A method for extracting and separating components of field inapplicable tobacco leaves

Abstract

The application provides a component extraction and separation method for field tobacco leaves, and the method comprises the following steps: 1) killing green treatment; 2) extracting solanesol; 3) extracting total chlorogenic acid and rutin; 4) separating solanesol; and 5) separating total chlorogenic acid and rutin. The method is used for killing green pretreatment of field tobacco leaves, so that the content of each main component is improved, the purity of solanesol, rutin and total chlorogenic acid obtained in the extraction process is more than 90%, and the utilization value of the active substances is increased.

Description

Technical Field

[0001] This invention relates to the field of tobacco leaf component extraction technology, and in particular to a method for extracting and separating components from tobacco leaves that are not suitable for field use. Background Technology

[0002] my country is a major tobacco producer, consumer, and grower. To optimize the grade structure of tobacco leaves and improve the effective supply of high-quality tobacco leaves, the bottom 2-3 leaves (those with poor sunlight exposure and nutrition) and the top 1-2 leaves (those with poor leaf opening) are removed during the field production process and are unsuitable for curing. These leaves are often discarded in the fields or destroyed in centralized locations, which can easily lead to pests and diseases in surrounding tobacco fields and negatively impact soil environmental purification. Therefore, the high-value comprehensive utilization of unsuitable tobacco leaves in the field has become an urgent problem for tobacco processing enterprises and tobacco farmers to solve.

[0003] Tobacco contains a wide variety of characteristic chemical components and bioactive substances. More than 3,000 compounds have been identified to date, among which important compounds such as solanesol, rutin, chlorogenic acid, tobacco essential oil, nicotine, tobacco protein, and tobacco polysaccharides have wide applications in chemical, pharmaceutical, and agricultural fields. Solanesol is a terpene compound with antibacterial, anti-inflammatory, anticancer, and hemostatic effects. It is also an important intermediate in the synthesis of coenzyme Q10, anti-ulcer drugs, and vitamin K2. Solanesol was first extracted and isolated from tobacco in 1956, yielding approximately 0.4% of the dry tobacco weight. Rutin, also known as rutin glycoside, is a rutin glycoside of the flavonol compound quercetin. Pharmacological experiments have shown it to have broad pharmacological activities, such as anti-free radical activity, anti-lipid peroxidation, antiviral activity, and anti-acute pancreatitis effects, making it useful in the treatment of various diseases. These numerous pharmacological activities give it multiple applications in the market. Chlorogenic acid, as a bioactive substance, has strong antioxidant capacity and also has functions such as anti-HIV, anti-tumor cell, antibacterial, enhancing central nervous system excitability, choleretic, anti-teratogenic, anti-allergic and regulating the activity of cytochrome P450 ligase.

[0004] The development of functional components in tobacco has attracted widespread attention. Patent CN104086425 uses ethanol as a solvent to simultaneously extract and separate chlorogenic acid, solanesol, nicotine, and rutin. Patent CN103342628 discloses a method using alkaline ethanol followed by extraction and separation to obtain nicotine and solanesol. Patent CN114534361A discloses a method for extracting tobacco essential oil, nicotine, and extract. However, all of these methods suffer from drawbacks such as complex procedures, high solvent consumption, low product extraction rates, and low purity. Furthermore, none of these methods are specifically designed for the treatment and extraction of tobacco leaves unsuitable for field use. Summary of the Invention

[0005] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a method for extracting and separating components from tobacco leaves that are not suitable for field use, in order to solve the problems in the prior art.

[0006] To achieve the above and other related objectives, the present invention provides a method for extracting and separating components from tobacco leaves unsuitable for field use, the extraction and separation method comprising the following steps:

[0007] 1) Blanching treatment: Hot air treatment is applied to unsuitable tobacco leaves in the field;

[0008] 2) Extraction of solanesol: The tobacco leaves obtained in step 1) are mixed with an organic solvent, heated under reflux, and the reflux liquid is collected. The organic solvent in the reflux liquid is removed by concentration to obtain the first extract for extracting solanesol.

[0009] 3) Extraction of total chlorogenic acid and rutin: Remove the organic solvent from the tobacco leaves after extracting solanesol, use an alcoholic solution as the extraction solvent, heat and reflux, collect the reflux liquid, concentrate and remove the alcoholic solution from the reflux liquid to obtain the second extract for extracting total chlorogenic acid and rutin.

[0010] 4) Separation of solanesol: Dissolve the first extract obtained in step 2) in an ethanol-NaOH aqueous solution, extract and recover the extractant to obtain the extract, separate it by normal phase silica gel column, collect the eluent to obtain the solanesol component;

[0011] 5) Separation of total chlorogenic acid and rutin: The second extract obtained in step 3) is separated by a resin column. The chlorogenic acid fraction is collected with a 10-25% (v / v) alcohol solution, and the rutin fraction is collected with a 30-50% (v / v) alcohol solution.

[0012] As described above, the method for extracting and separating components from field-unsuitable tobacco leaves of the present invention has the following beneficial effects:

[0013] (1) The utilization rate of unsuitable tobacco leaves in the field is greatly improved: This method pre-treats tobacco leaves in the field by killing the green, which increases the content of major components. The extract and separation of high-value active substances such as solanine, rutin, and chlorogenic acid from tobacco leaves improves the utilization rate of tobacco resources, reduces waste, and saves production costs;

[0014] (2) Improved product purity and yield: During the extraction process, the purity of solanesol, rutin and chlorogenic acid are all above 90%, which increases the utilization value of these active substances.

[0015] (3) The extraction method is simple and easy to scale up: almost no waste is generated during the extraction process, and organic solvents can be recycled and reused, reducing energy consumption and making it green and environmentally friendly. Attached Figure Description

[0016] Figure 1 The diagram shows the process flow of the method for extracting and separating components from field-unsuitable tobacco leaves according to the present invention.

[0017] Figure 2 The image shows the HPLC chromatogram of solanesol obtained in Example 2 of this invention.

[0018] Figure 3 The image shown is an HPLC chromatogram of the total chlorogenic acid lyophilized powder obtained in Example 2 of this invention.

[0019] Figure 4 The image shown is an HPLC chromatogram of the lyophilized rutin powder obtained in Example 2 of this invention. Detailed Implementation

[0020] This invention first provides a method for extracting and separating components from tobacco leaves that are not suitable for field use, the method comprising the following steps:

[0021] 1) Blanching treatment: Hot air treatment is applied to unsuitable tobacco leaves in the field;

[0022] 2) Extraction of solanesol: The tobacco leaves obtained in step 1) are mixed with an organic solvent, heated under reflux, and the reflux liquid is collected. The organic solvent in the reflux liquid is removed by concentration to obtain the first extract for extracting solanesol.

[0023] 3) Extraction of total chlorogenic acid and rutin: Remove the organic solvent from the tobacco leaves after extracting solanesol, use an alcoholic solution as the extraction solvent, heat and reflux, collect the reflux liquid, concentrate and remove the alcoholic solution from the reflux liquid to obtain the second extract for extracting total chlorogenic acid and rutin.

[0024] 4) Separation of solanesol: Dissolve the first extract obtained in step 2) in an ethanol-NaOH aqueous solution, extract and recover the extractant to obtain the extract, separate it by normal phase silica gel column, collect the eluent to obtain the solanesol component;

[0025] 5) Separation of total chlorogenic acid and rutin: The second extract obtained in step 3) is separated by a resin column. The chlorogenic acid fraction is collected with a 10-25% (v / v) alcohol solution, and the rutin fraction is collected with a 30-50% (v / v) alcohol solution.

[0026] In some embodiments of the present invention, in step 1), the equipment for the blanching process is an oven or a blanching machine.

[0027] During the withering process, the amount of tobacco leaves laid out is controlled or they are turned over regularly to ensure even heating and avoid local overheating or overdrying. After withering, the tobacco leaves turn bright green or a slightly darker green than before withering.

[0028] In some embodiments of the present invention, in step 1), the temperature of the hot air treatment is 80–100°C. The temperature of the hot air treatment is selected from any of the following ranges: 80–85°C, 85–90°C, 90–95°C, or 95–100°C.

[0029] In some embodiments of the present invention, in step 1), the hot air treatment time is 1 to 3 hours. The hot air treatment time is selected from any of the following ranges: 1 to 1.5 hours, 1.5 to 2 hours, 2 to 2.5 hours, and 2.5 to 3 hours.

[0030] In some embodiments of the present invention, in step 1), the hot air treatment is carried out until the moisture content of the tobacco leaves is 5% to 15%, which is suitable for grinding.

[0031] In some embodiments of the present invention, step 1) further includes cooling the tobacco leaves after hot air treatment.

[0032] In some embodiments of the present invention, step 1) further includes grinding and pulverizing the tobacco leaves after hot air treatment.

[0033] In some embodiments of the present invention, in step 2), the mixing ratio of the tobacco leaves to the organic solvent is 1 kg: (10-15) L. The mass ratio of the tobacco leaves to the organic solvent is selected from any of the following ranges: 1 kg: (10-12) L, 1 kg: (12-14) L, 1 kg: (14-15) L.

[0034] In a preferred embodiment of the present invention, the organic solvent is ethyl acetate.

[0035] In some embodiments of the present invention, step 2) further includes cooling the reflux liquid and then filtering it. The filtration method is filter paper filtration, filter cartridge filtration, or membrane filtration.

[0036] In some embodiments of the present invention, in step 2), the reflux time is 1 to 3 hours.

[0037] In some embodiments of the present invention, in step 2), the number of reflows is 1 to 3.

[0038] In some embodiments of the present invention, in step 2), the reflux temperature is 55-65°C.

[0039] In some embodiments of the present invention, in step 2), the concentration is vacuum concentration, atmospheric pressure concentration or freeze concentration, preferably vacuum concentration.

[0040] In some embodiments of the present invention, in step 2), the concentration temperature is 40-45°C.

[0041] In some embodiments of the present invention, the total chlorogenic acid is the sum of various chlorogenic acid isomers found in tobacco leaves.

[0042] In some embodiments of the present invention, in step 3), the volume fraction of the alcohol solution is 30-50%. The volume fraction of the alcohol solution is selected from any of the following ranges: 30-35%, 35-40%, 40-45%, and 45-50%.

[0043] In some embodiments of the present invention, in step 3), the alcohol solution is an ethanol solution.

[0044] In some embodiments of the present invention, in step 3), the ratio of tobacco leaves to alcohol solution is 1 kg:(10-20) L. The ratio of tobacco leaves to alcohol solution is selected from any of the following ranges: 1 kg:(10-12) L, 1 kg:(12-14) L, 1 kg:(14-16) L, 1 kg:(16-18) L, 1 kg:(18-20) L.

[0045] In some embodiments of the present invention, step 3) further includes cooling the reflux liquid and then filtering it. The filtration method is filter paper filtration, filter cartridge filtration, or membrane filtration.

[0046] In some embodiments of the present invention, in step 3), the reflux time is 1 to 3 hours.

[0047] In some embodiments of the present invention, in step 3), the number of reflows is 1 to 3.

[0048] In some embodiments of the present invention, in step 3), the concentration is vacuum concentration, atmospheric pressure concentration or freeze concentration, preferably vacuum concentration.

[0049] In some embodiments of the present invention, in step 3), the concentration temperature is 50–70°C. The concentration temperature is selected from any of the following ranges: 50–55°C, 55–60°C, 60–65°C, and 65–70°C.

[0050] In some embodiments of the present invention, in step 4), the ratio of the first extract to the ethanol-NaOH aqueous solution is 1:(1-2).

[0051] In some embodiments of the present invention, in step 4), the ethanol-NaOH aqueous solution has an ethanol volume fraction of 70-90%. The ethanol volume fraction is selected from any of the following ranges: 70-75%, 75-80%, 80-85%, and 85-90%.

[0052] In some embodiments of the present invention, in step 4), the concentration of the NaOH aqueous solution is 0.2–0.4 mol / L. The concentration of the NaOH solution is selected from any of the following ranges: 0.2–0.25 mol / L, 0.25–0.3 mol / L, 0.3–0.35 mol / L, or 0.35–0.4 mol / L.

[0053] In some embodiments of the present invention, in step 4), the extractant is petroleum ether.

[0054] The volume of the petroleum ether is equal to the volume of the ethanol-NaOH aqueous solution.

[0055] In some embodiments of the present invention, in step 4), the extraction is performed 2 to 4 times.

[0056] In some embodiments of the present invention, step 4) further includes combining the extractant.

[0057] In some embodiments of the present invention, in step 4), the method of recovering the extractant is distillation concentration.

[0058] In some embodiments of the present invention, in step 4), the temperature of the recovered extractant is 20–40°C. The temperature of the recovered extractant is selected from any of the following ranges: 20–25°C, 25–30°C, 30–35°C, or 35–40°C.

[0059] In some embodiments of the present invention, in step 4), the sample loading method of the normal phase silica gel column is dry loading.

[0060] In some embodiments of the present invention, in step 4), the volume ratio of the normal-phase silica column to the volume of the extract is (5-7):1.

[0061] In some embodiments of the present invention, step 4) further includes eluting with petroleum ether until a colored stream flows out before separation.

[0062] In some embodiments of the present invention, in step 4), the mobile phase for separation is petroleum ether:ethyl acetate = (20-40):1 (v / v). The mobile phase for separation is selected from any of the following ratios: petroleum ether:ethyl acetate = (20-25):1, petroleum ether:ethyl acetate = (25-30):1, petroleum ether:ethyl acetate = (30-35):1, petroleum ether:ethyl acetate = (35-40):1.

[0063] In some embodiments of the present invention, in step 4), isocratic elution is used during separation.

[0064] In some embodiments of the present invention, in step 4), one fraction is collected for each column volume during separation.

[0065] In some embodiments of the present invention, in step 4), the flow rate during separation is 2 to 5 BV / h.

[0066] In some embodiments of the present invention, in step 4), the target compound is detected by TLC during separation.

[0067] In some embodiments of the present invention, in step 4), the eluent is an eluent with an HPLC purity greater than 90%.

[0068] In some embodiments of the present invention, step 4) further includes post-processing the solanesol component to obtain solanesol solid.

[0069] The post-processing involves concentrating the solanesol component obtained in step 3).

[0070] In some embodiments of the present invention, the concentration is reduced pressure concentration.

[0071] In some embodiments of the present invention, in step 5), the volume ratio of the second extract to the resin column is 1 g to (1:3) mL.

[0072] In some embodiments of the present invention, in step 5), the loading rate of the second extract is 0.3 to 0.7 BV / h. The loading rate of the second extract is selected from any of the following ranges: 0.3 to 0.4 BV / h, 0.4 to 0.5 BV / h, 0.5 to 0.6 BV / h, or 0.6 to 0.7 BV / h.

[0073] In some embodiments of the present invention, in step 5), the filler of the resin column is D101 macroporous resin.

[0074] In some embodiments of the present invention, step 5) further includes washing the effluent with deionized water until it is clear and transparent before separation.

[0075] In some embodiments of the present invention, in step 5), when collecting the chlorogenic acid component, the volume fraction of the alcohol solution is selected from any of the following ranges: 10-15%, 15-20%, 20-25%.

[0076] In some embodiments of the present invention, in step 5), when collecting the chlorogenic acid component, the volume of the alcohol solution is 3 to 7 BV. The volume of the alcohol solution is selected from any of the following ranges: 3 to 4 BV, 4 to 5 BV, 5 to 6 BV, or 6 to 7 BV.

[0077] In some embodiments of the present invention, in step 5), when collecting the chlorogenic acid component, the flow rate of the alcohol solution is 1–3 BV / h. The flow rate of the alcohol solution is selected from any of the following ranges: 1–2 BV / h, 2–3 BV / h.

[0078] In a preferred embodiment, in step 5), when collecting the chlorogenic acid component, the alcohol solution is an ethanol solution.

[0079] In some embodiments of the present invention, step 5) includes eluting with 10-30% ethanol to remove some impurities after collecting the chlorogenic acid component and before collecting the rutin component.

[0080] In some embodiments of the present invention, the volume of ethanol is 3 to 7 BV when eluting to remove some impurities.

[0081] In some embodiments of the present invention, the ethanol flow rate is 1 to 3 BV / h when eluting to remove some impurities.

[0082] In some embodiments of the present invention, in step 5), when collecting the rutin component, the volume fraction of the alcohol solution is selected from any of the following ranges: 30-35%, 35-40%, 40-45%, 45-50%.

[0083] In some embodiments of the present invention, in step 5), when collecting the rutin component, the volume of the alcohol solution is 3 to 7 BV. The volume of the alcohol solution is selected from any of the following ranges: 3 to 4 BV, 4 to 5 BV, 5 to 6 BV, or 6 to 7 BV.

[0084] In some embodiments of the present invention, in step 5), when collecting the rutin component, the flow rate of the alcohol solution is 1–3 BV / h. The flow rate of the alcohol solution is selected from any of the following ranges: 1–2 BV / h, 2–3 BV / h.

[0085] In a preferred embodiment, in step 5), when collecting the rutin component, the alcohol solution is an ethanol solution.

[0086] In some embodiments of the present invention, step 5) further includes post-processing the chlorogenic acid component to obtain chlorogenic acid solid.

[0087] In some embodiments of the present invention, the post-processing is concentration followed by drying.

[0088] In some embodiments of the present invention, the concentration is reduced pressure concentration.

[0089] In some embodiments of the present invention, the concentration temperature is 50–70°C.

[0090] In some embodiments of the present invention, the drying is freeze drying.

[0091] In some embodiments of the present invention, step 5) further includes post-processing the rutin component to obtain rutin solid.

[0092] In some embodiments of the present invention, the specific steps of the post-processing are concentration, dissolution of the concentrate, filtration and refrigeration of the filtrate, precipitation of crystals, filtration and drying to obtain rutin solid.

[0093] In some embodiments of the present invention, the concentration is reduced pressure concentration.

[0094] In some embodiments of the present invention, the concentration temperature is 50–70°C.

[0095] In some embodiments of the present invention, the solvent for the dissolved concentrate is methanol or ethanol.

[0096] In some embodiments of the present invention, the filtration method is filter paper filtration, filter cartridge filtration, or membrane filtration.

[0097] In some embodiments of the present invention, the refrigeration temperature is 2 to 6°C.

[0098] In some embodiments of the present invention, the refrigeration time is 10 to 16 hours.

[0099] In some embodiments of the present invention, the drying is performed by forced-air drying.

[0100] In some embodiments of the present invention, the drying time is 8 to 14 hours.

[0101] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0102] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0103] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0104] Example 1: Comparison of pretreatments for tobacco leaves not suitable for field use

[0105] Two kilograms of discarded tobacco leaves were collected during the field growth process. One kilogram of these leaves was freeze-dried to a moisture content of 13%. The other 1 kilogram of leaves was placed individually in a 90°C oven for blanching. The color and moisture content of the leaves were checked every hour. After three hours, the leaves were removed and allowed to cool naturally. The moisture content of the blanched tobacco leaves was 13%.

[0106] The contents of solanesyl alcohol, rutin, and total chlorogenic acid in tobacco leaves obtained by the two pretreatment methods were determined by HPLC. As shown in Table 1, compared with freeze-drying, the content of each component in tobacco leaves obtained by blanching and drying is improved, and it is easier to operate in practice.

[0107] Table 1. Content of various components in tobacco leaves obtained by different pretreatment methods

[0108] processing method Solanine Rutin Total chlorogenic acid freeze-drying 0.02％ 0.03％ 0.01％ Blanching and drying 0.48％ 0.26％ 0.72％

[0109] Example 2: Extraction and separation of components from tobacco leaves unsuitable for field use

[0110] Step 1: Preprocessing

[0111] 10 kg of unsuitable tobacco leaves were collected from the field and subjected to blanching pretreatment as described in Example 1 to obtain 1.4 kg of dried tobacco leaves, which were then ground and pulverized.

[0112] Step 2: Extraction

[0113] Extraction of solanine: Take 1 kg of pretreated field-unsuitable tobacco leaves, reflux with 15 L of ethyl acetate at 60 °C for 2 h, extract twice, cool, filter, combine the two ethyl acetate extracts, concentrate under reduced pressure at 45 °C until ethyl acetate is removed, and obtain the first extract.

[0114] Extraction of chlorogenic acid and rutin: After extracting solanesol from tobacco leaves, evaporate ethyl acetate, then add 15L of 50% ethanol as the extraction solvent, reflux for 1h, and extract twice. After cooling and filtration, combine the two extracts and concentrate under reduced pressure at 60℃ until the ethanol is removed to obtain the second extract.

[0115] Step 3: Separation

[0116] Separation of solanesol: The first extract of tobacco leaves was dissolved in 200 ml of 85% ethanol-0.3 mol / L NaOH aqueous solution, and then extracted twice with 200 ml of petroleum ether. The petroleum ether extracts were combined and concentrated by distillation at 30 °C to obtain an extract containing solanesol. The extract was dry-loaded into a normal-phase silica gel column with a volume equal to 6 times the extract volume. It was first eluted with petroleum ether until colored elution occurred, and then isocratic eluted with a petroleum ether:ethyl acetate = 30:1 eluent. One fraction was collected for each column volume at a flow rate of 3 BV / h. The target compound was detected by TLC. The eluents with HPLC purity greater than 90% were combined and concentrated under reduced pressure until solanesol solid precipitated, yielding solanesol solid with a purity of 94.7%.

[0117] Separation of total chlorogenic acid and rutin:

[0118] 1 L of the second extract was loaded onto a 2 L D101 resin column for chromatography at a flow rate of 1.0 L / h. The eluent was first eluted with 6 L of deionized water until clear and transparent. Then, chlorogenic acid fraction was eluted with 10 L of 15% ethanol at a flow rate of 4 L / h and collected. Next, impurities were eluted with 10 L of 20% ethanol at a flow rate of 4 L / h. Finally, rutin fraction was eluted with 10 L of 40% ethanol at a flow rate of 4 L / h and collected.

[0119] The collected chlorogenic acid fraction was concentrated to a small volume under reduced pressure at 60°C and then freeze-dried to obtain chlorogenic acid freeze-dried powder solid.

[0120] The collected rutin fraction was concentrated to a small volume under reduced pressure at 60°C, dissolved in methanol, filtered, and the filtrate was placed in a refrigerator at 4°C for 12 hours to crystallize. After filtration, it was dried by forced air for 12 hours to obtain lyophilized rutin powder with a purity of 90.05%.

[0121] Table 1. List of waste generated from 1 kg of tobacco leaves

[0122]

[0123]

[0124]

[0125] Example 3: Extraction and separation of components from tobacco leaves unsuitable for field use

[0126] Step 1: Preprocessing

[0127] 10 kg of unsuitable tobacco leaves were collected from the field and subjected to blanching pretreatment as described in Example 1 to obtain 1.4 kg of dried tobacco leaves, which were then ground and pulverized.

[0128] Step 2: Extraction

[0129] Extraction of solanine: Take 1 kg of pretreated field-unsuitable tobacco leaves, reflux with 10 L of ethyl acetate at 65 °C for 2 h, extract twice, cool, filter, combine the two ethyl acetate extracts, concentrate under reduced pressure at 40 °C until ethyl acetate is removed, and obtain the first extract.

[0130] Extraction of chlorogenic acid and rutin: After extracting solanesol from tobacco leaves, evaporate ethyl acetate, then add 15L of 40% ethanol as the extraction solvent, reflux for 2 hours, and extract twice. After cooling and filtration, combine the two extracts and concentrate under reduced pressure at 60℃ until the ethanol is removed to obtain the second extract.

[0131] Step 3: Separation

[0132] Separation of solanesol: The first extract of tobacco leaves was dissolved in 200 ml of 75% ethanol-0.3 mol / L NaOH aqueous solution, and then extracted twice with 200 ml of petroleum ether. The petroleum ether extracts were combined and concentrated by distillation at 30 °C to obtain an extract containing solanesol. The extract was dry-loaded into a normal-phase silica gel column with a volume equal to 6 times the extract volume. It was first eluted with petroleum ether until colored elution occurred, and then isocratic eluted with a petroleum ether:ethyl acetate = 35:1 eluent. One fraction was collected for each column volume at a flow rate of 3 BV / h. The target compound was detected by TLC. The eluents with HPLC purity greater than 90% were combined and concentrated under reduced pressure until solanesol solid precipitated, yielding solanesol solid with a purity of 95.0%.

[0133] Separation of total chlorogenic acid and rutin:

[0134] 1 L of the second extract was loaded onto a 2 L D101 resin column for chromatography at a flow rate of 1.0 L / h. The eluent was first eluted with 6 L of deionized water until clear and transparent. Then, chlorogenic acid fraction was eluted with 10 L of 20% ethanol at a flow rate of 4 L / h and collected. Next, impurities were eluted with 10 L of 25% ethanol at a flow rate of 4 L / h. Finally, rutin fraction was eluted with 10 L of 45% ethanol at a flow rate of 4 L / h and collected.

[0135] The collected chlorogenic acid fraction was concentrated to a small volume under reduced pressure at 65°C and then freeze-dried to obtain chlorogenic acid freeze-dried powder solid.

[0136] The collected rutin fraction was concentrated to a small volume under reduced pressure at 65°C, dissolved in methanol, filtered, and the filtrate was placed in a refrigerator at 4°C for 12 hours to crystallize. After filtration, it was dried by forced air for 12 hours to obtain lyophilized rutin powder with a purity of 92.0%.

[0137] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications and variations of the methods listed herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. A method for extracting and separating components from tobacco leaves unsuitable for field use, characterized in that, The extraction and separation method includes the following steps: 1) Blanching treatment: Hot air treatment is applied to unsuitable tobacco leaves in the field; 2) Extraction of solanesol: The tobacco leaves obtained in step 1) are mixed with an organic solvent, heated under reflux, and the reflux liquid is collected. The organic solvent in the reflux liquid is removed by concentration to obtain the first extract for extracting solanesol. 3) Extraction of total chlorogenic acid and rutin: Remove the organic solvent from the tobacco leaves after extracting solanesol, use an alcoholic solution as the extraction solvent, heat and reflux, collect the reflux liquid, concentrate and remove the alcoholic solution from the reflux liquid to obtain the second extract for extracting total chlorogenic acid and rutin. 4) Separation of solanesol: Dissolve the first extract obtained in step 2) in an ethanol-NaOH aqueous solution, extract and recover the extractant to obtain the extract, separate it by normal phase silica gel column, collect the eluent to obtain the solanesol component; 5) Separation of total chlorogenic acid and rutin: The second extract obtained in step 3) is separated by a resin column. The chlorogenic acid fraction is collected with a 10-25% (v / v) alcohol solution, and the rutin fraction is collected with a 30-50% (v / v) alcohol solution.

2. The extraction and separation method according to claim 1, characterized in that, Step 1) includes one or more of the following conditions: 1) The temperature of the hot air treatment is 80–100°C; 2) The hot air treatment time is 1 to 3 hours; 3) Hot air treatment until the moisture content of the tobacco leaves is 5% to 15%; 4) Cool the tobacco leaves after hot air treatment; 5) Crush the tobacco leaves and dust after hot air treatment.

3. The extraction and separation method according to claim 1, characterized in that, Step 2) includes one or more of the following conditions: 1) The mixing ratio of tobacco leaves and organic solvent obtained in step 1) is 1 kg: 10-15 L; preferably, the organic solvent is ethyl acetate; 2) Filter the reflux liquid after cooling; preferably, the filtration method is filter paper filtration, filter cartridge filtration or membrane filtration; 3) The reflux time is 1 to 3 hours; 4) The number of refluxes is 1 to 3 times; 5) The concentration is vacuum concentration, atmospheric pressure concentration, or freeze concentration; preferably, the concentration is vacuum concentration; 6) The concentration temperature is 40-45℃.

4. The extraction and separation method according to claim 1, characterized in that, Step 3) includes one or more of the following conditions: 1) The volume fraction of the alcohol solution is 30-50%; preferably, the alcohol solution is an ethanol solution; 2) The ratio of tobacco leaves to alcohol solution after extraction of solanesol is 1 kg: 10-20 L; 3) Filter the reflux liquid after cooling; preferably, the filtration method is filter paper filtration, filter cartridge filtration or membrane filtration; 4) The reflux time is 1 to 3 hours; 5) The number of refluxes is 1 to 3; 6) The concentration is vacuum concentration, atmospheric pressure concentration, or freeze concentration; preferably, the concentration is vacuum concentration. 7) The concentration temperature is 50-70℃.

5. The extraction and separation method according to claim 1, characterized in that, Step 4) includes one or more of the following conditions: 1) The ratio of the first extract to the ethanol-NaOH aqueous solution is 1:1 to 2; 2) The ethanol-NaOH aqueous solution has an ethanol volume fraction of 70-90%; 3) The ethanol-NaOH aqueous solution has a NaOH aqueous solution concentration of 0.2–0.4 mol / L; 4) The extractant used in the extraction is petroleum ether; preferably, the volume of the petroleum ether is equal to the volume of the ethanol-NaOH aqueous solution; 5) The extraction should be performed 2 to 4 times; 6) The extractant is recovered by distillation and concentration; 7) The temperature for recovering the extractant is 20–40℃; 8) The loading method for the normal phase silica gel column is dry loading; 9) The volume ratio of the normal-phase silica gel column to the extract volume is 5–7:1; 10) Before separation, elute with petroleum ether until colored fluid flows out; 11) The mobile phase for separation is petroleum ether: ethyl acetate = 20-40: 1; 12) Isocratic elution is used during separation; 13) During separation, one fraction is collected for each column volume; 14) The flow rate during separation is 2–5 BV / h; 15) The target compound was tracked and detected using TLC during separation; 16) The eluent is an eluent with an HPLC purity greater than 90%.

6. The extraction and separation method according to claim 1, characterized in that, It also includes post-processing of the solanesol component obtained in step 4); preferably, the post-processing is to concentrate the solanesol component obtained in step 4); more preferably, the concentration is vacuum concentration.

7. The extraction and separation method according to claim 1, characterized in that, Step 5) includes one or more of the following conditions: 1) The volume ratio of the second extract to the resin column is 1g to 1:3mL; 2) The loading rate of the second extract is 0.3–0.7 BV / h; 3) The filler for the resin column is D101 macroporous resin; 4) Before separation, wash the effluent with deionized water until it is clear and transparent; 5) When collecting the chlorogenic acid component and / or rutin component, the alcohol solution is an ethanol solution; 6) When collecting chlorogenic acid and / or rutin components, the volume of the alcohol solution should be 3–7 BV; 7) When collecting chlorogenic acid and / or rutin components, the flow rate of the alcohol solution is 1-3 BV / h; 8) After collecting the chlorogenic acid fraction and before collecting the rutin fraction, some impurities were removed by elution with 10-30% ethanol (by volume).

8. The extraction and separation method according to claim 1, characterized in that, It also includes post-processing of the chlorogenic acid component obtained in step 5); preferably, the post-processing is to concentrate and then dry the chlorogenic acid component obtained in step 5); more preferably, the concentration is vacuum concentration; more preferably, the concentration temperature is 50-70°C; more preferably, the drying is freeze drying.

9. The extraction and separation method according to claim 1, characterized in that, It also includes post-processing of the rutin component obtained in step 5); preferably, the specific steps of the post-processing are concentration, dissolving the concentrate, filtration and refrigerating the filtrate, crystallization, filtration and drying.

10. The extraction and separation method according to claim 9, characterized in that, It also includes one or more of the following conditions: 1) Concentration is performed using reduced pressure concentration; 2) The concentration temperature is 50–70℃; 3) The solvent for dissolving the concentrate is methanol or ethanol; 4) The filtration method is filter paper filtration, filter cartridge filtration, or membrane filtration; 5) The refrigeration temperature is 2–6°C; 6) The refrigeration time is 10 to 16 hours; 7) The drying process is blower drying; 8) The drying time is 8 to 14 hours.

Images