A pharmacological basis of herbal medicines for epilepsy.

Epilepsy is the most common chronic neurological disease, affecting about 1% of the world's population during their lifetime. Most people with epilepsy can attain a seizure-free life upon treatment with antiepileptic drugs (AEDs). Unfortunately, seizures in up to 30% do not respond to treatment. It is estimated that 90% of people with epilepsy live in developing countries, and most of them receive no drug treatment for the disease. This treatment gap has motivated investigations into the effects of plants that have been used by traditional healers all over the world to treat seizures. Extracts of hundreds of plants have been shown to exhibit anticonvulsant activity in phenotypic screens performed in experimental animals. Some of those extracts appear to exhibit anticonvulsant efficacy similar to that of synthetic AEDs. Dozens of plant-derived chemical compounds have similarly been shown to act as anticonvulsants in various in vivo and in vitro assays. To a significant degree, anticonvulsant effects of plant extracts can be attributed to widely distributed flavonoids, (furano)coumarins, phenylpropanoids, and terpenoids. Flavonoids and coumarins have been shown to interact with the benzodiazepine site of the GABAA receptor and various voltage-gated ion channels, which are targets of synthetic AEDs. Modulation of the activity of ligand-gated and voltage-gated ion channels provides an explanatory basis of the anticonvulsant effects of plant secondary metabolites. Many complex extracts and single plant-derived compounds exhibit antiinflammatory, neuroprotective, and cognition-enhancing activities that may be beneficial in the treatment of epilepsy. Thus, botanicals provide a base for target-oriented antiepileptic drug discovery and development. In the future, preclinical work should focus on the characterization of the effects of plant extracts and plant-derived compounds on well-defined targets rather than on phenotypic screening using in vivo animal models of acute seizures. At the same time, available data provide ample justification for clinical studies with selected standardized botanical extracts and plant-derived compounds. This article is part of a Special Issue entitled "Botanicals for Epilepsy".

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.

Quality assessment of medicinal herbs and their extracts: Criteria and prerequisites for consistent safety and efficacy of herbal medicines.

Ingredients of commercial herbal medicines are assessed for quality primarily to ensure their safety. However, as complex mixtures of different groups of plant secondary metabolites, retention of overall phytochemical consistency of herbal medicines is pivotal to their efficacy. Authenticity and homogeneity of the herbs and strict regimes of physical processing and extract manufacturing are critical factors to maintain phytochemical consistency in commercial products. To ensure both safety and efficacy of herbal medicines, implementation of and adherence to good agricultural and collection practice (GACP), good plant authentication and identification practice (GPAIP), good manufacturing practice (GMP) before and during the manufacturing process, and good laboratory practice (GLP) in analysis are necessary. Establishment and application of harmonized multilaboratory-validated analytical methods and transparency in the supply (value) chain through vendor audits are additional requirements in quality assurance. In this article, we outline steps of a comprehensive quality assurance paradigm aimed at achieving and maintaining safety, consistent phytochemical composition, and clinical efficacy of ingredients of herbal medicines. This article is part of a Special Issue entitled Botanicals for Epilepsy.

Anti-inflammatory activity of cinnamon (C. zeylanicum and C. cassia) extracts - identification of E-cinnamaldehyde and o-methoxy cinnamaldehyde as the most potent bioactive compounds.

Chronic inflammation is a contributing factor in many age-related diseases. In a previous study, we have shown that Sri Lankan cinnamon (C. zeylanicum) was one of the most potent anti-inflammatory foods out of 115 foods tested. However, knowledge about the exact nature of the anti-inflammatory compounds and their distribution in the two major cinnamon species used for human consumption is limited. The aim of this investigation was to determine the anti-inflammatory activity of C. zeylanicum and C. cassia and elucidate their main phytochemical compounds. When extracts were tested in LPS and IFN-γ activated RAW 264.7 macrophages, most of the anti-inflammatory activity, measured by down-regulation of nitric oxide and TNF-α production, was observed in the organic extracts. The most abundant compounds in these extracts were E-cinnamaldehyde and o-methoxycinnamaldehyde. The highest concentration of E-cinnamaldehyde was found in the DCM extract of C. zeylanicum or C. cassia (31 and 34 mg g(-1) of cinnamon, respectively). When these and other constituents were tested for their anti-inflammatory activity in RAW 264.7 and J774A.1 macrophages, the most potent compounds were E-cinnamaldehyde and o-methoxycinnamaldehyde, which exhibited IC50 values for NO with RAW 264.7 cells of 55 ± 9 µM (7.3 ± 1.2 µg mL(-1)) and 35 ± 9 µM (5.7 ± 1.5 µg mL(-1)), respectively; and IC50 values for TNF-α of 63 ± 9 µM (8.3 ± 1.2 µg mL(-1)) and 78 ± 16 µM (12.6 ± 2.6 µg mL(-1)), respectively. If therapeutic concentrations can be achieved in target tissues, cinnamon and its components may be useful in the treatment of age-related inflammatory conditions.

An in vitro study of neuroprotective properties of traditional Chinese herbal medicines thought to promote healthy ageing and longevity.

BACKGROUND: Age is the leading risk factor for acute and chronic neurodegenerative diseases. The Shen Nong Ben Cao Jing, the oldest known compendium of Chinese materia media, lists herbal medicines that were believed to exert neither fast acting pharmacological effects nor discernible toxicity, but to promote general health and longevity. In modern terms, these herbal medicines could be considered as complementary health care products for prevention rather than treatment of diseases. In the present study, we examined whether a selection of 13 such herbal medicines exhibited neuroprotective activity. METHODS: The antioxidant capacity of the herbal extracts was determined using three non-cellular assays measuring the total phenol content (FCR assay), 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity and oxygen radical absorbance capacity (ORAC). Cytotoxic effects of the herbal extracts were assayed in cultured mouse cortical neurons and their neuroprotective activities were studied using staurosporine-induced apoptosis of the cultured neurons. RESULTS: Most of the herbal extracts showed negligible toxic effects at 100 µg/ml. However, Polygonum multiflorum and Rhodiola rosea exhibited some neurotoxicity at this concentration. Extracts of Ganoderma lucidum, Glycyrrhiza glabra, Schizandra chinensis, and Polygonum cuspidatum inhibited staurosporine-induced apoptosis by 30 - 50% in a dose-dependent manner. The neuroprotective effects of Polygonum cuspidatum were predominantly due to its major ingredient, resveratrol. The effective herbal extracts showed various levels of reactive oxygen species (ROS) scavenging capacity, which was significantly correlated with their neuro- protective activity. However, P. multiflorum and R. rosea extracts proved to be the exception as they exhibited a high level of antioxidant capacity, but did not exhibit neuroprotective effects in cell-based assay. CONCLUSIONS: This in vitro study provides evidence for neuroprotective activity of some Chinese herbal medicines traditionally used to promote healthy ageing and longevity. Our results provide a justification for further study of these herbal extracts in neurodegenerative animal models to assess their safety and effectiveness as a basis for subsequent clinical trials. These herbal medicines might potentially offer a novel preemptive neuroprotective approach in neurodegenerative diseases and might be developed for use in persons at risk.

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.

Effects of a novel herbal formulation JSK on acute spinal cord injury in rats.

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.

Cytoprotective properties of traditional Chinese medicinal herbal extracts in hydrogen peroxide challenged human U373 astroglia cells.

Age is the leading risk factor for many of the most prevalent and devastating diseases including neurodegenerative diseases. A number of herbal medicines have been used for centuries to ameliorate the deleterious effects of ageing-related diseases and increase longevity. Oxidative stress is believed to play a role in normal ageing as well as in neurodegenerative processes. Since many of the constituents of herbal extracts are known antioxidants, it is believed that restoring oxidative balance may be one of the underlying mechanisms by which medicinal herbs can protect against ageing and cognitive decline. Based on the premise that astrocytes are key modulators in the progression of oxidative stress associated neurodegenerative diseases, 13 herbal extracts purported to possess anti-ageing properties were tested for their ability to protect U373 human astrocytes from hydrogen peroxide induced cell death. To determine the contribution of antioxidant activity to the cytoprotective ability of extracts, total phenol content and radical scavenging capacities of extracts were examined. Polygonum multiflorum, amongst others, was identified as possessing potent antioxidant and cytoprotective properties. Not surprisingly, total phenol content of extracts was strongly correlated with antioxidant capacity. Interestingly, when total phenol content and radical scavenging capacities of extracts were compared to the cytoprotective properties of extracts, only moderately strong correlations were observed. This finding suggests the involvement of multiple protective mechanisms in the beneficial effects of these medicinal herbs.

The application of Chinese medicine to novel drug discovery.

INTRODUCTION: The apparent productivity crisis in the pharmaceutical industry and the economic and political rise of China have contributed to renewed interest in the application of Chinese medicine for drug discovery. AREAS COVERED: The author presents an overview of the historical development and basic principles of theory and practice of Chinese herbal medicine, its materia medica and prescription formulas, and discusses the motivation for and rationale of its application to drug discovery. Furthermore, the author distinguishes the five main approaches to drug discovery from Chinese herbal medicine, based on the decreasing amount and detail of historical and clinical Chinese medicine knowledge that informed the research effort. EXPERT OPINION: Many compounds that have been isolated from the Chinese materia medica exhibit pharmacological activities comparable to pharmaceutical drugs. With the exception of the antimalarial drug artemisinin, however, this knowledge has not led to the successful development of new drugs outside of China. The chance of success in a Chinese medicine-based drug discovery effort will be increased by consideration of the empirical knowledge that has been documented over many centuries in the historical materia medica and prescription literature. Most Chinese medicine-derived compounds affect more than one target and do not correspond to the one compound/one-target drug discovery paradigm. A new frontier is opening up with the development of drugs consisting of combinations of multiple compounds acting on multiple targets under the paradigm of network pharmacology. The ancient practice of combining multiple drugs in prescription formulas can serve as inspirational analogy and a practical guide.

From classical taxonomy to genome and metabolome: towards comprehensive quality standards for medicinal herb raw materials and extracts.

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.

DNA fingerprinting, DNA barcoding, and next generation sequencing technology in plants.

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.

Genomic DNA extraction and barcoding of endophytic fungi.

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.

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.

Association of the small GTPase Rheb with the NMDA receptor subunit NR3A.

The NMDAR subunit NR3A is most highly expressed during the second postnatal week, when synaptogenesis reaches peak levels. Genetic ablation or overexpression of the NR3A subunit negatively interferes with the maturation of cortical synapses and leads to changes in the shape and number of dendritic spines, the density of which is increased in NR3A knock-out mice and decreased in NR3A-overexpressing transgenic mice. Alterations in spine density have been linked to dysregulation of mTOR signaling and synaptic protein translation. Using a yeast two-hybrid system, we identified the mTOR-activating GTPase Rheb as an interacting protein of the NMDAR subunit NR3A. We confirmed the interaction in mammalian cells by expressing recombinant Rheb and NR3A and showed that Rheb and NR3A could be co-immunoprecipitated from synaptic plasma membranes from the developing rat brain. These data suggest that NR3A sequesters synaptic Rheb and might thus function as a break of the mTOR-dependent synaptic translation of protein.

A non-muscle myosin II motor links NR1 to retrograde trafficking and proteasomal degradation in PC12 cells.

Rat pheochromocytoma (PC12) cells have been shown to lack functional NMDA receptors; yet, these cells express NR1 subunits of the NMDA receptor. The reason for the lack of functional receptors has been attributed to the absence of significant levels of NR2 subunits to co-assemble with NR1. It is known that PC12 expresses very low levels of NR2C, with complete absence of other types of NR2 subunits. The purpose of the present study is to describe the molecular mechanism of trafficking and degradation of unassembled NR1 subunits in PC12 cells. The localization of NR1 subunits in PC12 cells were evaluated by immunofluorescence and co-immunoprecipitation, which showed that NR1 was present in the endoplasmic reticulum and cis-middle compartments of the Golgi apparatus. Upon treatment with a proteasome inhibitor, MG132, the ubiquitinylated species of NR1 subunit were detected, suggesting that NR1 is being targeted for endoplasmic reticulum-associated proteasomal degradation. Our previous studies suggest that NR1 subunits from the Golgi do not proceed to trans-Golgi, hence they will require re-routing to the endoplasmic reticulum for degradation. Further investigations on the factors involved in the trafficking of NR1 from Golgi to endoplasmic reticulum were performed using co-immunoprecipitation and matrix assisted laser desorption/ionization time-of-flight mass spectrometry. These revealed the co-association of NR1 with non-muscle myosin heavy chain II isoforms A and B. We also demonstrate the functional significance of this interaction through the use of a myosin inhibitor, blebbistatin, to disrupt brefeldin A-induced Golgi-to-endoplasmic reticulum trafficking of NR1. In conclusion, our results suggest that non-muscle myosin II is involved in the retrograde trafficking of NR1 subunits from the cis/middle-Golgi to the endoplasmic reticulum for proteasomal degradation in PC12.

Altered development of glutamatergic synapses in layer V pyramidal neurons in NR3A knockout mice.

Expression of the NMDA receptor (NMDAR) subunit NR3A reaches its highest level in layer V of the developing rodent cortex during the second postnatal week, a peak period of synaptogenesis. Incorporation of NR3A leads to the formation of non-canonical, Mg2+-insensitive NMDARs, but it is not known whether they participate in synaptic transmission and maturation. Here we show that in the second postnatal week, layer V pyramidal neurons in the somatosensory cortex of wild type (WT) mice exhibited evoked excitatory postsynaptic currents (eEPSCs) with 3- to 6-fold lower Mg2+ sensitivity than NR3A knockout (KO) mice and their reversal potential was approximately 2 mV more negative compared to KO mice consistent with decreased P(Ca) of NMDARs. Surprisingly, ablation of NR3A also led to a 20-fold reduction of the ratio of AMPAR- to NMDAR-mediated eEPSC amplitudes in KO mice. Insertion of AMPARs at the synapses of layer V pyramidal neurons appears to be facilitated by the expression of Mg2+-insensitive NMDARs. The data indicate that NR3A plays a significant role in the development of excitatory synapses in layer V of the developing neocortex.

Early alterations of AMPA receptors mediate synaptic potentiation induced by neonatal seizures.

The highest incidence of seizures during lifetime is found in the neonatal period and neonatal seizures lead to a propensity for epilepsy and long-term cognitive deficits. Here, we identify potential mechanisms that elucidate a critical role for AMPA receptors (AMPARs) in epileptogenesis during this critical period in the developing brain. In a rodent model of neonatal seizures, we have shown previously that administration of antagonists of the AMPARs during the 48 h after seizures prevents long-term increases in seizure susceptibility and seizure-induced neuronal injury. Hypoxia-induced seizures in postnatal day 10 rats induce rapid and reversible alterations in AMPAR signaling resembling changes implicated previously in models of synaptic potentiation in vitro. Hippocampal slices removed after hypoxic seizures exhibited potentiation of AMPAR-mediated synaptic currents, including an increase in the amplitude and frequency of spontaneous and miniature EPSCs as well as increased synaptic potency. This increased excitability was temporally associated with a rapid increase in phosphorylation at GluR1 S845/S831 and GluR2 S880 sites and increased activity of the protein kinases CaMKII (calcium/calmodulin-dependent protein kinase II), PKA, and PKC, which mediate the phosphorylation of these AMPAR subunits. Postseizure administration of AMPAR antagonists NBQX (2,3-dihydroxy-6-nitro-7-sulfonyl-benzo[f]quinoxaline), topiramate, or GYKI-53773 [(1)-1-(4-aminophenyl)-3-acetyl-4-methyl-7,8-methylenedioxy-3,4-dihydro-5H-2,3-benzodiazepine] attenuated the AMPAR potentiation, phosphorylation, and kinase activation and prevented the concurrent increase in in vivo seizure susceptibility. Thus, the potentiation of AMPAR-containing synapses is a reversible, early step in epileptogenesis that offers a novel therapeutic target in the highly seizure-prone developing brain.

Genome-based approaches to the authentication of medicinal plants.

Medicinal plants are the source of a large number of essential drugs in Western medicine and are the basis of herbal medicine, which is not only the primary source of health care for most of the world's population living in developing countries but also enjoys growing popularity in developed countries. The increased demand for botanical products is met by an expanding industry and accompanied by calls for assurance of quality, efficacy and safety. Plants used as drugs, dietary supplements and herbal medicines are identified at the species level. Unequivocal identification is a critical step at the beginning of an extensive process of quality assurance and is of importance for the characterization of the genetic diversity, phylogeny and phylogeography as well as the protection of endangered species. DNA-based methods have been developed for the identification of medicinal plants. Nuclear and chloroplast DNA is amplified by the polymerase chain reaction and the reaction products are analyzed by gel electrophoresis, sequencing, or hybridization with species-specific probes. Genomic fingerprinting can differentiate between individuals, species and populations and is useful for the detection of the homogeneity of the samples and presence of adulterants. Although sequences from single chloroplast or nuclear genes have been useful for differentiation of species, phylogenetic studies often require consideration of DNA sequence data from more than one gene or genomic region. Phytochemical and genetic data are correlated but only the latter normally allow for differentiation at the species level. The generation of molecular "barcodes" of medicinal plants will be worth the concerted effort of the medicinal plant research community and contribute to the ongoing effort of defining barcodes for every species on earth.

Characterization of mRNA expression in single neurons.

How neurons differ from each other is largely determined by their specific repertoire of mRNAs. The genes expressed by a given neuron reflect its developmental history, its interaction with other cells, and its synaptic activity. Since the introduction of reverse transcription polymerase chain reaction (RT-PCR), it has been possible to identify specific mRNAs present in small samples of total RNA. But isolating RNA from only those cells of interest, and not others, represents a significant challenge. Several approaches can be used to isolate RNA from selected neurons. Following whole-cell patch-clamp recording, mRNA can be harvested from living cells by aspirating the cytoplasm into the patch-clamp pipette. Transcripts expressed in the recorded neuron can then be amplified by RT-PCR. Another way of isolating identified neurons is to use cell-specific promoters to drive the expression of a marker gene such as green fluorescent protein (GFP). RNA can then be isolated from GFP-positive cells. In a tissue context, laser microdissection can also be used to excise the cells of interest directly into an RNA isolation solution. The above methods of RNA isolation can also be combined with RNA amplification and microarray technology to identify specific transcripts that are unique to the cell type being studied. Here we provide detailed protocols for harvesting RNA from single cells, methods for RNA purification, and PCR amplification.