot analysis displayed that OSA-PG tended to activate both nuclear factor-κB and mitogen-activated protein kinase cascades. Treatment of OSA-PG with polymyxin B revealed that cytokine production induced by OSA-PG was not caused by endotoxin contamination. Our findings also indicated that OSA-PG activates macrophages through not only Toll-like receptor (TLR) 4, but another receptor. Overall findings suggested that OSA-PG has a potential as an immunomodulatory food factor by stimulating macrophages.

Immunostimulatory effect of aqueous extract of Coriandrum sativum L. seed on macrophages.

BACKGROUND: Coriandrum sativum L. seed is generally used as a spice and crude drug. Although many functions of the various components in C. sativum L. seed have been reported, the immunostimulatory effect of water-soluble components in C. sativum L. seed has not been studied. In the present study, we focused on the immunostimulatory effect of C. sativum L. seed aqueous extract (CAE) on macrophages as a novel health function of C. sativum L. seed components. RESULTS: CAE significantly enhanced the production of tumor necrosis factor (TNF)-α and interleukin (IL)-6 in both RAW264.7 cells and peritoneal macrophages by enhancing the expression levels of these cytokine genes. CAE also stimulated nitric oxide (NO) production and the phagocytosis activity in RAW264.7 cells. We suggest that the activity of CAE is a result of the upregulation of mitogen-activated protein kinase and nuclear factor-κB cascades via TLR4. In addition, IL-6 production by peritoneal macrophages collected from CAE-administered mice was significantly enhanced, suggesting that CAE could stimulate macrophage activity in vivo. CONCLUSION: The findings of the present study suggest that CAE contains a novel water-soluble component with an immunostimulatory effect on macrophages. CAE would contribute to activating host defense against pathogens by stimulating the innate immunity. © 2017 Society of Chemical Industry.

Enantioselective syntheses of both enantiomers of 9'-dehydroxyimperanene and 7,8-dihydro-9'-dehydroxyimperanene and the comparison of biological activity between 9-norlignans and dihydroguaiaretic acids.

To estimate the effect of methyl group of dihydroguaiaretic acid, which shows many kinds of biological activities, on biological activity, both enantiomers of 9'-dehydroxyimperanene (5, 6) and 7,8-dihydro-9'-dehydroxyimperanene (7, 8) lacking one of the methyl groups of dihydroguaiaretic acid were synthesized. (S)-7,8-Dihydro-9'-dehydroxyimperanene (7) showed 4-6-fold higher cytotoxic activity than all stereoisomers of dihydroguaiaretic acid (2-4). The IC50 values of (S)-7,8-dihydro-9'-dehydroxyimperanene (7) against HL-60 and HeLa cells were 6.1µM and 5.6µM, respectively. Though only one of three stereoisomers of dihydroguaiaretic acid showed antibacterial activity against a gram negative bacterium, both enantiomers of 5-8 showed antibacterial activity against a gram negative bacterium. This is a Letter on biological activity of 9-norlignan, in which one of methyl groups of lignan is absent.

Immunostimulatory effect of spinach aqueous extract on mouse macrophage-like J774.1 cells and mouse primary peritoneal macrophages.

We herein report the immunostimulatory effect of spinach aqueous extract (SAE) on mouse macrophage-like J774.1 cells and mouse primary peritoneal macrophages. SAE significantly enhanced the production of interleukin (IL)-6 and tumor necrosis factor-α by both J774.1 cells and peritoneal macrophages by enhancing the expression levels of these cytokine genes. In addition, the phagocytosis activity of J774.1 cells was facilitated by SAE. Immunoblot analysis revealed that SAE activates mitogen-activated protein kinase and nuclear factor-κB cascades. It was found that SAE activates macrophages through not only TLR4, but also other receptors. The production of IL-6 was significantly enhanced by peritoneal macrophages from SAE-administered BALB/c mice, suggesting that SAE has a potential to stimulate macrophage activity in vivo. Taken together, these data indicate that SAE would be a beneficial functional food with immunostimulatory effects on macrophages.

Effect of the structure of dietary epoxylignan on its cytotoxic activity: relationship between the structure and the activity of 7,7'-epoxylignan and the introduction of apoptosis by caspase 3/7.

We compared the cytotoxic activities of dietary epoxylignans and their stereoisomers and found (-)-verrucosin, which is (7S,7'R,8R,8'R)-7,7'-epoxylignan, to be the most cytotoxic epoxylignan against HeLa cells (IC50 = 6.6 µM). On the other hand, the activity was about a factor of 10 less against HL-60. In this research on the relationship between the structure and cytotoxic activity of (-)-verrucosin 13, the 7-(4-methoxyphenyl)-7'-(3,4-dimethoxyphenyl) derivative 60, for which the activity (IC50 = 2.4 µM) is three times greater than (-)-verrucosin 13, was discovered. The induction of apoptosis by caspase 3/7 was observed upon treatment with the (-)-verrucosin derivative.

Structure-activity relationships of amide-phosphonate derivatives as inhibitors of the human soluble epoxide hydrolase.

Structure-activity relationships of amide-phosphonate derivatives as inhibitors of the human soluble epoxide hydrolase (sEH) were investigated. First, a series of alkyl or aryl groups were substituted on the carbon alpha to the phosphonate function in amide compounds to see whether substituted phosphonates can act as a secondary pharmacophore. A tert-butyl group (16) on the alpha carbon was found to yield most potent inhibition on the target enzyme. A 4-50-fold drop in inhibition was induced by other substituents such as aryls, substituted aryls, cycloalkyls, and alkyls. Then, the modification of the O-substituents on the phosphonate function revealed that diethyl groups (16 and 23) were preferable for inhibition to other longer alkyls or substituted alkyls. In amide compounds with the optimized diethylphosphonate moiety and an alkyl substitution such as adamantane (16), tetrahydronaphthalene (31), or adamantanemethane (36), highly potent inhibitions were gained. In addition, the resulting potent amide-phosphonate compounds had reasonable water solubility, suggesting that substituted phosphonates in amide inhibitors are effective for both inhibition potency on the human sEH and water solubility as a secondary pharmacophore.

Revised stereochemistry of ficifolidione and its biological activities against insects and cells.

Ficifolidione (1), a moderately active insecticidal compound from two species of Myrtaceae, and its derivatives were synthesized to evaluate their insecticidal activity. X-ray crystallographic analyses and specific rotation values of ficifolidione and its C-4 (2) demonstrated that the structure of ficifolidione differs from the reported absolute structure; that is, the C-4 configuration of ficifolidione should have an S configuration. The reported insecticidal activity of ficifolidione (1) and its C-4 epimer (2) against adult houseflies (Musca domestica), mosquito larvae (Culex pipiens), and cutworms (Spodoptera litura) was not observed. The cytotoxicities of ficifolidione and its derivatives (1-4) against four cell lines, Sf9, Colon26, HL60, and Vero, were also measured because ficifolidione has a phloroglucinol-derived moiety, a motif that is often present in the structure of cytotoxic chemicals. Compound 1 exhibited IC50 values of ca. 32, 9, 3, and 12 µM for Sf9, Colon26, HL60, and Vero cells, respectively, indicating that ficifolidione possesses selective cytotoxicity against the four cell lines. In HL60 cells treated with 1, DNA fragmentation and the activation of procaspase 3 were observed, suggesting that the cytotoxicity is induced by apoptosis.

Cytotoxic activity of butane type of 1,7-seco-2,7'-cyclolignanes and apoptosis induction by Caspase 9 and 3.

All stereoisomers of methoxybutane and fluorobutane type of 1,7-seco-2,7'-cyclolignane were synthesized and cytotoxic activities of these compounds were compared with those of all stereoisomers of butane and butanol type compounds. Both enantiomers of butane type secocyclolignane showed higher cytotoxic activity (IC50=16-20 µM) than methoxy type compounds, whereas none was observed for all the stereoisomers of butanol type secocyclolignane, however, (-)-Kadangustin J showed stereospecific cytotoxic activity (IC50=47-67 µM). Since (R)-9'-fluoro derivative 23 was most potent (IC50=19 µM) among the corresponding fluoro stereoisomers, (R)-9'-alkyl derivatives were synthesized, hydrophobic 9'-heptyl derivative 27 showing highest activity (IC50=3.7 µM against HL-60, IC50=3.1 µM against HeLa) in this experiment. Apoptosis induction caused by Caspase 3 and 9 for (R)-9'-heptyl derivative 27 was observed in the research on the mechanism. A degradation of DNA into small fragments was also shown by DNA ladder assay.

Structure-activity relationships of substituted oxyoxalamides as inhibitors of the human soluble epoxide hydrolase.

We explored both structure-activity relationships among substituted oxyoxalamides used as the primary pharmacophore of inhibitors of the human sEH and as a secondary pharmacophore to improve water solubility of inhibitors. When the oxyoxalamide function was modified with a variety of alkyls or substituted alkyls, compound 6 with a 2-adamantyl group and a benzyl group was found to be a potent sEH inhibitor, suggesting that the substituted oxyoxalamide function is a promising primary pharmacophore for the human sEH, and compound 6 can be a novel lead structure for the development of further improved oxyoxalamide or other related derivatives. In addition, introduction of substituted oxyoxalamide to inhibitors with an amide or urea primary pharmacophore produced significant improvements in inhibition potency and water solubility. In particular, the N,N,O-trimethyloxyoxalamide group in amide or urea inhibitors (26 and 31) was most effective among those tested for both inhibition and solubility. The results indicate that substituted oxyoxalamide function incorporated into amide or urea inhibitors is a useful secondary pharmacophore, and the resulting structures will be an important basis for the development of bioavailable sEH inhibitors.

Cross-species amplification from non-human primate DNAs in commercial human DNA assay kits.

Species specificity of commercial human DNA quantification kits and short tandem repeat (STR) profiling kits was examined using primate DNA samples. These samples comprised 33 individuals from eight primate species, each with gender and kinship data, including human (Homo sapiens), chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla), and orangutan (Pongo pygmaeus) of Hominidae family, and Japanese macaque (Macaca fuscata), long-tailed macaque (Macaca fascicularis), hamadryas baboon (Papio hamadryas), and savannah monkey (Chlorocebus sp.) of Cercopithecidae family. The findings revealed varying levels of cross-species amplifications in all non-human DNA samples that correlated with their evolutionary proximity to humans, both kit types. Moreover, cross-species amplification, including female DNA samples, was observed in a Y-chromosomal STR profiling kit. Additionally, species specificity differed among the commercial kits examined. The cross-species amplification data presented in this study offer valuable assistance in interpreting the results of individual human identification in forensic cases involving non-human primates.

Nobiletin suppresses adipocyte differentiation of 3T3-L1 cells by an insulin and IBMX mixture induction.

BACKGROUND: Nobiletin is a citrus flavonoid which possesses the flavone structure with six methoxy groups. Although nobiletin has been reported to display anti-inflammatory, anti-tumor, and anti-diabetes activities, its effect on adipocyte differentiation remained unclear. In the present study, we investigated the effect of nobiletin on the differentiation of 3T3-L1 preadipocytes into adipocytes. METHODS: 3T3-L1 preadipocytes were treated with nobiletin under various differentiation conditions. The effect of nobiletin on adipocyte differentiation was evaluated by oil red O staining, real-time RT-PCR, and Western blotting. RESULTS: Nobiletin significantly suppressed the differentiation of 3T3-L1 preadipocytes into adipocytes, upon induction with insulin together with a cAMP elevator such as 3-isobutyl-1-methylxanthine (IBMX), by downregulating the expression of the gene encoding peroxisome proliferator-activated receptor (PPAR) γ2. In addition, nobiletin decreased the phosphorylation of cAMP-response element-binding protein (CREB) and strongly enhanced the phophorylation of signal transducer and activator of transcription (STAT) 5. GENERAL SIGNIFICANCE: Nobiletin has a suppressive effect on the differentiation of preadipocytes into adipocytes when cells were induced with a general differentiation cocktail such as insulin, IBMX, and dexamethasone.

Structure-cytotoxic activity relationship of sesquilignan, morinol A.

The cytotoxic activities of sesquilignans, (7S,8S,7'R,8'R)- and (7R,8R,7'S,8'S)-morinol A and (7S,8S,7'S,8'S)- and (7R,8R,7'R,8'R)-morinol B were compared, showing no significant difference between stereoisomers (IC50=24-35 µM). As a next stage, the effect of substituents at 7, 7', and 7"-aromatic ring on the activity was evaluated to find out the higher activity of (7S,8S,7'R,8'R)-7,7',7"-phenyl derivative 18 (IC50=6-7 µM). In the research on the structure-activity relationship of 7"-position of (7S,8S,7'R,8'R)-7,7',7"-phenyl derivative 18, the most potent compounds were 7,7',7"-phenyl derivative 18 (IC50=6 µM) against HeLa cells. Against HL-60 cells, 7"-(4-nitrophenyl)-7,7'-phenyl derivative 33 and 7"-hexyl-7,7'-phenyl derivative 37 (IC50=5 µM) showed highest activity. We discovered the compounds showed four to sevenfold potent activity than that of natural (7S,8S,7'R,8'R)-morinol A. It was also confirmed that the 7'-benzylic hydroxy group have an important role for exhibiting activity, on the other hand, the resonance system of cinnamyl structure is not crucial for the potent activity.

Structural characterization of a modification subunit of a putative type I restriction enzyme from Vibrio vulnificus YJ016.

In multifunctional type I restriction enzymes, active methyltransferases (MTases) are constituted of methylation (HsdM) and specificity (HsdS) subunits. In this study, the crystal structure of a putative HsdM subunit from Vibrio vulnificus YJ016 (vvHsdM) was elucidated at a resolution of 1.80 Å. A cofactor-binding site for S-adenosyl-L-methionine (SAM, a methyl-group donor) is formed within the C-terminal domain of an α/ß-fold, in which a number of residues are conserved, including the GxGG and (N/D)PP(F/Y) motifs, which are likely to interact with several functional moieties of the SAM methyl-group donor. Comparison with the N6 DNA MTase of Thermus aquaticus and other HsdM structures suggests that two aromatic rings (Phe199 and Phe312) in the motifs that are conserved among the HsdMs may sandwich both sides of the adenine ring of the recognition sequence so that a conserved Asn residue (Asn309) can interact with the N6 atom of the target adenine base (a methyl-group acceptor) and locate the target adenine base close to the transferred SAM methyl group.

Biologically active ester derivatives as potent inhibitors of the soluble epoxide hydrolase.

Substituted ureas with a carboxylic acid ester as a secondary pharmacophore are potent soluble epoxide hydrolase (sEH) inhibitors. Although the ester substituent imparts better physical properties, such compounds are quickly metabolized to the corresponding less potent acids. Toward producing biologically active ester compounds, a series of esters were prepared and evaluated for potency on the human enzyme, stability in human liver microsomes, and physical properties. Modifications around the ester function enhanced in vitro metabolic stability of the ester inhibitors up to 32-fold without a decrease in inhibition potency. Further, several compounds had improved physical properties.