Content and Structure :� Quality of Herbal Materials, Biodiversity, Factors affecting Quality,

Adulterations� Quality Assessment and Control� Quality Assurance Systems: GAP, GCP, ISSC-MAP, HACCP, GMP

1

Genetic Identity and PhytochemicalQuality of Plants and BotanicalsCh. FRANZ and J. NOVAK, Vetmeduni Vienna

�Tools in phytochemistry: chromatographic methods (TLC/HPTLC, GLC, GC-MS, HPLC, HPLC-MS, etc.)

� In-vitro methods as e.g. antioxidantactivity

�Tools in molecular biology/genetics: RAPD, PCR, HRM, new generationsequencing…

2

DNA-based Identification in Everyday Life

3

From Plant Raw Materials to Final Products:

C. Schmiderer, J. Ruzicka, B. Lukas, J. Novak, Ch. F ranzInstitute for Animal Nutrition and Functional Plant CompoundsUniversity of Veterinary Medicine, A-1210 Vienna, Austria

4

Processed Materials / Final Products

5

Tissue

Isolation of genomic DNA

Amplification of markergenes with PCR

DNA sequence

Cycle sequencing PCR and automatic DNA-sequencing

Molecular Plant Taxonomy Today

6

Identification approach for admixturesPCR: Finding the needle in a haystack easily!

http://nl.ijs.si/et/talks/esslli02/metadata_files/Haystack-FINALb.jpg

forward primer

reverse primer

7

DNA based identification systems for plants and PFS

Species investigated :�Calendula officinalis�Ginkgo biloba�Matricaria recutita (adulteration with Anthemis sp.)�Plantago lanceolata�Plantago ovata�Valeriana officinalis�and others

8

Extraction

• releasing DNA• purifying DNA from compounds interacting with

subsequent PCR

Amplification

• producing multiple copies of a specific part of the DNA

• PCR

Sequencing

• ‚classical‘ Sanger (cycle) sequencing• next generation sequencing

Analysis & Interpretation

• data correction• alignment• bioinformatic approaches (phylogeny, etc.)

„DNA -Barcoding “

9

„DNA -Barcoding “

Database

� Taxon 1 ATG CAT GGG CTT TAA GGC CT

� Taxon 2 ATG CAT GGT CTA GAA GGC CT

� Taxon 3 ATG CAA GGT CTA GAA GGT CT

� Taxon 4 ATG CAT GGT CTA TAA GGT CT

� Taxon 5 ATG CAA GGG CTT TAA GGT CT

� Unknown sample ATG CAA GGT CTA GAA GGT CT

10

Intra- vs. Interspecific Variability

individuum 1individuum 2individuum 3individuum 4individuum 5

C T T A T T C T C T T A T C A C A A G T C T T G T. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . N. . - - - - - - - - . . . . . . . . - - - - . . .. . - - - - - - - - . . . . . . . . - - - - . . .. . - - - - - - - - . . . . . . . . . . . . . . .. . - - - - - - - - . . . . . . . . . . . . . . .. . - - - - - - - - . . . . . . . . - - - - . . .

Species 1

Species 2

consensus sequence

intraspecific variation����interspecific variation����

individuum 1individuum 2individuum 3individuum 4individuum 5

11

Identification of Passiflora incarnata by Sequencing and a Phylogenetic Tree

Neighbour-Joining tree based on ITS sequences

Sequence of Passiflora incarnata

12

Routine Assays

� Hybridization-based methods� dynamic allele-specific hybridization, molecular beacons, SNP

microarrays� Enzyme-based methods

� restriction fragment length polymorphism, PCR-based methods (e.g. �ARMS (amplification refractory mutation system) ), flapendonuclease, primer extension, 5’- nuclease, oligonucleotide ligation assay

� Other post-amplification methods based on physical properties of DNA� Single strand conformation polymorphism, temperature gradient gel

electrophoresis, denaturing high performance liquid chromatography, high-resolution melting of the entire amplicon , use of DNA mismatch-binding proteins, SNPlex

13

ARMS-Assay

C T T A T T C T C T T A T C A C A A G T C T T G T. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . N. . - - - - - - - - . . . . . . . . - - - - . . .. . - - - - - - - - . . . . . . . . - - - - . . .. . - - - - - - - - . . . . . . . . . . . . . . .. . - - - - - - - - . . . . . . . . . . . . . . .. . - - - - - - - - . . . . . . . . - - - - . . .

Species 1

other species

consensus sequence

species specific primer

14

Identification of Matricaria recutita and Anthemis spp.

Alignment excerpt; empty columns symbolize cut out sequence parts15

MARMS Multiple ARMS (amplification refractorymutation system )Combining multiple PCR reactions in one tube

16

MARMS for Matricaria recutita and Anthemis spp.

17

MARMS for Matricaria recutita and Anthemis spp.

18

MARMS discrimination:Matricaria recutita, Anthemis spp. and other Asteraceae

19

Detection of adulterations of Valeriana officinalis with other Valeriana species bymultiplex PCR

Specific marker for the genus Valeriana

Specific for the species V. officinalis

Principle of multiplex PCR

Lanes (from the left): 1 and 13…molecular ladder2,4,5,6,12…V. officinalis3,7,10,11…species of Valeriana other than V. officinalis7,8…other species (not from the genus Valeriana)

Novak, J. et al.., unpubl.) 20

Detection of adulterations ofValeriana officinalis with otherValeriana species by multiplex PCR

Specific for the genus Valeriana

Specific for the species V. officinalis

Lanes (from the left): 1 and 13…molecular ladder2,4,5,6,12…V. officinalis3,7,10,11…species of Valeriana other than V. officinalis7,8…other species (not from the genus Valeriana)

� Samples� 1…V. officinalis� 2…Valeriana sp.*

� 3…V. officinalis� 4…V. officinalis� 5…V. officinalis� 6…Valeriana sp. *

� 7…unknown adulteration� 8…unknown adulteration� 9…Valeriana sp. *

� 10…Valeriana sp. *

� 11…Valeriana officinalis*…another Valeriana species, but not V. officinalis

21

HRM (High Resolution Melting)

http://www.gene-quantification.de

Invitrogen

22

Identification of Matricaria recutitaDiscrimination of Anthemis spp. and other Asteraceae species by high resolution melting curve analysis ( HRM)

Matricaria recutita can be distinguished from M. discoidea by a second primer combination

PlantLIBRA PlantLIBRA PlantLIBRA PlantLIBRA

23

High Resolution Melting Curves(HRM) of Calendula sp. DNA

Novak, J. et al.., unpubl.)24

Detection of admixtures of Digitalis spp. in Plantago lanceolata

1) Plantago lanceolata (pure)2) 10% Digitalis3) 30% Digitalis4) 50% Digitalis5) 70% Digitalis6) 90% Digitalis7) Digitalis lanata (pure)NTC – non template control

Multiplex-PCR with primers specific for Plantago, P. lanceolata and Digitalis

High resolution melting curve analysis with primers specific for P. lanceolata and Digitalis

25

DNA – Methods applicable at:

�Family, Genus and Species level (inter-and intraspecific)

�Population level

�Chemotype level

�but also in the authentification of intermediate and final products

26

From Herbal Materials to Final Products

27

fresh plant material

drug

drugconc.

oilmacerate

mother tincture

pressjuice

powder

oilmacerate

spissumextract

siccumextract

tea cuttings

crushing

pressingextraction

drying

cutting

grinding cutting

fluid extracttincture

extraction

dryinggrinding

drying

Processing of Herbal Materials to PFS / HMPs

28

DNA Degradation

�DNA degradation is a process by which DNA breaks down into smaller fragments.

�Factors promoting DNA degradation:• physical shearing• boiling• nucleases• freezing• acid treatment• alkali treatment• UV exposure 29

DNA-Extraction of Sage (Salvia officinalis)

1

10

100

'fresh' material Salviaeofficinalis folium;

pharmacy

TeekanneFixSalbei

AlnaturaSalbeitee

DN

A-c

onc.

[ng/

µl]

(Quiagen DNeasy Plant Mini Kit; ng/µl)

30

amplification successSpecies Product ITS psbA-trnHValerianae radix Reference HM ���� ����

Pharmacy HMP ���� ����

Doskar PFS ���� ����

Ginseng radix pharmacy HMP ���� ����

Aurita PFS ���� ����

Doskar PFS ���� ����

Menthae folium Reference HM ���� ����

pharmacy HMP ���� ����

Alnatura Herbal tea ���� ����

Teekanne Herbal tea ���� ����

Salviae folium Reference HM ���� ����

pharmacy HMP ���� ����

Alnatura Herbal tea ���� ����

Teekanne Herbal tea ���� ����

(Schmiderer, Lukas, Novak, ZAG 18:65-72, 2013)

PCR-Successin different HM, HMP and PFS Products

HM: Herbal Material, HMP: Herbal Medicinal Product,PFS: Plant Food Supplement

31

amplification successSpecies Product ITS psbA-trnHValerianae radix Reference HM ���� ����

Pharmacy HMP ���� ����

Doskar PFS ���� ����

Ginseng radix pharmacy HM ���� ����

Aurita PFS ���� ����

Doskar PFS ���� ����

Menthae folium Reference HM ���� ����

pharmacy HMP ���� ����

Alnatura Herbal tea ���� ����

Teekanne Herbal tea ���� ����

Salviae folium Reference HM ���� ����

pharmacy HMP ���� ����

Alnatura Herbal tea ���� ����

Teekanne Herbal tea ���� ����

(Schmiderer, Lukas, Novak, ZAG 18:65-72, 2013)

PCR-Successin Different Drugs and Products

32

amplification successSpecies Product ITS psbA-trnHMatricariae flos Reference HM ���� ����

Pharmacy HMP ���� ����

Alnatura Herbal tea ���� ����

Teekanne Herbal tea ���� ����

Foeniculi fructus Reference HM ���� ����

Pharmacy HMP ���� ����

Alnatura Herbal tea ���� ����

Teekanne Herbal tea ���� ����

Juniperi pseudo-fructus Pharmacy HM ���� ����

Fuchs Spice ���� ����

Kotanyi Spice ���� ����

Salicis cortex Reference HM ���� ����

Pharmacy HMP ���� ����

Cinnamomi cortex Pharmacy HMP ���� ����

Alnatura PFS ���� ����

Kotanyi Spice ���� ����

(Schmiderer, Lukas, Novak, ZAG 18:65-72, 2013)

PCR-Successin Different Drugs and Products

33

DNA-based identification of an ethanolic extract„Echinacea sp. – spissum extract“

130 140 150 160 170 180

TTAAAGGGCTTGTGCTGTTATGCCCCGTCA-TTGGTGTGCATAGT-GTGCGTTGCTTCTTT plant extract..............................-..............-............... Echinacea purpurea..............................-............C.-............... Echinacea paradoxa..............................-............C.-..-............ Echinacea pallida..............................A............C.-............... Echinacea atrorubens..............................A............C.-............... Echinacea simulata..............................-............C.-............... Echinacea tennesseensisA.G...T..CC...T.........NN..T.-GC.......-..T.-.CA...GTG..... Rudbeckia fulgida var. fulgida..G...T..CC.....A...C...NN..TT-GC.......G-.T.-..A...G.A...... Rudbeckia subtomentosa.........CC.....A........-....-CC.......G.GT.-......G........ Sanvitalia fruticosaA.G...T..CC...T.............T.-GC.......G..T.-.CA...GTG...... Rudbeckia missouriensis.-G...T..CA..-......C.......T--GC.......G..TA-..A...G....TC. Rudbeckia heliopsidis....G.-.........T.G.C......CTT-GC.......GA.T.T.C....G........ Oblivia mikanioides......A.........A.G.C.......TT-GC.......GA.T.-..A..CGAA...... Idiopappus quitensis

34

DNA-based identification of a tincture and fluid extract of chamomile

Novak et al.: DNA-Based Authentication of Plant Extracts. Food Research International 40: 388-392, 2007.

10 20 30 40 50 60

G A T TG C A G A A T CC C G T G A A CC A T C G A G TT T T T G A A CG C A A G T T GC G C C C G A AG C C T T T T GG C C G

. . . .. . . . . . . .. . . . . . . .. . . . . . . .. . . . . . . .. . . . . . . .. . . . . . . .. . . . . . . .. . . .

. . . .. . . . . . . .. . . . . . . .. . . . . . . .. . . . . . . .. . . . . . . .. . . . . . . .. . . . . . . .. . . .

A . . .. . . . . . . .. . . . . . . .. . . . . . . .C . . . . . . .. . . . . . . .. . . . . . . .. . . A . . A .. . . .

70 80 90 100 110 120

A G G GC A C G T C T GC C T G G G C GT C A C G C A TC G C G T C G CC C C C A - - -- - - - - - - -- - - A C A A AC T A T

. . . .. . . . . . . .. . . . . . . .. . . . . . . .. . . . . . . .. . . . . - - -- - - - - - - -- - - . . . . .. . . .

. . . .. . . . . . . .. . . . . . . .. . . . . . . .. . . . . . . .. . . . . - - -- - - - - - - -- - - . . . . .. . . .

. . . .. . . . . . . .. . . . . . . .. . . . . . . .. . . . . . . .. . . . C C G CC C T C C G T GC G C . . . G CG C C C

130 140 150 160 170 180 190

G T T GG G G G - - - -- - - - C G G AT A T T G G T CT C C C G T G CT T A T G G - -- C G T G G T TG G C C A A A AT A G G

. . . .. . . . - - - -- - - - . . . .. . . . . . . .. . . . . . . .. . . . . . - -- . . . . . . .. . . . . . . .. . . .

. . . .. . . . - - - -- - - - . . . .. . . . . . . .. . . . . . . .. . . . . . - -- . . . . . . .. . . . . . . .. . . .

. . . .. . . . A G G GG G G G . . . .. . . . . . C .. . . . . . . .. C C . C . G CG . . C . . C .. . . . C . . .. G C .

200 210 220 230 240 250

A G T CC C T T - C G AT G G A C G C AC G A A C T A GT G G T G G T CG T A A A A A CC - - - C T C GT T C T T T G TT T T G

. . . .. . . . - . . .. . . . . . . .. . . . . . . .. . . . . . . .. . . . . . . .. - - - . . . .. . . . . . . .. . . .

. . . .. . . . - . . .. . . . . . . .. . . . . . . .. . . . . . . .. . . . . . . .. - - - . . . .. . . . . . . .. . . .

. T C .. T C G G . . .C T C . T . T CA C G . . A . .. . . . . . . T. A . C . . C T. A A T . . . .C G . G C C . .C G . .

Tin

ctur

e

Ref

eren

ce

Flu

idex

trac

t

35

Passiflora incarnata drug and productsIdentification success using a simple standard approach

Denomination no. of samples success [%]Drugs (raw material) 36 36 [100%]

Liquids 7 0 [0%]Dry extract 1 0 [0%]Extract 1 0 [0%]Extractum ethanoleum 5 0 [0%]

Pills 37 21 [57%]Raw material 2 2 [100%]Dry extract 14 10 [71%]Extract 16 7 [44%]not specified 5 2 [40%]

Dry extracts (powder) 2 1 [50%]36

Future PerspectivesPart 1: ‚Low Tech‘

� Victor M. Ugaz, A Pocket-Sized Convective PCR Thermocycler, Angewandte Chemie International Edition, doi: 10.1002/anie.200700306

� Open Source Biotechnology

37

Future PerspectivesPart 2: ‚High Tech‘ – Lab-on-a-Chip

http://lab-on-chip.gene-quantification.info/ 38

Future PerspectivesPart 2: ‚High Tech‘ – Lab-on-a-Chip

Microchip-based one step DNA extraction and real-time PCR in one chamber for rapid pathogen identificationJeong-Gun Lee, Kwang Ho Cheong, Nam Huh, Suhyeon Kim, Jeong-Woo Choib and Christopher KoaLab Chip, 2006, 6, 886–895

39

Future PerspectivesPart 2: ‚High Tech‘ Next Generation Sequencing (NGS)

Method ‚Classical‘ Sanger-Sequencing

NGS - Illumina

Read length 600 - 1000 bp 50 – 300 bp

Sequences per run 96 Up to 3 billion

Price per 1 million bp 1700.-€ 0.07.-€

40

41

Notopterygium/Hansenia Identity, Phylogenesis and Admixture Identification

Notopterygium incisumnew nomencl.: Hansenia

?

42

Genetic Identity and PhytochemicalQuality of Plants and Botanicals

�Tools in phytochemistry: chromatographic methods (TLC/HPTLC, GLC, GC-MS, HPLC, HPLC-MS, etc.)

� In-vitro methods as e.g. antioxidantactivity

�Tools in molecular biology/genetics: RAPD, PCR, HRM, new generationsequencing…

43

Kabelitz 2003

Atractylodis macrocephalae Rhizoma, TLC on flavonoid constituents, UV -light 366 nm

44

HPTLC phytochemical profile of Camellia sinensisEC C EGCG ECG EGC CAF GT2 GT1 DGT BT DBT T GA Q K

EC=Epicatechin GT2=Green tea 2

C=Catechin GT1=Green tea 1

EGCG=Epigallocatechin-3-gallate DGT=Decaffeinated green tea

EGC=Epigallocatechin BT=Black tea

CAF=Caffeine DBT=Decaffeinated black tea

T=Theaflavin Q=Quercetin

GA=Gallic acid K=Kaemferol

HPTLC of tea samples after derivatization with Fast Blue

Salt B reagent

45

� Analytical profiles for caffeine and catechin in Camellia sinensisproducts were obtained by gradient RP-HPLC/UV

� Analytical profiles for active amines (octopamine and synephrine) in PFS containing Citrus aurantium and other ingredients were performed using the same chromatographic conditions coupled with a fluorimetric detector

Figure 1 – Caffeine and catechin profile in green te a infusion

Figure 2 – Octopamine and synephrine in PFS containi ng Citrus aurantium

Phase A (v/v) Phase B (v/v)

Water 99.5% Acetonitrile 99.5%

Formic acid 0.5% Formic acid 0.5%

Phase A (v/v) Phase B (v/v)

SDS 0.288 g/L 100%

SDS 0.288 g/L 50%

Acetonitrile 50%

Partner: UMIL

46

0

10

20

30

40

50

60

70

EC

ECG

EGC

ECCG

Caffeine

Green-Tea-ExtractsGreen-Tea-Extracts combined with

other botanical extracts

Variability in Caffeine ofGreen Tea containing PFS

47

Rosmarinic acid in various Thymus vulgaris accessions and its contribution to theantioxidant activity

Waste: leaves after hydrodistillationChizzola et al. 2008, JAFC

48

AU

-0,05

0,00

0,05

0,10

0,15

0,20

0,25

Minutes5,00 10,00 15,00 20,00 25,00 30,00 35,00 40,00 45,00 50,00 55,00 60,00 65,00

P. lagopusA

U

0,00

0,02

0,04

0,06

0,08

0,10

0,12

0,14

0,16

0,18

0,20

0,22

nm200,00220,00240,00260,00280,00300,00320,00340,00360,00380,00400,00420,00440,00460,00480,00500,00520,00540,00560,00580,00

198,0

241,4

326,7

Chlorogenic acid

Species Origin IPK number

P. afra San Marino di Urbino, Italy IPK-PLA 16

P. albicans Gene Bank of IRA, Medenine, Tunisia IPK-PLA 19

P. arenaria Institute for breeding research Braunschweig, Germany IPK-PLA 40

P. cornuti Botanical garden University Bologna, Italy IPK-PLA 52

P. coronopus "Rühlemann" seed producing industry, Germany Rühl-Pla 18x

P. coronopus Madeira, Portugal IPK-PLA 55

P. coronopus seed producing industry, north Italy IPK-PLA 18

P. lagopus Botanical garden University Maputo, Mozambique IPK-PLA 50

P. lagopus France IPK-PLA 42

P. lanceolata wild collected Vienna, Austria

P. lanceolata Elverdamskroen, Denmark IPK-PLA 51

P. major wild collected Vienna, Austria

P. maritima Botanical garden Univ. Halle/Saale, Germany IPK-PLA 27

P. media wild collected Vienna, Austria

P. sempervirens Creissels, Aveyron, France IPK-PLA 64

P. subulata Pont-de-Montvert, Lozere, France IPK-PLA 65

DPPH- assayHPLC-DAD

Fast- GC-FID

Plantago sp.:Diversity of iridoids profile between species and plant parts resp.Diversity of the polyphenols profile and the antioxidant activity between species

Sponza, S. et al., unpubl.49

Harpagophytum procumbensPhytochemical profiling of plant raw material (prov ided by PhytoLab)

Pla

nt s

ourc

e co

mpa

rison

Hotellings T2 Range comparison: >95% similarity but clear differences detected

Marker compounds identified by accurate mass detection

50

Harpagophytum procumbensMethod development - Waters Acquity UPLC and Synapt HDMS

Standards profiles

MeOH/ACN based extraction

51

Volatile profiles of Matricaria and Anthemis species(German Chamomile and confused species)

Matricaria recutita:Chamazulene, bisabololoxide A, dicyloethers

Matricaria matricarioides:dicycloethers

Matricaria trichophylla,M. disciforms, M. perforata:Polyacetylenes: Matricariaesters

Anthemis austriaca: chrysanthenylacetate

Anthemis altissima, A. arvensis and A. cotula: very low in volatiles, mainly common sesquiterpenoids (e .g. anthecotulide: allergenic!)

Species morphological similar: flower heads with wh ite ray florets and yellow disc florets

VUW-Bot52

Misidentification / Adulteration!(Problem: PAs and Allergenic Compounds in Herbal In fusions

and………… PFS!)

Anthemis sp.

True Chamomile

53

Calendula officinalis

54

New varietywith high faradiol monoester content

55

Influence of Processing

56

Influence of the Solvent Ethanol-Water-Ratio on the Extraction of Bioactive Compoundsof Sage ( Salvia off.)

Durling 2006 Food Chemistry 101, 1417

TSS: Total soluble

Solids

TP: Total Phenolics

RA: Rosmarinic

acid

CT: Carnosol etc.

EO: Essential Oil

57

Influence of Temperature on the Extractionof Phenolic Compounds in Sage ( Salvia off.)

Durling 2006 Food Chemistry 101, 1417

TSS: Total soluble

Solids

TP: Total Phenolics

RA: Rosmarinic

acid

CT: Carnosol etc.

EO: Essential Oil

58

Conclusion and Prospects

Quality Assessment and Quality Assurance of PFS- Identity / Authenticity of the material (PCR,…)- Phytochemical Quality: from starting material to

final product (variation / range within species andbetween products!)

- Contaminations, Residues, Toxic Compounds- Quality Guidelines for herbal materials and

extracts used in PFS already elaborated in co-operation with European Botanical Forum/FSE

59

Supply Chain Management requested !

� Good Agricultural and Wild Collection Practice (GACP)

� Good Manufacturing Practice (GMP)

� Identification / Authentication� Quality Control: Phytochemistry� Quality Control: Contaminations, Residues

60