A composition containing phellodendri cortex and atractylodis rhizoma, and uses thereof, and products containing the composition and methods of making

Abstract

The present application relates to the technical field of medicine, in particular to a composition containing phellodendri chinensis and atractylodis and application and products containing the composition and preparation method. The preparation method comprises the following steps: taking dry phellodendri chinensis and atractylodis, crushing to obtain a powder mixture, adding water to the powder mixture, boiling and then keeping warm, and then cooling to obtain decoction material liquid; adding enzymes to the decoction material liquid, enzyme hydrolysis, enzyme inactivation, to obtain enzyme hydrolysate; sterilizing the enzyme hydrolysate, adding glucose, dipotassium hydrogen phosphate and fermentation bacteria, fermenting, sterilizing to obtain fermentation material; adding ethanol to the fermentation material, adjusting the ethanol concentration, heating and stirring to obtain extract material; filtering, ultrafiltering, concentrating and drying the extract material to obtain the composition containing phellodendri chinensis and atractylodis. The composition prepared by the present application has the effect of inhibiting Candida albicans biofilm with high efficiency, low toxicity, safety and effectiveness.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of pharmaceutical technology, in particular to a composition containing phellodendri chinensis and atractylodis and application and products containing the composition and preparation method. BACKGROUND

[0002] Vulvovaginal candidiasis, also known as vulvovaginal candidiasis (VVC), is the second most common vaginal infectious disease after bacterial vaginitis, mainly caused by opportunistic mucosal infection of Candida albicans, mainly manifested as tofu slag-like leucorrhea and vulva itching, and is one of the most common diseases of female vaginal infection, and is one of the risk factors for a series of high-risk diseases such as AIDS, cervicitis and abortion. Candida albicans is a conditional pathogenic fungus, which does not cause infection under normal circumstances, but when the body's immunity is low or the balance of normal flora is broken, Candida albicans turns into pathogenic bacteria, grows and reproduces in large quantities locally, and causes local mucosa and even systemic diseases.

[0003] The pathogenicity of Candida albicans mainly manifests as the generation of biofilm, the secretion of hydrolase, the secretion of adhesin, and the secretion of candidin. Among them, the generation of biofilm can protect the bacteria from the influence of the host immune response, help the bacteria escape the killing effect of antibacterial drugs and resist fungal drugs to produce drug resistance, causing chronic and difficult-to-cure infections. At the same time, the white Candida albicans gathered in the biofilm forms a quorum sensing system, which further regulates the generation cycle of the white Candida albicans biofilm and regulates the secretion of virulence factors.

[0004] At present, the drug library resources for inhibiting Candida albicans are limited, and azole drugs are the first choice for clinical treatment and prevention of Candida infection. The large-scale application of azole drugs not only treats Candida infection, but also increases the drug resistance of Candida albicans. In addition, the drugs for clinical treatment of Candida infection may cause irreversible harm to the human body due to their toxic side effects. In order to solve this series of problems, it is urgent to find new bacteriostatic agents for inhibiting the biofilm of Candida albicans from natural plant medicines with high efficiency, low toxicity and safety. SUMMARY

[0005] The present application is directed to the above technical problems, and the technical scheme of the present application is provided, which includes a composition containing phellodendri chinensis and atractylodis and application and products containing the composition and preparation method.

[0006] In one aspect, the present application provides a preparation method of a composition containing phellodendri chinensis and atractylodis, comprising the following steps:

[0007] Step 1: Take dry phellodendri chinensis and atractylodis, crush to obtain a powder mixture, add water to the powder mixture, boil and then keep warm, and then cool to obtain a decoction liquid;

[0008] Step 2: adding enzymes to the decoction, enzymolysis, enzyme inactivation, to obtain the enzymolysis material;

[0009] Step 3: sterilizing the enzymolysis material, adding glucose, dipotassium hydrogen phosphate and fermentation bacteria, fermentation, sterilization, to obtain the fermentation material;

[0010] Step 4: adding ethanol to the fermentation material, adjusting the ethanol concentration, heating and stirring, to obtain the extraction material;

[0011] Step 5: filtering, ultrafiltrating, concentrating and drying the extraction material, to obtain the composition containing Cortex Phellodendri and Atractylodes lancea.

[0012] Preferably, in step 1, the mass ratio of Cortex Phellodendri and Atractylodes lancea is 1:2-6.

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

[0014] Preferably, in step 1, the temperature of the incubation is 72-85℃, and the time is 1-6 hours.

[0015] Preferably, in step 2, the enzymes consist of Serratiopeptidase and Bromelain.

[0016] Preferably, in step 2, the dosages of Serratiopeptidase and Bromelain are 400-800 U / g decoction and 200-500 U / g decoction, respectively.

[0017] Preferably, in step 2, the enzymolysis temperature is 40-65℃, and the time is 1-5 hours.

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

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

[0020] Preferably, in step 3, the dosages of the fermentation bacteria are 1×10 9 -9×10 9 cfu / g enzymolysis material.

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

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

[0023] Preferably, in step 4, the heating temperature is 50-60℃, 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 no less than 0.1 microns.

[0025] Preferably, in step 5, the ultrafiltration is performed using a lung ultrafiltration membrane with a molecular weight cut-off of less 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 to a water content of less than 6%.

[0028] The application also provides a composition containing cortex phellodendri and atractylodes rhizome prepared according to the preparation method.

[0029] The application also provides an article containing the composition, wherein the mass percentage of the composition in the article is 0.1-30%, and the remaining part is excipients.

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

[0031] The application also provides a preparation method of the article, comprising the step of mixing the composition with excipients.

[0032] The application has the following advantages:

[0033] The application selects dry cortex phellodendri and atractylodes rhizome, grinds to obtain a powder mixture, adds water to the powder mixture, boils and keeps warm, then cools to obtain a decoction; adds enzymes to the decoction, performs enzymatic hydrolysis, inactivates the enzymes, and obtains an enzymatic hydrolysis product; sterilizes the enzymatic hydrolysis product, adds glucose, dipotassium hydrogen phosphate, and fermentation bacteria, performs fermentation, sterilizes, and obtains a fermentation product; adds ethanol to the fermentation product, adjusts the ethanol concentration, and heats and stirs to obtain an extract; filters, ultrafilters, concentrates, and dries the extract to obtain a composition containing cortex phellodendri and atractylodes rhizome. The composition prepared by the application has the effect of inhibiting Candida albicans biofilm with high efficiency, low toxicity, and safety.

[0034] The composition prepared by the application has a low MIC for Candida albicans, and has inhibitory activity on Candida albicans; the MIC of the composition for Candida albicans is close to the MIC of phenoxyethanol for Candida albicans.

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

[0036] The composition prepared by the application can reduce the metabolic activity of Candida albicans biofilm in the aggregation stage to different degrees, and can reduce the metabolic activity of Candida albicans biofilm in the mature stage to different degrees.

[0037] The composition of the present application can reduce the adhesion period of Candida albicans biofilm, reduce the aggregation period of Candida albicans biofilm and reduce the mature period of Candida albicans biofilm to different degrees.

[0038] In the preparation of the present application, decoction is used to better promote the extraction of active ingredients. The combination of serralysin and bromelain enzyme hydrolysis promotes each other to improve the types and concentration of active ingredients of the drug, and increases the drug efficacy. The combination of Lactobacillus reuteri CICC 6119 and Bacillus coagulans CICC 23843 fermentation enhances the drug property of the fermentation product, gives the composition more types and concentrations of active ingredients, and enhances the inhibitory effect of the concentrated and dried composition on Candida albicans. At the same time, when the ratio of Lactobacillus reuteri CICC 6119 and Bacillus coagulans CICC 23843 in the fermentation bacteria is 1:2-4, the composition obtained has better effect. In order to further improve the drug property of the composition, the present application uses ethanol aqueous solution for leaching and ultrafiltration for enrichment and impurity removal after fermentation, improves the drug activity of the composition, and gives the composition better drug efficacy.

[0039] The preparation method of the composition of the present application is simple, green and environmentally friendly, the obtained composition has excellent effect and is safe, and can effectively retain the active ingredients of Phellodendri Chinensis Cortex and Atractylodis Rhizoma. DETAILED DESCRIPTION

[0040] The present application will be described below in conjunction with specific embodiments, and various effects of the present application will be more clearly presented. Those skilled in the art should understand that these specific embodiments are used to illustrate the present application, not to limit the present application.

[0041] Phellodendri Chinensis Cortex was purchased from Kangmei Pharmaceutical Co., Ltd., and the medicinal material batch number was 230501491. Atractylodis Rhizoma was purchased from Beijing Kangmei Pharmaceutical Co., Ltd., and the medicinal material batch number was 220961031. Serralysin and bromelain were purchased from Hubei Ruibosin Chemical Co., Ltd. and Sigma, respectively. Lactobacillus reuteri CICC 6119 and Bacillus coagulans CICC 23843 were purchased from China Industrial Microbial Culture Collection Center.

[0042] I. Preparation of the composition:

[0043] Composition A:

[0044] The preparation method is as follows: step 1: dry Phellodendri Chinensis Cortex and Atractylodis Rhizoma are crushed to a particle size of less than 0.5 mm at a mass ratio of 1:4 to obtain a powder mixture, 25.5 times as much water as the mass of the powder mixture is added, boiled, then 78℃ is kept and stirred at 60rpm for 3.2 hours, then cooled to room temperature to obtain a decoction liquid;

[0045] Step 2: enzymes are added to the decoction liquid, and enzyme hydrolysis is carried out at 55℃ for 2.5 hours, and the enzyme is inactivated to obtain an enzyme hydrolysate;

[0046] wherein the enzyme consists of serralysin and bromelain; the serralysin and the bromelain are used in an amount of 600 U / g of the decoction material liquid and 400 U / g of the decoction material liquid, respectively;

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

[0048] wherein the glucose and the dipotassium hydrogen phosphate are added in an amount of 3.2% and 0.085% of the mass of the enzymatic hydrolysate;

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

[0050] The fermentation bacteria are used in an amount of 6.80 x 10 9 cfu / g of the enzymatic hydrolysate;

[0051] Step 4: add ethanol to the fermentation material, adjust the mass concentration of ethanol to 32%, heat at 55°C and stir at 70 rpm for 4 hours, and obtain an extraction material;

[0052] Step 5: filter the extraction material using a filter membrane with a pore size of 1 micrometer, perform ultrafiltration on the filtrate using an ultrafiltration membrane with a molecular weight cut-off of 2000 Da, remove ethanol from the ultrafiltrate by distillation under reduced pressure at 54°C, freeze-dry the ultrafiltrate at -40°C to a water content of 3.28 wt%, and obtain a composition containing Phellodendri Chinensis Cortex and Atractylodis Rhizoma.

[0053] Composition B:

[0054] The preparation method is as follows: Step 1: dry Phellodendri Chinensis Cortex and Atractylodis Rhizoma, crush to a particle size of less than 0.5 mm in a mass ratio of 1:2, obtain a powder mixture, add water in an amount of 27 times the mass of the powder mixture, boil, and then heat at 75°C and stir at 60 rpm for 3 hours, and then cool to room temperature, and obtain a decoction material liquid;

[0055] Step 2: add enzyme to the decoction material liquid, perform enzymatic hydrolysis at 45°C for 2 hours, and inactivate the enzyme, and obtain an enzymatic hydrolysate;

[0056] wherein the enzyme consists of serralysin and bromelain; the serralysin and the bromelain are used in an amount of 500 U / g of the decoction material liquid and 500 U / g of the decoction material liquid, respectively;

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

[0058] wherein the glucose and the dipotassium hydrogen phosphate are added in an amount of 3.5% and 0.087% of the mass of the enzymatic hydrolysate;

[0059] The fermenting bacteria are composed of Lactobacillus reuteri CICC6119 and Bacillus coagulans CICC23843 in a quantity ratio of 1:2.

[0060] The fermenting bacteria are used in an amount of 6.33 x 10 9 cfu / g of the enzymolysis material.

[0061] Step 4: The fermentation material is added with ethanol to adjust the mass concentration of ethanol to 25%, heated at 57°C and stirred 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 micrometer, the filtrate is subjected to ultrafiltration using an ultrafiltration membrane with a molecular weight cut-off of 2000 Da, the ultrafiltrate is subjected to distillation under reduced pressure to remove ethanol at 52°C, and freeze-dried at -35°C to a water content of 3.32 wt% to obtain a composition containing Phellodendri chinensis and Atractylodis rhizome.

[0063] Composition C:

[0064] Compared with Composition A, the only difference is that: Step 3: the enzymolysis material is sterilized, glucose, dipotassium hydrogen phosphate and fermenting bacteria are added, anaerobic fermentation is carried out at 35°C for 127 hours, sterilization is performed to obtain a fermentation material; wherein, the added amounts of glucose and dipotassium hydrogen phosphate are 3.2% and 0.085% of the mass of the enzymolysis material; the fermenting bacteria are composed of Lactobacillus reuteri CICC6119 and Bacillus coagulans CICC23843 in a quantity ratio of 1:1; the fermenting bacteria are used in an amount of 6.80 x 10 9 cfu / g of the enzymolysis material.

[0065] Composition D:

[0066] Compared with Composition A, the only difference is that: Step 3: the enzymolysis material is sterilized, glucose, dipotassium hydrogen phosphate and fermenting bacteria are added, anaerobic fermentation is carried out at 35°C for 127 hours, sterilization is performed to obtain a fermentation material; wherein, the added amounts of glucose and dipotassium hydrogen phosphate are 3.2% and 0.085% of the mass of the enzymolysis material; the fermenting bacteria are composed of Lactobacillus reuteri CICC6119 and Bacillus coagulans CICC23843 in a quantity ratio of 1:8; the fermenting bacteria are used in an amount of 6.80 x 10 9 cfu / g of the enzymolysis material.

[0067] Composition E:

[0068] Compared with Composition A, the only difference is that: Step 2: enzymes are added to the decoction liquid, enzymolysis is carried out at 55°C for 2.5 hours, and the enzymes are inactivated to obtain an enzymolysis material; wherein, the enzymes are protease Serratia; the protease Serratia is used in an amount of 1000 U / g of the decoction liquid.

[0069] Composition F:

[0070] The difference between the composition A is only that: step 2: add enzyme in decoction, enzyme hydrolysis for 2.5 hours at 55℃, enzyme inactivation, to obtain enzyme hydrolysate; wherein, the enzyme is bromelain; the dosage of bromelain is 1000 U / g decoction.

[0071] Composition G:

[0072] The difference between the composition A is only that: step 3: sterilize enzyme hydrolysate, add glucose, dipotassium hydrogen phosphate and fermentation bacteria, anaerobic fermentation for 127 hours at 35℃, sterilization, to obtain fermentation material; wherein, the addition amount of glucose and dipotassium hydrogen phosphate is 3.2% and 0.085% of the mass of enzyme hydrolysate; the fermentation bacteria is Lactobacillus reuteri CICC6119; the dosage of fermentation bacteria is 6.80 x 10 9 cfu / g enzyme hydrolysate.

[0073] Composition H:

[0074] The difference between the composition A is only that: step 3: sterilize enzyme hydrolysate, add glucose, dipotassium hydrogen phosphate and fermentation bacteria, anaerobic fermentation for 127 hours at 35℃, sterilization, to obtain fermentation material; wherein, the addition amount of glucose and dipotassium hydrogen phosphate is 3.2% and 0.085% of the mass of enzyme hydrolysate; the fermentation bacteria is Bacillus coagulans CICC23843; the dosage of fermentation bacteria is 6.80 x 10 9 cfu / g enzyme hydrolysate.

[0075] II. Effect test of composition

[0076] 1. Bacteriostatic activity

[0077] According to the M27-A3 scheme formulated by CLSI, the bacteriostatic activity of the composition is detected, that is, the MIC determination is carried out by microdilution method, which is briefly described as follows:

[0078] The composition is diluted with SDB medium, and the liquid of the composition is diluted in the first to tenth columns of the 96-well plate by microdilution method, to obtain 10 columns containing 100 μL of the liquid of the composition with concentration gradient, and 100 μL of blank medium is added to the 11th and 12th columns of the hole.

[0079] The white Candida suspension is diluted to 5 x 10 3 CFU / mL of inoculum liquid, and 100 μL of inoculum liquid is added to the first to eleventh columns of the 96-well plate containing 100 μL of the liquid of the composition, and 100 μL of blank medium is added to the 12th column, and the final volume of each well of the plate is 200 μL; wherein 1-10 columns are the administration group, the 11th column is the growth control group, and the 12th hole is the blank control group. Place the 96-well plate in a constant temperature incubator and incubate at 35℃ for 24-48h

[0080] The experimental results are evaluated by naked eye observation. The MIC corresponding to the well without fungus growth is just the right concentration. The test results are shown in Table 1.

[0081] Table 1 Antifungal activity of the compositions (n = 6)

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

[0083] The lower the MIC, the stronger the antifungal activity. The experimental results show that the MICs of compositions A-H to Candida albicans are relatively low, indicating that compositions A-H have antifungal activity to Candida albicans. Among them, the antifungal effect of composition A is the best, and that of composition B is the second.

[0084] Compared with the positive drug phenoxyethanol, the MICs of compositions A and B to Candida albicans are relatively close to that of phenoxyethanol. This indicates that the MICs of compositions A and B to Candida albicans are closest to the MIC of phenoxyethanol to Candida albicans.

[0085] 2. Inhibition of metabolism of bacteria in adherent biofilm

[0086] 200 μL of bacterial suspension with a concentration of 5 x 10 4 CFU / mL was inoculated in a flat-bottom 96-well plate and cultured at 37°C for 6 h. The planktonic bacteria were removed by washing with sterile PBS. 200 μL of compositions A-H and the positive control phenoxyethanol were added, respectively, and the drug concentration was MIC. The blank group was not added with active drug. Then, the culture was continued for 24 h, the well liquid was discarded, and the plate was washed with sterile PBS for 3 times. The operation was carried out according to the CCK-8 kit instructions: 100 μL of corresponding culture medium and 10 μL of CCK-8 solution were added to each well under light-proof condition, and then mixed and placed back in the incubator for 1 h of light-proof incubation. The OD450 value was measured by an enzyme marker instrument. The test results are shown in Table 2. Among them, the enhanced degree of inhibition compared with the blank group = 100% x (blank group OD 450 - group OD 450 / blank group OD 450 .

[0087] Table 2 OD of inhibition of metabolism of bacteria in adherent biofilm by the compositions 450

[0088]

[0089] The experimental results show that compositions A-H can reduce the metabolic activity of Candida albicans in adherent biofilm to different degrees.

[0090] Compared with the blank group, compositions A-H can enhance the inhibition of metabolic activity of Candida albicans in adherent biofilm. Among them, compositions A and B can most obviously enhance the inhibition of metabolic activity of Candida albicans in adherent biofilm compared with the blank group.

[0091] Compared with the positive group phenoxyethanol, compositions A and B showed no significant difference in inhibiting the metabolic activity of Candida albicans biofilm during the adhesion phase, and their inhibitory effects were comparable to those of the positive group phenoxyethanol.

[0092] 3. Inhibitory effect on the metabolism of bacteria within the biofilm during the aggregation phase.

[0093] Take 200 μL of a concentration of 5 × 10 4 CFU / mL bacterial suspension was inoculated into 96-well plates with a flat bottom and incubated at 37°C for 24 h. Airborne bacteria were removed by washing with sterile PBS. 200 μL each of the AH composition and the positive control phenoxyethanol were added to each well (MIC concentration). No active drug was added to the blank control. The plates were then incubated for another 24 h, the solution was discarded, and the plates were washed three times with sterile PBS. The CCK-8 kit was then administered according to the instructions: 100 μL of the corresponding culture medium and 10 μL of CCK-8 solution were added to each well under light-protected conditions, mixed thoroughly, and incubated for 1 h in the dark. OD was measured using a microplate reader. 450 Values. Test results are shown in Table 3. The degree of enhancement of inhibitory effect compared to the blank group = 100% × (OD value of blank group). 450 - OD of each group 450 ) / Blank group OD 450 .

[0094] Table 3. Compositions inhibit metabolic OD of biofilm bacteria during aggregation phase. 450

[0095]

[0096]

[0097] Experimental results show that the composition AH can reduce the metabolic activity of Candida albicans biofilm bacteria during the aggregation phase to varying degrees.

[0098] Compared with the control group, composition AH enhanced the inhibitory activity of Candida albicans biofilm bacteria during the aggregation phase. Among them, compositions A and B showed the most significant enhancement in inhibiting the metabolic activity of biofilm bacteria during the aggregation phase compared with the control group.

[0099] 4. Inhibitory effect on the metabolism of mature biofilm bacteria

[0100] Take 200 μL of a concentration of 5 × 10 4CFU / mL bacterial suspension was inoculated into 96-well plates with a flat bottom and incubated at 37°C for 48 h (the medium was changed every 24 hours). Airborne bacteria were removed by washing with sterile PBS. 200 μL each of the AH composition and the positive control phenoxyethanol were added to each well at the MIC concentration; no active drug was added to the blank control. The plates were then incubated for another 24 h, the solution was discarded, and the plates were washed three times with sterile PBS. The CCK-8 kit was then administered according to the instructions: 100 μL of the corresponding medium and 10 μL of CCK-8 solution were added to each well under light-protected conditions, mixed well, and incubated for 1 h in the dark. OD was measured using a microplate reader. 450 Values. Test results are shown in Table 4. The degree of enhancement of inhibitory effect compared to the blank group = 100% × (OD value of blank group). 450 - OD of each group 450 ) / Blank group OD 450 .

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

[0102]

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

[0104] Compared with the control group, composition AH enhanced the inhibitory activity of mature Candida albicans biofilm bacteria on metabolic activity. Among them, compositions A and B showed the most significant enhancement in inhibiting the metabolic activity of mature biofilm bacteria compared with the control group.

[0105] 5. Inhibitory effect on biofilm during the adhesion phase

[0106] Take 200 μL of a concentration of 5 × 10 4 CFU / mL bacterial suspension was inoculated into 96-well plates and incubated at 37°C for 6 h. Airborne bacteria were removed by washing with sterile PBS. 200 μL each of the AH composition and the positive control phenoxyethanol were added at the MIC concentration; no active drug was added to the blank control. The plates were then incubated for another 24 h, the wells were discarded, and the plates were washed three times with sterile PBS. 200 μL of methanol was added for fixation for 30 min; 200 μL of 0.1% crystal violet solution was added for staining for 15 min; unadhered stain was washed away with PBS; 200 μL of 95% ethanol was added and allowed to stand for 10 min to fully dissolve the stain; OD was measured using a microplate reader. 570 Values. Test results are shown in Table 5. The degree of inhibitory enhancement compared to the blank group = 100% × (OD value of the blank group). 570 - OD of each group 570 ) / Blank group OD 570 .

[0107] Table 5 OD of composition inhibiting adhesion phase biofilm 570

[0108]

[0109] The experimental results show that compositions A-H can reduce the adhesion phase biofilm of Candida albicans to different degrees.

[0110] Compared with the blank group, the compositions A-H all enhance the inhibition of the adhesion phase biofilm of Candida albicans. Among them, compositions A and B have the most obvious effect of enhancing the inhibition of the adhesion phase biofilm compared with the blank group.

[0111] 6, Inhibition of aggregation phase biofilm

[0112] Take 200 μL of the bacterial suspension with a concentration of 5 x 10 4 CFU / mL and inoculate it in a flat-bottom 96-well plate, and then incubate it at 37°C for 24 h. Wash off the floating bacteria with sterile PBS. Add 200 μL of compositions A-H and the positive control phenoxyethanol, respectively, and the dosing concentration is MIC. The blank group does not add active drugs. Then continue to incubate for 24 h, discard the well liquid, and wash it with sterile PBS for 3 times. Add 200 μL of methanol for fixation for 30 min. Add 200 μL of 0.1% crystal violet staining solution for staining for 15 min. Wash off the un-adhesion staining solution with PBS. Add 200 μL of 95% ethanol and stand for 10 min to fully dissolve the staining solution. Measure the OD value with an enzyme-labeled instrument. 570 The test results are shown in Table 6. Among them, the enhancement degree of the inhibition compared with the blank group = 100% x (blank group OD 570 - each group OD 570 / blank group OD 570 .

[0113] Table 6 OD of composition inhibiting aggregation phase biofilm 570

[0114]

[0115]

[0116] The experimental results show that compositions A-H can reduce the aggregation phase biofilm of Candida albicans to different degrees.

[0117] Compared with the blank group, the compositions A-H all enhance the inhibition of the aggregation phase biofilm of Candida albicans. Among them, compositions A and B have the most obvious effect of enhancing the inhibition of the aggregation phase biofilm compared with the blank group.

[0118] 7, Inhibition of mature phase biofilm

[0119] Take 200 μL of the bacterial suspension with a concentration of 5 x 10 4The bacterial suspension of CFU / mL was inoculated into flat-bottom 96-well plates and incubated at 37°C for 48 h (the medium was replaced every 24 h), and the planktonic bacteria were removed by washing with sterile PBS. 200 μL of each of compositions A-H and the positive control phenoxyethanol was added, with the dosing concentration being the MIC, and no active drug was added to the blank group. Then the incubation was continued for 24 h, the well liquid was discarded, and the plates were washed with sterile PBS for 3 times. 200 μL of methanol was added for fixation for 30 min, 200 μL of 0.1% crystal violet staining solution was added for staining for 15 min, the un-adhered staining solution was washed away with PBS, 200 μL of 95% ethanol was added for standing for 10 min to fully dissolve the staining solution, and the OD value was measured by a microplate reader 570 The test results are shown in Table 6. Wherein, the degree of enhancement of the inhibitory effect compared with the blank group = 100% x (blank group OD 570 - group OD 570 / blank group OD 570 .

[0120] Table 7 OD value of the mature biofilm inhibited by the compositions 570

[0121]

[0122]

[0123] The experimental results show that the compositions A-H can reduce the mature biofilm of Candida albicans to different degrees.

[0124] Compared with the blank group, the compositions A-H all enhance the inhibitory effect on the mature biofilm of Candida albicans. Among them, the compositions A and B have the most obvious enhancement of the inhibitory effect on the mature biofilm compared with the blank group.

[0125] Obviously, those skilled in the art can make various modifications and variations to the present application without departing from the spirit and scope of the present application. Thus, if these modifications and variations of the present application fall within the scope of the claims of the present application and their equivalent technologies, the present application also intends to include these modifications and variations.

Claims

1. A method of preparing a composition comprising Phellodendri chinensis cortex and Atractylodis lancea, characterized in that, The method comprises the following steps: Step 1: dry cortex phellodendri and rhizoma atractylodis are taken and pulverized to obtain a powder mixture, water is added to the powder mixture, and the mixture is boiled and then cooled to obtain a decoction; Step 2: enzymes are added to the decoction, and the mixture is subjected to enzymatic hydrolysis, enzyme inactivation, and then obtained as an enzymatic hydrolysate; Step 3: the enzymatic hydrolysate is sterilized, and glucose, dipotassium hydrogen phosphate, and fermentation bacteria are added to the mixture, which is then subjected to fermentation, sterilization, and then obtained as a fermentation product; Step 4: ethanol is added to the fermentation product, the concentration of ethanol is adjusted, and the mixture is heated and stirred to obtain an extract; Step 5: the extract is filtered, ultrafiltered, concentrated, and dried to obtain a composition containing cortex phellodendri and rhizoma atractylodis; The enzymes consist of serratia protease and bromelain, and the dosages of the serratia protease and the bromelain are 400-800 U / g of the decoction and 200-500 U / g of the decoction, respectively; The fermenting bacteria are composed of Lactobacillus reuteri CICC6119 and Bacillus coagulans CICC23843 in a quantity ratio of 1:2-4, and the fermenting bacteria are used in an amount of 1 x 10 9 -9 x 10 9 cfu / g of the enzymolysis material; In step 2, the enzymatic hydrolysis temperature is 40-65℃, and the time is 1-5 hours; In step 3, the fermentation temperature is 25-40℃, and the time is 100-150 hours.

2. The production method according to claim 1, characterized by, The mass ratio of cortex phellodendri to rhizoma atractylodis is 1:2-6.

3. The preparation method according to claim 1, characterized in that, The dosages of glucose and dipotassium hydrogen phosphate are 1.2-4% and 0.02-0.15% of the mass of the enzymatic hydrolysate, respectively.

4. The method of claim 1, wherein, In step 1, the mass ratio of the powder mixture to water is 1:10-50; and / or, the temperature for heat preservation is 72-85℃, and the time is 1-6 hours.

5. The preparation method according to claim 1, characterized in that, In step 4, the mass concentration of ethanol is adjusted to 20-40%, the heating temperature is 50-60℃, and the time is 2-6 hours.

6. The method of claim 1, wherein, In step 5, the filtration is performed using a filter membrane with a pore size not less than 0.1 microns.

7. The preparation method according to claim 1, characterized in that, In step 5, the ultrafiltration is performed using an ultrafiltration membrane with a molecular weight cut-off of less than 3 KDa.

8. A composition containing cortex phellodendri and rhizoma atractylodis prepared by the method of any one of claims 1-7.

9. An article comprising the composition of claim 8, wherein, The composition contains 0.1-30% of the composition and the remaining excipients.

10. A process for the preparation of the article of claim 9, characterized in that, The method comprises the step of mixing the composition with excipients. The method comprises the step of mixing the composition with excipients.

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