A method for extracting cephalosporin diterpenes from tobacco leaves

Abstract

This invention provides a method for extracting cephalothin diterpenes from tobacco leaves, comprising the following steps: extracting tobacco leaf raw materials and concentrating the extract to obtain an extract; decolorizing the extract using activated carbon and collecting the supernatant; extracting the extract using petroleum ether to obtain crude cephalothin diterpenes; eluting the crude cephalothin diterpenes with a first eluent until no fraction eluent is obtained, then eluting with a second eluent, collecting the eluent from the second eluent to obtain a crude extract; separating the crude extract with a third eluent to obtain separated β-cephalothin-4,6-diol and α-cephalothin-4,6-diol. This invention achieves precise separation of α-cephalothin-4,6-diol and β-cephalothin-4,6-diol, with a high extraction rate, simple operation, and low extraction cost.

Description

Technical Field

[0001] This invention belongs to the field of tobacco extraction technology, and specifically relates to a method for extracting cephalosporin diterpenes from tobacco leaves. Background Technology

[0002] Siperanes are a novel, unique class of natural products with important biological activities. They belong to the monocyclic diterpenoid class of compounds and are major components of glandular trichome secretions in tobacco leaves. These substances exist in tobacco as a tasteless surface wax, but they degrade during tobacco curing, aging, and processing, producing a series of aromatic degradation products. These degradation products have a significant impact on the aroma and taste of cigarettes and are important aroma precursors in tobacco.

[0003] Cembratriene-4,6-diols (CBT-diol) are a type of cembratriene diterpenoid compound with two isomers: α-cembriatriene-4,6-diol and β-cembriatriene-4,6-diol. They are abundant in tobacco gland secretions, accounting for approximately 60% of the total secretion content of tobacco leaves. Among the degradation products of cembratriene-4,6-diol, solanone, norsolanone, and solanifuran are considered the main sources of tobacco aroma. Studies have shown that adding cembratriene-4,6-diol to cigarettes can significantly enhance the aroma and taste of tobacco. Furthermore, cembratriene-4,6-diol has broad application prospects in anti-tumor and nervous system protection, and can be used in the development of drugs for Parkinson's disease, Alzheimer's disease, and anti-tumor purposes.

[0004] Patent application CN201510382060.9 discloses a method for extracting cephalothin diterpenes from tobacco inflorescences, comprising the following steps: 1. Organic solvent extraction and concentration to obtain a crude extract of glandular trichome secretions from the surface of tobacco flowers; 2. Removing higher alkanes from the crude extract using solvent precipitation and removing pigments with activated carbon to obtain a tobacco diterpenes extract. The above method can obtain β-4,8,13-cephalothin-1,3-diol and α-4,8,13-cephalothin-1,3-diol from tobacco inflorescence raw materials, but the separation and extraction of cephalothin-4,6-diol remains a gap. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a method for extracting cephalotriene diterpenoids from tobacco leaves. This method can specifically extract and separate cephalotriene-4,6-diol from tobacco leaves, which is beneficial for downstream industry applications.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A method for extracting cephalosporin diterpenes from tobacco leaves includes the following steps:

[0008] (1) Extract the tobacco raw material with an organic extractant to obtain an extract containing secretions from the glandular trichomes on the surface of the tobacco leaves. After drying and concentration under reduced pressure, an extract containing secretions from the glandular trichomes on the surface of the tobacco leaves is obtained.

[0009] (2) Dissolve the extract in an ethanol-water solution, add activated carbon for decolorization, and collect the supernatant;

[0010] (3) Concentrate the supernatant under reduced pressure to 20-30% volume, then add water to it, and then extract it with petroleum ether. Concentrate the extracted petroleum ether phase under reduced pressure to obtain crude cephalosporin diterpenes.

[0011] (4) Take the crude cephalosporin diterpenes and dry-load it onto the first silica gel column for column chromatography. The silica gel column used is filled with 200-300 mesh silica gel. The mass ratio of the amount of silica gel to the crude cephalosporin diterpenes is (25-35):1. After loading, elute with n-hexane / methyl tert-butyl ether at a volume ratio of (7.8-8.2):1 until no fraction flows out. Then elute with n-hexane / methyl tert-butyl ether at a volume ratio of (4.8-5.2):1. Collect the eluent from the second eluent and concentrate it under reduced pressure to obtain the crude extract.

[0012] (5) Take the crude extract and dry-load it onto the second silica gel column for column chromatography. The silica gel used for the second silica gel column is 200~300 mesh. The mass ratio of silica gel to crude extract is (25~35):1. Use dichloromethane / methyl acetate with a volume ratio of (5.5~6.5):1 as the eluent to separate and elute the crude extract to obtain the separated β-cephalitol-4,6-diol and α-cephalitol-4,6-diol.

[0013] Furthermore, in step (1), the extractant is selected from one or more of dichloromethane, trichloromethane, and tetrachloromethane.

[0014] Furthermore, in step (1), the mass ratio of the extractant to the tobacco raw material is (5~8):1.

[0015] Furthermore, in step (1), the tobacco raw material is tobacco powder with a particle size of 35-45 mesh and a moisture content of ≤10%. The tobacco powder is obtained by destemming and crushing dried tobacco leaves or fresh tobacco that has been roasted. The varieties of fresh tobacco leaves include, but are not limited to, Honghua Dajinyuan, Yunyan 87 or K327, etc., and the types of dried tobacco leaves include, but are not limited to, flue-cured tobacco, Burley tobacco, aromatic tobacco, etc.

[0016] Furthermore, in step (2), the volume percentage concentration of the ethanol aqueous solution is 73-78%, and the ratio of the ethanol aqueous solution to the extract is 50-60 mL: 10 g.

[0017] Furthermore, in step (2), the activated carbon is 200 mesh, and the mass ratio of activated carbon to extract is 1:(8~12).

[0018] Further, in step (2), after decolorization, the solution is centrifuged to obtain supernatant and precipitate. The precipitate is washed 3 to 5 times with 75% ethanol aqueous solution, and the effluent obtained from washing is combined with the supernatant.

[0019] Furthermore, in step (3), the amount of water injected is 25-35% of the volume of the supernatant after concentration.

[0020] Furthermore, in step (4), the elution rate of n-hexane / methyl tert-butyl ether is 1.4~1.6 mL / min.

[0021] Further, in step (5), the elution rate is controlled at 0.9~1.1 mL / min to obtain the separated β-cephalitol-4,6-diol and α-cephalitol; or the elution rates are controlled at 1.5 mL / min and 2 mL / min respectively to obtain β-cephalitol-4,6-diol and α-cephalitol respectively.

[0022] The following beneficial effects can be achieved by applying this invention:

[0023] 1. The method of this invention employs simultaneous distillation extraction and activated carbon adsorption to selectively remove higher alkanes and pigment impurities from the surface extract. Subsequently, silica gel column chromatography is used to achieve efficient purification and separation of tobacco-characteristic cephalosporin diterpenoids. This method overcomes the dependence of traditional processes on complex operating procedures and expensive equipment (such as preparative chromatography systems). Through a three-step integrated process, it achieves precise separation of the target product, significantly simplifying the process and improving separation efficiency. Compared to existing technologies, this extraction method features a higher extraction rate, simpler operation, and lower equipment costs.

[0024] 2. The recovery rate of cephalotriene diterpenes obtained by the method of this invention is approximately 25.7% relative to the surface secretions of tobacco leaves, and 1.88% relative to the tobacco leaves (dry weight). The recoveries of α-cephalotrien-4,6-diol and β-cephalotrien-4,6-diol in the cephalotriene diterpenes relative to the tobacco leaves (dry weight) are 1.46% and 0.42%, respectively, with purities exceeding 99%, which is beneficial for downstream industry applications. Attached Figure Description

[0025] Figure 1 The liquid chromatogram of α-cephalitol-4,6-diol;

[0026] Figure 2The liquid chromatogram is for β-cephalitol-4,6-diol. Detailed Implementation

[0027] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0028] Unless otherwise specified below, all raw materials and reagents used in the various embodiments of this invention are commercially available:

[0029]

[0030] The instruments used for testing in the various embodiments of the present invention:

[0031] Nuclear magnetic resonance spectrometer, AVANCENEO600, Bruker GmbH, Germany;

[0032] Liquid chromatography-mass spectrometry, model Agilent-1290InfinityII+Agilent-6460C, Agilent Technologies;

[0033] During the detection process of the liquid chromatography-mass spectrometry (LC-MS / MS): LC conditions: DB-5 capillary column (60m×250μm×0.25μm); vaporization chamber temperature 280℃; carrier gas: high-purity helium (99.999%); flow rate 1.0mL / min; injection volume 1.0µL; injection port temperature 180℃; split ratio: 6:1; column temperature 120℃ held for 2 min, then increased to 180℃ at 5℃ / min, then increased to 240℃ at 2℃ / min, held for 5 min, and finally increased to 280℃ at 5℃ / min, held for 20 min before termination.

[0034] Mass spectrometry conditions: transfer line temperature 280℃, electron multiplier voltage 1635V, EI source electron energy 70eV, mass scan range 30-550amu, ion source temperature 230℃, quadrupole temperature 150℃.

[0035] The method for determining the moisture content of tobacco powder used in each implementation shall be in accordance with YC / T31-1996 Preparation and Moisture Determination of Tobacco and Tobacco Products Samples - Oven Method;

[0036] The α-cipertriene-4,6-diol / β-cipertriene-4,6-diol standards used in the testing of the various embodiments of the present invention were obtained from Shanghai Huicheng Biotechnology Co., Ltd., with a specification of 99%.

[0037] Example 1

[0038] This embodiment provides a method for extracting cephalosporin diterpenes from tobacco leaves, specifically including the following steps:

[0039] S1. Place 137g of tobacco leaf powder with a particle size of 40 mesh into a distillation flask (1000mL, Mingke Experimental Instrument Co., Ltd.) of a simultaneous distillation and extraction apparatus, add distilled water with a pH of 7.0 (liquid-to-solid ratio 5-8:1), add dichloromethane (liquid-to-solid mass ratio 7:1) as the extractant, and extract for 3 hours to obtain an extract containing secretions from the glandular trichomes on the surface of the tobacco leaves; the tobacco leaf powder is made from safflower Dajinyuan flue-cured tobacco (from Shanghai Tobacco Group) after initial curing, and the tobacco leaves are pulverized to a particle size of 40 mesh after removing the stems, and the moisture content is tested to be 10%.

[0040] S2. The extract containing secretions from the glandular trichomes on the surface of tobacco leaves was dried with anhydrous sodium sulfate, and then filtered through a 0.22 μm organotin filter membrane to remove hydrated sodium sulfate. The filtrate was concentrated under reduced pressure at 40-50 °C and controlled at -0.01 MPa to obtain 10 g of extract containing secretions from the glandular trichomes on the surface of tobacco leaves.

[0041] S3. Take 10g of extract containing secretions from the glandular trichomes on the surface of tobacco leaves, add 60mL of 75v / v% ethanol solution, shake thoroughly to dissolve, add 1g of activated carbon for decolorization, then centrifuge and take the supernatant; the activated carbon used is 200 mesh; the precipitate obtained by centrifugation is washed 4 times with 60mL of 75v / v% ethanol, and the washing effluent is combined with the supernatant above.

[0042] S4. The combined supernatant was concentrated under reduced pressure (50℃, -0.01MPa) to 100mL. 33mL of deionized water (volume ratio 3:1) was added to the concentrate, and after mixing, it was extracted 4 times with 100mL of petroleum ether (1:1 volume each time). The petroleum ether phases were combined, and the concentration was carried out under reduced pressure at a controlled temperature of 55℃ and a pressure of -0.01MPa to obtain 6.7g of crude cephalotriene diterpenes. At this time, the content of cephalotriene-4,6-diol was found to be above 90%.

[0043] S5. Take 3g of the crude cephalosporin diterpenoid from step S4 and dry-load it onto a first silica gel column (200-300 mesh) (6cm×61cm). After loading, first elute with n-hexane / methyl tert-butyl ether (8:1) at a flow rate of 1.5mL / min until no fraction flows out. Then elute a second time with n-hexane / methyl tert-butyl ether (5:1) at a flow rate of 1.5mL / min until no fraction flows out. Collect the eluent from the second eluent, combine the two eluents, and concentrate under reduced pressure (temperature controlled at 45℃, pressure controlled at -0.01MPa) to obtain 1.8g of crude extract.

[0044] S6. Take 300 mg of the crude extract from step S5 and dry-load it onto the second silica gel column (200-300 mesh, 6 cm × 61 cm). After loading, elute with dichloromethane / methyl acetate at a volume ratio of 6:1. Control the elution rate at 1 mL / min during elution. Collect one tube of effluent every 10 mL and distinguish the different fractions by TLC plate, which are recorded as effluent A and effluent B, respectively.

[0045] The above-obtained effluents A and B were first concentrated under reduced pressure to remove most of the solvent at a temperature of 30°C and a vacuum degree of less than -0.01 MPa until a small amount (about 5-10 mL) of viscous liquid or oily / solid mixture remained at the bottom of the bottle. Then, the residual solvent was completely removed by vacuum drying at a temperature of 25°C or lower and a vacuum degree of less than -0.01 MPa. 1,998.97 mg (corresponding to 300 mg of extract in step (4)) of substance A corresponding to effluent A and 571.33 mg of substance B corresponding to effluent B were obtained. The final amount of substance A (1,998.97 mg) and substance B (571.33 mg) of the 10 g extract containing glandular secretions from the surface of tobacco leaves (hereinafter referred to as extract) obtained in step (2) was calculated to be 1,998.97 mg and 571.33 mg, respectively.

[0046] Table 1 shows the proton and carbon NMR spectra of substances A and B as described above. Figure 1 and Figure 2 .

[0047] Table 1. NMR detection data of substances A and B

[0048]

[0049] Based on the comprehensive analysis of the above-mentioned NMR hydrogen and carbon spectrum data and liquid chromatograms, it can be concluded that substance A is α-cephalitol-4,6-diol with CAS number 57605-80-8 and substance B is β-cephalitol-4,6-diol with CAS number 57605-81-9.

[0050] The α-cipertrien-4,6-diol and β-cipertrien-4,6-diol obtained above were analyzed by liquid chromatography-mass spectrometry, and their purities were both higher than 99%. Compared with the extract containing surface gland secretions (10g extract yielded 1,998.97mg of substance A and 571.33mg of substance B, with an α-cipertrien-4,6-diol:β-cipertrien-4,6-diol ratio of 3.50:1), the recoveries of α-cipertrien-4,6-diol and β-cipertrien-4,6-diol were 19.99% and 5.71%, respectively.

[0051] The tobacco leaves (from the Honghua Dajinyuan flue-cured tobacco variety) used to prepare 10g of extract weighed 137g (dry weight). The yield of the extract relative to the dry weight of the tobacco leaves was 10 / 137*100%=7.3%. The recoveries of α-cephalitol-4,6-diol and β-cephalitol relative to the dry weight of the tobacco leaves (recovery rate = mass of cephalitol diterpenes (α-cephalitol-4,6-diol and β-cephalitol) / mass of tobacco leaves (dry weight) × 100%) were 1.46% and 0.42%, respectively.

[0052] The above results show that the extraction method of this application is efficient enough to extract and separate α-cephalitol-4,6-diol and β-cephalitol-4,6-diol.

[0053] Example 2

[0054] This embodiment provides a method for extracting cephalosporin diterpenes from tobacco leaves, specifically including the following steps:

[0055] S1. Place 137g of tobacco leaf powder with a particle size of 40 mesh into a distillation flask of a simultaneous distillation and extraction apparatus (specification 1000mL, Mingke Experimental Instrument Co., Ltd.), add distilled water with a pH of 7.0 (liquid-to-solid ratio 5-8:1), add dichloromethane (liquid-to-solid mass ratio 7:1) as the extractant, and extract for 3 hours to obtain an extract containing secretions from the glandular trichomes on the surface of the tobacco leaves; the tobacco leaf powder is selected from fresh tobacco leaves of the 'Honghua Dajinyuan' variety, which are cut into pieces with a particle size of 5-10mm (the moisture content is tested to be 70%-80%).

[0056] S2. The extract containing secretions from the glandular trichomes on the surface of tobacco leaves was dried with anhydrous sodium sulfate, and then filtered through a 0.22μm organotin filter membrane to remove hydrated sodium sulfate. The filtrate was concentrated under reduced pressure at 40-50℃ and controlled at -0.01MPa to obtain 3.7g of extract containing secretions from the glandular trichomes on the surface of tobacco leaves.

[0057] S3. Take 3.7g of extract containing secretions from the glandular trichomes on the surface of tobacco leaves, add 25mL of 75v / v% ethanol solution, shake thoroughly to dissolve, and add 0.4g of activated carbon (200 mesh) for decolorization treatment. Then centrifuge and take the supernatant. The precipitate obtained by centrifugation is washed 4 times with 25mL of 75v / v% ethanol. The washing effluent and the supernatant are combined.

[0058] S4. The combined supernatant was concentrated under reduced pressure (50℃, -0.01MPa) to approximately 37mL (approximately 25-30% of the original volume). Approximately 12mL of deionized water (approximately 30-35% of the concentrated volume) was added to the concentrate, and after mixing, it was extracted four times with approximately 40mL of petroleum ether (each time the volume ratio of petroleum ether to concentrate was approximately 1:1). The petroleum ether phases were combined, and the concentrate was concentrated under reduced pressure at 55℃ and -0.01MPa to obtain 2.4g of crude cephalotriene diterpenoid. The content of cephalotriene-4,6-diol was found to be above 90%.

[0059] S5. Take 2.0 g of the crude cephalosporin diterpenoid from step S4 and dry-load it onto the first silica gel column (200-300 mesh, 6 cm × 61 cm). After loading, first elute with n-hexane / methyl tert-butyl ether (8:1) at a flow rate of 1.5 mL / min until no fraction flows out. Then elute with n-hexane / methyl tert-butyl ether (5:1) at a flow rate of 1.5 mL / min until no fraction flows out. Collect the eluent from the second eluent, combine the two eluents, and concentrate under reduced pressure (temperature controlled at 45℃, pressure controlled at -0.01 MPa) to obtain 1.2 g of crude extract.

[0060] S6, Same as in Example 1.

[0061] In this embodiment, the recovery rates of the obtained α-cephalitol-4,6-diol and β-cephalitol relative to the extract containing surface glandular secretions of tobacco leaves were 19.8% and 4.83%, respectively, and the purity of both was higher than 99%.

[0062] Based on the results of Examples 1 and 2, when using the method of the present invention to extract cephalosporin diterpenes, the recovery rate of pre-cured tobacco leaves is higher than that of fresh tobacco leaves. Therefore, in practical applications, curing fresh tobacco leaves before extraction can yield higher economic benefits.

[0063] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for extracting cephalosporin diterpenes from tobacco leaves, characterized in that, Includes the following steps: (1) Extract the tobacco raw material with an organic extractant to obtain an extract containing secretions from the glandular trichomes on the surface of the tobacco leaves. After drying and concentration under reduced pressure, an extract containing secretions from the glandular trichomes on the surface of the tobacco leaves is obtained. (2) Dissolve the extract in an ethanol-water solution, add activated carbon for decolorization, and collect the supernatant; (3) Concentrate the supernatant under reduced pressure to 20-30% volume, then add water to it, and then extract it with petroleum ether. Concentrate the extracted petroleum ether phase under reduced pressure to obtain crude cephalosporin diterpenes. (4) Take the crude cephalosporin diterpenes and dry-load it onto the first silica gel column for column chromatography. The silica gel column used is filled with 200-300 mesh silica gel. The mass ratio of the amount of silica gel to the crude cephalosporin diterpenes is (25-35):

1. After loading, elute with n-hexane / methyl tert-butyl ether at a volume ratio of (7.8-8.2):1 until no fraction flows out. Then elute with n-hexane / methyl tert-butyl ether at a volume ratio of (4.8-5.2):

1. Collect the eluent from the second eluent and concentrate it under reduced pressure to obtain the crude extract. (5) Take the crude extract and dry-load it onto the second silica gel column for column chromatography. The silica gel used for the second silica gel column is 200~300 mesh. The mass ratio of silica gel to crude extract is (25~35):

1. Use dichloromethane / methyl acetate with a volume ratio of (5.5~6.5):1 as the eluent to separate and elute the crude extract to obtain the separated β-cephalitol-4,6-diol and α-cephalitol-4,6-diol.

2. The method for extracting cephalosporin diterpenes from tobacco leaves according to claim 1, characterized in that, In step (1), the extractant is selected from one or more of dichloromethane, trichloromethane, and tetrachloromethane; the mass ratio of the extractant to the tobacco raw material is (5~8):

1.

3. The method for extracting cephalosporin diterpenes from tobacco leaves according to claim 1, characterized in that, In step (2), the volume percentage concentration of the ethanol aqueous solution is 73-78%, and the ratio of the ethanol aqueous solution to the extract is 50-60 mL: 10 g.

4. The method for extracting cephalosporin diterpenes from tobacco leaves according to claim 1, characterized in that, In step (2), the activated carbon is 200 mesh and the mass ratio of activated carbon to extract is 1:(8~12).

5. The method for extracting cephalosporin diterpenes from tobacco leaves according to claim 1, characterized in that, In step (2), after decolorization, the solution is centrifuged to obtain supernatant and precipitate. The precipitate is washed 3 to 5 times with 75% ethanol aqueous solution. The effluent obtained from washing is combined with the supernatant.

6. The method for extracting cephalosporin diterpenes from tobacco leaves according to claim 1, characterized in that, In step (3), the water injection volume is 25-35% of the volume of the concentrated supernatant.

7. The method for extracting cephalosporin diterpenes from tobacco leaves according to claim 1, characterized in that, In step (4), the elution rate of n-hexane / methyl tert-butyl ether is 1.4~1.6 mL / min and the elution rate of n-hexane / methyl tert-butyl ether is 1.4~1.6 mL / min.

8. The method for extracting cephalosporin diterpenes from tobacco leaves according to claim 1, characterized in that, In step (5), the elution rate is controlled at 0.9~1.1 mL / min to obtain the separated β-cephalitol-4,6-diol and α-cephalitol; or the elution rates are controlled at 1.5 mL / min and 2 mL / min to obtain β-cephalitol-4,6-diol and α-cephalitol, respectively.

9. The method for extracting cephalosporin diterpenes from tobacco leaves according to claim 1, characterized in that, The raw material for tobacco leaves is tobacco powder with a particle size of 35-45 mesh and a moisture content of ≤10%.

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