HPLC ESI-MSn Research Articles

Impact of different extraction solvents and techniques on the biological activities of Cirsium yildizianum (Asteraceae: Cynareae)

Several Cirsium species have been used in folk medicine for the management of human ailments. However, there is a paucity of scientific data regarding their biological activity as in the case of the new species, C. yildizianum, from Anatolia, Turkey. The present study endeavours for the first time to appraise the antioxidant and enzyme inhibitory activity of C. yildizianum. The phytochemical profiles of C. yildizianum extracts obtained using homogeniser-assisted extraction (HAE), ultrasound-assisted extraction (UAE), Soxhlet extraction, maceration, decoction, and infusion, using methanol or water as extraction solvents, was determined by high-performance liquid chromatography with electrospray ionization mass spectrometric detection (HPLC–ESI-MSn) analysis. In all extracts, flavonoids were the most abundant compounds, particularly luteolin and apigenin glycosides. HAE methanol extract presented the highest phenolic (37.10 mg gallic acid equivalent/g) and flavonoid (46.78 mg rutin equivalent/g) contents. Likewise, HAE-methanol extract showed potent radical scavenging (40.76 and 68.13 mg Trolox equivalent [TE]/g, for DPPH and ABTS, respectively) and reducing properties (127.62 and 89.95 mg TE/g, for CUPRAC and FRAP, respectively). HAE-methanol extract showed inhibitory activity against acetylcholinesterase (AChE) (3.57 mg galantamine equivalent [GALAE]/g). UAE-methanol extract was a potent inhibitor of butrylcholinesterase (BChE) (2.72 mg GALAE/g) and tyrosinase (121.06 mg kojic acid equivalent/g) However, poor inhibition was recorded for enzymes targeted in the management of diabetes type II, namely α-amylase and α-glucosidase. On the other hand, potent metal chelating property was observed for water extracts. This study provides comprehensive scientific information on the phytochemical profile of C. yildizianum extracted using different procedures and extraction solvents, which might be considered as valuable baseline data for future bioproducts development.

Screening bioactive components of Glycyrrhiza uralensis Fisch. with isolated perfused lung extraction and HPLC-ESI-MSn analysis

The isolated perfused rat lung (IPL), coupled with high performance liquid chromatography\ andem mass spectrometry analysis (HPLC–ESI-MSn), has been developed as a tool for screening bioactive components in Glycyrrhiza uralensis Fisch. (GU). First, IPL was perfused with the water extract of GU (EGU), the bioactive components in the EGU would selectively combine to the receptors or channels of lung. By changing the pH of perfused solution, the combined components were eluated and then detected by HPLC-ESI-MSn. Four compounds were detected in the desorption eluate of IPL, among these compounds, liquiritin (1), ononin (2) and glycyrrhizic acid (4) were identified by comparing with the chromatography of the standards, while licorice-saponin G2 (3) were determined by analysis of the structure clearage characterization of mass spectrometry. Then, due to the lack of compound 3 sample, compounds 1, 2 and 4 with respective concentrations of 50 μM, 5 μM, 500 nM, 50 nM and 5 nM were applied to evaluate the protective effect of pulmonary epithelial cells (PEC, A549 cell) injury induced by lipopolysaccharide (LPS) for anti-inflammatory activity assessment. The results showed that except the 5 nM group of compound 1, 5 nM and 50 nM groups of compound 2, all other groups could remarkably inhibit the PEC injury (vs LPS group, 2-500 nM groups: p < 0.05; other groups: p < 0.01), all compound showed the dose-dependent effect. In conclusion, IPL coupled with HPLC-ESI-MSn was successfully used to screen the anti-inflammatory components of GU for the first time. The application of IPL coupled with HPLC-ESI-MSn for screening bioactive components of TCMs is rapid, convenient and reliable, and the isolated perfused technology could be extended to isolated heart, liver, kidney, and so on.

Characterization of the Phytochemical Profiles and Biological Activities of Ajuga chamaepitys subsp. chia var. chia and Ajuga bombycina by High-Performance Liquid Chromatography–Electrospray Ionization–Tandem Mass Spectrometry (HPLC–ESI–MSn)

This study investigates into the pharmacological potential of three solvent extracts (ethyl acetate, methanol, and water) of two Ajuga species (Ajuga chamaepitys subsp. chia var. chia and Ajuga bombycina) based on their antioxidant activity and enzyme inhibitory effects along with establishing the phytochemical profile. Spectrophotometric and high-performance liquid chromatography–electrospray ionization–tandem mass spectrometry (HPLC–ESI–MSn) were used to determine the total and individual phytocompounds, respectively. Antioxidant potential was assessed using different assays such as DPPH, ABTS, CUPRAC, FRAP, phosphomolybdenum, and metal chelation. Enzyme inhibitory effects were studied against acetylcholinesterase, butyrylcholinesterase, tyrosinase, α-amylase, and α-glucosidase. The aqueous extract of both plants showed better ABTS scavenging, FRAP, and metal chelating activities. The methanol extracts displayed the highest tyrosinase inhibitory and antioxidant activity in the phosphomolybdenum assay while the ethyl acetate extracts of both plants showed better butyrylcholinesterase (BChE), α-amylase, and α-glucosidase inhibition. The total phenolic content was highest in the aqueous extract of A. chamaepitys while the methanolic extract of A. bombycina showed the highest flavonoid content. Identification by HPLC–ESI–MSn revealed the presence of some individual compounds including phenolic acids, flavonoids, iridoid glycosides, phenylethanoid glycosides, and other compounds. To conclude, both A. chamaepitys and A. bombycina can be considered as rich sources of phytocompounds to manage chronic diseases.

A new method based on supercritical fluid extraction for polyacetylenes and polyenes from Echinacea pallida (Nutt.) Nutt. roots

The genus Echinacea (Asteraceae) includes species traditionally used in phytotherapy. Among them, Echinacea pallida (Nutt.) Nutt. root extracts are characterized by a representative antiproliferative activity, due to the presence of acetylenic compounds.In this study, supercritical fluid extraction (SFE) was applied and compared with conventional Soxhlet extraction (SE) in order to obtain a bioactive extract highly rich in polyacetylenes and polyenes from E. pallida roots. The composition of the extracts was monitored by means of HPLC–UV/DAD and HPLC–ESI–MSn by using an Ascentis Express C18 column (150mm×3.0mm I.D., 2.7μm, Supelco, Bellefonte, PA, USA) with a mobile phase composed of (A) water and (B) acetonitrile, under gradient elution. By keeping SFE time at the threshold of 1h (15min static and 45min dynamic for 1 cycle) with the oven temperature set at 40–45°C and 90bar of pressure, an overall extraction yield of 1.18–1.21% (w/w) was obtained, with a high selectivity for not oxidized lipophilic compounds.The biological activity of the extracts was evaluated against human non-small lung A549 and breast carcinoma MCF-7 cancer cell lines. The cytotoxic effect of the SFE extract was more pronounced towards the MCF-7 than the A549 cancer cells, with IC50 values ranging from 21.01±2.89 to 31.11±2.l4μg/mL; cell viability was affected mainly between 24 and 48h of exposure.The results show the possibility of a new “green” approach to obtain extracts highly rich in genuine polyacetylenes and polyenes from E. pallida roots. The bioactivity evaluation confirmed the cytotoxicity of E. pallida extracts against the considered cancer cell lines, especially against MCF-7 cells, thus suggesting to represent a valuable tool for applicative purposes in cancer prevention.

Phenolic profiles of Lauraceae plant species endemic to Laurisilva forest: A chemotaxonomic survey

In this work, the phenolic composition of several trees endemic to Madeira archipelago (Portugal) was studied. Specifically, the leaves of the most relevant species of the Lauraceae family (Laurus novocanariensis, Apollonias barbujana, Ocotea foetens, and Persea indica) have been analyzed. The screening of the main phenolic compounds in their methanol extracts has been performed by high-performance liquid chromatography with electrospray ionization mass spectrometric detection (HPLC-ESI–MSn), identifying or tentatively characterizing almost 100 compounds, including a high number of proanthocyanidins (A- and B-type), which have been reported to present remarkable health benefits. Thirty-four compounds have been quantified, observing total individual phenolic contents (TIPCs) between 18.43 and 88.99mgg−1 dry extract, with the lowest TIPC in O. foetens and the highest in A. barbujana.

Structural deduction of pregnane glycosides from Dregea sinensis Hemsl by high-performance liquid chromatography and electrospray ionization-tandem mass spectrometry

In the search for novel natural products in the plant, particularly those with potential bioactivity, the MS fragmentation behavior of nine pregnane glycosides previously isolated from Dregea sinensis Hemsl was investigated by electrospray ionization-multistage tandem mass spectrometry (ESI–MSn) equipped with an ion trap analyzer. Furthermore, by high-performance liquid chromtography (HPLC) coupled to ESI–MSn, the thirty-eight unknown pregnane glycosides, combined with six known pregnane glycosides were deduced on the basis of the fragmentation patterns observed from the crude EtOAc extract of D. sinensis Hemsl. This study shows that the HPLC-ESI–MSn is an effective method to rapid and accurate online identification of pregnane glycosides from plant extracts.

Analysis of ginsenoside content, functional genes involved in ginsenosides biosynthesis, and activities of antioxidant enzymes in Panax quinquefolium L. adventitious roots by fungal elicitors

In this study, Candida tropicalis, Aspergillus niger, and a mixture of their cultures were previously used as fungal elicitors in Panax quinquefolium adventitious root culture to enhance ginsenoside production. The result showed that ginsenoside content (11.47 ± 0.54 mg g−1) was significantly enhanced to 3.25-fold of the control group after treatment with 400 mg L−1 C. tropicalis. The polysaccharide content (213.01 ± 18.63 mg g−1) reached the highest when A. niger was at the concentration of 200 mg L−1. HPLC–ESI-MSn analysis was performed, showing that ginsenosides Rh1 and Ro were identified after treatment with fungal elicitors. Furthermore, we found that C. tropicalis, A. niger, and mix elicitor significantly up-regulated the expression of the squalene synthase, squalene epoxidase, dammarenediol synthase, β-amyrin synthase, protopanaxadiol synthase, protopanaxatriol synthase, and UDP-glycosyltransferase. SDS-polyacrylamide gel electrophoresis was used to analysis the changes of protein. A new band was discovered at the size of 75 kDa. In addition, it found that fungal elicitors significantly increased the activities of catalases and peroxidase.

Fungal elicitors enhance ginsenosides biosynthesis, expression of functional genes as well as signal molecules accumulation in adventitious roots of Panax ginseng C. A. Mey

In this work, we selected three fungi strains (Aspergillus niger, Aspergillus flavus and Aspergillus oryzae) as elicitors prepared from mycelium or fermentation broth to improve ginsenosides production in adventitious roots culture. The results showed that ginsenosides production (29.90±4.67mgg−1) was significantly enhanced upon elicitation with 200mgL−1A. niger elicitor prepared from mycelium, which was 3.52-fold of untreated group. HPLC-ESI–MSn analysis was performed, showing that ginsenoside Rb3 was present after treatment with the A. niger. Furthermore, we found that A. niger significantly enhanced accumulation of Nitric oxide (NO), salicylic acid (SA) and jasmonic acid (JA) involved in plant defense response, and significantly up-regulated the expression of the geranyl diphosphate synthase (GPS), farnesyl diphosphate synthase (FPS), squalene synthase (SS), squalene epoxidase (SE), dammarenediol synthase (DS), Two cytochrome P450 (CYP) genes (CYP716A47 and CYP716A53v2) and three UDP-glycosyltransferases (UGT) genes (UGTAE2, UGT94Q2 and UGTpg100).

Ulex europaeus: from noxious weed to source of valuable isoflavones and flavanones

The screening and quantification of the main phenolic compounds in leaves and flowers of Ulex europaeus (gorse) was carried out by high-performance liquid chromatography with electrospray ionization mass spectrometric detection (HPLC-ESI–MSn) after ultrasound-assisted extraction with methanol. About 98% of compounds corresponded to flavonoids, distributed as flavonols, flavones, isoflavones and flavanones. Flavonols were mainly quercetin glucosides; most of the found flavones were apigenin derivatives and the isoflavone group was dominated by glycitin. The flavanone group was composed mainly of liquiritigenin derivatives, substances usually found in liquorice (Glycyrrhiza ssp) and associated with high pharmacological relevance; in Ulex they represent about 25% of total polyphenols content. Phenolic acids and saponins were also detected, as minor components. In vitro antioxidant activity (nitric oxide, superoxide assays, ABTS and DPPH assays) of leaves and flowers, and their inhibitory effects towards digestive enzymes related to carbohydrate metabolism (α-glucosidase and α-amylase) were also studied.

Phytochemicals from Camellia nitidissima Chi inhibited the formation of advanced glycation end-products by scavenging methylglyoxal

The objective of this study was to investigate the inhibitory effects of Camellia nitidissima Chi (CNC) on the advanced glycation end-product (AGE) formation. CNC was extracted with ethanol and further separated into dichloromethane, ethyl acetate, n-butanol, and water soluble fractions. Ethyl acetate fraction had the highest total phenolic and quercetin content compared with other fractions. Sixteen phenolic compounds were identified using HPLC Triple TOF MS/MS. Bovine serum albumin (BSA)–glucose assay showed that dichloromethane and ethyl acetate fraction inhibited AGE formation by 88.1% and 87.5% at 2.5mg/mL. BSA–methylglyoxal assay showed that ethyl acetate fraction inhibited 54.1% AGE formation while dichloromethane fraction inhibited 28.1%. Over 96.0% of methylglyoxal was scavenged by different fractions within 12h. Both mono- and di-methylglyoxal quercetin adducts were identified after incubating quercetin with methylglyoxal using HPLC–ESI-MSn. The results in this study suggest that CNC extracts inhibited AGEs formation in part through scavenging methylglyoxal by phenolic compounds.

HPLC-ESI-MS(n) Analysis, Fed-Batch Cultivation Enhances Bioactive Compound Biosynthesis and Immune-Regulative Effect of Adventitious Roots in Pseudostellaria heterophylla.

A electrospray ionization tandem mass spectrometry (ESI-MS(n)) analysis was performed in order to identify the active composition in Pseudostellaria heterophylla adventitious roots. Pseudostellarin A, C, D, and G were identified from P. heterophylla adventitious roots on the basis of LC-MS(n) analysis. The culture conditions of adventitious roots were optimized, and datasets were subjected to a partial least squares discriminant analysis (PLS-DA), in which the growth ratio and some compounds showed a positive correlation with an aeration volume of 0.3 vvm and inoculum density of 0.15 %. Fed-batch cultivation enhanced the contents of total saponin, polysaccharides, and specific oxygen uptaker rate (SOUR). The maximum dry root weight (4.728 g l(-1)) was achieved in the 3/4 Murashige and Skoog (MS) medium group. PLS-DA showed that polysaccharides contributed significantly to the clustering of different groups and showed a positive correlation in the MS medium group. The delayed-type hypersensitivity (DTH) reaction on the mice induced by 2,4-dinitrofluorobenzene (DNFB) was applied to compare the immunocompetence effects of adventitious roots (AR) with field native roots (NR) of P. heterophylla. As a result, AR possessed a similar immunoregulation function as NR.

Identification and simultaneous quantification of five alkaloids in Piper longum L. by HPLC–ESI-MSn and UFLC–ESI-MS/MS and their application to Piper nigrum L.

A simple, effective and suitable UFLC–ESI-MS/MS method was firstly developed to simultaneously determine five characteristic constituents (piperine, piperlonguminine, Δα,β-dihydropiperlonguminine, pellitorine and piperanine) of Piper longum L. The total alkaloids of P. longum L. was prepared. The alkaloid contents of Piper nigrum L. and P. longum L. were compared. The analysis was carried out in multiple reaction monitoring scan mode. The method showed a good specificity, linearity (R2>0.995), stability (RSD<2.53%), repeatability (RSD<2.58%), and recovery (90.0–103.5%). The limits of detection and limits of quantification of five alkaloids were in the range of 0.02–0.03 and 0.05–0.10ng/mL, respectively. The intra- and inter-day precision was less than 9.30% and 9.55%, respectively. The validation results confirmed that the method could simultaneously determine the target alkaloids in the sample. Furthermore, the identities of the alkaloids were verified by HPLC–ESI-MS/MS. Compared with P. nigrum, P. longum had lower piperine content but was enriched in the other four alkaloids.

Influence of cultivar and origin on the flavonol profile of fruits and cladodes from cactus Opuntia ficus-indica

Flavonols are hypothesized to be the most important polyphenolic antioxidants. This present study aimed at investigating flavonols in cactus Opuntia ficus-indica fruits from two Egyptian cultivars in comparison to three common Sicilian cultivars, and two further cultivars from South Africa. Besides, cactus O. ficus-indica cladodes from Egyptian cultivars have been investigated as well. HPLC–DAD analyses showed that cactus O. ficus-indica fruits and cladodes are mainly characterized by isorhamnetin glycosides. These flavonols were found only in the fruit's peel and the cladodes, but not in the pulp. However, all analyzed cultivars exhibited the same flavonol profile which might therefore serve as a chemical fingerprint with regard to genuineness of cactus O. ficus-indica fruits and cladodes or even food products containing whole cactus fruits as ingredients. The findings obtained have been confirmed both by enzymatic hydrolysis of flavonol glycosides following analysis of the aglycons and by HPLC–ESI-MSn. The concentration of total flavonols ranged from 2.2 to 4.1 and 6.3 to 7.6mg/g (dw) in the fruit peels and the cladodes, respectively. Further, fruit peels and cladodes exhibited high total phenolic contents and antioxidant activities compared to those of the fruit pulps. These investigations valorize cactus fruit's peel and cladode in comparison to fruit's pulp, and may further provide additional data for using flavonols in chemotaxonomic studies of cactus Opuntia spp. and authenticity of cactus O. ficus-indica products.

Macrophage biospecific extraction and HPLC–ESI-MSn analysis for screening immunological active components in Smilacis Glabrae Rhizoma

A cell-permeable membrane, as typified by Transwell insert Permeable Supports, permit accurate repeatable invasion assays, has been developed as a tool for screening immunological active components in Smilacis Glabrae Rhizoma (SGR). In this research, components in the water extract of SGR (ESGR) might conjugate with the receptors or other targets on macrophages which invaded Transwell inserts, and then the eluate which contained components biospecific binding to macrophages was identified by HPLC–ESI-MSn analysis. Six compounds, which could interact with macrophages, were detected and identified. Among these compounds, taxifolin (2) and astilbin (4) were identified by comparing with the chromatography of standards, while the four others including 5-O-caffeoylshikimic acid (1), neoastilbin (3), neoisoastilbin (5) and isoastilbin (6), were elucidated by their structure clearage characterizations of tandem mass spectrometry. Then compound 1 was isolated and purified from SGR, along with 2 and 4, was applied to the macrophage migration and adhesion assay in HUVEC (Human Umbilical Vein Endothelial Cells) -macrophages co-incultured Transwell system for immunological activity assessment. The results showed that compounds 1, 2 and 4 with concentration of 5μM (H), 500nM (M) and 50nM (L) could remarkably inhibit the macrophage migration and adhesion (Vs AGEs (Advanced Glycation End Produces) group, 1-L, 2-H and 4-L groups: p<0.05; other groups: p<0.01). Moreover, 1 and 4 showed satisfactory dose–effect relationship. In conclusion, the application of macrophage biospecific extraction coupled with HPLC–ESI-MSn analysis is a rapid, simple and reliable method for screening immunological active components from Traditional Chinese Medicine.

Comparison of ginsenoside composition in native roots and cultured callus cells of Panax quinquefolium L.

In order to compare the ginsenoside composition in native Panax quinquefolium and in suspension cultured cells derived from root callus, HPLC–ESI-MSn analysis was performed. Under the present HPLC–ESI-MSn conditions, ten ginsenosides from native root were acquired in the positive and negative ion modes, namely Rg1, Re, Ro, malonyl-Rb1, Rf, Rb1, Rc, Rb2, Rb3 and Rd. Only four ginsenosides (Rg1, Re, Rf and Rb1) were identified from callus cells. Radical scavenging activity of P. quinquefolium callus cells with 250 mg l−1 methanolic extract on 1,1-diphenyl-2-picrylhydrazyl (DPPH) was 55.72 %, while only 6.31 % DPPH inhibition was obtained in native root.

Widespread distribution and molecular diversity of diatom frustule bound aliphatic long chain polyamines (LCPAs) in marine sediments

The siliceous skeletal remains of diatoms are a primary component of sediments, covering vast areas of the sea floor. We used high performance liquid chromatography–electrospray ionization–tandem mass spectrometry (HPLC–ESI–MSn) under high resolution time-of-flight MS (TOF-MS) conditions, as well as nuclear magnetic resonance spectroscopy (NMR) and Fourier transform infrared spectroscopy (FTIR), to investigate the distribution and molecular diversity of long chain polyamines (LCPAs) entrapped in sedimentary diatom biosilica in sediments from the Indian sector of the Southern Ocean, the Bering Sea and the Northeast Pacific. This revealed the existence of complex polyamine populations with both known and unknown structural features, including the presence of ketone and carboxylic acid groups in the LCPA backbone, functional groups that likely modulate the silica precipitating properties of LCPAs in the environment and which may constitute points of initial silica deposition (Wallace, A.F., De Yoreso, J.J., Dove, P.M., 2009. Kinetics of silica nucleation on carboxy- and amine-terminated surfaces: insights for biomineralization. Journal of the American Chemical Society 131, 5244–5250). Although various LCPA species overlapped between samples (putrescine-based LCPAs with various degrees of methylation and N-methyl propylamine repeat units were ubiquitous in all samples), clear regional differences were shown in composition and structural characteristics, likely reflecting the various diatom species contributing to the total LCPA pool for each sedimentary environment. We further show, using scanning electron microscopy and energy dispersive X-ray analysis (SEM–EDX) that LCPAs purified from diatom skeletal remains rapidly re-direct the formation of silica nanospheres in vitro, and become embedded in the precipitated silicified matrix, forming an LCPA–silica composite material. The results show that aliphatic polyamines produced by diatoms for morphogenesis of their cell walls are widespread in sediments where the fossil frustules are preserved. These newly assigned components constitute useful biological markers of diatom input to sediments.

An MC3T3-E1 Cell Line Biomembrane Extraction and HPLC–ESI-MS n Method for Simultaneous Analysis of Potential Anti-Osteoporosis Components of Epimedium koreanum

Aerial parts of Epimedium koreanum Nakai have been used as Herba Epimedii in China. Its anti-osteoporosis effect has attracted much attention in recent years. In this study, a method involving osteoblastic cell (MC3T3-E1 cell line) extraction and high-performance liquid chromatography–electrospray ionisation–mass spectrometry (HPLC–ESI-MS n ) was developed for screening of potential anti-osteoporosis agents in E. koreanum. Four compounds identified as epimedin A, epimedin B, epimedin C and icariin were found to interact with MC3T3-E1 cells and possessed potent osteoblast-stimulating activity as evaluated by cell proliferation [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay] and differentiation [alkaline phosphatase (ALP) activity and Ca content] in vitro. The results suggest that these four flavonoids are the anti-osteoporosis constituents of Herba Epimedii and that the method combining MC3T3-E1 cell extraction with HPLC–ESI-MS n is rapid and effective in screening anti-osteoporosis agents from traditional Chinese medicines.

Molecular interactions of the phytotoxins destruxin B and sirodesmin PL with crucifers and cereals: Metabolism and elicitation of plant defenses

Destruxin B and sirodesmin PL are phytotoxins produced by the phytopathogenic fungi Alternaria brassicae (Berk.) Sacc. and Leptosphaeria maculans (asexual stage Phoma lingam), respectively. The molecular interaction of destruxin B and sirodesmin PL with cruciferous and cereal species was investigated using HPLC–ESI-MSn. It was determined that crucifers transformed destruxin B to hydroxydestruxin B, but sirodesmin PL was not transformed. Overall, the results suggest that the five cruciferous species Arabidopsis thaliana, Thellungiella salsuginea, Erucastrum gallicum, Brassica rapa and Brassica napus are likely to produce a destruxin B detoxifying enzyme (destruxin B hydroxylase), similar to other cruciferous species reported previously. In addition, HPLC analyses and quantification of the phytoalexins elicited in each cruciferous species by these phytotoxins indicates that sirodesmin PL elicits a larger number of phytoalexins than destruxin B. Interestingly, transformation of destruxin B appears to occur also in the cereals Avena sativa and Triticum aestivum; however, the various destruxin metabolites detected in these cereals suggest that these reactions are non-specific enzymatic transformations, contrary to those observed in crucifers, where only a main transformation pathway is detectable. None of the toxins appear to elicit production of metabolites in either A. sativa or T. aestivum.

Analysis of natural diatom communities reveals novel insights into the diversity of long chain polyamine (LCPA) structures involved in silica precipitation

The intricately nanopatterned siliceous frustules of diatoms are formed under the control of template organic molecules, some of which are tightly incorporated into the frustule during formation. Long chain polyamines (LCPAs) attract dissolved silicic acid species, enhance silica precipitation and play a role in frustule nanopattern formation. LCPAs thus play a central biogeochemical role, linking the cycling of organic matter with the cycling of silicic acid in the photic zone of the upper ocean. Their distribution and molecular diversity in the environment are as yet unknown. We used high performance liquid chromatography electrospray ionization–mass spectrometry (HPLC–ESI–MSn) to investigate the distribution and molecular diversity of aliphatic LCPAs extracted from the frustules of diatoms in mixed marine plankton communities collected within the euphotic zone of the northeastern Pacific coast, Bering Sea, and Puget Sound estuary. An astonishing variety of over 100 natural LCPAS with complex distribution patterns was found in each sample. Structural variation included degree of methylation, overall chain length, presence of secondary amine functionalities, incorporation of quaternary ammonium and occurrence of sulfonated derivatives. Putrescine-based LCPAs with various degrees of methylation and N-methyl propylamine repeat units were ubiquitous. Although some LCPA species overlapped between samples, there were regional differences in composition and structural characteristics. The structural variety of LCPAs employed by diatoms to produce their nanopatterned silicified exoskeleton could inform biomimetic studies of silica precipitation.

Phlorotannins from brown algae (Fucus vesiculosus) inhibited the formation of advanced glycation endproducts by scavenging reactive carbonyls.

Accumulation of advanced glycation end products (AGEs) in vivo is associated with aging, diabetes, Alzheimer's disease, renal failure, etc. The objective of this study was to investigate the inhibitory effects of brown algae Fucus vesiculosus phlorotannins on the formation of AGEs. F. vesiculosus phlorotannins were extracted using 70% acetone. The resultant extract was fractionated into dichloromethane, ethyl acetate, butanol, and water fractions. The ethyl acetate fraction was further fractionated into four subfractions (Ethyl-F1 to -F4) using a Sephadex LH-20 column. F. vesiculosus acetone extract or fractions significantly inhibited the formation of AGEs mediated by glucose and methylglyoxal in a concentration-dependent manner. The concentrations of F. vesiculosus extracts required to inhibit 50% of albumin glycation (EC(50)) in the bovine serum albumin (BSA)-methylglyoxal assay were lower than those of aminoguanidine (a drug candidate for diabetic complication), except for F. vesiculosus acetone extract and dichloromethane fraction. In the BSA-glucose assay, F. vesiculosus extracts inhibited BSA glycation more than or as effectively as aminoguanidine, except for Ethyl-F3 and -F4. The ethyl acetate fraction and its four subfractions scavenged more than 50% of methylglyoxal in two hours. The hypothesis whether F. vesiculosus phlorotannins scavenged reactive carbonyls by forming adducts was tested. Phloroglucinol, the constituent unit of phlorotannins, reacted with glyoxal and methylglyoxal. Five phloroglucinol-carbonyl adducts were detected and tentatively identified using HPLC-ESI-MS(n).