A glue grass extract, a preparation method and application thereof, an atomized essence and a CYP2A6 enzyme inhibitor

Abstract

The application discloses a gelatinous grass extract and a preparation method and application thereof, atomized essence and CYP2A6 enzyme inhibitor, and belongs to the technical field of extracts. The preparation method of the gelatinous grass extract comprises the following steps: providing gelatinous grass powder; extracting the gelatinous grass powder by using a mixed solvent of ethanol and limonene, and obtaining an extraction liquid after filtration; and concentrating and drying the extraction liquid to obtain the gelatinous grass extract. Through extraction of the gelatinous grass extract, the gelatinous grass extract has the advantages of good thermal stability and no aroma, so that when the gelatinous grass extract is applied in the atomized essence, the gelatinous grass extract has the function of inhibiting nicotine metabolism, can reduce the absorption of nicotine, and reduces the frequency of smoking.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of extraction, in particular to a marsh grass extract, a preparation method and application thereof, an atomized essence and a CYP2A6 enzyme inhibitor. BACKGROUND

[0002] Nicotine is a colorless, fat-soluble small molecule substance, which is usually taken into the body by oral inhalation. The half-life of nicotine in nicotine is 1-2 hours, which is mainly metabolized by CYP2A6 enzyme in the liver, and the main metabolite is cotinine.

[0003] CYP2A6 enzyme is the main metabolic enzyme of nicotine, and its activity is closely related to smoking behavior. CYP2A6 enzyme activity is divided into strong metabolic individuals (EMs) and weak metabolic individuals (PMs), which is used to evaluate the difference of CYP2A6 enzyme activity among individuals. EMs have stronger ability to metabolize nicotine than PMs, and for smokers, EM individuals usually smoke more cigarettes than PM individuals to maintain the body's demand for nicotine levels. Therefore, for the smoking population, factors that reduce CYP2A6 enzyme activity are protective factors.

[0004] Electronic atomization contains nicotine for users to take into the body by oral inhalation. However, due to the rapid metabolism of nicotine, users will continuously repeat the action of taking nicotine to maintain the nicotine content in the body. Under the social consensus of continuously reducing the nicotine content in electronic atomization, the behavior of the user is strengthened. In order to reduce the frequency of the user's action of taking nicotine, effective measures can be taken from the CYP2A6 enzyme activity that affects nicotine metabolism.

[0005] At present, there are many CYP2A6 enzyme activity inhibitors, such as amiodarone, amlodipine, buprenorphine, cholestyramine, clotrimazole, desipramine, disulfiram, entacapone, fenofibrate, gabapentin, isoniazid, ketoconazole, letrozole, methimazole, methoxsalen, metyrapone, miconazole, modafinil, orphenadrine, pregabalin, selegiline, sulconazole, thiocorazole, tranylcypromine, bupropion, etc. However, these components correspond to the use in atomized essence, all have various inapplicability, such as having a medicinal aroma, having a strong interference with the inherent aroma of the atomized essence; or poor thermal stability, invalid at the working temperature of 250-300℃ of the atomized essence; or problems such as slow onset time, large side effects, large use amount, poor inhalation safety, etc. SUMMARY

[0006] The main purpose of the present application is to provide a glue grass extract and a preparation method and application thereof, and an atomized essence and a CYP2A6 enzyme inhibitor, so as to overcome the problems of the prior art, such as strong odor, poor stability, and large side effects of the CYP2A6 enzyme activity inhibitor.

[0007] To achieve the above-mentioned purpose, the present application provides a preparation method of a glue grass extract, comprising the following steps:

[0008] providing glue grass powder;

[0009] extracting the glue grass powder by using a mixed solvent of ethanol and limonene, and obtaining an extract after filtration;

[0010] concentrating and drying the extract to obtain the glue grass extract.

[0011] Optionally, in the step of providing the glue grass powder, the moisture content of the glue grass powder is 5% to 8%.

[0012] Optionally, in the step of extracting the glue grass powder by using a mixed solvent of ethanol and limonene, and obtaining an extract after filtration:

[0013] the mass ratio of ethanol to limonene is (1 to 9) to 1; and / or,

[0014] the mesh number of the filter screen is 300 to 350.

[0015] Optionally, in the step of concentrating and drying the extract to obtain the glue grass extract:

[0016] the concentration temperature is 30 to 55°C; and / or,

[0017] the concentration pressure is 20 to 70 mbar; and / or,

[0018] the drying temperature is 50 to 60°C; and / or,

[0019] the drying pressure is 100 to 200 pa; and / or,

[0020] the drying time is 1 to 6 hours.

[0021] The present application also provides a glue grass extract prepared by the preparation method of the glue grass extract.

[0022] The present application also provides an atomized essence, comprising nicotine, an organic acid, and the glue grass extract.

[0023] Optionally, the mass ratio of the glue grass extract in the atomized essence is 0.001% to 0.1%.

[0024] Optionally, the organic acid comprises at least one of benzoic acid, glacial acetic acid, citric acid, malic acid, tartaric acid, oxalic acid, succinic acid, lactic acid, and acetylenic acid.

[0025] The application also provides an application of the extract of the marsh grass in reducing nicotine intake.

[0026] The application also provides a CYP2A6 enzyme inhibitor, which comprises the extract of the marsh grass.

[0027] The extract of the marsh grass provided by the application has the advantages of good thermal stability and no aroma, so that when the extract of the marsh grass is applied in atomized essence, the extract of the marsh grass has the function of inhibiting nicotine metabolism, can reduce the absorption of nicotine, and reduces the frequency of smoking. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical scheme and advantages of the embodiments of the application more clear, the technical scheme in the embodiments of the application will be clearly and completely described below. If the specific conditions are not specified in the embodiments, the conventional conditions or the conditions suggested by the manufacturer are adopted. If the reagents or instruments used are not specified by the manufacturer, they are all conventional products that can be purchased in the market. In addition, the meaning of "and / or" in the full text includes three parallel schemes. Taking "A and / or B" as an example, it includes the A scheme, or the B scheme, or the scheme that A and B are satisfied at the same time. In addition, the technical schemes of various embodiments can be combined with each other, but it must be based on the fact that a person skilled in the art can realize it. When the combination of technical schemes appears contradictory or unachievable, it should be considered that the combination of technical schemes does not exist and is not within the protection scope of the application. Based on the embodiments in the application, all other embodiments obtained by a person skilled in the art without creative labor are within the protection scope of the application.

[0029] Nicotine is a colorless, fat-soluble small molecule substance, which is usually taken into the body by oral inhalation. The half-life of nicotine in nicotine is 1-2 hours, which is mainly metabolized by CYP2A6 enzyme in the liver, and the main metabolite is cotinine.

[0030] CYP2A6 enzyme is the main metabolic enzyme of nicotine, and its activity is closely related to smoking behavior. The activity of CYP2A6 enzyme is divided into strong metabolic individuals (EMs) and weak metabolic individuals (PMs), which is used to evaluate the difference of CYP2A6 enzyme activity among individuals. EMs have stronger ability to metabolize nicotine than PMs. For smokers, generally, EM individuals smoke more cigarettes than PM individuals to maintain the body's demand for nicotine levels. Therefore, for the smoking population, factors that reduce the activity of CYP2A6 enzyme are protective factors.

[0031] The electronic atomization product contains nicotine for a user to intake into the body by oral inhalation, but due to the fast metabolism of nicotine, the user will continuously repeat the action of nicotine intake to maintain the nicotine content in the body. Under the social consensus of continuously reducing the nicotine content in the electronic atomization product, the user's behavior is strengthened. In order to reduce the frequency of the user's nicotine intake action, effective measures can be taken from the CYP2A6 enzyme activity affecting the metabolism of nicotine.

[0032] At present, there are many CYP2A6 enzyme activity inhibitors, such as amiodarone, amlodipine, buprenorphine, clofibrate, clotrimazole, desipramine, disulfiram, entacapone, fenofibrate, gabapentin, isoniazid, ketoconazole, letrozole, methimazole, methoxsalen, metyrapone, miconazole, modafinil, orphenadrine, pregabalin, selegiline, sulconazole, thiocorazole, tranylcypromine, bupropion and the like. However, when these components are used in atomized flavors, they all have various inadaptabilities, such as having a medicinal aroma, having a strong interference with the aroma of the atomized flavor itself, or having poor thermal stability, being invalid at the working temperature of 250-300 DEG C of the atomized flavor, or having problems such as slow onset time, large side effects, large use amount, poor inhalation safety and the like.

[0033] In view of this, the present application proposes a preparation method of grindelia extract, aiming to overcome the problems of the CYP2A6 enzyme inhibitors in the prior art, such as odor, poor stability, large side effects, slow action time and low safety.

[0034] Grindelia Willd was first used by Indians for cooking, self-made beverages and other fields. It has the characteristics of good thermal stability and no aroma.

[0035] A preparation method of grindelia extract, comprising the following steps:

[0036] Providing grindelia powder;

[0037] Extracting the grindelia powder with a mixed solvent of ethanol and limonene, and obtaining an extract after filtration;

[0038] Concentrating and drying the extract to obtain the grindelia extract.

[0039] It should be noted that the grindelia powder can be purchased grindelia powder or self-made grindelia powder. In order to improve the yield of the grindelia extract, the grindelia powder can be sieved through a 20-60 mesh sieve.

[0040] In the technical solution of the present application, through the extraction of the grindelia extract, the grindelia extract has the advantages of good thermal stability and no aroma, so that when it is applied in the atomized flavor, it has the function of inhibiting the metabolism of nicotine, can reduce the absorption of nicotine and reduce the number of smoking.

[0041] In some embodiments of the present application, the step provides that the moisture content of the glue grass powder is 5% to 8%. Within this range of moisture content, the dissolution rate of the effective substances in the glue grass can be effectively improved during extraction.

[0042] In some embodiments of the present application, the step uses a mixed solvent of ethanol and limonene to extract the glue grass powder, and after filtration, the mass ratio of ethanol to limonene in the extracted liquid is (1-9):1. The mixed solvent of ethanol and limonene can take into account the extraction needs of medium-polar and extremely low-polar components, and the mass ratio of ethanol to limonene (1-9):1 can adjust the extraction rate of medium-polar and extremely low-polar components, ultimately producing different use effects. It should be noted that the mass ratio of the extracted liquid to the glue grass powder can be (1-10):1.

[0043] In some embodiments of the present application, the step uses a mixed solvent of ethanol and limonene to extract the glue grass powder, and after filtration, the mesh size of the filter screen used for filtration is 300-350 mesh. The filter residue can be removed, the purity of the glue grass extract can be improved, and the influence of solid insoluble substances on the purity of the glue grass extract can be reduced. The filtrate after 300-350 mesh filter screen filtration can also be filtered with a 30-50 μm filter membrane to further remove the filter residue, improve the purity of the glue grass extract, and reduce the influence of solid insoluble substances on the purity of the glue grass extract.

[0044] In some embodiments of the present application, the step concentrates and dries the extracted liquid to obtain glue grass extract, and the concentration temperature is 30-55°C. Within this range of concentration temperature, the effective components can be effectively protected, and the denaturation and side reactions of the effective components during processing can be reduced.

[0045] In some embodiments of the present application, the step concentrates and dries the extracted liquid to obtain glue grass extract, and the concentration pressure is 20-70 mbar. Under this concentration pressure, the concentration time can be reduced, the concentration temperature can be reduced, and the effective components can be protected.

[0046] In some embodiments of the present application, the step concentrates and dries the extracted liquid to obtain glue grass extract, and the drying temperature is 50-60°C. Under this drying temperature, the effective components can be protected, and at the same time, the water in the dried sample can be sufficiently removed, and the stability of the glue grass extract can be improved.

[0047] In some embodiments of the present application, the step concentrates and dries the extracted liquid to obtain glue grass extract, and the drying pressure is 100-200 pa. Under this drying pressure, the water in the dried sample can be sufficiently removed, and the stability of the glue grass extract can be improved.

[0048] In some embodiments of the present application, the step of concentrating and drying the extract to obtain the extract of the marshmallow includes a drying time of 1-6 hours. Within this range of drying time, the water in the sample to be dried can be sufficiently removed, and the stability of the extract of the marshmallow can be improved.

[0049] The present application also provides an extract of the marshmallow prepared by the method.

[0050] The present application also provides an aerosol flavoring including nicotine, an organic acid, and the extract of the marshmallow.

[0051] In some embodiments of the present application, the mass ratio of the extract of the marshmallow in the aerosol flavoring is 0.001%-0.1%. Within this range of the mass ratio of the extract of the marshmallow in the aerosol flavoring, the intake of nicotine can be reduced, the cost can be lowered, and the mass ratio of other components in the aerosol flavoring can be reduced.

[0052] In some embodiments of the present application, the organic acid includes at least one of benzoic acid, acetic acid, citric acid, malic acid, tartaric acid, oxalic acid, succinic acid, lactic acid, and levulinic acid. At least one of the above-mentioned organic acids can be used to achieve the proton balance of nicotine-organic acid, and the thermal stability and odor compatibility of the organic acid can be considered.

[0053] The present application also provides an application of the extract of the marshmallow in reducing the intake of nicotine.

[0054] The present application also provides a CYP2A6 enzyme inhibitor including the extract of the marshmallow.

[0055] The technical solutions of the present application will be further described in detail below in combination with specific embodiments. It should be understood that the following embodiments are only used to explain the present application, and are not used to limit the present application.

[0056] Embodiment 1

[0057] A method for preparing an extract of the marshmallow includes the following steps:

[0058] Providing the powder of the marshmallow;

[0059] Extracting the powder of the marshmallow by using a mixed solvent of ethanol and limonene with a mass ratio of 1:1, filtering through a 300-mesh sieve, and filtering through a 30-μm filter membrane to obtain an extract, and the mass ratio of the powder of the marshmallow to the extract is 1:1;

[0060] Concentrating the extract at 30°C and 20 mbar, and vacuum drying at 50°C and 100 pa for 1 hour to obtain the extract of the marshmallow.

[0061] Embodiment 2

[0062] A method for preparing an extract of the marshmallow includes the following steps:

[0063] Providing a marshmallow powder;

[0064] Extracting the marshmallow powder with a mixed solvent of ethanol and limonene in a mass ratio of 9:1, filtering with a 350-mesh sieve, and filtering with a 50-μm filter membrane to obtain an extract, the mass ratio of the marshmallow powder to the extract being 1:10;

[0065] Concentrating the extract at 55°C and 70 mbar, and vacuum drying at 60°C and 200 pa for 6 h to obtain a marshmallow extract.

[0066] Example 3

[0067] A preparation method of a marshmallow extract, comprising the following steps:

[0068] Providing a marshmallow powder;

[0069] Extracting the marshmallow powder with a mixed solvent of ethanol and limonene in a mass ratio of 5:1, filtering with a 330-mesh sieve, and filtering with a 40-μm filter membrane to obtain an extract, the mass ratio of the marshmallow powder to the extract being 1:5;

[0070] Concentrating the extract at 45°C and 50 mbar, and vacuum drying at 55°C and 150 pa for 3 h to obtain a marshmallow extract.

[0071] Example 4

[0072] An atomized essence, comprising nicotine, lactic acid, and the marshmallow extract of Example 3, the mass fractions of the nicotine, the lactic acid, and the marshmallow extract being 3.000%, 2.080%, and 0.001% respectively.

[0073] Example 5

[0074] An atomized essence, comprising nicotine, lactic acid, and the marshmallow extract of Example 3, the mass fractions of the nicotine, the lactic acid, and the marshmallow extract being 3.000%, 2.080%, and 0.01% respectively.

[0075] Example 6

[0076] An atomized essence, comprising nicotine, lactic acid, and the marshmallow extract of Example 3, the mass fractions of the nicotine, the lactic acid, and the marshmallow extract being 3.000%, 2.080%, and 0.1% respectively.

[0077] Example 7

[0078] An atomized essence, comprising nicotine, lactic acid, and the marshmallow extract of Example 1, the mass fractions of the nicotine, the lactic acid, and the marshmallow extract being 3.000%, 2.080%, and 0.005% respectively.

[0079] Example 8

[0080] An aerosol flavouring comprising nicotine, lactic acid and the extract of example 2, comprising nicotine 3.000%, lactic acid 2.080%, extract of Galium aparine 0.05% by mass.

[0081] Comparative example 1

[0082] The same as example 4 except that the extract of Galium aparine was not included.

[0083] Comparative example 2

[0084] The same as example 4 except that the extract of Galium aparine was not included and methoxsalen was included.

[0085] Test method and results

[0086] In vitro test of metabolic inhibition of CYP2A6 enzyme by extract of Galium aparine:

[0087] The inhibitory effect of the extract of Galium aparine of examples 1 to 3 on the metabolic activity of CYP2A6 enzyme was studied using an in vitro enzyme incubation method with CYP2A6 enzyme as the CYP metabolic enzyme. In the in vitro enzyme reaction experiment, nicotine was used as the specific substrate of CYP2A6 enzyme, and the content of the metabolic product cotinine was determined by HPLC method, and the amount of metabolic product was used to calculate the CYP enzyme activity. IC50 value was used to evaluate the size of the inhibitory effect, and methoxsalen was used as the positive control inhibitor of CYP2A6.

[0088] The incubation system was 250 μL, with 100 mM potassium dihydrogen phosphate as the buffer, pH maintained at 7.4, mixed liver microsomal concentration of 0.5 mg / ml, and mixed liver cytoplasmic solution concentration of 0.4 mg / ml. Nicotine and extract of Galium aparine were dissolved in methanol. The mixture containing mixed liver microsomes, mixed liver cytoplasmic solution, nicotine (final concentration 1 μM), different concentrations of extract of Galium aparine was pre-incubated at 37°C for 5 minutes, then NADPH system (3.3 mM G6P, 1.3 mM-NADP+, 3.3 mM MgCl2, 1.0 unit / ml G6PDH) was added as the start of nicotine metabolism, and incubated for 30 minutes, then ice sodium chloride and sodium bicarbonate mixture containing internal standard caffeine was added as the reaction termination liquid, dichloromethane was used as the extraction agent, and after shaking well on the shaker for 10 minutes, centrifugation was performed at 4°C and 20000g for 10 minutes, the supernatant was removed, the liquid phase was transferred to a new EP tube, 0.01 N hydrochloric acid was added and shaken on the shaker for 10 minutes, then centrifugation was performed at 4°C and 20000g for 10 minutes, the supernatant was removed for detection, and IC50 was calculated. The results are shown in Table 1.

[0089] Table 1 Metabolic inhibition of CYP2A6 enzyme by extract of Galium aparine

[0090] Experimental group IC50 Example 1 0.82 μg / g Example 2 0.65 μg / g Example 3 0.51 μg / g Methoxy saalin (positive control) 0.70 μg / g Methanol (negative control) 75.33 μg / g

[0091] IC50(half maximal inhibitory concentration) is an index for measuring the effect of an inhibitor on biological activity. The lower the IC50 value of the extract of the marsh grass, the stronger the inhibitory effect. The IC50 values of the extract of the marsh grass of Examples 1 to 3 were comparable to that of methoxsalen and much smaller than that of methanol, indicating that the extract of the marsh grass had a CYP2A6 enzyme inhibitory effect.

[0092] Sensory evaluation of atomized flavors

[0093] Twenty-seven volunteers aged 25 to 45 years were selected, and 24 hours before the test, they did not smoke e-cigarettes or cigarettes and did not use other nicotine delivery devices. The atomized flavors of Examples 4 to 8 and Comparative Examples 1 and 2 were subjected to smoking tests, and when the testers had a smoking urge, they made the mouth full of smoke for 3 seconds. The number of smoking urges within 48 hours was recorded, and the average value was calculated. Each time the material was changed, the test was conducted 24 hours later. The test results are shown in Table 2.

[0094] Table 2 Sensory evaluation of atomized flavors

[0095]

[0096]

[0097] The nicotine content in Examples 4 to 8 and Comparative Example 1 was the same (30 mg / mL), so the difference in the number of urges was more related to other ingredients. Comparative Example 1 can cause a greater desire or urge to smoke, and Examples 4 to 8 and Comparative Example 2 have an effect on smoking behavior, so the average number of urges of Comparative Example 2 and Examples 4 to 8 is different from that of Comparative Example 1. The number of urges of Examples 4 to 8 is lower than that of Comparative Example 2, and the addition of the extract of the marsh grass in Examples 4 to 8 is more effective in inhibiting the urge to smoke.

[0098] The above are only preferred embodiments of the present application, and do not limit the patent scope of the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the patent protection scope of the present application.

Claims

1. An atomized essence characterized in that, It includes nicotine, organic acids, and bulrush extract, wherein the bulrush extract accounts for 0.001% to 0.1% by mass in the atomized flavoring; The bulrush extract was prepared by the following steps: Provide rubber grass powder; The powder of *Gynostemma pentaphyllum* was extracted using a mixed solvent of ethanol and limonene, and the extract was obtained after filtration. The extract was concentrated and dried to obtain the gum grass extract; In the step of concentrating and drying the extract to obtain the guarana extract: the concentration temperature is 30~55℃; the concentration pressure is 20~70mbar; the drying temperature is 50~60℃; the drying pressure is 100~200pa; and the drying time is 1~6h.

2. The atomized perfume of claim 1, wherein, The organic acids include lactic acid.

3. The atomized flavoring of claim 1, wherein In the step of providing rubber grass powder, the moisture content of the rubber grass powder is 5% to 8%.

4. The atomized flavoring of claim 1, wherein In the step of extracting the gum grass powder with a mixed solvent of ethanol and limonene, and then filtering to obtain the extract: The mass ratio of ethanol to limonene is (1~9):1; and / or, The filter screen has a mesh size of 300-350.

5. Use of a extract of Myrica gale in reducing nicotine intake, characterized in that, The bulrush extract was prepared by the following steps: Provide rubber grass powder; The powder of *Gynostemma pentaphyllum* was extracted using a mixed solvent of ethanol and limonene, and the extract was obtained after filtration. The extract was concentrated and dried to obtain the gum grass extract; In the step of concentrating and drying the extract to obtain the guarana extract: the concentration temperature is 30~55℃; the concentration pressure is 20~70mbar; the drying temperature is 50~60℃; the drying pressure is 100~200pa; and the drying time is 1~6h.

6. A CYP2A6 enzyme inhibitor, characterized in that, Includes a bulrush extract, which is prepared by the following steps: Provide rubber grass powder; The powder of *Gynostemma pentaphyllum* was extracted using a mixed solvent of ethanol and limonene, and the extract was obtained after filtration. The extract was concentrated and dried to obtain the gum grass extract; In the step of concentrating and drying the extract to obtain the guarana extract: the concentration temperature is 30~55℃; the concentration pressure is 20~70mbar; the drying temperature is 50~60℃; the drying pressure is 100~200pa; and the drying time is 1~6h.

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