KR20110108150A - Composition for preventing and treating neurological disease of brain or for the enhancement of memory comprising an extract of silk yarn - Google Patents

Abstract

The present invention relates to a composition for preventing and treating cerebral neurological disease or memory enhancement comprising a silkworm extract, the composition for preventing and treating cerebral neurological disease or a memory for containing a silkworm extract of the present invention and a pharmaceutically acceptable carrier. The composition exhibits the effect of inducing the growth and differentiation of neurons and maintaining survival, and thus can be usefully used as a memory enhancer and a preventive and therapeutic agent for neurological diseases.

Description

COMPOSITION FOR PREVENTING AND TREATING NEUROLOGICAL DISEASE OF BRAIN OR FOR THE ENHANCEMENT OF MEMORY COMPRISING AN EXTRACT OF SILK YARN}

The present invention relates to a composition for preventing and treating cerebral neurological diseases or enhancing memory, including a silkworm extract.

In the twentieth century, the rapid development of life sciences and medicine has led to an increase in the average life expectancy of humans and a growing proportion of the elderly population, highlighting new social problems, especially senile neurological diseases such as stroke and Alzheimer's disease. AD) and Parkinson's disease (PD) have been shown to be fatal neurological dysfunctions.

Growth, differentiation and cell death of neurons in the nervous system are important regulatory processes in normal development and intrinsic function of tissues and in maintaining homeostasis, respectively. There are two forms of neuronal cell death: apoptosis and necrosis. Necrotic necrosis is caused by damage leading to a rapid imbalance of intracellular ions. After the cytoplasm and the mitochondrial swelling and cytolysis, Induced by the destruction of the nuclear membrane, which refers to acute death of cells due to sudden injury such as ischemic, hypothermia, stroke, physical or chemical trauma (Tomei et al .; Proc. Natl Acad. Sci. USA, 90, pp853-857, 1993), unaffected by protein synthesis inhibitors and a representative example of what occurs in the nervous system when neurons are damaged by activation of ionic glutamic acid receptors. Apoptosis, also known as scheduled cell death, is characterized by membrane changes such as cell shrinkage, membrane blebbing, disruption of the cytoskeleton, and nuclear changes such as chromatin condensation. (Kerr JF, J. Pathol., 107 (3), pp 217-219, 1972: Arends MJ amp; Morris RG, Am. J. Pathol., 136 (3), pp 593-608, 1990). With the loss of mitochondrial function, cells form apoptotic bodies, which are membrane-structured substructures, which are phagocytosis completely removed by neighboring cells or macrophages in animal models. It is removed from the tissue without an inflammatory response due to the outflow of the cellular contents. In multicellular organisms, all actions such as cell growth, differentiation and migration are regulated by regulatory factors that exist outside the cell. Neurons also need these regulatory factors, which are neuro-regulatory factors, which are proteins that are released from target cells and affect the growth differentiation survival of neurons in the Central Nervous System (CNS) and the Peripheral Nervous System (PNS). All are called nerve growth factors. There are five related factors: nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurofactor NT-3 (Neurotrophin-3), NT-4, NT-5 These neurons are each synthesized, differentiated, expressed, and expressed at different target sites, but the amino acid sequence constituting each is very similar between species. Nerve factors suppress neuronal cell death in the nervous system. Neuronal cell death occurs when a neuronal factor is deficient, and is a form of cell death that relies mostly on cell death related genes. It is well established that neurofactors inhibit neuronal cell death during neuronal development.

Among these neuronal factors, nerve growth factor (NGF) was isolated by Levi-Montalcini from snake venom or mouse sarcoma in the early 1950's. It has been reported that the growth of axons is several times accelerated (Levi-Montalcini R., Birth Defects Orig Artic Ser, 19 (4), pp3-22, 1983). In the past, nerve growth factors were thought to promote neuronal division, but now known to prevent neuronal degeneration by inhibiting neuronal degeneration. These receptors are present in the dorsal root ganglion, brain, and sympathetic dominant organs. In vivo, sympathetic dominant organs, such as the heart, biosynthesize nerve growth factors, which are absorbed at the nerve endings and transported in the axon direction. Reaching neuronal cells has been reported to promote protein synthesis here (Mahalik TJ, Investig Dermatol Symp Proc., Aug; 2 (1), pp 14-18, 1997).

In the central nervous system, there are many reports that NGF protects against neuronal damage. For example, cholinergic axotomy is the cholinergic axotomy of the pituitary basal cholinergic nerve. It prevents cholinergic addition from the cells to the hippocampus, which causes the cholinergic neurons to degenerate slowly, and when the nerve growth factor is inserted after cutting, the degeneration of cholinergic neurons is almost completely suppressed. Administration of cholinergic antagonist may have a similar effect to nerve growth factor (Hefti FJ, Neurosci, Aug; 6 (8), pp2155-2162, 1986). In addition, the relationship between nerve growth factors and peripheral nerves, the role of nerve growth factors in mature animals has not yet been clearly identified, but recent reports show that the injection of antibodies of nerve growth factors causes sympathetic ganglion destruction and norepinephrine The activity of synthesizing tyrosine hydroxylase and dopamine beta-hydroxylase is also reported to be reduced (Levi-Montalcinin et al .; Bull. Soc. Sci. Med Grand Duche Luxemb, 115 (2), pp69-74). , 1978). On the other hand, the injection of nerve growth factors from the outside increases the survival of NGF-sensitive nerves and increases the degree of nerve dominance in the tissues, and reports that externally injected nerve growth factors weaken the developmental changes after peripheral nerve injury (Zettler). C, Head RJ, Rush RA, Brain Res., 538 (2), pp251-262, 1991), suggest that neuronal growth factor may play an important role in neuronal regeneration after nerve injury. These results show that the organization's nerve growth factor and sympathetic control are closely related. About 50% of neurons in development during the normal development of the nervous system are eliminated by apoptosis (Barres BA, Schmid R, Sendnter M, Raff MC., Development, 118 (1), pp283-95, 1993), and target cells. The neuronal factors secreted by are known to determine the survival of neurons. Growth factors, such as nerve growth factors, are essential for neurons to survive, grow, and differentiate in their normal state. However, these nerve growth factors do not cross the BBB (Blood brain barrier) in the brain because of their molecular weight. Therefore, it is difficult to see the therapeutic effect in degenerative brain diseases. Therefore, the synthesis of endogenous nerve growth factors should be further induced, and further development of materials that play the same role as nerve growth factors is urgently needed.

Degenerative brain disease is a disease caused by the death of nerve cells in the brain, and includes various diseases such as dementia, Parkinson's disease, stroke, and Huntington's disease. In addition, in modern society, the increase of degenerative brain diseases such as senile dementia due to the rapid increase of the elderly population is a serious social problem, but until now, effective drugs or treatments for the prevention and treatment of the disease have been developed. I can't.

Dementia, a representative degenerative brain disease, is a brain disease that indicates a general cognitive impairment. It is usually caused by chronic or progressive brain disease, and many senior cerebral functions such as memory, thinking, understanding, calculation, learning, and language judgment are impaired.

However, the cause of this dementia is not known exactly. Some presumed causes include damage to choline neurons at the base of the cerebral base, reduction of neurotransmitters, accumulation of beta-amyloid proteins by inflammatory responses, and oxidative stress. Davies P., et al., Lancet, 21, 1403 (1976); Rocher, AE, et al., J. Biol. Chem., 273, 29719 (1988); Coyle, JT, et al., Science, 262 , 689 (1993). The most desirable treatment for dementia is to delay brain cell breakdown and aging to protect brain cells, restore cognitive function, and promote brain cell regeneration, but none of them have been developed to date.

Therefore, the present inventors studied to develop an effective drug for the prevention and treatment of dementia, safe, non-toxic and various pharmacological extracts of silkworms inhibit neuronal cell death and increase the synthesis of nerve growth factors. The present invention was completed by discovering that learning ability can be improved.

The silkworm is a dry feces, or dung, of the larvae of the silkworm moth and insect houseworm moth (Bombyx mori L.). It is mainly used to collect the dung of sleeping silkworms from June to August and dry it two or three times in the sun before sifting the soil. Jamsa tastes sweet and very warm. It also contains a lot of phosphorus, potassium and calcium, and contains 2% of chlorophyll. The drug is used for rheumatism, neuralgia, low back pain and paralysis due to paralysis because it smoothes blood circulation and removes wind and reduces pain. In addition, it is effective in diabetes, urticaria, conjunctivitis, uterine bleeding, pruritus, etc. (Park, Young-Jun, Oriental Animal Bogam, Blue Wave, 2000) tirocin), serine, aspartic acid, alanine, alanine, moracin, glucosidase inhibitor, chlorophyll a, b, mulberrofuran F. G, guanon, moracin, demoracin, phytol, It contains β-sistol, cholesterol, ergosterol and tetracosanol and lupeol, and β-cistol-β-glucoside has been isolated (Chinese Dictionary, 1990).

However, Korean Patent Nos. 10-0615532 (Aug. 17, 2006) and No. 10-0592489 of pharmaceutical compositions having sleep enhancement, analgesic alleviation, anticonvulsion, weight loss and hyperlipidemia, using up to now as an active ingredient (Jun. 15, 2006), etc., there have been no reports on the effects of preventing and treating cerebral neurological diseases and enhancing memory.

Accordingly, it is an object of the present invention to provide a composition capable of effectively preventing and treating cerebral neurological diseases.

Another object of the present invention is to provide a composition that can effectively enhance memory.

In accordance with the above object, the present invention provides a composition for the prevention and treatment of neurological diseases of the brain comprising an extract of nasa as an active ingredient with a pharmaceutically acceptable carrier.

According to another object of the present invention, there is provided a composition for enhancing memory, comprising an extract of silkworm together with a pharmaceutically acceptable carrier.

The silkworm extract and the herbal medicine-containing silkworm extract of the present invention induce the growth and differentiation of neurons and exhibit the effect of maintaining survival, thereby promoting neuronal axon growth and nerves which play an important role in cerebral neurological diseases, especially degenerative brain diseases. It can replace the role of cell growth factor and can be used as a nontoxic natural medicine.

Figures 1a and 1b shows the growth rate (NGF) secretion and cells of the silkworm extract (K1) obtained in Example 1 (K1), k2 (vaccin) and the herbal extract containing the herbal medicine (K3) obtained in Example 2 using C6 cells It is the result of evaluating survival rate.
Figures 2a and 2b is a result of evaluating the NO (Nitric oxide) production rate and cell viability of the silkworm extract (K1) and herbal medicine containing silkworm extract (K3) using primary cultured neuroglia cells (primary microglia cells).
Figure 3 is a result of propidium iodide (PI) assay of silkworm extract (K1) and herbal medicine containing silkworm extract (K3) using organotype hippocampal sections.
FIG. 4 shows the results of evaluating the memory-improving effect of the scopolamine-induced forgetfulness model in the passive avoidance test of the silkworm extract (K1) and herbal medicine containing silkworm extract (K3) using ICR mice. to be.
5 is a memory improvement effect on the forgetfulness model induced by scopolamine in the Y-maze test of the silkworm extract (K1) and herbal medicine containing silkworm extract (K3) using ICR mice (modification) Results of evaluating behavior (a) and entry frequency (b)).
FIG. 6 is a result of passive avoidance test of silkworm extract (K1) and herbal medicine containing silkworm extract (K3) using ICR mice, and the cognitive ability of the silkworm extract (K1) and herbal medicine containing silkworm extract (K3) was improved. The result of evaluating the effect.
7 is a result of the Y-maze test of the silkworm extract (K1) and herbal medicine containing silkworm extract (K3) using ICR mice, recognition of the silkworm extract (K1) and herbal medicine containing silkworm extract (K3) This is the result of evaluating ability improvement effects (change behavior (a) and entry frequency (b)).

The silkworms that can be used in the present invention include silkworm moths and feces of insect houseworm moths (Bombyx mori L.).

The silkworm extract of the present invention can be obtained by extracting the silkworm with water or an organic solvent, water is preferably distilled distilled water, the organic solvent is lower alcohol, acetone, chloroform, methylene chloride, ether, ethyl acetate, hexane Etc. can be illustrated. Lower alcohols include methanol, ethanol, propanol and butanol, with ethanol being most preferred.

At the time of extraction, silkworms can be used in the form of dry silkworms or silkworm powder. It is preferable to use a silkworm powder. More preferred.

Specifically, the extract may be prepared by ultrasonic extraction by adding 5 to 15 times, preferably 10 times, water to the silk yarn powder. The organic solvent extract of the silkworm may be prepared by adding 1 to 5 times the organic solvent to the silkworm powder, preferably 3 times the organic solvent, extracting at room temperature, and then filtrating the filtrate under reduced pressure. In the above extraction methods, the extraction process may be repeated two or more times, preferably three times as necessary. The extract obtained after filtration may be dried under reduced pressure to form a powder.

In addition, the composition of the present invention may further include other medicinal herbs or extracts thereof that are pharmaceutically acceptable to enhance the pharmacological effect. In this case, an extract of the herbal medicine may be prepared according to the extraction method and then added to the composition, or the extract obtained by extracting by the method after mixing the silkworm and the herbal medicine may be included in the composition.

The herbal medicine that may be added to the composition of the present invention may be any pharmaceutically acceptable herbal medicine, and may be used, for example, alone or in combination with silkworm, rhubarb, tea, gardenia, baekxin and earth and sand.

The composition of the present invention may comprise a silkworm extract in an amount of 0.1 to 100% by weight, preferably 1 to 70% by weight, most preferably 10 to 20% by weight of the total weight of the composition, the herbal extract is the total weight of the composition It can be added in an amount of 0 to 99.1% by weight, preferably 30 to 70% by weight of.

The silkworm extract of the present invention induces differentiation and growth of neurons and maintains the survival of neurons. This role is played by a nerve growth factor synthesized in the body, the nerve growth factor, such as NGF (Nerve growth factor), brain-derived neurotrophic factor (BDNF), nerve factor (NT-3; Neurotrophin-3), NT-4 and NT-5. The silkworm extract of the present invention may play a role of nerve growth factor. In addition, the silkworm extract of the present invention increases the synthesis of nerve growth factor protein and nerve growth factor receptor in the body.

Therefore, the silkworm extract of the present invention can be used as a therapeutic agent capable of treating neurological diseases related to the growth, differentiation and survival of neurons. In particular, as an agent for the treatment and prevention of cerebral neurological diseases caused by neuronal cell death, specifically, it exhibits excellent preventive and therapeutic effects against degenerative brain diseases such as stroke, Parkinson's disease, senile dementia or Alzheimer's disease.

In addition, the mice administered the composition of the present invention exhibits remarkable effects of learning and memory.

Despite the effectiveness as described above, the composition of the present invention containing a silkworm extract does not show any toxicity, such as acute toxicity in the test using rats and does not show side effects on liver function.

The pharmaceutical formulations can be prepared according to conventional methods using the compositions of the present invention. In the preparation of the formulation, it is preferred to mix or dilute the active silkworm extract with the carrier or to enclose it in a container form. When the carrier is used as a diluent, it may be a solid, semisolid or liquid substance which acts as a vehicle, excipient or medium for the active ingredient.

Thus, the formulations may be in the form of tablets, pills, powders, assays, elixirs, suspensions, emulsions, solutions, syrups, aerosols, soft or hard gelatin capsules, sterile injectables, sterile powders and the like. Examples of suitable carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, Propylhydroxybenzoate, talc, magnesium stearate and mineral oil. The formulation may further comprise fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives and the like. Compositions according to the invention can be formulated using methods well known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal.

The composition according to the invention can be administered via several routes including oral, transdermal, subcutaneous, intravenous or intramuscular. For humans, typical daily dosages can range from 5 to 500 mg / kg body weight, preferably 100 to 250 mg / kg body weight, and can be administered once or in divided doses. However, the actual dosage should be determined in light of several relevant factors such as the route of administration, the age, sex and weight of the patient, the state of health and the severity of the disease.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these Examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

In addition, in the examples below, the following percentages for solids and solid mixtures, liquids and liquids, and liquids and solids are based on weight / weight, volume / volume and weight / volume, respectively, and all reactions are carried out at room temperature unless otherwise noted. It was.

Example 1 Preparation of Silkworm Extract

40 g of dried silkworm feces was added to 500 ml of distilled distilled water 3 times, and ultrasonic extraction was repeated three times. The extract was filtered, concentrated under reduced pressure, and dried to give 2.427 g of silk extract (yield 6%).

Example 2: Preparation of Jasa Extract Containing Medicinal Herbs

Buy silkworm, rhubarb, tea, gardenia, baekboksin, earthenware, mix in a ratio of 1.5: 0.5: 1.5: 1: 1: 1 to make 39g, and then extract ultrasonic waves three times in the same manner as in Example 1. Repeatedly obtained 2.329 g of Nasa water extract containing herbal medicine (yield 6%).

Example 3: Preparation of a composition comprising a silkworm extract

After diluting the silk extracts and the crude extracts prepared in Examples 1 and 2 in PBS (Phosphate buffered saline pH 7.2) buffer solution to a final concentration of 1.0 mg / ㎖ using a disposable syringe Filtration with membrane filter paper (0.2 µm, millipore), aseptic and concentrated under reduced pressure to obtain a composition.

Test Example 1: Assay of nerve growth factor secretion of the silkworm extract and crude extract containing the herbal medicine

(Step 1)

C6 cells, a mouse brain neuron line, were treated with DMEM (10% fetal bovine serum (FBS; Hyclone, South Logan, UT, USA)) and 1% penicillin / streptomycin (GibcoBRL, Life Technologies Inc, Gaitherburg, MD, USA). Hyclone) medium was used as a culture medium and cultured at 37 ° C and 5% CO ₂ .

(Step 2)

C6 cells were incubated overnight at 37 ° C., 5% CO ₂ conditions with 1 ml DMEM medium in 24-well culture plates (1 × 10 ⁵ cells / well), then at various concentrations (0.01, 0.1 and 1 μg / ml). It was incubated for 24 hours by treating the silkworm extract and the crude extract containing the herbal medicine. After one day, the medium was collected and returned to the centrifuge at 7500 rpm, 4 ° C for 5 minutes, and the supernatant was separated and quantitated using a Rat β-NGF kit (R & D systems, Duoset, Raf β-NGF). It was.

The amount of β-NGF, a neuronal growth factor (NGF) generated in C6 cells, was measured using DuoSet (ELISA Development System R & D Systems). First, 72 μg / ml of capture mAb was diluted in 1 × phosphate buffer solution (PBS, pH 7.2-7.4) and put into 100 μl of each well of a 96-well plate (Nunc coating plate), followed by overnight reaction at room temperature. This was washed three times with 1x PBS (PBS-T) containing 0.05% Tween-20, and then added with a blocking solution containing 1% bovine serum albumin (BSA). The part was blocked. After removing the diluting solution (blocking solution), 100 μl of the standard protein for each NGF and various concentrations of the cell culture cultured with or without drugs were reacted at room temperature for 2 hours. This was washed 3 times with 1 × PBS-T and 100 μl of 100 ng / ml detection mAb was added and reacted at room temperature for 2 hours. After completion of the reaction, the plate was washed three times with 1 × PBS-T, and then strepavidin-HRP mAb was added and allowed to react at room temperature for 20 minutes. This was again washed 3 times with 1 × PBS-T solution, tetramethyl bensidine (TMB) substrate solution was added to the dark reaction at room temperature for 20 minutes. To terminate the reaction, 50 μl of 2N H ₂ SO ₄ solution was added and the absorbance was measured at 450 nm and 540 nm with a microplate reader. The concentration of each NGF was calculated based on the standard protein quantitative curve. At this time, the untreated group was used as a control.

(Step 3) MTT assay of silkworm extract and herbal extract containing herbal medicine

200 μl of 0.1 mg / ml MTT solution was added to each well of the 24-well culture plate collecting the drug-treated medium, and incubated for 1 hour to induce a reduction reaction. Then, the MTT solution was removed and 400 Formazan crystals were completely dissolved by adding μl of DMSO solution. 200 μl of this was put in a 96-well plate. The color development was measured by absorbance at 570 nm using a microplate reader. Cytotoxicity was calculated based on the relative cell viability (%) based on the 100% survival rate of the control cells cultured only cells without drug treatment. This result is shown in FIG. Figures 1a and b shows the growth rate (NGF) and the cell growth rate of the NSA extract (K1), k2 (vaccin) and the herbal extract containing the herbal medicine (K3) obtained in Example 1 using C6 cells It is the result of evaluating survival rate. As shown here, it can be seen that the silkworm extract containing the silkworm extract and the herbal medicine of the present invention has a higher rate of nerve growth factor secretion compared to the control group, and thus the cell survival rate is also excellent.

Test Example 2: Determination of nitric oxide of silkworm extract and herbal extract containing herbal medicine

(Step 1)

Primary cultured neuroglial cells (primary microglia cells) were tested using 1-day-old SD mice purchased from Orient Bio, Kyunggido, Korea. Briefly, minimal essential medium-α (MEM-α) added with 10% fetal bovine serum (FBS), 1% HEPES, 1% penicillin / streptomycin, 1% L-glutamine inactivated at 50 ° C. for 30 minutes. Only the cerebral cortex was separated and dropped into single cells with Pasteur pipettes, and then passed through a nylon mesh. Pass all through 37-C, 5% CO ₂ into 75-cm 2 tissue culture flasks Cultured under conditions.

(Step 2)

Lipopolysaccharise (LPS; E. coli) B0111: B4; The concentration of nitric oxide (NO), an active nitrogen species produced from microglia activated by Sigma), was detected using Griess reagent in the form of NO ₂ − present in the cell culture. After culturing the primary cultured microglia cells for 12 days, the neuroglial cells were isolated and cultured overnight in a 96-well plate at a concentration of 3 × 10 ⁴ cells / ml, and a variety of silkworm extracts and herbal medicine-containing silkworm extracts were prepared. The cells were incubated for 30 minutes and then treated with 1 μg / ml LPS for 24 hours to induce cell activation. The amount of NO generated from small neuroglial cells was collected from the cell culture medium, and then 100 μl of Gries reagent (0.1% NED & 1% sulfanilamide in 5% H ₃ PO ₄ ) was added to 100 μl of the culture medium, followed by cancer reaction for 15 minutes. Absorbance was measured at 570 nm using. The concentration of NO in the cell culture (μM) was calculated based on the quantitative curve of NaNO ₂ standard solution.

(Step 3) MTT assay of silkworm extract and herbal extract containing herbal medicine

Each well of the drug-treated 96-well culture plate was added with 1 mg / ml MTT solution twice the amount of the culture medium and incubated for 1 hour to induce a reduction reaction, and then remove the MTT solution. 100 μl of DMSO solution was added to completely dissolve the formazan crystals. The degree of color development was measured at 570 nm using a microplate reader. Cytotoxicity was calculated based on the relative cell viability (%) based on the 100% survival rate of the control cells cultured only cells without drug treatment.

(Step 4) Nitric Oxide Measurement

Lipopolysaccharide (LPS; E. coli B0111: B4; The concentration of nitric oxide (NO), an active nitrogen species produced from small neuroglial cells activated by Sigma), was detected using a Gries reagent in the form of NO ₂ − present in the cell culture. That is, small neuroglial cells were incubated overnight in 96-well plates at a concentration of 3 × 10 ⁴ cells / ml, treated with various concentrations of silkworm extracts and herbal medicine-containing silkworm extracts. Time incubation induced cell activation. The amount of NO generated from small neuroglial cells was collected from the cell culture medium, and then 100 μl of Gries reagent (0.1% NED & 1% sulfanilamide in 5% H ₃ PO ₄ ) was added to 100 μl of the culture medium, followed by cancer reaction for 15 minutes. Absorbance was measured at 570 nm using. The concentration of NO in the cell culture (μM) was calculated based on the quantitative curve of NaNO ₂ standard solution.

The results are shown in FIG. Figures 2a and 2b is a result of evaluating the NO (Nitric oxide) production rate and cell viability of the silkworm extract (K1) and herbal medicine containing silkworm extract (K3) using primary cultured neuroglia cells (primary microglia cells).

Test Example 3: Propidium iodide (PI) assay of silkworm extract and herbal extract

In order to see the effects of the drug, the nutrient medium of organotype hippocampal sections cultured for 2 weeks was changed to a nutrient-free medium. Subsequently, the drug treatment was treated with 100 μM aβ, aβ + nap extract, and herbal extract containing 10mg / ml and 1μg / ml. At this time, after pretreatment of the silkworm extract and herbal medicine-containing silkworm extract in each medium for about 1 hour, 100μM aβ treatment was observed at 72 hours. Observation was carried out by placing PI (5 μg / ml) in each medium, and after 1-2 hours, PI staining images were obtained by a digital CCD camera (Axiocam, Zeiss, Oberko, Germany) using a fluorescence inverted microscope (Axiovert S 100, Zeiss, Oberko, Germany)). Observed PI uptake areas were measured with a 'density slice' in an image analysis program (Scion image beta 4.02 win, Scion Co., Maryland, USA) and the rate at which neurons died in each subfield. Was calculated. Figure 3 is a result of propidium iodide (PI) assay of silkworm extract (K1) and herbal medicine containing silkworm extract (K3) using an organotype hippocampal section.

Experimental Example 4: Cognitive-improving effect of silkworm extract and herbal medicine containing silkworm extract in scopolamine-induced forgetfulness model

(Step 1)

Male ICR mice (6 weeks old, 25-28g) were supplied from Daehan Biolink Co., Ltd. (Chungbuk, Korea) and used for 5 days in a clean cage at Kyung Hee University College of Pharmacy. The feed (Dae Biolink, Chungbuk, Korea) was freely ingested, and the temperature (22 ± 2 ℃), humidity (53 ± 3%) and contrast cycle (12 hours) were automatically adjusted.

(Step 2)

Mice were divided into 8 groups, 10 of each group. Groups 1 and 2 (control, negative control) orally administered saline at 5 mL per kg body weight for 1 day, and group 3 (positive control) 1 mg / kg of donepezil dissolved in saline. It was orally administered for 1 day. Groups 4 and 5 were orally administered for 1 day orally for 20 days, 100 mg / kg of saline extract dissolved in saline, and groups 6, 7, and 8 were 50 and 100, respectively. , Orally administered in an amount of 200 mg / kg for 1 day. Oral administration once a day and 1 hour after the oral administration (passive avoidance test and Y-maze test (Y-maze test) was performed as follows.

Passive avoidance tests were performed on a test rig in which the internal structure was divided into two identical spaces. Thirty minutes after drug administration, 1 mg of scopolamine was dissolved in 5 ml of saline solution and intraperitoneally administered to the mouse at a dose of 1 mg / kg. In a bright room, the dark room was turned off and the dark room was turned off. After 10 seconds stay, the time until the guillotine door was opened to enter the dark room was measured. When the test animal moved to the dark space, the guillotine door was closed and the sole of the foot was given 0.7 mA of electrical stimulation for 3 seconds. After 24 hours, a manual avoidance test was conducted in the same manner, and the latency time was measured.

FIG. 4 shows the results of evaluating the memory-improving effect of the scopolamine-induced forgetfulness model in the passive avoidance test of the silkworm extract (K1) and herbal medicine containing silkworm extract (K3) using ICR mice. to be. As shown here, the control group exhibited 266.56 ± 14.17 seconds, and the retention time of the negative control group was statistically significantly reduced to 54.86 ㅁ 3.85 seconds compared to the memory maintained by the electric shock, resulting in memory loss. This decrease was increased by the administration of the silkworm extract and the herbal extract containing the herbal medicine. In particular, the 100mg / kg-administrated group of the Chinese herbal medicine containing silkworm extract showed 158.79 11 11.44 seconds, which improved memory by significantly increasing the retention time.

The Y-shaped labyrinth test was carried out in an experimental apparatus with three arms at intervals of 120 microseconds and each branch was 40 cm long, 12 cm high, and 3 cm apart. After 30 minutes of drug administration, dissolve 1 mg of scopolamine in 5 ml of saline solution and intraperitoneally administer it to the mouse at a dose of 1 mg / kg, and set each branch of the experimental device as A, B, and C. Place it on a branch and record its movement to another branch for 8 minutes. Only when the tail is completely entered, it is also recorded when the branch went back. The resulting value is measured as: spontaneous alternation (%) = {actual alternation / total arm entries-2} x 100.

5 is a memory improvement effect on the forgetfulness model induced by scopolamine in the Y-maze test of the silkworm extract (K1) and herbal medicine containing silkworm extract (K3) using ICR mice (modification) This is a result of evaluating behavior (a) and the number of times of entry (b). As shown here, the control behavior decreased statistically significantly (52.48 ± 2.79%) by the administration of scopolamine, whereas the control group exhibited 73.58 ± 2.04% alteration behavior. The decrease in the altered behavioral power was increased statistically by the administration of the herbal extract containing silkworm extract. Increasing the altered behavioral power means that the learning and memory were restored, while there was no change in the number of entry, indicating the total number of entry into each branch, indicating that the altered behavioral power was not caused by the change in the activity of the mouse.

Test Example 5: Cognitive-improving effect of silkworm extract and herbal extract containing herbal medicine

Mice were divided into 7 groups, 10 of each group. The first group (control) orally administered saline solution at 5 mL / kg of body weight for 7 days, and the second and third groups orally administered saline extract dissolved in physiological saline for 20 days and 100 mg / kg, respectively, for 7 days. It was. Groups 5, 6, and 7 were orally administered with narcotic extracts containing herbal medicines dissolved in saline in amounts of 50, 100, and 200 mg / kg for 7 days. The Y-maze test and the passive avoidance test were performed in the same manner except for the intraperitoneal administration of scopolamine in Test Example 4-1, 1 hour after oral administration and 1 hour after oral administration. It was.

6 is a result of the passive avoidance test of the silkworm extract (K1) and herbal medicine containing silkworm extract (K3) using ICR mice, improves the cognitive ability of the silkworm extract (K1) and herbal medicine containing silkworm extract (K3) The result of evaluating the effect. As shown here, in the passive avoidance test, the control group exhibited 116.44 ± 5.61 seconds to maintain memory due to electric shock and to increase the retention time statistically significant compared to the control group in all drug administration groups except the 100 mg / kg group of silkworm extracts. This improves memory. In particular, the 200 mg / kg administration group containing crude herb extracts 296.50 ± 42.50 seconds showed a memory effect of about 2 times the control group.

Figure 7 is a result of the Y-maze test (Y-maze test) of the silkworm extract (K1) and herbal medicine containing silkworm extract (K3) using ICR mice, of the silkworm extract (K1) and herbal medicine containing silkworm extract (K3) This is a result of evaluating cognitive ability improvement effects (change behavior (a) and entry frequency (b)). As shown here, in the Y-shaped maze test, the control group exhibited 73.58 ± 2.04% alteration behavior, and 20 mg / kg of the silkworm extract and 100 mg / kg of the herbal extract containing the herb extract were 62.74 ± 2.76 and 67.84 ± 2.13%, respectively. The altered behavioral power was statistically significant compared to the control group. On the other hand, there was no change in the number of entry, indicating the total number of entry into each branch, indicating that the altered behavior was not caused by the change in activity of the mouse.

Formulation Example

The compositions of the present invention can be formulated in a variety of formulation forms according to conventional methods, either alone or in combination with excipients used pharmaceutically. Formulation examples are shown below, but the present invention is not limited thereto.

Preparation Example 1 Preparation of Powder

2 g of silkworm extract

1 g lactose

The above components were mixed and packed in airtight bags to prepare powders.

≪ Formulation Example 2 > Preparation of tablet

100 mg of silkworm extract

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

The tablets were prepared by mixing the above components and tableting according to a conventional method for producing tablets.

Preparation Example 3 Preparation of Capsule

100 mg of silkworm extract

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above ingredients was filled into the gelatin capsules in accordance with the conventional method for producing a gelatin capsule to prepare a capsule.

Preparation Example 4 Preparation of Injection

100 mg of silkworm extract

Suitable amount of distilled water for injection

pH adjuster

Injectables were prepared by dissolving the active ingredient in distilled water for injection and adjusting the pH to about 7.5 according to a conventional method for preparing an injectable drug, and then filling the whole with a 2 ml ampoule with sterilized water for injection.

As described above, the silkworm extract and the herbal medicine-containing silkworm extract have the effect of inducing the growth and differentiation of neurons and maintaining survival, and thus can be usefully used as a memory enhancing agent and also as a memory enhancer for the prevention and treatment of cerebral neurological diseases, especially degenerative brain diseases. .

Claims (14)

A composition for preventing or treating cranial nerve disease, comprising a silkworm extract and a pharmaceutically acceptable carrier.
Memory enhancement composition comprising a silkworm extract and a pharmaceutically acceptable carrier.
The method of claim 1,
Cranial nerve disease is a degenerative brain disease, characterized in that the composition.
The method of claim 3, wherein
The degenerative brain disease is a stroke, Parkinson's disease, senile dementia or Alzheimer's composition.
The method according to claim 1 or 2,
Composition is characterized in that the silkworm moth or insect houseworm moth (Bombyx mori L.) feces.
The method according to claim 1 or 2,
The composition of the silkworm extract is extracting the silkworm with water or an organic solvent.
The method according to claim 1 or 2,
The composition of claim 1, wherein the silk extract is obtained by adding 5 to 15 times the third distilled distilled water to the silk powder and ultrasonically extracting three times.
The method according to claim 1 or 2,
A pharmaceutically acceptable herbal medicine or composition further comprising the same.
The method of claim 8,
The herbal medicine is a composition characterized in that at least one selected from the group consisting of rhubarb, chacha, gardenia, baeksinsin and earth and sand.
The method according to claim 1 or 2,
Composition comprising a silkworm extract in an amount of 0.1 to 100% by weight.
The method of claim 10,
Composition comprising a silkworm extract in an amount of 10 to 20% by weight.
The method of claim 8,
Method comprising the herbal extract in an amount of 0 to 99.1% by weight of the total weight of the composition.
The method according to claim 1 or 2,
Compositions in the form of tablets, pills, powders, assays, elixirs, suspensions, emulsions, solutions, syrups, aerosols, soft or hard gelatin capsules, sterile injectables or sterile powders.
The method according to claim 1 or 2,
The daily dosage of the silkworm extract is 5 mg to 500 mg per kg body weight.

Images