Standard Operating Procedure to Optimize Resazurin-Based Viability Assays.

The resazurin assay, also known as the Alamar Blue assay, stands as a cornerstone technique in cell biology, microbiology, and drug development. It assesses the viability of cells through the conversion of resazurin into highly fluorescent resorufin. The resulting fluorescence intensity provides a reliable estimate of viable cell numbers. Cytotoxicity assays, such as the resazurin-based method, play a crucial role in the screening of potential drug candidates and in the assessment of pharmaceutical and chemical toxicity. In recent years, inconsistencies have arisen in pharmacogenomic studies, often due to poorly optimized laboratory protocols. These inconsistencies hinder progress in understanding how substances affect cell health, leading to unreliable findings. Thus, the need for standardized and rigorously optimized protocols is evident to ensure consistent and accurate results in cytotoxicity studies. This manuscript describes a standardized procedure for optimizing resazurin-based viability assays to improve the reliability of cytotoxicity data. This optimization approach focuses on critical experimental parameters and data quality, aiming to achieve a level of measurement imprecision of less than 20%. In conclusion, to address the critical issues of reproducibility and reliability, protocol standardization, such as the one described in this manuscript, can greatly enhance the credibility of cytotoxicity studies, ultimately advancing drug safety assessments.

Comparison of Radiation Response between 2D and 3D Cell Culture Models of Different Human Cancer Cell Lines.

Radiation therapy is one of the most effective tools in cancer therapy. However, success varies individually, necessitating improved understanding of radiobiology. Three-dimensional (3D) tumor spheroids are increasingly gaining attention, being a superior in vitro cancer model compared to 2D cell cultures. This in vitro study aimed at comparing radiation responses in 2D and 3D cell culture models of different human cancer cell lines (PC-3, LNCaP and T-47D) irradiated with varying doses (1, 2, 4, 6, 8 or 20 Gy) of X-ray beams. Radiation response was analyzed by growth analysis, various cell viability assays (e.g., clonogenic assay, resazurin assay) and amount of DNA damage (γH2AX Western Blot). Results showed decreasing cell proliferation with the increase of radiation doses for all cell lines in monolayers and spheroids of LNCaP and T-47D. However, significantly lower radiosensitivity was detected in spheroids, most pronounced in PC-3, evincing radiation resistance of PC-3 spheroids up to 8 Gy and significant growth inhibition only by a dose escalation of 20 Gy. Cell line comparison showed highest radiosensitivity in LNCaP, followed by T-47D and PC-3 in 2D, whereas, in 3D, T-47D showed highest sensitivity. The results substantiate the significant differences in radiobiological response to X-rays between 2D and 3D cell culture models.

Development and characterization of Spodoptera mauritia ovarian primary cell culture and evaluation of fenoxycarb toxicity.

The rice swarming caterpillar, Spodoptera mauritia (Biosduval, 1833) (Lepidoptera: Noctuidae), has been considered a severe pest and has caused the extreme loss of rice paddies in the nursery stages. The massive reproductive efficiency of pests results from balanced endocrine functioning. Insect treatments with exogenous either juvenile hormones (JH) or juvenile hormone analogues (JHAs) during low endogenous JH titers disrupt metamorphosis and ovarian development. Hence, it was thought worthwhile to develop a primary ovarian cell culture of Spodoptera mauritia to study the biological efficiency of JHA, fenoxycarb, at the cellular level. The study envisioned the cell characteristics and growth properties and found that most cells were spherical. Spindle-shaped cells were also present during the initial stage of active cell division. The majority of the cells grew attached to the bottom of the culture plates, and a few grew in suspension. The cell doubling time was 144 ± 14 h. The growth curve exhibited a sigmoid pattern of cell proliferation at first, followed by a declining phase. These properties indicate that the primary ovarian culture is a suitable candidate for developing a novel cell line of S. mauritia. The preliminary study investigated the cytotoxicity of the insect growth regulator fenoxycarb in ovarian, primary cell culture. Incubated cells showed noticeable morphological changes, including cell shrinkage and apoptotic body formation; inhibition of cellular proliferation; and apoptosis-inducing effects in ovarian, primary cell culture of S. mauritia. Evidently, from these results, it is clear that juvenile hormone mimics can react in a meaningful way to control the rice pest S. mauritia by inducing cellular apoptosis in ovarian cells, which may cause adult females to become sterile.

Screening and evaluating honokiol from Magnolia officinalis against Nocardia seriolae infection in largemouth bass (Micropterus Salmoides).

Nocardiosis caused by Nocardia seriolae is a major threat to the aquaculture industry. Given that prolonged therapy administration can lead to a growth of antibiotic resistant strains, new antibacterial agents and alternative strategies are urgently needed. In this study, 80 medicinal plants were selected for antibacterial screening to obtain potent bioactive compounds against N. seriolae infection. The methanolic extracts of Magnolia officinalis exhibited the strongest antibacterial activity against N. seriolae with the minimal inhibitory concentration (MIC) of 12.5 µg/ml. Honokiol and magnolol as the main bioactive components of M. officinalis showed higher activity with the MIC value of 3.12 and 6.25 µg/ml, respectively. Sequentially, the evaluation of antibacterial activity of honokiol in vivo showed that honokiol had good biosafety, and could significantly reduce the bacterial load of nocardia-infected largemouth bass (p < .001). Furthermore, the survival rate of nocardia-infected fish fed with 100 mg/kg honokiol was obviously improved (p < .05). Collectively, these results suggest that medicinal plants represent a promising reservoir for discovering active components against Nocardia, and honokiol has great potential to be developed as therapeutic agents to control nocardiosis in aquaculture.

Study of antimycobacterial, cytotoxic, and mutagenic potential of polymeric nanoparticles of copper (II) complex.

This study aimed to encapsulate and characterise a potential anti-tuberculosis copper complex (CuCl2(INH)2.H2O:I1) into polymeric nanoparticles (PNs) of polymethacrylate copolymers (Eudragit®, Eu) developed by nanoprecipitation method. NE30D, S100 and, E100 polymers were tested. The physicochemical characterisations were performed by DLS, TEM, FTIR, encapsulation efficiency and, in vitro release studies. Encapsulation of I1 in PN-NE30D, PN-E100, and PN-S100 was 26.3%, 94.5%, 22.6%, respectively. The particle size and zeta potentials were 82.3 nm and -24.5 mV for PNs-NE30D, 304.4 nm and +18.7 mV for PNs-E100, and 517.9 nm and -6.9 mV for PNs-S100, respectively. All PDIs were under 0.5. The formulations showed an I1 controlled release at alkaline pH with 29.7% from PNs-NE30D, 7.9% from PNs-E100 and, 28.1% from PNs-S100 at 1 h incubation. PNs were stable for at least 3 months. Particularly, PNs-NE30D demonstrated moderate inhibition of M. tuberculosis and low cytotoxic activity. None of the PNs induced mutagenicity.

A simple and low-cost resazurin assay for vitality assessment across species.

Working with biological organisms requires knowledge about the state of their viability and vitality to ascertain efficient processes. The phenoxazine dye resazurin is routinely used for viability assessment of many different species. Here, a novel use for resazurin as an indicator for vitality assessment across several species is proposed. Different amounts of biomass as well as mixtures of live/dead biomass were investigated for their capabilities of metabolizing resazurin and monitored over time. Increasing (live) biomass was found to increase reaction rate in a linear fashion, giving information about the cells' vitality. In an application example, stored suspension cultures of Sporosarcina pasteurii were found to decrease in viability over time, while urease activity decreased as well. For the first time, the assessment of vitality by one technique was demonstrated for several species in parallel.

High throughput method to determine the surface activity of antimicrobial polymeric materials.

Surface colonization by microorganisms, combined with the rise in antibiotic resistance, is the main cause of production failures in various industries. Self-sterilising materials are deemed the best prevention of surface colonization. However, current screening methods for these sterilising materials are laborious and time-consuming. The disk diffusion antimicrobial assay and the Japanese industrial standard method for antimicrobial activity on solid surfaces, JIS Z 2801, were compared to our modified solid surface antimicrobial assay in terms of detecting the activity of antibiotic-containing cellulose disks against four bacterial pathogens. Our novel assay circumvents the long incubation times by utilising the metabolic active dye, resazurin, to shorten the time in which antibacterial results are obtained to less than 4 h. This assay allows for increased screening to identify novel sterilising materials for combatting surface colonisation.•Disk diffusion assay could only detect the activity of small compounds that leached from the material over 20-24 h.•JIS Z 2801 was also able to detect the surface activity of non-polar compounds, thought to be inactive based on the disk diffusion results.•The resazurin solid surface antimicrobial assay could obtain the same results as the JIS Z 2801, within a shorter time and in a high-throughput 96-well plate setup.

Antimycobacterial screening of selected medicinal plants against Mycobacterium tuberculosis H37Rv using agar dilution method and the microplate resazurin assay.

Background: Tuberculosis (TB), has been serious disease to the global human population causing millions of deaths worldwide. The recent increase in the number of multi-drug resistant clinical isolates of Mycobacterium tuberculosis has created an urgent need for the discovery and development of new anti-TB drugs. Medicinal plants have had a great influence on the daily lives of people living in developing countries, particularly in India. Medicinal plants were selected, and they were evaluated for its anti-TB activity against the pathogenic strain of M. tuberculosis H37Rv. Methods: Eleven medicinal plants were selected on the basis of its literature survey, and three different extracts were prepared. Antimycobacterial activities were screened using two in vitro assays, namely agar dilution assay and microplate resazurin assay against M. tuberculosis H37Rv at different concentrations of prepared extracts. We analyzed minimal inhibition concentrations and percentage of inhibition of the used strain of Mycobacteria. Isoniazid was used as a standard anti-TB drug. Results: The results of this study showed that aqueous extracts four selected medicinal plants Ocimum sanctum, Adhatoda vasica, Leptadenia reticulata, and Cocculus hirsutus having minimum inhibitory concentration at 500 µg/ml, 500 µg/ml, 250 µg/ml, and 250 µg/ml, respectively, and O. sanctum (60.24%), A. vasica (62.89%), L. reticulata (74.26%), and C. hirsutus (81.67%) showed significant anti-TB activity against M. tuberculosis. Conclusion: This study helps society to found new anti-TB agents having better anti-TB activity with lesser or no side effects.

Gold nanoparticle-assisted enhancement in bioactive properties of Australian native plant extracts, Tasmannia lanceolata and Backhousia citriodora.

Green nanotechnology plays a significant role in developing effective treatment strategies for numerous diseases. The biological synthesis of metal nanoparticles (M-NPs) possesses suitable alternatives than chemical techniques. Using plant extract to synthesis M-NPs is an eco-friendly, non-toxic, and cost-effective that are suitable for biological applications and efforts are directed to explore the efficacy of these materials in cancer management. In this study, gold nanoparticles (Au-NPs) were synthesised by following a one-step green synthesis, a reaction between HAuCl4 and biological molecules present in Tasmannia lanceolata leaf extract as a sole agent for both reduction and stabilisation. The characterisation techniques confirmed the successful synthesis of Au-NPs. TEM photograph revealed spherical shape nanoparticles with an average size of 7.10 ± 0.66 nm. The in-vitro cytotoxicity of Au-NPs was performed by analysing the percentage inhibition of cell viability using Resazurin assay on human liver cancer (HepG2), melanoma cancer (MM418 C1) and breast cancer (MCF-7) cell lines and compared with Au-NPs synthesised by using Backhousia citriodora leaf extract. The results showed that biosynthesised Au-NPs displayed greater inhibitory activity towards MCF-7 cancer cells proliferation compared to HepG2 and MM418 cancer cells. In addition, synthesised Au-NPs@ Tasmannia lanceolata leaf extract indicated higher inhibitory activity towards cancer cells compared to Au-NPs@ Backhousia citriodora leaf extract.

Antibacterial Activities of Echinops kebericho Mesfin Tuber Extracts and Isolation of the Most Active Compound, Dehydrocostus Lactone.

Echinops kebericho Mesfin is traditionally used for the treatment of various infectious diseases. This study investigated antibacterial activity of the essential oil (EO) and the different fractions of ethanol extract. The most active component was isolated and identified. Isolation and purification was accomplished using chromatographic techniques while identification was done by spectroscopic method. Minimum inhibitory concentration (MIC) was determined using the broth micro-dilution method. In bioactive-guided isolation, percent inhibition was determined using optical density (OD) measurement. The MICs of the essential oil ranged from 78.125 µg/ml to 625 µg/ml, and its activity was observed against methicillin-resistant Staphylococcus aureus (MRSA, NCTC 12493). Ethyl acetate fraction showed high activity against MRSA (NCTC 12493), MIC = 39.075 µg/ml followed by Enterococcus faecalis (ATCC 49532), MIC = 78.125 µg/ml and was least active against Klebsiella pneumoniae (ATCC 700603), MIC = 1,250 µg/ml. MIC of hexane fraction ranged from 156.2 µg/ml to Escherichia coli (ATCC 49532) to 1,250 µg/ml to E. coli (NCTC 11954). The MICs of chloroform fraction ranged from 312.5 to 2500 µg/ml; while butanol fraction could be considered pharmacologically inactive as its MIC value was 2,500 µg/ml for all and no activity against E. coli (NCTC 11954). Dehydrocostus lactone was successfully isolated and identified whose MIC was 19.53 µg/ml against MRSA. Dehydrocostus lactone isolated from E. kebericho M. showed noteworthy antibacterial activity which lends support to ethnopharmacological use of the plant. Further optimization should be done to improve its antibacterial activities and pharmacokinetic profile.

Evaluation of synergy between host and pathogen-directed therapies against intracellular Leishmania donovani.

Visceral leishmaniasis (VL) is associated with treatment complications due to the continued growth of resistant parasites toward currently available pathogen-directed therapeutics. To limit the emergence and combat resistant parasites there is a need to develop new anti-leishmanial drugs and alternative treatment approaches, such as host-directed therapeutics (HDTs). Discovery of new anti-leishmanial drugs including HDTs requires suitable in vitro assay systems. Herein, we modified and evaluated a series of resazurin assays against different life-stages of the VL causing parasite, Leishmania donovani to identify novel HDTs. We further analyzed the synergy of combinatorial interactions between traditionally used pathogen-directed drugs and HDTs for clearance of intracellular L. donovani. The inhibitory concentration at 50% (IC50) of the five evaluated therapies [amphotericin B (AMB), miltefosine, paromomycin, DNER-4, and AR-12 (OSU-03012)] was determined against promastigotes, extracellular amastigotes, and intracellular amastigotes of L. donovani via a resazurin-based assay and compared to image-based microscopy. Using the resazurin-based assay, all evaluated therapies showed reproducible anti-leishmanial activity against the parasite's different life-stages. These results were consistent to the traditional image-based technique. The gold standard of therapy, AMB, showed the highest potency against intracellular L. donovani, and was further evaluated for combinatorial effects with the HDTs. Among the combinations analyzed, pathogen-directed AMB and host-directed AR-12 showed a synergistic reduction of intracellular L. donovani compared to individual treatments. The modified resazurin assay used in this study demonstrated a useful technique to measure new anti-leishmanial drugs against both intracellular and extracellular parasites. The synergistic interactions between pathogen-directed AMB and host-directed AR-12 showed a great promise to combat VL, with the potential to reduce the emergence of drug-resistant strains.

Standardization of a resazurin-based assay for the evaluation of metabolic activity in oral squamous carcinoma and glioblastoma cells.

The widely accepted resazurin-based assay can be used, prior to in vivo studies, as an inexpensive method to determine cytotoxicity. The aim of this study was to evaluate and standardize the assay conditions for oral squamous cancer cell (OSCC) and glioblastoma (U87-MG) lines by UV-vis spectroscopy. The cells were treated with 25 µgmL-1 of resazurin sodium salt and then incubated for 4 h, 6 h, and 6.5 h. All absorbance measurements were carried out at 21 ± 1 °C on a spectrophotometer with a microplate reader. After 4-hs of incubation, resazurin was completely reduced by OSCC cells, as demonstrated by the suppression of the absorbance at 380 nm. However, the U87-MG cells needed 6.5 h of incubation to demonstrate the same behavior. The Statistical analysis did not indicate significant differences between the OSCC and U87-MG cell lines' viability after 4 and 6.5 h respectively. We concluded that spectroscopic analysis is an efficient method for the standardization of the resazurin assay. In addition, without the implementation of suitable protocols, there could be an increase in the chance of errors or false positives or negatives that would reduce the usefulness of the data.

In vitro activity of 12 antimicrobial peptides against Mycobacterium tuberculosis and Mycobacterium avium clinical isolates.

Tuberculosis (TB) remains a major threat to human health worldwide. The increasing incidence of non-tuberculous mycobacterial infections and particularly those produced by Mycobacterium avium has emphasized the need to develop new drugs. Additionally, high levels of natural drug resistance in non-tuberculous mycobacteria (NTM) and the emergence of multidrug-resistant (MDR) TB is of great concern. Antimicrobial peptides (AMPs) are antibiotics with broad-spectrum antimicrobial activity. The objective was to assess the activity of AMPs against Mycobacterium tuberculosis and M. avium clinical isolates. MICs were determined using microtitre plates and the resazurin assay. Mastoparan and melittin showed the greatest activity against M. tuberculosis, while indolicidin had the lowest MIC against M. avium. In conclusion, AMPs could be alternatives for the treatment of mycobacterial infections. Further investigation of AMPs' activity in combination and associated with conventional antibiotics and their loading into drug-delivery systems could lead to their use in clinical practice.

Optimization of a resazurin-based microplate assay for large-scale compound screenings against Klebsiella pneumoniae.

A new resazurin-based assay was evaluated and optimized using a microplate (384-well) format for high-throughput screening of antibacterial molecules against Klebsiella pneumoniae. Growth of the bacteria in 384-well plates was more effectively measured and had a > sixfold higher signal-to-background ratio using the resazurin-based assay compared with absorbance measurements at 600 nm. Determination of minimum inhibitory concentrations of the antibiotics revealed that the optimized assay quantitatively measured antibacterial activity of various antibiotics. An edge effect observed in the initial assay was significantly reduced using a 1-h incubation of the bacteria-containing plates at room temperature. There was an approximately 10% decrease in signal variability between the edge and the middle wells along with improvement in the assay robustness (Z' = 0.99). This optimized resazurin-based assay is an efficient, inexpensive, and robust assay that can quantitatively measure antibacterial activity using a high-throughput screening system to assess a large number of compounds for discovery of new antibiotics against K. pneumoniae.

Comparison of mucoadhesive and cohesive features of poly(acrylic acid)-conjugates respective their molecular mass.

OBJECTIVES: This study aimed to assess the impact of molecular mass as well as the differences between poly(acrylic acid)-thiol-conjugates (PAA100,250,450KDa) on their mucoadhesive and cohesive qualities. METHODS: Covalent attachment of cysteine (CYS), cysteamine (CYSM) and l-gluthathione (GSH) to poly(acrylic acid) was achieved by formation of amide bonds between primary amino group of the amino acid (in the case of cysteine and glutathione), respectively the amino group of the aminothiol cysteamine and carboxylic acid group of the polymer. Obtained polymer conjugates were evaluated in regard to their safety profile, mucoadhesive properties on the buccal mucosa by rotating cylinder, tensile strength and rheological investigations, respectively. Furthermore, stability, cohesive and water uptake studies were performed. KEY FINDINGS: Mucoadhesive studies revealed that maximum detachment force of PAACYS450 was 24.3-fold higher in comparison to the respective controls. Stability studies revealed for PAACYS450 a 50.2-fold higher stability compared to controls. CONCLUSION: Taken together, among all polymers tested, PAACYS450 evinced the most favorable qualities regarding mucoadhesion and cohesion, followed by PAACYSM450 and PAACYS250.

A Microplate-Based System as In Vitro Model of Biofilm Growth and Quantification.

We describe a 96-well microtiter plate-based system as an in vitro model for biofilm formation and quantification. Although in vitro assays are artificial systems and thus significantly differ from in vivo conditions, they represent an important tool to evaluate biofilm formation and the effect of compounds on biofilms. Stainings to evaluate the amount of biomass (crystal violet staining) and the number of metabolically active cells (resazurin assay) are discussed and specific attention is paid to the use of this model to quantify persisters in sessile populations.

Screening antimycobacterial activity of Baccharis dracunculifolia, Centella asiatica, Lantana camara and Pterodon emarginatus / Atividade anti-micobacteriana de Baccharis dracunculifolia, Centella asiatica, Lantana camara e Pterodon emarginatus

ABSTRACT The permanent investigation of new antimycobacterial drugs is necessary for the eradication programs of tuberculosis and other mycobacterium-related diseases. The aim of the present study is to search for new sources of antimycobacterial drugs using plant materials. In this study, 11 plant materials (extracts, essential oils and some fractions) obtained from 4 species of medicinal plants traditionally used as general therapeutics for different illnesses and specifically as treatment of tuberculosis, were evaluated using the microplate resazurin assay against 2 species of the Mycobacterium tuberculosis Complex and 3 nontuberculous mycobacteria. The results showed the hexane extract and the essential oil from fruits of Pterodonemarginatus (Vogel) as potential sources of antimycobacterial drugs against 4 species of tested mycobacteria. The hexane fraction of methanol extract from leaves of Centella asiatica also presented significant mycobacterial growth inhibition, but against M. chelonae only. In conclusion, it was possible to contribute to the antimycobacterial investigations by presenting three new samples of plants with significant antimicrobial activity against four Mycobacteriumspp and suggest future studies about the antimycobacterial properties of fruits from P. emarginatus.

RESUMO A investigação permanente de novas drogas antimicobacterianas é necessária no programa de erradicação da tuberculose e de outras doenças relacionadas com micobactérias. O objetivo deste estudo foi buscar novas fontes de drogas antimicobacterianas usando material vegetal. Neste estudo, 11 materiais de base vegetal (extratos, óleos essenciais e algumas frações) foram avaliados contra 5 espécies de micobactérias. Estes materiais foram obtidos a partir de 4 espécies de plantas medicinais tradicionalmente utilizadas como terapêutica geral para diferentes doenças e, especificamente, no tratamento de tuberculose (Baccharis dracunculifolia, Centella asiatica, Lantana camara, Pterodon emarginatus). Os ensaios foram realizados em microplacas com resazurina contra duas espécies do Complexo Mycobacteriumtuberculosis e 3 espécies de micobactérias não tuberculosas. Os resultados mostraram o extrato hexânico e o óleo essencial de frutos de P.emarginatus como potenciais fontes para drogas antimicobacterianas contra quatro espécies de micobactérias testadas. A fração hexânica do extrato metanólico das folhas de C. asiatica também apresentou significativa inibição do crescimento de micobactérias apenas contra M.chelonae. Em conclusão, foi possível contribuir para as investigações de antimicobacterianos por apresentar três novas amostras de plantas com atividade antimicrobiana significativa contra quatro Mycobacterium spp e sugerir a realização de estudos futuros sobre as propriedades antimicobacterianas de frutos de P. emarginatus.

Optimisation of the microplate resazurin assay for screening and bioassay-guided fractionation of phytochemical extracts against Mycobacterium tuberculosis.

INTRODUCTION: Because of increased resistance to current drugs, there is an urgent need to discover new anti-mycobacterial compounds for the development of novel anti-tuberculosis drugs. The microplate resazurin assay (MRA) is commonly used to evaluate natural products and synthetic compounds for anti-mycobacterial activity. However, the assay can be problematic and unreliable when screening methanolic phytochemical extracts. OBJECTIVE: To optimise the MRA for the screening and bioassay-guided fractionation of phytochemical extracts using Mycobacterium tuberculosis H37Ra. METHODS: The effects of varying assay duration, resazurin solution composition, solvent (dimethyl sulphoxide - DMSO) concentration and type of microtitre plate used on the results and reliability of the MRA were investigated. The optimal bioassay protocol was applied to methanolic extracts of medicinal plants that have been reported to possess anti-mycobacterial activity. RESULTS: The variables investigated were found to have significant effects on the results obtained with the MRA. A standardised procedure that can reliably quantify anti-mycobacterial activity of phytochemical extracts in as little as 48 h was identified. The optimised MRA uses 2% aqueous DMSO, with an indicator solution of 62.5 µg/mL resazurin in 5% aqueous Tween 80 over 96 h incubation. CONCLUSION: The study has identified an optimal procedure for the MRA when used with M. tuberculosis H37Ra that gives rapid, reliable and consistent results. The assay procedure has been used successfully for the screening and bioassay-guided fractionation of anti-mycobacterial compounds from methanol extracts of Canadian medicinal plants.

Resazurin microtiter assay for isoniazid, rifampicin, ethambutol and streptomycin resistance detection in Mycobacterium tuberculosis: Updated meta-analysis.

AIMS: The present meta-analysis aims to assess the evidence regarding the diagnostic accuracy and performance characteristics of the colorimetric redox indicator (CRI) assay with a special emphasis on the use of the resazurin microtiter assay (REMA) for determination of primary anti-tuberculosis drug resistance. SUBJECT AND METHODS: By updating previous literature searches in Medline PubMed, ISI Web, Web of Science and Google academic databases of the REMA test for determination of primary anti-tuberculosis drug resistance, this meta-analysis includes 14 studies for isoniazid (INH); 15 studies for rifampicin (RIF); 6 studies for streptomycin (STR); and 5 studies for ethambutol (EMB). SROC curve analysis was performed for meta-analysis and diagnostic accuracy was summarized. RESULTS: Pooled sensitivity was 96% (94-98%) for INH, 97% (95-98%) for RIF, 92% (87-96%) for EMB and 92% (88-95%) for STR. Pooled specificity for INH, RIF, EMB and STR was 96% (95-98%), 99% (98-99%), 86% (81-89%) and 90% (87-93%), respectively. Susceptibility testing results had been obtained in 8-9days. CONCLUSION: In conclusion, REMA seems to be a reliable test for the determination of multi-drug resistant (MDR) isolates in laboratories with limited resources. However, few studies for STR and EMB have been found, and cost-effectiveness studies need to be determined to recommend its widespread use.

Rosmarinus officinalis essential oil: antiproliferative, antioxidant and antibacterial activities

The aim of this work was to investigate and compare the antiproliferative, antioxidant and antibacterial activities of Rosmarinus officinalis essential oil, native to Pakistan. The essential oil content from the leaves of R. officinalis was 0.93 g 100g-1. The GC and GC-MS analysis revealed that the major components determined in R. officinalis essential oil were 1,8-cineol (38.5 percent), camphor (17.1 percent), α-pinene (12.3 percent), limonene (6.23 percent), camphene (6.00 percent) and linalool (5.70 percent). The antiproliferative activity was tested against two cancer (MCF-7 and LNCaP) and one fibroblast cell line (NIH-3T3) using the MTT assay, while, the antioxidant activity was evaluated by the reduction of 2, 2-diphenyl-1-picryl hydrazyl (DPPH) and measuring percent inhibition of peroxidation in linoleic acid system. The disc diffusion and modified resazurin microtitre-plate assays were used to evaluate the inhibition zones (IZ) and minimum inhibitory concentration (MIC) of R. officinalis essential oil, respectively. It is concluded from the results that Rosmarinus officinalis essential oil exhibited antiproliferative, antioxidant and antibacterial activities.