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A high-performance liquid chromatography analysis of plasma artemisinin using a glassy carbon electrode for reductive electrochemical detection

Chan KL, Yuen KH, Jinadasa S, Peh KK, Toh WT

Planta Med, 1997 Feb;63(1):66-9.
PMID: 9063097

A high-performance liquid chromatography assay equipped with a glassy carbon electrode for electrochemical detection (HPLC-ECD) was developed at reductive mode for the analysis of artemisinin, the antimalarial drug from Artemisia annua (Asteraceae) in human plasma. This method was selective, sensitive, and produced satisfactory recovery, precision, and accuracy. Analysis of plasma samples from 8 male volunteers given 10 mg kg-1 of artemisinin orally as an aqueous suspension showed a mean peak plasma concentration (Cmax) of 580.89 ng ml-1 +/- 88.64 SD at 2.5 h +/- 0.5 SD after dosing, and the mean area under the plasma concentration-time curve (AUC0-infinity) was 2227.57 ng h ml-1 +/- 677.22 SD. In addition, the elimination rate constant (Ke), elimination half-life (t1/2), and apparent volume of distribution (Vd) were calculated to be 0.2971 h-1 +/- 0.0644 SD, 2.42 h +/- 0.46 SD, and 16.26 l kg-1 +/- 3.44 SD, respectively.

Matched MeSH terms: Chromatography, High Pressure Liquid/methods*
Improvement of COD removal by controlling the substrate degradability during the anaerobic digestion of recalcitrant wastewater

Kawai M, Nagao N, Kawasaki N, Imai A, Toda T

J Environ Manage, 2016 Oct 01;181:838-846.
PMID: 27449962 DOI: 10.1016/j.jenvman.2016.06.057

The recalcitrant landfill leachate was anaerobically digested at various mixing ratios with labile synthetic wastewater to evaluate the degradation properties of recalcitrant wastewater. The proportion of leachate to the digestion system was increased in three equal steps, starting from 0% to 100%, and later decreased back to 0% with the same steps. The chemical oxygen demand (COD) for organic carbon and other components were calculated by analyzing the COD and dissolved organic carbon (DOC), and the removal efficiencies of COD carbon and COD others were evaluated separately. The degradation properties of COD carbon and COD others shifted owing to changing of substrate degradability, and the removal efficiencies of COD carbon and COD others were improved after supplying 100% recalcitrant wastewater. The UV absorptive property and total organic carbon (TOC) of each molecular size using high performance liquid chromatography (HPLC)-size exclusion chromatography (SEC) with UVA and TOC detectors were also investigated, and the degradability of different molecular sizes was determined. Although the SEC system detected extracellular polymeric substances (EPS), which are produced by microbes in stressful environments, during early stages of the experiment, EPS were not detected after feeding 100% recalcitrant wastewater. These results suggest that the microbes had acclimatized to the recalcitrant wastewater degradation. The high removal rates of both COD carbon and COD others were sustained when the proportion of labile wastewater in the substrate was 33%, indicating that the effective removal of recalcitrant COD might be controlled by changing the substrate's degradability.

Matched MeSH terms: Chromatography, High Pressure Liquid/methods
New Determination Method of Two Bioactive Saponins in Lysimachia capillipes Hemsl. by Quantitative Analysis of Multi-Components by Single-Marker

Zhang X, Chen X, Jin J, Gong M, He Q, Li S, et al.

J Chromatogr Sci, 2021 Oct 29;59(10):941-948.
PMID: 33728454 DOI: 10.1093/chromsci/bmab028

Capilliposide B (CPS-B) and Capilliposide C (CPS-C), as the key components in Lysimachia capillipes Hemsl., increasingly aroused the interest and research concern of many researchers due to the good bioactivities. Nowadays, the reference standards of CPS-B and CPS-C yield were very limited. Due to the deficit of reference standards, the determination could be difficult to carry out, and the quality control and evaluation would be restrained afterwards. To solve this urgent problem, a quantitative analysis of multi-components by single-marker (QAMS) method was proposed and established based on high-performance liquid-chromatography tandem evaporative light-scattering detector. In this QAMS method, the content of the two bioactive components could be calculated by buddlejasaponin IV, which is applied as an external standard and readily obtained. And the methodological experiments were evaluated and indicated accuracy, stability and feasibility of this QAMS method. Therefore, in this study, this built method would properly meet the requirement of determination of CPS-B, CPS-C and quality control of the L. capillipes Hemsl. plant.

Matched MeSH terms: Chromatography, High Pressure Liquid
Constructed wetland-microbial fuel cell for azo dyes degradation and energy recovery: Influence of molecular structure, kinetics, mechanisms and degradation pathways

Oon YL, Ong SA, Ho LN, Wong YS, Dahalan FA, Oon YS, et al.

Sci Total Environ, 2020 Jun 10;720:137370.
PMID: 32325554 DOI: 10.1016/j.scitotenv.2020.137370

Complete degradation of azo dye has always been a challenge due to the refractory nature of azo dye. An innovative hybrid system, constructed wetland-microbial fuel cell (CW-MFC) was developed for simultaneous azo dye remediation and energy recovery. This study investigated the effect of circuit connection and the influence of azo dye molecular structures on the degradation rate of azo dye and bioelectricity generation. The closed circuit system exhibited higher chemical oxygen demand (COD) removal and decolourisation efficiencies compared to the open circuit system. The wastewater treatment performances of different operating systems were ranked in the decreasing order of CW-MFC (R1 planted-closed circuit) > MFC (R2 plant-free-closed circuit) > CW (R1 planted-open circuit) > bioreactor (R2 plant-free-open circuit). The highest decolourisation rate was achieved by Acid Red 18 (AR18), 96%, followed by Acid Orange 7 (AO7), 67% and Congo Red (CR), 60%. The voltage outputs of the three azo dyes were ranked in the decreasing order of AR18 > AO7 > CR. The results disclosed that the decolourisation performance was significantly influenced by the azo dye structure and the moieties at the proximity of azo bond; the naphthol type azo dye with a lower number of azo bond and more electron-withdrawing groups could cause azo bond to be more electrophilic and more reductive for decolourisation. Moreover, the degradation pathway of AR18, AO7 and CR were elucidated based on the respective dye intermediate products identified through UV-Vis spectrophotometry, high-performance liquid chromatography (HPLC), and gas chromatograph-mass spectrometer (GC-MS) analyses. The CW-MFC system demonstrated high capability of decolouring azo dyes at the anaerobic anodic region and further mineralising dye intermediates at the aerobic cathodic region to less harmful or non-toxic products.

Matched MeSH terms: Chromatography, High Pressure Liquid
Fulltext Development and validation of a bioanalytical method for quantification of 2,6-bis-(4-hydroxy-3-methoxybenzylidene)-cyclohexanone (BHMC) in rat plasma

Lee YZ, Ming-Tatt L, Lajis NH, Sulaiman MR, Israf DA, Tham CL

Molecules, 2012 Dec 07;17(12):14555-64.
PMID: 23222902 DOI: 10.3390/molecules171214555

A sensitive and accurate high performance liquid chromatography with ultraviolet/visible light detection (HPLC-UV/VIS) method for the quantification of 2,6-bis-(4-hydroxy-3-methoxybenzylidene)-cyclohexanone (BHMC) in rat plasma was developed and validated. BHMC and the internal standard, harmaline, were extracted from plasma samples by a simple liquid-liquid extraction using 95% ethyl acetate and 5% methanol. Plasma concentration of BHMC and internal standard were analyzed by reversed phase chromatography using a C₁₈ column (150 × 4.6 mm I.D., particle size 5 µm) and elution with a gradient mobile phase of water and methanol at a flow rate of 1.0 mL/min. Detection of BHMC and internal standard was done at a wavelength of 380 nm. The limit of quantification was 0.02 µg/mL. The calibration curves was linear (R² > 0.999) over the concentration range of 0.02-2.5 µg/mL. Intra- and inter-day precision were less than 2% coefficient of variation. The validated method was then applied to a pharmacokinetic study in rats by intravenous administration of BHMC at a single dose of 10 mg/kg. Pharmacokinetic parameters such as half-life, maximum plasma concentration, volume of distribution, clearance and elimination rate constant for BHMC were calculated.

Matched MeSH terms: Chromatography, High Pressure Liquid*
A validated size exclusion chromatography method coupled with fluorescence detection for rapid quantification of bevacizumab in ophthalmic formulations

Jirjees F, Soliman K, Wang Y, Sonawane R, Sheshala R, Jones D, et al.

J Pharm Biomed Anal, 2019 Sep 10;174:145-150.
PMID: 31167158 DOI: 10.1016/j.jpba.2019.05.038

Bevacizumab is a full-length human monoclonal antibody used to treat various neovascular diseases such as wet age-related macular degeneration (AMD), diabetic eye disease and other problems of the retina. Monthly intravitreal injections of bevacizumab (Avastin®) are effective in the treatment of wet AMD. However, there is a growing demand in the development of sustained release ophthalmic formulations. Therefore, this study aims, for the first time, to develop a rapid, simple, and sensitive method using size exclusion chromatography coupled with fluorescence detection for routine quantification of bevacizumab in ophthalmic formulations and during in vitro release studies. The selected chromatographic conditions included an aqueous mobile phase composed of 35 mM sodium phosphate buffer and 300 mM sodium chloride (pH 6.8), a flow rate of 0.5 mL/min, and the fluorescence detector was operated at excitation and emission wavelengths of 280 and 340 nm, respectively. The peak area-concentration relationship maintained its linearity over concentration range of 0.1-20 μg/mL (R2 = 0.9993), and the quantitation limit was 100 ng/mL. The method was validated for specificity, accuracy, precision, and robustness. The developed method had a run time of 6 min at temperature 25 °C, making it a unique validated method for rapid and cost-effective quantification of bevacizumab.

Matched MeSH terms: Chromatography, High Pressure Liquid
Determination and confirmation of isopropyl p-toluenesulfonate in cosmetics by HPLC-diode array detector method and GC-MS

Tay BY, Yung SC, Teoh TY

Int J Cosmet Sci, 2016 Dec;38(6):627-633.
PMID: 27169828 DOI: 10.1111/ics.12342

OBJECTIVE: Isopropyl p-toluenesulfonate (IPTS) is a potentially genotoxic by-product formed during the esterification of palm oil-based palmitic and palm kernel oil-based myristic acid with isopropanol to produce isopropyl palmitate or isopropyl myristate. There are no methods described for the analysis of IPTS in cosmetic products. In this work, we have established a simple, precise and accurate method to determine the presence and level of IPTS in various finished cosmetic products which contain palm-based esters in their formulations.
METHODS: An Agilent 1200 series high-performance liquid chromatography (HPLC) unit using a diode-array detector (DAD) has been employed and optimized to detect IPTS in cosmetic products. For the separation, a reverse-phase Hypersil Gold C8 column (5 μm, 4.6 mm i.d. 250 mm) 5 mM tetrabutylammonium phosphate buffer 50 : 50, (v/v) solution in acetonitrile as mobile phase, in isocratic mode and a flow rate of 0.8 mL min(-1) were used. A second method using a gas chromatography/mass selective detector GC-MSD was also developed to confirm the IPTS identity in the cosmetic products.
RESULTS: Recoveries of IPTS from cosmetic matrices such as a lotion, cleansing milk and a cream ranged from 94.0% to 101.1% with <5% relative standard deviation (%RSD) showing good accuracy and repeatability of the method. The six-point calibration curves (determined over the range 0.5-50 μg mL(-1) ) have a correlation coefficient of 0.9999 (based on HPLC peak area) and 0.9998 (based on HPLC peak height). The intra- and interday precisions (measured by the %RSD) of the method were <2% and <5%, respectively, indicating that the developed method is reliable, precise and reproducible. The detection and quantification limit of the method were found to be 0.5 μg mL(-1) and 1.6 μg mL(-1) , respectively. Analyses of 83 commercial cosmetics showed no presence of IPTS.
CONCLUSIONS: The validation data indicated that this method was suitable for the quantitative analysis of IPTS in commercial cosmetics. This method is applicable for analyses of trace levels of IPTS in cosmetics and has the advantage of using only simple sample preparation steps.

Matched MeSH terms: Chromatography, High Pressure Liquid/methods*
Fulltext Formation of 6-, 8- and 10-Shogaol in Ginger through Application of Different Drying Methods: Altered Antioxidant and Antimicrobial Activity

Ghasemzadeh A, Jaafar HZE, Baghdadi A, Tayebi-Meigooni A

Molecules, 2018 07 05;23(7).
PMID: 29976903 DOI: 10.3390/molecules23071646

Gingerols and shogaols are compounds found in ginger (Zingiber officinale Roscoe); shogaols are found in lower concentration than gingerols but exhibit higher biological activities. This work studied the effects of different drying methods including open sun drying (OSD) solar tunnel drying (STD) and hot air drying (HAD) with various temperature on the formation of six main active compounds in ginger rhizomes, namely 6-, 8-, and 10-gingerols and 6-, 8-, and 10-shogaols, as well as essential oil content. Antioxidant and antimicrobial activity of dried ginger was also evaluated. High performance liquid chromatography (HPLC) analysis showed that after HAD with variable temperature (120, 150 and 180 °C), contents of 6-, 8-, and 10-gingerols decreased, while contents of 6-, 8-, and 10-shogaol increased. High formation of 6-, 8-, and 10-shogaol contents were observed in HAD (at 150 °C for 6 h) followed by STD and OSD, respectively. OSD exhibited high content of essential oil followed by STD and HAD method. Ginger-treated with HAD exhibited the highest DPPH (IC50 of 57.8 mg/g DW) and FRAP (493.8 µM of Fe(II)/g DM) activity, compared to STD and OSD method. HAD ginger exhibited potent antimicrobial activity with lower minimum inhibition concentration (MIC) value against bacteria strains followed by STD and OSD, respectively. Ginger extracts showed more potent antimicrobial activity against Gram positive bacteria than Gram negative bacteria strains. Result of this study confirmed that conversion of gingerols to shogaols was significantly affected by different drying temperature and time. HAD at 150 °C for 6 h, provides a method for enhancing shogaols content in ginger rhizomes with improving antioxidant and antimicrobial activities.

Matched MeSH terms: Chromatography, High Pressure Liquid
Effect of HbE heterozygosity on the measurement of HbA1c

Sthaneshwar P, Shanmugam H, Swan VG, Nasurdin N, Tanggaiah K

Pathology, 2013 06;45(4):417-9.
PMID: 23635828 DOI: 10.1097/PAT.0b013e32836142eb

AIM: Measurement of HbA1c provides an excellent measure of glycaemic control for diabetic patients. However, haemoglobin (Hb) variants are known to interfere with HbA1c analysis. In our laboratory HbA1c measurement is performed by Variant II turbo 2.0. The aim of this study is to investigate the influence of HbE trait on HbA1c analysis.
METHODS: Haemoglobin variants were identified by HbA1c analysis in 93 of 3522 samples sent to our laboratory in a period of 1 month. Haemoglobin analysis identified HbE trait in 81 of 93 samples. To determine the influence of HbE trait on HbA1c analysis by Variant II Tubo 2.0, boronate affinity high performance liquid chromatography (HPLC) method (Primus PDQ) was used as the comparison method. Two stage linear regression analysis, Bland Altman plot and Deming regression analysis were performed to analyse whether the presence of HbE trait produced a statistically significant difference in the results. The total allowable error for HbA1c by the Royal Australasian College of Pathologists (RCPA) external quality assurance is 5%. Hence clinically significant difference is more than 5% at the medical decision level of 6% and 9%.
RESULTS: Statistically and clinically significant higher results were observed in Variant II Turbo 2.0 due to the presence of HbE trait. A positive bias of ∼10% was observed at the medical decision levels.
CONCLUSION: Laboratories should be cautious when evaluating HbA1c results in the presence of haemoglobin variants.

Matched MeSH terms: Chromatography, High Pressure Liquid
A simple and rapid HPLC-DAD method for simultaneously monitoring the accumulation of alkaloids and precursors in different parts and different developmental stages of Catharanthus roseus plants

Pan Q, Saiman MZ, Mustafa NR, Verpoorte R, Tang K

J Chromatogr B Analyt Technol Biomed Life Sci, 2016 Mar 1;1014:10-6.
PMID: 26854826 DOI: 10.1016/j.jchromb.2016.01.034

A rapid and simple reversed phase liquid chromatographic system has been developed for simultaneous analysis of terpenoid indole alkaloids (TIAs) and their precursors. This method allowed separation of 11 compounds consisting of eight TIAs (ajmalicine, serpentine, catharanthine, vindoline, vindolinine, vincristine, vinblastine, and anhydrovinblastine) and three related precursors i.e., tryptophan, tryptamine and loganin. The system has been applied for screening the TIAs and precursors in Catharanthus roseus plant extracts. In this study, different organs i.e., flowers, leaves, stems, and roots of C. roseus were investigated. The results indicate that TIAs and precursor accumulation varies qualitatively and quantitatively in different organs of C. roseus. The precursors showed much lower levels than TIAs in all organs. Leaves and flowers accumulate higher level of vindoline, catharanthine and anhydrovinblastine while roots have higher level of ajmalicine, vindolinine and serpentine. Moreover, the alkaloid profiles of leaves harvested at different ages and different growth stages were studied. The results show that the levels of monoindole alkaloids decreased while bisindole alkaloids increased with leaf aging and upon plant growth. The HPLC method has been successfully applied to detect TIAs and precursors in different types of C. roseus samples to facilitate further study of the TIA pathway and its regulation in C. roseus plants.

Matched MeSH terms: Chromatography, High Pressure Liquid
Fulltext Identification of αo-thalassaemia (–SEA) using an Enzyme- Linked Immunosorbent Assay (ELISA) for embryonic zeta-globin chain detection – a preliminary study

Tan, J. A. M. A., George, E., Lim, E. J., Zakaria, Z., Hassan, R., Wee, Y. C., et al.

Malaysian Journal of Medicine and Health Sciences, 2006;2(2):81-87.
MyJurnal

Objectives: This study aimed to evaluate the UBI MAGIWELTM ζ-GLOBIN ELISA Kit for the presumptive diagnosis of αo-thalassaemia. The ELISA results obtained were confirmed by molecular characterisation of αo-thalassaemia using a Duplex-PCR. Methods: Routine peripheral blood counts and red cell indices were determined in 94 blood samples sent for Hb analysis. Hb subtypes were quantified by high performance liquid chromatography (HPLC) and Hb electrophoresis conducted on agarose gel at pH 8.5. Zeta-globin chain levels were determined using the UBI MAGIWELTM ζ-GLOBIN ELISA Kit. Molecular analysis was performed using a duplex-PCR which simultaneously amplifies
a normal 136 bp sequence between the ψα−α2-globin genes and a 730 bp Southeast Asian deletion-specific sequence (–SEA) between the ψα2−θ1-globin genes. Results: Using the ELISA assay kit, 20 blood samples were presumptively identified as α-thalassaemia carriers from elevated ζ-globin chains (OD>0.3) while the remaining 74 blood samples showed OD

Matched MeSH terms: Chromatography, High Pressure Liquid
Extraction and microencapsulation of khat: effects on sexual motivation and estradiol level in female rats

Aziz HA, Peh KK, Tan YT

J Sex Med, 2009 Mar;6(3):682-95.
PMID: 19143913 DOI: 10.1111/j.1743-6109.2008.01157.x

Khat (Catha edulis) is an evergreen tree/shrub that is thought to affect sexual motivation or libido. Its positive effect on sexual desire is more frequently observed in females than in males and occurs when khat is chewed. Thus, khat's effects on sexual behavior may depend on the release mode of its active constituent.

Matched MeSH terms: Chromatography, High Pressure Liquid
Simple high-performance liquid chromatographic method for simultaneous determination of fusidic Acid and betamethasone dipropionate in a cream formulation

Peh KK, Tan YT

Int J Pharm Compd, 2000 May-Jun;4(3):229-31.
PMID: 23986007

A simple and selective high-performance liquid chromatography (HPLC) method using ultraviolet detection was developed for simultaneous determination of fusidic acid and betamethasone dipropionate in a cream formulation. A Supelcosil LC18 column was used for chromatographic separation. The mobile phase consisted of acetonitrile and 0.01 M disodium hydrogen orthophosphate (70:30, % v/v) adjusted to pH 6 with glacial acetic acid. Analysis was run at a flow rate of 1.0 mL/minute with the detector operating at 235 nm. The standard calibration curve was linear over a concentration range of 0.3 to 1.2 mg/mL for fusidic acid and 9.6 to 38.4 micrograms/mL for betamethasone dipropionate. The average recovery values for fusidic acid and betamethasone dipropionate were almost 100%. The within-run and between-run coefficient of variation and percent error values for the two drugs were all less than 2% and +/- 3%, respectively.

Matched MeSH terms: Chromatography, High Pressure Liquid
Characterization of edible bird's nest of different production, species and geographical origins using nutritional composition, physicochemical properties and antioxidant activities

Quek MC, Chin NL, Yusof YA, Law CL, Tan SW

Food Res Int, 2018 07;109:35-43.
PMID: 29803459 DOI: 10.1016/j.foodres.2018.03.078

Edible bird's nest (EBN) is a precious food made from the solidified saliva of swiftlets. EBN from three types of origin, namely production, swiftlet species and geographical were characterised based on its nutritional composition, physicochemical properties and antioxidant properties. Proximate composition, total phenolic content (TPC) and antioxidant activities were determined following official methods, while mineral and heavy metal contents were obtained by respective atomic adsorption spectrometry (AAS) and inductively coupled plasma-mass spectrometry (ICP-MS). Amino acids profile and sialic acid were determined using high performance liquid chromatography (HPLC). Calcium and sodium were the major elements in EBN samples at averages of 17,267 mg/kg and 13,681 mg/kg, respectively. Despite protein contents were not significantly different; interestingly the total amino acids in A. fuciphagus EBN, 64.57 g/100 g was found to be 23% higher than in A. maximus EBN. EBN from house, A. fuciphagus and Peninsular Malaysia had greater antioxidant activities, 2.33-3.49 mg AAE/g and higher sialic acid, 13.57 g/100 g while those from cave, A. maximus and East Malaysia contained more minerals like calcium and magnesium. The 1, 1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity and ferric ion reducing antioxidant power (FRAP) of house, A. fuciphagus and Peninsular Malaysia EBNs were approximately 2 times greater than the others. All samples were complied with the Malaysian Standard MS 2334:2011, except for mercury and nitrite. The overall findings suggest that the quality of EBN was varied following the production, species and geographical origins.

Matched MeSH terms: Chromatography, High Pressure Liquid
Proteomic characterization of venom of the medically important Southeast Asian Naja sumatrana (Equatorial spitting cobra)

Yap MK, Fung SY, Tan KY, Tan NH

Acta Trop, 2014 May;133:15-25.
PMID: 24508616 DOI: 10.1016/j.actatropica.2014.01.014

The proteome of Naja sumatrana (Equatorial spitting cobra) venom was investigated by shotgun analysis and a combination of ion-exchange chromatography and reverse phase HPLC. Shotgun analysis revealed the presence of 39 proteins in the venom while the chromatographic approach identified 37 venom proteins. The results indicated that, like other Asiatic cobra venoms, N. sumatrana contains large number of three finger toxins and phospholipases A2, which together constitute 92.1% by weight of venom protein. However, only eight of the toxins can be considered as major venom toxins. These include two phospholipases A2, three neurotoxins (two long neurotoxins and a short neurotoxin) and three cardiotoxins. The eight major toxins have relative abundance of 1.6-27.2% venom proteins and together account for 89.8% (by weight) of total venom protein. Other venom proteins identified include Zn-metalloproteinase-disintegrin, Thaicobrin, CRISP, natriuretic peptide, complement depleting factors, cobra venom factors, venom nerve growth factor and cobra serum albumin. The proteome of N. sumatrana venom is similar to proteome of other Asiatic cobra venoms but differs from that of African spitting cobra venom. Our results confirm that the main toxic action of N. sumatrana venom is neurotoxic but the large amount of cardiotoxins and phospholipases A2 are likely to contribute significantly to the overall pathophysiological action of the venom. The differences in toxin distribution between N. sumatrana venom and African spitting cobra venoms suggest possible differences in the pathophysiological actions of N. sumatrana venom and the African spitting cobra venoms, and explain why antivenom raised against Asiatic cobra venom is not effective against African spitting cobra venoms.

Matched MeSH terms: Chromatography, High Pressure Liquid
Six isoforms of cardiotoxin in malayan spitting cobra (Naja naja sputatrix) venom: cloning and characterization of cDNAs

Jeyaseelan K, Armugam A, Lachumanan R, Tan CH, Tan NH

Biochim. Biophys. Acta, 1998 Apr 10;1380(2):209-22.
PMID: 9565688

Cardiotoxins are the most abundant toxin components of cobra venom. Although many cardiotoxins have been purified and characterized by amino acid sequencing and other pharmacological and biochemical studies, to date only five cardiotoxin cDNAs from Taiwan cobra (Naja naja atra), three cDNAs from Chinese cobra (Naja atra) and two more of uncertain origin (either Chinese or Taiwan cobra) have been reported. In this paper we show the existence of four isoforms of cardiotoxin by protein analysis and nine cDNA sequences encoding six isoforms of cardiotoxins (CTX 1-3, 4a, 4b and 5) from N. n. sputatrix by cDNA cloning. This forms the first report on the cloning and characterization of several cardiotoxin genes from a single species of a spitting cobra. The cDNAs encoding these isoforms, obtained by reverse transcription-polymerase chain reaction (RT-PCR), were subsequently expressed in Escherichia coli. The native and recombinant cardiotoxins were first characterized by Western blotting and N-terminal protein sequencing. These proteins were also found to have different levels of cytolytic activity on cultured baby hamster kidney cells. Four of the isoforms (CTX 1, 2, 4 and 5) are unique to N. n. sputatrix, with CTX 2 being the most abundant species constituting about 50% of the total cardiotoxins. The isoform CTX 3 (20% constitution) is highly homologous to the cardiotoxins of N. n. atra and N. n. naja, indicating that it may be universally present in all Naja naja subspecies. Our studies suggest that the most hydrophilic isoform (CTX 5) could have evolved first followed by the hydrophobic isoforms (CTX 1, 2, 3 and 4). We also speculate that Asiatic cobras could be the modern descendants of the African and Egyptian counterparts.

Matched MeSH terms: Chromatography, High Pressure Liquid
Isolation and characterization of the thrombin-like enzyme from Cryptelytrops purpureomaculatus venom

Tan NH

Comp Biochem Physiol C Toxicol Pharmacol, 2010 Jan;151(1):131-6.
PMID: 19770070 DOI: 10.1016/j.cbpc.2009.09.002

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.

Matched MeSH terms: Chromatography, High Pressure Liquid
Fulltext Venomics of Tropidolaemus wagleri, the sexually dimorphic temple pit viper: Unveiling a deeply conserved atypical toxin arsenal

Tan CH, Tan KY, Yap MK, Tan NH

Sci Rep, 2017 02 27;7:43237.
PMID: 28240232 DOI: 10.1038/srep43237

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.

Matched MeSH terms: Chromatography, High Pressure Liquid
A Protein Decomplexation Strategy in Snake Venom Proteomics

Tan CH, Tan KY, Tan NH

Methods Mol Biol, 2019;1871:83-92.
PMID: 30276733 DOI: 10.1007/978-1-4939-8814-3_5

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).

Matched MeSH terms: Chromatography, High Pressure Liquid
Fulltext Cytotoxic and anticancer properties of the Malaysian mangrove pit viper (Trimeresurus purpureomaculatus) venom and its disintegrin (purpureomaculin)

Tan CH, Liew JL, Navanesan S, Sim KS, Tan NH, Tan KY

J Venom Anim Toxins Incl Trop Dis, 2020;26:e20200013.
PMID: 32742279 DOI: 10.1590/1678-9199-JVATITD-2020-0013

Background: The Asiatic pit vipers from the Trimeresurus complex are medically important venomous snakes. These pit vipers are often associated with snakebite that leads to fatal coagulopathy and tissue necrosis. The cytotoxic venoms of Trimeresurus spp.; however, hold great potential for the development of peptide-based anticancer drugs.
Methods: This study investigated the cytotoxic effect of the venom from Trimeresurus purpureomaculatus, the mangrove pit viper (also known as shore pit viper) which is native in Malaysia, across a panel of human cancer cell lines from breast, lung, colon and prostate as well as the corresponding normal cell lines of each tissue.
Results: The venom exhibited dose-dependent cytotoxic activities on all cell lines tested, with median inhibition concentrations (IC50) ranging from 0.42 to 6.98 µg/mL. The venom has a high selectivity index (SI = 14.54) on breast cancer cell line (MCF7), indicating that it is significantly more cytotoxic toward the cancer than to normal cell lines. Furthermore, the venom was fractionated using C18 reversed-phase high-performance liquid chromatography and the anticancer effect of each protein fraction was examined. Fraction 1 that contains a hydrophilic low molecular weight (approximately 7.5 kDa) protein was found to be the most cytotoxic and selective toward the breast cancer cell line (MCF7). The protein was identified using liquid chromatography-tandem mass spectrometry as a venom disintegrin, termed purpureomaculin in this study.
Conclusion: Taken together, the findings revealed the potent and selective cytotoxicity of a disintegrin protein isolated from the Malaysian T. purpureomaculatus venom and suggested its anticancer potential in drug discovery.

Matched MeSH terms: Chromatography, High Pressure Liquid

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