An autism spectrum disorder improver and a preparation method and application thereof

Abstract

This invention relates to an autism spectrum disorder modifier, comprising the following raw materials in parts by weight: 20-40 parts of fructooligosaccharides, 20-40 parts of galactooligosaccharides, 10-30 parts of spinach powder, 1-20 parts of Poria cocos powder, and 1-20 parts of blueberry powder. The autism spectrum disorder modifier of this invention is safe and effective. The various components work synergistically through different pathways to reduce the secretion of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) and the inflammatory mediator NO, inhibit the release of inflammatory mediators and pro-inflammatory factors, and balance inhibitory and excitatory circuits; it also acts on the release and synthesis of neurotransmitters; regulates the intestinal flora, increases the relative abundance of lactic acid and acetate-producing Bifidobacteriaceae, Enterococciceae, and Lactobacilliceae, as well as lactic acid and acetate-utilizing Prevotellaceae and Lachnospiraceae genera, increases the content of short-chain fatty acids, and improves the clinical symptoms of autism spectrum disorder.

Description

Technical Field

[0001] This invention relates to an autism spectrum disorder improver, its preparation method and application, belonging to the field of functional foods. Background Technology

[0002] Autism spectrum disorder (ASD), also known as autism spectrum disorder, is one of the most common neurodevelopmental disorders in children. It is mainly characterized by varying degrees of social impairment, communication difficulties, repetitive and stereotyped behaviors, and narrow interests. It includes autism, Asperger's syndrome, and unclassified pervasive developmental disorder. Children with ASD typically develop symptoms between 6 and 24 months of age, but some may have normal development initially, gradually exhibiting regressive changes such as loss of language and social skills between 24 and 36 months. Most children with ASD have a poor prognosis, essentially losing their social abilities and being unable to live independently, requiring lifelong parental care and placing a significant economic burden on individuals, families, and society.

[0003] The pathogenesis of autism remains unclear, but genetic factors, parental mental illness, and fetal exposure to psychotropic drugs may all be involved. Evidence suggests that many mutated genes in ASD are key components of activity-dependent signaling networks that regulate synaptic development and plasticity. Therefore, dysregulation of activity-dependent signaling pathways in neurons may play a crucial role in the etiology of autism spectrum disorders. Research indicates that autism is associated with central nervous system inflammation, leading to imbalances in plasma levels of certain inflammatory biomarkers. Identifying and measuring autism-specific inflammatory biomarkers may aid in rapid diagnosis and the identification of new therapeutic targets. Among these biomarkers, autism is associated with tumor necrosis factor-α (TNF-α), interleukin-8 (IL-8), and interleukin-6 (IL-6). Some literature suggests that high levels of IL-6 in the brains of autistic children can alter neuronal adhesion and migration, leading to an imbalance between inhibitory and excitatory circuits. They found that increased plasma levels of TNF-α, IL-8, and IL-6 may be a contributing factor to the development of autism.

[0004] As research into the etiology and pathogenesis of ASD deepens, scholars have discovered that the gut microbiota plays a crucial role in the occurrence and development of ASD. Studies have shown that the dominant bacterial phyla in both the ASD and control groups are Bacteroidetes and Firmicutes, followed by Proteobacteria, Actinobacteria, and Fusobacteria. The ASD group showed a significantly lower level of Bacteroidetes compared to the control group, with a reduced ratio of Bacteroidetes to Firmicutes. Bacteroidetes play an important role in polysaccharide digestion. Other studies have indicated that the abnormal carbohydrate digestion and intestinal mucosal dysbiosis in children with ASD may be related to a decrease in Bacteroidetes. A recent study on an autistic mouse model showed that treatment with Bacteroidetes fragilis strains can restore autism-related behaviors and gastrointestinal abnormalities, and also reduce previously reported high levels of Trichophytonceae and 4-ethylphenylsulfate, the latter being a metabolic marker of autism associated with Trichophytonceae, highlighting the importance of Bacteroidetes in the pathophysiology of ASD.

[0005] Short-chain fatty acids (SCFAs) are the end products of dietary fiber fermentation by gut microbiota. They not only provide energy to the host but also regulate blood-brain barrier permeability and maintain homeostasis within the central nervous system. The main SCFAs include acetic acid, propionic acid, and butyric acid. Studies have shown that butyrate can enter the brain via the bloodstream and bind to receptors such as GPR109A, regulating neuronal excitability, synaptic plasticity, and neural development. Furthermore, butyrate can transmit signals to the brain via the vagus nerve, influencing the synthesis and release of neurotransmitters such as dopamine and serotonin. These neurotransmitters are closely related to mood, cognition, and social functions. Simultaneously, SCFAs play a crucial role in the immune system, brain function, and behavior. They can enter cells to regulate immune homeostasis by modulating the NF-κB signaling pathway, inhibiting the production of pro-inflammatory factors such as high-sensitivity C-reactive protein, tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), and interleukin (IL).

[0006] Currently, there is no specific cure for ASD. Clinical treatment primarily employs a comprehensive approach, including medication, behavioral modification, educational training, and physical therapy. Internationally, there are no approved medications specifically targeting the core symptoms of autism; however, medication may play a role in treating its complications and related symptoms. Currently, antipsychotics and antidepressants are mainly used clinically to treat autistic patients; additionally, the use of oxytocin or vasopressin receptor antagonists appears to have some therapeutic effect on autism. Drug treatment requires long-term use, which can cause side effects. Therefore, developing natural, safe, and side-effect-free products to improve autism spectrum disorders is particularly important.

[0007] Chinese patent document CN116530684A discloses a probiotic supplement for alleviating depression in autistic patients and its preparation method, aiming to address the technical problem that prolonged use of existing antidepressants can disrupt the gut microbiota, leading to intestinal ecological imbalance and gastrointestinal reactions. The supplement includes probiotics, prebiotics, and nutrients. The probiotics include *Bifidobacterium adolescentis*, *Bifidobacterium rennet*, *Bifidobacterium breve*, *Bifidobacterium infantis*, *Bifidobacterium longum*, *Lactobacillus casei*, *Lactobacillus helveticus*, *Lactobacillus paracasei*, *Lactobacillus plantarum*, *Lactobacillus rhamnosus*, and *Lactobacillus salivarius*. The prebiotics include fructooligosaccharides, lactitol, L-arabinose, and inulin. The nutrients include vitamin B6, vitamin B9, and ginseng extract. This probiotic supplement addresses the technical problem that prolonged use of existing antidepressants can disrupt the gut microbiota, leading to intestinal ecological imbalance and gastrointestinal reactions; however, its effectiveness on clinical symptoms such as behavioral disorders in autism spectrum disorders remains unclear.

[0008] Chinese patent document CN114732880A discloses a traditional Chinese medicine composition, preparation method, and application for treating childhood autism. The composition combines Poria cocos with other traditional Chinese medicinal materials to improve symptoms of autism spectrum disorder. However, this invention is a traditional Chinese medicine combination, and long-term use may have certain side effects.

[0009] Chinese patent document CN 113318198 B discloses a capsule for treating childhood autism and its preparation method, which includes galacto-oligosaccharides, succinic anhydride, pumpkin polysaccharide, rutin, rice fermentation filtrate, β-carotene, microbial strains, fillers, and binders. It has many components, a complex preparation method, and is a capsule, which is not conducive to children taking it.

[0010] Therefore, designing a product that effectively improves the clinical symptoms of autism spectrum disorder, regulates gut microbiota, has simple, natural, safe and side-effect-free components, is of significant clinical value. Summary of the Invention

[0011] In view of the shortcomings of existing technologies, especially the poor clinical symptom treatment effects, long treatment periods, and obvious side effects of drug treatment for autism spectrum disorder, one objective of this invention is to provide an autism spectrum disorder improver, and the second objective is to provide a method for preparing the improved autism spectrum disorder improver.

[0012] The autism spectrum disorder modifier of this invention achieves its effects in three ways: firstly, by reducing the secretion of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) and the inflammatory mediator NO, inhibiting the release of inflammatory mediators and pro-inflammatory factors, balancing inhibitory and excitatory circuits, and reducing depressive-like and anxiety-like behaviors; secondly, by acting on the release and synthesis of neurotransmitters; and thirdly, by regulating the intestinal flora, increasing the relative abundance of lactic acid and acetate-producing Bifidobacteriaceae, Enterococciceae, and Lactobacilliceae, as well as lactic acid and acetate-utilizing Prevotellaceae and Lachnospiraceae genera, and increasing the content of short-chain fatty acids. It is safe and has no side effects.

[0013] To solve the above technical problems, the present invention is achieved through the following technical solution:

[0014] An autism spectrum disorder improver comprises the following raw materials in parts by weight: 20-40 parts of fructooligosaccharide, 20-40 parts of galactooligosaccharide, 10-30 parts of spinach powder, 1-20 parts of Poria cocos powder, and 1-20 parts of blueberry powder.

[0015] According to a preferred embodiment of the present invention, the autism spectrum disorder improver comprises the following raw materials in parts by weight: 25-35 parts of fructooligosaccharide, 25-35 parts of galactooligosaccharide, 15-25 parts of spinach powder, 10-18 parts of Poria cocos powder, and 2-10 parts of blueberry powder.

[0016] According to a preferred embodiment of the present invention, the autism spectrum disorder improver comprises the following raw materials in parts by weight: 30 parts of fructooligosaccharide, 30 parts of galactooligosaccharide, 20 parts of spinach powder, 15 parts of Poria cocos powder, and 5 parts of blueberry powder.

[0017] According to a preferred embodiment of the present invention, the spinach powder is prepared by the following method:

[0018] 1) After selecting, cleaning, and washing the spinach, remove the roots and cut it into sections, each section being 3-4cm long;

[0019] 2) Place the chopped spinach in hot water at 70-100℃, and add 0.2wt% anhydrous sodium bicarbonate solution at the same time, and blanch for 3-7 minutes;

[0020] 3) After blanching, drain the water, add purified water in a 1:1 ratio by mass, grind in a crusher, and then process with a colloid mill to obtain spinach pulp. Homogenize the spinach pulp 2 to 3 times.

[0021] 4) The homogenized spinach pulp is spray-dried until the moisture content is less than or equal to 8 wt% (dry basis), and then passed through an 80-mesh sieve to obtain spinach powder.

[0022] According to a preferred embodiment of the present invention, in step 3), the homogenization pressure is 25 MPa.

[0023] According to a preferred embodiment of the present invention, in step 4), the spray drying conditions are: inlet temperature 160-180°C, outlet temperature 70°C, peristaltic pump flow rate 50 mL / min, and fan frequency 40 Hz.

[0024] According to a preferred embodiment of the present invention, the Poria cocos powder is prepared by the following method:

[0025] Pulverize Poria cocos to 60-80 mesh, add deionized water to obtain a mixture with a water content of 5-10 wt%, then puff the mixture, place the puffed product in an oven at 60-70℃ and dry for 20-60 minutes to reduce its moisture content to 2-6 wt%, then pulverize it again and microwave it to obtain Poria cocos powder.

[0026] According to a preferred embodiment of the present invention, the puffing is performed using a twin-screw extruder, which is divided into three puffing zones: zone one, zone two, and zone three. During puffing, zone one is closed, the temperature of zone two is 110–120°C, and the temperature of zone three is 140–150°C. The screw speed of the twin-screw extruder is 45–50 rpm, the feeding speed is 8–13 rpm, and the rotary cutting speed is 16–20 rpm.

[0027] According to a preferred embodiment of the present invention, the particle size after secondary pulverization is 80-100 mesh; the microwave sterilization conditions are: temperature 60-70℃, time 2-9 min.

[0028] According to a preferred embodiment of the present invention, the blueberry powder is prepared by the following method:

[0029] (1) Grind the selected and cleaned blueberries in a colloid mill until they are evenly ground to obtain blueberry pulp;

[0030] (2) Freeze the blueberry pulp at -18℃ for 9 to 12 hours to ensure that the blueberry pulp is completely frozen;

[0031] (3) The frozen fruit pulp was placed in an in-situ vacuum freeze dryer and dried. The moisture content was measured every 0.5 hours until the moisture content was less than or equal to 8 wt% (dry basis).

[0032] (4) Place the freeze-dried blueberry pulp in a grinder and grind it. Then pass it through an 80-mesh sieve to obtain blueberry powder.

[0033] According to a preferred embodiment of the present invention, in step (3), the vacuum freeze-drying conditions are: vacuum degree 1.0 Pa, cold trap temperature -58.7℃ to -59.3℃.

[0034] According to the present invention, the preparation method of the above-mentioned autism spectrum disorder improver includes the following steps:

[0035] a. Pass fructooligosaccharides, galactooligosaccharides, spinach powder, poria cocos powder, and blueberry powder through an 80-mesh sieve, and mix them evenly according to the proportions to obtain a mixture;

[0036] b. Add alcohol to the mixture to obtain a soft material; then granulate it through a 20-mesh sieve, dry it at 50-60℃ for 2-3 hours after granulation, and then granulate and sieve to obtain an autism spectrum disorder improver.

[0037] According to a preferred embodiment of the present invention, in step b, the volume concentration of alcohol is 40% to 60%, and the amount of alcohol added is 5% to 10% of the weight of the mixture.

[0038] According to a preferred embodiment of the present invention, the above-mentioned autism spectrum disorder improver is used in the preparation of food, pharmaceutical or health products that improve autism spectrum disorder.

[0039] Technical features and advantages of the present invention:

[0040] The fructo-oligosaccharides (FOS) of this invention are non-reducing sugars, a natural active ingredient, and an excellent water-soluble dietary fiber. As one of the most studied and widely used prebiotics, FOS has been proven to be the only all-natural ingredient possessing the dual physiological properties of both a powerful bifidogenic factor and water-soluble dietary fiber. FOS can be fermented by beneficial bacteria such as Bifidobacteria, partially converting into short-chain fatty acids (mainly acetic acid and butyric acid) and a small amount of gas, which is beneficial to the host's health. Short-chain fatty acids can lower the pH of the gut microbiota, increase the growth of beneficial bacteria, and inhibit the growth of pathogens. Furthermore, the presence of short-chain fatty acids in the central nervous system contributes to the release and synthesis of neurotransmitters. Therefore, FOS improves the clinical symptoms of autism spectrum disorder through the gut-microbiota axis by regulating gut microbiota composition and its metabolites, increasing neurochemicals and short-chain fatty acids, and reducing intestinal permeability.

[0041] The galacto-oligosaccharide (GOS) of this invention is a functional oligosaccharide, belonging to a class of functional factors of prebiotics, possessing excellent physicochemical properties and unique physiological functions. On the one hand, GOS can promote the proliferation of Bifidobacteria and Lactobacillus, inhibit the proliferation of Clostridium, Bacteroides, Veillonella, and other microorganisms, regulate the intestinal flora, alter the production and content of short-chain fatty acids, regulate blood-brain barrier permeability, maintain the homeostasis of the central nervous system, and improve intestinal health and social behavior in autism spectrum disorder. On the other hand, GOS competitively binds to the hydrophobic cavity of MD-2, significantly reducing the secretion of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) and the inflammatory mediator NO, inhibiting the release of inflammatory mediators and pro-inflammatory factors, and balancing inhibitory and excitatory circuits. Simultaneously, GOS can reduce stress-induced corticosterone release; lower isobutyrate concentration; reduce pro-inflammatory cytokine levels and depressive-like and anxiety-like behaviors, and improve clinical symptoms of autism spectrum disorder.

[0042] The spinach powder of this invention is rich in nutrients, characterized by low fat, low calories, and high dietary fiber. It contains 18 essential amino acids, various vitamins, and abundant minerals. Spinach also contains a balanced ratio of omega-3 and omega-6 fatty acids, as well as various bioactive substances such as flavonoids, and has been named one of the "Top Ten Foods Beneficial to Humans" by Time magazine. The vitamins A, D, K, C, and B in spinach, along with minerals like zinc and iron, coumarins, and omega-3 fatty acids, can act on neurotransmitters, protect the nervous system, and thus improve the clinical symptoms of autism spectrum disorder. Furthermore, spinach powder can enhance the levels of bacteria in the genera *Prevotellaceae* and *Lachnospiraceae*, while *Lachnospiraceae* can produce butyrate. Butyrate regulates tyrosine hydroxylase (TH) mRNA levels, potentially affecting the catecholamine pathway in the brain and positively impacting ASD. Therefore, spinach powder can indirectly affect the central nervous system and improve abnormal behaviors associated with autism spectrum disorder by regulating the gut microbiota.

[0043] The Poria cocos powder of this invention contains abundant Poria cocos polysaccharides, which have functions such as enhancing immunity, regulating intestinal flora, improving glucose and lipid metabolism levels, and mitigating oxidative damage. These polysaccharides are of great significance for preventing and alleviating human diseases and sub-health conditions. On the one hand, Poria cocos polysaccharides increase the thickness of the small intestinal mucosa and the height of villi, reduce epithelial damage, lower TNF-α, IL-1β, and IL-6 levels, and inhibit the phosphorylation of JAK2 and STAT3. On the other hand, Poria cocos polysaccharides can significantly increase the abundance of lactic acid-producing bacteria and regulate intestinal flora. Therefore, Poria cocos polysaccharides improve social behavior in autism spectrum disorder through these two mechanisms of action.

[0044] The blueberry powder of this invention is rich in blueberry polyphenols, including extractable polyphenols (EPP) and non-extractable polyphenols (NEPP). Both EPP and NEPP can inhibit the expression of IL-1β and IL-6 mRNA in cells, reducing neuroinflammation. Simultaneously, blueberry polyphenols inhibit microRNAs (miRNAs) (miR-21, miR-146a, and miR-125b) to varying degrees, reduce tumor necrosis factor-α and interleukin-6 activity, and improve abnormal behaviors associated with autism spectrum disorder. Furthermore, blueberry polyphenols inhibit the growth of harmful bacteria, promote the proliferation of beneficial bacteria, reduce the abundance of Prevotella, and increase the abundance of Bifidobacterium, Desulfovibrio, Helicobacter, and other genera. Therefore, blueberry polyphenols can also improve the clinical symptoms of autism spectrum disorder by influencing the gut microbiota.

[0045] 1. The autism spectrum disorder modifier provided by this invention utilizes fructooligosaccharides to regulate gut microbiota and short-chain fatty acids to act on the release and synthesis of neurotransmitters, thereby improving clinical symptoms of autism spectrum disorder through the gut-brain axis; galactooligosaccharides improve gut health and social behavior in autism spectrum disorder by inhibiting the release of inflammatory mediators and pro-inflammatory factors, promoting the growth of beneficial bacteria, and reducing harmful bacteria; spinach powder, rich in vitamins, minerals, coumarins, and omega-3 fatty acids, acts on neurotransmitters and protects the nervous system, while also enhancing the function of the Prevotellaceae genus. Like the Lachnospiraceae genus, it improves the gut microbiota, thereby improving abnormal behaviors in autism spectrum disorder; Poria cocos powder is rich in Poria cocos polysaccharides, which reduce the levels of TNF-α, IL-1β and IL-6, inhibit the phosphorylation of JAK2 and STAT3, and regulate the gut microbiota to improve the clinical symptoms of autism spectrum disorder; Blueberry powder is rich in blueberry polyphenols, which improve the gut health and clinical symptoms of autism spectrum disorder by inhibiting the activity of inflammatory factors in cells: tumor necrosis factor-α and interleukin-6 and regulating the gut microbiota.

[0046] 2. The autism spectrum disorder improver provided by this invention utilizes fructooligosaccharides and spinach powder to synergistically promote the release and synthesis of neurotransmitters, protect the nervous system, and improve the clinical symptoms of autism spectrum disorder. Galuctooligosaccharides, Poria cocos powder, and blueberry powder synergistically reduce the expression levels of TNF-α, IL-1β, and IL-6, reducing neuroinflammation and thus improving the clinical symptoms of autism spectrum disorder. Furthermore, fructooligosaccharides, galuctooligosaccharides, spinach powder, Poria cocos powder, and blueberry powder synergistically regulate the intestinal flora, increasing the relative abundance of beneficial bacteria, inhibiting the growth of harmful bacteria, and increasing the content of short-chain fatty acids, thereby improving intestinal health and clinical symptoms of autism spectrum disorder.

[0047] 3. The autism spectrum disorder improver provided by this invention combines the effects of the above five ingredients. It is rationally formulated, safe and effective. The various ingredients work synergistically through different pathways, acting on the release and synthesis of neurotransmitters, protecting the nervous system, reducing the expression levels of TNF-α, IL-1β and IL-6, reducing neuroinflammation, regulating intestinal flora, increasing the relative abundance of beneficial bacteria and the content of short-chain fatty acids, especially butyrate, and thus significantly improving the symptoms of autism spectrum disorder.

[0048] 4. The spinach powder of the present invention is treated by spray drying. Spray drying can effectively preserve the color, chlorophyll, total phenols, total flavonoids and other nutrients and antioxidant activity of the spinach powder. The retention of more nutrients is beneficial to reducing the amount of spinach powder added under the same nutrient requirements.

[0049] 5. The Poria cocos powder of the present invention is processed by extrusion puffing. Extrusion puffing can significantly increase the content of water-soluble polysaccharides in Poria cocos, while having little effect on the three main triterpenoid acids in Poria cocos: 3-epi-dehydropachyric acid, dehydropachyric acid, and dehydrotomolic acid, thereby effectively improving the clinical symptoms of autism spectrum disorder.

[0050] 6. The blueberry powder of the present invention is processed by vacuum freeze-drying, which can avoid significant changes in the color, texture, nutrition and efficacy of the material, which is conducive to the preservation of total phenols, thereby improving the intestinal flora, reducing the activity of tumor necrosis factor-α and interleukin-6, alleviating neuroinflammation and improving abnormal behaviors of autism spectrum disorder. Attached Figure Description

[0051] Figure 1 This is a graph showing the self-grooming time analysis of Experiment Example 1 in the mine.

[0052] Figure 2 This is a time analysis diagram of the mine experiment in Experiment Example 1 during the middle of the open field.

[0053] Figure 3 This is a time analysis chart for the three-room experiment in Experiment Example 1, specifically in the toy box and the unfamiliar mouse box.

[0054] Figure 4 This is a time analysis chart for the pole climbing experiment in Experiment Example 1.

[0055] Figure 5 This is a time analysis diagram for the suspension rope experiment in Experiment Example 1.

[0056] Figure 6 The graph shows the time analysis of the rotating rod experiment in Experiment Example 1.

[0057] Figure 7 This is a graph showing the self-grooming time analysis in the mine experiment of Experiment Example 2.

[0058] Figure 8This is a time analysis diagram of the mine experiment in Experiment Example 2, taken in the middle of an open field.

[0059] Figure 9 This is a time analysis chart for the three-room experiment in Experiment Example 2, specifically in the toy box and the unfamiliar mouse box.

[0060] Figure 10 This is a time analysis chart for the pole climbing experiment in Experiment Example 2.

[0061] Figure 11 This is a time analysis diagram for the suspension rope experiment in Experiment Example 2.

[0062] Figure 12 This is a time analysis graph for the rotating rod experiment in Experiment Example 2. Note: Figures 1-12 In the figure, * indicates that compared with the model control group, *P<0.05, **P<0.01.

[0063] Figure 13 This is a graph showing the relative abundance of the target bacterial community in Experiment Example 3.

[0064] Figure 14 Butyric acid spectrum of the blank control group.

[0065] Figure 15 Butyrate spectrum of the model control group.

[0066] Figure 16 Butyric acid spectrum of experimental group 1.

[0067] Figure 17 Butyric acid spectrum of experimental group 2.

[0068] Figure 18 Butyric acid spectrum of experimental group 3.

[0069] Figure 19 Butyric acid spectrum of experimental group 4.

[0070] Figure 20 Butyric acid spectrum of experimental group 5.

[0071] Figure 21 The image shows the butyric acid analysis result for Experiment Example 3. Detailed Implementation

[0072] The present invention will be described in detail below with reference to specific embodiments. The following embodiments are only for the purpose of enabling those skilled in the art to understand the technical solutions of the present invention, implement or use the present invention, and are not intended to limit the scope of protection of the present invention.

[0073] Fructooligosaccharides are available from Baolingbao Biotechnology Co., Ltd.; galactooligosaccharides are available from Baolingbao Biotechnology Co., Ltd.; spinach and blueberries are available in chain supermarkets; Poria cocos is available in chain pharmacies.

[0074] Example 1

[0075] An autism spectrum disorder improver, the raw materials are as follows by weight: 30 parts fructooligosaccharide, 30 parts galactooligosaccharide, 20 parts spinach powder, 15 parts Poria cocos powder, and 5 parts blueberry powder.

[0076] Spinach powder is prepared according to the following method:

[0077] 1) Select fresh, organic spinach free from pests and diseases, and remove yellow leaves and broken spinach;

[0078] 2) Wash the selected spinach to remove dirt and impurities.

[0079] 3) Remove the roots of the spinach and cut it into sections, each section being 4cm long;

[0080] 4) Place the cut pieces in hot water at 90°C, and add an anhydrous sodium bicarbonate solution with a mass concentration of 0.2wt% for 4 minutes.

[0081] 5) After blanching, remove the spinach and drain the water. Add purified water at a mass ratio of 1:1 and grind it in a pulverizer. Then process it with a colloid mill. Homogenize the processed spinach pulp three times with a homogenizer at a pressure of 25 MPa.

[0082] 6) The homogenized slurry is spray-dried under the following conditions: inlet temperature 170℃, outlet temperature 70℃, peristaltic pump flow rate 50mL / min, fan frequency 40Hz, until the raw material moisture content is less than 8wt% (dry basis), and then passed through an 80-mesh sieve to obtain spinach powder.

[0083] Poria cocos powder is prepared according to the following method:

[0084] Poria cocos was pulverized to 80 mesh, added to deionized water, and mixed evenly to obtain a mixture with a water content of 8 wt%. The mixture was then puffed under the following conditions: zone one was closed, zone two temperature was 110℃, and zone three temperature was 140℃; the screw speed of the twin-screw extruder was 45 rpm, the feeding speed was 10 rpm, and the rotary cutting speed was 18 rpm; the puffed product was dried in a 65℃ oven for 30 minutes to reduce its moisture content to 3%, then pulverized again to 80 mesh, and finally microwaved at 60℃ for 6 minutes to obtain the final product.

[0085] Blueberry powder is prepared according to the following method:

[0086] (1) Select blueberries that are uniform in size, intact, and uniform in color, and remove rotten or damaged blueberries;

[0087] (2) Wash the blueberries.

[0088] (3) Grind the cleaned blueberries in a colloid mill until they are evenly ground to form blueberry pulp;

[0089] (4) Place the ground blueberry pulp in a tray and freeze it in a refrigerator at -18°C for 10 hours until the blueberry pulp is completely frozen.

[0090] (5) The frozen fruit pulp was placed in an in-situ vacuum freeze dryer for drying. The vacuum freeze drying conditions were: vacuum degree 1.0 Pa, cold trap temperature -59℃, moisture content measured every 0.5 h, and dried until the moisture content of the raw material was less than 8 wt% (dry basis).

[0091] (6) Place the freeze-dried blueberry pulp in a grinder and grind it. Then pass it through an 80-mesh sieve to obtain blueberry powder.

[0092] Preparation method of autism spectrum disorder improver:

[0093] Fructooligosaccharides, galactooligosaccharides, spinach powder, poria cocos powder, and blueberry powder were each passed through an 80-mesh sieve and mixed evenly in proportion to obtain a mixture. Alcohol with a volume concentration of 45% was added to the mixture, with the amount of alcohol added being 8% of the weight of the mixture, to obtain a soft material. This material was then granulated by passing it through a 20-mesh sieve. After granulation, it was dried at 55℃ for 2.5 hours, then granulated again and sieved to obtain an autism spectrum disorder improver.

[0094] Example 2

[0095] An autism spectrum disorder improver, the raw materials are as follows by weight: 40 parts fructooligosaccharide, 20 parts galactooligosaccharide, 30 parts spinach powder, 20 parts poria cocos powder, and 1 part blueberry powder.

[0096] The preparation of spinach powder, poria powder, and blueberry powder was carried out according to Example 1.

[0097] The preparation method of the autism spectrum disorder improver was carried out according to Example 1.

[0098] Example 3

[0099] An autism spectrum disorder improver, comprising the following ingredients by weight: 20 parts fructooligosaccharide, 40 parts galactooligosaccharide, 10 parts spinach powder, 1 part Poria cocos powder, and 20 parts blueberry powder.

[0100] The preparation of spinach powder, poria powder, and blueberry powder was carried out according to Example 1.

[0101] The preparation method of the autism spectrum disorder improver was carried out according to Example 1.

[0102] Comparative Example 1

[0103] An autism spectrum disorder improver, the raw materials are as follows by weight: 50 parts fructooligosaccharide, 50 parts galactooligosaccharide, 40 parts spinach powder, 30 parts Poria cocos powder, and 25 parts blueberry powder.

[0104] The preparation of spinach powder, poria powder, and blueberry powder was carried out according to Example 1.

[0105] The preparation method of the autism spectrum disorder improver was carried out according to Example 1.

[0106] Comparative Example 2

[0107] An autism spectrum disorder improver, the raw materials are as follows by weight: 15 parts fructooligosaccharide, 15 parts galactooligosaccharide, 7 parts spinach powder, 0.8 parts Poria cocos powder, and 0.8 parts blueberry powder.

[0108] The preparation of spinach powder, poria powder, and blueberry powder was carried out according to Example 1.

[0109] The preparation method of the autism spectrum disorder improver was carried out according to Example 1.

[0110] Comparative Example 3

[0111] An autism spectrum disorder improver, which does not contain blueberry powder, contains the following ingredients by weight: 30 parts fructooligosaccharides, 30 parts galactooligosaccharides, 20 parts spinach powder, and 15 parts Poria cocos powder.

[0112] The preparation of spinach powder and poria powder was carried out according to Example 1.

[0113] The preparation method of the autism spectrum disorder improver was carried out according to Example 1.

[0114] Comparative Example 4

[0115] An autism spectrum disorder improver, which does not contain Poria cocos powder, contains the following ingredients by weight: 30 parts fructooligosaccharides, 30 parts galactooligosaccharides, 20 parts spinach powder, and 5 parts blueberry powder.

[0116] The preparation of spinach powder and blueberry powder was carried out according to Example 1.

[0117] The preparation method of the autism spectrum disorder improver was carried out according to Example 1.

[0118] Comparative Example 5

[0119] An autism spectrum disorder improver, which does not contain spinach powder, contains the following ingredients by weight: 30 parts fructooligosaccharides, 30 parts galactooligosaccharides, 15 parts Poria cocos powder, and 5 parts blueberry powder.

[0120] The preparation of Poria cocos powder and blueberry powder was carried out according to Example 1.

[0121] The preparation method of the autism spectrum disorder improver was carried out according to Example 1.

[0122] Comparative Example 6

[0123] An agent for improving autism spectrum disorder, without containing galactooligosaccharide, and the raw material parts by weight are as follows: 30 parts of fructooligosaccharide, 20 parts of spinach powder, 15 parts of poria cocos powder, and 5 parts of blueberry powder.

[0124] The preparation of spinach powder, poria cocos powder, and blueberry powder is carried out according to Example 1.

[0125] The preparation method of the agent for improving autism spectrum disorder is carried out according to Example 1.

[0126] Comparative Example 7

[0127] An agent for improving autism spectrum disorder, without containing fructooligosaccharide, and the raw material parts by weight are as follows: 30 parts of galactooligosaccharide, 20 parts of spinach powder, 15 parts of poria cocos powder, and 5 parts of blueberry powder.

[0128] The preparation of spinach powder, poria cocos powder, and blueberry powder is carried out according to Example 1.

[0129] The preparation method of the agent for improving autism spectrum disorder is carried out according to Example 1.

[0130] Experimental Example 1

[0131] The efficacy of improving autism spectrum disorder was evaluated using a VPA-induced autism-like model.

[0132] Experimental subjects: SPF-grade ICR mice were selected in the mouse experiment.

[0133] Location of the experimental animal center: The 1st floor of Building 2, No. 519, South Road, Gangxing 2nd Road, High-tech Zone, Jinan City, Shandong Province.

[0134] Qualification of the animal center: License number: SYXK(Lu)2023 0030.

[0135] Experimental method:

[0136] At 7:00 pm daily, an 8-week-old female mouse and an adult male mouse were placed together overnight. The following 8:00 am, pregnant mice with vaginal plugs were separated and housed individually, and this was recorded as gestation day 0.5 (E0.5d). The pregnant mice were randomly divided into two groups. On E12.5d, one group received an intraperitoneal injection of 600 mg / kg VPA (VPA dissolved in 0.9% physiological saline to a concentration of 250 mg / mL), while the other group received an intraperitoneal injection of the same volume of physiological saline. The male offspring from the two groups were designated as the ASD model group and the blank control group, respectively. Day 1 of birth for offspring mice was designated P1. The ASD model group was randomly divided into 6 groups: a model control group and experimental groups 1-5. Starting from P7, experimental groups 1-3 received the improvers from Examples 1-3, while experimental groups 4-5 received the improvers from Comparative Examples 1-2. The recommended human dose was 6g / 60kg bw daily (main dose only). The intermediate dose group received 1000mg / kg bw (equivalent to 10 times the recommended human intake). The solution was prepared to the required concentration using physiological saline. Each mouse was administered 0.2mL / 10g bw via gavage. The blank control group and model control group were administered physiological saline via gavage. Gavage was performed at 20:00 every night for 7 consecutive days. Offspring mice in all groups were weaned at P21, and behavioral experiments began at P35. At P49, orbital vein blood was collected from three groups of offspring mice, and serum tumor necrosis factor-α and interleukin-6 levels were measured using enzyme-linked immunosorbent assay (ELISA).

[0137] Behavioral experiments:

[0138] (1) Pole Climbing Test: The pole climbing test was used to assess the motor coordination of mice. P35 offspring mice were used in the experiment. A cylindrical pole with a diameter of 1.5 cm and a length of 50 cm was used. A ball with a diameter of 2.5 cm was fixed to the top of the pole and wrapped with tape to prevent the mice from slipping. Each mouse was placed on top of the pole, face down, and the time it took for each mouse to reach the bottom of the pole was measured. The experiment was conducted over two days. On the first day, each mouse underwent two rounds of training. On the second day, each mouse was tested three times, with each test spaced two hours apart. The average time to reach the bottom of the pole was calculated. If a mouse failed to climb down within 180 seconds, it was excluded from the experiment. The pole was cleaned with 75% ethanol between tests to eliminate interference.

[0139] (2) Suspension test: The suspension test can be used to detect the motor coordination of mice. P38 offspring mice were used for the experiment. A 120cm long hemp rope, 40cm above the ground, was horizontally fixed between two platforms. Each mouse's forepaw was placed in the middle of the two platform lines, and the time it took for each mouse to fall to the ground was recorded. The experiment was conducted over two days. On the first day, each mouse underwent two rounds of training. On the second day, each mouse was tested three times, with a two-hour interval between each test. The average time to fall to the ground was analyzed. If a mouse did not fall for more than 180 seconds, the result was recorded as 180 seconds. Between tests, the hemp rope and the floor were cleaned with 75% ethanol to eliminate interference.

[0140] (3) Open Field Test: The open field test can be used to detect exploratory behavior and repetitive stereotyped behaviors in mice in unfamiliar environments. P42 offspring mice were used for the experiment. The open field box was 60cm long, 60cm wide, and 60cm high. Its bottom was artificially divided into 9 identical square areas, with the central area being the central area and the surrounding 8 areas collectively referred to as the peripheral areas. One day before the experiment, the mice were placed in the test room to acclimatize. During the test, each mouse was placed in the open field box with its head facing the box wall in the same position and allowed to move freely for 5 minutes. The ANY-Maze animal behavior video analysis system automatically recorded the time the mice spent in the central area and analyzed the self-grooming time of each mouse. The surrounding environment was kept quiet during the test, and the open field box was cleaned with 75% ethanol after each mouse was tested.

[0141] (4) Three-box test: The three-box test is a test of the social abilities of mice. P43 offspring mice were used for the experiment. The three-box equipment consisted of a 60cm×40cm×20cm transparent box, divided into three interconnected chambers by two movable transparent panels. The experiment lasted for two days. On the first day, no objects were placed in the three boxes. The mice were placed in the middle box and allowed to move freely in the three boxes for 5 minutes to adapt. On the second day, a male unfamiliar mouse (of similar age to the test mouse) was placed in one box on one side of the three boxes, and a toy was placed on the other side. No objects were placed in the middle box. The test mouse was then placed in the middle box and allowed to move freely for 5 minutes. The ANY-Maze animal behavior video analysis system automatically recorded the activity time of the mice in each box. The surrounding environment was kept quiet during the test. The three-box equipment was cleaned with 75% ethanol after each mouse was tested.

[0142] (5) Rotating bar test: The rotating bar test can be used to detect the motor coordination of mice. P46 offspring mice were used for the experiment. The experiment was conducted over 4 days. The rotating bar speed was set to 30 rpm and the rotating time was set to 3 min. The mice were given adaptive training for the first 3 days and the formal test was conducted on the 4th day. Each mouse was tested 3 times, with an interval of 2 hours between each test. The time it took for the mouse to fall off the rotating bar was recorded. The surrounding environment was kept quiet during the test. The rotating bar equipment was cleaned with 75% ethanol after each mouse was tested.

[0143] Data were processed using SPSS statistical software. t-tests were used to analyze significant differences between groups; P < 0.05 was considered statistically significant, and P < 0.01 was considered highly statistically significant. All data for the tested indicators are expressed as mean ± standard deviation. The test results are shown below. Figure 1-7 See Table 1.

[0144] Table 1: Cytokine levels in experimental groups 1-5, model control group, and blank control group mice

[0145] Serial Number Group TNF-α (pg / mL) IL-6 (pg / mL) 1 Blank control group 38.28±5.23** 15.05±4.40** 2 Model control group 71.80±7.53 42.04±7.17 3 Experimental group 1 53.99±4.16** 25.72±5.08** 4 Experimental group 2 58.66±4.59** 29.27±5.09** 5 Experimental group 3 60.41±4.53** 32.80±7.31* 6 Experimental group 4 65.15±6.78 35.27±7.76 7 Experimental group 5 66.47±6.72 37.76 5.56

[0146] Note: * indicates comparison with the model control group, *P<0.05, **P<0.01;

[0147] Tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6).

[0148] Depend on Figure 1-2 It was found that in the open field experiment, compared with the blank control group, the self-grooming time of the model control group mice within 5 minutes was significantly increased (P<0.01), indicating that the model control group mice exhibited obvious repetitive and stereotyped behaviors; the activity time of the model control group mice in the central area of ​​the open field was significantly reduced compared with the blank control group (P<0.01), indicating that the model control group mice exhibited reduced autonomous exploration behavior in unfamiliar environments and a decreased ability to explore new environments. Figure 3 The results showed that, in the three-box experiment, compared with the blank control group, the model control group mice spent significantly less time in the unfamiliar mouse box (P<0.01), while spending significantly more time in the toy box (P<0.01), indicating a significant reduction in social interaction behavior in the model control group mice. In conclusion, the model control group mice exhibited significantly reduced social interaction behavior, lacked social novelty towards unfamiliar mice, showed reduced autonomous exploration behavior in unfamiliar environments, and displayed obvious stereotyped and repetitive activities, suggesting successful ASD modeling.

[0149] Depend on Figure 1-6It was found that, compared with the model control group, experimental groups 1-2 had a significantly reduced self-grooming time (P<0.01), a significantly increased activity time in the central area of ​​the open field (P<0.01), a significantly increased time spent in the unfamiliar mouse box (P<0.01), and a significantly reduced time spent in the toy box (P<0.01); in the pole climbing experiment, they spent less time climbing to the bottom of the pole (P<0.05); in the rope suspension experiment, they spent more time falling from the rope (P<0.01); and in the spinner experiment, they spent more time on the spinner (P<0.01). Compared with the model control group, experimental group 3 showed a decrease in self-grooming time (P<0.05), a highly significant increase in activity time in the central area of ​​the open field (P<0.01), a highly significant increase in time spent in the unfamiliar mouse box (P<0.01), a decrease in time spent in the toy box (P<0.05), less time spent climbing to the bottom of the pole in the pole climbing experiment (P<0.05), a longer time spent falling from the rope in the suspension experiment (P<0.05), and a longer time spent on the spinner in the spinner experiment (P<0.01).

[0150] Compared with the model control group, experimental groups 4-5 showed no significant differences in self-grooming time, time spent in the central area of ​​the open field, time spent in the unfamiliar mouse box, time spent in the toy box, time spent climbing to the bottom of the pole, time spent falling from the suspension rope, and time spent on the spinning rod (P>0.05).

[0151] As shown in Table 1, compared with the blank control group, the serum TNF-α and IL-6 levels in the model control group were significantly increased (P < 0.01). In experimental groups 1 and 2, the serum TNF-α and IL-6 levels in mice were significantly decreased (P < 0.01). Compared with the model control group, the serum TNF-α level in experimental group 3 was significantly decreased (P < 0.01), and the IL-6 level was significantly decreased (P < 0.05). In experimental groups 4 and 5, there were no significant differences in the values ​​of any indicator (TNF-α level and IL-6 level) compared with the model control group (P > 0.05).

[0152] The above experimental results show that, compared with the improvers in comparative examples 1 to 2, the improvers formulated in examples 1 to 3 of the present invention have a significantly enhanced effect on improving autism spectrum disorder in mice.

[0153] Experimental Example 2

[0154] Based on the experimental results of Example 1, the improver from Example 1 was selected as the experimental sample. The recommended human dose is 6g / 60kg bw per day (main drug dose only). Three dose groups were set up: low, medium, and high, at 500mg / kg bw, 1000mg / kg bw, and 3000mg / kg bw, respectively. The doses of the low, medium, and high groups are equivalent to 5 times, 10 times, and 30 times the recommended human intake, respectively. Evaluation was still conducted using mouse experiments. The improver from Comparative Examples 3-7 was administered via gavage at the high dose. The experimental method was the same as in Example 1. Mice in the experimental groups were randomly divided into 12 groups: a model control group, a blank control group, and experimental groups 1-8. Experimental groups 1-3 were administered the low, medium, and high doses of Example 1, respectively. Experimental groups 4-8 were administered the improver from Comparative Examples 3-7 in the required proportions, using physiological saline as the solvent to the desired concentration. Each mouse was administered 0.2mL / 10g bw via gavage. The blank control group and the model control group were administered physiological saline via gavage. Mouse behavior and the determination and data processing of tumor necrosis factor-α and interleukin-6 levels were the same as in Experiment 1. The experimental indicators for each group are as follows: Figure 7-12 As shown in Table 2:

[0155] Table 2: Cytokine levels in experimental groups 1-9, model control group, and blank control group mice

[0156] Serial Number Group TNF-α (pg / mL) IL-6 (pg / mL) 1 Blank control group 37.48±7.84* 14.88±4.57** 2 Model control group 70.31±8.60 40.30±5.88 3 Experimental group 1 58.39±7.11** 28.13±4.31** 4 Experimental group 2 53.22±7.97** 25.07±4.69** 5 Experimental group 3 49.22±5.70** 20.13±4.17** 6 Experimental group 4 63.85±6.11 35.00±4.86 7 Experimental group 5 64.81±5.98 36.00±3.99 8 Experimental group 6 66.10±7.21 37.03±6.36 9 Experimental group 7 67.03±8.55 38.01±3.82 10 Experimental group 8 68.12±6.15 39.02±4.48

[0157] Note: * indicates comparison with the model control group, *P<0.05, **P<0.01;

[0158] Tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6).

[0159] Depend on Figure 7-8 It was found that in the open field experiment, compared with the blank control group, the self-grooming time of the model control group mice within 5 minutes was significantly increased (P<0.01), indicating that the model control group mice exhibited obvious repetitive and stereotyped behaviors; the activity time of the model control group mice in the central area of ​​the open field was significantly reduced compared with the blank control group (P<0.01), indicating that the model control group mice exhibited reduced autonomous exploration behavior in unfamiliar environments and a decreased ability to explore new environments. Figure 9 The results showed that, in the three-box experiment, compared with the blank control group, the model control group mice spent significantly less time in the unfamiliar mouse box (P<0.01), while spending significantly more time in the toy box (P<0.01), indicating a significant reduction in social interaction behavior in the model control group mice. In conclusion, the model control group mice exhibited significantly reduced social interaction behavior, lacked social novelty towards unfamiliar mice, showed reduced autonomous exploration behavior in unfamiliar environments, and displayed obvious stereotyped and repetitive activities, suggesting successful ASD modeling.

[0160] Compared with the model control group, experimental groups 1-3 showed highly significant differences in the following behaviors (time spent grooming, time spent in the central area of ​​the open field, time spent in the unfamiliar mouse cage, time spent in the toy box, time spent climbing to the bottom of the pole, time spent falling from the suspension rope, and time spent on the spinning rod) (P < 0.01). In experimental groups 4-9, there were no significant differences in the following behaviors (time spent grooming, time spent in the central area of ​​the open field, time spent in the unfamiliar mouse cage, time spent in the toy box, time spent climbing to the bottom of the pole, time spent falling from the suspension rope, and time spent on the spinning rod) compared with the model control group (P > 0.05).

[0161] Table 2 shows that, compared with the blank control group, the serum TNF-α and IL-6 levels in the model control group were significantly higher (P < 0.01). In experimental groups 1–3, the serum TNF-α and IL-6 levels were significantly lower than those in the model control group (P < 0.01); in experimental groups 4–8, the serum TNF-α and IL-6 levels showed no significant difference compared with the model control group (P > 0.05).

[0162] The above data shows that the absence of any one of the five components reduces the improvement of clinical symptoms of autism spectrum disorder. The fructooligosaccharides, galactooligosaccharides, spinach powder, poria cocos powder and blueberry powder of this invention work synergistically to improve the clinical symptoms of autism spectrum disorder.

[0163] Experiment Example 3: Regulation of Gut Microbiota and Butyrate

[0164] 1. Analysis of mouse gut microbiota

[0165] At the end of the mouse rearing period in Experiment 1, after removing the bedding from each cage of mice, the middle section of fresh feces excreted by each cage of mice was collected, placed in a sampling box, and immediately placed on dry ice. It was then stored in an ultra-low temperature freezer at -80℃ for DNA extraction and intestinal flora detection.

[0166] Illumina Miseq PE250 sequencing

[0167] Fecal samples stored at -80℃ were subjected to DNA extraction according to the description in the literature "Molecular microbial diversity of ananaerobic digestor as determined by small-subunit rDNA sequence analysis" (J JGodon 1, E Zumstein, P Dabert, F Habouzit, R Moletta). All DNA samples were sequenced on the Illumina Miseq platform using the Miseq kit V3 (600 cycles), and the sequencing data were analyzed. The results are as follows: Figure 13 As shown.

[0168] The gut microbiota comprises nine phyla, with 98% of the bacteria belonging to four: Bacteroidetes, Firmicutes, Proteobacteria, and Actinobacteria. Figure 13 Analysis of the relative abundance of different phyla in the gut microbiota of the experimental and control groups clearly showed that Firmicutes and Bacteroidetes were dominant in all samples, accounting for approximately 90% of the gut microbiota. Compared to the blank control group, the B / F ratio in the model control group decreased significantly (P < 0.01). The B / F ratios in experimental groups 1-3 were significantly higher than those in the model control group (P < 0.05), while the B / F ratios in experimental groups 4-5 showed no significant difference from the model control. This indicates that the ratios of the modifiers in experimental groups 1-3 can regulate the gut microbiota structure in mice, thereby improving the clinical symptoms of autism spectrum disorder.

[0169] 2. Analysis of mouse butyrate

[0170] At the end of the feeding period for mice in Experiment 1, orbital vein blood was collected using AB SCIEX TripleQuad. TM Butyrate concentration was detected using 4500MD.

[0171] Experimental conditions:

[0172] (1) Liquid phase conditions:

[0173] Column: Waters BEH C18 (100mm × 2.1mm, 1.7μm)

[0174] Mobile phase A: Water containing 0.1% formic acid

[0175] Mobile phase B: Methanol / isopropanol = 4:1, v / v

[0176] Column temperature: 40℃

[0177] Flow rate: 0.3 mL / min

[0178] Injection volume: 2 μL

[0179] The gradient elution conditions are shown in Table 3.

[0180] Table 3: Gradient elution conditions

[0181]

[0182] (2) Mass spectrometry conditions:

[0183] Ion source: ESI

[0184] Scan type: Positive ion mode

[0185] Scan mode: MRM

[0186] Other: Air curtain gas: 35psi; Voltage -4500V; Temperature 550℃; Spray gas (GS1) 50psi; Auxiliary heating gas (GS2) 50psi; Collision gas (CAD) 6psi; Atomizing gas current (NC): 5μA.

[0187] Experimental graphs as follows Figures 14-20 As shown. The mass spectrometry data were analyzed, and the results are as follows. Figure 21 As shown.

[0188] Depend on Figures 14-20 It can be seen that the peak elution time of butyric acid is 4 min. The relative ionic strength of the model control group is lower than that of the blank control group, while the relative ionic strength of experimental groups 1-5 is higher than that of the model control group, and the relative ionic strength of experimental groups 1-5 decreases sequentially. Further analysis... Figure 21 It was found that, compared to the blank control group, the butyrate concentration in the model control group decreased, while the butyrate concentration in experimental groups 1-5 was higher than that in the model control group, and the butyrate concentration in experimental groups 1-5 decreased sequentially. This indicates that the modifier ratios in experimental groups 1-3 were more effective than those in experimental groups 4-5. These results further demonstrate that the gut microbiota changed in the model control group compared to the blank control group, and that experimental groups 1-5 played a role in regulating the gut microbiota. These results correspond to the regulatory effect on the gut microbiota structure in mice.

[0189] The above description is merely an embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principle of this application should be included within the scope of the claims of this application.

Claims

1. A pharmaceutical preparation for improving autism spectrum disorder, comprising the following raw materials in parts by weight: 20-40 parts of fructooligosaccharide, 20-40 parts of galactooligosaccharide, 10-30 parts of spinach powder, 1-20 parts of Poria cocos powder, and 1-20 parts of blueberry powder; Spinach powder is prepared according to the following method: 1) After selecting, cleaning, and washing the spinach, remove the roots and cut it into sections, each section being 3-4cm long; 2) Place the chopped spinach in hot water at 70-100℃, and add 0.2wt% anhydrous sodium bicarbonate solution at the same time, and blanch for 3-7 minutes; 3) After blanching, drain the water, add purified water in a 1:1 mass ratio, grind in a crusher, and then process with a colloid mill to obtain spinach pulp. Homogenize the spinach pulp 2 to 3 times. 4) The homogenized spinach pulp is spray-dried until the moisture content is less than or equal to 8 wt% (dry basis), and then passed through an 80-mesh sieve to obtain spinach powder. Poria cocos powder is prepared according to the following method: Pulverize Poria cocos to 60-80 mesh, add deionized water to obtain a mixture with a water content of 5-10 wt%, then puff the mixture, place the puffed product in an oven at 60-70℃ and dry for 20-60 minutes to reduce its moisture content to 2-6 wt%, then pulverize it again and microwave sterilize it to obtain Poria cocos powder. The puffing process utilizes a twin-screw extruder, which consists of three puffing zones: Zone 1, Zone 2, and Zone 3. During puffing, Zone 1 is closed, Zone 2 maintains a temperature of 110–120℃, and Zone 3 maintains a temperature of 140–150℃. The screw speed of the twin-screw extruder is 45–50 rpm, the feeding speed is 8–13 rpm, and the rotary cutting speed is 16–20 rpm. The particle size after secondary crushing is 80–100 mesh. Microwave sterilization is performed at a temperature of 60–70℃ for 2–9 minutes. Blueberry powder is prepared according to the following method: (1) Grind the selected and cleaned blueberries in a colloid mill until they are evenly ground to obtain blueberry pulp; (2) Freeze the blueberry pulp at -18℃ for 9 to 12 hours to ensure that the blueberry pulp is completely frozen; (3) The frozen fruit pulp was placed in an in-situ vacuum freeze dryer and dried. The moisture content was measured every 0.5 hours until the moisture content was less than or equal to 8 wt% (dry basis). The vacuum freeze drying conditions were: vacuum degree 1.0 Pa, cold trap temperature -58.7℃ to -59.3℃. (4) Place the freeze-dried blueberry pulp in a grinder and grind it. Then pass it through an 80-mesh sieve to obtain blueberry powder.

2. The autism spectrum disorder improvement drug formulation according to claim 1, characterized in that, The autism spectrum disorder improvement drug formulation comprises the following raw materials in parts by weight: 25-35 parts of fructooligosaccharide, 25-35 parts of galactooligosaccharide, 15-25 parts of spinach powder, 10-18 parts of Poria cocos powder, and 2-10 parts of blueberry powder.

3. The autism spectrum disorder improvement drug formulation according to claim 1, characterized in that, The autism spectrum disorder improvement drug formulation comprises the following raw materials in parts by weight: 30 parts of fructooligosaccharide, 30 parts of galactooligosaccharide, 20 parts of spinach powder, 15 parts of Poria cocos powder, and 5 parts of blueberry powder.

4. The autism spectrum disorder improvement drug formulation according to claim 1, characterized in that, In the preparation of spinach powder, in step 3), the homogenization pressure is 25 MPa, and in step 4), the spray drying conditions are: inlet temperature 160-180℃, outlet temperature 70℃, peristaltic pump flow rate 50 mL / min, and fan frequency 40 Hz.

5. A method for preparing the autism spectrum disorder improvement drug formulation according to any one of claims 1-4, comprising the following steps: a. Pass fructooligosaccharides, galactooligosaccharides, spinach powder, poria cocos powder, and blueberry powder through an 80-mesh sieve, and mix them evenly according to the proportions to obtain a mixture; b. Add alcohol to the mixture to obtain a soft material; then granulate it through a 20-mesh sieve. After granulation, dry it at 50-60℃ for 2-3 hours, then granulate and sieve to obtain an autism spectrum disorder improver. The volume concentration of alcohol is 40%-60%, and the amount of alcohol added is 5%-10% of the weight of the mixture.

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