CN113390810B - Nicotine extraction method and nicotine content determination method for fresh tobacco leaves - Google Patents
Nicotine extraction method and nicotine content determination method for fresh tobacco leaves Download PDFInfo
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- SNICXCGAKADSCV-JTQLQIEISA-N (-)-Nicotine Chemical compound CN1CCC[C@H]1C1=CC=CN=C1 SNICXCGAKADSCV-JTQLQIEISA-N 0.000 title claims abstract description 67
- SNICXCGAKADSCV-UHFFFAOYSA-N nicotine Natural products CN1CCCC1C1=CC=CN=C1 SNICXCGAKADSCV-UHFFFAOYSA-N 0.000 title claims abstract description 67
- 229960002715 nicotine Drugs 0.000 title claims abstract description 67
- 238000000034 method Methods 0.000 title claims abstract description 60
- 235000002637 Nicotiana tabacum Nutrition 0.000 title claims abstract description 50
- 241000208125 Nicotiana Species 0.000 title claims abstract description 47
- 238000000605 extraction Methods 0.000 title description 24
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 44
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 34
- 239000006228 supernatant Substances 0.000 claims description 24
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 18
- 238000002156 mixing Methods 0.000 claims description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- 238000000227 grinding Methods 0.000 claims description 10
- 239000007788 liquid Substances 0.000 claims description 9
- 229910052757 nitrogen Inorganic materials 0.000 claims description 9
- 239000000243 solution Substances 0.000 claims description 9
- 239000002253 acid Substances 0.000 claims description 7
- 238000002835 absorbance Methods 0.000 claims description 6
- 238000005119 centrifugation Methods 0.000 claims description 6
- 238000007865 diluting Methods 0.000 claims description 4
- 238000011481 absorbance measurement Methods 0.000 claims description 2
- 239000007864 aqueous solution Substances 0.000 claims description 2
- 230000008033 biological extinction Effects 0.000 claims description 2
- 229910052799 carbon Inorganic materials 0.000 claims description 2
- 238000012937 correction Methods 0.000 claims description 2
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 7
- 238000005259 measurement Methods 0.000 description 7
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 239000008367 deionised water Substances 0.000 description 5
- 229910021641 deionized water Inorganic materials 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 238000002834 transmittance Methods 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 239000012530 fluid Substances 0.000 description 4
- 238000012546 transfer Methods 0.000 description 4
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 3
- 244000061176 Nicotiana tabacum Species 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 230000009849 deactivation Effects 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 241000208292 Solanaceae Species 0.000 description 1
- 238000003916 acid precipitation Methods 0.000 description 1
- 229930013930 alkaloid Natural products 0.000 description 1
- 150000003797 alkaloid derivatives Chemical class 0.000 description 1
- 239000012159 carrier gas Substances 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 239000001307 helium Substances 0.000 description 1
- 229910052734 helium Inorganic materials 0.000 description 1
- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000000703 high-speed centrifugation Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 1
- 239000012044 organic layer Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000001953 sensory effect Effects 0.000 description 1
- 230000014860 sensory perception of taste Effects 0.000 description 1
- CGFYHILWFSGVJS-UHFFFAOYSA-N silicic acid;trioxotungsten Chemical compound O[Si](O)(O)O.O=[W]1(=O)O[W](=O)(=O)O[W](=O)(=O)O1.O=[W]1(=O)O[W](=O)(=O)O[W](=O)(=O)O1.O=[W]1(=O)O[W](=O)(=O)O[W](=O)(=O)O1.O=[W]1(=O)O[W](=O)(=O)O[W](=O)(=O)O1 CGFYHILWFSGVJS-UHFFFAOYSA-N 0.000 description 1
- 239000000779 smoke Substances 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 238000001256 steam distillation Methods 0.000 description 1
- 235000019505 tobacco product Nutrition 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
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Abstract
The invention belongs to the technical field of nicotine content determination. Aiming at the problems that the prior method for determining the nicotine content needs to bake or de-enzyme fresh tobacco leaves, has complicated steps, long time consumption and low efficiency, the invention provides a method for extracting nicotine from fresh tobacco leaves and a method for determining the nicotine content.
Description
Technical Field
The invention belongs to the technical field of nicotine content determination, and particularly relates to a nicotine extraction method and a nicotine content determination method for fresh tobacco leaves.
Background
Nicotine, also known as nicotine, is a nitrogenous alkaloid present in solanaceae plants and accounts for 90-95% of the total alkaloid content of tobacco cultivars. Nicotine is a source of tobacco flavor, can stimulate the sense of taste of smokers to ensure that the smokers obtain physiological satisfaction, and is an important factor for determining the sensory quality and industrial usability of tobacco. The nicotine is one of important factors for measuring the quality of the tobacco leaves, directly influences the physiological strength, the smoke characteristics and the safety of tobacco products, has important influence on the quality of the aroma of the tobacco leaves, and has extremely obvious positive correlation with the content of the nicotine and the aroma. Currently, the nicotine content of high-quality tobacco leaves is about 1.5% to 3.5%.
Based on the important influence of nicotine on tobacco leaves, the determination of the content of nicotine becomes a relatively important content in the tobacco industry. The method for measuring the nicotine content in the tobacco leaves comprises a plurality of methods, such as an ultraviolet spectrophotometry method, a silicotungstic acid precipitation method, a GC/MS method, a high performance liquid chromatography and the like, wherein the methods have the disadvantages of complex sample treatment, need of baking or enzyme deactivation of dry leaves, multiple experimental steps, long time consumption and higher requirements on instruments and equipment. The current general acid extraction method is to add active carbon and sulfuric acid into a crushed dry sample for shaking extraction, and needs to de-enzyme or bake dry leaves, so that the required determination time is longer. At present, the fixation treatment of fresh tobacco leaves in a laboratory needs to be finished by using an oven, the space of the oven is limited, when the number of samples to be treated is large, the fixation of all tobacco leaves cannot be finished at one time, and the promotion of the experiment progress is greatly limited. In addition, the grinding process after the enzyme deactivation is a time-consuming and labor-consuming process, and if the tobacco leaves after the enzyme deactivation are very easy to regain moisture in the timely treatment, the moisture in the tobacco leaves needs to be dried again. In addition, a method for measuring the nicotine content by a non-steam distillation method is also proposed, and relatively speaking, the process is complicated and the efficiency is low. Therefore, it is important to develop a method for rapidly and efficiently determining nicotine in fresh tobacco leaves without deactivating enzymes.
Disclosure of Invention
Aiming at the problems that the prior method for measuring the nicotine content needs to bake or de-enzyme fresh tobacco leaves, has complex steps, long consumed time and low efficiency, the invention provides a method for extracting nicotine from fresh tobacco leaves and a method for measuring the nicotine content.
The invention is realized by the following technical scheme:
the invention provides a nicotine extraction method of fresh tobacco leaves, which adopts an acid extraction double-release method and comprises the following steps:
(1) Grinding fresh tobacco leaves by using liquid nitrogen, adding hydrochloric acid with the volume of vmL being 16% -22% of the concentration, uniformly mixing, and extracting at room temperature (namely acid extraction);
(2) Centrifuging at high speed, collecting supernatant, diluting with water of the same volume (i.e. diluting by multiple times), adding activated carbon with final concentration of 5%, and mixing;
(3) Centrifuging at high speed, taking supernatant, adding 2mL of hydrochloric acid with the concentration of 8% -11% into each 1mL of supernatant, uniformly mixing, and performing secondary extraction at room temperature;
(4) High speed centrifugation is carried out, and supernatant is taken, namely nicotine extracting solution.
On the other hand, the invention provides a method for measuring the nicotine content of fresh tobacco leaves, which is based on the acid-extracting double-release method and also comprises the following steps:
(5) Measuring the absorbance of the nicotine extract at 259nm,236nm and 282 nm; the water content of the leaves is set to be 90% consistently, and the nicotine content of the sample is calculated according to the following formula:
in the formula, 1.059 is a correction coefficient; 34.3 is the specific extinction coefficient of nicotine in aqueous solution; a259, a236, and a282 represent absorbance measurements.
Further, the concentration of the hydrochloric acid in the step (1) is 18.5%.
Further, in the step (1), after grinding every 0.3g of fresh tobacco leaves by liquid nitrogen, diluting with 2mL of hydrochloric acid, and extracting at room temperature for 3min.
Further, the concentration of the hydrochloric acid in the step (3) is 9%.
Further, the centrifugation speed of the steps (2), (3) and (4) is 12000-14000rpm.
Further, the centrifugation speeds of the steps (2), (3) and (4) are 13000rpm, and the centrifugation time is 5min.
The method can realize the rapid extraction and content determination of nicotine in the fresh tobacco leaf sample, can eliminate the influence of impurities in the fresh tobacco leaf on the determination result, has simple and convenient operation, can complete the nicotine content determination of the fresh tobacco leaf within 30min, and can be applied to the nicotine content determination of the fresh tobacco leaf sample on a large scale. The actual measurement shows that more than 500 parts of fresh tobacco samples can be extracted and the nicotine content can be measured by 3 persons in one day, and the time of more than 10 days is needed if the GC/MS method is used for extracting and measuring the nicotine content with the same sample quantity.
The method is a simple method for measuring the nicotine content of fresh leaves, and samples for comparison are collected from the same part as much as possible (the influence of the water content of the samples is reduced to the minimum): the n-th slice from the top is generally used for consistency marking. If necessary, one more sample can be taken for the determination of the water content.
The sample extracting solution processed by the method provided by the invention has good stability, and the final measuring result is still stable no matter the dosage of each reagent in the extracting process is enlarged or reduced in an equal ratio or the extracting solution is placed at room temperature overnight.
Drawings
FIG. 1 is a comparison of the results of the detection of nicotine content in fresh tobacco leaves by the method of the present invention and the GC/MS method in example 1;
FIG. 2 is a comparison of the results of the measurements of example 2 on an equal scale down (0.5X system) and the original system (1X system);
FIG. 3 is a comparison of the results of the measurement of example 2 on an equal scale (2X system) and the original system (1X system);
FIG. 4 is a comparison of the results of the measurement at the completion of the extraction (0 h) and after standing overnight (24 h) in example 3;
FIG. 5 shows the difference between the extraction effect and the transmittance in different processing manners of example 4.
FIG. 6 is a graph of the effect of different activated carbon concentrations on nicotine content determination in example 4.
Detailed Description
The present invention will be described in further detail with reference to the following embodiments and the accompanying drawings.
It is to be noted that the experimental methods described in the following embodiments are all conventional methods unless otherwise specified, and the reagents and materials are commercially available unless otherwise specified.
Example 1: rapid determination of nicotine content in tobacco plant leaves in the field
Taking tobacco leaf samples of a conventional variety and a low nicotine variety, taking the fourth tobacco leaf of each tobacco from top to bottom as a mark, randomly taking 10 samples of each variety, and respectively treating each fresh leaf by adopting the display method disclosed by the invention, wherein the specific steps are as follows: taking 0.3g of fresh leaves, fully grinding in liquid nitrogen, completely transferring into 2mL of 18.5% hydrochloric acid, fully mixing uniformly, and extracting at room temperature for 3min.13000rpm for 5min, transfer all supernatant into a new centrifuge tube, add 2mL deionized water, add 0.06g activated carbon powder (final concentration 5%). 13000rpm for 5min, will absorb 1mL supernatant fluid to the new centrifugal tube, adding 2mL 9% hydrochloric acid, fully mixing. 13000rpm for 5min, the supernatant was transferred to a new centrifuge tube, and the absorbance at 259nm (A259), 236nm (A236) and 282nm (A282) was measured, respectively, and according to the formula:
the nicotine content of these samples was calculated and is specified in the following table:
table 1: results of measurement of Nicotine content in 10 field samples (measurement method of the present invention)
Meanwhile, a GC/MS method is adopted for comparative verification. The GC/MS method was performed as follows: fresh tobacco leaves were de-enzymed, 10mg of a homogeneous dry leaf sample was soaked in 1mL of 10% NaOH (w/v) solution for 20 minutes, then an equal amount of dichloromethane was added, vortexed, centrifuged and the organic layer was collected. Nicotine content was determined using an Agilent Technologies 7890A chromatograph using a DB 5MS column and Agilent Technologies 5975C MSD detector with helium as the carrier gas. The column was first held at 100 ℃ for 5min, then increased to 210 ℃ at a rate of 50 ℃/min, and then held at 210 ℃ for 4min. The ion source temperature was 230 ℃ and the quadrupole temperature was 150 ℃. The pure nicotine product of Sigma-Aldrich is used as a standard control.
The comparison of the detection results of the determination method of the present invention and the existing GC/MS method is shown in FIG. 1. The determination method of the invention has little difference with the GC/MS method in the nicotine detection result, and the difference is within 10 percent, so the method for determining the nicotine in the fresh tobacco leaves is reliable.
Example 2: geometric scaling experiment
The determination method of the invention has higher stability after the reagent dosage is scaled proportionally, thus the determination of nicotine content can be completed under a smaller or larger system.
The method for rapidly determining the nicotine content in the fresh leaves comprises the following steps of (a) determining the nicotine content in certain field tobacco plant leaves after the dosage of reagents used in the method for rapidly determining the nicotine content in the fresh leaves is reduced in an equal proportion.
Taking the fourth tobacco leaf of each tobacco from top to bottom as a mark, taking a plurality of plants, and respectively treating each fresh leaf by adopting the method shown by the invention: grinding 0.15g fresh leaves in liquid nitrogen, transferring into 1mL 18.5% hydrochloric acid, mixing, and extracting at room temperature for 3min.13000rpm for 5min, transfer all supernatant into a new centrifuge tube, add 1mL deionized water, add 0.03g activated carbon powder (final concentration 5%). 13000rpm for 5min, will absorb 1mL supernatant fluid to the new centrifugal tube, adding 2mL 9% hydrochloric acid, fully mixing. 13000rpm for 5min, the supernatant was transferred to a new centrifuge tube, and the absorbance at 259nm (A259), 236nm (A236) and 282nm (A282) was determined, respectively, according to the formula:
the nicotine content of these samples was calculated. The comparison of the isometric scale-down (0.5X system) with the original system (1X system) is shown in FIG. 2, and there is no significant difference between the two. This shows that the extract has higher stability after scaling, and has little difference with the original system, further explaining the reliability of the extraction method.
(II) the following shows that the nicotine content in the tobacco plant leaves in a certain field is measured after the dosage of the reagent used in the method for rapidly measuring the nicotine content in the fresh leaves is amplified in equal proportion.
Taking the fourth tobacco leaf of each tobacco from top to bottom as a mark, taking a plurality of plants, and respectively treating each fresh leaf by adopting the method shown by the invention: taking 0.6g of fresh leaves, fully grinding in liquid nitrogen, transferring all the fresh leaves into 4mL of 18.5% hydrochloric acid, fully and uniformly mixing, and extracting at room temperature for 3min.13000rpm for 5min, transfer all supernatant into a new centrifuge tube, add 1mL deionized water, add 0.03g activated carbon powder (final concentration 5%). 13000rpm for 5min, will absorb 1mL supernatant fluid to the new centrifugal tube, adding 2mL 9% hydrochloric acid, fully mixing. 13000rpm for 5min, the supernatant was transferred to a new centrifuge tube, and the absorbance at 259nm (A259), 236nm (A236) and 282nm (A282) was determined, respectively, according to the formula:
the nicotine content of these samples was calculated.
The comparison of the 2 × system and the original system (1 × system) is shown in fig. 3, and there is no significant difference therebetween. This shows that the extract has higher stability after scaling, and has little difference with the original system, further explaining the reliability of the extraction method.
Example 3: stability test
The sample extracting solution treated by the method provided by the invention has good stability, and the final determination result is still stable after the extracting solution is placed at room temperature overnight. The following shows the measurement results after the extract is left for 24 hours in the method for rapidly measuring the nicotine content in fresh leaves. The method comprises the following specific steps: the fourth tobacco leaf from top to bottom of each tobacco is taken as a mark, and each fresh leaf is respectively treated by adopting the display method disclosed by the invention, and the method comprises the following specific steps: grinding 0.3g fresh leaves in liquid nitrogen, transferring into 2mL 18.5% hydrochloric acid, mixing, and extracting at room temperature for 3min.13000rpm for 5min, transfer all supernatant into a new centrifuge tube, add 2mL deionized water, add 0.06g activated carbon powder (final concentration 5%). 13000rpm for 5min, will absorb 1mL supernatant fluid to the new centrifugal tube, adding 2mL 9% hydrochloric acid, fully mixing. 13000rpm for 5min, the supernatant was transferred to a new centrifuge tube, its absorbance at 259nm (A259), 236nm (A236) and 282nm (A282) was determined at the completion of extraction (0 h) and left overnight (24 h), respectively, and according to the formula:
the nicotine content of these samples was calculated. The statistics of nicotine content at the completion of extraction (0 h) and overnight standing (24 h) are shown in fig. 4, and there is no significant difference between the two. This indicates that the extract has a higher stability and that more accurate nicotine content can still be determined after standing overnight, further indicating the reliability of the extraction method.
Example 4 Effect of activated carbon on transmittance and measurement of Structure
The method comprises the following operation steps:
(1) Grinding fresh tobacco leaf 0.3g with liquid nitrogen, adding 2mL of 18.5% hydrochloric acid, mixing, and extracting at room temperature for 3min.
(2) 13000rpm for 5min, the supernatant was poured into a centrifuge tube containing about 0.2mL volume of activated charcoal (0.06 g) 2mL deionized water and mixed well.
(3) 13000rpm for 5min, and 1mL of the supernatant was transferred to a centrifuge tube to which 2mL of 9% hydrochloric acid had been added.
(4) And centrifuging at 13000rpm for 5min, pouring the supernatant into a new centrifuge tube, and avoiding pouring out the residual activated carbon as much as possible.
FIG. 5 shows the difference between the extraction effect and the transmittance in different treatment methods.
Wherein, the acid extraction once refers to only adding 18.5 percent hydrochloric acid once for extraction without the operation step (3), and the acid extraction twice refers to adding hydrochloric acid again for extraction on the basis of the acid extraction once for extraction, and performing the operation step (3). After the activated carbon is added, the transmittance of a sample can be obviously improved; the extract has good stability, and the transmittance of the sample is not influenced after the extract is placed for 24 hours.
Figure 6 is a graph showing the effect of different activated carbon concentrations on nicotine content determination.
Wherein the control is nicotine sample with known concentration, and 2%, 5%, 8%, and 10% activated carbon are respectively added into the extractive solution to obtain final concentration of 2%, 5%, 8%, and 10%. The measured value of the sample added with the activated carbon with the final concentration of 2% is higher, which is related to the poorer adsorption capacity of the sample, the measured values of the sample added with the activated carbon with the final concentration of 8% and the sample added with the activated carbon with the final concentration of 10% are lower, which is related to the nicotine adsorption amount of the activated carbon, and the content of the sample added with the activated carbon with the final concentration of 5% is close to the actual sample content, so that the activated carbon with the final concentration of 5% is the optimal experimental dosage.
The above-mentioned embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solution of the present invention by those skilled in the art should fall within the protection scope defined by the claims of the present invention without departing from the spirit of the present invention.
Claims (6)
1. A method for measuring nicotine content of fresh tobacco leaves is characterized in that an acid double-release method is adopted, and the measuring method comprises the following steps:
(1) Grinding fresh tobacco leaves by using liquid nitrogen, adding 16-22% hydrochloric acid with the volume of v mL, uniformly mixing, and extracting at room temperature;
(2) Centrifuging at high speed, collecting supernatant, diluting the supernatant with water of the same volume, adding active carbon with final concentration of 5%, and mixing;
(3) Centrifuging at high speed, taking supernatant, adding 2mL of hydrochloric acid with the concentration of 8% -11% into every 1mL of supernatant, and uniformly mixing;
(4) Centrifuging at high speed, and collecting supernatant to obtain nicotine extractive solution;
(5) Measuring the absorbance of the nicotine extract at 259nm,236nm and 282 nm; the water content of the leaves is set to be 90% consistently, and the nicotine content of the sample is calculated according to the following formula:
in the formula, 1.059 is a correction coefficient; 34.3 is the specific extinction coefficient of nicotine in aqueous solution; a259, a236, and a282 represent absorbance measurements.
2. The method of claim 1, wherein the hydrochloric acid of step (1) has a concentration of 18.5%.
3. The method according to claim 2, wherein the step (1) comprises extracting the tobacco leaves with 2mL hydrochloric acid for 3min at room temperature after grinding with liquid nitrogen for every 0.3g of fresh tobacco leaves.
4. The method according to claim 3, wherein the hydrochloric acid concentration of the step (3) is 9%.
5. The method of claim 1, wherein the centrifugation speeds of steps (2), (3) and (4) are 12000-14000rpm.
6. The method of claim 5, wherein the centrifugation speeds of steps (2), (3) and (4) are 13000rpm and the centrifugation time is 5min.
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