The Shikimate Pathway It’s berry good for you! Important products of the shikimate pathway • Animals do not operate shikimate pathway, only plants & microbes do • Essential aromatic amino acids (phenylalanine, tyrosine & tryptophan) must be obtained through our diet! • Vitamins E and K have precursors from shikimate pathway Important products of the shikimate pathway • • • • • • • • • • Hydroxybenzoic acids (e.g. gallic acid) Coumarins Phenylpropanoids (e.g. cinnamic acid derivatives) Lignans Stilbenes Flavonoids Catechins Tannins Isoflavonoids Phylloquinones Formation of shikimic acid, which can be converted to an aromatic ring by reduction and dehydration, provides a precursor for many phenolics Conversion of shikimic acid to aromatic amino acids: precursors to C6C3 unit In most plants & microbes, the phenylpyruvic acids are precursors to L-Phe and Tyr. Animals do not operate the shikimate pathway and must get Phe and Tyr from their diet. In plants, L-Phe and L-Tyr are often deaminated by the enzyme PAL (phenyl ammonium lyase) to form the C6C3 cinnamic starter unit Cinnamic and p-coumaric (p-hydroxycinnamic) acids and their derivatives are very common in plants They may be functionalized on aromatic ring with additional OH or OMe groups, or the carboxylic acid group may be reduced to aldehyde or alcohol They may be found esterified with sugars, quinic acid, or terpenes. They also serve as precursors for biosynthesis of flavonoids and stilbenes Many essential oils of spices are derived from the cinnamates Coumarins are derived from o- or p-hydroxycinnamic (coumaric) acid Formation of a blood-thinning dimer consumption of warfarin-treated bait, rats die from internal haemorrhage. Other coumarin derivatives employed as rodenticides include coumachlor and coumatetralyl (Figure 4.32). In an increasing number of cases, rodents are becoming resistant to warfarin, an ability which has been traced to elevated production of vitamin K by their intestinal microflora. Origins of hydroxycinnamic acid derivatives and lignans 1) 2) 3) HSCoA NADPH NADPH HCAs may be functionalized with sugars, quinic acid amines, etc. Assembly of lignans occurs by phenolic oxidative coupling J. Agric. Food Chem (2004) 52:5843-48 Flavonoids, friends and relatives C6C3 from tyrosine + C6 from acetate The shikimate and acetate pathways combine to produce some key 2o metabolites. 4-hydroxycinnamoyl CoA is the precursor to hydroxycinnamic acids, stilbenes & flavonoids Pathway to stilbenes involves a different folding pattern than the path leading to chalcones and and flavonoids Flavanones are precursors to the other flavonoids such as flavonols Biosynthetic routes to flavonoid skeletons ALL are antioxidants; some are more effective than others based on structure Catechins (flavan-3-ols) found in tea, chocolate, berries --Antiproliferative against epidermal carcinomas --Iron chelation --Antiinflammatory --Hypolipidemic Flavonols (citrus, berries, onions) --Antiproliferative (breast, colon cancer) --Reduces platelet aggregation Anthocyanins – found in dark red foods --Reduce oxidative damage to neurons, vascular cells, antiinflammatory Polyphenols • Term encompasses all flavonoids, tannins, stilbenoids, and phenolic acids • All have varying levels of antioxidant and antiinflammatory activities as well as effects on specific biological pathways/diseases • Bioavailability of polyphenolics is a complex issue and depends on structure, metabolic transformation in small intestine, colon and liver • Berry polyphenols study (Koli, et al, 2010) reports on a clinical trial of 72 adults consuming berry products 2x daily (837mg/day total, including 62 mg phenolic acids) . Results • Plasma quercetin levels increased by 51-84% in treatment group; plasma phenolic acids (p-coumaric, caffeic, vanillic, protocatechuic) increased by 21-40% • Flavonoid metabolites (hydroxyphenylacetic acids, homovanillic acid) increased 7-31% in plasma • Urinary excretion of quercetin, p-coumaric acid and 3hydroxyphenyl acetic acid increased, but not the other phenolic acids. • The high concentrations of some metabolites suggest that their biological effects should be studied separately • Anthocyanins and PACs were not analyzed nor were tissue concentrations analyzed. Tannins Proanthocyanidins (poly-flavan-3-ols) or “condensed tannins” are oligomers of catechin or epicatechin units formed by phenolic radical coupling Ellagic acid Formed by radical coupling of two gallic acids and lactone formation A “B-type” proanthocyanidin, like those found in cacao. More on A-type PACs later… Ellagitannins contain a central ellagic acid unit esterified with sugars or more gallic acid units Tannins Pentagalloylglucose – a hydrolyzable tannin in which glucose is esterified with gallic acid Theaflavins & thearubigens: Found in fermented tea are dimeric catechin structures in which oxidative processes catalyzed by enzyme polyphenol oxidase have led to the formation of a seven membered tropolone ring. Cinchonains “Flavonolignan” Cinchonains were recently identified in leaves of bilberry, the European blueberry (Vaccinium myrtillus) A study in Journal of Ethnopharmacology (Qa’dan, et al, 2009) reported that cinchonain 1b enhances the release of insulin from pancreatic Islet cells – may play a role in treating type-2 diabetes Radical coupling of caffeic acid with epicatechin, followed by lactonization Leaves of other Vaccinium species (V. vitis-idaea and V. angustifolium) are being investigated for their mechanisms of anti-diabetic activity based on their use to treat diabetes in Cree traditional medicine in Northern Quebec. Green tea catechins (Camellia sinensis) Galloylated catechins (flavan-3-ols) from tea are some of the most wellstudied polyphenolics Content/200 mL cup EGCG = epigallocatechin gallate (142 mg) EGC = epigallocatechin (65 mg) ECG = epicatechin gallate (28 mg) GCG = gallocatechin gallate (17 mg) caffeine (76 mg) Fresh tea leaves are steamed or dried at elevated temps green tea Black tea is fermented and tends to keep longer. Catechins are oxidized to theaflavins and thearubigens. Biological activities of green tea/ EGCG • Reduces risk of esophageal, skin, stomach & other cancers • induces apoptosis in tumor cells • lowers total cholesterol, improves HDL/LDL ratio • strong antioxidant, mild antiinflammatory • brain and bone protection • accelerates weight loss? • Rat study: protection of eye from oxidative damage (J. Agric. Food Chem. 2010, 58:1523-1534) Bioavailability of tea catechins to the eye (Chu, et al, 2010) • 11 groups of rats (n=6) were fed green tea extract at dose of 550 mg/kg • Rats were sacrificed @ various timepoints • Tissues homogenized, extracted & analyzed by HPLC (no details given!) • Pharmacokinetics of biodistribution varied between the catechins vs. catechin gallates and between the tissues • Catechins were present in much higher quantity in most tissues than catechin gallates • Maximum conc. reached in 1 h for plasma, 3 – 5 h for humor, 5 – 10 h for retina, lens & cornea • Marker of oxidative stress (8-iso-PGF2a) decreased in humors, cornea & lens • Distribution routes may differ between compounds Isoflavonoids: radical rearrangement of flavonoid skeleton produces isoflavonoids, found widely in legumes, soy products Coumestrol, a coumestan found in clovers Phytoestrogenic activity estradiol • “Phytoestrogen” refers to non-steroidal plant materials having estrogenic properties. • The planar isoflavonoids and coumestans mimic the shape and polarity of the steroid hormone estradiol and are able to bind to estrogen receptors • They stimulate estrogenic response in some tissues, have opposite effect in others • Known to cause reproductive problems in grazing animals • As part of the diet they can influence overall estrogenic activity in the body • Isoflavonoid-rich foods/herbs may counter some of the side-effects of menopause • There is mounting evidence that phytoestrogens provide other beneficial effects: helping prevent heart attacks/CVD, protect against osteoporosis, lower the risk of breast and uterine cancer, etc. Antioxidants and more…Phylloquinones and tocopherols are part phenolic, part isoprenoid Coenzyme Q10: Redox carrier for electrons in human mitochondrial ETS Vitamin K1 Sources: plants, primarily green veggies Role: blood clotting – needed for carboxylation of Glutamate residues in prothrombin Inhibited by warfarins (coumadin) Vitamin E Sources: cereals, seed oils eggs, soybean, corn oil, barley --Free radical scavenger --Protects lipids in LDL and cell membranes from oxidation --decreases coronary artery lesions, but effect on CVD mortality still not proven Biosynthesis of Vitamin E: Shikimate pathway-derived 4-hydroxyphenyl pyruvic acid is alkylated with isoprenoid chain from mevalonate pathway Rings are methylated by SAM Cyclization of phenol with chain forms chroman ring Tocopherols differ in pattern of methylation on the ring Oxidation of phenolic ring to quinone forms plastoquinones, ubiquinones. Less common in food: phenolics made exclusively through acetate pathway Starter unit acetyl-CoA + Formation of non-shikimate, polyketide phenolics via the acetate pathway from a poly-b-keto ester precursor Formation of anthrones and anthraquinones (polyketide phenolics) Don’t be fooled! Compare these structures to that of the flavonoids – polyketide vs. shikimate-derived St. John’s Wort Hypericum perforatum Anthrones and their derivatives are found in numerous herbs/botanicals including Senna, Cascara and Aloe. They act as laxatives/purgatives a constituent of St. John’s Wort