WP7: Characterization of Botanicals...b) with possible risks Environmental contaminants and residues...
Transcript of 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
PLANTLIBRA WP7 Roma Sept. 2012
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
PLANTLIBRA WP7 Roma Sept. 2012
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?
PLANTLIBRA WP7 Roma Sept. 2012
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
PLANTLIBRA WP7 Roma Sept. 2012
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
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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
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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
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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
PLANTLIBRA WP7 Roma Sept. 2012
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
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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
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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!
PLANTLIBRA WP7 Roma Sept. 2012
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
PLANTLIBRA WP7 Roma Sept. 2012
…thank you for your interest…!