Development of an alternative method for determination of terpene lactones in ginkgo dry extract.

A new liquid chromatographic mass spectrometric (LC-MS) method for determination of terpene lactones in ginkgo dry extract has been developed. The new method has several advantages over the existing European Pharmacopoeia (Ph. Eur.) method for analysis of terpene lactones in ginkgo dry extract, the major ones being a very simple sample pre-treatment and an excellent selectivity. 5 terpene lactones were analysed with a precision expressed as relative standard deviation (RSD) of 0.4-3.1% and a mean relative error (RE) within +/-4.6%. The method was used to analyse 9 samples of ginkgo dry extracts from 3 different extract producers. The content of bilobalide was found to be in the range of 2.6-3.4% in all samples, whereas the sum of ginkgolides A, B and C was found to be in the range of 3.0-3.6%. Ginkgolide J was found in the range of 0.3-0.6%.

Key role of glutamic acid for the cytotoxic activity of the cyclotide cycloviolacin O2.

Cyclotides are cyclic plant proteins with potent cytotoxic effects. Here we systematically probed the importance of surface-exposed charged amino acid residues of the cyclotide cycloviolacin O2, using a strategy involving chemical modifications. We show that the single glutamic acid plays a key role for the cytotoxicity: methylation of this residue produced a 48-fold decrease in potency. Virtually no change in potency was observed when masking the single arginine residue using 1,2-cyclohexanedione, while acetylation of the two lysine residues reduced the potency 3-fold. The derivative with modifications at both arginine and lysine residues showed a 7-fold loss of potency. In addition, we show that the activity is dependent on an intact disulfide network and that the short sequences between the six cysteine residues, that is, the backbone loops, are devoid of cytotoxic activity.

Small, novel proteins from the mistletoe Phoradendron tomentosum exhibit highly selective cytotoxicity to human breast cancer cells.

Four novel proteins (phoratoxins C-F) have been isolated from the North American mistletoe Phoradendron tomentosum. The amino acid sequences of these phoratoxins were determined unambiguously using a combination of Edman degradation and trypsin enzymatic digestion, and by electrospray ionization tandem mass spectrometry sequencing. Phoratoxins C, E and F consist of 46 amino acid residues; and phoratoxin D of 41. All proteins had six cysteines, similar to the earlier described phoratoxins A and B, which are thionins. The cytotoxicity of each protein was evaluated in a human cell line panel that represented several cytotoxic drug-resistance mechanisms. For the half-maximal inhibitory concentrations (IC50 values) of the different cell lines in the panel, correlation with those of standard drugs was low. The most potent cytotoxic phoratoxin C was further tested on primary cultures of human tumor cells from patients. The solid tumor samples from breast cancer cells were 18 times more sensitive to phoratoxin C than the tested hematological tumor samples.

Spasmolytic effects of baccharis conferta and some of its constituents.

The Nahua of the Mexican state of Veracruz use Baccharis conferta in the treatment of a variety of gastrointestinal illnesses, especially diarrhoea associated with gastrointestinal cramps. The aerial parts of B. conferta were investigated phytochemically and pharmacologically using the guinea pig ileum assay as a model (histamine, KCI and electric stimulation). The crude ethanolic extract showed a dose-dependent antispasmodic effect that was particularly strong in flavonoid-rich fractions (e.g. IC50 value for fraction E.3.1 from the ethyl acetate fraction, in histamine-induced contraction, 10 microg mL(-1)). Several flavonoids (apigenin-4',7-dimethylether, naringenin-4',7-dimethylether, pectolinarigenin and cirsimaritin) were isolated, while others were identified in complex fractions by GC-MS. The flavonoids play an important role in the antispasmodic activity of this indigenous drug. Additionally, oleanolic acid and its methyl ester as well as erythrodiol were isolated. Oleanolic acid methyl ester shows weak antibacterial activity against M. luteusand E. coli (20 microg/spot in a TLC assay). The phytochemical as well as the pharmacological data provide some in-vitro evidence forthe use of B. conferta in thetreatment of gastrointestinal cramps.

First cyclotide from Hybanthus (Violaceae).

Hypa A, a novel macrocyclic polypeptide containing 30 amino acid residues, has been isolated from the n-butanol extract of the Argentine plant Hybanthus parviflorus. The sequence, cyclo-(SCVYIPCTITALLGCSCKNKVCYNGIPCAE), was determined by automated Edman degradation, quantitative amino acid analysis and nanospray MS/MS(2). Three intramolecular disulfide bridges stabilize the cyclic peptide backbone of hypa A. Using these structural features to classify the peptide as a cyclotide, we extended the distribution of that substance class to a new genus, and now propose a uniform nomenclature for cyclotides.

Cytotoxicity of digitoxin and related cardiac glycosides in human tumor cells.

The saponin digitonin, the aglycone digitoxigenin and five cardiac glycosides were evaluated for cytotoxicity using primary cultures of tumor cells from patients and a human cell line panel (representing different cytotoxic drug-resistance patterns). Of these seven compounds, proscillaridin A was the most potent (IC(50): 6.4--76 nM), followed by digitoxin, and then ouabain, digoxin, lanatoside C, digitoxigenin and digitonin. Correlation analysis of the log IC(50) values for the cell lines in the panel showed that compound cytotoxicity was only slightly influenced by resistance mechanisms that involved P-glycoprotein, topoisomerase II, multidrug resistance-associated protein and glutathione-mediated drug resistance. Digitoxin and digoxin expressed selective toxicity against solid tumor cells from patients, while proscillaridin A expressed no selective toxicity against either solid or hematological tumor cells. The results revealed marked differences in cytotoxicity between the cardiac glycosides, both in potency and selectivity, and modes of action for cytotoxicity that differ from that of commonly used anticancer drugs.

Seven novel macrocyclic polypeptides from Viola arvensis.

Seven novel macrocyclic polypeptides, designated as varv peptides B-H, have been isolated from the aerial parts of Viola arvensis. Their primary structures have been elucidated by automated Edman degradation and mass spectrometry. They all consist of 29 or 30 amino acid residues, covalently cyclized via the amide backbone and by three internal disulfide bridges. Their amino acid sequences are as follows: varv peptide B, cyclo-(TCFGGTCNTPGCSCDPWPMCSRNGLPVCGE); varv peptide C, cyclo-(TCVGGTCNTPGCSCSWPVCTRNGVPICGE); varv peptide D, cyclo-(TCVGGSCNTPGCSCSWPVCTRNGLPICGE); varv peptide E, cyclo-(TCVGGTCNTPGCSCSWPVCTRNGLPICGE); varv peptide F, cyclo-(TCTLGTCYTAGCSCSWPVCTRNGVPICGE); varv peptide G, cyclo-(TCFGGTCNTPGCSCDPWPVCSRNGVPVCGE); and varv peptide H, cyclo-(TCFGGTCNTPGCSCETWPVCSRNGLPVCGE). The varv peptides B-H exhibited high degrees of homology with the hitherto known macrocyclic peptides varv peptide A, kalata B1, violapeptide I, circulins A and B, and cyclopsychotride A.

Fractionation Protocol for the Isolation of Polypeptides from Plant Biomass

A fractionation protocol for the isolation of a highly purified polypeptide fraction from plant biomass is described. The procedure dereplicates ubiquitous substance classes known to interfere with bioassays often used in natural product-based drug discovery programs. The protocol involves pre-extraction with dichloromethane, extraction with ethanol (50%), removal of tannins with polyamide, removal of low-molecular-weight components with size-exclusion chromatography over Sephadex G-10, and final removal of salts and polysaccharides with solid-phase extraction using reversed-phase cartridges. The method has been applied to the aerial parts of Viola arvensis, resulting in the isolation of a peptide fraction that on further separation yielded a novel 29-residue macrocyclic polypeptide named varv peptide A, cyclo(-TCVGGTCNTPGCSCSWPVCTRNGLPVCGE-).

Some aspects of bioassay methods in natural-product research aimed at drug lead discovery.

The objective of natural-product research aimed at drug lead discovery is to find unique bioactive molecules with improved biopharmaceutical properties or new modes of action. In order to discover such bioactive substances, different bioassays are used for detection, for guidance of the chemical isolation procedures and for preliminary biological characterization of new compounds. This article gives a brief update on current biological activities and discusses some essential, but occasionally neglected, aspects of bioassay methods. Some future perspectives on the use of bioassay methods in the drug-discovery process are also discussed.

Topical antiinflammatory activity of the major lipophilic constituents of the rhizome of Zingiber cassumunar. Part I: The essential oil.

The essential oil of the rhizome of Zingiber cassumunar was found to exhibit a topical antiinflammatory effect, when tested using the model of carrageenan-induced hind paw edema in rats (ID(50) = 22 mg oil/paw). Individual assessment of topical antiinflammatory activity of the five major components of the oil demonstrated that (E)-1-(3,4-dimethoxyphenyl)butadiene (DMPBD), terpinen-4-ol and α-terpinene significantly inhibited edema formation, whereas sabinene and γ-terpinene were inactive up to 6 mg/paw. The most active compound, DMPBD, was found to be an antiinflammatory agent twice as potent as the reference drug diclofenac (ID(50) = 3 vs 6 mg/paw, respectively).

Topical antiinflammatory activity of the major lipophilic constituents of the rhizome of Zingiber cassumunar. Part II: Hexane extractives.

A hexane extract of the rhizome of Zingiber cassumunar was found to exhibit topical antiinflammatory activity, when tested in the model of 12-O-tetradecanoylphorbol-13-acetate-induced ear edema in rats (ID(50) = 854 µg/ear). Bioassay-guided fractionation (by MPLC on silica gel) of the hexane extract led to the isolation and identification of (E)-4-(3',4'-dimethoxyphenyl)but-3-enyl acetate (1), cis-3-(3',4'-dimethoxyphenyl)-4-[(E)-3,‴,4‴- dimethoxystyryl]cyclohex-l-ene (2), cis-3-(3',4'-dimethoxyphenyl)-4-[(E)-2‴,4‴,5‴- trimethoxystyryl]cyclohex-1-ene (3), cis-3-(2',4',5'-trimethoxyphenyl)-4-[(E)-2‴,4‴,5‴- trimethoxystyryl]cyclohex-l-ene (4) and (E)-4-(3'-4'-dime-thoxyphenyl)but-3-en-l-ol (5). Compounds 1-5 exerted potent topical antiinflammatory activities with ID(50)-values of 62, 21, 20, 2 and 47µg/ear, respectively. The ID(50) of the reference drug diclofenac was determined to be 61 µg/ear.

Non-phenolic linear diarylheptanoids from Curcuma xanthorrhiza: a novel type of topical anti-inflammatory agents: structure-activity relationship.

The topical anti-inflammatory activity of three non-phenolic linear 1,7-diarylheptanoids, previously isolated from a Thai medicinal plant, Curcuma xanthorrhiza (Zingiberaceae) and four new semi-synthetic derivatives of the naturally occurring compounds were assessed in the murine model of ethyl phenylpropiolate-induced ear edema. The naturally occurring compound 1E,3E,1,7-diphenylheptadien-5-one (6) exerted the most potent anti-inflammatory activity, with an ID50 value of similar magnitude to that of the reference drug oxyphenbutazone (67 vs. 46 micrograms/ear, respectively). None of the semi-synthetic diarylheptanoids was more active than 6. The chemical structures and pharmacological data of the natural and semi-synthetic derivatives identified a distinct structure-activity relationship. The degree of unsaturation in positions 1 and 3, and the nature of the oxygenated functional group in position 5 of the C7-chain were found to play significant roles in determining the observed in vivo activity. Based on these findings, the non-phenolic linear 1,7-diarylheptanoids-are proposed to represent a novel class of topical anti-inflammatory agents.

Three non-phenolic diarylheptanoids with anti-inflammatory activity from Curcuma xanthorrhiza.

Bioassay-guided fractionation of a hexane extract of the rhizomes of Curcuma xanthorrhiza Roxb. (Zingiberaceae) led to the isolation of three non-phenolic diarylheptanoids, identified mainly by high-field 1H-NMR as trans-trans-1,7-diphenyl-1,3-heptadien-4-one (alnustone), trans-1,7-diphenyl-1-hepten-5-ol, and trans,trans-1,7-diphenyl-1,3-heptadien-5-ol. The latter is reported for the first time as a plant constituent. Germacrone, curzerenone, and cinnamaldehyde were also isolated and identified. The three diarylheptanoids all exerted significant anti-inflammatory activity in the assay of carrageenin-induced hind paw edema in rats.

Inventory of plants used in traditional medicine in Somalia. IV. Plants of the families Passifloraceae-Zygophyllaceae.

Thirty-seven plants are listed, which are used by traditional healers in the central and southern parts of Somalia. For each species are listed: the botanical name with synonyms, collection number, vernacular name, medicinal use, preparation of remedy and dosage. Results of a literature survey are also reported including medicinal use, substances isolated and pharmacological effects. Three plants which should have been included in Part I of the series have been added and some corrections to that paper have been made. With these additions the series comprises 180 different plant species, distributed in 59 plant families.

Inventory of plants used in traditional medicine in Somalia. III. Plants of the families Lauraceae-Papilionaceae.

Thirty-five plants are listed, which are used by traditional healers in the central and southern parts of Somalia. For each species are listed: the botanical name with synonyms, collection number, vernacular name, medicinal use, preparation of remedy and dosage. Results of a literature survey are also reported including medicinal use, substances isolated and pharmacological effects.

Inventory of plants used in traditional medicine in Somalia. II. Plants of the families Combretaceae to Labiatae.

Fifty-nine plants are listed, which are used by traditional healers in the central and southern parts of Somalia. For each species are listed: the botanical name with synonyms, collection number, vernacular name, medicinal use, preparation of remedy and dosage. Results of a literature survey are also reported including medicinal use, substances isolated and pharmacological effects.

Inventory of plants used in traditional medicine in Somalia. I. Plants of the families Acanthaceae-Chenopodiaceae.

Thirty-eight plants are listed, which are used by traditional healers in the central and southern parts of Somalia. For each species are listed: the botanical name with synonyms, collection number, vernacular name, medicinal use, preparation of remedy and dosage. Results of a literature survey are also reported including medicinal use, substances isolated and pharmacological effects.

Calcium antagonistic properties of the sesquiterpene T-cadinol: a comparison with nimodipine in the isolated rat aorta.

(+)-T-Cadinol is a sesquiterpene with smooth muscle relaxing properties. In the isolated rat aorta, T-cadinol relaxed contractions induced by 60 mM K+ in a concentration-dependent fashion. The dihydropyridine calcium antagonist nimodipine was approximately 4,000 times more potent than T-cadinol. While both drugs nearly abolished the K(+)-induced contractions, they only partially relaxed contractions induced by phenylephrine. The relaxation induced by T-cadinol and nimodipine in K(+)-contracted aortic rings, was completely reversed by the calcium channel activator Bay K8644. In aortic preparations partially depolarized by 20 mM K+, Bay K8644 induced a concentration-dependent contraction. Nimodipine shifted the Bay K8644 concentration-response curve to the right in a parallel manner, consistent with a competitive mode of inhibition. T-cadinol at concentrations less than 10(-3.5) M also produced a right-ward shift of the Bay K8644 concentration-response curve with a maintained maximum response. However, the highest T-cadinol concentration used 10(-3.5 M) significantly reduced the maximum response. In conclusion, although T-cadinol and nimodipine display marked structural differences, their pharmacological profiles of action have several features in common, suggesting that T-cadinol is a calcium antagonist, possibly interacting with the dihydropyridine binding sites on the calcium channels.

T-cadinol: a pharmacologically active constituent of scented myrrh: introductory pharmacological characterization and high field 1H- and 13C-NMR data.

Fractionation of an ethyl acetate extract of scented myrrh (resin of Commiphora guidottii Chiov., Burseraceae), using the guinea pig ileum test to monitor pharmacological activity, resulted in isolation of the sesquiterpene (+)-T-cadinol. High field NMR spectroscopy yielded new detailed 1H- and 13C-NMR data for the compound. T-cadinol was shown to have a concentration-dependent smooth muscle relaxing effect on the isolated guinea pig ileum and a dose-dependent inhibitory effect on cholera toxin-induced intestinal hypersecretion in mice.