3. HEDGE FUNDS Y RIESGO SISTÉMICO
3.2 El Riesgo Sistémico y la Industria de los Hedge Funds
Correct identification of medicinal plants is very important and is essential for maintaining herbal drug quality, effective therapy, and patient safety.30 In the 1990s, a mix-up between two plants in China led to a dramatic inci-dent. The plant Stephania tetrandra (“han fung ji”) was an ingredient in an herbal slimming mixture in Europe. However, Aristolochia fangchi (“guang
Biodiversity of Medicinal Plants 19
fang ji”) has a similar Chinese name and was accidently mixed with Stepha-nia tetrandra. This led to severe nephrotoxic and carcinogenic side effects caused by the aristolochic acids in Stephania tetrandra. The confusion of the two plants caused severe renal failure in more than 100 cases necessi-tating renal transplants. Many affected patients also developed urothelial carcinoma.31 This example demonstrates why correct identification and nomenclature of plants is crucial.
Three main methods are used to identify plants: morphology or histol-ogy, chemotaxonomy and DNA-based authentication.
6.2.1. Identification of medicinal plants by morphology
Traditionally, morphology has been used to distinguish plants from one another. Shape, color, texture, size, smell, flowers and floral formula are some of the characteristics used by experienced botanists for correct identification.32,33Histological techniques include microscopic studies of tissue structure and the arrangement of cortex, cork cells, sieve tubes, xylem vessels or cell compounds.32 The identification and authentication of medicinal plants requires raw plant material and well-trained experts.
The identification of plant mixtures and dried or processed plant parts is rather difficult.30,34
6.2.2. Identification of medicinal plants by chemotaxonomy
Chemical identification of medicinal plants is based on analysis of chemical constituents. Such chemical analysis commonly makes use of techniques such as thin layer chromatography (TLC) and high performance liquid chromatography (HPLC).32
The constituents of a solution can be separated by TLC. The stationary phase forms an absorbance layer. Adsorbent agents like silica-gel, cellulose or aluminium oxide are put on a plate made of glass, aluminium or a syn-thetic material. The samples are applied to one end of the plate, which is transferred to a jar with a solvent (known as the mobile phase).35 Depend-ing on the mobile phase, constituents of the original solution migrate at different speeds through the stationary phase.36 At the end of the pro-cess, the solvent front is marked. Then, Rf values (retention factors) are
calculated and compared with those of reference substances to identify the constituents.35
HPLC is a form of column chromatography. The sample and the mobile phase are driven through a thin column containing a stationary phase. Depending on the interaction between a given compound and the stationary phase, the compound will elute from the column at a differ-ent time. If the compound interacts vigorously with the stationary phase, it will remain for a longer time in the column as compared to a com-pound with less affinity for the stationary phase. A detector can verify the different compounds eluted. The primary function of HPLC is the separation, identification and quantification of samples. It requires only small sample volumes.36 Examples of other chemical approaches include reversed-phase HPLC, ultra spectroscopy, infrared spectroscopy and gas chromatography32 as well as liquid chromatography coupled mass spec-trometry (LC-MS).
The composition and yield of plant chemicals depends on growing conditions, harvesting, post-harvest processing and storage. Variation in chemical composition can hinder identification. Furthermore, distinguish-ing between closely related species with similar chemical components is difficult.32
6.2.3. DNA-based authentication of medicinal plants
Morphological features and chemotaxonomy do not always lead to defi-nite or correct authentication of a species. An alternative used to identify medicinal plants is DNA technology. DNA-based molecular marker tech-niques are useful for the identification of plants, which are morphologically and/or chemically indistinguishable. The advantage of DNA is its stability.
DNA can be isolated from fresh, dried or processed material. In contrast to chemo-profiles, DNA markers are species-specific; yet they do not dif-fer among tissues of the same species. Another advantage of DNA-based methods is that analysis can be performed on very small amounts of plant material.30
There are two general approaches for genome-based identification of medicinal plants. In the first approach, the nucleotide sequence of the plant of interest at one or more gene loci is determined to assign the test
Biodiversity of Medicinal Plants 21
sample to its species.33 Technical methods used in this approach include allele-specific diagnostic PCR, amplification refractory mutation system (ARMS), multiplex amplification refractory mutation system (MARMS), DNA microarray and DNA sequencing.
In allele specific PCR, primers with allele specific 3ends labeled with different fluorochromes at their 5 ends are applied along with a general primer in a polymerase chain reaction (PCR). Analysis of the resulting amplified DNA can be performed by gel electrophoresis or capillary elec-trophoresis using an automated DNA sequencer.33
The principle underlying ARMS is that primers require complemen-tarity at their 3ends to bind to target DNA. Oligonucleotides with mutated 3ends cannot bind to the normal target sequence and consequently DNA amplification will not take place. Thus, the presence or absence of cer-tain PCR products translates to the presence or absence of the target allele. In multiplex ARMS (MARMS), several primer combinations are used simultaneously.
To accelerate the often slow and labor-intensive molecular analyses, miniaturized chip-based analytical tools have been developed.33 Simulta-neous analyses of multiple genes across many taxa are possible with a single DNA microarray. For authentication of medicinal plants, DNA sequences are identified that are unique to each species.30In DNA microarrays, DNA of known species is synthesized and immobilized on glass slides or on sil-icon or nylon membranes. Then, the DNA extracted from a test sample is labeled with a specific fluorescent dye and applied to the microarray. The sample DNA hybridizes with the complementary DNA on the microarray.
After a washing step, the fluorescence on the microchip is analyzed by a microarray reader.30,33
DNA is sequenced using cycling process of polymerization. A heat stable DNA polymerase, nucleotides, and dideoxynucleotides labeled with a fluorescent dye are combined in a thermocycler. In capillary electrophoresis, the resulting PCR amplicons are divided. Detection is carried out using laser-induced fluorescence and analysis by computer software.33The DNA sequence that is obtained can be compared to known sequences in genome databases; for example using BLAST (Basic Local Alignment Search Tool) in the GenBank database of NCBI (National Center for Biotechnology Information).30
Another possibility is the use of sequence-specific markers for loci such as the internal transcribed spacer 2 (ITS2) region of nuclear ribosomal DNA. ITS2 has the advantage of having a highly conserved region that can be used for universal primers, yet amplification of this region also pro-vides adequate variability to distinguish between species that are closely related.37Yao et al.37propose use of the ITS2 locus as a barcode for authen-tication of plant species. A DNA barcode is a short DNA segment that can be used to distinguish one species from another.38 The Consortium for the Barcode of Life (CBOL) uses standard DNA markers from nuclear, plastidal and mitochondrial regions for correct taxonomic identification of species.30
In the second approach to DNA-based identification, species-specific variations (polymorphisms) of nucleotide sequences of entire genome are used for characteristic fingerprinting of genomic DNA.33 Techniques for the generation of genomic fingerprints include restriction frag-ment length polymorphism (RFLP), randomly amplified polymorphic DNA (RAPD), arbitrarily primed PCR (AP-PCR), inter-simple sequence repeat-PCR (ISSR), amplified fragment length polymorphism (AFLP) and multiplex-PCR.
Determination of restriction fragment length polymorphisms is a non-PCR based method. Genomic DNA is digested with selected restriction endonucleases and the resulting DNA fragments are separated by gel elec-trophoresis. After transferring the digested sample to a matrix (such as a nitrocellulose or nylon membrane), the band pattern is hybridized with chemically labeled DNA. RFLP requires large amounts of high quality DNA and is time-consuming.30,33
Randomly amplified polymorphic DNA PCR uses short primers with arbitrary sequences of 10 nucleotides and results in DNA fragments of dif-ferent lengths, which are analyzed by gel electrophoresis. In comparison to RAPD, arbitrary polymerase chain reaction uses two larger arbitrary primers (10–50 nucleotides) and the amplicons are analyzed by polyacry-lamide gel electrophoresis.30
In inter-simple sequence repeat PCR, primers carry simple sequence repeats (SSRs). The primers are 1 to 7 nucleotides long and are ubiquitous and highly polymorphic.
Biodiversity of Medicinal Plants 23
For AFLP, genomic DNA is digested with restriction enzymes. The resulting restriction fragments are ligated with specific oligonucleotide adapters, which are amplified by PCR.30,33
Another method is multiplex-PCR. There are various sets of forward and reverse primers resulting in parallel amplification.33
The use of DNA-based authentication has some advantages, e.g. its requirement of only small quantities of plant material. Furthermore, DNA authentication is not impaired by environmental factors.30However, DNA methods are not without limitations; the success of DNA-based methods is contingent on DNA quality and quantity. Furthermore, high concentra-tions of secondary metabolites can interfere with DNA extraction and PCR.
Additionally, molecular authentication methods cannot be used for plant extracts.30
6.3. Geographical Biodiversity of Medicinal Plants