KR100291524B1 - Cinnamate derivatives with neuroprotective activity - Google Patents

Abstract

The present invention relates to a new use of the cinnamate derivative of the formula (1), specifically, extract the medicinal plant Hyunsam with a mixed solvent of chloroform, methanol and then fractionated again with an organic solvent containing dichloromethane, methanol and the like to protect neurons Cinnamate derivatives of the present invention obtained by separating and purifying cinnamate derivatives, which are active substances, are prepared by extracting and extracting ginseng extract having column chromatography and recrystallization using the same, and have excellent neuroprotective activity to prevent stroke and degenerative neurological diseases. It can be usefully used for treatment.

Wherein R ₁ is CH ₃ or H, and R ₂ is H or α-L-Rhamnose.

Description

Cinnamate derivatives with neuroprotective activity

The present invention relates to a novel use of the cinnamate derivative of formula (I).

In more detail, the extract of medicinal plants, Hyunsam, is extracted with a mixed solvent of chloroform and methanol, and then fractionated again with an organic solvent containing dichloromethane, methanol, etc. to prepare Hyunsam extract having neuronal cell-protective activity, using the column chromatography and recrystallization. Therefore, the cinnamate derivative of the present invention, which is obtained by separating and purifying the active substance cinnamate derivative, has excellent neuronal protective activity and may be usefully used for the prevention and treatment of stroke and degenerative neurological diseases.

Formula 1

Wherein R ₁ is CH ₃ or H, and R ₂ is H or α-L-Rhamnose.

Recently, as the elderly population increases, the number of patients with degenerative neurological diseases, especially senile dementia, is increasing rapidly. Geriatric dementia is a disease in which memory loss and deterioration of learning ability are the main symptoms and patients and their families have to suffer a lot together.It has become a social problem and the interest in prevention and treatment of it is increasing day by day. There is no treatment to develop such a seriously serious disease is highlighted.

Degenerative neurological diseases occur when the brain is damaged by secondary symptoms caused by adult diseases such as structural degeneration of the neuronal cells due to aging, circulatory disorders, or physical and mechanical factors such as traffic accidents, industrial accidents, and carbon monoxide poisoning. (Rothman. SM (1984) J. Neurosci. 4 : 1884-1891; Weiloch, T. (1985) Science. 230 : 681-683). In other words, when the blood flow to the brain is reduced or blocked, brain tissue becomes hypoxic and low glucose, so that normal metabolism cannot be performed, resulting in irreparable damage to brain tissue. This case is very common in degenerative neurological diseases as well as stroke, trauma and ischemic injury (Benveniste, H. et al. (1984) J. Neurochem. 43 : 1369- Hagberg, H. et al. (1985) J. Cereb. Blood Flow & Metab. 5 : 413-419).

In general, brain tissue damage is known to be largely due to two mechanisms. The first is due to the free release of excitatory amino acids by the change in cell membrane voltage, and the second is due to direct oxidative stress (Choi, DW (1988) Neuron. 1 : 623-634; Coyle , JT et al. (1993) Science. 262 : 689-695). Recent studies have shown that in the case of stroke, trauma, and ischemic injury, glutamate activates ionotrophic receptors, causing oxidative stress, causing abnormalities in glutamate neurons. Halliwell, B. et al. (1985a) Trends Neurosci. 5 : 22-26; Halliwell, B. et al. (1985b) Mol. Aspects Med. 8 : 89-193).

When blood flow in brain tissues decreases, glutamate levels rapidly increase in nerve junctions, causing toxicity and eventually killing neurons (Benveniste, H. et al. (1984) J. Neurochem. 43 : 1369-1374; Hagberg, H. et al. (1985) J. Cereb. Blood Flow & Metab. 5 : 413-419). Neurotoxicity caused by glutamate is divided into "acute toxicity" and "chronic toxicity" (Choi, DW (1988) Neuron. 1 : 623-634).

Acute toxicity begins with the influx of extracellular Ca ²⁺ into the cell, followed by the influx of ions such as Na ⁺ , K ⁺ and Cl ^- to maintain the osmotic pressure of the cell, causing the cell to swell and eventually burst and die. (Kauppisen, RA (1988) Neurosci. 27 : 175-182; Garthwaite, G. et al. (1988) J. Neurosci. 17 : 755-767).

Chronic toxicity by glutamate occurs by activating the glutamate NMDA receptor and the non-NMDA receptor kainic acid / AMPA receptor (Coyle, JT et al. (1993) Science. 262 : 689-695). That is, by the NMDA glutamate receptor and non -NMDA is activated Ca ²⁺ influx is an intracellular calcium-dependent if (calcium-dependent) enzyme phospholipase A ₂ (phospholipase A ₂₎ is abnormally activated by polyunsaturated glass The production of polyunsaturated free fatty acids is increased, and excess free radicals such as superoxide radicals and hydroxyl radicals are produced to promote cell peroxidation, which eventually leads to cell Chronic toxicity to death occurs (Coyle, JT et al. (1993) Science. 262 : 689-695; Halliwell, B. et al. (1985a) Trends Neurosci. 5 : 22-26; Halliwell, B. et al. . (1985b) Mol Aspects Med 8 :.. 89-193; Siesjo, BK et al (1985) Br J. Anaesth 47:.... 47-62; Sladeczek, F. et al (1985) Nature 317:. 717-719; Choi, DW et al. (1987) J. Neurosci. 7 : 369-379). Activation of the NMDA receptor also increases the activity of nitric oxide synthase, resulting in excessive production of nitric oxide, which also promotes cell peroxidation (Strijbos, PJLM et al. (1996) J. Neurosci. 16 5004-5013).

Neurotoxicity by glutamate is evaluated by xantine from glutamate antagonists, Na ⁺ and Ca ²⁺ channel blockers, antioxidants, free radical chain breakers and xanthine dehydrogenase. antiproteases, which are enzymes that inhibit the conversion to oxidase, but are particularly effective in antagonists to NMDA receptors (Choi, DW et al. (1988) J. Neurosci. 8 : 185-196; Olney, JW et al. (1969) Science. 166 : 368-388).

Hyunsam ( Scrophularia buergeriana ) is a perennial herb from Hyunsam and has a height of 1 to 1.5 m, leaves are opposite, leafy, obovate or egg-shaped lanceolate, slightly rounded or slightly straight, with sharp and sharp teeth. Flower is pale yellow green and blooms from August to September. It grows on stem end bracts as a wheat plant. Corollas are diseased, oblique, posterior, bilateral, calyxes 5 rows, lobes ovate or ovoid. Fruits are capsules, ovate, bilateral. Dried root is called ssam ( Scrophulariae Radix ) and is used for bleeding consonants, detoxification, fever swelling, and hunger. The genus of the present genus is distributed in about 300 species worldwide, and there are five species in Korea. As the study component antipyretic component is mainly composed of p - methoxy Shinagawa mixer acid (p -methoxycinnamic acid) and yirido Id (iridoid) based glycosides to the digging side (harpagoside), Haffa azide (harpagide), 8- (O - Methyl- p -coumaryl) hapazide [8- ( O- methyl- p- coumaroyl) harpagide] has been reported, but no systematic studies have been conducted to correlate its activities with individual components (Woo, WS (1968)). J. pharm Sci. 57 (1) : 27-30; Okano, T. (1967) Chem. Pharm. Bull. 15 (8) : 1254; Weinges, K. et al. (1978) Ann. Chem. 1968) .

Therefore, the present inventors artificially induced glutamate to glutamate in rat cerebral cortical cells from herbal medicine which has been known to be effective for forgetfulness or stroke in the past to isolate substances with new neuronal cell protective activity. While searching for natural products that weaken these neurotoxicities by using a screening system, they found that extracts of Hyunsam had significant neuroprotective activity, and isolated and purified cinnamate derivatives from them to confirm their structure and their new neurons. The present invention has been completed by confirming protective activity.

An object of the present invention is to provide a new use that can be used to prevent or treat degenerative neurological diseases such as stroke or senile dementia, cinnamate derivatives showing neuronal protective activity.

Figure 1 illustrates the process of producing a Hyunsam extract of the present invention,

Figure 2 illustrates the process of separating and purifying Compounds 1, 2 and 3 of the present invention from a 90% methanol fraction.

The present invention provides a cinnamate derivative having the neuroprotective activity and a new use in which pharmaceutically acceptable inorganic or organic salts, hydrates, solvates and isomers thereof can be used as neuronal protective agents.

Formula 1

Wherein R ₁ is CH ₃ or H, and R ₂ is H or α-L-Rhamnose.

Specifically, the cinnamate derivative of Formula 1 to Formula 2 p - methoxy - trans - cinnamic acid (p -methoxy- trans -cinnamic acid), the formula 3, p - methoxy - trans - cinnamoyl -4 '-α-L-rhamnose ester ( p- methoxy- trans- cinnamoyl-4'-α-L-rhamnose ester) or Cinnamic acid (cinnamic acid) of the formula (4).

In another aspect, the present invention is obtained by column chromatography and recrystallization of the ginseng extract having a nerve cell protective activity extracted with a mixed solvent of chloroform-methanol and then extracted again with an organic solvent containing dichloromethane, hexane, butanol, etc. Provided is a method of preparing a cinnamate derivative of Formula 1.

In another aspect, the present invention provides a pharmaceutical composition for neuronal cell protective agent comprising a ginseng extract extracted with a mixed solvent of chloroform-methanol and then extracted again with an organic solvent including dichloromethane, hexane, butanol and the like.

Hereinafter, the present invention will be described in detail.

The present invention provides a novel use in which cinnamate derivatives of the general formula (1) below and pharmaceutically acceptable inorganic or organic salts, hydrates, solvates and isomers thereof can be used as neuronal cell protective agents.

Formula 1

Wherein R ₁ is CH ₃ or H, and R ₂ is H or α-L-Rhamnose.

Specifically, the cinnamate derivative of Formula 1 to Formula 2 p - methoxy - trans - cinnamic acid (p -methoxy- trans -cinnamic acid), the formula 3, p - methoxy - trans - cinnamoyl -4 '-α-L-rhamnose ester ( p- methoxy- trans- cinnamoyl-4'-α-L-rhamnose ester) or Cinnamic acid (cinnamic acid) of the formula (4).

In the present invention, cinnamate derivatives having neuronal protective activity are isolated and purified from medicinal plants, Hyunsam.

First, the present invention provides a ginseng extract having a nerve cell protective activity extracted from an organic solvent. At this time, it is preferable to use a mixed solvent of chloroform and methanol as the organic solvent.

Specifically, in the present invention, the root of Hyunsam was extracted by boiling water with a mixed solvent of chloroform-methanol (CHCl ₃ -MeOH), and then concentrated under reduced pressure to obtain a crude extract.

In addition to the above process, the present invention provides an extract of Hyunsam with neuronal cell protective activity extracted again with an organic solvent such as dichloromethane (CH ₂ Cl ₂ ), hexane (hexane) or butanol (BuOH).

Specifically, in addition to the above process, the crude extract was suspended in water and extracted with dichloromethane to obtain a water fraction and a dichloromethane fraction having neuronal protective activity. The dichloromethane fraction was further suspended with methanol (MeOH) and extracted with hexane to obtain a methanol fraction and a hexane fraction with neuroprotective activity.

In order to confirm that the present ginseng extract of the present invention has neuronal protective activity, the cerebral cortical cells of rats were treated with the ginseng extract and then MTT assay was performed.

Specifically, the cerebral cortical cells of the rats were cultured, and the present ginseng extract of the present invention was administered, and after 1 hour, glutamate was acted to artificially induce toxicity and continued culturing (Throughout treatment). MTT (3- (4,5-dimethyl-thiazol-2-yl) -2,5-diphenyltetrazolium bromide) was added to the culture of cerebral cortical cells, and the resulting formazan was dissolved in DMSO (dimethylsulfoxide). MTT analysis was then performed by measuring absorbance.

As a result, crude extract, dichloromethane fraction and methanol fraction of Hyunsam significantly blocked neurotoxicity by glutamate, which reduced neuronal cell death.

In the present invention, in order to purely separate the compound having nerve cell protective activity from the Hyunsam extract, the Hyunsam extract was subjected to column chromatography and recrystallized.

Specifically, the methanol fraction of Hyunsam was hung on a silica gel column and chromatographed with a mixed solvent of chloroform and methanol, and the fraction having excellent neuronal protective activity was separated. : Compound 1 which was colorless needle crystal and compound 3 which was colorless prism was obtained by recrystallization with a mixed solvent of methanol.

The physicochemical and spectroscopic properties of Compound 1 were identified as follows.

(1) Appearance of substance: Colorless needle crystal (in EtOH)

(2) Molecular weight: EIMS (m / z) 179 [M ⁺ ]

(3) Molecular formula: C ₁₀ H ₁₀ O ₃

(4) Melting Point (mp): 188-189.5 °

(5) Hydrogen Nuclear Magnetic Resonance Spectrum ¹ H-NMR (CD ₃ OD, 300 MHz) δ 3.81 (3H, s, OCH ₃ ), 6.31 (1H, d, J = 15.93, H-α), 6.94 (1H × 2) , d, J = 9.51, H-3, 5), 7.53 (1H x 2, d, J = 9.51, H-2, 6), 7.61 (1H, d, J = 15.93, H-β).

(6) Carbon nuclear magnetic resonance spectra ¹³ C NMR (CD ₃ OD, 100 MHz) δ 56.65 (OCH ₃ ), 116.21 (C-3, 5), 117.39 (C-α), 129.22 (C-1), 131.68 ( C-2, 6), 146.99 (C-β), 163.89 (C-4), 171.59 (COOH).

Or more physicochemical and compound 1 minute based on the optical data p - methoxy - trans -. Was estimated to be thinner acid (p -methoxy- trans -cinnamic acid) reference value and (Kajimoto, T. et al (1989 ) Phytochem . 28 (10): 2701-2704; Falsone, G. et al (1982) Verbascun sinuatum Planta Med 44:...... 150-153; Fernandez, L. et al (1995) Scrophularia scorodonia Phytochem 34: 1569 -1571) compared and identified.

(7) chemical structure

Formula 2

The methanol fraction of Hyunsam was purified by silica gel column chromatography with a mixed solvent of chloroform and methanol, and then recrystallized with a mixed solvent of hexane and dichloromethane to separate and purify Compound 2 having another neuronal cell protective activity.

The physicochemical and spectroscopic properties of Compound 2 were identified as follows.

(1) Appearance of substance: Light yellow amorphous powder

(2) Molecular weight: FabMS (m / z) 347 [M + Na]

(3) Molecular formula: C ₁₆ H ₂₀ O ₇

(4) Hydrogen Nuclear Magnetic Resonance Spectrum ¹ H-NMR (CDCl ₃ : CD ₃ OD = 2: 1, 300 MHz) δ 1.20 (3H, d, J = 6.33, H-6 ′), 3.62 (3H, s, OCH ₃ ), 3.67 (1H, dd, J = 1.71, 3.42, H-2 '), 3.75 (1H, dd, J = 9.75, 3.3, H-3'), 3.86 (1H, dq, J = 6.09, 9.75, H-5 '), 4.76 (1H, t, J = 9.75, H-4'), 4.88 (1H, d, J = 1.71, H-1 '), 6.13 (1H, d, J = 15.84, H-α), 6.69 (1H × 2, d, J = 8.79, H-3, 5), 7.28 (1H × 2, d, J = 8.79, H-2, 6), 7.44 (1H, d, J = 15.84, H-β)

(5) Carbon nuclear magnetic resonance spectra ¹³ C NMR (CDCl ₃ : CD ₃ OD = 2: 1, 100 MHz) δ 16.95 (C-6 '), 54.86 (OCH ₃ ), 65.52 (C-5'), 68.94 ( C-3 '), 71.28 (C-4'), 74.34 (C-4 '), 93.83 (C-1'), 113.96 (C-3, 5), 114.55 (C-α), 126.54 (C- 1), 129.49 (C-2, 6), 145.05 (C-β), 161.26 (C-4), 167.51 (COO)

Determining a cinnamoyl -4'-α-L- rhamnose ester (p -methoxy- trans -cinnamoyl-4'- α-L-rhamnose ester) - at least a second compound on the basis of spectral data p - methoxy - trans It was.

(6) chemical structural formula

Formula 3

In addition, the physicochemical and spectroscopic properties of Compound 3 were identified as follows.

(1) Appearance of matter: Colorless

(2) Molecular weight: EIMS (m / z) 149 [M ⁺ ]

(3) Molecular formula: C ₉ H ₈ O ₂

(4) Melting Point (mp): 133 °

(5) UV absorption spectrum UV (nm) 205, 224, 311

(6) Hydrogen Nuclear Magnetic Resonance Spectrum ¹ H-NMR (CD ₃ OD, 300 MHz) δ 6.47 (1H, d, J = 15.93, H-α), 7.39 (1H, × 3, m, H-3, 4 , 5), 7.59 (1H × 2, m, H-2, 6), 7.67 (1H, d, J = 15.93, H-β)

(7) Carbon nuclear magnetic resonance spectra ¹³ C NMR (CD ₃ OD, 100 MHz) δ 1198.38 (C-α), 129.18 (C-2, 6), 130.01 (C-3, 5), 131.40 (C-4 ), 13.86 (C-1), 146.30 (C-β), 170.33 (COOH)

Based on the above physicochemical and spectroscopic results, Compound 3 was estimated as cinnamic acid and was determined by Calis, I. et al. (1993) Scrophularia ilwensis.J. Nat. Prod. 56 (4) : 606 -609; Calis, I. et al. (1988) Scrophularia scopolii.Planta Med. 168-180).

(8) chemical structural formula

Formula 4

The neuroprotective activity of Compounds 1, 2, and 3 isolated and purified from Hyunsam ginseng was the same as the method of searching for the neuronal protection activity of Hyunsam extract using the cerebral cortical cells of primary cultured rats that induced neurotoxicity with glutamate. Search.

As a result, Compounds 1, 2 and 3 showed significant neuroprotective activity.

In addition, in the present invention, in order to find out which mechanism of the compound of the present invention has been confirmed that neuronal protective activity blocks neurotoxicity by glutamate, first the administration time of the compound to the primary cultured cerebral cortical cells before the treatment of glutamate ( Each was divided into pretreatment and recovery treatments.

As a result, Compounds 1 and 2 showed a significant effect of protecting the neurons when both before and after the glutamate action (throughout treatment) or pretreatment, but when the post-treatment was found to be inferior neuroprotective activity. On the other hand, Compound 3 was found to be significantly maintained even if the administration time is different without increasing or decreasing the effect, it can be seen that it exhibits neuronal protective activity by a mechanism different from that of Compounds 1 and 2.

In addition, to investigate whether the compounds 1, 2 and 3 of the present invention act on glutamate receptors and exhibit neuronal protective activity, N-methyl-D-aspartate (NMDA) in glutamate receptors. And its action on non-NMDA receptors.

As a result, Compound 2 significantly reduced neurotoxicity induced by NMDA, but lacked the effect of blocking neurotoxicity induced by non-NMDA agonist kainic acid (abbreviated as 'KA'). Among the receptors, the selectivity of NMDA receptors was superior to that of non-NMDA receptors. Compounds 1 and 3 also significantly reduced neurotoxicity induced by NMDA and neurotoxicity induced by KA, thereby reducing the NMDA or non-NMDA receptors. It was confirmed that the selectivity was inferior to that of Compound 2.

On the other hand, the neurotoxicity of glutamate is due to the activation of various enzymes due to the influx of Ca ²⁺ which occurs initially when glutamate is acted on, so the neuronal protective activity of compounds 1, 2 and 3 of the present invention affects the influx of calcium. It is thought that these compounds will influence the Ca ²⁺ uptake.

The concentration of Ca ²⁺ in cerebral cortical cells was measured according to the method of Grynkiewicz et al. As a result, compounds 1, 2 and 3 significantly decreased the concentration of Ca ²⁺ introduced into the cells by glutamate. In particular, the compounds 1 and 2 was found to have a lower EC ₅₀ than MK-801 which is a known Ca ²⁺ channel blocker (Ca ²⁺ channel blocker).

In the present invention, the Ca ²⁺ - dependent enzyme present (Ca ²⁺ -dependent enzyme) excessive production of nitric oxide (nitric oxide, NO) and free radicals (free radical) that follows in accordance with the activation shall also be expected to some extent decreases the invention The effects of compounds 1, 2 and 3 on the production of nitric oxide were examined.

Specifically, compounds 1, 2, and 3 were treated with pre- or post-treatment in the cerebral cortical cells of rats cultured for 20 days at different concentrations to determine the change in NO production increased by the treatment of glutamate. At this time, the amount of nitrite released from the primary cultured cerebral cortical cells into the culture was measured by Dowson et al. (Kohno, K. et al. (1995) Neurosci. Lett. 199 : 65-68 ).

As a result, pretreatment with Compound 2 reduced the amount of NO increased by glutamate to 95% at steady-state, and decreased to 80% even after post-treatment, resulting in the reduction of NO production by lowering the Ca ²⁺ concentration. It could be confirmed that the reduction effect. On the other hand, compounds 1 and 3 also significantly reduced the amount of NO produced excessively by glutamate, but the effect was much lower compared to compound 2.

As a result, the compounds 1, 2 and 3 isolated from the present ginseng have significant neuroprotective activity against glutamate-induced neurotoxicity in the primary cerebral cortical cells of rats. It was confirmed that the neuronal protective activity was primarily exhibited by lowering the concentration of Ca ²⁺ excessively introduced. In addition, compound 2 was confirmed to reduce the amount of NO produced excessively due to glutamate to block neuronal cell death.

In another aspect, the present invention provides a pharmaceutical composition for protecting nerve cells comprising the present extract and / or the compound of Formula 1 as an active ingredient.

When using the ginseng extract of the present invention or the compound of the formula (1) as a therapeutic agent, it can be prepared by a known method in the pharmaceutical field, itself or a pharmaceutically acceptable carrier, excipient (forming agent) ), Mixed with diluents, etc., may be prepared and used in the form of powders, granules, tablets, capsules, or injections.

In addition, the pharmaceutical composition comprising the present ginseng extract of the present invention or the compound of Formula 1 as an active ingredient may be administered by dividing about three times a day at a concentration of 0.2 mg / kg.

Hereinafter, the present invention will be described in detail in the following examples.

The following examples illustrate the invention and are not intended to limit the scope of the invention.

Example 1 Extraction and Fraction of Hyunsam

24 kg of ginseng was extracted with hot water three times for 2 hours with a mixed solvent of chloroform-methanol (CHCl ₃ -MeOH, 1: 1), and the extract was concentrated under reduced pressure to obtain 1.8 kg of crude extract. Hyunsam extract (1.8 kg) was suspended in distilled water, and then fractionated with dichloromethane (CH ₂ Cl ₂ ) and concentrated under reduced pressure to obtain a dichloromethane fraction (350g) and water fraction. Was re-suspended in 90% methanol in dichloromethane fractions were concentrated, then n-hexane (n -hexane) portionwise to obtain a 90% methanol fraction (273 g) and n-hexane fraction (65 g). The water fraction was again partitioned with normal butanol ( n- BuOH) to give 562 g of normal butanol fraction (see FIG. 1).

Example 2 Activity Study of Hyunsam Extract

In order to investigate the effect of glutamate on the neurotoxicity of glutamate prepared in Example 1, treated with the ginseng extract in the primary cultured cerebral cortical cells of rats and then MTT assay (assay) was performed.

As the experimental animals, Sprague-Dawley rats were supplied from Seoul National University Animal Breeding Farm and were bred in the experimental animal laboratory of the College of Pharmacy, Seoul National University. The environment of the kennel was kept at 22 ± 5 ℃ and the lighting time was fixed from 7 am to 7 pm. The feed was 23.2% crude protein, 4.0% crude fat, 6.0% crude fiber, 10.0% crude ash, 0.6% crude calcium. Solid feed (Samyang) containing 0.4% join was used.

In order to separate the cerebral cortical cells from the used rats, only the cerebral part of the fetus is removed, the meninges are carefully removed under a dissecting microscope, and the cerebral tissue is treated with 0.25% trypsin for 30 minutes to soften the tissue. It was allowed to liberate into individual cells.

Culture of the isolated cerebral cortical cells was 2.0 × 10 ⁵ cells / ml, and streptomycin 100 was added to a culture vessel (Falcon, 15 × 24 mm) coated with poly-L-lysine. A culture consisting of μg / ml was used. Cultivation of the cells was carried out by continuously supplying a mixture of air (95%) and CO ₂ (5%) in a 37 ℃ incubator and after 4 days of culture 5 × 10 ^-5 M 5-fluorodeoxyuridine ( fluorodeoxyuridine) inhibited the growth of cells other than neurons (Choi, DW (1985) Neurosci. Lett. 58 : 293-297).

The crude extract and fractions of Hyunsam ginseng were dissolved in DMSO (within 0.1% final concentration), diluted with distilled water, and prepared at a concentration of 1 mg / ml, and then passed through a millipore membrane (0.22 μm; Millex-GV, USA). After aseptic administration, the cells were inoculated into the cerebral cortical cells of rats incubated for 20 days with the above procedure. After 1 hour, 50 μM glutamate was reacted for 15 minutes, and then the culture medium was removed and changed into DMEM alone. Was further incubated. At this time, the crude extract of each ginseng and each fraction were continuously treated (Throughout treatment).

MTT (5 mg / mL) was added to 10% of the culture medium in the cultured cerebral cortical cells, followed by further incubation for 3 hours, and the resulting formazan was dissolved in DMSO and absorbance was measured at 540 nm. MTT analysis was performed by the method (Mosmann, T. (1983) J. Immuno. Methods. 65 : 55-61).

Concentration (µg / ml) Survival rate (%) ^c Control ^a 100.0 ± 4.2 Comparative example ^b 0.0 ± 1.3 Crude extract 50 68.2 ± 5.5 ^*** 100 35.8 ± 3.1 ^* Dichloromethane fraction 5 46.2 ± 1.9 ^** 10 68.7 ± 1.2 ^*** Normal Hexane Fraction 5 0.3 ± 2.5 10 -10.1 ± 7.3 90% methanol 5 77.5 ± 5.0 ^*** 10 67.4 ± 2.2 ^*** Water fraction - Control ^a : primary cultured cerebral cortical cells Comparative example ^b : cerebral cortical cells exposed to glutamate for 15 minutes, control and p <0.001 survival rate ^c : 100 × (sample OD-comparative OD) / (control OD-comparative OD Significance: ^* p> 0.05, ^** p <0.01, ^*** p <0.001

For statistical significance review, the number of each experimental group was 3 (n = 3) and the variation from the control was "ANOVA test". When the P value was less than 5%, it was determined to be statistically significant.

As a result, as shown in Table 1, crude extract, dichloromethane fraction, and 90% methanol fraction of Hyunsam significantly blocked neurotoxicity by glutamate, which reduced neuronal cell death and consequently increased the number of surviving cells. I was.

Example 3 Isolation and Purification of the Compound from Hyunsam Extract

90% methanol fraction (270 g) showing significant neuronal protective activity in Example 2 was subjected to silica gel column chromatography (silicagel column chromatography) with a mixed solvent of chloroform: methanol (100: 1 → 0: 1). The fractions were divided into small fractions (fractions I to VII) and the neuroprotective activity of each was measured.

Concentration (µg / ml) Survival rate (%) Control 100.0 ± 4.2 Comparative example 0.0 ± 1.3 Fraction I One 2.0 ± 2.2 10 -5.8 ± 4.3 Fraction II One 56.2 ± 2.5 ^*** 10 38.1 ± 3.8 ^** Fraction Ⅲ One 0.3 ± 1.4 10 -10.1 ± 1.0 Fraction IV One 16.5 ± 3.1 10 24.4 ± 2.8 ^* Fraction Ⅴ One 59.7 ± 1.2 ^*** 10 82.4 ± 4.0 ^*** Fraction VI One -4.5 ± 0.3 10 21.1 ± 1.6 ^* Fraction Ⅶ One 0.5 ± 3.3 10 5.3 ± 2.1 Fraction Ⅷ Not determined

As a result, as shown in Table 2, it can be seen that small fraction 2 (fraction II) and small fraction 5 (fraction V) have significant neuroprotective activity.

In order to separate compounds having cerebral neuronal cell protective activity from subfraction 2 (fraction II, 20 g), which showed significant neuronal neuroprotective activity in a small fraction of 90% methanol of Hyunsam, normal hexane: ethyl acetate (EtOAc). Was purified by silica gel column chromatography with a mixed solvent (100: 1 to 0: 1) and divided into seven subfractions (fractions II-1 to II-7), and the double fraction II-2 was divided into normal hexane: dichloromethane: methanol (5: 1: 1) was recrystallized with a mixed solvent to obtain 980 mg of Compound 1, which is colorless acicular crystal, and 50 mg of Compound 3, which is colorless prism (see FIG. 2).

In addition, the precipitate obtained in the small fraction 4 (fraction IV, 11 g) of 90% methanol of Hyunsam was recrystallized with a mixed solvent of normal hexane: dichloromethane (1: 4) to give 642 mg of Compound 2 as a slightly yellow amorphous powder (Fig. 2).

Example 4 Structure Determination of Compound 1

The physicochemical and spectroscopic properties of Compound 1 isolated and purified in Example 3 were as follows.

(1) Appearance of substance: Colorless needle crystal (in EtOH)

(2) Molecular weight: EIMS (m / z) 179 [M ⁺ ]

(3) Molecular formula: C ₁₀ H ₁₀ O ₃

(4) Melting Point (mp): 188-189.5 °

(5) Hydrogen Nuclear Magnetic Resonance Spectrum ¹ H-NMR (CD ₃ OD, 300 MHz) δ 3.81 (3H, s, OCH ₃ ), 6.31 (1H, d, J = 15.93, H-α), 6.94 (1H × 2) , d, J = 9.51, H-3, 5), 7.53 (1H x 2, d, J = 9.51, H-2, 6), 7.61 (1H, d, J = 15.93, H-β).

(6) Carbon nuclear magnetic resonance spectra ¹³ C NMR (CD ₃ OD, 100 MHz) δ 56.65 (OCH ₃ ), 116.21 (C-3, 5), 117.39 (C-α), 129.22 (C-1), 131.68 ( C-2, 6), 146.99 (C-β), 163.89 (C-4), 171.59 (COOH).

Compound 1 based on the above physicochemical and spectroscopic data p - methoxy - trans - was estimated to be thinner mixer acid (p -methoxy- trans -cinnamic acid) was identified by comparison with the literature values.

(7) chemical structure

Formula 2

Example 5 Structure Determination of Compound 2

The physicochemical and spectroscopic properties of Compound 2 isolated and purified in Example 3 were as follows.

(1) Appearance of substance: Light yellow amorphous powder

(2) Molecular weight: FabMS (m / z) 347 [M + Na]

(3) Molecular formula: C ₁₆ H ₂₀ O ₇

(4) Hydrogen Nuclear Magnetic Resonance Spectrum ¹ H-NMR (CDCl ₃ : CD ₃ OD = 2: 1, 300 MHz) δ 1.20 (3H, d, J = 6.33, H-6 ′), 3.62 (3H, s, OCH ₃ ), 3.67 (1H, dd, J = 1.71, 3.42, H-2 '), 3.75 (1H, dd, J = 9.75, 3.3, H-3'), 3.86 (1H, dq, J = 6.09, 9.75, H-5 '), 4.76 (1H, t, J = 9.75, H-4'), 4.88 (1H, d, J = 1.71, H-1 '), 6.13 (1H, d, J = 15.84, H-α), 6.69 (1H × 2, d, J = 8.79, H-3, 5), 7.28 (1H × 2, d, J = 8.79, H-2, 6), 7.44 (1H, d, J = 15.84, H-β)

(5) Carbon nuclear magnetic resonance spectra ¹³ C NMR (CDCl ₃ : CD ₃ OD = 2: 1, 100 MHz) δ 16.95 (C-6 '), 54.86 (OCH ₃ ), 65.52 (C-5'), 68.94 ( C-3 '), 71.28 (C-4'), 74.34 (C-4 '), 93.83 (C-1'), 113.96 (C-3, 5), 114.55 (C-α), 126.54 (C- 1), 129.49 (C-2, 6), 145.05 (C-β), 161.26 (C-4), 167.51 (COO)

Determining a cinnamoyl -4'-α-L- rhamnose ester (p -methoxy- trans -cinnamoyl-4'- α-L-rhamnose ester) - at least a second compound on the basis of spectral data p - methoxy - trans It was.

(6) chemical structural formula

Formula 3

Example 6 Structure Determination of Compound 3

The physicochemical and spectroscopic properties of Compound 3 isolated and purified in Example 3 were as follows.

(1) Appearance of matter: Colorless

(2) Molecular weight: EIMS (m / z) 149 [M ⁺ ]

(3) Molecular formula: C ₉ H ₈ O ₂

(4) Melting Point (mp): 133 °

(5) UV absorption spectrum UV (nm) 205, 224, 311

(6) Hydrogen Nuclear Magnetic Resonance Spectrum ¹ H-NMR (CD ₃ OD, 300 MHz) δ 6.47 (1H, d, J = 15.93, H-α), 7.39 (1H, × 3, m, H-3, 4 , 5), 7.59 (1H × 2, m, H-2, 6), 7.67 (1H, d, J = 15.93, H-β)

(7) Carbon nuclear magnetic resonance spectra ¹³ C NMR (CD ₃ OD, 100 MHz) δ 1198.38 (C-α), 129.18 (C-2, 6), 130.01 (C-3, 5), 131.40 (C-4 ), 13.86 (C-1), 146.30 (C-β), 170.33 (COOH)

Based on the above physicochemical and spectroscopic results, compound 3 was estimated as cinnamic acid and compared with literature.

(8) chemical structural formula

Formula 3

Example 7 Investigation of Neuronal Protective Activity of Compounds 1, 2, and 3

Neuroprotective activity of the isolated compounds was investigated using cerebral cortical cells of primary cultured rats that induced neurotoxicity with glutamate.

Concentration (μM) Survival rate (%) Control 100.0 ± 4.2 Comparative example 0.0 ± 1.3 Compound 1 0.1 66.4 ± 2.6 ^*** 1.0 78.8 ± 3.9 ^*** 10.0 72.5 ± 1.1 ^*** Compound 2 0.1 48.3 ± 3.3 ^** 1.0 77.6 ± 3.6 ^*** 10.0 36.6 ± 2.2 ^** Compound 3 0.1 38.5 ± 1.5 ^** 1.0 28.2 ± 2.9 ^* 10.0 24.7 ± 3.0

In the same manner as in Example 2, compounds 1, 2, and 3 were first-cultured in the screening system using the cerebral cortical cells of rats. As a result, as shown in Table 3, Compounds 1, 2, and 3 exhibited significant neuroprotective activity, showing 78.8%, 77.6%, and 28.4%, respectively, at concentrations of 1 μM.

Example 8 Investigation of Action Steps of Compounds 1, 2, and 3

In Example 7, the administration time of the compound was examined in order to determine the mechanism by which the compound of the present invention confirmed the neuronal protective activity to block neurotoxicity by glutamate.

That is, compounds 1, 2, and 3 were administered to the cerebral cortical cells of rats at different concentrations from 1 hour before the glutamate treatment to just before the glutamate treatment (pretreatment), the culture medium was exchanged with DMEM, and cultured for another 24 hours, followed by MTT analysis. Was implemented. The results are shown in Table 4 below.

Concentration (μM) Survival rate (%) Control 100.0 ± 4.2 Comparative example 0.0 ± 1.3 Compound 1 0.1 73.5 ± 5.1 ^*** 1.0 81.2 ± 2.6 ^*** 10.0 74.4 ± 4.3 ^*** Compound 2 0.1 69.5 ± 1.5 ^*** 1.0 89.6 ± 0.8 ^*** 10.0 50.0 ± 2.0 ^** Compound 3 0.1 39.4 ± 1.1 ^** 1.0 49.3 ± 4.2 ^*** 10.0 32.7 ± 1.9 ^*

In addition, after incubating the cerebral cortical cells of rats for 20 days, the glutamate for 15 minutes, and then incubated with DMEM while incubating the compound with concentrations of DMEM for 24 hours (recovery only) and MTT analysis The results are shown in Table 5 below.

Concentration (μM) Survival rate (%) Control 100.0 ± 4.2 Comparative example 0.0 ± 1.3 Compound 1 0.1 16.0 ± 0.6 1.0 19.8 ± 1.9 10.0 0.0 ± 3.2 Compound 2 0.1 3.3 ± 0.3 1.0 34.1 ± 0.9 ^* 10.0 22.0 ± 1.1 Compound 3 0.1 33.5 ± 5.1 ^* 1.0 42.1 ± 2.6 ^** 10.0 34.4 ± 2.3 ^*

As shown in Table 4 and Table 5, Compounds 1 and 2 showed an effect of protecting the neurons significantly when both before and after the treatment (throughout) or pre-treatment, but when the post-treatment treatment was inferior in neuroprotective activity I could confirm that. Compound 3, on the other hand, was thought to exhibit neuroprotective activity in a different mechanism from compounds 1 and 2, even though the time of administration was maintained significantly without increasing or decreasing its effect.

Example 9 Investigation of the Actions of Compounds 1, 2 and 3 with Glutamate Receptors

In order to determine whether the compounds 1, 2 and 3 of the present invention act on glutamate receptors and exhibit neuronal protective activity, N-methyl-D-aspartate (NMDA) and The action on non-NMDA receptors was examined.

That is, compounds 1, 2 and 3 were acted on the cerebral cortical cells of primary cultured rats at different concentrations, and after 1 hour, NMDA agonist and KA, a non-NMDA agonist, were administered to induce neurotoxicity. The effect of 3 on the induced neurotoxicity was examined (Table 6 and Table 7).

Concentration (μM) Survival rate (%) Control 100.0 ± 4.2 Comparative Example (NMDA-treated zone) ^a 0.0 ± 1.3 Compound 1 0.1 36.3 ± 1.3 ^** 1.0 60.0 ± 1.8 ^*** Compound 2 0.1 52.5 ± 1.7 ^*** 1.0 67.8 ± 2.6 ^*** Compound 3 0.1 36.1 ± 2.2 ^** 1.0 33.3 ± 3.1 ^*

Concentration (μM) Survival rate (%) Control 100.0 ± 4.2 Comparative Example (KA-treated Tool) ^a 0.0 ± 1.3 Compound 1 0.1 33.9 ± 2.3 ^* 1.0 29.7 ± 1.9 ^* Compound 2 0.1 5.0 ± 1.6 1.0 18.4 ± 1.1 Compound 3 0.1 17.5 ± 1.1 1.0 24.6 ± 0.6 ^*

As a result, Compound 2 significantly reduced the neurotoxicity induced by NMDA, but the effect of blocking the neurotoxicity induced by KA was insignificant, resulting in better selectivity of NMDA receptor than non-NMDA receptor among glutamate receptors. On the other hand, Compounds 1 and 3 showed an effect of significantly blocking neurotoxicity induced by NMDA, and significantly reduced neurotoxicity by KA, resulting in poor selectivity to NMDA or non-NMDA receptors compared to Compound 2. I could confirm that.

Example 10 Investigation of the Effects of Compounds 1, 2, and 3 on Intracellular Calcium Influx

In order to determine whether the compounds 1, 2 and 3 of the present invention affect the influx of calcium into the cells, the compounds were treated in rat cerebral cortical cells and then in the cortical cells according to the method of Grynkiewicz et al. The concentration of Ca ²⁺ was measured (Grynkiewicz, G. et al. (1985) J. Biol. Chem. 260 : 3440-3450).

Specifically, compounds 1, 2, and 3 were administered to the cerebral cortical cells cultured on glass cover slides for 20 days, and after 1 hour, 50 μM of glutamate was reacted for 15 minutes, and the culture solution was again phosphate buffered saline. The reaction was carried out at 5 μM FURA-2 AM for 1 hour, and then the slides were removed and placed in a spectrophotometer cuvettes containing 2.5 ml of physiological saline (excitation 340/380 nm, emission 520 nm). .

Concentration (μM) Intracellular [Ca ²⁺ ] (nM) Relative protection activity (%) Control 75.0 ± 15.0 100.0 Comparative Example (Glutamate-treatment) ^b 423.0 ± 20.0 0.0 Compound 1 0.1 160.6 ± 9.1 ^*** 75.4 1.0 174.8 ± 7.8 ^*** 71.6 Compound 2 0.1 178.0 ± 6.0 ^*** 70.4 1.0 159.9 ± 3.7 ^*** 75.6 Compound 3 0.1 256.2 ± 5.1 ^** 47.9 1.0 265.3 ± 9.2 ^** 45.3 MK-801 ^d 0.1 294.4 ± 4.1 ^** 37.0 1.0 136.7 ± 2.9 ^** 82.3

As shown in Table 8, Compounds 1, 2, and 3 significantly reduced the concentration of calcium introduced into cells due to glutamate (Table 8), in particular, Compounds 1 and 2 were known Ca ²⁺ channel blockers. It was found to have a lower EC ₅₀ than MK-801 (Ca ²⁺ channel blocker).

Example 11 Investigation of the Effect of Compounds 1, 2 and 3 on the Production of Nitric Oxide

As a result of Example 10, the compound of the present invention is expected to reduce to some extent the excessive generation of nitric oxide (NO) or free radicals following the activation of Ca ²⁺ -dependent enzyme. The effects on NO production were examined.

By pretreatment or post-treatment of cerebral cortical cells of rats incubated for 20 days at different concentrations of compounds 1, 2 and 3 by quantifying how it affects the amount of NO produced by glutamate treatment (Table 9 and Table 10).

Concentration (μM) Nitrite (nM) Relative protective activity (%) Control 65.5 ± 2.7 100.0 Comparative Example (Glutamate-treatment) ^b 153.7 ± 5.2 0.0 Compound 1 0.1 106.5 ± 8.9 ^** 53.5 1.0 111.2 ± 5.7 ^** 48.2 Compound 2 0.1 91.7 ± 6.3 ^*** 70.3 1.0 69.9 ± 2.4 ^*** 95.0 Compound 3 0.1 109.8 ± 1.7 ^** 49.8 1.0 104.1 ± 8.6 ^** 56.2 MK-801 ^d 0.1 103.9 ± 1.5 ^** 56.5 1.0 88.5 ± 4.1 ^*** 74.0

Concentration (μM) Nitrite (nM) Relative protective activity (%) Control 65.5 ± 2.7 100.0 Comparative Example (Glutamate-treatment) ^b 153.7 ± 5.2 0.0 Compound 1 0.1 116.3 ± 2.5 ^** 42.4 1.0 136.9 ± 3.0 19.0 Compound 2 0.1 102.6 ± 1.5 ^** 57.9 1.0 87.0 ± 2.0 ^*** 81.1 Compound 3 0.1 137.2 ± 1.2 18.7 1.0 115.3 ± 6.6 ^** 43.5 MK-801 ^d 0.1 100.7 ± 3.2 ^** 60.1 1.0 79.7 ± 3.5 ^*** 83.9

The amount of nitrite released from the primary cultured cerebral cortical cells into the culture was measured by Dawson et al.

As a result, Compound 2 reduced the amount of NO increased by glutamate to 95% at steady state when pretreated at 1 μM, and decreased to 80% at 1 μM even after ^{posttreatment} . In addition to the reduction effect of NO production due to the degradation, it was assumed that it would have an effect of directly acting on nitric oxide synthase (NOS) to reduce NO production. On the other hand, compounds 1 and 3 also significantly reduced the amount of NO produced excessively by glutamate, but the effect was much lower compared to compound 2.

As described above, the present ginseng extract and the cinnamate derivative of Formula 1 have a concentration-dependent activity to significantly protect neurons from neurotoxicity by glutamate, thereby preventing degenerative neurological diseases such as stroke and dementia. It can be useful for treatment.

In addition, the cinnamate derivatives of the compounds 1, 2 and 3 of the present invention exhibit neuroprotective activity primarily by decreasing the concentration of Ca ²⁺ excessively introduced into cells due to glutamate. By reducing the amount of NO produced excessively, there is an effect that blocks the continuous death of neurons.

Claims (5)

Cinnamate derivatives for neuronal cell protective agents having a structure of Formula 1 and pharmaceutically acceptable inorganic or organic salts, hydrates, solvates and isomers thereof. Formula 1 R ₁ is CH ₃ , or H, and R ₂ is H or α-L-Rhamnose. The method of claim 1, wherein the cinnamate derivative to the general formula (2) p-methoxy-trans-cinnamic acid to (p -methoxy- trans -cinnamic acid), of formula 3 p-methoxy-trans-cinnamoyl-4 ' Cinnamate derivatives for neuronal cell protective agents, characterized in that the p- methoxy- trans- cinnamoyl-4'-α-L-rhamnose ester or cinnamic acid of the formula And pharmaceutically acceptable inorganic or organic salts, hydrates, solvates and isomers thereof. Formula 2 Formula 3 Formula 4 Brown ginseng is extracted with a mixed solvent of chloroform-methanol, fractionated with dichloromethane, and then suspended in methanol. A method for producing a cinnamate derivative according to claim 1. A pharmaceutical composition for preventing or treating stroke and degenerative nervous system diseases, comprising the cinnamate derivative of claim 1 as an active ingredient. A pharmaceutical composition for neuroprotective agents, wherein the extract is extracted with a mixed solvent of chloroform-methanol, or fractionated with dichloromethane, suspended in methanol, and extracted with hexane.