Determination method of mulberry phellinus igniarius polyphenol compound and application thereof

Abstract

The invention relates to the technical field of component analysis, in particular to a determination method of a mulberry phellinus igniarius polyphenol compound and application thereof. According to the method, the components in the mulberry phellinus igniarius polyphenol compound are determined by adopting an ultra-high performance liquid chromatography-mass spectrometry method, more than 50 chemical components in the mulberry phellinus igniarius polyphenol compound are identified by controlling chromatographic conditions and mass spectrometry conditions, and a powerful theoretical basis is provided for subsequent further research on the polyphenol compound. Meanwhile, the invention also finds that the mulberry phellinus igniarius polyphenol compound has a remarkable effect in the aspect of anti-tumor activity, especially has a remarkable inhibition effect on the growth of human lung cancer cells A549, and the application field of the mulberry phellinus igniarius polyphenol compound is widened.

Description

Technical Field

[0001] The present invention relates to the technical field of component analysis, and in particular to a method for determining polyphenol compounds in mulberry linterus and an application thereof. Background Art

[0002] Common methods for determining and analyzing compounds in plants include gas chromatography (GC) coupled with MS / MS (GC-MS), high-performance liquid chromatography coupled with fluorescence detection or ultraviolet (UV) detection, and immunological methods (ELISA). However, these methods suffer from disadvantages and technical limitations, such as long sample run times, large interfering peaks, and low sensitivity. In recent years, ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-ESI-MS) has gained widespread application for broadly targeted metabolite analysis due to its high throughput, ultra-sensitivity, wide coverage, and accurate qualitative and quantitative analysis. Compared with traditional HPLC, UHPLC-ESI-MS offers advantages such as high separation efficiency, short analysis time, and reduced solvent consumption. Furthermore, this method can utilize different ion modes to obtain more comprehensive chemical profiles and quantification, and has higher sensitivity.

[0003] Polyphenols in Phellinus igniarius (commonly referring to the fungus Phellinus igniarius, which parasitizes mulberry trees) are one of its important active ingredients, exhibiting potential biological activities such as antioxidant, anti-inflammatory, and anti-tumor activities. Phellinus igniarius contains a variety of polyphenols, but currently there is no method to analyze and measure the specific polyphenols in this fungus. Summary of the Invention

[0004] In view of this, the present invention provides a method for determining polyphenol compounds in mulberry linterus and its application. The determination method realizes comprehensive analysis and determination of polyphenol compounds in mulberry linterus, can analyze and determine a variety of polyphenol compounds in a short time, and has high sensitivity and good accuracy.

[0005] To solve the above technical problems, the first aspect of the present invention provides a method for determining polyphenol compounds in mulberry linterus, which is determined by ultra-high performance liquid chromatography-mass spectrometry, wherein the chromatographic conditions of the ultra-high performance liquid chromatography include:

[0006] Column: ACQUITY HSS T3;

[0007] Column temperature: 38-42°C;

[0008] Injection volume: 1-3 μL;

[0009] Mobile phase flow rate: 0.2-0.3 mL / min;

[0010] Mobile phases in positive ion mode: mobile phase B1: acetonitrile containing 0.1% formic acid, mobile phase A1: water containing 0.1% formic acid;

[0011] Mobile phases in negative ion mode: mobile phase B2: acetonitrile, mobile phase A2: 5 mM ammonium formate aqueous solution.

[0012] In combination with the first aspect, the elution mode of the ultra-high performance liquid chromatography is gradient elution:

[0013] The gradient elution program in positive ion mode was:

[0014] Time (min) Mobile phase B1 (%) Mobile phase A1 (%) 0～1 2 98 1～9 2→50 98→50 9～12 50→98 50→2 12～13.5 98 2 13.5～14 98→2 2→98 14～20 2 98

[0015] ; The gradient elution program in negative ion mode is:

[0016] Time (min) Mobile phase B2 (%) Mobile phase A2 (%) 0～1 2 98 1～9 2→50 98→50 9～12 50→98 50→2 12～13.5 98 2 13.5～14 98→2 2→98 14～17 2 98 .

[0017] In combination with the first aspect, the specifications of the chromatographic column are 2.1×150 mm, 1.8 μm.

[0018] In combination with the first aspect, the mass spectrometry conditions in the ultra-high performance liquid chromatography-mass spectrometry method include:

[0019] Detector: mass spectrometer;

[0020] Ion source: ESI ion source;

[0021] Spray voltage: positive ion spray voltage is 3.50 kV, negative ion spray voltage is -2.50 kV;

[0022] Primary scan: resolution 70000, ion scanning range m / z 100-1000;

[0023] Secondary fragmentation: resolution 17500, collision energy 30eV, collecting the first 10 ions for fragmentation.

[0024] In combination with the first aspect, the mass spectrometry conditions in the ultra-high performance liquid chromatography-mass spectrometry method further include: sheath gas 30 arb, auxiliary gas 10 arb, capillary temperature: 325°C.

[0025] In combination with the first aspect, the determination steps of the above-mentioned method for determining mulberry ignia polyphenol compounds include: accurately weighing an appropriate amount of mulberry ignia polyphenol compounds into a centrifuge tube, adding a methanol solution containing 2-chloro-L-phenylalanine as an internal standard, vortexing, adding glass beads, and grinding in a tissue grinder, followed by ultrasonication, solid-liquid separation, and filtration of the liquid phase, and the filtrate is tested on a machine.

[0026] Preferably, accurately weigh an appropriate amount of mulberry linterinary polyphenols into a 2 mL centrifuge tube, add 600 μL of methanol (containing 4 ppm of 2-chloro-L-phenylalanine), vortex for 30 seconds, add 100 mg of glass beads, place in a tissue grinder, and grind at 60 Hz for 90 seconds. Ultrasonicate at room temperature for 15 minutes, then centrifuge at 12,000 rpm at 4°C for 10 minutes. Filter the supernatant through a 0.22 μm membrane and add the filtrate to a test bottle for analysis.

[0027] A second aspect of the present invention provides a use of a mulberry linterus polyphenol compound in the preparation of an anti-tumor drug.

[0028] A third aspect of the present invention provides an application of mulberry linterinary polyphenol compounds in inhibiting the growth of tumor cells.

[0029] Preferably, the tumor cells include human lung cancer cells A549, human liver cancer cells HepG2 and human prostate cancer cells PC3.

[0030] The present invention has the beneficial effects of using ultra-high performance liquid chromatography-mass spectrometry to determine the components of mulberry ignia polyphenol compounds. By controlling the chromatographic and mass spectrometry conditions, over 50 chemical components in the mulberry ignia polyphenol compounds were identified, providing a strong theoretical basis for further research on polyphenol compounds. Furthermore, the present invention also found that the mulberry ignia polyphenol compounds have significant anti-tumor activity, particularly a significant inhibitory effect on the growth of human lung cancer A549 cells, broadening the application field of the mulberry ignia polyphenol compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0032] Figure 1 is the cation flow diagram of the measured polyphenol compounds of mulberry linterus;

[0033] Figure 2 is the anion chromatogram of the measured polyphenol compounds of mulberry linterus;

[0034] Figure 3 is a line graph showing the inhibitory effects of SSPIP and 5-FU on A549 cells as a function of concentration;

[0035] Figure 4Figure 3 shows the effects of different added concentrations of SSPIP and 5-FU on the morphology of A549 cells. A shows the morphology of A549 cells without drug addition, B shows the morphology of A549 cells after adding 150 μg / mL 5-Fu for 24 h, C shows the morphology of A549 cells after adding 50 μg / mL SSPIP for 24 h, D shows the morphology of A549 cells after adding 75 μg / mL SSPIP for 24 h, E shows the morphology of A549 cells after adding 100 μg / mL SSPIP for 24 h, and F shows the morphology of A549 cells after adding 150 μg / mL SSPIP for 24 h. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. 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.

[0037] Those skilled in the art will understand that, unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as commonly understood by those skilled in the art in the art to which the present invention pertains. It should also be understood that terms such as those defined in common dictionaries should be understood to have meanings consistent with those in the context of the prior art and, unless specifically defined, will not be interpreted in an idealized or overly formal sense.

[0038] Example 1

[0039] This embodiment provides a method for determining polyphenol compounds in mulberry linterus, which is determined by ultra-high performance liquid chromatography-mass spectrometry. The specific determination steps include:

[0040] 1. Sample processing

[0041] Accurately weigh an appropriate amount of mulberry linterinary polyphenols (previously purified) into a 2 mL centrifuge tube. Add 600 μL of methanol (containing 4 ppm of the internal standard 2-chloro-L-phenylalanine). Vortex for 30 seconds, add 100 mg of glass beads, and grind in a tissue grinder at 60 Hz for 90 seconds. Ultrasonicate at room temperature for 15 minutes, then centrifuge at 12,000 rpm at 4°C for 10 minutes. Remove the supernatant and filter through a 0.22 μm filter. Add the filtrate to a test vial for later use.

[0042] 2. Sample determination

[0043] The components of the treated filtrate were determined by ultra-performance liquid chromatography-mass spectrometry.

[0044] 2.1. The test conditions of the UPLC system are as follows:

[0045] Column: ACQUITY HSS T3 (2.1×150mm, 1.8μm);

[0046] Column temperature: 40°C;

[0047] Injection volume: 2 μL;

[0048] Mobile phase flow rate: 0.25 mL / min;

[0049] Mobile phases in positive ion mode: mobile phase B1: acetonitrile containing 0.1% formic acid, mobile phase A1: water containing 0.1% formic acid;

[0050] The gradient elution program in positive ion mode is shown in Table 1:

[0051] Table 1

[0052] Time (min) Mobile phase B1 (%) Mobile phase A1 (%) 0～1 2 98 1～9 2→50 98→50 9～12 50→98 50→2 12～13.5 98 2 13.5～14 98→2 2→98 14～20 2 98

[0053] ; Mobile phase in negative ion mode: Mobile phase B2: acetonitrile, mobile phase A2: 5 mM ammonium formate aqueous solution;

[0054] The gradient elution program in negative ion mode is shown in Table 2:

[0055] Table 2

[0056] Time (min) Mobile phase B2 (%) Mobile phase A2 (%) 0～1 2 98 1～9 2→50 98→50 9～12 50→98 50→2 12～13.5 98 2 13.5～14 98→2 2→98 14～17 2 98 ;

[0057] 2.2. Mass spectrometry conditions are as follows:

[0058] Data were collected using a Thermo Q Exactive mass spectrometer (Thermo Fisher Scientific, USA) with an electrospray ionization (ESI) source in both positive and negative ion modes. The positive ion spray voltage was 3.50 kV, the negative ion spray voltage was -2.50 kV, the sheath gas was 30 arb, and the auxiliary gas was 10 arb. The capillary temperature was 325°C, and a full scan was performed at a resolution of 70,000. The primary ion scan range was m / z 100 to 1000. Secondary fragmentation was performed using HCD with a collision energy of 30 eV and a secondary resolution of 17,500. The top 10 ions were fragmented, and dynamic exclusion was used to remove unnecessary MS / MS information.

[0059] 3. Conduct qualitative analysis on the results of step 2:

[0060] The data after UHPLC-ESI-MS detection were spectrally processed and database searched. Metabolites were first determined based on accurate molecular weight (error ≤ 30 ppm). Then, the metabolites of polyphenols were determined by comparing the MS / MS fragmentation patterns with mzCloud (http: / / www.macloud.org), LipidMaps (http: / / www.lipidmaps.org), Human MetabolomeDatabase (http: / / www.hmdb.ca), and standards.

[0061] Among them, the cation flow diagram obtained by the test is as follows Figure 1 As shown, the anion current diagram is as follows Figure 2 The components of the polyphenol compounds were identified by UHPLC-ESI-MS component detection and identification of positive and negative ions. The identification results are shown in Table 3.

[0062] Table 3

[0063]

[0064]

[0065] As can be seen from Table 3, after UHPLC-ESI-MS analysis, a total of 52 polyphenolic compounds including chlorogenic acid, epicatechin, gallic acid, hesperetin, etc. were detected from the polyphenol compounds of mulberry ignia. Among them, there are 15 flavonoids, 11 phenolic acid compounds, and 4 coumarin compounds, which provides a comprehensive theoretical basis for the study of the composition and function of polyphenolic compounds in mulberry ignia.

[0066] Example 2

[0067] This example studies the inhibitory effect of mulberry linterinary polyphenols on tumor cells. The specific experimental research is as follows:

[0068] 1. Experimental materials

[0069] Human hepatocellular carcinoma cell line (HepG2) and human lung cancer cell line (A549) were cultured in DEM / F 1:1 medium. Human gastric cancer cells (SGC-7901) and human prostate cancer cells (PC3) were cultured in F-12K medium, and human colon adenocarcinoma cells (HCT-116) were cultured in RPMI 1640 medium. Human breast cancer cells (MDA-MB-231) were cultured in high-glucose DMEM. All cell lines were provided by the Institute of Biology, Hebei Academy of Sciences.

[0070] 2. Experimental methods

[0071] 2.1. Solution preparation: According to the preset concentration of the experiment, weigh appropriate amounts of crude mulberry linterinary polyphenol compound (denoted as SSP) and purified mulberry linterinary polyphenol compound (denoted as SSPIP) and dissolve them in DMSO. Then take the corresponding amount and dissolve it in culture medium (wherein the volume ratio of DMSO solution: culture medium is ≤1:1000), filter the bacteria and set aside.

[0072] 2.2 Cell Culture

[0073] Thaw the cells in the cryovial by rapidly shaking in a 37°C water bath. Centrifuge at 1000 rpm for 10 minutes, resuspend the cells, and place in an incubator. Transfer the trypsinized cells to a centrifuge tube and centrifuge at 1000 rpm for 10 minutes. Discard the supernatant, resuspend the cells, transfer to a cryovial, and place in a cryopreservation box. Refrigerate at -80°C overnight and store in liquid nitrogen the next day. Remove the cells from the incubator, rinse with PBS, and digest. When the cells change morphology and detach from the flask wall, add culture medium to terminate the reaction. Gently blow off the cells, centrifuge at 1300 rpm for 10 minutes, discard the supernatant, resuspend the cells, and inoculate them into a culture flask in a CO2 incubator.

[0074] 2.3. CCK8 assay for anti-tumor activity

[0075] The cell suspension prepared according to the above cell culture method was mixed and plated into a 96-well plate. In a volume of 100 μL, 5×10 3 3 wells were reserved for culture medium instead of cell suspension. PBS solution was added to the surrounding wells and incubated at 37°C for 24 hours.

[0076] After the incubation, the old culture medium in the 96-well plate was discarded, and 100 μL of SSP and SSPIP at different concentrations (5, 10, 20, 40, 75, 100, 150 μg / mL) were added respectively. The solvent was culture medium. A blank group and a control group (5-fluorouracil, denoted as 5-Fu) were set up at the same time, and incubated in the incubator for 24 h.

[0077] After the incubation, the 96-well plate was removed, the supernatant was discarded, and 100 μL of 10% CCK8 PBS solution was added to each well. After gentle shaking, the cells were cultured at 37°C for 2 h. The results were detected using an enzyme-labeled instrument at 450 nm and the IC values were obtained. 50 .

[0078] The calculation formula of cell inhibition rate is as follows:

[0079] “A” is the cell viability.

[0080] 3. Experimental results

[0081] 3.1 Effects of mulberry linterinary polyphenols before and after purification on tumor cell growth

[0082] The inhibitory effects of SSP and SSPIP on different types of tumor cells are shown in Table 4.

[0083] Table 4

[0084]

[0085]

[0086] Note: A, B, and C indicate that the IC50 values of different treatments for the same tumor cell line are significantly different (P<0.05); a, b, c, d, e, and f indicate that the IC50 values of the same treatment for different tumor cell lines are significantly different (P<0.05).

[0087] As can be seen from Table 4, the inhibitory effect of SSPIP on different tumor cells is generally higher than that of SSP, and the IC values of both SSP and SSPIP on the inhibition rate of A549 tumor cell line are 50 The value is the lowest, lower than the IC of inhibition rate of other cell lines 50 The value was significantly different (P < 0.05), but higher than the positive control 5-FU (P < 0.05).

[0088] 3.2. Effects of different concentrations of SSPIP on the growth of A549

[0089] Through the screening of the above six tumor cell lines, the results showed that SSPIP had a good inhibitory effect on A549 cell line. Therefore, we further explored the inhibitory rate of SSPIP at different concentrations on A549 cells. Figure 3 shown.

[0090] Depend on Figure 3 It can be seen that the inhibitory effect of SSPIP on A549 cells is positively correlated with its concentration. Among them, in the concentration range of 10-20 μg / mL, the inhibition rate of SSPIP on A549 is better than that of 5-Fu. The inhibition rate increases with the increase of concentration. The inhibitory effect is better at a concentration of 150 μg / mL, and there is no significant difference with 5-Fu (P>0.05).

[0091] 3.3 Study on the effect of SSPIP on A549 cell morphology

[0092] After confirming that SSPIP has a good inhibitory effect on A549 cells, we further studied the effect of SSPIP on A549 cell morphology. Figure 4As shown, A is the morphology of A549 cells without drug addition, B is the morphology of A549 cells after adding 150 μg / mL 5-Fu for 24 hours, C is the morphology of A549 cells after adding 50 μg / mL SSPIP for 24 hours, D is the morphology of A549 cells after adding 75 μg / mL SSPIP for 24 hours, E is the morphology of A549 cells after adding 100 μg / mL SSPIP for 24 hours, and F is the morphology of A549 cells after adding 150 μg / mL SSPIP for 24 hours.

[0093] Figure 4 It can be seen that SSPIP has a certain inhibitory effect on A549 cells when added at low concentrations, but the inhibition rate is low. As the concentration of SSPIP increases, the number of dead cells gradually increases. When the concentration reaches 150 μg / mL, the death rate of A549 cells is high.

[0094] In summary, the present invention explored the inhibitory effects of SSP, SSPIP and 5-Fu on different tumor cell lines by the CCK8 method. The experimental results showed that SSP and SSPIP had certain inhibitory effects on A549, MDA, PC3, HepG2, HCT-116, and SGC-7901, and the inhibition rate of SSPIP on the above 6 tumor cells was significantly higher than that of SSP, and it had a better inhibitory effect on the A549 cell line. When SSPIP was added at 150 μg / mL, the inhibition rate was 80.63%, which was not significantly different from the same concentration of 5-FU (inhibition rate of 85.60%) (P>0.05). This shows that SSPIP has a good tumor inhibition rate, especially for the A549 cell line, and is expected to be used in the preparation of tumor drugs.

[0095] The above is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with this technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solutions and inventive concepts of the present invention, should be covered by the scope of protection of the present invention.

Claims

1. A method for determining polyphenol compounds in mulberry linterus, characterized in that: Ultra high performance liquid chromatography-mass spectrometry is used for determination, wherein the chromatographic conditions of ultra high performance liquid chromatography include: Column: ACQUITY HSS T3; Column temperature: 38-42°C; Injection volume: 1-3 μL; Mobile phase flow rate: 0.2-0.3 mL / min; Mobile phases in positive ion mode: mobile phase B1: acetonitrile containing 0.1% formic acid, mobile phase A1: water containing 0.1% formic acid; Mobile phases in negative ion mode: mobile phase B2: acetonitrile, mobile phase A2: 5 mM ammonium formate aqueous solution.

2. The method for determining polyphenols in mulberry linterus according to claim 1, wherein: The elution mode of the ultra-high performance liquid chromatography is gradient elution: The gradient elution program in positive ion mode was: The gradient elution program in negative ion mode is: 。 3. The method for determining polyphenols in mulberry linterinary polyphenols according to claim 1, wherein: The specifications of the chromatographic column are 2.1×150 mm and 1.8 μm.

4. The method for determining polyphenols in mulberry linterinary polyphenols according to any one of claims 1 to 3, wherein: The mass spectrometry conditions in the ultra-high performance liquid chromatography-mass spectrometry method include: Detector: mass spectrometer; Ion source: ESI ion source; Spray voltage: positive ion spray voltage is 3.50 kV, negative ion spray voltage is -2.50 kV; Primary scan: resolution 70000, ion scanning range m / z 100-1000; Secondary fragmentation: resolution 17500, collision energy 30eV, collecting the first 10 ions for fragmentation.

5. The method for determining polyphenols in mulberry linterinary medicine according to claim 4, wherein: The mass spectrometry conditions in the ultra-high performance liquid chromatography-mass spectrometry method further include: sheath gas 30 arb, auxiliary gas 10 arb, and capillary temperature: 325°C.

6. The method for determining polyphenols in mulberry linterus according to claim 5, wherein: The determination steps include: accurately weighing an appropriate amount of mulberry linterinary polyphenol compounds into a centrifuge tube, adding a methanol solution containing 2-chloro-L-phenylalanine as an internal standard, vortexing, adding glass beads, grinding in a tissue grinder, and then ultrasonicating, solid-liquid separation, and filtering the liquid phase, and the filtrate is tested on a machine.

7. Use of a mulberry linterus polyphenol compound in the preparation of anti-tumor drugs.

8. Use of a mulberry linterinary polyphenol compound in inhibiting tumor cell growth.

9. The use according to claim 8, characterized in that The tumor cells include human lung cancer cells A549, human liver cancer cells HepG2 and human prostate cancer cells PC3.

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