Amit Chandra, Ph.D

AMWAY R&D

amitabh.chandra@amway.com

Phytochemical Fingerprint

Botanical Integrity: Significance of Fit for Purpose Tests During

Botanical Product Development Process

WHY BOTANICALS IN SUPPLEMENTS:

Colorful fruits and vegetables are healthy sources of beneficial nutrients.

WHO recommends five servings (about 450g) of fruits and vegetables every day to meet these requirements.

75% of adults fail to meet this recommendation.

Supplements containing vitamins, minerals and botanicals help to close this gap.

Hence “quality of” and “choice of” right botanical for the right health benefit is extremely important !

(Nutrilite Health Institute-Global Phytonutrient Report,

British J. Nutrn, 2014)

TYPICAL FORM OF BOTANICAL INGREDIENTS, ACTIVES v/s MARKERS:

STANDARDIZED EXTRACT: Ingredient with certified / consistent levels of

phytonutrients produced via controlled manufacturing / extraction process

MARKERS (are typically quality indicating): Phytonutrients that are “unique to” and used as analytes for

identifying and quantifying the botanical ingredient.

Please note: It is typically the whole ingredient that offers / maintains health benefit (in a supplement)

not specific phytonutrients !

ADULTERATION OF BOTANICALS IN DIETARY SUPPLEMENT COMPROMISING ITS INTEGRITY:

(1)If any valuable constituent has been in whole or in part omitted or abstracted therefrom; or (2) if any substance has been substituted wholly or in part therefor; or (3) if damage or inferiority has been concealed in any manner; or (4) if any substance has been added thereto or mixed or packed therewith so as to increase its bulk or weight, or reduce its quality or strength, or make it appear better or of greater value than it is.

21 US Code 342 – Adulterated Food

TYPICALLY OBSERVED

ADULTERATIONS IN BOTANICALS

ECONOMICALLY MOTIVATED ADULTERATION (Intentional): Fortifying botanical ingredients by adding “markers” to pass quantitative tests !

It is by design, not accident ! Markers can be from synthetic / natural source

ACCIDENTAL ADULTERATION (Incidental): Due to carelessness in chain of custody

No traceability ! supply chain, violation of cGMP, cross contamination etc.

FORTIFYING OR REPLACING BOTANICALS WITH PRESCRIPTION DRUGS (Intentional)

For quick results ! In weight loss, body building, sexual enhancement

A holistic / qualitative analytical approach to identify the correct / intended botanical source SHOULD ALWAYS PRECEED its quantification in the product.

HENCE, THE CHOICE OF FIT FOR PURPOSE ANALYTICAL METHOD IS OF PRIME IMPORTANCE HERE

Chandra, A., in Korea-China Joint International Phytonutrient Symposium, Seoul, 2016, 147-156.

What is the purpose of method OR what is the question that is being asked (ID or Quant) ?

Is there some benchmark sample (authentic sample/control/reference) for comparison ?

Nature of botanical INGREDIENT (dehydrate or extract) ?

ID of whole plant or a specific plant part / processed ingredient ?

QUANTIFICATION OF INGREDIENT ? Genus spp and / or phytonutrients (markers) ….?

ANSWERS TO ABOVE WILL HELP TO CHOOSE THE PROPER ANALYTICAL TEST METHOD

AKA “FIT FOR PURPOSE”

CRITERIA TO CHOOSE FIT FOR PURPOSE METHODS?

AUTHENTIC BOTANICAL SAMPLE / CONTROLS / REFERENCE

• CERTIFIED REFERENCE MATERIALS

• VOUCHERED SPECIMENS,

• SELF COLLECTED / GENERATED BASED ON HISTORICAL USE, (with

documentation)

• ETC…..

DEPENDS ON YOUR PURPOSE.

SAMPLE MUST REPRESENT MATERIAL OF INTEREST

Example: Matrix, Harvest time, specific plant part, Standardization level

IT HAS TO

BE APPLES

TO APPLES

COMPARISON

QUALITATIVE METHODS USED FOR BOTANICAL ID

GENETIC: Full genomic sequence

DNA barcoding

Next generation sequencing (NGS)

BIOLOGICAL: Micro and Macroscopic, organoleptic…

CHEMICAL: (Spectroscopic) Patterns, Phytochemical Fingerprints

(Qualitative, Holistic)

Selected method must be

appropriate (fit for purpose) for

answering the question that is

being asked!

Khan, I.A., Smillie, T. J.Nat. Prod., 2012, 75 (9), 1665-1673.Betz, J.M., et al. Anal. Bioanal. Chem., 2007, 389, 19-25.

TIPS FOR FLAGGING BOTANICALS THAT ARE

VULNERABLE FOR THEIR INTEGRITY (PRONE TO

ADULTERATION)

• RESTRICTED / LIMITED SUPPLY CHAIN

• COMPLEX STANDARDIZATION PROCESS

• HIGH COST

• GENERIC PHYTONUTRIENT MARKERS

• ONE GENUS, MULTIPLE SPECIES

• LACK OF MANF. – SUPPLIER PARTNERSHIP

IN THIS CONTEXT,

I WILL NOW SHARE WITH YOU

TYPICAL EXAMPLES OF BOTANICAL SUPPLEMENTS IN MARKET THAT ARE VULNERABLE FOR ADULTERATION !

SIGNIFICANT INCREASE IN SUPPLY CHAIN

LOWERING OF COST

GENERIC PHYTONUTRIENT TARGET FOR STANDARDIZATION (Total flavonol-glycosides)

EXAMPLE 1: GINKGO BILOBA LEAF EXTRACT

Chandra A; Li Y; Rana J; Persons K; Hyun C; Shen S; Mulder T., J. Func. Foods, 3, 2011, 107-114

FIT FOR PURPOSE METHOD: PHYTOCHEMICAL FINGERPRINT

OF FLAVONOL GLYCOSIDES BEFORE HYDROLYSIS

RQ, K and I

This is the signature profile / fingerprint of

“authentic ginkgo leaf extract 24/6”

RESULTS AND CONCLUSIONS

All

Passed

24%

spec

Chandra A; Li Y; Rana J; Persons K; Hyun C; Shen S; Mulder T., J. Func. Foods, 3, 2011, 107-114

FLAGS:

SIGNIFICANT INCREASE IN SUPPLY CHAIN

LOWERING OF COST

GENERIC STANDARDIZATION (Total Anthocyanins)

ONE GENUS (Vaccinium spp) MANY SPECIES

EXAMPLE 2: BILBERRY (Vaccinium myrtillus)

Chandra A., Rana J., Li Y. J Agric Food Chem., 49 (8), 2001, 3515-3521

Li Y; Chandra A, Persons K, Stern N, Shen S. 127th AOAC International, 2013, Aug 25-28, Chicago IL, USA

FIT FOR PURPOSE TEST: HPLC Fingerprint of

anthocyanins in authentic Bilberry (as is )

This is the signature profile / fingerprint of anthocyanins in

“authentic bilberry extract”

Chandra A., Rana J., Li Y. J Agric Food Chem., 49 (8), 2001, 3515-3521

Looks like black rice

Looks like elderberry

Could be amaranth dye !

Li Y; Chandra A, Persons K, Stern N, Shen S. 127th AOAC International, 2013, Aug 25-28, Chicago IL, USA

RESULTS AND CONCLUSIONS

AUTHENTIC BILBERRY

BC BR BB1 BB2 GR BB BBn CG A

RECOMMENDED FIT FOR PURPOSE ANALYTICAL TESTS FOR ENSURING BOTANICAL INTEGRITY

DNA ORGANOLEPTIC

MACROSCOPIC

&

MICROSCOPIC

PHYTOCHEMICAL

WHOLE

DEHYD.

EXTRACT

STD. EXTRACT

SPECIFIC

PLANT PART * *

BLENDED

BOTANICALS

TEST

BO

TA

NIC

AL

*: RED FOR

EXTRACTS

* *

RED: Not fit for purpose

YELLOW: Limited fit

GREEN: Fit for purpose

RED FOR

UNKNOWN

BOTANICALS

Chandra, A., in Korea-China Joint International Phytonutrient

Symposium, Seoul, 2016, 147-156.

Quality By Design QbD ?

• Quality by Design (QbD) is a proactive and

systematic approach to development that begins

with predefined objectives and emphasizes

product and process understanding and process

control, based on sound science and quality risk

management.

WHY: FOR Advancement of botanical integrity,

safety and quality in the industry !

Khan, I.A., Smillie, T. J.Nat. Prod., 2012, 75 (9), 1665-1673.

Example of QbD for addressing botanical integrity (from

our latest market launch of Ayurvedic products in India)

Deployment of fit for purpose tests

(in that order):

1. Confirmation of correct genus and species by DNA

fingerprinting of whole plant

2. Verification of Non GMO status by DNA – PCR

3. Botanical ID (of specific plant part) by phytochemical

fingerprinting method using HP-TLC

4. Quantification of marker in extract and tablets by

chemical methods (HPLC) for claims, QA, stability etc.

5. Establishing quality standards with supply chain for each

production lot.

QbD, Right tests at the right stage-gates with full

documentation and partnership with supply chain and

compliance with regulatory = BOTANICAL INTEGRITY.

Example of QbD for Botanical

Integrity in development process

PRODUCT

DNA Fingerprinting on botanicals:

Where are we at ?

• DNA is the holy grail to address all problems !!!!! So much

confusion on the application of this technology in conjunction to

botanical integrity

• Industry trying to work to come to a harmonized approach for

recommending appropriate use of DNA test methods. AHPA-NIST

is leading an initiative in this direction with Industry

• FDA and USP do not recommend / endorse DNA testing as the only

test for genus and species. However, its use in conjunction to

relevant phytochemical methods is OK

• DNA is being legitimized as a test for botanical drugs in Korean

Pharmacopeia. However only at whole plant / herbal stage.

STEPS TO ENSURE INTEGRITY OF BOTANICALS

IN SUPPLEMENTS AND FOODS:

1. Full traceability of botanicals from farm to product

with audits and documentation

2. Active partnership with supply chain

3. Create and follow QbD process keeping end goal in

mind

4. Mandate use of fit for purpose analytical test

methods at all stage-gates during the development

process (seed to supplement)

5. Last but not the least, share – educate – publish –

talk – promote good practices !

ACKNOWLEDGEMENTS

Amway R&D

Amway-Nutrilite Botanical Authentication Team

Ole Miss NCNPR – Awesome Hosts

Industry Partners:

AHPA, AOAC, CRN, ABC, USP

….. and you the ELITE AUDIENCE !