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 This slideshow of a total of 23 slides has been automatically timed for total viewing durtaion of 20 mins Simply sit and watch !! 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 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? S.Aravamudhan, National Seminar,NEICSSR,Shillong

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

4. 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

5. Example of Spectra of small molecules Simulated Spectrum/ Not experimental S.Aravamudhan, National Seminar,NEICSSR,Shillong

6. 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. 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. try to interpret Next slide S.Aravamudhan, National Seminar,NEICSSR,Shillong

7. An experimental spectrum of Ethylalcohol PMR of Ethyl Benzene in next slide How will the spectrum differ? This –OH group is replaced by…. Phenyl (Benzene) group Note NO absorption from 4 to 11 S.Aravamudhan, National Seminar,NEICSSR,Shillong

8. An experimental spectrum of Ethylbenzene This peak is due to the benzene protons S.Aravamudhan, National Seminar,NEICSSR,Shillong

9. 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 Can be 10000 times larger, then all these protons/ hydrogen atom nuclei would give lines in this typical range 0-11ppm absent S.Aravamudhan, National Seminar,NEICSSR,Shillong

10. 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

11. The introductory way to describe METABOLISM This slide has the description of METABOLISM in the most general context. In the next three slides PLANT METABOLISM is described. 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

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

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

14. 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

15. 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

16. 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

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

18. 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

19. 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

20. 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

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

22. 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:- • ‘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 • http://aravamudhan-s.ucoz.com/BICProject_NEHU.html#OPSidhu S.Aravamudhan, National Seminar,NEICSSR,Shillong

23. 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 • http://aravamudhan-s.ucoz.com/BICProject_NEHU.html#NMRS_BIC • http://aravamudhan-s.ucoz.com/BICProject_NEHU.html#OPSidhu • a, b, & c at http://aravamudhan-s.ucoz.com/BICProject_NEHU.html#PLANTA_MEDICA S.Aravamudhan, National Seminar,NEICSSR,Shillong