This document discusses techniques used to study biosynthetic pathways in plants. It describes the shikimic acid pathway and mevalonate pathway, which are important for producing aromatic compounds and terpenes respectively. It also summarizes several techniques used in biogenic studies including the use of isolated organs, grafting, mutant strains, tracer techniques, and enzymatic studies. Tracer techniques involve using radioactive isotopes or stable isotopes to trace metabolic pathways and locate compounds. Proper labeling and introduction of tracers is required.

1. UNIT-I
Prepared By-
Smita V. Nhawkar
Assistant Professor at Ashokrao Mane college of Pharmacy, Pethvadgaon
Department- Pharmacognosy

2. Shikimic Acid Pathway
• Shikimic acid is commonly known as its anionic form shikimate.
• It is a cyclohexane, a cyclitol and cyclohexane-carboxylic acid.
• It is a important biochemical metabolite in plants and microorganism.
• Its name comes from the Japanese flower shikimi from which it was first isolated in 1885 by Johan
Fredrik Eykman.
• Shikimic acid is also the glycoside part of some hydrolysable tannins.
• Majority of the aromatic compounds are biosynthesied via shikimic acid pathways.
• It is important in the genesis of the aromatic building blocks of lignins and also leds to formation of
phenylpropane units like flavons, isoflavonone,coumarins, tannins, vanillin and terpenoid quinones
• It is an important route for biosynthesis of C6- C3 units (phenylpropane derivaties)
•

3. Role of the shikimic acid pathway
1. Starting point in the biosynthesis of some phonolies-
 Phenylalanine and tyrosin are precursors used in the biosynthesis of phenylpropanoids
 phenylpropanoids are then used to produce the flavonoids, coumarins,tannins,lignin
2. Gallic acid biosynthesis-
 Gallic acid formed from 3-dehydroshikimate by action of the enzyme shikimate dehydrogenase to
produce 3,5 didehydroshikimate.
 Latter compound spontaneously rearrange to gallic acid.
3. Shikimic acid is a precursor-
 Precursor for indol, indol derivatives and aromatic amino acid tryptophan

4.  Shikimic acid is a key intermediate for biosynthesis of C6- C3 units
The important steps involved in genesis of shikimic acid pathway are as follow-
 Shikimic acid pathway starts with the Erythrose 4-phosphate and
phospoenolpyruvate on aldol condensation to yield DHAP (2-Keto-3-Deoxy –D-
arabinoheptonic acid-7-phosphate)
 DHAP on removal of phosphoric acid cyclise to form 3-Dehydroquinic acid which
on dehydration to form 3-dehydroshikimic acid which yields shikimic acid by
reduction.
 Shikimic acid through phosphorylation and elimination reaction forms a very
important intermediate compound chorismic acid.
Chorismic acid is an important branching point, anthranilate synthase uses
chorismic acid as substrate to give anthranilic acid which is a precursor for
Tryptophan.
 Chorismic acid via simple rearrangement gives prephenic acid. It is on
dehydration and decarboxylation yields precursor of phenylalanine i,e
phenylpyruvic acid.

5.  On dehydrogenation and decarboxylation, prephenic acid yields p-hydroxy phenylpyruvic acid which is a
direct precursor of Tyrosin.
2. Isoprenoid pathway/ Acetate Mevalonate Pathway
Introduction-
 Isoprenoid pathway is also known as terpenoid pathway or acetate mevalonate pathway.
 The isoprene units from this pathway is contributed in biosynthesis of many other metabolite such as
anthraquinones, terpenoids, and indol alkaloids
 The important steps involved in the biogenesis of mevalonate pathway as follow
1. Acetate mevalonate pathway start with molecule of acetyl CoA which is produced from pyruvic acid which
is a end product of glycolysis.

6. 2. First two molecule of acetyl CoA forms acetoacetyl CoA through claisen condensation.
3. Third molecule of acetyl CoA forms an important intermediate B-hydroxyl B-methylglutaryl –CoA by aldol
addition
4. Next two step reduction gives rise to mevalonic acid which is a main precursor for biosynthesis of
terpenoids.
5. Mevalonic acid on ATP mediate phosphorylation results in mevalonic acid diphosphate which on
decarboxylation provides first isoprene unit, Isopentenyl pyrophosphate(IPP)
6. Mevalonate kinase and mevalonate-5-phosphate kinase form mevalonate diphosphate which is
decarboxylated via ATP to form IPP
7. By the isomerase enzyme the IPP gives rise to second isoprene unit Dimethyl allyl pyrophosphate (DMAPP)
8. The allylic phosphate group of DMAPP is an excellent leaving group which yields carbanium ion.

7. 9. Enzyme prenyltransferase yields C10,C15,C20 units C10,C20 and C40 compounds are synthesized in
plastids via D-L-deoxyxylulose-3- phosphate pathway and C15,C30 compounds are formed in cytosol via
mevalonate pathway.
10. Electrophilic addition of IPP with DMAPP via enzymeprenyl yields C10 unit, Geranyl diphosphate (GPP),
which is precursor for synthesis of monoterpenes.
11. Combination of another IPP unite again with GPP give rise to form farnesyl diphosphate (FPP), C15 unite
which acts as a precursor for the synthesis of sesquiterpene.
12. Further addition of another molecule of IPP unit yields C20 Geranyl diphosphate to produce range of
diterpenes.
13. on further addition of IPP unit yields terpenoids C25 unit called sesterterpenes
14. The tail to tail addition of two FPP units yields C30 unit, triterpene.

8.  Investigation of Biogenetic studies/Major technique
generally used for biogenetic studies of primary and
secondary metaboliotes
Biosynthetic pathway in plants can be investigated by means of
following techniques: -
» Use of isolated organ
» Grafting methods
» Use of mutant strain
» Tracer technique
» Enzymatic studies

9.  Use of isolated organs and tissues
This method is based on using isolated parts of plant e.g., stem,
roots.
This technique is useful in the determination of site of biosynthesis
of particular compounds. ex. Roots and leaves for the study of
Nicotiana and Datura, petal disc for the study of rose oil, tropane
alkaloids in the root of solanaceae family.
 Grafting methods: This method is used for the study of alkaloid
formation by grafted plants.
ex. Tomato scions grafted on Datura produce alkaloids, while Datura
scion grafted on Tomato produce less quantity of alkaloids. This
shows that main site of alkaloid biosynthesis is root.

10. Use of mutant strain-
 In this mutant strains microorganisms are produced with the lack of certain
enzymes. • ex. Gibberella mutant is used to produce isoprenoid
compounds, Lactobacillus acidophillus is used for mevalonic acid pathway
for isoprenoid biosynthesis
 Enzymatic study: Phyto hormone auxin plays critical roles in the regulation
of plant growth and development. • Indole-3-acetic acid (IAA) has been
recognized as the major auxin which is synthesized in plants is still unclear.
Previous genetic and enzymatic studies demonstrated that both
TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA) and YUCCA (YUC)
flavinmono oxygenase-like proteins are required for biosynthesis of IAA
during plant development. • TAA family produces indole-3-pyruvic acid (IPA)
and the YUC family functions in the conversion of IPA to IAA in
Arabidopsis (Arabidopsis thaliana).

11.  Tracer technique-
It can be defined as technique which utilizes a labelled compound to
find out or to trace the different intermediates and various steps in
biosynthetic pathways in plants, at a given rate & time. OR
In this technique different isotope, mainly the radioactive isotopes
which are incorporated into presumed precursor of plant metabolites
and are used as marker in biogenic experiments.
 Significance of tracer technique
1. Tracing of biosynthetic pathway: e.g. by incorporation of
radioactive isotope of 14C into phenylalanine, the biosynthetic
cyanogenetic glycoside prunasin, can be detected.
2. Location & quantity of compound containing tracer: 14C labelled
glucose is used for determination of glucose in biological system

12. 3. Different tracers for different studies: For studies on alkaloids,
proteins nitrogen and amino acid (Labelled nitrogen give specific
information than carbon). For terpenoids O atom and glycosides O, N,
S & C atom used
4. Convenient and suitable technique
 Criteria for tracer technique-
• The starting concentration of tracer must be sufficient withstand
resistance with dilution in course of metabolism
• Proper labeling: For proper labeling physical & chemical nature of
compound must be known
• Labelled compound should involve in the synthesis reaction
Labelled should not damage the system to which it is used

13.  Advantages
• High sensitivity.
• Applicable to all living organism.
• Wide ranges of isotopes are available.
• More reliable, easily administration & isolation procedure.
• Gives accurate result, if proper metabolic time & technique
applied.
 Limitations
• Kinetic effect
• Chemical effect
• Radiation effect

14. • Radiochemical purity
• High concentration distorting the result, expensive, short half life,
hazardous
Requirment for tracer technique
1. Preparation of labelled compound
• The labelled compound produce by growing chlorella in atmosphere
of 14CO2 All carbon compounds 14C labelled.
• The 3H (tritium) labelled compound are commercially available.
Tritium labeling is effected by catalytic exchange in aqueous media by
hydrogenation of unsaturated compound with tritium gas.
Tritium is pure β-emitter of low intensity & its radiation energy is
lower than 14C.

15.  Labelled compound can be prepared by use of two type of isotope
1. Radioactive isotopes: • [e.g. 1H, 14C, 24Na, 42K, 35P, 131I decay
with emission of radiation]
• 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.
• 3H compound is commercially available.

16. 2. Stable isotope
• [e.g. 2H, 13C, 15N, 18O]
• Used for labeling compounds as possible intermediates in biosynthetic
pathways.
• Usual method of detection are: – Mass spectroscopy [15N, 18O] • NMR
spectroscopy [2H, 13C]
 2. Introduction of labelled compounds
• Precautions: • •
1. The precursor should react at necessary site of synthesis in plant.
2. Plant at the experiment time should synthesize the compound under
investigation
3. The dose given is for short period.

17.  Methods used for introduction to labelled compounds into
plant tissue
1. Root feeding-
The plant in which roots are biosynthetic sites e.g Tobacco
The plants are hydroponically cultivated to avoid microbial
contamination.
2. Stem feeding:
Substrate can be administered through cut ends of stem
immersed in solution. For latex containing plant this
method is not suitable.

18.  Direct injection: Suitable for plant with hollow stem e.g., umbelliferous
and opium.
 Infiltration: Suitable for plant rooted in soil or other support without
disturbing the roots
 Floating method: When small amount of material is available this
method is used. This technique is used in conjugation with vacuum
infiltration to remove gases.
 Spray technique: Compound has been absorbed after being sprayed on
leaves in aqueous solution e.g., Steroids
3. Separation of compounds
Some ways are used for separation of compounds
• Soft & fresh tissue: Maceration, infusion
• Hard tissue: Decoction, hot percolation

19. • Unorganized drug: Maceration with adjustment
• Choice of solvent for extraction
• Fat & oil: Non-polar solvents • Alkaloids, glycosides, flavonoids
4. Detection and assay of compounds/radio isotope labelled
compounds
• Geiger – Muller counter
• Liquid Scintillation counter
• Gas ionization chamber
• Bernstein – Bellentine counter

20. • Mass spectroscopy
• NMR eletrodemeter
• Autoradiography
• Radio paper chromatography

21.  Geiger – Muller counter
 A Geiger counter (Geiger-Muller tube) is a device used for the
detection and measurement of all types of radiation: alpha, beta
and gamma radiation.
 Basically it consists of a pair of electrodes surrounded by a gas.
 The electrodes have a high voltage across them.
 The gas used is usually Helium or Argon.
 When radiation enters the tube it can ionize the gas.
 The ions (and electrons) are attracted to the electrodes and an
electric current is produced.

22.  A scaler counts the current pulses, and one obtains a "count"
whenever radiation ionizes the gas.
• Advantages
They are relatively inexpensive, durable, easily portable, detect all
types of radiations
 • Disadvantages •
 a)They cannot differentiate which type of radiation is being
detected.
 b)They cannot be used to determine the exact energy of the
detected radiation & have a very low efficiency

24.  Liquid scintillation counter
The liquid scintillation counter is used for detecting radioactivity.
It is an analytical method used for measuring the radioactivity when
light photons are emitted by the sample.
The liquid scintillation counter is a sophisticated laboratory counting
system used the quantify the activity of particulate emitting
radioactive samples, it can also detect the auger electrons emitted
from 51Cr and 125I samples.
The conversion of kinetic energy of a partial into a fletting pluse of
light as the result of its penetrating a suitable luminescent
substance.
The flashes of light produced bombardment with alpha particles on a
fluorescent screen prepared from zinc sulphide.

25. Liquid scintillation media consist of a solvent in which the
excitation occurs and a fluorescent solute which emits the light to
actuate the photo multiplier.
Advantages-
1. For the soft and easily absorbed radiation from 3H and 14C
labeled compound
2. Modern methods are full automatic i,e 100 samples at a time are
measured
3. To measure the mixed radiation such as 3H and 14 C because
both are beta emitters, which different radiation energies

26.  Auto radiography
Detecting radioactive compounds with a photographic emulsion (x-ray
film)
Types of autoradiography
1. In-vivo autoradiography - receptors are labelled in intact living
tissue by systemic administration of the radioligand (PET)
2. In-vitro autoradiography - slide-mounted tissue sections are
incubated with radioligand so that receptors are labelled under
very controlled conditions
Uses :
• Map anatomical location of radiolabelled ligands to visualize and
quantify receptors in tissue

27. • Trace neurons by axonal transport of radioactively labelled amino
acids, certain sugars, or transmitter substances
• Measure DNA production (e.g., 3H-thymidine)
Advantages
 Highly specific tool to pharmacologically characterize receptors in
tissue
 • Provides location of receptor (etc) in tissue • Enables
characterization of receptors in different tissues
 • Technically easy

28.  Disadvantages: - • Everything binds to everything (easy to
misinterpret results)
• There are no biochemical or physiological criteria to assess the •
binding specificity (i.e., to determine whether the binding site
really
• corresponds to an actual receptor)
• The presence of a high-affinity radiolabelled receptor does not
necessarily imply that the receptor has physiological significance
• Ligands are not always very specific

29.  Methods of tracer technique
 Precursor- product sequence
 Competitive feeding
 Sequential analysis
 Use of stable isotope
 Precursor- product sequence-
In this method the constituent is labeled it is fed to the plant for
specific period of time.
The constituents produced in plant are isolated and purified and its
radioactivity is determined

30.  Competitive feeding
In this method is used to differentiate the normal intermediate
between two different process to form mother compound to
product.
Therefor any isotope incorporation if done it is necessary to consider
the normal route of synthesis of secondary metabolite in plant not
the subsidiary pathway
This method is used for biosynthesis of propane alkaloids, hemlock
alkaloid.
e.g- C14 labelled compound is used for biosynthesis of conium
maculactum.

31.  Sequential analysis
A second method of investigation with 14C is to grow plants in an
atmosphere of 14CO2 and by analysis of the plants at given time
interval to obtain the sequence in which various related compound
become labeled.
Degradation of the isolated radioactive compound is important bcoz
some units of the molecule may become labeled more rapidly than
other.

32.  Mass spectrophotometer-
Mass spectrometry (MS) is an analytical technique that measures
the mass-to-charge ratio of charged particles.
• It is used for determining masses of particles, for determining
the elemental composition of a sample or molecule , and for
elucidating the chemical structures of molecules, such as peptides
and other chemical compounds.
 NMR Spectrophotometer: • NMR spectroscopy, is a research
technique that exploits the magnetic properties of certain atomic
nuclei to determine physical and chemical properties of atoms or
the molecules in which they are contained. •

33. It relies on the phenomenon of nuclear magnetic resonance and can
provide detailed information about the structure, dynamics, reaction
state, and chemical environment of molecules.
 Application of tracer technique
1. 14C 3H labelled mevalonic acid are used for study of cyclization
2. 14C and 15N labelled precursor is used to detect origin of carbon
and nitrogen atom respectively of purine ring system.
3. Labelled coumarin is used to study the formation of cinnamic acid
in coumarin biosynthesis pathway
4. Labelled phenylalanine is used to study the formation of
scopoletin

34. 5. 14C acetate is used for interrelation among 4-methyl sterol and
4,4 dimethyl sterols.
6. 2-14C mevalonate is used for terpenoids biosynthesis by
chloroplast isolated in organic solvent.
7. Ca-uptake by plant from the soil is identified by using labelled
45C