SECTION 1

A PROLOGUE TO PLANT DERIVED

SECONDARY METABOLITES

1

1. METABOLISM IN PLANTS

The organisms in order to live, grow and reproduce need a source of

energy and provisions of building blocks to construct their own tissues. This

they achieve by transforming and inter converting a vast number of organic

compounds through an integrated network of enzyme mediated and carefully

regulated chemical reactions. The sum of all the chemical reactions occurring

in living cells that provides the organism with matter and energy for its vital

activities is collectively termed as metabolism. Each organized sequence of

chemical and energy transformations by which one molecule is converted to

another is termed a metabolic pathway. Organic molecules involved in these

processes are called metabolites, which include the primary and secondary

metabolites, and they are interconverted during the metabolic processes as the

requirement arises1

.

The reactions involved in the primary metabolism are common to all

organisms and are either catabolic or anabolic.

Catabolism involves reactions in the cell that degrade an organic

matter into smaller or simpler products. Some of the important Catabolic

Pathways are:

1. Glycolysis is the set of reactions where a glucose molecule after

phosphorylation breakdown to a pyruvate molecule. It takes place in

cytoplasm.

2. Krebs or tricarboxylic acid (TCA) cycle takes place in mitochondria

and converts the pyruvate formed during the above process to CO2 and

water. This cycle provides electrons and ATP and also intermediates

for amino acid synthesis.

2

3. Respiration or electron transport chain also takes place in

mitochondria. This is a series of redox reactions for formation of 3

molecules of ATP and water by transferring electrons from

Nicotinamide Adenine Dihydrogenase (NADH) to an electron

acceptor.

Anabolism results in the formation of larger or more complex

molecules from smaller organic moiety. This includes the biosynthesis of the

molecules such as nucleotides, amino acids, hexosamines, fatty acids and

sugars and polymerization reactions which lead to the formation of larger

molecules such as DNA, RNA, proteins, peptidoglycans, lipids,

lipopolysaccharides and glycogen2.

The primary metabolites therefor include the nucleic acids and the

common amino acids and sugars and also the high molecular weight

polymeric materials such as cellulose, lignins and the proteins from which the

cellular structures are formed. In plants, compounds derived from primary

pathways make up the bulk of the plant. Some of the intermediates in the

primary metabolism act as the precursor molecules for a different series of

reaction that give end products which are usually the characteristic of a

particular species. These are the secondary metabolites, the molecules of our

concern, which are biosynthetically restricted to a selection of plants or even

to specific species. These appear to have little influence on the growth and

development of plant3.

Secondary metabolites unlike the primary metabolites are found to be

accumulated in particular tissues at high concentration, some of them being

toxic to the plant themselves if they are mislocalized4. Biosynthetic genes

responsible for the formation of those secondary metabolites may be highly

expressed in such tissues. Translocations of these compounds occur as well,

e.g., biosynthetic genes for nicotine in Nicotiana species are mostly expressed

3

in root tissues where as it is transported to the aerial part and is accumulated

in leaves5.

Fig. 1a Major Pathways of biosynthesis of secondary metabolites6

4

The dividing line between primary and secondary metabolism is

indistinct because many of the intermediates in primary metabolism are also

intermediates in secondary metabolism6. For example, several obscure amino

acids are definitely secondary metabolites, whereas many sterols play an

essential structural role in most organisms and must therefore be considered

as primary metabolites. The overlapping role of many compounds ensures a

close interconnection between primary and secondary metabolism7. A further

view is that the secondary compounds may be a convenient sink, into which

excess carbon and nitrogen can be diverted away from an inactive part of

primary metabolism. The secondary compounds are then degraded and the

stored carbon and nitrogen recycled back into the primary metabolism, when

there is a demand. The balance between the activities of the primary and

secondary metabolism is a dynamic one, which will be largely affected by

growth, tissue differentiation and development of the plant body and also

external pressures3.

5

2. SECONDARY METABOLITES

Plant Secondary Metabolites were frequently considered as

extravaganzas that serve no obvious biological purpose for the plant that

produce them. Their physiological role has not been completely elucidated.

However it is becoming increasingly clear that many plant secondary products

are involved in the interaction of the plant with its environment to cope with

various stress factors and the level of these phytochemicals is largely

determined by environmental conditions8,9

. A wide array of external stimuli

are capable of triggering changes in the plant cell which leads to a cascade of

reactions, ultimately resulting in the formation and accumulation of secondary

metabolites which help the plant to overcome stress factors10,11

. Secondary

metabolites play a significant role in plant survival and are important for plant

interactions with natural enemies including herbivores12,13

, pathogens14

and

competitors15

and with pollinators and seed dispersers16

. Secondary

metabolites are also involved in a number of physiological functions such as

toxic nitrogen storage and transport and UV-protectants17

.

Three basic metabolic processes governed by photosynthesis, i.e.,

nitrogen metabolism, fatty acid metabolism, and carbohydrate metabolism,

are responsible for the synthesis of secondary metabolites which are classified

into several groups based on their chemical structure and biosynthetic routes

providing them.

1.2.1. Phenolic compounds

The term phenolic compound embraces a wide range of plant

substances which possess in common an aromatic ring bearing one or more

hydroxyl groups. Phenolic compounds are generally synthesized via the

shikimate pathway but the polyketide pathway can also provide some

6

phenolics, such asorcinols and quinones. Phenolic compounds derived from

both pathways are quite common, e.g. flavonoids, stilbenes, pyrones and

xanthones. They most frequently occur combined with sugar as glycosides

and are usually located in cell vacuole. They are phenols or phenolic acids,

phenylpropanoids, flavonoids, stylbenes, lignans or xanthenes and tannins as

polymers of polyphenols that are classified into two types, i.e., hydrolyzable

and condensed Tannins. Among these the flavonoids form the largest group

and are brightly coloured compounds which have two benzene rings attached

by a propane unit. Different classes within this group differ by additional

oxygen-containing heterocyclic rings and hydroxyl groups and include the

chalcones, flavones, flavonols, flavanones, anthocyanins and isoflavones.

Anthocyanins impart red and blue pigment to flowers and fruits .The

isoflavonoids are rearranged flavonoids, in which this rearrangement is

brought about by a cytochrome P-450-dependent enzyme. Simple isoflavones

such as daidzein, and coumestans such as coumestrol, have sufficient

estrogenic activity to seriously affect the reproduction of grazing animals and

are known as phytoestrogens18

. Isoflavones exhibit estrogenic,

antiangeogenic, antioxidant and anticancer properties19, 20

. Epidemiological

studies suggest a link between consumption of soy isoflavones and reduced

risks of breast and prostate cancers21

. Isoflavones also possess other health-

promoting activities, such as chemoprevention of osteoporosis, and

prevention of postmenopausal disorders and cardiovascular diseases22, 23

. The

roles of flavonoids in plants also include intracellular and extracellular

signalling, male fertility, and pathogen defence24, 25

. Flavonoids also function

as allelochemicals in plant-plant interaction26

. A prime example of a flavanoid

used in defence is resveratrol, a substance found in the skin of grapes which

inhibits the growth of fungi27

. Resveratrol is also implicated in the prevention

of cancer and cardiovascular diseases in vasoprotection and neuroprotection28-

7

30. The phenylpropenes are important components of many essential oils, e.g.

eugenol in clove oil and anethole and myristicin in nutmeg31

.

Even though the functions of some of the phenolic compounds like

lignans and anthocyanins are well established, the biological role of many

others is not. They are reported to protect the plant against adverse factors

which threaten its survival in an unfavorable environment, such as drought,

infections or physical damage32

. The resistance of plants to UV radiations is

due to the phenolic compounds especially the phenylpropanoids present in

them33

. It is widely recognized that the beneficial influence of many

foodstuffs and beverages including fruits, vegetables, tea, red wine, coffee

and cacao on human health is associated to the antioxidant activity.

Antioxidants are hypothesized to play an important role in disease prevention,

since they may be able to avoid oxidative damage caused by reactive oxidant

species to vital biomolecules such as DNA, lipids and proteins. This type of

oxidative mechanism is accepted to be involved in numerous pathological

processes, such as cardiovascular and neurodegenerative diseases,

inflammation and carcinogenesis34-36

. The phenolic compounds and their

derivatives, due to their activity as antioxidants, play a significant role in the

prevention of diseases by being efficient in protecting cells from oxidative

stress37, 38

. The main mechanism of action of phenolic antioxidants is

considered to be the scavenging of free radicals by hydrogen-atom donation,

although other mechanisms may be involved39

. Thus, the evaluation and

molecular level interpretation of the biological activity of phenolic

compounds is presently the object of intense research. The action phenolic

compounds as neuroprotective40

, fungicidal41

bactericidal42

and their anti-

atherosclerosic effects43

and anticancer activity44, 45

is well documented. The

lignans, secoisolariciresinol and matairesinol from rye or linseed as well as

pinoresinol and lariciresionol are converted by intestinal microorganisms in

humans into the phyto-estrogens enterodiol and enterolactone which are

8

supposed to protect against estrogen-dependent cancers46

. Plant phenolics are

also known to protect food against lipid oxidation, which leads to the

production of undesirable off flavors47

.

Apart from its entire beneficial role these compounds are also

responsible for certain harmful effects. An excessive content of polyphenols,

in particular tannins, may have adverse consequences because it inhibits the

bioavailability of iron48, 49

and thiamine50

and blocks digestive enzymes in the

gastrointestinal tract51

. Phenolic compounds can also limit the bioavailability

of proteins with which they form insoluble complexes in the gastrointestinal

tract52

. Moreover, interactions between tannins and proteins lead to

astringency53

.

1.2.2. Terpenoids

Terpenes are the largest group of phytochemicals and the functional

assortment of chemicals within plants is best demonstrated by them as they

exhibit diverse functions in mediating antagonistic and beneficial interactions

in, and among, organisms54

. The terpenoids are lipid soluble and are located

in the cytoplasm of the cell. Their structures are hypothetically derived from

the isoprene molecule and their carbon skeletons are built up from the union

of two or more of these C5 units joined head to tail. Accordingly they are

classified as hemiterpenes (C5), monoterpenes (C10), sesquiterpenes (C15),

diterpenes (C20), sesterterpenes (C25), triterpenes (C30) and tetraterpenes (C40).

The polymerisation of the isoprene unit gives the rubbers and latex.

Two different biosynthetic pathways produce the main terpene

building block, isopentenyl diphosphate (IPP). The first classical biosynthetic

route is known as the MVA (mevalonic acid) pathway. This takes place in the

cytosol, producing sesquiterpenes55, 56

. The second biosynthetic route to

terpenes is referred to as either the MEP (methylerythriol-4-phosphate) or

9

DOX (1-deoxy-D-xylulose) pathway and takes place in plastid57

. The

evidence indicates that there may be sharing of intermediates across these

pathways, a sort of biosynthetic crosstalk58

.

The volatile essential oils are the mono and sesqui terpenes and are

important as the basis of natural perfumes and also of spices and flavourings

in the food industry. The diterpenes are less volatile and are not considered

essential oils and constitute a component of plant resins because of their

higher boiling point. Gibberellic acid, a plant growth regulator, and taxol are

diterpenes. Some diterpenes that are toxic and are responsible for the

poisonous nature of the plants bearing them.There are examples of diterpenes

that exhibited cytotoxic, antitumor and antimicrobial activities in vitro59

.

Triterpenes are non volatile, high melting and optically active substances and

either are true triterpenes, saponins and sterols or are cardiac glycosides. The

true triterpenoids include the lanostanes, dammaranes, cycloartanol, lupanes,

hopanes etc which occur in various plant parts and cucurbitacins, oleananes

and ursanes occurring in plants of Cucurbitaceae family60

. The limonins and

the cucurbitacins are potent insect steroid hormone antagonists61

. Sterols in

plants generally called the phytosterols are based on the cyclopentane

perhydrophenanthrene ring system. All plant steroids hydroxylated at C3 are

sterols and have profound importance as hormones (androgens such as

testosterone and estrogens such as progesterone), coenzymes and provitamins

in animals. The progesterones are derived semi synthetically from

diosgenin62

. They are also employed in the protection from the predators.

Saponins are glycosides of both triterpenes and sterols and are surface active

agents with soap like properties and can be detected by their ability to cause

foaming and to haemolyse blood cells. The cardiac glycosides have complex

structures and are mostly toxic and are pharmacologically active. The tetra

terpenoids are the carotenoids which are found in all kinds of plants and

10

function as an accessory pigment in photosynthesis and as coloring matters in

fruits and flowers63

.

Terpenes are vital for life in most organisms exerting metabolic control

and mediating inter and intra species interactions, for example, pollination

and defense in plants. Aside from the facts that plants manufacture these

compounds in response to herbivory or stress factors, it has also been shown

that flowers can emit terpenoids to attract pollinating insects and even attract

beneficial mites, which feed on herbivorous insects64

. Kessler and Baldwin65

have reported that herbivorous insects can cause the release of terpenes from

plants and also induce the release of signals that attract predatory species.

Cheng and coworkers66

have reported that terpenes may act as chemical

messengers influencing the expression of genes involved in plant defensive

functions or influence gene expression of neighboring plants. Many terpenes

are reported to act as toxins, growth inhibitors or deterrents to

microorganisms and animals67

.

1.2.3. Organic Acids, Polyketides and Sulphur containing compounds

Plants accumulate organic acids like citric acid, succinic acid, acetic

acid and tartaric acid in their cell vacuole leading to their acidity. Many of the

acids are important to the plant in their primary metabolic pathways. Majority

are non volatile, water soluble, colourless liquids or low melting solids68

.

The polyketides constitute the compounds derived from poly-β-keto

chains and include fatty acids, polyacetylenes, prostaglandins, macrolide

antibiotics and many aromatic compounds like anthraquinones and

tetracyclins. The organic acids and the polyketides are built via the acetate

pathway69

.

Secondary metabolites containing sulphur are rather unusual in plant

and are biosynthesized from sulphur bearing amino acids. They are involved

11

in several different types of chemical defenses; including constitutive,

induced and activated defenses in a broad range of higher plant species as

well as mosses and algae70-72

. They are volatile and carry an acrid taste or an

obnoxious smell. The flavours of mustard, garlic, onion and radish are due to

the sulphur containing components present in them73

.

Among the sulfur-containing secondary metabolites, two groups of

compounds are involved in so-called activated plant defense systems, the

glucosinolates and the alliins. In the intact plant tissue, these compounds,

which are relatively physiologically inert by themselves, are spatially

separated from their hydrolyzing enzymes, the myrosinases and alliinases,

respectively. When the tissue is damaged, for example upon herbivore attack,

the parent compounds are converted to biologically active products by the

action of the hydrolyzing enzymes74,75

. Besides their role in plant defense,

many studies on both glucosinolates and alliins have also been motivated by

the health-promoting effects of these compounds in the human diet. Some

examples are camalexin and related compounds from the Brassicaceae, the

glucosinolates from the Brassicales, the alliines from the Alliaceae, and the

thiophenes from the Asteraceae.

1.2.4. Nitrogen Compounds

Nitrogen containing compounds include amines, alkaloids, cyanogenic

glycosides, indoles, purins, pyrimidines, cytokinins, chlorophylls. They are

mainly associated with the protection of plant from predators.

Simple bases, such as methylamine, trimethylamine and other straight-

chain alkylamines and compounds such as betaines, choline and muscarine

are synthesized from amino acids and categorized as biological amines or

protoalkaloids. As in alkaloids, their nitrogen is not involved in a heterocycle

system. The polyamines, putrescine, spermine, spermidine and the

12

phenylalkylamines, such as β-phenylethylamine, dopamine, ephedrine,

mescaline and tryptamine belong to this class of compounds. An exception to

the above definition is the widely distributed vitamin B1 (thiamine) which

contains nitrogen in heterocycle and has physiological activity76

.

Cytokinins have been correlated to enhanced pathogenic resistance in

plants77,78

. The pain relieving and narcotic properties of opium are known

from ancient times and galanthamine has its use in the treatment of

Alzheimer’s disease.

Alkaloids are a structurally diverse class of nitrogen-containing

compounds, which often possess a strong physiological activity and, over the

centuries, have found many clinical applications. They are regarded as reserve

materials for protein synthesis, as protective substances discouraging animal

or insect attacks, as plant stimulants or regulators or simply as detoxication

products. They occupy an important position in applied chemistry and play an

indispensable role in medicinal chemistry79

. More than 12,000 alkaloids have

been identified in the plant kingdom80

. They occur naturally not only in plants

but also in microorganisms, marine organisms, and animals. Many kinds of

alkaloids with extraordinary structures and significant biological activities

have been isolated from marine organisms81, 82

. In plants, alkaloids generally

exist as salts of organic acids like acetic, oxalic, citric, malic, lactic, tartaric,

tannic and other acids. Some weak basic alkaloids (such as nicotine) occur

freely in nature. A few alkaloids also occur as glycosides of sugar such as

glucose, rhamnose and galactose, e.g. alkaloids of the solanum group

(solanine), as amides (piperine), and as esters (atropine, cocaine) of organic

acids83,84

. They may be present systematically in whole plants, or they may be

accumulated in large amounts in specific organs like roots, stem bark and

seeds. The alkaloids may also be the end product of detoxification reactions

13

and are capable of supplying nitrogen and other necessary fragments to the

plant development85

.

The potent physiological activity of many alkaloids has also led to

their use as pharmaceuticals, stimulants, narcotics and poisons. Alkaloids

currently in clinical use include the analgesics morphine and codeine, the

anticancer agent vinblastine, the gout suppressant colchicine, the muscle

relaxant (+) tubocurarine, the antiarrythemic ajmalicine, the antibiotic

sanguinarine and the sedative scopolamine. The plant alkaloids like caffeine

in tea and coffee and nicotine in all preparations (smoking, chewing) of

tobacco are widely consumed daily.83

1.2.5. Sugars and their derivatives

Sugars are the first complex organic compounds formed in the plant as

the result of photosynthesis and are the major sources of respiratory energy.

They also play a number of ecological roles, in plant- animal interaction, in

protection from wounding and infection and in the detoxification of foreign

substances. They can be classified as monosaccharides, oligosaccharides,

sugar alcohols and cyclitols86

. They are usually the food reservoirs in plant.

Glycerol is a building block of plant lipids, mannitol is common in higher

plants and the main function of sugar alcohols is as storage of energy and

osmo-regulation87

. The cyclitols function as intermediates in the synthesis of

different oligosachcharides88

.

The bulk of the carbohydrates occur in plants in the bound form,

attached to a range of different aglycones as the glycosides. Many studies

have shown that glycosylation reactions could be involved in the biosynthesis,

modification, transportation and storage of other secondary metabolites. It is

now recognized that the glycosylation of low-molecular-weight compounds of

plants, by adding a sugar moiety to the acceptors, usually changes acceptors

14

in terms of their bioactivity, stability, solubility, subcellular localization and

binding property to other molecules, and this possibly reduces the toxicity of

endogenous and exogenous toxic substances89

. Hence it is thought to be one

of the most important modification reactions towards plant secondary

metabolites, and plays a key role in maintaining cell homeostasis, thus likely

participating in the regulation of plant growth, development and in defense

responses to stress environments90,91

. For example during fungal attack, plants

form glycosidic bonds to detoxify the toxicity of pathogens92

. In the lignin

biosynthesis pathway, lignin monomers (coumaryl, coniferyl and sinapyl

alcohols) need to be translocated from the cytosol to the cell wall, where they

are polymerized into lignin. Here the glucosides of lignin monomers have

been considered as the transport forms93

. Glycosylation is usually the last step

of flavonol biosynthesis metabolism, probably indicating a requirement of

stabilization, reactivity or translocation. Multiple additions of sugar moieties

to a given compound, in parallel or in chains, give rise to a broad spectrum of

secondary metabolites, thus contributing to their unique properties. An

example is that one single flavonol, quercetin, has 300 different glycosides

naturally occurring in plants94

.

The plant secondary metabolites have large economical importance

because, these compounds are connected with the important traits of plant

itself, e.g., colour or fragrance of flowers, taste and colour of food, and

resistance against pests and diseases and also for the production of fine

chemicals such as drugs, antioxidants, flavors, fragrances, dyes, insecticides

and pheromones. For centuries, India, China, Egypt and Greece have led the

world in the use of natural products for healing. The folkloric medicine based

on judicious use of different plant parts has played a key role in reducing

human sufferings and counteracting diseases. In fact, modern pharmacology

arose from the traditional use of plant products as medicines, and even today

many therapeutic drugs are the plant secondary metabolites (e.g., morphine,

15

digoxin, atropine, ephedrine, atremisinin, vincristine,paclitaxel) or plant

secondary metabolite derivatives (e.g., codeine, buprenorphine, warfarin,

docetaxel)

According to an analysis conducted by Newman and coworkers, 42%

of all drugs approved from 1983 to 1994 originated from natural products and

more than 60% of all approved anti-infective and anti-cancer drugs in the

same period were derived from natural products95

. Natural products provide

greater structural diversity than standard combinatorial chemistry and the

chemical novelty associated with natural products is higher than that of any

other source. Indeed, 40% of the chemical scaffolds in a published database of

natural products are absent from synthetic chemistry96

. Industrially, secondary

metabolites serve as the sources of oils, both volatile and fixed, flavour and

fragrances, resins, gums, natural rubber, waxes, saponins and their

surfactants, dyes, pharmaceuticals, plants and insect growth regulators, and

many other specialty products.

Plant being a very complex organism producing millions of

compounds at a time, the isolation of the secondary metabolites becomes a

tedious task. Until recently, large-scale studies of plant metabolites have been

limited by the time-consuming and costly nature of available technology.

With the tremendous advance in the natural product chemistry and the

development of sophisticated techniques and improved chromatographic

separation methods, this difficulty is overcome. The newer spectroscopic

techniques such as two-dimensional high resolution NMR Spectroscopy, IR

and Raman Spectroscopy, Mass Spectroscopy and X-ray Crystallographic

Analysis have simplified the structural elucidation of new natural products.

16

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