KR20080114077A - Composition for preventing and treating cerebrovascular disease comprising a compound isolated from alpinia officinarum hance - Google Patents

Abstract

A composition for preventing and treating cerebrovascular disease, containing a compound isolated from alpinia officinarum hance is provided to be used as prevention and therapeutic agent for brain disorders by controlling the neural apoptosis at the ischemic area. A composition for preventing and treating cerebrovascular disease comprises a compound of the chemical formula 1 and a compound of the chemical formula 2 as an active ingredient. The compound is separated from the chloroform, ethylacetate or acetone fractionation of alpinia officinarum hance.

Description

Composition for preventing and treating cerebrovascular disease comprising a compound isolated from Alpinia officinarum Hance}

1 shows a solvent fraction of a mild steel.

2 shows the effect of the methanol fraction of the Yanggang on LPS induced PGE ₂ production.

Figure 3 shows the effect of the methanol fraction of the Yanggang on COX-2 production.

Figure 4 shows the effect of the solvent fraction of the Yanggang on LPS-induced NO production, it shows Methanol (M), hexane (Hx), chloroform (Hx), ethylacetate (EA) in the figure.

Figure 5 shows the effect of the solvent fraction of the Yanggang on the production of LPS-induced PGE ₂ , in the figure shows Methanol (M), hexane (Hx), chloroform (Hx), ethylacetate (EA).

Figure 6 shows the effect of chloroform fraction of the Yanggang on LPS-induced PGE ₂ production.

Figure 7 shows the effect of chloroform fraction of the Yanggang on LPS induced COX activity.

8 shows the effect of chloroform fraction of the Yanggang on COX-2 activity.

Figure 9 shows the effect of chloroform fraction of Yanggang on COX-1 activity.

Figure 10 shows the effect of chloroform fraction of the Yanggang on LPS induced COX-2 expression, in Figure A) RT-PCR: 1: control; 2: LPS treated group; 3: LPS + NS398; 4: LPS + AOC (10 ug / ml); 5: AOC (1 ug / ml); 6: AOC (0.1 ug / ml)

B) Western blot 1: 1: control; 2: LPS; 3: LPS + AOC (10 ug / ml); 4: AOC (1 ug / ml); 5: AOC (0.1 ug / ml); 6: LPS + dexamethasone is shown respectively.

11 shows the active material isolated from the Yanggang.

FIG. 12 shows the effects of AO-A and AO-B on iNOS protein (top) and mRNA (bottom) isolated from Yang Yang.

FIG. 13 shows the effect of Compound AO-A isolated from Yang Yang on LPS-induced PGE ₂ production.

14 is In in vitro Represents a BBB Model.

Figure 15 shows the morphology of bEnd.3 cells and HUVEC cells under phase contrast microscopy and co-cultured with mouse primary cultured astrocytes.

16 shows changes in TEER in several in vitro BBB models.

17 is the chloroform fraction of the Yanggang in Influence on TEER change in vitro BBB Model.

 Figure 18 shows the effect of chloroform fraction of the Yanggang on leukocyte-vascular endothelial cell adhesion by inflammatory stimulation.

19 shows the effect of the chloroform fraction of the Yanggang on the expression of adhesion molecules of vascular endothelial cells.

20 shows the effect of the methanol fraction of the Yanggang on matrix metalloproteinase (MMP) activity.

The present invention relates to a composition for the prevention and treatment of brain diseases associated with encephalitis or cerebrovascular inflammation, and more particularly, to a composition for the prevention and treatment of brain diseases comprising a compound isolated from the Yang Yang.

According to the latest statistics, the mortality rate from cerebrovascular disease reaches 73.8 per 100,000, leading the cause of death among all Koreans, and death from cerebrovascular disease increases every year. Even if the death of cerebrovascular disease does not lead to death, the accompanying disorder is severe, resulting in serious loss of life as well as significant social and economic losses. The incidence of cerebrovascular disease increases with age, and considering the current situation in Korea, which is rapidly transitioning to an aging society, the prevention or treatment of cerebrovascular disease can contribute to the national economy as well as to the national economy. Could be.

The domestic market for brain disease treatments is known to be 100 billion won per year for stroke (infarction) and more than 20 billion per year for degenerative brain disease. In addition, as of 2000, an estimated 41 million stroke patients and 37 million Alzheimer's disease patients in the world (WHO survey report) can generate enormous economic added value.

The treatment of stroke is a requirement for the development of drugs that can effectively control neuronal cell death in the ischemic area, but despite the efforts of leading research institutes around the world, the development of drugs with proven efficacy has not been made yet. Drugs used for the treatment and prophylaxis of stroke include hypertension, cerebral blood flow improver, and hyperlipidemia. However, after stroke, except for thrombolytics, appropriate treatments that can protect against brain injury caused by ischemia are currently available. Almost none. Recent research trends in drug development to control brain damage include a multifaceted approach to developing new therapeutics that can block or inhibit irreversible damage based on neuronal cell damage caused by cerebral ischemia. Due to the complexity of the mechanisms involved in damage, studies on various mechanisms of action rather than specific mechanisms of action are being conducted. Attempts to mitigate brain damage by regulating encephalitis have been actively conducted by several researchers.

Encephalitis reactions occur with injury to nerve cells by bruises and strokes, and are one of the pathological characteristics of degenerative and ischemic brain diseases. Encephalitis reactions are also known as risk factors for neurodegenerative diseases, and are known to increase neuronal damage in brain injury and to involve secondary brain damage after stroke. Encephalitis responses include increased expression of cell adhesion molecules of vascular endothelial cells and leukocytes, intracellular brain migration, changes in brain-vascular barrier function, increased innate immune response, and increased apoptosis-induced responses. Therefore, encephalitis response is an important factor that causes brain and cerebrovascular dysfunction in various brain diseases, and elucidation of mechanism and regulation principle of encephalopathy response can provide core technology to protect brain from various brain diseases. There will be.

Drugs used for the treatment and prophylaxis of stroke include hypertension, cerebral blood flow improver, and hyperlipidemia. However, after stroke, except for thrombolytics, appropriate treatments that can protect against brain injury caused by ischemia are currently available. Almost none. Stroke treatments currently used in the clinic include tPA and thrombolytic agents, as well as drugs of various mechanisms such as platelet inhibitors, anticoagulants, cerebrovascular agents, calcium blockers, and brain edema inhibitors. Recently, a multifaceted approach has been attempted to develop new therapeutic agents that can block or inhibit irreversible damage based on neuronal cell damage caused by cerebral ischemia. In addition, glutamate receptor antagonists such as NMDA and AMPA, Ca ⁺⁺ -channel blocker, Na ⁺ -channel blocker, free radical scavenger, calpain inhibitor, NOS inhibitor, COX-2 inhibitor, etc. are the main research targets. In other words, due to the complexity of the mechanisms associated with brain injury due to stroke, studies on various mechanisms of action are being conducted rather than specific mechanisms of action.

Attempts to mitigate brain damage by regulating encephalitis have been actively conducted by several researchers. The mechanism by which the inflammatory response contributes to cerebral ischemia is not yet fully understood, but the most convincing hypothesis is the decrease in blood flow rate due to adhesion of leukocytes to the cerebrovascular wall and the secretion of toxic inflammatory factors from damaged or dying cells. It has been presented as. Recent studies have shown that increased expression of inflammation-related enzymes, such as iNOS and COX-2, is a very important mechanism in the development of ischemic brain injury, and through their control, there is a clear improvement in ischemic brain injury, at least in experimental animals. At the level, it has been reported to be obtainable. In addition, intracellular adhesion of leukocytes and infiltration into brain tissue during ischemia is also an important mechanism that causes tissue damage. Early treatment of antibody to ICAM-1 among cell adhesion molecules has been performed. Nevertheless, most clinical trials have resulted in failure. This seems to be due to the reason that the applied antibody is a heterologous protein. Therefore, in order to utilize the usefulness of this approach, appropriate countermeasures such as the development of human antibodies or the application of small molecules should be taken. Vascular or cellular cerebral edema may also be caused by impaired function of the brain-brain barrier, which is associated with various brain injuries, which may be a direct cause of brain loss or death, thereby normalizing the function of the cerebrovascular barrier. This is also an important research direction.

Considering the fact that most patients are treated only 6 hours after the onset of the ischemic brain injury through the control of the inflammatory response, the therapeutic window is considered to be very significant in that the therapeutic window can be extended. If thrombolysis is used in combination with these treatments, the usefulness of early reperfusion can be increased and overall better treatment results can be expected.

The present invention has been made in view of the above necessity, and an object of the present invention is to provide an agent for preventing and treating brain diseases.

In order to achieve the above object, the present invention provides a composition for the prevention and / or treatment of brain diseases comprising the compound of formula 1 or a compound of formula 2 as an active ingredient.

                       [Formula 1]

                       [Formula 2]

It is preferable that the compound of the present invention is separated from the Yanggang, chloroform, ethyl acetate, or acetone of Yanggang Most preferably separated from fractions.

In the present invention, the brain disease is characterized by various brain diseases accompanied by encephalitis and cerebrovascular inflammation, the brain disease is stroke, Parkinson's disease, Alzheimer's disease, brain dysfunction due to traumatic injury, brain immune system abnormalities Brain diseases selected from the group consisting of dysfunction, progressive neurodegenerative diseases, metabolic brain diseases, and brain dysfunction caused by bacterial and viral infections.

Hereinafter, the present invention will be described.

The inventors of the present invention aimed to identify signaling systems involved in the activation of the inflammatory response, to investigate the effects of the inflammatory response on neuronal survival, and to search for substances that can effectively regulate the inflammatory response and to determine their mechanism of action. A series of studies have been conducted. In the present invention, by searching for a substance that can effectively control the encephalopathy response from the Yang Yang extract and to determine the mechanism of action of the new drug leading material for the prevention or treatment of stroke development.

As a result, Methanol (AOM) and Chloroform (AOC) fractions of Yanggang were strongly inhibited NO and PGE ₂ inflammatory mediators in LPS-treated brain microglia. -A and AO-B were separated purely to check their activity. The activity of AOC was not related to the induction of expression of COX-2 and was confirmed to be due to the direct inhibitory action on COX-2, and it was confirmed that it was relatively selective compared to COX-1.

In addition, AOC inhibited the increased BBB permeability induced by inflammatory stimuli in the in vitro cerebrovascular barrier (BBB) model and also effectively inhibited leukocyte-cerebrovascular endothelial cell adhesion induced by inflammatory stimuli. Inhibition of leukocyte infiltration into brain edema and parenchyma observed during brain injury was found to be related to inhibition.

Therefore, the compounds of the present invention isolated from AOC are various brain diseases involving encephalitis and cerebrovascular inflammation such as stroke, Parkinson's disease, Alzheimer's disease, brain dysfunction by traumatic injury, brain dysfunction by bacterial or viral infection, etc. It is expected to be effective for prevention or treatment.

On the other hand, Yanggang (良 姜, Alpinia officinarum Hance (also known as the Goyang River), dried perennial herbaceous roots belonging to the Zingiberaceae columnar or conical, sometimes with several side roots. The surface is reddish brown or dark brown with gray white nodes, vertical wrinkles between nodes, and the material is hard and not easily bent. It is a herbal medicine applied to indigestion, vomiting, abdominal pain and hari as a medicinal agent for odor, stomach pain, and anti-nausea. 2004, Herbology, 388-389, Yeonglimsa], is known to be used for rheumatic diseases in Southeast Asia. In addition, in China, it is known to exhibit excellent therapeutic effects against spleen and gastric diseases. Essential oils contain 0.5-1% of essential oils such as 1, 8-cineol, methyl cinnamate, α-cadinene and galangol as flavonoids, and galangin, kaempferide and alpinin as flavonoids. [The digestive tract, 1985. Chinese Medicine Dictionary, Vol. 2, 782. Shanghai Science and Technology Press]

Studies on the pharmacological activity of Yanggang have included inhibitory effects of pandreatic lipase [Shin, J.E .; Han, M. J .; Kim, D. H. 2003, Biol. Pharm. Bull. 26, 854-857], inhibiting fatty acid synthesis [Li, B.H .; Tian, W.X. 2003, J. Enzyme Inhib. Med. Chem. 18, 349-356, and the like.

 The German Commission E Monographs describe it as being used for loss of appetite and indigestion, and the US FDA regulates it as generically regarded as safe (21 CFR section 182.10, 182. 20).

Hereinafter, the present invention will be described in more detail with reference to non-limiting examples.

Example 1: Preparation of Yanggang Crude Extract

Yanggang (4.5 kg) purchased from Jinheung Construction Pharmaceutical Co., Ltd. in Gyeongdong Market was extracted for 2 hours with reflux cooling. After repeating this process three times, the extract was filtered and the filtrate was concentrated under reduced pressure using a rotary vacuum concentrator to give 421 g of methanol extract.

Example 2: Preparation of Yangtze Fraction

400 g of the methanol extract obtained in Example 1 was suspended in 2 liters of water, dissolved by adding 2 liters of hexane, followed by dissolving only the components dissolved in the hexane layer, and repeating the process three times. g was obtained. 2 liters of chloroform was added to the remaining aqueous layer to dissolve. Only the components dissolved in chloroform were obtained, and the process was repeated three times. The mixture was dried in vacuo to give 26 g of a soluble chloroform fraction. 18 g of an ethyl acetate soluble fraction was obtained in the same manner as above, and the remaining aqueous layer was concentrated under reduced pressure to obtain an aqueous layer fraction.

Example 3: Separation of Compounds A and B from Soluble Chloroform Soluble Fraction

The chloroform soluble fraction (2 g) was subjected to silica gel column chromatography under Hexane: EtOAc (30: 1-> 10: 1), and the eluate was largely divided into 6 fractions. From the C-4 fraction (874 mg) and the C-6 fraction (400 mg), silica gel column chromatography was further performed, and the mixture was further divided into three fractions. Among them, C-4-1 (19 mg) and C-4- Compound A (10.9 mg) and Compound B (96.3 mg) were isolated from two fractions (124 mg), respectively.

Example 4 Structure Determination of Compound A Isolated from Acute Steel

Compound A, isolated from the chloroform-soluble fraction of the Yanggang, was dark brown in crystalline form and colored blue green by 10% sulfuric acid on silica gel TLC. 8 aromatic protons (H-2 ', H-3', H-4 ', H-5', H-6 'and H-2 ", H near δ6.6-δ7.3 in ¹ H-NMR -5 ", H6") and 8 saturated type protons (H-1, H-2, H-) between OH at δ5.50 and 1 methoxy at δ3.80, δ2.4 -δ2.8 6, H-7) and two double-linked protons (H-4, H-5) in trans form at δ6.1 and δ6.8, and one at δ200 from ¹³ C-NMR. Observing typical methoxy carbons from ketonic carbons (C-3) and δ56.0, it is assumed that a typical enone-type diarylheptanoid structure with one methoxy group is used to obtain 7- (4-hydoxy-3-methoxy phenyl) -1- phenylhept-4-en-3-one [Fumiyuki, K., Satoshi, I., Masaaki, S., Fumio, H., Ushio, S., 1992. Chem . Pharm . Bull . 40, 387 -391]

Molecular Formula C ₂₀ H ₂₂ O ₃ (mw 310.39)

7- (4'-Hydroxy-3'-methoxyphenyl) -1-phenylhept-4-en-3 -one

¹ H-NMR (CDCl ₃ , 400 MHz): δ 2.47 (2H, dd , H-6,), δ2.68 (2H , t , H-7), δ2.82 (2H , m , H-2), δ2.89 ( 1H , m , H-1), δ3.80 (3H, s , OCH ₃ ), δ5.48 (1H, s , OH), δ 6.10 (1H, dd , H-4), δ6.62 (1H, s, H-2 ″), δ6.64 (1H, s , H-6 ″), δ6.70 (1H, m , H-5 ″), δ6.82 (1H, m, H-5), δ7.107.30 (5H, m, H-2 ′ , H-3 ′, H-4 ′, H-5 ′, H-6 ′).

¹³ C-NMR (CDCl ₃ , 100 MHz): δ30.02 ( t , C-1), δ34.11 ( t , C-7), δ34.46 ( t , C-6), δ41.70 ( t , C-2), δ 55.83 ( q , -OCH ₃ ), δ 110.81 ( d , C-2 ″), δ 114.29 ( d, C-5 ″), δ 120.85 ( d , C-6 ″), Δ 126.06 ( d , C-4 ′), δ 128.33 ( d , C-2 ′, −6 ′), δ 128.45 ( d, C-3 ′, C-5 ′), δ 130. 62 ( d , C-4), δ 132.57 ( s , C-1 ″), 141.19 ( s , C-1 ′), δ 143.92 ( s , C-4 ″), δ 146.38 ( d , C-5), δ146.41 ( s , C-3 "), δ199.46 ( s , C-3).

Example 5 Structure Determination of Compound B Isolated from Acute Steel

Compound B, isolated from the chloroform soluble fraction of the Yanggang, was brownish crystalline and developed dark green by 10% sulfuric acid on TLC. 8 aromatic protons (H-2 ', H-3', H-4 ', H-5', H-6 'and H-2 ", H near δ6.6-δ7.3 in ¹ H-NMR -5 ", H6") and one saturated OH group between δ5.64 and δ3.18, one methoxy group from δ3.80, and δ2.4-δ2.9, respectively. , H-2, H-6, H-7), two protons were observed at δ1.6-δ1.8, and one ketonic carbon (C-3) and δ55 at δ211 from ¹³ C-NMR. Typical methoxy carbon was observed at 7, and hydroxycarbon (C-5) bound to OH group at δ 66.8 was assumed to be a typical diarylheptanoid structure with one methoxy group, which was assumed as 5-Hydroxy -7- (4-hydroxy-3 It was confirmed that -methoxyphenyl) -1-phenylheptan-3-one.

Molecular Formula C ₂₀ H ₂₄ O ₄ (mw 328.40)

5-Hydroxy-7- (4-hydroxy-3-methoxyphenyl) -1-phenylheptan-3-one

¹ H-NMR (CDCl ₃ , 400 MHz): δ1.78 (2H, m , H-4), δ2.402.90 (4H , m , H-1, H-2), δ2.502.90 (4H , m , H-6, H-7), δ3.18 ( 1H , s , 5-OH), δ3.86 (3H, s , OCH ₃ ), δ4.04 (1H, dd , H-5), δ5.63 (1H, s , 4 ″ -OH), δ6.58 (1H, d, H-5 ″), δ6.64 (1H, s , H-6 ″), δ6.70 (1H, d , H-2 ″), Δ7.147.30 (5H, m, H-2 ′, H-3 ′, −4 ′, −5 ′, H-6 ′).

¹³ C-NMR (CDCl ₃ , 100 MHz): δ29.39 ( t , C-7), δ31.33 ( t , C-1), δ38.26 ( t , C-6), δ44.93 ( t , C-2), δ49.17 ( t , C-4), δ55.77 ( q , -OCH ₃ ), δ66.77 ( d, C-5), δ111.01 ( d , C-2 ″) , δ 114.22 ( d, C-5 ″), δ 120.82 ( d , C-6 ″), 126.15 ( d , C-4 ′) δ 128.19 ( d , C-2 ′, C-6 ′), Δ128.48 ( d, C-3 ′, C-5 ′), δ133.61 ( s, C-1 ″), δ140.57 ( s , C-1 ′), δ143.64 ( s , C-4 ″), δ146.35 ( s , C-3 ″), δ 211.15 ( s , C-3).

Experimental Example

(1) Securing extract library and separating and purifying active ingredient

The active ingredient was separated and purified using iNOS and COX-2 activity inhibitory extracts as a starting point. That is, extracts were obtained by standard extraction using MeOH, and then solvent fractions (hexane, ethylacetate, BuOH) were prepared according to polarity. Fractions with excellent activity were purified by various column chromatography and structural analysis was performed using spectroscopic data.

(2) iNOS and COX-2 inhibitory activity

Cell culture and treatment

After dispensing the brain microglia BV-2 cells into a 96 well plate (2-3 x 10 ⁵ / well), 10% fetal bovine serum (Hyclone) and 10% penicilline (100U / ml) / streptomycin (100U / ml DMEM (Gibco) medium containing (Gibco) was added thereto and incubated for 24 hours at 37 ° C. with 5% CO ₂ /95% air. After that, the cells were changed to fresh DMEM medium and treated with LPS (10 μg / ml) to activate macrophages. At this time, the amount of PGE ₂ and nitrite produced by quantifying the concentration of the natural product extract in the culture solution and then incubated for 24 hours.

-Nitrite (NO ₂ ^-) Determination

The amount of nitrite was quantified using the Griess reaction. Take 100 μl of the above LPS-treated medium, and add 1% sulfanilamide and 0.1% N-1-naphthylethylene dihydrochloride / 50% H ₃ PO ₄ to the medium in the same amount for 10 minutes to develop the medium at 540 nm. Absorbance was measured and the amount of nitrite was calculated from the calibration curve.

-PGE ₂ Inhibition and Cyclooxygenase Activity

RPMI medium containing 3% fetal calf serum (FCS, GIBCO) was added to the cells, and LPS and the samples were incubated together at 37 ° C. for a certain time. After incubation for a certain time, the whole amount of the medium was taken and stored at -20 ° C, and the amount of PGE ₂ produced was measured by EIA. Intracellular activity of COX was measured according to the experimental method by Fu et al. That is, PBS containing excess arachidonic acid (30 μM, Sigma) was added to the cells from which the medium was removed to measure PGE ₂ production ability, and the cells were incubated for 10 minutes. Thereafter, the medium was taken, and the amount of PGE ₂ contained therein was quantified by EIA and expressed as COX activity.

-wetern blot of iNOS, COX-2

After culturing the raw 264.7 cells confluent, LPS treatment and 24 hours after the whole lysate was obtained. Cell lysate was electrophoretically transferred to PVDF transfer membrane after electrophoresis with 10% SDS-PAGE. After blocking the membrane with PBS containing 3% non-fat dried milk for 90 minutes, polyclonal anti iNOS- or COX-2 rabbit IgG was diluted 1: 600 in PBS for overnight incubation. After washing 3-4 times with PBS, incubated with goat antirabbit immunoglobulin-alkaline phosphatase (1: 600) for 3-4 hours. Then, bromo-4-chloro-3-indoyl phosphate (5 mg / ml, 70% ethanol) and nitro blue tetrazolium (5 mg / ml, 100% dimethylformamide) were added at an alkaline ratio of 1: 600 and 1: 300, respectively. The solution diluted in buffer was added to the membrane to develop color, and the bands of iNOS and COX-2 were confirmed.

iNOS, COX-2 mRNA Quantitation

  Total RNA was isolated using easy BLUE (intron, Korea). 1 ml of easy BLUE and 200ul of chloroform were added thereto, incubated at 4 ° C. for 5 minutes to lyse the cells, and centrifuged at 12,000 × G and 4 ° C. for 15 minutes to extract only the supernatant. Add the same amount of isopropanol and incubation for 15min 4 ℃, centrifuge at 12000G, 15min, 4 ℃ to obtain RNA precipitate, wash with 70% ethanol and dry in air for 10 minutes, then use RNA spectrophotometer at 260nm Was measured. RT-PCR confirmed the change of iNOS, COX-2 and ICAM-1 by amplifying the corresponding mRNA. Reverse transcription was performed using AMV revers-transcriptase at 25 ° C for 10 minutes → 42 ° C for 60 minutes → 97 ° C for 5 minutes → 4 ° C for ever, and PCR was performed for COX-2 at 94 ° C for 1 minute → 60 ° C for 1 minute → 72 degreeC 1 minute) x28cycle, iNOS was performed on the conditions of 94 degreeC 1 minute → 60 degreeC 1 minute → 72 degreeC 1 minute) x28cycle.

iNOS primer

Sense (SEQ ID NO: 1): 5'-GTGTTCCACCAGGAGATGTTG-3 '

Antisense (SEQ ID NO: 2): 5'-CTCCTGCCCACTGAGTTCGTC-3 '

COX-2 primer

Sense (SEQ ID NO: 3): 5'-ACTCACTCAGTTTGTTGAGTCATTC-3 '

Antisense (SEQ ID NO: 4): 5'-TTTGATTAGTACTGTAGGGTTAATG-3 '

(3) In vitro cerebrovascular function screening

Cerebrovascular function was detected using bEnd.3 cells, a mouse cerebrovascular endothelial cell line.

-ICAM-1 mRNA Quantitation

Total RNA was isolated using easy BLUE (intron, Korea). 1 ml of easy BLUE and 200ul of chloroform were added thereto, incubated at 4 ° C. for 5 minutes to lyse the cells, and centrifuged at 12,000 × G and 4 ° C. for 15 minutes to extract only the supernatant. Add the same amount of isopropanol and incubation for 15min 4 ℃, centrifuge at 12000G, 15min, 4 ℃ to obtain RNA precipitate, wash with 70% ethanol and dry in air for 10 minutes, then use RNA spectrophotometer at 260nm Was measured. RT-PCR confirmed the change of iNOS, COX-2 and ICAM-1 by amplifying the corresponding mRNA. Reverse transcription was carried out using AMV revers-transcriptase at 25 ° C for 10 minutes → 42 ° C for 60 minutes → 97 ° C for 5 minutes → 4 ° C for ever. PCR was performed at 94 ° C for 3 minutes → (94 ° C for 30 seconds → 60% for GAPDH). ICAM-1 was performed on the conditions of 94 degreeC 3 minutes → (94 degreeC 1 minute → 60 degreeC 1 minute → 72 degreeC 2 minutes) x 30 cycles on conditions of 30 degreeC-72 degreeC 1 minute (s) 30 cycles.

ICAM-1 primer

Sense (SEQ ID NO: 5): 5'-GTCTGCTGAGACCCCTCTTG-3 '

Antisense (SEQ ID NO: 6): 5'-GAAGGTGGTTCTTCTGAGCG-3 '

GAPDH

Sense (SEQ ID NO: 7): 5'-GTGAAGGTCGGTGTGAACGGATTT-3 '

Antisense (SEQ ID NO: 8): 5'-CACAGTCTTCTGAGTGGCAGTGAT-3 '

Leukocyte-endothelial cell adhesion assay

To determine the degree of adhesion of leukocyte-endothelial cells induced by the inflammatory response, bEnd.3 cells were cultured in 24 wells with a cover glass, and the drugs were treated. U937, a human monocyte cell line, was stained in a cell incubator for 30 minutes with cellTracker ^TM Orange CMTMR, and then sprayed on a cover glass incubated with 500ul (1 × 10 ⁶ cells / well) of bEnd.3 for about 1 hour. After attaching at, it was washed with 1 × PBS, mounted with VECTA Shield and fixed on slide glass. The degree of adhesion was confirmed using confocal laser scanning microscopy CLSM.

-Zymography

In order to measure the activity of MMP, electrophoresis was performed on SDS-PAGE gel containing gelatin, and gelatin degradation by MMP was confirmed by staining. Electrophoresis was run at 4 ° C. at 90V, and after the gel was finished, the gel was removed and washed twice with 30 minutes of 2.5% Triton-X-100 gel. After washing, the gel was added to the incubation buffer, shaken incubation for 48 hours at 37 ℃ and stained with Coomassie Blue R-250 for 40 minutes. The destaining solution (Methanol: Acetic acid: Water = 50: 10: 40) was then destained for about 40 minutes, and the decomposed band was identified as a measure of MMP activity.

-In vitro Blood-Brain-Barrier (BBB) Model Establishment and BBB Permeability Measurement

To test the functional integrity of cerebrovascular vessels, we developed an in vitro BBB model in which bEnd.3 cells, a mouse cerebrovascular endothelial cell line, and mouse primary astrocytes cultured in gelatin-coated Transwells (ICN). It was established and compared with the existing model system. First, primary astrocytes were added with the transwell insert inverted and incubated for 5 hours, and then the insert was returned to its original position and cultured for 1 day. bEnd.3 or primary HUVECs were added to the insertso and incubated for 2 weeks. Transendothelial electrical resistance (TEER) was measured daily using Millcell-ERS (Millipore, Bedford, Mass., USA). For inflammatory stimulation, TNFa (20 ng / ml) or LPS (1 ug / ml) was added and then TEER was measured.

Results through the examples and experimental examples are as follows.

(1) active fraction In brain microglia NO  And PGE ₂  Impact on generation

A series of experiments were carried out to identify the active body by purifying the fraction having anti-inflammatory activity from Yanggang, among which fractions obtained by performing solvent fractionation on a sample with code name AO (FIG. 1) NO and PGE _{2 The} effect on production was examined. The methanol solvent fraction inhibited LPS-induced PGE ₂ production in a concentration-dependent manner, which could be reconfirmed in methanol fractions (AOIIM) obtained from other Yangtze samples (Fig. 2). The schematic IC ₅₀ was about 2 ug / ml. The methanol fraction of the Yangtze also inhibited cyclooxygenase-2 (COX-2) activity concentration dependently (FIG. 3). Among the solvent fractions after methanol, the chloroform fraction showed the strongest NO inhibitory activity (FIG. 4), but PGE ₂ production did not show a significant difference between the solvent fractions (FIG. 5).

(2) Effect of Chloroform Fraction from Yanggang on LPS-induced PGE ₂ Production and COX Activity

The most potent chloroform fractions were examined for their effects on LPS-induced PGE ₂ production and total COX activity. Total PGE ₂ production induced by LPS was concentration dependently inhibited by AOC and the schematic IC ₅₀ was about 0.1 ug / ml (FIG. 6). However, AOC did not significantly affect the COX activity induced by LPS except in the case of 10 ug / ml (FIG. 7). However, AOC was found to directly inhibit COX-2 activity in a concentration-dependent manner (FIG. 8), whereas AOC did not significantly affect COX-1 activity except in the case of 10 ug / ml (FIG. 9). . AOC had no significant effect on LPS-induced COX-2 expression (FIG. 10). These results indicate that inhibition of LPS-induced PGE ₂ production by AOC has a direct effect on COX-2 activity rather than attributable to COX-2 induction. It seems to be a result of, and it was confirmed that COX-2 was more selectively inhibited compared to COX-1.

(3) iNOS  And COX -2 Inhibitory Activity Identification

The chloroform fraction, which showed the strongest activity in the solvent fraction, was separated into a single component using silica gel column chromatography and preparatory HPLC, and structural analysis was performed using spectroscopic data. In all, seven types of compound structures were identified, and the structure of two types of compounds is shown in FIG. 11. These compounds were found to inhibit iNOS expression at the protein and mRNA levels (FIG. 12). Dual AO-A also strongly inhibited COX-2 activity (FIG. 13).

(4) In in vitro Blood - Brain - Barrier Model  Establishment BBB Permeability  Effect of Yanggang Solvent Fraction

To test the functional integrity of cerebrovascular vessels, we established an in vitro BBB model (FIG. 14) in which bEnd.3 cells, a cerebrovascular endothelial cell line, and mouse primary astrocytes are cultured together in gelatin-coated transwells (ICN). And compared. In the existing model, HUVEC and rat-derived C6 glioma cells are used instead of cerebrovascular cell lines. This study suggests that endothelial cells are not cerebral blood vessels, and that co-cultured astrocytes are different. In this study, we developed a co-culture system of astrocytes of the same species with cerebrovascular endothelial cell lines.

As shown in FIG. 15, the bEnd.3 + primary astrocyte model maintained a form with little cell spacing after 14 days of incubation, indicating that a tighter tight junction could be formed than the HUVEC + C6 glioma model. have. On the other hand, in the case of bEnd.3 + C6 glioma or bEnd.3 alone, the state of the cells rapidly deteriorated after 4 days, and many cells fell off about 7 days. Under the same conditions, the measurement of transendothlial electric resistance (TEER) through cultured vascular endothelial cells showed a generally consistent result. That is, the bEnd.3 + primary astrocyte model continuously increased TEER until 15 days, which is almost comparable to the existing HUVEC + C6 glioma model (FIG. 16). On the other hand, in other models, TEER did not increase significantly and tended to decrease rapidly from 4 days. Since HUVEC is a cord vascular endothelial cell, its origin is different from cerebrovascular endothelial cells, and it is difficult to obtain a large amount due to the condition of primary culture. In addition, C6 glioma has an advantage in culture, but it is not a differentiated astrocytic cell but also a mouse-derived cell, which limits the model. On the other hand, the bEnd.3 + primary astrocyte model attempted in this study uses the cerebrovascular endothelial cells, the cell line makes it possible to easily obtain the required cell volume, and the primary astrocytes can be obtained in large quantities and easily. In addition, the origin of both cells is mouse, and can be used as an advantage in constructing a stable BBB model. These results suggest that the bEnd.3 + primary astrocyte model attempted in this study has the advantage of replacing the existing model.

In the established bEnd.3 + primary astrocyte model, LPS was applied to induce an inflammatory condition, and then BBB permeability was measured. A significant increase (decrease in TEER) was observed compared to the control group, and AOC significantly improved permeability in a concentration-dependent manner. (Fig. 17)

(5) Effect of Solvent Fraction of Alumina on Leukocyte-vascular Endothelial Cell Adhesion and Cell Adhesion Molecule Expression

AOC in the solvent fraction of the Yangtze concentration-dependently inhibited leukocyte-vascular endothelial cell adhesion by inflammatory stimulation (FIG. 18) and significantly inhibited the expression of ICAM-1 (FIG. 19).

(6) Solvent fraction of Yangtze matrix metalloproteinase  Effect on activity

Metalloproteinase activity is also known as a very important factor in the incorporation of leukocytes into the brain. Therefore, we established a method for measuring MMP activity through zymography, and as shown in FIG. It was confirmed that MMP activity was inhibited, and the Yanggang methanol fraction which significantly inhibited PGE ₂ production showed the same result.

As can be seen from the above configuration, the compound of the present invention can be used as an effective prophylactic and therapeutic agent for various brain diseases involving encephalitis and cerebrovascular inflammation.

<110> Ajou University Industry Cooperation Foundation          KYONGGI PROVINCIAL GOVERNOR <120> Composition for preventing and treating cerebrovascular disease          comprising a compound isolated from Alpinia officinarum Hance <160> 8 <170> KopatentIn 1.71 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> iNOS primer <400> 1 gtgttccacc aggagatgtt g 21 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> iNOS primer <400> 2 ctcctgccca ctgagttcgt c 21 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> COX-2 primer <400> 3 actcactcag tttgttgagt cattc 25 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> COX-2 primer <400> 4 tttgattagt actgtagggt taatg 25 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ICAM-1 primer <400> 5 gtctgctgag acccctcttg 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ICAM-1 primer <400> 6 gaaggtggtt cttctgagcg 20 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> GAPDH primer <400> 7 gtgaaggtcg gtgtgaacgg attt 24 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> GAPDH primer <400> 8 cacagtcttc tgagtggcag tgat 24  

Claims (5)

A composition for preventing and / or treating brain diseases, comprising the compound of Formula 1 or a compound of Formula 2 as an active ingredient.

                       [Formula 1]

                       [Formula 2] The composition of claim 1, wherein the compound is isolated from the lumen. The composition of claim 1 or 2, wherein the compound is isolated from the chloroform, ethyl acetate, or acetone fraction of the lumen. According to claim 1, wherein the brain disease is a composition characterized in that the various brain diseases accompanied by encephalitis and cerebrovascular inflammation. The brain disease according to claim 4, wherein the brain disease is stroke, Parkinson's disease, Alzheimer's disease, brain dysfunction due to traumatic injury, immune system dysfunction, progressive neurodegenerative disease, metabolic brain disease, brain dysfunction due to bacterial and viral infections. Composition comprising a brain disease selected from the group consisting of.

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