Reviews

Identification of botanicals using molecular biotechnology

Mohd Imran1,A–F, Haya Majid2,D,E, Tasha Riaz1,D, Shayan Maqsood1,B
1
Department of Pharmacognosy and Phytochemistry, School of Pharmaceutical Education and Research, Jamia Hamdard University, New Delhi, India
2
Centre for Translational and Clinical Research, School of Chemical and Life Sciences, Jamia Hamdard University, New Delhi, India

A – research concept and design; B – collection and/or assembly of data; C – data analysis and interpretation;
D – writing the article; E – critical revision of the article; F – final approval of the article

Polymers in Medicine, ISSN 0370-0747 (print), ISSN 2451-2699 (online)  Polim Med. 2023;53(1):69–79

Address for correspondence

Mohd Imran
Abstract
E-mail: imranidrisi00786@gmail.com The available information on the abundance of restorative plants on earth is incomplete, and the data re-
garding botanicals from various countries differ significantly. The substantial development of the worldwide
Funding sources
None declared
natural botanical market is attributable to the expanding revenue of global drug companies trading herbal
medicines. This essential type of traditional medical care is depended on by approx. 72–80% of individuals.
Conflict of interest Even though numerous restorative plants are readily used, they have never been subject to the same strict
None declared quality guidelines as conventional drugs. Nonetheless, it is vital to have specific organic, phytochemical,
and molecular tools and methods for identifying restorative plant species so that traditional and novel plant
products can be safely used in modern medicine. Molecular biotechnology approaches provide a reliable and
Received on March 5, 2023 accurate way to identify botanicals and can be used to ensure the safety and efficacy of plant-based products.
Reviewed on April 5, 2023
Accepted on April 7, 2023 This review explores various molecular biotechnology approaches and methods for identifying botanicals.
Published online on June 20, 2023 Key words: genomic DNA, herbal drugs, phytotechnology

Cite as
Imran M, Majid H, Riaz T, Maqsood S. Identification
of botanicals using molecular biotechnology.
Polim Med. 2023;53(1):69–79. doi:10.17219/pim/163119

DOI
10.17219/pim/163119

Copyright
Copyright by Author(s)
This is an article distributed under the terms of the
Creative Commons Attribution 3.0 Unported (CC BY 3.0)
(https://creativecommons.org/licenses/by/3.0/)
70 M. Imran et al. Identification of botanicals with molecular biotechnology

Introduction and quantitative synthetic structure of constituents can

be created.4
Evidence on character and safety is of primary impor- Consequently, we should reason that surveying the natu-
tance among different essential factors in the quality ral properties of herbal medications is a fundamental ne-
verification of plant-based medications. Character refers cessity and should be the primary aim before a plant can
to botanical identity, authenticity and quality of the plant be formally included into an authoritative Pharmacopeia.5
material used, which requires the avoidance of quality re-
duction and misrepresentation of herbal medications. Such
factors include the presence of harmful constituents, low DNA-based confirmation
efficacy or anything else that is related to the fundamental
claim that the plant has health-promoting properties.1
of natural plants
In the context of herbal medications, misrepresenta- Recently, therapeutic plants have become popular in West-
tion refers to any deceptive or misleading practices that ern nations, where the desire to use natural remedies has
can occur during the production, labeling, or marketing developed and such use is now considered an option by doc-
of herbal products. This can include various forms of mis- tors and patients. There has been evidence of increasing
representation, such as: interest in herbal drugs (natural preparations) as they are
Substitution: The intentional or unintentional substitu- easily available and can be generally utilized as medicines
tion of one plant species with another that may have differ- with possibly lesser side effects in comparison to syntheti-
ent therapeutic properties or potential safety concerns. For cally derived drugs. As such, they are protective and do
example, using a cheaper or more readily available plant not cause any antagonistic effects. Nonetheless, the natural
as a substitute for the desired plant species. origin of herbs does not guarantee their safety. Some herbs
Adulteration: The addition of undeclared substances may have intrinsic toxicity, and there is a risk of substitution,
or contaminants to the herbal product, which can affect its contamination, and adulteration of herbal products. Addi-
quality, efficacy, or safety. Adulteration can occur by adding tionally, misidentification of plant species can occur, espe-
lower quality plant material, synthetic compounds, or other cially considering the wide variety of species used in herbal
substances that are not disclosed on the product label. medicine. These factors can have potential adverse effects
False labeling: Providing incorrect or misleading infor- on the quality and safety of botanical products.6
mation on the product label, such as inaccurate botanical It has been demonstrated that contamination or substi-
names, misleading claims about the product’s composi- tution of herbs has caused illness and even deaths. A case
tion or therapeutic effects, or incorrect information about of neuropathy and encephalopathy was recorded after the ad-
the geographical origin or processing methods. ministration of a preparation derived from the herb Gentiana
The morphology (visible with a naked eye) and appear- rigescens radix. Further examination revealed that poisoning
ance of plant-based medications have been demonstrated had occurred due to contamination by Podophyllum emodi
in the literature, yet it should be considered that the va- or Podophyllum hexandrum, which contain the neuro-
lidity of a medication cannot be ensured with certainty toxin podophyllotoxin and were found growing underneath
by identification of plants alone. Indeed, significant experi- the original preparation. A few instances of renal injury related
ence in pharmacognostics and the utilization of legitimate to Stephania tetrandra in a weight reduction formulation were
standardized drugs are also required.2 caused by Aristolochia fangchi, which contains the highly poi-
Scientists can distinguish the complicated ingredients sonous aristolochic acid, a nephrotoxin that causes urethral
of all basic molecular elements of a natural supplement cancer, among others. In this case, the confusion arose from
using imaging modalities and automated high-perfor- the similarity of the 2 plants in Chinese nomenclature.7
mance liquid chromatography (HPLC) analysis. Indeed, Consequently, to guarantee the quality and efficacy
well-defined HPLC methods using specific markers meet of natural medications, and ensure the wellbeing of the pa-
the prerequisites of legitimate science-based verification tients, identifying the plant species is essential. However,
of natural medications, and fulfilling strict requirements distinguishing natural components, which regularly
of the International Council for Harmonization Rejec- comprise dried or prepared parts, is by and large trouble-
tion (ICHR) regarding potential misrepresentations and some due to the change of characteristics through drying,
corruption of herbal medications for wellbeing reasons particularly when a herb has more than 1 name or when
should likewise be possible employing such procedures.3 a common name is used for more than 1 herb.8
All plants in the world are assigned a genus and spe-
cies name for portrayal and references in herbaria since
the founding of binomial terminology by Carl von Linne Validation of natural material
in 1753, such as Chamomilla (genus), and recutita and
inodora (species). Based on genetic, morphological and
on the DNA level
structural attributes, subspecies and substance races Genetics are thought to provide unwavering quality
should also be discerned so that an alternate subjective in the verification of natural materials at the DNA level due
Polim Med. 2023;53(1):69–79 71

to the advances in molecular biotechnology and plant ge- of high-throughput DNA microarrays have allowed for
netics. Moreover, the use of DNA-based molecular marker the definitive identification of taxon on a larger scale.11
methods for understanding species of therapeutic plants
is still expanding. Such improvements are particularly Random amplified polymorphic DNA
helpful for identifying species that are regularly confused
or adulterated with other plants, and for species that are Random amplified polymorphic DNA (RAPD) method
morphologically or phytochemically indistinguishable.9 employs arbitrarily arranged short-engineered oligonucle-
The DNA is a remarkably stable macromolecule that otides (10 bp long) as primers to produce countless random
is not influenced by external factors, meaning that it can be DNA fragments using PCR. The enhanced DNA segments are
recovered from fresh, dried and prepared natural materi- separated with the use of agarose gel electrophoresis and visu-
als. In contrast, substance fingerprinting is unequivocally alized using ethidium bromide dye or SyBRgreen. The poly-
influenced by the sample age, physiological conditions, morphisms in the enhanced fragments are brought about by:
environmental impact development region, reaping period, 1) base replacement or excision at the preparation sites,12
drying, and storage conditions. Moreover, the DNA marker 2) primer insertions at loci too far apart to result in am-
particles are not tissue-specific and, in this way, can be dis- plification or13
tinguished at all phases of organic growth. Also, a modest 3) insertions or deletions that change the size of the am-
quantity of a sample is adequate for examination.10 Table 1 plified fragment.14
presents a comparison of DNA profiling techniques for Due to its ease of use (no prior DNA sequence informa-
herbal authentication and quality control. tion is required), low expense, ability to produce a high
volume of DNA markers within a brief time frame, and
the use of non-complex equipment, RAPD has a wide scope
Types of DNA markers utilized of applications.15
in plant genome analysis Arbitrarily primed PCR
The DNA-based molecular strategies that have been uti-
lized to assess DNA polymorphisms for plant taxa verifica- Arbitrarily primed PCR (AP-PCR) is a distinct form
tion include hybridization polymerase chain reaction (PCR) of RAPD that uses single primers of 10–50 bps in length
and sequencing techniques. Currently, multi-locus se- during a 3-stage amplification process. The initial 2 an-
quence analysis (MLSA) is frequently used in phylogenetic nealing cycles are completed in a low-stringent environ-
investigations and has demonstrated its discriminatory ment, with the main cycle employing high-stringency
ability. In addition, recent improvements in the sensitivity conditions and primers of various length. Polyamide gel

Table 1. Comparison of DNA profiling techniques for herbal authentication and quality control.1 The table is based on the most frequent molecular tools
and does not represent all DNA-based molecular techniques27
Importance Quantity Sequence Devel-
Molecular Repro- Level of poly- Locus Technical Auto- Running
of authen- of DNA information opment
technique ducibility morphism specificity demand mation cost
ticity required required cost
ISSR ++ medium low medium no low no yes low low
AFLP ++ high medium medium no medium no yes medium low
ARMS ++ high high low yes low yes yes low medium
RAPD + low low medium no low no yes low low
RFLP + high high medium yes high yes no high medium
SSR + high low high no low yes yes medium high
RAMPO + medium low medium yes high no yes medium medium
CAPS ++ high low low yes low yes yes low medium
SCAR ++ high low low yes low yes yes low medium
CAPS ++ high low low yes low no difficult medium medium
LAMP + high low low yes medium yes yes low medium
SNP + high low high yes medium yes yes low high
Microarray +++ high low high yes high yes yes high high
Barcoding +++ high low low yes medium yes yes medium medium

ISSR – inter simple sequence repeat; AFLP – amplified fragment length polymorphism; ARMS – amplification-refractory mutation system; RAPD – random
amplified polymorphic DNA; RFLP – restriction fragment length polymorphism; SSR – simple sequence repeats; RAMPO – random amplified microsatellite
polymorphism; CAPS – cleaved amplified polymorphic sequences; SCAR – sequence-characterized amplified regions; LAMP – loop-mediated isothermal
amplification; SNP – single nucleotide polymorphism.
72 M. Imran et al. Identification of botanicals with molecular biotechnology

electrophoresis is chiefly used to examine the obtained of arbitrarily prepared PCR microsatellites (RAPDs) used
products. This method is utilized in a variety of genetic for hybridization and production of polymorphic heredi-
investigations and for species identification. However, sim- tary fingerprints, and does not require prior genomic in-
ilar to RAPDs, fingerprints produced using single primers formation. As in RAPDs, the genomic DNA is initially
can cause reproducibility issues since slight changes in cy- amplified with a single random 10-mer primer or micro-
cling conditions can affect the appearance of the bands.16 satellite-complimentary 15- to 16-mer PCR primer. This
method produces highly reproducible banding patterns,
DNA amplification fingerprinting which allow for clear identification of species-specific
groups. Furthermore, random amplified microsatellite
As an adaptation of the RAPD strategy, the technique polymorphism (RAMPO) fragments are less sensitive
of DNA amplification fingerprinting (DAF) in thermosta- to error than RAPD fragments due to their homology and
ble conditions allows primers to efficiently enhance am- the strong signal produced from the hybridization of dis-
plification at multiple random sites on each DNA strand tinct polymorphisms (i.e., the existence of repeat sequences
and to produce fingerprints with novel DNA patterns. in a particular microsatellite). The RAMPO is, for the most
Polyacrylamide gels and silver staining are used to resolve part, utilized for the identification and separation of geno-
the amplicons.17 types within germplasms and populaces, such as Ficus and
Phoenix dactylifera.20
Inter simple sequence repeats
Restriction fragment length polymorphism
Inter simple sequence repeats (ISSRs), or single primer
amplification reaction (SPAR) markers, are exceptionally Restriction fragment length polymorphism (RFLP)
adequate, replicable, profoundly polymorphic, and advan- is widely utilized for recognizing changes at the DNA level
tageous to use in higher plants. They are utilized in genetic and depends on the correlation of banding patterns from
fingerprinting, quality labeling, phylogenetic investigation, DNA sequences digested by specific restriction enzymes
species and cultivator identification, and the evaluation (e.g., HaeIII, EcoRI, BamHI). These enzymes are a class
of hybridization. The IISRs resemble RAPDs, though ISSR of endonucleases produced by microorganisms that can
primers are located in microsatellite (SSR) regions and are digest specific DNA sequences, with each protein recog-
longer in length (around 14 bp) than RAPD primers. The pro- nizing a particular palindromic sequence. They detect
cedure uses basic sequence repeats (the ISSRs) with mono-, and digest the DNA in a predictable manner, which gener-
di- or trinucleotide repeats of 4–10 repeating units. The SSRs ates restriction fragments of defined lengths. The length
are abundant and highly polymorphic, and are distributed of these fragments varies between species if there are dif-
throughout the genome. Moreover, this method is efficient ferences between the cleavage loci of a specific restriction
and more conservative. Also, the technique does not require endonuclease. Restriction sites are detected and then al-
sequencing data and does not use radioactive material.18 tered by point mutation, insertion, deletion, or transloca-
tion to produce distinct fragments that are separated and
Amplified fragment length polymorphism detected using gel electrophoresis. A few drawbacks of this
method are that it is time-consuming, expensive, requires
An amplified fragment length polymorphism (AFLP) extensive labor, and needs large amounts of DNA.21
is a powerful tool that combines RflM and PCR for DNA
fingerprinting of the organismal genome and can screen Microsatellites or single sequence repeats)
countless loci (ca. 50–100 parts for each response) for poly-
morphisms. Similar to ISSR, AFLP does not require prior Microsatellites, short tandem repeats (STRs), or simple
sequencing data for the targeted genome and is exceptionally sequence length polymorphisms (SSLPs), are the smallest
reproducible. It is utilized for DNA fingerprinting, usually class of monotonous DNA sequences and have 2–6 bps.
when very little data is available on the plant genome under They are dispersed throughout most eukaryotic genomes,
study. Some advantages of AFLP include the rapid procedure, meaning there is a wide variety in the quantity of repeat-
high genomic bounty, low labor requirements, abundant data, ing units that are profoundly polymorphic. Microsatellites
high reproducibility, and the ability to detect a high number have a broad assortment of uses, such as marker-guided
of loci in a single reaction. Furthermore, no information about reproduction, population genetics and genome mapping.
the target genome is required for preliminary development.19 Indeed, the marker sequence and the extensive number
of alleles allow for the detection of significant degrees
Random amplified microsatellite of similarities and differences between closely related spe-
polymorphism cies or strains. A few downsides of microsatellites are that
their use is time-consuming, and when the target DNA
Random amplified microsatellites (RAMS), or random is unknown, there are high costs associated with the isola-
amplified hybridization microsatellite (RAHM), is a mix tion and separation of each locus.22
Polim Med. 2023;53(1):69–79 73

Selectively amplified microsatellite Amplification-refractory mutation system

polymorphic loci
Amplification-refractory mutation system, or allele-spe-
The selectively amplified microsatellite polymorphic cific polymerase chain response (AS-PCR), is a method
loci (SAMPL) strategy encompasses the high multiplexing for separating alleles. It is a simple, versatile and powerful
ratio of the AFLP method and the high degree of mic- technique for the detection of minor deletions or trans-
rosatellite polymorphisms. The method uses AFLP-type formations, including SNPs. It uses a dynamic screening
primers matched to a complimentary microsatellite primer test that does not require any type of marking, because
to detect polymorphisms using restriction digestion and agarose gel electrophoresis and ethidium bromide staining
selective amplification. The microsatellite primer restric- are utilized for basic visualization of the amplified product.
tion fragments that have linked adapter sequences are then However, oligonucleotides with a random 30-mer sequence
amplified through primers complementary to the adapter will not function as a PCR primer in the ARMS. Another
sequences. A restriction of multi-locus SSR profiling is that disadvantage of the ARMS method is that it only amplifies
it only detects a portion of the microsatellite polymor- the test DNA at the objective allele and does not enhance
phisms due to the prevalence of predominant markers, the non-target allele. In this procedure, the enhance-
which makes it difficult to distinguish allelic sections ment and approval steps are joined to such an extent that
in complex DNA fingerprints.23 the presence or absence of the objective allele is analyzed
by the presence or absence of a PCR product. Nonethe-
Directed amplification less, this procedure has been applied to the validation
of minisatellite-region DNA of Alisma, Panax, Rheum, Dendrobium, and Curcuma.26

The directed amplification of minisatellite-region DNA Cleaved amplified polymorphic sequence

(DAMD) strategy is essentially a DNA fingerprinting
technique in which the loci of variable number tandem Cleaved amplified polymorphic sequence (CAPS) detects
repeats (VNTR) of hypervariable repeats are enhanced polymorphisms using a combination of target DNA am-
at moderately high stringencies. The double repeat DNA plification and restriction enzyme digestion in a 2-stage
sequences in minisatellites and VNTR are longer (around process. In the initial step, amplification of a character-
10–60 bp themes) than microsatellites, though micro- ized sequence uses specific 22–25-bp primers and is fol-
satellites display extensive polymorphisms due to their lowed by restriction of the PCR product using suitable
duplicate number of double repeat loci. The VNTR cen- enzymes. Agarose gels and ethidium bromide are then
tral sequence is utilized as a primer to coordinate the am- used to isolate the separated restriction fragments. The ca-
plification of minisatellite loci DNA using PCR and can pacity to distinguish DNA polymorphisms is higher for
provide data similar to RAPD for species identification.9 SSRs and AFLPs than for CAPS due to the limited size
However, with the utilization of longer primers in DAMD, of the restriction fragments and the nucleotide changes
enhanced DNA fingerprinting is produced, and conse- that are fundamental for CAPS identification. For valida-
quently, DAMD is more reproducible than RAPD.24 tion of Astragalus, Alisma, Duboisia, Sinopodophyllum,
Dysosma, Fritillaria, Ephedra, Artemisia, Panax, Actin-
Single nucleotide polymorphisms idia, Atractylodes, Glehnia, Dendrobium, and Codonopsis,
PCR-RFLP has been used.27
Single-base pairing locations in the genotypes of a mini-
mum of 2 persons are termed single nucleotide polymor- Sequence-characterized amplified regions
phisms (SNPs), at which various alleles are available. Single-
base pair contrasts are created between DNA sequences Sequence-characterized amplified regions (SCAR) are
through polymorphisms caused by point transformation. another type of RAPD-determined molecular that avoids
The SNPs are used for identifying people, ecotypes and many of the shortcomings of RAPDs. Cloned polymorphic
species, and are proficient molecular markers for genetic ex- RAPD or ISSR fragments with specific oligonucleotide
amination and rearing projects. Environmental and trans- primers are utilized alongside SCAR markers to rapidly
formative investigations also use SNPs. Furthermore, some enhance nucleic acids indicative of natural materials.
molecular markers depend on SNPs, such as RFLPs, cleaved Polymorphic regions from the cloned RAPDs or ISSRs
amplified polymorphic sequences (CAPS), amplification- are selected from known sequences and used as primers
refractory mutation system (ARMS), and single-strand to amplify and map SCAR markers. The SCAR recognize
conformation polymorphism (SSCP). Many great SNP se- just a single locus, their amplification is less sensitive to re-
quences in exemplar DNA have been demonstrated in DNA action conditions, and they tend to exhibit dominance due
microarrays (biotechnology organizations). Furthermore, to a partial overlap with the primers, so they are invalu-
SNPs are applied to the verification of Perilla, Dendrobium able over RAPD markers. This strategy has been used for
officinale, Panax cultivars, and Boehmeria species.25 validation of Panax and separation of types of Artemisia,
74 M. Imran et al. Identification of botanicals with molecular biotechnology

Phyllanthus, Pueraria, Sinocalycanthus, Embelia, and plants in which all non-angiosperm genomic DNA was
Lycium.28 extracted using the Clontech® PCR-Select™ complemen-
tary DNA (cDNA) Deduction unit (Takara, Shiga, Japan).
Single-strand conformational The SDA is superior to regular molecular identification
polymorphism techniques in terms of exactness, versatility and efficacy,
as well as being high-throughput with expansive applica-
Single-strand conformational polymorphism is a trans- tions. Moreover, the technique exhibits substantial po-
formation procedure. Under denaturing conditions, this tential for genotyping, as demonstrated by the extraction
method results in single-stranded DNA (ssDNA) folding of the non-angiosperm DNA. This review demonstrates
into a tertiary conformation. The ssDNA is changed into the capability of setting up a high-throughput microarray-
the tertiary structure through the interaction of nucleo- based autonomous genotyping procedure of significant
tides in the DNA sequences, which also influence the mo- angiosperm clades. The SDA procedure was suitable for
bility of the ssDNA during gel separation, with differences 2 separate ginseng species, P. ginseng and Panax quin-
in bands indicating SNPs. The F-SSCP is a modified form quefolius, which are often blended and contaminated.
of SSCP that includes theenhancement of the target se- Furthermore, SDA was sufficiently sensitive to iden-
quence using fluorescent probes, and although this tech- tify contamination debasement of 10% P. quinquefolius
nique is not often applied for verification, it has been used in P. ginseng. 31
for validating Boesenbergia.29
Multiplex ligation-dependent
Methylation-sensitive amplified probe amplification
polymorphism
Multiplex ligation-dependent probe amplification
Methylation-sensitive amplified polymorphism (MSAP) (MLPA) is a semi-quantitative PCR-based method suit-
is a modified AFLP method developed to screen genomic able for identifying clinical plants, and is a popular re-
DNA methylation. The methylation-sensitive chemicals search tool because of its minimal expense and versatility.
HpaII and MspI are used twice to digest genomic DNA, fol- It utilizes the versatility of the PCR, though it broadens
lowed by the methylation-insensitive EcoRI. The digested the specificity by including an important ligation step
samples are ligated using double-stranded DNA (dsDNA) which ensures only DNA sequences that have been hy-
adapters, pre-amplified using pre-selective or non-selective bridized are detected. A typical PCR primer is used for
primers, and then amplified using a pair of selective prim- the amplification of all target sequences, which is a critical
ers. The MSAP was first developed to identify DNA meth- element that ensures the general evaluation of each focus
ylation processes in dimorphic parasites and later adjusted against a control sample.32
for the recognition of cytosine methylation in the rice ge-
nome, pepper, apples, and Siberian ginseng.7 Ongoing polymerase chain reaction
Loop-mediated isothermal amplification Continuous quantitative PCR is a widely used method
for measuring nucleic acids in molecular diagnostics. It of-
Loop-mediated isothermal amplification (LAMP) fers advantages such as sensitivity, specificity and repro-
is used to enhance target nucleic acids with high speci- ducibility. However, like any diagnostic method, it has its
ficity and efficacy and is done under isothermal condi- limitations. These limitations may include issues related
tions. The method uses an auto-cycling process to segment to primer design, sample quality, amplification efficiency,
and displace DNA, followed by hybridization carried out and the potential for false-positive or false-negative re-
by a DNA enzyme with high helix displacement activity. sults. It is important to carefully consider these limita-
It was applied to identify Panax ginseng and to distinguish tions and optimize the experimental conditions in or-
the plant from Panax japonicus using Ginseng radix. It was der to ensure accurate and reliable results when using
subsequently utilized to pinpoint sites of Lophophora wil- continuous quantitative PCR in molecular diagnostics.
liamsii and Curcuma longa.30 It has become the principal specialized stage for nucleic
acid discovery both in research and routine diagnostics.
Strand displacement amplification By estimating the buildup of amplified products during
the PCR using fluorescent technology, it can identify a spe-
Constrains of PCR-based methods for differentiating cific DNA sequence in a sample. The process has been
plants come from their low latency, though hybridization- used for the routine validation of the Chinese medicinal
based microarrays offer a rapid, high-throughput tool for plant Cimicifuga foetida and its 4 substitutes: C. simplex,
genotype identification. In this regard, strand displacement C. dahurica, C. heracleifolia, and C. acerina, through ex-
amplification (SDA) is a technique created using combined amination of recombinant DNA (rDNA) using an inner
genomics. The DNA library consists of 49 angiosperm translated spacer (ITS) and fluorescence liquefying bend
Polim Med. 2023;53(1):69–79 75

investigation. In addition, continuous PCR has been used The ideal DNA marker should be useful for a wide scope
to investigate debasements of the genera Euphorbia, Gen- of taxa (expansiveness of order application), readily retriev-
tiana and Drynaria.33 able with an all-inclusive preliminary duo, be sufficiently
brief to be open to bidirectional sequencing, and provide
DNA sequencing analysis exceptional grouping and maximal separation among spe-
cies. The key example is the genus Dendrobium (Orchida-
The Sanger dideoxy-sequencing method was designed ceae), which is represented by 78 varieties within Chinese
in the 1970s and is used for DNA sequencing. It is the most botanical system, with 14 endemics. However, wide surface
common way of generating reads of the bases or nucleo- areas coupled with great morphological variability render
tides, such as A, T, G, and C, of a specific molecule of DNA. species identification challenging.16
The DNA is duplicated through chromosomal replication In light of preparation techniques, Herba dendrobii
and can be carried out using capillary electrophoresis, is characterized as a new Dendrobium as well as “Fengdou
in a cylinder, or on a microtiter plate with very small sam- Shihu”, along with “Huangcao Shihu”, as it is the domi-
ples. The sequence read length is 50–1000 bps. If the loci nant type of Herba dendrobii in traditional Chinese medi-
to be sequenced outperforms the length of a standard se- cine (TCM). A further noteworthy model where differ-
quencing read, internal reactions must be utilized to repro- ent varieties are utilized in TCM is the genus Fritillaria
duce the total sequence of a more drawn-out DNA region.34 (Liliaceae), which incorporates 24 varieties and 15 species
Most DNA sequencing methods utilize slim electropho- and is endemic in China. Bulbus fritillariae is the most
resis to isolate DNA particles dependent on their size and famous homegrown prescription in China and is being
fluorescent probes for labeling and identifying the 4 bases. utilized as an antitussive and expectorant herb.35 The Frit-
Currently, Sanger dideoxy-sequencing is a widely used illaria genus encompasses several species that are utilized
method in molecular biology with various applications. in TCM, including F. thunbergii, F. cirrhosa, F. unibrac-
It is particularly valuable in studying phylogenetic relation- teata, F. przewalskii, F. delavayi, F. ussuriensis, F. walu-
ships, population genetics, systematics, and advancing our jewii, F. pallidiflora, F. hupehensis, F. anhuiensis, and F.
understanding of genetic diversity. By sequencing DNA puqiensis. These species may have similar names, making
samples using the Sanger method, researchers can com- it challenging for buyers to distinguish between them.
pare sequences and analyze genetic variations within and Additionally, there may be a lack of specific and distinct
between populations. This information helps in elucidat- attributes or characteristics associated with each species,
ing evolutionary relationships, determining genetic mark- further adding to the confusion. It is important to ensure
ers, and studying the genetic basis of various traits or dis- accurate identification and quality control of these sub-
eases. The versatility of Sanger dideoxy-sequencing allows stances (the plant components) used in TCM to maintain
researchers to explore a wide range of genetic questions their safety and efficacy. Therefore, for quality control and
and make significant contributions to the fields of phylo- normalization of Fritillaria as a natural medication, DNA
genetics, population genetics, systematics, and molecular barcoding is the main reliable technique for the identifica-
biology as a whole. The sequencing examinations depen- tion of accurate species.36
dent on molecular ITS have been applied to Panax, Yamaji,
Fritillaria, Asarum, Astragalus, Dendrobium, Leonurus, DNA microarrays (DNA chip technology)
Perilla, Phyllanthus, Rehmannia, Salvia, Swertia, Plantago,
Bupleurum, and Euphorbia. For validation of Adenophora, The innovative “biochip” technology is intended to rec-
Aconitum, Angelica, Astragalus, Curcuma, Epimedium, ognize fluorescently marked DNA or ribonucleic acid
Fritillaria, Crocus, Ligularia, Pueraria, and Saussurea, (RNA) fragments through their hybridization to oligo-
a 5S rDNA intergenic spacer marker has been utilized. nucleotides. The DNA microarray innovation offers a high
Also, 18S rDNA has been evaluated in Dioscorea, Pinellia pace of creation for the simultaneous assessment of nu-
and Panax from atomic DNA.3 merous qualities in many taxa. This innovation is utilized
for the identification and confirmation of natural medi-
DNA barcoding cations, though we need to distinguish particular DNA
sequences of interest in every species to design a stan-
DNA barcoding is a novel molecular and bioinformatics dard test on a chip to use for comparisons. The DNA frag-
tool with enormous scope. It is intended to provide fast, pre- ments of a normal sample are immobilized and organized
cise, automatable, and accurate species identification with on a microarray and attached with glass slides, silicon
high validity. This innovation requires a novel nucleotide or nylon layers.37
sequence of small DNA fragments (400–800 bps), which Presently, DNA chip technology has been applied for
are then used with specific reference sequences to distin- the identification of different types of Fritillaria, Den-
guish samples and find unknown taxa. Also, it utilizes drobium and Bupleurum. Species-specific oligonucleotide
a shorter hereditary indicator from a regular locus (simi- assays were obtained through the 5S ribosomal RNA
lar to a creature’s plastidial DNA, atom or mitochondria). quality of Euphorbia kansui, Pinellia ternata, Teucrium
76 M. Imran et al. Identification of botanicals with molecular biotechnology

divaricatum, Pinellia cordata, Aconiti kusnezoffii, Typho- Withania somnifera, and Moricandia sinaica, gathered
nium giganteum, Croton tiglium, Dysosma tatula, Dysosma in various areas of Saudi Arabia. Five primers were used
pleiantha, Dysosma versipellis, Hyoscyamus niger, Pinellia to amplify the plant species DNA, and the RAPD profiles
pedatisecta, Datura inoxia, Rhododendron molle, Strych- increased from 307 to 1772 bps. Pairwise comparisons
nos nux-vomica, Alocasia macrorrhiza, Datura metel, and between promotions, given the extent of shared homol-
Aconitum carmichaeli.38 ogy created by the primers used, were determined with
the assistance of the StatistiXL program v. 1.7 (https://
www.statistixl.com/download/) using Jaccard’s simili-
Examples of DNA-based validation tude coefficient. A significant value of 0.32 was shown
between L. shawii and A. fragrantissima, and a least pair-
of natural plants wise likeness of 0 was seen between A. telephioides, Z. spi-
Olive oil is sold with 20% or more fake oils, and as artifi- nose, B. eriophora, B. eriophora, and Z. propinquum when
cial and unsaturated fats are similar, it is difficult to sepa- the 5 primers were consolidated.41
rate them using standard methods. However, the com- Boerhavia diffusa, also called Punarnava, is a herb na-
plete genomic DNA was isolated using olive oil mixed with tive to India and is frequently used in traditional Indian
sunflower oil and canola, and was investigated for SNPs medicine. Precise identification and clustering of B. dif-
in non-coding loci of psbA-trnH and the incomplete cod- fusa are critical for improving the adequacy and biosafety
ing area of the matK plastid. The amplification of these of the medications prepared with the use of this herb.
DNA loci was completed with specific primers using PCR, The DNA barcoding strategies are used to identify and iso-
and the obtained DNA sequences were aligned to stan- late B. diffusa through closely related species. The phyloge-
dardized tag sequences of the canola and sunflower oil netic examination of the 4 types of Boerhavia with poten-
DNA. This combination of molecular science methodology tial for tagging included ribosomal DNA locales ITS, ITS2,
and bioinformatics technology is a practical strategy for ITS1, and the chloroplast plastid psbA-trnH. Grouping
guaranteeing the purity of olive oil by aligning contami- arrangement uncovered 26% polymorphic regions in ITS,
nated DNA fragments with their standardized DNA after 30% in ITS1, 16% in ITS2, and 6% in psbA-trnH, separately.
blending canola and sunflower oil into olive oil. Adultera- A phylogenetic tree was built for 15 varieties using the ITS
tion of up to 5% in olive oil can be quickly detected using sequences that differentiated B. diffusa from different spe-
this plastid-based sub-atomic DNA innovation.39 cies. The ITS1 had a higher change/transversion propor-
The genome sequence of Parthenium argentatum plastid tion, level of variety and pairwise distance, which separate
was shown to have 152,803 bps. In addition, it was demon- B. diffusa from different types of Boerhavia. The concen-
strated that the genomic configuration of the P. argentatum trate uncovered that the potential candidate regions that
chloroplast is similar to that of Helianthus annuus, based could be utilized for distinguishing B. diffusa and verifying
on the general connection of specific protein expression pat- its natural products were ITS and ITS1.42
terns. Unlike Guizotia abyssinica, H. annuus and Lactuca Valerian (Valeriana officinalis) is a therapeutic herb gen-
sativa, P. argentatum (guayule) is a latex-producing woody erally utilized as a gentle sedative and anxiolytic. Among
shrub and was marketed to individuals with type I latex hy- the vast number of synthetic constituents (such as fla-
persensitivity as a hypoallergenic protectant. The total chlo- vonoids, alkaloids, terpenoids, and iridoids) found in va-
roplast genomes of L. sativa, H. annuus and G. abyssinica, lerian root, the active ingredients that are liable to have
along with the plastid genome of P. argentatum, have more a soothing effect are valerenic acid (C15 sesquiterpenoid)
modest 3.4 kb reversals compared to the larger 23 kb matK and valerena-4,7(11)-diene. The NGS (Roche 454 pyro-
and psbA-trnH spacer chloroplast DNA used for the stan- sequencing; Roche Diagnostics, Basel, Switzerland) was
dard identification of other Parthenium species: P. schot- used to generate 1 million record reads of valerian roots
tii, P. argentatum, P. hysterophorus, and P. tomentosum. to identify an active terpene synthase. Two sesquiterpene
When contrasted with the sequences of plastid genomes, synthases were identified (VoTPS1 and VoTPS2) from
namely L. sativa, G. abyssinica, H. annuus, and Asteraceae, the collected records, both of which demonstrated domi-
the DNA-based validation method revealed distinction nating articulation designs in the root. Transgenic yeasts
through the development of the 4 genomes. The improve- VoTPS2 and VoTPS1 delivered germacrene C/germacrene
ment in chloroplast-specific DNA standardized probes was D and valerena-4,7(11)-diene, separately, as significant ter-
then used to identify Parthenium varieties.40 pene products. Purified VoTPS1 and VoTPS2 recombinant
The RAPD has been widely used for the detection proteins affirmed these actions in vitro with pharmaco-
of genetic inconsistencies within plants. This approach kinetics (Km of ~10 μM and kcat of 0.01 s−1) shown for
was initially utilized to genetically identify 11 arid spe- the 2 chemicals. The design of the valerena-4,7(11)-diene
cies of plants, including Bassia eriophora, Caylusea hex- created from the VoTPS2 signals was additionally validated
agyna, Sonchus oleraceus, Zilla spinosa, Lycium shawii, through 13C-nuclear magnetic resonance (NMR) along
Rumex vesicarius, Zygophyllum propinquum ssp. miga- with gas chromatography-mass spectrometry (GC-MS)
hidii, Andrachne telephioides, Achillea fragrantissima, in correlation with an engineered standard. Kumar et al.
Polim Med. 2023;53(1):69–79 77

described a methodology that includes cutting-edge se- sequencing, microsphere-based suspension cluster, and
quencing and the use of metabolically designed organisms cutting-edge sequencing, will bring about further innova-
to understand terpenoid variety in restorative plants.43 tions. Other promising advancements include nanopore
Phyllanthus (Euphorbiaceae) varieties, well known due technology for the highly precise identification of DNA
to their hepatic defensive action, are used in a few tradi- bases, and Zenith, an enzymatic genotyping tool for
tional medicines in native medical services in India. They investigating tens to thousands of genomic variations
are traded as crude homegrown medications. Samples in a single multiplexed response. Another emerging
of Phyllanthus that are used in crude drugs were acquired technique is the oligonucleotide ligation assay (OLA),
from 25 shops in south India and consisted of 6 unique va- which has a broad scope in the multiplex examination
rieties, such as Phyllanthus amarus, which were identified of nucleic acid sequences, and can discover known single
by examining their morpho-taxonomical properties and SNPs and separate alleles in polymorphic genes. These
through molecular investigations. Testing revealed that novel ways to deal with DNA sequencing guarantee deep
76% of the pharmaceutical assays consisted of P. amarus genomic investigation, exhibit a high multiplexing limit,
at a purity >95%, without admixtures. The remaining 24% and open up many new ways for genotyping and future
of shops had 5 distinct species of Phyllanthus, including taxon identification.46
P. maderaspatensis, P. fraternus, P. debilis, P. kozhikodia- The new high-throughput sequencing (HTS) advances
nus, and P. urinaria. The chloroplast DNA region, psbA- allow for the development of new molecular methodolo-
trnH, was used to generate species-specific DNA standard- gies. An equal sequencing innovation, delivering a large
ized tag marks for Phyllanthus species. The DNA scanner number of DNA sequences (altogether 0.5–60 giga base
tag psbA-trnH locus of the chloroplast can successfully sets) in a solitary run, has transformed genomic research
isolate Phyllanthus species and can be subsequently uti- in science and medicine. These cutting-edge sequencing
lized to determine species admixtures in the raw drugs stages, such as the Roche 454, Illumina, Solexa, Helicos,
made from Phyllanthus.44 and Applied Biosystems framework, can arrange DNA
In this specific case, Ruta graveolens, which is sold quicker and at much lower costs than the standard 96-well
as a dried restorative herb, was found to be contaminated setup of Sanger sequencing.47
with Euphorbia dracunculoides. The contamination Cutting-edge sequencing strategies such as NGS can
of Ruta graveolens with Euphorbia dracunculoides repre- help answer new and long-standing questions. Even though
sents an instance where the intended herb was adulterated no “third generation” methods have been made monetarily
or substituted with another species. This contamination accessible yet, a few organizations have advanced models
highlights the importance of quality control and proper that propose dynamic concepts.48
identification of herbs to ensure their safety and efficacy
for consumers. The genomic DNA was extracted from
leaves (100 mg each) using a modified cetyltrimethylam- Conclusions
monium bromide (CTAB) protocol. The ITS sequences
of ribosomal DNA (nrDNA-ITS) and chloroplast spacer The effort to identify specific types of medicinal plants
sequences (rpoB and rpoC1) have favorable plant DNA and produce both conventional and novel natural com-
barcoding qualities. These spacer groupings were ampli- pounds suitable for use in medical science requires accu-
fied, sequenced and confirmed with a Basic Local Align- rate botanical, phytochemical and biochemical recognition
ment Search Tool (BLAST) search. The sequence arrange- methods.
ments were made using ClustalX (http://www.clustal.org/
clustal2/) to search for contrasts in the groupings. A DNA ORCID iDs
marker was then created dependent on rpoB and rpoC1 Mohd Imran  https://orcid.org/0000-0001-7349-3019
of the nrDNA-ITS to detect the E. dracunculoides contami- Haya Majid  https://orcid.org/0000-0003-1439-1172
nants in the R. graveolens samples. Ruta graveolens (289 and Tasha Riaz  https://orcid.org/0000-0002-4785-9715
Shayan Maqsood  https://orcid.org/0000-0003-1985-6581
264 bps) and E. dracunculoides (424 bps) sequences were
created through different groupings of the nrDNA-ITS and
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