KR102361719B1 - A compound having an ability to inhibit alpha-glucosidase and a composition for prevention, improvement and treatment of carbohydrate-mediated diseases - Google Patents

Abstract

이때, R은 -OR¹이고, R¹은 수소, 치환 또는 비치환된 C_1-10의 알킬기, 치환 또는 비치환된 C_6-20의 아릴기, 치환 또는 비치환된 C_2-10의 아케닐기 또는 치환 또는 비치환된 C_2-20의 알키닐기이다. The present invention relates to a compound represented by the following formula (15) or a pharmaceutically acceptable salt thereof having an alpha-glucosidase inhibitory activity and/or an ability to promote an increase in nerve growth factor, and a pharmaceutical composition, a food composition or a feed composition comprising the same. it is about
<Formula 15>

In this case, R is -OR ¹ , R ¹ is hydrogen, a substituted or unsubstituted C _1-10 alkyl group, a substituted or unsubstituted C _6-20 aryl group, or a substituted or unsubstituted C _2-10 ace It is a nyl group or a substituted or unsubstituted C _2-20 alkynyl group.

Description

A compound having an alpha-glucosidase inhibitory effect, and a composition for preventing, improving and treating a carbohydrate-mediated disease comprising the same DISEASES}

The present invention relates to a compound having an alpha-glucosidase inhibitory effect and a composition for preventing, ameliorating, and treating a carbohydrate-mediated-disease comprising the same.

Helicopterus is a white, wood-rotting fungus that is an edible mushroom belonging to the Hericiaceae genus Hericium. It is distributed in Korea, Japan, and China, mainly in East Asia, and occurs on old or raw trees of broad-leaved trees in autumn. The white hairs around the mushroom look like a roe deer's butt, so it is called a roe deer mushroom, and its English name is Lion's mane mushroom or Monkey's head mushroom. The hemisphere mushroom is a white hemispherical with a diameter of 5 to 20 cm, with short hairs on the upper side, and countless needles with a length of 1 to 5 cm on the sides and bottom. The vertical cut consists of the upper part made of porous flesh and the lower part of the needle group. It is white at first, but turns yellow or light yellow as it grows, and the tissue is white (FIG. 1).

On the other hand, cerebrovascular disorders and cranial nerve diseases are known to be closely related to the functional decline of cranial nerve cells and the death of cranial nerve cells (Korean Patent No. 10-0935615). Also, alpha-glucosidase is an intestinal enzyme that catalyzes the final step of carbohydrate digestion by converting carbohydrates into monosaccharides (Pharmacology of alpha-glucosidase inhibition, Bischoff, H. Eur. J. Clin. Invest. 1994). , 24, 3-10., Re-exploring promising α-glucosidase inhibitors for potential development into oral anti-diabetic drugs: Finding needle in the haystack., Ghani, U. Eur. J. Med. Chem. 2015, 103, 133 -162). Therefore, inhibition of alpha-glucosidase delays digestion and absorption of carbohydrates, thereby controlling postprandial hyperglycemia, resulting in hypoglycemic effects.

The present inventors confirmed that certain compounds have excellent effects on promoting nerve cell growth and increasing nerve growth factors, and that certain compounds have alpha-glucosidase inhibitory activity, The present invention was completed.

It is an object of the present invention to provide a compound and a pharmaceutically acceptable salt thereof having a nerve growth factor increase promoting ability and nerve cell growth promoting ability.

It is also an object of the present invention to provide a composition for preventing, treating or improving neurological diseases comprising the compound and a pharmaceutically acceptable salt thereof.

Another object of the present invention is to provide a compound having an alpha-glucosidase inhibitory activity and a pharmaceutically acceptable salt thereof.

Another object of the present invention is to provide a composition for preventing, treating or ameliorating a carbohydrate-mediated disease comprising the compound and a pharmaceutically acceptable salt thereof.

In order to achieve the above object, the present invention

Provided is a compound represented by the following formula (15) or a pharmaceutically acceptable salt thereof having the ability to promote increase in nerve growth factor:

Also, the present invention

Provided is a compound represented by the following formula (15) or a pharmaceutically acceptable salt thereof having alpha-glucosidase inhibitory activity:

In this case, R is -OR ¹ , R ¹ is hydrogen, a substituted or unsubstituted C _1-10 alkyl group, a substituted or unsubstituted C _6-20 aryl group, or a substituted or unsubstituted C _2-10 ace It is a nyl group or a substituted or unsubstituted C _2-20 alkynyl group.

The present invention also provides a pharmaceutical composition for preventing and treating neurological diseases, comprising the compound represented by Formula 15 or a pharmaceutically acceptable salt thereof.

In addition, the present invention provides a food composition for preventing and improving neurological diseases comprising the compound represented by Formula 15 or a pharmaceutically acceptable salt thereof.

In addition, the present invention provides a feed composition for preventing and improving neurological diseases comprising the compound represented by Formula 15 or a pharmaceutically acceptable salt thereof.

The present invention also provides a method for preventing and treating neurological diseases comprising administering the pharmaceutical composition to a subject.

The present invention also provides a method for preventing and improving neurological diseases comprising administering the food composition to a subject.

The present invention also provides a method for preventing and improving neurological diseases comprising administering the feed composition to an animal.

The present invention also provides a pharmaceutical composition for preventing and treating carbohydrate-mediated diseases, comprising the compound represented by Formula 15 or a pharmaceutically acceptable salt thereof.

In addition, the present invention provides a food composition for preventing and improving carbohydrate-mediated diseases, comprising the compound represented by Formula 15 or a pharmaceutically acceptable salt thereof.

The present invention also provides a feed composition for preventing and improving carbohydrate-mediated diseases, comprising the compound represented by Formula 15 or a pharmaceutically acceptable salt thereof.

The present invention also provides a method for preventing and treating carbohydrate-mediated diseases, comprising administering the pharmaceutical composition to a subject.

The present invention also provides a method for preventing and improving carbohydrate-mediated diseases comprising administering the food composition to a subject.

The present invention also provides a method for preventing and improving carbohydrate-mediated diseases, comprising administering the feed composition to an animal.

The compound represented by Formula 15 or a pharmaceutically acceptable salt thereof of the present invention has alpha-glucosidase inhibitory activity, nerve growth factor increase promoting ability, and nerve cell growth promoting ability.

The pharmaceutical composition of the present invention has the effect of preventing and treating carbohydrate-mediated-diseases and/or neurological diseases.

The food composition and feed composition of the present invention have an effect of preventing and improving carbohydrate-mediated diseases and/or neurological diseases.

1 is a photograph of a roe deer fungus.
Figure 2 is a photograph of the fruiting body powder of Hericium rhododendron mushroom.
Figure 3 shows the extraction and fractionation of fruiting body powder derived from Hericium oleracea.
4 is a schematic diagram showing a method for separating compounds 4 to 6 from the n-hexane fraction of the fruiting body powder of Heucerus oleracea.
5 is a schematic diagram showing a method for separating Compounds 1 to 3 and Compounds 7 to 14 from the n-hexane fraction of the fruiting body powder of Hericus oleracea.
6 is a ¹ H-NMR spectrum of compound 8. (CD3OD, 500 MHz)
7 is a ¹³ C-NMR spectrum of compound 8. (CD3OD, 100 MHz)
8 is a HSQC spectrum of compound 8. (CD3OD, 100 MHz)
9 is an HMBC spectrum of compound 8. (CD3OD, 100 MHz)
Figure 10 shows the Key HMBC correlations of compound 8.
11 is an HRESI-TOF-MS spectrum of compound 8.
12 is a ¹ H-NMR spectrum of compound 10. (CD3OD, 500 MHz)
13 is a ¹³ C-NMR spectrum of compound 10. (CD3OD, 100 MHz)
14 is a HSQC spectrum of compound 10. (CD3OD, 100 MHz)
15 is an HMBC spectrum of compound 10. (CD3OD, 100 MHz)
16 shows the Key HMBC correlations of compound 10.
17 is an HRESI-TOF-MS spectrum of compound 10.
18 is a ¹ H-NMR spectrum of compound 11. (CD3OD, 500 MHz)
19 is a ¹³ C-NMR spectrum of compound 11. (CD3OD, 100 MHz)
20 is the HSQC spectrum of compound 11. (CD3OD, 100 MHz)
21 is an HMBC spectrum of compound 11. (CD3OD, 100 MHz)
22 shows the Key HMBC correlations of compound 11.
23 is an HRESI-TOF-MS spectrum of compound 11.
24 is a ¹ H-NMR spectrum of compound 12. (CD3OD, 500 MHz)
25 is a ¹³ C-NMR spectrum of compound 12. (CD3OD, 100 MHz)
26 is the HSQC spectrum of compound 12. (CD3OD, 100 MHz)
27 is an HMBC spectrum of compound 12. (CD3OD, 100 MHz)
28 shows the Key HMBC correlations of compound 12.
29 is an HRESI-TOF-MS spectrum of compound 12.
30 is a 1H-NMR spectrum of compound 13. (CD3OD, 500 MHz)
31 is a ¹³ C-NMR spectrum of compound 13. (CD3OD, 125 MHz)
Figure 32 is the HSQC spectrum of compound 13. (CD3OD, 125 MHz)
33 is an HMBC spectrum of compound 13. (CD3OD, 125 MHz)
34 shows the Key HMBC correlations of compound 13.
35 is an HRESI-TOF-MS spectrum of compound 13.
36 is a ¹ H-NMR spectrum of compound 14. (CD3OD, 500 MHz)
37 is a ¹³ C-NMR spectrum of compound 14. (CD3OD, 125 MHz)
38 is the HSQC spectrum of compound 14. (CD3OD, 125 MHz)
39 is an HMBC spectrum of compound 14. (CD3OD, 125 MHz)
40 shows the Key HMBC correlations of compound 14.
41 is an HRESI-TOF-MS spectrum of compound 14.
42 shows the NGF-producing ability of compound 7, compound 8, and collarocin A as a positive control.
43 shows the nerve growth factor protein production ability of compound 7, compound 8, and collarocin A as a positive control.

The present invention is

To a compound represented by the following formula (15), or a pharmaceutically acceptable salt thereof, having an alpha-glucosidase inhibitory activity and/or an ability to promote an increase in nerve growth factor.

<Formula 15>

In this case, R is -OR ¹ , R ¹ is hydrogen, a substituted or unsubstituted C _1-10 alkyl group, a substituted or unsubstituted C _6-20 aryl group, or a substituted or unsubstituted C _2-10 ace It is a nyl group or a substituted or unsubstituted C _2-20 alkynyl group.

The present invention also relates to a pharmaceutical composition for preventing and treating carbohydrate-mediated diseases and/or neurological diseases, comprising the compound represented by Formula 15 or a pharmaceutically acceptable salt thereof.

The present invention also relates to a food composition for preventing and improving carbohydrate-mediated diseases and/or neurological diseases, comprising the compound represented by Formula 15 or a pharmaceutically acceptable salt thereof.

The present invention also relates to a feed composition for preventing and improving carbohydrate-mediated diseases and/or neurological diseases, comprising the compound represented by Formula 15 or a pharmaceutically acceptable salt thereof.

The present invention also relates to a method for preventing and treating carbohydrate-mediated diseases and/or neurological diseases, comprising administering the pharmaceutical composition to a subject.

The present invention also relates to a method for preventing and improving carbohydrate-mediated diseases and/or neurological diseases, comprising administering the food composition to a subject.

The present invention also relates to a method for preventing and improving carbohydrate-mediated diseases and/or neurological diseases, comprising administering the feed composition to an animal.

Hereinafter, the present invention will be described in detail.

A compound represented by Formula 15 or a pharmaceutically acceptable salt thereof

The present invention relates to a compound represented by Formula 15 or a pharmaceutically acceptable salt thereof.

<Formula 15>

In this case, R is -OR ¹ , R ¹ is hydrogen, a substituted or unsubstituted C _1-10 alkyl group, a substituted or unsubstituted C _6-20 aryl group, or a substituted or unsubstituted C _2-10 ace It is a nyl group or a substituted or unsubstituted C _2-20 alkynyl group. Preferably, R ¹ is hydrogen, a substituted or unsubstituted C _1-10 alkyl group, a substituted or unsubstituted C _6-10 aryl group, a substituted or unsubstituted C _2-10 akenyl group, or a substituted or unsubstituted It is a cyclic C _2-10 alkynyl group. More preferably, R ¹ is hydrogen, chichuan, or an unsubstituted C _1-10 alkyl group. Even more preferably, R ¹ is hydrogen.

The compound has an alpha-glucosidase inhibitory activity, an ability to promote an increase in nerve growth factor, and/or an ability to promote nerve cell growth. In addition, the compound has the ability to delay the digestion of carbohydrates or the absorption of carbohydrates. The compound may be of natural origin or may be chemically synthesized. For example, the compound may be a mushroom-derived compound, and more preferably, a compound derived from Hericus oleifera.

neurological disease

The present invention relates to a pharmaceutical composition for preventing and treating neurological diseases, and a food and/or feed composition for preventing and improving neurological diseases. The neurological disease is a neurological disease associated with or accompanied by a decrease in nerve growth factor activity, a decrease in nerve growth factor, nerve cell death or a decrease in nerve cell function. For example, the neurological disease may be at least one selected from the group consisting of Alzheimer's disease, Parkinson's disease, epilepsy, neuropathy, peripheral neuropathy, stroke, and ischemic brain disease.

Carbohydrate-borne diseases

The present invention relates to a pharmaceutical composition for preventing and treating carbohydrate-mediated diseases, and to a food and/or feed composition for preventing and ameliorating carbohydrate-mediated diseases. The carbohydrate-mediated disease refers to a disease in which the degree of disease progression is affected by blood carbohydrates, that is, blood sugar. The carbohydrate-mediated disease may be diabetes, type 1 diabetes or digestive diabetes.

pharmaceutical composition

The present invention relates to a pharmaceutical composition for preventing and treating neurological diseases, comprising the compound represented by Formula 15 or a pharmaceutically acceptable salt thereof. The present invention also relates to a pharmaceutical composition for preventing and treating carbohydrate-mediated diseases, comprising the compound represented by Formula 15 or a pharmaceutically acceptable salt thereof. It also relates to a method for preventing and treating a neurological disease comprising administering the pharmaceutical composition of the present invention to a subject. It also relates to a method for preventing and treating carbohydrate-mediated diseases comprising administering the pharmaceutical composition of the present invention to a subject. The subject is a mammal, including a human, who has been diagnosed with a carbohydrate-mediated disease and/or a neurological disease or is likely to develop a carbohydrate-mediated disease and/or a neurological disease.

The pharmaceutical composition of the present invention may contain 0.01 to 80% by weight of the compound or a pharmaceutically acceptable salt thereof, preferably 0.02 to 65% by weight. However, it can be increased or decreased according to the needs of the user, and can be appropriately increased or decreased according to the circumstances such as the age, diet, nutritional status, and disease progression of the subject.

The pharmaceutical composition of the present invention can be administered orally or parenterally, and can be used in the form of general pharmaceutical formulations. Preferred pharmaceutical formulations include formulations for oral administration such as tablets, hard or soft capsules, solutions, suspensions, etc., and these pharmaceutical formulations are pharmaceutically acceptable conventional carriers, for example, excipients in the case of oral formulations; It can be prepared using a binder, a disintegrant, a lubricant, a solubilizer, a suspending agent, a preservative, or an extender.

The dosage of the pharmaceutical composition comprising the compound of the present invention or a pharmaceutically acceptable salt thereof may be determined by an expert according to various factors such as the patient's condition, age, sex, and complications, but in general, per kg of adult It may be administered in a dose of 0.1 mg to 10 g, preferably 10 mg to 5 g. In addition, the daily dose of the pharmaceutical composition or a dose of 1/2, 1/3, or 1/4 thereof is contained per unit dosage form, and may be administered 1 to 6 times a day. However, in the case of long-term intake for health and hygiene or health control, the amount may be less than the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety.

food composition

The present invention relates to a food composition for preventing and improving neurological diseases, comprising the compound represented by Formula 15 or a pharmaceutically acceptable salt thereof. The present invention also relates to a food composition for preventing and improving carbohydrate-mediated diseases, comprising the compound represented by Formula 15 or a pharmaceutically acceptable salt thereof. The present invention also relates to a method for preventing and improving neurological diseases comprising administering the food composition to a subject. The present invention also relates to a method for preventing and improving carbohydrate-mediated diseases comprising administering the food composition to a subject. The subject is a mammal, including a human, who has been diagnosed with a carbohydrate-mediated disease and/or a neurological disease or is likely to develop a carbohydrate-mediated disease and/or a neurological disease.

The food of the present invention may be a health supplement, health functional food, functional food, etc., but is not limited thereto, and contains the compound of the present invention or a pharmaceutically acceptable salt thereof, such as natural food, processed food, general food material, etc. also includes

The food composition of the present invention, the compound of the present invention or a pharmaceutically acceptable salt thereof may be added as it is, or may be used together with other food or food compositions, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of its use (prevention, improvement or therapeutic treatment). In general, the compound of the present invention or a pharmaceutically acceptable salt thereof is added in an amount of 0.1 to 5% by weight, preferably 2 to 3% by weight, based on 100% by weight of the raw material of the food or beverage when preparing food or beverage. can be The effective dose of the mixed composition of the present invention may be used according to the effective dose of the pharmaceutical composition, but in the case of long-term intake for health and hygiene purposes or health control, it may be less than the above range, Since the active ingredient has no problem in terms of safety, it can be used in an amount greater than the above range.

There is no particular limitation on the type of the food. The food composition may be used in the form of preparations for oral administration such as tablets, hard or soft capsules, solutions, suspensions, etc., and these preparations are acceptable conventional carriers, for example, excipients, It can be prepared using a binder, a disintegrant, a lubricant, a solubilizer, a suspending agent, a preservative, or an extender.

Examples of foods to which the mixed composition of the present invention can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages and vitamin complexes, and other nutritional supplements, but are not limited to these types of foods.

feed composition

The present invention relates to a feed composition for preventing and improving neurological diseases, comprising the compound represented by Formula 15 or a pharmaceutically acceptable salt thereof. The present invention relates to a feed composition for preventing and improving carbohydrate-mediated diseases, comprising the compound represented by Formula 15 or a pharmaceutically acceptable salt thereof. The present invention also relates to a method for preventing and improving neurological diseases comprising administering the feed composition to an animal. The present invention also relates to a method for preventing and improving carbohydrate-mediated diseases, comprising administering the feed composition to an animal. The subject is a mammal that has been diagnosed with a carbohydrate-mediated disease and/or a neurological disease or is likely to develop a carbohydrate-mediated disease and/or a neurological disease.

The feed composition of the present invention contains 0.01-5.00% by weight of the compound or a pharmaceutically acceptable salt thereof, preferably 0.03-3.00% by weight, and more preferably 0.05-0.10% by weight on a dry matter basis.

The feed composition of the present invention may be a feed composition for vertebrates such as cattle and pigs, poultry such as chickens and ducks, fish, crustaceans, etc., but is not limited thereto. The feed composition of the present invention can be used regardless of the mixing ratio in the feed composition. For example, there is no problem in using the present invention even in a feed with a high protein content.

abbreviation

Abbreviations used in the present invention have the following meanings.

C.C.: Column Chromatography

CD3OD: Methanol-d4

CH2Cl2 : Methylene Chloride

CH3CN: Acetonitrile

DMSO: Dimethylsulfoxide

ESI-MS: Electrospray Ionization Mass Spectroscopy

EtOAc: Ethylacetate

ext. :Extract

Fr. : Fraction

HMBC: Heteronuclear Multiple Bond Correlation

HPLC: High Performance Liquid Chromatography

HRESI-TOF-MS: High Resolution Electrospray Time-of-Flight Ionization Mass Spectroscopy

HSQC: Heteronuclear Single Quantum Correlation

IR: Infrared Spectroscopy

m/z: Mass-to-charge Ratio

MeOH: Methanol

MPLC: Medium Pressure Liquid Chromatography

NMR: Nuclear Magnetic Resonance Spectroscopy

Prep. : Preparative

RP: Reverse Phase

UV: Ultra Violet Absorption

λmax: Maximum Wavelength

Advantages and features of the present invention, and methods for achieving them, will become apparent with reference to the embodiments described below in detail. However, the present invention is not limited to the embodiments disclosed below, but will be embodied in various different forms, and only these embodiments allow the disclosure of the present invention to be complete, and common knowledge in the art to which the present invention pertains It is provided to fully inform those who have the scope of the invention, and the present invention is only defined by the scope of the claims.

<Material>

Hericium erinaceum (Hericium erinaceum) samples were grown in Yeondong-myeon, Sejong Special Self-Governing City, and were provided by CNG. In this experiment, the powder of the fruiting body of the heiferous mushroom was used (FIG. 2).

The instruments and reagents used in this experiment are as follows.

Physicochemical Identification and Separation and Purification

device

Polarimeter : JASCO DIP-1000 polarimeter (Tokyo, Japan)

IR : JASCO FT-IR 4100 (Tokyo, Japan)

UV : JASCO UV-550 (Tokyo, Japan)

ESI-MS: LCQ Fleet (Thermo Scientific, USA)

HRESI-TOF-MS : High-Resolution Electrospray-ionization Time-of-flight mass spectrometry (maXis 4G 20207)

1D-NMR ( ¹ H-, ¹³ C-NMR), 2D-NMR (HSQC, HMBC, COSY)

: BRUKER (AVANCE Ⅲ 400 MHz, AVANCE 500 MHz, Germany).

: JEOL (400 MHz, Japan).

Rotary vacuum evaporator : IKA RV10 (IKA, Germany)

Low Temp. Circulator: EYELA CA-1112 (Tokyo, Rikakikai Co., Ltd., Japan)

UV lamp detector (254 nm, 365 nm)

: UVGL-25 (UVP. INC. San Gabriel, CA 91778, USA)

Vacuum Dry Oven : VO-10X (JEIO TECH. Co., Ltd.)

Vacuum Pump : GLD-050 (Sinku Kiko Co., Ltd.)

Cold Trap Bath : CTD-10 (JEIO TECH. Co., Ltd, Korea)

Speed Vac: Crist RVC 2-25 CD Plus, Germany

Prep HPLC:

Waters HPLC system (USA)

515 HPLC Pumps

Waters 996 Photodiode-array detector

Column : Phenomenex Gemini-NX 5μ C18 110A (150×10 mm, USA)

: Phenomenex Gemini-NX 5μ C18 110A (150×21.2 mm, USA)

Software : Empower system

Column Chromatography

Silica gel (200-400 Mesh, Fisher Scientific)

Diaion HP-20P (Mitsubishi Kasie. Chemical Co., Japan)

Sephadex LH-20 (25-100 mm, Pharmacia Fine Chemical Industries Co.)

Lichroprep RP-18 (75 mm, YMC, USA)

Kieselgel 60 F254 plate (0.25 mm, Merck, USA)

Spray reagent : 10% Vanillin-H2SO4 10% H2SO4 (in EtOH)

Other reagents and solvents for this experiment were used as analytical grade or first-class reagents, and HPLC grade was used as the solvent for HPLC.

<Experimental Example 1>

<1-1> Extraction and solvent fractionation

The dried, powdered, Hee-beetle mushroom (3.8 kg) was repeatedly extracted twice with 100% ethyl acetate (72 L) at room temperature. The extract was filtered with a reduced pressure filter, and the filtered filtrate was concentrated with a rotary vacuum concentrator to obtain an extract (180 g). This was suspended in 90% methanol: methylene chloride (CH2Cl2) (1:1 v/v), followed by n-hexane fractionation to obtain an n-hexane fraction (100.2 g) and a 90% methanol: methylene chloride fraction (46.9 g). obtained (Fig. 3).

<1-2> Separation of components from the n-hexane fraction of the heiferous mushroom

The n-hexane fraction (100.2 g) layer was silica open C.C. (n-hexane: EtOAc = 100: 0 → 0: 100, EtOAc: CH3OH = 100: 0 → 0: 100 gradient) was performed to divide a total of 14 fractions (H1-H14). The H7 fraction (8.2 g) was purified by MPLC RP silica C.C. (CH3OH: H2O = 10: 90 → 100: 0, gradient) was performed and divided into a total of 10 small fractions (H7-1 - H7-10). Compounds 5 (4.7 mg) and 6 (4.4 mg) were isolated by performing preparative HPLC (CH3CN 100%) on the H7-10 fraction. The H9 fraction (4.6 g) was purified by MPLC RP silica C.C. (CH3OH: H2O = 10: 90 → 100: 0, gradient) was performed to divide a total of 15 small fractions (H9-1 - H9-15), and the H9-14 fraction was subjected to preparative HPLC (CH3CN 100%) was carried out to isolate compound 4 (1.6 mg). Compound 1 (13.0 mg) was obtained through recrystallization from the H10 fraction (5.3 g), and MPLC RP silica C.C. (CH3OH: H2O = 10: 90 → 100: 0, gradient) was performed and divided into 11 small fractions (H10-1 - H10-11). Compounds 11 (2.2 mg), 12 (32.0 mg), 13 (1.3 mg), and 14 (1.0 mg) were separated by performing preparative HPLC (CH3CN:H2O=40:60) for the H10-4 fraction. In addition, compound 9 (22.6 mg) was obtained by performing preparative HPLC (CH3CN: H2O = 65:35) on the H10-5 fraction, and compound 7 (2.2 mg) and compound 3 (2.5 mg) were separated, and compound 2 (23.9 mg) was obtained through recrystallization from H10-8 fraction. The H13 fraction (1.7 g) was purified by MPLC RP silica C.C. (CH3OH: H2O = 10: 90 → 100: 0, gradient) was performed and divided into 11 small fractions (H13-1 - H13-11). Compound 8 (1.1 mg) was isolated by performing preparative HPLC (MeOH:H2O=50:50) on the H13-3 fraction. In addition, the H13-4 fraction was subjected to preparative HPLC (MeOH: H 2 O = 50: 50) to obtain compound 10 (1.2 mg) ( FIGS. 4 and 5 ).

<1-3> Physical and chemical properties and spectroscopic data analysis

Ergosterol (Compound 1)

White amorphous powder; ESI-MS m/z 397 [M+Na] ⁺ ; ¹ H-NMR (CDCl ₃ , 500 MHz), see Table 1.

Ergosterol peroxide (Compound 2)

White amorphous powder; ESI-MS m/z 451 [M+Na] ⁺ ; ¹ H-NMR (CDCl ₃ , 400 MHz), see Table 1.

9,11-Dehydroergosterol peroxide (Compound 3)

Colorless needles; ESI-MS m/z 449 [M+Na] ⁺ ; ¹ H-NMR (CD ₃ OD, 500 MHz) and ¹³ C-NMR (CD3OD, 125 MHz), see Table 2.

(22E)-Ergosta-4,6,8,22-tetraen-3-one (Compound 4)

Yellow syrup; ESI-MS m/z 393 [M+H] ⁺ ; ¹ H-NMR (CDCl ₃ , 500 MHz) and ¹³ C-NMR (CDCl ₃ , 100 MHz), see Table 2.

Hericene D (Compound 5)

Light yellow amorphous powder; ESI-MS m/z 603 [M+Na] ⁺ ; ¹ H-NMR (CDCl ₃ , 500 MHz) and ¹³ C-NMR (CDCl ₃ , 100 MHz), see Table 3.

Hericene A (Compound 6)

Light yellow amorphous powder; ESI-MS m/z 557 [M+H] ⁺ ; ¹ H-NMR (CDCl ₃ , 500 MHz) and ¹³ C-NMR (CDCl ₃ , 100 MHz), see Table 3.

Hercerin (Compound 7)

yellow syrup; ESI-MS m/z 442 [M+Na] ⁺ ; ¹ H-NMR (CD3OD, 500 MHz) and ¹³ C-NMR (CD ₃ OD, 100 MHz), see Table 4

Hercerinol A (Compound 8)

+16° (c 0.01, MeOH); FT-IR ν_max 3340, 1644 cm^-1; HRESI-TOF-MS m/z 350.1361 [M+Na]⁺ (calcd. for C₁₉H₂₁NNaO₄ 350.1368); ¹H-NMR (CD₃OD, 500 MHz) and ¹³C-NMR (CD₃OD, 100 MHz), 표 4 참조.Light yellow syrup;

+16° (c 0.01, MeOH); FT-IR ν _max 3340, 1644 cm ^-1 ; HRESI-TOF-MS m/z 350.1361 [M+Na] ⁺ (calcd. for C ₁₉ H ₂₁ NNaO ₄ 350.1368); ¹ H-NMR (CD ₃ OD, 500 MHz) and ¹³ C-NMR (CD ₃ OD, 100 MHz), see Table 4.

Hericenone J (Compound 9)

Light yellow amorphous powder; ESI-MS m/z 339 [M+Na] ⁺ ; ¹ H-NMR (CDCl ₃ , 500 MHz), see Table 5.

Hericenol A (Compound 10)

+17.8° (c 0.01, MeOH); FT-IR ν_max 3367, 1641 cm^-1; HRESI-TOF-MS m/z 373.1621 [M+Na]⁺ (calcd. for C₁₉H₂₆NaO₆ 373.1627); ¹H-NMR (CD₃OD, 500 MHz) and ¹³C-NMR (CD₃OD, 100 MHz), 표 5 참조.Yellow syrup;

+17.8° (c 0.01, MeOH); FT-IR ν _max 3367, 1641 cm ^-1 ; HRESI-TOF-MS m/z 373.1621 [M+Na] ⁺ (calcd. for C ₁₉ H ₂₆ NaO ₆ 373.1627); ¹ H-NMR (CD ₃ OD, 500 MHz) and ¹³ C-NMR (CD ₃ OD, 100 MHz), see Table 5.

Hericenol B (Compound 11)

-2.70° (c 0.01, MeOH); FT-IR ν_max 3364, 1644 cm^-1; HRESI-TOF-MS m/z 355.1514 [M+Na]+ (calcd. for C₁₉H₂₄NaO₅ 355.1521); ¹H-NMR (CD₃OD, 500 MHz) and ¹³C-NMR (CD₃OD, 100 MHz), 표 6 참조.light brown syrup;

-2.70° (c 0.01, MeOH); FT-IR ν _max 3364, 1644 cm ^-1 ; HRESI-TOF-MS m/z 355.1514 [M+Na]+ (calcd. for C ₁₉ H ₂₄ NaO ₅ 355.1521); ¹ H-NMR (CD ₃ OD, 500 MHz) and ¹³ C-NMR (CD ₃ OD, 100 MHz), see Table 6.

Hericenol D (Compound 12)

-2.74° (c 0.01, MeOH); FT-IR ν_max 3340, 1670 cm^-1; HRESI-TOF-MS m/z 371.1465 [M+Na]⁺ (calcd. for C₁₉H₂₄NaO₆ 371.1471); ¹H-NMR (CD₃OD, 500 MHz) and ¹³C-NMR (CD₃OD, 100 MHz), 표 7 참조.Yellow syrup;

-2.74° (c 0.01, MeOH); FT-IR ν _max 3340, 1670 cm ^-1 ; HRESI-TOF-MS m/z 371.1465 [M+Na] ⁺ (calcd. for C ₁₉ H ₂₄ NaO ₆ 371.1471); ¹ H-NMR (CD ₃ OD, 500 MHz) and ¹³ C-NMR (CD ₃ OD, 100 MHz), see Table 7.

Hericenol C (Compound 13)

-1.68° (c 0.01, MeOH); FT-IR ν_max 3324, 1733 cm^-1; HRESI-TOF-MS m/z 355.1515 [M+Na]⁺ (calcd. for C₁₉H₂₄NaO₅ 355.1521); ¹H-NMR (CD₃OD, 500 MHz) and ¹³C-NMR (CD₃OD, 125 MHz), 표 7 참조.light brown syrup;

-1.68° (c 0.01, MeOH); FT-IR ν _max 3324, 1733 cm ^-1 ; HRESI-TOF-MS m/z 355.1515 [M+Na] ⁺ (calcd. for C ₁₉ H ₂₄ NaO ₅ 355.1521); ¹ H-NMR (CD ₃ OD, 500 MHz) and ¹³ C-NMR (CD ₃ OD, 125 MHz), see Table 7.

Hericenol E (Compound 14)

+66.2 (c 0.01, MeOH); FT-IR ν_max 3868, 1644 cm^-1; HRESI-TOF-MS m/z 387.1782 [M+Na]⁺ (calcd. for C₁₉H₂₄NaO₇ 387.1420); ¹H-NMR (CD₃OD, 500 MHz) and ¹³C-NMR (CD₃OD, 125 MHz), 표 8 참조.Yellow syrup;

+66.2 (c 0.01, MeOH); FT-IR ν _max 3868, 1644 cm ^-1 ; HRESI-TOF-MS m/z 387.1782 [M+Na] ⁺ (calcd. for C ₁₉ H ₂₄ NaO ₇ 387.1420); ¹ H-NMR (CD ₃ OD, 500 MHz) and ¹³ C-NMR (CD ₃ OD, 125 MHz), see Table 8.

<Result>

<Experimental Example 2> Identification of the structure of the separated and purified compound

The structures of the compounds isolated and purified in Experimental Example 1 were identified.

<2-1> Ergosterol (Compound 1) and Ergosterol peroxide (Compound 2)

Compound 1 was isolated and purified as white amorphous powder, and showed a molecular ion peak (m/z 397 [M+H]+) in the ESI-MS spectrum, from which the molecular formula could be expected to be C28H44O. 5 methine proton peaks in 1H-NMR spectrum δH 3.66 (1H, m, H-3), δH 5.59 (1H, dd, J = 2.5, 5.5 Hz, H-6), δH 5.40 (1H, dd, J = 2.5, 5.5 Hz, H-7), δH 5.17 (1H, m, H-22), δH 5.27 (1H, m, H-23) and six methyl proton peaks δH 0.66 (3H, s, H-18) , δH 0.95 (3H, s, H-19), δH 1.06 (3H, d, J = 7.0 Hz, H-21), δH 0.87 (3H, d, J = 7.0 Hz, H-26), δδH 0.84 ( 3H, d, J = 7.0 Hz, H-27) and δH 0.95 (3H, d, J = 7.0 Hz, H-28) were observed. The above 1H-NMR spectrum data and 1. Huong, L.; Kiem, P.; Nghi, D.; Cuong, N.; Ha, T.; Minh, C. Chemical constituents of the fungus Hericium erinaceus SH1. Journal of chemistry. 2008, 46 (1), 96-101, Compound 1 was structurally identified as ergosterol having the structure of Formula 1.

Compound 2 was separated and purified as white amorphous powder, and the molecular weight was confirmed through ESI-MS spectrum (m/z 451 [M+Na]+), and the molecular formula was expected to be C28H44O3. The structure of compound 2 was very similar to that of compound 1, and in 1H-NMR spectrum, δH 6.24 (1H, d, J = 10.5 Hz, H-6), δH 6.50 (1H, d, J = 10.5 Hz, H- 7) confirmed the chemical shift difference and predicted that the structure had a peroxide group. The above 1H-NMR spectrum data and 1. Huong, L.; Kiem, P.; Nghi, D.; Cuong, N.; Ha, T.; Minh, C. Chemical constituents of the fungus Hericium erinaceus SH1. Journal of chemistry. With reference to 2008, 46 (1), 96-101, compound 2 was structurally identified as ergosterol peroxide having the structure of Formula 2.

1H-NMR spectroscopic data of Compound 1 and Compound 2 are shown in Table 1.

a Recorded at 500 MHz in CD3OD.

b Recorded at 400 MHz in CDCl3.

<2-2> 9,11-Dehydroergosterol peroxide (Compound 3) and (22E)-Ergosta-4,6,8,22-tetraen-3-one (Compound 4)

Compound 3 was separated and purified with colorless needles, and showed a molecular ion peak (m/z 449 [M+Na]+) in the ESI-MS spectrum, from which the molecular formula could be expected to be C28H42O3. The structure of compound 3 was very similar to that of compound 2, with δH 5.48 (1H, d, J = 6.0 Hz, H-11) in 1H-NMR spectrum and chemical shift 3C-NMR spectrum δC 144.3 (C-9), The structure of dehydroergosterol peroxide was predicted by confirming the difference at δC 120.8 (C-11). The above 1H-NMR spectrum data and Chen et al., Journal of agricultural and food chemistry. With reference to 2009, 57, 5713-5719, compound 3 was structurally identified as 9,11-dehydroergosterol peroxide having the structure of Chemical Formula 3.

Compound 4 was separated and purified with yellow syrup, and the molecular weight was confirmed through ESI-MS spectrum (m/z 393 [M+H]+), and the molecular formula could be expected to be C28H40O. In the structure of Compound 4, it was observed that the methine proton peak at position 3 did not exist in the 1H-NMR spectrum, and δC 199.6 (C-3) carbonyl carbon was confirmed in the 13C-NMR spectrum. The above 1H-NMR and 13C-NMR spectrum data and Li, W. et al. Biochemical systematics and ecology. With reference to 2017, 70, 254-259, Compound 4 was structurally identified as (22E)-ergosta-4,6,8,22-tetraen-3-one having the structure of Formula 4.

1H-NMR and 13C-NMR spectral data of compounds 3 and 4 are shown in Table 2.

a Recorded at 500 MHz in CD3OD.

b Recorded at 500 MHz in CDCl3.

<2-3> Hericene D (Compound 5) and Hericene A (Compound 6)

Compound 5 was isolated and purified as light yellow amorphous powder, and showed a molecular ion peak (m/z 603 [M+Na]+) in the ESI-MS spectrum, from which the molecular formula could be expected to be C19H24O4. In 1H-NMR spectrum, δH 6.52 (1H, s, H-6) corresponding to the aromatic proton was identified, one methoxy group δH 3.91 (3H, s, OMe), one CHO group δH 10.10 (1H, s, H-8) was confirmed. One carbonyl group δC 173.1 (C-1'') was identified in 13C-NMR spectrum, and δC 127.9 (C-9''), δC 128.1 (C-10'') corresponding to the olefinic CH group in the aliphatic-chain. ), δC 130.0 (C-12''), and δC 130.2 (C-13'') carbon peaks could be observed. At this time, it was confirmed that the proton peak appeared as δH 5.39-5.32 (1H, m, H-9'', 10'', 12'', 13''), thereby demonstrating that it was a compound containing linoleic ester. The above 1H-NMR spectrum data and Ma, B. et. Al., Journal of antibiotics. With reference to 2010, 63, 713-715, compound 5 was structurally identified as hericene D having the structure of Formula 5.

Compound 6 was separated and purified as light yellow amorphous powder, and the molecular weight was confirmed through ESI-MS spectrum (m/z 557 [M+H]+), and the molecular formula was expected to be C35H56O5. The structure of compound 6 was very similar to compound 5, but it showed a difference in that it did not have a double bond in the long aliphatic-chain following carbonyl carbon δC 173.2 (C-1'') in the 13C-NMR spectrum. 14 methylene carbons and 1 methyl carbon were identified in the aliphatic-chain, and one methyl proton at δH 0.88 (3H, t, J = 7.0 Hz, H-16'') could be observed in the 1H-NMR spectrum. The above 1H-NMR spectrum data and Miyazawa, M. et al., 1. 1. Tetrahedron. With reference to 2012, 68, 2007-2010, compound 6 was structurally identified as hericene A having the structure of Formula 6.

1H-NMR and 13C-NMR spectral data of compounds 5 and 6 are shown in Table 3.

a Recorded at 500 MHz in CDCl3.

b Recorded at 500 MHz in CDCl3.

<2-4> Hericerin (Compound 7) and Hericenol A (Compound 8)

Compound 7 was isolated and purified with yellow syrup, and showed a molecular ion peak in the ESI-MS spectrum (m/z 442 [M+Na]+), from which the molecular formula was predicted to be C27H33NO3. One aromatic proton of 1H-NMR spectrum δH 6.60 (1H, s, H-4), one methoxy proton δH 3.84 (3H, s, OMe), one methylene proton δH 4.23 (2H, s, H-3) The isoindolinone skeleton structure was predicted through the peak of From 1H-NMR spectrum, 5 aromatic protons, 2 trisubstituted olefinic protons, 5 methylene groups, and 3 methyl groups present in the side-chain were confirmed. From 13C-NMR spectrum, side-chain 6 aromatic carbons δC 140.2 (C-3''), δC 129.9 (C-4''), δC 129.7 (C-5''), δC 129.9 (C-6' '), δC 129.7 (C-7''), δC 129.9 (C-8'') and two methylene groups δC 44.7 (C-1''), δC 35.8 (C-2'') It was found that the phenylethyl group was substituted at the position. The nitrogen at position 2 was predicted through the carbon value δC 52.4 (C-3) of position 3 and the proton signal δH 4.23 (2H, s, H-3), and ESI-MS spectrum confirmed that it was a compound with nitrogen. The above 1H-NMR and 13C-NMR spectrum data and Li, W et al., Biochemical systematics and ecology. With reference to 2017, 70, 254-259, compound 7 was structurally identified as hericerin having the structure of Formula 7.

Compound 8 was isolated and purified with light yellow syrup, and molecular weight was confirmed through m/z 350.1361 [M+Na]+ (calcd. for C19H21NNaO4 350.1368) in HRESI-TOF-MS spectrum (FIG. 11). The isoindolinone skeleton structure of compound 8 was similar to compound 7, and in 1H-NMR spectrum, aromatic proton peak δH 6.88 (1H, s, H-7), methoxy group δH 3.85 (3H, s, OMe), methylene group δH 4.18 (2H) , s, H-3) peak and the carbonyl group δC 171.1 (C-1) of the 13C-NMR spectrum could be predicted ( FIGS. 6 and 7 ). The structure of isoindolinone skeleton is H-7/C-1, H-7/C-3a, H-7/C-5, H-3/C-3a, H-3/C-7a, H-3 in HMBC spectrum. It was confirmed through /C-4 and H-3/C-1 correlation. In the side-chain part, two methylene protons δH 2.99 (2H, overlap, H-1'), δH 3.66 (2H, t, J = 7.0 Hz, H-2') and methylene carbon δC 28.0 (C-1') , δC 62.5 (C-2') was confirmed, and the presence of oxygenated carbon was confirmed through the carbon value of C-2' to confirm substitution of a hydroxy group at the 2' position (FIG. 9). At this time, in HMBC spectrum, H-2'/C-1', H-2'/C-5, H-1'/C-6, H-1'/C-4, H-1'/C-5 The hydroxy group substitution position was determined through the correlation of (FIG. 10). Similar to compound 7, nitrogen at position 2 was predicted through carbon value δC 49.3 (C-3) at position 3 and proton signal δH 4.18 (2H, s, H-3), and from 13C-NMR spectrum, side-chain 6 aromatic carbons and 5 aromatic protons were identified in 1H-NMR spectrum. It was found that the phenylethyl group was substituted for nitrogen 2 through the H-1''/C-3 and H-1''/C-1 correlations of the HMBC spectrum. In the HSQC spectrum, two carbon peaks overlapped with one proton in δH 3.82 (2H, overlap, H-1'') and δH 2.99 (2H, overlap, H-1') were confirmed (FIG. 8). The above instrumental analysis results and Wang, X et al., Journal of asian natural products research. Based on comparison with 2016, 1183653, and 1028-6020, Compound 8 was identified as the structure of Formula 8, which was named Hericerinol A (Compound 8) as a compound reported for the first time in nature.

1H-NMR and 13C-NMR spectral data of compounds 7 and 8 are shown in Table 4.

a Recorded at 500 MHz in CD3OD.

b Recorded at 500 MHz in CD3OD.

<2-5> Hericenone J (Compound 9) and Hericenol A (Compound 10)

Compound 9 was separated and purified as light yellow amorphous powder, and showed a molecular ion peak (m/z 339 [M+Na]+) in the ESI-MS spectrum, from which the molecular formula could be expected to be C19H24O4. In 1H-NMR spectrum, δH 6.49 (1H, s, H-4) corresponding to the aromatic proton was confirmed, and one methoxy group and δH 5.23 (2H, s, H-) through δH 3.90 (3H, s, OMe) In 3), one methylene group was identified. Two trisubstituted olefinic protons present in the side-chain were identified through δH 5.18 (1H, t, J = 7.5 Hz, H-2') and δH 5.05 (1H, t, J = 8.0 Hz, H-6') 3 methylene groups in δH 3.35 (2H, d, J = 7.5 Hz, H-1'), δH 1.96 (2H, m, H-4'), δH 2.05 (2H, m, H-5'), Three methyl groups were observed: δH 1.64 (3H, s, H-8'), δH 1.77 (3H, s, H-9'), and δH 1.57 (3H, s, H-10'). The above 1H-NMR spectrum data and Li, W. et al., food chemistry. With reference to 2015, 170, 336-342, compound 9 was structurally identified as Hericenone J having the structure of Chemical Formula 9.

Compound 10 was separated and purified with yellow syrup, and molecular weight was confirmed through m/z 373.1621 [M+Na]+ (calcd. for C19H26NaO6 373.1627) in HRESI-TOF-MS spectrum, and hydroxy group (3367 cm in IR spectrum) The absorption wavelength corresponding to -1) was confirmed (FIG. 17). In the 1H-NMR spectrum of compound 10, aromatic proton peak δH 6.70 (1H, s, H-4), methoxy group δH 3.90 (3H, s, OMe), methylene group δH 5.25 (2H, s, H-3) through It could be expected that it had a benzolactone structure similar to that of compound 9 (Fig. 12), and the benzolactone portion was H-4/C-6, H-4/C-7a, H-4/C-3, H in the HMBC spectrum. It was confirmed through -3/C-3a and H-3/C-1 correlation (FIG. 16). 3 methylene protons in the side-chain part δH 3.40 (2H, overlap, H-1'), δH 2.26/1.97 (2H, m, H-4'), δH 1.32 (2H, m, H-5'), Three methyl groups δH 1.13 (3H, s, H-8'), δH 1.78 (3H, s, H-9'), and δH 1.09 (3H, s, H-10') were identified. From 13C-NMR spectrum, carbonyl group δC 174.1 (C-1) could be observed, δC 148.9 (C-3a), δC 97.5 (C-4), δC 166.2 (C-5), δC 118.2 (C-6) ), δC 155.7 (C-7), δC 105.5 (C-7a) six aromatic carbons and two olefinic carbons δC 123.3 (C-2'), δC 136.3 (C-3') were identified. In addition, δC 79.0 (C-6'), δC 73.8 (C-7') carbon peaks and Hinkley S. et al., Journal of antibiotics. By comparing 1999, 52 (11), and 988-997, oxygenated carbon in the side-chain part could be confirmed (FIG. 13). In the HSQC spectrum, it was confirmed that two proton peaks of δC 37.9 (C-4') were observed in each of δH 2.26/1.97 (2H, m, H-4') (FIG. 14), and H-9' of the HMBC spectrum C-4', H-5'/C-3', H-5'/C-10', H-6'/C-7', H-8'/C-6', H-10'/ By confirming the C-8' correlation, positions 6' and 7' where OH groups are substituted were confirmed (FIG. 15). Based on the above instrumental analysis results, the structure of compound 10 having the structure of Chemical Formula 10 was identified, and it was named Hericenol A (10) as a compound reported for the first time in nature.

1H-NMR and 13C-NMR spectral data of compounds 9 and 10 are shown in Table 5.

a Recorded at 500 MHz in CD3OD.

b Recorded at 500 MHz in CD3OD.

<2-6> Hericenol B (Compound 11)

Compound 11 was isolated and purified with light brown syrup, and the molecular weight was confirmed through HRESI-TOF-MS spectrum at m/z 355.1514 [M+Na]+ (calcd. for C19H24NaO5 355.1521) (FIG. 23). Compound 11 was very similar to compound 10, and in 1H-NMR spectrum, aromatic proton peak δH 6.70 (1H, s, H-4), methoxy group δH 3.90 (3H, s, OMe), methylene group δH 5.25 (2H, s) , H-3) could be expected to have a benzolactone structure similar to that of compound 10. From 1H-NMR and 13C-NMR spectrum, one carbonyl group, six aromatic carbons, one methoxy group, four methylene groups, and two methyl groups were confirmed. In HMBC spectrum, the δH 1.96 (2H, m, H-4') proton peak corresponding to the methylene group is H-4'/C-5', H-4'/C-2', H-4'/C- 3', and H-5'/C-4', H-5'/C-6', H-5'/C-7' of δH 1.57 (2H, m, H-5') peak Positions 4' and 5' were confirmed through correlation. In addition, it was possible to predict the presence of a hydroxy group through the proton peak δH 5.55 (1H, m, H-6') of 6' and the carbon value δC 74.7 (C-6') ( FIGS. 18 and 19 ). In the HSQC spectrum, δC 110.1 (C-8') two proton peaks were confirmed on one carbon, and it could be confirmed that the peak was due to exomethylene through the carbon value (FIG. 20). From H-8'/C-6', H-8'/C-10', H-8'/C-7' correlation of HMBC spectrum, the substitution position of exomethylene group, H-10'/C-8' , the position of the δH 1.66 (3H, s, H-10') methyl group was confirmed through the correlation of H-10'/C-6' ( FIGS. 21 and 22 ). The above instrumental analysis results and Siedlecka, J. et al., Lipids. Based on comparison with 2016, 51, and 229-244, compound 11 having the structure of Formula 11 was structurally identified, which was named Hericenol B (Compound 11) as a compound reported for the first time in nature.

1H-NMR and 13C-NMR spectral data of compound 11 are shown in Table 6.

a Recorded at 500 MHz in CD3OD.

<2-7> Hericenol D (Compound 12) and Hericenol C (Compound 13)

Compound 12 was isolated and purified with yellow syrup, and it showed a molecular ion peak at m/z 371.1465 [M+Na]+ (calcd. for C19H24NaO6 371.1471) in HRESI-TOF-MS spectrum, from which the molecular formula could be predicted to be C19H24O6. There was (Fig. 29). 1H-NMR spectrum 1 aromatic proton δH 6.70 (1H, s, H-4), 1 methoxy proton δH 3.90 (3H, s, OMe), 1 methylene proton δH 5.25 (2H, s, H-3) The presence of the benzolactone structure confirmed in compounds 10 and 11 could be predicted through the characteristic proton peak of (FIG. 24), and the corresponding carbon was confirmed in the HSQC spectrum (FIG. 26). 1' proton peak δH 3.36 (2H, d, J = 7.5 Hz, H-1') is H-1'/C-5, H-1'/C-6, H-1'/C in HMBC spectrum -7, showed correlation with H-1'/C-2', confirming the position of δC 21.2 (C-1') (FIG. 27). Exists in the side-chain through δH 5.25 (1H, t, J = 7.5 Hz, H-2'), δH 5.56 (1H, m, H-5'), δH 5.55 (1H, m, H-6') three trisubstituted olefinic protons were identified, and H-2'/C-4', H-4'/C-5', H-4'/C-6', H-8'/C- 6', δC 42.3 (C-4'), δC 128.2 (C-5'), δC 135.4 (C-6'), δC 81.0 (C-7') through 6', H-8'/C-7' correlation , the position of δC 23.5 (C-8') was confirmed (FIG. 28). Carbon peak of δC 81.0 (C-7') in 13C-NMR spectrum, molecular formula C19H24O6, Siedlecka, J et al., Lipids. With reference to 2016, 51, 229-244, it was possible to confirm the substitution of the 7' hydroperoxyl group (FIG. 25). Also, 3 methyl groups were observed in 1H-NMR spectrum: δH 1.26 (3H, s, H-8'), δH 1.75 (3H, s, H-9'), δH 1.26 (3H, s, H-10') H-8'/C-10', H-10'/C-8', H-10'/C-7', H-9'/C-4', H-9' through HMBC spectrum The position of the methyl group was confirmed through /C-2' correlation. Based on the above instrumental analysis results, the structure of compound 12 having the structure of Formula 12 was identified, which was named Hericenol D (Formula 12) as a compound reported for the first time in nature.

Compound 13 was separated and purified with light brown syrup, and molecular weight was confirmed through m/z 355.1515 [M+Na]+ (calcd. for C19H24NaO5 355.1521) in HRESI-TOF-MS spectrum, and hydroxy group (3324) in IR spectrum. cm-1) was confirmed (FIG. 35). 1H NMR spectrum, 13C NMR spectrum, HSQC spectrum, and HMBC spectrum (Figs. 30, 31, 32, 33, respectively) of compound 13 are similar to compound 12, so the structure of compound 13 is inferred to be very similar to compound 12 In the 13C-NMR spectrum of compound 13, the difference in carbon values of δC 71.4 (C-7') and δC 81.0 (C-7') of compound 12 could be confirmed. Carbon values and Siedlecka, J et al., Lipids. Through comparison with 2016, 51, 229-244, the presence of oxygenated carbon in the side-chain part was confirmed, and substitution of the hydroxy group at the 7' position could be predicted. H-8'/C-6', H-4'/C-6', H-9'/C-4', H-2'/C-4', H-1'/C- of HMBC spectrum 7 The position of the double bond and the position of the hydroxyl group were confirmed through correlation (FIG. 34). Based on the above instrumental analysis results, the structure of compound 13 having the structure of Formula 13 was identified, which was named Hericenol C (Formula 13) as a compound reported for the first time in nature.

1H-NMR and 13C-NMR spectral data of compound 12 and compound 13 are shown in Table 7.

a Recorded at 500 MHz in CD3OD.

b Recorded at 500 MHz in CD3OD.

<2-8> Hericenol E (Compound 14)

Compound 14 was isolated and purified with yellow syrup, and showed a molecular ion peak at m/z 387.1782 [M+Na]+ (calcd. for C19H24NaO7 387.1420) in HRESI-TOF-MS spectrum, from which the molecular formula could be predicted to be C19H24O7. there was. 1H-NMR spectrum 1 aromatic proton δH 6.70 (1H, s, H-4), 1 methoxy proton δH 3.90 (3H, s, OMe), 1 methylene proton δH 5.25 (2H, s, H-3) The presence of the benzolactone structure identified in Compound 12 could be predicted through the characteristic proton peak of , and the carbonyl group δC 173.3 (C-1) of the 13C-NMR spectrum. Compound 14 was very similar to the structure of compound 12, but in 13C-NMR spectrum, the difference between δC 182.1 (C-5') and δC 75.6 (C-6') was confirmed. structure was confirmed. In the HMBC spectrum, δC 36.9 (C- 4'), δC 182.1 (C-5'), δC 75.6 (C-6'), δC 77.8 (C-7'), δC 20.5 (C-8'), δC 19.8 (C-10') was confirmed. The above instrumental analysis results and Li, W. et al., Biochemical systematics and ecology. 2017, 70, Compound 14 having the structure of Formula 14 was structurally identified based on comparison with 254-259, which was named Hericenol E (Compound 14) as a compound reported for the first time in nature.

1H-NMR and 13C-NMR spectral data of compound 14 are shown in Table 8.

a Recorded at 500 MHz in CD3OD.

<Experimental Example 3> Evaluation of the ability to promote nerve cell generation and increase in nerve growth factor

For compound 7 (Hericerin) and compound 8 (Hericenol A), the ability to promote nerve cell generation and increase in nerve growth factor was evaluated. In this case, Corallocin A was used as a positive control. NGF (Nerve Growth Factor) production analysis was performed as follows. In order to measure the concentration of soluble NGF C6 glioma cells, a 24-well plate was seeded at a density of 1 x 10 5 cells/well. Cells were treated with samples at specific concentrations for 24 h, then conditioned medium was collected and centrifuged. NGF produced from C6 glioma culture supernatant was measured using a competitive enzyme linked immunoassay kit (R & D systems, Minneapolis, MN, USA) according to the manufacturer's protocol. Control cells that were not treated with the sample were concomitantly maintained.

Meanwhile, neurite growth analysis was performed as follows. In the case of N2a, the wells of a 24-well plate containing Poly-D-lysin (100 μg / ml) solution were first coated, incubated overnight, aspirated, washed with sterile water, and incubated at room temperature for 1 hour. Then, the neurons were seeded (15 to 30 X 103 cells/well) in high glucose DMEM supplemented with 10% FBS, incubated at 37 °C in an atmosphere of 5% CO2, and the cells were allowed to adhere overnight. Then, neurite outgrowth was analyzed after applying different concentrations of samples in 2% serum-free medium. Phase contrast images were captured every 2 h for 24 h with all monitored wells in the INCUCYTE ZOOM live cell analysis system.

Western blot analysis was performed as follows. C6 cells were seeded in 6-well plates at a density of 1×10 5 cells/well and treated with samples of different concentrations for 12 hours. N2a cells were also inoculated under the same conditions, and 12 hours later, the conditioned medium was transferred from the previously treated C6 glioma cells to M2a and cultured for 12 hours. After treatment, cells were collected and lysed in lysis buffer on ice for at least 2 h. Protein content was assessed using Bradford analysis. The same amount of protein (30 μg) was separated using SDS-PAGE and transferred to a nitrocellulose membrane. Membranes were ckeksehldjTrh rm in 5% skim milk in Tris-buffered saline containing Tween-20 (TBST) for 1 h followed by incubation with primary antibodies against GAPDH, NGF, Pro-BDNF, Synaptophysin for 12 h followed by horseradish ( horseradish) hydrogen peroxide (peroxidase)-conjugated secondary antibodies were incubated for 1 hour. Blots were developed using ECL Western Blotting Detection Reagents. “Concentration analysis of the codes was performed using ImageMaster™ 2D Elite software (version 3.1, Amersham Pharmacia Biotech).

As a result, the compounds isolated from the roe deer mushroom induced the production of NGF in astrocytes, and in particular, hericerinol A induced the production of NGF with the highest level ( FIG. 42 ). In addition, these compounds significantly increased the number and length of neurites in N2a cells and induced highly branched neurites. It exhibited neurophysiological activity by increasing the release of neurotrophic factors (NGF and BDNF), important precursors of synaptophysin, presynaptically and at the synapse leading to synapse formation (Fig. 43). Therefore, it was confirmed that the compounds isolated from roe deer mushroom can act as NGF mimetics, which may be useful for prophylactic and therapeutic uses in neurodegenerative diseases.

<Experimental Example 4>

The alpha-glucosidase inhibitory activity was evaluated for Compound 5, Compound 6, and Compounds 9 to 14. Alpha-glucosidase is an intestinal enzyme that catalyzes the final step of carbohydrate digestion by converting carbohydrates into single monosaccharides. Therefore, inhibition of alpha-glucosidase delays digestion and absorption of carbohydrates, thereby controlling postprandial hyperglycemia, resulting in hypoglycemic effects. Several alpha-glucosidase inhibitors, such as acarbose and voglibose, are used for the treatment of carbohydrate-mediated diseases.

Alpha-glucosidase inhibition assay was performed as follows. To confirm the ability of triterpenes to inhibit alpha-glucosidase, an inhibition assay was performed using a solution of the enzyme in deionized water. Test samples were mixed in 10 μl alpha-glucosidase (1 U/mL) and 80 μl enzyme buffer and incubated at 37° C. for 15 minutes. Then, 10 μL of a solution of p-nitrophenyl α-D-glucopyranoside diluted to 10 mM in deionized water was added, and the enzymatic reaction was performed at 37° C. for 20 minutes. After incubation, the amount of p-nitrophenyl cleaved by the enzyme was determined by measuring the absorbance at 405 nm in a 96-well microplate reader. In this case, DMSO (dimethyl sulfoxide) was used as a negative control, and 100 μM of Acarbose was used as a positive control.

As a result, compounds 5 and 6, which have structurally long chains, exhibited more than 90% inhibitory activity at a concentration of 100 uM. Compounds 9 to 14 also exhibited alpha-glucosidase inhibitory activity, and in particular, compounds having peroxy (-O-OH) exhibited an inhibitory effect of 50% or more. Therefore, these compounds inhibited the absorption of carbohydrates through the inhibition of the degradation of sugar, thereby inhibiting the rise of postprandial blood glucose and it was judged to be effective in the treatment of diabetes (Table 9).

Sample Alpha-glucosidase inhibition (%) Standard Deviation compound 5 91.4 0.3 compound 6 96.9 1.6 compound 9 44.7 8.6 compound 10 17.0 6.3 compound 11 58.6 3.4 compound 12 82.4 7.4 compound 13 42.7 6.6 compound 14 53.9 4.5 negative control (DMSO) 0.0 positive control (100 μM) 78.6 3.4

Claims (10)

delete delete delete delete delete A pharmaceutical composition for the prevention and treatment of diabetes comprising a compound of Formula 5 or Formula 6 or a pharmaceutically acceptable salt thereof:

<Formula 5>

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delete A food composition for preventing and improving diabetes comprising a compound of Formula 5 or Formula 6 or a pharmaceutically acceptable salt thereof:
<Formula 5>

<Formula 6>

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delete A feed composition for preventing and improving diabetes comprising a compound of Formula 5 or Formula 6 or a pharmaceutically acceptable salt thereof:
<Formula 5>

<Formula 6>

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