Lignan-acyl quinic acid heterodimer compound and application thereof

Abstract

The invention relates to the technical field of chemistry, and discloses a lignan-acyl quinic acid heterodimer compound and application thereof. The lignan-acyl quinic acid heterodimer compound comprises a new compound 1, and the compound is obtained by extracting, separating and purifying a traditional Chinese medicine cortex acanthopanacis, and is named as acanthopacid (1). A biological activity test shows that the compound 1 has obvious inhibitory activity on neutrophil elastinase (NE) and epoxidase-2 (COX-2), which indicates that the compound 1 can be used as a new anti-inflammatory drug component or a lead compound. The invention also provides a pharmaceutically acceptable pharmaceutical composition containing a therapeutically effective amount of the compound 1, and application of the compound in preparation of anti-inflammatory drugs. The preparation method, structural identification and pharmacological action of the compound 1 are described in detail through the embodiment, and a new strategy is provided for treatment of inflammation-related diseases.

Description

Technical Field

[0001] The present invention relates to the field of chemical technology, and in particular to a lignan-acyl quinic acid heterodimer compound and its application. Background Art

[0002] Rheumatic diseases refer to a class of chronic inflammatory diseases related to the human muscle, bone, and joint systems, such as rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, and gout. Currently, about 200 types of rheumatic diseases have been discovered, and the global prevalence rate exceeds 6%. Rheumatic diseases have become one of the major public health challenges faced by the world today. Anti-inflammatory is one of the main strategies recognized for the treatment of rheumatic diseases. Neutrophil elastase (NE) and cyclooxygenase inhibitors, especially selective cyclooxygenase-2 (COX-2) inhibitors, have been approved by the US Food and Drug Administration for the clinical treatment of rheumatic diseases because they can inhibit the synthesis of prostaglandins and have weaker side effects than non-steroidal anti-inflammatory drugs. Traditional Chinese medicine often has the characteristics of multiple effects and bidirectional regulation in the treatment of inflammation, as well as advantages such as fewer adverse reactions and richer sources. It has attracted more and more attention, and the search for and development of traditional Chinese medicine with anti-inflammatory effects has gradually become a hot topic.

[0003] Traditional Chinese medicine often has the characteristics of multiple effects and bidirectional regulation in the treatment of inflammation, as well as advantages such as fewer adverse reactions and richer sources. It has attracted more and more attention, and the search for and development of traditional Chinese medicine with anti-inflammatory effects has gradually become a hot topic.

[0004] The cortex acanthopanacis is the dried root bark of Acanthopanax gracilistylus W.W.Smith of the Araliaceae family. The 2015 edition of the Chinese Pharmacopoeia records that it has the effects of dispelling wind and dampness, and strengthening muscles and bones, and is mainly used for treating rheumatic arthralgia and flaccidity of muscles and bones. It is clinically commonly used for the treatment of rheumatoid arthritis. In the past 30 years, domestic and foreign studies on the chemical constituents of the cortex acanthopanacis have shown that it mainly contains kaurane-type diterpenoids; so far, more than 30 such compounds have been isolated and identified from the cortex acanthopanacis, and the sesquiterpenoid components in the cortex acanthopanacis have not been reported before.

[0005] In the process of systematically and deeply studying the chemical constituents and pharmacological effects of the cortex acanthopanacis, the present invention first discovered a lignan-acyl quinic acid heterodimer that has obvious inhibitory activity on both neutrophil elastase (NE) and cyclooxygenase-2 (COX-2), and identified the structure of this new compound. So far, there is no report on acanthopacid (1), no report on a pharmaceutical composition with compound 1 as an active ingredient, and no report on the use of compound 1 and its composition for anti-inflammatory. Summary of the Invention

[0006] The object of the present invention is to provide a new lignan-acyl quinic acid heterodimer with medicinal value and the application of compound 1 and its pharmaceutical composition in the treatment or improvement of inflammation-related diseases.

[0007] A kind of lignan-acyl quinic acid heterodimer compound provided by the present invention includes a new compound 1, named acanthopacid (1). This compound 1 has obvious inhibitory activities on neutrophil elastase (NE) and cyclooxygenase-2 (COX-2) at the same time. The structural formula of this compound 1 is

[0008]

[0009] Its molecular formula is C 26 H 28 O 14 .

[0010] Preferably, the compound 1 is extracted and separated from Cortex Acanthopanacis Radicis.

[0011] The present invention provides a preparation method of the above-mentioned compound 1, including the following steps:

[0012] S1. Crush the dried Cortex Acanthopanacis Radicis and soak it in 70% acetone at room temperature for 3 times, each time for 72 hours;

[0013] S2. Combine the soaking solutions, concentrate under reduced pressure to an extract, suspend the extract in an appropriate amount of water, and extract it 3 times with equal volumes of ethyl acetate and n-butanol respectively to obtain an ethyl acetate part (Fr.A) and a n-butanol part (Fr.B);

[0014] S3. Subject the n-butanol part to D101 macroporous resin column chromatography, elute it successively with water–methanol in proportion (v / v, 100:0, 50:50, 0:100), check by TLC and combine the same fractions to obtain 3 fractions of Frs.B-1 to Frs.B-3;

[0015] S4. Fr.B-2 (109.5 g) is subjected to silica gel column chromatography (gradient elution with dichloromethane–methanol) to obtain 4 fractions (Frs.B-2-1 to Frs.B-2-4);

[0016] S5. After concentration, purify it by semi-preparative liquid phase (Kromasil 100-5-Phenyl, 10×250 mm 2 , 3 mL / min), then analyze it by silica gel column (gradient elution with dichloromethane–methanol), and perform semi-preparative HPLC with acetonitrile–water (20:80) as the mobile phase to obtain compound 1;

[0017] Preferably, in the step S1, the amount of Cortex Acanthopanacis Radicis used is 15 kg.

[0018] Preferably, in the step S1, when the Acanthopanax cortex powder is soaked in 70% acetone at room temperature, the amount of acetone used is preferably 80 L each time.

[0019] Preferably, in the step S2, the amount of water used when the extract is suspended in an appropriate amount of water is preferably 5 L.

[0020] The present invention provides an application of a lignan-acyl quinic acid heterodimer compound as described above in anti-inflammatory activity. The compound 1 has obvious inhibitory activities on neutrophil elastase (NE) and cyclooxygenase-2 (COX-2), and can be used as a new drug ingredient or lead compound with anti-inflammatory effects.

[0021] Compared with the related technologies, the lignan-acyl quinic acid heterodimer compound provided by the present invention has the following beneficial effects:

[0022] The in vitro anti-inflammatory activity test results of the compound described in the present invention show that the compound 1 has good inhibitory effects on both NE and COX-2. When the concentration of the compound 1 is 200 μM, the inhibitory activities on NE and COX-2 are 80% and 93.7% respectively, and the IC 50 of COX-2 is 0.45 ± 0.018 μM. The results show that the compound 1 can be used as a new drug ingredient or lead compound with anti-inflammatory effects;

[0023] The compound 1 described in the present invention can be made into any pharmaceutically acceptable dosage form, such as tablets, dripping pills, ointments, sprays, capsules, etc., and excipients commonly used in the pharmaceutical industry, such as flavoring agents, disintegrants, solubilizers, buffers, etc., can be added. Description of the Drawings

[0024] Figure 1 is the structural formula of the compound 1 proposed by the present invention;

[0025] Figure 2 is the 1 1H-NMR spectrum of the compound 1 proposed by the present invention;

[0026] Figure 3 is the 13 13C-NMR spectrum of the compound 1 proposed by the present invention;

[0027] Figure 4 is the HSQC spectrum of the compound 1 proposed by the present invention;

[0028] Figure 5 is the HMBC spectrum of the compound 1 proposed by the present invention;

[0029] Figure 6It is the COSY spectrum of Compound 1 proposed by the present invention;

[0030] Figure 7 It is the ROESY spectrum of Compound 1 proposed by the present invention;

[0031] Figure 8 It is the HR-ESI-MS spectrum of Compound 1 proposed by the present invention. Detailed implementation manners

[0032] To better understand the present invention, the preparation method, structure identification, and pharmacological effects of the compound acanthopacid (1) of the present invention will be further described below in combination with test data and drawings using the test examples and examples of the present invention, but the present invention is not limited by these test examples and examples.

[0033] A lignan-acyl quinic acid heterodimer compound proposed by the present invention includes Compound 1, and the structural formula of this Compound 1 is as Figure 1 shown, and its molecular formula is C 26 H 28 O 14 .

[0034] Example 1:

[0035] Preparation of Compound 1:

[0036] 15 kg of dried Acanthopanax cortex was crushed and soaked in 70% acetone at room temperature 3 times (80 L / time), and each soaking was for 72 h. The soaking solutions were combined and concentrated under reduced pressure to an extract, and then the extract was suspended in an appropriate amount of water (5 L). After extracting 3 times with equal amounts of ethyl acetate and n-butanol respectively, the ethyl acetate part (Fr.A) and the n-butanol part (Fr.B) were obtained. Fr.B was separated by a D101 macroporous resin column [eluted successively with water–methanol in proportion (v / v, 100:0, 50:50, 0:100)] to obtain Frs.B-1–3. Fr.B-2 (109.5 g) was subjected to silica gel column chromatography (gradient elution with dichloromethane–methanol) to obtain 4 components (Frs.B-2-1 to Frs.B-2-4). After concentration, it was purified by semi-preparative liquid phase (Kromasil 100-5-Phenyl, 10×250 mm 2 , 3 mL / min), and then analyzed by a silica gel column (gradient elution with dichloromethane–methanol). After semi-preparative HPLC with acetonitrile–water (20:80) as the mobile phase, Compound 1 was obtained.

[0037] Example 2:

[0038] Structure identification of Compound 1:

[0039] The high-resolution mass spectrometry was determined by an IT-TOF-LC-MS (Shimadzu, Kyoto, Japan) mass spectrometer. The nuclear magnetic resonance spectrum was determined by a Brucker Avance III-600 superconducting nuclear magnetic resonance spectrometer (Bruker, Bremerhaven, Germany). TMS (tetramethylsilane) was used as the internal standard. The optical rotation was measured by a Jasco model 1020 polarimeter (Horiba, Tokyo, Japan). The ECD spectrum was measured by a circular dichroism spectrometer (Chirascan, Applied Photophysics Ltd, UK).

[0040] Compound 1, with the molecular formula C 26 H 28 O 14 , and the HRESIMS (+) m / z was 565.1708 [M+H] + .

[0041] 1 The 1H-NMR and 13 13C-NMR data are shown in Table 1.

[0042] Table 1. The 1 1H-NMR and 13 13C-NMR data of Compound 1

[0043]

[0044]

[0045] 1a: UV (MeOH) λ max (logε): 337, 292.

[0046] Example 3

[0047] (1) In vitro inhibitory activity test of the compound against NE:

[0048] 100 μL of phosphate buffer (0.05 M, pH = 7.8), 50 μL of the sample (200 μM, 10% DMSO), and 50 μL of N-succinyl-alanine-alanine-alanine-p-nitroaniline (100 μM, the above phosphate buffer) were sequentially added to a 96-well cell culture plate. Incubate at room temperature for 5 minutes, and then quickly add 50 μL of NE solution (0.02 U / mL, 0.1 M phosphate buffer, pH = 6.0) to the 96-well cell culture plate to initiate the reaction. After adding NE, the ultraviolet absorbance was detected, and the absorbance value was set at 405 nm. The inhibition percentage was calculated by the following formula:

[0049]

[0050] wherein, ΔA C and ΔA S are the differences in absorbance between the control group and the sample group between 0 minute and 5 minutes, respectively.

[0051] In vitro inhibitory activity test of compounds against COX-2

[0052] According to the operation instructions, prepare the working solutions of COX-2, COX-2 Cofactor, and COX-2 Substrate. Add 80 μL of COX-2 Assay Buffer, 5 μL of COX-2 Cofactor, and 5 μL of methanol to the 96-well black plate in sequence as the blank control group; add 75 μL of COX-2 Assay Buffer, 5 μL of COX-2 Cofactor, 5 μL of COX-2, and 5 μL of (methanol, celecoxib, sample) to the wells in sequence as the 100% enzyme activity control, positive inhibitor control, and sample group, respectively. Subsequently, incubate in an oven at 37 °C for 10 minutes, and then add 5 μL of COX-2 Probe (fluorescent probe) and the COX-2 Substrate working solution to each well in sequence. After incubating in the dark at 37 °C for 5 minutes, measure the fluorescence value using a microplate reader. The excitation wavelength is 560 nm, and the emission wavelength is 590 nm. The inhibition rate (%) is calculated by the following formula:

[0053]

[0054] where RFU E , RFU S and RFU B are the fluorescence values of the control group, the sample group, and the blank group, respectively.

[0055] The results of the activity test show that when the concentration of Compound 1 is 200 μM, it can produce inhibitory activities of 80% and 93.7% against NE and COX-2, respectively, and the IC 50 of Compound 1 against COX-2 is 0.45 ± 0.018 μM. The results indicate that Compound 1 can be used as a new drug ingredient or lead compound with anti-inflammatory effects.

[0056] In addition to the above embodiments, the present invention may have other embodiments. Any equivalent replacements and improvements made within the spirit and principle of the present invention shall be included within the scope of protection required by the present invention.

Claims

1. A lignan-acylquinic acid heterodimer compound, characterized in that: The novel compound 1 has a significant inhibitory activity on both neutrophil elastase (NE) and cyclooxygenase-2 (COX-2). The structural formula of the compound 1 is Its molecular formula is C 26 H 28 O 14 .

2. The lignan-acylquinic acid heterodimer compound according to claim 1, characterized in that: The compound 1 is extracted and separated from the Cortex Acanthopanacis.

3. A method for preparing compound 1 according to claim 1-2, characterized in that: The steps include: S1. Crush the dried Acanthopanax acanthopanax bark and soak it in 70% acetone at room temperature for 3 times, each time for 72 hours; S2, combine the soaking liquid, concentrate under reduced pressure to obtain an extract, suspend the extract in an appropriate amount of water, and extract with equal amounts of ethyl acetate and n-butanol three times respectively to obtain the ethyl acetate part, i.e., Fr.A, and the n-butanol part, i.e., Fr.B; S3. The n-butanol portion was chromatographed on a D101 macroporous resin column and eluted with water-methanol in a ratio of 100:0, 50:50, and 0:100 (v / v). After TLC inspection and merging of the same fractions, three components, Frs.B-1 to Frs.B-3, were obtained. S4 and Fr.B-2 (109.5 g) were chromatographed on a silica gel column (gradient elution with dichloromethane-methanol) to obtain four components (Frs.B-2-1 to Frs.B-2-4); S5, after concentration, semi-preparative liquid purification (Kromasil 100-5-Phenyl, 10×250mm 2 , 3 mL / min), and then analyzed by silica gel column (dichloromethane-methanol gradient elution). Semi-preparative HPLC with acetonitrile-water (20:80) as the mobile phase gave compound 1.

4. The method for preparing compound 1 according to claim 3, characterized in that: In the step S1, the amount of Acanthopanax acanthopanax used is 15 kg.

5. The method for preparing compound 1 according to claim 3, characterized in that: In the step S1, when the powdered Acanthopanax acanthopanax bark is soaked in 70% acetone at room temperature, the amount of acetone used is preferably 80 L / time.

6. The method for preparing compound 1 according to claim 3, characterized in that: In the step S2, the amount of water used when the extract is suspended in a suitable amount of water is preferably 5 L.

7. Use of the lignan-acylquinic acid heterodimer compound as claimed in claim 1 in anti-inflammatory activity, characterized in that: The compound 1 has obvious inhibitory activity on neutrophil elastase (NE) and cyclooxygenase-2 (COX-2), and can be used as a new drug component or lead compound with anti-inflammatory effect.

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