A study was carried out to compare the composition and thermal profiles of the fat component of six brands of commercial biscuits (BA, BB, BC, BD, BE & BF) with those of lard and palm oil. Extraction of fat from biscuit samples was done using petroleum ether according to the soxhlet extraction procedure. The isolated fat samples along with lard and palm oil were analyzed using gas liquid chromatography (GLC), reversed-phase high performance liquid chromatography (RP-HPLC), and differential scanning calorimetry (DSC). According to GLC analysis, palm oil, lard and all six biscuit brands had either palmitic or oleic acid as major fatty acids. Sn-2 positional analysis of fatty acids showed that oleic (> 60%) as the most dominant fatty acid of palm oil and biscuit brands BA, BB, BC, and BD while palmitic (> 60%) as the most dominant fatty acid of lard and biscuit brands BE and BF. RP-HPLC analysis showed that the triacylglycerol (TAG) profiles of lard and biscuit brands BE and BF were closely similar while those of brands BA, BB, BC, and BD and palm oil were similar. DSC analysis showed that the cooling and heating profiles of lard and brands BE and BF were similar, while those of palm oil and brands BA, BB, BC, and BD were similar. Hence, this study concluded that biscuit brands BE and BF are not suitable for consumers whose religious restriction prohibit the use of lard as food ingredient.
A sensitive and rapid reversed-phase ultra performance liquid chromatographic (UPLC) method for the simultaneous determination of tocopherols (α-, β-, γ-, δ-), tocotrienols (α-, β-, γ-, δ-), α-tocopherol acetate and α-tocopherol nicotinate is described. The separation was achieved using a Kinetex pentafluorophenyl (PFP) column (150 × 2.1mm, 2.6 µm) with both photodiode array (PDA) and fluorescence (FL) detectors that were connected in series. Column was thermostated at 42°C. Under a gradient system consisting of methanol and water at a constant flow rate of 0.38 mL min(-1), all the ten analytes were well separated in less than 9.5 min. The method was validated in terms of linearity, limits of detection and quantitation, precision and recoveries. Calibration curves of the ten compounds were well correlated (r(2)>0.999) within the range of 100 to 25,000 μg L(-1) for α-tocopherol acetate and α-tocopherol nicotinate, 10 to 25,000 μg L(-1) for α-tocotrienol and 5 to 25,000 μg L(-1) for the other components. The method is simple and sensitive with detection limits (S/N, 3) of 1.0 to 3.0 μg L(-1) (FL detection) and 30 to 74 μg L(-1) (PDA detection). Relative standard deviations for intra- and inter-day retention times (<1%) and peak areas (≤ 4%) were obtained. The method was successfully applied to the determination of vitamin E in vegetable oils (extra virgin olive, virgin olive, pomace olive, blended virgin and refined olive, sunflower, soybean, palm olein, carotino, crude palm, walnut, rice bran and grape seed), margarines and supplements.
Pleurotus pulmonarius has been reported to have a potent remedial effect on diabetic property and considered to be an alternative for type 2 diabetes mellitus treatment. This study aimed to investigate the antidiabetic properties of ammonium sulphate precipitated protein fractions from P. pulmonarius basidiocarps. Preliminary results demonstrated that 30% (NH4)2SO4 precipitated fraction (F30) inhibited Saccharomyces cerevisiae α-glucosidase activity (24.18%), and 100% (NH4)2SO4 precipitated fraction (F100) inhibited porcine pancreatic α-amylase activity (41.80%). Following RP-HPLC purification, peak 3 from F30 fraction demonstrated inhibition towards α-glucosidase at the same time with meagre inhibition towards α-amylase activity. Characterisation of proteins using MALDI-TOF/TOF MS demonstrated the presence of four different proteins, which could be implicated in the regulation of blood glucose level via various mechanisms. Therefore, this study revealed the presence of four antidiabetic-related proteins which are profilin-like protein, glyceraldehyde-3-phosphate dehydrogenase-like protein, trehalose phosphorylase-like (TP-like) protein, and catalase-like protein. Hence, P. pulmonarius basidiocarps have high potential in lowering blood glucose level, reducing insulin resistance and vascular complications.
A thrombin-like enzyme, purpurase, was purified from the Cryptelytrops purpureomaculatus (mangrove pit viper) venom using high performance ion-exchange and gel filtration chromatography. The purified sample (termed purpurase) yielded a homogeneous band in SDS-polyacrylamide gel electrophoresis with a molecular weight of 35,000. The N-terminal sequence of purpurase was determined to be VVGGDECNINDHRSLVRIF and is homologous to many other venom thrombin-like enzymes. Purpurase exhibits both arginine ester hydrolase and amidase activities. Kinetic studies using tripeptide chromogenic anilide substrates showed that purpurase is not fastidious towards its substrate. The clotting times of fibrinogen by purpurase were concentration dependent, with optimum clotting activity at 3mg fibronogen/mL. The clotting activity by purpurase was in the following decreasing order: cat fibrinogen>human fibrinogen>dog fibrinogen>goat fibrinogen>rabbit fibrinogen. Reversed-phase HPLC analysis of the products of action of purpurase on bovine fibrinogen showed that only fibrinopeptide A was released. Indirect ELISA studies showed that anti-purpurase cross-reacted strongly with venoms of most crotalid venoms, indicating the snake venom thrombin-like enzymes generally possess similar epitopes. In the more specific double-sandwich ELISA, however, anti-purpurase cross-reacted only with venoms of certain species of the Trimeresurus complex, and the results support the recent proposed taxonomy changes concerning the Trimeresurus complex.
We developed a simple and sensitive method for the simultaneous detection of imatinib mesylate (IM) and its active metabolite, N-desmethyl imatinib (M1), in human serum samples. Separation was successfully achieved using an Agilent(®) ZORBAX Eclipse plus C(18) reversed phase column (50 mm × 2.1 mm, i.d.; 1.8 μm) under isocratic mobile phase conditions consisting of acetonitrile: 0.02 M potassium dihydrogen phosphate with 0.2% triethylamine at pH 3 (25:75, v/v) and ultra-violet detection was achieved at 235 nm. Extraction of the target compounds was completed using 100% cold acetonitrile. Good linearities (r(2)>0.99) for both IM and M1 were achieved for the concentration ranges of 50-1800 ng/mL and 50-360 ng/mL, respectively. The detection limits were 20 ng/mL and 10 ng/mL for M1 and IM, respectively. The intra- and inter-day precisions were less than 1% with percent recoveries of more than 90%. The method was successfully applied to calculate the pharmacokinetic parameters of chronic myeloid leukemia patients receiving imatinib. The method is suitable to be routinely applied for determination of IM and M1 in serum.
Tropidolaemus wagleri (temple pit viper) is a medically important snake in Southeast Asia. It displays distinct sexual dimorphism and prey specificity, however its venomics and inter-sex venom variation have not been thoroughly investigated. Applying reverse-phase HPLC, we demonstrated that the venom profiles were not significantly affected by sex and geographical locality (Peninsular Malaya, insular Penang, insular Sumatra) of the snakes. Essentially, venoms of both sexes share comparable intravenous median lethal dose (LD50) (0.56-0.63 μg/g) and cause neurotoxic envenomation in mice. LCMS/MS identified six waglerin forms as the predominant lethal principles, comprising 38.2% of total venom proteins. Fourteen other toxin-protein families identified include phospholipase A2, serine proteinase, snaclec and metalloproteinase. In mice, HPLC fractions containing these proteins showed insignificant contribution to the overall venom lethality. Besides, the unique elution pattern of approximately 34.5% of non-lethal, low molecular mass proteins (3-5 kDa) on HPLC could be potential biomarker for this primitive crotalid species. Together, the study unveiled the venom proteome of T. wagleri that is atypical among many pit vipers as it comprises abundant neurotoxic peptides (waglerins) but little hemotoxic proteinases. The findings also revealed that the venom is relatively well conserved intraspecifically despite the drastic morphological differences between sexes.
Snake venoms are complex mixtures of proteins and peptides that play vital roles in the survival of venomous snakes. As with their diverse pharmacological activities, snake venoms can be highly variable, hence the importance of understanding the compositional details of different snake venoms. However, profiling venom protein mixtures is challenging, in particular when dealing with the diversity of protein subtypes and their abundances. Here we described an optimized strategy combining a protein decomplexation method with in-solution trypsin digestion and mass spectrometry of snake venom proteins. The approach involves the integrated use of C18 reverse-phase high-performance liquid chromatography (RP-HPLC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and nano-electrospray ionization tandem mass spectrometry (nano-ESI-LC-MS/MS).
The palm oil industry generates several byproducts, and more than half of the dry weight of the waste is of oil palm leaf whereby the tissue is underutilized. Recently, several research studies found promising potential of oil palm fronds as a source of nutraceutical due to its bioactive properties. However, the chemical composition of the tissue is still not deciphered. Using reversed-phase liquid chromatography (LC) electrospray mass spectrometry (ESI-MS), glycosylated apigenin and luteolin were separated and identified from oil palm (Elaeis guineensis Jacq.) leaf and structures of the constituents were elucidated by collision-induced dissociation (CID) tandem MS. From 28 derivatives of the flavones, 9 compounds were conjugated with hydroxymethylglutaric (HMG) acid. Improved knowledge on oil palm especially on bioactive component of the leaf tissue will allow correlation of its beneficial effects and further promotes efficient utilization of this agriculture byproduct.
The absence of chromophore and/or conjugated system, prerequisite for UV and florescent light detection, or absorbance at very low wavelength necessitates the development of simple and reliable methods for the determination of amikacin sulphate. Therefore, the present study describes for the first time dynamics of the drug derivatization using ninhydrin reagent and development and validation of a simple RP-HPLC method, using diode array detector (DAD). The variables such as heating time, heating type, drug-reagent ratio, reagent composition and storage temperature of the derivative were optimized. The analyte and aqueous ninhydrin solution upon heating for 2.00-5.00 min produced the colored drug-derivative which was stable for one month at refrigeration. The derivatized drug (20.00μL) was eluted through a column - Eclipse DB-C18 (5.00 µm, 4.60×150.00 mm), maintained at 25°C- using isocratic mobile phase comprising water and acetonitrile (70:30, v/v) at a flow rate of 1.00 mL/min, and detected at 400 nm. The method was found to be reliable (98.08-100.72% recovery), repeatable (98.02-100.72% intraday accuracy) and reproducible (98.47-101.27% inter day accuracy) with relative standard deviation less than 5%. The results of the present study indicate that the method is easy to perform, specific and sensitive, and suitable to be used for the determination of amikacin sulphate in bulk and pharmaceutical preparations using less expensive/laborious derivatization.
The present study examined the contents of tocopherols and tocotrienols and their distribution in 58 different varieties of whole rice cultivated in Malaysia. The analytical method used was saponification of samples followed by dispersive liquid-liquid microextraction and reverse phase high-performance liquid chromatography.
A simple sample preparation technique coupled with reversed-phase high-performance liquid chromatography was developed for the determination of tocopherols and tocotrienols in cereals. The sample preparation procedure involved a small-scale hydrolysis of 0.5g cereal sample by saponification, followed by the extraction and concentration of tocopherols and tocotrienols from saponified extract using dispersive liquid-liquid microextraction (DLLME). Parameters affecting the DLLME performance were optimized to achieve the highest extraction efficiency and the performance of the developed DLLME method was evaluated. Good linearity was observed over the range assayed (0.031-4.0μg/mL) with regression coefficients greater than 0.9989 for all tocopherols and tocotrienols. Limits of detection and enrichment factors ranged from 0.01 to 0.11μg/mL and 50 to 73, respectively. Intra- and inter-day precision were lower than 8.9% and the recoveries were around 85.5-116.6% for all tocopherols and tocotrienols. The developed DLLME method was successfully applied to cereals: rice, barley, oat, wheat, corn and millet. This new sample preparation approach represents an inexpensive, rapid, simple and precise sample cleanup and concentration method for the determination of tocopherols and tocotrienols in cereals.
A rapid reversed-phase high performance liquid chromatographic method using a monolithic column for the determination of eight catechin monomers and caffeine was developed. Using a mobile phase of water:acetonitrile:methanol (83:6:11) at a flow rate of 1.4 mL min(-1), the catechins and caffeine were isocratically separated in about 7 min. The limits of detection and quantification were in the range of 0.11-0.29 and 0.33-0.87 mg L(-1), respectively. Satisfactory recoveries were obtained (94.2-105.2 ± 1.8%) for all samples when spiked at three concentrations (5, 40 and 70 mg L(-1)). In combination with microwave-assisted extraction (MAE), the method was applied to the determination of the catechins and caffeine in eleven tea samples (6 green, 3 black and 2 oolong teas). Relatively high levels of caffeine were found in black tea, but higher levels of the catechins, especially epigallocatechin gallate (EGCG) were found in green teas.
Globally, peptide-based anticancer therapies have drawn much attention. Marine organisms are a reservoir of anticancer peptides that await discovery. In this study, we aimed to identify cytotoxic oligopeptides from Sarcophyton glaucum. Peptides were purified from among the S. glaucum hydrolysates produced by alcalase, chymotrypsin, papain, and trypsin, guided by a methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay on the human cervical cancer (HeLa) cell line for cytotoxicity evaluation. Purification techniques adopted were membrane ultrafiltration, gel filtration chromatography, solid phase extraction (SPE), and reversed-phase high-performance liquid chromatography (RP-HPLC). Purified peptides were identified by de novo peptide sequencing. From papain hydrolysate, three peptide sequences were identified: AGAPGG, AERQ, and RDTQ (428.45, 502.53, and 518.53 Da, respectively). Peptides synthesized from these sequences exhibited cytotoxicity on HeLa cells with median effect concentration (EC50) values of 8.6, 4.9, and 5.6 mmol/L, respectively, up to 5.8-fold stronger than the anticancer drug 5-fluorouracil. When tested at their respective EC50, AGAPGG, AERQ, and RDTQ showed only 16%, 25%, and 11% cytotoxicity to non-cancerous Hek293 cells, respectively. In conclusion, AERQ, AGAPGG, and RDTQ are promising candidates for future development as peptide-based anticancer drugs.
In this study, a simple and reliable reverse-phase high-performance liquid chromatography (RP-HPLC) method was established and validated to analyze S-mephenytoin 4-hydroxylase activity of a recombinant CYP2C19 system. This system was obtained by co-expressing CYP2C19 and NADPH-CYP oxidoreductase (OxR) proteins in Escherichia coli (E. coli) cells. In addition to RP-HPLC, the expressed proteins were evaluated by immunoblotting and reduced CO difference spectral scanning. The RP-HPLC assay showed good linearity (r(2) = 1.00) with 4-hydroxymephenytoin concentration from 0.100 to 50.0 μm and the limit of detection was 5.00 × 10(-2) μm. Intraday and interday precisions determined were from 1.90 to 8.19% and from 2.20 to 14.9%, respectively. Recovery and accuracy of the assay were from 83.5 to 85.8% and from 95.0 to 105%. Enzyme kinetic parameters (Km , Vmax and Ki ) were comparable to reported values. The presence of CYP2C19 in bacterial membranes was confirmed by immunoblotting and the characteristic absorbance peak at 450 nm was determined in the reduced CO difference spectral assay. Moreover, the activity level of co-expressed OxR was found to be comparable to that of the literature. As a conclusion, the procedures described here have generated catalytically active CYP2C19 and the RP-HPLC assay developed is able to serve as CYP2C19 activity marker for pharmacokinetic drug interaction study in vitro.
A cloud point extraction (CPE) process using non-ionic surfactant (DC193C) to extract selected paraben compounds from water samples was investigated using reversed phase high performance liquid chromatography (RP-HPLC). The CPE process with the presence of β-cyclodextrin (βCD) functionalized ionic liquid as a modifier (CPE-DC193C-βCD-IL) is a new extraction technique that has been applied on the optimization of parameters, i.e., pH, βCD-IL concentration and phase volume ratio. This CPE-DC193C-βCD-IL method is facilitated at 30 °C, showing great losses of water content in the surfactant-rich phase, resulting in a high pre-concentration factor and high distribution coefficient. The developed method CPE-DC193C-βCD-IL did show enhanced properties compared to the CPE method without the modifier (CPE-DC193C). The developed method of CPE-DC193C-βCD-IL gives an excellent performance on the detection of parabens from water samples with the limit of detection falling in the range of 0.013-0.038 µg mL-1. Finally, the inclusion complex formation, hydrogen bonding, and π-π interaction between the βCD-IL, benzyl paraben (ArP), and DC 193C were proven using 1H NMR and 2D NOESY spectroscopy.
The seeds of C. cleomifolia (locally known as kacang hantu) collected along Simpang Pulai - Berinchang Road, Cameron Highlands, was defatted with hexane and the resulting oil was analysed for their physico-chemical properties. The percentage yield of the oil was calculated as 5.3%. The acid value (1.2%), iodine value (85), peroxide value (0.6), saponification value (192.0) and unsaponifiable matter (2.3%) were determined to assess the quality of the oil. The physico-chemical characterisation showed that C. cleomifolia seeds oil is unsaturated semi-drying oil, with high saponifi cation and acidic values. The fatty acid composition of C. cleomifolia seed oil was determined by Gas Chromatography and Gas Chromatography-Mass Spectrometry (ToF). The seed oil of C. cleomifolia contained linoleic acid (57.59%) and palmitic acid (5.07%), the most abundant unsaturated and saturated fatty acids, respectively. The polyunsaturated triacylglycerol (TAG) in C. cleomifolia seed oil determined by reverse phase High performance Liquid Chromatography; contained as PLL (18.04%) followed by POL + SLL (11.92%), OOL (7.04%) and PLLn (6.31%). The melting and cooling point of the oil were 16.22°C and -33.54°C, respectively
This work describes a RP-HPLC method for the determination and interaction studies of cefpirome with ACE-inhibitors (captopril, enalapril and lisinopril) in various buffers. The separation and interaction of cefpirome with ACE-inhibitors was achieved on a Purospher Star, C18 (5 μm, 250×4.6 mm) column. Mobile phase consisted of methanol: water (80:20, v/v, pH 3.3); however, for the separation of lisinopril, it was modified to methanol-water (40:60, v/v, pH 3.3) and pumped at a flow rate of 1 mL min(-1). In all cases, UV detection was performed at 225 nm. Interactions were carried out in physiological pH i.e., pH 1 (simulated gastric juice), 4 (simulated full stomach), 7.4 (blood pH) and 9 (simulated GI), drug contents were analyzed by reverse phase high performance liquid chromatography. Method was found linear in the concentration range of 1.0-50.0 μg mL(-1) with correlation coefficient (r(2)) of 0.999. Precision (RSD%) was less than 2.0%, indicating good precision of the method and accuracy was 98.0-100.0%. Furthermore, cefpirome-ACE-inhibitors' complexes were also synthesized and results were elucidated on the basis of FT-IR, and (1)H NMR. The interaction results show that these interactions are pH dependent and for the co-administration of cefpirome and ACE-inhibitors, a proper interval should be given.
Sinigrin, a precursor of allyl isothiocyanate, present in the Raphanus sativus exhibits diverse biological activities, and has an immense role against cancer proliferation. Therefore, the objective of this study was to quantify the sinigrin in the R. sativus roots using developed and validated RP-HPLC method and further evaluated its' anticancer activity. To achieve the objective, the roots of R. sativus were lyophilized to obtain a stable powder, which were extracted and passed through an ion-exchange column to obtain sinigrin-rich fraction. The RP-HPLC method using C18 analytical column was used for chromatographic separation and quantification of sinigrin in the prepared fraction, which was attained using the mobile phase consisting of 20 mM tetrabutylammonium: acetonitrile (80:20%, v/v at pH 7.0) at a flow rate of 0.5 mL/min. The chromatographic peak for sinigrin was showed at 3.592 min for pure sinigrin, where a good linearity was achieved within the concentration range of 50 to 800 µg/mL (R2 > 0.99), with an excellent accuracy (-1.37% and -1.29%) and precision (1.43% and 0.94%), for intra and inter-day, respectively. Finally, the MTT assay was performed for the sinigrin-rich fraction using three different human cancer cell lines, viz. prostate cancer (DU-145), colon adenocarcinoma (HCT-15), and melanoma (A-375). The cell-based assays were extended to conduct apoptotic and caspase-3 activities, to determine the mechanism of action of sinigrin in the treatment of cancer. MTT assay showed IC50 values of 15.88, 21.42, and 24.58 µg/mL for DU-145, HCT-15, and A-375 cell lines, respectively. Increased cellular apoptosis and caspase-3 expression were observed with sinigrin-rich fraction, indicating significant increase in overexpression of caspase-3 in DU-145 cells. In conclusion, a simple, sensitive, fast, and accurate RP-HPLC method was developed for the estimation of sinigrin in the prepared fraction. The data observed here indicate that sinigrin can be beneficial in treating prostate cancer possibly by inducing apoptosis.
The effect of different drying methods on the degradation of flavonoids in Centella asiatica was evaluated. C. asiatica leaf, root and petiole were dried using air-oven, vacuum oven and freeze drier. Flavonoid was determined utilizing reverse-phase high performance liquid chromatography (RP-HPLC). Results of the study revealed the presence of high concentration of flavonoids in C. asiatica leaf, root and petiole, which include, naringin (4688.8 ± 69 μg/100 g, 3561.3 ± 205 μg/ 100 g, and 978.3 ± 96 μg/ 100 g), rutin (905.6 ± 123 μg/ 100 g, 756.07 ± 95 μg/ 100 g, and 557.25 ± 58 μg/ 100 g), quercetin (3501.1 ± 107 μg/ 100 g, 1086.31 ± 90 μg/ 100 g, and 947.63 ± 83 μg/ 100 g) and catechin (915.87 ± 6.01 μg/ 100 g, 400.6 ± 67 μg/ 100 g, and 250.56 ± 18 μg/ 100g). Luteolin, kaempferol and apigenin on the other hand, were inconsistently present in some parts of C. asiatica. Air-oven treatment resulted in the highest total flavonoids degradation followed by vacuum oven and freeze dried with percent degradation of 97%, 87.6% and 73%, respectively. Catechin and rutin were found to be the most stable flavonoids with percent degradation up to 35%, 66% and 76% for freeze dried, vacuum oven and air oven, respectively.
This study was conducted to determine radical scavenging activity and vitamin antioxidant composition in red pitaya from organic plantation. For antioxidant vitamins analysis, a reverse-phase high performance liquid chromatography was used and radical scavenging activity of methanolic and water extract were determined using 1,1-diphenyl-2-pircrylhydrazyl assay. Results for radical scavenging activity, red pitaya methanolic extract achieved the highest percentage 70.13% compared with water extract (47.13%). Antioxidant vitamins composition in red pitaya showed that the concentration of vitamin A is 120.13 ± 0.69 μg/100 g freeze-dried sample, vitamin C is 540.27 ± 0.59 μg/100 g fresh samples and vitamin E is 105.67 ± 0.56 μg/100 g freeze-dried samples. This shows that red pitaya may become an alternative and potential source of natural antioxidant.