A complex nutrient composition and its use in intervention of autism and mental retardation

Abstract

The application discloses a kind of composite nutrient composition and its application in intervention autism and mental retardation, the composite nutrient composition is composed of composite vitamin, mineral, enterobacterium, apple fruit powder and the substance for improving taste;Wherein, the procyanidin B1 content in the apple fruit powder is more than 30mg / kg, the composite nutrient composition of the application can effectively improve the repetitive stereotyped behavior of autistic children, the degree of anxiety, the degree of cognition, and social skills, and effectively improve the intestinal flora imbalance phenomenon of autistic children.It can also effectively improve the cognitive ability of children with mental retardation.

Description

TECHNICAL FIELD

[0001] The present application relates to a kind of composite nutrient composition and its application in intervention autism and mental retardation. BACKGROUND

[0002] Autism, also known as autism spectrum disorder (ASD), is a neurodevelopmental disorder. According to a survey report by Italian scholars, the global incidence of autism is about 0.6% per year, with a male-to-female ratio of 4:1, and the incidence is showing an increasing trend year by year. Autistic patients usually show social disorders, language expression disorders, repetitive stereotyped behaviors or other non-verbal communication problems. In recent years, although scholars have made further exploration of the pathology and physiology of autism, the cause and mechanism of autism are still unclear. At present, scholars generally believe that the cause of autism may be caused by the interaction of genetic factors and environmental factors. Due to the complexity of the mechanism of autism, there is currently no drug that can completely cure it, and ASD patients usually improve symptoms through behavior intervention and education. However, the above measures ignore certain physiological states specific to ASD patients, and whether the adjustment of the physiological state of ASD patients will alleviate the symptoms of ASD patients needs further research.

[0003] At present, the intervention effect of nutritional supplements on ASD patients is not consistent in reports, and some reports show that simple nutritional intervention has poor effect. How to effectively improve the intervention effect of various nutritional supplements on ASD patients is an important problem in the treatment of ASD. SUMMARY

[0004] The purpose of this section is to summarize some aspects of the embodiments of the present application and briefly introduce some preferred embodiments.

[0005] As one aspect of the present application, the present application provides a composite nutrient composition, wherein the composite nutrient composition is composed of a composite vitamin, a mineral, an intestinal bacillus, an apple fruit powder and a taste improving substance; wherein the content of procyanidin B1 in the apple fruit powder is more than 30 mg / kg.

[0006] As a preferred scheme of the composite nutrient composition according to the present application: the composite vitamin includes vitamin B2, vitamin B6, vitamin B 12 , vitamin C, vitamin D3 and folic acid.

[0007] As a preferred scheme of the composite nutrient composition according to the present application: the vitamin B2, vitamin B6, vitamin B 12The mass ratio of vitamin C, vitamin D3 and folic acid is: (0.5-1.0mg):(0.5-1.0mg):(1-2ug):(20-50mg):(2-5ug):(65-130ug).

[0008] As a preferred scheme of the composite nutrient composition of the present application: the content of procyanidin B1 is 30-200 mg / kg.

[0009] As a preferred scheme of the composite nutrient composition of the present application: the mass ratio of the composite vitamin and the enterobacterium is 50mg:(0.5-1.5g); the mass ratio of the composite vitamin and the apple fruit powder is 50mg:(3-5g); the mass ratio of the composite vitamin and the mineral is 1:(5-12).

[0010] As a preferred scheme of the composite nutrient composition of the present application: the mineral includes calcium and magnesium; the enterobacterium includes bifidobacterium.

[0011] As a preferred scheme of the composite nutrient composition of the present application: the mineral is composed of calcium gluconate and magnesium gluconate, and the mass ratio of the composite vitamin, the calcium gluconate and the magnesium gluconate is 1:(4-8):(2-3).

[0012] As a preferred scheme of the composite nutrient composition of the present application: the taste improving substance includes one or more of prune fruit powder, cherry fruit powder, xylitol and sucralose.

[0013] The beneficial effects of the present application: the composite nutrient composition of the present application can effectively improve the repetitive stereotyped behavior, anxiety degree, cognitive degree and social ability of children with autism, and effectively improve the intestinal flora imbalance phenomenon of children with autism. It can also effectively improve the cognitive ability of children with mental retardation. BRIEF DESCRIPTION OF DRAWINGS

[0014] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor. Among them:

[0015] Figure 1 Results of the rat bead burying experiment.

[0016] Figure 2 Results of the rat open field experiment.

[0017] Figure 3 Schematic diagram of three-box experiment principle.

[0018] Figure 4 For three-box social experiment results.

[0019] Figure 5 For nest-building experiment results.

[0020] Figure 6 For the correlation between the content of several different chemicals in apple fruit powder and the rat burying beads.

[0021] Figure 7 For the effect of several substances in apple fruit powder on the stereotyped behavior of autism rats.

[0022] Figure 8 For the synergistic effect of procyanidin B1 and complex nutrients on the improvement of stereotyped behavior and cognitive behavior of rats. DETAILED DESCRIPTION

[0023] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the specific embodiments of the present application will be described in detail below.

[0024] Example 1:

[0025] Take vitamin C 50mg, vitamin B2 1mg, vitamin B12 2ug, vitamin D3 5ug, vitamin B6 1mg, folic acid 130ug, calcium gluconate 400mg, magnesium gluconate 150mg, animal bifidobacterium (100 billion active) 1g, apple fruit powder 5g (wherein the concentration of procyanidin B1 is 60mg / kg), prune fruit powder 2g, xylitol 1.4g.

[0026] Example 2:

[0027] Take vitamin C 50mg, vitamin B2 1mg, vitamin B12 2ug, vitamin D3 5ug, vitamin B6 1mg, folic acid 130ug, calcium gluconate 400mg, magnesium gluconate 150mg, animal bifidobacterium (100 billion active) 1g, apple fruit powder 3g, xylitol 0.4g, and mix evenly.

[0028] Comparative Example 1:

[0029] Take vitamin C 50mg, vitamin B2 1mg, vitamin B12 2ug, vitamin D3 5ug, vitamin B6 1mg, folic acid 130ug, calcium gluconate 400mg, magnesium gluconate 150mg, animal bifidobacterium (100 billion active) 1g, prune fruit powder 7g, xylitol 1.4g, and mix evenly.

[0030] Comparative Example 2:

[0031] Take vitamin C 50 mg, vitamin B2 1 mg, vitamin B12 2 μg, vitamin D3 5 μg, vitamin B6 1 mg, folic acid 130 μg, calcium gluconate 400 mg, magnesium gluconate 150 mg, animal bifidobacterium (10 billion active) 1 g, apple fruit powder 8.4 g, xylitol 1.4 g, and mix evenly to obtain.

[0032] Example 3:

[0033] Take the sample of Example 1, and measure the particle size and particle size distribution according to the method (Chinese Pharmacopoeia 2020 edition general 0982 single screening method). Single screening method: weigh the test sample specified in each variety, place it in a drug sieve of the specified size (with a tightly fitting receiving container below the sieve), and cover the sieve above. Rotate and shake horizontally for at least 3 minutes, and occasionally tap the sieve vertically. Take the particles and powder below the sieve, weigh and calculate the proportion (%). The results show that the prepared finished product has a passing rate of 100% on No. 7 sieve, and the particle size meets the requirements.

[0034] Example 4:

[0035] Autism rat model (VPA model)

[0036] After one week of adaptation, SD female rats were caged with male rats at a ratio of 2:1 at 5:00 pm for the night. The next morning at 9:00 am, the female rats were observed for the presence of vaginal plugs. If a vaginal plug was found, the female rat was considered pregnant. If not, a vaginal smear was taken. If sperm was found in the smear, the female rat was considered pregnant. The pregnant female rats were then separated and fed individually. Both female rats were successfully impregnated. One group of pregnant rats was injected intraperitoneally with sodium valproate (VPA) at 12.5 days of gestation, with a dose of 600 mg / kg (diluted with normal saline to 60 mg / mL, 0.1 mL VPA per 10 g). The offspring of the VPA group were randomly selected as the VPA model. The tails of the VPA model offspring were observed 42 days after birth. If the tail was curved, the model was considered successful.

[0037] The experiment was divided into six groups: normal group (Normal group), autism model group (VPA group), Example 1 intervention group (Example 1 group), Example 2 intervention group (Example 2 group), Comparative Example 1 intervention group (Comparative Example 1 group), and Comparative Example 2 intervention group (Comparative Example 2 group). The normal group and the VPA group were given distilled water by gavage at a dose of 20 mL / kg / d, and the other four groups were given compound nutrients by gavage at a dose of 1.575 g / kg / d for 3 months. After the gavage was completed, the behavior was evaluated.

[0038] Marble embedding experiment to evaluate the repetitive stereotypy of VPA rats:

[0039] Principle: rodents have the nature of burying and digging, in the past, the buried bead experiment is often used to determine the anxiety of rodents as a behavioral evaluation method, but now it has been widely used in the evaluation of autism model and repetitive stereotypic behavior in rodents. This behavioral evaluation method is simple to operate, high in accuracy, and reliable in experimental record.

[0040] Before the experiment, clean the mouse cage (35 cm x 15 cm x 20 cm), fresh bedding and 20 ordinary 14 mm glass beads (color can be different), during the experiment, add fresh bedding to the clean mouse cage, the thickness is about 5 cm, and press the bedding flat; 20 glass beads are divided into five rows, each row is placed 4 glass beads at equal intervals, the experimental time is 10 min, more than 80% of the buried beads are recorded as buried successfully, in order to get more accurate data, the experimenter leaves the experimental site during the experiment, and counts the number of buried beads when the time is up. Between each experiment, the beads are cleaned and the next experiment is performed.

[0041] Figure 1 The influence of examples 1, 2, comparative example 1 and comparative example 2 on the stereotypic behavior of autistic rats (buried bead experiment).**: P<0.05;**: P<0.01; all compared with the VPA group (n=6). The number of buried beads is positively correlated with the repetitive stereotypic behavior of rats, as shown in Figure 1 The number of buried beads in the VPA group is significantly increased compared with the normal group, indicating that the autistic rats have obvious stereotypic behavior. After intervention with example 1 or example 2, the number of buried beads in the above two groups of rats is significantly reduced compared with the VPA group, which indicates that the compound vitamin powder can improve the repetitive stereotypic behavior of autistic rats. Compared with examples 1 and 2, comparative example 1 does not add apple pomace, and the number of buried beads in this group of rats is reduced compared with the VPA group, but is significantly higher than examples 1 and 2, which indicates that the addition of apple pomace effectively enhances the improvement of the nutrient composition of the application on the stereotypic behavior of autistic rats. Compared with examples 1 and 2, the amount of apple pomace in comparative example 2 is increased to 8.4 g, but the improvement of the stereotypic behavior of rats is not further enhanced, but its effect is weaker than examples 1 and 2. It is considered that apple pomace plays a synergistic role at the same time, and the excessive addition of sugar intake of rats will increase the occurrence of stereotypic behavior of autism, which has been reported in previous studies.

[0042] The open field experiment was used to evaluate the anxiety state and curiosity psychology of VPA rats:

[0043] Principle: Open field test is also called open box test. Rats will show nervous and fearful emotions in an open and bright unfamiliar environment, but they also have the psychological exploration of the surrounding environment, so open field test is widely used in behavioral experiments of anxiety state of rodents.

[0044] Method: The experimental time is from 9:00 to 12:00 in the morning, and a square gray open box with a size of 50 cm x 50 cm x 40 cm is used for the experiment. Video tracking system software (Grobel Biotechnology Co., Ltd., model: ANYmaze) is used to record the behavior of rats in the open box. A camera is installed above the open box, and the relevant computer program of the open field test is set. The bottom of the open box is divided into 16 small squares, and the middle 4 small squares are the central area, and the others are the outer area. Before the experiment, the rats are moved to the experimental room for 30 minutes to adapt. At the beginning of the experiment, the experimental rats are placed in the central area, and the ANYmaze program is used to track the total distance of the rats in the open box, the time of staying in the central area, the time of staying still, and the number of standing times, and the data are counted. The experimental time of each rat is 3 minutes, and the statistical time is 1 minute. After each experiment, the feces and urine of the rats in the open box are cleaned, and 75% alcohol is sprayed to remove the odor. After the alcohol evaporates, the next experiment is carried out.

[0045] Figure 2 Effects of Example 1, Example 2, Comparative Example 1 and Comparative Example 2 on the anxiety behavior of autistic rats (open field test). **: P<0.05; **: P<0.01; ns: no statistical difference; compared with VPA group (n=6). Figure 2 In the open field test, the time of staying in the central area of the open field is negatively correlated with the anxiety level of the rats, that is, the shorter the staying time, the higher the anxiety level. As shown in the figure, compared with the normal group, the VPA group has a shorter central staying time, indicating that autistic rats have a higher anxiety level. Compared with the VPA group, the central area staying time of Example 1 and Example 2 groups is significantly improved, indicating that the complex nutrients prepared by Example 1 and Example 2 can effectively alleviate the anxiety level of autistic rats. Although the staying time of the rats in the central area of Comparative Example 1 and Comparative Example 2 is longer than that of the VPA group, the staying time of the two groups is significantly smaller than that of Example 1 and Example 2, indicating that apple pomace can improve the alleviation of complex vitamins, probiotics and minerals on the anxiety level of autistic rats within the range of Example 1 and Example 2. Lower or higher than the above range, the intervention effect on the anxiety level of autistic rats will decrease.

[0046] The total distance of movement and the number of standing probes are positively correlated with the degree of curiosity of rats, as Figure 2As shown, compared with the normal group, the VPA group had a reduced total distance of movement and a reduced standing probe time. Compared with the VPA group, the total distance of movement and the standing probe time were increased in Example 1 and Example 2, indicating that the curiosity of the rats was improved after intervention by Example 1 and Example 2. The effect of Comparative Example 1 and Comparative Example 2 was weaker than that of Example 1 and Example 2, again proving that the addition of apple pomace is conducive to the complex nutrients to improve the curiosity level of autistic rats, but excessive intake of apple pomace will bring negative effects.

[0047] The three-chamber social interaction experiment was used to evaluate the social behavior of VPA rats:

[0048] Principle: Rodents have a group living characteristic. For a normal rat, when a peer approaches, he is willing to contact it and exhibit certain social behavior. However, the rat has a novelty-seeking characteristic, and for a familiar rat, it tends to contact a new stranger, which is manifested as social novelty. Through the two characteristics of the rat, a three-chamber social interaction experiment is established to evaluate the social behavior and social novelty of the test rat.

[0049] Method (as shown): Figure 3 The device is self-made and consists of three equal-sized open boxes (45 cm x 40 cm x 45 cm), which are divided into two side rooms and a center room. The partition of the two side rooms has a rectangular door (9 cm x 9 cm) for the rat to pass through, and a camera is installed above the device. The first stage is the familiarization stage: close the small door of the two side rooms, put the rat into the center room, and make it adapt to the environment. The adaptation time is 5 min, and no shooting is required. The second stage is the social stage: put two size restraint cages in the two side rooms, randomly put 1 unfamiliar rat (same sex, different cages, recorded as Stranger1) in one of the restraint cages, and put an empty cage in the other, recorded as Empty. Open the rectangular door of the two side rooms to allow the experimental rat to freely move in the two side rooms and the center room. Turn on the camera and shoot for 10 min. The third stage is the social novelty stage: put another new unfamiliar rat (same sex, different cages, recorded as Strangger2) in the empty cage, and open the rectangular door of the two side rooms to allow the experimental rat to freely move in the two side rooms and the center room. The time spent by the experimental rat in interacting with Stranger1 and Strangger2 is recorded to quantify the preference of the test rat for social novelty. After each experiment, clean the box and the restraint cage with 75% alcohol, and then test the next rat after the odor is volatilized.

[0050] Figure 3 The three-chamber experiment is a schematic diagram of the principle. Figure 4The ratio of the time of interaction with the Stranger 2 cage to the time of interaction with the Stranger 1 cage in the third phase for each group of rats. **: P < 0.05; **: P < 0.01; ns: no statistical difference; all compared with the VPA group (n = 6). The second phase of the three-chamber social interaction reflects the social ability of the rats, and the longer the time, the stronger the social ability of the rats. As shown in the figure, the social ability of the VPA group is reduced compared with the normal group. The social ability of the Example 1 and Example 2 groups is improved compared with the VPA group. There is no statistical difference in the social ability of the Comparative Example 1 and Comparative Example 2 groups compared with the VPA group.

[0051] The third phase of the three-chamber social interaction reflects the social novelty of the rats, and the higher the ratio, the stronger the social novelty ability of the rats. The social novelty ability of the VPA group is reduced compared with the normal group. The social novelty ability of the Example 1 and Example 2 groups is improved compared with the VPA group. The social ability of the Comparative Example 1 and Comparative Example 2 groups is stronger than that of the VPA group, but is significantly lower than that of the Example 1 and Example 2 groups.

[0052] The nesting experiment was used to evaluate the cognitive ability of the rats:

[0053] Principle: Rodents have the instinct of building nests, which serves the purpose of warmth and defense. The ability to build nests is related to reproduction, social cognition behavior, and the nesting experiment

[15] has been widely used in the social cognition behavior of rats.

[0054] Method: A clean standard rat cage was taken and about 2 cm thick clean bedding was placed in it, and the bedding was compacted. Seven clean wind sterile paper towels (32 cm x 18 cm) were placed in the cage. At 18:00 in the evening, the tested rats were placed in the cage (only one tested rat was placed in each cage), and sufficient feed and water were provided. The experiment was carried out according to the normal light rhythm, and the light source was turned off at 20:30 in the evening. The next morning at about 9:00, the nest built by the tested rat was scored according to the shape and height of the nest according to Table 1, and the score range was 1-5 points. After scoring, the tested rat was taken out, the clean bedding and paper towels were replaced, and the next rat was tested in the evening.

[0055] Figure 5Effect of Examples 1, 2, Comparative Example 1 and Comparative Example 2 on cognitive behavior of autism rats (nest building experiment). *: P < 0.05; **: P < 0.01; ns: no statistical difference; all compared with VPA group (n = 6). Nest building ability is positively correlated with cognitive ability of rats. The results show that the score of nest building ability of VPA group is reduced compared with the normal group, and the score of nest building ability of Examples 1 and 2 groups is increased compared with the VPA group, which indicates that the compound vitamin powder prepared by Examples 1 and 2 can improve the cognitive ability of autism rats. There is no statistical difference in cognitive ability between Comparative Example 1 and Comparative Example 2 groups and the VPA group.

[0056] Example 5:

[0057] Effect of content of special chemical components in apple fruit powder on intervention effect on autism rats:

[0058] Purchase commercially available apple fruit powder (used in Examples 1-4), determine the contents of procyanidin B1, procyanidin B2, procyanidin B4, quercitrin and querci-galactoside in the apple fruit powder by liquid chromatography-mass spectrometry, and prepare the compound nutrients according to the method in Examples 1 and 2. Perform the bead burying experiment to evaluate the effect of different chemical component contents in apples on the stereotyped behavior of autism rats. The results are shown in Table 1, which shows that among the several chemical components in apple fruit powder, only the change trend of procyanidin B1 content is consistent with the improvement trend of the stereotyped behavior of rats, Figure 6 which shows that procyanidin B1 has the highest correlation between the number of beads buried by rats among several chemical components, indicating that procyanidin B1 plays an important role in improving autism in rats. Especially when the content of procyanidin B1 in apple fruit powder is higher than 30 mg / kg, the intervention effect on the stereotyped behavior of rats is obviously improved. This indicates that apple procyanidin B1 plays a synergistic effect in the intervention of autism by compound vitamins, minerals and probiotics. Therefore, the content of procyanidin B1 in apple fruit powder of the present application should be higher than 30 mg / kg.

[0059] Table 1 Effect of several substances in apple fruit powder on stereotyped behavior of autism rats

[0060]

[0061] Example 6:

[0062] Verification of the improvement effect of procyanidin B1 on autism in rats:

[0063] Pure procyanidin B1, procyanidin B2, procyanidin B4, quercitrin and querci-galactoside (dose of 10 mg / kg) were used to intervene in autism rats for 90 days, and then the bead burying experiment was performed. The results are shown in Figure 7 , Figure 7For the influence of several substances in apple powder on the stereotyped behavior of autism rats, *: P<0.05; **: P<0.01; ns: no statistical difference; all compared with the VPA group (n=6). The experimental results prove that compared with procyanidin B2, procyanidin B4, quercitrin, and quercetin-galactoside, procyanidin B1 has a more significant intervention on the stereotyped behavior of autism rats.

[0064] Example 7

[0065] Synergistic effect of procyanidin B1 in apple powder on the intervention of complex nutrients on autism and mental retardation:

[0066] Sample 1, sample 2 and sample 3 were prepared according to Table 2. Sample 1, 2 and 3 were used to intervene in autism rats respectively, and the average bead number and nest building score were determined. Figure 8 It is shown that compared with procyanidin B1, the intervention effect of complex nutrients on the stereotyped behavior of autism rats and mental retardation is significantly improved.

[0067] According to the King's formula, q=E(A+B) / (EA+EB-EA·EB), where EA, EB, and E(A+B) are the effects of using A or B alone and combined medication respectively. When the q value is greater than 1, it is determined that A and B have a synergistic effect. Figure 8 Synergistic effect of procyanidin B1 and complex nutrients on the improvement of stereotyped behavior and cognitive behavior of rats. *: P<0.05; **: P<0.01; ns: no statistical difference; all compared with the VPA group (n=6). According to Figure 8 Results, the q value of procyanidin B1 and complex nutrients combined for the bead number and nest building score of rats was 1.5 and 1.4 respectively, indicating that procyanidin B1 combined with complex nutrients has a significant synergistic effect on the improvement of stereotyped behavior and cognitive ability of autism rats, and the mechanism may be that procyanidin B1 participates in the promotion of neural development by complex vitamins.

[0068] Table 2 sample grouping of Example 7

[0069]

[0070] It should be noted that the above examples are only used to illustrate the technical solutions of the present application and not to limit it. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the spirit and scope of the present application, and they should be covered in the scope of the claims of the present application.

Claims

1. A combination of nutrients for intervention in autism and mental retardation, characterized in that: The complex nutrient composition is composed of complex vitamins, minerals, enteric bacilli, apple fruit powder and taste improving substances; wherein the procyanidin B1 content in the apple fruit powder is 30-200 mg / kg; The complex vitamin consists of vitamin B2, vitamin B6, vitamin B 12 , vitamin C, vitamin D3 and folic acid; The mass ratio of the vitamin B2, vitamin B6, vitamin B 12 , vitamin C, vitamin D3 and folic acid is: (0.5-1.0 mg):(0.5-1.0 mg):(1-2 μg):(20-50 mg):(2-5 μg):(65-130 μg); The mass ratio of the complex vitamins to the enteric bacilli is 50mg:(0.5-1.5g); the mass ratio of the complex vitamins to the apple fruit powder is 50mg:(3-5g); The enteric bacilli are bifidobacterium; The minerals are composed of calcium gluconate and magnesium gluconate, wherein the mass ratio of the complex vitamins to the calcium gluconate and the magnesium gluconate is 1:(4-8):(2-3).

2. The composition of claim 1, wherein the composition is a combination of nutrients for intervention of autism and mental retardation. The taste improving substances include one or more of prune fruit powder, cherry fruit powder, xylitol and sucralose.

Images