CN103304522A - Compound with neuroinflammation inhibition activity, as well as preparation method and application thereof - Google Patents

Abstract

The invention discloses a compound with neuroinflammation inhibition activity, as well as a preparation method and an application thereof. The compound provided by the invention is as shown in the structural formula I and is separated from polygala tricornis cagnep roots for the first time. Pharmacology experiments prove that the compound has obvious neuroinflammation inhibition activity, can be used for preparing pharmaceuticals for preventing and/or treating neuroinflammation and related diseases and thus has a high clinical application value and a development prospect. Formula I is shown in the specification.

Description

A compound with neuroinflammation inhibitory activity and its preparation method and application

technical field

The invention relates to a compound with activity of inhibiting neuroinflammation and its preparation method and application.

Background technique

With the improvement of people's living standards and the aggravation of population aging, diseases such as Parkinson's disease (PD), Alzheimer's disease (Alzheimer's disease, AD), multiple sclerosis (multiple sclerosis, MS) and Hang Acute and chronic brain neurodegenerative diseases represented by Huntington's disease have become the leading cause of death after cardiovascular diseases, malignant tumors and strokes, causing serious burdens to patients and society. Therefore, it is of great significance to develop new drugs for the treatment of neurodegenerative diseases.

A large number of clinical studies and animal experiments have shown that neuroinflammation in the brain is closely related to the occurrence and development of various acute and chronic neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, multiple sclerosis and Huntington's disease . Inflammatory response-mediated neuronal degeneration is mainly caused by the activation of glial cells and the excessive release of inflammatory factors such as NO, iNOS, IL-1β, IL-6, TNF-α, and COX-2. In general, inhibiting neuroinflammation can attenuate neuronal pathology in damaged brain regions or delay the progression of neurodegenerative diseases. Therefore, finding and studying inhibitors of neuroinflammation in the brain is of great significance for the development of new drugs for the treatment of various acute and chronic neurodegenerative diseases.

Contents of the invention

The object of the present invention is to provide a compound with activity of inhibiting neuroinflammation and its preparation method.

The compound provided by the present invention has a structural formula as shown in formula I:

(Formula I)

Chemical name: 4-hydroxy-2-methylenebutanoic acid (5-formylfuran-2-methyl) ester, which is a brown oil with a molecular formula of C ₁₁ H ₁₂ O ₅ and a molecular weight of 224.

The above compounds were isolated from Polygala densiflora for the first time.

Polygala tricornis Gagnep. is a Polygala plant of the genus Polygala of the family Polygala, also known as chicken flower, Polygala multiflora, and its medicinal part is the root. Contained in "China Traditional Chinese Medicine Resources Chronicle", "Yunnan Botanical Research". The taste is bitter, pungent and warm. It has the effects of calming the nerves, invigorating the heart, nourishing the kidney, relaxing tendons and activating blood circulation. It is used for infirmity, kidney deficiency, bruises, etc.

The method for the compound shown in the present invention to prepare formula I specifically comprises the following steps:

1) after pulverizing Polygala densiflora root, reflux extraction with ethanol aqueous solution with a volume fraction of 50-100%, collecting the extract and recovering ethanol to obtain extract;

2) passing the extract through a macroporous adsorption resin chromatographic column, washing with water and carrying out gradient elution with ethanol aqueous solutions of different concentrations, collecting ethanol eluents with a volume fraction of 20-60% and concentrating to obtain concentrates; The compound was purified by silica gel column chromatography, and eluted with a mixed solution with a volume ratio of chloroform-methanol-water of 7:(1-3):0.1 to obtain the compound shown in formula I.

Wherein, the reflux extraction described in step 1) can be 1-5 times; during each extraction, the ratio of the addition of the aqueous ethanol solution to the root mass of Polygala densiflora is (4～10)L: 1kg; The extraction time is 0.5 to 3 hours.

The specific conditions are as follows: the reflux extraction described in step 1) is carried out 3 times, and the extracts are combined for 3 times; during each extraction, the ratio of the addition of the aqueous ethanol solution to the root mass of Polygala densiflora is 7L: 1kg; The extraction time was 2 hours.

The resin used in step 2) refers to macroporous adsorption resins cross-linked and polymerized with styrene, including but not limited to D series, HPD series, HP series, XAD series and AB-8 macroporous adsorption resins.

The D series macroporous resin is preferably D101; the HPD series macroporous resin is preferably HPD-500; the HP series macroporous resin is preferably HP-20; the XAD series macroporous resin is preferably XAD-4.

When the extract passes through the macroporous adsorption resin chromatographic column, the specific elution conditions are as follows: sequentially elute with H ₂ O, 10% volume fraction, and 30% volume fraction ethanol solution, and collect the 30% ethanol eluate;

When the concentrate is purified by silica gel column chromatography, it is specifically eluted with a mixed solution of chloroform-methanol-water with a volume ratio of 7:1:0.1.

Another object of the present invention is to provide the application of the compound of formula I.

The application of the compound of formula I provided by the present invention includes two aspects: 1) its application in the preparation of products for inhibiting neuroinflammation; Disease application in the product.

It is specifically manifested in the release of nitric oxide (NO), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α) and epoxy The expression of Cox-2 (COX-2) protein can be inhibited.

The invention also protects a product for inhibiting neuroinflammation, the active ingredient of which is the compound represented by formula I.

In addition, the products for the prevention and/or treatment of acute and chronic neurodegenerative diseases caused by neuroinflammation prepared with the compound represented by formula I as active ingredients also belong to the protection scope of the present invention.

The product can be medicine and/or health product.

The neurodegenerative diseases include: Parkinson's disease, Alzheimer's disease, multiple sclerosis and Huntington's disease, etc.

In the present invention, the drugs for inhibiting neuroinflammation and the drugs for preventing and/or treating acute and chronic neurodegenerative diseases caused by neuroinflammation can all be mediated by injection, spray, nasal drop, eye drop, penetration, absorption, physical or chemical Methods Introduce into the body such as muscle, intradermal, subcutaneous, vein, mucosal tissue; or introduce into the body after being mixed or wrapped with other substances.

When necessary, one or more pharmaceutically acceptable carriers can also be added to the above drugs. The carrier includes conventional diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants and the like in the pharmaceutical field.

The above-mentioned medicines can be made into various forms such as injections, tablets, powders, granules, capsules, oral liquids, ointments, and creams. The medicines in the above various dosage forms can be prepared according to conventional methods in the field of pharmacy.

The inventor of the present invention isolates and obtains formula I compound 4-hydroxyl-2-methylene butyrate (5-formylfuran-2-methyl) ester from Polygala densiflora for the first time, and proves through pharmacological experiment: this compound has significant The inhibitory activity of neuroinflammation can be used to prepare medicines for preventing and/or treating neuroinflammation and related diseases, so it has high clinical application value and development prospect.

Description of drawings

FIG. 1 is a histogram of NO concentration in microglia of different treatment groups in Example 5. FIG.

FIG. 2 shows the expression of inducible nitric oxide synthase (iNOS) protein in microglia of different treatment groups in Example 6. FIG.

FIG. 3 is the content of interleukin-1β in microglia of different treatment groups in Example 7. FIG.

FIG. 4 shows the content of interleukin-6 in microglia of different treatment groups in Example 8. FIG.

FIG. 5 shows the content of tumor necrosis factor α in microglial cells of different treatment groups in Example 9. FIG.

FIG. 6 shows the expression of cyclooxygenase-2 protein in microglial cells of different treatment groups in Example 10. FIG.

Detailed ways

The present invention will be described below through specific examples, but the present invention is not limited thereto.

The experimental methods described in the following examples, unless otherwise specified, are conventional methods; the reagents and biological materials, unless otherwise specified, can be obtained from commercial sources.

Example 1, Extraction and Identification of 4-Hydroxy-2-Methylene Butyrate (5-Formylfuran-2-M) Ester

The coarse powder of Polygala densiflora (3.0kg) was reflux extracted with 21L of 95% EtOH solution for 2 hours, extracted 3 times in total, filtered, combined the filtrates from 3 times, recovered the solvent under reduced pressure, and obtained 850g of solid extract. Take about 800g of the extract and suspend it in 1000mL of water, and pass it through the macroporous adsorption resin D101 chromatographic column. Use H ₂ O, 10% volume fraction, 30% volume fraction, 50% volume fraction and 70% volume fraction ethanol solution to elute sequentially, each gradient elution is 20L (or 3 times column volume). Collect the 30% ethanol eluate, evaporate to dryness under reduced pressure, purify by silica gel column chromatography, and elute with chloroform-methanol-water (7:1:0.1, v/v/v) to obtain 4-hydroxy-2-methylene (5-formylfuran-2-methyl)ylbutanoate (16 mg).

This compound is brown oil, and structural formula is as shown in formula I:

(Formula I)

The structural identification data are as follows:

Positive ion mode HR-ESIMS m/z 247.0573[M+Na] ⁺ (theoretical value C ₁₁ H ₁₂ O ₅ Na, 247.0577);

(logε)265(3.85)，218(2.02)nm；UV

(logε)265(3.85), 218(2.02)nm;

IR (film, KBr) v _max 3400, 2937, 1718, 1674, 1522, 1191, 1022cm ^-1 ;

¹ H NMR (500MHz, deuterated acetone) δ: 9.64 (1H, s, H-7), 7.41 (1H, d, J=3.5Hz, H-4), 6.77 (1H, d, J=3.5Hz, H-3), 6.19 (1H, d, J=1.5Hz, H-5′a), 5.74 (1H, d, J=1.5Hz, H-5′b), 5.26 (2H, s, H-6 ), 3.65(2H, t, J=6.5Hz, H-4'), 2.51(2H, t, J=6.5Hz, H-3');

¹³ C NMR (125MHz, deuterated acetone) δ: 178.5(C-7), 166.8(C-1'), 156.5(C-2), 154.0(C-5), 138.3(C-2'), 127.7 (C-5'), 123.2 (C-4), 113.3 (C-3), 61.1 (C-4'), 58.7 (C-6), 36.1 (C-3').

Example 2, Extraction and Identification of 4-Hydroxy-2-Methylene Butyrate (5-Formylfuran-2-M) Ester

The coarse powder of Polygala densiflora root (500g) was reflux extracted with 50% EtOH solution 3L for 1.5h, extracted 2 times in total, filtered, combined the filtrates 3 times, and recovered the solvent under reduced pressure to obtain 155g of solid extract. Take about 130g of the extract and suspend in 250mL of water, and pass through the macroporous adsorption resin HPD-500 chromatographic column. Elute with H ₂ O, 10% volume fraction, 40% volume fraction and 80% volume fraction ethanol solution in sequence, each gradient elution is 2.5 L (or 3 times column volume). Collect 40% ethanol eluate, evaporate to dryness under reduced pressure, purify by silica gel column chromatography, and elute with chloroform-methanol-water (7:1:0.1, v/v/v) to obtain 4-hydroxy-2-methylene (5-formylfuran-2-methyl)ylbutanoate (4.5 mg). The compound is a brown oil. The structural formula is shown in formula I, and the spectrum and spectral data are the same as in Example 1.

The extraction of embodiment 3,4-hydroxyl-2-methylene butyric acid (5-formylfuran-2-methyl) ester

The coarse powder of Polygala densiflora (500g) was reflux extracted with 70% EtOH solution 4L for 2h, extracted 3 times in total, filtered, combined the filtrates from 3 times, recovered the solvent under reduced pressure, and obtained 170g of solid extract. Take about 150g of the extract and suspend in 300mL of water, and pass through the macroporous adsorption resin HP-20 chromatographic column. Elute with H ₂ O, 10% volume fraction, 60% volume fraction and 80% volume fraction ethanol solution successively, and each gradient elution is 4L (or 3 times column volume). Collect the 60% ethanol eluate, evaporate to dryness under reduced pressure, purify by silica gel column chromatography, and elute with chloroform-methanol-water (7:2:0.1, v/v/v) to obtain 4-hydroxy-2-methylene (5-formylfuran-2-methyl)ylbutanoate (6.3 mg). The compound is a brown oil. The structural formula is shown in formula I, and the spectrum and spectral data are the same as in Example 1.

The extraction of embodiment 4,4-hydroxyl-2-methylene butyric acid (5-formylfuran-2-methyl) ester

The coarse powder of Polygala densiflora (500g) was reflux extracted with 100% EtOH solution 5L for 3h, extracted 5 times in total, filtered, combined the filtrates 5 times, recovered the solvent under reduced pressure, and obtained 185g of solid extract. Take about 150g of the extract and suspend in 300mL of water, and pass through the macroporous adsorption resin XAD-4 chromatographic column. Elute with H ₂ O, 10% volume fraction, 60% volume fraction and 80% volume fraction ethanol solution successively, and each gradient elution is 4L (or 3 times column volume). Collect the 60% ethanol eluate, evaporate to dryness under reduced pressure, purify by silica gel column chromatography, and elute with chloroform-methanol-water (7:3:0.1, v/v/v) to obtain 4-hydroxy-2-methylene (5-formylfuran-2-methyl)ylbutanoate (7.2 mg). The compound is a brown oil. The structural formula is shown in formula I, and the spectrum and spectral data are the same as in Example 1.

Example 5. Inhibition of 4-hydroxy-2-methylenebutyrate (5-formylfuran-2-methyl) ester on the release of nitric oxide (NO) in microglial cells induced by bacterial lipopolysaccharide (LPS) effect

BV-2 mouse microglial cells (5×10 ⁴ cells/well) were seeded in 48-well plates for 24 hours, and different concentrations of 4-hydroxy-2-methylenebutyric acid (5-formylfuran- The cells were treated with 2-methyl) ester (0.1, 1, and 10 μM final concentration) and 1 μg/mL LPS for 24 hours, the cell supernatant was collected, centrifuged at 8000 r/min, 4 °C for 10 minutes, the supernatant was taken, and the NO content was determined according to the Griess method , and the specific determination method was carried out according to the instructions of the kit. The results showed (see Figure 1), the concentration of NO in the cells of the 1 μg/mL LPS treatment group increased significantly. Nitric oxide concentrations in cells treated with different concentrations of 4-hydroxy-2-methylenebutyric acid (5-formylfuran-2-methyl) ester (final concentrations of 1 and 10 μM) compared with LPS treated group Significantly decreased (P<0.05), indicating that this compound has obvious anti-inflammatory activity.

Example 6, 4-hydroxy-2-methylenebutyrate (5-formylfuran-2-methyl) ester inhibits inducible nitric oxide synthase (iNOS) in microglial cells induced by bacterial lipopolysaccharide (LPS) ) protein expression

BV-2 mouse microglial cells (3.0× ¹⁰⁵ cells/well) were seeded in 6-well plates for 24 hours, and different concentrations of 4-hydroxy-2-methylenebutyric acid (5-formylfuran- The cells were treated with 2-methyl) ester (0.1, 1 and 10 μM final concentration) and 1 μg/mL LPS for 24 h. Add RIPA lysate and centrifuge at 12000g for 30min to collect the supernatant. Western blotting was used to detect the expression of inducible nitric oxide synthase (iNOS) protein in the cells. Western blot protein bands were semi-quantitatively detected with a grayscale scanner. The results showed (see FIG. 2 ), the expression level of iNOS protein in BV-2 cells treated with 1 μg/mL LPS was significantly increased. Compared with the LPS treatment group, the expression of iNOS protein in the cells of different concentrations of 4-hydroxy-2-methylenebutyric acid (5-formylfuran-2-methyl) ester (final concentration of 1 and 10 μM) treatment decreased (P<0.05), indicating that this compound has obvious anti-inflammatory activity.

Example 7, 4-hydroxy-2-methylenebutyrate (5-formylfuran-2-methyl) ester inhibits the production of interleukin-1β (IL-1β) in microglial cells induced by bacterial lipopolysaccharide (LPS) freed

BV-2 mouse microglial cells (2.0× ¹⁰⁵ cells/well) were seeded in 24-well plates, and different concentrations of 4-hydroxy-2-methylenebutyric acid (5-formylfuran-2 Cells were treated with -methyl) ester (0.1, 1 and 10 μM final concentration) and 1 μg/mL LPS for 24 h. The cell supernatant was collected, and the content of interleukin-1β (IL-1β) was determined by ELISA method. The results showed (see FIG. 3 ), the release of IL-1β from cells in the 1 μg/mL LPS treatment group was significantly increased. Compared with the LPS treatment group, the release of IL-1β from the cells treated with different concentrations of 4-hydroxy-2-methylenebutyrate (5-formylfuran-2-methyl) ester (final concentration 1 μM and 10 μM) was similar. There is a significant decrease (P<0.05), indicating that this compound has obvious anti-inflammatory activity.

Example 8, 4-hydroxy-2-methylenebutanoic acid (5-formylfuran-2-methyl) ester on the release of interleukin-6 (IL-6) in microglial cells induced by bacterial lipopolysaccharide (LPS) inhibition of

BV-2 mouse microglial cells (2.0× ¹⁰⁵ cells/well) were seeded in 24-well plates, and different concentrations of 4-hydroxy-2-methylenebutyric acid (5-formylfuran-2 Cells were treated with -methyl) ester (0.1, 1 and 10 μM final concentration) and 1 μg/mL LPS for 24 h. The cell supernatant was collected, and the content of interleukin-6 (IL-6) was determined by ELISA. The results showed (see FIG. 4 ), the IL-6 release of cells in the 1 μg/mL LPS treatment group was significantly increased. Compared with the LPS treatment group, the release of IL-6 from the cells treated with different concentrations of 4-hydroxy-2-methylenebutyrate (5-formylfuran-2-methyl) ester (final concentration 1 μM and 10 μM) was similar. There is a significant decrease (P<0.05), indicating that this compound has obvious anti-inflammatory activity.

Example 9, 4-hydroxy-2-methylenebutyrate (5-formylfuran-2-methyl) ester inhibits tumor necrosis factor α (TNF-α) in microglial cells induced by bacterial lipopolysaccharide (LPS) release

BV-2 mouse microglial cells (2.0× ¹⁰⁵ cells/well) were seeded in 24-well plates, and different concentrations of 4-hydroxy-2-methylenebutyric acid (5-formylfuran-2 Cells were treated with -methyl) ester (0.1, 1 and 10 μM final concentration) and 1 μg/mL LPS for 24 h. The cell supernatant was collected, and the content of tumor necrosis factor α (TNF-α) was determined by ELISA. The results showed (see FIG. 5 ), the release of TNF-α from cells in the 1 μg/mL LPS treatment group was significantly increased. Compared with the LPS treatment group, the TNF-α release amount of the cells treated with different concentrations of 4-hydroxy-2-methylenebutyric acid (5-formylfuran-2-methyl) ester (final concentration of 1 μM and 10 μM) was the same. decreased (P<0.05), indicating that this compound has obvious anti-inflammatory activity.

Example 10, 4-hydroxy-2-methylenebutyrate (5-formylfuran-2-methyl) ester inhibits cyclooxygenase-2 (COX-2) in microglial cells induced by bacterial lipopolysaccharide (LPS) protein expression

BV-2 mouse microglial cells (3.0× ¹⁰⁵ cells/well) were seeded in 6-well plates for 24 hours, and different concentrations of 4-hydroxy-2-methylenebutyric acid (5-formylfuran- The cells were treated with 2-methyl) ester (0.1, 1 and 10 μM final concentration) and 1 μg/mL LPS for 24 h. Add RIPA lysate and centrifuge at 12000g for 30min to collect the supernatant. Western blotting was used to detect the expression level of cyclooxygenase-2 (COX-2) protein in the cells. Western blot protein bands were semi-quantitatively detected with a grayscale scanner. The results showed (see FIG. 6 ), the expression of COX-2 protein in BV-2 cells treated with 1 μg/mL LPS was significantly increased. COX-2 protein expression in cells treated with different concentrations of 4-hydroxy-2-methylenebutyric acid (5-formylfuran-2-methyl) ester (final concentration 1 and 10 μM) compared with LPS treated group The amount decreased (P<0.05), indicating that this compound has obvious anti-inflammatory activity.

Claims (10)

1. The compound shown in formula I:

(Formula I). 2. the method for compound shown in formula I among the preparation claim 1, comprises the following steps: 1) After pulverizing the root of Polygala densiflora, reflux extraction with ethanol aqueous solution with a volume fraction of 50-100%, collecting and concentrating the extract to obtain an extract; 2) passing the extract through a macroporous adsorption resin chromatographic column, washing with water, and carrying out gradient elution with ethanol aqueous solutions of different concentrations, collecting and concentrating the ethanol eluent with a volume concentration of 20-60% to obtain a concentrate; Purified by silica gel column chromatography, eluting with a mixed solution with a volume ratio of chloroform-methanol-water of 7:(1-3):0.1 to obtain the compound represented by formula I. 3. method according to claim 2, is characterized in that: step 1) described in reflux extraction carries out 1-5 time; When extracting at every turn, the proportioning of the add-on of described ethanol aqueous solution and Polygala densiflora root quality It is (4~10)L: 1kg; the time for each extraction is 0.5~3 hours. 4. method according to claim 3, it is characterized in that: step 1) described in reflux extraction carries out 3 times, merges 3 times extracting liquid; The mass ratio is 7L:1kg; the time for each extraction is 2 hours. 5. The method according to any one of claims 2-4, characterized in that: when the medicinal extract in step 2) passes through the macroporous adsorption resin chromatographic column, the conditions for elution are as follows: successively use H ₂ O, 10% volume fraction, 30% volume fraction ethanol solution elution, collect 30% ethanol eluate; When the concentrate was purified by silica gel column chromatography, it was eluted with a mixed solution of chloroform-methanol-water with a volume ratio of 7:1:0.1. 6. The application of the compound shown in formula I in claim 1 in the preparation of the following products: 1) products for inhibiting neuroinflammation; 2) products for preventing and/or treating acute and chronic neurodegenerative diseases caused by neuroinflammation. 7. The application according to claim 6, characterized in that: the product is a medicine and/or health product; the neurodegenerative disease includes: Parkinson's disease, Alzheimer's disease, multiple sclerosis and Huntington's disease Dun's chorea. 8. A product whose active ingredient is a compound shown in formula I in claim 1; said product is the following 1) or 2): 1) a product that inhibits neuroinflammation; 2) prevention and/or treatment of neuroinflammation caused by products of acute and chronic neurodegenerative diseases. 9. The application according to claim 8, characterized in that: the product is medicine and/or health product. 10. The application according to claim 8 or 9, characterized in that: the neurodegenerative diseases include: Parkinson's disease, Alzheimer's disease, multiple sclerosis and Huntington's disease.

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