The chemically complex nature of botanically-derived ingredients calls for unique quality control processes by suppliers, manufacturers, and producers of herbal products. One of the regulatory requirements in industrialized nations is the appropriate testing for identity and authenticity of botanical materials used in consumer products. Nevertheless, there have been numerous recent cases of accidental or intentional adulteration. Intentional adulteration (economically motivated adulteration, EMA) is where raw materials are intentionally substituted or diluted with undisclosed lower-quality ingredients for financial gain of the seller. Examples are being compiled and published by an independent consortium consisting of the American Botanical Council (ABC), the American Herbal Pharmacopoeia (AHP), and the National Center for Natural Product Research (NCNPR) at the University of Mississippi as part of the ABC-AHP-NCNPR Botanical Adulterants Prevention Program.

Currently two types of Chamomile are used for medicinal purposes and beauty products. Both, Matricaria recutita L. and Anthemis nobilis L., belong to the familyAsteraceae (Compositae) and are used to treat similar health problems. However, they differ in their biologically active compounds. The use of DNA barcoding was explored to distinguish between the various Chamomile species. The trnL-trnF and psbA-trnH genomic regions were investigated for their utility to distinguish between Chamomile species using PCR-restriction fragment length polymorphism (PCR-RFLP) and designed oligonucleotides. A new set of markers for Chamomile was identified to distinguish between Matricaria recutita (German chamomile) and Anthemis nobilis (Roman chamomile). The PCR-RFLP and the designed oligonucleotides have the potential to be an informative tool to help distinguish between German (Matricaria recutita) and Roman chamomiles (Anthemis nobilis) plant samples.

Plant pathogenic fungi produce secondary metabolites that are phytotoxic to the host plant. These metabolites can be used as bioherbicides or lead molecules or templates in developing agrochemicals [1].From an infected leaf of Hedera helix (English Ivy) that showed necrosis, a fungus was isolated and identified as Diaporthe eres by molecular techniques. The ITS1 – 5.8S-ITS2 genomic region (ITS) was amplified from genomic DNA using the forward primer ITS1 (5'- TCCGTAGGTGAACCTGCGG-3') and the reverse primer ITS4 (5'- TCCTCCGCTTATTGATATGC-3') [2]. The primers cylh3f (5'- AGGTCCACTGGTGGCAAG-3') [3] and H3 – 1b (5′- GCGGGCGAGCTGGATGTCCTT-3′) [4] were used to amplify part of the histone H3 (HIS) gene, and the primers T1 (5′- AACATGCGTGAGATTGTAAGT-3′) [5] and Bt-2b (5′- ACCCTCAGTGTAGTGACCCTTGGC-3′)4 to amplify part of the beta-tubulin gene (TUB). This fungus was grown in Czapek Dox broth culture medium for two weeks. The ethyl acetate extract of the liquid culture medium showed phytotoxic activity against monocots (Agrostis stolinifera) and dicots (Lactuca sativa) with 90% and 80% germination inhibition respectively. The two known metabolites were isolated via bioassay guided isolation and identified by NMR spectroscopy. This is the first report of isolation of phytotoxins from D.eres. One of the phytotoxic constituents was identified as 8-hydroxy-3, 7 -dimethylisochroman-1-one. We have synthesized this phytotoxin as well as some analogs that showed phytotoxic activity in seed germination bioassays and Lemna pausicostata (duckweed) growth bioassay. The IC50 values of these compounds ranged from 82µM to 288 µm in L. pausicostata bioassay. Some analogs showed hormesis in L. pausicostata growth bioassay.

Oxidosqualene cyclases (OSCs) are the key enzymes responsible for cyclization of 2, 3-oxidosqualene to varied triterpenoids and phytosterols. Hoodia gordonii (Asclepiadacecae) a native of Kalahari deserts of South Africa, Namibia, and Botswana is one of the most prevalent herbal supplements for weight loss. The appetite suppressant properties are attributed to P57AS3, an oxypregnane steroidal glycoside. At the molecular level, the enzymes involved in biosynthesis of triterpenes and phytosterols from H. gordonii have not been reported in the literature. In the present investigation, predicted transcripts potentially encoding oxidosqualene cyclases, were first identified by mining publicly-available H. gordonii RNA-seq data sets. Recombinant enzyme studies with two of the OSCs-like sequences led to the identification of HgCS1 and HgCS2, encoding lupeol and cycloartenol synthases, respectively. GC-MS predicted the HgCS1 reaction product as lupeol, and the structure is confirmed using NMR.

The genetic stability of Stevia rebaudiana Bertoni plants, propagated through in vitro stored synthetic seeds was assessed. In vitro grown micro shoots of Stevia rebaudiana were encapsulated in 5% sodium alginate and 50 mM CaCl2. The synthetic seeds were stored in vitro for 4 months and re-grown under the tissue culture conditions (16-h photoperiod, 25 oC) on Murashige and Skoog (MS) medium supplemented with thidiazuron (TDZ 0.2 mg L-1) [1]. Well rooted plantlets were successfully transferred to a climatic controlled indoor cultivation facility for further cultivation with mother plant. Leaf samples were taken from nine, randomly selected, fully developed, healthy in vitro propagated plants and a mother plant for the molecular analysis of genetic fidelity using eight inter simple sequence repeat (ISSR) markers. Each tested primer produced clear and scorable amplification products ranging in size from about 176 bp (UBC 817) to 1354 bp (UBC 826) with an average of 5.1 products per primer. A total of 41 bands were generated, giving rise to monomorphic patterns across all 10 plantlets analyzed. No ISSR polymorphism was observed in the micropropagated plants. Furthermore, leaf samples from the plants used for molecular analysis were also evaluated for rebaudioside A and stevioside content using high performance liquid chromatography (HPLC) [2]. Our data show no significant differences (p < 0.05) in rebaudioside A and stevioside content among re-grown plants and the mother plant following four months of in vitro storage, thus confirming the clonal fidelity of Stevia rebaudiana plants.

The identification of raw plant material can be done by organoleptic characterization, macroscopic, microscopic and chemical analysis, and DNA barcoding. On the other hand the identification of processed plant material, for example in dietary supplements, can be difficult as the plant material often loses characteristic morphological features during the manufacturing process.

The strong flavor of licorice, which comes from the Glycyrrhiza root extract, is either loved or very much disliked. Licorice flavor is found in probably half of the many traditional Chinese medicine prescription to improve their flavor. The extract is made from three authentic licorice drug plants: G. uralensis, G. glabra and G. inflata. G. uralensis being the most important one but its habitats have been diminished severely due to overharvesting; it is now an endangered and nationally-protected medicinal plant in China. Consequently most of the Glycyrrhiza plant material is now cultivated. Large amounts of glycyrrhizin, a sweetener, is present in the three species G. uralensis, G. glabra and G. inflata but absent in other species. The formation of hybrids between the various species makes it difficult to clearly identify Glycyrrhiza plant material based on the analysis of the barcoding regions ITS, matK, psbA-trH, and rbcL. We have successfully analyzed a different genomic region and its ability to reveal the differences between various Glycyrrhiza samples.

Hoodia gordonii (Asclepiadacecae) a native of Kalahari deserts of South Africa, Namibia, and Botswana is one of the most prevalent herbal supplements for weight loss. The appetite suppressant properties are attributed to P57AS3, an oxypregnane steroidal glycoside. At the molecular level, the enzymes involved in biosynthesis of triterpenes and steroidal glycosides in H. gordonii have not been reported in the literature. In an attempt to isolate genes encoding enzymes involved in this biosynthesis process, we identified and cloned two putative oxidosqualene cyclases, designated HgCS1 and HgCS2, from publicly-available RNA-seq data prepared from H. gordonii. Sequence analysis and initial observations suggest that the two cyclases belong to lupeol synthase and cycloartenol synthase.

Ginkgo biloba is one of the oldest living tree species and its leaves are among the most extensively studied herbs. The leaves are used to treat blood disorders and to enhance memory. The flavonoids, diterpene lactones, terpenoids and polysaccharides are the bioactive compounds in Ginkgo leaves. Till date only 12 the genes responsible for ginkgolides/bilobalides and the flavonoids biosynthetic pathways from Ginkgo have been cloned and characterized [1]. This is due to lack of genome sequences/ESTs available in GenBank. Recently, Lin et al. 2011 reported functional analysis of Ginkgo biloba leaves using NGS (Next Generation Sequencing) technology and generated a total of 64,057 ESTs. Five and 11 ESTs for diterpenoid and terpenoids, respectively, were identified. In the present investigation, we initiated our attempt to analyze these ESTs. Two out of 16 ESTs were identified as genes coding for terpene synthases. These short EST sequences were then used for homolog search from the recently assembled Ginkgo transcriptome dataset at http://medicinalplantgenomics.msu.edu. Here, we describe the results of molecular cloning and characterization of these terpene synthase genes from G. biloba. The two genes were characterized as Farnescene synthase and Bisabolene synthase. This is the first report of α-Bisabolene compound which is obtained from yeast after expressing bisabolene synthase gene, exhibiting mild insect repellent activity against Aedes aegypti.

Few studies have addressed the presence and bioactivity of endophytic fungi living in plantlets growing under in vitro conditions. The objectives of the study were: 1) to identify a fungus UM 109 growing as endophytes of autotrophic cultures of the medicinal plant Smallanthus sonchifolius (yacon), and 2) to isolate the compounds produced by this endophyte that showed antifungal properties. The species was identified as Coniochaeta ligniaria using molecular and morphological methods. The crude extract of C. ligniaria displayed antifungal activity. The identification of the active constituents was done by systematic bioactivity-guided fractionation of the dichloromethane extract against the phytopathogenic Colletotrichum species, NMR spectroscopy and GC-FID analysis. The antifungal fractions from the C. ligniaria were identified as a mixture of 12 antifungal fatty acids, including caproic, caprylic, myristic, palmitic, heptadecanoic, stearic, oleic, linoleic and stearic. The presence of antifungal fatty acids was not unique to the fractions of C. ligniaria culture and caproic, caprylic, myristic, palmitic, heptadecanoic, stearic, oleic and linoleic acids were isolated and detected in the antifungal fractions of S. sonchifolius. In conclusion, the identification of the fungus C. ligniaria isolated from in vitro S. sonchifolius plantlets suggest that autotrophic cultures can shelter specific endophytic fungal communities in a special symbiosis with their plant hosts. Furthermore, the detection of the antifungal fatty acids produced by C. ligniaria in the plant fractions suggests that fungus and host together produced compounds that may help S. sonchifolius to resist to phytopathogenic fungal attacks. Acknowledgements: This work received partial support of the Conselho Nacional de Desenvolvimento Científico e Tecnológico (processo 200774/2011 – 5). The authors thank Ms. J.L. Robertson, Ms. R. Pace, Ms. Amber Reichley and Mr. Solomon Green III for technical support.

A simple and efficient micropropagation protocol was developed for Stevia rebaudiana Bert. using nodal segment. Inter-simple sequence repeat (ISSR) markers were used to evaluate the genetic stability of the micropropagated and hardened plants. Total genomic DNA was extracted from 20mg dried leaves of mother and randomly selected micropropagated plants with DNeasy Plant Mini Kit (Qiagen Valencia, CA; cat no.659104). Out of fifteen arbitrary primers tested, each produced clear and scorable amplification products ranging in size from about 216 bp in UBC 811 to 1917 bp in (GGGGT)3M with an average of 4.5 products per primer. A total of 49 bands (number of plantlets analyzed multiplied by number of bands with all primers) were generated by the ISSR method, giving rise to monomorphic patterns across all the gels prepared using Bio-Rad Gel Imaging System and analyzed by Quantity One analysis software version 4.3.0 (Bio-Rad Laboratories Inc., Hercules, CA). All the ISSR profiles from micropropagated plants were monomorphic and comparable to mother plants (Fig.1A and B), confirming the genetic stability among micropropagated plants and mother plant. These results suggest that the micropropagation protocol developed by us for rapid in vitro multiplication is appropriate and applicable for clonal mass propagation of elite Stevia rebaudiana.

Genetic markers separating Cannabis varieties have a practical utility for drug enforcement to connect marijuana samples with source populations and for pharmaceutical research to determine the identity of screened elite clones acquired from various sources. In the present study, we have reported simple sequence repeat (SSR) markers to differentiate Cannabis varieties. High THC yielding (drug type) varieties of Cannabis sativa, collected from different agro-climatic regions worldwide were grown from seeds, their high THC yielding female clones were screened and identified as mother plants using gas chromatography-flame ionization detection (GC-FID). Clones from the mother plants of different varieties were used as plant material to be tested. Using Cannabis sativa germplasm, a microsatellite-enriched library was created, 159 contigs were assembled and 93 repeats were detected. Thirty-nine markers were designed and screened against the clones of three different drug type varieties of Cannabis sativa. Using a set of seven amplified SSR markers, all the clones belonging to the different varieties tested were easily identified. The SSR markers developed here could assist in investigations for breeding programs and in the authentication of cannabis varieties used for pharmaceutical development.

Plant-based medicines play an important role in the lives of human beings. Identification of active principles and their molecular targets from traditional medicine provides an enormous opportunity for modern drug development. Using modern biotechnology, plants of specific chemical composition can be mass propagated for the extraction of bulk active pharmaceuticals. In the present study, we have used Cannabis sativa, as a model to assess the potential of the applications of biotechnology for the multiplication of elite medicinal plants. Cannabis contains Δ9-tetrahydrocannabinol (THC), a unique terpeno-phenolic compound which accumulates mainly in the glandular trichomes of the plant. Besides its psychoactivity, THC possesses analgesic, anti-inflammatory, appetite stimulant and anti-emetic properties making it a very promising therapeutic drug especially for cancer and AIDS patients. Because of the allogamous (cross fertilization) nature of this species it is difficult to maintain the reproducibility of the plants with unique chemical profile, if grown from seeds. Therefore, screening of elite clones and their propagation using biotechnological tools is the most suitable way to maintain the quality and the efficacy of the product. This report describes our recent biotechnological advancements in Cannabis propagation using tissue culture, synthetic seeds and temporary immersion bioreactors and, in-vitro conservation of elite propagules using slow growth conditions [1–5].

Fungal endophytes associated with leaves, lateral shoots, and roots of Echinacea purpurea (Asteraceae), a medicinal plant used by Native Americans, were evaluated for antifungal activity. Four different bioactive specimens of E. purpurea plants, grown by Solid State Fermentation technique at the University of Mississippi Field Station greenhouse led to a total of 39 fungi isolates. These were identified morphologically and by sequence analysis of the ITS region of the rRNA genes to determine their phylogenetic affinities. The 39 crude extracts from the isolates were screened for antifungal activity against the plant pathogen's targets Colletotrichum fragariae, C. gloeosporioides, and C. acutatum. Eleven endophytic taxa represented by species of the genera Ceratobasidium, Cladosporium, Colletotrichum, Fusarium, Glomerella, Mycoleptodiscus, and one taxa of order Pleosporales were identified. The most abundant taxa isolated from these plants were Cladosporium cladosporioides, Fusarium oxysporum, and Colletotrichum gloeosporioides. A total of 16 extracts (41%) showed antifungal activities against at least one phytopathogenic target; among them, seven were strongly active against C. acutatum, six against C. fragariae, and eight against C. gloeosporioides. Therefore, the fungal assemblage recovered from E. purpurea is a promising source of molecules with antifungal activities. These results open perspectives to discover new bioactive fungal communities of Echinacea species. Isolated active constituents produced by endophytic fungi could be used to develop new environmentally friendly pesticides against worldwide phytopathogenic fungi.

Centella asiatica (L.) Urb. (Apiaceae/Umbelliferae) is an important medicinal herb used in the Oriental and Indian system of herbal medicine. It has been used extensively in medicinal, pharmaceutical and cosmetic industry to improve memory, and for its wound healing properties. It is now becoming increasingly popular in the United States as dietary supplement. It is commonly available and sold as Gotu kola, Asiatic penny, Hydrocotyle or Brahmi. Use of common names in its place of the scientific binomial system of nomenclature often leads to its substitution with totally unrelated species. Bacopa monnieri (L.) is also commonly referred in Ayurvedic medicine as Brahmi and is often interchanged with Centella in traditional preparations.

Endophytic microorganisms living in tissues of ethno-medicinal plants appear to be potential new sources of bioactive compounds because of possible chemical contribution to the host's defense. In this study, two hosts of fungal endophytes, S. sonchifolius and S. uvedalius were evaluated for potential sources of compounds with antifungal properties. The genus Smallanthus sensu Robinson includes 21 species distributed from Canada to the Andes Mountains in South America. Smallanthus sonchifolius, also known as Yacon, is the most widely studied species due to its functional properties, while S. uvedalius, the North American species had medicinal value for the North American Cherokees and Iroquois. Fungal endophytes obtained from S. sonchifolius were identified by sequence analysis of the ITS region of the rRNA genes as Cladosporium, Colletotrichum, Coniothyrium, Didymella, Fusarium/Gibberella, Nigrospora, Plectosphaerella, and Trichoderma. Ethanolic extracts were screened for antifungal activity against the phytopathogenic fungi Colletotrichum fragariae, C. gloeosporioides, and C. acutatum. A total of 21 extracts showed antifungal activity to at least against one phytopathogenic test fungi. Trichoderma asparelllum, Gibberella sp., and Fusarium oxysporum produced extracts with the most antifungal activity against the Colletotrichum test species. Our results indicate that three genera Cladosporium, Fusarium and Trichoderma are common to both S. sonchifolius and S. uvedalius. These endophytes are possible sources of bioactive antifungal compounds that might be used to develop new environmental friendly pesticides.

Quality of genomic DNA is an important issue for the development of molecular markers because, in order for DNA to serve as a template for Polymerase Chain reaction (PCR), the entire sequence to be amplified must be present on a single DNA molecule. If the DNA is broken down into very small fragments, then the target sequence in the genomic DNA template is more likely to be disrupted by random breaks.

Cannabis sativa is an interesting crop for several industrial uses. It has been used for fiber (hemp), for medicinal purposes, and as a psychoactive. Although the main psychoactive chemical compound in Cannabis is Δ9-tetrahydrocannabinol (THC), the plant is known to contain about sixty cannabinoids, however, most of these “minor” cannabinoids are produced in trace amounts. Short Single Repeat (SSR) Microsatellite loci are highly informative genetic markers useful for population genetic studies, linkage mapping and parentage determination. Methods to identify novel microsatellite loci commonly use subtractive hybridization to enrich small-insert genomic libraries for repeat sequences. We have developed a method that allows highly efficient ligation to genomic DNA and improves recovery of sequences after subtractive hybridization to biotinylated oligos. The method improves current repeat-enrichment strategies, resulting in representative small-insert libraries with a very high proportion of positive clones. The effectiveness of genetic marker associated to determining three different chemotypes in Cannabis was evaluated and discussed, as possible method in marker-assisted breeding of Cannabis in the pharmaceutical field.

The leaves of Salvia (Labiatae) species have a reputed use in traditional medicine. They are known as ‘ada cayi’ in Turkey and consumed as a hot drink. Sage leaves are used traditionally as a tonic, stimulant, carminative, antiseptic, for inflammations in the mouth and for infections in Turkey [1]. Salvia madrensis, Salvia longispicata x farinacea, Salvia greggii, Salvia roemeriana, Salvia farinaceae, Salvia leucantha, Salvia splendens, Salvia coccinea from Dallas Arboretum & Botanical Garden and Salvia candidissima, S. forskahlei, S. tchihatcheffii, S. wiedemanni, S. napifolia, S. cryptantha, S. fruticosa from Turkey were subjected to microdistillation technique and their chemical compositions were analyzed using both gas chromatography (GC-FID) and gas chromatography–mass spectrometry (GC-MS) techniques. The differences in chemical composition of 15 Salvia species will be presented in this study. Short Single Repeat (SSR) Microsatellite loci are highly informative genetic markers useful for population genetic studies, molecular breeding and parentage determination. Microsatellites, short nucleotide (1–6 bp) sequences, are the current DNA marker of choice because of their highly polymorphic distribution within the genome. In this study we also report the isolation and characterization of microsatellites from 15 Salvia species from Turkey and other countries. The utility of SSR loci as possible method in determining chemotype and authentication of plant species was evaluated and discussed. References: [1] Demirci B, Tabanca N, Baser KHC (2002) Flavour Fragr. J. 17: 54–58.

Herbal teas prepared from selected Achillea (Asteraeceae) species are used in traditional Turkish medicine as diuretic, emmenagogue (menstrual flow stimulant), aid in wound healing, treatments for abdominal pain, and used to counteract diarrhea and flatulence [1]. Achillea biebersteinii is locally known as “Ayvadana, Sari civanpercemi” in Turkey. The aerial parts of five Achillea biebersteinii accessions were collected from different locations in Central Turkey to study the essential oil composition and their genetic fingerprinting. Hydrodistilled essential oils were analyzed by GC-FID and GC/MS techniques. Essential oils from plants obtained from Konya region were rich in 34–37% 1,8-cineole and oil from plants obtained from the Ankara region contained 27% p-cymene as the major constituent. Achillea oils were also evaluated for their antimalarial, antimicrobial and antifungal activities. Detailed chemical profile will be presented in this study. An increasing application of DNA fingerprinting is the use of marker assisted breeding and authentication/identification of (plant) species used in pharmacology or in commercial available food products. In this study we also describe the construction of a genomic library from Achillea biebersteinii enriched for Short Single Repeat (SSR) microsatellite loci. We have isolated several hundred clones with distinct SSRs fragments and designed oligonucleotides based on the identified sequence. The effectiveness of genetic markers as possible methods in determining specific chemotypes and authentication of plant species from Turkey and USA was evaluated and discussed in this study. References: [1] Konyalioglu S, Karamenderes C (2005) Journal of Ethnopharmacology, 102: 221–227.