KR20160125339A - A composition comprising extract of Lycopodiella cernua or compound isolated therefrom for preventing or treating of Alzheimer disease-Ⅰ - Google Patents

Abstract

The present invention relates to a composition for preventing or treating Alzheimers disease comprising a Lycopodiopsida extract or a compound isolated from the same. The Lycopodiopsida extract or a compound isolated from the same has an excellent effect of suppressing acetylcholinesterase, butyryl cholinesterase, and-secretase, thereby being useful for a composition for preventing or treating Alzheimers disease.

Description

A composition for preventing or treating Alzheimer's disease comprising a calcineurin extract or a compound isolated therefrom is provided. The composition for extracting or preventing Alzheimer '

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, 3?, 21?, 29-trihydroxyethyl-14-en-24-dioic acid-3? -Yl- (7'-hydroxycinnamate) (Compound 1), 21? (4'-methoxy-5 ', 4'-methoxy-4'-methoxyphenyl) -dihydro when benzoate) (compound 3), 3β, 21α- dia setok price Ratan -14β- ol (compound 4), 3β, 21β, 29- tree hydroxyl price Eilat 14-yen -3β- one p-di (Compound 5), cerat-14-en-3 ?, 21? -Diol (Compound 6), 3 ?, 21?, 29- trihydroxy- (Compound 7) and (2 E , 4 E , 6 R ) -6-hydroxydeca-2,4-dienoic acid (Compound 8) ( Lycopodiella cernua ) extract containing the extract of the present invention.

[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, chloroform and ethyl acetate.

The invention also lycopodiopsida (Lycopodiella cernua) of 3β, 21β, 29- tree hydroxyl price Eilat -14- of the formula (1) isolated from five yen -24- acid -3β- one - (7'-hydroxy-cinnamate (Compound 1), 21? -Hydroxyethyl-14-ene-3,16-dione (Compound 2), 3 ?, 14 ?, 15 ?, 21? -Tetrahydroxycellatane- - (4'-methoxy-5'-hydrosibenzoate) (Compound 3), 3β, 21α-diacetoxyseraTan- 14β-ol (Compound 4), 3β, 21β, 29-trihydroxy- 14-yen -3β- one p-dihydro Kumar (compound 5), 14-yen serat -3α, 21α- diol (compound 6), 3β, 21β, 29- tree hydroxyl price Eilat 14-ene- (Compound 7) and (2 E , 4 E , 6 R ) -6-hydroxydeca-2,4-dienoic acid (Compound 8) And a composition for preventing or treating Alzheimer's disease, which comprises at least one compound selected from the group consisting of

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

In another aspect, the present invention provides a pharmaceutical composition comprising 3β, 21β, 29-trihydroxycerat-14-en-24-dioic acid-3β-yl- (7'-hydroxycinnamate) ), 21? -Hydroxyethyl-14-ene-3,16-dione (Compound 2), 3?, 14 ?, 15 ?, 21? -Tetrahydroxyceratane- (Compound 3), 3?, 21? -Diacetoxyseratan-14? -Ol (Compound 4), 3?, 21?, 29-trihydroxycerat-14-ene-3? -yl p - dihydro Kumar (compound 5), 14-yen serat -3α, 21α- diol (compound 6), 3β, 21β, 29- tree hydroxyl price Eilat 14-yen acid -24- O - (Compound 7) and (2 E , 4 E , 6 R ) -6-hydroxydeca-2,4-dienoic acid (Compound 8) were selected from the group consisting of 3? -Dihydroxybenzoate ( Lycopodiella cernua ) extract comprising at least one compound selected from the group consisting of:

The present invention relates to a method for producing Lycopodiella cernua , comprising the steps of: preparing Lycopodiella cernua with water, C1 to C4 lower alcohol or a mixed solvent thereof; Sequentially fractionating the calcined gum extract using n-hexane and ethyl acetate; And each of the above fractions was chromatographed to give 3β, 21β, 29-trihydroxyethyl-14-en-24-oxacan-3β-yl- (7'-hydroxycinnamate) , 21? -Hydroxycerat-14-ene-3,16-dione (Compound 2), 3 ?, 14 ?, 15 ?, 21? -Tetrahydroxyceratane- (Compound 3), 3?, 21? -Diacetoxysera-14? -Ol (Compound 4), 3?, 21?, 29-trihydroxy- one p - dihydro Kumar (compound 5), 14-yen serat -3α, 21α- diol (compound 6), 3β, 21β, 29- tree hydroxyl price Eilat 14-yen -24- O acid -3β (Compound 7) and (2 E , 4 E , 6 R ) -6-hydroxydeca-2,4-dienoic acid (Compound 8) To a method for separating one or more compounds.

The present invention also provides a novel compound of Formula 1, 3?, 21?, 29-trihydroxyethyl-14-ene-24-oxacan-3? -Yl- (7'-hydroxycinnamate) , 21? -Hydroxycerat-14-ene-3,16-dione (Compound 2), 3 ?, 14 ?, 15 ?, 21? -Tetrahydroxyceratane- (Compound 3) and (2 E , 4 E , 6 R ) -6-hydroxydeca-2,4-dienoic acid (Compound 8).

Hereinafter, the present invention will be described in detail.

The present invention relates to a composition for preventing or treating Alzheimer's disease comprising Lycopodiella cernua extract or at least one compound selected from the group of compounds of formula (1) isolated therefrom.

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 re-fractionation with an organic solvent. As the organic solvent, acetone, n-hexane, chloroform and ethyl acetate may be used.

In addition, the compound separated from the calcined gum of the present invention can be separated by chromatography using the calcined gum extract, and the chromatography is carried out by silica gel column chromatography, RP-18 column chromatography (RP -18 column chromatography, thin layer chromatography (TLC), ion exchange resin chromatography, medium pressure liquid chromatography and high performance liquid chromatography HPLC) 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.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram of a novel compound of the present invention, 3?, 21?, 29-trihydroxycerat-14-ene-24-dioic acid-3? -Yl- (7'-hydroxycinnamate) Dihydroxy-3, 4-di (compound 2), 3?, 14 ?, 15 ?, 21? -Tetrahydroxyceratane- '- dihydro when benzoate) (compound 3) and (2 E, 4 E, 6 R) -6- hydroxy-deca-2,4-diene acid (compound ^{8) 1 H- 1 H COSY (} bold lines for ) And ¹ H- ¹³ C HMBC correlation (arrows).

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: Preparation of calcined gangrene extract and separation of 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.

The ethyl acetate fraction was fractionated by silica gel column chromatography (column size: 9.0 x 100 cm) according to a gradient elution gradient of n-hexane: acetone (25: 1 → 0: 1 [v: v] Fifteen fractions (E1-E15) were obtained.

The fraction E5 was fractionated by silica gel column chromatography (column size: 3.5 x 50 cm) according to the concentration gradient elution condition of n-hexane: ethyl acetate (20: 1 to 0: 1 [v: v] (E5.1-E5.3). E5.1 (280 mg) of the above fraction was purified by silica gel column chromatography (column size: 1.5 × 25 cm) according to a gradient elution gradient of chloroform: methanol (20: 1 → 0: 1 [v: v] Compound 1 (20.3 mg) was isolated. Further, the small fraction E5.2 (600 mg) was subjected to RP-18 column chromatography (YMC RP-18 column, column (1)) according to the concentration gradient elution condition of methanol: water (85:15 → 100: 0 [v: v] Size: 3.0 x 50 cm) to obtain Compound 2 (7.4 mg). The small fraction, E5.3, was purified by high performance liquid chromatography (RP-HPLC, Gilson Trilution system, YMC Pak ODS-A column, column size: 20) according to isocratic elution conditions of methanol: water (85:15 [v: × 250㎜, particle size: were isolated 5㎖ / min) with compound _{3 (8.3㎎, t R = 36.4min} ): 5㎛, UV detection: 254㎚, flow rate.

The fraction E6 (2.1 g) was fractionated by silica gel column chromatography (column size: 1.5 x 25 cm) according to a gradient elution gradient of n-hexane: acetone (15: 1 → 0: 1 [v: v] 4 (7.2 mg), Compound 5 (5.3 mg) and Compound 6 (6.2 mg).

The fraction E13 (1.4 g) was fractionated by silica gel column chromatography (column size: 60 x 6.5 cm) according to a gradient elution gradient of chloroform: acetone (20: 1 to 0: 1 [v: v] (E13.1-E13.5). E13.1 (225 mg) of the above fraction was purified by silica gel column chromatography (column size: 1.5 × 25 cm) according to the gradient elution gradient of chloroform: ethyl acetate (8: 1 → 2: 1 [v: To obtain Compound 7 (6.8 mg). Further, the small fraction E13.3 (340 mg) was subjected to RP-18 column chromatography (YMC RP-18 column (HPLC)) according to the concentration gradient elution condition of acetonitrile: water (50:50 to 75:25 [v: v] , Column size: 3.0 x 50 cm) to obtain Compound 8 (5.6 mg).

<Example 2> Identification of physicochemical structure of calcined calcium-derived compound>

Example  2-1. 3 [beta], 21 [beta], 29- Trihydroxycerat -14- yen -24- Oh Iksan -3 [beta] -yl- (7 ' - Hydroxy cinnamate ) (Compound 1)

3β, 21β, 29-trihydroxyserate-14-ene-24-oic acid-3β-yl- (7'-hydroxycinnamate);

White amorphous powder;

mp: 312-314 [deg.] C;

: +12.4 (c 0.9, MeOH);

: +12.4 ( c 0.9, MeOH);

UV (MeOH)? _Max (log ? ) 312 (4.56) nm;

IR (KBr) v _max : 3378, 1716, 1608 and 1519, 1347, 1142, 926 cm &lt; ^{-1 &} gt ;;

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

HR-FAB-MS m / z 657.3764 [M + Na] ⁺ (calcd for C ₃₉ H ₅₄ O ₇ Na _, 657.3768).

Example 2-2. 21-hydroxyethyl-14-ene-3,16-dione (Compound 2)

21β-hydroxyserate-14-ene-3,16-dione;

White amorphous powder;

mp: 176-178 [deg.] C;

: -26.8 (c 0.2, MeOH);

: -26.8 ( c 0.2, MeOH);

UV (MeOH)? _Max (log ? ) 248 (3.32) nm;

IR (KBr) v _max : 3465, 2927, 1723, 1126, 924 cm &lt; ^{-1 &} gt ;;

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

HR-FAB-MS m / z 455.3526 ^{[M + H] + (calcd} for C 30 H 46 O 3, 455.3527).

Examples 2-3. (3'-methoxy-5'-hyd rocibenzoate) (Compound 3) The title compound was prepared in the same manner as in Example 1,

3 [beta], 14 [alpha], 15 [alpha], 21 [beta] -tetrahydroxysulphonate-24-oic acid-3 [beta] -yl- (4'-methoxy-5'-hydroxybenzoate);

White amorphous powder;

mp: 343-345 [deg.] C;

: +8.4 (c 1.2, MeOH);

: +8.4 ( c 1.2, MeOH);

UV (MeOH)? _Max (log ? ) 324 (3.58), 281 (4.08) nm;

IR (KBr) v _max : 3428, 2963, 1712, 1648, 1592, 1361, 1120, 924 cm ^-1 ;

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

HR-FAB-MS m / z 679.3819 [M + Na] ⁺ (calcd for C ₃₈ H ₅₆ O ₉ Na _, 679.3822).

Examples 2-4. 3?, 21? -Diacetoxyseratan-14? -Ol (Compound 4)

3 [beta], 21 [alpha] -diacetoxysilatan-14 [beta] -ol;

White powder;

UV (MeOH)? _Max (log ? ) 254 (2.95) nm;

IR (KBr)? _Max 3364, 2938, 1714, 1263, 1021 cm ^-1 ;

&Lt; ¹ &gt; H NMR and &lt; ¹³ &gt; C NMR data are shown in Table 3 below;

FAB-MS m / z 567 [M + Na] &lt; ⁺ &gt;.

Examples 2-5. 3?, 21?, 29-trihydroxyethyl-14-ene-3? -Yl p - dihydrocommarate (Compound 5)

3 [beta], 21 [beta], 29-trihydroxyserate-14-ene-3 [beta] -yl p- dihydrocoumarate;

White powder;

mp: 343-345 [deg.] C;

: +8.4 (c 1.2, MeOH);

: +8.4 ( c 1.2, MeOH);

UV (MeOH)? _Max (log ? ) 324 (3.58), 281 (4.08) nm;

IR (KBr) v _max 3428, 2963, 1712, 1648, 1592, 1361, 1120, 924 cm ^-1 ;

FAB-MS m / z 629 [M + Na] &lt; ⁺ &gt;.

&Lt; ¹ &gt; H NMR and &lt; ¹³ &gt; C NMR data are shown in Table 4 below;

Examples 2-6. 14-ene-3 [alpha], 21 [alpha] -diol (Compound 6)

serrat-14-en-3?, 21? -diol;

White powder;

mp: 343-345 [deg.] C;

: +23.4 (c 1.2, CHCl₃);

_{: +23.4 (c 1.2, CHCl 3} );

UV (MeOH)? _Max (log ? ) 324 (3.58), 281 (4.08) nm;

IR (KBr) v _max 1719, 1248, 1108 cm &lt; ^{-1 &} gt ;;

EI-MS m / z 679 [M] ^&lt; ⁺ & gt ^; .

&Lt; ¹ &gt; H NMR and &lt; ¹³ &gt; C NMR data are shown in Table 3 below;

Example  2-7. 3 [beta], 21 [beta], 29- Trihydroxycerat -14- yen -24- Oh Iksan -3 [beta] -yl- (4'- Hydroxybenzoate ) (Compound 7)

3β, 21β, 29-trihydroxyserate-14-ene-24-oic acid-3β-yl- (4'-hydroxybenzoate);

Colorless powder;

mp: 335-337 [deg.] C;

: + 4.8 (c 1.2, MeOH);

: + 4.8 ( c 1.2, MeOH);

IR (KBr)? _Max 3567 (OH), 2970, 1724, 1620, 1590, 1517, 1385, 1230, 1120, 820, 690 cm ^-1 ;

FAB-MS m / z 607 [MH] ^- ;

&Lt; ¹ &gt; H NMR and &lt; ¹³ &gt; C NMR data are shown in Table 4 below;

Examples 2-8. (2 E ,4 E , 6 R ) -6-hydroxydeca-2,4-dienoic acid (Compound 8)

(2 E, 4 E, 6 R) -6-hydroxydeca-2,4-dienoicacid;

Light amorphous solid;

UV (MeOH) [lambda] _max (log 竜 ) 254 (2.95) nm;

IR (KBr)? _Max 3364, 2938, 1714, 1263, 1021 cm ^-1 ;

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

HR-FAB-MS m / z 207.0997 [M + Na] + (calcd for C 10 H 16 O 3 Na, 207.0997).

Position Compound 1 ^a
δ _H  ( J in Hz) Compound 2 ^a
δ _H  ( J in Hz) Compound 3 ^b
δ _H  ( J in Hz) Compound 8 ^a
δ _H  ( J in Hz) One 1.11, m
1.92, m 1.48, m
1.94, m 1.10, m
1.86, m 2 1.99, m
2.20, m 2.50, m
2.55, m 2.00, m
2.48, m 6.33, d (15.2) 3 5.11, dd (12.0, 4.0) 4.58, dd (12.0, 4.8) 7.80, dd (15.2, 11.2) 4 6.80, dd (15.2, 11.2) 5 1.20, m 1.40, m 1.20, m 6.40, dd (15.2, 5.6) 6 1.99, m
2.15, m 1.45, m 2.01, m
2.55, m 4.48, m 7 1.25, m
1.45, m 1.20, m
1.38, m 1.40, m
1.51, m 1.75, m 8 1.55, m 9 0.85, m 0.90, m 1.20, m 1.35, m 10 0.87, t (7.2) 11 1.15, m
1.85, m 1.11, m
1.85, m 1.22, m
1.77, m 12 1.94, m
2.01, m 1.13, m
1.90, m 1.90, m
2.04, m 13 2.10, m 2.55, m 1.19, m 15 5.51, br s 5.98, s 3.37, ovl ^c 16 2.14, m
2.25, m 2.01, m 17 2.40, m 3.06, s 1.87, m 19 1.70, m
2.05, m 1.60, m
2.30, m 1.65, m
1.86, m 20 1.85, m
2.11, m 1.95, m
2.05, m 1.94, m
2.11, m 21 4.61, br s 3.62, s 3.36, ovl ^c 23 1.61, s 1.14, s 1.14, s 24 1.08, s 25 1.15, s 0.74, s 0.86, s 26 0.91, s 0.92, s 0.90, s 27 1.87, m
2.33, m 1.90, m
2.40, m 1.50, m
1.70, m 28 0.87, s 0.82, s 0.78, s 29 3.99, d (10.8)
4.22, d (10.8) 1.40, s 0.75, s 30 1.60, s 1.73, s 0.83, s 2' 6.77, d (16.0) 3 ' 8.11, d (16.0) 7.44, s 5 ' 7.55, d (8.4) 6 ' 7.15, d (8.4) 7.45, d (8.1) 7 ' 6.72, d (8.1) 8' 7.15, d (8.4) 9 ' 7.55, d (8.4) 4'-OCH ₃ 3.77, s a: ¹ H NMR measured at 400 MHz in pyridine- d ₅
b: ¹ H NMR measured at 400 MHz in methanol- d ₄
c: Overlapped with other signals

Position Compound 1 ^a
δ _C , type Compound 2 ^a
δ _C , type Compound 3 ^b
δ _C , type Compound 8 ^a
δ _C , type One 39.4, CH ₂ 39.9, CH ₂ 40.0, CH ₂ 169.8, C 2 27.1, CH ₂ 34.7, CH ₂ 25.8, CH ₂ 123.3, CH 3 80.1, CH 217.0, C 81.0, CH 145.0, CH 4 49.0, C 47.7, C 50.0, C 127.5, CH 5 57.1, CH 55.3, CH 57.9, CH 148.0, CH 6 21.6, CH ₂ 20.7, CH ₂ 21.6, CH ₂ 71.7, CH 7 45.9, CH ₂ 44.7, CH ₂ 45.9, CH ₂ 30.8, CH ₂ 8 37.8, C 38.3, C 38.6, C 28.6, CH ₂ 9 63.1, CH 62.2, CH 61.6, CH 23.8, CH ₂ 10 39.6, C 38.4, C 39.8, C 14.7, CH ₃ 11 27.1, CH ₂ 25.6, CH ₂ 25.6, CH ₂ 12 28.2, CH ₂ 27.6, CH ₂ 27.3, CH ₂ 13 58.0, CH 59.4, CH 59.9, CH 14 139.3, C 163.6, C 78.9, C 15 124.5, CH 129.6, CH 77.5, CH 16 28.5, CH ₂ 201.8, C 28.2, CH ₂ 17 45.3, CH 60.0, CH 47.4, CH 18 36.9, C 45.1, C 39.6, C 19 32.3, CH ₂ 32.5, CH ₂ 34.0, CH ₂ 20 25.0, CH ₂ 26.3, CH ₂ 26.4, CH ₂ 21 70.2, CH 76.3, CH 76.8, CH 22 44.0, C 38.0, C 38.7, C 23 23.5, CH ₃ 27.5, CH ₃ 24.7, CH ₃ 24 177.5, C 21.6, CH ₃ 178.1, C 25 14.6, CH ₃ 15.7, CH ₃ 16.4, CH ₃ 26 20.3, CH ₃ 19.8, CH ₃ 22.5, CH ₃ 27 57.1, CH ₂ 56.1, CH ₂ 55.2, CH ₂ 28 14.6, CH ₃ 16.4, CH ₃ 14.7, CH ₃ 29 65.6, CH ₂ 22.6, CH ₃ 22.9, CH ₃ 30 23.5, CH ₃ 29.5, CH ₃ 29.2, CH ₃ One' 168.2, C 168.1, C 2' 116.4, CH 125.3, C 3 ' 145.7, CH 113.8, CH 4' 126.7, C 148.8, C 5 ' 131.2, CH 153.0, C 6 ' 117.3, CH 116.0, CH 7 ' 161.9, C 123.1, CH 8' 117.3, CH 9 ' 131.2, CH OCH ₃ 56.5, CH ₃ ^{a: 13 C NMR measured at100 ㎒} in pyridine- d 5
b: ¹³ C NMR measured at 100 MHz in methanol- d ₄

Compound 4 Compound 6 Position δ _H  (ppm, J in Hz) ^a δ _C  (ppm) ^b Position δ _H  (ppm, J in Hz) ^a δ _C  (ppm) ^b One 1.04, m; 1.74, m 38.4 One 1.06, m; 1.70, m 38.9 2 1.72, m; 1.94, m 26.4 2 1.73, m; 1.99, m 25.1 3 4.78, dd (10.4, 5.6) 81.4 3 4.69, m 78.4 4 38.5 4 37.9 5 0.90, m 55.9 5 0.94, m 49.4 6 1.50, m; 1.64, m 19.4 6 1.50, m; 1.64, m 18.8 7 1.49, m; 1.86, m 45.0 7 1.49, m; 1.80, m 44.9 8 38.4 8 38.2 9 1.68, m 60.0 9 1.68, m 56.9 10 38.4 10 35.9 11 1.26, m; 1.78, m 26.1 11 1.26, m; 1.78, m 25.4 12 1.73, m; 1.84, m 24.5 12 1.73, m; 1.80, m 28.4 13 1.18, m 60.4 13 1.18, m 62.7 14 75.0 14 139.2 15 2.03, m; 1.71, m 45.9 15 5.50, s 123.3 16 1.63, m; 1.50, m 19.5 16 1.50, m; 1.63, m 24.0 17 1.06, m 56.2 17 1.06, m 43.4 18 38.5 18 37.3 19 1.21, m; 1.97, m 38.5 19 1.21, m; 1.97, m 31.2 20 1.58, m; 2.03, m; 28.5 20 1.60, m; 2.03, m 27.1 21 4.61, dd (11.6, 4.8) 81.1 21 3.52, brs 78.7 22 39.2 22 37.4 23 0.85, s 28.4 23 0.93, s 16.6 24 0.92, s 17.4 24 1.08, s 19.8 25 0.87, s 17.1 25 1.07, s 30.0 26 1.10, s 23.5 26 1.20, s 16.0 27 1.78, m; 1.60, m 62.5 27 1.79, m; 1.63, m 56.3 28 1.29, s 16.7 28 1.36, s 14.3 29 1.00, s 17.0 29 1.15, s 21.8 30 1.03, s 30.4 30 1.31, s 27.7 3-O C OCH ₃ 171.2 3-OCO C H ₃ 2.11, s 21.6 21-O C OCH ₃ 171.0 21-OCO C H ₃ 2.07, s 21.62 a: ¹ H NMR (400 MHz in pyridine- d ₅ ) spectroscopic data
^{b: 13 C NMR (100 ㎒} in pyridine- d 5) spectroscopic data

Compound 5 Compound 7 Position δ _H  (ppm, J in Hz) ^a δ _C  (ppm) ^b Position δ _H  ( ppm , J in  ㎐) ^a δ _C  (ppm) ^b One 1.06, m; 1.74, m 39.4 One 1.11, m; 1.93, m 38.9 2 1.73, m; 1.99, m 27.1 2 1.99, m; 2.30, m 26.7 3 5.16, dd (3.8, 11.8) 80.3 3 5.13, dd (3.8, 11.8) 79.9 4 49.0 4 49.4 5 1.13, m 57.1 5 1.12, m 57.5 6 1.97, m; 2.33, m 21.6 6 1.98, m; 2.32, m 21.2 7 1.26, m; 1.41, m 45.9 7 1.25, m; 1.40, m 45.8 8 37.8 8 37.3 9 0.82, m 63.1 9 0.82, m 62.2 10 39.6 10 39.1 11 1.85 (1 H, m); 2.10 (1H, m) 27.1 11 1.85, m; 2.10, m 25.7 12 1.95, m; 2.01, m 28.2 12 1.95, m; 2.01, m 27.7 13 2.08, m 58.0 13 2.08, m 56.9 14 139.3 14 138.9 15 5.52, s 124.5 15 5.49, s 123.1 16 2.18, m 28.5 16 2.18, m 24.7 17 1.30, s 45.3 17 2.35, s 43.5 18 36.9 18 36.4 19 1.70, m; 1.98, m 32.3 19 1.70, m; 2.03, m 32.0 20 1.00, m; 1.86, m 25.0 20 1.15, m; 1.85, m 25.2 21 4.61, s 70.2 21 4.58, s 69.7 22 44.0 22 43.5 23 1.60, s 23.5 23 1.57, s 24.5 24 177.5 24 177.0 25 1.15, s 15.2 25 1.12, s 14.1 26 0.92, s 20.3 26 0.88, s 19.8 27 1.86, m; 2.40, m 57.4 27 1.86, m; 2.36, m 56.9 28 0.86, s 14.6 28 0.85, s 14.7 29 4.21, d (10.8)
3.99, d (10.8) 65.5 29 4.19, d (10.8)
3.96, d (10.8) 65.8 30 1.59, s 23.5 30 1.57, s 23.0 One' 167.2 One' 124.4 2' 2.40, t (7.5) 36.9 2' 7.12, d (8.5) 132.6 3 ' 3.06, t (7.5) 31.0 3 ' 8.42, d (8.5) 116.2 4' 134.0 4' 163.6 5 ' 8.44, d (8.4) 133.1 5 ' 8.42, d (8.5) 116.2 6 ' 7.14, d (8.4) 116.6 6 ' 7.12, d (8.5) 132.6 7 ' 164.0 7 ' 166.8 8' 7.14, d (8.4) 116.6 9 ' 8.44, d (8.4) 133.1 a: ¹ H NMR (400 MHz in pyridine- d ₅ ) spectroscopic data
^{b: 13 C NMR (100 ㎒} in pyridine- d 5) spectroscopic data

&Lt; Example 3: Measurement of inhibitory activity of acetylcholinesterase and butyrylcholinesterase >

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 by 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 2 10.67 ± 0.66 μM > 30 μM Compound 3 9.98 ± 0.29 μM > 30 μM Compound 4 0.91 ± 0.01 μM > 30 μM Compound 5 1.69 ± 0.10 μM 0.42 ± 0.01 μM Compound 6 > 30 μM 1.37 ± 0.22 μM Compound 7 0.22 ± 0.03 μM > 30 μM Berber 0.10 ± 0.01 μM 1.09 ± 0.17 μM

As shown in the results of Table 5, it was confirmed that the compound isolated from the calcined ginseng extract of the present invention has an inhibitory activity of acetylcholinesterase or butyrylcholinesterase, which is similar to berberine, which is a positive control, and thus is a preventive and therapeutic agent for Alzheimer's disease Can be used effectively.

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

When the substrate was cleaved by? -secretase 1, fluorescence appeared. The activity of the enzyme was confirmed by measuring it with a BACE1 FRET assay kit ( ? -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 6 below.

Condition β-secretase inhibitory activity (IC ₅₀ ) Compound 1 1.07 ± 0.01 μM Compound 2 0.23 ± 0.33 μM Compound 3 0.98 ± 0.21 μM Compound 7 0.31 ± 0.09 μM Compound 8 6.91 ± 0.44 μM Quercetin 6.50 ± 0.39 μM

Referring to the results in Table 6, the compound isolated from the calcine of the present invention showed an inhibitory activity of? -Secretase 1 which is 6 to 28 times stronger than the positive control quercetin, showing excellent therapeutic effect on Alzheimer's disease Can be confirmed.

<Example 5: Toxicity test>

Example 5-1. Acute toxicity

(1) of the present invention or the 3?, 21?, 29-trihydroxycerat-14-en-24-oxacan-3? -Yl- (7'-hydroxycinnamate) This study was conducted to investigate the toxicity to the animal body by acute (within 24 hours) when ingested and to determine the mortality rate. Twenty ICR mice were prepared, and 10 mice were assigned to each group. In the control group, 30% PEG-400 alone was administered, and the experimental group was orally administered with the polysaccharide 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, the control group and the control group treated with the CSF extract or compound 1 survived.

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

Long-term toxicity experiments were carried out on C57BL / 6J mice in order to investigate the effect of the present invention on the organs (tissues) of the animals. Blood was collected from the test group administered with 1.0 g / kg of dioxane-3β-yl- (7'-hydroxycinnamate) (Compound 1) and the control group treated with only the solvent at 8 weeks, glutamate-pyruvate transferase) and BUN (blood urea nitrogen) were measured by Select E (vital scientific NV, Netherland). 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

200 g of the calcined calcium extract of the present invention was 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

1 g of the calcined gangrene extract of the present invention, 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

The safflower extract of the present invention was added to the cooking sauce in an amount of 1% by weight to prepare a cooking sauce for health promotion.

Formulation Example 2-2. Manufacture of flour food products

A food for health promotion was prepared by adding breads, cakes, cookies, crackers and noodles using the mixture of 0.1% by weight to the wheat flour of the present invention.

Preparation Example 2-3. Manufacture of soups and gravies

Health enhancing soup and juice were prepared by adding the calcined ginseng extract of the present invention to soup and juice at 0.1 wt%.

Formulation Example 2-4. Manufacture of dairy products

The extract of the present invention was added to milk in an amount of 0.1% by weight, and various dairy products such as butter and ice cream were prepared using the milk.

Formulation Example 2-5. Vegetable juice manufacturing

The vegetable juice for health promotion was prepared by adding 0.5 g of the calcined ginseng extract of the present invention to 1,000 ml of tomato juice or carrot juice.

Formulation example  2-6. Fruit juice  Produce

0.1 g of the calcined ginseng extract of the present invention was added to 1,000 ml of apple juice or grape juice to prepare fruit juice for health promotion.

Claims (10)

(2 E , 4 E , 6 R ) -6-hydroxydeca-2,4-dienoic acid (Compound 8) of the following formula 1: Lycopodiella The present invention relates to a composition for preventing or treating Alzheimer's disease.
[Chemical Formula 1]

The method according to claim 1,
Wherein the calcined ginseng 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, chloroform and ethyl acetate. &Lt; / RTI &gt; (2 E , 4 E , 6 R ) -6-hydroxydeca-2,4-dienoic acid (Compound 8) of the following formula 1 isolated from Lycopodiella cernua Or a pharmaceutically acceptable salt thereof.
[Chemical Formula 1]

The method according to claim 1 or 3,
Wherein said compound inhibits the activity of at least one enzyme selected from the group consisting of acetylcholinesterase, butyrylcholinesterase and? -Secretase 1. &Lt; / RTI &gt; (2 E , 4 E , 6 R ) -6-hydroxydeca-2,4-dienoic acid (Compound 8) of the following formula 1: Lycopodiella cernua extract of the present invention for preventing or ameliorating Alzheimer's disease.
[Chemical Formula 1]

6. The method of claim 5,
Wherein the calcined ginseng 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, chloroform and ethyl acetate. Health functional foods for improvement. (2 E , 4 E , 6 R ) -6-hydroxydeca-2,4-dienoic acid (Compound 8) of the following formula 1 isolated from Lycopodiella cernua Health functional food for prevention or improvement.
[Chemical Formula 1]

The method according to claim 5 or 7,
Wherein said compound inhibits the activity of at least one enzyme selected from the group consisting of acetylcholinesterase, butyrylcholinesterase and? -Secretase 1. For the prevention or amelioration of the health functional food. Preparing Lycopodiella cernua by calcination with water, C1 to C4 lower alcohols or a mixed solvent thereof; Sequentially fractionating the calcined gum extract using n-hexane and ethyl acetate; And a method for separating of formula 1 (2 E, 4 E, 6 R) -6- hydroxy-deca-2,4-diene acid (Compound 8), the respective fractions by chromatography.
[Chemical Formula 1]

A novel compound (2 E , 4 E , 6 R ) -6-hydroxydeca-2,4-dienoic acid (Compound 8) having the following chemical structure:

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