Method for extracting hericium erinaceus extract and application thereof

Abstract

The application discloses a method for extracting Hericium erinaceus extract, and comprises the following steps: drying and crushing Hericium erinaceus to obtain Hericium erinaceus powder; putting the Hericium erinaceus powder into a carbon dioxide supercritical extraction kettle; and under the conditions of a pressure of 25-35 MP, a temperature of 30-40 DEG C, a time of 1-2 h and an ethanol content of 0-4 mL / g as a entraining agent, a Hericium erinaceus crude extract is obtained. The method is simple and environment-friendly, and the obtained monomer compound has the effect of preventing and treating type II diabetes.

Description

Technical Field

[0001] This invention belongs to the field of extraction technology of Hericium erinaceus extract. Background Technology

[0002] Hericium erinaceum, also known as monkey head mushroom, is a traditional and highly prized edible and medicinal fungus belonging to the Hericaceae family and the Hericium genus. It is named for its resemblance to a monkey's head. During the Ming and Qing dynasties, Hericium erinaceum was a tribute item and one of the four famous dishes of the Manchu Han Imperial Feast (along with bear's paw, bird's nest, and shark fin), earning it the title of "vegetarian meat." Traditional Chinese medicine believes that Hericium erinaceum benefits the five internal organs and aids digestion. Studies have shown that Hericium erinaceum produces a variety of bioactive secondary metabolites, including diterpenes, phenols, alkaloids, and fatty acids, with diverse chemical structures. Activity evaluation reveals that these compounds have broad effects such as liver protection, anti-cancer properties, promotion of nerve growth factor synthesis, and antioxidant activity.

[0003] Diabetes mellitus is a group of metabolic diseases characterized by hyperglycemia, which can lead to serious complications such as cardiovascular disease, kidney disease, and blindness. Currently, alpha-glucosidase inhibitors are an important class of drugs for the clinical treatment of diabetes. They slow down the breakdown of starch hydrolysates such as maltose and sucrose into glucose, further slowing the entry of glucose into the bloodstream, thereby controlling blood sugar levels. However, clinically used alpha-glucosidase inhibitors have significant side effects. They inhibit alpha-amylase, prolonging the breakdown time of starch in the small intestine, thus increasing the breakdown of starch by intestinal flora and easily causing adverse reactions such as bloating. There are reports that Hericium erinaceus (monkey head mushroom) has a certain hypoglycemic effect. Therefore, research on the alpha-glucosidase inhibitory activity of Hericium erinaceus extract and monomeric compounds provides new possibilities for the development of hypoglycemic drugs and health products.

[0004] Supercritical state is a state of matter that is neither gaseous nor liquid, existing only when its temperature and pressure exceed a critical point. Supercritical fluids possess gas-like diffusivity and liquid-like dissolving power, while also exhibiting low viscosity and low surface tension. This allows supercritical fluids to rapidly penetrate into micropores, making them faster and more efficient than liquids for extraction. In particular, their dissolving power varies with temperature, pressure, and polarity.

[0005] Carbon dioxide is the most commonly used supercritical fluid, its critical temperature is low, 31.05 DEG C can reach supercritical state, the critical pressure is not high, most solute has large solubility, and carbon dioxide is not flammable, non-toxic, safe, cheap and easy to get. At present, carbon dioxide supercritical fluid extraction is the most commonly used supercritical extraction technology. In the extraction process, the supercritical fluid is contacted with the material to be extracted, so that the components with different polarity, boiling point and molecular weight are selectively extracted in turn, and then the supercritical carbon dioxide is changed into gas by means of reducing pressure and temperature, and the extracted material is automatically and completely separated, so as to achieve the purpose of separation and purification.

[0006] At present, the existing extraction technology of active substances of Hericium erinaceus mainly includes ultrasonic and organic solvent reflux methods, because some toxic organic solvents and high temperature, ultrasonic operation are involved, which can easily cause product oxidation and denaturation. When using non-toxic solvents such as ethanol and water for extraction, small polarity substances cannot be well extracted. Carbon dioxide supercritical extraction technology can well avoid the above shortcomings, and can extract active substances under mild conditions without toxicity, harm and residue, thereby providing a good processing technology for high-value utilization of Hericium erinaceus. SUMMARY

[0007] The purpose of the present application is to provide a method for extracting Hericium erinaceus extract, comprising: drying and crushing Hericium erinaceus to obtain Hericium erinaceus powder; putting the Hericium erinaceus powder into a carbon dioxide supercritical extraction kettle; under the conditions of pressure 25-35 MPa, temperature 30-40 DEG C, time 1-2 h and entraining agent ethanol content 0-4 mL / g, Hericium erinaceus crude extract is obtained.

[0008] Further, the temperature is preferably 30 DEG C.

[0009] Further, the pressure is preferably 25 MPa.

[0010] Further, the time is preferably 1.5 h.

[0011] Further, the entraining agent ethanol content is preferably 4 mL / g.

[0012] The second purpose of the present application is a compound used in the preparation of a drug for treating or preventing diabetes, and the structure formula is as follows:

[0013]

[0014]

[0015] The present application has the advantages that the method is simple and environment-friendly, and the obtained monomer compound has the effect of preventing and treating type II diabetes. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 The liquid chromatogram of the extract obtained in Example 3.

[0017] Figure 2 The compound of Formula 1 1 H NMR spectrum (500 Hz).

[0018] Figure 3 The compound of Formula 1 13 C NMR spectrum (125 Hz).

[0019] Figure 4 The compound of Formula 2 1 H NMR spectrum (500 Hz).

[0020] Figure 5 The compound of Formula 2 13 C NMR spectrum (125 Hz).

[0021] Figure 6 The compound of Formula 3 1 H NMR spectrum (500 Hz).

[0022] Figure 7 The compound of Formula 3 13 C NMR spectrum (125 Hz).

[0023] Figure 8 The compound of Formula 4 1 H NMR spectrum (500 Hz).

[0024] Figure 9 The compound of Formula 4 13 C NMR spectrum (125 Hz).

[0025] Figure 10 The compound of Formula 5 1 H NMR spectrum (500 Hz).

[0026] Figure 11 The compound of Formula 5 13 C NMR spectrum (125 Hz).

[0027] Figure 12 The compound of Formula 6 1 H NMR spectrum (500 Hz).

[0028] Figure 13 The compound of Formula 6 13 C NMR spectrum (125 Hz).

[0029] Figure 14 The compound of Formula 7 1H NMR spectrum (500 Hz).

[0030] Figure 15 . Formula 7 13 C NMR spectrum (125 Hz).

[0031] Figure 16 . Formula 8 1 H NMR spectrum (500 Hz).

[0032] Figure 17 . Formula 8 13 C NMR spectrum (125 Hz).

[0033] Figure 18 . Formula 9 1 H NMR spectrum (500 Hz).

[0034] Figure 19 . Formula 9 13 C NMR spectrum (125 Hz).

[0035] Figure 20 . Formula 10 1 H NMR spectrum (500 Hz).

[0036] Figure 21 . Formula 10 13 C NMR spectrum (125 Hz).

[0037] Figure 22 . Formula 11 1 H NMR spectrum (500 Hz).

[0038] Figure 23 . Formula 11 13 C NMR spectrum (125 Hz).

[0039] Figure 24 . Formula 12 1 H NMR spectrum (500 Hz).

[0040] Figure 25 . Formula 12 13 C NMR spectrum (125 Hz).

[0041] Figure 26 . Formula 13 1 H NMR spectrum (500 Hz).

[0042] Figure 27 . Formula 13 13 C NMR spectrum (125 Hz).

[0043] Figure 28 . Formula 14 shown in the compound of 1 H NMR spectrum (500 Hz).

[0044] Figure 29 . Formula 14 shown in the compound of 13 C NMR spectrum (125 Hz).

[0045] Figure 30 . New compound 1-4 mass spectrum. DETAILED DESCRIPTION

[0046] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0047] Example 1

[0048] The Hericium erinaceus was dried thoroughly, crushed and sieved through an 80 mesh screen to obtain Hericium erinaceus powder;

[0049] 100 g of Hericium erinaceus powder was placed in a carbon dioxide supercritical extraction kettle, and 99.9% pure carbon dioxide was connected. The carbon dioxide flow rate was set to 15 kg / h, the extraction pressure was 25 MPa, the extraction temperature was 30°C, the ethanol entrainer was 0, and the extraction time was 1.0 h. In the separation kettle connected to the carbon dioxide supercritical extraction kettle, the final extract was obtained. The extract was weighed to be 1.663 g. HPLC analysis was performed.

[0050] Analysis method: HPLC instrument is Shimadzu LC-20AT liquid chromatograph with SPD-M20A detector, ethyl acetate as solvent, sample preparation as 1 mg / mL solution, sample loading amount 10 μL, chromatographic column Kromasil 4.5x250mm C8(5 μm), mobile phase A MeOH, B H2O; elution program 0-10 min, 85% MeOH, 10-50 min 85%-100% MeOH, 50-80 min 100% MeOH. Column temperature 40°C, detection wavelength 294 nm, HPLC analysis results see Figure 1 .

[0051] Example 2

[0052] The Hericium erinaceus was dried thoroughly, crushed and sieved through an 80 mesh screen to obtain Hericium erinaceus powder;

[0053] Put 100 g Hericium erinaceus powder into the supercritical carbon dioxide extraction kettle, and connect the carbon dioxide with purity of 99.9%. Set the carbon dioxide flow rate to 15 kg / h, the extraction pressure to 25 MPa, the extraction temperature to 40°C, and introduce 2 mL / g of ethanol entrainer. Extract for 1.5 h. In the separation kettle, the final extract is obtained. After the excess ethanol is evaporated, the extract is weighed to be 3.357 g. HPLC analysis is performed.

[0054] The analysis method is HPLC instrument is Shimadzu LC-20AT liquid chromatograph with SPD-M20A detector, ethyl acetate as solvent, sample preparation to 1 mg / mL solution, sample size 10 μL, chromatographic column Kromasil 4.5 x 250 mm C8 (5 μm), mobile phase A is MeOH, B is H2O; elution program is 0-10 min, 85% MeOH, 10-50 min 85%-100% MeOH, 50-80 min 100% MeOH. Column temperature 40°C, detection wavelength 294 nm, HPLC analysis results are shown in Table 1. Figure 1 .

[0055] Example 3

[0056] According to the design of Table 1, and the operation of Examples 1-2, put 100 g Hericium erinaceus powder into the supercritical carbon dioxide extraction kettle, and connect the carbon dioxide with purity of 99.9%. Set the carbon dioxide flow rate to 15 kg / h, set the extraction pressure to 25-35 MPa, set the extraction temperature to 30-50°C, and introduce 0-4 mL / g of ethanol entrainer. Extract for 1-2 h. In the separation kettle, the final extract is obtained. After the excess ethanol is evaporated, the extract is weighed to be 3.357 g. HPLC analysis is performed.

[0057] The analysis method is HPLC instrument is Shimadzu LC-20AT liquid chromatograph with SPD-M20A detector, ethyl acetate as solvent, sample preparation to 1 mg / mL solution, sample size 10 μL, chromatographic column Kromasil 4.5 x 250 mm C8 (5 μm), mobile phase A is MeOH, B is H2O; elution program is 0-10 min, 85% MeOH, 10-50 min 85%-100% MeOH, 50-80 min 100% MeOH. Column temperature 40°C, detection wavelength 294 nm, HPLC analysis results are shown in Table 1. Figure 1 .

[0058] Table 1 Extract content obtained by supercritical carbon dioxide extraction

[0059]

[0060] Note: The selection of extraction conditions is based on the orthogonal test design.

[0061] Example 4

[0062] According to the determination of the optimal extraction conditions of carbon dioxide supercritical extraction, the extraction temperature is 30℃, the pressure is 25 MPa, the time is 1.5h, and the content of the entraining agent ethanol is 4mL / g, the maximum yield can be obtained. According to the above conditions, a large amount of Hericium erinaceus carbon dioxide supercritical extract was prepared.

[0063] After the Hericium erinaceus was dried, it was crushed and sieved through an 80-mesh screen to obtain Hericium erinaceus powder;

[0064] 1000g of Hericium erinaceus powder was added to the carbon dioxide supercritical extraction kettle, and carbon dioxide with a purity of 99.9% was connected. The carbon dioxide flow rate was set to 15kg / h, the extraction pressure was 30MPa, the extraction temperature was 30℃, the content of the entraining agent ethanol was 4mL / g, and the extraction time was 1.5h. Finally, 33.9g of extract was obtained.

[0065] Example 5

[0066] Separation of monomer compounds in Hericium erinaceus carbon dioxide supercritical extract

[0067] 20g of Hericium erinaceus extract was mixed and dried with 15g of 200-300 mesh silica gel, and separated by silica gel normal phase column chromatography (chromatography column length 500mm, inner diameter 30mm). Petroleum ether (P): ethyl acetate (E) system (volume ratio 1:0, 50:1, 30:1, 20:1, 15:1, 10:1, 4:1, 2:1) was used for gradient elution, and each gradient elution was 2L.

[0068] According to the TLC results, 10 fractions (F-1~F-10) were obtained.

[0069] Fraction F-5 (0.85g, P-E 100:2) was further separated by reverse phase column chromatography, and 5 sub-fractions (F-5-1~F-5-5) were obtained by elution with 85% methanol / water.

[0070] In fraction F-5-3 (204mg), compound 1-4 (5.1, 3.2, 3.5 and 5.2mg, t R =62.1, 59.5, 70.1 and 80.2min).

[0071] Fraction F-6 (1.05g, P-E 100:5) was separated by Sephadex LH-20 column chromatography, and 7 sub-fractions (F-6-1~F-6-7) were obtained by elution with MeOH-CH2Cl2(50%, v / v).

[0072] Stream F-6-3 was further separated by reverse phase column to give 6 sub-streams (F-6-3-1 ~ F-6-3-6). Stream F-6-3-3, which was eluted by 80% MeOH, was further separated by HPLC prep to give compound 5-8 (25.4, 43.1, 33.9 and 51.2 mg, t R 52.1, 64.3, 70.5 and 49.3 min).

[0073] Stream F-7 (2.3 g, P-E 100:10) was similarly separated by gel Sephadex LH-20 column to give eight sub-streams (F-7-1 ~ F-7-8). Stream F-7-3 was further separated by HPLC prep to give compounds 9, 11, 12, 14 (15.5, 31.4, 22.3, 22.2 mg, t R 63.3, 48.8, 73.4, 59.3 min). By reverse phase HPLC, stream F-7-4 was separated to give compounds 10, 13 (31.8, 42.9 mg, t R 64.4, 55.9 min).

[0074] The compound was confirmed as the structure of Formula 1-14 by NMR and mass spectrometry.

[0075] By analyzing mass spectrometry and NMR data, the compound shown in Formula 1 was confirmed as (E)-4-(3,7-dimethyl-5-(palmitoyl)octa-2,6-dien-1-yl)-2-formyl-3-hydroxy-5-methoxyphenyl palmitate, (hericene E), and its spectral data are as follows: negative ion mode HRTOFMS mass spectrum m / z 809.6274 [M-H] - (calculated value C 51 H 85 O7, 809.6295), and its molecular formula was C 51 H 86 O7; 1 H NMR (500 MHz, CDCl3) δ H6.51 (s), 5.31 (s), 10.10 (s), 3.90 (s), 12.34 (s), 3.28 (dd, J = 14.1, 7.2 Hz), 3.33 (dd, J = 14.1, 7.2 Hz), 5.19 (t, J = 7.2 Hz), 2.27 (dd, J = 13.5, 7.4 Hz), 2.08 (dd, J = 13.5, 6.3 Hz), 5.59 (m), 5.04 (d, J = 9.2 Hz), 1.64 (s), 1.63 (s), 1.79 (s), 2.33 (m), 1.61 (m), 2.15 (m), 1.52 (m), 0.88 (t, J = 6.8 Hz), 1.24 (m); 13 CNMR (125 MHz, CDC13) δ C 138.5, 112.83, 162.86, 117.64, 163.38, 105.5, 62.91, 193.04, 55.89, 21.43, 124.86, 131.28, 45.31, 69.65, 123.77, 136.69, 25.61, 18.34, 16.47, 173.18, 34.23, 24.87, 173.07, 34.54, 24.99, 31.93, 22.74, 14.13, 29.13-29.70.

[0076]

[0077] By analysis of mass spectrometry and nuclear magnetic data, the compound shown in formula 2 is (E)-8-(3-formyl-2-hydroxy-6-methoxy-4-((palmitoyloxy) methyl) phenyl)-2,6-dimethyl octa-2,6-diene-4-yl (9Z,12Z)-octadecan-9,12-dienoic acid, (hericene F), the spectral data is as follows: negative ion mode HRTOFMS mass spectrum m / z 833.6285 [M-H] - (calcd C 53 H 85 O7, 833.6295), obtaining its molecular formula as C 53 H 86 O7; 1 H NMR (500 MHz, CDC13) δ H6.51 (s), 5.31 (s), 10.10 (s), 3.90 (s), 12.34 (s), 3.28 (dd, J = 14.1, 7.2 Hz), 3.33 (dd, J = 14.1, 7.2 Hz), 5.19 (t, J = 7.2 Hz), 2.27 (dd, J = 13.5, 7.4 Hz), 2.08 (dd, J = 13.5, 6.3 Hz), 5.59 (m), 5.04 (d, J = 9.2 Hz), 1.64 (s), 1.63 (s), 1.79 (s), 2.33 (m), 1.61 (m), 2.15 (m), 1.52 (m), 2.04 (m), 5.34 (m), 2.76 (t, J = 6.8), 0.88 (m), 1.24 (m); 13 C NMR (125 MHz, CDC13) δ C 138.65, 112.98, 163.01, 117.78, 163.53, 105.65, 63.06, 193.19, 56.04, 21.57, 125.02, 131.42, 45.45, 69.85, 123.9, 136.85, 25.57, 18.49, 16.62, 173.32, 34.38, 25.02, 173.18, 34.67, 25.12, 27.35, 130.35, 130.22, 128.16, 128.04, 25.63, 31.67, 32.08, 22.74, 22.85, 14.28, 14.23, 29.23-29.85.

[0078]

[0079] By analyzing the mass spectrum and nuclear magnetic data, the compound shown as formula 3 is (E)-8-(3-formyl-2-hydroxy-6-methoxy-4-((palmitoyloxy) methyl) phenyl)-2, 6-dimethyl octa-2, 6-diene-4-yl oleate, hericene G, and its spectral data are as follows: negative ion mode HRTOFMS mass spectrum m / z 835.6464 [M-H] - (calcd C 53 H 87 O7, 835.6452), obtaining its molecular formula as C 53 H 88 O7; 1 H NMR (500MHz, CDC13) δ H6.51 (s), 5.31 (s), 10.10 (s), 3.90 (s), 12.34 (s), 3.28 (dd, J = 14.1, 7.2 Hz), 3.33 (dd, J = 14.1, 7.2 Hz), 5.19 (t, J = 7.2 Hz), 2.27 (dd, J = 13.5, 7.4 Hz), 2.08 (dd, J = 13.5, 6.3 Hz), 5.59 (m), 5.04 (d, J = 9.2 Hz), 1.64 (s), 1.63 (s), 1.79 (s), 2.33 (m), 1.61 (m), 2.15 (m), 1.52 (m), 2.04 (m), 5.34 (m), 0.88 (m), 1.24 (m'); 13 C NMR (125 MHz, CDC13) δ C 138.67, 113.04, 163.03, 117.81, 163.55, 105.76, 63.11, 193.27, 56.04, 21.56, 125.04, 131.44, 45.45, 69.92, 123.92, 136.85, 25.74, 18.48, 16.61, 173.40, 34.36, 25.01, 173.36, 34.67, 25.11, 27.36, 130.18, 127.88, 32.05, 32.07, 22.83, 22.84, 14.26, 29.20-29.90.

[0080]

[0081] By analyzing the mass spectrum and nuclear magnetic data, the compound shown in formula 4 is (E)-8-(3-formyl-2-hydroxy-6-methoxy-4-((stearoyloxy)methyl)phenyl)-2,6-dimethyl-4- octadien-2,6-dienyl (9Z,12Z)-octadecan-9,12-dienoate (hericene H), and its spectral data are as follows: negative ion mode HRTOFMS mass spectrum m / z 861.6597 [M-H] - (calculated value C 55 H 89 O7, 861.6608), obtaining its molecular formula as C5H 90 O7; 1 H NMR (500MHz, CDC13) δ H6.51 (s), 5.31 (s), 10.10 (s), 3.90 (s), 12.34 (s), 3.28 (dd, J = 14.1, 7.2 Hz), 3.33 (dd, J = 14.1, 7.2 Hz), 5.19 (t, J = 7.2 Hz), 2.27 (dd, J = 13.5, 7.4 Hz), 2.08 (dd, J = 13.5, 6.3 Hz), 5.59 (m), 5.04 (d, J = 9.2 Hz), 1.64 (s), 1.63 (s), 1.79 (s), 2.33 (m), 1.61 (m), 2.15 (m), 1.52 (m), 2.04 (m), 5.34 (m), 2.76 (t, J = 6.8), 0.88 (m), 1.24 (m); 13 C NMR (125 MHz, CDC13) δ C 138.65, 112.98, 163.01, 117.78, 163.53, 105.65, 63.06, 193.19, 56.04, 21.57, 125.02, 131.42, 45.45, 69.85, 123.90, 136.85, 25.57, 18.49, 16.62, 173.32, 34.38, 25.02, 173.18, 34.67, 25.12, 27.35, 130.35, 130.22, 128.16, 128.05, 25.63, 31.67, 32.08, 22.74, 22.85, 14.28, 14.23, 29.23-29.85.

[0082]

[0083] By analyzing the NMR data and comparing with the literature, the compound shown as formula 5 was confirmed as (E)-4-(3,7-dimethylocta-2,6-dien-1-yl)-2-formyl-3-hydroxy-5-methoxyphenyl palmitate, (hericene A). Its spectral data are as follows: negative ion mode HRTOFMS mass spectrum m / z 555.4053 [M-H] - (CaIc. for C 35 H 55 O 555.4053), obtaining its molecular formula as C 35 H 56 O 555.4053); 1 H NMR (500 MHz, CDC13) δ H6.52 (s), 5.32 (s, 2H), 10.1 (s), 3.91 (s), 12.36 (s), 3.34 (d, J = 7.1 Hz), 5.17 (t, J = 7.1 Hz), 1.97 (m), 2.04 (m), 5.05 (t, J = 7.5 Hz), 1.63 (s), 1.57 (s), 1.76 (s), 2.33 (t, J = 7.6 Hz), 0.88 (t, J = 6.9 Hz), 1.61 (m), 1.25 (m); 13 C NMR (125 MHz, CDC13) δ C 138.4, 112.9, 162.9, 118, 163.5, 105.6, 63, 193.1, 55.9, 21.4, 121.2, 131.2, 39.8, 26.7, 124.3, 135.8, 25.7, 17.7, 16.1, 173.2, 34.2, 14.1, 31.9, 29.7, 29.7, 29.7, 29.6, 29.6, 29.4, 29.4, 29.2, 29.1, 24.9, 22.7.

[0084]

[0085] By analyzing the NMR data and comparing with the literature, the compound shown in formula 6 was confirmed as 4-((E)-3,7-dimethyloct-2,6-dien-1-yl)-2-formyl-3-hydroxy-5-methoxyphenyl oleate, (hericene B). Its spectral data are as follows: negative ion mode HRTOFMS mass spectrum m / z 581.4209 [M-H] - (CaIc. for C 37 H 57 O5, 581.4206), obtaining its molecular formula as C 37 H 58 O5; 1 H NMR (500 MHz, CDC13) δ H 6.52 (s), 5.32 (s), 10.1 (s), 3.91 (s), 12.36 (s), 3.34 (d, J = 7.0 Hz), 5.17 (t, J = 7.2 Hz), 1.97 (m), 2.03 (m), 5.05 (t, J = 7.3 Hz), 1.63 (s), 1.57 (s), 1.77 (s), 2.34 (t, J = 7.5 Hz), 0.88 (t, J = 6.7 Hz, 9H), 5.34 (m), 5.34 (m), 1.61 (m), 1.25 (m); 13 C NMR (125 MHz, CDC13) δ C138.4, 112.9, 162.9, 118.1, 163.5, 105.6, 62.9, 193.1, 55.9, 21.4, 121.2, 131.3, 39.7, 26.7, 124.4, 135.8, 25.7, 17.7, 16.1, 173.2, 34.2, 14.1, 130.0, 129.7, 31.9, 29.7, 29.6, 29.6, 29.5, 29.2, 29.1, 24.9, 22.7.

[0086]

[0087] By analyzing the NMR data and comparing with the literature, the compound of formula 7 was confirmed to be (E)-4-(3,7-dimethyloct-2,6-dien-1-yl)-2-formyl-3-hydroxy-5-methoxyphenyl stearate, (hericene C). Its spectral data are as follows: negative ion mode HRTOFMS mass spectrum m / z 583.4358 [M-H] - (CaIc. for C 37 H 59 O5, 583.4362), obtaining its molecular formula as C 37 H 60 O5; 1 H NMR (500 MHz, CDCI3) δ H 6.52 (s), 5.31 (s), 10.1 (s), 3.91 (s), 12.36 (s), 3.33 (d, J = 7.1 Hz), 5.15 (d, J = 7.0 Hz), 1.95 (m), 2.04 (m), 5.05 (t, J = 6.9 Hz), 1.63 (s), 1.56 (s), 1.76 (s), 2.32 (t, J = 7.6 Hz), 0.87 (t, J = 6.8 Hz, 4H), 1.60 (m) 1.25 (m); 13 CNMR (125 MHz, CDCI3) δ C 138.4, 112.9, 162.9, 118.0, 163.5, 105.6, 63.0, 193.1, 55.9, 21.4, 121.2, 131.2, 39.8, 26.7, 124.3, 135.8, 25.7, 17.7, 16.1, 173.2, 34.2, 14.1, 31.9, 29.7, 29.7, 29.7, 29.6, 29.6, 29.4, 29.4, 29.2, 29.1, 24.9, 22.7.

[0088]

[0089] By analyzing the NMR data and comparing with the literature, the compound shown in formula 8 was confirmed as 4-((E)-3,7-dimethylocta-2,6-dien-1-yl)-2-formyl-3-hydroxy-5-methoxyphenyl (9Z,12Z)-octadeca-9,12-dienoate, (hericene D). Its spectral data are as follows: negative ion mode HRTOFMS mass spectrum m / z 579.4057 [M-H] - (calcd C 37 H 55 O5,579.4049), obtaining its molecular formula as C 37 H 56 O5; 1 H NMR (500 MHz, CDCl3) δ H 6.52 (s), 5.32 (s), 10.11 (s), 3.91 (s), 12.36 (s), 3.34 (d, J = 7.1 Hz), 5.17 (t, J = 8.0 Hz), 1.95 (m), 2.05 (m), 5.05 (t, J = 7.1), 1.64 (s), 1.57 (s), 1.77 (s), 2.33 (t, J = 7.5 Hz), 0.88 (t, J = 7.0 Hz), 5.37 (m), 2.77 (t, J = 6.8 Hz), 2.05 (m), 1.61 (m), 1.25 (m); 13 C NMR (125 MHz, CDCl3) δ C 138.4, 112.9, 162.9, 118.1, 163.5, 105.6, 63.0, 193.1, 55.9, 21.4, 121.2, 131.2, 39.8, 26.7, 124.4, 135.7, 25.6, 17.6, 16.1, 173.1, 34.2, 14.1, 127.9, 128.1, 130.0, 130.2, 31.5, 29.6, 29.3, 29.7, 29.4, 29.2, 29.1, 24.9, 22.7.

[0090]

[0091] By analyzing the NMR data and comparing with the literature, the compound shown in formula 9 was confirmed as (E)-4-(3,7-dimethyl-5-oxooct-2,6-dien-1-yl)-2-formyl-3-hydroxy-5-methoxyphenyl palmitate, (hericenone C). Its spectral data are as follows: negative ion mode HRTOFMS mass spectrum m / z 593.5850 [M-H] - (calcd C 37 H 53O6, 593.3842), giving a molecular formula of C 37 H 54 O6; 1 H NMR (500 MHz, CDC13) δ H 6.53 (s), 10.11 (s), 3.91 (s), 5.32 (s), 12.38 (s), 3.4 (d, J = 7.2 Hz), 5.32 (m), 3.00 (s), 6.09 (s), 1.84 (s), 1.78 (s), 2.12 (s), 2.33 (t, J = 7.6 Hz), 0.88 (t, J = 6.9 Hz), 1.61 (m), 1.25 (m); 13 C NMR (125 MHz, CDC13) δ C 138.7, 112.9, 162.9, 117.3, 163.5, 105.6, 193.1, 55.9, 62.9, 21.6, 126.2, 130.3, 55.6, 199.6, 122.8, 155.5, 27.7, 16.4, 20.7, 173.2, 34.2, 14.1, 31.9, 29.7, 29.6, 29.5, 29.4, 29.2, 29.1, 24.9, 22.7.

[0092]

[0093] By analyzing the NMR data and comparing with the literature, the compound shown in formula 10 was confirmed as (E)-4-(3,7-dimethyl-5-oxo-2,6-octadien-1-yl)-2-formyl-3-hydroxy-5-methoxyphenyl stearate, (hericenone D). Its spectral data are as follows: negative ion mode HRTOFMS mass spectrum m / z 597.4148 [M-H] - (Found C 37 H 57 O6, 597.4155), giving a molecular formula of C 37 H 58 O6; 1 H NMR (500 MHz, CDC13) δ H 6.53 (s), 10.11 (s), 3.91 (s), 5.32 (s), 12.38 (s), 3.4 (d, J = 7.2 Hz), 5.32 (m), 3.00 (s), 6.09 (s), 1.84 (s), 1.78 (s), 2.12 (s), 2.33 (t, J = 7.6 Hz), 0.88 (t, J = 6.9 Hz), 1.61 (m), 1.25 (m); 13 C NMR (125 MHz, CDC13) δC 138.7, 112.9, 162.9, 117.3, 163.5, 105.6, 193.1, 55.9, 62.9, 21.6, 126.2, 130.3, 55.6, 199.6, 122.8, 155.5, 27.7, 16.4, 20.7, 173.2, 34.2, 14.1, 31.9, 29.7, 29.6, 29.5, 29.4, 29.2, 29.1, 24.9, 22.7.

[0094]

[0095] By analyzing the NMR data and comparing with the literature, the compound of formula 11 was confirmed as 4-((E)-3,7-dimethyl-5-oxo oct-2,6-dienyl)-2-formyl-3-hydroxy-5-methoxy phenyl (9Z,12Z)-octadeca-9,12-dienoate, (hericenone E). Its spectral data are as follows: negative ion mode HRTOFMS mass spectrum m / z 593.3849 [M-H] - Calcd for C 37 H 53 O6, 593.3842), the molecular formula of which was C 37 H 54 O6; 1 H NMR (500 MHz, CDC13) δ H 6.56 (s), 5.32 (s), 10.14 (s), 12.41 (s), 3.95 (s), 3.43 (d, J = 7.2 Hz), 5.32 (m), 3.04 (s), 6.12 (s), 1.87 (s), 1.81 (s), 2.16 (s), 2.37 (t, J = 7.6 Hz), 0.92 (t, J = 6.9 Hz), 5.34 (m), 2.8 (t, J = 6.5 Hz), 2.08 (m), 1.62 (m), 1.68 (m), 1.25 (m); 13 C NMR (125 MHz, CDC13) δ C 138.6, 112.9, 162.9, 117.3, 163.4, 105.6, 62.9, 193.1, 55.9, 21.6, 126.2, 130.3, 55.6, 199.5, 122.8, 155.5, 27.7, 16.4, 20.7, 173.2, 34.2, 14.1, 127.9, 128.1, 130.0, 130.2, 31.5, 29.6, 29.3, 29.1, 29.1, 27.2, 27.2, 25.6, 24.8, 22.6.

[0096]

[0097] By analyzing the NMR data and comparing with the literature, the compound shown in formula 12 was confirmed to be (8-formyl-5-methoxy-2-methyl-2-(4-methyl-2-oxovaleridene-1-yl)dihydrobenzofuran-7-yl)methyl palmitate, (hericenone F). Its spectral data are as follows: negative ion mode HRTOF MS mass spectrum m / z 569.3850 [M-H] - (calcd C 35 H 53 O6, 569.3842), obtaining its molecular formula as C 35 H 54 O6; 1 H NMR (500 MHz, CDC13) δ H 2.01 (dd, J = 13.8, 6.0 Hz), 1.92 (dd, J = 13.8, 6.9 Hz), 2.63 (t, J = 6.8 Hz), 6.54 (s), 1.44 (s), 2.82 (d, J = 14.1 Hz), 2.66 (d, J = 13.9 Hz), 6.06 (s), 2.14 (s), 1.86 (s), 10.42 (s), 3.88 (s), 5.51 (s), 2.41 (t, J = 7.6 Hz), 1.69 (m), 0.88 (t, J = 6.9 Hz), 1.25 (m); 13 C NMR (125 MHz, CDC13) δ C 76.9, 30.0, 16.4, 109.2, 161.9, 100.8, 139.7, 115.7, 158.3, 24.5, 52.5, 197.9, 124.9, 156.4, 20.8, 27.8, 190.3, 55.6, 64.5, 173.3, 34.4, 31.9, 14.1, 29.7, 29.7, 29.6, 29.5, 29.4, 29.3, 25.1, 22.7.

[0098]

[0099] By analyzing the NMR data and comparing with the literature, the compound shown in formula 13 was confirmed to be (8-formyl-5-methoxy-2-methyl-2-(4-methyl-2-oxovaleridene-1-yl)dihydrobenzofuran-7-yl)methyl palmitate, (hericenone F). Its spectral data are as follows: negative ion mode HRTOF MS mass spectrum m / z 569.3850 [M-H] - (calcd C 37 H57 O6,597.4155), giving a molecular formula of C 37 H 58 O6; 1 H NMR (500 MHz, CDC13) δ H 2.01 (m), 1.92 (m), 2.63 (t, J = 6.8 Hz), 6.54 (s), 1.44 (s), 2.82 (d, J = 14.1 Hz), 2.67 (d, J = 14.1 Hz), 6.06 (s), 2.14 (s, 3H), 1.86 (s), 10.41 (s), 3.88 (s), 5.51 (s), 2.41 (t, J = 7.6 Hz), 0.88 (t, J = 6.9 Hz), 1.69 (m), 1.25 (m); 13 C NMR (125 MHz, CDC13) δ C 76.9, 30.0, 16.4, 109.2, 161.9, 100.8, 139.7, 115.7, 158.3, 24.5, 52.5, 197.9, 124.9, 156.5, 20.8, 27.8, 190.4, 55.6, 64.5, 173.3, 34.4, 14.1, 31.9, 29.7, 29.7, 29.7, 29.6, 29.5, 29.4, 29.3, 29.3, 22.7, 25.1.

[0100]

[0101] By analyzing the NMR data and comparing with the literature, the compound shown in formula 14 was confirmed as (8-formyl-5-methoxy-2-methyl-2-(4-methyl-2-oxovaleryl-3-en-1-yl)dihydrobenzofuran-7-yl)methyl (9Z,12Z)-octadeca-9,12-dienoate (hericenone H). Its spectral data are as follows: negative ion mode HRTOFMS mass spectrum m / z 593.3853 [M-H] - (calcd C 37 H 53 O6,593.3842), giving a molecular formula of C 37 H 54 O6; 1 H NMR (500 MHz, CDC13) δ H2.01 (m), 1.92 (m), 2.63 (t, J = 6.8 Hz), 6.54 (s), 2.82 (d, J = 14.2 Hz), 2.66 (d, J = 14.2 Hz), 6.06 (s), 2.14 (s), 1.86 (s), 10.41 (s), 3.88 (s), 5.50 (s), 2.41 (t, J = 7.6 Hz), 0.88 (t, J = 6.8 Hz), 5.34 (m), 2.77 (t, J = 6.7 Hz), 2.04 (m), 1.69 (m); 13 C NMR (125 MHz, CDC13) δ C 76.9, 30.0, 16.5, 109.2, 161.9, 100.8, 139.6, 115.8, 158.3, 24.5, 52.5, 197.9, 124.9, 156.4, 20.8, 27.8, 190.4, 55.6, 64.6, 173.2, 34.4, 14.1, 127.9, 128.1, 130.0, 130.2, 31.5, 29.6, 29.4, 29.2, 29.1, 27.2, 27.2, 25.1, 22.6, 53.4, 25.6, 50.9, 29.2.

[0102]

[0103] Example 6

[0104] Evaluation of the α-glucosidase inhibitory activity of Hericium erinaceus monomeric compounds and extracts in vitro

[0105] The inhibitory activity of the drugs on α-glucosidase was evaluated using three substrates, p-nitrophenyl-α-D-glucoside (PNPG), maltose, and sucrose.

[0106] 1.0 g of mouse intestinal mucosa homogenate was suspended in 3 mL of 0.9% normal saline, and then ultrasonicated (12 times, 30 s each) at 4°C. The supernatant was obtained by centrifugation at 10000 g for 30 min at 4°C, to obtain a crude extract of α-glucosidase.

[0107] 10 μL of the test compound at different concentrations, 20 μL of 2.0 mM p-nitrophenyl-α-D-glucoside, and 60 μL of phosphate buffer (50 mM, pH 6.8) were mixed, and the reaction was carried out in a water bath at 37°C for 40 min. Subsequently, the absorbance of the product p-nitrophenol was detected at 405 nm using an enzyme marker.

[0108] 10 μL of different concentrations of test compounds or extract, 20 μL of 2.0 mM maltose or 2.0 mM sucrose were mixed with 60 μL of phosphate buffer (50 mM, pH 6.8), and reacted in a 37 °C water bath for 40 min. The glucose content produced by the hydrolysis of sucrose and maltose was determined using a glucose assay kit (GOD, glucose oxidase method), and the absorbance was measured at 550 nm to determine the amount of glucose produced.

[0109] The control group of the above three groups of experiments was replaced with an equal volume of phosphate buffer instead of the sample solution; the sample blank group used an equal volume of phosphate buffer instead of α-glucosidase, and acarbose was used as a positive control. The inhibition rate was calculated according to the following formula, and the IC 50 value was calculated according to the relationship between the inhibition rate and the concentration.

[0110] Inhibition rate (%) = [(OD 对照组 - OD 对照空白组 )-(OD 样品组 - OD 样品空白组 )] / (OD 对照组 - OD 对照空白组 ) x 100%.

[0111] According to the results in Table 2, the inhibitory activity of compounds 6, 9, 11, 12, 13, and 14 on the hydrolysis of PNPG by α-glucosidase was stronger than that of acarbose, with IC 50 values of 15.4 and 21.1 μM for 13 and 14, and an IC 50 value of 38.1 μM for acarbose. The IC 50 values of compounds 9 and 13 for the inhibition of the hydrolysis of sucrose by α-glucosidase were 13.5 and 12.1 μM, respectively, and the IC 50 value of acarbose was 20.2 μM. In addition, compound 9 had strong inhibitory activity on the hydrolysis of maltose, with an IC 50 value of 15.1 μM, and the IC 50 value of acarbose was 16.1 μM. The extract also had good inhibitory activity on α-glucosidase, with IC 50 values of 37.5, 39.0, and 71.2 μg / mL for the inhibition of the hydrolysis of PNPG, sucrose, and maltose, respectively, by α-glucosidase.

[0112] Table 2 Inhibitory activity (IC 50 , μM) of Hericium erinaceus compounds and extract on α-glucosidase

[0113]

[0114] Example 7

[0115] Evaluation of the in vitro α-amylase inhibitory activity of Hericium erinaceus monomeric compounds and extract

[0116] Pig pancreas-derived α-amylase was purchased from Shanghai Yuan Ye. The test compound was dissolved in DMSO, 1 μL of the test compound was added to 199 μL of starch solution (containing 1% starch and 17 mM NaCl), followed by 10 μL of α-amylase (26 U / mL dissolved in 0.1 M phosphate buffer, pH 7.0) and incubated on a shaker for 30 min. Then 20 μL of 2 M NaOH solution was added to stop the enzyme activity reaction. Next, 20 μL of dinitrosalicylic acid solution (44 mM 3,5-dinitrosalicylic acid, 106 mM potassium sodium tartrate, 40 mM NaOH) was added and reacted at 90°C for 20 min. The enzyme reader was read at 540 nm. The control group of the experiment was washed with an equal volume of DMSO instead of the sample solution; the sample blank group used an equal volume of phosphate buffer instead of α-amylase, and acarbose was used as a positive control. The inhibition rate was calculated according to the following formula, and the IC 50 value was calculated according to the relationship between the inhibition rate and the concentration.

[0117] Inhibition rate (%) = [(OD 对照组 - OD 对照空白组 )-(OD 样品组 - OD 样品空白组 )] / (OD 对照组 - OD 对照空白组 ) x 100%.

[0118] According to the test, the IC 50 of the monomer compound on α-amylase inhibition activity was >100 μM, the IC 50 of the extract was >200 μg / mL, and the IC 50 of acarbose was 56.1 μM. Since the side effects of acarbose are due to the strong inhibition activity of acarbose on α-amylase, and the inhibition activity of the monomer compound isolated from Hericium erinaceus on α-amylase is weak, the occurrence of side effects similar to acarbose can be avoided to some extent in the application process.

[0119] Table 3 Inhibition activity (IC 50 , μM) of Hericium erinaceus compounds and extracts on α-amylase

[0120]

[0121] Example 8

[0122] Preparation of Hericium erinaceus extract by ultrasonic extraction with ethyl acetate

[0123] The dried Hericium erinaceus was crushed and passed through an 80-mesh sieve. 10 g of the Hericium erinaceus powder was suspended in 200 mL of ethyl acetate, and ultrasonicated for 60 min. The filtrate was collected by filtration, and the residue was extracted twice more. The filtrates were combined and concentrated to a paste. The paste was dissolved in 20 mL of a 1:1 methanol-dichloromethane solvent, and the soluble portion was collected, concentrated, and dried to obtain 0.359 g of the compound.

[0124] Example 9

[0125] The extraction was performed using petroleum ether, n-hexane, dichloromethane, and ethanol as the extraction solvents, respectively, according to the above extraction method. Specifically, the dried Hericium erinaceus was crushed and passed through an 80-mesh sieve. 10 g of the Hericium erinaceus powder was suspended in 200 mL of the extraction solvent, and ultrasonicated for 60 min. The filtrate was collected by filtration, and the residue was extracted twice more. The filtrates were combined and concentrated to a paste. The paste was dissolved in 20 mL of a 1:1 methanol-dichloromethane solvent, and the soluble portion was collected, concentrated, and dried to obtain 0.228, 0.224, 0.308, and 0.148 g of the compound, respectively.

[0126] Example 10

[0127] Ethyl acetate Soxhlet extraction to prepare Hericium erinaceus extract

[0128] The dried Hericium erinaceus was crushed and passed through an 80-mesh sieve. 10 g of the Hericium erinaceus powder was suspended in 200 mL of ethyl acetate, and added to a Soxhlet extractor for refluxing for 10 min. The filtrate was collected and concentrated to a paste. The paste was dissolved in 20 mL of a 1:1 methanol-dichloromethane solvent, and the soluble portion was collected, concentrated, and dried to obtain 0.423 g of the compound.

[0129] Example 11

[0130] The extraction was performed using petroleum ether, n-hexane, dichloromethane, and ethanol as the extraction solvents, respectively, according to the above extraction method. Specifically, the dried Hericium erinaceus was crushed and passed through an 80-mesh sieve. 10 g of the Hericium erinaceus powder was suspended in 200 mL of the extraction solvent, and added to a Soxhlet extractor for refluxing for 10 min. The filtrate was collected and concentrated to a paste. The paste was dissolved in 20 mL of a 1:1 methanol-dichloromethane solvent, and the soluble portion was collected, concentrated, and dried to obtain 0.137, 0.138, 0.208, and 0.311 g of the compound, respectively.

[0131] Example 12

[0132] Quantitative analysis of active ingredients in Hericium erinaceus extract

[0133] According to the liquid phase analysis, compound 9 with α-glucosidase inhibitory activity is the main component in Hericium erinaceus extract. Therefore, quantitative analysis was carried out on compound 9 (hericeone C, formula-9) as a standard in the extracts obtained in Examples 1-9 and Examples 14-23.

[0134] HPLC analysis method

[0135] The HPLC instrument was Shimadzu LC-20AT liquid chromatograph equipped with an SPD-M20A detector. The sample was prepared into a 1 mg / mL solution with ethyl acetate as the solvent, the sample loading amount was 10 μL, the chromatographic column was Kromasil 4.5 x 250 mm C8 (5 μm), the mobile phase A was MeOH and the mobile phase B was H2O; the elution program was 0-10 min, 85% MeOH, 10-50 min 85%-100% MeOH. The column temperature was 40°C, and the detection wavelength was 294 nm. Under this method, the retention time of compound 9 was t R = 27.2 min, and baseline separation was achieved in the extract, indicating that the method was well applicable.

[0136] Preparation of standard curve

[0137] The ethyl acetate solution containing 2 mg / mL of compound 9 was precisely prepared, and half-gradient dilution was performed to obtain a series of mixed control solution with different concentrations. The sample was injected and determined according to the above chromatographic conditions, and the peak area was recorded. The standard curve was plotted with the peak area value as the vertical coordinate and the concentration as the horizontal coordinate, and the linear regression was performed to obtain the regression equation Y = 4.4520x + 0.1033, and the correlation coefficient R 2 was 0.9997, indicating that the linear relationship was good within the configured sample concentration range.

[0138] The extracts obtained by different methods were fully dried, dissolved and diluted with ethyl acetate to prepare sample solutions, and then analyzed according to the above analysis conditions. According to the integral area, the content of compound 9 in the sample was calculated, and the content in the crude extract was further calculated. It can be found by comparison that the content of compound 9 in the extract obtained by the carbon dioxide supercritical extraction method is the highest, which is 4.73%, so this method not only has high extraction rate, but also is environmentally friendly.

[0139] Table 4 Quantitative analysis results of compound 9 in extract

[0140]

Claims

1. The use of a compound in the preparation of an α-glucosidase inhibitor, having one of the following structural formulas: , or .

2. The use of a compound in the preparation of a drug for treating diabetes, having one of the following structural formulas: , or .

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