Bioautography Method Research Articles

Phenolic compounds of Tagetes lucida Cav. with antibacterial effect due to membrane damage

Tagetes lucida Cav. (Asteraceae=Compositae) is used for treating stomach infections. The study focused on evaluating the composition and antimicrobial effect of an extract of T. lucida Cav. The plant extracted with ethanol at 10% w/v, and the extract composition analyzed by Rp-HPLC-MS. The antibacterial effect was evaluated against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Salmonella choleraesuis using disk diffusion, microdilution and bioautography methods. The sytox and comet assays were used to evaluate the mechanism of action. In this way, nine phenolic compounds were detected in the extract of T. lucida. The extract exhibited activity only on S. aureus (MIC of 4.000 mg/ml). The bioautography revealed that the phenolic compounds could act synergistically. The sytox and comet tests showed an antibacterial action of the extract on the bacterial membrane and DNA of this bacterial strain.

Tetradenia riparia (Lamiaceae) essential oil: an alternative to Rhipicephalus sanguineus

In Brazil, Rhipicephalus sanguineus resistance to some pyrethroids have been detected, motivating research on new phyto-insecticides such as essential oil from Tetradenia riparia leaves (EOL) and flower buds (EOFB). The essential oils were obtained by hydrodistillation (3h) and identified by GC/MS. In addition, a multivariate exploratory analysis was done to determine the analysis of the major compounds (PCA). The bioassays on R. sanguineus larvae were done by immersion test at different EO concentrations which ranged from 50,000 to 0.47 mg/mL (v/v). The action mechanism of EOs were determined by bioautographic method evaluating the inhibitory potential on the acetylcholinesterase enzyme. The EO yield in leaves was 0.29±0.22 (%) and in flower buds 0.38±0.17 (%). The class projections showed oxygenated sesquiterpenes (43.62%) and diterpenes (15.60%) in EOFB, and hydrocarbon sesquiterpenes (26.44%) and oxygenated monoterpenes (16.44%) in EOL. Four components presented a greater distancing of mass flow: fenchone (11.57 and 6.01 %), α-cadinol (12.21 and 13.69 %), 14-hidroxy-9-epi-caryophyllene (8.56 and 15.38 %), and caryophyllene oxide (1.32 and 4.50 %) in EOL and EOFB, respectively. The lethal concentrations (LCs) to kill R. sanguineus larvae were (LC50: 2.18±0.24 and LC99.9: 9.98±0.10 mg/mL) for EOL, and (LC50: 5.36±2.50 and LC99.9: 20.12±0.54 mg/mL) for EOFB. The action mechanism of EOs by bioautographic methods indicated an inhibition of 0.70 mg/mL (EOL) and 1.40 mg/mL (EOFB) on the acetylcholinesterase enzyme (AChE). Therefore, this species can be considered promising to be part of the chemical larvicides to control this ectoparasite

Validated TLC-Contact Bioautography Method for Identification of Kanamycin Sulfate in Injection Preparation

Background: TLC-contact bioautography is one of an effective method for identification antibiotics, by which many antibiotics could be identification and determination simultaneously. Objective: To evaluate kanamycin sulfate in injection preparations based on its inhibitory activity against Escherichia coli ATCC 8739 as test organism. Methods: Sample and standard solutions were spotted onto TLC silica gel 60 F254 plate and developed in 10% potassium dihydrogen phosphate solutionas as mobile phase. The TLC-contact bioautography method was validated according to USP guidelines by considering specificity, LOD, LOQ, linearity, accuracy and precision parameters. Results: The TLC-contact bioautography method was found to be high sensitivity with LOD of 0.75 µg and LOQ 2.31 µg. Linearity range of 100-350 µg/mL with r = 0.9993 and linear regression equation was y = 0.0019x + 0.0338. The recovery obtained from addition of blank samples by three different concentrations of kanamycin sulfate standard was 101.40% ¬+ 2.02%. The precision of the method was good with coefficient of variation 0.080%. The TLC-contact bioautography method was supported by determination of kanamycin sulfate potency ratio in the injection preparation and kanamycin sulfate standard using 3-3 design. Random block design obtained the potential for kanamycin sulfate in injection preparations compared to kanamycin sulfate standard was 100.6%. Conclusion: The TLC-contact bioautography for kanamycin sulfate in injection preparations could be applied to the quality control analysis of the investigated drugs.

Isolation of Flavonoids from Anaphalis Busua and their Antibacterial Activity

Anaphalis busua (Buch-Ham ex D. Don) is traditionally used to treat cuts and wounds. In the agar dilution method, the hexane extract of the aerial part of A. busua exhibited potent antibacterial activity with a minimal inhibitory concentration of 25 μg/ml against Bacillus subtilis and 500 μg/ml against Staphylococcus aureus. For the isolation of antibacterial compounds, the hexane extract was fractionated by silica gel and Sephadex LH 20 column chromatography. The direct bioautography method was used to determine the antibacterial activity of the fractions. The active fractions were finally purified by semi-preparative HPLC on C18 Phenomenex column under gradient condition. Four flavones derivatives of 3,5-dihydroxy-6,7,8-trimethoxyflavone (1), 3,5,7-trihydroxy-6-methoxyflavone (alnusin) (2), 3,5,7-trihydroxy-8-methoxyflavone (3) and pinocembrin (4) were isolated, and their structures were established by 1H, 13C, DEPT-135, and ESI-MS spectroscopy. These compounds were isolated for the first time from A. busua. The fatty acid profile of the hexane extract was analyzed by gas chromatography-mass spectrometry (GC-MS) by silylation with N-methyl-N-trimethylsilyl-trifluoro-acetamide. Pinocembrin showed antibacterial activity with the minimum inhibitory concentration of 60 μg/mL against Bacillus subtilis and 420 μg/mL against Staphylococcus aureus.

Bioassay screening of 12 Iranian plants and detection of antibacterial compounds from Heracleum persicum using a TLC bioautography method

Allium paradoxum, Buxus hyrcana, Convolvulus persicus, Cyclamen coum, Eryngium caucasicum, Heracleum persicum, Pimpinella affinis, Parrotia persica, Primula heterochroma, Pyrus boissieriana, Ruscus hyrcanus, and Smilax excelsa are some medicinal and food flavoring plants found in the Hyrcanian forests in Iran. This study was designed to determine the most potent antibacterial compounds from the plants. The MIC values of n-hexane, ethyl acetate, and methanol extracts of different parts of the plants (63 extracts) were determined against Staphylococcus aureus, B. cereus, Pseudomonas aeruginosa, and Salmonella entritidis. The antibacterial compounds of the most potent extracts were determined by TLC bioautography, against S. aureus. According to the results, the n-hexane extract of the roots of H. persicum was selected to identify its bioactive phytochemicals. In the following, eight furanocoumarins, heratomin, xanthotoxin, isobergapten, bergapten, pimpinellin, isopimpinellin, moellendorffilin, and sphondin were identified by comparing with purified standards in different solvent systems. Among these compounds, moellendorffilin played a dominant role and exhibited significant activities against S. aureus, B. cereus, P. aeruginosa, and S. entritidis, with MIC values 31.25, 31.25, 62.5, and 15.62 µg/mL, respectively. Accordingly, H. persicum could be regarded as a promising high-potential plant and inexpensive source of antibacterial compounds for the pharmaceutical and food industries.

A new thin-layer chromatography–direct bioautography assay for the qualitative and quantitative determination of peroxidase inhibitors in plant extracts

Thin-layer chromatography (TLC) hyphenated to bioassays is a modern tool used for discovery of drugs with diverse biological activities from plant extracts which justifies the continuous need for developing such methods. A new TLC–bioautography assay for detecting peroxidase enzyme inhibitors was developed and validated in this study. Peroxidases are related to pathogenesis of many diseases such as neurodegenerative disorders and rheumatoid arthritis and are considered as inflammatory markers. The developed TLC–bioautography method was based on reaction of peroxidase with hydrogen peroxide and the subsequent formation of blue color by the reaction between the released oxygen and benzidine. Compounds with peroxidase inhibition activity are observed as yellowish spots against a blue background. The developed method was validated according to the ICH guidelines. Quantitative estimation of peroxidase inhibitors was achieved by applying image analyses techniques, showing good linearity (R2 = 0.998), accuracy (% recovery 96.6–99.7%), intra-day precisions (RSD% = 0.95–2.35) and inter-day precision (RSD% = 1.80–3.20) as well as low LOD (0.28 μg/spot) and LOQ (0.85 μg/spot). The developed method was applied to different Juniperus species fractions as a demonstrative illustration. Overall, the method proved to be efficient, simple and rapid for identification of peroxidase inhibitors in complex matrices and can be applied in high-throughput screening of plant extracts.

Peroxidase inhibitory and antioxidant constituents from Juniperus L. species guided by HPTLC-bioautography and molecular docking studies

The application of a newly developed HPTLC-bioautography assay for detecting peroxidase enzyme inhibitors in plant extracts in addition to bioautography methods for detecting antioxidant compounds resulted in the isolation of a new biflavonoid 3′-methoxy sahranflavone along with two known biflavonoids and three flavonoids from the leaves and cones of Juniperus communis, J. horizontalis and J. chinensis. The structures of all compounds were elucidated by means of 1 D and 2 D NMR and MALDI-TOF MS technique in addition to comparison to literature data. Quantitative estimation of antiperoxidase and antioxidative capacity based on DPPH free radical scavenging activity and β-carotene bleaching of extracts, active fraction and constituents was achieved by applying validated high resolution image analyses techniques. 3′-methoxy sahranflavone and quercetrin possessed high mutual antiperoxidase and antioxidant activities. Molecular docking simulations were performed to reveal the interaction of isolated compounds with human myeloperoxidase enzyme on the molecular level indicating the potential anti-inflammatory activity of 3′-methoxy sahranflavone and quercetrin.

Potensi Fungi Endofit Biji Pinang Sebagai Antibakteri Terhadap Escherichia coli dan Salmonella thypi

Endophyte fungi are microbe that living inside the plant tissue without harming the host plant. Endophyte fungi can produce secondary metabolite which can be used as antioxidant, anticancer and antimicobes compound. The aims of this study are to isolate and identify endophyte fungi from seed of Areca catechu L which is potential as an antibacterial producer. Antibacterial activity testing method used in this study is thin layer chromatography (TLC) bioautography method. In this work we had isolated five endophyte fungi from Areca catechu L. seed and investigated its antibacterial properties. Isolates IFBP_1 has the greatest antibacterial activity againts Escherichia coli dan Salmonella thypi. Areca catechu L seed endophyte fungi can inhibit Escherichia coli bacteria with Rf values 0.45, 0.65 and 0.92, and Salmonella thypi bacteria with Rf values 0.61 and 0.85. Identification of the chemical components Areca catechu L seed endophyte fungi that inhibit the growth of Escherichia coli and Salmonella thypi are alkaloid, tannin, and polyphenol compounds. This result suggest that endophyte fungi isolates from seed of Areca catechu L. can be further explored as new sources of antibacterial compounds

Enzymatic Bioautographic Methods.

Enzymatic bioautography enables the detection of enzyme inhibitors absorbed on a thin-layer chromatography plate. Therefore, it is an assay format that is particularly useful for the detection of inhibitors present in complex mixtures. The inhibition properties of compounds separated by thin-layer chromatography can be directly analyzed to produce an inhibition profile. Here, we describe the conditions to detect inhibitor of the enzymes xanthine oxidase and β-glucosidase immobilized on agar gel.

PROFIL BIOAUTOGRAM EKSTRAK FERMENTAT ISOLAT FUNGI ENDOFIT DARI DAUN GALING-GALING (Cayratia trifolia L) SEBAGAI ANTIBAKTERI

The research bioautogram profile of fermentate extract endophytic fungi from galing-galing ( Cayratia trifolia L) leaf as an antibacterial has been to find the activity of the extract fermentat endophytic fungi isolates against bacterial test using TLC Bioautography. This research begins with the isolation of leaves galing-galing using Potato Dextrose Agar Chloramfenicol (PDAC) media, isolates do purification and macroscopic test. Subsquently isolates were followed by screening endophytic fungi isolates against test bacteria. Isolates that give the most excellent activity followed by fermentation with Maltose Yeast Broth (MYB) media using a shaker speed of 200 rpm at room temperature for 21 days. After the fermented liquid is separated to obtain fermentat and mycelia. The fermented liquid was then extracted using a liquid-liquid extraction method using ethyl acetate solvents after which it was evaporated with a rotary evaporator until a thick extract was obtained. Extracts obtained followed by TLC Bioautography method using ethyl acetate : n-hexane (4: 1) eluent. Endophytic fungi were obtained as many as 6 isolates and the best activity was endophytic fungi with IDGG-03 code against test bacteria. The results from the TLC Bioautography profile 2 Bioautography provide active spot with Rf values of 0.09 and 0.54 against the bacteria B. subtilis , V. cholerae and P. aeruginosa.

Antimicrobial, DPPH scavenging and tyrosinase inhibitory activities of Thymus vulgaris, Helichrysum arenarium and Rosa damascena mill. ethanol extracts by using TLC bioautography and chemical screening methods

Antimicrobial, DPPH scavenging and tyrosinase inhibitory activities of Thymus vulgaris, Helichrysum arenarium and Rosa damascena Mill. ethanol extracts by using TLC bioautography and chemical screening methods. The ethanol extracts of Thymus vulgaris (Tv), Helichrysum arenarium (Ha) and Rosa damascena Mill. (Rm) (red) were screened for their antimicrobial, 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging and tyrosinase inhibitory activities. The test microorganisms included bacteria of Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 25923). Thin Layer Chromatography (TLC) - bioautography, disk diffusion and well diffusion methods were used for the antimicrobial activity assays. Rosa damascena Mill. extract was effective against E. coli and all plant extracts showed antimicrobial activity against S. aureus. The phenolic acids in the structure of the extracts were also identified by LC-MS analysis. Human blood agar well diffusion method and TLC-DPPH assays were used to identify the hemolytic and antioxidant activity of plant extracts, respectively, along with 10 compounds including phenolic acids as a standard. Among these compounds, caffeic acid (Rf = 0.68) was detected in all extracts while vanillic acid (Rf = 0.75), and gallic acid (Rf = 0.51) was found in Tv extract. Kojic acid (Rf = 0.36), on the other hand, was detected in Rm extract as a tyrosinase inhibitor. All plant extracts presented tyrosinase inhibitory activities on TLC-bioautography assay.

Antibacterial activity of the lichens Usnea Florida and Flavoparmelia caperata (Parmeliaceae)

Acetone extracts of the two common epiphytes lichens Usnea florida and Flavoparmelia caperata have been evaluated for their antimicrobial activities against Staphylococcus aureus, Candida albicans and Aspergillus brasiliensis. The dibenzofuran derivative (+)-usnic acid (1) was the main metabolite in these two species. Thamnolic (5), evernic (6), physodic (7) and 3-hydroxyphysodic acids (8) were isolated from U. florida, as well as 5,7-dihydroxy-6-methylphtalide (2) which was newly identified in this Genus. Protocetraric (3) and caperatic acids (4) and ergosterol peroxide (9) are usually biosynthezised by F. caperata. Antibacterial activity was determined for the four main compounds against Staphylococcus aureus using bioautography and broth dilution method. Minimal inhibitory concentrations of usnic acid, caperatic acid and protocetraric acid were comprised between 7.25 and 12.5 µg/mL.

Comparison of Essential Oils of Ferulago pachyloba (Fenzl) Boiss., F. trachycarpa Boiss. and F. bracteata Boiss. & Hausskn. Species (Apiaceae) Growing in Turkey and Determination of Their Antimicrobial Activities

Ferulago species have been utilized since ancient times as antihelmentic, digestive, sedative and aphrodisiac, along with in salads or as a seasoning in view of their special odours. F. pachyloba (Fenzl) Boiss., F. trachycarpa Boiss. and F. bracteata Boiss. & Hausskn. species were investigated for their chemical compositions of essential oils and their antimicrobial activities. Antimicrobial activities of essential oils were performed via microbroth-dilution and TLC bioautography methods. GC and GC/MS were utilized to analyse the essential oils. Primary components of aerial parts, flowers, roots and fruits of F. pachyloba were found as, sabinene (16.0 %), (Z)-β-ocimene (15.1 %); sabinene (25.8 %), (Z)-β-ocimene (27.5 %); (E)-2-decenal (14.3 %); bicyclogermacrene (11.1 %), respectively. Primary components of aerial parts, roots and fruits of F. trachycarpa were found as, (Z)-β-ocimene (13.8 %), spathulenol (25.0 %); (E)-2-decenal (11.9 %); spathulenol (32.8 %), bicyclogermacrene (23.0 %). Primary components of aerial parts, flowers and roots of F. bracteata were found as, 7-methoxy-6-(3-methyl-2-butenyl)-coumarin (86.7 %); α-pinene (12.1 %), α-phellandrene (22.8 %); (E)-2- decenal (13.9 %), hexadecanoic acid (40.4 %). Essential oils were particularly more effective against Staphylococcus aureus, with MIC values of between 0.39 and 6.25 μg/mL, respectively. They did not give significant inhibition against Pseudomonas aeruginosa by TLC bioautography assay. In contrast, they had active components against S. aureus and showed significant inhibition. The antimicrobial activities against these microorganisms from these species may be attributed to the presence of the main components in the essential oils.

Antimicrobial and antioxidant properties of novel octa-substituted phthalocyanines bearing (trifluoromethoxy) phenoxy groups on peripheral positions

This study presents a novel phthalonitrile derivative (2) bearing (trifluoromethoxy)phenoxy groups in 4,5 positions. Cyclotetramerization of (2) in [Formula: see text],[Formula: see text]-dimethylaminoethanol (DMAE) gave a series of peripherally octa-substituted metallophthalocyanines (3-Zn, 3-Co and 3-Cu). The newly synthesized phthalocyanines have been characterized by a combination of various spectroscopic techniques. Effects of solvent nature on aggregation behavior of 3-Zn were studied using different solvents such as acetone, CHCl3 and dichloromethane (DCM). In addition, the aggregation behavior of the phthalocyanine complex 3-Zn was examined in DCM at different concentrations ranging from 4 × 10[Formula: see text]–14 × 10[Formula: see text] M. Antimicrobial activities of synthesized compounds were tested by using the thin layer chromotography (TLC)-direct bioautography and disk diffusion methods. In both assays, the molecules showed activity on the tested Gram (+) bacteria. Antioxidant activities of the molecules, on the other hand, were determined by using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging method and a reducing power assay. The highest activity was obtained with 3-ZnPc for both methods.

Bioproducts against food-borne pathogenic bacteria

Advances in biotechnology research show the rising generation of a variety of products derived from microbial, plants and animal sources. These products are known as “bioproducts” or “natural products”. The preservation of the microbiological quality of foods without the use of chemical preservatives has become a challenge stimulating new researches on conservation alternatives. The aims of this study were the assessment of the the antibacterial activity of several bioactive compounds: essential oils of orange, lavender, green and red mandarins; ethanol extract of oregano and protein hydrolyzed from shrimp shell against Escherichia coli and Staphylococcus aureus isolated from foods, and the comparison between two different methods used in the screening of natural products with potential antibacterial activity. The antibacterial activities of the natural compounds were determined using agar diffusions tests and bioautography methods. The Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) were determined for the essential oils of orange and lavender, and the ethanolic oregano extract. The essential oils of orange and lavender and the ethanolic oregano extract showed antimicrobial activity against all bacteria tested in the study. The agar disk diffusion and the bioautographic methods showed no significant difference in the evaluation of the biological activity of natural products.

Comparative Analysis of Antimicrobial Activity of Herbal Extracts against Pathogenic Microbes

Antimicrobial activities of Spinacia oleracealeaf, Zingiberofficinalerhizome, Coriandrum sativum leaf, Allium sativum clove, Aloe vera gel and leaf was carried out against the multi-drug resistant strains using the minimum inhibitory concentration method. The direct TLC bioautography method is performed to identify the bioactive compounds present in the extracts exhibiting the antimicrobial property.All the plant extracts effectively inhibited the growth of pathogenic strains used in the study at a concentration of 250 to 31.25mg/ml. These results provide evidence that the tested plant extracts possess antimicrobial properties which can be tested further in the development of novel antimicrobial agents.

Analisis Bioautografi Kromatografi Lapis Tipis dan Aktivitas Antibakteri Ekstrak Etanol Bawang Tiwai (Eleutherine bulbosa (Mill.) Urb.) terhadap Methicillin-resistant Staphylococcus aureus (MRSA)

Methicillin-resistant Staphylococcus aureus (MRSA) infections are the major health problem in Indonesia; this is mainly due to their resistance to antibiotics. Based on this, the quest for new sources of natural based antimicrobial drugs is necessary. Bawang tiwai (Eleutherine bulbosa (Mill.) Urb.) is one of the natural materials that have potential as an antibacterial natural-based of Indonesia origin. Bawang tiwai already used empirically by local communities in East Kalimantan to treat various diseases. The aim of this research is to know the antibacterial activity of E. bulbosa ethanol extract on MRSA and to know the type of secondary metabolite with antibacterial activity tested by TLC bioautography method. The experiment was conducted on Research Laboratory, Faculty of Medicine, Mulawarman University, Samarinda. TLC results showed that ethanol extract of E. bulbosa has compounds containing sugar, alkaloids, steroids, and flavonoids. The results of TLC bioautography show that ethanol extract of E. bulbosa can inhibit the growth of MRSA. MIC results show that ethanol extract of E. bulbosa has potent antibacterial activity against MRSA with MIC 1 mg/mL. The conclusion of this research is all the results support the potential of bawang tiwai as the alternative component of natural-based antibacterial agents against MRSA.

Isolation of bioactive compounds of Clausena anisata (Willd.) Hook. growing in South Africa by liquid chromatography–mass spectroscopy profiling, and their antibacterial activities

Clausena anisata possesses several ethnopharmacological properties. It is used for the treatment of several diseases and health conditions, including oxidative stress and respiratory infections. This study was aimed at isolating and validating the potency of some bioactive compounds in this plant using Fourier transform infrared spectroscopy, liquid chromatography–mass spectroscopy and nuclear magnetic resonance techniques. A bioautography method was used for the antibacterial assay. Quercetin 3,4’-dimethyl ether was identified with a molecular mass of 330.2288 Da at a retention time of 0.72, clausine B was identified at a retention time of 24.31 and 24.37 with a molecular mass of 271.0844 Da, clausenocoumarine was identified at a retention time of 5.42 with a molecular weight of 366.1011 Da, while terpineol was also identified with the inhibition of the growth of the test organisms (Escherichia coli, Staphylococcus aureus and Subtilis cereus) to quercetin 3,4’-dimethyl ether, clausine B and clausenocoumarin observed at Rf values of 0.24, 0.52 and 0.54, respectively, on the plate using hexane/ethyl acetate (2:1) as mobile phase. A novel enantiomer of anisocoumarin derivatives and three known compounds were isolated and identified from a fraction showing a single band in the acetone leaf extract of C. anisata. Their chemical compounds were elucidated by nuclear magnetic resonance. The pharmacological potency of these compounds was discussed.

Antimicrobial activity of extracts and phthalides occurring in Apiaceae plants.

Apiaceae plants exhibit a broad spectrum of activities, for instance, antithrombotic, hypotensive, antioxidant, and insecticidal. They also provide a source of phthalides, which display antimicrobial activity. Considering the fact of rising resistance of both bacteria and fungi against commonly used antibiotics, developing of new naturally derived compounds is undeniably attractive approach. To our best knowledge, there are no other reviews concerning this subject in the literature. In view of above, an attempt to summarize an antimicrobial potential of isolated compounds and extracts from Apiaceae plants has been made, by specifying techniques of activity determination and methods of extraction. Techniques of antimicrobial activity evaluation are mainly based on bioautography, diffusion, and dilution methods. Therefore, we focused on in vitro data described in literature so far.

MÉTODOS PARA AVALIAÇÃO IN VITRO DA ATIVIDADE ANTIMICROBIANA DE PLANTAS MEDICINAIS: A NECESSIDADE DA PADRONIZAÇÃO

A crescente incidência de patógenos resistentes aos medicamentos atuais incentiva a busca de novos agentes antimicrobianos. Neste contexto, as plantas medicinais se destacam, sendo uma importante fonte de novos fármacos. Existem diversos métodos para avaliar a atividade antibacteriana e antifúngica de extratos, frações, óleos essenciais e substâncias isoladas de vegetais. Os mais conhecidos incluem métodos de difusão, diluição e bioautografia. A proposta desse trabalho é apresentar os métodos mais utilizados atualmente, juntamente com suas vantagens, desvantagens e fatores interferentes. Entre os artigos indexados na biblioteca SciELO, abrangendo os últimos dez anos, somente 4,4% das pesquisas com plantas medicinais estão relacionadas com atividade antimicrobiana. O método mais utilizado foi a microdiluição (57,9%), o mais recomendado devido à alta sensibilidade, à quantidade mínima de reagentes e amostra e à possibilidade de um maior número de réplicas. Nos trabalhos que utilizaram esse método, foram verificadas divergências de fatores que podem interferir nos resultados. A fim de facilitar a obtenção de resultados comparáveis e reprodutíveis, destaca-se a necessidade da padronização dos métodos utilizadas pelos pesquisadores. Recomenda-se utilizar como referência as normas estabelecidas pelo CLSI para meio de cultura e concentração de inóculo nos testes. Além disso, também recomenda-se a inclusão de um controle negativo da forma de solubilização das amostras, com quantificação do crescimento microbiano, para evitar a interferência nos resultados.