A method for extracting chlorogenic acid from stevia residue and its application.

Abstract

This invention discloses a method for extracting chlorogenic acids from stevia waste and its applications, including: extracting chlorogenic acids from stevia powder using an alkaline extraction method; and determining whether the chlorogenic acids contain chlorogenic acid, isochlorogenic acid A, and cryptochlorogenic acid. Using stevia waste as raw material, this invention involves solvent extraction, centrifugal filtration, reflux extraction, and chromatographic separation to obtain a fat-soluble extract. This extract is then dissolved and filtered with ethanol, evaporated and concentrated, and freeze-dried to obtain the chlorogenic acid product. This invention extracts and develops chlorogenic acids from stevia waste, solving the problems of environmental pollution and resource waste. The extracted chlorogenic acids contain chlorogenic acid, cryptochlorogenic acid, and isochlorogenic acid A, and have low sugar content. They possess antioxidant, anti-inflammatory, and blood pressure-lowering functions, and can be widely used in food, pharmaceuticals, and health products, demonstrating high application value and realizing the comprehensive utilization of stevia.

Description

Technical Field

[0001] This invention belongs to the field of natural product extraction and separation technology. More specifically, this invention relates to a method for extracting chlorogenic acid from stevia waste and the application of chlorogenic acid. Background Technology

[0002] In recent years, with the rapid development of the social economy and the continuous improvement of people's consumption capacity for health products, the market demand for traditional Chinese medicine extracts has been expanding, and the generation of solid waste from traditional Chinese medicine has also been increasing year by year, becoming one of the bottlenecks restricting the development of the traditional Chinese medicine pharmaceutical industry. Traditional disposal methods such as landfill, incineration, and fixed-area stockpiling seriously affect environmental quality, consume a lot of funds, and affect the normal production of pharmaceutical factories. How to achieve effective treatment and resource utilization of solid waste from traditional Chinese medicine is an unavoidable problem in the green development of the traditional Chinese medicine resource industry. Stevia repens, as a natural sugar-free sweetener, has been widely used in the food and pharmaceutical fields. Fermentation treatment of stevia repens has produced bio-fertilizers and feed additives, achieving certain economic benefits. Studies have found that stevia repens is rich in chlorogenic acid substances. my country has abundant stevia resources, but the comprehensive utilization rate of stevia repens is low.

[0003] Stevia contains a variety of functional components and is widely used in food, pharmaceuticals, and other fields. Besides steviol glycosides, stevia is also rich in chlorogenic acids. Currently, the utilization of stevia mainly focuses on the production of steviol glycosides, neglecting the development and utilization of other functional components such as chlorogenic acids. The waste residue after steviol glycoside extraction contains abundant chlorogenic acids and other functional components. How to separate and purify chlorogenic acids still requires active exploration. Therefore, extracting and developing chlorogenic acids from stevia waste can not only solve problems such as environmental pollution and resource waste, but also alleviate the problem of excessively high costs associated with chlorogenic acids, thereby achieving comprehensive utilization of stevia. Summary of the Invention

[0004] One object of the present invention is to solve at least the above-mentioned problems and / or defects, and to provide at least the advantages described below.

[0005] To achieve these and other advantages according to the present invention, the present invention provides a method for extracting chlorogenic acid compounds from stevia residue, characterized by comprising the following steps: Step 1: Extract chlorogenic acid from stevia powder using alkaline extraction. Step 2: Use column chromatography to analyze the extracted chlorogenic acid substances to determine whether the chlorogenic acid substances contain chlorogenic acid, cryptochlorogenic acid, or isochlorogenic acid A.

[0006] Preferably, in step one, the method for preparing the stevia powder includes: removing the roots and stems from the stevia residue, crushing the leaves in a ball mill, and passing them through a 60-mesh sieve to obtain stevia powder.

[0007] Preferably, the specific method for extracting chlorogenic acid substances from stevia powder using the alkaline extraction method in step one includes: S11. The sieved stevia powder is extracted twice by reflux with an alkaline solution of a certain concentration. The second extract is centrifuged and filtered, the filtrate is retained and the filter residue is removed. S12. Adjust the pH of the filtrate to 6-7 with a prepared dilute acid of a certain concentration. Perform rotary concentration and evaporation at a temperature of 40-50℃ and a rotation speed of 20-60 rpm. Retain the concentrate. Mix the concentrate with ethyl acetate for the first extraction. Then mix the extract from the first extraction with ethyl acetate for the second extraction. Retain the ethyl acetate layer. Concentrate by rotary evaporation. Pour the retained concentrate into an evaporating dish. Place the evaporating dish on a water bath to evaporate and dry to obtain a fat-soluble extract. S13. Dissolve the fat-soluble extract in a short-chain alcohol solution, filter, retain the filtrate, and freeze-dry to obtain the filtrate extract, which is a chlorogenic acid.

[0008] Preferably, in step S11, during the first reflux extraction, the mass ratio of stevia powder to alkaline solution is 1:5~10, the reflux time is 1~5h, and the extraction temperature is 45~55℃. During the second reflux extraction, the mass ratio of stevia powder to alkaline solution was 1:5~10, the reflux time was 1~5h, and the extraction temperature was 45~55℃.

[0009] Preferably, the alkaline solution in S11 includes one of the following: a sodium carbonate solution with a mass fraction of 0.1% to 0.5%, an ammonia solution with a mass fraction of 1.5% to 1.9%, or a sodium alkoxide solution with a mass fraction of 90% to 95%, wherein the sodium alkoxide solution includes sodium methoxide solution or sodium ethoxide solution.

[0010] Preferably, the dilute acid in S12 includes any one of the following: dilute hydrochloric acid (0.3646 mg / L to 36.46 mg / L), dilute sulfuric acid (0.4904 mg / L to 490.4 mg / L), or citric acid (0.2593 mg / L to 259.36 mg / L). During the first extraction, the volume ratio of the concentrate to ethyl acetate was 1:2; during the second extraction, the volume ratio of the extract to ethyl acetate was 1:1.

[0011] Preferably, the short-chain alcohol solution in S13 includes any one of methanol solution, ethanol solution or propanol solution.

[0012] Preferably, in step two, the specific method for performing chromatography on the extracted chlorogenic acid compounds includes: Add cotton to the chromatography column, then add quartz sand until it is level with the cotton. Prepare the eluent by mixing the silica gel in the chromatography column with the eluent and stirring it into a homogenate. Pour the mixture into the chromatography column and open it to allow the eluent to flow out slowly. The chlorogenic acid-like substances were dissolved in the first gradient eluent and added dropwise to the chromatography column to start the first gradient elution. Thin-layer chromatography was then used to confirm whether the chlorogenic acid-like substances contained chlorogenic acid, cryptochlorogenic acid, or isochlorogenic acid A.

[0013] Preferably, the eluent comprises ethyl acetate and petroleum ether, wherein the volume ratio of ethyl acetate to petroleum ether is 1:1 to 5; The first gradient eluent comprises ethyl acetate, petroleum ether, and formic acid, wherein the volume ratio of ethyl acetate, petroleum ether, and formic acid is 1:1~6:0.1~0.3.

[0014] An application of chlorogenic acid substances in stevia waste, wherein the chlorogenic acid substances are used to prepare chlorogenic acid gel, the specific method including: S1. Carboxylated graphene oxide was ultrasonically dispersed in deionized water to obtain a suspension. The pH of the suspension was adjusted to 4.5-6.0 using a mixed solution of 2-morpholine ethanesulfonic acid buffer and dilute hydrochloric acid (volume ratio 1:1, 2-morpholine ethanesulfonic acid buffer concentration 0.1 mol / L, dilute hydrochloric acid concentration 0.5~1 mol / L). 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide were added, and the mixture was magnetically stirred at 100~250 rpm for 10~30 min to obtain an activated carboxylated graphene oxide dispersion. S2. Add the chlorogenic acid extracted in step one and 2wt%~10wt% polyvinyl alcohol solution to the activated carboxylated graphene oxide dispersion, heat to 40~50℃ under light-protected conditions, and magnetically stir at 50~100rpm for 10~30min to obtain a gel system. S3. Freeze the gel system in a -18~-25℃ freezer for 6~24h, take it out and thaw it at 25~35℃ for 6~24h, repeat the freeze-thaw cycle multiple times, soak and rinse the thawed hydrogel with deionized water for 12~24h, and dry it at 50~70℃ for 1~12h to obtain chlorogenic acid gel. In S1, the ratio of carboxylated graphene oxide, deionized water, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide is 1~5g:100~250mL:1~4g:1~3g. In S2, the ratio of chlorogenic acid and polyvinyl alcohol solution is 5~10g:200~500mL.

[0015] This invention offers at least the following beneficial effects: Using stevia waste as raw material, the process involves centrifugal filtration, reflux extraction, and rotary concentration to obtain a fat-soluble extract. This extract is then dissolved and filtered with ethanol, and the filtrate is retained and freeze-dried to obtain the filtrate extract, namely, chlorogenic acid products. Extracting and developing chlorogenic acids from stevia waste can solve problems such as environmental pollution and resource waste. Extracting chlorogenic acids from stevia significantly improves the comprehensive utilization rate of stevia, reduces the waste of natural stevia resources, lowers resource consumption during production, and reduces waste emissions. This is a high-efficiency, green, and environmentally friendly extraction process for chlorogenic acids from stevia waste, which can advance the chlorogenic acid extraction industry.

[0016] The chlorogenic acid substances extracted by this invention contain isochlorogenic acid A and chlorogenic acid, and have low sugar content. They have antioxidant, anti-inflammatory and blood pressure lowering functions, and are widely used in food, medicine and health products and other fields. They have high application value and realize the comprehensive utilization of stevia.

[0017] The chlorogenic acid substance obtained in this invention is used to prepare chlorogenic acid gel with carboxylated graphene oxide and polyvinyl alcohol. After activation with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride / N-hydroxysuccinimide, the carboxylated graphene oxide can form a stable covalent bond with chlorogenic acid and polyvinyl alcohol, effectively preventing chlorogenic acid loss and achieving efficient loading and controlled release of chlorogenic acid, ensuring the long-term and stable antioxidant effect of the gel. At the same time, the activated carboxylated graphene oxide also has excellent system compatibility with polyvinyl alcohol and chlorogenic acid-like substances. The overall performance is balanced, ensuring high antioxidant performance while the prepared gel has good formability and mechanical strength. The antioxidant performance and mechanical properties are synergistically improved, which can meet the dual requirements of strength and bioactivity for medical dressings and antioxidant functional materials.

[0018] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0019] Figure 1 Fourier transform infrared spectroscopy (FT-IR) analysis of the alkaline extraction sample in Example 1. Figure 2 Fourier transform infrared spectrum of standard isochlorogenic acid A; Figure 3 The Fourier transform infrared spectrum of chlorogenic acid; Figure 4Fourier transform infrared spectrum of cryptochlorogenic acid; Figure 5 The high-performance liquid chromatograms (HPLC) of the alkaline extraction samples and standards in Example 1 are shown. Figure 6 for Figure 5 A magnified view of a portion of the image; Figure 7 This is a thin-layer chromatogram (TLC) of the alkaline extraction sample from Example 1. Detailed Implementation

[0021] The present invention will now be described in further detail with reference to the accompanying drawings, so that those skilled in the art can implement it based on the description.

[0022] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof. Example 1: A method for extracting chlorogenic acid from stevia residue includes the following steps: (1) Remove the roots and stems from the stevia residue, take the leaves and place them in a ball mill to crush them, and pass them through a 60-mesh sieve to obtain stevia powder.

[0023] Weigh a certain amount of the above-mentioned stevia powder and place it in a round-bottom flask. Set up a reflux apparatus. For the first reflux, the stevia powder and 0.1wt% sodium carbonate solution were refluxed at a mass ratio of 1:8, the temperature was 40℃, and the reflux time was 4.5 h. For the second reflux, the stevia powder and 0.1wt% sodium carbonate solution were refluxed at a mass ratio of 1:7, the temperature was 45℃, and the reflux time was 4 h. After two split extractions, the filtrate was retained.

[0024] (2) Adjust the pH of the filtrate to 7 with a prepared 25 mg / L dilute nitric acid solution, then concentrate it by rotary evaporation and retain the concentrate.

[0025] (3) Extract the concentrate and ethyl acetate separately at a ratio of concentrate:ethyl acetate = 1:2 for 10 h, then extract the concentrate after extraction at a ratio of concentrate:ethyl acetate = 1:1 for 2.5 h, retain the ester layer, and evaporate it by rotary evaporation and water bath to obtain a fat-soluble extract.

[0026] (4) Dissolve all the above fat-soluble extracts in 200 mL of 20 wt% methanol solution, filter, retain the filtrate, freeze dry to obtain the product, namely chlorogenic acid substances.

[0027] (5) The yield of the first extraction was 0.2901% and the yield of the second extraction was 0.20% by calculating the yield.

[0028] (6) The chemical structure of the alkaline extraction samples was characterized using infrared absorption spectroscopy. The specific results are as follows: Figures 1-4 As shown. Figures 1-4 These are, respectively, alkaline extraction samples, standard isochlorogenic acid A, chlorogenic acid, and cryptochlorogenic acid, 1703 cm. -1 This corresponds to the stretching vibration peak of the C=O double bond, at 3397 cm⁻¹. -1 This corresponds to the stretching vibration peak of -OH, at 977 cm⁻¹. -1 This corresponds to the stretching vibration peak of the benzene ring, at 1244 cm⁻¹. -1 This corresponds to the stretching vibration peak of the CO single bond. This indicates that the sample extracted by the alkaline extraction method contains isochlorogenic acid A, chlorogenic acid, etc.

[0029] (7) The presence of isochlorogenic acid in the alkaline extraction sample was determined by high performance liquid chromatography. The results are as follows: Figure 5 , Figure 6 As shown. By observing the characteristic peaks of the sample and the standard, it can be found that there are characteristic peaks of chlorogenic acid and isochlorogenic acid A in the sample, which confirms the presence of chlorogenic acid and isochlorogenic acid A in the alkaline extraction sample. However, the characteristic peak of cryptochlorogenic acid is not obvious, with only one small peak. Further determination of the content of cryptochlorogenic acid in the sample is needed.

[0030] (8) The presence of chlorogenic acid, isochlorogenic acid, and cryptochlorogenic acid in the alkaline extraction sample was further confirmed by column chromatography and TLC. The results are as follows: Figure 7 As shown. Elution in both the first and second stages revealed the presence of isochlorogenic acid A and chlorogenic acid in the sample, while the content of cryptochlorogenic acid was low, requiring further investigation.

[0031] (9) The presence of cryptochlorogenic acid in the sample was further confirmed by high performance liquid chromatography and liquid chromatography-mass spectrometry, and the results are as follows: Figure 6 , Figure 7 As shown.

[0032] Example 2 A method for extracting chlorogenic acid from stevia residue includes the following steps: (1) Remove the roots and stems from the stevia residue, take the leaves and place them in a ball mill to crush them, and then sieve them to obtain stevia powder.

[0033] Weigh a certain amount of the above-mentioned stevia powder and place it in a round-bottom flask. Set up a reflux apparatus and reflux twice with 0.5wt% ammonia water. The first reflux is carried out at a material-to-liquid ratio of 1:8, the reflux temperature is 40℃, and the reflux time is 3h. The second reflux is carried out at a material-to-liquid ratio of 1:7, the reflux temperature is 45℃, and the reflux time is 2h. After two reflux extractions, retain the filtrate and discard the filter residue.

[0034] (2) Adjust the pH of the filtrate to 7 with 19.616 mg / L dilute sulfuric acid, then concentrate it by rotary evaporation and retain the concentrate.

[0035] (3) Extract the concentrate and ethyl acetate separately at a ratio of concentrate:ethyl acetate = 1:2 for 10 h, then extract the concentrate after extraction at a ratio of concentrate:ethyl acetate = 1:1 for 5 h, retain the ester layer, and evaporate it by rotary evaporation and water bath to obtain a fat-soluble extract.

[0036] (4) Dissolve all the fat-soluble extract in 200 mL of 35 wt% methanol solution, filter, retain the filtrate, freeze dry to obtain the product, namely chlorogenic acid substances.

[0037] (5) Calculate the yield, and implement the same method as step (5) in Example 1.

[0038] (6) The implementation method is the same as step (6) in Example 1.

[0039] (7) The implementation method is the same as step (7) in Example 1.

[0040] (8) The implementation method is the same as step (8) in Example 1.

[0041] (9) The implementation method is the same as step (9) in Example 1.

[0042] Example 3 A method for extracting chlorogenic acid from stevia residue includes the following steps: (1) Remove the roots and stems from the stevia residue, take the leaves and place them in a ball mill to crush them, and then sieve them to obtain stevia powder.

[0043] Weigh a certain amount of the above-mentioned stevia powder and place it in a round-bottom flask. Set up a reflux apparatus and reflux twice with anhydrous ethanol. The first reflux is performed at a material-to-liquid ratio of 1:8, a reflux temperature of 50°C, and a reflux time of 4 hours. The second reflux is performed at a material-to-liquid ratio of 1:7, a reflux temperature of 55°C, and a reflux time of 2 hours. After two reflux extractions, retain the filtrate and discard the filter residue.

[0044] (2) Adjust the pH of the filtrate to 7 with the prepared 25 mg / L dilute nitric acid, then concentrate it by rotary evaporation and retain the concentrate.

[0045] (3) Extract the concentrate and ethyl acetate separately at a ratio of concentrate:ethyl acetate = 1:2 for 12 hours. Then extract the concentrate after extraction at a ratio of concentrate:ethyl acetate = 1:1 for 3 hours. Retain the ester layer, and evaporate it by rotary evaporation and water bath to obtain a fat-soluble extract.

[0046] (4) Dissolve all the above fat-soluble extracts in 25 mL of 20 wt% methanol solution, filter, retain the filtrate, freeze dry to obtain the product, namely chlorogenic acid substances.

[0047] (5) Calculate the yield, and implement the same method as step (5) in Example 1.

[0048] (6) The implementation method is the same as step (6) in Example 1.

[0049] (7) The implementation method is the same as step (7) in Example 1.

[0050] (8) The implementation method is the same as step (8) in Example 1.

[0051] (9) The implementation method is the same as step (9) in Example 1.

[0052] Example 4 (1) Remove the roots and stems from the stevia residue, take the leaves and place them in a ball mill to crush them, and then sieve them to obtain stevia powder.

[0053] Weigh a certain amount of the above-mentioned stevia powder and place it in a round-bottom flask. Set up a reflux apparatus and reflux twice with 95wt% ethanol solution. The first reflux was performed at a material-to-liquid ratio of 1:8, a reflux temperature of 55℃, and a reflux time of 4 hours. The second reflux was performed at a material-to-liquid ratio of 1:7, a reflux temperature of 50℃, and a reflux time of 3 hours. After two reflux extractions, retain the filtrate and discard the filter residue.

[0054] (2) Adjust the pH of the filtrate to 7 with the prepared 25.936 mg / L citric acid, then concentrate it by rotary evaporation and retain the concentrate.

[0055] (3) Extract the concentrate and ethyl acetate separately at a ratio of concentrate:ethyl acetate = 1:2 for 12 hours. Then extract the concentrate after extraction at a ratio of concentrate:ethyl acetate = 1:1 for 5 hours. Retain the ester layer, and evaporate it by rotary evaporation and water bath to obtain a fat-soluble extract.

[0056] (4) Dissolve all the fat-soluble extracts obtained above in 200 mL of 30 wt% methanol solution, filter, retain the filtrate, freeze dry to obtain the product, namely chlorogenic acid substances.

[0057] (5) Calculate the yield, and implement the same method as step (5) in Example 1.

[0058] (6) The implementation method is the same as step (6) in Example 1.

[0059] (7) The implementation method is the same as step (7) in Example 1.

[0060] (8) The implementation method is the same as step (8) in Example 1.

[0061] (9) The implementation method is the same as step (9) in Example 1.

[0062] Application Example 1 The chlorogenic acid compounds extracted in Example 1 were used to prepare chlorogenic acid gels. The specific method included: S1. Disperse 3g of carboxylated graphene oxide in 100mL of deionized water using ultrasonication to obtain a suspension. Adjust the pH of the suspension to 5.5 using a mixed solution of 2-morpholine ethanesulfonic acid buffer and dilute hydrochloric acid (volume ratio 1:1, 2-morpholine ethanesulfonic acid buffer concentration 0.1mol / L, dilute hydrochloric acid concentration 0.5mol / L). Add 3.5g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 2g of N-hydroxysuccinimide. Stir magnetically at 250rpm for 30min to obtain an activated carboxylated graphene oxide dispersion. The preparation method of carboxylated graphene oxide includes: adding 5g of graphene oxide and 1.5g of sodium nitrate to 40mL of 78% concentrated sulfuric acid, stirring evenly, slowly adding 10g of potassium permanganate, controlling the temperature not to exceed 20℃, stirring for 2h, raising the temperature to 35℃, continuing to stir for 30min, slowly adding 80mL of deionized water, raising the temperature to 95℃, reacting for 15min, finally adding 100mL of deionized water and 20mL of 30wt% hydrogen peroxide solution, washing three times with 5wt% hydrochloric acid, then washing with deionized water until neutral, ultrasonically dispersing, centrifuging to collect the solid, and drying the solid to obtain carboxylated graphene oxide; S2. Add 8g of chlorogenic acid extracted in step one and 100mL of 10wt% polyvinyl alcohol solution to the activated carboxylated graphene oxide dispersion, heat to 40℃ under light-protected conditions, and magnetically stir at 50rpm for 30min to obtain a gel system. S3. Freeze the gel system in a -25℃ freezer for 12 hours, then thaw it at 25℃ for 8 hours. Repeat the freeze-thaw cycle 5 times. Soak and rinse the thawed hydrogel with deionized water for 12 hours, and dry it at 55℃ for 5 hours to obtain chlorogenic acid gel.

[0063] Application Example 2 The chlorogenic acid compounds extracted in Example 1 were used to prepare chlorogenic acid gels. The specific method included: S1. 1g of carboxylated graphene oxide was ultrasonically dispersed in 100mL of deionized water to obtain a suspension. The pH of the suspension was adjusted to 5.5 using a mixed solution of 2-morpholine ethanesulfonic acid buffer and dilute hydrochloric acid (volume ratio 1:1, 2-morpholine ethanesulfonic acid buffer concentration 0.1mol / L, dilute hydrochloric acid concentration 0.5mol / L). 2g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1g of N-hydroxysuccinimide were added, and the mixture was magnetically stirred at 250rpm for 30min to obtain an activated carboxylated graphene oxide dispersion. The preparation method of carboxylated graphene oxide was the same as in Application Example 1. S2. Add 5g of chlorogenic acid extracted in step one and 100mL of 5wt% polyvinyl alcohol solution to the activated carboxylated graphene oxide dispersion, heat to 40℃ under light-protected conditions, and magnetically stir at 50rpm for 30min to obtain a gel system. S3. Freeze the gel system in a -25℃ freezer for 12 hours, then thaw it at 25℃ for 8 hours. Repeat the freeze-thaw cycle 5 times. Soak and rinse the thawed hydrogel with deionized water for 12 hours, and dry it at 55℃ for 5 hours to obtain chlorogenic acid gel.

[0064] Application Example 3 The chlorogenic acid compounds extracted in Example 1 were used to prepare chlorogenic acid gels. The specific method included: S1. 5g of carboxylated graphene oxide was ultrasonically dispersed in 150mL of deionized water to obtain a suspension. The pH of the suspension was adjusted to 5.5 using a mixed solution of 2-morpholine ethanesulfonic acid buffer and dilute hydrochloric acid (volume ratio 1:1, 2-morpholine ethanesulfonic acid buffer concentration 0.1mol / L, dilute hydrochloric acid concentration 0.5mol / L). 3.5g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 2.5g of N-hydroxysuccinimide were added, and the mixture was magnetically stirred at 250rpm for 30min to obtain an activated carboxylated graphene oxide dispersion. The preparation method of carboxylated graphene oxide was the same as in Application Example 1. S2. Add 10g of chlorogenic acid extracted in step one and 200mL of 10wt% polyvinyl alcohol solution to the activated carboxylated graphene oxide dispersion, heat to 40℃ under light-protected conditions, and magnetically stir at 50rpm for 30min to obtain a gel system. S3. Freeze the gel system in a -25℃ freezer for 12 hours, then thaw it at 25℃ for 8 hours. Repeat the freeze-thaw cycle 5 times. Soak and rinse the thawed hydrogel with deionized water for 12 hours, and dry it at 55℃ for 5 hours to obtain chlorogenic acid gel.

[0065] Comparative Example 1 The chlorogenic acid compounds extracted in Example 1 were used to prepare chlorogenic acid gels. The specific method included: S1. 3g of graphene oxide was ultrasonically dispersed in 100mL of deionized water to obtain a suspension. The pH of the suspension was adjusted to 5.5 using a mixed solution of 2-morpholine ethanesulfonic acid buffer and dilute hydrochloric acid (volume ratio 1:1, 2-morpholine ethanesulfonic acid buffer concentration 0.1mol / L, dilute hydrochloric acid concentration 0.5mol / L). 3.5g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 2g of N-hydroxysuccinimide were added, and the mixture was magnetically stirred at 250rpm for 30min to obtain an activated graphene oxide dispersion. S2. Add 8g of chlorogenic acid extracted in step one and 100mL of 10wt% polyvinyl alcohol solution to the activated graphene oxide dispersion, heat to 40℃ under light-protected conditions, and magnetically stir at 50rpm for 30min to obtain a gel system. S3. Freeze the gel system in a -25℃ freezer for 12 hours, then thaw it at 25℃ for 8 hours. Repeat the freeze-thaw cycle 5 times. Soak and rinse the thawed hydrogel with deionized water for 12 hours, and dry it at 55℃ for 5 hours to obtain chlorogenic acid gel.

[0066] Comparative Example 2 The chlorogenic acid compounds extracted in Example 1 were used to prepare chlorogenic acid gels. The specific method included: S1. Mix 8g of chlorogenic acid extracted in step one with 100mL of 10wt% polyvinyl alcohol solution, heat to 40℃ under light-protected conditions, and magnetically stir at 50rpm for 30min to obtain a gel system. S3. Freeze the gel system in a -25℃ freezer for 12 hours, then thaw it at 25℃ for 8 hours. Repeat the freeze-thaw cycle 5 times. Soak and rinse the thawed hydrogel with deionized water for 12 hours, and dry it at 55℃ for 5 hours to obtain chlorogenic acid gel.

[0067] Comparative Example 3 The chlorogenic acid compounds extracted in Example 1 were used to prepare chlorogenic acid gels. The specific method included: S1. 3g of carboxylated graphene oxide was ultrasonically dispersed in 100mL of deionized water to obtain a suspension; wherein, the preparation method of carboxylated graphene oxide was the same as in application example 1. S2. Add 8g of chlorogenic acid extracted in step one and 100mL of 10wt% polyvinyl alcohol solution to the carboxylated graphene oxide dispersion, heat to 40℃ under light-protected conditions, and magnetically stir at 50rpm for 30min to obtain a gel system. S3. Freeze the gel system in a -25℃ freezer for 12 hours, then thaw it at 25℃ for 8 hours. Repeat the freeze-thaw cycle 5 times. Soak and rinse the thawed hydrogel with deionized water for 12 hours, and dry it at 55℃ for 5 hours to obtain chlorogenic acid gel.

[0068] The DPPH free radical scavenging rate, tensile strength, and yield strength of the chlorogenic acid gels prepared in Application Examples 1-3 and Comparative Examples 1-3 were determined respectively. The specific experimental method for the DPPH free radical scavenging rate was as follows: 0.5 g of each chlorogenic acid gel sample was weighed and placed in a 50 mL centrifuge tube. 20 mL of ethanol-water mixture (volume ratio 1:1) was added, and the mixture was ultrasonically extracted at 30 °C and 40 kHz for 30 min. After extraction, the mixture was centrifuged at 8000 rpm for 10 min, and the supernatant was collected. The residue was added to 10 mL of the above mixture and the extraction was repeated once. The two supernatants were combined and the volume was adjusted to 50 mL with ethanol-water mixture to obtain the sample extract. The extract was sealed and protected from light for later use.

[0069] Weigh 2.4 mg of DPPH powder, dissolve it in anhydrous ethanol and dilute to 50 mL to prepare a 0.1 mmol / L DPPH ethanol solution. Store in the dark and refrigerated. Weigh 0.0100 g of VC, dissolve it in deionized water and dilute to 100 mL to prepare a 1.0 mg / mL VC standard stock solution. Store in the dark.

[0070] Add 2.0 mL of 0.1 mmol / L DPPH ethanol solution and 2.0 mL of sample extract to a 10 mL stoppered colorimetric tube, shake gently, and incubate in a 25°C water bath in the dark for 30 min. Use 2.0 mL of anhydrous ethanol and 2.0 mL of sample extract as blank samples, and 2.0 mL of 0.1 mmol / L DPPH ethanol solution and 2.0 mL of anhydrous ethanol as blank controls. Immediately after the reaction, measure the absorbance of each tube at 517 nm and record it as A. x (Sample group), A1 (blank sample), A0 (blank control). All samples were tested in 3 parallel groups, and the average value was calculated.

[0071] Scavenging rate calculation formula: DPPH free radical scavenging rate (%) = (A x -A1) / A0×100%: Among them, A x A1 represents the absorbance of the sample extract after reacting with DPPH solution; A2 represents the absorbance of the blank sample; A3 represents the absorbance of the blank control.

[0072] The test methods for tensile strength and yield strength are specified in GB / T 1040.1-2025; the test data are summarized in the table below: Table 1 Performance test data of various chlorogenic acid gels As can be seen from Table 1, the chlorogenic acid gels prepared in Application Examples 1-3 all showed significantly higher DPPH free radical scavenging rates than those in Comparative Examples 1-3. This is because after the carboxylated graphene oxide was activated by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, it formed a stable covalent bond with chlorogenic acid and polyvinyl alcohol (PVA), resulting in a high chlorogenic acid loading rate and stable release, thus exhibiting significant antioxidant properties. In Comparative Example 2, no graphene oxide was used, and only chlorogenic acid and PVA were physically mixed. Chlorogenic acid was easily lost, resulting in the lowest scavenging rate (65.4%).

[0073] The chlorogenic acid gel prepared in Comparative Example 1 (graphene oxide without carboxylation) showed a lower DPPH scavenging rate than that in Application Example 1, indicating that the surface functional groups of carboxylated graphene oxide can enhance the chlorogenic acid loading capacity. The chlorogenic acid gel prepared in Comparative Example 3 (carboxylated graphene oxide without activation) showed a much lower DPPH scavenging rate than that in Application Example 1, proving that the activation of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide is the core to achieve covalent cross-linking of carboxylated graphene oxide with chlorogenic acid and PVA. Without activation, it is a physical mixture with a low chlorogenic acid loading rate.

[0074] Consistent with the clearance rate trend, the chlorogenic acid gels prepared in Application Examples 1-3 exhibited the best tensile strength and elongation at break, as the three-dimensional network structure formed by covalent cross-linking enhanced the mechanical stability of the gel. In Comparative Example 2, without graphene oxide, the gel relied solely on the freeze-thaw cross-linking of PVA, resulting in a loose structure and the worst mechanical properties. Furthermore, the bottom walls of Application Examples 1 and Comparative Example 2 indicate that the addition of carboxylated graphene oxide played a crucial role in improving the mechanical properties of the chlorogenic acid gel.

[0075] The number of devices and processing scale described herein are for the purpose of simplifying the description of the invention. Applications, modifications, and variations of the invention will be readily apparent to those skilled in the art.

[0076] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A method for extracting chlorogenic acid from stevia waste, characterized in that, Includes the following steps: Step 1: Extract chlorogenic acid from stevia powder using alkaline extraction. Step 2: Use column chromatography to analyze the extracted chlorogenic acid substances to determine whether the chlorogenic acid substances contain chlorogenic acid, cryptochlorogenic acid, or isochlorogenic acid A.

2. The method for extracting chlorogenic acid from stevia residue according to claim 1, characterized in that, In step one, the method for preparing stevia powder includes: removing the roots and stems from stevia residue, placing the leaves in a ball mill to crush them, and passing them through a 60-mesh sieve to obtain stevia powder.

3. The method for extracting chlorogenic acid from stevia residue according to claim 1, characterized in that, In step one, the specific method for extracting chlorogenic acid substances from stevia powder using alkaline extraction includes: S11. The sieved stevia powder is extracted twice by reflux with an alkaline solution of a certain concentration. The second extract is centrifuged and filtered, the filtrate is retained and the filter residue is removed. S12. Adjust the pH of the filtrate to 6-7 with a prepared dilute acid of a certain concentration. Perform rotary concentration and evaporation at a temperature of 40-50℃ and a rotation speed of 20-60rpm. Retain the concentrate. Mix the concentrate with ethyl acetate for the first extraction. Then mix the extract from the first extraction with ethyl acetate for the second extraction. Retain the ethyl acetate layer. Concentrate by rotary evaporation. Pour the retained concentrate into an evaporating dish. Place the evaporating dish on a water bath to evaporate and dry to obtain a fat-soluble extract. S13. Dissolve the fat-soluble extract in a short-chain alcohol solution, filter, retain the filtrate, and freeze-dry to obtain the filtrate extract, which is a chlorogenic acid compound.

4. The method for extracting chlorogenic acid from stevia residue according to claim 3, characterized in that, In S11, during the first reflux extraction, the mass ratio of stevia powder to alkaline solution is 1:5~10, the reflux time is 1~5h, and the extraction temperature is 45~55℃. During the second reflux extraction, the mass ratio of stevia powder to alkaline solution was 1:5~10, the reflux time was 1~5h, and the extraction temperature was 45~55℃.

5. The method for extracting chlorogenic acid from stevia residue according to claim 3, characterized in that, The alkaline solution in S11 includes one of the following: a sodium carbonate solution with a mass fraction of 0.1% to 0.5%, an ammonia solution with a mass fraction of 1.5% to 1.9%, or a sodium alkoxide solution with a mass fraction of 90% to 95%. The sodium alkoxide solution includes sodium methoxide solution and sodium ethoxide solution.

6. The method for extracting chlorogenic acid from stevia residue according to claim 3, characterized in that, The dilute acid in S12 includes any one of the following: dilute hydrochloric acid (0.3646 mg / L to 36.46 mg / L), dilute sulfuric acid (0.4904 mg / L to 490.4 mg / L), or citric acid (0.2593 mg / L to 259.36 mg / L). During the first extraction, the volume ratio of the concentrate to ethyl acetate was 1:2; during the second extraction, the volume ratio of the extract to ethyl acetate was 1:

1.

7. The method for extracting chlorogenic acid from stevia residue according to claim 3, characterized in that, The short-chain alcohol solution in S13 includes any one of methanol solution, ethanol solution or propanol solution.

8. The method for extracting chlorogenic acid from stevia residue according to claim 1, characterized in that, In step two, the specific method for performing chromatography on the extracted chlorogenic acid compounds using a chromatography column includes: Add cotton to the chromatography column, then add quartz sand until it is level with the cotton. Prepare the eluent by mixing the silica gel in the chromatography column with the eluent and stirring it into a homogenate. Pour the mixture into the chromatography column and open it to allow the eluent to flow out slowly. The chlorogenic acid-like substances were dissolved in the first gradient eluent and added dropwise to the chromatography column to start the first gradient elution. Thin-layer chromatography was then used to confirm whether the chlorogenic acid-like substances contained chlorogenic acid, cryptochlorogenic acid, or isochlorogenic acid A.

9. The method for extracting chlorogenic acid from stevia residue according to claim 8, characterized in that, The eluent includes ethyl acetate and petroleum ether, with a volume ratio of ethyl acetate to petroleum ether of 1:1 to 5. The first gradient eluent comprises ethyl acetate, petroleum ether, and formic acid, wherein the volume ratio of ethyl acetate, petroleum ether, and formic acid is 1:1~6:0.1~0.

3.

10. An application of chlorogenic acid substances in stevia waste residue, wherein the chlorogenic acid substances in the stevia waste residue are extracted by the extraction method for chlorogenic acid substances in stevia waste residue according to any one of claims 1-9, characterized in that, The chlorogenic acid-like substances are used in the preparation of chlorogenic acid gels, and the specific methods include: S1. Carboxylated graphene oxide is ultrasonically dispersed in deionized water to obtain a suspension; the pH of the suspension is adjusted to 4.5-6.0 using a mixed solution of 2-morpholine ethanesulfonic acid buffer and dilute hydrochloric acid; 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide are added; the mixture is magnetically stirred at 100-250 rpm for 10-30 min to obtain an activated carboxylated graphene oxide dispersion. S2. Add the chlorogenic acid extracted in step one and 2wt%~10wt% polyvinyl alcohol solution to the activated carboxylated graphene oxide dispersion, heat to 40~50℃ under light-protected conditions, and magnetically stir at 50~100rpm for 10~30min to obtain a gel system. S3. Freeze the gel system in a freezer at -18~-25℃ for 6~24h, then thaw it at 25~35℃ for 6~24h. Repeat the freeze-thaw cycle multiple times. Soak and rinse the thawed hydrogel with deionized water for 12~24h, and dry it at 50~70℃ for 1~12h to obtain chlorogenic acid gel.

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