Anatomical study of secondary tuberized roots of Harpagophytum procumbens DC and quantification of harpagoside by high-performance liquid chromatography method.

AIM AND BACKGROUND: A botanical study is conducted to provide a standard diagnostic tool. In order to improve the quality assurance of the secondary tuberized roots of Harpagophytum procumbens, derived extract and phytomedicine, a simple, rapid, and accurate high-performance liquid chromatography (HPLC) method was developed to assess the harpagoside. MATERIAL AND MEHODS: This HPLC assay was performed on a reversedphase C18 column with methanol and water (50/50-V/V) as the mobile phase with a flow rate of 1.5 mL/min and using a monitoring wavelength at 278 nm. RESULTS AND CONCLUSION: This method was successfully applied to quantify these bioactive iridoid in an aqueous extract of H. procumbens and in its related phytomedicine "harpagophyton". The result demonstrated that the quantification of harpagoside, indicating that the quality control of the bioactive ingredient in H. procumbens, derived extract and phytomedicine, is critical to ensure its clinical benefits.

In vitro anthelmintic activity of the essential oils of Zanthoxylum zanthoxyloides and Newbouldia laevis against Strongyloides ratti.

The need for new anthelmintic with no chemical residues is becoming urgent. In a program aiming at the evaluation of plant as sources of new active molecules, the anthelmintic activities of the essential oils (EOs) obtained from either Zanthoxylum zanthoxyloides seeds or Newbouldia laevis leaves were evaluated against Strongyloides ratti by analyzing the results of two in vitro bioassays. These two plants and their tested parts were retained after an ethnopharmacology survey that confirmed their use by small-scale farmers for treatment of small ruminants affected by digestive helminths. The plants were harvested in Benin, and their EO were obtained by hydrodistillation. The EO yield of extraction was 0.65% (w/w) of for Z. zanthoxyloides seeds and 0.05% (w/w) for N. laevis. The chemical compositions of the two EOs were analyzed by gas chromatography coupled with mass spectrometry. The major constituents of the EO from Z. zanthoxyloides consisted of the following compounds: γ-terpinene (18 %), undecane (15 %), valencene (8.3 %), decanal (8.3 %), and 3-carene (6.7 %). In contrast, the major constituents of the EO from N. laevis leaves consisted of the following compounds: ß-caryophyllene (36 %) and eugenol (5.8 %). An egg-hatching inhibition (EHI) assay was developed and a larval migration inhibition assay was used on S. ratti to examine the effects of the EOs and to evidence their inhibitory concentrations (IC(50) and IC(90)) values on this nematode. Furthermore, the toxicity of the two EOs on Vero cell line was evaluated. When tested on S. ratti egg hatching, the two EOs resulted in similar IC(50) values (19.5 and 18.2 µg/ml for Z. zanthoxyloides and N. laevis, respectively), which were about sevenfold higher than that of the control (thiabendazole, IC(50) = 2.5 µg/ml). Larval migration was inhibited at similar concentrations for: Z. zanthoxyloides (IC(50) = 46 µg/ml), N. laevis (IC(50) = 51 µg/ml), and the control [levamisole (IC(50) = 36 µg/ml)]. No cytotoxicity was found on Vero cells because both EOs had IC(50) values higher than 50 µg/ml. Therefore, we have concluded that the EOs from two plants, used in folk medicine, may contain compounds with anthelmintic activity and could be used as improved traditional medicines or, at least, as food additives in a combined treatment for the control of helminth infections.

Amazonian plants from Peru used by Quechua and Mestizo to treat malaria with evaluation of their activity.

Indigenous Quechua and Mestizo populations from distinct areas in Loreto, Peru, were interviewed about traditional medication for the treatment of malaria. An ethnographic survey concerning the native theory of illness aetiology in the specific case of malaria permitted the elaboration of an efficient ethnopharmacological enquiry. The survey took place on three main zones corresponding to villages on the Napo and the Pastaza rivers (for the Quechua), and in the surroundings of Iquitos (for the Mestizos) and led to the collection of 14 plants. Serial extractions in hexane, dichloromethane, and methanol were performed on the different parts of the plants collected. The extracts were then tested for antiplasmodial activity in vitro. Seven plants displayed antiplasmodial activity (IC(50) from 2 to 25 microg/mL) and usually low cytotoxicity, indicating their antiplasmodial specificity. The results give scientific validation to the traditional medical knowledge of Quechua and Mestizo populations from Loreto and confirm a source of potentially active plants.

Molluscicidal activity against Bulinus truncatus of Croton campestris.

The bark of Croton campestris provided three furano-clerodane named, respectively, velamone, velamolone acetate and velamolone. Fractions containing these diterpenoids were investigated for their molluscicidal activity against Bulinus truncatus, one of the aquatic snail vectors of schistosomiasis. A dichloromethanic extract of root barks, rich in furano-clerodane and molluscicidal at 20ppm was promising as natural molluscicide. Enriched fraction with velamone gave 80% mortality from 25ppm, while its LD(100) was at 50ppm. The LD(80) was ca. at 45ppm, while LD(100) was at 60ppm for the enriched fraction containing velamolone acetate. The velamolone-enriched fraction was less efficient and gave a mortality of 90% at 90ppm. The pure compounds, velamone and velamolone acetate were active at 100% at 3 and 6ppm, respectively. Velamolone showed a molluscicidal activity at 20ppm.

Molluscicidal and radical scavenging activity of quinones from the root bark of Caryopteris x clandonensis.

In the search for new molluscicidal natural products, the activity of the chloroform extract of the root barks of Caryopteris x clandonensis was tested. The LC(100) was <5 ppm. The fractionation and purification of the extract afforded 15-deoxyfuerstione, fuerstione and alpha-caryopterone as the main compounds. These compounds were tested against the snail Bulinus truncatus, an intermediate host snail of a schistosomiasis parasite and showed strong molluscicidal activity with LC(100)<4 ppm. In addition, they were found to have potent radical scavenging properties on superoxide radical.

Identification of the alkaloids of Galipea officinalis by gas chromatography-mass spectrometry.

Galipea officinalis Hancock, a Venezuelan shrubby tree which is acclaimed in folk medicine for its many healing properties, is the only species of the genus to contain tetrahydroquinoline alkaloids. A GC-MS method has been developed in order to analyse the essential oil, hexane and chloroform extracts of the trunk bark of this plant, without prior derivatisation of the alkaloidal components. A study of the MS fragmentation patterns of the components permitted the identification of five new minor quinoline alkaloids together with the known alkaloids. In addition, the method could also be used for the characterisation of alkaloids within the genus Galipea.

Constituents of Pilocarpus trachylophus.

Four flavonoids, two terpenoids and a mixture of three polyprenols have been isolated from the leaves of Pilocarpus trachylophus.

Three clerodane diterpenoids from Croton eluteria Bennett.

Three furanoid clerodanes have been isolated from the stem bark of Croton eluteria Bennett. Their structures have been established by spectroscopic methods. The compounds were named cascarillin B (7alpha-acetoxy-3,4,15,16-diepoxy-12-oxo-cleroda-13(16),14-dien-20-al), cascarillin C (7alpha-acetoxy-15,16,12,20-diepoxy-20-hydroxy-cleroda-3,4,13(16),14-triene) and cascarillin D (7alpha-acetoxy-3,4,15,16-diepoxy-cleroda-13(16),14-dien-20-al).

Iridoids from Caryopteris x clandonensis.

In continuation of our phytochemical studies on Caryopteris x clandonensis (Lamiaceae), three further iridoids were isolated from the methanolic extract of the stems. Their structures were established by 1D and 2D NMR and MS analysis as a C-6 epimer of 8-O-acetylharpagide (6-epi-8-O-acetylharpagide), a derivative of harpagide which contained the unusual feature of a 3',4' seco-glycopyranosyl moiety (clandonoside II) and a methyl cetal of 8-O-acetylharpagide aglucone hydrate named clandonensine.

Human microsomal epoxide hydrolase is the target of germander-induced autoantibodies on the surface of human hepatocytes.

Germander, a plant used in folk medicine, caused an epidemic of cytolytic hepatitis in France. In about half of these patients, a rechallenge caused early recurrence, suggesting an immunoallergic type of hepatitis. Teucrin A (TA) was found responsible for the hepatotoxicity via metabolic activation by CYP3A. In this study, we describe the presence of anti-microsomal epoxide hydrolase (EH) autoantibodies in the sera of patients who drank germander teas for a long period of time. By Western blotting and immunocytochemistry, human microsomal EH was shown to be present in purified plasma membranes of both human hepatocytes and transformed spheroplasts and to be exposed on the cell surface where affinity-purified germander autoantibodies recognized it as their autoantigen. Immunoprecipitation of EH activity by germander-induced autoantibodies confirmed this finding. These autoantibodies were not immunoinhibitory. The plasma membrane-located EH was catalytically competent and may act as target for reactive metabolites from TA. To test this hypothesis CYP3A4 and EH were expressed with human cytochrome P450 reductase and cytochrome b(5) in a "humanized" yeast strain. In the absence of EH only one metabolite was formed. In the presence of EH, two additional metabolites were formed, and a time-dependent inactivation of EH was detected, suggesting that a reactive oxide derived from TA could alkylate the enzyme and trigger an immune response. Antibodies were found to recognize TA-alkylated EH. Recognition of EH present at the surface of human hepatocytes could suggest an (auto)antibody participation in an immune cell destruction.

An antioxidant sinapic acid ester isolated from Iberis amara.

The isolation of 6-O-sinapoyl sucrose (1) from Iberis amara seeds and an evaluation of its antioxidative properties in comparison with sinapic acid and ascorbic acid are reported.

Cytochrome P450-generated reactive metabolites cause mitochondrial permeability transition, caspase activation, and apoptosis in rat hepatocytes.

Although cytochrome P-450 (CYP)-generated reactive metabolites can cause hepatocyte apoptosis, the mechanism of this effect is incompletely understood. In the present study, we assessed the hepatotoxicity of skullcap, a diterpenoid-containing herbal remedy. Male rat hepatocytes were incubated for 2 hours with skullcap diterpenoids (100 microg/mL). This treatment decreased cell glutathione and protein thiols and increased cell [Ca(2+)]. This activated Ca(2+)-dependent tissue transglutaminase, forming a cross-linked protein scaffold, and also opened the mitochondrial permeability transition pore, causing outer mitochondrial membrane rupture, increased cytosolic cytochrome c, activation of procaspase 3, internucleosomal DNA fragmentation, and ultrastructural features of apoptosis. Cell death was increased by a CYP3A inducer (dexamethasone) or a sulfur amino acid-deficient diet increasing glutathione depletion. In contrast, cell death was prevented by decreasing CYP3A activity (with troleandomycin), preventing glutathione depletion (with cysteine or cystine), blocking Ca(2+)-modulated events (with calmidazolium), preventing mitochondrial permeability transition (with cyclosporin A), or inhibiting caspase 3 (with acetyl-Asp-G u-Va-Asp-a dehyde). Both calmidazolium and cyclosporin A also prevented the increase in cytosolic cytochrome c and procaspase 3 activation. In conclusion, CYP3A activates skullcap diterpenoids into reactive metabolites that deplete cellular thiols and increase cell [Ca(2+)]. This activates Ca(2+)-dependent transglutaminase and also opens the mitochondrial permeability transition pore, causing outer mitochondrial membrane rupture, cytochrome c release, and caspase activation. Preventing mitochondrial permeability transition pore opening and/or caspase activity blocks apoptosis, showing the fundamental role of these final events in metabolite-mediated hepatotoxicity.

Direct characterization of isoquinoline alkaloids in a crude plant extract by ion-pair liquid chromatography-electrospray ionization tandem mass spectrometry: example of Eschscholtzia californica.

An ion-pair HPLC-ESI-MS-MS method has been developed for the direct and rapid characterization of isoquinoline alkaloids in a crudely purified extract of the aerial parts of Eschscholtzia californica (Papaveraceae). This plant was chosen because of its increasing use in pharmaceutical industries and because its well known alkaloid composition allows the optimization of the experimental procedure through an on-line analytical sequence. Thus, 14 isoquinoline alkaloids of different types were detected and characterized. The identities of these compounds were confirmed unambigously by their fragmentation and UV spectra obtained by LC-diode-array detection. Various experiments including tandem mass spectrometry and in-orifice collision induced dissociation were performed and prove that MS-MS is a very efficient technique to identify these compounds. An explanation for each isoquinoline alkaloid type MS-MS fragmentation pattern is proposed and indicates similar neutral and/or radical losses. The order of the fragmentation depended on the type of compound but the lost fragments were similar.

Novel quinoline alkaloid from trunk bark of Galipea officinalis.

The isolation of N-methyl-4-hydroxy-3-(2',3'-epoxyisobutyl)-2-quinolone (1), a new natural compound, and candicine (2) from Galipea officinalis trunk bark is reported. This is the first report of candicine in the genus Galipea.

Alkaloids from Galipea officinalis.

Galipea officinalis Hancock is a shrubby tree reputed in folk medicine for its many properties which may be related to its alkaloid composition. We present in this paper the structural elucidation of five quinoline alkaloids: cuspareine, cusparine, and galipine, that have been previously described, demethoxycusparine, newly isolated in this species of Galipea and a new quinoline alkaloid named galipinine which is the 3',4'-methylenedioxycuspareine.

In vitro antimalarial activity of penduline, a bisbenzylisoquinoline from Isopyrum thalictroides.

Two bisbenzylisoquinolines, tetrandrine and penduline, were purified from Isopyrum thalictroides. When tested for antimalarial activity in vitro, penduline was efficient at concentrations fivefold lower than those of tetrandrine. In highly synchronized parasite cultures, penduline mostly interfered between the 8th and the 32nd hours of the parasite cycle.

Diterpenoids from germander, an herbal medicine, induce apoptosis in isolated rat hepatocytes.

BACKGROUND & AIMS: Germander was withdrawn from the market after its use for weight control caused an epidemic of hepatitis. Its toxicity was shown to be caused by diterpenoids and their cytochrome P4503A-mediated metabolic activation into electrophilic metabolites that deplete cellular thiols. The aim of the present study was to determine the mechanisms of cell death. METHODS: Isolated rat hepatocytes were incubated for 2 hours with germander diterpenoids (100 micrograms/mL). RESULTS: Diterpenoids decreased cell glutathione, increased cytosolic [Ca2+], activated Ca(2+)-dependent tissue transglutaminase forming a cross-linked protein scaffold, and caused internucleosomal DNA fragmentation and the ultrastructural features of apoptosis. Cell death was prevented by decreasing metabolic activation (with troleandomycin), preventing depletion of glutathione (with cystine), blocking activation of Ca(2+)-modulated enzymes (with calmidazolium), or inhibiting internucleosomal DNA fragmentation (with aurintricarboxylic acid). Apoptosis was increased and diterpenoids caused overexpression of p53 and interleukin 1 beta-converting enzyme in rats treated with dexamethasone (cytochrome P4503A inducer). Apoptosis was also increased by a diet deficient in sulfur amino acids. CONCLUSIONS: The germander furano diterpenoids cause apoptosis within 2 hours in isolated rat hepatocytes. Electrophilic metabolites may stimulate apoptosis by decreasing thiols, increasing [Ca2+], and activating Ca(2+)-dependent transglutaminase and endonucleases.

Hepatotoxicity of the herbal medicine germander: metabolic activation of its furano diterpenoids by cytochrome P450 3A Depletes cytoskeleton-associated protein thiols and forms plasma membrane blebs in rat hepatocytes.

Several herbal remedies have produced hepatitis in humans. The medicinal plant, germander, was recalled after its use as an adjuvant to slimming diets resulted in an epidemic of hepatitis in France. We studied the hepatotoxicity of germander in isolated rat hepatocytes. A crude fraction containing the diverse furano diterpenoids of germander, or the purified main constituents of this fraction, teucrin A and teuchamaedryn A, were hepatotoxic (correction for hepatototoxic), but not fractions containing more polar or lipophilic constituents. [3H]Teucrin A covalently bound to hepatocyte proteins. The furano diterpenoid fraction decreased cell glutathione and cytoskeleton-associated protein thiols, and led to formation of plasma membrane blebs and cell demise. Pretreatment of male rats with troleandomycin, an inhibitor of cytochrome P450 3A (CYP3A), slowed the depletion of glutathione and decreased toxicity, whereas dexamethasone, an inducer of CYP3A, had opposite effects. Female rat hepatocytes, which poorly express CYP3A, exhibited little toxicity, unless the animals were treated with dexamethasone. Feeding male rats with a sulfur amino acid-deficient diet decreased cell glutathione and enhanced toxicity, whereas supplementation of the standard diet with cystine had opposite effects. We conclude that the furano diterpenoids of germander are activated by CYP3A into electrophilic metabolites that deplete glutathione and cytoskeleton-associated protein thiols and form plasma membrane blebs. We suggest that studies in isolated hepatocytes be included in the preclinical assessment of herbal remedies.