Activation of intrinsic apoptotic signaling pathway in cancer cells by Cymbopogon citratus polysaccharide fractions.

Essential oils of Cymbopogon citratus were already reported to have wide ranging medical and industrial applications. However, information on polysaccharides from the plant and their anticancer activities are limited. In the present study, polysaccharides from C. citratus were extracted and fractionated by anion exchange and gel filtration chromatography. Two different polysaccharide fractions such as F1 and F2 were obtained, and these fractions were found to have distinct acidic polysaccharides as characterized by their molecular weight and sugar content. NMR spectral analysis revealed the presence of (1→4) linked b-d-Xylofuranose moiety in these polysaccharides. Using these polysaccharide fractions F1 and F2, anti-inflammatory and anticancer activities were evaluated against cancer cells in vitro and the mechanism of action of the polysaccharides in inducing apoptosis in cancer cells via intrinsic pathway was also proposed. Two different reproductive cancer cells such as Siha and LNCap were employed for in vitro studies on cytotoxicity, induction of apoptosis and apoptotic DNA fragmentation, changes in mitochondrial membrane potential, and profiles of gene and protein expression in response to treatment of cells by the polysaccharide fractions. These polysaccharide fractions exhibited potential cytotoxic and apoptotic effects on carcinoma cells, and they induced apoptosis in these cells through the events of up-regulation of caspase 3, down-regulation of bcl-2 family genes followed by cytochrome c release.

Induction of ROS-dependent mitochondria-mediated intrinsic apoptosis in MDA-MB-231 cells by glycoprotein from Codium decorticatum.

Marine macroalgae consist of a range of bioactive molecules exhibiting different biological activities, and many of these properties are attributed to sulfated polysaccharides, fucoxanthin, phycobiliproteins, and halogenated compounds. In this study, a glycoprotein (GLP) with a molecular mass of ∼48 kDa was extracted and purified from Codium decorticatum and investigated for its cytotoxic properties against human MDA-MB-231 breast cancer cells. The IC50 values of GLP against MDA-MB-231 and normal breast HBL-100 cells (control) were 75 ± 0.23 µg/mL (IC25), 55 ± 0.32 µg/mL (IC50), and 30 ± 0.43 µg/mL (IC75) and 90 ± 0.57 µg/mL (IC25), 80 ± 0.48 µg/mL (IC50), and 60 ± 0.26 µg/mL (IC75), respectively. Chromatin condensation and poly(ADP-ribose) polymerase (PARP) cleavage studies showed that the GLP inhibited cell viability by inducing apoptosis in MDA-MB-231 cells. Induction of mitochondria-mediated intrinsic apoptotic pathway by GLP was evidenced by the events of loss of mitochondrial membrane potential (ΔΨ(m)), bax/bcl-2 dysregulation, cytochrome c release, and activation of caspases 3 and 9. Apoptosis-associated factors such as reactive oxygen species (ROS) formation and loss of ΔΨ(m) were evaluated by DCFH-DA staining and flow cytometry, respectively. Cell cycle arrest of G2/M phase and expression of apoptosis associated proteins were determined using flow cytometry and Western blotting, respectively.

Optimized extraction of polysaccharides from Cymbopogon citratus and its biological activities.

In this study the extraction of hot water soluble polysaccharides (HWSPs) from Cymbopogon citratus using hot water decoction was discussed. Response surface methodology (RSM) based on a three level, three variable central composite rotatable design (CCRD), was employed to obtain best possible combination of extraction time (X1: 30-180 min), extraction temperature (X2: 70-100 °C) and water to the raw material ratio (X3: 10-60) for maximum HWSPs extraction. The optimum extraction conditions were as follows: extraction time was around 113.81 min, extraction temperature at 99.66 °C and the ratio of water to raw material was 33.11 g/mL. Under these conditions, the experimental yield was 13.24±0.23%, which is well in close agreement with the value predicted by RSM model yield (13.19%). The basic characterization of HWSPs was determined by using the FTIR. These preliminary in vitro biological studies indicated that lemongrass polysaccharides were useful for anticancer therapy.

Further studies and biological activities of macromolecular protein R-Phycoerythrin from Portieria hornemannii.

In the present study, the purified R-Phycoerythrin (R-PE) from a red alga Portieria hornemannii was subjected to the analysis of stability under the influence of different agents. Among the various inhibitors tested on R-PE EDTA at lower concentrations (<1 mM) supported the activity of R-PE. When R-PE was exposed to different organic solvents, ethanol supported the activity at the maximum followed by acetone, ethyl acetate, chloroform and methanol. Citric acid, as a preservative maintained the stability of R-PE both under 0 ± 5 °C and 30 ± 5 °C with 59.34% and 56.23% respectively, on 30th day. Thermal decomposition of the R-PE began near 60 °C. Maximum weight loss was occurred between 150 °C and 500 °C. Complete weight loss was recorded around 875 °C. Thermal denaturation was observed between 19 °C and 40 °C. Moderate to low antioxidant activities were observed in R-PE in relation to total antioxidant activities. After characterization, R-PE was taken for in vitro anticancer studies against selected cancer cell lines. Further studies involving AO/EB fluorescence staining and phase contrast microscope revealed characteristic apoptotic features like cell shrinkage, membrane blebbing, and nuclear DNA fragmentation, etc. Likewise, FACS analysis revealed the cell cycle distribution pattern of A549 and HepG2 cells.

Stabilization of mitochondrial and microsomal function of fucoidan from Sargassum plagiophyllum in diethylnitrosamine induced hepatocarcinogenesis.

Crude fucoidan from Sargassum plagiophyllum extracted from blade and purified by Q-Sepharose fast flow anion-exchange chromatography and three fucoidan fractions were obtained. Maximum sulphate containing fucoidan fraction was considered as purified fucoidan and purity was checked with agarose gel electrophoresis. The monosaccharides of purified fucoidan analysed by HPLC revealed the presence of the sugars such as fucose as a major sugar were 70.8 mol%. The percentages of other sugars were galactose (13.5%), xylose (2.5%) and mannose (11.2%). GPC was used to analyse molecular weight of purified fucoidan and it was found to be 35 kDa. The levels of ICDH, SDH, MDH, a-KGDH, Phase-I biotransformation enzymes, and Phase-II biotransformation enzymes were decreased in cancer bearing animals which may be due to oxidative stress and mitochondrial damage and fucoidan restored these enzyme activities. The inhibition of carcinogen metabolic activation indicates the anticancer activity of fucoidan in DEN induced liver cancer.

Isolation and characterization of macromolecular protein R-Phycoerythrin from Portieria hornemannii.

R-Phycoerythrin (R-PE) is one of the three phycobiliproteins which are extensively used as fluorescent probes, and it is prepared from red macro-algae. This macromolecular protein has gained importance in many biotechnological applications in food science, immunodiagnostic, therapy, cosmetics, protein and cell labeling, and analytical processes. In the present investigation, R-PE was isolated and purified from a red alga Portieria hornemannii. R-PE extracted and purified through ammonium sulfate precipitation (55%) followed by Q-Sepharose column chromatography had yielded a maximum purity of 5.2%. R-PE exhibited a typical "three-peak" with absorption maxima at 499, 545 and 565 nm. CD spectrum of R-PE yielded the following secondary structure data: alpha helix (14.30%), beta helix (28.10%), turn helix (19.20%) and random coil helix (38.40%). The molecular mass of R-PE was 240 kDa under Native-PAGE. Three different subunits such as α, ß and γ of 16 kDa, 21 kDa and 39 kDa were segregated under SDS-PAGE. On two dimensional gel electrophoresis, one basic and four acidic subunits were detected. Five different tryptic peptides were assigned under MALDI-TOF. The sequences of N-terminus of R-PE of 10 different amino acids are Met Lys Gln Met Trp Asp Arg Met Val Val. The preparative procedures of the R-PE extraction and purification established based on the experiments exhibit advantages and can offer a reference for R-PE preparation from other marine red macro-alga P. hornemannii.

Antioxidant and in vitro anticancer effect of 2-pyrrolidinone rich fraction of Brassica oleracea var. capitata through induction of apoptosis in human cancer cells.

The aim of this study was to analyze if the 2-pyrrolidinone rich fraction of Brassica oleracea var. capitata exhibiting antioxidant and in vitro anticancer activities. 2-Pyrrolidinone is an active compound present in Brassica oleracea var. capitata. Our findings explored the potential use of 2-pyrrolidinone in cancer treatment. This compound was identified and isolated by gas chromatography-mass spectrometry and high-performance liquid chromatography from the leaf of Brassica oleracea var. capitata. The resultant rich active compound exhibited in vitro cytotoxicity in HeLa and PC-3 human cancer cell lines, and it also exhibited antioxidant activity in cell free assays. DAPI staining, an apoptotic analysis and cell cycle analysis were performed to evaluate the anticancer activity of 2-pyrrolidinone against the above cell lines. The IC50 value of 2-pyrrolidinone was determined to be of 2.5 µg/ml for HeLa, 3 µg/ml for PC-3 cells at 24 h and 1.5 µg/ml for HeLa and 2 µg/ml for PC-3 cells at 48 h, respectively. However, cell cycle analysis revealed that the anti-proliferative effects of the 2-pyrrolidinone were mediated through cell cycle arrest in the G0/G1 phase. These results from the current study suggest that the 2-pyrrolidinone have potential anticancer effects, which will lead to the development of new anticancer agents for arresting cancer cells growth in vitro.