Protocorm culture of Dendrobium candidum in balloon type bubble bioreactors

Protocorm cultures of Dendrobium candidum were established in balloon type bubble bioreactors using Murashige and Skoog (MS) medium with 0.5 mg l⁻¹ α-naphthaleneacetic acid (NAA), 2.5% (w/v) sucrose, 5:25 mM NH₄:NO₃ and 1% (v/v) banana homogenate for the production of biomass and bioactive compounds. In 3 l bioreactor containing 2 l medium, a maximum protocorm biomass (21.0 g l⁻¹ dry biomass) and also optimum quantities of total polysaccharides (389.3 mg g⁻¹ DW), coumarins (18.0 mg g⁻¹ DW), polyphenolics (11.9 mg g⁻¹ DW), and flavonoids (4.5 mg g⁻¹ DW) were achieved after 7 weeks of culture. Based on these studies, 5 and 10 l bioreactor cultures were established to harvest 80 g and 160 g dry biomass. In 10 l bioreactors, the protocorms grown were accumulated with optimal levels of polysaccharides (424.1 mg g⁻¹ DW), coumarins (15.8 mg g⁻¹ DW), polyphenols (9.03 mg g⁻¹ DW) and flavonoids (4.7 mg g⁻¹ DW). The bioreactor technology developed here will be useful for the production of important bioactive compounds from D. candidum.     

Coumarins from the roots of Prangos uloptera

Three new coumarins, 6-O-[β-d-apiofuranosyl-(1 → 6)-β-d-glucopyranosyl]-prenyletin, 3″-O-[β-d-apiofuranosyl-(1 → 6)-β-d-glucopyranosyl]-oxypeucedanin hydrate and 2″-O-[β-d-apiofuranosyl-(1 → 6)-β-d-glucopyranosyl]-oxypeucedanin hydrate, together with six known coumarins, 3″-O-[β-d-apiofuranosyl-(1 → 6)-β-d-glucopyranosyl]-heraclenol, 3″-O-(β-d-glucopyranosyl)-heraclenol, tortuoside, 3″-O-(β-d-glucopyranosyl)-oxypeucedanin hydrate, heraclenol and oxypeucedanin hydrate, have been isolated from the roots of Prangos uloptera, and the structures of these coumarins were unequivocally determined by spectroscopic means, notably UV, HRESIMS, and 1D and 2D NMR spectroscopy.     

In vitro antituberculosis activities of the constituents isolated from Haloxylon salicornicum

In vitro antituberculosis activities of fractions and pure compounds (1–20) including seven triterpenes, two alkaloids, two cycloheximide derivatives, two coumarins six sterol derivatives and a long chain alcohol, respectively, isolated from Haloxylon salicornicum were determined against Mycobecterium tuberculosis H37Rv. Actively growing cultures were tested by rapid colorimetric method while the stationary phase cultures were tested by drug exposure methods for bactericidal activity. The MIC values were found to be 50 μg/ml for compounds 15, 19 and 20 where as rest of the compounds invariably showed MIC value of 100 μg/ml against the logarithmic phase culture. These were compare to Isoniazid as a control drug. The compounds exhibited no activity against the stationary phase culture of M. tuberculosis H37Rv up to 200 μg/ml. Further studies are required to investigate the in vivo efficacies and activities of the compounds in combination with antimicrobials that are already being used for TB therapy.     

Studies on the accumulation of phenolic acids and flavonoids in different in vitro culture systems of Schisandra chinensis (Turcz.) Baill. using a DAD-HPLC method

Different in vitro culture systems of the East-Asian origin medicinal plant species − Schisandra chinensis, were tested in order to investigate their potential for the accumulation of two groups of phenolic compounds. In vitro cultures were maintained on the Murashige and Skoog (MS) medium supplemented with 3 mg/l BA and 1 mg/l NAA in an agar system (30- and 60-day growth cycles), and also in two different liquid systems: stationary and agitated. Stationary liquid cultures were grown in batch (30- and 60-day growth cycles) and fed-batch modes. Of the twenty compounds, seven free phenolic acids and of the eleven compounds, five flavonoids were quantified in methanolic extracts from lyophilized biomass and in the growth media using the RP-HPLC-DAD method. For comparison purposes, phytochemical analyses of leaf and fruit extracts from the parent plant were also conducted. The estimated compounds were not detected in the growth media. The highest total amounts of phenolic acids (71.48 mg/100 g DW) and flavonoids (29.36 mg/100 g DW) were found in extracts from the biomass of agar cultures harvested after 30 days of cultivation. The main metabolites in all the tested systems were: protocatechuic acid (max. 35.69 mg/100 g DW), chlorogenic acid (max. 13.05 mg/100 g DW), and quercitrin (max. 27.43 mg/100 g DW).     

Retinoid compounds associated with water blooms dominated by Microcystis species

Retinoic acids play a critical role in vital physiological processes and vertebrate development, and their derivatives can be produced by some cyanobacterial species into surface waters. This study presents important environmentally-relevant information on total retinoid-like activity of field cyanobacterial biomasses and their surrounding waters. Intracellular and extracellular levels of total retinoid-like activity and retinoic acids have been investigated at a set of independent sites with the occurrence of water bloom dominated by widespread species Microcystis aeruginosa. Twelve samples of biomass and surrounding water from seven localities affected by blooms were studied in comparison with samples from M. aeruginosa laboratory cultures. The method for biomass extraction was optimized and final extracts and samples of surrounding water concentrated by solid phase extraction were assessed using in vitro reporter gene bioassay and chemical analyses for all-trans-retinoic acid (ATRA), 9-cis retinoic acid (9-cis RA) and microcystins RR, LR and YR. Methanol was the most efficient solvent for the extraction of compounds with retinoid-like activity. An in vitro bioassay with the P19/A15 transgenic cell line revealed retinoid-like activity in all cyanobacterial biomasses in the range of 356–2838 ng of retinoid acid equivalents (REQ)/g dry mass (dm), while only three of surrounding water samples exhibited detectable retinoid-like activity, in the range of 12.8–28.7 ng REQ/L. Microcystins were detected in all samples, but they elicited no detectable retinoid-like activity up to 10 mg/L. Chemical analyses detected concentrations up to 340 ng/g dm of all-trans-retinoic acid (ATRA) and 84 ng/g dm 9-cis retinoic acid (9-cis RA) in bloom extracts, and up to 19 ng/L ATRA and 2.2 ng/L 9-cis RA in surrounding water. In most samples, ATRA and 9-cis RA contributed relatively little to the total REQs, which indicates the presence of significant amounts of other compounds with retinoic acid receptor-mediated modes of action. The impact of retinoid-like cyanobacterial metabolites could be of importance namely in smaller water bodies with dense water blooms and low dilution.     

Binaphthyl scaffolded peptoids via ring-closing metathesis reactions and their anti-bacterial activities

An efficient synthesis of four new acyclic and four new cyclic binaphthyl-based cationic peptoids is described. These compounds had anti-bacterial activities with MIC values of 4–62 μg/mL against Staphylococcus aureus.     

Molecular characterization of trypsinogens and development of trypsinogen gene expression and tryptic activities in grass carp (Ctenopharyngodon idellus) and topmouth culter (Culter alburnus)

This study examined the gene structures and expression of trypsinogens, as well as the trypsin activities of the grass carp Ctenopharyngodon idellus (herbivorous) and the topmouth culter Culter alburnus (carnivorous), which are commercially important freshwater species of the family Cyprinidae in China. Isolated full-length trypsinogen cDNA clones were 869 bp and 857 bp. The deduced amino acid sequences were 242 aa and 247 aa long, both containing the highly conserved residues essential for serine protease catalytic and conformational maintenance. The results from isoelectric and phylogenetic analyses suggest that grass carp trypsinogen is grouped with teleost trypsinogen group I, while topmouth culter trypsinogen is grouped with group II. The expression pattern of trypsinogen mRNA was similar between these two species, appearing 2 days post-hatching (dph) and reaching peaks at 11 and 23 dph. The trypsin-specific activities in both species were detected 2 dph and reached the major peaks at 8 dph, however the minor peaks were observed at 20 dph in the grass carp and 17 dph in the topmouth culter. The trypsin-specific activity was significantly higher in the grass carp than in the topmouth culter, which may be attributed to the nature of their different nutritional habits.     

Cloning and sequencing of three new putative toxin genes from Clostridium bifermentans CH18

Three new open reading frames were found downstream from cbm71, a toxin gene from Clostridium bifermentans malaysia (Cbm) strain CH18. The first one (91 bp downstream) called cbm72, is 1857 bp long and encodes a 71 727-Da protein (Cbm72) with a sequence similar to that of Bacillus thuringiensis delta-endotoxins. This protein shows no significant toxicity to mosquito larvae. The two others, cbm17.1 (462 bp) and cbm17.2 (459 bp), are copies of the same gene encoding Cbm P18 and P16 polypeptides and located 426 bp and 1022 bp downstream from cbm72, respectively. They encode 17 189-Da and 17 451-Da proteins with sequences 44.6% similar to that of Aspergillus fumigatus hemolysin; however, they were not hemolytic in the conditions tested.     

Analysis of anti-bacterial and anti oxidative activity of Azadirachta indica bark using various solvents extracts

Herbal medications have been used for relief of symptoms of disease. Regardless of the great advances observed in current medicine in recent decades, plants still make a significant contribution to health care. An alarming increase in bacterial strains resistant to a number of antimicrobial agents demands that a renewed effort be made to seek antibacterial agents effective against pathogenic bacteria resistant to or less sensitive to current antibiotics. Anti-bacterial activity of Azadirachta indica stem bark was tested against pathogenic Salmonella paratyphi and Salmonella typhi using various solvent extracts. The in vitro anti-bacterial activity was performed by agar well diffusion method and the results were expressed as the average diameter of zone of inhibition of bacterial growth around the well. The ethanol and methanol extracts showed better anti-bacterial activity with zone of inhibition (20–25 mm) when compared with other tested extracts and standard antibiotic Erythromycin (15 mcg) with zone of inhibition (13–14 mm). Using Fisher’s exact test of significance difference was found between two Salmonella strains sensitivity patterns against tested extracts (P ⩽ 0.035). Extracts of A. indica stem bark also exhibited significant antioxidant activity, thus establishing the extracts as an antioxidant. The results obtained in this study give some scientific support to the A. indica stem bark for further investigation of compounds and in future could be used as drug.     

Coumarins incorporating hydroxy- and chloro-moieties selectively inhibit the transmembrane,tumor-associated carbonic anhydrase isoforms IX and XII over the cytosolic ones I and II

A series of coumarins incorporating hydroxy-, chloro- and/or chloromethyl-moieties in positions 3-, 4-, 6- and 7- of the heterocyclic ring were investigated for the inhibition of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1). These coumarins were very weak or ineffective as inhibitors of the house-keeping, offtarget isoforms CA I and II, but showed effective, submicromolar inhibition of the transmembrane, tumor-associated isoforms CA IX and XII. The nature and position of the groups substituting the coumarin ring greatly influenced CA inhibitory properties. 6-Hydroxycoumarin showed K_Is >100 μM against CA I and II, of 0.198 μM against CA IX and of 0.683 μM against CA XII, being thus a selective, efficient inhibitor for the tumor-associated over cytosolic isoforms. These compounds are also excellent leads for designing isoform-selective enzyme inhibitors.     

Flavonoid and stilbenoid production in callus cultures of Artocarpus lakoocha

Callus cultures of Artocarpus lakoocha Roxb., established from seedling explants and maintained on woody plant medium containing 1 mg/l 2,4-dichlorophenoxyacetic acid and 1 mg/l benzyladenine, were studied for their chemical constituents and biosynthetic potential of secondary metabolites. Four prenylflavones and prenylated stilbenes, along with nine known polyphenolic compounds, were isolated and elucidated for their structures through extensive analysis of their NMR and MS data. Among the 13 isolates, it appeared that seven of them are prenylated derivatives of 5,7,2′,4′-tetrahydroxyflavones, and four are prenylated derivatives of 2,4,3′,5′-tetrahydroxystilbene (oxyresveratrol), suggesting that the biosynthetic pathways of these two polyphenolic groups and their prenylating enzymes are highly expressed in A. lakoocha callus cultures. A study on the growth-product relationship of the callus cultures showed that the secondary metabolites were all formed simultaneously during the rapid growth phase of the culture cycle, with various prenylflavones, and a prenylated stilbene as major constituents. In assays for DPPH free radical scavenging activity and tyrosinase inhibitory potential, the stilbenoids appeared to possess moderate effects, whereas the flavonoids showed only weak activity.     

Nematicidal effect of volatiles produced by Trichoderma sp.

Trichoderma is an important biocontrol agent that produces metabolites harmful to nematodes. We investigated the volatile organic compounds (VOCs) of Trichoderma sp. YMF 1.00416 and examined their abilities to kill nematodes. Chemical investigations of the VOCs from this strain led to the isolation and identification of three metabolites: a new compound, 1β-vinylcyclopentane-1α,3α-diol (1) and two known metabolites, 6-pentyl-2H-pyran-2-one (2) and 4-(2-hydroxyethyl)phenol (3). Nematicidal activity assays showed that compound 2 was nematicidal, and killed > 85% of Panagrellus redivivus, Caenorhabditis elegans, and Bursaphelenchus xylophilus in 48 h at 200 mg/L in a 2 mL vial. Our results will help identify new nematicides.     

Constituents of Leonotis leonurus flowering tops

Phytochemical investigation of flowering tops of Leonotis leonurus, yielded a new diterpene ester, 1,2,3-trihydroxy-3,7,11,15-tetramethylhexadecan-1-yl-palmitate along with five known metabolites. The structures of all compounds were determined by spectroscopic methods including 1D- and 2D NMR spectroscopy. All the isolated compounds were evaluated for antimalarial, cytotoxicity and for antimicrobial activities. Antimalarial activity for luteolin 7-O-β-d-glucopyranoside (4) (IC₅₀ = 2.2 μg/mL for the D6 clone and 1.8 μg/mL for the W2 clone) was observed. Chloroquine and artemisinin were used as positive controls which showed IC₅₀ of 0.016 and 0.0048 μg/mL for the D6 clone, respectively, and IC₅₀ of 0.14 and 0.0047 μg/mL for the W2 clone, respectively. None of the compounds were cytotoxic to Vero cells up to a concentration of 4.76 μg/mL.     

Discovery of inhibitors of brassinin oxidase based on the scaffolds of the phytoalexins brassilexin and wasalexin

Inhibitors of brassinin oxidase (BOLm), a unique phytoalexin detoxifying enzyme produced by the plant pathogenic fungus Leptosphaeria maculans (asexual stage Phoma lingam), were designed based on scaffolds of the phytoalexins brassilexin and wasalexin. Evaluation of these compounds using purified BOLm established that the inhibitory effect of brassilexin and derivatives decreased as follows: 6-chlorobrassilexin ≈ 6-bromobrassilexin > 5-bromobrassilexin ≈ 5-chlorobrassilexin ≈ 6-fluorobrassilexin > 8-methylbrassilexin > brassilexin ≈ 5-fluorobrassilexin. 6-Chlorobrassilexin was determined to be the best competitive inhibitor of BOLm discovered to date, with a K_i = 31 μM. Importantly, brassilexin and derivatives did not appear to induce BOLm in fungal cultures. Overall, these results suggest that the brassilexin scaffold is a good lead for further development of paldoxins against L. maculans, as it inhibits competitively BOLm without apparent induction.     

Identification and simultaneous determination of p-FHA and p-FBA,two metabolites of anti-tumor agent—Fluorapacin in rat urine

Fluorapacin, bis(4-fluorobenzyl)trisulfide, a small molecule natural product derivative of trisulfide, has revealed a broad spectrum of anti-proliferative activity and in vivo anti-tumor efficacy in human xenograft mice models with excellent safety profile. In the present study, two new metabolites, para-fluorohippuric acid (p-FHA) and para-fluorobenzoic acid (p-FBA), were identified by GC–MS and HPLC as the main metabolites in urine of rats after intravenous administration of fluorapacin. A simple HPLC-UV method for simultaneous determination of these two metabolites in urine has been developed and validated. The newly developed method demonstrated excellent specificity, accuracy, precision, and stability. This method was successfully employed to study the urinary excretion of fluorapacin in rats. The results indicated that p-FHA was the major metabolite in urine, and the total excretion recovery of p-FHA and p-FBA was 67.6 ± 4.9% (mean ± SE, n = 6) of dosage after 48 h of administration.     

Design,synthesis, antiviral and cytostatic activity of ω-(1H-1,2,3-triazol-1-yl)(polyhydroxy)alkylphosphonates as acyclic nucleotide analogues

The efficient synthesis of a new series of polyhydroxylated dibenzyl ω-(1H-1,2,3-triazol-1-yl)alkylphosphonates as acyclic nucleotide analogues is described starting from dibenzyl ω-azido(polyhydroxy)alkylphosphonates and selected alkynes under microwave irradiation. Selected O,O-dibenzylphosphonate acyclonucleotides were transformed into the respective phosphonic acids. All compounds were evaluated in vitro for activity against a broad variety of DNA and RNA viruses and for cytostatic activity against murine leukemia L1210, human T-lymphocyte CEM and human cervix carcinoma HeLa cells. Compound (1S,2S)-16b exhibited antiviral activity against Influenza A H3N2 subtype (EC₅₀ = 20 μM—visual CPE score; EC₅₀ = 18 μM—MTS method; MCC >100 μM, CC₅₀ >100 μM) in Madin Darby canine kidney cell cultures (MDCK), and (1S,2S)-16k was active against vesicular stomatitis virus and respiratory syncytial virus in HeLa cells (EC₅₀ = 9 and 12 μM, respectively). Moreover, compound (1R,2S)-16l showed activity against both herpes simplex viruses (HSV-1, HSV-2) in HEL cell cultures (EC₅₀ = 2.9 and 4 μM, respectively) and feline herpes virus in CRFK cells (EC₅₀ = 4 μM) but at the same time it exhibited cytotoxicity toward uninfected cell (MCC ⩾ 4 μM). Several other compounds have been found to inhibit proliferation of L1210, CEM as well as HeLa cells with IC₅₀ in the 4–50 μM range. Among them compounds (1S,2S)- and (1R,2S)-16l were the most active (IC₅₀ in the 4–7 μM range).