Continuing our interest in the Uncaria genus, the phytochemistry and the in-vitro α-glucosidase inhibitory activities of Malaysian Uncaria cordata var. ferruginea were investigated. The phytochemical study of this plant, which employed various chromatographic techniques including recycling preparative HPLC, led to the isolation of ten compounds with diverse structures comprising three phenolic acids, two coumarins, three flavonoids, a terpene and an iridoid glycoside. These constituents were identified as 2-hydroxybenzoic acid or salicylic acid (1), 2,4-dihydroxybenzoic acid (2), 3,4-dihydroxybenzoic acid (3), scopoletin or 7-hydroxy-6-methoxy-coumarin (4), 3,4-dihydroxy-7-methoxycoumarin (5), quercetin (6), kaempferol (7), taxifolin (8), loganin (9) and β-sitosterol (10). Structure elucidation of the compounds was accomplished with the aid of 1D and 2D Nuclear Magnetic Resonance (NMR) spectral data and Ultraviolet-Visible (UV-Vis), Fourier Transform Infrared (FTIR) spectroscopy and mass spectrometry (MS). In the α-glucosidase inhibitory assay, the crude methanolic extract of the stems of the plant and its acetone fraction exhibited strong α-glucosidase inhibition activity of 87.7% and 89.2%, respectively, while its DCM fraction exhibited only moderate inhibition (75.3%) at a concentration of 1 mg/mL. The IC50 values of both fractions were found to be significantly lower than the standard acarbose suggesting the presence of potential α-glucosidase inhibitors. Selected compounds isolated from the active fractions were then subjected to α-glucosidase assay in which 2,4-dihydroxybenzoic acid and quercetin showed strong inhibitory effects against the enzyme with IC50 values of 549 and 556 μg/mL compared to acarbose (IC50 580 μg/mL) while loganin and scopoletin only showed weak α-glucosidase inhibition of 44.9% and 34.5%, respectively. This is the first report of the isolation of 2-hydroxybenzoic acid, 2,4-dihydroxybenzoic acid and loganin from the genus and the first report of the α-glucosidase inhibitory potential of 2,4-dihydroxybenzoic acid.
Matched MeSH terms: Chromatography, High Pressure Liquid
In the present study, a series of 2-benzoyl-6-benzylidenecyclohexanone analogs have been synthesized and evaluated for their anti-cholinesterase activity. Among the forty-one analogs, four compounds (38, 39, 40 and 41) have been identified as lead compounds due to their highest inhibition on both AChE and BChE activities. Compounds 39 and 40 in particular exhibited highest inhibition on both AChE and BChE with IC50 values of 1.6μM and 0.6μM, respectively. Further structure-activity relationship study suggested that presence of a long-chain heterocyclic in one of the rings played a critical role in the dual enzymes' inhibition. The Lineweaver-Burk plots and docking results suggest that both compounds could simultaneously bind to the PAS and CAS regions of the enzyme. ADMET analysis further confirmed the therapeutic potential of both compounds based upon their high BBB-penetrating. Thus, 2-benzoyl-6-benzylidenecyclohexanone containing long-chain heterocyclic amine analogs represent a new class of cholinesterase inhibitor, which deserve further investigation for their development into therapeutic agents for cognitive diseases such as Alzheimer.
Matched MeSH terms: Gas Chromatography-Mass Spectrometry
In this paper, we investigated the antibacterial mechanism and potential therapeutic targets of three antibacterial medicinal plants. Upon treatment with the plant extracts, bacterial proteins were extracted and resolved using denaturing gel electrophoresis. Differentially-expressed bacterial proteins were excised from the gels and subjected to sequence analysis by MALDI TOF-TOF mass spectrometry. From our study, seven differentially expressed bacterial proteins (triacylglycerol lipase, N-acetylmuramoyl-L-alanine amidase, flagellin, outer membrane protein A, stringent starvation protein A, 30S ribosomal protein s1 and 60 kDa chaperonin) were identified. Additionally, scanning electron microscope study indicated morphological damages induced on bacterial cell surfaces. To the best of our knowledge, this represents the first time these bacterial proteins are being reported, following treatments with the antibacterial plant extracts. Further studies in this direction could lead to the detailed understanding of their inhibition mechanism and discovery of target-specific antibacterial agents.
Matched MeSH terms: Gas Chromatography-Mass Spectrometry
Effective management of patients with diabetic foot infection is a crucial concern. A delay in prescribing appropriate antimicrobial agent can lead to amputation or life threatening complications. Thus, this electronic nose (e-nose) technique will provide a diagnostic tool that will allow for rapid and accurate identification of a pathogen.
Matched MeSH terms: Gas Chromatography-Mass Spectrometry
BACKGROUND: A simple and sensitive hollow fiber-liquid phase microextraction with in situ derivatization method was developed for the determination of α-ketoglutaric (α-KG) and pyruvic acids (PA) in small-volume urine samples. 2,4,6-trichloro phenyl hydrazine was used as derivatization agent.
RESULTS: Under the optimum extraction conditions, enrichment factors of 742 and 400 for α-KG and PA, respectively, were achieved. Calibration curves were linear over the range 1 to 1000 ng/ml (r(2) ≥ 0.998). Detection and quantitation limits were 0.03 and 0.02, and 0.10 and 0.05 ng/ml for α-KG and PA, respectively.
CONCLUSION: The concentrations in diabetic II and liver cancer samples were significantly lower than those from healthy people, showing their potential as biomarkers for these diseases.
Matched MeSH terms: Chromatography, High Pressure Liquid
Our group has previously reported the isolation, partial characterisation, and application of a Galbeta1-3GalNAc- and IgA1-reactive lectin from the seeds of champedak (Artocarpus integer). In the present study, we have subjected the purified lectin to reverse-phase high performance liquid chromatography and sequenced its subunits. Determination of the N-terminal sequence of the first 47 residues of the large subunit demonstrated at least 95% homology to the N-terminal sequence of the alpha chains of a few other galactose-binding Artocarpus lectins. The two smaller subunits of the lectin, each comprised of 21 amino acid residues, demonstrated minor sequence variability. Their sequences were generally comparable to the beta chains of the other galactose-binding Artocarpus lectins. When used to probe human serum glycopeptides that were separated by two-dimensional gel electrophoresis, the lectin demonstrated strong apparent interactions with glycopeptides of IgA1, hemopexin, alpha2-HS glycoprotein, alpha1-antichymotrypsin, and a few unknown glycoproteins. Immobilisation of the lectin to Sepharose generated an affinity column that may be used to isolate the O-glycosylated serum glycoproteins.
Matched MeSH terms: Chromatography, High Pressure Liquid
A chromatographic immunoassay cholera antigen detection kit, the Cholera Spot test, was evaluated. The test was found to be specific with a sensitivity of 10(6) cfu/ml for the direct detection of V. cholerae in simulated stool specimens and 10 cfu/ml in simulated cotton-tipped swab specimens after overnight incubation in alkaline peptone water. This enables early recognition of cholera cases and their contacts so that prevention and control measures can be promptly instituted.
The plant Typhonium flagelliforme (Araceae), commonly known as the 'rodent tuber', is often included as an essential ingredient in various herbal remedies recommended for cancer therapies in Malaysia. Various extracts prepared from either the roots, tubers, stems or leaves were tested for cytotoxic activity on murine P388 leukaemia cells using the MTT assay method. Both the chloroform (IC50 = 6.0 microg/mL) and hexane (IC50 = 15.0 microg/mL) extract from the 'roots and tubers' exhibited weak cytotoxic activity. The hexane extract (IC50 = 65.0 microg/mL) from the 'stems and leaves' exhibited weaker cytotoxic activity than the chloroform extract (IC50 = 8.0 microg/mL). Although the juice extract from the 'roots and tubers' is frequently consumed for cancer treatment, it exhibited poor cytotoxic activity. Further analysis using an amino acid analyser revealed that the juice extract contained a high concentration of arginine (0.874%). A high tryptophan content (0.800%) was confirmed by NMR and HPLC analysis.
Matched MeSH terms: Chromatography, High Pressure Liquid
Nine 3,4-secoapotirucallanes, argentinic acids A-I, were isolated from the bark of Aglaia argentea and transformed to their methyl esters 1-9. The structures were determined by spectral and chemical means. Compounds 1-8 showed moderate cytotoxic activity against KB cells (IC50 1.0-3.5 microg/mL).
The L-amino acid oxidase of Malayan pit viper (Calloselasma rhodostoma) venom was purified to electrophoretic homogeneity. The molecular weight of the enzyme was 132,000 as determined by Sephadex G-200 gel filtration chromatography and 66,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is a glycoprotein, has an isoelectric point of 4.4, and contains 2 mol of flavin mononucleotide per mole of enzyme. The N-terminal amino acid sequence of the enzyme was A-D-D-R-N-P-L-A-E-E-F-Q-E-N-N-Y-E-E-F-L. Kinetic studies suggest the presence of a alkyl side-chain binding site in the enzyme and that the binding site comprises at least four hydrophobic subsites. The characteristics of the binding site differ slightly from those of cobra venom L-amino acid oxidases.
Toxin production of a Malaysian isolate of the toxic red tide dinoflagellate Pyrodinium bahamense var. compressum was investigated at various stages of the batch culture growth cycle and under growth conditions affected by temperature, salinity, and light intensity variations. In all the experiments conducted, only 5 toxins were ever detected. Neosaxitoxin (NEO) and gonyautoxin V (GTX5) made up 80 mole percent or more of the cellular toxin content and saxitoxin (STX), GTX6 and decarbamoylsaxitoxin (dcSTX) made up the remainder. No gonyautoxins I-IV or C toxins were ever detected. In nutrient-replete batch cultures, toxin content rapidly peaked during early exponential phase and just as rapidly declined prior to the onset of plateau phase. Temperature had a marked effect on toxin content, which increased 3-fold as the temperature decreased from the optimum of 28 degrees C to 22 degrees C. Toxin content was constant at salinities of 24% or higher, but increased 3-fold at 20%. Toxin content decreased 2-fold and chlorophyll content increased 3-fold when light intensity was reduced from 90 to 15 microE m-2 s-1. This accompanied a 30% decrease in growth rate. Toxin composition (mole % individual toxin cell-1) remained constant throughout the course of the nutrient-replete culture and during growth at various salinities, but varied significantly with temperature and light intensity changes. At 22 degrees C, GTX5 was 25 mole % and NEO was 65 mole %, while at 34 degrees C, GTX5 increased to 55 mole % and NEO decreased proportionally to 40 mole %. When light intensity was reduced from 90 to 15 microE m-2 s-1, NEO decreased from 55 to 38 mole %, while GTX5 increased from 40 to 58 mole %. These data suggest that low light and high temperature both somehow enhance sulfo-transferase activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Matched MeSH terms: Chromatography, High Pressure Liquid
1. The two major phospholipase A2 enzymes (OHPLA-DE1 and OHPLA-DE2) of king cobra (Ophiophagus hannah) venom have been purified to electrophoretic homogeneity. 2. The isoelectric points of OHPLA-DE1 and OHPLA-DE2 were 3.81 and 3.89, respectively and the Mws were 14,000 and 15,000, respectively, as estimated by Sephadex G-75 gel filtration chromatography; and 14,000 as estimated by SDS-PAGE. 3. The enzymes were not lethal to mice at a dosage of 10 micrograms/g body wt by i.v. route. Both phospholipase A2 enzymes, however, exhibited moderate edema-inducing and anti-coagulant activities. 4. Bromophenacylation of the enzymes reduced the enzymatic activity drastically but did not affect the edema-inducing activity of the enzymes.
1. The hemorrhagic, procoagulant, anticoagulant, phosphodiesterase, hyaluronidase, alkaline phosphomonoesterase, 5'-nucleotidase, arginine ester hydrolase, phospholipase A, L-amino acid oxidase and protease activities of 26 samples of venoms of 13 taxa of Vipera were determined and the Sephadex G-75 gel filtration patterns for some of the venoms were also examined. 2. The results indicate the presence of certain common characteristics among the venoms, particularly if V. russelli is excluded from the comparison. The results also support the recently proposed reassignment of V. russelli to a separate genus. 3. The data show that information on venom biological properties can be used for differentiation of venoms of many species of Vipera. Particularly useful for this purpose are the protease, phosphodiesterase, phospholipase A and the procoagulant activities and the Sephadex G-75 gel filtration patterns of the venoms.
Capillary gas chromatographic 'steroid profiling' has been utilised to separate and quantify the metabolites (derivatized as methyloximes and/or trimethylsilyl ethers) formed from pregnenolone after incubation with rat testicular microsomes. A wide range of steroid metabolites was found, indicating that both the 5-ene and 4-ene pathways of testosterone biosynthesis were operating, as well as 16 alpha-hydroxylation, 20 beta-reduction and the formation of several C19 steroids (the 16-androstenes). At the concentration used, Metyrapone markedly inhibited 16 alpha- and 17-hydroxylation and side-chain cleavage of 17-hydroxylated C21 steroids. 16-Androstene production was also markedly inhibited and the formation of other metabolites was affected to lesser extents. Oxytocin abolished the formation of all C21 and C19 metabolites of pregnenolone.
Twenty authentic steroids, derivatized as O-methyl oximes (MO), trimethylsilyl (TMS) ethers or as MO-TMS ethers have been subjected to capillary gas chromatography using two different columns. Virtually all of the steroid derivatives have been resolved, one difficult pair to separate being 5,16-androstadien-3 beta-ol and 5 alpha-androst-16-en-3 beta-ol on the non-selective phase OV-1. Where syn and anti forms of MO derivatives arose, these were also resolved under the conditions utilised. This technique of 'steroid profiling' has been applied to the separation and quantification of metabolites of pregnenolone which were formed during incubations of the microsomal and cytosolic fractions from rat testes. The majority of the metabolites were found in the microsomal incubation. These compounds included some odorous 16-androstenes as well as other C21 and C19 steroids, the formation of which was consistent with the 5-ene and 4-ene pathways of testosterone biosynthesis being operative. In addition, evidence was obtained for 16 alpha-hydroxylation of C21 steroids. Very much less metabolic activity was found in the cytosolic fraction of rat testes. Metabolic pathways have been proposed which both confirm and extend earlier work. We conclude that the rat testis can only form some of the odorous, possibly pheromonal, 16-androstenes and that these are quantitatively less important than in the porcine testis.
In this post genomic era, there are a great number of in silico annotated hypothetical genes. However, experimental validation of the functionality of these genes remains tentative. Two of the major challenges faced by researcher are whether these hypothetical genes are protein-coding genes and whether their corresponding predicted translational start codons are correct. In this report, we demonstrate a convenient procedure to validate the presence of a hypothetical gene product of BPSS1356 from Burkholderia pseudomallei as well as its start codon. It was done by integration of a His-Tag coding sequence into C-terminal end of BPSS1356 gene via homologous recombination. We then purified the native protein using affinity chromatography. The genuine start codon of BPSS1356 was then determined by protein N-terminal sequencing.
Andrographis paniculata (Burm. F.) Nees. is considered as the herb of the future due to its precious chemical compounds, andrographolide (ANDRO), neoandrographolide (NAG) and 14-deoxyandrographolide (DAG). This study aims to profile the metabolites in young and mature leaf at six different harvest ages using 1HNMR-based metabolomics combined with multivariate data analysis. Principal component analysis (PCA) indicated noticeable and clear discrimination between young and mature leaves. A comparison of the leaves stage indicated that young leaves were separated from mature leaves due to its larger quantity of ANDRO, NAG, DAG, glucose and sucrose. These similar metabolites are also responsible for the PCA separation into five clusters representing the harvest age at 14, 16, 18, 20, 22 weeks of leaves extract. Loading plots revealed that most of the ANDRO and NAG signals were present when the plant reached at the pre-flowering stage or 18 weeks after sowing (WAS). As a conclusion, A. paniculata young leaves at pre-flowering harvest age were found to be richer in ANDRO, NAG and DAG compared to mature leaves while glucose and choline increased with harvest age. Therefore, young leaves of A. paniculata should be harvested at 18 WAS in order to produce superior quality plant extracts for further applications by the herbal, nutraceutical and pharmaceutical industries.
Matched MeSH terms: Chromatography, High Pressure Liquid
The role of aldose reductase (ALR2) in diabetes mellitus is well-established. Our interest in finding ALR2 inhibitors led us to explore the inhibitory potential of new thiosemicarbazones. In this study, we have synthesized adamantyl-thiosemicarbazones and screened them as aldehyde reductase (ALR1) and aldose reductase (ALR2) inhibitors. The compounds bearing phenyl 3a, 2-methylphenyl 3g and 2,6-dimethylphenyl 3m have been identified as most potent ALR2 inhibitors with IC50 values of 3.99 ± 0.38, 3.55 ± 0.26 and 1.37 ± 0.92 µM, respectively, compared with sorbinil (IC50 = 3.14 ± 0.02 μM). The compounds 3a, 3g, and 3m also inhibit ALR1 with IC50 value of 7.75 ± 0.28, 7.26 ± 0.39 and 7.04 ± 2.23 µM, respectively. Molecular docking was also performed for putative binding of potent inhibitors with target enzyme ALR2. The most potent 2,6-dimethylphenyl bearing thiosemicarbazone 3m (IC50 = 1.37 ± 0.92 µM for ALR2) and other two compound 3a and 3g could potentially lead for the development of new therapeutic agents.
Conventional anticoagulant therapy is the mainstay of medical treatment for deep vein thrombosis disorders. However,there are many complications associated with these agents such as bleeding. Hence, the search for novel anticoagulant derived from natural substances such as plants origin is in high demand nowadays. Ocimum sanctum(O.sanctum) also known as Ocimum tenuiform (OT), tulsi or holy basil from the family of Lamiaceae has been widely used for thousands of years in Ayurveda and Unani systems to cure or prevent a number of illnessessuch as headache, malaria, ulcers, bronchitis, cough, flu, sore throat and asthma. The objective is to investigate theeffect ofO. sanctum(Tulsi) aqueous leaf extract on prothrombin time (PT), activated partial thromboplastin time (APTT) and thrombin time (TT) in human plasma. Coagulation activity of O. sanctum was measured via PT, APTT and TT assay in citrated plasma collected from thirty-six healthy regular blood donors. The plasma was tested against different concentrations of O. sanctum aqueous extract as follows: 0.1mg/ml, 0.5 mg/ml and 1.0 mg/ml. Result shows the aqueous extract of O. sanctum prolonged the PT and APTT assays (p0.05). The gas chromatography-mass spectrometry (GC-MS) analysis had identified the linolenic acid at 1-10% of ethanol and aqueousconcentration at different retention time which was responsible for the coagulation activities of O. sanctumin human plasma. This study suggests that O. sanctum does affect coagulation activity in human plasma and can be potentially used as naturally derived anticoagulant products in the future.
Matched MeSH terms: Gas Chromatography-Mass Spectrometry
Poly(β-cyclodextrin functionalized ionic liquid) immobilized magnetic nanoparticles (Fe3O4@βCD-Vinyl-TDI) as sorbent in magnetic µ-SPE was developed for the determination of selected polycyclic aromatic hydrocarbons (PAHs) in rice samples coupled with gas chromatographic-flame ionization detector (GC-FID). The nanocomposite was characterized by various tools and significant parameters that affected the extraction efficiency of PAHs were investigated. The calibration curves were linear for the concentration ranging between 0.1 and 500 μg kg-1 with correlation determinations (R2) from 0.9970 to 0.9982 for all analytes. Detection limits ranged at 0.01-0.18 μg kg-1 in real matrix. The RSD values ranged at 2.95%-5.34% (intra-day) and 4.37%-7.05% (inter-day) precision for six varied days. The sorbents showed satisfactory reproducibility in 2.9% to 9.9% range and acceptable recovery values at 80.4%-112.4% were obtained for the real sample analysis. The optimized method was successfully applied to access content safety of selected PAHs for 24 kinds of commercial rice available in Malaysia.