TERPENOIDS

OKPAKO, ESEOGHENE

What are Terpenoids?

• Terpenoids are defined as hydrocarbons of plant origin having the general formula (C₅H₈)n; where n is the number of linked isoprene units
• Isoprene units are 5 carbon which could be IPP or DMAPP arranged in a head to tail fashion ( isoprene rule) to give terpenoids
• They are regarded as derivatives of polymers of isoprene
• i.e when terpenes are modified chemically such as by oxidation or rearrangement of carbon skeleton → TERPENOIDS
• They represent the essence or active constituent of plants known as essential oils

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Isoprene units

DMAPP

IPP

Isoprene Rule

• It states two or more isoprene units are joined in a head to tail fashion
• It can only be a guiding principle but not a fixed rule
• Carotenoids are joined in a tail to tail at their central and there are some terpenoids whose carbon content is not a multiple of five

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Occurrence

• They occur in fruits, flowers, leaves, stem barks and roots of nearly all the plants that have a pleasant smell
• Animals
• Microbes
• Insect
• Plant pathogens
• Endophytes
• marine

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Citral --- lemon

Geraniol – rose

Santalal --- sandal wood

Zinziberene ----- ginger

Cineole ----- eucalyptus

Pinene ----- turpentine

Camphor ----- camphor

Linalool ----- jasmine

Terpineol ----cardamon

Physical Properties of Terpenoids

• Odorous
• Volatile (monoterpenes and sesquiterpenes) at room temperature (triterpenes & diterpenes are not steam volatile)
• Soluble in organic solvent and usually insoluble in water
• They represent the essence or active constituent of plants known as essential oils
• Lighter than water
• Have high refractive index
• Are optically active

Chemical Properties

• They are unsaturated
• They undergo addition reaction with hydrogen, halogen, halogen acids to form addition products
• They undergo polymerization and dehydrogenation in the ring
• On thermal decomposition terpenoid gives terpene as one of its products
• Terpenoids are modified terpenes where in methyl groups have been moved or removed or oxygen atoms added
• Oxidised by nearly all oxidising agents

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Extraction of terpenes

• Expression
• Hydrodistillation
• Extraction by means of volatile solvents
• Enfluerage
• Fractional distillation
• Hot maceration process

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Biosynthesis of terpenoids

• The isoprene units are provided from dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP), which exist in equilibrium.
• DMAPP and IPP are structural isomers to each other.
• This pair of building blocks are produced by two distinct metabolic pathways:
• the mevalonate pathway and
• the non-mevalonate pathway.
• These two pathways are mutually exclusive in most organisms, except for some bacteria and land plants

Examples of pathways for organisms

Organism Pathways

• Bacteria MVA or MEP
• Archaea MVA
• Green Algae MEP
• Plants MVA and MEP
• Animals MVA
• Fungi MVA

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• The cytosol localized Mevalonate pathway provides C5 units for sesquiterpene and triterpene biosynthesis.
• The methylerythritol phosphate (MEP or non-mevalonate) pathway, localized in the plastids, is thought to provide IPP and dimethylallyl diphosphate for hemiterpene, monoterpene, and diterpene and tetraterpene biosynthesis.
• The non-mevalonate pathway or the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway starts with pyruvate and glyceraldehyde 3-phosphate (G3P) as the carbon source.
• The mevalonate pathway begins with acetyl CoA as the carbon source.

Mevalonate pathway

• 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 incorporated via a stereospecific aldol addition giving the branched-chain ester β-Hydroxy-β-MethylGlutaryl-CoA (HMG-CoA).
• 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 noted that, on purely chemical grounds, acetoacetyl-CoA is the more acidic substrate, and might be expected to act as the nucleophile rather than the third acetyl-CoA molecule.
• The enzyme thus achieves what is a less favourable reaction.
• The conversion of HMG CoA into (3R)- MVA involves a two-step reduction of the thioester group to a primary alcohol.
• The six-carbon compound MVA is transformed into the five-carbon phosphorylated isoprene units in a series of reactions, beginning with phosphorylation of the primary alcohol group.
• Decarboxylation / dehydration then give IPP.

• IPP is isomerized to the other isoprene unit, DMAPP, by an isomerase enzyme which stereospecifically removes the pro-R proton (HR) from C-2, and incorporates a proton from water on to C-4.
• Whilst the isomerization is reversible, the equilibrium lies heavily on the side of DMAPP.

Non mevelonate pathway

• This pathway is called the non-mevalonate route or alternatively, the 1-deoxy-d-xylulose-5-phosphate (DOXP) or 2C-methyl-d-erythritol-4-phosphate (MEP) pathway
• This pathway occurs in plant chloroplasts, algae, cyanobacteria, eubacteria, and apicomplexan parasites
• The non-mevalonate pathway starts with condensation of pyruvate and glyceraldehyde 3-phosphate to produce DOXP, catalyzed by 1-deoxy-d-xylulose-5-phosphate synthase (DXS) using thiamine pyrophosphate as cofactor
• DOXP is subsequently reduced into MEP by DOXP reductoisomerase (IspC).

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• DOXP is subsequently reduced into MEP by DOXP reductase (dxr).
• MEP is converted into 4-diphosphocytidyl-2-C-methylD-erythritol (CDPME) by CDPME synthase (ispD).
• CDPME is subsequently transformed into 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate (HMBPP) through two intermediates, 4-(cytidine 5′-diphospho)-2-C-methyl-D-erythritol (CDPMEP) and 2-C-methyl-D-erythritol2,4-cyclodiphosphate (MEC) by CDPME kinase (ispE), MEC synthase (ispF) and HMBPP synthase (ispG), respectively.
• Finally, HMBPP reductase catalyzes HMBPP into a 5-6:1 ratio of IPP and DMAPP,
• while IPP isomerase (idi) inter-converts IPP and DMAPP to adjust the ratio according to the cellular requirements

Condensation/Classification of isoprene units

• IPP + DMAPP → geranylpyrophosphate GPP
• GPP + IPP/DMAPP → farnesylpyrophosphate FPP
• GPP+ GPP → geranylgeranyl pyrophosphate
• GPP + FPP → geranylfarnesylpyrophosphate
• FPP+IPP/DMAPP → GPP/ Diterpene
• FPP+FPP → farnesylfarnesylpyrophosphate/triterpene

Classification of terpenes

Class Number of carbon atoms

• Hemiterpenes 5
• Monoterpene 10
• Sesquiterpene 15
• Diterpene 20
• Sesterpene 25
• Triterpene 30
• Sesquaterpene 35
• Tetraterpene 40

• Each class can be further sub-divided into subclasses according to the number of rings present in the structure.
• Acyclic
• Monocyclic
• Bicyclic
• Tricyclic
• Tetracyclic

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Hemiterpenes

• consist of a single isoprene unit. Isoprene itself is the only hemiterpene, but oxygen-containing derivatives such as prenol and isovaleric acid are hemiterpenoids.

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Prenol

• Prenol occurs naturally in citrus fruits, cranberry, bilberry, currants, grapes, raspberry, blackberry, tomato, white bread, hop oil, coffee, arctic bramble, cloudberry and passion fruit.
• Prenol is a building block of isoprenoid alcohol

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Isovaleric acid

• classified as a short-chain fatty acid.
• it has an unpleasant odor.
• The compound occurs naturally and can be found in many foods, such as cheese, soy milk, and apple juice.
• are widely used in perfumery

Monoterpenes

• consist of two isoprene units and have the molecular formula C₁₀H₁₆.
• They can be classified as thus:
• Acyclic e.g myrcene  (present in hops), linalool (present in lavender),
• Monocyclic e.g limonene (present in citrus fruits) menthol
• Bicyclic e.g camphor, eucalyptus
• Other Examples of monoterpenes and monoterpenoids include geraniol, terpineol (present in lilacs),
• Iridoids derive from monoterpenes.
• Examples of iridoids include aucubin and catalpol.

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Myrcene

• extracted from any number of plants, such as verbena or wild thyme, the leaves of which contain up to 40% by weight of myrcene
• Other plants include:  cannabis, Houttuynia, lemon grass, mango, Myrcia, West Indian bay tree, and cardamom.
• used in the perfumery industry
• contributes a peppery and balsam aroma in beer.
• As of October 2018, the U.S. FDA withdrew authorization for the use of myrcene as a synthetic flavoring substance for use in food

Limonene

• is the majorcomponent in the oil of citrus fruit peels.
• used in chemical synthesis as a precursor to carvone 
• formed from geranyl pyrophosphate
• is common as a dietary supplement and as a fragrance ingredient for cosmetics products

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Camphor�

•  found in the wood of the camphor laurel (Cinnamomum camphora),
•  commonly applied as a topical medication as a skin cream or ointment to relieve itching from insect bites, minor skin irritation, or joint pain. 
• The action on nerve endings also induces a slight local analgesia.
•  typically by steam inhalation, to inhibit coughing and relieve upper airway congestion due to the common cold

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Iridoids�

• monoterpenoids in the general form of cyclopentanopyran, found in a wide variety of plants and some animals
•  Iridoids are typically found in plants as glycosides, most often bound to glucose
• Structurally, they are bicyclic cis-fused cyclopentane-pyrans.
• Cleavage of a bond in the cyclopentane ring gives rise to a subclass known as secoiridoids, such as oleuropein and amarogentin.
• Aucubin and catalpol are two of the most common iridoids in the plant kingdom.
•  Iridoids are prevalent in the plant subclass Asteridae, such as Ericaceae, Loganiaceae, Gentianaceae, Rubiaceae, Verbenaceae, Lamiaceae, Oleaceae, Plantaginaceae, Scrophulariaceae, Valerianaceae, and Menyanthaceae
•  Iridoids can consist of ten, nine, or rarely eight carbons in which C11 is more frequently missing than C10.

Aucubin

• Aucubin, as other iridoids, is found in asterids such as Aucuba japonica (Garryaceae) etc
• These plants are used in traditional Chinese and folk medicine
• Aucubin was found to protect against liver damage induced by carbon tetrachloride or alpha-amanitin in mice and rats when 80 mg/kg was dosed intraperitoneally
• Aucubin has 10 carbons with the C11 carbon missing

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Amarogentin�

• is a chemical compound found in gentian (Gentiana lutea) or in Swertia chirata
• Gentian root is used as a herbal bitter in the treatment of digestive disorders and is an ingredient of many proprietary medicines
•  is one of the most bitter natural compounds known and is used as a scientific basis for measuring bitterness.
•  shows an antileishmanial activity

Sesquiterpenes

• consist of three isoprene units and have the molecular formula C₁₅H₂₄.
• Examples of sesquiterpenes and sesquiterpenoids include humulene, farnesenes, farnesol, geosmin. 
• (The sesqui- prefix means one and a half.)

Farnesol

• Farnesol is present in many essential oils such as citronella, neroli, cyclamen, lemon grass, tuberose, rose, musk, balsam, and tolu
• It is used in perfumery to emphasize the odors of sweet, floral perfumes.
• It enhances perfume scent by acting as a co-solvent that regulates the volatility of the odorants.
• It is especially used in lilac perfumes.
• Farnesol is a natural pesticide for mites and is a pheromone for several other insects.

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Humulene

• derived from farnesyl diphosphate (FPP)
•  It was first found in the essential oils of Humulus lupulus (hops),
•  is an isomer of β-caryophyllene, and the two are often found together as a mixture in many aromatic plants
• Is an active contributer to the insect repellent properties of commiphora leptophloeos leaf oil
•  is being studied for potential anti-inflammatory effects

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Diterpenes

• are composed of four isoprene units and have the molecular formula C₂₀H₃₂.
• They derive from geranylgeranyl pyrophosphate. Examples of diterpenes and diterpenoids are cafestol, kahweol, cembrene and taxadiene  (precursor of taxol).
• Diterpenes also form the basis for biologically important compounds such as phytol, tanshinone, carnosol,
• Commonly present resins and trees of Leguminosae, Buseraceae, Lamiaceae, Euphorbiaceae, Solanaceae and Meliaceae

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Cembrene

•  monocyclic diterpene isolated from corals of the genus Nephthea
• biosynthesized by macrocyclization of geranylgeranyl pyrophosphate
• Is a trail pheromone for termites

Carnosol

• found in the herbs rosemary (Rosmarinus officinalis)
• It has been studied in-vitro for anti-cancer effects in various cancer cell types

Sesterterpene

• terpenes having 25 carbons and five isoprene units, are rare relative to the other sizes.
• (The sester- prefix means two and a half.) An example of a sesterterpenoid is geranylfarnesol.
• Rarely found.
• Found in protective waxes of insects and fungi
• They are phytotoxic

Triterpene

• 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 precursors to all the steroids.
• Saponins belong to this class
• Commonly found in Leguminosae, Cucurbitaceae, Meliaceae, Apocynaceae and Moraceae
• Found in any part of the plant as free or glycosidic form

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Squalene

•  It is a colourless oil although impure samples appear yellow.
• It was originally obtained from shark liver oil (hence its name, as Squalus is a genus of sharks).
• All plants and animals produce squalene as a biochemical intermediate.
• biosynthesised by coupling two molecules of farnesyl pyrophosphate

Lanosterol

• is the compound from which all animal and fungal steroids are derived
• Lanosterol was shown to improve lens clarity in subjects with lens clouding due to aging or physical stressors
•  is an ingredient in over-the-counter ophthalmic products to prevent cataracts

Lupeol

• pharmacologically active pentacyclic triterpenoid
• Lupeol is found in a variety of plants, including mango, Acacia visco and Abronia villosa
• It has several potential medicinal properties, like anticancer and anti-inflammatory activity
• It also has antiprotozoal, antimicrobial properties

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Sesquaterpene

• are composed of seven isoprene units and have the molecular formula C₃₅H₅₆. Sesquarterpenes are typically microbial in their origin. Examples of sesquarterpenoids are ferrugicadiol and tetraprenylcurcumene.

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Tetraterpene

• carotenoids
• contain eight isoprene units and have the molecular formula C₄₀H₆₄.
• Biologically important tetraterpenoids include the acyclic lycopene, the monocyclic gamma-carotene, and the bicyclic alpha- and beta-carotenes

Lycopene

• is a bright red carotenoid hydrocarbon found in tomatoes and other red fruits and vegetables, such as red carrots, watermelons, grapefruits, and papayas. It is not present in strawberries or cherries.
• In plants, algae, and other photosynthetic organisms, lycopene is an intermediate in the biosynthesis of many carotenoids, including beta-carotene, which is responsible for yellow, orange, or red pigmentation, photosynthesis, and photoprotection
• Lycopene may increase the risk of bleeding when taken with anticoagulant drugs

Polyterpene

• 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

Apocarotenoids

• “apocarotenoid” refers to any compound derived from the oxidative cleavage of carotenoids by enzymatic (carotenoid cleavage dioxygenases (CCD’s)) or non-enzymatic means.
• Their formation is regulated by:-
• developmental stimuli
• environmental stimuli
• sub-cellular compartmentalization
• They are more soluble and volatile than carotenoids
• They are not simply breakdown products as there can be modifications post cleavage and their functions include:
• hormones (they regulate plant development)
• volatiles and
• signals in response to stress
• they fine tune carotenoid biosynthesis flux
• they famous for the pleasant aromas and give rise to fragrance and palatable flavors of many flowers and fruits
• Examples include : abscisic acid, strigolactone, vitamin A (all retinoids), crocin

Retinol

• also called vitamin A
• Dietary sources include fish, dairy products, and meat and orange coloured fruits and vegetables
• Retinol is used to treat vitamin A deficiency.

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Abscisic acid

•  is a plant hormone
• ABA functions in many plant developmental processes, including seed and bud dormancy
•  the control of organ size and stomatal closure.

Test for terpenes/terpenoids

• Salkowski test is used to detect terpenoids. Extract (5 ml) was mixed with chloroform (2 ml), and concentrated H₂SO₄(3 ml) was carefully added to form a layer.
• A reddish brown colouration of the inter face indicates the presence of terpenoids.

Importance of terpenoids

• Many terpenoid or terpenoid derivatives are used as traditional drugs with different medicinal values identified from different natural sources

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