Transcript An Advanced Spectroscopic Technique

An Advanced Spectroscopic Technique Convenient to Adapt to
Enrich Inventory of Medicinal Plant Resources of North East
Several of the spectra and figures reproduced are from the links at the webpage;
http://aravamudhan-s.ucoz.com/BICProject_NEHU.html
S.Aravamudhan
Department of Chemistry
North Eastern Hill University
SHILLONG 793022
Meghalaya
National Seminar
on
ENVIRONMENTAL MANAGEMENT AND
DEVELOPMENT OF NORTH EAST INDIA
JUNE 3rd and 4th, 2011
NORTH EAST INDIA COUNCIL FOR
SOCIAL SCIENCE RESEARCH
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Seminar,NEICSSR,Shillong
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This subject requires two distinct aspects to be elaborated.
1. What is Spectroscopy and how it can be
described for the grasping by non specialists and
common public.
Find a pedestrian approach to describe peripheral NMR in Sections III & IV of this article:-
http://aravamudhan-s.ucoz.com/nmr_for_oil_and_coal.html
2. How this technique becomes simple enough
for common people’s use? If it is for Metabolic
profiling of plant extracts and biological fluids,
what is metabolic profiling in this context?
It is the sequence as above which has been followed in the postings at the
webpage: http://aravamudhan-s.ucoz.com/BICProject_NEHU.html
But the two aspects would be taken up in the reverse order during this
presentation.
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Solar Energy is
the input energy
Used for Photosynthesis /
Synthesis of molecules for the
food value of the plants
From any of these parts of
plants extracts can be
obtained by treating them
with simple chemical
solvents, aqueous or non
aqueous organic solvents
FOOD Materials for
plants are from SOIL &
ATMOSPHERE
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If one makes an effort to isolate out a single chemical
compound of interest and characterize the molecule it can be a
tedious and laborious task. Such natural products which are
found useful will have to be made available in such bulk
quantities for use that it may be necessary to device a
laboratory synthetic route.
Before such a venture on commercial production of a natural
product, it is important to have an inventory of the plant species
with certain signatures by which the plant species gets completely
identified; after the validation by such a data base, the plant
material can be explored further for details.
Thus the plant extracts which may be a mixture of several
chemical compounds occurring in the plant system are
subjected to statistical procedures of analysis with the
extracted samples collected from all possible variety of
sources varying in the place and time of the occurrence of the
plants.
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Such a statistical variation in sampling the plant materials must be
allowed so that an inference would be possible as to identify the
invariant characteristics which uniquely identify the plant species. The
combinatorial or the method of principal component analysis is the
popular statistical approach.
Such a statistical approach can be applied on a collection of
samples each of which can be subjected to an analysis indicative
of the constituent chemicals in the extract and based on the
quantitative and qualitative tags on the contents a statistical
procedure can be possible to identify the common characteristic..
Thus even before any complete characterization of all constituents of a
plant extract, it is necessary to get the information that the constituents
and the method of extraction from sources are dependable for
characterizing the source plant and then explore for the complete details
of the advantages/disadvantages that are caused by the plant materials.
It is this aspect which makes possible for a perpetual activity of scientific nature to collect data
on materials from natural resources without having to know how and for what purpose. The
guidelines for utilizing these data are well stipulated and there are established centers facilities
to provide the required output from analytical instruments on submission of a sample
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This is the process of building an inventory so as to be able to reach for
the plant material any time with full assurance that it is authentically the
same plant source which is sought after.
Such fingure-print information can be gathered in terms of several
parameters, assignable to the constituents of plant extracts, obtained
from several analytical techniques;
Spectroscopy is one such highly reliable technique to build fingure-prints
for plant species. The way the extracts are analyzed for contents is called
“metabolic profiling”
Metabolism refers to the processes governing the biological changes which occur in living
organisms resulting thus in growth and development of the organism and evolution of
species. From the beginning till the end of its life time what all happens to the typical
specimen of a species would be required to unequivocally characterize the species to
which a specimen belongs.
There could be differences in the characteristic of organisms which are capable of motion
(displacement & migration from one place to another) as compared to living organisms
which exhibit certain movements of the parts but as a whole are not capable of a
displacement on their own. Plant species versus animal species and the humans are all
to be distinguished.
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Botanical ID (identification) method are defined as methods used to determine, with a high
degree of reliability, that a sample of unknown content and origin is a true example of a
given botanical species
The guidelines will describe the steps needed to validate these methods
1H
NMR and multivariate analysis techniques were applied to almost 60 plant samples collected in
different places in China. Using 1H NMR metabolomics, it was possible, without previous
evaporation or separation steps, to obtain metabolic fingerprints to distinguish between the species
The ability to quickly screen extracts for novel compounds, new sources or higher levels of known
compounds, monitoring of „ripeness“, and source would greatly optimize the selection of natural
product sources for possible efficacy. Similarly, the ability to screen extracts at multiple locations
allows analysis on a global scale
NMR's high reproducibility imparts cross platform quantification of individual compounds and
qualitative assessment (chemometric modeling), which makes it possible to compare data at
different sites
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It is hereby pointed out that the plant extracts and medical formulations
from medicinal plants consist in general of the chemical molecules (small
and large) which occur during the metabolic pathways.
Database of possible such molecules have been compiled and available in
the documentation.
What is required is, to identify a metabolite, may be a (medical
formulation) and these formulations as and when prepared and distributed
with prescriptions, take a small portion and gather a spectrum. Since such
medicines are made any number of times as there are patients to be cured,
these formulations each time can be ascertained by a NMR spectrum by
comparing it with previous time samples. This spectral information along
with the history and response of patients with their biodata would go a
long way by statistical correlations in improving the quality and establish
the value of traditional practices by modern scientific analysis.
There are several facilities in Institutions far and near, where these
spectrometers are housed and people can obtain spectra at nominal
charges by submitting their samples. No particular prerequisit knowledge
or expertise is necessary for submitting samples for spectra.
More about NMR tools at the link for SAIF, NEHU in
Dr.Aravamudhan's website: http://www.ugc-inno-nehu.com
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The introductory way to describe
METABOLISM
Breakdown
This slide has the description of
METABOLISM in the most general
context. In the next three slides PLANT
METABOLISM is described.
Proteins to Amino Acids, Starch to Glucose
The chemical processes occurring within a living cell or organism
that are necessary for the maintenance of life. In metabolism
some substances are broken down to yield energy for vital
processes while other substances, necessary for life, are
METABOLISM
synthesized. Catabolism
breaks down organic matter, for example
to harvest energy in cellular respiration. Anabolism uses energy to
construct components of cells such as proteins and nucleic acids
Synthesis
Amino Acids to Proteins, Glucose to Starch
The chemical processes occurring within a living cell or organism that are necessary for the maintenance of life.
In metabolism some substances are broken down to yield energy for vital processes while other substances,
necessary for life, are synthesized. Catabolism breaks down organic matter, for example to harvest energy in
cellular respiration. Anabolism uses energy to construct components of cells such as proteins and nucleic acids
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Plant Metabolism
Solar Energy is
the input energy
Used for Photosynthesis /
Synthesis of molecules for the
food value of the plants
FOOD Materials for plants
are from SOIL &
ATMOSPHERE
From any of these parts of plants extracts can be
obtained by treating them with simple chemical
solvents, aqueous or non aqueous organic
solvents
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Seminar,NEICSSR,Shillong
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Primary Metabolites
• Primary metabolites are compounds that are
commonly produced by all plants and that are
directly used in plant growth and
development.
• The main primary metabolites are
carbohydrates, proteins, nucleic acids, and
lipids.
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Amino Acids
typical small molecules of biological importance
•
•
•
•
Building blocks of proteins
Carboxylic acid group
Amino group
Side group R gives unique characteristics
R
I
CH3
I
H2N—C —COOH
I
H alanine
side chain
H2H—C —COOH
I
H
H
I
H2N—C —COOH
I
H glycine
H
I
H3N+—C —COOI
H glycine
Zwitter ion
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A BIOLOGOCAL MACROMOLECULE
To compare with small molecule a small peptide below
A TETRA PEPTIDE
Peptide
bond
Peptide
bond
Peptide
bondCH 3
CH3
CH3 O
S
CH CH3
SH
CH2
CH O
CH2 O
CH2 O
H3N CH C N CH C N CH C N CH C O
H
H
H
Ala-Leu-Cys-Met
Proteins which can be a metabolic constituent may be
consisting of several such small peptide units as above
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Analysis
A
Spectrum
on the
is a basis
graphical
of these
plot spectral
recordedfeatures
with frequency
is the Spectroscopic
on the X-axis with
nonzero amplitudes
technique;
Spectroscopy
at specific
thus isfrequency
an analytical
values.
technique
On the Y-axis
Amplitude
Frequency
A college level introductory description of Nuclear Magnetic Resonance [N.M.R.] can be found in
the Playlist : "NMR Videos“ at YOUTUBE link: 1_NMR_ElementaryDefinition.WMV
Most often the frequency is that of radiation applied to the sample. And Amplitude is
the measure of energy absorbed at any specified frequency of the applied radiation.
Such spectra obtained from specimen are found to be characteristic fingure
print information of the chemical molecules. Hence such spectra can be
helpful in identifying and characterizing the chemical species.
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Depending upon the frequency range of the radiations used,
several kinds of Spectroscopic techniques are in vogue.
For each kind of spectroscopy the present day advances in
instrumentation has made possible availability of sophisticated
spectrometers and all those instrumental aspects are beyond
the scope of this presentation.
Nuclear Magnetic Resonance Technique is one such
Spectroscopic technique and without going into the details
of the detailed spectroscopic techniques, in this
presentation an effort would be made to convince how
this technique can be useful for common people to
acquire information to improve the quality of plant
resource management.
There are several facilities in Institutions far and near,
where these spectrometers are housed and people can
obtain spectra at nominal charges by submitting their
samples. No particular prerequisit knowledge or
expertise is necessary for submitting samples for
spectra.
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Then, What makes NMR useful?
Living
Livingsystems
systemsconsists
consistsof
ofmany
manycomponents;
components;for
forthis
thisreason
reasonone
onewould
wouldexpect
expect
an
anNMR
NMRspectrum
spectrumto
toconsist
consistof
ofsuperposition
superpositionof
ofspectra
spectraof
ofmany
manydifferent
different
molecules
moleculeswhich
whichare
arehard
hardto
toanalyze
analyzeand
andto
toassign.
assign Fortunately, in practice the
situation
situationisisfrequently
frequently more
morefavorable:
favorable: only a few of the components have
such concentration that their NMR signals can be distinguished from
background. Hence, the spectra are usually comparatively simple and can be
interpreted without effort. From: NMR in Medicine and Biology, KH Hauser & HR
Kalbitzer, Physics in life series, Springer, Chapter 5, Page 173
What is metabolism and an illustration of the above situation of
Metabolite NMR spectrum in the next few slides
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Elementary article on NMR Author: S.Aravamudhan
http://aravamudhan-s.ucoz.com/nmr_article.html
Portions of 1H NMR spectra of lipid and sterol profile in
Jatropha curcas L. seeds (a) USFA, (b) TAG, (c) FAME,
(d) PUFA and (e) sterols
saturated fatty acids (SFA)
methyl ester of fatty
acids (FAME)
triglycerol esters (TAG)
polyunsaturated
fatty acids (PUFA)
unsaturated fatty
acids (USFA)
Diallylic signal at 2.78 ppm was used for estimation of
mono (USFA) and poly unsaturated fatty acids ( PUFA)
Signals around 2.76–2.82 ppm was of diallylic methylene
fatty acid, containing di-unsaturated and tri-unsaturated
fatty acids at stage I appeared as multiplet.
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polyunsaturated
fatty acids (PUFA)
Percentage contribution of PUFA such as linoleic acid (2.77
ppm) and linolenic (2.81 ppm) was estimated by conducting
homodecoupling experiment.
Integral value for the C18 protons signal of the sterols
was used for estimation of sterols with respect to total
fatty acids.
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Seminar,NEICSSR,Shillong
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Increase
amplification
No lines are visible
Selected region
6. 0 to 9.0 ppm
Selected region
3. 0 to 6.0 ppm
Selected region
0 to3.0 ppm
Three expanded regions
B,C and D would appear
Expanded
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MES buffer
MES buffer
29-deoxymugineic acid (DMA)
29-deoxymugineic acid (DMA)
spectrum of a wheat (Triticum
aestivum) exudate.
exudate was collected under conditions that enhanced the production of 29-deoxymugineic acid (DMA; structure top right). The normal onedimensional 1H NMR spectrum is shown in the projection at the top of the figure, and the TOCSY spectrum distributes the intensity in two
dimensions in a way that reflects the structure of the detected metabolites. In this exanple the TOCSY spectrum links the hydrogen atoms in
three isolated groups, revealing the expected correlations between H2, H3, and H4, between H19, H29, and H39, and between H1$, H2$, and
H3$. The spectrum also contains signals from MES buffer (structure top left) apparently as a result of uptake and re-secretion. Adapted from
Fan et al. (2001) with the permission of Elsevier Science.
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End of presentation…….
Thank You
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