Flavonoid compound separated from pinus massoniana cone and preparation method and application thereof

Abstract

The invention relates to the technical field of natural product separation, in particular to a flavonoid compound separated from a pinus massoniana cone and a preparation method and application of the flavonoid compound, and the preparation method of the flavonoid compound comprises the steps that the pinus massoniana cone is subjected to alcohol extraction, macroporous resin adsorption and column separation. The compound disclosed by the invention can be used for resisting inflammation, and can be applied to preparation of RAW 264.7 cell inhibitors and preparation of TNF-alpha, IL-1beta and IL-6 protein expression inhibitors. # imgabs0 #

Description

Technical Field

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

[0002] Pine cone is the cone of the genus Pinus of the Pinaceae family, also known as pine cone, pine cone, pine yuan, and is a traditional Chinese medicine. It tastes bitter and is warm in nature. It has the effects of antitussive, expectorant, antiasthmatic, wind-dispelling, laxative, and tranquilizing. Modern research shows that pine cones mainly contain volatile oils, terpenes, polysaccharides, polyphenols, lignin and other substances, which have antiviral, antioxidant, immune-enhancing, anti-tumor, and antibacterial pharmacological effects. In addition, it can also regulate the body's immune function, improve resistance, and reduce the occurrence and development of diseases. The diversity of pine cone natural products determines the multi-biological activity of pine cones. In-depth research on the activity of pine cones is carried out to develop their good medicinal value and maximize the resource utilization of pine cone natural products. Summary of the invention

[0003] One object of the present invention is to provide a flavonoid 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 pine cone 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 the second aspect of the present invention, the preparation method of the above 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 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 flavonoid compounds are used in the preparation of TNF-α, IL-1β, 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] According to an eighth aspect of the present invention, the above-mentioned flavonoid compounds are used in the preparation of health products or foods with anti-inflammatory activity.

[0017] The present invention has the following beneficial effects:

[0018] The present invention separates and extracts flavonoid compounds from masson pine cones. After experimental detection, compound 1 has certain inhibitory activity on RAW264.7 cells and TNF-α, IL-1β, and IL-6 protein expression, and can be used for reference use in inflammation treatment and for the preparation of related drugs. The preparation method of the present invention has simple steps and low cost, promotes the development of 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

[0019] Figure 1 HR-ESI-MS of compound 1 of the present invention is shown;

[0020] Figure 2 The compound 1 of the present invention is shown 1 H-NMR spectrum;

[0021] Figure 3 The compound 1 of the present invention is shown 13 C-NMR spectrum;

[0022] Figure 4 The HMQC nuclear magnetic resonance spectrum of compound 1 of the present invention is shown;

[0023] Figure 5 The HMBC nuclear magnetic resonance spectrum of compound 1 of the present invention is shown;

[0024] Figure 6 The NMR of compound 1 of the present invention is shown 1 H- 1 H COSY spectrum. DETAILED DESCRIPTION

[0025] 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.

[0026] Example 1

[0027] Step 1, take the dried and crushed Masson pine cone medicinal material (35kg), extract with 85% ethanol reflux, repeat the extraction 3 times, each time for 1.5 hours, use 8 times the amount of 85% ethanol for the first time, add 6 times the amount of 85% ethanol for the second and third times, filter the alcohol extract, combine the filtrate and recover the ethanol under reduced pressure, directly heat and concentrate, add water and evaporate the ethanol until there is no obvious ethanol smell, and obtain 2115g of total extract;

[0028] Step 2, 2115g of the total 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 turn to obtain different component segments (water segment, 80% ethanol segment);

[0029] Step 3, apply 80% ethanol extract (1685g) to a normal phase silica gel column, and gradient elute with 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 eluted parts with color, and combine by TLC detection to obtain 10 components (Fr.1-10). Concentrate the combined eluted parts to dryness under reduced pressure and set aside;

[0030] Step 4. Load Fr.5 (52.0 g) in step 3 onto a normal phase silica gel column and gradiently elute with chloroform-methanol (10:0-8:2). Detect by thin layer chromatography, develop color, combine the eluted parts with color, and combine by TLC detection 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.

[0031] The present invention conducts structural identification of compound 1: 1 H NMR, 13 C NMR nuclear magnetic spectrum, two-dimensional nuclear magnetic spectrum, and high-resolution mass spectrometry were used to identify the structure of the isolated monomer compound 1. Figure 1-6 shown.

[0032] Class 1, 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 chemical formula with labels is shown in the following formula.

[0033]

[0034] Table 1 Compound 1 1 H(600MHz) and 13 C (150 MHz) NMR data

[0035]

[0036] Test Example 1

[0037] To achieve the above-mentioned purpose of the present invention, the present invention studies the anti-inflammatory mechanism of flavonoid compounds in masson pine cones. NO (nitric oxide) is an important endogenous signal molecule that plays a key role in inflammatory reactions. By detecting the NO level, the inhibitory effect of flavonoid compounds in masson pine cones on inflammation can be intuitively evaluated. At the same time, cytokines such as TNF-α (tumor necrosis factor-α), IL-1β (interleukin-1β) and IL-6 (interleukin-6) are key mediators in inflammatory reactions, and their level changes can reflect the intensity and range of inflammatory reactions. The ELISA method can detect the generation of these cytokines with high sensitivity and high specificity, thereby further verifying the anti-inflammatory effect of masson pine cone compounds. The specific steps are as follows:

[0038] 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.

[0039] Step 2, cell culture and passage: observe the growth of cells every day. If the cell growth is slow and has not grown to 70-80%, only the liquid needs to be changed, that is, the cell culture bottle or culture dish is taken out of the incubator, sprayed with 75% medical alcohol, and transferred to the clean bench for subsequent operations. The culture solution in the culture bottle or culture dish is gently sucked out, and about 3mL of complete culture solution is added after washing twice with physiological saline to continue culturing. If the cells grow to 70-80%, they can be passaged, that is, the cell culture bottle or culture dish is taken out of the incubator, sprayed with 75% medical alcohol, and transferred to the clean bench for subsequent operations. The culture solution in the culture bottle or culture dish is gently sucked out, and about 1mL of trypsin is added after washing twice with physiological saline for about 2min, and the trypsin digestion solution is transferred to a centrifuge tube, and the cells are washed with culture solution and transferred to a centrifuge tube for centrifugation for 3min (1200rpm / min), and the supernatant is discarded. The cells attached to the tube wall are added with complete culture solution and repeatedly blown evenly, and then divided into bottles for culture.

[0040] 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.

[0041] 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.

[0042] Step 5: Preliminary screening of Masson pine cone compounds with anti-inflammatory activity: Take RAW 264.7 cells in the logarithmic growth phase and adjust the cell concentration to 3×10 5 / mL, and inoculated in 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 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 group. The final concentration of DEX was 25μmol / L, the final concentration of compound was 6.25μmol / L, 12.5μmol / L, 25μmol / L, 50μmol / L, 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 group. After 24h of culture, the culture supernatant was collected, and the NO level of the supernatant was determined according to the instructions of the NO detection kit; the effect of the supernatant on the production of TNF-α, IL-1β, and IL-6 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.

[0043] Table 2 IC of compound 1 against NO, TNF-α, IL-1β and IL-6 50 value

[0044]

[0045] 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.

[0046] 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 different treatment times, according to the IC value of compound 1 for NO 50 From the values, it can be concluded that compound 1 has significant inhibitory activity on RAW264.7 cells and NO, TNF-α, IL-1β, and IL-6 protein expression. It can be preliminarily inferred that flavonoid compounds can significantly reduce the levels of NO and cytokines, so it may exert anti-inflammatory effects by inhibiting the synthesis and release of inflammatory mediators or by regulating inflammatory signaling pathways. The revelation of this mechanism provides a direction for subsequent in-depth research. It shows that the flavonoid 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.

[0047] The present invention uses LPS-induced mouse macrophage RAW 264.7 as an inflammation 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 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 from Masson pine 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, the compounds of Masson pine cone have a wide range of biological activities and low toxic side effects, so they have great potential for drug development. In the future, with the deepening of research and the continuous advancement of technology, pine cone flavonoid compounds from Masson pine are expected to become a new class of anti-inflammatory drugs, providing more options for clinical treatment.

[0048] 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 flavonoid compound isolated from a masson pine cone, characterized in that: The compound has a structure shown in the following formula:

2. 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.

3. The method for preparing the compound according to claim 2, characterized in that: The alcohol extraction method comprises: the Masson pine cones are subjected to alcohol precipitation, filtration and concentration.

4. An inhibitor containing the flavonoid compound according to claim 1.

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

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

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

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

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