Power of fingerprints for Botanical Authentication QUALITY BEFRE QUANTITY. Amit Chandra, Ph.D Analytical Sciences, AMWAY R & D
Interesting Quotes since last two days : nce a CSIR always a CSIR! Knowledge base in India should combine with latest technology Where there is a will there is a way, where there is a way there are SHRTCUTS! Convert money to knowledge and then knowledge to money What is huge is not what is always great HPLC is not important, it is the biological aspect that is more important Food lying on the plate when consumed becomes us Lacking in regulation is availability of standard analytical methods Self Regulation, think about what you are feeding to your own people You can see light better from the dark side
Botanicals are the most interesting and complex ingredients in nature They are chosen to be part of food and dietary supplements because they afford unique health benefits as we already know Today we will be discussing the role of phytochemical fingerprinting technology in addressing botanical quality, specially when they are procured in powder and extract forms QUALITY BEFRE QUANTITY?
Phytochemical Fingerprint Profile A chromatogram (trace) that depicts the separation and detection of the phytochemicals that are the best / unique representatives of a specific botanical material (under the applied experimental conditions) can be deemed as its phytochemical fingerprint profile. In other words, this visual pattern of separated phytochemicals serves as the unique signature ID of the specific botanical material. Most common techniques used for generating phytochemical fingerprint profiles include HPLC, GC, MS and TLC. All the applications discussed in this presentation are from data developed and generated by us during the development of our products.
Here are some of the most important applications of phytochemical fingerprints: Those are in the areas of : AUTHENTICATIN of Botanicals (botanical ID) BENCHMARKING for efficacy (fingerprint profile showing quality indicating markers of an active botanical ingredient) TRACKING ADULTERATIN (accidental or deliberate! )
1. Authentication of Botanicals HW IT IS DNE : By creating signature fingerprint profiles on true to type botanicals on their respective genus, species, variety / cultivar (obtained from credible sources) Use data as qualitative ID on botanicals (sold as powders) form). UTILITY / BENEFIT : Ensure the use of correct (authentic) botanical material as claimed and fit for purpose
Citrus Fruits Compound Class Represented in Fingerprint: Bioflavonoids (aka flavanones and flavones)
Mandarin range hesperidin
Lemon hesperidin eriocitrin
Grapefruit naringin
Differentiation by Fingerprint Typical assay for specs: Total bioflavonoids as hesperidin equiv. Chandra, A et al; ICNPR-ASP, 2004 (Phoenix, AZ, USA)
Berries Compound Class Represented in Fingerprint: Anthocyanins Typical assay for specs is UV-Vis
Elderberry Cyd-gluc Cyd-samb Chandra, A., Rana, J., Li, Y. J Agric Food Chem., 49 (8), 2001, 3515-3521
Acai Cyd-rut Cyd-gluc
Differentiation by Fingerprint Elderberry Acai
Echinacea H C H C C H C CICHRIC ACID H C C H C CAFETARIC ACID R H C R 1 ECHINACSIDE R = Glucose (1,6-) & R 1 = Rhamnose (1,3-) C - C-CH=CH CHLRGENIC ACID Compound Class Represented in Fingerprint: Echinacea Polyphenols (hydroxy-cinnamates)
Echinacea angustifolia echinacoside
Echinacea purpurea Cichoric acid
Differentiation by Fingerprint E. angustifolia E. purpurea Typical assay for specs: total Echinacea polyphenols by HPLC Rana, J., Chandra, A. ACS Symposium series 925, 2006, 110-116
Difference in avocado leaves from same species grown in California versus Mexico (cultivars) X a: α-pinene, b: β-pinene, c: estragole, d: Me-estragole, e: caryophyllene Chandra, A et al; ASP, 2005 (Corvallis, R, USA)
Benchmarking of Botanicals HW IT IS DNE: By creating signature fingerprint profiles on clinical / active / proprietary / standardized botanicals (and extracts). UTILITY / BENEFIT: Maintain qualitative consistency of the in spec standardized extract, clinical materials and fractions for EFFICACY / HEALTH BENEFITS
Tianlife TM TLCT-Plus (Cistance tubulosa ext for memory enhancement) Compound Class Captured in Fingerprint of patended extract by Supplier (Sinphar) : Cistanche tubulosa glycosides (CTGs) R 4 R 3 R 5 R 2 R 1 Echinacoside : R1 =H, R2 = Rha, R3 = Cf, R4 = Gluc, R5 = Acteoside: R1 = H, R2 = Rha, R3 = Cf, R4 = H, R5 = Isoacteoside: R1 = H, R2 = Rha, R3 = H, R4 = Cf, R5 = Tubuloside A: R1 = AC, R2 = Rha, R3 = CF, R4 = Gluc, R5 =
Tianlife TM TLCT-Plus (Cistance tubulosa ext for memory enhancement) Benchmark Fingerprint of active material 1)Cistanbuloside C1/C2, 2) Echinacoside, 3) Cistanbuloside A, 4) Tubuloside A, 5) Acteoside and 6) Isoacteoside Quantitative spec is on echinacoside and total CTGs by UV
Teaflavin R (blend of green tea and black tea ext for cholesterol lowering effect ) Compound Class Captured in Fingerprint of patended tea extract by Supplier (Nashai Biotech) : Theaflavins
Teaflavin R (blend of green tea and black tea ext for cholesterol lowering effect ) Benchmark Fingerprint of active material Quantitative spec is total tea polyphenols by UV Chandra, A et al; ASP, 2003 (Chapel Hill, NC, USA)
How it is done: Tracking Adulteration (typically botanical to botanical) Design experiments and gather information to develop fingerprint info from benchmark / authentic samples and (as possible) some adulterated samples. Derive QUALITATIVE conclusions that are indicative of deliberate and/or accidental cross contamination (or fortification) of botanicals (and its extracts) in herbal products (that most often pass quantitative specs!). Create / recommend specs that can check such occurrence on a qualitative basis. UTILITY / BENEFIT: Maintain authenticity of the claimed botanical material by establishing critical parameters as part of specification as check points EFFICACY and CLAIMS! Comply with cgmp and Regulatory environment (global) REPUTATIN! Gain confidence in material that is procured and processed by third parties The greed or desire to adulterate is to meet the numerical specs in expensive botanicals by spiking them with enriched phytochemicals (and synthetic compounds). People who adulterate are very knowledgeable in analytical sciences
Ginkgo biloba!
Ginkgo biloba! STD PANEL H 3 + TYPICAL GINKG
Ginkgo biloba contd Category A Authentic Category B Category C Category D Typical spec is 24% flavone glyc after hydrolysis Chandra et al, 2011, Journal of Functional Foods
Bilberry Authentic Elderberry Black Rice Black Bean Red Dye? Typical Chromatography-Chemistry Bioassay Microbiology Advanced spec is 25% by UV method Imaging
Natural versus fermented / processed / hydrolyzed Pomegranate Natural Fermented / Processed? punicalagins Ellagic acid H H H H H H H
Synthetic versus natural β- carotene (from Dunaliella algae) Spec is based on UV method Natural Synthetic Curt Emenhiser et al., J. Ag. Food Chem., 1996, 44, 3887-3893.
Natural versus fermented / processed / hydrolyzed Blackcurrant Delph-gluc & rut Cyd-gluc & rut Fermented / Processed? Natural Industry Chromatography-Chemistry Bioassay Microbiology Advanced spec tot antho by UV-Vis Imaging
Qualitative fingerprint is a good and essential complement to numeric specifications (numbers v/s quality) It addresses and substantiates total quality from CNSISTENCY, AUTHENTICITY and EFFICACY PERSPECTIVE This aspect of phytochemistry aligns with best of nature best of science theory I strongly suggest that quality becomes an integral part of regulations in addition to quantity in nutraceuticals and botanicals (traditional medicines included)
ACKNWLEDGEMENTS AMWAY INDIA NUTRA INDIA SUMMIT TEAM MANAGEMENT AND STAFF ANALYTICAL SCIENCES NUTRILITE SPD and QA AMWAY FARMS And You the audience and young scientists / future CEs