Preparation method of sialic acid extracting solution and application of sialic acid extracting solution in cigarette filter stick

Abstract

The invention provides a preparation method of a sialic acid extracting solution and a cigarette filter stick containing sialic acid obtained by the preparation method. The preparation method of the sialic acid extracting solution comprises the following steps: soaking cubilose raw materials with water to form cubilose feed liquid; the cubilose feed liquid is subjected to heat extraction, the heat extraction temperature ranges from 80 DEG C to 90 DEG C, and the heat preservation time ranges from 50 min to 70 min; and carrying out solid-liquid separation after the thermal extraction is finished to obtain a sialic acid extracting solution. According to the preparation method of the sialic acid extracting solution, a relatively high sialic acid extraction rate can be obtained, the dissolution of impurities is effectively inhibited, and the purity of the sialic acid extracting solution is favorably improved. The cigarette filter stick containing the sialic acid can more effectively adsorb and intercept harmful substances in smoke, meanwhile, the irritation of the smoke can be reduced, the taste of the smoke is milder, and the effects of reducing harm of the smoke and improving the sensory quality are considered.

Description

Technical Field

[0001] This application belongs to the tobacco industry and specifically relates to a method for preparing sialic acid extract and a cigarette filter rod comprising sialic acid obtained by the preparation method. Background Technology

[0002] As consumers increasingly demand both safety and comfort in cigarette smoking, the tobacco industry has conducted extensive and continuous research on traditional cigarette harm reduction technologies. Among various technical approaches, introducing natural plant extracts (such as functional additives from traditional Chinese medicine) into cigarette products has become an important direction, aiming to reduce harmful components in smoke using plant active ingredients or to endow cigarettes with certain physiological regulatory functions. However, the core bottleneck currently facing this technology is that while the introduction of natural plant extracts has a certain potential for harm reduction, their combustion and pyrolysis products can easily alter the original chemical balance of the smoke. These additives often struggle to maintain the original smoothness and aroma characteristics of the smoke while achieving harm reduction effects. Specifically, they can easily introduce off-flavors, increase irritation, or affect the comfort of the aftertaste, making it difficult to effectively unify harm reduction goals with sensory quality. How to coordinate the balance between the two remains a key challenge in the current research and development of tobacco harm reduction technologies. Summary of the Invention

[0003] This application provides a method for preparing sialic acid extract, and a cigarette filter rod comprising sialic acid obtained by the method.

[0004] The first aspect of this application provides a method for preparing sialic acid extract, the method comprising: soaking bird's nest raw material in water to form bird's nest liquid; performing thermal extraction on the bird's nest liquid at a temperature of 80℃-90℃ and a holding time of 50min-70min; and performing solid-liquid separation after thermal extraction to obtain sialic acid extract.

[0005] The above-mentioned method for preparing sialic acid extract involves pre-soaking the bird's nest raw material to ensure sufficient moisture diffusion, providing a foundation for subsequent thermal extraction. Then, a gentle thermal extraction process (80℃-90℃) accelerates the movement of small molecules like sialic acid within the bird's nest particles, thereby increasing its extraction rate. Glycoproteins are less prone to denaturation at this gentle temperature, thus their dissolution rate can be effectively controlled. Larger glycolipid molecules are more difficult to dissolve under these conditions. In summary, extraction at the aforementioned temperature and holding time yields a high sialic acid extraction rate and effectively inhibits the dissolution of impurities, contributing to improved purity of the sialic acid extract.

[0006] In any embodiment of the first aspect, stirring is performed during the soaking process.

[0007] In any embodiment of the first aspect, the ratio of bird's nest raw material to water in the bird's nest liquid is 1g:45mL-1g:60mL, and can be selected as 1g:45mL-1g:50mL.

[0008] In any embodiment of the first aspect, the immersion temperature is 20°C-40°C, optionally 25°C-30°C.

[0009] In any embodiment of the first aspect, the soaking treatment lasts for 2-4 hours, optionally 2.5-3.5 hours.

[0010] In any embodiment of the first aspect, the aforementioned preparation method further includes a process of repeatedly performing thermal extraction n times on the precipitate obtained from solid-liquid separation, where 1 ≤ n ≤ 3.

[0011] In any embodiment of the first aspect, thermal extraction is performed by heating to a target temperature in a water bath.

[0012] In any embodiment of the first aspect, the solid-liquid separation process is centrifugation, and optionally, the centrifugation time is 8 min-12 min and the centrifugation speed is 8000 rpm-12000 rpm.

[0013] In any embodiment of the first aspect, the bird's nest raw material is bird's nest block material that has been crushed and screened, and the average particle size of the bird's nest block material is in the range of 0.5mm-1mm.

[0014] The second aspect of this application provides a cigarette filter rod comprising sialic acid obtained by any of the preparation methods provided in the first aspect.

[0015] Compared to conventional cigarette filters, the cigarette filters containing sialic acid can more effectively adsorb and retain harmful substances (such as aldehydes and carboxylic acids) in cigarette smoke, while reducing irritation and making the smoke taste milder, thus achieving both the effect of reducing the harm of cigarette smoke and improving sensory quality. Attached Figure Description

[0016] To more clearly illustrate the specific embodiments of the present invention, the accompanying drawings used in the specific embodiments will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0017] Figure 1 Volcano diagrams of the differences in cigarette extracts from cigarettes #1 and #3 in this application are shown.

[0018] Figure 2The effect of cigarette extracts (10% concentration) of cigarettes #1, #2 and #3 from this application on the cell viability of human bronchial epithelial cell lines after 48 hours is shown. ns indicates no statistically significant difference, *** indicates p<0.001, and **** indicates p<0.0001.

[0019] Figure 3 The apoptosis rate of human bronchial epithelial cell lines after 48 hours of treatment with cigarette extracts (10% concentration) of cigarettes #1, #2 and #3 in this application is shown. * indicates p<0.1, ** indicates p<0.01, and **** indicates p<0.0001. Detailed Implementation

[0020] The embodiments of this application will be described in further detail below with reference to the examples. The detailed description of the following embodiments is used to illustrate the principles of this application, but should not be used to limit the scope of this application, that is, this application is not limited to the described embodiments.

[0021] Unless otherwise specified, all embodiments and optional embodiments of this application can be combined to form new technical solutions.

[0022] Unless otherwise specified, all technical features and optional technical features of this application may be combined to form new technical solutions.

[0023] Sialic acid is a derivative of neuraminic acid, widely found in various biological tissues with vastly different concentrations. It is most abundant in bird's nest and is also an important bioactive component. Studies have shown that sialic acid has health benefits in areas such as nerve nutrition, immune regulation, and antioxidant activity. Current research on the health value of sialic acid mainly focuses on its effects on gut microbiota improvement, antiviral activity, and anti-aging, with limited application research in the tobacco industry, particularly in reducing harm from cigarettes. In particular, sialic acid in bird's nest is mostly bound to proteins or lipids, existing stably as glycoproteins or glycolipids. Therefore, effectively extracting sialic acid from bird's nest is a crucial prerequisite for its application in cigarette products.

[0024] Currently, the conventional method for extracting sialic acid involves heating water, specifically soaking the bird's nest raw material in hot water, typically at a temperature above 95°C for more than one hour to increase the extraction rate. The applicant's research found that while increasing the temperature improved the extraction rate of sialic acid, it also significantly increased the denaturation and dissolution rate of other components in the bird's nest, such as glycoproteins, and led to the release of more glycolipids. This resulted in a decrease in the purity of the sialic acid in the extracted solution, thus affecting its harm-reducing effect on cigarette smoke.

[0025] In the first embodiment of this application, a method for preparing sialic acid extract is provided. The method includes: soaking bird's nest raw material in water to form bird's nest liquid; performing hot extraction on the bird's nest liquid at a temperature of 80℃-90℃ and a holding time of 50min-70min; and performing solid-liquid separation after hot extraction to obtain sialic acid extract.

[0026] The above-mentioned method for preparing sialic acid extract involves pre-soaking the bird's nest raw material to ensure sufficient moisture diffusion, providing a foundation for subsequent thermal extraction. Then, a gentle thermal extraction process (80℃-90℃) accelerates the movement of small molecules like sialic acid within the bird's nest particles, thereby increasing its extraction rate. Glycoproteins are less prone to denaturation at this gentle temperature, thus their dissolution rate can be effectively controlled. Larger glycolipid molecules are more difficult to dissolve under these conditions. In summary, extraction at the aforementioned temperature and holding time yields a high sialic acid extraction rate and effectively inhibits impurity dissolution, improving the purity of the sialic acid extract. This enhances its tobacco harm reduction effect, more effectively retaining harmful substances in smoke (such as aldehydes and carboxylic acids), while reducing irritation and resulting in a milder, smoother smoke taste, thus better balancing the effects of smoke harm reduction and sensory quality improvement.

[0027] For example, the temperature of the thermal extraction can be 80°C, 82°C, 85°C, 88°C, or 90°C, or any range thereof.

[0028] For example, the duration of thermal extraction can be 50 min, 52 min, 55 min, 60 min, 65 min or 70 min, or any numerical range formed by any two of the above.

[0029] In some embodiments, the bird's nest raw material is crushed and sieved bird's nest chunks, with an average particle size in the range of 0.5mm-1mm. For example, the average particle size of the bird's nest chunks can be 0.5mm, 0.6mm, 0.7mm, 0.8mm, 0.9mm, or 1mm, or any range formed by any two of the above. The average particle size of a single bird's nest chunk refers to the average of its maximum and minimum particle sizes. When the average particle size of the bird's nest chunks obtained from crushing and sieving the raw material is within the above range, it allows for a larger contact area with water, which is beneficial for shortening the soaking time and accelerating the dissolution of sialic acid from the bird's nest chunks; at the same time, the bird's nest chunks are not too finely broken, reducing raw material loss during the crushing and sieving process.

[0030] In some embodiments, the ratio of bird's nest raw material to water in the bird's nest extract is 1g:45mL-1g:60mL. For example, the ratio of bird's nest raw material to water can be 1g:45mL, 1g:50mL, 1g:55mL, or 1g:60mL, or any range thereof. This ratio of bird's nest raw material to water in the above-mentioned bird's nest extract is the material-to-liquid ratio used during hot extraction, and therefore it is related to both the degree of soaking of the bird's nest raw material and the extraction efficiency during hot extraction. When the ratio of bird's nest raw material to water in the bird's nest extract is within the above range, the bird's nest raw material is fully soaked after the soaking treatment, promoting the dissolution of sialic acid. This not only helps to increase the extraction rate of sialic acid but also results in a relatively high concentration of sialic acid in the obtained extract, which is beneficial for direct use.

[0031] In some implementations, the ratio of bird's nest raw material to water in the bird's nest extract is 1g:45mL-1g:50mL, which can better balance the sialic acid extraction rate and the concentration of sialic acid in the extract.

[0032] In some embodiments, the soaking temperature is 20°C-40°C, optionally 25°C-30°C. For example, the soaking temperature can be 20°C, 25°C, 30°C, 35°C, or 40°C, or any range thereof. Soaking at this temperature range allows the bird's nest material to be fully soaked, facilitating subsequent thermal extraction, and also helps dissolve a certain amount of sialic acid. Exemplarily, the soaking process can be completed at room temperature without special temperature control.

[0033] In some embodiments, the soaking treatment lasts for 2-4 hours. For example, the soaking treatment duration can be 2 hours, 2.5 hours, 3 hours, 3.5 hours, or 4 hours, or any range thereof. Optionally, the soaking treatment duration is 2.5-3.5 hours, and more preferably 3 hours, to achieve relatively good extraction rate and extraction efficiency.

[0034] In some embodiments, stirring is performed during the soaking process. Stirring increases the effective contact area between the bird's nest blocks and water, promoting the dissolution and diffusion of sialic acid, which is beneficial for improving the sialic acid extraction rate. For example, and not limitingly, gentle, low-amplitude stirring is performed during the soaking process; alternatively, the soaking process may employ static soaking supplemented with intermittent stirring. Those skilled in the art can select suitable stirring devices according to actual needs, and specific stirring operations can be performed in a manner well known to those skilled in the art, which will not be elaborated here.

[0035] In some embodiments, the preparation method further includes repeating the aforementioned thermal extraction process n times on the precipitate obtained from solid-liquid separation, where 1 ≤ n ≤ 3. Repeating the extraction of the precipitate obtained from solid-liquid separation can further improve the extraction rate of sialic acid. In some preferred embodiments, after obtaining the sialic acid extract and precipitate from solid-liquid separation, a second extraction of the precipitate is performed (i.e., n=1), which can better balance the cost (including raw material cost and time cost) and the extraction effect (including sialic acid extraction rate and extract purity).

[0036] In some embodiments, the aforementioned thermal extraction is performed by heating to the target temperature in a water bath. Gentle and uniform heating via a water bath better suppresses localized overheating and the dissolution of impurities.

[0037] In some embodiments, the solid-liquid separation process is centrifugation. Specifically, a centrifugal processing device (e.g., a high-speed benchtop centrifuge) is used to perform solid-liquid separation on the heat-extracted liquid, the supernatant is sialic acid extract, and the solid precipitate can be used for secondary heat extraction.

[0038] In some implementations, the centrifugation time is 8-12 minutes and the centrifugation speed is 8000-12000 rpm. For example, centrifugation is performed at 10000 rpm for 10 minutes.

[0039] In a second embodiment of this application, a cigarette filter rod is provided, comprising sialic acid obtained according to any of the preparation methods in the first embodiment. This application does not impose any specific limitations on the method of applying sialic acid to the cigarette filter rod. Compared to conventional cigarette filters, the above-mentioned cigarette filter rod containing sialic acid can more effectively adsorb and retain harmful substances (such as aldehydes and carboxylic acids) in cigarette smoke, while reducing irritation and making the smoke taste milder and smoother. Cell model experiments show that the cigarette filter rod containing sialic acid can effectively alleviate the damaging effects of cigarette extracts on cells and reduce cell apoptosis induced by cigarette extracts, thus achieving both the effect of reducing harmful substances in cigarette smoke and improving sensory quality.

[0040] The beneficial effects of this application will be further illustrated below with reference to embodiments and comparative examples, but the scope of the invention is not limited to these embodiments. Unless otherwise specified, the raw materials involved in the following embodiments can all be obtained commercially.

[0041] The instrument models and manufacturers involved in the preparation or testing processes of the following embodiments are as follows:

[0042] Example 1

[0043] In this embodiment, the preparation method of sialic acid extract includes the following steps:

[0044] S1. After crushing the bird's nest using a multi-functional crusher, pass it through a 24-mesh sieve to obtain bird's nest chunks with an average particle size of 0.85mm.

[0045] S2. Weigh 0.5000g of bird's nest material using an electronic balance and place it in a container. Add pure water to the container according to the ratio of bird's nest material to water of 1g:45mL. Soak the bird's nest material for 3 hours at room temperature (25℃ ± 2℃) to form bird's nest liquid.

[0046] S3. Transfer the above bird's nest liquid into a digital display constant temperature water bath and heat it at 90℃ for hot extraction. The heat preservation time during the hot extraction is 1 hour. After the solid-liquid mixture after hot extraction is allowed to stand at room temperature, it is separated into solid and liquid using a high-speed benchtop centrifuge at a speed of 10000 rpm for 10 min to obtain sialic acid extract.

[0047] [Testing Method]

[0048] The sialic acid content is quantitatively detected by high-performance liquid chromatography (HPLC), and the sialic acid extraction rate can then be calculated. Specifically:

[0049] Preparation of the test sample: Transfer the above V mL sialic acid extract to a volumetric flask and make up to a volume of V1 mL; take V3 mL of the extract and mix it with glacial acetic acid at a volume ratio of 1:1, and perform hydrolysis treatment at 100℃ for 10 min; transfer the hydrolyzed sialic acid extract to a volumetric flask and make up to a volume of V2 mL, filter the above hydrolyzed sialic acid extract after volume adjustment using a 0.22 μm microporous membrane, and use the obtained filtrate for high performance liquid chromatography detection, and determine the peak area as A.

[0050] Preparation of standard samples: Weigh 2.00 mg of sialic acid standard (purity greater than 90.0%, purchased from Shanghai Yuanye Biotechnology Co., Ltd.) into a 10 mL volumetric flask, dilute it to the mark with pure water to obtain a sialic acid standard solution with a concentration of C0 = 200 μg / mL; filter the above sialic acid standard solution through a 0.22 μm microporous membrane, and use the filtrate for high performance liquid chromatography detection, and determine the peak area as A0.

[0051] The HPLC detection conditions included: a Thermo BioBasic SCX 250mm*4.6mm, 5μm column; a mobile phase of acetonitrile-0.1% phosphate buffer at a volume ratio of 90:10; a detection wavelength of 205nm; a column temperature of 30℃; an injection volume of 10μL; and a detection time of 15min.

[0052] The sialic acid concentration in the sample to be tested is C = C0 × A / A0, with units of μg / mL; furthermore, the sialic acid extraction rate in this embodiment can be calculated using the following formula:

[0053]

[0054] The concentration of sialic acid in the obtained sialic acid extract is C. m =M×Y×10 6 / V, in μg / mL;

[0055] Where M represents the mass of the bird's nest block, in grams.

[0056] The content of glycoproteins in the prepared sialic acid extract can be determined by the Kjeldahl method. Specifically:

[0057] 1 mL of sialic acid extract sample was placed in a digestion tube, and one catalyst (composed of 0.5 g copper sulfate and 4.5 g potassium sulfate) and 10 mL of concentrated sulfuric acid were added. The tube was then placed in a digestion furnace for digestion. After the temperature of the digestion furnace reached 420℃, digestion continued for 40 min. At this point, the liquid in the digestion tube was green and transparent. After cooling, the tube was connected to an automated Kjeldahl nitrogen analyzer (protein conversion factor of 6.25). Sodium hydroxide solution (concentration of 40%, volume of 10 mL) was added for alkaline distillation. During distillation, ammonia was absorbed by boric acid. The sample was then titrated with a 0.01 mol / L hydrochloric acid standard titration solution. The instrument automatically recorded the titration volume and calculated the protein content.

[0058] The boric acid mentioned above is derived from a boric acid solution containing a mixed indicator of methyl red and bromocresol green (concentration of 2%, volume of 20 mL). The preparation method is as follows: weigh 0.1 g of methyl red and dilute to 100 mL with anhydrous ethanol; weigh 0.1 g of bromocresol green and dilute to 100 mL with anhydrous ethanol; mix 1 part of the above methyl red ethanol solution with 5 parts of the above bromocresol green ethanol solution immediately before use, and then mix the mixed indicator with the boric acid solution at a volume ratio of 1:100.

[0059] Comparative Example 1

[0060] The only difference between this comparative example and Example 1 is that:

[0061] Step S2: Weigh 0.5000g of bird's nest material using an electronic balance and place it in a container. Add pure water to the container according to the ratio of bird's nest material to water of 1g:45mL. After mixing the bird's nest material with water, do not soak it. Proceed directly to step S3.

[0062] The operations and conditions for steps S1 and S3 are the same as in Example 1.

[0063] Example 2

[0064] The only difference between this embodiment and Embodiment 1 is that:

[0065] Step S2: Weigh 0.5000g of bird's nest material using an electronic balance and place it in a container. Add pure water to the container according to the ratio of bird's nest material to water of 1g:45mL. Soak the bird's nest material at room temperature (25℃ ± 2℃) for 1 hour to form bird's nest liquid.

[0066] The operations and conditions for steps S1 and S3 are the same as in Example 1.

[0067] Example 3

[0068] The only difference between this embodiment and Embodiment 1 is that:

[0069] Step S2: Weigh 0.5000g of bird's nest material using an electronic balance and place it in a container. Add pure water to the container according to the ratio of bird's nest material to water of 1g:45mL. Soak the bird's nest material at room temperature (25℃ ± 2℃) for 2 hours to form bird's nest liquid.

[0070] The operations and conditions for steps S1 and S3 are the same as in Example 1.

[0071] Example 4

[0072] The only difference between this embodiment and Embodiment 1 is that:

[0073] Step S2: Weigh 0.5000g of bird's nest material using an electronic balance and place it in a container. Add pure water to the container according to the ratio of bird's nest material to water of 1g:45mL. Soak the bird's nest material at room temperature (25℃ ± 2℃) for 4 hours to form bird's nest liquid.

[0074] The operations and conditions for steps S1 and S3 are the same as in Example 1.

[0075] Example 5

[0076] The only difference between this embodiment and Embodiment 1 is that:

[0077] Step S2: Weigh 0.5000g of bird's nest material using an electronic balance and place it in a container. Add pure water to the container according to the ratio of bird's nest material to water of 1g:45mL. Soak the bird's nest material at room temperature (25℃ ± 2℃) for 5 hours to form bird's nest liquid.

[0078] The operations and conditions for steps S1 and S3 are the same as in Example 1.

[0079] The sialic acid concentration was determined according to the test method in Example 1, and the sialic acid extraction rate of Comparative Example 1 and Examples 2-5 was calculated. The results are shown in Table 1.

[0080] Table 1

[0081] As shown in Table 1, soaking the bird's nest raw materials significantly improves the sialic acid extraction rate. Soaking time is typically between 1 and 3 hours. Increased soaking time promotes thorough saturation of the bird's nest raw materials, leading to a higher sialic acid extraction rate. However, after soaking for more than 3 hours, the sialic acid extraction rate plateaus and no longer increases further. Furthermore, the protein content in the sialic acid extracts from Examples 1 to 5 and Comparative Example 1 was found to be essentially the same.

[0082] Comparative Example 2

[0083] The only difference between this comparative example and Example 1 is that:

[0084] Step S3: Transfer the above bird's nest liquid into a digital display constant temperature water bath and heat it at 100°C for hot extraction. The hot extraction time is 1 hour. The subsequent processing is the same as in Example 1.

[0085] The operations and conditions for steps S1 and S2 are the same as in Example 1.

[0086] Comparative Example 3

[0087] The only difference between this comparative example and Example 1 is that:

[0088] Step S3: Transfer the above bird's nest liquid into a digital display constant temperature water bath and heat it at 70°C for hot extraction. The hot extraction time is 1 hour. The subsequent processing is the same as in Example 1.

[0089] The operations and conditions for steps S1 and S2 are the same as in Example 1.

[0090] Example 6

[0091] The only difference between this embodiment and Embodiment 1 is that:

[0092] Step S3: Transfer the above bird's nest liquid into a digital display constant temperature water bath and heat it at 80°C for hot extraction. The hot extraction time is 1 hour. The subsequent processing is the same as in Example 1.

[0093] The operations and conditions for steps S1 and S2 are the same as in Example 1.

[0094] The sialic acid concentration was determined according to the test method in Example 1, and the sialic acid extraction rates of Comparative Examples 2, 3 and Example 6 were calculated. The results are shown in Table 2.

[0095] Table 2

[0096] As shown in Table 2, the selection of thermal extraction temperature significantly affects the sialic acid extraction rate. When the thermal extraction temperature is between 80℃ and 90℃, the sialic acid extraction rate is significantly higher than that of Comparative Example 3, while the protein concentration is significantly lower than that of Comparative Example 2. This indicates that the temperatures in Examples 1 and 2 are beneficial for increasing the dissolution of sialic acid and reducing the dissolution of protein.

[0097] Example 7

[0098] The only difference between this embodiment and Embodiment 1 is that:

[0099] Step S2: Weigh 0.5000g of bird's nest material using an electronic balance and place it in a container. Add pure water to the container according to the ratio of bird's nest material to water of 1g:40mL. Soak the bird's nest material at room temperature (25℃ ± 2℃) for 3 hours to form bird's nest liquid.

[0100] The operations and conditions for steps S1 and S3 are the same as in Example 1.

[0101] Example 8

[0102] The only difference between this embodiment and Embodiment 1 is that:

[0103] Step S2: Weigh 0.5000g of bird's nest material using an electronic balance and place it in a container. Add pure water to the container according to the ratio of bird's nest material to water of 1g:50mL. Soak the bird's nest material at room temperature (25℃ ± 2℃) for 3 hours to form bird's nest liquid.

[0104] The operations and conditions for steps S1 and S3 are the same as in Example 1.

[0105] Example 9

[0106] The only difference between this embodiment and Embodiment 1 is that:

[0107] Step S2: Weigh 0.5000g of bird's nest material using an electronic balance and place it in a container. Add pure water to the container according to the ratio of bird's nest material to water of 1g:60mL. Soak the bird's nest material at room temperature (25℃ ± 2℃) for 3 hours to form bird's nest liquid.

[0108] The operations and conditions for steps S1 and S3 are the same as in Example 1.

[0109] The sialic acid concentration was determined according to the test method in Example 1, and the sialic acid extraction rate of Examples 7-9 was calculated. The results are shown in Table 3.

[0110] Table 3

[0111] The ratio of bird's nest material to water, i.e., the material-liquid ratio used in thermal extraction, is shown in Table 3. As the ratio of bird's nest material to water is adjusted from 1g:40mL to 1g:45mL and then to 1g:50mL, the relative amount of water gradually increases, and the extraction rate of sialic acid also continuously improves. This may be related to the more thorough soaking of the bird's nest material, resulting in higher extraction efficiency. However, when the relative amount of water is further increased, for example, compared with Example 8 in Example 9, the difference in the extraction rate of sialic acid is minimal, meaning the amount of sialic acid extracted is basically the same. Moreover, in Example 9, the relative amount of water is greater, resulting in a lower concentration of sialic acid in the extract. This indicates that there is a suitable range for the material-liquid ratio, within which both the extraction rate and the concentration of sialic acid in the extract can be considered.

[0112] Comparative Example 4

[0113] The only difference between this comparative example and Example 1 is that:

[0114] Step S3: Transfer the above bird's nest liquid into a digital display constant temperature water bath and heat it at 90°C for hot extraction. The hot extraction holding time is 0.5 hours. The subsequent processing is the same as in Example 1.

[0115] The operations and conditions for steps S1 and S2 are the same as in Example 1.

[0116] Comparative Example 5

[0117] The only difference between this comparative example and Example 1 is that:

[0118] Step S3: Transfer the above bird's nest liquid into a digital display constant temperature water bath and heat it at 90°C for hot extraction. The hot extraction time is 1.5 hours. The subsequent processing is the same as in Example 1.

[0119] The operations and conditions for steps S1 and S2 are the same as in Example 1.

[0120] The sialic acid concentration was determined according to the test method in Example 1, and the sialic acid extraction rates of Comparative Examples 4 and 5 were calculated. The results are shown in Table 4.

[0121] Table 4

[0122] As shown in Table 4, a heat extraction holding time of about 1 hour is preferred to obtain a high sialic acid extraction rate. A shorter holding time (such as in Comparative Example 4) may result in incomplete leaching of sialic acid, while a longer holding time does not improve the sialic acid extraction rate but instead increases the risk of glycoprotein dissolution.

[0123] Example 10

[0124] In this embodiment, the preparation method of sialic acid extract includes the following steps:

[0125] Step S1 is the same as in Example 1;

[0126] Step S2: Weigh 0.5000g of bird's nest material using an electronic balance and place it in a container. Add pure water to the container according to the ratio of bird's nest material to water of 1g:50mL. Soak the bird's nest material at room temperature (25℃ ± 2℃) for 3 hours to form bird's nest liquid.

[0127] Step S3: Transfer the above bird's nest liquid into a digital display constant temperature water bath and heat it at 90℃ for hot extraction. The heat preservation time during the hot extraction is 1 hour. After the solid-liquid mixture after hot extraction is allowed to stand at room temperature, it is separated into solid and liquid by using a high-speed benchtop centrifuge at a speed of 10000 rpm for 10 min to obtain sialic acid extract and precipitate.

[0128] Step S4: Reserve the sialic acid extract for later use. Mix the precipitate and water at a ratio of 1g:50mL to form a secondary bird's nest extract. Transfer the extract to a digital display constant temperature water bath and heat it at 90℃ for 1 hour. After the solid-liquid mixture is allowed to stand at room temperature, use a high-speed benchtop centrifuge to separate the solid and liquid components. The centrifugation speed is 10000rpm and the centrifugation time is 10min to obtain the secondary sialic acid extract and the secondary precipitate.

[0129] Step S5: Reserve the secondary sialic acid extract for later use. Mix the secondary precipitate and water at a ratio of 1g:50mL to form the tertiary bird's nest extract. Transfer the extract to a digital display constant temperature water bath and heat it at 90℃ for 1 hour. After the solid-liquid mixture is allowed to stand at room temperature, it is then separated into solid and liquid components using a high-speed benchtop centrifuge at 10000rpm for 10min to obtain the tertiary sialic acid extract and the tertiary precipitate.

[0130] The sialic acid concentration in the three sialic acid extracts in Example 10 was determined according to the test method in Example 1, and the sialic acid extraction rate was calculated for each of the three extractions, as shown in Table 5.

[0131] Table 5

[0132] As shown in Table 5, under constant process conditions, the sialic acid extraction rate gradually decreases with increasing extraction frequency, and the decrease is significant. Using a two-stage extraction method can better balance cost (including raw material cost and time cost) and extraction efficiency.

[0133] Cigarette Harm Reduction Effect Test

[0134] The sialic acid extracts obtained in the examples and comparative examples were loaded onto particles and then placed in cigarette filter rods, specifically including:

[0135] Cellulose diacetate powder, hydroxypropyl methylcellulose, and water were mixed to form matrix particles with a particle size of 0.5 mm. Sialic acid extract was sprayed onto the surface of the matrix particles using an atomization and fragrance method at a pressure of 0.1 MPa and a mass ratio of sialic acid extract to matrix particles of 1:4. The particles coated with sialic acid extract were then equilibrated at 30°C and 30% RH for 72 hours, and then sieved through a 20-40 mesh sieve to obtain sialic acid-loaded particles with a moisture content of 7.5%.

[0136] Sialic acid-loaded particles are applied to the filter rod tow at a ratio of 2 mg / mm to form a sialic acid-loaded material rod. This material rod is then combined with a cellulose acetate filter rod using a molding device to obtain a composite filter rod with a total length of 30 mm. The sialic acid-loaded material rod is 10 mm long, and the cellulose acetate filter rod is 20 mm long. This composite filter rod is then assembled into cigarettes for later use.

[0137] Blank samples were prepared as controls using the same processing method: the sialic acid extract was replaced with pure water, and sialic acid-free particles were prepared using the same matrix particles. The moisture content of the particles was kept at 7.5%. The sialic acid-free particles were applied to the filter rod bundle at a ratio of 2 mg / mm to obtain a sialic acid-free filter rod. This filter rod was then combined with a cellulose acetate filter rod to obtain a composite filter rod with a total length of 30 mm. The sialic acid-loaded material rod was 10 mm long, and the cellulose acetate filter rod was 20 mm long. The composite filter rod was then assembled into cigarettes for later use.

[0138] The prepared cigarettes were subjected to sensory evaluation according to the standard method of GB 5606.4-2005 "Cigarettes Part 4: Sensory Technical Requirements". The evaluation results showed that the cigarettes had a better smoothness and the dryness and irritation were effectively suppressed compared with the blank sample. In particular, the cigarettes containing the sialic acid extract obtained in Example 1 had a stronger smoothness and a lighter dryness and irritation.

[0139] Cigarettes prepared using the sialic acid extracts from Example 1 and Comparative Example 2 were designated as Cigarette #1 and Cigarette #2, respectively, while the cigarette prepared from the blank sample was designated as Cigarette #3. Cigarette extracts were prepared using Cigarette #1, Cigarette #2, and Cigarette #3, and smoke component analysis and cell experiments were performed.

[0140] Preparation of Cigarette Cigarette Extract (CSE):

[0141] Add 10 mL of culture medium to the drainage bag on a clean bench, seal the drainage bag, and connect the device; light a cigarette and draw a 50 mL syringe at a constant speed. Turn the three-way valve to ensure unobstructed flow between the syringe and the drainage bag, injecting smoke into the drainage bag until the cigarette burns out within approximately 5 minutes. Seal the drainage bag and use sealing film to seal it. Shake the sealed drainage bag in the dark for 90 minutes at 100 rpm; collect the cigarette extract from the drainage bag, test its pH value, and adjust the pH to approximately 7.4 with NaOH; transfer to a clean bench and filter through a 0.22 μm filter membrane. At this point, the concentration of the cigarette extract should be 100%. Aliquot the extract into 300 μL brown 1.5 mL EP tubes and store at -80°C.

[0142] 1) Flue gas composition analysis

[0143] The main differences in the components of the three flue gases were detected by GC / MS, and the classification results of the compound types are shown in Table 6.

[0144] Table 6

[0145] Compared to the blank sample cigarette #3, the number of hydrocarbon species in the cigarette extracts of cigarettes #1 and #2 increased, while the number of aldehydes and carboxylic acids decreased. The difference in smoke composition between cigarettes #1 and #3 was more pronounced; cigarette #1 showed an increase of 45 hydrocarbon species compared to #3, while the number of aldehydes and carboxylic acids decreased by 14 and 10 species, respectively. Aldehydes (especially lower aldehydes) are the main components causing irritation and spiciness in the mouth and throat, while carboxylic acids contribute to the sour taste and irritation. A decrease in these two types of substances generally indicates a milder, less irritating cigarette smoke, but may also result in a slight reduction in the richness of the cigarette's aroma.

[0146] in addition, Figure 1 A volcano plot of the differential substances in the smoke of cigarettes #1 and #3 is provided, where the horizontal axis is log2(FC), where FC represents the difference factor, and the vertical axis is -lg(p-value). The smaller the p-value, the larger the statistical difference between the two. Figure 1 It was found that there were 185 statistically significant differences between cigarette #1 and cigarette #3, accounting for approximately 1.52% of the total 12,201 substances detected in the two types of smoke. Among them, 58 substances were upregulated and 127 substances were downregulated.

[0147] Among the substances whose concentrations were increased was sorbic acid, possibly due to the addition of sialic acid-loaded particles to the filter rod, which altered the retention efficiency of sorbic acid. The substance with a significant decrease was undecenoal, which may have been adsorbed by sialic acid-loaded particles, or the particles may have inhibited its formation. A reduction in the content of these long-chain aldehydes usually indicates a decrease in irritating components in the smoke. Indoline, triazole, and undecenoal require higher temperatures to form; therefore, the decrease in the concentration of nitrogen-containing heterocyclic compounds such as 1,1,3-trimethyl-2-methyleneindoline and 1-methyl-1h-1,2,4-triazole-5-amine suggests a possible reduction in combustion temperature. This, in turn, reduces harmful substances produced by high-temperature pyrolysis in the smoke, making the cigarette smoke chemically milder and potentially reducing direct irritation to the respiratory system and subsequent inflammatory burden. The above analysis of smoke components indicates that adding sialic acid-loaded particles to the filter rod can, to some extent, reduce some irritating and harmful substances in the smoke.

[0148] 2) Cell model experiments

[0149] A human bronchial epithelial cell line model (HBE135-E6E7) was constructed. Cigarette extracts from cigarettes #1, #2, and #3 were applied to the human bronchial epithelial cell line to observe the effects of cigarette smoke on cellular oxidative stress and intracellular environment.

[0150] Cell viability assay

[0151] Cell viability was determined using the CCK-8 assay. HBE135-E6E7 cells were cultured at approximately 1.0 × 10⁻⁶ cells per cell line. 4 Cells were seeded at a density of 10 cells / well in 96-well plates. After overnight adhesion, cigarette extracts (10% concentration each) from cigarette #1, cigarette #2, and cigarette #3 were added to each well for 48 h. After treatment, 10 μL of CCK-8 working solution was added to each well, and the plates were incubated at 37°C and 5% CO2 for 2 h. The absorbance (OD) value was read at 450 nm using a microplate reader. Cells not treated with cigarette extract were designated as the control group. Cell viability was calculated as (OD of treated group / OD of control group) × 100%. Results are shown in the figure. Figure 2 .

[0152] Figure 2The cell viability test data shown indicate that the cell viability after treatment with cigarette extract from cigarette #1 was higher than that of cigarette #2 and significantly higher than that of cigarette #3. Under the same cigarette extract concentration, the cell viability after treatment with cigarette extract from cigarette #1 remained at approximately 64%, which was statistically significantly different from that after treatment with cigarette extracts from cigarettes #2 and #3. These results suggest that the particles prepared from the sialic acid extract of Example 1, when used in cigarette filters, can alleviate cell damage caused by cigarette extracts and reduce oxidative stress in cells.

[0153] Apoptosis detection

[0154] Apoptosis rate was detected by Annexin V-FITC / PI double staining flow cytometry. HBE135-E6E7 cells were cultured at 2×10⁻⁶ cells / year. 5 Cells were seeded at a density of 10 cells / well in 6-well plates. After adhesion, cigarette extracts (10% each) of cigarette #1, #2, and #3 were added for 48 h. Suspension and adherent cells were collected and combined, washed twice with PBS (0.01 M, pH 7.4), resuspended in 1× binding buffer, and incubated with 5 μL Annexin V-FITC reagent and 5 μL propidium iodide (PI) at room temperature in the dark for 10 min. Samples were analyzed by flow cytometry within 1 h. The specific procedures for analysis followed standard methods. The apoptosis rate was calculated as (number of apoptotic cells / total number of cells) × 100%. Results are shown in [Figure number missing]. Figure 3 .

[0155] Figure 3 The apoptosis detection data shown indicate that, under the same concentration of cigarette extracts, the apoptosis rates after treatment with cigarette extracts of cigarette #1, cigarette #2, and cigarette #3 were 11%, 29%, and 43%, respectively (corresponding to...). Figure 3 Among the samples (1#, 2#, and 3#), the apoptosis rate of cells in sample 1# was significantly different from that in sample 3#, and the apoptosis rate of cells in sample 1# was also significantly lower than that in sample 2#, with the difference also being statistically significant. These results indicate that the particles prepared from the sialic acid extract of Example 1, when used in cigarette filter rods, can significantly reduce cigarette extract-induced apoptosis and alleviate cell damage.

[0156] It should be noted that this application is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments with the same structure and effect as the technical concept within the scope of this application are included in the technical scope of this application. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of this application, are also included in the scope of this application.

Claims

1. A method for preparing sialic acid extract, characterized in that, The preparation method includes: The raw bird's nest is soaked in water to form a bird's nest solution; The bird's nest liquid is subjected to thermal extraction at a temperature of 80℃-90℃ and a holding time of 50min-70min. After the thermal extraction is completed, solid-liquid separation is performed to obtain sialic acid extract.

2. The preparation method according to claim 1, characterized in that, Stirring is performed during the soaking process.

3. The preparation method according to claim 1 or 2, characterized in that, The ratio of bird's nest raw material to water in the bird's nest liquid is 1g:45mL-1g:60mL, and can be selected as 1g:45mL-1g:50mL.

4. The preparation method according to any one of claims 1 to 3, characterized in that, The soaking temperature is 20℃-40℃, and can be selected as 25℃-30℃.

5. The preparation method according to any one of claims 1 to 4, characterized in that, The soaking treatment lasts for 2-4 hours, and can be selected as 2.5-3.5 hours.

6. The preparation method according to any one of claims 1 to 5, characterized in that, The preparation method further includes repeatedly performing the thermal extraction process on the precipitate obtained from the solid-liquid separation n times, where 1≤n≤3.

7. The preparation method according to any one of claims 1 to 6, characterized in that, The thermal extraction is performed by heating to the target temperature in a water bath.

8. The preparation method according to any one of claims 1 to 7, characterized in that, The solid-liquid separation process is centrifugation. Optionally, the centrifugation time is 8 min-12 min and the centrifugation speed is 8000 rpm-12000 rpm.

9. The preparation method according to any one of claims 1 to 8, characterized in that, The bird's nest raw material is bird's nest block material that has been crushed and screened, and the average particle size of the bird's nest block material is in the range of 0.5mm-1mm.

10. A cigarette filter rod comprising sialic acid obtained by any one of claims 1 to 9.

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