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The present study focuses on investigating 60 strains of yeast isolated from the natural fermentation broth of Vitis labruscana Baily × Vitis vinifera L. These strains underwent screening using lysine culture medium and esculin culture medium, resulting in the identification of 27 local non-Saccharomyces yeast strains exhibiting high β-glucosidase production. Subsequent analysis of their fermentation characteristics led to the selection of four superior strains (Z-6, Z-11, Z-25, and Z-58) with excellent β-glucosidase production and fermentation performance. Notably, these selected strains displayed a dark coloration on esculin medium and exhibited robust gas production during Duchenne tubules' fermentation test. Furthermore, all four non-Saccharomyces yeast strains demonstrated normal growth under specific conditions including SO 2 mass concentration ranging from 0.1 to 0.3 g/L, temperature between 25 and 30 °C, glucose mass concentration ranging from 200 to 400 g/L, and ethanol concentration at approximately 4%. Molecular biology identification confirmed that all selected strains belonged to Pichia kudriavzevii species which holds great potential for wine production., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Chicory, renowned for its multifaceted benefits, houses two vital coumarins, esculetin and esculin, both instrumental in reducing uric acid. This study emphasizes the metabolic pathways of esculetin and esculin under both standard and hyperuricemia conditions. Hyperuricemia was induced in Sprague-Dawley rats using oxonic acid potassium salt (300 mg·kg -1 ) and a 10% fructose water regimen over 21 days. Leveraging the ultra-high-performance liquid chromatography-Q Exactive hybrid quadrupole-orbitrap high resolution mass spectrometry, we analyzed the fragmentation behaviors of esculetin and esculin in rat bio-samples. Post oral-intake of esculetin or esculin, a notable dip in serum uric acid levels was observed in hyperuricemia rats. The investigation unveiled 24 esculetin metabolites and 14 for esculin. The metabolic pathways of both compounds were hydrolysis, hydroxylation, hydrogenation, dehydroxylation, glucuronidation, sulfation, and methylation. Interestingly, certain metabolites presented variations between standard and hyperuricemia rats, indicating that elevated levels of uric acid may affect enzyme activity linked to these metabolic reactions. This is the first systematic study on comparison of metabolic profiles of esculetin and esculin in both normal and hyperuricemia states, which was helpful to enrich our understanding of the complicated structure-activity relationships between esculin and esculetin and shed light to their action mechanism., (© 2023 Wiley-VCH GmbH.)

Fraxini Cortex is a traditional Chinese herbal medicine that has been used for thousands of years to treat dampness-heat diarrhea, dysentery, red or white vaginal discharge, painful swelling or redness of the eyes, and nebula. It contains various chemical components, including coumarins, iridoids, phenolic acids, and flavonoids. Coumarins are important active ingredients in Fraxini Cortex and have antibacterial, anti-inflammatory, antioxidant, antitumor, and antiviral activities. Aesculin and aesculetin are two major coumarin components of Fraxini Cortex that are widely used in its quality evaluation. Previous HPLC methods for determination of aesculin and aesculetin present several limitations, such as long analysis times and high solvent and reference compound consumption. In this study, a rapid, eco-friendly and cost saving HPLC method for the determination of aesculin and aesculetin in Fraxini Cortex was established by using the core-shell column and equal absorption wavelength (EAW). Different factors influencing the extraction process, such as the extraction solvent, temperature, and time, were assessed to obtain the optimal extraction conditions. The results showed that Fraxini Cortex samples could be well extracted by ultrasonic extraction for 5 min with a 25% ethanol aqueous solution. A core-shell column was used, and different mobile phases and flow rates were investigated to obtain the best rapid-HPLC separation conditions. The optimized HPLC conditions were as follows: a Poroshell 120 EC-C 18 column (50 mm×4.6 mm, 2.7 μm), acetonitrile-0.1% formic acid aqueous solution (6∶94, v/v) as the eluent, a flow rate of 1.5 mL/min, and a column temperature of 25 ℃. The EAW of aesculin and aesculetin was a key factor in their determination using a single reference compound. EAW selection was performed in two steps. First, the UV spectra of two equimolar concentrations of the reference compounds (aesculin and aesculetin) were compared to determine the EAW of the two analytes. The EAW results were then verified by the HPLC analysis of the reference compound solutions. The final EAW of aesculin and aesculetin was 341 nm. The determination of aesculin and aesculetin using only one reference compound (i. e., aesculin) was achieved by HPLC-UV at this EAW. The newly developed HPLC method revealed a good linear relationship between the two target analytes ( r =1.0000). The limits of detection (LODs) and limits of quantification (LOQs) were 1.5 μmol/L and 3.0 μmol/L, respectively, and the average recoveries of aesculin and aesculetin were 99.0% and 97.5%. The stabilities of the sample solutions were examined, and the two analytes demonstrated good stability for 24 h. The contents of the target analytes in 10 batches of Fraxini Cortex were determined using the proposed EAW method and the classic external standard method (ESM), and comparable concentrations were obtained. The contents of aesculin and aesculetin in the 10 batches of Fraxini Cortex were 0.26%-2.80% and 0.11%-1.47%, respectively. A t -test was conducted to compare the results of the proposed EAW technique with those obtained via the method reported in the Chinese Pharmacopoeia, and no significant difference between the two assay methods was noted ( P >0.05). Comparison of the newly established EAW method with those reported in the literature revealed that our method required only 10 min to complete and used as little as 0.5 mL of the solvent and only one standard. Therefore, the developed EAW method is a rapid, simple, eco-friendly, and cost-effective analytical method that is suitable for the determination of aesculin and aesculetin in Fraxini Cortex and its related products. The proposed technique is an improved method for determining aesculin and aesculetin and contributes to the enhancement of the quality evaluation of Fraxini Cortex.

Bunium species have been reported to be used both as food and in traditional medicines. The scientific community has attempted to probe into the pharmacological and chemical profiles of this genus. Nonetheless, many species have not been investigated fully to date. In this study, we determined the phenolic components, antimicrobial, antioxidant, and enzyme inhibitory activities of aerial parts of four Bunium species (B. sayai, B. pinnatifolium, B. brachyactis and B. macrocarpum). Results showed that B. microcarpum and B. pinnatifolium were strong antioxidants as evidenced in the DPPH, ABTS, CUPRAC, and FRAP assays. B. brachyactis was the most effective metal chelator, and displayed high enzyme inhibition against cholinesterase, tyrosinase, amylase, glucosidase, and lipase. The four species showed varied antimicrobial activity against each microorganism. Overall, they showed high activity against P. mirabilis and E. coli (MIC and MBC <1 mg mL -1 ). B. brachyactis was more effective against Aspergillus versicolor compared to the standard drug ketoconazole. B. brachyactis was also more effective than both ketoconazole and bifonazole against Trichoderma viride. B. sayai was more effective than ketoconazole in inhibiting A. fumigatus. B. sayai was most non-toxic to HEK 293 (cellular viability = 117%) and HepG2 (cellular viability = 104%). The highest level of TPC was observed in B. pinnatifolium (35.94 mg GAE g -1 ) while B. microcarpum possessed the highest TFC (39.21 mg RE g -1 ). Seventy four compounds were detected in B. microcarpum, 70 in B. brachyactis, 66 in B. sayai, and 51 in B. pinnatifolium. Quinic acid, chlorogenic acid, pantothenic acid, esculin, isoquercitrin, rutin, apigenin, and scopoletin were present in all the four species. This study showed that the four Bunium species are good sources of biologically active compounds with pharmaceutical and nutraceutical potential., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

A fast and precise qNMR method was developed for quantification of major bioactive constituents in the bark of horse chestnut and dry extracts prepared thereof. The method was optimised using 600 MHz spectrometer, and the final acquisition parameters (90°-pulse, acquisition time - 3.0 s, relaxation delay - 27 s, number of transients - 16) allowed for performing of quantitative experiments in under 15 min. The contents of three analytes were determined using specific 1 H resonances at δ7.45 ppm for esculin, δ5.00 ppm for fraxin, and δ5.94 ppm for (-)-epicatechin. The validation showed good precision (RSD < 1.5%) and accuracy (95-103%), and adequate sensitivity (LODs in the range of 3.3-5.9 µg) of the measurements. The determined levels in commercial samples of Hippocastani cortex were in the range of 25.89-38.94 mg/g dry weight (dw) of the bark for esculin, 12.58-17.13 mg/g dw for fraxin and 10.42-13.96 mg/g dw for (-)-epicatechin, and in the dry extracts prepared thereof 97.02-143.51 mg/g, 45.78-58.92 mg/g and 28.07-46.29 mg/g, respectively. The obtained results were cross-validated by a HPLC-PDA method with the use of a fused-core column, and no statistical differences were found between the results obtained by both methodologies, but with the advantage of higher precision of the qNMR assay. The relevant variability in quantitative composition of the commercial samples emphasise the need to introduce quality control studies in production of preparations containing horse chestnut bark and the developed method was proved suitable for this purpose., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Fraxini Cortex (also known as Qinpi, QP) has been used for the treatment of hyperuricemia with a significant difference on efficacy of QP from different regions. However, it`s still unknown whether proportion of components is the key and why same kind of herbs have different therapeutic effects. In this study, different sources of QP were collected from Shaanxi Qinpi extracts (SQPE), Henan Qinpi extracts (HQPE), Hebei Qinpi extracts (GQPE) provinces in China. Rat model of hyperuricemia with hypoxanthine combined with potassium oxonate were established to determine the levels of blood urea nitrogen (BUN), serum uric acid (SUA), urine uric acid (UUA) and creatinine (Cr). Hematoxylin-eosin staining (H&E) and Periodic Acid-Schiff staining (PAS) were performed for renal pathology while Western blot analysis and real-time PCR analysis for proteins and mRNA expression levels. High-performance liquid chromatograph (HPLC) was used for components and composition analysis. Our results demonstrated that QPE from different regions could alleviate hyperuricemia via increasing significantly the SCr and BUN levels whereas decreasing markedly UCr, SUA and UUA levels. Additionally, QPE could also improve the pathological changes of the kidneys. The protein and mRNA levels of urate reabsorption transporter 1 (URAT1) and glucose transporter 9 (GLUT9) were down-regulated by QPE treatment. SQPE hold a better activity on improving hyperuricemia and regulating URAT1 and GLUT9. HPLC analysis showed that the proportion of four components aesculin, aesculetin, fraxin, fraxetin were 9.002: 0.350: 8.980: 0.154 (SQPE); 0.526: 0.164: 7.938: 0.102 (HQPE); 12.022: 1.65: 0.878: 1.064 (GQPE). These data indicate that this proportion of effective components may be an important factor for efficacy of QP and had implications for the treatment of hyperuricemia., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

A sensitive and rapid ultra-performance liquid chromatography-electrospray ionization-mass spectrometry (UPLC-ESI-MS/MS) method was developed for the simultaneous analysis of anemoside B4, phellodendrine, berberine, palmatine, obakunone, esculin, esculetin, toosendanin (IS 1 of anemoside B4), tetrahydropalmatine (IS 2 of phellodendrine, berberine, palmatine and obakunone) and scopoletin (IS 3 of esculin and esculetin) and to compare the pharmacokinetics of these active ingredients in normal and ulcerative colitis rats. After methanol deproteinization, solvents were evaporated at 40°C under a gentle stream of nitrogen. Chromatography was performed using a C18 column with a gradient elution of 0.1% aqueous formic acid and acetonitrile at 0.4ml/min. Detection and measurement were performed on a 4000 QTRAP UPLC-MS/MS system from AB Sciex in the multiple reaction monitoring (MRM) mode. Phellodendrine, berberine, palmatine, obakunone, esculin, esculetin, tetrahydropalmatine (IS 2 ) and scopoletin (IS 3 ) were monitored under positive ionization conditions. Anemoside B4, and toosendanin (IS 1 ) were monitored under negative ionization conditions. The optimized mass transition ion-pairs (m/z) were 1221.1/750.7 for anemoside B4, 343.2/193.2 for phellodendrine, 337.1/321.0 for berberine, 353.0/336.9 for palmatine, 455.1/161.1 for obakunone, 341.2/179.2 for esculin, 179.1/123.0 for esculetin, 573.4/531.4 for toosendanin (IS 1 ), 356.2/192.2 for tetrahydropalmatine (IS 2 ) and 193.0/133.1 for scopoletin (IS 3 )., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Esculin, a coumarin derivative found in Fraxinus rhynchophylla, has been reported to possess multiple biological activities. This present study is designed to investigate the metabolic profile of esculin in vivo based on ultra high performance liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry (UHPLC-FT-ICR-MS) for the first time. After oral administration of esculin (100 mg/kg) for rats, plasma, urine, feces and bile samples were collected to screen metabolites. As a result, a total of 19 metabolites (10 phase I metabolites and 9 phase II metabolites) were found and identified. Results showed that metabolic pathways of esculin included hydrolysis, dehydrogenation, hydroxylation, methylation, dehydrogenation, glucuronidation, sulfation, and glycine conjugation. It was also found that after oral administration of esculin, the esculin could be metabolized to esculetin in vivo via deglycosylation, and esculetin was found in all biological samples. This study also laid solid basis for in-depth development of esculin and provided important information for clarifying the biotransformation process of esculin in vivo., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The location of individual coumarins in leaves of Fraxinus ornus acclimated at full solar irradiance was estimated using their specific UV- and fluorescence spectral features. Using a combination of UV-induced fluorescence and blue light-induced fluorescence of tissues stained with diphenylborinic acid 2-amino-ethylester, in wide field or confocal laser scanning microscopy, we were able to visualize the distribution of esculetin and esculetin 6-O-glucoside (esculin) in palisade cells. Coumarins are not uniformly distributed in the cell vacuole, but accumulate mostly in the adaxial portion of palisade cells. Our study indeed shows, for the first time, that coumarins in palisade cells accumulate as vacuolar inclusions, as previously reported in the pertinent literature only for anthocyanins. Furthermore, esculetin and esculin have a different vacuolar distribution: esculetin largely predominates in the first 15 μm from the adaxial epidermis. This leads to hypothesize for esculetin and esculin different transport mechanisms from the endoplasmic reticulum to the vacuole as well as potentially different roles in photoprotection. Our study open to new experiments aimed at exploring the mechanisms that deliver coumarins to the vacuole using different fluorescence signatures of coumarin aglycones and coumarin glycosides., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The acetyl esterase of Trichoderma reesei belonging to carbohydrate esterase (CE) family 16 catalyzes transacylations to carbohydrate moieties of flavonoid glycosides, esculin and rutin. The enzyme recognizes as acyl donors vinyl esters of short carboxylic acids. Esculin was acylated at position 3 of the glucosyl residue in aqueous solutions saturated with vinyl acetate and vinyl propionate. The yields of esculin monoacetate and monopropionate of esculin in aqueous medium (esculin 40 mM, enzyme 40 µg/ml, 40 °C, 3 days) were 67 and 55 %, respectively. Replacement of water by 2-propanol was required for a similar acylation of rutin at 4 mM concentration. The yields of rutin monoacetate and propionate were 60 and 30 %, respectively. The results indicate that the enzyme could be used for an easy modification of solubility and hydrophobicity of glycosylated compounds, including drugs and functional food additives.

In this paper, a chromatographic method for isolation and purification of coumarin compounds from Cortex fraxinus was established by using Superose 12 as the separation media for the first time. The conditions for separation were optimized. Four kinds of coumarin compounds including aesuletin, aesculin, fraxetin and fraxin were obtained. The purity of these compounds were 98.5, 99.1, 97.9 and 97.3%, respectively, which were determined by HPLC area normalization method. The chemical structures of the separated compounds were identified according to (1)H and (13)C nuclear magnetic resonance data. The retention behavior of the separated coumarin compounds on Superose 12 was also discussed. The retention is based on a mixture of hydrogen bonding and hydrophobic interactions between the coumarin compounds and the residues of the cross-linking reagents used in the manufacturing process of Superose 12. The results of this paper indicate that Superose 12 is not only suitable for size-exclusion chromatography of proteins and other biological macromolecules but also for low-molecular-weight natural products., (© The Author [2013]. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Actinomycosis is a relatively rare infection caused by saprophytic bacteria of the oral cavity and gastrointestinal tract that can become pathogenic. The chronic hyperglycemia of diabetes mellitus induces events that promote structural changes in various tissues and are associated with problems in wound healing. This infection remains largely unknown to most clinicians because of its different presentations, and palatal involvement is extremely rare. This report describes the case of a 46-year-old woman who was diagnosed with actinomycosis involving the hard palate. The main clinical, histopathologic, and therapeutic characteristics and differential diagnosis of actinomycosis are reviewed. To date, 3 cases of actinomycosis involving the hard palate have been reported., (Copyright © 2014 American Association of Oral and Maxillofacial Surgeons. Published by Elsevier Inc. All rights reserved.)

In this report, rapid and effective shoot as well as root regeneration system through direct multiplication was successfully developed for Cichorium intybus L. Furthermore, the effect of exogenous growth regulators (TDZ and IAA) at different concentrations on the regulation process of the plant was also studied. Enhanced production of esculin in developed C. intybus L. was evaluated using leaf extract. Only on the expense of 20 days, regeneration was seen and very low dose of TDZ was seen to be more effective. When 0.02 mg/L of TDZ was combined with 1.5mg/L of IAA, nearly 100% of explants produced shoots with the highest number of regenerated shoots (85.37). With further increase in concentration (≥ 0.05 mg/L), the number of shoots per explants get decreased. A lower NAA to IBA ratio (1.0mg/L of IBA and 0.5mg/L of NAA) seemed to be more effective for root generation and considered to be the most effective combination among the tried groups. IBA was more effective in root development than NAA, but both were comparatively effective. On quantitative analysis by RP-HPLC, the 76.23% of Esculin were found in leaf extract of the in vitro developed C. intybus L. This amount was 26.77% higher than normal grown plants.

Objective: To establish a new quantitative method for simultaneous determination of multi-coumarins in Fraxini Cortex by using one chemical reference substance, and validate its feasibilities., Method: The new quality evaluation method, quantitative analysis of multi-components by singer-marker (QAMS), was established and validated with Fraxini Cortex. Four main coumarins were selected as analytes to evaluate the quality and their relative correlation factors (RCF) were determined by HPLC-DAD. Within the linear range, the values of RCF at 340 nm of aesculin to asculetin, fraxin and fraxetin were 1.771, 0.799, 1.409, respectively. And the contents of aesculin in samples of Fraxini Cortex were authentically determined by the external standard method, and the contents of the three other coumarins were calculated by their RCF. The contents of these four coumarins in all samples were also determined by the external standard method., Result: Within a certain range, the RCF had a good reproducibility (RSD 2.5%-3.9%). Significant differences were not observed between the quantitative results of two methods., Conclusion: It is feasible and suitable to evaluate the quality of Fraxini Cortex and its Yinpian by QAMS.

In this report, a novel carbon nanotube/poly(ethylene-co-vinyl acetate) (CNT/EVA) composite electrode was developed for the amperometric detection in CE. The composite electrode was fabricated by packing a mixture of CNTs and melted EVA in a piece of fused-silica capillary under heat. It was coupled with CE for the separation and detection of esculin and esculetin in Cortex Fraxini, a traditional Chinese medicine, to demonstrate its feasibility and performance. Esculin and esculetin have been well separated within 9 min in a 40 cm long capillary at a separation voltage of 12 kV using a 50 mM borate buffer (pH 9.2). The new CNT-based CE detector offered significantly lower detection potentials, yielded enhanced S/N characteristics, and exhibited high resistance to surface fouling and enhanced stability. It showed long-term stability and reproducibility with relative standard deviations of less than 5% for the peak current (n=15) and should also find a wide range of applications in other microfluidic analysis systems.

In this paper, a micelle-mediated extraction and cloud point preconcentration method was developed for the determination of less hydrophobic compounds aesculin and aesculetin in Cortex fraxini by HPLC. Non-ionic surfactant oligoethylene glycol monoalkyl ether (Genapol X-080) was employed as the extraction solvent. Various experimental conditions were investigated to optimize the extraction process. Under optimum conditions, i.e. 5% Genapol X-080 (w/v), pH 1.0, liquid/solid ratio of 400:1 (ml/g), ultrasonic-assisted extraction for 30 min, the extraction yield reached the highest value. For the preconcentration of aesculin and aesculetin by cloud point extraction (CPE), the solution was incubated in a thermostatic water bath at 55 degrees C for 30 min, and 20% NaCl (w/v) was added to the solution to facilitate the phase separation and increase the preconcentration factor during the CPE process. Compared with methanol, which was used in Chinese Pharmacopoeia (2005 edition) for the extraction of C. fraxini, the extraction efficiency of 5% Genapol X-080 reached higher value.

Objective: To study the optimum extracting and dried process of Qinxiangzhixie enteric-coated tablet., Methods: With the content of Aesculin and Aescaletin as factor, the optimum extraction process was selected with the orthogonal design., Results: The optimum extraction process conditions were as: decocting for 2 times, with water of 8, 6 times of the drug mixture respectively 1.5 h, 1.0 h respectively. The optimum decompression dried process conditions was as follows :60 degrees C, -0.08 pa., Conclusions: The experimental results provide the basis for the ascertainment of extraction process of Qinxiang enteric-coated tablet.

A capillary zone electrophoresis (CZE) with indirect laser-induced fluorescence detection (ILIFD) method is described for the simultaneous determination of esculin, esculetin, isofraxidin, genistein, naringin and sophoricoside. The baseline separation was achieved within 5 min with running buffer (pH 9.4) composed of 5mM borate, 20% methanol (v/v) as organic modifier, 10(-7)M fluorescein sodium as background fluorophore and 20 kV of applied voltage at 30 degrees C of cartridge temperature. Good linearity relationships (correlation coefficients >0.9900) between the second-order derivative peak-heights (RFU) and concentrations of the analytes (mol L(-1)) were obtained. The detection limits for all analytes in second-order derivative electrophoregrams were in the range of 3.8-15 microM. The RSD data of intra-day for migration times and second-order derivative peak-height were less than 0.95 and 5.02%, respectively. This developed method was applied to the analysis of the courmin compounds in herb plants with recoveries in the range of 94.7-102.1%. In this work, although the detection sensitivity was lower than that of direct LIF, yet the method would extend the application range of LIF detection.

A fast, accurate and convenient method for the simultaneous determination of multi-component in the Chinese herbal medicine was proposed by using ultraviolet absorption spectrum. In this method, dummy components were added to training sample, and a double artificial neural network (DANN) that has the function of high self-revision and self-simulation was used. Effect of other interference components could be eliminated by adjusting concentration of dummy components. Therefore, the accuracy of concentration prediction for multi-component in the complicated Chinese herbal medicine was improved. It has been realized that two effective components of Cortex Fraxini, aesculin and aesculetin, were simultaneously determined, without any separation. The predicted accuracy was 92% within the permitted relative errors. The measurement precisions of the aesculin and aesculetin were 0.37% and 1.5%, respectively.

A non-aqueous capillary electrophoresis method has been developed for the separation and simultaneous determination of fraxin, esculin and esculetin in Cortex fraxini and its preparation for the first time. Optimum separation of the analytes was obtained on a 47 cm x 75 microm i.d. fused-silica capillary using a non-aqueous buffer system of 60 mM sodium cholate, 20 mM ammonium acetate, 20% acetonitrile and 3% acetic acid at 20 kV and 292 K, respectively. The relative standard deviations (RSDs) of the migration times and the peak heights of the three analytes were in the range of 0.23-0.28 and 2.12-2.60%, respectively. Detection limits of fraxin, esculin and esculetin were 0.1557, 0.4073 and 0.5382 microg/mL, respectively. In the tested concentration range, good linear relationships (correlation coefficients 0.9995 for fraxin, 0.9999 for esculin and 0.9992 for esculetin) between peak heights and concentrations of the analytes were observed. This method has been successfully applied to simultaneous determination of the three bioactive components with the recoveries from 90.2 to 109.2% in the five samples.

A simple method for the simultaneous determination of five bioactive components (rutin, puerarin, daidzein esculin and esculetin) in traditional medicinal preparations by non-aqueous capillary electrophoresis with UV detection has been developed for the first time. A running buffer composed of 15% acetonitrile, 2.5% acetic acid and 90 mM sodium cholate in methanol was found to be the most suitable for this separation. The limits of detection for five analytes were over the range of 0.050-1.216 microg ml(-1). The relative standard deviations (R.S.Ds.) of the migration times and the peak areas of the analytes were in the range of 1.3-2.9% and 2.2-2.7% (intraday), 1.7-1.9% and 2.8-3.6% (interday), respectively. In the tested concentration range, linear relationships (correlation coefficients: 0.9974 for rutin, 0.9976 for puerarin, 0.9981 for daidzein, 0.9972 for esculin and 0.9929 for esculetin) between peak areas and concentrations of the analytes were obtained. This method has been successfully applied to simultaneous determination of the five bioactive components with recoveries over the range of 89.4-107.4%.

One hundred and twenty lipid dependent Malassezia spp. isolates were obtained from the clinically normal skin of 38 healthy adult volunteers by swabbing three different body sites (back, chest and scalp). Ninety-six percent of these strains could be grouped into three biotypes on the basis of microscopic, cultural, metabolic and biochemical (catalase, esculin and lipase (C-14)) characteristics. The differential features were simple to determine and easily reproduced. Moreover, the three biotypes were referable to the species M. globosa (biotype 1), M. sympodialis (biotype 2) and M. restricta (biotype 3). Based on their microscopic features, cultural properties and body site locations, we suggest that biotype 1 /M. globosa corresponds to the description of Pityrosporum orbiculare (round yeast cells with a narrow base, very frequently found on the upper trunk), and biotype 3/M. restricta corresponds to the concept of P. ovale (oval yeast cells with a broad budding base, located mainly on the scalp). Pleomorphic biotype 2/M. sympodialis, most frequently found in the back, does not clearly fit into any of the Pityrosporum species.

A chromatographic analysis of the hay charge used for balneological and thermotherapy which contains coumarin, the hydroxycoumarins umbelliferone and esculin and also the furanocoumarin imperatorin. The sedative effect of the hay can be explained by the coumarin and the imperatorin; the absorption site is to be found in the respiratory tract.

31 samples of potato varieties with slow, medium and fast rates of browning were studies. Characteristic enzyme patterns were obtained from polyacrylamide gel electrophoresic of the phenoloxidases of varieties with different discolouration rates. The differences lie mainly in the intensities of the enzyme bands. The qualitative determinaiton of the phenols showed no significant differences. Tyrosine, chlorogenic acid and caffeic acid produce coloured oxidation products; the characteristic colour gradations of in vivo browning were only observed in the presence of tyrosine. It is concluded that the same reactions take place during the discolouration of all the varieties.