Method for identifying cinnabar tobacco leaves

Abstract

The invention discloses a method for identifying cinnabar tobacco leaves, and particularly discloses a method for identifying cinnabar tobacco leaves, which adopts a compound in a formula 1 (# imgabs0 #) and / or a compound in a formula 2 (# imgabs1 #) as a marker for identifying the cinnabar tobacco leaves. The identification index is high in accuracy.

Description

Technical Field

[0001] This technology belongs to the field of tobacco leaf raw materials, and specifically relates to a method for identifying cinnabar tobacco leaves. Background Art

[0002] Common tobacco (Nicotiana tobacum) is an allopolyploid formed by the interspecific hybridization of Nicotiana tomentosiformis and Nicotiana sylvestris, but its alkaloid spectrum is very different from that of the two ancestors. In the green and senescent leaves of most common tobacco, nicotine accounts for about 95% of the total alkaloids. N. tomentosiformis accumulates nor-nicotine in both green and senescent leaves; while N. sylvestris mainly accumulates nicotine in green leaves, but most of the nicotine is converted to nor-nicotine during leaf senescence. Some mutant strains of flue-cured tobacco varieties show red spots on the cured leaves, and most of the nicotine in their tobacco leaves is demethylated to form nor-nicotine. Therefore, the phenotype of nor-nicotine as the dominant alkaloid in this flue-cured tobacco is similar to "atavism", which is a very meaningful evolutionary problem. Such studies have been carried out in the United States and Japan, and were first reported in the 1950s and 1960s. The natural occurrence probability of this mutation is very low. Some studies have shown that 3 mutant strains were found among 538 individual plants, with a probability of about 0.56%. In tobacco leaf production, this variant strain is called "vermilion tobacco", and its aroma quality is unique, with strong development and application value. Through in-depth research on the nicotine demethylase gene family, the mechanism of nicotine conversion to nor-nicotine has been analyzed, providing a support point for further research on the formation mechanism of vermilion tobacco. So far, a total of 4 functional genes in the nicotine demethylase gene family have been cloned, namely CYP82E4, CYP82E5V2, CYP82E10, and CYP82E21. Although the CYP82E21 gene can encode an active nicotine demethylase, it is only expressed in anthers and has little effect on the nor-nicotine content in leaves. The research focus is mainly on the first three. CYP82E4 is the major gene in burley tobacco. The nicotine conversion rate of its EMS mutant strain is 2.2%, which is much lower than that of the starting control material (>60%). This result is basically consistent with that of Julio et al. There is no significant difference in the nicotine conversion rate between the single and double mutant materials of CYP82E5V2 and CYP82E10 genes and the control, but the nicotine conversion rate of the E4 / E5 / E10 triple mutant material is 0.55%, which is lower than that of the E4 single mutant and is equivalent to that of the RNAi transgenic plants. Analysis of the CYP82E4 gene promoter shows that it is only expressed when leaf senescence is induced by ethylene and TMV infection. The expression level of the CYP82E4 gene in the green leaves of both the transformed and non-transformed burley tobacco plants is relatively low, and it is significantly induced by an unknown mechanism only in the senescent leaves of the transformed plants, resulting in a high nicotine conversion rate. Because the alkaloid spectrum is similar to that of the "transformed strain" of burley tobacco, we speculate that the CYP82E4 gene in the "transformed strain" (vermilion tobacco) of flue-cured tobacco may also be induced to be highly expressed, resulting in the accumulation of nor-nicotine as the main alkaloid in its tobacco leaves.

[0003] Some methods for identifying cinnabar tobacco have been reported in the prior art. For example, the Chinese patent document with the publication number CN113584210A discloses a method for screening cinnabar tobacco plants based on the expression level of the NtPIF1 gene. The Chinese patent document with the publication number CN113584211A discloses a method for screening cinnabar tobacco plants based on the expression level of the CYP82E4 gene. The Chinese patent document with the publication number CN112666316A discloses a method for distinguishing cinnabar tobacco from ordinary tobacco by leaf phenotype or measuring the SPAD value characterizing chlorophyll content. The Chinese patent document with the publication number CN111380812A discloses a method for rapidly identifying cinnabar tobacco leaves of flue-cured tobacco by the amplitude variation of the color parameters lightness (L*), redness (a*), yellowness (b*), and saturation (C*) corresponding to the tobacco leaves. Although some identification methods have been disclosed in the prior art, the identification accuracy still needs to be improved. Summary of the Invention

[0004] In view of the lack of identification methods for cinnabar tobacco leaves and the problems of unsatisfactory accuracy and efficiency in the few existing identification methods, the purpose of the present invention is to provide a brand-new identification method for cinnabar tobacco leaves, aiming to improve the identification efficiency, objectivity, and accuracy.

[0005] An identification method for cinnabar tobacco leaves uses the compound of formula 1 and / or formula 2 as a marker for identifying cinnabar tobacco leaves;

[0006]

[0007] Research in the present invention shows that using the compound of formula 1 and / or formula 2 as an identification marker can efficiently and accurately identify cinnabar tobacco leaves.

[0008] In the identification method for cinnabar tobacco leaves of the present invention, the marker in the tobacco leaves to be identified is measured. When the tobacco leaves to be identified contain the marker, they are identified as cinnabar tobacco leaves. Conversely, they are not cinnabar tobacco leaves.

[0009] In the identification method for cinnabar tobacco leaves of the present invention, the marker is measured by HPLC-MS.

[0010] Preferably, in HPLC-MS, the HPLC mobile phase includes mobile phase A and mobile phase B. Among them, mobile phase A is water, and mobile phase B is acetonitrile;

[0011] The elution gradient is:

[0012] Time Mobile Phase B v%

[0013] 0 - 20 min 80 - 90;

[0014] 21 - 22 min 10 - 20;

[0015] 21 - 28 min, 10 - 20;

[0016] 28 - 29 min, 80 - 90;

[0017] 29 - 35 min, 80 - 90;

[0018] The preferred elution gradient is:

[0019] Time Mobile phase B v%

[0020] 0 - 20 min, 89 - 90;

[0021] 21 - 22 min, 9 - 10;

[0022] 21 - 28 min, 9 - 10;

[0023] 28 - 29 min, 89 - 90;

[0024] 29 - 35 min, 89 - 90.

[0025] In the elution stage described above, the total volume of mobile phases A and B is 100%.

[0026] In the present invention, the chromatographic column is: Alphasil XD Amide, 5 μm, 4.6×250 mm, produced by Huapu Keyi (Beijing) Technology Co., Ltd.;

[0027] In the present invention, the detection wavelength is 255 - 265 nm, further preferably 259 - 261 nm.

[0028] In the present invention, the tobacco leaves to be identified can be pre - extracted with a solvent and then subjected to the above - mentioned HPLC - MS determination.

[0029] In the present invention, the solvent is alcohol and / or water; the alcohol is an alcohol with 1 - 4 carbon atoms. Preferably, the solvent is a mixed solvent of alcohol and water. Further preferably, in the solvent, the volume ratio of alcohol to water is 75 - 85:15 - 25.

[0030] Research in the present invention shows that by using this process for extraction and then combining with the subsequent HPLC - MS determination, the test sensitivity and accuracy of the markers can be improved, false positives can be reduced, and thus the identification accuracy of cinnabar tobacco leaves can be further improved.

[0031] In the present invention, the solvent is at least one of methanol and ethanol.

[0032] In the present invention, in the solvent extraction stage, the liquid - solid ratio is 10 - 50 mL / g.

[0033] In the present invention, the solvent extraction stage is carried out under ultrasonic assistance.

[0034] In the present invention, the time for solution extraction is more than 10 min, preferably 30 - 120 min.

[0035] Beneficial effects

[0036] Research in the present invention shows that the compound of formula 1 and / or formula 2 as a marker can significantly improve the discrimination accuracy of Cinnabar tobacco leaves. On this basis, further using the combined alcohol extraction HPLC-MS determination process can further improve the test sensitivity of the marker, which helps to further synergistically improve the discrimination accuracy of Cinnabar tobacco leaves. Description of the drawings

[0037] Figure 1 It is the HPLC-MS test chart of Cinnabar tobacco leaves (2021 - YN - ZSY - ZS - 1), non - Cinnabar tobacco leaves (2019 - YNCX - KY - PY - C1F - GC) and solvent in the present invention; (black: Cinnabar tobacco, red: non - Cinnabar tobacco, blue: 80% methanol - water (solvent)). Detailed implementation manners

[0038] Example 1:

[0039] 0.20 g of tobacco sample powder (20 mesh) was ultrasonically extracted with 5 mL of a mixture of methanol and water (80 + 20, v / v) for 20 min. Then, after centrifugation at 3000 rpm, the supernatant was filtered through a 0.22 μm membrane and injected, with an injection volume of 10 μL. High - performance liquid chromatography conditions: Alphasil XDAmide, 5 μm, 4.6×250 mm chromatographic column (Huapu Keyi (Beijing) Technology Co., Ltd.). The mobile phase was water (A) and acetonitrile (B), and gradient elution was used. The gradient was 80% B for 0 - 20 min, 20% B for 20 - 21 min, 20% B for 21 - 28 min, 80% B for 28 - 29 min, 80% B for 29 - 35 min, and the detection wavelength was 260 nm. The standard solution concentrations of the calibration curve were 20, 400, 800, 1200, 1600, 2000, 2400 μg / mL respectively. A blind test was conducted on a mixed sample library containing Cinnabar tobacco leaves and non - Cinnabar tobacco leaves. The tobacco leaf category was judged through the test results, and the test accuracy was statistically analyzed. The detection accuracy of Cinnabar tobacco leaves by this method was 100%. The results are shown in Table 1:

[0040] Table 1

[0041]

[0042]

[0043]

[0044] Note: In Table 1, (a) is the total content of Formula 1 and Formula 2;

[0045] Example 2:

[0046] Compared with Example 1, the only difference is that the ultrasonic extraction is extended from 20 min to 30 min, and the gradient elution conditions are changed to Condition 2: (gradient is 0-20 min 90% B, 20-21 min 10% B, 21-28 min 10% B, 28-29 min 90% B, 29-35 min 90% B). The results are shown in Table 2, and the expression of characteristic substances is significantly improved.

[0047] Table 2

[0048]

[0049]

[0050] Note: In Table 2, (a) is the total content of Formula 1 and Formula 2;

[0051] As shown in Table 2, the use of the preferred elution mechanism can obtain better determination results of Formula 1 and Formula 2. This helps to further reduce the missed detection rate of cinnabar smoke.

[0052] Comparative Example 3

[0053] The test results show that there is a large difference in the content of β-pyridine, 3-vinylpyridine, 3-methylcyclopentane-1,2-dione, and mysmine in cinnabar tobacco and non-cinnabar tobacco. The markers β-pyridine, 3-vinylpyridine, 3-methylcyclopentane-1,2-dione, and mysmine were selected for a joint identification experiment to replace Formula 1 / Formula 2 of this application. The relative content of the four substances in cinnabar tobacco leaves was significantly higher than that of non-cinnabar tobacco leaves. The four substances in the tobacco leaves were tested and the test results were compared with the minimum content of cinnabar tobacco leaves. The content was significantly less than the minimum value for non-cinnabar tobacco leaves, and the content was significantly higher than the minimum value for non-cinnabar tobacco leaves. The results are shown in Table 3. It was found that this method has certain feasibility, but the accuracy of the test results will be reduced to a certain extent for some tobacco leaves with less obvious cinnabar characteristics, and there will be a certain result deviation for the analysis of oriental tobacco leaves and cinnabar tobacco leaves.

[0054] Table 3

[0055]

[0056]

[0057]

[0058] From the above results, it can be seen that by using the markers of Formula 1 and Formula 2 of the present invention, the judgment accuracy for cinnabar cigarettes can reach 100%, which can avoid the problems of insufficient accuracy and easy occurrence of false positives existing in the prior art.

Claims

1. A method for identifying cinnabar tobacco leaves, characterized in that: Using the compound of formula 1 and / or formula 2 as a marker for identifying cinnabar tobacco leaves; 2. The identification method of cinnabar tobacco leaves according to claim 1, characterized in that: Determine the marker in the tobacco leaves to be identified. When the tobacco leaves to be identified contain the marker, they are identified as cinnabar tobacco leaves.

3. The identification method of cinnabar tobacco leaves according to claim 2, wherein: The determination method is HPLC-MS.

4. The identification method of cinnabar tobacco leaves according to claim 3, characterized in that: In HPLC-MS, the HPLC mobile phase includes mobile phase A and mobile phase B. Among them, mobile phase A is water and mobile phase B is acetonitrile; The elution gradient is:

5. The identification method of cinnabar tobacco leaves according to claim 4, characterized in that: The HPLC column is: Alphasil XDAmide produced by ChromCore (Beijing) Technology Co., Ltd., 5μm, 4.6×250mm.

6. The identification method of cinnabar tobacco leaves according to claim 4, characterized in that: The detection wavelength of HPLC is 255 - 265nm.

7. The identification method of cinnabar tobacco leaves according to any one of claims 3 to 6, characterized in that: The tobacco leaves to be identified are pre-extracted with a solvent and then subjected to the above HPLC-MS determination.

8. The identification method of cinnabar tobacco leaves according to claim 7, characterized in that: The solvent is alcohol and / or water; the alcohol is C1 - C4 alcohol.

9. The identification method of cinnabar tobacco leaves according to claim 8, characterized in that: The solvent is a mixed solvent of alcohol and water.

10. The identification method of cinnabar tobacco leaves according to claim 9, characterized in that: In the solvent, the volume ratio of alcohol to water is 75 - 85:15 - 25.

11. The method for identifying cinnabar tobacco leaves according to claim 8, characterized in that: In the solvent extraction stage, the liquid-solid ratio is 10 - 50 mL / g.

12. The identification method of cinnabar tobacco leaves according to claim 7, characterized in that: The solvent extraction stage is carried out under ultrasonic assistance.

13. The identification method of cinnabar tobacco leaves according to claim 7, characterized in that: The extraction time of the solution is more than 10 minutes, preferably 30 - 120 minutes.

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