Novel cleaved iridoid compound and preparation method, pharmaceutical composition and application thereof

Abstract

The present invention belongs to the field of medical technology, and specifically discloses a novel scissoric terpenoid compound and a preparation method, a pharmaceutical composition and an application thereof. The scissoric terpenoid compound (±)-Cornuscone provided by the present invention has not been reported yet. Experimental results show that the scissoric terpenoid compound provided by the present invention has significant anti-inflammatory activity on lipopolysaccharide-induced RAW 264.7 cells, which is significantly better than the positive drug indomethacin, and can be used to develop drugs for the treatment of various inflammation-related diseases, including but not limited to arthritis, conjunctivitis, pneumonia, hepatitis, nephritis, gastroenteritis, metabolic syndrome, diabetes, cancer, etc.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and more specifically, relates to a novel secoiridoid compound and a preparation method thereof, a pharmaceutical composition and application thereof in drugs for treating various inflammation-related diseases. Background Art

[0002] Inflammation is a basic pathological process that occurs when living tissues with vascular systems are stimulated by various damaging factors and is mainly a defensive response. It is an indispensable protective function of the human body and can be accompanied by changes in multiple systems such as metabolism, endocrine, respiratory, circulatory function, and psychology. Inflammation and related diseases are serious public health issues and the main cause of human death.

[0003] Cornus officinalis is the dried mature pulp of Cornus officinalis Sieb.et Zucc., a plant of the Cornaceae family. It is widely distributed in Foping, Shaanxi, Xixia, Henan, Linqi, Zhejiang, and other places in my country, and is also distributed in Japan and South Korea. In order to find drugs for treating inflammation-related diseases, the inventors isolated a new type of cleaved iridoid compound (±)-Cornuscone from Cornus officinalis, and found that it has significant anti-inflammatory activity, which is significantly better than the positive drug Indometacin, and can be used to develop drugs for treating various inflammation-related diseases. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention provides novel cleaved iridoid compounds and preparation methods thereof, pharmaceutical compositions and applications thereof in drugs for treating various inflammation-related diseases.

[0005] To achieve the above object, the technical solution of the present invention is as follows:

[0006] A novel cleaved-cornuscone compound (referred to as compound (±)-Cornuscone) has a structure as shown in formula (1):

[0007]

[0008] Among them, stereoisomers, tautomers or pharmaceutically acceptable salts of the cleaved iridoid compounds also fall within the protection scope of the present invention.

[0009] Another aspect of the present invention provides a method for preparing the novel secoiridoid compounds. The novel secoiridoid compounds are obtained by extraction and separation from Cornus officinalis.

[0010] Furthermore, the preparation method of the novel cleaved iridoid compound comprises the following steps:

[0011] S1, extracting Cornus officinalis by refluxing with a solvent, combining the extracts and concentrating them to obtain an extract;

[0012] S2, adding water to fully dissolve the extract through macroporous adsorption resin, using ethanol-water gradient elution, and obtaining the target elution part after concentration;

[0013] S3, using chloroform-methanol solution to gradiently elute the target elution part through silica gel column chromatography to obtain fraction C; when using silica gel thin layer chromatography to identify, the Rf value corresponding to fraction C is 0.71-0.73;

[0014] S4. Fraction C is subjected to gradient elution by ODS column chromatography using methanol-water solution to obtain fraction C1-1. When detected by silica gel thin layer chromatography, the Rf value corresponding to fraction C1-1 is 0.21-0.30;

[0015] S5. Fraction C1-1 is subjected to gradient elution by Sephadex LH-20 gel column chromatography using methanol-water solution to obtain fraction C1-1.10. When detected by silica gel thin layer chromatography, the Rf value corresponding to fraction C1-1.10 is 0.61-0.68;

[0016] S6. Separating a novel cleaved iridoid compound having a structure as shown in formula (1) from fraction C1-1.10 by HPLC;

[0017] The mobile phase used in HPLC was a methanol-water mixture.

[0018] This application uses methanol-water as the mobile phase and adopts HPLC method using C 18 The chromatographic column produces a split ring terpene compound having a structural formula as shown in formula (1) in fraction C1-1.10.

[0019] Furthermore, in step S6, the volume ratio of methanol to water in the methanol-water mixed solution is (22:78)-(27:73).

[0020] Preferably, the volume ratio of methanol to water in the methanol-water mixed solution is 25:75.

[0021] Further, in step S6, the characteristic ultraviolet absorption (λ max =208nm, 262nm) to further separate the fraction C1-1.10, including the split ring cyclopentadiene ether terpenoid compound represented by the structural formula (1), with a retention time of 20-25min.

[0022] Preferably, when the volume ratio of methanol to water in the methanol-water mixed solution is 25:75, the retention time of the split ring cyclopentadiene ether terpene compound with the structural formula shown in formula (1) is 21 minutes.

[0023] Furthermore, in step S1, the solvent is water; the mass of the added solvent is 8-10 times that of the Cornus officinalis, the number of reflux extractions is 2-4 times, and each extraction lasts 1-3 hours.

[0024] Furthermore, in step S2, the extract is passed through D101 macroporous adsorption resin and eluted using ethanol-water gradient elution and concentrated to obtain an elution portion. When the elution portion is eluted out, the volume ratio of ethanol to water in the ethanol-water mixed solution is (50:50)-(60:40) based on the total volume of 100; preferably, the volume ratio of ethanol to water is 55:45.

[0025] Furthermore, in step S3, the volume ratio of chloroform to methanol in the chloroform-methanol mixed solution when washing out fraction C is (80:20)-(90:10); preferably, the volume ratio of chloroform to methanol is 85:15.

[0026] The Rf value in this application is the Rf value of the fluorescent spot observed at a scanning wavelength of 254 nm.

[0027] Furthermore, in step S4, the volume ratio of methanol to water in the methanol-water solution when washing out fraction C1-1 is (15:85)-(25:75); preferably, the volume ratio of methanol to water is 20:80.

[0028] Furthermore, in step S5, the volume ratio of methanol to water in the methanol-water solution when washing out fraction C1-1.10 is (55:45)-(65:35); preferably, the volume ratio of methanol to water is 60:40.

[0029] In another aspect, the present invention provides a pharmaceutical composition containing the novel secoiridoid compound.

[0030] Furthermore, the pharmaceutical composition comprises a synergist and a pharmacodynamically acceptable carrier or excipient.

[0031] That is to say, the pharmaceutical composition containing the secoiridoid compound of the present invention as an active ingredient and conventional pharmaceutical excipients or adjuvants or carriers is also included in the present invention.

[0032] Furthermore, the synergist is one or more of the following substances:

[0033] Aspirin, ibuprofen, indomethacin (indomethacin), naproxen, nabumetone (nabumetone), piroxicam (piroxicam), phenylbutazone, diclofenac, fenprofen, ketoprofen, ketorolac, tetraclofenamic acid, sulindac, tolmetin, fenbufen, sulindac, celecoxib, and cortisone.

[0034] Furthermore, the dosage form of the pharmaceutical composition is tablets, capsules, granules, oral liquid, granules, pills or micropills.

[0035] In another aspect, the present invention also provides the use of the novel cleaved iridoid compound or pharmaceutical composition in drugs for treating inflammatory diseases, wherein the inflammation includes but is not limited to arthritis, conjunctivitis, pneumonia, hepatitis, nephritis, gastroenteritis, metabolic syndrome, diabetes or cancer.

[0036] Compared with the prior art, the present invention has the following advantages:

[0037] (1) The present invention provides a novel secoiridoid compound (±)-Cornuscone, which has not been reported so far. Experimental results show that the secoiridoid compound provided by the present invention can inhibit the release of NO in RAW 264.7 cells induced by lipopolysaccharide, exhibiting significant anti-inflammatory activity, which is significantly better than the first-line anti-inflammatory drug indomethacin, and can be used to develop drugs for the treatment of various inflammatory-related diseases.

[0038] (2) The preparation method of the novel split-ring cyclopentadiene terpene compound (±)-Cornuscone is simple to operate, has good reproducibility and high extraction purity. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The compound 1 prepared in Example 1 of the present invention 1 H NMR spectrum (400 MHz, Methanol-d4);

[0040] Figure 2 The compound 1 prepared in Example 1 of the present invention 13 C NMR spectrum (100 MHz, Methanol-d4);

[0041] Figure 3 This is the HMBC spectrum of compound 1 prepared in Example 1 of the present invention. DETAILED DESCRIPTION

[0042] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments.

[0043] It should be understood that the specific embodiments described herein are only used to explain the present invention, and are not used to limit the present invention.

[0044] In the examples, unless otherwise specified, all the means used are conventional means in the art.

[0045] As used herein, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that comprises the listed elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, step, method, article, or apparatus.

[0046] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0047] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0048] In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0049] Example 1

[0050] This embodiment discloses a method for preparing a novel cleaved iridoid compound, comprising the following steps:

[0051] S1. Extract Cornus officinalis with a solvent by refluxing, and concentrate the combined extracts to obtain an extract; specifically, the solvent is water; the mass of the added solvent is 8-10 times that of the Cornus officinalis, the number of reflux extractions is 2-4 times, and each extraction takes 1-3 hours.

[0052] The specific implementation scheme is as follows: Weigh cornus officinalis (63.0 kg), add water 10 times the mass of cornus officinalis as solvent, reflux and extract three times, each extraction is 2 hours, and the extracts are combined and concentrated to obtain an extract (28.9 kg).

[0053] S2, the extract was fully dissolved in water and subjected to macroporous adsorption resin column chromatography, eluted with ethanol-water gradient, and concentrated to obtain the target elution site; according to the characteristic ultraviolet absorption (λ max =208, 262nm) for detection, based on the total volume of 100, the volume ratio of ethanol to water in the ethanol-water solution when eluting the target elution site is (50:50)-(60:40); preferably, the ethanol-water volume ratio is 55:45, at which time the elution efficiency is higher.

[0054] The specific steps are as follows: the extract is subjected to D101 macroporous adsorption resin column chromatography, using ethanol-water gradient elution with volume ratios of 0:100, 30:70, 55:45, and 95:5, and each elution fraction is concentrated. The total volume is taken as 100 as the basis, and the ethanol-water volume ratio in the ethanol-water mixed solution when the target elution fraction is eluted is 55:45.

[0055] S3. Use chloroform-methanol solution to perform gradient elution on the target elution part through silica gel column chromatography to obtain fraction C; when detected by silica gel thin layer chromatography, the Rf value corresponding to fraction C is 0.71-0.73; based on the total volume of 100, the volume ratio of chloroform to methanol in the chloroform-methanol solution when eluting fraction C is (80:20)-(90:10); preferably, the volume ratio of chloroform-methanol is 85:15.

[0056] It should be noted that, during gradient elution, chloroform-methanol solutions with a volume ratio of (80:20)-(90:10) can elute the elution site, and the fraction with an Rf value of 0.71-0.73 is fraction C using silica gel thin layer chromatography. Among them, the elution efficiency of chloroform-methanol solution with a volume ratio of 85:15 is the highest. The principle of "preferred volume ratio" in other gradient elution processes is the same.

[0057] The Rf value in this application is the Rf value of the fluorescent spot observed at a scanning wavelength of 254 nm.

[0058] The specific steps are as follows: the extract is subjected to silica gel column chromatography, using chloroform-methanol gradient elution with a volume ratio of 100:0, 95:5, 85:15, 75:25, 65:35, 50:50, 25:75, 0:100, and each 500 mL volume is one fraction, and fractions 1-15, 16-30, 31-40, 41-50, 51-60, 61-70, 71-80, 81-90 are obtained in sequence, and a total of 90 fractions are collected. The Rf values ​​observed at 254 nm are 0.82-0.84 (fractions 1-11) , 0.73-0.81 (fraction 12-30), 0.71-0.73 (fraction 31-40), 0.67-0.70 (fraction 41-46), 0.55-0.65 (fraction 47-52), 0.52-0.54 (fraction 53-61), 0.40-0.47 (fraction 62-70), and 0.25-0.37 (fraction 71-90) were combined with similar fractions to obtain 8 fractions, which were named fraction A, B, C, D, E, F, G and H respectively; among them, fraction C was obtained after combining fractions 31-40.

[0059] According to the characteristic ultraviolet absorption (λ max=208, 262nm) Fraction C was selected for the next separation.

[0060] S4. Use methanol-water solution to perform gradient elution on fraction C through ODS column chromatography to obtain fraction C1-1; when detected by silica gel thin layer chromatography, the Rf value corresponding to fraction C1-1 is 0.21-0.30; the volume ratio of methanol to water in the methanol-water solution when eluting fraction C1-1 (i.e., corresponding to fraction C1-1) is (15:85)-(25:75); preferably, the volume ratio of methanol to water is 20:80.

[0061] The specific operation includes: according to the characteristic ultraviolet absorption (λ max =208,262nm) C was selected for further separation, and fraction C was subjected to ODS column chromatography, using a methanol-water gradient elution with a volume ratio of 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 100:0 to obtain fractions 1-10, 11-15, 16-25, 26-35, 36-45, 46-50, 51-55, 56-60, a total of 60 fractions, and detected by silica gel thin layer chromatography. According to the Rf values ​​observed at a wavelength of 254nm, they were 0.21-0. The fluorescent spot results of 0.30 (fraction 1-10), 0.30-0.35 (fraction 11-15), 0.36-0.40 (fraction 16-28), 0.41-0.47 (fraction 19-27), 0.48-0.55 (fraction 28-36), 0.56-0.61 (fraction 37-44), 0.63-0.68 (fraction 45-54), and 0.73-0.80 (fraction 55-60) were combined with similar fractions to obtain 8 fractions C1-1 to C1-8 in turn; among them, fraction C1-1 was obtained after combining fractions 1-10.

[0062] S5. Gradient elution of fraction C1-1 by Sephadex LH-20 gel column chromatography using a methanol-water solution to obtain fraction C1-1.10; when detected by silica gel thin layer chromatography, the Rf value corresponding to fraction C1-1.10 is 0.61-0.68; the volume ratio of methanol to water in the methanol-water solution when eluting fraction C1-1.10 is (55:45)-(65:35); preferably, the volume ratio of methanol to water is 60:40.

[0063] The specific operation includes: according to the characteristic ultraviolet absorption (λ max=208, 262nm) C1-1 was selected for further separation, and fraction C1-1 was purified by Sephadex LH-20 gel column chromatography, using a methanol-water gradient elution with a volume ratio of 0:100, 10:90, 15:85, 20:80, 25:75, 30:70, 40:60, 45:55, 50:50, 60:40, 70:30, 80:20, 100:0, and collecting fractions 1-9, 10-14, 15-21, 22-26, 27-33, 34-38, 39-40, 41-47, 48-52, 53-63, 64-69, 70-77, 78-86, a total of 86 fractions, detected by silica gel thin layer chromatography, according to the fluorescence spot Rf values ​​observed at a wavelength of 254nm, they were 0.20-0.25 fractions (1-9), 0.26-0.29 (fractions 10-14), 0.30-0.34 (fraction 15-21), 0.35-0.39 (fraction 22-26), 0.40-0.46 (fraction 27-33), 0.47-0.50 (fraction 34-38), 0.47-0.51 (fraction 39-40) 0.53-0.57 (fraction 41-47), 0.58-0.60 (fraction 48-52), 0.61-0.68 (fraction 53-63), 0.70-0.74 (fraction 64-69), 0.76-0.79 (fraction 70-77), 0.80-0.82 (fraction 78-86), similar fractions were combined to obtain 13 fractions C1-1.1 to C1-1.13 in sequence; among them, fraction C1-1.10 was obtained by combining fractions 53-63.

[0064] S6. Separating the split ring iridoid compound having the structure shown in formula (1) from fraction C1-1.10 by HPLC;

[0065] The mobile phase used in HPLC was a methanol-water mixture.

[0066] This application uses methanol-water as the mobile phase and adopts HPLC method using C 18 The chromatographic column produces a split ring cyclopentane ether compound having a structural formula as shown in formula (1) in fraction C1-1.10.

[0067] Wherein, the volume ratio of methanol to water in the methanol-water mixed solution is (22:78)-(27:73). Preferably, the volume ratio of methanol to water in the methanol-water mixed solution is 25:75.

[0068] According to the characteristic ultraviolet absorption (λ max=208, 262nm) and the fraction C1-1.10 was further separated, including the novel split ring cyclopentane ether compound with the structural formula shown in formula (1) with a retention time of 20-25min and a purity of 99%.

[0069] The physical properties and test data of compound 1 prepared in Example 1 are as follows:

[0070] Light yellow powder, easily soluble in methanol. According to high resolution mass spectrometry (HR-ESI-MS), m / z 365.0876 ([MH] + The calculated value is 365.0867), and its molecular weight is estimated to be 366, and its molecular formula is determined to be C 17 H 18 O9, its unsaturation is calculated to be 9.

[0071] 1 H NMR (Methanol-d4, 400MHz) spectrum ( Figure 1 ) shows that there is a pair of symmetrical aromatic proton signals in the low field region. H 7.00 (2H, s, H-2′, 6′), indicating that it contains a benzene ring fragment; 1 olefinic hydrogen proton signal δ H 7.64 (1H, d, J = 0.7 Hz, H-3), indicating the presence of one oxygen-linked double bond; one oxygen-linked methine proton signal δ H 4.85 (1H, overlap, H-1); 2 oxymethylene proton signals δ H 4.47 (2H, q, J = 6.5 Hz, H-7); 1 methoxy proton signal δ H 3.79 (3H, s, H-11); 2 methylene proton signals δ H 3.25(1H,m,H-6a) and δ H 3.30 (1H, overlap, H-6b); a methyl proton signal δ can be observed in the high field region H 1.51 (3H, d, J = 6.5 Hz, H-9).

[0072] 13 C NMR (Methanol-d4, 100MHz) spectrum ( Figure 2 ) shows a total of 15 carbon signals, including 2 carbonyl carbon signals (δ C 168.4) and (δ C 161.4); 1 group of carbon signals on the benzene ring (δ C 121.7,110.2,146.6,139.9); 2 groups of olefinic carbon signals (δ C 131.5,133.9), (δ C143.0,144.0); 1 oxygen-linked methylene carbon signal (δ C 62.4); 1 oxymethylene carbon signal (δ C 64.8); 1 methoxy carbon signal (δ C 52.3); 1 methylene carbon signal (δ C 25.1); 1 methyl carbon signal (δ C 23.7).

[0073] 1 H NMR, 13 The signal assignments of C NMR are shown in Table 1.

[0074] Table 1 NMR data of compound 1 δ (ppm)

[0075]

[0076]

[0077] In the HMBC spectrum ( Figure 3 ), H-1(δ H 4.85) and C-3(δ C 144.0), C-5(δ C 131.5), C-8(δ C 133.9), H-3(δ H 7.64) and C-4(δ C 143.0) and C-5 (δ C 131.5), thus confirming the connection mode of the ring; in addition, H-9 (δ H 1.51) and C-8(δ C 133.9) and C-1(δ C 62.4), confirming that the methyl group is attached at the C-1 position; H-3 (δ H 7.64) and C-10 (δ C 161.4), H-11(δ H 3.79) and C-10 (δ C 161.4), indicating that the methyl ester group is attached at the C-4 position; in addition, H-7 (δ H 4.47) and C-6 (δ C 25.1) and C-5(δ C 131.5), indicating that an ethyl segment is connected to the iridoid segment; H-7 (δ H 4.47) and C-7′(δ C168.4), indicating that the galloyl fragment is connected to the ethyl fragment through an ester bond.

[0078] In summary, the structure of the new compound 1 (compound (±)-Cornuscone) was determined as follows:

[0079]

[0080] Comparative Example 1

[0081] Comparative Example 1 of the present invention provides a method for preparing a novel split-ring cyclopentadiene ether terpene compound, and its steps are similar to those of Example 1, except that in step S6, in the mobile phase used for separating fraction C1-1.10 by HPLC, the volume ratio of methanol to water is adjusted to be lower than (22:78) and higher than (27:73).

[0082] The results showed that compound 1 could not be prepared.

[0083] Comparative Example 2

[0084] Comparative Example 2 of the present invention provides a method for preparing a novel split ring cyclopentadiene ether terpene compound, and its steps are similar to those of Example 1, except that in step S4, when fraction C is subjected to ODS column chromatography, the volume ratio of methanol-water solution is lower than (15:85) and higher than (25:75).

[0085] The results showed that compound 1 could not be prepared.

[0086] In order to better understand the essence of the present invention, the new application of the above-mentioned cleocyclic iridoid compounds in the pharmaceutical field is explained below in combination with pharmacological tests and results.

[0087] Test Example 1

[0088] This test example discloses an experiment on the NO release inhibition activity of the above compound (±)-Cornuscone.

[0089] (1) Experimental materials and instruments

[0090] RAW 264.7 cell line was purchased from the Cell Collection of the Chinese Academy of Sciences (Shanghai);

[0091] RPMI 1640 medium (Beijing Solebow Technology Co., Ltd.);

[0092] PBS buffer (Gibco, USA);

[0093] Fetal bovine serum (FBS, Gibco, USA);

[0094] Trypsin (Gibco, USA);

[0095] NO detection kit (Beyotime, USA);

[0096] Indomethacin (Shanghai MacLean Biochemical Technology Co., Ltd.);

[0097] Normal saline (Beijing Puli Zhicheng Biotechnology Co., Ltd.);

[0098] 96-well plate (Corning, USA);

[0099] (±)-Cornuscone (made in the laboratory);

[0100] 10-(3,5-dimethoxy-4-hydroxybenzoate)-iridrol glycoside (prepared in the laboratory);

[0101] Carbon dioxide cell culture incubator (Thermo Corporation, USA);

[0102] Full wavelength microplate reader (Thermo Corporation, USA);

[0103] BIOFUGE STRATOS centrifuge (Thermo Corporation, USA);

[0104] IX73 inverted electron microscope (Olympus, Japan);

[0105] Pipette (Eppendorf, Germany);

[0106] Clean bench, centrifuge tubes, pipettes and other related consumables.

[0107] (2) Experimental methods

[0108] Cell recovery and culture: Take out the marked RAW 264.7 cell cryopreservation tube from liquid nitrogen and immediately put it into a 37°C water bath, and try to complete rapid thawing within 1 minute; after disinfecting the cryopreservation tube with alcohol, transfer the cryopreservation solution in the tube into a 15mL sterile centrifuge tube, add the corresponding culture medium, mix it, and centrifuge it at 1000rpm for 5min to remove the supernatant; repeat the above steps once for washing, then add 10mL of culture medium to mix and resuspend the cells, then transfer them to a 10mL culture dish, and culture them in a constant temperature incubator at 37°C and 5% CO2.

[0109] Cell passaging: When the cell density reaches 70%-80%, it will be passaged; first remove the old culture medium, add PBS to wash twice, add trypsin containing EDTA to the culture dish and place it in a 37℃ incubator for 3 minutes, then add culture medium to terminate digestion, centrifuge at 1000rpm for 5 minutes, remove the digestion solution, re-add serum-containing culture medium and repeatedly blow the cells to form a cell suspension, and finally transfer them to a new culture dish according to the number of cells to complete cell passaging.

[0110] Griess method was used to evaluate cell viability: RAW 264.7 cells were cultured at 2×10 4 The cells / well were plated in 96-well plates, with 6 replicates in each group. After plating, they were cultured for 24 hours. The positive drugs indomethacin, 10-(3,5-dimethoxy-4-hydroxybenzoate)-iridoid glycosides, and compound (±)-Cornuscone were dissolved and serially diluted (1.26, 3.13, 6.25, 12.5, 25, 50, 100 μM) and then administered respectively. After adding 10 μL / well to treat the cells, they were incubated at 37°C for 48 hours, and then 10 μL of the NO detection kit was added. After 2 hours, the absorbance at 540 nm was detected by an enzyme reader, and the IC of inhibition of NO release was calculated. 50 value.

[0111] (3) Experimental results

[0112] As shown in Table 2, (±)-Cornuscone inhibited LPS-induced NO release in RAW 264.7 cells and exhibited significant anti-inflammatory activity. 50 The value was 31.15±1.29μM, which was significantly better than the positive drug indomethacin (IC 50 value was 48.32±1.66μM) and 10-(3,5-dimethoxy-4-hydroxybenzoate)-iridoid glycosides (IC 50 The value was 54.66±0.91μM).

[0113] Table 2 Inhibitory activity of compounds on NO release from LPS-induced RAW 264.7 cells

[0114]

[0115] In summary, the novel split-cyclic iridoid compound (±)-Cornuscone described in the present invention has significant anti-inflammatory activity, which is significantly better than the first-line anti-inflammatory drug indomethacin, and can be used to treat various inflammation-related diseases, including but not limited to arthritis, conjunctivitis, pneumonia, hepatitis, nephritis, gastroenteritis, metabolic syndrome, diabetes, cancer, etc.

[0116] Application Example 1

[0117] The application example of the present invention discloses a capsule preparation using (±)-Cornuscone as a raw material drug, and its components are as follows:

[0118]

[0119] The specific preparation process is as follows:

[0120] (±)-Cornuscone, starch and sodium metabisulfite were mixed evenly, anhydrous ethanol was added to make a soft material, the mixture was passed through a 24-mesh sieve to make granules, the granules were dried, magnesium stearate was added, the mixture was mixed evenly and the granules were put into capsules.

[0121] Application Example 2

[0122] The application example of the present invention discloses a granule preparation using the compound (±)-Cornuscone as a raw material drug, and the components thereof are as follows:

[0123]

[0124]

[0125] The specific preparation process is as follows:

[0126] (±)-Cornuscone was mixed with starch and sodium bisulfite, and then anhydrous ethanol was added to make a soft material. The mixture was passed through a 24-mesh sieve to make granules, dried, magnesium stearate was added, mixed, and bagged.

[0127] Application Example 3

[0128] The application example of the present invention discloses an oral solution using the compound (±)-Cornuscone as a raw material drug, and its components are as follows:

[0129]

[0130] The specific preparation process is as follows:

[0131] After the above components are mixed evenly, they can be packaged using a conventional oral liquid preparation method.

[0132] Application Example 4

[0133] The application example of the present invention discloses an injection with the compound (±)-Cornuscone as the raw material drug, and its components are as follows:

[0134]

[0135] The specific preparation process is as follows:

[0136] After the above components are mixed evenly, 100 vials can be obtained using a conventional injection preparation method.

[0137] Application Example 5

[0138] The application example of the present invention discloses a tablet with compound (±)-Cornuscone and aspirin as raw materials, and its components are as follows:

[0139]

[0140] The specific preparation process is as follows:

[0141] Take (±)-Cornuscone, aspirin, hydroxypropyl methylcellulose, talc, lactose and magnesium stearate, mix them evenly, add anhydrous ethanol to make a soft material, pass through a 24-mesh sieve, make granules, dry, add magnesium stearate, mix evenly, and press into tablets.

[0142] Application Example 6

[0143] The application example of the present invention discloses a capsule preparation using compound (±)-Cornuscone and ibuprofen as raw materials, and the components thereof are as follows:

[0144]

[0145] The specific preparation process is as follows:

[0146] (±)-Cornuscone, ibuprofen, starch and sodium metabisulfite were mixed, anhydrous ethanol was added to make a soft material, the mixture was passed through a 24-mesh sieve to make granules, the granules were dried, magnesium stearate was added, the mixture was mixed, and the granules were filled into capsules.

[0147] Application Example 7

[0148] The application example of the present invention discloses an injection with compound (±)-Cornuscone and cortisone as raw materials, and its components are as follows:

[0149]

[0150]

[0151] The specific preparation process is as follows:

[0152] After the above components are mixed evenly, 100 vials can be obtained using a conventional injection preparation method.

[0153] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A novel cleaved iridoid compound, characterized in that: It has the structure shown in formula (1):

2. A method for preparing the novel cleaved iridoid compound as claimed in claim 1, characterized in that: The cleaved iridoid compound is extracted and separated from Cornus officinalis. The preparation steps are as follows: S1, extracting Cornus officinalis by refluxing with a solvent, combining the extracts and concentrating them to obtain an extract; the solvent is water; S2. The extract was fully dissolved in water, and the extract was eluted with D101 macroporous adsorption resin using a gradient elution of ethanol and water in a volume ratio of (50:50)-(60:40) based on a total volume of 100, and then concentrated to obtain an elution fraction; S3. Using a chloroform-methanol solution with a volume ratio of (80:20)-(90:10) to perform gradient elution on the elution portion through silica gel column chromatography to obtain fraction C; when detected by silica gel thin layer chromatography, the Rf value corresponding to fraction C is 0.71-0.73; S4. Fraction C was gradient eluted by ODS column chromatography using a methanol-water solution with a volume ratio of (15:85)-(25:75) to obtain fraction C1-1; when detected by silica gel thin layer chromatography, the Rf value corresponding to fraction C1-1 was 0.21-0.30; S5. Fraction C1-1 was subjected to gradient elution by Sephadex LH-20 gel column chromatography using a methanol-water solution with a volume ratio of (55:45)-(65:35) to obtain fraction C1-1.

10. When detected by silica gel thin layer chromatography, the Rf value of fraction C1-1.10 was 0.61-0.68; S6, using HPLC method, using C 18 The chromatographic column separates the novel split ring iridoid compound having the structure shown in formula (1) from fraction C1-1.10; The mobile phase used in the HPLC method was a methanol-water mixed solution with a volume ratio of (22:78)-(27:73).

3. The preparation method according to claim 2, characterized in that: In step S6, fraction C1-1.10 is further separated according to the characteristic ultraviolet absorption of the cleaved iridoids observed by HPLC analysis, and the retention time of the novel cleaved iridoid compound having the structural formula shown in formula (1) is 20-25 min.

4. The preparation method according to claim 2, characterized in that: In step S1, the mass of the added solvent is 8-10 times that of the Cornus officinalis, the number of reflux extractions is 2-4 times, and each extraction lasts 1-3 hours.

5. The preparation method according to claim 2, characterized in that: In step S2, when washing out the elution part, the volume ratio of ethanol to water in the ethanol-water mixed solution is 55:

45.

6. The preparation method according to claim 2, characterized in that: In step S3, the volume ratio of chloroform to methanol in the chloroform-methanol mixed solution when fraction C is eluted is 85:

15.

7. The preparation method according to claim 2, characterized in that: In step S4, the volume ratio of methanol to water in the methanol-water solution when washing out fraction C1-1 is 20:

80.

8. The preparation method according to claim 2, characterized in that: In step S5, the volume ratio of methanol to water in the methanol-water solution when fraction C1-1.10 is eluted is 60:

40.

9. A pharmaceutical composition, characterized in that The invention comprises the novel split ring iridoid ether terpenoid compound as claimed in claim 1.

10. The pharmaceutical composition according to claim 9, characterized in that The pharmaceutical composition comprises a synergist and a pharmacologically acceptable excipient.

11. The pharmaceutical composition according to claim 10, characterized in that The synergist is one or more of the following substances: Aspirin, ibuprofen, indomethacin, naproxen, nabumetone, piroxicam, phenylbutazone, diclofenac, fenprofen, ketoprofen, ketorolac, tetraclofenamic acid, sulindac, tolmetin, fenbufen, celecoxib, cortisone.

12. The pharmaceutical composition according to claim 10, characterized in that The dosage form of the pharmaceutical composition is tablets, capsules, granules, oral liquid, granules, dropping pills or micropills.

13. Use of the novel secoiridoid compound according to claim 1 and / or the pharmaceutical composition according to any one of claims 9 to 12 in the preparation of drugs for treating inflammatory diseases.

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