High isoflavone compound with anti-inflammatory activity extracted from fibrous roots of radix ophiopogonis as well as preparation method and application of high isoflavone compound

Abstract

The invention belongs to the field of chemical medicines, and particularly relates to a homoisoflavone compound with anti-inflammatory activity, which is extracted from fibrous roots of ophiopogon japonicus and has a structure as shown in a formula (I), and a preparation method and application of the homoisoflavone compound. Particularly, dry fibrous roots of ophiopogon plant radix ophiopogonis are used as raw materials, a compound is obtained through separation by certain preparation steps, the specific structure is clarified according to multiple spectrum technologies such as NMR, HRESI and IR, and the compound is reported for the first time. The invention discloses a specific structure of the novel compound, the anti-inflammatory and analgesic activity of the novel compound is evaluated, and a result shows that the compound has obvious anti-inflammatory and analgesic activity.

Description

Technical Field

[0001] The invention belongs to the field of chemical medicine, and specifically relates to a high isoflavone compound with anti-inflammatory and analgesic activity separated from ophiopogon japonicus root hairy roots, and a preparation method and application thereof. Background Art

[0002] Ophiopogon japonicas (L. f) Ker-Gawl. is a dried root of Ophiopogon japonicas (L. f) Ker-Gawl., a plant of the genus Ophiopogon in the family Liliaceae. It is sweet, slightly bitter, and slightly cold in nature. It enters the heart, lung, and stomach meridians. It has the effects of nourishing yin and promoting fluid production, moistening the lungs and clearing the heart. It is used for dry cough due to lung dryness, tuberculosis due to yin deficiency, sore throat, thirst due to fluid loss, internal heat and thirst, restlessness and insomnia, and constipation due to dry intestines. Ophiopogon japonicus is a traditional Chinese medicine. It was first recorded in the "Shen Nong's Herbal Classic". It is known for its long-term use to make the body light and not old or hungry. Ophiopogon japonicus grows in the shade and wet places on the slopes below 2,000 meters above sea level, under the forest or beside the stream. It is cultivated in Zhejiang, Sichuan, Guangxi and other places. At present, the main secondary metabolites isolated from this plant are steroidal saponins, homoisoflavonoids, polysaccharides, etc.

[0003] In traditional Chinese medicine, only the tuberous roots of Ophiopogon japonicus are used as medicine, while the fibrous roots are used as livestock feed or directly discarded. Existing studies have shown that the nutritional components and active substances of the fibrous roots of Ophiopogon japonicus are similar to those of the tuberous roots of Ophiopogon japonicus, and some are even higher than those of the tuberous roots. Therefore, its medicinal value may be similar to that of the tuberous roots of Ophiopogon japonicus, and it should have a good development and application prospect. The present invention takes the fibrous roots of Ophiopogon japonicus, a non-traditional medicinal part of Ophiopogon japonicus, as the research object, and discovers new active compounds therefrom, which is of great significance for fully tapping and utilizing Ophiopogon japonicus resources and promoting the sustainable, rapid and healthy development of the Ophiopogon japonicus industry. Summary of the invention

[0004] One of the purposes of the present invention is to discover a new compound as shown in formula (I) from the fibrous roots of Ophiopogon japonicus, the chemical structure of which is as follows:

[0005]

[0006] The second object of the present invention is to disclose a method for preparing a new compound, comprising the following steps:

[0007] 1) After the dried Ophiopogon japonicus root is crushed, it is refluxed and extracted with 50% to 80% ethanol solution. The amount of extraction solvent is 8 to 15 times the mass of the raw material. The number of extractions is 2 to 4 times, each time for 1 to 3 hours. The extracts are combined, filtered, concentrated to a solid-liquid ratio of 1:5 to 1:15, and allowed to stand for 3 to 12 hours for use;

[0008] 2) The supernatant obtained in step 1) is subjected to a macroporous adsorption resin, and eluted with 2 to 10 column volumes of 50% to 70% ethanol and 90% ethanol, respectively, and the eluate is collected, and the 90% ethanol eluate is concentrated to dryness to obtain a crude extract containing the target compound, which is set aside;

[0009] 3) The crude extract obtained in step 2) was subjected to silica gel column chromatography, and gradient elution was performed using chloroform: methanol solvent system of 100:1, 50:1, 20:1, 10:1, 5:1, 1:1, and 0:1 as eluents, and the eluents were collected, and the eluents were combined by TLC spot plate detection to obtain 18 components Fr.1-18; Fr.10 was subjected to silica gel column chromatography, and gradient elution was performed using petroleum ether: ethyl acetate solvent system of 50:1, 20:1, 10:1, 5:1, and 1:1 as eluents, and the eluents were collected, and the eluents were combined by TLC spot plate detection to obtain 5 subcomponent segments Fr.10A-Fr.10E; Fr.10D was subjected to Sephadex LH-20 column chromatography, eluted with methanol, and 4 sample segments Fr.10D1-Fr.10D4 were separated; Fr.10D2 was subjected to ODS column chromatography, and eluted with 60% ethanol aqueous solution to obtain compound I.

[0010] The macroporous adsorption resin in step 2) is one of HPD-100, D-101 and AB-8.

[0011] The structures of the new compounds are 1 H-NMR, 13 C-NMR, DEPT, HSQC, COSY, HMBC, and HR-ESI-MS were used for identification.

[0012] The third object of the present invention also relates to the use of the new compound in the preparation of anti-inflammatory analgesic drugs. Among them, the compound has a significant inhibitory effect on the release of inflammatory factors IL-6 and TNF-α in RAW264.7 cells induced by LPS, and has a significant inhibitory effect on the pain caused by peritoneal inflammation in mice stimulated by acetic acid.

[0013] The structure, preparation method and pharmacological activity of the new compound described in the present invention have not been reported in the literature. Through the effect experiment on the release of inflammatory factors of RAW264.7 cells induced by LPS and the acetic acid writhing experiment in mice, it is proved that the new high isoflavone compound has significant anti-inflammatory and analgesic activity, and can be used as a natural product to develop new Chinese medicine drugs, which has broad prospects. In addition, the preparation of the new compound described in the present invention comes from the non-traditional medicinal part of Ophiopogon japonicus, the root of Ophiopogon japonicus, which is of great significance for the comprehensive utilization of Ophiopogon japonicus medicinal materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a graph showing the effect of the compounds of the present invention on the release of inflammatory factors in RAW264.7 cells induced by LPS;

[0015] Figure 2 This is a graph showing the effect of the compounds of the present invention on the number of writhing times induced by acetic acid in mice;

[0016] Figure 3 is the hydrogen nuclear magnetic resonance spectrum of the compound of formula I of the present invention;

[0017] Figure 4 The carbon-NMR spectrum of the compound of formula I of the present invention;

[0018] Figure 5 is the HRESI spectrum of the compound of formula I of the present invention;

[0019] Figure 6 The compounds of the present invention 1 H- 1 H COSY and main HMBC signal correlation diagram;

[0020] Figure 7 It is the structural formula of compound I of the present invention. DETAILED DESCRIPTION

[0021] The present invention will be further described in detail below by way of examples, and those skilled in the art can refer to the content of the present invention and appropriately improve the relevant parameters according to the specific actual equipment. The protection scope of the present invention is not limited to the content, and the methods in the embodiments are conventional methods unless otherwise specified, and the reagents used are conventional commercial reagents or reagents prepared according to conventional methods unless otherwise specified.

[0022] Example 1: Extraction and separation of compounds

[0023] 1) Take 15 kg of dried Ophiopogon japonicus root, crush it, put it in a multifunctional extraction tank, add 70% ethanol 10 times the mass of the raw material, reflux extraction, add 10 times the amount of 70% ethanol each time to extract for 2 h, extract 3 times in total, filter the extract, combine the filtrate and place it in a double-effect concentrator (60-70 ℃) to concentrate it to a solid-liquid ratio of 1:10, let it stand for 8 h, and set aside;

[0024] 2) The supernatant obtained in step 1) was separated using HPD-100 macroporous adsorption resin, and eluted with 5 column volumes of 70% ethanol and 90% ethanol in sequence, the eluate was collected, and the 90% ethanol eluate was concentrated to dryness to obtain 150 g of a crude extract containing the target compound, which was set aside;

[0025] 3) The crude extract obtained in step 2) was ultrasonically dissolved with an appropriate amount of ethanol, mixed with 100-200 mesh silica gel at a 2-fold mass ratio, evaporated to dryness, and dry-packed with 200-300 mesh silica gel at a 50-fold mass ratio, and gradient eluted with chloroform: methanol solvent system of 100:1, 50:1, 20:1, 10:1, 5:1, 1:1, 0:1 as eluent, and the eluent was collected, and the eluent was combined after TLC spot plate detection to obtain 18 components Fr.1-18; Fr.10 was subjected to silica gel column chromatography, and gradient eluted with petroleum ether: ethyl acetate solvent system of 50:1, 20:1, 10:1, 5:1, 1:1 as eluent, and the eluent was collected, and the eluent was combined after TLC spot plate detection to obtain 5 subcomponent segments Fr.10A- Fr.10E and Fr.10D were separated by Sephadex LH-20 column chromatography and eluted with methanol to obtain four sample segments Fr.10D1-Fr.10D4; Fr.10D2 was separated by ODS column chromatography and eluted with 60% ethanol aqueous solution to obtain compound Ⅰ (78.5 mg).

[0026] Example 2: Extraction and separation of compounds

[0027] 1) Take 15 kg of dried Ophiopogon japonicus root, crush it, put it in a multifunctional extraction tank, add 80% ethanol in an amount of 8 times the mass of the raw material, reflux extract, add 8 times the amount of 80% ethanol each time to extract for 3 hours, extract 4 times in total, filter the extract, combine the filtrate, and place it in a double-effect concentrator (60-70 ℃) to concentrate it to a solid-liquid ratio of 1:5, let it stand for 3 hours, and set aside;

[0028] 2) The supernatant obtained in step 1) was separated with AB-8 macroporous adsorption resin, and eluted with 2 column volumes of 60% ethanol and 90% ethanol in sequence, the eluate was collected, and the 90% ethanol eluate was concentrated to dryness to obtain 155 g of a crude extract containing the target compound, which was set aside;

[0029] 3) Same as step 3) in Example 1.

[0030] Example 3: Extraction and separation of compounds

[0031] 1) Take 15 kg of dried Ophiopogon japonicus root, crush it, put it in a multifunctional extraction tank, add 50% ethanol 15 times the mass of the raw material, reflux extraction, add 15 times the amount of 50% ethanol each time to extract for 1 hour, extract twice in total, filter the extract, combine the filtrate and place it in a double-effect concentrator (60-70 ℃) to concentrate it to a solid-liquid ratio of 1:15, let it stand for 12 hours, and set aside;

[0032] 2) The supernatant obtained in step 1) was separated with D101 macroporous adsorption resin, and eluted with 10 column volumes of 50% ethanol and 90% ethanol in sequence, the eluate was collected, and the 90% ethanol eluate was concentrated to dryness to obtain 146 g of a crude extract containing the target compound, which was set aside;

[0033] 3) Same as step 3) in Example 1.

[0034] Example 4: Structural Identification of New Compounds

[0035] Compound Ⅰ, yellow amorphous powder, was characterized by negative ion peak m / z 367.0819 (calculated value 367.0818) and 13 C-NMR spectral data, the chemical formula was inferred to be C 20 H 16 O7. IR spectrum showed the presence of hydroxyl groups (3432 cm −1 ) and carbonyl (1646 cm −1 ) related absorption bands. 1 The H-NMR spectrum (Table 1) showed the presence of methylenedioxy (δ 5.91, 2H, s), aldehyde (δ 10.35), methoxy (δ 3.90, 3H, s), benzylmethylene (δ 3.68, 2H, s), a methyl group attached to the aromatic nucleus (δ 2.16), three aromatic protons at δ 6.70 - 6.78 (3H, m) and a chelated hydroxyl group at δ 12.92 (1H, s), suggesting that compound Ⅰ may be a 2,3-unsaturated-3-benzylchroman-4-one type homoisoflavonoid compound. 13 The presence of 20 carbon signals in the C-NMR spectrum (including the DEPT-135 spectrum) confirms the above hypothesis. The typical aldehyde and α,β-unsaturated ketone signals appear at δ C 191.7 and 174.6. The HMBC data confirmed the substituents on ring A: hydroxyl (δ H 12.92) related to C-6 and C-8, methyl (δ H 2.16) related to C-5 and C-7, methoxy (δ 3.90) related to C-5, aldehyde (δ H 10.35) are associated with C-7 and C-10. In addition, the methylenedioxy protons (δ H 5.91) and C-3′ (δ C 147.9) and C-4′ (δ C146.3) showed that ring B was 1,3,4-trisubstituted. All the above results showed that the structure of compound I was 5-methoxy-6-methyl-7-hydroxy-8-formyl-3-(3, 4'-methylenedioxybenzyl)chroman-4-one, a new compound named ophiopogonone F. Its structural formula with atomic number is:

[0036]

[0037]

[0038] Example 5: LPS-induced release of inflammatory factors from RAW264.7 cells

[0039] RAW264.7 macrophages in the logarithmic growth phase were seeded in 6-well culture plates at a cell density of 2×10 5 / mL, 2mL per well, cultured at 37℃, 5% CO2. The cell supernatant was removed every other day, and the model control group, test group (ophiopogonone F) and positive control group (indomethacin) were all modeled with 200ng / mL LPS. At the same time, the test group (ophiopogonone F) and positive control group (indomethacin, 10μg / mL) were administered at the corresponding concentrations. The normal control group was not modeled, and the serum-free DMEM medium was replaced, 1mL per well, and 3 replicates were set for each group. After incubation for 24h, the cell supernatant was collected, and the IL-6 and TNF-α contents in the cell supernatant were detected by ELISA, and the results were statistically analyzed.

[0040] like Figure 1 As shown in the results, compared with the normal control group, LPS-induced RAW264.7 cells could significantly increase the levels of IL-6 and TNF-α in the cell supernatant (p<0.01); compared with the model control group, ophiopogonone F in each dose group could significantly inhibit the levels of IL-6 and TNF-α in the cell supernatant (p<0.01), and it was dose-dependent, indicating that ophiopogonone F has good anti-inflammatory activity.

[0041] Example 6: Acetic acid writhing experiment in mice

[0042] 72 SPF KM mice, all male, weighing 18-22 g, were randomly divided into a vehicle control group, an aspirin 0.4 g / kg group, an ophiopogonone F low-dose group (5 mg / kg), and an ophiopogonone F high-dose group (10 mg / kg), with 12 mice in each group. Each group of mice was intragastrically administered with the corresponding test substance at a dosage volume of 20 mL / kg, and the vehicle control group was given an equal volume of solvent. One hour after administration, 0.62% glacial acetic acid was intraperitoneally injected at a dosage volume of 10 mL / kg. Ten minutes later, the number of twisting reactions of the mice within 15 minutes was observed (based on the reaction of the mice showing abdominal concavity, extension of the trunk and hind limbs, and lifting of the buttocks), and the results were statistically analyzed.

[0043] like Figure 2 As shown, compared with the vehicle control group, the writhing times of mice in each dose group of compound ophiopogonone F were significantly reduced (p<0.05 or p<0.01) in a dose-dependent manner, indicating that ophiopogonone F has good analgesic activity.

Claims

1. A high isoflavone compound extracted from the fibrous roots of Ophiopogon japonicus, characterized in that: The structure is shown in formula (I): 。 2. The method for preparing high isoflavone compounds extracted from the fibrous roots of Ophiopogon japonicus according to claim 1, characterized in that: The following steps are involved: 1) After the dried Ophiopogon japonicus root is crushed, it is refluxed and extracted with 50% to 80% ethanol solution. The amount of extraction solvent is 8 to 15 times the mass of the raw material. The number of extractions is 2 to 4 times, each time for 1 to 3 hours. The extracts are combined, filtered, concentrated to a solid-liquid ratio of 1:5 to 1:15, and allowed to stand for 3 to 12 hours for use; 2) The supernatant obtained in step 1) is subjected to a macroporous adsorption resin, and eluted with 2 to 10 column volumes of 50% to 70% ethanol and 90% ethanol, respectively, and the eluate is collected, and the 90% ethanol eluate is concentrated to dryness to obtain a crude extract containing the target compound, which is set aside; 3) The crude extract obtained in step 2) was subjected to silica gel column chromatography, and gradient elution was performed using chloroform: methanol solvent system of 100:1, 50:1, 20:1, 10:1, 5:1, 1:1, and 0:1 as eluents, and the eluents were collected, and the eluents were combined by TLC spot plate detection to obtain 18 components Fr.1-18; Fr.10 was subjected to silica gel column chromatography, and gradient elution was performed using petroleum ether: ethyl acetate solvent system of 50:1, 20:1, 10:1, 5:1, and 1:1 as eluents, and the eluents were collected, and the eluents were combined by TLC spot plate detection to obtain 5 subcomponent segments Fr.10A-Fr.10E; Fr.10D was subjected to Sephadex LH-20 column chromatography, eluted with methanol, and 4 sample segments Fr.10D1-Fr.10D4 were separated; Fr.10D2 was subjected to ODS column chromatography, and eluted with 60% ethanol aqueous solution to obtain compound I.

3. The method for preparing high isoflavone compounds extracted from the fibrous roots of Ophiopogon japonicus according to claim 2, characterized in that: The macroporous adsorption resin in step 2) is one of HPD-100, D-101 and AB-8.

4. The high isoflavone compound extracted from the fibrous roots of Ophiopogon japonicus according to claim 1, characterized in that: The structure of the compound is 1 H-NMR, 13 The proteins were identified by C-NMR, DEPT, HSQC, COSY, HMBC and HR-ESI-MS spectroscopy techniques.

5. The high isoflavone compound extracted from the fibrous roots of Ophiopogon japonicus according to claim 1, characterized in that: Application of the compound in the preparation of anti-inflammatory and analgesic drugs.

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