KR101359760B1 - The pharmaceutical composition comprising the extract of Astilbe chinesis for preventing and treating the degenerative brain disease - Google Patents

Abstract

The present invention provides a pharmaceutical composition for the prevention and treatment of degenerative brain diseases containing the roe deer urinary extract as an active ingredient. The roe deer urinary extract of the present invention has a neuroprotective effect on the nerve cells, and thus exhibits the effect of preventing and / or treating various degenerative brain diseases.

Description

The pharmaceutical composition comprising the extract of Astilbe chinesis for preventing and treating the degenerative brain disease}

The present invention relates to a pharmaceutical composition for preventing and treating degenerative brain disease containing roe deer urinary tract extract.

According to the National Statistical Office (NSO) released in October 2003, the proportion of the elderly population in Korea has reached 7.2% of the total population in 2000, It is expected to enter the aging society. Thus, as the aging problem becomes a social issue, the public's interest in the characteristics of the elderly population, the welfare of the elderly such as housing, health, culture, and leisure is increased, and the demand for statistics is also increasing. The key to this change is that chronic degenerative diseases are becoming more of a problem than acute communicable diseases, which have been the cause of death for the past 50 years due to the increase in the aging population. In particular, among chronic degenerative diseases, cerebrovascular death is a very important disease, which is the second most common cause of single-disease mortality.

These cerebrovascular diseases can be classified into two major types. One is hemorrhagic brain disease seen in cerebral hemorrhage, and the other is ischemic brain disease caused by cerebral vascular occlusion. Hemorrhagic brain disease is mainly caused by traffic accidents, and ischemic brain disease is a disease that is frequently seen in elderly people.

If transient cerebral ischemia is induced in the cerebrum, the supply of oxygen and glucose is blocked, resulting in ATP depression and edema in nerve cells, resulting in extensive damage of the brain. Neuronal death occurs after a considerable period of time following cerebral ischemia, which is called delayed neuronal death. In the transient forebrain ischemic model using the Mongolian gerbil, the neuronal cell death was observed in the CA1 region of the hippocampus 4 days after induction of cerebral ischemia (Kirino T, Sano K. Acta Neuropathol . , 62: 201-208, 1984; Kirino T. Brain Res. , 239: 57-69, 1982).

The most widely known mechanisms of neuronal death due to cerebral ischemia are two. One is the accumulation of excess glutamate outside the cell by cerebral ischemia. This glutamate enters the cell and eventually causes excessive accumulation of intracellular calcium, resulting in neuronal death (Kang TC, et al. . J. Neurocytol, 30: 945-955, 2001) and the other is ischemia-abrupt oxidation resistance due to the care that the supply of oxygen due to the increase in the bio-radical-induced wear damage to the DNA, and a cytoplasmic cell cider upon reperfusion ( Won MH, et al., Brain Res . , 836: 70-78, 1999; Sub AY., Chen YM., J. Biomed . Sci . , ≪ / RTI > 5: 401-414, 1998; Flowers F, Zimmerman JJ. New Horiz . 6: 169-180,1998).

Based on these meticulous studies, many studies have been conducted to search for substances that effectively inhibit neuronal cell death in cerebral ischemia, and to clarify the mechanism of the substance. However, there are few substances that effectively inhibit neuronal cell death due to cerebral ischemia.

Tissue plasminogen activator (Tissue plasminogen activator) is currently the only FDA-approved drug for treating cerebral ischemia, and it is a substance that induces rapid supply of oxygen and glucose by melting blood clots that cause cerebral ischemia. Therefore, it is not necessary to protect the nerve cells directly. Therefore, it is necessary to use it fast. Due to the characteristic of the thrombolytic agent, when used excessively or frequently, the blood vessel wall becomes thinner and eventually causes hemorrhagic cerebrovascular disease.

In addition, a clinical test was conducted in the case of MK-801, a calcium channel blocker for effectively inhibiting early influx of calcium, but the drug was discarded due to its side effects.

On the other hand, in Korea, many natural substances are marketed as health foods effective for stroke prevention, but most of them are not subjected to scientific validation, and they become social problems because they cause abuse of health food.

Therefore, there is an urgent need for efforts to develop natural materials that have long-term use and have proven their stability by objectively verifying the effects of treating and preventing brain diseases.

Astilbe chinesis is a plant belonging to the genus Rosaceae, distributed in Korea, Japan, Manchuria, China, Amur, and Usulli. The height is 30 ~ 70 cm, long brown hairs, and the rhizome is thick and shortly stretched laterally. Flowers bloom in July and August, reddish purple, and conical flowers hang at the end of the main stem and are about 30 cm long, with many flowers running, with short hairs. Other names of roe deer urine are called Geummo Samchil (金 毛 三七), Hong Seung-ma (紅 升麻), Homa (虎 麻), and Hongdu-woo (紅 頭牛). The part called red horse is used as medicine.

The outpost on the ground contains cyanide and the flowers contain quercetin. In general, it is known that a plant of the genus of roe deer contains 2-hydroxyphenylacetic acid, astilbic acid as a triterpenic acid, and astilbin as a flavonoid.

This pressurizing urine improves blood circulation, releases blood, releases heat, releases poisons, stops cramps, and suppresses pain. It is used for painful symptoms, bruises, joint pain, postoperative pain, and poisoning of snake bites (Korean Medicinal Plants, Kyohaksa, 2000, p204, Bae Hwan-hwan).

However, there have been no studies on the neuroprotective effect of roe deer urinary extract, especially on the neuroprotective effect using animal model.

An object of the present invention to provide a pharmaceutical composition for the prevention and treatment of degenerative brain disease containing roe deer urinary extract as an active ingredient.

An object of the present invention is to provide a functional food for preventing and improving degenerative brain disease containing roe deer urinary extract as an active ingredient.

The present invention provides a pharmaceutical composition for the prevention and treatment of degenerative brain diseases containing the roe deer urinary extract as an active ingredient.

The degenerative brain disease may be any one or more selected from the group consisting of stroke, stroke, dementia, Alzheimer's disease, Parkinson's disease, Huntington's chorea, Pick's disease, and Creutzfeld-Jacob's disease.

The roe deer urine may use the whole roe deer urine or a part consisting of flowers, fruits, leaves, stems and / or roots, root is preferred.

The extract may be extracted with water, alcohol or a mixture thereof as a solvent, the alcohol is preferably a lower alcohol of C1 ~ C4.

The present invention also provides a dietary supplement for the prevention and improvement of degenerative brain diseases containing the roe deer urinary extract as an active ingredient.

The roe deer urinary extract of the present invention has a neuroprotective effect on neurons, and has an effect of improving, preventing or treating degenerative brain diseases.

FIG. 1 shows the results of observing brain tissue sections of rats in a 4-vessel occlusion model, a control group administered with 5% DMSO, and an experimental group administered with the roe deer urinary extract of the present invention by cresyl violet staining. It is photograph to show.
Figure 2 is a graph comparing the number of normal brain cells in the normal group, the control group and the experimental group in the 4-vascular occlusion model.
3 is a photograph showing the results of observing the brain tissue sections of the control group administered 5% DMSO to the mouse in the photochemical embolic mouse model, and the experimental group to which the roe deer urine extract of the present invention was administered by TTC staining.
Figure 4 is a graph comparing the brain tissue damage rate (infarction rate) in the control group and the experimental group in the photochemical embolic mouse model.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for the prevention and treatment of degenerative brain diseases containing the roe deer urinary extract as an active ingredient.

Method for preparing roe deer urine extract

The roe deer urine extract is preferably prepared by a manufacturing method comprising the following steps, but is not limited thereto:

(1) crushing after drying the roe deer urine;

(2) extracting the extract by adding an extraction solvent to the crushed roe urine;

(3) cooling the extract and then filtering; And

(4) A step of concentrating the filtered extract under reduced pressure and drying.

The roe deer urine used in the step (1) can be used without limitation, such as grown or commercially available. In addition, the roe deer urine may use the whole roe deer urine or a part consisting of flowers, fruits, leaves, stems, and / or roots, the root is most preferred.

The extraction of step (2) may be performed by shaking extraction, Soxhlet extraction, or a reflux extraction method, but is not limited thereto. The extraction temperature is preferably 40 to 100 캜, more preferably 60 to 80 캜. In addition, the extraction time is preferably 2 to 24 hours, the extraction frequency is preferably 1 to 5 times, and more preferably extracted three times.

The extraction solvent used in step (2) may be water, alcohol or a mixture thereof. The alcohol is preferably a C1-C4 lower alcohol, and it is most preferable to use methanol or ethanol. In addition, it is preferable to extract by adding an extraction solvent of 5 to 15 times the weight or volume of the roe deer urine prepared in the step (1), more preferably by adding 10 times.

The reduced pressure concentration of step (4) is preferably using a vacuum pressure concentrator or vacuum rotary evaporator, and the drying is preferably using a reduced pressure drying, vacuum drying, boiling drying, spray drying or freeze drying method. However, the present invention is not limited thereto.

Pharmaceutical composition

The roe deer urine extract obtained by the above method was found to have superior neuroprotective effect against neuronal cell damage caused by cerebral ischemia through 4-vascular occlusion model (4-VO) and photochemical embolic mouse model (PTB).

Accordingly, the present invention may include a roe deer urinary tract extract having a neuroprotective effect as an active ingredient, thereby providing a pharmaceutical composition for preventing and treating degenerative brain diseases caused by neuronal cell death.

The degenerative brain disease may be any one or more selected from the group consisting of stroke, stroke, dementia, Alzheimer's disease, Parkinson's disease, Huntington's chorea, Pick's disease, and Creutzfeld-Jacob's disease.

The pharmaceutical composition containing the roe deer urinary extract of the present invention as an active ingredient is preferably included in an amount of 0.1 to 50% by weight based on the total weight of the composition, but is not limited thereto.

The composition of the present invention may be administered in various formulations, oral and parenteral, at the time of actual clinical administration. In the case of formulation, it may be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants which are usually used.

Solid preparations for oral administration include tablets, pills, powders, granules, and capsules, and the like, which may be used in the pharmaceutical compositions of the present invention at least one excipient such as starch, calcium carbonate, sucrose, It is prepared by mixing lactose and gelatin. In addition to simple excipients, lubricants such as magnesium, stearide, and talc may also be used.

Liquid preparations for oral administration include suspensions, solutions, emulsions and syrups. Various excipients such as wetting agents, sweeteners, fragrances and preservatives may be included in addition to water and liquid paraffin, which are commonly used simple diluents. have.

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories. Examples of the non-aqueous solvent and the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Witepsol, macrogol, tween 61, cacao paper, laurin, glycerol, gelatin and the like may be used as a base for suppositories. The pharmaceutical composition of the present invention can be administered by parenteral administration by subcutaneous injection, intravenous injection, or intramuscular injection.

The dosage of the pharmaceutical composition of the present invention varies depending on the condition and body weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, and can be appropriately selected by those skilled in the art. However, for the desired effect, the extract of the present invention is preferably administered at 0.0001 to 100 mg / kg per day. The administration may be carried out once a day or divided into several doses. However, the scope of the present invention is not limited to these dosages.

The composition of the present invention can be used alone or in combination with methods using surgery, hormone therapy, chemotherapy and biological response modifiers for the prevention and treatment of degenerative brain diseases.

Hereinafter, preferred embodiments and experimental examples are provided to facilitate understanding of the present invention. However, the following examples and experimental examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples and experimental examples.

< Example > Roe pee Astilbe chinensis ) Preparation of extract

100 g of roots of annual roe deer urine collected from Pocheon, Gyeonggi-do, were sliced, immersed in 1 L of 70% ethanol aqueous solution, shaken once for 3 hours at 80 ° C., and filtered through a filter paper. 6.44 g of dried roe deer urine extract was prepared by concentrating the extract under reduced pressure using a vacuum concentrator.

< Experimental Example  1> 4-vascular occlusion model (4- VO Of roe deer urinary extract Neuronal cell  Confirmation of protective effect

To measure the neuroprotective effects of roe deer urinary tract extracts against neuronal cell damage caused by cerebral ischemia, a 4-vessel occlusion model developed by Pulsinelli in 1979 was used (Pulsinelli WA, Brierley). JB, Stroke . , 10, 267-272, 1979)

As a test animal, male rats of 5 weeks old (Wistar, Samtako, Korea) male rats of about 170g were purchased and supplied with sufficient feed and water to adapt to the experimental environment. After one week of adaptation, the experiment was performed.

The rat was anesthetized with 5% isoflurane contained in a mixture of nitrogen and oxygen (70% nitrogen and 30% oxygen), and then the head was horizontally placed on a stereotaxic apparatus. Fixed at an angle of about 30 ° down. The nose and around the mouth were connected to the anesthesia with a plastic cone cone and kept anesthetized with 1.5% isoflurane.

The tail was fixed to the operating table and the cervical spine was extended. First, the incision of the neck of the rat was performed, and the cervical vertebra at the lower part of the occipital bone was treated with a surgical loupe to make sure that the muscle was not injured. A fine electrocautery of less than 1 mm was made through the alar foramina of the cervical vertebra Into the tunnel through which the vertebral artery passes, and the vertebral artery was cauterized by intermittent current conduction. Using a surgical microscope, the vertebral artery in the tunnel passing through the bone was completely cauterized and confirmed to be closed, and then closed with a surgical clip.

Then, in order to operate the throat area, a loop was made in the common carotid artery with a suture to induce ischemia and reperfusion. In order to block the circulation of the microvessel during ischemia, the cervical anterior chamber is located so that trachea, esophagus, external jugular vein and common carotid arteries are located behind. (cervical), paravertebral muscles were penetrated and then wound closed with surgical clips.

Twenty-four hours after the suture, the surgical clips were removed and the whole carotid artery was tightened with an aneurysm clip for 10 minutes to induce ischemia. If the light reflex is not lost within 1 minute, the cervical suture is made firm. At this time, the rats with no loss of the light reflection are completely unilateral CA1 nerves in the parallel hippocampus It was excluded because the damage was not induced, and the rats causing the seizures were also excluded. Ten minutes after induction of ischemia, the aneurysm clip was removed from the common carotid artery to stop ischemia and reperfusion. Immediately and 90 minutes after reperfusion, the roe deer urine extract prepared in <Example> was intraperitoneally injected at a concentration of 100 mg / kg in 5 ml DMSO at a volume of 0.1 ml per 10 g of rat weight, respectively. An equal volume of 5% DMSO was administered.

One week after induction of ischemia, rats were anesthetized by intraperitoneal administration of 1.2 g / kg of urethane, and then incisions were made in the right ventricle of the heart, and a 5.0% sodium nitrate saline solution treated with heparin was applied to the heart for 10 minutes. Perfusion and perfusion with 4.0% formalin fixative. The brains of the rats were then detached and post-fixed in formalin fixative buffered with 0.1 M phosphoric acid for 2 hours, then placed in 30% sucrose solution and left overnight. Coronal blocks in the dorsal hippocampus region between the bregma -2.5 mm and -4.0 mm in the fixed brain were dissected, frozen at -70 ° C, To prepare a tissue section containing hippocampus.

Sections prepared by the above method were stained and fixed in cresyl violet, and then the parts most likely to be damaged by delayed neuronal death among dorsal hippocampal CA1 (Crain BJ et al., Neuroscience , 27, 387-402, 1988) The number of damaged neurons present in the 1,000 μm portion of the middle zone was observed. The number of cells was observed by three observers in two brains, 6 left and right sides of three different tissue sections in one brain tissue, with the number of pyramidal cells showing normal morphology at high magnification (× 400). Values were averaged.

The brain hippocampal tissues of rats that did not cause cerebral ischemia in the normal group, the brain hippocampal tissues of the rats treated with 5% DMSO in the control group, and the brain hippocampal tissues of the rats administered the roe deer urinary extract of the present invention in the experimental group. Used.

As a result, as shown in Figure 1, the control group was able to observe the phenomenon that the cells are released from the surrounding cells as the tissue is relaxed, unlike the normal group, the cell body also (pyramidal) form of the original It was lost and condensed and found to be in the form of a single cell. In addition, it was confirmed that apoptosis was achieved by the concentration of nuclear chromatin and the collapse of the nuclear membrane.

On the other hand, the experimental group intraperitoneally administered 100 mg / kg of the roe deer urinary extract of the present invention showed a similar form to the neurons of the normal group, and is easily distinguished due to the protruding perikaryon and the central round nucleus. It became. In addition, it was confirmed that the majority of cells were protected from apoptosis to form a normal vertebral form, and continued to maintain junctions with surrounding cells.

In addition, as shown in the graph of Figure 2, the average number of cells per 1 mm 2 horizontal × length of the normal group was 378.00 ± 4.15 cells / mm ² , the control group was 74.00 ± 5.96 cells / mm ² to show neuronal cell death It could be confirmed that a lot happened. On the other hand, in the experimental group administered 100 mg / kg of roe deer urinary extract of the present invention it was confirmed that the cell number average is 190.00 ± 62.26 cell number / mm ² exhibited an excellent neuronal cell protective effect of 38.16% compared to the control group.

< Experimental Example  2> photochemical Embolization  Mouse model ( PTB Of roe deer urinary extract Neuronal cell  Confirmation of protection effect

An applied photochemical embolism model was used to measure the neuroprotective effect of roe deer urinary tract extract against nerve cell damage caused by focal cerebral ischemia (Lee, et al., Surgical Neurology , 67: 620-625, 2007).

Male C57BL / 6 (Samtaco, Korea) male mice, each weighing 22 g or less, were purchased from the experimental animals. After one week of adaptation, the experiment was performed.

The mice are anesthetized by intraperitoneal injection of 450 mg / kg chloral hydrate, and the skull is fixed to a stereotactic head fixation device (SG-4N, Narishige, Tokyo, Japan). Incision about 1.5 cm of the scalp at the site where the lesion is to be made, and the periosteum were removed to expose the skull, and then the center of the bregma was centered at the center of a 4 mm diameter fiber light source (CL 6000 LED; Carl Zeiss, Gottingen, Germany). Mark it with the oil pen at the mm point. Rose bengal (Sigma-Aldrich, St. Louis, Mo.), one of the dyes, was intraperitoneally injected at a concentration of 10 mg / ml for 0.1 ml per mouse. After 5 minutes, the illuminator was closely attached to the skull and irradiated for 15 minutes using an LED light source without heat generation. Immediately after induction, the roe deer urine extract prepared in <Example> was injected intraperitoneally with 5% DMSO at a volume of 0.1 ml per 10 g of mouse body, respectively, at a concentration of 200 mg / kg, and the control group was 5 % DMSO was administered. Reseal the skin and restore it naturally. Mice were sacrificed 24 hours after challenge and brains were excised within 2 minutes and 5 sections were obtained with a thickness of 2 mm. The tissues were sufficiently immersed in a plate containing 2% triphenyltetrazolium chloride (TTC) solution, left at 37 ° C. for 30 minutes, and fixed with 4% paraformaldehyde to observe the tissues. The stained brain tissues were taken one by one with a digital camera and the images were transferred to a computer. The volume of cerebral infarction was measured using an image analysis program (Axiovision Rel. 4.7).

As a result of measuring the protective effect of the brain tissue and nerve cells of the roe deer urine extract, as shown in Figure 3, the experimental group administered the roe deer urine extract of the present invention compared to the control group had a small area of the area not stained with TTC, It was found that the area of the dyed black-red site was large. For reference, when brain tissue is stained with TTC, the areas where tissues are killed due to damage are not stained and appear white, and the tissues of normal areas are dyed and appear black.

In addition, as shown in the graph of Figure 4, the control group administered with 5% DMSO showed a cerebral infarction volume of 60.49 ± 3.77 mm ³ , 40.27 ± 2.68 mm ³ in the experimental group administered 200 mg / kg of roe deer urine extract. The cerebral infarction volume of the experimental group was 33.4% lower than that of the control group, thereby confirming the superior neuronal protective effect of the roe deer urine extract.

< Manufacturing example  > Preparation of Pharmaceutical Formulations

1. Manufacturing of powder

Example of roe deer urinary extract 300mg

Lactose 100mg

Talc 10mg

The above components are mixed and filled in airtight bags to prepare powders.

2. Preparation of tablets

Example of roe deer urine extract 50mg

Corn starch 100 mg

Lactose 100mg

Magnesium stearate 2 mg

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

3. Preparation of Capsule

Example of roe deer urine extract 50mg

Corn starch 100 mg

Lactose 100mg

Magnesium stearate 2 mg

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare tablets.

4. Preparation of injections

Example of roe deer urine extract 50mg

Sterile sterilized water for injection

pH adjuster

According to the conventional method for preparing an injection, the amount of the above ingredient is prepared per ampoule (2 ml).

5. Manufacture of liquids

Example of roe deer urine extract 1,000 mg

Sugar 20g

20g per isomer

Lemon incense quantity

Purified water was added to adjust the total volume to 1,00 ml. According to the conventional method for preparing a liquid, the above components are mixed, and then filled into a brown bottle and sterilized to prepare a liquid.

Claims (4)

A pharmaceutical composition for the prevention and treatment of stroke, stroke, peak disease or Creutzfeld-Jacob disease, which contains Astilbe chinesis root extract as an active ingredient.
delete delete The pharmaceutical composition of claim 1, wherein the roe deer urine extract is extracted with an extraction solvent selected from water, C1 ~ C4 alcohol, or a mixture thereof.

Images