Supporting Information DNA-Based Authentication of ...€¦ · Supporting Information . DNA-Based Authentication of Botanicals and Plant-Derived Dietary Supplements: Where Have We

1

Supporting Information

DNA-Based Authentication of Botanicals and Plant-Derived Dietary

Supplements: Where Have We Been and Where Are We Going?

Denise F. Coutinho Moraes1*, David W. Still2*, Michelle R. Lum3, Ann M. Hirsch4,

5

*Joint first authors.

Affiliations

1Federal University of Maranhão (UFMA), Department of Pharmacy, São Luis,

Maranhão, Brazil

2Department of Horticulture/Plant and Soil Science, California State Polytechnic

University, Pomona, CA, USA

3Department of Biology, Loyola Marymount University, Los Angeles, USA

4Department of Molecular, Cell and Developmental Biology, University of California-

Los Angeles, Los Angeles, CA, USA

5Molecular Biology Institute, University of California-Los Angeles, Los Angeles, CA,

USA

Correspondence

Ann M. Hirsch

Department of Molecular Cell and Developmental Biology and Molecular Biology

Institute

University of California-Los Angeles

Los Angeles, CA 90095-1606, USA

© Georg Thieme Verlag KG · DOI 10.1055/s-0035-1545843 · Planta Med · Moraes DFC et al.

2

Phone: +1 310 206 8673

Fax: +1 310 206 5413

[email protected]

Table 1S Some examples of DNA-based techniques used to evaluate herbal products,

their advantages and disadvantages, and the results obtained.

PCR-

based

DNA

methods

Commercial

product/major

medicinal plant

studied

Referenc

e

Advantages

of method

Disadvantages Results/remark

s

RFLP with

PCR

sequencin

g

Red clover

(Trifolium

pretense) and

alfalfa

(Medicago

sativa)

[1] Relatively

rapid. Can be

used with

dried

specimens.

Species-

specific

primers can

be designed.

Relatively high

cost, numerous

steps, esp. if

DNA needs

repairing,

requires a

relatively large

amount of high

quality DNA.

Both alfalfa and

red clover

commercial

products were

confirmed as

authentic.

Siberian ginseng

(Eleutherococcus

. senticosus)

[2] Of 40 samples,

12 (30%) were

not

authenticated.

Espinheira-santa

component

(Maytenus

aquifolia or M.

ilicifolia)

[3] Fifty-five % of

the commercial

samples

contained

another plant,

mainly Sorocea

bonplandii.

RAPD Shankhpushpi

(Clitoria

ternatea,

Convolvulus

pluricaulis and

Evolvulus

alsinoides)

[4] Relatively

low cost,

small amount

of genomic

DNA needed,

relatively

easy to

perform.

Does not

require prior

sequence

knowledge.

Requires the

purchase of

commercial

primers and

frequently

numerous

primers have

to be tested.

Among six

samples, all

were found to

have either

adulterants or

were

misidentified.

Turmeric powder

(Curcuma

longa)

[5] All samples (3)

were mixed with

large amounts of

Curcuma

zedoaria (white

turmeric).

Pingwei San and

Wuling San:

[6] Pingwei San was

identified as A.


3

products contain

Atractylodes spp.

(A. ovata, A.

japonica or A.

laucea).

japonica and

Wuling San as

A. ovata.

Cuscuta reflexa [7] In a market in

India, the

samples of C.

reflexa

contained C.

chinensis.

Senna

angustifolia

[8] Adulteration was

found among the

samples of S.

angustifolia

purchased in

various markets.

Verbenae Herba

(Verbena

officinalis)

[9] To identify V.

officinalis and

differentiate it

from possible

adulterants of

Verbenae Herba,

the authors also

used other

methods,

including ITS

and SCAR

markers. The

commercial

samples were

authenticated

with RAPD.

Rasayana churna

(Tinospora

cordifolia,

Emblica

officinalis and

Tribulus terestris

[10] The only

commercial

sample analyzed

contained the

three correct

species.

Glycyrrhiza

glabra

[11] G. glabra was

distinguished

from its

adulterant Abrus

precatorius

SSCP Individual and

mixed samples of

alfalfa

(Medicago

sativa); red

clover (Trifolium

[12] Reproducible

, rapid, and

relatively

simple.

Separates

adulterants

Requires

electrophoresis

.

All commercial

samples

analyzed had

additional plant

material in

addition to what


4

pretense); woad

(Isatis

indigotica);

European licorice

(Glycyrrhiza

glabra); Chinese

licorice (G.

uralensis) as well

as commercial

samples of the

legume plants.

from the

main plant of

interest.

was specified on

the label.

AP-PCR

and RAPD

Herba Taraxaci

(Taraxacum

mongolicum)

[13] Simple, low

cost, small

amount of

DNA

required.

Sometimes low

reproducibility.

Taraxacum

mongolicum was

distinguished

from adulterants.

“Ku-Di-Dan”

(herba

elephantopi)

(Elephantopus

scaber)

[14] The results

confirmed that

the samples

analyzed were

related to E.

scaber. Thus,

they were

authenticated.

Multiplex

DNA PCR

using 5.8S

RNA and

ITS

Commercial

capsules and

tablets containing

either St John’s

wort extract and

a mixture of

plant extracts or

powdered

Hypericum

perforatum

[15] Discriminate

s among

closely

related

species in

one reaction.

Labor

intensive.

Primers need

to be

specifically

aligned to

distinctive

regions of

nrITS and

rigorously

tested.

Among the three

samples

analyzed, two

were H.

perforatum,

whereas the

other did not

show identity to

Hypericum.

DNA

microarray

Herba Dendrobii

(Dendrobium

species)

[16] Provides

information

about many

genes in one

experiment;

fast and easy.

Possibility of

cross-

hybridization

occurring.

Of the two

samples

analyzed, one

contained D.

nobile, which is

authorized by

Chinese

Pharmacopoeia,

whereas the

second

contained D.

lohohense,

which is not.

Multiplex

ARMS

Ginseng (Panax

ginseng);

Chikusetsu-

[17] Using

multiple

primers,

All four samples

of ginseng were

authenticated.


5

Ninjin (P.

japonicas);

American

ginseng (P.

quinquefolius);

Notoginseng (P.

notoginseng)

plants with a

similar

sequence in

one locus,

but different

in another

one can be

identified.

SCAR Phyllanthus

emblica

[18] RAPD-based

method, but

more

specific,

sensitive, and

reproducible.

See

disadvantages

for RAPD

procedure.

The three

samples were

authenticated.

Abbreviations: AP-PCR = Arbitrarily Primed Polymerase Chain Reaction; ARMS = Amplification

Refractory Mutation System; RAPD = Random Amplified Polymorphic DNA; RFLP = Restriction

Fragment Length Polymorphism; SCAR = Sequence Characterized Amplified Regions; SSCP = Single-

Strand Conformation Polymorphism.

References for Table 1S

[1] Lum MR, Potter E, Dang T, Heber D, Hardy, Hirsch AM. Identification of botanicals

and potential contaminants through RFLP and sequencing. Planta Med 2005; 71: 841-846

[2] Zhu S, Bai Y, Oya M, Tanaka K, Komatsuko K, Maruyama T, Goda Y, Kawasaki T,

Fujita M, Shibata T. Genetic and chemical diversity of Eleutherococcus senticosus and

molecular identification of Siberian ginseng by PCR-RFLP analysis based on chloroplast

trnK intron sequence. Food Chem 2011; 129: 1844-1850

[3] Nakamura SS, do Valle JS, Jacomassi E, Linde GA, Colauto NB. Molecular

authentication of Maytenus sp. by PCR-RFLP. Semina: Ciências Agrárias 2013; 34: 627-

634

[4] Ganie SH, Srivastava PS, Narula A, Ali Z, Sharma MP. Authentication of

shankhpushpi by RAPD markers. Eurasia J Biosci 2012; 6: 34-96

[5] Sasikumar B, Syamkumar S, Remya R, Zachariah TJ. PCR based detection of

adulteration in the market samples of turmeric powder. Food Biotech 2004; 18: 299-306

[6] Chen KU, Su YC, Lin JG, Hsin LH, Su YP, Su CH, Li SY, Cheng JH, It Mao S.

Identification of Atractylodes plants in Chinese herbs and formulations by random

amplified polymorphic DNA. Acta Pharmacol Sin 2001; 22: 493-497

[7] Khan S, Mirza KJ, Abdin MZ. Development of RAPD markers for authentication of

medicinal plant Cuscuta reflexa Eurasia J BioSci 2010; 4: 1-7

[8] Khan S, Mirza KJ, Al-Qurainy F, Abdin MZ. Authentication of the medicinal plant

Senna angustifolia by RAPD profiling. Saudi J Biol Sci 2011; 18: 287-292


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[9] Ruzicka J, Lukas B, Merza L, Gohler I, Abel G, Popp M, Novak J. Identification of

Verbena officinalis based on ITS sequence analysis and RAPD-derived molecular

markers. Planta Med 2009; 75: 1271-1276

[10] Shinde VM, Dhalwal K, Mahadik KR, Joshi KS, Patwardhan BK. RAPD Analysis

for Determination of Components in Herbal Medicine. eCAM 2007; 4: 21-23

[11] Khan S, Mirza KJ, Tayaab M, Abdin MZ. RAPD profile for authentication of

medicinal plant Glycyrrhiza glabra Linn. Internet J Food Safety 2009; 11: 24-28

[12] Lum MR, Hirsch AM. Detecting the components of botanical mixtures by single-strand conformation polymorphism analysis. In: Ebeler SE, Takeoka GR, Winterhalter P, editors. Authentication of Food and Wine, ACS Symposium Series, Vol. 1081; 2011: 351-362

[13] Hui C, But PPH, Pangtchui S. Identification of Herba taraxaci and its adulterants in

Hong Kong market by DNA fingerprinting with random primed PCR. China J Chinese

Mat Med 1997; 4: 197

[14] Cao H, But PPH, Shaw PC. Authentication of the Chinese drug “ku-di-dan” (Herba

elephantopi) and its substitutes using random-primed polymerase chain reaction (PCR).

Acta Pharmacol Sin 1996; 31: 543-553

[15] Howard C, Socratous E, Williams S, Graham E, Fowler MR, Scott NW, Bremner

PD, Slater A. PlantID–DNA-based identification of multiple medicinal plants in complex

mixtures. Chinese Med 2012; 7: 1-9

[16] Zhang YB, But PPH, Wang ZT, Shaw PC. Current approaches for the authentication

of medicinal Dendrobium species and its products. Plant Genet Res 2003; 3: 144-148

[17] Zhu S, Bai Y, Oya M, Tanaka K, Komatsu K, Maruyama T, Goda Y, Kawasaki T,

Fujita M, Shibata T. Genetic and chemical diversity of Eleutherococcus senticosus and

molecular identification of Siberian ginseng by PCR-RFLP analysis based on chloroplast

trnK intron sequence. Food Chem 2011; 129: 1844-850

[18] Dnyaneshwar W, Preeti C, Kalpana J, Bhushan J. Development and application of

RAPD-SCAR marker for identification of Phyllanthus emblica Linn. Biol Pharm Bull

2006; 29: 2313-2316


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Table 2S Examples of DNA barcoding-based studies to evaluate the authenticity of

herbal products. Several of these investigations also included chemical analyses, but

these are not discussed here.

Commercial

product and its

medicinal plant

Reference Gene or intergenic

space used

Country

where the

product

came from

or was

purchased

DNA-based

results

Dendrobrii Herba.

Various

pharmacopeoia

have authorized

only certain

species of

Dendrobium for

herbal medicines.

[1] ITS China A number of

unauthorized

species of

Dendrobium were

identified in the

various Dendobrii

Herba samples

analyzed. A

Hedera species was

found in one

sample.

Herbal products of

Maytenus ilicifolia

and Mikania

glomerata

[2] rbcL, matK, and ITS2 Brazil Of 17 samples of

Maytenis ilicifolia

products, only

62.5% were

authenticated. Of

the 15 samples of

Mikania glomerata,

one contained

another plant.

Different products

from both plant

and animal

sources

[3] trnL p-loop region

and the mitochondrial

16S ribosomal gene

China Of the commercial

samples analyzed

by high-throughput

sequencing

methods, several of

them contained

heavy metals and

plants that produce

toxic compounds or

are endangered

species.

Baiying (Solanum

lyrati) and

Xungunfeg

(Aristolochia

mollissima)

[4] ITS, rbcL, matK,

trnH-psbA

China In an analysis of

five different

products of

Baiying, two were

found to be A.

mollissima. Two of


8

the five Xungunfeg

products were

identified as S.

lyrati instead of A.

mollissima.

Dang gui

(Angelica sinensis)

[5] ITS China Of the eight

samples tested,

four were

adulterated with

other species of

Angelica.

Coco de mer or

sea coconut

(Lodoicea

maldivica), often

substituted with

Cocos nucifera

(coconut)

[6] PCR primers for a

nuclear

phosphoribulokinase

(PRK) region,

specific to palms,

were used for L.

maldivica. Plastid

sequences were used

to verify quality of

the DNA.

Republic of

Seychelles

Among the seven

samples, only one

was identified as L.

maldivica. The

Cocos nucifera

PCR product was

only 80%

homologous to the

sequence of L.

maldivica.

Shigoka (SGK)

(Eleutherococcus

senticosus)

[7] ITS Japan and

China

Of the 34 samples,

68% were

authenticated and

the rest contained

other species.

Notoginseng

Radix (Panax

notoginseng)

[8] 18S RNA and matK Japan and

China

Samples from

Japanese and

Chinese markets

were identified as

P. notoginseng,

although two

genetic groups

were apparent.

Baihuasheshecao

(Hedyotis diffusa)

[9] ITS China and

USA

Four of seven

samples of

Baihuasheshecao

were adulterated

with H. corymbosa.

Hong Dangshen (a

TCM from Hong

Kong) with an

unknown source

plant, but similar

to Radix

Codonopsis,

which consists of

three Codonopsis

species.

[10] ITS Hong Kong

(China)

The two samples of

Hong Dangshen

were authenticated

as Codonopsis

pilosula var.

modesta.1


9

Plant roots from

different species

[11] rpoC1, psbA-trnH,

matK, and ITS

Morocco Problems with

extracting,

amplification, and

sequencing of the

DNA resulted in a

low species level

identification.

Some authentic

roots were found in

the markets.

Leigongteng

(Tripterygium

wilfordii)

[12] 5S RNA and ITS China Two of the five

commercial

samples were

closely related to

Celestus angulatus

and C. hypoleucus

instead of the

correct species.

Madouling Herb

from Taiwan,

where the

Aristolochia

species that was

the original herb in

this product, was

banned and

replaced by

Cardiocrinum and

Lilium species.

[13] ITS China and

USA

Two of the

sequences from the

samples clustered

with Aristolochia

contorta whereas

the others were

identified as

Cardiocrinum

giganteum.

Four common

European herbal

mixtures, although

listed as “not for

human

consumption”, are

smoked as an

alternative to

cannabis or used

as incense. The

mixtures are

marketed as “legal

highs” and sold in

stores or online2.

[14] rbcL and trnH-psbA Products

seized by the

Genoa, Italy

police

(investigation

on illicit drug

marketing

activities).

The commercial

herbal blends

consisted of

different plant

species. Three of

the blends were

adulterated with

cannabinoid-like

compounds as

shown by HPLC.

DNA-barcoding

demonstrated that

the blends

contained aromatic

plants to mask the

cannabinoids. The

fourth blend

contained

Mitragyna speciosa

and was also

enriched with

alkaloids from this


10

plant.

Radix Astragali

(Astragalus

membranaceus

and A.

mongholicus)

[15] ITS and 5.8S RNA China and

USA

Samples showed

genetic sequence

variation similar to

the identified

samples,

suggesting

variation among

the taxa.

Radix Astragali

(Astragalus

membranaceus

and A.

membranaceus

var. mongholicus)

[16] ITS, matK, rbcL, and

coxI

China Only two out of

four samples

analyzed were

reliably

authenticated.

Chuanxiong has

different plant

sources on labels

in China

(Ligusticum

chuanxiong)

versus Japan

(Cnidium

officinale).

[17] matK and ITS China and

Japan

The products from

China and Japan

are from an

identical plant

source (Ligusticum

chuanxiong).

Green Tea

(Camellia

sinensis)

[18] ITS and matK USA Both commercial

products contained

C. sinensis.

Capsules of St.

John’s wort

containing dried,

powdered material

or the extract of

Hypericum

perforatum alone

and mixed with

other plants

[19] 5.8S and ITS UK Among three

samples analyzed,

two were H.

perforatum and one

was not identified

as a Hypericum

species.

1This was a study to identify the source plant of a TCM.

2This study combined morphological, molecular, and chemical techniques in order to identify the plant and

its chemical composition.

References for Table 2S

[1] Takamiya T, Wongsawad P, Tajina N, Shioda N, Lu JF, Wen CL, Wu JB, Handa T,

Iiima H, Kitanaka S, Yukawa T. Identification of Dendrobium species used for herbal

medicines based on ribosomal DNA internal transcribed spacer sequence. Biol Pharm

Bull 2011; 34: 779-782


11

[2] Palhares RM, Mügge FLB, Brasil BSAF, Drummond MG, Cosenza GP, Brandão

MGL, Oliveira GC. Application of the DNA Barcode technology and thin layer

chromatography (TLC) for the identification of fraud in marketed vegetal drugs and

herbal medicines approved by ANVISA. Simpósio Brasileiro de Identificação Molecular

de Espécies, Foz do Iguaçu, 2012

[3] Coghlan ML, Haile J, Houston J, Murray DC, White NE, Moolhuijzen P, Bellgard

MI, Bunce M. Deep sequencing of plant and animal DNA contained within traditional

Chinese medicines reveals legality issues and health safety concerns. PLoS Genetics

2012; 8: 1-11

[4] Li M, Au KY, Lam H, Cheng L, Jiang RW, But PPH, Shaw PC. Identification of

Baiying (Herba Solani Lyrati) commodity and its toxic substitute Xungufeng (Herba

Aristolochiae Mollissimae) using DNA barcoding and chemical profiling techniques.

Food Chem 2012; 135: 1653-1658

[5] Feng T, Liu S, He XJ. Molecular authentication of the traditional Chinese medicinal

plant Angelica sinensis based on internal transcribed spacer of nrDNA. Elect J Biotech

2010; 13: 1-10

[6] Mak CY, Mok CS. Molecular identification of Lodoicea maldivica (coco de mer)

seeds. Chinese Med 2011; 6: 2-5

[7] Maruyama T, Kamakura H, Miyai M, Komatsu K, Kawasaki T, Fujita M, Shimada H,

Yamamoto Y, Shibata T, Goda Y. Authentication of the traditional medicinal plant

Eleutherococcus senticosus by DNA and chemical analyses. Planta Med 2008; 74: 787–

789

[8] Fushimi H, Komatsu K, Namba T, Isobe M. Genetic heterogeneity of ribosomal RNA

gene and matK gene in Panax notoginseng. Planta Med 2000; 66: 659-661

[9] Li M, Jiang RW, Hon PM, Cheng L, Li LL, Zhou JR, Shaw PC, But PPH.

Authentication of the anti-tumor herb Baihuasheshecao with bioactive marker

compounds and molecular sequences. Food Chem 2010; 119: 1239-1245a

[10] Zhang YB, Jiang RW, Li SL, Qiao CF, Han QB, Xu HX, Wong KL, But PPH, Shaw

PC. Chemical and molecular characterization of Hong Dangshen, a unique medicinal

material for diarrhea in Hong Kong. J Chin Pharm Sci 2007; 3: 202-206

[11] Kool A, Boer HJ de, Kruger A, Rydberg A, Abbad A, Bjork L, Martin G. Molecular

identification of commercialized medicinal plants in Southern Morocco. PLoS One 2012;

7: 1-13


12

[12] Law SKY, Simmons MP, Techen N, Khan IA, He MF, Shaw PC, But PPH.

Molecular analyses of the Chinese herb Leigongteng (Tripterygium wilfordii Hook.f.).

Phytochemistry 2011; 72: 21-26

[13] Li M, Ling KH, Lam H, Shaw PC, Cheng L, Techen N, Khan IA, Chang YS, But

PPH. Cardiocrinum seeds as a replacement for Aristolochia fruits in treating cough. J

Ethnopharm 2010; 130: 429-432

[14] Cornara L, Borghesi B, Canali C, Andrenacci M, Basso M, Federici S, Labra M.

Smart drugs: green shuttle or real drug? Int J Legal Med 2013; 127: 1109-1123

[15] Xiao WL, Motley TJ, Unachukwu UJ, Lau CBS, Jiang B, Hong F, Leung PC, Wang

QF, Livingston PO, Cassileth BR, Kennelly EJ. Chemical and genetic assessment of

variability in commercial Radix Astragali (Astragalus spp.) by ion trap LC-MS and

nuclear ribosomal DNA Barcoding sequence analyses. J Agric Food Chem 2011; 59:

1548-1556

[16] Guo HY, Wang WW, Yang N, Guo BL, Zhang S, Yang RJ, Yuan Y, Yu JL, Hu SN,

Sun QS, Yu J. DNA barcoding provides distinction between Radix Astragali and its

adulterants. Sci China Life Sci 2010; 53: 992-999

[17] Liu YP, Cao H, Han GR, Fushimi H, Komatsu K. matK and ITS nucleotide

sequencing of crude drug Chuanxiong and phylogenetic relationship between their

species from China and Japan. Acta Pharm Sinica 2002; 37: 63-68

[18] Lum MR, Baycher A, Prigge BA, Hardy M, Heber D, Hirsch AM. Identification of

green tea (Camellia sinensis L.) and tea oil (Camellia oleifera Abel.) by molecular

biological and anatomical methods. In: Ebeler SE, Takeoka GR, Winterhalter P, editors.

Authentication of food and wine. Washington, D.C.: ACS and Oxford University Press;

2007: 290-304

[19] Howard C, Bremner PD, Fowler MR, Isodo B, Scott NW, Slater A. Molecular

identification of Hypericum perforatum by PCR amplification of the ITS and 5.8S rDNA

region. Planta Med 2009; 75: 864-869


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Supporting Information DNA-Based Authentication of ...€¦ · Supporting Information . DNA-Based Authentication of Botanicals and Plant-Derived Dietary Supplements: Where Have We