Pharmaceutical composition and application thereof in preparation of drugs for treating autism spectrum disorder

Abstract

The invention discloses a pharmaceutical composition and application thereof in preparation of a medicine for treating autism spectrum disorder, and relates to the field of biomedicine.The pharmaceutical composition is composed of pediococcus acidilactici and radix bupleuri decoction, and when the pharmaceutical composition is used for preparing the medicine for treating autism spectrum disorder, the pediococcus acidilactici and the radix bupleuri decoction are added. The medicine for treating the autism spectrum disorder takes the pharmaceutical composition as an active ingredient for resisting the autism spectrum disorder, and also contains pharmaceutically acceptable carriers or auxiliary materials to be prepared into pharmaceutically acceptable dosage forms. According to the invention, the traditional Chinese medicine big bupleurum decoction and pediococcus acidilactici are combined to prepare the autism spectrum disorder drug for the first time, and the autism spectrum disorder is efficiently and safely treated through drug combination synergistically. The prepared medicine enhances the gastrointestinal function of autism children, effectively relieves autism core symptoms, has the application prospect of being developed into autism spectrum disorder treatment medicine, and can provide new thinking and selection for development and production of autism spectrum disorder related clinical treatment medicine.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and specifically to a pharmaceutical composition and its application in the preparation of a therapeutic drug for autism spectrum disorder. Background Art

[0002] Autism spectrum disorder (ASD), simply referred to as autism, is a severe neurodevelopmental disorder that occurs in early childhood. Its main clinical features include social communication and interaction disorders, restricted interests or activities, and repetitive stereotyped behaviors. It often co-occurs with intellectual disabilities, attention deficit hyperactivity disorder, etc., seriously affecting the healthy growth of children.

[0003] Currently, drugs such as risperidone are often used clinically for adjuvant treatment of ASD. However, long-term clinical treatment results show that most patients will develop immune adaptation to these drugs after long-term use, and the treatment effect is very limited. In addition, these drugs also have many side effects, causing some other damages to the patient's body.

[0004] Studies have shown that the gut microbiota plays a key role in brain development, and there is a two-way information exchange between the gut microbiota and the brain. The gut microbiota can affect brain function through the neuroendocrine system, neuroimmune system, and autonomic nervous system of the "microbiota-gut-brain axis". On this basis, some reports in the prior art also disclose the use of probiotics to regulate the gut microbiota of patients to alleviate the core symptoms of ASD, such as Lactobacillus plantarum PS128, Lactobacillus rhamnosus GG, Bifidobacterium longum NCC3001, but the therapeutic effect of these probiotics on ASD is relatively general.

[0005] Therefore, there is still a severe shortage of highly effective and safe therapeutic drugs for ASD, and there is an urgent need to develop more effective and efficient therapeutic drugs for ASD. Summary of the Invention

[0006] The present invention aims to provide a pharmaceutical composition and its application in the preparation of a therapeutic drug for autism spectrum disorder to effectively and safely treat autism.

[0007] To solve the above technical problems, the specific solution adopted by the present invention is: a pharmaceutical composition for treating autism spectrum disorder, which is composed of Pediococcus acidilactici and Dacaihu Decoction.

[0008] As a further optimization of the above technical solution, the addition ratio of Pediococcus acidilactici to Dacaihu Decoction is 10 10 cfu: 10 g, where the mass of Dacaihu Decoction is recorded according to the mass of the medicinal materials.

[0009] As a further optimization of the above technical solution, the preparation method of the Major Bupleurum Decoction is as follows: By mass, take 12 - 16 parts of Bupleurum chinense, 8 - 10 parts of Scutellaria baicalensis, 8 - 10 parts of Paeonia lactiflora, 8 - 10 parts of Pinellia ternata, 8 - 10 parts of Fructus Aurantii Immaturus, 5 - 7 parts of Rheum officinale, 11 - 13 parts of Ziziphus jujuba, and 14 - 16 parts of Zingiber officinale, and decoct them with water to make it.

[0010] As a further optimization of the above technical solution, Pediococcus acidilactici is formulated into a Pediococcus acidilactici solution, and then mixed with the Major Bupleurum Decoction. The viable count of Pediococcus acidilactici in the Pediococcus acidilactici solution is 10 12 cfu / ml.

[0011] As a further optimization of the above technical solution, the name of the Pediococcus acidilactici is Pediococcus acidilactici QZ - 01, and the deposit number is CCTCC M 2019930.

[0012] Use of a pharmaceutical composition in the preparation of a therapeutic drug for autism spectrum disorder.

[0013] As a further optimization of the above technical solution, the drug uses the pharmaceutical composition as the active ingredient for treating autism spectrum disorder, and also contains a pharmaceutically acceptable carrier or excipient, and is made into a pharmaceutically acceptable dosage form.

[0014] As a further optimization of the above technical solution, the carrier or excipient is solid, liquid or semi - solid.

[0015] As a further optimization of the above technical solution, the dosage form includes tablets, granules, capsules, pills, transdermal microneedle preparations, oral liquids and injections.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0017] The present invention combines the traditional Chinese medicine Major Bupleurum Decoction and Pediococcus acidilactici for the first time to prepare a drug for autism spectrum disorder, and realizes the efficient and safe treatment of autism spectrum disorder through combined medication.

[0018] The drug prepared by the present invention enhances the gastrointestinal function of autistic children, effectively alleviates the core symptoms of autism, has the application prospect of being developed into a therapeutic drug for autism spectrum disorder, and can provide new ideas and choices for the development and production of clinical therapeutic drugs related to autism spectrum disorder. Description of the Drawings

[0019] Figure 1 It is the isolation, identification and phylogenetic analysis diagram of Pediococcus acidilactici QZ - 01;

[0020] Figure 2 It is the behavioral analysis of mice;

[0021] Figure 3 For the compatibility analysis of Dachengqi Decoction and probiotics;

[0022] Figure 4 For the analysis of oxidative stress and inflammation levels in mice;

[0023] Figure 5 For the levels of intestinal barrier function markers in mice;

[0024] Figure 6 For the HE staining analysis of hippocampal tissues in mice. Specific implementation mode

[0025] The technical solutions of the present invention will be further elaborated in detail below in conjunction with specific embodiments. For the parts not detailedly recorded and disclosed in the following embodiments of the present invention, they should all be understood as the prior art known or should be known to those skilled in the art. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field; unless otherwise specified, the reagents and materials used in the present invention are all commercially available.

[0026] The present invention discloses a pharmaceutical composition for treating autism spectrum disorder. The pharmaceutical composition is composed of probiotics and traditional Chinese medicine. Among them, the probiotic is Pediococcus acidilactici, and the traditional Chinese medicine is Dachengqi Decoction. The quality of Dachengqi Decoction is recorded according to the quality of the medicinal materials. The addition ratio of Pediococcus acidilactici to Dachengqi Decoction is 10 10 cfu: 10 g.

[0027] Pediococcus acidilactici is Pediococcus acidilactici QZ-01 with the function of regulating intestinal homeostasis, which was independently isolated and screened by the applicant from traditional fermented foods in the early stage. The preservation number is CCTCC M2019930. The phylogenetic characteristics of the isolated strain were identified by 16S rRNA gene sequencing, as Figure 1 shown.

[0028] Dachengqi Decoction comes from Treatise on Febrile and Miscellaneous Diseases by Zhang Zhongjing. It is composed of Bupleurum chinense, Scutellaria baicalensis, Paeonia lactiflora, Pinellia ternata, Aurantii Fructus Immaturus, Rheum palmatum, Jujube Fruit and Fresh Ginger. It is a formula for relieving exterior and interior syndromes, with the effects of reconciling Shaoyang and purging heat and stasis. In addition, modern pharmacology shows that Dachengqi Decoction has the effects of enhancing gastrointestinal motility, regulating immunity and anti-inflammation.

[0029] In the present invention, the preparation method of Dachengqi Decoction is as follows: according to parts by mass, take 12 - 16 parts of Bupleurum chinense, 8 - 10 parts of Scutellaria baicalensis, 8 - 10 parts of Paeonia lactiflora, 8 - 10 parts of Pinellia ternata, 8 - 10 parts of Aurantii Fructus Immaturus, 5 - 7 parts of Rheum palmatum, 11 - 13 parts of Jujube Fruit and 14 - 16 parts of Fresh Ginger, and decoct with water to make it.

[0030] Dissolve Pediococcus acidilactici in water to prepare a Pediococcus acidilactici solution, and then mix the Pediococcus acidilactici solution with the Major Bupleurum Decoction. The viable count of Pediococcus acidilactici in the Pediococcus acidilactici solution is 10 12 cfu / ml.

[0031] The present invention also discloses the use of the said pharmaceutical composition in the preparation of a therapeutic drug for children with autism spectrum disorder. The therapeutic drug for children uses the said pharmaceutical composition as the active ingredient against autism spectrum disorder and further contains a pharmaceutically acceptable carrier or excipient, and is made into a pharmaceutically acceptable dosage form. The carrier or excipient is solid, liquid or semi-solid, and the dosage forms include tablets, granules, capsules, pills, transdermal microneedle preparations, oral liquids and injections.

[0032] In order to verify the application of the said pharmaceutical composition of the present invention in the treatment of autism spectrum disorder, the applicant conducted the following experiments:

[0033] To avoid repetition, the materials required in the following experimental procedures are first described uniformly as follows:

[0034] Pediococcus acidilactici solution: Dissolve Pediococcus acidilactici with the name of Pediococcus acidilactici QZ-01 and the preservation number of CCTCC M2019930 in water to prepare a Pediococcus acidilactici solution. The viable count of Pediococcus acidilactici in the Pediococcus acidilactici solution is 10 12 cfu / ml. The commercially available Pediococcus acidilactici is prepared in the same way (brand: Heta, model: HTSW-1102-379, purchase link: https: / / detail.1688.com / offer / 849511629757.html?src=zhanwai&pid=301012_1000&ptid=hr5a266048d7f340&exp=enquiry%3AB%3BqueryMobilePhone%3AA%3Bxlyx%3AB&_force_exp_buckets_=11803%2C2024061701%2C2024011602&spm=a312h.2018_new_sem.dh_002.5.133b6a8a6lz4Ql&cosite=360jj_sc&tracelog=p4p&_p_isad=1&clickid=b60026b124f34e759547e4613f88644a&sessionid=e2fced052ceab6a8fee9e5c6ff651e96&a=1417&e=gJUt7J-4uON9LhEpsmeihq1BSelSgz6axIjDFT2DCthc.8GlOiSl553gRLGoKYujB7K3YAv-BtPNSgj-zs.KvUrUcLiLCDMYC.wkkaFN1IfNEt0kAH0Sq7P6Jty7epA9luB74Pog8-gNt5qIR3wBN594NeIuhMTsFn5sbeQTxCiRSP4yt-mUQ0kjBM95GFl0JaiZWHFLajHdMgOwZdyAKfswczFDPKtnBAsUcTWapvwWjdBhvE9UubBYdDF7z2vlAVIi0m5KLO0fa7Tn0iqbS32S7B8r4B7VlJM2hrOcICKJ.UviC5biO30bNCeVllpr&sk=sem&style=1).

[0035] Major Bupleurum Decoction: Take 15 g of Bupleurum chinense, 9 g of Scutellaria baicalensis, 9 g of Paeonia lactiflora, 9 g of Pinellia ternata, 9 g of Aurantii Fructus Immaturus, 6 g of Rheum palmatum, 12 g of Jujube Fruit, and 15 g of Fresh Ginger, and decoct them in water to make a 125-ml solution. The specific decocting method is as follows: Add 500 ml of water, bring the solution to a boil over high heat, and then simmer it over low heat for 25 minutes. Filter the solution to obtain 250 ml of the decoction, and continue to decoct it over low heat for 25 minutes to obtain 125 ml of the decoction.

[0036] I. Experimental Methods

[0037] (1) Establishment and grouping of autism mouse models.

[0038] Adult C57BL / 6 mice were selected and housed in a clean - grade animal room, with free access to food and water. An autism mouse model was established using sodium valproate according to the method of T Schneider et al.

[0039] Adult C57BL / 6 mice with a male - to - female ratio of 1:3 were caged together overnight. After the female mouse was detected with a vaginal plug, it was marked as embryonic day 1. Pregnant mice were intraperitoneally injected with sodium valproate for modeling from embryonic days 10 - 13, and the pups were weaned at 21 days after birth.

[0040] After behavioral assessment, 150 autistic pups were selected and randomly divided into five groups: autism model group (ASD), group of mice with autism treated with Bupleurum and Major Decoction (TCM + ASD), group of mice with autism treated with Pediococcus acidilactici QZ - 01 (PCM + ASD), group of mice with autism treated with the combination of Bupleurum and Major Decoction and Pediococcus acidilactici QZ - 01 (TPCM + ASD), and group of mice with autism treated with the combination of Bupleurum and Major Decoction and commercially available Pediococcus acidilactici HT (HTCM + ASD).

[0041] After the autism mouse model was successfully established, the mice in the autism model group were gavaged with 0.5 mL of normal saline every day, and the experimental groups were respectively gavaged with the same dose of traditional Chinese medicine decoction, probiotic solution, and the combination of traditional Chinese medicine decoction and probiotic solution (Table 1).

[0042] Table 1 Classification settings of experimental groups.

[0043] (2) Analysis of synergistically improving the core symptoms of autistic mice.

[0044] Through autism behavioral tests (hot plate test, open - field test, three - chamber test, water maze test), the differences in social interaction, stereotyped behavior, self - exploration, and cognitive abilities of mice in the group of mice with autism treated with the combination of Bupleurum and Major Decoction and Pediococcus acidilactici QZ - 01 (TPCM + ASD), the group of mice with autism treated with the combination of Bupleurum and Major Decoction and commercially available Pediococcus acidilactici HT (HTCM + ASD), autism model group (ASD), group of mice with autism treated with Bupleurum and Major Decoction (TCM + ASD), and group of mice with autism treated with Pediococcus acidilactici QZ - 01 (PCM + ASD) were compared and analyzed.

[0045] (3) Observation of intestinal tissue morphology.

[0046] After 3 months of intervention in each group, 5 experimental mice were sacrificed by cervical dislocation, and the small intestine and large intestine tissues were isolated, fixed with 4% paraformaldehyde solution, and then comprehensively observed for morphological characteristics by HE staining, immunohistochemistry, immunofluorescence, etc. after dehydration, clearing, wax infiltration, embedding, sectioning, and staining steps using an FSX100 intelligent biological image navigator.

[0047] (4) Evaluation of the intestinal health of mice.

[0048] After the mice were sacrificed by cervical dislocation, blood was collected, and humoral immune indexes such as IgG, TNF-α, and TGF-β in the serum were detected by ELISA; a commercial standard kit was used to detect oxidative stress markers ROS, antioxidant markers GSH, CAT, and intestinal injury markers fatty acid binding protein, D-lactic acid, diamine oxidase, and LPS by spectrophotometry.

[0049] (5) Evaluation of the synergistic effect of two substances, Dachengqi Decoction and Pediococcus acidilactici

[0050] The evaluation of the synergistic effect of Dachengqi Decoction and Pediococcus acidilactici was carried out by the equiradiant analysis method recognized in the field. Taking the three-chamber social behavior obtained from the core symptoms of ASD as an index, Dachengqi Decoction and Pediococcus acidilactici were formulated in a ratio of 1:1. Calculate the median effective dose (IC50mix) of Dachengqi Decoction, Pediococcus acidilactici, and the combination of Dachengqi Decoction and Pediococcus acidilactici on the improvement of three-chamber social behavior respectively. The results of the combination of the two drugs were analyzed using an equiradiant analysis diagram: (1) The IC50 values of the two drugs when acting alone were marked on the X and Y axes respectively, and the two points were connected to obtain an additive line, that is, the equivalent curve of the two drugs showing a simple additive effect; (2) The upper and lower limits of the 95% confidence interval of the IC50 values of the two drugs were marked and connected with a dotted line respectively, and the range within the dotted line was the 95% confidence limit of the additive effect of the two drugs; (3) Draw the ratio line of the combination of the two drugs and calculate the theoretical median improvement concentration (IC50add) of the combination of the two drugs; (4) Mark the coordinate points representing the IC50mix value and the IC50add value on the equiradiant analysis diagram respectively. If IC50mix < IC50add and the coordinate point of IC50mix falls on the left side of the 95% confidence limit, it indicates that the interaction between the two drugs is synergistic.

[0051] (6) Data collection and analysis.

[0052] GraphPad Prism 9 was used for data statistical analysis and the production of various statistical charts. All data were expressed as mean ± SD, and normality and homogeneity of variance tests were performed. If they conformed to normal distribution and homogeneity of variance, an independent sample T-test was used for comparison between data groups; if they were non-normal or had unequal variances, the Wilcoxon rank sum test was used, with P < 0.05 as the significance test level. Behavioral analysis was carried out using the Anymaze video acquisition and analysis system of Global Biotech Inc. in the United States. This system has a built-in comparison function, can locate the movement trajectory of mice, record the movement time, and automatically analyze by region. All analyses were offline analyses. The data were exported to Excel, sorted, imported into Graphpad prism 9 for plotting, and statistical analysis was performed using its built-in analysis software.

[0053] II. Result Analysis

[0054] As Figure 2 shown, after the treatment with the combination of Bupleurum and Pinococcus acidilactici (TPCM+ASD) and the combination of Bupleurum and commercially available Pinococcus acidilactici HT (HTCM+ASD), compared with the autism model group (ASD), the single Bupleurum treatment group (TCM+ASD), and the single Pinococcus acidilactici treatment group (PCM+ASD), the autistic-like behaviors of autistic mice were significantly improved, indicating that the combination of Bupleurum and Pinococcus acidilactici has a better therapeutic effect than the single use of Bupleurum or Pinococcus acidilactici, and there is a synergistic effect in improving the core symptoms of ASD between the two.

[0055] As Figure 3 shown, the results of the isobolographic analysis of traditional Chinese medicine Bupleurum and Pinococcus acidilactici QZ-01 are as follows. Point A represents the IC50 value of the single action of Pinococcus acidilactici QZ-01; Point B represents the IC50 value of the single action of Bupleurum; the blue solid line is the additive line of the interaction between the two drugs; the dotted line range is the 95% confidence limit; the green solid line is the proportional line of the compatibility of Bupleurum and probiotic QZ-01 in a ratio of 1:1; Point C is the intersection point of the additive line and the proportional line, that is, the theoretical half inhibitory concentration IC50add of the compatibility group; Point D represents the measured half inhibitory concentration IC50mix of the compatibility group. It can be seen from Figure 3 this that the IC50mix of the compatibility of traditional Chinese medicine Bupleurum and Pinococcus acidilactici QZ-01 in a ratio of 1:1 falls on the left side of the 95% confidence limit of the additive line, and IC50mix < IC50add, which indicates that there is a synergistic effect after the compatibility of traditional Chinese medicine Bupleurum and probiotic QZ-01 in a ratio of 1:1.

[0056] As Figure 4 shown, after the treatment with the combination of Bupleurum and Pinococcus acidilactici (TPCM+ASD) and (HTCM+ASD), compared with the autism model group (ASD), the single Bupleurum treatment group (TCM+ASD), and the single Pinococcus acidilactici treatment group (PCM+ASD), the oxidative stress and immune function of the autistic mice were significantly improved, and the effect of the combined drug and bacteria group was significantly higher than that of the single Bupleurum treatment and the single Pinococcus acidilactici treatment group, indicating that the combination of Bupleurum and Pinococcus acidilactici has a better therapeutic effect than the single use of traditional Chinese medicine or probiotics, and there is a synergistic effect between the two.

[0057] As Figure 5As shown, after the treatment with the combination of Bupleurum and Pinococcus acidilactici (TPCM+ASD) and (HTCM+ASD), compared with the autism model group (ASD), the single treatment group of Bupleurum (TCM+ASD), and the single treatment group of Pinococcus acidilactici (PCM+ASD), the intestinal barrier function of the autistic mice has been significantly improved. The effect of the combined treatment group of medicine and bacteria is significantly higher than that of the single treatment group of Bupleurum and the single treatment group of Pinococcus acidilactici, indicating that the combination of Bupleurum and Pinococcus acidilactici has a better therapeutic effect than the single use of Bupleurum or Pinococcus acidilactici, and there is a synergistic effect between the two.

[0058] As Figure 6 Shown is the HE staining of the hippocampus. The results show that compared with the control group, the neurons in the DG area of the hippocampus in the ASD group are arranged loosely and there are neuron necroses. After the combined treatment of medicine and bacteria, the cell atrophy is slowed down and the neurons are arranged tightly, indicating that the combined treatment of medicine and bacteria may improve the hippocampal function through the gut-brain axis.

[0059] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A pharmaceutical composition for treating autism spectrum disorder, characterized in that: It is composed of Pediococcus acidilactici and Da Chaihu Decoction.

2. A pharmaceutical composition for treating autism spectrum disorder according to claim 1, characterized in that: The ratio of adding Pediococcus acidilactici to Da Chaihu Tang is 10 10 cfu: 10g, of which the mass of Da Chaihu Tang is recorded according to the mass of medicinal materials.

3. A pharmaceutical composition for treating autism spectrum disorder according to claim 1, characterized in that: The preparation method of Da Chaihu Decoction is as follows: according to weight parts, take 12-16 parts of Bupleurum, 8-10 parts of Scutellaria, 8-10 parts of Paeonia, 8-10 parts of Pinellia, 8-10 parts of Citrus aurantium, 5-7 parts of Rhubarb, 11-13 parts of Jujube, and 14-16 parts of ginger, and boil them in water.

4. A pharmaceutical composition for treating autism spectrum disorder according to claim 1, characterized in that: The lactic acid Pediococcus is prepared into a lactic acid Pediococcus liquid, which is then mixed with Da Chaihu Decoction. The number of viable lactic acid Pediococcus in the lactic acid Pediococcus liquid is 10 12 cfu / ml.

5. A pharmaceutical composition for treating autism spectrum disorder according to claim 1, characterized in that: The name of Pediococcus acidilactici is Pediococcus acidilactici QZ-01, and the preservation number is CCTCC M2019930.

6. Use of the pharmaceutical composition according to any one of claims 1 to 5 in the preparation of a drug for treating autism spectrum disorders.

7. Use of the pharmaceutical composition according to claim 6 in the preparation of a drug for treating autism spectrum disorders, characterized in that: The drug uses the drug composition as an active ingredient for treating autism spectrum disorders, and also contains a pharmaceutically acceptable carrier or auxiliary material to be prepared into a pharmaceutically acceptable dosage form.

8. Use of the pharmaceutical composition according to claim 7 in the preparation of a drug for treating autism spectrum disorders, characterized in that: The carrier or auxiliary material is solid, liquid or semi-solid.

9. Use of the pharmaceutical composition according to claim 7 in the preparation of a drug for treating autism spectrum disorders, characterized in that: The dosage forms include tablets, granules, capsules, pills, transdermal microneedle preparations, oral solutions and injections.

Images