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Edible fungi are a valuable resource in the search for sustainable solutions to environmental pollution. Their ability to degrade organic pollutants, extract heavy metals, and restore ecological balance has a huge potential for bioremediation. They are also sustainable food resources. Edible fungi (basidiomycetes or fungi from other divisions) represent an underutilized resource in the field of bioremediation. By maximizing their unique capabilities, it is possible to develop innovative approaches for addressing environmental contamination. The aim of the present study was to find selective chemical agents suppressing the growth of microfungi and bacteria, but not suppressing white-rot fungi, in order to perform large-scale cultivation of white-rot fungi in natural unsterile substrates and use it for different purposes. One application could be the preparation of a matrix composed of wooden sleeper (contaminated with PAHs) and soil for further hazardous waste bioremediation using white-rot fungi. In vitro microbiological methods were applied, such as, firstly, compatibility tests between bacteria and white-rot fungi or microfungi, allowing us to evaluate the interaction between different organisms, and secondly, the addition of chemicals on the surface of a Petri dish with a test strain of microorganisms of white-rot fungi, allowing us to determine the impact of chemicals on the growth of organisms. This study shows that white-rot fungi are not compatible to grow with several rhizobacteria or bacteria isolated from soil and bioremediated waste. Therefore, the impact of several inorganic materials, such as lime (hydrated form), charcoal, dolomite powder, ash, gypsum, phosphogypsum, hydrogen peroxide, potassium permanganate, and sodium hydroxide, was evaluated on the growth of microfungi (sixteen strains), white-rot fungi (three strains), and bacteria (nine strains) in vitro . Charcoal, dolomite powder, gypsum, and phosphogypsum did not suppress the growth either of microfungi or of bacteria in the tested substrate, and even acted as promoters of their growth. The effects of the other agents tested were strain dependent. Potassium permanganate could be used for bacteria and Candida spp. growth suppression, but not for other microfungi. Lime showed promising results by suppressing the growth of microfungi and bacteria, but it also suppressed the growth of white-rot fungi. Hydrogen peroxide showed strong suppression of microfungi, and even had a bactericidal effect on some bacteria, but did not have an impact on white-rot fungi. The study highlights the practical utility of using hydrogen peroxide up to 3% as an effective biota-suppressing chemical agent prior to inoculating white-rot fungi in the large-scale bioremediation of polluted substrates, or in the large-scale cultivation for mushroom production as a foodstuff.

We report here the first fully automated capillary electrophoresis (CE) system that can be operated underwater. The system performs sample acquisition and analysis by coupling CE to contactless conductivity detection. Using 5 M acetic acid as the background electrolyte (BGE), inorganic cations and amino acids at concentrations as low as 5.2 μM can be separated and identified. This technology could be augmented to include a variety of other detection modes. This system serves as an early prototype for potential future underwater explorers on ocean worlds of the outer solar system such as Europa or Enceladus. This work documents the first step in the development of this general-purpose technology platform.

Capillary electrophoresis (CE) holds great promise as an in situ analytical technique for a variety of applications. However, typical instrumentation operates with open reservoirs (e.g., vials) to accommodate reagents and samples, which is problematic for automated instruments designed for space or underwater applications that may be operated in various orientations. Microgravity conditions add an additional challenge due to the unpredictable position of the headspace (air layer above the liquid) in any two-phase reservoir. One potential solution for these applications is to use a headspace-free, flow-through reservoir design that is sealed and connected to the necessary reagents and samples. Here, we demonstrate a flow-through high-voltage (HV) reservoir for CE that is compatible with automated in situ exploration needs, and which can be electrically isolated from its source fluidics (in order to prevent unwanted leakage current). We also demonstrate how the overall system can be rationally designed based on the operational parameters for CE to prevent electrolysis products generated at the electrode from entering the capillary and interfering with the CE separation. A reservoir was demonstrated with a 19 mm long, 1.8 mm inner diameter channel connecting the separation capillary and the HV electrode. Tests of these reservoirs integrated into a CE system show reproducible CE system operation with a variety of background electrolytes at voltages up to 25 kV. Rotation of the reservoirs, and the system, showed that their performance was independent of the direction of the gravity vector., (© 2023 Wiley-VCH GmbH. California Institute of Technology. Government sponsorship acknowledged. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

Regenerative medicine is a fast expanding scientific topic. One of the main areas of development directions in this field is the usage of additive manufacturing to fabricate functional components that would be later integrated directly into the human body. One such structure could be a microfluidic valve which could replace its biological counterpart in veins as it is worn out over the lifetime of a patient. In this work, we explore the possibility to produce such a structure by using multiphoton polymerization (MPP). This technology allows the creation of 3D structures on a micro- and nanometric scale. In this work, the fabrication of microfluidic systems by direct laser writing was carried out. These devices consist of a 100 μ m diameter channel and within it a 200 μ m long three-dimensional one-way mechanical valve. The idea of this device is to have a single flow direction for a fluid. For testing purposes, the valve was integrated into a femtosecond laser-made glass microfluidic system. Such a system acts as a platform for testing such small and delicate devices. Measurements of the dimensions of the device within such a testing platform were taken and the repeatability of this process was analyzed. The capability to use it for flow direction control is measured. Possible implications to the field of regenerative medicine are discussed., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2022 Lucero Hernandez-Cedillo et al.)

Expansion of the microfluidics field dictates the necessity to constantly improve technologies used to produce such systems. One of the approaches which are used more and more is femtosecond (fs) direct laser writing (DLW). The subtractive model of DLW allows for directly producing microfluidic channels via ablation in an extremely simple and cost-effective manner. However, channel surface roughens are always a concern when direct fs ablation is used, as it normally yields an RMS value in the range of a few µm. One solution to improve it is the usage of fs bursts. Thus, in this work, we show how fs burst mode ablation can be optimized to achieve sub-µm surface roughness in glass channel fabrication. It is done without compromising on manufacturing throughput. Furthermore, we show that a simple and cost-effective channel sealing methodology of thermal bonding can be employed. Together, it allows for production functional Tesla valves, which are tested. Demonstrated capabilities are discussed.

This work involves a comprehensive chemical composition analysis of leaf and cone samples of Lithuanian hop varieties. This study aimed to determine the chemometric properties of the leaves and cones of five Lithuanian hop varieties. Determined properties were the following: (a) xanthohumol content, (b) phenolic compounds, (c) flavonoids, (d) radical scavenging activity, and (e) the qualitative composition of volatile compounds. The total content of phenolic compounds in aqueous 75% methanolic extracts varied between 31.4-78.2 mg of rutin equivalents (RE)/g, and the concentration of flavonoids was between 11.0-23.3 mg RE/g. Radical scavenging activity varied between 34.4-87.2 mg RE/g. A QUENCHER analysis procedure showed 91.7-168.5 mg RE/g of the total phenolic compound content, 12.7-21.4 mg RE/g of flavonoids, and 48.4-121.0 mg RE/g of radical scavenging activity. 'Fredos taurieji' and 'Fredos derlingieji' varieties have shown maximum values of phenolic compounds and radical scavenging activity both in leaf and cone suspensions. These varieties accumulated a higher amount of xanthohumol in leaves. The concentration of xanthohumol in the samples varied between 0.0014-0.2136% of dry mass, with the highest concentration in the cones of 'Kauno gražieji'. We identified 19 volatile compounds in leaves, and in cones, we identified 32. In both of them, α-humulene and β caryophyllene dominated. 'Raudoniai' leaves were exceptional in their aroma due to dominating compound nagina ketone (Kovats index 1306). The QUENCHER procedure has shown a great potential for the unextractable residue of hop raw material. Further investigation and valorization of different hop biomass components, not only cones, are essential.

In capillary electrophoresis (CE), analyte identification is primarily based on migration time, which is a function of the analyte's electrophoretic mobility and the electro-osmotic flow (EOF). The migration time can be impacted by the presence of parasitic flow from changes in temperature or pressure during the run. Presented here is a high-voltage-compatible flow sensor capable of monitoring the volumetric flow inside the capillary during a separation with nL/min resolution. The direct measurement of both flow and time allows for compensation of flow instabilities. By expressing the electropherogram in terms of signal versus electromigration velocity instead of time, it is possible to improve the run-to-run reproducibility up to 25×.

Unknown extraction recovery from solid matrix samples leads to meaningless chemical analysis results. It cannot always be determined, and it depends on the complexity of the matrix and properties of the extracted substances. This paper combines a mathematical model with the machine learning method-neural networks that predict liquid extraction recovery from solid matrices. The prediction of the three-stage extraction recovery of polycyclic aromatic hydrocarbons from a wooden railway sleeper matrix is demonstrated. Calculation of the extraction recovery requires the extract's volume to be measured and the polycyclic aromatic hydrocarbons' concentration to be determined for each stage. These data are used to calculate the input values for a neural network model. Lowest mean-squared error (0.014) and smallest retraining relative standard deviation (20.7%) were achieved with the neural network setup 6:5:5:4:1 (six inputs, three hidden layers with five, five, and four neurons in a layer, and one output). To train such a neural network, it took less than 8000 steps-less than a second--using an average-performance laptop. The relative standard deviation of the extraction recovery predictions ranged between 1.13 and 5.15%. The three-stage recovery of the extracted dry sample showed 104% of three different polycyclic aromatic hydrocarbons. The extracted wet sample recovery was 71, 98, and 55% for phenanthrene, anthracene, and pyrene, respectively. This method is applicable in the environmental, food processing, pharmaceutical, biochemical, biotechnology, and space research areas where extraction should be performed autonomously without human interference., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)

In this work, the design and characterization of a multi-cell capacitively coupled contactless conductivity detection system are described. The operation and simultaneous acquisition from 3 detector cells are demonstrated, however, the system is capable of supplying 8 detection cells and can be easily upgraded to maintain 64 capacitively coupled contactless conductivity detection cells. On performing flow-injection analysis, the system recorded as low as 0.01 mM of acetic acid, phosphoric acid, NaH 2 PO 4, and Na 2 B 4 O 7 solutions in water. The instrument was also capable of recording and distinguishing different mixtures of organic solvents: (a) methanol-acetonitrile, (b) hexane-acetone. The designed detection system is expected to be used coupled with multi-channel separation devices for monitoring simultaneous processes.

Hazardous remote places exist in the world. Why should health or life be risked sending a scientist to the investigation site, as the remote analytical instrumentation exists? Different scientific fields require instruments that could be used on-site (in situ), therefore the purpose of this work was to design a fully automated chemical analysis system small enough to be mountable on a drone. Here we show an autonomous analytical system with sampling capability on a drone. The system is suited for the remote and autonomous analysis of volatile and non-volatile chemicals in the air. The designed system weighs less than 800 g. Data are transmitted wirelessly. Collected substances are separated automatically without the intervention of the operator using the method of capillary zone electrophoresis. The analytes are detected using a miniaturized contactless conductivity detector quantifying them down to less than 1 μM. In this work, we demonstrated sampling and separation of volatile amines (triethylamine and diethylamine) and organic acids (acetic and formic acids), non-volatile inorganic cations (K + , Ca 2+ , Na + ), and protein (bovine serum albumin) in the aerosol state. It was shown that the capillary electrophoretic analysis can be performed on a hovering drone. We anticipate our work to be a starting point for more sophisticated, autonomous complex sample analysis. We believe that our designed instrument will enable the investigation of hazardous places in different research fields.

Background: Avian infectious bronchitis (IB) is a disease that can result in huge economic losses in the poultry industry. The high level of mutations of the IB virus (IBV) leads to the emergence of new serotypes and genotypes, and limits the efficacy of routine prevention. Medicinal plants, or substances derived from them, are being tested as options in the prevention of infectious diseases such as IB in many countries. The objective of this study was to investigate extracts of 15 selected medicinal plants for anti-IBV activity., Results: Extracts of S. montana, O. vulgare, M. piperita, M. officinalis, T. vulgaris, H. officinalis, S. officinalis and D. canadense showed anti-IBV activity prior to and during infection, while S. montana showed activity prior to and after infection. M. piperita, O. vulgare and T. vulgaris extracts had > 60 SI. In further studies no virus plaques (plaque reduction rate 100%) or cytopathogenic effect (decrease of TCID 50 from 2.0 to 5.0 log 10 ) were detected after IBV treatment with extracts of M. piperita, D. canadense and T. vulgaris at concentrations of extracts ≥0.25 cytotoxic concentration (CC 50 ) (P < 0.05). Both PFU number and TCID 50 increased after the use of M. piperita, D. canadense, T. vulgaris and M. officinalis extracts, the concentrations of which were 0.125 CC 50 and 0.25 CC 50 (P < 0.05). Real-time PCR detected IBV RNA after treatment with all plant extracts using concentrations of 1:2 CC 50 , 1:4 CC 50 and 1:8 CC 50 . Delta cycle threshold (Ct) values decreased significantly comparing Ct values of 1:2 CC 50 and 1:8 CC 50 dilutions (P < 0.05)., Conclusions: Many extracts of plants acted against IBV prior to and during infection, but the most effective were those of M. piperita, T. vulgaris and D. canadense .

The methodology described in this article will significantly reduce the time required for understanding the relations between chromatographic data and bioactivity assays. The methodology is a hybrid of hypothesis-based and data-driven scientific approaches. In this work, a novel chromatographic data segmentation method is proposed, which demonstrates the capability of finding what volatile substances are responsible for antiviral and cytotoxic effects in the medicinal plant extracts. Up until now, the full potential of the separation methods has not been exploited in the life sciences. This was due to the lack of data ordering methods capable of adequately preparing the chromatographic information. Furthermore, the data analysis methods suffer from multidimensionality, requiring a large number of investigated data points. A new method is described for processing any chromatographic information into a vector. The obtained vectors of highly complex and different origin samples can be compared mathematically. The proposed method, efficient with relatively small sized data sets, does not suffer from multidimensionality. In this novel analytical approach, the samples did not need fractionation and purification, which is typically used in hypothesis-based scientific research. All investigations were performed using crude extracts possessing hundreds of phyto-substances. The antiviral properties of medicinal plant extracts were investigated using gas chromatography-mass spectrometry, antiviral tests, and proposed data analysis methods. The findings suggested that (i) β- cis-caryophyllene, linalool, and eucalyptol possess antiviral activity, while (ii) thujones do not, and (iii) α-thujone, β-thujone, cis- p-menthan-3-one, and estragole show cytotoxic effects.

One of the main problems of the remote complex sample analysis instrumentation is that such systems are susceptible to temperature fluctuations. Temperature regulation is energetically ineffective, and it is not used in most of the field portable analytical systems. Separations performed in a changing temperature environment provide electropherograms with considerable baseline fluctuations, resulting in significant errors in detection and integration of the peaks. This paper describes electropherogram baseline compensation that is suitable for the capillary electrophoresis-contactless conductivity detection analytical method. The baseline compensation utilizes linear or polynomial data processing methods, and can be programmed in-line using simple microcontroller, or on-line and off-line in data acquisition software. This method is targeted for field portable and autonomous analytical systems that are utilized in a fluctuating environment., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Determination of natural preservatives using electrophoretic or chromatographic techniques in fermented milk products is a complex task due to the following reasons: (i) the concentrations of the analytes can be below the detection limits, (ii) complex matrix and comigrating/coeluting compounds in the sample can interfere with the analytes of the interest, (iii) low recovery of the analytes, and (iv) the necessity of complex sample preparation. The aim of this study was to apply capillary zone electrophoresis coupled with contactless conductivity detection for the separation and determination of nisin in fermented milk products. In this work, separation and determination of natural preservative-nisin in fermented milk products is described. Optimized conditions using capillary zone electrophoresis coupled with capacitance-to-digital technology based contactless conductivity detector and data conditioning, which filter the noise of the electropherogram adaptively to the peak migration time, allowed precise, accurate, sensitive (limit of quantification: 0.02 μg/mL), and most importantly requiring very minute sample preparation, determination of nisin. Sample preparation includes following steps: (i) extraction/dilution and (ii) centrifugation. This method was applied for the determination of nisin in real samples, i.e. fermented milk products. The values of different nisin forms were ranging from 0.056 ± 0.003 μg/mL to 9.307 ± 0.437 μg/g., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In the present work, we demonstrate a novel approach to improve the sensitivity of the "out of lab" portable capillary electrophoretic measurements. Nowadays, many signal enhancement methods are (i) underused (nonoptimal), (ii) overused (distorts the data), or (iii) inapplicable in field-portable instrumentation because of a lack of computational power. The described innovative migration velocity-adaptive moving average method uses an optimal averaging window size and can be easily implemented with a microcontroller. The contactless conductivity detection was used as a model for the development of a signal processing method and the demonstration of its impact on the sensitivity. The frequency characteristics of the recorded electropherograms and peaks were clarified. Higher electrophoretic mobility analytes exhibit higher-frequency peaks, whereas lower electrophoretic mobility analytes exhibit lower-frequency peaks. On the basis of the obtained data, a migration velocity-adaptive moving average algorithm was created, adapted, and programmed into capillary electrophoresis data-processing software. Employing the developed algorithm, each data point is processed depending on a certain migration time of the analyte. Because of the implemented migration velocity-adaptive moving average method, the signal-to-noise ratio improved up to 11 times for sampling frequency of 4.6 Hz and up to 22 times for sampling frequency of 25 Hz. This paper could potentially be used as a methodological guideline for the development of new smoothing algorithms that require adaptive conditions in capillary electrophoresis and other separation methods.

The kiwi fruit, Actinidia kolomikta, has valuable properties such as high antioxidant activity, high vitamin C, polyphenols, chlorophylls and organic acids content, but the species are hardly commercialized due to their short shelf life (less than two days). In this study three different cultivars of A. kolomikta (Anykšta, Sentiabrskaya and VIR2) were coated with low, medium and high molecular weight chitosan bio-polymer with the aim to extend the shelf life. The changes in fruit firmness, mass, phenolic compound content, vitamin C content and subjective criteria (withering level, decoloration level and aesthetic appearance) were monitored. It was observed that high molecular weight chitosan had higher positive effect on the shelf life of Sentiabrskaya and Anykšta cultivars than VIR2. Low molecular weight chitosan was found effective on VIR2., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Purpose: To evaluate the antiproliferative effect of the aerial part of Chamerion angustifolium (L.) Holub. (Onagraceae) extract and its fractions in vitro. This is the first study on the anti-proliferative effect of C. angustifolium on 3 distinct breast cancer cell lines., Material/methods: Breast cancer cell lines MCF7, MDA-MB-468 and MDA-MB-231 were exposed to different concentrations of the water extract of C. angustifolium, where DPPH radical scavenging activity was 0.018-0.443mg/ml, expressed in rutin equivalents. Cell growth was analyzed after 24, 48 and 72h of incubation. Solid-phase extraction was applied for the fractionation of C. angustifolium water extract and MDA-MB-468 cell line growth was tested using different fractions., Results: The concentrations corresponding to radical scavenging activity of 0.117 and 0.266mg/ml caused MCF7 cells growth inhibition, while in the samples exposed to the highest concentration (0.355 and 0.443mg/ml) no proliferation was register, suggesting cell death. MDA-MB-468 cell analysis showed similar responses. MDA-MB-231 demonstrated cell growth inhibition following the exposure to all analyzed high extract doses (0.117-0.443mg/ml). MDA-MB-468 cells were selected to evaluate the effect of fractions. In the samples exposed to the fraction containing the highest amount (91%) of oenothein B, at the concentration of 0.117mg/ml a pronounced cell growth inhibition while at higher concentrations (0.266 and 0.443mg/ml) no cell proliferation was observed., Conclusions: The consumption of C. angustifolium herb can be advantageous, alongside with conventional breast cancer treatment., (Copyright © 2017. Published by Elsevier B.V.)

The miniaturization and optimization of a white rot fungal bioremediation experiment is described in this paper. The optimized procedure allows determination of the degradation kinetics of anthracene. The miniaturized procedure requires only 2.5 ml of culture medium. The experiment is more precise, robust, and better controlled comparing it to classical tests in flasks. Using this technique, different parts, i.e., the culture medium, the fungi, and the cotton seal, can be analyzed. A simple sample preparation speeds up the analytical process. Experiments performed show degradation of anthracene up to approximately 60% by Irpex lacteus and up to approximately 40% by Pleurotus ostreatus in 25 days. Bioremediation of anthracene by the consortium of I. lacteus and P. ostreatus shows the biodegradation of anthracene up to approximately 56% in 23 days. At the end of the experiment, the surface tension of culture medium decreased comparing it to the blank, indicating generation of surfactant compounds.

Due to the wide spectrum of biological activities, Chamerion angustifolium L. as medicinal plant is used for the production of food supplements. However, it should be kept in mind that quality (biological activity) of the herb depends on its geographic origin, the way of raw material preparation or extraction and chemotype. The purpose of this study was to evaluate and compare the compositions of volatile, non-volatile compounds and antioxidant activities of C. angustifolium grown in Kaunas Botanical Garden after the introduction from different locations in Lithuania. The compositions of fresh and air-dried samples were compared. The profile of volatile compounds was analyzed using headspace solid phase microextraction coupled with GC/MS. trans-2-Hexenal (16.0-55.9% of all volatiles) and trans-anethole (2.6-46.2%) were determined only in the dried samples, while cis-3-hexenol (17.5-68.6%) only in fresh samples. Caryophyllenes (α- and β-) were found in all analyzed samples, contributing together from 2.4% to 52.3% of all volatiles according to the origin and preparation (fresh or dried) of a sample. Total amount of phenolic compounds, total content of flavonoids and radical scavenging activity (using 2,2-diphenyl-1-picrylhydrazyl (DPPH)) were determined using spectrophotometric assays. The variation of total phenolic compounds content was dependent on the sample origin, moreover, drying reduced amount of phenolics 1.5-3.5 times. The DPPH free radical scavenging activity was in the range of 238.6-557.1mg/g (expressed in rutin equivalents) in the fresh samples and drastically reduced to 119.9-124.8 mg/g after drying. The qualitative analysis of phenolic compounds in the aqueous methanolic extracts of C. angustifolium was performed by means of HPLC with UV detection. Oenothein B and rutin were predominant in the samples; also caffeic and chlorogenic acids, and quercetin were determined. Chemometric methods, namely principal component analysis, hierarchical cluster analysis and K-means clustering analysis, were applied for evaluation of the results. Chemometric analysis showed existence of different chemotypes of C. angustifolium L. and their relation to the geographic origin., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The capacitance-to-digital single chip detector was upgraded. The paper discusses hardware issues and benefits of the designed/upgraded detector. The device can be operated from rechargeable lithium-ion battery as stand-alone, portable system and is capable of transmitting real-time data wirelessly. The detector and additional modules (battery, battery holder, microcontroller board, wireless module) weight is less than 85 g. Electrophoretic separation in low conductivity 20 mM MES/L-His buffer, pH 6.1, was performed in order to evaluate detection parameters. The system is capable of quantification of potassium ions down to 0.31 μM. Investigation of differential signal acquisition configuration showed improved performance regarding external noise and temperature fluctuations. The system can be a solution for stand-alone, field-portable capillary format separation detector., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A capacitance-to-digital converter integrated circuit was implemented in an automated CE device as a single chip detector. In this paper, design and hardware issues related to the fabrication and application of a miniature detector for contactless measurement of complex impedance are discussed. The capacitance-to-digital converter integrated circuit was used as the whole detector. The advantage of this setup is that the single integrated circuit provides digital data and neither additional signal conditioning nor analog-to-digital converter is required. Different separation conditions were used to evaluate the detection characteristics of the constructed detection unit. A 1 μM LOD for sodium and a 1.6 μM LOD for potassium ions were revealed for the detector. The detection system designed is competitive with miniaturized contactless conductivity detectors or UV absorbance detectors with respect to overall parameters (sensitivity, resolution, power consumption properties, and size). The obtained separation and detection results show that such detection technique can be used as an extremely low power consuming and space saving solution for CE detection with potential applications in environmental monitoring, process control, and various analytical measurements.

Five fatty acids (oleic, linoleic, myristic, lauric and capric) were incorporated in 10% (w/w) into ointment formulation and their influence on lipophilic model drug tolnaftate release in vitro and enhancing effect on tolnaftate penetration into epidermis and dermis of human skin ex vivo were investigated. The prepared ointments were tested for homogeneity, pH and theological properties. In vitro release studies and ex vivo skin penetration experiments were carried out using Hanson and Bronaugh-type flow-through diffusion cells, respectively. Tolnaftate cumulative amount liberated from semisolids was assayed using UV-Vis spectrophotometer. After in vitro skin penetration studies, appropriately extracted human skin layers were analyzed for tolnaftate content using a validated HPLC method. Statistical analysis revealed that release rate of tolnaftate from control ointment and ointments with fatty acids was not significantly different and only 7.34-8.98% of drug was liberated into an acceptor medium after 6 h. Tolnaftate amount penetrating into 1 cm2 of epidermis from ointments containing oleic, linoleic, myristic and lauric acids was significantly greater (p < 0.05) than from the control ointment. Penetration enhancing ratios for these fatty acids for tolnaftate penetration into epidermis ranged from 1.48 to 1.75. In conclusion, fatty acids did not increase the liberation of tolnaftate from ointment formulation, but demonstrated their enhancing effect on tolnaftate penetration into human epidermis in vitro. Results from in vitro release experiments do not suit for prediction of the situation in the skin in vitro, if chemical penetration enhancers are incorporated into the ointment formulation.