Ultra-high Performance Liquid Chromatography-diode Array Research Articles

Ameliorative effects of aqueous extract of Forsythiae suspensa fruits on oxaliplatin-induced neurotoxicity in vitro and in vivo

BackgroundThe dried fruits of Forsythia suspensa has generally been used to clear heat and detoxify in traditional Korean and Chinese medicine. Oxaliplatin is a first-line treatment chemotherapeutic agent for advanced colorectal cancer, but it induces peripheral neuropathy as an adverse side effect affecting the treatment regimen and the patient’s quality of life. The present study was conducted to evaluate the neuroprotective effects of an aqueous extract of F. suspensa fruits (EFSF) on oxaliplatin-induced peripheral neuropathy.MethodsThe chemical components from EFSF were characterized and quantified using the ultra-high performance liquid chromatography-diode array detector system. The cytotoxicities of anticancer drugs in cancer cells and PC12 cells were assessed by the Ez-Cytox viability assay. To measure the in vitro neurotoxicity, the neurite outgrowth was analyzed in the primary dorsal root ganglion (DRG) cells, and neural PC12 cells that were differentiated with nerve growth factor. To evaluate the in vivo neuroprotective activity, the von Frey test was performed in six-week-old male mice (C57BL/6) receiving EFSF (60–600 mg/kg) in the presence of 20–30 mg/kg cumulative doses of oxaliplatin. Thereafter, the mice were euthanized for immunohistochemical staining analysis with an antibody against PGP9.5.ResultsEFSF attenuated the cytotoxic activities of the various anticancer drugs in neural PC12 cells, but did not affect the anticancer activity of oxaliplatin in human cancer cells. Oxaliplatin remarkably induced neurotoxicities including cytotoxicity and the inhibited neurite outgrowth of DRG and neural PC12 cells. However, the co-treatment of EFSF (100 μg/ml) with oxaliplatin completely reversed the oxaliplatin-induced neurotoxicity. Forsythoside A, the major component of EFSF, also exerted remarkable neuroprotective effects against the oxaliplatin-induced neurotoxicity. In addition, EFSF (60–200 mg/kg) significantly alleviated the oxaliplatin-induced mechanical allodynia and loss of intra-epidermal nerve fiber to the levels of the vehicle control in the mouse peripheral neuropathy model.ConclusionsEFSF could be considered a useful herbal medicine for the treatment of peripheral neuropathy in cancer patients receiving chemotherapy with oxaliplatin.

Relationship between hydroxycinnamic acids and the resistance of apple cultivars to rosy apple aphid

The phenolic profiles of apple cultivars from the SERIDA Asturian cider apple breeding program, including parents and progenies, were determined by ultrahigh-performance liquid chromatography-diode array detector-electrospray ionization-quadrupole time of flight/mass spectrometer in order to study the relationship between phenols and the resistance of apple tree cultivars to rosy apple aphid (RAA). A pattern recognition technique named partial least square discriminant analysis (PLS-DA) was used to classify apple cultivars based on resistance to RAA, resistant and susceptible, reaching scores with accuracy higher than 97% and 91% respectively. Hydroxycinnamic acids, particularly 4-caffeoylquinic acid (4-CQA) and 4-p-coumaroylquinic acid (4-pCoQA), were identified as the major player in RAA resistance by the PLS-DA model. Indeed, the isomerisation 5-CQA → 4-CQA is favoured in resistant cultivars, whereas the isomerisation 5-pCoQA → 4-pCoQA is favoured in susceptible cultivars. As a result, resistant cultivars accumulate higher amounts of 4-CQA than susceptible ones, and the opposite occurs for 4-pCoQA. Also, minor isomerisations of 5-CQA to 1-CQA or 3-CQA show opposite behaviour for resistant and susceptible cultivars. Cultivar resistance to RAA is concluded to be related with the phenylpropanoid pathway, the isomerisation reactions being the key metabolic reaction for a cultivar to be resistant or susceptible to RAA.

Variability in strength of red yeast rice supplements purchased from mainstream retailers.

The United States Food and Drug Administration (FDA) has introduced manufacturing standards for dietary supplements, including red yeast rice, to assure their identity, purity, strength, and composition. One supplement commonly used to self-treat high cholesterol, red yeast rice, may contain monacolin K, an ingredient identical to prescription lovastatin. We examined whether FDA's manufacturing standards led to standard concentrations of the statin monacolin K in red yeast rice supplements. We analyzed 28 brands of red yeast rice supplements by ultra-high performance liquid chromatography-diode array detector-quadrupole time-of-flight mass spectrometry for monacolin K content. Monacolin K was not detected in two brands. In the 26 brands that contained monacolin K, the quantity ranged more than 60-fold from 0.09 to 5.48 mg per 1200 mg of red yeast rice. Following the manufacturers' recommendations for daily servings, the quantity of monacolin K consumed per day would range more than 120-fold from 0.09 to 10.94 mg. Despite FDA manufacturing standards, strength and composition of red yeast rice supplements sold at mainstream retail stores in the United States remains unpredictable.

Metabolic profiling of nuciferine in rat urine, plasma, bile and feces after oral administration using ultra-high performance liquid chromatography-diode array detection-quadrupole time-of-flight mass spectrometry

Nuciferine, a major alkaloid found in Nelumbinis Folium, exhibits a broad spectrum of bioactivities, such as antiobesity, anti-diabetes and anti-inflammatory. However, many research regarding nuciferine focused on the extraction, isolation and biological activity, the metabolism is not comprehensively explained in vivo. Thence, the present of this paper is to establish a simple method for speculating metabolites of nuciferine. A total of 15 metabolites were detected and tentatively identified through ultra high performance liquid chromatography-diode array detection-quadrupole time-of-flight mass spectrometry (UHPLC-DAD-QTOF-MS), including 7 new metabolites. Among them, we also discovered a previously unmentioned metabolically active site at the C1-OCH3 position. These metabolites suggested that demethylation, oxidation, glucuronidation and sulfation were major metabolic pathways. This study provided significant experiment basis for its safety estimate and valuable information about the metabolism of nuciferine, which will be advantageous for new drug development.

EFFICACY OF PHENOLIC-RICH EXTRACTS FROM LEAVES OF PEPPER LANDRACES AGAINST ALTERNARIA LEAF BLIGHT OF TOMATO

Plant-derived antifungals have an interesting potential to be used for the sustainable management of plant diseases. In this work, seven different local genotypes of Capsicum annuum, diffused in agricultural areas of Campania Region (Italy), were explored for their potential in providing phytochemical extracts suitable for antifungal applications. Bioactive hydro-methanolic extracts has been assessed for phenolic composition and antioxidant capacity and, then, assayed both in vitro and in planta for the suppression of Alternaria alternata, the causal agent of early blight of tomato. Ultra high-performance liquid chromatography-diode array detection (UPLC-DAD) analysis showed phenolic profiles of each extract, identifying and quantifying the individual known components, including gallic acid, chlorogenic acid, epicatechin, cumaric acid, rutin, ferulic acid and luteolin. The phenolic-rich extracts of all peppers varieties proved to be active against in vitro mycelial development of A. alternata. Dipping artificially infected tomato leaves using extracts at 10 µg GAE ml -1 of total phenolics, reduced foliar early blight disease severity closely to 50%, on average. The role played by the phenolic complex in the antifungal activity and in disease control efficacy of the extracts, has been discussed.

Simultaneous quantitation of 14 active components in Yinchenhao decoction with an ultrahigh performance liquid chromatography-diode array detector: Method development and ingredient analysis of different commonly prepared samples.

J. Sep. Sci. 2016, 39, 4147-4157 DOI: 10.1002/jssc.201600284 Yinchenhao decoction (YCHD) is a famous Chinese herbal formula recorded in the Shang Han Lun which was prescribed by Zhongjing Zhang during 150-219 AD. A novel quantitative analysis method was developed, based on ultrahigh performance liquid chromatography coupled with a diode array detector for the simultaneous determination of 14 main active components in Yinchenhao decoction. Furthermore, the method has been applied for compositional difference analysis of the 14 components in eight normal extraction samples of Yinchenhao decoction, with the aid of hierarchical clustering analysis and similarity analysis. The present research could help hospital, factory and lab choose the best way to make Yinchenhao decoction with better efficacy.

Qualitative and quantitative analysis of an alkaloid fraction from Piper longum L. using ultra-high performance liquid chromatography-diode array detector–electrospray ionization mass spectrometry

In a previous research, an alkaloid fraction and 18 alkaloid compounds were prepared from Piper longum L. by series of purification process. In this paper, a qualitative and quantitative analysis method using ultra-high performance liquid chromatography-diode array detector–mass spectrometry (UHPLC-DAD–MS) was developed to evaluate the alkaloid fraction. Qualitative analysis of the alkaloid fraction was firstly completed by UHPLC-DAD method and 18 amide alkaloid compounds were identified. A further qualitative analysis of the alkaloid fraction was accomplished by UHPLC–MS/MS method. Another 25 amide alkaloids were identified according to their characteristic ions and neutral losses. At last, a quantitative method for the alkaloid fraction was established using four marker compounds including piperine, pipernonatine, guineensine and N-isobutyl-2E,4E-octadecadienamide. After the validation of this method, the contents of above four marker compounds in the alkaloid fraction were 57.5mg/g, 65.6mg/g, 17.7mg/g and 23.9mg/g, respectively. Moreover, the relative response factors of other three compounds to piperine were calculated. A comparative study between external standard quantification and relative response factor quantification proved no remarkable difference. UHPLC-DAD–MS method was demonstrated to be a powerful tool for the characterization of the alkaloid fraction from P. longum L. and the result proved that the quality of alkaloid fraction was efficiently improved after appropriate purification.

Sequence analysis of porothramycin biosynthetic gene cluster.

The biosynthetic gene cluster of porothramycin, a sequence-selective DNA alkylating compound, was identified in the genome of producing strain Streptomyces albus subsp. albus (ATCC 39897) and sequentially characterized. A 39.7 kb long DNA region contains 27 putative genes, 18 of them revealing high similarity with homologous genes from biosynthetic gene cluster of closely related pyrrolobenzodiazepine (PBD) compound anthramycin. However, considering the structures of both compounds, the number of differences in the gene composition of compared biosynthetic gene clusters was unexpectedly high, indicating participation of alternative enzymes in biosynthesis of both porothramycin precursors, anthranilate, and branched L-proline derivative. Based on the sequence analysis of putative NRPS modules Por20 and Por21, we suppose that in porothramycin biosynthesis, the methylation of anthranilate unit occurs prior to the condensation reaction, while modifications of branched proline derivative, oxidation, and dimethylation of the side chain occur on already condensed PBD core. Corresponding two specific methyltransferase encoding genes por26 and por25 were identified in the porothramycin gene cluster. Surprisingly, also methyltransferase gene por18 homologous to orf19 from anthramycin biosynthesis was detected in porothramycin gene cluster even though the appropriate biosynthetic step is missing, as suggested by ultra high-performance liquid chromatography-diode array detection-mass spectrometry (UHPLC-DAD-MS) analysis of the product in the S. albus culture broth.

Accurate dereplication of bioactive secondary metabolites from marine-derived fungi by UHPLC-DAD-QTOFMS and a MS/HRMS library.

In drug discovery, reliable and fast dereplication of known compounds is essential for identification of novel bioactive compounds. Here, we show an integrated approach using ultra-high performance liquid chromatography-diode array detection-quadrupole time of flight mass spectrometry (UHPLC-DAD-QTOFMS) providing both accurate mass full-scan mass spectrometry (MS) and tandem high resolution MS (MS/HRMS) data. The methodology was demonstrated on compounds from bioactive marine-derived strains of Aspergillus, Penicillium, and Emericellopsis, including small polyketides, non-ribosomal peptides, terpenes, and meroterpenoids. The MS/HRMS data were then searched against an in-house MS/HRMS library of ~1300 compounds for unambiguous identification. The full scan MS data was used for dereplication of compounds not in the MS/HRMS library, combined with ultraviolet/visual (UV/Vis) and MS/HRMS data for faster exclusion of database search results. This led to the identification of four novel isomers of the known anticancer compound, asperphenamate. Except for very low intensity peaks, no false negatives were found using the MS/HRMS approach, which proved to be robust against poor data quality caused by system overload or loss of lock-mass. Only for small polyketides, like patulin, were both retention time and UV/Vis spectra necessary for unambiguous identification. For the ophiobolin family with many structurally similar analogues partly co-eluting, the peaks could be assigned correctly by combining MS/HRMS data and m/z of the [M + Na]+ ions.

Effect of enzyme-assisted extraction on the chilled storage stability of bilberry (Vaccinium myrtillus L.) anthocyanins in skin extracts and freshly pressed juices

Three different commercial enzyme preparations were evaluated with respect to the extractability and chilled storage (10°C, 9days) stability of bilberry skin anthocyanins in model systems. Using ultra high-performance liquid chromatography–diode array detection, 15 individual anthocyanins were characterized, with delphinidin and cyanidin glycosides being the predominant compounds. Except for the galactosides of cyanidin and peonidin, 1.2–4.7 times higher concentrations of individual anthocyanins were observed for the extracts from enzymatically treated bilberry skin suspensions. Anthocyanin degradation followed well (R2=0.89–0.98) a first-order reaction kinetics and the half-life values increased (12–64%) due to enzyme-assisted extraction. The results obtained demonstrate an improved storage stability of anthocyanins, enzymatically released from the bilberry skin matrix. On the basis of the enzyme screening assay, a novel process, composed of partial dejuicing (pressing, ~40% juice yield) of the bilberry mash followed by enzymatic maceration (EMPD), was developed. Interestingly, enzymatic treatment resulted in a decrease among counts of mesophilic and psychrotrophic microorganisms and moulds and yeasts in the bilberry juice upon chilled storage (10°C, 9days), implying an inhibition of the microbial growth. Therefore, applying the EMPD process, this two-step juice extraction technology may be very helpful, allowing both enhanced contents of bioactive compounds and extended microbiological shelf-life of “fresh-like” berry juices.

Determination of 10 Caffeoylquinic Acids and 22 Flavonoids in Chrysanthemum Samples by Ultra-high Performance Liquid Chromatography-Diode Array Detection-Tandem Mass Spectrometry

An ultra-high performance liquid chromatography-diode array detection-tandem mass spectrometric( UPLC-DAD-MS/MS) method was developed for determination of phenolic compounds in chrysanthemum samples.The chromatographic separation was performed on an UPLC HSS T3 column by gradient elution with mobile phases of acetonitrile and water both containing 0.1% formic acid at a flow rate of 0.3 mL/min.The samples were extracted with aqueous methanol solution under ultrasonic condition,and the crude extracts were directly assayed without any further pretreatment.Total 10 caffeyolquinic acids( CQAs) and 22 flavonoids were positively identified in Boju,Chuju,Gongju and Hangju by comparing retention times and mass spectra of samples with standards and publications.The results showed that the amounts of phenolic compounds in four chrysanthemum samples all were agreed with the criteria of China Pharmacopoeia( 2010),but there were significant differences for the content and constitute distribution of each caffeyolquinic acid and flavonoid.Except chlorogenic acid( 3-CQA),3,5-dicaffeoylquinic acid( 3,5-DCQA) and luteolin-7-O-glucoside( L-7-O-Glu),other compounds with high content,such as apigenin-7-O-6″-malonylglucoside( AP-7-O-6″-MGlu),4,5-dicaffeoylquinic acid( 4,5-DCQA),1,5-dicaffeoylquinic acid( 1,5-DCQA),luteolin-7-O-6″-malonylglucoside( L-7-O-6″-MGlu) and apigenin-7-O-glucoside( AP-7-O-Glu),were also found in chrysanthemum samples.Among the four chrysanthemum samples,there were big variances for the amounts of total flavonoids,but total CQAs were found at the same level; as for total phenolic compounds in four chrysanthemum samples,Gongju showed the highest content of 66.85 mg/g,followed by Hangju,Chuju and Boju with the contents of 54.69,44.59 and 37.10 mg/g,respectively.