Extraction method for directionally improving content of antioxidant and anti-inflammatory active ingredients of traditional Chinese medicine and antioxidant and anti-inflammatory active ingredients

Abstract

The invention belongs to the technical field of traditional Chinese medicine processing, and particularly relates to an extraction method for directionally improving the content of antioxidant and anti-inflammatory active ingredients of traditional Chinese medicine and the antioxidant and anti-inflammatory active ingredients. The method comprises the following steps: grinding traditional Chinese medicinal materials into powder in a grinding machine, adding the powder into an MRS bouillon liquid culture medium, inoculating lactobacillus plantarum after high-pressure sterilization, standing for fermentation, and centrifuging to take supernate, so as to obtain the traditional Chinese medicinal fermentation liquor. According to the method, a single lactobacillus plantarum scheme is adopted to focus on efficient conversion of flavonoid components, single advantages of beta-glucosidase and cell wall degrading enzyme are relied on, combined flavonoids can be converted into free flavonoids through short-time fermentation, the content of total sugar, flavonoids and polyphenols in the medicinal materials is remarkably increased, the oxidation resistance is remarkably improved, meanwhile, the process is simple and short, consumed time is short, and the method is suitable for industrial production. The method is suitable for large-scale production, free of organic solvent residues, green and safe.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of traditional Chinese medicine processing, and particularly relates to an extraction method for improving the content of antioxidant and anti-inflammatory active ingredients of traditional Chinese medicine in a targeted manner and antioxidant and anti-inflammatory active ingredients. BACKGROUND

[0002] Pericarpium Citri Reticulatae Viride, Fructus Aurantii and Fructus Aurantii Immaturus are traditional Chinese medicinal materials with a long history of use and wide clinical application value. All of the three medicinal materials are derived from plants of the Rutaceae family and have similar chemical components and pharmacological activities. They are classified as qi-regulating medicinal materials in traditional Chinese medicine theory and are mainly used for treating chest and hypochondrium distending pain, food stagnation, phlegm stagnation and qi obstruction, etc. All of the three medicinal materials are rich in flavonoids, and flavonoids have significant antioxidant, anti-inflammatory and anti-tumor biological activities. Therefore, the three medicinal materials have certain free radical scavenging ability and can inhibit lipid peroxidation and protect cells from oxidative damage.

[0003] At present, the traditional extraction methods for the compounds contained in traditional Chinese medicines often include water extraction, organic solvent extraction (such as ethanol and methanol), acid-base extraction and distillation (for volatile oil), etc. Among them, the water extraction method causes the oxidation or hydrolysis of heat-sensitive components (such as volatile oil and part of flavonoid glycosides) due to high-temperature decoction; in the organic solvent extraction, the organic solvents such as ethanol and methanol can damage the polar-sensitive compounds, and the residual solvents need to be removed and treated separately, resulting in complicated operation; in the acid-base extraction, the treatment with strong acid / alkali causes the degradation of glycosides, polysaccharides and other components, for example, hesperidin is easily hydrolyzed under acidic conditions, which affects the quality of the product; the distillation method is mainly used for extracting volatile oil, but high temperature can easily cause the isomerization or oxidation of terpene compounds. Flavonoids (such as hesperidin and naringin) and volatile oil (such as limonene) are easily decomposed under high temperature, and the solubility of polysaccharides and phenolic acid components in organic solvents is low or they react, so the bioavailability of the active ingredients obtained by the traditional extraction methods is not high, and part of the effective components are easily destroyed in the processing process, which limits the full play of their efficacy.

[0004] The prior art discloses the application of lactic acid bacteria in the fermentation modification of traditional Chinese medicines. The lactic acid bacteria have the characteristics of high safety, strong fermentation ability and good acid tolerance. At present, compound lactic acid bacteria fermentation is often used. The compound lactic acid bacteria fermentation uses multiple lactic acid bacteria, such as mixed fermentation of Lactobacillus acidophilus, Bifidobacterium and Lactobacillus plantarum, to realize the generation of active products through the synergistic effect of multiple different strains. However, the above-mentioned compound lactic acid bacteria fermentation process has the problems of complicated operation, low controllability and low production efficiency on the one hand, and cannot efficiently and directionally improve the content of specific components in traditional Chinese medicines under the intervention of multiple lactic acid bacteria, which is not conducive to improving the production benefit. In addition, even if the existing technology can extract flavonoids with antioxidant or anti-inflammatory activity, the flavonoids obtained by the traditional method are only conjugated flavonoids, which have low efficacy.

[0005] In order to further improve the medicinal value of pericarpium citri reticulatae, fructus aurantii and fructus aurantii immaturus, it is necessary to further optimize the traditional Chinese medicine processing technology based on fermentation technology of pericarpium citri reticulatae, fructus aurantii and fructus aurantii immaturus. SUMMARY

[0006] The present application overcomes the shortcomings of the prior art and provides an extraction method for directional improvement of the content of active ingredients of traditional Chinese medicine against oxygen and inflammation, which can effectively improve the production efficiency of flavonoids and has the advantages of simple process and short fermentation time.

[0007] In order to solve the above technical problems, an extraction method for directional improvement of the content of active ingredients of traditional Chinese medicine against oxygen and inflammation is provided, which directional improves the content of total sugar, copper or polyphenol in the active ingredients against oxygen and inflammation, and the extraction method comprises the following steps:

[0008] S1: one or more than two kinds of medicinal materials of pericarpium citri reticulatae, fructus aurantii or fructus aurantii immaturus are put into a powder machine to grind into powder, the powder is added to MRS broth liquid medium, and then high-pressure steam sterilization is carried out to obtain a medicinal powder liquid medium;

[0009] S2: the medicinal powder liquid medium is inoculated with lactobacillus plantarum in an anaerobic environment and fermented, the fermentation temperature is 36-38 DEG C, the fermentation time is 20-25 h, and a fermentation mixture is obtained;

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

[0011] S3: the fermentation mixture is subjected to centrifugal treatment, and then the supernatant is collected, the supernatant is a fermentation liquid containing total sugar, copper or polyphenol, and the fermentation liquid is used for preparing active ingredients against oxygen and inflammation.

[0012] Further, the inoculation amount of the lactobacillus plantarum is 1wt%-4wt%. Preferably, the inoculation amount of the lactobacillus plantarum is 3%.

[0013] Further, the treatment temperature of the high-pressure steam sterilization is 100-130 DEG C, the treatment time is 10-20 min, and the treatment pressure is 0.05-0.15 MPa. Preferably, the treatment temperature of the high-pressure steam sterilization is 121 DEG C, the treatment time is 15 min, and the treatment pressure is 0.1 MPa.

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

[0015] Further, the number of viable bacteria in the lactobacillus plantarum bacterial liquid is >10 8 CFU / ml.

[0016] Further, in the centrifugal treatment of the fermentation mixture, the centrifugal speed is 5000 rpm to 9000 rpm, and the centrifugal time is 6 min to 15 min.

[0017] The application also provides an anti-oxidation and anti-inflammation active ingredient prepared by the above-mentioned method for increasing the content of the anti-oxidation and anti-inflammation active ingredient of traditional Chinese medicine.

[0018] Compared with the prior art, the application has the following beneficial effects:

[0019] (1) In the application, the fermentation temperature is only 36-38 DEG C, which is lower than the boiling temperature of 100 DEG C in the traditional water extraction method, and thus the heat-sensitive components of the medicinal materials are not damaged, and no organic solvent or strong acid / base is needed, so that the risk of solvent residue is eliminated.

[0020] (2) In the application, only Lactobacillus plantarum is used as a single strain, which is specifically used for the high-efficiency conversion of flavonoids, which are active components with anti-oxidation, anti-inflammation and anti-tumor effects, in pericarpium citri reticulatae, fructus aurantii and fructus aurantii immaturus.

[0021] (3) In the present application, the single Lactobacillus plantarum is used for directional fermentation of green orange peel, immature bitter orange and immature Chinese orange, which significantly improves the content of active ingredients of total sugar, flavonoids and polyphenols, and enhances the antioxidant capacity of flavonoids. The experimental data shows that after fermentation, the total sugar of green orange peel increases by 357%, the flavonoids increase by 107%, the polyphenols increase by 134%, the DPPH free radical scavenging capacity increases by 114%, the superoxide anion scavenging capacity increases by 48%, and the hydroxyl radical scavenging capacity increases by 1421%; the total sugar of immature bitter orange increases by 212%, the flavonoids increase by 87%, the polyphenols increase by 103%, the DPPH free radical scavenging capacity increases by 65%, the superoxide anion scavenging capacity increases by 37%, and the hydroxyl radical scavenging capacity increases by 542%; the total sugar of immature Chinese orange increases by 339%, the flavonoids increase by 72%, the polyphenols increase by 123%, the DPPH free radical scavenging capacity increases by 110%, the superoxide anion scavenging capacity increases by 27%, and the hydroxyl radical scavenging capacity increases by 664%.

[0022] (4) In the present application, the single strain advantage is exerted, and there is no need to coordinate the proportion of multiple strains. Only the inoculation amount, temperature and time of Lactobacillus plantarum need to be controlled, and the supernatant can be directly taken after centrifugation without complex purification steps. The process flow is simplified, the process is controllable, the enzymatic reaction is rapid, the flavonoid aglycone conversion can be completed in a short time fermentation, the product activity is significantly improved, the fermentation parameter control is simple, and the standardization control is easy. Therefore, the standardized production can be realized by controlling the fermentation conditions of temperature and fermentation time, the quality consistency of each batch of products is ensured, the overall quality and the stability of the medicinal effect of green orange peel, immature bitter orange and immature Chinese orange are improved, and the large-scale industrial production is suitable.

[0023] It should be understood that the above general description and the following detailed description are only exemplary and explanatory, and cannot limit the present application. BRIEF DESCRIPTION OF DRAWINGS

[0024] The above and other objects, features and advantages of the present application will become more apparent from the following detailed description when taken in conjunction with the accompanying drawings.

[0025] Figure 1 Content diagram of the same concentration of flavonoids in the medicinal liquid before fermentation (comparative example) and after fermentation (experimental example);

[0026] Figure 2 Content diagram of the same concentration of total sugar in the medicinal liquid before fermentation (comparative example) and after fermentation (experimental example);

[0027] Figure 3 Content diagram of the same concentration of polyphenols in the medicinal liquid before fermentation (comparative example) and after fermentation (experimental example);

[0028] Figure 4DPPH free radical scavenging rate diagram of the same concentration of medicinal liquid before fermentation (comparative example) and after fermentation (experimental example) ;

[0029] Figure 5 Superoxide anion scavenging rate diagram of the same concentration of medicinal liquid before fermentation (comparative example) and after fermentation (experimental example) ;

[0030] Figure 6 Hydroxyl radical scavenging rate diagram of the same concentration of medicinal liquid before fermentation (comparative example) and after fermentation (experimental example). DETAILED DESCRIPTION

[0031] Preferred embodiments of the present application will be described in more detail below. Although preferred embodiments of the present application are shown, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments set forth herein. Rather, these embodiments are provided so that the present application will be thorough and complete, and will fully convey the scope of the present application 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. As used in this application and the appended claims, the singular forms "a," "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will also be understood that the term "and / or" as used herein refers to and encompasses any and all possible combinations of one or more of the associated listed items.

[0033] It will be understood that, although the terms "first," "second," "third," etc. can be used herein to describe various information, these terms are not intended to denote a particular order or hierarchy. These terms are used only to distinguish one from another. For example, a first information can also be referred to as a second information, and similarly, a second information can also be referred to as a first information, without departing from the scope of the present application. Thus, the features defined with "first," "second" can explicitly or implicitly include one or more of the features. In the description of the present application, the meaning of "a plurality of" is two or more, unless otherwise specifically limited.

[0034] Example 1

[0035] The present embodiment discloses an extraction method for improving the content of antioxidant and anti-inflammatory active ingredients of green skin, comprising the following steps:

[0036] S1: Put the green skin into a powder machine to grind into powder, filter with a 50 mesh screen, add to MRS broth liquid medium, perform high pressure steam sterilization, the sterilization treatment temperature is 121℃, the treatment pressure is 0.1MPa, the mass volume ratio of the medicinal powder to MRS broth is 1g:20mL;

[0037] The fine powder filtered by the 50 mesh screen can increase the contact area of the medicinal material and the strain, and improve the fermentation efficiency; the autoclaving can eliminate the competition of miscellaneous bacteria, and ensure the pure strain fermentation of Lactobacillus plantarum, and assist the release of the components of the medicinal material.

[0038] S2: The medicinal powder liquid medium obtained in step S1 is placed in an anaerobic environment, and 3wt% of Lactobacillus plantarum is inoculated for fermentation, the temperature in the fermentation process is 37℃, and the time is 24h, and the viable bacterial count in the Lactobacillus plantarum bacterial liquid is >10 8 CFU / ml;

[0039] Here, the Lactobacillus plantarum fermentation temperature is 37℃, the bacterial proliferation is the fastest, and the enzyme synthesis rate is the highest, such as β-glucosidase. If the fermentation temperature is too low, the bacterial metabolism is slow, the enzyme activity is low, and the fermentation period is prolonged; if the temperature is too high, the protein is denatured, the bacteria are dead, and the key enzyme is inactivated.

[0040] S3: The fermentation liquid obtained in step S2 is centrifuged at a speed of 5000rpm, in actual application, it can also be selected according to the actual situation. The time is 10min, the supernatant is taken, and the traditional Chinese medicine fermentation liquid is obtained.

[0041] Example 2

[0042] The embodiment discloses an extraction method for directionally increasing the content of antioxidant and anti-inflammatory active ingredients of Fructus Aurantii, comprising the following steps:

[0043] S1: Put Fructus Aurantii into a powder machine to grind into powder, filter with a 50 mesh screen, add to MRS broth liquid medium, and perform high-pressure steam sterilization, the time is 10min, the sterilization treatment temperature is 100℃, the treatment pressure is 0.05MPa, and the mass-volume ratio of the medicinal powder to the MRS broth is 0.5g:10mL;

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

[0045] S3: The fermentation liquid obtained in step S2 is centrifuged at a speed of 5000rpm, in actual application, it can also be selected according to the actual situation. The time is 10min, the supernatant is taken, and the traditional Chinese medicine fermentation liquid is obtained.

[0046] Example 3

[0047] The embodiment discloses an extraction method for directionally increasing the content of antioxidant and anti-inflammatory active ingredients of Fructus Aurantii, comprising the following steps:

[0048] S1: put the Citrus aurantium into a powder machine to grind into powder, filter with a 50 mesh screen, add to a MRS broth liquid culture medium, perform high pressure steam sterilization, the sterilization treatment temperature is 130 DEG C, the treatment pressure is 0.15 Mpa, the mass volume ratio of the medicine powder and the MRS broth is 2g:40ml;

[0049] S2: place the medicine powder liquid culture medium obtained in step S1 in an anaerobic environment, inoculate 4wt% of Lactobacillus plantarum to perform fermentation, the temperature during fermentation is 38 DEG C, the time is 25h, the viable cell count in the Lactobacillus plantarum liquid is >10 8 CFU / ml;

[0050] S3: centrifuge the fermentation liquid obtained in step S2, the rotation speed is 9000rpm, in actual application, it can also be selected according to actual conditions, the time is 15min, take the supernatant to obtain the traditional Chinese medicine fermentation liquid.

[0051] Effect verification:

[0052] In order to further illustrate the effect of the present application, the following experiments and comparisons are carried out:

[0053] Experimental example 1

[0054] The embodiment discloses an extraction method for directionally improving the content of antioxidant and anti-inflammatory active ingredients of Citrus aurantium, comprising the following steps:

[0055] S1: put the Citrus aurantium into a powder machine to grind into powder, filter with a 50 mesh screen, add to a MRS broth liquid culture medium, perform high pressure steam sterilization, the sterilization treatment temperature is 130 DEG C, the treatment pressure is 0.15 Mpa, the mass volume ratio of the medicine powder and the MRS broth is 2g:40ml;

[0056] S2: place the medicine powder liquid culture medium obtained in step S1 in an anaerobic environment, inoculate 4wt% of Lactobacillus plantarum to perform fermentation, the temperature during fermentation is 38 DEG C, the time is 25h, the viable cell count in the Lactobacillus plantarum liquid is >10

[0057] S3: centrifuge the fermentation liquid obtained in step S2, the rotation speed is 9000rpm, in actual application, it can also be selected according to actual conditions, the time is 15min, take the supernatant to obtain the traditional Chinese medicine fermentation liquid.

[0058] Experimental example 2

[0059] The embodiment discloses an extraction method for directionally improving the content of antioxidant and anti-inflammatory active ingredients of Citrus aurantium, comprising the following steps:

[0060] S1: Put the Fructus Aurantii in a powder machine to grind into powder, filter with a 50 mesh sieve, add to the MRS broth liquid medium, high-pressure steam sterilization, time 10 min, sterilization treatment temperature 100℃, treatment pressure 0.05 MPa, the mass volume ratio of the medicine powder and MRS broth is 0.5g:10mL;

[0061] S2: Put the medicine powder liquid medium obtained in step S1 in an anaerobic environment, inoculate 1wt% of Lactobacillus plantarum for fermentation, the temperature during fermentation is 36℃, the time is 20h, the viable count of the Lactobacillus plantarum bacteria liquid is >108CFU / ml;

[0062] S3: Centrifuge the fermentation liquid obtained in step S2, the speed is 5000rpm, in actual application, it can also be selected according to actual conditions, the time is 10min, take the supernatant, and obtain the traditional Chinese medicine fermentation liquid.

[0063] Experimental example 3

[0064] The embodiment discloses an extraction method for improving the content of anti-oxidation and anti-inflammatory active ingredients of Fructus Aurantii, comprising the following steps:

[0065] S1: Put the Fructus Aurantii in a powder machine to grind into powder, filter with a 50 mesh sieve, add to the MRS broth liquid medium, high-pressure steam sterilization, time 20 min, sterilization treatment temperature 130℃, treatment pressure 0.15 MPa, the mass volume ratio of the medicine powder and MRS broth is 2g:40mL;

[0066] S2: Put the medicine powder liquid medium obtained in step S1 in an anaerobic environment, inoculate 4wt% of Lactobacillus plantarum for fermentation, the temperature during fermentation is 38℃, the time is 25h, the viable count of the Lactobacillus plantarum bacteria liquid is >108CFU / ml;

[0067] S3: Centrifuge the fermentation liquid obtained in step S2, the speed is 9000rpm, in actual application, it can also be selected according to actual conditions, the time is 15min, take the supernatant, and obtain the traditional Chinese medicine fermentation liquid.

[0068] Comparative example 1

[0069] Put the Pericarpium Citri Reticulatae in a powder machine to grind into powder, filter with a 50 mesh sieve, the medicine powder is extracted by water, rotary evaporated, and freeze-dried to obtain freeze-dried powder, which is subsequently reconstituted into corresponding concentrations for comparative detection.

[0070] Comparative example 2

[0071] Put the Fructus Aurantii in a powder machine to grind into powder, filter with a 50 mesh sieve, the medicine powder is extracted by water, rotary evaporated, and freeze-dried to obtain freeze-dried powder, which is subsequently reconstituted into corresponding concentrations for comparative detection.

[0072] Comparative Example 3

[0073] Put the immature bitter orange into a powder grinder and grind it into powder, then filter it with a 50-mesh sieve. The powder is extracted with water, rotary evaporated, and freeze-dried to obtain freeze-dried powder, which is then re-dissolved into the corresponding concentration for comparative testing.

[0074] 1. Verification of the effect of flavonoid ingredients:

[0075] The flavonoid contents of Experimental Examples 1 to 3 and Comparative Examples 1 to 3 were respectively measured according to the following flavonoid content determination method, wherein Experimental Examples 1 to 3 refer to the flavonoid determination after fermentation, and Comparative Examples 1 to 3 refer to the flavonoid determination before fermentation.

[0076] Experimental method: The fermentation broth and water extract were dissolved and diluted to 25 mg / ml using 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 in the dark at room temperature for 15 minutes. The mixture was measured at 510 nm. A standard curve was drawn with the absorbance value as the vertical coordinate and rutin as the horizontal coordinate to calculate the total flavonoid content. The linear regression equation for the standard curve of total flavonoid content was fitted: y = 0.0075x + 0.0077, R 2 =0.9999.

[0077] Experimental results: Figure 1 The flavonoid content of the fermented Citrus aurantium, Citrus aurantium fructus and Citrus aurantium immaturus was significantly improved. As shown in Table 1, the flavonoid content of the fermented Citrus aurantium increased by 107%, the flavonoid content of Citrus aurantium fructus increased by 87%, and the flavonoid content of Citrus aurantium immaturus increased by 72%.

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

[0079] Medicinal materials Flavonoid content (μg / mL) of comparative example Flavonoid content (μg / mL) of experimental example Pericarpium citri reticulatae viride 204.4 423.8 Fructus aurantii 437.6 819.8 Fructus aurantii immaturus 232.0 398.3

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

[0081] The total sugar contents of Experimental Examples 1 to 3 and Comparative Examples 1 to 3 were respectively measured according to the following total sugar content determination method, wherein Experimental Examples 1 to 3 refer to the total sugar determination after fermentation, and Comparative Examples 1 to 3 refer to the total sugar determination 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 vortex, 5 mL of concentrated sulfuric acid was added to the mixture, vortexed again, and left at room temperature for 30 min (from the start of adding concentrated sulfuric acid). The mixture was measured at 490 nm. The standard curve was plotted with absorbance value as the vertical coordinate and glucose as the horizontal coordinate, and the total sugar content was calculated. The linear regression equation of the total sugar content standard curve was y = 0.0042x + 0.0143, R 2 = 0.9984.

[0083] Experimental results: as Figure 2 , the total sugar content of the fermented green peel, bitter orange and immature bitter orange was significantly improved. In Table 2, the total sugar content of the experimental example green peel after fermentation was increased by 357%, the total sugar content of the bitter orange was increased by 212%, and the total sugar content of the immature bitter orange was increased by 339%.

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

[0085] Medicinal materials Total sugar content (μg / mL) of comparative example Total sugar content (μg / mL) of experimental example Pericarpium citri reticulatae viride 0.47 2.12 Fructus aurantii 1.05 3.29 Fructus aurantii immaturus 0.45 1.96

[0086] 3, Effect verification of polyphenol component:

[0087] The polyphenol content of experimental examples 1-3 and comparative examples 1-3 was determined by the following polyphenol content determination method, wherein experimental examples 1-3 refer to polyphenol determination after fermentation, and comparative examples 1-3 refer to polyphenol determination before fermentation.

[0088] Experimental method: the fermentation broth and water extract were dissolved and diluted to 25 mg / ml with distilled water, 50 μL of sample was taken and 100 μL of Folin phenol reagent was added, and then 1000 μL of 7% sodium carbonate and 850 μL of pure water were added, and the mixture was placed in the dark for 45 min. The mixture was measured at 750 nm, and the standard curve was plotted with absorbance value as the vertical coordinate and polyphenol as the horizontal coordinate, and the polyphenol content was calculated. The linear regression equation of the polyphenol content standard curve was y = 0.0042x + 0.027, R 2 = 0.9949.

[0089] Experimental results: as Figure 3 , the total sugar content of the fermented green peel, bitter orange and immature bitter orange was significantly improved. In Table 2, the total sugar content of the experimental example green peel after fermentation was increased by 357%, the total sugar content of the bitter orange was increased by 212%, and the total sugar content of the immature bitter orange was increased by 339%.

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

[0091] Medicinal materials Polyphenol content (μg / mL) of comparative example Polyphenol content (μg / mL) of experimental example Pericarpium citri reticulatae viride 159.8 373.7 Fructus aurantii 230.7 468.9 Fructus aurantii immaturus 167.4 373.6

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

[0093] The DPPH radical scavenging abilities of Experimental Examples 1 to 3 and Comparative Examples 1 to 3 were respectively measured according to the following DPPH radical scavenging rate determination method, wherein Experimental Examples 1 to 3 refer to the DPPH radical scavenging rate determination after fermentation, and Comparative Examples 1 to 3 refer to the DPPH radical scavenging rate determination before fermentation.

[0094] Experimental Method: Fermentation broth and aqueous extract were diluted to 25 mg / mL with distilled water. 50 μL of fermentation supernatant was collected in a 5 mL brown centrifuge tube. 1.5 mL of 0.07 mg / mL DPPH was added to the tube, mixed thoroughly, and incubated in the dark for 30 minutes. Anhydrous ethanol was used as a blank control, and the aqueous extract was used as a control. Each sample was measured in triplicate, and the resulting mixture was measured at 517 nm. The calculation formula was:

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

[0096] Experimental results: Figure 4 As shown in Table 4, the DPPH radical scavenging rates of fermented Citrus aurantium, Citrus aurantium fructus, and Citrus aurantium immaturus were all higher than those of the unfermented group, indicating that the DPPH radical scavenging abilities of Citrus aurantium, Citrus aurantium fructus, and Citrus aurantium immaturus were significantly enhanced after bioprocessing with Lactobacillus plantarum. As shown in Table 4, the DPPH radical scavenging abilities of Citrus aurantium, Citrus aurantium fructus, and Citrus aurantium immaturus increased by 114%, 65%, and 110% after fermentation.

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

[0098] Medicinal materials DPPH radical scavenging rate (%) of comparative example DPPH radical scavenging rate (%) of experimental example Pericarpium citri reticulatae viride 37.8 81.0 Fructus aurantii 36.4 60.1 Fructus aurantii immaturus 31.5 66.3

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

[0100] The superoxide anion scavenging abilities of Experimental Examples 1 to 3 and Comparative Examples 1 to 3 were respectively measured according to the following superoxide anion scavenging rate determination method, wherein Experimental Examples 1 to 3 refer to the superoxide anion scavenging rate determination after fermentation, and Comparative Examples 1 to 3 refer to the superoxide anion scavenging rate determination before fermentation.

[0101] Experimental method: The fermentation broth and water extract were dissolved and diluted to 25 mg / ml with distilled water. The supernatant was taken and the experimental steps of the hydroxyl free radical scavenging ability test kit were followed. The calculation formula was:

[0102] Hydroxyl free radical scavenging rate D% = (A test - A pair) ÷ (A blank - A pair) x 100%

[0103] Experimental results: as Figure 5 , the superoxide anion scavenging rate of fermented green peel, bitter orange and immature bitter orange is higher than that of the non-fermented group, indicating that the superoxide anion scavenging capacity of green peel, bitter orange and immature bitter orange is significantly improved after being bio-processed by lactobacillus plantarum. In Table 5, it can be seen that the superoxide anion scavenging capacity of the fermented experimental example green peel is increased by 48%, the superoxide anion scavenging capacity of the fermented experimental example bitter orange is increased by 37%, and the superoxide anion scavenging capacity of the fermented experimental example immature bitter orange is increased by 27%.

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

[0105] Medicinal materials Superoxide anion scavenging rate (%) of comparative example Superoxide anion scavenging rate (%) of experimental example Pericarpium citri reticulatae viride 53.0 78.5 Fructus aurantii 58.8 80.7 Fructus aurantii immaturus 59.4 75.5

[0106] 6. Hydroxyl radical scavenging capacity effect verification:

[0107] The hydroxyl radical scavenging capacity of experimental examples 1-3 and comparative examples 1-3 was determined by the following hydroxyl radical scavenging rate determination method, wherein experimental examples 1-3 refer to the determination of hydroxyl radical scavenging rate after fermentation, and comparative examples 1-3 refer to the determination of hydroxyl radical scavenging rate before fermentation.

[0108] Experimental method: The fermentation broth and water extract were dissolved and diluted to 25 mg / ml with distilled water, 0.5 mL of fermentation supernatant was added to 1 mL of 6 mM ferrous sulfate and 1 mL of 6 mM salicylic acid-ethanol mixture. Mix well and stand for 10 minutes. Add 0.5 mL of 8.8 mM hydrogen peroxide to the mixture, mix well again and stand in the dark for 30 min. Each sample was measured three times, the mixture was measured at 517 nm, and distilled water was used as a blank control and water extract as a control. According to the calculation formula:

[0109] Hydroxyl radical scavenging rate D% = (A 测 -A 对 ) ÷ (A 空 -A 对 ) x 100%

[0110] Experimental results: as Figure 6 , the hydroxyl radical scavenging rate of fermented green peel, bitter orange and immature bitter orange is higher than that of the non-fermented group, indicating that the hydroxyl radical scavenging capacity of green peel, bitter orange and immature bitter orange is significantly improved after being bio-processed by lactobacillus plantarum. In Table 6, it can be seen that the hydroxyl radical scavenging capacity of the fermented experimental example green peel is increased by 1421%, the hydroxyl radical scavenging capacity of the fermented experimental example bitter orange is increased by 542%, and the hydroxyl radical scavenging capacity of the fermented experimental example immature bitter orange is increased by 664%.

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

[0112] Medicinal materials Hydroxyl radical scavenging rate (%) of comparative example Hydroxyl radical scavenging rate (%) of experimental example Pericarpium citri reticulatae viride 1.4 21.3 Fructus aurantii 3.3 21.2 Fructus aurantii immaturus 2.8 21.4

[0113] Having described various embodiments of the application, it is to be understood that the above description is meant to be illustrative only and not exhaustive. Modifications, changes and variations which are obvious to those of ordinary skill in the art are intended to be within the scope of the application. The foregoing description details certain embodiments of the application. It will be appreciated, however, that no matter how detailed the above appears in text, the application can be practiced in many ways, as will be understood by those skilled in the art. Therefore, the application should not be construed as limited to the embodiments described above. Rather, the scope of the application should be measured by the broadest interpretation of the claims that are included below.

Claims

1. A method for extracting traditional Chinese medicine to increase the content of antioxidant and anti-inflammatory active ingredients, characterized in that: The extraction method for increasing the content of total sugar, brass or polyphenols in the antioxidant and anti-inflammatory active ingredients comprises the following steps: S1: Grind one or more medicinal materials selected from Citrus aurantium, Citrus aurantium fructus, or Citrus aurantium immaturum into powder in a powder grinder, add the powder into MRS broth liquid culture medium, and then sterilize with high-pressure steam to obtain a medicinal powder liquid culture medium; S2: inoculating the powder liquid culture medium with Lactobacillus plantarum in an anaerobic environment and fermenting the culture medium at a temperature of 36° C. to 38° C. for 20 to 25 hours to obtain a fermentation mixture; S3: centrifuging the fermentation mixture to collect the supernatant, which is a fermentation broth containing total sugars, brass or polyphenols. The fermentation broth is used to prepare antioxidant and anti-inflammatory active ingredients.

2. The method for extracting the anti-oxidative and anti-inflammatory active ingredients of traditional Chinese medicine according to claim 1, wherein: The inoculation amount of the Lactobacillus plantarum is 1 wt% to 4 wt%.

3. The method for extracting the anti-oxidative and anti-inflammatory active ingredients of traditional Chinese medicine according to claim 1, wherein: The high-pressure steam sterilization treatment temperature is 100° C. to 130° C., the treatment time is 10 min to 20 min, and the treatment pressure is 0.05 MPa to 0.15 MPa.

4. The method for extracting the anti-oxidative and anti-inflammatory active ingredients of traditional Chinese medicine according to claim 1, wherein: The mass volume ratio of the medicinal powder to the MRS broth liquid culture medium is 0.5 g to 2 g: 10 mL to 40 mL.

5. The method for extracting the anti-oxidative and anti-inflammatory active ingredients of traditional Chinese medicine according to claim 1, characterized in that: The number of viable bacteria in the plant lactobacillus liquid is>10 8 CFU / ml.

6. The method for extracting the anti-oxidative and anti-inflammatory active ingredients of traditional Chinese medicine according to claim 1, characterized in that: During the centrifugal treatment of the fermentation mixture, the centrifugal speed is 5000 rpm to 9000 rpm, and the centrifugal time is 6 min to 15 min.

7. An antioxidant and anti-inflammatory active ingredient, characterized in that: The invention is prepared by the extraction method for directionally increasing the content of the antioxidant and anti-inflammatory active ingredients of traditional Chinese medicine according to any one of claims 1 to 6.

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