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
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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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S.Aravamudhan, National
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 pedastrian 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?
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S.Aravamudhan, National
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A Spectrum is a graphical plot recorded with frequency on the X-axis with
nonzero amplitudes at specific frequency values. 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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S.Aravamudhan, National
Seminar,NEICSSR,Shillong
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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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S.Aravamudhan, National
Seminar,NEICSSR,Shillong
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Example of Spectra of small molecules
Simulated Spectrum/ Not experimental
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Seminar,NEICSSR,Shillong
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After taking the spectra, the spectroscopist would try to interpret the
spectra for the identification and the characterization of the chemical
molecule which gave this spectrum.
It is this stage which makes the demand on knowing the details of
the phenomena which gives rise to such spectral features. This can
be a tedious task even for certain small molecules.
Next slide
When the molecules become larger as in the case of biological samples the spectra may not
be so simple; there will be a lot more lines within the same 0 to 11 ppm range and the
spectrum would have overlapping crowded line patterns from which it will be difficult
characterize the large molecule completely. It is a time consuming process but current
spectroscopic techniques have considerably advanced to provide enough details even for
biological molecules.
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S.Aravamudhan, National
Seminar,NEICSSR,Shillong
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An experimental
spectrum of
Ethylalcohol
PMR of Ethyl
Benzene in next
slide
How will the
spectrum
differ?
Phenyl
This
–OH
group is
(Benzene)
replaced
group
by….
Notefrom 4 to 11
NO absorption
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S.Aravamudhan, National
Seminar,NEICSSR,Shillong
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An experimental
spectrum of
Ethylbenzene
This peak is due
to the benzene
protons
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S.Aravamudhan, National
Seminar,NEICSSR,Shillong
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For the CH3-CH2- the
alcohol has range 1 to 4
ppm where as for ethyl
benzene it is only from
1 to 3.
This is a small molecule since
the number of atoms in this
molecule are less, which reflects
in its molecular mass /Molecular
Weight
Macromolecules can
be several orders of
magnitude larger than
these small molecules
absent
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S.Aravamudhan, National
Seminar,NEICSSR,Shillong
Can be 10000 times
larger, then all these
protons/ hydrogen
atom nuclei would
give lines in this
typical range 0-11ppm
9
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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S.Aravamudhan, National
Seminar,NEICSSR,Shillong
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The introductory way to describe METABOLISM
Breakdown
Proteins to Amino Acids, Starch to Glucose
This slide has the description
of METABOLISM in the most
general context.
In the next three slides PLANT
METABOLISM is described.
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
METABOLISM
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
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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Seminar,NEICSSR,Shillong
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Plant Metabolism
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Seminar,NEICSSR,Shillong
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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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Seminar,NEICSSR,Shillong
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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
H2N—C —COOH
I
H alanine
side chain
H2H—C —COOH
I
H
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CH3
I
H
I
H2N—C —COOH
I
H
glycine
H
I
H3N+—C —COOI
H
glycine
Zwitter ion
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Seminar,NEICSSR,Shillong
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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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Seminar,NEICSSR,Shillong
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Seminar,NEICSSR,Shillong
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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.
S.Aravamudhan, National
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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Seminar,NEICSSR,Shillong
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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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Seminar,NEICSSR,Shillong
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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
pre-requisit 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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Seminar,NEICSSR,Shillong
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ABSTRACT
Advanced Technology can gain a popular appreciation only by extraneous efforts by the specialists to make it useful to
the public. This requires creating awareness among all sections of society about the advantages of the technological
tool and providing prescriptions to use the technique advantageously to improve the standard of living. This precisely
was the context for an earlier contribution (1) on the use of spectroscopic technique for the mining operations for the oil
and coal. It is again the same spectroscopic tool – Nuclear Magnetic Resonance Spectroscopy-which is being now
brought to the forum to point out that it has become a viable technique for Biological context for the “Metabolic Profiling”.
The term metabolic profiling may be popularly explained as the way one can get a “fingure-print” information about the
constituents of a biofluid. Typically those fluids which are available as excreta or extracted by special methods in clinics
for tests, may be termed as bio-fluids. In the same way, the plant extracts which are for natural product isolations or such
of those formulations from medicinal plants used by traditional medical practitioners have constituents which are not far
different from those of the biofluids as defined before and hence the metabolic profiling is applicable in the contexts of
identifying the plant characteristics and defining standards for the formulations in traditional medicines.
This metabolic profiling is becoming more and more viable with the Nuclear Magnetic Resonance technique because of
the advances being made in the spectroscopic techniques and the instrumentation. Sophisticated spectrometers are
available on commercial basis and the use of the instruments require only specially trained operators who can deliver
spectra on submitted sample, even without any knowledge about the instrumentation or spectroscopic technique on the
part of the user. It only requires to gather such spectra for several specimens from different sources and at different
times as per the requirement and simply tabulate the parameters from the spectral patterns which seems a merely a
cataloguing exercise (tabulating the ‘y-axis’ values corresponding to “X-axis” values) rather than a analysis on a
molecular level basis for a characterization. Automation of the operating procedures makes possible an automated
accumulation of spectra of several specimens without any human intervention. And this Profiling technique has been
used for Boatnical research(2) and is convenient for adapting for popular activities to make inventories.
The results of such research activities and its advantages in the context of North East resources would be elaborated
and the convenience of using such a technique would be highlighted.
References:1‘Technological Strategies for Effective and Safe Mining Activity’’, by S.Aravamudhan, Pages 31-49, in
Development and Environment, Editors: Zahid Husain and S.K. Barik, Year 2004, Regency Publications, New
Delhi
2http://aravamudhan-s.ucoz.com/BICProject_NEHU.html#OPSidhu
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Seminar,NEICSSR,Shillong
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Scope of the Contents of Full Paper:It has been pointed out in an earlier article: “Technical Education for Industrial Growth and Prosperity in North East India” by
S.Aravamudhan (in “In Defence of Regional Economic Development in India”, A case for the North East edited by P.M.Passah,
Akansha Publications, New Delhi, Year 2006, Pages 149-145) the importance of popularizing advanced scientific methods and
techniques for development, has been elaborated. In a preceding article of the year 2004, an article by S.Aravamudhan (in
“Development and Environment”, Editors: Zahid Husain and S.K. Barik, Regency Publications, New Delhi, Pages: 31-49) a
consideration of the strategy to utilize the Spectroscopic Tools for Effective Safe mining activities was discussed. To be reinforcing the
importance of such efforts and the public at large would be able to mobilize support for such initiatives, in the present contribution
entitled: An Advanced Spectroscopic Technique Convenient to Adapt to Enrich Inventory of Medicinal Plant Resources
of North East, the following results of research studies would be elaborated at a popular level to intensify the awareness that
growth can be faster if these techniques are adapted at the earliest.
The following excerpts from publications would be elaborated as per the outlines given in the abstract for this contribution.
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 (5a). 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 (5b). 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 (5c).
It is intended, through this contributed paper, to stimulate useful discussion over various issues pertinent to the environment
and its management so as to find out the proper guidelines for the management of natural resources without hampering the growth
process of the country, particularly the North East India .
http://www.ugc-inno-nehu.com/ToxicHE.html
http://aravamudhan-s.ucoz.com/nmr_for_oil_and_coal.html
3http://aravamudhan-s.ucoz.com/BICProject_NEHU.html#NMRS_BIC
4http://aravamudhan-s.ucoz.com/BICProject_NEHU.html#OPSidhu
5a, b, & c at http://aravamudhan-s.ucoz.com/BICProject_NEHU.html#PLANTA_MEDICA
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