Polymerase chain reaction-restriction fragment length...

Details Polymerase chain reaction-restriction fragment length... Polymerase chain reaction-restriction fragment length...

2000-04-03

2001-10-30

Fredman, Jeffrey (Department: 1656)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091200, C536S022100, C536S024300

Reexamination Certificate

active

06309840

ABSTRACT:

FIELD OF THE INVENTION
This invention is directed to the authentication of herbal and animal Chinese medicinal materials based upon RFLP patterns of the PCR-amplified rDNA.
BACKGROUND OF THE INVENTION
Traditional Chinese medicine refers to the medicinal materials and clinical application of such materials in the framework of the theoretical and empirical parameters circumscribed by the Chinese people in the last 2-3 millennia. This medical system and many of the medicinal materials have spread to and have been adopted by other Oriental countries such as Japan and Korea and evolved into Oriental medicine in those countries. As a result, traditional Chinese medicine should not be limited to only the herbs and other natural products used in Chinese medicine, but also to Oriental medicine. Traditional Chinese medicine currently in mainland China also covers the practice and medicinal materials used by Tibetan, Mongolian and other ethnic minorities.
The herbs and other natural products (animals and minerals) used in Chinese medicine have been recorded in a) classical herbals, e.g. Bencao Gangmu
and Bencao Gangmu Shiyi
the two together contain about 2,500 items; b) pharmacopoeia, e.g. Pharmacopoeia of the People's Republic of China
which contains some 600 items; and c) treaties, e.g. Encyclopedia of Chinese Materia Medica Zhongyao Dacidian
, which contains 5,767 items.
Traditionally the authentication of Chinese herbs relied upon morphological and histological inspection. In many cases, such as in the authentication of different ginseng species, and in the authentication of Acorus species, this method is unreliable. An effective program of authentication of Chinese herbs is essential and central issue in the healthy development of the herbal industry. It provides a necessary protection for consumers, minimises unfair business competition and prevents the health hazard of many adulterants.
In plant, animals and insect nuclear genomes, genes for ribosomal RNA (rDNA) are normally clustered in an array of multiple tandemly repeated copies of the cistron of 18S-ITS1-5.8S-ITS2-28S (Hillis, D. M and Dixon, M. T., 1991, The Quarterly Review of Biology, 66: 411-453). The sequence separates the 18S and 5.8S rRNA genes is designated as ITS1 (
I
nternal
T
ranscribed
S
pacer 1) and the sequence between 5.8S and 28S is designated as ITS2. The coding regions of the three rDNA genes are highly conserved, whereas the sequence homology within the ITS1 and ITS2 regions are lower across the plant kingdom. Furthermore within a given individual organism or species, the rDNA sequence is usually very similar due to the homogenization of the sequence by gene conversion and crossing over. This invention takes advantage of these features of plant rDNA, and use PCR to amplify the DNA of ITS1-5.8S-ITS2 regions with the conserved DNA sequences flanking to the regions as primers, and explores the DNA polymorphism in different plant species within the ITS1-5.8S-ITS2 region as a mean of authentication.
The roots of
Panax quinquefolius
(American Ginseng) and
P. ginseng
(Oriental ginseng) are important herbal medicinal materials widely applied in the Orient as tonic, prophylactic and anti-aging agents. In recent years the American ginseng, cultivated mainly in Wisconsin, USA, and British Columbia and Ontario, Canada, enjoys increasing popularity as a health food in Western countries. The ginseng trade is a big industry, in 1993 Hong Kong imports more than HK$1,500 million worthy of American and Oriental ginsengs. The retail price of cultivated American ginseng is usually much more expensive than that of cultivated Oriental ginseng produced in China, and that prompts wild-spread practice of disguising Orient ginseng as American ginseng by dishonest merchants. Tremendous financial incentive is also responsible for the imitation or adulteration of ginsengs with some herbal products including several poisonous plants that bear morphological similarity with ginsengs. The two ginsengs also have different medical values and potency.
Both American and Oriental ginsengs, together with several important Chinese medicines including Sanchi (
P. notoginseng
), belong to the genus of Panax in the family of Araliaceae. American ginseng and Oriental ginseng have similar morphological appearance. Furthermore many commercial ginseng products exist in the forms of powder or shredded slice, rendering their authentication by morphological and histological methods difficult and unpractical. In recent years, techniques have been developed to authenticate ginseng samples by examination of their ginsenoside profiles (Lang, Z., Lou, W S. and But, P P H, 1993, J. Clin. Pharm. Sci., 2:133-143). However, the application of chemical analysis may be limited as the amount of ginsenosides are significantly affected by many environmental factors such as the storage condition, the freshness of the products and the different post-harvest processing. In addition, the chemical method demands large quantity of materials for proper analysis.
SUMMARY OF THE INVENTION
This invention is based upon the DNA polymorphism in the ITS1-5.8S-ITS2 region of rDNA. Accordingly, plant or animal genomic DNA was isolated and the ITS regions of rDNA were selectively amplified using pairs of primers that correspond to the consensus DNA sequence within the rDNA. The resultant PCR products were then subject to the fragmentation by selected restriction endonuclease to generate, after electrophoresis, discrete and species-specific RFLP patterns. Application of this invention to authenticate American ginseng from Oriental ginseng and several common adulterants are detailed as examples. This invention is suitable to authenticate herbal and animal materials used in traditional Chinese medicine and differentiate them from various adulterants.


REFERENCES:
patent: 4965188 (1990-10-01), Mullis et al.
patent: 5876977 (1999-03-01), Wang et al.
David M. Hillis and Michael T. Dixon “Ribosomal DNA: Molecular Evolution And Phylogenetic Inference”The Quarterly Review of Biology. (1991) vol. 66 No. 4:410-449.
Wen-Sheng Lang, Zhi-Cen Lou and Paul Pui-Hay But “High-Performance Liquid Chromatographical Analysis of Ginsenosides inPanax ginseng,P. quinquefaliumandP. notoginseng”Journal of Chinese Pharmaceutical Sciences(1993) pp. 133-143.
Jun Wen and Elizabeth A. Zimmer “Phylogeny and Biogeography of Panax L. (the Ginseng Genus, Araliaceae): Inferences from ITS Sequences of Nuclear Ribosomal DNA”Molecular Phylogenetics And Evolution(1996) vol. 6, No. 2, pp. 167-177, Article No. 0069.

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