A method for selectively increasing the content of antioxidant active ingredients in traditional Chinese medicine and the antioxidant active ingredients.

Abstract

This invention belongs to the field of traditional Chinese medicine processing technology, specifically relating to an extraction method and antioxidant active ingredients for targeted improvement of the content of antioxidant active ingredients in traditional Chinese medicine. The method includes the following steps: grinding the traditional Chinese medicine materials into powder using a grinder, adding the powder to MRS broth liquid culture medium, sterilizing under high pressure, inoculating with *Lactobacillus plantarum*, allowing static fermentation, centrifuging to collect the supernatant, thus obtaining the fermented broth of the traditional Chinese medicine. This method uses a single *Lactobacillus plantarum* scheme to focus on the efficient conversion of flavonoid components, relying on the single advantage of β-glucosidase and cell wall degrading enzymes. Through short-term fermentation, bound flavonoids can be converted into free flavonoids, significantly increasing the content of total sugar, flavonoids, and polyphenols, as well as the antioxidant capacity of the medicinal materials. Simultaneously, the process is concise, time-saving, suitable for large-scale production, and leaves no organic solvent residue, making it green and safe.

Description

Technical Field

[0001] This application belongs to the field of traditional Chinese medicine processing technology, specifically relating to an extraction method for targeted improvement of the content of antioxidant active ingredients in traditional Chinese medicine and the antioxidant active ingredients themselves. Background Technology

[0002] Green tangerine peel, immature bitter orange peel, and immature bitter orange fruit are traditional Chinese medicinal herbs with a long history of use and wide clinical application value. These three herbs all originate from plants in the Rutaceae family, possessing similar chemical compositions and pharmacological activities. In traditional Chinese medicine theory, they are classified as qi-regulating herbs, primarily used to treat symptoms such as chest and rib pain, indigestion, and phlegm stagnation. All three herbs are rich in various flavonoids, which exhibit significant antioxidant, anti-inflammatory, and anti-tumor biological activities. Therefore, all three herbs possess a certain ability to scavenge free radicals, inhibit lipid peroxidation, and protect cells from oxidative damage.

[0003] Currently, traditional extraction methods for compounds contained in traditional Chinese medicine often employ water extraction, organic solvent extraction (such as ethanol and methanol), acid-base extraction, and distillation (for volatile oils). Among these, water extraction, due to high-temperature decoction, leads to the oxidation or hydrolysis of heat-sensitive components (such as volatile oils and some flavonoid glycosides); organic solvent extraction, with solvents like ethanol and methanol, can damage polar and sensitive compounds, and residual solvents require separate removal, making the process complex; acid-base extraction, with strong acid / base treatment, causes the degradation of glycosides and polysaccharides, such as hesperidin, which is easily hydrolyzed under acidic conditions, affecting product quality; distillation is mainly used to extract volatile oils, but high temperatures easily lead to the isomerization or oxidation of terpenes. Flavonoids (such as hesperidin and naringin) and volatile oils (such as limonene) are easily decomposed at high temperatures, while polysaccharides and phenolic acids have low solubility in organic solvents or react with them. Therefore, the active ingredients obtained by traditional extraction methods often have low bioavailability, and some effective components are easily destroyed during processing, limiting their full efficacy.

[0004] Existing technologies disclose the application of lactic acid bacteria in the fermentation and modification of traditional Chinese medicine (TCM). These lactic acid bacteria are characterized by high safety, strong fermentation capacity, and good acid resistance. Currently, compound lactic acid bacteria fermentation is commonly used, employing a variety of lactic acid bacteria, such as Lactobacillus acidophilus, Bifidobacterium, and Lactobacillus plantarum, to achieve the generation of active products through the synergistic effect of multiple strains. However, the aforementioned compound lactic acid bacteria fermentation process is complex to operate, has low controllability, and affects production efficiency. Furthermore, the intervention of multiple lactic acid bacteria cannot efficiently and directionally increase the content of specific components in TCM, which is not conducive to improving production efficiency. In addition, even if existing technologies can extract flavonoids, which are antioxidants or anti-inflammatory components, the flavonoids obtained traditionally are only conjugated flavonoids, resulting in lower efficacy.

[0005] To further enhance the medicinal value of green tangerine peel, it is necessary to further optimize the traditional Chinese medicine processing technology based on fermentation for green tangerine peel. Summary of the Invention

[0006] This invention overcomes the shortcomings of existing technologies by providing a method for selectively increasing the content of antioxidant active ingredients in traditional Chinese medicine. This method can effectively improve the production efficiency of flavonoids and has the advantages of simple process and short fermentation time.

[0007] To address the aforementioned technical problems, a method for extracting and selectively increasing the content of antioxidant and anti-inflammatory active ingredients in traditional Chinese medicine is provided. This method specifically increases the content of total sugars, flavonoids, or polyphenols in the antioxidant active ingredients. The extraction method includes the following steps:

[0008] S1: Grind the green tangerine peel into powder using a grinder, add the powder to MRS broth liquid culture medium, and then autoclave to obtain the drug powder liquid culture medium.

[0009] S2: The liquid culture medium containing the powder is inoculated with Lactobacillus plantarum in an anaerobic environment and fermented at a temperature of 36℃~38℃ for 20h~25h to obtain a fermentation mixture.

[0010] Preferably, the fermentation temperature is 37℃ and the fermentation time is 24h.

[0011] S3: The fermentation mixture is centrifuged and the supernatant is collected. The supernatant is a fermentation broth containing total sugar, flavonoids or polyphenols. The fermentation broth is used to prepare antioxidant active ingredients.

[0012] Further, the inoculum amount of *Lactobacillus plantarum* is 1 wt% to 4 wt%. Preferably, the inoculum amount of *Lactobacillus plantarum* is 3%.

[0013] Further, the high-pressure steam sterilization process is performed at a temperature of 100℃~130℃, a time of 10min~20min, and a pressure of 0.05MPa~0.15MPa. Preferably, the high-pressure steam sterilization process is performed at a temperature of 121℃, a time of 15min, and a pressure of 0.1MPa.

[0014] Further, the mass-to-volume ratio of the drug powder to the MRS broth liquid culture medium is 0.5g~2g:10mL~40mL. Preferably, the mass-to-volume ratio of the drug powder to the MRS broth liquid culture medium is 1g:20mL.

[0015] Furthermore, the viable count of the *Lactobacillus plantarum* bacterial solution is >10. 8 CFU / ml.

[0016] Furthermore, in the centrifugation treatment of the fermentation mixture, the centrifugation speed is 5000 rpm to 9000 rpm, and the centrifugation time is 6 min to 15 min. Preferably, in the centrifugation treatment of the fermentation mixture, the centrifugation speed is 8000 rpm, and the centrifugation time is 10 min.

[0017] An antioxidant active ingredient is also provided, which is prepared by the above-mentioned extraction method for specifically increasing the content of antioxidant active ingredients in traditional Chinese medicine.

[0018] Compared with the prior art, the beneficial effects of the present invention are:

[0019] (1) In this invention, compared with the traditional water extraction method of boiling at 100°C, the fermentation temperature of this invention is only 36°C~38°C, which avoids high temperature damage to the heat-sensitive components of the medicinal materials. There is no need to add organic solvents or strong acids / alkalis, eliminating the risk of solvent residue. Therefore, this invention is green and safe, the fermentation process is mild, and no harmful chemicals are introduced, which is in line with the modern green pharmaceutical concept.

[0020] (2) In this invention, only *Lactobacillus plantarum* is used as a single strain. On one hand, it focuses on the efficient conversion of flavonoids, the active components in *Citrus reticulata* (green peel) that have effective antioxidant, anti-inflammatory, and anti-tumor properties. Specifically, *Lactobacillus plantarum* can specifically secrete β-glucosidase and cell wall degrading enzymes. β-glucosidase efficiently hydrolyzes flavonoid glycosides in *Citrus reticulata*, such as hesperidin and naringin, into more active aglycones, such as hesperidin and naringin. The phenolic hydroxyl groups of the aglycones are more likely to provide electrons to scavenge free radicals (such as DPPH), significantly enhancing the antioxidant capacity of flavonoids. Cellulase and pectinase in the cell wall degrading enzymes destroy the cell walls in *Citrus reticulata*, *Citrus aurantium*, and *Citrus aurantium*, releasing encapsulated antioxidant components, such as polysaccharides and polyphenols, effectively increasing the extraction yield of polysaccharides and polyphenols. Therefore, this invention converts bound flavonoids into free flavonoid aglycones through a microbial enzyme system, significantly increasing the content of active components and antioxidant capacity in *Citrus reticulata*. This invention achieves a synergistic effect between *Lactobacillus plantarum* and its metabolites and the components in *Citrus reticulata*.

[0021] (3) In this invention, a single Lactobacillus plantarum was used for directional fermentation of green tangerine peel, which significantly increased the content of total sugar, flavonoids, and polyphenols, and enhanced the antioxidant capacity of flavonoids. Experimental data showed that after fermentation, the total sugar content of green tangerine peel increased by 357%, flavonoids by 107%, polyphenols by 134%, DPPH free radical scavenging capacity by 114%, superoxide anion scavenging capacity by 48%, and hydroxyl free radical scavenging capacity by 1421%; after fermentation, the total sugar content of trifoliate orange peel increased by 212%, flavonoids by 87%, polyphenols by 103%, DPPH free radical scavenging capacity by 65%, superoxide anion scavenging capacity by 37%, and hydroxyl free radical scavenging capacity by 542%; after fermentation, the total sugar content of trifoliate orange fruit increased by 339%, flavonoids by 72%, polyphenols by 123%, DPPH free radical scavenging capacity by 110%, superoxide anion scavenging capacity by 27%, and hydroxyl free radical scavenging capacity by 664%.

[0022] (4) In this invention, the advantages of a single bacterial strain are utilized, eliminating the need to coordinate the proportion of multiple strains. Only the inoculation amount, temperature, and time of *Lactobacillus plantarum* need to be controlled. After centrifugation, the supernatant is directly taken, eliminating the need for complex purification steps. The process is simplified and controllable. The enzymatic reaction is rapid, and the conversion of flavonoid aglycones can be completed in a short fermentation time, resulting in a significant increase in product activity. The fermentation parameters are simple to control and easy to standardize. Therefore, standardized production can be achieved by controlling the fermentation conditions of temperature and fermentation time, ensuring the consistency of quality of each batch of products, improving the overall quality and efficacy stability of green tangerine peel, and making it suitable for large-scale industrial production.

[0023] It should be understood that the above general description and the following detailed description are exemplary and explanatory only, and do not limit this application. Attached Figure Description

[0024] The above and other objects, features and advantages of this application will become more apparent from the more detailed description of exemplary embodiments thereof in conjunction with the accompanying drawings.

[0025] Figure 1 A schematic diagram showing the flavonoid content at the same concentration in the drug solution before fermentation (comparative example) and after fermentation (experimental example); where *** indicates p-value ≤ 0.001, indicating a highly statistically significant difference, **** indicates p-value ≤ 0.0001, indicating a highly statistically significant difference, and the bubble points on the column represent data points from multiple repeated experiments to reflect the degree of data dispersion.

[0026] Figure 2A schematic diagram showing the total sugar content of the same concentration in the drug solution before fermentation (comparative example) and after fermentation (experimental example); where **** indicates a p-value ≤ 0.0001, indicating a highly statistically significant difference, and the bubble points on the column represent data points from multiple repeated experiments to reflect the degree of data dispersion.

[0027] Figure 3 A schematic diagram showing the content of polyphenols at the same concentration in the drug solution before fermentation (comparative example) and after fermentation (experimental example); where **** indicates a p-value ≤ 0.0001, indicating a highly statistically significant difference, and the bubble points on the column represent data points from multiple repeated experiments to reflect the degree of data dispersion.

[0028] Figure 4 Schematic diagram of DPPH free radical scavenging rate of the same concentration of drug solution before fermentation (comparative example) and after fermentation (experimental example); where ** indicates p value ≤ 0.01, the difference is highly statistically significant, and *** indicates p value ≤ 0.001, the difference is extremely statistically significant. The bubble points on the column are data points from multiple repeated experiments to reflect the degree of data dispersion.

[0029] Figure 5 Schematic diagram of superoxide anion scavenging rate of the same concentration of drug solution before fermentation (comparative example) and after fermentation (experimental example); where **** indicates p value ≤ 0.0001, the difference is highly statistically significant, and the bubble points on the column are data points from multiple repeated experiments to reflect the degree of data dispersion.

[0030] Figure 6 Schematic diagram of hydroxyl radical scavenging rate of the same concentration of drug solution before fermentation (comparative example) and after fermentation (experimental example); where *** indicates p value ≤ 0.001, the difference is highly statistically significant, **** indicates p value ≤ 0.0001, the difference is highly statistically significant, and the bubble points on the column are data points from multiple repeated experiments to reflect the degree of data dispersion. Detailed Implementation

[0031] Preferred embodiments of the invention will now be described in more detail. While preferred embodiments of the invention have been shown, it should be understood that the invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that the invention will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0032] The terminology used in this application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The singular forms “a,” “the,” and “the” used in this application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise. It should also be understood that the term “and / or” as used herein refers to and includes any or all possible combinations of one or more of the associated listed items.

[0033] It should be understood that although the terms "first," "second," "third," etc., may be used in this application to describe various information, this information should not be limited to these terms. These terms are only used to distinguish information of the same type from one another. For example, without departing from the scope of this application, first information may also be referred to as second information, and similarly, second information may also be referred to as first information. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this application, "multiple" means two or more, unless otherwise explicitly specified.

[0034] Example 1

[0035] This embodiment discloses a method for selectively increasing the content of antioxidant and anti-inflammatory active ingredients in tangerine peel, comprising the following steps:

[0036] S1: Grind the green tangerine peel into powder using a grinder, filter it through a 50-mesh sieve, add it to MRS broth liquid culture medium, and perform high-pressure steam sterilization for 15 minutes at a temperature of 121°C and a pressure of 0.1 MPa. The mass-volume ratio of the powder to the MRS broth is 1 g: 20 mL.

[0037] Filtering fine powder through a 50-mesh sieve increases the contact area between the medicinal materials and the microbial culture, improving fermentation efficiency; high-pressure sterilization eliminates competition from other microorganisms, ensuring pure fermentation of Lactobacillus plantarum and aiding in the release of medicinal components.

[0038] S2: The liquid culture medium containing the medicinal powder obtained in step S1 is placed in an anaerobic environment and inoculated with 3 wt% *Lactobacillus plantarum* for fermentation. The fermentation temperature is 37°C and the time is 24 hours. The viable count of *Lactobacillus plantarum* in the culture solution is >10. 8 CFU / ml;

[0039] Here, *Lactobacillus plantarum* exhibits the fastest cell proliferation and the highest enzyme synthesis rate, such as β-glucosidase, at a fermentation temperature of 37℃. If the fermentation temperature is too low, cell metabolism slows down, enzyme activity is low, and the fermentation cycle is prolonged; if the temperature is too high, proteins denature, cells die, and key enzymes are inactivated.

[0040] S3: Centrifuge the fermentation broth obtained in step S2 at 5000 rpm for 10 minutes. The supernatant is then collected to obtain the traditional Chinese medicine fermentation broth. (In practical applications, the speed can be adjusted according to the specific situation.)

[0041] Example 2

[0042] This embodiment discloses a method for selectively increasing the content of antioxidant and anti-inflammatory active ingredients in Citrus aurantium, comprising the following steps:

[0043] S1: Grind the Citrus aurantium into powder using a grinder, filter it through a 50-mesh sieve, add it to MRS broth liquid culture medium, and perform high-pressure steam sterilization for 10 minutes at a temperature of 100°C and a pressure of 0.05 MPa. The mass-to-volume ratio of the powder to the MRS broth is 0.5 g: 10 mL.

[0044] S2: The liquid culture medium containing the medicinal powder obtained in step S1 is placed in an anaerobic environment and inoculated with 1 wt% *Lactobacillus plantarum* for fermentation. The fermentation temperature is 36℃ and the time is 20 hours. The viable count of *Lactobacillus plantarum* in the culture solution is >10. 8 CFU / ml;

[0045] S3: Centrifuge the fermentation broth obtained in step S2 at a speed of 5000 rpm. In practical applications, the speed can be adjusted according to the actual situation. The time is 10 minutes. Take the supernatant to obtain the traditional Chinese medicine fermentation broth.

[0046] Example 3

[0047] This embodiment discloses a method for selectively increasing the content of antioxidant and anti-inflammatory active ingredients in Citrus aurantium, comprising the following steps:

[0048] S1: Grind the Citrus aurantium into powder using a grinder, filter it through a 50-mesh sieve, add it to MRS broth liquid culture medium, and perform high-pressure steam sterilization for 20 minutes at a temperature of 130°C and a pressure of 0.15 MPa. The mass-to-volume ratio of the powder to the MRS broth is 2 g: 40 mL.

[0049] S2: The liquid culture medium containing the medicinal powder obtained in step S1 is placed in an anaerobic environment and inoculated with 4 wt% *Lactobacillus plantarum* for fermentation. The fermentation temperature is 38°C and the time is 25 hours. The viable count of *Lactobacillus plantarum* in the culture solution is >10. 8 CFU / ml;

[0050] S3: Centrifuge the fermentation broth obtained in step S2 at a speed of 9000 rpm. In practical applications, the speed can be adjusted according to the actual situation. The time is 15 minutes. Take the supernatant to obtain the traditional Chinese medicine fermentation broth.

[0051] Effect verification:

[0052] To further illustrate the effects of the present invention, the following experiments and comparisons were conducted:

[0053] Experimental Example 1

[0054] This embodiment discloses a method for selectively increasing the content of antioxidant and anti-inflammatory active ingredients in tangerine peel, comprising the following steps:

[0055] S1: Grind the green tangerine peel into powder using a grinder, filter it through a 50-mesh sieve, add it to MRS broth liquid culture medium, and perform high-pressure steam sterilization for 15 minutes at a temperature of 121°C and a pressure of 0.1 MPa. The mass-volume ratio of the powder to the MRS broth is 1 g: 20 mL.

[0056] S2: The liquid culture medium containing the medicinal powder obtained in step S1 is placed in an anaerobic environment and inoculated with 3 wt% *Lactobacillus plantarum* for fermentation. The fermentation temperature is 37°C and the time is 24 hours. The viable count of *Lactobacillus plantarum* in the culture solution is >10. 8 CFU / ml;

[0057] S3: Centrifuge the fermentation broth obtained in step S2 at 5000 rpm for 10 minutes. The supernatant is then collected to obtain the traditional Chinese medicine fermentation broth. (In practical applications, the speed can be adjusted according to the specific situation.)

[0058] Experimental Example 2

[0059] This embodiment discloses a method for selectively increasing the content of antioxidant and anti-inflammatory active ingredients in Citrus aurantium, comprising the following steps:

[0060] S1: Grind the Citrus aurantium into powder using a grinder, filter it through a 50-mesh sieve, add it to MRS broth liquid culture medium, and perform high-pressure steam sterilization for 10 minutes at a temperature of 100°C and a pressure of 0.05 MPa. The mass-to-volume ratio of the powder to the MRS broth is 0.5 g: 10 mL.

[0061] S2: The liquid culture medium containing the medicinal powder obtained in step S1 is placed in an anaerobic environment and inoculated with 1 wt% *Lactobacillus plantarum* for fermentation. The fermentation temperature is 36℃ and the time is 20 hours. The viable count of *Lactobacillus plantarum* in the culture solution is >10. 8 CFU / ml;

[0062] S3: Centrifuge the fermentation broth obtained in step S2 at a speed of 5000 rpm. In practical applications, the speed can be adjusted according to the actual situation. The time is 10 minutes. Take the supernatant to obtain the traditional Chinese medicine fermentation broth.

[0063] Experimental Example 3

[0064] This embodiment discloses a method for selectively increasing the content of antioxidant and anti-inflammatory active ingredients in Citrus aurantium, comprising the following steps:

[0065] S1: Grind the Citrus aurantium into powder using a grinder, filter it through a 50-mesh sieve, add it to MRS broth liquid culture medium, and perform high-pressure steam sterilization for 20 minutes at a temperature of 130°C and a pressure of 0.15 MPa. The mass-to-volume ratio of the powder to the MRS broth is 2 g: 40 mL.

[0066] S2: The liquid culture medium containing the medicinal powder obtained in step S1 is placed in an anaerobic environment and inoculated with 4 wt% *Lactobacillus plantarum* for fermentation. The fermentation temperature is 38°C and the time is 25 hours. The viable count of *Lactobacillus plantarum* in the culture solution is >10. 8 CFU / ml;

[0067] S3: Centrifuge the fermentation broth obtained in step S2 at a speed of 9000 rpm. In practical applications, the speed can be adjusted according to the actual situation. The time is 15 minutes. Take the supernatant to obtain the traditional Chinese medicine fermentation broth.

[0068] Comparative Example 1

[0069] The green tangerine peel was ground into powder using a grinder and filtered through a 50-mesh sieve. The powder was then extracted with water, evaporated by rotary evaporation, and freeze-dried to obtain freeze-dried powder. Subsequently, it was reconstituted to the corresponding concentration for comparative testing.

[0070] Comparative Example 2

[0071] The bitter orange peel was ground into powder using a grinder and filtered through a 50-mesh sieve. The powder was then extracted with water, evaporated by rotary evaporation, and freeze-dried to obtain freeze-dried powder. Subsequently, it was reconstituted to the corresponding concentration for comparative testing.

[0072] Comparative Example 3

[0073] The immature bitter orange was ground into powder using a grinder and filtered through a 50-mesh sieve. The powder was then extracted with water, evaporated by rotary evaporation, and freeze-dried to obtain freeze-dried powder. Subsequently, it was reconstituted to the corresponding concentration for comparative testing.

[0074] 1. Verification of the effects of flavonoid components:

[0075] The flavonoid content of Experimental Examples 1-3 and Comparative Examples 1-3 was determined according to the following method for determining flavonoid content. Among them, Experimental Examples 1-3 refer to the flavonoid content determination after fermentation, and Comparative Examples 1-3 refer to the flavonoid content determination before fermentation.

[0076] Experimental Method: The fermentation broth and water extract were dissolved and diluted to 25 mg / ml with distilled water. 1 mL of fermentation broth and 150 μL of 5% (v / v) sodium nitrite were added to a 5 mL test tube, and the mixture was allowed to stand for 6 minutes. 150 μL of 10% (v / v) aluminum nitrate solution was added to the test tube, mixed, and allowed to stand for 6 minutes. 2 mL of 4% (v / v) sodium hydroxide solution was added to the test tube. Finally, anhydrous ethanol was added to the test tube to bring the final volume to 5 mL, and the mixture was placed at room temperature in the dark for 15 minutes. The absorbance was measured at 510 nm. A standard curve was plotted with absorbance as the ordinate and rutin as the abscissa, and the total flavonoid content was calculated. The linear regression equation fitted to the standard curve of total flavonoid content was: y = 0.0075x + 0.0077, R0 2 =0.9999.

[0077] Experimental results: such as Figure 1 Fermentation significantly increased the flavonoid content of green tangerine peel, bitter orange peel, and bitter orange fruit. Table 1 shows that the flavonoid content of the fermented green tangerine peel increased by 107%, that of the bitter orange peel by 87%, and that of the bitter orange fruit by 72%.

[0078] Table 1. Comparison of flavonoid content in solutions of the same concentration before and after fermentation.

[0079]

[0080] 2. Verification of the effect of total sugar content:

[0081] The total sugar content of Experimental Examples 1-3 and Comparative Examples 1-3 was determined according to the following method for determining total sugar content. In Experimental Examples 1-3, the total sugar content was determined after fermentation, and in Comparative Examples 1-3, the total sugar content was determined before fermentation.

[0082] Experimental Method: The fermentation broth and water extract were dissolved and diluted to 25 mg / ml with distilled water. 1 mL of 6% (v / v) phenol solution was added to 1 mL of fermentation broth. After vortexing, 5 mL of concentrated sulfuric acid was added to the mixture, and the mixture was vortexed again. The mixture was allowed to stand at room temperature for 30 min (starting from the addition of concentrated sulfuric acid). The absorbance of the mixture was measured at 490 nm. A standard curve was plotted with absorbance on the ordinate and glucose on the abscissa, and the total sugar content was calculated. The linear regression equation fitted to the total sugar content standard curve was: y = 0.0042x + 0.0143, R0 2 =0.9984.

[0083] Experimental results: such as Figure 2 Fermentation significantly increased the total sugar content of the green tangerine peel, bitter orange peel, and bitter orange fruit. Table 2 shows that the total sugar content of the green tangerine peel increased by 357%, the total sugar content of the bitter orange peel increased by 212%, and the total sugar content of the bitter orange fruit increased by 339% after fermentation.

[0084] Table 2 Comparison of total sugar content in solutions of the same concentration before and after fermentation

[0085]

[0086] 3. Verification of the effects of polyphenol components:

[0087] The polyphenol content of Experimental Examples 1-3 and Comparative Examples 1-3 was determined according to the polyphenol content determination method described below. Among them, Experimental Examples 1-3 refer to the polyphenol determination after fermentation, and Comparative Examples 1-3 refer to the polyphenol determination before fermentation.

[0088] Experimental Methods: The fermentation broth and water extract were dissolved and diluted with distilled water to 25 mg / ml. 50 μL of each sample was added to 100 μL of Folin-Ciocalteu reagent, allowed to stand for 10 min, then 1000 μL of 7% sodium carbonate and 850 μL of pure water were added. The mixture was allowed to stand in the dark for 45 min. The absorbance was measured at 750 nm. A standard curve was plotted with absorbance as the ordinate and polyphenol content as the abscissa. The polyphenol content was calculated. The linear regression equation fitted to the polyphenol content standard curve was: y = 0.0042x + 0.027, R² = 0.9949.

[0089] Experimental results: such as Figure 3 Fermentation significantly increased the polyphenol content of green tangerine peel, bitter orange peel, and bitter orange fruit. Table 3 shows that the polyphenol content of the fermented green tangerine peel increased by 134%, that of the bitter orange peel by 103%, and that of the bitter orange fruit by 123%.

[0090] Table 3. Comparison of polyphenol content in solutions of the same concentration before and after fermentation

[0091]

[0092] 4. Verification of DPPH free radical scavenging ability:

[0093] The DPPH free radical scavenging capacity of Experimental Examples 1-3 and Comparative Examples 1-3 was determined according to the DPPH free radical scavenging rate determination method described below. Among them, Experimental Examples 1-3 refer to the DPPH free radical scavenging rate determination after fermentation, and Comparative Examples 1-3 refer to the DPPH free radical scavenging rate determination before fermentation.

[0094] Experimental Method: The fermentation broth and water extract were dissolved and diluted with distilled water to 25 mg / mL. 50 μL of the fermentation supernatant was collected in a 5 mL brown centrifuge tube. Then, 1.5 mL of 0.07 mg / mL DPPH was added to the centrifuge tube, mixed well, and incubated in the dark for 30 min. Anhydrous ethanol was used as a blank control, and the water extract as a control. Each sample was measured three times, and the mixture was measured at 517 nm. The result was calculated using the following formula:

[0095] DPPH free radical scavenging rate D% = [[A blank - (A assay - A control)] ÷ A blank] x 100%

[0096] Experimental results: such as Figure 4 As shown, the DPPH free radical scavenging rates of fermented green tangerine peel, bitter orange peel, and bitter orange fruit were all higher than those of the unfermented group, indicating that the DPPH free radical scavenging ability of green tangerine peel, bitter orange peel, and bitter orange fruit was significantly improved after being processed by *Lactobacillus plantarum*. Table 4 shows that the DPPH free radical scavenging ability of fermented green tangerine peel increased by 114%, that of bitter orange peel increased by 65%, and that of bitter orange fruit increased by 110%.

[0097] Table 4 DPPH free radical scavenging capacity before and after fermentation

[0098]

[0099] 5. Verification of superoxide anion scavenging ability:

[0100] The superoxide anion scavenging capacity of Experimental Examples 1-3 and Comparative Examples 1-3 was determined according to the superoxide anion scavenging rate determination method described below. Among them, Experimental Examples 1-3 refer to the superoxide anion scavenging rate determination after fermentation, and Comparative Examples 1-3 refer to the superoxide anion scavenging rate determination before fermentation.

[0101] Experimental method: The fermentation broth and water extract were dissolved and diluted with distilled water to 25 mg / ml. After taking the supernatant, the experimental procedure of the hydroxyl radical scavenging capacity test kit was followed. The result was obtained according to the calculation formula:

[0102] Hydroxyl radical scavenging rate D% = (Atest - Ap) ÷ (Ablank - Ap) x 100%

[0103] Experimental results: such as Figure 5 The fermented tangerine peel, immature bitter orange peel, and immature bitter orange fruit all showed higher superoxide anion scavenging rates than the unfermented group, indicating that the superoxide anion scavenging ability of these three fruits was significantly enhanced after processing with *Lactobacillus plantarum*. Table 5 shows that the superoxide anion scavenging ability of the fermented tangerine peel increased by 48%, that of the immature bitter orange peel by 37%, and that of the immature bitter orange fruit by 27%.

[0104] Table 5 Superoxide anion scavenging capacity before and after fermentation

[0105]

[0106] 6. Verification of hydroxyl radical scavenging ability:

[0107] The hydroxyl radical scavenging capacity of Experimental Examples 1-3 and Comparative Examples 1-3 was determined according to the following method for determining hydroxyl radical scavenging rate. In Experimental Examples 1-3, the hydroxyl radical scavenging rate was determined after fermentation, and in Comparative Examples 1-3, the hydroxyl radical scavenging rate was determined before fermentation.

[0108] Experimental Method: The fermentation broth and aqueous extract were dissolved and diluted to 25 mg / ml with distilled water. 0.5 mL of the fermentation supernatant was added to a mixture of 1 mL of 6 mM ferrous sulfate and 1 mL of 6 mM salicylic acid-ethanol. The mixture was vortexed until homogeneous and allowed to stand for 10 minutes. 0.5 mL of 8.8 mM hydrogen peroxide was added to the mixture, and the mixture was vortexed again until homogeneous and allowed to stand in the dark for 30 minutes. Each sample was measured three times. The mixture was measured at 517 nm. Distilled water was used as a blank control, and the aqueous extract as a control. The result was calculated using the following formula:

[0109] Hydroxyl radical scavenging rate D% = (A 测 -A 对 )÷(A 空 -A 对 )x100%

[0110] Experimental results: such as Figure 6 The fermented tangerine peel, immature bitter orange peel, and immature bitter orange fruit all showed higher hydroxyl radical scavenging rates than the unfermented group, indicating that the hydroxyl radical scavenging ability of these three fruits was significantly enhanced after processing with *Lactobacillus plantarum*. Table 6 shows that the hydroxyl radical scavenging ability of the fermented tangerine peel increased by 1421%, that of the immature bitter orange peel by 542%, and that of the immature bitter orange fruit by 664%.

[0111] Table 6 Hydroxyl radical scavenging capacity before and after fermentation

[0112]

[0113] The embodiments of this application have been described above. These embodiments are merely examples for clearly illustrating the invention and are exemplary, not exhaustive, and are not limited to the disclosed embodiments. Those skilled in the art can make other variations or modifications based on the above description. It is neither necessary nor possible to exhaustively describe all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this invention should be included within the scope of protection of the claims of this invention. The terminology used herein is chosen to best explain the principles, practical applications, or improvements to the technology in the market of the embodiments, or to enable other those skilled in the art to understand the embodiments disclosed herein.

Claims

1. A method for selectively increasing the content of antioxidant active ingredients in traditional Chinese medicine, characterized in that, To specifically increase the content of total sugars, flavonoids, and polyphenols in the antioxidant active ingredients, the extraction method includes the following steps: S1: Grind the green tangerine peel into powder using a grinder, add the powder to MRS broth liquid culture medium, and then autoclave to obtain the drug powder liquid culture medium. S2: The liquid culture medium containing the powder is inoculated with Lactobacillus plantarum in an anaerobic environment and fermented at a temperature of 36℃~38℃ for 20h~25h to obtain a fermentation mixture. S3: Centrifuge the fermentation mixture and collect the supernatant, which is a fermentation broth containing total sugar, flavonoids and polyphenols, and is used to prepare antioxidant active ingredients; The inoculation amount of *Lactobacillus plantarum* is 1 wt% to 4 wt%.

2. The extraction method for targeted enhancement of the content of antioxidant active ingredients in traditional Chinese medicine according to claim 1, characterized in that, The high-pressure steam sterilization process involves a temperature of 100℃~130℃, a processing time of 10min~20min, and a processing pressure of 0.05MPa~0.15MPa.

3. The extraction method for targeted enhancement of the content of antioxidant active ingredients in traditional Chinese medicine according to claim 1, characterized in that, The mass-to-volume ratio of the powder to the MRS broth liquid culture medium is 0.5g~2g:10mL~40mL.

4. The extraction method for targeted enhancement of the content of antioxidant active ingredients in traditional Chinese medicine according to claim 1, characterized in that, The viable count of *Lactobacillus plantarum* bacterial solution is >10. 8 CFU / ml.

5. The extraction method for targeted enhancement of the content of antioxidant active ingredients in traditional Chinese medicine according to claim 1, characterized in that, In the centrifugation treatment of the fermentation mixture, the centrifugation speed is 5000rpm~9000rpm and the centrifugation time is 6min~15min.

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