Protective Effect of Flavonoids from Mulberry Leaf on AAPH-Induced Oxidative Damage in Sheep Erythrocytes.

To evaluate the antioxidant activity of flavonoids extracted from Chinese herb mulberry leaves (ML), flavonoids from mulberry leaves (FML) were extracted and purified by using ultrasonic-assisted enzymatic extraction and D101 macroporous resin. Using LC-MS/MS-Liquid Chromatography with tandem mass spectrometry analysis, hesperidin, rutoside, hyperoside, cyanidin-3-o-glucoside, myricitrin, cyanidin, and quercetin were identified, and NMR and UV were consistent with the verification of IR flavonoid characteristics. The antioxidant activity of FML has also been evaluated as well as the protective effect on 2,2 0-azobis (2-amidinopropane) dihydrochloride (AAPH)-induced oxidative stress. The results showed that FML exhibited powerful antioxidant activity. Moreover, FML showed dose-dependent protection against AAPH-induced sheep erythrocytes' oxidative hemolysis. In the enzymatic antioxidant system, pretreatment with high FML maintained the balance of SOD, CAT, and GSH-Px; in the non-enzymatic antioxidant system, the content of MDA can be effectively reduced after FML treatment. This study provides a research basis for the development of natural products from mulberry leaves.

[Effect of Shelf Life on the Physical Stability of Suspended Ophthalmic Solutions].

Quality changes associated with physical changes in suspended eye drops are difficult to predict. In this study, we attempted to evaluate the aggregation and redispersability in commercially available suspended eye drops (fluorometholone ophthalmic solutions). The 0.1% fluorometholone ophthalmic solutions (the original product and 4 generic products) were gently mixed by hand after short-term (4 months) or long-term (40 months) storage, and the drug concentration in the first drop and physical stability (redispersability and particle size) were measured. All eye drops produced a cloudy precipitate on the bottom surface of the container, and the amount of precipitate decreased with mixing time. The drug concentration per drop in the original product was approximately 70% of the labeled value after mixing 10 times, and the drug particle size was approximately 4 µm. After mixing the generic products stored short-term 10 times, the concentration ranged from less than 50% to almost 100%. In addition, some generic products after long-term storage had a reduced redispersion ability and labeled concentration. These results suggested that at least 10 mixing were required before the using of fluorometholone original product. In addition, some generic products may not provide sufficient drug exposure even when mixed in the same manner as the original products.

Dose Uniformity of Topical Corticosteroids: A Simulated Trial of Fluorometholone Acetate 0.1% and Loteprednol Etabonate Gel 0.5.

PURPOSE: The purpose of the study was to determine the concentrations of Flarex® and Lotemax® when shaken and not shaken. Many patients fail to shake or inappropriately shake suspensions of corticosteroids before instillation as directed. This study was designed to help determine what concentration of corticosteroid these patients are receiving. In addition, independent confirmation of loteprednol etabonate ophthalmic gel dose uniformity was determined and compared as a possible alternative. METHODS: Drug concentrations of shaken versus unshaken Flarex and Lotemax were determined over a 20-day simulated tapered course in our institutional laboratory. Collected samples were analyzed by reversed-phase high-performance liquid chromatography with photodiode array detection at 240 nm. RESULTS: Flarex had a mean concentration of 93.7% of the declared concentration when shaken and 7.25% when not shaken. The difference between these groups was statistically significant (P = 0.0001). Lotemax had a mean concentration of 96.74% of the declared concentration when shaken and a mean concentration of 98.97% when not shaken. The difference between these groups was not statistically significant (P = 0.194). CONCLUSIONS: Flarex maintains dose uniformity when shaken. When not shaken, it has poor dose uniformity. Lotemax was consistent whether shaken or not in our study and can be considered to eliminate the variability of poor patient compliance with shaking. The manufacturers of both drugs recommend shaking before application.

Stability-Indicating RP-HPLC Methods for the Determination of Fluorometholone in Its Mixtures with Sodium Cromoglycate and Tetrahydrozoline Hydrochloride.

Two stability-indicating reversed-phase liquid chromatographic methods were developed and validated for the determination of fluorometholone (FLU) in its mixtures with sodium cromoglycate (SCG) and tetrahydrozoline hydrochloride (THZ). The first HPLC method (Method 1) was based on isocratic elution of FLU and SCG along with their alkaline degradation products on a reversed phase C18 column (250 × 4.6 mm id)-ACE Generix 5, using a mobile phase consisting of methanol-water (70 : 30, v/v), pH adjusted to 2.5 using orthophosphoric acid at a flow rate of 1.2 mL min(-1) Quantitation was achieved with UV detection at 240 nm. The second HPLC method (Method 2) was based on isocratic elution of FLU, its alkaline degradation product and THZ on a reversed phase C8 column (250 × 4.6 mm)-ACE Generix 5, using a mobile phase consisting of acetonitrile-50 mM potassium dihydrogen orthophosphate (40 : 60, v/v) at a flow rate of 2 mL min(-1) Quantitation was achieved by applying dual-wavelength detection, where FLU and its alkaline degradation product were detected at 240 nm and THZ was detected at 215 nm at ambient temperatures. Linearity, accuracy and precision were found to be acceptable over the concentration range of 5-50 and 10-500 µg mL(-1) for FLU and SCG (Method 1) and over the concentration range of 5-80 and 5-60 µg mL(-1) for FLU and THZ (Method 2), respectively. Besides, the FLU alkaline degradation product was verified using IR, NMR and LC-MS spectroscopy. The two proposed methods could be successfully applied for the routine analysis of the studied drugs either in their pure bulk powders or in their pharmaceutical preparations without any preliminary separation step.

[Fluorometholone Reference Standard (control 011) of National Institute of Health Sciences].

The raw material of fluorometholone was examined for the preparation of the "Fluorometholone Reference Standard (Control 011)". The analytical data obtained were: optical rotation, [alpha]20D = .5 degrees; UV spectrum, lambda max of 240 nm and specific absorbance in methanol at 240 nm = 350.7; IR spectrum, same as that of the Fluorometholone Reference Standard (Control 864); high-performance liquid chromatography (HPLC), total amount of impurities estimated to be less than 0.5%; loss on drying, 0.01%. Based on the above results, the raw material was authorized as the Fluorometholone Reference Standard (Control 011) of the National Institute of Health Sciences.

Determination of tetrahydrozoline hydrochloride and fluorometholone in pharmaceutical formulations by HPLC and derivative UV spectrophotometry.

Two methods for the quantitative determination of tetrahydrozoline hydrochloride (1) and fluorometholone (2) in pharmaceutical eye drops (Efemoline) are described. The procedures are based on derivative UV spectrophotometry and HPLC. In the former method, d2A/d lambda 2 values were measured in methanol at 226 and 282 nm for 1 and 2, respectively. The relative standard deviations for the method were found to be 1.06% for 1 and 0.98% for 2. The latter method based on a reversed phase HPLC system using a Partisil 5 ODS analytical column. The mobile phase used for the separation of 1, 2 and internal standard (lidocaine) was methanol/acetonitrile/water (50:50:10 v/v) and the compounds in the eye drops were detected at 220 nm using an UV detector. The relative standard deviations for the HPLC method were determined to be 0.61% and 0.50% for 1 and 2, respectively. The proposed methods, which give thoroughly comparable data, are simple, rapid, and allow precise and accurate results and could be used for commercial formulations containing tetrahydrozoline hydrochloride and fluorometholone in combination.

Mapping of intracellular halogenous molecules by low and high resolution SIMS microscopy.

The subcellular distribution of halogenous molecules has been studied by SIMS microscopy in cultured cells of a human breast carcinoma (MCF-7 cell line). Two instruments of microanalysis were used. A low lateral resolution ion microscope (SMI 300 CAMECA) and a prototype scanning ion microscope equipped with a cesium gun that gives high lateral resolution images. This apparatus has been developed by G Slodzian, in Onera Laboratories (Office National d'Etudes et de Recherches Aérospatiales). Molecules studied by low lateral resolution ion microscope were halogenous steroids: fluorometholone, triamcinolone, bromocriptine and bromoandrosterone. Analytical images show that the first two compounds are mainly localized in the nuclear structure of MCF-7 cells whereas the last two molecules are localized in cytoplasm of these cells. Images were obtained with a resolution of 1 micron. With the scanning ion microscope, it is now possible to obtain images at the ultrastructural level. Four analytical images can be simultaneously obtained by a single scan of the imaged area, corresponding to a depth of erosion of the section of ten nm. The intranuclear distributions of three pyrimidine analogs, 5-bromo-2'-deoxyuridine, 5-iodo-2'-deoxyuridine and 5-fluorouracil have been studied in phase S and M of MCF-7 cells and these images have been compared to the distribution of sulfur, nitrogen and phosphorus. All these images have been obtained with a lateral resolution better than 100 nm.

Laboratory automation of high-pressure liquid chromatography.

An automated system for high-pressure liquid chromatography was developed. The system is built around commercial modules wherever possible, modified to varying degrees. An automatic sampler, a sample pump, a high-pressure sampling valve, a recorder with an integrator, and a high-pressure liquid chromatograph comprise the commercial instruments. Relays, solenoid valves, and timers control chromatographic events, i.e., duration of sampling and rinse, mobile phase pump refill, sample injection, and chromatographic time. The automated system is dependable over long periods of unattended operation. With the 40-sample capacity of the sample tray and the last sample stop capability, the automated system produces, for example, 40 20-min chromatograms in approximately 13 hr of unattended operation. Data demonstrate the reliability and utility of the system.