ABSTRACT
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.
Subject(s)
Antiprotozoal Agents , Benzofurans , Dalbergia , Leishmaniasis , Antiprotozoal Agents/pharmacology , Humans , Leishmaniasis/drug therapy , Phenols/pharmacology , Phenols/therapeutic useABSTRACT
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.
Subject(s)
Foodborne Diseases , Plants, Toxic , Humans , Japan , Polymerase Chain Reaction , Polymorphism, Restriction Fragment LengthABSTRACT
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.
ABSTRACT
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)
Subject(s)
Animals , Peptides , Bee Venoms , Biological ProductsABSTRACT
Abstract 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 -helical peptides; rich in hydrophobic and basic amino acids with no disulfide bond, and accordingly, it can be predicted to adopt an amphipathic -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 -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.
ABSTRACT
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.
ABSTRACT
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.
Subject(s)
Coumarins/chemistry , Coumarins/classification , Crataegus/chemistry , Flavanones/chemistry , Flavanones/classification , Fruit/chemistry , Coumarins/isolation & purification , Flavanones/isolation & purification , Molecular StructureABSTRACT
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.
Subject(s)
Cinnamomum/chemistry , Lactones/analysis , Monoterpenes/analysis , Plant Extracts/chemistry , Fruit/chemistry , Gas Chromatography-Mass Spectrometry , Lactones/isolation & purification , Models, Molecular , Monoterpenes/isolation & purificationABSTRACT
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).
Subject(s)
Fabaceae/chemistry , Leishmania/drug effects , Stilbenes/chemistry , Stilbenes/pharmacology , Trypanocidal Agents/chemistry , Trypanocidal Agents/pharmacology , Humans , Leishmaniasis/drug therapy , Medicine, Traditional , Plant Leaves/chemistry , Plant Stems/chemistry , Stilbenes/isolation & purification , Trypanocidal Agents/isolation & purificationABSTRACT
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.
Subject(s)
Aconitine/analysis , Aconitum/chemistry , Aconitum/poisoning , Aconitine/analogs & derivatives , Adult , Aged , Chromatography, High Pressure Liquid/methods , Fatal Outcome , Female , Heart Arrest/etiology , Humans , Male , Middle Aged , Plant Leaves/chemistry , Plant Leaves/poisoning , Shock, Cardiogenic/etiology , Tachycardia, Ventricular/etiology , Tandem Mass Spectrometry/methodsABSTRACT
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.
Subject(s)
Antiprotozoal Agents/pharmacology , Diospyros/chemistry , Leishmania major/drug effects , Naphthols/pharmacology , Naphthoquinones/pharmacology , Animals , Antiprotozoal Agents/chemistry , Antiprotozoal Agents/isolation & purification , Cell Survival/drug effects , Cells, Cultured , Crystallography, X-Ray , Dose-Response Relationship, Drug , Leishmania major/metabolism , Mice , Models, Molecular , Molecular Structure , Myanmar , Naphthols/chemistry , Naphthols/isolation & purification , Naphthoquinones/chemistry , Naphthoquinones/isolation & purification , Parasitic Sensitivity Tests , Plant Extracts/chemistry , Stereoisomerism , Structure-Activity Relationship , Wood/chemistryABSTRACT
Four novel peptides were isolated from the venoms of the solitary eumenine wasps Eumenes rubrofemoratus and Eumenes fraterculus. Their sequences were determined by MALDI-TOF/TOF (matrix assisted laser desorption/ionization time-of-flight mass spectrometry) analysis, Edman degradation and solid-phase synthesis. Two of them, eumenitin-R (LNLKGLIKKVASLLN) and eumenitin-F (LNLKGLFKKVASLLT), are highly homologous to eumenitin, an antimicrobial peptide from a solitary eumenine wasp, whereas the other two, EMP-ER (FDIMGLIKKVAGAL-NH(2)) and EMP-EF (FDVMGIIKKIAGAL-NH(2)), are similar to eumenine mastoparan-AF (EMP-AF), a mast cell degranulating peptide from a solitary eumenine wasp. These sequences have the characteristic features of linear cationic cytolytic peptides; rich in hydrophobic and basic amino acids with no disulfide bond, and accordingly, they can be predicted to adopt an amphipathic α-helix secondary structure. In fact, the CD (circular dichroism) spectra of these peptides showed significant α-helical conformation content in the presence of TFE (trifluoroethanol), SDS (sodium dodecylsulfate) and asolectin vesicles. In the biological evaluation, all the peptides exhibited a significant broad-spectrum antimicrobial activity, and moderate mast cell degranulation and leishmanicidal activities, but showed virtually no hemolytic activity.
Subject(s)
Peptides/pharmacology , Venoms/pharmacology , Wasps/metabolism , Amino Acid Sequence , Animals , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Cations/chemistry , Circular Dichroism , Mass Spectrometry , Molecular Sequence Data , Peptides/chemistry , Peptides/isolation & purification , Protein Structure, Secondary , Venoms/chemistry , Wasps/chemistryABSTRACT
Four novel peptides were isolated from the venoms of the solitary eumeninewasps Eumenes rubrofemoratus and Eumenes fraterculus. Their sequences were determined by MALDI-TOF/ TOF (matrix assisted laser desorption/ionization time-of-flight mass spectrometry)analysis, Edman degradation and solid-phase synthesis. Two of them, eumenitin-R (LNLKGLIKKVASLLN) and eumenitin-F (LNLKGLFKKVASLLT), are highly homologous to eumenitin, an antimicrobial peptide from a solitary eumeninewasp, whereas the other two, EMP-ER (FDIMGLIKKVAGAL-NH2) and EMP-EF (FDVMGIIKKIAGAL-NH2), are similar to eumenine mastoparan-AF (EMP-AF), a mast cell degranulating peptide from a solitary eumeninewasp. These sequences have the characteristic features of linear cationic cytolyticpeptides; rich in hydrophobic and basic amino acids with no disulfide bond, and accordingly, they can be predicted to adopt an amphipathic a-helix secondary structure. In fact, the CD (circular dichroism) spectra of these peptides showed significant a-helical conformation content in the presence of TFE (trifluoroethanol), SDS (sodium dodecylsulfate) and asolectin vesicles. In the biological evaluation, all the peptides exhibited a significant broad-spectrum antimicrobial activity, and moderate mast cell degranulation and leishmanicidal activities, but showed virtually no hemolytic activity.
Subject(s)
Animals , Peptides/isolation & purification , Wasp Venoms/analysis , Wasp Venoms/toxicity , Linear Models , Products with Antimicrobial ActionABSTRACT
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.
Subject(s)
Asteraceae/chemistry , Plants, Medicinal/chemistry , Sesquiterpenes, Eudesmane/isolation & purification , Sesquiterpenes, Guaiane/isolation & purification , Molecular Structure , Nuclear Magnetic Resonance, Biomolecular , Philippines , Plant Leaves/chemistry , Sesquiterpenes, Eudesmane/chemistry , Sesquiterpenes, Guaiane/chemistryABSTRACT
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.
Subject(s)
Analgesics/isolation & purification , Analgesics/pharmacology , Sea Anemones/chemistry , Analgesics/chemistry , Animals , Cnidarian Venoms/chemical synthesis , Cnidarian Venoms/chemistry , Cnidarian Venoms/isolation & purification , Cnidarian Venoms/pharmacology , Edema/chemically induced , Edema/drug therapy , Hindlimb/drug effects , Male , Molecular Structure , Naloxone/pharmacology , Narcotic Antagonists/pharmacology , Nuclear Magnetic Resonance, Biomolecular , Rats , Rats, Wistar , Receptors, Serotonin/drug effects , Receptors, Serotonin/metabolismABSTRACT
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.
Subject(s)
Cnidarian Venoms , Cnidarian Venoms/toxicity , Cnidarian Venoms/therapeutic use , Chromatography/methods , Hemolysin Factors , NeurotoxinsABSTRACT
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.
Subject(s)
Alkaloids/pharmacology , Antiprotozoal Agents/pharmacology , Benzophenanthridines/pharmacology , Fruit/chemistry , Leishmania/drug effects , Papaveraceae/chemistry , Alkaloids/chemistry , Alkaloids/isolation & purification , Animals , Antiprotozoal Agents/chemistry , Antiprotozoal Agents/isolation & purification , Benzophenanthridines/chemistry , Benzophenanthridines/isolation & purification , Leishmania/growth & development , Molecular Structure , Parasitic Sensitivity Tests , Plant Extracts/chemistry , Plant Extracts/isolation & purification , Plant Extracts/pharmacology , StereoisomerismABSTRACT
Species identification of five Dendrobium plants was conducted using phylogenetic analysis and the validity of the method was verified. Some Dendrobium plants (Orchidaceae) have been used as herbal medicines but the difficulty in identifying their botanical origin by traditional methods prevented their full modern utilization. Based on the emerging field of molecular systematics as a powerful classification tool, a phylogenetic analysis was conducted using sequences of two plastid genes, the maturase-coding gene (matK) and the large subunit of ribulose 1,5-bisphosphate carboxylase-coding gene (rbcL), as DNA barcodes for species identification of Dendrobium plants. We investigated five medicinal Dendrobium species, Dendrobium fimbriatum, D. moniliforme, D. nobile, D. pulchellum, and D. tosaense. The phylogenetic trees constructed from matK data successfully distinguished each species from each other. On the other hand, rbcL, as a single-locus barcode, offered less species discriminating power than matK, possibly due to its being present with little variation. When results using matK sequences of D. officinale that was deposited in the DNA database were combined, D. officinale and D. tosaense showed a close genetic relationship, which brought us closer to resolving the question of their taxonomic identity. Identification of the plant source as well as the uniformity of the chemical components is critical for the quality control of herbal medicines and it is important that the processed materials be validated. The methods presented here could be applied to the analysis of processed Dendrobium plants and be a promising tool for the identification of botanical origins of crude drugs.
Subject(s)
DNA, Plant/genetics , Dendrobium/genetics , Drugs, Chinese Herbal , Endoribonucleases/genetics , Nucleotidyltransferases/genetics , Phylogeny , Ribulose-Bisphosphate Carboxylase/genetics , Drugs, Chinese Herbal/isolation & purification , Plant Leaves/genetics , Plant Stems/genetics , Sequence Analysis, DNA/methodsABSTRACT
Simple and rapid analysis of aristolochic acid (AA) in crude drugs and Kampo extracts using a solid-phase extraction method and HPLC-PDA analysis was investigated. Extraction of AA from samples was accomplished by adding methanol containing 1% ammonia. The addition of ammonia ionized the AA of acidic substances so that they adhered to an acrylamide copolymer of a strong anion exchange resin (Sep-Pak QMA) coupled to diol silica easily. Furthermore, a mixture of acetonitrile-water-phosphoric acid (75:25:2, v/v) was effective in isolating AA from its carrier. Since almost all interfering peaks originating from contaminants in crude drugs and Kampo extract formulations could be removed, a satisfactory HPLC chromatogram of AA was obtained. A good result was also obtained when Aristolochiaceae and crude drugs containing AA were tested. Particularly in the case of the medicinal parts of Asarum, several interfering peaks and a ghost peak detected near the AA peak were eliminated. The AA contents of two Kampo extract formulations, tokishigyakukagoshuyushokyoto and ryutanshakanto, were calculated by HPLC analysis. The AA content (the sum of AA-I and AA-II) was 1.25-6.13 mg per daily dose. From an additional recovery experiment for Kampo formulations, high recovery rates of AA were obtained. Neither LC/MS nor special instrumentation was necessary. Our results suggest that this simple, quick, and sensitive analytical method to detect AA in crude drugs and Kampo extract formulations would be valuable in safety inspections of AA in crude drugs and their products.