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Optimum conditions have been established for culture of the fungus Candida albicans (ATCC 90028) for microbial detection of zones in direct bioautographic TLC. Bioluminescent ATP assay is a highly sensitive method for optimizing the viability of Candida albicans test fungus in bioautographic TLC detection. A suspension of microbes (OD 600nm = 0.5–0.7, in Mueller-Hinton broth with 5% glucose) in the log phase of growth can be used for dipping TLC plates. On the basis of our results with Candida albicans , we can differentiate between microbiostatic (bacteriostatic or fungistatic) and microbiocidal (bactericidal or fungicidal) effects on TLC plates. Our micrographs clearly show the borders of inhibition zones in bioautograms. This technique leads to new possibilities in studies of the interactions between microbes and antimicrobial compounds on bioautographic silica gel TLC plates by using scanning electron microscopy. In this paper we describe an optimization procedure for bioautographic TLC detection.

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Abstract  

Using a LKB-2277 bioactivity monitor, stop-flow mode, the power–time curves of Candida albicans growth at 37 °C affected by berberine were measured. The check experiments were studied based on agar cup method to observe the inhibitory diameter and serial dilution method to determine the minimal inhibitory concentration (MIC) of berberine on C. albicans growth. By analyzing the quantitative thermogenic parameters taken from the power–time curves using correspondence analysis (CA), we could find that berberine at a low concentration (5.0 μg mL−1) began to inhibit the growth of C. albicans and at a high concentration (75.0 μg mL−1) completely inhibited C. albicans growth. The anti-fungal activity of berberine could also be expressed as half-inhibitory concentration IC50, i.e., 50% effective in this inhibition. The value of IC50 of berberine on C. albicans was 34.52 μg mL−1. The inhibitory diameters all exceeded 10 mm in test range and the MIC was 500 μg mL−1. Berberine had strong anti-fungal effect on C. albicans growth. This work provided an important idea of the combination of microcalorimetry and CA for the study on anti-fungal effect of berberine and other compounds. Compared with the agar cup method and serial dilution method, microcalorimetry not only offered a useful way for evaluating the bioactivity of drugs, but also provides more information about the microbial growth and all this information was significant for the synthesis and searching of antibiotics.

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Thin-layer chromatography with microbiological detection (direct bioautography) of amphotericin B has never been reported. The combination of these methods can be used advantageously, especially when not only chemical identification of samples is required, but also when antifungal activity is of interest. In this paper a fast and easy-to-perform method is introduced in which major ( R F 0.46) and minor ( R F 0.31) components can be separated from amphotericin B, which itself is not a homogenous substance but mixture of polyenes. Thin-layer chromatography is performed on silica gel layers with chloroform-methanol-borate buffer 4:5:1 ( v/v ) as optimized mobile phase, and the microbiological activity of amphotericin B can be measured sensitively by direct bioautography. Candida albicans (ATCC 90028) and Saccharomyces cerevisiae (ATCC 9763) fungus strains were tested. Among the detection methods investigated, direct bioautography with Candida albicans proved to be the most sensitive, with a detection limit of 0.8 ng per spot. For densitometric evaluation of plates with (385 nm) ten times more substance is required, and with a UV lamp (366 nm) as much as 50 ng AmB per spot is needed to visualize the main component.

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, Bacillus subtilis; Gram (−) bacteria as Escherichia coli , Pseudomonas aeuroginosa . They were incubated at 35–37 °C for 24–48 h and yeast as Candida albicans incubated at 30 °C for 24–48 h and then the diameters of the inhibition zones were measured

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A previous study on Solidago virgaurea extracts showed an inhibiting activity of Candida albicans yeast—hyphal transition due to a mixture of triterpene saponins, leading to applications in the field of oral care products. Such applications require the development of an efficient, fast, and simple quantification method of S. virgaurea total saponins. Two methods were developed: the first was based on high-performance liquid chromatography (HPLC) separation with gradient elution and evaporative light scattering detection (ELSD); the second was based on high-performance thin-layer chromatography (HPTLC) separation with in-situ hydrolysis followed by densitometric measurements at 366 nm. Both calibration curves showed good linear regressions (R 2 > 0.99) within the range of the concentrations tested. The limit of detection (LOD) and the limit of quantitation (LOQ) were 2–4 μg and 10–16 μg, respectively. The intra- and inter-day variations were studied and found to remain below 5.6% in terms of relative standard deviation (RSD). The recoveries were 97.02–101.91% with RSD of 0.3–1.96% for spiked sample. Both validated methods were successfully applied to the analysis of total saponins in different S. virgaurea samples. In particular, a harvesting study could be supported by these methods to identify the most relevant vegetal parts to extract, and the locations and vintage to collect, to obtain the desired active saponins. HPLC is the recommended method for precise analyses, but HPTLC is the most efficient for the fast analysis of multiple samples.

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In this study, we have developed a validated high-performance thin-layer chromatographic (HPTLC) method for the simultaneous quantification of two phenolic biomarkers, protocatechuic acid (compound 1) and quercetin 4ʹ-O-β-d-glucopyranoside (compound 2) in antimicrobial and antioxidant active A. cepa ethyl acetate extract (ACEAE). The quantitative analysis was carried out on normal-phase HPTLC (glass-backed silica gel 60 F254) plates with solvents toluene, ethyl acetate, and formic acid in the ratio of 3:6:1, v/v (as the mobile phase). Well-resolved, compact, and intense peaks of compound 1 (R F = 0.56 ± 0.001) and compound 2 (R F = 0.05 ± 0.001) were found at λ max = 275 nm. The linear regression equation / r 2 for compound 1 (Y = 8.89X + 250.71 / 0.994) and compound 2 (Y = 6.64X + 209.34 / 0.998) in the concentration range of 100–700 ng spot−1 indicated good linear relationship. The low values of percent relative standard deviation (%RSD) for intra-day / inter-day precision of compound 1 (1.14–1.26 / 1.08–1.23) and compound 2 (0.97–1.18 / 0.93–1.16) suggested that the method is precise. The (%) recoveries for compound 1 / compound 2 were found as 98.07–99.55 / 98.20–99.89 which confirms the good accuracy of the proposed method. The quantities (%w/w) of compound 1 / compound 2 in ACEAE were found as 18.84% / 13.64% of the dried weight of the extract. In vitro 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay showed the promising free radical scavenging activity of ACEAE (69.00 ± 2.99%) and compound 2 (63.86 ± 2.02%) which were comparable to ascorbic acid tested at 400 μg mL−1. ACEAE was found to be highly active against all tested bacterial strains, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa; however, Candida albicans was found to be susceptible to both compound 2 and ACEAE. The presence of compound 2 in high quantity in the ethyl acetate fractions of A. cepa peel (ACEAE) validated its antimicrobial and antioxidant property. The above developed HPTLC method can be further employed in the analysis of these markers in marketed formulations and in the quality control of herbal drugs.

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An important bioactive molecule, ursolic acid was isolated from the leaves of Diospyros melanoxylon and characterized with help of physical and spectroscopic data viz. m. p, IR, 1H, and 13C NMR. A high-performance thin-layer chromatography method has also been developed and validated for its quantification in D. melanoxylon leaves. The high-performance thin-layer chromatography analysis was performed on high performance thin-layer chromatography plates using chloroform-methanol (9.5:0.5, v/v) as mobile phase. The compound was quantified at 540 nm after derivatzation with sulphuric acid reagent. The sensitivity of the method with respect to limit of detection and limit of quantification were found to be 20 and 40 ng per spot. The response was obtained as a linear function of peak area and concentration in the range of 50 to 450 ng per spot with correlation coefficient of r 2 = 0.9998. The method showed excellent accuracy greater than 97.54% with acceptable precession and was successfully validated according to International Conference of Harmonization protocols. Antimicrobial screenings of ursolic acid revealed potent activity against two Gram-positive bacteria viz. Staphylococcus aureus and Enterococcus faecalis and three fungal starins viz. Aspergillus niger, Candida tropicalis, and Candida albicans.

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Journal of Thermal Analysis and Calorimetry
Authors: Mihaela Badea, Rodica Olar, Dana Marinescu, Veronica Lazar, Carmen Chifiriuc, and Gina Vasile

Abstract  

This paper reports the investigation on the thermal stability of new complexes with mixed ligands of the type [Cd(NN)(C3H3O2)2(H2O)m]·nH2O [(1) NN: 1,10-phenantroline, m = 1, n = 0; (2) NN: 2,2′-bipyridine, m = 0, n = 1.5 and (C3H3O2): acrylate anion]. The IR data indicate a bidentate coordination mode for both heterocyclic amine and acrylate. The in vitro qualitative and quantitative antimicrobial activity assays showed that the complexes exhibited variable antimicrobial activity against planktonic as well as biofilm embedded Gram-negative (Escherichia coli, Klebsiella sp., Proteus sp., Salmonella sp., Shigella sp., Acinetobacter boumani, Pseudomonas aeruginosa), Gram-positive (Bacillus subtilis, Staphylococcus aureus) and fungal (Candida albicans) strains, reference and isolated ones from the hospital environment. The thermal behaviour steps were investigated in synthetic air flow. The thermal transformations are complex processes according to TG and DTA curves including dehydration, amine as well as acrylate thermolysis. The final products of decomposition are the most stable metal oxides.

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Metal complexes of fenoterol drug

Preparation, spectroscopic, thermal, and biological activity characterization

Journal of Thermal Analysis and Calorimetry
Authors: M. Soliman, Gehad Mohamed, and Eman Mohamed

Abstract

Metal complexes of fenoterol (FEN) drug are prepared and characterized based on elemental analyses, IR, 1H NMR, magnetic moment, molar conductance, and thermal analyses (TG and DTA) techniques. From the elemental analyses data, the complexes are formed in 1:2 [Metal]:[FEN] ratio and they are proposed to have the general formula [Cu(FEN)2]·2H2O; [M(FEN)2(H2O)2yH2O (where M = Mn(II) (y = 2), Co(II) (y = 4), Ni(II) (y = 4), and Zn(II) (y = 0) and [Cr(FEN)2(H2O)2]Cl·H2O. The molar conductance data reveal that all the metal chelates are non-electrolytes except Cr(III) complex, having 1:1 electrolyte. IR spectra show that FEN is coordinated to the metal ions in a uninegative bidentate manner with ON donor sites of the aliphatic –OH and secondary amine –NH. From the magnetic moment measurements, it is found that the geometrical structures of these complexes are octahedral (Cr(III), Mn(II), Co(II), Ni(II), and Zn(II)) and square planar (Cu(II)). The thermal behavior of these chelates is studied using thermogravimetric and differential thermal analyses (TG and DTA) techniques. The results obtained show that the hydrated complexes lose water molecules of hydration followed immediately by decomposition of the coordinated water and ligand molecules in the successive unseparate steps. The FEN drug, in comparison to its metal complexes is also screened for their antibacterial activity against bacterial species (Bacillus subtilis, Staphylococcus aureus, Escherichia coli, and Salmonella typhi), Yeasts (Candida albicans and Saccharomyces cervisiae), and Fungi (Aspergillus niger and Aspergillus flavus). The activity data show that the metal complexes have antibacterial activity like that of the parent FEN drug against one or more species.

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Given the large applicability in both pharmaceutical and cosmetic field, this work was aimed at analytical (high-performance thin-layer chromatography [HPTLC] method), antioxidant (2,2-diphenyl-1-picrylhydrazyl [DPPH] method), and antimicrobial (diffusion method on Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 8739, and Candida albicans ATCC 10231) studies on a series of whole vegetal extracts and corresponding aqueous, ethyl acetate and chloroform fractions (selective extracts) prepared from eight plant species growing in Romania. Briefly, it was revealed moderate to certain activity against S. aureus ATCC 6538 and E. coli ATCC 8739 in the case of greater burdock leaves (Arctium lappa), beech leaves (Fagus sylvatica) and great willowherb aerial part (Epilobium hirsutum) whole extracts. Purple loosestrife aerial part (Lythrum salicaria) and sea-buckthorn leaves (Hippophae rhamnoides) whole extracts showed only weak activity against these bacteria, while tarragon aerial part (Artemisia dracunculoides), chokeberries leaves (Aronia melanocarpa) and quince fruit (Cydonia oblonga) whole extracts have not shown activity. Subsequently studies on the selective extracts have revealed that combinations of glycosides of the same aglycones lead to very different antimicrobial properties indicating synergistic effects between polyphenols (demonstrated in the case of Fagus sylvatica extracts) or the contribution of other phytocompounds to the final effect (demonstrated in the case of Arctium lappa extracts) and, secondly, that the glycosyl chain in which they occur may also contribute to the final antimicrobial effect (demonstrated by the fact that the most active vegetal extracts emphasized the dominance of flavonoid monoglycosides). Moreover, inhibitory antimicrobial effects were revealed (Arctium lappa extracts). Finally, none of the studied extracts acted against C. albicans ATCC 10231. Concerning antioxidant activity, DPPH tests indicated high potency of all tested extracts (IC50 measuring from 2.66 to 4.80). Further studies on the most active vegetal extracts which aim to reveal MICs values and potential inhibitory or activatory effects in combination with chemical antibiotics are ongoing.

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