Method for purifying polycyclic aromatic hydrocarbons by using gynostemma pentaphyllum extract

Abstract

The application provides a method for purifying polycyclic aromatic hydrocarbons (PAHs) in gynostemma extract, and the presence of PAHs in gynostemma extract is found for the first time, and the PAHs in the gynostemma extract are removed in a targeted manner, wherein the purification process is to remove the PAHs in the extract by using activated carbon under specific conditions, and the gynostemma extract is dissolved more fully by selecting ethanol solution as a solvent and controlling the dissolving process, so as to provide a basis for subsequent purification, and the activated carbon purification process is optimized, the amount of activated carbon and the purification temperature are controlled, and the content of PAHs in the gynostemma extract is greatly reduced by adjusting the overall solvent process and the purification process, and the loss of the gynostemma extract is reduced. The whole process is simple, cost-effective, environmentally friendly, can obviously improve the quality of the extract, and meets the strict quality standards.

Description

Technical Field

[0001] This application relates to the field of natural plant extraction, and in particular to a method for purifying Gynostemma pentaphyllum extract, specifically a method for purifying polycyclic aromatic hydrocarbons from Gynostemma pentaphyllum extract. Background Technology

[0002] Gynostemma pentaphyllum, a traditional Chinese medicine, has gained widespread recognition for its medicinal value. This plant contains various bioactive components, including saponins, flavonoids, and polysaccharides, which have demonstrated significant antioxidant, anti-inflammatory, and anti-tumor properties in modern medical research. Furthermore, Gynostemma pentaphyllum is also believed to enhance immunity and improve cardiovascular health, potentially playing a positive role in promoting human health and preventing disease. Given these health benefits, Gynostemma pentaphyllum has attracted widespread attention from researchers worldwide and has become a research hotspot.

[0003] Due to its strong adaptability and short growth cycle, Gynostemma pentaphyllum absorbs polycyclic aromatic hydrocarbons (PAHs) from the soil during cultivation. Solvents used to extract saponins from Gynostemma pentaphyllum include water, ethanol, and methanol. During the extraction process, PAHs are extracted from the plant, resulting in a small amount of PAHs in the final extract.

[0004] Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous organic pollutants in the environment, primarily originating from the incomplete combustion of fossil fuels, including coal, oil, and natural gas. These compounds consist of two or more benzene rings, exhibiting high chemical stability and hydrophobicity, resulting in extremely slow degradation in the natural environment and easy accumulation in organisms. PAHs pose a potential threat to human health due to their carcinogenic, mutagenic, and teratogenic properties.

[0005] Currently, no existing technology has proposed a process for removing polycyclic aromatic hydrocarbons (PAHs) from Gynostemma pentaphyllum extract. The inventors intend to remove PAHs from Gynostemma pentaphyllum extract to obtain a higher quality extract while avoiding loss of extract during purification. Summary of the Invention

[0006] To address the shortcomings of existing extraction processes for Gynostemma pentaphyllum and the presence of impurities such as polycyclic aromatic hydrocarbons (PAHs) in the extracts, this invention provides a method for purifying PAHs from Gynostemma pentaphyllum extract. The method involves purifying the extracted Gynostemma pentaphyllum to remove PAHs, making the extract more environmentally friendly and healthier. Furthermore, this purification process minimizes the loss of Gynostemma pentaphyllum extract during purification.

[0007] One of the concepts of this invention is to provide an extraction process for Gynostemma pentaphyllum, which uses pulverized Gynostemma pentaphyllum powder to extract it multiple times with ethanol (stepwise extraction). The extract is then filtered and concentrated to finally obtain Gynostemma pentaphyllum extract. The provided extraction method for Gynostemma pentaphyllum has high extraction efficiency and minimal loss of Gynostemma pentaphyllum saponins.

[0008] Another aspect of this invention is the provision of a method for purifying polycyclic aromatic hydrocarbons (PAHs) from Gynostemma pentaphyllum extract. This purification method employs a comprehensive adsorption technique, effectively removing PAHs from the extract by adjusting adsorption conditions, thus achieving the goal of purifying the extract and avoiding loss of extract during the purification process. This purification method not only removes contaminants (PAHs) from the extract but also has minimal impact on other beneficial components. The method is simple and ensures the quality and efficacy of the extract.

[0009] Specifically, the present invention provides a method for extracting Gynostemma pentaphyllum, comprising the following steps:

[0010] Step S1: Extraction. Add Gynostemma pentaphyllum to an ethanol solution, heat, and reflux to extract the extract.

[0011] Step S2: Concentration, concentrate the extract from step S1 to 1.5 to 2.0 times the weight volume of the raw material, and control the Baumé degree to be >8;

[0012] Step S3: Dilution and centrifugation. Dilute the concentrated liquid obtained in step S2 with hot water at a volume of 6 to 12 times the weight of the raw material. After dilution, centrifuge and collect the centrifuged liquid for later use.

[0013] Step S4: Column loading and elution: Load the centrifuged liquid obtained in step S3 onto the column at a flow rate of 0.5–0.75 column volumes / h; after the feed liquid has been completely loaded, wash with water at a flow rate of 0.5–0.8 column volumes / h for 1.5 column volumes; after washing, elute with 2.0–2.5 column volumes of ethanol at a flow rate of 0.5–0.65 column volumes / h.

[0014] Step S5: Concentration and drying of the eluent. The eluent is transferred to a single-effect concentrator for concentration. After concentration, the concentrate is transferred to a spray tank for spray drying to obtain Gynostemma pentaphyllum extract.

[0015] Furthermore, in step S1, the Gynostemma pentaphyllum is pulverized Gynostemma pentaphyllum;

[0016] The extraction in step S1 is a stepwise extraction, which includes at least two reflux extractions;

[0017] In the reflux extraction, 6 to 12 times the volume of ethanol extract based on the weight of the raw material is added.

[0018] The concentration of the ethanol is 65-75%;

[0019] The extraction time is 1 to 3 hours;

[0020] Furthermore, the extraction in step S1 includes three reflux extractions;

[0021] The first reflux extraction was performed by adding 70±2% ethanol at 8 times the weight of the raw material, stirring, heating to a gentle boil, controlling the temperature at 75-85℃, reflux extraction for 1.5 hours, filtering through a 300-mesh filter, and collecting the filtrate.

[0022] The second reflux extraction was performed by adding 70±2% ethanol (6 times the weight of the raw material) to the residue from the first reflux extraction, stirring, heating to a gentle boil, controlling the temperature at 70-85℃, reflux extraction for 1.0 h, filtering through a 300 mesh filter, and collecting the filtrate.

[0023] The third reflux extraction was performed by adding 70±2% ethanol (6 times the weight of the raw material) to the residue from the second reflux extraction, stirring, heating to a gentle boil, controlling the temperature at 70-85℃, reflux extraction for 1.0 h, and then filtering.

[0024] Furthermore, the specific operation of step S2 is to concentrate the extract obtained in step S1 into a single-effect concentrator. When the volume of the concentrate reaches 1.0 to 1.5 times the weight of the raw material and the Baumé degree is >8, water is added and concentrated until there is no alcohol odor. The amount of water added is 3 to 4 times the volume of the concentrate. Finally, the concentrate is concentrated to 1.5 to 2.0 times the weight of the raw material and the Baumé degree is >8. The concentration temperature does not exceed 70°C and the vacuum degree is -0.04 to -0.10 MPa.

[0025] Furthermore, the specific operation of step S3 is as follows: transfer the concentrated liquid obtained in step S2 to a preparation tank, circulate hot water (60-100℃) at 5-6 times the weight of the raw material in the concentrator for 5-10 minutes, make up to 8 times the weight of the raw material, stir evenly, centrifuge, and collect the centrifuged liquid for later use.

[0026] Furthermore, in step S5, the vacuum degree is controlled at -0.04 to -0.10 MPa during concentration, and the temperature is controlled not to exceed 70°C; for spray drying, the inlet air temperature of the spray dryer is 180 to 190°C, and the outlet air temperature is 85 to 110°C.

[0027] On the other hand, the present invention also provides a method for purifying polycyclic aromatic hydrocarbons (PAHs) from Gynostemma pentaphyllum extract, wherein the purification method involves dissolving the Gynostemma pentaphyllum extract in an ethanol solution, adding activated carbon, and adsorbing the PAHs.

[0028] The concentration of the ethanol solution is 60-80%, preferably 60-75%, more preferably 60-70%, and even more preferably 70%.

[0029] The adsorption time is 0.1 to 1.5 hours, preferably 0.5 to 1 hour, and more preferably 0.5 hours.

[0030] The amount of activated carbon used is 1-3% relative to the mass of the Gynostemma pentaphyllum extract alcohol solution, preferably 2-3%, and more preferably 2.5%.

[0031] The adsorption temperature is 45–55°C; preferably 50°C.

[0032] The volume of the ethanol solution used is 40 to 50 times the mass of the Gynostemma pentaphyllum extract, preferably 40 times.

[0033] Furthermore, the method for purifying polycyclic aromatic hydrocarbons from Gynostemma pentaphyllum extract involves dissolving the extract in an ethanol solution. The dissolution of the extract in the ethanol solution is a stepwise dissolution, including at least two dissolution operations.

[0034] The aforementioned at least two dissolution operations involve dissolving the Gynostemma pentaphyllum extract in a portion of an ethanol solution, and then adding the remaining ethanol solution in batches after dissolution is complete.

[0035] The dissolution of the Gynostemma pentaphyllum extract in the ethanol solution involves two dissolution processes, which include:

[0036] The ethanol solution is divided into solvent A and solvent B, with a volume ratio of solvent A to solvent B of 1:0.5 to 1.5.

[0037] The Gynostemma pentaphyllum extract was dissolved in solvent A. After dissolution, an intermediate state Gynostemma pentaphyllum extract solution was obtained. Then, solvent B was added to the intermediate state Gynostemma pentaphyllum extract solution to dissolve it, and finally, a Gynostemma pentaphyllum extract solution was obtained.

[0038] Specifically, this invention provides a method for purifying polycyclic aromatic hydrocarbons (PAHs) from Gynostemma pentaphyllum extract. The method involves dissolving the Gynostemma pentaphyllum extract in a 60-70% ethanol solution to obtain an alcoholic solution of the extract, with a mass-to-volume ratio of extract to ethanol of 1:40-50. Activated carbon is then added, with the amount of activated carbon being 2-3% of the mass of the dissolved Gynostemma pentaphyllum extract in the ethanol solution. The mixture is then shaken for adsorption.

[0039] Furthermore, the operation of dissolving the Gynostemma pentaphyllum extract in a 60-70% ethanol solution is a stepwise dissolution, specifically including at least two dissolutions;

[0040] Furthermore, the at least two dissolutions involve dissolving the Gynostemma pentaphyllum extract in a portion of a 60-70% ethanol solution, and after complete dissolution, adding the remaining portion of the 60-70% ethanol solution in batches to complete the dissolution.

[0041] Furthermore, the dissolution of the Gynostemma pentaphyllum extract in a 60-70% ethanol solution is a two-stage dissolution process. The two-stage dissolution process involves dividing the 60-70% ethanol solution into solvent A and solvent B, with a volume ratio of solvent A to solvent B of 1:0.5-1.5.

[0042] The Gynostemma pentaphyllum extract is dissolved in solvent A. After dissolution, an intermediate state Gynostemma pentaphyllum extract solution is obtained. Then, solvent B is added to the aforementioned intermediate state Gynostemma pentaphyllum extract solution for dissolution, and finally, a Gynostemma pentaphyllum extract solution is obtained.

[0043] Furthermore, the volume of the 60-70% ethanol solution used is 40-50 times the mass of the Gynostemma pentaphyllum extract.

[0044] Preferably, the 60-70% ethanol is divided into solvent A and solvent B, wherein the volume ratio of solvent A to solvent B is 1:1.

[0045] More preferably, the volume of the 60-70% ethanol solution used is 40 times the mass of the Gynostemma pentaphyllum extract; the 60-70% ethanol solution is divided into solvent A and solvent B, wherein the volume of solvent A is 20 times the mass of the Gynostemma pentaphyllum extract, and the volume of solvent B is 20 times the mass of the Gynostemma pentaphyllum extract.

[0046] Compared to existing technologies, this invention reveals for the first time the presence of polycyclic aromatic hydrocarbons (PAHs) in Gynostemma pentaphyllum extract and conducts in-depth research on them. This study proposes a method for adsorbing and removing PAHs from Gynostemma pentaphyllum extract using activated carbon, particularly targeting benzo[a]pyrene, to achieve purification of the extract. In the purification process, by adjusting key process parameters such as extraction time, activated carbon dosage, and extraction temperature, PAHs in the Gynostemma pentaphyllum extract are effectively removed while minimizing extract loss.

[0047] The implementation of this purification process not only significantly improved the quality of Gynostemma pentaphyllum extract but also provided innovative ideas and methods for the removal of polycyclic aromatic hydrocarbons (PAHs) from other natural plant extracts. Through the optimized purification process, the content of PAHs in the product was significantly reduced, thereby meeting more stringent food safety and pharmaceutical quality standards. Furthermore, the development of this process offers advantages such as high cost-effectiveness, ease of operation, and environmental friendliness, providing practical technical support for industrial production. Attached Figure Description

[0048] Figure 1 Photographs showing the dissolution of Gynostemma pentaphyllum extract in ethanol solvents of different concentrations in Example 3.

[0049] Figure 2 Photographs showing the solubility of *Gynostemma pentaphyllum* extract in ethanol of different concentrations and multiples in Example 4. Detailed Implementation

[0050] To enable those skilled in the art to better understand the technical solutions of this disclosure, the following detailed, clear, and complete description of this disclosure is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of this disclosure.

[0051] This invention provides an extraction process for Gynostemma pentaphyllum, comprising the following steps:

[0052] Step S1: Extraction, add ethanol solution to Gynostemma pentaphyllum, heat, reflux and extract to obtain extract;

[0053] In step S1, the Gynostemma pentaphyllum raw material is first sieved to remove mud and sand, and then crushed in a pulverizer. This operation can effectively improve the extraction efficiency and shorten the extraction time. However, this crushing process is a routine operation in the field.

[0054] Furthermore, in step S1, the concentration of ethanol is selected to be 65-75%, for example, 68%, 70% or 72%; at this concentration, the extract can ensure both production safety and extraction efficiency.

[0055] Furthermore, in step S1, the volume of ethanol used is 6 to 12 times the mass of Gynostemma pentaphyllum, for example, it can be 6 times, 7 times, 8 times, 9 times, or 12 times.

[0056] It should be noted that in actual production, the amount of liquid used is generally calculated by volume, while the amount of solid material used is generally calculated by mass. The unit of liquid material usage is mL or L, and the unit of solid material mass is g or Kg. Here, the volume of ethanol used being 6 to 9 times the mass of Gynostemma pentaphyllum means that the ratio of the volume of ethanol used to the mass of Gynostemma pentaphyllum is between 6 and 9 times (mL / g, or L / Kg).

[0057] Furthermore, the extraction time is 1 to 3 hours, for example, 1 hour, 2 hours, or 3 hours. The extraction time will affect the extraction efficiency of Gynostemma pentaphyllum. For example, with a longer extraction time, the extraction efficiency of Gynostemma pentaphyllum will be improved, but the problem of too many impurities in the extract will also be faced.

[0058] In some embodiments, the extraction in step S1 is a stepwise extraction, including at least two reflux extractions; for example, ethanol can be used to extract two, three, or four times. The number of extractions will affect the extraction yield of Gynostemma pentaphyllum, and the number of extractions can be adjusted according to the extraction residue and cost considerations.

[0059] In some embodiments, the extraction of Gynostemma pentaphyllum in step S1 involves three extractions, wherein...

[0060] The first reflux extraction was performed by adding 70% ethanol at 8 times the weight of the raw material, stirring, heating to a gentle boil, controlling the temperature at 70℃, reflux extraction for 1.5 hours, filtering through a 300-mesh filter, and collecting the filtrate.

[0061] The second reflux extraction was performed by adding 70% ethanol at 6 times the weight of the raw material to the residue from the first reflux extraction, stirring, heating to a gentle boil, controlling the temperature at 80℃, reflux extraction for 1.0 h, filtering through a 300 mesh filter, and collecting the filtrate.

[0062] The third reflux extraction was performed by adding 70% ethanol at 6 times the weight of the raw material to the residue from the second reflux extraction, stirring, heating to a gentle boil, controlling the temperature at 80℃, and refluxing for 1.0 h, followed by filtration.

[0063] Step S2: Concentration. The extract from step S1 is concentrated to 1.5 to 2.0 times the weight and volume of the raw material, with the Baumé degree controlled to be >8.

[0064] In the concentration step, the main task is to remove the solvent ethanol from the extract. During concentration, the operation is carried out according to the Baumé degree of the extract, and the Baumé degree of the solution needs to be continuously monitored.

[0065] Specifically, the extract obtained in step S1 is concentrated in a single-effect concentrator. When the volume of the concentrate reaches 1.0 to 1.5 times the weight of the raw material and the Baumé degree is >8, hot water is added at this point, with the amount of water replenished being 3 to 4 times the volume of the concentrate. Concentration continues until there is no alcohol taste, and finally the concentrate is concentrated to 1.5 to 2.0 times the weight of the raw material and the Baumé degree is >8.

[0066] During the concentration process, the concentration temperature is less than 70℃ and the vacuum degree is -0.04~-0.10Mpa.

[0067] Step S3: Dilution and centrifugation. Dilute the concentrated liquid obtained in step S2 with hot water at a volume of 6 to 9 times the weight of the raw material. After dilution, centrifuge and collect the centrifuged liquid for later use.

[0068] Furthermore, the amount of hot water used can be 6, 7, 8, or 9 times that of the raw materials.

[0069] Step S4: Column loading and elution: Load the centrifuged liquid obtained in step S3 onto the column at a flow rate of 0.5–0.75 column volumes / h; after the feed liquid has been completely loaded, wash with water at a flow rate of 0.5–0.8 column volumes / h for 1.5 column volumes; after washing, elute with 2.0–2.5 column volumes of ethanol at a flow rate of 0.5–0.65 column volumes / h.

[0070] Step S5: Concentration and drying of the eluent. The eluent is transferred to a single-effect concentrator for concentration. After concentration, the concentrate is transferred to a spray tank for spray drying to obtain Gynostemma pentaphyllum extract.

[0071] Regarding the Gynostemma pentaphyllum extract extracted in the previous embodiment, this embodiment also provides a purification method for the Gynostemma pentaphyllum extract to remove polycyclic aromatic hydrocarbons (PAHs) from the extract. The purification method involves dissolving the Gynostemma pentaphyllum extract in an ethanol solution, adding activated carbon, shaking, and adsorption.

[0072] The concentration of the ethanol solution is 60-70%.

[0073] The volume of the ethanol solution used is 30 to 50 times the mass of the extract, preferably 40 to 50 times, and more preferably 40 times.

[0074] This invention uses ethanol as a purification solvent. The concentration and amount of ethanol affect the solubility of the extract, which in turn affects the removal efficiency of the adsorbent for polycyclic aromatic hydrocarbons (PAHs), especially benzo[a]pyrene. Furthermore, due to the differences in the solubility of Gynostemma pentaphyllum extract caused by the solvent, the recovery efficiency of Gynostemma pentaphyllum after final adsorption and purification will also be affected during the recovery process. Therefore, it is necessary to control the dissolution conditions well to avoid the problem of low recovery rate of Gynostemma pentaphyllum due to changes in dissolution conditions.

[0075] The adsorption time is 0.1 to 1 h, preferably 0.1 to 0.6 h, and more preferably 0.5 h.

[0076] The amount of activated carbon used is 2-3% relative to the mass of the Gynostemma pentaphyllum extract alcohol solution, preferably 2.5%;

[0077] The adsorption temperature is 45–55℃; preferably 50℃.

[0078] In this invention, the amount of adsorbent used and the adsorption conditions (temperature and time) both affect the yield of Gynostemma pentaphyllum and the removal efficiency of polycyclic aromatic hydrocarbons (PAHs).

[0079] Specifically, this invention provides a purification method for Gynostemma pentaphyllum extract to remove polycyclic aromatic hydrocarbons (PAHs) from the extract. The purification method involves dissolving the extract in a 60-70% ethanol solution to obtain an alcoholic solution of the extract, with a mass-to-volume ratio of extract to ethanol of 1:40-50; adding activated carbon, wherein the amount of activated carbon is 2-3% of the mass of the alcoholic solution; shaking; and adsorption.

[0080] Furthermore, the present invention provides the following embodiments to illustrate the specific solutions of this application. It should be noted that in each embodiment, since there are various types of testing instruments in the laboratory, the testing instruments used in the embodiments of the same group are generally of the same type. In different groups, the test results may have different decimal places due to differences in the instrument models used. Such differences are normal and do not affect the authenticity of the experimental results in the embodiments of the present invention.

[0081] Example 1

[0082] Extraction methods of Gynostemma pentaphyllum

[0083] In Example 1, the extraction steps for 7.8% total saponins from the rhizome of Gynostemma pentaphyllum were as follows:

[0084] Step S1: Extraction. Accurately weigh 100g of pulverized Gynostemma pentaphyllum raw material (7.8% total saponins, rhizome), add 1000mL (10 times) of 70% ethanol for the first extraction and reflux for 1.5h, then filter through a 300-mesh filter.

[0085] The residue was then refluxed with 800 mL (8 times) of 70% ethanol for 1.0 h, filtered through a 300 mesh to obtain the extract.

[0086] Step S2, concentration: The extract obtained in step S1 is concentrated at 70°C to 1.0 to 1.2 times the weight of the medicinal material (100 mL to 120 mL). 150 mL (1.5 times the volume of the concentrated liquid) of water is added to continue concentration to 100 mL (1.0 times). The concentration is repeated once by adding water, and finally concentrated to 110 mL (1.1 times). The concentration degree of Baume is >8.

[0087] Step S3: dilute and centrifuge. Add water at 50-60℃ to a total volume of 800mL (8.0 times). Stir well and centrifuge at 4000 rpm for 10 min. Wash the precipitate with 100mL of water at 50-60℃, centrifuge again, and combine the supernatants.

[0088] Step S4: Column loading and elution. The supernatant was passed through column D101 (165g) at a flow rate of 4mL / min (0.92BV / h). After the feed solution was completely fed, the column was washed with 500mL (1.92BV) of water at a flow rate of 4mL / min (0.86BV / h). Then, the supernatant was eluted with 750mL of 70% ethanol at a flow rate of 3mL / min (0.692BV / h). The solution was concentrated at 70℃ and dried.

[0089] Step S5: Concentrate and dry the eluent. Transfer the eluent to a single-effect concentrator for concentration. After concentration, transfer the concentrate to a spray tank for spray drying to obtain Gynostemma pentaphyllum extract.

[0090] The feed solutions were tested at each stage of the experiment, and the relative yield and total saponin content were calculated.

[0091] ;

[0092] Where: m1—content of the test sample in the standard curve (ug)

[0093] m—Sample weight (g)

[0094] V1—Dilution volume of the test sample (mL)

[0095] V2—Volume (mL) of sample taken for testing.

[0096] The results are shown in the table below:

[0097] Table 1. Relative raw material yield and total saponin content of extracts for each extraction stage in Example 1.

[0098]

[0099] Based on the extraction experiment in the example, when the content of the raw material is 7.8%, the relative raw material yield is 10.4% and the content is 113.9%. In other words, the higher the content of the raw material, the higher the yield of the qualified finished product and the lower the cost.

[0100] Example 2

[0101] In Example 2, the Gynostemma pentaphyllum raw material contained 6.3% total saponins. The extraction steps for the rhizome are as follows:

[0102] Step S1: Extraction. Accurately weigh 200g of the pulverized raw material. First, add 2400mL (12 times) of 70% ethanol and reflux for 1.5h, then filter through a 300-mesh filter. Second, add 2000mL (10 times) of 70% ethanol to the residue and reflux for 1.0h, then filter through a 300-mesh filter.

[0103] Step S2, concentration: Concentrate the extract obtained in step S1 at 70°C to 0.8 to 1.2 times the weight of the medicinal material (150 mL), add 1.0 to 1.5 times the volume of the concentrated liquid (300 mL) of water and continue to concentrate to 0.8 to 1.2 times the volume of the raw material (160 mL), repeat the water addition and concentration once, and finally concentrate to 160 mL (0.8 times).

[0104] Step S3: dilute and centrifuge, add water at 50-60℃ to a total volume of 1600mL (8 times), stir well, centrifuge at 4000 rpm for 10 min, wash the precipitate with 200mL of water at 50-60℃, centrifuge again, and combine the supernatants.

[0105] Step S4: Column loading and elution. The supernatant is passed through the column (165g) at a flow rate of 4mL / min (0.86BV / h). After the feed solution is completely loaded, the column is washed with 500mL (1.8BV) of water at a flow rate of 3-4mL / min (0.65-0.86BV / h). Then, the column is eluted with 750mL of 70% ethanol at a flow rate of 3mL / min (0.692BV / h). The column is then concentrated and dried at 70℃.

[0106] The relevant parameters of the extract were obtained according to the calculation method provided in Example 1, as shown in the table below.

[0107] Table 2. Relative raw material yield and total saponin content of extracts for each extraction stage in Example 2.

[0108]

[0109] Based on the extraction experiment in Example 2, when the content of the raw material is 6.3%, the relative yield of the raw material is 9.3%, and the content is 99.3%. In other words, the higher the content of the raw material, the higher the yield of the extracted product and the lower the cost.

[0110] Example 3

[0111] The solubility of Gynostemma pentaphyllum extract in ethanol of different concentrations was determined as follows:

[0112] (1) Take 20 mL of each of 0%, 20%, 40%, 60% and 80% ethanol into a 50 mL centrifuge tube, add about 1 g of Gynostemma pentaphyllum extract to each, sonicate to dissolve for about 1 hour, centrifuge, and pour out the supernatant.

[0113] (2) Add 10 mL of each concentration of ethanol to each centrifuge tube, sonicate to dissolve, centrifuge, and pour out the supernatant to mix with the previous mixture;

[0114] (3) After drying and weighing the centrifuge tubes and removing the residue, the centrifuge tubes are washed, dried and weighed again, and the weight of the residue is calculated.

[0115] Table 3. Solubility in ethanol at different concentrations in Example 3

[0116]

[0117] See Figure 1 The images show the dissolution of Gynostemma pentaphyllum extract in ethanol solvents of different concentrations in Example 3. From the centrifuged liquids in the images, it can be seen that as the ethanol concentration increases, the turbidity decreases, while the color shows no significant difference.

[0118] This shows that when dissolving the Gynostemma pentaphyllum extract twice—first with 20 times the amount of ethanol, then again with 10 times the amount of ethanol—undissolved extract was still present when using ultrasonic dissolution. Using water as a solvent resulted in the least amount of dissolution compared to using an ethanol-water solution. Significant residue remained at ethanol concentrations of 20% and 40%, while the amount of insoluble matter was significantly reduced at 60-80% ethanol concentration. However, further increasing the ethanol concentration did not significantly improve the dissolving power of the mixed solvent. Therefore, to minimize the loss of saponins in the Gynostemma pentaphyllum extract, and considering production safety and cost, an ethanol concentration of 60-80% is most suitable.

[0119] Example 4

[0120] The solubility of the *Gynostemma pentaphyllum* extract in ethanol of different concentrations at the same level was compared to that in Example 3, where the dissolution method used vortexing and sonication.

[0121] (1) Take five centrifuge tubes AE, weigh them (with caps), weigh about 1g of Gynostemma pentaphyllum extract into each centrifuge tube, and add 20 / 25 / 30 / 35 / 40mL of 70% ethanol to the centrifuge tubes respectively.

[0122] (2) Vortex, sonicate for about 1 hour, centrifuge, and pour out the supernatant;

[0123] (3) Dry and weigh the centrifuge tubes (with lids);

[0124] Table 4. Solubility of *Gynostemma pentaphyllum* extract in ethanol at different concentrations and multiples in Example 4

[0125]

[0126] See Figure 2 , Figure 2 The images show the dissolution of the *Gynostemma pentaphyllum* extract in different concentrations of ethanol in Example 4. As can be seen from the images, the color of the centrifuged liquid becomes lighter as the volume of dissolved liquid increases. A comparison of the colors in this set of examples with Example 3 shows that the amount dissolved decreases significantly with increasing solvent volume.

[0127] Further analysis revealed that when using 70% ethanol as a solvent to dissolve Gynostemma pentaphyllum extract at different concentrations, group B (25 times) dissolved the most extract. However, as the solvent volume increased (e.g., group D at 35 times and group E at 40 times), the mass of dissolved extract decreased despite the increased solvent volume. This may be related to the degree of polarity matching between the extract and the solvent.

[0128] Furthermore, analysis showed that the solubility of 30 mL of 70% ethanol solvent in Example 4 was compared with that of 60% and 80% ethanol solvents in Example 3. As analyzed in Example 3, stepwise ultrasonic dissolution achieved good dissolution results at 60-80% ethanol concentrations. Example 4 involved a single dissolution, while Example 3 involved stepwise dissolution. In Example 4, the undissolved residue weight of the 35-fold C group was 0.202 g (1.074-0.872 g), significantly greater than the undissolved residue after dissolution with the same volume of 60% and 80% ethanol solvents in Example 3. This demonstrates that the amount of solvent and the dissolution method have a significant impact on the performance of extract dissolution.

[0129] Example 5

[0130] Based on Example 4, the dissolution method of Gynostemma pentaphyllum was changed, and a combination of vortexing and ultrasound was used for dissolution.

[0131] The specific steps are as follows:

[0132] (1) Take five centrifuge tubes AE, weigh them (with caps), weigh about 1g of Gynostemma pentaphyllum extract into each centrifuge tube, and add 20mL of 70% ethanol to each centrifuge tube.

[0133] (2) Vortex to dissolve completely, then add 0 / 5 / 10 / 15 / 20 mL of 70% ethanol, sonicate to dissolve for about 1 hour, centrifuge, and pour out the supernatant. Among them, the amount of ethanol used in centrifuge tube A is 20 mL, the amount of ethanol used in centrifuge tube B is 25 mL, the amount of ethanol used in centrifuge tube C is 30 mL, the amount of ethanol used in centrifuge tube D is 40 mL, and the amount of ethanol used in centrifuge tube E is 45 mL.

[0134] (3) Dry and weigh the centrifuge tubes (with lids);

[0135] Table 5. Solubility of *Gynostemma pentaphyllum* extract in ethanol of the same concentration but different multiples of ethanol in Example 5

[0136]

[0137] As shown in Table 5, this embodiment used different multiples of 70% ethanol to dissolve Gynostemma pentaphyllum extract, employing a stepwise dissolution method. In the first dissolution step, 20 times the volume of solvent was used, and the dissolution method was vortexing. Then, the solvent for the second dissolution step was added, with the solvent multiple in the second step being a variable. Regarding the dissolution effect, all centrifuge tubes contained a certain amount of undissolved material. The group with the most dissolved Gynostemma pentaphyllum extract was group E (40 times). In terms of the mass of dissolution, 20 times the volume of solvent was sufficient to dissolve most of the Gynostemma pentaphyllum extract. Regarding the solvent multiple added in the second step, the amount of Gynostemma pentaphyllum extract dissolved increased with the increase of the ethanol volume added in the second step. When 20 times the volume of solvent was added in the second step, the overall undissolved rate of Gynostemma pentaphyllum extract was 2.4%. It can be seen that as the volume of solvent added in the second dissolution step increases, the mass of dissolution also increases. The volume of solvent used to dissolve the extract can be considered appropriately during production.

[0138] Example 6

[0139] Activated carbon adsorption was used to remove benzo[a]pyrene and polycyclic aromatic hydrocarbons from Gynostemma pentaphyllum extract;

[0140] The raw material used was the Gynostemma pentaphyllum extract obtained in Example 1. The dissolution procedure is as follows:

[0141] (1) Take 400 mL of 60%, 70% and 80% ethanol into a 1000 mL centrifuge bottle, add 10 g of Gynostemma pentaphyllum extract to each, sonicate to dissolve for about 1 hour, centrifuge and pour out the supernatant;

[0142] (2) Add 0.5g of activated carbon to each of the centrifuged liquids of 60%, 70% and 80% ethanol, shake well and let it sit overnight (more than 12 hours).

[0143] (3) Centrifugation, concentration, drying, powdering and testing of the centrifuged liquid; ultrasonic removal and drying of activated carbon residue with a small amount of water and testing.

[0144] (4) Testing: One raw material of Gynostemma pentaphyllum extract and three processed extracts were sent for testing for Gynostemma pentaphyllum saponin content, loss on drying, benzo[a]pyrene, and polycyclic aromatic hydrocarbons;

[0145] 1g of activated carbon raw material and 3 activated carbon samples after adsorption and drying were sent for testing for benzo[a]pyrene and polycyclic aromatic hydrocarbons.

[0146] Gynostemma pentaphyllum extract was dissolved and centrifuged with 40 times its volume of 60%, 70%, and 80% ethanol, respectively, and each solution produced a small amount of residue and color.

[0147] The test results are shown in the table below:

[0148] Table 6. Test results of activated carbon adsorption for the removal of benzo[a]pyrene and polycyclic aromatic hydrocarbons from Gynostemma pentaphyllum extract in Example 6.

[0149]

[0150] As shown in Table 6, the results of Example 6 indicate that activated carbon effectively adsorbs benzo[a]pyrene and polycyclic aromatic hydrocarbons (PAHs) from Gynostemma pentaphyllum extract. Except for the experimental group with 80% alcohol-dissolved PAHs, which showed a concentration of 60.4 ppb, none of the others were detected. Furthermore, the saponin content of the Gynostemma pentaphyllum extract did not decrease significantly after adsorption by activated carbon, with a minimum yield of 89%. However, it can also be seen that when using the same amount of activated carbon, the adsorption capacity of activated carbon significantly affects the adsorption capacity of Gynostemma pentaphyllum extract.

[0151] The final product extract had a color similar to the raw material, though slightly lighter. However, specifically, under 80% ethanol conditions, activated carbon could not completely remove polycyclic aromatic hydrocarbons (PAHs), and it also reduced the yield of Gynostemma pentaphyllum extract. Furthermore, under 60% ethanol dissolution conditions, although benzo[a]pyrene or other PAHs were not detected, the yield of purified Gynostemma pentaphyllum was reduced. This suggests that during purification, the solvent affects the adsorption capacity of activated carbon, even with the same amount of activated carbon. Higher ethanol concentrations increase the adsorption capacity of activated carbon for the extract, leading to a lower yield and reduced PAH adsorption. Conversely, lower ethanol concentrations ensure the adsorption of benzo[a]pyrene and PAHs, but increase the adsorption capacity of activated carbon for the extract, resulting in a lower yield. Therefore, based on the primary objective of removing benzo[a]pyrene and PAHs, the amount of ethanol used is 60-70%, but 70% is preferred.

[0152] Example 7

[0153] The effect of low activated carbon dosage on the purification effect of Gynostemma pentaphyllum extract was investigated, and the experimental procedure is as follows:

[0154] (1) Take 400 mL of 70% ethanol and add 10 g of Gynostemma pentaphyllum extract. Dissolve in steps using the method in Example 4. The first step is to use 20 times the amount of solvent to dissolve in steps. Dissolve by sonication for about 1 hour, centrifuge, and pour out the supernatant (transfer the residual liquid with a dropper).

[0155] (2) The precipitate is mixed and dried, and the saponin content is measured;

[0156] (3) Add 50 mg of activated carbon (accurately weighed) to each centrifuged liquid, shake well, and let it sit overnight (more than 12 hours).

[0157] (4) Centrifuge, concentrate, dry, and grind the centrifuged liquid to detect the content of saponins, benzo[a]pyrene and polycyclic aromatic hydrocarbons;

[0158] Detection:

[0159] One raw material of Gynostemma pentaphyllum extract, one processed extract, and one centrifuged residue were prepared. The content of Gynostemma pentaphyllum saponins, loss on drying, benzo[a]pyrene, and polycyclic aromatic hydrocarbons were determined.

[0160] The test results are shown in the table below:

[0161] Table 7. Test results of activated carbon adsorption for removing benzo[a]pyrene and polycyclic aromatic hydrocarbons from Gynostemma pentaphyllum extract in Example 7.

[0162]

[0163] In Example 7, the amount of activated carbon was reduced (from 5% to 0.5% of the raw material mass), resulting in less loss of Gynostemma pentaphyllum extract. The mass yield after activated carbon adsorption was 98%, higher than the 89-96% in the previous experiment. This indicates that appropriately reducing the use of activated carbon can reduce the loss of Gynostemma pentaphyllum extract. However, it also faces the possibility of detecting polycyclic aromatic hydrocarbons (PAHs). However, the amount of PAHs relative to the raw material was reduced from 1480.8 ppm (as measured in Example 6) to 53.8 ppm.

[0164] In this embodiment, reducing the amount of activated carbon resulted in complete adsorption of benzo[a]pyrene, but not complete adsorption of polycyclic aromatic hydrocarbons. Appropriately increasing the amount of activated carbon can remove both.

[0165] Example 8

[0166] The time required for activated carbon adsorption to remove benzo[a]pyrene and polycyclic aromatic hydrocarbons from Gynostemma pentaphyllum extract was determined experimentally as follows:

[0167] (1) Ethanol dissolution:

[0168] Add 800 mL of 70% ethanol to a 1000 mL centrifuge bottle, add 20 g of Gynostemma pentaphyllum extract, sonicate to dissolve for about 1 hour, centrifuge, and pour off the supernatant for later use.

[0169] (2) Add 0.1g of activated carbon to each of the centrifuged liquids, shake well, and then oscillate.

[0170] After 0, 0.5, 1, 2, and 3 hours, 40 mL of solution was taken, filtered, the filtrate was dried, and the remaining liquid was shaken overnight. The next day, it was centrifuged, the centrifuged liquid was concentrated, dried, and powdered, and benzo[a]pyrene and polycyclic aromatic hydrocarbons were detected.

[0171] (3) Testing:

[0172] The sample was tested for benzo[a]pyrene and polycyclic aromatic hydrocarbons in Gynostemma pentaphyllum.

[0173] The test results are shown in the table below:

[0174] Table 8. Test results of activated carbon adsorption for the removal of benzo[a]pyrene and polycyclic aromatic hydrocarbons from Gynostemma pentaphyllum extract in Example 8.

[0175]

[0176] Analysis of the test results obtained from Example 8 shows that the experimental data of the group with an adsorption time of 0.5 h were the best. The detection values ​​of benzo[a]pyrene and polycyclic aromatic hydrocarbons were the lowest among all experimental groups. In the experiment, after the adsorption time of 0.5 h, the adsorption effect did not improve with a longer adsorption time; on the contrary, more benzo[a]pyrene and polycyclic aromatic hydrocarbons were detected. This may be because the upper limit of activated carbon adsorption was reached, and with the increase of time, benzo[a]pyrene and polycyclic aromatic hydrocarbons were shaken out of the activated carbon. In future experiments, increasing the amount of activated carbon could be considered. In addition, compared with Example 7, the dissolution operation in Example 8 was direct dissolution, while that in Example 7 was stepwise dissolution. It can be further seen that stepwise dissolution also has a significant impact on the adsorption capacity of activated carbon. That is, the two-step dissolution in Example 7 still showed a better adsorption effect than the one-step dissolution in Example 8 after overnight incubation.

[0177] Example 9

[0178] The experiment on the amount of activated carbon used for adsorption and removal of benzo[a]pyrene and polycyclic aromatic hydrocarbons from Gynostemma pentaphyllum extract was conducted, and the experimental procedure is as follows:

[0179] (1) Ethanol dissolution:

[0180] Add 400 mL of 70% ethanol to five 1000 mL centrifuge bottles, add 10 g of Gynostemma pentaphyllum extract, sonicate to dissolve for about 1 hour, centrifuge, and pour off the supernatant for later use.

[0181] (2) Activated carbon adsorption:

[0182] Add 1%, 1.5%, 2%, 2.5%, and 3% (0.1g, 0.15g, 0.2g, 0.25g, and 0.3g) of activated carbon to the centrifuged liquid, respectively, shake well, and incubate for 0.5h.

[0183] Centrifugation, concentration, drying, and powdering of the centrifuged liquid; detection of benzo[a]pyrene and polycyclic aromatic hydrocarbons.

[0184] (3) Detection:

[0185] Detection of benzo[a]pyrene and polycyclic aromatic hydrocarbons in Gynostemma pentaphyllum;

[0186] Table 9. Test results of activated carbon adsorption for the removal of benzo[a]pyrene and polycyclic aromatic hydrocarbons from Gynostemma pentaphyllum extract in Example 9.

[0187]

[0188] As can be seen from the five groups of experiments in Example 9, none of the groups met the detection requirements (benzo[a]pyrene ≤ 10 ppb, polycyclic aromatic hydrocarbons ≤ 50 ppb). The benzo[a]pyrene content of the 2.5% activated carbon group was qualified, and although the polycyclic aromatic hydrocarbon content was not qualified, it was the lowest among all groups.

[0189] It should be noted that, in the overall comparison of the results of Example 9 and Example 8, the adsorption in Example 9 only lasted for 30 minutes, and the benzo[a]pyrene and polycyclic aromatic hydrocarbons were not completely adsorbed. In the time curve experiment of Example 8, the data at 30 minutes was the lowest among all groups. This may be because in the time curve experiment, a small portion of the upper liquid was taken for concentration and drying, while in this experiment, all the liquid was concentrated and dried. However, this data does not affect the overall relationship between adsorption time and adsorption amount obtained in Example 8.

[0190] Example 10

[0191] Study on the optimal temperature for adsorption and removal of benzo[a]pyrene and polycyclic aromatic hydrocarbons from Gynostemma pentaphyllum extract by activated carbon

[0192] Experimental procedure:

[0193] (1) Ethanol dissolution:

[0194] Add 200 mL of 70% ethanol to three 1000 mL centrifuge bottles, add 5 g of Gynostemma pentaphyllum extract, sonicate to dissolve for about 1 hour, centrifuge, and pour off the supernatant for later use.

[0195] (2) Activated carbon adsorption:

[0196] Add 2.5% g of activated carbon to the centrifuged liquid, shake well, and then shake for 0.5 h at room temperature, 50℃, and 70℃ respectively.

[0197] Centrifugation, concentration, drying, and powdering of the centrifuged liquid;

[0198] (3) Testing:

[0199] Detection of benzo[a]pyrene and polycyclic aromatic hydrocarbons in Gynostemma pentaphyllum;

[0200] Table 10 Test results of activated carbon adsorption for the removal of benzo[a]pyrene and polycyclic aromatic hydrocarbons from Gynostemma pentaphyllum extract in Example 10

[0201]

[0202] Based on the experiment in Example 10, all groups were qualified and met the detection requirements (benzo[a]pyrene ≤ 10 ppb, polycyclic aromatic hydrocarbons ≤ 50 ppb). The polycyclic aromatic hydrocarbon content was lowest in the 50℃ group, and the content of benzo[a]pyrene was not detected in any group. Therefore, 50℃ can be selected as the adsorption temperature, and the adsorption time can be appropriately extended.

[0203] It should be noted that in the experimental results of Examples 10 and 9, in the adsorption experimental data of 2.5% activated carbon in Example 9, the detection results of the room temperature group were benzo[a]pyrene not detected and polycyclic aromatic hydrocarbons 103.87. This result may be due to the instability of instrument detection data.

[0204] It should be noted that the various embodiments of this application can be arbitrarily combined into new embodiments, provided that the solutions do not conflict and the technical solutions can coexist.

[0205] The present application has been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present application. The descriptions of the embodiments above are only for the purpose of helping to understand the present application and its core ideas. It should be noted that those skilled in the art can make several improvements and modifications to the present application without departing from the principles of the present application, and these improvements and modifications also fall within the protection scope of the claims of the present application.

Claims

1. A method for purifying polycyclic aromatic hydrocarbons from Gynostemma pentaphyllum extract, used to remove polycyclic aromatic hydrocarbons from Gynostemma pentaphyllum extract, characterized in that, The method for purifying polycyclic aromatic hydrocarbons from Gynostemma pentaphyllum extract involves dissolving the extract in an ethanol solution, adding activated carbon, and adsorbing the hydrocarbons. The concentration of the ethanol solution is 60-70%. The adsorption time is 0.5 to 1 hour; The amount of activated carbon used is 2-3% relative to the mass of the Gynostemma pentaphyllum extract; The adsorption temperature is 45–55°C; The volume of the ethanol solution used is 40 to 50 times the mass of the Gynostemma pentaphyllum extract; The operation of dissolving Gynostemma pentaphyllum extract in ethanol solution involves two dissolution operations; The two dissolution operations include dividing the ethanol solution into solvent A and solvent B, with a volume ratio of solvent A to solvent B of 1:0.5-1.

5. The Gynostemma pentaphyllum extract is dissolved in solvent A. After dissolution, an intermediate state Gynostemma pentaphyllum extract solution is obtained. Then, solvent B is added to the aforementioned intermediate state Gynostemma pentaphyllum extract solution for dissolution, and finally, a Gynostemma pentaphyllum extract solution is obtained. The extraction method of the Gynostemma pentaphyllum extract is as follows: Step S1: Add Gynostemma pentaphyllum to an ethanol solution, heat, and reflux to extract to obtain an extract; Step S2: Concentrate the extract from step S1 to 1.5 to 2.0 times the weight volume of the raw material, and control the Baumé degree to be >8; Step S3: Dilute the concentrated liquid obtained in step S2 with 6 to 12 times the volume of hot water at 60-100°C, centrifuge after dilution, and collect the centrifuged liquid for later use. Step S4: Control the flow rate of the centrifuged liquid obtained in step S3 to 0.5–0.75 column volumes / h and load it onto the column; after the feed liquid has been completely fed, wash with water at a flow rate of 0.5–0.8 column volumes / h for 1.5 column volumes; after washing with water, elute with 2.0–2.5 column volumes of ethanol at a flow rate controlled at 0.5–0.65 column volumes / h. Step S5: The eluent is transferred to a single-effect concentrator for concentration. After concentration, the concentrate is transferred to a spray tank for spray drying to obtain Gynostemma pentaphyllum extract.

2. The method for purifying polycyclic aromatic hydrocarbons from Gynostemma pentaphyllum extract as described in claim 1, characterized in that, The ethanol solution is divided into solvent A and solvent B, wherein the volume ratio of solvent A to solvent B is 1:

1.

3. The method for purifying polycyclic aromatic hydrocarbons from Gynostemma pentaphyllum extract as described in claim 2, characterized in that, The volume of the ethanol solution used is 40 times the mass of the Gynostemma pentaphyllum extract; the ethanol solution is divided into solvent A and solvent B, the volume of solvent A is 20 times the mass of the Gynostemma pentaphyllum extract, and the volume of solvent B is 20 times the mass of the Gynostemma pentaphyllum extract.

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