A method for extracting forsythiaside and forsythin from forsythia and forsythia extract thereof

Abstract

The application provides a method for extracting forsythiaside and forsythin from forsythia suspensa, comprising the following steps: (1) preparation of an extraction reagent, adopting a chitosan-organic acid solution as the extraction reagent; (2) treatment of the top tender leaves of forsythia suspensa; (3) reduced-pressure filtration; and (4) pH adjustment. The application also discloses a forsythia suspensa extract obtained by the above method, comprising target active ingredients, forsythiaside and forsythin, the extraction rate of forsythiaside being 0.058-0.071%, and the extraction rate of forsythin being 6.1-7.6%. The application has the following advantages: (1) the application innovatively adds chitosan and organic acid as the extraction reagent, and research finds that the extraction rate of the target active ingredients can be effectively improved; (2) the forsythia suspensa extract obtained by the application has good pH response controlled release effect; and (3) the raw material adopted by the application is the top tender leaf part of forsythia suspensa which is pruned during forsythia suspensa planting, does not compete for resources in the field of traditional Chinese medicines, and realizes resource recycling and reuse while improving the extraction rate of active ingredients and the bioavailability, thereby saving forsythia suspensa plant resources.

Description

Technical Field

[0001] This invention belongs to the field of skin care technology, specifically relating to a method for the efficient combined extraction of forsythoside and forsythoside A from Forsythia suspensa and the Forsythia suspensa extract thereof. Background Technology

[0002] In recent years, Forsythia suspensa extract, as a natural plant ingredient, has received widespread attention in the skincare industry due to its excellent anti-inflammatory and antioxidant effects, as well as its high safety profile. Currently, most skincare products utilize Forsythia suspensa extract by extracting the medicinal active ingredients from the fruit, which creates a disadvantageous situation of competing with the traditional Chinese medicine field for resources. Therefore, in recent years, the cosmetics industry has focused its research on the non-medicinal part of Forsythia suspensa, the leaves, and has made some progress. Currently, existing technologies use traditional reagents such as water and ethanol to extract Forsythia suspensa leaves, but the extraction rate is significantly limited. For example, Chinese patent CN102219813B uses water as the extraction reagent to extract forsythoside and forsythoside A from Forsythia suspensa leaves, but the extraction rate has not yet been significantly improved.

[0003] Furthermore, practical research has revealed that the cultivation of Forsythia also results in a significant waste of resources. To promote vigorous plant growth and improve fruit quality, the tender leaves at the top of the Forsythia plant are typically pinched off periodically to remove apical dominance. These pinched leaves are often overlooked and discarded. However, these tender leaves are also rich in various medicinal active ingredients, and at higher levels than ordinary Forsythia leaves. This wastes Forsythia plant resources and fails to fully utilize them.

[0004] Therefore, how to efficiently extract the active ingredients from Forsythia suspensa, reduce resource waste, and further improve its bioavailability are urgent technical problems to be solved. Summary of the Invention

[0005] The purpose of this invention is to provide a method for the efficient combined extraction of forsythoside and forsythoside A from Forsythia suspensa, and the resulting Forsythia suspensa extract. This method introduces a novel extraction reagent that can simultaneously and efficiently extract the active ingredients for skincare benefits from Forsythia suspensa: forsythoside and forsythoside A. The final Forsythia suspensa extract exhibits pH-responsive controlled-release properties. Specifically, during the preparation process, the degree of chitosan molecular chain relaxation is adjusted by bidirectional pH regulation, achieving efficient extraction and encapsulation of active substances. When exposed to the acidic environment of human skin (pH approximately 5.4), the chitosan molecular chains relax again, allowing for the slow release of active ingredients and reducing the irritation of the plant extract. This method improves bioavailability while achieving efficient utilization of plant resources and conserving Forsythia suspensa plant resources.

[0006] To achieve the above objectives, the present invention provides a method for the efficient combined extraction of forsythoside A and forsythoside A from Forsythia suspensa, comprising the following steps: (1) Preparation of extraction reagent: Prepare an organic acid-water solution to obtain an organic acid solution, add chitosan to the organic acid solution, heat and stir to obtain a chitosan-organic acid solution, and thus obtain the extraction reagent; (2) Treatment of tender leaves at the top of Forsythia: The tender leaves at the top of Forsythia after the "pinching" treatment during the planting process are collected and crushed and sieved. They are then added to the extraction reagent obtained in step (1) and stirred for extraction. (3) Vacuum filtration: After extraction in step (2), vacuum filtration is performed to obtain Forsythia extract; (4) pH adjustment: Add sodium bicarbonate to adjust the Forsythia extract in step (3) to obtain Forsythia extract. The extracted Forsythia extract includes forsythoside and forsythoside A. The extraction rate of forsythoside is 0.058-0.071%, and the extraction rate of forsythoside A is 6.1-7.6%.

[0007] This invention innovatively incorporates chitosan and organic acids as extraction reagents. Chitosan can serve as a carrier for the active ingredients of plant extracts. This invention also innovatively uses chitosan dissolved in organic acid solution to extract the active ingredients from Forsythia suspensa. Studies have shown that this can effectively improve the extraction rate of the active ingredients.

[0008] Furthermore, research shows that the content of active ingredients in the tender leaves at the top of Forsythia suspensa is higher than that in ordinary Forsythia suspensa leaves. Therefore, by using the tender leaves at the top of Forsythia suspensa in combination with the novel extraction reagent of this invention, the tender leaves at the top that are ignored and discarded in the prior art can be recycled and reused, saving Forsythia suspensa plant resources to the greatest extent, while significantly improving the extraction rate of active ingredients. This results in the novel extraction method of this invention achieving a variety of beneficial effects.

[0009] Preferably, in step (1), the chitosan is any one of deacetylated chitosan, carboxymethyl deacetylated chitosan, or hydroxyethyl deacetylated chitosan, and the organic acid is any one of glycyrrhizic acid, malic acid, lactic acid, or citric acid. This invention has found that chitosan exhibits high molecular network expansion and high exposure of active functional groups under acidic conditions, which facilitates the extraction of plant active ingredients. By increasing the pH of the extract, the chitosan network structure contracts and encapsulates the active ingredients. When exposed to the acidic environment of human skin (pH approximately 5.4), the chitosan molecular chains expand again, resulting in high permeability of the active ingredients, allowing for slow release of the active ingredients and reducing the irritation of plant extracts upon skin contact. Furthermore, in step (1), the pH value of the organic acid solution is 2.5-5.5, the mass percentage of chitosan in the organic acid solution is 0.5-2.5%, and the chitosan-organic acid solution is obtained by stirring at 25-80℃ and 200-600r / min for 30-240min.

[0010] Furthermore, in step (2), the mass ratio of the tender leaves at the top of Forsythia suspensa to the extraction reagent is 1:12-1:20, and the extraction is carried out by stirring for 30-240 minutes at 25-80℃ and 200-600r / min.

[0011] Furthermore, in step (3), after extraction in step (2), the mixture is cooled to below 35°C and filtered under reduced pressure at 0.08-0.1 MPa for 3-5 times to obtain Forsythia extract.

[0012] Furthermore, in step (4), sodium bicarbonate is added to adjust the Forsythia extract in step (3) to adjust the pH to 6.5-8.5.

[0013] By using the specific conditions and parameters of each of the above steps, the novel extraction reagent can extract the active ingredients of Forsythia suspensa to the maximum extent. The reagents used are easy to purchase, the extraction process is easy to implement, and it is easy to scale up production.

[0014] Furthermore, the method of the present invention also includes step (5) constructing a stable system: combining the forsythia extract obtained in step (4) with a polyol, wherein the mass percentage of the polyol in the forsythia extract is 20-80%.

[0015] Preferably, the polyol in step (5) is glycerol or butanediol.

[0016] The novel extraction method of this invention yields a Forsythia extract containing forsythoside and forsythoside A. This invention also found that adding glycerol or butylene glycol can increase the stability of the Forsythia extract and extend the storage period of the product.

[0017] Compared with existing technologies, the Forsythia extract prepared by the novel extraction method of this invention has a significantly different content of active ingredients, therefore the extract obtained by this invention is also within the scope of protection.

[0018] In summary, the beneficial effects of this invention are as follows: (1) This invention innovatively adds chitosan and organic acids as extraction reagents, which can effectively improve the extraction rate of active ingredients; (2) The Forsythia extract obtained by this invention has a good pH-responsive controlled release effect, that is, in the preparation process, the degree of relaxation of chitosan molecular chains is adjusted by bidirectional pH regulation. Under acidic conditions, the degree of relaxation of the polymer network is high and the exposure of active functional groups is high, which helps to extract plant active ingredients; by increasing the pH value of the extract, the chitosan network structure shrinks and encapsulates the active ingredients. When it comes into contact with the acidic environment of human skin (pH is about 5.4), the chitosan molecular chains relax again, so that the active ingredients can be released slowly, reducing the irritation of plant extracts when they come into contact with the skin; (3) The raw material used in this invention is the tender leaf part at the top of Forsythia that has been pinched off during the planting process, which does not compete for Chinese medicinal resources; and the novel extraction method of this invention improves the extraction rate of active ingredients and enhances bioavailability, while realizing the efficient utilization of plant resources and saving Forsythia plant resources. Attached Figure Description

[0019] Figure 1 The release rate of forsythoside A from D01, D05, and A02 under different pH conditions; Figure 2 The graph shows the cell viability of keratinocytes D01, A01, A02, and A03; * indicates P < 0.05, indicating a significant difference compared to the control group, and ** indicates P < 0.01, indicating a highly significant difference compared to the control group. Figure 3 The graph shows the results of the soothing effects of the sample group and the blank group; * indicates P < 0.05, indicating a significant difference compared with the blank group, and ** indicates P < 0.01, indicating a highly significant difference compared with the blank group. Detailed Implementation

[0020] The specific embodiments of the present invention will be described in detail below. It should be noted that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0021] Example 1: First, a malic acid aqueous solution with a pH of 2.5 was prepared by adjusting the malic acid content. Using this solution as a solvent, a carboxymethyl deacetylated chitosan-malic acid solution with a solute mass percentage of 0.5% was prepared. The solution was stirred at 25°C and 200 rpm for 30 minutes to obtain a carboxymethyl deacetylated chitosan-malic acid composite solution. 5g of crushed tender leaves from the top of *Forsythia suspensa* were weighed and added to the above carboxymethyl deacetylated chitosan-malic acid solution, making the ratio of *Forsythia suspensa* leaves to carboxymethyl deacetylated chitosan-malic acid solution 1:12. The solution was stirred at 25°C and 200 rpm for 30 minutes for extraction. After extraction, the solution was cooled to below 35°C and filtered three times under 0.08 MPa to obtain the *Forsythia suspensa* extract. Sodium carbonate solution was added to adjust the pH to 6.5. After adjustment, *Forsythia suspensa* extract A01 was obtained.

[0022] Example 2: First, a glycyrrhizic acid-water solution with a pH of 4.0 was prepared by adjusting the glycyrrhizic acid content. Using this solution as a solvent, a deacetylated chitosan-glycyrrhizic acid solution with a solute mass percentage of 1.25% was prepared. The solution was stirred at 53°C and 400 rpm for 135 minutes to obtain a deacetylated chitosan-glycyrrhizic acid composite solution. 5g of pulverized Forsythia suspensa leaves were weighed and added to the above deacetylated chitosan-glycyrrhizic acid composite solution, making the ratio of Forsythia suspensa leaves to deacetylated chitosan-glycyrrhizic acid solution 1:16. The solution was stirred at 53°C and 400 rpm for 135 minutes for extraction. After extraction, the solution was cooled to below 35°C and filtered four times under 0.09 MPa to obtain the Forsythia suspensa extract. Sodium carbonate solution was added to adjust the pH to 7.5. After adjustment, Forsythia suspensa extract A02 was obtained.

[0023] Example 3: First, a lactic acid-water solution with a pH of 5.5 was prepared by adjusting the lactic acid content. This solution was then used as a solvent to prepare a 2.5% hydroxyethyl deacetylated chitosan-lactic acid solution. The solution was stirred at 80°C and 600 rpm for 240 minutes to obtain a hydroxyethyl deacetylated chitosan-lactic acid composite solution. 5g of pulverized Forsythia suspensa leaves were weighed and added to the above hydroxyethyl deacetylated chitosan-lactic acid composite solution, making the ratio of Forsythia suspensa leaves to hydroxyethyl deacetylated chitosan-lactic acid solution 1:20. The solution was stirred at 80°C and 600 rpm for 240 minutes for extraction. After extraction, the solution was cooled to below 35°C and filtered five times under 0.1 MPa to obtain the Forsythia suspensa extract. Sodium carbonate solution was added to adjust the pH to 8.5. After adjustment, Forsythia suspensa extract A03 was obtained.

[0024] Example 4: First, a citric acid-water solution with a pH of 3.0 was prepared by adjusting the citric acid content. Using this solution as a solvent, a 2% deacetylated chitosan-citric acid solution was prepared. The solution was stirred at 45°C and 350 rpm for 60 minutes to obtain a deacetylated chitosan-citric acid composite solution. 5g of pulverized Forsythia suspensa leaves were weighed and added to the above deacetylated chitosan-citric acid composite solution, making the ratio of Forsythia suspensa leaves to deacetylated chitosan-citric acid solution 1:13. The solution was stirred at 30°C and 350 rpm for 50 minutes for extraction. After extraction, the solution was cooled to below 35°C and filtered four times under 0.08 MPa to obtain the Forsythia suspensa extract. Sodium carbonate solution was added to adjust the pH to 6.8. After adjustment, glycerol was added so that the proportion of glycerol in the Forsythia suspensa extract was 20%, resulting in the compounded Forsythia suspensa extract A04.

[0025] Example 5: First, a glycyrrhizic acid-water solution with a pH of 5.0 was prepared by adjusting the glycyrrhizic acid content. This solution was then used as a solvent to prepare a 1% carboxymethyl deacetylated chitosan-glycyrrhizic acid solution. The solution was stirred at 70°C and 500 rpm for 230 min to obtain a carboxymethyl deacetylated chitosan-glycyrrhizic acid composite solution. 5 g of pulverized Forsythia suspensa leaves were weighed and added to the above carboxymethyl deacetylated chitosan-glycyrrhizic acid composite solution, making the ratio of Forsythia suspensa leaves to carboxymethyl deacetylated chitosan-glycyrrhizic acid solution 1:19. The solution was stirred at 70°C and 500 rpm for 230 min for extraction. After extraction, the solution was cooled to below 35°C and filtered five times under 0.09 MPa to obtain the Forsythia suspensa extract. Sodium carbonate solution was added to adjust the pH to 8. After adjustment, butanediol was added, making the butanediol content in the Forsythia suspensa extract 20%, resulting in the compounded Forsythia suspensa extract A05.

[0026] Example 6: First, a lactic acid-water solution with a pH of 2.6 was prepared by adjusting the lactic acid content. This solution was then used as a solvent to prepare a 2.4% carboxymethyl deacetylated chitosan-lactic acid solution. The solution was stirred at 35°C and 400 rpm for 40 minutes to obtain a carboxymethyl deacetylated chitosan-lactic acid composite solution. 5g of pulverized Forsythia suspensa leaves were weighed and added to the above composite carboxymethyl deacetylated chitosan-lactic acid solution, making the ratio of Forsythia suspensa leaves to carboxymethyl deacetylated chitosan-lactic acid solution 1:14. The solution was stirred at 35°C and 400 rpm for 40 minutes for extraction. After extraction, the solution was cooled to below 35°C and filtered three times under 0.08 MPa to obtain the Forsythia suspensa extract. Sodium carbonate solution was added to adjust the pH to 6.6. After adjustment, glycerol was added so that the proportion of glycerol in the Forsythia suspensa extract was 50%, resulting in the compounded Forsythia suspensa extract A06.

[0027] Example 7: First, a citric acid-water solution with a pH of 5.4 was prepared by adjusting the citric acid content. Using this solution as a solvent, a 0.6% deacetylated chitosan-citric acid solution was prepared. The solution was stirred at 60°C and 550 rpm for 220 min to obtain a deacetylated chitosan-citric acid composite solution. 5 g of pulverized Forsythia suspensa leaves were weighed and added to the above deacetylated chitosan-citric acid solution, making the ratio of Forsythia suspensa leaves to deacetylated chitosan-citric acid solution 1:18. The solution was stirred at 60°C and 550 rpm for 220 min for extraction. After extraction, the solution was cooled to below 35°C and filtered five times under 0.09 MPa to obtain the Forsythia suspensa extract. Sodium carbonate solution was added to adjust the pH to 8.4. After adjustment, butanediol was added so that the proportion of butanediol in the Forsythia suspensa extract was 50%, resulting in the compounded Forsythia suspensa extract A07.

[0028] Example 8: First, a malic acid-water solution with a pH of 2.7 was prepared by adjusting the malic acid content. Using this solution as a solvent, a 2.3% deacetylated chitosan-malic acid solution was prepared. The solution was stirred at 50°C and 250 rpm for 70 minutes to obtain a deacetylated chitosan-malic acid composite solution. 5g of pulverized Forsythia suspensa leaves were weighed and added to the above deacetylated chitosan-malic acid composite solution, making the ratio of Forsythia suspensa leaves to the deacetylated chitosan-malic acid composite solution 1:15. The solution was stirred at 50°C and 250 rpm for 70 minutes for extraction. After extraction, the solution was cooled to below 35°C and filtered four times under 0.08 MPa to obtain the Forsythia suspensa extract. Sodium carbonate solution was added to adjust the pH to 6.7. After adjustment, glycerol was added so that the glycerol content in the Forsythia suspensa extract was 80%, resulting in the compounded Forsythia suspensa extract A08.

[0029] Example 9: First, a glycyrrhizic acid-water solution with a pH of 5.3 was prepared by adjusting the glycyrrhizic acid content. This solution was then used as a solvent to prepare a 0.7% hydroxyethyl deacetylated chitosan-glycyrrhizic acid solution. The solution was stirred at 55°C and 350 rpm for 100 min to obtain a hydroxyethyl deacetylated chitosan-glycyrrhizic acid composite solution. 5 g of pulverized Forsythia suspensa leaves were weighed and added to the above hydroxyethyl deacetylated chitosan-glycyrrhizic acid composite solution, making the ratio of Forsythia suspensa leaves to the hydroxyethyl deacetylated chitosan-glycyrrhizic acid composite solution 1:17. The solution was stirred at 65°C and 350 rpm for 100 min for extraction. After extraction, the solution was cooled to below 35°C and filtered five times under 0.09 MPa to obtain the Forsythia suspensa extract. Sodium carbonate solution was added to adjust the pH to 7.5. After adjustment, butanediol was added so that the butanediol content in the Forsythia suspensa extract was 80%, resulting in the compounded Forsythia suspensa extract A09.

[0030] The following lists some of the experimental procedures for the technical solution of this invention. This invention can be configured with various combinations of extraction reagents, extraction methods, and condition parameters. Furthermore, due to space limitations, this patent only lists some of the experimental procedures to illustrate the inventive effort the applicant made in finally obtaining the technical solution.

[0031] Comparative Example 1 (Water Extraction Control Group) Weigh 5g of crushed Forsythia leaves and add them to deionized water to make the ratio of Forsythia leaves to deionized water 1:16. Stir and extract at 45℃ and 350r / min for 120min. After extraction, cool to below 35℃ and filter under reduced pressure to obtain Forsythia extract. Add sodium carbonate solution to adjust the pH to 7.0. After adjustment, Forsythia extract DO1 is obtained.

[0032] Comparative Example 2 First, a glycyrrhizic acid-water solution with a pH of 1.5 was prepared by adjusting the glycyrrhizic acid content. Using this solution as a solvent, a deacetylated chitosan-glycyrrhizic acid solution with a solute concentration of 0.25% was prepared. The solution was stirred at 45℃ and 350 rpm for 60 minutes to obtain a deacetylated chitosan-glycyrrhizic acid composite solution. 5g of pulverized Forsythia suspensa leaves were weighed and added to the above deacetylated chitosan-glycyrrhizic acid composite solution, making the ratio of Forsythia suspensa leaves to the deacetylated chitosan-glycyrrhizic acid composite solution 1:10. The solution was stirred at 25℃ and 350 rpm for 15 minutes for extraction. After extraction, the solution was cooled to below 35℃ and filtered under reduced pressure to obtain the Forsythia suspensa extract. Sodium carbonate solution was added to adjust the pH to 6.0. After adjustment, Forsythia suspensa extract DO2 was obtained.

[0033] Comparative Example 3 First, a glycyrrhizic acid-water solution with a pH of 6.0 was prepared by adjusting the glycyrrhizic acid content. Using this solution as a solvent, a 3.5% deacetylated chitosan-glycyrrhizic acid solution was prepared. The solution was stirred at 45℃ and 350 rpm for 60 minutes to obtain a deacetylated chitosan-glycyrrhizic acid composite solution. 5g of pulverized Forsythia suspensa leaves were weighed and added to the above deacetylated chitosan-glycyrrhizic acid composite solution, making the ratio of Forsythia suspensa leaves to deacetylated chitosan-glycyrrhizic acid solution 1:22. The solution was stirred at 90℃ and 350 rpm for 300 minutes for extraction. After extraction, the solution was cooled to below 35℃ and filtered under reduced pressure to obtain the Forsythia suspensa extract. Sodium carbonate solution was added to adjust the pH to 9.0. After adjustment, Forsythia suspensa extract DO3 was obtained.

[0034] Comparative Example 4 (Alcohol Extraction Control Group) Weigh 5g of crushed Forsythia leaves and add them to anhydrous ethanol, making the ratio of Forsythia leaves to anhydrous ethanol 1:16. Stir and extract at 45℃ and 350r / min for 120min. After extraction, cool to below 35℃ and filter under reduced pressure to obtain Forsythia extract. Add sodium carbonate solution to adjust the pH to 7.0. After adjustment, Forsythia extract DO4 is obtained.

[0035] The extraction rate and antioxidant efficacy of the proportions and examples will be tested to further determine the preparation method conditions and parameters that work in conjunction with the extraction reagents.

[0036] Methods for testing extraction rate and antioxidant activity: The contents of forsythoside and forsythoside A in Forsythia suspensa extract were determined using the high-performance liquid chromatography (HPLC) method as described in the Chinese Pharmacopoeia. HPLC is an analytical technique used to separate, identify, and quantify components in a mixture. It utilizes a chromatographic column to separate the mixture, followed by spectroscopic identification and quantification. By determining the peak times and peak areas of forsythoside and forsythoside A standards using HPLC, and then employing peak area regression localization, the contents of forsythoside and forsythoside A in the sample were determined. Since this detection method is existing technology, it is only briefly described here.

[0037] The antioxidant capacity of Forsythia suspensa extract was determined using a DPPH free radical scavenging assay. 1,1-Diphenyl-2-trinitrophenylhydrazine (DPPH) is a stable, long-lived free radical; its ethanol solution is deep purple and exhibits strong absorption near 517 nm. In the presence of a free radical scavenger, the light absorption of the DPPH ethanol solution is reduced due to single electron pairing. The degree of fading of the DPPH ethanol solution is linearly related to the number of electrons it accepts. The absorbance was measured using a UV-1600 spectrophotometer, and the free radical scavenging rate at different concentrations was calculated. Curve fitting was then performed to calculate the IC50 of the sample. 50 The concentration required to scavenge 50% of free radicals (i.e., the concentration required to remove 50% of free radicals) can be used to evaluate the antioxidant activity of Forsythia suspensa extract. Since this detection method is existing technology, it will be described briefly.

[0038] Table 1. Extraction rates and antioxidant activity of forsythoside A and forsythoside A from Forsythia suspensa extract.

[0039] As shown in Table 1, the results indicate that: (1) The extraction rate of forsythoside A in all groups was higher than that in the water extraction control group and the alcohol extraction control group; (2) The extraction rate of forsythoside A in the groups within the range of Examples 1 to 3 of the present invention is higher than that in the groups of other comparative examples; (3) The antioxidant effect is positively correlated with the extraction rate of forsythoside A. The forsythia extract obtained in this invention has excellent antioxidant activity.

[0040] To ensure the ease of storage and application of the Forsythia extract of the present invention, further stability tests were conducted on the extract. Stability testing methods: The stability of Forsythia suspensa extract was studied according to ISO / TR 18811:2018(E) Guidelines for Stability Testing in Cosmetics. The main focus was on detecting factors that easily change during storage and affect the quality of the raw materials. This experiment focused on observing the changes in the appearance (color, odor, etc.) and physicochemical properties (turbidity value) of the raw materials over time after the addition of polyols, thereby reflecting the effect of polyols on the stability of Forsythia suspensa extract. Turbidity refers to the degree of obstruction of light transmission by a solution, including light scattering by suspended matter and light absorption by solute molecules; therefore, changes in solution turbidity directly reflect its stability.

[0041] As shown in Table 2, the stability test results of Forsythia suspensa extract showed that compared with the water extraction control group D01 and the alcohol extraction control group D04, all groups maintained good stability within 0-7 days. The solution properties remained unchanged, remained clear and transparent, and the turbidity change rate did not change significantly. Therefore, adding 20%-80% of polyols (glycerol and butylene glycol) can effectively improve the stability of the solution.

[0042] Table 2. Results of stability test of Forsythia extract

[0043] The following are the experimental results of the pH response of the extract obtained in this invention: The pH response of Forsythia suspensa extract was tested using the method described by Li Yingren et al. in their study on "Preparation and Sustained-Release Performance of pH-Response Chitosan Maleate Hydrogel". The method involved sealing the pH-responsive material in a dialysis bag, then immersing the entire bag in a sustained-release medium of different pH values ​​and shaking it. The drug content in the external medium was measured at fixed intervals to reflect the pH responsiveness of the test material. At 0, 1, 2, 4, 6, 8, 24, and 36 hours, 1 mL of the release solution was collected to detect forsythoside A. The release concentration at different time points was compared with the complete release concentration to calculate the release rate. The formula for calculating the release rate is as follows:

[0044] Set up comparison 5 First, a glycyrrhizic acid-water solution with a pH of 4.0 was prepared by adjusting the glycyrrhizic acid content. Using this solution as a solvent, a deacetylated chitosan-glycyrrhizic acid solution with a solute mass percentage of 1.25% was prepared. The solution was stirred at 45℃ and 350 rpm for 60 minutes, and then heated to obtain a deacetylated chitosan-glycyrrhizic acid composite solution. 5g of pulverized Forsythia suspensa leaves were weighed and added to the above deacetylated chitosan-glycyrrhizic acid composite solution, making the ratio of Forsythia suspensa leaves to deacetylated chitosan-glycyrrhizic acid solution 1:16. The solution was stirred at 45℃ and 350 rpm for 120 minutes for extraction. After extraction, the solution was cooled to below 35℃ and filtered under reduced pressure to obtain Forsythia suspensa extract D05.

[0045] Table 3. Release rates of forsythoside A from D01, D05, and A02 under different pH conditions.

[0046] According to Table 3 and Figure 1 The results show that: (1) D01 has no pH response and no sustained release effect. Its release behavior is the same under pH conditions of 5.5 and 8.5, and it bursts within 0-1h, reaching the maximum release rate at 2h. (2) D05 has no pH response and has a sustained-release effect. Its release behavior is the same under pH conditions of 5.5 and 8.5, with no pH response. It is slowly released from 0 to 36 hours, with a maximum release rate of 67.32%. (3) A02 has pH response and sustained release effect. Compared with pH 8.5, the release effect is better at pH 5.5, and the maximum release rate (90.73%) is reached at 8h.

[0047] The following are verification tests demonstrating the repairing and soothing effects of the extract of this invention: The in vitro repair assay for Forsythia suspensa extract was performed using a keratinocyte proliferation assay. Keratinocytes are an important cell model; their proliferation and migration capabilities indicate the active recovery and regeneration function of the skin barrier. Cell proliferation refers to the ability of cells to divide and increase in number. Cell viability was detected using the CCK8 assay on samples D01, A01, A02, and A03. A statistically significant difference (P<0.05) was observed when the cell proliferation rate of sample A02 was higher than that of the negative group, indicating that the test substance at that concentration could promote keratinocyte proliferation and stratum corneum metabolism, thus verifying the repair efficacy of the test substance.

[0048] according to Figure 2It can be seen that, compared with the blank group, 0.016%–0.4% D01 (water-extracted control group) reduced the cell survival rate of keratinocytes; compared with the blank group, 0.016%–0.4% A01, A02, and A03 all improved the cell survival rate of keratinocytes, and the differences were significant; therefore, it can be inferred that A01, A02, and A03 have repair effects, and the concentration here refers to the concentration of the sample in the cell culture medium.

[0049] The efficacy testing method for Forsythia suspensa extract adopted T / BDCA 2022-0004 "Cosmetic Soothing Efficacy Test - Human Lactic Acid Sting Test Method". The lactic acid sting test is a widely used evaluation method: at room temperature, 50 μL of 10% lactic acid solution was applied to the nasolabial fold and one cheek. Subjects were asked about their subjective symptoms (itching and stinging) at 2.5 min and 5 min, respectively, and scored on a 4-point scale. The two scores were then added together; a total score ≥3 points was considered positive for lactic acid stinging. Then, samples were applied. The sample group received a hydrogel containing 5% Forsythia suspensa extract (A02), while the control group received a blank hydrogel without Forsythia suspensa extract (A02). Subjects were asked about their subjective symptoms and scored them over a certain period to analyze the soothing ability of the samples. Results are as follows: Figure 3 As shown, the results indicate that: (1) The scores of stinging and itching in the sample group continued to decrease within 0-15 minutes, and dropped to 0 points at 10 minutes, with no stinging or itching.

[0050] (2) Although the scores of stinging and itching in the blank group decreased continuously from 0 to 15 minutes, they did not drop to 0 within 15 minutes and still had stinging and itching.

[0051] (3) Compared with the blank group, the sample group can significantly reduce the stinging and itching caused by lactic acid stimulation, and has a significant difference from the blank group, indicating that it has excellent soothing effect.

[0052] The following experiments verify that, in the embodiments of the present invention, the optimal extraction reagent is A02. Stable extraction reagent combinations were screened using zeta potential (the larger the absolute value of the zeta potential, the more stable the solution system). Experimental Example 1: (Deacetylated chitosan + malic acid) First, a malic acid solution with a pH of 4.0 was prepared by adjusting the malic acid content. Then, a 1.25% deacetylated chitosan-malic acid solution was prepared using this solution as a solvent. The solution was stirred at 45°C and 350 rpm for 60 minutes. The resulting solution was a deacetylated chitosan-malic acid composite solution.

[0053] Experimental Example 2 (Deacetylated chitosan + glycyrrhizic acid) First, a glycyrrhizic acid solution with a pH of 4.0 was prepared by adjusting the glycyrrhizic acid content. Then, a 1.25% deacetylated chitosan-glycyrrhizic acid solution was prepared using this solution as a solvent. The solution was stirred at 45°C and 350 rpm for 60 minutes. The resulting solution was a deacetylated chitosan-glycyrrhizic acid composite solution.

[0054] Experimental Example 3 (Deacetylated chitosan + lactic acid) First, a lactic acid solution with a pH of 4.0 was prepared by adjusting the lactic acid content. Then, a 1.25% deacetylated chitosan-lactic acid solution was prepared using this solution as a solvent. The solution was stirred at 45°C and 350 r / min for 60 min. The resulting solution was a deacetylated chitosan-lactic acid composite solution.

[0055] Experimental Example 4 (Deacetylated chitosan + citric acid) First, a citric acid solution with a pH of 4.0 was prepared by adjusting the citric acid content. Then, a 1.25% deacetylated chitosan-citric acid solution was prepared using this solution as a solvent. The solution was stirred at 45°C and 350 rpm for 60 minutes. The resulting solution was a deacetylated chitosan-citric acid composite solution.

[0056] Experimental Example 5 (Carboxymethyl deacetylated chitosan + malic acid) First, a malic acid solution with a pH of 4.0 was prepared by adjusting the malic acid content. Then, a 1.25% carboxymethyl deacetylated chitosan-malic acid solution was prepared using this solution as a solvent. The solution was stirred at 45°C and 350 rpm for 60 minutes. The resulting solution was a deacetylated chitosan-malic acid composite solution.

[0057] Experimental Example 6 (Carboxymethyl deacetylated chitosan + glycyrrhizic acid) First, a glycyrrhizic acid solution with a pH of 4.0 was prepared by adjusting the glycyrrhizic acid content. Then, a 1.25% carboxymethyl deacetylated chitosan-glycyrrhizic acid solution was prepared using this solution as a solvent. The solution was stirred at 45°C and 350 rpm for 60 minutes. The resulting solution was a deacetylated chitosan-glycyrrhizic acid composite solution.

[0058] Experimental Example 7 (Carboxymethyl deacetylated chitosan + lactic acid) First, a lactic acid solution with a pH of 4.0 was prepared by adjusting the lactic acid content. Then, a 1.25% carboxymethyl deacetylated chitosan-lactic acid solution was prepared using this solution as a solvent. The solution was stirred at 45°C and 350 r / min for 60 min, and the resulting solution was obtained by stirring and heating.

[0059] Experimental Example 8 (Carboxymethyl deacetylated chitosan + citric acid) First, a citric acid solution with a pH of 4.0 was prepared by adjusting the citric acid content. Then, a 1.25% carboxymethyl deacetylated chitosan-citric acid solution was prepared using this solution as a solvent. The solution was stirred at 45°C and 350 rpm for 60 minutes. The resulting solution was a deacetylated chitosan-citric acid composite solution.

[0060] Experimental Example 9 (Hydroxyethyl deacetylated chitosan + malic acid) First, a malic acid solution with a pH of 4.0 was prepared by adjusting the malic acid content. Then, a 1.25% hydroxyethyl deacetylated chitosan-malic acid solution was prepared using this solution as a solvent. The solution was stirred at 45°C and 350 rpm for 60 minutes. The resulting solution was a deacetylated chitosan-malic acid composite solution.

[0061] Experimental Example 10 (Hydroxyethyl deacetylated chitosan + glycyrrhizic acid) First, a glycyrrhizic acid solution with a pH of 4.0 was prepared by adjusting the glycyrrhizic acid content. Then, a 1.25% hydroxyethyl deacetylated chitosan-glycyrrhizic acid solution was prepared using this solution as a solvent. The solution was stirred at 45°C and 350 rpm for 60 minutes, and then heated to obtain a deacetylated chitosan-glycyrrhizic acid composite solution.

[0062] Experimental Example 11 (Hydroxyethyl deacetylated chitosan + lactic acid) First, a lactic acid solution with a pH of 4.0 was prepared by adjusting the lactic acid content. Then, a 1.25% hydroxyethyl deacetylated chitosan-lactic acid solution was prepared using this solution as a solvent. The solution was stirred at 45°C and 350 r / min for 60 min, and the resulting solution was obtained by stirring and heating.

[0063] Experimental Example 12 (Hydroxyethyl deacetylated chitosan + citric acid) First, a citric acid solution with a pH of 4.0 was prepared by adjusting the citric acid content. Then, a 1.25% hydroxyethyl deacetylated chitosan-citric acid solution was prepared using this solution as a solvent. The solution was stirred at 45°C and 350 rpm for 60 minutes. The resulting solution was a deacetylated chitosan-citric acid composite solution.

[0064] Table 4. Detection results of zeta potential of different chitosan-organic acid groups

[0065] As shown in Table 4, the results show that Comparative Example 2 (i.e., the deacetylated chitosan + glycyrrhizic acid group) has the highest absolute value of zeta potential, indicating that its solution stability is the highest. Therefore, the extraction reagent used in Example A02 is the optimal choice.

[0066] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various specific technical features in any suitable manner. To avoid unnecessary repetition, the present invention will not describe the various possible combinations separately. However, these simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A method for extracting forsythoside A and forsythoside A from Forsythia suspensa, characterized in that, The method includes the following steps: (1) Preparation of extraction reagent: Prepare an organic acid-water solution to obtain an organic acid solution, add chitosan to the organic acid solution, heat and stir to obtain a chitosan-organic acid solution, and thus obtain the extraction reagent; (2) Treatment of tender leaves at the top of Forsythia: Collect the tender leaves at the top of Forsythia after pinching during the planting process, crush and sieve the tender leaves, add them to the extraction reagent obtained in step (1), and stir to extract. (3) Vacuum filtration: After extraction in step (2), vacuum filtration is performed to obtain Forsythia extract; (4) pH adjustment: Sodium bicarbonate was added to adjust the Forsythia extract in step (3) to obtain Forsythia extract. The extracted Forsythia extract includes forsythoside and forsythoside A. The extraction rate of forsythoside is 0.058-0.071%, and the extraction rate of forsythoside A is 6.1-7.6%. In step (1), the pH value of the organic acid solution is 2.5-5.5, and the mass percentage of chitosan in the organic acid solution is 0.5-2.5%. The chitosan-organic acid solution can be obtained by stirring at 25-80℃ and 200-600r / min for 30-240min. In step (4), sodium bicarbonate is added to adjust the pH of the Forsythia extract in step (3) to 6.5-8.

5. The organic acid is any one of glycyrrhizic acid, malic acid, lactic acid, and citric acid. In step (2), the mass ratio of the tender leaves at the top of Forsythia suspensa to the extraction reagent is 1:12-1:20, and the extraction is carried out by stirring for 30-240 minutes at 25-80℃ and 200-600r / min.

2. The extraction method according to claim 1, characterized in that, In step (1), the chitosan is any one of deacetylated chitosan, carboxymethyl deacetylated chitosan, or hydroxyethyl deacetylated chitosan.

3. The extraction method according to claim 1, characterized in that, In step (3), after extraction in step (2), the mixture is cooled to below 35°C and filtered under reduced pressure for 3-5 times at 0.08-0.1 MPa to obtain Forsythia extract.

4. The extraction method according to claim 1, characterized in that, It also includes step (5) to build a stable system: the Forsythia extract obtained in step (4) is compounded with a polyol, and the mass percentage of the polyol in the Forsythia extract is 20-80%.

5. The extraction method according to claim 4, characterized in that, The polyol in step (5) is glycerol or butanediol.

6. A forsythia extract, characterized in that, The Forsythia extract was prepared by any one of the methods described in claims 1-5. The Forsythia extract includes forsythoside and forsythoside A. The extraction rate of forsythoside is 0.058-0.071%, and the extraction rate of forsythoside A is 6.1-7.6%.

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