Beta-gamma '-type lignan compound as well as preparation method and application thereof

Abstract

The invention discloses two beta-gamma'type lignan compounds as well as a preparation method and application thereof. The two beta-gamma '-type lignan compounds are separated and obtained from the nature for the first time, are respectively named as notopterygium root lignan A and notopterygium root lignan B, and are extracted and separated from notopterygium root rhizomes. Pharmacological test researches show that the two compounds have excellent anti-inflammatory activity and have the potential of being developed into drugs for treating inflammation-related diseases.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and relates to β-γ'-type lignan compounds, their preparation methods and applications. Specifically, it relates to two β-γ'-type lignan compounds isolated from the rhizome of Notopterygium incisum, their preparation methods and applications in the preparation of anti-inflammatory drugs. Background Art

[0002] Notopterygium incisium belongs to the genus Notopterygium in the Apiaceae family. This genus only contains two species, Notopterygium incisium and Notopterygium franchetii, both of which are endemic to China and are mainly distributed in high-altitude mountainous areas such as Tibet, Sichuan, Qinghai, Gansu, and Shaanxi. As a traditional Chinese medicine, Notopterygium incisium has a very wide range of applications in the field of traditional Chinese medicine. Notopterygium incisium has a strong effect of dispelling wind and removing dampness, and is often used to treat diseases such as arthritis, rheumatic joint pain, and rheumatoid arthritis.

[0003] Chemical research shows that the chemical composition of Notopterygium incisium is complex and its structural types are diverse. It mainly contains coumarins, organic acids and their glycosides, polyynes, steroids, volatile oils, etc. The pharmacological research on Notopterygium incisium focuses on the coumarin compounds in it, which have effects such as anti-inflammatory, antioxidant, antithrombotic, anticancer, and memory improvement. Notopterygium incisium has unique advantages in the treatment of inflammatory diseases. Therefore, discovering new anti-inflammatory compounds from it is of great significance for the further development and utilization of Notopterygium incisium medicinal materials. Summary of the Invention

[0004] Object of the Invention: The technical problem to be solved by the present invention is to provide two β-γ'-type lignan compounds with effective anti-inflammatory effects.

[0005] Another technical problem to be solved by the present invention is to provide a preparation method for the β-γ'-type lignan compounds.

[0006] The last technical problem to be solved by the present invention is to provide the application of β-γ'-type lignan compounds in the preparation of anti-inflammatory drugs or drugs for preventing or treating inflammation-related diseases.

[0007] Technical Solution: To solve the above technical problems, the present invention provides β-γ'-type lignan compounds, and the structures of the β-γ'-type lignan compounds are shown in Formula I or Formula II:

[0008]

[0009] The two C6-C3 structural units of the compounds of the present invention are connected through the β-position and γ-position of the side chain, and the structure contains a phenylethanol structural unit. In the present invention, Compound 1 and Compound 2 are respectively named Notopterygium lignan A and Notopterygium lignan B.

[0010] The present invention also includes a preparation method of the β-γ'-type lignan compounds, which is characterized by comprising the following steps:

[0011] 1) The rhizome of Notopterygium incisum is crushed and extracted with an ethanol solution. After the extraction solutions are combined, they are concentrated under reduced pressure to obtain a brown extract. The extract is suspended in pure water and successively and sufficiently extracted with petroleum ether, ethyl acetate, and n-butanol respectively to obtain an ethyl acetate extract.

[0012] 2) The ethyl acetate extract is subjected to a first gradient elution with petroleum ether-ethyl acetate and combined by thin-layer chromatography. A total of 3 components are obtained. Component 3 is subjected to silica gel column chromatography again and subjected to a second gradient elution with petroleum ether-ethyl acetate and combined by thin-layer chromatography. A total of 3 sub-components 3.1 to 3.3 are obtained. Sub-component 3.2 is subjected to silica gel column chromatography again and subjected to a third gradient elution with petroleum ether-ethyl acetate and combined by thin-layer chromatography to obtain 4 sub-components 3.2.1 to 3.2.4; Component 3.2.3 is prepared by preparative high performance liquid chromatography to obtain 9 sub-components 3.2.3.1 to 3.2.3.9. Component 3.2.3.8 is further prepared by preparative high performance liquid chromatography to obtain Compound 1 of Formula I, and Component 3.2.3.9 is further prepared by preparative high performance liquid chromatography to obtain Compound 2 of Formula II.

[0013] Among them, the mass-volume ratio of the rhizome of Notopterygium incisum to the ethanol solution is 1:2 to 1:10 kg / L.

[0014] Among them, the volume ratios of petroleum ether to ethyl acetate in the first gradient elution with petroleum ether-ethyl acetate are successively 100:0, 90:10, 80:20, 60:40, 40:60, and 0:100; the volume ratios of petroleum ether to ethyl acetate in the second gradient elution with petroleum ether-ethyl acetate are successively 80:1, 50:1, 30:1, 15:

[0015] 1, 8:1, 6:1, 4:1, 3:1, 2:1, and 0:100; the volume ratios of petroleum ether to ethyl acetate in the third gradient elution with petroleum ether-ethyl acetate are successively 80:1, 50:1, 30:1, 15:1, 8:1, 6:1, 4:1, 3:1, 2:1, and 0:100.

[0016] Among them, the preparation conditions using preparative high performance liquid chromatography include: the detection wavelength is 254 nm, the chromatographic column is a reverse-phase C-18 chromatographic column, and methanol and water are used as the mobile phase; preferably, the elution time of Compound 1 is 28.1 min, and the elution time of Compound 2 is 31.3 min.

[0017] The present invention also includes the application of the β-γ'-type lignan compounds in preparing anti-inflammatory drugs or in preparing for preventing or treating inflammation or related diseases thereof.

[0018] Among them, the inflammation includes the inflammation formed by LPS-induced RAW264.7 macrophages.

[0019] The present invention also includes an anti-inflammatory drug, and the active ingredient thereof includes one or two of the β-γ'-type lignan compounds described in claim 1.

[0020] Among them, the anti-inflammatory drug includes a single-agent or compound preparation.

[0021] Among them, the dosage form of the anti-inflammatory drug includes granules, capsules, tablets, powders, liquid preparations or sprays.

[0022] Beneficial effects: The two β-γ'-type lignan compounds of the present invention are isolated from nature for the first time, and their chemical structures are analyzed and identified by various modern spectroscopic techniques, which is original. At the same time, the two β-γ'-type lignan compounds of the present invention have strong anti-inflammatory activity and have the potential to be developed into drugs for treating inflammation-related diseases. Description of the Drawings

[0023] Figure 1 1H NMR spectrum of Compound 1;

[0024] Figure 2 13C NMR spectrum of Compound 1;

[0025] Figure 3 1H NMR spectrum of Compound 2;

[0026] Figure 4 13C NMR spectrum of Compound 2;

[0027] Figure 5 a shows the toxicity of the compound, and 5b shows the anti-inflammatory activity. Detailed Embodiments

[0028] Example 1 Preparation of Compound 1 and Compound 2

[0029] Plant source: The rhizome of Notopterygium incisum was collected from the Notopterygium incisum planting base in Xiaojin County, Aba Prefecture, Sichuan Province in June 2022.

[0030] Separation and Extraction of Notopterygii Rhizoma: The rhizome of Notopterygii Rhizoma (120 Kg) was crushed and extracted 4 times with 250 L of 80% ethanol at 60°C for 12 hours each time. After combining the extracts, they were concentrated under reduced pressure to obtain a brown extract (19.3 Kg). This extract was suspended in 40 L of pure water and successively extracted 5 times with equal volumes of petroleum ether, ethyl acetate, and n-butanol, yielding a petroleum ether extract (2.56 Kg), an ethyl acetate extract (5.02 Kg), and an n-butanol extract (4.22 Kg). The ethyl acetate extract (5.02 Kg) was subjected to silica gel (100 - 200 mesh, 30 Kg) (produced by Qingdao Marine Chemical Factory) column chromatography and eluted with petroleum ether - ethyl acetate in 6 gradients (100:0, 90:10, 80:20, 60:40, 40:60, and 0:100, v / v). The volume of the eluent for each gradient was 5 column volumes (the column volume was 25 L). Through thin-layer chromatography combination, a total of 3 crude components (Component 1 - Component 3) were obtained. Component 3 (1.03 Kg) was again subjected to silica gel (100 - 200 mesh, 10 Kg) column chromatography and eluted with petroleum ether - ethyl acetate in 6 gradients (80:1, 20:1, 10:1, 5:1, 2:1, and 0:100). The volume of the eluent for each gradient was 5 column volumes (the column volume was 8 L). Through thin-layer chromatography combination, a total of 3 sub-components (Component 3.1 - Component 3.3) were obtained. Component 3.2 (232.0 g) was again subjected to silica gel (100 - 200 mesh, 4 Kg) column chromatography and eluted with petroleum ether - ethyl acetate in 10 gradients (80:1, 50:1, 30:1, 15:1, 8:1, 6:1, 4:1, 3:1, 2:1, and 0:100). The volume of the eluent for each gradient was 8 column volumes (the column volume was 3 L). Through thin-layer chromatography combination, 4 sub-components (Component 3.2.1, Component 3.2.2 - Component 3.2.4) were obtained. Component 3.2.3 (35.4 g) was prepared by preparative high-performance liquid chromatography. The preparation conditions were: detection wavelength 254 nm, chromatographic column was a reversed-phase C-18 chromatographic column (Unisil-10-120-C18, 250 mm × 50 mm, 9 μm), and methanol and water were used as the mobile phase (80:20, v / v), obtaining 9 sub-components (Component 3.2.3.1 - Component 3.2.3.9). Component 3.2.3.8 (1.59 g) was further prepared by preparative high-performance liquid chromatography. The preparation conditions were: detection wavelength 254 nm, chromatographic column was a reversed-phase C-18 chromatographic column (Unisil-10-120-C18, 250 mm × 50 mm, 9 μm), and methanol and water were used as the mobile phase (75:25, v / v), yielding monomer compound 1 (231.3 mg, t R(= 28.1 min); Component 3.2.3.9 (2.20 g) was further prepared by preparative high performance liquid chromatography. The preparation conditions were as follows: the detection wavelength was 254 nm, the chromatographic column was a reverse-phase C-18 chromatographic column (Unisil-10-120-C18, 250 mm × 50 mm, 9 μm), and methanol and water were used as the mobile phase (75:25, v / v), to obtain Compound 2 (68.8 mg, t R = 31.3 min).

[0031] Example 2

[0032] Compound 1 was a light yellow oil. The HR-ESI-MS spectrum in the positive ion mode gave a quasi-molecular ion peak at m / z 529.2198 [M+Na] + (the calculated value was 529.2197). Combining 1 1H NMR and 13 13C NMR data (Table 1), it was determined that the molecular formula of Compound 1 was C 30 21 34 H 1 37 H O7. In the 1H NMR spectrum, there were two sets of trisubstituted aromatic proton signals in the low field region: δ H 6.87, 1H, s, H-2; 6.79, 1H, d, J = 8.1 Hz, H-6; 6.89, 1H, d, J = 8.1 Hz, H-5 and δ 1 6.72, 1H, s, H-2′; 6.71, 1H, d, J = 8.1 Hz, H-6′; 6.79, 1H, d, J = 8.1 Hz, H-5′, indicating that the structure of Compound 1 contained two 1,3,4-trisubstituted aromatic ring structural fragments. H The 1H NMR spectrum also gave five monosubstituted aromatic proton signals: δ H 7.20, 2H, d, J = 6.4 Hz, H-2″ / 6″; 7.25, 2H, t, J = 6.4 Hz, H-3″ / 5″; 7.22, 1H, overlap, H-4″; two trans-olefinic proton signals: δ H 6.13, 1H, d, J = 15.7 Hz, H-7′; 5.74, 1H, m, H-8′; three pairs of methylene proton signals: δ H 1.98 and 2.23, 2H, m, H-9′; 2.94, 2H, t, J = 7.2 Hz, H-7″; 4.34, 2H, td, J = 7.2, 2.5 Hz, H-8″; one ethoxy proton signal: δ H3.87, 3H, s, H-10; 3.82, 3H, s, H-10'. In the HMBC spectrum, the correlations of H-2,6 / C-4, H-5,10 / C-3, H-2',6' / C-4' and H-5',10' / C-3' suggest that compound 1 contains two 3-methoxy-4-hydroxybenzene ring structural units. 1 H- 1 In the H COSY spectrum, the correlation signals of H-7″ / H-8″ and H-2" / H-3" / H-4" / H-5" / H-6″, combined with the correlation signals of H-3′,′5",8" / C-1″, H-4",7″ / C-2″ and H-2",6" / C-7" in the HMBC spectrum, indicate that compound 1 contains one phenethyl structural unit. By comparing the hydrogen spectrum and carbon spectrum data of compound 1 with the literature data, it is confirmed that the chemical structure of compound 1 is similar to that of the known compound 7S*,8R*-phenethyl-(7-methoxy-8-isoeugenol)-ferulate, except that there is a methoxy group at the 7-position of the known compound, while there is an ethoxy group at the 7-position of compound 1. HSQC and HMBC further confirmed the planar structure of compound 1. 3 J H-7,H-8 A relatively large coupling constant of J = 10.3 Hz, and the absence of a correlation signal between H-11 and H-9′ in the NOESY spectrum indicate that the relative configurations at C-7 and C-8 in compound 1 are 7S* and 8R*. Based on the above various evidences, the chemical structure of compound 1 was determined and named as notopterygium lignan A. The NMR of compound 1 was fully assigned according to 1D NMR and 2D NMR (as shown in Table 1).

[0033] Example 3

[0034] Compound 2 is a light yellow oily substance. In the positive ion mode, the HR-ESI-MS spectrum gives a quasi-molecular ion peak at m / z 459.1815 [M - H] - (calculated value is 459.1813). Combining 1 the H NMR and 13 C NMR data (Table 1) determines that the molecular formula of compound 2 is C 28 H 28 O6. The hydrogen spectrum and carbon spectrum data of compound 2 are similar to those of compound 1, except that there is no ethoxy nuclear magnetic resonance signal in compound 2. At the same time, the carbon signals of C-7 (δ C 82.9) and C-8 (δ C 53.5) in compound 1 shift to lower field to C-7 (δ C 140.9) and C-8 (δ C(128.0), indicating the presence of a double bond between C-7 and C-8 in Compound 2. The long-range correlations between H-7 and C-2, C-6, C-9 and C-9′ in the HMBC spectrum further confirmed the above speculation. The chemical structure of Compound 2 was further confirmed by 1 H- 1 H COSY, HSQC and HMBC spectra, and named Notopterygium lignan B. The NMR of Compound 2 was fully assigned based on 1D NMR and 2D NMR (Table 1).

[0035] Table 1 1 H-NMR (400 MHz) and 13 C-NMR (100 MHz) NMR data (CDCl3)

[0036]

[0037]

[0038] Example 4

[0039] 1. Results and analysis of cell viability determination by MTT method

[0040] The effects of monomeric Compound 1 and Compound 2 on the viability of RAW 264.7 cells at different concentrations (10 μM, 20 μM, 30 μM, 40 μM, 50 μM) were studied (the results are shown in Figure 5 a). RAW264.7 cells in the logarithmic growth phase were seeded in 96-well plates at a seeding density of 1×10^5 cells / 100 μL / well. Different concentration drug groups were set: 0 μM (control group), 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, with 3 replicates in each group. After seeding, the cells were incubated overnight in a cell culture incubator. The next day, different concentrations of drugs were added to each experimental group and incubated for 24 h. The supernatant was discarded, MTT (5 μg / mL) was added, and 100 μL of Stopping buffer (10% SDS + 0.01 M HCl) was added 4 h later. The OD550 was measured 16 h later. The experimental results showed that when the drug concentrations of Compound 1 and Compound 2 were 50 μM, there was no significant toxic effect compared with the control group (0 μM), and the cell survival rates were both greater than 90%, without obvious cytotoxic effect. At 50 μM, the cell survival rate of Compound 1 was 90.51%, and the cell survival rate of Compound 2 was 98.03%.

[0041] 2. Results and analysis of NO release amount determination

[0042] The release amount of NO is one of the important indicators for measuring the cell inflammation level. Therefore, NO inhibitors usually have the potential to be developed into anti-inflammatory drugs. The inhibitory effects of monomeric compound 1 and compound 2 on the release amount of NO in LPS-induced RAW264.7 cells at different concentrations (10 μM, 20 μM, 30 μM, 40 μM, 50 μM) were studied (the results are shown in Figure 5 b). RAW264.7 cells in the logarithmic growth phase were seeded in 96-well plates at an inoculation quantity of 1×10^5 cells / 100 μL / well. Different concentration drug groups were set as 0 μM (LPS group, model group), 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, with 3 replicates in each group. After inoculation, the cells were incubated overnight in a cell culture incubator. The next day, different concentrations of drugs (compound 1, compound 2 or positive drug L-NMA) were added to each test group. After 30 min, LPS (1 μg / mL) was added. After 24 h, 100 μL of the supernatant was taken from each well, 100 μL of Griess working solution (Sigma, catalog number MAK367) was added, and the OD 570 was measured after reacting at room temperature for 10 min. The experimental results showed that when the concentrations of compound 1 and compound 2 were 30 μM, the release amounts of NO in RAW264.7 cells were both greater than 50% and less than 75%, showing a certain inhibitory effect on the release of NO in RAW264.7 cells, and there was a significant difference compared with the model group (p < 0.05).

[0043] When the concentration increased to 50 μM, compound 1 and compound 2 significantly inhibited the release amount of NO in RAW264.7 cells, and their release amounts were both less than 61% (p < 0.05). The release amounts of NO in the compound 1 and compound 2 groups were 28.45% and 60.78% respectively. Compared with the model group, they could significantly inhibit the release amount of NO (p < 0.05). The release amounts of NO of the positive drug L-NMA at 30 μM, 40 μM and 50 μM were 66.27%, 58.73% and 55.28% respectively, all significantly higher than 55.28%, 43.75% and 28.45% of compound 1, indicating that the anti-inflammatory effect of compound 1 was significantly better than that of the positive drug L-NMA (Aladdin, catalog number N133898).

[0044] 3. Results and analysis of in vitro anti-inflammatory activity

[0045] In summary, the present invention uses an LPS-induced RAW264.7 macrophage inflammation model to measure the inhibitory effect of each compound on NO production and evaluate its anti-inflammatory activity. First, the cytotoxic effects of Compound 1 and Compound 2 on RAW264.7 cells at 0 μM, 10 μM, 20 μM, 30 μM, 40 μM, and 50 μM were evaluated, and combined with the inhibitory effects of Compound 1 and Compound 2 on NO production in RAW264.7 cells at 30 μM, 40 μM, and 50 μM. To ensure that the inhibitory effect of the monomeric compound on NO production is not caused by cytotoxic activity, based on this, the in vitro anti-inflammatory activities of Compound 1 and Compound 2 at 50 μM were further explored. The results showed that when the concentration of Compound 1 and Compound 2 was 50 μM, the cell viability was greater than 90%, and there was no significant difference compared with the blank control group( Figure 5 a), indicating that the above compounds had no obvious cytotoxicity at this concentration. As Figure 5 b, compared with the LPS group, Compound 1 and Compound 2 could significantly inhibit NO production at a concentration of 50 μM.

[0046] In summary, the cell viability tests of monomeric compounds 1 and 2 in mouse mononuclear macrophages (RAW264.7) at different concentrations (30 μM, 40 μM, and 50 μM) showed that the two compounds were non-toxic to cells at 50 μM. The results showed that Compound 1 had anti-inflammatory effects at 10 μM, while Compound 2 had anti-inflammatory effects at 20 μM. At 50 μM, both Compound 1 and Compound 2 had good anti-inflammatory effects. Among them, the anti-inflammatory effect of Compound 1 at 30-50 μM was better than that of the positive drug L-NMA.

Claims

1. A β-γ′ type lignan compound, characterized in that, The structure of the β-γ'-type lignan compounds is shown in Formula I or Formula II:

2. The preparation method of the β-γ′ type lignan compound according to claim 1, characterized in that, It includes the following steps: 1) The rhizome of Notopterygium incisum is crushed, extracted with an ethanol solution, and the extracts are combined and concentrated under reduced pressure to obtain a brown extract. The extract is suspended in pure water and successively extracted thoroughly with petroleum ether, ethyl acetate, and n-butanol to obtain an ethyl acetate extract. 2) The ethyl acetate extract is subjected to the first gradient elution with petroleum ether - ethyl acetate, and components are combined by thin-layer chromatography, a total of 3 components are obtained. Component 3 is subjected to silica gel column chromatography again and eluted with petroleum ether - ethyl acetate for the second gradient elution, and components are combined by thin-layer chromatography, a total of 3 sub-components 3.1 - 3.3 are obtained. Sub-component 3.2 is subjected to silica gel column chromatography again and eluted with petroleum ether - ethyl acetate for the third gradient elution, and components are combined by thin-layer chromatography to obtain 4 sub-components 3.2.1 - 3.2.4; Component 3.2.3 is prepared by preparative high-performance liquid chromatography to obtain 9 sub-components 3.2.3.1 - 3.2.3.

9. Component 3.2.3.8 is further prepared by preparative high-performance liquid chromatography to obtain Compound 1 of Formula I, and Component 3.2.3.9 is further prepared by preparative high-performance liquid chromatography to obtain Compound 2 of Formula II.

3. The preparation method of the β-γ'-type lignan compound according to claim 2, wherein, The mass-volume ratio of the rhizome of Notopterygium incisum to the ethanol solution is 1:2 - 1:10 kg / L.

4. The preparation method of the β-γ'-type lignan compound according to claim 2, wherein For the first gradient elution of petroleum ether - ethyl acetate, the volume ratios of petroleum ether to ethyl acetate are successively 100:0, 90:10, 80:20, 60:40, 40:60, and 0:100; for the second gradient elution of petroleum ether - ethyl acetate, the volume ratios of petroleum ether to ethyl acetate are successively 80:1, 50:1, 30:1, 15:1, 8:1, 6:1, 4:1, 3:1, 2:1, and 0:100; for the third gradient elution of petroleum ether - ethyl acetate, the volume ratios of petroleum ether to ethyl acetate are successively 80:1, 50:1, 30:1, 15:1, 8:1, 6:1, 4:1, 3:1, 2:1, and 0:

100.

5. The preparation method of the β-γ'-type lignan compound according to claim 2, characterized in that, The conditions for preparation by preparative high-performance liquid chromatography include: the detection wavelength is 254 nm, the chromatographic column is a reverse-phase C-18 chromatographic column, and methanol and water are the mobile phase; preferably, the peak time of Compound 1 is 28.1 min, and the peak time of Compound 2 is 31.3 min.

6. Use of the β-γ'-type lignan compound according to claim 1 in the preparation of an anti-inflammatory drug or in the preparation for preventing or treating inflammation or its related diseases.

7. The application according to claim 6, characterized in that, The inflammation includes the inflammation formed by LPS-induced RAW264.7 macrophages.

8. An anti-inflammatory drug, characterized in that, Its active ingredient includes one or two of the β-γ'-type lignan compounds according to claim 1.

9. The anti-inflammatory drug according to claim 8, wherein, The anti-inflammatory drug includes a single-agent or compound preparation.

10. The anti-inflammatory drug according to claim 8, characterized in that, The dosage form of the anti-inflammatory drug includes granules, capsules, tablets, powders, liquid preparations, or sprays.

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