Dihydroisoflavone dimer compound as well as preparation method and application thereof
By isolating and purifying dihydroisoflavone dimers from Astragalus membranaceus roots, the problems of penetration and side effects of existing anti-neuroinflammatory drugs have been solved, achieving effective inhibition of neuroinflammation and treatment of multi-stage diseases.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-23
- Publication Date
- 2026-04-14
AI Technical Summary
Existing anti-neuroinflammatory drugs have difficulty penetrating the blood-brain barrier, have significant side effects with long-term use, and cannot fully regulate the neuroinflammatory network, lacking adaptability to different disease stages.
Dihydroisoflavone dimers were isolated from the roots of Astragalus membranaceus. Compounds 1-4 were obtained through extraction, separation and purification, and were used in the preparation of drugs to inhibit LPS-induced NO release from BV2 cells.
Compounds 1-4 exhibit significant anti-neuroinflammatory activity and can effectively inhibit LPS-induced NO release from BV2 cells, showing potential applications in the treatment of Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, and Parkinson's disease.
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Figure CN121850971A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of traditional Chinese medicine manufacturing, specifically to a dihydroisoflavone dimer compound, and also to its uses and preparation methods. Background Technology
[0002] Neuroinflammation is one of the core pathological responses in central nervous system diseases. Its essence lies in the overactivation of microglia and astrocytes, releasing large amounts of pro-inflammatory factors and forming a vicious cycle of "neuroinflammation-neuronal damage." This chronic inflammatory state not only accelerates neuronal death and blood-brain barrier disruption but also leads to decreased synaptic plasticity and cognitive decline through pathways such as activating the NLRP3 inflammasome and promoting oxidative stress. For example, in Alzheimer's disease, the neuroinflammatory response triggered by β-amyloid deposition can persist for decades, ultimately leading to irreversible neuronal loss; while ischemia-reperfusion injury after stroke activates microglia by releasing DAMPs, causing secondary neurological damage. Currently used clinical drugs (such as nonsteroidal anti-inflammatory drugs and glucocorticoids) have poor blood-brain barrier penetration and are prone to side effects such as gastrointestinal bleeding or immunosuppression with long-term use, making them unsuitable for precise treatment. Furthermore, existing anti-inflammatory drugs often target single signaling pathways, failing to comprehensively regulate the neuroinflammatory network and lacking adaptability to different stages of the disease. Therefore, the development of novel anti-neuroinflammatory drugs is an urgent clinical need.
[0003] Astragalus is derived from the legume Astragalus membranaceus. Astragalus membranaceus var. mongholicus Or Astragalus membranaceus Astragalus membranaceus The dried root of Astragalus membranaceus (Huangqi) is primarily used for its functions of tonifying Qi and raising Yang, consolidating the exterior and stopping sweating, promoting diuresis and reducing swelling, generating fluids and nourishing blood, promoting blood circulation and relieving pain, promoting pus drainage and detoxification, and promoting tissue regeneration and wound healing. In clinical practice, Astragalus membranaceus is also widely used as an adjunct treatment for ischemic stroke, and the correlation between its pharmacodynamic material basis and neuroprotective effects has attracted attention from the academic community. Therefore, using Astragalus membranaceus as a research vehicle to screen and develop highly effective and low-toxicity neuroinflammatory inhibitors has significant academic value and clinical translational significance. Summary of the Invention
[0004] This invention isolates a class of dihydroisoflavone dimers from the root of Astragalus membranaceus, which has a novel structure and exhibits activity in inhibiting neuroinflammation.
[0005] Therefore, in a first aspect, the present invention provides a dihydroisoflavone dimer compound having the structure shown in Formula I, Formula II, Formula III or Formula IV: Formula I Formula II Formula III Formula IV R1-R8 may be the same or different, and each is independently selected from: hydroxyl and methoxy.
[0006] Preferably, in Formula I, two, three, or four of R1-R7 are hydroxyl groups, and the remainder are methoxy groups. More preferably, R1 and R4 are hydroxyl groups; and zero, one, or two of R2, R3, R5, R6, and R7 are hydroxyl groups, and the remainder are methoxy groups. Even more preferably, R1 and R4 are hydroxyl groups, R2 and R3 are methoxy groups, and one of R5, R6, and R7 is a hydroxyl group, and the remainder are methoxy groups.
[0007] Preferably, in Formula II, 3, 4, or 5 of R1-R8 are hydroxyl groups, and the remainder are methoxy groups. More preferably, R1 and R5 are hydroxyl groups; 1, 2, or 3 of R2, R3, R4, R6, R7, and R8 are hydroxyl groups, and the remainder are methoxy groups. Even more preferably, R1 and R5 are hydroxyl groups; 1 of R2, R3, and R4 is a hydroxyl group, and the remainder are methoxy groups; and 1 of R6, R7, and R8 is a hydroxyl group, and the remainder are methoxy groups.
[0008] Preferably, in Formula III, 3, 4, or 5 of R1-R8 are hydroxyl groups, and the remainder are methoxy groups. More preferably, R1 and R5 are hydroxyl groups; 1, 2, or 3 of R2, R3, R4, R6, R7, and R8 are hydroxyl groups, and the remainder are methoxy groups. Even more preferably, R1 and R5 are hydroxyl groups; 1 of R2, R3, and R4 is a hydroxyl group, and the remainder are methoxy groups; and 1 of R6, R7, and R8 is a hydroxyl group, and the remainder are methoxy groups.
[0009] Preferably, in Formula IV, two, three, or four of R1-R7 are hydroxyl groups, and the remainder are methoxy groups. More preferably, R1 is a hydroxyl group; one, two, or three of R2, R3, R4, R5, R6, and R7 are hydroxyl groups, and the remainder are methoxy groups. Even more preferably, R1 is a hydroxyl group; one of R2, R3, and R4 is a hydroxyl group, and the remainder are methoxy groups; and one of R5, R6, and R7 is a hydroxyl group, and the remainder are methoxy groups.
[0010] Preferably, the dihydroisoflavone dimer compounds of the present invention are selected from the following compounds 1-4: A second aspect of the invention relates to a method for preparing the aforementioned dihydroisoflavone dimers, particularly compounds 1-4, comprising: a. Extraction steps: Extract Mongolian Astragalus ( Astragalus membranaceus var. mongholicus The roots of the plant were extracted successively with anhydrous ethanol and ethanol-water by heating and reflux. The extracts were combined and concentrated under reduced pressure to obtain an extract. b. Separation steps: The suspension was added to water and then extracted with petroleum ether and ethyl acetate to obtain petroleum ether extract, ethyl acetate extract and water fraction. These fractions were then separated by column chromatography to obtain compounds 1-4.
[0011] In one embodiment of the present invention, the extraction step includes: extracting the root of Astragalus membranaceus by heating and refluxing with anhydrous ethanol 2-3 times, extracting the residue by heating and refluxing with ethanol-water at a volume concentration of 30-70% 2-3 times, combining the extracts, and concentrating under reduced pressure to obtain an extract.
[0012] Preferably, the residue is further extracted by heating and reflux with ethanol-water at a volume concentration of 50-60% 2-3 times.
[0013] Preferably, the column chromatography includes one or more combinations of silica gel column chromatography, medium-low pressure ODS column chromatography, reversed-phase semi-preparative HPLC, and preparative HPLC.
[0014] Preferably, the separation step includes: The ethyl acetate extract was separated by silica gel column chromatography using petroleum ether-ethyl acetate ratios of 5:1, 1:1, and 0:1. The B2 fraction was separated by gradient elution with methanol-water (20:85, 40:60, 60:40, 80:20, 100:0, v / v) to obtain 29 fractions B2A-B2Z and B2a-B2c. The B2T fraction was separated by reversed-phase semi-preparative HPLC with acetonitrile-water (43.5:56.5, v / v) as the mobile phase and eluted isocratically to obtain compounds 2 and 1. The B2U fraction was separated by reversed-phase semi-preparative HPLC with acetonitrile-water (48:52, v / v) as the mobile phase and eluted isocratically to obtain compound 3. The B2W fraction was separated by reversed-phase semi-preparative HPLC with acetonitrile-water (65:35, v / v) as the mobile phase and eluted isocratically to obtain compound 4.
[0015] This invention has discovered that the dihydroisoflavone dimer compounds have good effects in anti-neuroinflammatory activity, and can inhibit LPS-induced NO release from BV2 cells.
[0016] Therefore, a third aspect of the present invention is to provide the use of the dihydroisoflavone dimer in the preparation of a medicament for the treatment of neuroinflammation.
[0017] Preferably, the drug is used to treat neurodegenerative diseases. Preferably, the neurodegenerative diseases include Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, or Parkinson's disease.
[0018] A fourth aspect of the present invention is to provide a pharmaceutical composition comprising the dihydroisoflavone dimer compound.
[0019] The beneficial effects of this invention are: This invention isolates a novel class of dihydroisoflavone dimers from the root of *Astragalus membranaceus*. These dimers possess a novel structure. The anti-inflammatory activity of these compounds was evaluated using an LPS-induced BV2 cell model. The results showed that the dihydroisoflavone dimers of this invention exhibit significant anti-neuroinflammatory activity. Therefore, the dihydroisoflavone dimers of this invention can serve as novel anti-neuroinflammatory drugs. Attached Figure Description
[0020] 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: Figure 1 For compound 1 1 H- 1 H COSY spectrum; Figure 2 The HSQC spectrum of compound 1; Figure 3 The HMBC spectrum of compound 1; Figure 4 The NOESY spectrum of compound 1; Figure 5 For compound 2 1 H- 1 H COSY spectrum; Figure 6 The HSQC spectrum of compound 2; Figure 7 The HMBC spectrum of compound 2; Figure 8 The NOESY spectrum of compound 2; Figure 9 This is a schematic diagram of the crystal structure of compound 2; Figure 10 For compound 3 1 H- 1 H COSY spectrum; Figure 11 The HSQC spectrum of compound 3; Figure 12 The HMBC spectrum of compound 3; Figure 13 The NOESY spectrum of compound 3; Figure 14 For compound 4 1 H- 1 H COSY spectrum; Figure 15 The HSQC spectrum of compound 4; Figure 16 The HMBC spectrum of compound 4; Figure 17 The NOESY spectrum of compound 4; Figure 18 The anti-neuroinflammatory activity of compounds 1-4 was evaluated. Results are expressed as mean ± SD, where... This indicates that p < 0.05 compared to the model group; This indicates that p < 0.01 compared to the model group; This indicates that p < 0.001 compared to the model group. Detailed Implementation
[0021] The embodiments of the present invention are described in detail below. The embodiments are provided to better illustrate the content of the present invention and are only used to explain the present invention, and should not be construed as limiting the present invention.
[0022] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.
[0023] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.
[0024] Example 1: Preparation of compounds 1-4 50 kg of Astragalus membranaceus root was mechanically pulverized and extracted twice with 500 L of anhydrous ethanol under reflux for 2 hours each time. The extracts were filtered, and the residue was further extracted twice with 400 L of ethanol-water (1:1) under reflux for 2 hours each time. The extracts were combined and concentrated under reduced pressure to obtain a paste. The paste was suspended in water and extracted sequentially with petroleum ether and ethyl acetate to obtain 565 g of petroleum ether extract, 213 g of ethyl acetate extract, and 10847 g of aqueous fraction.
[0025] 200 g of ethyl acetate extract was separated by silica gel column chromatography with a gradient elution of petroleum ether-ethyl acetate (5:1, 1:1, 0:1, v / v) to obtain four fractions B1–B4. Fraction B2 (12.5 g) was separated by medium-pressure ODS column chromatography with a gradient elution of methanol-water (20:85, 40:60, 60:40, 80:20, 100:0, v / v) to obtain 29 fractions B2A–B2Z and B2a–B2c. Fraction B2T (0.280 g) was subjected to reversed-phase semi-preparative HPLC with isocratic elution of acetonitrile-water (43.5:56.5, v / v) to obtain compound 2 (t). R =36.2 min, 13.64 mg) and 1 (t R =45.0 min, 12.47 mg). The B2U fraction (0.221 g) was subjected to reversed-phase semi-preparative HPLC with acetonitrile-water (48:52, v / v) as the mobile phase and eluted isocratically to give compound 3 (t). R =38.0 min, 14.10 mg). The B2W fraction (0.067 g) was subjected to reversed-phase semi-preparative HPLC with acetonitrile-water (65:35, v / v) as the mobile phase and eluted isocratically to give compound 4 (t R =17.9 min, 6.21 mg).
[0026] Compound 1 Light yellow powder, -10 ( c 0.1, CH3OH); UV (CH3OH) λ max (log ε 203 (2.28)nm; ECD (MeOH) λ max (Δ ε 213 (-5.64) nm; IR (KBr) ν max 3204, 2938, 1620, 1500, and1459 cm -1 HRESIMS m / z 601.2078 ([M - H] - The calculated value is C. 34 H 33 O 10 , 601.2079); 1 H NMR (600 MHz, DMSO-) d 6) δ H: 4.00 (1H, m, H-2a), 3.89 (1H, m, H-2b), 3.08 (1H, m, H-3), 2.82 (1H, m, H-4a), 2.59 (1H, m, H-4b), 6.82 (1H, d, J =8.3 Hz, H-5), 6.26(1H, dd, J =8.3, 2.4 Hz, H-6), 6.14 (1H, d, J =2.4 Hz, H-8), 6.88 (1H, d, J =8.7Hz, H-5′), 6.96 (1H, d, J =8.7 Hz, H-6′), 4.06 (1H, m, H-2′′a), 3.85 (1H, m, H-2′′b), 3.33 (1H, m, H-3′′), 2.75 (1H, m, H-4′′a), 2.53 (1H, m, H-4′′b), 6.69(1H, d, J =8.3 Hz, H-5′′), 6.19 (1H, dd, J =8.3, 2.4 Hz, H-6′′), 6.02 (1H, d, J =2.4 Hz, H-8′′), 5.97 (1H, s, H-6′′′), 3.38 (3H, s, 3′-OCH3), 3.80 (3H, s, 4′-OCH3), 3.77 (3H, s, 2′′′-OCH3), 3.80 (3H, s, 3′′′-OCH3), 9.16 (1H, s, 7-OH),9.11 (1H, s, 7′′-OH), 8.67 (1H, s, 2′′′-OH) ; 13 C NMR (150 MHz, DMSO- d 6), δ C:69.3 (C-2), 31.3 (C-3), 30.6 (C-4), 130.0 (C-5), 108.1 (C-6), 156.5 (C-7), 102.5 (C-8), 154.3 (C-9), 112.4 (C-10), 127.1 (C-1′), 145.8 (C-2′), 141.0 (C-3′), 152.4 (C-4′), 109.6 (C-5′), 121.8 (C-6′), 68.4 (C-2′′), 31.0 (C-3′′), 29.1 (C-4′′), 129.7 (C-5′′), 108.1 (C-6′′), 156.4 (C-7′′), 102.5 (C-8′′), 154.4 (C-9′′), 111.7 (C-10′′), 123.0 (C-1′′′), 142.6 (C-2′′′), 141.4 (C-3′′′), 140.4 (C-4′′′), 144.8 (C-5′′′), 107.8 (C-6′′′), 59.8 (3′-OCH3), 60.4(4′-OCH3), 60.8 (3′′′-OCH3), 55.8 (4′′′-OCH3).
[0027] By HRESIMS m / z 601.2078 ([M - H] - The calculated value is C. 34 H 33 O 10 (601.2079), the molecular formula of compound 1 was determined to be C 34 H 34 O 10 It has 18 degrees of unsaturation. 1 H NMR showed two sets of ABX hydrogen signals from the benzene ring, one pair of ortho-hydrogen signals from the benzene ring, one independent hydrogen signal from the benzene ring, and four methyl hydrogen signals. 13 C10 NMR and HSQC spectra revealed 24 benzene ring carbon signals, 4 oxymethyl carbon signals, 4 methylene carbon signals, and 2 methine carbon signals. These characteristic signals suggest that compound 1 is a dihydroisoflavone dimer. Further analysis of key signals from the COSY, HSQC, and HMBC spectra of compound 1 confirmed its chemical structure. Based on the single-crystal diffraction structure data of compound 2, the absolute configuration of compound 2 was determined to be 3... R 3′′ R Then, the specific rotation and ECD data of compound 1 were compared and analyzed with those of compound 2. They were found to be basically consistent, and the absolute configuration of compound 1 was determined to be 3.R , 3′′ R .
[0028] Compound 2 Light yellow powder, -20 ( c 0.1, CH3OH); UV (CH3OH) λ max (log ε 203 (2.45)nm; ECD (MeOH) λ max (Δ ε 211 (-6.02) nm; IR (KBr) ν max 3388, 2935, 2850, 1620,1469 and 1154 cm -1 HRESIMS m / z 601.2078 ([M - H] - The calculated value is C. 34 H 33 O 10 , 601.2079); 1 H NMR (600 MHz, DMSO- d 6) δ 4.19 (1H, dd, J =10.0, 2.9 Hz, H-2a), 3.95 (1H, t, J =10.0 Hz, H-2b), 3.38 (1H, m, H-3), 2.87 (1H, dd, J =15.4, 10.8 Hz, H-4a),2.76 (1H, dd, J =15.4, 3.8 Hz, H-4b), 6.86 (1H, d, J =8.3 Hz, H-5), 6.27 (1H, dd, J =8.3, 2.4 Hz, H-6), 6.17 (1H, d, J =2.4 Hz, H-8), 6.65 (1H, s, H-6′), 4.19(1H, dd, J =10.0, 2.9 Hz, H-2′′a), 3.95 (1H, t, J=10.0 Hz, H-2′′b), 3.38 (1H, m,H-3′′), 2.87 (1H, dd, J =15.4, 10.8 Hz, H-4′′a), 2.76 (1H, dd, J =15.4, 3.8 Hz,H-4′′b), 6.86 (1H, d, J =8.3 Hz, H-5′′), 6.27 (1H, dd, J =8.3, 2.4 Hz, H-6′′),6.17 (1H, d, J =2.4 Hz, H-8′′), 6.65 (1H, s, H-6′′′), 3.75 (3H, s, 3′-OCH3),3.55 (3H, s, 4′-OCH3), 3.75 (3H, s, 3′′′-OCH3), 3.55 (3H, s, 4′′′-OCH3), 9.15 (1H, s, 7-OH), 9.04 (1H, s, 2′′′-OH), 9.15 (1H, s, 7′′-OH), 9.04 (1H, s, 2′′′-OH); 13 C NMR (150 MHz, DMSO- d 6), δ C: 69.0 (C-2), 31.7 (C-3), 29.8 (C-4), 130.1(C-5), 108.0 (C-6), 156.5 (C-7), 102.5 (C-8), 154.6 (C-9), 112.7 (C-10),123.0 (C-1′), 147.6 (C-2′), 140.3 (C-3′), 149.6 (C-4′), 122.7 (C-5′), 123.7(C-6′), 69.0 (C-2′′), 31.7 (C-3′′), 29.8 (C-4′′), 130.1 (C-5′′), 108.0 (C-6′′), 156.5 (C-7′′), 102.5 (C-8′′), 154.6 (C-9′′), 112.7 (C-10′′), 123.0 (C-1′′′), 147.6 (C-2′′′), 140.3 (C-3′′′), 149.6 (C-4′′′), 122.7 (C-5′′′), 123.7(C-6′′′), 60.4 (3′-OCH3), 60.0 (4′-OCH3), 60.4 (3′′′-OCH3), 60.0 (4′′′-OCH3).
[0029] Single-crystal data for compound 2: C 34 H 34 O 10 , monoclinic, Space groupI2, a = 12.0859(5) Å, b = 5.5247(2) Å, c = 21.7305(9) Å, α = 90°, β = 102.390(4)°, γ = 90°, V =1417.18(10) Å3, Z = 2, Cu Kα (λ = 1.54184), 12966 reflections collected, Independent reflections 2809 [R int = 0.0558, R sigma = 0.0445], Final R indexes [I>=2σ (I)]R1= 0.0409, wR2= 0.0960, Final R indexes [all data]R1= 0.0557, wR2=0.1027, Flack parameter-0.01(14).
[0030] By HRESIMS m / z 601.2078 ([M - H] - The calculated value is C. 34 H 33 O 10 (601.2079), the molecular formula of compound 2 was determined to be C 34 H 34 O 10 It has 18 degrees of unsaturation. 1 1H NMR showed two sets of ABX hydrogen signals from the benzene ring, two independent hydrogen signals from the benzene ring, and four hydrogen signals from the methyl ring. 13 C10 NMR and HSQC spectra revealed 24 carbon signals on the benzene ring, 4 carbon signals on the oxymethyl ring, 4 carbon signals on the methylene ring, and 2 carbon signals on the methine ring. These characteristic signals suggest that compound 2 is a symmetrical dihydroisoflavone dimer. Further analysis of the key signals from the COSY, HSQC, and HMBC spectra of compound 2 confirmed its chemical structure. Based on the single-crystal diffraction data of compound 2 (prepared by solution evaporation in dichloromethane-cyclohexane (2:1), its absolute configuration was determined to be 3. R , 3′′ R .
[0031] Compound 3 Light yellow powder, -20 ( c 0.1, CH3OH); UV (CH3OH) λ max (log ε 204 (2.30)nm; ECD (MeOH) λ max (Δ ε 211 (-5.42) nm; IR (KBr) ν max 3416, 2939, 1622, 1508,1462 and 1159 cm -1 HRESIMS m / z 601.2068 ([M - H] - The calculated value is C. 34 H 33 O 10 , 601.2079). 1 H NMR (600 MHz, DMSO- d 6) δ H: 4.17 (1H, m, H-2a), 3.95 (1H, m, H-2b), 3.37(1H, m, H-3), 2.89 (1H, m, H-4a), 2.74 (1H, m, H-4b), 6.75 (1H, s, H-5), 6.30(1H, s, H-8), 6.47 (1H, d, J =8.7 Hz, H-5′), 6.80 (1H, d, J =8.7 Hz, H-6′), 4.21(1H, m, H-2′′a), 3.92 (1H, m, H-2′′b), 3.37 (1H, m, H-3′′), 2.89 (1H, m, H-4′′a), 2.74 (1H, m, H-4′′b), 6.86 (1H, d, J =8.2 Hz, H-5′′), 6.27 (1H, dd, J =8.2, 2.5 Hz, H-6′′), 6.18 (1H, d, J =2.5 Hz, H-8′′), 6.64 (1H, s, H-6′′′), 3.68(3H, s, 3′-OCH3), 3.75 (3H, s, 4′-OCH3), 3.75 (3H, s, 3′′′-OCH3), 3.60 (3H, s,4′′′-OCH3), 9.12 (1H, s, 7-OH), 8.93 (1H, s, 2′-OH), 8.92 (1H, s, 7′′-OH),8.92 (1H, s, 2′′′-OH) ; 13 C NMR (150 MHz, DMSO- d 6), δ C: 69.2 (C-2), 31.6 (C-3), 29.7 (C-4), 131.8 (C-5), 118.4 (C-6), 153.7 (C-7), 102.5 (C-8), 153.5 (C-9), 112.4 (C-10), 121.1 (C-1′), 148.1 (C-2′), 136.1 (C-3′), 151.6 (C-4′), 103.2(C-5′), 121.5 (C-6′), 69.1 (C-2′′), 31.9 (C-3′′), 29.7 (C-4′′), 130.1 (C-5′′), 107.9 (C-6′′), 156.4 (C-7′′), 102.5 (C-8′′), 154.5 (C-9′′), 112.7 (C-10′′), 122.7 (C-1′′′), 147.3 (C-2′′′), 140.2 (C-3′′′), 149.7 (C-4′′′), 123.2(C-5′′′), 123.9 (C-6′′′), 60.2 (3′-OCH3), 55.6 (4′-OCH3), 60.3 (3′′′-OCH3), 59.8 (4′′′-OCH3).
[0032] By HRESIMS m / z 601.2068 ([M - H] - The calculated value is C. 34 H 33 O 10 (601.2079), the molecular formula of compound 3 was determined to be C 34 H 34 O 10 It has 18 degrees of unsaturation. 1 1H NMR showed one group of ABX hydrogen signals from the benzene ring, two ortho-hydrogen signals from the benzene ring, three independent hydrogen signals from the benzene ring, and four methyl hydrogen signals. 13 C10 NMR and HSQC spectra revealed 24 benzene ring carbon signals, 4 oxymethyl carbon signals, 4 methylene carbon signals, and 2 methine carbon signals. These characteristic signals suggest that compound 3 is a dihydroisoflavone dimer. Further analysis of key signals from the COSY, HSQC, and HMBC spectra of compound 3 confirmed its chemical structure. Comparison of the specific rotation and ECD data of compound 3 with those of compound 2 showed they were essentially identical, confirming the absolute configuration of compound 3 as 3. R , 3′′ R .
[0033] Compound 4 Light yellow powder, -10 ( c 0.1, CH3OH); UV (CH3OH) λ max (log ε ) 205 (2.38) nm; ECD (MeOH) λ max (Δ ε ) 211 (-7.57) nm; IR (KBr) ν max 3408, 2935, 1619, 1500, 1462, 1153 and 1102 cm -1 ; HRESIMS m / z 601.2072 ([M - H] - , calculated for C 34 H 33 O 10 , 601.2079). 1 1H NMR (600 MHz, DMSO- d 6), δ H δ: 4.18 (1H, m, H-2a), 3.95 (1H, m, H-2b), 3.41 (1H, m, H-3), 2.93 (1H, m, H-4a), 2.80 (1H, m, H-4b), 7.00 (1H, d, J J = 8.4 Hz, H-5), 6.34 (1H, dd, J J = 8.4, 2.6 Hz, H-6), 6.18 (1H, d, J J = 2.6 Hz, H-8), 6.47 (1H, d, J J = 8.7 Hz, H-5′), 6.78 (1H, d, J J = 8.7 Hz, H-6′), 4.16 (1H, m, H-2′′a), 3.93 (1H, m, H-2′′b), 3.37 (1H, m, H-3′′), 2.83 (1H, m, H-4′′a), 2.81(1H, m, H-4′′b), 6.85 (1H, d, J J = 8.3 Hz, H-5′′), 6.27 (1H, dd, J=8.3, 2.4 Hz, H-6′′), 6.17 (1H, d, J =2.4 Hz, H-8′′), 6.63 (1H, s, H-6′′′), 3.68 (3H, s, 3′-OCH3), 3.74 (3H, s, 4′-OCH3), 3.77 (3H, s, 3′′′-OCH3), 3.70 (3H, s, 4′′′-OCH3), 8.94 (1H, s, 2′-OH), 9.13 (1H, s, 7′′-OH), 9.01 (1H, s, 2′′′-OH); 13 CNMR (150 MHz, DMSO- d 6) δ C : 69.3 (C-2), 31.3 (C-3), 29.7 (C-4), 130.3 (C-5), 107.8 (C-6), 157.8 (C-7), 102.9 (C-8), 154.6 (C-9), 115.9 (C-10), 120.7 (C-1′), 148.1 (C-2′), 136.1 (C-3′), 151.7 (C-4′), 103.2 (C-5′), 121.4 (C-6′), 68.7 (C-2′′), 31.6 (C-3′′), 29.6 (C-4′′), 130.0 (C-5′′), 108.0 (C-6′′), 156.5(C-7′′), 102.5 (C-8′′), 154.5 (C-9′′), 112.4 (C-10′′), 123.5 (C-1′′′), 145.4 (C-2′′′), 141.2 (C-3′′′), 144.2 (C-4′′′), 139.6 (C-5′′′), 115.4 (C-6′′′), 60.2 (3′-OCH3), 55.6 (4′-OCH3), 60.9 (3′′′-OCH3), 60.5 (4′′′-OCH3).
[0034] By HRESIMS m / z 601.2072 ([M - H] - The calculated value is C. 34 H 33 O 10 (601.2079), the molecular formula of compound 4 was determined to be C 34 H 34 O10 It has 18 degrees of unsaturation. 1 1H NMR showed two sets of ABX hydrogen signals from the benzene ring, two ortho-hydrogen signals from the benzene ring, one independent hydrogen signal from the benzene ring, and four methyl hydrogen signals. 13 C10 NMR and HSQC spectra revealed 24 benzene ring carbon signals, 4 oxymethyl carbon signals, 4 methylene carbon signals, and 2 methine carbon signals. These characteristic signals suggest that compound 4 is a dihydroisoflavone dimer. Further analysis of key signals from the COSY, HSQC, and HMBC spectra of compound 4 confirmed its chemical structure. Comparison of the specific rotation and ECD data of compound 4 with those of compound 2 showed they were essentially identical, confirming the absolute configuration of compound 4 as 32. R , 3′′ R .
[0035] Example 2: Anti-neuroinflammatory activity test of compounds 1-4 (1) Cell culture BV2 cells were obtained from the Cell Bank of the Chinese Academy of Sciences and cultured in DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 50 mg / mL streptomycin. All cells were cultured in an incubator at 37°C and 5% CO2.
[0036] (2) Determination of NO concentration released by LPS-induced BV2 cells BV2 cells in the logarithmic growth phase were seeded in 96-well cell culture plates at a cell density of 2 × 10⁶ cells / well. 4 Cells per well. After 12 hours of culture, the culture medium in the 96-well plate was discarded, and control, model, and drug treatment groups (compounds 1, 2, 3, and 4) were set up. The positive control was dexamethasone (DX), and each sample was set up in triplicate. The control group was added to complete DMEM medium, the model group was added to complete DMEM medium containing 1 μg / mL LPS, and the drug treatment groups were added to complete DMEM medium containing 1 μg / mL LPS and 10 μM of drug. The cells were cultured for another 24 hours. Then, 50 μL of cell supernatant was taken from each well, and the NO content in the cell supernatant was measured according to the instructions of the nitric oxide content detection kit.
[0037] For details, please see [link / details]. Figure 18 Compounds 1-4 significantly reduced LPS-induced NO release from BV2 cells and inhibited neuroinflammatory responses.
[0038] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.
Claims
1. A dihydroisoflavone dimer compound having the structure shown in Formula I, Formula II, Formula III or Formula IV: Formula I Formula II Formula III Formula IV R1-R8 may be the same or different, and each is independently selected from: hydroxyl and methoxy.
2. The dihydroisoflavone dimer compound according to claim 1, characterized in that, In Formula I, two, three, or four of R1-R7 are hydroxyl groups, and the rest are methoxy groups; preferably, R1 and R4 are hydroxyl groups; 0, 1, or 2 of R2, R3, R5, R6, and R7 are hydroxyl groups, and the rest are methoxy groups; even more preferably, R1 and R4 are hydroxyl groups, R2 and R3 are methoxy groups, and one of R5, R6, and R7 is a hydroxyl group, and the rest are methoxy groups; In Formula II, 3, 4, or 5 of R1-R8 are hydroxyl groups, and the rest are methoxy groups; preferably, R1 and R5 are hydroxyl groups; 1, 2, or 3 of R2, R3, R4, R6, R7, and R8 are hydroxyl groups, and the rest are methoxy groups; even more preferably, R1 and R5 are hydroxyl groups; 1 of R2, R3, and R4 is a hydroxyl group, and the rest are methoxy groups; 1 of R6, R7, and R8 is a hydroxyl group, and the rest are methoxy groups. In Formula III, three, four, or five of R1-R8 are hydroxyl groups, and the rest are methoxy groups; preferably, R1 and R5 are hydroxyl groups; one, two, or three of R2, R3, R4, R6, R7, and R8 are hydroxyl groups, and the rest are methoxy groups; even more preferably, R1 and R5 are hydroxyl groups; one of R2, R3, and R4 is a hydroxyl group, and the rest are methoxy groups; one of R6, R7, and R8 is a hydroxyl group, and the rest are methoxy groups. In Formula IV, two, three, or four of R1-R7 are hydroxyl groups, and the rest are methoxy groups; preferably, R1 is a hydroxyl group; one, two, or three of R2, R3, R4, R5, R6, and R7 are hydroxyl groups, and the rest are methoxy groups; even more preferably, R1 is a hydroxyl group, one of R2, R3, and R4 is a hydroxyl group, and the rest are methoxy groups, and one of R5, R6, and R7 is a hydroxyl group, and the rest are methoxy groups.
3. The dihydroisoflavone dimer compound according to claim 1, characterized in that, The compound is selected from the following compounds 1-4: 。 4. Use of the dihydroisoflavone dimer compound according to any one of claims 1-3 in the preparation of a medicament for use against neuroinflammation.
5. The use according to claim 4, characterized in that, The drug is used to treat neurodegenerative diseases.
6. The use according to claim 5, characterized in that, The neurodegenerative diseases mentioned include Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, or Parkinson's disease.
7. A pharmaceutical composition comprising the dihydroisoflavone dimer compound according to any one of claims 1-3.
8. The method for preparing the dihydroisoflavone dimer compound according to claim 3, comprising: a. Extraction steps: Extract Mongolian Astragalus ( Astragalus membranaceus var. mongholicus The roots of the plant were extracted successively with anhydrous ethanol and ethanol-water by heating and reflux. The extracts were combined and concentrated under reduced pressure to obtain an extract. b. Separation steps: The extract was suspended in water and extracted with petroleum ether and ethyl acetate to obtain petroleum ether extract, ethyl acetate extract and water fraction, which were then separated by column chromatography to obtain compounds 1-4 respectively.
9. The method for preparing the dihydroisoflavone dimer compound according to claim 8, characterized in that, The extraction steps include: extracting the roots of Astragalus membranaceus by heating and refluxing with anhydrous ethanol 2-3 times, extracting the residue by heating and refluxing with ethanol-water at a volume concentration of 30-70% 2-3 times, combining the extracts, and concentrating under reduced pressure to obtain an extract. The column chromatography includes one or more combinations of silica gel column chromatography, medium-low pressure ODS column chromatography, reversed-phase semi-preparative HPLC, and preparative HPLC.