Mpharm ( p.analysis)herbal and cosmetic analysis adulteration and deterioration

1. DNA Fingerprinting of Herbal
Drugs
G Jayaprakash Narayana
Sri Padmavathi School of Pharmacy

2. Definitions:
 Herbal Drugs:
 DNA Finger printing:
 Chemical marker

3. Introduction
DNA based molecular technology has a potential utility in the herbal drug analysis and widely used for the authentication of
plant species of medicinal importance , as each plant is like a factory, capable of synthesizing unlimited number of complex
and un usual chemical substances whose structures could otherwise escape the imagination forever.
A chemical marker is a group of chemical compounds, it is an unique identity for a plant material to correlate with biological
efficacy as an herbal drug.
In some cases don't have any chemical and pharmacological data of chemical markers
effect of Temp, light, solvents
use of genetic technology for authentication
Degradation of
chemical
marker
Confusion for
authenticity
DNA profile used
as fingerprinting
To study the DNA profiles ,some of the finger printing
techniques are developed in order to get high level of
resolution ,throughput and reliability
DNA Forms
Geno type(genetic identity)
Phenotype(physical features)

4. DNA fingerprinting
It is an a barcode like patterns generated by amplification of chromosomal DNA of an individual which can distinguish the
uniqueness of this individual from another. Also known as DNA typing, genetic fingerprinting, DNA profiling.
The basic technique of DNA fingerprinting was discovered by Great Britain geneticist
Alec J. Jeffrey in 1984
Primarily used in botanicals for protection of biodiversity, identifying markers for traits , identification of
gene diversity and variation.
DNA markers are used in molecular biology and biotechnology experiments where they are used to
identify a particular sequence of DNA ,as these are very highly specific they can be identified with the help of molecular
markers .
Types of DNA based markers
DNA Barcode
Hybridization based
• Restriction Fragment Length Polymorphism (RFLP)
• Variable Number Tandem Repeat (VNTR)
• Probe hybridization with Micro and Minisatellite
• Random Genomic Clone
• cDNA Clone
PCR based
• Inter Simple Sequence Repeat (ISSR)
• Random Amplification Polymorphic DNA
(RAPD)/Arbitrary Primed PCR
• Amplified Fragment Length Polymorphism (AFLP)
• DNA Amplification Fingerprinting (DAF)
Sequence based
• Simple Sequence Repeats (SSR)
• Sequence Characterized Amplified Region (SCAR)
• Cleaved Amplified Polymorphic Sequence (CAPS)
• Single Nucleotide Polymorphism (SNP).

5. Methodology of DNA fingerprinting for identification of herbal drugs
The basic methodology of DNA profiling in plants involve first the isolation of DNA from plant cells, quantification and quality
assessment of isolation. The important steps involved in DNA fingerprinting
Identification of herbal drugs
DNA analysis
Stored at (-20 C) 3-5 Years
sample
Sequence methodPCR Hybridization methods
DNA profiling
Isolation of DNA from plant parts like
leaves,root,stem
Check quality and quantity of isolated DNA
Run Polymerase chain reaction for amplify
the specifi
Respective Genotyping
Matching has to be done with sample
recovered and control sample of suspected
herb.
Result interpretation

6. PCR (polymerase chain reaction)
PCR was invented by Kary Mullis , 1983 is a method used to generate
billions of copies of genomic DNA within a very short time . This
amplification is useful in cases where there are miniscule amounts of
DNA available
PCR finds application in almost all aspects of biomedicine. PCR has
been used for the detection of many pathogenic oraganisms from
bacteria to viruses.
The DNA amplification by thermal cycling called Polymerase Chain
Reaction is in vitro method that can be used to amplify a specific DNA
segment from small amounts of DNA template or duplex into millions
of copies.
Steps involved in PCR are:
• Heat Denaturation.
• Annealing.
• Primer Extension.
Heat denaturation: This temperature denatures the double stranded
DNA into two individual stands. Denaturation temperature is 95° C for
30 seconds or is 97° C for 15 seconds, however higher temperature
may be appropriate, especially for Guanine and Cytosine rich
nucleotides.

7. Annealing: During this time one primer binds with the 5’end of one DNA strand and the other primer binds with 3’end of its
complementary strand. Annealing is hybridization of primers to single stranded DNA and the length of time required for primer
annealing depends on the base composition, length and concentration of primers.
Primer Extension: This temperature varies for Taq DNA polymerase which adds complementary nucleotides one by one to the
3’ OH group of the primer. Estimates for the rate of nucleotide incorporation at 72° C vary from 35-100 nucleotides per second
depending upon the buffer, pH salt concentration and nature of DNA template
Simple Sequence Repeats (SSRs)
also known as microsatellites , where 1-6 nucleotides in length, which have a high degree of polymorphism, are isolated and
using hybridized probes followed by their sequencing. Like any DNA fragment, SSRs can be detected by specific dyes or radio
labelling using gel electrophoresis. The advantage of using SSRs as molecular markers is the extent of polymorphism, which
enables the detection of differences at multiple loci between strains. From a sample we can identify the plant species or strain
of interest by using the coupled chemical and morphological data.

8. Restriction Fragment Length Polymorphisms

9. Amplified fragment length polymorphism
It is PCR based derivative method of RFLP in which sequences are selectively amplified using primers.
DNA is cut with two restriction enzymes to
generate specific sequences
amplified
Deletion or addition of bases at the 3 prime end
Determines the selectivity
and complexity of the
amplification
The amplified fragments are
visualized on denaturing
polyacrylamide gels either
through autoradiography or
fluorescence method
Keygene
discovered this
technique in 1990’s
AFLP-PCR
Procedure: 1) digestion of total cellular DNA with one or more restriction enzymes
and ligation of restriction half site specific adaptors to all restriction fragments
2) selective amplification of some of these fragments with two PCR primers
that have corresponding adaptor and restriction site specific sequences.
3) Electrophoretic separation of amplicons on a gel matrix followed by visualization of band pattern

10. Random amplified polymorphism DNA (RAPD)
It is one of the most commonly used primary assays for screening the differences in DNA sequences of two species of plants.
 RAPD consists of fishing for the sequence using random amplification.
Here plant genomic DNA is cut and amplified using short single primers at low annealing temperatures, resulting
in amplification at multiple loci.
By running 2 dimensional electrophoresis gel, it is possible to determine the change in sequence pattern by
superimposing the 2gels .
Once band is identified , the gel is cut , and the DNA is isolated and sequenced.
Using this target DNA from other cultivators can be assessed using other techniques such as AFLP or SSRs.
EXAMPLE :
1) Authentication of traditional formulations
In this case study the technique employed for determination of claims for the presence of herbs in ayurvedic
formulations named Rasayana churna ( polyherbal formulation)
the formulation claims for the presence of three herbs named dried stem of Tinospora cordifolia
dried fruit of Emblica officinalis powder form
dried fruit of Tribulus terristris
To establish the presence of these herbs in the formulation , 120 decamer oligonucleotide primers were screened
in the RAPD analysis. By using RAPD reaction with OPC-6 in the formulation.
By performing analytical technique we prove claims for the presence of ingredients, In addition to chromatographic
fingerprints.

11. Procedure :
The dried plant parts were washed with 70% ethanol for 5 min. and then with sterile deionized water for 1min. Following
ethanol, using sonication to avoid surface contamination after being air dried, then sample was cut into pieces and ground into a
powder with liquid nitrogen using a mortar and pestle , where rasayana churna was directly ground with liquid nitrogen.
DNA was extracted from each of the samples using a modified CTAB( cetyl trimethylammonium bromide ) procedure. OPC-6
simultaneously generated three distinct amplicons each specific to one component.
The 600 base pairs is specific to T.cardifolia , the marker with 500 base pairs is specific to E.officinalis, the marker with
greater than 1000 base pairs is specific to T.terrestris
RAPD reaction
commercially available kits – OPA,OPC,OPG(operon technologies ),CA,USA.
120 primers screened for presence of distinct and consistent bands in this 4 pairs were
found most useful (OPA-16,OPC-7,OPC-13,and OPG- 5)
REACTION MIXTURE: 1)Buffer tris-KCL(20mM Tris –HCL, pH 8.4 and 50mM KCL)
2)2mM MgCl2, 0.8µM primer, 1 mM dNTP, 5U/reaction of Taq DNA polymerase
3) 15 ng template DNA and sterile deionized