Flavonoid compound and extraction method thereof

Abstract

The invention belongs to the technical field of natural product chemistry and medicine extraction, and provides a flavonoid compound and an extraction method thereof. The flavonoid compound provided by the invention has a structure as shown in the following formula. The flavonoid compound provided by the invention is novel in structure, and the variety of natural bioactive substances is widened. Meanwhile, the flavonoid compound provided by the invention has a rapid anti-depression effect. The invention further provides an extraction method of the flavonoid compound. The extraction method has the advantages of being high in extraction efficiency, reasonable in purification route, high in product purity and good in reproducibility.

Description

Technical Field

[0001] This invention relates to the fields of natural product chemistry and drug extraction technology, and in particular to a flavonoid compound and its extraction method. Background Technology

[0002] Lanmaoa asiatica is a unique edible wild fungus native to Yunnan Province, known for its distinctive flavor and rich content of various bioactive natural products. Studies have shown that certain secondary metabolites of this fungus, such as strophasterol E, have inhibitory effects on Staphylococcus aureus and Bacillus subtilis, while another compound, 6β-methoxyergosterol-7,9(11),22E-triene-3β,5α-diol, exhibits antitumor activity. Exploring the active ingredients in Lanmaoa asiatica is of great significance for expanding the range of natural bioactive substances. Summary of the Invention

[0003] In view of this, the purpose of this invention is to provide a flavonoid compound and its extraction method. The flavonoid compound provided by this invention has a novel structure, expanding the range of natural bioactive substances.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a flavonoid compound having the structure shown in Formula 1: Formula 1.

[0005] This invention also provides a method for extracting flavonoids as described in the above technical solution, comprising the following steps: The fruiting bodies of Boletus lanmoides were mixed with an ethanol-water solution and subjected to ultrasonic extraction, solid-liquid separation and liquid-phase concentration in sequence to obtain a crude extract. The crude extract was reconstituted to obtain a crude extract dispersion; impurities in the crude extract dispersion were removed by extraction with petroleum ether and chloroform in sequence to obtain a purified aqueous phase; the purified aqueous phase was extracted with ethyl acetate, and the obtained ethyl acetate phase was concentrated to obtain the ethyl acetate fraction extract; The ethyl acetate extract was subjected to silica gel column chromatography, with the eluent being a chloroform-methanol system. The volume ratio of chloroform to methanol in the chloroform-methanol system was 1:0, 2:0.8~1.2, 1:0.8~1.2, and 0:1, respectively. The eluent fraction with a chloroform to methanol volume ratio of 1:0.8~1.2 was concentrated to obtain the crude product. The crude product was subjected to high-performance liquid chromatography (HPLC) separation to obtain the flavonoids; The high-performance liquid chromatography separation includes performing a first high-performance liquid chromatography separation and a second high-performance liquid chromatography separation sequentially; The conditions for the first high-performance liquid chromatography separation include: The chromatographic column was a Waters XBridge BEH OBD C18 preparative column; The mobile phase includes mobile phase A and mobile phase B, wherein mobile phase A is water and mobile phase B is acetonitrile; The elution method is gradient elution; The gradient elution procedure is as follows: 0-5 min: The volume fraction of mobile phase A is 95%; 5-30 min: The volume fraction of mobile phase A changes uniformly from 95% to 5%; The conditions for the second high-performance liquid chromatography separation include: The chromatographic column was a Waters XSelect CSH C18 column; The mobile phase includes mobile phase A and mobile phase B, wherein mobile phase A is water and mobile phase B is acetonitrile; The elution method is gradient elution; The gradient elution procedure is as follows: 0-5 min: The volume fraction of mobile phase A is 90%; 5-15 min: The volume fraction of mobile phase A changes from 90% to 40% at a constant rate.

[0006] Preferably, the volume concentration of the ethanol aqueous solution is 80-95%; The ratio of the fruiting body of Boletus lanmoides to the ethanol aqueous solution is 1g:3~10mL.

[0007] Preferably, the ultrasonic extraction is performed 1 to 3 times; The ultrasonic extraction power is 80~120W, and the frequency is 28~40kHz; The ultrasonic extraction is performed in an intermittent mode, which consists of 4-6 minutes of ultrasound followed by a 4-6 minute pause; a single ultrasonic extraction session includes 2-4 intervals.

[0008] Preferably, the extraction of petroleum ether, chloroform, and ethyl acetate is performed independently 2 to 4 times.

[0009] Preferably, the sample loading method for silica gel column chromatography is dry loading; the dry loading includes the following steps: mixing the ethyl acetate extract with silica gel, and loading the sample after the solvent has evaporated; the mass ratio of the ethyl acetate extract to silica gel is 1:8~12; the particle size of the silica gel is 100~200 mesh; The silica gel used in the silica gel column chromatography has a particle size of 80-100 mesh.

[0010] Preferably, during the silica gel column chromatography process, the flow rate of the eluent is 0.8~1.2 mL / min.

[0011] Preferably, the conditions for the first high-performance liquid chromatography separation include: The flow rate is 1~2 mL / min; The column temperature is 25~35℃; The ultraviolet detection wavelengths are 215nm and 254nm; Collect the components that are retained for 15 to 18 minutes.

[0012] Preferably, the conditions for the second high-performance liquid chromatography separation include: The flow rate is 0.4~0.6 mL / min; The column temperature is 25~35℃; The ultraviolet detection wavelengths are 215nm and 254nm; Collect the main peak flow.

[0013] Preferably, after the target fraction is obtained by high performance liquid chromatography, the process further includes post-processing, which includes: sequentially concentrating and freeze-drying the target fraction to obtain the flavonoid compound.

[0014] This invention provides a flavonoid compound. The flavonoid compound provided by this invention has a novel structure, broadening the range of natural bioactive substances. Furthermore, the flavonoid compound provided by this invention exhibits rapid antidepressant effects.

[0015] This invention also provides a method for extracting flavonoids as described in the above technical solution, which has the following advantages: (1) High extraction efficiency: Ultrasonic-assisted extraction is time-saving and low-temperature, which can effectively protect thermally unstable components and improve the extraction rate.

[0016] (2) Reasonable purification route: Combining normal phase silica column chromatography and reversed phase HPLC, the purification route design has a clear logic, removes impurities step by step, and is highly targeted while taking into account universality.

[0017] (3) High product purity: The (8,9,10-tris(hydroxymethyl)-11H-[1,4]dioxa[2,3-b]xanthon-2,3,11-trione obtained by the extraction method provided by this invention has a purity of more than 98%, which meets the strict requirements for compound purity in drug research and development.

[0018] (4) Good reproducibility: The process parameters of each step are clear, the operation is simple, and it has good reproducibility and scale-up potential. Attached Figure Description

[0019] Figure 1 The ESI+ spectrum of the final product obtained in Example 1; Figure 2 ESI spectrum of the final product obtained in Example 1 Figure 3 The ESI-MS / MS spectrum of the final product obtained in Example 1; Figure 4 The final product obtained in Example 1 1 H NMR spectrum; Figure 5 The final product obtained in Example 1 13 C NMR spectrum; Figure 6 The flavonoids obtained in Example 1 inhibited DBH enzyme activity; Figure 7 The effect of the flavonoids obtained in Example 1 on the activity of DDC enzyme; Figure 8 The effect of flavonoids obtained in Example 1 on MAO enzyme activity; Figure 9 This study describes the antidepressant effect of the flavonoids obtained in Example 1 on a mouse model of chronic social frustration stress. Detailed Implementation

[0020] This invention provides a flavonoid compound having the structure shown in Formula 1: Formula 1.

[0021] The flavonoids provided by this invention have novel structures, broadening the range of natural bioactive substances. Furthermore, the flavonoids provided by this invention exhibit rapid antidepressant effects.

[0022] This invention also provides a method for extracting flavonoids as described in the above technical solution, comprising the following steps: The fruiting bodies of Boletus lanmoides were mixed with an ethanol-water solution and subjected to ultrasonic extraction, solid-liquid separation and liquid-phase concentration in sequence to obtain a crude extract. The crude extract was reconstituted to obtain a crude extract dispersion; impurities in the crude extract dispersion were removed by extraction with petroleum ether and chloroform in sequence to obtain a purified aqueous phase; the purified aqueous phase was extracted with ethyl acetate, and the obtained ethyl acetate phase was concentrated to obtain the ethyl acetate fraction extract; The ethyl acetate extract was subjected to silica gel column chromatography, with the eluent being a chloroform-methanol system. The volume ratio of chloroform to methanol in the chloroform-methanol system was 1:0, 2:0.8~1.2, 1:0.8~1.2, and 0:1, respectively. The eluent fraction with a chloroform to methanol volume ratio of 1:0.8~1.2 was concentrated to obtain the crude product. The crude product was subjected to high-performance liquid chromatography (HPLC) separation to obtain the flavonoids; The high-performance liquid chromatography separation includes performing a first high-performance liquid chromatography separation and a second high-performance liquid chromatography separation sequentially; The conditions for the first high-performance liquid chromatography separation include: The chromatographic column was a Waters XBridge BEH OBD C18 preparative column; The mobile phase includes mobile phase A and mobile phase B, wherein mobile phase A is water and mobile phase B is acetonitrile; The elution method is gradient elution; The gradient elution procedure is as follows: 0-5 min: The volume fraction of mobile phase A is 95%; 5-30 min: The volume fraction of mobile phase A changes uniformly from 95% to 5%; The conditions for the second high-performance liquid chromatography separation include: The chromatographic column was an XSelect CSH C18 column; The mobile phase includes mobile phase A and mobile phase B, wherein mobile phase A is water and mobile phase B is acetonitrile; The elution method is gradient elution; The gradient elution procedure is as follows: 0-5 min: The volume fraction of mobile phase A is 90%; 5-15 min: The volume fraction of mobile phase A changes from 90% to 40% at a constant rate.

[0023] Unless otherwise specified, the raw materials used in this invention are preferably commercially available products.

[0024] This invention involves mixing *Boletus edulis* fruiting bodies with an ethanol-water solution, followed by sequential ultrasonic extraction, solid-liquid separation, and liquid-phase concentration to obtain a crude extract. In this invention, the *Boletus edulis* fruiting bodies are preferably fresh. In this invention, the *Boletus edulis* fruiting bodies preferably undergo pretreatment before ultrasonic extraction; the pretreatment preferably includes washing and pulverizing the *Boletus edulis* fruiting bodies sequentially. This invention does not specifically limit the washing operation and parameters, as long as the *Boletus edulis* fruiting bodies are cleaned. In this invention, the pulverization is preferably mechanical pulverization. In this invention, the volume concentration of the ethanol-water solution is preferably 80-95%, more preferably 85-90%, and specifically preferably 80%, 85%, 90%, or 95%. In this invention, the ratio of fresh *Boletus edulis* fruiting bodies to the ethanol-water solution is preferably 1g:3-10mL, more preferably 1g:5mL.

[0025] In this invention, the power of the ultrasonic extraction is preferably 80-120W, more preferably 100W; the frequency is preferably 28-40kHz, specifically 28kHz, 30kHz, 35kHz, or 40kHz. In this invention, the ultrasonic extraction is preferably performed in an intermittent mode, preferably with 4-6 minutes of ultrasonic extraction followed by a 4-6 minute pause, more preferably 5 minutes of ultrasonic extraction followed by a 5-minute pause; a single ultrasonic extraction preferably includes 2-4 intervals, more preferably 3 intervals; each interval specifically refers to 4-6 minutes of ultrasonic extraction followed by a 4-6 minute pause. In this invention, the number of ultrasonic extractions is preferably 1-3 times, more preferably 2 times. In this invention, the ultrasonic extraction is preferably performed in an ultrasonic cleaner.

[0026] In this invention, the solid-liquid separation method is preferably filtration, and the filtration is preferably Buchner funnel filtration.

[0027] In this invention, the liquid-phase concentration method is preferably vacuum rotary evaporation, and the vacuum rotary evaporation temperature is preferably 55°C. In this invention, the ethanol produced by the vacuum rotary evaporation is recovered.

[0028] In this invention, an aqueous ethanol solution of a specific concentration is used as the extractant, and the target components are extracted efficiently and at low temperature from the pulverized Boletus edulis fruiting bodies under optimized ultrasonic power, frequency and intermittent mode.

[0029] After obtaining the crude extract, the present invention reconstitutes the crude extract to obtain a crude extract dispersion; extracts the crude extract dispersion with petroleum ether and chloroform in sequence to remove impurities, and obtains a purified aqueous phase; extracts the purified aqueous phase with ethyl acetate, concentrates the obtained ethyl acetate phase, and obtains the ethyl acetate fraction extract.

[0030] In this invention, the solvent used for redissolution is preferably water, and more preferably distilled water.

[0031] In this invention, the petroleum ether extraction is preferably performed 2 to 4 times, more preferably 3 times. In this invention, the petroleum ether extraction is preferably performed using equal-volume extraction. In this invention, the volume ratio of petroleum ether to crude extract dispersion is preferably 1:1. After petroleum ether extraction, the aqueous phase is collected and recorded as the initial pure aqueous phase.

[0032] In this invention, the extraction of chloroform is preferably performed 2 to 4 times, more preferably 3 times. In this invention, the extraction of chloroform is preferably performed using an equal volume extraction method. In this invention, the volume ratio of chloroform to the initial purified aqueous phase is preferably 1:1. After the chloroform extraction, the aqueous phase is collected and designated as the purified aqueous phase.

[0033] In this invention, the ethyl acetate is preferably extracted 2 to 4 times, more preferably 3 times. In this invention, the ethyl acetate extraction is preferably performed using an equal volume extraction method. In this invention, the volume ratio of ethyl acetate to purified aqueous phase is preferably 1:1. After the ethyl acetate extraction, the ethyl acetate phase is collected.

[0034] In this invention, the concentration is preferably vacuum concentration. This invention does not specifically limit the parameters of the vacuum concentration; the ethyl acetate phase can be concentrated to dryness under vacuum.

[0035] In this invention, by sequential extraction with petroleum ether, chloroform, and ethyl acetate, weakly polar impurities such as lipids and chlorophyll, as well as strongly polar impurities, are effectively removed, and the target flavonoid compounds are enriched in the ethyl acetate fraction.

[0036] After obtaining the ethyl acetate fraction extract, the present invention performs silica gel column chromatography on the ethyl acetate fraction extract. The eluent for the silica gel column chromatography is a chloroform-methanol system, wherein the volume ratio of chloroform to methanol in the chloroform-methanol system is 1:0, 2:0.8~1.2, 1:0.8~1.2, and 0:1, respectively. The eluting fraction with a chloroform to methanol volume ratio of 1:0.8~1.2 is concentrated to obtain the crude product.

[0037] In this invention, the sample loading method for silica gel column chromatography is preferably dry loading; the dry loading method preferably includes the following steps: mixing the ethyl acetate extract and silica gel, and loading the sample after the solvent has evaporated. In this invention, the mass ratio of the ethyl acetate extract to silica gel is preferably 1:8~12, more preferably 1:10; the particle size of the silica gel is preferably 100~200 mesh. In this invention, the particle size of the silica gel packed in the chromatography column used for silica gel column chromatography is preferably 80~100 mesh. In a specific embodiment of this invention, the weight of the silica gel packed in the chromatography column used for silica gel column chromatography is preferably 200~400g, more preferably 300g. In this invention, the diameter of the chromatography column used for silica gel column chromatography is preferably 4~6cm, more preferably 5cm.

[0038] In this invention, the volume ratio of chloroform to methanol in the chloroform-methanol system is preferably 1:0, 2:1, 1:1, and 0:1, respectively.

[0039] In this invention, during the silica gel column chromatography process, the flow rate of the eluent is preferably 0.8~1.2 mL / min, and more preferably 1 mL / min.

[0040] In this invention, the silica gel column chromatography process is preferably monitored by thin-layer chromatography (TLC).

[0041] In this invention, a dry loading method and a specific chloroform-methanol gradient elution system are used to initially separate fractions containing the target compound from complex ethyl acetate fraction extracts.

[0042] After obtaining the crude product, the present invention performs high performance liquid chromatography separation on the crude product to obtain the flavonoid compounds.

[0043] In this invention, the high-performance liquid chromatography separation includes performing a first high-performance liquid chromatography separation and a second high-performance liquid chromatography separation in sequence.

[0044] In this invention, the conditions for the first high-performance liquid chromatography separation include: The chromatographic column is a Waters XBridge BEH OBD C18 preparative column, and the preferred dimensions of the Waters XBridge BEH OBDC18 preparative column are 5 μm and 10 × 250 mm. The mobile phase includes mobile phase A and mobile phase B, wherein mobile phase A is water, and the water is ultrapure water; mobile phase B is acetonitrile. The elution method is gradient elution; The gradient elution procedure is as follows: 0-5 min: The volume fraction of mobile phase A is 95%; 5-30 min: The volume fraction of mobile phase A changes uniformly from 95% to 5%; The flow rate is preferably 1~2 mL / min, more preferably 1.5 mL / min; The column temperature is preferably 25~35℃, and more preferably 30℃; The preferred ultraviolet detection wavelengths are 215 nm and 254 nm; Collect the components that are retained for 15 to 18 minutes.

[0045] In this invention, before the crude product undergoes the first high-performance liquid chromatography (HPLC) separation, it preferably further includes: dissolving the crude product, filtering it, and then performing the first HPLC separation. In this invention, the reagent used for dissolution is preferably methanol. In this invention, the pore size of the filter membrane used for filtration is preferably 0.22 μm.

[0046] In this invention, the conditions for the second high-performance liquid chromatography separation include: The chromatographic column is a Waters XSelect CSH C18 column, and the preferred size of the XSelect CSH C18 column is 5μm, 3×250mm; The mobile phase includes mobile phase A and mobile phase B, wherein mobile phase A is water, and the water is ultrapure water; mobile phase B is acetonitrile. The elution method is gradient elution; The gradient elution procedure is as follows: 0-5 min: The volume fraction of mobile phase A is 90%; 5-15 min: The volume fraction of mobile phase A changes uniformly from 90% to 40%; The flow rate is preferably 0.4~0.6 mL / min, more preferably 0.5 mL / min; The column temperature is preferably 25~35℃, and more preferably 30℃; The preferred ultraviolet detection wavelengths are 215 nm and 254 nm; Collect the main peak flow.

[0047] In this invention, after the first high-performance liquid chromatography separation, a corresponding fraction is preferably obtained; before the corresponding fraction is separated by the second high-performance liquid chromatography, it is preferable to further include: concentrating the corresponding fraction.

[0048] After the target fraction is obtained by high-performance liquid chromatography (HPLC), post-processing is preferably included. The post-processing preferably includes: sequentially concentrating and freeze-drying the target fraction to obtain the flavonoid compound. In this invention, the concentration is preferably vacuum concentration, and the vacuum concentration temperature is preferably 40°C.

[0049] In this invention, a reversed-phase C18 column is used, and through two consecutive semi-preparative / analytical HPLC steps, the target compound with a chromatographic purity of over 98% is finally obtained using a precisely optimized mobile phase gradient.

[0050] In this invention, the flavonoids exhibit a rapid antidepressant effect and have a rapid and significant therapeutic effect on depression.

[0051] The following detailed description of the flavonoids and their extraction methods provided by the present invention, with reference to specific examples, should not be construed as limiting the scope of protection of the present invention.

[0052] Example 1 (1) Raw material processing and extraction: Take 1.0 kg of fresh Boletus edulis fruiting bodies, wash them, and mechanically crush them; add 5 L of 90% ethanol aqueous solution to 1 kg of Boletus edulis fruiting body residue, and perform ultrasonic extraction in an ultrasonic cleaner (power 100W, frequency 28kHz); the ultrasonic extraction adopts intermittent mode: ultrasonic extraction for 5 min, pause for 5 min, this is one cycle, and a total of 3 cycles are performed. After ultrasonic extraction, filter with a Buchner funnel and collect the filtrate; repeat ultrasonic extraction on the residue once; combine all the filtrates, and evaporate under reduced pressure at 55℃ to recover ethanol and obtain crude extract.

[0053] (2) Solvent extraction: Add an appropriate amount of distilled water to the crude extract above, suspend and disperse it, extract it three times with an equal volume of petroleum ether, and collect the initial pure aqueous phase; extract the initial pure aqueous phase three times with an equal volume of chloroform, and collect the purified aqueous phase; extract the purified aqueous phase three times with an equal volume of ethyl acetate, collect the ethyl acetate phase, concentrate it to dryness under reduced pressure, and obtain a brownish-yellow ethyl acetate extract (about 10g).

[0054] (3) Silica gel column chromatography: Weigh 100~200 mesh silica gel (about 200 g) and mix thoroughly with the ethyl acetate extract obtained in step (2) (about 20 g). After the solvent evaporates, load the sample into a chromatography column (5 cm in diameter) containing 300 g of silica gel (80~100 mesh). Use a chloroform-methanol system for gradient elution. The elution order is: pure chloroform → chloroform:methanol = 2:1 (v / v) → chloroform:methanol = 1:1 (v / v) → pure methanol. Control the flow rate at 1 mL / min. Use thin layer chromatography (TLC) to monitor the flow. Collect the chloroform:methanol = 1:1 (v / v) fraction, combine them, and concentrate under reduced pressure to obtain a pale yellow crude product (about 0.5 g).

[0055] (5) High-performance liquid chromatography purification: Step 1 HPLC: The crude product obtained in step (3) was dissolved in methanol, filtered through a 0.22 μm microporous membrane, and separated using a Waters XBridge BEH OBD C18 preparative column (5 μm, 10 × 250 mm); Mobile phase: A was ultrapure water, B was acetonitrile; Elution gradient: 0-5 min, 95% A; 5-30 min, the volume fraction of mobile phase B changed linearly to 95%; Flow rate was 1.5 mL / min, column temperature was 30℃; UV detection wavelength: 215 nm, 254 nm. The fractions corresponding to the target peak (retention time about 15-18 minutes) were collected according to the chromatogram.

[0056] Step 2 HPLC: After appropriately concentrating the fraction collected in Step 1, the final purification was performed using a Waters XSelect CSH C18 analytical column (5μm, 3×250mm): Mobile phase: A is ultrapure water, B is acetonitrile; Elution gradient: 0-5min, 90% A; 5-15min, the volume fraction of mobile phase B changes linearly to 60%; Flow rate is 0.5mL / min, and the main peak fraction is collected.

[0057] The final collected fraction was concentrated under reduced pressure at 40°C and freeze-dried to obtain a light yellow powder. The purity was determined to be greater than 98% by HPLC area normalization method.

[0058] The structure of the final product was characterized, and the results are shown in [the table below]. Figures 1-5 ,from Figures 1-5 It can be seen that the molecular weight of the final product is 372.04, and the molecular formula is C. 18 H 12 O9, the structural formula is shown in Equation 1: Formula 1.

[0059] The chemical name is (8,9,10-tris(hydroxymethyl)-11H-[1,4]dioxino[2,3-b]xanthene-2,3,11-trione, (8,9,10-tris(hydroxymethyl)-11H-[1,4]dioxino[2,3-b]xanthene-2,3,11-trione).

[0060] Example 2 In vitro enzyme activity test of the flavonoids obtained in Example 1 1. DBH enzyme activity assay Following the methods described in the literature, DBH enzyme activity was determined using continuous spectroscopy. N,N-dimethyl-p-phenylenediamine dihydrochloride (DMPD) was used as the electron donor to replace the essential cosubstrate ascorbic acid. Recombinant DBH protein purchased from a commercial company was used. The effect of different concentrations of the flavonoids obtained in Example 1 on DBH activity was evaluated by monitoring the degradation rate of the substrate DMPD. A blank control was used without the flavonoids obtained in Example 1. Absorbance was measured at 515 nm using a microplate reader, and the kinetic curves were recorded over 15 minutes. The results are as follows: Figure 6 As shown, from Figure 6 It can be seen that at the concentration of the obtained compound at 26 μM, the DBH enzyme activity was completely inhibited, and there was a significant activity gradient effect at different concentration gradients.

[0061] 2. DDC enzyme activity assay DDC catalyzes the decarboxylation of L-DOPA and 5-hydroxytryptophan (5-HTP) to generate dopamine (DA) and serotonin (5-HT). Enzyme activity was assessed by measuring the degradation of these two substrates. The procedure was as follows: 50 μL of mouse brain homogenate (prepared by freezing and grinding intact brain tissue with 1 mL PBS at 10,000 rpm, and collecting the supernatant as mouse brain homogenate) or recombinant DDC protein (2 μM), 26–3.25 μM of the flavonoids obtained in Example 1, 50 μL of pyridoxal phosphate, and 300 μL of PBS buffer (0.01 M, pH 7.2) containing 39 mM dithiothreitol (DTT) and 0.167 mM EDTA-2Na) were mixed and pre-incubated at 37°C in the dark for 2 h. Subsequently, 100 μL of 2 mM L-DOPA or 5-HTP was added, and the mixture was incubated at 37°C in the dark for 2 h (L-DOPA) or 18 h (5-HTP), respectively. After the reaction, the mixture was centrifuged and ultrafiltered, and the filtrate was analyzed by reversed-phase high-performance liquid chromatography (RP-HPLC). The relative degradation rate of the substrate was calculated by peak area integration, thereby calculating the DDC activity. The results are as follows: Figure 7 As shown, the left graph represents the degradation rate of L-DOPA by different concentrations of flavonoids, and the right graph represents the degradation rate of 5-HTP by different concentrations of flavonoids. Figure 7 It can be seen that the obtained flavonoids have a good promoting effect on DDC enzyme activity at different concentrations, which upregulates the conversion rate of substrates L-Doap and 5-HTP, thereby increasing the content of dopamine and serotonin.

[0062] 3. MAO enzyme activity assay A commercial MAO assay kit was used. The procedure was as follows: Brain tissue was homogenized on ice, and crude mitochondrial enzyme solution was extracted by differential centrifugation. In a 96-well plate, 20 μL of different concentrations of the flavonoid solution obtained in Example 1, 160 μL of Reagent I, and 20 μL of Reagent II were added to the assay wells to achieve concentrations of 13 μM, 6.5 μM, and 3.25 μM of the flavonoids obtained in Example 1, respectively; 180 μL of Reagent I and 20 μL of Reagent II were added to the blank wells. The initial absorbance (A1) at 360 nm was recorded immediately after mixing, and the final absorbance (A2) was recorded after precise incubation at 37°C for 60 min. MAO activity was calculated using the absorbance change (ΔA = ΔA1 - ΔA2), with 1 nmol of substrate catalyzed to convert into 1 enzyme activity unit (U) per minute. The results are expressed as U / mg protein. Figure 8 As shown. From Figure 8 It can be seen that the obtained flavonoids have a good inhibitory effect on monoamine oxidase, and have a significant inhibitory gradient at different concentrations, thereby reducing the reduction of neurotransmitters such as dopamine and serotonin.

[0063] In vitro enzyme activity experiments showed that the flavonoids obtained in Example 1 could significantly inhibit the activities of DBH and MAO at concentrations of 26–6.5 μM, while significantly promoting the activity of DDC, and exhibited a significant concentration gradient effect. These three enzymes, as important rate-limiting / synthetic enzymes in the monoamine synthesis pathway, significantly upregulated / downregulated the synthesis of dopamine, serotonin, and norepinephrine.

[0064] Example 3 Antidepressant effect of flavonoids obtained in Example 1 on a mouse model of chronic social frustration stress 1. Animal models and drug administration Seven-week-old male C57BL / 6J mice were randomly assigned to groups. Mice in the model group and the drug-treated group underwent 10 consecutive days of chronic social frustration stress (SDS), which involved daily interaction with aggressive CD1 mice for 10 minutes followed by 24-hour isolation. During the SDS modeling period, the flavonoid-treated groups received daily intraperitoneal injections of different doses of flavonoids (1 mg / kg, 0.2 mg / kg, and 0.04 mg / kg).

[0065] 2. Behavioral Testing: After 10 days of model establishment and drug administration, a series of behavioral tests will be conducted: Tail Suspension Test (TST): The mouse tail is suspended and the immobility time for the last 5 minutes within a 6-minute period is recorded.

[0066] Forced swimming test (FST): Mice are placed in a cylindrical water tank and the immobility time is recorded for 6 minutes.

[0067] Social Interaction Test (SIT): In an open area, empty cages and cages containing unfamiliar CD1 mice were placed one after the other, and the time the mice spent in the interaction area was recorded.

[0068] Sucrose Preference Test (SPT): Mice were given the option to drink water with 1% sucrose or plain water, and the percentage of sucrose preference was calculated.

[0069] All behaviors are automatically analyzed using the SMART video tracking system.

[0070] The results are as follows Figure 9 As shown, A is the sucrose preference graph, B is the social interaction graph, C is the tail suspension rest time graph, and D is the forced swimming rest time graph; the SDS group is the social stress depression model group, and saline is the control group of model mice injected with physiological saline. From Figure 9 It can be seen that, compared with the model group, intraperitoneal injection of 1 mg / kg flavonoids for 7 consecutive days can significantly improve the depressive-like behavior of CSDS mice in SIT, FST, TST and SPT, confirming its rapid antidepressant effect.

[0071] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A flavonoid compound, characterized in that, It has the structure shown in Equation 1: Formula 1.

2. The method for extracting flavonoids according to claim 1, characterized in that, Includes the following steps: The fruiting bodies of Boletus lanmoides were mixed with an ethanol-water solution and subjected to ultrasonic extraction, solid-liquid separation and liquid-phase concentration in sequence to obtain a crude extract. The crude extract was reconstituted to obtain a crude extract dispersion; impurities in the crude extract dispersion were removed by extraction with petroleum ether and chloroform in sequence to obtain a purified aqueous phase; the purified aqueous phase was extracted with ethyl acetate, and the obtained ethyl acetate phase was concentrated to obtain the ethyl acetate fraction extract; The ethyl acetate extract was subjected to silica gel column chromatography, with the eluent being a chloroform-methanol system. The volume ratio of chloroform to methanol in the chloroform-methanol system was 1:0, 2:0.8~1.2, 1:0.8~1.2, and 0:1, respectively. The eluent fraction with a chloroform to methanol volume ratio of 1:0.8~1.2 was concentrated to obtain the crude product. The crude product was subjected to high performance liquid chromatography to separate the flavonoids. The high-performance liquid chromatography separation includes performing a first high-performance liquid chromatography separation and a second high-performance liquid chromatography separation sequentially; The conditions for the first high-performance liquid chromatography separation include: The chromatographic column was a Waters XBridge BEH OBD C18 preparative column; The mobile phase includes mobile phase A and mobile phase B, wherein mobile phase A is water and mobile phase B is acetonitrile; The elution method is gradient elution; The gradient elution procedure is as follows: 0-5 min: The volume fraction of mobile phase A is 95%; 5-30 min: The volume fraction of mobile phase A changes uniformly from 95% to 5%; The conditions for the second high-performance liquid chromatography separation include: The chromatographic column was a Waters XSelect CSH C18 column; The mobile phase includes mobile phase A and mobile phase B, wherein mobile phase A is water and mobile phase B is acetonitrile; The elution method is gradient elution; The gradient elution procedure is as follows: 0-5 min: The volume fraction of mobile phase A is 90%; 5-15 min: The volume fraction of mobile phase A changes from 90% to 40% at a constant rate.

3. The extraction method according to claim 2, characterized in that, The volume concentration of the ethanol aqueous solution is 80-95%; The ratio of the fruiting body of Boletus lanmoides to the ethanol aqueous solution is 1g:3~10mL.

4. The extraction method according to claim 2 or 3, characterized in that, The ultrasonic extraction is performed 1 to 3 times; The ultrasonic extraction power is 80~120W, and the frequency is 28~40kHz; The ultrasonic extraction is performed in an intermittent mode, which consists of 4-6 minutes of ultrasound followed by a 4-6 minute pause; a single ultrasonic extraction session includes 2-4 intervals.

5. The extraction method according to claim 2, characterized in that, The extraction times for petroleum ether, chloroform, and ethyl acetate were independently 2 to 4 times.

6. The extraction method according to claim 2, characterized in that, The sample loading method for silica gel column chromatography is dry loading; the dry loading includes the following steps: mixing the ethyl acetate extract with silica gel, and loading the sample after the solvent has evaporated; the mass ratio of the ethyl acetate extract to silica gel is 1:8~12; the particle size of the silica gel is 100~200 mesh; The silica gel used in the silica gel column chromatography has a particle size of 80-100 mesh.

7. The extraction method according to claim 2 or 6, characterized in that, During the silica gel column chromatography process, the eluent flow rate is 0.8~1.2 mL / min.

8. The extraction method according to claim 2, characterized in that, The conditions for the first high-performance liquid chromatography separation include: The flow rate is 1~2 mL / min; The column temperature is 25~35℃; The ultraviolet detection wavelengths are 215nm and 254nm; Collect the components that are retained for 15 to 18 minutes.

9. The extraction method according to claim 8, characterized in that, The conditions for the second high-performance liquid chromatography separation include: The flow rate is 0.4~0.6 mL / min; The column temperature is 25~35℃; The ultraviolet detection wavelengths are 215nm and 254nm; Collect the main peak flow.

10. The extraction method according to claim 2, 8, or 9, characterized in that, After the target fraction is obtained by high performance liquid chromatography, the post-processing includes: concentrating and freeze-drying the target fraction sequentially to obtain the flavonoid compound.

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