Dilip K. Rai

Characterization of Phenolic Composition in Lamiaceae Spices by LC-ESI-MS/MS

A total of 38 phenolic compounds in the solid/liquid extracts of five Lamiaceae spices, rosemary, oregano, sage, basil, and thyme, were identified in the present study using LC-ESI-MS/MS. These compounds were distributed in four major categories, namely, hydroxycinnamic acid derivatives, hydroxybenzoic acid derivatives, flavonoids, and phenolic terpenes. Among them, the category of flavonoids was the largest, with 17 compounds. Identification of the phenolic compounds was carried out by comparing retention times and mass spectra with those of authentic standards. If standards were unavailable, phenolic compounds were identified on the basis of accurate mass of pseudomolecular [M - H](-) ions and tandem mass spectrometry (MS/MS) data. The results of accurate mass measurements fit well with the elemental composition of the compounds. The diagnostic fragmentation patterns of the compounds during collision-induced dissociation (CID) elucidated the structural information of the compounds analyzed.

Enrichment of polyphenol contents and antioxidant activities of Irish brown macroalgae using food-friendly techniques based on polarity and molecular size

An efficient, food-friendly process for the enrichment of macroalgal phlorotannins from solid-liquid extracts (SLE) of three brown macroalgae, namely Fucus spiralis Linnaeus, Pelvetia canaliculata (Linnaeus) Decaisne & Thuret and Ascophyllum nodosum (Linnaeus) Le Jolis, has been demonstrated. The initial utilisation of molecular weight cut-off (MWCO) dialysis generated fractions of low molecular weight (LMW) (<3.5 kDa) and of high molecular weight (HMW) (3.5-100 kDa and >100 kDa) from cold water, hot water and aqueous ethanolic SLE extracts. An enhancement of the total phenolic content (TPC), radical scavenging abilities (RSA) and ferric reducing antioxidant power (FRAP) in the HMW fractions of 3.5-100 kDa and/or >100 kDa from the cold water and aqueous ethanolic extracts was observed. The initial weak TPC, RSA and FRAP observed in the LMW fractions relative to the HMW fractions were substantially enhanced following a reverse-phase flash chromatography fractionation method. Quadrupole time-of-flight mass spectrometry (Q-Tof-MS) suggests that phlorotannins of varying degrees of phloroglucinol polymerisation are present in LMW fractions of the three brown macroalgal species. The development of a food-friendly process for the extraction and enrichment of phlorotannins from Irish macroalgae is vital to facilitate the use of this valuable resource in future developments of macroalgal-based functional foods.

Laminarin from Irish Brown Seaweeds Ascophyllum nodosum and Laminaria hyperborea: Ultrasound Assisted Extraction, Characterization and Bioactivity

Ultrasound assisted extraction (UAE), purification, characterization and antioxidant activity of laminarin from Irish brown seaweeds Ascophyllum nodosum and Laminarina hyperborea were investigated. UAE was carried out using 60% ultrasonic power amplitude and 0.1 M hydrochloric acid for 15 min. Separately, solid-liquid extraction was carried in an orbital shaker using 0.1 M hydrochloric acid at 70 °C for 2.5 h. UAE with hydrochloric acid resulted in the highest concentration of laminarin, 5.82% and 6.24% on dry weight basis from A. nodosum and L. hyperborea, respectively. Purification of all extracts was carried out using molecular weight cut off dialysis at 10 kDa. Characterization of the laminarin fraction was carried out using matrix assisted laser desorption/ionization time-of-flight mass spectrometry. Antioxidant activity of A. nodosum and L. hyperborea extracts had 2,2-diphenyl-1-picrylhydrazyl (DPPH) inhibition levels of 93.23% and 87.57%, respectively. Moreover, these extracts have shown inihibition of bacterial growth of Staphylcoccus aureus, Listeria monocytogenes, Escherichia coli and Salmonella typhimurium.

Water at room temperature as a solvent for the extraction of apple pomace phenolic compounds.

A fractionation method was used to extract phenolic compounds from apple pomace (AP) involving a first extraction with water and subsequent extractions of the same residue with two different organic solvents. The water extracts obtained contained high amounts of phenolic compounds with high antioxidant capacity. However, the second and third extractions of the same residue still extracted considerable amounts of remaining phenolic compounds, both with significant antioxidant capacities. Liquid chromatography-electrospray ionisation mass spectrometry (LC-ESI/MS) studies showed water to be a good solvent to extract hydroxycinnamic acids, flavonols, flavanols, dihydrochalcones and flavones present in the AP. However, water was not the ideal solvent to extract the quercetin glycosides.

Engineered Synthesis of 7-Oxo- and 15-Deoxy-15-Oxo-Amphotericins: Insights into Structure-Activity Relationships in Polyene Antibiotics

Site-directed mutagenesis and gene replacement were used to inactivate two ketoreductase (KR) domains within the amphotericin polyketide synthase in Streptomyces nodosus. The KR12 domain was inactivated in the DeltaamphNM strain, which produces 16-descarboxyl-16-methyl-amphotericins. The resulting mutant produced low levels of the expected 15-deoxy-15-oxo analogs that retained antifungal activity. These compounds can be useful for further chemical modification. Inactivation of the KR16 domain in the wild-type strain led to production of 7-oxo-amphotericin A and 7-oxo-amphotericin B in good yield. 7-oxo-amphotericin B was isolated, purified, and characterized as the N-acetyl methyl ester derivative. 7-oxo-amphotericin B had good antifungal activity and was less hemolytic than amphotericin B. These results indicate that modification at the C-7 position can improve the therapeutic index of amphotericin B.

Isolation, purification and characterization of antioxidant peptidic fractions from a bovine liver sarcoplasmic protein thermolysin hydrolyzate.

Sarcoplasmic proteins isolated from bovine livers were hydrolyzed using the enzyme thermolysin at 37°C for 2h. The hydrolyzates were filtered through molecular weight cut off membranes (MWCO) and filtrates were obtained. The water activity (a(w)) of unhydrolysed sarcoplasmic protein, full hydrolyzates, 10-kDa and 3-kDa filtrates were below the limit necessary for microbial growth. The antioxidant activities of both filtrates and fractions were assessed using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity assay, the ferric ion reducing antioxidant power (FRAP) assay and the Fe(2+) chelating ability assay. RP-HPLC was used for purification of the full hydrolyzates, the 10-kDa and the 3-kDa filtrates. The peptidic content of the full hydrolyzates, the 10-kDa and the 3-kDa filtrates were assessed using the Dumas method and peptide contents of each fraction were characterized using electrospray quadrupole time-of-flight (ESI-Q-TOF) mass spectrometry with the resultant spectrum analysed using the software programmes Protein Lynx Global Server 2.4. and TurboSEQUEST. Similarities between the amino acid composition of characterized peptides from each fraction and previously reported antioxidant peptides were found. This study demonstrates that meat by-product such as liver can be utilised as raw material for the generation of bioactive peptides with demonstrated antioxidant activities in vitro using the enzyme thermolysin. It is significant as it presents a potential opportunity for meat processors to use their waste streams for the generation of bioactive peptides for potential functional food use.

A Review of Extraction and Analysis of Bioactives in Oat and Barley and Scope for Use of Novel Food Processing Technologies

Oat and barely are cereal crops mainly used as animal feed and for the purposes of malting and brewing, respectively. Some studies have indicated that consumption of oat and barley rich foods may reduce the risk of some chronic diseases such as coronary heart disease, type II diabetes and cancer. Whilst there is no absolute consensus, some of these benefits may be linked to presence of compounds such as phenolics, vitamin E and β-glucan in these cereals. A number of benefits have also been linked to the lipid component (sterols, fatty acids) and the proteins and bioactive peptides in oats and barley. Since the available evidence is pointing toward the possible health benefits of oat and barley components, a number of authors have examined techniques for recovering them from their native sources. In the present review, we summarise and examine the range of conventional techniques that have been used for the purpose of extraction and detection of these bioactives. In addition, the recent advances in use of novel food processing technologies as a substitute to conventional processes for extraction of bioactives from oats and barley, has been discussed.

Anti-inflammatory properties of potato glycoalkaloids in stimulated Jurkat and Raw 264.7 mouse macrophages.

AIMS The potato glycoalkaloids, α-chaconine, α-solanine and solanidine, along with potato peel extracts were investigated for potential anti-inflammatory effects in vitro. Their potential to reduce two biomarkers of inflammation, cytokine and nitric oxide (NO) productions, were assessed in the stimulated Jurkat and macrophage models, respectively. MAIN METHODS Cytokine and nitric oxide productions were stimulated in Jurkat and Raw 264.7 macrophages with Concanavalin A (Con A; 25 μg/ml) and lipopolysaccaride (LPS; 1 μg/ml), respectively. Selective concentrations of glycoalkaloids and potato peel extracts were added simultaneously with Con A or LPS for 24h to investigate their potential to reduce inflammatory activity. KEY FINDINGS α-Chaconine and solanidine significantly reduced interleukin-2 (IL-2) and interleukin-8 (IL-8) productions in Con A-induced Jurkat cells. The potato peel extracts did not influence cytokine production. In LPS-stimulated Raw macrophages, α-solanine, solanidine and two potato peel extracts significantly reduced induced NO production. SIGNIFICANCE Our findings suggest that sub-cytotoxic concentrations of potato glycoalkaloids and potato peel extracts possess anti-inflammatory effects in vitro and with further investigation may be useful in the prevention of anti-inflammatory diseases.

Potential of a renin inhibitory peptide from the red seaweed Palmaria palmata as a functional food ingredient following confirmation and characterization of a hypotensive effect in spontaneously hypertensive rats.

This work examined the resistance of the renin inhibitory, tridecapeptide IRLIIVLMPILMA derived previously from a Palmaria palmata papain hydrolysate, during gastrointestinal (GI) transit. Following simulated GI digestion, breakdown products were identified using mass spectrometry analysis and the known renin and angiotensin I converting enzyme inhibitory dipeptide IR was identified. In vivo animal studies using spontaneously hypertensive rats (SHRs) were used to confirm the antihypertensive effects of both the tridecapeptide IRLIIVLMPILMA and the seaweed protein hydrolysate from which this peptide was isolated. After 24 h, the SHR group fed the P. palmata protein hydrolysate recorded a drop of 34 mm Hg in systolic blood pressure (SBP) from 187 (±0.25) to 153 (± 0.64) mm Hg SBP, while the group fed the tridecapeptide IRLIIVLMPLIMA presented a drop of 33 mm Hg in blood pressure from 187 (±0.95) to 154 (±0.94) mm Hg SBP compared to the SBP recorded at time zero. The results of this study indicate that the seaweed protein derived hydrolysate has potential for use as antihypertensive agents and that the tridecapeptide is cleaved and activated to the dipeptide IR when it travels through the GI tract. Both the hydrolysate and peptide reduced SHR blood pressure when administered orally over a 24 h period.

Mechanistic insight into the formation of tetraarylazadipyrromethenes.

The tetraarylazadipyrromethene chromophore class has gained increasing attention in the past decade for a diverse set of scientific interests and applications. The most direct synthetic route available for their generation is heating of 4-nitro-1,3-diarylbutan-1-ones with an ammonium source in an alcohol solvent. Despite the practical simplicity, the reaction pathway(s) for these conversions are lengthy and unclear. To gain insight into the steps involved, (15)N labeling experiments with MS and NMR analysis were utilized for conversion of 4-nitro-1,3-diphenylbutan-1-one 1 into tetraphenylazadipyrromethene 2 with (15)NH(4)OAc. To permit examination of later stages of the reaction sequence to 2, the (15)N-labeled potential intermediate 3,5-diphenyl-1H-pyrrol-2-amine 10 was synthesized. A study of the dimerization pathway utilizing (15)N-labeled 10 revealed an unprecedented nitrogen rearrangement in the final stages of the pathway involving a ring-opening/closing of a pyrrole ring. Utilizing (15)N labeling experiments we have shown that 2,4-diphenylpyrrole 8 can also react under the reaction conditions with 3,5-diphenyl-2H-pyrrol-2-imine 7 (from oxidation of 10) to produce 2. Overall in the conversion of 1 into 2, two related pathways are ongoing concurrently; the first involves a dimerization of 3,5-diphenyl-2H-pyrrol-2-imine 7, and the other a reaction of 7 with 2,4-diphenylpyrrole 8.