Title of Invention

HEAVY METAL COLLECTION SYSTEM

Abstract

ABSTRACT The present invention provides solution to the problems associated with quality control systems for bioactive molecules obtained from plant source. The invention is successful in providing an HPLC method of profiling the active moieties of plant extracts. Additionally, it has developed a visual user interface which allows user to pictorially visualize differences in chromatographic profile of an extract and provides unique and consistent chromatographic peaks.

Full Text

FIELD OF THE INVENTION The present invention is in relation to a process of profiling plant extracts. More particularly, the present invention provides a quality control system to assess the profile of extracts, efficacy of plant extracts or bioactives which are prepared from the raw materials (e.g, seeds) from different geographic origin and harvested at different time points of the year. The method of profiling so developed is named as MetaGrid ^^. BACK GROUND AND PRIOR ART OF THE INVENTION Metabolomics is defined as the simultaneous analysis of all metabolites in a given biological system. This is a technically challenging task, as no single analytical method is capable of extracting and detecting all metabolites at once due to enormous chemical variety of metabolites and large range of concentrations at which metabolites can be present. Therefore, the characterization of a complete metabolome or an extract as such, requires different complementary analytical technologies. Currently, reverse phase - high performance liquid (HPLC) coupled with photodiode array (PDA) or / and with mass spectrometry is the most sensitive method enabling the detecfion of hundreds of compounds from given single extract. OBJECTIVES OF THE PRESENT INVENTION The principle objective of the present invention is to develop a method for profiling of plant extracts. Another objective of the present invention is to provide a quality control system to assess the efficacy of plant extracts or bioactives. Yet another objective of the present invention is to provide a visual interface for displaying plant extract analysis. BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS Figure 1: Chromatogram profile of AGT006 seed extract, extracted at 200nm from PDA Chromatogram. Figure 2: Java applet showing the reproducibility of triplicate runs in one window [the data is for AGT006 seed extract extracted at 200nm from PDA chromatogram]. Figure 3: Java applet showing the complete constituent profile (Signature) of AGT006 seed extract fi-om PDA chromatogram. STATEMENT OF THE INVENTION Accordingly, the present invention is in relation to a method of profiling plant extract(s), comprising steps of: preparing sample solution of plant part(s); injecting the sample solution into HPLC column; detecting eluant(s) from the HPLC column; and analyzing the eluant(s) via visual interface to profile the plant extract(s); and visual interface for displaying plant extract analysis having predetermined colored data points for depicting retention time and tolerance range. DETAILED DESCRIPTION OF THE PRESENT INVENTION The present invention is in relation to a method of profiling plant extract(s), comprising steps of: preparing sample solution of plant part(s); injecting the sample solution into HPLC column; detecting eluant(s) from the HPLC column; and analyzing the eluant(s) via visual interface to profile the plant extract(s). In another embodiment of the present invention said profiling involves analyzing chromatographic data, assessing efficacy and bioactivity screening of plant extract(s). In yet another embodiment of the present invention said plant parts are selected from a group comprising root, shoot, leaf, seeds or the whole plant. In still another embodiment of the present invention the preparation of sample solution comprises steps of: sieving pulverized plant parts, extracting the pulverized plant parts to obtain extract; concentrating the extract followed by drying; and dissolving the dried extract in a solvent to obtain the sample solution. In still another embodiment of the present invention the HPLC column is maintained at a temperature of about 37° C. In still another embodiment of the present invention said HPLC is carried by gradient technique using water, methanol and acetonitrile as mobile phase solvents. In still another embodiment of the present invention the chromatographic data is obtained after every second from wavelength ranging from 190 nm to 700 nm with a resolution of about 1.2 nm. In still anbther embodiment of the present invention said HPLC comprises reverse phase liquid chromatography coupled with photo diode array detector or reverse phase liquid chromatography and photo diode array detector along with mass spectrometer. In still another embodiment of the present invention said visual interface allows user to pictorially visualize differences in chromatographic profile of an extract and provides unique and consistent chromatographic peaks. The present invention is in relation to visual interface for displaying plant extract analysis having predetermined colored data points for depicting retention time and tolerance range. Extraction and resolution of components present in complex phyto-extracts is an area that has been plagued by a lack of standard operating procedures that permit adequate standardization and quantitative estimation of metabolites at a comprehensive level. In this regard, Avesthagen has developed and optimized novel algorithms that enable metabolite fingerprinting of multi-constituent plant extracts. The major limiting factors that need to be circumvented in this context are listed below: A. Poor coverage of the metabolite diversity typical of plant extracts by commercially available standard metabolites B. Occasional variations in degree of interaction of metabolite constituents with a given chromatographic matrix resulting in concomitant changes in the retention time The metabolite fingerprinting methods commonly employed in the herbal industries are focused on less than 10 metabolites that occur in significant proportions (> 0.1%) within a given extract that may contain several thousand metabolites. Therefore, a metabolite profiling approach that is solely focused on major constituents alone will not be able to address the batch to batch variations observed in bioactivity especially in those situations where the minor constituents ( Standardization of medicinal plant extracts to check the variation in metabolite profile of the raw material, extraction and the stability of extracts over time is utmost importance. Here we present the MetaGrid^"^, the proprietary technology of Avesthagen to answer some of the today's problem faced by herbal and nutritional industry worldwide. The proprietary technology is a reproducible analytical approach consisting of reverse phase liquid chromatography (single or multiple dimensional chromatography) coupled with photodiode array (PDA). Chromatogram data's were processed using validated software tools (Millennium32, Empower pro and LCsolutions) from the instrument maker to get the information on relative time and lambda max for UV and visible spectrum from liquid chromatography and PDA. The objective behind the development of MetaGrid^'^ technology was to use MetaGrid^"^ as a quality control system to assess the efficacy of plant extracts or bioactives, which are prepared from the raw materials (for example AGT006 seeds) from different geographic origin and harvested at different time points of the year. The method of metabolite profiling comprising of: • Simultaneous profiling of >90% resolvable constituents of a phytoextracts (with respect to each given set of chromatographic column matrix and associated run parameters) across 50 different wavelength windows in the UV-Visible range (190-450 nm) of the absorption spectrum • Linear representation of all validated data points generated using instrument specific data acquisition software allowing the run-time and absorption maxima based visualization of all resolved constituents • Novel algorithm that searches for all retention times that are within the tolerance range provided by the user that accommodates for possible variations in retention time of similar / identical constituents across multiple runs of the same extract or multiple runs of different extracts that contain the said constituents. • The chromatographic data analysis that enables the analyst to focus on the development of metabolite resolution methodologies that capture the chemo-diversity of complex phytoextracts even in the absence of information pertaining to the identities of the individual metabolite components. The process as in Claim 1, wherein said process of bioactivity-guided fractionation allows the correlation of observed bioactivity with specific sections of the chromatographic spectrum that represent a fraction of the total metabolite content. The reproducibility of plant material extractions from batch to batch will be tested using in-house metabolite fingerprinting QA/QC method based on HPLC equipped with PDA. For analysis, conserved region of the specific wavelength will be considered and the standard deviation for batch-to-batch extraction will be less than ± 10 % will be maintained through out and any deviated extracts will be removed and destroyed. With MetaGrid™ technology one can have proper control over the quality of the plant extracts interns of efficacy and magnitude of activity, when the proven biologically active standard are not available. The technology of the instant Application is further elaborated with the help of following examples. However, the examples should not be construed to limit the scope of the invention. Example: 1 Seeds of AGT006, which were grown in natural habituate, were collected from local vendor. Good quality seeds, free from contaminants like foreign organic matter, soil and diseased were used as a source for extraction after pulverizing the material to get 200 mesh size seed power. The clean and dried powder of AGT006 (lOOgm) was extracted with 1500ml of ethanol in a soxhlet extractor at 65°C for 4 hours. The extract was clarified by filtration and concentrated and dried using roto-evaporator. Dried extracts were stored in airtight bottles at room temperature, away from direct sunlight and damp conditions till the analysis. Chromatography Conditions HPLC was carried out using quaternary gradient Waters Alliance 2695 system fitted with column oven. Chromatography separations were carried out using Xterra (CI8) precolumn (4.6 x 20 mm) and analytical column (4.6 x 150mm, 100A°, and particle size 3nm) from Waters were used. The column temperature was maintained at STV, where the sample tray and room temperature are 22'^C. Degassed solutions of water, methanol and acetonitrile were used with a flow rate of 750 ml (0.75ml) per minute. The gradient profile is as follows with specific curves. Detection of constituents by PDA The HPLC system was connected online to a Waters 2996 PDA detector for Millennium32 or Empower pro software or Shimadzu SPD-M20A for LC solutions, set to acquire data every second from 190 to 700nm with a resolution of 1.2nm. Example: 2 MetaGrid^"^ analysis For Metagrid analysis, the chromatogram data from 190 to 700nm was extracted at lOnm difference across the runs in triplicate from the PDA chromatogram and thus collected data (output of Millenium32 or Empower pro or LC solutions) is converted to suitable format (for example: excel sheet). Thus prepared data sheet was submitted to MetaGrid^"^ software for analysis. PDA chromatogram is a 3-dimensional chromatogram, where the retention times of the data points are represented by x-axis, the nm of analysis on z-axis and the intensity of nm absorption on y-axis. The data points are extracted at lOnm difference from first data point to the second and from second to third, similarly till the last data point. Bioactivity screening of extracts Medicinal plant extracts that are analyzed using MetaGrid^"^ technology were also screened for functionality / bioactivity using standard protocols. For example, assays utilized to prove the blood glucose modulation property of bioactives are alpha-glucosidase inhibition and dipeptidyl peptidase-IV inhibition potential using enzyme based assays. Similarly, the insulin similar (mimetic) activity and insulin sensitization (reduction in insulin resistance) activity were assayed using cell based glucose uptake assays, in absence and in presence of known concentrations of insulin in the experiments, respectively. Glucose uptake assays are carried out using 3T3-L1, C2C12 and Hep-G2 cell lines under standard conditions using in-house standardized standard protocols for the respective assays. Example: 3 RESULTS: MetaGrid^'^ data extraction To test the reproducibility of MetaGrid^"^ analysis, chromatogram of AGT006 seed extract were performed using HPLC in triplicates. The parameters that are considered for the chromatogram (data) consistency are relative time (Rt) and lambda maxima (A, max) of the chromatogram peaks. The HPLC analysis of AGT006 seed extract was performed at one-month intervals for a period of one year and the data was extracted and analyzed using MetaGrid^'^ software to prove the robustness of technology. Figure 1 depicts one such chromatogram profile of AGT006 seed extracts extracted at 200nm from PDA chromatogram. Such three HPLC runs for AGT006 seed extract forms one set of data points and that data set will be analyzed MetaGrid^"^ technology. MetaGrid^"^ analysis MetaGrid^'^ analysis will be carried out in two steps; the first step of MetaGrid^"^ analysis of three runs of AGT006 seed extract is depicted in Figure 2. In Figure 2, the red colored data points represents the constituents having retention time and X max with the tolerable range, that is ±5 seconds for retention time and ±1.2 for A, max. The fluorescent green colored data points represent the constituents having the retention time with in the tolerance range but the X. max, out side the tolerance range. The black colored data points represent the constituents having retention time and X max beyond the tolerance range. This runs on a in-house developed Java applet, with mouse over on data points displays the retention time and X max in the empty boxes shown in the left-hand corner of the picture, the right hand corner of the Java applet displays the number of constituents present per run along with percent conservation of the data points across the runs. The line joining the three data points across three runs represents the chromatographic peaks / constituents having retention time and X max with in the tolerance range. The output of first step MetaGrid^'^ analysis is that the technology allows the user to pictorially visualize any difference in the constituent profile or chromatography profile of an extract across three runs for a given nm (190 to 700nm at lOnm difference) from PDA extracted chromatogram. The second step of MetaGrid^^ consists of displaying the output of 51 chromatographic profiles (190 to 700nm at lOnm difference from one to the next) in one picture. The second step of MetaGrid^'^ analysis software runs on Java platform and considers only unique (non-repeating) and consistent chromatographic peaks (data points joined by line across three chromatographic runs) from the step one of MetaGrid^'^ analysis for display. The pictorial representation of total constituent profile of the AGT006 seed extract is depicted in Figure 3, where the y-axis of the representation represent the retention time of the constituent (Clmax is hidden but visible upon mouse over on to the data point) and the x-axis represent the nm at which the chromatograms are extracted from PDA chromatogram. The MetaGrid^'^ result window displays other important data details like number of common constituents across three runs, average number of constituents across three runs and conserved data points across three runs along with pictorial representation of the details. Example: 4 MetaGrid^*^ analysis and bioactivity of the plant extracts The objective behind the development of MetaGrid^"^ technology was to use MetaGrid^"^ as a quality control system to assess the efficacy of plant extracts or bioactives, which are prepared from the raw materials (for example AGT006 seeds) from different geographic origin and harvested at different time points of the year. From results of well-planned metabolite fingerprinting experiments it is known that the extracts prepared from the seeds of same plant at different time points of the year are different. Similarly, the extracts prepared from the seeds procured from different geographic locations vary significantly. Due to variation in the metabolite composition, assessing the efficacy or bioactivity of extract is much difficult with out checking the variability. In herbal industry, due to absence of standards and the activity or functionality of the biomolecules are not ascertained, it is very difficult monitor the efficacy of extracts thus prepared. To answer this problem to some extent, Avesthagen come out with a break through technology called MetaGrid. The efficacy assessing of the plant extract using MetaGrid technology involve database building, which involves reference signature (for example, AGT006 seed extract signature), like one depicted in figure 3 for number of extracts prepared for a raw material, which is collected from different geographic locations and different time points of the year. The same plant extracts are screened for bioactivity using standard protocols. The bioactivity or efficacy of the plant extracts are linked to specific plant extract signature and then the grading of the extract interns of no-bioactivity to low-bioactivity to high-bioactivity. Thus prepared grading system is used as a standard and the signature of the extract in question is superimposed against the standards to know the magnitude of bioactivity or efficacy. MetaGrid^ is a reproducible analytical system consisting of reverse phase liquid chromatography (single or multiple dimensional chromatography) coupled with Quadrapole trap mass system or/and photodiode array (PDA). MetaGrid had two kinds of analysis outputs one being from the combination of reverse phase HPLC and PDA and the other being from the combination of reverse phase HPLC and PDA along with mass spectrometer. The output of these results were analyzed further separately by in-house developed software, where the results are depicted in the form of grid called as a metabolite signature or MetaGrid. In this article, we present MetaGrid^"^ analysis-using reverse-phase HPLC coupled PDA based results, where the end points are chromatography peaks with specific retention time and lambda maxima. Reference: Sofia Moco, Raoul J. Bino, Oscar Vorst, Harrie A. Verhoeven, Joost de Groot, Teris A. van Beek, Jacques Vervoort, and C.H. Ric de Vos "A Liquid Chromatography-Mass Spectrometry-Based Metabolome Database for Tomato" Plant Research International, Plant Physiology, August 2006, Vol. 141, pp. 1205-1218. We Claim: 1) A method of profiling plant extract(s), comprising steps of: a) preparing sample solution of plant part(s); b) injecting the sample solution into HPLC column; c) detecting eluant(s) from the HPLC column; and d) analyzing the eluant(s) via visual interface to profile the plant extract(s). 2) The method as claimed in claim 1, wherein said profiling involves analyzing chromatographic data, assessing efficacy and bioactivity screening of plant extract(s). 3) The method as claimed in claim 1, wherein said plant parts are selected from a group comprising root, shoot, leaf, seeds or the whole plant. 4) The method as claimed in claim 1, wherein the preparation of sample solution comprises steps of: a) sieving pulverized plant parts, b) extracting the pulverized plant parts to obtain extract; c) concentrating the extract followed by drying; and d) dissolving the dried extract in a solvent to obtain the sample solution. 5) The method as claimed in claim 1, wherein the HPLC column is maintained at a temperature of about 37° C. 6) The method as claimed in claim 1, wherein said HPLC is carried by gradient technique using water, methanol and acetonitrile as mobile phase solvents. 7) The method as claimed in claim 2, wherein the chromatographic data is obtained after every second from wavelength ranging from 190 nm to 700 nm with a resolution of about 1.2 nm. 8) The method as claimed in claim 1, wherein said HPLC comprises reverse phase liquid chromatography coupled with photo diode array detector or reverse phase liquid chromatography and photo diode array detector along with mass spectrometer. 9) The method as claimed in claim 1, wherein said visual interface allows user to pictorially visualize differences in chromatographic profile of an extract and provides unique and consistent chromatographic peaks. 10) Visual interface for displaying plant extract analysis having predetermined colored data points for depicting retention time and tolerance range. 11) A method of profiling plant extract and visual interface are substantially as herein described along with accompanying examples and drawings.

Documents:

2160-CHE-2008 CORRESPONDENCE OTHERS 04-02-2014.pdf

2160-CHE-2008 ABSTRACT.pdf

2160-CHE-2008 AMENDED CLAIMS 14-08-2014.pdf

2160-CHE-2008 AMENDED PAGES OF SPECIFICATION 14-08-2014.pdf

2160-CHE-2008 CLAIMS.pdf

2160-CHE-2008 CORRESPONDENCE OTHERS.pdf

2160-CHE-2008 DESCRIPTION (COMPLETE).pdf

2160-CHE-2008 DRAWINGS.pdf

2160-CHE-2008 EXAMINATION REPORT REPLY RECEIVED 14-08-2014.pdf

2160-CHE-2008 FORM-1 14-08-2014.pdf

2160-CHE-2008 FORM-1.pdf

2160-CHE-2008 FORM-18.pdf

2160-CHE-2008 FORM-3 14-08-2014.pdf

2160-CHE-2008 FORM-3.pdf

2160-CHE-2008 FORM-5.pdf

2160-CHE-2008 OTHERS.pdf

2160-CHE-2008 POWER OF ATTORNEY.pdf