KR20150092038A - A composition comprising extract of Japanese apricot or compounds isolated therefrom for preventing or treating osteoporosis - Google Patents

Abstract

The present invention relates to a composition comprising compounds isolated from a Japanese apricot extract for preventing or treating osteoporosis. The compounds excellently effective in inducing osteoblast differentiation and suppressing osteoclast differentiation can be used easily for treating osteoporosis.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a composition for preventing or treating osteoporosis, which comprises a plum extract or a compound isolated therefrom,

The present invention relates to a composition for preventing or treating osteoporosis containing a compound isolated from a mussel extract.

Osteoporosis is a typical geriatric bone metabolic disorder caused by a decrease in osteoblast activity or an increase in the relative activity of osteoclasts. It is associated with aging, hormone abnormalities (estrogen reduction in women), inflammatory rheumatoid arthritis, metabolic diseases, Cancer (breast cancer, prostate cancer, etc.), resulting in severe pain and difficulty in walking. Currently, hormone therapy and bisphosphonate preparations are mainly used to treat osteoporosis. However, side effects such as breast cancer and stroke have been reported in the case of hormone therapy, and bisphosphonate preparations have been reported to be effective in suppressing osteonecrosis osteonecrosis of the jaw), there is a strong demand for the development of a natural osteoporosis therapeutic agent and a functional food for prevention.

The bone is a special type of connective tissue, consisting of calciumapple phosphate (hydroxyapatite) and calcification of other ions, and consists of a matrix of collagen, glycoprotein and proteoglycan. The cells that constitute the bone include osteoclasts that perform bone resorption, osteoblasts that are osteogenic cells, osteocytes that are present in the completed bone tissue, and cartilage cells that function as joints at the ends of bones (chondrocyte).

Bone is a characteristic feature of vertebrate and is one of the living tissues in vivo. Bone not only protects organs, it also acts as a hematopoietic cell which produces blood cells carrying nutrients and waste materials necessary for each tissue throughout the body. And accumulating minerals. The mature osteoclast is a polynuclear cell and originates from hematopoietic stem cells. The osteoblast differentiated from the mesenchymal stem cells survive for about 3 to 4 months, and the activated osteoclasts decompose the old bone. Bones are formed. When a large number of osteoblasts form a bone matrix and gradually become mineralized, osteogenesis is completed, then about 70% or more of osteoblasts are apoptosed and some are differentiated into bone cells and bone surface cells.

Bone growth begins with the formation of a primary ossification center and ends at the end of puberty when the growth plate is closed. Bone growth begins with the formation of a primary ossification center, cartilage formation, cell enlargement, ) → calcification of non-chondrocytes → some apoptosis → formation of a bone collar → infiltration of blood into the chondrocyte cell layer → formation of a bone marrow with vascular infiltration It can be divided into stages.

In bone health, skeletal metabolism is regulated by bone resorption by osteoclasts and osteoblast formation during bone development, resulting in the formation of bones. This process continues throughout the lifespan, following the absorption of bone in a healthy state, This process is called bone remodeling and bone remodeling contributes to mineral homeostasis and skeletal homeostasis. These bone reshapes are initiated by chemical and mechanical signals. The main chemical signals are parathyroid hormone (PHT) and female hormone (estrogen), which are secreted in response to low levels of plasma calcium. In the absence of osteoclasts, osteoclast activity exacerbates the activity of osteoblasts, resulting in a decrease in bone mass, leading to postmenopausal osteoporosis.

The osteoblasts differentiated from the mesenchymal stem cells survive for about 3 to 4 months. The activated osteoclasts form new bone at the site where the old bone is degraded. As a result, numerous osteoblasts form bone matrix and the matrix gradually becomes mineralized The bone formation is completed. When MC3T3-E1 subclone4 cell, which is an osteoblast cell, is supplemented with a medium containing? -Glycerophosphate and ascorbic acid,? -Glycerophosphate induces differentiation into osteoblasts, And ascorbic acid is involved in cell growth and collagen formation.

Bone mineralization is a phenomenon in which calcium and minerals are introduced into cells or extracellular matrix. Collagen fibers form a network of calcium and phosphorus, and are mixed with water to form a hydroxyapatite to maintain bone firmness (Gkimcher, M.J. et al., 1989).

RANK (Receptor-Activator of Nuclear Factor Kappa B) is a TNF receptor family protein that plays a crucial role in osteoclast differentiation and activity. It binds to RANKL (RANK ligand) and plays a role in osteoclast signaling. do. The signaling pathway by the RANK / RANKL signal transduction pathway is regulated by recruitment of TRAFs (TNF-receptor-associated factor protein), activation of transcription factors (NF-κB, AP-1, NFATc1) And activation of MAPK pathway (ERK, JNK, p38) (Steven, L. et al., 2003).

In osteoclast differentiation, V-ATPase pumping-out H ⁺ to the outside of the cell produces osteoclasts favorable environment for decomposing bone matrix (acid conditions). Tartrate-resistant acid phosphatase (TRAP) is an enzyme secreted by cells during osteoclast differentiation, which is not inhibited by tartrate. It plays a role in degrading bone matrix phosphorus (Kim, HH et al., 2005).

Prunus mume is a fruit of plum tree belonging to Rosaceae widely distributed in Korea, Japan and China. It is mainly used for processing because it contains a lot of organic acids such as citric acid, malic acid, succinic acid and tartaric acid, , Magnesium, and sodium. It is known that plum is effective for digestion by facilitating the secretion of gastric juice, facilitating metabolism, effective for restoring fatigue, detoxifying, restoring liver function, improving constitution, preventing diabetes and blood pressure increase. Since the content of citric acid which helps the absorption of calcium is higher than other fruits, plum is considered to be an easy raw material for the production of calcium carbonate with high intestinal absorption rate. In addition, it is harvested in early summer (June to July), and most of it is processed into pickles such as pickles, pickles, salads, etc., and is manufactured as an extract or concentrate and sold as a raw material type product. , But the most common form is the plum extract, which is added to the plum and aged for a long time.

Meanwhile, the inventors of the present invention completed the present invention by confirming that compounds isolated from plum extract activates osteoblast differentiation and inhibits the activity of osteoclast while proceeding with research on compositions for treating osteoporosis.

Japanese Patent Application Laid-Open No. 2009-137929 discloses a method for producing a plumice derived from a plumice derived from plum gypsum which comprises plum gypsum in contact with a styrene-divinylbenzene synthetic absorption resin, eluting it with an aqueous solvent, It has been disclosed that a polyphenol extract can be prepared and used as a composition for preventing osteoporosis. In Kono, R. et al., It has already been disclosed that a mussel extract is effective for osteoblast differentiation. However, It is not mentioned in the literature that certain compounds derived from plums derived from the present invention are effective in treating osteoporosis.

Japanese Patent Application Laid-Open No. 2009-137929 (a method for producing a polyphenol extract, an osteoporosis prevention agent, a carbohydrase inhibitor, a functional composition using the same, a food composition containing the functional composition, a specific food composition for health use, June 25, 2009)

Kono, R. et al., A Prunus mume extract stimulated the proliferation and differentiation of osteoblastic MC3T3-E1 cells., Biosci. Biotechnol. Biochem., 2011, 75 (10), 1907-1911. Gkimcher, M.J. et al., Mechanism of calcification: role of collagen fibrils and collagen-phosphoprotein complexes in vitro and in vivo., Anat Rec., 1989, 224, 139-153. Steven, L. et al., Genetic regulation of osteoclast development and function., Nat. Rev. Genet., 2003,4, 638-649. Kim, H.H. et al., Proteomic identification of the TRAF6 regulation of V-ATPase for osteoclast function, Proteomics., 2005, 5, 4152-4160.

It is an object of the present invention to provide a composition for preventing or treating osteoporosis which contains a compound isolated from a plum extract.

The present invention relates to a method for producing

6'-O-acetyl-3-O- (E) -p-coumaroyl sucrose (Compound 1)

3'-O-acetyl-3-O- (E) -p-coumaroyl sucrose (Compound 2)

3'-O-acetyl-3-O- (Z) -p-coumaroyl sucrose (Compound 3)

3,4,5-trimethoxyphenyl -? - D-glucopyranoside (Compound 4),

Benzoyl- [beta] -D-glucopyranoside (Compound 5),

Benzyl-beta-D-glucopyranoside (Compound 6),

Benzyl-? -L-arabinopyranosyl- (1? 6) -? - D-glucopyranoside (Compound 7),

Benzyl-? -L-arabinofuranosyl- (1? 6) -? - D-glucopyranoside (Compound 8),

Benzyl- beta -D-xylopyranosyl- (1 → 6) - β-D-glucopyranoside (Compound 9),

Prunacin (Compound 10),

Amigallatine (Compound 11),

Chlorogenic acid methyl ester (Compound 12),

Cryptochlorogenic acid methyl ester (Compound 13),

Neochlorogenic acid methyl ester (Compound 14),

Isochlorogenic acid (compound 15),

(+) - pinorecinol (Compound 17),

(+) - medioresinol (Compound 18),

(+) - Cyringaredinol (Compound 19),

1,5-dimethyl citrate (Compound 20),

3-hydroxy-3-methoxycarbonylglutaric acid (Compound 21),

(2E) -7-hydroxy-3,7-dimethyl-2-octenyl α-L-arabinopyranosyl (1 → 6) - β-D-glucopyranoside (Compound 22)

Squalene (Compound 23),

2β, 3β-epoxy-5,7,4'-trihydroxyplazane- (4α → 8) -epicathecene (Compound 24)

Quercetin 3-O-neohesperidoside (Compound 28)

And a composition for prevention or treatment of osteoporosis, which comprises at least one compound selected from the group consisting of

[Chemical Formula 1]

(2)

(3)

Such compounds may have an effect of promoting differentiation of osteoblasts or inhibiting osteoclast differentiation.

The present invention also relates to a novel compound of the following formulas (4) to

6'-O-acetyl-3-O- (E) -p-coumaroyl sucrose (Compound 1)

3'-O-acetyl-3-O- (E) -p-coumaroyl sucrose (Compound 2)

3'-O-acetyl-3-O- (Z) -p-coumaroyl sucrose (Compound 3)

2?, 3? -Epoxy-5,7,4'-trihydroxyflaguan- (4? 8) -epiquathecin (Compound 24).

[Chemical Formula 4]

[Chemical Formula 5]

[Chemical Formula 6]

(7)

Hereinafter, the present invention will be described in detail.

The compounds of the present invention can be obtained by extracting the mussel extract with an organic solvent (alcohol, ether, acetone, etc.), partitioning with an organic solvent such as hexane, dichloromethane, ethyl acetate or the like, Can be fractionated or purified and separated by a known or a suitable combination method.

The chromatography was performed by silica gel column chromatography, HP-20 column chromatography, LH-20 column chromatography, ion exchange resin chromatography ( ion exchange resin chromatography, medium pressure liquid chromatography, thin layer chromatography (TLC), silica gel vacuum liquid chromatography and high performance liquid chromatography ). ≪ / RTI >

The present invention also provides a pharmaceutical composition for preventing or treating osteoporosis, which comprises at least one compound selected from the compounds 1 to 28 separated from the mussel extract.

The one or more compounds selected from the compounds 1 to 28 in the pharmaceutical composition may be contained in an amount of 0.001 to 99.9%. The pharmaceutical compositions 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 according to conventional methods. 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 form preparations 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, 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 will depend on the age, sex, body weight of the subject to be treated, the particular disease or condition to be treated, the severity of the disease or condition, the route of administration and the judgment of the prescriber. Dosage determinations based on these factors are within the level of ordinary skill in the art, and dosages generally range from 0.01 mg / kg / day to approximately 2000 mg / kg / day, based on the compound content. 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 of the present invention 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.

The present invention relates to a composition for preventing or treating osteoporosis, which contains a compound isolated from a mussel extract, wherein the compound induces osteoblast differentiation, whereas osteoclast is excellent in the effect of inhibiting differentiation, As shown in FIG.

FIG. 1 is a graph showing the results (using Sirius Red) of a change in collagen content induced by the differentiation of osteoblast from compounds 1 to 28 isolated from plum extract at the time of osteoblast differentiation. From left to right, osteoblast differentiation group (negative control group, Con), osteoblast differentiation group (positive control group, Di) and then osteoblast differentiation group were treated with compounds 1 to 28, 2 < / RTI >
FIG. 2 is a graph showing a result of mineralization (using Alizarin Red S) of calcium content induced by Compounds 1 to 28 isolated from plum extract at osteoblast differentiation. FIG. Compound treatment conditions are shown in Fig.
FIG. 3 shows the results of confirming the inhibitory effect of TRAP induced by compounds 1 to 28 isolated from plum extract at osteoclast differentiation. From left to right, the group treated with Compound 1 to 28 in the osteoclast differentiation group (negative control, Con), the osteoclast differentiation group (positive control, TC) and the osteoclast differentiation group, respectively, And 3, respectively.

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 compounds 1 to 28 from plum extract >

20 kg of fresh raw plums were finely pulverized and then extracted three times at 30 캜 in 60 캜 methanol for 8 hours. After the solvent was completely removed by distillation under reduced pressure, 1150 g of the extract was obtained, suspended in 5 L of water, and then fractionated by repeating 3 times with 5 L of dichloromethane to obtain 38 g of a dichloromethane fraction. The dichloromethane fraction was removed, and the filtrate was repeated three times with 5 L each of ethyl acetate to obtain 170 g of an ethyl acetate fraction. The final filtrate (water layer) was eluted with water, methanol and mixed solvent using HP-20 column chromatography (adsorptive macroporous resins HP-20 column chromatography) to obtain a resin eluate under each solvent condition Water, 25% MeOH methanol eluate, 50% methanol eluate, 75% methanol eluate, methanol eluate).

The dichloromethane fractions were fractionated using silica gel column chromatography (n-hexane / EtOAc, 12: 1-1: 1, v / v) (Fr. C1-C10). Fr. C1 was subjected to silica gel column chromatography (n-hexane / EtOAc, 60: 1-12: 1, v / v) to obtain Compound 23 (108 mg).

Fr. A C7 fraction was purified by silica gel column chromatography to obtain:: - - (C7-11 Fr. C7-1 ) (CHCl 3 / MeOH, 50 1 6 1, v / v) 11 of minor fraction using. Compound 17 (5 mg), Compound 18 (7 mg) and Compound 19 (44 mg) were obtained from Fr. From C7-3 YMC RP-C18 column chromatography _{(MeOH / H 2 O, 1} : 1.3, v / v) and purified by silica gel column chromatography (n-hexane / acetone, 2 : 1, v / v) was obtained using . Compound 16 (27㎎) was YMC RP-C18 column chromatography _{(MeOH / H 2 O, 1} : 3, v / v) and purified by silica gel column chromatography _{(CHCl 3/94% EtOH,} 16: 1, v / v) Through Fr. C7-9.

Five fractions (Fr. E1-E5) were obtained from the ethyl acetate fractions using silica gel column chromatography (CH ₂ Cl ₂ / MeOH, 25: 1-4: 1, v / v)

Fr. E3 was fractionated by silica gel column chromatography (CHCl ₃ /94% EtOH, 8: 1 - 4: 1, v / v) and separated into 6 small fractions (Fr. E3-1 - E3-6). Fr. E3-1 was filtered through a poly prep column to obtain Compound 21 (840 mg). Fr. E3-3 was purified by silica gel column chromatography (CHCl ₃ / MeOH / H ₂ O, 10: 1: 0-6: 1: 0.1, v / v / v) and YMC RP-C18 column chromatography (acetone / H ₂ O , 1: 15-1: 5, v / v) to obtain Compound 10 (25 mg) and Compound 13 (18 mg).

Fr. E3-4 was separated into six small fractions using silica gel column chromatography (CH ₂ Cl ₂ / MeOH / H ₂ O, 10: 1: 0.1-4: 1: 0.1, v / v / v) E3-4-1 - E3-4-6). Fr. E3-4-1 was filtered through a poly prep column to obtain Compound 20 (85 mg). Compound 1 (47 mg), a mixture of compounds 2 and 3 (62 mg) was obtained from Fr. E3-4-5 was obtained by YMC RP-C18 column chromatography (MeOH / H ₂ O, 1: 6-1: 4, v / v). Fr. E3-5 was purified by silica gel column chromatography (CHCl ₃ / MeOH / H ₂ O, 6: 1: 0.1-3: 1: 0.1, v / v / v) and YMC RP-C18 column chromatography (acetone / H ₂ O , 2: 5, v / v) to obtain Compound 24 (8.6 mg). Fr. E3-6 was purified by silica gel column chromatography (CHCl ₃ / MeOH / H ₂ O, 4: 1: 0.1-1.8: 1: 0.1, v / v / v) and YMC RP-C18 column chromatography (MeOH / H ₂ O (10 mg), Compound 25 (4.7 mg) and Compound 26 (14 mg) were obtained by using 1: 10-1: 1.3, v / v.

25% methanol aqueous solution eluate (30 g) was purified by silica gel column chromatography (CH ₂ Cl ₂ / MeOH / H ₂ O, 9: 1: 0.1-4: 1: 0.1, v / v / v) Fractions were obtained (Fr. W1 - W5).

Fr. Six small fractions were obtained using silica gel column chromatography (n-hexane / EtOAc / 94% EtOH, 4: 20: 1-0: 10: 1, v / v / v) - W1-6). Fr. W1-1 was purified by YMC RP-C18 column chromatography (MeOH / H ₂ O, 1: 3-1: 2, v / v) to obtain Compound 27 (11 mg). Fr. W1-3, Fr. W1-4, and Fr. W1-6 was filtered to obtain Compound 5 (30 mg), Compound 6 (445 mg) and Compound 4 (79 mg).

Fr. W3 was fractionated by silica gel column chromatography (EtOAc / 94% EtOH, 10: 1-3: 1, v / v) to give 7 small fractions (Fr. W3-1 - W3-7). Fr. In W3-2 YMC RP-C18 column chromatography _{(MeOH / H 2 O, 1} : 10-1: 3, v / v) and purified by silica gel column chromatography _{(CH 2 Cl 2 / MeOH /} H 2 O, 7: 1 : 0.1, v / v / v) to obtain Compound 14 (89 mg). Fr. W3-5 a silica gel column chromatography _{(CHCl 3 / MeOH, 6:} 1, v / v) and YMC RP-C18 column chromatography (MeOH / H2O, 1: 3 , v / v) to give compound 8 (25 in Mg). Fr. W3-6 was filtered through a poly prep column to obtain Compound 9 (484 mg). Fr. Silica gel column to W3-7 Purification by chromatography _{(CHCl 3 / MeOH / H 2} O, 4:: 1 0.1, v / v / v) gave Compound 7 (1767㎎). Compound 11 (245 mg) was obtained from Fr. W4 was recrystallized and obtained by silica gel column chromatography (CH ₂ Cl ₂ /94% EtOH, 3.5: 1 - 3: 1, v / v).

Seven fractions were obtained from silica gel column chromatography (CHCl ₃ / MeOH, 8: 1 - 3: 1, v / v) in 75% aqueous methanol solution (16 g) (Fr. Fr. The M5 YMC RP-C18 column chromatography _{(MeOH / H 2 O, 1} : 1.8, v / v) using a to give the compound 12 (9㎎) and compound 22 (17㎎). Fr. M6 a YMC PR-C18 chromatography (MeOH / H2O, 1: 2 , v / v) and purified by silica gel column chromatography _{(CH 2 Cl 2 / MeOH /} H 2 O, 4: 1: 0.1, v / v / v) To obtain Compound 28 (18 mg).

<Example 2> Identification of physicochemical properties of the compounds 1 to 28>

Example 2-1. 6'-O-acetyl-3-O- (E) -p-coumaroyl sucrose ; Mumeose P (compound 1)

6'- O- acetyl-3- O - (E) -p-coumaroylsucrose; Mumeose P;

Yield 0.00024%;

White amorphous powder;

Molecular formula C ₂₃ H ₃₀ O ₁₄ ;

IR (KBr): 3366, 1709, 1631, 1605, 1515 cm &lt; ^{-1 &} gt ;;

_{^{1 H-NMR (CD 3 OD}} ) δ 2.09 (3H, s), 3.27 - 3.30 (1H, m), 3.44 (1H, dd, J = 9.6, 3.4 Hz), 3.58 (1H, d, J = 11.7 Hz Dd, J = 11.7, 5.5 Hz), 3.94 (1H, d, J = - 3.97 (1H, m), 4.10 - 4.13 (1H, m), 4.16 (1H, dd, J = 11.7, 6.2 Hz), 4.34 (1H, t, J = 7.6 Hz), 4.47 (1H, dd, J = 11.7, 1.4 Hz), 5.44 (1H, d, J = 3.4 Hz), 5.47 (1H, d, J = 7.6 Hz), 6.41 (1H, d, J = 15.8 Hz), 6.81 (2H, d, J = 8.2 Hz), 7.52 (2H, d, J = 8.2 Hz), 7.71 (1H, d, J = 15.8 Hz);

_{^{13 C NMR (CD 3 OD)}} δ 173.0, 168.5, 161.5, 147.6, 131.6, 127.3, 117.0, 114.8, 104.9, 93.1, 84.3, 79.7, 75.0, 74.3, 73.2, 72.3, 71.8, 65.6, 65.4, 63.8, 21.0 ;

HRMS: calculated for C ₂₃ H ₃₀ O ₁₄ : m / z 553.1528 [M + Na] ⁺ , Found: 553.1480.

Example 2-2. 3'-O-acetyl-3-O- (E) -p-coumaroyl sucrose; Mumeose Q ( compound 2)

3'- O- acetyl-3- O - (E) -p- coumaroylsucrose; Mumeose Q;

Yield 0.00021%;

White amorphous powder;

IR (KBr): 3369, 1710, 1631, 1605, 1514 cm &lt; ^{-1 &} gt ;;

_{^{1 H NMR (CD 3 OD)}} : δ 2.09 (3H, s), 3.58 - 3.61 (3H, m), 3.68 (1H, d, J = 12.4 Hz), 3.77 - 3.81 (2H, m), 3.82 - 3.85 (2H, m), 3.91 - 3.96 (1H, m), 3.94 - 3.98 (1H, m), 4.37 (1H, t, J = 8.2 Hz), 5.22 (1H, t, J = 9.6 Hz), 5.50 ( 1H, d, J = 3.5 Hz ), 5.50 (1H, d, J = 8.2 Hz), 6.44 (1H, d, J = 15.8 Hz), 6.80 (2H, d, J = 8.2 Hz), 7.53 (2H, d, J = 8.2 Hz), 7.72 (1H, d, J = 15.8 Hz);

_{^{13 C NMR (CD 3 OD)}} δ 173.0, 168.7, 161.4, 147.7, 131.7, 127.4, 116.9, 114.9, 105.0, 93.4, 84.2, 79.7, 77.2, 74.6, 74.0, 71.5, 69.3, 65.2, 63.2, 62.0, 21.3 ;

HRMS: calculated for C ₂₃ H ₃₀ O ₁₄ : m / z 553.1528 [M + Na] ⁺ , Found: 553.1512.

Examples 2-3. 3'-O-acetyl-3-O- (Z) -p -coumaroyl sucrose; Mumeose R (Compound 3)

3'- O -acetyl-3- O - ( Z) - p -coumaroylsucrose; Mumeose R;

Yield 0.00011%;

White amorphous powder;

IR (KBr): 3369, 1710, 1631, 1605, 1514 cm &lt; ^{-1 &} gt ;;

_{^{1 H NMR (CD 3 OD)}} : δ 2.10 (3H, s), 3.56 - 3.59 (3H, m), 3.66 (1H, d, J = 12.4 Hz), 3.77 - 3.81 (2H, m), 3.82 - 3.85 (2H, m), 3.89 - 3.93 (1H, m), 3.91 - 3.96 (1H, m), 4.36 (1H, t, J = 8.2 Hz), 5.16 (1H, t, J = 9.6 Hz), 5.46 ( 1H, d, J = 8.2 Hz ), 5.49 (1H, d, J = 3.5 Hz), 5.92 (1H, d, J = 12.4 Hz), 6.75 (2H, d, J = 8.2 Hz), 6.93 (1H, d, J = 12.4 Hz), 7.72 (2H, d, J = 8.2 Hz);

_{^{13 C NMR (CD 3 OD)}} : δ 173.1, 167.5, 160.3, 146.5, 134.2, 127.7, 116.2, 116.0, 104.9, 93.3, 84.2, 79.3, 77.2, 74.4, 73.7, 71.5, 69.2, 65.3, 63.1, 61.8, 21.3;

HRMS: calculated for C ₂₃ H ₃₀ O ₁₄ : m / z 553.1528 [M + Na] ⁺ , Found: 553.1512.

Examples 2-4. 3,4,5-trimethoxyphenyl -? - D-glucopyranoside (Compound 4)

3,4,5-trimethoxyphenyl - ? - D-glucopyranoside;

Yield: 0.00040%;

* White crystals;

Molecular formula C ₁₅ H ₂₂ O ₉ ;

_{^{1 H NMR (DMSO-d 6}} ): δ 3.10 (1H, m), 3.21 (1H, m), 3.26 (1H, m), 3.33 (1H, m), 3.42, 3.71 (2H, m), 3.59 ( 3H, s), 3.74 (6H, s), 4.78 (1H, d, J = 7.6Hz), 6.38 (2H, s);

_{^{13 C NMR (DMSO-d 6}} ): δ 154.00, 153.11, 132.39, 101.02, 94.34, 77.31, 76.84, 73.26, 70.11, 60.90, 60.12, 55.74.

Examples 2-5. Benzoyl- beta -D-glucopyranoside (Compound 5)

benzoyl- [ beta] -D-glucopyranoside;

Yield 0.00015%;

Colorless crystals;

Molecular formula C ₁₃ H ₁₆ O ₇ ;

_{^{1 H NMR (CD 3 OD)}} : δ 3.44 - 3.54 (4H, m), 3.72 (1H, dd, J = 4.6, 12.2 Hz), 3.88 (1H, dd, J = 2.0, 12.2 Hz), 5.75 (1H d, J = 7.8 Hz), 7.51 (2H, t, J = 8.1 Hz), 7.65 (1H, tt, J = 1.2, 8.1 Hz), 8.11 (2H, dd, J = 1.2, 8.1 Hz);

¹³ C NMR (CD ₃ OD):? 166.83, 134.78, 130.96, 130.84, 129.66, 96.27, 78.88, 78.03, 74.00, 71.01, 62.25.

Examples 2-6. Benzyl- beta -D-glucopyranoside (Compound 6)

benzyl- [ beta] -D-glucopyranoside;

Yield 0.00223%;

White crystals;

The molecular formula C ₁₃ H ₁₈ O ₆

_{^{1 H NMR (DMSO-d 6}} ): δ 3.01 - 3.17 (4H, m), 3.47 (1H, m), 3.71 (1H, dd, J = 11.5, 6.3 Hz), 4.23 (1H, d, J = 7.8 Hz), 4.58 (1H, d, J = 12.2Hz), 4.84 (1H, d, J = 12.2Hz), 7.28-7.41 (5H, m);

_{^{13 C NMR (DMSO-d 6}} ): δ 138.21, 128.27, 127.75, 127.48, 102.18, 77.04, 76.81, 73.58, 70.18, 69.52, 61.19.

Examples 2-7. Benzyl-? -L-arabinopyranosyl- (1? 6) -? - D-glucopyranoside ( Compound 7)

benzyl- [ alpha] -L-arabinopyranosyl- (1- &gt; 6) - [ beta] -D-glucopyranoside;

Yield 0.00884%;

Colorless liquid material;

Molecular formula C ₁₈ H ₂₆ O ₁₀ ;

_{^{1 H NMR (CD 3 OD)}} : δ 3.24 - 3.48 (4H, m), 3.50 - 3.54 (1H, m), 3.50 - 3.54 (1H, m), 3.61 (1H, dd, J = 9.04, 6.84 Hz) , 3.76 (1H, dd, J = 11.44, 5.84 Hz), 3.80 (1H, m), 3.87 (1H, dd, J = 12.44, 3.20 Hz), 4.13 (1H, dd, J = 11.44, 2.20 Hz), 4.35 (1H, d, J = 6.84 Hz), 4.37 (1H, d, J = 7.56 Hz), 4.67 (1H, d, J = 11.96 Hz), 4.92 (1H, d, J = 11.96 Hz), 7.24 - 7.29 (1H, m), 7.31-7.35 (2H, m), 7.43 (2H, dd, J = 8.32, 1.48 Hz);

¹³ C NMR (CD ₃ OD): δ 139.36, 129.52, 129.43, 128.90, 105.42, 103.59, 78.11, 77.14, 75.25, 74.36, 72.56, 72.07, 71.83, 69.68, 69.65, 66.88.

Examples 2-8. Benzyl-? -L-arabinofuranosyl- (1? 6) -? - D-glucopyranoside ( Compound 8)

benzyl- [alpha] -L-arabinofuranosyl- (1- &gt; 6) - [ beta] -D-glucopyranoside;

Yield 0.00013%;

Colorless crystals;

Molecular formula C ₁₈ H ₂₆ O ₁₀ ;

_{^{1 H NMR (CD 3 OD)}} : δ 3.22 - 3.37 (3H, m), 3.44 (1H, m), 3.62 - 3.67 (2H, m), 3.75 (1H, dd, J = 3.2, 12.0 Hz), 3.84 (1H, dd, J = 3.4 , 5.8 Hz), 3.99 (1H, m), 4.03 (1H, m), 4.05 (1H, dd, J = 2.2, 10.8 Hz), 4.35 (1H, d, J = 7.8 Hz), 4.65 (1H, d , J = 11.7 Hz), 4.90 (1H, d, J = 11.7 Hz), 5.00 (1H, d, J = 1.2 Hz), 7.27 (1H, t, J = 8.1 Hz) , 7.33 (2H, t, J = 8.1 Hz), 7.42 (2H, d, J = 8.1 Hz);

¹³ C NMR (CD ₃ OD): δ 139.17, 129.54, 129.53, 128.98, 110.21, 103.48, 86.04, 83.45, 79.11, 78.17, 77.00, 75.27, 72.15, 72.07, 68.28, 63.23.

Examples 2-9. Benzyl- beta -D-xylopyranosyl- (1 → 6) -β-D-glucopyranoside (Compound 9)

benzyl- β -D-xylopyranosyl- (1 → 6) - β -D-glucopyranoside;

Yield 0.00242%;

White powder;

Molecular formula C ₁₈ H ₂₆ O ₁₀ ;

_{^{1 H NMR (DMSO-d 6}} ): δ 2.95 - 3.34 (8H, m), 3.56 (1H, dd, J = 6.8, 11.5 Hz), 3.70 (1H, dd, J = 5.4, 11.2 Hz), 3.97 ( 1H, d, J = 11.5 Hz ), 4.21 (1H, d, J = 7.6 Hz), 4.25 (1H, d, J = 7.6 Hz), 4.58 (1H, d, J = 12.2 Hz), 4.80 (1H, d, J = 12.2 Hz), 7.26-7.41 (5H, m);

_{^{13 C NMR (DMSO-d 6}} ): δ 138.13, 128.27, 127.92, 127.52, 104.22, 102.00, 76.68, 76.65, 75.96, 73.46, 73.41, 70.10, 69.64, 69.58, 68.48, 65.73.

Examples 2-10. Prunacin (compound 10)

prunasin;

Yield 0.00013%;

White powder;

Molecular formula C ₁₄ H ₁₇ NO ₆ ;

¹ H NMR (CD ₃ OD):? 3.10-3.20 (4H, m), 3.60 (1H, dd, J = 11.96, 6.32 Hz), 3.82 (1H, dd, J = 11.96, 1.96 Hz) , d, J = 7.08Hz), 5.84 (1H, s), 7.36-7.39 (3H, m), 7.50 (2H, m);

¹³ C NMR (CD ₃ OD):? 135.10, 131.29, 130.42, 129.28, 119.65, 102.01, 78.55, 77.98, 74.89, 71.60, 68.41, 62.92.

Examples 2-11. Amigraldine (Compound 11)

amygdalin;

Yield 0.00123%;

White crystals C ₂₀ H ₂₇ NO ₁₁ ;

_{^{1 H NMR (DMSO-d 6}} ): δ 2.99-3.36 (8H, m), 3.45 (1H, m), 3.62 (1H, m), 3.68 (1H, m), 4.02 (1H, m), 4.24 ( M), 7.47-7.49 (2H, m), 7.57-7.59 (2H, m), 4.42 (1H, d, J = 7.84Hz) ;

_{^{13 C NMR (DMSO-d 6}} ): δ 134.04, 129.78, 129.15, 127.47, 119.01, 103.83, 101.73, 76.86, 76.67, 76.61, 76.56, 73.83, 73.21, 70.15, 70.13, 68.54, 66.85, 61.12.

Examples 2-12. Chlorogenic acid methyl ester (Compound 12)

chlorogenic acid methyl ester;

Yield 0.00005%;

White amorphous powder;

Molecular formula C ₁₇ H ₂₀ O ₉ ;

¹ H NMR (CD ₃ OD):? 1.97-2.22 (4H, m), 3.68 (3H, s), 3.73 (1H, m), 4.12 , d, J = 15.8 Hz) , 6.77 (1H, d, J = 8.04 Hz), 6.94 (1H, dd, J = 1.4, 8.04 Hz), 7.03 (1H, d, J = 1.4 Hz), 7.51 (1H , &lt; / RTI &gt; d, J = 15.8 Hz);

¹³ C NMR (CD ₃ OD): δ 175.74, 168.59, 149.99, 147.48, 147.15, 127.86, 123.22, 116.77, 115.33, 115.26, 76.01, 72.70, 72.28, 70.49, 53.12, 38.16, 37.94.

Examples 2-13. Cryptochlorogenic acid methyl ester (Compound 13)

cryptochlorogenic acid methyl ester;

Yield 0.00009%;

White amorphous powder;

Molecular formula C ₁₇ H ₂₀ O ₉ ;

_{^{1 H NMR (CD 3 OD)}} : δ 1.92 - 2.15 (4H, m), 3.67 (3H, s), 4.15 - 4.23 (2H, m), 4.75 (1H, dd, J = 3.2, 8.8 Hz), 6.29 (1H, d, J = 15.9 Hz), 6.71 (1H, d, J = 8.3 Hz), 6.89 (1H, dd, J = 2.0, 8.3 Hz), 6.99 (1H, d, J = 2.0 Hz), 7.56 (1 H, d, J = 15.9 Hz);

¹³ C NMR (CD ₃ OD): δ 175.95, 169.21, 149.86, 147.43, 147.08, 128.03, 123.24, 116.67, 115.48, 115.31, 78.64, 76.54, 69.12, 65.88, 53.11, 42.21, 38.52.

Examples 2-14. Neochlorogenetic acid methyl ester (Compound 14)

neochlorogenic acid methyl ester;

Yield 0.00045%;

White amorphous powder;

Molecular formula C ₁₇ H ₂₀ O ₉ ;

_{^{1 H NMR (CD 3 OD)}} : δ 2.00 (1H, dd, J = 8.6, 14.4 Hz), 2.08 - 2.14 (2H, m), 2.21 (1H, dd, J = 3.6, 14.4 Hz), 3.68 (1H , dd, J = 3.2, 7.6 Hz), 3.72 (3H, s), 4.12 (1H, m), 5.35 (1H, m), 6.30 (1H, d, J = 15.8 Hz), 6.77 (1H, d, J = 8.3 Hz), 6.94 (1H, dd, J = 2.0,8.3 Hz), 7.04 (1H, d, J = 2.0 Hz), 7.59 (1H, d, J = 15.8 Hz);

¹³ C NMR (CD ₃ OD): δ 176.74, 169.23, 149.72, 147.12, 147.05, 128.18, 123.13, 116.70, 115.99, 115.34, 75.50, 74.04, 72.78, 68.78, 53.02, 40.95, 36.50.

Examples 2-15. The isochlorogenic acid (compound 15)

isochlorogenic acid;

Yield 0.00005%;

Yellow powder;

Molecular formula C ₁₆ H ₁₈ O ₉ ;

_{^{1 H NMR (CD 3 OD)}} : δ 1.89-2.09 (4H, m), 3.81 (1H, m), 3.98 (1H, m), 5.40 (1H, m), 6.31 (1H, d, J = 15.8 Hz ), 6.77 (1H, d, J = 8.3 Hz), 6.94 (1H, dd, J = 8.3, 1.7 Hz), 7.05 (1H, d, J = 1.7 Hz), 7.58 (1H, d, J = 15.8 Hz );

¹³ C NMR (CD ₃ OD): δ 182.45, 168.92, 149.58, 146.90, 146.90, 128.11, 123.02, 116.60, 115.90, 115.24, 75.99, 73.32, 72.53, 70.35, 37.60, 37.60.

Examples 2-16. (+) - lionidecinol (Compound 16)

(+) - lyroiresinol;

Yield 0.00014%;

Yellow amorphous powder;

Molecular formula C ₂₂ H ₂₈ O ₈ ;

^{1 H NMR (Pyridine-d5)} : δ 2.24 (1H, m), 2.67 (1H, m), 3.12 (2H, m), 3.66 (6H, s), 3.79 (6H, s), 4.08 - 4.18 (4H , m), 5.08 (1H, d, J = 5.4 Hz), 6.79 (1H, s), 6.94 (2H, s);

¹³ C NMR (Pyridine-d5):? 149.24, 148.57, 148.29, 139.76, 139.31, 135.65, 129.90, 127.01, 107.74, 107.58, 66.71, 64.35, 59.98, 56.69, 56.37, 49.70, 42.70, 41.96, 34.18.

Examples 2-17. (+) - Pinorecinol (Compound 17)

(+) - pinoresinol;

Yield 0.00003%;

White amorphous powder C ₂₀ H ₂₂ O ₆

_{^{1 H NMR (Acetone-d 6}} ): δ 3.09 (2H, m), 3.80 (2H, dd, J = 9.3, 3.9 Hz), 3.84 (6H, s), 4.20 (2H, dd, J = 9.3, 6.8 Hz), 4.67 (2H, d , J = 4.4 Hz), 6.78 (2H, d, J = 7.8 Hz), 6.83 (2H, dd, J = 7.8, 2.0 Hz), 6.99 (2H, d, J = 2.0 Hz);

¹³ C NMR (Acetone-d ₆ ):? 148.42, 146.95, 134.27, 119.69, 115.62, 110.68, 86.72, 72.30, 56.33, 55.33.

Examples 2-18. (+) - Medioresinol (Compound 18)

(+) - medioresinol;

Yield 0.00004%;

White amorphous powder C ₂₁ H ₂₄ O ₇ ;

_{^{1 H NMR (Acetone-d 6}} ): δ 3.09 (2H, m), 3.80 - 3.84 (2H, m), 3.82 (6H, s), 3.84 (3H, s), 4.21 (1H, dd, J = 8.8 , 6.3 Hz), 4.20 (1H , dd, J = 8.8, 6.3 Hz) 4.67 (2H, d, J = 4.4 Hz), 6.69 (2H, s), 6.78 (1H, d, J = 7.8 Hz), 6.83 (1H, dd, J = 7.8, 2.0 Hz), 6.98 (1H, d, J = 2.0 Hz);

¹³ C NMR (Acetone-d ₆ ):? 148.78, 148.42, 146.95, 136.28, 134.27, 133.30, 119.68, 115.63, 110.67, 104.56, 86.90, 86.73, 72.39, 72.35, 56.73, 56.33, 55.45,

Example 2-19. (+) - Cyringaredinol (Compound 19)

(+) - syringaresinol;

Yield 0.00022%;

Colorless needle crystals;

Molecular formula C ₂₂ H ₂₆ O ₈ ;

_{^{1 H NMR (Acetone-d 6}} ): δ 3.10 (2H, m), 3.82 (12H, s), 3.84 (2H, dd, J = 9.3, 3.9 Hz), 4.23 (2H, dd, J = 9.3, 6.8 Hz), 4.67 (2H, d, J = 4.4 Hz), 6.69 (4H, s);

¹³ C NMR (Acetone-d ₆ ):? 148.76, 136.26, 133.29, 104.51, 86.88, 72.43, 56.72, 55.39.

Examples 2-20. 1,5-dimethyl citrate (Compound 20)

1,5-dimethyl citrate;

Yield 0.00043%;

Colorless crystals;

Molecular formula C ₈ H ₁₂ O ₇ ;

¹ H NMR (CD ₃ OD):? 2.81 (2H, d, J = 15.4 Hz), 2.95 (2H, d, J = 15.4 Hz), 3.66 (6H, s);

_{^{13 C NMR (CD 3 OD)}} : δ 176.72, 172.14, 74.38, 52.34, 44.15.

Examples 2-21. 3-hydroxy-3-methoxycarbonylglutaric acid (Compound 21)

3-hydroxy-3-methoxycarbonyl glutaric acid;

Yield 0.00420%;

Colorless crystals;

Molecular formula C ₇ H ₁₀ O ₇ ;

¹ H NMR (CD ₃ OD):? 2.76 (2H, d, J = 15.6 Hz), 2.91 (2H, d, J = 15.6 Hz), 3.75 (3H, s);

_{^{13 C NMR (CD 3 OD)}} : δ 175.80, 173.58, 74.72, 53.21, 44.29.

Example 2-22. (1E6) - ? - D-glucopyranoside (Compound 22) was obtained in the same manner as in Example 1, except that (2E) -7-hydroxy-3,7-

(2E) -7-hydroxy-3,7-dimethyl-2-octenyl [alpha] -L-arabinopyranosyl (1- &gt; 6) - [ beta] -D-glucopyranoside;

Yield 0.00009%;

Colorless liquid;

Molecular formula C ₂₁ H ₃₈ O ₁₁ ;

_{^{1 H NMR (CD 3 OD)}} : δ 1.16 (6H, s), 1.41 (2H, m), 1.49 (2H, m), 1.68 (3H, s), 2.03 (2H, t, J = 7.56 Hz), 3.17 (1H, m), 3.32 (1H, m), 3.32 (1H, m), 3.39 (1H, m), 3.51 (1H, m), 3.52 (1H, dd, J = 12.36, 1.38 Hz), 3.58 (1H, m), 3.72 ( 1H, dd, J = 11.04, 5.52 Hz), 3.79 (1H, m), 3.85 (1H, dd, J = 12.36, 2.70 Hz), 4.08 (1H, dd, J = 11.04 , 2.10 Hz), 4.19 (1H , dd, J = 11.70, 7.56 Hz), 4.27 (1H, d, J = 7.56 Hz), 4.30 (1H, d, J = 6.84 Hz), 4.34 (1H, dd, J = 11.70, 6.90 Hz), 5.37 (1H, t, J = 6.87 Hz);

¹³ C NMR (CD ₃ OD):? 142.12, 121.74, 105.27, 103.09, 78.09, 76.99, 75.15, 74.32, 72.49, 71.74, 71.49, 69.59, 69.55, 66.81, 66.70, 44.42, 41.20, 29.34, 29.34, 16.59.

Examples 2-23. Squalene (Compound 23)

squalene;

Yield 0.00054%;

Colorless oil;

Molecular formula C ₃₀ H ₅₀ ;

¹ H NMR (CD ₃ OD):? 1.62 (18H, s), 1.70 (6H, s), 1.97-2.13 (20H, m), 5.09-5.17 (6H, m);

¹³ C NMR (CD ₃ OD):? 135.30, 135.10, 131.44, 124.63, 124.53, 124.50, 39.98, 39.96, 28.50, 27.00, 26.89, 25.91, 17.89, 16.26, 16.22.

Example 2-24. 2β, 3β-epoxy-5,7,4'-trihydroxyplazane- (4α → 8) -epicathelin (Compound 24)

2 β, 3 β -epoxy-5,7,4' -trihydroxyflavan- (4 α → 8) -epicatechin;

Yield 0.00004%;

Yellow powder;

UV (MeOH) [lambda] _max (log [ epsilon] ): 280 nm;

_{CD (MeOH) λ max (Δ} ε): 204 (+1.23), 215 (-4.06), 229 (-7.60), 235 (+5.62), 246 (-92.31), 270 (+34.42), 284 (- 24.84) nm;

IR (KBr)? _Max : 3382, 1613, 1514, 1451, 1238, 1142, 1115 cm ^-1 ;

_{^{1 H NMR (CD 3 OD)}} : δ 2.85 (1H, dd, J = 17.2, 2.1 Hz), 2.91 (1H, dd, J = 17.2, 4.1 Hz), 4.17 (1H, d, J = 3.4 Hz), 4.24 (1H, m), 4.41 (1H, d, J = 3.4 Hz), 5.03 (1H, br s), 5.89 (1H, d, J = 2.0 Hz), 6.06 (1H, d, J = 2.0 Hz) , 6.09 (1H, s), 6.84 (1H, d, J = 8.3 Hz), 6.84 (2H, d, J = 8.9 Hz), 6.95 (1H, dd, J = 8.3, 2.0 Hz), 7.13 (1H, d, J = 2.0 Hz), 7.51 (2H, d, J = 8.9 Hz);

¹³ C NMR (CD ₃ OD):? 159.1, 158.3, 156.9, 156.8, 154.3, 152.3, 151.5, 146.4, 131.9, 131.6, 129.7, 119.6, 116.3, 115.7, 115.4, 107.1, 104.2, 102.1, 100.7, 96.7, 96.6, 81.0, 67.9, 67.3, 29.6, 29.5;

_{HRMS: calculated for C 30 H 24} O 11: m / z 559.1246, Found: 559.1205.

Examples 2-25. 2β, 3β-epoxy-5,7,3 ', 4'-tetrahydroxyflavan- (4α → 8 )

2 β, 3 β -epoxy-5,7,3 ', 4'-tetrahydroxyflavan- (4 α → 8) -epicatechin;

Yield 0.00002%;

Yellow powder C ₃₀ H ₂₄ O ₁₂ ;

_{^{1 H NMR (CD 3 OD)}} : δ 2.85 (1H, dd, J = 17.2, 2.2 Hz), 2.92 (1H, dd, J = 17.2, 3.9 Hz), 4.15 (1H, d, J = 3.4 Hz), 4.24 (1H, m), 4.40 (1H, d, J = 3.4 Hz), 5.03 (1H, br s), 5.88 (1H, d, J = 2.2 Hz), 6.06 (1H, d, J = 2.2 Hz) , 6.08 (1H, s), 6.82 (1H, d, J = 8.3 Hz), 6.84 (1H, d, J = 8.3 Hz), 6.95 (1H, dd, J = 8.3, 2.0 Hz), 7.03 (1H, dd, J = 8.3, 2.0 Hz), 7.13 (1H, d, J = 2.0 Hz), 7.14 (1H, d, J = 2.0 Hz);

_{^{13 C NMR (CD 3 OD)}} : δ 158.4, 157.0, 154.4, 152.4, 151.6, 147.1, 146.5, 132.6, 131.7, 120.1, 119.6, 116.4, 115.9, 115.8, 115.4, 107.1, 104.2, 102.1, 100.6, 98.1, 96.7, 81.0, 67.9, 67.3, 29.6, 29.4.

Example 2-26. Isoquercitrin (Compound 26)

isoquercitrin;

Yield 0.00007%;

Yellow powder C ₂₁ H ₂₀ O ₁₂ ;

_{^{1 H NMR (CD 3 OD)}} : δ 3.22 (1H, m), 3.36 (1H, m), 3.42 (1H, m), 3.48 (1H, m), 3.58 (1H, dd, J = 5.5, 12.4 Hz ), 3.71 (1H, dd, J = 2.8, 12.4 Hz), 5.13 (1H, d, J = 7.6 Hz), 6.07 (1H, s), 6.23 (1H, s), 6.85 (1H, d, J = 8.3 Hz), 7.57 (1H, dd, J = 8.3, 2.0 Hz), 7.71 (1H, d, J = 2.0 Hz);

¹³ C NMR (CD ₃ OD): δ 178.92, 166.76, 162.91, 159.23, 158.40, 150.48, 146.30, 135.63, 123.31, 123.16, 117.51, 116.24, 105.20, 103.89, 102.37, 96.52, 78.50, 78.33, 75.86, 62.59.

Examples 2-27. Routine (Compound 27)

routine;

Yield 0.00006%;

Yellow powder C ₂₇ H ₃₀ O ₁₆ ;

_{^{1 H NMR (CD 3 OD)}} : δ 1.11 (3H, d, J = 6.1 Hz), 3.23 - 3.49 (7H, m), 3.53 (1H, dd, J = 3.2, 9.6 Hz), 3.62 (1H, m ), 3.79 (1H, dd, J = 10.7 Hz), 4.51 (1H, s), 5.10 (1H, d, J = 7.3 Hz), 6.20 d, J = 8.3 Hz), 7.62 (1H, dd, J = 8.3, 2.1 Hz), 7.66 (1H, d, J = 2.1 Hz);

¹³ C NMR (CD ₃ OD): δ 179.71, 166.48, 163.27, 159.61, 158.81, 150.10, 146.12, 135.88, 123.79, 123.36, 117.92, 116.29, 105.81, 104.95, 102.64, 100.21, 95.11, 78.37, 77.40, 74.11, 72.41, 72.28, 71.57, 69.88, 68.73, 17.99

Example 2-28. Quercetin 3-O-neohesperidoside (Compound 28)

quercetin 3- O- neohesperidoside;

Yield 0.00009%;

Yellow powder;

Molecular formula C ₂₇ H ₃₀ O ₁₆

_{^{1 H NMR (CD 3 OD)}} : δ 0.97 (3H, d, J = 6.1 Hz), 3.21 (1H, m), 3.22 - 3.79 (2H, m), 3.50 - 3.79 (5H, m), 3.99 (1H , m), 4.04 (1H, m), 5.21 (1H, s), 5.70 (1H, d, J = 7.6 Hz), 6.08 (1H, d, J = 2.1 Hz), 6.24 (1H, d, J = 2.1 Hz), 6.84 (1H, d, J = 8.7 Hz), 7.59 (1H, d, J = 2.2 Hz), 7.59 (1H, dd, J = 8.7, 2.2 Hz);

¹³ C NMR (CD ₃ OD): δ 179.21, 166.00, 163.17, 158.99, 158.08, 150.15, 146.42, 134.48, 123.66, 123.41, 117.16, 116.25, 104.82, 102.82, 101.48, 100.57, 95.83, 80.28, 79.12, 74.28, 72.57, 72.45, 71.84, 70.14, 62.67, 17.58.

On the other hand, the compounds 1, 2, 3, 4, 5, 7, 8, 14, 21, 22, 24 and 25 are compounds initially separated from plum.

The analytical results of the novel compounds 1, 2 and 3 and 24 are as follows, and are also shown in Tables 1 and 2 below.

= 31.3(c = 1.0, MeOH)의 파지티브 옵티컬 로테이션(positive optical rotation)을 갖는 흰색의 무정형 분말이다. HR-ESI-MS(High resolution electrospray ionization mass spectrum, m/z: 553.1480([M+Na]⁺)) 분석을 통해 분자식 C₂₃H₃₀O₁₄를 갖는 것으로 확인된다. Compound 1 (Mumeose P)

= 31.3 (c = 1.0, MeOH) with a positive optical rotation. It is confirmed to have the molecular formula C ₂₃ H ₃₀ O ₁₄ by HR-ESI-MS (high resolution electrospray ionization mass spectrum, m / z: 553.1480 ([M + Na] ⁺ )) analysis.

It is via an IR spectrum of compound 1 confirmed that the hydroxyl group (3366cm ^-1), carbonyl group (1709cm ^-1), double bond (1631cm ^-1), an aromatic ring (1605cm ^-1, 1515 cm ^-1) .

¹ H-NMR spectra showed typical (E) -p-coumaroyl groups [delta] 7.71 and 6.41 (1H, d, J = 15.8 Hz, respectively); 7.52 and 6.81 (2H, d, J = 8.2 Hz, respectively)] and the acetyl group [delta 2.09 (3H, s)].

^In the analysis of the ¹³ C NMR, DEPT and HMQC spectral data, Compound 1 contained two ester carbonyl carbons, six aromatic carbons, two olefinic carbons, twelve oxygen-bearing carbons, one methyl It is confirmed to contain 23 carbon atoms having carbon atoms. This carbon signal is consistent with the (E) -p-coumaroyl group, the acetyl group and the disaccharide.

Sucrose (Compound 1a) is formed through the alkali treatment with an aqueous solution of Compound 1 in a 10% KOH-1,4-dioxane (1: 1, v / v) mixture (see Example 3) Spectral data and Rf value (0.21, normal-phase TLC, CH ₂ Cl ₂ / MeOH / H ₂ O, 2: 1: 0.1, v / v / v).

Thus, Compound 1 is identified as having (E) -p-coumaroyl group, acetyl group and sucrose moiety. (E) -p-coumaroyl group was found to bind to the carbon atom 3 of the fructose moiety through the HMBC (heteronuclear multiple bond correlation) analysis [H-3 (δ 5.47) and C-9 " 168.5)], and the acetyl group is found to be bonded to the 6-carbon of glucose [H-6 '(δ 4.16 and 4.47) and acetylcarbonyl (δ 173.0)].

Thus, Compound 1 has the structure of 6'-O-acetyl-3-0- (E) -p-coumaroyl sucrose (6'-O-acetyl-3-0- Respectively.

Compounds 2 and 3 (Mumeose Q and Mumeose R) were obtained in the form of cis-trans isomers as a white amorphous powder (approximately 2: 1, ¹ H-NMR And ¹³ C NMR) and the molecular formula is C ₂₃ H ₃₀ O ₁₄ (HR-ESI-MS at m / z: 553.1512 ([M + Na] ⁺ )).

IR spectral results indicated that compounds 2 and 3 had hydroxyl groups (3369 cm ^-1 ), carbonyl groups (1710 cm ^-1 ), double bonds (1631 cm ^-1 ), aromatic rings (1605 and 1514 cm ^-1 ) . It is also confirmed that Compound 1 has a sucrose moiety.

The ¹ H NMR and &lt; ¹³ &gt; C NMR spectra of Compound 2 showed p-coumaroyl groups [delta] 7.72 and 6.44 (1H, d, J = 15.8 Hz, respectively); 7.53 and 6.80 (each 2H, d, J = 8.2 Hz )], sucrose moieties [δ 3.58 - 3.61 (3H, m), 3.68 (1H, d, J = 12.4 Hz), 3.77 - 3.85 (4H, m) , 3.91 - 3.98 (2H, m ), 4.37 (1H, t, J = 8.2 Hz), 5.22 (1H, t, J = 9.6 Hz), 5.50 (1H, d, J = 3.5 Hz), and 5.50 (1H , d, J = 8.2 Hz) and an acetyl group [delta 2.09 (3H, s)], and the results are confirmed by DEPT, 2D-NMR analysis (COZY, HMQC and HMBC spectra).

The coupling constant ( J = 15.8 Hz) of the double bond indicates that the p-coumaroyl group is in E form.

HMBC analysis confirmed that the (E) -p-coumaroyl group was bonded to the 3-carbon of the sucrose moiety and the acetyl group was bonded to the 3 'position of the sucrose moiety [H-3 ? 5.50) and C-9 '' (? 168.7), H-3 '(? 5.22) and acetyl carbonyl (? 173.0).

Based on the above results, Compound 2 (mumeose Q) was synthesized from 3'-O-acetyl-3-0- (E) -p-coumaroyl sucrose (3'- -coumaroylsucrose).

&Lt; ¹ &gt; H NMR and &lt; ¹³ &gt; C NMR of compound 3 have a Z-shaped (cis) double bond instead of the E-type (trans) double bond of p-coumaroyl group as compared to compound 2. [ ¹ H-NMR of the compound 3 shows the small coupling constants, which means that it has a δ 6.93 and 5.92 proton signal, Z forms a double bond of the (each 1H, d, J = 12.4 Hz ).

The HMBC spectrum of Compound 3 shows that H-7 '' (δ 6.93) and C-9 '' (δ 167.5), H-3 (δ 5.46) and C-9 ' And carbon (delta 173.1). Thus, compound 3 is analyzed with 3'-O-acetyl-3-O- (Z) -p-coumaroyl sucrose.

= -73.5 (c = 1.0, MeOH)의 네거티브 옵티컬 로테이션(negative optical rotation)을 갖는 황색 분말이며, 분자식 C₃₀H₂₄O₁₁(HR-ESI-MS (m/z: 559.1205, [M-H]^-))을 갖는다. Compound 24 is [?]

= -73.5 is a yellow powder with a negative optical rotation (negative optical rotation) of the (c = 1.0, MeOH), molecular formula _{C 30 H 24 O 11 (HR} -ESI-MS (m / z: 559.1205, [MH] -) ).

Compound 24 was found to have hydroxyl groups (3382 cm ^-1 ), aromatic rings (1613 and 1514 cm ^-1 ), and methylene moieties (1451 cm ^-1 ) via IR spectrum.

&Lt; ¹ &gt; H NMR analysis of Compound 24 showed 10 aromatic signals and 1,4-disubstituted aromatic ring [delta] in spin-coupling patterns 7.51 and 6.84 (each 2H, d, J = 8.9 Hz )], 1,3,4- tree sub Altitude styryl aromatic ring [δ 7.13 (1H, d, J = 2.0 Hz), 6.95 (1H, dd, J = 8.3, 2.0 Hz), and 6.84 (1H, d, J = 8.3 Hz)], and 1,2,3,5- tetrahydro sub Altitude styryl aromatic ring [δ 6.06 (1H, d, J = 2.0 Hz), 5.89 (1H, d, J = 2.0 Hz)).

^{The 1} H NMR spectrum showed the presence of four aliphatic proton atom signals [delta 5.03 (1H, br s), 4.24 (1H, m), 2.91 (1H, dd, J = 17.2, 4.1 Hz) (1H, dd, J = 17.2, 2.1 Hz), which means having a typical signal of flavan-3-ol with a 2,3-cis-configuration (Epicatechin type). The ¹ H NMR spectral data means that compound 24 has a plasmodium dimer. In this result, the 1,4-di-stistilled aromatic ring and the 1,3,4-tri-stistined aromatic ring represent the B-ring and the E-ring of the dimeric plabane, and 1,2,3,5- The substi- tuted ring is attributed to the A-ring. The remaining Aromatic proton [delta 6.09 (1H, s)] is the H-6 of the D-ring, which is identified as the pentasubstisted aromatic ring and analyzed by HMBC (Table 2) Coupling of C-10 (delta 102.1), H-6 and C-8 (delta 107.1) can be observed. ^A pair of doublets [delta 4.17 and 4.41 (1H, d, J = 3.4 Hz, respectively) according to an AB-type spin system confirmed by ¹ H NMR spectroscopy (H-3) and delta 67.9 (C-3), delta 4.41 (H-4) and delta 29.5 (C-3) (C-4). &Lt; / RTI &gt; The coupling constant ( J = 3.4 Hz) means that compound 24 has 3,4-trans-configuration. All of these ¹ H-NMR analyzes are confirmed by analysis of ¹ H- ¹ H COZY (Table 2).

Compound 24 was identified to have 30 carbon atoms with one methylene group, 14 methine groups, and 15 quaternary carbon atoms via ¹³ C NMR, DEPT and HMQC spectral data. Chemical shifts at δ 81.0, 67.3, and 29.6 mean that there is an epicatechinic moiety and δ 100.7 and 67.9 mean that there is an epoxy group between C-2 and C-3 (C-ring). That is, compound 24 has a 2,3-epoxy-5,7,4'-trihydroxyflavone moiety and an epicatechin moiety, which is confirmed by HMBC. Also, HMBC analysis confirms the coupling between C-7, C-8 and C-9 of H-4 and D-ring of C-ring.

It can be seen that the CD spectrum of Compound 24 has a negative Cotton effect at a wavelength of [-4.06 (215 nm)], which indicates that the C-4 (C-ring) and C-8 (D-ring Orientation of the interflavan bond between the interfacial bonds. Compared to compound 25, compound 24 has very similar NMR data except for the chemical shift of the B-ring. Thus, the structure of Compound 24 is 2?, 3? -Epoxy-5,7,4'-trihydroxyflavan- (4? 8) -epicatechin (2 ?, 3? (4α → 8) -epicatechin).

&Lt; Example 3: Analysis by alkaline hydrolysis of compounds 1 to 3 >

Compound 1 (10 mg) was dissolved in 5 ml of a 10% aqueous KOH-1,4-dioxane mixture (10% aqueous KOH-1,4-dioxane mixture, 1: 1, v / v) and stirred at room temperature for 24 hours Lt; / RTI &gt; The reaction solution was neutralized with Dowex HCR W2 (H ⁺ form) and filtered to remove the resin. Thereafter, the reaction solution was distilled under reduced pressure to evaporate the solvent to obtain a crude product. Sucrose (Compound 1a, 2 mg) was obtained from the crude product using silica gel column chromatography (CH ₂ Cl ₂ / MeOH / H ₂ O, 3: 1: 0.1, v / v / v) The compound 1a was confirmed to be sucrose by analysis (see Table 1) and Rf value (0.21, normal-phase TLC, CH ₂ Cl ₂ / MeOH / H ₂ O, 2: 1: 0.1, v / v / v). Compounds 2 and 3 were also able to identify sucrose through such alkaline digestion.

&Lt; Example 4: Differentiation induction of osteoblast or osteoclast &

MC3T3-E1 cells (preosteoblasts from C57BL / 6 mouse) is 37 ℃, 5% CO α- MEM in a _second state (α-modified minimum essential medium, 10% heat-inactivated fetal bovine serum, 100 U / ㎖ penicillin and 100㎍ / Ml streptomycin. &Lt; / RTI &gt; When the MC3T3-E1 cells were 90% grown in the culture dish, 10 mM β-glycerophosphate and 50 μg / ml ascorbic acid were added to the cell culture medium to induce differentiation into osteoblasts Respectively. The differentiation medium containing? -glycerophosphate and ascorbic acid was changed once every three days.

RAW 264.7 cells (macrophages [preosteoclasts] from BALB / c mouse) were cultured for 2 days at a concentration of 1 × 10 ⁴ cells / ml in 96-well plates using DMEM (Dulbecco's Modified Eagle's Medium, 10% Respectively. Thereafter, the medium was replaced with a medium containing 50ng / ml RANKL to induce differentiation into osteoclasts. The differentiation medium containing the RANKL was changed once every two days.

&Lt; Example 5: Confirmation of collagen content >

Collagen content was confirmed by Sirius Red-based colorimetric assay.

MC3T3-E1 cells were induced to differentiate into osteoblasts for 8 days and then treated with 5 μM of each compound in a medium containing 0.3% BSA (bovine serum albumin), followed by culturing for 2 days. Thereafter, the medium was removed, the cells were washed with PBS and fixed with Bouin's fluid for 1 hour. After cell fixation, the cells were immersed in tap water flowing through a cell culture dish for 15 minutes, and then the cell culture dish was dried and stained with Sirius Red solution for 1 hour. After removing the solution, the cells were washed twice with acetic acid / distilled water (1: 200, v / v). The stained cells were dissolved in 0.1 N NaOH and the absorbance was measured at 540 nm in an ELISA reader The results are shown in FIG.

FIG. 1 is a graph showing the results (using Sirius Red) of a change in collagen content induced by osteoblast differentiation of Compounds 1 to 28 isolated from plum extract at the time of osteoblast differentiation. From left to right, osteoblast differentiation group (negative control group, Con), osteoblast differentiation group (positive control group, Di) and then osteoblast differentiation group were treated with compounds 1 to 28, 2 &lt; / RTI &gt;

Referring to FIG. 1, it was confirmed that the collagen content was increased at 5 μM of Compound 1, 2, 3, 5, 7, 8, 10, 12, 13, 15 and 19, have.

&Lt; Example 6: Calcium content determination >

Alizarin Red S was used to confirm calcium deposition (mineralization) in osteoblasts treated with compounds 1 to 28.

MC3T3-E1 cells were induced to differentiate for 15 days, treated with 5 μM of each compound in a medium containing 0.3% BSA (bovine serum albumin), and cultured for 2 days. After the cells were washed with PBS, the cells were fixed in 70% aqueous ethanol solution for 1 hour, and 40 mM Alizarin Red S was allowed to react for 10 minutes.

To quantify the staining, 1 ml of a 10% (w / v) cetylpyridinium chloride solution was added for 15 minutes while the light was blocked, and absorbance was measured at 562 nm using an ELISA reader , And the results thereof are shown in Fig.

FIG. 2 is a graph showing a result of mineralization (using Alizarin Red S) of calcium content induced by Compounds 1 to 28 isolated from plum extract at osteoblast differentiation. FIG. From left to right, osteoblast differentiation group (negative control group, Con), osteoblast differentiation group (positive control group, Di) and then osteoblast differentiation group were treated with compounds 1 to 28, 2 &lt; / RTI &gt;

Referring to FIG. 2, it can be seen that the calcium content is increased at 5 μM of Compound 2, 3, 5, 7, 8, 12, 21 and 28 and the compounds have the ability to differentiate into osteoblasts. Results similar to the results.

Example 7. Confirmation of TRAP (Tartrate-resistant acid phosphatase) activity [

RAW 264.7 cells were induced to differentiate into osteoclasts for 5 days, followed by addition of 1 and 10 μM of Compound 1 to 28 (containing 0.3% BSA) for 2 days. The medium was then removed and the cells were washed with cold phosphate buffered saline And washed. Cells were then fixed with 3.5% formaldehyde solution (3.5% formaldehyde) for 10 min and ethanol-acetone (1: 1) solution for 1 min. Next, the fixed cells were washed with PBS, dried, and reacted in 50 mM citrate buffer (pH 4.5) containing 10 mM sodium tartrate and 6 mM para-nitrophenylphosphate for 1 hour. The reaction was then transferred to a new well plate containing 0.1 N NaOH and the absorbance was confirmed at 405 nm using an ELISA reader. The TRAP activity was expressed as a percentage as compared with the untreated group, and the results are shown in Fig.

FIG. 3 shows the results of confirming the activity of TRAP induced by Compounds 1 to 28 isolated from plum extract at osteoclast differentiation. From left to right, the group treated with Compound 1 to 28 in the osteoclast differentiation group (negative control, Con), the osteoclast differentiation group (positive control, TC) and the osteoclast differentiation group, respectively, And 3, respectively.

Referring to FIG. 3, it was confirmed that all the compounds except compounds 1 and 28 had an excellent effect of inhibiting the activity of TRAP at a concentration of 10 μM, and thus, the compounds were excellent in inhibiting osteoclast differentiation.

Thus, the results of Examples 5 to 7 show that the compounds of the present invention have excellent therapeutic effects on osteoporosis. The results are summarized in Table 3 below.

Condition Compound No. Compounds with osteoblast differentiation ability 1, 2, 3, 5, 7, 8, 10, 12, 13, 15, 19, 21, 28 Compounds that inhibit osteoclast differentiation 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 23, 24 Compound having both osteoblast differentiation ability and osteoclast differentiation inhibiting ability 2, 3, 5, 7, 8, 10, 12, 13, 15, 19, 21

<Example 8> Toxicity test>

Example 8-1. Acute toxicity

This experiment was conducted to examine the toxicity of the compound of the present invention 14 (neochlorogenic acid methyl ester) to animals in an acute (within 24 hours) toxicity when an excessive amount of the compound of the present invention was ingested in a short period of time and to determine the mortality. Twenty ICR mice were injected into each of the control and experimental groups. The control group was treated with PEG-400 / tween-80 / EtOH (8/1/1, v / v / v) , v / v / v). After 24 hours of administration, the respective mortality rates were examined. As a result, it was confirmed that mice were all alive in the control group and the test group to which the compound 14 of the present invention was administered at a concentration of 2 g / kg / day.

Example 8-2. Organ organs toxicity test in experimental group and control group

In the long-term toxicity test, Compound 14 of the present invention was administered to C57BL / 6J mice (10 mice per group) for 8 weeks at each concentration. In order to investigate the effect on the organs (tissues) of animals, a test group to which Compound 14 of the present invention was administered and a control group to which only PEG-400 / tween-80 / EtOH (8/1/1, v / v / Blood samples were collected from the animals at 8 weeks post-transfusion. Glutamate-pyruvate transferase (GPT) and blood urea nitrogen (BUN) concentrations were measured using 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, followed by a general tissue section preparation, histological observation with an optical microscope, and no abnormalities were observed in all tissues.

&Lt; Formulation Example 1 >

Formulation Example 1-1. Manufacture of tablets

200 g of the compound 14 of the present invention (neochlorogenic acid methyl ester) 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 compound 14 of the present invention (neochlorogenic acid methyl ester), 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.

Claims (3)

(1) to (3), which are separated from the plum extract
3,4,5-trimethoxyphenyl -? - D-glucopyranoside (Compound 4),
Benzoyl- [beta] -D-glucopyranoside (Compound 5),
Benzyl-beta-D-glucopyranoside (Compound 6),
Benzyl-? -L-arabinopyranosyl- (1? 6) -? - D-glucopyranoside (Compound 7),
Benzyl-? -L-arabinofuranosyl- (1? 6) -? - D-glucopyranoside (Compound 8),
Benzyl- beta -D-xylopyranosyl- (1 → 6) - β-D-glucopyranoside (Compound 9),
Prunacin (Compound 10),
Amigallatine (Compound 11),
Chlorogenic acid methyl ester (Compound 12),
Cryptochlorogenic acid methyl ester (Compound 13),
Neochlorogenic acid methyl ester (Compound 14),
Isochlorogenic acid (compound 15),
(+) - pinorecinol (Compound 17),
(+) - medioresinol (Compound 18),
(+) - Cyringaredinol (Compound 19),
1,5-dimethyl citrate (Compound 20),
3-hydroxy-3-methoxycarbonylglutaric acid (Compound 21),
(2E) -7-hydroxy-3,7-dimethyl-2-octenyl α-L-arabinopyranosyl (1 → 6) - β-D-glucopyranoside (Compound 22)
Squalene (Compound 23), and
(2), 3? -Epoxy-5,7,4'-trihydroxyplazane- (4? 8) -epicatechin (Compound 24) Or a pharmaceutically acceptable salt thereof.
[Chemical Formula 1]

(2)

(3)

The method according to claim 1,
Wherein said compounds have an effect of promoting the induction of osteoblast differentiation or inhibiting osteoclast differentiation.
The novel compounds 2?, 3? -Epoxy-5,7,4'-trihydroxyplazane- (4? 8) -epicathecine (Compound 24)
(7)

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