TW544286B - Process for quality control and standardisation of tobacco - Google Patents

Abstract

A process for establishing a standard specification for a tobacco plant material, comprises: (i) preparing a test solution or test extract of a sample of the tobacco plant material which is known to possess the or each property required for the standard; (ii) submitting the said solution or extract to analysis by a combination of NMR spectroscopy and a computer-based pattern recognition technique; (iii) obtaining results from the analytical methods used in step (ii); and (iv) establishing a standard specification for the said tobacco plant material on the basis of the results obtained in step (iii). Candidate samples of the tobacco plant material may subsequently be tested for compliance with the standard. They can be accepted or rejected depending on whether they give analytical results which fall within or outside either part or all of the specification established in step (iv).

Description

544286 ^ A7 B7 V. Description of the invention (1) The present invention relates to the use of nuclear magnetic resonance spectroscopy and computer-based statistical programs to integrate tobacco standardization and quality control. Smoking, inhalation or ingestion is an entertaining activity enjoyed by an important part of most social entertainment in the society. Leaf tobacco such as cigarettes, cigars or pipes is typically smoked, but can also be chewed. Alternatively, but not commonly, tobacco leaves can be ground into powder for snuff. Tobacco cultivation in many parts of the world includes the Americas, South Africa, Russia and India as cash crops. Tobacco leaves are typically stacked, rated, and matured for one or two years after being packaged for industrial processing. ^ Yucao "refers to the Nicotiana genus. N. tabacum is now the main species for commercial grass use, although it was original in the 17th century. Introduced into Europe as N.rustica. But N.rustica contains high concentration in thieves and is currently only planted on a large scale in Russia and North India. Nicotiana species are highly polymorphic, and a specific species is best recognized as a species ( Cultivars). Categories can be classified according to their curing methods described below, (ii) fire-smoked, (iii) air-dried, or (iv) sun-cured. The main example of a smoked category is Orinoco , Resulting in the following categories: White stem Orinoco; Virginia Bright; 400; Oxford; Dixie Bright; Vamorr; Vesta; Coker; White Gold; and Kutsaga 51. The main smoked category is Pryor, which originated from the smoked Orinico. Another The fire-fighting category is Heavy Western. Burley is the main category in the air-dried category. Others include Southern Maryland and Maryland Mammoth. 0 Smoke is typically used in brick farmhouses to block wood, oil, gas or other heat sources in closed smoke. Internal heating. It is important to control the temperature and humidity in the warehouse. Fire fumigation is usually at -4- This paper size is applicable to Chinese National Standard (CNS) A4 specifications (210X 297 mm) 2 544286 V. Description of the invention Actually. Tobacco suspensions will grow mixed with oils and special aromas in the ground pits after turning yellow to 4-7. Air-drying system-natural: process, under the condition, it is completed in wood or pasture bunkers. Tobacco leaves grow yellow after the sun grows. The internal ventilation gradually increases the drying speed. Finally, the tobacco leaves are air-dried in the queen day industry for 2 to 3 days, and stacked for 24 to 36 hours. The sun is effective after fermentation. The tobacco is used for drying in the tobacco manufacturing industry. .Typically put a weight on the stack with 0. Use it on top. The stacking should be large enough to allow some fermentation to occur in the tobacco). Etc./Use J © for at least one month (according to size, color and texture). Specially introduced. The grade of tobacco leaves depends on the position of the leaves on the plant. The bottom f ^ is called 1 feed (protuberance), the lower middle leaves are cnmers (cut off), and the upper, middle and top leaves are called clustered tobacco . :: Category Appraisal, Final Leaf Rating, and Depositors Many properties can be determined by the suitable final use of grass. Erzhuo's main products are cigarettes, cigars and pipe tobacco. '::: Most of the grass is smoked and cured by the flue, or some light-colored air-dried or soil is added; Manufactured by Yuzhuo. Many kinds of additives can be used during manufacturing: these are typically used as "harmony flavors", which can be freely dipped in tobacco leaves. Types of additives used include glycerol molasses, menthol, and dungite beans (t〇nkabean, Dipterix〇d〇 =. Tangtang Xuezhuang is typically made from air-dried tobacco leaves, and special varieties are made from blended tobacco glow and air-dried tobacco reeds) ^ ' The grass, yuetong or bright dry finish. The nose is also typically made from dark air-drying and / from the above shows that many factors affect the total quality and binding of finished grass products such as incense, etc. 1 ---____________- 5-This Paper standard uses CNS standard A4 specification (210 X 297S) 544286 A7 B7 Five invention description = taste is helpful. This includes all supplies (such as), growth and harvest knowledge and preservation and processing Technology and additives (if used), etc. Catering, grass product manufacturers set a large amount of storage in order to obtain special brand names coming out-shell flavor and quality. In fact, two brands choose, because of strength, flavor and product: two: : 2: ^ Exclusive love for excuses. It is the duty to set the trademark—Xiang Zhong, Xuezhuang Tobacco hopes to have loyal consumer support.

Therefore, due to the nature of the grass product market, the stomach brand becomes a real manufacturer. Don't threaten it. 4 _ When the imitation stomach manufacturer also hurts the reliability and consistency of the forage product, because of its fragrance, snow, and end of materials. The grass is similar but its quality and properties are different from the original. &-A related issue is adulteration. Unauthorized manufacturers supplement the necessary grade and quality of genuine products with low-quality, inexpensive tobacco. As discussed above, Zuo February & harm consumers ’perception of the trust and consistency of brand products and detract from the real t tobacco today can do three chemical analyses of its ingredients. That is, nicotine, gas (ion and total sugar). These three analyses are completed by HPLC and GLC. Commodity blending of tobacco is often performed with organoleptic evaluation, the purpose of which is to distinguish between different batches of a given pusher or different blend However, this point is purely subjective based on the quality identification of aroma sensitivity differences in individuals. Neither chemical analysis nor organoleptic evaluation can provide a true and consistent method for identifying tobacco and grass product types and blends. Therefore, to date, the useless technology has been able to reliably and consistently distinguish under the same brand name the same as the real grass product from the counterfeit producers and sellers. There is no convenient way to prove one-6-544286

V. Description of the invention (4 kinds of names, the authenticity of tobacco products or confirming that a suspicious counterfeit product is a counterfeit. — In fact, it is impossible to provide any guarantee today that a certain herbaceous material obtained from different sources has the same _ A f Or—Different batches designated for grass blends—the same cannot reliably distinguish the differences between different tobacco blends or between different batches of a designated tobacco product. The private grass industry is the king of post-harvest treatment of grass plant materials and commercial production. Yang Duan needs—a method to facilitate the review of its quality; to distinguish between different batches and different blends of grass plant material; and to track the source, type and other properties of tobacco plant material. The present invention is directed to this—need to provide —Standardization and quality of seed plant materials: a method of control. This study considers the entire tobacco plant components without studying the internal nature of the components themselves or the biochemistry of the terminal grass plant. For the purpose of this article ^ terminal grass plant material "It covers the original state of the plant, the processed final leaves and the finished tobacco products such as cigarettes, cigars and pipe tobacco. Therefore, one measure of the method of the present invention is based on The required special properties of the sample provide a standard that defines the designated tobacco plant material. The standard specification = by extracting known counties as a combination of nuclear magnetic co-subtraction spectroscopy and computer-based cognitive technology and explaining the results obtained as standard specifications. This: ::::: ΐΐ "candidate" can then comply with the standard test: depending on whether it falls into some or all of the established specifications or extraordinarily == provide-a method of ㈧ to develop the final plant material Standard Regulations for Preparation-Known to have the standard requirements of this or f

544286 A7 B7 V. Description of the invention (Test solution or test extract of grass plant material sample; (ii) The solution or extract is used for combined analysis of nuclear magnetic resonance spectroscopy and computer-based cognitive technology; (ill) obtained (ii ) Step analysis results; and (IV) define the standard specifications of the plant material based on the results obtained in step (1U). (Iv) The standard specifications obtained in step are then recognized based on nuclear magnetic resonance spectroscopy and computer-based types. ^ ^ Hair: further extraction (B) Method Supply-Final grass plant material sample, the sample conforms to the previously defined standard for plant materials, the procedure includes: ⑺ preparing a test solution 3 or test substance of a standby sample of grass plant material; ⑴,) The solution or extract is used for the combined analysis of nuclear magnetic resonance spectroscopy and computer-based basic technologies; obtaining the results of (ii,) step analysis; and the results obtained in step 1 are in accordance with the aforementioned procedure (step), and the material “standard specifications are selected for future use” Samples: The sample invention extraction (c) method identifies and rejects samples of tobacco materials that do not meet the standards and specifications of the materials defined above. The procedures include ... Tobacco plant material test samples, test materials, test solutions, or test extracts for nuclear magnetic resonance spectroscopy and computer basis (iii) obtain the results of step (ii) analysis; and determine the steps The results obtained do not meet the aforementioned procedures. Liaobu Township System ^ Zhang Shiji Shicai V. Description of the invention (Material standard specifications do not accept equipment and procedures. 〇 Procedures pass through high materials, and the samples are the same-in each batch of grass plant material, To complete. The standby test method is particularly suitable for suspected counterfeiting or there are steps to (iv,). (C) The same is called investigation. The product can be, for example ,? Brand Zheng: Xiang Zhuo Cao. | ° 0 Brand of Xiang Yu , Cigar or Pipe Tobacco In the full text of the above-mentioned method of the present invention, "the standard is required for any property or property of grass plant material ^ due to the quality of the product, for example, it is a trademarked grass product. This: === other; true source ( Regarding the grass planting place or commodity batch :: = = state. The relevant pathological state can be a set level of maturity, for example = time, the position of tobacco leaves on the Zhuo plant or the potential damage to the relevant terminal grass plants. Parasites, except Requirements for rust or other agents Such as the nature of the month of the car; brother towel [known to have the standard or this nature]: grass plant material samples are known and certified tobacco plants or Yuancheng tobacco products Samples. For example, a specific tobacco variety can be tested with a designated tobacco leaf grade, a sample of tobacco grown in a specific geographic location, or a tobacco sample matured with a special technique. It can be selected as a genuine finished tobacco. Samples of the product are sent to the above-mentioned nuclear magnetic resonance spectroscopy / type certification to develop standard specifications. Subsequent samples of the same tobacco plant material, whose source or quality is unknown or suspected, can then be tested to meet the requirements for such certification and audit materials. Standard Specifications The procedure of the present invention represents a significant distance from the traditional techniques of analyzing tobacco with special independent ingredients such as nicotine or sugar. Therefore, consider the data generated under this program to consider tobacco-9-This paper size is applicable to the Chinese National Standard (CNS) A4 specification (210X297 public love) V. Description of the invention (Plant material: more than one or one type of compound exists. Examination and extraction The solvent used in the product is typically soluble = one or more types of plant materials contained in the plant material: the compounds mentioned above & & A @ Μ ^ Μ ^ With other types of compounds, they can be, for example, physical factors: scoop I1㊣, etc.-Test solution or test extraction in specific examples = This package contains more than one compound in grass plant materials. Test solution in another eight cases Or the test compound contains the compound compounds present in the plant material such as more than one structural or functional molecule. The test bath or test extract preferably contains the largest number of species that are present in the grass plant material complex in response to the nuclear magnetic resonance technique used. The NMR data obtained in the program of the present invention may therefore reflect the total number of the better compounds or the maximum number of classes in the NMR technology used in the plant material response. 4 The test extract is typically a total extract. The preferred solvent for the test solution or extract is formazan. From the above description, it is suggested that the use of separate isolated compounds or compound extracts should be avoided in the procedures of the present invention. However, this application It is still included in the scope of the present invention. Although this method is not necessary, the special components of the grass plant material are not pre-selected, optimized or isolated. Nuclear magnetic resonance spectroscopy (NMR) itself is known as the research of plant materials An analysis tool. One of its application examples is to verify the authenticity of fruit and vegetable-derived beverages. One way is to use Η2 nuclear magnetic resonance spectroscopy to determine the authenticity of fruit juices using position-specific natural isotope segmentation technology (SNIF-NMR). Examples include jaqac engineering This 1996, July-August, 79 (4) ··· 917-928 Martin described the use of Η2 nuclear magnetic resonance spectroscopy (SNIF-NMR method) to detect the use of beet sugar-added fruit juice. 544286 A7 B7 V. Description of the invention (8) This technology relies on the fact that the proportion of ethanol molecules that are deuterated at the methyl position when the fruit juice or concentrated fruit juice is fermented decreases with the addition of beet sugar. Any juice sample added with beet bran will have a significantly lower (D / Η) isotope ratio than a fairly real sample. This technique has also been applied to the determination of wine Chaptalisation by H2-NMR spectroscopy, such as Martin et al.]. 〇1 丨 111.?11}^-Chim Biol. 1983, Vol. 80, pages 293-297. H1-Nuclear Magnetic Resonance Spectroscopy itself is not suitable for plant materials because their generated spectra are too complicated to be visually analyzed. Solve this Questions such as those reported by Kowalski and Bender in J. Am. Chem. Soc. 1972, M, 5632-5639 are based on appropriate multivariate statistical analysis such as principal component analysis (PCA). This is a type of cognitive technology in which the size of data is formed by combining related variable numbers (spikes in the spectrum) to form independent orthogonal variables called smaller new forms of main components (PCs). These PCs are arranged according to the variation contained in their interpretation of the original data. Specimens protrude in the space originally erected by the PCs, producing similar or different views on the biochemical composition of the specimens. Unknown or test samples can also stand out in this space, and can often be compared visually with reference samples (Vogels et al., J. Agric. Food Chem. 44, 175-180, 1996) 〇1 · NMR spectroscopy and type recognition The technology portfolio has been used as a detection tool in determining orange juice certification (Vogels et al., Cited in 1996). Suspicious adulteration can be detected by this method. The identification of the corresponding contaminants was then measured from the PCA relationship of the particular resonance results contained in the original nuclear magnetic resonance spectrum. The combination of H1-NMR spectroscopy and C13-NMR spectroscopy for PCA has also been used to distinguish wines based on their origin (Vogels et al., Chemometrics and Intelligent Laboratory Systems ·· Laboratory Information __ -11-This paper standard applies to China Standard (CNS) A4 specification (210 X 297 mm) 5442 · V. Description of invention (9)

Management, 21 (1993) 249-258). The regional performance of different samples from different wine-producing regions in Germany is based on the statistical analysis and supervision method of entertainment groups that take samples at different times. Recombined spectra are then prepared from the PCA data to reveal spikes in the NMR spectrum of specific wine components that can be distinguished (e.g., monosaccharides such as glucose, mannose, rhamnose, and galactose). For other examples of similar research reports, see “The Trend of Fragrance Research”, such as v0 yarn. Maarse & Van de Heij (eds.), Ehevier,

Amsterdam (1994) pp 99-106.

Hl · Nuclear Magnetic Resonance Spectroscopy and the Analysis of the Main Components-Application by Trevisan et al. In "Characterisation of cell suspensi〇n 职 职 〇f

Humulus lupulus L. ”Chapter 8, page 95_122 is a report published by i997 at the University of Leiden, the Netherlands. The authors performed H1-NMR and PCA on the treated cell extracts to identify the speciality of cell accumulation after treatment. Metabolites. Therefore, their attention is in the pursuit of nuclear magnetic resonance spectroscopy. The special punches are known to be due to individual cell components. Comparing the nuclear magnetic resonance spectroscopy used in the method of the present invention with the types of cognitive processes, these reporters & The analysis of the biochemistry of terminal plant materials has no cognitive relationship with the concurrency type of special nuclear magnetic resonance spectroscopic resonance results of special components (etc.) of terminal plant materials. On the other hand, it depends on the inherent types of clusters derived from nuclear magnetic resonance data. The information in turn reflects the totality of the compounds in the NMR spectroscopic technology used in the response of the plant material of the final plant. ^ The nuclear magnetic resonance spectroscopy used in the invention program combined with computer-based type knowledge (hereinafter referred to as nuclear magnetic resonance spectroscopy / Type cognition) typically includes: test solution or test extract for nuclear magnetic resonance spectroscopy Recording a nuclear magnetic resonance spectroscopy or item; and 54if6

, ι ^ · ΐ iy I —Μ, Κ. 丨 丨 _ — S clearly states (() provides each obtained NMR spectrum or each NMR spectrum to a multivariate analysis to generate one or more points in the score Figure 1. I—The acceptability range is typically defined on the scoring chart generated in the above (π) step: or more points around 'then nuclear magnetic resonance spectroscopy / type cognitive analysis is set to the standard specifications of grass plant materials. The range then constitutes the specification part = after being rejected or rejected, the above-mentioned nuclear magnetic resonance must be provided = two in / type, and whether the point obtained in step (ii) in the analysis is in the range = resonance Plant material ^ sample-method. Therefore, the present invention is extracted in the characteristics-a kind of procedure making = Zhuo plant material in accordance with the standard specifications previously formulated by its material, the program package (η = test solution for the standby sample of the tobacco plant material or Test extraction order (π ") = test sample or sample for _ spectroscopy and nuclear magnetic resonance spectroscopy; (iii ") provide the information obtained by the nuclear magnetic resonance or per-spectrum to the multivariate points in the plot Yield one or more points; and ㈣ As long as the (Hi ") step is included in the definition of the standard specifications, a spare sample is selected as a sample that meets the standard specifications. Also, & includes the standard specification procedures prepared in this feature of the present invention, including / 、 This property or each property required for private approval; I _ 13 This paper size applies to Chinese National Standards (cns) 544286 ^ :, ζ. &Quot; χ '31 Wu Ya Piao ^ Ming (11) (i 〇 Present the test solution or test extract to nuclear magnetic resonance spectroscopy and record one or several spectra; ㈤ ...) Extract this spectrum or f-data obtained from this spectrum to computer-based multivariate analysis to generate a score on the graph Or more points, and (iv) define the acceptable range of this standard specification or part of the standard specification for the tobacco plant material around the points produced in the step. Ordinary multivariate analysis theoretically the total data is the score multiplied by the load The load graph can be used to explain the contribution of each variable (spectral description). "Score graph is a graphical representation of the space in which a sample is cast by: more than one main component axis. The main component Analysis (PCA) system-a special method to Analyze the analysis data contained in multivariate analysis. The position of the sample in the PCA can be easily clustered in the scoring diagram at the edge of the two-dimensional degree, while the dissimilar samples will tend to scatter. 2) Distance (Kowalski & Bender, 1972, and Trevisan, 1997 are all cited above. The environment of the points generated on the score chart in the program of the present invention must be the same when formulating the standard specifications and analyzing the standby samples in accordance with the standards. The methodological components used to define the positions of the points on the knife chart must be presented when processing the NMR spectrum data of the standby sample. In fact, the data derived from the ^ sample NMR spectrum used by the standard are subject to multivariate statistical methods such as Group injury analysis or regular variation together with the appropriate treatment of the standard. Acceptability range Limits are defined in the multi-point scoring chart derived from more than one extract of a known sample based on location. The multivariate analysis in a preferred situation of the present invention is performed using unsupervised methodologies. The nuclear magnetic resonance spectroscopy technique used in the present invention can be included in the high electric field and multivariate scores 544286 V. Description of the invention (12) The analysis is completed by H1-nuclear magnetic resonance spectroscopy. In this particular case, the NMR spectrum is typically measured at 400 to 700 MHz. The data derived from it is then analyzed by multivariate analysis software, such as the commercially available "Pirouette," a complete set of equipment. Two-resolution H1-NMR spectra and type-cognition analysis are performed by M · l

For discussions by Anthony et al., See Biomarkers 1996, 13543 and Mowular Pharmacology 46, 199-211, 1994, and J.O.T. Yi et al. ~ Biochem. Physiol. Vol. 118B No. 3, pp 587_598, 1997. As a choice of 1-dimensional high electric field Η1-nuclear magnetic resonance spectroscopy, the present invention can use other nuclear magnetic resonance-active nucleus such as C 13 _ or Η 2_ of __ dimensional NMR spectroscopy. A series of 2-dimensional pulsation sequence spectra can also be used to explore the above-mentioned h, or other nuclear magnetic resonance-active nuclei. Although the same principle is practical in each case, the results are analyzed by appropriate multivariate analysis. NMR spectroscopy can be regularized or irregularized before completing the basic cognitive types of computers. Regularization has the effect of eliminating the highest intensity, and is a pure parameter of a spectrum as a difference factor. Therefore, when the main goal of the program is to highlight the quality difference between the spectra obtained from different samples, it is typically standardized. However, sometimes if absolute quantitative values such as power are required, the intensity of the spike is required as a recognition factor. In such cases, the spectrum is irregular. A significant advantage of NMR spectroscopy / type recognition is that it is not limited by selective derivation or inspection systems. The test solution or test extract does not need to be purified in advance and the & redundant spectrum ' then allows one component of all components of the tobacco material sample to have a proton as a factor of the entire nuclear magnetic resonance spectrum. The spectroscopic analysis using the multivariate analysis technique in the above discussion shows that spectral identification features that may be valuable can be used to illustrate the complex mixture of components contained in tobacco plant materials with high accuracy. Paper size is applicable to China National Standard (CNS) μ specifications (Café 297)

There may be some cases where the sample of tobacco plant material is not distinguishable on the score chart. The self-determined grass plant material is derived from the same sample and extended at multiple points instead of forming clusters. This loss of similarity occurs when the NMR spectrum of individual plant material components changes. This change can be caused by, for example, a special compound containing an overwhelmingly high concentration in tobacco or by shifting the shift value due to the influence of ipH or metal ions. If this problem occurs, the test solution or test extract extracted from the grass plant material for nuclear magnetic resonance spectroscopy can be pretreated to eliminate the source of error to obtain a better cluster in the score chart. A feature of the procedure of the present invention therefore includes an additional initial step to purify a test solution or test extract of a sample of a spare tobacco plant material and then provide nuclear magnetic resonance spectroscopy. Tea provides a simple example of this principle. Caffeine signals dominate the NMR spectrum of tea, so when the NMR spectrum of tea is analyzed by multiple variables, the points on the tea-like score chart do not form groups. If the caffeine in the tea is removed first, for example, by reverse phase chromatography and then subjected to nuclear magnetic resonance spectroscopy, a unique cluster of samples appears, showing similar points of similar samples on the sigmagram. Exemplified in Example 2 and Figures 4A and 4B. Figure 4 A is a score chart of untreated tea samples, in which the hazy clusters. Figure 4B is a score chart of pretreated tea. In contrast, there are clear clusters to clearly distinguish them. Nuclear magnetic resonance spectroscopy / type cognitive analysis is highly sensitive and capable of identifying samples of tobacco plant material that appear to be identical by other methods. This principle has been repeatedly exemplified by tea. Comparative Example 1 illustrates the analysis of two different types of tea extracts by high performance liquid chromatography (HPLC). The obtained chromatographic separation spectrum is shown in FIGS. 2 and 3. One experiment used untreated samples (each shown in Figures 2 and 3), and the other used a treated sample (see Figure B in each figure). ___ -16- This paper size is in accordance with Chinese National Standard (CNS) A4 (210 X 297 mm) 544286 -17- A7 B7 V. Description of the invention (14) The figures first show that HPLC does not clearly distinguish between treated and untreated The tea-like analysis 2A and 2B are actually the same, as the color spectrum 3 and 36. Secondly, each figure shows that HPLC cannot distinguish different tea types. The color spectrum of Figure 2 is essentially the same as that of Figure 3. These two aspects of HPLC and the nuclear magnetic resonance spectroscopy / type recognition technique used in the present invention are illustrated in, for example, Example 2 and Figures 4 A and 4B. . Many finished commercial tobacco products contain blends of more than two different types of tobacco. For example, as mentioned earlier, cigarettes can mainly contain flue-cured tobacco and add a part of soil to the leaves. Yu Dou Yu Cao is also often smoked and air-dried. As discussed above, the nuclear magnetic resonance spectroscopy and type recognition technology of the present invention can be used for the standardization and differentiation of blends of end-of-grass plant materials. In addition, this technology can be particularly effective in identifying a given tobacco blend and identifying different tobacco blends. Mix. The present invention has thus enabled the differentiation and standardization of tobacco swaps. The principle of the present invention applied to tobacco plant material is exemplified in Example 5. Those who say that the traditional Chinese medicine treatment sample is plant material can be distinguished according to the present invention. The tobacco plant material in the method of the present invention typically includes or is derived from a whole plant, which is a part of a grass plant, a plant extract, and a grass plant. 'Or the finished product is commercially used in grass products such as Xiangyu, Xuezhuang, Silk or Biming. The principle of the present invention will be described in the following reference examples and examples with reference to the drawings-step by step, the general formula is simple. # < Sample Element 3 Axis) Key points :): PCA score chart), from different suppliers such as the description in the heart. The symbols used in the figure are as follows: * = supplier! ; For the standard applicable to China National Standard (CNS) M specifications (⑽X297 public love)

544286 6 (A7 I: _ B7 to Ershi il description (15) 2; Supplier 3; ▽ = Supplier 4; □ = Supplier 5; and 0 = Supplier 6 〇1? A and 2 B performance Kemmun The two-HPLC chromatogram of the tea was optimized using a catechin separation system described in Comparative Example 1, wherein FIG. 2A represents an untreated residue, and FIG. 2B represents a tea sample pretreated by a solid-phase extraction tower. 13 A and 3 B show the dichroic layer spectrum of Lapsang Souchong tea. The catechin separation optimization system described in Comparative Example 1 is used. Figure 3 A represents an untreated tea sample and Figure 3B represents a solid phase extraction. The tea sample pretreated by the tower. 14 A and 4B are the PC A 3 plots of element 2 (y-axis) versus element 1 (x-axis) of six different types of tea, obtained from Example 2 below. The type of tea mark is as follows: □ = Oolong; + = Kemmun; V = LapSang Souchong;. = Dai: jeeling; * = Gunpowder; ♦ = Assam o is the Tanacetum parthenium (small white chrysanthemum) capsules obtained in Example 3 from five different commercial samples Element 3 (y-axis) versus element! (X-axis) Pca score chart. The symbols used for each sample are as follows: ® = sample 1; * = sample 2; mouth = sample 3; · = sample 4 ; And ▽ = Sample 5. The above is the pCA score chart of factor 2 (y-axis) versus element! (Χ-axis) of the seven samples that Tanacetum parthenium harvested at different times after transplantation as described in Example 4. Used. The symbols are as follows: · = Sample TO; ▽ == Sample τι; 0 = Sample T2, * = Sample T3, + = Sample T4; □ = Sample T5; and * = Sample T6 ° JL Series Score chart of element 2 (y-axis) vs. element ^ (x-axis) in the analysis of traditional Chinese medicine treatment in Example 5. The notation used is as follows: · = supplier 1; * = supplier 2; and + = supplier 3. __________- 18-This paper size is in accordance with China National Standard (CNS) A4 (210X297 public attack) 544286 A7 B7 V. Description of the invention (16) Reference example 1: Identifying Panax by nuclear magnetic resonance spectroscopy Source of ginseng The extract was obtained from six different commercial suppliers. White cockroach samples were derived from the roots of Panax ginseng CA · Meyer. The roots were briefly placed in cockroach water and dipped in sugar juice. Then dried in the sun. White Ravioli is also known as sugar mandarin. Dried plant root material is milled with an "Illico" blender for 5 minutes to form a fine powder. 1 g of powder is mixed with 20 ml of cold water in a room. The temperature (22 ° C) was continuously stirred on a shaker at 150 rpm for 2 hours. The extract was filtered through Whatman No. 1 filter paper and collected and freeze-dried overnight. A 10 mg sample was dissolved in 1 ml of deuterated water for NMR spectral analysis. 800 μL was used for nuclear magnetic resonance spectroscopy analysis. The sample was intrinsically compared with TSP at 0.00 ppm. The nuclear magnetic resonance spectroscopy was planned. The H1-NMR spectrum was recorded at 600.13 MHz on a Brilker DRX 600 spectrometer operated with a proton frequency. The spectrum is the result of a 64 ppm sweep with a sweep width of 20 ppm, which is aggregated into 49152 data points. The uptake time was 2.0 seconds per scan. Before the conversion, a line broadening of 0.3 Hz is performed, and the spectrum is transformed by Fourier. Refer to TSP at 0.00 ppm. The nuclear magnetic resonance spectrum was analyzed with AMIX software to reduce the spectrum to a histograms with a bucket width of 0.4 ppm. ○ Analysis of the main components The obtained records were expanded in Excel to complete the normalization with the sum of all spectra. The data obtained was then subjected to multivariate analysis using the Pirouette software suite. Complete unsupervised PC A with average concentration (and automatic measurement). -19-This paper size applies the Chinese National Standard (CNS) A4 specification (210 X 297 mm) 544286 A7 Β7 V. Description of the invention (17) Result The data is converted into points on a PCA score chart. This is shown in Figure 1. The figure clearly shows six different point groups attributed to each Panax ginseng sample, thus exemplifying that this technique provides a discriminating measure between identical samples on the surface of a plant material. Comparative Example 1: HPLC analysis of tea extract Two teas, Kemmun and Lapsang Souchong, were used. The extracts prepared for HPLC analysis were prepared as follows: Untreated samples Dry commercial tea samples (50 g) were milled with a Moulinex "Illico" blender for about 1 minute to produce a fine powder to produce a homogeneous sample. Take 5 grams of powder into a 100 ml conical glass flask at room temperature (22 ° C). Pour 50 ml of boiling distilled water on the tea sample and stir with a plastic stick, and leave it at room temperature for 30 minutes. The extract was filtered through Whatman No. 1 filter paper, and the filtrate was collected and lyophilized overnight. Dissolve 10 mg of the sample in 1 ml of distilled water. HPLC equipment: Hewlett Packard Series II 1090 liquid chromatography. HPLC column: C18 RP Hypersil 5μ, 150x4.6 mm. HPLC mobile phase: methanol: water: orthophosphoric acid 20: 79.9: 0.1 v / v, flow rate 0.9 ml / min. The absorption wavelength is 210mm. The obtained chromatogram is shown in Figures 2A (Kemmun) and 3A (Lapsang Souchong). The processed samples were ground with a Moulinex “Illico” blender to dry powder (50 g) for about 1 minute to obtain a fine powder. Sample. Tea samples are poured up to 50 ml of boiling distilled water, using plastic sticks 4 -20- This paper size applies Chinese National Standard (CNS) A4 (210 X 297 mm) 544286 A7 B7 V. Description of the invention (18) After mixing 30 minutes at room temperature. The extract was filtered through Whatman No. 1 filter paper, and the filtrate was collected and lyophilized overnight. Dissolve 10 g of sample in 1 ml of distilled water and flow through a reverse phase (RP) C18 IsoluteR column under vacuum. Using 20 µI > as HPLC analysis, the aforementioned method was performed on an untreated sample. The chromatograms are shown in Figures 2B (Kemmun) and 3B (Lapsang Souchong). The illustrated HPLC does not clearly distinguish between treated and untreated tea samples, because the chromatograms 2A and 2B are virtually identical, as are the chromatograms 3A and 3B. Each figure also proves that HPLC is incapable of distinguishing between different tea types. The chromatograms shown in Figure 2 and Figure 3 are actually the same. Reference Example 2: Differences between NMR and NMR spectroscopy and variability analysis. The tea types used are Gunpowder, Darjeeling, Kemmun, Assam, Lapsang Souchong, and 〇 〇 〇ng. The untreated and treated samples of these teas were prepared as described in Comparative Example i. Preparation of NMR spectroscopy and analysis of main components. Dissolve 10 mg of tea in 1 liter of deuterated water (DA). 8000 is called NMR resonance spectroscopy, and the inside of the sample is compared to TSP at 0.00 ppm. The procedure described in Reference Example 1 was performed for each tea extract. Results The data were converted to points on the two-score chart, which are shown in Figures 4A and 4B. Figure 4B shows six different spot clusters attributable to each tea extract after treatment, while Figure 4A shows poor clustering before treatment. Nuclear magnetic resonance spectroscopy / PCA technology proved to have discriminating ability, which was in sharp contrast to the inability of HPLC to distinguish tea types as shown in Comparative Example 1. Reference Example 3: I Nuclear Magnetic Resonance Spectroscopy and Multivariate Analysis Identification of Shaoyaozhi-21-This paper size is applicable to China @ ^^ (CNS) 八 44 (21〇x 297 公 爱) " 544286 A7 B7 ο 5. Description of the invention (19) Product sample extracts are prepared from commercial capsules of Feverfew from three different manufacturers. The surface of the product is the same, all of which are hard gelatin capsules containing "standardized" white inulin. Take five samples each containing 10 capsules. Take the same from manufacturer A. Take two samples from manufacturer B with the same lot number. The other two lot numbers are different and are taken from the manufacturer 3. Sample number Manufacturer batch number Capsule content 1 AI 500 g 2 B II 384 mg 3 B II 384 mg 4 C III 380 mg 5 C IV 380 mg Place the capsule content in each sample in a Moulinex “Illico” blend Grinding to fine powder in the combined machine produces a uniform sample. Dissolve 500 mg of powder in 50 ml of cold water and stir the extract on a 150 i: pm shaker for 4 hours at room temperature (22 ° C) to make an extracted aqueous solution for nuclear magnetic resonance spectroscopic analysis. The extract was filtered through Whatman No. 1 filter paper, and the filtrate was collected and freeze-dried overnight. Formulated speckles * components based on wide resonance spectroscopy The procedure described in Reference Example 1 was performed for each extract. Results The transformed data are the points on the score chart (average concentration). This is shown in Figure 5, where each point is equivalent to a capsule. The samples 1, 4, and 5 shown in the figure are well clustered, reflecting the consistency between the individual capsules in each batch. The clusters of samples 2 and 3 are relatively weak, reflecting that each capsule in each special product varies greatly. This figure also highlights the significant differences between batches of the same product claimed by one manufacturer (Sample 4 and 5). The standard of this paper is -22- 544286 A7 B7 V. Description of the invention (20) Reference example 4 · Li _ 糸 Magnetic resonance 糸 f good study and main component split stems different maturity Uanacetum parthen 彳 um 詖 birch plant test Samples of this plant were collected from CN. Seeds, Ely, UK, and the guaranteed seed medicinal plant species Tanacetum parthenium was cultivated in the United Kingdom. The seeds were sown on April 18 and cultivated in the greenhouse. It sprouted on April 25th, transplanted into a small ground on a checkered base on May 24th, and developed an arbitrary sampling tissue. After transplantation, five samples containing 4 plantlets were often collected from the small ground for analysis. Two additional samples were taken during the final harvest. Frozen storage, package in January and February after harvest. Interval analysis. The sampling harvest date is as follows: Sample number harvest date T0 _ June 30th ___ T1 July 14th T2 August 4th T3 August 19th T4 September 18th-T5 ---- September 18th to October + 3rd — ____T6 September 18th to November 10th to collect the dried plant material, and immediately put it into the _ 2 0 freezer. The cold beam post-material was placed in a freeze dryer for 12 hours, and then the sample was checked to ensure that it was dry. The material was ground in a Moulinex “Illico” blender for 2 minutes to produce a fine powder to produce a uniform sample. 500 mg of plant material was dissolved in 50 ml of cold water to prepare a water extract for nuclear magnetic resonance resonance spectroscopy. The extract was stirred at MO ΓΠΠ on a shaker at room temperature (22 ° C) for 4 hours. The extract was filtered through Whatman No. 1 filter paper, and the filtrate was collected and freeze-dried overnight. ° -23- 544286 A7 B7 V. Description of the invention (21) Preparation and analysis of nuclear magnetic resonance spectroscopy The above sample 10 mg was dissolved in 1 ml of deuterated water for NMR spectroscopic analysis. Using 800 μ]: for nuclear magnetic resonance spectroscopic analysis, the sample was internally referenced to TSP at 0.0000 ppnl. Follow the procedure described in Reference Example 1. Results The data were transformed into points on the PCA score map (mean concentration). This is shown in Figure 6. There are clear clusters of related different samples, for example, the maturation process can be shown on the pCA chart. The method of the present invention thus provides a method for discriminating between samples of known plant materials having different physiological states. Reference Example 5 · NMR spectroscopy and multivariate analysis of samples for distinguishing plant materials from oblique plots. Traditional Chinese medicine prescriptions are called Liu Wei Huang Wan (六味 广 丸) containing six plant components. Different suppliers (indicated by 丨, 2 and 3). Extract M method Each supplier weighed 100 tablets in 10 tablets. Grind with a pestle and bowl, and transfer 1 g of the obtained powder to a glass bottle. Pour 100 ml of cold water on the powder and reflux the mixture for 30 minutes. The cooled solution was lyophilized after filtration. Oral controlled magnetic resonance spectroscopy and main component analysis Take 10 ml each of the samples prepared above and dissolve in 1 ml deuterated water. ^ Test case 1 illustrates the procedure. The results are transformed into points on the PCA score map (average set). This has clear clusters based on three supplier samples. These results prove that: samples of multicomponent plant materials with identical surfaces can be successfully identified. law

544286 A7 B7 V. Description of the Invention (22) Example 1: Differentiation of Multiple Batches of Tobacco and Comet Extraction by Nuclear Magnetic Resonance Spectroscopy and Multivariate Analysis Preparation of the following eight commercial brand cigarette samples: Sample number package name on each package Fragrant lot number 1 Country 20 8998127533124 2 Star Mild 16 8998127311173 3 PrinslP 12 8998127323138 4 PrinslP 12 8998127323039 5 Bentoel 12 8998127123035 6 Bentoel Mild 16 8998127301617 7 Bentoel International 12 8998127111032 8 Inter Biru Export Quality 12 8998127312033 Since the sample 1-8 Take out 10 cigarettes each in the box and remove the cigarette paper from the tobacco material. The tobacco material was then extracted by refluxing in 70% alcohol for 30 minutes. The resulting solution was filtered, and the solvent was removed by rotary evaporation, followed by freeze-drying. Analysis of the proposed constituents of nuclear magnetic resonance spectroscopy Take 10 mg of each of the above samples and dissolve them in 1 ml of fully deuterated fluorenyl alcohol for NMR resonance spectroscopic analysis. Complete each of the eight samples as described in Reference Example 1. Results The data were transformed into points on the PCA score chart. A clear point cluster is equivalent to each of the eight samples. These results demonstrate that the method of the present invention can successfully distinguish tobacco from cigarettes under different brand names. This method also recognizes different batches of designated commercial brand cigarettes (see samples 3 and 4, the ratio of which is "PrinslP" brand, but the number of batches is different). Application of stubborn technique ___ -25- This paper size is applicable to Chinese National Standard (CNS) A4 specification (210X297 mm) 544286 A7 B7 V. Description of the invention (23) Based on the results obtained above, each Standard specifications for a brand of cigarettes. The subsequent cigarette samples sold under these brand names can then use the aforementioned trendy extraction test to verify the authenticity and compare the results with the relevant standard specifications. This measure may identify, for example, counterfeit or counterfeit cigarettes that have been adulterated with non-authentic tobacco materials. • 26- This paper size applies to China National Standard (CNS) A4 (210 X 297 mm)

Claims (1)

544286 1! Shenshen patented the method of the second standard of the plant material of the grass 1 standard method, the method includes: a test solution or a test extract of an early plant material sample, the enemy knows that this standard or various properties are necessary 11; it is used for 11+ duplex analysis of NMR spectroscopy and 5 types of Helio solution or extracts and computer-type cognitive technology; r- * machine basis (out) to obtain the results of the analysis method used in step ⑼; and (iv) The standard specifications obtained in step (iii). Based on the spoon, the early plant material was prepared. 2: A method for formulating a sample of tobacco plant material, the aforementioned standard specification 'The method includes: /, material ⑻ = test solution or test 备用 of a standby sample of the final grass plant material ( Η,) The solution or extract is analyzed by a combination of nuclear magnetic resonance spectroscopy-type cognitive technology; the basic (ii〇 obtains the result used in step (ii '); and (iv) the result obtained in the right step (111') is in line with A sample of the standard specifications of the tobacco material formulated in step (iV) of one item is selected. 3 · Species and non-acceptance benefits, 'Ji. ^ A J + I »… A standard specification method for a smoke material sample, comprising: ((pi prepared from a test solution or test substance of a grass plant material backup sample; ") using S solution or extract as a nuclear magnetic resonance spectroscopy and computer-based model Cognitive technology combination analysis; Extraction process extraction category-27- 544286 Patent application scope (11. Obtain the analysis result of step (ii '); and (iv ») If the result obtained in step (iii,) matches the taste Special The target specification of item 1 of the target range is not accepted as a supplementary standard when the standard specification of the material is established in the second step of the method. 4. According to the method of item 3 of the patent application scope, the backup sample is one yuan Zhuo products, suspected to be imitated stomach or faked by non-genuine materials. 5. = The method of item 4 of the material range, in which the grass product is commercially available incense, cigar or pipe tobacco. 6. According to the application The scope of patents is any one of the methods from to 5 items, where # MRI money science and computer basics _ cognitive ㈣ combination includes / ⑷ extract test solution or test extract for nuclear magnetic resonance spectroscopy and record one or more nuclear magnetic resonance Spectrum; and ⑻ = this: magnetic resonance spectroscopy or multivariate analysis of each spectral data to generate one or more points on a juice plot. 7. According to the method of any one of the mountain items in the scope of the patent application, the computer basis Type recognition technology includes main component analysis (PC eight) 8. According to the method of any of the U5 items in the scope of the patent application, it is recommended that the test solution or test extract of the reserve sample be subjected to nuclear magnetic resonance spectroscopy Joint technology with computer basic type recognition. 9. Method according to patent application scope No.! To 5 orders / items, wherein the plant material includes, or is derived from the whole terminal grass plant, J part of the terminal grass plant, tobacco plant extract Or a tobacco plant fragment. 10. A method for providing standard specifications of tobacco plant materials =, including procedures (...) (i " 't) Preparation of test solutions or test extracts of grass plant material samples-28 a National Standard (⑽) A4 Specification (21GX297 Public Ding Ding Ministry, the scope of the patent application is known to have this or each property required by this standard; present the test solution or test extract for nuclear magnetic resonance spectroscopy and record one or more spectra; (iii " ') extract the spectrum or each A multivariate analysis of the data obtained by nuclear magnetic resonance spectroscopy to generate one or more points on the score chart; and defining the eligibility range around the multiple points generated in the step (in " ') as the standard specification for the plant material Or part of it. U. The method according to item 10 of the scope of patent application, wherein the multivariate analysis of the (;;,) step is performed using an unsupervised methodology. 12. A method for preparing a material plant material sample, the material standard specification before the test, the method comprising: (η preparing a test solution or a test extract of the standby sample of the tobacco plant material; (Π ") presenting the test solution or Test the extract for nuclear magnetic resonance spectroscopy and record one or more nuclear magnetic resonance spectra; (iii & quot) extract the nuclear magnetic resonance or data obtained from each spectrum for multivariate analysis to generate one or more points on the score chart; and (1V ") If the points generated in the step (di ") score chart belong only to the qualified range in the standard specifications established in the method step (iv ") described in the patent scope of item 10, select this spare sample to meet the Samples of standard specifications. Η. According to the method of scope of application for patent No. u10, # 中 终 草 植物 材料 试样 料 has the standard requirements of the nature of f or every _ nature of which is known for 5 to 55 «or audit Samples of grass plant materials. VI. Patent Application Park 14. Tobacco of this nature or each nature according to item i or 丨 0 of the scope of patent application, among which the sample system of which is the standard of Zhuojia Branch A specific Specimens for 4 members, —species grown on grass in a particular geographical location, —species ^ on leaf grade secret grass samples, or __species ^ on grass samples matured by special techniques' or a designated A sample of a finished tobacco product. 15_ The method according to any one of claims 1 to 5, ", 丨 丨, and ^ ', wherein the herbaceous plant material is derived from or contains two or more different types of herbaceous grass. It is a mixture. -30- This paper size applies to China National Standard (CNS) A4 (210 X 297 mm)