Dried blood spots (DBS) are currently the recommended sample collection method for newborn screening programmes in America. Early diagnosis of beta-thalassaemia screening is essential as it provides an added advantage especially in sickle cell disease. Beta-thalassaemia frequency is high in many poor countries, and the cost of using commercial DNA extraction kits can be prohibitive. Our study assessed three methods that use minimal reagents and materials to extract DNA from DBS for beta-thalassaemia identification.
Optimization process is an important aspect in the natural product extractions. Herein, an alternative approach is proposed for the optimization in extraction, namely, the Generalized Likelihood Uncertainty Estimation (GLUE). The approach combines the Latin hypercube sampling, the feasible range of independent variables, the Monte Carlo simulation, and the threshold criteria of response variables. The GLUE method is tested in three different techniques including the ultrasound, the microwave, and the supercritical CO2 assisted extractions utilizing the data from previously published reports. The study found that this method can: provide more information on the combined effects of the independent variables on the response variables in the dotty plots; deal with unlimited number of independent and response variables; consider combined multiple threshold criteria, which is subjective depending on the target of the investigation for response variables; and provide a range of values with their distribution for the optimization.
Matched MeSH terms: Chemical Fractionation/methods*
Four different methods were evaluated to extract proteins from "Musang King" durian pulps and subsequently proteins with different abundance between fresh and long term frozen storage were identified using two-dimensional polyacrylamide gel electrophoresis coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometer analyses. The acetone-phenol method was found to produce good protein yields and gave the highest gel resolution and reproducibility. Differential protein analyses of the durian pulp revealed that 15 proteins were down-regulated and three other proteins were up-regulated after a year of frozen storage. Isoflavone reductase-like protein, S-adenosyl methionine synthase, and cysteine synthase isoform were up-regulated during frozen storage. The down-regulation of proteins in frozen durian pulps indicated that frozen storage has affected proteins in many ways, especially in their functions related to carbohydrate and energy metabolisms, cellular components, and transport processes. This study will enable future detailed investigations of proteins associated with quality attributes of durians to be studied.
Matched MeSH terms: Chemical Fractionation/methods*
Hollow fibre liquid-phase microextraction with in situ derivatization using dansyl chloride has been successfully developed for the high-performance liquid chromatography-ultraviolet (HPLC-UV) determination of the biogenic amines (tryptamine, putrescine, cadaverine, histamine, tyramine, spermidine) in food samples. Parameters affecting the performance of the in situ derivatization process such as type of extraction solvent, temperature, extraction time, stirring speed and salt addition were studied and optimized. Under the optimized conditions (extraction solvent, dihexyl ether; acceptor phase, 0.1M HCl; extraction time, 30 min; extraction temperature, 26 degrees C; without addition of salt), enrichment factors varying from 47 to 456 were achieved. Good linearity of the analytes was obtained over a concentration range of 0.1-5 microg mL(-1) (with correlation coefficients of 0.9901-0.9974). The limits of detection and quantification based on a signal-to-noise ratio of 3-10, ranged from 0.0075 to 0.030 microg mL(-1) and 0.03 to 0.10 microg mL(-1), respectively. The relative standard deviations based on the peak areas for six replicate analysis of water spiked with 0.5 microg mL(-1) of each biogenic amine were lower than 7.5%. The method was successfully applied to shrimp sauce and tomato ketchup samples, offering an interesting alternative to liquid-liquid extraction and solid phase extraction for the analysis of biogenic amines in food samples.
Matched MeSH terms: Chemical Fractionation/instrumentation; Chemical Fractionation/methods*
Brown seaweeds are rich source of functional polysaccharides that exhibit various bioactivities. However, Malaysian seaweeds are under-utilised, leading to low revenue throughout the supply chain of the seaweed industry. The aims of this study were to extract the functional polysaccharides, namely fucoidan (F), laminaran (L) and alginate (A) from Malaysian brown seaweeds (Sargassum polycystum, Turbinaria ornata and Padina boryana) and subsequently evaluate the properties of the extracted polysaccharides. P. boryana recorded the significantly (p ≤ 0.05) highest carbohydrate content (74.78 ± 1.63%) with highest fucoidan yield (Fpad = 1.59 ± 0.16%) while T. ornata contained significantly (p ≤ 0.05) highest alginate yield (Atur = 105.19 ± 3.45%). Water activities of these extracted polysaccharides varied from 0.63-0.71 with average score of browning indexes (~40). Fourier transform infrared (FTIR) spectroscopy analysis demonstrated that the extracted polysaccharides exhibited similar spectral pattern of spectra with the respective standards. Meanwhile, laminaran extracts showed the significantly highest (p ≤ 0.05) total phenolic contents (Lsar = 43.29 ± 0.43 mgGAE/g) and superoxide anion scavenging activity (Lsig = 21.7 ± 3.6%). On the other hand, the significantly highest (p ≤ 0.05) DPPH scavenging activity was recorded in alginate with Asar at 85.3 ± 0.8%. These findings reported the properties and bioactivities of natural polysaccharides from Malaysian brown seaweeds that revealed the potential to develop high-value functional ingredients from Malaysian brown seaweeds.
The lipase-catalyzed interesterification of refined, bleached, deodorized palm olein with iodine value (IV) of 62 was studied in a pilot continuous packed-bed reactor operating at 65 degrees C. Sn-1,3 specific immobilized enzyme; Lipozyme TL IM (Thermomyces Lanuginosa) from Novozyme A/S was used in this study. The interesterification reaction produced fully solidified fats at ambient temperature due to the production of trisaturated triacylglycerols (TAG) (PPP and PPS, where P = palmitic acid, S = stearic acid). The reaction also increased the percentage of triunsaturated TAG (OLL, OLO, and OOO, where O = oleic acid, L = linoleic acid). The interesterified product was then dry fractionated at temperatures of 9, 12, 15, 18, and 21 degrees C to separate the saturated fats from the unsaturated. The results show that IV of olein increased when the fractionation temperature (T(FN)) decreased. The highest IV of olein was 72, obtained from T(FN) at 9 degrees C. After interesterification and laboratory-scale fractionation, the olein fractions contained higher unsaturation content ranging from 64.7% to 67.7% compared to the starting material (58.3%), while the saturation content was reduced from 41.7% to the range of 32.3% to 35.3%. The yields of these oleins were low with the range of 24.8% to 51.8% due to the limitation of the vacuum filtration. Ten kilograms of pilot-scale fractionation with membrane press filter was used to determine the exact olein yield. At T(FN) of 12 degrees C, 67.1% of olein with saturation content of 33.9% was obtained.
The partitioning behavior of immunoglobulin G (IgG) in the aqueous two-phase system (ATPS) composed of poly(ethylene glycol) (PEG) and phosphate was studied. The parameters of ATPS exhibiting the pronounced effects on the partitioning behavior of IgG include phase composition, PEG molecular weight, and the addition of sodium chloride (NaCl). The accumulation of IgG at the interface of the ATPS increased drastically as the tie-line length (TLL) was increased. This trend was correlated with a linear relationship relating the natural logarithm of interfacial partition coefficient (ln G) to the difference of PEG concentration between the top phase and the bottom phase (Δ[PEG]), and a good fit was obtained. An attempt was made to correlate the natural logarithm of partition coefficient (ln K) to the presence of NaCl with the proposed linear relationship, ln K = α″ ln [Cl(-)] + β″. The proposed relationship, which serves as a better description of the underlying mechanics of the protein partitioning behavior in the polymer-salt ATPS, provides a good fit (r(2) > 0.95) for the data of IgG partitioning. An optimum recovery of 99.97% was achieved in an ATPS (pH 7.5) composed of 14.0% (w/w) PEG 1450, 12.5% (w/w) phosphate and 5.0% (w/w) NaCl.
Matched MeSH terms: Chemical Fractionation/methods*
Increasing levels of antibiotic resistance by Staphylococcus aureus have posed a need to search for non-antibiotic alternatives. This study aimed to assess the inhibitory effects of crude and fractionated cell-free supernatants (CFS) of locally isolated lactic acid bacteria (LAB) against a clinical strain of S. aureus. A total of 42 LAB strains were isolated and identified from fresh vegetables, fresh fruits and fermented products prior to evaluation of inhibitory activities. CFS of LAB strains exhibiting a stronger inhibitive effect against S. aureus were fractionated into crude protein, polysaccharide and lipid fractions. Crude protein fractions showed greater inhibition against S. aureus compared to polysaccharide and lipid fractions, with a more prevalent effect from Lactobacillus plantarum 8513 and L. plantarum BT8513. Crude protein, polysaccharide and lipid fractions were also characterised with glycine, mannose and oleic acid being detected as the major component of each fraction, respectively. Scanning electron microscopy revealed roughed and wrinkled membrane morphology of S. aureus upon treatment with crude protein fractions of LAB, suggesting an inhibitory effect via the destruction of cellular membrane. This research illustrated the potential application of fractionated extracts from LAB to inhibit S. aureus for use in the food and health industry.
Oil palm empty fruit bunch (EFB) was pretreated by Formiline process to overcome biomass recalcitrance and obtain hemicellulosic syrup and lignin. Higher formic acid concentration led to more lignin removal but also higher degree of cellulose formylation. Cellulose digestibility could be well recovered after deformylation with a small amount of lime. After digested by enzyme loading of 15 FPU+10 CBU/g solid for 48 h, the polysaccharide conversion could be over 90%. Simultaneous saccharification and fermentation (SSF) results demonstrated that ethanol concentration reached 83.6 g/L with approximate 85% of theoretic yield when performed at an initial dry solid consistency of 20%. A mass balance showed that via Formiline pretreatment 0.166 kg of ethanol could be produced from 1 kg of dry EFB with co-production of 0.14 kg of high-purity lignin and 5.26 kg hemicellulosic syrup containing 2.8% xylose. Formiline pretreatment thus can be employed as an entry for biorefining of EFB.
Matched MeSH terms: Chemical Fractionation/methods
In recent years, essential oils have received a growing interest because of the positive health effects of their novel characteristics such as antibacterial, antifungal, and antioxidant activities. For the extraction of plant-derived essential oils, there is the need of advanced analytical techniques and innovative methodologies. An exhaustive study of hydrodistillation, supercritical fluid extraction, ultrasound- and microwave-assisted extraction, solid-phase microextraction, pressurized liquid extraction, pressurized hot water extraction, liquid-liquid extraction, liquid-phase microextraction, matrix solid-phase dispersion, and gas chromatography (one- and two-dimensional) hyphenated with mass spectrometry for the extraction through various plant species and analysis of essential oils has been provided in this review. Essential oils are composed of mainly terpenes and terpenoids with low-molecular-weight aromatic and aliphatic constituents that are particularly important for public health.
A study was carried out to investigate the fractionation of Cd, Cr, Cu, Fe, Mn, Pb, and Zn in shrimp aquaculture sludge from Selangor, Malaysia, using original (unmodified) and modified four-steps BCR (European Community Bureau of Reference, now known as the Standards Measurements and Testing Program) sequential extraction scheme. Step 2 of the unmodified BCR procedure (subsequently called Method A) involves treatment with 0.1 M hydroxylammonium chloride at pH 2, whereas 0.5 M hydroxylammonium chloride at pH 1.5 was used in the modified BCR procedure (subsequently called Method B). Metal analyses were carried out by flame atomic absorption spectrometry. A pseudo-total aqua-regia digest of BCR CRM 701 has also been undertaken for quality assurance purposes. The recovery of Method A for all metals studied ranges from 96.14% to 105.26%, while the recovery for Method B ranges from 95.94% to 122.40%. Our results reveal that Method A underestimated the proportion of metals bound to the easily reducible fraction except for copper. Therefore, the potential mobility of these elements is higher than others. Thus, to use this sludge as a fertilizer we have to first find a remediation for reduction of heavy metal contamination.
Matched MeSH terms: Chemical Fractionation/methods*
A three-phase hollow fiber liquid-phase microextraction (HF-LPME) coupled either with capillary electrophoresis (CE) or high performance liquid chromatography (HPLC) with UV detection methods was successfully developed for the determination of trace levels of the anti-diabetic drug, rosiglitazone (ROSI) in biological fluids. The analyte was extracted into dihexyl ether that was immobilized in the wall pores of a porous hollow fiber from 10 mL of aqueous sample, pH 9.5 (donor phase), and was back extracted into the acceptor phase that contained 0.1M HCl located in the lumen of the hollow fiber. Parameters affecting the extraction process such as type of extraction solvent, HCl concentration, donor phase pH, extraction time, stirring speed, and salt addition were studied and optimized. Under the optimized conditions (extraction solvent, dihexyl ether; donor phase pH, 9.5; acceptor phase, 0.1M HCl; stirring speed, 600 rpm; extraction time, 30 min; without addition of salt), enrichment factor of 280 was obtained. Good linearity and correlation coefficients of the analyte was obtained over the concentration ranges of 1.0-500 and 5.0-500 ng mL(-1) for the HPLC (r(2)=0.9988) and CE (r(2)=0.9967) methods, respectively. The limits of detection (LOD) and limits of quantitation (LOQ) for the HPLC and CE methods were (0.18, 2.83) and (0.56, 5.00) ng mL(-1), respectively. The percent relative standard deviation (n=6) for the extraction and determination of three concentration levels (10, 250, 500 ng mL(-1)) of ROSI using the HPLC and CE methods were less than 10.9% and 13.2%, respectively. The developed methods are simple, rapid, sensitive and are suitable for the determination of trace amounts of ROSI in biological fluids.
Matched MeSH terms: Chemical Fractionation/methods*
Anthocyanins not just have various benefits in food industry but also have been used as natural colourants in cosmetic, coating products and as potential natural photosensitizers in solar cell. Thus, the main purpose of this study was to obtain information on the maximum yield of anthocyanin that can be recovered from Melastoma malabathricum fruit. Factors such as extraction temperature, extraction time, and solid to liquid ratio were identified to be significantly affecting anthocyanin extraction efficiency. By using three-level three-factor Box-Behnken design, the optimized conditions for anthocyanin extraction by acidified methanol (R (2) = 0.972) were temperature of 60°C, time of 86.82 min, and 0.5 : 35 (g/mL) solid to liquid ratio while the optimum extraction conditions by acidified ethanol (R (2) = 0.954) were temperature of 60°C, time of 120 min, and 0.5 : 23.06 (g/mL) solid to liquid ratio. The crude anthocyanin extract was further purified by using Amberlite XAD-7 and Sephadex LH-20 column chromatography. Identification of anthocyanins revealed the presence of cyanidin dihexoside, cyanidin hexoside, and delphinidin hexoside as the main anthocyanins in M. malabathricum fruit.
Two types of palm oil and sal fat based cocoa butter equivalents, namely fCBE (produced by using co-fractionation method) and mCBE (produced by using conventional method) were prepared. Results showed that the fCBE had triglyceride composition and solidification characteristics closer to the Malaysian cocoa butter than the mCBE produced at the same yield percentage. Increasing acetone washing time had little effect on the fCBE if compared to the effect of increasing palm olein to sal fat blend ratio. Co-fractionation technique increase the compatibility between CBE component triglycerides. Thus, more palm oil can be incorporated in the preparation and the process can be carried out at not low temperature as compared to the conventional method.
Recently, the quality-by-design concept has been widely implemented in the optimization of pharmaceutical processes to improve batch-to-batch consistency. As flavonoid compounds in pigmented rice bran may provide natural antioxidants, extraction of flavonoid components from red and brown rice bran was optimized using central composite design (CCD) and response surface methodology (RSM). Among the solvents tested, ethanol was most efficient for extracting flavonoids from rice bran. The examined parameters were temperature, solvent percentage, extraction time, and solvent-to-solid ratio. The highest total flavonoid content (TFC) in red rice bran was predicted as 958.14 mg quercetin equivalents (QE)/100 g dry matter (DM) at 58.5 °C, 71.5% (v/v), 36.2 min, and 7.94 mL/g, respectively, whereas the highest TFC in brown rice bran was predicted as 782.52 mg QE/100 g DM at 56.7 °C, 74.4% (v/v), 36.9 min, and 7.18 mL/g, respectively. Verification experiment results under these optimized conditions showed that the TFC values for red and brown rice bran were 962.38 and 788.21 mg QE/100 g DM, respectively. No significant differences were observed between the predicted and experimental TFC values, indicating that the developed models are accurate. Analysis of the extracts showed that apigenin and p-coumaric acid are abundant in red and brown rice bran. Further, red rice bran with its higher flavonoid content exhibited higher nitric oxide and 2,2-diphenyl-1-picrylhydrazyl scavenging activities (EC50 values of 41.3 and 33.6 μg/mL, respectively) than brown rice bran. In this study, an extraction process for flavonoid compounds from red and brown rice bran was successfully optimized. The accuracy of the developed models indicated that the approach is applicable to larger-scale extraction processes.
Matched MeSH terms: Chemical Fractionation/methods
Environmental impact assessment of glucose production from paddy milling waste, known as empty and partially filled paddy grain (EPFG) in Malaysia, was performed using life cycle assessment (LCA). Three scenarios were conducted based on system expansion of the process. The LCA was conducted using ReCiPe methodology at midpoint and endpoint levels. The results indicate that enzymatic hydrolysis phase is the hotspot in the conversion system due to enzyme production. In addition, the agriculture phase also contributed to negative impacts, especially towards climate change. An improved environmental load was observed in scenario 2 when all EPFG fractionation was utilised to replace fossil-based electricity. Sensitivity analysis showed an increase in glucose yield leads to reduced environmental impact. Thus, the LCA study suggests that the conversion process of EPFG could further benefit and improve the paddy industry waste management with low impact contribution to the environment compared to other feedstock used for glucose production.
A series of experiments were carried out to determine the best medium for the recovery of cobalt by means of an electrogenerative system. Use of the electrogenerative system with a chloride medium had shown promising performance with the highest free energy of -389.8 kJ mol(-1) compared to that with sulphate and nitrate media. Subsequently, the influence of catholyte concentrations on cobalt recovery using the electrogenerative process was carried out by varying the initial cobalt concentration and sodium chloride concentration. The results showed that almost 100% recovery was attained within 1-4 h of the recovery process. Influence of pH was investigated where the electrogenerative system performed best between pH 5.0 and 7.0. Maximum cell performance of 83% with 99% cobalt removal was obtained at 90 min when 100 mg L(-1) of Co(2+) in 0.5 M NaCl was taken as catholyte solution. The values of ΔH(o) and ΔS(o) of the process were evaluated as 33.41 kJ mol(-1) and 0.13 kJ mol(-1), respectively.
Matched MeSH terms: Chemical Fractionation/methods*
In earlier ex vivo studies, we reported the biphasic effect of a methanolic extract of unripe Morinda citrifolia fruit (MMC) on dopamine-induced contractility in isolated rat vas deferens preparations. The present in vivo study was designed and undertaken to further explore our earlier ex vivo findings. This study examined the effect of the ethyl acetate fraction of a methanolic extract of unripe Morinda citrifolia Linn. fruit (EA-MMC; 5-100 mg/kg, p.o.) on the dopaminergic system using mouse models of apomorphine-induced climbing time and climbing behavior, methamphetamine-induced stereotypy (sniffing, biting, gnawing, and licking) and haloperidol-induced catalepsy using the bar test. Acute treatment with EA-MMC at a low dose (25 mg/kg, p.o.) significantly attenuated the apomorphine-induced climbing time and climbing behavior in mice. Similarly, EA-MMC (5 and 10 mg/kg, p.o.) significantly inhibited methamphetamine-induced stereotyped behavior in mice. These results demonstrated that the antidopaminergic effect of EA-MMC was observed at relatively lower doses (<25 mg/kg, p.o.). On the other hand, EA-MMC showed dopaminergic agonistic activity at a high dose (3,000 mg/kg, p.o.), which was evident from alleviation of haloperidol (a dopamine D2 blocker)-induced catalepsy in mice. Therefore, it is concluded that EA-MMC might possess a biphasic effect on the dopaminergic system, i.e., an antagonistic effect at lower doses (<25 mg/kg, p.o.) and an agonistic effect at higher doses (>1,000 mg/kg, p.o.). However, further receptor-ligand binding assays are necessary to confirm the biphasic effects of M. citrifolia fruit on the dopaminergic system.
Drastic increment of skin cancer incidence has driven natural product-based chemoprevention as a promising approach in anticancer drug development. Apart from its traditional usages against various ailments, Ardisia crispa (Family: Myrsinaceae) specifically its triterpene-quinone fraction (TQF) which was isolated from the root hexane extract (ACRH) was recently reported to exert antitumor promoting activity in vitro. This study aimed at determining chemopreventive effect of TQF against chemically-induced mouse skin tumorigenesis as well as elucidating its possible pathway(s).
Dracaena cinnabari Balf.f. is a red resin endemic to Socotra Island, Yemen. Although there have been several reports on its therapeutic properties, information on its cytotoxicity and anticancer effects is very limited. This study utilized a bioassay-guided fractionation approach to determine the cytotoxic and apoptosis-inducing effects of D. cinnabari on human oral squamous cell carcinoma (OSCC). The cytotoxic effects of D. cinnabari crude extract were observed in a panel of OSCC cell lines and were most pronounced in H400. Only fractions DCc and DCd were active on H400 cells; subfractions DCc15 and DCd16 exhibited the greatest cytotoxicity against H400 cells and D. cinnabari inhibited cells proliferation in a time-dependent manner. This was achieved primarily via apoptosis where externalization of phospholipid phosphatidylserine was observed using DAPI/Annexin V fluorescence double staining mechanism studied through mitochondrial membrane potential assay cytochrome c enzyme-linked immunosorbent and caspases activities revealed depolarization of mitochondrial membrane potential (MMP) and significant activation of caspases 9 and 3/7, concomitant with S phase arrest. Apoptotic proteins array suggested that MMP was regulated by Bcl-2 proteins family as results demonstrated an upregulation of Bax, Bad, and Bid as well as downregulation of Bcl-2. Hence, D. cinnabari has the potential to be developed as an anticancer agent.