wp7: characterization of botanicals...b) with possible risks environmental contaminants and residues...

Investigation on Botanical Ingredients of / and Plant Food Supplements (PFS):

plant identity, methods, new compounds; network of laboratories

Chlodwig FRANZ, WP7 leaderon behalf of the Partners of WP7

PLANTLIBRA WP7 Roma Sept. 2012

WP7: Characterization of Botanicals

The objectives of WP7 are:

to provide new methods and experimental data to support a sustainable integrated meta-database on the basis of gaps identified by other WPs

to create an international network of laboratories that can perform reliable analytical assays concerning PFS


Main Items WP 7:providing experimental data concerning

Identification of Plant Material Bioactive constituents a) with beneficial role,

b) with possible risks Environmental contaminants and residues Identification of methods for bio-marker deter-

mination of a) (pos.) physiolog. activity, b) adverse effects, c) misidentification of plants

WP 7 Meeting – Roma 19 Sept . 2012

Plant Material

Herbal medicinalProducts (HMP)

Plant FoodSupplements (PFS)

Pharm. Legislation‚activity driven‘

Food Legislation‚safety driven‘

Active Ingredients !(Single substance /Entire Extract?)

Residues(herbicides, pesticides)

Contaminations(microbial, heavy metals,

Irradiation)

Residues(herbicides pesticides,…)

Contaminations(irradiation, toxic subst.)

Quality???(functional substances?)

FOODPHARMA

• Electrochemical biosensors have been developed based on a screen-printed electrode: can be used for development of short-time-use or as a disposable biosensors for heavy metal ions detection.

• Actually, studies for detection of e.g. Cd in real PFS samples are performed.

EVALUATION OF AMPEROMETRIC ENZYME-BASED BIOSENSORS FOR HEAVY METAL IONS DETECTION IN

FOOD AND FOOD SUPPLEMENTS

Development and testing of methods for contaminant and residue determination on enzyme- based biosensors


SENSORS and BIOSENSORSSENSORS and BIOSENSORSPartner: UTBVPartner: UTBV

ELECTROCHEMICAL BIOSENSORSELECTROCHEMICAL BIOSENSORS

http://www.google.ro/search?q=microfluidics&hl=ro&client=firefox-a&hs=3ys&rls=org.mozilla:en-US:official&prmd=imvnslb&tbm=isch&tbo=u&source=univ&sa=X&ei=oE-RT4agKNDS4QTKpbyKBA&ved=0CG4QsAQ&biw=1366&bih=665&sei=r0-RT5rZAvT04QSgoqCiBA

Microfluidics

I.Švancara, M. Florescu et al.: Sensing in Electroanalysis 5, 2010, 109

Cd and Pb detectionCd and Pb detection

LoDLoD

Limits of detections (LoDs) could be estimated to be:

Zn : 19 µg/L, Sn: 53 µg/L, Tl: 35 µg/L,

approx. 5 µg/L for both Cd and Pb

I.Švancara, M. Florescu et al.: Sensing in Electroanalysis 5, 2010, 109

PFS

NORISK!SafetyFirst!

QUALITY ?Species only?

HEALTH CLAIMS?


3 Main Errors about Plants and Plant Products:

Plants are always available Plants as natural products are

always safe Plants (and plant products) have

always the same composition


Frequency Distribution of Chemotypes in 3 Chamomile-Populations

(70 clones each x 4 repetitions)

Bisabolol type

Bisabolol oxide A type Bisabolon oxide type

Azulene type

Bisabolol oxide B type

CH 29

30%

37%

29%

4%

H 29

96%

2%2%

Menemen

12%

88%

(Schroeder & Franz 1981)

Example of a heterogeneous vs. homogeneous populationof Thymus vulgaris

Each column represents one individual plant

0

10

20

30

40

50

60

70

80

90

100

rty1932.d

rty1926.d

rty1921.d

rty1934.d

rty1930.d

rty1929.d

rty1908.d

rty1913.d

rty1931.d

rty1911.d

rty1923.d

rty1916.d

rty1917.d

rty1922.d

rty1927.d

rty1928.d

rty1915.d

Caryo phyllene OxideSpathuleno ldelta-Cadinenegamma-Cadineneß-Bisabo lenegermacrene Dbicyclo germacreneX151/166ß-Caryo phyllenea-terpinyl acetateCarvacro lThymo lBo rnyl-AcetateLinalyl-acetateCarvacro l MethyletherThymo l M ethylethert-dihydro carvo nea-Terpineo lTerpinen-4-o lBo rneo lCampho rc is-Sabinene HydrateLinalo o lt-Linalo ol-Oxidec is-Linaloo l-Oxidet-Sabinene Hydrateg-Terpinene1,8-Cineo leLimo nenep-Cymenea-Terpinenemyrceneß-PineneSabineneCamphenea-P inenea-ThujeneTricyclene

0

10

20

30

40

50

60

70

80

90

100

4406

4409

4407

4405

4418

4420

4416

4412

4429

4410

4421

4417

4413

4403

4402

4401

4422

4424

4404

4414

4408

4419

4423

4425

4411

4430

4428

4415

4426

4427

Caryophyllene Oxide

Geranyl-butyrat

Elemol

ß-Bis abolene

germacrene D

bicyc logermacrene

X151/166

ß-Caryophy llene

Geranyl-Acetat

ß_Bourbonene

a-terpinyl acetate

Carvacrol

Thymol

Bornyl-Acetate

Terpendiol II

Geranial

Linalyl-acetate

Geraniol

Thymoquinone

Carvacrol Methylether

Neral

Nerol

a-Terpineol

Terpinen-4-ol

Borneol

Camphor

cis-Sabinene Hydrate

Linalool

t-Linalool-Oxide

cis-Linalool-Oxide

t-Sabinene Hydrate

g-Terpinene

1,8-Cineole

0

10

20

30

40

50

60

Early March Late March Late May

%

Confusion / Admixture:Plantain (Plantago lanceolata) with

Foxglove (Digitalis lanata)

(Kabelitz 2003)

Plantago lanceolata Digitalis lanata

Digitalis glycoside

Aucubin

…a dangerousconfusion!

Kabelitz 2003

HPTLC Test on Aristolochic Acid in Stephania tetrandra (‘Han Fangchi’) due to adulteration

with Aristolochia clematitis (‘Guang Fangchi’)

PLANTLIBRA WP7 Roma Sept.2012

These results show: that occasionally herbal drugs are adulterated with foreign

or even toxic plants. This applies especially to plants from collection in wild habitats, where confusion with similar plants as well as poorly qualified personnel presents a risk for the quality of the herbal material,

that especially the identity/authenticity and purity of herbal materials is problematic and

that regular tests for confusions/admixtures/adulterations as well as on the phytochemical quality on one side,

but also on heavy metals, pesticides, mycotoxins and microbiological counts on the other side are required

to get appropriate quality information.

DNA-based Identification in Everyday Life

Tissue

Isolation of genomic DNA

Amplification of markergenes with PCR

DNA sequence

Cycle sequencing PCR and automatic DNA-sequencing

Molecular Plant Taxonomy Today

DNA based identification systems for plants and PFS (Partner: VUW-Bot)

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


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

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

PLANTLIBRA Strasbourg Meeting 4 July 2012 Novak, J. et al.., unpubl.)

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

Processed Materials / Final Products

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

‚Lab-on-a-Chip‘

Pal et al., Lab Chip 5:1024–1032, 2005

1.6 cm

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. lagopus

AU

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

3 26, 7

Chlorogenic acid

Species Origin IPK numberP. afra San Marino di Urbino, Italy IPK-PLA 16P. albicans Gene Bank of IRA, Medenine, Tunisia IPK-PLA 19P. arenaria Institute for breeding research Braunschweig, Germany IPK-PLA 40P. cornuti Botanical garden University Bologna, Italy IPK-PLA 52P. coronopus "Rühlemann" seed producing industry, Germany Rühl-Pla 18xP. coronopus Madeira, Portugal IPK-PLA 55P. coronopus seed producing industry, north Italy IPK-PLA 18P. lagopus Botanical garden University Maputo, Mozambique IPK-PLA 50P. lagopus France IPK-PLA 42P. lanceolata wild collected Vienna, AustriaP. lanceolata Elverdamskroen, Denmark IPK-PLA 51P. major wild collected Vienna, AustriaP. maritima Botanical garden Univ. Halle/Saale, Germany IPK-PLA 27P. media wild collected Vienna, AustriaP. sempervirens Creissels, Aveyron, France IPK-PLA 64P. subulata Pont-de-Montvert, Lozere, France IPK-PLA 65

DPPH- assayHPLC-DAD

Fast- GC-FIDPlantago sp.:Diversity of iridoids profile between species, plant partsDiversity of the polyphenols profile and the antioxidant activity between species

VUW-Bot: Sponza, S. et al., unpubl.

Analytical profiles for caffeine and catechin in Camellia sinensis products 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 tea infusion

Figure 2 – Octopamine and synephrine in PFS containing 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

Harpagophytum procumbensMethod development - Waters Acquity UPLC and Synapt HDMS

Standards profiles

MeOH/ACN based extraction

Harpagophytum procumbensPhytochemical profiling of plant raw material (provided by PhytoLab)

Plan

t sou

rce

com

paris

on

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

Marker compounds identified by accurate mass detection

Anthrones in Frangula bark(Partner: PL FIN)

In some EU countries Frangula bark products are very popular in the weight loss and GI health segment

Elevated anthrone levels in improperly stored material may lead to side effects

No selective quantitative method available today First steps have been carried out to develop a selective

method Next steps: structural identification of relevant peaks,

establishment of specific reference substances, method validation and screening of raw materials and products

PLANTLIBRA Strasbourg Meeting 4 July 2012

Anthrones in Frangula bark

anthraquinones can be distinguished by the UV spectrum, e.g. emodin:

max 359 nm missing

max >400 nm typical

Peaks 3 and 5: glycosides of frangula emodine anthrones

…and finally, there is a 4th error:

Knowledge on main compounds and their activity is sufficient… (?!)

…but what‘s about metabolom matrix effects (‚phytocomplex‘), type of preparation (powdered entire plant/part, solvent, extraction technology, a.s.o.)?

Systems Biological Approach requested!


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 and between products!)

- Contaminations, Residues, Toxic Compounds- Pool of Reference Laboratories (‚Network‘)- Quality Guidelines for PFS to be elaborated in co-

operation with the European Botanical Forum- …‘work in progress‘PLANTLIBRA WP7 Roma Sept. 2012

Many Thanks to the 12 Participantsof WP 7

1 UMIL

3 CSIR

9 ICC

10 IFR

12 ISS

13 PLFIN

15 STIC

17 UBA

20 USP

21 UTBV

22 VUW-Bot

23 WUR

Università degli Studi di Milano Italy

Council for Scientific and Industrial Research S.A.

International Association for Cereal Science and

Technology Austria

Institute of Food Research United Kingdom

Istituto Superiore di Sanità Italy

Phytolab GmbH & Co. KG Germany

Swiss Toxicological Information Center Switzerland

Hospital de Clinicas "José de San Martín",

University of Buenos Aires Argentina

Universidade de São Paulo Brazil

Universitatea Transilvania DIN Brasov Romania

Vetmed University Vienna Austria (WP7 Leader)

Wageningen University The Netherlands


…thank you for your interest…!

wp7: characterization of botanicals...b) with possible risks environmental contaminants and residues...

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