KR20110041711A - Pharmaceutical composition for inhibiting aging comprising epifriedelanol - Google Patents

Abstract

PURPOSE: A pharmaceutical composition containing Salicis radicis cortex-derived terepenes or catechin glycoside compounds for anti-aging is provided to suppress cell senescence and to treat aging-related diseases. CONSTITUTION: A pharmaceutical composition for anti-aging contains Salicis radicis cortex-isolated terepenes or catechin glycosides as an active ingredient. The terepenes compound is isolated by adding distilled water and hexane to Salicis radicis cortex methanol extract and separating from hexane layer. The terepenes compound is friedelin or epifridelanol. The pharmaceutical composition is used for treating skin aging, rheumatic arthritis, hepatitis, chronic skin lesion, arteriosclerosis, glandula prostatica vegetation, or liver cancer.

Description

Anti-aging pharmaceutical composition containing epipriederanol as an active ingredient {Pharmaceutical composition for inhibiting aging comprising epifriedelanol}

The present invention relates to a pharmaceutical composition for inhibiting aging containing one or two or more compounds of terpene-based compounds or catechin glycoside-based compounds isolated from Salicis radicis cortex, in particular epipriederanol as an active ingredient.

Cell aging is a phenomenon in which normal somatic cells can no longer divide after a certain number of divisions, which contributes to aging of individuals and tissues, and is an important mechanism for inhibiting abnormal proliferation and cancer formation of cells.

Cell aging is caused by shortening of telomeres, the ends of chromosomes due to repeated division of somatic cells, increased activity of oncogenes or cancer suppressor genes, excessive oxidative stress, UV or radiation, cytotoxic substances such as anticancer agents, and inflammatory reactions. It is also caused by the back.

Aged cells increase in shape, flatten, increase heterochromatin in the nucleus, increase fear in the cytoplasm, increase SA-β-gal (senescence-associated β-galatosidase) activity, Increasing amounts of proteins that inhibit cell growth, such as p53, p16INK4, and p21, insulin-like growth factor binding proteins (IGGFBPs), interleukin-6, and conversion growth factor It secretes several inflammatory proteins, such as transforming growth factor-β (TGF-β) and interferon.

Although cell aging is induced by various factors as described above, retinoblastoma (Rb) and p53 cancer suppressor signaling are known to be important mechanisms for regulating cell aging.

Cell aging not only contributes to aging of an individual or tissue, but also plays an important role in the pathogenesis of various diseases. Aging cells are known to increase with age in human tissues, especially the skin and liver. Ultraviolet rays are representative stimuli that induce skin aging, and the aging of fibroblasts and keratinocytes of the skin is induced by ultraviolet rays, and aging of these cells contributes to the generation of skin damage caused by ultraviolet rays.

In skin ulcers, the degree of aging of fibroblasts plays an important role in the treatment and prognosis of skin ulcers. In addition, senescent cells are frequently observed in inflammatory lesion tissues such as rheumatoid arthritis, osteoarthritis, hepatitis, chronic skin damage tissue, and atherosclerotic vascular tissue. In addition, cell aging is observed in prostate hyperplasia, hepatitis, liver cancer and the like.

As the senescent cells accumulate, the senescent cells do not divide well, and thus, damaged tissues are not properly repaired. Also, as the senescent cells accumulate enzymes or inflammatory cytokines that break down surrounding tissues, they accelerate tissue damage. Contribute to the etiology of the disease.

As it is known that cell aging plays an important role in the pathogenesis of various diseases associated with aging, researches to prevent and treat related diseases through the regulation of cell aging are active. Among them, some studies have been conducted to search for substances that can regulate cell aging and to use them in the prevention and treatment of diseases.

Examples of substances known to regulate cell aging include antioxidants such as N-acetylcysteine and resveratrol, which is found in red wine, which is known to increase SIRT1 activity.

In addition, the research on identifying and separating substances capable of regulating cell aging from plant extracts and developing them as anti-aging ingredients has been reported to apply them to cosmetics and health functional foods, for example, Persicaria hydropiper (whole plant), battery Grass (Filipendula glaberrima; root), water lily (Nymphaea tetragona; root) and camellia (Camellia japonica; leaf) extracts have been reported.

However, most of these studies have searched for a substance that inhibits the activity of matrix metalloproteinase (MMP), which is known to cause tissue damage after irradiating UV rays to fibroblasts. There is no direct evidence that you have it.

Adriamycin is a type of anticancer agent that kills cells at high concentrations but is known to induce cell aging at low concentrations. And the most common method of cell aging is SA-β-gal staining.

Elm ( Ulmus davidiana var. Japonica) is a houseplant and mainly distributed in Korea, Japan and China, and has been used as an anti-inflammatory agent to treat species, gonorrhea, mastitis, etc. The analgesic, anti-inflammatory, antibacterial and anticancer effects of gastric and colorectal cancers have been reported.

Therefore, in the present invention, cell aging is performed by adriamycin in each cell in order to find a substance that inhibits cell aging in various cells such as fibroblasts, vascular endothelial cells, and vascular smooth muscle cells from the root skin of the elm root bark. By investigating substances that inhibit SA-β-gal activity staining in the process of induction, we attempted to find compounds having the effect of directly inhibiting cell aging.

The present inventors completed the present invention by selecting a compound having an effect of specifically inhibiting the cellular aging of human fibroblasts, vascular endothelial cells or vascular smooth muscle cells from the root extract, the fraction obtained therefrom and the compounds separated therefrom. It was.

Accordingly, an object of the present invention is a terpene-based compound or catechin glycoside-based compound isolated from Salicis radicis cortex having the effect of specifically inhibiting cell aging of human fibroblasts, vascular endothelial cells or vascular smooth muscle cells, In particular, to provide an anti-aging pharmaceutical composition and health food containing epipriedelanol as an active ingredient.

In order to achieve the above object, the present invention provides a pharmaceutical composition for inhibiting aging containing one or two or more compounds of terpene-based compounds or catechin glycoside-based compounds isolated from Salicis radicis cortex as an active ingredient.

The terpene-based compound may be separated from the hexane layer fractionated by adding distilled water and hexane to a Salicis radicis cortex methanol extract, and preferably selected from fredelin or epipriedelanol. Can be.

The catechin glycoside-based compound may be separated from the fractionated butanol layer by sequentially adding ethyl acetate and butanol to the distilled water layer fractionated by adding distilled water and hexane to the Salicis radicis cortex methanol extract. Can be selected from catechin-7-O-β-apiofuranoside or catechin-7-O-β-D-glupyraniside (catechin-7-Ob-apiofuranoside). Can be.

The compound may inhibit aging of any one or two or more cells selected from the group consisting of fibroblasts, vascular endothelial cells and vascular squamous muscle cells, and in particular, may effectively inhibit cell aging by adriamycin.

The cell aging inhibitory effect was SA-b-gal activity staining, cytotoxicity was investigated by measuring the MTT method and the number of cells. In addition, Western blots showed the inhibition of p53 protein expression, which is increased during cell aging by adriamycin. Investigate.

In addition, the present invention provides a pharmaceutical composition for inhibiting aging containing epipriedelanol as an active ingredient.

The pharmaceutical composition may treat or prevent aging-related diseases, and in particular, may treat any one disease selected from the group consisting of skin aging, rheumatoid arthritis, osteoarthritis, hepatitis, chronic skin damage tissue, arteriosclerosis, prostatic hyperplasia and liver cancer. However, the present invention is not limited thereto.

In addition, the pharmaceutical composition of the present invention may further comprise a suitable carrier, excipient or diluent commonly used in the manufacture of the pharmaceutical composition.

Carriers, excipients or diluents that may be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, Calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The pharmaceutical compositions of the present invention may be used in the form of oral dosage forms, external preparations, suppositories, and sterile injectable solutions, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, respectively, according to conventional methods. have.

When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, which form at least one excipient such as starch, calcium carbonate, sucrose or Prepare by mixing lactose, gelatin, etc.

In addition to simple excipients, lubricants such as magnesium styrate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. .

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The amount of epipriedellanol of the present invention may vary depending on the age, sex, and weight of the patient, but may be administered once to several times daily in an amount of 0.1 to 100 mg / kg.

In addition, the dosage of epipriederanol may be increased or decreased depending on the route of administration, the severity of the disease, sex, weight, age, and the like. Thus, the dosage amounts are not intended to limit the scope of the invention in any manner.

The pharmaceutical composition may be administered to various mammals such as mice, mice, livestock, humans, and the like. All modes of administration can be expected, for example by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

In particular, when administering epipriedellanol according to the present invention to the human body, there is no concern of side effects as compared to the synthetic drug, because the compound is isolated from natural extracts, and performing the toxicity test by orally administering the herbal extract of the present invention to rats As a result, the 50% lethal dose (LD50) by the toxicity test proved to be a safe substance of at least 1 g / kg or more, and its stability is secured.

The present invention also provides a health food for inhibiting aging containing one or two or more compounds of a terpene-based compound or catechin glycoside-based compound isolated from Salicis radicis cortex as an active ingredient. In particular, the present invention provides an anti-aging health food containing epipriedelanol as an active ingredient.

The health food is used with other food or food additives in addition to the compound, and may be suitably used according to a conventional method. The mixed amount of the active ingredient may be appropriately determined depending on the purpose of use thereof, for example, prophylactic, health or therapeutic treatment.

The effective dose of the compound contained in the health food may be used in accordance with the effective dose of the pharmaceutical composition, but may be less than the above range for long term intake for health and hygiene purposes or health control purposes. However, since the active ingredient has no problem in terms of safety, it is evident that it can be used in an amount above the above range.

There is no particular limitation on the kind of the health food, for example, meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, drinks, tea, Drinks, alcoholic drinks, vitamin complexes, etc. are mentioned.

According to the present invention, the compound isolated from the myodermal bark inhibits cell aging by adramycin and inhibits p53 protein expression, which is increased during this cell aging process, thereby aging-related diseases such as skin aging and rheumatoid arthritis. , Osteoarthritis, hepatitis, chronic skin damage tissue, arteriosclerosis, prostate hyperplasia and liver cancer can be usefully used in the treatment of diseases.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the present invention is not limited by these examples.

Example 1 Material Preparation

Human fibroblasts, umbilical vascular endothelial cells, and vascular smooth muscle cells were purchased from Lonza (Basel, Switzerland). Dubeccos-Modified Eagle's medium (DMEM), fetal calf serum, antibiotic solution (Penicillin-Streptomycin), endothelial cell growth medium-2 (EGM-2), vascular smooth muscle cell growth culture-2 (smooth muscle) cell growth medium-2, SmGM-2) was purchased from Cambrex Bio Science (Walkersvill, MD, USA).

Antibodies against p21 and p53 are described by SantaCruz Biotech, Inc. (SantaCruz, CA, USA) and GAPDH antibody was distributed by Dr. Ki-sun Kwon, Korea Research Institute of Bioscience and Biotechnology. Adriamycin used products of Ildong Pharmaceutical Co., Ltd.

Example 2 Separation of Rhizome Extract and Compound Separation

1.Yugi Skin Extract Fraction

10 kg of dried root skin was extracted with 70% methanol (13 L × 3 times) at reflux for about 24 hours at reflux. The total extract was concentrated under reduced pressure to obtain 1.1 kg of methanol extract. Distilled water (1.4 L) and n-hexane (1.4 L) were added to the methanol extract, and the distilled water layer and the n-hexane layer were fractionated three times using a separatory funnel, and each fraction was concentrated under reduced pressure to obtain a distilled water extract and n-hexane. An extract was obtained.

Distilled water was extracted with ethyl acetate (EtOAc) and butanol (BuOH) in the same manner as above to obtain 67.6 g of n-hexane extract, 70.5 g of ethyl acetate extract, 320 g of butanol extract and 555 g of distilled water extract.

67 g of n-hexane extracts were subjected to normal phase column chromatography. After filling the column (100 × 11 cm) with silica gel (No.9385, 230-400 mesh, Merck) about 30 cm and washing through 3 L of n-hexane, 67 g of the sample was silica gel (No.7734, 70- 230 mesh, Merck) was loaded onto the column by adsorption on 200 g.

Then eluted with n-hexane-ethyl acetate (gradient from n-hexane 100% to ethyl acetate 100%) and ethyl acetate-methanol (gradient from ethyl acetate 100% to methanol 100%) to give 34 fractions (UDH1 to 34). Got it.

Substances U6 (80 mg), U7 (120 mg), U8 (100 mg), U15 (20 mg), U10 (1 g) in UDH1, 3, 6, 9, 22, 23, 31 and 34 in the fraction, U11 (20 mg), U16 (33 mg) and U12 (1.5 g) were obtained, respectively. Ten fractions of UDH18 (1.5 g) were obtained by secondary normal phase column chromatography. Among them, UDH18-8 was used for reverse phase column (4 × 50 cm, LiChroprep RP-18). Eluting with methanol-H ₂ O (gradient from 80:20 to 100% methanol) to give the substance U9 (15 mg). UDH23 (300 mg) was flowed into 100% methanol using a Sephades LH-20 column (3 × 90 cm, Sephadex LH-20) to obtain substance U13 (30 mg), and UDH30 was a Sephades LH-20 column (3 × 90). cm, Sephadex LH-20) and a reversed phase column (4 × 50 cm, LiChroprep RP-18) were used repeatedly to obtain substance U14 (27 mg).

150 g of n-butanol fraction (100 × 11 cm) was eluted with silica gel (No.9385, 230-400 mesh, Merck) with CH ₂ Cl ₂ -methanol (gradient from CH ₂ Cl ₂ 100% to methanol 100%). 30 fractions (UDB1-30) were obtained. Among them, UDA was eluted with 10% methanol using a reversed phase column (4 × 50 cm, LiChroprep RP-18) to obtain a substance U26 (45 mg). Substances U17 (20 mg), U21 (30 mg), U28 (16 mg), and U20 (11.6 mg) were obtained from UDB15 using a secondary, tertiary reversed phase column (4 × 50 cm, LiChroprep RP-18). .

Sub-fraction UDB15-18 was obtained by HPLC (ODS column, 20 × 250 mm) using methanol-distilled water (isocratic elution 33:67) to obtain substances U18 (39.8 mg) and U19 (23 mg). Substances U22 (32 mg) and U23 (500 mg) were obtained by eluting the fraction UDB19 with 100% methanol using a Sephades LH-20 column (3 × 90 cm, Sephadex LH-20) and U25 (200 mg) was UDB25. It was obtained by recrystallization with methanol. Substances U29 (17 mg), U24 (25 mg) and U27 (20 mg) were obtained separately from UDB23 in the same manner and conditions.

2. Determination of Structure of Separation Compound

The chemical structures of the compounds separated as described above were measured by 1 H-, 13 C-NMR, and mass spectra of the compounds as follows, and were determined by comparing them with previously reported literature values.

1) 24-ethylcholesta-5,22-diene-3'-ol-palmitic acid ester (U6)

Colorless needles: ¹ H-NMR (250 MHz, acetone) δ 0.72 (3H, s, H-18), 0.85 (d, J = 6.5 Hz, H-21), 0.96, 0.87 (6H, d, J = 6.5 Hz, H-26, H-27), 1.04 (3H, s, H-19), 4.53 (1H, m, H-3), 5.10 (1H, dd, J = 17.2, 7.6 Hz, H-22) , 5.26 (1H, doublet of doublets, J = 17.2, 7.6 Hz, H-23), 5.37 (1H, doublet, J = 5.6 Hz, H-6). ¹³ C-NMR (62.5 MHz, acetone) δ 39.3 (C-1), 28.1 (C-2), 74.4 (C-3), 37.7 (C-4), 141.1 (C-5), 123.3 (C- 6), 32.9 (C-7), 33.0 (C-8), 51.3 (C-9), 37.6 (C-10), 22.1 (C-11), 38.2 (C-12), 43.4 (C-13 ), 57.2 (C-14), 23.6 (C-15), 28.9 (C-16), 57.9 (C-17), 12.5 (C-18), 19.6 (C-19), 47.0 (C-20) , 19.7 (C-21), 130.9 (C-22), 128.3 (C-23), 29.8 (C-24), 30.4 (C-25), 20.0 (C-26), 20.5 (C-27), 27.0 (C-28), 12.6 (C-29), 173.4 (C = O), 34.6 (CH ₂ CO), 23.7-35.2 (13 ㅧ CH ₂ ), 14.7 (CH ₃ ). Positive FABMS for C ₄₅ H ₇₈ O ₂ m / z 675.0 [M + Na] ⁺ .

2) priedelin (U7)

Colorless needles: mp 260-263 ° C. ¹ H-NMR (250 MHz, pyridine-d ₅ ) δ 0.67 (3H, s, CH ₃ -24), 0.75 (3H, s, CH ₃ -25), 0.92 (3H, s, CH ₃ -30), 0.94 (3H, d, J = 6.5 Hz, CH ₃ -23), 0.99 (3H, s, CH ₃ -26), 1.02 (3H, s, CH ₃ -29), 1.05 (3H, s, CH _3- 27), 1.17 (3H, s, CH ₃ -28), 2.39 (1H, m, H-2), 2.24 (1H, d, J = 6.4 Hz, H-4). ¹³ C-NMR (62.5 MHz, pyridine-d ₅ ) δ 22.4 (C-1), 41.6 (C-2), 211.8 (C-3), 58.0 (C-4), 42.1 (C-5), 41.1 (C-6), 18.4 (C-7), 53.1 (C-8), 37.5 (C-9), 59.2 (C-10), 35.7 (C-11), 30.7 (C-12), 39.8 ( C-13), 38.4 (C-14), 33.0 (C-15), 36.3 (C-16), 30.1 (C-17), 43.0 (C-18), 35.5 (C-19), 28.3 (C -20), 32.5 (C-21), 39.4 (C-22), 7.3 (C-23), 14.6 (C-24), 17.9 (C-25), 20.3 (C-26), 18.8 (C- 27), 32.3 (C-28), 32.0 (C-29), 35.1 (C-30). Positive FABMS for C ₃₀ H ₅₀ O m / z 426.7 [M ⁺ ].

3) Epipriederanol (U8)

White crystals: mp 280-284. ^{1 H-NMR (250 MHz,} CDCl 3) δ 3.71 (1H, br d, H-3), 1.14 (3H, s, CH 3 -30), 0.98 (3H, s, CH 3 -26), 0.97 ( 3H, s, CH ₃ -28), 0.96 (3H, s, CH ₃ -27), 0.94 (3H, s, CH ₃ -24), 0.92 (3H, s, CH ₃ -29), 0.89 (3H, d, J = 7.0 Hz, CH 3 -23), 0.83 (3H, s, CH 3 -25). ¹³ C-NMR (62.5 MHz, pyridine-d ₅ ) δ 16.6 (C-1), 35.9 (C-2), 71.3 (C-3), 49.9 (C-4), 37.4 (C-5), 42.3 (C-6), 18.0 (C-7), 53.5 (C-8), 38.5 (C-9), 61.9 (C-10), 35.5 (C-11), 30.9 (C-12), 38.5 ( C-13), 39.9 (C-14), 33.1 (C-15), 36.6 (C-16), 30.2 (C-17), 43.1 (C-18), 36.3 (C-19), 28.3 (C -20), 32.5 (C-21), 39.4 (C-22), 12.6 (C-23), 17.0 (C-24), 18.6 (C-25), 18.9 (C-26), 20.3 (C- 27), 32.2 (C-28), 35.1 (C-29), 32.0 (C-30). Negative FABMS for C ₃₀ H ₅₂ O m / z 427 [M ⁻ H] ⁻ .

4) Acorusol (U9)

Viscous oil: ¹ H-NMR (250 MHz, CDCl ₃ ) δ 3.59 (1H, dd, J = 10.5, 5.3 Hz, H-1), 2.28 (1H, m, H-11), 2.17 (1H, m, H-9α), 2.03 (1H, m, H-3), 1.90 (1H, m, H-8α), 1.57 (1H, m, H-8β), 1.47 (1H, m, H-9β), 1.66 _{(3H, s, CH 3 -14} ), 0.93 (3H, d, J = 7.0 Hz, CH 3 -12), 0.90 (3H, s, CH 3 -15), 0.87 (3H, d, J = 7.0 Hz , CH ₃ -13). ¹³ C-NMR (CDCl ₃ , 62.9 MHz) δ 76.8 (C-1), 26.7 (C-2), 32.1 (C-3), 139.1 (C-4), 136.5 (C-5), 206.8 (C -6), 57.4 (C-7), 21.6 (C-8), 36.8 (C-9), 42.9 (C-10), 25.7 (C-11), 18.1 (C-12), 21.0 (C- 13), 20.7 (C-14), 18.3 (C-15). 11.8 (C-29). Positive FABMS for C ₁₅ H ₂₄ O ₂ m / z 236 [M] ⁺ .

5) β-sitosterol (U10)

White crystals: ¹ H-NMR (CDCl ₃ , 250 MHz) δ 5.35 (1H, d, J = 5.3 Hz, 6-H), 3.54 (1H, m, 3-H), 2.29 (2H, d, J = 8.5 Hz, 4-H), 2.00 (2H, t, J = 6.6 Hz, 12-H), 1.00 (3H, s, 19-CH ₃ ), 0.93 (3H, d, J = 6.4 Hz, 21-CH ₃ ), 0.86 (3H, d, J = 6.7 Hz, 27-CH ₃ ), 0.83 (3H, d, J = 6.4 Hz, 26-CH ₃ ), 0.82 (3H, t, J = 5.5 Hz, 29- CH ₃ ), 0.68 (3H, s, 18-CH ₃ ). ¹³ C-NMR (CDCl ₃ , 62.9 MHz) δ 37.2 (C-1), 31.6 (C-2), 71.8 (C-3), 42.3 (C-4, C-13), 140.7 (C-5) , 121.7 (C-6), 31.9 (C-7, C-8), 50.1 (C-9), 36.5 (C-10), 21.0 (C-11), 39.8 (C-12), 56.8 (C -14), 24.2 (C-15), 28.2 (C-16), 56.0 (C-17), 12.0 (C-18), 19.0 (C-19), 36.1 (C-20), 18.7 (C- 21), 33.9 (C-22), 23.0 (C-23), 45.8 (C-24), 29.1 (C-25), 19.8 (C-26), 19.3 (C-27), 26.0 (C-28 ), 11.8 (C-29). Positive FABMS for C ₂₉ H ₄₈ O m / z 414.4 [M] ⁺ .

6) Betulinic Acid (U11)

White crystals: mp 314-316. [α] ²⁵ _D +9 μs (c 0.1, MeOH). ¹ H-NMR (250 MHz, pyridine-d ₅ ) δ 4.93 (1H, br s, H-29α), 4.76 (1H, br s, H-29β), 3.53 (1H, m, H-3), 2.64 (1H, m, H-19 ), 1.77 (3H, s, CH 3 -30), 1.21 (3H, s, CH 3 -23), 1.04 (3H, s, CH 3 -27), 1.03 (3H, s, CH ₃ -26), 0.99 (3H, s, CH ₃ -24), 0.79 (3H, s, CH ₃ -25). ¹³ C-NMR (62.9 MHz, pyridine-d ₅ ) δ 39.2 (C-1), 28.3 (C-2), 78.1 (C-3), 39.5 (C-4), 55.8 (C-5), 18.7 (C-6), 34.6 (C-7), 41.0 (C-8), 50.9 (C-9), 37.5 (C-10), 21.1 (C-11), 26.0 (C-12), 38.5 ( C-13), 42.8 (C-14), 31.1 (C-15), 32.8 (C-16), 56.6 (C-17), 49.7 (C-18), 47.7 (C-19), 151.3 (C -20), 30.2 (C-21), 37.5 (C-22), 28.6 (C-23), 16.3 (C-24), 16.4 (C-25), 16.3 (C-26), 14.8 (C- 27), 178.9 (C-28), 110.0 (C-29), 19.4 (C-30). Positive FABMS m / z 456.7 [M ⁺ ].

7) cytosterol-3-O-β-D-glucoside (U12)

Brown soild: ¹ H-NMR (250 MHz, pyridine-d ₅ ) δ 5.35 (1H, d, J = 5.3 Hz, H-6), 3.54 (1H, m, H-3), 2.29 (2H, d, J = 8.5 Hz, H-4), 2.00 (2H, t, J = 6.6 Hz, H-12), 1.00 (3H, s, H-19), 0.93 (3H, d, J = 6.4 Hz, H- 21), 0.86 (3H, d, J = 6.7 Hz, H-27), 0.83 (3H, d, J = 6.4 Hz, H-26), 0.82 (3H, t, J = 5.5 Hz, H-29) , 0.68 (3H, s, H-18). ¹³ C-NMR (62.9 MHz, pyridine-d ₅ ) δ 140.7 (C-5), 121.7 (C-6), 102.5 (C-1 ′), 78.6 (C-3 ′), 78.5 (C-5 ′ ), 78.0 (C-4 ′), 75.3 (C-2 ′), 71.6 (C-3), 62.8 (C-6 ′), 56.8 (C-14), 56.0 (C-17), 50.1 (C -9), 45.8 (C-24), 42.3 (C-13, C-4), 39.8 (C-12), 37.2 (C-1), 36.5 (C-10), 36.1 (C-20), 33.9 (C-22), 31.9 (C-7, C-8), 31.6 (C-2), 29.1 (C-25), 28.2 (C-16), 26.0 (C-28), 24.2 (C- 15), 23.0 (C-23), 21.0 (C-11), 19.8 (C-26), 19.3 (C-27), 19.0 (C-19), 18.7 (C-21), 12.0 (C-18) ), 11.8 (C-29). Positive FABMS for C ₃₅ H ₆₀ O ₆ m / z 572.4 [MH ₂ O] ⁺ .

8) oleanolinic acid (U13)

White crystals: mp 310-312 ° C. ¹ H-NMR (250 MHz, pyridine-d ₅ ) 5.49 (1H, br s, H-12), 3.46 (1H, t, J = 8.2 Hz, H-3), 3.32 (1H, dd, J = 10.5 , 4,4 Hz, H-18), 1.26 (3H, s, CH ₃ -27), 1.23 (3H, s, CH ₃ -29), 1.01 (3H, s, CH ₃ -25, 30), 0.99 (3H, s, CH ₃ -23), 0.93 (3H, s, CH ₃ -24), 0.83 (3H, s, CH ₃ -26). ¹³ C-NMR (62.5 MHz, pyridine-d ₅ ) 39.4 (C-1), 27.1 (C-2), 78.0 (C-3), 39.9 (C-4), 55.7 (C-5), 18.7 ( C-6), 33.2 (C-7), 46.4 (C-8), 48.1 (C-9), 37.3 (C-10), 23.7 (C-11), 122.5 (C-12), 144.8 (C -13), 42.1 (C-14), 26.9 (C-15), 23.6 (C-16), 46.6 (C-17), 30.9 (C-20), 33.2 (C-21), 28.7 (C- 23), 16.5 (C-24), 15.5 (C-25), 17.4 (C-26), 180.2 (C-28), 32.9 (C-29), 26.1 (C-30). Positive FABMS m / z 438.3 [M-OH] ⁺ .

9) Maslinic acid (U14)

White powder: mp 310-312 ° C. [α] ²⁵ _D + 60 ° (c 0.01, CHCl ₃ ). ¹ H-NMR (250 MHz, MeOD) 5.24 (1H, broad s, H-12), 3.61 (1H, m, H-2), 2.90 (1H, d, J = 9.5 Hz, H-3), 2.81 (1H, m, H-18 ), 1.15 (3H, s, CH 3 -26), 1.00 (3H, s, CH 3 -24, 29), 0.93 (3H, s, CH 3 -30), 0.90 ( _{3H, s, CH 3 -25)} , 0.80 (6H, s, CH 3 -23, 27). ¹³ C-NMR (62.5 MHz, MeOD) 48.1 (C-1), 69.5 (C-2), 84.4 (C-3), 40.6 (C-4), 56.7 (C-5), 19.6 (C-6 ), 33.8 (C-7), 40.5 (C-8), 48.6 (C-9), 39.2 (C-10), 24.1 (C-11), 123.4 (C-12), 145.3 (C-13) , 42.9 (C-14), 28.8 (C-15), 24.6 (C-16), 47.6 (C-17), 42.7 (C-18), 47.2 (C-19), 31.6 (C-20), 34.9 (C-21), 33.8 (C-22), 29.3 (C-23), 17.7 (C-24), 17.1 (C-25), 17.5 (C-26), 26.4 (C-27), 182.1 (C-28), 33.6 (C-29), 24.0 (C-30). Positive FABMS for C ₃₀ H ₄₈ O ₄ m / z 472.3 [M] ⁺ .

10) 3-O- (6-O-palmityryl) -β-D-glucopyranosyl stigmasterol (U16)

White powder: [α] ²⁵ _D -25.4 ° (c 0.1 pyridine). ¹ H-NMR (250 MHz, pyridine-d ₅ ) δ 5.47 (1H, dd, J = 4.5, 3.0 Hz, H-6), 5.35 (1H, dd, J = 15.0, 8.4 Hz, H-22), 5.00 (1H, dd, J = 15.0, 8.4 Hz, H-23), 4.48 (1H, dd, J = 11.7, 4.5 Hz, H-glu-6), 4.39 (1H, d, J = 7.6 Hz, H -glu-1), 4.28 (1H, d, J = 11.7 Hz, H-glu-6), 3.39-3.58 (4H, m, H-glu-2-5), 3.56 (1H, m, H-3 ), 1.02 (3H, d, J = 7.0 Hz, H-21), 1.01 (3H, s, H-19), 0.88 (3H, d, J = 8.0 Hz, H-26), 0.87 (3H, d , J = 6.0 Hz, H-27), 0.70 (3H, s, H-18). ¹³ C-NMR (62.9 MHz, pyridine-d ₅ ) δ 37.2 (C-1), 31.7 (C-2), 71.8 (C-3), 42.3 (C-4), 140.3 (C-5), 121.7 (C-6), 31.7 (C-7), 50.1 (C-9), 36.5 (C-10), 22.7 (C-11), 38.9 (C-12), 39.7 (C-13), 56.8 ( C-14), 24.3 (C-15), 28.9 (C-16), 56.0 (C-17), 12.2 (C-18), 19.0 (C-19), 40.5 (C-20), 21.3 (C -21), 138.8 (C-22), 129.3 (C-23), 51.2 (C-24), 29.1 (C-25), 21.1 (C-26), 19.3 (C-27), 25.4 (C- 28), 12.0 (C-29), 101.2 (glu-1), 74.0 (glu-2), 76.0 (glu-3), 70.1 (glu-4), 73.5 (glu-5), 64.7 (glu-6 ), 174.6 (palmitate-1), 34.2 (palmitate-2), 24.9 (palmitate-3), 29.3 (palmitate 4-15), 27.2 (palmitate-17), 14.1 (palmitate-18), 31.9 (palmitate-8) , 16,25). Positive FABMS for C ₅₁ H ₈₈ O ₇ m / z 835 [M + Na] ⁺ .

11) (+)-5-methoxyisolarishresinol-9-O-β-D-xylpyranoside (U17)

Yellowish amorphous powder: [α] ²⁵ _D : + 35.1 ° (c 0.001, MeOH). ¹ H NMR (MeOD, 250 MHz) δ 6.99 (2H, s, H-2 ′, 6 ′), 6.88 (1H, s, H-2), 6.70 (1H, s, H-5), 4.04 (1H , d, J = 6.8 Hz, H-7 ′), 3.89 (1H, d, J = 7.5 Hz, H-1 ″), 3.85 (1H, m, H-9b), 3.72 (3H, s, OCH ₃ ), 3.67 (6H, s, OCH ₃ ), 3.65 (1H, m, H-5 ″ b), 3.57 (1H, m, H-9b), 3.48 (1H, m, H-9a), 3.29 (1H , m, H-4 ″), 3.05 (1H, m, H-3 ″), 3.0 (1H, m, H-2 ″), 2.98 (1H, m, H-5 ″ a), 2.95 (1H, m, H-9′a), 2.72 (2H, m, H-7), 1.89 (1H, m, H-8), 1.70 (1H, m, H-8 ′). ¹³ C NMR (MeOD, 62.9 MHz) δ 148.3 (C-3 ′, 5 ′), 146.8 (C-3), 145.8 (C-4), 136.5 (C-1 ′), 133.8 (C-4 ′) , 133.8 (C-6), 127.9 (C-1), 117.7 (C-5), 112.3 (C-2), 107.5 (C-2 ′, 6 ′), 106.0 (C-1 ″), 78.3 ( C-3 ″), 75.0 (C-2 ″), 71.0 (C-4 ″), 68.2 (C-9 ′), 67.0 (C-5 ″), 63.9 (C-9), 56.2 (3 ′, 5′-OMe), 55.8 (3-OMe), 47.7 (C-8 ′), 45.1 (C-7 ′), 38.8 (C-8), 33.6 (C-7). Positive FABMS for C ₂₆ H ₃₄ O ₁₁ m / z 522.2 [M] ⁺ .

12) Lyoniside (U18)

Colorless needles: [a] ²⁵ _D + 23.5 ° (c 0.002 MeOH). ¹ H NMR (MeOD, 250 MHz) δ 6.54 (1H, s, H-6), 6.40 (2H, s, H-2 ′, 6 ′), 4.22 (1H, d, J = 7.0 Hz, H-7 ′), 4.10 (1H, d, J = 7.2 Hz, H-1 ″), 3.83 (3H, s, OCH ₃ ), 3.79 (1H, m, H-9′b), 3.78 (1H, m, H -5 ″ b), 3.74 (6H, s, OCH ₃ ㅧ 2), 3.63 (1H, m, H-9), 3.55 (1H, m, H-4 ″), 3.48 (1H, m, H-9 ′ A), 3.23 (3H, s, OCH ₃ ), 3.23 (1H, m, H-3 ″), 3.16 (1H, m, H-5 ″ a), 3.12 (1H, m, H-2 ″) , 2.67 (2H, m, H-7), 1.98 (1H, m, H-8 ′), 1.70 (1H, m, H-8). ¹³ C NMR (MeOD, 62.9 MHz) δ 148.9 (C-3 ′, 5 ′), 148.6 (C-3), 147.5 (C-5), 139.6 (C-1 ′), 138.9 (C-4), 134.4 (C-4 ′), 130.2 (C-1), 126.4 (C-2), 107.7 (C-6), 106.8 (C-2 ′, 6 ′), 105.5 (C-1 ″), 78.0 ( C-3 ″), 74.8 (C-2 ″), 71.2 (C-4 ″), 70.8 (C-9 ′), 67.0 (C-5 ″), 65.9 (C-9), 60.0 (3-OMe ), 56.7 (3 ′, 5′-OMe), 56.5 (3-OMe), 46.8 (C-8 ′), 43.0 (C-7 ′), 40.4 (C-8), 34.1 (C-7). Positive FABMS for C ₂₇ H ₃₆ O ₁₂ m / z 552.3 [M] ⁺ .

13) Nudiposide (U19)

Colorless needles: [a] ²⁵ _D -32.5 ° (c 0.002 MeOH). ¹ H NMR (MeOD, 250 MHz) δ 6.55 (1H, s, H-6), 6.40 (2H, s, H-2 ′, 6 ′), 4.22 (1H, d, J = 7.0 Hz, H-7 ′), 4.10 (1H, d, J = 7.2 Hz, H-1 ″), 3.84 (3H, s, OCH ₃ ), 3.80 (1H, m, H-9′b), 3.78 (1H, m, H -5 ″ b), 3.74 (6H, s, OCH ₃ × 2), 3.63 (1H, m, H-9), 3.54 (1H, m, H-4 ″), 3.46 (1H, m, H-9 ′ A), 3.23 (3H, s, OCH ₃ ), 3.23 (1H, m, H-3 ″), 3.16 (1H, m, H-5 ″ a), 3.12 (1H, m, H-2 ″) , 2.67 (2H, m, H-7), 1.98 (1H, m, H-8 ′), 1.70 (1H, m, H-8). ¹³ C NMR (MeOD, 62.9 MHz) δ 149.2 (C-3 ′, 5 ′), 148.6 (C-3), 147.5 (C-5), 139.6 (C-1 ′), 138.9 (C-4), 134.4 (C-4 ′), 130.1 (C-1), 126.3 (C-2), 107.6 (C-6), 106.8 (C-2 ′, 6 ′), 105.0 (C-1 ″), 78.0 ( C-3 ″), 74.9 (C-2 ″), 71.3 (C-4 ″), 70.9 (C-9 ′), 67.1 (C-5 ″), 66.0 (C-9), 59.9 (3-OMe ), 56.7 (3 ′, 5′-OMe), 56.5 (3-OMe), 46.9 (C-8 ′), 43.4 (C-7 ′), 40.6 (C-8), 34.1 (C-7). Positive FABMS for C ₂₇ H ₃₆ O ₁₂ m / z 552.3 [M] ⁺ .

14) Ssioriside (U20)

Yellow amorphous powder: [α] ²⁵ _D + 20.5 ° (c 0.001, MeOH). ¹ H NMR (MeOD, 250 MHz) δ 6.33 (2H, s, H-2,6), 6.31 (2H, s, H-2 ′, 6 ′), 4.18 (1H, d, J = 7.4 Hz, H -1 '), 3.99 (1H, m, H-9), 3.67 and 3.54 (1H, dd, J = 11.0, 6.0 Hz, H-9'), 3.15-3.85 (5H, overlapping, H-2 ', 3 ′, 4 ′, 5 ′), 2.54 ~ 2.68 (4H, overlapping, H-7,7 ′), 2.06 ~ 1.95 (4H, overlapping, H-8,8 ′). ¹³ C NMR (MeOD, 62.9 MHz) δ 148.9 (C-3,5,3 ′, 5 ′), 134.1 (C-4,4 ′), 133.2 (C-1 ′), 133.0 (C-1), 107.3 (C-2 ′, 6 ′), 107.2 (C-2,6), 105.3 (C-1 ″), 78.0 (C-3 ″), 75.1 (C-2 ″), 71.3 (C-4 ″ ), 70.8 (C-9), 67.0 (C-5 ″), 62.6 (C-9 ′), 56.6 (3,5-OMe), 56.5 (3 ′, 5′-OMe), 43.9 (C-8 ′), 41.4 (C-8), 36.3 (C-7), 36.1 (C-7 ′). Positive FABMS for C ₂₇ H ₃₈ O ₁₂ m / z 554.2 [M] ⁺ .

15) (-)-Catechin (U21)

Brown amorphous powder: [α] ²⁵ _D -20.5 ° (c 0.02 MeOH). ¹ H NMR (250 MHz, MeOD) δ 2.50 (1H, dd, J = 16.3, 8.9 Hz, H-4α), 2.86 (1H, dd, J = 16.3, 5.3 Hz, H-4β), 3.95 (1H, m, H-3), 4.55 (1H, d, J = 7.4 Hz, H-2), 5.84 (1H, d, J = 2.3 Hz, H-8), 5.90 (1H, d, J = 2.3 Hz, H-6), 6.68-6.77 (2H, m, H-5 ', H-6'), 6.82 (1H, d, J = 1.8 Hz, H-2 '). ¹³ C NMR (62.9 MHz, MeOD) δ 28.5 (C-4), 68.8 (C-3), 82.8 (C-2), 95.5 (C-8), 96.3 (C-6), 100.8 (C-10 ), 115.2 (C-2 ′), 116.1 (C-5 ′), 120.0 (C-6 ′), 132.2 (C-1 ′), 146.2 (C-3 ′, 4 ′), 156.9 (C-9 ), 157.6 (C-5), 157.8 (C-7). Positive FABMS for C ₁₅ H ₁₄ O ₆ m / z 290.1 [M] ⁺ .

16) Catechin-7-O-α-L-Rhamnopyranoside (U22)

Yellowish amorphous solid: [α] ²⁵ _D -96.1 ° (c 0.001 MeOH). ¹ H NMR (250 MHz, MeOD) δ 1.22 (3H, d, J = 6.1 Hz, H-6 ″), 2.55 (1H, dd, J = 16.3, 7.9 Hz, H-4α), 2.87 (1H, dd , J = 16.3, 5.3 Hz, H-4β), 3.46 (1H, t, J = 5.3 Hz, H-5 ″), 3.63 (1H, m, H-4 ″), 3.79 (1H, dd, J = 9.5, 3.4 Hz, H-3 ″), 3.93 (1H, m, H-2 ″), 4.01 (1H, m, H-3), 4.59 (1H, d, J = 7.4 Hz, H-2), 5.30 (1H, d, J = 1.2 Hz, H-1 "), 6.10 (1H, d, J = 2.3 Hz, H-6), 6.15 (1H, d, J = 2.3 Hz, H-8), 6.71 6.77 (2H, m, H-5 ', H-6'), 6.81 (1H, d, J = 1.8 Hz, H-2 '). ¹³ C NMR (62.9 MHz, MeOD) δ 18.0 (C-6 ″), 28.4 (C-4), 68.5 (C-3), 70.5 (C-5 ″), 72.1 (C-2 ″), 72.3 ( C-3 ″), 73.9 (C-4 ″), 82.9 (C-2), 96.7 (C-8), 97.2 (C-6), 99.9 (C-1 ″), 103.4 (C-10), 115.2 (C-2 ′), 116.1 (C-5 ′), 119.9 (C-6 ′), 132.0 (C-1 ′), 146.2 (C-3 ′, 4 ′), 156.9 (C-9), 157.4 (C-5), 157.7 (C-7). Positive FABMS for C ₂₁ H ₂₄ O ₁₀ m / z 436.1 [M] ⁺ .

17) Catechin-7-O-β-Apiofuranoside (U23)

Colorless needles: [a] ²⁵ _D 31.6 ° (c 0.001 MeOH). ¹ H NMR (250 MHz, MeOD) δ 2.48 (1H, dd, J = 16.3, 8.9 Hz, H-4α), 2.55 (1H, dd, J = 16.3, 5.3 Hz, H-4β), 3.60 (1H, br, H-5 ″), 3.83 (1H, J = 9.7 Hz, H-4 ″ α), 3.96 (1H, m, H-3), 4.07 (1H, J = 9.7 Hz, H-4 ″ β) , 4.12 (1H, J = 3.0 Hz, H-2 "), 4.58 (1H, d, J = 7.4 Hz, H-2), 5.46 (1H, d, J = 3.0 Hz, H-1"), 6.05 (1H, d, J = 2.3 Hz, H-8), 6.12 (1H, d, J = 2.3 Hz, H-6), 6.71-6.77 (2H, m, H-5 ′, H-6 ′), 6.81 (1H, doublet, J = 1.8 Hz, H-2 ′). ¹³ C NMR (62.9 MHz, MeOD) δ 28.4 (C-4), 64.9 (C-5 ″), 68.6 (C-3), 75.4 (C-4 ″), 78.3 (C-2 ″), 80.3 ( C-3 ″), 82.9 (C-2), 96.8 (C-8), 97.3 (C-6), 103.2 (C-10), 108.7 (C-1 ″), 115.2 (C-2 ′), 116.1 (C-5 '), 119.9 (C-6'), 132.1 (C-1 '), 146.2 (C-3', 4 '), 156.8 (C-9), 157.6 (C-5), 158.2 (C-7). Positive FABMS for C ₂₀ H ₂₂ O ₁₀ m / z 423.1 [M + H] ⁺ .

18) Catechin-3-O-α-L-Rhamnopyranoside (U24)

Colorless needles: [a] ²⁵ _D -56.4 ° (c 0.001 MeOH). ¹ H NMR (250 MHz, MeOD) δ 1.22 (3H, d, J = 6.1 Hz, H-6 ″), 2.57 (1H, dd, J = 16.3, 7.9 Hz, H-4α), 2.82 (1H, dd , J = 16.3, 5.3 Hz, H-4β), 3.49-3.72 (4H, overlapping, H-2 ″, 3 ″, 4 ″, 5 ″), 3.92 (1H, m, H-3), 4.26 (1H , d, J = 1.2 Hz, H-1 ''), 4.58 (1H, d, J = 7.4 Hz, H-2), 5.83 (1H, d, J = 2.3 Hz, H-6), 5.91 (1H, d, J = 2.3 Hz, H-8), 6.68-6.72 (2H, m, H-5 ', H-6'), 6.82 (1H, d, J = 1.8 Hz, H-2 '). ¹³ C NMR (62.9 MHz, MeOD) δ 17.9 (C-6 ″), 27.9 (C-4), 70.3 (C-5 ″), 72.0 (C-2 ″), 72.2 (C-3 ″), 73.9 (C-4 ″), 75.9 (C-3), 81.1 (C-2), 95.5 (C-6), 96.4 (C-8), 100.6 (C-1 ″), 102.1 (C-10), 115.0 (C-2 ′), 116.1 (C-5 ′), 119.8 (C-6 ′), 131.9 (C-1 ′), 146.2 (C-3 ′, 4 ′), 156.9 (C-9), 157.4 (C-5), 157.7 (C-7). Positive FABMS for C ₂₁ H ₂₄ O ₁₀ m / z 437.2 [M + H] ⁺ .

19) Butyl α-D-Plactofuranoside (U26)

Amorphous powder: [α] ²⁵ _D + 31.0 ° (c 0.002 MeOH). ¹ H NMR (250 MHz, CDCl ₃ ) δ 0.89 (3H, t, J = 7.3 Hz, 4-H), 1.35 (2H, m, H-3), 1.53 (2H, m, H-2), 3.50 (2H, m, H-1), 3.58 (1H, dd, J = 10.8, 2.0 Hz, H-6′a), 3.60 (1H, d, J = 11.0 Hz, H-1′a), 3.64 ( 1H, m, H-6′b), 3.72 (1H, d, J = 11.0 Hz, H-1′b), 3.82 (1H, m, H-5 ′), 3.88 (1H, dd, J = 10.0 , 4.0 Hz, H-4 ′), 4. 04 (1H, d, J = 12.0 Hz, H-3 ′). ¹³ C NMR (62.9 MHz, CDCl ₃ ) δ 14.2 (C-4), 20.4 (C-3), 33.5 (C-2), 61.6 (C-1), 61.9 (C-1 ′), 62.6 (C -6 '), 78.4 (C-4'), 83.2 (C-3 '), 83.8 (C-5'), 108.7 (C-2 '). Positive FABMS for C ₁₀ H ₂₀ O ₆ m / z 259 [M + Na] ⁺ .

20) Ampelopsisionoside (U28)

White powder: [α] ²⁵ _D -32.5 ° (c 0.001, MeOH). ¹ H NMR (250 MHz, MeOD) δ 5.92 (1H, dd, J = 15.9, 6.4 Hz, H-8), 5.74 (1H, d, J = 15.9 Hz, H-7), 4.43 (1H, m, H-9), 4.34 (1H, J = 7.7 Hz, H-1 '), 3.84 (1H, J = 11.9, 2.4 Hz, H-6'β), 3.64 (1H, J = 11.9, 5.3 Hz, H -6'α), 3.13 to 3.34 (41H, m, H-2 ', 3', 4 ', 5'), 2.85 (1H, d, J = 13.4 Hz, H-2α), 2.44 (1H, t , J = 13.2 Hz, H-4α), 2.25 (1H, m, H-5), 2.13 (1H, m, H-4β), 1.82 (1H, dd, J = 13.4, 2.1 Hz, H-2β) , 1.31 (3H, d, J = 6.4 Hz, H-10), 0.97 (3H, s, H-11), 0.92 (3H, s, H-12), 0.90 (3H, d, J = 6.4 Hz, H-13). ¹³ C NMR (62.9 MHz, MeOD) δ 214.8 (C-3), 134.9 (C-8), 133.9 (C-7), 102.6 (C-1 ′), 78.1 (C-6), 78.0 (C- 3 ′), 77.9 (C-5 ′), 77.7 (C-9), 75.3 (C-2 ′), 71.5 (C-4 ′), 62.7 (C-6 ′), 52.4 (C-2), 46.1 (C-4), 43.9 (C-1), 37.7 (C-5), 25.3 (C-12), 24.9 (C-11), 21.4 (C-10), 16.4 (C-13). Positive FABMS for C ₁₉ H ₃₂ O ₈ m / z 411.2 [M + Na] ⁺ .

21) Catechin-7-O-β-D-glucopyranoside (U29)

Colorless solid: [α] ²⁵ _D -86.5 ° (c 0.001 MeOH). ¹ H NMR (250 MHz, MeOD) δ 2.47 to 2.57 (1H, dd, J = 16.4, 8.0 Hz, H-4α), 2.82 (1H, dd, J = 16.4, 5.5 Hz, H-4β), 3.38 to 3.41 (4H, overlapping, H-2 ″, 3 ″, 4 ″, 5 ″), 3.70 (1H, dd, J = 11.0, 3.0 Hz, H-6 ″ α), 3.85 (1H, d, J = 12.0 Hz, H-6 ″ β), 3.97 (1H, m, H-3), 4.57 (1H, d, J = 7.4 Hz, H-2), 4.80 (1H, d, J = 7.0 Hz, H-1 ″), 6.14 (1H, d, J = 2.3 Hz, H-8), 6.18 (1H, d, J = 2.3 Hz, H-6), 6.71-6.77 (2H, m, H-5 ′, H− 6 '), 6.82 (1H, d, J = 1.7 Hz, H-2'). ¹³ C NMR (62.9 MHz, MeOD) δ 28.5 (C-4), 62.4 (C-6 ″), 68.6 (C-3), 71.3 (C-4 ″), 74.8 (C-2 ″), 78.0 ( C-3 ″), 78.0 (C-5 ″), 82.9 (C-2), 96.8 (C-8), 97.3 (C-6), 102.2 (C-1 ″), 103.5 (C-10), 115.2 (C-2 ′), 116.0 (C-5 ′), 120.0 (C-6 ′), 132.0 (C-1 ′), 146.2 (C-3 ′, 4 ′), 156.8 (C-9), 157.5 (C-5), 158.7 (C-7). Positive FABMS for C ₂₁ H ₂₄ O ₁₁ m / z 452.1 [M] ⁺ .

Example 3 Induction of Cell Aging by Adriamycin Treatment

1. Cell Culture

Human fibroblasts, umbilical cord endothelial cells, and vascular smooth muscle cells were used. Human fibroblasts were divided into 2 X 10 ⁵ cells in a 100 mm diameter dish using DMEM medium containing 10% fetal bovine serum and 1% antibiotics (penicillin 10,000unit / ml, stretomycin 10,000ug / ml). Incubated in a 5% carbon dioxide incubator at 37 ° C. When 80-90% of the cells grew at the bottom of the petri dish, the cells were separated by treatment with trypsin-EDTA solution (2.5%), followed by subculture.

Umbilical cord vascular endothelial cells were cultured in the same manner using EGM-2 and vascular smooth muscle cells using SmGM-2 as a culture medium. Each time the cells are passaged, the number of cells is measured to determine how many times the cells divide. The number of cell division (population doubling, PD) was investigated by the following equation. PD = log ₂ F / log ₂ I (F = last cell number, I = first cell number). The cells used in the experiment were divided into 40 times for human fibroblasts, 30 times for umbilical vascular endothelial cells, and 30 times for vascular smooth muscle cells.

2. Induction of Cell Aging by Adriamycin Treatment

100 mm a human fibroblasts in culture dishes 1 X 10 ⁵ gae diameter, cord blood in vitro blood cells and vascular smooth muscle cells, 2 X 10 ⁵ gae was dispensed. After incubation in a 5% carbon dioxide incubator at 37 ° C. for 3 days, the cell culture was removed. The cells were washed twice with DMEM broth containing antibiotics and then treated with 500 nM adriamycin for 4 hours. After washing the cells three times with DMEM medium containing antibiotics, human fibroblasts were treated with 10% fetal bovine serum and DMEM medium containing antibiotics, cord blood vessel endothelial cells were EGM-2, and vascular smooth muscle cells were SmGM-2 medium. Incubated with. After 4 days, cell senescence was induced by senescence-associated β-galactosidase (SA-β-gal) activity staining.

Example 4 Cytotoxicity Review

1. Cell culture and herbal extract pretreatment

It was examined whether the extracts from the roots of the roots and the compounds isolated therefrom were effective for the cell senescence induced by adriamycin. After 4 hours of treatment with adriamycin, trypsin-EDTA was isolated from the culture plate, and then fibroblasts (5000 cells / ml) and umbilical cord endothelium in DMEM medium containing 10% fetal bovine serum and antibiotics in 96 well cell culture vessels. Cells and vascular smooth muscle cells were each made to 10,000 / ml, and then 100 μl were dispensed into each well.

Finally, 500 fibroblasts and umbilical vascular endothelial cells and vascular smooth muscle cells were dispensed for each well. Incubated at 37 ° C., 5% carbon dioxide incubator for one day. After adding 100 μl of DMEM medium containing 10% fetal bovine serum and 1% antibiotic to each well, the root extract and fractions U1 to U5 were 100 μl / ml, and the single compound U6 to U29 separated therefrom was 10 Treated with μl / ml.

DMSO as a negative control and N-acetylcysteine as a positive control were added at 10 mg / ml. After incubation in a 37%, 5% carbon dioxide incubator for 3 days, the growth rate of the cells was examined by MTT assay and cell number measurement as follows.

2. MTT Analysis

50 μl of 0.1% MTT (3- (4,5-dimethylthiazol-2yl) -2,5-diphenyltetrazolium bromide) solution was added to each well of the 96 well culture vessel and reacted at 37 ° C. and 5% carbon dioxide incubator for 3 hours. . After removing the culture solution and the MTT solution, 100 μl of DMSO was added to dissolve the crystals formed. Absorbance was measured at 550 nm using a microplate reader.

3. Results

As shown in Figure 3, U4 in human fibroblasts, U1, U2, U3, U4, U18 in the umbilical cord endothelial cells showed cytotoxicity, the other fractions did not show cytotoxicity.

Example 5 Examination of Adriamycin Induced Cell Aging Inhibition Effect

1. Cell culture and herbal extract pretreatment

It was examined whether the extracts from the roots of the roots and the compounds isolated therefrom were effective for the cell senescence induced by adriamycin. After 4 hours of treatment with adriamycin, trypsin-EDTA was isolated from the culture plate, and then fibroblasts (5000 cells / ml) and umbilical cord endothelium in DMEM medium containing 10% fetal bovine serum and antibiotics in 96 well cell culture vessels. Cells and vascular smooth muscle cells were each made to 10,000 / ml, and then 100 μl were dispensed into each well.

Finally, 500 fibroblasts and umbilical vascular endothelial cells and vascular smooth muscle cells were dispensed for each well. Incubated at 37 ° C., 5% carbon dioxide incubator for one day. After adding 100 μl of DMEM medium containing 10% fetal bovine serum and 1% antibiotic to each well, the root extract and fractions U1 to U5 were 100 μl / ml, and the single compounds U6 to U29 separated therefrom were 10 Treated with μl / ml.

DMSO as a negative control and N-acetylcysteine as a positive control were added at 10 mg / ml. After culturing in a 5% carbon dioxide incubator at 37 ° C. for 3 days, the degree of cell aging was examined by SA-β-gal (SA-β-galsenescence-associated β-galactosidase) activity staining as follows.

2. SA-β-gal active staining

After treatment with the herbal extract for 3 days in 96 well culture vessel or 24 well culture vessel, the cells were washed with phosphate buffer. After fixing 3.7% paraformaldehyde cells, the fixative was removed.

SA-β-gal staining solution (40 mM citric acid / phosphate [pH 5.85], 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl, 2 mM MgCl ₂ , X-gal 1 mg / ml) 100 ㎕ of each well was put into a 96 well culture vessel and 500 ㎕ of each well was put into a 24 well culture vessel. Wrapped in tinfoil and reacted at 37 ° C. for 16 to 18 hours.

After washing twice with phosphate buffer (PBS), it was stained for 5 minutes with 1% eogene solution. After washing twice with phosphate buffer solution, the cells stained blue with an optical microscope were observed. The SA-β-gal activity level was expressed as a percentage (%) by measuring the number of cells stained blue in the cytoplasm out of a total of 50 to 100 cells.

3. Results

As shown in Figure 4, U7, U8, U9, U13, U14, U15, U17, U19, U20, U21, U22, U23 fractions in human fibroblasts, U8, U10 fractions in cord blood vessel endothelial cells are adriamycin It was observed to inhibit cell aging by.

Ten fractions of primary screening fractions with relatively low cytotoxic effects and observed to inhibit SA-b-gal activity (U3, U7, U8, U15, U19, U20, U22, U23, U27, U29) Further experiments were carried out, and as a result, U8 (epifriedelanol), U20 (ssioriside), U29 (catechin-7-ObD-glucopyraniside) fraction (figure 5) in fibroblasts, U7 (friedelin) in umbilical vascular endothelial cells, U8 (epifriedelanol), U23 (catechin-7-Ob-apiofuranoside) fractions (FIG. 6) were found to inhibit cell aging.

The effect of U8 fraction confirmed to inhibit cell aging in both cells was examined in vascular smooth muscle cells. As shown in FIG. 7, the U8 fraction also inhibited cell aging by adriamycin in vascular smooth muscle cells.

Example 6 Western Blot Analysis

U8 (epifriedelanol) not only inhibited SA-b-gal activity staining, but also investigated how it affects the expression of p53, a biochemical marker of cell aging.

1. Cell Protein Extraction

Each cell was aliquoted into 1 × 10 ⁵ in a 60 mm culture dish and cultured in a 37 ° C., 5% carbon dioxide incubator. The root extract, fractions and the compounds separated therefrom were pretreated for 1 hour by concentration and adriamycin 500 nM for 4 hours. After removing the culture solution, it was washed once with phosphate buffer.

Cell lysis solution (25 mM Tris-HCl [pH 7.6], 150 mM NaCl, 1% tryptone X-100, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM sodium vanadate, 5 mM NaF, protease inhibitor or 1 mM PMSF) 50 μl was added. The cell scraper was used to collect the solution and cells and transferred to the microacupuncture tube. The solution was shaken every 10 minutes while reacting for 30 minutes on ice. The supernatant was transferred to a new tube by centrifugation at 12,000 xg for 10 minutes.

The amount of protein in the solution was quantified by BCA (bicinchoninic acid) method (Pierce Biotechnology Inc., Rockford IL, USA) using bovine serum albumin as a standard protein.

2. Western Blot Analysis

Protein (30 μg) was isolated by electrophoresis on 10% SDS-polyacrylamide gel. After transferring the protein to the nitrocellulose membrane, it was reacted for 1 hour in Tween-20-Tris buffered saline (TTBS) containing 5% whole milk powder. The nitrocellulose membrane was reacted overnight in a 5% whole milk powder-TTBS solution containing primary antibodies against p53 or p21.

After washing three times with TTBS solution, and reacted with a horseradish peroxidase-conjugated secondary antibody for 3 hours. After washing the membrane five times for 5 minutes with TTBS, the amount of p53 or p21 was measured using an enhanced chemiluminescence solution.

The amount of specific protein reacted with each antibody was measured using a LAS-3000 imaging device (Fujifilm Corp., Stanford, CT, USA). The same amount of protein was used in each experiment using a glyceraldehyde-3-phosphate dehydrogease (GAPDH) antibody.

3. Results

As shown in FIG. 8, epipriedellanolan concentration decreased p53 expression increased by adriamycin treatment. In addition, as a result of examining the cytotoxicity of the U8 fraction, no cytotoxicity was observed up to a concentration of 50 μg / ml (see FIG. 9).

From the above results, it was confirmed that epipriedellanol is a substance that directly and effectively inhibits apoptosis induced by adriamycin in fibroblasts, umbilical vascular endothelial cells, and vascular smooth muscle cells.

Example 7 Oral Acute Toxicity in Rats

Acute toxicity test was performed using 6-week-old specific pathogen-free (SPF) SD rats. Three animals per group were suspended epipriederanol in 0.5% methylcellulose solution and administered once orally at doses of 100 mg / kg, 500 mg / kg and 1 g / kg.

After the administration of epipriedellanol, the mortality, clinical symptoms, and weight changes of the animals were observed. Hematological and hematological examinations were performed. The necropsy was performed to visually observe the abdominal and thoracic organ abnormalities.

As a result, all the animals treated with epipriedellanol had no clinical symptoms or dead animals, and no toxic changes were observed in weight changes, blood tests, blood biochemistry tests, and autopsy findings.

As a result, the epipriedelanol of the present invention did not show a toxicity change in the rat up to 1 g / kg, it was determined to be a safe substance.

Hereinafter, an example of the preparation of a pharmaceutical composition containing epipriederanol of the present invention will be described, but the present invention is not intended to be limited thereto but merely to be described in detail.

Preparation Example 1 Preparation of Powder

A powder was prepared by mixing 10 mg epipriedellanol, 100 mg lactose and 10 mg talc and filling into an airtight bag.

≪ Formulation Example 2 > Preparation of tablet

Tablets were prepared by mixing epipriedellanol 10 mg, corn starch 100 mg, lactose 100 mg, and magnesium stearate 2 mg and then tableting according to a conventional method for preparing tablets.

Preparation Example 3 Preparation of Capsule

According to a conventional capsule preparation method, epipreedelanol 10 mg, corn starch 100 mg, lactose 100 mg and magnesium stearate 2 mg were mixed and filled into gelatin capsules to prepare a capsule.

Preparation Example 4 Preparation of Injection

According to the conventional method for preparing an injection, it was prepared by containing 10 mg of epipriedellanol per ampoule, a sterile distilled water injection amount and a pH adjusting agent amount per injection.

Preparation Example 5 Preparation of Liquid

After dissolving 10 mg of epipriedellanol, 10 g of isomerized sugar and 5 g of mannitol in an appropriate amount of purified water according to a conventional method of preparing a liquid, adding a proper amount of lemon flavor, mixing the mixture, and adjusting it to 100 ml by adding purified water. The solution was prepared by sterilization by filling in a brown bottle.

Preparation Example 6 Preparation of Health Food

Epipriedellanol 50 mg, vitamin mixture (70 mg of vitamin A acetate, 1.0 mg of vitamin E, 0.13 mg of vitamin B 1, 0.15 mg of vitamin B 2, 0.5 mg of vitamin B 6, 0.2 μg of vitamin B 12, vitamin C 10 mg) 10 μg biotin, 1.7 mg nicotinamide, 50 μg folic acid, 0.5 mg calcium pantothenate and an appropriate amount of mineral mixture (1.75 mg ferrous sulfate, 0.82 mg zinc oxide, 25.3 mg magnesium carbonate, 15 mg potassium monophosphate, second 55 mg of calcium phosphate, 90 mg of potassium citrate, 100 mg of calcium carbonate, 24.8 mg of magnesium chloride) were mixed, and then granules were prepared and health food was prepared according to a conventional method.

Figure 1 shows a schematic diagram of separating the active ingredient from the root extract methanol extract,

Figure 2 shows the chemical structures of the active ingredients isolated from the extract of the roots of methanol,

Figure 3 shows the effect of the compounds according to the invention on cell aging by adriamycin using MTT assay,

Figure 4 shows the effect of the compounds according to the invention on cell aging by adriamycin using SA-β-gal active staining,

Figure 5 shows the effect of the compounds according to the invention on cell aging by adriamycin in human fibroblasts,

Figure 6 shows the effect of the compounds according to the invention on cell aging by adriamycin in human umbilical vascular endothelial cells,

Figure 7 shows the effect of the compounds according to the invention on cell aging by adriamycin in human vascular smooth muscle cells,

Figure 8 shows the effect of the compounds according to the invention on cell aging by adriamycin using p53 protein expression change analysis,

Figure 9 shows the cytotoxic effect of epipriedellanol (U8) using MTT assay.

Claims (9)

An anti-aging pharmaceutical composition containing one or two or more of a terpene-based compound or catechin glycoside-based compound isolated from Salicis radicis cortex as an active ingredient. The pharmaceutical composition for inhibiting aging according to claim 1, wherein the terpene-based compound is separated from a hexane layer fractionated by adding distilled water and hexane to a Salicis radicis cortex methanol extract. The pharmaceutical composition for inhibiting aging according to claim 2, wherein the terpene-based compound is selected from friedelin or epipriedelanol. The catechin glycoside compound is separated from the butanol layer fractionated by sequentially adding ethyl acetate and butanol to the distilled water layer fractionated by adding distilled water and hexane to a methanol extract of Salicis radicis cortex. Anti-aging pharmaceutical composition, characterized in that. The method according to claim 4, wherein the catechin glycoside-based compound is catechin-7-O-β-apiofuranoside (catechin-7-Ob-apiofuranoside) or catechin-7-O-β-D-glupyraniside ( catechin-7-ObD-glucopyraniside). The pharmaceutical composition for inhibiting aging according to claim 3 or 5, wherein the compound inhibits aging of any one or two or more cells selected from the group consisting of fibroblasts, vascular endothelial cells and vascular squamous muscle cells. The method according to claim 6, wherein the pharmaceutical composition inhibits aging, characterized in that to treat any one disease selected from the group consisting of skin aging, rheumatoid arthritis, osteoarthritis, hepatitis, chronic skin damage tissue, arteriosclerosis, prostate hyperplasia and liver cancer Pharmaceutical composition. Anti-aging pharmaceutical composition containing epipriedelanol as an active ingredient. Anti-aging health food containing epipriedelanol as an active ingredient.

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