Solid-phase Extraction Fractions Research Articles

Evaluation of resazurin microtiter plate assay and HPLC- photodiode array analysis of the roots of Asparagus adscendens

Asparagus adscendens Roxb. (Asparagaceae), is native to the Himalayas. The present study, for the first time, was undertaken to explore the antimicrobial potential, to determine the minimum inhibitory concentration (MIC) values of the methanol extract of the roots of A. adscendens and its solid-phase extraction (SPE) fractions using resazurin microtitre assay against Gram-positive and negative bacterial-registered strains and to carry out HPLC-photodiode array analysis of the SPE fractions. The methanol extract and all SPE exhibited considerable level of antibacterial potential against Gram-positive bacteria (MIC: 2.5–0.009 mg/mL) than against Gram-negative bacteria (MIC: 1.25–2.5 mg/mL). The use of microtitre plates has the advantage of lower cost, fast and quantitative results. Like other Asparagus species, the presence of phenolic compounds in all SPE fractions was evident in the HPLC-PDA data.

Liquid Chromatography Mass Spectrometry Analysis and Cytotoxicity of Asparagus adscendens Roots against Human Cancer Cell Lines.

Background:Asparagus adscendens Roxb. (Asparagaceae), is native to the Himalayas. This plant has been used in the prevention and effective treatment of various forms of cancers.Objective:This paper reports, for the first time, on the cytotoxicity of the methanol (MeOH) extract of the roots of A. adscendens and its solid-phase extraction (SPE) fractions against four human carcinoma cell lines and LC-ESI-QTOF-MS analysis of the SPE fractions.Materials and Methods:Finely powdered roots of A. adscendens were macerated in methanol and extracted through SPE using gradient solvent system (water: methanol) proceeded for analysis on LC-ESI-QTOF-MS and cytotoxicity against four human carcinoma cell lines: breast (MCF7), liver (HEPG2), lung (A549), and urinary bladder (EJ138), using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide assay.Results:The MeOH extract and four SPE fractions exhibited cytotoxicity against all cell lines with the IC50 values ranging from 6 to 79 μg/mL. As observed in other Asparagus species, the presence of saponins and sapogenins in the SPE fractions was evident in the liquid chromatography-mass spectrometry data.Conclusion:It is reasonable to assume that the cytotoxicity of the MeOH extract of the roots of A. adscendens and its SPE fractions, at least partly, due to the presence of saponins and their aglycones. This suggests that A. adscendens could be exploited as a potential source of cytotoxic compounds with putative anticancer potential.SUMMARY The MeOH extract and all solid-phase extraction (SPE) fractions exhibited various levels of cytotoxicity against all cell lines with the IC50 values ranging from 6 to 79 μg/mLThe presence of saponins and sapogenins in the SPE fractions was evident in the Liquid chromatography-mass spectrometry dataDue to the presence of saponins and their aglycones, suggest that A. adscendens could be exploited as a potential source of cytotoxic compounds with putative anticancer potential. Abbreviation used: SPE: Solid-phase extraction, MCF7: Breast cancer cell line, HEPG2: Liver cancer cell line, A549: Lung liver cancer cell line, EJ138: Urinary bladder cancer cell line, MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide, LC-MS: Liquid chromatography-mass spectrometry.

Anti-Proliferative and Antimicrobial Activity of Methanolic Extract and SPE Fractions of Artemisia spicigera

Objectives and Methods: This study was conducted to investigate in vitro, methanolic extract and methanol/water fractions obtained from the arial parts of Artemisia spicigera C. Koch (Asteraceae family) and for evaluation of their antiproliferative effects against HT-29, L-929 and A 549 cell lines by MTT assay at different concentrations (1, 10, 100, 1000 µg/mL). Furthermore, this study aimed to detect antimicrobial activities of the mentioned samples: two Gram-positive (Staphylococcus epidermidis and Staphylococcus aureus), two Gram-negative bacteria (Pseudomonas aeruginosa and Escherichia coli) and a fungi (Candida albicanse), using agar well diffusion method. Results: The IC50 values for antiproliferative activity of the methanolic extract, 20%, 40% and 60% SPE (solid phase extraction) fractions, found to be 345.91 ± 28.77, 442.44 ± 83.22, 220.19 ± 43.13 and 579.90 ± 153.19 μg/mL, respectively; in this case, the maximum inhibition percentage belonged to 40% SPE fraction (220.19 ± 43.13 μg/mL). The total methanolic extract of A. spicigera indicated inhibitory activity against Gram- positive strains, S. epidermidis and S. aureus, with MIC values of 150 mg/mL for both bacteria. Among SPE fractions, 20% was the only active one against Gram possitive species with MIC values of 35 mg/mL for both strains. Conclusions: This study revealed that methanolic extract of A. spicigera and its SPE fractions might be regarded as bioactive natural products, which deserves to be further identification and isolation of cytotoxic and antibacterial phytochemicals from them.

Anti-Proliferative and Antimicrobial Activity of Methanolic Extract and SPE Fractions of Artemisia spicigera

Objectives and Methods: This study was conducted to investigate in vitro, methanolic extract and methanol/water fractions obtained from the arial parts of Artemisia spicigera C. Koch (Asteraceae family) and for evaluation of their antiproliferative effects against HT-29, L-929 and A 549 cell lines by MTT assay at different concentrations (1, 10, 100, 1000 µg/mL). Furthermore, this study aimed to detect antimicrobial activities of the mentioned samples: two Gram-positive (Staphylococcus epidermidis and Staphylococcus aureus), two Gram-negative bacteria (Pseudomonas aeruginosa and Escherichia coli) and a fungi (Candida albicanse), using agar well diffusion method. Results: The IC50 values for antiproliferative activity of the methanolic extract, 20%, 40% and 60% SPE (solid phase extraction) fractions, found to be 345.91 ± 28.77, 442.44 ± 83.22, 220.19 ± 43.13 and 579.90 ± 153.19 μg/mL, respectively; in this case, the maximum inhibition percentage belonged to 40% SPE fraction (220.19 ± 43.13 μg/mL). The total methanolic extract of A. spicigera indicated inhibitory activity against Gram- positive strains, S. epidermidis and S. aureus, with MIC values of 150 mg/mL for both bacteria. Among SPE fractions, 20% was the only active one against Gram possitive species with MIC values of 35 mg/mL for both strains. Conclusions: This study revealed that methanolic extract of A. spicigera and its SPE fractions might be regarded as bioactive natural products, which deserves to be further identification and isolation of cytotoxic and antibacterial phytochemicals from them.

Cytotoxicity of the Roots of Trillium govanianum Against Breast (MCF7), Liver (HepG2), Lung (A549) and Urinary Bladder (EJ138) Carcinoma Cells.

Trillium govanianum Wall. (Melanthiaceae alt. Trilliaceae), commonly known as 'nag chhatri' or 'teen patra', is a native species of the Himalayas. It is used in various traditional medicines containing both steroids and sex hormones. In folk medicine, the rhizomes of T. govanianum are used to treat boils, dysentery, inflammation, menstrual and sexual disorders, as an antiseptic and in wound healing. With the only exception of the recent report on the isolation of a new steroidal saponin, govanoside A, together with three known steroidal compounds with antifungal property from this plant, there has been no systematic pharmacological and phytochemical work performed on T. govanianum. This paper reports, for the first time, on the cytotoxicity of the methanol extract of the roots of T. govanianum and its solid-phase extraction (SPE) fractions against four human carcinoma cell lines: breast (MCF7), liver (HEPG2), lung (A549) and urinary bladder (EJ138), using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide cytotoxicity assay and liquid chromatography and electrospray ionization quadrupole time-of-flight mass spectrometry analysis of the SPE fractions. The methanol extract and all SPE fractions exhibited considerable levels of cytotoxicity against all cell lines, with the IC50 values ranging between 5 and 16 µg/mL. Like other Trillium species, presence of saponins and sapogenins in the SPE fractions was evident in the liquid chromatography mass spectrometry data. Copyright © 2016 John Wiley & Sons, Ltd.

Silver-Ion Solid Phase Extraction Separation of Classical, Aromatic, Oxidized, and Heteroatomic Naphthenic Acids from Oil Sands Process-Affected Water.

The separation of classical, aromatic, oxidized, and heteroatomic (sulfur-containing) naphthenic acid (NA) species from unprocessed and ozone-treated oil sands process-affected water (OSPW) was performed using silver-ion (Ag-ion) solid phase extraction (SPE) without the requirement of pre-methylation for NAs. OSPW samples before SPE and SPE fractions were characterized using ultra performance liquid chromatography ion mobility time-of-flight mass spectrometry (UPLC-IM-TOFMS) to corroborate the separation of distinct NA species. The mass spectrum identification applied a mass tolerance of ±1.5 mDa due to the mass errors of NAs were measured within this range, allowing the identification of O2S-NAs from O2-NAs. Moreover, separated NA species facilitated the tandem mass spectrometry (MS/MS) characterization of NA compounds due to the removal of matrix and a simplified composition. MS/MS results showed that classical, aromatic, oxidized, and sulfur-containing NA compounds were eluted into individual SPE fractions. Overall results indicated that the separation of NA species using Ag-ion SPE is a valuable method for extracting individual NA species that are of great interest for environmental toxicology and wastewater treatment research, to conduct species-specific studies. Furthermore, the separated NA species on the milligram level could be widely used as the standard materials for environmental monitoring of NAs from various contamination sites.

Growth inhibitory effect of Microcystis on Aphanizomenon flos-aquae isolated from cyanobacteria bloom in Lake Dianchi, China

Severe eutrophication of freshwater ecosystem promotes frequent and long-lasting cyanobacteria blooms. In many lakes, the population dynamics of the dominant cyanobacteria species follows seasonal shifts. In Lake Dianchi, a plateau lake in China, blooms involve the alternating seasonal succession of two dominant cyanobacteria species, Aphanizomenon and Microcystis. In this study, we investigated the inhibitory effects of five Microcystis strains on a strain of Aphanizomenon flos-aquae, all isolated during blooms in Lake Dianchi. We used dialysis tubing involved batch and semi-continuous co-culture bioassays to investigate the interaction between these two genera. Our results revealed that different Microcystis inhibited the growth of A. flos-aquae to various degrees, with the strains Microcystis ichthyoblabe kutz 1313 and Microcystis aeruginosa 905 (Ma905) exhibiting the strongest inhibitory effects. The axenic Ma905 did not differ from a non-axenic strain in the inhibitory effect, demonstrating that the growth inhibitory effects we observed were due to Microcystis rather than bacteria. Filtrate of most Microcystis monoculture in different growth phases had a negligible effect on A. flos-aquae. However, the filtrate of Microcystis viridis 1337 (Mv1337) and Ma905 co-cultured with A. flos-aquae from day 21 and day 28, respectively, exhibited stronger inhibitory effects than did their corresponding monoculture filtrates, suggesting that the allelopathic effect of some Microcystis strains is inducible. Results of experiments using solid phase extraction (SPE) fractions of Microcystis monoculture were similar to those from the dialysis tubing bioassays. The microcystin content of the SPE fractions of Ma905 and Mv1337 from day 28 contained 380μgL−1 and 290μgL−1 microcystin-LR (MC-LR), respectively. However, a further bioassay with pure MC-LR with concentrations of 250μgL−1 and 500μgL−1 did not inhibit A. flos-aquae, and further excluded MC-LR as a candidate allelochemical. In summary, our research demonstrates that some Microcystis strains are able to suppress the growth of A. flos-aquae. This finding indicates that allelopathic effects likely play a role in driving the alternating seasonal succession of these dominant cyanobacteria blooms in Lake Dianchi.

Purification and identification of dipeptidyl peptidase (DPP) IV inhibitory peptides from the macroalga Palmaria palmata

Dipeptidyl peptidase (DPP)-IV inhibitory peptides were purified and identified from an aqueous Palmaria palmata protein extract hydrolysed with Corolase PP. The hydrolysate was fractionated by solid phase extraction (SPE) using a C18 matrix followed by semi-preparative reverse phase-high performance liquid chromatography (SP RP-HPLC). IC50 values of 1.47±0.09, 0.54±0.03 and 0.36±0.03mg/ml were obtained for the hydrolysate, the 25% – acetonitrile (ACN) SPE fraction and the most active SP RP-HPLC peptide fraction (SP RP-HPLC 25_F28), respectively. Thirteen peptide sequences were identified following UPLC–ESI MS/MS analysis of SP RP-HPLC 25_F28. Three novel DPP-IV inhibitory peptides, Ile-Leu-Ala-Pro, Leu-Leu-Ala-Pro and Met-Ala-Gly-Val-Asp-His-Ile, with IC50 values in the range 43–159μM were identified. The results indicate that P. palmata derived peptides may have potential as functional food ingredients in the prevention and management of type 2 diabetes.

Molecularly imprinted polymer dedicated to the extraction of glyphosate in natural waters

Three molecularly imprinted polymers (MIPs) have been synthesized in order to bind efficiently glyphosate (GLY) in natural waters (mineral and underground). Since the target analyte is polar and hydrophilic, electrostatic interactions and hydrogen bonds have been favored with two templates (phenylphosphonic acid and diethyl(α-aminobenzyl)-phosphonic acid) and two functional monomers (1-allyl-2-thiourea and methacrylic acid). MIPs have been assessed by comparison of the recoveries obtained with MIP and NIP (non imprinted polymer) by solid-phase extraction (SPE). The selectivity of MIP versus NIP was satisfactory for the three imprinted polymers with a very straightforward protocol: conditioning of 250mg of MIP or NIP packed in 3-mL polypropylene cartridges with 3mL Milli-Q water, loading of Milli-Q water (15mL) spiked with 5mgL−1 of GLY and its metabolite, aminomethylphosphonic acid (AMPA) and elution by 3mL NH4OH (10mM) or 3mL HCl (100mM). SPE fractions were directly analyzed by capillary electrophoresis (CE). Thus, the recoveries of both analytes were greater than 80% for all MIPs and less than 25% for most NIPs. Moreover, the MIP prepared with 1-allyl-2-thiourea as functional monomer and phenylphosphonic acid as template displayed a capacity of 0.033μmol/mg for GLY. However, the substitution of Milli-Q water by mineral water caused the decrease of MIP recoveries, for that, a pretreatment of the sample by ionic exchange resins was set up and succeeded in improving recoveries (about 50% for GLY and 25% for AMPA). Then, groundwaters were spiked with low concentrations of GLY and AMPA (0.5μgL−1) and directly percolated through MIP cartridges. The extractions were carried out by triplicate and the elution fractions were analyzed by UPLC–MS/MS. The results showed no retention of AMPA but a total retention of GLY by MIP.

Assessment of free-radical scavenging activity of Gypsophila pilulifera: assay-guided isolation of verbascoside as the main active component

Gypsophila pilulifera, Boiss & Heldr, Caryophyllaceae, is a perennial medicinal herb that grows in the southwestern region of Turkey. Except for only one report on the isolation of cytotoxic saponins from the underground parts of G. pilulifera, there are no published thorough phytochemical or bioactivity studies on this species. In the present study, the free-radical scavenging activity of extracts and fractions of the stems of G. pilulifera was evaluated, using a slightly modified and more precise version of the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, reported here for the first time. The DPPH assay-guided HPLC-PDA-purification of the active solid-phase extraction fraction (50% methanol in water) of the methanolic extract exhibited verbascoside as the main free-radical scavenger present in this species. The structure of this active compound was resolved by spectroscopy, and the free-radical scavenging potential of verbascoside was determined.

The occurrence of polycyclic aromatic hydrocarbons and their derivatives and the proinflammatory potential of fractionated extracts of diesel exhaust and wood smoke particles

Exposure to combustion emissions, including diesel engine exhaust and wood smoke particles (DEPs and WSPs), has been associated with inflammatory responses. To investigate the possible role of polycyclic aromatic hydrocarbons (PAHs) and PAH-derivatives, the DEPs and WSPs methanol extracts were fractionated by solid phase extraction (SPE), and the fractions were analyzed for more than ∼120 compounds. The pro-inflammatory effects of the fractionated extracts were characterized by exposure of bronchial epithelial lung cells (BEAS-2B). Both native DEPs and WSPs caused a concentration-dependent increase in IL-6 and IL-8 release and cytotoxicity. This is consistent with the finding of a rather similar total content of PAHs and PAH-derivatives. Yet, the samples differed in specific components, suggesting that different species contribute to the toxicological response in these two types of particles. The majority of the IL-6 release and cytotoxicity was induced upon exposure to the most polar (methanol) SPE fraction of extracts from both samples. In these fractions hydroxy-PAHs, carboxy-PAHs were observed along with nitro-amino-PAHs in DEP. However, the biological effects induced by the polar fractions could not be attributed only to the occurrence of PAH-derivatives. The present findings indicate a need for further characterization of organic extracts, beyond an extensive analysis of commonly suspected PAH and PAH-derivatives. Supplemental materials are available for this article. Go to the publisher's online edition of Journal of Environmental Science and Health, Part A, to view the supplemental file.

Red wines attenuate TNFα production in human histiocytic lymphoma cell line: An NMR spectroscopy and chemometrics based study

Nuclear magnetic resonance (NMR) spectroscopy and multivariate data analyses methods are applied to the metabolic profiling of different red wines from Portugal. The water, methanol–water (1:1), and methanol fractions from solid phase extraction (SPE) with C18 resin were subjected to in vitro TNFα activity assay. Principal component analysis allowed the clear separation among the different SPE fractions according to the activity. Various supervised data reduction algorithms were tested and compared to highlight the TNFα inhibition by SPE fractions of wines. Partial least squares-discriminant analysis and orthogonal bidirectional PLS-DA were found most effective in discriminating the samples with different activities. By calculating variable importance in the projection, the active ingredients in the high activity samples have been identified as caftaric acid, quercetin, and catechin. Among the different vintages, samples from 2010 vintage were found with maximum anti-TNFα activity. The effectiveness of NMR spectroscopy in combination with chemometrics to identify the possible bioactivity in the several crude extracts is highlighted.

In Vitro Inhibition of the Transcription Factor NF‐κB and Cyclooxygenase by Bamboo Extracts

Several bamboo species have been used in traditional medicine for the treatment of inflammatory conditions. The present study evaluates the in vitro anti-inflammatory properties of the traditionally used bamboo species Phyllostachys nigra (Lodd.) Munro and Sasa veitchii (Carr.) Rehder to explore their future research opportunities and therapeutic potential as anti-inflammatory agents. The extracts were evaluated for their potential inhibitory activity at the level of NF-κB-induced gene expression and suppression of cyclooxygenase (COX)-1 and COX-2 enzyme activities, representative pharmacological targets for the anti-inflammatory action of glucocorticoids and non-steroidal anti-inflammatory drugs, respectively. The activity of P. nigra (Lodd.) Munro and S. veitchii (Carr.) Rehder was compared with bamboo species without traditional anti-inflammatory indications. High-performance liquid chromatography with diode-array detection and liquid chromatography-tandem mass spectrometry analyses were performed to phytochemically characterize the extracts. P. nigra (Lodd.) Munro leaf extract potently inhibited NF-κB-induced gene expression, while S. veitchii (Carr.) Rehder leaf extract exerted a selective COX-2 inhibition. The crude extracts consistently showed a more potent bioactivity than the solid phase extraction fractions. P. nigra (Lodd.) Munro and S. veitchii (Carr.) Rehder both exert anti-inflammatory properties, but act via a different molecular mechanism.

Antioxidative Properties of Defatted Dabai Pulp and Peel Prepared by Solid Phase Extraction

Solid phase extraction (SPE) using Sep-Pak® cartridges is one of the techniques used for fractionation of antioxidant compounds in waste of dabai oil extraction (defatted dabai parts). The aim of this study was to determine the phenolic compounds and antioxidant capacity in crude extracts and several SPE fractions from methanolic extract of defatted dabai pulp and peel. Based on SPE, Sep-Pak® cyanopropyl and C18 cartridges were used to fractionate the antioxidant-rich crude extracts into water and methanolic fractions. Analyzed using LC-MS, flavonoids, anthocyanins, saponin derivatives and other unknown antioxidative compounds were detected in the defatted dabai crude extracts and their SPE fractions. Anthocyanins were the major phenolic compounds identified in the defatted dabai peel and detected in most of the SPE fractions. Methanolic fractions of defatted dabai parts embraced higher total phenolics and antioxidant capacity than water fractions. This finding also revealed the crude extracts of defatted dabai peel have the most significant antioxidant properties compared to the methanolic and water fractions studied. The crude extract of defatted dabai parts remain as the most potent antioxidant as it contains mixture of flavonoids, anthocyanins and other potential antioxidants.

Isolation and estimation of the ‘aromatic’ naphthenic acid content of an oil sands process-affected water extract

The naphthenic acids of oil sands process-affected water (OSPW) are said to be important toxicants. The major acids are stated to have alicyclic structures and recently, numerous of these have been identified, but some evidence suggests ‘aromatic’ acids are also present. The proportions of such acids have not been reported because they exist in so-called supercomplex mixtures with the alicyclic species. Their contribution to the toxicity of OSPW, if any, is therefore unknown. Here we report the use of multidimensional comprehensive gas chromatography–mass spectrometry (GC×GC–MS) with polar first dimension and non-polar second dimension GC columns and argentation solid phase extraction, to separate methyl esters of the acids of an OSPW supercomplex, into distinct fractions. A major fraction (ca 70%) was shown to contain acids (methyl esters) previously identified as alicyclic species. Authentic adamantane acid methyl esters were shown to chromatograph in this fraction. This fraction was isolated by argentation solid phase extraction (SPE) by elution with hexane. GC–MS and GC×GC–MS confirmed this to be the major fraction in the original supercomplex containing alicyclic acids (methyl esters). A second fraction shown to contain monoaromatic acids (methyl esters) by GC×GC–MS was unexpectedly abundant (ca 30% relative to the acyclic acids). The naphtheno-aromatic dehydroabietic acid was confirmed by co-injection with an authentic compound and several acids previously tentatively identified as naphtheno-monoaromatics were present. This fraction was isolated by argentation SPE by elution with more polar 5% diethyl ether in hexane. GC–MS and GC×GC–MS confirmed that the fraction represented a significant proportion of the original supercomplex. A further fraction, eluting from the argentation SPE column with more 5% diethyl ether in hexane in the same retention volume as authentic methyl naphthoate, contained, in addition to some of the second fraction, a third, much more minor complex. This had somewhat similar GC×GC retention characteristics to that of methyl naphthoate and the methyl ester of authentic fluorene-9-carboxylic acid. Spectra are reported. Non-alicyclic, mono- and possibly diaromatic acids are a much more quantitatively significant proportion of OSPW than previously realised. The individual acids need to be better identified and the toxicity of these and other SPE fractions studied in order to assess any possible environmental effects of OSPW.

Glycomic Analysis of Tear and Saliva in Ocular Rosacea Patients: The Search for a Biomarker

The purpose of this study was to study changes in glycosylation in tear and saliva obtained from control and ocular rosacea patients in order to identify potential biomarkers for rosacea. Tear fluid was collected from 51 subjects (28 healthy controls and 23 patients with ocular rosacea). Saliva was collected from 42 of the same subjects (25 controls and 17 patients). Pooled and individual samples were examined to determine overall glycan profiles and individual variations in glycosylation. O-and N- glycans were released from both patients and control subjects. Released glycans were purified and enriched by solid-phase extraction (SPE) with graphitized carbon. Glycans were eluted based on glycan size and polarity. SPE fractions were then analyzed by high-resolution mass spectrometry. Glycan compositions were assigned by accurate masses. Their structures were further elucidated by tandem mass spectrometric using collision-induced dissociation (CID), and specific linkage information was obtained by exoglycosidase digestion. N- and O-glycans were released from 20-μL samples without protein identification, separation, and purification. Approximately 50 N-glycans and 70 O-glycans were globally profiled by mass spectrometry. Most N-glycans were highly fucosylated, while O-glycans were sulfated. Normal tear fluid and saliva contain highly fucosylated glycans. The numbers of sulfated glycans were dramatically increased in tear and saliva of rosacea patients compared to controls. Glycans found in tear and saliva from roseatic patients present highly quantitative similarity. The abundance of highly fucosylated N-glycans in the control samples and sulfated O-glycans in ocular rosacea patient samples may lead to the discovery of an objective diagnostic marker for the disease.

Bioactivity of Centaurea persica boiss. (Asteraceae)

The free-radical-scavenging property, antibacterial activity and brine shrimp toxicity of petroleum ether (PE), dichloromethane (DCM) and methanol (MeOH) extracts of Centaurea persica, a Turkish medicinal plant, were assessed using the 2,2-diphenyl-1-picryl-hydrazyl (DPPH) assay, the resazurin microtiter plate based assay, and the brine shrimp lethality assay, respectively. Additionally, the disc diffusion assay was also used to assess antibacterial activity. Only the MeOH extract of C. persica exhibited a significant free-radical-scavenging property in the DPPH assay, with an RC50 value of 0.025 mg/mL. However, in the TLC-based qualitative assay, petroleum ether and DCM extracts showed an extremely low level of free-radical-scavenging property. Among the solid-phase extraction fractions of the MeOH extract, the fractions eluted with 60% and 80% MeOH in water exhibited the highest level of free-radicalscavenging activity (RC50 = 0.010 and 0.015 mg/mL, respectively). While DCM extract showed reasonable antibacterial activity against five out of the nine test strains both in the disc diffusion assay and in the resazurin assay, the MeOH extract was highly active against both Escherichia coli and ampicillin-resistant E. coli strains. Among the solid-phase extraction fractions of the MeOH extract, fractions eluted with 80% MeOH in water and 100% MeOH displayed significant antibacterial potencies against both E. coli species. None of the extracts showed any significant toxicity towards brine shrimps (LD50 = >1.00 mg/mL).

Isolation of activity and partial characterization of large non-proteinaceous lytic allelochemicals produced by the marine dinoflagellate Alexandrium tamarense

Certain strains of the toxigenic dinoflagellate Alexandrium tamarense produce potent allelochemicals with lytic activity against a wide variety of marine microorganisms. Our efforts to characterize these allelochemicals from a lytic strain focused on the less polar components because of their higher lytic activity. Fractionation and partial purification after solid phase extraction (SPE) were achieved via alternative chromatographic methods, namely HPLC separation on C8 and HILIC phases. Through MALDI-TOF mass spectrometry we compared the mass differences in SPE, C8 HPLC, and HILIC fractions between a lytic and non-lytic strain of A. tamarense. Several large species with masses between 7 kDa and 15 kDa were found in the HILIC lytic fraction by MALDI-TOF MS. Tryptic digestion and tryptic digestion-coupled size-exclusion chromatography (SEC) suggested that the lytic compounds are large non-proteinaceous molecules (<23.3 kDa, trypsin). Although there is no direct proof that the large molecules found in the lytic HILIC fraction are responsible for the lytic activity of this fraction, the mass range deduced from SEC strongly supports this hypothesis. Total sugar content analysis showed that the lytic HILIC fraction contained two-fold more sugar than the non-lytic one. Nevertheless, the low percentage of saccharide per dry mass equivalent (0.18 ± 0.01%) indicates that sugar residues are likely not a major component of the lytic compounds. We concluded that at least one group of lytic allelochemicals produced by A. tamarense comprise a suite of large non-proteinaceous and probably non-polysaccharide compounds between 7 kDa and 15 kDa.

Different Solid Phase Extraction Fractions of Tualang (Koompassia excelsa) Honey Demonstrated Diverse Antibacterial Properties Against Wound and Enteric Bacteria

Different Solid Phase Extraction Fractions of Tualang (Koompassia excelsa) Honey Demonstrated Diverse Antibacterial Properties Against Wound and Enteric Bacteria

Identification and inhibitory properties of multifunctional peptides from pea protein hydrolysate.

Pea protein isolate was hydrolyzed with alcalase, and the hydrolysate passed through a 1 kDa cutoff ultrafiltration membrane. The permeate was freeze-dried and fractionated on a cationic solid-phase extraction (SPE) column. All fractions were tested for their inhibitory activities against angiotensin-converting enzyme (ACE), renin, and calmodulin-dependent phosphodiesterase 1 (CaMPDE). With the exception of the first eluted fraction, inhibitory properties of the SPE fractions against CaMPDE (but not ACE and renin) were directly related to cationic character (residence time on the column). However, the fraction that eluted with 1% ammonium hydroxide (SPE 1%) had the highest peptide yield and was subsequently fractionated using two consecutive rounds of reversed-phase high-performance liquid chromatography to obtain three peaks with major peptides identified as IR, KF, and EF by ultra performance liquid chromatography-tandem mass spectrometry. The three dipeptides showed weak inhibitory properties toward CaMPDE but strong inhibitions (IC50 values <25 mM) of ACE and renin. In general, the peptides had higher potency against ACE than against renin. It is indicated from our results that these peptides may be used as potential ingredients to formulate multifunctional food products and nutraceuticals.