BY: PRABHAT KHARE

M.Pharm (PHARMACOGNOSY)
Asst. Professor 1
 Living plants considered as biosynthetic
laboratory for production of primary as
well as secondary metabolite.
Various intermediate and steps are
involved in biosynthetic pathway in
plants that can be investigated by
means of following techniques:
Using Isolated Organs / Tissues
Grafting Method
Using Mutant Strains
Using Tracer Techniques

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 i) Using Isolated Organ / Tissues:
This method is based on using isolated parts of plant ( eg. Stem, roots
etc.).This technique is useful in determination of site of synthesis of particular
compound.
Ex.: Roots and Leaves for study of Nicotiana and Datura, Petal disc for study of oil
of rose, Tropane Alkaloids formed in roots of Solanaceae Family.

ii) Grafting methods:

This method is used for study of alkaloid formation by Grafted Plants.
Ex.: Tomato Scions grafted on datura accumulate alkaloids, while Datura Scion
grafted on Tomato contained very small amount of Alkaloids. This suggests that
main site for formation of Datura Alkaloids is Root

iii) Use of mutant strains:

In this method Mutant Strains of micro organisms are produced which lack certain
enzymes.
Ex.: Gibberella mutant is used to produce isoprenoid compounds, Lactobacillus
acidophilus is used for Mevoloinic Acid Pathway of Isoprenoid compound synthesis.

iv) Tracer technique:

This technique utilises labelled compound to find out/ trace different intermediates
& various steps in Biosynthetic Pathway

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 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.
Also this technique utilises the labelled
compound which when introduced into plant
system, they become part of general
metabolic pool & undergo reactions
associated with that particular plant system

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 High sensitivity.
Applicable to living system.
Wide ranges of isotopes are available.
More reliable, easy administration & isolation
procedure.
Gives accurate result, if proper metabolic time &
technique applied.
Location & Quantity of compound containing tracer
14C labelled glucose is used for determination of

glucose in biological system.

Different tracers can be used for different studies.
Ex. For studies on nitrogen and amino acid, Labelled
nitrogen give specific information than carbon.

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 Two types of isotopes are generally used for
labelling
1. Radioactive isotopes.
2. Stable isotopes.
Following points must be considered before
selection
1. The starting concentration of tracer must be
sufficient enough to withstand dilution in
course of metabolism.
2. Physical & chemical nature of compound must
be known for proper labelling.
3. Higher Half-life.
4. Should actively participate during synthesis.
5. Should not damage the system.

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1. Radioactive isotopes: - [e.g. 1H, 14C, 24Na,
42K, 35S, 35P, 131I ]

For biological investigation C & H.

For metabolic studies S, P, and alkali and
alkaline earth metals are used.
For studies on protein, alkaloids, and amino
acid - labelled N-atom.

2. Stable isotopes: - [e.g. 2H, 13C, 15N, 18O]

Used for labelling compounds as possible
intermediates in biosynthetic pathways.
Usual method of detection are: MASS
spectroscopy [15N, 18O]
N.M.R spectroscopy [2H, 13C]

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I. Preparation of labelled compound.
II. Introduction of labelled compound
into a biological system.
III. Separation & determination of
labelled compound in various
biochemical fractions.
IV. Methods for tracer technique

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 The labelled compound produce by growing
them in atmosphere of 14CO2. All carbon
compounds get 14C labelled.
The 3H (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 14C.
By the use of organic synthesis:
CH3MgBr + 14CO2 CH314COOHMgBr + H2O

CH314COOH + Mg(OH)Br

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 Root feeding
Stem feeding
Direct injection
Infiltration
Floating method
Spray technique

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 GM Counters
Liquid Scintillation Chamber
Gas Ionization Chamber
Mass Spectrophotometer
NMR Spectrophotometer
Auto-Radiography

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 A GeigerMuller counter, is a type of particle detector that
measures ionizing radiation e.g. alpha, beta particles, or gamma
rays by the ionization produced in a low-pressure gas usually
helium, neon or argon with halogens added in a GeigerMuller
tube which briefly conducts electrical charge when
a particle or photon of radiation makes the gas conductive by
ionization. This charge has been detected in form of current
pulse.

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 A scintillation detector or scintillation counter is obtained when
a scintillator is coupled to an electronic light sensor such as
a photomultiplier tube (PMT) or a photodiode.
A scintillator is a material that exhibits scintillation the
property of luminescence when excited by ionizing radiation.
Samples are dissolved or suspended in a "cocktail" containing
a solvent (aromatic organics such as benzene or toluene),
typically some form of a surfactant, and small amounts of
scintillators.

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 The ionization chamber is the simplest of all gas-filled radiation
detectors, and is widely used for ionizing radiation; X-
rays, gamma rays and beta particles.
Conventionally, the term "ionization chamber" is used exclusively
to describe those detectors which collect all the charges created
by direct ionization within the gas through the application of an
electric field.

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 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.

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 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.
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.

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 Autoradiography is a method for investigating the
distribution of radioactive material in a plant object, e.g.
histological tissues, a chromatography plate.
This techniques uses a photographic film or emulsion as
detector of ionizing radiation. The sample is in close contact
with emulsion for a certain period of time (exposure period)

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 Precursor Product Sequences
Double & Multiple Labelling
Competitive Feeding
Isotope Incorporation
Sequential Analysis

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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.
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

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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 labelled lysine used to determine
which hydrogen of lysine molecule was involved
in formation of piperidine ring of anabasine in
Nicotina glauca.

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Study of formation of Piperidine ring using doubly labelled Lysine

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3. COMPETITIVE FEEDING: -
This method provides the possible
intermediates that plant normally used during
biogenesis.

Application: -
This method is used for elucidation of
biogenesis of propane alkaloids.
Biosynthesis of hemlock alkaloids (conline,
conhydrine etc) using 14C labelled
compounds.
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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.

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5. SEQUENTIAL ANALYSIS: -
The principle of this method of investigation is
to grow plant in atmosphere of 14CO2 & then
analyze the plant at given time interval to
obtain the sequence in which various
correlated compound become labelled.
Application: -
14CO2 sequential analysis has been very
successfully used in elucidation of carbon in
photosynthesis.
Determination of sequential formation of
opium hemlock and tobacco alkaloids.

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1. Tracing of bio-synthetic pathway of cyanogenetic glycoside
prunacine; by incorporating 14C into phenylaniline
2. Interrelationship among 4 methyl sterols & 4, 4 dimethyl sterols,
by use of 14C acetate.
3. Study of squalene cyclization by use of 14C, 3H labelled mevalonic
acid.
4. Terpenoid biosynthesis by chloroplast isolated in organic solvent,
by use of 2- 14C mevalonate.
5. Study the formation of cinnamic acid in pathway of coumarin from
labelled coumarin.
6. Origin of carbon & nitrogen atoms of purine ring system by use of
14C or 15N labelled precursor.

7. Study of formation of scopoletin by use of labelled phenylalanine.

8. By use of 45Ca as tracer, - found that the uptake of calcium by
plants from the soil. (CaO & CaCO2).
9. By adding ammonium phosphate labelled with 32P 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.
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