Revision of the 'Candidatus Phytoplasma' species description guidelines.

The genus 'Candidatus Phytoplasma' was proposed to accommodate cell wall-less bacteria that are molecularly and biochemically incompletely characterized, and colonize plant phloem and insect vector tissues. This provisional classification is highly relevant due to its application in epidemiological and ecological studies, mainly aimed at keeping the severe phytoplasma plant diseases under control worldwide. Given the increasing discovery of molecular diversity within the genus 'Ca. Phytoplasma', the proposed guidelines were revised and clarified to accommodate those 'Ca. Phytoplasma' species strains sharing >98.65 % sequence identity of their full or nearly full 16S rRNA gene sequences, obtained with at least twofold coverage of the sequence, compared with those of the reference strain of such species. Strains sharing <98.65 % sequence identity with the reference strain but >98.65 % with other strain(s) within the same 'Ca. Phytoplasma' species should be considered related strains to that 'Ca. Phytoplasma' species. The guidelines herein, keep the original published reference strains. However, to improve 'Ca. Phytoplasma' species assignment, complementary strains are suggested as an alternative to the reference strains. This will be implemented when only a partial 16S rRNA gene and/or a few other genes have been sequenced, or the strain is no longer available for further molecular characterization. Lists of 'Ca. Phytoplasma' species and alternative reference strains described are reported. For new 'Ca. Phytoplasma' species that will be assigned with identity ≥98.65 % of their 16S rRNA gene sequences, a threshold of 95 % genome-wide average nucleotide identity is suggested. When the whole genome sequences are unavailable, two among conserved housekeeping genes could be used. There are 49 officially published 'Candidatus Phytoplasma' species, including 'Ca. P. cocostanzaniae' and 'Ca. P. palmae' described in this manuscript.

Plectranthus zeylanicus: A Rich Source of Secondary Metabolites with Antimicrobial, Disinfectant and Anti-Inflammatory Activities.

Plectranthus zeylanicus Benth is used in Sri Lankan folk medicine as a remedy for inflammatory conditions and microbial infections. Our previous investigations revealed potent 5-lipoxygenase (5-LO) inhibitory activity in lipophilic extracts of this plant, supporting its anti-inflammatory potential. In-depth studies on the antimicrobial activity have not been conducted and the bioactive ingredients remained elusive. As a continuation of our previous work, the present investigation was undertaken to evaluate the antimicrobial activity of different extracts of P. zeylanicus and to isolate and characterize bioactive secondary metabolites. Different organic extracts of this plant were analyzed for their antibacterial activity, and the most active extract, i.e., dichloromethane extract, was subjected to bioactivity-guided fractionation, which led to the isolation of 7α-acetoxy-6ß-hydroxyroyleanone. This compound displayed strong antibacterial activity against methicillin-resistant Staphylococcus aureus with a minimum inhibitory concentration of 62.5 µg/mL, and its disinfectant capacity was comparable to the potency of a commercial disinfectant. Moreover, 7α-acetoxy-6ß-hydroxyroyleanone inhibits 5-LO with IC50 values of 1.3 and 5.1 µg/mL in cell-free and cell-based assays, respectively. These findings rationalize the ethnopharmacological use of P. zeylanicus as antimicrobial and anti-inflammatory remedy.

Cytotoxic abietane-type diterpenoids from roots of Salvia spinosa and their in Silico pharmacophore modeling.

The roots of Salvia spinosa L. (Lamiaceae) were extracted with hexane, dichloromethane (DCM) and ethyl acetate. The DCM extract exhibited cytotoxic activity (IC50 32.7 µg/mL) against MFC-7 breast cancer cell line in MTT colorimetric bioassay. Ferruginol (1), taxodione (2), 12-deoxy-6-hydroxy-6,7-dehydroroyleanone (3), 14-deoxycoleon U (4), 15-deoxyfuerstione (5) and taxodone (6) were isolated from the DCM roots extract. Their structures were elucidated by a combination of spectroscopic analyses including EIMS and 1H- and 13C NMR spectra. The cytotoxicity of compound 3 was determined against MCF-7 and K562 cell lines and compared with the other compounds. A pharmacophore model was built based on potent input compounds to resolve important pharmacophore features responsible for cytotoxic activity of the isolated compounds.

Duckweed for Human Nutrition: No Cytotoxic and No Anti-Proliferative Effects on Human Cell Lines.

Duckweeds (Lemnaceae) possess good qualitative and quantitative profiles of nutritional components for its use as human food. However, no studies have been conducted on the probable presence or absence of any adverse effects. The extracts from seven duckweed species (Spirodela polyrhiza, Landoltia punctata, Lemna gibba, Lemna minor, Wolffiella hyalina, Wolffia globosa, and Wolffia microscopica) covering all five genera of the plant family were herewith tested for cytotoxic effects on the human cell lines HUVEC, K-562, and HeLa and for anti-proliferative activity on HUVEC and K-562 cell lines. From these assays, it is evident that duckweeds do not possess any detectable anti-proliferative or cytotoxic effects, thus, the high nutritional value is not diminished by such detrimental factors. The present result is a first step to exclude any harmful effects of highly nutritious duckweed for human.

Precursor-Directed Biosynthesis of Phenylbenzoisoquinolindione Alkaloids and the Discovery of a Phenylphenalenone-Based Plant Defense Mechanism.

Phenylbenzoisochromenone glucosides (oxa-phenylphenalenone glucosides) occurring in some phenylphenalenone-producing plants of the Haemodoraceae undergo conversion to phenylbenzoisoquinolindiones (aza-phenylphenalenones) in extracts of Xiphidium caeruleum. Precursor-directed biosynthetic experiments were used to generate a series of new phenylbenzoisoquinolindiones from native phenylbenzoisochromenone glucosides and external amines, amino acids, and peptides. Intermediates of the conversion were isolated, incubated with cell-free extracts, and exposed to reactions under oxidative or inert conditions, respectively, to elucidate the entire pathway from phenylbenzoisochromenones to phenylbenzoisoquinolindiones. An intermediate in this pathway, a reactive hydroxylactone/aldehyde, readily binds not only to amines in vitro but may also bind to the N-terminus of biogenic peptides and proteins of herbivores and pathogens in vivo. The deactivation of biogenic amino compounds by N-terminal modification is discussed as the key reaction of a novel phenylphenalenone-based plant defense mechanism. According to these data, the ecological function of phenylphenalenone-type compounds in the Haemodoraceae, subfamily Haemodoroideae, has been substantiated.

Sulfur-containing compounds from the roots of Ferula latisecta and their cytotoxic activities.

Five new sulfur-containing compounds, with a new geometry (cis) of the propenyl moiety, and five known compounds were isolated from the roots of Ferula latisecta. The structures of these compounds, were elucidated on the basis of spectroscopic data including 1D and 2D NMR experiments and HRMS. All of the isolated compounds were tested against A2780, A549, HeLa, and HCT116 human cancer cell lines and some of them showed moderate cytotoxic activities.

Application of the Crystalline Sponge Method to Revise the Structure of the Phenalenone Fuliginone.

The structure of fuliginone was revised from a phenyl substituted phenalenone to a hydroxyl substituted phenalenone as a result of its re-purification via HPLC with subsequent NMR analysis together with an independent synthesis and analysis of the crystal structure, which was secured via the crystalline sponge method. On-flow High Performance Liquid Chromatography coupled to Nuclear Magnetic Resonance spectroscopy (HPLC-NMR) was employed to confirm the presence of the natural product in the plant extract and to monitor for any possible degradation or conversion of the compound.

The roots of Salvia rhytidea: a rich source of biologically active diterpenoids.

Sahandinone (1), 12-deoxysalvipisone (2), miltirone (3), 7α-acetoxyroyleanone (4), and labda-7,14-dien-13-ol (5) were isolated from the roots of Salvia rhytidea Benth. (Lamiaceae). Their structures were elucidated by a combination of spectroscopic analyses including EIMS and NMR. The 13C NMR spectroscopic data were revised for the quaternary carbons of both 1 and 3 with the help of HMBC spectra in respect to the spectral data previously reported in the literature. Compounds 1 and 3, two very potent anticancer agents, were isolated in high yields from the roots of the plant. The biological activities of the plants' constituents were reported in the literature as antimicrobial, cytotoxic and antimalarial are discussed in this article.

Cytotoxic activity and chemical constituents of Anthemis mirheydari.

Context The genus Anthemis L. (Asteraceae) comprises about 195 species which are widely used in the pharmaceutical, cosmetic and food industries. Objective Anthemis mirheydari Iranshar, an endemic plant from Iran, was investigated for its cytotoxic properties and chemical constituents. Materials and methods The whole parts of the plant (320 g) were extracted by dichloromethane and methanol for four days, successively. The cytotoxic activity of both dichloromethane and methanol extracts were assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric methods against three human cancer cell lines including LS180, MCF-7 and MOLT-4. Different concentrations (10-100 µg/mL) of the plant extracts were tested to obtain IC50 values. The dichloromethane extract of A. mirheydari was subjected to silica gel-column and thin layer chromatography for purification of its chemical constituents and the isolated compounds were further tested against MOLT-4 cells. The structures of the pure compounds were elucidated using different spectral data including nuclear magnetic resonance and electron impact mass spectra. Results The IC50 values of the dichloromethane extract were 30.8 ± 6.7, 25.2 ± 6.5 and 8.6 ± 1.1 µg/mL (means ± standard error) for the above-mentioned cell lines, respectively. Two triterpenoids, taraxasterol (1) and pseudotaraxasterol (2), one sterol, ß-sitosterol (3) and one coumarin, 7-methoxycoumarin (4) were isolated from the extract. The IC50 of the mixture of compounds 1 and 2 as well as compounds 3 and 4 were higher (>100 µM) than that reported for the dichloromethane extract against MOLT-4 cells. Conclusion The dichloromethane extract was the most active one among the tested material.

Chemical Composition and Antimicrobial Activity of Populus nigra Shoot Resin.

The chemical composition of Populus nigra shoot resin has been investigated by chromatographic and spectroscopic methods. The analyses resulted in identification of 19 known compounds. The resin exhibited low activity against selected microorganisms.

Highly Oxygenated Sesquiterpene Lactones from Cousinia aitchisonii and their Cytotoxic Properties: Rhaserolide Induces Apoptosis in Human T Lymphocyte (Jurkat) Cells via the Activation of c-Jun n-terminal Kinase Phosphorylation.

Infrared-guided chromatographic fractionation of sesquiterpene lactones from the extracts of Cousinia aitchisonii and Cousinia concolor led to the isolation of five pure compounds. A new sesquiterpene lactone, namely, aitchisonolide, and two known sesquiterpene lactones (desoxyjanerin and rhaserolide) were isolated from C. aitchisonii and two known lignans (arctiin and arctigenin) from C. concolor. The structures of these compounds were elucidated by one-dimensional and two-dimensional nuclear magnetic resonance techniques, as well as high-resolution mass spectrometry. The purified and characterized compounds were subjected to cytotoxicity assay. The sesquiterpene lactones desoxyjanerin and rhaserolide showed significant cytotoxic activities against five different cancer cell lines and the normal human embryonic kidney cell line. Rhaserolide was chosen to evaluate the possible mechanism of action. Western blot analysis revealed that rhaserolide could induce apoptosis in Jurkat cells via the activation of c-Jun n-terminal kinase phosphorylation.

Biotransformation of Flavokawains A, B, and C, Chalcones from Kava (Piper methysticum), by Human Liver Microsomes.

The in vitro metabolism of flavokawains A, B, and C (FKA, FKB, FKC), methoxylated chalcones from Piper methysticum, was examined using human liver microsomes. Phase I metabolism and phase II metabolism (glucuronidation) as well as combined phase I+II metabolism were studied. For identification and structure elucidation of microsomal metabolites, LC-HRESIMS and NMR techniques were applied. Major phase I metabolites were generated by demethylation in position C-4 or C-4' and hydroxylation predominantly in position C-4, yielding FKC as phase I metabolite of FKA and FKB, helichrysetin as metabolite of FKA and FKC, and cardamonin as metabolite of FKC. To an even greater extent, flavokawains were metabolized in the presence of uridine diphosphate (UDP) glucuronic acid by microsomal UDP-glucuronosyl transferases. For all flavokawains, monoglucuronides (FKA-2'-O-glucuronide, FKB-2'-O-glucuronide, FKC-2'-O-glucuronide, FKC-4-O-glucuronide) were found as major phase II metabolites. The dominance of generated glucuronides suggests a role of conjugated chalcones as potential active compounds in vivo.

Foetithiophenes C-F, thiophene derivatives from the roots of Ferula foetida.

CONTEXT: Ferula foetida Regel (Apiaceae) is an Iranian medicinal plant with various biological activities including antispasmodic and anthelmintic. OBJECTIVE: The sulfur compounds from the roots of F. foetida were isolated and characterized to test their cytotoxic and antimicrobial activities. MATERIALS AND METHODS: The methanolic extract of the roots of F. foetida was fractionated using silica column chromatography. The components of each fraction were further purified using RP-HPLC. Their structures were elucidated by 1- and 2-D NMR spectroscopy as well as HREIMS. Their cytotoxic and antimicrobial activities were evaluated using Alamar Blue assay and broth microdilution method, respectively. RESULTS: Four new thiophene derivatives, namely foetithiophenes C-F (3-6), together with four known compounds, foetithiophenes A (1) and B (2), coniferaldehyde, and sinapic aldehyde, were isolated from the roots of F. foetida. Antimicrobial activities were observed in particular against the Gram-positive bacteria. The best antimicrobial activity was observed for compound 6 against B. cereus with a MIC value 50 µg/mL. The tested compounds did not show cytotoxic properties against MCF-7 and K562 cells. CONCLUSION: Four new thiophene derivatives including foetithiophenes C-F (3-6) were characterized from the roots of F. foetida. Foetithiophene F (6) exhibited the most potent activity against the Gram-positive bacteria B. cereus.

Phytochemical profile of aerial parts and roots of Wachendorfia thyrsiflora L. studied by LC-DAD-SPE-NMR.

Hyphenated liquid chromatography - diode array detection - solid phase extraction - nuclear magnetic resonance spectroscopy (LC-DAD-SPE-NMR) was used to investigate the phytochemical composition of aerial parts and roots of Wachendorfia thyrsiflora (Haemodoraceae). Eleven phenylphenalenones and related compounds were identified in the aerial parts of the plant, ten compounds were found in the roots, and four additional compounds occurred in both plant parts. Twelve compounds are previously unreported natural products including five alkaloids (phenylbenzoisoquinolinones) are described here for the first time. In the work presented here, phenylphenalenones with an intact C(19) core structure were found only in the roots. Oxa analogs with a C(18)O scaffold occurred both in the roots and in the aerial plant parts, while most of the aza analogs with a C(18)N scaffold were detected in the aerial plant parts. This distribution pattern suggests that phenylphenalenones form in the roots, then the intact C(19) skeleton is converted into oxa analogs in the roots, translocated into the leaves and further reacted with amines or amino acids to form aza analogs (phenylbenzoisoquinolin-1,6-dione alkaloids).

Metabolic detoxification of capsaicin by UDP-glycosyltransferase in three Helicoverpa species.

Capsaicin ß-glucoside was isolated from the feces of Helicoverpa armigera, Helicoverpa assulta, and Helicoverpa zea that fed on capsaicin-supplemented artificial diet. The chemical structure was identified by NMR spectroscopic analysis as well as by enzymatic hydrolysis. The excretion rates of the glucoside were different among the three species; those in the two generalists, H. armigera and H. zea, were higher than in a specialist, H. assulta. UDP-glycosyltransferases (UGT) enzyme activities measured from the whole larval homogenate of the three species with capsaicin and UDP-glucose as substrates were also higher in the two generalists. Compared among five different larval tissues (labial glands, testes from male larvae, midgut, the Malpighian tubules (MT), and fat body) from the three species, the formation of the capsaicin glucoside by one or more UGT is high in the fat body of all the three species as expected, as well as in H. assulta MT. Optimization of the enzyme assay method is also described in detail. Although the lower excretion rate of the unaltered capsaicin in H. assulta indicates higher metabolic capacity toward capsacin than in the other two generalists, the glucosylation per se seems to be insufficient to explain the decrease in capsaicin in the specialist, suggesting that H. assulta might have another important mechanism to deal with capsaicin more specifically.

(8R)-3ß,8-dihydroxypolypoda-13E,17E,21-triene induces cell cycle arrest and apoptosis in treatment-resistant prostate cancer cells.

Mastic, a resinous exudate from Pistacia lentiscus, has been reported to exhibit selective cytotoxicity against different cancer cell lines. There are, however, no data published correlating distinct mastic-derived compounds with the postulated cytotoxic activity. A polypodane-type bicyclic triterpenoid, (8R)-3ß,8-dihydroxypolypoda-13E,17E,21-triene (1), was isolated from P. lentiscus oleogum resin. In androgen-independent PC-3 prostate cancer cells, 1 potently inhibited the expression of cyclins D1 and E, but had no effect on the expression of the cyclin kinase inhibitor p21(Waf1/Cip1). Inhibition of the expression of cell cycle-regulating cyclins resulted in cell cycle arrest in the G0/G1 phase, reduction in the number of cells in the S phase, and the triggering of apoptosis, as detected by increased expression of phosphatidylserine on the cell surface and by formation of DNA laddering. In addition, 1 suppressed the formation of prostate cancer colonies in soft agar and inhibited proliferation, angiogenesis, and the growth of prostate tumors xenografted onto chick chorioallantoic membranes without overt systemic toxicity. Taken together, these data show that 1 triggers apoptosis in chemoresistant, androgen-independent human prostate cancer cells in vitro and in vivo. Thus, 1 may serve as a lead compound for targeting so far incurable androgen-insensitive prostate cancers.

Antitrypanosomal and cytotoxic activities of 22-Hydroxyclerosterol, a new sterol from Allexis cauliflora (Violaceae).

In the search for new antiparasitic natural compounds from the medicinal plants from Cameroon, the new 22-hydroxyclerosterol, established as such on the basis of detailed chemical and spectroscopic analysis, was isolated from the hexane extract of the stem bark of Allexis cauliflora together with the known clerosterol. 22-Hydroxyclerosterol inhibited the growth of Trypanosoma brucei brucei cells with an ED(50) value of 1.56 µM. The compound was also established as an uncompetitive inhibitor of the glycolytic enzyme PGI of T. brucei (Ki'= 3 ± 1 µM), an uncompetitive inhibitor of mammalian rabbit muscles' enzyme PyK (Ki'= 26 ± 3 µM) and a mixed inhibitor of PyK of Leishmania mexicana (Ki'= 65 ± 10 µM; Ki= 24 ± 5 µM).

Identification of spathulenol in Salvia mirzayanii and the immunomodulatory effects.

The methanol extract of Salvia mirzayanii has shown an immunomodulatory effect on peripheral blood lymphocytes. Bioassay-guided fractionation using a lymphocyte proliferation assay on Salvia mirzayanii was performed in order to purify and identify the active compounds. Fractionation of the methanol extract and purification of the components using normal column chromatography and preparative thin layer chromatography resulted in identification of the bioactive compound, spathulenol, with an immunoinhibitory effect. Identification of this compound was performed by 1D and 2D NMR methods and HRMS. Treatment of activated lymphocytes with a concentrated fraction containing 62% of spathulenol (SP) showed a decrease in the proliferation of lymphocytes with an IC(50) of 85.4 ± 11.08 µg/mL. Flow cytometry analysis using annexin V and propidium iodide staining of the stimulated peripheral blood lymphocytes in the presence of SP demonstrated a dose dependent increase in the percentage of apoptotic cells (IC(50) ; 77.2 ± 5.31 µg/mL). No significant increase in caspase 3 activity in a 20 h treatment of stimulated lymphocytes compared with the control was observed. In conclusion, this study identified the possible activity of spathulenol as one of the immunomodulatory compounds present in Salvia mirzayanii. SP showed the capacity to inhibit proliferation in the lymphocytes and to induce apoptosis in these cells possibly through a caspase-3 independent pathway.

New sesquiterpene coumarins from the roots of Ferula flabelliloba.

Three new sesquiterpene coumarins, namely, farnesiferone B (1), flabellilobin A (2) and flabellilobin B (3), together with nine known compounds, ligupersin A, 7-epi-gamma-eudesmol, persicasulfide A, conferdione, umbelliprenin, conferone, feselol, lehmferin and farnesiferol B were isolated from the roots of Ferula flabelliloba Rech. f. & Aell. (Apiaceae). The structures of these compounds were elucidated by various 1- and 2-D NMR techniques as well as HREIMS.

Antitrypanosomal alkaloids from Polyalthia suaveolens (Annonaceae): their effects on three selected glycolytic enzymes of Trypanosoma brucei.

In continuation of our study on medicinal plants of Cameroon, stem barks of Polyalthia suaveolens were phytochemically studied. This investigation yielded a new indolosesquiterpene alkaloid, named polysin (1) and four hitherto known alkaloids (2-5). Polysin (1) appeared as a competitive reversible inhibitor (K(i)=10 microM) of phosphofructo kinase (PFK) of Trypanosoma brucei with respect to fructose-6-phosphate (K(i)/K(M)=0.05) and could be used in the design of new trypanocidal drugs. The other isolated compounds (2-5) also exhibited interesting inhibitory effects on selected glycolytic enzymes (PFK, glyceraldehyde-3-phosphate dehydrogenase and aldolase).