Phytochemistry II Lecture (1)

VOLATILE OILS
Synonymous: Ethereal oils, Essential oils.
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Volatile oils are

complex liquid mixtures of odoriferous principles of varying chemical composition, which easily evaporate when exposed to air at ordinary temperature, and which are used for either their specific therapeutic activity or their aroma.

Volatile or ethereal oils to indicate that they easily evaporate on exposure to air at ordinary temperature (volatile, from Latin volare) Essential oils after the Latin essentia meaning a liquid easily changed to a gas and most probably because they represent the essences or odoriferous constituents 2 of the plants.

In general, volatile oils represent the essences or odorous principles found in various plant parts. With the exception of oils such as oil winter green, oil of mustard and oil of almond, which are produced by the enzymatic hydrolysis of the corresponding glycosides, these oils are contained largely as such in plant.

Differences between volatile and fixed oils:

Volatile oils
Volatile Terpenoids Non-greasy Resinify (O2)

Fixed oils
non- volatile Triglycerides. Greasy Rancid (O2)

Concrete: extract of characteristic odor, obtained from a fresh starting material of vegetable origin. by extraction with non-aqueous solvent. Pomade: perfumed fat obtained from flowers either by cold enfleurage (i.e. Diffusion of the odoriferous constituents of the flowers into the fat), or by hot enfleurage (i.e. the digestion or immersion of the flowers in the melted fat). Resinoid: extract of characteristic odor, obtained from a dried starting material of natural origin, by extraction with non aqueous solvent. Here , starting material of natural origin means: vegetable, animal, or microbial origin, including products of enzymatic 5 hydrolysis.

Absolute: product of characteristic odor, obtained from a concrete, a pomade, or a resinoid, by ethanol extraction at ambient temperature. The resulting ethanol extract cooled and filtered in order to eliminate waxes; the ethanol is removed by distillation. Spices: natural plant products, free of foreign matter, that are used to impart flavour and aroma, and to season food; the term applies both to the entire product and to the powder

Aroma: the concept of aroma is at the same time different from, and broader than, that of essential oil, since it applies to any fragrant principle emanating from natural substances or which is generated by a physical, chemical, or enzymatic process (example include roasted coffee, grilled meat, fish, cheese). Flavors are products or substances designed to be added to
medicines to mask or improve the taste or smell, excluding substances that only have a sweet, acidic, or salty taste

Examples of Flavors in the current European Legislation:

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

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: Oil cell (on or near the surface of the plant) in ( Lauraceae or Zingiberaceae) Glandular trichomes (lamiaceae) Secretory cavities (Myrtaceae or Rutaceae) Secretory canals (Apiaceae or Astraceae) Function of Essential oils: The biological function of essential oils remains obscure Allelopathic, particularly germination inhibitors Protection against predators (insect and fungi) Attraction of pollinating species
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Economical Values of Volatile oils:

Large quantities of volatile oil are produced annually;, it is estimated that the total world production, in metric tons, was: for sweet orange 26,000, lemon oil 2,200,and grapefruit 700 for cineole-type eucalyptus oil 3,300, for clove oil 2,000, for cinnamon bark oil 5, and for dill oil 2.5 tons and for peppermint oil 3,700. In the early 1990s, 21 essential oils had a world market greater than 500t. The fabrication of perfumes is a multimillion-dollar industry. Fine perfumes are considered works of art . The formulas for these perfumes are generally well-guarded secrets, and the perfumer must have an intimate knowledge of perhaps 1000 aromatic substances when formulating a perfume. perfume
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Economical Values of Volatile oils: Perfume formulation has been compared to the creation of a musical composition;

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

Perfumes contain elements of all 3 categories, which are blended to provide a unique, harmonious odour.
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The top notes are the most volatile products. They leave the skin rapidly and include lemon, lavender, and ainse oils.

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

Base notes are products with low volatility and high tenacity and are also described as fixatives because they provide staying power for the perfume. vanillin; musk
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Musk, the dried secretion from the preputial follicles of the male musk deer of Asia Civet, a glandular secretion appearing in an outwardly discharging pocket underneath the posterior appendage of both the male and female civet cats and Ambergris, one of the most valuable materials used by the perfumer. It is a pathologic product formed in the stomach of the sperm whale when it is feeding on squid or cuttlefish. It is thought that the indigestible beaks of these animals irritate the whales stomach, which in turn stimulates the formation of the ambergris.
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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.

Civet

Whale Genuine Ambergris Oil

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Therapeutic action:
Oil of eucalyptus: Bronchitis and Mucolytic. Oil of lemon: flavouring Oil of turpentine: starting materials for the synthesis of other compounds Those oils with a high phenol content, e.g. clove and thyme have antiseptic properties,

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Therapeutic action:
Carminatives, antispasmodic activity are much used in popular medicine as those of Rosmarinus officinalis, Mentha piperita Matricaria chamomilla, Foeniculum vulgare, Carum carvi and Citrus aurantium. Volatile oils are stated to interfere with respiration and electron transport in a variety of bacteria, hence accounting for their use in food preservation and in cosmetic preparations. Flavours for foods and confections and in the spice, perfume, and cosmetic trades.
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Physical properties of Essential Oils

1. Liquid at ambient temperature 2. Volatile, (c.f. fixed oils) 3. Colourless liquids with the exception of chamomile oil ( violet in colour) 4. Their density is lower than water, with the exception clove or cinnamon heavier than water) 5. They have a high refractive index and most of them rotate the plane polarized light. 6. They are soluble in common organic solvent and liposoluble (lipophilic) 7. Steam distilled and sparingly soluble in water; they are soluble enough, however, to impart a distinct fragrance to 17 water /aromatic water)

Phytochemistry II Lecture 2 Chemical composition of Essential oils

Summary of the Previous Lecture

What 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 Lecture

Lecture Content:
Chemical composition of Essential oils
Biosynthesis

of terpenoids

Mevalonate Pathway Biosynthesis of mono -, sesquiterpenes and cyclization mechanisms

Biosynthesis of phenylpropanoids (Shikimate 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.

Essential oils are complex and highly variable mixtures of constituents which belong to two groups 1. The group of

Some essential oils contain Degradation products of non-volatile constituents (results of enzymatic hydrolysis) Nitrogen containing compounds Sulphur containing compounds Polyacetylenes
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terpenoids
2. The group, far less common, of aromatic compounds derived from

phenylpropane

Biosynthesis of terpenoids:
What are terpenes ?
Terpenes are defined as natural products whose structures may be divided into isoprene units.

Isoprene units:
arise from acetate via mevalonic acid
branched-chain, 5-carbon units containing 2

unsaturated bonds

Isoprene unit

Isoprene 26

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 have more 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.

Isoprene units arise from acetate via mevalonic acid

HO Me
2 1 3 4 5
4 3 2 1

OPP

CH2OH (R)-Mevalonic acid

HOCO

3-Methylbut-3-enyl pyrophosphate

(Isopentenyl pyrophosphate)
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During the formation of terpenes, the isoprene units are linked in a head-totail fashion
Head Tail Isoprene Head

Tail
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Terpene Classification
Class Name Carbon Number Hemiterpene 5 Monoterpene 10 Sesquiterpene 15 Diterpene 20 Triterpene 30 No. of Isoprene Units & Molecular Formula 1 (C5 H8) 2 3 4 6 (C10 H16) (C15 H24) (C20 H32) (C30 H48)
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The terpenes found most often in volatile oils

are monoterpenes and sesquiterpens.

They either occur in acyclic or cyclic (monocyclic, bicyclic or tricyclic) forms as hydrocarbons and as oxygenated derivatives, such as alcohols, aldehydes, ketones, phenols, oxides, peroxides and esters.

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 Eleoptene, the hydrocarbon portion of the oil, which is liquid, and Stearoptenes, the oxidised hydrocarbon portions of the oil, which are usually solid The odour and taste of volatile oils are mainly determined by these oxygenated constituents, which are usually appreciably soluble in water (e.g: orange-flower water, rose water, etc.) but more soluble in alcohol (e.g: tinctures or essences of lemon, etc.)
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The Olfactory System

Volatile Oil and Functional Groups

The functional group names can be used in characterizing and describing the odours 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: TERPENOIDS AND STEROIDS

The six-carbon compound MVA is transformed into the five-carbon phosphorylated isoprene units in a series of reactions. First phosphorylation of the primary alcohol group (2 ATP-dependant enzymes are involved (MAPP). Decarboxylation / dehydration then follow to give IPP (A third ATP) IPP is isomerized to the other isoprene unit, (DMAPP), by an isomerase enzyme which stereospecifically removes the proton from C2, and 35 incorporates a proton from water on to C4

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THE MEVALONATE PATHWAY

Sugars (2) CH3COSCoA Acetyl co A
CH3COSCoA
and hydrolysis

Biosynthesis of MVA

Fats CH3COCH2COSCoA Aceto acetyl co A HO Me

2 NADPH

HO Me

HOCO

COSCoA HOCO

CH2OH

S Hydroxy methylglutaryl Co enzyme A

R Mevalonic acid
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THE MEVALONATE PATHWAY (continued)

HO Me
2 ATP

HO

Me
ATP

Conversion of MVA into IPP

HOCO P O H O C

CH2OH

HOCO

OPP

Me
3 4 2 1

OPP

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

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Not isolable

THE MEVALONATE PATHWAY (continued)

Isomerization of IPP into DMAPP

OPP 3-Methylbut-3-enyl PP (isopentenylpp) (IPP) (C5)

isomerase

OPP Dimethylallyl pp (DMAPP) (C5)

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THE MEVALONATE PATHWAY (continued)

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

OPP DMAPP Head H IPP

OPP trans-Geranylpyrophosphate Monoterpene (C10)

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THE MEVALONATE PATHWAY (continued)

OPP
Third IPP gives sesquiterpene (C15)

OPP

OPP trans, trans, Farnesyl pp = Sesquiterpene (C15)

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MONOTERPENES (C10)
1. ACYCLIC STRUCTURES
OPP trans-Geranylpyrophosphate Monoterpene (C10)
OR

E
OPP

Geranyl PP (GPP)

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 Geranyl PP, in which the new double bond is trans (E)

E
OPP

OPP

OPP Geranyl PP (GPP) Linalyl PP (LPP) Neryl PP (NPP)

Linaly PP and neryl PP are isomers of geranyl PP, and are likely to be formed from geranyl PP by ionization to the allylic cation (see next slide) This allow a change in the attachment of the diphosphate group (to the tertiary carbon in linalyl PP) Or a change in stereochemistry at the double bond (to Z in neryl PP) 43

Formation of Linaly PP and Neryl PP from Geranyl PP by ionization to the allylic cation

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 Geranyl PP, Linaly PP and Neryl PP can give rise to a range of linear ( ACYLIC) monoterpenes found as components of volatile oils The resulting compounds may be hydrocarbons, alcohols, aldehydes, or ester especially acetates. See the next slide

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2. MONO-CYCLIC MONOTERPENES BIOSYNTHESIS

The range of monoterpenes encountered is

extended by cyclization reactions, and mono, bi or tricyclic systems can be created. Such cyclization would not be expected to occur with the precursor geranyl PP E stereochemistry of the double bond being unfavourable for ring formation. Neryl PP or linalyl PP, however, do have favourable stereochemistry Either or both would seem more immediate precursors of monocyclic menthane system
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Mechanism of cyclization
Monoterpene cyclase enzymes are able to accept all three diphosphate. Linalyl PP being the best substrate These enzymes first isomerize the substrate as well as to cyclize them So the species involved in the cyclization as the delocalized allylic cation tightly bound to the diphosphate anion
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Cyclization of Linalyl PP into menthyl/-terpinyl cation

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The possible fates of carbocations (menthyl /-terpinyl cation)

1. Quenching with nucleophiles (specially water) 2. Loss of a proton 3. The possibility for Wagner-Meerwein rearrangements (1,3-hydride shift). 4. Folding the cationic side chain towards the double bond (via the surface characteristics of the enzyme) Bicylicmonoterpenes. Bicylicmonoterpenes
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The possible fates of carbocations (menthyl /terpinyl cation)

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3. BICYCLIC
MONOTERPENES BIOSYNTHESIS
Folding the cationic side chain towards the double bond (via the surface characteristics of the enzyme)

Bicylicmonoterpenes. terpenes
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4. Biosynthesis of aromatic monoterpenes

Mevalonate Pathway resulting in aliphatic HC or

oxygenated terpenes. However, aromatic monoterpenes are formed through dehydrogenation of monocyclic terpenes. Aromatic volatile components are biosynthesised through SKIMATE PATHWAY (Phenyl Propane)

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Aromatic mono-cyclic-monoterpenes biosynthesis through dehydrogenation of mono-cyclic monoterpene HC

Dehydrogenation

P-Menthane

P-Cymene

OH OH

Thymol

Carvacrol

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A few compounds (e.g. P-Cymene, thymol, carvacrol), although aromatic in structure, are terpenoid in origin.

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SESQUITERPENES (C15)
FPP ( farnesyl PP) can give rise to linear and cyclic

sesquiterpenes, Because of the increased chain length and additional double bond, the number of possible cyclization modes is also increased, and a huge range of mono-, bi- and tri-cyclic structures can result. The double bond nearest the diphosphate can adopt an E, E configuration ( in FPP), or a E, Z configuration ( in FPP )
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Biosynthesis of phenylpropanoid compounds shikimic acid pathway

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Biosynthesis of phenylpropanoids
Shikimic acid pathway 2 glucose metabolite Shikimic acid Prephenic acid

Dehydration and decarboxylation Into phenylpyruvic acid

Dehydrogenation and decarboxylation into p-hydroxyphenylpyruvic acid

L-phenylalanine
Enzymatic deamination

L-Tyrosine

Cinnamic acid

P-Hydroxy cinnamic acid (p-Coumaric acid)

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Separation of the different constituting volatile oil.

components

The components of volatile oils can be separated in various ways: 1.Low temperatures, which crystallize out the stearoptenes. 2.Fractional distillation. 3.Fractional crystallization from poor solvents. 4.Different forms GC/MS, and HPLC). of chromatography (GC,

5. Removal by chemical action.

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In the last group, Removal by chemical action compounds with free acidic groups may be removed from the oil with sodium carbonate, basic compounds may be removed with hydrochloric acid, phenols with sodium hydroxide, aldehydes with sodium bisulfite

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