[PCR-RFLP Method for Rapid Discrimination of Toxic Plants Involved in Food Poisoning].

Food poisoning caused by natural toxins, especially poisonous plants, is characterized by severe symptoms and a relatively high mortality rate. Therefore, rapid and accurate identification of the causative agent is extremely important. From plant toxin food poisoning data published by the Ministry of Health, Labour and Welfare of Japan from 1989 to 2015, we selected five plants (Veratrum spp., Datura spp., Aconitum spp., Narcissus spp. and Colchicum spp.) that are frequently involved in poisoning outbreaks, and developed a PCR-RFLP assay to discriminate them. Separation of the PCR-RFLP products by electrophoresis resulted in detection of two fragments from poisonous plants and one from edible plants. The PCR-RFLP method is rapid and straightforward and does not require expensive analytical devices. This assay was also confirmed to be applicable to cooked samples.

Novel Monoterpene Lactones from Cinnamomum inunctum.

Four new monoterpene lactones, 5-(2,3-dihydroxy-3-methylbutyl)-4-hydroxy-4-methyldihydrofuran-2(3H)-one (1), 5-(2,3-dihydroxy-3-methylbutyl)-4-methylfuran-2(5H)-one (2), 8-hydroxy-4,7,7-trimethyl-1,6-dioxaspiro[4.4]non-3-en-2-one (3) and 8-hydroxy-4,7,7-trimethyl-1,6-dioxaspiro[4.4]non-3-en-2-one (4), were isolated from the methanolic extract of the fruit of Cinnamomum inunctum, a folk medicine in Myanmar, together with a known compound, 3-hydroxy-4,4-dimethyl-4-butyrolactone (5). Their chemical structures were determined by spectral methods. Among these, 3 and 4 possessed unique spirolactone moieties.

New leishmanicidal stilbenes from a Peruvian folk medicine, Lonchocarpus nicou.

Two new stilbenes, 1 and 2, were isolated as leishmanicidal constituents from the methanolic extract of Lonchocarpus nicou leaves and stem, together with five known stilbenes and rotenoids. Their chemical structures were determined by spectral methods. Among them, the cis stilbene-type compounds 1 and 4 showed potent leishmanicidal activity (IC50: 5.5, 3.9 µg/mL), while the trans stilbene-type compounds 2 and 5, and rotenoids 6 and 7, showed moderate activities (IC50: 9.9, 12.8, 22.6, 19.6 µg/mL, respectively).

Leishmanicidal activities and cytotoxicities of bisnaphthoquinone analogues and naphthol derivatives from Burman Diospyros burmanica.

A methanol extract of the wood of Diospyros burmanica, collected in Burma (Myanmar), was found to exhibit significant activity against Leishmania major. Subsequent chromatographically resolved fractionation led to the isolation of three novel bisnaphthoquinone analogues, burmanin A, B, and C (1-3), together with nine known compounds (4-12). The structure of 1 was confirmed by X-ray crystallography, and those of 2 and 3 by spectroscopic techniques, including 1D and 2D NMR. The inhibitory activities of the isolates were evaluated against the promastigote forms of Leishmania major and the murine macrophage-like cell line, RAW264.7.

Sesquiterpenes from Blumea balsamifera.

Five new guaiane sesquiterpenes, blumeaenes E1 (1), E2 (2), K (3), L (4), and M (5), and one new eudesmane sesquiterpene, samboginone (6), along with three known compounds, cryptomeridiol, 3,3',5,7-tetrahydroxy-4'-methoxyflavanone, and austroinulin, were isolated from the leaves of the Philippine medicinal herb sambong, Blumea balsamifera. The absolute configuration of the new guaiane core was determined as 1S,7S,9S,10R by employing the modified Mosher's method. In the structure of 1, the absolute configuration of the epoxyangelic acid moiety was identified as 2S,3S using (R)-PGME as a chiral anisotropic auxiliary.

Bunodosine 391: an analgesic acylamino acid from the venom of the sea anemone Bunodosoma cangicum.

A new acylamino acid, bunodosine 391 (BDS 391), was isolated from the venom of the sea anemone Bunodosoma cangicum. The structure was elucidated by spectroscopic analyses (2D NMR, ESIMS/MS) and verified by its synthesis. Intraplantar injection of BDS 391 into the hind paw of a rat induced a potent analgesic effect. This effect was not altered by naloxone (an opioid receptor antagonist), but was completely reversed by methysergide (a serotonin receptor antagonist), indicating that the effect is mediated by activation of serotonin receptors.

Leishmanicidal phenolic compounds derived from Dalbergia cultrata.

Leishmaniasis is a protozoan tropical infection that is estimated to be more than 0.3 million new cases occur annually worldwide. A novel phenolic compound, cultratin A (1), was isolated as a leishmanicidal constituent from the timber of Dalbergia cultrata, along with three known neoflavanoids (2, 3, 4), two benzofurans (5, 6), and two phenolic compounds (7, 8). Their structures were determined using spectral methods. Among them, a new compound (1) and 4-(S)-methoxydalbergione (2) showed effective leishmanicidal activities (IC50: 2.0 and 2.6 µM, respectively), while compound 8 showed moderate activity (IC50: 11 µM). The cytotoxicity of compounds 1 and 2 was also weaker than that of the other compounds.

In vitro leishmanicidal activity of benzophenanthridine alkaloids from Bocconia pearcei and related compounds.

Leishmanicidal activities of benzophenanthridine alkaloids isolated from fruits of Bocconia pearcei and their derivatives were examined. Seven benzophenanthridine compounds were isolated from the methanolic extracts of B. pearcei. Among them, dihydrosanguinarine showed the most potent leishmanicidal activities (IC(50) value: 0.014 microg/ml, respectively). To examine the structure-activity relationship of the benzophenanthridine skeleton, the leishmanicidal activities for 32 synthetic samples were examined. The existence of bulky groups at the C(7)-C(8) position was found to enhance the activity. On the other hand, the bulkiness at the C(2)-C(3) position on the D-ring, a carbonyl group at C-6, substitution at C-6 and cleavage or saturation of the C(5)-C(6) bond reduced activity. A methyl group on nitrogen of the C-ring was thought to be necessary for significant activity.

Peptidomic analysis of the venom of the solitary bee Xylocopa appendiculata circumvolans.

BACKGROUND: Among the hymenopteran insect venoms, those from social wasps and bees - such as honeybee, hornets and paper wasps - have been well documented. Their venoms are composed of a number of peptides and proteins and used for defending their nests and themselves from predators. In contrast, the venoms of solitary wasps and bees have not been the object of further research. In case of solitary bees, only major peptide components in a few venoms have been addressed. Therefore, the aim of the present study was to explore the peptide component profile of the venom from the solitary bee Xylocopa appendiculata circumvolans by peptidomic analysis with using LC-MS. METHODS: A reverse-phase HPLC connected to ESI-OrbiTrap MS was used for LC-MS. On-line mass fingerprinting was made from TIC, and data-dependent tandem mass spectrometry gave MSMS spectra. A major peptide component was isolated by reverse-phase HPLC by conventional way, and its sequence was determined by Edman degradation, which was finally corroborated by solid phase synthesis. Using the synthetic specimen, biological activities (antimicrobial activity, mast cell devaluation, hemolysis, leishmanicidal activity) and pore formation in artificial lipid bilayer were evaluated. RESULTS: On-line mass fingerprinting revealed that the crude venom contained 124 components. MS/MS analysis gave 75 full sequences of the peptide components. Most of these are related to the major and novel peptide, xylopin. Its sequence, GFVALLKKLPLILKHLH-NH2, has characteristic features of linear cationic α-helical peptides; rich in hydrophobic and basic amino acids with no disulfide bond, and accordingly, it can be predicted to adopt an amphipathic α-helix secondary structure. In biological evaluation, xylopin exhibited broad-spectrum antimicrobial activity, and moderate mast cell degranulation and leishmanicidal activities, but showed virtually no hemolytic activity. Additionally, the peptide was able to incorporate pores in artificial lipid bilayers of azolectin, confirming the mechanism of the cytolytic activity by pore formation in biological membranes. CONCLUSIONS: LC-ESI-MS and MS/MS analysis of the crude venom extract from a solitary bee Xylocopa appendiculata circumvolans revealed that the component profile of this venom mostly consisted of small peptides. The major peptide components, xylopin and xylopinin, were purified and characterized in a conventional manner. Their chemical and biological characteristics, belonging to linear cationic α-helical peptides, are similar to the known solitary bee venom peptides, melectin and osmin. Pore formation in artificial lipid bilayers was demonstrated for the first time with a solitary bee peptide.

In vitro leishmanicidal constituents of Millettia pendula.

The in vitro leishmanicidal constituents of Millettia pendula were examined. Two new compounds, 1 (millettilone A) and 2 (millettilone B), were isolated from the methanol extract of M. pendula, together with six known compounds: 3R-claussequinone (3), pendulone (4), secundiflorol I (5), 3,8-dihydroxy-9-methoxypterocarpan (6), 3,10-dihydroxy-7,9-dimethoxypterocarpan (7), and formononetin (8). Among these, pendulone showed the most potent leishmanicidal activity. Compound 2 was found to be a purple pigment in this heartwood. Their chemical structures were elucidated using spectral methods.

Crataegusins A and B, New Flavanocoumarins from the Dried Fruits of Crataegus pinnatifida var. major (Rosaceae).

Crataegusins A (1) and B (2), new flavanocoumarins, were isolated from the crude drug Crataegus Frictus, i.e., the dried fruits of Crataegus pinnatifida var. major..Their structures were determined by spectroscopic methods. They were unique in terms of carrying a 3-(or 4-)substituted coumarin substructure while a flavanocoumarin generally does not carry any substituents in the 2-pyron ring. They showed a significant DPPH reducing activity compared with epicatechin Their production would be biosynthetically regulated considering the results of an LC-MS analysis of the dried and fresh fruits, fruit skin, hypanthia, and leaves. Their structures led the authors to consider a hypothetical general biosynthetic pathway of the flavanocoumarins, to which a flavan-3-ol is converted through a Michael addition and successive oxidative decarboxylation or dehydration pathway.

Phenylpropanoid derivatives from edible canna, Canna edulis.

Two phenylpropanoid sucrose esters were isolated from dry rhizomes of Canna edulis Ker Gawl., along with a known phenylpropanoid sucrose ester and four known phenylpropanoids. On the basis of analysis of spectroscopic data and chemical evidence, these two phenylpropanoid sucrose esters were shown to be 3-O-p-coumaroyl-6-O-feruloyl-beta-D-fructofuranosyl 6-O-acetyl-alpha-D-glucopyranoside and 3,6-di-O-p-coumaroyl-beta-D-fructofuranosyl 6-O-acetyl-alpha-D-glucopyranoside.

Diterpenoid from Salvia greggii.

The structure of a diterpenoid, designated salvigresin, that was isolated from the aerial parts of Salvia greggii, has been confirmed by spectroscopic investigation and X-ray analysis.

Diterpenoid glucosides from Salvia greggii.

The structure of four diterpenoid glucosides, designated as salvigresides A-D (1-4), isolated from the aerial parts of Salvia greggii, have been confirmed by spectroscopic and chemical investigation.

Nine new isoxuxuarine-type triterpene dimers from Maytenus chuchuhuasca.

Nine new isoxuxuarine-type triterpene dimers, named isoxuxuarines B alpha (1), B beta (2), 7,8-dihydro-B alpha (3), C alpha (4), C beta (5), 7,8-dihydro-C alpha (6), D alpha (7), D beta (8), and 7,8-dihydro-D alpha (9), were isolated from the South American medicinal plant 'xuxuá' (Maytenus chuchuhuasca). The structures were elucidated on the basis of several spectroscopic analyses, including 2D-NMR experiments, MS spectra, and CD spectral studies. The isolations and structure elucidations of the new triterpene dimers are presented, and a rationale for the divergent formation of the regiochemical and stereochemical isomers of triterpene dimers is discussed.

Tropane alkaloids in adventitious root cultures of Physochlaina physaloides.

Adventitious root cultures of Physochlaina physaloides were established and their growth and tropane alkaloid production were investigated. Although the roots cultured in Murashige-skoog liquid medium containing 1.0-3.0 mg/l IBA grew well, the addition of lower concentrations of auxins was more suitable for alkaloid production. The best alkaloid yield was obtained with 0.01 mg/l NAA in 1/2 MS liquid medium. In addition, the alkaloid differences between the cultured roots and the regenerated plant cultivated in the soil was studied. Cultured roots in this condition showed higher level of alkaloids when compared to leaf, stem and roots from the regenerated plant (3 months in field conditions).

Tropane alkaloids in auxin-independent root cultures of Physochlaina physaloides.

Adventitious and hairy root cultures of Physochlaina physaloides were established. These roots grew well and produced high amounts of tropane alkaloids (particularly hyoscyamine and 6 beta-hydroxyhyoscyamine) in auxin-free culture medium. The effects of basal media and temperature on the growth and alkaloid production of these roots were investigated. Both root cultures produced highest amount of tropane alkaloids in B5 medium though the optimum temperature for hairy roots were lower than that for adventitious roots.

[Case of fatal aconite poisoning, and its background].

Two people out of three who accidentally ate boiled aconite leaves died in 2012. This was a typical case of aconite poisoning in Japan: Aconite (Aconitum spp.) was mistakenly collected instead of Anemone flaccida, an edible wild plant. The leaves of these plants are quite similar to each other. Chemical analyses of the aconite plant left at the scene suggested intake of a fatal amount of aconitine alkaloids by each person. The collector, who died, had missed the botanical differences between the two plants, even though he owned a wild plant guidebook. A. flaccida should be collected with its flowers in order to aid positive indentification and avoid aconite poisoning.

Peptidomic analysis of the venom of the solitary bee Xylocopa appendiculata circumvolans

Background: Among the hymenopteran insect venoms, those from social wasps and bees - such as honeybee, hornets and paper wasps - have been well documented. Their venoms are composed of a number of peptides and proteins and used for defending their nests and themselves from predators. In contrast, the venoms of solitary wasps and bees have not been the object of further research. In case of solitary bees, only major peptide components in a few venoms have been addressed. Therefore, the aim of the present study was to explore the peptide component profile of the venom from the solitary bee Xylocopa appendiculata circumvolans by peptidomic analysis with using LC-MS. Methods: A reverse-phase HPLC connected to ESI-OrbiTrap MS was used for LC-MS. On-line mass fingerprinting was made from TIC, and data-dependent tandem mass spectrometry gave MSMS spectra. A major peptide component was isolated by reverse-phase HPLC by conventional way, and its sequence was determined by Edman degradation, which was finally corroborated by solid phase synthesis. Using the synthetic specimen, biological activities (antimicrobial activity, mast cell devaluation, hemolysis, leishmanicidal activity) and pore formation in artificial lipid bilayer were evaluated. Results: On-line mass fingerprinting revealed that the crude venom contained 124 components. MS/MS analysis gave 75 full sequences of the peptide components. Most of these are related to the major and novel peptide, xylopin. Its sequence, GFVALLKKLPLILKHLH-NH2, has characteristic features of linear cationic alpha-helical peptides; rich in hydrophobic and basic amino acids with no disulfide bond, and accordingly, it can be predicted to adopt an amphipathic alpha-helix secondary structure. In biological evaluation, xylopin exhibited broad-spectrum antimicrobial activity, and moderate mast cell degranulation and leishmanicidal activities, but showed virtually no hemolytic activity. Additionally, the peptide was able to incorporate pores in artificial lipid bilayers of azolectin, confirming the mechanism of the cytolytic activity by pore formation in biological membranes. Conclusions: LC-ESI-MS and MS/MS analysis of the crude venom extract from a solitary bee Xylocopa appendiculata circumvolans revealed that the component profile of this venom mostly consisted of small peptides. The major peptide components, xylopin and xylopinin, were purified and characterized in a conventional manner. Their chemical and biological characteristics, belonging to linear cationic alpha-helical peptides, are similar to the known solitary bee venom peptides, melectin and osmin. Pore formation in artificial lipid bilayers was demonstrated for the first time with a solitary bee peptide.

Peptidomic analysis of the venom of the solitary bee Xylocopa appendiculata circumvolans

Background: Among the hymenopteran insect venoms, those from social wasps and bees - such as honeybee, hornets and paper wasps - have been well documented. Their venoms are composed of a number of peptides and proteins and used for defending their nests and themselves from predators. In contrast, the venoms of solitary wasps and bees have not been the object of further research. In case of solitary bees, only major peptide components in a few venoms have been addressed. Therefore, the aim of the present study was to explore the peptide component profile of the venom from the solitary bee Xylocopa appendiculata circumvolans by peptidomic analysis with using LC-MS. Methods: A reverse-phase HPLC connected to ESI-OrbiTrap MS was used for LC-MS. On-line mass fingerprinting was made from TIC, and data-dependent tandem mass spectrometry gave MSMS spectra. A major peptide component was isolated by reverse-phase HPLC by conventional way, and its sequence was determined by Edman degradation, which was finally corroborated by solid phase synthesis. Using the synthetic specimen, biological activities (antimicrobial activity, mast cell devaluation, hemolysis, leishmanicidal activity) and pore formation in artificial lipid bilayer were evaluated. Results: On-line mass fingerprinting revealed that the crude venom contained 124 components. MS/MS analysis gave 75 full sequences of the peptide components. Most of these are related to the major and novel peptide, xylopin. Its sequence, GFVALLKKLPLILKHLH-NH2, has characteristic features of linear cationic α-helical peptides; rich in hydrophobic and basic amino acids with no disulfide bond, and accordingly, it can be predicted to adopt an amphipathic α-helix secondary structure. In biological evaluation, xylopin exhibited broad-spectrum antimicrobial activity, and moderate mast cell degranulation and leishmanicidal activities, but showed virtually no hemolytic activity. Additionally, the peptide was able to incorporate pores in artificial lipid bilayers of azolectin, confirming the mechanism of the cytolytic activity by pore formation in biological membranes. Conclusions: LC-ESI-MS and MS/MS analysis of the crude venom extract from a solitary bee Xylocopa appendiculata circumvolans revealed that the component profile of this venom mostly consisted of small peptides. The major peptide components, xylopin and xylopinin, were purified and characterized in a conventional manner. Their chemical and biological characteristics, belonging to linear cationic α-helical peptides, are similar to the known solitary bee venom peptides, melectin and osmin. Pore formation in artificial lipid bilayers was demonstrated for the first time with a solitary bee peptide.(AU)