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Optimization of protease extraction from horse mango (Mangifera foetida Lour) kernels by a response surface methodology

Ahmad MN, Liew SL, Yarmo MA, Said M

Biosci Biotechnol Biochem, 2012;76(8):1438-44.
PMID: 22878182

Protease is one of the most important industrial enzymes with a multitude of applications in both food and non-food sectors. Although most commercial proteases are microbial proteases, the potential of non-conventional protease sources, especially plants, should not be overlooked. In this study, horse mango (Mangifera foetida Lour) fruit, known to produce latex with a blistering effect upon contact with human skin, was chosen as a source of protease, and the effect of the extraction process on its protease activity evaluated. The crude enzyme was extracted from the kernels and extraction was optimized by a response surface methodology (RSM) using a central composite rotatable design (CCRD). The variables studied were pH (x(1)), CaCl(2) (x(2)), Triton X-100 (x(3)), and 1,4-dithryeitol (x(4)). The results obtained indicate that the quadratic model is significant for all the variables tested. Based on the RSM model generated, optimal extraction conditions were obtained at pH 6.0, 8.16 mM CaCl(2), 5.0% Triton X-100, and 10.0 mM DTT, and the estimated response was 95.5% (w/w). Verification test results showed that the difference between the calculated and the experimental protease activity value was only 2%. Based on the t-value, the effects of the variables arranged in ascending order of strength were CaCl(2) < pH < DTT < Triton X-100.

Matched MeSH terms: Liquid-Liquid Extraction/methods*
Response Surface Methodology Modelling of an Aqueous Two-Phase System for Purification of Protease from Penicillium candidum (PCA 1/TT031) under Solid State Fermentation and Its Biochemical Characterization

Alhelli AM, Abdul Manap MY, Mohammed AS, Mirhosseini H, Suliman E, Shad Z, et al.

Int J Mol Sci, 2016 Nov 11;17(11).
PMID: 27845736

Penicillium candidum (PCA 1/TT031) synthesizes different types of extracellular proteases. The objective of this study is to optimize polyethylene glycol (PEG)/citrate based on an aqueous two-phase system (ATPS) and Response Surface Methodology (RSM) to purify protease from Penicillium candidum (PCA 1/TT031). The effects of different PEG molecular weights (1500-10,000 g/mol), PEG concentration (9%-20%), concentrations of NaCl (0%-10%) and the citrate buffer (8%-16%) on protease were also studied. The best protease purification could be achieved under the conditions of 9.0% (w/w) PEG 8000, 5.2% NaCl, and 15.9% sodium citrate concentration, which resulted in a one-sided protease partitioning for the bottom phase with a partition coefficient of 0.2, a 6.8-fold protease purification factor, and a yield of 93%. The response surface models displayed a significant (p ≤ 0.05) response which was fit for the variables that were studied as well as a high coefficient of determination (R²). Similarly, the predicted and observed values displayed no significant (p > 0.05) differences. In addition, our enzyme characterization study revealed that Penicillium candidum (PCA 1/TT031) produced a slight neutral protease with a molecular weight between 100 and 140 kDa. The optimal activity of the purified enzyme occurred at a pH of 6.0 and at a temperature of 50 °C. The stability between different pH and temperature ranges along with the effect of chemical metal ions and inhibitors were also studied. Our results reveal that the purified enzyme could be used in the dairy industry such as in accelerated cheese ripening.

Matched MeSH terms: Liquid-Liquid Extraction
Use of response surface methodology for partitioning, one-step purification of alkaline extracellular lipase from Penicillium candidum (PCA 1/TT031)

Alhelli AM, Abdul Manap MY, Mohammed AS, Mirhosseini H, Suliman E, Shad Z, et al.

J Chromatogr B Analyt Technol Biomed Life Sci, 2016 Dec 15;1039:66-73.
PMID: 27836491 DOI: 10.1016/j.jchromb.2016.10.037

This report shows the partitioning and purification of alkaline extracellular lipase from Penicillium candidum (PCA 1/TT031) by solid-state fermentation (SSF). In the present analysis, some of the important parameters such as PEG concentration, PEG molecular mass, salt concentration and buffer concentration were optimised through the response surface methodology (RSM). The optimum aqueous two-phase systems (ATPS) environment consisted of 13.8% (w/w) phosphate buffer, 9.2% (w/w) PEG-3000 and 3.3% (w/w) NaCl at 25°C. The RSM approach was proved to be the most suitable methodology for the recovery of desired enzymes. In this method, the enzyme partitioned into the top phase of the PEG-buffer-NaCl ATPS. Under this experimental environment, the purification factor was found to be 33.9, the partition coefficient was 4.0 and the yield was found to be 84.0% of lipase. Moreover, the experimental and predicted results were in considerable agreement, which established the reliability and validity of the proposed model. The ATPS methodology is proven to be effective for the primary recovery of lipase at a low cost with a large loading capacity and possibility of linear scale up. In addition to using the existing methodologies for improving enzyme production, the use of statistical optimisation of the constituents of phases through RSM continues to be the basic and practical method.

Matched MeSH terms: Liquid-Liquid Extraction/methods*
Salting-out assisted liquid-liquid extraction coupled with hydrophilic interaction chromatography for the determination of biguanides in biological and environmental samples

Alshishani A, Salhimi SM, Saad B

J Chromatogr B Analyt Technol Biomed Life Sci, 2018 Jan 15;1073:51-59.
PMID: 29241085 DOI: 10.1016/j.jchromb.2017.12.013

A new salting-out assisted liquid-liquid extraction (SALLE) sample preparation method for the determination of the polar anti-diabetic biguanide drugs (metformin, buformin and phenformin) in blood plasma, urine and lake water samples were developed. The SALLE was performed by mixing samples (plasma (0.2mL), urine or lake water (1.0mL)) with acetonitrile (0.4mL for plasma, 0.5mL for urine or lake water), sodium hydroxide powder was then added for the phase separation. The effects of type of salting-out reagent, type of extraction solvent, volumes of acetonitrile and sample, amount of sodium hydroxide, vortexing and centrifugation times on the extraction efficiency were investigated. The upper layer, containing the biguanides, was directly injected into a HPLC unit using ZIC-HILIC column (150mm×2.1mm×3.5μm) and was detected at 236nm. The method was validated and calibration curves were linear with r2>0.99 over the range of 20-2000μgL-1for plasma and 5-2000μgL-1for urine and lake water samples. The limits of detection were in the range (3.8-5.6)μgL-1, (0.8-1.5)μgL-1and (0.3-0.8)μgL-1for plasma, urine and lake water, respectively. The accuracies in the three matrices were within 87.3-103%, 87.4-109%, 82.2-109% of the nominal concentration for metformin, buformin and phenformin, respectively. The relative standard deviation for inter- and intra -day precision were in the range of 1.0-17% for all analytes in the three matrices.

Matched MeSH terms: Liquid-Liquid Extraction/methods*
Fulltext A Novel Aqueous Two Phase System Composed of Surfactant and Xylitol for the Purification of Lipase from Pumpkin (Cucurbita moschata) Seeds and Recycling of Phase Components

Amid M, Manap MY, Hussin M, Mustafa S

Molecules, 2015 Jun 17;20(6):11184-201.
PMID: 26091076 DOI: 10.3390/molecules200611184

Lipase is one of the more important enzymes used in various industries such as the food, detergent, pharmaceutical, textile, and pulp and paper sectors. A novel aqueous two-phase system composed of surfactant and xylitol was employed for the first time to purify lipase from Cucurbita moschata. The influence of different parameters such as type and concentration of surfactants, and the composition of the surfactant/xylitol mixtures on the partitioning behavior and recovery of lipase was investigated. Moreover, the effect of system pH and crude load on the degree of purification and yield of the purified lipase were studied. The results indicated that the lipase was partitioned into the top surfactant rich phase while the impurities partitioned into the bottom xylitol-rich phase using an aqueous two phase system composed of 24% (w/w) Triton X-100 and 20% (w/w) xylitol, at 56.2% of tie line length (TLL), (TTL is one of the important parameters in this study and it is determined from a bimodal curve in which the tie-line connects two nodes on the bimodal, that represent concentration of phase components in the top and bottom phases) and a crude load of 25% (w/w) at pH 8.0. Recovery and recycling of components was also measured in each successive step process. The enzyme was successfully recovered by the proposed method with a high purification factor of 16.4 and yield of 97.4% while over 97% of the phase components were also recovered and recycled. This study demonstrated that the proposed novel aqueous two phase system method is more efficient and economical than the traditional aqueous two phase system method for the purification and recovery of the valuable enzyme lipase.

Matched MeSH terms: Liquid-Liquid Extraction*
A novel liquid/liquid extraction process composed of surfactant and acetonitrile for purification of polygalacturonase enzyme from Durio zibethinus

Amid M, Manap Y, Azmira F, Hussin M, Sarker ZI

J Chromatogr B Analyt Technol Biomed Life Sci, 2015 Jul 1;993-994:1-8.
PMID: 25973865 DOI: 10.1016/j.jchromb.2015.04.034

Polygalacturonase is one of the important enzymes used in various industries such as food, detergent, pharmaceutical, textile, pulp and paper. A novel liquid/liquid extraction process composed of surfactant and acetonitrile was employed for the first time to purify polygalacturonase from Durio zibethinus. The influences of different parameters such as type and concentration of surfactants, concentrations of acetonitrile and composition of surfactant/acetonitrile on partitioning behavior and recovery of polygalacturonase was investigated. Moreover, the effect of pH of system and crude load on purification fold and yield of purified polygalacturonase were studied. The results of the experiment indicated the polygalacturonase was partitioned into surfactant top rich phase with impurities being partitioned into acetonitrile bottom rich phase in the novel method of liquid/liquid process composed of 23% (w/w) Triton X-100 and 19% (w/w) acetonitrile, at 55.6% of TLL (tie line length) crude load of 25% (w/w) at pH 6.0. Recovery and recycling of components also was measured in each successive step of liquid/liquid extraction process. The enzyme was successfully recovered by the method with a high purification factor of 14.3 and yield of 97.3% while phase components were also recovered and recycled above 95%. This study demonstrated that the novel method of liquid/liquid extraction process can be used as an efficient and economical extraction method rather than the traditional methods of extraction for the purification and recovery of the valuable enzyme.

Matched MeSH terms: Liquid-Liquid Extraction/methods*
Anti-cholinesterase potential of diverse botanical families from Malaysia: Evaluation of crude extracts and fractions from liquid-liquid extraction and acid-base fractionation

Amir Rawa MS, Hassan Z, Murugaiyah V, Nogawa T, Wahab HA

J Ethnopharmacol, 2019 Dec 05;245:112160.
PMID: 31419500 DOI: 10.1016/j.jep.2019.112160

ETHNOPHARMACOLOGICAL RELEVANCE: Enhancement of cholinergic functions in the brain via acetylcholinesterase inhibition is one of the main therapeutic strategies to improve symptoms associated with Alzheimer's or related cognitive deficits. There is a pathophysiological correlation between Alzheimer's and Diabetes Mellitus, as well as inflammation and oxidative stress that may cause cognitive decline.
AIM OF THE STUDY: The present study was intended to evaluate anti-cholinesterase potential of 177 Malaysian plant extracts from 148 species known to have related ethnomedicinal uses such as anti-inflammatory, anti-oxidant, anti-diabetic, epilepsy, headache, memory enhancement and anti-aging.
MATERIALS AND METHODS: Anti-cholinesterase screening against both acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes was performed on the basis of in-vitro colorimetric 96-well microplate-based assay method. Potent active plant extracts were subjected to liquid-liquid extraction and acid-base fractionation for further analysis.
RESULTS: Fifty-seven plant extracts exhibited potent anti-cholinesterase activities (50-100% inhibition) at 200 μg/ml. Majority of the active plants originated from Fabaceae family. Coccoloba uvifera (L.) L. stem extract manifested the lowest IC50 of 3.78 μg/ml for AChE and 5.94 μg/ml for BChE. A few native species including Tetracera indica (Christm. & Panz.) Merr., Cyrtostachys renda Blume and Ixora javanica (Blume) DC. showed cholinesterase inhibition despite limited local medical applications. Further anti-AChE evaluation (50 μg/ml) of 18 potent plant extracts harbored active polar components in butanol and water fractions, except Senna pendula (Willd.) H.S.Irwin & Barneby (leaves and stems), Acacia auriculiformis Benth. (leaves), Artocarpus altilis (Parkinson ex F.A.Zorn) Fosberg (leaves), and Macaranga tanarius (L.) Mull.Arg. (leaves) that showed inhibitory activity in less polar fractions. The acidic extraction of these four plant species improved their inhibition level against AChE.
CONCLUSION: This study rendered a preliminary overview of anti-cholinesterase activity from diverse Malaysian botanical families in which provided the medical relevance toward these native plant species, especially ones with limited ethnobotanical record or practice.

Matched MeSH terms: Liquid-Liquid Extraction
Fulltext Development and validation of RP-HPLC-UV method for the determination of Glipizide in human plasma

Atif M, Khalid SH, Onn Kit GL, Sulaiman SA, Asif M, Chandersekaran A

J Young Pharm, 2013 Mar;5(1):26-9.
PMID: 24023449 DOI: 10.1016/j.jyp.2013.01.005

A simple, sensitive and selective HPLC method with UV detection for determination of Glipizide in human plasma was developed. Liquid-liquid extraction method was used to extract the drug from the plasma samples. Chromatographic separation of Glipizide was achieved using C18 column (ZORBAX ODS 4.6 × 150 mm). The mobile phase was comprised of 0.01 M potassium dihydrogen phosphate and acetonitrile (65:35, v/v) adjusted to pH 4.25 with glacial acetic acid. The analysis was run at a flow rate of 1.5 mL/min with an injection volume was 20 μL. The detector was operated at 275 nm. The calibration curve was linear over a concentration range of 50-1600 ng/mL. Intra-day and inter-day precision and accuracy values were below 15%. The limit of quantification was 50 ng/mL and the mean recovery was above 98%. Freeze-thaw, short-term, long-term and post-preparative stability studies showed that Glipizide in plasma sample was stable. The method may be successfully applied to analyze the Glipizide concentration in plasma samples for bioavailability and bioequivalence studies.

Matched MeSH terms: Liquid-Liquid Extraction
DNA extraction on bio-chip: history and preeminence over conventional and solid-phase extraction methods

Ayoib A, Hashim U, Gopinath SCB, Md Arshad MK

Appl Microbiol Biotechnol, 2017 Nov;101(22):8077-8088.
PMID: 28942548 DOI: 10.1007/s00253-017-8493-0

This review covers a developmental progression on early to modern taxonomy at cellular level following the advent of electron microscopy and the advancement in deoxyribonucleic acid (DNA) extraction for expatiation of biological classification at DNA level. Here, we discuss the fundamental values of conventional chemical methods of DNA extraction using liquid/liquid extraction (LLE) followed by development of solid-phase extraction (SPE) methods, as well as recent advances in microfluidics device-based system for DNA extraction on-chip. We also discuss the importance of DNA extraction as well as the advantages over conventional chemical methods, and how Lab-on-a-Chip (LOC) system plays a crucial role for the future achievements.

Matched MeSH terms: Liquid-Liquid Extraction
Fulltext Reverse micelle Extraction of Antibiotics using an Eco-friendly Sophorolipids Biosurfactant

Chuo SC, Abd-Talib N, Mohd-Setapar SH, Hassan H, Nasir HM, Ahmad A, et al.

Sci Rep, 2018 01 11;8(1):477.
PMID: 29323139 DOI: 10.1038/s41598-017-18279-w

Reverse micelles extraction of erythromycin and amoxicillin were carried out using the novel Sophorolipids biosurfactant. By replacing commonly used chemical surfactants with biosurfactant, reverse micelle extraction can be further improved in terms of environmental friendliness and sustainability. A central composite experimental design was used to investigate the effects of solution pH, KCl concentration, and sophorolipids concentration on the reverse micelle extraction of antibiotics. The most significant factor identified during the reverse micelle extraction of both antibiotics is the pH of aqueous solutions. Best forward extraction performance for erythromycin was found at feed phase pH of approximately 8.0 with low KCl and sophorolipids concentrations. Optimum recovery of erythromycin was obtained at stripping phase pH around 10.0 and with low KCl concentration. On the other hand, best forward extraction performance for amoxicillin was found at feed phase pH around 3.5 with low KCl concentration and high sophorolipids concentration. Optimum recovery of erythromycin was obtained at stripping phase pH around 6.0 with low KCl concentration. Both erythromycin and amoxicillin were found to be very sensitive toaqueous phase pH and can be easily degraded outside of their stable pH ranges.

Matched MeSH terms: Liquid-Liquid Extraction/methods*
Fulltext Bioguided Fractionation of Local Plants against Matrix Metalloproteinase9 and Its Cytotoxicity against Breast Cancer Cell Models: In Silico and In Vitro Study

Hariono M, Rollando R, Karamoy J, Hariyono P, Atmono M, Djohan M, et al.

Molecules, 2020 Oct 14;25(20).
PMID: 33066411 DOI: 10.3390/molecules25204691

Matrix metalloproteinase9 (MMP9) is known to be highly expressed during metastatic cancer where most known potential inhibitors failed in the clinical trials. This study aims to select local plants in our state, as anti-breast cancer agent with hemopexin-like domain of MMP9 (PEX9) as the selective protein target. In silico screening for PEX9 inhibitors was performed from our in house-natural compound database to identify the plants. The selected plants were extracted using methanol and then a step-by-step in vitro screening against MMP9 was performed from its crude extract, partitions until fractions using FRET-based assay. The partitions were obtained by performing liquid-liquid extraction on the methanol extract using n-hexane, ethylacetate, n-butanol, and water representing nonpolar to polar solvents. The fractions were made from the selected partition, which demonstrated the best inhibition percentage toward MMP9, using column chromatography. Of the 200 compounds screened, 20 compounds that scored the binding affinity -11.2 to -8.1 kcal/mol toward PEX9 were selected as top hits. The binding of these hits were thoroughly investigated and linked to the plants which they were reported to be isolated from. Six of the eight crude extracts demonstrated inhibition toward MMP9 with the IC50 24 to 823 µg/mL. The partitions (1 mg/mL) of Ageratum conyzoides aerial parts and Ixora coccinea leaves showed inhibition 94% and 96%, whereas their fractions showed IC50 43 and 116 µg/mL, respectively toward MMP9. Using MTT assay, the crude extract of Ageratum exhibited IC50 22 and 229 µg/mL against 4T1 and T47D cell proliferations, respectively with a high safety index concluding its potential anti-breast cancer from herbal.

Matched MeSH terms: Liquid-Liquid Extraction
Fulltext Benzyl and methyl fatty hydroxamic acids based on palm kernel oil as chelating agent for liquid-liquid iron(III) extraction

Haron MJ, Jahangirian H, Silong S, Yusof NA, Kassim A, Rafiee-Moghaddam R, et al.

Int J Mol Sci, 2012;13(2):2148-59.
PMID: 22408444 DOI: 10.3390/ijms13022148

Liquid-liquid iron(III) extraction was investigated using benzyl fatty hydroxamic acids (BFHAs) and methyl fatty hydroxamic acids (MFHAs) as chelating agents through the formation of iron(III) methyl fatty hydroxamate (Fe-MFHs) or iron(III) benzyl fatty hydroxamate (Fe-BFHs) in the organic phase. The results obtained under optimized conditions, showed that the chelating agents in hexane extract iron(III) at pH 1.9 were realized effectively with a high percentage of extraction (97.2% and 98.1% for MFHAs and BFHAs, respectively). The presence of a large amount of Mg(II), Ni(II), Al(III), Mn(II) and Co(II) ions did affect the iron(III) extraction. Finally stripping studies for recovering iron(III) from organic phase (Fe-MFHs or Fe-BFHs dissolved in hexane) were carried out at various concentrations of HCl, HNO(3) and H(2)SO(4). The results showed that the desired acid for recovery of iron(III) was 5 M HCl and quantitative recovery of iron(III) was achieved from Fe(III)-MFHs and Fe(III)-BFHs solutions in hexane containing 5 mg/L of Fe(III).

Matched MeSH terms: Liquid-Liquid Extraction/methods*
Electrochemically Modulated Liquid-Liquid Extraction for Sample Enrichment

Jajuli MN, Hussin MH, Saad B, Rahim AA, Hébrant M, Herzog G

Anal Chem, 2019 06 04;91(11):7466-7473.
PMID: 31050400 DOI: 10.1021/acs.analchem.9b01674

A new sample preparation method is proposed for the extraction of pharmaceutical compounds (Metformin, Phenyl biguanide, and Phenformin) of varied hydrophilicity, dissolved in an aqueous sample. When in contact with an organic phase, an interfacial potential is imposed by the presence of an ion, tetramethylammonium (TMA+), common to each phase. The interfacial potential difference drives the transfer of ionic analytes across the interface and allows it to reach up to nearly 100% extraction efficiency and a 60-fold enrichment factor in optimized extraction conditions as determined by HPLC analysis.

Matched MeSH terms: Liquid-Liquid Extraction*
Two-stage bile preparation with acetone for recovery of fluorescent aromatic compounds (FACs)

Karami A, Syed MA, Christianus A, Willett KL, Mazzeo JR, Courtenay SC

J Hazard Mater, 2012 Jul 15;223-224:84-93.
PMID: 22608400 DOI: 10.1016/j.jhazmat.2012.04.051

In this study we sought to optimize recovery of fluorescent aromatic compounds (FACs) from the bile of African catfish (Clarias gariepinus) injected with 10mg/kg benzo[a]pyrene (BaP). Fractions of pooled bile were hydrolyzed, combined with ten volumes of methanol, ethanol, acetonitrile, or acetone, centrifuged and supernatants were analyzed by high-performance liquid chromatography with fluorescent detection (HPLC/FL). As well, to test whether FACs were being lost in solids from the centrifugation, pellets were resuspended, hydrolyzed and mixed with six volumes of the organic solvent that produced best FAC recovery from the supernatant, and subjected to HPLC/FL. Highest FAC concentrations were obtained with 2000μl and 1250μl acetone for supernatants and resuspended pellets respectively. FACs concentrations were negatively correlated with biliary protein content but were unaffected by addition of bovine serum albumin (BSA) followed by no incubation indicating that the presence of proteins in the biliary mixture does not simply interfere with detection of FACs. In another experiment, efficiency of acetone addition was compared to two different liquid-liquid extractions (L-LEs). Acetone additions provided significantly higher biliary FACs than the L-LE methods. The new two-stage bile preparation with acetone is an efficient, inexpensive and easily performed method.

Matched MeSH terms: Liquid-Liquid Extraction
Recent development of unconventional aqueous biphasic system: characteristics, mechanisms and applications

Kee PE, Ng TC, Lan JC, Ng HS

Crit Rev Biotechnol, 2020 Jun;40(4):555-569.
PMID: 32283954 DOI: 10.1080/07388551.2020.1747388

Aqueous biphasic system (ABS) is widely used in the recovery, extraction, purification and separation of proteins, enzymes, nucleic acids and antibodies. The ABS with high water content and low interfacial tension offers a biocompatible environment for the recovery of labile biomolecules. Process integration can be achieved using ABS by incorporating multiple-steps of purification, concentration and purification of biomolecules in a single-step operation which often results in high product recovery yield and purity. Conventional ABS is usually formed by aqueous solutions of two polymers or a polymer and a salt above a critical concentration. The high viscosity of polymer-based ABS causes slow phase separation and hinders the mass transfer of biomolecules, whereas polymer/salt ABS is characterized by high ionic strength resulting in the loss of bioactivity of recovered biomolecules. These limitations have encouraged the development of novel ABS which is more cost-effective for various biotechnological applications. This review discusses the characteristics and mechanisms of several types of emerging unconventional ABS using phase-forming components such as hyperbranched polymers, special salts, surfactants, magnetic fields, the addition of nanoparticles and incorporation of various solvent. Moreover, several novel applications of ABS for different separation purposes such as microfluidic-based ABS, ABS bioreactors, application of ABS as an analytical tool, and ABS micropatterning are discussed in this review. In the last section of this review, a comprehensive summary of process integration using ABS for extractive fermentations, bioconversion, crystallization and precipitation is also supplemented for the comprehensive review of various types and applications of ABS in recent years.

Matched MeSH terms: Liquid-Liquid Extraction
Fulltext Antioxidative properties of an extract of Hygrocybe conica, a wild edible mushroom

Khoo, H.E., Chong, E.L., Sia, C.M., Chang, S.K., Yim, H.S.

Malays J Nutr, 2014;20(1):101-111.
MyJurnal

Introduction: Hygrocybe conica (HC), a wild mushroom commonly consumed by the indigenous people (Orang Asli) in Peninsular Malaysia, was assessed for its antioxidant content. Methods: The HC mushroom was extracted using distilled water and the crude extract partitioned using different solvents and open column chromatography to evaluate its potential antioxidant properties. The mushroom extract was partitioned using liquid-liquid extraction into the hexane (Fl), chloroform (F2), butanol (F3) and formic acid (F4) fractions. Based on solvent polarity, the water extract of the mushroom was fractionated into non-polar (FI), semi-polar (Fii), and polar fractions (Fiii) using open column chromato graphy. Antioxidant capacities were determined using DPPH, ABTS, and ferric reducing antioxidant power (FRAP) assays while Folin-Ciocalteu reagent assay was used to determine total phenolic content (TPC). Results: The HC extract had the highest TPC and DPPH scavenging capacity compared to its extract fractions. TE values (ABTS assay) of F2 and F4 were not significantly higher than the HC extract. Among the extract fractions of different polarities, Fiii had the highest antioxidant capacities (DPPH and FRAP) compared to FI and Fii while FRAP values of these fractions were not significantly lower than the FRAP value of HC extract. The HC extract had significantly lower antioxidant capacity than antioxidant standards (ascorbic acid and BHA). Tannie acid as the main bioactive component in HC mushroom was detected using HPLC method. The presence of phenolics in HC extract was also confirmed using TLC. Conclusion: Due to the presence of potent phenolic components, the mycelia of HC could be consumed for potential antioxidative benefits.

Matched MeSH terms: Liquid-Liquid Extraction
Fulltext Phosphorus Removal and Phytonutrients Retention in the Refining of Solvent Extracted Palm-Pressed Mesocarp Fiber Oil

Lau HLN, Tee YS, Chan MK, Teh SS

J Oleo Sci, 2022;71(2):177-185.
PMID: 35110462 DOI: 10.5650/jos.ess21256

Phosphoric acid is used in the refining of palm oil for the removal of phosphatides. The high concentration of phosphorus in solvent extracted palm-pressed mesocarp fiber oil hinders palm oil mills to recover this phytonutrients-rich residual oil in pressed fiber which typically contains 0.1 to 0.2% of total oil yield. This study aimed to refine the palm-pressed mesocarp fiber oil and determine the optimum dosage of phosphoric acid for acid-degumming of palm-pressed mesocarp fiber oil while retaining its phytonutrients. The refining process was carried out with combination of wet degumming, acid degumming, neutralisation, bleaching and deodorization. The optimum dose of phosphoric acid was identified as 0.05 wt.% by incorporating the wet degumming process. The refined palm-pressed mesocarp fiber oil showed a reduction in phosphorus content by 97% (from 901 ppm to 20 ppm) and 97% free fatty acid content removal (from 6.36% to 0.17%), while the Deterioration of Bleachability Index increased from 1.76 to 2.48, which showed an increment of 41%. The refined oil retained the key phytonutrients such as carotenoids (1,150 ppm) and vitamin E (1,540 ppm) that can be further developed into high-value products. The oil meets the quality specification of refined, bleached, and deodorized palm oil while preserving the heat-sensitive phytonutrients, which in turn provides a new resource of nutritious oil.

Matched MeSH terms: Liquid-Liquid Extraction/methods*
Fulltext Optimization for Liquid-Liquid Extraction of Cd(II) over Cu(II) Ions from Aqueous Solutions Using Ionic Liquid Aliquat 336 with Tributyl Phosphate

Lee LY, Morad N, Ismail N, Talebi A, Rafatullah M

Int J Mol Sci, 2020 Sep 18;21(18).
PMID: 32962106 DOI: 10.3390/ijms21186860

This study investigates the separation of two heavy metals, Cd(II) and Cu(II), from the mixed synthetic feed using a liquid-liquid extraction. The current study uses tri-octyl methylammonium chloride (Aliquat 336) as the extractant (with tributyl phosphate (TBP) as a phase modifier), diluted in toluene, in order to investigate the selective extraction of Cd(II) over Cu(II) ions. We investigate the use of ethylenediaminetetraacetic acid (EDTA) as a masking agent for Cu(II), when added in aqueous feed, for the selective extraction of Cd(II). Five factors that influence the selective extraction of Cd(II) over Cu(II) (the equilibrium pH (pHeq), Aliquat 336 concentration (Aliquat 336), TBP concentration (TBP), EDTA concentration (EDTA), and organic to aqueous ratio (O:A)) were analyzed. Results from a 25-1 fractional factorial design show that Aliquat 336 significantly influenced Cd(II) extraction, whereas EDTA was statistically significant for the antagonistic effect on the E% of Cu(II) in the same system. Moreover, results from optimization experiment showed that the optimum conditions are Aliquat 336 concentration of 99.64 mM and EDTA concentration of 48.86 mM-where 95.89% of Cd(II) was extracted with the least extracted Cu(II) of 0.59%. A second-order model was fitted for optimization of Cd(II) extraction with a R2 value of 0.998, and ANOVA results revealed that the model adequately fitted the data at a 5% significance level. Interaction between Aliquat 336 and Cd(II) has been proven via FTIR qualitative analysis, whereas the addition of TBP does not affect the extraction mechanism.

Matched MeSH terms: Liquid-Liquid Extraction/instrumentation; Liquid-Liquid Extraction/methods*
Novel protocol optimized for microalgae lutein used as food additives

Low KL, Idris A, Mohd Yusof N

Food Chem, 2020 Mar 01;307:125631.
PMID: 31634761 DOI: 10.1016/j.foodchem.2019.125631

Lutein available in the current market is derived from marigold petals. However, extensive studies showed that microalgae are rich in lutein content and potentially exploitable for its dietary and other industrial applications. In this study, microwave assisted binary phase solvent extraction method (MABS) was the novel protocol being developed and optimized to achieve maximum lutein recovery from microalgae Scenedesmus sp. biomass. Results showed that 60% potassium hydroxide solution with acetone in the ratio of 0.1 (ml/ml) was the ideal binary phase solvent composition. Empirical model developed using response surface methodology revealed highest lutein content can be recovered through MABS extraction method at 55 °C treatment temperature, 36 min in extraction time, 0.7 (mg/ml) for biomass to solvent ratio, 250 Watt microwave power and 250 rpm stirring speed. This optimized novel protocol had increased the amount of lutein recovered by 130% and shorten the overall extraction time by 3-folds.

Matched MeSH terms: Liquid-Liquid Extraction/methods*
Vasorelaxant effect of water fraction of Labisia Pumila and its mechanisms in spontaneously hypertensive rats aortic ring preparation

Manshor NM, Razali N, Jusoh RR, Asmawi MZ, Mohamed N, Zainol S, et al.

Int J Cardiol Hypertens, 2020 Mar;4:100024.
PMID: 33447753 DOI: 10.1016/j.ijchy.2020.100024

Introduction: Labisia pumila has been reported to possess activities including antioxidant, anti-aging and anti-cancer but there is no report on its vasorelaxant effects.
Objective: This study aims to fractionate water extract of Labisia pumila, identify the compound(s) involved and elucidate the possible mechanism(s) of its vasorelaxant effects.
Methods: Water extract of Labisia pumila was subjected to liquid-liquid extraction to obtain ethyl acetate, n-butanol and water fractions. In SHR aortic ring preparations, water fraction (WF-LPWE) was established as the most potent fraction for vasorelaxation. The pharmacological mechanisms of the vasorelaxant effect of WF-LPWE were investigated with and without the presence of various inhibitors. The cumulative dose-response curves of potassium chloride (KCl)-induced contractions were conducted to study the possible mechanisms of WF-LPWE in reducing vasoconstriction.
Results: WF-LPWE produced dose-dependent vasorelaxant effect in endothelium-denuded aortic ring and showed non-competitive inhibition of dose-response curves of PE-induced contraction, and at its higher concentrations reduced KCl-induced contraction. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) significantly inhibited vasorelaxant effect of WF-LPWE. WF-LPWE significantly reduced the release of intracellular calcium ion (Ca2+) from the intracellular stores and suppressed the calcium chloride (CaCal2)-induced contraction. Nω-nitro-L-arginine methyl ester (L-NAME), methylene blue, indomethacin and atropine did not influence the vasorelaxant effects of WF-LPWE.
Conclusion: WF-LPWE exerts its vasorelaxant effect independently of endothelium and possibly by inhibiting the release of calcium from intracellular calcium stores, receptor-operated calcium channels and formation of inositol 1,4,5- triphosphate. WF-LPWE vasorelaxant effect may also mediated via nitric oxide-independent direct involvement of soluble guanylate cyclase (sGC)/ cyclic guanosine monophosphate (cGMP) pathways.

Matched MeSH terms: Liquid-Liquid Extraction

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