A compound having a neuroinflammation inhibitory activity and an extraction method thereof

Abstract

The present application relates to the field of medicine, and particularly relates to a compound with neuroinflammation inhibiting activity and an extraction method thereof. A novel structure of withaferin compound is extracted and separated from Datura leaf, and a structural formula is as shown in formula I. Activity research shows that the compound has an effect of inhibiting release of inflammatory factor NO of nerve cells, has excellent inhibiting activity on LPS-induced inflammatory BV2 microgliacytes, and can be used for prevention or treatment of neurodegenerative diseases such as Alzheimer's disease, Parkinson and the like.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, specifically to a compound with neuroinflammatory inhibitory activity and its extraction method, and more specifically to a compound with neuroinflammatory inhibitory activity extracted and isolated from Datura leaves. Background Technology

[0002] Datura leaves, the dried leaves of the plant *Datura stramonium* L. (family Solanaceae), possess antitussive and analgesic properties and are commonly used to treat rheumatism and rheumatoid arthritis, asthma, gastrointestinal spasms, and migraines. They contain a large amount of solanolactone compounds. However, current research on datura leaves is not systematic or in-depth. Chemically, aside from alkaloids, only a small number of solanolactone compounds and other compounds have been reported. Therefore, further extraction, separation, and identification of the active ingredients in datura leaves are urgently needed to conduct more in-depth research on their medicinal activity and provide more bioactive medicinal components. Summary of the Invention

[0003] In one aspect, this invention provides compounds of Formula I or pharmaceutically acceptable salts thereof:

[0004]

[0005] The compound shown in Formula I above was prepared by a method comprising the following steps:

[0006] 1) Extract the leaves of Datura stramonium by cold soaking in methanol, and recover the solvent to obtain the extract;

[0007] 2) After dissolving the extract in water, the extract was separated by column chromatography using macroporous adsorption resin, silica gel, ODS, gel chromatography, and preparative high performance liquid chromatography to obtain the solanacolin compounds.

[0008] In step 1) of the above method, the weight ratio of datura leaves to methanol is 1:1-12, preferably 1:8-12, and more preferably 1:10;

[0009] The cold maceration extraction can be performed multiple times, such as 2-6 times, preferably 2-4 times, and more preferably 3 times;

[0010] The extraction time is 1-7 days / time, preferably 2-4 days / time, and more preferably 3 days / time;

[0011] In step 2), the macroporous adsorption resin is selected from HPD-BJQH, HP-20, and AB-8, with HPD-BJQH being preferred;

[0012] Step 2) includes the following operations:

[0013] a) The dissolved extract was added to the macroporous adsorption resin and eluted sequentially with water, 30% ethanol (volume concentration), and 95% ethanol (volume concentration). The eluents were collected and concentrated under reduced pressure to obtain the water-eluted fraction, the 30% ethanol-eluted fraction, and the 95% ethanol-eluted fraction.

[0014] b) The 95% ethanol eluent was separated by silica gel column chromatography with gradient elution using dichloromethane-methanol as the mobile phase (1:0-0:1, v / v) to obtain 8 fractions, Fr.A-Fr.H;

[0015] c) The Fr.G fraction was separated by ODS column chromatography with methanol-water gradient elution (30:70-80:20, 1:0, v / v) to obtain 14 fractions, Fr.G1-Fr.G14;

[0016] d) The Fr.G8 fraction was separated by vacuum silica gel column chromatography, eluted with dichloromethane-methanol, to obtain 5 fractions, Fr.G8-1 to Fr.G8-5;

[0017] e) The Fr.G8-3 fraction was eluted by preparative HPLC with methanol-water as the eluent to obtain the compound shown in Formula I.

[0018] In part a), the flow rate of the eluent is 1 BV·h. -1 ;

[0019] The dosage of water is 2 BV, the dosage of 30% ethanol is 2 BV, and the dosage of 95% ethanol is 4 BV.

[0020] In b), the elution gradient of the mobile phase dichloromethane-methanol (1:0-0:1) is as follows: mixed solutions with dichloromethane to methanol volume ratios of 1:0, 50:1, 20:1, 10:1, 5:1, and 0:1 elute 3, 5, 4, 3, 4, and 2 times the column volume, respectively.

[0021] In c), the elution gradient of the mobile phase methanol-water (30:70-80:20, 1:0) is as follows: mixed solutions with methanol to water volume ratios of 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, and 1:0 elute 2, 4, 5, 3, 3, 2, and 2 times the column volume, respectively.

[0022] In d), the elution gradient of the mobile phase dichloromethane-methanol (20:1-0:1) is as follows: mixed solutions with dichloromethane to methanol volume ratios of 20:1, 15:1, 10:1, and 0:1 elute 2, 4, 3, and 2 times the column volume, respectively.

[0023] In e), the volume ratio of methanol to water is 65:35.

[0024] Another aspect of the present invention provides a pharmaceutical composition comprising a compound of formula I or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[0025] In another aspect, the present invention provides the use of the compound of Formula I or a pharmaceutically acceptable salt thereof in the preparation of medicaments for the prevention and / or treatment of neuroinflammatory and neurodegenerative diseases.

[0026] In the application, the neurodegenerative diseases include Alzheimer's disease and Parkinson's disease.

[0027] Activity studies have shown that the compounds of this invention have the effect of inhibiting the release of inflammatory factor NO from nerve cells, and have excellent inhibitory activity against LPS-induced inflammatory BV2 microglia. They can be used for the prevention or treatment of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease.

[0028] This invention studies the solanolactone component in Datura stramonium leaves to expand medicinal resources, obtain more bioactive medicinal components, and broaden the scope of choices for finding new natural drugs. Through further isolation and research of the active components in Datura stramonium leaves, a new compound with neuroinflammatory inhibitory activity was discovered. Attached Figure Description

[0029] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings:

[0030] Figure 1 ESI-MS chromatogram of compound 7α,27-dihydroxy-3β-adenine-(20S,22R)-1-one-azolones-5,24-dienolactone-27-O-β-D-glucoside obtained in Example 1 of this invention.

[0031] Figure 2 The compound 7α,27-dihydroxy-3β-adenine-(20S,22R)-1-one-azolonesta-5,24-dienolactone-27-O-β-D-glucoside obtained in Example 1 of this invention 1 H-NMR spectrum.

[0032] Figure 3 The compound 7α,27-dihydroxy-3β-adenine-(20S,22R)-1-one-azolonesta-5,24-dienolactone-27-O-β-D-glucoside obtained in Example 1 of this invention 13 C-NMR spectrum.

[0033] Figure 4The DEPT 135 diagram shows the compound 7α,27-dihydroxy-3β-adenine-(20S,22R)-1-one-azolones-5,24-dienolactone-27-O-β-D-glucoside obtained in Example 1 of this invention.

[0034] Figure 5 The HSQC diagram is shown for the compound 7α,27-dihydroxy-3β-adenine-(20S,22R)-1-one-azolones-5,24-dienolactone-27-O-β-D-glucoside obtained in Example 1 of this invention.

[0035] Figure 6 The compound 7α,27-dihydroxy-3β-adenine-(20S,22R)-1-one-azolonesta-5,24-dienolactone-27-O-β-D-glucoside obtained in Example 1 of this invention 1 H- 1 H COSY diagram.

[0036] Figure 7 The image shows the HMBC diagram of the compound 7α,27-dihydroxy-3β-adenine-(20S,22R)-1-one-azolones-5,24-dienolactone-27-O-β-D-glucoside obtained in Example 1 of this invention.

[0037] Figure 8 The NOESY diagram shows the compound 7α,27-dihydroxy-3β-adenine-(20S,22R)-1-one-azolones-5,24-dienolactone-27-O-β-D-glucoside obtained in Example 1 of this invention.

[0038] Figure 9 The image shows the CD diagram of compound 7α,27-dihydroxy-3β-adenine-(20S,22R)-1-one-azolones-5,24-dienolactone-27-O-β-D-glucoside obtained in Example 1 of this invention. Detailed Implementation

[0039] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0040] Example 1: Preparation of compound 7α,27-dihydroxy-3β-adenine-(20S,22R)-1-one-azolones-5,24-dienolactone-27-O-β-D-glucoside (compound shown in Formula I)

[0041] 20 kg of dried Datura leaves were weighed and extracted with methanol at a ratio of 10:1 (by weight). The extraction was repeated three times, each time for three days. The extracts were combined and recovered under reduced pressure to obtain 2.6 kg of Datura leaf extract (yield 13.0%). The extract was then mixed with water at a 1:1 ratio and passed through HPD-BJQH macroporous adsorption resin at 1 BV·h⁻¹. -1 Column chromatography elution was performed at a flow rate of [flow rate missing]. The eluents were water (2 BV), 30% ethanol (2 BV), and 95% ethanol (4 BV) sequentially. Each eluent was collected and concentrated under reduced pressure, yielding 450 g of water eluent, 663 g of 30% ethanol eluent, and 1027 g of 95% ethanol eluent. High-performance liquid chromatography (HPLC) analysis identified the following components: water eluent as a glycoside (17.3%), 30% ethanol eluent as a flavonoid (25.5%), and 95% ethanol eluent as a solanolone (39.5%). Take 400.0 g of the 95% ethanol eluent and perform gradient elution using silica gel column chromatography with dichloromethane-methanol (1:0-0:1) as the mobile phase [1:0 (3 BV), 50:1 (5 BV), 20:1 (4 BV), 10:1 (3 BV), 5:1 (4 BV), 0:1 (2 BV)]. The eluents were identified by TLC analysis and then combined to obtain 8 fractions, Fr.AH. Fr.G (42.6 g) was separated by ODS column chromatography and eluted with methanol-water gradient (30:70-80:20, 1:0) [30:70 (2 BV), 40:60 (4 BV), 50:50 (5 BV), 60:40 (3 BV), 70:30 (3 BV), 80:20 (2 BV), 1:0 (2 BV)] to obtain 14 fractions, Fr.G1 to Fr.G14. Fr.G8 (2.6 g) was separated by silica gel column chromatography, eluted with dichloromethane-methanol (20:1-0:1) [(20:1 (2 BV), 15:1 (4 BV), 10:1 (3 BV), 0:1 (2 BV)], yielding 5 fractions, Fr.G8-1 to Fr.G8-5. Fr.G3 (82 mg) was purified by preparative HPLC (methanol:water volume ratio 65:35) to obtain compound 7α,27-dihydroxy-3β-adenine-(20S,22R)-1-one-azolon-5,24-dienolactone-27-O-β-D-glucoside (2.1 mg), the structural formula of which is shown in Formula I.

[0042] The characterization data of compound 7α,27-dihydroxy-3β-adenine-(20S,22R)-1-one-azolones-5,24-dienolactone-27-O-β-D-glucoside are as follows:

[0043] ESI-MS (e / z) of compound 7α,27-dihydroxy-3β-adenine-(20S,22R)-1-one-azolo-5,24-dienolactone-27-O-β-D-glucoside: ([M+HCOO) - 796.3760 (Calculated value 796.3774)

[0044] The structural formula and NMR data assignment of compound 7α,27-dihydroxy-3β-adenine-(20S,22R)-1-one-azolo-5,24-dienolactone-27-O-β-D-glucoside:

[0045]

[0046] Table 1. NMR spectra of compound 7α,27-dihydroxy-3β-adenine-(20S,22R)-1-one-azolo-5,24-dienolactone-27-O-β-D-glucoside (Pyridine-d6, δin ppm, J in Hz)

[0047]

[0048]

[0049] Example 2: Activity screening and results of compound 7α,27-dihydroxy-3β-adenine-(20S,22R)-1-one-azolones-5,24-dienolactone-27-O-β-D-glucoside

[0050] 2.1 Experimental Methods

[0051] Microglia (BV2) cells were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences and cultured in 10% DMEM medium at 37°C in a 5% CO2 cell culture incubator.

[0052] BV2 cells in logarithmic growth phase were planted at a density of 1×10⁻⁶. 5 Cells were seeded at 100 μL / well in 96-well plates, with 3 replicates per group, and incubated overnight at 37°C, 5% CO2. Cell status was observed, and once complete adhesion was confirmed, the culture medium was aspirated. Except for the control group, 100 μL of DMEM medium containing 100 ng / mL LPS was added to the remaining wells, and the cells were incubated for 24 h. The culture medium was then aspirated. 100 μL of complete DMEM medium was added to the control and model groups, while 100 μL of DMEM medium containing various concentrations (final concentrations of 0.15625, 0.3125, 0.625, 1.25, 2.5, 5, 10, and 20 μM) of the drug was added to the sample groups. After 24 h, the supernatant was collected, and the OD value of each well was measured at 540 nm using the Griess reagent method, and the NO inhibition rate was calculated.

[0053] Inhibition rate = (OD value of model group - OD value of sample group) / (OD value of model group - OD value of blank group) × 100%.

[0054] 2.2 Experimental Results

[0055] Table 2

[0056]

[0057] As shown in Table 2, the compound 7α,27-dihydroxy-3β-adenine-(20S,22R)-1-one-azolo-5,24-dienolactone-27-O-β-D-glucoside of this invention has an inhibitory IC50 value on NO in lipopolysaccharide-induced inflammatory microglia. 50 With a value of 1.86 μM, it showed extremely high inhibitory activity against inflammatory factors released by inflammatory microglia, and can be used to prevent or treat neuroinflammatory diseases such as Alzheimer's disease and Parkinson's disease.

[0058] In addition, we conducted inhibitory microglial inflammatory activity assays on compounds (Dmetelin A and Daturmetelide U) with similar structures to compound 7α,27-dihydroxy-3β-adenine-(20S,22R)-1-one-azolonesta-5,24-dienolactone-27-O-β-D-glucoside) using the method described above (experimental method as in 2.1). The results showed that the IC50 of both compounds was significantly lower than that of 7α,27-dihydroxy-3β-adenine-(20S,22R)-1-one-azolonesta-5,24-dienolactone-27-O-β-D-glucoside. 50 Both compounds have a molecular weight greater than 20 μM and do not possess anti-inflammatory drug activity. The specific structures of the two compounds are as follows:

[0059]

[0060] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. The compound represented by Formula I or a pharmaceutically acceptable salt thereof: 。 2. A method for preparing the compound shown in Formula I of claim 1, comprising the following steps: 1) extracting Datura leaves by cold maceration with methanol, and recovering the solvent to obtain an extract; 2) Includes the following steps: a) The dissolved extract was added to a macroporous adsorption resin and eluted sequentially with water, 30% ethanol, and 95% ethanol. The eluents were collected and concentrated under reduced pressure to obtain the water-eluted fraction, the 30% ethanol-eluted fraction, and the 95% ethanol-eluted fraction. b) The 95% ethanol eluent was separated by silica gel column chromatography with gradient elution using dichloromethane-methanol as the mobile phase to obtain 8 fractions, Fr.A-Fr.H; c) The Fr. G fraction was separated by ODS column chromatography and eluted with methanol-water gradient to obtain 14 fractions, Fr. G1-Fr. G14; d) The Fr. G8 fraction was separated by vacuum silica gel column chromatography with dichloromethane-methanol gradient elution to obtain 5 fractions, Fr. G8-1 to Fr. G8-5; e) The Fr. G8-3 fraction was eluted by preparative HPLC with methanol-water as the eluent to obtain the compound shown in Formula I.

3. The method as described in claim 2, characterized in that, In step 1), the weight ratio of datura leaves to methanol is 1:8-12; The cold maceration extraction is performed 2-6 times; Extraction time is 1-7 days / time.

4. The method as described in claim 2, characterized in that, In step 2), the macroporous adsorption resin is selected from HPD-BJQH, HP-20 or AB-8.

5. The method as described in claim 2, characterized in that, In (a), the flow rate of the eluent is 1 BV·h. -1 ; The dosage of water is 2 BV, the dosage of 30% ethanol is 2 BV, and the dosage of 95% ethanol is 4 BV. In b), the elution gradient of the mobile phase dichloromethane-methanol is as follows: mixed solutions with dichloromethane to methanol volume ratios of 1:0, 50:1, 20:1, 10:1, 5:1, and 0:1 elute 3, 5, 4, 3, 4, and 2 times the column volume, respectively. In c), the elution gradient of the mobile phase methanol-water is as follows: mixed solutions with methanol to water volume ratios of 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, and 1:0 elute 2, 4, 5, 3, 3, 2, and 2 times the column volume, respectively. In d), the elution gradient of the mobile phase dichloromethane-methanol is as follows: mixed solutions with dichloromethane to methanol volume ratios of 20:1, 15:1, 10:1, and 0:1 elute 2, 4, 3, and 2 times the column volume, respectively. In e), the volume ratio of methanol to water is 65:

35.

6. The use of the compound of formula I of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing and / or treating neuroinflammatory or neurodegenerative diseases by inhibiting the inflammatory activity of microglia, wherein the neurodegenerative disease is selected from Alzheimer's disease or Parkinson's disease.

7. A medicament for preventing and / or treating neuroinflammation or neurodegenerative diseases by inhibiting the inflammatory activity of microglia, comprising the compound of formula I of claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

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