Method for extracting and detecting polyphenol substances in tobacco leaves

Abstract

The invention relates to a method for extracting and detecting polyphenol substances in tobacco leaves, which comprises the following steps: mixing the tobacco leaves and a solvent, carrying out microwave treatment, carrying out solid-liquid separation to obtain an extracting solution of the polyphenol substances in the tobacco leaves, and detecting the extracting solution by liquid chromatography to obtain the content of the polyphenol substances in the tobacco leaves. The method is simple, convenient and easy to use, has a remarkable time advantage while ensuring the extraction efficiency, can realize rapid and accurate analysis of the polyphenol substances in the tobacco leaves, and has a very good application prospect in detection and analysis of the polyphenol substances in the tobacco leaves.

Description

Technical Field

[0001] The present invention relates to the technical field of tobacco leaf extraction and detection, and particularly relates to a method for extracting and detecting polyphenolic substances in tobacco leaves. Background Art

[0002] Polyphenolic compounds in tobacco not only play an important role in the growth and development process of tobacco, but also are important components affecting the quality of tobacco, having a great influence on tobacco aroma, color, safety, etc. Polyphenolic compounds in tobacco mainly exist in the forms of glucosides and esters, including tannins (such as chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, etc.) and flavonoids (such as rutin). The polyphenol content is related to the color of tobacco leaves after baking and the smoking taste of cigarettes. During the burning and smoking process, polyphenols and their pyrolysis products transfer into the smoke, directly affecting the smoke flavor, and are positively correlated with the grade of tobacco products. The higher the content of polyphenolic substances in cigarettes, the higher the grade, and vice versa. In the tobacco industrial production, it is of great significance to quickly and accurately extract and detect polyphenolic substances (such as chlorogenic acid, rutin, etc.) in tobacco leaves for optimizing the production process and realizing dynamic quality monitoring.

[0003] Currently, the determination of the content of polyphenolic substances in tobacco leaves is carried out based on the tobacco industry standard YC / T 202-2006 "Tobacco and Tobacco Products - Determination of Chlorogenic Acid, Scopoletin and Rutin in Polyphenolic Compounds". In this determination method, the extraction of polyphenolic substances is based on ultrasonic-assisted extraction method, which has problems such as long time consumption (usually dozens of minutes to several hours), large solvent consumption, and low extraction rate of target substances, and it is difficult to meet the requirements of high-throughput and real-time analysis in industrial production.

[0004] Therefore, there is still a need to develop a simpler and faster extraction method to determine the content of polyphenolic substances in tobacco leaves. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a method for extracting and detecting polyphenolic substances in tobacco leaves. This method is simple and easy to use. While ensuring the extraction efficiency, it also has a significant time advantage, can achieve rapid and accurate analysis of polyphenolic substances in tobacco leaves, and has good application prospects in the detection and analysis of polyphenolic substances in tobacco leaves.

[0006] To achieve this purpose, the present invention adopts the following technical solutions:

[0007] The present invention provides a method for extracting and detecting polyphenolic substances in tobacco leaves, the method comprising: mixing tobacco leaves and a solvent and performing microwave treatment, and after solid-liquid separation, obtaining an extraction solution of polyphenolic substances in tobacco leaves, and detecting the extraction solution by liquid chromatography to obtain the content of polyphenolic substances in tobacco leaves.

[0008] The present invention uses microwave assistance to extract polyphenolic substances from tobacco leaves, effectively shortening the extraction time and improving the extraction efficiency. At the same time, high-performance liquid chromatography is used to achieve the accurate detection of polyphenolic substances. The method provided by the present invention can further accelerate the extraction and analysis of polyphenolic substances from tobacco leaves, and establish a method suitable for rapid analysis and quantification of polyphenolic substances in tobacco leaves during the production process of cigarette products, providing reliable technical support for the quality control of the tobacco industry.

[0009] Preferably, the solvent comprises a combination of an organic solvent and water.

[0010] Preferably, the volume concentration of the organic solvent in the solvent is 40-80% (for example, it can be 40%, 50%, 60%, 70%, 80%, etc.).

[0011] In the prior art, pure organic solvents are used for extraction, which generally have low boiling points and certain toxicity. Pure organic solvents are prone to generate organic vapors under microwave radiation, presenting certain safety hazards. Based on this, the present invention has developed a compound extraction system of an organic solvent and water. As a green solvent, water can effectively reduce the proportion of the organic solvent. By regulating the solvent polarity, the selective dissolution ability of polyphenolic substances can be improved, while reducing volatility and toxicity, which is beneficial to enhancing the process safety.

[0012] Preferably, the organic solvent comprises any one or a combination of at least two of methanol, acetonitrile, tetrahydrofuran, 1,4-dioxane, dimethyl sulfoxide or N,N-dimethylformamide, preferably a combination of methanol and acetonitrile.

[0013] Preferably, the volume ratio of methanol to acetonitrile is 1:(0.5-2) (for example, it can be 1:0.5, 1:1, 1:1.5, 1:2, etc.).

[0014] In the present invention, when a compound solvent of methanol and acetonitrile is selected, the two solvent molecules interact with the polyphenolic substances in the tobacco leaves, having a synergistic effect on promoting the extraction of active substances, which is beneficial to improving the efficiency of microwave extraction.

[0015] Preferably, the tobacco leaves are subjected to a pulverization treatment before being mixed with the solvent.

[0016] Preferably, the dosage ratio of the tobacco leaves to the solvent is 100mg:(15-25)mL (for example, it can be 100mg:15mL, 100mg:16mL, 100mg:18mL, 100mg:20mL, 100mg:22mL, 100mg:24mL, 100mg:25mL, etc.).

[0017] Preferably, the power of the microwave treatment is 80 - 800 W (such as 80 W, 100 W, 200 W, 300 W, 400 W, 500 W, 600 W, 700 W, 800 W, etc.), and the time is 5 - 20 s (such as 5 s, 8 s, 10 s, 12 s, 14 s, 16 s, 18 s, 20 s, etc.).

[0018] By optimizing the power and time of microwave extraction, the present invention can further improve the efficiency of microwave extraction.

[0019] Preferably, the method for solid-liquid separation includes filtration.

[0020] Preferably, the pore size of the filter membrane used for filtration is 0.2 - 0.5 μm (such as 0.2 μm, 0.22 μm, 0.4 μm, 0.45 μm, 0.5 μm, etc.).

[0021] Preferably, the mobile phase used in the liquid chromatography includes phase A and phase B; phase A is an aqueous solution of formic acid with a volume concentration of 0.25 - 0.35% (such as 0.25%, 0.28%, 0.3%, 0.32%, 0.35%, etc.), and phase B is acetonitrile.

[0022] Preferably, the liquid chromatography adopts gradient elution, and the program of the gradient elution is as follows:

[0023] At the 0th minute, the volume ratio of phase A is 10 - 12% (e.g., it can be 10%, 10.5%, 11%, 11.5%, 12%, etc.), and the volume ratio of phase B is 88 - 90% (e.g., it can be 88%, 88.5%, 89%, 89.5%, 90%, etc.); then it uniformly changes to the 10th minute, the volume ratio of phase A is 13 - 14% (e.g., it can be 13%, 13.2%, 13.5%, 13.8%, 14%, etc.), and the volume ratio of phase B is 86 - 87% (e.g., it can be 86%, 86.2%, 86.5%, 86.8%, 87%, etc.); then it uniformly changes to the 20th minute, the volume ratio of phase A is 25 - 30% (e.g., it can be 25%, 26%, 27%, 28%, 29%, 30%, etc.), and the volume ratio of phase B is 70 - 75% (e.g., it can be 70%, 71%, 72%, 73%, 74%, 75%, etc.); then it uniformly changes to the 30th minute, the volume ratio of phase A is 70 - 80% (e.g., it can be 70%, 72%, 74%, 76%, 78%, 80%, etc.), and the volume ratio of phase B is 20 - 30% (e.g., it can be 20%, 22%, 24%, 26%, 28%, 30%, etc.); then it uniformly changes to the 35th minute, the volume ratio of phase A is 92 - 96% (e.g., it can be 92%, 93%, 94%, 95%, 96%, etc.), and the volume ratio of phase B is 4 - 8% (e.g., it can be 4%, 5%, 6%, 7%, 8%, etc.); then it uniformly changes to the 40th minute, the volume ratio of phase A is 10 - 12% (e.g., it can be 10%, 10.5%, 11%, 11.5%, 12%, etc.), and the volume ratio of phase B is 88 - 90% (e.g., it can be 88%, 88.5%, 89%, 89.5%, 90%, etc.).

[0024] In the present invention, when the above - mentioned specific elution procedure is adopted, the separation effect of chromatography is better, which is conducive to improving the precision and accuracy of detection.

[0025] Preferably, the stationary phase of the chromatographic column of the liquid chromatography is octadecylsilane.

[0026] Preferably, the column temperature of the liquid chromatography is 25 - 35 °C (e.g., it can be 25 °C, 28 °C, 30 °C, 32 °C, 34 °C, 35 °C, etc.).

[0027] Preferably, the flow rate of the liquid chromatography is 0.8 - 1.2 min / mL (e.g., it can be 0.8 min / mL, 0.9 min / mL, 1 min / mL, 1.1 min / mL, 1.2 min / mL, etc.).

[0028] Preferably, the detection wavelength in the liquid chromatography is 322 - 326 nm (for example, it can be 322 nm, 323 nm, 324 nm, 325 nm, 326 nm, etc.), and preferably 325 nm.

[0029] Compared with the prior art, the present invention has at least the following beneficial effects:

[0030] The present invention develops a method for extracting and detecting polyphenols in tobacco leaves. By using microwave-assisted extraction and combining with liquid chromatography, accurate detection of the content is achieved. This method is simple and easy to use. While ensuring the extraction efficiency, it also has significant time advantages and can achieve rapid and accurate analysis of polyphenols in tobacco leaves, having good application prospects in the detection and analysis of polyphenols in tobacco leaves. Detailed implementation manners

[0031] To facilitate the understanding of the present invention, the following examples are listed. Those skilled in the art should understand that the examples are only for helping to understand the present invention and should not be regarded as specific limitations on the present invention.

[0032] Example 1

[0033] This example provides a method for extracting and detecting polyphenols in tobacco leaves. The method includes: mixing 100 mg of tobacco leaf powder with 20 mL of solvent, performing microwave treatment at 800 W for 15 s, filtering with a 0.45 μm aqueous filter membrane after cooling to obtain an extract of polyphenols in tobacco leaves, and detecting the extract by liquid chromatography to obtain the content of polyphenols in tobacco leaves;

[0034] Among them, the solvent is a mixed solution of methanol, acetonitrile and water, the volume concentration of methanol is 25%, and the volume concentration of acetonitrile is 25%;

[0035] The conditions of the liquid chromatography are as follows:

[0036] Chromatographic column: Copsil C18 column (250 mm × 4.6 mm);

[0037] Mobile phase: Phase A is 0.3% (v / v) formic acid aqueous solution, and Phase B is anhydrous acetonitrile;

[0038] The gradient elution program is (calculated based on the sum of the volumes of Phase A and Phase B being 100%):

[0039] 0 - 10 min, the volume ratio of Phase A changes uniformly from 11% to 13%, 10 - 20 min, the volume ratio of Phase A changes uniformly from 13% to 28%, 20 - 30 min, the volume ratio of Phase A changes uniformly from 28% to 75%, 30 - 35 min, the volume ratio of Phase A changes uniformly from 75% to 95%, 35 - 40 min, the volume ratio of Phase A changes uniformly from 95% to 11%;

[0040] Column temperature: 30 °C;

[0041] Flow rate: 1.0 min / mL;

[0042] Sample injection volume: 10 μL;

[0043] Detection wavelength: 325 nm.

[0044] Example 2

[0045] This example provides a method for extracting and detecting polyphenolic substances in tobacco leaves. The method includes: mixing 100 mg of tobacco leaf powder with 15 mL of a solvent, subjecting it to microwave treatment at 240 W for 20 s, filtering with a 0.45 μm aqueous filter membrane after cooling to obtain an extract of polyphenolic substances in tobacco leaves, and detecting the extract by liquid chromatography to obtain the content of polyphenolic substances in tobacco leaves;

[0046] Among them, the solvent is a mixed solution of methanol, acetonitrile and water, the volume concentration of methanol is 40%, and the volume concentration of acetonitrile is 20%;

[0047] The conditions of liquid chromatography are as follows:

[0048] Chromatographic column: Copsil C18 column (250 mm × 4.6 mm);

[0049] Mobile phase: Phase A is 0.35% (v / v) formic acid aqueous solution, and phase B is anhydrous acetonitrile;

[0050] The gradient elution program is (based on the sum of the volumes of phase A and phase B being 100%):

[0051] 0 - 10 min, the volume percentage of phase A changes uniformly from 10% to 13%, 10 - 20 min, the volume percentage of phase A changes uniformly from 13% to 25%, 20 - 30 min, the volume percentage of phase A changes uniformly from 25% to 70%, 30 - 35 min, the volume percentage of phase A changes uniformly from 70% to 92%, 35 - 40 min, the volume percentage of phase A changes uniformly from 92% to 10%;

[0052] Column temperature: 25 °C;

[0053] Flow rate: 1.2 min / mL;

[0054] Sample injection volume: 10 μL;

[0055] Detection wavelength: 325 nm.

[0056] Example 3

[0057] This embodiment provides a method for extracting and detecting polyphenols in tobacco leaves. The method includes: mixing 100 mg of tobacco leaf powder with 25 mL of a solvent, performing microwave treatment at 800 W for 10 s, filtering with a 0.45-μm aqueous filter membrane after cooling to obtain an extract of polyphenols in tobacco leaves, and detecting the extract by liquid chromatography to obtain the content of polyphenols in tobacco leaves;

[0058] Among them, the solvent is a mixed solution of methanol, acetonitrile, and water, with the volume concentration of methanol being 25% and the volume concentration of acetonitrile being 50%;

[0059] The conditions of the liquid chromatography are as follows:

[0060] Chromatographic column: Copsil C18 column (250 mm × 4.6 mm);

[0061] Mobile phase: Phase A is an aqueous solution of 0.25% (v / v) formic acid, and Phase B is anhydrous acetonitrile;

[0062] The gradient elution program is (calculated based on the sum of the volumes of Phase A and Phase B being 100%):

[0063] 0 - 10 min, the volume ratio of Phase A changes uniformly from 12% to 14%, 10 - 20 min, the volume ratio of Phase A changes uniformly from 14% to 30%, 20 - 30 min, the volume ratio of Phase A changes uniformly from 30% to 80%, 30 - 35 min, the volume ratio of Phase A changes uniformly from 80% to 96%, 35 - 40 min, the volume ratio of Phase A changes uniformly from 96% to 12%;

[0064] Column temperature: 35°C;

[0065] Flow rate: 0.8 min / mL;

[0066] Injection volume: 10 μL;

[0067] Detection wavelength: 325 nm.

[0068] Example 4

[0069] This embodiment provides a method for extracting and detecting polyphenols in tobacco leaves. The difference from Example 1 is only that methanol is replaced by tetrahydrofuran, and the others refer to Example 1.

[0070] Example 5

[0071] This embodiment provides a method for extracting and detecting polyphenols in tobacco leaves. The difference from Example 1 is only that methanol is replaced by N,N-dimethylformamide, and the others refer to Example 1.

[0072] Example 6

[0073] This embodiment provides a method for extracting and detecting polyphenols in tobacco leaves. The only difference from Embodiment 1 is that the solvent is a mixed solution of methanol and water, where the volume concentration of methanol is 50%, and the rest refers to Embodiment 1.

[0074] Example 7

[0075] This embodiment provides a method for extracting and detecting polyphenols in tobacco leaves. The only difference from Embodiment 1 is that the solvent is a mixed solution of acetonitrile and water, where the volume concentration of acetonitrile is 50%, and the rest refers to Embodiment 1.

[0076] Example 8

[0077] This embodiment provides a method for extracting and detecting polyphenols in tobacco leaves. The only difference from Embodiment 1 is that the microwave power is adjusted to 80 W, the processing time remains unchanged, and the rest refers to Embodiment 1.

[0078] Example 9

[0079] This embodiment provides a method for extracting and detecting polyphenols in tobacco leaves. The only difference from Embodiment 1 is that the microwave time is adjusted to 5 s, the processing power remains unchanged, and the rest refers to Embodiment 1.

[0080] Example 10

[0081] This embodiment provides a method for extracting and detecting polyphenols in tobacco leaves. The only difference from Embodiment 1 is that the gradient elution program is adjusted to: 0 - 10 min, the volume ratio of phase A changes uniformly from 89% to 87%; 10 - 20 min, the volume ratio of phase A changes uniformly from 87% to 72%; 20 - 30 min, the volume ratio of phase A changes uniformly from 72% to 25%; 30 - 35 min, the volume ratio of phase A changes uniformly from 25% to 5%; 35 - 40 min, the volume ratio of phase A changes uniformly from 5% to 89%, and the rest refers to Embodiment 1.

[0082] Comparative Example 1

[0083] This comparative example provides a method for extracting and detecting polyphenols in tobacco leaves. The only difference from Embodiment 1 is that the microwave treatment is replaced by ultrasonic treatment at a power of 80 W for 25 min.

[0084] Test Example 1

[0085] Evaluation of extraction efficiency and precision:

[0086] The tobacco leaves from Dali Re-drying Factory are divided into 1 - 20 grid areas. Taking Area 3 as the detection sample, the methods provided in Examples 1 - 10 and Comparative Example 1 are used for extraction and detection, and each sample is tested 3 times repeatedly. The results are shown in Table 1.

[0087] Table 1

[0088]

[0089]

[0090] It can be seen from the test results that:

[0091] (1) It can be seen from Examples 1 to 10 that by adopting the method of microwave-assisted extraction, the extraction efficiency of polyphenolic substances in tobacco leaves can be improved. At the same time, combined with the liquid chromatography detection method, the precise detection of polyphenolic substances in tobacco leaves is realized.

[0092] (2) It can be seen from the comparison between Example 1 and Examples 6-9 that by optimizing the extraction solvent, the power and time of microwave treatment, better microwave extraction efficiency can be achieved, and the content of the obtained polyphenolic substances is further improved.

[0093] It can be seen from the comparison between Example 1 and Example 10 that by adopting a specific gradient elution program, the separation effect of liquid phase detection can be further improved, and the precision is further improved.

[0094] (3) It can be seen from the comparison between Example 1 and Comparative Example 1 that the method of microwave-assisted extraction adopted in the present invention can significantly shorten the extraction time and improve the extraction efficiency compared with ultrasonic extraction.

[0095] Test Example 2

[0096] Accuracy evaluation:

[0097] Standard sample solutions of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, rutin, and kaempferol-3-O-rutinoside at 5 mg / mL, 15 mg / mL, 5 mg / mL, 10 mg / mL, and 5 mg / mL were prepared respectively, and were detected by the liquid chromatography methods provided in Examples 1-3 and Example 10 respectively, and the recovery rates were calculated. The results are shown in Table 2.

[0098] Table 2

[0099]

[0100] It can be seen from the test results that the detection method provided by the present invention has high accuracy. It can be seen from the comparison between Example 1 and Example 10 that adopting the specific gradient elution program of the present invention can further improve the accuracy of detection.

[0101] Test Example 3

[0102] The tobacco leaves of Dali Re-drying Factory are divided into 1 - 20 regions in a grid pattern. Taking the 1 - 20 sub-regions as samples respectively, the extraction and detection are carried out by the method provided in Example 1. Each sample is tested 3 times, and the results of the contents of each polyphenol component are shown in Table 3.

[0103] Table 3

[0104]

[0105]

[0106] The results show that the extraction and detection method provided in this application can achieve the detection of tobacco leaves with different polyphenol contents.

[0107] The applicant declares that the above description is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by any person skilled in the art within the technical scope disclosed by the present invention fall within the protection scope and the disclosure scope of the present invention.

Claims

1. A method for extracting and detecting polyphenolic substances in tobacco leaves, characterized in that, The method includes: mixing tobacco leaves and a solvent for microwave treatment, and after solid-liquid separation, obtaining an extract of polyphenolic substances in the tobacco leaves, and detecting the extract by liquid chromatography to obtain the content of polyphenolic substances in the tobacco leaves.

2. The method according to claim 1, wherein The solvent includes a combination of an organic solvent and water; Preferably, the volume concentration of the organic solvent in the solvent is 40-80%.

3. The method according to claim 1 or 2, characterized in that, The organic solvent includes any one or a combination of at least two of methanol, acetonitrile, tetrahydrofuran, 1,4-dioxane, dimethyl sulfoxide, or N,N-dimethylformamide, and preferably a combination of methanol and acetonitrile; Preferably, the volume ratio of methanol to acetonitrile is 1:(0.5-2).

4. The method according to any one of claims 1 to 3, characterized in that, The tobacco leaves are pulverized before being mixed with the solvent; Preferably, the dosage ratio of the tobacco leaves to the solvent is 100 mg:(15-25) mL.

5. The method according to any one of claims 1-4, characterized in that, The power of the microwave treatment is 80-800 W, and the time is 5-20 s.

6. The method according to any one of claims 1-5, characterized in that, The method of solid-liquid separation includes filtration; Preferably, the pore size of the filter membrane used for filtration is 0.2-0.5 μm.

7. The method according to any one of claims 1-6, characterized in that, The mobile phase used in the liquid chromatography includes a phase A and a phase B; the phase A is an aqueous solution of formic acid with a volume concentration of 0.25-0.35%, and the phase B is acetonitrile.

8. The method according to claim 7, wherein The liquid chromatography uses gradient elution, and the program of the gradient elution is as follows: At the 0th minute, the volume ratio of the phase A is 10-12%, and the volume ratio of the phase B is 88-90%; then it changes uniformly to the 10th minute, the volume ratio of the phase A is 13-14%, and the volume ratio of the phase B is 86-87%; then it changes uniformly to the 20th minute, the volume ratio of the phase A is 25-30%, and the volume ratio of the phase B is 70-75%; then it changes uniformly to the 30th minute, the volume ratio of the phase A is 70-80%, and the volume ratio of the phase B is 20-30%; then it changes uniformly to the 35th minute, the volume ratio of the phase A is 92-96%, and the volume ratio of the phase B is 4-8%; then it changes uniformly to the 40th minute, the volume ratio of the phase A is 10-12%, and the volume ratio of the phase B is 88-90%.

9. The method according to any one of claims 1-8, characterized in that, The stationary phase of the chromatographic column in the liquid chromatography is octadecylsilane; Preferably, the column temperature of the liquid chromatography is 25-35 °C.

10. The method according to any one of claims 1-9, characterized in that The flow rate of the liquid chromatography is 0.8-1.2 min / mL; Preferably, the detection wavelength in the liquid chromatography is 322-326 nm.

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