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VOLATILE OILSVOLATILE OILS

Synonymous: Synonymous:

Ethereal oils, Essential oils.Ethereal oils, Essential oils.

Phytochemistry IIPhytochemistry II

Lecture (1)Lecture (1)

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Volatile oilsVolatile oils are are complex liquidcomplex liquidmixtures of mixtures of odoriferous principlesodoriferous principlesof varying of varying chemical chemical compositioncomposition, which , which easily evaporate when easily evaporate when exposed to air at exposed to air at ordinary temperature, ordinary temperature, and which are used for and which are used for either their specific either their specific therapeutic activitytherapeutic activity or or their aromatheir aroma..

VolatileVolatile or or ethereal ethereal oils oils to indicate that they to indicate that they easily easily evaporate on evaporate on exposure to air at exposure to air at ordinary temperatureordinary temperature(volatile, from Latin(volatile, from Latinvolarevolare))Essential oilsEssential oils after after the the Latin Latin essentia essentia meaning a meaning a liquid easily changed to a liquid easily changed to a gas and most probably gas and most probably because they represent because they represent the essences orthe essences orodoriferous constituentsodoriferous constituentsof the plants.

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In general, volatile oils represent In general, volatile oils represent the essences the essences or odorousor odorous principles found in various plant principles found in various plant parts. parts.

With the exceptionWith the exception of oils such as of oils such as oil winteroil wintergreengreen, , oil of mustardoil of mustard and and oil of almondoil of almond, which , which are produced by the enzymatic hydrolysis of are produced by the enzymatic hydrolysis of the corresponding glycosides, these oils are the corresponding glycosides, these oils are contained largely as such in plant.contained largely as such in plant.

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Differences between volatile and fixed oils:Differences between volatile and fixed oils:

Volatile oilsVolatile oils Fixed oilsFixed oilsVolatileVolatile nonnon-- volatilevolatile

TerpenoidsTerpenoids Triglycerides.Triglycerides.

NonNon--greasygreasy GreasyGreasy

Resinify (OResinify (O22)) Rancid (ORancid (O22))

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Concrete:Concrete: extractextract of characteristic odor, obtained of characteristic odor, obtained from a from a freshfresh starting material of vegetable origin. by starting material of vegetable origin. by extraction with nonextraction with non--aqueous solvent.aqueous solvent.

Pomade:Pomade: perfumed fatperfumed fat obtained from obtained from flowersflowers either by either by ‘’‘’cold enfleuragecold enfleurage’’’’ (i.e. Diffusion of the odoriferous (i.e. Diffusion of the odoriferous constituents of the flowers into the fat), or by constituents of the flowers into the fat), or by ‘’‘’hot hot enfleurageenfleurage’’’’ (i.e. the digestion or immersion(i.e. the digestion or immersion of the flowers in the melted fat).flowers in the melted fat).

Resinoid:Resinoid: extractextract of characteristic odor, obtained from of characteristic odor, obtained from a a drieddried starting material of natural origin, by starting material of natural origin, by extraction with extraction with non aqueous solventnon aqueous solvent. Here , starting . Here , starting material of natural origin means: material of natural origin means: vegetable, animal, or vegetable, animal, or microbial origin, including products of enzymatic microbial origin, including products of enzymatic hydrolysis.hydrolysis.

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Absolute:Absolute: product of characteristic odor, obtained from product of characteristic odor, obtained from a a concreteconcrete, a , a pomadepomade, or a , or a resinoidresinoid, by , by ethanol ethanol extraction at ambient temperature.extraction at ambient temperature.The resulting ethanol extract cooled and filtered in The resulting ethanol extract cooled and filtered in order to eliminate order to eliminate waxeswaxes; the ethanol is removed by ; the ethanol is removed by distillation.distillation.

Spices:Spices: natural plant products, free of foreign matter, natural plant products, free of foreign matter, that are used to impart flavour and aroma, and to that are used to impart flavour and aroma, and to season food; the term applies both to the season food; the term applies both to the entire entire product and to the powderproduct and to the powder

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Aroma:Aroma: the concept of aroma is at the same time the concept of aroma is at the same time different from, and broader than, that of essential oil, different from, and broader than, that of essential oil, since it applies to any since it applies to any fragrant principle emanating fragrant principle emanating from natural substances or which is generated by a from natural substances or which is generated by a physical, chemical, or enzymatic process (example physical, chemical, or enzymatic process (example include roasted coffee, grilled meat, fish, cheese).include roasted coffee, grilled meat, fish, cheese).

FlavorsFlavors are products or substances designed to be added to are products or substances designed to be added to medicines to mask or improve the taste or smell, excluding medicines to mask or improve the taste or smell, excluding substances that only have a sweet, acidic, or salty tastesubstances that only have a sweet, acidic, or salty taste

Examples of Flavors in the current European Examples of Flavors in the current European LegislationLegislation::

Natural flavors: menthol.Natural flavors: menthol.Identical flavors: synthetic vanillin.Identical flavors: synthetic vanillin.Artificial flavors: ethylvanillin.Artificial flavors: ethylvanillin.Flavoring preparationsFlavoring preparationsSmoke flavor.Smoke flavor.

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Distribution:Essential oils occur virtually only in higher plants, there are 17,500 aromatic species. Myrtaceae, Lauraceae, Rutaceae, Lamiaceae, Astraceae, Apiaceae. Cupressaceae, Poaceae, Zingiberaceae, and Piperaceae.

Essential oils accumulate in all of the types of vegetable organs:Flowers (bergamot, rose, jasmine), Leaves ( Citronella, eucalyptus)barks (cinnamon)Woods (rosewood, sandalwood)Roots (vetiver)Rhizomes (ginger)

Fruits ( anise, star anise, fennel)Seeds (nutmeg)

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Localization:Localization:Oil cellOil cell (on or near the surface of the plant) in ( (on or near the surface of the plant) in ( Lauraceae or Zingiberaceae)Lauraceae or Zingiberaceae)Glandular trichomesGlandular trichomes (lamiaceae)(lamiaceae)Secretory cavitiesSecretory cavities (Myrtaceae or Rutaceae)(Myrtaceae or Rutaceae)Secretory canalsSecretory canals (Apiaceae or Astraceae)(Apiaceae or Astraceae)

Function of Essential oils:Function of Essential oils:The biological function of essential oils remains obscureThe biological function of essential oils remains obscureAllelopathicAllelopathic, particularly germination inhibitors, particularly germination inhibitorsProtection against predatorsProtection against predators (insect and fungi)(insect and fungi)AttractionAttraction of pollinating species of pollinating species

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Economical Values of Volatile oils:Economical Values of Volatile oils:••Large quantities of volatile oil are produced annually;, it is Large quantities of volatile oil are produced annually;, it is estimated that the total world production, in metric tons, was: estimated that the total world production, in metric tons, was: for sweet for sweet orange 26,000orange 26,000, , lemon oil 2,200,and grapefruit 700 lemon oil 2,200,and grapefruit 700 for cineolefor cineole--type eucalyptus oil 3,300, for clove oil 2,000, for type eucalyptus oil 3,300, for clove oil 2,000, for cinnamon bark oil 5, and for dill oil 2.5 tons and for peppermincinnamon bark oil 5, and for dill oil 2.5 tons and for peppermint t oil 3,700. oil 3,700.

In the early 1990s, 21 essential oils had a world market greaterIn the early 1990s, 21 essential oils had a world market greaterthan 500t.than 500t.

•• The fabrication of perfumes The fabrication of perfumes is a multimillionis a multimillion--dollar industry. dollar industry. Fine perfumes are considered works of art .Fine perfumes are considered works of art . The formulasThe formulas for for these perfumes are generally wellthese perfumes are generally well--guarded secrets,guarded secrets, and and the perfumer must have an intimate knowledge of perhaps the perfumer must have an intimate knowledge of perhaps 1000 aromatic substances when formulating a perfume1000 aromatic substances when formulating a perfume.

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Economical Values of Volatile oils:Economical Values of Volatile oils:

Perfume formulationPerfume formulation has been compared to the has been compared to the creation of a creation of a musical compositionmusical composition; ;

The various odorants can be classified like musical The various odorants can be classified like musical notes into notes into top, medium, and base notes.top, medium, and base notes.

Perfumes contain elements of all 3 categoriesPerfumes contain elements of all 3 categories, which , which are blended to provide a unique, harmonious odour. are blended to provide a unique, harmonious odour.

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The top notesThe top notes are the most volatile productsare the most volatile products. They . They leave the skin rapidly and include leave the skin rapidly and include lemon, lavender, lemon, lavender, and ainse oils. and ainse oils.

Odorants with intermediate volatility and tenacity Odorants with intermediate volatility and tenacity are classifiedare classified as as middle notesmiddle notes and include and include thyme, thyme, neroli, and rose oils. neroli, and rose oils.

Base notesBase notes are products are products with low volatility and high with low volatility and high tenacity and are also describedtenacity and are also described as fixatives because as fixatives because they provide staying power for the perfume. they provide staying power for the perfume. vanillin; vanillin; musk musk

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AmbergrisAmbergris, one of the most valuable materials used by , one of the most valuable materials used by the perfumer. It is a pathologic product formed in the the perfumer. It is a pathologic product formed in the stomachstomach of of the sperm whalethe sperm whale when it is feeding on when it is feeding on squid or cuttlefish. It is thought that the indigestible squid or cuttlefish. It is thought that the indigestible beaks of these animals irritate the whalebeaks of these animals irritate the whale’’s stomach, s stomach, which in turn stimulates the formation of the which in turn stimulates the formation of the ambergris.ambergris.

MuskMusk, the dried secretion from the preputial follicles of , the dried secretion from the preputial follicles of the male the male musk deermusk deer of Asia of Asia

CivetCivet, a glandular secretion appearing in an outwardly , a glandular secretion appearing in an outwardly discharging pocket underneath the posterior appendage discharging pocket underneath the posterior appendage of both the of both the male and female civet catsmale and female civet cats and and

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Preputial Gland This gland is located on the inside of the buck's penal sheath and may be used for lubrication.

CivetCivet

Whale Genuine Ambergris Oil

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Therapeutic action: Therapeutic action:

Oil of eucalyptus: Oil of eucalyptus: Bronchitis and Mucolytic.Bronchitis and Mucolytic.

Oil of lemon:Oil of lemon: flavouringflavouring

Oil of turpentine:Oil of turpentine: starting materials for the synthesis starting materials for the synthesis of other compoundsof other compounds

Those oils with a high phenol contentThose oils with a high phenol content, e.g. , e.g. clove and clove and thymethyme have antiseptic properties, have antiseptic properties,

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Carminatives, antispasmodicCarminatives, antispasmodic activity are much used activity are much used in popular medicine as those of in popular medicine as those of Rosmarinus Rosmarinus officinalis, Mentha piperita Matricaria chamomilla, officinalis, Mentha piperita Matricaria chamomilla, Foeniculum vulgare, Carum carvi and Citrus Foeniculum vulgare, Carum carvi and Citrus aurantiumaurantium. .

Volatile oils are stated to interfere with respiration Volatile oils are stated to interfere with respiration and electron transport in a variety of bacteria, and electron transport in a variety of bacteria, hence accounting for their use in food hence accounting for their use in food preservation preservation and in cosmetic preparationsand in cosmetic preparations..

FlavoursFlavours for foods and confections and in the for foods and confections and in the spice, spice, perfume, and cosmetic trades.perfume, and cosmetic trades.

Therapeutic action: Therapeutic action:

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Physical properties of Essential OilsPhysical properties of Essential Oils1.1. Liquid at ambient temperatureLiquid at ambient temperature

2.2. Volatile, (c.f. fixed oils)Volatile, (c.f. fixed oils)

3.3. Colourless liquids with the exception of chamomile oil ( Colourless liquids with the exception of chamomile oil ( violet in colour)violet in colour)

4.4. Their density is lower than water, with the exception clove Their density is lower than water, with the exception clove or cinnamon heavier than water)or cinnamon heavier than water)

5.5. They have a high refractive index and most of them rotate They have a high refractive index and most of them rotate the plane polarized light.the plane polarized light.

6.6. They are soluble in common organic solvent and They are soluble in common organic solvent and lipolipo--soluble (soluble (lipophiliclipophilic))

7.7. Steam distilled and sparingly soluble in water; they are Steam distilled and sparingly soluble in water; they are soluble enough, however, to impart a distinct fragrance to soluble enough, however, to impart a distinct fragrance to water /aromatic water)water /aromatic water)

Chemical composition of Essential oilsChemical composition of Essential oils

Lecture 2Lecture 2

Phytochemistry II

Summary of the Previous LectureSummary of the Previous LectureWhat is volatile oil?

Volatile oil or essential oil?

Volatile oil VS Fixed oil

Concrete, Pomade, Resinoid, Absolute, Spices, Aroma, Flavors.

Essential oils accumulation:(Flowers, Leaves, barks, Woods, Roots, Rhizomes, Fruits, Seeds)

Localization: (Oil cell, Glandular trichomes, Secretory cavities, Secretory canals).

Function of Essential oils: (Allelopathic, Protection against predators, Attraction)

Economical Values of Volatile oils

The top notes, middle notes , base notes.

Musk, Civet, Ambergris.

Therapeutic action

Physical properties of Essential Oils

Summary of the Previous LectureSummary of the Previous Lecture

Lecture Content:Lecture Content:Chemical composition of Essential oilsChemical composition of Essential oils

•• Biosynthesis of Biosynthesis of terpenoidsterpenoids•• MevalonateMevalonate PathwayPathway

•• Biosynthesis of mono Biosynthesis of mono --, , sesquiterpenessesquiterpenes and and

cyclizationcyclization mechanismsmechanisms

•• Biosynthesis of Biosynthesis of phenylpropanoidsphenylpropanoids

((ShikimateShikimate Pathway)Pathway)

Volatile Oil Composition

•An essential oil is a mixture, often containing hundreds of compounds, and reflects the characteristics of a mixture.

•Individual compounds are covalently bonded volatile liquids that contribute their individual properties to the oil.

•An individual component may be harmful on its own but, when diluted and with other ingredients, it may have an additive and beneficial effect; this is an example of synergy.

•As with all mixtures, the composition of oils may vary and thus the overall characteristics of an oil may vary. The composition may also vary depending on the plant species and subspecies and such variations are genetically determined.

Volatile Oil Components

•Individual components or groups of components in a mixture can be separated by physical methods such as dissolving or distilling.

•The removal of particular useful compounds from essential oils is done on a large scale in the cosmetics, pharmaceutical and food industries, exploiting physical properties such as boiling point or solubility. This has a minimal effect on the chemical properties of the compound removed.

•An aromatherapy-grade oil should have no components removed, added or enhanced: this would constitute adulteration. Adding the blue compound chamazulene to Moroccan chamomile to make it look like the more expensive German chamomile would be such an adulteration.

Volatile Oil and Health•There is a very dangerous misconception that if a substance is natural it is not harmful, and can be used therapeutically without the fear of any side-effects.

•Essential oils are very complex mixtures of organic compounds, many of which should be used with great care.

•Their roles in the plant body are often protective and defensive, e.g. to repel invading organisms.

•In a genuine aromatherapy-grade essential oil, the more toxic components are often balanced by others that act as quenchers.

• A knowledge of the oil components is needed for their safe use and this is why it is vital to use high-grade essential oils in a controlled way.

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Essential oils are Essential oils are complex and highly complex and highly variable mixtures of variable mixtures of constituents which constituents which belong to two belong to two groupsgroups

1.1. The group of The group of terpenoidsterpenoids

2.2. The group, far less The group, far less common, of common, of aromaticaromaticcompounds derived compounds derived from from phenylpropanephenylpropane

Some essential oils Some essential oils containcontain

•• Degradation products Degradation products of nonof non--volatile volatile constituents (results constituents (results of enzymatic of enzymatic hydrolysis)hydrolysis)

•• NitrogenNitrogen containing containing compoundscompounds

•• SulphurSulphur containing containing compoundscompounds

•• PolyacetylenesPolyacetylenes

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What are What are terpenesterpenes ??

TerpenesTerpenes are defined as natural products whose are defined as natural products whose structures may be divided into structures may be divided into isoprene units.isoprene units.

Biosynthesis of Biosynthesis of terpenoidsterpenoids::

Isoprene units:Isoprene units:••arise from arise from acetateacetate via via mevalonicmevalonic acidacid

•• branchedbranched--chain, 5chain, 5--carbon units containing 2 carbon units containing 2 unsaturated bonds unsaturated bonds

IsopreneIsoprene unit

Volatile Oil and Oxidation

•The majority of essential oils have compounds that are unsaturated (i.e. they possess double or triple bonds). These multiple bonds tend to be very reactive

•Parts of the molecule that can often combine with oxygen if an oil is not stored correctly.

•In nature, many compounds are polyunsaturated, that is they havemore than one double or triple bond. They are found in vegetable oils and some vitamins, where they are thought to be beneficial in the diet for a variety of reasons.

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OPP

MeHO

CH2OH1

2543

HOCO(R)-Mevalonic acid 3-Methylbut-3-enyl

pyrophosphate

23

4 1

(Isopentenyl pyrophosphate)

Isoprene units arise fromIsoprene units arise from acetate acetate via via mevalonicmevalonic acidacid

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During the formation of During the formation of terpenesterpenes, ,

the isoprene units are linked in a headthe isoprene units are linked in a head--toto--tail fashiontail fashion

Head

Tail

Head

Tail

Isoprene

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TerpeneTerpene ClassificationClassification

••Class Name No. of Isoprene Units & Class Name No. of Isoprene Units & Carbon Number Molecular FormulaCarbon Number Molecular Formula•• Hemiterpene 1 (CHemiterpene 1 (C55 HH88))

5 5 •• MonoterpeneMonoterpene 2 (C2 (C1010 HH1616))

1010•• SesquiterpeneSesquiterpene 3 (C3 (C1515 HH2424))

1515•• DiterpeneDiterpene 4 (C4 (C2020 HH3232) )

2020•• TriterpeneTriterpene 6 (C6 (C3030 HH4848) )

3030

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••The The terpenesterpenes found most often in volatile oils found most often in volatile oils are are monoterpenesmonoterpenes and and sesquiterpenssesquiterpens. .

••They either occur in acyclic or cyclic (monocyclic, They either occur in acyclic or cyclic (monocyclic, bicyclicbicyclic or or tricyclictricyclic) forms as hydrocarbons and as ) forms as hydrocarbons and as oxygenated derivatives, such as alcohols, oxygenated derivatives, such as alcohols, aldehydesaldehydes, , ketonesketones, phenols, oxides, peroxides and , phenols, oxides, peroxides and esters.esters.

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• EleopteneEleoptene,, the hydrocarbon portion of the oil, the hydrocarbon portion of the oil, which is liquid, and which is liquid, and

•• StearoptenesStearoptenes,, the oxidised hydrocarbon portions the oxidised hydrocarbon portions of the oil, which are usually solidof the oil, which are usually solid

••The The odourodour and and tastetaste of volatile oils are mainly of volatile oils are mainly determined by these determined by these oxygenatedoxygenated constituents, constituents, which are usually appreciably soluble in water which are usually appreciably soluble in water ((e.ge.g: orange: orange--flower water, rose water, etc.) but flower water, rose water, etc.) but more soluble in alcohol (more soluble in alcohol (e.ge.g: tinctures or essences : tinctures or essences of lemon, etc.) of lemon, etc.)

The Olfactory System

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Volatile Oil and Functional Groups

•The functional group names can be used in characterizing and describing theodours of many perfumes and some of the essential oils.

•Aldehydic smelling oils are due to compounds with functional groups called aldehydes, and sweet and fruity smelling oils are due to compounds with functional groups called esters.

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THE MEVALONATE PATHWAY: THE MEVALONATE PATHWAY: TERPENOIDS AND STEROIDSTERPENOIDS AND STEROIDS••The The sixsix--carbon compound carbon compound MVAMVA is transformed into is transformed into the the fivefive--carbon carbon phosphorylatedphosphorylated isopreneisoprene units in a units in a series of reactions.series of reactions.

••First First phosphorylationphosphorylation of the primary alcohol group (2 of the primary alcohol group (2 ATPATP--dependant enzymes are involved dependant enzymes are involved (MAPP).(MAPP).

••DecarboxylationDecarboxylation / / dehydrationdehydration then follow to give then follow to give IPPIPP (A third ATP)(A third ATP)

••IPP is IPP is isomerizedisomerized to the other isoprene unit, to the other isoprene unit, (DMAPP)(DMAPP),, by an by an isomeraseisomerase enzyme which enzyme which stereospecificallystereospecifically removes the proton from removes the proton from CC22, and , and incorporates a proton from water on to incorporates a proton from water on to CC44

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CH3COSCoA)2( CH3COCH2COSCoA

FatsSugars

Acetyl co A Aceto acetyl co A

CH3COSCoA

and hydrolysisHO Me

CH2OHHOCO

2 NADPHHO Me

COSCoAHOCOS Hydroxy methylglutaryl Co enzyme A

R Mevalonic acid

THE MEVALONATE PATHWAY Biosynthesis of MVA

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CH2OH

HO Me

HOCO

HO Me

HOCO

2 ATP

OPP

ATP

O MeP

C OO

H

Not isolable

OPP3-Methylbut-3-enyl PP(isopentenyl PP) (IPP)OPP

1

23

4

THE MEVALONATE PATHWAY (continued)Conversion of MVA into IPP

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OPP3-Methylbut-3-enyl PP(isopentenylpp)

isomerase

OPP

(IPP) (C5)

Dimethylallyl pp(DMAPP) (C5)

THE MEVALONATE PATHWAY (continued)Isomerization of IPP into DMAPP

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THE MEVALONATE PATHWAY (continued)

OPP

OPP

HDMAPP IPP

OPP

trans-Geranylpyrophosphate Monoterpene (C10)

Tail

Head

DMAPP & IPP are linked in a head to tail fashion resulting in monoterpene (C10)

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THE MEVALONATE PATHWAY (continued)

OPP

OPP

trans, trans, Farnesyl pp = Sesquiterpene (C15)

OPP

Third IPP gives sesquiterpene (C15)

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MONOTERPENES (C10)MONOTERPENES (C10)

OPP

trans-Geranylpyrophosphate Monoterpene (C10)

OR

OPP

Geranyl PP (GPP)

E

1. ACYCLIC 1. ACYCLIC STRUCTURESSTRUCTURES

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•• GeranylGeranyl PP, in which the new PP, in which the new double bond isdouble bond is trans (E)trans (E)

•• LinalyLinaly PPPP and and nerylneryl PPPP are isomers of are isomers of geranylgeranyl PP, and are PP, and are likely to be formed from likely to be formed from geranylgeranyl PP by PP by ionization to theionization to theallylicallylic cationcation (see next slide)(see next slide)

•• This allow a This allow a change in the attachmentchange in the attachment of the of the diphosphatediphosphategroup group (to the tertiary carbon in (to the tertiary carbon in linalyllinalyl PP)PP)

•• Or a Or a change in stereochemistrychange in stereochemistry at the double bond at the double bond (to (to Z Z in in nerylneryl PP)PP)

OPP

Geranyl PP (GPP)

OPPE

OPP

Z

Linalyl PP (LPP)

Neryl PP (NPP)

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Formation of Formation of LinalyLinaly PP and PP and NerylNeryl PP from PP from GeranylGeranyl PP PP by ionization to by ionization to the the allylicallylic cationcation

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• GeranylGeranyl PPPP, , LinalyLinaly PPPP and and NerylNeryl PPPP can give rise can give rise to a range of to a range of linear ( ACYLIC)linear ( ACYLIC) monoterpenesmonoterpenes found found as components of volatile oilsas components of volatile oils

•• The resulting compounds may be hydrocarbons, The resulting compounds may be hydrocarbons, alcohols, alcohols, aldehydesaldehydes, or ester especially acetates., or ester especially acetates.

•• See the next slideSee the next slide

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22. MONO. MONO--CYCLIC MONOTERPENES BIOSYNTHESISCYCLIC MONOTERPENES BIOSYNTHESIS• The range of The range of monoterpenesmonoterpenes encountered is encountered is extended by extended by cyclizationcyclization reactions, and reactions, and mono, bi or mono, bi or tricyclictricyclic systems can be created.systems can be created.

•• Such Such cyclizationcyclization would not be expectedwould not be expected to occur to occur with the precursor with the precursor geranylgeranyl PPPP

•• EE stereochemistry of the stereochemistry of the double bonddouble bond being being unfavourableunfavourable for ring formation.for ring formation.

•• NerylNeryl PP or PP or linalyllinalyl PP, however, do have PP, however, do have favourablefavourable stereochemistrystereochemistry

•• Either or both would seem more immediate Either or both would seem more immediate precursors of precursors of monocyclic monocyclic menthanementhane systemsystem

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Mechanism of Mechanism of cyclizationcyclization

••MonoterpeneMonoterpene cyclasecyclase enzymesenzymes are able to are able to accept all three accept all three diphosphatediphosphate..

••LinalylLinalyl PPPP being the being the best substratebest substrate

••These enzymes first These enzymes first isomerizeisomerize the substrate as the substrate as well as to well as to cyclizecyclize themthem

••So the species involved in the So the species involved in the cyclizationcyclization as the as the delocalized delocalized allylicallylic cationcation tightly bound to the tightly bound to the diphosphatediphosphate anionanion

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CyclizationCyclization of of LinalylLinalyl PP into PP into menthyl/menthyl/αα--terpinylterpinyl cationcation

50

The possible fates of The possible fates of carbocationscarbocations

((menthylmenthyl //αα--terpinylterpinyl cationcation))1.1. Quenching with Quenching with nucleophilesnucleophiles (specially water)(specially water)

2.2. Loss of a Loss of a protonproton

3.3. The possibility for The possibility for WagnerWagner--MeerweinMeerweinrearrangements (1,3rearrangements (1,3--hydride shift).hydride shift).

4.4. Folding the cationic side chainFolding the cationic side chain towards the towards the double bond (via the double bond (via the surface characteristics of surface characteristics of the enzymethe enzyme) ) BicylicmonoterpenesBicylicmonoterpenes.

51

The possible The possible fates of fates of carbocationscarbocations

((menthylmenthyl //αα--terpinylterpinylcationcation))

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33. BICYCLIC . BICYCLIC MONOTERPENES MONOTERPENES BIOSYNTHESISBIOSYNTHESIS

Folding the Folding the cationic side cationic side chain towards the chain towards the double bond (via double bond (via the surface the surface characteristics of characteristics of the enzyme) the enzyme) BicylicmonoBicylicmono--terpenesterpenes.

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4. Biosynthesis of aromatic 4. Biosynthesis of aromatic monoterpenesmonoterpenes

• MevalonateMevalonate Pathway resulting in aliphatic HC or Pathway resulting in aliphatic HC or oxygenated oxygenated terpenesterpenes..

•• However, However, aromatic aromatic monoterpenesmonoterpenes are formed are formed through through dehydrogenation of monocyclic dehydrogenation of monocyclic terpenesterpenes..

•• Aromatic volatile components are biosynthesised Aromatic volatile components are biosynthesised through through SKIMATE PATHWAY (Phenyl Propane)SKIMATE PATHWAY (Phenyl Propane)

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P-Menthane

Dehydrogenation

P-Cymene

OH

OH

Thymol Carvacrol

Aromatic monoAromatic mono--cycliccyclic--monoterpenesmonoterpenes biosynthesis biosynthesis through dehydrogenation of monothrough dehydrogenation of mono--cyclic cyclic monoterpenemonoterpene HC HC

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A few compounds (e.g. A few compounds (e.g. PP--CymeneCymene, ,

thymolthymol, , carvacrolcarvacrol), although ), although aromaticaromatic in structure, are in structure, are terpenoidterpenoidin origin.in origin.

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SESQUITERPENES (C15)SESQUITERPENES (C15)

• FPP ( FPP ( farnesylfarnesyl PP) can give rise to linear and cyclic PP) can give rise to linear and cyclic sesquiterpenessesquiterpenes,,

••Because of the increased chain length and additional Because of the increased chain length and additional double bond, the number of possible double bond, the number of possible cyclizationcyclizationmodes is also increased, and a huge range of monomodes is also increased, and a huge range of mono--, , bibi-- and triand tri--cyclic structures can result.cyclic structures can result.

•• The double bond nearest the The double bond nearest the diphosphatediphosphate can adopt can adopt an an E, E E, E configuration ( in FPP), or a configuration ( in FPP), or a E, ZE, Zconfiguration ( in FPP )configuration ( in FPP )

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Biosynthesis of phenylpropanoid compoundsBiosynthesis of phenylpropanoid compounds

shikimic acid pathway shikimic acid pathway

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LL--phenylalaninephenylalanine LL--TyrosineTyrosine

Cinnamic acidCinnamic acid PP--Hydroxy cinnamic acidHydroxy cinnamic acid((pp--Coumaric acid)Coumaric acid)

2 glucose metabolite

Dehydration and decarboxylationInto phenylpyruvic acidphenylpyruvic acid

Dehydrogenation and decarboxylation into pp--hydroxyphenylpyruvic acidhydroxyphenylpyruvic acid

Enzymatic deamination

Biosynthesis of phenylpropanoidsBiosynthesis of phenylpropanoidsShikimic acid pathwayShikimic acid pathway

Shikimic acid Prephenic acidShikimic acid Prephenic acid

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Separation of the different components Separation of the different components constituting volatile oil. constituting volatile oil.

The components of volatile oils can be separated The components of volatile oils can be separated in various ways:in various ways:

1.Low temperatures, which crystallize out the 1.Low temperatures, which crystallize out the stearoptenes.stearoptenes.

2.Fractional distillation.2.Fractional distillation.

3.Fractional crystallization from poor solvents.3.Fractional crystallization from poor solvents.

4.Different forms of chromatography 4.Different forms of chromatography (GC, (GC, GC/MS, and HPLC).GC/MS, and HPLC).

5. Removal by chemical action.5. Removal by chemical action.

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In the last group, In the last group, Removal by chemical actionRemoval by chemical action

compounds with compounds with free acidicfree acidic groups may be groups may be removed from the oil with removed from the oil with sodium carbonatesodium carbonate, ,

basic compoundsbasic compounds may be removed with may be removed with hydrochloric acidhydrochloric acid, ,

phenolsphenols with with sodium hydroxidesodium hydroxide, ,

aldehydesaldehydes with with sodium bisulfitesodium bisulfite

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