Compounds isolated from senecio cruckshanksii and preparation method and use thereof

Abstract

The present application relates to the technical field of natural product separation, in particular to a compound separated from gerbera kikuchii, a preparation method and application thereof, the preparation method of the compound comprising the steps of alcohol extraction from gerbera kikuchii, resin adsorption separation, column chromatography and gel separation. The compound disclosed by the present application can be used for preparing a RAW264.7 cell inhibitor.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of natural product separation, in particular to a compound separated from Gnaphalium affine, and a preparation method and application thereof. BACKGROUND

[0002] Gnaphalium affine is the dry whole plant of Gnaphalium affine (Franch.) of the tribe of the family of Compositae. Oreoseris delavayi It has the effects of clearing heat and reducing dampness, eliminating accumulation and killing insects, and is used for treating dysentery, stomachache, indigestion and ascariasis. Modern studies show that the main compounds in Gnaphalium affine are coumarins, phenylacetone, phenylpropionic acid, organic acid and flavonoids, and pharmacological experiments show that coumarins and flavonoids in Gnaphalium affine are very important metabolites, have good anti-inflammatory, antibacterial and antiviral activities, and have broad development and application prospects.

[0003] At present, the research on Gnaphalium affine mainly focuses on cultivation and propagation techniques and cell cultures, and there are few studies on its chemical components and pharmacological activities. Therefore, Gnaphalium affine is separated and identified by using various chromatographic separation and purification techniques, and the anti-inflammatory activity of the separated compounds is studied, so as to clarify the pharmacodynamic material basis of Gnaphalium affine, expand the research on its pharmacological activities, and provide a basis for further development and utilization of Gnaphalium affine and quality control standards. SUMMARY

[0004] One object of the present application is to provide a phenolic dimer compound extracted from Gnaphalium affine, and another object of the present application is to provide a method for obtaining effective chemical components from Gnaphalium affine, and to provide the use of the phenolic dimer compound.

[0005] According to a first aspect of the present application, a compound separated from Gnaphalium affine has a structure as shown in formula 1:

[0006]

[0007] According to a second aspect of the present application, a compound separated from Gnaphalium affine has a structure as shown in formula 2:

[0008]

[0009] According to a third aspect of the present application, a compound separated from Gnaphalium affine has a structure as shown in formula 3:

[0010]

[0011] According to a fourth aspect of the present application, a preparation method of a compound separated from Senecio cruentus includes the following steps:

[0012] The Senecio cruentus is subjected to alcohol extraction to obtain a first alcohol extract, the first alcohol extract is subjected to adsorption by a macroporous resin and then elution by alcohol to obtain a first eluate, and the eluate is subjected to column chromatography to obtain the compounds 1-3.

[0013] The compound 1-3 has a structure as shown in formula 1 to formula 3.

[0014]

[0015] According to an embodiment of the present application, the preparation method of the first alcohol extract includes the following steps.

[0016] The dried and crushed Senecio cruentus is subjected to reflux extraction by an ethanol solution to obtain a first filtrate, and the first filtrate is concentrated to obtain the first alcohol extract.

[0017] According to an embodiment of the present application, the macroporous resin is a D101 macroporous resin, and the elution is performed by using water and an alcohol solution.

[0018] According to an embodiment of the present application, the column chromatography is performed by using a normal phase column and gel chromatography.

[0019] According to a fifth aspect of the present application, the above-mentioned phenolic dimer compound is applied to the preparation of an inhibitor of RAW264.7 cells.

[0020] According to an embodiment of the present application, the dosage form of the inhibitor is a tablet, a pill, a powder, a granule, a capsule, an oral solution, an infusion, a freeze-dried powder injection, an ointment, a gel or a spray.

[0021] The present application has the following beneficial effects:

[0022] The present application separates and extracts the phenolic dimer compound of formula 1-3 from Senecio cruentus, and the experimental detection shows that the compound 1-3 has certain inhibitory activity on RAW264.7 cells, can be used for reference use of an inflammation inhibitor, and can also be used for preparation of related drugs. The preparation method of the present application is simple in steps and low in cost, promotes the development of Senecio cruentus in medicine and other aspects, and plays an important role in promoting the sustainable development of the industry. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1a HR-ESI-MS of the compound 1 of the present application;

[0024] Figure 1b HR-ESI-MS of the compound 1 of the present application;

[0025] Figure 2 HR-ESI-MS for the compound 1 of the present application 1 H-NMR spectrum;

[0026] Figure 3 HR-ESI-MS for the compound 1 of the present application 13 C-NMR spectrum;

[0027] Figure 4 NMR HSQC spectrum for the compound 1 of the present application;

[0028] Figure 5 NMR HMBC spectrum for the compound 1 of the present application;

[0029] Figure 6a HR-ESI-MS for the compound 2 of the present application

[0030] Figure 6b HR-ESI-MS for the compound 2 of the present application

[0031] Figure 7 H-NMR spectrum for the compound 2 of the present application 1 H-NMR spectrum for the compound 2 of the present application

[0032] Figure 8 C-NMR spectrum for the compound 2 of the present application 13 C-NMR spectrum for the compound 2 of the present application

[0033] Figure 9 NMR HSQC spectrum for the compound 2 of the present application;

[0034] Figure 10 NMR HMBC spectrum for the compound 2 of the present application;

[0035] Figure 11a HR-ESI-MS for the compound 3 of the present application

[0036] Figure 11b HR-ESI-MS for the compound 3 of the present application

[0037] Figure 12 H-NMR spectrum for the compound 3 of the present application 1 H-NMR spectrum for the compound 3 of the present application

[0038] Figure 13 C-NMR spectrum for the compound 3 of the present application 13 C-NMR spectrum for the compound 3 of the present application

[0039] Figure 14 NMR HSQC spectrum for the compound 3 of the present application;

[0040] Figure 15 NMR HMBC spectrum for the compound 3 of the present application. DETAILED DESCRIPTION

[0041] In order to make the objectives, technical solutions and advantages of the present application clearer, the present application will be further described in detail. It should be understood that the specific embodiments described herein are only used to explain the present application and not used to limit the present application, i.e., the described embodiments are only a part of the embodiments of the present application, but not all the embodiments. Example 1

[0042] Step 1, take the hook big daisy medicinal materials (20 kg), use 50% ethanol reflux extraction 3 times, 1.5 hours each time, the first time use 8 times amount of 50% ethanol, the second time, the third time use 6 times amount of 50% ethanol, combine the alcohol extract after reducing pressure recovery of ethanol, direct heating concentration constantly add water to evaporate ethanol until no obvious ethanol taste;

[0043] Step 2, after the paste is mixed, the D101 macroporous adsorption resin is used for rough separation, the macroporous adsorption resin is adsorbed, and the water and 50% ethanol are eluted and concentrated in turn, to obtain different component segments (water segment, 50% ethanol segment);

[0044] Step 3, the 50% ethanol segment extract is added to the normal phase silica gel column, eluted with chloroform-methanol (10:0-6:4) gradient, combined by TLC detection, to obtain 8 components (Fr.1-8). The combined elution part is concentrated to dryness under reduced pressure, and used as needed.

[0045] Step 4, Fr.5 in step 3 is subjected to normal phase silica gel column (ethyl acetate-methanol 200:1-1:1), TLC detection and combination, and then concentrated to obtain 6 components (Fr.5.1-5.6). Fr.5.1 is subjected to normal phase silica gel column (dichloromethane-methanol system 1:0-5:1), Toyopearl HW-40F gel column chromatography (methanol), to obtain compound 1. Fr.5.4 is subjected to Sephadex LH-20 gel column chromatography (methanol), ODS reverse phase column (20%-100% methanol water), Toyopearl HW-40F gel column chromatography (methanol), and preparation HPLC (80% methanol water), to obtain compound 3.

[0046] Step 5, Fr.6 in step 3 is subjected to normal phase silica gel column (ethyl acetate-methanol 9.8:0.2-0:1), TLC detection and combination, and then concentrated to obtain 7 components (Fr.6.1-6.7). Fr.6.4 is subjected to MCI (10% methanol water), normal phase silica gel column (dichloromethane-methanol 9:1), Sephadex LH-20 gel column chromatography (dichloromethane-methanol 1:1), Toyopearl HW-40F gel column chromatography (methanol), Sephadex LH-20 gel column chromatography (50% acetone water), ODS reverse phase column (10% methanol water), to obtain compound 2.

[0047] The structure of the present application is identified by using 1 H NMR, 13 C NMR nuclear magnetic spectrum, two-dimensional nuclear magnetic spectrum, high resolution mass spectrum for structure identification of the separated monomer compound, as shown in Figures 1-15.

[0048] Among them, compound 1 and compound 2 are stereoisomers, compound 1 is levorotatory, and compound 2 is dextrorotatory.

[0049] Compound 1, yellow solid, molecular formula: C 25 H 20 O8; HR-ESI-MS m / z: 471.1074 [M+Na] ＋ (calculated value: 471.1050), 447.1083 [M-H] － (calculated value: 447.1074); the compound is determined to be (-)-Caesalstilbene C by spectral technology, and its nuclear magnetic data is shown in Table 1.

[0050] Compound 2, yellow solid, molecular formula: C 25 H 20 O8; HR-ESI-MS m / z: 471.1074 [M+Na] ＋ (calculated value: 471.1050), 447.1083 [M-H] － (calculated value: 447.1074); the compound is determined to be (+)-Caesalstilbene C by spectral technology, and its nuclear magnetic data is shown in Table 2.

[0051] Compound 3, white solid, molecular formula: C 14 H 16 O8; HR-ESI-MS m / z: 335.0742 [M+Na] ＋ (calculated value: 335.0737), 357.0824 [M+HCOO] － (calculated value: 357.0816); the compound is determined to be Ampelopsin J by spectral technology, and its nuclear magnetic data is shown in Table 3, and the corresponding labeled chemical formula is shown in the following formula 1-3.

[0052]

[0053] Table 1 Nuclear magnetic resonance data of compound 1 1 H (400 MHz) and 13 C (100 MHz) NMR data

[0054]

[0055] Table 2. Compound 2 1 H (400 MHz) and 13 C (100 MHz) NMR data

[0056]

[0057] Table 3. Compound 3 1 H (600 MHz) and 13 C (150 MHz) NMR data

[0058]

[0059] Test Example 1

[0060] To achieve the above-mentioned objects of the present application, the anti-inflammatory mechanism of the extract from Gerbera kikuchii is studied, and the specific steps are as follows:

[0061] Step 1, cell recovery: the frozen cells are quickly taken out from the -80°C refrigerator, and then thawed in a 37°C constant temperature water bath. The outer wall of the cryogenic tube is sterilized by spraying medical alcohol, and the cover is opened in the clean bench. The supernatant is discarded, and the cell precipitate is mixed with 1 mL of DMEM complete culture solution, and then transferred to a culture bottle. 1 mL of DMEM is used to wash the cryogenic tube, and then 3 mL of DMEM is added to the culture bottle. After mixing, it is placed in a 5% CO2, 37°C incubator for 24 hours, and then the culture solution is replaced.

[0062] Step 2, cell culture and subculture: the growth of cells is observed every day. If the cell growth is slow and does not reach 70-80%, only the liquid needs to be replaced, that is, the cell culture bottle or dish is taken out of the incubator, sprayed with 75% medical alcohol, transferred to the clean bench for subsequent operation, the culture solution in the culture bottle or dish is gently sucked out, washed twice with physiological saline, and then about 3 mL of complete culture solution is added for continuous culture. If the cells grow to 70-80%, the cell culture bottle or dish can be subcultured, that is, the cell culture bottle or dish is taken out of the incubator, sprayed with 75% medical alcohol, transferred to the clean bench for subsequent operation, the culture solution in the culture bottle or dish is gently sucked out, washed twice with physiological saline, and then about 2 mL of trypsin is added for digestion for about 3 min. The trypsin digestion solution is transferred to a centrifugal tube, and the cells are washed with culture solution and transferred to the centrifugal tube for centrifugation for 3 min (1200 rpm / min). The supernatant is discarded, and the cells adhering to the wall of the tube are obtained. After adding complete culture solution, they are uniformly blown and beaten, and then subcultured.

[0063] Step 3. The experiment was divided into a drug group and different concentrations of LPS groups. The control group was treated with complete DMEM medium, and the different concentrations of LPS groups were treated with medium containing final LPS concentrations of 0.25, 0.5, 1, 2, and 4 μg / mL, respectively.

[0064] Step 4: Collect cells in the logarithmic growth phase and adjust the cell concentration to about 3×10 5 The cells were seeded into 96-well plates at a concentration of approximately 100 μl per well. The plates were incubated at 37°C in a 5% CO2 incubator for 24 hours to allow them to adhere to the plate. After 24 hours of incubation, the plates were rinsed once with PBS and the OD values ​​were measured using the CCK-8 assay. Each concentration was replicated in triplicate in triplicate wells, and the experiment was repeated three times.

[0065] .

[0066] Step 5: Preliminary screening of Gerbera ulmoides compounds with anti-inflammatory activity: Mouse macrophage RAW264.7 cells in the logarithmic growth phase were taken and the cell concentration was adjusted to 3×10 5 Cells were plated in 96-well plates at a concentration of approximately 100 μL per well and cultured at 37°C in a 5% CO2 incubator for 24 hours. The cells were then divided into control, model, positive, and drug-treated groups. Complete DMEM medium was added to the control and model groups, DEX-containing medium was added to the positive group, and compound-containing medium was added to the drug-treated group. The final concentrations of DEX, compound, and LPS were 25 μmol / L, 100 μmol / L, and 0.25 μg / mL, respectively. After 3 hours of culture, complete DMEM medium was added to the control group, LPS-containing medium was added to the model group, DEX- and LPS-containing medium was added to the positive group, and LPS-containing medium was added to the drug-treated group. After 24 hours of culture, the supernatant was collected and NO levels were measured according to the NO detection kit instructions. Three replicates were performed at each concentration, and the experiment was repeated three times. NO concentrations were calculated according to the formula, and NO inhibition rates were calculated.

[0067] .

[0068] Table 4 Inhibitory effects of compounds 1-3 on RAW 264.7 cells

[0069]

[0070] From Table 4, according to the inhibitory effect of compounds 1-3 on RAW264.7 cells, it can be concluded that compounds 1-3 have certain inhibitory activity on mouse macrophage RAW264.7, and compound 3 has a significant inhibitory effect on RAW264.7 cells, indicating that the compound of the present application can be used as a reference for the use of an inhibitor of inflammation, and used for the preparation of related drugs, and laid a foundation for the study of drug mechanism.

[0071] The present application reveals the anti-inflammatory activity of compounds 1-3 by using LPS to induce mouse macrophage RAW 264.7 as an inflammatory factor, detecting the cell survival rate by CCK8 method, and detecting the level of inflammatory mediator NO by using a kit, and lays a foundation for the study of the mechanism of compounds 1-3.

[0072] The above-described embodiments only express the specific implementation of the present application, which is described in detail, but it cannot be understood as a limitation on the protection scope of the present application. It should be noted that for ordinary skilled persons in the art, without departing from the technical concept of the present application, a number of modifications and improvements can be made, which are within the protection scope of the present application.

Claims

1. A compound isolated from Gerbera ulmoides, characterized in that: The compound has a structure as shown in Formula 1:

2. A compound isolated from Gerbera ulmoides, characterized in that: The compound has a structure as shown in Formula 2:

3. A compound isolated from Gerbera ulmoides, characterized in that: The compound has a structure as shown in Formula 3:

4. A method for preparing a compound isolated from Gerbera ulmoides, characterized in that: The steps include: Extracting Gerbera glomerata with alcohol to obtain a first alcohol extract, adsorbing the first alcohol extract with a macroporous resin, and then eluting with alcohol to obtain a first eluate, and separating the eluate with column chromatography to obtain compound 1-3; The compounds 1-3 have structures as shown in Formula 1 to Formula 3:

5. A method for preparing a compound isolated from Gerbera scoparia as claimed in claim 4, characterized in that: The preparation method of the first alcohol extract comprises the following steps: The Gerbera scoparia is extracted by reflux using an ethanol solution to obtain a first filtrate, and the first filtrate is concentrated to obtain a first alcohol extract.

6. The method for preparing a compound isolated from Gerbera ulmoides according to claim 4, wherein: The macroporous resin is D101 macroporous resin, and the elution is performed using water and alcohol solution.

7. The method for preparing a compound isolated from Gerbera scoparia according to claim 4, wherein: The column chromatography was performed using a normal phase column, a reverse phase column and gel chromatography.

8. Use of the compound according to any one of claims 1 to 3 in the preparation of RAW264.7 cell inhibitors.

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