SECONDARY METABOLITE PRODUCTION

INTRODUCTION

Higher plants synthesize a wide variety of low molecular weight compounds in addition to the essential primary metabolites. These are called secondary metabolites

These secondary metabolites offer protection against pests,they act as attractatnts and as the plant’s own hormones

WHY SECONDARY METABOLITES

Chemically warfare to protect plants from the attacks by predators, pathogens, or competitors

Attract pollinators are seed dispersal agentImportant for abiotic stressMedicineIndustrial additives

CLASSIFICATION OF SECONDARY METABOLITES

They are classified on the basis of 1)chemical structure 2)chemical composition 3)solubility in various solvents 4)pathways by which they are

synthsized

A simple classification of secondary metabolites includes three main groups

1)the terpenes- made from mevalonic acid, composed almost entirely of carbon and hygrogen

2) phenolics – made from simple sugars, containing benzene rings, hydrogen and oxygen

3) nitrogen-containing compounds – extremely diverse, may also contain sulphur

ALKALOIDS

Most are derived from a few common amino acids (i.e..tyrosine , tryptophan , ornithine or argenine and lysine

Compounds have a ring structure and a nitrogen residue

Indole alkaloids is the largest group in this family , derived from tryptophan

Widely used as medicine

CHARACTERISTICS

They are bitter in taste. Derived from amino acids.The amino acids that are

most often serve as alkaloidal precursors are: phenylalanine, tyrosine, tryptophan, histidine, anthranilic acid, lysine and ornithine.

Alkaloids form double salts with compounds of mercury, gold, platinum and other heavy metals. These salts are obtained as precipitate which are microcrystals.

Insoluble or sparingly soluble in water, but the salts formed on reaction with acids are usually freely soluble.

Most are crystalline solids although a few are amorphous.

Free alkaloids are usually soluble in polar solvents like ether, chloroform

Some alkaloids are liquid because of lacking of oxygen in their molecules. (e.g coniine, nicotine, spartenine)

Sources and Occurrence of Alkaloids

Alkaloids can occur in plant kingdoms; among the angiosperms,

Leguminosae, Papaveraceae, Ranunculaceae, Rubiaceae, Solanaceae,

Berberidaceae are outstanding alkaloid-yielding plants.

Uses of Alkaloids in Plants:

Poisonous agents which protect plants against insects

and herbivores

End products of detoxification reactions representing a

metabolic locking-up of compounds otherwise harmful to

the plants.

For regulatory growth factors

Reserve substance capable of supplying nitrogen or

other elements necessary to the plant’s economy

TERPENES

Terpenes are natural products that are structurally related to isoprene

Isopropene (2-methyl-1,3-butadiene)

H2C C

CH3

CH CH2OR

1. The number of C atoms is a multiple of 5, C5

C10 C15 C20 C25 C30 C35 C40

2. Each group of 5 C is an isoprene subunit3. They can be saturated or unsaturated4. Many contain O atoms as well.

PHENOLICS

• Derived from aromatic amino acids, such as phenylalanine, tyrosin, and trytophan.

• All contain structures derived from phenol• Some examples:

Coumarins: antimicrobial agents, feeding deterrents, and germination inhibitors.

Lignin: abundant in secondary cell wall, rigid and resistant to extraction or many degradation reagents

Anthocyanins Flavones Flavnols Phenols are present in every plant they attract pollinators to the plant

and even impact how these plants act with one another. .

GLYCOSIDES

• Compounds that contain a carbonhydrate and a noncarbohydrate

• Glycosides are present in vacuoles in inactive form

• Glucosinolates: found primarily in the mustard family to give the pungent taste

There are four type of linkages present between glycone and aglycone:

C-linkage/glycosidic bond, O-linkage/glycosidic bondN-linkage/glycosidic bondS-linkage/glycosidic bond

Sources and effects of major secondary metabolites

carotenoids are organic pigments occuring in plants and are mostly found in red orange and yellow fruits and vegetables .

they have antioxidative effects and prevents cancer

They boost the immune system and reduce the risk of heart attacks

PHYTOSTEROLS

Found in- sunflower seeds ,sesame,nuts and soy beans

Protect against colon cancerand low cholestrol level

SAPONINS

Flavour additives found in- legumes and spinach

Boost the immune system

Lower the cholestrol level in blood

Reduce the risk of intestinal cancer

FLAVONOIDS

Organic pigment

Gives plant red violet or blue colour

Inhibit the growth of bacteria and viruses

Protect the cells against the damage of freeradicals

Influence blood coagulation

SULPHIDES

Contain sulphur

Belongs to the lily family onions ,leeks, asparagus, garlic

Inhibit the growth of bacteria

Lower cholestrol levels

Preventive efect against cancer

CURCUMIN

•Extracted from turmeric.•Coloring and medicinal uses•Wound healing•Antiulcer•Anti inflammatory•Antimicrobial & antiviral •Hepatoprotective•Antioxidant•No toxicity.

FLAVORSCocoa seedsVennilaStrawberryCinnamonCardamomClovesPepperareca

PRODUCTION OF SECONDARY METABOLITES IN CULTURES/BIOREACTORS

1)choice of explants any part of the plant can be used dicotelydons are rather amenable for

callus tissue induction procured explants are sterilized prior

to culture by using ethanol

2)NUTIENT CULTURE MEDIA

To induce callus from an explant and to cultivate the callus and cells in suspension

3)Callus induction

A callus cell culture is usually sustained on gel medium. Callus induction medium consists of agar and a mixture of macronutrients and micronutrients for the given cell type. There are several types of basal salt mixtures used in plant tissue culture, but most notably modified Murashige and Skoog medium White's medium, and woody plant medium. Vitamins are also provided to enhance growth such as Gamborg B5 vitamins. For plant cells, enrichment with nitrogen phosphorus, and potassium is especially important.

SUSPENSION CULTURE

The growth rate of suspension cultured cell is generally faster than that of the solid culture. The former is more desirable, particularly in the production of useful metabolites on a large scale

FERMENTORS OR BIOREACTORS

Carboy system-described by tulecke and nickell in 1959,consists of a rubber-stoppered 20 litre carboy fitted with four tubes. Filtered compressed air is employed for oxygen supply, aeration and agitation of the medium

Stirred jar fermetor- used for microbial cultivation

Agitation speed of 50 to 100 rpm

HAIRY ROOT CULTURE

It is the culture produced after the infection of explants and cultures by the gram negative soil bacteria Agrobacterium rhizogens

This process take advantage of the naturally occuring hairy root disease in dicotyledons

Agrobacterium recognizes some signal molecules exuded by wonded plant cells and becomes attached to it.The bacteria contain the Root inducing plasmid ( Ri-plasmid). The bacteria Genetically trasfer part of the

Ri-plasmid called the trasfer DNA (T-DNA) to the plant genome.

The plant cell to proliferate by increasing the rate of cell division ( cytokine expression ) and cell elongation (auxin expression) to produce the hairy roots.

Produce the opines which is a type of unusual amino acids ( octopine, agropine, nopaline, mannopine amd cucumopine) which is used by the bacterium as a carbon , nitrogen and energy source.

Induction of hairy root cultures In Vitro

Explants are wounded and then inoculated with Agrobacterium rhizogenes.

Usually two or three days later, the explant can be transferred into solid media with antibiotics, such as cefotaxime , vancomycine or ampicillin to kill or eliminate redundant bacteria.

The hairy roots will be induced within a short period of time, which varies from one week to over a month depending on different plant species.

The decontaminated hairy roots can be subcultured on phytohormone –free medium.

Commercial production of secondary metabolites

higher plants contain a variety of metabolic products which are useful as medicines ,food additives,perfumes,etc. however , non-compromising reduction in plant sources,increases in labour cost and other problems in order to obtain these high value added substances from natural plants ,have forced to develop analternative approachtowards the use of plant cell culture for production of these metabolic products .since ptc is not affected by changes in environmental conditions such as climate or natural depredation, improved production is possible

PHARMACEUTICALLY IMPORTANT CHEMICALS

1)Morphine alkaloids

Codeine is an anlagesic and cough suppressing drug from Papaver somniferum

also morphine can be converted into codeine

many workers have tried to produce codeine by undifferentiated cells of these plants

BERBERINE

it is an isoquinoline alkaloid which is produced and distibuted in roots of Coptis japonica and cortex of Phellonderon amurense Berberine chloride is used in treatment of intestinal disorders

TORPANE ALKALOIDS

Scopolamine and hyoscyamine areused commercially as anesthetic and antispasmodic drugs.

Occur in the leaves of members of the family Solanaceace including Duboisia myoporoides Datura leichhartdtti, Scopolia, Atropa,and Hyoscyamus

ANTITUMOUR COMPOUNDS

VINCA ALKALOIDS

Extracted from Catharanthus roseusTreat leukemias, hodgkin’s disease and antitumor activity

GINSENG

Roots of Panax ginseng is widely used as tonicEffective for genetic disorders, diabetes and weak circulationRoot contains saponins and sapogeninis

FOOD COLORING AGENTS

SAFFRON

Made from the stamens of Crocus sativus and used as flavouring agent and colourant

Plant contains crocin(yellow pigment), safranal(fragrance), picocrocin(bitter substance)

Baked goods, soaps, cheese

Medicinal value for stomach ailments

SAFFLOWER YELLOW

Yellow pigment obtained from the floret of the Carthamus tinctorius

Major pigment is carthamin

Carthamin is a quinoid form of isocarthamin

Used in baked goods and beverages

CHEWING GUMS CHICLE

Chicle a raw material is obtained from the latex of Achras sapota

Chicle contains 60% resin and 15% of rubber

Resin consisits of lupeo,α-amyrin andβ-amyrin

Rubber consists of cis and trans- 1,4-polyisoprene

Synthesized through mevalonic acid pathway

TAXOL

It is a ditrpene amideExtracted from the bark of wild grown Taxus brevifoliaTreats breast and lung cancer