Assessment of physio-biochemical assessment and gene expression analysis of sugarcane genotypes under water stress.

BACKGROUND: Sugarcane, an economically important crop cultivated for its unique character of accumulating sucrose into its stalk and the world's major crop according to production quantity. Sugarcane production is negatively influenced by abiotic stresses because it faces all types of environments due to its long-life cycle period. Among the various abiotic stresses, drought is one of the major limiting factors creates obstacle in sugarcane production. Thus, an attempt was made to assess the molecular insights into sugarcane genotypes under water stress. A preliminary screening was done in ten sugarcane genotypes grown under semi-arid region of India through physiological, biochemical and antioxidant responses of these genotypes under two water deficit levels. METHODS: In the current study, drought was imposed on ten sugarcane genotypes during their formative stage (110 DAP) by depriving them of irrigation. A pot experiment was carried out to see how several commercial sugarcane genotypes responded to water scarcity. Sugarcane received two treatments, the first after 125 days and the second after 140 days. The physio-biochemical and antioxidant responses recorded were RWC, MSI, SCMR, Proline accumulation, SOD, Catalase, Peroxidase and Lipid peroxidation. The significant variations were recorded in responses of all genotypes. On the basis of physio-biochemical, three genotypes Cos 98,014, Cos 13,235 and Colk 14,201 were selected for differential gene expression pattern analysis. The total RNA was isolated and reverse transcribe to cDNA and real time PCR was performed for expression analysis under 10 genes. RESULTS: Under drought conditions, all sugarcane genotypes showed significantly decreased RWC, chlorophyll content, and MSI. However, when water was scarce, proline buildup, malondialdehyde (MDA) contents, enzymatic antioxidant activity (CAT, POD, and SOD), and contents all increased dramatically. Finally, in all physiological and biochemical parameters, Co 98,014 genotype displayed superior adaptation responses to drought stress, followed by Co 018, Cos 13,235, and Colk 14,201. For gene expression analysis out of 21 genes, 10 genes were expressed in sugarcane genotypes, in which 7 genes (Shbbx2, Shbbx3, Shbbx4, Shbbx5, Shbbx8, Shbbx15 and Shbbx20) were upregulated and 3 genes (Shbbx1, Shbbx16 and Shbbx17) were downregulated. CONCLUSION: The statistical analysis conducted in this study demonstrated that drought stress had a negative impact on physiological responses, including RWC, SPAD, and MSI, in sugarcane crops. However, it was found that the crops were able to survive in these stress conditions by increasing their biochemical parameters, all while maintaining their growth and function.

Strategies for mitigation of pesticides from the environment through alternative approaches: A review of recent developments and future prospects.

Chemical-based peticides are having negative impacts on both the healths of human beings and plants as well. The World Health Organisation (WHO), reported that each year, >25 million individuals in poor nations are having acute pesticide poisoning cases along with 20,000 fatal injuries at global level. Normally, only ∼0.1% of the pesticide reaches to the intended targets, and rest amount is expected to come into the food chain/environment for a longer period of time. Therefore, it is crucial to reduce the amounts of pesticides present in the soil. Physical or chemical treatments are either expensive or incapable to do so. Hence, pesticide detoxification can be achieved through bioremediation/biotechnologies, including nano-based methodologies, integrated approaches etc. These are relatively affordable, efficient and environmentally sound methods. Therefore, alternate strategies like as advanced biotechnological tools like as CRISPR Cas system, RNAi and genetic engineering for development of insects and pest resistant plants which are directly involved in the development of disease- and pest-resistant plants and indirectly reduce the use of pesticides. Omics tools and multi omics approaches like metagenomics, genomics, transcriptomics, proteomics, and metabolomics for the efficient functional gene mining and their validation for bioremediation of pesticides also discussed from the literatures. Overall, the review focuses on the most recent advancements in bioremediation methods to lessen the effects of pesticides along with the role of microorganisms in pesticides elimination. Further, pesticide detection is also a big challenge which can be done by using HPLC, GC, SERS, and LSPR ELISA etc. which have also been described in this review.

Exogenous application of nanocarrier-mediated double-stranded RNA manipulates physiological traits and defence response against bacterial diseases.

Stability and delivery are major challenges associated with exogenous double-stranded RNA (dsRNA) application into plants. We report the encapsulation and delivery of dsRNA in cationic poly-aspartic acid-derived polymer (CPP6) into plant cells. CPP6 stabilizes the dsRNAs during long exposure at varied temperatures and pH, and protects against RNase A degradation. CPP6 helps dsRNA uptake through roots or foliar spray and facilitates systemic movement to induce endogenous gene silencing. The fluorescence of Arabidopsis GFP-overexpressing transgenic plants was significantly reduced after infiltration with gfp-dsRNA-CPP6 by silencing of the transgene compared to plants treated only with gfp-dsRNA. The plant endogenous genes flowering locus T (FT) and phytochrome interacting factor 4 (PIF4) were downregulated by a foliar spray of ft-dsRNA-CPP6 and pif4-dsRNA-CPP6 in Arabidopsis, with delayed flowering and enhanced biomass. The rice PDS gene targeted by pds-dsRNA-CPP6 through root uptake was effectively silenced and plants showed a dwarf and albino phenotype. The NaCl-induced OsbZIP23 was targeted through root uptake of bzip23-dsRNA-CPP6 and showed reduced transcripts and seedling growth compared to treatment with naked dsRNA. The negative regulators of plant defence SDIR1 and SWEET14 were targeted through foliar spray to provide durable resistance against bacterial leaf blight disease caused by Xanthomonas oryzae pv. oryzae (Xoo). Overall, the study demonstrates that transient silencing of plant endogenous genes using polymer-encapsulated dsRNA provides prolonged and durable resistance against Xoo, which could be a promising tool for crop protection and for sustaining productivity.

In-situ gel: A smart carrier for drug delivery.

In-situ gel technology is a promising drug delivery strategy that undergoes a 'sol to gel' transition upon administration, providing controlled and prolonged drug release. These gels are composed of cross-linked 3D networks of polymers, with hydrogels being a specific type of absorbing water while retaining their shape. Gelation can be triggered by various stimuli, such as temperature, pH, ions, and light. They offer several advantages like improved patient compliance, extended drug residence time, localized drug delivery, etc, but also have some disadvantages like drug degradation and limited mechanical strength. In-situ gel falls into three categories: temperature-sensitive, ion-sensitive, and pH-sensitive, but multi-responsive gels that respond to multiple stimuli have better drug release characteristics. The mechanism of in-situ gel formation involves physical and chemical mechanisms. There are various applications of in-situ gel, like ocular drug delivery, nose-to-brain delivery, etc. In this review, we have discussed the types, and mechanisms of in-situ gel & use of in-situ gel in the treatment of different diseases through various routes like buccal, vaginal, ocular, nasal, etc., along with its use in targeted drug delivery.

Synthesis, characterization, biological activity and computation-based efficacy of cobalt(II) complexes of biphenyl-2-ol against SARS-CoV-2 virus.

Cobalt(II) complexes of biphenyl-2-ol of composition, CoCl2-n(OC6H4C6H5-2)n(H2O)4 (where n = 1 or 2), were prepared by reacting cobaltous(II) chloride with equi- and bimolar ratios of sodium salt of biphenyl-2-ol. The structural characterization of the synthesized complexes was accomplished by NMR, FTIR, thermogravimetry (TGA), high resolution mass spectroscopy (HRMS), electronic spectroscopic techniques coupled with density functional theory (DFT). The stability of the complexes in different pH media of solvent was studied. Chemical reactivity parameters of the newly synthesized complexes, computed using DFT, indicated greater reactivity of complex 2 over complex 1 and free ligand as indicated by its low HOMO-LUMO energy gap corresponding to 1.71 eV. Molecular docking (MD) studies were carried out in order to study the binding affinities between amino acid residues of DNA duplex (PDB ID: 1BNA) and SARS-CoV-2 (PDB ID: 7T9K) with newly synthesized complexes. Complex 2 has shown promising antivirus behaviour with an inhibition constant value of 0.0423 µmol-1 with amino acid residues of SARS-CoV-2 virus. Toxicity of the complexes was predicted using ProTox-II online server. Antibacterial studies have indicated the complexes to exhibit greater efficacy than the free ligand, while the antioxidant activities have suggested them to display enhanced antioxidant behaviour as compared to reference compounds.Communicated by Ramaswamy H. Sarma.

PLGA-based microspheres loaded with metformin hydrochloride: Modified double emulsion method preparation, optimization, characterization, and in vitro evaluation.

The modified solvent removal method was used to encapsulate metformin hydrochloride (MH) within poly(lactic-co-glycolic acid) (PLGA) microspheres. The study investigated the effect of varying polymer concentrations on the loading and release of the drug from the microspheres. The encapsulation process involved using a double emulsion method, resulting in microspheres with particle diameters ranging from approximately 4.4µm to 2.7µm. The study achieved high encapsulation efficiencies, ranging from 81% to 90%, with drug loadings ranging from 18% to 11%. The release of the drug from the microspheres followed a biphasic pattern over 24 days, with nearly complete release by the end of the study period. Fourier transform infrared spectroscopy (FTIR) analysis indicated that there were no notable differences between PLGA and MH-loaded microspheres, suggesting minimal interactions between MH and PLGA. Differential scanning calorimetry (DSC) and X-ray diffraction (XRD) techniques were used to investigate the state of the MH within the microspheres. The results suggested that the MH was dispersed at a molecular level within the spheres and existed in an amorphous state. This amorphous state of the drug may explain the slow and prolonged release observed in the study.

(+)-Cyanidan-3-ol inhibits epidermoid squamous cell carcinoma growth via inhibiting AKT/mTOR signaling through modulating CIP2A-PP2A axis.

BACKGROUND: Despite recent advances in the treatment of squamous cell skin cancer (SCSC), the disease persists, and treatment resistance develops. Thus, identifying new targets and developing new therapeutic approaches showing low vulnerability to drug resistance is highly needed. PURPOSE: This study aimed to reveal a novel targeted phytotherapeutic strategy for SCSC treatment alone or in combination with standard targeted anticancer molecules. STUDY DESIGN: A library of natural products was utilized to identify molecules that inhibit the growth of skin cancer cells. The anticancer potential of the selected compound was evaluated in human skin squamous carcinoma models, in vitro and in vivo. A comprehensive ingenuity pathway analysis (IPA) strategy and molecular biology technology was adopted to investigate the therapeutic mechanisms in human SCSC. METHODS: The Matrigel invasion chamber, foci formation and soft agar colony formation assays were employed to study the cells invasion and migration potential in vitro. In vivo antitumor effects were evaluated in DMBA/TPA-induced skin papilloma and A431 human skin squamous carcinoma xenograft tumor models. An integrative IPA was employed to identify mechanisms and protein targets in human SCSC.Compounds synergies were determined by the bliss model and evaluated using human SCSC cell lines and xenograft tumors. Histological staining, immunofluorescence imaging, real-time PCR, Western blots, and flow cytometric analyses were employed to analyze apoptosis and cell signaling mechanisms. RESULTS: We identified (+)-cyanidan-3-ol (CD-3) as a selective compound for inhibiting the growth of SCSC cell lines. CD-3 inhibited tumor growth and burden without apparent toxicity and prolonged the survival of tumor-bearing mice. CD-3 inhibitory effects on SCSC growth are mediated via cell cycle arrest and caspase-dependent apoptosis induction. Mechanistic studies showed that CD-3 activates PP2A via inhibiting CIP2A and produces tumor growth inhibitory effects via promoting dephosphorylation of oncogenic AKT/mTOR signaling proteins in SCSC cells and xenograft tumors in a PP2A dependent manner. Furthermore, the combination of CD-3 and mTOR inhibitors (mTORi) synergistically reduced oncogenic phenotypes. CONCLUSIONS: Our study suggests that PP2A activation is an effective strategy for SCSC treatment and the CD-3 and mTORi combination may serve as a promising treatment for SCSC.

Sequence and functional analysis of MIR319 promoter homologs from Brassica juncea reveals regulatory diversification and altered expression under stress.

KEY MESSAGE: Extensive regulatory divergence during development, abiotic stress and ABA regime observed amongst promoter homologs and homeologs of MIR319 from Brassica juncea. Gene duplication followed by sub-functionalization, neo-functionalization, and pseudogenization are routes to functional and adaptive diversification. The influence of polyploidy on protein-coding genes is well investigated but little is known about their impact on transcriptional regulation of MIRNA gene family. The present study was therefore performed with an aim to uncover regulatory diversification of MIR319 homologs and homeologs in Brassica juncea. We employed comparative genomics to identify and isolate six promoter homologs of MIR319 from B. juncea. Regulatory diversification was studied using analysis of reporter activity driven by BjMIR319 promoters in a heterologous system employing promoter-reporter fusion constructs. MIR319 is known to play important roles in leaf and flower development, and multiple stress responses. Reporter activity was therefore monitored during development, hormonal and stress regimes. In-silico analyses revealed differential distribution of cis-regulatory motifs and functional analysis revealed distinct spatiotemporal expression patterns. The significance of presence of selected cis-regulatory motifs corresponding to heat, cold, salt and ABA stress were further functionally validated. It was observed that promoter of Bj -MIR319a-A01 was upregulated in response to cold and salt stress, while promoter of Bj -MIR319c-A04 (D1) and Bj -MIR319c-A05 (FL) were downregulated in response to high temperature. In summary, comparative analysis of homologous promoters from Brassica juncea, an allopolyploid revealed extensive sequence and functional diversity. Spatiotemporal activity of reporter gene driven by BjMIR319 promoter was distinct, and partially overlapping with from those reported previously for A. thaliana. The present study clearly demonstrates regulatory divergence amongst promoter homologs of MIR319 in Brassica juncea during development and stress response, and underlines the urgent need for dissection of promoter function and detailed characterization including identification of interacting trans-factors. Genbank accession numbers: MT379853-MT379858.

Microsynteny analysis to understand evolution and impact of polyploidization on MIR319 family within Brassicaceae.

The availability of a large number of whole-genome sequences allows comparative genomic analysis to reveal and understand evolution of regulatory regions and elements. The role played by events such as whole-genome and segmental duplications followed by genome fractionation in shaping genomic landscape and in expansion of gene families is crucial toward developing insights into evolutionary trends and consequences such as sequence and functional diversification. Members of Brassicaceae are known to have experienced several rounds of whole-genome duplication (WGD) that have been termed as paleopolyploidy, mesopolyploidy, and neopolyploidy. Such repeated events led to the creation and expansion of a large number of gene families. MIR319 is reported to be one of the most ancient and conserved plant MIRNA families and plays a role in growth and development including leaf development, seedling development, and embryo patterning. We have previously reported functional diversification of members of miR319 in Brassica oleracea affecting leaf architecture; however, the evolutionary history of the MIR319 gene family across Brassicaceae remains unknown and requires investigation. We therefore identified homologous and homeologous segments of ca. 100 kb, with or without MIR319, performed comparative synteny analysis and genome fractionation studies. We detected variable rates of gene retention across members of Brassicaceae when genomic blocks of MIR319a, MIR319b, and MIR319c were compared either between themselves or against Arabidopsis thaliana genome which was taken as the base genome. The highest levels of shared genes were found between A. thaliana and Capsella rubella in both MIR319b- and MIR319c-containing genomic segments, and with the closest species of A. thaliana, A. lyrata, only in MIR319a-containing segment. Synteny analysis across 12 genomes (with 30 sub-genomes) revealed MIR319c to be the most conserved MIRNA loci (present in 27 genomes/sub-genomes) followed by MIR319a (present in 23 genomes/sub-genomes); MIR319b was found to be frequently lost (present in 20 genomes/sub-genomes) and thus is under least selection pressure for retention. Genome fractionation revealed extensive and differential loss of MIRNA homeologous loci and flanking genes from various sub-genomes of Brassica species that is in accordance with their older history of polyploidy when compared to Camelina sativa, a recent neopolyploid, where the effect of genome fractionation was least. Finally, estimation of phylogenetic relationship using precursor sequences of MIR319 reveals MIR319a and MIR319b form sister clades, with MIR319c forming a separate clade. An intra-species synteny analysis between MIR319a-, MIR319b-, and MIR319c-containing genomic segments suggests segmental duplications at the base of Brassicaceae to be responsible for the origin of MIR319a and MIR319b.

An improved method for rapid analysis of promoters using modified sonication-assisted transient assay.

We present here a modified, sonication-assisted transient transformation assay for rapid analysis of cis-regulatory elements. We tested promoter elements from MIR159B locus of Brassica juncea by generating stable transgenic lines and compared the transcriptional activity of GUS reporter with that of the transient assay method. To obtain reliable and repeatable results, and to omit false-positive data, we optimized several parameters including sonication duration and cycle and concentration of Agrobacterium tumefaciens measured as optical density (O.D.) at 600 nm. To the best of our knowledge, this is the first report of promoter characterization of MIR159B from Brassica juncea, and comparative analysis of stable and transient lines. Our analysis shows that the protocol described herein allows understanding promoter activity/transcriptional control in tissues other than leaf or protoplast which have remained the mainstay for transient analysis thus far. We tested reporter gene GUS under the control of constitutive promoter, CaMV 35S, and MIR159b from Brassica juncea. We optimized the duration of sonication (5-, 10- and 15-min cycle), bacterial density (measured as O.D at 600 nm = 0.6/0.8/1.0) and Agro-infection time (5, 10, 15 min), and co-cultivation (12-, and 24-h). Sonication cycle of 10-min, followed by Agro-infection and co-cultivation with Agrobacterium tumefaciens with O.D. 600 nm = 0.8 and for 12 h was found to be optimum. We could successfully express reporter genes in deep-seated tissues such as floral organs and pollen grains where it was previously not possible to perform transient assay. Constitutive GUS activity was observed when reporter was placed under control of the constitutive promoter of CaMV 35S. Reporter GUS when placed under transcriptional control of MIR159b promoter from Brassica juncea showed reporter activity in floral tissues, in mature pollen grains. Comparative analysis of reporter activity from stable transgenic lines at T2 generation with that of transient assay system reveals identical to near-identical reporter activity. Transient assay could be successfully performed in tissues collected not only from Arabidopsis thaliana, but also from Brassica juncea and Brassica nigra to demonstrate its wide applicability. Our modified method thus has the potential of quick and rapid analysis of promoter activity and allows us to record the developmental dynamics and spatio-temporal expression pattern driven by specific promoters. Suitable modification and controls should also allow analysis of hormonal regulation and identification of trans-factors via DNA-protein interactions. Furthermore, this method can also be extended to study promoters under various environmental conditions that otherwise do not allow growth and complete life cycle of healthy plants and can be modified to test reporter activity in other non-model plants or plants with long life cycle.

Diversity-oriented sustainable synthesis of antimicrobial spiropyrrolidine/thiapyrrolizidine oxindole derivatives: New ligands for a metallo-ß-lactamase from Klebsiella pneumonia.

A simple, environmentally benign and highly proficient microwave assisted one-pot approach for the synthesis of antimicrobial spiropyrrolidine/thiapyrrolizidine oxindole derivatives is reported assembling two pharmacophoric moieties (1,3-indanedione and pyrrolidine/thiapyrrolizidine) in a single molecular framework via three-component 1,3-dipolar cycloaddition reaction of substituted isatin, sarcosine/1,3-thiazoles-4-carboxylic acid and Knoevenagel adduct (2-Cyano-3-phenyl-acrylic acid ethyl ester or 2-Benzylidene-malononitrile) in 2,2,2-trifluoroethanol as a reusable green solvent. Good functional group tolerance and broad scope of usable substrates are other prominent features of the present methodology with high degree of chemo-, regio- and stereoselectivity. The stereochemistry of synthesized compounds was confirmed by single crystal X-ray analysis. All the synthetic compounds were examined for their antimicrobial potential. The synthesized compounds having pyrrolothiazole moiety showed excellent activity against K. pneumoniae as compared to others and even more inhibitory activity than the mentioned drugs, i.e. compounds 6a (MIC=0.09µg/mL), 6b (MIC=0.045µg/mL), 6c (MIC=0.005µg/mL), 6d (MIC=0.19µg/mL). Additionally, compound 6c has shown better binding affinity against New Delhi Metallo-beta-Lactamase-1 (NDM-1) protein in computational docking studies.

In Vitro and In Vivo Evaluation of Small Cationic Abiotic Lipopeptides as Novel Antifungal Agents.

We investigated the antifungal potential of short lipopeptides against clinical fungal isolates with an objective to evaluate their clinical feasibility. All tested lipopeptides exhibit good antifungal activity with negligible difference between the MICs against susceptible and drug-resistant clinical fungal isolates. The MTT assay results revealed the lower cytotoxicity of lipopeptides toward mammalian cells (NRK-52E). In particular, LP24 displayed highest potency against most of the tested fungal isolates with MICs in the range of 1.5-4.5 µg/mL. Calcein dye leakage experiments with model membrane suggested the membrane-active mode of action for LP24. Extending our work from model membranes to intact Aspergillus fumigatus in scanning electron micrographs, we could visualize surface perturbation caused by LP24. LP24 (5 mg/kg) significantly reduces the A. fumigatus burden among the various organs of infected animals, and 70% of the infected mice survived when observed for 28 days. This study underscores the potential of small cationic abiotic lipopeptides to develop into the next-generation antimicrobial therapy.

Treatment of refractory nano-filtration reject from a tannery using Pd-catalyzed wet air oxidation.

We attempted catalytic wet air oxidation (CWAO) of nanofiltration (NF)-reject using Pd based catalyst viz., Pd/activated charcoal (AC) and PdCl2 with the objective of degradation of refractory organic pollutants. Refractory organic pollutants in NF-reject before and after WAO and CWAO were confirmed by GC-MS analysis. Experiments were conducted to investigate the effects of temperature, catalyst dosage and air partial pressure on the rate of removal of total organic carbon (TOC). The reaction kinetics can be conveniently described by considering two-stage first order kinetics. The use of Pd/AC afforded 85% TOC removal, the corresponding rate constant (k) was 2.90 ± 0.075 × 10(-3)min(-1) (Pd/AC, 100mg/L; T, 473.15K; Pair, 0.69 MPa). On the other hand, 75% TOC was removed with k=2.31 ± 0.075 × 10(-3)min(-1) using Pd(2+) catalyst (Pd(2+), 16.66 mg/L; T, 473.15K; Pair, 0.69 MPa). The observed rate of mineralization under Pd-catalyzed conditions was significantly higher than that of the uncatalyzed oxidation (41%) under the similar experimental conditions. Catalyst stability experiments were performed and TEM, SEM, XRD, Raman and XPS characterization data collected. Despite some morphological transformation of support, Pd catalyst was stable under CWAO conditions.

Growth inhibition and apoptosis induction by (+)-Cyanidan-3-ol in hepatocellular carcinoma.

The objective of this study was to evaluate the cytotoxicity of (+)-cyanidan-3-ol (CD-3) in human hepatocellular carcinoma cell line (HepG2) and chemopreventive potential against hepatocellular carcinoma (HCC) in Balb/c mice. The HepG2 cell line was treated with CD-3 at various concentrations and the proliferation of the HepG2 cells was measure by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT), sulforhodamine B (SRB) and lactate dehydrogenase (LDH) assays. Cell apoptosis was detected by Hoechst 33258 (HO), Acridine orange/ethylene dibromide (AO/EB) staining, DNA fragmentation analysis and the apoptosis rate was detected by flow cytometry. The HCC tumor model was established in mice by injecting N-nitrosodiethylamine/carbon tetrachloride (NDEA/CCl4) and the effect of CD-3 on tumor growth in-vivo was studied. The levels of liver injury markers, tumor markers, and oxidative stress were measured. The expression levels of apoptosis-related genes in in-vitro and in vivo models were determined by RT-PCR and ELISA. The CD-3 induced cell death was considered to be apoptotic by observing the typical apoptotic morphological changes under fluorescent microscopy and DNA fragmentation analysis. Annexin V/PI assay demonstrated that apoptosis increased with increase in the concentration of CD-3. The expression levels of apoptosis-related genes that belong to bcl-2 and caspase family were increased and AP-1 and NF-κB activities were significantly suppressed by CD-3. Immunohistochemistry data revealed less localization of p53, p65 and c-jun in CD-3 treated tumors as compared to localization in NDEA/CCl4 treated tumors. Taken together, our data demonstrated that CD-3 could significantly inhibit the proliferation of HepG2 cells in-vitro and suppress HCC tumor growth in-vivo by apoptosis induction.

Cytotoxicity and apoptosis induction in human breast adenocarcinoma MCF-7 cells by (+)-cyanidan-3-ol.

The objective of this study was to evaluate the cytotoxicity and possible signalling pathway implicated in (+)-cyanidan-3-ol (CD-3) induced apoptosis in the human breast adenocarcinoma cell line (MCF-7). The effects of CD-3 on cell proliferation of MCF-7 cells were evaluated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT), sulforhodamine B (SRB) and lactate dehydrogenase (LDH) assays. Cell apoptosis was detected by Hoechst 33258 (HO) and acridine orange/ethylene dibromide (AO/EB) staining and DNA fragmentation analysis. The expressions of apoptosis-related genes were assessed by RT-PCR and ELISA. Our data revealed that CD-3 induced MCF-7 cell death in a dose-dependent manner. Marked changes in apoptotic morphology was clearly observed after CD-3 treatment. CD-3 induced cell death was considered to be apoptotic by observing the typical apoptotic morphological change under fluorescent microscopy and DNA fragmentation assays. The induction of apoptosis is correlated with the increased mRNA expressions of p53, Bax, and caspase-3, -7, -8 and -9 and decreased mRNA expressions of bcl-2. Subsequently, CD-3 decreased the mRNA expressions of mdm2, p65, c-jun, c-fos in MCF-7 cells. The protein levels of p53, Bax, and caspase-3 were increased, whereas, that of p65, c-jun and Bcl-2 were decreased in MCF-7 cells on CD-3 treatment. These results clearly demonstrated that CD-3 effectively induced growth inhibition and apoptosis in MCF-7 cells.

Human breast adenocarcinoma cytotoxicity and modulation of 7,12-dimethylbenz[a]anthracene-induced mammary carcinoma in Balb/c mice by Acacia catechu (L.f.) Wild heartwood.

OBJECTIVE: The chemopreventive potential of (+)-catechin-rich extract of Acacia catechu (L.f.) Willd. heartwood (AQCE) was evaluated against human breast adenocarcinoma cell line (MCF-7) and 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary carcinoma in Balb/c mice. METHODS: Cell cytotoxicity was investigated using different colorimetric assays. Apoptosis was observed using diphenylamine assay and fluorescent microscopy. AQCE was further evaluated for antitumor activity against DMBA-induced mammary carcinoma. The levels of tumor markers and oxidative stress were measured. Furthermore, level of transcription factors was measured by enzyme-linked immunosorbent assay. RESULTS: The results showed that administration of AQCE showed a dose-dependent growth inhibition response and DNA fragmentation in MCF-7 cells. Tumor multiplicity was significantly decreased to 42.91% with AQCE when compared with DMBA-treated animals. The levels of tumor markers such as total sialic acid and lipid-associated sialic acid were substantially increased after DMBA treatment. However, AQCE treatment restored tumor markers level. AQCE also significantly reduced elevated levels of nitrite and malondialdehyde in DMBA-treated animals. Additionally, AQCE also increased the activities of antioxidant enzymes, viz., catalase, superoxide dismutase, total thiol, reduced glutathione, protein thiol, glutathione peroxidase, glutathione reductase, and glutathione-S-transferase in the mammary tissue and liver mitochondria of DMBA-administered animals. Significant increase in the protein levels of p53, c-jun, and p65 were observed in DMBA-treated mice, whereas less expression was observed in AQCE-treated animals. Eventually, AQCE also significantly improved body weight and maintained the mammary tissue architecture in normal range. CONCLUSIONS: The present data strongly suggest that anticancer potentiality of (+)-catechin-rich AQCE may be attributable to its ability to positively modulate tumor markers as well as the antioxidant system that could decompose the peroxides and, thereby, offer a protection against lipid peroxidation and linked to the expression of transcription factors during DMBA-induced mammary carcinoma.

Chemopreventive efficacy of (+)-catechin-rich aqueous extract of Acacia catechu Willd. Heartwood against 7,12-dimethylbenz[a]anthracene-induced hepatocarcinoma in Balb/c mice.

The objective of this study was to investigate the chemopreventive potential of (+)-catechin-rich extract of Acacia catechu heartwood (AQCE) against 7,12-dimethylbenz[a]anthracene (DMBA)-induced hepatocellular carcinoma in Balb/c mice. The levels of liver injury markers, tumor markers, and oxidative stress were measured in serum and liver tissues. Furthermore, the levels of transcription factors were measured by ELISA. Tumor incidence was found to be 100% in DMBA-treated animals (group 2), whereas, in AQCE-treated animals (group 3), it was 37.5%. AQCE treatment reduced liver injury and restored tumor-marker levels. AQCE also significantly reduced elevated levels of nitrite and hepatic malondialdehyde (MDA) in DMBA-treated animals. Additionally, AQCE modulated the activity of different antioxidant enzymes in liver tissues. Eventually, AQCE also significantly improved body weight, prevented the increase of relative liver weight, and maintained the liver cellular architecture within the normal range of the control. A significant increase in the protein levels of p53, c-jun, and NF-κB (p65) were observed in DMBA-treated mice, whereas low levels of these markers were observed in DMBA+AQCE-treated animals. These findings strongly suggest (1) that (+)-catechin-rich AQCE exerts a chemopreventive effect by modulating the levels of lipid peroxidation and by promoting the enzymatic and non-enzymatic antioxidant defense system and (2) that this effect is linked to the expression of transcription factors during hepatocarcinogenesis.

Human epithelial carcinoma cytotoxicity and inhibition of DMBA/TPA induced squamous cell carcinoma in Balb/c mice by Acacia catechu heartwood.

OBJECTIVES: Acacia catechu heartwood contains significant amounts of polyphenolic compounds that exhibit powerful antioxidant activity. The purpose of this study was to evaluate the cytotoxicity of A. catechu heartwood extracts in a human epithelial carcinoma cell line (A431) and antitumour activity against DMBA/TPA induced squamous cell carcinoma in Balb/c mice. METHODS: Various extracts, including aqueous, ethyl acetate, chloroform and n-hexane, were tested for cytotoxic properties on a human epithelial carcinoma cell line (A431) by using MTT, sulforhodamine B and lactate dehydrogenase leakage assays. The standardized A. catechu heartwood aqueous extract (AQCE) was further evaluated for antitumour activity against 7,12-dimethylbenz[a]anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA) induced skin carcinoma in Balb/c mice. KEY FINDINGS: The results showed that administration of AQCE showed a dose-dependent growth inhibition response, with an IC50 value of 78.56 µg/ml. Tumour incidence was significantly decreased (P < 0.001) to 30% with AQCE compared with 100% in the DMBA/TPA group. The AQCE was also found to significantly upregulate different antioxidant enzymes in skin and liver tissue. CONCLUSIONS: The results suggest that AQCE may exert its chemopreventive activity by acting as an antioxidant.

Cellulose acetate microspheres as floating depot systems to increase gastric retention of antidiabetic drug: formulation, characterization and in vitro-in vivo evaluation.

Gastric emptying is a complex process that is highly variable and makes the in vivo performance of drug delivery systems uncertain. In order to avoid this variability, efforts have been made to increase the retention time of the drug delivery systems for more than 12 hours utilizing floating or hydrodynamically controlled drug delivery systems. The objective of this investigation was to develop a floating, depot-forming drug delivery system for an antidiabetic drug based on microparticulate technology to maintain constant plasma drug concentrations over a prolonged period of time for effective control of blood sugar levels. Formulations were optimized using cellulose acetate as the polymer and evaluated in vitro for physicochemical characteristics and drug release in phosphate buffered saline (pH 7.4), and evaluated in vivo in healthy male albino mice. The shape and the surface morphology of the prepared microspheres were characterized by optical microscopy and scanning electron microscopy. In vitro drug release studies were performed and drug release kinetics were calculated using the linear regression method. Effects of stirring rate during preparation and polymer concentration on the size of microspheres and drug release were observed. The prepared microspheres exhibited prolonged drug release (more than 10 hours) and remained buoyant for over 10 hours. Spherical and smooth-surfaced microspheres with encapsulation efficiency ranging from 73% to 98% were obtained. The release rate decreased and the mean particle size increased at higher polymer concentrations. Stirring speed affected the morphology of the microspheres. This investigation revealed that upon administration, the biocompatible depot-forming polymeric microspheres controlled the drug release and plasma sugar levels more efficiently than plain orally given drug. These formulations, with their reduced frequency of administration and better control over drug disposition, may provide an economic benefit to the user compared with products currently available for diabetes control.

Osmotic delivery of flurbiprofen through controlled porosity asymmetric membrane capsule.

The release of poorly water-soluble drug, flurbiprofen, through asymmetric membrane capsule of cellulose acetate containing different pore forming agents like glycerol, polyethylene glycol 400, and dibutyl phthalate, in presence of sodium lauryl sulfate was investigated. The asymmetric membrane was fabricated in the shape of capsule body and cap by phase inversion technique. The type of pore forming agent incorporated had a marked influence on the porosity of the asymmetric membrane. However flurbiprofen due to its poor solubility was unable to create enough osmotic pressure and hence less than 10% of drug was released from all the systems with out SLS. However when the study was conducted with SLS, a maximum release of 72% was observed from the capsule with 70% glycerol. The release rates were found to increase with the increase in the concentration of pore forming agent and the amount of SLS encapsulated.