Chromatographic Analysis and Anti-Oxidative Property of Naoxinqing Tablet, a Proprietary Preparation of Diospyros Kaki Leaves.

The Naoxinqing (NXQ) tablet is a standardised proprietary herbal product containing an extract of persimmon leaves (Diospyros kaki) for the management of cardio- and cerebrovascular diseases. Although previous reports suggested that the efficacy of NXQ is at least partly mediated by its anti-oxidative property, the anti-oxidative effect of the major components of NXQ has not been studied systematically. For quality control purposes, only analytical methods limited to 3 marker analytes have been reported, the extent to which the other components affect efficacy has not been explored. In this study, we developed an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC MS/MS) method for the identification of seven analytes (kaempferol-3-O-glucoside (astragalin), quercetin-3-O-galactoside (hypericin), quercetin-3-O-glucoside (isoquercitin), kaempferol, 3,4-dihydroxybenzoic acid (protocatechuic acid), and furan-2-carboxylic acid (pyromucic acid) and quercetin) in the NXQ. This is the first method reported and validated for the quantification of the seven major secondary metabolites in NXQ. The results for the quantified analytes were then compared in 15 different batches of NXQ. The variation observed in the seven components highlights the need to quantify key bioactive components to ensure product consistency. Radical scavenging activity and abundance was used to rank the analytes. The anti-oxidative effects of NXQ were examined using cultured human vascular endothelial cells (EA.hy926). Corrected 2,2-di(4-tert-octylphenyl)-1-picrylhydrazyl (DPPH) activity results revealed that quercetin and kaempferol have the strongest anti-oxidant capacity in the extract. Both quercetin and kaempferol significantly inhibited the hydrogen peroxide (H2O2)-induced EA.hy926 cell injury and intracellular reactive oxygen species (ROS) generation. In conclusion, we established and validated an UPLC-MS/MC method for the analysis of major bioactive components in the NXQ and demonstrated that its anti-oxidative property may play a critical role in cerebrovascular protection.

Analytical Method Validation and Quality Control of a Seven-Herb Chinese Medicine Formulation Used for the Treatment of Irritable Bowel Syndrome with Constipation.

There is a need for increased QC of complex herbal medicine formulations to ensure product consistency, efficacy, and safety. This study reports an HPLC with photodiode array and electrospray ionization-tandem MS method for quantifying selected analytes in a seven-herb formulation. Fourteen analytes were selected for quantification based on the criteria available from the Herbal Chemical Marker Ranking System, which takes into account the bioavailability, reported bioactivity, and physiological action related to its intended use, as well as commercial availability of the standard. After optimizing the columns and chromatographic conditions, 13 of the 14 analytes were able to be determined in one run, with the remaining analyte analyzed on its own. The method was successfully applied to two different extracts of the formulation, demonstrating an application for the QC of a complex herbal mixture with respect to their chemical characteristics.

An in vitro study of anti-inflammatory activity of standardised Andrographis paniculata extracts and pure andrographolide.

BACKGROUND: The anti-inflammatory activity of Andrographis paniculata (Acanthaceae), a traditional medicine widely used in Asia, is commonly attributed to andrographolide, its main secondary metabolite. Commercial A. paniculata extracts are standardised to andrographolide content. We undertook the present study to investigate 1) how selective enrichment of andrographolide in commercial A. paniculata extracts affects the variability of non-standardised phytochemical components and 2) if variability in the non-standardised components of the extract affects the pharmacological activity of andrographolide itself. METHODS: We characterized 12 commercial, standardised (≥30% andrographolide) batches of A. paniculata extracts from India by HPLC profiling. We determined the antioxidant capacity of the extracts using 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging, oxygen radical antioxidant capacity (ORAC) and a Folin-Ciocalteu (FC) antioxidant assays. Their anti-inflammatory activity was assessed by assaying their inhibitory effect on the release of tumor necrosis factor alpha (TNF-α) in the human monocytic cell line THP-1. RESULTS: The andrographolide content in the samples was close to the claimed value (32.2 ± 2.1%, range 27.5 to 35.9%). Twenty-one non-standardised constituents exhibited more than 2-fold variation in HPLC peak intensities in the tested batches. The chlorogenic acid content of the batches varied more than 30-fold. The DPPH free radical scavenging activity varied ~3-fold, the ORAC and FC antioxidant capacity varied ~1.5 fold among batches. In contrast, the TNF-α inhibitory activity of the extracts exhibited little variation and comparison with pure andrographolide indicated that it was mostly due to their andrographolide content. CONCLUSIONS: Standardised A. paniculata extracts contained the claimed amount of andrographolide but exhibited considerable phytochemical background variation. DPPH radical scavenging activity of the extracts was mostly due to the flavonoid/phenlycarboxylic acid compounds in the extracts. The inhibitory effect of andrographolide on the release of TNF-α was little affected by the quantitative variation of the non-standardised constituents.

The Saccharomyces cerevisiae transcriptome as a mirror of phytochemical variation in complex extracts of Equisetum arvense from America, China, Europe and India.

BACKGROUND: Pattern-oriented chemical profiling is increasingly being used to characterize the phytochemical composition of herbal medicines for quality control purposes. Ideally, a fingerprint of the biological effects should complement the chemical fingerprint. For ethical and practical reasons it is not possible to test each herbal extract in laboratory animals or humans. What is needed is a test system consisting of an organism with relevant biology and complexity that can serve as a surrogate in vitro system. The purpose of this study was to test the hypothesis that the Saccharomyces cerevisiae transcriptome might be used as an indicator of phytochemical variation of closely-related yet distinctly different extracts prepared from a single species of a phytogeographically widely distributed medicinal plant. We combined phytochemical profiling using chromatographic methods (HPTLC, HPLC-PDA-MS/MS) and gene expression studies using Affymetrix Yeast 2.0 gene chip with principal component analysis and k-nearest neighbor clustering analysis to test this hypothesis using extracts prepared from the phytogeographically widely distributed medicinal plant Equisetum arvense as a test case. RESULTS: We found that the Equisetum arvense extracts exhibited qualitative and quantitative differences in their phytochemical composition grouped along their phytogeographical origin. Exposure of yeast to the extracts led to changes in gene expression that reflected both the similarities and differences in the phytochemical composition of the extracts. The Equisetum arvense extracts elicited changes in the expression of genes involved in mRNA translation, drug transport, metabolism of energy reserves, phospholipid metabolism, and the cellular stress response. CONCLUSIONS: Our data show that functional genomics in S. cerevisiae may be developed as a sensitive bioassay for the scientific investigation of the interplay between phytochemical composition and transcriptional effects of complex mixtures of chemical compounds. S. cerevisiae transcriptomics may also be developed for testing of mixtures of conventional drugs ("polypills") to discover novel antagonistic or synergistic effects of those drug combinations.

Using GenBank® for genomic authentication: a tutorial.

The GenBank(®) database is perhaps one of the most important repositories of genetic information. A researcher working in the field of genomic authentication must therefore be equipped with the skills needed to competently access the required information from this database whilst ultimately contributing their own data to it. This chapter presents a practical guide to using GenBank(®) to search for sequences, search and align unknown sequences using BLAST, and uploading and maintaining your own sequences. This chapter also details some other software helpful in sequence manipulation.

Simultaneous determination of eight sympathomimetic amines in urine by gas chromatography/mass spectrometry.

A GC method was developed for the identification and quantitation of eight sympathomimetic amines in urine, i.e., amphetamine, methamphetamine, mephentermine, ephedrine, pseudoephedrine, methylenedioxyamphetamine, methylenedioxymethamphetamine, and methylenedioxyethylamphetamine. Methoxyphenamine was used as the internal standard (IS). The assay is rapid, sensitive, and simple to perform. It involves a liquid-liquid extraction procedure with simultaneous in-solution derivatization of the organic layer with pentafluorobenzoyl chloride (PFB-CI), followed by GC/MS analysis. These derivatives and the IS were extracted from 1 mL alkaline urine into hexane before derivatization with PFB-CI. The organic layer was then removed and evaporated to dryness before dissolution with hexane for GC/MS analysis. Calibration curves for each analyte showed linearity in the range of 25-5000 ng/mL (r2 > or = 0.997). Recoveries ranged from 88 to 99%, with the precision of recoveries typically < or = 5%. The LOD values ranged from 7 to 28 ng/mL, and the LOQ values ranged from 23 to 94 ng/mL. At least four ions were available for each analyte for confirmation of identity by MS.

LC determination of albiflorin and paeoniflorin in Bai Shao (Paeonia lactiflora) as a raw herb and dried aqueous extract.

A validated analytical method is reported for the analysis of paeoniflorin and albiflorin in Bai Shao (Paeonia lactiflora) as a dried raw herb and commercially prepared dried aqueous extract. The samples were extracted by sonication in methanol and the extract analyzed by LC-photodiode array with identity confirmation by electrospray ionization-tandem MS. A C18 column was used with an acetonitrile-water gradient mobile phase. Paeoniflorin and albiflorin were quantified at 230 nm. Ions with m/z 121 and 327 were produced with the MS detector, using the paeoniflorin precursor ion with m/z 479. For albiflorin, the precursor ion with m/z 479 produced the m/z 121 and 77 ions. The amounts of paeoniflorin and albiflorin found in the raw herb were 33.2 and 1.8 mg/g, respectively; and in the dried aqueous extract, the amounts were 34.8 and 15.7 mg/g, respectively. The LODs for paeoniflorin and albiflorin were 0.37 and 1.39 mg/g, respectively, for the raw herb and 0.25 and 0.06 mg/g, respectively, for the dried aqueous extract.

Liquid chromatographic determination of narirutin and hesperidin in Zhi Ke (Citrus aurantium L.) in the form of the raw herb and of the dried aqueous extract.

A validated analytical method is reported for the analysis of narirutin and hesperidin in Zhi Ke (Citrus aurantium L.) in the form of the dried raw herb and of the commercially prepared dried aqueous extract. The samples were extracted by sonication in methanol and the extract was analyzed by liquid chromatography-photodiode array (PDA) detection with identity confirmation by negative electrospray ionization-tandem mass spectrometry (MS). A C18 column was used with a methanol-water gradient mobile phase. Narirutin and hesperidin were quantified at 284 nm using the PDA detector. Using the MS detector, the narirutin precursor ion with m/z 579 produced daughter ions with m/z 271 and 151. For hesperidin, the precursor ion with m/z 609 produced the m/z 301, 285, and 164 ions. The amounts of narirutin and hesperidin found in the certified raw herb were 14.2 and 147.9 mg/g, respectively, and in the dried aqueous extract the amounts were 9.2 and 8.6 mg/g, respectively. For the raw herb, the average recovery across the three spike levels (50, 100, and 150%) for narirutin and hesperidin were 110.7 and 94.5%, respectively. For the dried aqueous extract, the average recovery across the three spike levels for narirutin and hesperidin were 85.8 and 98.9%, respectively.

Liquid chromatographic determination of flumetsulam in soybeans.

A liquid chromatography (LC) method with UV detection is reported for the determination of the sulfonamide herbicide flumetsulam in soybeans. The ground soybean sample was partitioned between methanol and hexane. The hexane removed the lipids, and the methanol layer containing the analyte was further partitioned between dichloromethane and aqueous phosphate buffer at pH 7.0. The aqueous layer, containing the analyte, was acidified to pH 2.2 and partitioned with fresh dichloromethane. The dichloromethane layer containing the analyte was evaporated, and the residue was dissolved in the LC mobile phase for analysis. A polar embedded C18 column was used with a mobile phase of pH 2.2 aqueous phosphate buffer-acetonitrile (68 + 32), run isocratically with detection at 225 nm. The average recovery was 82% with a relative standard deviation (RSD) of 10%. A coefficient of determination of R2 = 0.9992 was achieved for the analyte calibration curve, from 0.005 to 1 microg/mL. The limit of detection, determined from 3 times the standard deviation of 7 replicate extractions of the lowest fortification level (0.01 microg/g), was 0.005 microg/g with an RSD of 22%. LC/electrospray ionization/mass spectrometry in the positive-ion mode was used for identity confirmation of flumetsulam in the fortified soybean extract. The ions at m/z 326, 348, and 129 were observed.

Determination of clenbuterol, salbutamol, and cimaterol in bovine retina by electrospray ionization-liquid chromatography-tandem mass spectrometry.

An electrospray ionization-liquid chromatography-tandem mass spectrometry (ESI/LC/MS/MS) method was developed for the simultaneous determination of the beta-agonists clenbuterol, salbutamol, and cimaterol in bovine retina. The tissue was homogenized in alkaline buffer and spiked to give 10, 15, and 20 ng/g each of the 3 analytes together with the internal standards d6-salbutamol and d6-clenbuterol. The mixture was incubated with protease enzyme to release any protein-bound analytes and then made alkaline before extraction with isobutanol. The extract was dissolved in water and transferred to a clenbuterol immunoaffinity column. After washing, the analytes were eluted and analyzed by ESI/LC/MS/MS using a C18 column with acetic acid-methanol as mobile phase. No interferences were observed from the spiked retina extract at the various single-reaction monitoring modes. Average recoveries for clenbuterol, salbutamol, and cimaterol were 94, 85, and 87% with coefficients of variation (CVs) of 9.4, 9.9, and 8.6%, respectively. A correlation coefficient of r2 = 0.9999 was obtained for all analytes. The limits of detection for clenbuterol, salbutamol, and cimaterol, determined from 3 times the standard deviation of 7 replicates of the lowest spike, were 2.5, 3.5, and 2.0 ng/g with CVs of 8.9, 11.6, and 7.2%, respectively.