Anthraquinone compound as well as extraction method and application thereof

Abstract

The invention provides an anthraquinone compound which is a natural product extracted from fermentation liquor of soil streptomyces and has a hypoglycemic effect.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to an anthraquinone compound. Background Art

[0002] Diabetes mellitus (DM) is a metabolic disease characterized by abnormally high levels of glucose in the blood. It is estimated that by 2050, the number of diabetes patients worldwide will exceed 1.3 billion. Statistics from all continents of the world show that type 2 diabetes (T2DM) is the most common type of diabetes. α-Glucosidase inhibitors are the most effective drugs for the treatment of T2DM. Currently, Streptomyces is the main source of natural products. Acarbose, the first α-glucosidase inhibitor used in Europe and the United States to treat T2DM, is derived from Streptomyces. In addition, voglibose is also an α-glucosidase inhibitor. It is a synthetic derivative of valiolamine produced by Streptomyces hygroscopicus and was used clinically in 1994. In 1996, miglitol was found to be an inhibitor of α-glucosidase. Therefore, Streptomyces is an important source of α-glucosidase inhibitors.

[0003] Given the current situation that the prevalence of diabetes is increasing year by year and most of the glucose-lowering drugs used clinically have certain side effects, it is very important and has great potential to explore more novel natural products from microbial sources and develop natural drugs that have glucose-lowering effects and are safe and effective. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides an anthraquinone compound, which is extracted from the fermentation liquid of soil Streptomyces and has a blood sugar lowering effect.

[0005] In order to achieve the purpose of the present invention, the technical solution adopted by the present invention is:

[0006] An anthraquinone compound with the following structure:

[0007]

[0008] The method for extracting anthraquinone compounds of the present invention comprises the following steps:

[0009] A. Fermentation: fermenting Streptomyces sp. PH9030 in a culture medium to obtain a fermentation product for later use;

[0010] B. Separation: The fermented product was extracted three times with ethyl acetate / methanol and then concentrated to obtain a crude extract paste; the compound Resistomycin B was obtained by normal-phase silica gel column chromatography, ODS column chromatography gradient elution, and high-performance liquid chromatography separation.

[0011] Preferably, Streptomyces sp. PH9030 was first cultured in a conical flask containing TSB solution at 30 °C with shaking for 3 days to obtain a Streptomyces seed fermentation broth, and then cultured in a production medium at 30 °C with shaking for 7 days, after which fermentation was stopped and the resin in the fermentation broth was collected.

[0012] More preferably, by mass, each liter of the medium for production fermentation contains: soluble starch 20 g / L, corn starch 20 g / L, KH 2 PO 4 0.5 g / L, MgSO 4 ·7H 2 O 0.25 g / L and trace elements 0.001 g / L, which was sterilized and used for large-scale fermentation.

[0013] More preferably, the trace elements were added to the medium in the same mass of ZnSO 4 ·7H 2 O, FeSO 4 ·7H 2 O, MnCl 2 ·4H 2 O and CaCl 2 Added to the medium.

[0014] More preferably, the volume ratio of ethyl acetate to methanol is 1:1.

[0015] Preferably, in step B, the crude extract paste was subjected to normal-phase silica gel column chromatography with a mobile phase of petroleum ether / ethyl acetate / methanol, and 6 fractions Fr.A - F were collected. Fraction Fr.F was subjected to ODS column chromatography gradient elution with methanol / water to obtain 7 fractions Fr.F1 - 7. Fraction Fr.F6 was subjected to ODS column chromatography gradient elution with methanol / water to obtain 4 fractions Fr.F6 - 1 - Fr.F6 - 4. Then, fraction Fr.F6 - 3 was separated by high-performance liquid chromatography to obtain the compound Resistomycin B.

[0016] More preferably, the volume ratio change of the petroleum ether / ethyl acetate gradient elution is 20:1, 10:1, 1:1, 1:5, 1:20; the volume ratio change of the ethyl acetate / methanol gradient elution is 20:1, 1:1, 1:20, 0:100;

[0017] The volume ratio change of the methanol / water ODS gradient elution is as follows: 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, 0:10;

[0018] The chromatographic conditions of the high performance liquid chromatography are as follows:

[0019] Chromatographic column: Yuexu AQ-C18, with a specification of 250×10mm, 5μm

[0020] Mobile phase: acetonitrile (A): 0.1% formic acid in water (B); gradient elution is adopted. From 0 to 11.0 min, the mobile phase ratio A:B increases from 20:80 to 95:5. From 11.0 to 11.8 min, the mobile phase ratio remains 95:5. From 11.8 to 12.0 min, the mobile phase ratio changes from 95:5 to 5:95. From 12.0 to 15.0 min, the mobile phase ratio remains 5:95;

[0021] Flow rate: 3 mL / min;

[0022] Column temperature: 35°C.

[0023] The application of the anthraquinone compound described in the present invention in the preparation of hypoglycemic products, and the products include drugs and health foods. Description of the drawings

[0024] Figure 1 is the 1 1H NMR of compound Resistomycin B;

[0025] Figure 2 is the 13 13C NMR of compound Resistomycin B;

[0026] Figure 3 is the DEPT90 of compound Resistomycin B;

[0027] Figure 4 is the DEPT135 of compound Resistomycin B;

[0028] Figure 5 is the two-dimensional nuclear magnetic resonance spectrum HSQC of compound Resistomycin B;

[0029] Figure 6 is the two-dimensional nuclear magnetic resonance spectrum HMBC of compound Resistomycin B;

[0030] Figure 7 is the two-dimensional nuclear magnetic resonance spectrum 1 1H- 1 1H COSY of compound Resistomycin B;

[0031] Figure 8 It is the NOESY of the two-dimensional nuclear magnetic resonance spectrum of the compound Resistomycin B;

[0032] Figure 9 It is the high-resolution mass spectrum of the compound Resistomycin B;

[0033] Figure 10 It is the ultraviolet absorption spectrum of the compound Resistomycin B. Detailed implementation mode

[0034] In order to more clearly and detailedly illustrate the technical solutions of the present invention, the present invention will be further described below through relevant embodiments. The following embodiments are only for specifically illustrating the implementation methods of the present invention and do not limit the protection scope of the present invention.

[0035] Example 1

[0036] An anthraquinone compound has the following structure:

[0037]

[0038] Example 2

[0039] The extraction method of the anthraquinone compound of the present invention includes the following steps:

[0040] A. Fermentation: Ferment Streptomyces sp. PH9030 in a culture medium to obtain a fermented product for standby;

[0041] B. Separation: Extract the fermented product three times with ethyl acetate / methanol (1:1, v / v) and then concentrate to obtain a crude extract paste. Use normal-phase silica gel column chromatography with the mobile phase of petroleum ether / ethyl acetate / methanol, and collect 6 fractions Fr.A - F. Subject fraction Fr.F to gradient elution with methanol / water ODS column chromatography to obtain 7 fractions Fr.F1 - 7. Subject fraction Fr.F6 to gradient elution with methanol / water ODS column chromatography to obtain 4 fractions Fr.F6 - 1 - Fr.F6 - 4. Then subject fraction Fr.F6 - 3 to high-performance liquid chromatography separation to obtain the compound Resistomycin B.

[0042] The genomic sequence of the Streptomyces sp. PH9030 of the present invention is deposited in the NCBI database.

[0043] The strain Streptomyces sp. PH9030 was isolated from the rhizosphere soil of the medicinal plant Kadsura coccinea in Huaihua City, Hunan Province (recorded in Molecules 2024, 29(15), 3450). This strain was preserved on a slant of growth medium. The composition of the production medium is as follows: glucose 0.5 g / L, yeast powder 0.5 g / L, soy peptone 0.5 g / L, soluble starch 0.5 g / L, acid hydrolyzed casein 0.5 g / L, sodium pyruvate 0.5 g / L, K2HPO4 0.3 g / L, MgSO4·7H2O 0.024 g / L, and agar 20 g / L. After sterilization, it is ready for use.

[0044] Example 3

[0045] The method for extracting the anthraquinone compounds of the present invention comprises the following steps:

[0046] A. Fermentation: Ferment Streptomyces sp. PH9030 in a medium to obtain a fermented product for standby;

[0047] First, Streptomyces sp. PH9030 was cultured in a conical flask containing TSB solution at 30 °C with shaking at 220 rpm for 3 days to obtain a Streptomyces seed fermentation broth. Then, it was cultured in the production medium at 30 °C with shaking at 220 rpm for 7 days, after which fermentation was stopped, and the resin in the fermentation broth was collected.

[0048] Each liter of the medium for production fermentation contains: soluble starch 20 g / L, corn starch 20 g / L, KH 2 PO 4 0.5 g / L, MgSO 4 ·7H 2 O 0.25 g / L, and trace elements 0.001 g / L. After sterilization, it is used for large-scale fermentation.

[0049] B. Separation: Extract the fermented product three times with ethyl acetate / methanol (1:1, v / v), then concentrate to obtain a crude extract paste. Use normal-phase silica gel column chromatography with the mobile phase of petroleum ether / ethyl acetate / methanol, and collect 6 fractions, Fr.A - F. Fraction Fr.F was eluted with a methanol / water ODS column chromatography gradient to obtain 7 fractions, Fr.F1 - 7. Fraction Fr.F6 was eluted with a methanol / water ODS column chromatography gradient to obtain 4 fractions, Fr.F6 - 1 - Fr.F6 - 4. Then, fraction Fr.F6 - 3 was separated by high-performance liquid chromatography to obtain the compound Resistomycin B.

[0050] Example 4

[0051] The method for extracting the anthraquinone compounds of the present invention comprises the following steps:

[0052] A. Fermentation: First, Streptomyces sp. PH9030 was cultured in a 1000 mL flat-bottom conical flask containing 300 mL of TSB solution at 30 °C with shaking at 220 rpm for 3 days to obtain a Streptomyces seed fermentation broth. The Streptomyces seed fermentation broth was inoculated at a volume of 10% of the large fermentation medium volume. Then, it was cultured in the production medium at 30 °C with shaking at 220 rpm for 7 days, after which fermentation was stopped and the resin in the fermentation broth was collected.

[0053] Each liter of the medium for production fermentation contained: soluble starch 20 g / L, corn flour 20 g / L, KH 2 PO 4 0.5 g / L, MgSO 4 ·7H 2 O 0.25 g / L and trace elements 0.001 g / L. After sterilization, it was used for large-scale fermentation.

[0054] The trace elements were added to the medium in the same mass of ZnSO 4 ·7H 2 O, FeSO 4 ·7H 2 O, MnCl 2 ·4H 2 O and CaCl 2 added to the medium.

[0055] B. Isolation: The fermented product was extracted three times with ethyl acetate / methanol (1:1, v / v) and then concentrated to obtain a crude extract paste. Normal-phase silica gel column chromatography was used with the mobile phase of petroleum ether / ethyl acetate / methanol, and 6 fractions Fr.A - F were collected. Fraction Fr.F was eluted with a methanol / water ODS column chromatography gradient to obtain 7 fractions Fr.F1 - 7. Fraction Fr.F6 was eluted with a methanol / water ODS column chromatography gradient to obtain 4 fractions Fr.F6 - 1 - Fr.F6 - 4. Then, fraction Fr.F6 - 3 was separated by high-performance liquid chromatography to obtain compound Resistomycin B.

[0056] The volume ratio change of the petroleum ether / ethyl acetate gradient elution was 20:1, 10:1, 1:1, 1:5, 1:20; the volume ratio change of the ethyl acetate / methanol gradient elution was 20:1, 1:1, 1:20, 0:100;

[0057] The volume ratio change of the methanol / water ODS gradient elution was: 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, 0:10;

[0058] The chromatographic conditions for high-performance liquid chromatography were:

[0059] Chromatographic column: Welch AQ-C18, with specifications of 250×10 mm, 5 μm

[0060] Mobile phase: Acetonitrile (A): 0.1% formic acid in water (B); Gradient elution was used. From 0 - 11.0 min, the mobile phase ratio A:B increased from 20:80 to 95:5. From 11.0 - 11.8 min, the mobile phase ratio remained at 95:5. From 11.8 - 12.0 min, the mobile phase ratio changed from 95:5 to 5:95. From 12.0 - 15.0 min, the mobile phase ratio remained at 5:95;

[0061] Flow rate: 3 mL / min;

[0062] Column temperature: 35 °C.

[0063] Structural analysis description of Resistomycin B:

[0064] Resistomycin B was fully structurally characterized through comprehensive structural analysis.

[0065] (1) High-resolution mass spectrometry (HRESIMS) analysis yielded the [M+H] molecular ion peak (m / z) of Resistomycin B as 367.08087, which was consistent with the calculated value of 367.08123 for its standard molecular formula C + H 20 H 14 O 7 [C 20 H 15 O 7 +H] + , with a molecular weight of 367.08123 ( Figure 9 ).

[0066] (2) Ultraviolet spectral analysis demonstrated that Resistomycin B had five absorption peaks at 205.2 nm, 261.9 nm, 294.0 nm, 364.2 nm, and 464.6 nm ( Figure 10 ).

[0067] Resistomycin B is yellow in color; UV(MeOH)max 205.2 nm, 261.9 nm, 294.0 nm, 364.2 nm, 464.6 nm, see Figure 9 ; HRESIMS: Calculated value for the molecular formula C 20 H 15 O 7 : 367.08123, measured value: 367.08087, see Figure 9 ;

[0068] (3) NMR data: See Table 1. Figures 1-4 It is a one-dimensional nuclear magnetic resonance spectrum,Figures 5-8 is a two-dimensional nuclear magnetic resonance spectrum, where Figure 1 is of Resistomycin B 1 H NMR, pyridine-d 5 (500 MHz); Figure 2 is of Resistomycin B 13 C NMR, pyridine-d 5 (125 MHz); Figure 3 is the DEPT90 of Resistomycin B, Figure 4 is the DEPT135 of Resistomycin B and Figures 5-7 is a two-dimensional nuclear magnetic resonance spectrum, where Figure 5 is the HSQC of Resistomycin B, Figure 6 is the HMBC of Resistomycin B; Figure 7 is of Resistomycin B 1 H- 1 H COSY; Figure 8 is the two-dimensional nuclear magnetic resonance spectrum NOESY of compound Resistomycin B; Figure 9 is the high-resolution mass spectrum of compound Resistomycin B; Figure 10 is the ultraviolet absorption spectrum of compound Resistomycin B.

[0069]

[0070] Table 1

[0071]

[0072]

[0073] In vitro inhibitory activity experiment of α-glucosidase of compound Resistomycin B

[0074] Glucosidase exists on the microvilli of small intestinal brush border cells and is a membrane-bound enzyme of small intestinal epithelial cells, playing an important role in the process of carbohydrate digestion. Therefore, it can be used as a drug action target to treat diabetes. α-Glucosidase inhibitors can competitively inhibit the activity of α-glucosidase in the small intestine, delay or inhibit the absorption of glucose in the intestine, thereby effectively reducing the peak value of postprandial blood glucose and adjusting the blood glucose level, playing a crucial role in the process of inhibiting postprandial hyperglycemia.

[0075] The present invention measures the in vitro inhibitory activity of the compound Resistomycin B using an in vitro inhibitory activity test. In an in vitro enzyme reaction system, the compound interacts with the enzyme by adding a substrate, an enzyme, and a buffer. Finally, the ultraviolet absorption level of the enzyme is measured in an enzyme-linked immunosorbent assay (ELISA) reader, and then its IC 50 is used to determine the in vitro inhibitory activity of the target compound against α-glucosidase.

[0076] Experimental method

[0077] A 200 μL reaction system is set up. In the sample group, 130 μL of PBS buffer is added, followed by 10 μL of the compound Resistomycin B solution and 10 μL of a 0.1 U / mL enzyme solution. Then, it is incubated in a water bath at 37 °C for 20 min. 50 μL of 0.5 mM PNP-Glucose is added to each well, and then it is incubated in a water bath at 37 °C for 40 min. 100 μL of Na 2 CO 3 is added to terminate the reaction.

[0078]

[0079] In the formula, A s represents the background group (Resistomycin B + PBS), A t represents the sample group (Resistomycin B + enzyme + PBS), A c represents the control group (enzyme + PBS), and A b represents the blank group (PBS). Acarbose is used as a positive control.

[0080] Table 2 In vitro inhibitory activity experimental reaction system for α-glucosidase

[0081]

[0082] Table 3 In vitro inhibitory activity of Resistomycin B and the positive drug acarbose

[0083] Compound <![CDATA[IC 50 (μM)]]> Resistomycin B 5.60±0.09 Acarbose 671.50±0.17

[0084] As shown in Table 3, the inhibitory IC 50 of the compound Resistomycin B against α-glucosidase is 5.60 ± 0.09 μM, and the IC 50 of acarbose is 671.50 ± 0.17 μM. The activity of the compound Resistomycin B is significantly stronger than that of the positive drug acarbose.

[0085] Cytotoxic activity screening

[0086] The MTT method was used to screen the biological activities of compound Resistomycin B against mouse neuroblastoma cell line (Neuro-2a), lung cancer cell line (A-549), and mouse colon cancer cell line (MC38) in vitro. Doxorubicin was used as a positive control (the experimental results are shown in Table 4). Experimental method: A single cell suspension was prepared with a culture medium (MEM, DMEM, or RPMI-1640) containing 10% fetal bovine serum. 5000 - 10000 cells were seeded into each well of a 96-well plate, with a volume of 100 μL per well. Adherent cells were seeded 12 h in advance; the test compound solution was added (initial screening at a fixed concentration of 40 μM, and 5 concentrations were set for gradient re-screening for compounds with 50% growth inhibition of tumor cells at this concentration). The final volume in each well was 200 μL, and 3 replicates were set for each treatment; after culturing at 37 °C for 48 h, the culture supernatant in the wells was carefully aspirated. 10 μL of MTT solution and 100 μL of culture medium were added to each well, and incubation was continued for 4 h to allow the reaction to proceed fully; at a wavelength of 570 nm, the optical absorption value of each well was read using an enzyme-linked immunosorbent assay detector (Bio-Rad 680), and the results were recorded. The cell growth curve was plotted with the concentration as the abscissa and the cell survival rate as the ordinate, and the IC 50 value was calculated using the two-point method (Reed and Muench method).

[0087] Table 4 Half-maximal inhibitory concentration IC 50 (μM) of compound Resistomycin B on tumor cell proliferation

[0088]

[0089] Resistomycin B had a weak inhibitory effect on the mouse neuroblastoma cell line (Neuro-2a) and a poor inhibitory effect on the lung cancer cell line (A-549) and mouse colon cancer cell line (MC38), indicating that the cytotoxic activity of this compound was very poor.

[0090] The MEM medium was purchased from HyClone, USA, and the DMEM and RPMI-1640 media were purchased from Gibco, USA.

[0091] The above-described embodiments only represent the specific implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the patent of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention.

Claims

1. An anthraquinone compound, characterized in that: The structure is as follows:

2. The method for extracting anthraquinone compounds according to claim 1, characterized in that: The following steps are involved: A. Fermentation: fermenting Streptomyces sp. PH9030 in a culture medium to obtain a fermentation product for later use; B. Separation: The fermented product was extracted three times with ethyl acetate / methanol and concentrated to obtain a crude extract; normal phase silica gel column chromatography, ODS column chromatography gradient elution, and high performance liquid chromatography were used for separation to obtain the compound Resistomycin B.

3. The method for extracting anthraquinone compounds according to claim 2, characterized in that: First, Streptomyces sp. PH9030 was cultured in a conical flask containing TSB solution at 30°C with shaking for 3 days to obtain Streptomyces seed fermentation liquid, and then the fermentation was stopped after shaking culture at 30°C in a production medium for 7 days, and the resin in the fermentation liquid was collected.

4. The method for extracting anthraquinone compounds according to claim 3, characterized in that: By mass, each liter of culture medium for production fermentation contains: 20 g / L soluble starch, 20 g / L corn flour, 0.5 g / L KH2PO4, 0.25 g / L MgSO4·7H2O and 0.001 g / L trace elements, and is used for large-scale fermentation after sterilization.

5. The method for extracting anthraquinone compounds according to claim 4, characterized in that: The trace elements are added into the culture medium in the form of ZnSO4·7H2O, FeSO4·7H2O, MnCl2·4H2O and CaCl2 in the same mass.

6. The method for extracting anthraquinone compounds according to claim 2, characterized in that: The volume ratio of ethyl acetate to methanol is 1:

1.

7. The method for extracting anthraquinone compounds according to claim 2, characterized in that: In the step B, the crude extract is chromatographed on a normal phase silica gel column with a mobile phase of petroleum ether / ethyl acetate / methanol to collect 6 fractions Fr.AF, fraction Fr.F is eluted by gradient chromatography on a methanol / water ODS column to obtain 7 fractions Fr.F1-7, fraction Fr.F6 is eluted by gradient chromatography on a methanol / water ODS column to obtain 4 fractions Fr.F6-1-Fr.F6-4, and fraction Fr.F6-3 is separated by high performance liquid chromatography to obtain compound Resistomycin B.

8. The method for extracting anthraquinone compounds according to claim 7, characterized in that: The volume ratio of petroleum ether / ethyl acetate gradient elution was changed to 20:1, 10:1, 1:1, 1:5, 1:20; the volume ratio of ethyl acetate / methanol gradient elution was changed to 20:1, 1:1, 1:20, 0:100; The volume ratio of methanol / water ODS gradient elution varied as follows: 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, 0:10; The chromatographic conditions of HPLC were: Chromatographic column: Yuexu AQ-C18, specification: 250×10mm, 5μm Mobile phase: acetonitrile (A): 0.1% formic acid water (B); gradient elution was used, the mobile phase ratio A:B increased from 20:80 to 95:5 during 0-11.0 min, the mobile phase ratio was maintained at 95:5 during 11.0-11.8 min, the mobile phase ratio was changed from 95:5 to 5:95 during 11.8-12.0 min, and the mobile phase ratio was maintained at 5:95 during 12.0-15.0 min; Flow rate: 3 mL / min; Column temperature: 35°C.

9. Use of the anthraquinone compound according to claim 1 in the preparation of hypoglycemic products.

10. The use of anthraquinone compounds in the preparation of hypoglycemic products according to claim 9, characterized in that: The products include medicines and health foods.

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