CN103304522B - Compound with neuroinflammation inhibition activity, as well as preparation method and application thereof - Google Patents
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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
Technical field
The present invention relates to a kind of compound with the neural inflammatory activity of inhibition and preparation method thereof and application.
Background technology
Along with the raising of people's living standard and the aggravation of aging population, with as Parkinson's disease (Parkinson ' sdisease, PD), alzheimer's disease (Alzheimer ' s disease, AD), multiple sclerosis (multiple sclerosis, MS) and Hang Ting Dun Shi chorea (Huntington ' s disease) for the acute and chronic cerebral nerve degenerative disease of representative, oneself becomes the main lethal disease being only second to after cardiovascular disorder, malignant tumour and apoplexy, cause serious burden to patient and society.Therefore, exploitation have treatment nerve degenerative diseases newtype drug there is very great meaning.
Clinical studyes and experimentation on animals evidence show in a large number, and the interior neural inflammation of brain and multiple acute and chronic nerve degenerative diseases are as the generation of Parkinson's disease, alzheimer's disease, multiple sclerosis and Hang Ting Dun Shi chorea etc. and develop closely related.The neuron degeneration of inflammatory reaction mediation is mainly then to cause inflammatory factor as NO by the activation of spongiocyte, iNOS, and IL-1 β, IL-6, TNF-α, the excessive release such as COX-2 is caused.Generally speaking, suppress the process that Neuroinflammation can weaken the neurone pathology in impaired brain district or postpone nerve degenerative diseases.Therefore, find and the interior neural inflammation inhibitor of research brain, the newtype drug for exploitation with the multiple acute and chronic nerve degenerative diseases for the treatment of has great importance.
Summary of the invention
The object of this invention is to provide a kind of compound with the neural inflammatory activity of inhibition and preparation method thereof.
Compound provided by the present invention, its structural formula is suc as formula shown in I:
(formula I)
Chemical name: 4-hydroxyl-2-methylene radical butyric acid (5-formylfuran-2-first) ester, it is brown oil, molecular formula C
11h
12o
5, molecular weight is 224.
Above-claimed cpd separates and obtains first from root of Denselflower Milkwort.
Root of Denselflower Milkwort Polygala tricornis Gagnep. is Polygalaceae rogation flower Polygala plant, has another name called chicken flower, spends more polygala root, and its medicinal part is root.Be loaded in " Chinese medicine resource will is wanted ", " Yunnan plant research ".This product bitter, pungent, warm, the effect of have the bushing of calming the nerves, kidney tonifying, relaxing the muscles and stimulate the blood circulation, in poor health, suffers from a deficiency of the kidney, wound etc.
Shown in preparation formula I of the present invention, the method for compound specifically comprises the steps:
1) after root of Denselflower Milkwort root is pulverized, the aqueous ethanolic solution that is 50～100% by volume fraction carries out refluxing extraction, collects extracting solution and reclaims ethanol, obtains medicinal extract;
2) described medicinal extract is passed through to macroporous adsorbent resin chromatography post, after washing, carry out gradient elution with the aqueous ethanolic solution of different concns, the ethanol eluate that collected volume mark is 20～60% is also concentrated, obtains enriched material; Enriched material, by silica gel chromatography, taking chloroform-methanol-water volume ratio as 7: (1-3): 0.1 mixing solutions carries out wash-out, is obtained to compound shown in formula I.
Wherein, step 1) described in refluxing extraction can be 1-5 time; When each extraction, the proportioning of the add-on of described aqueous ethanolic solution and root of Denselflower Milkwort root quality is (4～10) L: 1kg; Each time of extracting is 0.5～3 hour.
Actual conditions is as follows: step 1) described in refluxing extraction carry out 3 times, merge No. 3 times extracting solution; When each extraction, the proportioning of the add-on of described aqueous ethanolic solution and root of Denselflower Milkwort root quality is 7L: 1kg; Each time of extracting is 2 hours.
Step 2) in the resin that adopts refer to that the macroporous adsorbent resin of vinylbenzene crosslinking polymerization includes but not limited to D series, HPD series, HP series, XAD series and AB-8 macroporous adsorbent resin.
The preferred D101 type of macroporous adsorbent resin of described D series; The preferred HPD-500 type of macroporous adsorbent resin of HPD series; The preferred HP-20 type of macroporous adsorbent resin of HP series; The preferred XAD-4 of XAD series macroporous adsorbent resin.
When described medicinal extract passes through macroporous adsorbent resin chromatography post, the condition of concrete wash-out is as follows: use successively H
2o, volume fraction 10%, volume fraction 30% ethanolic soln wash-out, collect 30% ethanol eluate;
When described enriched material passes through silica gel chromatography, specifically carry out wash-out taking chloroform-methanol-water volume ratio as the mixing solutions of 7: 1: 0.1.
A further object of the present invention is to provide the application of formula I compound.
The application of formula I compound provided by the present invention comprises two aspects: be 1) its application in the product of the neural inflammation of preparation inhibition; 2) for it prevents and/or treats the application in the product of the acute and chronic nerve degenerative diseases being caused by neural inflammation in preparation.
Be in particular in that formula I compound is all inhibited to nitrogen protoxide (NO), interleukin-6 (IL-6), the release of tumor necrosis factor alpha (TNF-α) and the expression of cyclooxygenase-2 (COX-2) albumen in the microglia of bacteria lipopolysaccharide (LPS) induction.
The present invention also protects a kind of product that suppresses neural inflammation, and its effective constituent is the compound shown in formula I.
In addition, the product that prevents and/or treats the acute and chronic nerve degenerative diseases being caused by neural inflammation of preparing taking the compound shown in formula I as effective constituent, also belongs to protection scope of the present invention.
Described product can be medicine and/or healthcare products.
Described nerve degenerative diseases comprises: Parkinson's disease, alzheimer's disease, multiple sclerosis and Hang Ting Dun Shi chorea etc.
The medicine that suppresses the medicine of neural inflammation in the present invention and prevent and/or treat the acute and chronic nerve degenerative diseases being caused by neural inflammation all can import body as muscle, intracutaneous, subcutaneous, vein, mucosal tissue by the method for injection, injection, collunarium, eye drip, infiltration, absorption, physics or chemistry mediation; Or mixed by other materials or wrap up after import body.
When needs, in said medicine, can also add one or more pharmaceutically acceptable carriers.Described carrier comprises thinner, vehicle, weighting agent, tackiness agent, wetting agent, disintegrating agent, absorption enhancer, tensio-active agent, absorption carrier, lubricant of pharmaceutical field routine etc.
Said medicine can be made the various ways such as injection liquid, tablet, pulvis, granule, capsule, oral liquid, paste, creme.The medicine of various formulations all can be according to the ordinary method preparation of pharmaceutical field above.
The present inventor separates and obtains formula I compound 4-hydroxy base-2-methylene radical butyric acid (5-formylfuran-2-first) ester first from root of Denselflower Milkwort; and prove through pharmacological evaluation: this compound has the neural inflammatory activity of significant inhibition; can be used for the medicine that preparation prevents and/or treats neural inflammation and relative disease thereof, therefore it has higher clinical value and DEVELOPMENT PROSPECT.
Brief description of the drawings
Fig. 1 is the column diagram of NO concentration in the microglia of different treatment group in embodiment 5.
Fig. 2 is the expression of inducible nitric oxide synthase (iNOS) albumen in the microglia of different treatment group in embodiment 6.
Fig. 3 is the content of il-1 β in the microglia of different treatment group in embodiment 7.
Fig. 4 is the content of interleukin-6 in the microglia of different treatment group in embodiment 8.
Fig. 5 is the content of tumor necrosis factor alpha in the microglia of different treatment group in embodiment 9.
Fig. 6 is the expression of cyclooxygenase-2 in the microglia of different treatment group in embodiment 10.
Embodiment
Below by specific embodiment, the present invention will be described, but the present invention is not limited thereto.
Experimental technique described in following embodiment, if no special instructions, is ordinary method; Described reagent and biomaterial, if no special instructions, all can obtain from commercial channels.
The extraction and identification of embodiment 1,4-hydroxyl-2-methylene radical butyric acid (5-formylfuran-2-first) ester
By volume fraction 95%EtOH solution 21L refluxing extraction 2h for root of Denselflower Milkwort root (3.0kg) meal, extract altogether 3 times, filter, merge 3 times filtrate, decompression and solvent recovery, obtains solid extract 850g.Get the about 800g of medicinal extract and be suspended in 1000mL water, by macroporous adsorbent resin D101 chromatographic column.Use successively H
2o, volume fraction 10%, volume fraction 30%, volume fraction 50% and volume fraction 70% ethanolic soln wash-out, each gradient elution 20L (or 3 times of column volumes).Collect 30% ethanol eluate; evaporated under reduced pressure, by silica gel chromatography, with chloroform-methanol-water (7: 1: 0.1; v/v/v) wash-out, obtains 4-hydroxyl-2-methylene radical butyric acid (5-formylfuran-2-first) ester (16mg).
This compound is brown oil, and structural formula is suc as formula shown in I:
(formula I)
Structural Identification data are as follows:
Positive ion mode HR-ESIMS m/z 247.0573[M+Na]
+(theoretical value C
11h
12o
5na, 247.0577);
UV
(logε)265(3.85)，218(2.02)nm；
IR(film，KBr)v
max?3400，2937，1718，1674，1522，1191，1022cm
-1；
1h 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 ' is 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 ');
13c 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 ').
The extraction and identification of embodiment 2,4-hydroxyl-2-methylene radical butyric acid (5-formylfuran-2-first) ester
By volume fraction 50%EtOH solution 3L refluxing extraction 1.5h for root of Denselflower Milkwort root (500g) meal, extract altogether 2 times, filter, merge 3 times filtrate, decompression and solvent recovery, obtains solid extract 155g.Get the about 130g of medicinal extract and be suspended in 250mL water, by macroporous adsorbent resin HPD-500 chromatographic column.Use successively H
2o, volume fraction 10%, volume fraction 40% and volume fraction 80% ethanolic soln wash-out, each gradient elution 2.5L (or 3 times of column volumes).Collect 40% ethanol eluate; evaporated under reduced pressure, by silica gel chromatography, with chloroform-methanol-water (7: 1: 0.1; v/v/v) wash-out, obtains 4-hydroxyl-2-methylene radical butyric acid (5-formylfuran-2-first) ester (4.5mg).This compound is brown oil.Structural formula is suc as formula shown in I, and spectrum and spectral data are with embodiment 1.
The extraction of embodiment 3,4-hydroxyl-2-methylene radical butyric acid (5-formylfuran-2-first) ester
By volume fraction 70%EtOH solution 4L refluxing extraction 2h for root of Denselflower Milkwort root (500g) meal, extract altogether 3 times, filter, merge 3 times filtrate, decompression and solvent recovery, obtains solid extract 170g.Get the about 150g of medicinal extract and be suspended in 300mL water, by macroporous adsorbent resin HP-20 chromatographic column.Use successively H
2o, volume fraction 10%, volume fraction 60% and volume fraction 80% ethanolic soln wash-out, each gradient elution 4L (or 3 times of column volumes).Collect 60% ethanol eluate; evaporated under reduced pressure, by silica gel chromatography, with chloroform-methanol-water (7: 2: 0.1; v/v/v) wash-out, obtains 4-hydroxyl-2-methylene radical butyric acid (5-formylfuran-2-first) ester (6.3mg).This compound is brown oil.Structural formula is suc as formula shown in I, and spectrum and spectral data are with embodiment 1.
The extraction of embodiment 4,4-hydroxyl-2-methylene radical butyric acid (5-formylfuran-2-first) ester
By volume fraction 100%EtOH solution 5L refluxing extraction 3h for root of Denselflower Milkwort root (500g) meal, extract altogether 5 times, filter, merge 5 times filtrate, decompression and solvent recovery, obtains solid extract 185g.Get the about 150g of medicinal extract and be suspended in 300mL water, by macroporous adsorbent resin XAD-4 chromatographic column.Use successively H
2o, volume fraction 10%, volume fraction 60% and volume fraction 80% ethanolic soln wash-out, each gradient elution 4L (or 3 times of column volumes).Collect 60% ethanol eluate; evaporated under reduced pressure, by silica gel chromatography, with chloroform-methanol-water (7: 3: 0.1; v/v/v) wash-out, obtains 4-hydroxyl-2-methylene radical butyric acid (5-formylfuran-2-first) ester (7.2mg).This compound is brown oil.Structural formula is suc as formula shown in I, and spectrum and spectral data are with embodiment 1.
The restraining effect of embodiment 5,4-hydroxyl-2-methylene radical butyric acid (5-formylfuran-2-first) ester to nitrogen protoxide (NO) release in the microglia of bacteria lipopolysaccharide (LPS) induction
By BV-2 mouse microglia (5 × 10
4individual/hole) be inoculated in 24h in 48 orifice plates; add respectively different concns 4-hydroxyl-2-methylene radical butyric acid (5-formylfuran-2-first) ester (final concentration 0.1,1 and 10 μ M) and 1 μ g/mL LPS to process 24h to cell simultaneously; collecting cell supernatant; 8000r/min; 4 DEG C of centrifugal 10min; get supernatant, measure the content of NO according to Griess method, concrete measuring method carries out according to test kit specification sheets.Result shows (seeing Fig. 1), and in 1 μ g/mLLPS treatment group cell, the concentration of NO significantly increases.Compared with LPS treatment group; in 4-hydroxyl-2-methylene radical butyric acid (5-formylfuran-2-first) ester (final concentration is 1 and 10 μ M) treatment group cell of different concns, nitric oxide production concentration significantly reduces (P < 0.05), illustrates that this compound has obvious anti-inflammatory activity.
The expression of inducible nitric oxide synthase (iNOS) albumen in the microglia of embodiment 6,4-hydroxyl-2-methylene radical butyric acid (5-formylfuran-2-first) ester anti-bacteria lipopolysaccharides (LPS) induction
By BV-2 mouse microglia (3.0 × 10
5individual/hole) be inoculated in 24h in 6 orifice plates, add respectively different concns 4-hydroxyl-2-methylene radical butyric acid (5-formylfuran-2-first) ester (final concentration 0.1,1 and 10 μ M) and 1 μ g/mLLPS to process 24h to cell simultaneously.Add RIPA lysate, the centrifugal 30min of 12000g collects supernatant liquor.Detect the expression amount of inducible nitric oxide synthase (iNOS) albumen in cell by detected by Western blot (Western blotting).The protein band of immunoblotting carries out half-quantitative detection with gray scale scanning instrument.Result shows (seeing Fig. 2), and in the BV-2 cell that 1 μ g/mLLPS processes, the expression level of iNOS albumen obviously rises.Compared with LPS treatment group; in different concns 4-hydroxyl-2-methylene radical butyric acid (5-formylfuran-2-first) ester (final concentration is 1 and 10 μ M) treatment group cell, the expression amount of iNOS albumen all declines (P < 0.05) to some extent, illustrates that this compound has obvious anti-inflammatory activity.
The release of il-1 β (IL-1 β) in the microglia of embodiment 7,4-hydroxyl-2-methylene radical butyric acid (5-formylfuran-2-first) ester anti-bacteria lipopolysaccharides (LPS) induction
By BV-2 mouse microglia (2.0 × 10
5individual/hole) be inoculated in 24 orifice plates, add respectively different concns 4-hydroxyl-2-methylene radical butyric acid (5-formylfuran-2-first) ester (final concentration 0.1,1 and 10 μ M) and 1 μ g/mL LPS to process 24h to cell simultaneously.Collecting cell supernatant, with the content of ELISA method mensuration il-1 β (IL-1 β).Result shows (seeing Fig. 3), and the IL-1 β burst size of 1 μ g/mL LPS treatment group cell significantly increases.Compared with LPS treatment group; the IL-1 β burst size of different concns 4-hydroxyl-2-methylene radical butyric acid (5-formylfuran-2-first) ester (final concentration is 1 μ M and 10 μ M) treatment group cell is all decreased significantly (P < 0.05), illustrates that this compound has obvious anti-inflammatory activity.
The restraining effect of embodiment 8,4-hydroxyl-2-methylene radical butyric acid (5-formylfuran-2-first) ester to interleukin-6 (IL-6) release in the microglia of bacteria lipopolysaccharide (LPS) induction
By BV-2 mouse microglia (2.0 × 10
5individual/hole) be inoculated in 24 orifice plates, add respectively different concns 4-hydroxyl-2-methylene radical butyric acid (5-formylfuran-2-first) ester (final concentration 0.1,1 and 10 μ M) and 1 μ g/mL LPS to process 24h to cell simultaneously.Collecting cell supernatant, with the content of ELISA method mensuration interleukin-6 (IL-6).Result shows (seeing Fig. 4), and the IL-6 burst size of 1 μ g/mL LPS treatment group cell significantly increases.Compared with LPS treatment group; the IL-6 burst size of different concns 4-hydroxyl-2-methylene radical butyric acid (5-formylfuran-2-first) ester (final concentration is 1 μ M and 10 μ M) treatment group cell is all decreased significantly (P < 0.05), illustrates that this compound has obvious anti-inflammatory activity.
The release of tumor necrosis factor alpha (TNF-α) in the microglia of embodiment 9,4-hydroxyl-2-methylene radical butyric acid (5-formylfuran-2-first) ester anti-bacteria lipopolysaccharides (LPS) induction
By BV-2 mouse microglia (2.0 × 10
5individual/hole) be inoculated in 24 orifice plates, add respectively different concns 4-hydroxyl-2-methylene radical butyric acid (5-formylfuran-2-first) ester (final concentration 0.1,1 and 10 μ M) and 1 μ g/mL LPS to process 24h to cell simultaneously.Collecting cell supernatant, with the content of ELISA method mensuration tumor necrosis factor alpha (TNF-α).Result shows (seeing Fig. 5), and the TNF-α burst size of 1 μ g/mL LPS treatment group cell obviously increases.Compared with LPS treatment group; the TNF-α burst size of different concns 4-hydroxyl-2-methylene radical butyric acid (5-formylfuran-2-first) ester (final concentration is 1 μ M and 10 μ M) treatment group cell all declines (P < 0.05) to some extent, illustrates that this compound has obvious anti-inflammatory activity.
The expression of cyclooxygenase-2 (COX-2) albumen in the microglia of embodiment 10,4-hydroxyl-2-methylene radical butyric acid (5-formylfuran-2-first) ester anti-bacteria lipopolysaccharides (LPS) induction
By BV-2 mouse microglia (3.0 × 10
5individual/hole) be inoculated in 24h in 6 orifice plates, add respectively different concns 4-hydroxyl-2-methylene radical butyric acid (5-formylfuran-2-first) ester (final concentration 0.1,1 and 10 μ M) and 1 μ g/mLLPS to process 24h to cell simultaneously.Add RIPA lysate, the centrifugal 30min of 12000g collects supernatant liquor.Detect the expression level of cyclooxygenase-2 (COX-2) albumen in cell by detected by Western blot (Western blotting).The protein band of immunoblotting carries out half-quantitative detection with gray scale scanning instrument.Result shows (seeing Fig. 6), and in the BV-2 cell that 1 μ g/mL LPS processes, the expression of COX-2 albumen is obviously risen.Compared with LPS treatment group; in different concns 4-hydroxyl-2-methylene radical butyric acid (5-formylfuran-2-first) ester (final concentration is 1 and 10 μ M) treatment group cell, the expression amount of COX-2 albumen all declines (P < 0.05) to some extent, illustrates that this compound has obvious anti-inflammatory activity.
Claims (10)
1. the compound shown in formula I:
2. the method for preparing compound shown in claim 1 Chinese style I, comprises the steps:
1) after root of Denselflower Milkwort root is pulverized, the aqueous ethanolic solution that is 50～100% by volume fraction carries out refluxing extraction, collects extracting solution concentrated, obtains medicinal extract;
2) described medicinal extract is passed through to macroporous adsorbent resin chromatography post, after washing, carry out gradient elution with the aqueous ethanolic solution of different concns, collected volume concentration is 20～60% ethanol eluates concentrated, obtains enriched material; Enriched material is passed through to silica gel chromatography, taking chloroform-methanol-water volume ratio as 7:(1-3): 0.1 mixing solutions carries out wash-out, obtains compound shown in formula I.
3. method according to claim 2, is characterized in that: step 1) described in refluxing extraction carry out 1-5 time; When each extraction, the proportioning of the add-on of described aqueous ethanolic solution and root of Denselflower Milkwort root quality is (4～10) L:1kg; Each time of extracting is 0.5～3 hour.
4. method according to claim 3, is characterized in that: step 1) described in refluxing extraction carry out 3 times, merge No. 3 times extracting solution; When each extraction, the proportioning of the add-on of described aqueous ethanolic solution and root of Denselflower Milkwort root quality is 7L:1kg; Each time of extracting is 2 hours.
5. according to the method described in any one in claim 2-4, it is characterized in that: step 2) described in medicinal extract when the macroporous adsorbent resin chromatography post, the condition of wash-out is as follows: use successively H
2o, volume fraction 10%, volume fraction 30% ethanolic soln wash-out, collect 30% ethanol eluate;
When described enriched material passes through silica gel chromatography, the mixing solutions taking chloroform-methanol-water volume ratio as 7:1:0.1 carries out wash-out.
6. the application of compound shown in claim 1 Chinese style I in the following product of preparation: the product that 1) suppresses neural inflammation; 2) prevent and/or treat the product of the acute and chronic nerve degenerative diseases being caused by neural inflammation.
7. application according to claim 6, is characterized in that: described product is medicine and/or healthcare products; Described nerve degenerative diseases comprises: Parkinson's disease, alzheimer's disease, multiple sclerosis and Hang Ting Dun Shi chorea.
8. a product, its activeconstituents is compound shown in claim 1 Chinese style I; Described product is following 1) or 2): the product that 1) suppresses neural inflammation; 2) prevent and/or treat the product of the acute and chronic nerve degenerative diseases being caused by neural inflammation.
9. product according to claim 8, is characterized in that: described product is medicine and/or healthcare products.
10. product according to claim 8 or claim 9, is characterized in that: described nerve degenerative diseases comprises: Parkinson's disease, alzheimer's disease, multiple sclerosis and Hang Ting Dun Shi chorea.
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