A probiotic composition having xanthine oxidase inhibitory effect and use thereof

Abstract

The present application belongs to the technical field of medicine, and particularly relates to a probiotic composition, a fermented traditional Chinese medicine composition and application. The probiotic composition with xanthine oxidase inhibiting effect comprises the following components: Lactobacillus plantarum, Enterococcus faecium 1 and Enterococcus faecium 2. The Lactobacillus plantarum has a preservation number of CGMCC 1.557, the Enterococcus faecium 1 has a preservation number of CICC 6078, and the Enterococcus faecium 2 has a preservation number of CGMCC 1.130. The present application utilizes the uric acid method to determine that the Lactobacillus plantarum, the Enterococcus faecium 1 and the Enterococcus faecium 2 have a strong inhibiting effect, and after the three kinds of bacteria are mixed, the inhibiting rate is 65%, which reflects that the three kinds of bacteria strains have good synergism in inhibiting the xanthine oxidase effect.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a probiotic composition with xanthine oxidase inhibitory activity and its application. Background Technology

[0002] Xanthine oxidase is a key enzyme in humans and animals involved in purine metabolism. Xanthine oxidase is a molybdenum flavin protease composed of approximately 1300 amino acids.

[0003] Abnormal xanthine oxidase activity can induce many diseases, the most common being gout. It can also cause cardiovascular and cerebrovascular inflammation. During nucleic acid and protein metabolism, xanthine oxidase converts hypoxanthine and xanthine into uric acid, which is then excreted. Reducing xanthine oxidase activity can decrease uric acid production, thus alleviating gout symptoms. Xanthine oxidase is a key target in gout treatment, and xanthine oxidase inhibitors have been shown to have therapeutic effects. Marketed Western medicines such as allopurinol inhibit xanthine oxidase activity, reducing uric acid production through competitive inhibition. However, the ingestion of allopurinol and similar drugs has significant side effects. Therefore, exploring green, safe, and effective xanthine oxidase inhibitors has become a research hotspot.

[0004] There are many related studies, but further research is needed on the effects of using probiotics alone and their therapeutic efficacy, and the efficacy of traditional Chinese medicine combinations fermented with probiotics also needs to be improved. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a probiotic composition with xanthine oxidase inhibitory activity.

[0006] To achieve the objectives of this invention, the following technical solution is adopted:

[0007] A probiotic composition with xanthine oxidase inhibitory activity comprises the following components: Lactobacillus plantarum, Enterococcus faecalis 1, and Enterococcus faecalis 2.

[0008] Preferably, the *Lactobacillus plantarum* has the accession number CGMCC 1.557, *Enterococcus faecalis* 1 has the accession number CICC6078, and *Enterococcus faecalis* 2 has the accession number CGMCC 1.130.

[0009] Preferably, the mass ratio of Lactobacillus plantarum, Enterococcus faecalis 1 and Enterococcus faecalis 2 is 1:0.5-1.5:0.5-1.5, and more preferably 1:1:1.

[0010] Preferably, the total viable count of the probiotic composition is 2 × 10⁻⁶. 8 -4×108 CFU / mL; preferably 2.5 × 10⁻⁶ 8 -3.5×10 8 CFU / mL, the optimal value is 2.8 × 10⁻⁶. 8 CFU / mL.

[0011] Another object of the present invention is to provide a fermented traditional Chinese medicine composition, wherein the raw materials of the composition include the following components: Angelica sinensis, Clematis chinensis, Fraxinus chinensis, Polygonum cuspidatum, Akebia trifoliata, Cinnamomum cassia, Alisma plantago-aquatica, and the above-mentioned probiotic composition.

[0012] Preferably, by weight, it comprises the following components: 8-12 parts Angelica sinensis, 8-12 parts Clematis chinensis, 18-22 parts Fraxinus chinensis, 18-22 parts Polygonum cuspidatum, 13-17 parts Akebia trifoliata, 13-17 parts Cinnamomum cassia, and 18-22 parts Alisma plantago-aquatica.

[0013] Another object of the present invention is to provide a method for preparing a fermented traditional Chinese medicine composition, comprising the following steps:

[0014] (1) Mix Angelica sinensis, Clematis chinensis, Fraxinus rhizome, Polygonum cuspidatum, Akebia trifoliata, Cinnamomum cassia, and Alisma plantago-aquatica, add water and extract and concentrate to obtain a Chinese medicine liquid;

[0015] (2) Add the probiotic composition to the Chinese medicine liquid and ferment it to obtain the product.

[0016] Preferably, the pH of the fermentation in step (2) is 6-8, the fermentation temperature is 35-39°C, and the inoculum size is 2-4%.

[0017] Another object of the present invention is to provide the use of probiotic compositions in the preparation of xanthine oxidase inhibitors or in the treatment of gout.

[0018] Another object of the present invention is to provide the use of fermented traditional Chinese medicine compositions in the preparation of products that have xanthine oxidase inhibitory effects, or for the treatment of gout or for lowering uric acid.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] (1) The uric acid method was used to determine that Lactobacillus plantarum, Enterococcus faecalis 1 and Enterococcus faecalis 2 had strong inhibitory effects on lactic acid bacteria. Under the same dosage, the inhibition rates were 61% for Lactobacillus plantarum, 55% for Enterococcus faecalis 1 and 51% for Enterococcus faecalis 2. After mixing the three bacteria with the same amount, their inhibitory effect on xanthine oxidase was measured. After mixing, the inhibition rate was 65%, which showed good synergy among the three strains.

[0021] (2) The half-maximal inhibitory concentration (IC50) of the Chinese herbal medicine formulation before fermentation was 7 mg / mL; the half-maximal inhibitory concentration (IC50) of the Chinese herbal medicine formulation after fermentation was 5 mg / mL.

[0022] (3) The composition of the present invention has the effect of inhibiting xanthine oxidase and has a good therapeutic effect on gout in goslings. Detailed Implementation

[0023] The present invention will be further described below with reference to specific embodiments.

[0024] Lactobacillus plantarum (purchased from China General Microbiological Culture Collection Center, accession number CGMCC 1.557), Enterococcus faecium 1 (purchased from China Industrial Microbiological Culture Collection Center, accession number CICC 6078), and Enterococcus faecium 2 (purchased from China General Microbiological Culture Collection Center, accession number CGMCC 1.130).

[0025] Example 1

[0026] The probiotic composition in this embodiment includes a mixture of *Lactobacillus plantarum*, *Enterococcus faecium* 1, and *Enterococcus faecium* 2 in a mass ratio of 1:1:1. Water is added to prepare a total viable count of 2.8 × 10⁻⁶. 8 CFU / mL bacterial suspension.

[0027] Example 2

[0028] The probiotic composition in this embodiment includes a mixture of *Lactobacillus plantarum*, *Enterococcus faecium* 1, and *Enterococcus faecium* 2 in a mass ratio of 1:1:1. Water is added to prepare a total viable count of 2.6 × 10⁻⁶. 8 CFU / mL bacterial suspension.

[0029] Example 3

[0030] The probiotic composition in this embodiment includes a mixture of *Lactobacillus plantarum*, *Enterococcus faecium* 1, and *Enterococcus faecium* 2 in a mass ratio of 1:1:1. Water was added to prepare a total viable count of 3.1 × 10⁻⁶. 8 CFU / mL bacterial suspension.

[0031] Example 4

[0032] The fermented traditional Chinese medicine composition is prepared by the following method:

[0033] 1. Weigh out the components of the traditional Chinese medicine compound: 8 parts by weight of Angelica sinensis, 8 parts by weight of Clematis chinensis, 18 parts by weight of Fraxinus chinensis, 18 parts by weight of Polygonum cuspidatum, 13 parts by weight of Akebia trifoliata, 13 parts by weight of Cinnamomum cassia, and 18 parts by weight of Alisma plantago-aquatica.

[0034] 2. Add 6 times the volume of water for the first decoction, which takes 0.5 hours. Then centrifuge and collect the supernatant.

[0035] 3. After the first extraction, add 6 times the volume of water to the dregs and boil for a second time for 0.5 hours. Then centrifuge and collect the supernatant.

[0036] 4. Mix the supernatants from the two extractions to obtain the aqueous extract of the traditional Chinese medicine compound;

[0037] 5. Evaporate the water extract to obtain a concentrated solution, autoclave, and store at 4°C for later use.

[0038] 6. Inoculate the probiotic composition of Example 1 into the drug solution of step (5), set the pH to 7, the temperature to 37°C, the inoculum size to 3%, and incubate statically for 24 hours. Filter to obtain the fermented traditional Chinese medicine solution. The concentration of fermented traditional Chinese medicine bacteria (total number of the three types of bacteria) is 2.8 × 10⁻⁶. 8 CFU / mL.

[0039] Example 5

[0040] The fermented traditional Chinese medicine composition is prepared by the following method:

[0041] 1. Weigh each component of the Chinese herbal compound: Weigh each component of the Chinese herbal compound: Angelica sinensis 12 parts by weight, Clematis chinensis 12 parts by weight, Fraxinus chinensis 22 parts by weight, Polygonum cuspidatum 22 parts by weight, Akebia trifoliata 17 parts by weight, Cinnamomum cassia 17 parts by weight, and Alisma plantago-aquatica 22 parts by weight.

[0042] 2. Add 10 times the volume of water for the first decoction, which takes 0.5 hours. Then centrifuge and collect the supernatant.

[0043] 3. After the first extraction, add 10 times the volume of water to the dregs and boil for a second time for 0.5 hours. Then centrifuge and collect the supernatant.

[0044] 4. Mix the supernatants from the two extractions to obtain the aqueous extract of the traditional Chinese medicine compound;

[0045] 5. Evaporate the aqueous extract to obtain a more concentrated medicinal solution, place it in a container, autoclave, and store at 4°C for later use.

[0046] 6. Inoculate the probiotic composition of Example 1 into the drug solution of step (5), set the pH to 7, the temperature to 37°C, the inoculum size to 3%, and incubate statically for 24 hours. Filter to obtain the fermented traditional Chinese medicine solution. The final concentration of fermented traditional Chinese medicine bacteria is 2.8 × 10⁻⁶. 8 CFU / mL.

[0047] Comparative Example 1

[0048] The difference between this comparative example and Example 1 is the strain; specifically, it is Lactobacillus plantarum.

[0049] Comparative Example 2

[0050] The difference between this comparative example and Example 1 is the strain; specifically, it is Enterococcus faecalis 1.

[0051] Comparative Example 3

[0052] The difference between this comparative example and Example 1 is the strain; specifically, it is Enterococcus faecalis 2.

[0053] Comparative Example 4

[0054] The difference between this comparative example and Example 4 is the fermentation pH, specifically 5. The final concentration of the herb culture during fermentation was 1×10⁻⁶. 8 CFU / mL.

[0055] Comparative Example 5

[0056] The difference between this comparative example and Example 4 is the fermentation pH, specifically 9. The final concentration of the herb culture during fermentation was 0.8 × 10⁻⁶. 8 CFU / mL.

[0057] Comparative Example 6

[0058] The difference between this comparative example and Example 4 is that the fermentation temperature was 35℃. The final concentration of the herb culture during fermentation was 0.9 × 10⁻⁶. 8 CFU / mL.

[0059] Comparative Example 7

[0060] The difference between this comparative example and Example 4 is that the fermentation temperature was 39℃. The final concentration of the herb culture during fermentation was 0.8 × 10⁻⁶. 8 CFU / mL.

[0061] Comparative Example 8

[0062] The difference between this comparative example and Example 4 is that the inoculum size was 5%. The final concentration of the herb culture during fermentation was 1.3 × 10⁻⁶. 8 CFU / mL.

[0063] Comparative Example 9

[0064] The difference between this comparative example and Example 4 is that the inoculum size was 1%. The final concentration of the herb culture during fermentation was 0.9 × 10⁻⁶. 8 CFU / mL.

[0065] Experiment 1 used ultraviolet spectrophotometry to determine the in vitro inhibitory effect of different probiotics and their combinations on xanthine oxidase.

[0066] The steps are as follows:

[0067] In a 96-well plate, 50 μL of xanthine oxidase (0.25 U / mL) was added to each well, along with 10 μL of the probiotic composition from Example 1, Comparative Examples 1-3, or allopurinol (0.35 mg / mL). PBS buffer was added to fill the wells, bringing the final reaction volume to 200 μL. The plate was mixed and vortexed, then incubated for 1 hour. 100 μL of xanthine (0.1 g / mL) was added to initiate the reaction. The plate was placed in a microplate reader, and the program was set to 295 nm. The plate was vortexed for 10 seconds before the first measurement, with a 10-second delay for each subsequent measurement, and the test lasted 3 minutes. A control group without sample was included. Enzyme activity (295 nm) was measured using a microplate dynamic method, and Vmax was recorded.

[0068] Calculation: Sample inhibition rate (%) = (AB) / A × 100% (A is the control group Vmax, B is the sample group Vmax).

[0069] The results are as follows:

[0070] Allopurinol showed a 99% inhibition rate against XOD, while the blank control group showed no inhibitory effect; several strains of Bacillus showed weak inhibitory effects against XOD, less than 5%.

[0071] The names and sources of the probiotics used in this experiment, as well as their enzyme inhibition rates, are shown in Table 1 below. The probiotics used were Lactobacillus plantarum, Enterococcus faecalis, Lactobacillus rhamnosus, Bacillus subtilis, Bacillus coagulans, Bacillus belyssus, Bacillus henryi, Enterococcus faecalis 1, Enterococcus faecalis 2, Lactobacillus brevis, Lactobacillus fermentum, Lactobacillus paracasei, and Leuconostoc mesenteroides, provided by the Henan Provincial Microbial Biotransformation Laboratory of Henan University of Animal Husbandry and Economics.

[0072] The preparation of the bacterial strains for streak plate isolation is as follows: 1g of a compound probiotic product containing Bacillus subtilis, Bacillus coagulans, Bacillus belye, Bacillus henryi, and Enterococcus faecalis was dissolved in 100ml of water and inoculated onto ordinary agar plates and MRS plates, respectively. The plates were incubated at 37℃ for 24 hours. Bacillus subtilis was isolated from white colonies with rough surfaces and wrinkles; Bacillus coagulans was isolated from rough, opaque, and slightly yellow colonies; Bacillus belye was isolated from white colonies with neat edges and round shapes; Bacillus henryi was isolated from smooth white colonies; and Enterococcus faecalis was isolated from oval, smooth colonies.

[0073] Table 1

[0074]

[0075]

[0076] In Comparative Examples 1-3, *Lactobacillus plantarum*, *Enterococcus faecalis* 1, and *Enterococcus faecalis* 2 showed strong inhibitory effects, with inhibition rates of 61% for *Lactobacillus plantarum*, 55% for *Enterococcus faecalis* 1, and 51% for *Enterococcus faecalis* 2.

[0077] Example 1: After mixing the three bacteria evenly (Lactobacillus plantarum: Enterococcus faecalis 1: Enterococcus faecalis 2 = 1:1:1), the inhibitory effect on xanthine oxidase was measured. The total number of bacteria after mixing remained unchanged compared with the number of bacteria measured with single bacteria, and the inhibition rate was 65%.

[0078] Experiment 2 determined the half-maximal inhibitory concentration (IC50) of the fermented Chinese medicine composition on xanthine oxidase.

[0079] In a 96-well plate, first add 50 μL of PBS solution sequentially. Then, add 50 μL of the concentrated drug solution from Example 4 and the fermentation broth of the traditional Chinese medicine to well 1, mix thoroughly, and precisely pipette 50 μL into well 2. Repeat this serial dilution until well 8. The xanthine oxidase inhibition rate of each well is measured according to the method described above. Plot a curve with the concentration of the traditional Chinese medicine on the x-axis and the xanthine oxidase inhibition rate on the y-axis, and calculate the half-maximal inhibitory concentration (IC50).

[0080] Results: The half-maximal inhibitory concentration (IC50) of the concentrated herbal liquid before fermentation was 7 mg / mL; the half-maximal inhibitory concentration (IC50) of the fermented herbal liquid after fermentation was 5 mg / mL. The half-maximal inhibitory concentrations of specific Examples 4 and Comparative Examples 4-9 are shown in Table 2 below.

[0081] Table 2

[0082] Group Half-maximal inhibitory concentration (IC50) / mg / mL Example 4 5.0 Comparative Example 4 5.7 Comparative Example 5 5.9 Comparative Example 6 5.8 Comparative Example 7 5.7 Comparative Example 8 5.4 Comparative Example 9 5.8

[0083] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A probiotic composition with xanthine oxidase inhibitory activity, characterized in that, It consists of the following components: Lactobacillus plantarum, Enterococcus faecalis 1, and Enterococcus faecalis 2; The preservation number of *Lactobacillus plantarum* is CGMCC 1.557, the preservation number of *Enterococcus faecium* 1 is CICC 6078, and the preservation number of *Enterococcus faecium* 2 is CGMCC 1.

130. Both Enterococcus faecalis 1 and Enterococcus faecalis 2 are Enterococcus lactis; The mass ratio of Lactobacillus plantarum, Enterococcus faecalis 1, and Enterococcus faecalis 2 was 1:1:

1.

2. The composition according to claim 1, characterized in that, The total live bacteria count of the probiotic composition is 2 × 10⁻⁶. 8 -4×10 8 CFU / mL.

3. A fermented traditional Chinese medicine composition, wherein the raw materials of the composition are composed of the following components: Angelica sinensis, Clematis chinensis, Fraxinus chinensis, Polygonum cuspidatum, Akebia trifoliata, Cinnamomum cassia, Alisma plantago-aquatica, and the probiotic composition according to any one of claims 1-2; By weight, the ingredients are: Angelica sinensis 8-12 parts, Clematis chinensis 8-12 parts, Fraxinus chinensis 18-22 parts, Polygonum cuspidatum 18-22 parts, Akebia trifoliata 13-17 parts, Cinnamomum cassia 13-17 parts, and Alisma plantago-aquatica 18-22 parts.

4. A method for preparing the fermented traditional Chinese medicine composition according to claim 3, characterized in that, Includes the following steps: (1) Mix Angelica sinensis, Clematis chinensis, Fraxinus chinensis, Polygonum cuspidatum, Akebia trifoliata, Cinnamomum cassia and Alisma plantago-aquatica, add water and extract and concentrate to obtain Chinese medicine liquid; (2) Add the probiotic composition to the Chinese medicine liquid and ferment it to obtain the product.

5. The preparation method according to claim 4, characterized in that, The fermentation pH in step (2) is 6-8, the fermentation temperature is 36-38℃, and the inoculum size is 2-4%.

Images