KR100487112B1 - A new sphingolipid, (4E, 6E, 2S, 3R)-2-N-Eicosanoyl-4,6-tetradeca sphingenine of Beauveria bassiana 101A having enhancement of the neurite outgrowth - Google Patents

Abstract

The present invention (4E, 6E, 2S, 3R) -2-is a novel brain neuroactive substance isolated and purified from the extract of Beauveria bassiana 101A in order to treat and prevent degenerative brain diseases related to neuronal growth and differentiation. N-aicosanoyl-4,6-tetradecaspingenin and pharmaceutical preparations thereof.

In the present invention the white sleep The extract of 101A and its active ingredient were found to be used for PC augmentation and neurotrophic factor protein growth promotion and neurotrophic factor protein enhancement, which are important in degenerative brain diseases. Degeneratives such as Alzheimer's, Parkinson's, and strokes by providing (4E, 6E, 2S, 3R) -2-N-aicosanoyl-4,6-tetradecaspingenin, a new compound isolated from Alpase 101A that is not cytotoxic. It has reduced the nerve cell atrophy and damage caused by brain diseases, and is expected to improve the national health and serve as a new income source for sheep farmers.

Description

Cerebral nerve growth promoter (4E, 6E, 2S, 3R) -2-N-aicosanoyl-4,6-tetradecaspingenin, isolated from purified Alpase 101A (4E, 6E, 2S, 3R) ) -2-N-Eicosanoyl-4,6-tetradeca sphingenine of Beauveria bassiana 101A having enhancement of the neurite outgrowth}

The present invention (4E, 6E, 2S, 3R) -2-is a novel brain neuroactive substance isolated and purified from the extract of Beauveria bassiana 101A in order to treat and prevent degenerative brain diseases related to neuronal growth and differentiation. N-aicosanoyl-4,6-tetradecaspingenin ((4E, 6E, 2S, 3R) -2-N-Eicosanoyl-4,6-tetradeca sphingenine) and pharmaceutical preparations thereof.

Silkworm (Bombyx mori, nuegwa Bombycidae) when a period of time bred under then inoculated baekganggyun 101A to the skin of the larvae an appropriate temperature and humidity conditions, and the mycelium is formed at the silkworm worm indicates a degradation phenomenon there is die eventually baekgangjam the dead worms (Bombycis corpus ). As a prescriptive example of Paik sleep, it has been used to treat orbital wasabi, children's game, headache, pulmonary tuberculosis due to stroke, and ¹⁾ clinical reports include diabetes and urgent mastitis.

In addition, the white nap has excellent whitening effect and is used as a component of whitening agents such as cosmetic compositions, and is also used as a main raw material of microbial insecticides.

In Korean Patent Laid-Open Publication No. 99-85202, Baek sleep was used in combination with various herbal medicines to prepare a composition for preventing and treating dementia.

However, it has not been found that baekpja contains active substances for the treatment and prevention of degenerative brain diseases, and it has been reported to separate the various constituents of baekpjap extract, and to report the function of each active ingredient. It was not done.

In addition, the necessity of a substance having a function to treat and prevent degenerative brain diseases without cytotoxicity associated with cerebral nerve cells has emerged.

Nerve growth factor (NGF) is a neurodegenerative brain that stimulates the growth of neurites in nerve cells and plays an important role in the survival and maintenance of nerves in the central nervous system. It is estimated to have good efficacy in the treatment of diseases. Recently, animal studies have shown that NGF may stop or delay the progression of cholinergic whole brain degeneration associated with Alzheimer's disease. ^{4), 5) The} trophic factors, including NGF, are known to be the most powerful approaches to treat neurodegenerative diseases by engaging in processes such as neuronal differentiation and cell death. At the same time, NGF stimulates cholinergic nerves that are known to die during Alzheimer's disease, improving cognitive and behavioral capabilities and inhibiting the degeneration of the nervous system. Clinical studies have reported that injection of NGF into the skull improves the patient's verbal memory. ^{6), 7)} Therefore ^, it is a reasonable approach to select NGF or compounds with neuroactive effects as a means of treating Alzheimer's disease ^. Therefore, the new therapeutic potential and applicability of these similar compounds may be expanded. However, because endogenous NGF is a very high molecular weight peptide polymer (polypeptide) it does not cross the blood brain barrier (BBB). Therefore, in order to treat diseases caused by the brain due to NGF deficiency, it is necessary to inject the brain directly to maximize the therapeutic effect, which is not easy to use. In addition, when administered by a method such as intraperitoneal injection, it is easily metabolized by proteolytic enzymes, so the efficacy cannot be expected. Therefore, the derivation of low molecular weight compounds with neuronal activity from neurite outgrowth enhancement from natural products can be said to be meaningful as a search method for the treatment of degenerative brain diseases.

Accordingly, the inventors of the present invention confirmed that the baekpja 101A enhances neuronal growth with little cytotoxicity, and the present invention was completed based on this.

Accordingly, it is an object of the present invention to provide a novel compound having the effect of preventing and treating degenerative brain diseases related to neuronal growth and differentiation from purified Alpaca 101A extract.

That is, the object of the present invention is (4E, 6E, 2S, 3R) -2-N-aicosanoyl-4,6-tetradecaspinnin ((4E, 6E, 2S, 3R) -2-N -Eicosanoyl-4,6-tetradecasphingenine).

In addition, the present invention (4E, 6E, 2S, 3R) -2-N-aikosanoyl-4, which has neuroprotective activity such as senile dementia by removing the cytotoxicity of Baekja, while having neuronal growth promoting activity A pharmaceutical formulation of 6-tetradecaspinnin.

Hereinafter, the present invention will be described in detail.

Baekgangjam 101A used in the present invention was obtained by newly inoculating the skin of the larvae and breeding under a certain condition by newly developing Beauveria bassiana 101A, a subspecies of Baekganggyun, from the Provincial Insecticide Division of the Rural Development Administration of Korea.

Baekjaerum 101A extract according to the present invention is prepared by extracting concentrated and lyophilized Baekjaerum 101A with water, lower alcohol, ethyl acetate or a mixed solvent thereof. Ultrasonic extraction, filtration, reflux extraction may be used as a method for preparing the extract of the white nap 101A, and in addition, it may be prepared using a conventionally used extraction method. In addition, the concentration of the extract, fraction preparation and freeze-drying process can also be carried out by commonly known methods.

The extract of Alpase 101A is a physiologically active component extracted from water, alcohol or water and alcohol mixed solvent and then separated from fractions except for cytotoxic fraction, wherein one of the isolated and purified brain neurons is (4E, 6E, 2S, 3R) -2-N-aicosanoyl-4,6-tetradecaspinnin (C ₃₄ H ₆₅ NO ₃ ).

Therefore, the present invention relates to (4E, 6E, 2S, 3R) -2-N-aicosanoyl-4,6-tetradecaspingenin, which is a brain neuroactive substance isolated and purified from baekjapja extract represented by the following formula (1): .

The compound was an amorphous white powder and identified by the HR-FAB-MS spectrum ([M + Na] ⁺ m / z 558.4874) as having a molecular formula of C ₃₄ H ₆₅ NO _3. The chemical structure of the active material was determined using NMR techniques ( 4E, 6E, 2S, 3R) -2-N-aicosanoyl-4,6-tetradecaspingenin. This is the first substance reported in nature. The compounds extracted in the present invention can be applied to neurodegenerative brain diseases related to neuronal nutrients by showing neuronal growth enhancing effects such as neurotrophic factor and also replacing their role.

PC12 cells are a noradrenergic cell line of rat pheochromocytoma origin, which are sensitive to NGF and have many characteristics of normal sympathetic neurons, thus in vitro in NGF mechanism research. It is used as a model. In other words, PC12 cell line divides in the medium containing serum and shows neuronal axon growth when NGF is added to the medium, but when killed in the absence of serum, it dies. The cell death is also known to be inhibited by NGF. ought. ^{8) The} treatment of PC12 cells with methanol extracts of baekpseum 101A and its small fractions, ie, hexane fractions, showed significant neuronal growth promoting effects.

In addition, the present invention relates to a pharmaceutical preparation of (4E, 6E, 2S, 3R) -2-N-aicosanoyl-4,6-tetradecaspingenin, which is a novel compound isolated and purified from the extract of Alpase 101A. .

Pharmaceutical preparations for the treatment and prevention of brain diseases of the present invention containing (4E, 6E, 2S, 3R) -2-N-aicosanoyl-4,6-tetradecaspingenin as active ingredients Fillers, extenders, binders, which are commonly used in the formulation, can be administered orally or parenterally. It may be prepared using diluents or excipients such as disintegrants and surfactants. Such solid preparations include tablets, powders, pills, granules, capsules, and the like, which may be prepared by mixing at least one excipient with a sample of Bacillus subtilis. Oral liquid preparations include suspensions, liquid solutions, emulsions, syrups, etc. Various excipients may be used in addition to commonly used simple diluents such as water and liquid paraffin. As a base of suppositories, Uittepsol and Macrogol tween 61. may be used.

Hereinafter, the present invention will be described in detail with reference to the following examples, but the scope of the present invention is not limited thereto.

Example 1 Preparation of Baeksijam 101A Extract

Baekgangjam 101A was supplied by the Rural Development Administration's Sasa Insects. 1.5 kg of dry powder 101A dry powder was repeatedly extracted with methanol at room temperature five times and extracted three times at 60 ° C. to obtain 120 g of methanol extract. The average yield was over 8%.

Example 2 White Sleep Fraction preparation of 101A suspension

The methanol extract, concentrated under reduced pressure, was suspended in H ₂ O, and 55 g of a fraction was obtained with hexane (800 mL × 3). As a result of measuring the NGF activity on the fraction (Table 1), it was confirmed that the activity is excellent in the hexane fraction, and the active component was separated from the fraction.

Example 3 Single Compound Separation from Beauveria bassiana 101A

Hexane-ethyl acetate-methanol mixed solvent (5: 1: 0, 3: 1: 0, 1: 1: 0, 10: 10: 1, 5: 5: 1, 2: 5: 1 and 55 g of hexane fraction) Silica gel chromatography was carried out with increasing methanol content, divided into four small fractions (H-1 to H-4), and then the H-1 fraction (30 g) was added to hexane-ethyl acetate (3: Three small fractions (H-11 to H-13) were obtained by subjecting 1) to silica gel column chromatography using effluent solvent. Separdex LH-20 chromatography was performed on H-13 fraction (4.6 g) using dichloromethane-methanol (1: 1) as an eluent ^. A column chromatography (90% MeOH) was carried out to give compound (15 mg).

Example 4 Instrumental Analysis of a Single Compound

¹ H- and ¹³ C-NMR spectra were measured on a Brucker AMX 500 spectrometer and MS spectra on a VG70-VSEQ (VG Analytical, UK). Solvents for extraction and column chromatography were used as primary reagents and other reagents as either primary or express. Silica gel for column chromatography was Kiesel gel 60 (70-230 and 230-400mesh, ASTM Art. 7734 and 9385, Merck), and the filler for reverse phase column chromatography was LiChroprep RP-18 (40-63). Μm, Art. 13900, Merck). LPLC used a LiChroprep Si60 Lobar-B column (Art. 10401, Merck). TLC plate was used Kiesel gel 60 F ₂₅₄ precoated plate (Art. 5554, Merck).

Example 5 Structural Name of Compound

Through the instrument analysis, the structure was determined as follows.

(4E, 6E, 2S, 3R) -2-N-aicosanoyl-4,6-tetradecaspinnin

White powder, mp. 74 ° C., [α] _D ²⁰ + 3.2 ° (c. 0.05, CHCl ₃ ), nm (logε): 233 (4.23), cm ^-1 : 3340, 2930, 2850, 1648, 1625, 1535, 1470, 1070, 1050, EI-MS m / z (rel. int.): 535 (M +, 1), 517 (10), 487 (19 ), 388 (15), 354 (17), 337 (90), 312 (24), 306 (20), 224 (38), 206 (22), 193 (21), 60 (100), HR-FAB -MS: C ₃₄ H ₆₅ NO ₃ Na Found: 558.4874 Calcd. : 558.4862, FAB-CID MS m / z (rel. Int.): 558 (100), 537 (1.9), 528 (0.9), 514 (0.7), 500 (0.7), 486 (1.0), 472 (0.9 ), 458 (1.1), 444 (0.8), 430 (1.0), 416 (0.8), 402 (0.7), 388 (0.8), 374 (1.0), 358 (1.2), 332 (1.0), 318 (2.0) ), 206 (0.6), ¹ H-NMR (500 MHz, CDCl ₃ ): 0.88 (6H, t, J = 7.0 Hz, H-14 and H-20), 1.20-1.39 (42H, m, H-9 -H-13, H-4-H-19), 1.61 (2H, br.sep, J ≒ 7.5 Hz, H-3), 2.06 (2H, m, H-8), 2.22 (2H, t, J = 7.5 Hz, H-2), 2.74 (1H, br.s, OH), 2.88 (1H, br.s, OH), 3.70 (1H, br.d, J = 10.5 Hz, H-1), 3.89 -3.96 (2H, m, H-1 and H-2), 4.39 (1H, br.s, H-3), 5.61 (1H, dd, J = 15.0, 6.0 Hz, H-4), 5.73 (1H , dt, J = 15.0, 7.5 Hz, H-7), 6.03 (1H, dd, J = 15.0, 10.5 Hz, H-6), 6.26 (1H, br.d, J = 7.0 Hz, NH), 6.29 (1H, dd, J = 15.0, 10.5 Hz, H-5), 13C-NMR (125 MHz, CDCl3): 14.78, 14.81 (C-14, C-20), 23.35, 23.39 (C-13, C- 19), 26.46 (C-3), 29.85, 29.87, 29.90, 29.98, 30.03, 30.06, 30.20, 30.27, 30.34, 30.36, 30.40 (C-9 to C-13, C-4 to C-17), 32.51 , 32.64 (C-12, C-18), 33.35 (C-8), 37.55 (C-2), 55.16 (C-2), 63.21 (C-1), 75.26 (C-3), 129.62, 129.64 (C-5, C-6), 133.46 (C-4), 137.50 (C-7), 174.60 ( C-1)

The compound was identified as a molecular formula C ₃₄ H ₆₅ NO _{3 by the} HR-FAB-MS spectrum ([M + Na] ⁺ m / z 558.4874) as an amorphous white powder. In the IR spectrum it was observed the absorption band of the amide group at 1648cm ^-1 and the absorption band of hydroxyl groups at 3340cm ^{-1. 1} H-NMR spectra at δ3.70 (1H, br.d, J = 10.5 Hz), 3.89~3.96 (2H, m) and 4.39 δ55 at the peak and ¹³ C-NMR spectra (1H, br.s) Peaks of .16, 63.21 and 75.26 are typical peaks of 2-amino-1,3-diol groups in the hydrocarbon chain, suggesting that the compounds have a structure similar to sphingosine derivatives. . ¹⁰⁾ Except for the peaks above, in the ¹ H-NMR spectrum, δ 0.88 (6H, t, J = 7.0 Hz), 1.20-1.39 (42H, m), 1.61 (2H, br. Sep, J ≒ 7.5 Hz), Peaks of aliphatic hydrocarbon chains at 2.06 (2H, m) and 2.22 (2H, t, J = 7.5 Hz), δ 5.61 (1H, dd, J = 15.0, 6.0 Hz), 5.73 (1H, dt, J = 15.0 , 7.5 Hz), 6.03 (1H, dd, J = 15.0, 10.5 Hz), 6.29 (1H, dd, J = 15.0, 10.5 Hz) were able to observe the peaks of the four unsaturated hydrogens conjugated to each other. ^{In the 13} C-NMR spectrum, two terminal methyl peaks at δ14.78 and 14.81, double bond carbon peaks at δ 129.62, 129.64, 133.46 and 137.50, and amide carbon peaks at 174.60 were observed. The location and geometry of the double bond can be determined from the coupling constant and ¹ H- ¹ H COSY data. Fatty acid esters obtained through acid methanolysis of the compounds were identified as eicosanoyl methyl esters by GC-MS analysis. The CID (Collision-induced dissociation) spectrum of the compound's [M + Na] ⁺ ion in FAB-MS showed a main ion peak at m / z 318 (296 + Na-H). Through this, it could be estimated that the compound is a fatty acid amide derivative of C ₁₄ amino alcohol. ¹¹⁾ The optical rotation of the compound (+ 3.2 °) sphingosine derivatives ^{12), 13) as} a result of the three-dimensional structure of the compound was confirmed to be 2S, 3R. Based on the above data, the structure of the compound was determined to be (4E, 6E, 2S, 3R) -2-N-aicosanoyl-4,6-tetradecaspingenin.

[Test Example 1]

<Cell Culture>

Inoculated with 3X10 ⁴ PC12 cells in a 100 mm culture dish (TPP, Switzerland), followed by 2 g / L sodium bicarbonate, 5% horse serum, 10% fetal bovine serum (FBS) And penicillin-streptomycin (10000 U / ml) 1% in RPMI1640 medium. The FBS used at this time was used by heat inactivation (heat-inactivation) at 56 ℃ 30 minutes before use. In addition, 5% CO ₂ and O ₂ was incubated after maintaining the conditions of 70% humidity and 37 ℃ temperature in 95% incubator. The medium was replaced with fresh medium every 10 ml, and all reagents used for cell culture were GIBCO BRL.

<Process of sample>

When cells became 1x10 ⁷ cells / well in a 100 mm culture vessel, 500 μl of 0.25% Trypsin-EDTA (Gobco BRL, USA) solution was used to separate the cells from the culture vessel and trypsin-EDTA with 10 ml fresh medium. The solution was neutralized and cell suspension was obtained. 10 μl of 0.4% Tryphan blue (in PBS buffer) was added to 20 μl of the cell suspension, and the number of living cells was counted using a hemocytometer. Poly-D-lysine (Sigma, USA, 50 ug / ㎖) was diluted with DPBS, and then added to the 2 well in 6 well incubator and left at 37 ℃ for more than 1 hour. After 1 hour, the cells were washed with DPBS (pH.4), and then inoculated in a culture vessel at a concentration of 1x10 ⁵ cells / well in 6well dried. The sample was dissolved in DMSO (Dimethylsulfoxide, Sigma, USA) to make its final concentration less than 0.1% and filtered using a 0.22μM sterilization filter. After 24 hours of inoculation, the final concentrations of the compounds were treated at 10 μM and NGF at 50 ng / ml, respectively. After culturing for 6 days in the incubator maintained at a constant temperature and humidity under 5% CO ₂ and 95% O ₂ mixed air, the degree of neurites growth was observed and added to a fresh medium every 2 days.

Nerve dendritic growth measurement

24 hours after inoculation, the cells were replaced with fresh medium supplemented with 1% FBS and 2% HS. After 24 hours, the degree of neurites grown from the cell body was observed using a phase contrast microscope at a constant time every day, and the length was measured using a printed photograph after taking a photomicrograph using the phase contrast microscope. The effect on neurite outgrowth is set to 0 if the neurite length is not visible, + if the diameter of the cell body is the same as +, and ++ if it has grown to twice the diameter of the neuronal cell body, and more. When the length is digitized + +. Table 10 shows the results of measuring the growth rate of neurites of each cell by selecting 10 cells per well.

The results of treatment of PC12 cells with methanol extracts of Bacillus 101A and small fractions, hexane fractions, fractionated according to polarity according to the present invention are shown in Table 1.

TABLE 1

The effect of leuk sleep 101A extract on PC12 cell line was measured on the effect of neuronal growth.

sample Neuronal cell growth White Sleep 101A B. corpus 101A NGF (50 ng.ml) +++ Methanol (10 μg / ml) + Hexane fraction ++ Chlorform + Butanol fraction ++

PC12 cells in 6 well plates were treated with subfraction and NGF, and neurite outgrowth was measured under a microscope at 6 days post treatment. Fresh medium with compounds or NGF was changed every 2 days. Randomly selected fields were taken by the camera attached to light microscope. +; Processes with lengths equivalent to one diameters of the cell body, ++; Processes with lengths equivalent to two diameters of the cell body, +++; Processes with lengths equivalent to three diameters of the cell body

As a result of Table 1, it can be seen that the napja 101A methanol extract of the present invention has a neuronal growth promoting effect.

1 is a compound (4E, 6E, 2S, 3R) -2-N-aikosano, which is a compound which has been characterized by separating one species of sphingosine by conducting an active ingredient study from a hexane fraction having excellent neurotrophic activity. To show the results of searching for the effect of promoting neuronal growth against the day-4,6-tetradecaspinnin, (4E, 6E, 2S, 3R) -2-N-aicosanoyl-4,6 according to the present invention Tetradecaspinnin has a neuronal growth promoting effect.

<NGF gene expression confirmation>

RNA isolation

Total cellular RNA was isolated using Trizol solution (Gibco BRL). After lysing the cells by treatment with 1 ml of Trizol solution, the cell lysates obtained by scraping with a cell scraper (TPP, switzerland) were collected in a 1.5 ml microtube and homogenized by a 21G syringe. . Cell homogenate was added to the supernatant obtained by centrifuging at 12,000 rpm for 10 minutes at 4 ° C in the same amount, and then allowed to stand at room temperature for 15 minutes, and then centrifuged at 4 ° C and 12,000 rpm for 15 minutes. The supernatant, or RNA layer, was taken and transferred to a new microcentrifuge tube. An equal amount of 100% isopropyl alcohol was added and mixed, followed by centrifugation at 4 ° C. and 12,000 rpm for 15 minutes. The supernatant was removed and the RNA pellet was washed with 1.5 ml 75% ethanol and centrifuged at 12,000 rpm for 5 minutes at 4 ° C. After completely drying at room temperature for 10 minutes, RNA was dissolved in 100 μl of diethylpyrocarbonate (DEPC) solution, and then measured at UV 260 nm and 240 nm using a spectrophotometer (Hewlett Packard, U.S.A.).

Test Example 2 Acute Toxicity Test

Baekjap prepared in the above embodiment Before using 101A, put the bran in the hot pot until it turns yellow, remove it and sift to remove the bran, and then the rats were examined for toxicity by oral and intraperitoneal administration.

1. Oral administration

10 ICR mice and 10 SD rats 101A extract was administered orally at doses of 100, 250, 500 and 1000 mg / kg. Two weeks after oral administration, toxicity was not observed in any group and no symptoms were found in the control group.

2. Intraperitoneal administration

Oral administration of 10 ICR mice and 10 SD rats 101A extract was intraperitoneally administered at doses of 25, 100, 250 and 500 mg / kg. After 24 hours of intraperitoneal administration, two mice died at 500 mg / kg, and no surviving animals were found.

As confirmed through the above-described results, the effect of promoting the neuronal growth of the extract and its components of Baekseuk 101A was very excellent.

(4E, 6E, 2S, 3R) -2-N-aicosanoyl-4,6-tetradecaspingenin and its pharmaceutical preparations according to the present invention are neuronal axons importantly involved in degenerative brain diseases. Promotes dendritic growth and enhances neurotrophic factor protein and has no cytotoxicity, alleviating neuronal atrophy and damage caused by degenerative brain diseases such as Alzheimer's, Parkinson's, and stroke. The role is expected.

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Figure 1 Whooping on PC 12 Cell Line Shows the effect on neuronal growth after treatment of novel compounds isolated from 101A

PC 12 cells in 6-well plates were treated with compound and NGF, and neurite outgrowth was measured under a microscope at 6d-post treatment. Fresh medium with compounds or NGF was changed every day. Each value represents the mean + S.D. (n = 30). ** Significantly different from NGF value at the level of p <0.01. *** Significantly different from NGF value at the level of p <0.001.

Claims (2)

A novel compound, which is a brain nerve growth promoting substance, isolated and purified from the extract of Beauveria bassiana 101A, which is represented by the following formula (4E, 6E, 2S, 3R) -2-N-aicosanoyl-4,6 Tetradecaspingenin. [Formula 1] The method of claim 1, wherein the stroke, cerebral ischemia, Parkinson's, senile dementia, and Huntington's disease using the (4E, 6E, 2S, 3R) -2-N-aicosanoyl-4,6-tetradecaspingenin as an active ingredient Pharmaceutical formulations for the treatment of brain diseases, including.