Composition containing cortex phellodendri and rhizoma atractylodis, application of composition, product containing composition and preparation method of product

Abstract

The invention relates to the technical field of medicines, in particular to a composition containing cortex phellodendri and rhizoma atractylodis, application of the composition, a product containing the composition and a preparation method of the product. The preparation method comprises the following steps: taking dried cortex phellodendri and rhizoma atractylodis, crushing to obtain a powder mixture, adding water into the powder mixture, boiling, preserving heat, and cooling to obtain a decoction liquid; adding enzyme into the decocted material liquid, performing enzymolysis, and performing enzyme deactivation to obtain an enzymolysis material; sterilizing the enzymolysis material, adding glucose, dipotassium phosphate and zymophyte, fermenting, and sterilizing to obtain a fermented material; adding ethanol into the fermented material, adjusting the ethanol concentration, heating and stirring to obtain an extracted material; and filtering, ultra-filtering, concentrating and drying the extracted material to obtain the composition containing cortex phellodendri and rhizoma atractylodis. The composition prepared by the invention has the effects of high efficiency, low toxicity, safety and effectiveness in inhibition of a candida albicans biofilm.

Description

Technical Field

[0001] The invention relates to the technical field of medicines, and in particular to a composition containing Phellodendron chinense and Atractylodes lancea and an application thereof, and a product containing the composition and a preparation method thereof. Background Art

[0002] Vulvovaginal candidiasis (VVC) is the second most common vaginal infection after bacterial vaginitis. It is mainly caused by Candida albicans, an opportunistic mucosal infection, and is mainly manifested by tofu-like leucorrhea and vulvar itching. It is one of the most common vaginal infections in women and one of the risk factors for a series of high-risk diseases such as AIDS, cervicitis and miscarriage. Candida albicans is a conditionally pathogenic fungus that will not cause infection under normal circumstances. When the body's immunity is low or the balance of the body's normal flora is broken, Candida albicans will turn into a pathogenic bacterium, grow and reproduce locally, causing local mucosal or even systemic diseases.

[0003] The pathogenicity of Candida albicans is mainly reflected in the formation of biofilms, secretion of hydrolases, secretion of adhesins, secretion of candidiasis, etc. Among them, the formation of biofilms can protect the bacteria from the host's immune response, help the bacteria escape the killing effect of antibacterial drugs and develop resistance to antifungal drugs, causing chronic and difficult-to-cure infections. At the same time, a quorum sensing system is formed between the Candida albicans aggregated in the biofilm, which further regulates the biofilm generation cycle of Candida albicans and the secretion of virulence factors.

[0004] At present, the drug library resources for inhibiting Candida albicans are limited. Azoles are the first choice for clinical treatment and prevention of Candida albicans infection. The large-scale application of azoles increases the drug resistance of Candida albicans while treating Candida albicans infection. In addition, the toxic and side effects of drugs for clinical treatment of Candida albicans infection may cause irreversible damage to the human body. In order to solve this series of problems, it is urgent to find new antibacterial agents that are efficient, low-toxic, safe and effective in inhibiting Candida albicans biofilm from natural plant traditional Chinese medicine. Summary of the invention

[0005] The present invention aims at the above technical problems and proposes a technical solution of the present invention, which includes a composition containing Phellodendron chinense and Atractylodes lancea and its application, and a product containing the composition and a preparation method.

[0006] In one aspect, the present invention provides a method for preparing a composition containing Phellodendron chinense and Atractylodes lancea, comprising the following steps:

[0007] Step 1: Take dried Phellodendron chinense and Atractylodes lancea, crush them to obtain a powder mixture, add water to the powder mixture, boil it, keep it warm, and then cool it to obtain a decoction liquid;

[0008] Step 2: adding enzyme to the decoction liquid, performing enzymolysis, and inactivating the enzyme to obtain an enzymolysis material;

[0009] Step 3: sterilize the enzymatic hydrolysis material, add glucose, dipotassium hydrogen phosphate and fermentation bacteria, ferment, sterilize, and obtain the fermentation material;

[0010] Step 4: adding ethanol to the fermented material, adjusting the ethanol concentration, heating and stirring to obtain an extract material;

[0011] Step 5: Filter the extracted material, ultrafilter, concentrate, and dry to obtain a composition containing Phellodendron chinense and Atractylodes lancea.

[0012] Preferably, in step 1, the mass ratio of Phellodendron chinense to Atractylodes lancea is 1:2-6.

[0013] Preferably, in step 1, the mass ratio of the powder mixture to water is 1:10-50.

[0014] Preferably, in step 1, the insulation temperature is 72-85° C. and the insulation time is 1-6 hours.

[0015] Preferably, in step 2, the enzyme consists of Serratia protease and bromelain.

[0016] Preferably, in step 2, the amounts of serratia protease and bromelain are 400-800 U / g decoction liquid and 200-500 U / g decoction liquid, respectively.

[0017] Preferably, in step 2, the enzymatic hydrolysis temperature is 40-65°C and the time is 1-5 hours.

[0018] Preferably, in step 3, the added amounts of glucose and dipotassium hydrogen phosphate are 1.2-4% and 0.02-0.15% of the mass of the enzymatic hydrolysis material.

[0019] Preferably, in step 3, the fermentation bacteria are composed of Lactobacillus reuteri CICC6119 and Bacillus coagulans CICC23843 in a quantitative ratio of 1:2-4.

[0020] Preferably, in step 3, the amount of fermentation bacteria is 1×10 9 -9×10 9 cfu / g enzymatic material.

[0021] Preferably, in step 3, the fermentation temperature is 25-40°C and the fermentation time is 100-150 hours;

[0022] Preferably, in step 4, the ethanol mass concentration is adjusted to 20-40%.

[0023] Preferably, in step 4, the heating temperature is 50-60° C. and the time is 2-6 hours.

[0024] Preferably, in step 5, the filtration is performed using a filter membrane with a pore size of not less than 0.1 micron.

[0025] Preferably, in step 5, ultrafiltration is performed using a lung ultrafiltration membrane with a molecular weight cutoff lower than 3 KDa.

[0026] Preferably, in step 5, the drying is freeze-drying or vacuum drying.

[0027] Preferably, in step 5, the drying is performed until the moisture content is less than 6%.

[0028] The present invention also provides a composition containing Phellodendron chinense and Atractylodes lancea prepared according to the preparation method.

[0029] The present invention also provides a product comprising the composition, wherein the mass percentage content of the composition in the product is 0.1-30%, and the remaining auxiliary materials.

[0030] Preferably, the auxiliary materials include one or more of starch, cellulose and its derivatives, vegetable gum, and water.

[0031] The present invention also provides a method for preparing the product, comprising the step of mixing the composition with auxiliary materials.

[0032] Beneficial effects of the present invention:

[0033] The present invention selects dried Phellodendron chinense and Atractylodes lancea, crushes them to obtain a powder mixture, adds water to the powder mixture, boils and then keeps warm, and then cools to obtain a decoction liquid; adds enzyme to the decoction liquid, performs enzymolysis, inactivates the enzyme, obtains an enzymolysis material; sterilizes the enzymolysis material, adds glucose, dipotassium hydrogen phosphate and fermentation bacteria, ferments, sterilizes, obtains a fermentation material; adds ethanol to the fermentation material, adjusts the ethanol concentration, heats and stirs, obtains an extract material; filters the extract material, ultrafilters, concentrates, and dries to obtain a composition containing Phellodendron chinense and Atractylodes lancea. The composition prepared by the present invention has the efficacy of inhibiting Candida albicans biofilm with high efficiency, low toxicity, safety and effectiveness.

[0034] The MIC of the composition prepared by the present invention to Candida albicans is relatively low, and the composition of the present invention has antibacterial activity to Candida albicans; the MIC of the composition to Candida albicans is close to the MIC of phenoxyethanol to Candida albicans.

[0035] The composition prepared by the invention can reduce the metabolic activity of biofilm bacteria in the adhesion stage of Candida albicans to varying degrees.

[0036] The composition of the present invention can reduce the metabolic activity of biofilm bacteria in the aggregation stage of Candida albicans to varying degrees, and can reduce the metabolic activity of biofilm bacteria in the mature stage of Candida albicans to varying degrees.

[0037] The composition of the invention can reduce the biofilm of Candida albicans in the adhesion stage, reduce the biofilm of Candida albicans in the aggregation stage and reduce the biofilm of Candida albicans in the mature stage to different degrees.

[0038] The present invention adopts a decoction method during preparation, which can better promote the extraction of active ingredients. The combination of Serratia protease and bromelain is used for enzymolysis, and the types and concentrations of active ingredients of the medicine are mutually promoted to increase the efficacy. Lactobacillus reuteri CICC 6119 and Bacillus coagulans CICC 23843 are used for co-fermentation to enhance the medicinal properties of the fermentation product, give the composition a richer variety and concentration of active ingredients, and enhance the inhibitory effect of Candida albicans on the concentrated and dried composition. At the same time, when the ratio of Lactobacillus reuteri CICC 6119 to Bacillus coagulans CICC 23843 in the fermentation bacteria is 1:2-4, the obtained composition has a better effect. At the same time, in order to further enhance the medicinal properties of the composition, the present invention uses an ethanol aqueous solution for extraction and ultrafiltration for enrichment and impurity removal after fermentation, thereby enhancing the medicinal activity of the composition and giving the composition a better efficacy.

[0039] The preparation method of the composition of the invention is simple and green and environmentally friendly; the obtained composition has excellent effect and is safe, and can effectively retain the active ingredients of Phellodendron chinense and Atractylodes macrocephala. DETAILED DESCRIPTION

[0040] The present invention will be described below in conjunction with specific embodiments, and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments are used to illustrate the present invention, rather than to limit the present invention.

[0041] Phellodendron chinense was purchased from Kangmei Pharmaceutical Co., Ltd., with a batch number of 230501491. Atractylodes lancea was purchased from Beijing Kangmei Pharmaceutical Co., Ltd., with a batch number of 220961031. Serratia protease and bromelain were purchased from Hubei Ruiboxin Chemical Co., Ltd. and Sigma, respectively. Lactobacillus reuteri CICC6119 and Bacillus coagulans CICC23843 were purchased from China Industrial Microbiological Culture Collection Administration Center.

[0042] 1. Preparation of the composition:

[0043] Composition A:

[0044] The preparation method is as follows: Step 1: Take dried Phellodendron chinense and Atractylodes lancea, grind them into a particle size of less than 0.5 mm in a mass ratio of 1:4 to obtain a powder mixture, add 25.5 times the mass of water to the powder mixture, boil it, keep it warm at 78°C and stir it at 60rpm for 3.2 hours, and then cool it to room temperature to obtain a decoction liquid;

[0045] Step 2: adding enzyme to the decoction liquid, performing enzymolysis at 55°C for 2.5 hours, inactivating the enzyme, and obtaining enzymolysis material;

[0046] The enzymes are composed of serratia protease and bromelain; the dosages of serratia protease and bromelain are 600U / g decoction liquid and 400U / g decoction liquid respectively;

[0047] Step 3: sterilize the enzymatic hydrolysis material, add glucose, dipotassium hydrogen phosphate and fermentation bacteria, perform anaerobic fermentation at 35°C for 127 hours, sterilize and obtain the fermentation material;

[0048] The added amounts of glucose and dipotassium hydrogen phosphate are 3.2% and 0.085% of the mass of the enzymatic hydrolysis material;

[0049] The fermentation bacteria consisted of Lactobacillus reuteri CICC6119 and Bacillus coagulans CICC23843 in a quantitative ratio of 1:3;

[0050] The fermentation bacteria dosage is 6.80×10 9 cfu / g enzymatic material;

[0051] Step 4: adding ethanol to the fermentation material to adjust the ethanol mass concentration to 32%, heating at 55°C and stirring at 70 rpm for 4 hours to obtain an extract material;

[0052] Step 5: The extract material is filtered using a filter membrane with a pore size of 1 micron, and the filtrate is ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 2000Da. The ultrafiltrate is distilled under reduced pressure at 54°C to remove ethanol, and freeze-dried at -40°C to a water content of 3.28wt%, to obtain a composition containing Phellodendron amurense and Atractylodes lancea.

[0053] Composition B:

[0054] The preparation method is as follows: Step 1: Take dried Phellodendron chinense and Atractylodes lancea, grind them into a particle size of less than 0.5 mm in a mass ratio of 1:2 to obtain a powder mixture, add 27 times the mass of water to the powder mixture, boil it, keep it warm at 75°C and stir it at 60rpm for 3 hours, and then cool it to room temperature to obtain a decoction liquid;

[0055] Step 2: adding enzyme to the decoction liquid, performing enzymolysis at 45°C for 2 hours, inactivating the enzyme, and obtaining enzymolysis material;

[0056] The enzymes are composed of serratia protease and bromelain; the dosages of serratia protease and bromelain are 500U / g decoction liquid and 500U / g decoction liquid respectively;

[0057] Step 3: sterilize the enzymatic hydrolysis material, add glucose, dipotassium hydrogen phosphate and fermentation bacteria, perform anaerobic fermentation at 32°C for 120 hours, sterilize and obtain the fermentation material;

[0058] The added amounts of glucose and dipotassium hydrogen phosphate are 3.5% and 0.087% of the mass of the enzymatically hydrolyzed material;

[0059] The fermentation bacteria consisted of Lactobacillus reuteri CICC6119 and Bacillus coagulans CICC23843 in a quantitative ratio of 1:2;

[0060] The fermentation bacteria dosage is 6.33×10 9 cfu / g enzymatic material;

[0061] Step 4: adding ethanol to the fermentation material to adjust the ethanol mass concentration to 25%, heating at 57°C and stirring at 70 rpm for 3.8 hours to obtain an extract material;

[0062] Step 5: The extract material is filtered using a filter membrane with a pore size of 1 micron, and the filtrate is ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 2000Da. The ultrafiltrate is distilled under reduced pressure at 52°C to remove ethanol, and freeze-dried at -35°C to a water content of 3.32wt%, to obtain a composition containing Phellodendron amurense and Atractylodes lancea.

[0063] Composition C:

[0064] Compared with composition A, the only difference is that: step 3: sterilize the enzymatic material, add glucose, dipotassium hydrogen phosphate and fermentation bacteria, perform anaerobic fermentation at 35°C for 127 hours, sterilize, and obtain a fermentation material; wherein, the amount of glucose and dipotassium hydrogen phosphate added is 3.2% and 0.085% of the mass of the enzymatic material; the fermentation bacteria are composed of Lactobacillus reuteri CICC6119 and Bacillus coagulans CICC23843 in a quantitative ratio of 1:1; the amount of fermentation bacteria is 6.80×10 9 cfu / g enzymatic material.

[0065] Composition D:

[0066] Compared with composition A, the only difference is that: step 3: sterilize the enzymatic material, add glucose, dipotassium hydrogen phosphate and fermentation bacteria, perform anaerobic fermentation at 35°C for 127 hours, sterilize, and obtain a fermentation material; wherein, the amount of glucose and dipotassium hydrogen phosphate added is 3.2% and 0.085% of the mass of the enzymatic material; the fermentation bacteria are composed of Lactobacillus reuteri CICC6119 and Bacillus coagulans CICC23843 in a quantity ratio of 1:8; the amount of fermentation bacteria is 6.80×10 9 cfu / g enzymatic material.

[0067] Composition E:

[0068] Compared with composition A, the only difference is: step 2: adding enzyme to the decoction liquid, enzymolysis at 55°C for 2.5 hours, inactivating the enzyme, and obtaining an enzymolysis material; wherein the enzyme is Serratia protease; and the amount of Serratia protease used is 1000U / g decoction liquid.

[0069] Composition F:

[0070] Compared with composition A, the only difference is: step 2: adding enzyme to the decoction liquid, enzymolysis at 55°C for 2.5 hours, inactivating the enzyme, and obtaining an enzymolysis material; wherein the enzyme is bromelain; and the amount of bromelain used is 1000U / g decoction liquid.

[0071] Composition G:

[0072] Compared with composition A, the only difference is that: step 3: sterilize the enzymatic material, add glucose, dipotassium hydrogen phosphate and fermentation bacteria, perform anaerobic fermentation at 35°C for 127 hours, sterilize, and obtain a fermentation material; wherein, the amount of glucose and dipotassium hydrogen phosphate added is 3.2% and 0.085% of the mass of the enzymatic material; the fermentation bacteria is Lactobacillus reuteri CICC6119; the amount of fermentation bacteria is 6.80×10 9 cfu / g enzymatic material.

[0073] Composition H:

[0074] Compared with composition A, the only difference is that: step 3: sterilize the enzymatic material, add glucose, dipotassium hydrogen phosphate and fermentation bacteria, anaerobically ferment at 35°C for 127 hours, sterilize, and obtain the fermentation material; wherein, the amount of glucose and dipotassium hydrogen phosphate added is 3.2% and 0.085% of the mass of the enzymatic material; the fermentation bacteria is Bacillus coagulans CICC23843; the amount of fermentation bacteria is 6.80×10 9 cfu / g enzymatic material.

[0075] 2. Composition effect test.

[0076] 1. Antibacterial activity

[0077] According to the M27-A3 protocol developed by CLSI, the antibacterial activity of the composition was detected, that is, the MIC was determined by the microdilution method, which is briefly described as follows:

[0078] The composition was diluted with SDB medium, and the composition liquid was diluted in the 1st to 10th columns of a 96-well plate by the microdilution method to obtain 10 columns of concentration gradient wells containing 100 μL of the composition liquid, and 100 μL of blank culture medium was added to the wells in the 11th and 12th columns.

[0079] The suspension of Candida albicans was diluted to 5×10 3 CFU / mL of inoculated bacterial solution, and then add 100 μL of inoculated bacterial solution to the 1st to 11th columns of the 96-well plate containing 100 μL of the composition liquid, and add 100 μL of blank culture medium to the 12th column. The final volume of each well of the well plate is 200 μL; 1-10 columns are the drug administration group, the 11th column is the growth control group, and the 12th well is the blank control group. Place the 96 microplate in a constant temperature incubator and culture at 35°C for 24-48 hours.

[0080] Evaluation of experimental results: Observe with the naked eye, the corresponding concentration of the well where no bacteria grow is the MIC. The test results are shown in Table 1.

[0081] Table 1 Antibacterial activity of the composition (n=6)

[0082] Group MIC Phenoxyethanol 5.12mg / mL Composition A 6.25mg / mL Composition B 7.83mg / mL Composition C 14.59mg / mL Composition D 18.43mg / mL Composition E 25.96mg / mL Composition F 30.14mg / mL Composition G 48.57mg / mL Composition H 52.01mg / mL

[0083] The lower the MIC, the stronger the antibacterial activity. The experimental results show that the MICs of compositions AH against Candida albicans are all low, indicating that compositions AH have antibacterial activity against Candida albicans, among which composition A has the best antibacterial effect, followed by composition B.

[0084] Compared with the positive drug phenoxyethanol, the MIC of compositions A and B against Candida albicans is closer to that of phenoxyethanol, indicating that the MIC of compositions A and B against Candida albicans is closest to the MIC of phenoxyethanol against Candida albicans.

[0085] 2. Inhibitory effect on bacterial metabolism in biofilm during adhesion period

[0086] Take 200 μL of 5×10 4 CFU / mL of bacterial suspension was inoculated into a flat-bottom 96-well plate, cultured at 37°C for 6h, and rinsed with sterile PBS to remove floating bacteria. 200μL of composition AH and positive control phenoxyethanol were added respectively, and the drug concentration was MIC. No active drug was added to the blank group; then the culture was continued for 24h, the well liquid was discarded, and the wells were washed 3 times with sterile PBS. Follow the instructions of the CCK-8 kit: under light-proof conditions, add 100μL of the corresponding culture medium and 10μL CCK-8 solution to each well, mix well, and return to the incubator for incubation in the dark for 1h. The OD450 value was measured by an enzyme reader. The test results are shown in Table 2. Among them, the degree of inhibition enhancement compared with the blank group = 100% × (blank group OD 450 - OD of each group 450 ) / blank group OD 450 .

[0087] Table 2 Compositions inhibit metabolic OD of biofilm bacteria during the adhesion period 450

[0088]

[0089] The experimental results showed that the combination AH could reduce the metabolic activity of biofilm bacteria of Candida albicans in the adhesion stage to varying degrees.

[0090] Compared with the blank group, compositions AH enhanced the metabolic activity of Candida albicans biofilm bacteria in the adhesion period. Among them, compositions A and B enhanced the metabolic activity of biofilm bacteria in the adhesion period most significantly compared with the blank group.

[0091] Compared with the positive group phenoxyethanol, there was no significant difference in the inhibition of metabolic activity of Candida albicans biofilm bacteria during the adhesion period between compositions A and B, and the inhibitory effects of both were comparable to those of the positive group phenoxyethanol.

[0092] 3. Inhibitory effect on bacterial metabolism in biofilm during aggregation period

[0093] Take 200 μL of 5×10 4 The bacterial suspension of CFU / mL was inoculated into a flat-bottom 96-well plate, cultured at 37°C for 24 hours, and rinsed with sterile PBS to remove the floating bacteria. 200 μL of the composition AH and the positive control phenoxyethanol were added respectively, and the drug concentration was MIC. No active drug was added to the blank group; then the culture was continued for 24 hours, the well solution was discarded, and washed 3 times with sterile PBS. Follow the instructions of the CCK-8 kit: add 100 μL of the corresponding culture medium and 10 μL of CCK-8 solution to each well under light-proof conditions, mix well, and return to the incubator to incubate in the dark for 1 hour. Measure OD with an enzyme reader 450 The test results are shown in Table 3. Wherein, the degree of inhibition enhancement compared with the blank group = 100% × (blank group OD 450 - OD of each group 450 ) / blank group OD 450 .

[0094] Table 3 Compositions inhibit the metabolism of biofilm bacteria in the aggregation period OD 450

[0095]

[0096]

[0097] The experimental results show that the combination AH can reduce the metabolic activity of Candida albicans biofilm bacteria in the aggregation stage to varying degrees.

[0098] Compared with the blank group, compositions AH enhanced the metabolic activity of Candida albicans biofilm bacteria in the aggregation period. Among them, compositions A and B enhanced the metabolic activity of Candida albicans biofilm bacteria in the aggregation period most significantly compared with the blank group.

[0099] 4. Inhibitory effect on bacterial metabolism in mature biofilm

[0100] Take 200 μL of 5×10 4The bacterial suspension of CFU / mL was inoculated into a flat-bottom 96-well plate, cultured at 37°C for 48h (the culture medium was replaced every 24 hours), and washed with sterile PBS to remove the floating bacteria. 200μL of the composition AH and the positive control phenoxyethanol were added respectively, and the drug concentration was MIC. No active drug was added to the blank group; then the culture was continued for 24h, the well liquid was discarded, and the wells were washed 3 times with sterile PBS. Follow the instructions of the CCK-8 kit: add 100μL of the corresponding culture medium and 10μL CCK-8 solution to each well under light-proof conditions, mix well, and return to the incubator for incubation in the dark for 1h. Measure OD with an enzyme reader 450 The test results are shown in Table 4. Wherein, the degree of inhibition enhancement compared with the blank group = 100% × (blank group OD 450 - OD of each group 450 ) / blank group OD 450 .

[0101] Table 4 Compositions inhibit the metabolism of mature biofilm bacteria OD 450

[0102]

[0103] The experimental results show that the combination AH can reduce the metabolic activity of mature biofilm bacteria of Candida albicans to varying degrees.

[0104] Compared with the blank group, compositions AH enhanced the metabolic activity of Candida albicans biofilm bacteria in the mature stage. Among them, compositions A and B enhanced the metabolic activity of Candida albicans biofilm bacteria in the mature stage most significantly compared with the blank group.

[0105] 5. Inhibitory effect on biofilm during adhesion period

[0106] Take 200 μL of 5×10 4 CFU / mL bacterial suspension was inoculated into a flat-bottom 96-well plate, incubated at 37°C for 6 hours, and rinsed with sterile PBS to remove floating bacteria. 200 μL of composition AH and positive control phenoxyethanol were added respectively, and the drug concentration was MIC. No active drug was added to the blank group; then the culture was continued for 24 hours, the well solution was discarded, and washed 3 times with sterile PBS. Add 200 μL of methanol to fix for 30 minutes; add 200 μL of 0.1% crystal violet staining solution to stain for 15 minutes; wash away the unadhered staining solution with PBS; add 200 μL of 95% ethanol and let stand for 10 minutes to fully dissolve the staining solution; measure OD with an enzyme reader 570 The test results are shown in Table 5. Wherein, the degree of inhibition enhancement compared with the blank group = 100% × (blank group OD 570 - OD of each group 570 ) / blank group OD 570 .

[0107] Table 5 Compositions inhibit OD of biofilm during adhesion period 570

[0108]

[0109] The experimental results showed that the composition AH could reduce the biofilm of Candida albicans in the adhesion stage to varying degrees.

[0110] Compared with the blank group, the inhibitory effects of compositions A and H on biofilms of Candida albicans during the adhesion period were enhanced. Among them, the inhibitory effects of compositions A and B on biofilms during the adhesion period were most significantly enhanced compared with the blank group.

[0111] 6. Inhibition of biofilm during the aggregation period

[0112] Take 200 μL of 5×10 4 CFU / mL bacterial suspension was inoculated into a flat-bottom 96-well plate, incubated at 37°C for 24 hours, and rinsed with sterile PBS to remove floating bacteria. 200 μL of composition AH and positive control phenoxyethanol were added respectively, and the drug concentration was MIC. No active drug was added to the blank group; then the culture was continued for 24 hours, the well liquid was discarded, and washed 3 times with sterile PBS. Add 200 μL of methanol to fix for 30 minutes; add 200 μL of 0.1% crystal violet staining solution to stain for 15 minutes; wash away the unadhered stain with PBS; add 200 μL of 95% ethanol and let stand for 10 minutes to fully dissolve the stain; measure OD with an enzyme reader 570 The test results are shown in Table 6. Wherein, the degree of inhibition enhancement compared with the blank group = 100% × (blank group OD 570 - OD of each group 570 ) / blank group OD 570 .

[0113] Table 6 Compositions inhibiting biofilm OD during aggregation period 570

[0114]

[0115]

[0116] The experimental results show that the composition AH can reduce the biofilm of Candida albicans aggregation stage to varying degrees.

[0117] Compared with the blank group, the effects of compositions A and H on inhibiting biofilm in the aggregation stage of Candida albicans were enhanced. Among them, the effects of compositions A and B on inhibiting biofilm in the aggregation stage were most significantly enhanced compared with the blank group.

[0118] 7. Inhibition of mature biofilm

[0119] Take 200 μL of 5×10 4The bacterial suspension of CFU / mL was inoculated into a flat-bottom 96-well plate, cultured at 37°C for 48 hours (the culture medium was replaced every 24 hours), and washed with sterile PBS to remove the floating bacteria. 200 μL of the composition AH and the positive control phenoxyethanol were added respectively, and the drug concentration was MIC. No active drug was added to the blank group; then the culture was continued for 24 hours, the well liquid was discarded, and washed 3 times with sterile PBS. Add 200 μL of methanol to fix for 30 minutes; add 200 μL of 0.1% crystal violet stain for 15 minutes; wash away the non-adherent stain with PBS; add 200 μL of 95% ethanol and let stand for 10 minutes to fully dissolve the stain; measure OD with an enzyme reader 570 The test results are shown in Table 6. Among them, the degree of inhibition enhancement compared with the blank group = 100% × (blank group OD 570 - OD of each group 570 ) / blank group OD 570 .

[0120] Table 7 Compositions inhibiting mature biofilm OD 570

[0121]

[0122]

[0123] The experimental results showed that the combination AH could reduce the mature biofilm of Candida albicans to varying degrees.

[0124] Compared with the blank group, the inhibitory effects of compositions A and H on the mature biofilm of Candida albicans were enhanced. Among them, the inhibitory effects of compositions A and B on the mature biofilm were most significantly enhanced compared with the blank group.

[0125] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for preparing a composition containing Phellodendron chinense and Atractylodes lancea, characterized in that: The steps include: Step 1: Take dried Phellodendron chinense and Atractylodes lancea, crush them to obtain a powder mixture, add water to the powder mixture, boil it, keep it warm, and then cool it to obtain a decoction liquid; Step 2: adding enzyme to the decoction liquid, performing enzymolysis, and inactivating the enzyme to obtain an enzymolysis material; Step 3: sterilize the enzymatic hydrolysis material, add glucose, dipotassium hydrogen phosphate and fermentation bacteria, ferment, sterilize, and obtain the fermentation material; Step 4: adding ethanol to the fermented material, adjusting the ethanol concentration, heating and stirring to obtain an extract material; Step 5: Filter the extracted material, ultrafilter, concentrate, and dry to obtain a composition containing Phellodendron chinense and Atractylodes lancea.

2. The preparation method according to claim 1, characterized in that: The mass ratio of Phellodendron chinense to Atractylodes lancea is 1:2-6.

3. The preparation method according to claim 1, characterized in that: The enzyme is composed of serratia protease and bromelain, and the dosage of serratia protease and bromelain is 400-800U / g decoction liquid and 200-500U / g decoction liquid respectively; And / or, the fermentation bacteria are composed of Lactobacillus reuteri CICC6119 and Bacillus coagulans CICC23843 in a quantitative ratio of 1:2-4, and the amount of the fermentation bacteria is 1×10 9 -9×10 9 cfu / g enzymatic material; And / or, the added amounts of glucose and dipotassium hydrogen phosphate are 1.2-4% and 0.02-0.15% of the mass of the enzymatically hydrolyzed material.

4. The preparation method according to claim 1, characterized in that: In step 1, the mass ratio of the powder mixture to water is 1:10-50; And / or, the insulation temperature is 72-85° C. and the time is 1-6 hours.

5. The preparation method according to claim 1, characterized in that: In step 2, the enzymatic hydrolysis temperature is 40-65°C and the time is 1-5 hours; and / or, in step 3, the fermentation temperature is 25-40° C. and the fermentation time is 100-150 hours; And / or, in step 4, the ethanol mass concentration is adjusted to 20-40%, the heating temperature is 50-60° C., and the heating time is 2-6 hours.

6. The preparation method according to claim 1, characterized in that: In step 5, the filtration is performed using a filter membrane with a pore size of not less than 0.1 micron.

7. The preparation method according to claim 1, characterized in that: In step 5, ultrafiltration is performed using a lung ultrafiltration membrane with a molecular weight cutoff lower than 3 KDa.

8. A composition containing Phellodendron chinense and Atractylodes lancea prepared by the preparation method according to any one of claims 1 to 7.

9. A product comprising the composition of claim 8, characterized in that The mass percentage content of the composition in the product is 0.1-30%, and the remaining auxiliary materials.

10. A method for preparing the product according to claim 9, characterized in that: The method comprises the step of mixing the composition with auxiliary materials.

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