Biotransformation on nitazoxanide by filamentous fungi - a microbial model to mammalian metabolism

High performance liquid chromatography in pharmaceutical analyses

本試驗以不同劑量的254 nm短波紫外線(Ultraviolet-C; UV-C)照射柑桔類果實，然後觀察果皮中所生成之植物殺菌素scoparone (6,7-dimethoxycoumarin)在照射後的變化及UV-C處理對貯藏病害之影響。椪柑及柳橙經UV-C照射後之果皮萃取物以薄層色層分析(TLC)在螢光下可觀察到scoparone之生成；另以高效能液相層析(HPLC)及螢光檢測器分析，可分離並偵測到scoparone及scopoletin (6-methoxy, 7-hydroxycoumarin)二種生成物，但scopoletin之濃度很低，測得之濃度不超過1 μg/g Fwt.。柑桔果實只有受到UV-C照射的部位才會生成scoparone，而其生成之部位為外果皮(油胞層)；旦不論果皮黃綠均可生成。椪柑及柳橙果實經0.5 ~ 3.0 KJ/m2 UV-C照射後果皮中之scoparone生成量隨時間逐漸增加，至10天左右達最高量，然後又逐漸下降。照射劑量愈高，scoparone生成量也愈大，而同劑量UV-C照射誘導椪柑所生成scoparone量較柳橙為高。 椪柑及柳橙果實以人工接種綠黴菌24小時後進行UV-C照射，並未抑制病菌生長。若果實先經UV-C照射後再接種綠黴菌，則能明顯降低發病率及感染直徑。UV-C之抑菌效果以照射後24小時接種之感染率最低，隨照射後時間之增長，對所接種綠黴菌之抑菌效果逐漸下降；因此果皮中scoparone含量高低與抑菌效果間無明顯關係。 未經任何藥劑處理之椪柑以1.5及3.0 KJ/m2 UV-C照射處理，在15℃貯藏三個月後由綠黴病引起之腐爛率比對照組低；但是經UV-C處理之椪柑，其綠蒂率較低，且蒂腐病發生率增加，因而總腐爛率反而提高。椪柑以1.5 KJ/m2 UV-C照射後並在果蒂塗覆200 ppm 2,4-D，可有效維持其綠蒂率並降低貯藏期間之總腐爛率。

Fungal biotransformation of mosapride by Cunninghamella elegans

This study was conducted in the laboratories of the Department of Food Science / College of Agriculture/ University of Tikrit with the aim of isolating and diagnosing fungi contaminated by stored wheat grains and estimating the concentration of FB1 in it and determining its physiological effects in rats, The results of the study showed the presence of Aspergillus sp. molds by 38 in the tested samples, followed by Fusarium sp. by 28 and Alternaria alternata by 19 then the genus Penicillium sp. by 10 and Mucor sp. by 5. The presence of FB1 toxin in local and imported wheat samples was also investigated by estimating using ELISA (Enzyme-linked Immune Sorbent Assays) as its highest concentration was 2.240 mg/kg in local wheat from the A-Dour district, while the lowest concentration was 0.103 mg/kg in Australian wheat samples. The ability of F.monliforme mold isolates to produce FB1 was also studied, it was found that they were capable of producing toxin with concentrations of 4.264 mg / kg in local wheat samples taken from Al-Dour district and 3.597 mg / kg in local wheat samples taken from Baiji district. Using HPLC (High Performance Liquid Chromatography). With regard to the sensory evaluation of wheat flour used in the study, the best results for wheat flour of Australian wheat gave the highest results which reached 24 degrees while the worst results for wheat flour of Baiji which amounted to 14 degrees for the qualities studied: color, smell. With regard to the sensory evaluation of beard made from wheat samples in this study, the highest was recorded 33 degrees for Australian wheat and lowest evaluation for Dour wheat with 24 degrees with regard to the studied qualities of color, smell, taste. The effect of FB1 on the rate of weight gain in male rats has also been studied, the presence of this toxin in the rats' diet led to a significant decrease in weight after 21 days of the trial age, where the average weight in the T3 treatment was 141 g and the results showed a significant increase in the relative weight of the liver and Kidney in treatment T3 was 4.6g and 1.7g respectively.

In addition to predicting biotransformation in humans, drug biotransformation studies are important because they can generate active metabolites or new intermediates with possible use by the pharmaceutical industry. Endophytic fungi of the genus Cunninghamella can metabolize many drugs in a similar way to humans. The analysis of these metabolites requires prior treatment of the samples in order to obtain compatibility with the detection system and the separation technique. This work aimed to study the biotransformation of the drugs duloxetine (DLX), citalopram (CIT) and amlodipine (ANL) by endophytic fungi Cunninghamella elegans ATCC 9245, the development of micro extraction methods in the context of green chemistry, and the validation of the analytical methods for drugs and their respective metabolites. Bioanalytical method by HPLC was successfully developed and validated for both drugs (DLX, CIT and ANL). The metabolites of DLX and CIT obtained by biotransformation studies were not detected in the conditions of this study, but it was possible to visualize one additional peak in the chromatogram analysis of ANL indicate that this drug has been metabolized by Cunninghamella elegans. Furthermore, it has been developed two liquid-liquid micro extraction methods achieving success by removing drugs in both techniques, with recovery greater than 90%. The use of Quality by Design (QbD) through the experimental design provided the best conditions for carrying out biotransformation and micro extraction studies of drugs from the fungal matrix.

Effects of Solid Fermentation on Protein Content and Amino Acid Composition of Cassava. This study was carried out to assess the protein and amino acid quantity of solid fermentation of cassava (Manihot esculenta) using pure culture of the Rhizopus oligosporus and traditional inoculum (laru). The protein content of the fermented product was analyzed by Biuret method, and the animo acid composition by HPLC (high performance liquid chromatography) method. The results showed that solid fermentation of cassava increased the protein content from 2.1% to 4.0% and 4.7%. The animo acid contents of the fermented product increased 2.5 folds of that of cassava. Higher increase was seen in substrates fermented with traditional inoculum. This is due to the addition of coconut oil and ammonium to the cassava substrate which improved the growth of mold. Key words: cassava (Manihot esculenta), food fermentation, protein content, amino acid composition

Protein glycosylation can be vital for changing the function or physiochemical properties of a protein. Abnormal glycosylation can lead to protein malfunction, resulting in severe diseases. Therefore, it is important to develop techniques for characterization of such modifications in proteins at a sensitivity level comparable with state-of-the-art proteomics. Whereas techniques exist for characterization of high abundance glycoproteins, no single method is presently capable of providing information on both site occupancy and glycan structure on a single band excised from an electrophoretic gel. We present a new technique that allows characterization of low amounts of glycoproteins separated by gel electrophoresis. The method takes advantage of sequential specific and nonspecific enzymatic treatment followed by selective purification and characterization of the glycopeptides using graphite powder microcolumns in combination with mass spectrometry. The method is faster and more sensitive than previous approaches and is compatible with proteomic studies.

Background Intracameral or intracorneal administration of amphotericin B (AMB) can achieve significant therapeutic efficacy in the treatment of recalcitrant fungal keratitis in cases that do not respond to conventional antifungal therapy. However, the ocular pharmacokinetics of the two routes of administration is unclear.Objective The goal of this study was to investigate the level of amphotericin B in cornea and aqueous humor of rabbits after administration of AMB via three different routes. Methods Forty-five healthy domestic rabbits were randomly divided into three groups. 1% amphotericin B of 10 μg was intrastromally or intracamerally injected into 15 rabbits, respectively,in group A and group B. Topical 0. 25% amphotericin B was topically administered to the eyes with corneal epithelial debridement (group C). Experimental animals were sacrificed and the corneas and aqueous humor samples were obtained for the detection of levels of amphotericin B at 30 minutes,6 hours, 1 day,3 and 7 days by high-performance liquid chromatography (HPLC). Results Calibration curves were linear over the range of 0. 10-100. 00 mg/L. The concentration of 0. 10 mg/L was the lowest quantifiable limit. The recovery of amphotericin B ranged from 89. 1% -95.7% from aqueous humor samples and 81.4% -83.6% from the cornea samples. After a single injection,effective drug levels were achieved and maintained for 7 days in cornea in group A, exceeding the minimum inhibitory concentration at which 90% of isolates are inhibited (MIC90) for a wide spectrum of fungi and molds with significant differences in comparison with group B and group C ( P＜0. 05 ). Effective drug levels were achieved in the aqueous humor in group B at 30 minutes after a single injection, but drug levels decreased dramatically within 6 hours. The evident differences were found between group B and group A or group C (P＜ 0.05). A considerable amount of amphotericin B was detected in the cornea and aqueous humor in group C within 1 day.Conclusion Effective high drug levels can be reached in rabbit cornea and aqueous humor after intrastromal and intracameral injection, respectively. Penetration of topical amphotericin B was greatly elevated after epithelial debridement. Key words: Amphotericin B; Chromatography; High pressure liquid; Pharmacokinetics

Methods in Protein Sequence Analysis

Since the classical studies of Moore and Stein [1,2] the separation of amino acids on cation exchange resins has been the method for their quantitation in both hydrolysates and physiological fluids. Subsequently the ease with which this could be accomplished was greatly improved by the development of an automatic analyser [3]. However over the last few years the technologies associated with high performance liquid chromatography (HPLQ have been applied with increasing success to the determination of amino acids and their derivatives. Much early work in HPLC involved investigating and ‘re-discovering’ much that was already well known to users and manufacturers of amino acid analysers. For example the comprehensive studies on the chromatography of amino acids performed by Hamilton [4–6] in the late 1950s are reflected a decade later in the HPLC literature. Similarly it is only in the 1980s that HPLC manufacturers and users are advocating post-column derivatization to enhance both selectivity and sensitivity and developing a theoretical understanding of the mechanisms involved. Modern HPLC equipment has only recently reached the level of sophistication common in amino acid analysers ten years ago. It should be stressed that cation-exchange chromatography of amino acids represents one specific application of the general analytical technique of liquid column chromatography and that high performance (or high pressure) liquid chromatography is simply the terminology applied to the technique as currently optimized.

Antenatal screening/testing of pregnant women should be carried out according to the guidelines of the NHS Sickle Cell and Thalassaemia Screening programme. Newborn screening and, when necessary, follow up testing and referral, should be carried out according to the guidelines of the NHS Sickle Cell and Thalassaemia Screening programme. All babies under 1 year of age arriving in the UK should be offered screening for sickle cell disease. Preoperative screening for sickle cell disease should be carried out in patients from ethnic groups in which there is a significant prevalence of the condition. Emergency screening with sickle solubility tests must always be followed by definitive analysis. Laboratories performing antenatal screening should utilize methods capable of detecting significant variants and be capable of quantitating haemoglobins A 2 and F at the cut-off points required by the national antenatal screening programme. The laboratory must ensure a provisional report is available within three working days from sample receipt.

An high performance liquid chromatography (HPLC) method for the enantioselective determination of donepezil (DPZ), 5-O-desmethyl donepezil (5-ODD), and 6-O-desmethyl donepezil (6-ODD) in Czapek culture medium to be applied to biotransformation studies with fungi is described for the first time. The HPLC analysis was carried out using a Chiralpak AD-H column with hexane/ethanol/methanol (75:20:5, v/v/v) plus 0.3 % triethylamine as mobile phase and UV detection at 270 nm. Sample preparation was carried out by liquid-liquid extraction using ethyl acetate as extractor solvent. The method was linear over the concentration range of 100-10,000 ng mL(-1) for each enantiomer of DPZ (r ≥ 0.9985) and of 100-5,000 ng mL(-1) for each enantiomer of 5-ODD (r ≥ 0.9977) and 6-ODD (r ≥ 0.9951). Within-day and between-day precision and accuracy evaluated by relative standard deviations and relative errors, respectively, were lower than 15 % for all analytes. The validated method was used to assess DPZ biotransformation by the fungi Beauveria bassiana American Type Culture Collection (ATCC) 7159 and Cunninghamella elegans ATCC 10028B. Using the fungus B. bassiana ATCC 7159, a predominant formation of (R)-5-ODD was observed while for the fungus C. elegans ATCC 10028B, DPZ was biotransformed to (R)-6-ODD with an enantiomeric excess of 100 %.

The unique ability of lactic acid bacteria (LAB) to convert unsaturated fatty acids to saturated fatty acids via the production of unsaturated intermediates is known as bio-hydrogenation, which is responsible for the conversion or the bio-transformation of unsaturated fatty acids like linoleic acid to saturated fatty acids like stearic acid via the intermediate compounds such as conjugated linoleic acid (CLA). Realizing the health benefits of CLA, lactic acid bacteria were isolated from curd in order to study their ability of CLA production. Initially, four bacterial strains isolated from curd and designated as 1s, 13s, 15s, and 17s were screened and found to be gram-positive, catalase negative and acid producers. Out of these four strains, 1s and 13s showed rapid adaptation to the change in the substrate from glucose to linseed oil hydrolysate. The fermentation study conducted in shake flask mode revealed that the maximum concentration of trans-10, cis-12 CLA produced in medium enriched with 0.4% (w/v) castor oil hydrolysate (mCOH) by isolate 1s was 620 mg·L-1 at 4h while that produced by isolate 13s in mCOH was 430mg·L-1 at 8h as assessed by gas chromatography (GC) performed using a flame ionization detector (FID). Thus, the study proved the ability of LAB to synthesize CLA. Furthermore, in order to test the feasibility of CLA production on a large scale, an additional study was conducted using Lactobacillus delbreukii NCIM 2025 in a continuously stirred tank reactor in both batch and continuous modes using linseed oil hydrolysate as the fatty acid substrate. The maximum concentration of CLA produced by Lactobacillus delbreukii NCIM 2025 in medium enriched with 0.4% linseed oil hydrolysate (mLOH) was 380µg·L-1 at 6 h in batch mode and 590 µg·L-1 at 1h in continuous mode as analyzed using highperformance liquid chromatography (HPLC) performed using an ultraviolet (UV) detector at 233nm. Thus, the study also reveals that there is a vast scope for improvement in the scale-up of CLA production from the laboratory scale to a commercial scale.

The purpose of this paper is to describe the glycosylation of ambrisentan (AMB) by cultures of Cunninghamella elegans ATCC 9245. AMB is an endothelin receptor antagonist, which is used to treat pulmonary arterial hypertension. Filamentous fungi are morphologically complex and may exhibit different forms depending on the species and the nature of the culture medium. A biotransformation study was conducted to investigate the ability of C. elegans to metabolize AMB. Parameters were optimized by testing on different culture media and concentrations, pH, drug concentration, static and shaking conditions. Ambrisentan's metabolite, obtained after 240 h of incubation as a result of glycosylation pathway, was separated by HPLC and determined by high-resolution mass spectrometry. The method showed linearity over 300-1000 μg mL-1 (r = 0.998). Accuracy, precision, robustness and stability studies agree with international guidelines. Results are consistent in accordance with the principles of green chemistry as the experimental conditions had a low environmental impact, and used little solvent.

Drug biotransformation studies appear as an alternative to pharmacological investigations of metabolites, development of new drug candidates with reduced investment and most efficient production. The objective of this study was to evaluate the capacity of biotransformation of Rifampicin (RIF) by the filamentous fungus Cunninghamella elegans as a microbial model of mammalian metabolism. In 120 h, C. elegans transformed the drug into the following two metabolites: rifampicin quinone and novel metabolite. The products of rifampicin formed in vitro were monitored by HPLC-PDA, being identified through UHPLC–QTOF/MS. Metabolites were characterized according to their chromatographic profile, mass fragments and UV spectral data. The major metabolic pathways of rifampicin transformed by the fungus were oxidation, demethylation and mono-oxidation. The microbial transformation of RIF showed the potential of Cunninghamella species to produce RIF metabolites. This process can be used for a cost effective method for both known and unknown metabolite production.