apis of botanical origin quality and adulteration · pdf filecontaminants are the impurities...

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

http://www.edqm.eu/en/European-Pharmacopoeia-1401.html

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


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


NATURAL VARIABILITY OF METABOLITES

enviroment

phenotypes (traits)

biochemical circuit

proteins

translation

transcription

(storage) DNA

gene expression

proteomics

metabolomics

http://www.google.it/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&uact=8&ved=0CAcQjRw&url=http://cliparts.co/photos-of-arrows&ei=6F1cVbPcHIbaywOvjoHADg&bvm=bv.93756505,d.bGQ&psig=AFQjCNGMwAW5bLWyyzYugc8pH-DpPIdtiw&ust=1432203107181285

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



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

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

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 (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 (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

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


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

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


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

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

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


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


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


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


typical HPLC profiles of willow extract

before and after the hydrolysis

willow extract after hydrolisys willow extract before hydrolisys


hplc profiles of willow extracts available on the market

willow extract after hydrolisys willow extract before hydrolisys


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


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


adulteration: accidental or deliberate?

cimicifuga foetida cimicifuga racemosa


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


Minutes

10.00 20.00 30.00 40.00

Cimifugin 17.6 min

Cimifugin

glucoside 9.8 min

cimicifuga foetida

HPLC-MS profile


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


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)


PCA was invented in 1901 by karl pearson

it was later developed (and named) by

harold hotelling in the 1930s


http://en.wikipedia.org/wiki/File:Portrait_of_Karl_Pearson.jpg

http://en.wikipedia.org/wiki/File:Harold_Hotelling.jpg


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


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


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


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


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


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


panax ginseng genus


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


the PCA evaluation of the HPLC data disclose clusters

related to the country of origin: nigeria and china


pc1 accounts for the origin

variable 6-gingerol:

high PC1 values =>nigeria

variable 10-gingerol:

low PC1 values => china



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


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

MIXTURES


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




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




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

apis of botanical origin quality and adulteration · pdf filecontaminants are the impurities...

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