Pharmaceutical combination for treating or preventing epilepsy and use thereof

Abstract

To provide pharmaceutical combinations containing Betula platyphylla sap extract for treating or preventing epilepsy while reducing a dosage of conventional anti-epileptic agents.SOLUTION: The present disclosure provides use of Betula platyphylla sap extract in producing compositions and medicines for treating or preventing epilepsy. The compositions further include other ingredients to enhance anti-epileptic effects. The compositions of the present disclosure have characteristics of high efficiency, safety, and convenience, and can effectively treat or prevent epilepsy seizure while reducing side effects of the anti-epileptic agents. The present disclosure also provides methods for treating or preventing epilepsy seizure with the compositions.SELECTED DRAWING: Figure 1A

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 of a birch sap extract and at least one drug and its use. [Background technology]

[0002] The description of the background art includes information that is helpful for understanding the present invention, and any information provided herein should not be considered background art or art related to the present invention, nor should any publications cited explicitly or implicitly be considered background art.

[0003] Epilepsy is a chronic disease of the nervous system, and is the fourth most common neurological disease after stroke, migraine and Alzheimer's disease, affecting approximately 70 million people worldwide. The disease is characterized by recurrent, spontaneous and unpredictable seizures caused by abnormal discharges of brain neurons, mainly occurring in the cerebral cortex and hippocampus. The cause of epilepsy is related to congenital or acquired brain lesions, such as brain damage, infection, stroke and brain tumors, but the cause of onset in approximately 70% of epilepsy patients is unknown. Although the cause of most epilepsy is unknown, it is generally believed to be due to excessive excitation of brain neurons caused by a disruption in the balance between the brain's inhibitory nervous system (i.e., the GABA system) and excitatory nervous system (i.e., the glutamate system).

[0004] Currently, the mechanism of action of antiepileptic drugs to suppress epilepsy seizures is mainly to directly suppress the excitation of brain neurons by blocking sodium channels (e.g., phenytoin, carbamazepine), or to regulate the stability of brain neurons by suppressing glutamate (e.g., lamotrigine, felbamate), or to increase GABA (e.g., benzodiazepines, tiagabine, vigabatrin). However, although there are currently nearly 30 types of epilepsy drugs, about one-third of epilepsy patients have drug resistance to the current drugs, and the side effects of the drugs affect patients' adherence to the drugs they are currently using.

[0005] Currently, it is known that commonly used antiepileptic drugs have many side effects. For example, the package insert for carbamazepine lists side effects such as drowsiness, loss of appetite, diarrhea, dizziness, heavy head, ataxia (coordination disorder), leukopenia, aplastic anemia, liver dysfunction, arrhythmia, and rash. The package insert for phenytoin lists side effects such as nausea, vomiting, heartburn, loss of appetite, diplopia, nystagmus, ataxia (coordination disorder), gingival hyperplasia, hirsutism, rough face, involuntary movement, lymph node swelling, deformity, and rash. The usual treatment after the occurrence of side effects is to suppress the side effects by changing the drug, reducing the dosage, or adding other drugs, but reducing the dosage makes it easier for epilepsy to occur again, which is painful both physically and mentally for the patient.

[0006] Therefore, it is necessary to search for and develop new, more effective and safer drugs for the treatment of epilepsy, and medicinal plants have been attracting attention because they are widely used in traditional medicine and folk medicine to treat and prevent different nervous system diseases (including epilepsy). In recent years, it has been proven that many plant-derived bioactive molecules can exert antiepileptic effects by affecting ion channels, GABA or glutamate, and medicinal plants, such as flavonoids, alkaloids, terpenoids, and cannabidiol, have become research targets for antiepileptic drugs.

[0007] Birch (Betula platyphylla) is a member of the Betula genus in the Betulaceae family and is distributed in Siberia, Finland, Hokkaido, Mongolia, and other regions. According to the Materia Medica, birch bark is used to treat arthritis, skin diseases, injuries, and to clear heat and detoxify. Currently, birch is widely used in folk medicine to treat inflammatory diseases, such as hepatitis, acute tonsillitis, pneumonia, chronic bronchitis, cholecystitis, urinary tract infections, and burns. Research has shown that birch has many biological activities, including anti-inflammatory, antioxidant, antiviral, anticancer, anti-fatigue, and neuroprotective effects, and these activities are related to the triterpenoid betulin, the main active ingredient in birch. However, there is little research on the relationship of birch to the central nervous system, so a combination of medicines containing birch extracts is needed to treat or prevent epilepsy. Summary of the Invention [Problem to be solved by the invention]

[0008] The technical problem that the present invention seeks to solve is to provide a pharmaceutical combination containing birch extract that can treat or prevent epilepsy and reduce the dose of conventional antiepileptic drugs. [Means for solving the problem]

[0009] In order to solve the above problems and needs, the present invention provides a use of birch (Betula platyphylla) sap extract in preparing a medicament for treating or preventing epilepsy.

[0010] In one embodiment, the birch sap extract of the present invention is collected from birch trees grown in Hokkaido, Japan.

[0011] In one embodiment, epilepsy is caused by damage to brain neurons. In another embodiment, epilepsy is caused by activation of glial cells. In another embodiment, epilepsy is caused by an inflammatory response in the brain caused by inflammatory molecules. In yet another embodiment, the inflammatory molecules are selected from the group consisting of Interleukin-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).

[0012] In one embodiment, the birch sap extract of the present invention is further combined with at least one other antiepileptic drug to manufacture a medicament for treating or preventing epilepsy. In another embodiment, the at least one other antiepileptic drug is a Western medicine preparation. In yet another embodiment, the at least one other antiepileptic drug is a Western medicine preparation selected from the group consisting of phenytoin, carbamazepine, lamotrigine, felbamate, benzodiazepine, tiagabine, and vigabatrin. In another embodiment, the at least one other antiepileptic drug is a traditional Chinese medicine preparation or prescription. In another embodiment, the at least one antiepileptic drug is a traditional Chinese medicine preparation or prescription selected from the group consisting of Tianma, Diao Teng Gao, Seki Jie Ming, Zhen Gan Xifeng Tang, and Tianma Gang Teng Yin.

[0013] The present invention provides a method for producing a birch sap extract, comprising the following steps: Step 1: Select a mature birch tree and cut and treat the bark to allow the sap to flow easily; Step 2: Use a collector to collect the flowing sap; Step 3: Filter the sap at room temperature to remove bark fragments and other foreign matter; Step 4: Using low-temperature concentration technology, a highly concentrated birch sap extract is obtained by removing most of the water in the sap. The above steps may be increased or decreased depending on the extraction of specific components or other practical needs.

[0014] In one embodiment, the birch is grown in Hokkaido, Japan. In another embodiment, the birch sap is collected in the winter or spring. In another embodiment, the birch sap is collected in early spring.

[0015] The present invention also provides a pharmaceutical combination for treating or preventing epilepsy, comprising birch sap extract, at least one other antiepileptic drug, a pharma- ceutically acceptable carrier and / or a stabilizer, the dosage of which is less than that of the other antiepileptic drug administered alone. In one embodiment, the at least one other antiepileptic drug is selected from the group consisting of phenytoin, carbamazepine, lamotrigine, felbamate, benzodiazepine, tiagabine and vigabatrin. In one embodiment, the birch sap extract and the at least one other antiepileptic drug are administered to a patient at the same time, sequence or interval.

[0016] Various objects, features, aspects and advantages of the present invention will become apparent from the following detailed description of the preferred embodiment and the accompanying drawings. Effect of the Invention

[0017] The pharmaceutical combination containing birch extract of the present invention can effectively treat or prevent epilepsy, and at the same time, it allows the dosage of conventional antiepileptic drugs to be reduced, thereby suppressing side effects. [Brief description of the drawings]

[0018] The features and advantages of the present invention will be more apparent from the following description of the preferred embodiments with reference to the drawings, in which similar reference words refer to the same parts or components in the text.

[0019] [Figure 1A] FIG. 2 shows the chemical properties of birch sap extract (HCF02) and the monosaccharide composition. [Figure 1B] Chemical properties of birch sap extract (HCF02), showing the monosaccharide composition. [Figure 1C] FIG. 2 shows the chemical properties of birch sap extract (HCF02) and the molecular weight distribution. [Figure 1D] FIG. 2 is a chemical characterization of birch sap extract (HCF02) and shows the 1H NMR spectrum. [Figure 2A] 1 is a bar graph showing that pretreatment with birch sap extract reduces KA-induced epileptic behavior in animals. Data are mean ± SEM (n = 2-13 rats / group). *, p < 0.05 compared with KA group; ***, p < 0.001 compared with KA group. [Figure 2B] 1 is a bar graph showing that pretreatment with birch sap extract reduces KA-induced epileptic behavior in animals. Data are mean ± SEM (n = 2-13 rats / group). *, p < 0.05 compared with KA group; ***, p < 0.001 compared with KA group. [Figure 3A] Figure 1. Pretreatment with birch sap extract and betulin prevents KA-induced death of cerebral cortex and hippocampal neurons. Data are mean ± SEM. *, p<0.01 compared with control group; ***, p<0.001 compared with control group; ♯, p<0.05 compared with KA group; n=3 for each group. [Figure 3B]Figure 1. Pretreatment with birch sap extract and betulin prevents KA-induced death of cerebral cortex and hippocampal neurons. Data are mean ± SEM. *, p<0.01 compared with control group; ***, p<0.001 compared with control group; ♯, p<0.05 compared with KA group; n=3 for each group. [Figure 4A] Figure 1. Pretreatment with birch sap extract and betulin attenuates KA-induced degeneration of cerebral cortex and hippocampal neurons. Data are mean ± SEM. ***, p<0.001 compared with control group; ♯, p<0.05 compared with KA group; n=3 for each group. [Figure 4B] Figure 1. Pretreatment with birch sap extract and betulin attenuates KA-induced degeneration of cerebral cortex and hippocampal neurons. Data are mean ± SEM. ***, p<0.001 compared with control group; ♯, p<0.05 compared with KA group; n=3 for each group. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] The following description contains information that is helpful in understanding the present invention, and any information provided herein should not be considered prior art or relevant to the scope of the present invention, and any publications cited explicitly or implicitly should not be considered prior art.

[0021] The present invention is not limited to the specific example materials, structures, processes, methods or constructions described herein, and therefore, although some options are described herein, all similar or effective options may be applied to the practice or embodiment of the present invention, and only the best materials and methods are described herein.

[0022] The following detailed description in conjunction with the accompanying drawings is a description of an exemplary embodiment of the present invention, and is not intended to represent the only exemplary embodiment for carrying out the present invention. The technical term "exemplary" used in this specification means "one embodiment, example, or example," and it is not necessary to explain that the exemplary embodiment is better or superior to other possible exemplary embodiments. The detailed description includes specific parts and is provided to understand the embodiments of the present specification. It is understood by those skilled in the art that the embodiments of the present specification can be carried out without using these specific parts.

[0023] As used herein, each of the following terms has the meaning set forth in the paragraph. The terminology used in the present invention is for the purpose of describing specific embodiments of the present invention and is not intended to be limiting.

[0024] Unless otherwise defined, technical and scientific terms used herein are the same as those commonly understood by those skilled in the art. In general, the nomenclature used herein is that known and commonly used in the art of animal pharmacology, pharmaceutical science, separation science, and organic chemistry laboratory processes. In the methods described herein, actions can be performed in any order unless the time and order of operations are clearly stated. Furthermore, actions can be performed simultaneously unless specifically stated to be performed separately in the claim language. For example, an action to perform X and an action to perform Y can be performed simultaneously in a single operation, and the resulting method is within the scope of the claimed method.

[0025] The following non-limiting acronyms 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.

[0026] Unless the context clearly dictates otherwise, as used in the specification and claims, the words "a," "an," "a type," and "the" include the plural. For example, "a type of component" means one component or one or more components.

[0027] As used herein, the technical term "about" should be understood by those of ordinary skill in the art and may vary to some extent depending on the context. As used herein, in the case of measurable values, such as quantities, lengths of time, and the like, "about" is meant to include a variation of ±20%, ±10%, ±5%, ±1% or ±0.1% of the particular value. These variations are appropriate for the disclosed method. Thus, in some embodiments, the numerical parameters in the specification and claims are approximations and may vary to obtain the desired properties in a particular implementation. In some embodiments, the numerical parameters are interpreted using ordinary numerical rounding techniques in accordance with the stated significance. Numerical values ​​in some embodiments of the present invention may contain errors that may be accounted for by the standard deviation in individual test measurements.

[0028] In one embodiment, the technical term "co-administration" refers to administering to a subject other drugs to treat or prevent the disease considered herein along with the birch sap extract of the present invention. In some embodiments, the birch sap extract and other drugs to be co-administered are administered separately or in any combination to provide a sole therapeutic regimen.

[0029] As used herein, a "disease" is a state of health in a subject where the subject is unable to maintain a stable internal state and, if the disease is not improved, the subject's health continues to deteriorate.

[0030] As used herein, a "disease condition" of a subject is a state of health in which the subject's health is no better than it would be if the disease condition were not present, but the subject is able to maintain an internal stable state. Failure to treat a disease condition does not necessarily result in a worsening of the subject's health.

[0031] As used herein, the technical term "pharmaceutical acceptable" refers to a substance, such as a vehicle or diluent, that does not destroy the biological activity or properties of a compound useful in the present invention. Moreover, the substance is non-toxic, i.e., the substance does not produce an unexpected biological reaction when administered to a subject or interact in a deleterious manner with any of the components contained in the composition.

[0032] As used herein, the technical terms "pharmacologically effective dose," "therapeutically effective dose," or "effective dose" of a compound is an amount of the compound that confers a beneficial effect on a subject to which the compound is administered.

[0033] As used herein, the technical terms "subject," "individual," and "patient" are used interchangeably and include human or non-human mammals. Non-human mammals include, for example, farm animals and pets, such as sheep, pigs, canines, felines, and murine mammals. In some embodiments, the subject is a human.

[0034] As used herein, the technical terms "prophylaxis" or "prevention" refer to the administration of an agent or compound prior to the subject's onset of a disease or condition, thereby avoiding or delaying the onset of these symptoms.

[0035] As used herein, the technical term "treatment" refers to the administration of a drug or compound to a subject, thereby reducing the frequency or progression of a disease or condition experienced by the subject.

[0036] Throughout the disclosure of the present invention, each aspect of the invention is expressed in the form of a range. The description in range format is merely for convenience and brevity, and is not a limitation on the scope of the present invention. Thus, the description of a range should be considered specifically as all possible subranges and single numerical values ​​within that range. For example, the description of a range of 1 to 6 is considered to specify subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and single and partial numbers within the range are, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. All ranges apply regardless of the range.

[0037] [About birch sap] The birch sap of the present invention is a colorless or light yellow transparent liquid extracted from birch bark and / or trunk. Birch sap contains active substances such as betulin, polysaccharides, saponin, protein, and minerals, and has relatively high medicinal value. In the theory of traditional Chinese medicine, birch sap belongs to the medicine for clearing heat and detoxifying, and the therapeutic effects and advantages disclosed at present are summarized as follows: Clearing heat and detoxifying: Birch sap has the effect of clearing heat and detoxifying, which can not only remove heat and dampness from the body, but also relieve symptoms such as fever, thirst, sore throat, and rashes. Diuretic and anti-swelling: Birch sap can promote the excretory function of the kidneys and increase urine volume, helping to eliminate excess fluid from the body and reducing symptoms such as swelling. Anti-inflammatory and analgesic: Birch sap contains ingredients with anti-inflammatory and analgesic properties, such as menthol and salicylic acid, which can relieve pain and inflammation. Improves digestion: Birch sap can stimulate gastrointestinal motility and increase the secretion of digestive juices, which can improve gastrointestinal problems such as indigestion and diarrhea. Nutritional supplementation: Birch sap contains a wide variety of nutrients, such as vitamin C, B vitamins, iron, potassium, etc., which can replenish the nutrients needed by the body and strengthen the body's immune system and ability to fight diseases.

[0038] In one embodiment, the birch sap is collected annually in winter or spring, preferably in early spring. In another embodiment, the birch sap is collected from birch trees that are 20-50 years old. In another embodiment, the birch trees are grown in Hokkaido, Japan.

[0039] In one embodiment, the birch sap extract of the present invention can be used or manufactured into a combination of one or more other pharmaceutical agents to treat or prevent epilepsy, including known compounds (e.g., commercially available compounds) that treat, prevent, or alleviate the symptoms of epilepsy.

[0040] In one embodiment, the present invention provides a method for treating or preventing epilepsy in a subject. The method includes administering to the subject a therapeutically effective amount of the birch sap extract of the present invention. In one embodiment, the subject is further co-administered with at least one other drug effective for treating or preventing epilepsy. In another embodiment, the at least one other drug is a traditional Chinese medicine preparation or prescription, such as Tianma, Diao Teng Gao, Shi Jie Ming, Zhen Gan Xifeng Tang, Tianma Gouteng Yin, etc., and the dosage of the traditional Chinese medicine preparation can be adjusted by a traditional Chinese medicine practitioner according to the conventional dosage. In another embodiment, the other drug is a traditional Chinese medicine preparation or prescription, which is co-formulated with the birch sap extract. In another embodiment, the at least one other drug is a Western medicine preparation, such as phenytoin, carbamazepine, lamotrigine, felbamate, benzodiazepine, tiagabine, vigabatrin, etc. The dosage of the Western medicine preparation can be adjusted by a physician according to the recommended dosage of each drug.

[0041] In one embodiment, the subject with epilepsy is a person who has just been diagnosed with epilepsy or is taking at least one antiepileptic drug. In one embodiment, the subject who has just been diagnosed with epilepsy is given a combination of birch sap extract and at least one antiepileptic drug, and the at least one antiepileptic drug is administered in a lower dose when administered in combination than when administered alone. In another embodiment, the birch sap extract is given to a subject who is taking at least one antiepileptic drug, thereby reducing the dose of the antiepileptic drug.

[0042] The method of treatment involves the combination of effective amounts. The therapeutic compound can be administered to the patient before or after the onset of the disease or condition. Furthermore, the therapeutic compound can be administered daily or in several divided doses and staggered doses in sequence, or the doses can be administered continuously. In addition, the therapeutic compound can be increased or decreased in proportion to the exigencies of the treatment or prevention situation.

[0043] By administering the birch sap extract of the present invention to a patient in a known sequence, dosage and duration, epilepsy is effectively treated or prevented. The effective amount of birch sap extract required for the effect of treating and / or preventing epilepsy varies depending on many factors, such as the activity of the particular 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 medical condition of the patient being treated, the age, sex, weight, symptoms, general health and previous medical history of the patient; and similar factors well known in the medical field, and the dosage, etc. can be adjusted to provide a good therapeutic response. For example, several divided doses can be administered daily, or the dosage can be reduced in proportion to the urgency of the treatment situation.

[0044] The birch sap extract of the present invention is described as being administered to humans in a manner that is in accordance with medical ethics, but those skilled in the art should understand that the composition is generally suitable for a variety of animals. Modifications of the birch sap extract of the present invention for administration to a variety of animals are well known, and veterinary pharmacologists skilled in the art can design and carry out such modifications through general experimentation (if necessary). Subjects to which the birch sap extract of the present invention can be administered include, but are not limited to, humans and other primates, non-mammals, and mammals of commercial value, such as cows, pigs, horses, sheep, cats and dogs.

[0045] Humans and animals have different tolerances to the same drug, and generally speaking, animals have stronger tolerance than humans. When calculated by unit body weight or unit surface area, animals use more drugs than humans. The amount of drug used per unit body weight for animals and humans can be calculated by referring to the following conversion table 1.

[0046] [Table 1]

[0047] Source: International Life Sciences Institute Taiwan, ILSI Taiwan

[0048] For example, taking the rat model of the embodiment of the present invention as an example, the birch sap extract of the present invention is orally administered at a dose of 3mg / kg / time to 20mg / kg / time, preferably 6mg / kg / time to 10mg / kg / time, for example 6mg / kg / time to 10mg / kg / time. After conversion according to the above unit weight dose conversion table, the dose of the birch sap extract of the present invention given to humans is 0.48mg / kg / time to 3.2mg / kg / time, preferably 0.96mg / kg / time to 1.6mg / kg / time. The dose of the birch sap extract of the present invention suitable for other animals can also be obtained according to the above unit weight dose conversion table or other unit body surface area conversion tables.

[0049] In some embodiments, the birch sap extract of the present invention is administered to a patient in a dosage range of 1 to 5 or more times daily. In other embodiments, the birch sap extract of the present invention is 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 other day, every 3 days to a week, or once every two weeks. Those skilled in the art will appreciate that the frequency of administration of each combination composition of the present invention will vary from subject to subject, and this will depend on a number of factors, including, but not limited to, age, the disease or condition being treated, sex, overall health, and other factors. Thus, the present invention should not be construed as being limited to any particular dosing schedule, and the attending physician should ascertain the exact dosage and composition to be administered to the patient after considering all factors relevant to the patient.

[0050] In one embodiment, when the birch sap extract of the present invention is administered in combination with at least one other antiepileptic drug, the birch sap extract and the at least one other antiepileptic drug can be administered to the subject simultaneously. In another embodiment, the birch sap extract and the at least one other antiepileptic drug can be administered to the subject sequentially, before or after each other or with an interval therebetween.

[0051] Oral Administration: Liquid preparations for oral administration may be in the form of solutions, syrups or suspensions. The liquid preparations may be prepared by conventional methods with pharma- ceutically acceptable additives, such as syrups or pharma-ceutically acceptable preservatives.

[0052] Those skilled in the art will recognize and, using routine experimentation, will be able to ascertain equivalents to the specific sequences, methods, claims, and examples described herein. These equivalents are recognized as being within the scope of the present invention and are further encompassed by the claims. For example, modifications of reaction conditions, such as reaction times, reaction conditions / volumes, and experimental reagents (e.g., solvents, catalysts), pressure, atmospheric conditions, and reducing / oxidizing agents, as well as art-recognized substitutions and routine experimentation, are all within the scope of the present invention.

[0053] For any numerical values ​​and ranges recited herein, the description of the ranges is for convenience and brevity and should not be construed as a limitation on the scope of the invention. Thus, all numerical values ​​and ranges contained within the numerical values ​​and ranges are included within the scope of the invention. Also, all numerical values ​​and upper or lower limits of numerical ranges falling within these ranges are contemplated by the invention. All descriptions of ranges specifically disclose possible subranges and individual numerical values ​​within the ranges, and where appropriate, integers within the numerical values ​​are also considered to be included within the range. For example, the description of a range of 1 to 6 is considered to be specifically disclosed as a subrange, such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and individual numbers within the range, such as 1, 2, 2.7, 3, 4, 5, 5.3 and 6, all of which apply regardless of the range.

[0054] The following examples further illustrate various aspects of the present invention, but are not intended to be limitations on the teachings or disclosure of the invention set forth herein.

[0055] The present invention will now be described with reference to the following examples, which are intended for illustrative purposes only and are not intended to limit the invention, but rather to include all variations made evident by the teachings provided herein.

[0056] The birch sap extract exemplified in the present invention is prepared and / or tested according to the following process.

[0057] [Birch Sap Extract] Sap is collected from birch (Betula platyphylla) and birch sap extract is produced by the following steps. Step 1: Select a mature birch tree and cut and treat the bark to allow the sap to flow easily; Step 2: Collect the sap that flows out with a collector to avoid external contamination; Step 3: Filter the sap at room temperature to remove bark fragments and other foreign matter; Step 4: Using low-temperature concentration technology, a highly concentrated birch sap extract is obtained by removing most of the water in the sap.

[0058] [Animal experiments] Male Sprague-Dawley rats (SD, 150-200g) were purchased from BioLASCO TAIWAN Co., Ltd. and kept at the Fu Jen University Laboratory Animal Center. All animal experiments were approved by the Fu Jen University Laboratory Animal Care Committee. In accordance with the 3R rule, all experiments were performed with the minimum number of animals that would produce reliable results while minimizing animal suffering as much as possible.

[0059] [Example 1 - Birch sap component analysis] Sap extracted from birch is analyzed by monosaccharide analysis method. In the birch sap monosaccharide analysis, the ratio of three major monosaccharides, fructose, glucose and fucose, in the birch sap extract is determined to be 27:17:10 (Figures 1A and 1B). When analyzed by high pressure size exclusion chromatography (HPSEC), the molecular weight of birch sap is approximately 1.29 kDa or less (Figure 1C). 1 The H NMR spectrum (Figure 1D) shows two anomeric protons at δH5.13 and δH4.45. The signals at δH5.13 and δH4.45 show a bimodal profile with a ratio of 100:124, which corresponds to two monosaccharide residues and an α-anomeric structure ( 3 J 1,2 =3.4Hz) and β-anomeric structure ( 3 J 1,2 = 7.7 Hz. The results of the above analysis allow the amount of birch sap extract to be quantified for use in subsequent biological tests.

[0060] [Example 2 - KA-induced animal epilepsy model] The antiepileptic effect of birch sap extract is evaluated in an animal epilepsy model induced by kainic acid (KA). KA is a glutamate analogue, and the injection of KA into rats increases the release of glutamate and overactivates glutamate receptors, causing progressive limbic seizures in rats. This causes an increase in calcium ions in neurons, oxidative stress and loss of mitochondrial function, resulting in the death of neurons in many areas of the cerebral cortex, especially in the hippocampus area. Furthermore, KA-induced neuronal death and glial activation are associated with inflammatory responses mediated by inflammation-related 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)). Because these pathological changes induced by KA are similar to human epileptic behavior, KA-induced animal epilepsy models are widely used to screen novel compounds with antiepileptic activity.

[0061] After intraperitoneal injection of KA into rats, epileptic behavior was analyzed within 4 hours. The evaluation method for seizure severity was based on the Racine scale (1972) and is shown in Table 2.

[0062] [Table 2]

[0063] Example 3 - Evaluation of the effect of birch sap extract on KA-induced epileptic behavior in animals Sprague-Dawley rats were administered different test substances and divided into the following groups: 1, negative control group: saline. 2, KA group: kainic acid (KA) 15 mg / kg / ip / once. 3. Low dose group of birch sap extract: 6 mg / kg of birch sap extract. 4. High dose group of birch sap extract: 10 mg / kg of birch sap extract. 5. High-dose antiepileptic drug group: 100 mg / kg carbamazepine (CBZ). 6. Antiepileptic drug composition group: containing 6 mg / kg of birch sap extract and 50 mg / kg of carbamazepine (CBZ). 7. Low-dose antiepileptic drug group: 50 mg / kg carbamazepine (CBZ).

[0064] The rats in the control group, birch sap extract group, antiepileptic drug group and antiepileptic drug composition group were given saline, birch sap extract and / or carbamazepine at fixed times every day. After 7 days, the rats in each group were intraperitoneally injected with KA (15 mg / kg / ip / once) except for the negative control group. The dose of KA injection was based on the previous study. The epileptic behavior of the rats was analyzed based on the Racine scale (1972) within 4 hours after intraperitoneal injection of KA, and the results are shown in Table 3.

[0065] Table 3 shows that pretreatment with birch sap extract reduced epileptic behavior induced by KA in animals.

[0066] [Table 3]

[0067] Data shown are means ± SEM. **, p < 0.05 compared with KA group; ***, p < 0.001 compared with KA group.

[0068] As can be seen from the experimental results, in the KA group (positive control group), 91% of rats given KA were induced to have epileptic seizures. The duration and score of epileptic seizures were 66.6±6.4 minutes and 4.6±0.2, respectively. Compared with the KA group, when the birch sap extract of the present invention was pretreated by oral ingestion for 7 days, the duration of epileptic seizures induced by KA could be extended and the severity of epileptic seizures induced by KA could be reduced (p<0.05; Fig. 2A-2B). In addition, the high dose group of birch sap extract (10 mg / kg) had a good inhibitory effect on the severity of epileptic seizures induced by KA, which was similar to that of the high dose group of antiepileptic drugs (CBZ 100 mg / kg), proving that the birch sap extract of the present invention has an effect of preventing epilepsy caused by KA (Table 3). In other words, the birch sap extract of the present invention can be used as a substitute for antiepileptic drugs.

[0069] Furthermore, in the low-dose antiepileptic drug group, the duration and severity of the animal epileptic seizures are not statistically different from those in the KA group. However, if used in combination with low-dose birch sap extract, i.e., in the antiepileptic drug composition group (6 mg / kg birch sap extract + 50 mg / kg CBZ), the duration and severity of the animal epileptic seizures are significantly improved compared to the KA group (p<0.05; Figure 2A-2B), which can greatly improve the shortcoming that low-dose antiepileptic drugs cannot effectively suppress epileptic symptoms. The antiepileptic drug composition group has an effect of preventing epilepsy caused by KA similar to that of the high-dose antiepileptic drug group, so this result shows that the combination of low-dose (6 mg / kg) birch sap and low-dose (50 mg / kg) CBZ can prevent animal epileptic behavior induced by KA, and low-dose CBZ can also reduce the side effects caused by antiepileptic drugs.

[0070] According to rat experiments, the method provided by the present invention can be used to prevent epileptic seizures, and has obvious preventive effects, especially against epileptic symptoms that are difficult to control. When the birch sap extract of the present invention is used alone, it has an anti-epileptic effect equivalent to that of anti-epileptic drugs (e.g., CBZ). In other words, the birch sap extract can replace anti-epileptic drugs. When the birch sap extract of the present invention is used in combination with an anti-epileptic drug, it can achieve an anti-epileptic effect and reduce the dose of the anti-epileptic drug (e.g., CBZ). Therefore, whether the birch sap extract of the present invention is used alone or in combination with an anti-epileptic drug, the side effects of the anti-epileptic drug can be reduced. The method provided by the present invention has relatively low toxicity and side effects, making it a safe and effective method for preventing epilepsy.

[0071] [Example 4 - Pathological analysis of birch sap extract on brain and nervous tissue] Following Example 3, the experimental animals were intraperitoneally injected with KA (15 mg / kg) and killed three days later, and brain tissue was removed for biopsy and other correlation experimental analyses.

[0072] Three days after intraperitoneal injection of KA into rats, Nissl staining revealed a clear decrease in the number of neurons in the cerebral cortex and hippocampal CA1 / CA3 in the KA group, and pretreatment with birch sap extract and betulin could reduce the decrease in neuronal cell number caused by KA (p<0.05; Figures 3A and 3B).

[0073] Furthermore, Fluoro-Jade B (FJB) staining also revealed an increase in the number of degenerated cells in the brain cortex and hippocampus CA1 / CA3 in the KA group, which was also reduced by pretreatment with birch sap extract and betulin (p<0.05; Fig. 4A and Fig. 4B). These preliminary results indicate that pretreatment with birch sap extract and / or betulin can prevent KA-induced neuronal damage.

[0074] The results of the above animal experiments show that the birch sap extract of the present invention can extend the time of suppressing epilepsy seizures induced by KA and reduce the severity of epilepsy induced by KA, confirming that the birch sap extract has the effect of preventing epilepsy. Therefore, birch sap has the potential to be used in the development of antiepileptic drugs and / or related products.

[0075] The following are illustrative examples, the numbering of which should not be construed as a matter of importance.

[0076] Example 1 provides the use of birch sap extract in the manufacture of a medicament for treating or preventing epilepsy.

[0077] Example 2 provides the use of Example 1, where the birch sap extract is collected from birch grown in Hokkaido, Japan.

[0078] Example 3 provides a use of Example 1, in which the epilepsy is caused by damage to brain nerve cells.

[0079] Example 4 provides a use of Example 1, wherein the epilepsy is caused by activation of glial cells.

[0080] Example 5 provides a use of Example 1, wherein the epilepsy is caused by an inflammatory response in the brain due to inflammatory molecules.

[0081] Example 6 provides a use of Example 5, wherein the inflammatory molecule is selected from the group consisting of interleukin 1 beta (IL-1β), interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), high mobility group protein (HMGB1), interleukin 1 receptor 1 (IL-1R1), and toll-like receptor 4 (TLR-4).

[0082] Example 7 provides the use of Example 1, further comprising combining at least one other anti-epileptic drug with the birch sap extract to manufacture a medicament for treating or preventing epilepsy.

[0083] Example 8 provides the use of Example 7, wherein the at least one other antiepileptic drug is a Western medicine preparation.

[0084] Example 9 provides the use of Example 8, wherein the at least one other antiepileptic drug is a Western drug preparation selected from the group consisting of phenytoin, carbamazepine, lamotrigine, felbamate, benzodiazepine, tiagabine, and vigabatrin.

[0085] Example 10 provides the use of Example 7, wherein the at least one other antiepileptic drug is a herbal medicine preparation or formula.

[0086] Example 11 provides the use of Example 10, wherein the at least one antiepileptic drug is a Chinese herbal medicine or formula selected from the combined group consisting of Tianma, Diao Teng Gao, Shi Jie Ming, Zhen Gan Xifeng Tang and Tian Ma Gong Teng Yin.

[0087] Example 12 provides the use of Example 1, wherein the medicament is used to treat or prevent epilepsy in humans.

[0088] Example 13 provides a use of Example 1, wherein the medicament is used to treat or prevent epilepsy in a non-human mammal.

[0089] Example 14 provides a method for producing a birch sap extract, which includes the following steps: Step 1: Select a mature birch tree and cut and treat the bark to allow the sap to flow easily; Step 2: Use a collector to collect the flowing sap; Step 3: Filter the sap at room temperature to remove bark fragments and other foreign matter; Step 4: Using low-temperature concentration technology, a highly concentrated birch sap extract is obtained by removing most of the water in the sap.

[0090] Example 15 provides the method of Example 14, where the birch is grown in Hokkaido, Japan.

[0091] Example 16 provides the method of Example 15, wherein the birch sap is collected in winter or spring.

[0092] Example 17 provides the method of Example 15, where the birch sap is collected in early spring.

[0093] Example 18 provides a pharmaceutical combination for treating or preventing epilepsy, comprising a birch sap extract prepared in any of Examples 14 to 17 and at least one other antiepileptic drug, wherein the dose of the other antiepileptic drug is less than the dose when administered alone.

[0094] Example 19 provides the pharmaceutical combination of Example 18, wherein the birch sap extract comprises a pharma- ceutically acceptable carrier and / or stabilizer.

[0095] Example 20 provides the pharmaceutical combination of example 18, wherein the at least one other antiepileptic drug is selected from the group consisting of phenytoin, carbamazepine, lamotrigine, felbamate, benzodiazepines, tiagabine, and vigabatrin.

[0096] Example 21 provides the pharmaceutical combination of Example 18, wherein the at least one other antiepileptic drug is carbamazepine.

[0097] Example 22 provides the pharmaceutical combination of Example 18, wherein the birch sap extract and the at least one other antiepileptic drug are administered to a patient simultaneously, sequentially or at intervals.

[0098] While the present invention has disclosed certain embodiments, other embodiments and variations may be designed by those skilled in the art without departing from the spirit and scope of the present invention, and the claims are intended to cover any and all modifications of the embodiments to the same effect. [Industrial Applicability]

[0099] The pharmaceutical combination of birch extract provided by the present invention has the characteristics of high efficacy, safety and convenience, which can effectively treat or prevent epilepsy, and reduce the side effects caused by the long-term use of traditional drugs.

Claims

1. A use of a birch (Betula platyphylla) sap extract and carbamazepine for the manufacture of a drug for treating or preventing epilepsy, the birch sap extract being obtained by filtering and concentrating birch sap collected from winter to spring at room temperature, and the birch sap extract is analyzed by a monosaccharide analysis method to find that the ratio of fructose, glucose and fucose contained therein is 27:17:

10.

2. 2. The use according to claim 1, characterized in that the birch sap extract is collected from birch grown in Hokkaido, Japan.

3. 2. The use according to claim 1, characterized in that the epilepsy is caused by damage to brain nerve cells.

4. 2. The use according to claim 1, characterized in that said epilepsy is caused by activation of glial cells.

5. 2. The use according to claim 1, characterized in that the epilepsy is caused by an inflammatory response in the brain due to inflammatory molecules.

6. The use according to claim 1, 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 Box 1 (HMGB1), interleukin-1 receptor 1 (IL-1R1), and Toll-like receptor 4 (TLR-4).

7. A pharmaceutical combination for treating or preventing epilepsy, comprising a birch sap extract and at least one other antiepileptic drug, said birch sap extract being obtained by filtering and concentrating birch sap collected from winter to spring at room temperature, and analyzed by a monosaccharide analysis method to find that the concentration ratio of three main monosaccharides, fructose, glucose and fucose, contained in said birch sap extract is 27:17:10, said at least one other antiepileptic drug is carbamazepine, and said dosage of said other antiepileptic drug is less than the dosage when administered alone.

8. 8. A pharmaceutical combination according to claim 7, characterized in that said birch sap extract and said at least one other antiepileptic drug are administered to a patient simultaneously, sequentially or at intervals.

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