asaph aharoni, department of plant sciences ulmann ... · 12 – cinnamic acid 13 – naringenin...

Frontiers in METABOLOME Sciences

Asaph Aharoni, Department of Plant SciencesUlmann Building, room 105Tel: 3643E.mail: [email protected]

Frontiers in METABOLOME Sciences

Code: 20063212Credits: 1.5Thursday 14:00-16:00, Wolfson Room 18

- Every class, seminar on a topic- 40-45 min. followed by questions and discussion

- End of semester, minireview 3-6 pages on the topic

The MetabolomeThe Metabolome::

Is a comprehensive profile of all the Is a comprehensive profile of all the low molecular weight compounds low molecular weight compounds of an organism, a tissue, or a of an organism, a tissue, or a cellcell

Oliver et al., (1998)Oliver et al., (1998)

GENOMEGENOME

Transcriptome

Proteome

Metabolome

PHENOTYPEPHENOTYPE

Gene

mRNA

Protein

Product

Levels of regulation

Life’s Complexity PyramidOltavai & Barabasi,

Science, 2002

The cell is a complex network in which the components are connected by functional links

What will we hear and talk about?

Recent advances in Metabolome:

- ANALYSIS / MEASURMNET

- REGULATION

- EVOLUTION

- APPLICATIONS

- ENGINEERING

Network of Metabolic Pathways in a Single Cell

Estimated Size of the MetabolomeEstimated Size of the Metabolome

-- In yeast In yeast 550550 compounds in a cellcompounds in a cell

-- In plants In plants 4,000 to 20,0004,000 to 20,000 compounds in a compounds in a cell cell

-- To date, a total of To date, a total of 200,000200,000 structures structures identified from plantsidentified from plants

KEGG: 13,803 compounds, 6579 reactions KEGG: 13,803 compounds, 6579 reactions

The MetabolomeThe Metabolome

1.1. The Metabolome is organism, The Metabolome is organism, tissue and cell specifictissue and cell specific

2.2. Influenced by the environmentInfluenced by the environment

The Metabolome of Human, E.coli & ArabidopsisThe Metabolome of Human, E.coli & Arabidopsis

According to HumanCyc ):http://HumanCyc.org/(

35 metabolic pathways are common to all three organisms

ANALYSING the METABOLOME, ANALYSING the METABOLOME, WHY?WHY?-- Assessing gene function and relationship to phenotypesAssessing gene function and relationship to phenotypes

-- Understanding metabolism and predicting novel pathwaysUnderstanding metabolism and predicting novel pathways

-- To increase metabolite fluxes into valuable biochemical To increase metabolite fluxes into valuable biochemical pathways using metabolic engineeringpathways using metabolic engineering

-- To compare genetically modified organisms to nonTo compare genetically modified organisms to non--modifiedmodified

-- To measure flux of carbon under varying conditionsTo measure flux of carbon under varying conditions

-- To asses the effect of environmental changesTo asses the effect of environmental changes

ANALYSINGANALYSING the METABOLOMEthe METABOLOME

-- MoreMore complicated compared to complicated compared to protein and nucleic acids:protein and nucleic acids:

•• Unknown Unknown pathwayspathways•• Often difficult to purifyOften difficult to purify•• Can be impossible to synthesizeCan be impossible to synthesize•• No No amplificationamplification

ANALYSING the METABOLOMEANALYSING the METABOLOME

Different metabolites, different characteristics:Different metabolites, different characteristics:•• volatilevolatile•• non volatile: polar, semi polar and apolar non volatile: polar, semi polar and apolar


Making the difference:Elemental compositionOrder of the atomsType of bondsStereochemical orientation

(+)-Carvone

Oקימל

(-)-Carvone

Oנענה


-- Ideally:Ideally: The The comprehensivecomprehensive, , quantitative and quantitative and qualitativequalitative analysis of all metabolites within a cell, analysis of all metabolites within a cell, tissue or an organismtissue or an organism

-- Far from reality in any systemFar from reality in any system

-- Multiple technologies are required Multiple technologies are required

Analysing the METABOLOMEAnalysing the METABOLOME

1.1. Metabolite ExtractionMetabolite Extraction2.2. Metabolite Metabolite (separation not(separation not always) always)

detectiondetection3.3. Data analysis:Data analysis:-- From raw data to information which is From raw data to information which is

ready for miningready for mining-- Extraction of biological relevanceExtraction of biological relevance-- Data visualization (maps, tables, charts Data visualization (maps, tables, charts

etc..)etc..)

Analysing the METABOLOMEAnalysing the METABOLOME

• Metabolite target analysis: just a few specific compounds

• Metabolic profiling: classes of compounds

• Metabolic fingerprinting: with pattern recognition

• Metabolomics: as comprehensive as possible

Fiehn, Comp. Func. Genomics 2,155-168 (2001)

Metabolome Analyses Technologies- Infrared spectroscopy (IR)- Nuclear magnetic resonance (NMR)- Thin layer chromatography (TLC)- High performance liquid chromatography (UV & photodiode array)(LC/UV/PDA)- Capillary electrophoresis coupled to UV absorbance detection (CE/UV)- Capillary electrophoresis coupled to laser induced fluoresence detection(CE/LIF)- Capillary electrophoresis coupled to mass spectrometry (CE/MS)- Gas chromatography mass spectrometry (GC/MS)- Liquid chromatography mass spectrometry (LC/MS)- Liquid chromatography tandem mass spectrometry (LC/MS/MS)- Fourier transform ion cyclotron mass spectrometry (FTMS)- HPLC coupled with MS and NMR detection (LC/NMR/MS)- LC/NMR/MS/MS

Metabolite Target Analysis

Tomato peel

s mixture’Standard Naringenin Chalcone Standard

12

13

13 14

15

Tomato peel

12 – Cinnamic acid13 – Naringenin Chalcone14 – Naringenin15 – Kaempferol

m/z100 200 300 400 500 600 700 800 900 1000 1100

%

0

100

%

0

1001: TOF MS ES+

2.35e4273.0797

147.0500

274.0554

275.0623357.0263 583.1375 855.1465629.0711 987.4974 1151.2333

1: TOF MS ES+ 7.90e3273.0789

153.0202

274.0617

357.0297 583.1320 855.1419651.2269 1011.27051060.0111Time5.70 5.80 5.90 6.00 6.10 6.20 6.30 6.40

%

0

100

5.70 5.80 5.90 6.00 6.10 6.20 6.30 6.40

%

0

100Tom_8_WTa3 1: TOF MS ES+

TIC1.37e4

6.02

5.75

MixAllStand_b1 1: TOF MS ES+ TIC

8.16e36.02

5.93

5.69

6.106.15

6.466.41

Metabolic profilingHPLC chromatograms (recorded at 520 nm) of anthocyanins of red ripe strawberries ACHENES: 1 = cyanidin-3-glucoside; 2 = pelargonidin-3-glucoside; 3 = pelargonidin-3-rutinoside; 4 = cyanidin-3-glucoside-malonate; 5 = pelargonidin-3-glucoside-malonate .

Metabolic FingerprintingInvestigation of the Mode of Action of the Phytotoxin (5S,8R,13S,16R)-(-)-Pyrenophorol Using (1)H Nuclear Magnetic Resonance Fingerprinting (Konstantinos A. Aliferis and Maria Chrysayi-Tokousbalides, JAFC, 2006)

Phyt

otox

ic c

ompo

unds

Metabolic FOOTprinting or Exometabolome

“A Strategy for analyzing the properties of cells or tissues by looking in a high-throughput manner at the metabolites that they excrete or fail to take up from their surroundings (Kell et al, 2005).”

• Similar to fingerprinting, based on pattern recognition• In Fingerprinting intracellular metabolites analyzed while in Footprinting the culture media (as in the case of yeast).• Stimulating metabolic changes by adding various carbon compounds or inhibitors

Metabolomics and Other OMICS ApproachesMetabolomics and Other OMICS Approaches

• Transcriptomics: Monitor the expression levels of tens of thousands of genes

• Proteomics: Monitor abundance patterns of thousands of proteins

• Metabolomics: Monitor thousands of low molecular weight metabolites simultaneously

• Bio-informatics: Processing data and extracting biological meaning

Metabolomics: A. Thaliana (QA. Thaliana (Q--TofTof--MS)MS)

a ra b L E R

1 0 0 1 5 0 2 0 0 2 5 0 3 0 0 3 5 0 4 0 0 4 5 0 5 0 0 5 5 0 6 0 0 6 5 0 7 0 0 7 5 0 8 0 0 8 5 0 9 0 0 9 5 0 1 0 0 0m /z0

1 0 0

%

a ra b 3 5 8 3 1 (0 .6 5 9 ) C m (1 7 :8 7 ) T O F M S E S + 5 .3 7 e 31 0 4 .1 1 0 2

5 3 6 9

1 5 4 .9 7 1 84 5 7 1

1 2 2 .0 8 6 42 9 0 0

1 7 2 .9 7 9 94 4 6 2

2 3 0 .9 8 4 54 3 5 1

2 1 9 .0 3 2 51 9 8 4

2 9 8 .0 4 5 43 3 0 0

2 8 2 .0 7 0 81 3 1 7

3 8 1 .0 8 5 92 9 1 9

3 7 9 .0 4 9 51 7 6 6

3 1 6 .0 5 3 81 3 4 6

3 6 3 .0 6 3 46 4 7

4 2 7 .0 8 2 11 0 7 5

4 4 4 .0 7 0 58 6 9

6 1 7 .1 3 6 96 0 4

4 9 7 .0 8 7 84 0 6 5 4 1 .2 6 8 2

2 9 7 5 8 5 .3 0 3 82 4 2

6 6 3 .1 3 1 84 0 8

m/z

124.0100699

123.0848198

126.9717219124.9933

180127.0315

128

m/z

1000100

inte

nsity

MetaboMetabo--ll--omics and Metaboomics and Metabo--nn--omicsomicsNicholson et al. 1999• Metabonomics: “the quantitative measurement of the time –

related multi-parametric response of living systems to pathophysioloical stimuli or genetic modification”

Fiehn,• Metabolomics: “the comprehensive and quantitative analysis of

all metabolites”Approaches are the same, Nomics more metabolic response to

drugs and diseases (animal systems and with NMR) while Lomics more bacterial/plants (with MS (GC, LC)

Metabolome Analysis and NutritionMetabolome Analysis and Nutrition- Nutritional Metabolomics

- Regulation of metabolic pathways and networks by nutrients and other food components

- Establishment of analytical methods that profile human serum and urinary metabolites to assess nutritional imbalances and disease risk

- Coordination between FOOD SCIENCE and METABOLOMICS

Metabolome Analysis and NutritionMetabolome Analysis and Nutrition

Extrinsic Factors

DietDrugs

Physical activity

Colonic floraStress

Body compositionTissue turnoverAge

Health statusReproductive status

Intrinsic Factors

Tissue turnover

Resting metabolic rate

Genotype

Diurnal cycleThe Human Metabolome

Factors that could effect the HUMAN MetabolomeFactors that could effect the HUMAN Metabolome

Metabolome Analysis and NutritionMetabolome Analysis and Nutrition

The Human METBOLOME According to HumanCyc Prediction:

- Enzyme genes: 2,742- Compounds: 661

- If the numbers are so:

Personal Metabolomics is possible

Metabolome Analysis and CancerMetabolome Analysis and CancerTumor Metabolomics in recent years it was based on NMR (non invasive) for clinical diagnosis.

Metabolic profiles of human brain tumors using quantitative in vivo 1H magnetic resonance spectroscopy (MRS; Howe et al., 2003).

Metabolome Analysis and CancerMetabolome Analysis and Cancer• Try to understand the metabolic phenotype of tumor cells

• Interrelationship between respiration and glycolysis investigated for decades

• Hypoxia Inducible Factor-1 (HIF-1), widely expressed in a range of cancers, seems to be of large importance

• HIF-1 controls the response to oxygen and coordinated the up-regulation of genes involved in many metabolic pathways concerned with tumor growth and metabolism

• Metabolomics might explain the differences between tumor and normal cells

MetabolomicsMetabolomics--

Next weekNext week’’s class and visit to s class and visit to GCGC--MS and LCMS and LC--MS instruments MS instruments in WISin WIS

Metabolome Diversity and EvolutionMetabolome Diversity and Evolution

The role of Promiscuous Protein FunctionThe role of Promiscuous Protein Function

Promiscuous activity in a protein is also Promiscuous activity in a protein is also termed:termed:

substrate ambiguitysubstrate ambiguitycross reactivitycross reactivitymoonlighting activitymoonlighting activity

Too few genes, too many metabolites?Too few genes, too many metabolites?-- In plants more than 200,000 structures have been In plants more than 200,000 structures have been identifiedidentified-- Every plant estimated to containEvery plant estimated to contain-- 4k4k--20k20k-- Diversity in modification of the same backbone Diversity in modification of the same backbone structurestructure-- Example: 300 different glycosides of the Example: 300 different glycosides of the flavonol quercitinflavonol quercitin-- In Arabidopsis according to AraCyc:In Arabidopsis according to AraCyc:1900 genes encoding enzymes with defined 1900 genes encoding enzymes with defined functionfunction

Too few genes, too many metabolites?Too few genes, too many metabolites?

How to explain METABOLOME size/diversity?How to explain METABOLOME size/diversity?

a.a. DNA level: Alternative reading frames and gene DNA level: Alternative reading frames and gene fusionfusion

b.b. mRNA level: Alternative splicingmRNA level: Alternative splicingc.c. Protein: Post translational modification and Protein: Post translational modification and

Hetrodimer formationHetrodimer formation

AndAnd………… Enzyme Specificity!Enzyme Specificity!

Plant Secondary Metabolism: Plant Secondary Metabolism: An Excellent ExampleAn Excellent Example

Alcohol acylAlcohol acyl--CoA transferasesCoA transferases

One One enzymeenzyme……....MultipleMultiple substratessubstrates……....Multiple Multiple productsproducts

Volatile Composition of Cultivated StrawberryVolatile Composition of Cultivated Strawberry

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Ester Furan Terpene Lactone

Volatile Esters Contribute to the Aroma of Most Fruit

>100 esters92 esters

• Some are responsible for a particular fruit aroma (e.g. banana)

Metabolic Route for the Formation of Esters in Fruit

Precursors

Alcohols

Aldehydes

Acids (e.g acetic acid)

Alcohols Acyltransferase

Fatty acids

Amino acids

ReductionOxidation

Beta-oxidation

CoA

Adapted and modified from Olias Et al. In : R. L. Rouseff and M. M. Leahy (eds.) Fruit Flavours. Biogenesis, charachterization and authentication (1995).

ESTERS

Ester Formation by Alcohol Acyltransferase (AAT)Ester Formation by Alcohol Acyltransferase (AAT)

OSR1

CoAR1 O

OR2+ OH R2

AAT

Acetyl-CoA +Ethylalcohol

Isoamylalcohol

Ethylacetate

Isoamylacetate

AAT

Ester Formation by Ester Formation by SAATSAAT Expressed in Expressed in E.coli E.coli CellsCells

.

4 107

Retention Time (min)

Det

ecto

r R

espo

nse

(%)

F

E

G

D

C

B

AButanol Butyl acetate

100

100

100

100

100

100

100

0

0

0

0

0

0

0

In B:

+Butanol Acetyl CoA

Butylacetate

Broad substrate specificity (alcohols)

0

10

20

30

40

50

60

70

80

90

100

methano

leth

anol

1-propanol

iso-pr

opano

l1-buta

nol2-buta

nol

isoam

ylalcoh

ol1-hexa

nol

trans-

2-hexen-1-ol

cis-2-

hexen-1-ol

trans-

3-hexen-1-ol

cis-3-

hexen-1-ol

heptanol

octanol

nonanol

decanol

linalo

ol

2-phenyle

thylalco

holbenzyl

alcoho

l

furfur

ylalco

hol

Act

ivity

, % o

f max

imum

(oct

anol

)

Alcohol

Broad Substrate Specificity (acyl CoAs)

Acyl CoAs (Carbon no.) Alcohol Ester formed Ester propertiesn-propionyl CoA (C3:0) 1-butanol 1-butyl propanoate ethereal, bananan-butyryl CoA (C4:0) 1-propanol 1-propyl butyrate sharp, pungent, rancid, sweaty,

sickeningn-butyryl CoA (C4:0) 1-butanol 1-butyl butyrate fruity, pineappleisobutyryl CoA (C4:0) 1-butanol 1-butyl isobutyrate fruity, apple, banana and

pineapplen-crotonoyl CoA (C4:1) 1-butanol 1-butyl crotoate not describedn-hexanoyl CoA (C6:0) 1-propanol 1-propyl hexanoate wine-like, cheesen-decanoyl CoA (C10:0) 1-butanol 1-butyl decanoate Brandy (Whisky-Cognac)-like

odorbenzoyl CoA (C7:0) 1-butanol 1-butyl benzoate mild floral-balsamic odor

SAAT is a member of a small gene family in strawberry responsible for generating more than 100 different type of esters in the ripe fruit

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Ester Furan Terpene Lactone


Terpene CyclasesTerpene Cyclases

One One enzymeenzyme……....OneOne substratesubstrate……....Multiple Multiple productsproducts


Trees such as Grand Fir) אשוח :(--Produce oleoresin in response to stem wounding Produce oleoresin in response to stem wounding and insect attackand insect attack--The turpentine fraction of the oleoresin contains The turpentine fraction of the oleoresin contains terpenes, mainly mono, sesqui and diterpenesterpenes, mainly mono, sesqui and diterpenes-- In Grand Fir, 38 sesquiterpenes (12.5% of In Grand Fir, 38 sesquiterpenes (12.5% of turpentine) and the remaining monoterpenesturpentine) and the remaining monoterpenes-- Two terpene synthases expressed in E.coli could Two terpene synthases expressed in E.coli could synthesize three major products but in total 35 and synthesize three major products but in total 35 and 53 total sesquiterpenes.53 total sesquiterpenes.

Steele et al., 1998

Metabolome Diversity and EvolutionMetabolome Diversity and Evolution

-- Promiscuous function of proteins seems to be a Promiscuous function of proteins seems to be a major step in the evolution of new protein major step in the evolution of new protein functions (Aharoni et al., 2005)functions (Aharoni et al., 2005)-- Conflict between the need for mutations for a Conflict between the need for mutations for a new function and the deleterious effect of new function and the deleterious effect of mutations on survivalmutations on survival-- Therefore mutations causing promiscuous Therefore mutations causing promiscuous functions are staring points (no loss of original functions are staring points (no loss of original function)function)-- Subsequently gene duplication and a new Subsequently gene duplication and a new protein occursprotein occurs

Metabolome Metabolome RegulationRegulation and Evolutionand Evolution

--Past and recent years several discoveries of RNA Past and recent years several discoveries of RNA molecules as regulatory elements in cellular processes:molecules as regulatory elements in cellular processes:

-- antisenseantisense-- tRNA tRNA –– mRNA interactionsmRNA interactions-- translation control by temp. dependent translation control by temp. dependent

modulation of RNA structuremodulation of RNA structure-- microRNAs, siRNAsmicroRNAs, siRNAs

Metabolome Metabolome RegulationRegulation by by Riboswitches Riboswitches

-- Newly discovered natural elements located at Newly discovered natural elements located at untranslated regions of mRNAs called untranslated regions of mRNAs called ““RiboswichesRiboswiches””

-- Mainly at the 5Mainly at the 5’’ endend

-- Regulate gene expression and translation by binding Regulate gene expression and translation by binding small metabolitessmall metabolites

-- Up to date, none of them required protein factors for Up to date, none of them required protein factors for metabolite bindingmetabolite binding

Metabolome Metabolome RegulationRegulation by by RiboswitchesRiboswitches- Suggested to be descendents of the ancient metabolite sensors in the RNA world

- In the RNA world, RNA handled storage of genetic information (instead of DNA) and also expression (instead of proteins)

- Genes controlled by Riboswitches (RBS) most often encode proteins involved in the biosynthesis or transport of the metabolites being sensed (feedback loop)

The Two Components of Riboswitch Functions

• Direct (protein-free) binding of metabolite to RNA (aptamer)• Metabolite-dependent regulation of genes (expression platform)

… an ‘expression platform’ for gene regulation.

A metabolite-binding‘aptamer’ domain and …

UUUUU

X

3 421

Metabolome Metabolome RegulationRegulation by by RiboswitchesRiboswitches

Ribosome Binding Site Sequestration

Terminator of transcription formed

Riboswitch Ligands

Metabolome Metabolome RegulationRegulation by by RiboswitchesRiboswitchesNearly 2% of the genes of the model organism Bacillus subtilisappear to be controlled by riboswitches

Metabolome in a Specific Cell types & Single CellMetabolome in a Specific Cell types & Single Cell

The Metabolome profile could be very different The Metabolome profile could be very different between cellbetween cell--typestypes

Could even be different between cells of the same Could even be different between cells of the same typetype

Can we measure the differences in such a Can we measure the differences in such a resolution?resolution?

OH

R-Linalool R = OCH 3Eugenol

OH

R

R = H Estragole R = OCH3 Methyl Eugenol

OCH3

R

GLANDS in BasilGLANDS in Basil

BALUTOT

Old leavesYoung leaves

Most secondary metabolites in Basil are produced in the Peltate Glands

Peltate Glands Capitate Glands

Peltate Glands Isolated From Sweet Basil

Active Pathways in Peltate Glands (Sweet Basil)

(Iijima et al, 2004)

Terpenoids in Peltate Glands (Sweet Basil)

Monoterpenes Sesquiterpenes

Phenylpropans in Peltate Glands (Sweet Basil)

Methyl Eugenol R = OCH 3Eugenol

OH

R

R = H Estragole R = OCH3

OCH3

R

The Metabolome of Vascular Bundles (Schad et al., 2005)The Metabolome of Vascular Bundles (Schad et al., 2005)

• Using laser microdissection for collecting tissue specific samples

• Standard tissue fixation and embedding protocols do not allow metabolite analysis(fix and dehydrate steps results in loss of small molecules)

• Used cryosectioning- preserving cellular structure without solute exchange steps

The Metabolome of Vascular Bundles (Arabidopsis)The Metabolome of Vascular Bundles (Arabidopsis)

The Metabolome of Vascular BundlesThe Metabolome of Vascular Bundles


Vascular bundles tissue Sections without vascular bundles


• From 100 collected vascular bundles (approx. 5,000 cells), 68 metabolites identified

• Half of the identified, enriched or depleted in vascular bundles compared to the surrounding tissue

Metabolic EngineeringMetabolic Engineering

Systematically induce biological changes that will produce desired cellular properties

Metabolic EngineeringMetabolic EngineeringJust a few of the possibilities:- Block enzyme activity- Enhance enzyme activity- Use transcription factors- Use feedback insensitive genes- Ectopic gene expression- Introduce/block multiple genes- Use directly chemicals/proteins/RNA and not genes

Metabolic Engineering of Flavors from Metabolic Engineering of Flavors from Strawberry fruit to ArabidopsisStrawberry fruit to Arabidopsis

800

900

1000

1100

1200

1300

1400

0

10000

20000

ime

nero

lido

l

800

900

10 00 11 00 12 00 13 00 14 000

10000

20000

ime

Abu

ndan

ce

ransgenicrabidopsis

ild typerabidopsis

Abu

ndan

ce

lina

lool

Linalool

Nerolidol

Introduced product: Introduced product: linaloollinaloolModified by endogenous Modified by endogenous enzymes:enzymes:–– P450 hydroxylation (2P450 hydroxylation (2--

3)3)–– Double bond reductionDouble bond reduction–– Glycosylation (2Glycosylation (2--3)3)

OH

OH

OH

OH

OH

OH

OH

S-Linalool

E-8-Hydroxylinalool Z-8-Hydroxylinalool

E-8-Hydroxy-6,7-dihydrolinalool

GlycosideGlycoside

Glycoside

Un Expected Effects When Conducting Un Expected Effects When Conducting Metabolic EngineeringMetabolic Engineering

Metabolic Engineering Needs Fine Tuning

Metabolic Engineering and MetabolomicsMetabolic Engineering and Metabolomics

-- Metabolic analysis to identify key points Metabolic analysis to identify key points of metabolic regulationof metabolic regulation

-- Metabolic analysis to check predictions Metabolic analysis to check predictions and generate hypothesis and strategyand generate hypothesis and strategy

Genetically Modified Organisms (GMOs)Genetically Modified Organisms (GMOs)

- Public concern about GMOs

- Can we use Metabolome Analysis to determine similarities or differences between GMO to non-GMOs


'substantial equivalence'

This means that if a GM food can be characterized as substantially equivalent to its 'natural' antecedent (chemically), it can be assumed to pose no new health risks and hence to be acceptable for commercial use


GMO tomato fruit overproducing

flavonoids

NMR profiles discriminates between GMO and Non-GMO

Le Gall et al., 2003

See you next week !

asaph aharoni, department of plant sciences ulmann ... · 12 – cinnamic acid 13 – naringenin...

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