Lorenz Lo Sauer / 2010

Flickr photos: lezumbalaberenjena, CCBYSA

Part 1: Introduction ◦ Gingko – the living fossil ◦ Gingko – the favorite ◦ Ingredients ◦ Egb 761 ◦ NTP studies ◦ Health benefits? / adverse effects

Part 2: Primary active compounds ◦ Recapitulation: Isoprenoids ◦ Ginkgolide B

MNP ◦ Flavonol ◦ Biflavones ◦ Polyprenols ◦ Alkylphenols

Q:

How

?

Common Names ◦ ginkgo, ginkgo biloba, fossil tree, baiguo, bai guo

ye...

Own genus ◦ Ginkgophyto

(pre-gymnosperm)

Living fossil ◦ Longlived: 2.500+yrs

Tall: 20-35m (66-115ft)! Resistance to disease; insect-resistant wood,

aerial roots and sprouts, some fire resistance Survivalist: „rough“ settings such as stream

banks, rocky slopes, cliff edges…

A: T

ough!

Last decade ◦ 3000+ papers published

◦ 400+ about chemical analysis

◦ Over 3200 ginkgo-related patents filed

Interests for triterpene lactones (TTLs) ◦ Ginkgolides (memory enhancers?)

◦ Bilobalide

◦ Biloba extract

Ginkgo industry ◦ Revenue >1 billion $

Fig 1. Ginkgolides: structure

Brackets(..) list important precursors terpene trilactones (Isoprenoids., IPP)

flavonol glycosides (ACoA + PP)

Biflavones (ACoA + PP)

Proanthocyanidins (Flavonoid Synth.)

Alkylphenols (PP, Shikimpath.)

simple phenolic acids (PP, Shikimpath.)

6-hydroxykynurenic acid (PP+AA)

4-O-methylpyridoxine (PP)

Polyprenols (IPP)

ACoA: AcylCoA (incl. AcetylCoA ) IPP: Isopentyl pyrophosphate PP: pentose phosphate AA: Aminoacids

(−)-Bilobalide from Ginkgo biloba leaves ≥93% (HPLC) (−)-Epicatechin ≥98% (HPLC), from green tea (−)-Epicatechin purum, ≥95.0% (HPLC) (−)-Epigallocatechin ≥95% (HPLC), from green tea (−)-Epigallocatechin analytical standard (−)-Gallocatechin ≥98% (HPLC) (−)-Riboflavin ≥98% (−)-Riboflavin BioReagent, suitable for cell culture, insect cell culture tested, ≥99% (+)-Catechin hydrate ≥98% (HPLC), powder (+)-γ-Tocopherol ≥96% (HPLC) 1-Docosanol 98% 1-Docosanol purum, ≥97.0% (GC) 1-Heptacosanol ≥98% 1-Hexacosanol ≥97% (capillary GC) 1-Hexacosanol synthetic, ≥97.0% (GC) 1-Octacosanol ≥99% (GC) 2-Isopropylphenol 98% 3,4-Dihydroxybenzoic acid purum, ≥97.0% (T) 4-Hydroxybenzoic acid ≥99% D-(−)-Fructose ≥99% D-(−)-Fructose SAJ special grade, ≥98.0% D-(+)-Xylose ≥99% D-(+)-Xylose SAJ special grade, 98.0-101.0% D-(−)-Quinic acid 98% D-(−)-Quinic acid purum, ≥98.0% (T) D-(+)-Galactose ≥99% D-(+)-Galactose BioUltra, ≥99.5% (sum of enantiomers, HPLC) D-(+)-Galactose anhydrous, cell culture tested, insect cell culture tested, powder D-(+)-Glucose ≥99.5% (GC) D-(+)-Glucose BioUltra, anhydrous, ≥99.5% (sum of enantiomers, HPLC) D-(+)-Mannose BioUltra, ≥99.5% (sum of enantiomers, HPLC) D-(+)-Mannose from wood, ≥99% D-(+)-Raffinose pentahydrate ≥98.0% D-(+)-Raffinose pentahydrate for microbiology, ≥99.0% D-(+)-Raffinose pentahydrate powder, BioReagent, suitable for cell culture D-Pantothenic acid hemicalcium salt cell culture tested, insect cell culture tested, plant cell culture tested D-Pinitol 95% L-(−)-Malic acid 95-100% (enzymatic) L-(−)-Malic acid 97%, optical purity ee: 99% (GLC) L-(−)-Malic acid SAJ special grade, ≥99.0% L-(−)-Malic acid puriss., ≥99.5% (T) L-(+)-Arabinose ≥99% L-(+)-Arabinose 98% L-(+)-Lactic acid ~98% L-Alanine ≥98% (TLC) L-Alanine ≥99.0% (NT) L-Alanine SAJ special grade, ≥99.0% L-Arginine SAJ special grade, ≥98.0% L-Arginine meets EP, USP testing specifications, BioXtra, cell culture tested, 98.5-101.0%, from non-animal source L-Arginine reagent grade, ≥98% (TLC), powder L-Ascorbic acid ≥99.0% L-Ascorbic acid 99% L-Ascorbic acid JIS special grade, ≥99.0% L-Ascorbic acid puriss. p.a., ≥99.0% (RT) L-Ascorbic acid reagent grade, crystalline, ~325 mesh L-Asparagine monohydrate ≥99% (TLC) L-Aspartic acid ≥98% L-Aspartic acid ≥99.0% (T) L-Aspartic acid SAJ special grade, ≥99.0% L-Aspartic acid reagent grade, ≥98% (TLC) L-Cysteine 97% L-Cysteine SAJ special grade, ≥98.5% L-Cysteine hydrochloride BioReagent, cell culture tested, anhydrous, from non-animal source, ≥98.0% L-Cysteine hydrochloride monohydrate JIS special grade, ≥99.0% L-Cysteine hydrochloride monohydrate from non-animal source, cell culture tested, meets EP, USP testing specifications L-Cystine ≥99.0% (T) L-Cystine SAJ special grade, ≥99.0% L-Cystine crystalline L-Glutamic acid JIS special grade, ≥99.0% L-Glutamic acid ReagentPlus®, ≥99% (TLC) L-Histidine BioUltra, ≥99.5% (NT) L-Histidine ReagentPlus®, ≥99% (TLC) L-Histidine SAJ special grade, ≥98.5% L-Histidine cell culture tested, meets EP, USP testing specifications, from non-animal source L-Homoserine L-Homoserine ≥98.0% (NT) L-Isoleucine BioUltra, ≥99.5% (NT) L-Isoleucine SAJ special grade, ≥99.0% L-Isoleucine meets EP, JP, USP testing specifications, BioXtra, cell culture tested, 98.5-101.0%, from non-animal source L-Isoleucine reagent grade, ≥98% (TLC) L-Leucine SAJ special grade, ≥99.0% L-Leucine reagent grade, ≥98% (TLC) L-Lysine ≥98% (TLC) L-Lysine SAJ special grade, ≥98.0% L-Lysine purum, crystallized, ≥98.0% (NT) L-Methionine BioXtra, meets EP, JP, USP testing specifications, cell culture tested, 99.0-101.0%, from non-animal source L-Methionine SAJ special grade, ≥98.5% L-Methionine reagent grade, ≥98% (TLC) L-Phenylalanine ≥99.0% (NT) L-Phenylalanine BioXtra, meets EP, JP, USP testing specifications, suitable for cell culture, 98.5-101.0%, from non-animal source L-Phenylalanine SAJ special grade, ≥99.0% L-Phenylalanine reagent grade, ≥98% L-Proline ≥99.0% (NT) L-Proline ReagentPlus®, ≥99% (TLC) L-Proline SAJ special grade, ≥99.0% L-Proline meets EP, USP testing specifications, cell culture tested, from non-animal source L-Serine ≥99.0% (NT) L-Serine ReagentPlus®, ≥99% (TLC) L-Threonine BioXtra, cell culture tested, meets EP, JP, USP testing specifications, from non-animal source, 99.0-101.0% L-Threonine reagent grade, ≥98% (TLC) L-Tryptophan BioUltra, ≥99.5% (NT) L-Tryptophan meets EP, JP, USP testing specifications, BioXtra, cell culture tested, ≥99.0%, from non-animal source L-Tryptophan reagent grade, ≥98% (TLC) L-Tyrosine BioXtra, cell culture tested, meets EP, USP testing specifications, ≥99.0%, from non-animal source L-Tyrosine SAJ special grade, ≥99.0% L-Valine SAJ special grade, ≥98.5% L-Valine meets EP, JP, USP testing specifications, cell culture tested, 98.5-101.0%, from non-animal source L-Valine reagent grade, ≥98% (TLC) Acacetin ≥97.0% (HPLC) Acenaphthene 99% Acetic acid ≥99.7% Acetic acid ≥99.7% Acetic acid ≥99.7%, SAJ super special grade Acetic acid 99.5-100.0% Acetic acid JIS special grade, ≥99.7% Acetic acid ReagentPlus®, ≥99% Acetic acid SAJ first grade, ≥99.0% Acetic acid purum, ≥99.0% (T) Amentoflavone ≥99.0% (HPLC) Apigenin ≥95.0% (HPLC) Apigenin 95% Apigenin ~95% (TLC), from parsley, powder Arachidic acid ≥99% Arachidic acid puriss., synthetic, ≥99.0% (GC) Behenic acid 99% Behenic acid puriss., ≥99.0% (GC) Benzoic acid ReagentPlus®, 99% Benzoic acid SAJ first grade, ≥99.5% Benzoic acid SAJ special grade, ≥99.5% Benzoic acid purum, ≥99.0% (T) Butyric acid ≥99% Butyric acid SAJ special grade, ≥99.5% Campesterol ~65%, crystalline Choline chloride BioReagent, suitable for cell culture, suitable for insect cell culture, ≥98%, powder Citric acid ≥99% Citric acid 99% Citric acid BioUltra, anhydrous, ≥99.5% (T) Citric acid SAJ first grade, ≥99.5% Citric acid cell culture tested, anhydrous Dodecanoic acid ≥99% (GC/titration) Ergosterol ≥95.0% (HPLC) Formic acid JIS special grade, ≥98.0% Formic acid SAJ first grade, 88.0-89.5% Formic acid puriss. p.a., ~98% (T) Formic acid reagent grade, ≥95% Ginkgolide A purum, analytical standard, ≥98.0% (GC) Ginkgolide C purum, analytical standard, ≥96.0% (GC) Glycine ReagentPlus®, ≥99% (TLC) Glycine SAJ special grade, ≥99.0% Heptacosane purum, ≥98.0% (GC) Hexanoic acid ≥99.5% Hexanoic acid purum, ≥98.0% (GC) Isorhamnetin ≥95.0% (HPLC) Kaempferol ≥97.0% (HPLC) Linalool oxide purum, mixture of isomers, ≥97.0% (GC) Linoleic acid ≥99% Linolenic acid ≥99% Luteolin ≥98% (TLC), powder Myristic acid Sigma Grade, ≥99% Nicotinic acid ≥98% Nicotinic acid ≥99.5% (HPLC) Nicotinic acid SAJ special grade, ≥99.5% Nicotinic acid cell culture tested, insect cell culture tested, ~98% Nonacosane 99% Octanoic acid ≥96.0% Octanoic acid ≥98% Octanoic acid ≥99% Oleic acid ≥99% (GC) Oleic acid SAJ first grade, ≥70.0% Oleic acid suitable for cell culture, BioReagent Oxalic acid SAJ first grade, ≥97.0% Oxalic acid puriss. p.a., anhydrous, ≥99.0% (RT) Palmitic acid ≥99% Palmitic acid SAJ first grade, ≥95.0% Palmitoleic acid ≥99% (capillary GC), liquid Procyanidin B1 analytical standard Procyanidin B2 analytical standard Propionic acid 99.5% Propionic acid SAJ first grade, ≥98.0% Propionic acid insect cell culture tested, ~99% Propionic acid puriss. p.a., ≥99.5% (GC) Propionic acid reagent grade, ~99% Pyrocatechol ≥99%, powder Pyrocatechol ReagentPlus®, ≥99% Pyruvic acid 98% Quercetin 3-β-D-glucoside ≥90% (HPLC) Rutin hydrate ≥94% (HPLC), powder Rutin trihydrate purum, ≥95.0% (HPLC) Sesamin ≥95%, crystalline Shikimic acid ≥99% Sodium pyruvate ≥99%, powder, cell culture tested, insect cell culture tested Sodium pyruvate SAJ special grade, ≥95.0% Sodium succinate dibasic hexahydrate SAJ first grade, 98.0-102.0% Sodium succinate dibasic hexahydrate cell culture tested, insect cell culture tested Stearic acid CP Stearic acid Grade I, ≥98.5% (capillary GC) Stearic acid SAJ first grade, ≥90.0% Stearic acid SAJ first grade, ≥90.0%, powder Stearic acid SAJ special grade, ≥95.0% Stearic acid puriss., ≥98.5% (GC) Stigmasterol ~95% Succinic acid ReagentPlus®, ≥99.0% Succinic acid SAJ first grade, ≥99.0% Succinic acid SAJ special grade, ≥99.5% Succinic acid purum p.a., ≥99.0% (T) Sucrose ≥99.5% (GC) Sucrose JIS special grade Sucrose SAJ first grade Sucrose reagent grade Tannic acid SAJ first grade Tannic acid Source: Chinese natural gall nuts (from grass) Thiamine hydrochloride BioReagent, suitable for insect cell culture, suitable for cell culture Thiamine hydrochloride SAJ special grade, ≥98.5% Thymol ≥99.5% Thymol SAJ first grade, ≥98.0% Tricosane 99% Tricosane puriss., ≥99.0% (GC) Valeric acid ≥99% Valeric acid puriss., ≥99.0% (GC) Vanillic acid 97% Vanillic acid purum, ≥97.0% (HPLC) Xanthophyll from marigold Zeaxanthin purum p.a., ≥95.0% (TLC) α-Ionone technical grade, 90% β-Carotene Type II, synthetic, ≥95% (HPLC), crystalline β-Carotene purum, ≥97.0% (UV) β-Eudesmol ≥90% (HPLC) β-Ionone 96% β-Sitosterol synthetic, ≥95% γ-Aminobutyric acid ≥99% p-Coumaric acid ≥98.0% (HPLC) p-Coumaric acid purum, ≥98.0% (HPLC) p-Cymene 99% … etc

Merck

Chemical analysis and quality control of Ginkgo biloba leaves, extracts, and phytopharmaceuticals, T.A. van Beek, P. Montoro / J. Chromatogr. A 1216 (2009) 2002–2032

EGb 761: a standardized Ginkgo b. extract

Extract is made from Ginkgo leaves

EGb 761: ◦ 5% to 7 % Terpenlactones

◦ 22% to 27 % Flavonglykosides

◦ useful subst. (Ginkgolides, Terpenlactones,…)

◦ toxic ginkgolic acid (>5ppm),...

Standardized Extracts for Dementia treatment: ◦ EGb 761, LI 1370

traditional ◦ asthma, bronchitis, fatigue, and tinnitus

recent: ◦ improve memory; treat / prevent Alzheimer’s

disease ◦ Dementia ◦ decrease intermittent claudication (lame, limping,

leg pain b/c o. narr. arteries);

sexual dysfunction multiple sclerosis Other health conditions

National Toxicology Program: NTP ◦ Looked into- and carried out own studies: “NTP

studies” ( http://ntp-server.niehs.nih.gov/ )

best researched ◦ dementia, memory impairment,

intermittent claudication, tinnitus

Egb 761 ineffective vs ◦ Dementia, Alzheimer's ◦ slowing cognitive decline ◦ lowering blood pressure ◦ reducing hypertension

NTP Flyer (June/2010) ◦ No clear benefits of Egb 761 thus far! ◦ www.niehs.nih.gov/health/assets/docs_f_o/ntp_gingko.pdf

headache, nausea, gastrointestinal upset, diarrhea, dizziness, and/or

allergic skin reactions ◦ occasionally severe allergic reactions

Potentiates anticoagulants (slide 14)

Fresh (raw) ginkgo seeds contain ginkgotoxin ◦ Discarded in Egb 761

Flavonoids (- glycosides)

terpenoids (ginkgolide, bilobalide)

14C markers in Ginkgolide =terpenoid origin(polycyclic Hydroxylactone)

Chemical analysis and quality control of Ginkgo biloba leaves, extracts, and phytopharmaceuticals, T.A. van Beek, P. Montoro / J. Chromatogr. A 1216 (2009) 2002–2032

Fig 2. Structural formulas of ginkgolides and biolbalide

Color Atlas of Biochemistry, 3rd Edition, Jan Koolman et al, Thieme 2005

PAF: Platelet-Activating-Factor

Ginkgolide B is a strong PAF antagonist ◦ Potent & selective

possible bleeding complications in accidents?

Mostly in-vitro studies

No drug development as of 2010

May inhibit prion neurotoxicity

Bleeding and Intracerebral hemorrhage

Vascular wall decreases

Potentiates Anticoagulants (e.g. Aspirin)

Definitive

PAF antagonist

Color Atlas of Pharmacology, 3rd edition, Albrecht Ziegler, et al, Thieme

MPN short for 4- methoxypyridoxine

In Ginkgo biloba Seeds

Low amount in leaves

Toxic Convulsant ◦ Inhibits Glutamic Acid Decarboxylase

◦ GAD decarboxylates glutamate to GABA + CO2

ID: H2O extract, IPC (C18, Fluores., [fmol])

MPN a competitive inhibitor of glutamate decarboxylase GAD and other enzymes

Chemical analysis and quality control of Ginkgo biloba leaves, extracts, and phytopharmaceuticals, T.A. van Beek, P. Montoro / J. Chromatogr. A 1216 (2009) 2002–2032

In many fruits and vegetables

Human health effects?

Antifungal

Lipolysis?

UVB Prot.?

Human antitumor effects?

Present in many plants, incl. Ginkgo leaves

Sugar carriers in protein glycosylation

linearly linked 10–30 isoprene units

Common moiety: acetate, ω-units, two trans isoprene units and 10 to 20 cis-isoprene units terminal cis-isoprene unit w. ester group

Chemical analysis and quality control of Ginkgo biloba leaves, extracts, and phytopharmaceuticals, T.A. van Beek, P. Montoro / J. Chromatogr. A 1216 (2009) 2002–2032

Cytotoxic

Toxicity ◦ in HET (Hen‘s egg test) studies ◦ in vivo studies

Ginkgo presence ◦ Leaves 0.5%-5%, sarcotesta 5-10%, nut ~0.02%

ginkgolic acids:

Health effects: antitumour, antidepressant, antistressant, antiamnestic (inh. prolyl endopeptidase), antifungal, antifeedant, acaricidal, molluscicidal, insecticidal

Side effects: contact allergenic, cytotoxic, embryotoxic , immunotoxic, mutagenic, probably neurotoxic!

Wikimedia Commons, Riosnestor, 2012