Automated Chloroform-Methanol Protein Extraction on the Biomek-FX Liquid Handler System v2

This protocol details steps to extract protein from Gram-negative bacterial or fungal cells (that have been pretreated with zymolyase) in quantitative proteomic workflows by using a Biomek FX liquid handler system. It is a semi-automated protocol that includes several 'pause' steps for centrifugation steps that are conducted manually "off-deck". This protocol works best as part of an automated proteomic sample preparation workflow with: Automated Protein Quantitation with the Biomek-FX liquid handler system and Automated Protein Normalization and Tryptic Digestion on a Biomek-NX Liquid Handler System

Automated Protein Normalization and Tryptic Digestion on a Biomek-NX Liquid Handler System v2

This protocol details steps to normalize the amount of protein for tryptic digestion in quantitative proteomic workflows by using a Biomek NX liquid handler system. It is optimized to normalize protein concentrations in a 96-well plate format and add TCEP, IAA, and trypsin. This protocol works best as part of a semi-automated proteomic sample preparation workflow with: Automated Chloroform-Methanol Protein Extraction on the Biomek-FX Liquid Handler System and Automated Protein Quantitation with the Biomek-FX liquid handler system

Automated Protein Quantification with the Biomek-FX Liquid Handler System v2

This protocol details steps to perform the protein quantification (Lowry-based) assay by using a Biomek FX liquid handler system. It is optimized to assay a full 96-well plate of protein samples in duplicate with a separate (control) plate for BSA standards. You will need a plate reader to measure the samples and standards. This protocol works best as part of a full proteomic sample preparation workflow with: Automated Chloroform-Methanol Protein Extraction on the Biomek-FX Liquid Handler System and Automated Protein Normalization and Tryptic Digestion on a Biomek-FX Liquid Handler System

Application of Central Composite Design for screening and Optimization of HPTLC method for simultaneous quantitation of Aprepitant, Dexamethasone and Ondansetron in their synthetic mixtures

New HPTLC method was developed and optimized for estimation of Ondansetron (OND), Dexamethasone (DEX) and Aprepitant (APT) in laboratory prepared ternary mixtures by using Central composite design (CCD). The independent variables used for the optimization were the acetone content in mobile phase (%mL), distance of developing solvent (cm) and saturation time (min). HPTLC Separation was performed on Precoated silica gel F254 aluminum plate (10X10 cm, 100μm thickness) with a mobile phase consisting of chloroform: methanol: acetone: ethyl acetate: ammonia (9:4:2:5:0.2 % v/v/v/v). Quantification of OND, APT and DEX were achieved based on a Densitometric analysis over the concentration range of 200-1200 ng/band, 500-1000 ng/band and 1000-2000 ng/band, respectively, at 254nm. The method was yielded dense and well-resolved bands at Rf values of 0.54± 0.02, 0.79±0.02 and 0.23±0.01 for OND, APT and DEX, respectively. The linear regression analysis for the calibration plots produced r2= 0.9997, r2= 0.9998 and r2=0.9997 for OND, APT and DEX, respectively. The method was validated according to the ICH guidelines. The robustness test was determined that the selected factors have an insignificant effect on the responses. The results indicated that the method is suitable for the routine quality control testing of OND, APT and DEX in their bulk form.

Assessment of Fatty Acid-Specific Lipolysis by In Vitro Digestion and GC-FID

The nutritional relevance of food compositional data could be improved by taking the bioaccessibility of these constituents into account. A lack of routine methods to assess the bioaccessibility of fatty acids (FAs) in food is one of the limiting factors of doing so. An analytical protocol is proposed for routine assessment of the extent of lipolysis via in vitro digestion simulation methods in food products. The established method provides specific information on each FA individually. Steps of the protocol including the Bligh and Dyer chloroform/methanol/water extraction of esterified and free FAs from in vitro digesta, methyl ester derivatization, and GC-FID analysis were specifically tailored to help routine work and were harmonized with the Infogest in vitro digestion simulation protocol (both v1.0 and v2.0). The method was applied to assess the degree of FA-specific lipolysis in a baked fish (carp) meal and the results showed that the FA composition of the original food significantly differed from that of the distribution of FFAs in the digesta. The use of gastric lipase (in Infogest v2.0 protocol) increased total FA release by 9.5% and its specific impact on palmitic acid was the most prominent.

Rapid, Highly-Sensitive and Ecologically Greener Reversed-Phase/Normal-Phase HPTLC Technique with Univariate Calibration for the Determination of Trans-Resveratrol

The rapid, highly-sensitive and ecologically greener reversed-phase (RP)/normal-phase (NP) high-performance thin-layer chromatography (HPTLC) densitometric technique has been developed and validated for the determination of trans-resveratrol (TRV). The reversed-phase HPTLC-based analysis of TRV was performed using ethanol–water (65:35, v v−1) combination as the greener mobile phase, while, the normal-phase HPTLC-based estimation of TRV was performed using chloroform–methanol (85:15, v v−1) combination as the routine mobile phase. The TRV detection was carried out at 302 nm for RP/NP densitometric assay. The linearity was recorded as 10–1200 and 30–400 ng band−1 for RP and NP HPTLC techniques, respectively. The RP densitometric assay was observed as highly-sensitive, accurate, precise and robust for TRV detection in comparison with the NP densitometric assay. The contents of TRV in commercial formulation were recorded as 101.21% utilizing the RP densitometric assay, while, the contents of TRV in commercial formulation were found to be 91.64% utilizing the NP densitometric assay. The greener profile of RP/NP technique was obtained using the analytical GREEnness (AGREE) approach. The AGREE scales for RP and NP densitometric assays were estimated 0.75 and 0.48, respectively. The recorded AGREE scale for the RP densitometric assay indicated that this technique was highly green/the ecologically greener compared to the NP densitometric assay. After successful optimization of analytical conditions, validation parameters, AGREE scale and chromatography performance, the RP densitometric assay with univariate calibration was found to be better than the NP densitometric assay for the analysis of TRV.

Optimization of a novel lipid extraction process from microalgae

Previous study found that the solvent extraction efficiency of lipid in microalgae could be greatly improved by washing algae cells before the second time extraction. Based on the "organic solvents–water–organic solvents" method, this research further studied the effect of four solvent systems (acetone, chloroform/methanol, chloroform/methanol/water, dichloromethane/methanol), two types of water treatment (vortex and ultrasonic), three water treatment time gradient (0 s, 30 s, 120 s) on the lipid extraction at three different microalgae growth stages (3rd day, 5th day, 9th day). The results show that the combination of water treatment type, treatment time and solvent is very important to the efficiency of lipid extraction. The total lipid extracted was generally increased by 10–30% after water treatment. Especially under the condition of 120 s vortex water treatment with dichloromethane/methanol as extraction solvent, the total lipid extracted increased by 61.14%. In addition, microalgae cells at different culture stages had different sensitivity to water treatment. In this study, under the combination of chloroform/methanol/water as extraction solvent and vortex water treatment for 120 s, the highest lipid yield was obtained on the ninth day of cell culture, which accounts 47.88% of the cell dry weight (478 mg/g cell dry weight). The changes of cell morphology and structure after water treatment were studied by scanning electron microscope, and it was found that water treatment could seriously destroy the cell membrane damaged by solvent, thus promoting the release of lipids. This study further optimizes the "solvent–water–solvent" lipid extraction method, which neither produces impurities nor damages the lipid quality, and can reduce the amount of organic solvent applied in the classical lipid extraction method with the same lipid yield, so it has a broad application prospect.

THE MOLLUSCICIDAL EFFECT OF THE STEM EXTRACTS OF Tinospora crispa IN CONTROLLING THE GOLDEN APPLE SNAIL Pomacea canaliculata

This study investigated the molluscicidal effect of the stem extracts of Tinospora crispa in controlling the golden apple snail Pomacea canaliculata. Extracts were prepared in four solvents, i.e., hexane, chloroform, methanol, and distilled water at three concentrations (1,000, 5,000, and 10,000 ppm) per solvent. The phytochemical contents of the extracts were qualitatively identified, and the lethal concentration (LC50) of the extracts for mollusicicidal potential was determined using the probit analysis. The effect of T. crispa extracts on the snail was monitored for three days and the snail mortality was recorded every 24 h. The stem extract prepared in methanol at 10,000 ppm showed the highest molluscicidal effect with a mortality of 80% at 72 h. Phytochemicals identified in the stem extracts included alkaloids, flavonoids, saponin, tannin, and terpenoids. Based on the probit analysis, stem extracts of T. crispa prepared in methanol showed the lowest LC50 value of 3,428 ppm for mollusicicidal potential and followed by extracts prepared in chloroform, hexane, and distilled water at 5,888, 14,771, and 14,993 ppm, respectively.

Two Validated Chromatographic Methods for Determination of Ciprofloxacin HCl, One of its Specified Impurities and Fluocinolone Acetonide in Newly Approved Otic Solution

Two sensitive, selective and precise chromatographic methods have been established for concomitant quantification of ciprofloxacin HCl (CIP), fluocinolone acetonide (FLU) along with ciprofloxacin impurity A (CIP-imp A). The first method was thin-layer chromatography (TLC-densitometry) where separation was accomplished using TLC silica plates 60 G.F254 as a stationary phase and chloroform–methanol–33%ammonia (4.6:4.4:1, by volume) as a developing system. The obtained plates were scanned at 260 nm over concentration ranges of 1.0–40.0, 0.6–20.0 and 1.0–40.0 μg band−1 for CIP, FLU and CIP-imp A, respectively. The second method was based on high-performance liquid chromatography using a Zorbax ODS column (5 μm, 150 × 4.6 mm i.d.) where adequate separation was achieved through a mobile phase composed of phosphate buffer pH 3.6–acetonitrile (45:55, v/v) at flow rate 1.0 mL min−1 with ultraviolet detection at 254 nm. Linear regressions were obtained in the range of 1.0–40.0 μg mL−1 for CIP, 0.6–20.0 μg mL−1 for FLU and 1.0–40.0 μg mL−1 for CIP-imp A. The suggested methods were validated in compliance with the International Conference on Harmonization guidelines and were successfully applied for determination of CIP and FLU in bulk powder and newly marketed otic solution.

Isolation and Characterization of Flavonoids of the Ethanolic extracts of stems of Mimosa hamata (Willd.) by Chromatographic techniques

Since there are many bioactive compounds present in plant material containing various multi-component mixtures, their separation and determination is important to identify the active phytoconstituents responsible for pharmacological activity. Practically most of them have to be separated by column chromatographic techniques. The present study deals with the identification and characterization of bioactive principles from the stems of Mimosa hamata. The isolated fractions from the ethanolic extracts of stems of Mimosa hamata was carried out by column chromatography. For separation of a bioactive compound, the solvent system tried for column chromatography was Chloroform: Methanol in various ratios like 90:10, 80:20, 70:30, and 60:40 amongst these we could separate the first fraction at 80:20 and second fraction at 70:30. Two flavonoids compounds were isolated from the ethanolic extracts of stems of the medicinal plant Mimosa hamata (Willd). Based on chemical and spectral analyses their structures were elucidated as Quercetin and Cirsimaritin. From the above study it reveals the presence of flavonoids in the ethanolic extracts of stems of Mimosa hamata (Willd.) was isolated using column chromatography further subjected to characterization of isolated compounds including UV, IR, NMR and Mass spectroscopic study for elucidating the structure of the two separated compounds. The interpreted data concluded that, both isolated compounds are flavonoids i.e. Cirsimaritin and Quercetin. The pharmacological effect of Mimosa hamata stems may be due to the presence of its phytoconstituents flavonoids.