Chapter Two

TERPENES
2.1 INTRODUCTION

Terpenes are a large and diverse class of organic compounds, produced by

a variety of plants, particularly conifers though also by some insects such as
termites or swallowtail butterflies, which emit terpenes from their osmeterium.
They are often strong smelling and thus may have had a protective function. They
are the major components of resin, and of turpentine produced from resin. The
name "terpene" is derived from the word "turpentine". When terpenes are
modified chemically (by oxidation or rearrangement of the carbon skeleton), the
resulting compounds are generally referred to as terpenoids. Terpenoids are also
known as isoprenoids.
Some authors use the term "terpenes" more broadly, to include the
terpenoids. These terpenes are frequently found in plant essential oils which
contain the "Quinta essentia", the plant fragrance. They are universally present in
small amounts in living organisms, where they play numerous vital roles in plant
physiology as well as important functions in all cellular membranes. Terpenes and
terpenoids are the primary constituents of the essential oils of many types of
plants and flowers. Essential oils are used widely as natural flavor additives for
food, as fragrances in perfumery, and in traditional and alternative medicines such
as aromatherapy. Synthetic variations and derivatives of natural terpenes and
terpenoids also greatly expand the variety of aromas used in perfumery and
flavors used in food additives. Vitamin A is an example of a terpene. Terpenes are

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released by trees more actively in warmer weather, acting as a natural form of
cloud seeding. The clouds reflect sunlight, allowing the forest to regulate its
temperature. The aroma and flavor of hops, highly desirable in some beers, comes
from terpenes.

Terpenes may be defined as a group of molecules whose structure is based on various

but definite number of isoprene units (2-methylbuta-1,3-diene, named hemiterpene,
with 5 carbon atoms).

The terpenes have been prized for their essential oils and their use as fragrances for
over two thousand years. An archaeological investigation in Egypt in 1997 unearthed
boswellic acids from the resin of frankincense (Boswellia spp.) dating from 400 to
700 AD. Records from the Middle Ages of terpene-based essential oils were
preserved, and chemical analysis of the oils began early in the nineteenth century.
Commerce in essential oils and aromatherapy continues today. For example, rose
(Rosa spp.) fragrance has enchanted many. Bulgarian rose oil requires over 4000 kg of
petals to produce 1 kg of steam-distilled oil. Over 260 constituents have been
identified, many of which are olfactory relevant. It should be apparent that even the
simple terpenes found in fragrances have a considerable amount of structural
diversity.Fortunately, despite their diversity, the terpenes have a simple unifying
feature by which they are defined and by which they may be easily classified. This

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generality, referred to as the isoprene rule, was postulated by Otto Wallach and it
states that Terpene skeleton are formed by linking together isoprene units through
carbon atoms 1 and 4 (head- to-tail), carbon atoms 1 and 1 (head-to-head), carbon
atoms 4 and 4 (tail-to-tail) or combination thereof.
This rule describes all terpenes as having fundamental repeating five- carbon
isoprene units. The head-to-tail arrangement is most common. This isoprene rule has
proved to be of great value in deriving the structure of terpenes. Thus, terpenes are
defined as a unique group of hydrocarbon- based natural products that possess a
structure that may be hypothetically derived from isoprene, giving rise to structures
that may be divided into isopentane (2-methylbutane) units. The actual biosynthetic
route to terpenes is not quite so simple. Two different biosynthetic pathways
produce the main terpene building block, isopentenyl diphosphate (IPP). The first is
referred to as either the MEP (methylerythritolphosphate) or DOX (1- deoxy-D-
xylulose) pathway. Here, IPP is formed in the chloroplast, mainly for the more
volatile mono and diterpenes. The second biosynthetic route is known as the MVA
(mevalonic acid) pathway. This takes place in the cytosol, producing
sesquiterpenes. Terpenoids are extraordinarily diverse but they all originate through
the condensation of the universal phosphorylated derivative of hemiterpene,
isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) giving
geranyl pyrophosphate (GPP).

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2.2. Isolation of Terpenes

Terpenes are isolated from essential oils present in plants. The essential oil is
extracted from the plant tissues by four methods: (1) steam distillation; (2)
digestion with solvents; (3) expression; and (4) adsorptionin purified fats.
The steam distillation is the one most widely used. The plant tissue is macerated and
then steam distilled. If a particular terpene is decomposed under these conditions, it
may be removed by extracting with light petrol at 50°C and the solvent distilled
under reduced pressure. Alternatively, the method of adsorption in fats may be
employed. For example, the flower petals are spread over molten fat until the

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latter is saturated with essential oil. The fat is then digested with ethanol to
remove the essential oil from it. Essential oils obtained from plants as above
usually contain a number of terpenes which are separated by fractional distillation
or chromatography. Gas chromatography has been particularly useful.

2.3. Properties of Terpenes

Physical
Most of the terpenes are colourless fragrant liquids having a boiling point between
150°C and 200°C. They are lighter than water and are readily volatile in steam.
They dissolve in organic solvents, but usually not in water. Most of them are
optically active although there is no prevailing direction of rotation; some are
dextro, others are laevo.

Chemical
Most of the terpenes being unsaturated hydrocarbons, are highly reactive. They
undergo addition reactions with hydrogen bromide, bromine, hydrogen, nitrosyl
chloride and ozone. They also form characteristic addition compounds with NO2
and NOBr, which are used in their identification. Most terpenes are oxidised easily
and tend to resinify upon exposure to air.

2.4 Classification of Terpenes

A rational classification of the terpenes has been established based upon the number
of isoprene (or isopentane) units incorporated in the basic molecular skeleton; a
prefix in the name indicates the number of terpene units needed to assemble the
molecule. The number of five-carbon units they contain thus classifies terpenes:

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 Hemiterpenes (C5) consist of a single isoprene unit. Isoprene itself is considered
the only hemiterpene, but oxygen-containing derivatives such as prenol and
isovaleric acid are hemiterpenoids.

 Monoterpenes (C10) consist of two isoprene units and have the

molecular formula C10H16. Examples of monoterpenes are:
geraniol, limonene and terpineol.

 Sesquiterpenes (C15) consist of three isoprene units and have the molecular formula
C15H24. Examples of sesquiterpenes are: farnesenes, farnesol. (The sesqui prefix
means one and a half).

 Diterpenes (C20) are composed for four isoprene units and have the molecular
formula C20H32. They are derived from geranylgeranyl pyrophosphate. Examples
of diterpenes are cafestol, kahweol, cembrene and taxadiene (precursor of taxol).
Diterpenes also form the basis for biologically important compounds such as retinol,
retinal, and phytol. They are known tobe antimicrobial and antiinflammatory.

 Sesterterpenes (C25), terpenes having 25 carbons and five isoprene units, are rare
relative to the other sizes. (The sester prefix means half to three, i.e. two and a half).
An example of a sesterterpene is geranylfarnesol.

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 Triterpenes (C30) Consist of six isoprene units and have the molecular formula
C30H48. The linear triterpene squalene, the major constituent of shark liver oil, is
derived from the reductive coupling of two molecules of farnesyl pyrophosphate.
Squalene is then processed biosynthetically to generate either lanosterol or
cycloartenol, the structural precursor to all the steroids.

 Tetraterpenes (C40) Contain eight isoprene units and have the molecular
formula C40H64. Biologically important tetraterpenes include the acyclic lycopene,
the monocyclic gamma-carotene, andthe bicyclic alpha-carotene and beta-carotene.

 Polyterpenes Consist of long chains of many isoprene units. Natural rubber

consists of polyisoprene in which the double bonds are cis. Some plants produce a
polyisoprene with trans double bonds, known as gutta-percha.

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2.4.1 Monoterpenes: C10
A bewildering assortment of isoprene-based decane arrangements exist in
nature. This gives the term “terpenoid” a particularly elastic meaning and is
reminiscent of some of the current combinatorial efforts employed in the
pharmaceutical industry. The monoterpenoids are the major component of many
essential oils and, as such, have economic importance as flavors and perfumes. They
are the terpenes that have been known for several centuries as components of the
fragrant oils obtained from leaves, flowers and fruits. Monoterpenes, with
sesquiterpenes, are the main constituents of essential oils. While a few, such as
camphor, occur in a near pure form, most occur as complex mixtures, often of isomers
difficult to separate. These essential oils have numerous actions, such as
allelochemical functions between plants as well as between plants and predators, a
role in wound healing and many monoterpenes possess antitumor activity in animals.
Common acyclic examples include myrcene, geraniol, and linalool. Cyclic
structures include many well-known compounds, including menthol, camphor,
pinene, and limonene. Most of the monoterpenes come from common sources with
which most of us are familiar.
The thujone diastereomers are rapidly metabolised convulsants. They act as
noncompetitive blockers of the γ-aminobutyric acid (GABA) gated chloride channel.
Myrcene is found in the essential oil of bay leaves (Laurus nobilis) as well as hops
(Humulus lupulus). It is used as an intermediate in the manufacture of perfumes.
Geraniol, which is isomeric with linalool, constitutes the major part of the oil of
geraniums (Pelargonium graveolens) and is also found in essential oils of citronella
(Cymbopogon nardus), lemongrass (Cymbopogon citratus or C. flexuosus), and
others. Lavandulol is one of the principal ingredients of oil of lavender (Lavandula

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augustifolia), commonly used in male perfumes. Perillene can be found in the perilla
(Perilla frutescens), native to South and East Asia. Menthol is a well-known
monoterpene that is found in the essential oil of peppermint (Mentha piperita) and
other members of the mint family (Lamiaceae). Carvone is a common monoterpene.
It is one of the main olfactory components of caraway seed (Carum carvi), and it
shows antifungal activity. 3-Carene is a cyclopropane containing monoterpene,
derivatives of which have shown anesthetic activity. α-Pinene, the major ingredient in
turpentine, may play a significant role in the activity of hydrocarbon-degrading
bacteria in nature. Linalool is one of the principle constituents of coriander
(Coriandrum sativum), a common spice. It is also one of the most common floral
scent compounds found in flowering plants, and it is a common flavor compound in
various teas. Safranal is chiefly responsible for the characteristic odor of saffron
(Crocus sativus). Eucalyptol (1,8- cineole) is the main component of the essential oil
of eucalyptus leaf (Eucalyptus globulus). Eucalyptol along with camphor, form the
major constituents of rosemary oil. Recent research showed that eucalyptol is
effective in reducing inflammation and pain and in promoting leukemia cell death.
2.4.1.1 Acyclic monoterpenes
They can be considered as derivatives of 2,6-dimethyloctane.

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Among natural molecules, the followings are well known and have several
structural isomers.

2.4.1.2 Monocyclic monoterpenes

They are derived from cyclohexane with an isopropyl substituent. The most
typical are:

2.4.1.3 Bicyclic monoterpenes

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2.4.2 Sesquiterpenes: C15
Sesquiterpenoids are defined as the group of 15 carbon compounds derived from
three isoprene units, the C15 sesquiterpenes exist in aliphatic, bicyclic, and
tricyclic frameworks. Like the monoterpenes, most of the sesquiterpenes are
components of the essential oil of the plant from which they are derived. An
important member of this series is farnesol, with pyrophosphate that serves as a
key intermediate in terpenoid biosynthesis. They are found mainly in higher
plants but also in invertebrates. Sesquiterpenes, with monoterpenes, are an
important constituent of essential oils in plants. They are the most diverse group
of isoprenoids. In plants, they function as pheromones and juvenile hormones.
They can be classified as:

2.4.2.1 Acyclic compounds

The acyclic representative are also called farnesans, term derived from the basic
structure, farnesol. Farnesol and nerolidol are very common and are isolated from
essential oils of various sources.

Farnesol is widely distributed in many essential oils such as citronella, neroli,

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cyclamen, lemon grass, tuberose, rose, musk, and balsam. It is used in perfumery to
emphasise the odors of perfumes. Moreover, it is a natural pesticide for mites and is
also a pheromone for several insects and mammals, including elephants
(territorial marking, individual recognition, mate attraction).
2.4.2.2 Cyclic compounds
Abscisic acid plays a key role in plants in the regulation of stomatal closure by
regulating ion channel activities and water exchanges across the plasma membrane
of guard cells. Abscisic acid has also a variety of roles in plant development, bud
and seed dormancy, germination, cell division and movement. It induces also
storage protein synthesis in seeds and may be involved in defense against insect
attack.

2.4.3 Diterpenes: C20

They have 20 carbon atoms and are derived from geranylgeraniol pyrophosphate.
They are of fungal or plant origin and are found in resins, gummy exudates, and in
the resinous high-boiling fractions remaining after distillation of essential oils.
Diterpenoid groups that are physiologically active include: vitamin A (retinol),
phytohormones that regulate plant growth and germination, e.g. gibberellin, fungal
hormones that stimulate the switch from asexual to sexual reproduction, e.g.
trisporic acid; disease resistance agents (phytoalexins), e.g. casbene and podocarpic

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acid, the anticancer drug, taxol, from the bark of the yew tree, the cancer promoter,
phorbol, and natural cannabinoids. The diterpenes have exceptionally open chain,
as found in geranylgeraniol or phytol, which forms a part of chlorophyll and the
side chain of vitamin E and K, and crocetin, which is a diacid diterpenoid, and the
lipid part of the crocins, glycosylated derivatives present in saffron. Examples of
diterpene substances are given below:

The diterpenes are a widely varied group of compounds based on four isoprene
groups. Because of their higher boiling points, they are not considered to be
essential oils. Instead, they are classically considered to be resins, the material that
remains after steam distillation of a plant extract. Many interesting examples may
be mentioned here. The cyclic ether zoapatanol is derived from the Mexican
zoapatle plant (Montanoa tomentosa). It has been used as an abortifacient.

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Marrubiin is a diterpene lactone from white horehound (Marrubium vulgare). It
has been used as a vasorelaxant. Taxol® or paclitaxol (derived from needles and
bark of Taxus spp., yews) is a wholly unique antimitotic agent used to treat breast
cancer. Chemically, it is made up of a diterpenoid core with an alkaloid side group.
It binds to microtubules and stabilises them, as opposed to all other antimitotics of
the tubulin-binding type, such as vincristine, the podophyllotoxins, and
colchicines.

Marrubiin
2.4.4 Sesterpenes: C25
They are derived from geranylfarnesol pyrophosphate and have 25 arbon atoms. They
were isolated from insect protective waxes and from fungal sources.

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These examples of sesterpenes are shown below.

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2.4.5 Triterpenes: C30
They form a large group of natural substances, such as:

Squalene is also the immediate biological precursor of all triterpenoids. which

includes steroids and consequently sterols. Squalene is the immediate biological
precursor of all triterpenoids.

2.4.5.Tetraterpenes: C40
The most common tetraterpenoids are the carotenoids a widely distributed group

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of C40 compounds. Whereas the structures of the di- and triterpenes can have a
wide variety of fascinating structures, the carotenoids are generally derived from
lycopene. Cyclisation at one end gives γ-carotene and at both ends provides β-
carotene. This pigment was first isolated in 1831. The nature of these compounds
was discovered during the 19th century. In 1831, Wachenroder H. proposed the
term "carotene" for the hydrocarbon pigment he had crystallised from carrot roots.
Berzelius J. called the more polar yellow pigments extracted from autumn leaves
"xanthophylls" and Tswett M., who separated many pigments by column
chromatography, called the whole group "carotenoids". It is virtually universal in
the leaves of higher plants. As is evident from this polyene structure, numerous
double-bond isomers are possible for these basic structures, all of which can
provide brightly colored pigments. In plants, carotenoids serve as necessary
pigments in photosynthesis, where they are believed to protect plants from over
oxidation catalysed by other light-absorbing pigments, such asthe chlorophylls.
They are also responsible for colours varying from yellow to red in both flowers
and fruits. This colouration attracts pollinators (flowers) and serves as a source of
food for animal herbivores (fruits), thus aiding in seed dispersal. Among this
important group, the numerous compounds consist of C40 chains (tetraterpenes)
with conjugated double bonds, they show strong light absorption and often are
brightly coloured (red, orange). They occur as pigments in bacteria, algae and
higher plants. The hydrocarbon carotenoids are known as carotenes, while
oxygenated derivatives of these hydrocarbons are known as xanthophylls.
Carotenoids are important components of the light harvesting in plants, expanding
the absorption spectra of photosynthesis. The major carotenoids in this context are

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lutein, violaxanthin and neoxanthin. Additionally, there is considerable evidence
which indicates a photoprotective role of xanthophylls preventing damage by
dissipating excess light. In mammals, carotenoids exhibit immunomodulatory
actions, likely related to their anticarcinogenic effects. β-Carotene was thus shown
to enhance cell-mediated immune responses. The decrease in prostate cancer risk
has been linked to the consumption of tomatoes, vegetable rich in lycopene, as
prostatic tissues. While there is yet limited direct evidence linking lycopene and
prostate cancer, several observations, including the ability of the prostate to
concentrate lycopene, suggest a special protection of lycopene against that
pathology. Carotenoids consist of eight isoprenoid units joined in such a manner
that the arrangement of isoprenoid units is reversed at the centre of the molecule so
that the two central methyl groups are in a 1,6-position relationship and the
remaining non-terminal methyl groups are in a 1,5- position relationship. They are,
by far the predominant class of tetraterpenes. They may be also classified in the
terpenoids. Carotenoids can be considered derivatives of lycopene, found in
tomatoes, fruits and flowers. Its long straight chain is highly unsaturated and
composed of two identical units joined by a double bond between carbon 15 and
15'. Each of these 20 carbon units may be considered to be derived from 4 isoprene
units. Lycopene is a bioactive red coloured pigment naturally occurring in plants.
Interest in lycopene is increasing due to increasing evidence proving its antioxidant
activities and its preventive properties toward numerous diseases. In vitro, in vivo
and ex vivo studies have demonstrated that lycopene-rich foods are inversely
associated to diseases such as cancers, cardiovascular diseases, diabetes, and
others. Carotenoids may be acyclic (seco-carotenoids) or cyclic (mono- or bi-,

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alicyclic or aryl). Oxyfunctionalisation of various carotenoids leads to a large
number of xanthophylls in which the function may be a hydroxyl, methoxyl,
carbonyl, oxo, formyl or epoxy group. Only some of the most common carotenes
and xanthophylls are given below:

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 Some examples of xanthophylls

In human serum, several carotenes and xanthophylls have been detected. If α-, β-
carotene and lycopene are frequently quoted in specialised papers, some others are
now determined with precise HPLC methods (lutein, zeaxanthin, cantaxanthin and
β-cryptoxanthin). These compounds originate from ingested fruit, green leaves,
berries and yellow corn.

2.4.6.Polyterpenes: (Cn)
The two most important polyterpenes are natural rubber and guttapercha. In
natural rubber, the double bonds of the repeating isoprene units are cis; the molecules
are able to bend back and forth a little so that the rubber can stretch. Gutta-percha
has the trans structure. It is hard and non- rubbery. Gutta-percha is extensively used

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in electrical insulation and dentistry.

2.5. Biosynthesis of Terpenes

The most important biosynthetic pathways of natural products are derived from:
1. Mevalonic acid
2. Amino acids
3. Shikimic acid
4. Acetyl coenzyme A (acetyl-CoA)

2.5.1 Mevalonic acid pathway for Terpenes Biosynthesis

Three molecules of acetyl-coenzyme A are used to form mevalonic acid. Two
molecules combine initially in a Claisen condensation to give acetoacetyl-CoA,
and a third is incor- porated via a stereospecific aldol addition giving the
branched-chain ester β-hydroxy-β- methylglutaryl-CoA (HMG-CoA). This third
acetyl-CoA molecule appears to be bound to the enzyme via a thiol group, and this
linkage is subsequently hydrolysed to form the free acid group of HMG-CoA. In
the acetate pathway, an acetoacetic acid thioester (bound to the acyl carrier protein)
would have been formed using the more nucleophilic thioester of malonic acid.
The mevalonate pathway does not use malonyl derivatives and it thus diverges
from the acetate pathway at the very first step. In the second step, it should be

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