Use of enterococcus in combination with berberine in the treatment of phenylketonuria

Abstract

The application relates to the technical field of medicines, and provides application of a composition of enterococcus and berberine or a salt thereof in preparation of medicines for preventing and / or treating phenylketonuria. Specifically, the enterococcus can significantly metabolize phenylalanine in an in-vitro culture medium system; the enterococcus belongs to the firmicutes, the bacilli class, the lactobacillus order and the enterococcus family. Meanwhile, the enterococcus and the berberine can be combined to significantly treat the phenylketonuria; the plasma phenylalanine of the phenylketonuria milk mouse can be reduced by 34.2% after 4 weeks of treatment, which indicates that the enterococcus and the berberine have an outstanding treatment effect in preventing and / or treating the phenylketonuria.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, more particularly to the application of a composition of Enterococcus and berberine or its salts in the preparation of a medicine for preventing and / or treating phenylketonuria. BACKGROUND

[0002] Phenylketonuria (PKU) is a common amino acid metabolic disease, mainly due to the deficiency of phenylalanine hydroxylase in the body, which makes phenylalanine in the metabolic pathway of phenylalanine unable to be converted into tyrosine, so that a large amount of phenylalanine and phenylpyruvic acid accumulates in the body. Phenylketonuria mainly occurs in children aged 3-6 months, and the symptoms worsen at 1 year old, which is a common autosomal recessive genetic disease. The main clinical features are low intelligence level in children, poor mental state, eczema / scratched skin signs, and abnormal electroencephalogram, etc. If early diagnosis and early intervention treatment can be obtained, the above-mentioned clinical manifestations will not occur, the intelligence level of children is normal, and the abnormal electroencephalogram can also be relieved accordingly. At present, most of the treatment methods are to reduce the content of phenylalanine in breast milk or infant formula milk, and there is no medicine for prevention / treatment in clinic.

[0003] In recent years, the concept of gut-brain axis has attracted the attention of many scholars to the role of intestinal flora or oral probiotics in the central nervous system of animals. Changes in intestinal flora can affect the changes in the central nervous system of animals, thereby changing the behavior.

[0004] Intestinal probiotics are active microorganisms that colonize in the human body, change the composition of host flora, and produce benefits to the host. They have many benefits such as promoting the digestion and absorption of nutrients in the host, improving the immunity of the body, maintaining the balance of intestinal flora structure, and can improve diseases to a certain extent. The patent described herein is a lactic acid bacteria belonging to Enterococcus, which has many biological functions, such as safe and non-toxic, can adhere to the intestinal mucosa and colonize, has the dual functions of exerting probiotic effect and expressing foreign proteins, can survive in the body for a long time and continuously express foreign proteins, and a single inoculation can produce a persistent immune response. Lactic acid bacteria can be added to milk products or feed containing lactic acid bacteria to prevent and treat various diseases. At the same time, it was found in the laboratory that berberine has the effect of treating phenylketonuria. It is found that the combination of berberine and Enterococcus can significantly improve phenylketonuria, and the effect is better than that of berberine or Enterococcus alone. SUMMARY

[0005] The technical problem to be solved by the present application is to provide the application of a composition of Enterococcus and berberine or its salts in the preparation of a medicine for preventing and / or treating phenylketonuria.

[0006] To solve the technical problem of the present application, the present application provides the following technical solutions:

[0007] The first aspect of the technical solution of the present application is to provide a use of a composition of Enterococcus and berberine or its salts in the preparation of a medicine for preventing and / or treating phenylketonuria.

[0008] The berberine is as shown in structural formula (I), and the berberine salts include hydrochloride, sulfate, hydrobromide, hydroiodide, formate, acetate or oxalate.

[0009]

[0010] The Enterococcus belongs to Firmicutes, Bacilli, Lactobacillales and Enterococcaceae.

[0011] Further, the Enterococcus includes Enterococcus faecalis and Enterococcus faecium.

[0012] The Enterococcus can effectively degrade phenylalanine in an in-vitro culture system.

[0013] The phenylketonuria is caused by an increase in blood phenylalanine.

[0014] Preferably, the quantity and quality ratio of the composition of the Enterococcus and berberine or its salts ranges from 10 9 to 10 mg.

[0015] Further preferably, the more preferable quantity and quality ratio of the composition of the Enterococcus and berberine or its salts ranges from 10 9 to 2 mg.

[0016] Most preferably, the most preferable quantity and quality ratio of the composition of the Enterococcus and berberine or its salts ranges from 10 9 to 1.5 mg.

[0017] Beneficial technical effects

[0018] The present patent describes that the Enterococcus can significantly degrade phenylalanine in vitro, and the combination of the Enterococcus and berberine can significantly treat phenylketonuria. The Enterococcus can significantly reduce the content of phenylalanine in the in-vitro phenylalanine conversion experiment. The α-methylphenylalanine-induced phenylketonuria model mouse is one of the important experiments for evaluating phenylketonuria. After the α-methylphenylalanine induction, the blood phenylalanine concentration of the mouse is significantly higher than that of the normal control group. The present application uses the combination of the Enterococcus and berberine to decompose the phenylalanine existing in the intestinal cavity, thereby reducing the acquisition of phenylalanine from the intestinal tract, reducing the content of phenylalanine in the blood plasma, and effectively improving phenylketonuria.

[0019] Currently, there is a lack of effective drugs for the treatment of pediatric phenylketonuria in the clinic, and the combined administration of Enterococcus and berberine can effectively improve the phenylalanine metabolic pathway, so that the phenylalanine decomposition pathway in the intestinal flora is enhanced, and less phenylalanine penetrates the intestine into the body, reducing the content of plasma phenylalanine. Therefore, the combined administration of Enterococcus and berberine can reduce the content of phenylalanine in the blood of SD mice, suggesting the application of Enterococcus and berberine in the prevention and / or treatment of phenylketonuria drugs. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 . Enterococcus faecalis phenylalanine in vitro conversion experiment

[0021] Figure 2 . After 5 days of treatment with Enterococcus faecalis, berberine and the combined administration of Enterococcus faecalis and berberine, the content of phenylalanine in the blood of phenylketonuria mice

[0022] Figure 3 . After 2 weeks of treatment with Enterococcus faecalis, berberine and the combined administration of Enterococcus faecalis and berberine, the content of phenylalanine in the blood of phenylketonuria mice

[0023] Figure 4 . After 3 weeks of treatment with Enterococcus faecalis, berberine and the combined administration of Enterococcus faecalis and berberine, the content of phenylalanine in the blood of phenylketonuria mice

[0024] Figure 5 . After 4 weeks of treatment with Enterococcus faecalis, berberine and the combined administration of Enterococcus faecalis and berberine, the content of phenylalanine in the blood of phenylketonuria mice DETAILED DESCRIPTION

[0025] Example 1. Enterococcus faecalis phenylalanine in vitro conversion experiment

[0026] The concentration of phenylalanine in the in vitro culture system is one of the important indicators for evaluating the ability of Enterococcus faecalis to degrade phenylalanine.

[0027] 1. Experimental strains, instruments and reagents

[0028] Phenylalanine was purchased from Beijing Solabio Technology Co., Ltd. Enterococcus faecalis (ATCC47077) was from ATCC Company, and MRS culture medium was from Solabio Company.

[0029] 2. Experimental instruments and analysis methods

[0030] High performance liquid chromatography-triple quadrupole mass spectrometry (LC-MS / MS 8050, Shimadzu Corporation, Japan) was used to quantitatively determine the content of phenylalanine in the blood. Alltima C 18(5 μm, 4.6 x 150 mm) column at 40 °C. The mobile phase was water-formic acid (100:0.1 v / v) and methanol. Gradient elution (A:B, 0 min, 90:10; 1 min, 90:10; 1.01 min, 60:40; 5 min, 5:95; 7 min, 5:95; 7.01 min 90:10; 10 min 90:10) at a flow rate of 0.8 mL / min. Quantification was performed in the multiple reaction monitoring (MRM) mode with the quantification ion pair phenylalanine 165.85→120.20.

[0031] 3. Experimental design

[0032] Experimental design: the strain was inoculated in MRS liquid medium overnight, and the next day was inoculated in new MRS liquid medium at a ratio of 2%, and cultured at 37 °C to OD 600 = 0.8-1.0, centrifuged in a large centrifuge tube to collect the bacterial cells, and added MRS liquid medium containing 1 mg / L phenylalanine to keep the bacterial concentration at 3 x 10 8 per mL, and then distributed into small tubes, and sampled at 0 h, 12 h, and 24 h, and the phenylalanine concentration was determined.

[0033] 4. Results

[0034] As shown in Table 1, Figure 1 after 12 h and 24 h of co-culture of Enterococcus faecalis with MRS liquid medium containing 1 mg / L phenylalanine, the concentration was reduced by 17.9% and 56.5%, respectively, and the results showed that Enterococcus faecalis has excellent ability to degrade phenylalanine, and can significantly reduce the content of phenylalanine in the medium.

[0035] Table 1 Concentration of phenylalanine in the system after metabolism by Enterococcus faecalis (μg / mL, n = 5)

[0036]

[0037] Example 2. Therapeutic effect of Enterococcus faecalis and combined use of berberine in SD rat models of phenylketonuria induced by α-methylphenylalanine.

[0038] The concentration of phenylalanine in the blood is one of the important indicators for evaluating the therapeutic effect of SD rat models of phenylketonuria.

[0039] 1. Experimental animals, instruments, and pharmaceutical reagents

[0040] SD rats (1 day old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The animals were housed with their dams in a SPF environment (21 ± 2 °C, 12-hour light cycle) and had free access to food and water during the experiment. Phenylalanine was purchased from Beijing Solabio Technology Co., Ltd. Alpha-methylphenylalanine was purchased from Nanjing Sunny Kay Co., Ltd. Enterococcus faecalis (ATCC 47077) was from the ATCC strain library and was cultured and expanded by the laboratory. Berberine was purchased from Bailingwei Co., Ltd.

[0041] 2. Experimental instruments and analysis methods

[0042] The content of phenylalanine in blood was quantitatively determined by high performance liquid chromatography-triple quadrupole tandem mass spectrometry (LC-MS / MS 8050, Shimadzu, Japan). The determination of phenylalanine used an Alltima C 18 (5 μm, 4.6 x 150 mm) chromatographic column, and the column temperature was 40 °C. The mobile phase was water-formic acid (100:0.1 v / v) and methanol. Gradient elution (A:B, 0 min, 90:10; 1 min, 90:10; 1.01 min, 60:40; 5 min, 5:95; 7 min, 5:95; 7.01 min 90:10; 10 min 90:10) was used at a flow rate of 0.8 mL / min. Quantitative determination was performed in multiple reaction monitoring (MRM) mode, and the quantitative ion pair was phenylalanine 165.85→120.20.

[0043] 3. Experimental design and animal grouping

[0044] Experimental design: The experimental animals were divided into 5 groups, including a normal group (N), a model control group (M), a model group + berberine treatment group (B), a model group + Enterococcus faecalis treatment group (F), and a model group + Enterococcus faecalis + berberine treatment group (F+B). The model was established by subcutaneously injecting alpha-methyl-L-phenylalanine 0.05 g / kg and Phe 0.2 g / kg to SD rats from 6 days old for 10 days, and then treated for 30 days. The content of plasma phenylalanine was determined at 1 week, 2 weeks, 3 weeks and 4 weeks after administration.

[0045] Animal grouping:

[0046] (1) Normal group (N); subcutaneous injection of normal saline 0.1 mL;

[0047] (2) Model control group (M): subcutaneous injection of alpha-methylphenylalanine (50 mg / kg / day) + subcutaneous injection of phenylalanine (200 mg / kg / day);

[0048] (3) Model group + berberine treatment group (B): subcutaneous injection of α-methylphenylalanine (50 mg / kg / day) + subcutaneous injection of phenylalanine (200 mg / kg / day), oral administration of berberine (100 mg / kg).

[0049] (4) Model group + Enterococcus faecalis (F): subcutaneous injection of α-methylphenylalanine (50 mg / kg / day) + subcutaneous injection of phenylalanine (200 mg / kg / day), oral administration of Enterococcus faecalis (F) (1x10 9

[0050] (5) Model group + Enterococcus faecalis + berberine (F+B): subcutaneous injection of α-methylphenylalanine (50 mg / kg / day) + subcutaneous injection of phenylalanine (200 mg / kg / day), oral administration of berberine (100 mg / kg), oral administration of Enterococcus faecalis (F) (1x10 9

[0051] 4. Results

[0052] As shown in Table 2, Figure 2 the content of phenylalanine in the blood of the model group of suckling mice after 5 days of oral treatment was significantly higher than that of the normal control group, indicating that the model was successfully established; the content of phenylalanine in the blood of the oral berberine group (B) was significantly reduced by 16.7%, the oral Enterococcus faecalis group (F) was reduced by 4.6%, and the oral berberine and Enterococcus faecalis group (B+F) was significantly reduced by 55.6%, indicating that berberine, Enterococcus faecalis, and combined administration of berberine and Enterococcus faecalis all had certain therapeutic effects on phenylketonuria, among which the combined administration of berberine and Enterococcus faecalis had the best therapeutic effect.

[0053] Table 2 Content of phenylalanine in the blood of phenylketonuria suckling mice after 5 days of treatment with Enterococcus faecalis, berberine, and combined administration of Enterococcus faecalis and berberine (x 20 ng / mL, n=8)

[0054]

[0055] Note: * is an abnormal value

[0056] As shown in Table 3, Figure 3 and Table 4, Figure 4 and Table 5, Figure 5 ​​As shown, the serum phenylalanine level in the model group was significantly higher than that in the normal group, indicating that the model was successfully established. Similarly, on day 14 after oral treatment, the serum phenylalanine level in the berberine treatment group decreased significantly by 34.7%; on days 14, 21, and 30 after oral treatment, the serum phenylalanine level in the combined berberine and Enterococcus faecalis treatment group decreased significantly by 18.6%, 30.8%, and 34.2%, respectively. This indicates that oral berberine and Enterococcus faecalis treatment is effective and has a certain time dependence, significantly reducing serum phenylalanine levels. Specific experimental data are summarized in Table 6.

[0057] Table 3. Serum phenylalanine levels (×20 ng / mL, n=8) in neonatal rats with phenylketonuria after 14 days of treatment with Enterococcus faecalis, berberine, and a combination of Enterococcus faecalis and berberine.

[0058]

[0059] Note: * indicates an outlier.

[0060] Table 4. Serum phenylalanine levels (×20 ng / mL, n=8) in suckling mice with phenylketonuria after 21 days of treatment with Enterococcus faecalis, berberine, and a combination of Enterococcus faecalis and berberine.

[0061]

[0062] Note: * indicates an outlier.

[0063] Table 5. Serum phenylalanine levels (×20 ng / mL, n=8) in suckling mice with phenylketonuria after 30 days of treatment with Enterococcus faecalis, berberine, and a combination of Enterococcus faecalis and berberine.

[0064]

[0065] Note: * indicates an outlier.

[0066] Table 6. Summary of the reduction in serum phenylalanine levels in neonatal rats with phenylketonuria after 5 days, 2 weeks, 3 weeks, and 4 weeks of treatment with Enterococcus faecalis, berberine, and a combination of Enterococcus faecalis and berberine.

[0067]

[0068] Note: *** indicates p < 0.001, ** indicates p < 0.01, and * indicates p < 0.05.

Claims

1. The use of a composition of an enterococcus and berberine or its salts in the preparation of a medicament for the prevention and / or treatment of phenylketonuria, wherein the enterococcus is Enterococcus faecalis ATCC47077.

2. The application according to claim 1, characterized in that, The berberine mentioned is berberine as shown in structural formula (I), and the berberine salts include its hydrochloride, sulfate, hydrobromide, hydroiodide, formate, acetate or oxalate; 3. The application according to claim 1, characterized in that, The Enterococcus mentioned belongs to the phylum Firmicutes, class Bacillus, order Lactobacilliles, and family Enterococci.

4. The application according to claim 1, characterized in that, The Enterococcus described can effectively degrade phenylalanine in an in vitro culture system.

5. The application according to claim 1, characterized in that, The aforementioned phenylketonuria is caused by elevated levels of phenylalanine in the blood.

6. The application according to any one of claims 1-5, characterized in that, The quantity and mass ratio range of the aforementioned combination of Enterococcus and berberine or its salts is:

10. 9 Individual doses: (0.1–10 mg).

7. The application according to claim 6, characterized in that, The quantity and mass ratio range of the aforementioned combination of Enterococcus and berberine or its salts is:

10. 9 Individual doses: (0.1–2 mg).

8. The application according to claim 7, characterized in that, The quantity and mass ratio range of the aforementioned combination of Enterococcus and berberine or its salts is:

10. 9 Dosage: (0.5–1.5 mg).

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