Lecture 10 Flavonoids, BIOL 439

BIOL 439 FLAVONOIDS 11/7/2024 Okechukwu Igboeli, Department of Biology https://www.openfit.com/what-are-flavonoids  Flavonoids ▪ One of the largest and most important groups of allelochemicals ▪ Some allelopathic – Some mutually beneficial ▪ Large family of chemical...

BIOL 439 FLAVONOIDS 11/7/2024 Okechukwu Igboeli, Department of Biology https://www.openfit.com/what-are-flavonoids  Flavonoids ▪ One of the largest and most important groups of allelochemicals ▪ Some allelopathic – Some mutually beneficial ▪ Large family of chemicals with many functions ▪ > 3000 compounds characterized to date ▪ From acetate (via malonate) and phenylalanine ▪ Most from higher plants; animals do not make them (e.g., flavonoids that give butterflies their color come from diet) ▪ Most are water soluble ▪ Absorb UV and visible light Flavonoids PAGE 1  Flavonoids ▪ Important class of plant-derived natural products ▪ Have polyphenolic structure and widely found in fruits, vegetables and certain beverages ▪ Possess several favourable biochemical and antioxidant effects relevant in cancer, Alzheimer’s disease, atherosclerosis, etc. ▪ Also has a broad spectrum of general health-promoting effects investigated and exploited in nutraceutical, pharmaceutical, medicinal and cosmetic industries challenge since not single chemical ▪ Based on their antioxidative, anti-inflammatory, anti-mutagenic and anti- carcinogenic properties and ability to modulate some important cellular enzyme functions Flavonoids Hibisus used in kombutha PAGE 2 Flavanoidssunersistic  Flavonoids In all parts of the plant Flower pigments UV absorbing compounds in leaf epidermis radiation Free radical scavengers Protect againstdamaging amor then conferneath Phytoalexins – fungicides (allelopathy) fi i Can be used for taxonomy iiieTompound tia bantinif can be allelopathic innaming Flavonoids PAGE 3  Flavonoids ▪ Are potent inhibitors of some enzymes e.g., xanthine oxidase (XO), cyclooxygenase (COX), lipoxygenase and phosphoinositide3-kinase inflammation formins ▪ Used by plants for growth and defense cut acid ▪ Belong to class of low-molecular-weight phenolic compounds widely distributed in the plant kingdom ▪ Very common in higher plants ▪ A lot of them are easily recognized as flower pigments but are found in all parts of plants almost everything we consume ▪ Commonly found in plant-derived foods and beverages (hence referred to as dietary flavonoids) e.g., fruits, vegetables, tea, cocoa and wine rich source for medicinal Flavonoids PAGE 4 isoflavanoid in legumes  Flavonoids ▪ There are several subgroups: chalcones, flavones, flavonols and isoflavones unique to specific sources ▪ Onions and tea are major food sources of flavonols and flavones isoflavanes ▪ Have several biological roles in plants, animals and bacteria ▪ Responsible for colour and aroma of flowers, attract pollinators, promote fruit dispersion, seed & spore germination, growth & development of seedlings ▪ Protect plants from different biotic and abiotic stresses acting as unique UV filters signal transduction ▪ Function as signal molecules, allopathic compounds, phytoalexins, detoxifying agents & antimicrobial defensive compounds dependingon exposure to lightproducescompound Flavonoids PAGE 5 inducingcompound  Flavonoids ▪ Play a role in drought resistance as well as heat acclimatization and freezing tolerance ▪ General positive effects on human and animal health and huge opportunity for disease treatment an itdeficient it eietenstmant.tt ▪ Up to 6000 flavonoids are known to contribute to the colourful pigments of fruits, herbs, vegetables and medicinal plants Flavonoids PAGE 6  Flavonoids Scarlet + UV Absorbing The > 3000 Crimson flavonoids Purple l share the same skeletons colour Bring about UV Absorbing UV Absorbing UV Absorbing UV Absorbing Flavonoids PAGE 7  Flavonoids Precursor to The > 3000 all flavonoids flavonoids share the same skeletons Flavonoids PAGE 8  The major classes of flavonoid Class # of known Role of pigmentation structures1 Anthocyanins 260 Provides cyanic colour from scarlet, red, mauve to blue Aurones 20 Visible yellow colour and/or UV patterning Chalcones 90 in yellow flowers Yellow flavonols 40 Colourless flavones and 1600 Copigments to anthocyanins. Body to flavonols cream/ivory flowers. UV patterning in some yellow flowers. Flavanones 200 Mostly colourless but may interact with Dihydroflavonols 70 other flavonoids; may act as UV screen in Dihydrochalcones 35 leaves; some are anti-feedant or anti- microbial in activity. Leucoanthocyanidins 40 Catechins 70 Flavans 30 Isoflavonoids 650 1Including aglycones and glycosides but excluding oligomers and polymers. Flavonoids PAGE 9  > 3000 flavonoids from 24 skeletons Quiz slide Most common modifications ▪ Most are conjugated to sugars at the hydroxy groups on the rings Glycosides give water solubility Many of the anthocyanins, flavones, flavonols and flavonones are glycosylated ▪ Many are O-methylated and O-acylated at hydroxyls Makes the compounds lipophilic want to be in the middle to be Many flavonols and anthocyanins a good duns ▪ Many chelate metals use as anti to Affects their spectral properties Smetals ▪ Can be dimerized and oligomerized Flavonoids PAGE 10 Charcoal used for chelate to toxin  Flavonoids Classification ▪ Subdivided into subgroups based on the carbon of the C ring on which the B ring is linked, degree of unsaturation and the oxidation of the C ring ▪ When the B ring is linked in position 3 of the C ring → isoflavones ▪ If the B ring is linked in position 4 → neoflavonoids ▪ If the B ring is linked in position 2 → several subgroups based on structural features of the C ring. Flavonoids anthocyanins and chalcones PAGE 11 3T ▪ flavones, flavonols, flavanones, flavanonols, flavanols or catechins, subgroups could be  Flavonoids 2 2 Classification 3 3 Flavanone Flavonoids PAGE 12 mn  Flavonoids – Correction Classification Flavonoids PAGE 13  Flavonoids Classification: Flavones ▪ Found in leaves, flowers & fruits as glucosides ▪ Sources include celery, parsley, red peppers, chamomile, mint and ginkgo biloba ▪ Examples: luteolin, apigenin and tangeritin ▪ Citrus fruit peels contain high concentrations of poly-methoxylated flavones: tageretin, nobiletin and sinensetin Flavonoids PAGE 14  Flavonoids Classification: Flavanones ▪ Also known as dihydroflavones and mostly present in citrus fruits such as oranges, lemons and grapes ▪ Examples include hesperitin, naringenin & eriodictyol antioxidant ▪ Linked with several health benefits due to their free radical-scavenging activities ▪ Responsible for the bitter taste of the juice and peel of citrus fruits ▪ Beneficial health effects: antioxidant, anti-inflammatory, blood lipid-lowering & cholesterol-lowering agents Flavonoids PAGE 15  Flavonoids Quiz slide Classification: Isoflavonoids ▪ Very unique subgroup of flavonoids ▪ Limited distribution in the plant kingdom and commonly found in soyabeans and other legumes ▪ Have been reported in microbes ▪ Play important role as precursors for synthesis of phytoalexins in plant microbe interactions ▪ Has significant potential to fight several diseases ▪ Genistein and daidzein shown to possess oestrogenic activity in certain animal models stimulate Flavonoids PAGE 16  Flavonoids Classification: Neoflavonoids ▪ Different structurally, because flavonoids have a 2-phenylchromen-4-one backbone, while neoflavonoids have a 4-phenylchromen backbone with no hydroxyl group substitution at position 2. ▪ First member isolated from natural sources in 1951 was calophyllolide (Calophyllum inophyllum seeds). ▪ Also found in bark and timber of the common Sri Lankan tree, Mesua thwaitesii Flavonoids PAGE 17  Flavonoids Classification: Flavanols ▪ Also called dihydroflavonols or catechins ▪ They are 3-hydroxy derivatives of flavanones ▪ Unlike many flavonoids, there is no double bond between positions 2 and 3 x▪ Abundant in bananas, apples, blueberries, peaches & pears Flavonoids PAGE 18  Flavonoids Classification: Anthocyanins ▪ Pigments responsible for colours in plants, flowers and fruits. ▪ Cyanidin, delphinidin, malvidin, pelargonidin & peonidin are the most studied ▪ Occur predominantly in the outer cell layers of various fruits - cranberries, black currants, red grapes, merlot grapes, raspberries, strawberries, blueberries, bilberries and blackberries ▪ Used widely in the food industry fruitsuitnTocolour ▪ The colour depends on the pH as well as methylation or acylation at the hydroxyl groups on the A and B rings Flavonoids PAGE 19  Flavonoids Quiz slide Classification: Chalcones ▪ Characterized by the absence of ‘ring C’ of the basic flavonoid skeleton structure ▪ Also referred to as open-chain flavonoids ▪ Examples: phloridzin, arbutin, phloretin & chalconaringenin ▪ Abundantly found in tomatoes, pears, strawberries, bearberries & wheat products ▪ Have numerous nutritional and biological benefits Flavonoids PAGE 20 Flavonoid classes Don't memorize Flavonoids Panche, Diwan & Chandra 20216 PAGE 21 Ii eatin fits Flavonoids: classes & dietary sources Flavonoids PAGE 22 Panche, Diwan & Chandra 20216  Flavonoids: research opportunities ▪ Anti-cholinesterase activity ▪ Anti-inflammatory activity ▪ Steroid-genesis modulators ▪ Xanthine oxidase modulators ▪ Countering antibiotic resistance ▪ Disease-combating activity Flavonoids PAGE 23  Flavonoids: research opportunities Mechanisms ▪ Radical scavenging: Flavonoid (OH) + r(O) + RH ▪ Xanthine oxidase inhibition ▪ Anti-inflammation ▪ Combating neurodegenerative diseases combat amyloiddeposition Flavonoids PAGE 24 Flavonoids: Functions and applications diabetestreatment Flavonoids Panche, Diwan & Chandra 20216 PAGE 25  Flavonoids The > 3000 flavonoids share the same skeletons 12 classes of compounds - Based on oxidation state of central pyran ring 24 different skeletons remember commital step and starting point Flavonoids PAGE 26  Biosynthesis of flavonoids Production of the >3000 compounds can be classified into four groups of reactions 1. Phenylpropanoid pathway a) Phenylalanine to 4-coumaroyl-CoA b) 4-coumaroyl-CoA is a precursor to a number products – Including flavonoids and lignins allelochemical structurehardtodigest 2. 4-coumaroyl-CoA + 3 malonyl-CoA to chalcone (committal step) COO- OH NH3+ O HO OH Other + 3 -OOC CH2CSCoA Flavonoids Malonyl-CoA OH O Phe Chalcone normallytonsiluclization 3. Oxidation and reduction of the central pyran ring to the 24 flavonoid skeletons 4. Modification of flavonoids by glycosylation, methylation, acylation, metalation and oligomerization PAGE 27 Flavonoids  nm Biosynthesis of flavonoids 1. Steps to 4-Coumaroyl-CoA 0 0 Can be used to make flavonoids or lignin Flavonoids mn PAGE 28  Biosynthesis of flavonoids CoAS Condensation of 3 malonyl- Carboxylation of Acetyl-CoA CoA with 1 coumaroyl-CoA or Caffoyl-CoA (1 extra hydroxy) by chalcone synthase 2. Formation of Chalcone CoAS- Concerted Reaction on one enzyme – Chalcone synthase Committal step in Flavonoid synthesis Flavonoids  Biosynthesis of flavonoids 2. Formation of Chalcone completeYsed Hugging hiEmplethose Flavonoids  Biosynthesis of flavonoids Flavonoids PAGE 31  Biosynthesis of flavonoids Flavonoids  Biosynthesis of flavonoids 1 rearrangement 4 3. Oxidation and reduction of 3 [O] pyran ring 5 P450 6 wherehappens [O] Series of oxidations and reductions of the central reduction 7 pyran ring to form the 12 8 groups (and 24 skeletons) reduction 4. Flavonoids now modified by [O] glycosylation, methylation, metalation, oligomerization Flavonoids PAGE 33 9  Biosynthesis of Enzymes flavonoids Precursor biosynthesis I Acetyl-CoA carboxylase II Phenylalanine ammonia-lyase III Cinnamate 4-hydroxylase IV 4-Coumarate:CoA ligase Synthesis of flavonoid classes 1 Chalcone synthase 2 Chalcone isomerase 3 2-Hydroxyisoflavanone synthase 4 Flavone synthase 5 Flavonone 3-hydroxylase 6 Flavonol synthase 7 Dihydroflavonol 4-reductase 8 Flavan-3,4-cis-diol 4-reductase 9 Anthocyanidin/flavonol 3-D-glucosyl transferase Flavonoid modification 1 B-ring hydroxylases 2 Glycosyl transferases 3 Methyl transferases 4 Acyl transferases Flavonoids 5 Sulphate transferases  Flavonoid occurrence and function Localization in Plants ▪ Flowers Anthocyanins Flavonones as co-pigments Usually in epidermal layers of petals Close to surface where they can contribute best to color Flavonoids PAGE 35  Flavonoid occurrence and function Localization in Plants ▪ Leaves Anthocyanin pigmentation and other flavonoids Purplish color; e.g., in some ornamental plants Red cabbage, red onions Usually in epidermal layers They reflect blue and red with interference of chlorophyll to give purple color ▪ UV absorbing pigments In epidermal layers of leaves Also, in mesophyll as free radical scavengers Flavonoids PAGE 36  Flavonoid occurrence and function cps Localization within the plant cell ▪ Usually in the vacuole of the cell – especially Vacuole for flower coloring and UV protection ▪ However, biosynthesis occurs in the cytoplasm ▪ Enzymes may be associated with tonoplast membrane (the membrane around the vacuole) Nucleus Flavonoids PAGE 37  Flavonoid occurrence and function Localization within the plant cell ▪ During the final stage of biosynthesis, flavonoids transported into the vacuole (e.g., when the glycosyl group is attached) ▪ In vacuole, aggregates of flavonoids may form – anthocyanoplasts for flower coloring ▪ Flavonoids also in chloroplasts (cps) and cytoplasm – important for free radical scavenging ▪ Extracellular flavonoids – in waxy layers and cuticles (methylated flavonoids) – fungicides & bactericides T.se EEttoTetitintocell Flavonoids PAGE 38  Flavonoid function Flower coloring ▪ Cyanic color – Stability of color and basis of color variety OH ▪ Main pigments - anthocyanins HO O + OH OH ▪ Positive charge in pyran ring can be attacked by OH- ▪ Color lost if attacked ▪ pH of vacuole is acidic (pH ~ 4.5), somewhat protecting the + charge ▪ However, even at pH 4.5, still enough OH- to attack + charge ▪ To preserve color, additional protection of + charge is necessary – done by aggregation of the molecules Flavonoids PAGE 39  Flavonoid function Quiz Mechanisms of slide anthocyanin stabilization in vivo via aggregation 2 4 types of aggregates 1 3 4 Pilots anerate me charge Eng Flavonoids  Flower coloring Self-association ▪ Solutions of anthocyanins deviate from Beer’s Law as anthocyanin concentration increases ▪ A = cl  = extinction coefficient; c = concentration; l = path length ▪ As concentration increases, Absorbance (A) saturates ▪ Either number of molecules in solution is not increasing with concentration or  is changing or both A does not increase with of male rules Flavonoids PAGE 41  Flower coloring Self-association ▪ If aggregation (self-association) is occurring, effective concentration does not increase in proportion to number of molecules (dimers, trimers, etc., absorb as single molecules) ▪ That is, aggregates absorb as a single molecule. A Abs does not go up with each molecule added into solution (also  is changing as aggregates have absorbance spectrum shifted to longer wavelength, max) [Anthocyanin] ▪ For aggregates to form, 4’ hydroxy must be free ▪ Sugars at 3 and 5 positions increase association properties Flavonoids PAGE 42  Flower coloring Quiz slide Self-association ▪ They stack in a planer fashion; promoting − interactions and hydrophobic interactions ▪ Association protects the + charge from OH- (hydroxide) attack Intermolecular co-pigmentation ▪ Variation on a theme – e.g., anthocyanins associate with flavonones ▪ Still protects anthocyanin + charge ▪ Will also change spectral properties of the pigments in the complexes – shifts max just like with anthocyanin aggregates ▪ Again, held together by − interactions and hydrophobic interactions ▪ Glycosyls again promote association Flavonoids PAGE 43  Flower coloring Intermolecular co-pigmentation ▪ Exceptionally stable pigments ▪ Acylated through glycosyl groups ▪ Acyl group is an aromatic ring (e.g., coumaroyl, caffoyal or sinapoyl) ▪ Still protects anthocyanin + charge ▪ Will also change spectral properties of the pigments in the complexes – shifts max just like with anthocyanin aggregates ▪ Have very deep blue colors – Heavenly blue Metal Complexes ▪ Same idea as above, with metals (usually Mg +2) forming even bigger complexes Flavonoids Each different aggregate will have a different color  Cyanoplasts Anthocyanin w/acyl group Blue color of Flavonone A Gentian Sage (Salvia patens) F Meta Intermolecular co-pigmentation From: Chiral Molecular Recognition on Formation of a Metalloanthocyanin: A Supramolecular Metal Complex Pigment from Blue Flowers of Salvia patens. T Kondo,* K-I Oyama, and K Yoshida. Angew. Chem. Int. Ed. Flavonoids 2001, 40, 894-897.  Cyanoplasts Anthocyanin – Blue Flavonone – Yellow Mg – Red dot Flavonoids PAGE 46  Flower coloring Yellow flower coloring Antho abs 400 to 500 nm Carot abs 450 to 550 nm ▪ From a combination of anthocyanins and A A carotenoids 400 500 600 700 400 500 600 700 Abs together 400 to 550 nm Transmission is A 550 to 600 nm; ▪ Combination gives a sharp yellow sharp yellow color 400 500 600 700 Flavonoids  UV protection in plants ▪ Flavonoids that absorb UVB (especially flavonols) are synthesized in response to UVB ▪ In epidermal layers of leaves ▪ Absorb strongly at 290 to 350 nm, screening mesophyll from UVB ▪ Sunlight Photosynthetically Active Radiation (also visible) 400 nm to 700 nm ▪ 2000 mol m-2 s-1 UVA 320 nm to 400 nm ▪ 200 mol m-2 s-1 UVB 290 nm to 320 nm ▪ 20 mol m-2 s-1 ▪ UVB is increasing from loss of ozone layer ▪ Paper from reading list: Wilson et al. Flavonoids PAGE 48  Sunlight spectrum Flavonoids Wavelength (nm)  Cross section of a leaf EE Upper Flavanols epidermis anthmycin bestdefense neefriptnittosynthes don't let the Mesophyll thingin Lower Flavonoids PAGE 50 epidermis Artificial sunlight meetrese meet Effitas based made on what y lightitisto exposed Flavonoids UVB induction of flavonoids in Brassica napus (canola) originallymade Grow plants under these three from Rapeseed light Treatments for 2 weeks Extract flavonoids from leaves didn'ttaste Analyze for amounts and ID of good structures with HPLC and MS gteneotf.FI aifigt.ci fi itcotpdiidmakecanoiao to Flavonoids HPLC analysis of flavonoids HPLC analysis of flavonoids Increases in peaks 1, 2,11, and 12 Loss of other peaks Flavonoids Mass spec identification of flavonoids Flavonoids PAGE 54 Example of MS fragmentation pattern of a flavonoid Kaempferol-3G-Fer Fragments and where wy aven the tracing Flavonoids PAGE 55 Flavonoid identification with authentic material used known compound to identify Flavonoids PAGE 56 Spectral quality of flavonols Flavonoids PAGE 57 UVB induction of flavonoids in Brassica napus (canola) Inotinduced Flavonoids PAGE 58 Flavonoids PAGE 59