Standards in the Botanical Testing Industry · 2019. 5. 28. · 789 N. Dixboro Road, Ann Arbor, Michigan 48105 USA alicephoto/123RF Graphic elements sourced from 123RF.com Jesse D

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NSF INTERNATIONAL789 N. Dixboro Road, Ann Arbor, Michigan 48105 USA

alicephoto/123RFGraphic elements sourced from 123RF.com

Standards in the Botanical Testing Industry


Topics

kasto/123RF

• Molecular Assays and Standards Needs• Chemical Analysis of Botanicals• Wish List

NSF INTERNATIONAL | 3NSF INTERNATIONAL | 3Next Generation Sequencing for Food Authenticity

DNA Methods

There are many factors to consider when selecting an appropriate DNA-based method for botanical authentication such as its ability to:

• Differentiate between close relatives

• Detect and identify expected and/or unexpected contaminants/adulterants

• Identify more than one species in a mixture

The most commonly used DNA-based method in botanical taxonomic studies is DNA sequencing, commonly known as “DNA-barcoding”.


What is DNA Barcoding?

The process of extracting genomic DNA from an Organism

Targeting regions of the genome that allow differentiation of the organism from other non-target organisms with primers

Typically, this involves choosing one gene (ITS, matK, rbcL, psbA, or trnL)

Amplification of the regions of interest (amplicon generation)

Sequencing of amplicons to determine sequence

Comparison of sequenced amplicons to reference database

Next Generation Sequencing for Food Authenticity

Sergey Nivens /123RF

NSF INTERNATIONAL | 5NSF INTERNATIONAL | 5Next Generation Sequencing for Food Authenticity

The Importance of Quality References

Need to be careful with publicly available reference data

Databases like Genbank can be helpful in some situations, but not all of it is reliable

GENE SELECTION AND REFERENCE DATABASE ARE OF UTMOST IMPORTANCE


Chloroplast Sequencing

Allows a deeper look into the genome, but not as deep as WGS

Barcoding genes are on the chloroplast as well

• trnL, psbA, rbcL, more


By Kelvinsong - Own work, CC BY 3.0, https://commons.wikimedia.org/w/index.php?curid=26247252


Sequencing Standards

Sequencing doesn’t care about the plant part

Inclusion and exclusion panels!!

• Molecular methods can detect all species in a sample

• Need to tune the method to encompass closely related species and common adulterants

Vouchered samples are important

• Smithsonian

• Harvard

• Missouri

BUT…Can you have vouchered samples you have made from your own plant stock?

• N of 20 at a minimum?

• Vertically integrated?

• Is it OK to run experiments with your sample (that you want to test positive) as the control?



Analytical Chemistry Approaches to Botanical AnalysisAndrew Erickson, Senior Analytical Chemist, NSF International

Analytical Chemistry Approaches to Botanical Analysis

Alena Brozova/123RF


Complexity of Natural Products


O

OHO

OH

O

O

OH

OH

OH

OH

OH

OH

OH

OH

CH3CH3

CH3

CH3 CH3

CH3

CH3CH3

ginsenoside Rg1 801 amu

Epigallocatechin gallate

Panax quinquefolius

Camillia sinensisPapaver somniferum

byrdyak/123RF

Originoo Stock/123RF

Valerii Kirsanov /123RF

Serg_v /123RF


Constituent complexity within a plant Plant Part

• Aerial parts (herba)

• Leaf (folia)

• Fruit (fructus)

• Bark (cortex)

• Root (radix)

• Rhizome (rhizoma)

• Bulb (bulbus)


Sandra Cunningham/123RF

Panax trifolius L. dwarf ginseng

Britton, N.L., and A. Brown. 1913. An illustrated flora of the northern United States, Canada and the British Possessions. 3 vols. Charles Scribner's Sons, New York. Vol. 2: 619. Courtesy of Kentucky Native Plant Society. Scanned by Omnitek Inc. Usage Requirements. http://plants.usda.gov/java/profile?symbol=PATR2&photoID=patr2_001_avd.tif

http://www.knps.org/

http://www.omnitekinc.com/

http://plants.usda.gov/java/usageGuidelines?imageID=patr2_001_avd.tif


Natural Variability• Opportunities and Challenges

• Encompasses vast diversity

• Millions of different species

• Within species variation: “Chemotypes” or “Chemovars”

• Subject to natural variability

• Location/climate

• Soil

• Season

• Subject to cultivation variables

• Fertilization

• Irrigation

• Subject to harvesting and storage variables

• Time of day

• Sun/shade

• Dried/heated

• Shipping

• Subject to misidentification


Iakov Kalinin/123RF


EXAMPLE: Ginkgo Biloba


Is it the right plant and the

right plant part?

• Leaf extracts (more

common form)

• Seed

What about the profile of the

marker compounds?

• Flavonoids (antioxidants)

• Terpenoids (improve blood

flow)

• May be standardized 24/6

• What about ginkgolic acid?

– Cytotoxic and allergenic

– EU has a 5ppm limit

– Not controlled in US

marketplace

• What about ginkgotoxin

– Can naturally occur in the

seed

– Could cause seizures


Ginkgolic Acids by HPLC


min2.5 5 7.5 10 12.5 15 17.5 20 22.5

mAU

0

10

20

30

40

DAD1 A, Sig=242,8 Ref=400,100 (GINKGOLIC ACIDS_15\GINKGOACD031117.D)

Are

a: 367

.961

Are

a: 411

.098

2.4

85

3.4

05

3.7

75

4.3

03

4.9

20

5.2

11

6.0

73

7.0

20

8.7

57

9.7

92

10

.14

5

11

.25

8

12

.01

4 -

G

inkg

olic

aci

d I

13

.28

5

14

.34

3

15

.11

0

16

.33

0

17

.37

2 -

G

inkg

olic

aci

d II

21

.30

7

min2.5 5 7.5 10 12.5 15 17.5 20 22.5

mAU

-10

0

10

20

30


3.4

26

3.9

71

11.

978

- G

inkg

olic

aci

d I

16.

386

17.

391

- G

inkg

olic

aci

d II

min2.5 5 7.5 10 12.5 15 17.5 20 22.5

mAU

0

20

40

60

80

100

120

140


2.1

23

2.6

14

3.5

08

4.1

70

4.8

59 5

.283

5.8

77

7.1

11

8.5

73

10.

711

14.

009

16.

466

Ginkgolic acids –reference standard chromatogram High quality ginkgo extract –

No Ginkgolic acids detected

Poor quality ginkgo extract –Significant levels of Ginkgolic acids


EXAMPLE: Ginkgo Biloba


HPLC chromatogram of a tablet sample

containing Ginkgo biloba leaf extract

• Markers are ginkgo flavonoids

• quercetin (readily available, cheap)

• isorhamnetin (expensive)

• kaempferol (expensive)

• Test should be designed to show spiking with quercetin

• Meets the ginkgo flavonoid total specification

• But if not in the natural ratio—it is adulterated !

• Natural ratio Q:K:I is ~5:5:1


Adulteration with synthetic products


▪ Ginkgoflavonoids ratio in ginko biloba extracts to avoid spiked products

By Pilar Pais, Supply Side, Las Vegas NV, November 6, 2012.

E U R O M E D S . A .

Q u a l i t y C o n t r o l D e p a r t m e n t

P r o je c t : G in k g o B i lo b a 2

D a t e : 2 / 2 0 / 2 0 0 7

1 o f 1

G I N K G O

B a t c h : 3 6 0 3 0 3 6 8 N a m e : G in k g o B i lo b a E . S - 1

T y p e : U n k n o w n R e f . S t d . S o lu t io n :

D a t e A c q u i r e d : 1 0 / 1 2 / 2 0 0 6 1 : 4 7 : 2 6 A M V ia l : 6 In je c t io n : 1

D a t e P r o c e s s e d 1 0 / 1 6 / 2 0 0 6 9 : 4 7 : 0 6 A M In je c t io n V o lu m e : 1 0 . 0 0 u l

S y s t e m : H P L C 3 R u n T im e : 2 2 . 0 0 M in u t e s

A c q M e t h o d : H P L C 4 3 8 _ S 3 W a v e le n g h t : 3 7 0 n m

S a m p le C o n c e n t r a t io n ( m g / m l ) : 1 . 0 3 0 0 A n a ly s t : L D

AU

0 . 0 0

0 . 0 2

0 . 0 4

0 . 0 6

0 . 0 8

0 . 1 0

0 . 1 2

M i n u t e s

2 . 0 0 4 . 0 0 6 . 0 0 8 . 0 0 1 0 . 0 0 1 2 . 0 0 1 4 . 0 0 1 6 . 0 0 1 8 . 0 0 2 0 . 0 0 2 2 . 0 0Q

uercetin - 10.547

Kaem

pferol - 15.617

Isorham

netin - 17.106

1

2

3

S u m

N a m e R T A r e a % A r e a H e i g h t A m o u n t

Q u e r c e t i n

K a e m p f e r o l

I s o r h a m n e t i n

1 0 . 5 4 7

1 5 . 6 1 7

1 7 . 1 0 6

2 4 4 7 7 9 0

5 4 7 0 8 3

1 6 5 7 0 4

7 7 . 4 5

1 7 . 3 1

5 . 2 4

1 1 4 0 1 9

2 5 2 7 7

7 5 3 8

5 . 9 9 9 0 2 4

1 . 4 3 3 9 8 4

1 . 2 3 6 0 1 2

8 . 6 6 9 0 1 9

E U R O M E D S . A .

Q u a l i t y C o n t r o l D e p a r t m e n t

P r o je c t : G in k g o B i lo b a 2

D a t e : 2 / 2 0 / 2 0 0 7

1 o f 1

G I N K G O

B a t c h : 3 6 7 0 6 1

N a m e : G in k g o B i lo b a E . S S E - 1

T y p e : U n k n o w n R e f . S t d . S o lu t io n :

D a t e A c q u i r e d : 1 0 / 5 / 2 0 0 6 2 : 1 0 : 2 6 P M V ia l : 2 In je c t io n : 1

D a t e P r o c e s s e d 1 0 / 6 / 2 0 0 6 1 1 : 3 2 : 0 7 A M In je c t io n V o lu m e : 1 0 . 0 0 u l

S y s t e m : H P L C 3 R u n T im e : 2 2 . 0 0 M in u t e s

A c q M e t h o d : H P L C 4 3 8 _ S 3 W a v e le n g h t : 3 7 0 n m

S a m p le C o n c e n t r a t io n ( m g / m l ) : 1 . 0 8 6 0 A n a ly s t : L D

AU

0 . 0 0 0

0 . 0 1 0

0 . 0 2 0

0 . 0 3 0

0 . 0 4 0

0 . 0 5 0

0 . 0 6 0

0 . 0 7 0

0 . 0 8 0

0 . 0 9 0

M i n u t e s

2 . 0 0 4 . 0 0 6 . 0 0 8 . 0 0 1 0 . 0 0 1 2 . 0 0 1 4 . 0 0 1 6 . 0 0 1 8 . 0 0 2 0 . 0 0 2 2 . 0 0

9.350

Quercetin - 11.185

12.652

13.429

Kaem

pferol - 16.192

Isorham

netin - 17.623

18.515

19.146

1

2

3

S u m

N a m e R T A r e a % A r e a H e i g h t A m o u n t

Q u e r c e t i n

K a e m p f e r o l

I s o r h a m n e t i n

1 1 . 1 8 5

1 6 . 1 9 2

1 7 . 6 2 3

1 8 5 4 6 3 3

1 6 3 0 9 3 1

5 2 0 6 8 5

4 5 . 2 0

3 9 . 7 5

1 2 . 6 9

8 4 8 8 4

7 7 6 4 5

2 4 8 7 8

4 . 2 8 8 3 4 3

4 . 0 6 3 7 6 7

3 . 7 6 8 8 6 4

1 2 . 1 2 0 9 7 4

Authentic

ginkgo biloba

Spiked ginkgo

biloba

HPTLC Fingerprint HPLC-UV ginkoflavonoids

Addition of rutin

O

O-Glc-Rha

HO

OH O

OH

OH


TLC of Ginko w/ rutin


Rutin

Gly

cosi

des

&Tr

iter

pen

oid

sag

lyco

nes


Botanical Standards for Chemistry

Different chemical profiles in different parts of the plant

Expression of chemicals varies based on geography and environmental conditions

Synthetics and spiked ingredients need to be accounted for

NSF Sources individual reference material from Chromadex or Mountain Rose Herbs

• For TLC, we source the plant part specifically

Wish List

• A standards “Kit”

• Reference material

• Market compunds

• Common Adulterants



NSF INTERNATIONAL789 N. Dixboro Road, Ann Arbor, Michigan 48105 USA

alicephoto/123RFGraphic elements sourced from 123RF.com

Jesse D. Miller, [email protected]

Andrew [email protected] x2371

Standards in the Botanical Testing Industry

Documents

Standards in the Botanical Testing Industry · 2019. 5. 28. · 789 N. Dixboro Road, Ann Arbor, Michigan 48105 USA alicephoto/123RF Graphic elements sourced from 123RF.com Jesse D