An Advanced Spectroscopic Technique Convenient to Adapt to

1. 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 S.Aravamudhan, National Seminar,NEICSSR,Shillong

2. 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. S.Aravamudhan, National Seminar,NEICSSR,Shillong

3. Solar Energy is the input energy Used for Photosynthesis / Synthesis of molecules for the food value of the plants LEAF FRUIT STEM ROOT From any of these parts of plants extracts can be obtained by treating them with simple chemical solvents, aqueous or non aqueous organic solvents FOODMaterials for plants are from SOIL & ATMOSPHERE S.Aravamudhan, National Seminar,NEICSSR,Shillong

4. 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. S.Aravamudhan, National Seminar,NEICSSR,Shillong

5. 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 S.Aravamudhan, National Seminar,NEICSSR,Shillong

6. 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-printsfor 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. S.Aravamudhan, National Seminar,NEICSSR,Shillong

7. 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 S.Aravamudhan, National Seminar,NEICSSR,Shillong

8. 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 S.Aravamudhan, National Seminar,NEICSSR,Shillong

9. This slide has the description of METABOLISM in the most general context. In the next three slides PLANT METABOLISM is described. The introductory way to describe METABOLISM Breakdown 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 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 METABOLISM 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 S.Aravamudhan, National Seminar,NEICSSR,Shillong

10. Plant Metabolism Solar Energy is the input energy Used for Photosynthesis / Synthesis of molecules for the food value of the plants LEAF FRUIT STEM ROOT FOODMaterials 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 S.Aravamudhan, National Seminar,NEICSSR,Shillong

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12. 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. S.Aravamudhan, National Seminar,NEICSSR,Shillong

13. Amino Acidstypical small molecules of biological importance • Building blocks of proteins • Carboxylic acid group • Amino group • Side group R gives unique characteristics Rside chain I H2H—C —COOH I H CH3 I H2N—C —COOH I H alanine H I H2N—C —COOH I H glycine H I H3N+—C —COO- I H glycine Zwitter ion S.Aravamudhan, National Seminar,NEICSSR,Shillong

14. A BIOLOGOCAL MACROMOLECULETo compare with small molecule a small peptide belowA TETRA PEPTIDE Peptide bond Peptide bond Ala-Leu-Cys-Met Peptide bond Proteins which can be a metabolic constituent may be consisting of several such small peptide units as above S.Aravamudhan, National Seminar,NEICSSR,Shillong

15. A Spectrum is a graphical plot recorded with frequency on the X-axis with nonzero amplitudes at specific frequency values. On the Y-axis Analysis on the basis of these spectral features is the Spectroscopic technique; Spectroscopy thus is an analytical technique 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. S.Aravamudhan, National Seminar,NEICSSR,Shillong

16. 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. S.Aravamudhan, National Seminar,NEICSSR,Shillong

17. Then, What makes NMR useful? Living systems consists of many components; for this reason one would expect an NMR spectrum to consist of superposition of spectra of many different molecules which are hard to analyze and to assign. Fortunately, in practice the situation is frequently more 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 Living systems consists of many components; for this reason one would expect an NMR spectrum to consist of superposition of spectra of many different molecules which are hard to analyze and to assign situation is frequently more favorable: What is metabolism and an illustration of the above situation of Metabolite NMR spectrum in the next few slides S.Aravamudhan, National Seminar,NEICSSR,Shillong

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19. 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) polyunsaturated fatty acids (PUFA) 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) Percentage contribution of PUFAsuch as linoleic acid (2.77 ppm) and linolenic (2.81 ppm) was estimated by conducting homodecoupling experiment. 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. 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

20. Increase amplification No lines are visible Selected region 0 to3.0 ppm Selected region 6. 0 to 9.0 ppm Selected region 3. 0 to 6.0 ppm Three expanded regions B,C and D would appear Expanded S.Aravamudhan, National Seminar,NEICSSR,Shillong

21. MES buffer 29-deoxymugineic acid (DMA) MES buffer 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 one-dimensional 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. S.Aravamudhan, National Seminar,NEICSSR,Shillong

22. End of presentation……. Thank You S.Aravamudhan, National Seminar,NEICSSR,Shillong