Compound Aromasalvin H and application thereof in preparation of medicine for treating chronic inflammation

Abstract

The invention discloses a compound Aromasalvin H and application of the compound Aromasalvin H in preparation of a medicine for treating chronic inflammation, and belongs to the technical field of medicinal compounds. The invention discloses a compound Aromasalvin H, and the chemical formula of the compound Aromasalvin H is C21H24O5. The structure of the novel monomeric compound is identified by comprehensively adopting high-resolution mass spectrum, spectrum analysis and ECD calculation. A biological activity test result shows that the compound has the capability of inhibiting release of NO in RAW 264.7 macrophages induced by LPS, so that the compound has a good anti-inflammatory effect and has a good development and application prospect in treatment of inflammatory diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of medicinal compounds, and more specifically, to a new compound Aromasalvin H isolated from Salvia przewalskii Maxim. and its application in the preparation of drugs for treating chronic inflammation. Background Art

[0002] Salvia przewalskii Maxim., a perennial herb of the genus Salvia L. in the Lamiaceae family, is distributed in western Gansu, western Sichuan, northwestern Yunnan, Tibet and other places in China. As one of the medicinal material sources of traditional Chinese medicine Salvia miltiorrhiza Bunge, its root is used as medicine, also known as purple Salvia miltiorrhiza, which has the effects of promoting blood circulation to remove blood stasis, calming the mind, expelling pus and relieving pain, and can be used to treat angina pectoris, irregular menstruation, dysmenorrhea, amenorrhea, metrorrhagia and leukorrhagia, mass in the abdomen, accumulation, pain due to blood stasis, pain in joints, palpitation and insomnia, malignant sores and toxic swelling and other diseases.

[0003] Inflammatory response is a self-regulatory mechanism in the body aimed at combating microbial infection or local injury. Excessive inflammatory response can lead to tissue damage and the onset of autoimmune diseases, such as rheumatoid arthritis, inflammatory bowel disease, atherosclerosis, and even cancer. During the pathogenic process, monocytes in the blood differentiate into macrophages. Activated macrophages release pro-inflammatory factors, such as TNF-α, IL-6 and the inflammatory mediator nitric oxide (NO) to regulate the host defense mechanism. Based on this, it is of great significance to invent a drug for treating chronic inflammatory diseases by inhibiting the release of pro-inflammatory factors.

[0004] Therefore, it is an urgent problem for those skilled in the art to provide compound Aromasalvin H and its application in the preparation of drugs for treating chronic inflammation. Summary of the Invention

[0005] In view of this, the present invention provides compound Aromasalvin H and its application in the preparation of drugs for treating chronic inflammation.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] Compound Aromasalvin H, its chemical formula is C 21 H 24 O5; the structural formula is as follows:

[0008]

[0009] Furthermore, the preparation method of the compound Aromasalvin H is as follows in specific steps:

[0010] (1) Take 20.0 kg of dry Salvia przewalskii roots, crush them, and extract with 95% ethanol 3 times at room temperature, 200 L each time for 24 hours. Absorb the supernatant, combine the extracts, and concentrate under reduced pressure to obtain the total extract;

[0011] (2) Suspend the total extract obtained in step (1) in 5 L of water, first extract with petroleum ether 3 times, 5 L each time, to obtain the petroleum ether-soluble fraction; then extract with ethyl acetate 3 times, 5 L each time, to obtain the ethyl acetate-soluble fraction;

[0012] (3) Separate the ethyl acetate-soluble fraction obtained in step (2) by silica gel column chromatography, and perform gradient elution successively with petroleum ether - ethyl acetate at a volume ratio of 40:1, 20:1, 10:1, 5:1, 1:1, 0:1; by TLC spotting, combine similar components to obtain eight components, Fr.EA~EH, and concentrate and dry them under reduced pressure respectively for standby;

[0013] (4) The component Fr.EA obtained in step (3) is separated by silica gel column chromatography, and gradient elution is performed successively with petroleum ether - ethyl acetate at a volume ratio of 80:1, 40:1, 20:1, 10:1, 5:1, 0:1. By TLC spotting, combine similar components to obtain twelve components, Fr.EA1~EA12;

[0014] (5) The component FrEA8 obtained in step (4) is separated by silica gel column chromatography and eluted with petroleum ether - ethyl acetate at a volume ratio of 20:1, and then eluted by Sephadex LH-20 gel chromatography with pure methanol to obtain the extract to be purified. Subsequently, it is purified by preparative high-performance liquid chromatography with 75:25 v / v methanol - water as the mobile phase for elution, and the chromatographic peak with a retention time of 9.8 min is collected to obtain the purified new monomer compound Aromasalvin H.

[0015] Furthermore, the application of the compound Aromasalvin H in the preparation of drugs for treating chronic inflammation.

[0016] Through the above technical solutions, compared with the prior art, the present invention discloses the compound Aromasalvin H and its application in the preparation of drugs for treating chronic inflammation. A brand-new compound Aromasalvin H is isolated from Salvia przewalskii. The compound Aromasalvin H has an inhibitory effect on the release of NO in lipopolysaccharide-induced RAW 264.7 macrophages, and has good development and application prospects for the preparation of drugs for treating chronic inflammation. Description of the Drawings

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on the provided drawings.

[0018] Figure 1 It is the molecular structure diagram of the novel compound Aromasalvin H of the present invention;

[0019] Figure 2 It is the HRESIMS spectrum of the novel compound Aromasalvin H of the present invention;

[0020] Figure 3 It is for the novel compound Aromasalvin H of the present invention 1 1H-NMR spectrum (CD3OD, 500 MHz);

[0021] Figure 4 It is for the novel compound Aromasalvin H of the present invention 13 13C-NMR spectrum (CD3OD, 125 MHz);

[0022] Figure 5 It is the HSQC spectrum of the novel compound Aromasalvin H of the present invention;

[0023] Figure 6 It is the HMBC spectrum of the novel compound Aromasalvin H of the present invention;

[0024] Figure 7 It is the NOESY spectrum of the novel compound Aromasalvin H of the present invention;

[0025] Figure 8 It is the detection results of TNF-α, IL-1β and IL-6 of the present invention. Detailed implementation manners

[0026] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0027] Example 1 Preparation of a novel compound

[0028] The preparation process of the compound Aromasalvin H is as follows:

[0029] Step 1: Take 20.0 kg of dry Salvia przewalskii roots, crush them, extract with 95% ethanol (200 L each time) at room temperature for 3 times, 24 hours each time. Absorb the supernatant, combine the extracts, and concentrate under reduced pressure to obtain 1226 g of total extract.

[0030] Step 2: Suspend the total extract obtained in Step 1 in 5 L of water. First, extract with petroleum ether three times, 5 L each time, to obtain the petroleum ether-soluble fraction (412.1 g); then extract with ethyl acetate three times, 5 L each time, to obtain the ethyl acetate-soluble fraction (491.9 g).

[0031] Step 3: Separate the ethyl acetate-soluble fraction (491.9 g) obtained in Step 2 by silica gel column chromatography, and perform gradient elution successively with petroleum ether-ethyl acetate at a volume ratio of 40:1, 20:1, 10:1, 5:1, 1:1, 0:1. After TLC (thin-layer chromatography) spotting, combine similar components to obtain eight fractions, Fr.EA~EH, and concentrate and dry them under reduced pressure respectively for standby.

[0032] Step 4: The fraction Fr.EA (5.9 g) obtained in Step 3 is separated by silica gel column chromatography, and gradient elution is performed successively with petroleum ether-ethyl acetate at a volume ratio of 80:1, 40:1, 20:1, 10:1, 5:1, 0:1. After TLC spotting, combine similar components to obtain twelve fractions, Fr.EA1~EA12.

[0033] Step 5: The fraction FrEA8 (0.9 g) obtained in Step 4 is separated by silica gel column chromatography with petroleum ether-ethyl acetate elution at a volume ratio of 20:1, and then eluted by Sephadex LH-20 gel chromatography with pure methanol to obtain the extract to be purified. Subsequently, it is purified by preparative high-performance liquid chromatography with methanol-water (75:25 v / v) as the mobile phase for elution, and the chromatographic peak with a retention time of 9.8 min is collected to obtain the purified new monomer compound Aromasalvin H (4.2 mg).

[0034] Structure identification: High-resolution mass spectrometry (HRESIMS), nuclear magnetic spectra 1 HNMR, 13 C NMR, HMQC and HMBC, as well as calculated ECD are used to identify the structure of the new compound Aromasalvin H obtained in Step 5, and the results are shown in Figures 2 to 7 ; The nuclear magnetic spectrum data are shown in Table 1.

[0035] Table 1: 1 HNMR and 13 CNMR data of compound Aromasalvin H

[0036]

[0037]

[0038] After testing, the quasi-molecular ion peak of Aromasalvin H obtained in step 5 was m / z 355.1551 (calculated value 355.1540), and the speculated molecular formula was C 21 H 24 O5. The 1H NMR and 13C NMR data were analyzed, and the planar structure was determined by combining with 2D NMR spectra. The stereostructure of the compound was determined according to the NOESY spectrum, and a new compound Aromasalvin H was obtained. The structural formula was as shown in Figure 1 shown.

[0039] Detection of the inhibitory effect of the new compound Aromasalvin H on the release of NO in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages in Example 2

[0040] Step 1: Take RAW 264.7 macrophages in the exponential growth phase with good condition, add 0.25% trypsin digestion solution to digest and make the adherent cells detached, count to 1×10 5 cells / mL to prepare a cell suspension. Take the cell suspension and inoculate it on a 24-well plate, 500 μL / well, place it at 37 °C, and culture it in a CO2 incubator for 24 hours. After changing the medium, add the test drugs at different concentrations, 20 μL / well (final concentrations: 40, 20, 10, 5, 2.5 μM), and culture for 2 hours. Add LPS (final concentration 2 μg / ml) to the 24-well plate, 20 μL / well, culture in the incubator for 24 hours, and take the culture supernatant and store it at -20 °C for later use. L-NMMA (non-selective nitric oxide synthase inhibitor) was used as a positive control.

[0041] Step 2: Take 50 μL of the culture supernatant and mix it with an equal volume of Griess reagent (composed of 0.1% naphthylamine and 1.0% sulfanilamide in 2.5% phosphoric acid solution), and incubate at room temperature for 10 minutes. Measure the absorbance at 550 nm with an automatic microplate reader. The concentration of nitric oxide (NO) was determined by referring to the sodium nitrite standard curve. The test results are shown in Table 2.

[0042] Table 2: Inhibitory effect on the release of NO in LPS-induced RAW 264.7 phagocytes:

[0043]

[0044] The results showed that the compound Aromasalvin H had an inhibitory effect on the release of NO in LPS-induced RAW 264.7 macrophages, and had good application prospects in the treatment of chronic inflammation.

[0045] Example 3

[0046] RAW 264.7 macrophages in the exponential growth phase and in good condition were taken, and 0.25% trypsin digestion solution was added for digestion to make the adherent cells detached. The cells were counted at 1×10 5 cells / mL to prepare a cell suspension. The cell suspension was inoculated on a 24-well plate, 500 μL / well, and cultured in a CO2 incubator at 37 °C for 24 hours. After changing the medium, different concentrations of the test drug were added, 20 μL / well (final concentration: 10, 5, 2.5 μM), and cultured for 2 hours. LPS (final concentration 2 μg / mL) was added to the 24-well plate, 20 μL / well, and cultured in the incubator for 24 hours. 100 μL of the supernatant was taken from each well, and the operation process of the ELISA kits for TNF-α, IL-1β and IL-6 (Wuhan elabscinence) was followed, and the absorbance was measured at 450 nm. A standard curve was plotted, and then the concentration was calculated. The results are shown in Figure 8 .

[0047] Figure 8 The results showed that compound Aromasalvin H had a strong inhibitory ability on the expression of the three inflammatory factors, namely TNF-α, IL-1β and IL-6, exhibited strong anti-inflammatory activity, and had extremely high potential value for the treatment of chronic inflammatory diseases.

[0048] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Compound Aromasalvin H, characterized in that, Its chemical formula is C 21 H 24 O5; The structural formula is as follows:

2. The preparation method of the compound Aromasalvin H according to claim 1, characterized in that, The specific steps are as follows: (1) Take 20.0 kg of dry Salvia przewalskii roots, crush them, and extract them with 95% ethanol 3 times at room temperature, 200 L each time for 24 hours. Absorb the supernatant, combine the extracts, and concentrate under reduced pressure to obtain the total extract. (2) Suspend the total extract obtained in step (1) in 5 L of water. First, extract with petroleum ether 3 times, 5 L each time, to obtain the petroleum ether-soluble part; then extract with ethyl acetate 3 times, 5 L each time, to obtain the ethyl acetate-soluble part. (3) Separate the ethyl acetate-soluble part obtained in step (2) by silica gel column chromatography, and perform gradient elution successively with petroleum ether-ethyl acetate at a volume ratio of 40:1, 20:1, 10:1, 5:1, 1:1, and 0:

1. After TLC spotting, combine similar components to obtain eight components, Fr.EA~EH, and concentrate and dry them under reduced pressure respectively for standby. (4) The component Fr.EA obtained in step (3) is separated by silica gel column chromatography, and gradient elution is performed successively with petroleum ether-ethyl acetate at a volume ratio of 80:1, 40:1, 20:1, 10:1, 5:1, and 0:

1. After TLC spotting, combine similar components to obtain twelve components, Fr.EA1~EA12. (5) The component FrEA8 obtained in step (4) is separated by a silica gel chromatographic column and eluted with petroleum ether-ethyl acetate at a volume ratio of 20:1, and then eluted with Sephadex LH-20 gel chromatography with pure methanol to obtain the extract to be purified. Subsequently, it is purified by preparative high-performance liquid chromatography with 75:25 v / v methanol-water as the mobile phase for elution, and the chromatographic peak with a retention time of 9.8 min is collected to obtain the purified new monomer compound Aromasalvin H.

3. Use of the compound Aromasalvin H according to claim 1 in the preparation of a drug for treating chronic inflammation.

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