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Current Trends in Biotechnology and PharmacyVol. 5 (2) 1163-1172 April 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)1163Molecular Identification and Development of Nuclear DNA ITSSequence Based Marker to Distinguish Coscinium fenestratumGaertn. (Menispermaceae) from its AdulterantsS. P. Balasubramani and Padma Venkatasubramanian*Centre for Pharmacognosy, Pharmaceutics & PharmacologyInstitute of Ayurveda & Integrative Medicine (I-AIM)Foundation for Revitalisation of Local Health Traditions (FRLHT)Bangalore-560106, India.*For Correspondence - padma.venkat@frlht.orgAbstractCoscinium fenestratum Gaertn.(Menispermaceae), commonly known as ‘treeturmeric’, is an endangered plant species used inTraditional Systems of Medicine in Asia to treatdiabetes, hypercholesterolemia, tetany etc. Marketsurvey in India indicates Berberis aristata, B.asiatica, B. lycium of family Berberidaceae andMorinda umbellata (Rubiaceae) are traded assubstitutes or adulterants of C. fenestratum. Tostrengthen the pharmacognosy parameters,molecular identification of C. fenestratum wasperformed based on the nuclear DNA ITS(internal transcribed spacer) sequence.Subsequently, a species-specific DNA markerwas developed for easy identification of C.fenestratum. The marker developed can be usedas a molecular pharmacognosy tool in qualitycontrol of herbal raw drugs.Key words: Coscinium fenestratum, ITSsequence, Adulterants, DNA marker,Pharmacognosy, Quality ControlIntroductionCoscinium fenestratum (Gaertn.) is a seedpropagated dioecious, large, woody climber withcylindrical stem. It is commonly called as ‘TreeTurmeric’ and belongs to the familyMenispermaceae. This plant is indigenous to theIndo-Malayan region. In India, it is restricted toWestern Ghats at 500 – 750 m altitude (1). IUCNRed list of threatened plants indicates that C.fenestratum as endangered in India, vulnerablein Vietnam, rare in Singapore and intermediate inSri Lanka (2).For centuries the stems of Cosciniumfenestratum is used in the Traditional System ofMedicine in Asia. It is called as ‘Kaliyaka’ inAyurveda and is used for treating diabetes (3), inSri Lanka as an anti-dote for tetanus (4, 5). C.fenestratum is called ‘Hamm’ in Thai language,the stem is very popularly used for balancing bloodpressure, being a detoxifying and anti-diabeticagent and for treatment of hypercholesterolemia(6).Pharmacological studies have shown thatC. fenestratum has anti-fungal, anti-yeast, antibacterial,hypotensive and antiproliferativeactivities (1). The major components in wood androot of C. fenestratum are isoquinoline alkaloidssuch as berberine, palmatine, tetrahydropalmatine,crebanine and jatrorhizine, while berberine is foundMolecular Marker to distinguish Coscinium fenestratum
Current Trends in Biotechnology and PharmacyVol. 5 (2) 1163-1172 April 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)1164to be the major and active constituent. It has beenshown to bind to DNA and inhibit its cleavage(7). Antibacterial, antileishmanial, antiplatelet, antidiarrhoeal,anti-protozoan, cardioprotective, anticancer,anti-inflammatory, anti-hepatotoxic,immunostimulatory and anti HIV properties havebeen reported with berberine (7).In India, market survey indicates thatBerberis aristata DC, B. asiatica Roxb., B.lycium Royle, and Morinda umbellata L. aretraded as adulterants or substitutes of C.fenestratum (1, 8). The annual trade combiningall the five species is estimated to be 500 – 1000MT (8). Morphologically intact C. fenestratumstems can be distinguished from the other ‘TreeTurmeric’ candidates using the radical array inthe transverse section (3). However, when tradedas chips or in powder form it is not distinguishable.Biotechnology-driven applications play animportant role in modern pharmacognosy analysisfor herbal drug development and discovery.Current focus on chemotype-driven fingerprintingand related techniques requires integration withgenotype-driven molecular techniques so that anoptimal characterization of botanical materials ispossible (9). Chinese researchers have appliedDNA markers extensively for characterization ofbotanicals from the Chinese materia medica.These markers have shown remarkable utility inquality control of commercially importantbotanicals like Ginseng, Echinacea, Atractylodesetc (10).DNA markers to distinguish some of theauthentic and the adulterant medicinal plants usedin Indian System of medicine have also beenreported from our lab (11 – 13). ITS (InternalTranscribed Spacer) sequences have been shownto be a valuable source of evidence to resolvephylogenetic relationships in many angiospermgroups (14). ITS1 separates 18S and 5.8S, whileITS2 separates 5.8S and 26S rRNA genes.Because of their different rates of evolution, ITSregions have become the favored markers inevolutionary studies at different taxonomic levels(15). In recent times, these sequence variationsare used to develop species-specific markers foridentification and authentication of raw drugs andherbal formulations (16 – 18).The objective of the present study was toidentify C. fenestratum at molecular level and todevelop a species-specific marker based on ITSregion to distinguish from its adulterants.Materials and MethodsPlant material: Field and market samples ofBerberis aristata, Berberis asiatica, Berberislycium, Coscinium fenestratum and Morindaumbellata, were collected from differentgeographical locations of India. The authenticityof the samples was confirmed by Dr. K.Ravikumar, botanist at FRLHT, Bangalore. Eachsample was assigned with a specific laboratoryidentification number as indicated in Table 1.Voucher specimens were deposited in theHerbarium and Raw Drug Repository (FRLH),Bangalore, India.Chemicals: Tris-HCl, EDTA, NaCl, CTAB(Cethyltrimethylammonium bromide), LiCl, PVP(Polyvinyl pyrrolidone), Beta mercaptoethanol,isopropanol, agarose, boric acid and ethanol werepurchased from Sigma Chemicals (USA).Enzymes (Taq Polymerase and RNase A), buffer,MgCl 2and dNTP’s for PCR amplification werepurchased from Bangalore Genei (Bangalore,India).Genomic DNA Extraction: Dried stem samplesof C. fenestratum, B. aristata, B. asiatica, B.lycium and M. umbellata were surface sterilizedand cut in small pieces. The pieces were groundinto coarse powder in a domestic blender. Theprotocol described by Milligan (19) was used forDNA extraction with modifications.Approximately, 100 mg of the coarse powder wasBalasubramani and Venkatasubramanian
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