Pharmaceutical composition for treating or preventing epilepsy and application thereof

Abstract

The invention discloses a pharmaceutical composition for treating or preventing epilepsy and application thereof. The invention discloses a composition of a betula platyphylla sap extract for treating or preventing epilepsy and an application of the composition in preparation of a medicine. The composition is prepared from a betula platyphylla sap extract, a betula platyphylla sap extract, a betula platyphylla sap extract and a betula platyphylla sap extract, the composition may further include other ingredients to enhance its anti-epileptic effect. The composition provided by the invention has the characteristics of high efficiency, safety and convenience, can effectively treat or prevent epilepsy, and reduces the side effects of drugs at the same time. The invention also provides a method for treating or preventing epilepsy by using the composition.

Description

Technical Field

[0001] The present invention relates to a pharmaceutical combination for treating or preventing epilepsy and its use, in particular, to a pharmaceutical combination comprising birch sap extract and at least one other medicament and its use. Background Art

[0002] The description of the prior art includes content that is helpful for understanding the present invention. This does not admit that any content provided herein is prior art or related to the invention claimed in this case, nor does it admit that any publication explicitly or implicitly cited is prior art.

[0003] Epilepsy is a chronic neurological disease. It is the fourth largest neurological disease after stroke, migraine and Alzheimer's disease, affecting up to 70 million people worldwide. The disease is characterized by abnormal discharges of brain nerve cells leading to repetitive, spontaneous and unpredictable seizures, which mainly occur in the cerebral cortex and hippocampus. The cause of epilepsy may be related to congenital or acquired brain lesions, such as brain trauma, infection, stroke and tumor, but about 70% of epilepsy patients cannot find any cause of the disease. Although the cause of most epilepsy is unknown, it is generally believed that it may be due to an imbalance between the inhibitory nervous system (i.e., GABA system) and the excitatory nervous system (i.e., glutamate system) in the brain, resulting in excessive excitement of nerve cells.

[0004] The mechanism of action of anti-epileptic drugs currently used to control epileptic seizures is mainly to directly inhibit the excitability of nerve cells by blocking sodium ion channels (e.g., phenytoin, carbamazepine); inhibit glutamate (e.g., lamotrigine, felbamate); or enhance GABA (e.g., benzodiazepines, tiagabine, vigabatrin) to regulate the stability of nerve cells. However, although there are nearly thirty drugs currently used to treat epilepsy, about one-third of epilepsy patients are still resistant to existing drugs. In addition, the side effects of the drugs also limit patients' compliance with existing drugs.

[0005] It is known that commonly used anti-epileptic drugs have many side effects. For example, the instructions for use of carbazapine list possible side effects such as drowsiness, stomach discomfort, diarrhea, dizziness, heavy head, unsteady gait (ataxia), leukocytopenia, aplastic anemia, abnormal liver function, irregular heartbeat, skin rash, etc. The instructions for use of phenytoin list side effects such as nausea, vomiting, burning sensation in the stomach, loss of appetite, diplopia, eye tremors, unsteady gait (ataxia), gingival hypertrophy, hirsutism, rough face, athetosis, lymphadenopathy, teratogenesis, skin rash, etc. When side effects occur, the treatment usually involves changing the medication, reducing the dosage, or adding other drugs to suppress the side effects. However, reducing the dosage can easily lead to another seizure, causing physical and mental pain to epilepsy patients.

[0006] Therefore, the search and development of new, more effective and safer drugs is necessary for the treatment of epilepsy, and medicinal plants have attracted attention because they are widely used in traditional and folk medicine to treat and prevent different neurological diseases (including epilepsy). In recent years, many plant-derived bioactive molecules have been shown to produce anti-epileptic effects by affecting ion channels, GABA or glutamate, such as flavonoids, alkaloids, terpenoids and cannabidiol. Therefore, medicinal plants have become the target of anti-epileptic drug research.

[0007] Betula platyphylla belongs to the genus Betula of the Betulaceae family and is distributed in Siberia, Finland, Hokkaido, Mongolia and other places. In the Compendium of Materia Medica, birch bark is recorded to be used to treat arthritis, skin diseases, physical trauma and clear away heat and detoxify. At present, birch has been widely used in folk medicine to treat a variety of inflammatory diseases, including hepatitis, acute tonsillitis, pneumonia, chronic bronchitis, cholecystitis, urinary tract infection and burns. Studies have shown that birch has many biological activities, such as anti-inflammatory, antioxidant, antiviral, anticancer, anti-fatigue and neuroprotective effects, and these activities are related to the main active ingredient of birch, triterpenoids - betulin. However, there are not many studies on birch in the central nervous system. Therefore, there is still a need for a pharmaceutical composition containing birch extract to treat or prevent epilepsy. Summary of the invention

[0008] The present disclosure provides an application of a birch sap extract in preparing a medicine for treating or preventing epilepsy.

[0009] In one embodiment, the birch sap extract of the present disclosure is obtained from birch trees grown in Hokkaido, Japan.

[0010] In one embodiment, epilepsy is caused by damage to brain nerve cells. In another embodiment, epilepsy is caused by activation of glial cells. In another embodiment, epilepsy is caused by brain inflammation caused by inflammatory molecules. In another embodiment, the inflammatory molecules are selected from the group consisting of interleukin 1β (linterleukin-1β, IL-1β), interleukin 6 (IL-6), tumor necrosis factor-α (tumor necrosis factor-α, TNF-α), high mobility group box 1 (high mobility group Box 1, HMGB1), interleukin 1 receptor 1 (IL-1R1), and Toll-like receptor-4 (Toll-like receptor-4, TLR-4).

[0011] In one embodiment, the birch sap extract of the present disclosure is further combined with at least one other anti-epileptic agent for the preparation of a drug for treating or preventing epilepsy. In another embodiment, the at least one other anti-epileptic agent is a Western medicine preparation. In another embodiment, the at least one other anti-epileptic agent is a Western medicine preparation selected from the group consisting of phenytoin, carbazapine, lamotrigine, febamazepine, benzodiazepines, tiagabine, and vigabatrin. In another embodiment, the at least one other anti-epileptic agent is a Chinese medicine preparation or prescription. In another embodiment, the at least one other anti-epileptic agent is a Chinese medicine preparation or prescription selected from the group consisting of Gastrodia elata, Uncaria rhynchophylla, Semen Cassiae, Zhengan Xifeng Decoction, and Gastrodia Uncaria rhynchophylla.

[0012] The present disclosure also provides a method for preparing birch sap extract, which comprises

[0013] Step 1: Select a mature birch tree and cut the bark to promote sap flow;

[0014] Step 2: Use a collector to collect the sap that flows out;

[0015] Step 3: Filter the sap at room temperature to remove bark fragments and other impurities; and

[0016] Step 4: Use low-temperature concentration technology to remove most of the water in the sap to obtain a highly concentrated birch sap extract.

[0017] The above steps can be increased or decreased according to the extraction of specific components or other actual needs.

[0018] In one embodiment, the birch tree is a birch tree grown in Hokkaido, Japan. In another embodiment, the birch sap is harvested in winter or spring. In another embodiment, the birch sap is harvested in early spring.

[0019] The present disclosure also provides a pharmaceutical composition for treating or preventing epilepsy, comprising birch sap extract and at least one other anti-epileptic drug and a pharmaceutically acceptable carrier and / or stabilizer, wherein the dosage of the other anti-epileptic drug is lower than the dosage when it is administered alone. In one embodiment, the at least one other anti-epileptic drug is selected from the group consisting of phenytoin, carbazapine, lamotrigine, febamazepine, benzodiazepines, tiagabine, and vigabatrin. In one embodiment, the birch sap extract and the at least one other anti-epileptic drug are administered to the patient simultaneously, sequentially, or at intervals.

[0020] Various objects, features, aspects and advantages of the present invention will become more apparent from the following detailed description of preferred embodiments and the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Further features and advantages of the present invention will become apparent from the following more particular description of preferred embodiments, as illustrated in the accompanying drawings accompanying the present invention, and in which like reference numerals generally refer to the same parts or components throughout, and wherein:

[0022] Figures 1A-1D Chemical properties of birch sap extract (HCF02), Figure 1A and 1B Shows the monosaccharide composition, Figure 1C is the molecular weight distribution, Figure 1D for 1 H NMR spectroscopy.

[0023] Figure 2A and Figure 2B The bar graph shows that birch sap extract pretreatment alleviated the manifestation of KA-induced epileptic behavior in animals. Data are mean ± SEM (n = 2-13 rats / group). *, compared with the KA group, p < 0.05; ***, compared with the KA group, p < 0.001.

[0024] Figure 3A and Figure 3B Pretreatment with birch sap extract and birch brain prevented KA-induced neuronal cell death in the cerebral cortex and hippocampus. Data are mean ± SEM. *, compared with the control group, p < 0.01; ***, compared with the control group, p < 0.001; #, compared with the KA group, p < 0.05; n = 3 per group.

[0025] Figure 4A and Figure 4B Birch sap extract and birch brain pretreatment alleviated the degeneration of cortical and hippocampal neurons induced by KA. Data are mean ± SEM. ***, compared with the control group, p < 0.001; #, compared with the KA group, p < 0.05; n = 3 per group. DETAILED DESCRIPTION

[0026] The following description includes content that helps to understand the present invention. This does not admit that any content provided herein is prior art or related to the invention claimed in this case, nor does it admit that any publication explicitly or implicitly cited is prior art.

[0027] It should be understood that the present invention is not limited to the specific exemplary materials, structures, procedures, methods or configurations listed herein. Therefore, although some options are listed herein, those similar or with the same effects can all be applied to the implementation or embodiment of the present invention, and only the preferred materials and methods are described herein.

[0028] The detailed description set forth below in connection with the accompanying drawings accompanying the present invention is considered to be a description of an exemplary embodiment of the present invention and is not intended to represent the only exemplary embodiment in which the present invention may be implemented. The term "exemplary" as used throughout the present specification means "serving as an example, instance, or illustration" and should not necessarily be construed as being better or superior to other exemplary embodiments. The detailed description includes specific details for the purpose of providing a comprehensive understanding of the exemplary embodiments of the present specification. It may be apparent to a person of ordinary skill in the art to which the present invention belongs that the exemplary embodiments of the present specification may be implemented without these specific details.

[0029] definition

[0030] As used herein, the following terms have the meanings as described in this paragraph. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the present invention only and is not intended to be limiting.

[0031] Unless otherwise defined, all technical and scientific terms used herein generally have the same meanings as those understood by those with ordinary knowledge in the art to which the present invention belongs. In general, the nomenclature used herein and laboratory procedures in animal pharmacology, pharmaceutical science, separation science, and organic chemistry are well known and commonly used in the art. In the methods described herein, except for the explicit description of the time or order of operation, the actions can be performed in any order. In addition, unless the explicit request terms are semantically described as being performed separately, the actions referred to can be performed simultaneously. For example, the requested action of implementing X and the requested action of implementing Y can be performed simultaneously in a single operation, and the resulting method will fall within the scope of the text of the requested method.

[0032] The following non-limiting abbreviations are used herein: KA, kainic acid; IL-1β, interleukin-1β; IL-6, interleukin-6; TNF-α, tumor necrosis factor-α; HMGB1, high mobility group Box 1; IL-1R1, interleukin-1 receptor 1; CBZ, carbamazepine; CABA, gamma-aminobutyric acid; and TLR-4, Toll-like receptor-4.

[0033] As used in this specification and the claims appended hereto, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, "an element" means one element or more than one element.

[0034] As used herein, the term "about" will be understood by those with ordinary knowledge in the art and will vary to some extent according to the context in which it is used. As used herein, when referring to measurable values ​​such as quantity, length of time, "about" means including changes of ±20%, ±10%, ±5%, ±1% or ±0.1% of the specific value. Because these changes are suitable for carrying out the disclosed method. Therefore, in some embodiments, the numerical parameters proposed in this specification and the attached claims are approximate values, which can be changed according to the desired characteristics attempted to be obtained by a specific embodiment. In some embodiments, the numerical parameters should be interpreted according to the recorded significant digits and applying general numerical rounding techniques. The numerical values ​​presented in some embodiments of the present invention may contain certain errors, which are inevitably caused by the standard deviations present in their respective experimental measurements.

[0035] In one aspect, the term "co-administered" with respect to a subject refers to administering to the subject the birch sap extract of the present disclosure together with other agents that can also treat or prevent the diseases contemplated herein. In certain embodiments, the co-administered birch sap extract and other agents are administered separately, or in any kind of combination as part of a single treatment method.

[0036] As used herein, a "disease" is a condition in a subject's health in which the subject cannot maintain homeostasis and in which the subject's health will continue to deteriorate if the disease is not ameliorated.

[0037] As used herein, a "disorder" in a subject is a state of health in which the subject is able to maintain homeostasis, but the subject's health is not as good as it would be without the disorder. Without treatment, the disease does not necessarily lead to a further deterioration in the subject's health.

[0038] As used herein, the term "pharmaceutically acceptable" refers to a substance, such as a carrier or diluent, that does not abrogate the biological activity or properties of the useful compounds of the present invention and is relatively non-toxic, i.e., the substance can be administered to a subject without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition.

[0039] As used herein, the term "pharmaceutically effective amount," "therapeutically effective amount," or "effective amount" of a compound is an amount of the compound sufficient to provide a beneficial effect to a subject to which the compound is administered.

[0040] As used herein, the terms "subject," "individual," and "patient" are used interchangeably and refer to a human or non-human mammal. Non-human mammals include, for example, livestock and pets, such as sheep, pigs, canines, felines, and murine mammals. In certain embodiments, the subject is a human.

[0041] As used herein, the term "prevent" or "preventing" means avoiding or delaying the onset of symptoms associated with a disease or condition in a subject who has not yet developed such symptoms at the time administration of the agent or compound is initiated.

[0042] As used herein, the term "treating" means reducing the frequency or severity of symptoms experienced by a subject of a disease or disorder by administering an agent or compound to the subject.

[0043] Range: Throughout this disclosure, various aspects of the invention may be presented in the form of a range. It should be understood that the description in range form is for convenience and brevity only and should not be construed as limiting the scope of the invention. Therefore, the description of a range should be considered to specifically disclose all possible sub-ranges and single values ​​within the range. For example, a range description from 1 to 6 should be considered to have specifically disclosed sub-ranges, such as from 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as single and partial numbers within the range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the range.

[0044] Birch sap

[0045] The birch sap disclosed in the present invention is a colorless or slightly yellowish transparent liquid collected and extracted from birch bark and / or branches. Birch sap contains active substances such as betulin, polysaccharides, saponins, proteins, minerals, etc., and has high medicinal value. In traditional Chinese medicine theory, birch sap is classified as a heat-clearing and detoxifying medicine. The currently disclosed efficacy and benefits are:

[0046] Clearing away heat and detoxifying: Birch sap has the effect of clearing away heat and detoxifying. It can help remove heat and dampness from the body and relieve symptoms such as fever, thirst, sore throat, and rash.

[0047] Diuretic and detumescent: Birch sap can promote the excretion function of the kidneys, increase urine volume, help eliminate excess water in the body, and relieve symptoms such as edema.

[0048] Anti-inflammatory and analgesic: Birch sap contains some ingredients with anti-inflammatory and analgesic effects, such as menthol and salicylic acid, which can help relieve pain and inflammation symptoms.

[0049] Improve digestion: Birch sap can promote gastrointestinal motility, increase the secretion of digestive juices, and improve gastrointestinal problems such as indigestion and diarrhea.

[0050] Nutritional supplement: Birch sap contains a variety of nutrients, such as vitamin C, B vitamins, iron, potassium, etc., which can help supplement the nutrients needed by the body and enhance the body's immunity and disease resistance.

[0051] In one embodiment, birch sap is collected in winter or spring every year, preferably in early spring. In another embodiment, birch sap is collected from birch trees aged 20 to 50 years. In another embodiment, the birch trees are birch trees grown in Hokkaido, Japan.

[0052] Combination therapy

[0053] In one aspect, the birch sap extract of the present disclosure can be used in combination with one or more other agents for treating or preventing epilepsy, or prepared into a pharmaceutical composition. These other agents may include compounds known to treat, prevent or alleviate epileptic symptoms (e.g., commercially available compounds).

[0054] In one embodiment, the present disclosure provides a method for treating or preventing epilepsy in a subject, the method comprising administering a therapeutically effective amount of the birch sap extract of the present disclosure to the desired subject. In one embodiment, the subject is further co-administered with at least one other agent that helps treat or prevent epilepsy. In another embodiment, the at least one other agent is a traditional Chinese medicine preparation or prescription, such as Gastrodia elata, Uncaria rhynchophylla, Semen Cassiae, Zhengan Xifeng Decoction, Gastrodia elata Uncariae Drink, etc., wherein the dosage of the traditional Chinese medicine preparation can be adjusted by a traditional Chinese medicine practitioner according to a well-known dosage. In another embodiment, the other agent is a traditional Chinese medicine preparation or prescription, and is co-prepared with the birch sap extract. In another embodiment, the at least one other agent is a Western medicine preparation, such as phenytoin, carbazapine, lamotrigine, febamet, benzodiazepines, tiagabine, vigabatrin, etc., wherein the dosage of the Western medicine preparation can be adjusted by a physician according to the recommended dosage of each agent.

[0055] In one embodiment, the subject suffering from epilepsy may be a subject who has been diagnosed with epilepsy for the first time, or is taking at least one anti-epileptic drug. In one embodiment, a combination of birch sap extract and at least one anti-epileptic drug may be provided to a subject who has been diagnosed with epilepsy for the first time, wherein the dosage of the at least one anti-epileptic drug when administered in combination is lower than the dosage when administered alone. In another embodiment, birch sap extract may be further provided to a subject who is taking at least one anti-epileptic drug, and the dosage of the at least one anti-epileptic drug may be reduced.

[0056] Drug administration / dosage

[0057] The treatment regimen may affect the composition of the effective amount. The therapeutic formulation may be administered to the patient before or after the onset of the disease or condition. In addition, several separate doses and staggered doses may be administered daily or sequentially, or the doses may be administered continuously. In addition, the dose of the therapeutic formulation may be increased or decreased proportionally depending on the urgency of the treatment or prevention situation.

[0058] The birch sap extract of the present disclosure can be administered to a patient using known procedures, dosages, and time periods to effectively treat or prevent epilepsy. The effective amount of birch sap extract necessary to achieve the therapeutic and / or prophylactic effect of epilepsy can vary depending on many factors, such as the activity of the specific birch sap extract used; the time of administration; the excretion rate of the extract; the duration of treatment; other drugs, compounds, or substances used in combination with the extract; the state of the disease or condition of the patient being treated, age, sex, weight, symptoms, general health, and previous medical history; and similar factors well known in the medical field. The dosage regimen can be adjusted to provide an optimized therapeutic response. For example, several divided doses may be administered daily, or the dose may be proportionally reduced as described, such as depending on the urgency of the treatment situation.

[0059] Although the description of birch sap extract provided herein is primarily directed to being suitable for administration to humans in a medically ethical manner, it will be understood by those skilled in the art that the composition is generally suitable for administration to a variety of animals. Modifications of birch sap extracts are well known in order to make the composition suitable for administration to a variety of animals, and such modifications can be designed and implemented by a veterinary pharmacologist skilled in the art with only routine experimentation (if necessary). Subjects to which the birch sap extract of the present disclosure may be administered include, but are not limited to, humans and other primates, mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, and dogs.

[0060] Humans and animals have different tolerances to the same drug. Generally speaking, animals have a greater tolerance than humans. The dosage per unit body weight or per unit body surface area is greater for animals than for humans. The dosage per unit body weight for animals and humans can be calculated by referring to the following general conversion table.

[0061]

[0062] Table source: International Life Science Society of Taiwan, China

[0063] For example, taking the rat model of the embodiment of the present disclosure as an example, the birch sap extract of the present disclosure can be orally administered to deliver a dose of 3 mg / kg / time to 20 mg / kg / time, preferably, the birch sap extract can be orally administered at a dose of 6 mg / kg / time to 10 mg / kg / time, for example, the birch sap extract can be orally administered at a dose of 6 mg / kg / time or 10 mg / kg / time. After conversion according to the above-mentioned unit body weight dose conversion table, the dose of the birch sap extract of the present disclosure that can be administered to humans is 0.48 mg / kg / time to 3.2 mg / kg / time, preferably 0.96 mg / kg / time to 1.6 mg / kg / time. The applicable dose of the birch sap extract of the present disclosure for other animals can also be obtained through the above-mentioned unit body weight conversion table or other unit body surface area conversion tables.

[0064] In certain embodiments, the birch sap extract of the present disclosure is administered to a patient in a dosage range of 1 to 5 times per day or more. In other embodiments, the birch sap extract of the present disclosure may be administered to a patient in a dosage range including, but not limited to, once a day, twice a day, three times a day, four times a day, once every two days, every three days to one week, or once every two weeks. Those skilled in the art will readily appreciate that the frequency of administration of the various combinations of the compositions of the present invention will vary with the subject, depending on many factors, including, but not limited to, age, the disease or condition to be treated, sex, overall health, and other factors. Therefore, the present invention should not be construed as being limited to any particular dosage regimen, and the exact dosage and composition to be administered to any patient should be determined by the attending physician taking into account all other factors concerning the patient.

[0065] In one embodiment, when administering the birch sap extract of the present disclosure and the at least one other anti-epileptic agent, the birch sap extract and the at least one other anti-epileptic agent can be administered to the subject simultaneously. In another embodiment, the birch sap extract and the at least one other anti-epileptic agent can be administered to the subject sequentially, successively or intermittently.

[0066] Route of administration

[0067] Oral administration

[0068] Liquid preparations for oral administration may be in the form of solutions, syrup preparations or suspensions. The liquid preparations may be prepared by well-known methods with pharmaceutically acceptable additives, such as syrup or pharmaceutically acceptable preservatives.

[0069] Those skilled in the art will appreciate, or be able to ascertain using no more than routine experimentation, many equivalents to the specific procedures, embodiments, claims, and examples described herein. Such equivalents are considered to be within the scope of the present invention and are covered by the claims appended hereto. For example, it should be understood that modifications and art-recognized substitutions for reaction conditions, including but not limited to reaction times, reaction sizes / volumes, and experimental reagents (e.g., solvents, catalysts), pressures, atmospheric conditions, and reducing / oxidizing agents, and the like, and using no more than routine experimentation are within the scope of this application.

[0070] It should be understood that, no matter where the numerical value and range are provided herein, the description in the form of range is only for convenience and simplicity, and should not be interpreted as limiting the scope of the invention. Therefore, all numerical values ​​and ranges covered by the numerical value and range are included in the scope of the invention. In addition, all numerical values ​​falling within these ranges and the upper or lower limit of the numerical range are also expected by the present application. The description of the range should be considered to specifically disclose all possible sub-ranges and individual values ​​within the range, and when appropriate, the partial integer of the numerical value is also included in the range. For example, the description of the range from 1 to 6 should be considered to have specifically disclosed sub-ranges, such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, from 3 to 6, etc., and individual numbers within the range, such as 1, 2, 2.7, 3, 4, 5, 5.3 and 6, regardless of the width of the range, all apply.

[0071] The following examples further illustrate various aspects of the present invention. However, they are not intended to limit the teachings or disclosures of the present invention described herein.

[0072] Example

[0073] The present invention is now described with reference to the following examples, which are provided for illustrative purposes only, and the present invention is not limited to these examples but encompasses all variations which become apparent as a result of the teachings provided herein.

[0074] Materials and Methods

[0075] The following procedures can be used to prepare and / or test exemplary birch sap extracts of the present invention.

[0076] Birch Sap Extract

[0077] The following steps are used to extract sap from birch trees and make birch sap extract:

[0078] Step 1: Select a mature birch tree and cut the bark to promote sap flow.

[0079] Step 2: Use a collector to collect the sap that flows out and avoid external contamination.

[0080] Step 3: Filter the sap at room temperature to remove bark fragments and other impurities.

[0081] Step 4: Use low-temperature concentration technology to remove most of the water in the sap to obtain a highly concentrated birch sap extract.

[0082] Experimental animals

[0083] Male Sprague-Dawley rats (SD, 150-200 g) were purchased from Lesco and housed in the Experimental Animal Center of Fu Jen Catholic University. All animal experiments were approved by the Experimental Animal Management Committee of Fu Jen Catholic University. According to the 3R rules, all experiments were conducted to minimize animal suffering and use the minimum number of animals required to produce reliable results.

[0084] Example 1 - Analysis of birch sap components

[0085] The sap extracted from birch trees was analyzed using a monosaccharide analysis method. In the monosaccharide analysis of birch sap, the ratio of the three main monosaccharides, fructose, glucose and fucose, in the birch sap extract was determined to be 27:17:10 ( Figure 1A and 1B ). According to single-column high-performance molecular sieve chromatography (HPSEC), the molecular weight of birch sap is about or less than 1.29 kDa ( Figure 1C ). 1 H NMR spectroscopy ( Figure 1D ) shows two anomeric protons at δH 5.13 and 4.45. The signals at δH 5.13 and δH 4.45 appear as double peaks with a ratio of 100:124, which are two monosaccharide residues and α-anomeric configuration ( 3 J 1,2 =3.4 Hz) and β-anomeric configuration ( 3 J 1,2 =7.7 Hz). Based on the above analysis results, the amount of birch sap extract used in subsequent biological tests can be further quantified.

[0086] Example 2 - KA-induced epilepsy animal model

[0087] The anti-epileptic effect of birch sap extract was evaluated in an animal model of epilepsy induced by kainic acid (KA). KA is a glutamate analog. Injection of KA into rats causes increased glutamate release and overactivation of glutamate receptors, leading to progressive limbic seizures in rats. This phenomenon leads to increased calcium ions in neurons, oxidative stress, and mitochondrial dysfunction, which in turn causes neuronal cell death in many areas of the brain, especially in the cortex and hippocampus. In addition, KA-induced neuronal cell death is associated with inflammatory responses caused by glial cell activation and inflammatory molecules [e.g., interleukin 1β (IL-1β), interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), high-mobility group protein 1 (HMGB1), interleukin 1 receptor 1 (IL-1R1), and toll-like receptor 4 (TLR-4)]. Since these pathological changes induced by KA are similar to the behavior and pathology of human epilepsy, the KA-induced animal epilepsy model is widely used to screen new compounds with anti-epileptic activity.

[0088] The epileptic behavior was analyzed within 4 hours after intraperitoneal injection of KA in rats, and the severity of the seizure was assessed according to the Racine scale (1972), as shown in the following table.

[0089]

[0090] Example 3 - Evaluation of the effect of birch sap extract on KA-induced epileptic behavior in animals

[0091] Sprague-Dawley rats were divided into the following groups according to the administration of different test substances:

[0092] 1. Negative control group: physiological saline

[0093] 2. KA group: 15 mg / kg / ip / single dose of kainate (KA)

[0094] 3. Low-dose group of birch sap extract: 6 mg / kg of birch sap extract

[0095] 4. High-dose birch sap extract group: 10 mg / kg birch sap extract

[0096] 5. High-dose anti-epileptic drug group: 100 mg / kg carbazapine (CBZ)

[0097] 6. Anti-epileptic pharmaceutical composition group: containing 6 mg / kg of birch sap extract and 50 mg / kg of carbazapine (CBZ)

[0098] 7. Low-dose anti-epileptic drug group: 50 mg / kg carbazapine (CBZ)

[0099] The control group, birch sap extract group, anti-epileptic drug group and anti-epileptic pharmaceutical composition group were fed with normal saline, birch sap extract and / or carbazapine by feeding tube at regular intervals every day. After seven days, except for the negative control group, each group of rats was intraperitoneally injected with KA (15 mg / kg / ip / single). The dose of KA injection was based on previous studies. The epileptic behavior of rats was analyzed according to the Racine scale (1972) within 4 hours after the intraperitoneal injection of KA. The results are shown in Table 1.

[0100] Table 1. Pretreatment with birch sap extract alleviates the manifestations of KA-induced epileptic behavior in animals.

[0101]

[0102] Data are mean ± SEM. **, p < 0.05 compared with the KA group; ***, p < 0.001 compared with the KA group.

[0103] The experimental results showed that KA administration induced epileptic seizures in 91% of the injected rats in the KA group (positive control group). The duration and score of epileptic seizures were 66.6±6.4 minutes and 4.6±0.2, respectively. Compared with the KA group, the pretreatment with the birch sap extract of the present invention for seven days can significantly prolong the duration of KA-induced epileptic seizures and reduce the severity of KA-induced epileptic seizures (p<0.05; Figure 2A and Figure 2B ), and the high-dose group (10 mg / kg) of the birch sap extract group had a better inhibitory effect on the severity of KA-induced epileptic seizures, which was similar to the high-dose group of anti-epileptic drugs (CBZ 100 mg / kg). This result proves that the birch sap extract of the present invention has the effect of preventing KA from causing epilepsy (Table 1). In other words, the birch sap extract of the present invention can be used to replace anti-epileptic drugs.

[0104] In addition, in the low-dose anti-epileptic drug group, the duration and severity of epileptic seizures of animals were not statistically different from those of the KA group. However, when used in combination with a low dose of birch sap extract, i.e., the anti-epileptic pharmaceutical composition group (6 mg / kg birch sap extract + 50 mg / kg CBZ), the duration and severity of epileptic seizures of animals were significantly improved compared with the KA group (p<0.05; Figure 2A and Figure 2B), which can greatly improve the disadvantage that low-dose anti-epileptic drugs cannot effectively suppress epileptic symptoms. Since the anti-epileptic pharmaceutical composition group can produce similar effects of preventing KA-induced epilepsy as the high-dose anti-epileptic drug group, this result shows that the combination of low-dose (6 mg / kg) birch sap and low-dose (50 mg / kg) CBZ can be used to prevent KA-induced epileptic behavior in animals, and low-dose CBZ can further reduce the side effects of anti-epileptic drugs.

[0105] Experiments on rats have confirmed that the method provided by the present invention can be used to prevent epileptic seizures, especially for difficult-to-control epilepsy symptoms. When the birch sap extract of the present invention is used alone, an anti-epileptic effect comparable to that of anti-epileptic drugs (such as CBZ) can be obtained. In other words, the birch sap extract can be used to replace anti-epileptic drugs. In addition, when the birch sap extract of the present invention is used in combination with anti-epileptic drugs, the dosage of anti-epileptic drugs (such as CBZ) can be further reduced while achieving the anti-epileptic effect. Therefore, whether the birch sap extract of the present invention is used alone or in combination with anti-epileptic drugs, the side effects of anti-epileptic drugs can be reduced. The method provided by the present invention has lower toxicity and side effects, making it a safe and effective method for preventing epilepsy.

[0106] Example 4 - Histopathological analysis of birch sap extract on cranial nerves

[0107] Continuing from Example 3, the experimental animals were sacrificed three days after intraperitoneal injection of KA (15 mg / kg), and the brain tissues were removed for sectioning and other related experimental analyses.

[0108] Three days after KA was intraperitoneally injected into rats, Nissl staining revealed that the number of neurons in the CA1 / CA3 of the cerebral cortex and hippocampus of the KA group was significantly reduced. Birch sap extract and birch brain pretreatment could reduce the reduction in the number of neurons caused by KA (p<0.05; Figure 3A and Figure 3B ).

[0109] In addition, Fluoro-Jade B (FJB) staining also revealed an increase in the number of degenerative cells in the cerebral cortex and hippocampal CA1 / CA3 of the KA group, which was also reduced by birch extract and birch brain pretreatment (p<0.05; Figure 4A and Figure 4B ), these preliminary results suggest that pretreatment with birch sap extract and / or betulin can prevent KA-induced neuronal damage.

[0110] The above animal experiment results confirm that the birch sap extract disclosed in the present invention can significantly prolong the inhibition time of KA-induced epileptic seizures and reduce the severity of KA-induced epilepsy, which can confirm that the birch sap extract has the effect of preventing epilepsy. Therefore, birch sap has the prospect of being used in the development of anti-epileptic drugs and / or related products.

[0111] Examples of implementations:

[0112] The following illustrative embodiments are provided, and their numbering should not be construed as designating importance.

[0113] Embodiment 1 provides a use of a birch sap extract in preparing a drug for treating or preventing epilepsy.

[0114] Embodiment 2 provides the use of embodiment 1, wherein the birch sap extract is taken from birch trees grown in Hokkaido, Japan.

[0115] Embodiment 3 provides the use of embodiment 1, wherein the epilepsy is caused by brain nerve cell damage.

[0116] Embodiment 4 provides the use of embodiment 1, wherein the epilepsy is triggered by activation of glial cells.

[0117] Embodiment 5 provides the use of embodiment 1, wherein the epilepsy is triggered by a brain inflammatory response caused by inflammatory molecules.

[0118] Embodiment 6 provides the use of embodiment 5, wherein the inflammatory molecule is selected from the group consisting of interleukin 1β (IL-1β), interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), high mobility group protein 1 (HMGB1), interleukin 1 receptor 1 (IL-1R1), and Toll-like receptor 4 (TLR-4).

[0119] Embodiment 7 provides the use of embodiment 1, further comprising at least one other anti-epileptic agent combined with the birch sap extract for preparing the medicament for treating or preventing epilepsy.

[0120] Embodiment 8 provides the use of embodiment 7, wherein the at least one other anti-epileptic drug is a western medicine preparation.

[0121] Embodiment 9 provides the use of embodiment 8, wherein the at least one other anti-epileptic drug is selected from a Western medicine preparation consisting of phenytoin, carbazapine, lamotrigine, febamazepine, benzodiazepines, tiagabine, and vigabatrin.

[0122] Embodiment 10 provides the use of embodiment 7, wherein the at least one other anti-epileptic agent is a traditional Chinese medicine preparation or prescription.

[0123] Embodiment 11 provides the use of embodiment 10, wherein the at least one other anti-epileptic agent is selected from the Chinese medicine preparation or prescription of the group consisting of Gastrodia elata, Uncaria rhynchophylla, Semen Cassiae, Zhengan Xifeng Decoction, and Gastrodia elata decoction.

[0124] Embodiment 12 provides the use of embodiment 1, wherein the medicament is for treating or preventing epilepsy in a human.

[0125] Embodiment 13 provides the use of embodiment 1, wherein the medicament is for treating or preventing epilepsy in a non-human mammal.

[0126] Embodiment 14 provides a method for preparing birch sap extract, comprising

[0127] Step 1: Select a mature birch tree and cut the bark to promote sap flow;

[0128] Step 2: Use a collector to collect the sap that flows out;

[0129] Step 3: Filter the sap at room temperature to remove bark fragments and other impurities; and

[0130] Step 4: Use low-temperature concentration technology to remove most of the water in the sap to obtain a highly concentrated birch sap extract.

[0131] Embodiment 15 provides the method of embodiment 14, wherein the birch tree is a birch tree grown in Hokkaido, Japan.

[0132] Embodiment 16 provides the method of embodiment 15, wherein the birch sap is harvested in winter or spring.

[0133] Embodiment 17 provides the method of embodiment 15, wherein the birch sap is harvested in early spring.

[0134] Embodiment 18 provides a pharmaceutical combination for treating or preventing epilepsy, comprising the birch sap extract prepared as described in any one of embodiments 14 to 17 and at least one other anti-epileptic agent, wherein the dosage of the other anti-epileptic agent is lower than that when the other anti-epileptic agent is administered alone.

[0135] Embodiment 19 provides the pharmaceutical combination of embodiment 18, wherein the birch sap extract further comprises a pharmaceutically acceptable carrier and / or stabilizer.

[0136] Embodiment 20 provides the pharmaceutical combination of embodiment 18, wherein the at least one other anti-epileptic agent is selected from the group consisting of phenytoin, carbazapine, lamotrigine, febamazepine, benzodiazepines, tiagabine, and vigabatrin.

[0137] Embodiment 21 provides the pharmaceutical combination of embodiment 18, wherein the at least one other anti-epileptic agent is carbazapine.

[0138] Embodiment 22 provides the pharmaceutical combination of embodiment 18, wherein the birch sap extract and the at least one other anti-epileptic agent are administered to a patient simultaneously, sequentially or intermittently.

[0139] Although the present invention has been disclosed with reference to specific embodiments, it is obvious to those with ordinary knowledge in the art to which the present invention belongs that other embodiments and variations can be designed without departing from the true spirit and scope of the present invention. The attached claims are intended to be interpreted as including all such embodiments and equivalent variations.

Claims

1. Use of a birch (Betula platyphylla) sap extract in the preparation of a drug for treating or preventing epilepsy.

2. The use according to claim 1, wherein the birch sap extract is taken from birch trees grown in Hokkaido, Japan.

3. The use according to claim 1, wherein the epilepsy is caused by brain nerve cell damage.

4. The use according to claim 1, wherein the epilepsy is triggered by activation of glial cells.

5. The use according to claim 1, wherein the epilepsy is triggered by a brain inflammatory response caused by inflammatory molecules.

6. The use according to claim 1, wherein the inflammatory molecule is selected from interleukin 1β The group consists of linterleukin-1β (IL-1β), interleukin 6 (IL-6), tumor necrosis factor-α (TNF-α), high mobility group Box 1 (HMGB1), interleukin 1 receptor 1 (IL-1R1), and Toll-like receptor-4 (TLR-4).

7. The use according to claim 1, further comprising at least one other anti-epileptic agent for combining with the birch sap extract for preparing the drug for treating or preventing epilepsy.

8. The use according to claim 7, wherein the at least one other anti-epileptic drug is a Western medicine preparation or a Chinese medicine preparation.

9. The use according to claim 8, wherein the at least one other anti-epileptic drug is selected from a Western medicine preparation consisting of phenytoin, carbamazepine, lamotrigine, felbamate, benzodiazepines, tiagabine, and vigabatrin.

10. The use according to claim 8, wherein the at least one other anti-epileptic drug is selected from the group consisting of Chinese medicine preparations of Gastrodia elata, Uncaria rhynchophylla, Semen Cassiae, Zhengan Xifeng Decoction and Gastrodia elata decoction.

11. A pharmaceutical combination for treating or preventing epilepsy, comprising birch sap extract and at least one other anti-epileptic agent, wherein the dosage of the other anti-epileptic agent is lower than the dosage of the other anti-epileptic agent when administered alone.

12. The pharmaceutical combination according to claim 11, wherein the at least one other anti-epileptic agent is selected from the group consisting of phenytoin, carbazapine, lamotrigine, febamazepine, benzodiazepines, tiagabine, and vigabatrin.

13. The pharmaceutical combination according to claim 11, wherein the at least one other anti-epileptic agent is carbazapine.

14. The pharmaceutical combination according to claim 11, wherein the birch sap extract and the at least one other anti-epileptic agent are administered to the patient simultaneously, sequentially or intermittently.

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