Method for separating biflavonoids from ironwood flower buds and application thereof

Abstract

The application provides a method for separating biflavonoids from Rhus potaninii flower buds, which specifically comprises the following steps: firstly, taking Rhus potaninii flower bud powder, and refluxing extraction by 95% ethanol, and filtering to obtain a filtrate; then purifying the filtrate by semi-preparative HPLC to obtain an eluate; after detecting the eluate at 300 nm, the eluate with a retention time of 20.5-23.0 min and 38.4-40.6 min is collected; and the eluate is concentrated and evaporated under reduced pressure to obtain biflavonoids Rhusflavanone and Rhusflavone. The separation method is simple, fast, efficient, environmentally friendly, and the obtained biflavonoids have high purity. The obtained biflavonoids can be used as a control sample to determine the content of the biflavonoids in Rhus potaninii and its pseudo-products Rhus succedaneum and Rhus succedaneum, and effectively identify whether the pseudo-products are replaced in the medicinal material or the preparation, and fill the technical blank.

Description

Technical Field

[0001] This invention belongs to the field of compound separation technology, specifically relating to a method for separating biflavonoid compounds from ironwood flower buds and its application. Background Technology

[0002] Mesuaferrea L., an evergreen tree belonging to the genus Mesuaferrea in the family Clusiaceae, is a traditional Uyghur medicinal herb known as Narmishik in Uyghur medicine. It is mainly distributed in India, Bangladesh, Pakistan, Thailand, and is also cultivated in Guangxi and Yunnan provinces of China. It is recorded in various ethnic medical texts and widely used in traditional preparations. Its main chemical components include flavonoids, coumarins, triterpenoids, and carboxylic acids, exhibiting strong anticancer and antioxidant activities. It is primarily used to treat damp-cold or phlegmatic diseases, cold-type heart deficiency, depression, neurasthenia, cold limbs and impotence, damp-type stomach deficiency, diarrhea, various damp sores, and hemorrhoidal bleeding. Currently, the misuse of other plants in the genus *Gnaphalium* as Mesuaferrea L. for medicinal purposes is widespread. Counterfeit Ironwood 1: *Mammea siamensis* (Miq.) T. Anderson, a plant belonging to the genus *Mammea* in the family Clusiaceae, is mainly distributed in Thailand, Myanmar, Laos, Cambodia, and Vietnam. It has anti-proliferation and anti-apoptotic effects, as well as inhibiting nitric oxide production. It is used to treat heart disease, clear heat and detoxify, and relieve pain. It can also increase appetite and is a substitute for ironwood. Counterfeit Ironwood 2: *Mammea yunnanensis* (HLLi) Kosterm., distributed in southern Yunnan, my country. Its properties, meridian tropism, and indications are unclear. However, there are still technical gaps in the determination of the content of effective components in ironwood and its counterfeit *Mammea* species, as well as in identifying whether counterfeit products are used in ironwood medicinal materials or preparations.

[0003] Biflavonoids belong to a subclass of flavonoids and are widely distributed in nature. Their chemical structure consists of two flavonoid molecules or their derivatives linked by CC or COO. Flavonoid molecules have numerous linking sites, and the variations in the position and type of these groups result in a vast number and diverse types of biflavonoids. Biflavonoids possess a wider range of biological activities and higher medicinal value than monomeric compounds. Natural biflavonoids exhibit a broad range of pharmacological effects, such as anti-inflammatory, antioxidant, anti-infective, antitumor, antibacterial, hypoglycemic, antiviral, and effects on the nervous system. Rhusflavanone and Rhusflavone are two natural biflavonoids found in ironwood. Rhusflavanone has the molecular formula CCO. 30 H 22 O 10It possesses antiviral activity, inhibiting the secretion activity of Salmonella effector proteins and exhibiting strong inhibitory activity against elastase and tyrosinase, but with low DPPH- free radical scavenging activity. Rhusflavone, with the molecular formula C... 30 H 20 O 10 It competitively inhibits influenza binding, shortens sleep latency, and leads to prolonged sleep duration. However, there is currently limited research on Rhusflavanone and Rhusflavone. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for separating biflavonoids from the flower buds of *Ilex cornuta* in view of the shortcomings of the prior art. The obtained biflavonoids, Rhusflavanone and Rhusflavone, are used as reference standards to determine their content in *Ilex cornuta* and its adulterants, *Ilex cornuta* and *Ilex cornuta*, and are used to identify the authenticity of *Ilex cornuta* medicinal materials and whether adulterants are used in the preparations, thus filling the technical gap.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0006] A method for isolating biflavonoids from ironwood flower buds, comprising the following steps:

[0007] S1. First, take dried ironwood flower buds, crush them, extract them by reflux with 95% ethanol, filter them, concentrate the extract under reduced pressure to obtain the extract.

[0008] S2. Dissolve the extract obtained in S1 in 95% ethanol, then add macroporous resin and stir thoroughly to adsorb, then concentrate under reduced pressure, dry, and grind into powder.

[0009] S3. The powder obtained in S2 was added to a chromatography column packed with macroporous resin and eluted sequentially with ethanol solutions of volume fractions of 0%, 10%, 30%, 50%, 70%, and 95%. The collected fractions were combined by HPLC analysis and concentrated under pressure to obtain four fractions: Fr.A, Fr.B, Fr.C, and Fr.D.

[0010] S4. The fraction Fr.D obtained in S3 was purified by semi-preparative HPLC, and the eluent was collected. After detecting the eluent at 300 nm, the eluent with a retention time of 20.5–23.0 min was collected, concentrated under reduced pressure and evaporated to dryness to obtain biflavonoid compound 1: Rhusflavanone; the eluent with a retention time of 38.4–40.6 min was collected, concentrated under reduced pressure and evaporated to dryness to obtain biflavonoid compound 2: Rhusflavone.

[0011] The molecular formula of Rhusflavanone is C 30 H 22 O 10 The structural formula is:

[0012]

[0013] The molecular formula of Rhusflavone is C 30 H 20 O 10 The structural formula is:

[0014]

[0015] Preferably, the ratio of ironwood flower buds to 95% ethanol in S1 is 1g:10-15mL, the extraction time is 1h each time, the extraction is performed 3 times, and the extracts are combined.

[0016] Preferably, the mass ratio of the extract to the macroporous resin in S2 is 1:1 to 1:1.5.

[0017] Preferably, the macroporous resin in S2 and S3 is D101 macroporous resin.

[0018] Preferably, the chromatographic column used for semi-preparative HPLC purification in S4 is octadecylsilane-bonded silica gel with dimensions of 20 mm × 250 mm and a diameter of 10 μm. The detection wavelength is 300 nm, the column temperature is 25 °C, and isocratic elution is performed using 0.1% glacial acetic acid as mobile phase A and methanol as mobile phase B at a flow rate of 5.0 mL / min. The isocratic elution program is 0-50 min, with mobile phase A at 35% and mobile phase B at 65%.

[0019] The present invention also provides an application of the aforementioned biflavonoid compound, which can be used as a reference standard for its content in ironwood and Siamese jasmine and jasmine buds, as well as for identifying the authenticity of ironwood medicinal materials or whether there are counterfeit substitutes in preparations.

[0020] The method for determining the content of biflavonoids is as follows: Rhusflavanone and Rhusflavone reference standards are dissolved in 50% methanol to prepare Rhusflavanone reference solution and Rhusflavone reference solution, respectively. Then, the sample powder to be tested is taken, 50% methanol is added, the mixture is sonicated, cooled, and filtered through a microporous membrane. The filtrate is collected to obtain the sample solution to be tested. The reference solution and the sample solution to be tested are then analyzed by high-performance liquid chromatography (HPLC). The content of biflavonoids Rhusflavanone and Rhusflavone in the sample is calculated based on the peak area. The method for calculating the content (mg / g) of Rhusflavanone and Rhusflavone in the sample to be tested is as follows:

[0021]

[0022] Preferably, the high-performance liquid chromatography (HPLC) conditions are as follows: a ShimNex WR C18 column with dimensions of 4.6 mm × 250 mm and a diameter of 5 μm; a detection wavelength of 300 nm; a column temperature of 25 °C; and gradient elution with 0.1% glacial acetic acid as mobile phase A and methanol as mobile phase B at a flow rate of 1.0 mL / min. The gradient elution program is as follows: 0-3 min, mobile phase A 80% and mobile phase B 20%; 3-8 min, mobile phase A decreasing from 80% to 70% and mobile phase B increasing from 20% to 30%; 8-10 min, mobile phase A decreasing from 70% to 62% and mobile phase B increasing from 30% to 38%; 10-25 min, mobile phase A decreasing from 60% to 72% and mobile phase B increasing from 30% to 38%; and 10-25 min, mobile phase A decreasing from 60% to 70% and mobile phase B increasing from 30% to 38%. From 25 to 33 min, mobile phase A decreased from 60% to 47%, while mobile phase B increased from 40% to 53%. From 33 to 35 min, mobile phase A decreased from 47% to 40%, while mobile phase B increased from 53% to 60%. From 35 to 44 min, mobile phase A decreased from 40% to 30%, while mobile phase B increased from 60% to 70%. From 44 to 44.1 min, mobile phase A increased from 30% to 80%, while mobile phase B decreased from 70% to 20%. From 44.1 to 54 min, mobile phase A was 80%, and mobile phase B was 20%.

[0023] The present invention has the following significant technical effects:

[0024] 1. This invention isolates and extracts two biflavonoid compounds, Rhusflavanone and Rhusflavone, from ironwood flower buds. The separation method of this invention is simple and rapid, the process is safe and environmentally friendly, and the isolated biflavonoid compounds have high purity.

[0025] 2. The biflavonoid compounds obtained by this invention can be used as reference standards to determine their content in ironwood, Siamese jasmine, and jasmine, and to identify the authenticity of ironwood medicinal powder and whether counterfeit products are used in the preparation. This fills a technological gap, and the identification method is simple, fast, and accurate, with great promotion and application value.

[0026] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. Attached Figure Description

[0027] Figure 1 This is a flowchart illustrating the separation of the biflavonoid compounds Rhusflavanone and Rhusflavone from ironwood flower buds according to the present invention;

[0028] Figure 2 Semi-preparative liquid chromatograms of Rhusflavanone and Rhusflavone;

[0029] Figure 3 The image shows the ultraviolet absorption spectrum of Rhusflavanone.

[0030] Figure 4 The high-performance liquid chromatogram of Rhusflavanone;

[0031] Figure 5 For Rhusflavanone 1 H-NMR spectrum;

[0032] Figure 6 For Rhusflavanone 13 C-NMR spectrum;

[0033] Figure 7 High-resolution time-of-flight mass spectrum of Rhusflavanone;

[0034] Figure 8 The image shows the ultraviolet absorption spectrum of Rhusflavone.

[0035] Figure 9 The high-performance liquid chromatogram of Rhusflavone;

[0036] Figure 10 For Rhusflavone 1 H-NMR spectrum;

[0037] Figure 11 For Rhusflavone 13 C-NMR spectrum;

[0038] Figure 12High-resolution time-of-flight mass spectrum of Rhusflavone;

[0039] Figure 13 The high performance liquid chromatogram of sample 1 (ironwood flower buds);

[0040] Figure 14 The high-performance liquid chromatogram of sample 2 (ironwood flower buds);

[0041] Figure 15 The high performance liquid chromatogram of sample 3 (ironwood flower buds);

[0042] Figure 16 The high performance liquid chromatogram of sample 4 (ironwood flower buds);

[0043] Figure 17 The high performance liquid chromatogram of sample 5 (ironwood flower buds);

[0044] Figure 18 The high performance liquid chromatogram of sample 6 (flower bud of Siamese gorse);

[0045] Figure 19 The high performance liquid chromatogram of sample 7 (flower buds of Siamese gorse);

[0046] Figure 20 The high-performance liquid chromatogram of sample 8 (flower bud of Siamese gorse);

[0047] Figure 21 The high performance liquid chromatogram of sample 9 (flower bud of Siamese gerbera);

[0048] Figure 22 The image shows the high-performance liquid chromatogram of sample 10 (flower buds of the Glechoma hederacea). Detailed Implementation

[0049] Instruments and reagents used in the embodiments of this invention:

[0050] Instruments: Shimadzu LC-2010AHT liquid chromatograph (analytical liquid chromatograph converted to semi-preparative liquid chromatograph); Shimadzu LC-20AD XR liquid chromatograph; Shimadzu UV-2600 ultraviolet spectrophotometer; Varian 400-MR nuclear magnetic resonance spectrometer; Waters Xevo G2-s QToF high-resolution time-of-flight mass spectrometer; Mettler XPR205 electronic balance.

[0051] Reagents: Methanol was of chromatographic grade, 95% ethanol and glacial acetic acid were of analytical grade, and water was ultrapure water.

[0052] Example 1

[0053] This embodiment describes a method for isolating biflavonoids from ironwood flower buds, comprising the following steps:

[0054] S1. Take 10g of dried ironwood flower buds, crush them through a No. 4 sieve (60 mesh), and extract them by reflux with 100mL of 95% ethanol for 1h. Extract them three times in total. Combine the extracts and concentrate them under reduced pressure using a rotary evaporator to obtain an extract.

[0055] S2. Take 5g of the extract obtained in S1 and dissolve it in 5mL of 95% ethanol. Add 5g of D101 macroporous resin and stir thoroughly for adsorption. Concentrate using a rotary evaporator and dry to granules using an electric hot air dryer. Grind into powder.

[0056] S3. The powder obtained in S2 is evenly sprinkled into a chromatography column packed with D101 macroporous resin. Eluent is slowly added along the column wall for elution. The eluent is successively 0%, 10%, 30%, 50%, 70%, and 95% ethanol solutions by volume fraction. The collected components are combined by HPLC analysis and concentrated under pressure using a rotary evaporator to obtain four components: Fr.A, Fr.B, Fr.C, and Fr.D.

[0057] S4. HPLC analysis showed that Rhusflavanone and Rhusflavone were mainly concentrated in the component Fr.D. Component Fr.D was purified by semi-preparative HPLC, and the eluent was collected. After detecting the eluent at 300 nm, the eluent with a retention time of 20.5–23.0 min was collected, concentrated under reduced pressure, and evaporated to dryness to obtain biflavonoid compound 1: Rhusflavanone; the eluent with a retention time of 38.4–40.6 min was collected, concentrated under reduced pressure, and evaporated to dryness to obtain biflavonoid compound 2: Rhusflavone. The semi-preparative HPLC purification column was an octadecylsilane-bonded silica column, 20 mm × 250 mm, 10 μm in diameter, with a detection wavelength of 300 nm and a column temperature of 25 °C. Isocratic elution was performed using 0.1% glacial acetic acid as mobile phase A and methanol as mobile phase B at a flow rate of 5.0 mL / min. The isocratic elution program was 0-50 min, with mobile phase A at 35% and mobile phase B at 65%. The semi-preparative HPLC chromatograms of Rhusflavanone and Rhusflavone are shown below. Figure 2 As shown.

[0058] The compounds isolated in this embodiment were identified. Rhusflavanone was a pale yellow powder with a purity of 95.0%. The specific structural identification spectrum is shown below. Figures 3-7 As shown, the molecular formula of the product is C. 30 H 22 O 10 The structural formula is:

[0059]

[0060] Figure 3 This is the UV absorption spectrum of Rhusflavanone, UV(EtOH)λ. max : 235nm, 295nm.

[0061] Figure 4 The image shows the high-performance liquid chromatogram of Rhusflavanone, with retention times ranging from 39.2 to 40.0 min.

[0062] Figure 5 For Rhusflavanone 1 H-NMR spectrum. 1 H NMR (400MHz, Methanol-d4): δ H 7.31(2H,d,J=8.1Hz,H-2′,H-6′),7.17(2H,d,J=8.1Hz,H-2″′,H-6″′), 6.81(2H,d,J=8.1Hz,H-3′,H-5′),6.72(2H,d,J=8.1Hz,H-3″′,H-5″′),6 .03(1H,s,H-6″),6.02(1H,s,H-8),5.33(1H,dd,J=12.7,3.0Hz,H-2″), 5.30 (1H, dd, J = 12.7, 3.0 Hz, H-2), 3.01 (2H, m, H-3b), 2.69 (2H, m, H-3a).

[0063] Figure 6 For Rhusflavanone 13 C-NMR spectrum. 13 C NMR (101MHz, cd3od): δ C198.27(C-4),197.92(C-4″),166.70(C-7),166.65(C-9),164.76(C-7″),164.08(C-9″),163.26(C-5″) ,162.80(C-4′),158.94(C-4″′),158.49(C-5),131.41(C-1′),131.20(C-1″′),129.09(C-2′,C-6′),12 8.56(C-2″′,6″′),116.29(C-3′,C-5′),116.14(C-3″′,C-5″′),103.65(C-10″),103.31(C-10),102.33 (C-8″),101.15(C-6),96.68(C-6″),95.71(C-8),80.52(C-2),79.86(C-2″),44.12(C-3),43.84(C-3″).

[0064] Figure 7 High-resolution time-of-flight mass spectrum of Rhusflavanone; ESI-MS m / z: 543.1588 [M+H] + .

[0065] The compound Rhusflavone isolated in this embodiment is a pale yellow powder with a purity of 96.0%. The specific structural identification spectrum is shown below. Figures 8-12 As shown, the molecular formula of the product is C. 30 H 20 O 10 The structural formula is:

[0066]

[0067] Figure 8 This is the UV absorption spectrum of Rhusflavone, UV(EtOH)λ. max : 233nm, 275nm, 295nm, 328nm.

[0068] Figure 9 The image shows the high-performance liquid chromatogram of Rhusflavone, with retention times ranging from 41.6 to 42.4 min.

[0069] Figure 10 For Rhusflavone 1 H-NMR spectrum. 1 H NMR (400MHz, Methanol-d4): δ H7.55(2H,d,J=8.8Hz,H-2″′,H-6″′),7.37(2H,d,J=8.5Hz,H-2′,H-6′),6.85(2H,d,J=8.5Hz,H-3′,H-5′),6.78(2H,d,J=8.8 Hz,H-3″′,H-5″′),6.57(1H,s,H-3″),6.36(1H,s,H-6″),6.16(1H,s,H-8),5.43(1H,dd,J=12.7,3.0Hz,H-2),3.22(2H,m,H-3 b ),2.78(2H,m,H-3 a ).

[0070] Figure 11 For Rhusflavone 13 C-NMR spectrum. 13 C NMR (101MHz, cd3od): δ C 197.95(C-4),184.30(C-4″),166.61(C-7),166.07(C-2″),164.53(C-9),164.16(C-7″),163.61(C-5),16 2.49(C-5″),162.38(C-4″′),159.01(C-4′),156.99(C-9″),131.06(C-1′),129.29(C-2″′,6″′),129.12(C -2′,C-6′),123.41(C-10″),116.90(C-3″′),116.86(C-5″′),116.37(C-3′,C-5′),105.51(C-1″′),103.45 (C-3″),103.36(C-6),101.38(C-10),100.54(C-8″),99.84(C-6″),95.84(C-8),80.59(C-2),43.99(C-3).

[0071] Figure 12 High-resolution time-of-flight mass spectrum of Rhusflavone; ESI-MS m / z: 541.3022 [M+H] + .

[0072] Example 2

[0073] This embodiment describes a method for determining the content of Rhusflavanone and Rhusflavone in dried flower buds of *Ilex cornuta* and *Gervais spp.* using Rhusflavanone and Rhusflavone isolated in the previous embodiment as reference standards, and for identifying whether *Gervais spp.* flower buds are mixed in.

[0074] Specifically:

[0075] 1. Preparation of reference solution

[0076] Accurately weigh Rhusflavanone (purity 95.0%) and Rhusflavone (purity 96.0%) reference standards, add 50% methanol, and prepare Rhusflavanone reference standard solutions with concentrations of 42.2 μg / mL and 47.4 μg / mL, respectively.

[0077] 2. Preparation of the sample solution to be tested

[0078] Accurately weigh 1g of the sample powder to be tested, add 100mL of 50% methanol, sonicate for 30min, cool, filter with a microporous membrane with a size of 0.45μm, and take the filtrate to obtain the sample solution to be tested.

[0079] 3. Measurement

[0080] Accurately pipette 10 μL each of the above-mentioned reference solution and the test sample solution, and inject them into the liquid chromatograph for detection. The chromatographic conditions are as follows:

[0081] A ShimNex WR C18 column (4.6 mm × 250 mm, 5 μm) was used with a detection wavelength of 300 nm and a column temperature of 25 °C. Mobile phase A was 0.1% glacial acetic acid, and mobile phase B was methanol. The flow rate was 1.0 mL / min. Gradient elution was performed: 0-3 min, mobile phase A 80%, mobile phase B 20%; 3-8 min, mobile phase A decreased from 80% to 70%, mobile phase B increased from 20% to 30%; 8-10 min, mobile phase A decreased from 70% to 62%, mobile phase B increased from 30% to 38%; 10-25 min, mobile phase A decreased from 62% to... 60%, mobile phase B increases from 38% to 40%; 25-33 min, mobile phase A decreases from 60% to 47%, mobile phase B increases from 40% to 53%; 33-35 min, mobile phase A decreases from 47% to 40%, mobile phase B increases from 53% to 60%; 35-44 min, mobile phase A decreases from 40% to 30%, mobile phase B increases from 60% to 70%; 44-44.1 min, mobile phase A increases from 30% to 80%, mobile phase B decreases from 70% to 20%; 44.1-54 min, mobile phase A is 80%, mobile phase B is 20%.

[0082] 4. Results Analysis

[0083] If no chromatographic peak with the same retention time as Rhusflavanone and Rhusflavone reference standards appears in the chromatogram of the test sample solution, then the test sample does not contain *Imperata cylindrica* flower buds. If a chromatographic peak with the same retention time as Rhusflavanone and Rhusflavone reference standards appears in the chromatogram of the test sample solution, and the content is not less than 0.90 mg / g, combined with the identification of medicinal materials, then the test sample is considered to contain only *Imperata cylindrica* flower buds. If a chromatographic peak with the same retention time as Rhusflavanone and Rhusflavone reference standards appears in the chromatogram of the test sample solution, but the content is between 0-0.90 mg / g, then the test sample is considered to contain not only *Imperata cylindrica* flower buds, i.e., there is a mixture. In the preparation feeding process, if there is no positive interference, this method is also applicable after examining the process recovery rate, etc.

[0084] The method for calculating the content (mg / g) of Rhusflavanone and Rhusflavone in the sample to be tested is as follows:

[0085]

[0086] Example 3

[0087] Five batches of dried flower buds from different locations were collected and tested to further verify the method of Example 2. These samples included *Mesuaferrea* L. (family Clusiaceae), four batches of *Mammea siamensis* (Miq.) T. Anderson (family Clusiaceae), and one batch of *Mammea yunnanensis* (family Clusiaceae).

[0088] The results showed that the chromatograms of five batches of *Ilex crenata* flower buds and four batches of *Gnaphalium affine* flower buds all showed peaks with the same retention times as the Rhusflavanone and Rhusflavone reference standards. However, the lowest content of the two substances in *Ilex crenata* was 0.98 mg / g, with an average of 2.3 mg / g; while the highest content in *Gnaphalium affine* was 0.66 mg / g, with an average of 0.53 mg / g. Therefore, setting a content limit of not less than 0.90 mg / g can effectively distinguish between *Ilex crenata* and *Gnaphalium* powders and extracts. The chromatogram of one batch of *Gnaphalium affine* flower buds did not show peaks with the same retention times as the Rhusflavanone and Rhusflavone reference standards. The HPLC chromatograms of samples 1-10 are shown below. Figures 13-22 As shown, the peak elution times of Rhusflavanone and Rhusflavone were 39.2–40.0 min and 41.6–42.4 min, respectively.

[0089] The results of the determination of Rhusflavanone and Rhusflavone content in samples 1-10 are shown in Table 1.

[0090] Table 1 Sample Measurement Results

[0091]

[0092] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. A method for isolating biflavonoid compounds from ironwood flower buds, characterized in that, Includes the following steps: S1. First, take dried ironwood flower buds, crush them, extract them by reflux with 95% ethanol, filter them, concentrate the extract under reduced pressure to obtain the extract. S2. Dissolve the extract obtained in S1 in 95% ethanol, then add macroporous resin and stir thoroughly to adsorb, then concentrate under reduced pressure, dry, and grind into powder. S3. The powder obtained in S2 was added to a chromatography column packed with macroporous resin and eluted sequentially with ethanol solutions of volume fractions of 0%, 10%, 30%, 50%, 70%, and 95%. The collected components were combined by HPLC analysis and concentrated under pressure to obtain four components: Fr.A, Fr.B, Fr.C, and Fr.D. S4. The fraction Fr.D obtained in S3 was purified by semi-preparative HPLC, and the eluent was collected. After detecting the eluent at 300 nm, the eluent with a retention time of 20.5~23.0 min was collected, concentrated under reduced pressure and evaporated to dryness to obtain biflavonoid compound 1: Rhusflavanone. The eluent with a retention time of 38.4–40.6 min was collected, concentrated under reduced pressure, and evaporated to dryness to obtain biflavonoid compound 2: Rhusflavone; the molecular formula of Rhusflavone is C 30 H 22 O 10 The structural formula is: ， The molecular formula of Rhusflavone is C 30 H 20 O 10 The structural formula is: 。 2. The method according to claim 1, characterized in that, The ratio of dried ironwood flower buds to 95% ethanol in S1 is 1g:10~15mL, the extraction time is 1h each time, the extraction is performed 3 times, and the extracts are combined.

3. The method according to claim 1, characterized in that, The mass ratio of the extract to the macroporous resin in S2 is 1:1 to 1:1.

5.

4. The method according to claim 1, characterized in that, The macroporous resins mentioned in S2 and S3 are D101 macroporous resins.

5. The method according to claim 1, characterized in that, The semi-preparative HPLC purification column described in S4 is an octadecylsilane-bonded silica gel column with dimensions of 20 mm × 250 mm and a diameter of 10 μm. The detection wavelength is 300 nm, and the column temperature is 25 °C. 0.1% glacial acetic acid is used as mobile phase A, methanol is used as mobile phase B, and the flow rate is 5.0 mL / min. Isocratic elution is performed. The isocratic elution program is: 0-50 min, mobile phase A is 35%, and mobile phase B is 65%.

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