Solid-phase Extraction Fractions Research Articles

Cryoprotective Activity of Different Characterized Fractions Isolated from Enzymatic Hydrolysates of Croceine Croaker (Pseudosciaena crocea).

In this study, ultrafiltration fractions (<3 k Da, LMH; >3 k Da, HMH) and solid-phase extraction fractions (hydrophilic hydrolysate, HIH; hydrophobic hydrolysate, HOH) from trypsin hydrolysate purified from croceine croaker (Pseudosciaena crocea) isolate were obtained to investigate the cryoprotective effects of the different fractions, achieved by means of maceration of turbot fish meat after three freeze-thaw cycles. Alterations in the texture, color, moisture loss, myofibrillar protein oxidation stability and conformation, and microstructure of the fish were analyzed after freezing and thawing. The results demonstrate that HIH maximized the retention of fish texture, reduced moisture loss, minimized the oxidation and aggregation of myofibrillar proteins, and stabilized the secondary and tertiary structures of myofibrillar proteins compared to the control group. In conclusion, the HIH component in the trypsin hydrolysates of croceine croaker significantly contributes to minimizing freeze damage in fish meat and acts as an anti-freezing agent with high industrial application potential.

An Eco-Friendly Extraction and Purification Approach for Obtaining Active Ingredients for Cosmetics from Two Marine Brown Seaweeds.

Brown seaweeds are attracting attention due to their richness in bioactive compounds, in particular, their phlorotannins. We present here a case study of two Fucales, Ascophyllum nodosum and Halidrys siliquosa, sustainably collected, to produce active polyphenols for the cosmetics sector. Phenolic contents of crude extracts, obtained by Accelerated Solvent Extraction (ASE), were more elevated in H. siliquosa at 100.05 mg/g dry weight (DW) than in A. nodosum (29.51 mg/g DW), considering 3 cycles with cell inversion. The temperature of extraction for a high phenolic content and high associated antioxidant activities close to positive controls was 150 °C for both algae and the use of only one cycle was enough. A semi-purification process using Solid-phase Extraction (SPE) was carried out on both ASE crude extracts (one per species). The majority of phlorotannins were found in the ethanolic SPE fraction for A. nodosum and the hydroethanolic one for H. siliquosa. The SPE process allowed us to obtain more concentrated fractions of active phenolic compounds (×1.8 and 2 in A. nodosum and H. siliquosa, respectively). Results are discussed in regard to the exploitation of seaweeds in Brittany and to the research of sustainable processes to produce active natural ingredients for cosmetics.

Two-step dual-layer SPE method to separate antibacterial and antioxidant mushroom compounds

We developed a two-step dual-layer solid-phase extraction (SPE) method to separate and concentrate minor bioactive compounds of golden oyster mushroom (Pleurotus citrinopileatus) methanol extract. The mushroom extract is rich in fatty acids, including the highly abundant (20–35 %) and antibacterial linoleic acid. To eliminate the masking effect of linoleic acid in the bioassay, its removal was achieved by homemade dual-layer SPE that consisted of a lower C18 phase and an upper silica gel phase with the dried-on extract. In the first step the elution was performed with water (aqueous eluate), and then the dried C18 and silica gel phases were eluted separately with methanol (C18 methanol eluate and silica gel methanol eluate, respectively). Thin-layer chromatography (TLC)-effect-directed analysis demonstrated that the aqueous eluate contained sugars and antioxidants (TLC-DPPH); fatty acids were present in the silica gel methanol eluate, in the C18 methanol eluate the biologically active minor secondary metabolites were concentrated (TLC–Bacillus subtilis assay) beside 2 % remained linoleic acid according to HPLC-UV measurement. As a result, the antioxidant and antibacterial metabolites divided, and the low-abundant antibacterial compounds could be detected. Based on this study, the substance found at RF 0.25 by TLC-DPPH played the most significant role in generating the antioxidant effect of the aqueous eluate, equivalent to 5.07 ± 0.09 µg of gallic acid. Moreover, each SPE fraction and raw extract were tested for antibacterial effect using a microplate assay with a non-pathogenic Gram-positive Bacillus subtilis. The P. citrinopileatus extract (MIC-value – 50 µg/mL) and C18 methanol eluate (MIC-value - 25 µg/mL) inhibited the growth of B. subtilis while the other eluates did not.

Bioactivity Assays of Eryngium thyrsoideum; Focusing on Cytotoxic Effects, Antioxidant Activity, and Antimalarial Properties

Background: The genus Eryngium, Apiaceae is widely distributed in the world and used in traditional medicine for different therapeutic purposes. Eryngium thyrsoideum was investigated due to a lack of phytochemical studies on the probable bioactivities of its compounds. Methods: The aerial parts of the plant were extracted using n-hexane, dichloromethane, and methanol (MeOH) by the soxhlet method. The MeOH extract was exposed to C18 Sep-Pak fractionation by a step gradient of MeOH-H2 O. The essential oil was obtained by the Clevenger apparatus. Free-radical scavenging activity of the extracts and fractions was assessed using the DPPH-scavenging activity method. Total flavonoid content was determined by Aluminum chloride colorimetric and phenolic content was determined by using the Folin-Ciocalteu reagent method. α-Glucosidase inhibitory and antimalarial activity of extracts and essential oil and cytotoxic activity of essential oils was also established in this study. Results: The 20% solid phase extraction fraction demonstrated high antioxidant activity (0.13±0.01 mg/mL) and also indicated the most total flavonoid and total phenolic contents (474.20±23.11 and 27.90±0.55 mg/100 g) respectively, in comparison to other fractions. We observed a moderate β-Hematin Formation Assay inhibitory effect of the essential oil (10.37±0.02 mg/mL) and DCM extract (7.64±0.01 mg/mL). Low levels of α-glucosidase inhibition were observed in n-hexane extract (9.78±1.1%) and essential oil of aerial parts (2.7±1.1%). Cytotoxic activity for the essential oils is confirmed by representing respectively IC50 with 2.51 mcg/mL and 1.07 mcg/mL in 24 and 48 hours in MCF-7 cell lines. Conclusion: The presence of phenolic compounds and flavonoids in MeOH extract and fractions is effective on the antioxidant potency of E. thyrsoideum. Essential oil of this plant presented noticeable effects on human breast cancer cell lines.

Gas chromatography with mass spectrometry analysis of 4,4-dimethylsterols and 4-methylsterols in edible oils after their enrichment by means of solid phase extraction

4-Methylsterols (4-M-sterols) and 4,4-dimethylsterols (4,4-D-sterols) are a group of underexplored minor sterols that occur in almost all living organisms. Here, we developed a strategy for the determination of the biochemical precursors of the predominant 4-desmethylsterols in edible oils. Due to their low contribution to the sterol content in the samples, a solid phase extraction (SPE) method was developed for the enrichment of 4-M- and 4,4-D-sterols in the hexane extracts of saponified oils. In a two-fold SPE procedure, the bulk of 4,4-D-sterols was collected in one fraction. The residual sample was subjected to a second SPE step which targeted all 4-M-sterols and low shares of 4,4-D-sterols in one fraction and the predominant 4-desmethylsterols in another one. After silylation of the SPE fractions, gas chromatography with mass spectrometry (GC/MS) was used to analyze 4,4-D- and 4-M-sterols. The results were used to define eight subgroups whose characteristic structural features could be linked with the presence of specific m/z values. These m/z values were measured sensitively by GC/MS operated in selected ion monitoring (SIM) mode. Application of the GC/MS method to eighteen edible oils enabled the detection of 55 mostly very low abundant 4-M- and 4,4-D-sterols. Twenty-four of the 4-M- and 4,4-D-sterols could be assigned and the remaining 31 unknown sterols could be traced back to their basic structures.

Investigating analyte breakthrough under non-linear isotherm conditions during solid phase extraction facilitated by non-targeted analysis with comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry

Solid phase extraction (SPE) sample preparation for the analysis of complex organic mixtures is often applied assuming all analytes of interest will preconcentrate on the stationary phase. This assumption ignores the reality that extraction is a dynamic interactive process and a diverse range of affinities for the stationary phase will result in equally diverse breakthrough volumes due to competitive sorption processes. To study this dynamic interactive process, and further to take advantage of it, we extracted a JP-8 jet fuel spiked with 40 ppm of a polar compound mix with silica and alumina SPE cartridges and analyzed sequential extracted fractions of the fuel to both assess the shifting chemical landscape present in the extraction and the impact of both SPE stationary phases on this process. Tile-based 1v1 comparative analysis (a recently reported extension of tile-based Fisher ratio analysis) was used to discover the (polar) compounds whose concentrations change between extracted fractions, discovering 21 compounds extracted with silica and 27 compounds extracted with alumina with at least a 2-fold change in concentration from the neat sample relative to the first 1 mL pass fraction sample. These compounds were quantified in each fraction to construct concentration ratio profiles, defined as the concentration ratio for a given SPE fraction per analyte compound relative to the analyte concentration in the neat fuel, for which the extraction behavior for each analyte could be assessed. These analyte compounds were found to breakthrough at different rates, with some analytes remaining on the column indefinitely (until extracted with a subsequent polar solvent) and other analytes eluting before the extraction is complete. Furthermore, in a comparison of the effect of selected stationary phase, alumina was found to retain oxygen-containing phenolic compounds to a greater extent than silica. Principal component analysis (PCA) was used to analyze the concentration ratio profiles of the various trace analytes in the JP8 fuel (phenols, indoles, etc.) in the context of their stationary phase affinity (silica or alumina) and competitive sorption behavior.

Prospects for Pharmaceutical of Zingiber officinale Extracts and Fractions by Analysis of the Chemical Markers, Safety Profile, Anti-Lipoxygenase, and Analgesic Activity

Ginger (Zingiber officinale) has gained recognition for its flavoring uses and its numerous health benefits. Ginger can be used in functional foods and nutraceuticals alone or combined with other herbal plants. This study aims to validate the pharmaceutical potential of the Zingiber officinale variety from the Philippines by examining the chemical composition, acute oral toxicity, anti lipoxygenase, and analgesic activities of its extracts and fractions. 6, 8, 10-Gingerols and 6, 8, 10-Shogaols were identified by high-performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) in methanol and hexane extracts. Acute oral toxicity in mice indicated the median lethal dose (LD50) of methanol and hexane extracts of Zingiber officinale, and the 1% IPA-hexane and 50% IPA-hexane fractions obtained by solid phase extraction (SPE) was greater than 2000 mg/kg body weight. The 1% IPA-hexane fraction altered the behavioral pattern of the animals. The anti-inflammatory activity was analyzed by lipoxygenase (LOX) inhibition assay. The IC50 value for the inhibitory action of crude methanol extract on LOX was 218.00 µg/mL. The crude hexane extract IC50 value was 57.40 µg/mL. SPE fractionation of hexane extracts resulted in higher IC50 values; 72.14 µg/mL for the 1% IPAhexane fraction and 98.65 µg/mL for the 50% IPA-hexane fraction. Through in-vitro suppression of LOX, the hexane crude extracts showed more effective anti-inflammatory properties. The analgesic effect was measured using acetic acid-induced writhing in mice. All doses of the methanol and hexane extract inhibited writhing by a percentage ranging from 91-100%. The SPE fractions of the hexane extract have 78-99% inhibition. Both extracts and fractions demonstrated strong analgesic effects in the mice models.

Chemical Composition, Anti-Inflammatory and Analgesic Activities of Extracts and Fractions of Vitex negundo

This study aims to determine chemical components and demonstrate the anti-inflammatory and analgesic activities of Vitex negundo species native to the Philippines. Using high-performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), eleven compounds were tentatively identified from the iridoids, flavonoids, and phenolics group. The anti-inflammatory activity was determined by measuring the inhibition of proinflammatory enzymes, lipoxygenase (LOX), and cyclooxygenase (COX-2). For LOX inhibitory effect, crude methanol extract has an IC50 value of 440.9 µg/mL. Solid-phase extraction (SPE) fractionation results in IC50 values of 196.6 µg/mL and 150.0 µg/mL for the 50% methanol-water and 100% methanol fractions, respectively. The IC50 value of crude hexane extract is 519.7 µg/mL. SPE produced lower IC50 values; 397.5 µg/mL for 2% IPA-hexane fraction and 142.9 µg/mL for 50% IPA-hexane fraction. The inhibition against COX-2 was effective in the 100% methanol fraction with an IC50 of 34.27 μg/mL and 2% IPA-hexane with an IC50 of 6.57 μg/mL. SPE fractions demonstrated more potent anti-inflammatory effects via in-vitro inhibition of LOX for the 100% methanol-water and 50% IPA-hexane or COX-2 for the 100% methanol and 2% IPA-hexane. The analgesic action was evaluated using acetic acid-induced writhing in mice. The percentage inhibition of writhing at all dose levels of methanol and hexane extracts ranged from 90-96% (p&lt;0.05). The SPE fractions from methanol extracts showed inhibition of 73-97% (p&lt;0.05). Both extracts and fractions showed strong analgesic effects in an in-vivo animal model.

Comparison of column chromatography and solid-phase extraction on virgin and aged bituminous maltene phases

Bitumen is a complex mixture consisting of different hydrocarbons, which can be categorised into substance classes called SARA (saturates, aromatics, resins and asphaltenes) fractions. The saturates, aromatics and resins are further referred to as maltenes or maltene phase. To determine the SARA fractions the asphaltenes were separated from the maltenes in a first step. A standard method to further separate the maltenes is column chromatography (CC). Since column chromatography is a time-consuming and solvent-intensive process, an alternative method for separating maltenes using solid-phase extraction (SPE) was investigated. A total of nine bitumen samples of the same type in different ageing states but from different manufacturers were separated with both methods to determine the mass percentages of the fractions for investigating their comparability. The results show that both methods led to different mass percentages of the fractions. However, with the SPE, separating smaller amounts of maltenes is possible while saving a large amount of material, costs, and workload. Linear regression analyses between the colloidal indices with conventional and rheological parameters revealed that the fractions obtained from SPE led to higher coefficients of determination of the linear regression model. The obtained fractions were further structurally compared with FTIR spectroscopy, whereby the spectra of the SPE fractions and the CC fractions did not show any significant differences. The overall consideration of the results concludes that the SPE can replace the CC.

Phytochemistry and Bioactivity of Nepeta racemosa Lam.

Background and objectives: The genus Nepeta is used in traditional medicine for the antiseptic and astringent properties in cutaneous eruptions and snake bites. Nepeta racemosa was investigated here due to the rich source of phytochemical compounds in the Nepeta genus and the lack of any phytochemical studies. Methods: The aerial parts were extracted successively using n-hexane, chloroform, and methanol (MeOH) by maceration method, respectively. The MeOH extract was exposed to C18 Sep-Pak fractionation by a step gradient of MeOH-H2O. Further purification of the fractions by preparative reversed-phase HPLC yielded three compounds. The chemical ingredients of essential oil were determined by GC-MS. Free-radical scavenging activity of the extracts, fractions, as well as their total flavonoid and phenolics contents, were assessed using the DPPH method, AlCl3, and Folin-Ciocalteu reagents, respectively. Results: Phytochemical study of 20% and 40% solid phase extraction fractions of MeOH extract yielded one iridoid (8,9-epi-7-deoxy-loganic acid) and two phenylethanoids (forsythoside B and verbascoside), correspondingly. The 4aα,7α,7aα-nepetalactone (31.70 %), germacrene D (7.39 %), n-hexadecanoic acid (6.47 %), were the main compounds of essentials oil. The MeOH extract demonstrated high activity in terms of antioxidant activity, total phenolics content along with total flavonoids content (0.09 ± 0.01 mg/mL, 1581.80 ± 10.28 mg/100g and 33.01 ± 0.02 mg/100g) as well as 40% SPE fraction (0.01 ± 0.00 mg/mL, 659.20 ± 40.32 mg/100g and 22.5 ± 0.37 mg/100g), respectively. Conclusion: The presence of phenylethanoid derivatives as phenolic compounds appears to be an important antioxidant compound in Nepeta racemosa.

Phytochemical and Antimalarial Effects of Ecballium elaterium (L.) Rich. Growing in Iran

Background: Malaria is a well-recognized parasitic disease and a serious public health problem worldwide, particularly in tropical and subtropical areas. Objectives: This study was conducted to investigate the antimalarial properties of extracts with different polarities from the various parts of Ecballium elaterium (L.) Rich. (or wild cucumber) as a perennial herbaceous plant growing in Gilan and Azerbaijan provinces of Iran. Methods: The air-dried and powdered fruits, seeds, and roots of E. elaterium were extracted using three solvents with different polarities, n-Hexane (n-Hex), dichloromethane (DCM), and methanol (MeOH). The MeOH extract of roots was subjected to fractionalizing by a C18 Sep-Pak cartridge. All extracts and fractions with different polarities were assessed for their antimalarial activity using the cell-free beta-hematin formation test, and the structural groups of the fractions were identified by Thin-Layer Chromatography (TLC). Results: According to our results, the MeOH extracts of the plant’s roots presented considerable antimalarial effects with an IC50 value of 0.124 ± 0.0002 mg/mL. Bioactivity-guided fractionation of root MeOH extract by solid phase extraction (SPE) afforded six fractions. The 20% fraction showed the most potent antimalarial effect with an IC50 value of 0.167 ± 0.002 mg/mL. Moreover, the three fractions of 80%, 60%, and 100% methanol/water demonstrated considerable antimalarial activities. Phytochemical analysis of potent fractions of E. elaterium suggested the presence of flavonoids in 20% and 60% fractions and flavonoids and triterpenoids in 80% and 100% fractions. Conclusions: According to our primary phytochemical investigation on the six SPE fractions, it is recommended to purify the active constituents of the most effective fractions and investigate their biological effects in animal models.

Supernatants of intestinal luminal contents from mice fed high-fat diet impair intestinal motility by injuring enteric neurons and smooth muscle cells.

Damage to enteric neurons and impaired gastrointestinal muscle contractions cause motility disorders in 70% of diabetic patients. It is thought that enteric neuropathy and dysmotility occur before overt diabetes, but triggers of these abnormalities are not fully known. We tested the hypothesis that intestinal contents of mice with and without high-fat diet- (HFD-) induced diabetic conditions contain molecules that impair gastrointestinal movements by damaging neurons and disrupting muscle contractions. Small and large intestinal segments were collected from healthy, standard chow diet (SCD) fed mice. Filtrates of ileocecal contents (ileocecal supernatants; ICS) from HFD or SCD mice were perfused through them. Cultured intact intestinal muscularis externa preparations were used to determine whether ICS and their fractions obtained by solid-phase extraction (SPE) and SPE subfractions collected by high-performance liquid chromatography (HPLC) disrupt muscle contractions by injuring neurons and smooth muscle cells. ICS from HFD mice reduced intestinal motility, but those from SCD mice had no effect. ICS, aqueous SPE fractions and two out of twenty HPLC subfractions of aqueous SPE fractions from HFD mice blocked muscle contractions, caused a loss of nitrergic myenteric neurons through inflammation, and reduced smooth muscle excitability. Lipopolysaccharide and palmitate caused a loss of nitrergic myenteric neurons but did not affect muscle contractions. Unknown molecules in intestinal contents of HFD mice trigger enteric neuropathy and dysmotility. Further studies are required to identify the toxic molecules and their mechanisms of action.

Antioxidant properties of extracts and SPE fractions from Annona muricata leaves

Annona muricata L. (Magnoliales: Annonaceae) is a tropical plant species known for its edible fruit and well known for the medicinal value. In this study, the antioxidant properties of A. muricata leaves is evaluated for different extracts and fractions. The airdried leaves of A. muricata leaves were successively extracted with n-hexane and methanol. The methanolic crude extract was further semi-purified by utilizing solid-phase extraction (SPE) in a mixture of mobile phase aqueous: methanol to methanol and resulted in four semi-purified fractions (ALM 1 – ALM 4). Antioxidant properties of extracts and fractions were evaluated by free radical scavenging activity against 1,1-Diphenyl-2- picrylhydrazyl (DPPH) radicals comparing with standard ascorbic acid, quercetin, and catechin. Results indicated that methanolic extracts showed exceptionally higher radical scavenging activity compared with n-hexane crude extract with IC50 44.21 µg/mL and 302.41 µg/mL, respectively. Meanwhile, fractions ALM 2 (aqueous: methanol; 50: 50) exhibited the highest scavenging activity with the IC50 of 12.46 µg/mL followed by ALM 1 (aqueous: methanol; 80: 20) with 51.44 µg/mL, ALM 3 (aqueous: methanol; 20: 80) with 54.39 µg/mL and ALM 4 (100% methanol) with 89.02 µg/mL, respectively. In conclusion, ALM 2 could be potentially applied as a natural antioxidant agent in both food, nutraceuticals or pharmaceuticals fields for health promotion. However, ALM 2 has to be further purified to study the constituents which possibly condensed mainly with phenolic and flavonoids that known to possess the highest antioxidant effects.

Flavonoid Profiling Analysis by Liquid Chromatography–Tandem Mass Spectrometry

Flavonoids are secondary metabolites with antioxidant, anticancer, and anti‐inflammatory properties. However, no method has yet been reported for the simultaneous profiling analysis for flavonoids of various types. Thus, simultaneous profiling analysis of various flavonoids was performed in this study. Under optimal conditions, the developed method showed good linearity (r ≥ 0.9973) in the range 25–1000 ng/μL with a limit of detection (LOD) of 0.9–12.1 pg. and a limit of quantification (LOQ) of 2.6–36.5 pg., respectively. The repeatability as a percentage of relative standard deviation (% RSD) varied from 0.5 to 5.9 (% RSD). And accuracy as a percentage of relative error (% RE) was from −11.8 to 10.5 (% RE). The validated method was applied to a solid phase extraction fraction of Orostachys japonicus (Maxim.) A. Berger. Six flavonoids of catechin, rutin, quercitrin, quercetin, luteolin, and kaempferol were positively identified and quantified. Therefore, the present method will be useful for the monitoring and screening of flavonoids in natural products.

Brisk Method for Fractionating and Separating Bio-Crude Oil

A new approach for the analysis of bio-crude oils (BCOs) has been qualitatively explored. The analytical scheme is based on the fractionation of BCO through precipitation in water, freeze-drying, solid phase extraction (SPE) and combinations of analytical techniques for the analysis of fractions. Monomeric components in BCO were characterised using gas chromatography coupled with mass spectrometer and flame ionisation detector (GC-MS/FID). The molecular mass distribution of oligomers was determined using gel permeation chromatography (GPC). The fractionation procedure appeared to have succeeded to a large extent as evident in the detection of various components along their expected fraction in the GC-MS/FID analysis. However, a quantitative analysis of the multiple components in different fractions appeared difficult owing to the loss of many volatile fractions through the fractionation procedure. And less than 7 wt. % of the sugar-related components in the acetylated polar SPE fraction were identified.

Biological Activities and Phytochemical Study of Pedicularis wilhelmsiana Fisch Ex. From Iran.

This research paper presents findings of an investigation about antibacterial effect of methanol/water fractions as well as cytotoxic activity of the extracts obtained from Pedicularis wilhelmsiana (Scrophulariaceae) which grows in Azarbaijan/Iran by agar well diffusion method and brine shrimp lethality test successively. Phytochemical study of this plant was determined as well. A combination of solid-phase extraction (SPE), preparative reversed-phase HPLC analysis and spectroscopic means were applied for fractionation, purification, and identification of ingredients respectively. Antimicrobial test demonstrated that 40% and 60% methanol/water fractions were more active than methanolic extract and other SPE fractions. No cytotoxic effect was detected from the extracts of this plant by brine shrimp lethality assay. Phytochemical study of aerial parts of Pedicularis wilhelmsiana (P. wilhelmsiana) afforded two phenylethanoids (verbascoside and martynoside), three iridoids (Aucubin, ipolamiid, 5-hydroxy-8-epi-loganin) and two flavonoids (luteolin, luteolin-7-glucoside) along with mannitol on the basis of spectral evidences (UV, 1H and 13CNMR) as well as comparison with literature data. The findings of this research supported further studies related to antibiotic potential of methanolic extract of P. wilhelmsiana.

Quorum sensing between Gram-negative bacteria responsible for methane production in a complex waste sewage sludge consortium.

Quorum sensing (QS) plays a key role in activating bacterial functions through small molecules called autoinducers. In this study, the QS of Gram-negative bacteria in waste sewage sludge (WSS) was downregulated by adding the quorum quenching enzyme, AiiM lactonase, which cleaved the acyl-homoserine lactone (AHL) autoinducer signals from Gram-negative bacteria, and subsequently methane production was inhibitedby over 400%. The pH was lowered after 2days in the anaerobic fermentation whereas protease activity at the hydrolysis step was almost the same with or without AiiM. The production of acetic acid significantly increased during the fermentation in the presence of AiiM. The bacterial community at day 2 indicated that the population of Gram-positive bacteria increased in the presence of AiiM, and the percentage of Gram-negative bacteria decreased in the WSS containing AiiM. The change in the bacterial community in the presence of AiiM may be due to the different antimicrobial agents produced in the WSS because some of the Gram-positive bacteria were killed by adding the solid-phase extraction (SPE) fraction from the WSS without AiiM. In contrast, the SPE fraction with AiiM had reduced bactericidal activity against Gram-negative bacteria. Thus, bacterial signaling between Gram-negative bacteria is critical for methane production by the microbial consortia.

Simultaneous quantification of epoxy and hydroxy fatty acids as oxidation products of triacylglycerols in edible oils

Epoxy and hydroxy fatty acids are important intermediates during lipid oxidation; quantification of both structures may help evaluate the extent of competition among various lipid oxidation pathways. This article describes a method to simultaneously determine saturated- and unsaturated- epoxy and hydroxy fatty acids derived from oxidation of vegetable oils. The experimental procedures employed transesterification with sodium methoxide, separation of epoxy and hydroxy fatty acid methyl esters (FAME) using solid-phase extraction (SPE), and trimethylsilyl (TMS) derivatization of hydroxy groups. GC–MS was used to identify the epoxy and hydroxy FAME in two different SPE fractions, while GC-flame ionization detection (GC-FID) was used to determine their quantities. Epoxy-octadecanoate/octadecenoate and hydroxy-octadecanoate/octadecenoate/octadecadienoate were determined as lipid oxidation products generated from oxidation of sunflower and canola oils. An isomer of methyl 13-hydroxyoctadeca-9,11-dienoate (13-HODE) TMS ether co-eluted with methyl 15-hydroxyoctadeca-9,12-dienoate TMS ether, which was only present in canola oil; thus, GC–MS-selected ion monitoring (GC–MS–SIM) was used to determine the concentration of 13-HODE. The proposed method has been successfully applied to monitor epoxy and hydroxy fatty acids in sunflower oil and canola oil oxidized at 40 °C.

Optimization and validation of reverse phase HPLC method for qualitative and quantitative assessment of polyphenols in seaweed

A simple reverse phase-high performance liquid chromatography (RP–HPLC) coupled to a diode array detector (DAD) and negative ion electrospray mass spectrometer (ESI–MS) method was developed for simultaneous identification and quantification of phenolic antioxidants in seaweed. The proposed method was validated in terms of linearity, limits of detection (LOD), limits of quantification (LOQ), recovery and intermediate precision. The calibration curves were linear with correlation coefficient ranging from 0.9909 to 0.9997 while the values of LOD (0.26–0.82mg/L), LOQ (0.77–2.50mg/L), recovery (≥97.2%) and precision in terms of retention time (%RSD ≤2.27) and peak area (% RSD ≤5.11) were satisfactory. Brown seaweed Himanthalia elongata used in this study was extracted with 60% methanol and the crude extract was cleaned with SPE (Solid Phase Extraction) cartridge. HPLC-DAD-MS/MS analysis of the SPE fraction allowed the identification of 7 phenolic compounds comprising phlorotannins, hydroxybenzoic acid, hydroxycinnamic acid and flavonols subclasses of polyphenols. Quantitative analysis of these compounds revealed the presence of phloroglucinol (394.1±4.33μg/g), gallic acid (96.3±3.12μg/g), chlorogenic acid (38.8±1.94μg/g), caffeic acid (44.4±2.72μg/g), ferulic acid (17.6±0.85μg/g), myricetin (8.6±0.85μg/g) and quercetin (4.2±0.15μg/g), in the extract. The SPE fraction were tested for antioxidant capacity which were significantly (P <0.05) higher (EC50; 14.5±0.57mg/g) than the ascorbic acid (EC50; 35.8±0.59mg/g) and the crude extract (EC50; 46.3±0.48mg/g). The occurrence of all these phenolic antioxidant compounds in H. elongata extract suggested that the developed method is sensitive enough and reproducible and could be used for qualitative and quantitative assessment of polyphenols in seaweed.

Cytotoxicity, In vitro anti-Leishmanial and fingerprint HPLC- photodiode array analysis of the roots of Trillium govanianum

Trillium govanianum Wall. ex D. Don (Melanthiaceae alt. Trilliaceae), commonly known as ‘nagchhatry’ or ‘teen patra’, distributed from Pakistan to Bhutan about 2500–3800 m altitude is indigenous to Himalayas region. In folk medicine the plant has been reported for the treatment of wound healing, sepsis and in various sexual disorders. This paper reports, for the first time, to evaluate the cytotoxicity, in vitro anti-leishmanial (promastigotes) and fingerprint HPLC-photodiode array analysis of the MeOH extract of the roots of T. govanianum and its solid phase extraction fractions. Reverse phase HPLC-PDA based quantification revealed the presence of significant amount of quercetin, myrecetin and kaemferol ranging from 0.221to 0.528 μg/mg DW. MeOH extract revealed distinguishable protein kinase inhibitory activity against Streptomyces 85E strain with 18 mm bald phenotype. The remarkable toxicity profile against brine shrimps and leishmanial was manifested by MeOH extract with LC50 10 and 38.5 μg/mL, respectively.