This document discusses metabolic pathways in higher plants and their determination. It outlines several key pathways including the shikimic acid pathway, acetate mevalonate pathway, acetate malonate pathway, and amino acid pathways that are involved in the biosynthesis of primary and secondary metabolites in plants. Techniques used to study these biogenetic pathways include using isolated organs/tissues, grafting, mutant strains, and tracer techniques with radioactive isotopes.

1. Pharmacognosy & Phytochemistry-II
Unit–I Metabolicpathwaysinhigherplantsandtheirdetermination
Presented by :- Miss. M A Joshi
Asst. Professor, IVM’s IIPER

2. Plant biosynthesis
• Plants are biosynthetic laboratory
• Produces no of metabolites
• Higher plants are also called as
solar powered biochemical factory
• This factory manufactures primary
and secondary metabolites

3. What are primary and secondary
metabolites???
• Primary metabolites- fulfill primary need of plants.
• Growth of plant
• Physiological development of plant

5. Secondary metabolites
• Biosynthetically derived from primary
metabolites
• Present only in higher plant or taxonomic group.
• They are chemical adaptation of plant to
environmental stress.
• They serves as defensive, protective or offensive
chemicals.
• Sometimes waste products of plant which
interrupt normal metabolism in plant are also
converted into secondary metabolites.

6. Plants defense offense and protection

7. Importance of secondary metabolites
• As medicines – as laxative, cardio tonic,
antihypertensive, sedatives, hypnotics,
Immuno-modulators etc.
• Flavors
• Perfumes
• For industrial purpose such as lubricant
protection etc
• Insecticides and pesticides eg. tobacco

8. General pathways of formation of secondary
metabolites

9. Different pathways of secondary metabolites
• Shikimic acid pathway:- formation of Phenylalanine, Tyrosine, Tryptophan,
Gallic acid, Anthranilic acid.
• Acetate Mevalonate pathway:- Squalene, triterpenes, steroids, Phytol ,
Gibberlins, Caretenoids
• Acetate Malonate pathway:- Palmitic acid, stearic acid, oleic acid, Linoleic
acid, Arachidonic acid.
• Amino Acid pathway:-Glutamine, Aspargine, Hydroxypyroline,
ornithine,methionine, Leiucine.

10. Shikimic acid Pathway

11. 1. conversion of erythrose -4-phosphate to 3-dehydroquinic acid
reaction catalysed by Phospo -2-oxo-3-deoxyheptonate aldolase
3-dehydroquinate synthase enzyme needs cobalt and NAD as co-
factor

12. 2.Formation of Shikimic acid

13. 3. Formation of Chorismic acid
Catalysed by shikamate kinase enzyme
Enolpyruvylshikimate phosphate synthase enzyme
chorismate synthase enzyme

14. 4. Formation of other intermediate
Chorismic acid to anthranilic acid in presence of glutamine
chorismic acid to Prephenic acid by chorismate mutase enzyme
chorismic acid to p-aminobenzioc acid

15. 5. Formation of Aromatic amino acid
Anthranilic acid to Phosphoribosyl anthranilic acid- anthranilate phosphoribosyl
transferase
Carboxyphenylaminodeoxyribulose-5-Phosphate-Phosphoribosyl anthranilate
isomarase
conversion to Indolyl-3-glycero phosphate – Indolyl-3-glycerol phosphate synthase
condensation of indol with serine to form tryptophan

16. Prephenic acid isomarise to 4-Hydroxyphenylpyruvic acid- prephenate
dehydrogenase enzyme
tyrosine- by tyrosine aminotransferase enzyme
prephennic acid to phenylpyruvic acid- prephenate dehydratase enzyme
Phenylalanine by phenylalanine aminotransferase enzyme.

17. Acetate Mevalonate Pathway
Role of acetic acid in biogenetic was discovered in 1950 after the discovery of
acetyl co-enzyme A (Active acetate).
Mevalonic acid is associated with acetate.
IPP and DMAPP are two chief isoprene unit as the basic building block of
isoprenoid compounds.
They produce geranyl pyrophosphate (C10 monoterpenes), which assciates with
IPP to produce Farnesyl pyrophosphate (C-15 sequiterpenes)
Farnesyl pyrophosphate multiply to form C30 compound and then after further
addition of IPP it forms Squalene , Triterpenes and Steroids.
Farnesyl pyrophosphate and IPP forms C-20 Geranyl geraniol pyrophosphate.
Geranyl Geraniol pyrophosphate and further additions of IPP forms Guttas, Phytol
and Gibberlins.
Two molecultes of Geranyl Geraniol pyrophosphate forms C-40 carotenoids.

21. Acetate Malonate Pathway
Involvement of Acyl carrier protein (ACP), this pathway produces fatty acyl
thioesters of ACP.
This ACP builds imp intermediates in fatty acid synthesis.

24. Amino acid pathway
Amino acid found in free state and they are basic unit of proteins
They are precursors for secondary metabolites eg. Alkaloids
Most of the amino acid found in nature in the form of alpha amino
acid.( functional group attached to alpha carbon both functional
groups carboxylic acid and amino group)
Amino acid pathway starts from Glycolysis and Krebs cycle
Plant synthesize all 20 amino acids by using different routes of
biosynthesis.
Nitrogen enter in reaction through reductive amination of alpha keto
acid.
The aliphatic and heterocyclic amino acid have different routes of
biogenesis.
We have to learn amino acid biogenesis by 5 different pathways.

25. Biosynthesis of Glutamine and Asparagine
Glutamine:-
Asparagine:-
NH3 NH3
α- Ketoglutaric acid
GlutamineGlutaminc acid
NH3
NH3
Oxaloacetic acid
Aspartic acid Asparagine

26. Biosynthesis of Ornithine, Proline and Hydroxyproline
Glutaminc acid Glutamic-γ-semialdehyde
Ornithine
Pyrroline-5-carboxilic acid
Proline Hydroxyproline

27. Biosynthesis of Serine and Glyceine

28. Biosynthesis of Methionine

29. Biosynthesis of Alanine, Valine and Leucine

30. Biogenetic studies
• Various intermediated steps involved in biogenetic pathways of
plant are investigated by following different techniques.
1. Using Isolated organs/tissues
2. Grafting method
3. Use of Mutant strains
4. Tracer Technique

31. Using Isolated organs/tissues

33. Grafting technique

35. Used for study of alkaloids
Tomato and Dhatura are grafted to locate the position of secondary
metabolite.
When tomato scion grafted of Dhatura root stock it shows accumulation of
alkaloids.
But when Dharura scion grafted on tomato root stock it do not show very
small amount of alkaloid.
It is conclude that main synthesis site for formation of Dhatura alkaloids is
root.

36. Use of Mutant strain technique
• Gibberela is used for synthesis of isopronoid compounds
• Lactobacillus acidophilus is used in mevalonic acid
pathway for synthesis of isoprenoid units.
• In this technique normal metabolism is blocked on any
stage to synthesise required compound.

37. Utilization of radioactive isotopes
Radio labeled compound is used to investigate biosynthetic
pathways.
To investigate different techniques
C14, H3, S35,P32 are mainly used compounds for the investigation
of biosynthetic pathways.
Highly effective technique
Can be used in living system.
Provides accurate results.

38. Steps involved in utilization of
Radiolabel compound
1.Criteria for Tracer/ Isotope selection :-
Concentration should be sufficient
Physical and chemical properties should be well known.
It should not interfere in reaction system.
2.Preparation of Labeled compound:-
Labeled compound can produce by allowing to grow them in co2(14) atmosphere.
CH3MgBr + 14CO2 ------ CH314COOHMgBr + H2O
CH314COOHMgBr ------ CH314COOH +Mg(OH)Br

39. Introduction of Labeled compound
into biological system
• Root feeding method
• Step feeding
• Direct injection
• Infiltration or wick feeding
• Floating method
• Spray technique

40. 4. Separation and detection technique
• Soft and fresh tissue :- Infusion and maceration
• Hard tissue:- Decoction and Hot percolation
• Unorganized tissue:- maceration with adjustment
• Fat and oil :- non-polar solvents
• Akaloids, Glycosides, and Flavonoids:- Slightly polar solvent
• Plants and Phenols:- Polar solvents
• GM counter
• Liquid scintillation Chember
• Gas ionisation chember
• Mass spectroscopy
• NMR spectroscopy
• Auto-radiography

41. Methods for Tracer Technique
• 1. Precursor Product Sequence:
• 2. Double and Multiple Labeling
• 3. Competitive Feeding
• 4. Isotope Incorporation
• 5. Sequential Analysis

46. Plants defense offense and protection