Katkam N. Gangadhar

Maritime Halophyte Species from Southern Portugal as Sources of Bioactive Molecules

Extracts of five halophytes from southern Portugal (Arthrocnemum macrostachyum, Mesembryanthemum edule, Juncus acutus, Plantago coronopus and Halimione portulacoides), were studied for antioxidant, anti-inflammatory and in vitro antitumor properties. The most active extracts towards the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical were the methanol extracts of M. edule (IC50 = 0.1 mg/mL) and J. acutus (IC50 = 0.4 mg/mL), and the ether extracts of J. acutus (IC50 = 0.2 mg/mL) and A. macrostachyum (IC50 = 0.3 mg/mL). The highest radical scavenging activity (RSA) against the 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical was obtained in the ether extract of J. acutus (IC50 = 0.4 mg/mL) and H. portulacoides (IC50 = 0.9 mg/mL). The maximum total phenolic content (TPC) was found in the methanol extract of M. edule (147 mg gallic acid equivalents (GAE)/g) and in the ether extract of J. acutus (94 mg GAE/g). Significant decreases in nitric oxide (NO) production were observed after incubation of macrophages with lipopolysaccharide (LPS) and the chloroform extract of H. portulacoides (IC50 = 109 µg/mL) and the hexane extract of P. coronopus (IC50 = 98.0 µg/mL). High in vitro cytotoxic activity and selectivity was obtained with the ether extract of J. acutus. Juncunol was identified as the active compound and for the first time was shown to display selective in vitro cytotoxicity towards various human cancer cells.

Synthesis and evaluation of sodium deoxycholate sulfate as a lipid drug carrier to enhance the solubility, stability and safety of an amphotericin B inhalation formulation.

Amphotericin B (AmB) is still used as the gold standard for therapy against invasive fungal diseases. However, the use of AmB through oral administration is restricted due to its low solubility and stability in aqueous solution, which is the cause for its poor bioavailability and highly varying absorption. Therefore, an attempt has been made to enhance the solubility and stability of AmB to evaluate its bioactivity and safety for use as an inhaler by using a new excipient sodium deoxycholate sulfate (SDS) with aim of using it as a drug carrier for AmB. Therefore, SDS was formulated together with AmB as a dry powder by lyophilization. The dry powder was reconstituted in distilled water and evaluated its physicochemical properties such as zeta potential, particle size and pH to compare its solubility and stability of the formulations with a SDC-AmB (i.e., known as Fungizone(®)). In vitro toxicity studies were carried out with red blood cells (RBC) and respiratory cell lines. Bioactivity was determined by a micro-dilution method against Candidaalbicans and Cryptococcusneoformans. We found that SDS-AmB had a zeta potential (-45.53 mV), which was higher than of Fungizone(®); and produced a stable particle size in solution (73.8 nm). The particle size distributions of both formulations were expressed as their mass median aerodynamic diameters (MMAD; 1.70 and 1.74 μm), their fine particle fractions (FPF; 70 and 80%) and geometric standard deviations (GSD; 2.3 and 2.0), respectively. These values indicated that the sizes were appropriate for use in an inhaler. Pure AmB was found to hemolyse RBC and was very toxic to alveolar macrophage cells, as their viability rapidly declined from 93 to 56% when the AmB concentration increased from 1 to 8 μg/mL. The SDS-AmB formulation had a significantly reduced toxicity compared to AmB. The results clearly indicated that the SDS-lipid based nanoparticles had the potential to be used as an alternative option to Fungizone(®) for an AmB formulation for inhalation.

Isololiolide, a carotenoid metabolite isolated from the brown alga Cystoseira tamariscifolia, is cytotoxic and able to induce apoptosis in hepatocarcinoma cells through caspase-3 activation, decreased Bcl-2 levels, increased p53 expression and PARP cleavage.

BACKGROUND Brown macroalgae have attracted attention because they display a wide range of biological activities, including antitumoral properties. Inthis study we isolated isololiolide from Cystoseira tamariscifolia for the first time. PURPOSE To examine the therapeutical potential of isololiolide against tumor cell lines. METHODS/STUDY DESIGN The structure of the compound was established and confirmed by 1D and 2D NMR as well as HRMS spectral analysis. The in vitro cytotoxicity was analyzed by colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay in tumoral as well as in non-tumoral cell lines. Cell cycle arrest and induction of apoptosis were assessed by flow cytometry. Alteration of expression levels in proteins important in the apoptotic cascade was analyzed by western blotting. RESULTS Isololiolidewas isolated for the first time from the brown macroalga C.tamariscifolia. Isololiolide exhibited significant cytotoxic activity against three human tumoral cell lines, namely hepatocarcinoma HepG2 cells, whereas no cytotoxicity was found in non-malignant MRC-5 and HFF-1 human fibroblasts. Isololiolide completely disrupted the HepG2 normal cell cycle and induced significant apoptosis. Moreover, western blot analysis showed that isololiolide altered the expression of proteins that are important in the apoptotic cascade, increasing PARP cleavage and p53 expression while decreasing procaspase-3 and Bcl-2 levels. CONCLUSION Isololiolide isolated from C. tamariscifolia is able to exert a selective cytotoxic activity on hepatocarcinoma HepG2 cells as well as induce apoptosis through the modulation of apoptosis-related proteins.

Urban wastewater treatment by Tetraselmis sp. CTP4 (Chlorophyta)

The ability of a recent isolate, Tetraselmis sp. CTP4, for nutrient removal from sewage effluents before and after the nitrification process under batch and continuous cultivation was studied. Biomass productivities in both wastewaters were similar under continuous conditions (0.343±0.053gL-1d-1) and nutrient uptake rates were maximal 31.4±0.4mgNL-1d-1 and 6.66±1.57mgP-PO43-L-1d-1 in WW before nitrification when cultivated in batch. Among batch treatments, cellular protein, carbohydrate and lipid levels shifted with aging cultures from 71.7±6.3 to 29.2±1.2%, 17.4±7.2 to 57.2±3.9% and 10.9±1.7 to 13.7±4.7%, respectively. In contrast, CTP4 cultivated continuously in Algal medium (control) showed lower biomass productivities (0.282gVSSL-1d-1) although improved lipid content (up to 20% lipids) in batch cultivation. Overall, Tetraselmis sp. CTP4 is promising for WW treatment as a replacement of the costly nitrification process, fixating more nutrients and providing a protein and carbohydrate-rich biomass as by-product.

Isolation of a euryhaline microalgal strain, Tetraselmis sp. CTP4, as a robust feedstock for biodiesel production

Bioprospecting for novel microalgal strains is key to improving the feasibility of microalgae-derived biodiesel production. Tetraselmis sp. CTP4 (Chlorophyta, Chlorodendrophyceae) was isolated using fluorescence activated cell sorting (FACS) in order to screen novel lipid-rich microalgae. CTP4 is a robust, euryhaline strain able to grow in seawater growth medium as well as in non-sterile urban wastewater. Because of its large cell size (9–22 μm), CTP4 settles down after a six-hour sedimentation step. This leads to a medium removal efficiency of 80%, allowing a significant decrease of biomass dewatering costs. Using a two-stage system, a 3-fold increase in lipid content (up to 33% of DW) and a 2-fold enhancement in lipid productivity (up to 52.1 mg L−1 d−1) were observed upon exposure to nutrient depletion for 7 days. The biodiesel synthesized from the lipids of CTP4 contained high levels of oleic acid (25.67% of total fatty acids content) and minor amounts of polyunsaturated fatty acids with ≥4 double bonds (<1%). As a result, this biofuel complies with most of the European (EN14214) and American (ASTM D6751) specifications, which commonly used microalgal feedstocks are usually unable to meet. In conclusion, Tetraselmis sp. CTP4 displays promising features as feedstock with lower downstream processing costs for biomass dewatering and biodiesel refining.

Can macroalgae provide promising anti-tumoral compounds? A closer look at Cystoseira tamariscifolia as a source for antioxidant and anti-hepatocarcinoma compounds

Marine organisms are a prolific source of drug leads in a variety of therapeutic areas. In the last few years, biomedical, pharmaceutical and nutraceutical industries have shown growing interest in novel compounds from marine organisms, including macroalgae. Cystoseira is a genus of Phaeophyceae (Fucales) macroalgae known to contain bioactive compounds. Organic extracts (hexane, diethyl ether, ethyl acetate and methanol extracts) from three Cystoseira species (C. humilis, C. tamariscifolia and C. usneoides) were evaluated for their total phenolic content, radical scavenging activity against 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radicals, and antiproliferative activity against a human hepatocarcinoma cell line (HepG2 cells). C. tamariscifolia had the highest TPC and RSA. The hexane extract of C. tamariscifolia (CTH) had the highest cytotoxic activity (IC50 = 2.31 µg/mL), and was further tested in four human tumor (cervical adenocarcinoma HeLa; gastric adenocarcinoma AGS; colorectal adenocarcinoma HCT-15; neuroblastoma SH-SY5Y), and two non-tumor (murine bone marrow stroma S17 and human umbilical vein endothelial HUVEC) cell lines in order to determine its selectivity. CTH strongly reduced viability of all tumor cell lines, especially of HepG2 cells. Cytotoxicity was particularly selective for the latter cells with a selectivity index = 12.6 as compared to non-tumor cells. Incubation with CTH led to a 2-fold decrease of HepG2 cell proliferation as shown by the bromodeoxyuridine (BrdU) incorporation assay. CTH-treated HepG2 cells presented also pro-apoptotic features, such as increased Annexin V/propidium iodide (PI) binding and dose-dependent morphological alterations in DAPI-stained cells. Moreover, it had a noticeable disaggregating effect on 3D multicellular tumor spheroids. Demethoxy cystoketal chromane, a derivative of the meroditerpenoid cystoketal, was identified as the active compound in CTH and was shown to display selective in vitro cytotoxicity towards HepG2 cells.

Assessment and comparison of the properties of biodiesel synthesized from three different types of wet microalgal biomass

In recent years, microalgae-based carbon-neutral biofuels (i.e., biodiesel) have gained considerable interest due to high growth rate and higher lipid productivity of microalgae during the whole year, delivering continuous biomass production as compared to vegetable-based feedstocks. Therefore, biodiesel was synthesized from three different microalgal species, namely Tetraselmis sp. (Chlorophyta) and Nannochloropsis oculata and Phaeodactylum tricornutum (Heterokontophyta), and the fuel properties of the biodiesel were analytically determined, unlike most studies which rely on estimates based on the lipid profile of the microalgae. These include density, kinematic viscosity, total and free glycerol, and high heating value (HHV), while cetane number (CN) and cold filter plugging point (CFPP) were estimated based on the fatty acid methyl ester profile of the biodiesel samples instead of the lipid profile of the microalgae. Most biodiesel properties abide by the ASTM D6751 and the EN 14214 specifications, although none of the biodiesel samples met the minimum CN or the maximum content of polyunsaturated fatty acids with ≥4 double bonds as required by the EN 14214 reference value. On the other hand, bomb calorimetric experiments revealed that the heat of combustion of all samples was on the upper limit expected for biodiesel fuels, actually being close to that of petrodiesel. Post-production processing may overcome the aforementioned limitations, enabling the production of biodiesel with high HHV obtained from lipids present in these microalgae.

Phase behavior of rifampicin in cholesterol-based liquid crystals and polyethylene glycol

The phase behavior of rifampicin in cholesteryl-based carbonate esters (CCEs), cholesterol and polyethylene glycol4000 (PEG4000) was determined to evaluate their potential in the formulation of dry powder inhalation dosage forms. To guide the identification of the most suitable liquid crystalline system for the solubilization of rifampicin, the dielectric constants were evaluated. Thereafter, the phase behavior of various mixtures were characterized by polarized light microscope (PLM), transmission electron microscopy (TEM), X-ray diffraction (XRD) and differential scanning calorimetry (DSC). Among the mixed-carrier systems, the cholesteryl cetyl carbonate (CCC) and PEG4000 system at 1:1 mole ratios yielded a homogeneous mixture with relatively high rifampicin content. This result was consistent with the prediction based on the dielectric constants. The phase diagram contained a eutectic point at CCC:PEG4000 (1:1) and 50 °C. Finally, the rifampicin drug was successfully incorporated into CCC and PEG system at a 1:6:6 mole ratio. Such a system would be amenable for oral inhalation.

In vitro and in silico approaches to unveil the mechanisms underlying the cytotoxic effect of juncunol on human hepatocarcinoma cells

BACKGROUND Juncunol is a phenanthrene isolated from the halophyte species Juncus acutus, with selective cytotoxic activity towards human hepatocarcinoma (HepG2) cells. However, its mechanism of action is unknown. METHODS The in vitro cytotoxic mechanism of juncunol was evaluated on HepG2 cells through several methods to elucidate its potential to induce apoptotic features, decrease mitochondrial membrane potential, promote internal ROS production and influence cell cycle. We also report its haemolytic activity on human erythrocytes and in silico DNA-binding studies. RESULTS Juncunol induced an increase in the number of apoptotic cells in a concentration-dependent manner, accompanied by a decrease in the mitochondrial membrane potential. No significant differences were observed in production of reactive oxygen species (ROS). Moreover, juncunol application at the IC50 value significantly induced cell cycle arrest in the G0/G1 phase comparatively to the control group. No significant haemolysis was detected. In silico studies indicate that juncunol seems to bind between GC base pairs. CONCLUSION Juncunol reduced HepG2 cells proliferation through the induction of apoptotic cellular death, in a concentration-dependent manner. Apoptosis induction seems to be related with a decrease of the mitochondrial membrane potential but not with ROS production. Juncunol had no haemolytic activity and may act as a DNA intercalator. Our data suggests juncunol as a suitable candidate for more detailed studies, including in vivo experiments, in order to completely characterize its mode of action.BACKGROUND Juncunol is a phenanthrene isolated from the halophyte species Juncus acutus, with selective cytotoxic activity towards human hepatocarcinoma (HepG2) cells. However, its mechanism of action is unknown. METHODS The in vitro cytotoxic mechanism of juncunol was evaluated on HepG2 cells through several methods to elucidate its potential to induce apoptotic features, decrease mitochondrial membrane potential, promote internal ROS production and influence cell cycle. We also report its haemolytic activity on human erythrocytes and in silico DNA-binding studies. RESULTS Juncunol induced an increase in the number of apoptotic cells in a concentration-dependent manner, accompanied by a decrease in the mitochondrial membrane potential. No significant differences were observed in production of reactive oxygen species (ROS). Moreover, juncunol application at the IC50 value significantly induced cell cycle arrest in the G0/G1 phase comparatively to the control group. No significant haemolysis was detected. In silico studies indicate that juncunol seems to bind between GC base pairs. CONCLUSION Juncunol reduced HepG2 cells proliferation through the induction of apoptotic cellular death, in a concentration-dependent manner. Apoptosis induction seems to be related with a decrease of the mitochondrial membrane potential but not with ROS production. Juncunol had no haemolytic activity and may act as a DNA intercalator. Our data suggests juncunol as a suitable candidate for more detailed studies, including in vivo experiments, in order to completely characterize its mode of action.

Microalgae-based unsaponifiable matter as source of natural antioxidants and metal chelators to enhance the value of wet Tetraselmis chuii biomass

Abstract The present work aimed to determine the antioxidant, metal chelating and neuroprotective potential of the unsaponifiable matter (UM) of Tetraselmis chuii to be applied to a biorefinery setting. The UM obtained via saponification from crude lipids extracted from microalgal wet biomass showed a radical scavenging activity (RSA) towards the DPPH radical of 90.7±1.3% and 57.1±1.2% at a concentration of 10 and 5 mg/mL, respectively. The UM fraction also displayed metal chelating capacity at a concentration of 5 mg/mL: 58.5±1.4% and 50.9±4.0% for copper and iron, respectively. The chemical characterization of the UM revealed significant levels of total phenolics (TPC, 13.61 mg GAE/g) and carotenoids (2.45 mg/g of β-carotene, lutein and violaxanthin). Overall, the separation of the UM containing high value metabolites might significantly upgrade the total wet biomass value in a biorefinery, allowing the exploitation of a stream with relevant antioxidant and metal chelating activities.