A natural active compound, pyridoxamine, preparation method and application thereof

Abstract

The present application relates to the technical field of compounds, in particular to a natural active compound pseudo-norfurane, a preparation method and application thereof. The present application provides a natural active compound pseudo-norfurane, which has the structure shown in the following formula (I), the compound formula (I) of the present application can significantly reduce the lipid accumulation in a HepG2 cell lipid model, has a hypolipidemic biological activity, can be used for preparing a hypolipidemic drug or a hypolipidemic health food, and has a good development and application prospect.

Description

Technical Field

[0001] This invention relates to the field of compound technology, specifically to a natural active compound called nofuran, its preparation method, and its applications. Background Technology

[0002] Cardiovascular disease remains the leading cause of death and its burden is becoming increasingly severe. Studies have shown that lowering blood lipid levels can significantly improve the quality of life for patients with cardiovascular disease and effectively reduce its incidence. Currently, statins are the first-line drugs for treating cardiovascular disease in clinical practice, but long-term use of these drugs can cause side effects such as elevated blood sugar, muscle pain, and liver and kidney toxicity in some patients. Therefore, the search for and development of safe, effective, and low-cost new cardiovascular disease drugs is urgently needed. Summary of the Invention

[0003] This invention marks the first time that *Nocardia* ZHD001 has been isolated from sediments collected from the waters surrounding Zhoushan, and its fermentation product, norofuran, has been further isolated. The chemical structure of the novel compound norofuran was determined by analyzing high-resolution mass spectrometry (HRESIMS, 1H, 1C, COSY, HMQC, and HMBC spectra). A series of studies have shown that norofuran can significantly reduce lipid accumulation in a HepG2 cell lipid model, exhibiting lipid-lowering bioactivity. It has promising potential for the development and application of lipid-lowering drugs.

[0004] On one hand, the present invention provides a natural active compound, norofuran, characterized in that it has the structure described in formula (I):

[0005] Furthermore, the molecular formula of the compound of formula (I) is C 19 H 23 NO6, molecular weight [M+Na] + 384.1413, yellow powder, soluble in methanol.

[0006] On the other hand, the present invention provides a method for preparing a natural active compound, norofuran, which includes the following steps:

[0007] 1) Obtain Nocardia strain Nocardiopsis sp ZHD001 was inoculated onto Gao's No. 1 solid medium for activation. Single colonies of the activated Nocardia strain ZHD001 were inoculated onto Gao's No. 1 liquid medium and cultured with shaking to obtain fermentation broth.

[0008] 2) Extract the fermentation broth with an equal volume of ethyl acetate. Remove the ethyl acetate solvent by vacuum distillation using a rotary evaporator to obtain a concentrated solution. Separate the concentrated solution by silica gel column chromatography with gradient elution. Collect the eluent and analyze each component by thin-layer chromatography. Combine the components containing norofuran.

[0009] 3) The components containing norofuran were separated and purified using a preparative high-performance liquid chromatograph to obtain the compound norofuran.

[0010] Preferably, the Nocardia strain in step 1) Nocardiopsis The accession number of sp ZHD001 is CCTCC NO:M2022921.

[0011] Preferably, the formula of Gao's No. 1 solid culture medium used in step 1) is as follows: 20g soluble starch, 1g potassium nitrate, 0.5g dipotassium hydrogen phosphate, 0.5g magnesium sulfate, 0.01g ferrous sulfate, 20g agar, 25g sea salt, and 1L water.

[0012] Preferably, the formula of Gao's No. 1 liquid culture medium used in step 1) is as follows: 20g soluble starch, 1g potassium nitrate, 0.5g dipotassium hydrogen phosphate, 0.5g magnesium sulfate, 0.01g ferrous sulfate, 25g sea salt, and 1L water.

[0013] Preferably, the mixed gradient elution in step 2) is a gradient elution of dichloromethane and methanol mixed solvents at ratios of 100:1, 80:1, 50:1, 30:1, 20:1, 10:1, 5:1, 1:1 and 0:1.

[0014] Preferably, in step 3), the high-performance liquid chromatography (HPLC) separation and purification column is an Agilent Pursuit C18 column (21.2 × 250 mm, 10 μm), with a detection wavelength of 210 nm. A methanol-water system with a volume percentage of 45%–60% is used for gradient elution at 10 mL / min for 40 minutes, and the eluent is collected after 12.7–14.5 minutes.

[0015] This invention provides the application of the above-mentioned compound formula (I) in the preparation of lipid-lowering drugs.

[0016] The compound of this invention (I) can significantly reduce lipid accumulation in the HepG2 cell lipid model, has lipid-lowering biological activity, and can be used to prepare lipid-lowering drugs, showing great development and application prospects. Attached Figure Description

[0017] Figure 1 This is the HRESIMS spectrum of the natural active compound norofuran of this invention.

[0018] Figure 2 This is the proton NMR spectrum of the natural active compound novofuran of this invention.

[0019] Figure 3 This is the carbon spectrum of the natural active compound norofuran of this invention.

[0020] Figure 4 This is the COSY spectrum of the natural active compound norofuran of this invention.

[0021] Figure 5 This is the HMQC spectrum of the natural active compound norofuran of this invention.

[0022] Figure 6 This is the HMBC spectrum of the natural active compound novofuran of this invention.

[0023] Figure 7 This is data from a model of the natural active compound novofuran inhibiting HepG2 lipids, as presented in this invention. Detailed Implementation

[0024] Example 1 Nocardia Nocardiopsis Preparation of fermentation broth for sp. ZHD001

[0025] Nocardia strain Nocardiopsis sp ZHD001 was inoculated onto Gao's No. 1 solid medium and then placed in a 37℃ incubator for 5 days of activation culture. Single colonies of the activated Nocardia strain ZHD001 were inoculated into 500mL Erlenmeyer flasks, each containing 250mL of Gao's No. 1 liquid medium, and cultured in a shaker at 28℃ and 180rpm for 10 days to obtain the fermentation broth.

[0026] The formula of Gao's No. 1 solid culture medium used is as follows: 20g soluble starch, 1g potassium nitrate, 0.5g dipotassium hydrogen phosphate, 0.5g magnesium sulfate, 0.01g ferrous sulfate, 20g agar, 25g sea salt, and 1L water.

[0027] The formula for the Gao's No. 1 liquid culture medium used is as follows: 20g soluble starch, 1g potassium nitrate, 0.5g dipotassium hydrogen phosphate, 0.5g magnesium sulfate, 0.01g ferrous sulfate, 25g sea salt, and 1L water.

[0028] Example 2: Preparation process of norofuran

[0029] The fermentation broth was extracted three times with an equal volume of ethyl acetate. The resulting ethyl acetate extract was then removed by vacuum distillation using a rotary evaporator to obtain a concentrated solution. The concentrated solution was separated by silica gel column chromatography using a mixed solvent gradient of dichloromethane and methanol at ratios of 100:1, 80:1, 50:1, 30:1, 20:1, 10:1, 5:1, 1:1, and 0:1. The eluents were collected, and each component was analyzed by thin-layer chromatography. Components containing norofuran were combined.

[0030] The fraction containing norofuran was separated and purified using a preparative Shimadzu LC-20AP high-performance liquid chromatograph. An Agilent Pursuit C18 column (21.2 × 250 mm, 10 μm) was used with a detection wavelength of 210 nm. A methanol-water system (45%–60% by volume) was used for gradient elution at 10 mL / min for 40 min. The eluent from 12.7 to 14.5 min was collected to obtain the compound norofuran.

[0031] Example 3: Confirmation of the structure of pseudofuran

[0032] Norifuran, a yellow powder, soluble in methanol. Its molecular formula, calculated using high-resolution mass spectrometry (HRESIMS), is C[2]. 19 H 23 NO6([M+Na)) + 384.1413), the high-resolution mass spectrum of norofuran is as follows: Figure 1 As shown. Figures 2-6 The 1H, 1C, COSY, HMQC, and HMBC spectra of norofuran were obtained. By analyzing these spectral data, the chemical structure of the natural active compound norofuran was determined as shown in formula (I), indicating that it is a new compound.

[0033] Example 4: Lipid-lowering activity of norofuran

[0034] HepG2 cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) at 37°C and 5% CO2 under saturated humidity. Every 3 days, the cells were digested with 0.25% trypsin and passaged at a ratio of 1:3. Cells in the logarithmic growth phase were collected, suspended, and seeded into 24-well plates. After incubation for 24 hours, the experiment was divided into 3 groups: ① Blank control group: cultured in DMEM medium containing 10% FBS for 24 hours; ② Model group: cultured in DMEM medium containing 1mM oleic acid for 24 hours; ③ Sample group: cultured in DMEM medium containing 1mM oleic acid and 10μM phenoxyfuran for 24 hours.

[0035] After removing the culture medium from each group, the cells were washed three times with PBS. 80 μL of 4% paraformaldehyde was added to each well for fixation for 40 min, followed by three washes with PBS. The cells were then treated with 60% isopropanol / water for 10 min, and then 50 μL of Oil Red working solution (prepared from a stock solution of 0.3 g / 100 mL Oil Red / isopropanol and water at a volume ratio of 3:2) was added for 20 min. The Oil Red working solution was then removed, and the cells were washed three times with PBS. Intracellular lipid droplet staining was observed using an inverted microscope. The stained lipid droplets were dissolved in 80 μL of isopropanol, and the OD value was measured at 520 nm using a microplate reader.

[0036] Experimental results are as follows Figure 7 As shown (compared with the model group, ***p<0.001; **p<0.001; *p<0.01). The results indicate that norifuran can significantly inhibit lipid accumulation in the HepG2 lipid model. Figure 7 ).

Claims

1. A natural active compound, pyriNoid, characterized in that, The compound has the structure as shown in formula (I) 。 2. The natural active compound of claim 1, wherein, ###0002### having the molecular formula C 19 H 23 NO6, molecular weight [M+Na] + 384.1413, yellow powder, soluble in methanol.

3. The method of claim 1 or 2 for the preparation of natural active compounds pseudoginsenosides, characterized in that, The preparation method comprises the following steps: 1) inoculate Nocardiopsis sp ZHD001 into Gao's No. 1 solid culture medium for activation, inoculate single colony of the activated Nocardiopsis sp ZHD001 into Gao's No. 1 liquid culture medium, and shake culture to obtain a fermentation liquor; 2) extract the fermentation liquor with an equal volume of ethyl acetate, remove the ethyl acetate solvent from the obtained ethyl acetate extract by rotary evaporation under reduced pressure to obtain a concentrated liquor, separate the concentrated liquor by silica gel column chromatography, gradient elution, collect the eluate, detect each component by thin layer chromatography, and combine components containing Nocardiopsidin; 3) separate and purify the components containing Nocardiopsidin by a preparative high performance liquid chromatograph to obtain the compound Nocardiopsidin; The preservation number of the Nocardiopsis sp ZHD001 in step 1) is CCTCC NO: M2022921; The formula ratio of the Gao's No. 1 solid culture medium used in step 1) is as follows: soluble starch 20 g, potassium nitrate 1 g, dipotassium hydrogen phosphate 0.5 g, magnesium sulfate 0.5 g, ferrous sulfate 0.01 g, agar 20 g, sea salt 25 g, and water 1 L; Or the formula ratio of the Gao's No. 1 liquid culture medium used in step 1) is as follows: soluble starch 20 g, potassium nitrate 1 g, dipotassium hydrogen phosphate 0.5 g, magnesium sulfate 0.5 g, ferrous sulfate 0.01 g, sea salt 25 g, and water 1 L; The gradient elution in step 2) is gradient elution of a mixed solvent of dichloromethane and methanol with a ratio of 100:1, 80:1, 50:1, 30:1, 20:1, 10:1, 5:1, 1:1 and 0:1; The high performance liquid chromatograph in step 3) is an Agilent pursuit C18 chromatographic column 21.2*250 mm, 10 μm, the detection wavelength is 210 nm, the methanol-water system with a volume percentage of 45%-60% is used for gradient elution at 10 mL / min for 40 minutes, and the eluate collected at 12.7-14.5 minutes.

4. Use of the compound of formula (I) in claim 1 or 2 in the preparation of a drug for reducing blood lipid.

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