Performance Liquid Chromatography Electrospray Ionization Research Articles

Bio-Functional Properties of Bee Pollen: The Case of “Bee Pollen Yoghurt”

The objectives of the present work were: (a) to characterize bee pollen from the region of Epirus in terms of biofunctional activity parameters as assessed by (i) the determination of specific polyphenols using high performance liquid chromatography electrospray ionization mass spectrometry (HPLC/ESI-MS), (ii) antioxidant capacity (DPPH assay), and (iii) total phenolic content (Folin-Ciocalteu assay), and (b) to prepare yoghurts from cow, goat, and sheep milk supplemented with different concentrations of grounded bee pollen (0.5, 1.0, 2.5 and 3.0%, w/v), and study afterwards the trend in antioxidant capacity and total phenolic content along with product’s sensory properties. Results showed that bee pollen ethanolic extracts are a rich source of phytochemicals based on the high total phenolic content and in vitro antioxidant activity that were monitored. The addition of grounded bee pollen in yoghurts resulted in a food matrix of a higher in vitro antioxidant capacity and total phenolic content, whereas it improved the yoghurt’s taste, odour, appearance, and cohesion; the latter indicates its beneficial use as a general food surface and interface material enhancer due to the possible formation of surface/interface active lipid-linked proteins. Based on the present findings, bee pollen yoghurt is proposed as a novel and costless functional food whereas it may comprise a research basis for food or material science in the scientific society of the future. Results were further supported by implementation of advanced chemometric analyses providing a full characterization of the product’s uniqueness.

Fast and sensitive determination of 10 forbidden phthalates in perfumes by ultra–high performance liquid chromatography–electrospray ionization–tandem mass spectrometry

A fast and sensitive ultra–high performance liquid chromatography–electrospray ionization–tandem mass spectrometry (UHPLC–ESI–MS/MS) method was developed for simultaneously analyzing 10 phthalates in perfume, which are forbidden by the hygienic standards for cosmetics in China (2007 edition). Matrix effect is significant on a phthalate when it is co–eluted with other phthalates. Improving the resolution between adjacent phthalate peaks is found effective in reducing the matrix effects. Thus, simultaneous analysis of the 10 phthalates requires successful resolutions of each phthalate. Nonetheless, a trade–off between the resolution and analysis time results either incomplete separation or prolonged analysis time. Here, the UHPLC elution gradient is optimized considering the predicted retention time of each phthalate. The resolutions and matrix effects of targeted compounds are evaluated to determine the optimal elution gradient for UHPLC-MS analysis method. Under the optimized gradient, the resolution between closest phthalate peaks is beyond 1.7, while the analysis time is merely 7 min. Except for dimethyl phthalate (DMP) and dicyclohexyl phthalate (DCHP), insignificant matrix effects have been found on all the phthalates. Direct quantifications through external calibration curve are appropriate for such analytes. Nonetheless, DMP and DCHP suffering obvious matrix effects require extra analyses of spiked samples for the quantifications through the standard addition method. Instrumental limits of quantitation (iLOQs) are 0.12–89 μg L−1 for the targeted phthalates. Meanwhile, the accuracy and precision of the analytical method are good. Finally, the forbidden phthalates in 26 sampled perfumes are successfully analyzed by the developed method.

Lipidomic differentiation of Graves' ophthalmopathy in plasma and urine from Graves' disease patients.

Approximately 50% of patients with Graves' disease (GD) develop retracted eyelids with bulging eyes, known as Graves' ophthalmopathy (GO). However, no simple diagnostic blood marker for distinguishing GO from GD has been developed yet. The objective of this study was to conduct comprehensive profiling of lipids using plasma and urine samples from patients with GD and GO undergoing antithyroid therapy using nanoflow ultrahigh performance liquid chromatography electrospray ionization tandem mass spectrometry. Plasma (n = 86) and urine (n = 75) samples were collected from 23 patients with GD without GO, 31 patients with GO, and 32 healthy controls. Among 389 plasma and 273 urinary lipids that were structurally identified, 281 plasma and 191 urinary lipids were quantified in selected reaction monitoring mode. High-abundance lipids were significantly altered, indicating that the development of GD is evidently related to altered lipid metabolism in both plasma and urine. Several urinary lysophosphatidylcholine species were found to be increased (3- to 10-fold) in both GD and GO. While the overall lipid profiles between GD and GO were similar, significant changes (area under receiver operating curve > 0.8) in GO vs. GD were observed in a few lipid profiles: 58:7-TG and (16:1,18:0)-DG from plasma, 16:1-PC and 50:1-TG from urine, and d18:1-S1P from both plasma and urine samples. An altered metabolism of lipids associated with the additional development of ophthalmopathy was confirmed with the discovery of several candidate markers. These can be suggested as candidate markers for differentiating the state of GO and GD patients based on plasma or urinary lipidomic analysis. Graphical abstract.

Diurnal Variations in Partitioning of Atmospheric Glyoxal and Methylglyoxal between Gas and Particles at the Ground Level and in the Free Troposphere

This work presents diurnal variations of gas- and particle-phase dicarbonyls (glyoxal (Gly) and methylglyoxal (Mgly)) in the atmosphere, which are important compounds that contribute to the formation and growth of atmospheric particulate matter. To obtain variations in partitioning, continuous collection of gaseous dicarbonyls was performed using a parallel plate wet denuder, and at the same time, the dicarbonyls in particle were collected using a spray-type particle collector downstream. Hourly samples were analyzed by high performance liquid chromatography–electrospray ionization–tandem mass spectrometry. This method is advantageous to monitor the gaseous and particulate carbonyls separately without loss during sampling. Sampling was performed in summer and winter in a midsize city (Kumamoto, Japan). The concentrations of the dicarbonyls increased in the summer daytime, which suggests that they are mostly formed by secondary production in the local atmosphere. The dicarbonyls and formaldehyde (HCHO) wer...

Laccase-catalyzed poly(ethylene glycol)-templated ‘zip’ polymerization of caffeic acid for functionalization of wool fabrics

Abstract A cleaner approach for functionalization of wool fabrics via laccase-catalyzed polymerization of caffeic acid was developed. Caffeic acid was first polymerized to poly(caffeic acid) in the system where poly(ethylene glycol) was used as a template under the catalysis of laccase/O2. Ultraviolet–Visible spectroscopic measurements verified that poly(ethylene glycol) formed a complex with caffeic acid though hydrogen bonds based on the ‘zip’ mechanism. During the catalysis of caffeic acid by laccase, poly(ethylene glycol) template promoted the enzymatic polymerization in term of the quantity of poly(caffeic acid) and the degree of polymerization. The data obtained from Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry and High Performance Liquid Chromatography-Electro Spray Ionization confirmed that poly(ethylene glycol) template aided the arrangement of the caffeic acid monomer, resulting in the increased degree of polymerization of caffeic acid in the laccase-catalyzed reaction. The poly(caffeic acid)/poly(ethylene glycol)-treated wool fabrics were prepared by laccase-catalyzed in-situ polymerization of caffeic acid under the same conditions. The treated wool fabrics showed good wettability and electroactivity. The results from fibre conductivity test and cyclic voltammetry showed that the wool fabric treated with poly(caffeic acid)/poly(ethylene glycol) complex was found to have better electrical conductivity. These data showed that the presence of poly(ethylene glycol) as a template in the laccase-catalyzed polymerization reaction greatly improved the characteristic of poly(caffeic acid). Moreover, higher color depth of poly(caffeic acid)/poly(ethylene glycol)-treated wool fabrics further validated the important role of the poly(ethylene glycol) as a template.

Biosynthesis of morin-3-O-rhamnopyranoside in a genetically engineered Escherichia coli

Flavonoids are significant secondary metabolites of vascular plants. They have been proven to possess numerous types of anti-bacterial, anti-tumor, anti-oxidant and anti-inflammatory bioactivities etc. Thereby, there are many flavonoids extracted and purified from plants and have been used for biological tests. Nevertheless, the traditional extraction methods require a large amount of initial sample, tedious and professional techniques and low yield of target compound. Until present, the advanced development of genetic engineerings and recombinant protein as platform allow synthesis of high amount of different types of flavoid in short time. Escherichia coli is one of the most popular host for whole-cell biotransformation of flavonoids and their derivatives due to its simple genetical, physiological system and fast growth. In this paper, we described the biosynthetic method of morin-3-O-rhamnopyranoside from a genetically engineered E. coli using morin as substrate. In particular, the E. coli harboring biosynthetic gene cluster of activated TDP-L-rhamnose and gene encoding for glycosyltransferase was used as host for biotransformation. Morin was fed into the culture broth of recombinant E. coli for rhamnosylation. The formation of morin-3-O-rhamnopyranoside was then quantitied and qualified using Thin Layer Chromatography (TLC), Reverse Phase High Performance Liquid Chromatography (RP-HPLC) and Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometric (LC-ESI-MS/MS) assays. The highest yield of product (56.5 µM) was achieved after 48h, 32 oC and pH = 7.2-8.0. This result showed that morin-3-O-rhamnopyranoside was succesfully synthesized in the genetically engineered E. coli. Furthermore, metabolic engineering of intra-cellular system for improving absorption of substrate as well as excretion of product or enhancement of glycosyltransferase activity may increase the final yield of biotransformation process.

Identification of an ACE-Inhibitory Peptide from Walnut Protein and Its Evaluation of the Inhibitory Mechanism.

In the present study, a novel angiotensin I-converting enzyme inhibitory (ACE inhibitory) peptide, EPNGLLLPQY, derived from walnut seed storage protein, fragment residues 80–89, was identified by ultra-high performance liquid chromatography electrospray ionization quadrupole time of flight mass spectrometry (UPLC-ESI-Q-TOF-MS/MS) from walnut protein hydrolysate. The IC50 value of the peptide was 233.178 μM, which was determined by the high performance liquid chromatography method by measuring the amount of hippuric acid (HA) generated from the ACE decomposition substrate (hippuryl-l-histidyl-l-leucine (HHL) to assess the ACE activity. Enzyme inhibitory kinetics of the peptide against ACE were also conducted, by which the inhibitory mechanism of ACE-inhibitory peptide was confirmed. Moreover, molecular docking was simulated by Discovery Studio 2017 R2 software to provide the potential mechanisms underlying the ACE-inhibitory activity of EPNGLLLPQY.

Target screening of 105 veterinary drug residues in milk using UHPLC/ESI Q-Orbitrap multiplexing data independent acquisition.

This paper presents a multi-class target screening method for the detection of 105 veterinary drug residues from 11 classes in milk using ultra-high performance liquid chromatography electrospray ionization quadrupole Orbitrap mass spectrometry (UHPLC/ESI Q-Orbitrap). The method is based on a non-target approach of full mass scan and multiplexing data-independent acquisition (Full MS/mDIA). The veterinary drugs include endectocides, fluoroquinolones, ionophores, macrolides, nitroimidazole, NSAIDs, β-lactams, penicillins, phenicols, sulfonamides, and tetracyclines. Veterinary drug residues were extracted from milk using a salting-out and solid-phase extraction (SOSPE) procedure, which entailed the precipitation of milk proteins by an extraction buffer (oxalic acid and EDTA, pH 3) and acetonitrile, a salting-out acetonitrile/water phase separation using ammonium sulfate, and solid-phase extraction for clean-up using polymeric reversed-phase sorbent cartridges. The Q-Orbitrap Full MS/dd-MS2 (data-dependent acquisition) was used to acquire product-ion spectra of individual veterinary drugs to build a compound database and a mass spectral library, whereas its Full MS/mDIA was utilized to acquire sample data from milk for target screening of veterinary drugs fortified at 1.0 or 10.0 μg/kg. The in-spectrum mass correction or solvent background lock-mass correction was used to minimize mass error when building the compound database from experimental dd-MS2 accurate mass data. Retention time alignment and response threshold adjustment were used to eliminate or reduce false negativesand/or false positive rates. The validated method was capable of screening 58% and 96% of 105 veterinary drugs at 1.0 and 10.0 μg/kg, respectively, without manually evaluating every compound during data processing, which will reduce the workload in routine practice.

The UPLC–ESI–QqQLIT–MS/MS method for quantitative determination of phytochemicals in ethanolic extracts of different parts of eight Ficus species: Development and validation

ABSTRACTFicus and validation of the ultra performance liquid chromatography–electrospray ionization hybrid triple quadrupole–linear ion trap–tandem mass spectrometry (UPLC–ESI–QqQLIT–MS/MS) method in a multiple-reaction monitoring (MRM) mode for the quantitative determination of 19 phytochemicals. The chromatographic separation of targeted phytochemicals was performed using the Waters ACQUITY UPLC BEH™ C18 column (1.7 μm, 2.1 mm × 50 mm) with 0.1% formic acid with water and acetonitrile as a mobile phase at a flow rate of 0.25 mL/min. The validation parameters showed the overall recoveries from 95.78−101.44% (RSD ≤ 3.25%), precision (intra-day: RSD ≤ 2.96%; inter-day: RSD ≤ 2.89%), linearity (R2 ≥ 0.9982), limit of detection (8.60 × 10–10−2.18 × 10–6 mg/mL), and the limit of quantitation (2.60 × 10–9–6.63 × 10–6 mg/mL) in the concentration range from 0.5 to 1000 × 10–6 mg/mL. This method was successfully applied in ethanolic extracts of different parts (fruits, leaves, and barks) of selected eight Ficus species. Quinic acid was predominant followed by rutin and chlorogenic acid among the studied nineteen phytochemicals. Ficus benjamina showed the maximum total content in fruits and leaves. The UPLC–ESI–QqQLIT–MS/MS method combined with principal component analysis (PCA) was successfully used for Ficus species discrimination on the basis of the contents of 15 compounds. The UPLC–ESI–QqQLIT–MS/MS method combined with PCA could be used for quality control.

Effect of bariatric surgery on urinary sphingolipids in adolescents with severe obesity

BackgroundUntreated severe obesity of adolescents is associated with abnormal kidney function and development of chronic kidney disease. Lipotoxicity due to lipid accumulation in glomeruli might be an important mechanism in the progression of kidney disease in obesity. ObjectiveTo assess subclinical glomerular injury by measuring urinary sphingolipids in adolescents with severe obesity before and after weight loss surgery. We hypothesized that the levels of urinary sphingolipids would be elevated at baseline and improve after weight reduction. SettingCincinnati Children’s Hospital Medical Center, University of Cincinnati. MethodsTen adolescents undergoing bariatric surgery with no microalbuminuria and normal kidney function were selected. Urinary sphingolipids (ceramides, glycosphingolipids, and sphingomyelins) were quantified using ultra performance liquid chromatography electrospray ionization tandem mass spectrometry at baseline and 1 year postoperatively. The levels of sphingolipids were compared with lean and moderately obese controls. ResultsParticipants with severe obesity had a mean baseline body mass index of 50 kg/m2 that decreased to 36 kg/m2 at 1 year postsurgery (28% reduction). Almost all urinary ceramides, glycosphingolipids, and sphingomyelin species were significantly elevated in participants with severe obesity compared with controls at baseline (P<.01). One year after weight loss surgery, levels of urinary sphingolipids improved but were still significantly elevated compared with controls. ConclusionsOur study indicates that severe obesity is associated with increased urinary excretion of sphingolipids despite the absence of microalbuminuria or decreased kidney function. Urinary sphingolipids may therefore represent a marker of early (subclinical) glomerular injury in adolescents with severe obesity.

Quantification of glycocholic acid in human serum by stable isotope dilution ultra performance liquid chromatography electrospray ionization tandem mass spectrometry.

A rapid, accurate and sensitive stable isotope dilution ultra performance liquidchromatography electrospray ionization tandem mass spectrometry (ID-UPLC-ESI-MS/MS) method for the determination of glycocholic acid (GCA) in human serum was developed and validated. Serum samples were spiked with D5-glycocholic acid and then pretreated with protein precipitation. The analysis was performed on a Waters BEH C18 column (100 mm×2.1mm, 1.7μm), followed by ESI-MS/MS detection in negative ion mode under multiple reaction monitoring mode. The calibration curves covered a concentration range from 0.2 to 400ng/mL. The limit of detection and limit of quantification was 0.01ng/mL and 0.05ng/mL, respectively. The method showed satisfactory precision on intra-day (2.3-6.1%) and inter-day (2.4-4.6%) analyses and achieved good recovery at three spiked levels (103.7-114.3%). Moreover, this established method was successfully applied for quantification of GCA in serum samples from healthy volunteers, patients with hepatocellular carcinoma (HCC) and patients with other cancers. We demonstrated that the level of GCA in patients with HCC was significantly higher not only than that in healthy controls, but also than that in patients with other cancer, whereas no significant difference of GCA level was observed between healthy control group and other cancers group.

Simultaneous Determination of Seven Phenolic Acids in Rat Plasma Using UHPLC-ESI-MS/MS after Oral Administration of Echinacea purpurea Extract.

A rapid and sensitive Ultra High Performance Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometry (UHPLC-ESI-MS/MS) method was developed and validated to simultaneously determine the concentration of seven phenolic acids (syringic acid, ferulic acid, caffeic acid, vanillic acid, p-coumaric acid, 3,4-dihydroxybenzoic acid and 4-hydroxybenzoic acid) in rat plasma after oral administration of Echinacea purpurea extract. After mixing with the internal standard (IS), butylparaben, plasma samples were prepared by liquid–liquid extraction with ethyl acetate. The separation was performed using the Agilent Eclipse Plus C18 column (1.8 μm, 2.1 mm × 50 mm) with a gradient system consisting of solution A (0.1% acetic acid in water) and solution B (methanol) at a flow rate of 0.3 mL/min. The detection was accomplished by a multiple reaction monitoring (MRM) mode with electrospray ionization (ESI). The method was validated in terms of linearity, precision, accuracy, extraction recovery, matrix effect and stability. This method was successfully applied to study the pharmacokinetic properties of the seven compounds after oral administration of Echinacea purpurea extract in rats.

Spatial Distribution Patterns of Root-Associated Bacterial Communities Mediated by Root Exudates in Different Aged Ratooning Tea Monoculture Systems.

Positive plant–soil feedback depends on beneficial interactions between roots and microbes for nutrient acquisition; growth promotion; and disease suppression. Recent pyrosequencing approaches have provided insight into the rhizosphere bacterial communities in various cropping systems. However; there is a scarcity of information about the influence of root exudates on the composition of root-associated bacterial communities in ratooning tea monocropping systems of different ages. In Southeastern China; tea cropping systems provide the unique natural experimental environment to compare the distribution of bacterial communities in different rhizo-compartments. High performance liquid chromatography–electrospray ionization–mass spectrometry (HPLC–ESI–MS) was performed to identify and quantify the allelochemicals in root exudates. A high-throughput sequence was used to determine the structural dynamics of the root-associated bacterial communities. Although soil physiochemical properties showed no significant differences in nutrients; long-term tea cultivation resulted in the accumulation of catechin-containing compounds in the rhizosphere and a lowering of pH. Moreover; distinct distribution patterns of bacterial taxa were observed in all three rhizo-compartments of two-year and 30-year monoculture tea; mediated strongly by soil pH and catechin-containing compounds. These results will help to explore the reasons why soil quality and fertility are disturbed in continuous ratooning tea monocropping systems; and to clarify the associated problems.

Analysis of individual and total microcystins in surface water by on-line preconcentration and desalting coupled to liquid chromatography tandem mass spectrometry

A fast and high-throughput method is proposed for the determination of total microcystins (ΣMC) in environmental surface waters. After a 1-h Lemieux-von Rudloff oxidation step to yield the 2-methyl-3-methoxy-4-phenylbutyric acid (MMPB) moiety, samples were quenched, filtered, and directly analyzed. This was achieved via solid phase extraction (SPE) coupled on-line to ultra-high performance liquid chromatography electrospray ionization triple stage quadrupole mass spectrometry. The choice of on-line SPE settings was conducted using experimental designs. Given the matrix complexity of oxidation extracts, the on-line desalting step was found to be a critical parameter to ensure suitable method robustness. The on-line sample loading volume was 5mL, and the wash volume applied for on-line desalting was 3mL. Instrumental analysis was performed in just 8min. The method limit of quantification was 0.5ngL−1 ΣMC (i.e. 2000 times lower than the current World Health Organization − WHO drinking water guideline). Excellent determination coefficients were observed for matrix-free and matrix-based calibration curves alike, and the linearity range tested spanned∼4 orders of magnitude. Accuracy and intermediate precision did not depend on the spike level and proved satisfactory (in the range of 93–110% and 3–6%, respectively). A thorough assessment of instrumental matrix effects was conducted by comparing standard additions curves in several lake and river oxidation extracts with the matrix-free reference. Regardless of the internal standard used (4-PB or D3-MMPB), instrumental matrix effects were efficiently compensated. The matrix effect that may occur at the earlier sample preparation stage was evaluated separately. While the oxidation step was generally not complete (yield ∼65%), the conversion rates of MCs into MMPB remained within a consistent range of values regardless of matrix type. No significant back-pressure was observed upon consecutive injections of oxidation-based samples, while the instrumental sensitivity remained unaffected. The herein described method could therefore be eligible for future large-scale monitoring surveys. The method was applied to a selection of surface water samples (n=30) collected across the province of Québec, Canada, and the results were compared to those achieved by an individual variant analysis of 8 MC congeners and a commercial ELISA kit.

Functional regulation of a UDP-glucosyltransferase gene (Pq3-O-UGT1) by RNA interference and overexpression in Panax quinquefolius

Ginsenosides have been thought to be the major pharmacological active ingredient in the roots of Panax quinquefolius, P. ginseng and other Panax plants. Glycosylation is the last step in the biosynthesis pathway of ginsenosides and leads to the formation of various secondary metabolites. As for P. quinquefolius, dammarenediol-II synthase (PqDS), protopanaxadiol synthase (PqD12H) and protopanaxatriol synthase (CYP6H) have been cloned and identified in the early time. Here, we cloned and identified a novel UDP-glycosyltransferase (UGT) gene from P. quinquefolius (Pq3-O-UGT1, GenBank accession no. KiR028477) for the first time, reverse transcription-PCR (RT-PCR) analysis showed a remarkable transcription increase of Pq3-O-UGT1 in the methyl jasmonate (MeJA)-treated hairy roots, in vitro enzymatic assay confirmed that Pq3-O-UGT1 catalyzed the glycosylation of protopanaxadiol to produce ginsenoside Rh2 whose chemical structure was confirmed using high performance liquid chromatography electrospray ionization mass spectrometry (HPLC/ESI-MS). Moreover, overexpression of Pq3-O-UGT1 led to increased accumulation of Pq3-O-UGT1 mRNA and a higher level of protopanaxadiol-group ginsenosides (especially for Rh2 and Rd) in transgenic hairy roots, whereas, RNA interference against Pq3-O-UGT1 in transgenic hairy roots resulted in the relative decrease of Pq3-O-UGT1 transcription and lower content of ginsenoside Rh2. Interestingly, both overexpression and RNAi upregulated the key upstream genes Pq-D12H and Pq-CYP6H, and there was no effect on the upstream gene of Pq-DS. Thus, these results indicate that Pq3-O-UGT1 is a key enzyme for the synthesis of ginsenoside Rh2 and plays a key role in the biosynthesis of protopanaxadiol-group ginsenosides in P. quinquefolius.

Proanthocyanidins purified from fruit pericarp of Clausena lansium (Lour.) Skeels as efficient tyrosinase inhibitors: structure evaluation, inhibitory activity and molecular mechanism.

Fruit pericarp of Clausena lansium (Lour.) Skeels, a food waste, was selected as a raw material for proanthocyanidins. The proanthocyanidins' structures were integrally analyzed using three methods: matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), high performance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESI-MS) and 13C nuclear magnetic resonance (NMR). The results elucidated that these compounds were composed of prodelphinidin (75%) and procyanidin (25%) with a degree of polymerization (DP) up to the 20-mers. They were proved to be remarkable, reversible and mixed competitive inhibitors of tyrosinase according to results from enzyme experiments. The IC50 values were calculated to be 23.6 ± 1.2 and 7.0 ± 0.2 μg mL-1 for the monophenolase and diphenolase activities, respectively. In addition, the proanthocyanidins had a good inhibitory effect on cell proliferation, cellular tyrosinase activity and melanin production of B16 mouse melanoma cells. Chelation between the hydroxyl group on the B ring of the proanthocyanidins and dicopper irons of the enzyme provided one of the feasible mechanisms for the inhibition on the basis of fluorescence quenching and molecular docking analyses. This research would supply the scientific basis to these compounds application in the pharmaceutical, insecticides, and preservative fields.

Functional regulation of ginsenoside biosynthesis by RNA interferences of a UDP-glycosyltransferase gene in Panax ginseng and Panax quinquefolius

Panax ginseng (Asian ginseng) and Panax quinquefolius (American ginseng) have been used as medicinal and functional herbal remedies worldwide. Different properties of P. ginseng and P. quinquefolius were confirmed not only in clinical findings, but also at cellular and molecular levels. The major pharmacological ingredients of P. ginseng and P. quinquefolius are the triterpene saponins known as ginsenosides. The P. ginseng roots contain a higher ratio of ginsenoside Rg1:Rb1 than that in P. quinquefolius. In ginseng plants, various ginsenosides are synthesized via three key reactions: cyclization, hydroxylation and glycosylation. To date, several genes including dammarenediol synthase (DS), protopanaxadiol synthase and protopanaxatriol synthase have been isolated in P. ginseng and P. quinquefolius. Although some glycosyltransferase genes have been isolated and identified association with ginsenoside synthesis in P. ginseng, little is known about the glycosylation mechanism in P. quinquefolius. In this paper, we cloned and identified a UDP-glycosyltransferase gene named Pq3-O-UGT2 from P. quinquefolius (GenBank accession No. KR106207). In vitro enzymatic activity experiments biochemically confirmed that Pq3-O-UGT2 catalyzed the glycosylation of Rh2 and F2 to produce Rg3 and Rd, and the chemical structure of the products were confirmed susing high performance liquid chromatography electrospray ionization mass spectrometry (HPLC/ESI-MS). High sequence similarity between Pq3-O-UGT2 and PgUGT94Q2 indicated a close evolutionary relationship between P. ginseng and P. quinquefolius. Moreover, we established both P. ginseng and P. quinquefolius RNAi transgenic roots lines. RNA interference of Pq3-O-UGT2 and PgUGT94Q2 led to reduce levels of ginsenoside Rd, protopanaxadiol-type and total ginsenosides. Expression of key genes including protopanaxadiol and protopanaxatriol synthases was up-regulated in RNAi lines, while expression of dammarenediol synthase gene was not obviously increased. These results revealed that P. quinquefolius was more sensitive to the RNAi of Pq3-O-UGT2 and PgUGT94Q2 when compared with P. ginseng.

Extraction and Identification of Black Rice Polyphenolic Compounds by Reversed Phase High Performance Liquid Chromatography-Electrospray Ionization Mass Spectrometry

Seven kinds of phenolic compounds in black rice were isolated and identified by reversed phase high performance liquid chromatography–electrospray ionization mass spectrometry. Proanthocyanidins glucoside which has the strongest ability of antioxidant and free radical scavenging was tentatively identified in black rice for the first time. By mass spectrometry in negative ion mode, three types of phenolic acids compounds which were caffeic acid hexose, procyanidinB2-3-O-gallate hexose, epiafzelchineepicatechin-O-dimethylgallate were first confirmed. In this experiment, liquid–mass spectrometry model was established to detect traces of polyphenolic compounds in black rice. This model can increase the efficiency of electron capture and the sensitivity of mass spectrometry detection. The sensitivity, mass accuracy legitimated the identification of phenolic compounds present in black rice extract. So the method of reversed phase high performance liquid chromatography (HPLC)–electrospray ionization mass spectrometry can rapidly detect traces of unknown phenolic compounds in grain. Practical Applications The plant phenols possess the ability to scavenge both active oxygen species and electrophiles. They are the most important group of natural antioxidants because of their diversity and extensive distribution. The interest in the exploitation of novel antioxidants from black rice is increasing accordingly due to abundant contents of polyphenols. Black rice which is a kind of typical whole grain is considered a good source of fiber, minerals, and phytochemicals as well as basic nutrients.

Quantitative determination of multi markers in five varieties of Withania somnifera using ultra-high performance liquid chromatography with hybrid triple quadrupole linear ion trap mass spectrometer combined with multivariate analysis: Application to pharmaceutical dosage forms

An ultra-high performance liquid chromatography electrospray ionization tandem mass spectrometry method has been developed and validated for simultaneous quantification of six major bioactive compounds in five varieties of Withania somnifera in various plant parts (leaf, stem and root). The analysis was accomplished on Waters ACQUITY UPLC BEH C18 column with linear gradient elution of water/formic acid (0.1%) and acetonitrile at a flow rate of 0.3mLmin−1. The proposed method was validated with acceptable linearity (r2, 0.9989–0.9998), precision (RSD, 0.16–2.01%), stability (RSD, 1.04–1.62%) and recovery (RSD ≤2.45%), under optimum conditions. The method was also successfully applied for the simultaneous determination of six marker compounds in twenty-six marketed formulations. Hierarchical cluster analysis and principal component analysis were applied to discriminate these twenty-six batches based on characteristics of the bioactive compounds. The results indicated that this method is advance, rapid, sensitive and suitable to reveal the quality of Withania somnifera and also capable of performing quality evaluation of polyherbal formulations having similar markers/raw herbs.

Proanthocyanidins Extracted from Rhododendron pulchrum Leaves as Source of Tyrosinase Inhibitors: Structure, Activity, and Mechanism.

The objective of this study was to assess the structure, anti-tyrosinase activity, and mechanism of proanthocyanidins extracted from Rhododendron pulchrum leaves. Results obtained from mass spectra of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and high performance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESI-MS) revealed that proanthocyanidins were complex mixtures of procyanidins, prodelphinidins, propelargonidins, and their derivatives, among which procyanidins were the main components. The anti-tyrosinase analysis results indicated that the mixtures were reversible and mixed competitive inhibitors of tyrosinase. Interactions between proanthocyanidins with substrate (L-tyrosine and 3,4-dihydroxyphenylalanine) and with copper ions were the important molecular mechanisms for explaining their efficient inhibition. This research would provide scientific evidence for the use of R. pulchrum leaf proanthocyanidins as new novel tyrosinase inhibitors.