A flavonoid compound isolated from masson pine cones, and its preparation method and use

Abstract

The present invention relates to the field of natural product separation technology, and more specifically, to a flavonoid compound isolated from Masson pine cones, a preparation method, and uses thereof. The preparation method of the flavonoid compound comprises alcohol extraction of the Masson pine cones, macroporous resin adsorption, and column separation. The compound disclosed in the present invention can be used for anti-inflammatory purposes, as well as in the preparation of RAW 264.7 cell inhibitors and in the preparation of TNF-α, IL-1β, and IL-6 protein expression inhibitors.

Description

Technical Field

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

[0002] Pine cones, also known as pine cones, pine cones, and pine nuts, are the cones of the Pinus genus in the Pinaceae family. They are a traditional Chinese medicinal material. They are bitter and warm in nature, and have antitussive, expectorant, antiasthmatic, wind-relieving, bowel-moistening, and tranquilizing properties. Modern research indicates that pine cones primarily contain volatile oils, terpenes, polysaccharides, polyphenols, and lignin, which exhibit antiviral, antioxidant, immune-enhancing, anti-tumor, and antibacterial pharmacological effects. They also regulate the body's immune system, enhance resistance, and reduce the incidence and progression of disease. The diverse range of pine cone natural products contributes to their diverse biological activities. In-depth research on these activities is needed to explore their medicinal value and maximize the resource utilization of these natural products. Summary of the Invention

[0003] One object of the present invention is to provide a flavonoid compound separated from the pine cones of Masson pine. Another object of the present invention is to provide a method for obtaining effective chemical components from the pine cones and to provide uses of the flavonoid compound.

[0004] According to a first aspect of the present invention, a flavonoid compound isolated from a masson pine cone has a structure shown in the following formula:

[0005]

[0006] According to a second aspect of the present invention, the preparation method of the above-mentioned compound comprises: alcohol extraction of Masson pine cones, macroporous resin adsorption and column separation.

[0007] According to one embodiment of the present invention, the alcohol extraction method comprises: the Masson pine cones are subjected to alcohol precipitation, filtration and concentration.

[0008] According to one embodiment of the present invention, the macroporous resin is D101 macroporous resin.

[0009] According to one embodiment of the present invention, column separation includes normal phase column and gel chromatography.

[0010] According to the third aspect of the present invention, an inhibitor contains the above-mentioned flavonoid compound.

[0011] According to a fourth aspect of the present invention, an anti-inflammatory inhibitor contains the above-mentioned flavonoid compound.

[0012] According to a fifth aspect of the present invention, the above flavonoid compounds are used in the preparation of anti-inflammatory inhibitors.

[0013] According to a sixth aspect of the present invention, the above flavonoid compounds are used in the preparation of RAW 264.7 cell inhibitors.

[0014] According to a seventh aspect of the present invention, the above-mentioned flavonoid compounds are used in the preparation of TNF-α, IL-1β, and IL-6 protein expression inhibitors.

[0015] According to one embodiment of the present invention, the dosage form of the compound is tablets, pills, powders, granules, capsules, oral liquids, infusions, lyophilized powder injections, ointments, gels or sprays.

[0016] The present invention has the following beneficial effects:

[0017] The present invention isolates and extracts flavonoid compounds from masson pine cones. Experimental testing shows that compound 1 has certain inhibitory activity against RAW264.7 cells and TNF-α, IL-1β, and IL-6 protein expression. It can be used as a reference for the treatment of inflammation and for the preparation of related drugs. The preparation method of the present invention is simple and low-cost, promoting the development of pine cones in medicine, health products, food, etc., and playing an important role in promoting the sustainable development of the industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 HR-ESI-MS of compound 1 of the present invention is shown;

[0019] Figure 2 The compound 1 of the present invention is shown 1 H-NMR spectrum;

[0020] Figure 3 The compound 1 of the present invention is shown 13 C-NMR spectrum;

[0021] Figure 4 The HMQC nuclear magnetic resonance spectrum of compound 1 of the present invention is shown;

[0022] Figure 5 The HMBC nuclear magnetic resonance spectrum of compound 1 of the present invention is shown;

[0023] Figure 6 The nuclear magnetic resonance of compound 1 of the present invention is shown 1 H- 1 H COSY spectrum. DETAILED DESCRIPTION

[0024] 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. Example 1

[0025] Step 1: Take 35 kg of dried and crushed Pinus massoniana pine cones, extract them with 85% ethanol under reflux, and repeat the extraction three 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. The alcohol extract is filtered, and the filtrate is combined and the ethanol is recovered under reduced pressure. The extract is directly heated and concentrated, and water is continuously added to evaporate the ethanol until there is no obvious ethanol smell, to obtain 2115 g of total extract;

[0026] Step 2: 2115 g of the total extract was mixed and subjected to coarse fractionation by D101 macroporous adsorption resin. The mixture was adsorbed by the macroporous adsorption resin, and then eluted and concentrated with water and 80% ethanol in sequence to obtain different component segments (water segment, 80% ethanol segment);

[0027] Step 3: Load the 80% ethanol extract (1685 g) onto 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 colored elution fractions, and detect and combine them by TLC to obtain 10 fractions (Fr. 1-10). Concentrate the combined elution fractions to dryness under reduced pressure and set aside.

[0028] Step 4. Load Fr.5 (52.0 g) in step 3 onto a normal phase silica gel column and elute with a gradient of chloroform-methanol (10:0-8:2). Detect by thin layer chromatography, develop color, combine the eluted fractions, and detect and combine them by TLC to obtain 8 components (Fr.5.1-Fr.5.8). Fr5.5 (1.4 g) was subjected to Toyopearl HW-40C gel column chromatography (methanol) to obtain compound 1.

[0029] The present invention carries out structural identification of compound 1: 1 H NMR, 13 The structure of the isolated monomer compound 1 was identified by C NMR, 2D NMR and high-resolution mass spectrometry. Figure 1-6 shown.

[0030] Class 1 compound, properties (yellow needle-shaped crystals), molecular formula: C 17 H 14 O6; HR-ESI-MS m / z: 315.0856[M+H] +, the calculated value is 315.0863; its NMR data are shown in Table 1, and the corresponding numbered chemical formulas are shown below.

[0031]

[0032]

[0033] Test Example 1

[0034] To achieve the above-mentioned objectives, the present invention investigated the anti-inflammatory mechanisms of flavonoid compounds in masson pine cones. NO (nitric oxide) is an important endogenous signaling molecule that plays a key role in inflammatory responses. By measuring NO levels, the inhibitory effects of masson pine cone flavonoid compounds on inflammation can be intuitively assessed. Furthermore, cytokines such as TNF-α (tumor necrosis factor-α), IL-1β (interleukin-1β), and IL-6 (interleukin-6) are key mediators of inflammatory responses, and changes in their levels can reflect the intensity and extent of the inflammatory response. The ELISA method can detect the production of these cytokines with high sensitivity and specificity, further validating the anti-inflammatory effects of masson pine cone compounds. The specific steps are as follows:

[0035] Step 1. Cell recovery: Quickly remove the frozen cells from the -80°C freezer and thaw them quickly in a 37°C constant temperature water bath. Spray the outer wall of the cryovial with medical alcohol to sterilize it. Open the lid on a clean bench and aspirate the cell suspension with a pipette. Place it into a centrifuge tube. Slowly add 4-5mL of culture medium dropwise and pipette to suspend the cells. Centrifuge at room temperature at 1200r / min for 3 minutes. Aspirate the supernatant, add 1-2mL of culture medium, pipette evenly, and then transfer to a culture dish containing 3-4mL of culture medium. Observe cell growth regularly.

[0036] Step 2. Cell Culture and Passaging: Observe cell growth daily. If cell growth is slow and has not reached 70-80% growth, only a medium change is required. Remove the cell culture flask or dish from the incubator, spray it with 75% medical alcohol, and transfer it to a clean bench for subsequent operations. Gently aspirate the culture medium in the flask or dish, rinse it twice with saline, and add approximately 3 mL of complete culture medium to continue culturing. If the cells have grown to 70-80%, they can be passaged. Remove the cell culture flask or dish from the incubator, spray it with 75% medical alcohol, and transfer it to a clean bench for subsequent operations. Gently aspirate the culture medium in the flask or dish, rinse it twice with saline, and add approximately 1 mL of trypsin to digest for approximately 2 minutes. Transfer the trypsinized solution to a centrifuge tube, then rinse the cells with culture medium and transfer them to a centrifuge tube for centrifugation for 3 minutes (1200 rpm / min). Discard the supernatant and remove cells attached to the tube wall. Add complete culture medium and repeatedly pipette to evenly distribute the cells before culturing them in separate flasks.

[0037] Step 3. The experiment was divided into control 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.

[0038] Step 4: Collect cells in the logarithmic growth phase and adjust the cell concentration to about 3×10 5 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 attachment. After 24 hours of incubation, the plates were rinsed once with PBS and the OD values ​​were measured using the CCK-8 assay. Five wells were plated for each concentration, and the experiment was repeated three times.

[0039] Step 5: Preliminary screening of pine cone compounds with anti-inflammatory activity: RAW 264.7 cells in the logarithmic growth phase were taken and the cell concentration was adjusted to 3×10 5 Cells were seeded 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 were 25 μmol / L, the compound concentrations were 6.25 μmol / L, 12.5 μmol / L, 25 μmol / L, 50 μmol / L, and 100 μmol / L, and the final LPS concentration was 0.25 μg / mL. 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 compound- and LPS-containing medium was added to the drug-treated group. After 24 hours of culture, the culture supernatant was collected and NO levels were measured according to the instructions of the NO detection kit. The production of TNF-α, IL-1β, and IL-6 by the supernatant was determined by ELISA. Each concentration was tested in triplicate and the experiment was repeated three times. IC values ​​of compound 1 for NO, TNF-α, IL-1β, and IL-6 50 See Table 3 for values.

[0040]

[0041] Among them, IC 50 The concentration of the compound when the proliferation inhibition rate is 50% is used to express the anti-inflammatory activity.

[0042] As shown in Table 3, by comparing the effects of pine cone flavonoids from Masson pine on NO levels and cytokine production at different concentrations or treatment times, according to the IC value of compound 1 for NO 50The results indicate that compound 1 has significant inhibitory activity against RAW264.7 cells and the expression of NO, TNF-α, IL-1β, and IL-6 proteins. It can be preliminarily inferred that flavonoids can significantly reduce the levels of NO and cytokines. Therefore, they may exert their anti-inflammatory effects by inhibiting the synthesis and release of inflammatory mediators or by regulating inflammatory signaling pathways. This mechanism provides a direction for further research. This indicates that the flavonoids 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 on drug action mechanisms.

[0043] The present invention uses LPS-induced mouse macrophage RAW 264.7 as an inflammatory model, uses the CCK8 method to test the effect of the compound on the survival rate of RAW 264.7 cells, determines its dosage concentration, uses a NO detection kit to detect the level of the inflammatory mediator NO, and uses the ELISA method to determine the effect of the supernatant on the production of TNF-α, IL-1β, and IL-6. The preliminary revelation of the anti-inflammatory activity of pine cone flavonoid compounds not only helps to understand its application in traditional medicine, but also provides new ideas and basis for modern drug development. As a natural resource, pine cones of masson pine have a wide range of biological activities and low toxic side effects, and therefore have great potential for drug development. In the future, with the deepening of research and the continuous advancement of technology, pine cone flavonoid compounds of masson pine are expected to become a new class of anti-inflammatory drugs, providing more options for clinical treatment.

[0044] The above-described embodiments merely represent specific implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of protection of the present application. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the technical concept of the present application, and all such variations and improvements fall within the scope of protection of the present application.

Claims

1. A flavonoid compound isolated from a masson pine cone, characterized in that: The compound has the structure shown in the following formula: 。 2. 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; The macroporous resin adsorption and column separation comprises: adsorption with D101 macroporous adsorption resin, elution and concentration with 80% ethanol to obtain an 80% ethanol fraction; The 80% ethanol segment was loaded onto a normal phase silica gel column and eluted with a gradient of petroleum ether-ethyl acetate 10:0-0:10 and ethyl acetate-methanol 10:0-3:7 to obtain 10 fractions Fr.1-10; Fr.5 was loaded onto a normal phase silica gel column and eluted with a gradient of chloroform-methanol 10:0-8:2 to obtain 8 components Fr.5.1-Fr.5.

8. Fr5.5 was purified by gel column chromatography to obtain compound 1.

3. The method for preparing the compound according to claim 2, wherein The alcohol extraction method comprises: alcohol precipitation, filtration and concentration of the masson pine cones.

4. An anti-inflammatory inhibitor comprising the flavonoid compound according to claim 1.

5. Use of the flavonoid compound according to claim 1 in the preparation of an anti-inflammatory inhibitor.

6. Use of the flavonoid compound according to claim 1 in the preparation of RAW 264.7 cell inhibitors.

7. Use of the flavonoid compound according to claim 1 in the preparation of a TNF-α, IL-1β or IL-6 protein expression inhibitor.

Images