An integrated web medicinal materials DNA database: MMDBD (Medicinal Materials DNA Barcode Database)
- Shao-Ke Lou†1, 3,
- Ka-Lok Wong†2,
- Ming Li2,
- Paul Pui-Hay But2,
- Stephen Kwok-Wing Tsui1, 3Email author and
- Pang-Chui Shaw1, 2Email author
© Lou et al; licensee BioMed Central Ltd. 2010
Received: 19 January 2010
Accepted: 24 June 2010
Published: 24 June 2010
Thousands of plants and animals possess pharmacological properties and there is an increased interest in using these materials for therapy and health maintenance. Efficacies of the application is critically dependent on the use of genuine materials. For time to time, life-threatening poisoning is found because toxic adulterant or substitute is administered. DNA barcoding provides a definitive means of authentication and for conducting molecular systematics studies. Owing to the reduced cost in DNA authentication, the volume of the DNA barcodes produced for medicinal materials is on the rise and necessitates the development of an integrated DNA database.
We have developed an integrated DNA barcode multimedia information platform- Medicinal Materials DNA Barcode Database (MMDBD) for data retrieval and similarity search. MMDBD contains over 1000 species of medicinal materials listed in the Chinese Pharmacopoeia and American Herbal Pharmacopoeia. MMDBD also contains useful information of the medicinal material, including resources, adulterant information, medical parts, photographs, primers used for obtaining the barcodes and key references. MMDBD can be accessed at http://www.cuhk.edu.hk/icm/mmdbd.htm.
This work provides a centralized medicinal materials DNA barcode database and bioinformatics tools for data storage, analysis and exchange for promoting the identification of medicinal materials. MMDBD has the largest collection of DNA barcodes of medicinal materials and is a useful resource for researchers in conservation, systematic study, forensic and herbal industry.
Herbal medicine is the ancient form of pharmaceutics, which is still used by many cultures for curing diseases. With a trend of living in harmony with Nature, the use of herbal materials for treatment and health maintenance is on the rise. At present, many plant, fungal and animal species are being used for treating diseases and the Chinese Pharmacopoeia has listed 670 commonly used species . Herbal medicine is a useful source of bioactive compounds, such as oils obtained from the evening primrose (Oenothera biennis) for treating atopic dermatitis , and hyperforin extracted from St. John's Wort (Hypericum perforatum) as an antidepressant drug . Nevertheless, their efficacy is critically dependent on the use of the correct material. If toxic adulterants or substitutes are administered, life-threatening poisoning may occur. In 2002, 63 people were reported with symptoms of general malaise, nausea and vomiting after consumption of herbal tea which was inadvertently mixed with neurotoxic Japanese star anise (Illicium anisatum) . Adulteration resulting in an epidemic of severe kidney damages caused by aristolochic acid was first reported in Belgium in 1993 , followed by Hong Kong and Korea [6, 7] in 2004. In these cases, the concerned herbs were substituted with the nephrotoxic Aristolochia species. A case of misusing Datura metel as Rhododendron molle was reported in Singapore in 2008 . These two species share the same Chinese herb name "Naoyanghua", but D. metel contains anticholinergic compound that causes confusion, dilated pupils, and absence of sweating. Traditionally, medicinal materials are identified by their organoleptic characteristics and physical properties such as shape, color, texture, and odor. However, the differences among related species or processed products are sometimes not obvious. Unique chemicals may serve as important markers for authentication, but chemical markers or profiles may be affected by the physiological and storage conditions.
With the advancement of molecular technology, DNA markers have now become a convenient means for species identification and molecular systematic study [9–11] and many DNA markers have in fact been patented for further development . With the help of polymerase chain reaction (PCR), specific DNA regions can be amplified from only a small amount of samples. An unequivocal identification of a tested sample can be reached by comparing its DNA sequences against the sequence of an authentic sample. To develop a universal identification platform, The Consortium for the Barcode of Life (CBOL) proposed to set up a standardized sampling method and experimental protocol to analyze agreed-upon 'DNA barcodes' .
DNA barcode is a short DNA sequence of an organism, which can be used to distinguish the organism from the other species. Mitochondrial cytochrome c oxidase subunit 1 (COI) is chosen as the standard for all groups of higher animals [14, 15]. For plant species, COI is not a suitable barcode because it evolves much slower than that of animals. Plant researchers examined several coding and non-coding regions, but they soon realized that a single DNA locus has limited resolving power for closely related species [16, 17]. Although more laborious than the single-locus approach, it is generally agree to combine two or more barcodes to increase the successful rate [18, 19]. Recently, members of the CBOL plant working group evaluated seven chloroplast genes and proposed to use mat K and rbc L as plant barcodes, based on the following criteria: easy to be amplified with a single primer pair, amenable to bidirectional sequencing with little manual editing, and high resolving power in species discrimination . rbc L offers high universality and good discriminating power, whereas mat K offers higher resolution. Nevertheless, the differentiation power of these two markers may not be high in closely related plant species . Also, experiences from our group and other researchers showed that chloroplast genes including trn H-psb A spacer, trn L-F, and nuclear regions such as internal transcribed spacer (ITS) and 5S rRNA intergenic spacer are also useful for the authentication purpose [22–24].
The Barcode of Life Data System (BOLD) is an online informatics workbench for the management, analysis and use of DNA barcodes , which is managed by the Canadian Center for DNA Barcoding, University of Guelph. Basically, this system utilizes COI and internal transcribed spacer (ITS) for animal and fungal identification, respectively. For plant species identification, 2-locus combination of mat K and rbc L are the default barcodes. The system also allows identification of unknown sequences up to species level provided by users.
Besides BOLD, some web-based barcode databases have been constructed to serve specific groups of organism. UNITE is an rDNA sequence database which contains 2842 ITS sequences from 1105 species of 152 genera of ectomycorrhizal fungi . The main target of UNITE is to facilitate the identification of environmental samples of fungal DNA. In addition to similarity searches, UNITE has built-in maximum parsimony heuristic and neighbor joining phylogenetic tools for online analysis. All Leps Barcode of Life is a database for the identification and discovery of Lepidoptera. This database now has 448,054 barcodes from 40,907 species . As insect has different morphological characters throughout its lifecycle, the DNA barcodes provide an accurate tool for the identification of the species. The Fish barcode of Life Initiative (FISH-BOL) addresses identification and natural history of various fish species through the use of COI sequence . This database contains DNA barcodes, images and geospatial coordinates of examined specimens. For plant species, Genome Database for Rosaceae (GDR) is an integrated web-based relational database contains genetic markers and ESTs of the Rosaceae [29, 30].
DNA barcodes are gaining popularity for authenticating medicinal materials [31–35]. Along with this trend, the 2010 edition of the Chinese Pharmacopoiea has included protocols for DNA extraction and DNA barcodes for selected medicinal materials. As a timely action, we set forth to establish the Medicinal Materials DNA Barcode Database (MMDBD) for recording the DNA barcode sequences, basic information and the key references of medicinal materials. The aims of the this database are: (1) to develop an organized and integrated web resource for DNA barcodes for medicinal species identification, (2) to collect and integrate the basic information of medicinal materials and their DNA barcodes, (3) to develop online tools and resources for sequence comparison. In this paper, we describe the structure and content of the database and reveal the database access utility and tools.
Construction and content
Medicinal materials information
Medicinal species listed in the Pharmacopoeia of the People's Republic of China , American Herbal Pharmacopoeia , and from prescriptions of folk medicine were chosen for including in the database. Substitutes, adulterants and closely related species were also included in the database for comparison. Currently, there are total 1259 species with 18,436 sequences available in this database. The scientific names, medicinal name, general information, and the classification of the materials in the Convention on International Trade in Endangered Species (CITES)  were collected. The voucher number of the samples, primer sequences and PCR conditions for generating barcode sequences are also provided as additional information. Photographs of live specimen or dried medical part of the medicinal materials were captured digitally. Live specimen images were mostly taken at the Chinese herbal garden of The Chinese University of Hong Kong (CUHK). Dried medicinal materials were provided by the Chinese Medicine Museum of the Institute of Chinese Medicine, CUHK. All images are of high resolution with 1200 × 800 pixels.
DNA sequences selected for MMDBD
DNA barcodes and supplementary barcodes recorded in MMDBD for authenticating medicinal materials
DNA barcode proposed by CBOL
Chloroplast matK & rbcL
All Chloroplast DNA regions
Nuclear ITS, ribosomal RNA
All mitochondrial DNA regions
All mitochondrial DNA regions
DNA sequences extracted from public databases
INSD Seq eXtensible Markup Language (XML) files that contained DNA sequences and related information were downloaded from GenBank. Scripts were used to extract and filter sequence data from XML files, by which irrelevant sequences such as microsatellite or mRNA sequences were excluded. To keep genes without standardized names, such as 'ITS', 'ITS1', 'ITS-1' or 'ITS-2' for ITS region, multiple alternative keywords were used to keep these genes in our dataset. Then all the extracted data were imported into the MYSQL database.
DNA sequences generated by our groups
Our group has generated 531 DNA for 189 medicinal materials, including ITS, 5S rRNA intergenic spacer, chloroplast trn L, trn L-F and mitochondrial cytochrome b regions. Samples and specimen were collected from various localities and authenticated by experts in pharmacognosy. Total DNA was extracted and selected DNA regions were amplified by polymerase chain reaction (PCR) using universal primers . The PCR products were sequenced directly or in case of unresolved sequences, the products were first cloned and sequenced. Primer flanking sites on the determined DNA sequences were removed.
Software design and implementation
Description of the three tables in MMDBD database
A) Barcode sequence
Sequence ID in MMDBD, which consists of identifier "mmdbd" and 9 digital number
DNA region of medicinal material
DNA sequence of a specific region
Voucher number of authentic sample
Location where sample was collected
Forward primer for PCR
Reverse primer for PCR
Source of the sequence: "Y" means that it was generated by our group; "N" means that it was extracted from paper or NCBI nucleotide database
NCBI accession number
Foreign key of "Medicinal material information" table
Foreign key of "Reference" table
B) Medicinal material information
Medicinal material ID
Species name of medicinal material
Variant name of medicinal material
Family name of medicinal material
Medicinal name of medicinal material
Type of medicinal material: "P" for plant, "A" for animal, "I" for insect, and "F" for fungus
Medical part of medicinal material
Appendices of CITES listed species
Listed species in Chinese Pharmacopoeia or American Herbal Pharmacopoeia
Adulterant of medicinal material
File name of medicinal material image
Stroke number of medicinal material name in simplified Chinese
PubMed Unique Identifier (PMID) from PubMed
Title of reference
Author name of reference
Journal title, publication volume, date and page number
Abstract of reference
Utility and discussion
Database access and tools
MMDBD has three major functions: database query by text-based interface, sequence similarity search, and data submission. Researchers can enrich the database by contributing their DNA barcode sequences to MMDBD. For data submission, we have provided a template file in EXCEL format, which enables users to upload batches of sequences and information via email. The administrator will then check the data quality before incorporating them to the database.
Database search interface
Sequence similarity search
The database has already covered 66.5% and 84.5% of the medicinal materials listed in the Chinese Pharmacopoeia and the American Herbal Pharmacopoeia, respectively. It provides an open forum for further addition of medicinal materials barcodes. In addition, efforts will focus on improving the tools and functionality of the web interface such as an advanced search site with options for search or display categories.
The MMDBD is initiated to support the DNA barcoding initiative, which includes numerous important medicinal materials. MMDBD contains DNA barcode sequences, basic information and references of the medicinal materials. The integrated database provides users with easy access and retrieval of the data and web tools for sequence comparison. MMDBD will play a timely and important role for the authentication and quality control of medicinal materials and benefit the herbal industry. It will also be useful to investigators for conservation, forensic and systematic analysis.
Availability and requirements
The MMDBD is publicly available and can be accessed at http://www.cuhk.edu.hk/icm/mmdbd.htm.
This work was supported by the Strategic Investments Scheme Fund (Scheme A) of the Chinese University of Hong Kong.
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