Tetrazolium-based Colorimetric Assay Research Articles

In vitro antiplasmodial and anticancer analyses of endophytic fungal extracts isolated from selected Nigerian medicinal plants

Ethnomedicinal plants are thought to have better prospects of harboring endophytes that produce natural products with pharmacological activities. This study aimed to investigate the antiplasmodial and anticancer properties of secondary metabolites of endophytic fungi from three medicinal plants. The endophytic fungi included Lasiodiplodia theobromae isolated from Cola acuminata, Curvularia lunata Bv4 isolated from Bambusa vulgaris, and Curvularia lunata Eg7 isolated from Elaeis guineensis. The identification of the fungi was based on the internal transcribed spacer (ITS-rDNA) sequence. The fungi were subjected to solid-state fermentation and the secondary metabolites were extracted with ethyl acetate. In vitro antiplasmodial screening of extracts was performed using the SYBR green I-based fluorescence assay on the chloroquine-resistant Plasmodium falciparum strain DD2. The cytotoxicity of the extracts on human red blood cells and Jurkat (leukemia) cells was assessed using the tetrazolium-based colorimetric MTT assay. Gas chromatography-mass spectrometry (GC–MS) analysis was used to identify the constituents of the fungal extracts. The extract of L. theobromae showed the best antiplasmodial activity against chloroquine-resistant P. falciparum (IC50 = 5.4 µg/mL) and was not harmful to erythrocytes (CC50 > 100 µg/mL). All three fungal extracts showed a weak cytotoxic effect against Jukart cell lines (CC50 > 100 µg/mL). GC–MS analysis of the three endophytic fungal extracts revealed the presence of forty major bioactive compounds, including: oxalic acid, isobutyl nonyl ester, 2,4-di-tert-butylphenol, and hexadecanoic acid, among others. The endophytic fungi from the medicinal plants in this study were promising sources of bioactive compounds that could be further evaluated as novel drugs for the treatment of malaria caused by P. falciparum-resistant strains.

Novel Tetrazolium-Based Colorimetric Assay for Helicase nsp13 in SARS-CoV-2

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a human pathogenic virus that encodes for a helicase (SC2Hel) that is essential for viral replication. SC2Hel has the ability to unravel dsRNA or dsDNA in an NTP-dependent manner from the 5′ to 3′ directionality. The standard helicase assay from studies involving SARS-CoV and SARS-CoV-2 have relied on the concept of fluorescence resonance energy transfer. Adding to the collection of helicase assays, herein, we have developed a novel tetrazolium-based colorimetric assay system for the detection of ADP that is produced via SC2Hel activity. This SC2Hel assay combines three enzyme-coupled steps involving the ADP-dependent Thermococcus litoralis glucokinase (TlGlcK), Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase (LmG6PDH), and Clostridium kluyveri diaphorase (CkDIA). Iodonitrotetrazolium chloride (INT), a colorimetric tetrazolium reagent, was used in the final step of the assay that converted into INT-formazan during reduction. INT-formazan in the assay’s buffered solution at pH 7.6 exhibited an intense colorimetric response at a wavelength maximum of 505 nm. The assay exhibited excellent performance characteristics as it revealed a Z’ factor of 0.87 and it has the potential to be further adopted into high-throughput screening studies for therapeutic drug discovery research.

Brucine Entrapped Titanium Oxide Nanoparticle for Anticancer Treatment: An In Vitro Study.

Backgroundand Objective. The public's health has been seriously threatened by cervical cancer during recent times. In terms of newly diagnosed cases worldwide, it ranks as the ninth most prevalent malignancy. Multiple investigations have proven that nanoparticles can effectively combat cancer due to their small dimensions and extensive surface area. In the meantime, bioactive compounds which are biocompatible are being loaded onto nanoparticles to promote cancer therapy. The current study investigates the anticancerous potential of Brucine-entrapped titanium oxide nanoparticles (TiO2 NPs) in cervical cancer cell line (HeLa). Materials and Methods. The physiochemical, structural, and morphological aspects of Brucine-entrapped TiO2 NPs were evaluated by UV-visible spectrophotometer, Fourier transform-infrared spectroscopy (FT-IR), dynamic light scattering (DLS), scanning electron microscopy (SEM), and energy dispersive X-ray (EDAX). The cytotoxic effect against the HeLa cell line was assessed using a tetrazolium-based colorimetric assay (MTT), a trypan blue exclusion (TBE) assay, phase contrast microscopic analysis, and a fluorescence assay including ROS and DAPI staining. Furthermore, estimation of antioxidant markers includes catalase (CAT), glutathione (GSH), and superoxide dismutase (SOD). Results. The UV spectrum at 266 nm revealed the formation of TiO NPs. The FT-IR peaks confirmed the effective entrapment of brucine with TiO2 NPs. The average size (100.0 nm) of Brucine-entrapped TiO2 NPs was revealed in DLS analysis. The micrograph of the SEM revealed the formation of ellipsoidal to tetragonal-shaped NPs. The Ti, O, and C signals were observed in EDAX. In MTT assay, Brucine-entrapped TiO2 NPs showed inhibition of cell proliferation in a dose-wise manner and IC50 was noticed at the concentration of 30 µg/mL. The percentage of viable cells gradually reduced in the trypan blue exclusion assay. The phase contrast microscopic analysis of Brucine-entrapped TiO2 NP-treated cells showed cell shrinkage, cell wall deterioration, and cell blebbing. The intracellular ROS level was increased in a dose-wise manner when compared to control cells in ROS staining. The condensed nuclei and apoptotic cells were increased in treated cells, as noted in DAPI staining. In treated cells, the antioxidant markers such as CAT, GSH, and SOD levels were substantially lower compared to the control cells. Conclusion. The synthesized Brucine entrapped TiO2 NPs exhibited remarkable anticancer activity against the HeLa cell line.

In vitro drug screening models derived from different PC12 cell lines for exploring Parkinson's disease based on electrochemical signals of catecholamine neurotransmitters.

Goldnanostructures anda Nafion modified screen-printed carbon electrode (Nafion/AuNS/SPCE) were developed to assess the cell viability of Parkinson's disease (PD) cell models. The electrochemical measurement of cell viability was reflected by catecholamine neurotransmitter (represented by dopamine) secretion capacity, followed by a traditional tetrazolium-based colorimetric assay for confirmation. Due to the capacity to synthesize, store, and release catecholamines as well as theirunlimited homogeneous proliferation, and ease of manipulation, pheochromocytoma (PC12) cells were used for PD cell modeling.Commercial low-differentiated and highly-differentiated PC12 cells, and home-made nerve growth factor (NGF) induced low-differentiated PC12 cells (NGF-differentiated PC12 cells)were included in the modeling. This approach achieved sensitive and rapid determination of cellular modeling and intervention states. Notably, among the three cell lines, NGF-differentiated PC12 cells displayed the enhanced neurotransmitter secretion level accompanied with attenuated growth rate, incremental dendrites in number and length that were highly resemble with neurons. Therefore, it was selected as the PD-tailorable modeling cell line. In short, the electrochemical sensor can be used to sensitively determine the biological function of neuron-like PC12 cells with negligible destruction and to explore the protective and regenerative impact of various substances on nerve cell model.

In vitro biological activities of Combretum molle R.Br. ex G. Don (Combretaceae) against mastitis-causing organisms

Bovine mastitis, caused mostly by Staphylococcus species, has gained global importance owing to the increasing prevalence of multi-drug resistant bacteria, which comprise a risk to food security and public health. In this study, the antibacterial, anti-biofilm and quorum quenching activities of six leaf extracts of Combretum molle R.Br. ex G. Don prepared using different solvents were assessed against clinical isolates of Staphylococcus aureus and two Staphylococcus ATCC strains (S. aureus ATCC 29213 and S. epidermidis ATCC 35984). The antioxidant and anti-inflammatory activity and cytotoxicity of the extracts was also evaluated. The antibacterial and anti-biofilm potential of the extracts was determined via serial microdilution and crystal violet and INT assays. Quorum quenching activity was ascertained via inhibition of violacein production in Chromobacterium violaceum ATCC 12472. The antioxidant activity was determined using in vitro chemical assays. The 15-lipoxygenase enzyme inhibition and the nitric oxide inhibition assays were utilized to ascertain the anti-inflammatory activity of the extracts. The tetrazolium-based colorimetric (MTT) reduction assay was used to determine the cytotoxicity of the extracts against Vero African green monkey kidney cells. The antibacterial activity of the extracts was moderate to good, with minimum inhibitory concentration (MIC) values of 0.02–0.63 mg/mL. The extracts had promising biofilm inhibition (≥50 %) against four of the test strains. The effects of the extracts on the metabolic activity of the test strains showed ≥ 50 % inhibition in most of the test strains at the different test times. All extracts had moderate to excellent quorum quenching activity at different concentrations with an inhibition ranging between 32.81- 96.32 %. The methanol and cold water extracts had the best antioxidant activity against the 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and 2, 2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radicals with IC50 of 7.79 and 2.34 µg/mL, respectively. The hot water extract (AQH) had the best anti-inflammatory activity in terms of 15-lipoxygenase enzyme inhibition with 85.26 % inhibition, while the methanol extract had the best nitric oxide inhibition in LPS-induced RAW 264.7 macrophages with inhibition of 96.75 % at 100 µg/mL. Selectivity index values of the extracts were as high as 50. Combretum molle, therefore, offers promising potential as a source of antibiofilm and anti-quorum sensing agents with the ability to disrupt microbial virulence factors. Additionally, the extract had good antioxidant and anti-inflammatory properties, supporting further research into its use in preventing and treating bovine mastitis.

Effect of Silica in the Manufacture of Autologous Serum Eye Drops on Corneal Stromal Cells.

The aim of this study was to evaluate the effect of serum clot activator, silica (SiO 2 ), which may be used for making autologous serum eye drops, on human corneal fibroblasts. Cultured human corneal fibroblasts were exposed to 10%, 20%, and 30% silica for 1, 6, and 24 hours; methyl thiazolyl tetrazolium-based colorimetric assay was performed to determine the survival rate of fibroblasts and lactate dehydrogenase leakage assay to assess the cytotoxicity. The apoptotic response was evaluated by flow cytometric analysis and fluorescence staining with Annexin V and propidium iodide. Cellular morphology was evaluated by inverted phase-contrast light microscopy and electron microscopy. The survival rate of human corneal fibroblasts and cytotoxicity showed both dose-dependent and time-dependent responses. The fluorescent micrograph and flow cytometry showed that as the exposure time increased, more cells underwent apoptosis or necrosis after treatment with 30% silica. When observed with light and electron microscopy, the number of corneal fibroblasts decreased and they were more detached from the dish. In addition, damaged corneal fibroblasts showed degenerative changes after exposure to 30% silica. Silica showed dose-dependent and time-dependent toxicity in human corneal fibroblasts. It is safer to keep the blood in tubes without a clot activator when manufacturing autologous serum eye drops to prevent possible corneal cytotoxicity.

In vitro and in silico anti-malarial activity and cytotoxicity of n-hexyl 1-O-rutinoside (a glycoside) isolated from Annickia polycarpa (DC.) Setten and Maas ex I.M. Turner (Annonaceae)

Ethnopharmacological relevanceAnnickia polycarpa leaf is an effective anti-malarial agent. However, its chemical constituents have not been isolated and assayed against any pathogen. Aim of the studyTo isolate and characterize anti-malarial compound(s) from the leaf of A. polycarpa. Materials and methodsBioassay-guided fractionation was employed to isolated the compound (AL1) from the chloroform fraction (ALCF) of the basified ethanol extract of A. polycarpa leaf (ALE). AL1 was characterized by LC-MS, 1D and 2D NMR spectroscopic analysis. Anti-malarial activity was evaluated against drug resistance Dd2 and drug sensitive 3D7 Plasmodium falciparum strains using the SYBR green assay. Cytotoxicity and mechanistic studies were determined using tetrazolium-based colorimetric assay and molecular docking respectively. ResultsAL1 was characterized as n-hexyl 1-O-rutinoside. The IC50 values of ALE and ALCF against 3D7 and Dd2 P. falciparum strains ranges from 3.441 (0.3389) - 4.255 (0.2246) μg/mL. The IC50s obtained for n-hexyl 1-O-rutinoside and Artesunate (standard drug) were 7.71 (0.5473) and 0.001 (0.00008) nM against the 3D7 parasite strain respectively. Also, the efficacy of n-hexyl 1-O-rutinoside increased by 24.40% against the chloroquine resistance Dd2 P. falciparum strain whiles that of Artesunate decreased by 98.96%. Furthermore, ALE, ALCF and n-hexyl 1-O-rutinoside were weakly cytotoxic to human RBCs with high selectivity indices. N-hexyl 1-O-rutinoside inhibits P. falciparum chloroquine resistance transporter (PfCRT) and dihydrofolate reductase-thymidylate synthase (PfDHFR-TS) better than chloroquine and pyrimethamine respectively. But, produced similar inhibition of P. falciparum 2-trans-enoyl -ACP-reductase (PfERN) as triclosan. ConclusionThese results show that A. polycarpa leaf and n-hexyl 1-O-rutinoside possessed profound anti-malarial activity and are not cytotoxic. N-hexyl 1-O-rutinoside could therefore, be developed into a new anti-malarial medicine. This is the first study to report the anti-malarial activity of n-hexyl 1-O-rutinoside and its isolation from the genus Annickia.

Antibacterial Activity of Different Artemisia dracunculus Extracts Against Dental Caries-Related Pathogens

Background: This study investigated the antibacterial properties of four types of tarragon (Artemisia dracunculus) extracts against two caries contributing bacteria, namely, Streptococcus mutans and Streptococcus sobrinus. Methods: The extracts of A. dracunculus were prepared using n-hexane, ethyl acetate, methanol, and water solvents. In addition, the diameter of inhibition zones, minimum inhibitory concentrations (MICs), and minimum bactericidal concentrations (MBCs) were determined, and finally, MTT (tetrazolium-based colorimetric assay) was used to analyze the cytotoxic effects of the extracts. Results: The well-diffusion method showed the antibacterial property of four tested extracts against bacteria. Methanol and water extracts made the highest inhibition zone diameters (P<0.001). This was true for both tested bacteria. The mIC of the methanol, water, n-hexane, and ethyl acetate extracts were 0.78, 1.5, 3.1, and 1.5 mg/mL, as well as 0.78, 0.78, 1.5, and 1.5 mg/mL against S. mutans and S. sobrinus, respectively. The half-maximal inhibitory concentration (IC50) values for methanolic, aqueous, ethyl acetate, and n-hexane extracts were 0.78, 0.78, 1.56, and 3.12 mg/mL, respectively. Conclusions: The results provided a rational reason for the traditional use of A. dracunculus extracts against anti-caries-related bacteria. The methanolic extract demonstrated better activity, thus methanol can probably extract a wider range of plant compounds with antibacterial effects.

ANTIOXIDANT AND INHIBITORY EFFECT OF SELECTED GHANAIAN VEGETABLES ON NITRIC OXIDE EXPRESSION IN LIPOPOLYSACCHARIDE-INDUCED MACROPHAGE CELLS

Objective: Nitric oxide (NO) is a signaling molecule that plays a key role in the pathogenesis of inflammation. Inhibitors of NO may be useful candidates for the treatment of inflammatory diseases. The study aimed to determine the antioxidant and inhibitory effect of commonly used Ghanaian vegetables, namely Corchorus olitorius (CO), Solanum melongena (SM), Solanum torvum (ST), Xanthosoma sagittifolia (XS) and Abelmoschus esculentus (AE) on NO expression in a Lipopolysaccharide (LPS)-induced RAW 264.7 macrophage cell line. Methods: The cytotoxic effects of the vegetables on the cell line were determined using a tetrazolium-based colorimetric assay. The inflammatory activity was determined by measuring the inhibition of NO production in LPS-induced RAW 264.7 macrophage cells. Total antioxidant activity, total phenolic, flavonoid, and reduced glutathione contents were evaluated using 2, 2-diphenyl-1-picrylhydrazyl (DPPH) assay, Folin-Ciocalteu, aluminium chloride, and O-Phthalaldehyde methods, respectively. Results: Our results showed that CO and ST significantly inhibited NO production in a concentration-dependent manner with good cell viability. Solanum torvum also exhibited strong antioxidant activity (IC50= 0.466±0.23 mg/ml) with total phenolic content of 230.73±1.84 mg/g GAE, while CO showed high flavonoid content (291.45±2.14 mg/g QUE). Abelmoschus esculentus recorded the highest glutathione content (58.6 µg/g GSH. Saponins, alkaloids, tannins, terpenoids, and cardiac glycosides were present in all the samples except SM and AE, which lacked terpenoids. Conclusion: These findings suggest that CO and ST possess anti-inflammatory and antioxidant activities that could be explored as potential therapeutic remedies for inflammatory disorders.

Caffeic Acid Inhibits Proliferation, Migration, and Stemness of DU-145 Prostate Cancer Cells

The issue of toxicity associated with existing cancer drugs necessitates the exploration of the anticancer potentials of natural products such as caffeic acid (CA). Here, we determined the effect of CA on the proliferation, migration, and stem cell-like properties of DU-145 prostate cancer cells. Tetrazolium-based colorimetric assay and flow cytometric analysis showed that CA decreased cell proliferation in a dose- and time-dependent manner without affecting cell cycle progression. CA also inhibited cell migration and repressed epithelial-to-mesenchymal transition by upregulating the expression of cadherin 1 (CDH1) and downregulating the expression of cadherin 2 (CDH2). Furthermore, CA reduced the cancer stem cell population from 95% to 63% and 47% at concentrations of 1.25 and 2.5 mg/mL, respectively; and inhibited stem cell-like properties by downregulating the expression of NANOG and octamer-binding transcription factor 4 (OCT4) genes. These findings suggest that CA could be considered in the development of improved chemotherapy against prostate cancer.

Tetrazolium–based colorimetric assays underestimat the direct antitumor effects of anti-VEGF agent bevacizumab

The direct antitumor effect of bevacizumab (BEV) has long been debated. Assessment of the direct cytotoxic activities of drugs is usually conducted via in vitro experiments, of which tetrazolium-based colorimetric assays are widely employed to measure the direct antitumor activity of BEV. This study aimed to investigate whether tetrazolium-based colorimetric assays are applicable when evaluating the cytotoxicity of BEV against tumor cells. Our results showed that BEV significantly augmented tumor-cell mitochondrial metabolism. Enhanced mitochondrial metabolism caused changes in cellular oxidation-and-reduction environment and upregulated succinate dehydrogenase, which in turn promoted the reduction of tetrazolium to produce formazan. Increased formazan formation resulted in underestimation of the in vitro direct antitumor effect of BEV. Furthermore, inhibition of mitochondrial hypermetabolism partially corrected the underestimation of colorimetric assays in evaluating the direct antitumor activity of BEV. Our findings suggest that tetrazolium-based colorimetric assays are unsuitable for accurately assessing the in vitro cytotoxicity of anti-VEGF drugs and may be the methodological reason for the controversial direct antitumor effect of BEV.

Safety assessment of surfactin-producing Bacillus strains and their lipopeptides extracts in vitro and in vivo.

Beneficial Bacillus strains can be administered to livestock as probiotics to improve animal health. Cyclic lipopeptides produced by Bacillus such as surfactins may be responsible for some of the beneficial effects due to their anti-inflammatory and immunomodulatory activity. The aim of the present study was to isolate and evaluate the biocompatibility of native Bacillus spp. strains and their surfactin-like lipopeptides in vitro and in vivo to determine their potential to be used on animals. Biocompatibility of endospore suspensions (108 UFC/mL), and different dilutions (1:10; 1:50; 1:100; 1:500, and 1:1000) of Bacillus lipopeptide extracts containing surfactin was tested on Caco-2 cells by microculture tetrazolium-based colorimetric assay. Genotoxicity was tested on BALB/c mice (n = 6) administered 0.2 mL of endospore suspensions by the bone marrow erythrocyte micronuclei assay. All the isolates tested produced between 26.96 and 239.97 µg mL- 1 of surfactin. The lipopeptide extract (LPE) from isolate MFF1.11 demonstrated significant cytotoxicity in vitro. In contrast, LPE from MFF 2.2; MFF 2.7, TL1.11, TL 2.5, and TC12 had no cytotoxic effect (V% > 70%) on Caco-2 cells, not affecting cell viability signifficantly in most treatments. Similarly, none of the endospore suspensions affected cell viability (V% > 80%). Likewise, endospores did not cause genotoxicity on BALB/c mice. This study was elementary as a first step for a new line of research, since it allowed us to choose the safest isolates to keep working on the search of new potentially probiotic strains destined to production animals to improve their performance and health.

Abstract 3694: Metabolic response in human cervical cancer cells to modifications in bioavailable ATP

Abstract Introduction: Cancer cells utilize a markedly reduced oxidative phosphorylation (OXPHOS) when experiencing conditions of high oxygen stress (Warburg Effect/aerobic glycolysis), a phenomenon first studied by Otto Warburg. While there have been many discoveries associated with metabolic differences across cancer cells, one of the most common alterations is the primary means of generating ATP. The importance of deciphering these cellular decisions will enable an understanding of the multifaceted role ATP has in cancer proliferation, senescence, and chemotherapeutic resistance. The use of synthetic protein DX, a man-made ATP chelator with high specificity and affinity, is a proposed method of investigating the metabolic response in cancer cells. Hypothesis: Following DX expression, HeLa cells respond to energy stress (reduced intracellular ATP) via metabolic adaptations, specifically inducing the spare respiratory capacity. Methods: The impact of DX on cell viability was determined using a tetrazolium-based colorimetric cell viability assay and a caspase 3/7 assay. To correlate phenotypic/viability change with DX activity, bioavailable ATP levels were measured at specific time points following DX expression. Additionally, the relative contribution of glycolysis and OXPHOS to the total ATP production rate was measured using the label-free XF Real Time ATP Rate Assay and XF Cell Mito Stress Test (XFe96 Seahorse, Agilent Technologies) over time post DX expression. To determine the impact of ATP stress on mitochondrial and nuclear content, both were quantified following DX-expression. Results: In a time- and dose- dependent manner, DX negatively impacted cell growth and induced cell death via apoptosis, at a time concomitant with a decrease in bioavailable ATP. In response to DX over time, the total ATP production rates in HeLa cells significantly decreased. Importantly, this reduction in the rate of ATP production was associated with a significant downregulation in both OXPHOS and glycolysis. Conclusion: Advances in synthetic biology have allowed for the intentional design of artificial proteins that maintain function in vivo. These proteins can be developed into powerful investigative tools to study biological questions, such as cellular decisions involved in cancer cell metabolism. In response to DX-potentiated ATP stress, the significant reduction in both glycolysis and oxidative phosphorylation suggests metabolic downregulation is required to maintain HeLa cell viability. Citation Format: Parth K. Jayaswal, Taha Muhammad, Ashley Brown, Jeffrey Norris, Shaleen B. Korch. Metabolic response in human cervical cancer cells to modifications in bioavailable ATP. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3694.

Interfering with Color Response by Porphyrin-Related Compounds in the MTT Tetrazolium-Based Colorimetric Assay.

Porphyrin compounds are widely distributed in various natural products and biological systems. In this study, effects of porphyrin-related compounds including zinc protoporphyrin (ZnPP), protoporphyrin IX (PPIX), cyanocobalamin (CBL), hemin, and zinc phthalocyanine (ZnPC) were analyzed on color response of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) tetrazolium-based assay, a commonly-used method for analyzing cell viability. Color responses of MTT formazan formed in cells treated with ZnPP, PPIX, or ZnPC were significantly reduced even at submicromolar concentrations without affecting cell viability, whereas hemin and CBL did not. ZnPP, PPIX, and ZnPC rapidly induced degradation of MTT formazan already-produced by cells when exposed to light, but not under a dark condition. Photosensitizing properties of the three compounds were also verified through extensive generation of reactive oxygen species under light. The porphyrins did not affect the stability of water-soluble formazans including XTT, WST-1, WST-8, and MTS formazans. Several factors including different light sources and antioxidants modulated the degradation process of MTT formazan by the porphyrins. The results suggest that certain porphyrin compounds could cause a severe artifact in the MTT assay through rapid degradation of formazan dye due to their photosensitizing property, which needs to be considered carefully in the related assays.

Phytochemical composition and cytotoxicity of ethanolic extracts of some selected plants

ABSTRACT This study determines the phytochemical contents of selected medicinal plant extracts quantitatively using spectrophotometry and to evaluate their safety on mammalian cells in vitro using tetrazolium-based colorimetric cytotoxicity assay. Twenty-two ethanolic plant extracts were assessed, and the result showed that Psidium guajava had the highest concentration of alkaloids (219.06±11.50mg/g dry-weight (dw)). In contrast, Acacia nilotica leaf extract with 191.60±9.07mg/gdw had the highest level of flavonoids. Vernonia amygdalina and Moringa oleifera contained more steroids than the other plant extracts while condensed tannin was highest in Carya illinoinensis (21.72 ± 0.84 mg/g dw). Coffea arabica, Acacia nilotica leaf, Vernonia amygdalina, Carya illinoinensis and Psidium guajava had more saponins than the other plant extracts. Regarding cytotoxicity evaluation, Allium cepa and Tulbaghia violacea with concentration killing 50% of cells (LC50) values of 0.5182±0.40 and 0.4909 ± 0.034 mg/ml, respectively, were the safest of all the plant extracts. Acacia nilotica pod and Camelia japonica leaf extracts were the most cytotoxic with LC50 values of 0.0101 ± 0.016 and 0.0151 ± 0.005 mg/ml, respectively, which are well below the recommended toxic cut-off point (0.03mg/ml). The best selectivity index (SI) value of 1.061 was obtained with Aloe ferox against S. aureus, which is a Gram-positive bacterium.

Anatomically and Biomechanically Relevant Monolithic Total Disc Replacement Made of 3D-Printed Thermoplastic Polyurethane.

Various implant treatments, including total disc replacements, have been tried to treat lumbar intervertebral disc (IVD) degeneration, which is claimed to be the main contributor of lower back pain. The treatments, however, come with peripheral issues. This study proposes a novel approach that complies with the anatomical features of IVD, the so-called monolithic total disc replacement (MTDR). As the name suggests, the MTDR is a one-part device that consists of lattice and rigid structures to mimic the nucleus pulposus and annulus fibrosus, respectively. The MTDR can be made of two types of thermoplastic polyurethane (TPU 87A and TPU 95A) and fabricated using a 3D printing approach: fused filament fabrication. The MTDR design involves two configurations—the full lattice (FLC) and anatomy-based (ABC) configurations. The MTDR is evaluated in terms of its physical, mechanical, and cytotoxicity properties. The physical characterization includes the geometrical evaluations, wettability measurements, degradability tests, and swelling tests. The mechanical characterization comprises compressive tests of the materials, an analytical approach using the Voigt model of composite, and a finite element analysis. The cytotoxicity assays include the direct assay using hemocytometry and the indirect assay using a tetrazolium-based colorimetric (MTS) assay. The geometrical evaluation shows that the fabrication results are tolerable, and the two materials have good wettability and low degradation rates. The mechanical characterization shows that the ABC-MTDR has more similar mechanical properties to an IVD than the FLC-MTDR. The cytotoxicity assays prove that the materials are non-cytotoxic, allowing cells to grow on the surfaces of the materials.

Evaluation of erythrocyte viability, antioxidant capacity and antiplasmodial activity induced by alkaloid extract of Phyllanthus amarus

Introduction: The production of large amounts of reactive oxygen species in severe malarial infection is due to parasite invasion to erythrocytes. Malaria resistance to medication has left malaria-endemic countries with no alternate source of medications but traditional medicine. One of such plants utilized by traditional healers is Phyllanthus amarus. Therefore, this study aims at ascertaining the antiplasmodial and cytotoxic activities of P. amarus and its specific actions on retaining erythrocyte viability and antioxidant activity. Methods: Antiplasmodial and erythrocyte viability activities were determined in vitro via parasite suppression and tetrazolium-based colorimetric assays, respectively. Antioxidant capacity was determined by measuring extract’s ability to inhibit lipid peroxidation, scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) and superoxide ions, reduce iron(III) ions, and chelate iron in vitro using documented methods. Results: Alkaloid extracts of P. amarus showed great antiplasmodial activity (IC50=0.52μg/mL) and low cytotoxic activity (CC50=54.95 μg/mL). Erythrocyte viability assay showed the minimal impact of the extract on the uninfected erythrocytes but improved viability of the infected RBC in a dose dependent manner, and antioxidant activity manifested mainly in its iron chelating activity (EC50=0.34 μg/mL). Conclusion: This study suggests that the alkaloid extract of P. amarus has significant antiplasmodial and antioxidant activities. These activities promoted the repair of parasite-induced free radical damage to the erythrocyte membrane but distorted the parasites redox balance and defense mechanism, and hence survival rate as indicated by the parasite suppression associated with alkaloid extract treatment of malarial infection.

Antibacterial and anti-inflammatory activity of plant species used in traditional poultry ethnomedicine in Zimbabwe: A first step to developing alternatives to antibiotic poultry feed additives

Ethnopharmacological relevanceGlobal interest in phytogenic feed additives as alternatives to antibiotics in feed has been spurred by the banning of antibiotic growth promoters by several countries. Suitable plant extracts for development of phytogenic feed additives should have therapeutic value and should also be safe. Aim of studyThe aim of this study was to evaluate the antibacterial, antioxidant and anti-lipoxygenase activities as well as cytotoxicity of selected plant species used in poultry ethnomedicine in Zimbabwe. MethodsAntibacterial activity was determined against three ATCC strains (Staphylococcus aureus, Escherichia coli, Salmonella Enteritidis) and two clinical strains isolated from chickens (Escherichia coli and Salmonella Gallinarum) using a two-fold serial microdilution assay. Qualitative antibacterial bioautography was also carried out using the ATCC strains. Antioxidant activities of crude acetone and methanol extracts were determined using free radical scavenging assays whilst anti-lipoxygenase activity was evaluated using a ferrous oxidation-xylenol orange (FOX) assay. Cytotoxicity was evaluated using a tetrazolium-based colorimetric assay (MTT assay) on Vero monkey kidney cells. ResultsErythrina abyssinica had the best antibacterial activity against both ATCC strains and clinical strains with minimum inhibitory concentration (MIC) values ranging from 0.02 to 0.156 mg/ml. Aloe greatheadii, Adenia gummifera (leaves), Senna singueana and Aloe chabaudii had moderate activity against the poultry pathogens. Bioautography showed that all ten plant species have antibacterial activity against the tested microorganisms with E. abyssinica and S. singueana having prominent bands of inhibition against both Gram-negative and Gram-positive bacteria. The acetone extract of S. singueana and the methanol extract of Euphorbia matabelensis had the most powerful antioxidant activities with mean IC50 values of 1.43 μg/ml and 1.31 μg/ml respectively in the ABTS assay which were comparable with those of the positive controls (ascorbic acid and trolox). Bobgunnia madagascariensis, A. chabaudii, E. abyssinica and Tridactyle bicaudata extracts had reasonable antioxidant activity. The S. singueana extract had the most potent anti-lipoxygenase activity with a mean IC50 value of 1.72 μg/ml. The cytotoxicity results showed that only the acetone extracts of A. greatheadii and S. singueana were relatively safe at concentrations that were active against the tested microorganisms (selective index >1). Regarding anti-lipoxygenase activity, extracts of B. madagascariensis, S. singueana, T. bicaudata and E. matabelensis were more active than toxic (selective index >5) indicating anti-inflammatory potential. ConclusionsThis study showed that S. singueana had a cocktail of therapeutic activity and supports further investigation of this plant species for development of phytogenic poultry feed additives. Other plant species with noteworthy biological activities include B. madagascariensis, E. abyssinica, A. greatheadii, T. bicaudata and E. matabelensis.

The effect of pore morphology and agarose coating on mechanical properties of tricalcium phosphate scaffolds

Three-dimensional biocompatible porous structures can be fabricated using different methods. However, the biological and mechanical behaviors of scaffolds are the center of focus in bone tissue engineering. In this study, tricalcium phosphate scaffolds with similar porosity contents but different pore morphologies were fabricated using two different techniques, namely, the replica method and the pore-forming agent method. The samples fabricated using the pore-forming agent showed more than two times higher compressive and bending strengths and more than three times higher compressive moduli. Furthermore, a thin layer of agarose coating improved the compressive and bending strength of both types of ceramic scaffolds. Subsequently, the samples’ capability to guide biomineralization was evaluated by immersion into a simulated body fluid that developed Ca-P nano-platelets formation and enhanced the compressive strength. Finally, the tetrazolium-based colorimetric (MTT) assay was used to evaluate L929 cell viability and proliferation on all the samples and confirmed that cell behavior was not affected by pore morphology or agarose coating. In summary, samples produced by the use of the pore-forming agent showed higher potential to be applied as bone scaffolds in tissue engineering applications.

Bee Venom Effect on Glioblastoma Cells Viability and Gelatinase Secretion.

BackgroundThe involvement of MMP-2 and MMP-9 in the pathogenesis of various kinds of cancers including glioblastoma is well documented. The evaluation of the anticancer potential of honey bee (Apis mellifera) venom (BV) consisting of the inhibition of MMP-2 and MMP-9 secretion in a glioblastoma cell culture model was the aim of the study.Methods8-MG-BA and GAMG human primary glioblastoma cell lines vs. HT-22 mouse hippocampal neuronal cells were applied for the study. The BV dose (0.5, 1.0, 1.25, 1.5, 1.75, 2.0, 2.5, and 5.0 μg/ml) and time-dependent (24, 48, 72 h) cytotoxicity was evaluated with the tetrazolium-based colorimetric assay (MTT test). MMP-2 and MMP-9 activities in the cell culture medium under different BV concentrations were determined by gelatin zymography.ResultsA dose and time-dependent BV effect on cytotoxicity of both glioblastoma cell lines and hippocampus line was observed. The weakest, but statistically important effect was exerted by BV on HT-22 cells. The greatest cytotoxic effect of BV was observed on the 8-MG-BA line, where a statistically significant reduction in viability was observed at the lowest BV dose and the shortest incubation time. The reduction of both gelatinases secretion was observed at 8-MG-BA and GAMG lines without significant effect of HT-22 cell line.ConclusionIn vitro studies indicate that BV has both cytotoxic and inhibitory effects on the secretion of MMP-2 and MMP-9 in selected lines of glioma, suggesting anticancer properties of BV.