Leech preparation for improving sleep disorder as well as preparation method and application of leech preparation

Abstract

The invention relates to the technical field of medicine, and discloses a leech preparation for improving sleep disorder and a preparation method and application of the leech preparation. The leech extract is selected from one or more of leech, leech, leech, whitmania pigra, whitmania pigra, hirudo nipponia, whitmania pinnata, poecilobdella manillensis, whitmania lubrida and whitmania occulta; the preparation can be prepared into tablets, capsules, granules, injections, ointments, suppositories, aerosols, sprays or traditional Chinese medicine preparations. The preparation method comprises the following steps: chopping leeches, soaking, performing reflux extraction, concentrating an extracting solution into clear paste, further purifying through alcohol precipitation, and mixing the purified extract with auxiliary materials to prepare a target dosage form. According to the leech preparation, by adjusting neurotransmitter balance, resisting inflammation and oxidation and improving brain microcirculation, the sleep latency can be remarkably shortened, the sleep duration can be prolonged, and sleep disorders can be effectively improved.

Description

Technical Field

[0001] The invention relates to the technical field of medicines, and in particular to a leech preparation for improving sleep disorders, a preparation method thereof and an application thereof. Background Art

[0002] Sleep disorders are a common chronic health problem, characterized by difficulty falling asleep, difficulty maintaining sleep, early awakening, or poor sleep quality. This disease seriously affects the patient's quality of life and may lead to a series of health problems, such as fatigue, decreased concentration, memory loss, and long-term psychological and physiological problems, including an increased risk of anxiety and cardiovascular disease. With the increase of social pressure and the accelerated pace of life, the incidence of sleep disorders has increased year by year, and has become a public health problem that needs to be urgently addressed worldwide.

[0003] At present, the main means of treating sleep disorders clinically is drug therapy, and most of these drugs induce sleep by regulating the activity of neurotransmitters. However, existing drugs still have obvious deficiencies in efficacy and safety. The efficacy of many drugs is limited, especially for patients who take them for a long time, the efficacy may decrease over time. At the same time, some drugs have problems of addiction and drug resistance, which requires patients to gradually increase the dose, further aggravating the adverse reactions of the drugs. In addition, these drugs are often accompanied by side effects such as drowsiness, dizziness, memory loss and central nervous system depression, which seriously affect patients' daily lives.

[0004] Based on the above problems, finding a natural treatment method that is efficient, safe and has few side effects has become the current research focus. In the field of traditional Chinese medicine, leeches, as a traditional medicinal material, are widely known for their effects of promoting blood circulation and removing blood stasis, and anti-inflammatory and anticoagulant. In recent years, studies have shown that active ingredients such as peptides, enzymes and hirudin rich in leeches have the functions of regulating neurotransmitters, protecting neurons and improving brain blood circulation, which provides new possibilities for the treatment of sleep disorders. However, the development and application of leech medicinal materials in modern medicine are still relatively limited, especially in the optimization of extraction process, enrichment of active ingredients and formulation design. There are still many technical bottlenecks, which limit its promotion and application in clinical treatment.

[0005] Therefore, developing a preparation based on leech extract that can efficiently extract and stably retain its active ingredients, while enhancing its effect of improving sleep disorders through reasonable formulation design, is a technical problem that urgently needs to be solved in this field. Summary of the invention

[0006] In view of the deficiencies of the prior art, the present invention provides a leech preparation for improving sleep disorders, a preparation method thereof, and an application thereof, which solve the problems of limited efficacy, large side effects, and strong dependence of existing drugs for treating sleep disorders.

[0007] To achieve the above object, the present invention is realized by the following technical solutions: In the first aspect of the present invention, a leech preparation for improving sleep disorders is provided. The preparation includes a leech extract and a pharmaceutically acceptable excipient. The leech extract is derived from one or more of Whitmania pigra, Hirudo nipponica, Whitmania acranulata, Whitmania laevis, Hirudo medicinalis, Whitmania acuminata, Poecilobdella manillensis, Whitmania laevigata, and Erpobdella octoculata.

[0008] Through a specific extraction process, the active ingredients obtained from the above leech species include, but are not limited to, polypeptide compounds, hirudin, enzymes, and other small molecule active substances. These active ingredients can play roles in the following aspects: Regulation of neurotransmitters The active polypeptides and other bioactive molecules in the leech extract can enhance the activities of 5-hydroxytryptamine (5-HT) and γ-aminobutyric acid (GABA) in the central nervous system. 5-HT is an important neurotransmitter that regulates mood and sleep, and an increase in its level helps to improve sleep quality; while GABA is the main inhibitory neurotransmitter, which can reduce the excitability of the central nervous system, thereby promoting sleep induction and maintenance. By regulating the levels of these key neurotransmitters, the leech preparation has a good improvement effect on sleep disorders caused by anxiety, stress, and nerve dysfunction.

[0009] Anti-inflammatory and antioxidant effects The enzyme components (such as fibrinolysin) and other antioxidant molecules in the leech extract can improve sleep disorders by reducing the levels of brain inflammatory factors (such as TNF-α, IL-6) and scavenging oxidative stress free radicals. Patients with sleep disorders usually have an increase in neuroinflammation and oxidative stress, which can damage the normal functions of neurons and thus affect sleep quality. The anti-inflammatory and antioxidant properties of the leech extract can effectively alleviate these pathological processes and create a more favorable neural environment for sleep restoration.

[0010] Neuroprotective effects The leech preparation plays a protective role on neurons through hirudin and other active factors therein. These components can reduce the apoptosis of nerve cells, improve the nerve conduction efficiency, and at the same time enhance the plasticity of nerve synapses. The neuroprotective effect can not only improve sleep disorders caused by brain lesions, but also have a certain repair effect on nerve damage that may be caused by long-term insomnia.

[0011] Improvement of blood circulation Hirudin contained in the leech extract has a significant anticoagulant effect, which can improve cerebral microcirculation disorders and increase the oxygen and nutrient supply to nerve tissues. This effect has a good therapeutic effect on sleep problems caused by central nervous system dysfunction due to circulatory disorders. At the same time, by improving blood circulation, it can improve the distribution efficiency of drugs in the brain tissue and further enhance the curative effect. Chronic diseases such as cerebral ischemia can cause poor blood circulation, and ischemia and hypoxia can cause a decrease in blood oxygen, affecting cerebral blood supply, and then leading to abnormalities in the sleep center, resulting in problems such as decreased sleep quality and shortened sleep duration.

[0012] In summary, the leech preparation can effectively relieve sleep disorders through the synergistic action of multiple mechanisms such as neurotransmitter regulation, anti-inflammatory and antioxidant, neuroprotection, and improvement of blood circulation. It is especially suitable for patients with sleep disorders caused by anxiety, stress, neuroinflammation, or circulatory disorders, and has higher safety and tolerance.

[0013] Preferably, the leech extract includes an extract solution, a freeze-dried powder, a raw powder, or a combination thereof. Different forms of leech extracts can meet the diverse requirements of formulation design while ensuring the stability and bioavailability of active ingredients: Extract solution The extract solution is a common form of leech extract, prepared by water extraction, alcohol extraction, or other extraction processes, which can fully enrich active ingredients such as polypeptides and hirudin in leeches. The extract solution can be directly used to prepare dosage forms such as injections and sprays. Its liquid characteristics make it easy to absorb and take effect quickly, and it is suitable for patients with acute or severe sleep disorders. In addition, the extract solution can improve the purity and stability of drugs through process optimization during the formulation process.

[0014] Freeze-dried powder The freeze-dried powder removes the water in the extract solution through freeze-drying technology, retains the active ingredients of the leech extract, and avoids the degradation of active ingredients caused by high temperature or other treatment methods. The freeze-dried powder has high stability, is convenient for storage and transportation, and is particularly suitable for various dosage forms such as tablets, capsules, and injections. In addition, the freeze-dried powder can achieve precise control of dosage through accurate weighing and dispensing during the formulation process.

[0015] Raw powder The raw powder is the powder of leeches without extraction, directly prepared by crushing leech raw materials, which retains the natural ingredients of leeches and their synergistic effect. The raw powder is usually used in the preparation of traditional Chinese medicine dosage forms (such as pills and decoctions), and can achieve multiple therapeutic effects when combined with other traditional Chinese medicine ingredients. At the same time, the raw powder can also be used to prepare dosage forms such as granules and tablets. Its simple processing technology can effectively reduce production costs and meet the needs of specific patients for natural drugs.

[0016] Combination The combination of the extract, freeze-dried powder, and raw powder can bring out the advantages of each and achieve multi-level and multi-target therapeutic effects. For example, when the extract and freeze-dried powder are combined and applied in the preparation of injections or tablets, it can achieve the dual effects of rapid onset and long-term stability; the combined use of the raw powder with the extract or freeze-dried powder can meet the requirements of synergistic effects in traditional Chinese medicine compound preparations.

[0017] Through the optimized selection and flexible combination of leech extracts in different forms, the active ingredients of leeches can be maximally retained and utilized, adapting to various dosage forms and clinical needs. The extract is suitable for treatment scenarios with rapid onset, the freeze-dried powder is suitable for the design of highly stable preparations, and the raw powder can balance the integrity of natural ingredients and cost-effectiveness.

[0018] Preferably, the dosage forms of the preparation include injections, tablets, capsules, granules, ointments, suppositories, aerosols, sprays, or traditional Chinese medicine dosage forms. The design of different dosage forms can meet the diverse needs of patients while ensuring the stability and bioavailability of leech extracts: Injections Injections are suitable for patients with acute or severe sleep disorders. Through intravenous or intramuscular injection, the leech extract can be directly delivered into the bloodstream, avoiding the destruction of active ingredients by gastrointestinal metabolism. The rapid onset characteristic of injections is very suitable for patients who need immediate improvement in sleep status, and at the same time, by precisely controlling the dosage, the risk of side effects caused by drug metabolism is reduced.

[0019] Tablets and capsules Tablets and capsules are common oral dosage forms, which are convenient for patients to take for a long time and have good dosage uniformity. Through coating technology, the release rate of leech extract in the gastrointestinal tract can be further controlled, improving the absorption efficiency of the drug. In addition, excipients in capsules (such as microcrystalline cellulose and starch) can stabilize the active ingredients of leech extract and extend the shelf life of the drug.

[0020] Granules Granules can be quickly dissolved in water, which is convenient for patients with difficulty swallowing. At the same time, they can quickly release active ingredients and are suitable for patient groups who need rapid onset but are not convenient for injection. Sweeteners or flavors can be added during the production process of granules to improve the taste and enhance patient compliance.

[0021] Ointments and suppositories Ointments are suitable for topical use. By percutaneous absorption of active ingredients, the systemic circulatory system can be indirectly improved, especially suitable for patients who are not convenient for oral administration or injection. Suppositories are mainly used for rectal administration, suitable for certain special patients (such as those with gastrointestinal discomfort) or specific clinical needs, and can avoid the degradation of leech extract by the gastrointestinal environment.

[0022] Aerosols and sprays Aerosols and sprays are administered by inhalation or nasal route, can act directly on the central nervous system, and have the characteristic of rapid onset. This dosage form is particularly suitable for patients who need to relieve anxiety and insomnia symptoms in a short time, and avoids the first-pass effect of the liver of oral preparations.

[0023] Traditional Chinese medicine dosage forms Including traditional forms such as decoctions and pills, which are convenient for synergistic use with other Chinese medicine ingredients, so as to achieve better therapeutic effects. This dosage form can be flexibly adjusted according to the needs of patients, retaining the characteristics of traditional Chinese medicine medication.

[0024] Through the design of the above-mentioned various dosage forms, it is possible to maximize the adaptation to the physiological status, disease degree and living habits of different patients, give full play to the medicinal effects of leech preparations, and at the same time ensure their stability and safety. These dosage forms can meet the diverse needs of clinical use and enhance the convenience and accessibility of treatment.

[0025] Preferably, the leech extract is prepared by water extraction, alcohol precipitation or steam distillation. Different extraction methods can effectively separate the active ingredients in leeches and are selected according to the use and target efficacy of the preparation: Water extraction Water extraction is a traditional and efficient extraction method. By crushing leeches and soaking or refluxing them with water, water-soluble components such as polypeptide compounds, enzymes and polysaccharides can be extracted. This method is simple, low-cost, and can retain the main active substances in leeches, making it an ideal choice for preparing traditional Chinese medicine dosage forms (such as decoctions and granules).

[0026] Water extraction can maximize the activity of water-soluble components, which play an important role in regulating neurotransmitters (such as 5-HT and GABA), thus directly improving sleep disorders. In addition, the water extraction method can also extract anti-inflammatory and antioxidant components, which helps to relieve sleep problems caused by neuroinflammation and oxidative stress.

[0027] Alcohol precipitation The alcohol precipitation method is to add ethanol to the extract to precipitate the impurities insoluble in alcohol, thereby further purifying the extract and enriching the active ingredients in leeches, such as hirudin, polypeptides and certain fat-soluble components. The extract after alcohol precipitation is more stable and suitable for making high-purity preparations such as tablets, capsules and injections.

[0028] The alcohol precipitation method effectively concentrates the components with neuroprotective effects in leeches. These components help to relieve neuron damage caused by long-term sleep disorders by protecting nerve cells and regulating the brain microenvironment. At the same time, alcohol precipitation can improve the stability of the drug, facilitating long-term storage and industrial production.

[0029] Steam distillation method Steam distillation is mainly used to extract volatile active ingredients from leeches. This method extracts small molecule active substances through the distillation process, while removing some impurities, making the final extract more suitable for dosage forms such as aerosols and sprays.

[0030] After being absorbed through the nasal cavity, the volatile components can directly enter the central nervous system, take effect quickly, and improve sleep disorders caused by emotional tension and anxiety. In addition, the components extracted by this method can relieve the over-excited state of the nervous system within a short time, and are suitable for scenarios where quick results are required.

[0031] Leech extracts prepared by water extraction, alcohol precipitation or steam distillation can separate and purify according to the characteristics of different active ingredients, providing diverse choices for the development of subsequent preparations. The preferred application of these extraction processes ensures the efficient extraction and maximization of the functionality of the active substances in leeches, providing high-quality core raw materials for improving sleep disorders.

[0032] The second aspect of the present invention provides a preparation method of the leech preparation in the first aspect, including the following steps: A. Chop the leeches and add 2-8 times the weight of water and soak for 2-8 hours; By chopping the leeches to increase their surface area, the release efficiency of active ingredients in the subsequent extraction process can be effectively improved. During the process of adding 2-8 times the weight of water and soaking for 2-8 hours, some water-soluble components in leeches (such as polypeptides, enzymes, polysaccharides, etc.) will gradually dissolve in water, thus laying a foundation for subsequent extraction.

[0033] The water soaking process can initially separate water-soluble components and, by softening the tissue structure of leeches, promote the full release of active substances during subsequent heating and reflux. In addition, the control of the soaking time avoids the destruction of the active ingredients in leeches due to hydrolysis or oxidation for too long a time.

[0034] B. Heat and reflux the soaked leeches for extraction 1-3 times, each time for 1-5 hours, and combine the extraction solutions; Heat reflux extraction can fully release the main active substances in leeches, including water-soluble polypeptides, enzymes and some small molecule substances, by controlling the extraction times and the duration of each time. During the reflux process, the extraction efficiency of active ingredients is further improved by continuous cyclic extraction.

[0035] The main purpose of heat reflux extraction is to accelerate the dissolution of active substances from leech tissues under the action of temperature, while maintaining the stability of the active ingredients in the solution. Multiple extractions can ensure the completeness of the extraction of active substances, avoid waste of resources, and protect some heat-sensitive components (such as enzymes and polypeptides).

[0036] C. Concentrate the extraction solution to 50-80 °C to obtain a clear extract with a relative density of 1.10-1.25; During the concentration process of the extract, by controlling the temperature (50 - 80 °C) and concentrating to a certain relative density, excess water can be removed, and water-soluble active ingredients can be effectively concentrated in the clear paste. This step ensures the stability and easy storage of the extract.

[0037] Through the concentration process, active ingredients (such as polypeptides and hirudin) are highly enriched. At the same time, the relatively low-temperature concentration reduces the risk of thermal degradation of the ingredients, ensuring the stable quality of the prepared clear paste. The relative density (1.10 - 1.25) in the clear paste can provide an ideal viscosity, facilitating subsequent separation and purification.

[0038] D. Add ethanol to the clear paste to make the alcohol content reach 60% - 80%, let it stand for 24 - 72 hours, and filter to obtain the filtrate; By adding ethanol to make the alcohol content of the solution reach 60% - 80%, some water-soluble impurities can be effectively precipitated, while alcohol-soluble active ingredients (such as polypeptides, hirudin, etc.) are retained. After standing, the impurities are separated from the active ingredients by filtration.

[0039] The ethanol precipitation method utilizes the polarity difference between water and ethanol. Under certain alcohol content conditions, impurities (such as protein fragments, ineffective polysaccharides, etc.) are precipitated, while the target active ingredients are enriched. The standing time is controlled within 24 - 72 hours, making the precipitation process more complete. The filtrate after filtration has a purer active ingredient, providing high-quality raw materials for the next concentration process.

[0040] E. Concentrate the filtrate to 30 - 50 °C to obtain an extract with a relative density of 1.30 - 1.45; The filtrate after ethanol filtration is concentrated again to further remove ethanol and water, making the extract reach a higher concentration level. The extract obtained in this step (with a relative density of 1.30 - 1.45) is a highly enriched concentrate of active ingredients, laying the foundation for the production of the final preparation.

[0041] This step uses low-temperature concentration (30 - 50 °C) to remove excessive ethanol while retaining the active ingredients in the extract, especially providing a good protection environment for heat-sensitive active ingredients such as polypeptides and enzymes. The strict control of the relative density (1.30 - 1.45) ensures the stability and consistency of the extract quality.

[0042] 1. Through conventional pharmaceutical preparation processes, mix the extract with pharmaceutically acceptable excipients to prepare a preparation; 2. Finally, according to the requirements of different preparations, mix the obtained extract with pharmaceutically acceptable excipients (such as microcrystalline cellulose, starch, dextrin, etc.) to prepare preparations in forms such as tablets, capsules, granules, etc.

[0043] This step, through the addition of excipients, can not only improve the stability and bioavailability of the preparation, but also regulate the release rate of the active ingredient according to the dosage form design to meet the clinical needs of different patients. The conventional preparation process is convenient for industrial production and provides guarantee for the long-term storage of drugs.

[0044] This preparation method ensures the efficient separation and stability of the active ingredients in leeches through multiple optimized processes such as water extraction, concentration, and ethanol precipitation, while removing impurities to the lowest level.

[0045] Preferably, in step B, the amount of water added each time during extraction is 4 to 6 times the weight of the leeches, and the reflux time is 1 to 3 hours; an appropriate amount of water helps to maintain the dissolution stability of the active ingredients, avoiding incomplete extraction of the active substances or degradation caused by too high local component concentration due to too little water. The optimization of the extraction time is a balance between the extraction efficiency of the active substances and their thermal stability. The efficient release of the active ingredients in leeches can be ensured within 1 to 3 hours, while avoiding an increase in impurities or inactivation of the target components due to too long extraction time.

[0046] Preferably, the standing time after adding ethanol in step D is 24 to 36 hours; The standing time of 24 to 36 hours provides sufficient time for the precipitation particles to settle fully, ensuring a clear separation between the precipitate layer and the supernatant. This time length enables the ethanol to act fully with the clear extract, while avoiding the degradation that may be caused by the long-term exposure of active ingredients such as enzymes and polypeptides in the solution. After standing for 24 to 36 hours, the obtained supernatant contains a high concentration of the target active ingredient, facilitating the operation of the subsequent concentration step. In addition, this time parameter has good repeatability and stability in production, can be adapted to preparation processes of different scales, and is suitable for industrial application.

[0047] Preferably, the pharmaceutically acceptable excipients are microcrystalline cellulose, starch, or dextrin; Microcrystalline cellulose Microcrystalline cellulose is a commonly used pharmaceutical excipient with good compressibility, stability, and fluidity, and is widely used in tablets, capsules, and granules.

[0048] As a diluent: Microcrystalline cellulose can dilute the viscous components in the leech extract, facilitating the preparation process and improving the uniformity of the preparation.

[0049] As a disintegrant: In oral preparations, microcrystalline cellulose helps the disintegration of tablets or capsules, ensures the rapid release of the leech extract in the gastrointestinal tract, and improves the bioavailability of the active ingredients.

[0050] Microcrystalline cellulose can absorb moisture and rapidly expand through its porous structure, promoting tablet disintegration, thus accelerating the release of active ingredients and enhancing their absorption effect.

[0051] Starch Starch is a natural excipient with good biocompatibility and a wide range of pharmaceutical functions.

[0052] As a binder: Starch can combine the leech extract with other excipients to form a uniform mixture, facilitating tablet compression or granule formation.

[0053] As a filler: In low-dose formulations, starch can be used as a filler to increase the volume of the formulation, making it more convenient for processing and administration.

[0054] Through its hygroscopic and adhesive properties, starch enhances the mechanical strength of the formulation, and at the same time, in the gastrointestinal tract, it can release monosaccharides or polysaccharides that contribute to drug absorption through hydrolysis, improving the stability and solubility of the formulation.

[0055] Dextrin Dextrin is a multifunctional excipient commonly used in the production of granules and tablets, with good water solubility and stability.

[0056] As a diluent and stabilizer: Dextrin can evenly distribute the active ingredients in the leech extract, preventing their decomposition or moisture absorption and deterioration during the production process.

[0057] As a sustained-release material: In granules or tablets, dextrin can delay the release of active ingredients, forming a sustained-release effect, which helps to maintain a long-term therapeutic effect.

[0058] Through its good hydration and colloidal properties, dextrin can regulate the release rate of active ingredients, while preventing the active ingredients from being affected by environmental factors (such as humidity, temperature) during storage or processing.

[0059] In tablets, microcrystalline cellulose and starch can be used as diluents and disintegrants to improve the hardness, uniformity, and dissolution rate of tablets.

[0060] In granules, dextrin can enhance the formability and solubility of granules, while achieving the sustained-release or long-term effect of active ingredients.

[0061] In capsules, microcrystalline cellulose and dextrin can prevent the moisture absorption and degradation of active ingredients during storage, extending the shelf life of the formulation.

[0062] By preferably using microcrystalline cellulose, starch, or dextrin as pharmaceutically acceptable excipients, not only the physical properties and processing efficiency of the leech extract formulation are improved, but also its stability and bioavailability are enhanced, providing reliable technical support for the design of different dosage forms.

[0063] In the third aspect of the present invention, there is provided the use of the leech preparation according to the first aspect in the preparation of a drug for improving sleep disorders, and the leech preparation is used for the preparation of a drug for improving sleep latency or prolonging sleep duration. The application of the leech preparation in this field is mainly based on its multiple pharmacological effects and significant biological activities.

[0064] Improving sleep latency The polypeptide and hirudin components in leeches can enhance the synthesis and release of serotonin, thereby increasing the transmission of sleep induction signals; at the same time, inhibiting the release of excitatory neurotransmitters (such as glutamate), reducing the over-excited state of neurons, and creating favorable conditions for falling asleep quickly.

[0065] Prolonging sleep duration The anti-inflammatory active substances (such as polypeptides and enzymes) in leeches can reduce brain neuroinflammation, thereby stabilizing the sleep maintenance mechanism; in addition, hirudin, through anticoagulation and improvement of microcirculation, increases the oxygen supply and nutrient supply to brain tissue, improves the sleep quality decline and shortened sleep duration caused by hypoxia, helps to improve sleep quality, and prolongs deep sleep time.

[0066] Sleep disorders caused by the environment (such as hypoxia) In sleep disorders caused by environmental factors (such as high altitude hypoxia, pollution, etc.), the leech preparation shows good therapeutic potential. The main characteristics of such sleep disorders are prolonged sleep latency, increased frequency of sleep interruption, and shortened deep sleep time, which are often related to brain hypoxia, microcirculation disorders, and neuronal metabolic disorders.

[0067] The hirudin, polypeptide components, and antioxidant substances in the leech preparation act on sleep disorders caused by the environment through the following mechanisms: Improving the hypoxic state: Through the anticoagulation and microcirculation improvement effects of hirudin, the blood supply and oxygen supply to brain tissue are increased, thereby alleviating neurotransmitter disorders and brain fatigue caused by hypoxia, and promoting sleep maintenance and recovery.

[0068] Relieving the inflammatory response: Environmental factors (such as pollution or hypoxia) are likely to trigger brain neuroinflammation, and the anti-inflammatory components (such as polypeptides and enzymes) in the leech preparation can reduce the inflammatory response, stabilize neuronal function, and improve sleep quality.

[0069] Regulating neurotransmitters: The hypoxic environment may affect the balance of excitatory neurotransmitters (such as glutamate) and inhibitory neurotransmitters (such as γ-aminobutyric acid). The leech preparation helps the nervous system to restore the balance state by regulating the release of neurotransmitters, thereby alleviating the problems of difficulty in falling asleep and nocturnal awakening.

[0070] The leech preparation has good therapeutic application prospects in the following types of sleep disorders: Primary insomnia: By regulating the balance of neurotransmitters, it improves the problems of difficulty falling asleep and nocturnal awakening.

[0071] Secondary sleep disorder: Applicable to sleep disorders caused by hypoxia resulting from anxiety, depression or other chronic diseases. It alleviates symptoms through multiple action mechanisms.

[0072] Sleep disorder caused by the environment (such as hypoxia): Aiming at sleep problems induced by high-altitude hypoxia or air pollution caused by the environment, it improves the brain hypoxia state, regulates the neurotransmitter balance, and significantly improves the sleep quality.

[0073] Sleep problems in the elderly: The safety and versatility of the leech preparation make it particularly suitable for elderly patients, and it can avoid drug dependence and side effects while improving sleep.

[0074] The present invention provides a leech preparation for improving sleep disorders, its preparation method and its application. It has the following beneficial effects: 1. The leech preparation of the present invention can effectively shorten the sleep latency and prolong the sleep duration by regulating the levels of key neurotransmitters (such as serotonin and gamma-aminobutyric acid) in the central nervous system, and significantly improve the sleep quality of patients. The action of multiple mechanisms ensures a wide adaptability to different types of sleep disorders (such as primary insomnia and secondary sleep problems).

[0075] 2. The leech extract of the present invention is a natural active substance with clear components and low toxicity and side effects, avoiding the dependence, addiction and other central nervous system inhibitory side effects of traditional chemical drugs. It is particularly suitable for long-term medication patients and the elderly, with higher safety and tolerance, and will not cause dependence and drug resistance problems after long-term use.

[0076] 3. The present invention uses simple and easy processes such as water extraction and alcohol precipitation, which not only retains the biological activity of the active ingredients of leeches, but also realizes efficient separation and purification. Such optimized process conditions are easy to promote industrially, can significantly reduce production costs, and at the same time ensure the consistency and stability of product quality.

[0077] 4. The leech preparation of the present invention can be prepared into various dosage forms such as tablets, capsules, granules, injections, etc., which is suitable for patients who need rapid onset as well as for situations that require long-acting and sustained release. The diverse dosage form design meets the individualized needs of different patients, improves the medication compliance of patients, and at the same time enhances the market competitiveness of the drug. Detailed implementation mode

[0078] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0079] Example 1: Take 100 g of leeches, cut them into pieces, add 2 times the amount of water and soak for 2 h, then add 4 times the amount and reflux for 2 h. Filter the extract, and concentrate the filtrate to a clear paste with a relative density of 1.10 - 1.15 at 50 °C. Add ethanol to the above clear paste to make the alcohol content reach 70%, let it stand for 24 h and then filter. Recover the filtrate and concentrate it to an extract with a relative density of 1.30 - 1.35 at 30 °C. Take the above extract and add microcrystalline cellulose to prepare capsules.

[0080] Example 2: Take 150 g of leeches, cut them into pieces, add 4 times the amount of water and soak for 4 h, then successively add 4 times the amount and 4 times the amount and reflux for 1 h and 2 h respectively. Combine the extracts, filter, and concentrate the filtrate to a clear paste with a relative density of 1.15 - 1.20 at 60 °C. Add ethanol to the above clear paste to make the alcohol content reach 75%, let it stand for 36 h and then filter. Recover the filtrate and concentrate it to an extract with a relative density of 1.40 - 1.45 at 40 °C. Take the above extract and add starch to prepare tablets.

[0081] Example 3: Take 200 g of leeches, cut them into pieces, add 8 times the amount of water and soak for 8 h, then successively add 4 times the amount, 8 times the amount, and 8 times the amount and reflux for 1 h, 2 h, and 2.5 h respectively. Combine the extracts, filter, and concentrate the filtrate to a clear paste with a relative density of 1.20 - 1.25 at 70 °C. Add ethanol to the above clear paste to make the alcohol content reach 80%, let it stand for 48 h and then filter. Recover the filtrate and concentrate it to an extract with a relative density of 1.30 - 1.40 at 50 °C. Take the above extract and add dextrin and sucrose to prepare granules.

[0082] Example 4: Pharmacodynamic experiment Experimental animals and experimental materials 1. Experimental animals SPF - level ICR mice, male, 8 - week - old, weighing 20 - 22 g, purchased from Beijing Huafukang Biotechnology Co., Ltd. The experimental animals are raised in an environment with a room temperature of 23 - 25 °C and a 12 - h light - dark cycle (light: 8:00 - 20:00; dark: 20:00 - 8:00), with free access to food and water, and are used for experiments after adapting to the environment for one week. 2. Drugs and reagents Normal saline (Shandong Hualu Pharmaceutical Co., Ltd.) Pentobarbital sodium (Sigma) p-Chlorophenylalanine (PCPA, Sigma) Zaoren Anshen Capsule (Tongjitang Pharmaceutical Co., Ltd., China National Pharmaceutical Corporation) 3. Instruments Small animal behavior video tracking system, mouse gavage needle, stopwatch

[0083] Grouping, model establishment and administration 1. Experimental grouping The mice were randomly divided into 6 groups with 12 mice in each group. The specific grouping is as follows: Control group: Normal mice, without modeling, only given pure water by gavage.

[0084] Model group: The sleep disorder model was established by intraperitoneal injection of PCPA, and the mice were given pure water by gavage.

[0085] Low-dose leech group: Model mice were gavaged with 0.3 g / kg leech extract.

[0086] Medium-dose leech group: Model mice were gavaged with 1 g / kg leech extract.

[0087] High-dose leech group: Model mice were gavaged with 3 g / kg leech extract.

[0088] Positive drug group: Model mice were gavaged with 0.75 g / kg Zaoren Anshen liquid medicine.

[0089] 2. Model establishment Except for the control group, the mice in the other groups were intraperitoneally injected with 300 mg / kg PCPA suspension for two consecutive days. The mice in the control group were intraperitoneally injected with an equal volume of weakly alkaline 0.9% sodium chloride injection. 12 hours after the injection of PCPA, the behavioral characteristics and diet of the mice were observed to confirm the success of the model.

[0090] 3. Administration 30 minutes after the intraperitoneal injection of PCPA on the second day, different test substances were gavaged to each group according to body weight: Control group and model group: Given pure water.

[0091] Low-, medium- and high-dose leech groups: Given 0.3, 1, 3 g / kg leech extract respectively.

[0092] Positive drug group: Given Zaoren Anshen liquid medicine (0.75 g / kg).

[0093] After continuous gavage for 6 days, the pentobarbital sodium-induced sleep experiment was carried out; the open field experiment was carried out on the 7th day.

[0094] Experimental procedures 1. Pentobarbital sodium-induced sleep experiment On the 6th day, the mice were intraperitoneally injected with a suprathreshold dose of pentobarbital sodium (65 mg / kg), and the following indicators were recorded: Sleep latency: The time (seconds) required from the injection of pentobarbital sodium to the disappearance of the righting reflex.

[0095] Sleep duration: The time (minutes) from the disappearance of the righting reflex to recovery.

[0096] 2. Open field test On the 7th day, the mice were placed in the open field test device, and the following indicators were recorded: Moving distance: The total movement distance (centimeters) of the mice within 10 minutes.

[0097] Movement time: The total movement duration (seconds) of the mice within 10 minutes.

[0098] Experimental data and statistical analysis 1. Data analysis method SPSS 26.0 software was used for statistical analysis, and the experimental data were expressed as mean ± standard error (mean ± SEM).

[0099] One-way analysis of variance (One-Way ANOVA) was used for comparison between data groups. Fisher's Least Significant Difference (LSD) test was used when the variances were homogeneous, and Dunnett's T3 test was used when the variances were heterogeneous.

[0100] The significance level was P < 0.05.

[0101] 2. Experimental results (1) Results of pentobarbital sodium-induced sleep experiment Group Sleep latency (seconds) Sleep duration (minutes) Control group 65.2±5.1 180.3±10.4 Model group 120.5±8.6** 85.4±8.7** Low-dose leech group 100.4±7.3* 110.8±9.1* Medium-dose leech group 85.6±6.8** 145.7±12.2** High-dose leech group 70.4±5.2** 170.2±11.7** Positive drug group (diazepam) 68.1±5.0** 175.4±10.9** Note: Compared with the model group, *P < 0.05, **P < 0.01.

[0102] (2) Results of open field test Group Moving distance (centimeters) Exercise time (seconds) Control group 1520±140 215±16 Model group 780±105** 95±12** Low-dose leech group 960±110* 120±13* Medium-dose leech group 1320±130** 180±15** High-dose leech group 1480±135** 200±14** Positive drug group (diazepam) 1500±140** 210±15** Note: Compared with the model group, *P < 0.05, **P < 0.01.

[0103] Experimental conclusion The effect of the leech extract on the sleep status of mice with sleep disorders induced by intraperitoneal injection of PCPA. The results showed that compared with the control group, the sleep latency of the mice in the model group was significantly increased, and the sleep duration was significantly decreased; compared with the model group, the sleep latency of the mice in the medium and high-dose groups of the leech extract and the positive drug group was significantly decreased, and the sleep duration was significantly increased. It indicates that the medium and high-dose groups of the leech extract can significantly improve the sleep status of mice with sleep disorders induced by intraperitoneal injection of PCPA.

[0104] Effect of leech extract on the locomotor activity of mice with sleep disorder model induced by intraperitoneal injection of PCPA. The results showed that compared with the control group, the moving distance and locomotor time of the mice in the model group were significantly decreased; compared with the model group, the moving distance and locomotor time of the mice in the medium-dose, high-dose groups of leech extract and the positive drug group were significantly increased, indicating that the medium-dose and high-dose groups of leech extract could significantly improve the locomotor activity of mice with sleep disorder model induced by intraperitoneal injection of PCPA.

[0105] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A leech preparation for improving sleep disorders, characterized in that: The preparation comprises leech extract and pharmaceutically acceptable excipients, wherein the leech extract is derived from one or more of leeches, leeches, willow leaf leeches, wide-bodied golden leeches, Japanese medicinal leeches, pointed golden leeches, Philippine cattle leeches, smooth golden leeches, and eight-mesh stone leeches.

2. The leech preparation for improving sleep disorders according to claim 1, characterized in that: The leech extract comprises an extract solution, freeze-dried powder, raw powder or a combination thereof.

3. The leech preparation for improving sleep disorders according to claim 1, characterized in that: The dosage forms of the preparation include injection, tablet, capsule, granule, ointment, suppository, aerosol, spray or traditional Chinese medicine dosage form.

4. The leech preparation for improving sleep disorders according to claim 1, characterized in that: The leech extract is prepared by water extraction, alcohol precipitation or steam distillation.

5. A method for preparing a leech preparation for improving sleep disorders, characterized in that: The following steps are involved: A. Chop the leeches into small pieces and soak them in 2-8 times the weight of water for 2-8 hours; B. Heat the soaked leeches and extract them under reflux for 1-3 times, each time for 1-5 hours, and combine the extracts; C. The extract is concentrated to 50-80°C to obtain a clear paste with a relative density of 1.10-1.25; D. Add ethanol to the clear paste to make the alcohol content reach 60%-80%, let it stand for 24-72 hours, and filter to obtain a filtrate; E. Concentrate the filtrate to 30-50°C to obtain an extract with a relative density of 1.30-1.45; F. The extract is mixed with pharmaceutically acceptable excipients to prepare a preparation through conventional preparation technology.

6. The method for preparing a leech preparation for improving sleep disorders according to claim 5, characterized in that: During the extraction in step B, the amount of water added each time is 4-6 times the weight of the leech, and the reflux time is 1-3 hours.

7. The method for preparing a leech preparation for improving sleep disorders according to claim 5, characterized in that: The standing time after adding ethanol in step D is 24-36 hours.

8. The method for preparing a leech preparation for improving sleep disorders according to claim 5, characterized in that: The pharmaceutically acceptable excipient is microcrystalline cellulose, starch or dextrin.

9. Use of the leech preparation according to any one of claims 1 to 4 in the preparation of a drug for improving sleep disorders, characterized in that: The leech preparation is used for preparing medicine for improving sleep latency or prolonging sleep duration.