A tombstone extract, its preparation method and use in the tobacco industry

Abstract

The present application relates to the tobacco technical field, provide a kind of applied in tobacco industry's tomb head back extract and its production preparation process.Tomb head back medicinal materials are extracted with small polarity solvent to obtain component 1;After extracting with alcohol-water solvent, component 2 is obtained by macroporous resin column chromatography;Two components are mixed, and the solvent is removed to prepare tomb head back extract.The tomb head back extract described in the present application can be applied to tobacco products, electronic cigarette production and manufacturing, and has the functions of enriching and coordinating smoke characteristics, increasing smoke sweetness, reducing smoke irritation, etc., and also has certain antibacterial and anti-inflammatory, sedative and sleep aid effects.

Description

Technical Field

[0001] This invention relates to the field of tobacco product manufacturing technology, specifically to a *Gnaphalium affine* extract, its preparation method, and its application in the tobacco industry. Background Technology

[0002] my country is a major producer and consumer of tobacco. Smoking causes serious harm to the human body, the most direct effect being irritation to the respiratory system, which can lead to coughing, sore throat, and upper respiratory tract inflammation. Furthermore, nicotine in cigarettes can excite the sympathetic nervous system, and long-term smoking can cause neurasthenia, sleep disorders, and other neurological symptoms. Therefore, developing natural flavorings that can enrich and harmonize the aroma of tobacco, reduce its irritation, and improve respiratory and nervous system functions is of significant value in enhancing product competitiveness, and there is a high market demand for such products.

[0003] *Patrinia rupestris* (Pall.) Juss. subsp. scabra (Bunge) HJWang or *Patrinia heterophylla* Bunge., belonging to the Valerianaceae family, is a wild plant resource with high development and medicinal value. *Patrinia rupestris* is rich in active ingredients and has wide applications. According to the *National Compendium of Traditional Chinese Medicine*, it has effects such as clearing heat and drying dampness, stopping bleeding, stopping leukorrhea, and treating malaria. Modern research shows that *Patrinia rupestris* is rich in volatile oils, triterpenoid saponins, flavonoids, and other active ingredients, possessing antibacterial, anti-inflammatory, sedative, sleep-aiding, tumor-inhibiting, and immune-enhancing activities. However, there are currently no related technologies or products using *Patrinia rupestris* in the tobacco industry. The technical solution and products involved in this invention patent are the first public disclosure and application of *Patrinia rupestris* in tobacco product manufacturing. Summary of the Invention

[0004] This invention is the first to use extracts of *Gynostemma pentaphyllum* in the flavoring and additive manufacturing process of tobacco products and e-cigarettes. It utilizes its unique aromatic components to enrich and harmonize the characteristics of smoke, increase the sweetness of smoke, and reduce the irritation of smoke. Furthermore, it utilizes its antibacterial, anti-inflammatory, sedative, and sleep-aiding effects to improve the stimulation and damage to the respiratory and nervous systems caused by nicotine and tar in tobacco.

[0005] The extract of Patrinia rupestris described in this invention is extracted from the traditional Chinese medicine Patrinia rupestris, which is the root of Patrinia rupestris (Pall.) Juss. subsp. scabra (Bunge) HJWang or Patrinia heterophylla Bunge., both belonging to the Valerianaceae family.

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

[0007] A method for preparing a *Cypripedium spp.* extract, characterized by comprising the following steps:

[0008] (a) Take dried medicinal materials from the tomb, crush them, and extract them with a low polarity solvent by cold soaking, infiltration, ultrasonication or hot reflux to obtain component 1;

[0009] (b) The residue obtained in step (a) is extracted with C1-C6 alcohol or alcohol-water solution as extraction solvent, and then extracted by cold soaking, infiltration, ultrasonication or hot reflux, and concentrated to obtain extract.

[0010] (c) The extract obtained in step (b) is separated by column chromatography using macroporous adsorption resin, eluted with a polar solvent, and the eluent is collected to obtain component 2;

[0011] (d) After mixing components 1 and 2, the mixture was concentrated and dried under reduced pressure to obtain the extract of *Cynanchum paniculatum*.

[0012] In the above preparation method, the small polar solvent in step (a) is one or more of diethyl ether, petroleum ether, n-pentane, n-hexane, and dichloromethane, with dichloromethane being preferred.

[0013] In the above preparation method, the C1-C6 alcohol in step (b) is methanol or ethanol, preferably 95% ethanol.

[0014] In the above preparation method, the macroporous adsorption resin in step (c) is selected from one or more of AB-8, SP-70, D101, DM-130, S-8, D3520, D4020, and HP-20, with D101 being preferred. The polar solvent used for elution is ethanol, methanol, or an aqueous solution of ethanol and methanol. Preferably, impurities are first eluted with a 10%-30% ethanol or methanol aqueous solution until no peaks are observed, followed by elution with a 60%-80% ethanol or methanol aqueous solution, and the eluent of the 60%-80% ethanol or methanol aqueous solution is collected. More preferably, impurities are first eluted with a 10%-30% ethanol aqueous solution until no peaks are observed, followed by elution with a 60%-80% ethanol aqueous solution, and the eluent of the 60%-80% ethanol aqueous solution is collected. Even more preferably, impurities are first eluted with a 20% ethanol aqueous solution until no peaks are observed, followed by elution with a 70% ethanol aqueous solution, and the eluent of the 70% ethanol aqueous solution is collected. (The percentages above are volume percentages).

[0015] A specific technical solution for the preparation method of the *Cypripedium spp.* extract according to the present invention includes the following steps:

[0016] (a) Take dried medicinal materials from the tomb, crush them, use dichloromethane as the extraction solvent, and extract by cold soaking, infiltration, ultrasound, or hot reflux. Combine the extracts to obtain component 1.

[0017] (b) Extract the residue obtained in step (a) by hot reflux with 95% ethanol to obtain an extract;

[0018] (c) The extract obtained in step (b) is separated by column chromatography using macroporous adsorption resin. First, impurities are eluted with 10%-30% aqueous ethanol solution, and then eluted with 60%-80% aqueous ethanol solution. The eluent of 60%-80% aqueous ethanol solution is collected to obtain component 2.

[0019] (d) After mixing components 1 and 2, the mixture was concentrated and dried under reduced pressure to obtain the extract of *Cynanchum paniculatum*.

[0020] Another object of the present invention is to provide a *Cynanchum paniculatum* extract prepared by the method described herein.

[0021] Another object of the present invention is to provide the application of the *Phyllostachys edulis* extract described herein in the tobacco industry. The *Phyllostachys edulis* extract can be formulated into flavoring preparations, the flavoring preparations being present in tobacco at a content of 0.001% to 10% (w / w).

[0022] The present invention has the following advantages:

[0023] 1. This invention employs a stepwise extraction method combined with macroporous resin column chromatography to selectively extract the aroma and active ingredients from *Gynostemma pentaphyllum*. The fragrance preparations made from the *Gynostemma pentaphyllum* extract are clear and transparent in appearance, have good flowability, and meet the requirements for use in the tobacco industry and e-cigarette flavorings.

[0024] 2. The extract of *Gnaphalium affine* described in this invention has a special herbal aroma, a refreshing fragrance, and also has woody and balsam-like aromas. When applied to tobacco products, it can enrich and harmonize the characteristics of smoke, increase the sweetness of smoke, and reduce the irritation of smoke.

[0025] 3. Tombstone has multiple activities such as antibacterial and anti-inflammatory, sedative and sleep-aiding effects. When the extract is applied to tobacco products, it can reduce the stimulation and damage of nicotine and tar in tobacco to the respiratory and nervous systems, and improve the function of the respiratory and nervous systems. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0027] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples are commercially available.

[0028] Example 1: Preparation of *Cypripedium spp.* extract

[0029] 1. Take 60g of the medicinal material from the tomb, crush it, and extract it with 480mL of dichloromethane using ultrasound for 1 hour. Filter the extract and extract the residue again with 480mL of dichloromethane using ultrasound for 1 hour. Filter the extract and combine the extracts to obtain component 1.

[0030] 2. Extract the medicinal residue from the tomb head by refluxing with 480 mL of 95% ethanol for 2 hours, filter the extract, and then extract the residue by refluxing with 480 mL of 95% ethanol for 2 hours, filter the extract, combine the extracts, and concentrate under reduced pressure to obtain the extract.

[0031] 3. The obtained extract was adsorbed using macroporous adsorption resin D101. First, impurities were eluted with 20% ethanol aqueous solution until no peaks were observed, and then eluted with 70% ethanol aqueous solution. The 70% ethanol eluent was collected to obtain component 2.

[0032] 4. After mixing components 1 and 2, concentrate and dry under reduced pressure to obtain Tombstone Extract 1.

[0033] Option 2

[0034] 1. Take 60g of the medicinal material from the tomb, crush it, and extract it with 600mL of dichloromethane using ultrasound for 1 hour. Filter the extract and extract the residue again with 600mL of dichloromethane using ultrasound for 1 hour. Filter the extract and combine the extracts to obtain component 1.

[0035] 2. Extract the medicinal residue from the tomb head by refluxing with 500 mL of methanol for 3 hours, filter the extract, and then extract the residue by refluxing with 500 mL of methanol for 3 hours, filter the extract, combine the extracts, and concentrate under reduced pressure to obtain the extract.

[0036] 3. The obtained extract was adsorbed using macroporous adsorption resin D101. First, impurities were eluted with 10% ethanol aqueous solution until no peaks were observed, and then eluted with 60% ethanol aqueous solution. The 60% ethanol eluent was collected to obtain component 2.

[0037] 4. After mixing components 1 and 2, concentrate and dry under reduced pressure to obtain Tombstone Extract 2.

[0038] Option 3

[0039] 1. Take 60g of the medicinal material from the tomb, crush it, and extract it with 400mL of petroleum ether using ultrasound for 1 hour. Filter the extract. Extract the residue with 400mL of petroleum ether using ultrasound for 1 hour. Filter the extract. Combine the extracts to obtain component 1.

[0040] 2. Extract the medicinal residue from the tomb head by refluxing with 550 mL of 95% ethanol for 3 hours, filter the extract, and then extract the residue by refluxing with 550 mL of 95% ethanol for 3 hours, filter the extract, combine the extracts, and concentrate under reduced pressure to obtain the extract.

[0041] 3. The obtained extract was adsorbed using macroporous adsorption resin D101. First, impurities were eluted with 30% ethanol aqueous solution until no peaks were observed, and then eluted with 80% ethanol aqueous solution. The 80% ethanol eluent was collected to obtain component 2.

[0042] 4. After mixing components 1 and 2, concentrate and dry under reduced pressure to obtain Tombstone Extract 3.

[0043] Comparative Example: Preparation of Tombstone Extract

[0044] Option 4

[0045] 1. Take 60g of the medicinal material from the tomb, crush it, and extract it with 500mL of ethyl acetate using ultrasound for 1 hour. Filter the extract. Extract the residue with 500mL of ethyl acetate using ultrasound for 1 hour. Filter the extract. Combine the extracts to obtain component 1.

[0046] 2. Extract the medicinal residue from the tomb head by refluxing with 500 mL of 95% ethanol for 2 hours, filter the extract, and then extract the residue by refluxing with 500 mL of dichloromethane and 95% ethanol for 2 hours, filter the extract, combine the extracts, and concentrate under reduced pressure to obtain the extract.

[0047] 3. The obtained extract was adsorbed using macroporous adsorption resin D101. First, impurities were eluted with 20% ethanol aqueous solution until no peaks were observed, and then eluted with 80% ethanol aqueous solution. The 80% ethanol eluent was collected to obtain component 2.

[0048] 4. After mixing components 1 and 2, concentrate and dry under reduced pressure to obtain Tombstone Extract 4.

[0049] Option 5

[0050] 1. Take 60g of the medicinal material from the tomb, crush it, and extract it with 600mL of petroleum ether using ultrasound for 1 hour. Filter the extract and extract the residue again with 600mL of petroleum ether using ultrasound for 1 hour. Filter the extract and combine the extracts to obtain component 1.

[0051] 2. The medicinal residue from the tomb was extracted by refluxing with 450 mL of 95% ethanol for 2 hours, and the extract was filtered off. The residue was then extracted by refluxing with 450 mL of 95% ethanol in dichloromethane for 2 hours, and the extract was filtered off. The extracts were combined and concentrated under reduced pressure to obtain the extract.

[0052] 3. The obtained extract was adsorbed using macroporous adsorption resin D101. First, impurities were eluted with 10% ethanol aqueous solution until no peaks were observed, and then eluted with 90% ethanol aqueous solution. The 90% ethanol eluent was collected to obtain component 2.

[0053] 4. After mixing components 1 and 2, concentrate and dry under reduced pressure to obtain Tombstone Extract 5.

[0054] Example 2: Preparation of a fragrance preparation made from *Cypripedium sibiricum* extract

[0055] The extracts of *Gnaphalium affine* 1-5 prepared in Example 1 and Comparative Example 1 were added to a mixing tank, and an emulsifier (composed of glyceryl monostearate, glyceryl caprylate, and Tween 80 in a weight ratio of 1:1:0.5) was added. The mixture was stirred and mixed for 1.0 h under a certain temperature condition. The liquid was then cooled and sent to a high-pressure homogenizer, and then uniformly mixed with 80% ethanol solution to obtain the fragrance preparation 1-5 of *Gnaphalium affine* extract.

[0056] The amount of the extract added is 50% of the mass of the fragrance preparation. The amount of emulsifier added is 2% of the mass of the fragrance preparation; the mixing conditions are a temperature of 60-80℃ and a stirring speed of 200-350 rpm; the high-pressure homogenization conditions are a feed temperature of 45℃ and a homogenization pressure of 10-20 MPa. The amount of 80% ethanol solution used is 48% of the mass of the fragrance preparation.

[0057] Example 3: Evaluation of the appearance, solubility, odor, and sensory properties of the fragrance preparation made from the extract of *Gnaphalium affine* according to the present invention.

[0058] Taking the fragrance formulations 1-5 of the extract from *Gnaphalium affine* from Example 2 as examples, their appearance, solubility, odor, and sensory evaluation were conducted, and the results are as follows:

[0059] Appearance: The product is transparent and clear, does not separate into layers when left to stand, and has no impurities or sediment.

[0060] Aroma: It has a unique herbal aroma, a refreshing fragrance, and notes of wood and balm.

[0061] Sensory evaluation test of tobacco coating: Flavoring preparations 1-3 are natural and refreshing, coordinated with the tobacco aroma, have a sweet taste, and can reduce the irritation of tobacco; Flavoring preparations 4 and 5 have a strong medicinal aroma, have a mixed smell, and are not well coordinated with the tobacco aroma.

[0062] The tobacco flavoring preparations 1-5 were uniformly sprayed into the tobacco shreds using an atomization method, with each flavoring preparation comprising 1% of the tobacco shreds by weight. The resulting tobacco shreds were then equilibrated in a constant temperature (22℃±2℃) and constant humidity (RH 60%±5%) chamber for 48 hours, and cigarettes were made accordingly, corresponding to cigarette products 1-5. Sensory quality assessment and stability evaluation were then conducted on each product. The main evaluation indicators were aroma characteristics, harmony, aroma quality, sweetness, aftertaste, off-flavors, and irritation, with each indicator having a maximum score of 10 points.

[0063] A higher aroma characteristic score indicates a more pronounced sensory change in the aroma of tobacco products.

[0064] The higher the score for harmony and aroma quality, the better the harmony between the aroma and the smoke, the purer, fresher, and more appealing the aroma, and the fewer off-flavors.

[0065] A higher sweetness score indicates that the smoke will be less harsh after adding it, resulting in a better comfort experience.

[0066] A higher irritation score indicates lower irritation and a smoother taste, while a low score indicates that the smoke is pungent and irritating to the throat.

[0067] A higher score for impurities indicates a purer smoke after addition, while a lower score indicates a cloudy smoke and unclear aroma.

[0068] A higher aftertaste score indicates that the aroma of the smoke lingers in the mouth for a longer period and has a greater aftertaste.

[0069] Table 1 Average scores of sensory evaluation results

[0070]

[0071] Comparison of blank samples and flavoring preparations 4 and 5 shows that the tobacco flavoring preparations 1-3 processed by the present invention, when added to the cigarette leaf formulation, have the effects of enriching and coordinating the characteristics of smoke, improving the quality and sweetness of smoke, prolonging the aftertaste of smoke, and reducing impurities and irritation. They can effectively improve the quality of smoke and enhance the quality of cigarettes.

[0072] Example 4: Study on the anti-inflammatory activity of the extract from *Cypripedium sibiricum* of the present invention

[0073] Take 1-3 grams of the *Euphorbia hirta* extract obtained in Example 1, dissolve it in DMSO, and use LPS-induced RAW264.7 cell inflammation model to determine the anti-inflammatory activity of the *Euphorbia hirta* extract using NO as an inflammation marker.

[0074] RAW264.7 cells in logarithmic growth phase were trypsinized into a single-cell suspension, seeded into 96-well plates, and cultured at 37°C in a 5% CO2 incubator for 24 hours. After washing the plates with PBS, the cells were divided into groups and administered drugs. The experiment consisted of a model group, a negative control group, and a sample test group. The negative control group received 0.1% DMSO; the model group received the inducing agent LPS; and the sample test group received the inducing agent and different concentrations of *Gnaphalium affine* extract. 24 hours after drug administration, the supernatant was collected, and the NO content in the culture medium was measured using a NO detection kit as an inflammatory marker. The results are shown in Table 2.

[0075] Table 2. Screening of anti-inflammatory activity of *Cynanchum paniculatum* extract obtained from different implementation schemes.

[0076]

[0077] As can be seen from Table 2, the extract of *Cynanchum paniculatum* obtained by this preparation method has certain anti-inflammatory activity.

[0078] Example 5: Study on the sleep-promoting effect of the extract from *Cypripedium sibiricum* of the present invention

[0079] Forty-eight healthy male Kunming mice were randomly divided into three groups: a normal group, a model group, a blank cigarette group, and three "graveyard cigarette" groups (n=8 per group), and housed in separate cages. Except for the normal group, the other mice were administered 300 mg / kg of p-chlorophenylalanine via gavage for two consecutive days to induce an insomnia model. During the modeling period, the normal and model groups were kept in normal conditions without exposure to smoke. Mice in the blank cigarette and graveyard cigarette groups were placed in separate smoking boxes. Two blank cigarette samples or graveyard cigarette products (1-3) from Example 3 were lit each time and placed on a mosquito coil dish in the box, with the lit cigarettes being relit every 10 minutes for 8 consecutive hours daily. After 7 days of smoking, each group of mice was injected intraperitoneally with 50 mg / kg of p-chlorophenylalanine. –1 Sodium pentobarbital. Observe the sleep status of mice. Sleep is indicated by the disappearance of the righting reflex; if the righting reflex disappears for more than 1 minute, the mouse is considered to have entered a sleep state. The mouse wakes up from sleep and rolls over. If the mouse can roll over independently and crawl slowly after being rolled over three times consecutively, it indicates that the mouse is awake. The time from the disappearance of the righting reflex to awakening is the sleep time. Record the sleep latency and sleep duration.

[0080] Table 3. Study on the sleep-inducing effect of *Pseudorasbane* extract on mice after exposure to cigarette smoke.

[0081]

[0082] As shown in Table 3, compared with the model group and the blank cigarette group, the sleep time of mice in the Tombstone cigarette group was significantly prolonged, indicating that the Tombstone extract obtained by this preparation method has a certain sleep-promoting effect.

[0083] The above description is only a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above-described embodiments. All technical solutions that fall within the scope of the present invention are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements or additions or subtractions of steps, changes in the quantity, proportion, or concentration of raw and auxiliary materials without departing from the principle of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. The application of a *Gnaphalium affine* extract in the tobacco industry, characterized in that... The extract from the tombstone was prepared using the following method: (a) Take dried medicinal materials from the tomb, crush them, and use dichloromethane as the extraction solvent. Extract by cold soaking, infiltration, ultrasonication or hot reflux. Combine the extracts to obtain component 1. (b) Extract the residue obtained in step (a) by hot reflux with 95% ethanol to obtain an extract; (c) The extract obtained in step (b) is separated by column chromatography using macroporous adsorption resin. First, impurities are eluted with 10%-30% aqueous ethanol solution, and then eluted with 60%-80% aqueous ethanol solution. The eluent of 60%-80% aqueous ethanol solution is collected to obtain component 2. (d) After mixing components 1 and 2, the mixture was concentrated and dried under reduced pressure to obtain the extract of *Euphorbia humifusa*. The obtained *Euphorbia hirta* extract was added to a mixing tank, and an emulsifier was added. The emulsifier consisted of glyceryl monostearate, glyceryl caprylate, and Tween 80 in a weight ratio of 1:1:0.

5. The mixture was stirred and mixed for 1.0 h under certain temperature conditions. The liquid was then cooled and sent to a high-pressure homogenizer. The mixing conditions were: temperature 60-80℃, stirring speed 200-350 rpm, and high-pressure homogenization conditions: feed temperature 45℃, homogenization pressure 10-20 MPa. The mixture was then uniformly mixed with 80% ethanol solution to obtain a flavoring preparation of the *Euphorbia hirta* extract. The flavoring preparation was applied to the flavoring and seasoning process of tobacco products. The flavoring preparation had a mass percentage content of 0.001%-10% in the tobacco.

2. A flavoring formulation of tobacco extract, characterized in that... It is prepared by the following method: (a) Take dried medicinal materials from the tomb, crush them, and use dichloromethane as the extraction solvent. Extract by cold soaking, infiltration, ultrasonication or hot reflux. Combine the extracts to obtain component 1. (b) Extract the residue obtained in step (a) by hot reflux with 95% ethanol to obtain an extract; (c) The extract obtained in step (b) is separated by column chromatography using macroporous adsorption resin. First, impurities are eluted with 10%-30% aqueous ethanol solution, and then eluted with 60%-80% aqueous ethanol solution. The eluent of 60%-80% aqueous ethanol solution is collected to obtain component 2. (d) After mixing components 1 and 2, the mixture was concentrated and dried under reduced pressure to obtain the extract of *Euphorbia humifusa*. (e) The obtained *Cynanchum paniculatum* extract is added to a mixing tank, and an emulsifier is added. The emulsifier is composed of glyceryl monostearate, glyceryl caprylate, and Tween 80 in a weight ratio of 1:1:0.

5. The mixture is stirred and mixed for 1.0 h under certain temperature conditions. The liquid is then cooled and sent to a high-pressure homogenizer. The mixing conditions are: temperature 60-80℃, stirring speed 200-350 rpm; the high-pressure homogenization conditions are: feed temperature 45℃, homogenization pressure 10-20 MPa; and then uniformly mixed with 80% ethanol solution to obtain the fragrance preparation of *Cynanchum paniculatum* extract.

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