Suitability of tetrazolium test for Tamarindus indica L. seeds

Seed quality is routinely assessed by direct tests, e.g, the germination test, or indirect tests like the tetrazolium test, which has shoown to be promising in the determine viability and vigor, allowing the diagnosis of the main problems that may affect seed quality, such as mechanic damages, field deterioration and storage. In this respect, this study was conducted to develop a tetrazolium test protocol to evaluate the viability and vigor of Tamarindus indica L. seeds. Before exposing the seeds to the tetrazolium solution, seed preconditioning studies were carried out in which seven soaking times were tested. The soaking time that did not cause damage to the seed embryo and allowed the removal of the seed coat to expose the seed structures to the tetrazolium salt was selected. Then, an experiment was set up in a completely randomized design with a 2x3x3 factorial arrangement involving two seed lots, three soaking times in tetrazolium salt (6, 12 and 16 h) and three salt concentrations (0.075, 0.1 and 0.5%), totaling 18 treatments with four replicates of 25 seeds, evaluated at 40 ºC. For each treatment, the seeds were divided into three classes, namely, viable and vigorous embryos (class 1); viable embryos (class 2) and non-viable or dead embryos (class 3). For a comparison with the tetrazolium test results, the two seed lots were analyzed for water content, germination, emergence, first count, germination speed index, emergence speed index, growth and seedling dry weight. The viability and vigor of T. indica seeds can be evaluated after a soaking period of 48 h and immersion for 6h in tetrazolium salt at the concentration of 0.1%, at 40°C, with provides results similar to conventional seed viability tests. The tetrazolium test proved to be adequate to differentiate T. indica seed lots in terms of viability.

The Impact of Fullerenes as Doxorubicin Nano-Transporters on Metallothionein and Superoxide Dismutase Status in MCF-10A Cells

This study aimed to synthesise C60–DOX complexes followed by the analysis of their effect on the concentration of metallothionein (MT) as a non-enzymatic antioxidant and on the concentration and activity of superoxide dismutase (SOD) as an antioxidant enzyme in healthy human mammary MCF-10A cells. Dynamic light scattering and electrophoretic light scattering were used to establish the size and zeta potential of the complexes. The MT and SOD concentrations were determined using the ELISA method; SOD activity was determined by tetrazolium salt reduction inhibition. Lower MT concentration following exposure of cells to both DOX and C60 fullerene compared to the control sample was found. However, the concentration of this protein increased as a consequence of the C60–DOX complexes action on MCF-10A cells compared to the control. C60 used alone did not affect the concentration and activity of SOD in MCF-10A cells. Application of free DOX did not activate cellular antioxidant defence in the form of an increase in SOD concentration or its activity. In contrast treatment of cells with the C60–DOX complex resulted in a decrease in SOD1 concentration and a significant increase in SOD activity compared to cells treated with free DOX, C60 and control. Thus, it was found that C60–DOX complexes showed potential for protective effects against DOX-induced toxicity to MCF-10A cells.

Improved Formazan Dissolution for Bacterial MTT Assay

Reduction of the water-soluble tetrazolium salt 3-(4,5-dimethylthiazol)-2,5 diphenyl-tetrazolium bromide (MTT) to purple, water-insoluble formazan is commonly used for assessment of cell viability and proliferation. Spectrophotometric detection of formazan requires its solubilization.

The Cytotoxicity and Nephroprotective Activity of the Ethanol Extracts of Angelica keiskei Koidzumi Stems and Leaves against the NAPQI-Induced Human Embryonic Kidney (HEK293) Cell Line

Ethnopharmacological Relevance. In Indonesia, Angelica keiskei Koidzumi (ashitaba or Japanese celery) has been traditionally used to maintain health and to achieve longevity. Previously, the chlorophyll-rich extract of A. keiskei planted in Korea exhibited a strong antioxidant activity. The objective of the present study was to investigate the cytotoxicity and nephroprotective activity of the ethanol extract of A. keiskei Koidzumi on the N-acetyl-p-benzoquinone imine (NAPQI) induced human embryonic kidney (HEK293) cell line. Materials and Methods. A. keiskei Koidzumi plant was collected from Mount Rinjani, Lombok, Indonesia, and was identified at the School of Biology Sciences and Technology, Bandung Institute of Technology, Indonesia. Extraction of the stems (ASE) and leaves (ALE) was performed by employing ethanol 70% for 3 × 24 h at 26°C. The cytotoxicity study of the extracts was assessed using the water-soluble tetrazolium salt-8 (WST-8) reagent on the HEK293 cell line, while the nephroprotective activity assay was determined on the NAPQI-induced HEK293 cell line. Results. The WST-8 assay showed that the cytotoxicity IC50 of ASE = 2322 μg/mL and IC50 of ALE = 2283 μg/mL. The nephroprotective activity assay revealed that ASE possesses nephroprotective activity against the NAPQI-induced HEK293 cell line at 1161 μg/mL, while ALE does not show the nephroprotective activity. Conclusion. Taken together, lower concentrations of ASE and ALE (<2000 μg/mL) are not toxic to the HEK293 cell line, and only ASE indicates the activity to protect the HEK293 cell line against NAPQI damage. This Japanese celery could be further explored for its potential as a plant-based nephroprotective drug.

The Synthesis of Europium-Doped Calcium Carbonate by an Eco-Method as Free Radical Generator Under Low-Intensity Ultrasonic Irradiation for Body Sculpture

Body sculpture is a common method to remove excessive fat. The diet and exercise are the first suggestion to keep body shape; however, those are difficult to keep adherence. Ultrasound has been developed for fat ablation; however, it could only serve as the side treatment along with liposuction. In the study, a sonosensitizer of europium-doped calcium carbonate (CaCO3: Eu) would be synthesized by an eco-method and combined with low-intensity ultrasound for lipolysis. The crystal structure of CaCO3: Eu was identified by x-ray diffractometer (XRD). The morphology of CaCO3: Eu was analyzed by scanning electron microscope (SEM). The chemical composition of CaCO3: Eu was evaluated by energy-dispersed spectrophotometer (EDS) and inductively coupled plasma mass spectrometer (ICP-MS). The electronic diffraction pattern was to further check crystal structure of the synthesized individual grain by transmission electron microscope (TEM). The particle size was determined by Zeta-sizer. Water-soluble tetrazolium salt (WST-1) were used to evaluate the cell viability. Chloromethyl-2′,7′-dichlorofluorescein diacetate (CM-H2DCFDA) and live/dead stain were used to evaluate feasibility in vitro. SD-rat was used to evaluate the safety and efficacy in vivo. The results showed that CaCO3: Eu had good biocompatibility and could produce reactive oxygen species (ROS) after treated with low-intensity ultrasound. After 4-weeks, the CaCO3: Eu exposed to ultrasound irradiation on SD rats could significantly decrease body weight, waistline, and subcutaneous adipose tissue. We believe that ROS from sonoluminescence, CO2-bomb and locally increasing Ca2+ level would be three major mechanisms to remove away adipo-tissue and inhibit adipogenesis. We could say that the combination of the CaCO3: Eu and low-intensity ultrasound would be a non-invasive treatment for the body sculpture.

Interference of Antioxidant Flavonoids with MTT Tetrazolium Assay in a Cell Free System

Introduction: The tetrazolium salt 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) used extensively to measure the quantitative survival and proliferation of mammalian cells. The analysis is based upon the reduction of MTT by metabolically active cells to insoluble formazan crystals. Flavonoids are a large group of natural compounds found in plants with variable phenolic structures. Flavonoids, as they are potential reducing agents, they act as a free radical scavenger. The aim of the study is to assess the reducing effect of some of the flavonoids on tetrazolium salt and their interference with the colorimetric analysis of MTT. The cell viability obtained from the MTT assay was compared with that of SRB assay in the determination of flavonoids cytotoxicity. Materials and Methods: The present study examined the effect of few bio-flavonoids like Quercetin, EGCG, Rutin and Resveratrol to reduce MTT in the absence of cells under different experimental conditions such as concentration of flavonoids, incubation time and results were compared with SRB assay findings. The study also involves the analysis of flavonoid cytotoxicity on lung cancer cells NCIH-460 and NCIH-522 by MTT and SRB assay to establish the suitable cell viability assay for flavonoids. Results: All the flavonoids showed the instant formation of the dark blue formazan salt in the absence of the cells with MTT assay. Whereas SRB assay of flavonoids in the absence of cells, results showed the absorbance similar to that of the blank, indicating that SRB did not interfere with flavonoids in a cell-free system. Conclusion: From the results, it is evident that MTT is not a suitable method to determine the effect of flavonoids on cell viability and proliferation as flavonoids itself reduces the MTT to formazan crystals. Study also suggests that SRB assay is more suitable method to determine the effect of flavonoids on cell viability.

Effects of Dedifferentiated Fat Cells on Neurogenic Differentiation and Cell Proliferation in Neuroblastoma Cells

Recent reports demonstrated that mesenchymal stem cells (MSCs) can induce differentiation of neuroblastoma (NB) cells. Dedifferentiated fat cells (DFAT) and MSCs have similar properties. The present study investigated whether DFAT can induce NB cell differentiation and suppress cell proliferation. DFAT was obtained from mature adipocytes isolated from adipose tissue from a ceiling culture. NB cells were cultured in a medium with/without DFAT, and subsequently in a DFAT-conditioned medium (CM) with/without phosphatidylinositol 3 kinase (PI3K) inhibitor. Length of neurites was measured, and the mRNA expression levels of the neurofilament (NF) and tubulin beta III (TUBβ3) were assessed using quantitative real-time reverse transcription polymerase chain reaction. Cell viability was assessed by the water-soluble tetrazolium salt-1 assay. NB cells cultured with DFAT elongated the neurites and upregulated the expression of NF and Tubβ3 compared with the control. However, NB cells cultured in DFAT-CM demonstrated increased cell viability compared with the control. NB cells cultured with DFAT-CM and PI3K inhibitor suppressed cell viability and demonstrated increased neurite length and expression and upregulation of Tubβ3. Therefore, the combined use of DFAT-CM and PI3K inhibitors suppresses the proliferation of NB cells and induces their differentiation. DFAT may offer new insights into therapeutic approaches in NB.

Wound Healing-Promoting and Melanogenesis-Inhibiting Activities of Angelica polymorpha Maxim. Flower Absolute In Vitro and Its Chemical Composition

Angelica polymorpha Maxim. (APM) is used in traditional medicine to treat chronic gastritis, rheumatic pain, and duodenal bulbar ulcers. However, it is not known whether APM has epidermis-associated biological activities. Here, we investigated the effects of APM flower absolute (APMFAb) on responses associated with skin wound healing and whitening using epidermal cells. APMFAb was obtained by solvent extraction and its composition was analyzed by GC/MS. Water-soluble tetrazolium salt, 5-bromo-2′-deoxyuridine incorporation, Boyden chamber, sprouting, and enzyme-linked immunosorbent assays and immunoblotting were used to examine the effects of APMFAb on HaCaT keratinocytes and B16BL6 melanoma cells. APMFAb contained five compounds and induced keratinocyte migration, proliferation, and type IV collagen synthesis. APMFAb also induced the phosphorylations of ERK1/2, JNK, p38 mitogen-activated protein kinase, and AKT in keratinocytes. In addition, APMFAb decreased serum-induced B16BL6 cell proliferation and inhibited tyrosinase expression, melanin contents, and microphthalmia-associated transcription factor expression in α-melanocyte-stimulating hormone-stimulated B16BL6 cells. These findings demonstrate that APMFAb has beneficial effects on skin wound healing by promoting the proliferation, migration, and collagen synthesis of keratinocytes and on skin whitening by inhibiting melanin synthesis in melanoma cells. Therefore, we suggest that APMFAb has potential use as a wound healing and skin whitening agent.

New Insights into the Potential Cytotoxic Role of Bacillus cytotoxicus Cytotoxin K-1

The thermotolerant representative of the Bacillus cereus group, Bacillus cytotoxicus, reliably harbors the coding gene of cytotoxin K-1 (CytK-1). This protein is a highly cytotoxic variant of CytK toxin, initially recovered from a diarrheal foodborne outbreak that caused the death of three people. In recent years, the cytotoxicity of B. cytotoxicus has become controversial, with some strains displaying a high cytotoxicity while others show no cytotoxicity towards cell lines. In order to better circumscribe the potential pathogenic role of CytK-1, knockout (KO) mutants were constructed in two B. cytotoxicus strains, E8.1 and E28.3. The complementation of the cytK-1 KO mutation was implemented in a mutant strain lacking in the cytK-1 gene. Using the tetrazolium salt (MTT) method, cytotoxicity tests of the cytK-1 KO and complemented mutants, as well as those of their wild-type strains, were carried out on Caco-2 cells. The results showed that cytK-1 KO mutants were significantly less cytotoxic than the parental wild-type strains. However, the complemented mutant was as cytotoxic as the wild-type, suggesting that CytK-1 is the major cytotoxicity factor in B. cytotoxicus.

Synthesis and Physicochemical Characterization of Novel Dicyclopropyl-Thiazole Compounds as Nontoxic and Promising Antifungals

There is a need to search for new antifungals, especially for the treatment of the invasive Candida infections, caused mainly by C. albicans. These infections are steadily increasing at an alarming rate, mostly among immunocompromised patients. The newly synthesized compounds (3a–3k) were characterized by physicochemical parameters and investigated for antimicrobial activity using the microdilution broth method to estimate minimal inhibitory concentration (MIC). Additionally, their antibiofilm activity and mode of action together with the effect on the membrane permeability in C. albicans were investigated. Biofilm biomass and its metabolic activity were quantitatively measured using crystal violet (CV) staining and tetrazolium salt (XTT) reduction assay. The cytotoxic effect on normal human lung fibroblasts and haemolytic effect were also evaluated. The results showed differential activity of the compounds against yeasts (MIC = 0.24–500 µg/mL) and bacteria (MIC = 125–1000 µg/mL). Most compounds possessed strong antifungal activity (MIC = 0.24–7.81 µg/mL). The compounds 3b, 3c and 3e, showed no inhibitory (at 1/2 × MIC) and eradication (at 8 × MIC) effect on C. albicans biofilm. Only slight decrease in the biofilm metabolic activity was observed for compound 3b. Moreover, the studied compounds increased the permeability of the membrane/cell wall of C. albicans and their mode of action may be related to action within the fungal cell wall structure and/or within the cell membrane. It is worth noting that the compounds had no cytotoxicity effect on pulmonary fibroblasts and erythrocytes at concentrations showing anticandidal activity. The present studies in vitro confirm that these derivatives appear to be a very promising group of antifungals for further preclinical studies.