Oyster enzymolysis ultrafiltration extract nasal spray and preparation method thereof

Abstract

The invention discloses an oyster enzymolysis ultrafiltration extract nasal spray and a preparation method thereof, the nasal spray comprises the following components: 5-10 wt% of oyster extract, 0.1-0.3 wt% of preservative, 0.05-0.15 wt% of sodium chloride and 0.1-0.3 wt% of emulsifier, the pH value is 5.5-6.5, the content of the preservative is 0.1-0.3 wt%, the content of the sodium chloride is 0.05-0.15 wt%, the content of the emulsifier is 0.1-0.3 wt%, and the balance is water. The preservative is one of benzalkonium chloride, sorbic acid and potassium sorbate, and the emulsifier is polysorbate 80. Through definite raw material composition, a step-by-step process and quality control, the bioavailability of active components is improved, the flavor of the product is optimized, the effect of safely and efficiently improving sleep is achieved, the problems of low bioavailability, poor flavor and potential safety hazards of an existing sleep improving product are solved, the process repeatability is high, and the preparation method is suitable for industrial production. The long-term use of people with chronic insomnia is facilitated.

Description

Technical Field

[0001] This invention belongs to the field of health product technology, and relates to a nasal spray, specifically to a sleep-improving health product made from oysters and using a nasal spray formulation, and its preparation method. Background Technology

[0002] With the fast pace and increasing pressure of modern life, sleep disorders have become a common problem affecting human health. According to relevant studies, about 30% of people worldwide suffer from insomnia, and about 300 million people in my country are troubled by sleep disorders, which manifest as difficulty falling asleep, poor sleep maintenance, and low sleep quality. In the long term, this can lead to fatigue, decreased attention, mood abnormalities, and even increase the risk of chronic diseases such as cardiovascular disease and diabetes.

[0003] Currently, methods for improving sleep mainly include medication, psychotherapy, and health supplements. Among medications, hypnotics such as benzodiazepines and diorhinone antagonists can provide short-term relief from insomnia, but long-term use can easily lead to tolerance and dependence, and may also cause side effects such as cognitive impairment, headaches, and drowsiness. Moreover, their efficacy for chronic insomnia is limited. Cognitive behavioral therapy, as a first-line non-pharmacological treatment, is difficult to widely promote due to its high cost, time-consuming nature, and lack of professional personnel.

[0004] In the health supplement industry, most existing products are in oral dosage form. While some ingredients (such as plant extracts and amino acids) have been proven to have some sleep-aiding potential, they suffer from low bioavailability. For example, protein and peptide components are easily destroyed by gastric juices and degraded by digestive enzymes after oral administration, making it difficult for them to exert their effects effectively. In addition, some products have problems such as fishy smell and poor taste due to the characteristics of their raw materials, which affects user acceptance.

[0005] Oysters, as one of the first batch of food and medicine homologous substances in my country, are rich in protein, amino acids (such as glycine and arginine), and minerals (such as zinc, selenium, and magnesium). Studies have confirmed that they have activities such as improving sleep, anti-oxidation, and anti-fatigue. Traditional Chinese medicine also records their ability to "nourish blood and calm the mind." However, current technologies for oyster development mainly focus on oral preparations, failing to fully address issues such as low bioavailability of active ingredients and a strong fishy smell, thus hindering the maximization of their sleep-improving effects. Therefore, developing a sleep-improving product based on oysters, with high bioavailability, good safety, and a suitable flavor has become an urgent need in the industry. Summary of the Invention

[0006] To address the problems of tolerance, dependence, and side effects associated with drug treatments in existing sleep improvement products, low bioavailability of oral supplements, and unpleasant flavors in some products (such as the noticeable fishy smell of oyster products), this invention provides a nasal spray of oyster enzymatic hydrolysis ultrafiltration extract and its preparation method. This invention, through clearly defined raw material composition, step-by-step processes, and quality control, improves the bioavailability of active ingredients, optimizes product flavor, and achieves safe and efficient sleep improvement. It solves the problems of low bioavailability, poor flavor, and safety concerns associated with existing sleep improvement products, and the process is highly reproducible.

[0007] The objective of this invention is achieved through the following technical solution: A nasal spray containing oyster enzymatic hydrolysis ultrafiltration extract comprises the following components: oyster extract, preservative, sodium chloride, and emulsifier. The oyster extract content is 5-10 wt%, the preservative content is 0.1-0.3 wt%, the sodium chloride content is 0.05-0.15 wt%, the emulsifier content is 0.1-0.3 wt%, and the pH is 5.5-6.5. The preservative is one of benzalkonium chloride, sorbic acid, and potassium sorbate, and the emulsifier is polysorbate 80.

[0008] A method for preparing the above-mentioned oyster enzymatic hydrolysis ultrafiltration extract nasal spray includes the following steps: Step 1: Enzymatic hydrolysis of oyster homogenate: Using oysters as raw material, oyster enzymatic hydrolysis products are obtained through enzymatic treatment. The specific steps are as follows: Step 1-1: Take fresh oyster meat, drain and homogenize it, adjust the pH to 6.0~6.8, add protease at a material-to-liquid ratio of 1:2~4, and enzymatically hydrolyze it in a constant temperature magnetic stirring water bath at 50~60℃ for 1~6 hours. The protease is bromelain or papain, and the amount of enzyme added is 500~700U / g. Steps 1-2: Inactivate enzymes in a boiling water bath at 100℃ for 5-15 minutes, then cool, centrifuge, and filter to obtain oyster enzymatic hydrolysate; Step 2: Ultrafiltration separation and purification: The oyster enzymatic hydrolysis products were separated by ultrafiltration to obtain oyster extract. The specific steps are as follows: Step 2-1: Use a 200μm ceramic membrane filtration system to perform preliminary filtration of the enzymatic hydrolysis products to remove macromolecular impurities; Step 2-2: Collect components <3 kDa by fractional ultrafiltration using a 3 kDa ultrafiltration membrane; Steps 2-3: Desalination treatment using ion exchange resin; Step 3, Remove fishy smell: The oyster extract after ultrafiltration is subjected to deodorization treatment. The deodorization method is Maillard reaction or β-cyclodextrin encapsulation method. The process conditions of Maillard reaction are: temperature 80~120℃, time 10~50min, pH=7~9, reducing sugar to amino acid ratio 1~1.5:1~1.5, and the reducing sugar is xylose, glucose or sucrose. Step 4, Spray drying: The deodorized enzymatic hydrolysate is concentrated to a solids concentration of 10-50 wt%, and then spray-dried into powder. The inlet air temperature of the spray dryer is 140-200℃, and the outlet air temperature is 70-100℃. Step 5: Nasal spray preparation and sterilization: Step 5-1: Purify the dried powder using alcohol precipitation, add preservatives, sodium chloride, and emulsifier, and adjust the pH to 5.5-6.5; Step 5-2: Sterilization. The sterilization method is either irradiation sterilization or steam sterilization. The specific steps for irradiation sterilization are as follows: A cobalt-60 gamma ray source is used, with a pre-verified minimum sterilization dose of 25 kGy and a maximum tolerated dose of 35 kGy. The irradiation dose is set to 25-30 kGy. A lithium fluoride dosimeter is used. 200 bottles of the product are neatly stacked in each box, with a 5 cm gap between the boxes. Dosimeters are placed at the four corners and center of each tray. The irradiation time is set to 120 minutes, and the target dose is 28 kGy. The temperature inside the chamber is controlled to ≤25℃. Parameters are monitored and recorded in real time. Sampling tests are conducted on sterility, active ingredient content, related substances, spray characteristics, and packaging compatibility. The product is released after passing the quality department's review. The steam sterilization temperature is 100℃, and the time is 20-40 minutes.

[0009] Compared with the prior art, the present invention has the following advantages: 1. Significantly safe with no risk of side effects: Acute toxicity tests, Ames tests, bone marrow cell micronucleus tests, mouse sperm abnormality tests, and 30-day feeding trials have verified that the product is non-toxic and has no mutagenic effects. It has no adverse effects on animal weight, food intake, hematological indicators, serum biochemical indicators, or organ coefficients.

[0010] The heavy metal content meets national standards (cadmium <0.03mg / kg, lead <0.50mg / kg, etc.), and the microbial limits and virus and mycoplasma tests all meet the standards, ensuring the safety of long-term use and avoiding the problems of tolerance, dependence and liver and kidney damage of traditional hypnotic drugs.

[0011] 2. Clearly improves sleep function, and the effect is quantifiable and controllable: Animal experiments: It can significantly prolong the sleep time induced by sodium pentobarbital in mice (the best dose group showed a significant prolongation effect, P<0.05), shorten the sleep latency, and increase the incidence of sleep (up to 50%). At the same time, it can upregulate the levels of inhibitory neurotransmitters (5-HT, GABA, melatonin) and downregulate the levels of excitatory neurotransmitters (DA, NE) in the brain, clarifying the mechanism of action.

[0012] Human trials: After use in 30 patients with chronic insomnia, the Pittsburgh Sleep Quality Index (PSQI) decreased from 17.53 points at baseline to 3.60 points after 4 weeks, the Athens Insomnia Scale (AIS) score decreased from 17.96 points to 3.21 points, and the average sleep duration increased by 1.08 hours, with no adverse reactions, demonstrating its significant effect on improving chronic insomnia (especially insomnia caused by excessive mental exertion).

[0013] 3. Significant advantages in dosage form and process, resulting in high bioavailability: Nasal spray formulation: It is absorbed through the nasal mucosa, avoiding the problem of low bioavailability caused by gastric juice and digestive enzyme degradation of oral preparations. It also has a fast onset of action, no first-pass effect in the liver, and can directly deliver the ingredients to the target site, solving the pain point of low utilization rate of active ingredients in traditional oral oyster products.

[0014] Process optimization: The process employs controlled enzymatic hydrolysis (optimal hydrolysis time of 4 hours), 3 kDa ultrafiltration separation (enrichment of active peptides), and Maillard reaction deodorization (100℃, 30 min) to effectively remove the fishy smell while retaining key components such as glycine, arginine, zinc, selenium, and magnesium (zinc content reaches 832 mg / kg) in oysters, thereby improving the palatability and activity stability of the product.

[0015] 4. Quality is controllable and meets standardized production requirements: The product has established clear quality standards, including sensory (clear and transparent, no sediment), physicochemical (pH 5.5~6.5, osmotic pressure 285~310mOsmol / kg), and microbiological (aerobic bacteria ≤200cfu / mL) indicators. The type test results all meet the requirements, ensuring batch-to-batch consistency and providing a guarantee for large-scale production.

[0016] In summary, this invention achieves technical advantages of "safety without side effects, clear function, high utilization, and controllable quality" through the naturalness of raw materials, advanced technology, and innovative dosage form. It effectively solves the problems of low bioavailability, poor flavor, and easy degradation after oral administration of oyster products in the prior art, making it suitable for long-term use by people with chronic insomnia. Attached Figure Description

[0017] Figure 1 Flowchart of the preparation process for oyster extract nasal spray.

[0018] Figure 2The degree of hydrolysis of oyster enzymatic hydrolysate.

[0019] Figure 3 This refers to the glutamic acid content of oyster hydrolysate.

[0020] Figure 4 The effect of this product on the sleep time of mice.

[0021] Figure 5 The effect of this product on the sleep latency of mice.

[0022] Figure 6 The effect of this product on the 5-HT content in the mouse brain.

[0023] Figure 7 The effect of this product on the GABA content in the mouse brain.

[0024] Figure 8 The effect of this product on the DA content in the mouse brain.

[0025] Figure 9 The effect of this product on the Glu content in the mouse brain.

[0026] Figure 10 The Pittsburgh Sleep Quality Index was used for evaluation.

[0027] Figure 11 Evaluation using the Athens Insomnia Scale.

[0028] Figure 12 Analysis of changes in sleep duration. Detailed Implementation

[0029] The technical solution of the present invention will be further described below with reference to the accompanying drawings, but it is not limited thereto. Any modifications or equivalent substitutions to the technical solution of the present invention that do not depart from the spirit and scope of the technical solution of the present invention should be covered within the protection scope of the present invention.

[0030] Example 1: This embodiment provides a nasal spray of oyster enzymatic hydrolysis ultrafiltration extract, the composition of which is as follows: Main ingredients: Oyster extract (containing <3 kDa oyster active peptides, oyster polysaccharides and mineral elements such as zinc, selenium, and magnesium), with a content of 7.5% (by mass). Excipients: Potassium sorbate (preservative): 0.20% (based on the mass percentage of the nasal spray), Sodium chloride (to adjust osmotic pressure): 0.1% (based on the mass percentage of the nasal spray), Polysorbate 80 (emulsification and thickening): 0.2% (based on the mass percentage of the nasal spray). This combination of excipients and parameter range ensures the stability, safety, and nasal adaptability of the nasal spray; and the pH is adjusted to 5.5~6.5 by NaOH and HCl.

[0031] like Figure 1 As shown, the preparation method of the above-mentioned oyster enzymatic hydrolysis ultrafiltration extract nasal spray includes the following steps: Step 1: Enzymatic hydrolysis of oyster homogenate: Procedure: Take fresh oyster meat, drain and homogenize it, adjust the pH to 6.5, add bromelain (600 U / g) at a material-to-liquid ratio of 1:3, and enzymatically hydrolyze in a 55℃ constant temperature magnetic stirring water bath for 4 hours to maximize the release of oyster active ingredients; then inactivate the enzyme in a 100℃ boiling water bath for 10 minutes, cool and centrifuge at 8000 rpm and 4℃ for 20 minutes, filter with gauze to remove impurities, and obtain the oyster enzymatic hydrolysis product.

[0032] Function: Controllable enzymatic hydrolysis breaks down large protein molecules in oysters into smaller peptides and amino acids (such as glycine and arginine), improving the release efficiency of active ingredients and laying the foundation for subsequent separation and purification. The degree of hydrolysis of the oyster enzymatic hydrolysate is as follows: Figure 2 As shown, the glutamic acid content of oyster hydrolysate is as follows: Figure 3 As shown.

[0033] Physicochemical composition analysis of oyster enzymatic hydrolysis products: The basic composition of oyster enzymatic hydrolysis products is shown in Table 1, the amino acid composition of oyster enzymatic hydrolysis products is shown in Table 2, and the mineral element composition of oyster enzymatic hydrolysis products is shown in Table 3.

[0034] Table 1. Basic Components of Oyster Enzymatic Hydrolysate (%)

[0035] Table 2 Amino acid composition of oyster enzymatic hydrolysates

[0036] Table 3. Mineral element content composition of oyster enzymatic hydrolysate (mg / kg)

[0037] Step 2: Ultrafiltration separation and purification: Procedure: The enzymatic hydrolysis products were initially filtered using a 200μm ceramic membrane filtration system to remove macromolecular impurities; then, fractional ultrafiltration was performed using a 3KDa ultrafiltration membrane to collect components <3KDa; finally, desalination was performed using ion exchange resin.

[0038] Function: To screen out active components with smaller molecular weights and easier absorption (<3KDa), avoid the influence of large molecules on absorption efficiency, reduce impurities and improve product purity.

[0039] Physicochemical composition analysis of ultrafiltration fractions of oyster enzymatic hydrolysis products: The basic composition of the ultrafiltration fraction of oyster enzymatic hydrolysis product is shown in Table 4, the amino acid composition of the ultrafiltration fraction of oyster enzymatic hydrolysis product is shown in Table 5, and the mineral element content of the ultrafiltration fraction of oyster enzymatic hydrolysis product is shown in Table 6.

[0040] Table 4. Basic Components of Ultrafiltration Fragments from Oyster Enzymatic Hydrolysate (%)

[0041] Table 5 Amino acid composition of ultrafiltration fractions from oyster enzymatic hydrolysis products

[0042] Table 6. Mineral element content composition of ultrafiltration fractions of oyster enzymatic hydrolysis products (mg / kg)

[0043] Step 3, Maillard reaction to remove fishy smell: Procedure: Based on the optimal process (temperature 100℃, time 30 min, pH 7.5, sucrose to amino acid mass ratio 1:1), the ultrafiltration oyster extract was subjected to the Maillard reaction, which effectively masked the fishy smell of oysters and improved the palatability of the product. The composition of free amino acids in the product was analyzed, and the results are shown in Table 7.

[0044] Table 7 Amino acid composition analysis

[0045] Function: By utilizing the amino acids naturally present in oysters to react with sucrose, flavor substances are generated to mask the fishy smell, thus solving the problem of low user acceptance of oyster products due to the fishy smell.

[0046] Step 4, Spray drying: Procedure: The deodorized enzymatic hydrolysate is concentrated to a solids concentration of 30% using a rotary evaporator, and then spray-dried (inlet air temperature 180℃, outlet air temperature 80℃) to form powder. These conditions can efficiently retain the active ingredients.

[0047] Function: Drying increases the concentration of active ingredients, facilitating subsequent formulation processing while preserving the bioactivity of oyster extract.

[0048] Step 5: Nasal spray preparation and sterilization: Procedure: The dried powder was purified using alcohol precipitation (ethanol was added to a concentration of 80%, allowed to stand for at least 12 hours, filtered, and concentrated to a concentration of approximately 1 g / mL of oyster powder). Excipients (potassium sorbate, sodium chloride, and polysorbate 80) were added, and the pH was adjusted to 5.5-6.5. Finally, irradiation sterilization was used to ensure effective sterilization while minimizing damage to active ingredients and ensuring compliance with microbiological standards. The finished product was tested and found to contain 900 mg of oyster active peptides and 325 mg of oyster glycogen per 100 ml, demonstrating good preservation of the natural active ingredients in oysters.

[0049] Function: Formulated as a nasal spray, it takes advantage of the rich blood supply and rapid absorption of the nasal mucosa to avoid gastric juice damage and the first-pass effect of the liver when taken orally, thus improving bioavailability; sterilization process ensures product safety.

[0050] In this embodiment, the following standards are set to ensure product effectiveness and safety: Sensory indicators: Clear and transparent in color, free of suspended matter, sediment and stratification, odorless or with a slight oyster smell; Physicochemical properties: osmotic pressure 285~310mOsmol / kg, pH 5.5~6.5, volume 5mL, total number of sprays per bottle 40, spray volume per spray 125μL, protein concentration ≥8μg / μL; Safety indicators: Total aerobic bacteria count ≤200 cfu / mL, total mold and yeast count ≤20 cfu / mL, Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa must not be detected; no mycoplasma or endogenous / endogenous viral sequences; heavy metal content meets national standards (Table 8).

[0051] Table 8 Heavy Metal Content

[0052] Function implementation principle: This product improves sleep by overcoming the side effects of existing medications and the shortcomings of oral formulations in the following ways: Synergistic effects of active ingredients: Glycine in oyster extract can cross the blood-brain barrier and act on NMDA receptors in the suprachiasmatic nucleus, while arginine helps improve sleep; zinc promotes melatonin synthesis, selenium regulates neurotransmitter secretion, and magnesium inhibits excessive excitation of nerve cells, all of which work together to regulate sleep-related pathways.

[0053] Neurotransmitter regulation: By increasing the levels of inhibitory neurotransmitters (5-hydroxytryptamine, γ-aminobutyric acid, melatonin) in the brain and decreasing the levels of excitatory neurotransmitters (dopamine, norepinephrine), sleep latency is shortened and sleep time is prolonged.

[0054] Advantages of nasal spray formulations: Nasal administration allows for direct absorption through the mucous membrane, resulting in rapid onset of action (avoiding oral degradation), high bioavailability, non-invasiveness (superior to injection), and convenient use.

[0055] Scope of application and uses: This product is derived from oyster extract, which contains various active substances, nutrients, and saline solution secreted by oysters. Studies have found that this product can calm the nerves, improve sleep patterns and quality in people with insomnia, and promote natural sleep, especially for chronic insomnia caused by excessive mental exertion.

[0056] This product contains natural active ingredients that can promote natural sleep. After 3-4 weeks of use, it can maintain natural sleep for a longer period of time. It is easy to use, non-addictive and non-dependent, and does not require long-term use.

[0057] Usage and dosage: For external use in the nasal cavity. One hour before bedtime, once a day, 4 sprays each time, 2 sprays per nostril.

[0058] Effectiveness evaluation: Safety assessment: Safety evaluation was conducted using C57BL / 6J and weaned SD rats. (1) Acute toxicity test: Mice and rats were given intranasal drops daily (dosage groups: 0.5 μl / g·bw, 1 μl / g·bw, 1.5 μl / g·bw) and observed for 30 days. No obvious poisoning symptoms were observed in either male or female mice or rats, and no deaths occurred. This proves that this product is non-toxic.

[0059] (2) The Ames test was conducted using the plate incorporation method. With or without liver microsomal enzyme S9, this product did not cause a significant increase in the number of reversion mutant colonies. The Ames test result was negative.

[0060] (3) The bone marrow cell micronucleus test was performed by administering the test substance over 30 hours. The difference between the positive control group and the solvent control group in the bone marrow cell micronucleus test was statistically significant. The micronucleus rate of this product was not statistically significant compared with that of the solvent control group. The mouse bone marrow cell micronucleus test was negative.

[0061] (4) The sperm abnormality rate of the positive control group in the mouse sperm abnormality test was significantly different from that of the solvent control group. The sperm abnormality rate of this product was not significantly different from that of the solvent control group, and the mouse sperm abnormality test was negative.

[0062] (5) 30-day feeding test: The animal was given this product via nasal drops. During the test, no obvious abnormalities were observed in the animal's activity, feeding, defecation, or other general conditions. Effect of this product on rat body weight: There was no statistically significant difference in body weight between the female and male dose groups and the control group, indicating that this product has no significant effect on the animal's body weight (Table 9).

[0063] Table 9 Animal weight changes

[0064] (6) Effects on food intake and food utilization in rats: There was no statistically significant difference in food intake between the female and male dose groups and the control group, indicating that the product had no significant effect on the food intake of the animals (Table 10).

[0065] Table 10 Changes in animal feed intake

[0066] (7) Hematological examination results: There was no statistically significant difference in the blood routine indicators of the female and male animals in each dose group compared with the control group, indicating that the product has no significant effect on the blood routine indicators of the animals (Table 11).

[0067] Table 11 Changes in routine blood parameters of animals

[0068] (8) Serum biochemical test results: There were no statistically significant differences in serum biochemical indicators (alanine aminotransferase, aspartate aminotransferase, creatinine, cholesterol, triglycerides, total protein, albumin, blood urea nitrogen, blood glucose) between the animals in each dose group and the control group, indicating that this product has no significant effect on the serum biochemical indicators of animals (Table 12).

[0069] Table 12 Changes in Serum Biochemical Indicators in Animals

[0070] (9) Effects of this product on organ weight and organ coefficient: There were no statistically significant differences in the weight and organ coefficient of the liver, kidney, spleen and gonads of animals in each dosage group compared with the control group, indicating that this product has no significant effect on the organ weight and organ coefficient of animals (Table 13).

[0071] Table 13 Changes in organ weight and organ coefficients in animals

[0072] Behavioral experiments Behavioral experiments were conducted using C57BL / 6J mice. The results showed that if the sample had an inhibitory effect on the central nervous system, it would induce direct sleep in the mice, indicating that the sample does not fall under the category of improving sleep. When no animals in the negative control group or experimental group slept, it was determined that the product had no inhibitory effect on the central nervous system, and the remaining three behavioral experiments were then conducted. Table 14 shows that after administering different doses of the product via nasal drops for 30 minutes, neither the negative control group nor the nasal drop group exhibited direct sleep, and the difference was not statistically significant (P>0.05), indicating that the product does not induce direct sleep in mice.

[0073] Table 14 Number of Hypnotized Mice

[0074] The length of sleep directly affects sleep quality. Sodium pentobarbital is a centrally acting drug with a hypnotic effect, effectively prolonging sleep time. Compared to the negative control group, the sleep time of mice treated with this product was significantly prolonged, indicating that the sample can synergistically prolong sleep time in mice with sodium pentobarbital. Based on... Figure 4 It was found that both the positive control group (diazepam: 2 mg / kg) and the experimental group prolonged the sleep time induced by sodium pentobarbital in mice. Compared with the negative control group, both the positive control group and the experimental group showed significantly prolonged sleep time. Administration of 12 μL of this product achieved the optimal prolongation time. Therefore, this product can enhance the effect of sodium pentobarbital and prolong the sleep time in mice.

[0075] Sodium pentobarbital is a central nervous system drug that is metabolized by liver enzymes. If the sample inhibits liver enzymes, prolonged sleep time may also occur, leading to false positive results. To rule out false positives, a subthreshold dose-induced hypnotic experiment with sodium pentobarbital should be conducted. Mice in each group were treated and then intraperitoneally injected with 30 mg / kg sodium pentobarbital (0.1 mL / 10 g). After 30 minutes, the number of mice falling asleep and the sleep incidence rate were measured. Sleep incidence rate = (number of animals falling asleep / number of animals per group) × 100%. The sleep incidence rates of the negative control group, positive control group, and experimental group were compared. Table 15 shows that compared with the negative control group, the sleep incidence rates of mice in both the positive control group and the experimental group were increased. These results indicate that this product can synergistically increase the sleep incidence rate with sodium pentobarbital.

[0076] Table 15. Effects of this product on the incidence of sleepiness in mice.

[0077] The speed of falling asleep is also an important indicator of sleep quality; a short sleep latency indicates rapid onset of sleep. Sodium barbital is a drug with central nervous system depressant effects. If, after intraperitoneal injection, the sleep latency in each group is significantly shortened, it indicates a synergistic effect between the sample and sodium barbital, and the experimental result is positive. One hour after the last gavage, mice in each group were intraperitoneally injected with 300 mg / kg sodium barbital (0.1 mL / 10 g). The time from sodium barbital injection to the disappearance of the righting reflex was defined as the sleep latency. The sleep latency of the negative control group, positive control group, and experimental group was compared. Figure 5 It was found that, compared with the negative control group, the sleep latency of both the positive control group and the experimental group was significantly shortened. The results indicate that this product, in synergy with sodium barbital, significantly shortens the sleep latency of mice. Based on the above experiments, administering 12 μL of this product achieves the best effect and is also the optimal dosage. Subsequent experiments will be conducted at this dosage.

[0078] Neurotransmitter content detection Sleep and wakefulness is a complex physiological process regulated by the activity of multiple parts of the brain, among which the neurotransmitter system plays a crucial role. The neurotransmitters most closely related to sleep are 5-HT and GABA. 5-HT and GABA are the most important inhibitory neurotransmitters in the brain; they can reduce neuronal activity and regulate nerve cell function. 5-HT is widely distributed in the brain and is currently one of the most extensively studied neurotransmitters. Increased 5-HT levels can promote sleep. To detect the levels of neurotransmitters in brain tissue, mice in each group were sacrificed 1 hour after the last gavage. The hypothalamus and cerebral cortex of the mice were quickly harvested on ice, weighed, and homogenized with PBS at a ratio of 1:9 (mass ratio). Centrifugation was performed according to the kit instructions at 3000 rpm at 4°C for 20 min. The supernatant was used to determine the levels of serotonin (5-HT), γ-aminobutyric acid (GABA), melatonin (MT), dopamine (DA), and norepinephrine (NE). Compared with the negative control group, the hypothalamic 5-HT levels in both the positive control group and the experimental group were significantly increased. Figure 6 (P<0.05).

[0079] GABA is an inhibitory neurotransmitter synthesized in neurons and widely distributed. GABAergic neurons are distributed in the basal forebrain, anterior hypothalamus, midbrain, and hindbrain, and can increase firing frequency during sleep. Furthermore, during REM sleep, GABAergic neurons can inhibit other types of neurons involved in wakefulness, improving sleep production. GABAergic neurons are distributed in all cortical structures, and the size and duration of GABA synaptic activity are determined by plasma membrane proteins, specifically GABA transporters. Compared with the negative control group, the GABA content in the hypothalamus of both the positive control and experimental groups was significantly increased (P<0.05), and the GABA content in the cerebral cortex of both the positive control and experimental groups was significantly higher than that of the negative control group. Figure 7 (P<0.05).

[0080] Dopamine (DA) is an excitatory neurotransmitter; increased DA levels promote sleep-wake cycles. The hypothalamic DA levels in both the positive control group and the experimental group were significantly lower than those in the negative control group (P<0.05). Compared to the negative control group, the DA levels in the cerebral cortex of both the positive control group and the experimental group were decreased. Figure 8 (P<0.05).

[0081] Glu is an excitatory neurotransmitter that promotes sleep-wake cycles. A significant decrease in Glu levels indicates that the sample has a sleep-improving effect. The hypothalamic Glu levels in both the positive control group and the experimental group were lower than those in the negative control group, with a significant decrease in Glu levels in both groups (P<0.05). Compared to the negative control group, the experimental group showed a significant decrease in Glu levels in the cerebral cortex. Figure 9 (P<0.05).

[0082] Evaluation of sleep improvement effects in humans In a mouse model, 12 μL of this product achieved the best results. Based on the animal-to-human dose conversion factor (Table 16), the nasal spray dose of this product in humans is approximately 108 μL. Next, we will investigate whether this product has an ameliorative effect on chronic insomnia in humans.

[0083] Table 16. Dosage Conversion Factors for Animals and Humans

[0084] (1) The diagnostic criteria for insomnia are as follows: Western medicine diagnostic criteria: Referring to the International Classification of Sleep Disorders, Third Edition (ICSD-3) developed by the American Academy of Sleep Medicine in 2014, the diagnostic criteria for chronic insomnia must simultaneously meet the following: A. The patient reports, or the patient's parents or caregivers observe, that the patient has one or more of the following: ① difficulty falling asleep; ② difficulty maintaining sleep; ③ waking up earlier than the expected wake-up time; ④ refusing to go to bed at the appropriate time; ⑤ difficulty falling asleep without intervention from parents or caregivers.

[0085] B. Patient reports, or the patient's parents or recruiters observe that the patient has one or more of the following symptoms associated with nighttime sleep difficulties: ① fatigue / mental fatigue; ② impaired attention, concentration, or memory; ③ impairment of social, family, or occupational functioning or academic performance; ④ mood instability / irritability; ⑤ daytime sleepiness; ⑥ behavioral problems (e.g., hyperactivity, impulsivity, or aggression); ⑦ decreased motivation, energy, or enthusiasm; ⑧ error-proneness / accident-proneness; ⑨ excessive concern or dissatisfaction with their sleep quality.

[0086] C. These sleep / wake difficulties cannot be entirely explained by unsuitable sleep opportunities (such as sufficient sleep time) or environment (such as a safe, comfortable, dark, and quiet environment).

[0087] D. These sleep difficulties and related daytime symptoms persist at least 3 times a week.

[0088] E. These sleep difficulties and related daytime symptoms persist for at least 3 months.

[0089] F. These sleep difficulties and related daytime symptoms cannot be better explained by other sleep disorders.

[0090] Traditional Chinese Medicine (TCM) diagnostic criteria: Referencing the diagnostic criteria for insomnia in the "Interpretation of Clinical Diagnosis and Treatment Guidelines in Traditional Chinese Medicine: Brain Diseases" jointly compiled by the China Association of Traditional Chinese Medicine and other organizations in 2015, the following criteria must be met simultaneously: 1) Difficulty falling asleep, or waking up easily and finding it difficult to fall back asleep, or even having trouble sleeping all night; 2) It is often accompanied by symptoms such as irritability, excessive dreaming, palpitations, forgetfulness, headache, dizziness, and fatigue; 3) For more than 4 consecutive weeks; 4) No other organic lesions or triggers that interfere with sleep.

[0091] (2) Inclusion criteria: 1) Meets the above diagnostic criteria; 2) Age 18-70; 3) PSQI score > 5; 4) Has not taken any sedative-hypnotic drugs in the past 3 months; 5) Participate voluntarily and sign the informed consent form.

[0092] (3) Exclusion criteria: 1) Does not meet the above diagnostic and inclusion criteria; 2) Individuals with respiratory-related sleep disorders, environmental sleep difficulties, drug-induced insomnia, mental disorders, or short sleep; 3) Patients with cardiovascular and cerebrovascular diseases (such as cognitive impairment, hypertension, etc.), endocrine and metabolic diseases (such as hypothyroidism, diabetes, etc.), and tumors; 4) Patients with hematological diseases and persistent pain caused by various reasons; 5) Pregnant women; 6) Individuals who have participated in other clinical trials within the past 3 months; 7) Patients with poor adherence, intolerance, or who drop out midway; 8) Other circumstances deemed unsuitable for inclusion by the researchers.

[0093] (4) Elimination criteria: 1) Those who were mistakenly included but did not meet the inclusion criteria; 2) Those who self-administered other insomnia intervention methods during the study period; 3) Those who have poor compliance during the study and do not receive treatment or assessment according to the study protocol.

[0094] (5) Shedding criteria: 1) Those who withdraw midway through the study due to poor compliance or failure to complete the treatment according to the study protocol because of various objective factors; 2) Individuals who experience serious adverse events or other special changes and are no longer suitable to receive intervention.

[0095] (6) Termination criteria: 1) A serious safety incident occurred during the experiment; 2) The experimental design deviated significantly during implementation, affecting the evaluation of the results.

[0096] The product was evaluated using the Pittsburgh Sleep Quality Index and the Athens Insomnia Scale, respectively.

[0097] Following the inclusion and exclusion criteria, a total of 30 participants were included in the Pittsburgh Sleep Quality Index (PSI) questionnaire survey to assess their sleep quality over the past month, including sleep onset time, sleep duration, sleep efficiency, sleep disturbances, use of hypnotics, and daytime dysfunction. Higher scores indicated poorer sleep quality. Scores ≥8 were considered poor sleep quality, and <8 were considered good sleep quality. This scale is commonly used in clinical psychiatric studies and sleep quality evaluation in my country, demonstrating significant reliability and validity. After using this product, insomnia patients experienced improved sleep quality within one week, and achieved better sleep quality after two weeks. Figure 10 ).

[0098] According to the inclusion and exclusion criteria, a total of 27 participants were included to evaluate this product using the Athens Insomnia Scale. The Athens Insomnia Scale was designed in 1985 by the Ohio State University School of Medicine to help people assess their insomnia. Due to its accurate self-test results and ease of use, it is widely used in clinical practice and has become an internationally recognized standard scale for evaluating insomnia. Experimental data suggests that after using this product for one week, the Athens Insomnia Scale scores of insomnia patients significantly decreased, and sleep quality improved. Continued use can lead to better sleep quality. Figure 11 ).

[0099] Sleep duration analysis suggests that this product can prolong sleep time and has a positive effect on improving sleep. Figure 12 Until the completion of this test, no adverse reactions were observed with this product, demonstrating its high safety.

[0100] Example 2: The difference between this embodiment and Example 1 is that bromelain is replaced with papain, and effective enzymatic hydrolysis is achieved under the same enzymatic hydrolysis conditions (pH 6.5, 55℃).

[0101] Example 3: The difference between this embodiment and Embodiment 1 is that the deodorization method uses β-cyclodextrin encapsulation.

[0102] Example 4: The difference between this embodiment and Embodiment 1 is that the sterilization method is flowing steam sterilization (100℃, 30min).

[0103] Example 5: The difference between this embodiment and embodiments 1-4 is that the content of oyster extract is 5 wt%, the content of potassium sorbate is 0.1 wt%, the content of sodium chloride is 0.05 wt%, and the content of polysorbate 80 is 0.1~0.3 wt%.

[0104] Example 6: The difference between this embodiment and embodiments 1-4 is that the content of oyster extract is 10wt%, the content of potassium sorbate is 0.3wt%, the content of sodium chloride is 0.15wt%, and the content of polysorbate 80 is 0.3wt%.

Claims

1. A nasal spray containing oyster enzymatic hydrolysis ultrafiltration extract, characterized in that... The nasal spray comprises the following ingredients: oyster extract, preservative, sodium chloride, and emulsifier. The content of oyster extract is 5-10 wt%, the content of preservative is 0.1-0.3 wt%, the content of sodium chloride is 0.05-0.15 wt%, and the content of emulsifier is 0.1-0.3 wt%.

2. The oyster enzymatic hydrolysis ultrafiltration extract nasal spray according to claim 1, characterized in that... The preservative is one of benzalkonium chloride, sorbic acid and potassium sorbate, and the emulsifier is polysorbate 80.

3. The oyster enzymatic hydrolysis ultrafiltration extract nasal spray according to claim 1, characterized in that... The nasal spray contains the following ingredients: oyster extract, potassium sorbate, sodium chloride, and polysorbate 80. The content of oyster extract is 5-10 wt%, the content of potassium sorbate is 0.2 wt%, the content of sodium chloride is 0.1 wt%, and the content of polysorbate 80 is 0.2 wt%.

4. A method for preparing a nasal spray of oyster enzymatic hydrolysis ultrafiltration extract according to any one of claims 1-3, characterized in that... The method includes the following steps: Step 1: Enzymatic hydrolysis of oyster homogenate: Oysters are used as raw materials, and oyster enzymatic hydrolysis products are obtained through enzymatic hydrolysis. Step 2: Ultrafiltration separation and purification: Oyster enzymatic hydrolysis products were separated by ultrafiltration to obtain oyster extract; Step 3, Remove fishy smell: The oyster extract after ultrafiltration was subjected to deodorization treatment; Step 4, Spray drying: The deodorized enzymatic hydrolysate was concentrated to a solids concentration of 10-50 wt% and then spray-dried to produce powder. Step 5: Nasal spray preparation and sterilization: Step 5-1: Purify the dried powder using alcohol precipitation, add preservatives, sodium chloride, and emulsifier, and adjust the pH to 5.5-6.5; Step 5-2: Sterilization.

5. The method for preparing the oyster enzymatic hydrolysis ultrafiltration extract nasal spray according to claim 4, characterized in that... The specific steps of step 1 are as follows: Step 1-1: Take fresh oyster meat, drain and homogenize it, adjust the pH to 6.0~6.8, add protease at a material-to-liquid ratio of 1:2~4, and enzymatically hydrolyze it in a 50~60℃ constant temperature magnetic stirring water bath for 1~6 hours. Steps 1-2: Inactivate enzymes in a boiling water bath at 100℃ for 5-15 minutes, then cool, centrifuge, and filter to obtain oyster enzymatic hydrolysis products.

6. The method for preparing the oyster enzymatic hydrolysis ultrafiltration extract nasal spray according to claim 5, characterized in that... The protease is bromelain or papain, and the amount of enzyme added is 500~700 U / g.

7. The method for preparing the oyster enzymatic hydrolysis ultrafiltration extract nasal spray according to claim 4, characterized in that... The specific steps of step 2 are as follows: Step 2-1: Use a 200μm ceramic membrane filtration system to perform preliminary filtration of the enzymatic hydrolysis products to remove macromolecular impurities; Step 2-2: Collect components < 3 kDa using fractional ultrafiltration through a 3 kDa ultrafiltration membrane; Steps 2-3: Desalination is performed using ion exchange resin.

8. The method for preparing the oyster enzymatic hydrolysis ultrafiltration extract nasal spray according to claim 4, characterized in that... In step 3, the deodorization method is the Maillard reaction or the β-cyclodextrin encapsulation method. The process conditions for the Maillard reaction are: temperature 80~120℃, time 10~50min, pH=7~9, reducing sugar to amino acid ratio 1~1.5:1~1.5, and the reducing sugar is xylose, glucose or sucrose.

9. The method for preparing the oyster enzymatic hydrolysis ultrafiltration extract nasal spray according to claim 4, characterized in that... In step 4, the inlet air temperature of the spray dryer is 140~200℃, and the outlet air temperature is 70~100℃.

10. The method for preparing the oyster enzymatic hydrolysis ultrafiltration extract nasal spray according to claim 4, characterized in that... In step 5-2, the sterilization method is either irradiation sterilization or steam sterilization.

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