Novel compound extracted and separated from salvia officinalis and application of novel compound in preparation of medicine for treating chronic inflammation

Abstract

The invention discloses a novel compound extracted and separated from salvia officinalis and application of the novel compound in preparation of a medicine for treating chronic inflammation, and belongs to the technical field of medicinal compounds. The invention discloses a novel compound which is extracted and separated from salvia japonica thunb. The novel compound is Aromasalvin A and Aromasalvin B. The novel compound is a novel compound which is extracted and separated from salvia japonica thunb. 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 new compound has the capability of inhibiting release of NO in RAW 264.7 macrophages induced by LPS, which indicates that the compounds have a good anti-inflammatory effect and have 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 particularly to new compounds isolated from Salvia przewalskii and their use in the preparation of drugs for treating chronic inflammation. Background Art

[0002] Salvia przewalskii, a perennial herb of the genus Salvia in the family Lamiaceae, is distributed in western Gansu, western Sichuan, northwestern Yunnan, Tibet and other places in China. As one of the medicinal material sources of Danshen in traditional Chinese medicine, its roots are used as medicine, also known as purple Danshen, which has the effects of promoting blood circulation to remove stasis, calming the mind, expelling pus and relieving pain, and can be used to treat angina pectoris, irregular menstruation, dysmenorrhea, amenorrhea, metrorrhagia, abdominal masses, stasis pain, bone pain, palpitation and insomnia, malignant sores and other diseases.

[0003] Inflammatory response is a self-regulatory mechanism in the body aimed at combating microbial infections or local injuries. Excessive inflammatory responses 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 new compounds isolated from Salvia przewalskii and their use in the preparation of drugs for treating chronic inflammation. Summary of the Invention

[0005] In view of this, the present invention provides new compounds isolated from Salvia przewalskii and their use in the preparation of drugs for treating chronic inflammation. Two new compounds with anti-inflammatory effects were isolated from Salvia przewalskii, named Aromasalvin A and Aromasalvin B; Aromasalvin A and Aromasalvin B are chiral compounds.

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

[0007] The new compound isolated from Salvia przewalskii, the new compound 1 is Aromasalvin A, and its chemical formula is C 21 H 26 O5; the new compound 2 is Aromasalvin B, and its chemical formula is C 21 H 26 O5; the structural formula is as follows:

[0008]

[0009] Furthermore, the preparation method of the new compound extracted and isolated from *Salvia przewalskii* Maxim. is as follows:

[0010] (1) Take 20.0 kg of dry roots of *Salvia przewalskii* Maxim., crush them, extract with 95% ethanol 3 times at room temperature, 200 L each time for 24 hours each time. 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 three times, 5 L each time, to obtain the petroleum ether-soluble part; then extract with ethyl acetate three times, 5 L each time, to obtain the ethyl acetate-soluble part;

[0012] (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, 0:1; by TLC plate 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 plate spotting, combine similar components to obtain twelve components, Fr.EA1~EA12;

[0014] (5) The component FrEA5 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 eluted with pure methanol by Sephadex LH-20 gel chromatography 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 11.7 min is collected to obtain the purified new monomer compound Aromasalvin A;

[0015] 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 eluted with pure methanol by Sephadex LH-20 gel chromatography to obtain the part to be purified. Finally, 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 7.9 min is collected to obtain the purified new monomer compound Aromasalvin B.

[0016] Furthermore, the use of the novel compound extracted and isolated from Salvia przewalskii Maxim. in the preparation of drugs for treating chronic inflammation.

[0017] As can be seen from the above technical solutions, compared with the prior art, the present invention discloses a novel compound extracted and isolated from Salvia przewalskii Maxim. and its use in the preparation of drugs for treating chronic inflammation. The present invention isolates two completely new compounds from Salvia przewalskii Maxim. These compounds have an inhibitory effect on the release of NO in lipopolysaccharide-induced RAW 264.7 macrophages, and have good development and application prospects for the preparation of drugs for treating chronic inflammation. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order 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 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, other drawings can be obtained according to the provided drawings without creative efforts.

[0019] Figure 1 It is the molecular structure diagram of the compounds Aromasalvin A and Aromasalvin B of the present invention; among them, 1 is Aromasalvin A, and 2 is Aromasalvin B;

[0020] Figure 2 It is the HRESIMS spectrum of the novel compound Aromasalvin A of the present invention;

[0021] Figure 3 It is the 1 1H-NMR spectrum (CD3OD, 500 MHz) of the novel compound Aromasalvin A of the present invention;

[0022] Figure 4 It is the 13 13C-NMR spectrum (CD3OD, 125 MHz) of the novel compound Aromasalvin A of the present invention;

[0023] Figure 5 It is the HSQC spectrum of the novel compound Aromasalvin A of the present invention;

[0024] Figure 6 It is the HMBC spectrum of the novel compound Aromasalvin A of the present invention;

[0025] Figure 7 It is the NOESY spectrum of the novel compound Aromasalvin A of the present invention;

[0026] Figure 8HRESIMS spectrum of the novel compound Aromasalvin B of the present invention;

[0027] Figure 9 of the novel compound Aromasalvin B of the present invention 1 1H-NMR spectrum (CD3OD, 500 MHz);

[0028] Figure 10 of the novel compound Aromasalvin B of the present invention 13 13C-NMR spectrum (CD3OD, 125 MHz);

[0029] Figure 11 HSQC spectrum of the novel compound Aromasalvin B of the present invention;

[0030] Figure 12 HMBC spectrum of the novel compound Aromasalvin B of the present invention;

[0031] Figure 13 NOESY spectrum of the novel compound Aromasalvin B of the present invention;

[0032] Figure 14 Experimental and calculated ECD spectra of the novel compounds Aromasalvin A and Aromasalvin B of the present invention; among them, 1 is Aromasalvin A and 2 is Aromasalvin B;

[0033] Figure 15 Detection results of TNF-α, IL-1β and IL-6 of the present invention. Detailed implementation manners

[0034] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying 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 shall fall within the protection scope of the present invention.

[0035] Example 1 Preparation of novel compounds

[0036] The preparation processes of compounds Aromasalvin A and Aromasalvin B are as follows:

[0037] Step 1: Take 20.0 kg of dried Salvia przewalskii Maxim. roots, crush them, extract with 95% ethanol (200 L each time) at room temperature for 3 times, 24 hours each time, draw the supernatant, combine the extracts, and concentrate under reduced pressure to obtain 1226 g of total extract;

[0038] Step 2: The total extract obtained in Step 1 was suspended in 5 L of water. It was first extracted three times with petroleum ether, 5 L each time, to obtain the petroleum ether-soluble fraction (412.1 g); then it was extracted three times with ethyl acetate, 5 L each time, to obtain the ethyl acetate-soluble fraction (491.9 g).

[0039] Step 3: The ethyl acetate-soluble fraction (491.9 g) obtained in Step 2 was separated by silica gel column chromatography, and gradient elution was carried out successively with petroleum ether-ethyl acetate at a volume ratio of 40:1, 20:1, 10:1, 5:1, 1:1, and 0:1. By TLC (thin-layer chromatography) spotting, similar components were combined to obtain eight fractions, Fr.EA~EH, which were separately concentrated under reduced pressure and dried for standby.

[0040] Step 4: The fraction Fr.EA (5.9 g) obtained in Step 3 was separated by silica gel column chromatography, and gradient elution was carried out successively with petroleum ether-ethyl acetate at a volume ratio of 80:1, 40:1, 20:1, 10:1, 5:1, and 0:1. By TLC spotting, similar components were combined to obtain twelve fractions, Fr.EA1~EA12.

[0041] Step 5: The fraction FrEA5 (0.9 g) obtained in Step 4 was separated by silica gel column chromatography 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 was purified by preparative high-performance liquid chromatography eluted with methanol-water (75:25 v / v) as the mobile phase, and the chromatographic peak with a retention time of 11.7 min was collected to obtain the purified new monomeric compound Aromasalvin A (4.4 mg).

[0042] The fraction FrEA8 (0.9 g) obtained in Step 4 was separated by silica gel column chromatography 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 fraction to be purified. Finally, it was purified by preparative high-performance liquid chromatography eluted with methanol-water (75:25 v / v) as the mobile phase, and the chromatographic peak with a retention time of 7.9 min was collected to obtain the purified new monomeric compound Aromasalvin B (6.6 mg).

[0043] Structure identification: High-resolution mass spectrometry (HRESIMS), nuclear magnetic spectra 1 HNMR, 13 13C NMR, HMQC, and HMBC, as well as calculated ECD were used to identify the structures of the two new compounds obtained in Step 5, and the results are shown in Figures 2 to 14 ; The nuclear magnetic spectrum data are shown in Table 1.

[0044] Table 1: 1 H NMR and 13 C NMR data ( 1 H 500 MHz, 13 C 125 MHz, in CD3OD, δ: ppm, J in Hz.)

[0045]

[0046]

[0047] Note: 1 is Aromasalvin A, 2 is Aromasalvin B.

[0048] After testing, the quasi-molecular ion peak of the new compound Aromasalvin A obtained in step 5 is m / z 359.1849 (calculated value 359.1853), and the speculated molecular formula is C 21 H 26 O5; the quasi-molecular ion peak of Aromasalvin B is m / z 359.1850 (calculated value 359.1853), and the speculated molecular formula is C 21 H 26 O5. Analyze the 1H NMR and 13C NMR data, determine the planar structure by combining with the 2D NMR spectra, and determine the stereostructure of the compound according to the NOESY spectra. The structural formulas of the two new compounds are as Figure 1 shown.

[0049] Example 2 Detection of the inhibitory effect of the new compounds Aromasalvin A and Aromasalvin B on the release of NO in LPS-induced RAW 264.7 macrophages

[0050] Step 1: Take RAW 264.7 macrophages in the exponential growth phase with good condition, add 0.25% trypsin digestive solution to digest and make the adherent cells detached, count to 1×10 5 cells / mL to make 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 concentration: 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) is used as a positive control.

[0051] Step 2: Take 50 μL of the culture supernatant and mix it with an equal volume of Griess reagent (composed of 0.1% naphthylamine, 1.0% sulfanilamide in 2.5% phosphoric acid solution), and incubate at room temperature for 10 minutes. Measure the absorbance at 550 nm using an automatic microplate reader. Determine the concentration of nitric oxide (NO) by referring to the sodium nitrite standard curve. The test results are shown in Table 2.

[0052] Table 2: Inhibitory effect of LPS-induced NO release in RAW 264.7 macrophages:

[0053]

[0054]

[0055] The results in Table 2 show that compounds Aromasalvin A and Aromasalvin B have an inhibitory effect on NO release in RAW264.7 macrophages induced by lipopolysaccharide (LPS), and have good application prospects in the treatment of chronic inflammation.

[0056] Example 3

[0057] Take RAW 264.7 macrophages in the exponential growth phase with good state, add 0.25% trypsin digestion solution to digest and make the adherent cells detach, count to 1×10 5 cells / mL to make a cell suspension. Inoculate the cell suspension 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 different concentrations of the test drugs, 20 μL / well (final concentration: 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, and culture in the incubator for 24 hours. Take 100 μL of the supernatant from each well, operate according to the operating procedure of the ELISA kits for TNF-α, IL-1β, and IL-6 (Wuhan elabscinence), measure the absorbance at 450 nm, draw a standard curve, and then calculate the concentration. The results are shown in Figure 15 .

[0058] Figure 15 The results show that both compounds 1 and 2 have a strong inhibitory ability on the expression of three inflammatory factors, namely TNF-α, IL-1β, and IL-6, showing strong anti-inflammatory activity and having extremely high potential value for the treatment of chronic inflammatory diseases.

[0059] The foregoing 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 readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A new compound extracted and isolated from Salvia przewalskii Maxim., characterized in that, The new compound 1 is Aromasalvin A, and its chemical formula is C 21 H 26 O5; the new compound 2 is Aromasalvin B, and its chemical formula is C 21 H 26 O5; the structural formula is as follows:

2. The preparation method of the new compound extracted and isolated from Salvia przewalskii Maxim., as described in claim 1, characterized in that, The specific steps are as follows: (1) Take 20.0 kg of dried 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, 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; (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; (5) The component FrEA5 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 11.7 min is collected to obtain the purified new monomer compound Aromasalvin A; 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 part to be purified. Finally, 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 7.9 min is collected to obtain the purified new monomer compound Aromasalvin B.

3. Use of the new compound extracted and separated from Salvia przewalskii described in claim 1 in the preparation of a drug for treating chronic inflammation.

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