Pharmacognosy and

Phytochemistry-II
Unit-1
[Metabolic Pathways in Higher Plants and their
determination]

(B.Pharma 5 th Sem)
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 Unit-I (Syllabus)

Metabolic pathways in higher plants and their determination

 Brief study of basic metabolic pathways and formation of different secondary

metabolites through these pathways- Shikimic acid pathway, Acetate
pathways and Amino acid pathway.

 Study of utilization of radioactive isotopes in the investigation of

Biogenetic studies.
Contents :
 Metabolism

 Metabolites

 Basic metabolic Pathways

 Formation of Secondary metabolites by these metabolic pathways

a. Shikimic acid pathway

b. Mevalonic acid pathway

c. Acetate malonate pathway

d. Amino acid pathway

 Metabolism
 Metabolism is a term that is used to describe all the chemical reactions involved
in maintaining the living state of the cells and organisms.

 They are divided into two categories:

1. Catabolism- Breakdown of molecules to obtain energy.
2. Anabolism-Synthesis of compounds needed by the cells.
 Metabolites
 Metabolites are the intermediates and the products of metabolism.
 E.g., A B C D

 Types of metabolites:
1. Primary metabolites
2. Secondary metabolites
 Types of Metabolites
S. No. Primary metabolites Secondary metabolites
1 They are directly synthesized in They are synthesized by the help
plants. of primary metabolites.
2 Widely distributed in nature. Present in specific parts of
plants.
3 Simple in nature. Complex in nature.
4 Directly involved in the growth, Not directly involved in growth
development, and reproduction. and development.
Play a key role in ecological
functions like defence, medicinal
use, flavourings, etc.
5 Produced in large quantities and Produced in small quantities and
its extraction is easy. its extraction is difficult.
6 Examples- Examples-
Starch, cellulose, amino acid, Alkaloids, Glycosides, Tannins,
Vitamins, protein, carbohydrate, Resins, Volatile oils, Flavonoids.
Lipids, etc.
Introduction to Basic Metabolic Pathway

 A metabolic pathway is a linked series of chemical reactions

occurring within a cell.

 The reactants, products and intermediates of an enzymatic reaction are known

as metabolites.

 The pathways are required for the maintenance of homeostasis.

Basic Metabolic Pathway
 Basic Metabolic Pathways
 Shikimic acid pathway

 Acetate Pathways

(a) Mevalonic acid pathway

(b) Acetate malonate pathway

 Amino acid pathway

 Shikimic Acid Pathway
 It is a metabolic pathway for the biosynthesis of Aromatic aminoacids
(Phenylalanine, Tyrosine and Tryptophan).

 Also gives phenyl-propanoids or phenyl-propane derivatives (C6-C3

compunds).

 It is also known as Aromatic pathway.

 Shikimic acid name is the chemical constituent which is present in

Japanese flower i.e. “Shikimi”(Star anise, Illicium anisatum).

 It was first isolated in 1885 by the scientist Johan Fredrik Eykman.

 Shikimic acid pathway is a seven step metabolic pathway that occurs in plant,
fungi and bacteria but not in animals.

 Biosynthetic precursors:
o Phospho-enol-pyruvate (Glycolysis)
o Erythrose-4-phosphate (Pentose phosphate pathway)

 Role of SAP:
Precursor for the biosynthesis of some phenolic compounds, flavonoids,
Coumarin, tannins, alkaloidal compounds.
Shikimic Acid Pathway
 Shikimic Acid Pathway
 The pathway starts with two substrates i.e. Phospho-enol-pyruvate and Erythrose-4-
phosphate and forms 2-keto-3-deoxy-7-phospo-D-glucoheptonic acid in the presence
of enzyme DAHP synthase.
 2-keto-3-deoxy-7-phospo-D-glucoheptonic acid is then transformed to 3-dehydroquinic
acid (DHQ), in a reaction catalyzed by 3-dehydroquinic acid synthase.

 3-dehydroquinic acid (DHQ) is dehydrated (removal of water molecule) to

dehydroshikimic acid by the enzyme 3-dehydroquinate dehydratase.

 3-dehydroshikimic acid is converted to Shikimic acid by the enzyme shikimate

dehydrogenase.

 Now the Shikimic acid is transformed to Shikimate 3-phosphate by the involvement of

enzyme shikimate kinase.
 Shikimate 3-phosphate is then coupled with phosphoenol pyruvate to give 5- enol-pyruvyl-
shikimate-3-phosphate via the enzyme 5-enol-pyruvyl-shikimate-3- phosphate (EPSP) synthase.

 Then 5-enol-pyruvyl-shikimate-3-phosphate is transformed into Chorismic acid by an

enzyme chorismate synthase.

 From the compound Chorismic acid, in the presence of enzyme Chorismate mutase Prephenic acid
is synthesized.

 Now the Prephenic acid is oxidatively decarboxylated with retention of the hydroxyl group by
Prephenate dehydrogenase to give p- hydroxy-phenyl pyruvate, which is transaminated using
glutamate as the nitrogen source to give Tyrosine.

 The Prephanic acid undergo aromatization to synthesize Phenyl pyruvic acid followed by
reductive amination to form Phenylalanine.

 On the other hand of Chorismic acid, Anthranilic acid is formed which is extended to
formphospho-ribosyl-anthranilic acid and serine in presence of tryptophan synthase enzyme and
it converts Tryptophan.
 Acetate Pathways
 Acetate occupies a central position in relation to the general metabolism
of plants.

 Two main route of Acetate pathway:

1. Acetate Mevalonate pathway or Isoprenoid

pathway
2. Acetate Malonate pathway.
Acetate Pathway
 Mevalonic Acid Pathway
 Also known as:
▪ Acetate mevalonate pathway
▪ Isoprenoid pathway

 Isoprenoid compounds are made up of isoprene unit (C5H8) and

forms monoterpenes, sesquiterpenes, diterpenes, triterpenes,
tetraterpenes.

 Mevalonic acid pathway begins with acetyl-CoA and ends with the
production of IPP (Isopentenyl pyrophosphate) and DMAPP
(Dimethyl-allyl-pyrophosphate).
Mevalonic Acid Pathway
 Mevalonic Acid Pathway
1. The first step condenses two Acetyl-CoA molecules to yieldAceto-acetyl-CoA in the
presence of enzyme Aceto-acetyl-CoAthiolase.

2. Now the Aceto-acetyl-CoA is condensed with another Acetyl CoA molecule to form
HMG-CoA (3-hydroxy-3- methyl-glutaryl-CoA) by the help of enzyme HMG CoA
synthase.

3. Reduction of HMG-CoA yields Mevalonate by the use of HMG CoA reductase.

 These first 3 enzymatic steps are called the upper mevalonate pathway.

4. The lower mevalonate pathway which converts mevalonate into Isopentanyl

Pyrophosphate (IPP) and Dimethylallylpyrophosphate (DMPP).

5. IPP than isomerizes to form DMPP. These isoprene unit is the universal
precursor for synthesis of different types of Terpenoids and Steroidal
compounds.
 Acetate Malonate Pathway
 By this pathway, fatty acids are produced like-
▪ Stearic acid
▪ Palmetic acid
▪ Lauric acid
▪ Myristic acid

 Precursor involved:
 Acetate and CoA-SH (Co-enzyme with sulpha-hydryl functional group).
Acetate Malonate Pathway
 Amino Acid Pathway
 Amino acid is the basic unit of protein, occupy a central position in the
metabolism of all organisms.

 Amino acid synthesis is the set of biochemical processes by which the amino
acids are produced.

 Not all organisms are able to synthesize all amino acids. For example,
humans can only synthesize 11 of the 20 standard amino acids (non-essential
amino acid), Whereas most bacteria and plants can synthesize all 20,
mammals can synthesize only about half of them.

 All amino acids are derived from intermediates in glycolysis, the citric acid
cycle, or the pentose phosphate pathway.

 Aromatic amino acids like phenylalanine, tyrosine and tryptophan in plants

are not only essential components of protein synthesis, but also serve as
precursors for a wide range of secondary metabolites that are important for
plant growth as well as for human nutrition and health.
 Standard Amino Acid
 A standard amino acid is an amino acid, organisms use in the synthesis
of peptides and are the basic building block of all the organisms.
 There are twenty standard amino acids.

S. No. SAA S.No. SAA

1. Alanine 11. Leucine
2. Arginine 12. Lysine
3. Asparagine 13. Methionine
4. Aspartic Acid 14. Phenylalanine
5. Cysteine 15. Proline
6. Glutamine 16. Serine
7. Glutamic Acid 17. Threonine
8. Glycine 18. Tryptophan
9. Histidine 19. Tyrosine
10. Isoleucine 20. Valine
 These standard amino acids are divided into three categories:

1. Essential amino acids

2. Semi-essential amino acids
3. Non-essential amino acids
 1. Essential Amino Acids
 As the body cannot produce them by themselves, so they have to be
supplied externally.

 Eight amino acids are essential for humans.

 They are:
isoleucine, leucine, lysine, methionine, phenylalanine, threonine,
tryptophan and valine.
 2.Semi-Essential Amino Acids
 Semi-essential amino acids are those that can be synthesized by the body's
metabolic pathways, but possibly not in sufficient quantity.

 They have to be consumed in the diet under certain circumstances.

 They are:

 Arginine and histidine.

 3. Non-Essential Amino Acid

 The ten non-essential amino acids are able to be produced in the body.

 The following amino acids fall into this category:

alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid,

glycine, proline, serin and tyrosine.
Study of Utilization of Radio Active
Isotopes in the investigation of
Biogenetic Studies
 Introduction
• Living plants were considered as biosynthetic laboratory, because it
can synthesize primary and secondary metabolites by the help of
biosynthetic pathway.

• Different biosynthetic pathways in plants for the production of

secondary metabolites are:

❑ Shikimic acid pathway

❑ Acetate pathways
➢ Mevalonic acid pathway
➢ Malonate pathway
❑ Amino acid pathway
 Radio-Tracer Technique
❑ It can be defined as-
A technique which utilizes a radio isotopes or radio-active isotopes
to find out or to trace the different intermediates and varioussteps in bio-
synthetic pathways in plants, at a given rate & time, is called as Radio-tracer
technique.

❑ Isotopes- Elements with the same atomic no. but different mass no.
❑ Isotopes used in tracer technique-
1. Radio-isotopes
2. Stable isotopes
1. Radio-isotopes:
▪ Also called as Radio-active isotopes.
▪ These are the isotopes that can emit radiation in the form of α, β, γ rays.
▪ e.g., 1H, 14C, 24Na, 42K, 35S, 35P, 131I .

❖ For biological investigation – carbon & hydrogen.

❖ For metabolic studies – S, P, and alkali and alkaline earth metals are used.
❖ For studies on protein, alkaloids, and amino acid -labelled nitrogen atom give more
specific information.

2. Stable isotopes:
▪ They do not emit radiation.
▪ They have no health hazard.
➢ e.g. 2H, 13C, 15N, 18O.
▪ Used for labeling compounds as possible intermediates in biosynthetic pathways.
Criteria for Tracer technique:-
▪ Physical and chemical nature of compound must be known.
▪ Labelled compound should involve in the synthesis reaction.
▪ Labelled compound should not damage the system.

Advantages:-
• Highly sensitive method.
• Applicable to all living system.
• Wide range of isotopes are available.
• More reliable.
• Easily administration and isolation procedure.
• Gives accurate result.
 Steps involved in Tracer techniques
I. Preparation of labelled compound.
Plant constituent precursor is presumed firstly.

Then, structure of presumed precursor is find out.

Now, the presumed precursor atom is replaced by radio-isotope.

Labelled compound is ready.

II. Introduction of labelled compound into a biological system:

▪ The precursor should react at necessary site of synthesis in plant.
▪ The dose given is for short period.
▪ Methods:-
1. Root Feeding
2. Stem Feeding
3. Floating Method
4. Spray Technique
5. Direct Injection
III. Separation and Determination of Labelled Compound

1. NMR Spectroscopy
2. Mass Spectroscopy
3. Geiger muller counter
4. Liquid scintillation counter
5. Gas ionization chamber, etc.
Methods in Tracer Techniques :

 Precursor product sequence

 Double and multiple labelling
 Competitive feeding
 Sequential analysis.
1. Precursor Product Sequence
Presumed precursor of plant constituent is taken

Labeling is done

Now, the labelled compound is fed/introduced in the plant After sometime constituent

is isolated

Purified Radioactivity is determined

2. Double and Multiple Labelling
• In this method, atleast 2 positions or more than two positions are
labelled with either a same isotope or with different isotope.

• Leete (Scientist), used double labelled lysine, to determine which H

of lysine molecule was involved in the formation of piperidine ring
of Anabasine in Nicotiana glauca.
3. Competitive Feeding Method

❑ This method is used to determine which of the two possible intermediates

is normally used by the plant in the normal route of synthesis.

❑ By competitive feeding, we can distinguish whether B or B’is normal

intermediate in the formation of C fromA.
e.g. B

A C

B*
 4. Sequential Analysis

❖ In this method, plant is allowed to grow in the atmosphere of

14CO2 at a given interval of time, after that the plant constituent
is isolated and analyzed to obtain the sequence.
 Applications
• To identify the sequence of intermediate compounds in the formation of
metabolites.

• Heavy metals can be detected.

• Stable isotopes like 13C can detect food adulteration.

• Used to trace biosynthetic pathway.

• It utilizes 14C, 3H labelled mevalonic acid for studying squalene
cyclization.
• It utilizes labelled phenylalanine to study Scopoletin formation.

• Nitrogen fixation can be quantified using tracer technique.

 Thank You

God Bless you All