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APIS OF BOTANICAL ORIGIN QUALITY AND ADULTERATION
ROBERTO PACE – ERNESTO MARCO MARTINELLI (INDENA SPA)
Herbal drug preparations: the quality scenario The key quality factors: starting vegetable material, manufacturing process and final specifications Accidental and deliberate adulteration: champagne taste for beer price? From genomics to metabomolomics: the emerging quality weapons
OUTLINE
Plant are incredible variable sources of substances that are
generally difficult to synthetize due to their chemical
complexity can be classified as
• Primary Metabolites
and
• Secondary metabolites
PRIMARY VS SECONDARY PLANT METABOLISM
Primary metabolism in a plant comprises all metabolic pathways that are essential to the
plant's survival: are directly involved in the growth
and development of a plant
Secondary metabolites are compounds produced in other not essential metabolic pathways ,
often for defence purposes
PRIMARY VS SECONDARY PLANT METABOLISM
PRYMARY METABOLITES (EXAMPLES)
SECONDARY METABOLITES (EXAMPLES)
• Generally Complex chemical structure • present a number of pharmacophors and an high level of
stereochemistry responsible of a specific activity • are natural metabolites, “biologically active” and substrates of
one or more of the natural transport systems • Many substances belonging to the same class (flavonoids,
flavons, sesquiterpenes etc.) • Are present in a characteristic pattern of constituents in the
same plant/part of the plant
THE PLANTS AS INTERESTING SOURCES OF ACTIVE PHARMACEUTICAL INGREDIENTS
SYNERGIC EFFECTS
Biological activity of these preparations might result from synergy of active compounds rather than from a single
chemical entity
e.g. The antimicrobial activity of alkaloid berberine is 100 times enhanced by 5’-methoxyhydnocarpin (5’MHC)
e.g. activity of Ginkgo biloba dry extracts for their anticlastogenic, antioxidant, vasoregulatory, cognition enhancing, stress alleviating
and gene-regulatory effects (Curtis et al., 1999)
PLANTS AS HISTORICAL SOURCE OF ACTIVE SUBSTANCE
ARE USED IN FORM OF
HERBS
EXTRACTS
ISOLATED COMPOUND/S
SEMISYNTHETIC COMPOUNDS
QUALITY STANDARDS Quality of the extracts is extremely important and this is the reason
why guidelines are mandatory to comply with:
Good Agriculture and Collection practices (GACP)
Good Manufacturing Practices (cGMP)
Pharmacopoeial Standard
Herbal Products Guidelines (EMA)
PART II
EXTRACTS
APIs
GMP
HERBAL DRUG PREPARATIONS
are active ingredients in their entirity
1
2 3
4 5
6
7 8 9
10
11 12
15
13 1
4 1
6
HERBAL DRUG PREPARATIONS (API)
Generally it is not possible to identify a substance responsible for the therapeutical/physiological effect:
all substances generally contribute in a synergic way to parmacological effect and / or mitigating toxic
effects Isolated individual constituents generally behave in different way
HERBAL DRUG PREPARATIONS
1
2 3
4 5
6
7 8 9
10
11 12
15 13
14
16
O
OCH2R
O
HO
OHOOH
HO
HO R
CH3
OHOOH
OHO
OH O
OH
OH
OH
HO
OH O
O
OHO
OH O
OR
OH
OH
HO COOH
O
OH
HO
O
OH
OH
HERBAL DRUG PREPARATIONS CLASSIFICATION
STANDARDIZED EXTRACTS
all actives are known
QUANTIFIED EXTRACTS
not all actives are known
OTHER EXTRACTS
actives are not known
MAY INCLUDE ‘PURIFICATION’ (*)
(*) During Production of extracts, purification procedures may be applied to increase
these proportions with respect of the expected values; such extracts are also referred
to as ‘refined‘
consistent quality of the herbal extracts depends on
• quality of starting vegetable material
• by the manufacturing process
• in-process controls
• final specifications
HERBAL DRUG PREPARATIONS
NATURAL VARIABILITY OF METABOLITES
enviroment
phenotypes (traits)
biochemical circuit
proteins
translation
transcription
(storage) DNA
gene expression
proteomics
metabolomics
STARTING HERBAL DRUG
origin: europe cultivation: wild
definition a qualitative statement of the botanical source, plant part used and its state
(e.g. whole, reduced, powdered, fresh, dry). it is also important to know the geographical source(s) and the conditions under which
the herbal substance is obtained
GENUS SPECIES AUTHOR PART OF THE PLANT
Vaccinium myrtillus L. FRESH FRUITS (FROZEN)
macroscopic id
microscopic id
vaccinium myrtillus l. – bilberry
STARTING HERBAL DRUG IDENTIFICATION
HPLC
TLC
vaccinium myrtillus l. - bilberry
STARTING HERBAL DRUG IDENTIFICATION
STARTING HERBAL DRUG IDENTIFICATION
genomic approach, a new frontier
DNA-based authentication of medicinal plants
many species investigated: echinacea, panax, equisetum, fritillaria, salvia, astragalus, actaea, etc
DNA-based authentication is a quickly, efficient, reliable and very economic (PCR)
contaminants are the impurities of botanicals
pesticides heavy metals
micotoxins microbial
radioactivity others (e.g. dioxines, PHAs, irradiation)
STARTING HERBAL DRUG POTENTIAL CONTAMINANTS
consistent quality of the herbal extracts depends on
• quality of starting vegetable material
• by the manufacturing process
• in-process controls
• final specifications
HERBAL DRUG PREPARATIONS
cutting, extracting
herbal starting material
primary extract
concentrated soft extract
dry extract
standardized, quantified, other dry extract, refined/purified
concentration purification
drying, milling
standardization quantification
TYPICAL MANUFACTURING SCHEME OF DRY EXTRACTS
extraction solvent
• extraction solvents • extraction conditions (temperature)
C C
C
C
C
C
C
C C
C
C
C C
C
C C
C
C
C C
C
C
C
C
C
C
C C
C
C
C C
SOLVENT 1
SOLVENT 2
different extraction solvent = different products
TYPICAL MANUFACTURING SCHEME OF DRY EXTRACTS
consistent quality of the herbal extracts depends on
• quality of starting vegetable material
• by the manufacturing process
• in-process controls
• final specifications
HERBAL DRUG PREPARATIONS
typical manufacturing process (ph. eur.)
QC testing
excipient (up to x % w/w)
extraction (2) extraction (n)
purification steps (2) purification steps (n)
extraction (1)
purification steps (1)
drying (1) (intermediate 1)
drying (2) (intermediate 2)
drying (n) (intermediate n)
mixing
(standardisation)
QC testing final standardised extract
the process may include the addition of excipient
STANDARDISED EXTRACTS
typical manufacturing process (ph. eur.)
QC testing
excipient (up to x % w/w)
extraction (2) extraction (n)
purification steps (2) purification steps (n)
extraction (1)
purification steps (1)
drying (1) (intermediate 1)
drying (2) (intermediate 2)
drying (n) (intermediate n)
mixing
(standardisation)
QC testing final quantified extract
the process can not include the addition of excipient except for technological reasons and in constant amount
QUANTIFIED EXTRACTS
mixing phases - example
starting materials (leaves) with high markers variability
mix
ing
of
vege
tab
le
mat
eria
l bat
che
s
FG TL
0.5%
2.0%
0.1%
0.5%
FG
FG
TL
TL TL FG
0.9%
1.3%
0.20%
0.35%
22%
27%
5%
7%
24% 25%
5.5% 6.5%
extraction/ purification
final mixing
ginkgo biloba purified dry extract, quantified
FG = flavonglucosides TL = terpenlattoni
IN-PROCESS CONTROLS
consistent quality of the herbal extracts depends on
• quality of starting vegetable material
• by the manufacturing process
• in-process controls
• final specifications
HERBAL DRUG PREPARATIONS
QUALITY CONTROLS
• identification (e.g. HPLC, TLC/HPTLC profiles) • chemical-physical assays • assay/s (e.g. HPLC) contaminants (impurities for herbals): • heavy metals • pesticides • microbiological control • micotoxins • unwanted toxic substances, residual solvents
HERBAL DRUG PREPARATIONS - TYPICAL QC
stability of the constituents with known therapeutic activity will not suffice:
other substances present in the herbal preparations, their proportional content remains comparable to the initial fingerprint (e.g., by means of
appropriate fingerprint chromatograms)
HERBAL DRUG PREPARATION - STABILITY
HERBAL DRUG PREPARATION - STABILITY
the variation in content during the proposed shelf-life should not exceed ± 5% of the
declared assay value (standardized extracts)
a variation in marker content during the proposed shelf-life of ±10% of the initial assay value can be accepted,
if justified by the applicant, for quantified extract
adulteration of botanical ingredients can be accidental or deliberate
accidental adulteration occurs as a result of poor quality-control
procedures, as well as the intentional adulteration of plant-based products for financial gain
the us food and drug administration defines
“economically motivated adulteration” (EMA)
counterfeiting has been the subject of a recent european directive (2011/62) both for GMP and falsification reasons
new proposed USP general chapter 2251 adulteration of dietary
supplements with drugs and drug analogs
ADULTERATION
CHAMPAGNE TASTE FOR BEER PRICE ?
I BARI (CARAVAGGIO)
VACCINIUM MYRTILLUS DRY EXTRACT
authentic bilberry extracts are expensive:
• are difficult to harvest
• can not be cultivated
• can not be processed unfrozen, since tissue damage triggers the
deglycosidation of anthocyanosides
• extract is obtained in low yield (ca 1% from the plant material)
• are within the most expensive botanical ingredients
VACCINIUM MYRTILLUS DRY EXTRACT
VACCINIUM MYRTILLUS DRY EXTRACT
bilberries are unique berries - HPLC method is suitable
in determining which fruit has been used in the
preparation of a particular extract Vaccinium corymbosum, Highbush blueberry
Vaccinium macrocarpon, American cranberry Vaccinium angustifolium, Lowbush blueberry
Vaccinium myrtillus
VACCINIUM MYRTILLUS DRY EXTRACT
VACCINIUM MYRTILLUS
additionally, deliberate adulteration
occurring in commercial bilberry
extract can be detected with this
method of analysis
the HPLC trace clearly show that
amaranth elutes in the first part of the
chromatogram (retention time about
4.3 minutes) in a region free of
anthocyanins showing that the
analytical methodology is adequate to
accurately determine this adulterant
AMARANTH
VACCINIUM MYRTILLUS DRY EXTRACT
presence of sambucus nigra
(elderberry) typical
anthocyanins was detected:
1:1 ratio between the two
kind of berries
Bilberry extract
Coelution of Cyanidin-3-O-sambubioside and cyanidin-3-O-glucoside
Cyanidin rutinoside
Elderberry markers
Cyanidin-5-O-glucoside
3-O-sambubioside
AU
-0.010
0.000
0.010
0.020
0.030
0.040
0.050
0.060
0.070
0.080
0.090
0.100
De
lfin
idin
a 3
-O-g
al
- 1
4.7
65
De
lfin
idin
a 3
-O-g
lu -
17
.77
4
Cia
nid
ina
3-O
-ga
l -
20
.08
3
De
lfin
idin
a 3
-O-a
ra -
20
.69
1
Cia
nid
ina
3-O
-glu
- 2
3.7
89
Pe
tun
idin
a 3
-O-g
al
- 2
5.5
92
C
ian
idin
a 3
-O-a
ra -
26
.14
1
Pe
tun
idin
a 3
-O-g
lu -
29
.01
9
De
lfin
idin
a -
29
.92
8
Pe
on
idin
a 3
-O-g
al
- 3
1.2
38
P
etu
nid
ina
3-O
-ara
- 3
1.9
06
Pe
on
idin
a 3
-O-g
lu -
35
.23
8
Ma
lvid
ina
3-O
-ga
l -
35
.89
4
Pe
on
idin
a 3
-O-a
ra -
37
.63
7
Ma
lvid
ina
3-O
-glu
- 3
9.3
36
Cia
nid
ina
- 4
0.8
54
Ma
lvid
ina
3-O
-ara
- 4
2.3
50
Pe
tun
idin
a -
45
.27
3
Pe
on
idin
a -
48
.85
7
Ma
lvid
ina
- 4
9.3
36
AU
-0.010
0.000
0.010
0.020
0.030
0.040
0.050
0.060
0.070
0.080
0.090
0.100
Minutes
0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00
De
lfin
idin
a 3
-O-g
al
- 1
4.6
67
Cia
nid
ina
5g
lu 3
-Sa
mb
ub
- 1
6.7
60
D
elf
inid
ina
3-O
-glu
- 1
7.6
54
Cia
nid
ina
3-O
-ga
l -
19
.94
3
De
lfin
idin
a 3
-O-a
ra -
20
.54
2
Cia
nid
ina
3-O
-glu
- 2
3.7
47
Pe
tun
idin
a 3
-O-g
al
- 2
5.3
88
C
ian
idin
a 3
-O-a
ra -
25
.94
0
Cia
nid
ina
ru
tin
osid
e -
27
.72
7
Pe
tun
idin
a 3
-O-g
lu -
28
.79
2
De
lfin
idin
a -
29
.69
9
Pe
on
idin
a 3
-O-g
al
- 3
1.0
00
P
etu
nid
ina
3-O
-ara
- 3
1.6
62
Pe
on
idin
a 3
-O-g
lu -
34
.97
1
Ma
lvid
ina
3-O
-ga
l -
35
.61
1
Pe
on
idin
a 3
-O-a
ra -
37
.35
0
Ma
lvid
ina
3-O
-glu
- 3
9.0
33
Cia
nid
ina
- 4
0.5
37
Ma
lvid
ina
3-O
-ara
- 4
2.0
20
Pe
tun
idin
a -
45
.10
9
Pe
on
idin
a -
48
.59
3
Ma
lvid
ina
- 4
9.2
25
VACCINIUM MYRTILLUS DRY EXTRACT
Vaccinium myrtillus
Bilberry softgel capsules
• the content of anthocyanins is about ten times less than the expected
• the anthocyanins pattern is not coherent with vaccinium myrtillus fruit
• similarly anomalous pattern has been previously found in some extracts manufactured in china
• analytical investigation outlines that black soybean hull extract has similar composition
VACCINIUM MYRTILLUS DRY EXTRACT
Vaccinium myrtillus
Bilberry capsules
Nutritional/food supplements
Label’s statement: “each 380
mg capsule contains 160 mg of
bilberry extract (from fruit)”
The content of Bilberry extract
in the product is almost
neglectable, charcoal was
detected
VACCINIUM MYRTILLUS DRY EXTRACT
24 commercially available vaccinium myrtillus brands were compared for for the anthocyanins and antocyanidins content
marketplace
europe, united states, japan
25 % of the analyzed products exhibited an HPLC profile different to typical HPLC profile of bilberry extract
VACCINIUM MYRTILLUS DRY EXTRACT
GINKGO BILOBA
common adulteration with flavoneglycosides from alternative sources:
e.g. quercetin and rutin
GINKGO BILOBA
Ginkgo Flavone Glycoside HPLC-Fingerprint
ruti
n
qu
erce
tin
kaem
pfe
rol
assay on ginkgo flavone glycosides
• assay on more than 20 individual ginkgo flavone glycosides is very complex and expensive
• is simplified by acid hydrolysis • the analysis is reduced to 3 aglycons
GINKGO BILOBA
AU
0 . 0 0
0 . 1 0
0 . 2 0
0 . 3 0
0 . 4 0
0 . 5 0
Qu
er
ce
ti
n -
1
3.5
46
Ka
em
pf
er
ol
-
1
9.2
40
Is
or
ha
mn
et
in
-
2
0.7
85
AU
0 . 0 0
0 . 1 0
0 . 2 0
0 . 3 0
0 . 4 0
0 . 5 0
Qu
er
ce
ti
n -
1
3.5
48
Ka
em
pf
er
ol
-
1
9.2
47
Is
or
ha
mn
et
in
-
2
0.8
02
AU
0 . 0 0
0 . 1 0
0 . 2 0
0 . 3 0
0 . 4 0
0 . 5 0
M i n u t e s
0 . 0 0 5 . 0 0 1 0 . 0 0 1 5 . 0 0 2 0 . 0 0 2 5 . 0 0 3 0 . 0 0 3 5 . 0 0
Qu
er
ce
ti
n -
1
3.5
66
Ka
em
pf
er
ol
-
1
9.2
67
Is
or
ha
mn
et
in
-
2
0.8
24
GINKGO BILOBA
ruti
n
qu
erce
tin
kae
mp
fero
l
GINKGO BILOBA
ruti
n
qu
erce
tin
kaem
pfe
rol
GINKGO BILOBA
the advantages of adulteration • 2 % quercetin => about 2.5 % of total gfg • 2 % kaemperol => about 5 % of total gfg • 2 % rutin => about 2,5 % of total gfg raw material used for spiking • rutin from sophora japonica or fagopyrum esculentum • kaemperol from beans
ECHINACEA
the genus echinacea
echinacea angustifolia
echinacea purpurea
echinacea paradoxa var. par.
echinacea sanguinea
echinacea pallida
echinacea simulata
echinacea atrorubens
ECHINACEA
echinacea is nice, but difficult to work with
diversity, instability and difficulty of purification
of its constituents
intraspecific taxonomic uncertainties
sophistication of the plant material and its
extracts
ECHINACEA
E. PALLIDA ECHINACOSIDE
PENTADECA-8Z-EN-2-ONE
MINUTES 10,00 20,00 30,00 40,00 50,00 60,00
ECHINACOSIDE ALKYLAMIDE 8
E. ANGUSTIFOLIA
HPLC fingerprint of echinacea pallida and angustifolia
ECHINACEA
HPLC fingerprint of echinacea species
ECHINACEA
E. angustifolia
E. pallida
E. atrorubvens
E. simulata
E. sanguinea
E.purpurea
E. paradoxa
a field research: developing an id method - DNA
echinacea genus investigation – a case study
ECHINACEA
Species A
Species B
ECHINACEA
EQUISETUM ARVENSE
the plant contains several substances which can be used medicinally:
healing, antihemorrhagic, diuretic, astrigent, diuretic
it is rich in the minerals silicon (10%), potassium, calcium, ascorbic acid, equisetonin, flavonoids, alkaloids
adulteration with other species; equisetum palustre
(toxic alkaloids – palustrine)
genetic ID allows to identify and quantify the possible contamination
EQUISETUM ARVENSE
horsetail: developing an id and quantitation method – DNA barcoding
EQUISETUM ARVENSE
WILLOW (Salix alba L.)
the bark is rich in phenolic compounds as proanthocyanidins,
flavonoids, and glycosides of phenols and of phenolic acids,
salicin, salicortin, tremulacin, populin
the anti-inflammatory properties are linked to salicylic acid,
which take origin from the oxidation of salicyl alcohol,
formed upon intestinal hydrolisis of salicin, itself either
native or produced by slow degradation of salicortin
ID by TLC and HPLC, typical sophistication of willow extract:
addition of synthetic salicin
WILLOW (Salix alba L.)
HPLC procedure foresees an hydrolysis of the extract
with sodium hydroxide in order to convert the
salicin-derivatives into salicin
HPLC profiles before and after the hydrolysis
procedure are different
WILLOW (Salix alba L.)
typical HPLC profiles of willow extract
before and after the hydrolysis
willow extract after hydrolisys willow extract before hydrolisys
WILLOW (Salix alba L.)
hplc profiles of willow extracts available on the market
willow extract after hydrolisys willow extract before hydrolisys
WILLOW (Salix alba L.)
ACTEA RACEMOSA (Cimicifuga)
black cohosh (actaea racemosa, formerly cimicifuga racemosa) is a herbal medicine used mainly to alleviate menopausal symptoms.
in recent years, several international regulatory agencies have monitored a possible relationship between black cohosh
and liver toxicity
investigation outline that products marketed in the united states as black cohosh were consistent with the presence of
other related herbal species
products not containing authentic black cohosh may be associated with liver adverse reactions
ACTEA RACEMOSA (Cimicifuga)
black cohosh (actaea racemosa syn. cimicifuga racemosa) root
the economic adulteration of black cohosh root and rhizome with other
species is well established
known adulterant are chinese cimicifuga root/rhizome,
also known as sheng ma
this material commonly consists of cimicifuga foetida, c. dahurica , c.
heracleifolia
ACTEA RACEMOSA (Cimicifuga)
adulteration: accidental or deliberate?
cimicifuga foetida cimicifuga racemosa
ACTEA RACEMOSA (Cimicifuga)
HPLC-MS techniques allows to detect possible contamination of
cimicifuga foetida in cimicifuga racemosa
the chromone cimifucin is a typical constituent of c. foetida
but absent in c. racemosa
the presence of cimicifugin in c. racemosa extract indicates a
contamination by c. foetida
ACTEA RACEMOSA (Cimicifuga)
Minutes
10.00 20.00 30.00 40.00
Cimifugin 17.6 min
Cimifugin
glucoside 9.8 min
cimicifuga foetida
HPLC-MS profile
ACTEA RACEMOSA (Cimicifuga)
1: isoferulic acid, 2: norcimifugin, 3: actein, 4: 23-epi-26 deoxyactein, 5: cimifugin, 6, 7: cimicifuga racemosa, 8: c. foetida, 9: c. heracleifolia, 10: c. dahurica, 11: c. americana
EDQM
black cohosh
monograph
n: 2069
TLC ID
ACTEA RACEMOSA (Cimicifuga)
PRUNUS AFRICANA
prunus africana (pygeum) is found in mountain regions of central, southern
africa and madagascar and may grow to a height of over 40 meters
placed in the red list, under the protection of cites since 1993
espensive raw material
pygeum native extract has a waxy consistency
sterol content in the range of 14-16% by spectrophotometry
cheaper samples on the market are in powder form
PRUNUS AFRICANA
Typical Pygeum extract GC
profile
Test solution
Reference solution
with GLC ID adulterated
product lacks of the typical
constituents :
docosanol, n-docosyl ferulate,
ursolic and behenic acids
adulterated product
chromatographic pattern
is coherent with the profile
of synthetic b-sitosterol
available on the market
(rich in stigmasterol)
PRUNUS AFRICANA
batch to batch consistency can not be represented using monovariate statistics (one variable each time) being
extracts complex multicomponent mixtures
European Pharmacopoeia has introduced the use of chemometrics as powerful tool for multivariate evaluation of the analytical data
PRE-PROCESSING
BOTANICAL EXTRACTS BATCH TO BATCH CONSISTENCY
the current best and powerful method to evaluate the
batch to batch consistency is the principal component analysis (PCA)
using different available analytical variables
the variables could be chromatographic data, chromatograms
or spectroscopic data like 1H-NMR data (takes into considerations
all extract constituents)
BOTANICAL EXTRACTS BATCH TO BATCH CONSISTENCY
PCA was invented in 1901 by karl pearson
it was later developed (and named) by
harold hotelling in the 1930s
BOTANICAL EXTRACTS BATCH TO BATCH CONSISTENCY
BOTANICAL EXTRACTS BATCH TO BATCH CONSISTENCY
principal component analysis (PCA) oldest and most widely used non-supervised multivariate statistical technique
reduce the dimension of the original data set
very useful for multicomponent mixtures and particularly for the evaluation of the vegetable extracts where the natural variability plays an
important role in the product quality
this technique can be applied on all the analytical variables involved with the different instrumental techniques such as
NIR, HPLC, GLC, NMR, UV or images (e.g. TLC)
PRINCIPAL COMPONENT ANALYSIS (PCA)
ANALYTICAL DATA HPLC, NMR, IR, etc
classifiability
PCA - PANAX GINSENG GENUS
HPLC IDENTIFICATION AND
FINGERPRINTING
PLANT MATERIAL
MULTIVARIATE
ANALYSIS
PATTERN
RECOGNITION
METHOD
CLASSIFIABILITY
Report Name: Area CdQ
12:33:32 Europe/RomeReported by User: Emanuele Annoni (w aters) Date Printed: 01/08/2014
Panax Ginseng E.S. Rad. Second.Sample Name: CoA: 57761
Batch: 31425/M1Analytical Method: PS9833
Identif ication: Sol. esame 1 CDQ_HPLC06_CONFASystem Name:
Column: 275/C18/QCSample Set Name: GInseng_ELSD_Lug2014
Balance: QC 27Processing Method: Ginseng_ELSD_2695_2420
Injection Volume: 15.00 ulProc. Chnl. Descr.: ELSD Signal
CDQ_SE\2014\HPLC\Ginseng_ELSDProject Name:
Date Acquired: 13/07/2014 04:13:17 CEST Injection #:Vial: 4 2
Date Processed: 14/07/2014 08:45:59 CEST Dilution:Sample Weight: 20.00 180.4300
Chrom atogram
Rg
1 -
21
.03
1R
e -
21
.36
5
Ma
lon
yl-R
b1
- 2
9.9
30
Ma
lon
yl R
b2
or
Rb
3 o
r R
c -
31
.21
9M
alo
nyl
-Ra
- 3
2.0
07
Ma
lon
yl-R
b2
or
Rb
3 o
r R
c -
32
.91
7
Rf
- 3
6.3
49
Ma
lon
yl-R
d -
37
.23
2
Ma
lon
yl-R
d is
om
er
- 4
1.8
56
Rb
1 -
43
.26
0R
g2
- 4
4.1
52
Rc
- 4
5.9
58
Ra
- 4
6.6
24
Rb
2 -
49
.73
5R
b3
- 5
0.8
10
Rd
- 5
6.3
44
LS
U
0.00
200.00
400.00
600.00
800.00
1000.00
1200.00
1400.00
1600.00
1800.00
2000.00
Minutes
0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00
1
2
3
4
5
6
7
8
9
10
11
Name RT (min.) RRT Area (uV*sec)
Rg1
Re
Malonyl-Rb1
Malonyl Rb2 or Rb3 or Rc
Malonyl-Ra
Malonyl-Rb2 or Rb3 or Rc
Rf
Malonyl-Rd
Malonyl-Rd isomer
Rb1
Rg2
21.031
21.365
29.930
31.219
32.007
32.917
36.349
37.232
41.856
43.260
44.152
0.486
0.494
0.692
0.722
0.740
0.761
0.840
0.861
0.968
1.021
1776387.48
19686984.63
11143960.13
4329815.62
656948.03
3889901.53
1956269.74
426790.08
387008.62
35711020.19
56505.38
12
13
14
15
16
Name RT (min.) RRT Area (uV*sec)
Rc
Ra
Rb2
Rb3
Rd
45.958
46.624
49.735
50.810
56.344
1.062
1.078
1.150
1.175
1.302
11018729.97
1223812.15
5154750.43
830323.94
7851005.51
Peak Table
PRINCIPAL COMPONENT ANALYSIS (PCA)
panax ginseng - HPLC fingerprinting
Report Name: Area CdQ
12:33:32 Europe/RomeReported by User: Emanuele Annoni (w aters) Date Printed: 01/08/2014
Panax Ginseng E.S. Rad. Second.Sample Name: CoA: 57761
Batch: 31425/M1Analytical Method: PS9833
Identif ication: Sol. esame 1 CDQ_HPLC06_CONFASystem Name:
Column: 275/C18/QCSample Set Name: GInseng_ELSD_Lug2014
Balance: QC 27Processing Method: Ginseng_ELSD_2695_2420
Injection Volume: 15.00 ulProc. Chnl. Descr.: ELSD Signal
CDQ_SE\2014\HPLC\Ginseng_ELSDProject Name:
Date Acquired: 13/07/2014 04:13:17 CEST Injection #:Vial: 4 2
Date Processed: 14/07/2014 08:45:59 CEST Dilution:Sample Weight: 20.00 180.4300
Chrom atogram
Rg
1 -
21
.03
1R
e -
21
.36
5
Ma
lon
yl-R
b1
- 2
9.9
30
Ma
lon
yl R
b2
or
Rb
3 o
r R
c -
31
.21
9M
alo
nyl
-Ra
- 3
2.0
07
Ma
lon
yl-R
b2
or
Rb
3 o
r R
c -
32
.91
7
Rf
- 3
6.3
49
Ma
lon
yl-R
d -
37
.23
2
Ma
lon
yl-R
d is
om
er
- 4
1.8
56
Rb
1 -
43
.26
0R
g2
- 4
4.1
52
Rc -
45
.95
8R
a -
46
.62
4
Rb
2 -
49
.73
5R
b3
- 5
0.8
10
Rd
- 5
6.3
44
LS
U
0.00
200.00
400.00
600.00
800.00
1000.00
1200.00
1400.00
1600.00
1800.00
2000.00
Minutes
0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00
1
2
3
4
5
6
7
8
9
10
11
Name RT (min.) RRT Area (uV*sec)
Rg1
Re
Malonyl-Rb1
Malonyl Rb2 or Rb3 or Rc
Malonyl-Ra
Malonyl-Rb2 or Rb3 or Rc
Rf
Malonyl-Rd
Malonyl-Rd isomer
Rb1
Rg2
21.031
21.365
29.930
31.219
32.007
32.917
36.349
37.232
41.856
43.260
44.152
0.486
0.494
0.692
0.722
0.740
0.761
0.840
0.861
0.968
1.021
1776387.48
19686984.63
11143960.13
4329815.62
656948.03
3889901.53
1956269.74
426790.08
387008.62
35711020.19
56505.38
12
13
14
15
16
Name RT (min.) RRT Area (uV*sec)
Rc
Ra
Rb2
Rb3
Rd
45.958
46.624
49.735
50.810
56.344
1.062
1.078
1.150
1.175
1.302
11018729.97
1223812.15
5154750.43
830323.94
7851005.51
Peak Table
RG1
RE
RB1
RG2
RC RB2
RB3
RD
typical hplc profile of panax ginseng roots
typical hplc profile of ginseng leaves
PANAX GINSENG
PRINCIPAL COMPONENT ANALYSIS (PCA)
panax ginseng parts of the plant HPLC fingerprinting PART OF THE PLANT RG1 RE …. …. …. RB2 RB3 RD
Q.G. green fruits 0,000 0,510 …. …. …. 0,290 0,890 0,070 Q.G. leaves 0,460 2,080 …. …. …. 0,610 1,820 0,910
Q.G. main roots 0,100 0,880 …. …. …. 0,000 0,030 0,260 Q.G. neck 0,190 1,200 …. …. …. 0,040 0,120 1,010
Q.G. red fruits 0,000 0,440 …. …. …. 0,090 0,320 0,000 Q.G. roots 0,260 1,770 …. …. …. 0,130 0,210 1,440 Q.G. stem 0,040 0,300 …. …. …. 0,030 0,100 0,100
P.G. flowers 0,650 4,270 …. …. …. 0,690 0,100 2,460 P.G. leaves 1,640 4,030 …. …. …. 0,930 0,130 2,150 P.G. leaves 1,250 2,720 …. …. …. 0,410 0,060 1,160 P.G. leaves 1,330 3,870 …. …. …. 0,510 0,070 1,250 P.G. leaves 1,760 3,440 …. …. …. 0,580 0,100 1,570 P.G. leaves 1,320 3,610 …. …. …. 0,530 0,080 1,440 P.G. leaves 1,950 3,500 …. …. …. 0,520 0,000 2,090 P.G. leaves 2,040 2,650 …. …. …. 0,350 0,000 1,470 P.G. leaves 1,550 2,570 …. …. …. 0,320 0,000 1,250 P.G. leaves 1,270 3,870 …. …. …. 0,490 0,000 1,670 P.G. leaves 1,740 4,200 …. …. …. 0,710 0,100 1,650 P.G. leaves 1,030 2,730 …. …. …. 0,420 0,000 1,680 P.G. leaves 1,440 3,260 …. …. …. 0,630 0,080 2,170
P.G. main roots 1,160 0,950 …. …. …. 0,680 0,090 0,320 P.G. main roots 1,320 0,790 …. …. …. 0,730 0,120 0,330 P.G. main roots 1,260 1,730 …. …. …. 1,930 0,330 1,250 P.G. main roots 1,350 0,850 …. …. …. 0,730 0,110 0,320
P.G. roots 1,160 2,430 …. …. …. 2,660 0,420 1,920 P.G. roots 1,230 2,140 …. …. …. 2,210 0,340 1,520 P.G. roots 1,090 2,570 …. …. …. 2,860 0,440 2,040 P.G. roots 1,110 2,790 …. …. …. 3,280 0,460 2,510 P.G. roots 1,150 2,310 …. …. …. 2,530 0,400 1,780 P.G. roots 1,210 2,500 …. …. …. 3,370 0,600 2,070 P.G. roots 1,200 2,350 …. …. …. 2,400 0,370 1,500 P.G. roots 1,250 2,440 …. …. …. 2,520 0,430 1,620 P.G. roots 1,100 2,920 …. …. …. 2,800 0,580 2,090 P.G. stem 0,120 0,250 …. …. …. 0,000 0,000 0,020 P.G. stem 0,150 0,260 …. …. …. 0,000 0,000 0,020 P.G. stem 0,120 0,290 …. …. …. 0,000 0,000 0,020 P.G. stem 0,160 0,290 …. …. …. 0,000 0,000 0,000 P.G. stem 0,130 0,220 …. …. …. 0,000 0,000 0,000 P.G. stem 0,130 0,240 …. …. …. 0,000 0,000 0,000
PRINCIPAL COMPONENT ANALYSIS (PCA)
PRINCIPAL COMPONENT ANALYSIS (PCA)
panax ginseng genus
PRINCIPAL COMPONENT ANALYSIS (PCA)
panax ginseng parts of the plant
PCA – PLANT MATERIAL ORIGIN
zingiber CO2 extracts
zingiber constituents
n=6: 6-gingerol n=8: 8-gingerol n=10: 10-gingerol
n=6: 6-shogaol n=8: 8-shogaol n=10: 10-shogaol
zingiber CO2 extracts - origin investigation
the HPLC analysis do not outline differences related to the country
of origin
PCA – PLANT MATERIAL ORIGIN
the PCA evaluation of the HPLC data disclose clusters
related to the country of origin: nigeria and china
PCA – PLANT MATERIAL ORIGIN
pc1 accounts for the origin
variable 6-gingerol:
high PC1 values =>nigeria
variable 10-gingerol:
low PC1 values => china
PCA – PLANT MATERIAL ORIGIN
PCA – PLANT MATERIAL ORIGIN
PC2 accounts for the quality
gingerols are the natural metabolites:
low PC2 values => high quality
shogaols originate from gingerols trasformation:
high PC2 values => lower quality
multivariate QC analysis of vaccinium myrtillus dry extracts
typical 1H-NMR spectra of vaccinium myrtillus dry extract
BATCH TO BATCH CONSISTENCY
polyphenols
(anthocyanins/anthocyanidins)
aligned and normalized NMR spectra
PC1 - 44.2 percent explained variance -400 -200 0 200 400 600 800P
C2 -
25
.3 p
erc
en
t expla
ined
vari
an
ce
-800
-600
-400
-200
0
200
400
600
VM-IND 1VM-IND 2
VM-IND 3
VM-IND 4
VM-IND 5VM-IND 6
VM-IND 7
VM-IND 8
VM-IND 9
VM-IND 10VM-IND 11
VM-IND 12
VM-IND 13
VM-IND 14
VM-IND 15
VM-IND 16VM-IND 17VM-IND 18
VM-IND 19
VM-IND 20
VM-IND 21
VM-IND 22
VM-IND 23
VM-IND 24VM-IND 25
VM-IND 26VM-IND 27VM-IND 28
BIL 1
BIL 2
BIL 3
BIL 4
BIL 5
BIL 6
BIL 7
BIL 8
BIL 9
BIL 10
BIL 11
BIL 12
95% conf ellipse95% conf ellipse
INDENA
OTHER ORIGINS
NMR SPECTRA - PCA EVALUATION
# 104
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2-0.1
-0.08
-0.06
-0.04
-0.02
0
0.02
0.04
0.06
0.08
LOADING 1
loadings of pc1
other origins
maltodextrins/sugars
sugars
NMR SPECTRA - PCA EVALUATION
serenoa repens smal
plant material
NMR identification and fingerprinting
multivariate analysis
pattern recognition
method classifiability
PCA – SERENOA REPENS
PRINCIPAL COMPONENT ANALYSIS (PCA)
serenoa repens alcoholic extract
batch to batch consistency NMR fingerprinting
NMR FINGERPRINTING – PCA EVALUATION
batch 1 batch 2 ………. ………. batch 26
freq. 1 190858,36 184027,63 …….. …….. 130894,31
freq. 2 123306,38 173300,17 …….. …….. 178673,50
…….. …….. …….. …….. …….. ……..
…….. …….. …….. …….. …….. ……..
…….. …….. …….. …….. …….. ……..
…….. …….. …….. …….. …….. ……..
…….. …….. …….. …….. …….. ……..
…….. …….. …….. …….. …….. ……..
…….. …….. …….. …….. …….. ……..
…….. …….. …….. …….. …….. ……..
…….. …….. …….. …….. …….. ……..
…….. …….. …….. …….. …….. ……..
…….. …….. …….. …….. …….. ……..
…….. …….. …….. …….. …….. ……..
freq. 33000 173500,66 …….. …….. …….. 139527,17
ABOUT 900.000 VALUES
NMR FINGERPRINTING – PCA EVALUATION
NMR FINGERPRINTING – PCA EVALUATION
MIXTURES
NMR FINGERPRINTING – PCA EVALUATION
SERENOA REPENS EXTRACTS
ALCOHOLIC, CARBON DIOXIDE & HEXANE EXTRACTS
PHYTOCHEMICAL EQUIVALENCE
PC1
PC
2
GREEN: CARBON DIOXIDE EXT. BLU: HEXANE EXT. RED: ALCOHOLIC EXT.
2 CH=CH UNSATURATED FATTY ACIDS 6 CH2OCOR ACYLGLYCEROLS 13 CH=CHCH2CH=CH LINOLEYL AND LINOLENYL 15 CH2COOH ACYL CHAINS 16 CH2CH=CH UNSATURATED FATTY ACIDS 18 CH2CH2COOH ACYL CHAINS 20 (CH2)N ACYL CHAINS 22 CH2CH2CH2CH3 ACIDS EXCEPT LINOLENYL
PC2
PC1
2
15
16
13
6
18
20
22
SERENOA REPENS EXTRACTS
ALCOHOLIC, CARBON DIOXIDE & HEXANE EXTRACTS
PHYTOCHEMICAL EQUIVALENCE
consistently similar composition of the saw palmetto extracts obtained with carbon dioxide and hexane
alcoholic extracts show minor differences ascribable to a major concentration of monounsaturated fatty acids and
diglycerides
the magnitude of these differences resulted lower to the natural variability
of the saw palmetto berries composition
SERENOA REPENS EXTRACTS
ALCOHOLIC, CARBON DIOXIDE & HEXANE EXTRACTS
PHYTOCHEMICAL EQUIVALENCE
BOTANICAL EXTRACTS QUALITY IS DESIGNED/BUILT
efficacy and safety depend on reproducible quality linked to starting plant material, manufacturing process,
in-process control, GMP/HACCP
CONSISTENT
QUALITY
SAFETY food or
Medicinal Products
regulations
EFFICACY physiological
or therapeutical
effects
QUALITY MEANS DOING IT RIGHT WHEN NO ONE IS LOOKING
HENRY FORD