Partial purification and characterization of antimicrobial peptide(s) from Mimosa pudica (Mimosaceae)

@#Plants have been a major source of natural products for sustaining human health. The use of the different parts of the plant as infusions, decoctions, extracts, and powders are being employed in the treatment of different diseases in humans, plants, and animals. One property of great significance in terms of therapeutic treatments, especially with the emergence of multi-drug resistant microbes, is the antimicrobial activity. A new promising source of antimicrobials that demonstrate novel mechanisms of therapeutic strategies is low molecular weight peptides. In this study, the antimicrobial activities of Mimosa pudica crude and partially purified peptide extracts against Gram-negative Enterobacter cloacae ATCC 23355 and Enterobacter aerogenes ATCC 13048, and Gram-positive Staphylococcus epidermidis ATCC 12228 using resazurin colorimetric assay and tricine SDS-PAGE bioautography were reported. M. pudica crude and partially purified extracts exhibited antimicrobial activity against all the bacteria tested. Specifically, the peptide that was partially purified from M. pudica with a molecular weight of 5.14 kDa inhibited the growth of Enterobacter cloacae.

Standardization of resazurin use in susceptibility testing of natural products against yeasts in planktonic cells and in biofilms formation

Natural products, such as the ethanolic propolis extract (PE), have been shown to be a safe and effective therapeutic alternative for the treatment of fungal skin and nail diseases. However, the presence of the resin and the physicochemical characteristics of the extract sometimes difficult the reading and determination of breakpoints of the in vitrotests, evidencing the need for alternatives that facilitate the reading. The present study aimed to standardize the use of resazurin in tests of susceptibility of PE with planktonic yeast cells and biofilm forms. The antifungal activity of PE was determined by minimum inhibitory concentration (MIC) and we observed that, for all Candida spp. tested, the most reproducible MIC results were obtained when resazurin was placed after 24 hours of incubation and remained more 24 hourswith yeasts plus PE. For encapsulated yeasts, there was no dye reduction and color transition. Resazurin was also used for the evaluation of minimal biofilm inhibitory concentration and minimal biofilm eradication concentration and it was metabolized and reproducedthe action of PE on Candida biofilms. In addition, microdilution checkerboard plates were made with the dye, which assisted reading the result of the interaction between PE and nystatin. We observed that the resin, the color and the turbidity of the PE slightly changed the color of the resazurin in high concentrations of the extract and did not impair the reading. Therefore, the resazurin standardization tests were proven to be efficient and grounds that it should be used as an auxiliary methodology for reading and interpretation of the susceptibility tests for non-encapsulated yeasts with natural products, which form turbidity or precipitation, such as propolis.

Cytotoxicity Activity and Phytochemical Screening of Anthocleista djalonensis Root Extracts against Cancer Cells

Aim: Several medicinal uses have been reported for Anthocleista djalonensis and many types of pure compounds have been isolated. However, the anti-cancer activity of this plant has not been proven. The aim of this study was to screen for the phytochemicals present in the rootn-hexane, ethyl acetate, and acetone extracts of Anthocleista djalonensis, and to evaluate its anticancer potential against human cervix adenocarcinoma cells (HeLa cells) in vitro. Place and Duration of Study: The study was carried out in Department of Organic Chemistry, Rhodes University, Grahamstown, South Africa. The duration period was between March and July, 2016. Methodology: Extracts were prepared by soaking the root powder in the respective solvents with continuous stirring; The extracts were filtered and evaporated to remove the solvents. The extracts were then screened for phytocompounds by preliminary screening methods. Anti-cancer potential was carried out by a Resazurin assay and CC50 values were determined. Results: The extracts showed the presence of carbohydrates, glucoside, alkaloids, flavonoids, terpenoids, tannins, saponins, sterols. All extracts demonstrated moderate cytotoxicity against HeLa cells. Conclusion: The hexane, ethyl acetate and acetone extracts showed anticancer property. The roots extracts of Anthocleista djalonesis were thus found to possess potential anticancer activities.

Desenvolvimento de método colorimétrico para determinação de sulfato de neomicina empregando os conceitos de qualidade por design analítico (AQbD) / Development of a colorimetric method for determination of neomycin sulphate using the concepts of quality by analytical design (AQbD)

Para efetivamente tratar uma infecção, é necessário que o antibiótico possua atividade antimicrobiana adequada e seja capaz de inibir o crescimento do microrganismo patogênico. O doseamento microbiológico é uma metodologia indicada para a análise do antimicrobiano de forma simples, quando comparado com outras metodologias. A Food and Drug Administration (FDA) tem encorajado uma abordagem proativa para introduzir inovações e benefícios associados ao processo de produção farmacêutica. A Qualidade por Design Analítico (AQbD) ajuda no desenvolvimento de métodos analíticos robustos e de baixo custo, que são aplicáveis durante todo ciclo de vida do produto. Os métodos microbiológicos tradicionais, de forma geral, apresentam baixa reprodutibilidade e alta incerteza. Desta forma, justifica-se o desenvolvimento de métodos microbiológicos alternativos para a análise de antimicrobianos empregando-se os conceitos de Qualidade por Design Analítico, com a finalidade de melhorar a reprodutibilidade e reduzir a incerteza final. O objetivo deste trabalho foi aplicar o conceito de Qualidade por Design Analítico (AQbD) no desenvolvimento de método colorimétrico para análise de sulfato de neomicina. O sulfato de neomicina é um antimicrobiano aminoglicosídeo amplamente empregado no tratamento de infecções cutâneas ou mucosas, tais como queimaduras, úlceras, e dermatites infecciosas. Métodos cromatográficos como a cromatografia líquida de alta eficiência em fase reversa, de pareamento iônico ou cromatografia iônica com derivatização (pré ou pós-coluna) são utilizados para a análise de aminoglicosídeos, inclusive sulfato de neomicina. Contudo, de acordo com as farmacopeias, o método microbiológico é o método analítico de escolha para a análise de sulfato de neomicina e outros aminoglicosídeos. A análise colorimétrica é um método amplamente utilizado para a detecção e quantificação de diferentes substâncias, incluindo o crescimento microbiano em estudos de eficácia terapêutica. Neste trabalho, propomos o uso de resazurina como marcador colorimétrico. O indicador sofre uma reação de oxido-redução na qual altera a coloração em resposta à redução química resultante do crescimento celular. O uso de microplacas para a análise colorimétrica é uma alternativa ao método realizado em tubos de ensaio. Uma alternativa ao uso de espectrofotômetros para a análise colorimétrica é o uso de aparelhos smartphones, pois são equipados com CPUs rápidas, câmeras de alta resolução e sensores de imagem. O processamento da imagem captada pela câmera do dispositivo é utilizado como um analisador colorimétrico. Portanto, a aplicação dos conceitos de Qualidade por Design Analítico (AQbD) possibilitou o desenvolvimento racional de método microbiológico colorimétrico para análise de sulfato de neomicina

o effectively treat an infection, the antibiotic must have adequate antimicrobial activity and be capable of inhibiting the growth of the pathogenic microorganism. The microbiological assay is an indicated methodology for the analysis of the antimicrobial in a simple way, when compared with other methodologies. The Food and Drug Administration (FDA) has encouraged a proactive approach to introducing innovations and benefits associated with the pharmaceutical production process. Analytical Design Quality (AQbD) assists in the development of robust, low cost analytical methods that are applicable throughout the product life cycle. Traditional microbiological methods, in general, have low reproducibility and high uncertainty. Thus, it is justified the development of alternative microbiological methods for the analysis of antimicrobials using the concepts of Quality by Analytical Design, in order to improve reproducibility and reduce final uncertainty. The objective of this work was to apply the concept of Quality by Analytical Design (AQbD) in the development of a colorimetric method for the analysis of neomycin sulfate. Neomycin Sulfate is an aminoglycoside antimicrobial widely used in the treatment of cutaneous or mucosal infections, such as burns, ulcers, and infectious dermatitis. Chromatographic methods such as reverse phase high performance liquid chromatography, ion-pairing or ion chromatography with derivatization (pre or post-column) are used for the analysis of aminoglycosides, including neomycin sulfate. However, according to pharmacopoeias, the microbiological method is the analytical method of choice for the analysis of neomycin sulphate and other aminoglycosides. Colorimetric analysis is a widely used method for the detection and quantification of different substances, including microbial growth in studies of therapeutic efficacy. In this work, we propose the use of resazurin as a colorimetric marker. The indicator undergoes an oxidation-reduction reaction in which it alters the coloration in response to the chemical reduction resulting from cell growth. The use of microplates for colorimetric analysis is an alternative to the method carried out in test tubes. An alternative to the use of spectrophotometers for colorimetric analysis is the use of smartphones because they are equipped with fast CPUs, high resolution cameras and image sensors. The image processing captured by the device's camera is used as a colorimetric analyzer. Therefore, the application of the concepts of Quality by Analytical Design (AQbD) allowed the rational development of a microbiological colorimetric method for analysis of neomycin sulfate

Evaluation of four colourimetric susceptibility tests for the rapid detection of multidrug-resistant Mycobacterium tuberculosisisolates

The purpose of this study is to evaluate four rapid colourimetric methods, including the resazurin microtitre assay (REMA), malachite green decolourisation assay (MGDA), microplate nitrate reductase assay (MNRA) and crystal violet decolourisation assay (CVDA), for the rapid detection of multidrug-resistant (MDR) tuberculosis. Fifty Mycobacterium tuberculosisisolates were used in this study. Eighteen isolates were MDR, two isolates were only resistant to isoniazid (INH) and the remaining isolates were susceptible to both INH and rifampicin (RIF). INH and RIF were tested in 0.25 µg/mL and 0.5 µg/mL, respectively. The agar proportion method was used as a reference method. MNRA and REMA were performed with some modifications. MGDA and CVDA were performed as defined in the literature. The agreements of the MNRA for INH and RIF were 96% and 94%, respectively, while the agreement of the other assays for INH and RIF were 98%. In this study, while the specificities of the REMA, MGDA and CVDA were 100%, the specificity of the MNRA was lower than the others (93.3% for INH and 90.9% for RIF). In addition, while the sensitivity of the MNRA was 100%, the sensitivities of the others were lower than that of the MNRA (from 94.1-95%). The results were reported on the seventh-10th day of the incubation. All methods are reliable, easy to perform, inexpensive and easy to evaluate and do not require special equipment.

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.

Antioxidant and Antibacterial Activity of Field Grown and Tissue Cultured Root Callus of Mangrove Species.

Aim: The main aim of this study is to evaluate the anti-oxidant and antimicrobial activity of mangrove species and the development of the callus biomass. Study Design: This is the first comparative evaluation report of field grown root and tissue cultured root callus of mangrove on anti-oxidant and anti-bacterial activity. Acanthus ilicifolius, Calophyllum inophyllum and Excoecaria agallocha were tested in this present investigation. Place and Duration of Study: Centre of Advanced Study in Marine Biology, Faculty of Marine Sciences, Annamalai University, Parangipettai 608 502, Tamil Nadu, India. Between February 2012 and July 2013 Methodology: In order to develop callus biomass, a number of growth hormones were supplemented with the MS medium. Following the callus development, anti-oxidant and anti-microbial activities were tested with field grown root and its tissue cultured root callus of Acanthus ilicifolius, Calophyllum inophyllum and Excoecaria agallocha. This was confirmed by different anti-oxidant, anti-bacterial and minimum inhibitory concentration assays. Results: Maximum (89%) root callus biomass was obtained from Acanthus ilicifolius on MS medium fortified with 0.3+0.3 mg/L of 2,4-D and KIN. 2,4-D 0.3mg/L and BAP 0.5 mg/L showed the maximum callus from both Calophyllum inophyllum (81%), Excoecaria agallocha (58%). In addition anti-oxidant and anti-bacterial effect of root and root callus of these three species were tested. In this study the root callus materials of all the three species showed the best anti-oxidant anti-bacterial activities. Conclusion: The metabolites from mangroves are good remedy for number of health problems especially the enhanced level of metabolites through the tissue culture techniques. The present study confirmed the anti-oxidant and anti-bacterial effect of Acanthus ilicifolius, Calophyllum inophyllum, Excoecaria agallocha n root extract. This study will be a key to develop a new drug to achieve healthy life. especially the root callus materials showed the better activity when compared to the field grown plants.

In Vitro Antimicrobial and Haemolytic Studies of Bambusa arundinaceae leaves.

Bambusa arundinacea locally known as Bans or bamboo, a perennial fastest growing plant. This plant is medicinally much famous. The biological activities of this plant were carried out by using standard procedures. The aim of this work was to investigate the in vitro antimicrobial and haemolytic activities of Bambusa arundinaceae leaves. The GC-MS analysis revealed that the major components determined in n-hexane extract were as n-Nonane (17.14%), 2,2,3-Trimethylbutane (5.85%), Tridecane (5.42%), and 3,4,5,6-Tetramethyloctane (5.32%). The disc diffusion and modified resazurin microtitre-plate assays were used to evaluate the inhibition zones (IZ) and minimum inhibitory concentration (MIC) of B. arundinaceae leaves extracts. The plant extracts showed considerable antimicrobial activity. The heamolytic activity against human blood erythrocytes (RBCs) was studied and the % lysis of RBCs was found to be in the range of 1.03 to 4.81. The results of the present study confirmed the considerable antimicrobial and haemolytic activities of the plant Bambusa arundinaceae.

Validation of a modified fluorimetric assay for the screening of trichomonacidal drugs

A fluorimetric microassay that uses a redox dye to determine the viability of the flagellate Trichomonas vaginalis has been optimised to provide a more sensitive method to evaluate potential trichomonacidal compounds. Resazurin has been used in recent years to test drugs against different parasites, including trichomonadid protozoa; however, the reproducibility of these resazurin-based methods in our laboratory has been limited because the flagellate culture medium spontaneously reduces the resazurin. The objective of this work was to refine the fluorimetric microassay method previously developed by other research groups to reduce the fluorescence background generated by the media and increase the sensitivity of the screening assay. The experimental conditions, time of incubation, resazurin concentration and media used in the microtitre plates were adjusted. Different drug sensitivity studies against T. vaginalis were developed using the 5-nitroimidazole reference drugs, new 5-nitroindazolinones and 5-nitroindazole synthetic derivatives. Haemocytometer count results were compared with the resazurin assay using a 10% solution of 3 mM resazurin dissolved in phosphate buffered saline with glucose (1 mg/mL). The fluorimetric assay and the haemocytometer counts resulted in similar percentages of trichomonacidal activity in all the experiments, demonstrating that the fluorimetric microtitre assay has the necessary accuracy for high-throughput screening of new drugs against T. vaginalis.

Antifungal susceptibility testing of Candida tropicalis biofilms against fluconazole using calorimetric indicator resazurin.

Background: C.tropicalis is an important cause of nosocomial infections particularly in immunocompromised patients. Infections caused by Candida spp. are often associated with biofilm formation on implanted medical devices or on epithelial cell surfaces. Phenotypic characteristics of sessile cells in biofilms are known to be different from those of their free-living, planktonic counterparts. Biofilm forming strains often show increased resistance to antimicrobial agents. Materials and Methods : We measured susceptibility to fluconazole of fifty C.tropicalis isolates from immunocompromised (29) and immunocompetent (21) patients by minimum inhibitory concentration (MIC) and minimum biofilm inhibitory concentration (MBIC) assays. MBIC was done using the calorimetric indicator resazurin, to measure the metabolically active cells. Results : Biofilm forming cells showed increased resistance to fluconazole. Conclusion : The resazurin dye test was found to be a good method for determining MBIC.

Comparison of LIVE/DEAD(R) BacLight(TM) Bacterial Viability Test and alamarBlue(R) Method for Enumeration of Live and Dead Bacteria for Oral Bacterial Species

LIVE/DEAD(R) BacLight(TM) and alamarBlue(R) are fluorescent materials used for the enumeration of live and dead bacteria. LIVE/DEAD(R) BacLight(TM) is generally used for confocal microscopy applications to differentiate live from dead bacteria in a biofilm or planktonic state. AlamarBlue(R) has also been used widely to assay live and dead bacteria in a planktonic state. Whilst these materials are successfully utilized in experiments to discriminate live from dead bacteria for several species of bacteria, the application of these techniques to oral bacteria is limited to the use of LIVE/DEAD(R) BacLight(TM) in biofilm studies. In our present study, we assessed whether these two methods could enumerate live and dead oral bacterial species in a planktonic state. We tested the reagents on Streptococcus mutans, Streptococcus sobrinus, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans and Enterococcus faecalis and found that only LIVE/DEAD(R) BacLight(TM) could differentiate live from dead cells for all five of these oral strains. AlamarBlue(R) was not effective in this regard for P. gingivalis or A. actinomycetemcomitans. In addition, the differentiation of live and dead bacterial cells by alamarBlue(R) could not be performed for concentrations lower than 2 x 10(6) cells/ml. Our data thus indicate that LIVE/DEAD(R) BacLight(TM) is a more effective reagent for this analysis.

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.

Growth kinetics of mycobacterium tuberculosis measured by quantitative resazurin reduction assay: a tool for fitness studies

We standardized a method to evaluate the growth kinetics of Mycobacterium tuberculosis by measuring quantitatively the reduction of resazurin by spectrophotometry. Growth curves and the rate of growth of twenty-one M. tuberculosis clinical isolates were determined. The method showed technical simplicity and is inexpensive to assess the fitness of each isolate.