Application of fresh potato juice in preparation of food or medicine for improving ADHD

Abstract

The invention discloses an application of fresh juice prepared from diploid potato'small yellow 'in improvement of attention deficit hyperactivity disorder (ADHD). The fresh juice is rich in various metabolic components such as amino acids, alkaloids and the like, and network pharmacological analysis shows that the fresh juice can regulate signal channels such as Neuroactive ligand-receptor interaction, cAMP, Calcium, Insulin and the like through target spots such as SCN2A, SLC6A1, CACNA1H, HTR2C and the like, so that the spatial learning and memory ability and hyperactivity behaviors of ADHD model rats are remarkably improved. The invention provides a safe and natural ADHD adjuvant therapy scheme which has important application value.

Description

Technical Field

[0001] The present invention relates to the field of food and pharmaceutical technology, and in particular to an application of fresh juice prepared from the diploid potato variety "Xiao Huang" in improving attention deficit hyperactivity disorder (ADHD). Background Art

[0002] Attention Deficit Hyperactivity Disorder (ADHD) is a common neurological and psychological developmental disorder in children, often developing before the age of 12. Its main manifestations are developmental disproportionate to children of the same age. Clinically, it is often accompanied by inattention, short attention span, hyperactivity, and impulsivity, often accompanied by learning difficulties, behavioral and emotional disorders, but with normal intelligence. Currently, the treatment of ADHD mainly relies on drug interventions such as methylphenidate and bupropion, but these drugs may have side effects such as insomnia, loss of appetite, and mood swings. Therefore, finding a safe and natural health treatment is of great significance.

[0003] The potato is an annual herbaceous plant of the Solanaceae family, native to South America. It possesses multiple characteristics, encompassing both food and vegetable and fruit, making it a vital source of food for many countries worldwide. Traditional potato breeding primarily relies on tetraploid potatoes, a method with limitations such as long breeding cycles, low genetic diversity, and low reproductive efficiency. The diploid potato "Xiao Huang" (Youshu No. 1), a new variety developed by Academician Huang Sanwen's team, is a late-maturing variety with a growing period of approximately 110 days. Its tubers are round, with yellow skin and flesh, and a smooth surface. It produces nearly 3 tons per mu (approximately 1600 sq ft), demonstrating significant heterotic vigor in yield. Furthermore, "Xiao Huang" boasts high dry matter and carotenoid content, resulting in excellent cooking quality. The diploid potato "Xiao Huang" not only represents an innovation in breeding technology, but its optimized composition has the potential to elevate potatoes from a staple food to a nutritionally functional food, combining safety and nutritional value. Favorite is a diploid potato introduced from the Netherlands. It is a medium-early maturing variety for fresh consumption, with a growing period of 60-70 days. Its tubers are oblong, light yellow in skin, dark yellow in flesh, and smooth in skin. The tubers are clustered in groups of 4-5, large and uniform, with a short dormancy period. The average yield per mu is 2,000-3,000 kg. Atlantic is a tetraploid potato introduced from the United States. It is a medium-maturing variety with a growing period of approximately 85-90 days. Its tubers are between round and oblong, with a flat crotch, light yellow skin, white flesh, and a slightly reticulated surface. It is storable.

[0004] Modern pharmacology has revealed that potatoes are highly nutritious, containing 76.3% water and 23.7% dry matter, including 9%–20% starch, 1.5%–2.3% protein, 0.1%–1.1% fat, and 0.6%–0.8% crude fiber. The nutritional content of potatoes per 100 g includes 318 kJ of energy, 5–8 mg of calcium, 15–40 mg of phosphorus, 0.4–0.8 mg of iron, 200–340 mg of potassium, 0.8–1.2 mg of iodine, 12–30 mg of carotene, 0.03–0.08 mg of thiamine, 0.01–0.04 mg of riboflavin, and 0.4–1.1 mg of niacin. Studies have shown that many of these components have antioxidant, anti-inflammatory, and neuroprotective properties. However, no studies have examined the effects of potatoes on the symptoms associated with attention deficit hyperactivity disorder. Summary of the Invention

[0005] ADHD is a common neurodevelopmental disorder in children, and existing medications carry the risk of side effects. Potato juice, a natural food ingredient, has untapped potential. Traditional tetraploid potatoes, due to genetic limitations, struggle to meet the demands of functional foods. However, the diploid potato ("Little Yellow"), with its high dry matter and carotenoid content, offers a new avenue for optimizing functional ingredients. Based on this, the present invention provides the use of fresh potato juice in the preparation of a food or medication for improving ADHD.

[0006] First, fresh juice was prepared from the diploid potato "Xiaohuang". Metabolomic analysis showed that it was rich in various metabolite components such as amino acids, alkaloids and phenols. Network pharmacology analysis showed that it may improve ADHD by regulating neuroactive ligand–receptor interaction, cAMP, calcium, insulin and other signaling pathways through targets such as SCN2A, SLC6A1, CACNA1H and HTR2C.

[0007] The preparation method of the potato fresh juice comprises the following steps: (1) Select fresh, disease-free and insect-free diploid potatoes (Xiaohuang), wash and peel them; (2) Cut the potatoes into pieces and squeeze the juice, then filter to remove the residue to obtain clear fresh juice; (3) Divide the fresh juice into smaller pieces and store at low temperature for later use.

[0008] Next, spontaneously hypertensive rats (SHR) were used as ADHD model rats, and the Morris water maze test and novel object recognition test were used to investigate the effects of fresh potato juice of different varieties on the learning, memory and cognitive abilities of the animal models. The results showed that compared with the model group, the rats in each experimental group that drank fresh potato juice could shorten the latency period in the water maze test to varying degrees, improve the novel object recognition index, and had a significant improvement on learning and memory abilities and hyperactive behavior.

[0009] Therefore, fresh potato juice can be used to prepare food or medicine for improving ADHD.

[0010] Potatoes are a natural food ingredient, and their fresh juice is safe and reliable, with no side effects if consumed long-term. The present invention provides a safe and natural supplementary prevention and treatment solution for ADHD, which has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 : Distribution of metabolite components in potato “little yellow” fresh juice.

[0012] Figure 2 : Network pharmacology analysis results of potato "Xiao Huang" fresh juice in improving ADHD, including: A: Venn diagram of the intersection of "Xiao Huang" fresh juice metabolite targets and ADHD targets; B: core target network diagram; C: GO enrichment analysis; D: KEGG pathway analysis results.

[0013] Figure 3 : Docking heat map of core metabolites and core target molecules in potato "little yellow" fresh juice.

[0014] Figure 4 : Results of water maze behavioral experiment on ADHD rats after drinking potato juice.

[0015] Figure 5 : Results of novel object recognition behavioral experiment in ADHD rats after drinking potato juice. DETAILED DESCRIPTION

[0016] The technical solution of the present invention is further described in detail below with reference to specific embodiments.

[0017] Example 1: Preparation of fresh potato juice (1) Select fresh, disease-free diploid potatoes "Xiao Huang", a commercial tetraploid potato variety Atlantic, and diploid Faureta, wash and peel them.

[0018] (2) Cut the potatoes into pieces and use a juicer to extract the juice.

[0019] (3) Filter the fresh juice to remove the residue and obtain clarified potato fresh juice.

[0020] (4) Divide the fresh juice into smaller pieces and store them in a -80℃ refrigerator for later use.

[0021] Example 2: Metabolite Analysis of Potato Juice 1. Sample Processing Remove samples from a -80°C freezer, thaw until ice-free, and vortex for 10 seconds to mix thoroughly. Transfer 200 μL of sample to the corresponding numbered 1.5 mL centrifuge tube and add 200 μL of 70% methanol-containing internal standard extract (if less than 200 μL, add the extract at a 1:1 (v / v) ratio). The internal standard extract was prepared by dissolving 1 mg of the standard in 1 mL of 70% methanol-water to prepare a 1000 μg / mL stock solution. This 1000 μg / mL stock solution was further diluted with 70% methanol to prepare a 250 μg / mL internal standard solution. Vortex for 15 minutes and centrifuge at 12,000 rpm at 4°C for 3 minutes. Remove the supernatant, filter through a 0.22 μm pore size microporous filter, and store in a vial for LC-MS / MS analysis.

[0022] 2. Chromatographic conditions The extracted samples were analyzed using LC-MS, a system consisting of ultra-high-performance liquid chromatography (UPLC) and tandem mass spectrometry (MS / MS). Mass spectrometry analysis was performed in positive (ESI+) and negative (ESI-) ionization modes. After reversed-phase separation, the sample was injected onto an Agilent SB-C18 column (2.1 mm × 1100 mm, 1.8 µm diameter). The injection volume was 2 μL, and the column temperature was 40°C.

[0023] During the analysis, a quality control sample was inserted every 10 samples to monitor analytical reproducibility. The mobile phase flow rate was 0.35 mL / min. Eluents A and B were ultrapure water containing 0.1% (v / v) formic acid and acetonitrile containing 0.1% (v / v) formic acid, respectively. The elution gradient was: 5% B (0 min); 95% B (0-9 min); 95% B (9-10 min); 5% B (10-11 min); and 5% B (11-14 min). The spray voltages were 5.5 kV and 4.5 kV in ESI+ and ESI- modes, respectively.

[0024] 3. Data Analysis The software Analyst 1.6.3 was used to process the mass spectrometry and chromatographic analysis results. The characteristic ions of each substance were screened by a triple quadrupole, and the signal intensity (CPS) of the characteristic ions was obtained in the detector. The mass spectrometry file of the sample was opened with MultiOut software to integrate and correct the chromatographic peaks. The peak area (Area) of each chromatographic peak represents the relative content of the corresponding substance. Finally, all chromatographic peak area integration data were exported and saved.

[0025] The results showed that the main metabolites of the "Little Yellow" potato and the other two potatoes were amino acids and alkaloids, accounting for 22.77% and 14.64% of the total metabolites, respectively. Phenolic and terpenoid compounds were second, accounting for 13.86% and 13.37%, respectively. In addition, it also contained higher levels of flavonoids, lignans, and coumarin compounds ( Figure 1 ).

[0026] Example 3: Network pharmacology analysis 1. Potential targets of differential metabolites of “Xiao Huang” Metabolite SMILE numbers were obtained from the PubChem database (https: / / pubchem.ncbi.nlm.nih.gov / ). Metabolite targets were predicted using the SuperPred (https: / / prediction.charite.de / index.php) and SwissTargetPrediction (https: / / www.swisstargetprediction.ch / ) databases. Targets with a probability ≥50% and ≥0.5 from these two databases were selected as potential targets of potato "Little Yellow." Data from the two databases were combined and duplicates removed to identify potato-related targets.

[0027] 2. Targets related to attention disorders The GeneCards database (https: / / www.genecards.org / ) was searched for targets related to ADHD using the keywords “ADHD” and “attention deficit hyperactivity disorder”, and the relevance score was set to ≥5.

[0028] 3. Network Analysis Venny 2.1.0 (https: / / bioinfogp.cnb.csic.es / tools / venny / ) was used to identify the intersectional targets of "Xiao Huang" and ADHD, and a Venn diagram was constructed. These intersectional targets of "Xiao Huang" and ADHD were imported into the STRING database (https: / / cn.string-db.org / ) to identify key targets. During the screening process, multiple proteins were selected, with human attributes, non-connected nodes hidden, and a confidence level set at ≥0.4 to generate a PPI network. Subsequently, Cytoscape 3.9.0 software was used for further analysis, and the Centiscape 2.2 plugin was used to calculate the screening criteria and identify and extract core targets.

[0029] 4. Enrichment Analysis The intersection targets were imported into the DAVID database (https: / / david.ncifcrf.gov / ) for gene ontology (GO) functional analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis.

[0030] 5. Molecular Docking The 2D or 3D structures of the small molecule ligands were downloaded from the PubChem database. The 2D structures were converted to PDB structures using Chemdraw3D. The core protein data (PDB) structure files were obtained from the RCSB PDB website (https: / / www.rcsb.org / ). The core target receptors and core metabolite ligands were processed using Pymol software. Molecular docking was performed using AutoDock Vina 1.1.2, and visualization analysis was performed using PyMOL 2.2.0. The docking maps between the core targets and the core metabolites in potato juice were obtained.

[0031] Result analysis: Figure 2 As shown in Figure 2, network pharmacology analysis confirmed that the target network of the active ingredients of "Xiao Huang" contains 385 target nodes significantly associated with the improvement of ADHD. KEGG pathway enrichment analysis showed that these targets are mainly distributed in the following signaling pathways: (a) neuroactive ligand-receptor interaction pathway; (b) cyclic AMP-mediated signaling pathway; (c) calcium ion signaling pathway; and (d) insulin signaling pathway. These targets achieve multi-dimensional regulation of ADHD pathology through the synergistic action of multiple pathways.

[0032] like Figure 3As shown, molecular docking verification showed that the binding energy of the top 10 differential metabolites with the core targets was less than -4 kcal / mol, among which SCN2A sodium ion channel protein formed a stable hydrogen bond network with M121 (Demissidine) and M379 (Pseudoprotodioscin), and the binding sites were located at THR at position 885 and LYS at position 1422, respectively.

[0033] Table 1: Corresponding numbers of potato metabolites

[0034] Example 4: Evaluation of the efficacy of fresh potato juice in improving attention deficit hyperactivity disorder 1. Establishment of the Attention Deficit Hyperactivity Disorder Rat Model Methods: Forty-eight SPF male spontaneously hypertensive rats (4 weeks old) were purchased and housed in a constant temperature and humidity animal room at the Jarvis Animal Experimental Center, with 4 rats per cage. The light-dark cycle was 12 h, and the animals had free access to food and water. After one week of adaptive feeding, the rats were randomly divided into three experimental groups (Atlantic group, Favorita group, and Small Yellow group; n = 12) and a control group (n = 12).

[0035] 2. Experimental Design Fresh potato juice of different varieties was thawed and diluted in sterile water in proportion. Rats in the experimental group were orally administered with fresh potato juice daily (5 mL / rat / day), while rats in the model group were given sterile water as usual. The experiment lasted for 8 weeks, during which the weight and behavioral changes of the rats were regularly observed.

[0036] 3. Behavioral testing Morris water maze test: used to assess the spatial learning and memory abilities of rats. Four groups, namely the model group, the Atlantic group, the Favorita group, and the Xiaohuang group, were trained and tested sequentially.

[0037] (1) Acquisition training: On the first day, a platform was placed 1 cm below the liquid surface. Rats were placed into the water, head facing the wall, in one of four random starting positions: east, west, south, or north. The time it took for the animal to find the submerged platform was recorded. If the time exceeded 60 seconds, the animal was guided to the platform. The animal remained on the platform for 10 seconds. The system camera recorded the total distance traveled in the water, average speed, latency (time to find the platform and land), distance and time spent in each quadrant, and a trajectory map.

[0038] Starting from the second day, the platform was placed 1 cm below the liquid surface. Each rat was trained 3 times a day, with an interval of 1.5-2 h between two training sessions. The training lasted for 5 consecutive days (including the first day). The above indicators were recorded every day of training for 5 consecutive days.

[0039] (2) Test: Place the animal into the water from the opposite side of the original platform quadrant and record the data and movement trajectory.

[0040] Preparation before the test: On the second day after the last acquisition training (day 6 of the experiment), the platform was removed and a 60-s exploration test was started.

[0041] Novel Object Recognition Test: This test assesses the memory and cognitive abilities of rats. Four groups (model, Atlantic, Favorita, and Little Yellow) were trained and tested sequentially.

[0042] (1) Training phase: Before starting training, rats were placed in the testing room and allowed to adapt to the environment for 3 h in advance. Before training, the training venue, objects and other experimental equipment were cleaned and disinfected with 70% ethanol.

[0043] Place two objects of identical texture and shape in the training arena, such as balls, blocks, cubes, etc. Allow the rat to freely explore the training arena for 5 minutes and observe whether it will touch the objects. After the training is completed, return the rat to its home cage.

[0044] (2) Testing: Before the test phase, an old object is replaced with a new object. This new object may be different from the original object in shape, color or material. Let the rats explore freely in the test area for 5 minutes, and record the exploration time and number of explorations of the old and new objects. The degree of preference of the rats for old and new objects is calculated by analyzing the exploration time and number of explorations. The parameters that need to be recorded include the animal's activity time, exploration time, number of explorations, exploration distance, exploration speed, and stay time during training and testing. The most important of these is the time and number of explorations of the animals for the old and new objects, which is the main indicator for evaluating the animal's cognition and memory ability for new and old objects.

[0045] 4. Results Analysis like Figure 4 As shown, the latency of rats in the potato juice drinking group in the water maze test was significantly shortened compared with the model group, indicating that their spatial learning and memory abilities were improved, and the latency of the small yellow group was shorter in each time period.

[0046] like Figure 5 As shown in the data, the new object recognition index (the ratio of new object exploration time to total exploration time) of the rats in the experimental group drinking fresh potato juice was significantly higher than that of the model group in the new object recognition experiment, suggesting that drinking fresh potato juice can enhance memory ability, among which the new object recognition index of the small yellow group was higher than that of other potato varieties.

[0047] The present invention verifies the improvement effect of potato juice on attention deficit disorder through metabolomics, network pharmacology, molecular docking and animal experiments, and can effectively enhance learning and memory abilities and reduce hyperactive behavior. This method is safe and natural, and has great application prospects in diet therapy and traditional Chinese medicine health care.

Claims

1. Application of fresh potato juice in preparing food or medicine for improving attention deficit hyperactivity disorder.

2. The use according to claim 1, characterized in that The fresh potato juice is prepared from the diploid potato variety "Xiao Huang" and contains a variety of metabolite components such as amino acids, alkaloids, phenols, terpenes, flavonoids, lignans and coumarins.

3. The use according to claim 2, characterized in that The preparation method of the potato fresh juice comprises the following steps: (1) Select fresh, disease-free and insect-free diploid potatoes "Xiaohuang", wash and peel them; (2) Cut the potatoes into pieces and squeeze the juice, then filter to remove the residue to obtain clear fresh juice; (3) Divide the fresh juice into smaller pieces and store at low temperature for later use.

4. The use according to claim 1, wherein The fresh juice can shorten the latency period of ADHD model rats in a Morris water maze test and improve the novel object recognition index in a novel object recognition test.

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