Abietane diterpenoid compound separated from pinus massoniana cone as well as preparation method and application of abietane diterpenoid compound

Abstract

The invention relates to the technical field of natural product separation, in particular to an abietane diterpenoid compound separated from a pinus massoniana cone and a preparation method and application of the abietane diterpenoid compound, and the preparation method of the abietane diterpenoid compound comprises the steps of alcohol extraction, macroporous resin adsorption and column separation of the pinus massoniana cone. The compound disclosed by the invention is applied to resisting inflammation and preparing a RAW 264.7 cell inhibitor. # imgabs0 #

Description

Technical Field

[0001] The invention relates to the technical field of natural product separation, and in particular to a rosinane diterpene compound separated from masson pine cones, and a preparation method and application thereof. Background Art

[0002] Pine cones are cones of plants in the Pinaceae family, also known as pine cones, pine cones, and pine cones. The chemical components in pine cones are numerous and complex. Studies have found that the main chemical components are polysaccharides, terpenes, flavonoids, polyphenols, lignin, volatile oils, etc. These compounds in pine cones help design and develop effective drugs for the treatment of various diseases including cancer. At present, many drugs based on natural compounds have been used in clinical practice or are being evaluated in clinical trials. Pine cones have shown potential for clinical treatment of various diseases, such as cough, enteritis, neurasthenia, viral infection, tumors, and anti-inflammatory. These potential effects of pine cones have attracted people's attention, and it is hoped that they can play a greater role. Summary of the invention

[0003] One object of the present invention is to provide a rosinane diterpenoid compound separated from a Masson pine cone, and another object of the present invention is to provide a method for obtaining effective chemical components from a Masson pine cone, and to provide uses of the rosinane diterpenoid compound.

[0004] According to a first aspect of the present invention, a rosinane diterpene 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 diterpene 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 Masson pine cones includes: drying and crushing the Masson 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 compounds in the preparation of RAW 264.7 cell inhibitors and the anti-inflammatory effects of the above-mentioned terpenoid compounds.

[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. After experimental detection, compound 1-2 has certain inhibitory activity on RAW264.7 cells, can be used as a reference for inhibitors of inflammation, and is used for the preparation of related drugs. The preparation method of the present invention has simple steps and low cost, promotes the development of 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 NMR of compound 1 of the present invention is shown 1 H- 1 H COSY spectrum;

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

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

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

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

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

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

[0032] Fig.13 The NMR of compound 2 of the present invention is shown 1 H- 1 H COSY spectrum;

[0033] Fig.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 solution and advantages of the present invention more clear, the present invention is further described in detail. 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, 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, taking the dried and crushed Masson pine cone medicinal material (35 kg), reflux extraction with 85% ethanol, repeating the extraction 3 times, each time for 1.5 hours, using 8 times the amount of 85% ethanol for the first time, and adding 6 times the amount of 85% ethanol for the second and third times respectively, filtering the alcohol extract, combining the filtrate and recovering ethanol under reduced pressure, directly heating and concentrating, and continuously adding water to evaporate the ethanol until there is no obvious ethanol smell;

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

[0038] Step 3, the 80% ethanol extract was loaded onto a normal phase silica gel column, and gradient eluted with petroleum ether-ethyl acetate (10:0-0:10) and ethyl acetate-methanol (10:0-3:7), and detected by thin layer chromatography, color was developed, and the eluted parts with color were combined, and the combined elution parts were detected by TLC to obtain 10 components (Fr.1-10). The combined elution parts were concentrated to dryness under reduced pressure and set aside;

[0039] Step 4, Fr.6 in step 3 was gradient eluted with MCI CHP20 / P120 (30%-95% ethanol), and the mixture was combined and concentrated after TLC detection to obtain 10 components (Fr.6.1-6.10). Among them, Fr.6.6 was subjected to normal phase silica gel column, and gradient eluted with petroleum ether-ethyl acetate (30:1-4:6), and the mixture was combined and concentrated after TLC detection to obtain 13 components (Fr.6.6.1-6.6.13). Among them, Fr.6.6.11 was subjected to Sephadex LH-20 gel column chromatography (methanol), Toyopearl HW-40F gel column chromatography (methanol), and then subjected to normal phase silica gel column, and gradient eluted with petroleum ether-ethyl acetate (30:1-5:5), and the mixture was combined and concentrated after TLC detection to obtain 2 components (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 in step 3 was subjected to gradient elution through a normal phase silica gel column (chloroform-ethyl acetate 10:0-3:7), and the mixture was combined and concentrated after TLC detection to obtain 7 components (Fr.4.1-4.7). Among them, Fr.4.1 was subjected to a normal phase silica gel column, and gradient eluted with petroleum ether-ethyl acetate (10:0-7:3), and the mixture was combined and concentrated after TLC detection to obtain 10 components (Fr.4.1.1-4.1.10). Among them, Fr.4.1.4 was subjected to Toyopearl HW-40C gel column chromatography (methanol), Toyopearl HW-40F gel column chromatography (methanol), and then subjected to ODS reverse phase column (40%-80% methanol-water) gradient elution, and the mixture was combined and concentrated after TLC detection to obtain 3 components (Fr.4.1.4.1-4.1.4.3). 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 structural 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 are used to identify the structure of the separated monomer compounds, such as 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(100MHz)NMR

[0046]

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

[0048]

[0049]

[0050] Test Example 1

[0051] To achieve the above-mentioned purpose of the present invention, the present invention studies the anti-inflammatory activity of abietane diterpenoid compounds in Masson pine cones, uses LPS (lipopolysaccharide)-induced mouse macrophages RAW264.7 as an inflammation model, combines the CCK-8 method to test the effect of the compound on the survival rate of RAW264.7 cells to determine its safe administration concentration, and uses the NO detection kit to evaluate the level change of inflammatory mediator nitric oxide (NO). This study has far-reaching significance in preliminarily revealing that the abietane diterpenoid compounds extracted from Masson pine cones have significant anti-inflammatory effects, and lays a solid foundation for subsequent drug action mechanism research. The specific steps are as follows:

[0052] Step 1. Cell recovery: Take the frozen cells out of the -80℃ freezer quickly and thaw them quickly in a 37℃ constant temperature water bath. Spray the outer wall of the cryotube with medical alcohol to sterilize it. Open the lid on the clean bench, suck out the cell suspension with a pipette, put it into a centrifuge tube, and then slowly add 4-5mL of culture solution drop by drop, and suspend the cells by blowing. Centrifuge at room temperature at 1200r / min for 3min. Aspirate the supernatant, add 1-2mL of culture solution, blow evenly, and then transfer to a culture dish containing 3-4mL of culture solution, and observe the cell growth status regularly.

[0053] Step 2, cell culture and passaging: Observe the growth of cells every day. If the cell growth is relatively slow and has not reached 70-80% fusion, only the fluid change operation is required, that is, the cell culture bottle or culture dish is taken out of the incubator, sprayed with 75% medical alcohol, and then transferred to the clean bench to complete the subsequent operations. In the clean bench, the culture fluid in the culture bottle or culture dish can be gently aspirated, and the cells are washed with saline. After washing twice, about 3mL of complete culture fluid is added to allow the cells to continue to be cultured. When the cells grow to a fusion of 70-80%, the passaging operation is required. Similarly, first take the cell culture bottle or culture dish out of the incubator, spray it with 75% medical alcohol, and then transfer it to the clean bench. Next, the culture fluid in the culture bottle or culture dish needs to be gently aspirated, and the cells are washed with saline. After washing twice, about 1mL of trypsin is added to digest the cells. The entire digestion process takes about 2 minutes. After the trypsin digestion is completed, the trypsin digestion solution needs to be transferred to a centrifuge tube, and the cells attached to the wall of the culture bottle or culture dish need to be washed off with culture medium, and then transferred to a centrifuge tube together and centrifuged at 1200r / min for 3 minutes. After the centrifugation is completed, the supernatant can be discarded to obtain the cells attached to the tube wall. At this time, it is necessary to add complete culture medium to the cells, and repeatedly blow to evenly disperse the cells, and then culture them in separate bottles.

[0054] Step 3. The experiment was divided into control group and different concentrations of LSP group. Complete DMEM medium was added to the control group, and medium with final LPS concentrations of 0.25, 0.5, 1, 2 and 4 μg / mL was added to the different concentrations of LPS groups.

[0055] Step 4: Collect cells in the logarithmic growth phase and adjust the cell concentration to about 3×10 5 The cells were inoculated into 96-well plates, 100 μL per well, and cultured in a 37°C, 5% CO2 incubator for 24 hours to allow them to adhere to the wall. After 24 hours of culture, the cells were rinsed once with PBS and the OD value was detected by CCK-8 method. Five wells were paralleled for each concentration, and the experiment was repeated 3 times.

[0056] Step 5: Preliminary screening of Masson pine cone compounds with anti-inflammatory activity: Take RAW264.7 cells in the logarithmic growth phase and adjust the cell concentration to 3×10 5 / mL, and inoculated in a 96-well plate, 100μL per well, cultured in a 37°C, 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 the compound was 6.25, 12.5, 25, 50, 100μmol / L, and the final concentration of LPS was 0.25μg / mL, the same below. After 3h of culture, 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 24h of culture, 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 compounds 1-2 for NO 50 value

[0058]

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

[0060] As shown in Table 3, the use of NO detection kit to detect the level changes of inflammatory mediator NO can intuitively reflect the degree of inhibition of the compound on the inflammatory response. 50 The experimental results preliminarily revealed that the abietane diterpenoid compounds extracted from the pine cones of Masson pine have significant anti-inflammatory effects, which provides strong evidence for the subsequent in-depth study of its anti-inflammatory mechanism. It shows that the compounds of the present invention can be used as reference for inhibitors of inflammation and for the preparation of related drugs, laying a foundation for the study of drug action mechanisms.

[0061] The present invention adopts LPS-induced mouse macrophage RAW264.7 as an inflammation model, tests the effect of the compound on the survival rate of RAW264.7 cells by the CCK8 method, determines its administration concentration, and uses a NO detection kit to detect the level of inflammatory mediator nitric oxide (NO). The preliminary research results of the present invention not only verify the anti-inflammatory activity of abietane diterpenoid compounds, but also lay the foundation for subsequent drug action mechanism research. By further exploring the effects of the compound on key links such as inflammatory signaling pathways, synthesis and release of inflammatory mediators, its anti-inflammatory mechanism can be gradually revealed, providing theoretical support for the development of new anti-inflammatory drugs. It lays a solid foundation for further exploring the specific mechanism of action of these abietane diterpenoid components in the treatment of inflammation-related diseases, and provides important clues for the development of new drugs.

[0062] The above-mentioned embodiments only express the specific implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the protection scope of the present application. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the technical solution concept of the present application, and these all belong to the protection scope of the present application.

Claims

1. A rosinane diterpenoid compound isolated from pine cones of Masson pine, characterized in that: The compound has a structure as shown in Formula 1:

2. A rosinane diterpenoid compound isolated from pine cones of Masson pine, characterized in that: The compound has a structure as shown in Formula 2:

3. The method for preparing the compound according to claim 1, characterized in that: include: The pine cones of Masson pine were extracted by alcohol, adsorbed by macroporous resin and separated by column.

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

5. An inhibitor containing the abietane diterpenoid compound as claimed in claim 1.

6. An inhibitor containing the abietane diterpenoid compound as claimed in claim 2.

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

8. Use of the abietane diterpenoid compound according to claim 1 or claim 2 in the preparation of RAW 264.7 cell inhibitors.

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