challenges in characterizing and identifying components in
TRANSCRIPT
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Challenges in Characterizing and Identifying Components in
Botanical ProductsJoseph M. BetzJoseph M. Betz
Office of Dietary SupplementsOffice of Dietary SupplementsU.S. National Institutes of HealthU.S. National Institutes of Health
Department of Health and Human ServicesDepartment of Health and Human Services
http://dietary-supplements.info.nih.gov/
March 25, 2005
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Disclaimer
Views expressed are my own and do not reflect the views of ODS, NIH, or HHS
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Known Bioactive Compounds in Foods
Inherent to Foods• Alkaloids• Cyanogens• Glucosinolates• Lectins• Carbohydrase Inhibitors• Vicine/Convicine
(Favism)• Phenolics• Non-protein Amino Acids• Peptides
Accidental Ingestion• Mushroom Toxins
Contaminants in Foods• Mycotoxins• Alkaloids• Shellfish toxins
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Alkaloids
N
N N
N
O
H3C
O
CH3
CH3
Caffeine-LD50-127 mg/kg* mice, 50-500 mg/kg*, humans
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Cassava (manioc)- Manihot esculentaCH3
CN
CH3O
O
HO
OH
HOOH
linamarin
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Biogenic Amines
NH2
HO CH3
HONH2
HN
CH3
OH
HO
N-methyltyramine
tyramine synephrine
HO
OH
NH2
octopamine
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Biogenic Aminessynephrine tyramine Me-tyramine Octapamine
C. reticulata(tangerine)
125 1 15 1
C. reticulata(Mandarin or.)
280 1 58 2
C. aurantium(sour orange)
19 - 1 -
Musaparadisiaca(banana)
- 29 - -
Mg/kg fresh
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6'7'-DihydroxybergamottinGrapefruit juice inhibits intestinal cytochromeP450 (CYP) 3A4
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GlucosinolatesS-C6H11O5
R-C
N-OSO3-
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Glucosinolates
3-Methylsulfinylpropyl-glucosinolate: Glucoiberin
Allyl-glucosinolate: Sinigrin
4-Methylsulfinylbutyl-glucosinolate: Glucoraphanin
3-Indolylmethyl-glucosinolate: Glucobrassicin
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GlucosinolatesHepatic, renal, Pancreatic lesions caused by OZT’s and nitriles
O
NH
S
H
CH2
GoitrinProgoitrin
Thiocyanates and L-5-vinyl-2-thiooxazolidone=Goitrogens
•Activity of goitrin not reversible by dietary iodine
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(CH2)5CH CHCH2CH3C C C C CH2CHCH
OH
CH2
Carotatoxin- 10-20 mg/kg: LD50-100 mg/kg (i.p.), mice
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Soy (Glycine max)
OHO
OOH
O
OOH
HO
OH
Daidzin=daidzein-7-glucoside
Genistin=genistein-7-glucoside
Soyasaponin II
Stachyose
Phytic AcidBowman-BirkProtease Inhibitor
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"What we observe is not nature itself, but nature exposed to our method of questioning."
- Werner Heisenberg
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Biological relevance
O
OH
O–rutinose
O
HO
OH
OH
Rutin
O
OH
OOH
HO
OH
OH
QuercetinCompound Rel. Efficiency
THBQ 1.00BHA 1.67Quercetin 0.18Rutin 0.01
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O
OH
OOH
HO
OH
OH
O
OH
O–rutinose
O
HO
OH
OH
Quercetin
Rutin
HO OH
O
OH
β-D-glucose–O
Cytotoxicity in Caco 2 cells(Inhibition of MTT reduction)
AJE Quercetin Rutin Phloridzin
1 mM 50 µM 5 µM 5 µM
Phloridzin
•Addition of ascorbate or α-tocopherol abolished cytoxicity
•Postulated that formation of reactive polyphenol oxidation products at high levels responsible for cytotoxicity
•Also postulated that AJE was less cytotoxic because the compounds protected each other
Pohl et al. 2004 The Toxicologist 78,S-1: 213
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Ubiquitous plant flavonoidsO
OH
OOH
HO
OH
OHO
OH
O–rutinose
O
HO
OH
OHO
O quercetinRutin
(quercetin-3-rutinoside)Flavone
OO
HO
CH3
HO OH HOOH
OOO
OH O
OCH3
OHOH
CH2
O
OCH3
O
HO
OH
OH
hesperidin
hesperetin
Hydroxylated flavonoids (quercetin, naringenin) usually inhibit P450 isozymes in vitro, nonhydroxylated flavonoids (flavone, nobiletin, tangeretin) stimulate P450 system in vivo and in vitro
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Quercetin
Quercetin reduces expression of the amiloride-sensitive epithelial Na+ channel (ENaC) in salt sensitive hypertensive rats (Aoi et al. 2004 Biochem BiophysRes Commun 315:892)
ENaC is rate limiting step in Na+ reabsorption in distal segment of renal tubule
High-salt diet rats have elevated αENaC expression and BPQuercetin blocked elevated expression of αENaC mRNA and BP elevationQuercetin effect on αENaC not found in colon
Aglycone (colon) vs. glucuronide, sulfate after absorption?
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Quercetin
Quercetin (10 µM) inhibits insulin-stimulated methylglucose uptake by direct interaction with the facilitative glucose transporter GLUT4 in rat adipocytes (Strobel et al. 2005 Biochem. J. 386:471)
Does not inhibit protein tyrosine phosphorylationQuercetin non-competitively antagonizes the γ-aminobutyric acid C receptor subunit GABAD1(Goutman & Calvo, 2004 Brit J Pharmacol 141:717)
Quercetin active at ionotropic GABA receptors and other ligand-gated ion channels
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St. John’s Wort
1814-1st American mention of toxicity of H. perforatum (to livestock)1o photosensitivity
hypericin + light = lipid hydroperoxide in vitroSJW induced neuropathy, erythroderma
Hypericin-induced phototoxicity in vitrolong term effects?
SJW/IndinavirHerb/drug interactions
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SJW Constituents
Seven accessions of SJW collected in Tuscany (Bergonzi et al. 2001; Drug Dev Ind Pharm 27:491)
Total flavonols: 4.6-15.9%Total hypericins: 0.05-0.11%Total hyperforins: 1.4-20.8%
H3C
OH O OH
OH
H3C
OH O OH
OH
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Hypericum perforatum L.DAC ’86 (German Drug Codex)- not < 0.4% dianthrones of hypericin group, as hypericinDAC ’91: method change to correct for chlorophyll interference: results are 20% lowerHypericin MAO inhibitor in vitro1996 trial showed hypericin-free but not hyperforin free products effectiveUSP ‘99-SJW extract, not < 0.2% hypericin and pseudohypericin combined , not < 3.0% hyperforin (LC)
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Are we having fun yet?
Bioassay directed H. perforatum prep HPLCFlavonoid rich fractions-Antidepressant (FST)
Hyperoside, isoquercitrin, miquelianin, quercitrin, some hyperoside, astilbinIsolated pure compounds tested
All pure cmpds except quercetin, quercitrin, and astilbin active in FST (Butterweck et al. (2000) Planta Med. 66:3-6)
2002: new proprietary SJW extract product
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SJW flavonoids
miquelianin(quercetin 3-O-β-D-glucuronopyranoside)
isoquercitrin(quercetin 3-O-β-D-glucopyranoside)
hyperoside(quercetin 3-O-β-D-galactopyranoside)
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Drug Discovery
Natural Products Cancer Drug DiscoveryBioassay Directed Fractionation to pure chemical entities
O O
CH3
CH3
O
H3CONHO
OH
O
H3C CH3OH
OOOH
O
CH3
O
O
H
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“Whole plant”
Berberis spp.-marker=berberineFlavonolignan: 5'-methoxyhydnocarpin (5'-MHC) inhibitor of the MDR pump in S. aureusPrevents bacterial cells from eliminating berberine
Stermitz et al. (2000) Proc Natl Acad Sci 97:1433-7Stermitz et al. (2000) J Nat Prod 63:1146-9
N
O
O
H3CO
OCH3
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Analytical Methods and Reference Materials Program
Quercetin dihydrate (7)Molecular Formula: C15H10O7 · 2H2O , Formula Weight: 338.27, CAS Number: 6151-25-3
Q0125 ≥98% (HPLC), powder (Sigma) Q-112 solid (Sigma) 83370 BioChemika, ≥98.0% (HPLC) (Fluka)33,795-1 ≥95% (Aldrich) 17,196-4 98% (Aldrich)32782 >99% (Riedel-de Haën)69249 ≥99.0% (HPLC) (Fluka)
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Quercetin Dihydrate(1200 mg)
Company HPLC Purity(%)
Water Content
(%)
Residual Content
(%)
Calculated Value(%)
Price
A 99.57 20.2 0 79.46 $550
B 99.46 0.41 0.4 99.12 $6500
C ca 90 $1250
D Not less than 98
NA NA $1320