A terpenoid compound with anti-inflammatory activity and its preparation method and application

Abstract

The present invention relates to a terpene compound with anti-inflammatory activity, a preparation method, and an application thereof. The terpene compound includes a compound represented by formula (I), or a pharmaceutically acceptable salt or pharmaceutically acceptable esterified derivative of formula (I): #imgabs0#(I). The novel terpene component with anti-inflammatory activity provided by the present invention is isolated from slugs for the first time. In vitro pharmacological experiments have confirmed that the terpene component Limaxol D has a NO production inhibition rate of 47.11% at a concentration of 25 μmol / L, indicating that it has good anti-inflammatory activity. Therefore, the terpene component Limaxol D, or its derivatives, or its pharmaceutically acceptable salts can be used to prepare anti-inflammatory drugs.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a terpenoid compound with anti-inflammatory activity, a preparation method and an application thereof. Background Art

[0002] Terpenoids are compounds and their derivatives derived from mevalonic acid, whose molecular skeletons are based on isoprene units (C5 units). They are widely found in nature and are the main components of the essences, resins, pigments, etc. of certain plants. They have multiple biological activities such as anti-inflammatory, antioxidant, antibacterial, antiviral, antitumor, liver protection, hypoglycemic, nervous system protection, and cardiovascular system protection. They also have minimal toxic side effects and are increasingly receiving widespread attention from natural medicine researchers.

[0003] Slugs are molluscs with lungs, belonging to the Gastropoda class, Slugidae family. Because their mucus-covered bodies resemble snails but without shells, they are also commonly known as "slugs" or "slugs." The main species found in Guangxi include the footfold slug, giant slug, yellow slug, wild slug, and two-lined slime slug. As a traditional Chinese medicine, according to medical texts such as "Shennong's Herbal Classic," "Compendium of Materia Medica," and "The Great Dictionary of Chinese Medicine," slugs are described as "salty and cold in nature, non-toxic. They clear heat and dispel wind, relieve edema and toxins, break up blood stasis and promote menstruation, and treat stroke, paralysis, muscle spasms, epilepsy, wheezing, throat numbness, pharyngeal swelling, and carbuncles." Modern pharmacological research has shown that slugs have a significant inhibitory effect on cancer cells such as ascites sarcoma, Lewis tumor, and human non-small cell lung cancer. They are also effective in treating chronic bronchitis and bronchial asthma. At present, there are some research reports on the chemical composition, but there are no reports on the isolation of terpenoid components with anti-inflammatory activity from slugs.

[0004] In view of this, the present invention provides a terpenoid compound with anti-inflammatory activity and a preparation method and application thereof, which provides a new choice for inflammatory therapeutic drugs and makes up for the shortcomings of the existing technology. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a terpenoid compound with anti-inflammatory activity and a preparation method and application thereof.

[0006] The technical solution of the present invention to solve the above technical problems is as follows:

[0007] In the first aspect, a terpenoid compound having anti-inflammatory activity, wherein the terpenoid compound comprises a compound represented by formula (I) or a pharmaceutically acceptable salt or pharmaceutically acceptable esterified derivative of formula (I):

[0008]

[0009] (Ⅰ).

[0010] The compound or pharmaceutically acceptable salt of formula (I) is selected from salts formed by terpenoid compounds and organic or inorganic acids. The terpenoid compound with anti-inflammatory activity of the present invention is named Limaxol D.

[0011] The present invention has the following beneficial effects: the new terpenoid component with anti-inflammatory activity provided by the present invention is isolated from slugs for the first time; the terpenoid component Limaxol D is confirmed by in vitro pharmacological experiments to have a NO production inhibition rate of 47.11% at a concentration of 25 μmol / L, and the results show that it has good anti-inflammatory activity; therefore, the terpenoid component Limaxol D or its pharmaceutically acceptable salts and esterified derivatives can be used in anti-inflammatory drugs.

[0012] On the basis of the above technical solution, the present invention can also be improved as follows.

[0013] In a second aspect, a terpenoid compound having anti-inflammatory activity is used to prepare an anti-inflammatory drug.

[0014] Furthermore, the terpenoid compound is used to prepare a drug for treating redness, swelling, heat, pain and / or functional disorders caused by inflammation due to external irritants and / or injuries.

[0015] In a third aspect, a drug with anti-inflammatory activity comprises the terpenoid compound. The present invention provides a pharmaceutical preparation for treating inflammation, comprising an effective dose of an active ingredient selected from a mixture of any one or more of the terpenoid compound with anti-inflammatory activity, its pharmaceutically acceptable salts, and pharmaceutically acceptable esterified derivatives.

[0016] Furthermore, it also includes pharmaceutically acceptable pharmaceutical excipients and / or carriers.

[0017] Furthermore, the dosage form of the drug is any one of granules, capsules, nanoformulations, controlled-release preparations, and sustained-release preparations.

[0018] In a fourth aspect, a method for preparing a terpenoid compound having anti-inflammatory activity comprises the following steps:

[0019] (1) After the dried slugs are crushed, reflux extraction is performed using an alkane compound, and the mixture is filtered and concentrated under reduced pressure to obtain a crude extract; the crude extract is floated with water and then extracted, and the extraction solution is concentrated under reduced pressure to obtain an extract; the extract is dissolved in methanol, crystallized, and a crude crystalline sample is obtained;

[0020] (2) The crude crystal sample is prepared by semi-preparative liquid chromatography to obtain terpenoid compounds.

[0021] The beneficial effects of this approach are: the potent anti-inflammatory active ingredient extracted and isolated from slugs is simple and easy to prepare, with promising potential economic benefits. Furthermore, the resulting anti-inflammatory active ingredient, Limaxol D, is stable and easy to store. It is expected to serve as a compound or lead compound for the development of new drugs for the treatment of inflammatory diseases.

[0022] Furthermore, in step (1), the ratio of the dried slug to the alkane compound during each reflux extraction is (250-1000) g: (1-5) L; the alkane compound comprises at least one of n-hexane, pentane, and petroleum ether;

[0023] In step (1), the extraction is specifically as follows: extracting with ethyl acetate and then floating the crude extract with water for at least 4 times.

[0024] Furthermore, the semi-preparative liquid chromatograph in step (3) uses an eluent having a volume fraction ratio of n-hexane:ethyl acetate:isopropanol of 90:8:2 for isocratic elution to obtain an eluent with a retention time of 7.35 min, which is concentrated and dried to obtain terpenoid compounds.

[0025] Furthermore, the slugs include at least one of the big slug (Limax maximus.), the yellow slug (L. flavus), the sheathed fold slug (Vaginulus alte), the wild slug (Agriolimax agrestis) and the two-lined slime slug (Phiolomycus bilineatus). BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is the compound LimaxolD 1 H-NMR (500 MHz, CD3OD) spectrum;

[0027] Figure 2 It is the compound LimaxolD 13 C-NMR (125 MHz, CD3OD) spectrum;

[0028] Figure 3 is the HSQC spectrum of compound Limaxol D;

[0029] Figure 4 is the HMBC spectrum of compound Limaxol D;

[0030] Figure 5 It is the compound LimaxolD 1 H- 1 H COSY spectrum;

[0031] Figure 6is the HR-EIMS spectrum of compound Limaxol D;

[0032] Figure 7 This is a diagram showing the effect of the compound Limaxol D on NO release from macrophages. DETAILED DESCRIPTION

[0033] The principles and features of the present invention are described below. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in this field or the product instructions are used. Where the manufacturers of the reagents or instruments used are not specified, they are all conventional products that can be purchased through regular channels.

[0034] Example 1

[0035] 1. Limaxol D is extracted, separated and purified from animal slugs

[0036] Limax maximus L. was purchased from a medicinal material market, and the separation and purification process for extracting, separating, and purifying terpenoid compounds therefrom was as follows:

[0037] (1) After 500 g of dried slugs were crushed, 2.5 L of the extracted solution was refluxed with n-hexane for 3 times, each extraction for 2 h, and filtered. The filtrates were combined and concentrated under reduced pressure to obtain a n-hexane extract. The n-hexane extract was floated with 0.1 L of water and extracted 4 times with ethyl acetate. 0.4 L of the combined ethyl acetate extracts were concentrated under reduced pressure to obtain an ethyl acetate extract. The ethyl acetate extract was dissolved in 0.05 L of anhydrous methanol by heating. After dissolution, the extract was cooled and allowed to stand to precipitate crystals. The crystals were filtered to obtain a crude crystalline sample. The crystalline sample was prepared by semi-preparative liquid phase to obtain Limaxol D.

[0038] (2) The crude crystal sample was dissolved in n-hexane and prepared by semi-preparative liquid chromatography (LC12 semi-preparative liquid chromatograph of Beijing Saipu Ruisi Technology Co., Ltd., HT-3501 C of Suzhou Huitong Company). 18 A semi-preparative column (10 mm × 250 mm) was used for isocratic elution with a solution of 90:8:2 (n-hexane:ethyl acetate:isopropanol). The eluate with a retention time of 7.35 min was collected and concentrated to dryness to obtain the compound shown in structural formula (I), which is the target terpenoid compound Limaxol D (63 mg).

[0039] 2. Identification of Limaxol D

[0040] Figure 1 It is the compound Limaxol D 1H-NMR spectrum; Figure 2 It is the compound Limaxol D 13 C-NMR spectrum; Figure 3 This is the HSQC spectrum of compound Limaxol D; Figure 4 is the HMBC spectrum of compound Limaxol D; Figure 5 It is the compound Limaxol D 1 H- 1 H COSY spectrum; Figure 6 This is the HR-EIMS spectrum of compound Limaxol D. Figure 6 ) shows that its quasi-molecular ion peak is [M+Na] + 631.3835 (calculated value 631.3817), suggesting that the molecular formula is C 34 H 56 O9, unsaturation is 7. 1 H-NMR (500 MHz, CD3OD), 13 C-NMR (125 MHz, CD3OD) data are shown in Table 1 below.

[0041] Table 1 NMR data of compound Limaxol D (in CD3OD)

[0042]

[0043] Based on the comprehensive analysis of the above spectral data, the structural formula of the compound was deduced as shown in formula (I), and named Limaxol D:

[0044]

[0045] Formula (I).

[0046] Example 2

[0047] 1. Activity detection

[0048] (1) Cell model: Mouse macrophage RAW264.7 cells, obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences. Complete culture medium: 90% DMEM medium + 10% fetal bovine serum + 1% P / S double antibody. Culture conditions: 37°C, 5% CO2, saturated humidity.

[0049] (2) Drugs and reagents: The compounds used in the experiment were prepared according to Example 1, accurately weighed, and dissolved in DMSO to a stock solution with a concentration of 20 μM for later use.

[0050] (3) Cell viability was determined by CCK-8 method. The steps are as follows:

[0051] The experimental groups were set as blank control group, background control group, and compound group.

[0052] RAW264.7 cells in the logarithmic growth phase (purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences) were diluted to 2×10 5 Single-cell suspensions of cells / mL were inoculated into 96-well plates at a volume of 100 μL / well (3 replicates per group), cultured in a CO2 incubator for 24 h, taken out, and the test samples were added respectively. The blank control group was added with complete culture medium, the background group was added with only 100 μL complete culture medium without cells, and the compound group was added with different concentrations of the test compound (final concentrations of 25 μM, 12.5 μM, and 6.25 μM). The plates were placed in a CO2 incubator and cultured for another 24 h. The plates were taken out, 10 μL of CCK-8 was added to each well, incubated at 37°C for 1 h, and the OD value of each well at a wavelength of 450 nm was detected using a microplate reader.

[0053] Calculation formula: Cell proliferation rate = (OD 样品 -OD 背景 ) / (OD 空白 -OD 背景 )×100%

[0054] (4) Griess method was used to detect the effect of compounds on NO release from macrophages. The steps are as follows:

[0055] The experiment set up blank control group, DMSO group, model group, positive drug group (parthenolide) and compound group.

[0056] RAW264.7 cells in the logarithmic growth phase (purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences) were diluted to 2×10 5 Single-cell suspensions of cells / mL were inoculated into 96-well plates at a volume of 100 μL / well (3 replicates per group) and cultured in a cell culture incubator at a concentration of 5% CO2 and a temperature of 37°C for 24 h. The plates were then taken out and complete culture medium was added to the blank control group and model group, DMSO dilution was added to the DMSO group, and different concentrations of positive drugs and test compounds were added to the positive drug group and compound group, respectively. After 1 h of drug action, LPS (final concentration of 1 μg / mL) was used to induce a cell inflammation model in all experimental groups except the blank control group. After culturing in a CO2 incubator for 24 h, the cell supernatant was collected and the OD value at a wavelength of 540 nm was measured using a microplate reader using the Griess method to detect the amount of NO released and calculate the NO inhibition rate.

[0057] Calculation formula: Inhibition rate = [1-(OD 样品 -OD 空白 ) / (OD 模型 -OD 空白 )]×100%

[0058] 2. Activity Results

[0059] The results of cell viability assays showed that when the concentration of compound Limaxol D was below 25 μM (the compound could be completely dissolved only at a concentration below 25 μM), the cell proliferation rate was greater than 90%, indicating that compound Limaxol D had no significant cytotoxic effect on RAW264.7 cells.

[0060] The results of NO release assay showed that Figure 7 As shown in the figure, the NO production inhibition rate of compound Limaxol D at a concentration of 25 μM was 47.11% (the IC 50 The value was 0.90 μM), and the results showed that it had good anti-inflammatory activity.

[0061] In summary, the present invention utilizes slugs as a raw material to extract and isolate Limaxol D, a potent anti-inflammatory ingredient. The preparation method is simple and has great potential economic benefits. Furthermore, the resulting novel anti-inflammatory ingredient, Limaxol D, is stable and easy to store. Its excellent anti-inflammatory activity makes it a promising lead compound for the development of new inflammatory therapeutics.

[0062] 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 terpenoid compound with anti-inflammatory activity, characterized in that: The terpenoid compound is selected from the compound represented by formula (I) or a pharmaceutically acceptable salt of formula (I): （Ⅰ）。 2. An application of a terpenoid compound with anti-inflammatory activity, characterized in that: The terpenoid compound according to claim 1 is used for preparing anti-inflammatory drugs.

3. The use of a terpenoid compound with anti-inflammatory activity according to claim 2, characterized in that: The terpenoid compound is used to prepare medicine for treating redness, swelling, heat, pain and / or functional disorder caused by inflammation caused by external irritants and / or injuries.

4. A drug with anti-inflammatory activity, characterized in that The invention comprises the terpenoid compound according to claim 1.

5. The drug with anti-inflammatory activity according to claim 4, characterized in that: Also included are pharmaceutically acceptable excipients and / or carriers.

6. The drug with anti-inflammatory activity according to claim 4, characterized in that: The dosage form of the medicine is any one of granules, capsules, nano preparations, controlled-release preparations and sustained-release preparations.

7. A method for preparing a terpenoid compound with anti-inflammatory activity according to claim 1, characterized in that: The steps include: (1) After the dried slugs are crushed, reflux extraction is performed using an alkane compound, and the mixture is filtered and concentrated under reduced pressure to obtain a crude extract; the crude extract is floated with water and then extracted, and the extraction solution is concentrated under reduced pressure to obtain an extract; the extract is dissolved in methanol, crystallized, and a crude crystalline sample is obtained; (2) The crude crystal sample is prepared by semi-preparative liquid chromatography to obtain terpenoid compounds.

8. The method for preparing a terpenoid compound with anti-inflammatory activity according to claim 7, characterized in that: In step (1), the ratio of the dried slug to the alkane compound during each reflux extraction is (250-1000) g: (1-5) L; the alkane compound comprises at least one of n-hexane, pentane, and petroleum ether; In step (1), the extraction is specifically as follows: the crude extract is floated with water and then extracted with ethyl acetate at least 4 times.

9. The method for preparing a terpenoid compound with anti-inflammatory activity according to claim 7, characterized in that: The semi-preparative liquid chromatograph in step (2) uses an eluent having a volume fraction ratio of n-hexane:ethyl acetate:isopropanol of 90:8:2 for isocratic elution to obtain an eluent with a retention time of 7.35 min, which is concentrated and dried to obtain terpenoid compounds.

10. The method for preparing a terpenoid compound with anti-inflammatory activity according to claim 7, characterized in that: The slugs include at least one of a large slug, a yellow slug, a sheathed slug, a wild slug and a two-lined slime slug.

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