1H NMR and MVA metabolomic profiles of urines from piglets fed with boluses contaminated with a mixture of five mycotoxins

Metabolic profile of urine from piglets administered with single boluses contaminated with mycotoxin mixture (deoxynivalenol, aflatoxin B₁, fumonisin B₁, zearalenone, and ochratoxin A) were studied by ¹H NMR spectroscopy and chemometrics (PCA, PLS-DA, and OPLS-DA). The mycotoxin levels were close to the established maximum and guidance levels for animal feed (2003/100/EC and 2006/576/EC). Urine samples were obtained from four groups of four piglets before (control, C) or within 24 h (treated, T) after receiving a contaminated boluses with increasing doses of mycotoxins (boluses 1–4). For the two highest dose groups, the urines were collected also after one week of wash out (W). For the two lowest doses groups no significant differences between the C and T samples were observed. By contrast, for the two highest doses groups the T urines separated from the controls for a higher relative content of creatinine, p-cresol glucuronide and phenyl acetyl glycine and lower concentration of betaine and TMAO. Interestingly, a similar profile was found for both W and T urines suggesting, at least for the highest doses used, serious alteration after a single bolus of mycotoxin mixture.     

Simultaneous occurrence of mycotoxins in human food commodities from Cameroon

Eighty-two samples of dried food commodities from Cameroon were screened and quantified for different mycotoxins, including fumonisin B₁ (FB₁), zearalenone (ZEA), deoxynivalenol (DON), aflatoxin (AF) and ochratoxin A (OTA), by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC), respectively. The percentage of positive samples was as follows: FB₁ 41%, AF 51%, ZEA 57%, DON 65% and OTA 3%. High FB₁ contents were found in maize, averaging 3,684 μg/kg (range: 37-24,225 μg/kg), whereas the highest average ZEA level was found in peanuts (70 μg/kg), followed by maize (69 μg/kg), rice (67 μg/kg) and beans (48 μg/kg) with no ZEA was detected in soybeans. DON contents were low, ranging from 13 to 273 μg/kg, and for AF the average content was 2.6 μg/kg with peanuts and maize as principal substrates. The incidence of OTA was low, with a mean level of 6.4 μg/kg recorded. The majority (79%) of samples contained more than one mycotoxin and the most frequent co-occurrence found was FB₁ + ZEA + DON, detected in 21% of samples (mainly maize) analysed. Co-contamination with FB₁ + ZEA + DON + AF was found in 11% of the samples. Although a large proportion of samples had fairly low levels of individual mycotoxins, this should be of concern as the co-occurrence of mycotoxins may generate additive or synergistic effect in humans, especially if the respective commodities are consumed almost on a daily basis.     

In Vitro Detoxification of Aflatoxin B1, Deoxynivalenol,Fumonisins, T-2 Toxin and Zearalenone by Probiotic Bacteria from Genus Lactobacillus and Saccharomyces cerevisiae Yeast

The aim of the following research was to determine the detoxification properties of probiotic Lactobacillus sp. bacteria (12 strains) and S. cerevisiae yeast (6 strains) towards mycotoxins, such as aflatoxin B₁, deoxynivalenol, fumonisins, T-2 toxin and zearalenone, which pose as frequent feed contamination. The experiment involved analysing changes in concentration of mycotoxins in PBS solutions, after 6, 12 and 24 h of incubation with monocultures of tested microorganisms, measured by high-performance liquid chromatography (HPLC). We found that all strains detoxified the mycotoxins, with the highest reduction in concentration observed for the fumonisin B₁ and B₂ mixture, ranging between 62 and 77% for bacterial strains and 67–74% for yeast. By contrast, deoxynivalenol was the most resistant mycotoxin: its concentration was reduced by 19–39% by Lactobacillus sp. strains and 22–43% by yeast after 24 h of incubation. High detoxification rates for aflatoxin B₁, T-2 toxin and zearalenone were also observed, with concentration reduced on average by 60%, 61% and 57% by Lactobacillus, respectively, and 65%, 69% and 52% by yeast, respectively. The greatest extent of reduction in the concentration for all mycotoxins was observed after 6 h of incubation; however, a decrease in concentration was noted even after 24 h of incubation. Thus, the tested microorganisms can potentially be used as additives to decrease the concentrations of toxins in animal feed.

     

Acylated phenylethanoid oligoglycosides with hepatoprotective activity from the desert plant Cistanche tubulosa

The methanolic extract from fresh stems of Cistanche tubulosa (Orobanchaceae) was found to show hepatoprotective effects against d-galactosamine (d-GalN)/lipopolysaccharide (LPS)-induced liver injury in mice. From the extract, three new phenylethanoid oligoglycosides, kankanosides H₁ (1), H₂ (2), and I (3), were isolated together with 16 phenylethanoid glycosides (4–19) and two acylated oligosugars (20, 21). The structures of 1–3 were determined on the basis of spectroscopic properties as well as of chemical evidence. Among the isolates, echinacoside (4, IC₅₀ = 10.2 μM), acteoside (5, 4.6 μM), isoacteoside (6, 5.3 μM), 2′-acetylacteoside (8, 4.8 μM), and tubuloside A (10, 8.6 μM) inhibited d-GalN-induced death of hepatocytes. These five isolates, 4 (31.1 μM), 5 (17.8 μM), 6 (22.7 μM), 8 (25.7 μM), and 10 (23.2 μM), and cistantubuloside B₁ (11, 21.4 μM) also reduced TNF-α-induced cytotoxicity in L929 cells. Moreover, principal constituents (4–6) exhibited in vivo hepatoprotective effects at doses of 25–100 mg/kg, po.     

The effect of inclusion of lupins/triticale whole crop silage in the diet of winter finishing beef cattle on their performance and meat quality at two levels of concentrates

This study investigated the performance and instrumental meat quality of finishing beef steers offered grass silage (GS), grass silage:maize silage (GS:MS) and grass silage:lupins/triticale silage (GS:LT). The lupins/triticale silage was grown as either two separate crops in the same field and harvested together (LT1) or grown and harvested as a mixture (LT2). The silages were offered to eighty continental cross beef steers, initial live weight 530 ± 47.7 kg and 18 ± 1.6 months of age and were supplemented with 3 or 6 kg concentrates fresh/head/d. Silage was fed ad libitum with the mixtures offered at a ratio of 60 GS:40 maize silage (MS), LT1 or LT2 on a dry matter (DM) basis and concentrates were offered once (3 kg) or twice (6 kg) daily on top of the silage. Animals were slaughtered in three batches after 100, 117 and 124 d on experiment. The LT1 and LT2 produced yields of 8.7 and 7.5 kg DM/ha and the silages were poorly fermented as demonstrated by high ammonia-N concentration (182 and 173 g/kg total N), low lactic acid (9 g/kg DM) and high pH (5.0 and 4.7). Silage type had no significant effect on slaughter live weight, liveweight gain, carcass gain, forage DM intake (DMI), total DMI or feed efficiency expressed as kg DMI/kg liveweight gain or kg DMI/kg carcass gain. Silage type had no effect on carcass characteristics or instrumental meat quality. The results of this study demonstrate that offering lupins/triticale silage in combination with high quality grass silage (D-value greater than 700) at a ratio of 60 grass silage:40 LT1 or LT2 on a DM basis had no effect on animal performance, carcass characteristics or meat quality parameters relative to high quality grass silage offered alone or in combination with maize silage.     

Pencitrin and pencitrinol,two new citrinin derivatives from an endophytic fungus Penicillium citrinum salicorn 46

Two new citrinin derivatives, pencitrin (1) and pencitrinol (2), and a known compound citrinin (3), together with its two known dimers, penicitrinone A (4), penicitrinone E (5), were isolated from an endophytic fungus P. citrinum 46 derived from Salicornia herbacea Torr. by adding CuCl₂ into fermentation medium. The structures of these compounds were elucidated by detailed spectroscopic analysis, and the absolute configurations of 1, 4, and 5 were determined by comparison of quantum chemical time-dependent density functional theory (TDDFT) calculated and electronic circular dichroism (ECD) spectra. Compound 1 exhibited potent cytotoxic activities towards A549 human lung cancer cells and HepG2 human liver cancer cells with IC₅₀ values of 23.73 ± 3.61 and 35.73 ± 2.15 μM, respectively, whereas compound 5 showed moderate cytotoxic activities towards A549 and HepG2 cancer cells with IC₅₀ values of 40.47 ± 4.52 and 53.57 ± 3.24 μM, respectively. The results from the current research highlighted the effectiveness and usefulness of the pipeline to discover novel bioactive fungal secondary metabolites by modification of the culture media.     

Morphology and bloom dynamics of Cochlodinium geminatum (Schütt) Schütt in the Pearl River Estuary,South China Sea

An unarmored dinoflagellate bloom of Cochlodinium geminatum (Schütt) Schütt has been identified in the Pearl River Estuary, South China Sea during the severe dry season, from late October to early November, 2009, when temperature and salinity ranged between 20.0–27.2 °C and 10.6–33.4, respectively. Light and scanning electron microscopy were used to identify the characteristics of C. geminatum and provided the clear morphological structure for this species. The organism was primarily found in chains of two cells or single cell, and no longer chains were observed. Cells were irregularly spherical or slightly dorso-ventrally, with size ranged between 28 and 36 μm and longer than wide. A large nucleus in the center with numerous golden chloroplasts was present, and the cingulum made 1.5 turns around the cell. The concentration of C. geminatum ranged from 10² to greater than 10⁷ cells l⁻¹ during the bloom period. Nutrient concentration ranges during the bloom were 1.29–81.00 μM NO₃, 0.14–12.14 μM NO₂, 0.21–6.29 μM NH₄, 0.23–6.26 μM PO₄ and 3.29–171.43 μM SiO₃, respectively. Total biomass expressed in terms of chlorophyll a ranged from 2.44 to 135.45 μg l⁻¹, with an average 19.9 μg l⁻¹ in surface water throughout the PRE. Two main clusters corresponding to the water sectors were defined with multivariate analysis (cluster and nMDS). Based on the composition and abundance of phytoplankton, spatial variations were observed at a significant level (ANOSIM, R = 0.44, P < 0.01). Although the pairwise correlation analysis detected no significant effect of any single environmental variable on the abundance of C. geminatum, the multivariate analysis (BIO-ENV) between biotic and abiotic variables resulted in the best variables combination with all measured factors involved (temperature, salinity, turbidity, NO₃, NO₂, NH₄, PO₄ and SiO₃) which showed a combined effect during the bloom of C. geminatum in the Pearl River Estuary (ρ_w = 0.477).     

Dimeric cyclohexane-1,3-dione oximes inhibit wheat acetyl-CoA carboxylase and show anti-malarial activity

A series of dimeric 1,3-cyclohexanedione oxime ethers were synthesized and found to have significant antiplasmodial activity with IC₅₀’s in the range 3–12 μM. The most active dimer was tested in the Plasmodium berghei mouse model of malaria and at a dose of 48 mg/kg gave a 45% reduction in parasitaemia. Several commercial herbicides, all known to be inhibitors of maize acetyl-CoA carboxylase, were also tested for antimalarial activity, but were essentially inactive with the exception of butroxydim which gave an IC₅₀ of 10 μM.     

Design,synthesis of 2,3-disubstitued 4(3H)-quinazolinone derivatives as anti-inflammatory and analgesic agents: COX-1/2 inhibitory activities and molecular docking studies

A new series, 2-substituted mercapto-3-[2-(pyridin-2-yl)ethyl]-4(3H)-quinazolinone 1–21, was synthesized and evaluated for in vivo anti-inflammatory and analgesic activities and in vitro COX-1/COX-2 inhibition. Compounds 1, 4, 5, 6, 8, 10, 13, 14, 15, 16, and 17 exhibited potent anti-inflammatory and analgesic properties, with ED₅₀ values of 50.3–112.1 mg/kg and 12.3–111.3 mg/kg, respectively. These values may be compared with those of diclofenac sodium (ED₅₀ = 112.2 and 100.4 mg/kg) and celecoxib (ED₅₀ = 84.3 and 71.6 mg/kg). Compounds 4 and 6 possessed strong COX-2 inhibitory activity with IC₅₀ (0.33 μM and 0.40 μM, respectively) and selectivity index (SI > 303.0 and >250.0, respectively) values that are similar to those of the reference drug celecoxib (IC₅₀ 0.30 μM and COX-2 SI > 333). Compounds 5, 8, and 13 demonstrated effective COX-2 inhibitory activity with IC₅₀ values of 0.70–0.80 μM and COX-2 SI > 125–142. Potent COX-2 inhibitors, such as compounds 4, 6, and 13, were docked into the active site pockets of COX-1 and COX-2, with the greatest recognition occurring at the COX-2 binding site and insignificant interactions at the binding site of the COX-1 pocket.     

Assessing the trypanocidal potential of natural and semi-synthetic diketopiperazines from two deep water marine-derived fungi

Human African trypanosomiasis (HAT, commonly known as African sleeping sickness) is categorized as a neglected disease, as it afflicts >50,000 people annually in sub-saharan Africa, and there are few formal programs in the world focused on drug discovery approaches for this disease. In this study, we examined the crude extracts of two fungal strains (Aspergillus fumigatus and Nectria inventa) isolated from deep water sediment which provided >99% growth inhibition at 1 μg/mL of Trypanosoma brucei, the causative parasite of HAT. A collection of fifteen natural products was supplemented with six semi-synthetic derivatives and one commercially available compound. Twelve of the compounds, each containing a diketopiperazine core, showed excellent activity against T. brucei (IC₅₀ = 0.002–40 μM), with selectivity over mammalian cells as great as 20-fold. The trypanocidal diketopiperazines were also tested against two cysteine protease targets Rhodesain and TbCatB, where five compounds showed inhibition activity at concentrations less than 20 μM. A preliminary activity pattern is described and analyzed.     

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).     

The effects of light intensity on the growth of Japanese Gambierdiscus spp. (Dinophyceae)

Marine toxic dinoflagellates of the genus Gambierdiscus are the causative agents of ciguatera fish poisoning (CFP), a form of seafood poisoning that is widespread in tropical, subtropical and temperate regions worldwide. The distributions of Gambierdiscus australes, Gambierdiscus scabrosus and two phylotypes of Gambierdiscus spp. type 2 and type 3 have been reported for the waters surrounding the main island of Japan. To explore the bloom dynamics and the vertical distribution of these Japanese species and phylotypes of Gambierdiscus, the effects of light intensity on their growth were tested, using a photoirradiation-culture system. The relationship between the observed growth rates and light intensity conditions for the four species/phylotypes were formulated at R > 0.92 (p < 0.01) using regression analysis and photosynthesis-light intensity (P-L) model. Based on this equation, the optimum light intensity (L_max) and the semi-optimum light intensity range (L_s-opt) that resulted in the maximum growth rate (μ_max) and ≥80% μ _max values of the four species/phylotypes, respectively, were as follows: (1) the L_max and L_s-opt of G. australes were 208 μmol photons m⁻² s⁻¹ and 91–422 μmol photons m⁻² s⁻¹, respectively; (2) those of G. scabrosus were 252 and 120–421 μmol photons m⁻² s⁻¹, respectively; (3) those of Gambierdiscus sp. type 2 were 192 and 75–430 μmol photons m⁻² s⁻¹, respectively; and (4) those of Gambierdiscus sp. type 3 were ≥427 and 73–427 μmol photons m⁻² s⁻¹, respectively. All four Gambierdiscus species/phylotypes required approximately 10 μmol photons m⁻² s⁻¹ to maintain growth. The light intensities in coastal waters at a site in Tosa Bay were measured vertically at 1 m intervals once per season. The relationships between the observed light intensity and depth were formulated using Beer’s Law. Based on these equations, the range of the attenuation coefficients at Tosa Bay site was determined to be 0.058–0.119 m⁻¹. The values 1700 μmol photons m⁻² s⁻¹, 500 μmol photons m⁻² s⁻¹, and 200 μmol photons m⁻² s⁻¹ were substituted into the equations to estimate the vertical profiles of light intensity at sunny midday, cloudy midday and rainy midday, respectively. Based on the regression equations coupled with the empirically determined attenuation coefficients for each of the four seasons, the ranges of the projected depths of L_max and L_s-opt for the four Gambierdiscus species/phylotypes under sunny midday conditions, cloudy midday conditions, and rainy midday conditions were 12–38 m and 12–54 m, 1–16 m and 1–33 m, and 0 m and 0–16 m, respectively. These results suggest that light intensity plays an important role in the bloom dynamics and vertical distribution of Gambierdiscus species/phylotypes in Japanese coastal waters.     

Structural changes that occur upon photolysis of the Fe(II)a3–CO complex in the cytochrome ba3-oxidase of Thermus thermophilus: A combined X-ray crystallographic and infrared spectral study demonstrates CO binding to CuB

The purpose of the work was to provide a crystallographic demonstration of the venerable idea that CO photolyzed from ferrous heme-a₃ moves to the nearby cuprous ion in the cytochrome c oxidases. Crystal structures of CO-bound cytochrome ba₃-oxidase from Thermus thermophilus, determined at ~ 2.8–3.2 Å resolution, reveal a Fe–C distance of ~ 2.0 Å, a Cu–O distance of 2.4 Å and a Fe–C–O angle of ~ 126°. Upon photodissociation at 100 K, X-ray structures indicate loss of Fe_a3–CO and appearance of Cu_B–CO having a Cu–C distance of ~ 1.9 Å and an O–Fe distance of ~ 2.3 Å. Absolute FTIR spectra recorded from single crystals of reduced ba₃–CO that had not been exposed to X-ray radiation, showed several peaks around 1975 cm^? 1; after photolysis at 100 K, the absolute FTIR spectra also showed a significant peak at 2050 cm^? 1. Analysis of the ‘light’ minus ‘dark’ difference spectra showed four very sharp CO stretching bands at 1970 cm^? 1, 1977 cm^? 1, 1981 cm^? 1, and 1985 cm^? 1, previously assigned to the Fe_a3–CO complex, and a significantly broader CO stretching band centered at ~ 2050 cm^? 1, previously assigned to the CO stretching frequency of Cu_B bound CO. As expected for light propagating along the tetragonal axis of the P4₃2₁2 space group, the single crystal spectra exhibit negligible dichroism. Absolute FTIR spectrometry of a CO-laden ba₃ crystal, exposed to an amount of X-ray radiation required to obtain structural data sets before FTIR characterization, showed a significant signal due to photogenerated CO₂ at 2337 cm^? 1 and one from traces of CO at 2133 cm^? 1; while bands associated with CO bound to either Fe_a3 or to Cu_B in “light” minus “dark” FTIR difference spectra shifted and broadened in response to X-ray exposure. In spite of considerable radiation damage to the crystals, both X-ray analysis at 2.8 and 3.2 Å and FTIR spectra support the long-held position that photolysis of Fe_a3–CO in cytochrome c oxidases leads to significant trapping of the CO on the Cu_B atom; Fe_a3 and Cu_B ligation, at the resolutions reported here, are otherwise unaltered. This article is part of a Special Issue entitled: Respiratory Oxidases.     

Acylated iridoid glycosides and acylated rhamnopyranoses from Gmelina arborea flowers

Nine acylated iridoid glycosides (1–9), five acylated rhamnopyranoses (10–14) and verbascoside (15) were isolated from Gmelina arborea flowers, including 5 new compounds (1, 2, and 10–12). The cytoprotective activity of 11 selected compounds (1–8, 10, 11, and 15) against CCl₄-induced cytotoxicity on liver was determined. Compounds 1, 2, 4, 7, 8 and 15 displayed hepatoprotective activity. 6-O-α-l-(2″, 3″-di-O-trans-p-hydroxycinnamoyl)rhamnopyranosylcatalpol (2) exhibited the most potent cytoprotective effect with an EC₅₀ value of 42.5 μM (SI = 19.3) compared with biphenyldimethylesterate (DDB, EC₅₀ = 277.3 μM, SI = 9.8) and bicylo-ethanol (EC₅₀ = 279.2 μM, SI = 12.2). Among the acylated iridoid glycosides, the compounds (2 and 8) containing phenolic hydroxy groups were more active than were those lacking them.     

Interaction between vitamin B6 and source of selenium on the response of the selenium-dependent glutathione peroxidase system to oxidative stress induced by oestrus in pubertal pig

This study aimed to assess the interaction between vitamin B₆ and selenium (Se) for the flow of Se towards the Se-dependent glutathione peroxidase (GPX) system in response to oxidative stress naturally induced by oestrus in a pubertal pig model. At first oestrus, forty-five gilts were randomly assigned to the experimental diets (n = 9/group): basal diet (CONT); CONT + 0.3 mg/kg of Na-selenite (MSeB₆0); MSeB₆0 + 10 mg/kg of HCl-B₆ (MSeB₆10); CONT + 0.3 mg/kg of Se-enriched yeast (OSeB₆0); and OSeB₆0 + 10 mg/kg of HCl-B₆ (OSeB₆10). Blood samples were collected at each oestrus (long-term profiles), and daily from day −4 to +3 (slaughter) of the fourth oestrus (peri-oestrus profiles) after which liver, kidneys, and ovaries were collected. For long-term profiles, CONT had lower blood Se than Se-supplemented gilts (p < 0.01) and OSe was higher than MSe (p < 0.01). Lower erythrocyte pyridoxal-5-phosphate was found in B₆0 than B₆10 (p < 0.01). No treatment effect was observed on GPX activity. For peri-oestrus profiles, treatment effects were similar to long-term profiles. Treatment effects on liver Se were similar to those for long-term blood Se profiles and OSe had higher renal Se concentrations than MSe gilts (p < 0.01). Gene expressions of GPX1, GPX3, GPX4, and selenocysteine lyase in liver and kidney were greatest in OSeB₆10 gilts (p < 0.05). These results suggest that dietary B₆ modulate the metabolic pathway of OSe towards the GPX system during the peri-oestrus period in pubertal pigs.     

Calcium protection of PS2 complex of Phaseolus coccineus from cadmium toxicity: In vitro study

The action of 10 and 20 mM Ca against harmful Cd effect on PS2 complex isolated from leaves of Phaseolus coccineus L. cv. Pi?kny Ja? was studied. The changes in fast chlorophyll a fluorescence induction kinetics and protein composition of PS2 complex were the symptoms of Cd toxicity and Ca protection of PS2 complex. Calcium applied at 10 mM concentration prevented F₀ reduction caused by the presence of 250–1000 μM Cd in the incubation mixture, but that of (the variable chlorophyll a fluorescence) F_v, F_m, F_v/F₀, and F_v/F_m only at 250 μM Cd. Ca concentration doubling in the incubation mixture resulted in complete overcoming the toxicity of 250–1000 μM Cd to F_v and F_m. However, the protection of F_v/F₀ and the photochemical efficiency of PS2 (F_v/F_m) from 1000 μM Cd was only partial even at 20 mM Ca. A protective effect of 10 mM Ca on D1, D2 and 17 kDa proteins was found in PS2 complex exposed to 250 μM Cd, and on 43 kDa protein in the complex treated with 500 μM Cd. However, 20 mM Ca counteracted the toxicity of 500 μM Cd to the 43, 47 and 17 kDa proteins, as well as the harmful effect of 1000 μM Cd on 47 and 17 kDa ones.     

Determination of asiatic acid in beagle dog plasma after oral administration of Centella asiatica extract by precolumn derivatization RP-HPLC

A novel precolumn derivatization reversed-phase high-performance liquid chromatography (RP-HPLC) method with UV–vis detection for the quantitative determination of total concentration of asiatic acid (AA) in beagle dog plasma is described. AA was extracted with n-hexane-dichloromethane-2-propanol (20:10:1, v/v/v) from plasma, which had been hydrolyzed by acid and derivatized with p-Toluidine. Chromatographic separation was achieved on a C₁₈ column using gradient elution in a water–methanol system. Detection was set at UV wavelength of 248 nm. A calibration curve ranging from 0.01 to 1.5 μg/mL was shown to be linear, and the lower limit of quantification (LLOQ) was 0.01 μg/mL. The intra- and inter-day precisions which were determined by three different concentrations (0.05, 0.2 and 0.8 μg/mL) ranged from 4.4% to 13.1% and 4.6% to 14.2%, respectively. Mean extraction recoveries were no less than 65% for AA and ursolic acid (IS). Plasma samples containing asiatic acid were stable for 30 days at ?20 °C. The method was successfully applied to a pharmacokinetic study in beagle dogs after oral administration of Centella asiatica extract, and the main pharmacokinetic parameters obtained were: T_1/2, 4.29 h; T_max, 2.70 h; C_max, 0.74 μg/mL; AUC_0–t and AUC_0–∞, 3.74 and 3.82 μg h/mL, respectively.     

Triterpenes from the roots of Lantana montevidensis with antiprotozoal activity

Bioassay guided fractionation of the roots of Lantana montevidensis (Verbenaceae) has resulted in the isolation and identification of three new triterpenoids; 13β-hydroxy-3-oxo-olean-11-en-28-oic acid (1), 12β,13β-dihydroxyolean-3-oxo-28-oic acid (2) and 12β,13β,22β-trihydroxyolean-3-oxo-28-oic acid (3) in addition to nine known compounds: oleanonic acid (4), oleanolic acid (5), 3β,25β-dihydroxy-olean-12-en-28-oic acid (6), lantadene A (7), 19α-hydroxy-3-oxo-olean-12-en-28-oic acid (8) pomolic acid (9), camaric acid (10) together with β-sitosterol (11) and β-sitosterol-3-O-β-d-glucoside (12). The structures of the isolated metabolites were elucidated based on comprehensive 1D and 2D NMR spectroscopic data as well as HR-ESI–MS. The extracts and the isolated metabolites were evaluated for their antiprotozoal and antimicrobial activities. Compound 2 showed antibacterial activity against Staphylococcus aureus and methicillin resistant S. aureus with IC₅₀ values against both organisms of 2.1 μM and compound 10 showed activity against same organisms with IC₅₀ values 8.74 and 8.09 μM, respectively, compared to the positive control ciprofloxacin (IC₅₀ = 0.3 μM against S. aureus and MRSA). Compounds 1, 4, 5, 6, and 10 showed moderate antileishmanial activity with IC₅₀ values ranging between (2.54–14.95 μM) and IC₉₀ values ranging between (11.90–19.47 μM), using pentamidine as a control (IC₅₀ values 2.09 ≥ 16.8 μM) and IC₉₀ values ranging between (4.72 ≥ 16.8 μM). These compounds also showed highly potent antitrypanosomal activity with IC₅₀ values ranging between (0.39–7.12 μM) and IC₉₀ values ranging between (1.91–10.51 μM), which are more efficient than the DFMO, the antitrypanosomal drug employed as positive control (IC₅₀ and IC₉₀values 11.82 and 30.82 μM).     

Comparing anti-HIV,antibacterial, antifungal,micellar, and cytotoxic properties of tricarboxylato dendritic amphiphiles

Three series of homologous dendritic amphiphiles—RCONHC(CH₂CH₂COOH)₃, 1(n); ROCONHC(CH₂CH₂COOH)₃, 2(n); RNHCONHC(CH₂CH₂COOH)₃, 3(n), where R = n-C_nH_2n+1 and n = 13–22 carbon atoms—were assayed for their potential to serve as antimicrobial components in a topical vaginal formulation. Comparing epithelial cytotoxicities to the ability of these homologues to inhibit HIV, Neisseria gonorrhoeae, and Candida albicans provided a measure of their prophylactic/therapeutic potential. Measurements of the ability to inhibit Lactobacillus plantarum, a beneficial bacterium in the vagina, and critical micelle concentrations (CMCs), an indicator of the potential detergency of these amphiphiles, provided additional assessments of safety. Several amphiphiles from each homologous series had modest anti-HIV activity (EC₅₀ = 110–130 μM). Amphiphile 2(18) had the best anti-Neisseria activity (MIC = 65 μM), while 1(19) and 1(21) had MICs against C. albicans of 16 and 7.7 μM, respectively. Two measures of safety showed promise as all compounds had relatively low cytotoxic activity (EC₅₀ = 210–940 μM) against epithelial cells and low activity against L. plantarum, 1(n), 2(n), and 3(n) had MICs ? 490, 1300, and 940 μM, respectively. CMCs measured in aqueous triethanolamine and in aqueous potassium hydroxide showed linear dependences on chain length. As expected, the longest chain in each series had the lowest CMC—in triethanolamine: 1(21), 1500 μM; 2(22), 320 μM; 3(22), 340 μM, and in potassium hydroxide: 1(21), 130 μM; 3(22), 40 μM. The CMC in triethanolamine adjusted to pH 7.4 was 400 μM for 1(21) and 3900 μM for 3(16). The promising antifungal activity, low activity against L. plantarum, relatively high CMCs, and modest epithelial cytotoxicity in addition to their anti-Neisseria properties warrant further design studies with dendritic amphiphiles to improve their safety indices to produce suitable candidates for antimicrobial vaginal products.     

Effects of major tanshinones isolated from Danshen (Salvia miltiorrhiza) on rat CYP1A2 expression and metabolism of model CYP1A2 probe substrates

This study explored the effects of Danshen on metabolism/pharmacokinetics of model CYP1A2 substrates and hepatic CYP1A2 expression in rats. The effects of Danshen and tanshinones on CYP1A2 activity was determined by metabolism of model substrates in vitro (phenacetin) and in vivo (caffeine). HPLC was used to determine model substrates/metabolites. The effect of Danshen on CYP1A2 expression was determined by Western blot. Tanshinones (1.25–50 μM) competitively inhibited phenacetin O-deethylation in vitro. Inhibition kinetics studies showed the K_i values were in the order: dihydrotanshinone (3.64 μM), cryptotanshinone (4.07 μM), tanshinone I (22.6 μM) and tanshinone IIA (23.8 μM), furafylline (35.8 μM), a CYP1A2 inhibitor. The Ki of Danshen extract (mainly tanshinones) was 72 μg/ml. Acute Danshen extract treatment (50–200 mg/kg, i.p.) decreased metabolism of caffeine to paraxanthine, with overall decrease in caffeine clearance (14–22%); increase in AUC (11–25%) and plasma T_1/2 (12–16%). Danshen treatment with (100 mg/kg/day, i.p. or 200 mg/kg/day, p.o.) for three or fourteen days showed similar pharmacokinetic changes of the CYP1A2 probe substrate without affecting CYP1A2 expression. This study demonstrated that major tanshinones competitively inhibited the metabolism of model CYP1A2 probe substrates but had no effect on rat CYP1A2 expression.