12/7/2012

Kratika
Kratika Daniel (Ph.D)
Assistant professor
MANDSAUR INSTITUTE OF PHARMACY,
Mandsaur
Mrs Kratika Daniel,Asst. Prof.
MIP Mandsaur
12/7/2012
INTRODUCTION
Living plants considered as biosynthetic laboratory primary
as well as secondary metabolite.
Different biosynthetic pathway: -
Shikmic acid pathway
Mevalonic acid pathway
Acetate pathway
Various intermediate and steps are involved in biosynthetic pathway
in plants can be investigated by means of following techniques: -
Tracer technique
Use of isolated organ
Grafting methods
Use of mutant strain
Mrs Kratika Daniel,Asst. Prof.
MIP Mandsaur
12/7/2012
Definition: - 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.
Mrs Kratika Daniel,Asst. Prof.
MIP Mandsaur
12/7/2012
The labelled compound can be prepared by use of
two types of isotopes.
Radioactive isotopes.
Stable isotopes.
Radioactive isotopes: - [e.g.
1
H,
14
C,
24
Na,
42
K,
35
S,
35
P,
131
I 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.

3
H compound is commercially available.
Stable isotopes: - [e.g.
2
H,
13
C,
15
N,
18
O]
Used for labelling compounds as possible intermediates in
biosynthetic pathways.
Usual method of detection are: MASS spectroscopy [
15
N,
18
O]
NMR spectroscopy [
2
H,
13
C]
Mrs Kratika Daniel,Asst. Prof.
MIP Mandsaur
12/7/2012
SIGNIFICANCE OF TRACER TECHNIQUE
Tracing of Biosynthetic Pathway: - e.g. By incorporation of radioactive
isotope of
14
C into phenylalanine, the biosynthetic cyanogenetic glycoside
prunasin, can be detected.
Location & Quantity of compound containing tracer: -
14
C labelled glucose is
used for determination of glucose in biological system
Different tracers for different studies: - For studies on nitrogen and amino
acid. (Labelled nitrogen give specific information than carbon)
Convenient and suitable technique
CRITERIA FOR TRACER TECHIQUE
The starting concentration of tracer must be sufficient withstand
resistance with dilution in course of metabolism.
Proper Labelling: - for proper labelling 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.
Mrs Kratika Daniel,Asst. Prof.
MIP Mandsaur
12/7/2012
ADVANTAGES
High sensitivity.
Applicable o all living organism.
Wide ranges of isotopes are available.
More reliable, easily administration & isolation procedure.
Gives accurate result, if proper metabolic time & technique applied.

LIMITATION
Kinetic effect
Chemical effect
Radiation effect
Radiochemical purity
High concentration distorting the result.
Mrs Kratika Daniel,Asst. Prof.
MIP Mandsaur
12/7/2012
REQUIREMENT FOR TRACER TECHNIQUE
Preparation of labelled compound.
Introduction of labelled compound into a biological system.
Separation & determination of labelled compound in various
biochemical fractions at later time.
I. Preparation of Labelled Compound: -
CH
3
MgBr
+
14
CO
2
CH
3
14
COOHMgBr
+
H
2
O
CH
3
14
COOH
+
Mg(OH)Br
The labelled compound produce by growing chlorella in atmosphere of
14
CO
2
.

All carbon compounds
14
C labelled.
The
3
H (tritium) labelled compound are commercially available. Tritium labelling is
effected by catalytic exchange in aqueous media by hydrogenation of unsaturated
compound with tritium gas. Tritium is pure emitter of low intensity & its
radiation energy is lower than
14
C.
By the use of organic synthesis: -
Mrs Kratika Daniel,Asst. Prof.
MIP Mandsaur
12/7/2012
PRECAUTION: -
The precursor should react at necessary site of synthesis in plant.
Plant at the experiment time should synthesize the compound under investigation
The dose given is for short period.
II. Introduction of labelled compound: -
1. Root feeding
2. Stem feeding
3. Direct injection
4. Infiltration
5. Floating method
6. Spray technique
III. Separation and detection of compound: -
a) Geiger Muller counter.
b) Liquid Scintillation counter.
c) Gas ionization chamber.
d) Bernstein Bellentine counter.
e) Mass spectroscopy.
f) NMR eletrodemeter.
g) Autoradiography.
h) Radio paper chromatography.
Mrs Kratika Daniel,Asst. Prof.
MIP Mandsaur
12/7/2012
METHODS IN TRACER TECHNIQUE
1. PRECURSOR PRODUCT SEQUENCE: - In this technique, the presumed
precursor of the constituent under investigation on a labelled form is fed
into the plant and after a suitable time the constituent is isolated, purified
and radioactivity is determined.
Disadvantage: - The radioactivity of isolated compound alone is not
usually sufficient evidence that the particular compound fed is direct
precursor, because substance may enter the general metabolic pathway
and from there may become randomly distributed through a whole range
of product.
Application: -
Stopping of hordenine production in barley seedling after 15 20 days
of germination.
Restricted synthesis of hyoscine, distinct from hyoscyamine in Datura
stramonium.
This method is applied to the biogenesis of morphine & ergot alkaloids
Mrs Kratika Daniel,Asst. Prof.
MIP Mandsaur
12/7/2012
2. DOUBLE & MULTIPLE LABELLING: - This method give the
evidence for nature of biochemical incorporation of precursor arises
double & triple labelling. In this method specifically labelled precursor
and their subsequent degradation of recover product are more employed.
Application: -
This method is extensively applied to study the biogenesis of plant secondary
metabolite.
Used for study of morphine alkaloid.
E.g. Leete, use Doubly labeled lysine used to determine which hydrogen of lysine
molecule was involved in formation of piperidine ring of anabasine in Nicotina glauca.

N H
2
N H
2
COO
-
Lysine - 2 -
14
C,
15

N. glauca
N
N
H
N H
2
N H
2
COOH
Lysine - 2 -
14
C,
15

N. glauca
N
N
H
Anabasine
Anabasine
Mrs Kratika Daniel,Asst. Prof.
MIP Mandsaur
12/7/2012
B
B'
C A
A C
A B
C
A B' C
N H
2
N H
2
COOH
N
H
CH
3
conline
O
O
HOOC CH
3
O
poly -

keto acid
N
CH
3
N
H
CH
3
OH
conhydrine
3. COMPETITIVE FEEDING: - If incorporation is obtained it is
necessary to consider whether this infact, the normal route of synthesis in
plant not the subsidiary pathway. Competitive feeding can distinguish
whether B & B is normal intermediate in the formation of C from A.
Application: -
This method is used for elucidation of biogenesis of propane alkaloids.
Biosynthesis of hemlock alkaloids (conline, conhydrine etc) e.g. biosynthesis of
alkaloids of Conium maculactum (hemlock) using
14
C labelled compounds.

OR

Mrs Kratika Daniel,Asst. Prof.
MIP Mandsaur
12/7/2012
4. ISOTOPE INCORPORATION: - This method provides
information about the position of bond cleavage & their formation
during reaction.
E.g. Glucose 1- phosphatase cleavage as catalyzed by alkaline
phosphatase this reaction occur with cleavage of either C O bond or
P O bond.
O
OPO
3
H
OH
OH
OH
CH
2
OH
+
H
2
18
O
O
OH
OH
OH
CH
2
OH
OH
+
H
2
PO
4
Mrs Kratika Daniel,Asst. Prof.
MIP Mandsaur
12/7/2012
5. SEQUENTIAL ANALYSIS: - The principle of this method of
investigation is to grow plant in atmosphere of
14
CO
2
& then analyze
the plant at given time interval to obtain the sequence in which various
correlated compound become labelled.

Application: -

14
CO
2
& sequential analysis has been very successfully used
in elucidation of carbon in photosynthesis.
Determination of sequential formation of opium hemlock and
tobacco alkaloids.
Exposure as less as 5 min.
14
CO
2
, is used in detecting
biosynthetic sequence as
Piperitone --------- (-) Menthone ---------- (-) Menthol in
Mentha piperita.
Mrs Kratika Daniel,Asst. Prof.
MIP Mandsaur
12/7/2012
APPLICATION OF TRACER TECHNIQUE
1. Study of squalene cyclization by use of
14
C,
3
H labelled mevalonic acid.
2. Interrelationship among 4 methyl sterols & 4, 4 dimethyl sterols, by use of
14
C acetate.
3. Terpenoid biosynthesis by chloroplast isolated in organic solvent, by use of
2-
14
C mevalonate.
4. Study the formation of cinnamic acid in pathway of coumarin from labelled
coumarin.
5. Origin of carbon & nitrogen atoms of purine ring system by use of
14
C or
15
N labelled precursor.
6. Study of formation of scopoletin by use of labelled phenylalanine.
7. By use of
45
Ca as tracer, - found that the uptake of calcium by plants from
the soil. (CaO & CaCO
2
).
8. By adding ammonium phosphate labelled with
32
P of known specific activity
the uptake of phosphorus is followed by measuring the radioactivity as label
reaches first in lower part of plant, than the upper part i.e. branches, leaves
etc.
Mrs Kratika Daniel,Asst. Prof.
MIP Mandsaur