KR101818084B1 - A composition comprising extract of Lycopodiella cernua or compound isolated therefrom for preventing or treating of Alzheimer disease-Ⅱ - Google Patents

Abstract

The present invention relates to a composition for preventing or treating Alzheimer's disease comprising a calcined ginger extract or a compound isolated therefrom, wherein the calcined ginger extract or the compound isolated therefrom is selected from the group consisting of acetylcholinesterase, butyrylcholinesterase, The first inhibitory effect of Crete is excellent and can be effectively used as a composition for preventing or treating Alzheimer's disease.

Description

A composition for preventing or treating Alzheimer's disease comprising a calcineurin extract or a compound isolated therefrom, the composition comprising extract of Lachopodiella cernua or compound isolated therefrom for treating or treating Alzheimer disease-

The present invention relates to a composition for preventing or treating Alzheimer's disease comprising Lycopodiella cernua extract or a compound isolated therefrom.

With the rapid development of life sciences and medicine, the average life span of humans has increased, and the proportion of the elderly population has increased, and new social problems are emerging. In particular, geriatric neurological diseases such as stroke, Alzheimer disease (AD), Parkinson disease (PD) and the like are manifest as dysfunctional nervous system dysfunctions. Until now, there is no effective way to prevent this, And a considerable amount of medical expenses.

In particular, the most common of the geriatric neurological diseases is Alzheimer's disease, the most common degenerative brain disease that causes dementia, and is a brain disease that causes memory, thinking and behavior problems. Alzheimer 's disease is estimated to account for 60 to 80 percent of cases of dementia, and dementia is a general term that means loss of memory and other intellectual abilities that are so severe as to interfere with daily life. In the United States, the number of Americans with Alzheimer's and other dementia is expected to grow each year as more than 5 million people have Alzheimer's disease and the proportion of people over 65 in the United States continues to grow.

The mechanism and the cause of Alzheimer's disease have not yet been well known. To date, there has been no report on reduction of the synthesis of acetylcholine, deposition of beta-amyloid, hyperphosphorylation of tau protein (2002) reported that the neuronal damage was mainly caused by injury to the nerve cells (Lee et al., 2008).

Acetylcholine is one of the neurotransmitters and is found mostly at the junction of the neurons, the neurons, and the skeletal muscle. In addition, the acetylcholine secreted from the nerve end is decomposed into choline and acetic acid by acetylcholinesterase (AChE) after the transmission of the stimulation is terminated, and the choline is released by the action of cholinacetylase And then acetylcholine. Acetylcholinesterase inhibitors support signal transduction between neurons by blocking the degradation of acetylcholine, an important chemical messenger for memory and learning, and maintaining high levels of acetylcholine. In addition, a recent study found that butyrylcholinesterase (BChE) degrades acetylcholine in the brain and plays a role in cholinergic transmission. Therefore, the inhibition of acetylcholinesterase and butyrylcholinesterase increases the concentration of acetylcholine in the synaptic gap, thereby enhancing neurotransmission of acetylcholine and improving deficiency.

Until now, the fundamental treatment method of Alzheimer's disease has not been developed yet. However, acetylcholinesterase inhibitor is most commonly used in various countries. It can not completely prevent progression of the disease, and alleviates or slows the progress of some pathological symptoms. It only has an effect. Drugs in this line include donepezil, rivastingmine, galantamine, and tacrine. Acetylcholinesterase inhibitors, which have fewer side effects and have more effective side effects, will develop drugs and functional materials using various natural products in accordance with research trends in which acetylcholinesterase is most widely used as a pharmacological action point in the development of therapeutic agents worldwide There is a need.

In addition, β-secretase 1 acts on amyloid-β-precursor protein (APP) to produce β-amyloid fragments. In addition, since accumulation of? -Amyloid is known to be a major cause of Alzheimer's disease, the development of a substance against the inhibitory activity of? -Secretase may prevent the amyloid precursor protein from being cleaved and may be useful for the treatment of Alzheimer's disease have. In the case of? -Secratratase inhibitors, various inhibitors can be easily developed because of their clear structure by x-ray crystallography, but it is not yet possible to use them as therapeutic agents to be. Therefore, the development of new β-secretagogue inhibitors will be of great help in the treatment of Alzheimer's disease.

Lycopodiella cernua ( Lycopodiella cernua ) is a river of spore-forming vascular plants belonging to the Lycopodium and other similar plants. It is widespread throughout the world and consists of 4 genera, about 1,000 species. The plants of this river are small in size and grow up on other plants by standing upright or using instruments. The stem is divided into two branches and pruned. The root, stem and leaf are distinct and photosynthesis is possible. The sporangium is a special leaf called a sporophyll, which collects into conical spores. It is an evergreen perennial plant that lives in Jeju, Jeonnam, Hambuk, Hamnam, and Gangwon. It is distributed from temperate regions of northern hemisphere such as Japan, Taiwan, China, Kuril, Europe and Africa.

In China, chrysanthemum has traditionally been used as a treatment for rheumatism, pertussis, hepatitis, kidney stones and bruises (Zhang, XC et al., 2004; Xiao, PG, 2002) . Korean Patent Laid-Open Publication No. 2011-0118145 also shows that lycopoduim clavatum extract or compounds isolated therefrom are effective for cancer treatment, and the antimemolytic effect of plant bases isolated from lycopersicin (Chuong, NN et al., 2014), and the anticholinesterase activity of plant bases isolated from the South American rice germplasm (Konrath, EL et al., 2013). However, it is not yet known that the compounds isolated from the calcined gut of the present invention have an inhibitory effect on acetylcholinesterase or butyrylcholinesterase or? -Secretase 1.

Korean Patent Publication No. 2011-0118145, composition and application method thereof, disclosed on October 28, 2011.

Zhang, X. C. et al., Flora of China; Science Press: Beijing, 6, 70, 2004. Xiao, P. G., Modern Chinese Material Medica; Chemistry and Industry Press: Beijing, 3, 135, 2003. Chuong, N. N. et al., Anti-amnesic effect of alkaloid fraction from Lycopodiella cernua (L.) Pic. Serm. on scopolamine-induced memory impairment in mice, Neuroscience letters, 575, 42, 2014. Konrath, E. L. et al., Alkaloid profiling and anticholinesterase activity of South American Lycopodiaceae species, J. Enzyme. Inhib. Med. Chem., 28, 218, 2013. Eunhee et al., Effects of acetylcholine ester race inhibitors on cerebrospinal β-amyloid 1-42 fraction and phosphorylated tau protein concentration in Korean Alzheimer's disease patients, Korean Journal of Neurology, 25, 224, 2008. Jonghyoung, Development of therapeutic drug for Alzheimer's disease

It is an object of the present invention to provide a composition for preventing or treating Alzheimer's disease comprising a calcined ginger extract or a compound isolated therefrom.

The present invention relates to a composition for preventing or treating Alzheimer's disease comprising Lycopodiella cernua extract or a compound isolated therefrom. More specifically, the present invention relates to a process for producing a compound represented by the following formula (1): rhamnazine (compound 1), quercetin 3,3'-dimethyl ether (compound 2), guaiacyl glycerol (compound 3), apigenin 5), Bahia genin -4'- O - (2 '', 6 '' - di-p -O- - Kumar in one) -β-D- gluconic nose Llano side (compound 6), spring Basin -4- O - β - D - Glu nose Llano side (compound 7), (7 R, 8 S) -7,8- dihydro-8-hydroxy-7- (3-methoxyphenyl -4- O - β - D- gluconic nose Llano side) 1'-benzofuran propionic acid (compound 8), Bahia genin -4'- O - (6 '' - O - p - one to Kumar) - β -D- gluconic nose Llano side ( (Compound 9), dihydrodihydroconidyl alcohol-4- O - ? - D-glucopyranoside (Compound 10) and Sharposide (Compound 11) For the prevention or treatment of Alzheimer's disease characterized by containing Lycopodiella cernua extract ≪ / RTI >

[Chemical Formula 1]

The calcined calcium extract is an extract obtained by extracting calcined steel with at least one solvent selected from the group consisting of water, C1 to C4 lower alcohols, acetone, n-hexane, dichloromethane and ethyl acetate.

The present invention relates to a pharmaceutical composition comprising Rhamnazin (Compound 1), Quercetin 3,3'-dimethyl ether (Compound 2), Guaiacyl Glycerol (Compound 3), Apigenin (Compound 4) isolated from Lycopodiella cernua ), Isosaphthoside (Compound 5), apigenin-4'- O- (2 '', 6 '' -di-O- p -coumaroyl) -? - D-glucopyranoside ), spring Basin -4- O - β - D - Glu nose Llano side (compound 7), (7 R, 8 S) -7,8- dihydro-8-hydroxy-7- (3-methoxy phenyl -4- O - β -D- gluconic nose Llano side) 1'-benzofuran propionic acid (compound 8), Bahia genin -4'- O - (6 '' - O - p - Kumar in one) - β -D-glucopyranoside (Compound 9), dihydrodihydroconiferyl alcohol-4- O- beta -D-glucopyranoside (Compound 10) and Sharposide (Compound 11) A composition for preventing or treating Alzheimer's disease characterized by comprising at least one compound .

The compound is characterized by inhibiting at least one enzyme activity selected from the group consisting of acetylcholinesterase, butyrylcholinesterase and? -Secretase 1.

The present invention also relates to a process for the production of quinacridone (compound 1), quercetin 3,3'-dimethyl ether (compound 2), guaiacyl glycerol (compound 3), apigenin (compound 4) iso Sharp sat side (compound 5), Bahia genin -4'- O - (2 '', 6 '' - di-p -O- - a daily Kumar) -β-D- gluconic nose Llano side (compound 6), Basin spring -4- O - β - D - Glu nose Llano side (compound 7), (7 R, 8 S) -7,8- dihydro-8-hydroxy-7- (3-methoxyphenyl- 4- O - β -D- gluconic nose Llano side) 1'-benzofuran propionic acid (compound 8), Bahia genin -4'- O - (6 '' - O - p - one to Kumar) - β -D -Glucopyranoside (Compound 9), dihydrodihydroconiferyl alcohol-4- O - ? - D-glucopyranoside (Compound 10) and Sharposide (Compound 11) The present invention relates to an Alzheimer ' s < RTI ID = 0.0 > ( Lycopodiella cernua ) And a health functional food for preventing or improving diseases.

In another aspect, the present invention provides a method for producing Lycopodiella cernua , comprising the steps of: preparing Lycopodiella cernua with water, C1-C4 lower alcohol or a mixed solvent thereof; Sequentially fractionating the calcined gum extract with n-hexane and ethyl acetate, and then fractionating the residue with dichloromethane; And the water layer excluding the re-fractionated dichloromethane was chromatographed on a column of rhamnazine (compound 1), quercetin 3,3'-dimethyl ether (compound 2), guaiacyl glycerol (compound 3), apigenin Compound 4), isosapphthaside (Compound 5), apigenin-4'- O- (2 '', 6 '' - di-O- p -coumaroyl) -? - D-glucopyranoside compound 6), spring Basin -4- O - β - D - Glu nose Llano side (compound 7), (7 R, 8 S) -7,8- dihydro-8-hydroxy-7- (3 methoxyphenyl -4- O - β -D- gluconic nose Llano side) 1'-benzofuran propionic acid (compound 8), Bahia genin -4'- O - (6 '' - O - p - one to Kumar) - β -D- gluconic nose Llano side (compound 9), dihydro-dihydro Coney perylene alcohol -4- O - from the group consisting of β -D- gluconic nose Llano side (compound 10) and the sharp side Sat (compound 11) To a method for separating at least one compound selected.

The present invention also provides for the novel compounds having the chemical structure (7 R, 8 S) -7,8- dihydro-8-hydroxy-7- (3-methoxyphenyl -4- O - β -D -Glucopyranoside) -1 ' -benzofuran < / RTI > propionic acid.

Hereinafter, the present invention will be described in detail.

The present invention relates to a composition for preventing or treating Alzheimer's disease comprising the compound of Chemical Formula 1 isolated from Lycopodiella cernua .

The calcined gut extract can be obtained by extracting calcined steel with water, C1 to C4 lower alcohols or a mixed solvent thereof. As the C1 to C4 lower alcohols, methanol, ethanol, propanol, isopropanol, have. In addition, the calcined gum extract may be a fraction obtained by fractionation with an organic solvent. As the organic solvent, n-hexane, ethyl acetate and dichloromethane may be used.

Compounds separated from the calcined gypsum can be obtained by fractionating the water layer extract of the lysozyme by chromatography. The chromatography is carried out by silica gel column chromatography, HP-20 column chromatography, , RP-18 column chromatography, LH-20 column chromatography, high-performance liquid chromatography, and the like.

Meanwhile, the compound of the present invention can be synthesized according to a conventional method in the art, and can also be prepared as a pharmaceutically acceptable salt.

In addition, the present invention provides a pharmaceutical composition for preventing or treating Alzheimer's disease comprising a calcined ginger extract or at least one compound selected from the group of compounds represented by the above formula (1) isolated therefrom. The pharmaceutical composition containing the extract or the compound may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions, Can be used. Examples of carriers, excipients and diluents that can be contained in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose , Methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient, such as starch, calcium carbonate, sucrose, Or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The dosage of the pharmaceutical composition comprising the calcitonin extract of the present invention or the compound isolated therefrom will depend on the age, sex, body weight, the specific disease or condition to be treated, the severity of the disease or condition, the route of administration, It will depend on judgment. Dosage determinations based on these factors are within the level of ordinary skill in the art and generally the dosage ranges from 0.01 mg / kg / day to approximately 2000 mg / kg / day. A more preferable dosage is 1 mg / kg / day to 500 mg / kg / day. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

The pharmaceutical composition comprising the calcitonin extract of the present invention or a compound isolated therefrom can be administered to mammals such as rats, livestock, humans, and the like in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine dural or intracerebral injection. Since the extract of the present invention or the compound isolated therefrom has little toxicity and side effects, it can be safely used even for long-term administration for preventive purposes.

The present invention also provides a health functional food for preventing or ameliorating Alzheimer's disease, which comprises a calcined ginger extract or at least one compound selected from the group of the compounds of the formula 1 isolated therefrom and a pharmaceutically acceptable food supplementary additive do. The extract or the compound may be added to the health functional food of the present invention in an amount of 0.001 to 100% by weight. The health functional food of the present invention includes forms such as tablets, capsules, pills, and liquids. Examples of the foods to which the extract or compound of the present invention can be added include various foods, beverages, gums, tea, Vitamin complexes and the like.

The present invention relates to a composition for preventing or treating Alzheimer's disease comprising a calcined ginger extract or a compound isolated therefrom, wherein the calcined ginger extract or the compound isolated therefrom is selected from the group consisting of acetylcholinesterase, butyrylcholinesterase, The first inhibitory effect of Crete is excellent and can be effectively used as a composition for preventing or treating Alzheimer's disease.

Figure 1 is a (7 R, 8 S) the novel compounds of the invention 7,8-dihydro-8-hydroxy-7- (3-methoxyphenyl -4- O - β -D- gluconic nose Llano side) is an illustration showing a 1'-benzofuran propionic acid ^{1 H- 1 H COSY (bold lines} ) and the ¹ H- ¹³ C HMBC correlation (arrow) on.

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein but may be embodied in other forms. Rather, the intention is to provide an exhaustive, complete, and complete disclosure of the principles of the invention to those skilled in the art.

≪ Example 1: Isolation of calcined gum derived compound >

The Lycopodium cernua used in the present invention was obtained in April 2012 in Rumdong Province, Vietnam and calcined (3 kg) was subjected to reflux extraction and filtration with methanol (2 L × 3 times), and the filtrate And distilled under reduced pressure to obtain 280 g of a methanol extract. Thereafter, the methanol extract was suspended in water (2 L) and sequentially fractionated with n-hexane (2 L × 3 times) and ethyl acetate (2 L × 3 times) to give a n-hexane fraction (48 g) and ethyl acetate fraction g) and finally a fraction (55 g) containing a water layer residue was obtained.

Thereafter, the fraction containing the residue of the water layer was suspended in 5% [v / v] hydrochloric acid and then fractionated with dichloromethane to obtain a dichloromethane fraction (1.9 g). Further, aqueous layers of the fractions obtained by fractionating with dichloromethane were adjusted to pH 11-12 with 0.1 N sodium hydroxide and then fractionated with dichloromethane. The organic layer was collected and dried with sodium sulfate and evaporated to obtain a crude alkaloid fraction. The residue containing the water layer was dissolved in a methanol-aqueous solution (0 to 100% [v / v] 20 column chromatography to obtain 12 fractions (W1-W12). Among the above fractions, W3 (1.24 g) was subjected to silica gel column chromatography (column size: 3.5 × 50 cm) according to the isocratic elution conditions of dichloromethane: methanol: water (4: 1: 0.2 [v: v: And fractionated to obtain 13 small fractions (W3.1-W3.13).

W3.4 (115 mg) of the above fraction was purified by silica gel column chromatography (column size: 1.5 x 25 (volume)) according to the elution conditions of dichloromethane: methanol: water (5: 1: 0.1 [v: v: Cm) to obtain Compound 3 (8.8 mg). Further, the small fraction W3.6 (65 mg) was purified by silica gel column chromatography (column size: 1.5 x 25 (volume)) according to the isocratic elution conditions of dichloromethane: methanol: water (4: 1: 0.2 [v: v: Cm) to obtain Compound 1 (28.7 mg). The small fraction, W3.8 (36 mg), was subjected to isocratic elution with 20% [v / v] acetonitrile in RP-C18 The residue was separated by silica gel column chromatography to obtain Compound 2 (7.7 mg).

The fraction W6 (1.05 g) was fractionated by silica gel column chromatography (column size: 3.0 x 50 cm) according to the isocratic elution conditions of dichloromethane: methanol: water (3: 1: 0.2 [v: v: Nine small fractions (W6.1-W6.9) were obtained. Of the above small fractions, W6.2 (25 mg) was filtered to obtain Compound 4 (6.2 mg). The small fraction W6.9 (167 mg) was fractionated by Sephadex LH-20 column chromatography (column size: 2.0 x 120 cm) according to the isocratic elution conditions of methanol: water (1: 1 [v: v] Compound 5 (7 mg) and Compound 6 (10.3 mg) were obtained.

W8 (1.52 g) in the fractions was fractionated by silica gel column chromatography (column size: 3.0 x 60 cm) according to isocratic elution conditions of dichloromethane: methanol: water (3: 1: 0.1 [v: v: To obtain eight small fractions (W8.1-W8.8). Among these fractions, W8.6 (125 mg) was purified by Sephadex LH-20 column chromatography (column size: 2.0 x 60 cm) according to the isocratic elution conditions of methanol: water (1: 1 [v: v]) And fractionated to obtain Compound 7 (3 mg). The small fraction W8.8 (150 mg) was subjected to Sephadex LH-20 column chromatography (column size: 1.5 x 120 cm) according to the isocratic elution conditions of methanol: water (1: 1 [v: v] Compound 8 (12.3 mg) was isolated.

Fraction W9 (1.36 g) was purified by silica gel column chromatography (column size: 3.0 x 60 cm) according to isocratic elution conditions of dichloromethane: methanol: water (3: 1: 0.1 [v: v: The small fraction (W9.1-W9.7) was isolated. W9.4 (45 mg) of the above small fractions was subjected to Sephadex LH-20 column chromatography (column size: 1.5 × 120 cm) according to the isocratic elution conditions of methanol: water (2: 1 [v: v]) And fractionated to obtain Compound 9 (6.7 mg). The small fraction W9.7 (65.7 mg) was further purified by Sephadex LH-20 column chromatography (column size: 1.5 x 120 cm) according to the isocratic elution conditions of methanol: water (2: 1 [v: v]) Followed by fractionation to obtain Compound 10 (14.1 mg) and Compound 11 (10.5 mg).

≪ Example 2: Identification of physicochemical structure of the compound separated from the calcined gypsum &

Example 2-1. Rhamnazine (compound 1)

rhamnazine;

Yellowish powder;

IR (KBr)? _Max 3545, 3400, 1658, 1130, 1117 cm ^-1 ;

¹ H NMR and ¹³ C NMR data, Table 1 and Table 2;

^{EI-MS [M] + 330.2} m / z, C 17 H 14 O 7.

Example 2-2. Quercetin 3,3'-dimethyl ether (Compound 2)

quercetin 3,3'-dimethyl ether;

Yellowish powder;

¹ H NMR and ¹³ C NMR data, Table 1 and Table 2;

^{EI-MS [M] + 330.2} m / z, C 17 H 14 O 7.

Example  2-3. Guaiacylglycerol (Compound 3)

guaiacylglycerol;

White powder;

¹ H NMR and ¹³ C NMR data, Table 1 and Table 2;

^{EI-MS [M] + 214.2} m / z, C 10 H 14 O 5.

Examples 2-4. Apigenin (Compound 4)

apigenin;

Yellowish powder;

¹ H NMR and ¹³ C NMR data, Table 1 and Table 2;

^{EI-MS [M] + 270.0} m / z, C 15 H 10 O 5.

Examples 2-5. Isosaphthoside (Compound 5)

isoschaftoside;

Yellowish powder;

≪ ¹ > H NMR and < ¹³ > C NMR data are shown in Table 3 below;

EI-MS [M] < ⁺ & gt ^; 564.4 m / z, C ₂₆ H ₂₈ O ₁₄ .

Examples 2-6. Apigenin-4'- O - (2 ", 6 " -di-O- p -Coumaroyl) -? - D-glucopyranoside (Compound 6)

apigenin-4'- O - (2 ' ', 6 '' - di- O - p -coumaroyl) - β -D-glucopyranoside;

White amorphous powder;

≪ ¹ > H NMR and < ¹³ > C NMR data are shown in Table 4 below;

ESI-MS [M + H] 579.1 m / z, C ₃₀ H ₂₇ O ₁₂ .

Examples 2-7. Spring Bashin-4- O - beta - D - glucopyranoside (Compound 7)

bombasin-4- O - ? - D -glucopyranoside;

White amorphous powder;

UV (MeOH): 231 (4.23), 283 (4.17) nm;

IR (KBr): 3367, 2920, 1651, 1601, 1514, 1464, 1427, 1265, 1223, 1159, 1126 cm ^-1 ;

≪ ¹ > H NMR and < ¹³ > C NMR data are shown in Table 4 below;

^{EI-MS [M] + 506.18} m / z, C 25 H 30 O 11.

Example  2-8. (7 R ,8 S ) -7,8- Dihydro -8- Hydroxymethyl -7- (3- Methoxyphenyl -4- O - beta -D-Glue Kopilano Di) -1'- Benzofuranpropionic acid (Compound 8)

(7R, 8S) -7,8-dihydro-8-hydroxymethyl-7- (3-methoxyphenyl-4-0-? -D- glucopyranoside) -1'-benzofuranpropanoic acid (also called "lycocernuaside A"

White amorphous powder;

: -36.2 ( c 0.53, MeOH);

IR (KBr)? _Max 3379, 2941, 2834, 1638, 1600, 1515, 1453, 1274, 1032 cm ^-1 ;

UV (MeOH) [lambda] _max (log 竜 ) 281 (2.50), 230 (3.12) nm;

ECD (c 0.030 mM, MeOH) : △ ε 278 -2.38, △ ε 232 -1.42, △ ε 216 +1.30;

≪ ¹ > H NMR and < ¹³ > C NMR data are shown in Table 5 below;

HR-FAB-MS m / z 559.1794 [M + Na] ⁺ (calcd for C ₂₆ H ₃₂ O ₁₂ Na, 559.1791).

Example  2-9. Apigenin -4'- O - (6 " - O - p - Kumaroto ) - beta -D- Glucopyranoside  (Compound 9)

apigenin-4 ' -O- (6 " -O - p- coumaroyl) - [ beta] -D-glucopyranoside;

White amorphous powder;

≪ ¹ > H NMR and < ¹³ > C NMR data are shown in Table 6 below;

ESI-MS [M + H] 579.1 m / z, C ₃₀ H ₂₇ O ₁₂ .

Examples 2-10. Dihydrodihydroconiferyl alcohol-4- O - beta -D-glucopyranoside (Compound 10)

dihydrodehydroconiferyl alcohol-4- O - ? - D-glucopyranoside;

Colorless rubber;

IR (KBr) v _max 3380, 2945, 2834, 1600, 1517, 1462, 1270, 1035 cm ^-1 ;

UV (MeOH)? _Max (log?) 280 (2.35), 232 (3.0) nm;

≪ ¹ > H NMR and < ¹³ > C NMR data are shown in Table 6 below;

EI-MS m / z 552 [M] ^< ⁺ & gt ^; (calcd for C ₂₆ H ₃₄ O _{11 ,} 552).

Examples 2-11. Sharp acid (Compound 11)

schaftoside;

Yellowish powder;

≪ ¹ > H NMR and < ¹³ > C NMR data are shown in Table 3 below;

EI-MS [M] < ⁺ & gt ^; 564.4 m / z, C ₂₆ H ₂₈ O ₁₄ .

Compound 1 Compound 2 Compound 3 Compound 4 Position [delta] _H (ppm) ^a [delta] _H (ppm) ^a [delta] _H (ppm) ^b [delta] _H (ppm) ^a One 4.43 (1H, d, J = 6.4 Hz) 2 3.59 (1H, m, H-2) 3 3.41 (1H, dd, J = 11.2, 4.0 Hz, H-3a) 3.27 6.68 (1 H, s) 4 5 6 6.77 (1H, s, J = 2.0 Hz) 6.75 (1H, s, J = 2.0 Hz) 6.54 (1H, s, J = 2.0 Hz) 7 8 6.38 (1H, s, J = 2.0 Hz) 6.40 (1H, s, J = 2.0 Hz) 6.25 (1H, s, J = 2.0 Hz) 9 10 One' 2' 7.68 (1H, d, J = 2.0 Hz) 7.70 (1H, d, J = 2.0 Hz) 6.91 (1H, d, J = 1.2H) 7.98 (1H, d, J = 8.7 Hz) 3 ' 7.20 (1H, d, J = 8.7 Hz) 4' 5 ' 6.92 (1H, s, J = 8.0 Hz) 6.92 (1H, s, J = 8.0 Hz) 6.69 (1H, d, J = 8.0 Hz) 7.20 (1H, d, J = 8.7 Hz) 6 ' 7.62 (1H, dd, J = 8.0, 2.0 Hz) 7.62 (1H, dd, J = 8.0, 2.0 Hz) 6.73 (1H, dd, J = 8.0, 1.2 D) 7.98 (1H, d, J = 8.7 Hz) 7-MeO 3.86 (3 H, s) 3-MeO 3.80 (3H, s) 3'-MeO 3.87 (3 H, s) 3.85 (3 H, s) 3.78 (3 H, s) 5-OH 12.05 (1H, s) 12.14 (1 H, s) a: ¹ H NMR (400 MHz in DMSO-d ₆ ) spectroscopy data
b: ¹ H NMR (400 MHz in MeOD-d ₄ ) spectroscopy data

Compound 1 Compound 2 Compound 3 Compound 4 Position [delta] _C (ppm) ^a [delta] _C (ppm) ^a [delta] _C (ppm) ^b [delta] _C (ppm) ^a One 75.6 2 156.3 156.5 77.7 164.2 3 133.6 136.5 64.3 105 4 177.7 178.2 182.5 5 160.8 161.2 158.7 6 97.9 97.3 94.7 7 165.2 164.4 164.8 8 92.4 92.6 99.6 9 157 157.7 163.2 10 105 106.2 105.2 One' 120.7 122.5 134.9 126 2' 112.6 112.4 111.6 128.9 3 ' 149.6 150.5 147.2 117.5 4' 147.2 147.7 149 161 5 ' 115.4 115.6 116 117.5 6 ' 122.9 121.4 120.8 128.9 7-MeO 55.4 3-MeO 55.2 3'-MeO 55.7 56.1 56.4 5-OH a: ¹³ C-NMR (100 M Hz in DMSO-d ₆ ) spectroscopy data
b: ¹³ C-NMR (100 MHz in MeOD-d ₄ ) spectroscopy data

Compound 5 Compound 11 Position δ _H ( ppm ) ^a δ _C ( ppm ) ^b Position δ _H ( ppm ) ^c δ _C ( ppm ) ^d 2 165.2 2 166.6 3 6.63 (1 H, s) 105.5 3 6.41 (1 H, s) 105.5 4 183.5 4 184.1 5 156.8 5 157.4 6 106.8 6 108.3 7 163.2 7 163 8 103.8 8 103.7 9 163.2 9 162.7 10 109.6 10 105.7 One' 123.5 One' 123.3 2' 8.32 (1H, d, J = 8.8 Hz) 129.7 2' 7.80 (1H, d, J = 8.0 Hz) 129.6 3 ' 7.27 (1H, d, J = 8.8 Hz) 117.5 3 ' 7.74 (1H, d, J = 8.0 Hz) 117.1 4' 160.5 4' 160.1 5 ' 7.27 (1H, d, J = 8.8 Hz) 117.5 5 ' 7.74 (1H, d, J = 8.0 Hz) 117.1 6 ' 8.32 (1H, d, J = 8.8 Hz) 129.7 6 ' 7.80 (1H, d, J = 8.0 Hz) 129.6 8-Glc 6-Glc One'' 5.86 (1H, d, J = 9.6 D) 75.7 One'' 4.93 (1H, d, J = 9.6 D) 75.5 2'' 4.68 (1 H, m) 81.4 2'' 3.75 (1 H, m) 82.6 3 '' 4.39 (1 H, m) 76.7 3 '' 3.72 (1 H, m) 80.3 4'' 4.28 (1 H, m) 71.9 4'' 3.49 (1 H, m) 71.5 5 '' 4.50 (1H, m) 72.8 5 '' 3.60 (1 H, m) 72.4 6 '' 4.57-4.28 (1H, m) 63.4 6 '' 3.32 (1 H, m), 3.84 (1 H, m) 63.2 6-Rha 8-Rha One''' 5.66 (1H, d, J = 9.6 D) 84 One''' 4.63 (1H, d, J = 9.6 D) 82.9 2''' 4.67 (1 H, m) 76.2 2''' 3.90 (1 H, m) 76.6 3 '' ' 4.48 (1 H, m) 73.4 3 '' ' 3.75 (1 H, m) 73.2 4''' 4.37 (1 H, m) 71.4 4''' 3.42 (1 H, m) 71.2 5 '' ' 4.33 (1 H, m) 72.2 5 '' ' 3.80 (1 H, m) 71.7 a: ¹ H NMR (400 MHz in pyridine- d ₅ ) spectroscopy data
^{b: 13 C-NMR (100} ㎒ in Pyridine- d 5) spectroscopy data
c: ¹ H NMR (400 MHz in DMSO- d ₆ ) spectroscopy data
^{d: 13 C-NMR (100} ㎒ in DMSO- d 6) spectroscopy data

Compound 6 Compound 7 Position δ _H ( ppm ) ^a δ _C ( ppm ) ^b δ _H ( ppm ) ^c δ _C ( ppm ) ^d One 130.8 2 163.8 7.33 (1H, d, J = 2.0 Hz) 111.9 3 6.71 (1 H, s) 105.7 150.6 4 182.6 148.4 5 158.3 7.19 (1H, dd, J = 2.0, 8.5 Hz) 116.6 6 6.53 (1H, s, J = 2.0 Hz) 94.5 7.59 (1H, d, J = 8.5 Hz) 119.5 7 165.1 6.16 (1H, d, J = 6.5 Hz) 89.3 8 6.27 (1H, s, J = 2.0 Hz) 100.2 3.96 (1 H, m) 54.2 9 163.7 4.27 (2H, m) 64 10 105.1 One' 124.6 7.78 (1H, brs) 120.1 2' 7.90 (1H, d, J = 8.7 Hz) 129.1 132.6 3 ' 7.18 (1H, d, J = 8.7 Hz) 117.5 7.92 (1H, brs) 113.6 4' 161.3 137.6 5 ' 7.18 (1H, d, J = 8.7 Hz) 117.4 147 6 ' 7.90 (1H, d, J = 8.7 Hz) 129.1 145.2 7 ' 196.7 8' 2.58 (3 H, s) 26.5 9 ' One'' 5.42 (1H, d, J = 8.1 Hz) 99.8 5.69 (1H, d, J = 7.0 Hz) 102.7 2'' 5.15 (1H, dd, J = 9.4, 8.1 Hz) 74.2 4.36 (1 H, m) 75 3 '' 3.80 (1H, t, J = 9.0 Hz) 73.9 4.37 (1 H, m) 78.6 4'' 3.70 (1H, t, J = 9.0 Hz) 77.3 4.32 (1 H, m) 71.4 5 '' 3.78 (1 H, m) 74.6 4.13 (1H, m) 79 6 '' 3.83 (1H, dd, J = 12.1, 2.0 Hz)
3.74 (1H, dd, J = 12.1, 5.5 Hz) 66.4 4.55 (1H, dd, J = 2.0, 12.0 Hz) 4.37 (1H, m) 62.5 One''' 126.7 2''' 7.54 (1H, d, J = 8.5 Hz) 131.2 3 '' ' 6.68 (1H, d, J = 8.5 Hz) 116.5 4''' 160.8 5 '' ' 6.68 (1H, d, J = 8.5 Hz) 115.4 6 '' ' 7.54 (1H, d, J = 8.5 Hz) 130.5 7 '' ' 7.66 (1H, d, J = 15.0 Hz) 146 8''' 6.37 (1H, d, J = 15.0 Hz) 114.8 9 '' ' 166.7 One'''' 125.8 2'''' 7.53 (1H, d, J = 8.5 Hz) 131.1 3 '' '' 6.86 (1H, d, J = 8.5 Hz) 116.6 4'''' 160.5 5 '' '' 6.86 (1H, d, J = 8.5 Hz) 116.6 6 '' '' 7.53 (1H, d, J = 8.5 Hz) 131.1 7 '' '' 7.60 (1H, d, J = 15.0 Hz) 145.6 8'''' 6.39 (1H, d, J = 15.0 Hz) 114.7 9 '' '' 167.8 3-OMe 3.86 (3 H, s) 56.2 6'-OMe 3.69 (3 H, s) 56.2 ^{a: 1 H NMR (400 ㎒} in DMSO- d 6) spectroscopy data
^{b: 13 C-NMR (100} ㎒ in DMSO- d 6) spectroscopy data
c: ¹ H NMR (400 MHz in MeOD- d ₄ ) spectroscopy data
^{d: 13 C-NMR (100} ㎒ in MeOD- d 4) spectroscopy data

Compound 8 Position δ _H  ( ppm , J in  ㎐) ^a δ _C ( ppm ) ^b One 129.6 2 7.06, br s 111.2 3 150.9 4 147.5 5 7.17, d (8.0) 118.1 6 6.96, brd (8.0) 119.4 7 5.55, d (6.0) 88.4 8 3.51, m 55.5 9 3.88, m
3.78, m 65.2 One' 136.1 2' 6.80, brs 114.1 3 ' 138.5 4' 147.7 5 ' 145.2 6 ' 6.76, brs 117.8 7 ' 2.84, t (7.6) 32.3 8' 2.53, t (7.6) 38.1 9 ' 178.1 One'' 4.89, d (7.6) 102.8 2'' 3.53, m 74.8 3 '' 3.51, m 77.8 4'' 3.43, m 71.3 5 '' 3.45, m 79.4 6 '' 3.72, dd (3.5, 12.0)
3.89, m 62.4 3-OMe 3.88, s 56.7 5'-OMe 3.85, s 56.8 a: ^{1 H NMR (500 ㎒ in Methanol-} d 4, d values) spectroscopic data
b: ¹³ C NMR (125 MHz in methanol- d ₄ , d values) spectroscopic data

Compound 9 Compound 10 Position δ _H ( ppm ) ^a δ _C ( ppm ) ^b δ _H ( ppm ) ^c δ _C ( ppm ) ^d One 129.4 2 164.2 7.01 (1H, brs) 111.3 3 6.68 (1 H, s) 105 150.7 4 182.5 147.4 5 158.7 6.87 (1H, d, J = 8.5 Hz) 117.9 6 6.54 (1H, s, J = 2.0 Hz) 94.7 6.99 (1H, dd, J = 1.5, 8.5 Hz) 119.2 7 164.8 5.61 (1H, d, 6.0 Hz) 88.3 8 6.25 (1H, s, J = 2.0 Hz) 99.6 3.55 (1H, m) 55.4 9 163.2 3.91 (1 H, m) 3.82 (1 H, m) 64.9 10 105.2 One' 126 6.79 (1H, brs) 120.3 2' 7.98 (1H, d, J = 8.7 Hz) 128.9 132.7 3 ' 7.20 (1H, d, J = 8.7 Hz) 117.5 7.20 (1H, brs) 114.9 4' 161 138.2 5 ' 7.20 (1H, d, J = 8.7 Hz) 117.5 147.3 6 ' 7.98 (1H, d, J = 8.7 Hz) 128.9 145 7 ' 2.69 (2H, t, J = 7.5 Hz) 32.6 8' 1.88 (2 H, m) 36 9 ' 3.63 (2H, t, J = 6.5 Hz) 62.3 One'' 5.39 (1H, d, J = 8.1 Hz) 99.4 4.95 (1H, d, J = 7.0 Hz) 102.6 2'' 5.17 (1H, dd, J = 9.4, 8.1 Hz) 74.1 3.53 (1H, m) 74.7 3 '' 3.82 (1H, t, J = 9.0 Hz) 75.6 3.45 (1 H, m) 77.6 4'' 3.61 (1H, t, J = 9.0 Hz) 71.2 3.46 (1 H, m) 71.1 5 '' 3.71 (1 H, m) 78.1 3.52 (1H, m) 77.9 6 '' 3.96 (1H, dd, J = 12.1, 5.8 Hz) 62.2 3.74 (1H, dd, J = 3.5, 12.0 D) 3.94 (1H, m) 62 One''' 126.8 2''' 7.54 (1H, d, J = 8.5 Hz) 130.9 3 '' ' 6.68 (1H, d, J = 8.5 Hz) 116.5 4''' 160.5 5 '' ' 6.68 (1H, d, J = 8.5 Hz) 116.5 6 '' ' 7.54 (1H, d, J = 8.5 Hz) 130.9 7 '' ' 7.66 (1H, d, J = 15.0 D) 145.5 8''' 6.37 (1H, d, J = 15.0 D) 115.2 9 '' ' 166.5 3-OMe 3.90 (3 H, s) 56.5 6'-OMe 3.94 (3 H, s) 56.6 ^{a: 1 H NMR (400 ㎒} in DMSO- d 6) spectroscopy data
^{b: 13 C NMR (100 ㎒} in DMSO- d 6) spectroscopy data
c: ¹ H NMR (400 MHz in MeOD- d ₄ ) spectroscopy data
^{d: 13 C NMR (100 ㎒} in MeOD- d 4) spectroscopy data

< Example  3. Acetylcholinesterase  And Butyrylcholinesterase  Measurement of inhibitory activity>

When acetylcholine or butyryl acetylcholine is hydrolyzed by cholinesterase, a yellow 5'-thio-2-nitrobenzoate anion is formed. Therefore, the change in absorbance at 412 nm And the enzyme activity was measured.

In 96-well 96-well plates, 140 μl of sodium phosphate buffer (pH 8.0) and 20 μl of each compound of the present invention at a final concentration of 100 μM, acetylcholinesterase or butyrylcholinesterase (5 units / ml ), 10 μl of dithiobisnitrobenzoate (DTNB), 10 μl of acetylcholine or butyrylcholine, and reacted at room temperature for 15 minutes. Berberine was used as a positive control, and the 5'-thio-2-nitrobenzoate anion formed after the reaction was confirmed at 412 nm with an absorbance meter (VERSA max, Molecular Devices, USA) ₅₀ (the half maximal inhibitory concentration).

Condition Acetylcholinesterase inhibitory activity (IC ₅₀ ) Butyrylcholinesterase inhibitory activity (IC ₅₀ ) Compound 1 8.69 ± 0.49 μM > 30 μM Compound 2 8.78 ± 0.97 μM 13.04 占 0.99 占 M Compound 3 1.56 ± 0.22 μM > 30 μM Compound 4 7.29 ± 0.23 μM 15.78 ± 0.95 μM Compound 5 0.23 ± 0.17 μM 3.08 ± 0.26 μM Compound 6 1.24 ± 0.31 μM 3.11 ± 0.77 μM Compound 7 11.25 ± 0.99 μM 12.23 ± 0.89 μM Compound 8 15.22 ± 0.89 μM 24.09 + - 0.33 [mu] M Compound 9 2.78 ± 0.42 μM 7.15 ± 0.19 μM Compound 10 11.93 ± 0.96 μM 7.02 + 0.29 [mu] M Compound 11 1.70 ± 0.22 μM 0.62 ± 0.47 μM Berber 0.71 ± 0.11 μM 7.08 ± 0.01 μM

As shown in the results of Table 7 above, it was confirmed that the compound isolated from the calcined ginseng extract of the present invention had an inhibitory activity of acetylcholinesterase or butyrylcholinesterase, and thus it can be effectively used as a preventive and therapeutic agent for Alzheimer's disease .

<Example 4: Measurement of inhibitory activity of? -Secretase 1>

When the substrate was cleaved by β-secretase 1, fluorescence was observed. The activity of the enzyme was confirmed by measuring with BACE1 FRET assay kit ( β- Secretase, human recombinant, PanVera Co. USA).

10 μl of each compound of 100 μM was added to each of 10 μl of 50 mM sodium acetate (pH 4.5), β-secretase 1 and substrate (750 nM Rh-EVNLDAEFK-Quencher) and reacted in the dark at 25 ° C. for 60 minutes . Quercetin was used as a positive control. Fluorescence was measured at an excitation wavelength of 545 nm and an emission wavelength of 585 nm in order to confirm the reaction result, and the IC ₅₀ (the half maximal inhibitory concentration) was shown in Table 8 below.

Condition β-secretase inhibitory activity (IC ₅₀ ) Compound 1 5.59 + - 0.33 [mu] M Compound 3 7.33 ± 1.77 μM Compound 10 26.86 ± 1.94 μM Compound 11 2.16 ± 0.37 μM Quercetin 6.76 ± 0.63 μM

As shown in Table 8, it was confirmed that the compound isolated from the calcined ginseng extract of the present invention inhibited the activity of? -Secretase 1, thus confirming the excellent therapeutic effect on Alzheimer's disease.

<Example 5: Toxicity test>

Example 5-1. Acute toxicity

Of the present invention lycopodiopsida extract or (7 R, 8 S) -7,8- dihydro-8-hydroxy-7- (3-methoxyphenyl -4- O - β -D- gluconic nose Llano side) -1'-benzofuranpropionic acid (Compound 1) in a short period of time was investigated to determine the toxicity to the animal body and to determine the mortality rate in an acute (within 24 hours). Twenty ICR mice were prepared, and 10 mice were assigned to each group. In the control group, only 30% PEG-400 was administered, and the test group was orally administered with the extract of the present invention or Compound 1 at a concentration of 1.0 g / kg, respectively. After 24 hours of administration, the respective mortality rates were examined. As a result, both the control and the extract or the test group to which Compound 1 was administered survived.

Example 5-2. Organ organs toxicity test in experimental group and control group

Long-term toxicity experiments lycopodiopsida extract or (7 R, 8 S) of the present invention to investigate the effect of each organ (tissue) of the animals intended for C57BL / 6J mice, 7,8-dihydro-8-hydroxy The experimental group administered with 1.0 g / kg of ricinomethyl-7- (3-methoxyphenyl-4- O - ? - D-glucopyranoside) -1'-benzofuranpropionic acid and the control group Blood samples were taken from the animals 8 weeks later and the concentrations of GPT (glutamate-pyruvate transferase) and BUN (blood urea nitrogen) in the blood were measured using Select E (vital scientific NV, Netherland) instrument. As a result, GPT, which is known to be related to hepatotoxicity, and BUN, which is known to be related to renal toxicity, showed no significant difference compared to the control group. In addition, liver and kidney were cut from each animal and histological observation was performed with an optical microscope through a conventional tissue section production process, but no abnormal abnormalities were observed.

&Lt; Formulation Example 1 >

Formulation Example 1-1. Manufacture of tablets

Of the present invention (7 R, 8 S) -7,8- dihydro-8-hydroxy-7- (3-methoxyphenyl -4- O - β -D- gluconic nose Llano side) 1' 200 g of benzofuranpropionic acid were mixed with 175.9 g of lactose, 180 g of potato starch and 32 g of colloidal silicic acid. To this mixture was added a 10% gelatin solution, which was pulverized and passed through a 14-mesh sieve. This was dried, and a mixture obtained by adding 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate was made into tablets.

Formulation Example 1-2. Injection preparation

Of the present invention (7 R, 8 S) -7,8- dihydro-8-hydroxy-7- (3-methoxyphenyl -4- O - β -D- gluconic nose Llano side) 1' 1 g of benzofuran propionic acid, 0.6 g of sodium chloride and 0.1 g of ascorbic acid were dissolved in distilled water to make 100 ml. This solution was placed in a bottle and sterilized by heating at 20 DEG C for 30 minutes.

<Formulation Example 2: Food Preparation>

Formulation Example 2-1. Manufacture of cooking seasonings

Of the present invention (7 R, 8 S) -7,8- dihydro-8-hydroxy-7- (3-methoxyphenyl -4- O - β -D- gluconic nose Llano side) 1' Benzoylfuranpropionic acid was added to the cooking seasoning at 1 wt% to prepare a cooking sauce for health promotion.

Formulation Example 2-2. Manufacture of flour food products

Of the present invention (7 R, 8 S) -7,8- dihydro-8-hydroxy-7- (3-methoxyphenyl -4- O - β -D- gluconic nose Llano side) 1' Benzoylfuranpropionic acid was added to flour at 0.1 wt%, and bread, cake, cookies, crackers and noodles were prepared using this mixture to prepare foods for health promotion.

Preparation Example 2-3. Manufacture of soups and gravies

Of the present invention (7 R, 8 S) -7,8- dihydro-8-hydroxy-7- (3-methoxyphenyl -4- O - β -D- gluconic nose Llano side) 1' Benzofuranpropionic acid was added to soup and juice at 0.1% by weight to prepare health promotion soup and juice.

Formulation Example 2-4. Manufacture of dairy products

Of the present invention (7 R, 8 S) -7,8- dihydro-8-hydroxy-7- (3-methoxyphenyl -4- O - β -D- gluconic nose Llano side) 1' Benzofuranpropionic acid was added to milk in an amount of 0.1 wt%, and various dairy products such as butter and ice cream were prepared using the milk.

Formulation Example 2-5. Vegetable juice manufacturing

Of the present invention (7 R, 8 S) -7,8- dihydro-8-hydroxy-7- (3-methoxyphenyl -4- O - β -D- gluconic nose Llano side) 1' 0.5 g of benzofuran propionic acid was added to 1,000 ml of tomato juice or carrot juice to prepare health promotion vegetable juice.

Formulation example  2-6. Fruit juice  Produce

Of the present invention (7 R, 8 S) -7,8- dihydro-8-hydroxy-7- (3-methoxyphenyl -4- O - β -D- gluconic nose Llano side) 1' 0.1 g of benzofuran propionic acid was added to 1,000 ml of apple juice or grape juice to prepare health promotion fruit juice.

Claims (10)

delete delete delete delete delete delete delete delete Preparing Lycopodiella cernua by calcination with water, C1 to C4 lower alcohols or a mixed solvent thereof; Sequentially fractionating the calcined gum extract with n-hexane and ethyl acetate, and then fractionating the residue with dichloromethane; And separating the sharp acid (compound 11) of the following formula 1 by chromatography on a water layer excluding the re-fractionated dichloromethane.
[Chemical Formula 1]

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