KR101805801B1 - Pharmaceutical composition for prevention or treatment of Parkinson's disease comprising tilianin - Google Patents

Abstract

The present invention relates to a pharmaceutical composition and a functional health food for preventing or treating Parkinsons disease, comprising a composite extract including Agastachis Herba containing tilianin, Paeonia lactiflora, Ligusticum chuanxiong, Angelica gigas, Bupleurum falcatum, Gardenia jasminoides, Paeonia suffruticosa, and Eugenia caryophyllata. The composition of the present invention alleviates MPTP-induced behavioral disorders, and restores a loss of dopaminergic neurons, and thus is effective in preventing, alleviating and treating Parkinsons disease.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a pharmaceutical composition for preventing or treating Parkinson's disease,

The present invention relates to the use of at least one of Paeonia lactiflora, Ligusticum chuanxiong, Angelica gigas, Bupleurum falcatum, Gardenia jasminoides, Paeonia suffruticosa, Agastachis Herba and Eugenia caryophyllata ), A pharmaceutical composition for preventing, ameliorating or treating Parkinson's disease, and a health functional food.

Parkinson disease (PD) is a common neuropathic disorder characterized by symptoms such as tremor, rigidity, bradykinesia, and postural instability (Parkinsonism IN: Merritt's neurology, 2000, Ed 10, 679-693). Parkinson's disease is also characterized by microgliosis, astrogliosis, progressive degeneration of dopaminergic neurons, presence of Lewy bodies in dopaminergic neurons, and substantia nigra pars compacta. (Neuron, 2003, 39, 889-909). In the present study, Although the use of drugs to alleviate the symptoms of Parkinson's disease is common, the chronic use of drugs results in side effects that weaken the mind and body (Neurology, 1991, 41, 202-205) It has not been developed yet. Although the exact cause of Parkinson 's disease is unknown, it is thought to be related to environmental toxins, genetic factors, and mitochondrial dysfunction.

As a model of Parkinson's disease, the neurotoxin 6-hydroxydopamine (6-OHDA) or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine phenyl-1,2,3,6-tetrahydropyridine, MPTP) are mainly used (Nat Rev Neurosci, 2001, 2 (5), 325-334). 6-OHDA is absorbed by the dopamine transporter (DAT) and produces free radicals. MPTP reduces dopaminergic neurons in humans and non-human primates, causes astrocytomas, and activates microglial cells in the cerebral substantia nigra (Nat Med, 1999, 5 (12), 1403-1409), leading to typical biochemical or pathological symptoms of Parkinson's disease (Neurosci, 2000, 16 (2), 135-142).

In traditional medicine Parkinson 's disease is also called shaking palsy because of trembling, numbness, numbness, and weakening of the limbs. According to the theory of traditional Asian medicine, the most important pathological features of Parkinson's disease are the stagnation of Qi and the stagnation of blood, the lack of liver-Yin and the lack of liver-Yin, And tile blood (Behav Pharmacol, 2006, 17 (5-6), 403-410).

As drugs for the treatment of Parkinson's disease, there are known l-dopa preparations, dopamine receptor agonists, anticholinergics, Eldepryl, and the like. However, most of these drugs are not causative treatments, but rather play a role in regulating symptoms, and therefore require constant, continuous drug use. The long-term administration of these drugs causes problems of drug side effects. For example, anticholinergic agents may have autonomic nervous system abnormalities or mental dysfunctions, which limits the continuous administration to older patients. In addition, in the case of the el-dopa preparation, the effects gradually deteriorate due to long-term use, side effects such as twisting of the body and abnormal movement in which the hands or feet move spontaneously occur. Other surgical treatments such as high frequency nerve stimulation, such as high frequency waves or deep brain stimulation, have also been performed, but they require invasive surgery and are costly.

On the other hand, there is a large amount of plant resources in China. More than 146,900 plant species have been found in China, of which more than 22,500 species have been identified as medicinal plants (Fitoterapia, 2013, 84, 273-285). Currently, medicinal plants capable of treating Parkinson's disease There is a need for an effective natural remedy.

Accordingly, the present inventors have made intensive efforts to develop a composition capable of preventing or treating Parkinson's disease. As a result, they have found that Paeonia lactiflora, Ligusticum chuanxiong, Angelica gigas, Bupleurum falcatum, Gardenia The present invention has been accomplished by confirming that a composition comprising a complex extract comprising jasminoides, Paeonia suffruticosa, Agastachis Herba, and Eugenia caryophyllata is effective for Parkinson's disease.

The present invention relates to the use of at least one of Paeonia lactiflora, Ligusticum chuanxiong, Angelica gigas, Bupleurum falcatum, Gardenia jasminoides, Paeonia suffruticosa, Agastachis Herba and Eugenia caryophyllata ). The present invention also provides a pharmaceutical composition for preventing or treating Parkinson's disease.

The present invention also relates to the use of a composition according to the invention for the preparation of a medicament for the treatment and / or prophylaxis of diseases of the genus Paeonia lactiflora, Ligusticum chuanxiong, Angelica gigas, Bupleurum falcatum, Gardenia jasminoides, Paeonia suffruticosa, Agastachis herba, Eugenia caryophyllata) for preventing or ameliorating Parkinson's disease.

In one aspect to accomplish the above object, the present invention provides a method for the treatment and / or prophylaxis of an infectious disease, such as Paeonia lactiflora, Ligusticum chuanxiong, Angelica gigas, Bupleurum falcatum, Gardenia jasminoides, Paeonia suffruticosa ), Agastachis Herba, and Eugenia caryophyllata. The present invention also provides a pharmaceutical composition for preventing or treating Parkinson's disease.

The term "peony" used in the present invention is a plant called "Paeonia lactiflora", which is a perennial dicotyledonous plant belonging to the family Ranunculaceae, and is distributed in Korea, Mongolia, and East Siberia. It is used for gardening because it is beautiful with flowers. It is used as medicine for pain, stomach pain, dysmenorrhea, amenorrhea, hematemesis, anemia, bruise and so on.

As used in the present invention, the term "Chengung" is a plant called "Ligusticum chuanxiong", a dicotyledonous plant belonging to the persimmon family. It is native to China and cultivated as a medicinal plant. It is effective for sedation, analgesia and tonic, and is used to treat headache, anemia, and women's diseases.

The term " Angelica gigas ", as used in the present invention, is referred to as the scientific name " Angelica gigas ", and the root of Angelica gigas is used in Korea. The efficacy of Angelica gigas is a blood-producing action that produces blood when blood is scarce. Pharmacologically, Angelica promotes the blood flow of the coronary arteries and stimulates red blood cell production.

The term "Seiho" used in the present invention refers to a plant with the scientific name of "Bupleurum falcatum". It is a perennial herbaceous perennial plant belonging to the family Acanthopanax melanogaster. The roots contain saponin and fatty oil, and they are used as herbal medicine for treating fever, pain, tonic, respiratory, digestive and circulatory diseases.

The term 'gardenia' used in the present invention is a plant which is called scientific name 'Gardenia jasminoides'. It is a green bamboo plant belonging to the macaques and has blood, blood, diuretic, tertiary, chelating and detoxifying effects.

As used herein, the term " Momchipi " is a root shell of pea (Paeonia Suffruticosa), which is harmless to human body which is used as a medicinal material in Oriental medicine. Anti-inflammatory, antimicrobial, and histamine-inhibiting action in mast cells.

The term "Gwakae" used in the present invention is a medicament made by using the ground part of Agastache rugosa. It is used only in Korea. It treats abdominal cavity, anorexia, nausea, vomiting and diarrhea. It is effective for cold with digestive disorder, vomiting due to summer diaper, diarrhea, halitosis, numbness and numbness.

The present invention may include tilianin as an active ingredient.

The 'tilianin' is a flavonoid having a structure represented by the following formula (1), and is one of the main components of the flavonoid, and inhibits accumulation of cholesterol in the body to improve arteriosclerosis.

[Chemical Formula 1]

As a result of HPLC analysis of the complex extract (CGT II) of the present invention, specific peaks were found in comparison with other complex extracts (CGT and CGT I), and further analysis revealed that the corresponding peak was tilylene Respectively. In addition, it was confirmed that the tilyanine was contained in the outline but not in the outline (Figs. 4 to 6).

The term 'clove' as used in the present invention refers to a plant of the scientific name 'Eugenia caryophyllata'. It is a tropical evergreen gourd, originating from the Moluka Islands and produced in Zanzibar Island in Tanzania, Sumatra Indonesia, Brazil and Malaysia. Vomiting, gastric cancer, abdominal pain, indigestion and increased sexual function, gingival inflammation and gum pain.

In the present invention, the commercially available products such as Gwak, Cheonjae, Cheonjae, Angelica gigas, Seicho, Rhizoma, Rhododendron, and Cloves can be purchased and used or cultivated or cultivated in nature.

The term "extract" used in the present invention means a substance obtained by separating from the above-mentioned Kwak, Kyojung, Gyunggung, Angelica gigas, Sephora, Rhizoma, Rhododendron or Clove, and may be a conventional fraction solvent known in the art, , Polar solvents such as ethanol and methanol such as C1-C4 alcohols such as methanol, ethanol and butanol, and nonpolar solvents such as hexane, ethyl acetate, chloroform and dichloromethane, or mixed solvents thereof have.

Specifically, the extract of the present invention may be prepared by extracting three or more kinds of the extracts of Ganoderma lucidum, Ganoderma lucidum, Ganoderma lucidum, Angelica gigas, Sephora, A lower alcohol having 1 to 4 carbon atoms or a mixed solvent thereof having a mixing ratio of about 1: 0.1 to 1: 10, preferably 1: 0.2 to 1: 3, at 20 to 100 DEG C, preferably 30 to 70 DEG C The extraction method such as hot water extraction, reflux cooling extraction, ultrasonic extraction, and the like, preferably the extraction method of the reflux cooling extraction, for about 1 hour to 2 days, preferably for 2 hours to 1 day at the extraction temperature of 1 to 5 times, Preferably 2 to 3 times, to obtain an extract. The extract is filtered under reduced pressure, and the filtered extract is mixed and concentrated under reduced pressure at 20 to 100 DEG C, preferably 50 to 70 DEG C with a rotary vacuum concentrator to obtain an organic solvent Water, and a solvent having a carbon number of 1 to 4 Crude extracts which are soluble extracts based on lower alcohols or mixed solvents thereof can be obtained.

In addition, the polar solvent-soluble extract may be prepared by adding 1 to 10 times, preferably about 1 to 5 times the volume of ethanol to the water-soluble fraction which has undergone the non-polar solvent-soluble fraction extraction process, 1 to 5 times, preferably 2 to 4 times Soluble fraction and water-soluble fraction can be obtained. The solvent fraction can be concentrated under reduced pressure using a rotary vacuum concentrator to remove the solvent to obtain the respective extracts.

Specifically, the extract of the present invention may be a water-soluble extract or an ethanol-soluble extract, more specifically, a 30% ethanol extract.

In one specific example, the extract according to the present invention is prepared by extracting 100 g of a mixture of Kwak, Kyojung, Taeungguk, Angelica gigas, Sephora, Rhizoma, Rhododendron and Clove at 100 ℃ with 30% ethanol using a reflux condenser, , Followed by concentration under reduced pressure and lyophilization to give 15.47 g of ethanol extract (CGT II, Example 1).

The term " Parkinson " disease used in the present invention is caused by the gradual disappearance of dopaminergic neurons distributed in the substantia nigra of the brain, and chronic progressive degeneration of the nervous system characterized by stable tremor, stiffness, ≪ / RTI >

As used herein, the term " prophylactic " means any action that inhibits or delays Parkinson's disease by administration of a composition comprising the CGT II. The term " treatment " used in the present invention means all the actions that improve or ameliorate symptoms of a disease upon administration of the composition containing CGT II.

The composition of the present invention may be characterized in that the effect of improving Parkinson's disease is enhanced by the addition of the extract and the clove extract.

In a specific experimental example, the CGT II demonstrated improved behavioral capacity and protection against dopaminergic melanoma cells compared to CGT and CGT I in MPTP-induced Parkinson's disease model mice (FIGS. 2 and 3).

Therefore, it has been found that the composition comprising the herbal mixed extract of the present invention or the fraction thereof as an active ingredient has antioxidative effect and protective effect on nerve cell, and thus can be effectively used for prevention and treatment of Parkinson's disease.

The composition of the present invention is administered in a pharmaceutically effective amount. The term " pharmaceutically effective amount " as used herein means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dosage level will vary depending on the species and severity, age, sex, The activity of the compound, the sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts. For example, the CGT II may be administered as an active ingredient at a dose of 0.01 to 500 mg / kg per day, preferably 10 to 100 mg / kg per day, and the administration may be administered once a day or divided into several times. In addition, the pharmaceutical composition of the present invention may contain the herbal mixed extract or the fraction thereof in an amount of 0.001 to 50% by weight based on the total weight of the composition.

The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And can be administered singly or multiply. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without adverse effect, and can be easily determined by those skilled in the art.

The pharmaceutical composition for preventing or treating Parkinson's disease of the present invention may contain a pharmaceutically acceptable carrier, excipient or diluent in addition to the above-described effective ingredient. Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The pharmaceutical compositions of the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols or the like, oral preparations, suppositories or sterilized injection solutions according to a conventional method . In detail, when formulating, it can be prepared by using diluents or excipients such as fillers, weighing agents, binders, humectants, disintegrants, surfactants and the like which are generally used. Solid formulations for oral administration include, but are not limited to, tablets, pills, powders, granules, capsules, and the like. Such a solid preparation may be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose, lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration, liquid paraffin, and various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like. Examples of the suppository base include withexol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The pharmaceutical composition of the present invention may be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) depending on the desired method, and the dose may be determined depending on the condition and the weight of the patient, The type of administration, the route of administration, and the time, but may be suitably selected by those skilled in the art.

In another aspect, the present invention provides a method of treating or preventing a disease selected from the group consisting of Paeonia lactiflora, Ligusticum chuanxiong, Angelica gigas, Bupleurum falcatum, Gardenia jasminoides, Paeonia suffruticosa, Agastachis herba ) And clove (Eugenia caryophyllata). The present invention also provides a health functional food for preventing or ameliorating Parkinson's disease.

The definitions of the extract and the Parkinson's disease are as described above.

The term " health functional food " used in the present invention means a food prepared and processed using raw materials or ingredients having useful functions in accordance with the Health Functional Food No. 6727, Means that the structure and function of the human body are ingested for the purpose of obtaining nutritional control effects or physiological effects and health effects.

The health functional food may contain flavoring agents such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and thickening agents (cheese, chocolate etc.), pectic acid and its salts, alginic acid and its salts, Organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. In addition, it may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The health functional food may be in the form of any one of meat, sausage, bread, chocolate, candy, confectionery, pizza, ramen, gum, ice cream, soup, beverage, tea, functional water, have.

In addition, the health functional food may further include food additives, and the suitability of the food functional food as a 'food additive' is not limited to those mentioned in the General Rules for Food Additives approved by the Food and Drug Administration, Standards and standards.

Examples of the above-mentioned 'food additives' include natural compounds such as ketones, glycine, potassium citrate, nicotinic acid, cinnamic acid and the like, sensory coloring matter, licorice extract, crystalline cellulose and guar gum, sodium L- A mixed preparation such as a preparation, a noodle-added alkali agent, a preservative preparation, a tar coloring agent, and the like.

The extract according to the present invention, which is added to foods containing beverages in the course of manufacturing the health functional food, can be appropriately added or decreased as needed.

In another embodiment, the present invention provides a method for the treatment of a disease selected from the group consisting of Paeonia lactiflora, Ligusticum chuanxiong, Angelica gigas, Bupleurum falcatum, Gardenia jasminoides, Paeonia suffruticosa, Agastachis Herba) and clove (Eugenia caryophyllata) to an individual other than a human. The present invention also provides a method for preventing or treating Parkinson's disease.

As used herein, the term "individual" may refer to all animals, including humans, who have or are at risk of developing Parkinson's disease. The animal may be, but is not limited to, a mammal such as a cow, a horse, a sheep, a pig, a goat, a camel, a nutrient, a dog, a cat,

The preventive or therapeutic method of the present invention may specifically include administering the composition in a pharmaceutically effective amount to a subject suffering from or at risk of developing Parkinson's disease.

The term "administering" as used herein refers to the introduction of a pharmaceutical composition of the present invention to a patient by any suitable method, and the route of administration of the composition of the present invention may be oral or parenteral May be administered via various routes.

The composition containing the tilianin of the present invention as an active ingredient has an excellent effect for preventing, improving and treating Parkinson's disease because it improves behavioral disturbance induced by MPTP and restores loss of dopaminergic neurons.

FIG. 1 is a graph showing the protective effect of nerve cell by administration of the complex extract in the nerve cell strain SH-SY5Y.
FIG. 2 is a graph showing behavioral analysis results for confirming the inhibitory effect of compound extract administration on behavioral capacity in an animal model of Parkinson's disease. FIG. a) Rotarod test, b) Rearing test, c) Open field test.
FIG. 3 shows the results of immunohistochemical staining of TH (tyrosine hydroxylase) in striatum (ST) and black substance (Substantia nigra, SN).
4 is a graph showing the results of HPLC analysis of the combined extract CGT, CGT I and CGT II.
5 is a graph showing the results of HPLC analysis for identifying specific peaks in CGT II.
FIG. 6 is a graph showing the results of HPLC analysis for confirming the contents of tilianin in the wastewater and optical waviness. FIG.
FIG. 7 is a graph showing the neuroprotective effect of wilting and tilianin in a Parkinson's disease cell model; a) neuroprotective effect, b) neuroprotective effect of tilylene.

Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited by the following examples.

Example  One. Wow  Added 30% ethanol complex extract ( CGT -II)

6: 4: 4: 3.2: 1.6: 6: 1.6: 6 in a ratio of 324 g, then put in a reflux extractor together with 10% of 30% ethanol, The mixture was heated to 95 캜 and further heated for 4 hours from the point when the bath liquid started to boil. After the heating was stopped and the mixture was cooled for 30 minutes, the extract was filtered under reduced pressure using two filter paper, and a powdery sample was prepared by using a freeze dryer.

Comparative Example  1. 30% ethanol composite extract ( CGT )

The mixture was mixed with 324g of 6: 4: 4: 3.2: 1.6: 1.6 at a ratio of 6: 4: 4: 3.2: 1.6: 1.6, and the mixture was heated at 95 ° C with boiling water From the starting point, the reaction mixture was further extracted with heating for 4 hours. After the heating was stopped and the mixture was cooled for 30 minutes, the extract was filtered under reduced pressure using two filter paper, and a powdery sample was prepared by using a freeze dryer.

Comparative Example  2. Photo Gallery  Added 30% ethanol complex extract ( CGT -I)

6: 4: 4: 3.2: 1.6: 6: 1.6: 6 at a ratio of 324 g, and then added to a reflux condenser at a rate of 95 ° C with 10% ethanol in 30% ethanol And further heated for 4 hours from the point when the bath liquid started to boil. After the heating was stopped and the mixture was cooled for 30 minutes, the extract was filtered under reduced pressure using two filter paper, and then a powdery sample was prepared using a freeze dryer.

Example  2. SH - SY5Y Nerve cell line  Ready

Human neuroblastoma SH-SY5Y cells were cultured in dulbecco's modified eagle's medium (Hyclone, Thermo, USA) containing 10% fetal bovine serum and antibiotic (Gibco-BRL, USA). The incubator maintained a temperature of 37 ° C, and gas containing 95% air and 5% CO 2 was continuously supplied to obtain appropriate conditions for cell culture. Cells were cultured 24 hours before the experiment to be 1x10 < ⁶ > in 96-well plate.

Example  3. Animals

12 week-old male C57BL / 6 mice (Central Lab. Animal Inc., Seoul, Republic of Korea) having a weight of 22-26 g were used. Animals were treated with a 12-hour, 12-hour night cycle of 23 ± 12 hours following NIH guidelines (NIH publication No. 85-23, revised 1985) and the Korean Academy of Medical Science (KHUASP (SE) RTI ID = 0.0 > 1 C. < / RTI >

For in vivo testing, mice were randomly divided into five groups: (1) control (n = 6; intraperitoneal saline injection), (2) MPTP (Sigma-Aldrich, St. Louis, (3) CGT (n = 5; intraperitoneal MPTP + CGT injection); (4) CGT I (n = 5; intraperitoneal MPTP + CGT I injection). (5) CGT II (n = 5; intraperitoneal MPTP + CGT II injection)

Experimental Example  1. Nerve cell line SH - From SY5Y  The 6- Hydroxydopamine  Analysis of cytoprotective activity by toxicity

In order to confirm the effect of protecting 6-hydroxydopamine-treated nerve cell damage, MTT reduction assay was performed using the SH-SY5Y cell line of Example 2. DMSO, in which each of the complex extracts (CGT, CGT I, CGT II) was dissolved in a 96-well plate in which the SH-SY5Y cell line was cultured, was treated for 30 minutes and then treated with 200 μM of 6-hydroxydodamine (6-OHDA). After 24 hours of 6-OHDA treatment, the MTT solution was added to each well such that the final concentration of the 96-well plate was 0.5 mg / ml. After incubation for 2 hours in the incubator, the media and MTT solution were removed, and DMSO was added and stirred. When completely dissolved, the UV absorbance was measured at 540 nm using a microplate reader (Molecular device, USA).

Cell viability was calculated using the following equation (1).

[Equation 1]

Cell survival rate (%) = [(control OD.- test group OD) / control group OD] 100

As a result, CGT II showed the best cell viability at each concentration as compared to CGT or CGT I in the presence of the neurotoxin 6-OHDA, as shown in Fig. Thus, it was confirmed that the composition of the present invention has nerve cell protecting activity in vitro.

Experimental Example  2. MPTP  Improvement of Behavioral Capacity of Combined Extracts in Animal Model of Induced Parkinson's Disease

Generally, within minutes of MPTP injection, dopamine is depleted and symptoms of severe Parkinsonism develop, including rigidity, convulsions, and bradykinesia. First, the following experiment was conducted to test the exercise functionality.

2-1. rotarod  test

This test measures the time it takes to run on a rounded bar, measuring the time the mouse ran on the rod and fell down. The total measurement time was measured at 480 seconds, and the speed at which the rod was rotated was set to increase gradually from 0 to 35 rpm

2-2. rearing test

Experimental animals were placed in a cylindrical plastic cylinder with a height of 20 cm and a diameter of 12 cm, and the camera was installed and taken for 5 minutes. The number of times that both the cylinder wall surface and both front feet contacted was recorded.

2-3. 개방 필드 시험

The open flied test was an experimental method to evaluate the walking ability of experimental animals. The number of times the experimental animals passed through the central area of the activity cage was measured.

The activity cage standard was 40 × 40 × 30 cm ², using a white color cage. On the activity cage, the free movement of the experimental animal was photographed through a camera for 5 minutes, and the moving distance was quantified by analyzing the captured image with the program. After the evaluation, the activity cage was wiped with 70% ethyl alcohol, dried sufficiently, and then the following experimental animals were evaluated.

As shown in FIG. 2, the behavioral tests of the MPTP group showed a decrease in the behavioral ability of the three groups of behavioral tests compared to the normal group. The CGT-I and CGT-II-treated groups showed rotarod test and rearing test showed significant recovery of behavioral ability. In particular, CGT-II showed significant differences in all behavioral tests compared to the control group, and showed the best restoration of behavioral capacity compared to CGT and CGT-I, confirming the therapeutic effect of CGT-II on Parkinson's disease.

Experimental Example  3. MPTP  Blackness of an animal model of induced Parkinson's disease From the striatum  Protective Effect of Compound Extract on Dopaminergic Cells

After the last MPTP injection in Example 3, the mice were sacrificed on the seventh day and cold 4% paraformaldehyde (PFA) in 0.2 M phosphate buffer was sprayed transcardially. Each brain was detached, fixed with 4% PFA at 4 ° C all day, and immersed in 30% sucrose solution to prevent freezing. Frozen brains were cut into 40 μm sections using a frozen flaky laminator (CM1850; Leica, Germany) and stored at 4 ° C in a cryorotectant (30% ethylene glycol, 30% glycol, 0.02M PB) Respectively. In immunohistochemistry, brain slices were washed with 0.05 M PBS containing 3% H2O2 and blocked with 1% BSA and standard goat serum. TH (1: 1000; Santa Cruz Biotechnology, CA, USA) was added to rabbit anti-tyrosine hydroxylase and incubated at room temperature overnight. Brain tissues were incubated with anti-rabbit IgG (Vector Laboratories Inc., CA, USA) with biotin attached for 1 hour, and finally incubated for 1 hour at room temperature with ABC reagent Vector Laboratories Inc., CA, USA). Peroxidase activity was incubated in 1 M TBS (tris-buffered saline, pH 7.5) for 2 minutes in a solution containing 0.02% diaminobenzidine and 0.003% hydrogen peroxide . The slices were placed on gelatin coated slides, dried to remove moisture, and then covered with a coverslip. Photographs of striatum and substantia nigra were taken using a bright field microscope (BX51; Olympus, Japan). The TH-immunopositivity in striatum was measured using Image Pro version 6.0 for Window (Media Cybernetics Inc., MD, USA). The number of TH-positive cells in the SN was counted.

As a result, as shown in Fig. 3, when the optical activity of TH in the striatum was compared with the control group, administration of MPTP caused dramatic degeneration of dopaminergic fibers. However, the administration of CGT II was found to protect the loss of fiber induced by MPTP, which was more prominent compared to other compound extracts (CGT and CGT I).

Compared with the control group, the black body was also able to identify neurons damaged by administration of MPTP, but it was confirmed that administration of CGT II protects neuronal cell death from neurotoxicity.

Taken together, CGT II inhibits dopaminergic neuronal loss in black and striatum, indicating the therapeutic effect of Parkinson's disease.

Experimental Example  4. Complex extract CGT , CGT -I, CGT Identification of constituents of -II

4-1. Complex extract Test solution  Produce

Specifically, 0.1 g of the 30% ethanol complex extract prepared in the same manner as in Example 1 and Comparative Examples 1 and 2 was placed in a volumetric flask, and 9 mL of 100% methanol was added thereto, followed by ultrasonic extraction for 60 minutes. After sonication, 10 mL of 100% methanol was added, mixed well with a magnetic stirrer for 60 minutes, and filtered through a 0.2 μm PVDF (polyvinylidene difluoride) filter. Prepare this solution as Test Solution 1, add 70% ethanol instead of 100% methanol in the same manner, and use the obtained reagent as Test Solution 2.

4-2. Ingredients of complex extracts HPLC  Pattern analysis

The components of the combined extracts extracted with 30% ethanol were subjected to HPLC (high performance liquid chromatography).

The standard substances used for the qualitative analysis of the combined extracts are Geniposide (gardenia), Paeoniflorin (peony), Tilianin (wahoo), Paonol Eugenol (clove), Saikosaponin A (Siho), Ligustilide (Tungkung) and Decursin (Angelica). The standard stock solutions were prepared by dissolving the standard stock solution in methanol from Geniposide to Decursin except for tilianin and then adding 0.15 mg / mL, 0.15 mg / mL, 0.1 mg / mL, 0.25 mg / mL, 0.1 mg / mL, 0.5 mg / mL, and 0.15 mg / mL, respectively. Tyllian was dissolved in 70% ethanol, sonicated for 60 minutes and dissolved well. The final concentration was 0.05 mg / mL.

The sample was separated into YMC Pack Pro C18 (250 × 4.6 mm, 5 μm) (Kyoto, Japan) at 30 ° C. The detection wavelengths are UV 210 nm (psychosaponin A, ligustilide) and 230 nm (geniposide, loniflorin, tilianin, faanol, eugenol, decurine). The mobile phase was composed of (A) acetonitrile and (B) water under the gradient condition shown in Table 1, and the flow rate was 1.0 mL / min.

As shown in FIG. 4, specific peaks were detected at a retention time (RT) of 31 minutes in 30% ethanol composite extract (CGT-II) differentiated from other complex extracts (CGT and CGT-I). The spectrum of the CGT-II mixed extracts was confirmed by PDA (Photodiode Array) detector. No specific peaks were found in CGT and CGT-I mixed extracts.

Experimental Example  5. Combined Extract ( CGT -II) specific Peak Derived herbal medicine  Confirm

5-1. Complex extract ( CGT -II) and a single herbal extract ( Wow ) Test solution  Produce

(CGT-II) and a 30% ethanol single herbal extract (Wako) prepared by the same extraction method as in Example 1 were prepared according to the preparation method of Test Solution 2 of Experimental Example 4-1.

5-2. Complex extract ( CGT -II) and a single herbal extract ( Wow ) Component HPLC  analysis

HPLC (high-performance liquid chromatography) was performed to confirm the origin of the specific peaks detected in the 30% ethanol composite extract (CGT-II).

In the same manner as in Experimental Example 4-2, tyllian was dissolved in 70% ethanol and dissolved to a final concentration of 0.05 mg / mL. The 30% ethanol combined extract (CGT -II) and 30% ethanol single herbal extracts (Wako) were also analyzed.

As a result, as shown in FIG. 5, it was confirmed that the RT of the specific Peak detected in the complex extract (CGT-II) was the same in Tilianin and 30% ethanol single herbal extracts.

In addition, the spectra were examined by a PDA (Photodiode Array) detector. As a result, it was confirmed that the specific peak of the 30% ethanol complex extract (CGT-II) was a tilianin component derived from the wastewater.

Experimental Example  6. In the optics Tilianin  Confirmation of content

6-1. 30% ethanol complex extract ( CGT -II) and a single herbal extract ( Wow , Photo Gallery ) Preparation of test liquid

Specifically, the 30% ethanol composite extract (CGT-II) and 0.1 g of the herbal extract prepared in the same manner as in Example 1 were placed in a volumetric flask, and 9 mL of 70% ethanol was added thereto, followed by sonication for 60 minutes. After sonication, 10 mL of 70% ethanol was added, mixed well with a magnetic stirrer for 60 minutes, and filtered through a 0.2-μm PVDF (polyvinylidene difluoride) filter. This solution is used as the sample solution.

6-2. 30% ethanol complex extract ( CGT -II) and a single herbal extract ( Wow , Photo Gallery ) Component HPLC  analysis

HPLC (high-performance liquid chromatography) was performed to confirm the presence or absence of tilyanine contained in the 30% ethanol composite extract (CGT-II) from the optical spectrum.

In the same manner as in Experimental Example 4-2, tyllian was dissolved in 70% ethanol and dissolved to a final concentration of 0.05 mg / mL to prepare a standard solution. The 30% ethanol mixed extract (CGT -II) and 30% ethanol single herbal extracts (Kwakyeong, Kwangwoo) were analyzed together.

As a result, as shown in FIG. 6, tilyanine detected in 30% ethanol composite extract (CGT-II) and 30% ethanol single herbal extract (Wako) was not found in 30% ethanol single herbal medicine extract.

Experimental Example  7. In the Parkinson's cell model Wow  And Tillian's  Neuroprotective effect

The MT-reduction assay was performed using SH-SY5Y cell line of Example 2 in order to confirm the effect of protecting the neuronal cell damage by the 6-hydroxydopamine treatment (30% ethanol) and tilianin. (6-OHDA) was treated with DMSO alone or 30 minutes after dissolving the extracts and the tilianin in a 96-well plate in which the SH-SY5Y cell line had been cultured. After 24 hours of 6-OHDA treatment, the MTT solution was added to each well such that the final concentration of the 96-well plate was 0.5 mg / ml. After incubation for 2 hours in the incubator, the media and MTT solution were removed, and DMSO was added and stirred. When completely dissolved, the UV absorbance was measured at 540 nm using a microplate reader (Molecular device, USA).

As a result, as shown in Fig. 7, the cell survival rate was the best in the presence of the neurotoxin, 6-OHDA, at a concentration of 50 ug / ml and a concentration of tilianine of 1 uM. Thus, it was confirmed that the active ingredient of the composition of the present invention had neuronal cell protection activity in vitro in the presence of tilylene.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

Claims (9)

It consists of Paeonia lactiflora, Ligusticum chuanxiong, Angelica gigas, Bupleurum falcatum, Gardenia jasminoides, Paeonia suffruticosa, Agastachis Herba and Eugenia caryophyllata. Or a pharmaceutically acceptable salt or solvate thereof, for the manufacture of a medicament for preventing, ameliorating or treating Parkinson's disease.
2. The pharmaceutical composition according to claim 1, wherein the wrinkle comprises tiliania as an active ingredient.
The method of claim 1, Wherein the composition is characterized in that the effect of treating Parkinson's disease is improved by the addition of the extract and the clove extract.
The pharmaceutical composition according to claim 1, wherein the extract is extracted with water, a C ₁ -C ₄ alcohol or a mixed solvent thereof.
It consists of Paeonia lactiflora, Ligusticum chuanxiong, Angelica gigas, Bupleurum falcatum, Gardenia jasminoides, Paeonia suffruticosa, Agastachis Herba and Eugenia caryophyllata. Or a pharmaceutically acceptable salt thereof.
[Claim 6] The health functional food according to claim 5, wherein the wrinkle contains tiliania as an active ingredient.
The method of claim 5, Wherein the health functional food is characterized in that the effect of improving Parkinson's disease is improved by the addition of the Wako and clove extracts.
The health functional food according to claim 5, wherein the extract is extracted with water, a C ₁ -C ₄ alcohol or a mixed solvent thereof.
It consists of Paeonia lactiflora, Ligusticum chuanxiong, Angelica gigas, Bupleurum falcatum, Gardenia jasminoides, Paeonia suffruticosa, Agastachis Herba and Eugenia caryophyllata. And administering the combined extract to a subject other than a human.

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