Varied pharmacological responses have been reported for mitragynine in the literature, but no supportive scientific explanations have been given for this. These studies have been undertaken without a sufficient understanding of the physicochemical properties of mitragynine. In this work a UV spectrophotometer approach and HPLC-UV method were employed to ascertain the physicochemical properties of mitragynine. The pKa of mitragynine measured by conventional UV (8.11 ± 0.11) was in agreement with the microplate reader determination (8.08 ± 0.04). Mitragynine is a lipophilic alkaloid, as indicated by a logP value of 1.73. Mitragynine had poor solubility in water and basic media, and conversely in acidic environments, but it is acid labile. In an in vitro dissolution the total drug release was higher for the simulated gastric fluid but was prolonged and incomplete for the simulated intestinal fluid. The hydrophobicity, poor water solubility, high variability of drug release in simulated biological fluids and acid degradable characteristics of mitragynine probably explain the large variability of its pharmacological responses reported in the literature. The determined physicochemical properties of mitragynine will provide a basis for developing a suitable formulation to further improve its solubility, stability and oral absorption for better assessment of this compound in preclinical studies.
Matched MeSH terms: Chromatography, High Pressure Liquid/methods
The colour pigments of five chili powders of different origins were separated and quantified by reversed-phase high-performance liquid chromatography (RP-HPLC). The similarities and dissimilarities of pigment composition of chili powders were elucidated by principal component analysis (PCA). RP-HPLC separated 50-100 pigment fractions depending on the detection wavelength and on the origin of chili powder. It was found that the pigment composition of chili powders from Malaysia and China and from India and Pakistan show marked similarities while the composition of colour pigments of chili powder from Thailand was different. It was further established that the chromatograms are similar in the first 5-35 min of development, they are highly different between 35 and 75 min and moderately different at the end of the chromatograms. It was concluded that RP-HPLC followed by PCA can be successfully used for the identification of chili powders according to the composition of their colour pigments.
Matched MeSH terms: Chromatography, High Pressure Liquid/methods*
For application to the analysis of aflatoxins (AF) in commercial peanut and corn products, the ISOLUTE multimode column (IMC, solid phase multifunctional column) method was validated by comparing with the modified Florisil column (MFC) method. Twenty-two peanut and eight corn products from Malaysia and the Philippines were analysed for AFB1, AFB2, AFG1 and AFG2 firstly by the MFC method and then by the IMC method. For peanut products, 14 out of 22 samples were positive by the two methods in the range of 1-378 micrograms/kg of AF, and correlation coefficients (r) for AFB1 and AFB2 were 0.987 and 0.997, respectively. For corn and corn products, all the samples were positive in the range of 1-130 micrograms/kg, and r values were 0.992 and 0.805 for AFB1 and AFB2 respectively. Thus, the results were significantly (p < 0.01) in close agreement, particularly for lower range of 1-50 micrograms/kg of AF concentrations in all the samples. For the occurrence of AF, 11 (65%) of peanut products from Malaysia were contaminated with AF at a mean level of 50 micrograms/kg (maximum 180 micrograms/kg) and two (40% products from the Philippines were contaminated with as high as 375 micrograms/kg and 177 micrograms/kg of AF, respectively. All the corn products from the Philippines were contaminated with AF at a mean level of 44 micrograms/kg (maximum 130 micrograms/kg). Contamination of commercial foods with high levels of AF is a very important issue to both the countries since these foods are very popular among children.
Matched MeSH terms: Chromatography, High Pressure Liquid/methods
A rapid and direct liquid chromatographic (LC) technique is described for the determination of a eutectic mixture of diphenyl oxide and biphenyl such as Dowtherm A thermal heating fluid (THF) in oleochemicals and palm olein. Analysis is performed with an RP-18 column with fluorescence detection (excitation at 247 nm and emission at 310 nm). The isocratic mobile phase (1.0 mL/min) consists of methanol and water (90 + 10, v/v). A linear calibration model (correlation coefficient = 0.9999) was developed directly from used Dowtherm A THF with the biphenyl peak (4.70 min) as a marker. Average recoveries from spiked glycerin, fatty alcohol mixture, methyl ester mixture, fatty acids, and palm olein were 90.9-108.7%, with a detection limit of 0.1 microgram/mL. The technique requires no prior sample cleanup nor extraction steps and is good for quality assurance purposes.
1. The L-amino acid oxidase of the monocellate cobra (Naja naja kaouthia) venom was purified to electrophoretic homogeneity. The molecular weight of the enzyme was 112,200 as determined by Sephadex G-200 gel filtration chromatography, and 57,400 as determined by SDS-polyacrylamide gel electrophoresis. 2. The enzyme had an isoelectric point of 8.12 and a pH optimum of 8.5. It showed remarkable thermal stability, and, unlike many venom L-amino acid oxidase, was also stable in alkaline medium. The enzyme was partially inactivated by freezing. 3. The enzyme was very active against L-phenylalanine and L-tyrosine, moderately active against L-tryptophan, L-methionine, L-leucine, L-norleucine, L-arginine and L-norvaline. Other L-amino acids were oxidized slowly or not oxidized. 4. Kinetic studies suggest the presence of a side-chain binding site in the enzyme, and that the binding site comprises of at least four hydrophobic subsites.
Matched MeSH terms: Chromatography, Gel; Chromatography, Ion Exchange
Using the rapid gas chromatographic steroid profiling technique, a number of metabolites of pregnenolone have been separated and quantified after incubation of this steroid with adult rat and neonatal porcine testicular homogenates. It was shown that the 5-ene-3 beta-hydroxy- and the 4-en-3-oxosteroid pathways for androgen biosynthesis were operating in both species, although the former pathway appeared to be more important in porcine testis. This tissue was characterised by the formation of several odorous, and pheromonal, 16-androstenes, which were quantitatively more important than the androgens. Three non-steroidal anti-inflammatory drugs (NSAIDS) caused dose-related inhibition of androgen and 16-androstene biosynthesis when co-incubated with pregnenolone. The order of potency was flurbiprofen > indomethacin > > > aspirin. The possibility that the NSAIDS may interfere with cytochrome P-450 is discussed, since several steroid-transforming enzymes, known to be dependent on this cytochrome for their activity, were markedly inhibited.
The fruit extracts of ripening cv. Harumanis mango contained a number of glycosidases and glycanases. Among the glycosidases, beta-D-galactosidase (EC 3.2.1.23) appeared to be the most significant. The enzyme activity increased in parallel with increase in tissue softness during ripening. Mango beta-galactosidase was fractionated into three isoforms, viz. beta-galactosidase I, II and III by a combination of chromatographic procedures on DEAE-Sepharose CL-6B, CM-Sepharose and Sephacryl S-200 columns. Apparent Km values for the respective beta-galactosidase isoforms for p-nitrophenyl beta-D-galactoside were 3.7, 3.3 and 2.7 mM, and their Vmax values were 209, 1024 and 62 nkat mg-1 protein. Optimum activity occurred at ca pH 3.2 for beta-galactosidase I and II, and pH 3.6 for beta-galactosidase III. Mango beta-galactosidase and its isoforms have galactanase activity, and the activity of the latter in the crude extracts generally increased during ripening. The close correlation between changes in beta-galactosidase activity, tissue softness, and increased pectin solubility and degradation suggests that beta-galactosidase might play an important role in cell wall pectin modification and softening of mango fruit during ripening.
Matched MeSH terms: Chromatography, Gel; Chromatography, Ion Exchange
An acidic, lethal phospholipase Az was purified to electrophoretic homogeneity from the venom of the Malayan cobra (Naja naja sputatrix). The enzyme has an isoelectric point of 5.58, a molecular weight of 12000, and a medium lethal dose (LD50) of 0.86 micrograms/g in mice by intravenous injection. The enzyme also exhibited weak anticoagulant and edema-forming activities. The amino acid composition of the enzyme is similar to those of other cobra venom phospholipases Az.
Matched MeSH terms: Chromatography, Gel; Chromatography, Ion Exchange
The abuse of phenylbutazone among rheumatoid arthritis patients has recently become a subject of interest. Unscrupulous manufacturers take advantage of the miraculous analgesic property of phenylbutazone and deliberately add this toxic drug in their preparations without declaring its presence on the label. In a recent survey, many such illicit preparations were seized from Chinese medical halls in Johor and sent to the Department of Chemistry, Johor Bahru for analysis. Here a Gas Chromatograph Mass Selective Detector (GC-MSD) method was developed for the determination of phenylbutazone in illicit traditional preparations.
Matched MeSH terms: Gas Chromatography-Mass Spectrometry/methods*
A high-performance liquid chromatographic method was developed to enable dapsone, monoacetyl dapsone and pyrimethamine to be measured simultaneously in plasma samples from volunteers in England and Malaysia who had been dosed with Maloprim. Mean half-lives of 25 and 80 h were calculated for dapsone and pyrimethamine, respectively, but there was wide individual variation. All subjects were found to be classifiable as "slow acetylators".
Matched MeSH terms: Chromatography, High Pressure Liquid/methods*
Venoms of cobras (Naja spp.) contain high abundances of cytotoxins, which contribute to tissue necrosis in cobra envenomation. The tissue-necrotizing activity of cobra cytotoxins, nevertheless, indicates anticancer potentials. This study set to explore the anticancer properties of the venoms and cytotoxins from Naja sumatrana (equatorial spitting cobra) and Naja kaouthia (monocled cobra), two highly venomous species in Southeast Asia. The cytotoxicity, selectivity, and cell death mechanisms of their venoms and cytotoxins (NS-CTX from N. sumatrana: NS-CTX; N. kaouthia: NK-CTX) were elucidated in human lung (A549), prostate (PC-3), and breast (MCF-7) cancer cell lines. Cytotoxins were purified through a sequential fractionation approach using cation-exchange chromatography, followed by C18 reverse-phase high-performance liquid chromatography (HPLC) to homogeneity validated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and identified by liquid chromatography-tandem mass spectrometry (LCMS/MS). The cobra venoms and their respective cytotoxins exhibited concentration-dependent growth inhibitory effects in all cell lines tested, with the cytotoxins being more potent compared to the corresponding whole venoms. NS-CTX and NK-CTX are, respectively, P-type and S-type isoforms of cytotoxin, based on the amino acid sequences as per LCMS/MS analysis. Both cytotoxins exhibited differential cytotoxic effects in the cell lines tested, with NS-CTX (P-type cytotoxin) being significantly more potent in inhibiting the growth of the cancer cells. Both cytotoxins demonstrated promising selectivity only for the A549 lung cancer cell line (selectivity index = 2.17 and 2.26, respectively) but not in prostate (PC-3) and breast (MCF-7) cancer cell lines (selectivity index < 1). Flow cytometry revealed that the A549 lung cancer cells treated with NS-CTX and NK-CTX underwent necrosis predominantly. Meanwhile, the cytotoxins induced mainly caspase-independent late apoptosis in the prostate (PC-3) and breast (MCF-7) cancer cells lines but lacked selectivity. The findings revealed the limitations and challenges that could be faced during the development of new cancer therapy from cobra cytotoxins, notwithstanding their potent anticancer effects. Further studies should aim to overcome these impediments to unleash the anticancer potentials of the cytotoxins.
Matched MeSH terms: Chromatography, High Pressure Liquid; Chromatography, Reverse-Phase
This paper presents the successful application of ultrasound-assisted packed-bed (UAE-PB) method for the extraction of hypericin from the Hypericum perfuratum L. The Soxhlet system was utilized for the determination of suitable solvent from ethanol, methanol or from the mixture of different proportions of ethanol-methanol. The mixture of 50:50 v/v ethanol-methanol was obtained to be the most suitable solvent since it led to the highest extraction amount of hypericin. The extraction amount of hypericin increased by 13.6% and 21.4% when the solvent changed from pure methanol to the mixture of 50:50 v/v ethanol-methanol for the extraction time of 3 and 8 h, respectively. Subsequently, the extraction was conducted through the UAE-PB, and the effects of temperature, time, and the ratio of solvent to the dried plant were studied. The response surface method (RSM) was used to investigate the effect of parameters on the extraction in the UAE-PB system. At the temperature of 60 °C, extraction time of 105 min, and the solvent to plant ratio of 15.3, the maximum extraction yield of hypericin was achieved. In the optimal conditions, the amount of extraction was 0.112 mg hypericin/g dried plant, which was in accordance with the optimized predicted value (0.111 mg hypericin/g dried plant) from Design-Expert software.
Matched MeSH terms: Chromatography, High Pressure Liquid/methods
As a widely consumed beverage, coffee tends to be a target for intentional adulteration. This study describes the application of modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) coupled to liquid chromatography-high-resolution mass spectrometry (LC-HRMS) for simultaneous screening, identification, and quantification of undeclared phosphodiesterase 5 (PDE5) inhibitors in instant coffee premixes (ICPs). The mass spectrometer was operated in auto MS/MS acquisition for simultaneous MS and MS/MS experiments. Qualitative establishments from the suspected-target screening and targeted identification processes led to an unambiguous analyte assignment from the protonated molecule ([M+H]+) precursor ion which is subsequently used for quantification of 23 targeted PDE5 inhibitors. The analytical method validation covered specificity, linearity, range, accuracy, limit of detection (LOD), limit of quantification (LOQ), precisions, matrix effect (ME), and extraction recovery (RE). The specificity was established using the optimised chromatographic separation as well as the distinguishable [M+H]+ precursor ion. The linearity of each target analyte was demonstrated with a coefficient of determination (r2) of >0.9960 over the expected range of sample concentrations. The accuracy ranged from 88.1%-119.3% with LOD and LOQ of <70 ng/mL and 80 ng/mL, respectively. Excellent precisions were established within 0.4%-9.1% of the relative standard deviation. An insignificant ME within -5.2% to +8.7% was achieved using three different strategies of chromatography, sample extraction, and sample dilution. The RE was good for all target analytes within 84.7%-123.5% except for N-desethylacetildenafil at low (53.8%) and medium (65.1%) quality control levels. The method was successfully applied to 25 samples of ICPs where 17 of them were found to be adulterated with PDE5 inhibitors and their analogues. Further quantification revealed the total amount of these adulterants ranged from 2.77 to 121.64 mg per sachet.
In the present work, supercritical fluid extraction (SFE) with CO2 as solvent and EtOH/water (v/v) as co-solvent was optimised by applying 23 factorial experimental design for the extraction of betacyanins from red pitaya fruit (Hylocereus polyrhizus) peel. Three independent variables of pressure (20-30 MPa), temperature (40-60°C) and co-solvent concentration (10-20%) were chosen for response variables. With the 2 mL/min flow rate of CO2, the dynamic time of extraction was found to be 90 min. The linear effects of main factors and interactions were evaluated. The calculated response surface model for the pressure/temperature was found to be significant for all the dependent variables. At optimal condition of SFE, the response variables were assessed as maximum extraction yield of 4.09 ± 0.69%, total betacyanins content of 25.49 ± 1.54 mg/100 mL, redness (a*) of 58.18 ± 0.82, and IC50 (antioxidant activity) of 1.34 ± 0.12 mg/mL for the experimental peel extracts. The optimal levels of independent variables were validated for the experimental responses as predicted by the mathematical model. The reliability of this method was confirmed as there was no significant difference between experimental and predicted values. The HPLC-MS profile of betacyanins extract comprised of both acylated and non-acylated betacyanins constituents.
Matched MeSH terms: Chromatography, High Pressure Liquid; Chromatography, Supercritical Fluid
The cholesterol-lowering properties of 12 lactic acid bacteria (LAB) in the absence or presence of 0.3% bile salts were assessed and compared quantitatively and qualitatively in vitro. A new, more sensitive and cost-effective high-performance thin-layer chromatography method combined with digital image evaluation of derivatised chromatographic plates was developed and validated to quantify cholesterol in LAB culture media. The performance of the method was compared with that of the o-phthalaldehyde method. For qualitative assessment, assimilated fluorescently tagged cholesterol was visualised by confocal microscopy. All LAB strains exhibited a cholesterol-lowering effect of various degrees (19-59% in the absence and 14-69% in the presence of bile salts). Lactobacillus plantarum LAB12 and Pentosaceus pentosaceus LAB6 were the two best strains of lactobacilli and pediococci. They lowered cholesterol levels by 59% and 54%, respectively, in the absence and by 69% and 58%, respectively, in the presence of bile salts. Confocal microscopy showed that cholesterol was localised at the outermost cell membranes of LAB12 and LAB6. The present findings warrant in-depth in vivo study. Graphical abstract (A) 3D plots based on scan at 525 nm of (B) derivatized HPTLC plate of separated cholesterol and (C) confocal microscopic image showing the localisation of NBD-cholesterol assimilated by LAB.
Different extraction processes were employed to extract bioactive metabolites from Salacca zalacca flesh by a range of aqueous and organic solvents. The highest extraction yield was obtained by 50% ethanol extract of SE (73.18 ± 4.35%), whereas SFE_1 showed the lowest yield (0.42 ± 0.08%). All extracts were evaluated for in vitro α-glucosidase inhibitory activity, measured by their IC50 values in comparison to that of quercetin, the positive control (IC50 = 2.7 ± 0.7 μg/mL). The lowest α-glucosidase inhibitory activity was indicated by water extract of SE (IC50 = 724.3 ± 42.9 μg/mL) and the highest activity was demonstrated by 60% ethanol extract by UAE (IC50 = 16.2 ± 2.4 μg/mL). All extracts were analysed by GC-MS and identified metabolites like carbohydrates, fatty acids, organic acids, phenolic acids, sterols and alkane-based compounds etcetera that may possess the potential as α-glucosidase inhibitor and may attribute to the α-glucosidase inhibitory activity.
Matched MeSH terms: Gas Chromatography-Mass Spectrometry; Chromatography, Supercritical Fluid
Nine derivatives of three natural diarylheptanoids, curcumin, demethoxycurcumin and bisdemethoxycurcumin, were prepared. Their antioxidant, free radical scavenging, nitric oxide (NO) inhibitory and cytotoxic activities were evaluated and compared with those of the respective natural compounds. Curcumin (1), demethoxycurcumin (2), demethyldemethoxy-curcumin (C3), diacetyldemethoxycurcumin (AC2) and triacetyldemethylcurcumin (AC5) exhibited higher antioxidant activity than quercetin while products from demethylation of 1 and 2 exhibited higher free radical scavenging activity. Compounds AC2 and AC5 were found to be most active in inhibiting breast cancer cells (MCF-7) proliferation with IC50 values of 6.7 and 3.6 microM, respectively. The activity of AC2 is almost doubled and of AC5 almost tripled as compared to curcumin. Their selectivity towards different cell lines is also more noticeable. Compounds AC2 and AC5 also showed increased activity against a human prostate cancer cell line (DU-145) and non-small lung cancer cell line (NCI-H460) with IC50 values of 20.4, 16.3 and 18.3, 10.7 microM, respectively.
The volatile compounds of pork, other meats and meat products were studied using an electronic nose and gas chromatography mass spectrometer with headspace analyzer (GCMS-HS) for halal verification. The zNose™ was successfully employed for identification and differentiation of pork and pork sausages from beef, mutton and chicken meats and sausages which were achieved using a visual odor pattern called VaporPrint™, derived from the frequency of the surface acoustic wave (SAW) detector of the electronic nose. GCMS-HS was employed to separate and analyze the headspace gasses from samples into peaks corresponding to individual compounds for the purpose of identification. Principal component analysis (PCA) was applied for data interpretation. Analysis by PCA was able to cluster and discriminate pork from other types of meats and sausages. It was shown that PCA could provide a good separation of the samples with 67% of the total variance accounted by PC1.
Matched MeSH terms: Gas Chromatography-Mass Spectrometry/methods*
Kratom (Mitragyna speciosa) is a psychoactive plant popular in the United States for the self-treatment of pain and opioid addiction. For standardization and quality control of raw and commercial kratom products, an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the quantification of ten key alkaloids, namely: corynantheidine, corynoxine, corynoxine B, 7-hydroxymitragynine, isocorynantheidine, mitragynine, mitraphylline, paynantheine, speciociliatine, and speciogynine. Chromatographic separation of diastereomers, or alkaloids sharing same ion transitions, was achieved on an Acquity BEH C18 column with a gradient elution using a mobile phase containing acetonitrile and aqueous ammonium acetate buffer (10mM, pH 3.5). The developed method was linear over a concentration range of 1-200 ng/mL for each alkaloid. The total analysis time per sample was 22.5 minutes. The analytical method was validated for accuracy, precision, robustness, and stability. After successful validation, the method was applied for the quantification of kratom alkaloids in alkaloid-rich fractions, ethanolic extracts, lyophilized teas, and commercial products. Mitragynine (0.7%-38.7% w/w), paynantheine (0.3%-12.8% w/w), speciociliatine (0.4%-12.3% w/w), and speciogynine (0.1%-5.3% w/w) were the major alkaloids in the analyzed kratom products/extracts. Minor kratom alkaloids (corynantheidine, corynoxine, corynoxine B, 7-hydroxymitragynine, isocorynantheidine) were also quantified (0.01%-2.8% w/w) in the analyzed products; however mitraphylline was below the lower limit of quantification in all analyses.
Matched MeSH terms: Chromatography, High Pressure Liquid/methods
Adsorption of lysozyme on the dye-affinity nanofiber membranes was investigated in batch and dynamic modes. The membrane matrix was made of electrospun polyacrylonitrile nanofibers that were grafted with ethylene diamine (EDA) and/or chitosan (CS) for the coupling of Reactive Blue 49 dye. The physicochemical properties of these dye-immobilized nanofiber membranes (P-EDA-Dye and P-CS-Dye) were characterized microscopically, spectroscopically and thermogravimetrically. The capacities of lysozyme adsorption by the dye-affinity nanofiber membranes were evaluated under various conditions, namely pH, dye immobilized density, and loading flow rate. The adsorption of lysozyme to the dye-affinity nanofiber membranes was well fitted by Langmuir isotherm and pseudo-second kinetic models. P-CS-Dye nanofiber membrane had a better performance in the dynamic adsorption of lysozyme from complex chicken egg white solution. It was observed that after five cycles of adsorption-desorption, the dye-affinity nanofiber membrane did not show a significant loss in its capacity for lysozyme adsorption. The robustness as well as high dynamic adsorption capability of P-CS-Dye nanofiber membrane are promising for the efficient recovery of lysozyme from complex feedstock via nanofiber membrane chromatography.