Xinjiang Ferula extract, preparation method and application thereof

Abstract

The present invention discloses new compounds (7R, 8R)-sinkiangenoneE and (7R, 8S)-sinkiangenoneE isolated from Xinjiang Ferula and their applications in the preparation of anti-inflammatory drugs, belonging to the field of medical technology. The compounds (7R, 8R)-sinkiangenoneE and (7R, 8S)-sinkiangenoneE disclosed in the present invention show good activity in anti-inflammatory aspects. Compared with L-NAME, the expression of pro-inflammatory cytokines TNF-α, IL-1β and IL-6 produced by LPS-induced RAW264.7 cells can be significantly inhibited. The above two compounds can be used to prepare TNF-α, IL-1β and IL-6 inhibitors, which have an important effect on the research and development of anti-inflammatory drugs and can be used to treat chronic inflammatory diseases such as gastritis, enteritis and rheumatoid arthritis.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, in particular to a compound obtained from Xinjiang Ferula extract, a preparation method thereof and an application thereof in preparing anti-inflammatory drugs. Background Art

[0002] Inflammation underlies many physiological and pathological processes. Inflammation-inducing pathogen-associated molecular patterns (PAMPs) include highly conserved structures such as lipopolysaccharide (LPS), heat shock proteins (HSPs), peptidoglycans (PGNs), and cytosine phosphate guanine motifs. PAMPs are recognized by pattern recognition receptors (PRRs), which, once activated, transduce signals within the cell. Furthermore, they induce, produce, and release proinflammatory mediators such as TNF-α, interleukin-1 (IL-1), and interleukin-6 (IL-6). These proinflammatory cytokines are associated with numerous diseases, such as cardiovascular disease, osteoporosis, diabetes, and cancer. Anti-inflammatory therapies are particularly important in these conditions. Clinically, steroidal anti-inflammatory drugs (SAIDS) and nonsteroidal anti-inflammatory drugs (NSAIDS) are commonly used to treat inflammatory diseases. SAIDS generally refer to steroid hormones, including corticosteroids and glucocorticoids. These drugs have potent anti-inflammatory effects but are associated with serious side effects, such as water and sodium retention and infection. Nonsteroidal anti-inflammatory drugs (NSAIDs) are a class of anti-inflammatory drugs that do not contain a steroidal structure and include aspirin, naproxen, diclofenac, and ibuprofen. While they are well tolerated, their long-term use is limited by severe gastrointestinal adverse reactions, including the potential for gastrointestinal bleeding and perforation. Therefore, the discovery of new, low-cost alternatives with minimal side effects is urgent.

[0003] Xinjiang Ferula is a perennial herbaceous plant of the genus Ferula in the family Apiaceae. Its medicinal part, listed in the Chinese Pharmacopoeia as its resin, is a traditional folk medicine native only to the arid desert regions of Xinjiang, my country. It is commonly used in the region to treat inflammatory conditions such as rheumatoid arthritis, bronchitis, and gastrointestinal digestion. In recent years, scholars both domestically and internationally have conducted preliminary research on the anti-inflammatory effects of Ferula, but its composition and mechanism remain unclear.

[0004] Therefore, extracting compounds with high activity of TNF-α, IL-1 and IL-6 inhibitory effects from Xinjiang Ferula to achieve drug development and treatment of chronic inflammatory diseases is a technical problem that needs to be solved urgently by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides a method for extracting compounds (7R, 8R)-sinkiangenoneE and (7R, 8S)-sinkiangenoneE from Xinjiang Ferula and application of the method in preparing anti-inflammatory drugs.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] A preparation method of Xinjiang Ferula extract comprises the following steps:

[0008] (1) extracting Xinjiang Ferula resin by reflux extraction with ethanol, combining the extracts and filtering, and concentrating the filtrate under reduced pressure to a thick extract to obtain Xinjiang Ferula resin alcohol extract;

[0009] (2) The alcohol extract obtained in step (1) was extracted with dichloromethane, and then subjected to silica gel column chromatography with a gradient elution of petroleum ether-ethyl acetate and dichloromethane-methanol to obtain six components Fr.1 to 6; Fr.6 was subjected to semi-preparative liquid chromatography with an isocratic elution of methanol-water to obtain two compounds;

[0010] (3) The two compounds were identified using modern spectral technology and named (7R,8R)-sinkiangenoneE and (7R,8S)-sinkiangenoneE, which are Xinjiang Ferula extracts.

[0011] Furthermore, the structure of the (7R, 8R)-sinkiangenoneE is shown in Formula I, and the structure of the (7R, 8S)-sinkiangenoneE is shown in Formula II:

[0012]

[0013] The ethanol described in step (1) is ethanol with a volume concentration of 95%, the number of ethanol extractions is 3, the amount of ethanol used each time is 8 times the amount, and the extraction time each time is 2 hours.

[0014] Furthermore, the petroleum ether-ethyl acetate elution gradient in step (2) is 20:1 to 0:1 (v / v);

[0015] The dichloromethane-methanol elution gradient is 1:0 to 0:1 (v / v);

[0016] Furthermore, the semi-preparative liquid chromatography conditions for Fr.6 are dichloromethane-methanol 1:1 to 0:1 (v / v);

[0017] The methanol-water isocratic elution ratio is 75%:25% (v / v).

[0018] The present invention also provides the use of the compounds (7R, 8R)-sinkiangenoneE and (7R, 8S)-sinkiangenoneE in the Xinjiang Ferula extract in the preparation of anti-inflammatory drugs.

[0019] Furthermore, the medicine is in the form of capsules, granules, tablets, solutions, pills or injections.

[0020] Furthermore, the drug is a soft capsule, a dry suspension, a dispersible tablet, an effervescent tablet, a chewable tablet, an orally disintegrating tablet, a syrup, a concentrated pill, a dropping pill or a micropill.

[0021] The present invention also provides a pharmaceutical composition, which is prepared from an effective amount of compounds (7R, 8R)-sinkiangenoneE and (7R, 8S)-sinkiangenoneE and pharmaceutically acceptable excipients.

[0022] It can be seen from the above technical solution that, compared with the prior art, the present invention discloses two new compounds (7R, 8R)-sinkiangenoneE and (7R, 8S)-sinkiangenoneE and their use in the preparation of anti-inflammatory drugs. The compounds (7R, 8R)-sinkiangenoneE and (7R, 8S)-sinkiangenoneE show good anti-inflammatory activity; and the compounds (7R, 8R)-sinkiangenoneE and (7R, 8S)-sinkiangenoneE show good anti-inflammatory activity. Compared with L-NAME, they can significantly inhibit the expression of pro-inflammatory cytokines TNF-α, IL-1β and IL-6 produced by RAW264.7 cells induced by LPS. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The accompanying figure is a hydrogen spectrum of the compound (7R,8R)-sinkiangenoneE of the present invention;

[0024] Figure 2 The accompanying figure is a carbon spectrum of the compound (7R,8R)-sinkiangenoneE of the present invention;

[0025] Figure 3 The accompanying drawings show the calculated ECD spectrum and experimental ECD spectrum of the compound (7R,8R)-sinkiangenoneE of the present invention;

[0026] Figure 4 The accompanying drawing is a hydrogen spectrum of the compound (7R,8S)-sinkiangenoneE of the present invention;

[0027] Figure 5 The accompanying drawing is a carbon spectrum of the compound (7R,8S)-sinkiangenoneE of the present invention;

[0028] Figure 6 The accompanying drawings show the calculated ECD spectrum and experimental ECD spectrum of the compound (7R,8S)-sinkiangenoneE of the present invention;

[0029] Figure 7The accompanying drawings show the effects of the compounds (7R,8R)-sinkiangenoneE and (7R,8S)-sinkiangenoneE of the present invention on the amount of NO produced by LPS-induced RAW264.7 cells;

[0030] Figure 8 The accompanying drawings show the effects of the compounds (7R,8R)-sinkiangenoneE and (7R,8S)-sinkiangenoneE of the present invention on the expression levels of TNF-α, IL-1β and IL-6 induced by LPS in RAW264.7 cells. DETAILED DESCRIPTION

[0031] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0032] In the examples of the present invention, the RAW264.7 cell line was provided by the Institute of Basic Medicine, Chinese Academy of Medical Sciences; RAW264.7 cells were cultured using DMEM medium (Gibco, USA); trypsin, double antibody, MTT, and FBS were purchased from Gibco, USA, dimethyl sulfoxide (DMSO), L-NAME, and LPS were purchased from Sigma-Aldrich, USA, and TNF-α, IL-1β, and IL-6 kits were purchased from Biolegend, USA.

[0033] Example 1

[0034] Preparation of compound Sinkiangenol E:

[0035] (1) Xinjiang Ferula resin was extracted with 8 times the amount of 95% reflux for 3 times, each time for 2 hours, the extracts were combined and filtered, and the filtrate was concentrated under reduced pressure to a thick extract to obtain Xinjiang Ferula resin alcohol extract;

[0036] (2) The alcohol extract obtained in step (1) was extracted with dichloromethane, and then subjected to silica gel column chromatography with a gradient elution of petroleum ether-ethyl acetate (20:1-0:1, v / v) and dichloromethane-methanol (1:0-0:1, v / v) to obtain six components Fr.1-6; Fr.6 was subjected to semi-preparative liquid chromatography (dichloromethane-methanol 1:1-0:1, v / v) and isocratic elution with methanol-water (75%:25%, v / v) to obtain the two compounds 1 and 2;

[0037] (3) Compounds 1 and 2 were identified as new compounds using modern spectroscopic techniques (IR, UV, 1D-, 2D-NMR, and HR-ESI-MS) and named (7R,8R)-sinkiangenoneE and (7R,8S)-sinkiangenoneE. Their structures are as follows:

[0038]

[0039] (7R,8R)-sinkiangenoneE is a new compound with the following physicochemical data: orange-red colloid, UV(MeOH)λ max (logε): 318 (4.64), 287 (4.64) nm; [α]25D: -17.9° (c = 0.084, MeOH); HR-ESI-MS (positive ion mode) observed a quasi-molecular ion peak of m / z: 573.2092 [M+Na] + (C 31 H 34 The theoretical molecular weight of O9Na is 573.2101), and its molecular formula is inferred to be C 31 H 34 O9, the degree of unsaturation is 15. Infrared spectrum (KBr) shows the presence of hydroxyl groups (3369 cm -1 )Signal.

[0040] (7R,8S)-sinkiangenoneE is a new compound. Its physicochemical data are as follows: orange-red colloid, UV(MeOH)λ max (logε): 322 (4.64) nm; [α]25D: +15.0° (c = 0.04, MeOH); HR-ESI-MS (positive ion mode) observed its quasi-molecular ion peak at m / z: 573.2092 [M+Na] + (C 31 H 34 The theoretical molecular weight of O9Na is 573.2101), and its molecular formula is inferred to be C 31 H 34 O9, the degree of unsaturation is 15. Infrared spectrum (KBr) shows the presence of hydroxyl groups (3437 cm -1 )Signal.

[0041] Table 1 Compounds (7R,8R)-sinkiangenoneE and (7R,8S)-sinkiangenoneE 1 HNMR (600 MHz, CD3OD) and 13 CNMR (150 MHz, CD3OD) data

[0042]

[0043]

[0044] Example 2

[0045] First, RAW264.7 cells in the logarithmic growth phase were cultured at 2.0×10 5 The cells were seeded at a density of 1 μg / well in a 96-well plate, and 100 μL of DMEM complete medium containing test samples at different concentrations were added to each well in the presence of LPS (1 μg / mL), with the concentrations being 50, 25, 12.5, 6.25, 3.125, and 1.5625 μM, respectively.

[0046] The blank group received a 0.02% DMEM solution instead of the test sample solution, without LPS (1 μg / mL) solution. The model group (LPS group) received a DMEM solution containing LPS (1 μg / mL) without the test sample solution. The sample group received both LPS (1 μg / mL) and DMEM solutions containing samples of varying concentrations. The positive control group received both LPS (1 μg / mL) solution and L-NAME solutions of varying concentrations. After incubation at 37°C for 24 hours, the NO level in the culture supernatant was determined using the Greiss reaction.

[0047] After the incubation, 50 μL of the culture supernatant from each well was transferred to a new 96-well plate and an equal volume of Griess reagent was added at room temperature. After 15 minutes, the absorbance of each well was measured at a wavelength of 540 nm using a microplate reader (and the amount of NO in each cell culture supernatant was calculated by measuring the standard curve of the kit used, and then the IC 50 The results showed that (7R,8R)-sinkiangenoneE and (7R,8S)-sinkiangenoneE could significantly reduce the NO content in LPS-induced RAW264.7 cells in a concentration-dependent manner. IC 50 The values ​​were 17.6 ± 0.4 μM and 14.9 ± 0.1 μM, respectively.

[0048] Example 3

[0049] Mouse TNF-α, mouse IL-1β and mouse IL-6 ELISA kits were used to determine their effects on the release of inflammatory factors TNF-α, IL-1β and IL-6.

[0050] RAW264.7 cells in the logarithmic growth phase were cultured at a rate of 2×10 5Cells were seeded at a density of 1 μg / mL per well in a 96-well plate. LPS (1 μg / mL) and DMEM solutions containing different sample concentrations were added to the sample group at concentrations of 1.5625, 3.125, and 6.25 μM, respectively. A blank group received 0.02% DMEM solution in place of the test sample solution, without the addition of LPS (1 μg / mL). The model group (LPS group) received DMEM solution containing LPS (1 μg / mL) without the addition of the test sample solution. A positive control group received both LPS (1 μg / mL) and L-NAME solution. After incubation at 37°C, 5% CO2 for 24 hours, the ELISA kit was followed according to the instructions. The absorbance (A) of each well was measured at a wavelength of 450 nm using a microplate reader. The release of inflammatory factors in the cell culture supernatant was calculated using the standard curve of the kit.

[0051] ELISA results showed that (7R,8R)-sinkiangenoneE and (7R,8S)-sinkiangenoneE effectively alleviated LPS-induced inflammation by inhibiting the expression of TNF-α, IL-1β and IL-6 and reducing the production of inflammatory molecules such as NO.

[0052] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A method for preparing Xinjiang Ferula extract, characterized in that: The following steps are involved: (1) extracting Xinjiang Ferula resin by reflux extraction with ethanol, combining the extracts and filtering, and concentrating the filtrate under reduced pressure to a thick extract to obtain Xinjiang Ferula resin alcohol extract; (2) The ethanol extract obtained in step (1) was extracted with dichloromethane, and then subjected to silica gel column chromatography with a gradient elution of petroleum ether-ethyl acetate and dichloromethane-methanol to obtain six components Fr.1 to Fr.6; Fr.6 was subjected to semi-preparative liquid chromatography with an isocratic elution of methanol-water to obtain two compounds; they were named (7R,8R)-sinkiangenone E and (7R,8S)-sinkiangenoneE, which are Xinjiang Ferula extracts; The structure of the (7R,8R)-sinkiangenone E is shown in Formula I, and the structure of the (7R,8S)-sinkiangenone E is shown in Formula II:

2. The method for preparing the Xinjiang Ferula extract according to claim 1, wherein: The ethanol described in step (1) is ethanol with a volume concentration of 95%, the number of ethanol extractions is 3, the amount of ethanol used each time is 8 times the amount, and the extraction time each time is 2 hours.

3. The method for preparing the Xinjiang Ferula extract according to claim 1, wherein: The petroleum ether-ethyl acetate elution gradient in step (2) is 20:1 to 0:1 v / v; The dichloromethane-methanol elution gradient v / v is 1:0 to 0:1; The semi-preparative liquid chromatography conditions for Fr.6 are dichloromethane-methanol v / v 1:1 to 0:1; The methanol-water isocratic elution ratio is 75%:25% v / v.

4. A Xinjiang Ferula extract, characterized in that The invention is prepared by the method according to any one of claims 1 to 3.

5. Use of compounds (7R,8R)-sinkiangenone E and (7R,8S)-sinkiangenone E in the preparation of anti-inflammatory drugs, characterized in that: The structure of the (7R,8R)-sinkiangenone E is shown in Formula I, and the structure of the (7R,8S)-sinkiangenone E is shown in Formula II:

6. The use according to claim 5, characterized in that The medicine is in the form of capsules, granules, tablets, solutions, pills or injections.

7. The use according to claim 6, characterized in that The medicine is a soft capsule, a dry suspension, a dispersible tablet, an effervescent tablet, a chewable tablet, an orally disintegrating tablet, a syrup, a concentrated pill, a dripping pill or a micropill.

8. A pharmaceutical composition, characterized in that The invention is prepared from the compounds (7R, 8R)-sinkiangenone E and (7R, 8S)-sinkiangenone E according to claim 1 and pharmaceutically acceptable excipients.

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