INTRODUCTION:

Recent development in molecular biology provided the basis for discovering almost infinite numbers of DNA markers. The utility of DNA-based markers is generally determined by the technology that is used to reveal DNA-based polymorphism. Currently, the restriction fragment length polymorphism (RFLP) assay has been the choice for many species to measure genetic diversity¹. However, an RFLP assay which detects DNA polymorphism through restriction enzyme digestion, coupled with DNA hybridization, is time consuming and laborious. Over the last decade, polymerase chain reaction (PCR) technology has become a widespread research technique and led to the development of several novel genetic assays based on selective amplification of DNA². This popularity of

PCR is primarily due to its apparent simplicity and high probability of success. Unfortunately, because of the need for DNA sequence information, PCR assays are limited in their application. The discovery that PCR with random primers can be used to amplify a set of randomly distributed loci in any genome facilitated the development of genetic markers for a variety of purposes^3,4. The simplicity and applicability of the RAPD technique have attracted many Pharmacognosists. The main reason for the success of RAPD analysis is the gain of a large number of genetic markers that require small amounts of DNA without the requirement for cloning, sequencing or any other form of the molecular characterization of the genome of the species in question. In this paper the principle and several most common applications of RAPD markers relevant to standardization and quality control are reviewed.

PRINCIPLE OF THE RAPD TECHNIQUE:

The standard RAPD technology³utilizes short synthetic oligonucleotide (10 bases long) of random sequences as primers to amplify nanogram amounts of total genomic DNA under low annealing temperatures by PCR.

Amplification products are generally separated on agarose gels and stained with ethidium bromide. Decamer primers are commercially available from various sources. Welsh and McClelland independently developed a similar methodology using primers about 15 nucleotides long and different amplification and electrophoretic conditions from RAPD and called it the arbitrarily primed polymerase chain reaction (AP-PCR) technique⁴. PCR amplification with primers shorter than 10 nucleotides [DNA amplification fingerprinting (DAF)] has also been used producing more complex DNA fingerprinting profiles⁵. Although these approaches are different with respect to the length of the random primers, amplification conditions and visualization methods, they all differ from the standard PCR condition in that only a single oligonucleotide of random sequence is employed and no prior knowledge of the genome subjected to analysis is required².

At an appropriate annealing temperature during the thermal cycle, oligonucleotide primers of random sequence bind several priming sites on the complementary sequences in the template genomic DNA and produce discrete DNA products if these priming sites are within an amplifiable distance of each other, the profile of amplified DNA primarily depends on nucleotide sequence homology between the template DNA and oligonucleotide primer at the end of each amplified product. Nucleotide variation between different sets of template DNAs will result in the presence or absence of bands because of changes in the priming sites. Recently, sequence characterized amplified regions (SCARs) analysis of RAPD polymorphisms showed that one cause of RAPD polymorphisms is chromosomal rearrangements such as insertions/deletions^6,7. Therefore, amplification products from same alleles in a heterozygote differ in length and will be detected as presence and absence of bands in the RAPD profile. The profile of RAPD bands is similar to that of low stringency minisatellite DNA fingerprinting patterns and therefore also termed as RAPD fingerprinting. On average, each primer directs amplification of several discrete loci in the genome so that allelism is not distinguishable in RAPD patterns.

APPLICATIONS OF RAPD TECHNOLOGY IN CHARACTERIZATION OF TRADITIONAL FORMULATIONS:

DNA-based molecular markers have proved their utility in fields like taxonomy, physiology, embryology, genetics, etc. As the science of plant genetics progressed, researchers have tried to explore these molecular marker techniques for their applications in medicinally important plants and recently in pharmacognostic characterization of Traditional medicine.

A). DNA markers as new pharmacognostic tool:

Traditionally, Pharmacognosy mainly addressed quality related issues using routine botanical and organoleptic parameters of crude drugs. Now pharmacognosy became more interdisciplinary because of subsequent advances in analytical chemistry. These development added emphasis on chemoprofiling-assisted characterization with chromatographic and spectroscopic techniques. The new Pharmacognosy includes all aspects of drug development and discovery, where biotechnology-driven applications will play an important role. Extensive research on DNA-based molecular markers is in progress in many research institutes all over the world. This technique remains important in plant genome research with its applications in pharmacognostic identification and analysis. Chinese researchers have applied DNA markers extensively for characterization of botanicals from the Chinese materia medica. These markers have shown remarkable utility in quality control of commercially important botanicals like Ginseng, Echinacea, Atractylodes. In India several agricultural universities and research institutes are actively involved in exploring DNA-based techniques in genotyping of medicinal plants.

Although considerable progress has been made in DNA marker technology, applications of these techniques for characterizing semi-processed and processed botanical formulations to, ensure the desirable quality remain underutilized. Although DNA analysis is currently considered to be cutting-edge technology, it has certain limitations due to which its use has been limited to academia. In order to establish a marker for identification of a particular species, DNA analysis of closely related species and/or varieties and common botanical contaminants and adulterants is necessary, which is a costly and time-consuming process. Isolation of good-quality DNA suitable for analysis from semi-processed or processed botanicals is also a challenge.

Another important issue is that DNA fingerprint will remain the same irrespective of the plant part used, while the phytochemical content will vary with the plant part used, physiology and environment. DNA fingerprinting ensures presence of the correct genotype but does not reveal the contents of the active principle or chemical constituents. Hence DNA analysis and pharmacognostic techniques for chemoprofiling such as TLC, HPTLC, etc. will have to be used hand in hand rather than in isolation. Identification of quantitative-trait loci⁸ that are closely linked to a biologically active phytochemical will prove to be useful. Several attempts have been made in recent years, to correlate DNA markers with qualitative and quantitative variations in phytochemical composition among closely related species^9-14. Proper integration of molecular techniques and analytical tools will lead to the development of a comprehensive system of botanical characterization that can be conveniently applied at the industry level for quality control of botanicals. Ayurvedic classification of medicinal plant is based on basic principles and therapeutic characters that may have a genetic

basis. The present review suggests a exploratory use of molecular markers for quick identification of botanical materials in crude, semi-processed and processed traditional formulations. The application of RAPD markers has been explored for standardization of botanical formulations containing Ayurvedic medicines like Emblica officinalis¹⁵ and Tinospora cordifolia¹⁶.

B). Authentication of Medicinal Plants:

DNA-based techniques have been widely used for authentication of plant species that are used in different traditional systems of medicine. This is especially useful in case of those that are frequently substituted or adulterated with other species or varieties that are morphologically and/or phytochemically indistinguishable. Dried fruit samples of Lycium barbarum were differentiated from its related species using RAPD markers⁸. The RAPD technique has also been used for determining the components of a Chinese traditional prescription, yu-pingfengsan. In this study the presences of three herbs (Astragalus membanaceus, Ledebouriella seseloides and Atractylodes macrocephala ) in the formulation have been detected using a single RAPD primer¹⁷.Three RAPD primers have been identified that could successfully distinguish between three species of Atractylodes, from Chinese formulation purchased from local markets¹⁸.

In another study, three random primers were used to reveal the genetic variability of Astragalus medicine. SSCP analysis was also conducted on PCR products from the ITS-1 region of rDNA in order to differentiate the two Astragalus species ⁽¹⁹⁾. Primers have been designed for hybridization with the hypervariable ends of microsatellite loci that could reveal DNA-polymorphism among five Eucalyptus species²⁰. DAF has been used to identify the Chinese traditional medicine, Magnoliae officinalis, its adultrants and substitutes²¹.

An RAPD primer that is selective for an elite strain Aizu K-111 of Panax ginseng, including its cultured tissues has been identified²². RAPD and PCR– RFLP analysis have been used for authentication of P. ginseng among ginseng populations²³. Some researchers have used a new approach called Direct Amplification of Length Polymorphism (DALP) for authentication of Panax ginseng and Panax quinquefolius²⁴.

C). Detection of adulteration/substitution:

RAPD technique was adopted to identify eight types of dried Coptis rhizomes and one type of Picrorrhiza rhizome, a substitute for the former in Chinese herbal market ⁽²⁵⁾. A molecular marker that is specific to medicinal rhubarb- based on chloroplast trnL/trnF sequence, which is absent in its adulterants, has been identified. DNA sequence analysis of rDNA ITS and PCR–RFLP were explored for their application in differentiating four medicinal Codonopsis species from

their related adulterants, Campanumoea javania and Platycodon grandiflorus. The technique allowed effective and reliable differentiation of Codonopsis from the adulterants. P. ginseng is often substituted by P. quinquefolius (American ginseng). Sequence characterized amplified region (SCAR), AP–PCR, RAPD and RFLP have been successfully applied for differentiation of these plants and to detect substitution by other closely related species^26-28. Characterization of Echinacea species and detection of possible adulterations have been done using RAPD technique²⁹. DNA fingerprinting and polymorphism in the Chinese drug ‘Ku-Di-Dan’ (herba elephantopi) and its substitutes were studied using AP–PCR and RAPD. The results were used for authentication of ‘Ku- Di-Dan’ and its substitutes ⁽³⁰⁾. DNA fingerprinting of Taraxacum mongolicum (herba taraxaci) and its adulterants of six species of Compositae was demonstrated using AP–PCR and RAPD²⁶. Bulb of Fritillaria cirrhosa, an official drug of Chinese Pharmacopoeia (1995), is commonly used as an antitussive and expectorant. It has often been adulterated with similar bulbs of other related species Specific DNA-based primers have been designed for authentication of F. cirrhosa at the genomic level³¹. A molecular marker that is specific to medicinal rhubarb-based on chloroplast trnL/trnF sequence, which is absent in its adulterants, has been identified³². DNA sequence analysis of rDNA ITS and PCR–RFLP were explored for their application in differentiating four medicinal Codonopsis species from their related adulterants, Campanumoea javania and Platycodon grandiflorus. The technique allowed effective and reliable differentiation of Codonopsis from the adulterants³³.

D) Marker assisted selection of desirable chemo type:

Along with authentication of species identity, prediction of the concentration of active phytochemicals may be required for quality control in the use of plant materials for pharmaceutical purposes. Identification of DNA markers that can correlate DNA fingerprinting data with quantity of selected phytochemical markers associated with that particular plant, would have extensive applications in quality control of raw materials. AFLP analysis has been found to be useful in predicting phytochemical markers in cultivated Echinacea purpurea germplasm and some related wild species³³. RAPD fingerprint has been developed to support the chemotypic differences in oil quality of three different genotypes of Pelargonium graveolens³⁴ and flavonoid composition of Aconitum species ⁽³⁵⁾. DNA profiling has been used to detect the phylogenetic relationship among Acorus calamus chemotypes differing in their essential-oil composition³⁶. Artemisia annua, a source of antimalarial compound artemisinin, shows variation in artemisinin content all over India. These chemotype variants of A. annua L. have been characterized using RAPD markers. This study also revealed existence of high levels of genetic variation in the Indian population despite geographical isolation and opens out a possibility of further genetic improvement for superior artemisinin content. An attempt has also been made to study variation in essential- oil components and interspecific variations using RAPD technique³⁷. Morphological, chemical and genetic differences in twelve basil (Ocimum gratissimum L.) accessions were studied to determine whether volatile oil and flavonoids can be used as taxonomical markers and to examine the relation between RAPDs and these chemical markers³⁸.

E). Medicinal plant breeding:

ISSR–PCR has been found to be an efficient and reliable technique for the identification of zygotic plantlets in citrus interploid crosses³⁹. Molecular markers have been used as a tool to verify sexual and apomictic offspring of intraspecific crosses in Hypericum perforatum, a well-known antihelminthic and diuretic⁴⁰. An attempt has been made towards marker-assisted selection of fertile clones of garlic with the help of RAPD markers⁴¹. RAPD markers have been successively used for selection of micropropogated plants of Piper longum for conservation⁴².

F). Applications in foods and nutraceuticals:

DNA-based molecular markers have been used extensively for a wide range of applications in food crops and horticultural plants^43-45. These applications include study of genetic variation, cultivar identification, genotyping, crossbreeding studies, identification of disease-resistant genes, and identification of quantitative-trait loci, diversity analysis of exotic germplasms, sex identification of dioeceous plants, phylogenetic analysis, etc.

Recently, the application of DNA-based molecular markers is being explored in the field of nutraceuticals. According to the new European Council legislation⁴⁶, the labelling of food or food ingredients produced from, or containing licensed genetically modified organisms must indicate the inclusion of these ingredients where they are present at or above a level of 1%. In compliance with the labelling regulation for GM foods, several countries in Europe such as Germany and Switzerland, have extensively developed PCR methods for both identification and quantification purposes. In response to reports of unlicensed GM ingredients in food in the international market, the Food Safety Authority of Ireland has completed a survey to determine the levels of GM maize ingredients in tortilla chips and taco shells on sale in Ireland, using the PCR technique⁴⁷. Where sufficient GM DNA was present in the sample, quantitative analysis was undertaken using real-time PCR. Primers specific for inserted genes in Roundup Ready TM soybean have been found to be suitable for detection and discrimination of GM soybean from non-GM products⁴⁸. In another study, Roundup Ready soybeans, Bt 176 maize and Cecropin D capsicum have been successfully discriminated from non-GM products using primers specific for inserted genes and crop endogenous genes⁴⁹.

G). Genetic variation/genotyping:

It has been well documented that geographical conditions affect the active constituents of the medicinal plant and hence their activity profiles⁵⁵. Many researchers have studied geographical variation at the genetic level. Estimates of genetic diversity are also important in designing crop improvement programmes for management of germplasm and evolving conservation strategies. RAPD-based molecular markers have been found to be useful in differentiating different accessions of Taxus wallichiana⁵⁶, Azadirachta indica⁵⁷, Juniperus communis⁵⁸, Codonopsis pilosula⁵⁹, Allium schoenoprasum⁽⁶⁰,Andrographis paniculata⁽⁶¹ collected from different geographical regions. Similarly, different accessions of Cannabis sativa⁶² have been discriminated using ISSR markers and those of Arabidopsis thaliana⁶³ Heynh, have been differentiated using cleaved amplified Polymorphic sequence and ISSR markers. Inter- and intra-species variation has also been studied using DNA-based molecular markers. Interspecies variation has been studied using RFLP and RAPD in different genera such as Glycerrhiza⁶⁴, Echinacea⁶⁵, Curcuma⁶⁶ and Arabidopsis⁶⁷. RAPD and RFLP have also been applied for characterization of Epimedium⁶⁸ species at the genetic level. Members of three different species of Scutellaria⁶⁹, Chinese medicinal plants and three subspecies of Melissa officinalis⁷⁰ have been discriminated using RAPD. Varietal characterization of Kenaf (Hibiscus cannabinus L)⁷¹ has been done with the help of agronomical and RAPD data. Varietal identification and genetic purity test in pepper and Capsicum annuum were carried out using RAPD markers⁷². RFLP technique was used for interspecific genetic variation within the genus Capsicum and also for DNA fingerprinting of pepper cultivars ⁽⁷³⁾. RAPD has served as a tool for the detection of variability in Jojoba (Simmondsia chinensis L. Schneider)⁷⁴, Vitis vinifera L⁷⁵ and tea (Camellia sinesis)⁷⁶. An attempt has been made to understand population structure of Podophyllum peltatum to establish commercial level propagation of useful secondary metabolites using molecular markers⁷⁷. Also, high genetic diversity has been shown in Podophyllum hexandrum species from Himachal Pradesh, India⁷⁸. Genetic variation and relationships among and within Withania species⁷⁹, and genetic relationships among papaya and its wild relatives (Caricaceae)⁸⁰ have been revealed using AFLP markers. Genetic variation within Brassica campestris cultivars has been studied using AFLP and RAPD markers⁸¹.

Phylogenetic relationship has been studied among citrus and its relatives using SSR markers⁸². RAPD has been used to construct genetic linkage maps of Eucalyptusgrandis and Eucalyptus urophylla⁸³. RAPD markers have been developed for genetic mapping of Pacific yew (Taxus bravifolia Nutt.)⁸⁴. An attempt has been made to develop a physical AFLP map of the complex Arabidopsis genome by combining gel-based AFLP analysis with in Silico restriction fragment analysis using the published genome sequence.

EPRODUCIBILITY OF RAPD MARKERS:

Although the RAPD method is relatively fast, economic and easy to perform in comparison with other methods that have been used as DNA markers, the issue of reproducibility has been of much concern since the publication of the technique. In fact, ordinary PCR is also sensitive to changes occurs in reaction conditions, but the RAPD reaction is far more sensitive than conventional PCR because of the length of a single and arbitrary primer used to amplify anonymous regions of a given genome. This reproducibility problem is usually the case for bands with lower intensity. The reason for bands with high or lower intensity is still not known. Perhaps some primers do not perfectly match the priming sequence, amplification in some cycles might not occur, and therefore bands remain fainter. The chance of these kinds of bands being sensitive to reaction conditions of course would be higher than those with higher intensity amplified with primers perfectly matching the priming sites. The most important factor for reproducibility of the RAPD profile has been found to be the result of inadequately prepared template DNA.

Differences between the template DNA concentration of 2 individuals’ DNA samples result in the loss or gain of some bands. Since RAPD amplification is directed with a single, arbitrary and short oligonucleotide primer, DNA from virtually from all sources is amenable to amplification. Therefore, DNA from the genome in question may include contaminant DNA from infections and parasites in the material from which the DNA has been isolated. Special care is needed for keeping out the DNA to be amplified from other sources of DNA.

CONCLUSION:

Standardization of traditional formulations is a difficult task due to variation in appearance, morphological characters and chemical constituents of crude drugs or crude material that are used in the manufacturing of Ayurvedic/Traditional formulations. The main reason of this variation is environmental condition, altitude, different techniques for cultivation collection storage and use of parts other than specified in traditional text. However the Random amplified DNA technology use oligonucleotide (10 bases long) of random sequences as primers that are impervious by any of the given factors, so it provide a better fingerprint for the identification of the plant material used in the Traditional formulation.

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Received on 03.04.2008 Modified on 20.04.2008

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