A cembranoid diterpenoid compound isolated from a masson pine cone and a preparation method and use thereof

Abstract

The present application relates to the technical field of natural product separation, and in particular to a cembranoid diterpene compound separated from a pine cone of Pinus massoniana and a preparation method and application thereof, the preparation method of the cembranoid diterpene compound comprising alcohol extraction, macroporous resin adsorption and column separation of the pine cone of Pinus massoniana. The application discloses the application of the compound in anti-inflammation and preparation of a RAW 264.7 cell inhibitor.

Description

Technical Field

[0001] The present invention relates to the technical field of natural product separation, and in particular to abietic acid diterpenoid compounds separated from masson pine cones, and a preparation method and application thereof. Background Art

[0002] Pine cones are the cones of plants in the Pinaceae family, also known as pine cones, pine cones, and pine cones. The chemical composition of pine cones is diverse and complex, with studies revealing that the main chemical components are polysaccharides, terpenes, flavonoids, polyphenols, lignin, and volatile oils. These compounds in pine cones may aid in the design and development of effective drugs for treating various diseases, including cancer. Currently, many drugs based on natural compounds are in clinical use or are being evaluated in clinical trials. Pine cones have demonstrated potential in the clinical treatment of various conditions, such as cough, enteritis, neurasthenia, viral infections, tumors, and anti-inflammatory effects. These potential benefits of pine cones have attracted considerable attention, and there is hope that their value can be further realized. Summary of the Invention

[0003] One object of the present invention is to provide a rosinane diterpenoid compound isolated from Pinus massoniana pine cones. Another object of the present invention is to provide a method for obtaining effective chemical components from Pinus massoniana pine cones and to provide uses of the rosinane diterpenoid compound.

[0004] According to a first aspect of the present invention, a rosinane diterpenoid compound isolated from a Masson pine cone has a structure as shown in Formula 1:

[0005]

[0006] According to a second aspect of the present invention, a rosinane diterpenoid compound isolated from a Masson pine cone has a structure as shown in Formula 2:

[0007]

[0008] According to a third aspect of the present invention, the preparation method of the compound of formula 1 comprises: alcohol extraction of masson pine cones, macroporous resin adsorption and column separation.

[0009] According to a fourth aspect of the present invention, the preparation method of the compound of formula 2 comprises: alcohol extraction of masson pine cones, macroporous resin adsorption and column separation.

[0010] According to one embodiment of the present invention, the step of alcohol extraction of Pinus massoniana pine cones includes: drying and crushing the Pinus massoniana pine cones, refluxing and extracting with an ethanol solution to obtain a first filtrate, and concentrating the first filtrate to obtain a first alcohol extract.

[0011] According to one embodiment of the present invention, column separation includes normal phase column, gel chromatography, reverse phase column and preparative liquid chromatography.

[0012] According to a fifth aspect of the present invention, an inhibitor containing the above-mentioned abietane diterpenoid compound of formula 1 is provided.

[0013] According to a sixth aspect of the present invention, an inhibitor containing the above-mentioned abietane diterpenoid compound of formula 2.

[0014] According to a seventh aspect of the present invention, the above-mentioned abietane diterpenoid compound is used in the preparation of an anti-inflammatory inhibitor.

[0015] According to an eighth aspect of the present invention, the use of the above-mentioned abietane diterpenoid compound in the preparation of RAW 264.7 cell inhibitors and the anti-inflammatory effect of the above-mentioned terpenoid compound.

[0016] According to one embodiment of the present invention, the dosage form of the inhibitor is tablets, pills, powders, granules, capsules, oral liquids, infusions, lyophilized powder injections, ointments, gels or sprays.

[0017] According to a ninth aspect of the present invention, the above-mentioned terpenoid compounds are used in the preparation of health products or foods with anti-inflammatory activity.

[0018] The present invention has the following beneficial effects:

[0019] The present invention separates and extracts abietane diterpenoid compounds of formula 1-2 from masson pine cones. Experimental testing shows that compound 1-2 has certain inhibitory activity against RAW264.7 cells and can be used as a reference for inhibitors of inflammation and for the preparation of related drugs. The preparation method of the present invention has simple steps and low cost, promotes the development of the seedling medicine masson pine cones in medicine, health products, food, etc., and plays an important role in promoting the sustainable development of the industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 HR-ESI-MS of compound 1 of the present invention is shown;

[0021] Figure 2 The compound 1 of the present invention is shown 1 H-NMR spectrum;

[0022] Figure 3 The compound 1 of the present invention is shown 13 C-NMR spectrum;

[0023] Figure 4 The HMQC nuclear magnetic resonance spectrum of compound 1 of the present invention is shown;

[0024] Figure 5The HMBC nuclear magnetic resonance spectrum of compound 1 of the present invention is shown;

[0025] Figure 6 The nuclear magnetic resonance of compound 1 of the present invention is shown 1 H- 1 H COSY spectrum;

[0026] Figure 7 shows the nuclear magnetic resonance DEPT 135 spectrum of compound 1 of the present invention;

[0027] Figure 8 HR-ESI-MS of compound 2 of the present invention is shown;

[0028] Figure 9 The compound 2 of the present invention is shown 1 H-NMR spectrum;

[0029] Figure 10 The compound 2 of the present invention is shown 13 C-NMR spectrum;

[0030] Figure 11 The HMQC nuclear magnetic resonance spectrum of compound 2 of the present invention is shown;

[0031] Figure 12 The HMBC nuclear magnetic resonance spectrum of compound 2 of the present invention is shown;

[0032] Figure 13 The nuclear magnetic resonance of compound 2 of the present invention is shown 1 H- 1 H COSY spectrum;

[0033] Figure 14 The nuclear magnetic resonance DEPT 135 spectrum of compound 2 of the present invention is shown. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be described in further detail. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. That is, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments.

[0035] Example 1

[0036] Step 1: Take 35 kg of dried and crushed Pinus massoniana pine cone medicinal materials, reflux extract with 85% ethanol, repeat the extraction 3 times, each time for 1.5 hours, use 8 times the amount of 85% ethanol for the first time, and add 6 times the amount of 85% ethanol for the second and third times respectively. The alcohol extract is filtered, the filtrate is combined and the ethanol is recovered under reduced pressure, directly heated and concentrated, and water is continuously added to evaporate the ethanol until there is no obvious ethanol smell;

[0037] Step 2, the extract was mixed and subjected to coarse fractionation by D101 macroporous adsorption resin, adsorbed by the macroporous adsorption resin, eluted and concentrated with water and 80% ethanol in sequence to obtain different component segments (water segment, 80% ethanol segment);

[0038] Step 3: Apply the 80% ethanol extract to a normal phase silica gel column and elute with a gradient of petroleum ether-ethyl acetate (10:0-0:10) and ethyl acetate-methanol (10:0-3:7). Detect by thin layer chromatography, develop color, combine the color-developed elution fractions, and combine them for TLC detection to obtain 10 fractions (Fr.1-10). Concentrate the combined elution fractions to dryness under reduced pressure and set aside.

[0039] Step 4: Fraction 6 from step 3 was eluted with a gradient of MCI CHP20 / P120 (30%-95% ethanol), analyzed by TLC, combined, and concentrated to obtain 10 fractions (Fr.6.1-6.10). Fraction 6.6 was eluted with a gradient of petroleum ether-ethyl acetate (30:1-4:6) on a normal phase silica gel column. The fractions were analyzed by TLC, combined, and concentrated to obtain 13 fractions (Fr.6.6.1-6.6.13). Fraction 6.6.11 was chromatographed on a Sephadex LH-20 gel column (methanol) and a Toyopearl HW-40F gel column (methanol), then eluted again on a normal phase silica gel column with a gradient of petroleum ether-ethyl acetate (30:1-5:5). The fractions were analyzed by TLC, combined, and concentrated to obtain two fractions (Fr.6.6.11.1-6.6.11.2). Among them, Fr.6.6.11.2 was eluted through an ODS reverse phase column (40%-75% methanol-water) to obtain compound 1.

[0040] Step 5. Fr.4 from step 3 was eluted with a normal phase silica gel column (chloroform-ethyl acetate 10:0-3:7) using a gradient elution method. After TLC analysis, the residues were combined and concentrated to obtain seven fractions (Fr.4.1-4.7). Among them, Fr.4.1 was eluted with a normal phase silica gel column using a gradient elution method of petroleum ether-ethyl acetate 10:0-7:3 ... Among them, Fr.4.1.4.2 was separated by preparative liquid phase (mobile phase: 77% methanol, chromatographic column: C18, flow rate: 21.5 ml / min), and the RT37.5min component was enriched and concentrated to obtain compound 2.

[0041] The structure identification performed by the present invention: 1 H NMR, 13 C NMR nuclear magnetic spectrum, two-dimensional nuclear magnetic spectrum, and high-resolution mass spectrometry were used to identify the structures of the isolated monomer compounds. Figure 1-14 shown.

[0042] Compound 1, properties (white needle-shaped crystals), molecular formula: C 20 H 32 O5; HR-ESI-MSm / z: 351.2179[MH] - , the calculated value is 351.2166; its NMR data are shown in Table 1.

[0043] Compound 2, properties (white solid), molecular formula: C 19 H 26 O; HR-ESI-MSm / z: 271.2065[M+H] + , the calculated value is 271.2056; its NMR data is shown in Table 2, and the corresponding chemical formulas with labels are shown in Formula 1-2 below.

[0044]

[0045] Table 1 Compound 1 1 H (400MHz) and 13 C (100 MHz) NMR

[0046]

[0047] Table 2 Compound 2 1 H (400MHz) and 13 C (100 MHz) NMR

[0048]

[0049]

[0050] Test Example 1

[0051] To achieve the above-mentioned objectives of the present invention, the present invention studied the anti-inflammatory activity of abietane diterpenoid compounds extracted from masson pine cones. LPS (lipopolysaccharide)-induced mouse macrophage RAW264.7 cells were used as an inflammation model. The effects of the compounds on the survival rate of RAW264.7 cells were tested in combination with the CCK-8 method to determine their safe dosing concentrations. The levels of nitric oxide (NO), a inflammatory mediator, were evaluated using a NO detection kit. This study has far-reaching significance in preliminarily revealing that abietane diterpenoid compounds extracted from masson pine cones have significant anti-inflammatory effects and lays a solid foundation for subsequent research on the mechanism of drug action. The specific steps are as follows:

[0052] Step 1, cell recovery: After the frozen cells were quickly taken out from the -80 °C refrigerator, they were quickly thawed in a 37 °C constant temperature water bath. The outer wall of the cryogenic tube was sterilized by spraying medical alcohol, the cover was opened in the clean bench, the cell suspension was sucked out with a pipette, and was transferred into a centrifuge tube. Then 4-5 mL of culture medium was slowly added drop by drop, and the cells were suspended by blowing. Centrifugation was performed at room temperature at 1200 r / min for 3 min. The supernatant was aspirated, 1-2 mL of culture medium was added, and the cells were uniformly dispersed by blowing. Then the cells were transferred into a culture dish containing 3-4 mL of culture medium, and the cell growth condition was observed at regular time intervals.

[0053] Step 2, cell culture and passage: The growth of the cells was observed every day. If the cell growth was relatively slow and the confluence reached 70-80%, only the medium was changed. The cell culture bottle or dish was taken out from the incubator and sprayed with 75% medical alcohol, and then transferred to the clean bench for subsequent operation. In the clean bench, the culture medium in the culture bottle or dish was gently aspirated, and the cells were washed with normal saline twice. Then about 3 mL of complete culture medium was added for continuous culture. When the cells grew to 70-80% confluence, the cells were passaged. Similarly, the cell culture bottle or dish was taken out from the incubator and sprayed with 75% medical alcohol, and then transferred to the clean bench. Then the culture medium in the culture bottle or dish was gently aspirated, and the cells were washed with normal saline twice. Then about 1 mL of trypsin was added for cell digestion. The whole digestion process took about 2 min. After trypsin digestion, the trypsin digestion solution was transferred to a centrifuge tube, and the cells adhering to the wall of the culture bottle or dish were washed down with culture medium and transferred to the centrifuge tube. Centrifugation was performed at 1200 r / min for 3 min. After centrifugation, the supernatant was discarded, and the cells adhering to the wall of the tube were obtained. At this time, complete culture medium was added to the cells, and the cells were uniformly dispersed by repeated blowing, and then the cells were subcultured.

[0054] Step 3, the experiment was divided into Control and different concentrations of LSP groups. The Control group was added with complete DMEM medium, and the different concentrations of LSP groups were added with LPS with a final concentration of 0.25, 0.5, 1, 2 and 4 μg / mL, respectively.

[0055] Step 4, the logarithmic growth phase cells were collected, and the cell concentration was adjusted to about 3×10 5

[0056] ​Step 5: Preliminary screening of pine cone compounds with anti-inflammatory activity: RAW264.7 cells in the logarithmic growth phase were taken and the cell concentration was adjusted to 3×10 5 / mL, and inoculated into 96-well plates, 100μL per well, cultured in a 37℃, 5% CO2 incubator for 24h, and the cells were divided into Control group, Model group, positive group and drug-treated group. Complete DMEM medium was added to the Control group and Model group, medium containing DEX was added to the positive group, and medium containing compound was added to the drug-treated group. The final concentration of DEX was 25μmol / L, the final concentration of compound was 6.25, 12.5, 25, 50, 100μmol / L, and the final concentration of LPS was 0.25μg / mL, the same below. After culturing for 3h, complete DMEM medium was added to the Control group, medium containing LPS was added to the Model group, mixed medium of DEX and LPS was added to the positive group, and mixed medium of compound and LPS was added to the drug-treated group. After culturing for 24h, the culture supernatant was collected, and the NO level in the supernatant was determined according to the instructions of the NO detection kit. Three parallel wells were added for each concentration, and the experiment was repeated 3 times. IC of compound 1-2 for NO 50 See Table 3 for values.

[0057] Table 3 IC of compound 1-2 for NO 50 value

[0058]

[0059] Among them, IC 50 The concentration of the compound when the proliferation inhibition rate is 50% is used to express the anti-inflammatory activity.

[0060] As shown in Table 3, the use of NO detection kit to detect the level of inflammatory mediator NO can intuitively reflect the degree of inhibition of the compound on the inflammatory response. 50 The experimental results preliminarily reveal that abietane diterpenoids extracted from Masson pine cones have significant anti-inflammatory effects, providing strong evidence for further research into their anti-inflammatory mechanisms. This indicates that the compounds of the present invention can be used as reference for inhibitors of inflammation and in the preparation of related drugs, laying the foundation for research into drug mechanisms of action.

[0061] The present application adopts LPS-induced mouse macrophage RAW264.7 as an inflammation model, tests the influence of the compound on the survival rate of RAW264.7 cells by CCK8 method, determines the administration concentration, and detects the level of inflammatory mediator nitric oxide (NO) by using a NO detection kit. The preliminary research results of the present application not only verify the anti-inflammatory activity of cembranoid diterpenes, but also lay a foundation for subsequent research on the mechanism of drug action. By further exploring the influence of the compound on key links such as inflammatory signal pathways and synthesis and release of inflammatory mediators, the anti-inflammatory mechanism can be gradually revealed, and theoretical support can be provided for the development of new anti-inflammatory drugs. The present application lays a solid foundation for further exploring the specific mechanism of these cembranoid diterpenes in the treatment of inflammation-related diseases, and provides an important clue for the development of new drugs.

[0062] The above-described embodiments only express the specific implementation of the present application, and the description is relatively specific and detailed, but it should not 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 all belong to the protection scope of the present application.

Claims

1. A rosinane diterpenoid compound isolated from Pinus massoniana pine cones, characterized in that: The compound has a structure as shown in Formula 1: 。 2. A rosinane diterpenoid compound isolated from Pinus massoniana pine cones, characterized in that: The compound has a structure as shown in Formula 2: 。 3. The method for preparing the compound according to claim 1, wherein include: The pine cones of Masson pine were extracted with alcohol, adsorbed with macroporous resin and separated by column.

4. The method for preparing the compound according to claim 2, wherein include: The pine cones of Masson pine were extracted with alcohol, adsorbed with macroporous resin and separated by column.

5. An inhibitor containing the abietane diterpenoid compound according to claim 1.

6. An inhibitor containing the abietane diterpenoid compound according to claim 2.

7. Use of the abietane diterpenoid compound according to claim 1 or claim 2 in the preparation of an anti-inflammatory preparation.

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