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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.

Purpose: Acute spinal cord injury (SCI) triggers multiple cellular and molecular pathways; therapy aimed at only one pathway is unlikely to succeed. Anecdotal reports indicate that a novel herbal formulation (JSK-Ji-Sui-Kang) may enhance recovery in humans with SCI. We investigated whether JSK's therapeutic effects could be verified in a well-established SCI model in rats., Methods: Therapeutic effects of JSK were tested using a standard behavioral assessment, histological, immunochemical and microarray analysis. Phytochemical fingerprinting of JSK was performed using high performance liquid chromatography coupled with photodiode array detection and electrospray ionization-mass spectrometry. JSK or vehicle was gavaged to rats 24 hours after SCI and daily thereafter for 3 weeks., Results: Locomotor function significantly improved (n = 12; p < 0.05), tissue damage was reduced (p < 0.01; n = 6) and more axons and myelin were observed in JSK-treated compared with vehicle control animals. JSK significantly enhanced expression of neuroglobin, vascular endothelial growth factor and growth-associated protein 43, and reduced the expression of caspase 3, cyclooxygenase-2, RhoA (p < 0.05; n = 6) and fibrinogen (p < 0.01; n = 6). RNA microarray indicated that JSK altered transcription of genes involved in ischemic and inflammatory/immune responses and apoptosis (p < 0.05; n = 3)., Conclusions: JSK appears to target multiple biochemical and cellular pathways to enhance functional recovery and improve outcomes of SCI. The results provide a basis for further investigation of JSK's effects following SCI.

Fundamental to herbal medicine quality is the use of 'authentic' medicinal herb species. Species, however, 'represent more or less arbitrary and subjective man-made units'. Against this background, we discuss, with illustrative examples, the importance of defining species boundaries by accommodating both the fixed (shared) diagnostic and varying (within-species) traits in medicinal herb populations. We emphasize the role of taxonomy, floristic information and genomic profiling in authenticating medicinal herb species, in addition to the need to include within species phytochemical profile variations while developing herbal extract identification protocols. We outline the application of species-specific genomic and phytochemical markers, chemoprofiling and chemometrics as additional tools to develop qualifying herbal extract references. We list the diagnostic traits available subsequent to each step during the medicinal herb extract manufacturing process and delineate limits to qualification of extract references., (Copyright © 2012 Elsevier B.V. All rights reserved.)

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.

DNA fingerprinting of plants has become an invaluable tool in forensic, scientific, and industrial laboratories all over the world. PCR has become part of virtually every variation of the plethora of approaches used for DNA fingerprinting today. DNA sequencing is increasingly used either in combination with or as a replacement for traditional DNA fingerprinting techniques. A prime example is the use of short, standardized regions of the genome as taxon barcodes for biological identification of plants. Rapid advances in "next generation sequencing" (NGS) technology are driving down the cost of sequencing and bringing large-scale sequencing projects into the reach of individual investigators. We present an overview of recent publications that demonstrate the use of "NGS" technology for DNA fingerprinting and DNA barcoding applications.

Endophytes live inter- and/or intracellularly inside healthy aboveground tissues of plants without causing disease. Endophytic fungi are found in virtually every vascular plant species examined. The origins of this symbiotic relationship between endophytes go back to the emergence of vascular plants. Endophytic fungi receive nutrition and protection from their hosts while the plants benefit from the production of fungal secondary metabolites, which enhance the host plants' resistance to herbivores, pathogens, and various abiotic stresses. Endophytic fungi have attracted increased interest as potential sources of secondary metabolites with agricultural, industrial, and medicinal use. This chapter provides detailed protocols for isolation of genomic DNA from fungal endophytes and its use in polymerase chain reaction-based amplification of the internal transcribed spacer region between the conserved flanking regions of the small and large subunit of ribosomal RNA for barcoding purposes.

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.

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.

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.

A rapid, sensitive and specific reversed phase high-performance liquid chromatographic (LC) method with photodiode array detection (DAD) has been developed for the determination of glycyrrhizic acid in both the raw herb and a commercially prepared dried aqueous extract of Glycyrrhiza uralensis Fisch. ex DC. root (Zhi Gan Cao, liquorice). It was determined that extracting the raw herb in aqueous methanol (50:50 v/v) by sonication for 2 x 30 min was the most efficient sample preparation. Baseline resolution of the glycyrrhizic acid peak was achieved on a Varian Polaris RP C18-A (250 mm x 4.6 mm, 5 microm packing) column using an isocratic mobile phase consisting of 0. v aqueous phosphoric acid and acetonitrile in the ratio 60:40 v/v. Chromatograms were monitored between 200 and 400 nm for peak purity assessments, with quantitation performed at 254 nm. Glycyrrhizic acid calibration curves in the concentration range of 14-558 microg/ml were prepared on the day of analysis. Curve fitting was by the least-squares method, with correlation coefficients of >0.9998 obtained each time. The average recovery at three spike levels (50, 100, 200%) was of 95.91+/-1.05% and 98.36+/-3.45% (+/-S.D., n=7) for the spiked raw herb and dried aqueous extract respectively. The limit of detection and limit of quantitation was 0.52 and 1.72 mg/g respectively for the raw herb, and 0.75 and 2.51 mg/g respectively for the dried aqueous extract. Identity confirmation of the chromatographic peak was achieved by (-) electrospray ionisation tandem mass spectrometry. The concentration of glycyrrhizic acid in the root and dried aqueous extract was found to be 31.1+/-0.2 and 40.4+/-0.3mg/g (+/-S.D., n=7) respectively.

A rapid and specific reversed-phase high performance liquid chromatography (RP-LC) method with photodiode array detection (DAD) was developed and validated for the determination of four common schisandra lignans, schisandrin (1), schisandrol B (2), deoxyshisandrin (3) and gamma-schisandrin (4), in raw herb materials and commercial dried aqueous extracts of Schisandra chinensis (Wu Wei Zi). The extraction solvent and extraction method were optimised where it was found that a 4 h Soxhlet extraction using methanol was successful at extracting >99.5% of each of the schisandra lignans analysed from the raw herb material. The sample preparation process for the dried aqueous extract samples involved sonication using methanol for 2 x 30 min. The herb and extract solutions were separated on a Varian Microsorb-MV 100-5 C18 column using a gradient mixture of 0.1% aqueous phosphoric acid and acetonitrile. Subsequent detection and quantitation of the schisandra lignans was performed at 210 nm. The correlation coefficients of the linear regression analysis performed on these calibration curves were >0.9996 for all four schisandra lignans assayed. The detection limits and quantification limits ranged from 0.12 to 0.57 and 0.41 to 1.89 mg g(-1), respectively. The mean recoveries of the various analytes ranged from 92.20 to 107.01%. The method was used to investigate the levels of the four mentioned components in herb samples and dried aqueous extracts. The identities of the chromatographic peaks were confirmed by (+) electrospray ionisation LC-MS/MS.