Method for separating biflavone compounds from ferrugina flower buds and application of biflavone compounds

Abstract

The invention provides a method for separating biflavone compounds from ferrugina flower buds, which specifically comprises the following steps: firstly, taking ferrugina flower bud powder, adding 95% ethanol for reflux extraction, and filtering to obtain filtrate; the preparation method comprises the following steps: firstly, filtering the raw materials to obtain a filtrate, then purifying the filtrate through semi-preparative HPLC (High Performance Liquid Chromatography) to obtain an eluent, detecting the eluent at 300nm, determining the eluent with the collection and retention time of 20.5-23.0 minutes and 38.4-40.6 minutes, and carrying out vacuum concentration and evaporation to dryness to obtain the biflavone compounds Rhusflavanone and Rhusflavanone. The separation method disclosed by the invention is simple, rapid, efficient and environment-friendly, and the obtained biflavone compound is high in purity. The biflavone compound obtained by the invention can be used as a reference substance, is used for measuring the content of the biflavone compound in the fermentum and counterfeit siamensis siamensis and siamensis siamensis and siamensis siamensis and siamensis siamensis, and effectively identifying the authenticity of a fermentum medicinal material or whether counterfeit replacement exists in a preparation or not, so that the technical blank is filled.

Description

Technical Field

[0001] The invention belongs to the technical field of compound separation, and particularly relates to a method for separating biflavonoid compounds from ironwood flower buds and application thereof. Background Art

[0002] The Uyghur medicinal material ironwood, the dried flower buds of the evergreen tree ironwood Mesuaferrea L. of the genus Ironwood of the family Garcinia, is a traditional medicine used by the Uyghurs. The Uyghur name is Narmisik. It is mainly distributed in India, Bangladesh, Pakistan, Thailand and other places. It is also cultivated in Guangxi and Yunnan, China. It is recorded in many ethnic medicine books and widely used in ethnic preparations. The main chemical components are flavonoids, coumarins, triterpenes, carboxylic acids, etc. It has strong anti-cancer and antioxidant activities. It is mainly used to treat damp-cold or mucous diseases, cold heart deficiency, depression, neurasthenia, cold body impotence, damp stomach deficiency, diarrhea and various wet sores, hemorrhoid bleeding, etc. At present, the phenomenon of plants of the genus Mesuaferrea being used as ironwood medicinal materials is very common. Ironwood counterfeit 1: Mammea siamensis (Miq.) T. Anderson, a plant of the genus Mammea of ​​the family Garcinia, mainly distributed in Thailand, Myanmar, Laos, Cambodia, Vietnam, etc., has anti-proliferation and apoptosis effects, and inhibits the production of nitric oxide. It is used to treat heart disease, clear away heat and detoxify, relieve pain, etc. It can increase appetite and is a substitute for ironwood. Ironwood counterfeit 2: Mammeayunnanensis (HLLi) Kosterm., distributed in southern Yunnan, my country, its nature, flavor, meridians, functions and indications are not clear. However, there are still technical gaps in the determination of the content of active ingredients in ironwood and its counterfeit Mammea plants, and in identifying whether counterfeit products are used in ironwood medicinal materials or preparations.

[0003] Biflavonoids are a subclass of flavonoids and are widely distributed in nature. Their chemical structure is composed of two flavonoid molecules or their derivatives connected by CC or COC. There are many connection sites on the flavonoid molecules. Coupled with the differences in the position and type of the groups, the number of biflavonoids is huge and the types are diverse. Biflavonoids have a wider range of biological activities than monomeric compounds and have higher medicinal value. Natural biflavonoids have a wide range of pharmacological effects, such as anti-inflammatory, antioxidant, anti-infective, anti-tumor, antibacterial, hypoglycemic, antiviral and effects on the nervous system. Rhusflavanone and Rhusflavone are two natural biflavonoids contained in ironwood. Rhusflavanone, with a molecular formula of C 30 H 22 O 10, has anti-toxic effects, can inhibit the secretion activity of Salmonella effector proteins, has strong inhibitory activity against elastase and tyrosinase, but has low scavenging activity against DPPH-free radicals. Rhusflavone, molecular formula C 30 H 20 O 10 , has a competitive inhibitory effect on influenza binding, can cause a shortened sleep latency, and lead to a longer sleep duration. However, there are few studies 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 biflavonoid compounds from the flower buds of the ironwood tree in view of the deficiencies of the above-mentioned prior art, and to use the obtained biflavonoid compounds Rhusflavanone and Rhusflavone as reference substances for determining their contents in the ironwood tree and its counterfeits, the Siamese galangal and galangal trees, and for identifying the authenticity of the ironwood medicinal material and whether counterfeits are used in the preparations, thereby filling the technical gap.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0006] A method for separating biflavonoid compounds from ironwood flower buds comprises the following steps:

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

[0008] S2, dissolving the extract obtained in S1 in 95% ethanol, then adding macroporous resin and stirring to absorb, then concentrating under reduced pressure, drying, and grinding into powder;

[0009] S3, adding the powder obtained in S2 to a chromatography column filled with a macroporous resin, eluting with ethanol solutions with volume fractions of 0%, 10%, 30%, 50%, 70%, and 95% in sequence, combining the collected components through HPLC analysis, and concentrating under pressure to obtain four components: Fr.A, Fr.B, Fr.C, and Fr.D;

[0010] S4, purifying the component Fr.D obtained in S3 by semi-preparative HPLC, collecting the eluate; after detecting the eluate at 300nm, determining to collect the eluate with a retention time of 20.5 to 23.0min, concentrating under reduced pressure and evaporating to dryness, to obtain the biflavonoid compound 1: Rhusflavanone; determining to collect the eluate with a retention time of 38.4 to 40.6min, concentrating under reduced pressure and evaporating to dryness, to obtain the 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 the ironwood flower buds to 95% ethanol in S1 is 1 g: 10-15 mL, each extraction time is 1 hour, the number of extractions is 3, and the extracts are combined.

[0016] Preferably, the mass ratio of the extract in S2 to the macroporous resin 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 for semi-preparative HPLC purification in S4 is octadecylsilane bonded silica gel with specifications of 20 mm×250 mm, 10 μm, a detection wavelength of 300 nm, a column temperature of 25°C, 0.1% glacial acetic acid as mobile phase A, methanol as mobile phase B, a flow rate of 5.0 mL / min, and isocratic elution; the isocratic elution program is: 0-50 min, mobile phase A is 35%, and mobile phase B is 65%.

[0019] The present invention also provides an application of the biflavonoid compound. The biflavonoid compound can be used as a reference substance to determine its content in the flower buds of ironwood, Siamese cycads, and cycads, as well as to identify the authenticity of ironwood medicinal materials or whether there are counterfeit substitutions in preparations.

[0020] The method for determining the content of biflavonoid compounds is as follows: taking reference substances Rhusflavanone and Rhusflavone, dissolving them in 50% methanol respectively, and preparing Rhusflavanone reference substance solution and Rhusflavone reference substance solution; then taking the sample powder to be tested, adding 50% methanol, ultrasonically treating, cooling, filtering with a microporous filter membrane, and taking the filtrate to obtain the sample solution to be tested; then taking the reference substance solution and the sample solution to be tested respectively, and detecting them by high performance liquid chromatography, and calculating the content of biflavonoid compounds Rhusflavanone and Rhusflavone in the sample to be tested according to 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 HPLC conditions are ShimNex WR C18 chromatographic column with specifications of 4.6 mm × 250 mm, 5 μm, a detection wavelength of 300 nm, a column temperature of 25°C, 0.1% glacial acetic acid as mobile phase A, methanol as mobile phase B, a flow rate of 1.0 mL / min, and gradient elution; the gradient elution program is: 0-3 min, mobile phase A is 80%, mobile phase B is 20%; 3-8 min, mobile phase A decreases from 80% to 70%, mobile phase B increases from 20% to 30%; 8-10 min, mobile phase A decreases from 70% to 62%, mobile phase B increases from 30% to 38%; 10-25 min, mobile phase A decreases from 6 From 25 to 33 min, mobile phase A decreases from 60% to 47%, and mobile phase B increases from 40% to 53%; from 33 to 35 min, mobile phase A decreases from 47% to 40%, and mobile phase B increases from 53% to 60%; from 35 to 44 min, mobile phase A decreases from 40% to 30%, and mobile phase B increases from 60% to 70%; from 44 to 44.1 min, mobile phase A increases from 30% to 80%, and mobile phase B decreases from 70% to 20%; from 44.1 to 54 min, mobile phase A is 80%, and mobile phase B is 20%.

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

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

[0025] 2. The biflavonoid compounds obtained by the present invention can be used as reference substances to determine their contents in ironwood, Siamese galangal and galangal, and to identify the authenticity of ironwood medicinal powder and whether there is a problem of counterfeit substitution in the preparation of raw materials, filling the gap in this technology. The identification method is simple, fast and highly accurate, and has great promotion and application value.

[0026] The present invention is further described in detail below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The present invention is a flow chart of separating the biflavonoid compounds Rhusflavanone and Rhusflavone from the flower buds of the ironwood tree;

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

[0029] Figure 3 is the UV absorption spectrum of Rhusflavanone;

[0030] Figure 4 is 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 This is the high-resolution time-of-flight mass spectrum of Rhusflavanone;

[0034] Figure 8 is the UV absorption spectrum of Rhusflavone;

[0035] Fig. 9 is the high performance liquid chromatogram of Rhusflavone;

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

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

[0038] Fig.12This is the high-resolution time-of-flight mass spectrum of Rhusflavone;

[0039] Fig.13 is the HPLC chromatogram of sample 1 (ironwood buds);

[0040] Fig.14 is the HPLC chromatogram of sample 2 (ironwood buds);

[0041] Fig.15 is the HPLC chromatogram of sample 3 (ironwood buds);

[0042] Fig.16 is the HPLC chromatogram of sample 4 (ironwood buds);

[0043] Fig.17 is the HPLC chromatogram of sample 5 (ironwood buds);

[0044] Fig.18 This is the HPLC chromatogram of sample 6 (flower buds of Siamese galangal);

[0045] Fig.19 is the HPLC chromatogram of sample 7 (flower buds of Siamese galangal);

[0046] Fig. 20 is the HPLC chromatogram of sample 8 (flower buds of Siamese galangal);

[0047] Fig.21 is the HPLC chromatogram of sample 9 (flower buds of Siamese galangal);

[0048] Fig. 22 This is the HPLC chromatogram of sample 10 (flower buds of Psoralea corylifolia). DETAILED DESCRIPTION

[0049] The instruments and reagents used in the embodiments of the present invention are:

[0050] Instruments: Shimadzu LC-2010AHT liquid chromatograph (analytical liquid phase converted into semi-preparative liquid chromatograph); Shimadzu LC-20AD XR liquid chromatograph; Shimadzu UV-2600 ultraviolet spectrophotometer; Varian 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 is a method for separating biflavonoid compounds from the flower buds of Tetrapanax ferox, comprising the following steps:

[0054] S1. Take 10 g of dried flower buds of Ironwood, crush them through a No. 4 sieve (60 mesh), and reflux extract them with 100 mL of 95% ethanol for 1 h, extract them 3 times in total, combine the extracts, and concentrate the extracts 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 well for adsorption, use a rotary evaporator to concentrate, use an electric hot air dryer to dry to granules, and grind to powder;

[0056] S3, sprinkle the powder obtained in S2 evenly into a chromatography column filled with D101 macroporous resin, slowly add eluent along the tube wall for elution, the eluents are 0%, 10%, 30%, 50%, 70%, 95% ethanol solutions by volume fraction, merge the collected components by HPLC analysis, and use a rotary evaporator to pressurize and concentrate to obtain 4 components: Fr.A, Fr.B, Fr.C, and Fr.D;

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

[0058] The compound separated in this example was identified. The compound Rhusflavanone was a light yellow powder with a purity of 95.0%. The specific structural identification spectrum is shown in FIG. Figure 3 to Figure 7 As shown, the molecular formula of the product is C 30 H 22 O 10 , the structural formula is:

[0059]

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

[0061] Figure 4 This is the HPLC chromatogram of Rhusflavanone, with a retention time of 39.2-40.0 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 example is a light yellow powder with a purity of 96.0%. The specific structural identification spectrum is as follows: Figure 8 to Figure 12 As shown, the molecular formula of the product is C 30 H 20 O 10 , the structural formula is:

[0066]

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

[0068] Fig. 9 This is the HPLC chromatogram of Rhusflavone, with a retention time of 41.6 to 42.4 min.

[0069] Fig.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] Fig.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] Fig.12 High-resolution time-of-flight mass spectrum of Rhusflavone; ESI-MS m / z: 541.3022 [M+H] + .

[0072] Example 2

[0073] This example is a method for determining the content of Rhusflavanone and Rhusflavone in the dried flower buds of Ironwood and Siamese Caryophyllaceae, and identifying whether the dried flower buds of Caryophyllaceae are mixed, using the biflavonoid compounds Rhusflavanone and Rhusflavone separated in the example as reference substances.

[0074] Specifically:

[0075] 1. Preparation of reference solution

[0076] Rhusflavanone (purity 95.0%) and Rhusflavone (purity 96.0%) reference substances were accurately weighed, and 50% methanol was added to prepare Rhusflavanone reference substance solutions with a concentration of 42.2 μg / mL and Rhusflavone reference substance solutions with a concentration of 47.4 μg / mL, respectively.

[0077] 2. Preparation of sample solution

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

[0079] 3. Determination

[0080] Accurately pipette 10 μL of the reference solution and the sample solution to be tested respectively, and inject into the liquid chromatograph for detection. The chromatographic conditions are as follows:

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

[0082] 4. Results Analysis

[0083] If the chromatographic peak with the same retention time as Rhusflavanone and Rhusflavone reference substances does not appear in the chromatogram of the sample solution to be tested, the sample to be tested does not contain ironwood buds; if the chromatographic peak with the same retention time as Rhusflavanone and Rhusflavone reference substances appears in the chromatogram of the sample solution to be tested, and the content is not less than 0.90 mg / g, combined with the pharmacognosy identification of medicinal materials, it is considered that all the samples to be tested are ironwood buds; if the chromatographic peak with the same retention time as Rhusflavanone and Rhusflavone reference substances appears in the chromatogram of the sample solution to be tested, but the content is between 0-0.90 mg / g, it is considered that not all the samples to be tested are ironwood buds, that is, there is a mixed situation. In the preparation feeding, if there is no positive interference, after examining the process recovery rate and other conditions, this method is also applicable.

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

[0085]

[0086] Example 3

[0087] Five batches of dried flower buds of ironwood (identified as Mesuaferrea L. of the genus Garcinia family), four batches of Mammea siamensis (identified as Mammea siamensis (Miq.) T. Anderson of the genus Garcinia family) and one batch of Mammea yunnanensis (identified as Mammea yunnanensis (HLLi) Kosterm.) from different places were collected for detection, and the method of Example 2 was further verified.

[0088] The results showed that the chromatograms of 5 batches of ironwood flower buds and 4 batches of Siamese strychnos nucifera flower buds showed chromatographic peaks with the same retention time as the Rhusflavanone and Rhusflavone reference substances, but the content of the two substances in ironwood was as low as 0.98 mg / g, with an average of 2.3 mg / g; the highest content in Siamese strychnos nucifera 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 ironwood and powders and extracts of strychnos nucifera plants. However, the chromatogram of 1 batch of strychnos nucifera flower buds did not show chromatographic peaks with the same retention time as the Rhusflavanone and Rhusflavone reference substances. The HPLC chromatograms of samples 1-10 are shown in Figure 1. Figure 13 to Figure 22 As shown, the peak times of Rhusflavanone and Rhusflavone are 39.2-40.0 min and 41.6-42.4 min, respectively.

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

[0090] Table 1 Sample measurement results

[0091]

[0092] The above is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent change made to the above embodiment according to the technical essence of the invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A method for separating biflavonoid compounds from the flower buds of Tetrapanax ferox, characterized in that: The following steps are involved: S1. First, take the dried flower buds of the ironwood tree, crush them, extract them with 95% ethanol by reflux, filter them, and concentrate the extract under reduced pressure to obtain an extract; S2, dissolving the extract obtained in S1 in 95% ethanol, then adding macroporous resin and stirring to absorb, then concentrating under reduced pressure, drying, and grinding into powder; S3, adding the powder obtained in S2 to a chromatography column filled with a macroporous resin, eluting with ethanol solutions with volume fractions of 0%, 10%, 30%, 50%, 70%, and 95% in sequence, combining the collected components through HPLC analysis, and concentrating under pressure to obtain four components: Fr.A, Fr.B, Fr.C, and Fr.D; S4, purifying the component Fr.D obtained in S3 by semi-preparative HPLC, collecting the eluate; after detecting the eluate at 300nm, determining to collect the eluate with a retention time of 20.5 to 23.0min, concentrating under reduced pressure and evaporating to dryness, to obtain the biflavonoid compound 1: Rhusflavanone; determining to collect the eluate with a retention time of 38.4 to 40.6min, concentrating under reduced pressure and evaporating to dryness, to obtain the biflavonoid compound 2: Rhusflavone; The molecular formula of Rhusflavanone 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 the ironwood flower buds to 95% ethanol in S1 is 1 g: 10-15 mL, the extraction time is 1 hour each time, the extraction times are 3 times, and the extracts are combined.

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

5.

4. The method according to claim 1, characterized in that: The macroporous resin in S2 and S3 is D101 macroporous resin.

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

6. An application of the biflavonoid compounds separated by the method of claim 1, characterized in that: The biflavonoid compounds can be used as reference substances to determine their contents in the flower buds of the ironwood, Siamese galangal and galangal trees, and to identify the authenticity of the ironwood medicinal materials or whether there are counterfeit substitutions in the preparations.

7. The use according to claim 6, characterized in that: The method for determining the content of biflavonoid compounds is as follows: taking reference substances Rhusflavanone and Rhusflavone, dissolving them in 50% methanol respectively, and preparing Rhusflavanone reference substance solution and Rhusflavone reference substance solution; then taking the powder of the sample to be tested, adding 50% methanol, ultrasonically treating, cooling, filtering with a microporous filter membrane, and taking the filtrate to obtain the sample solution to be tested; then taking the reference substance solution and the sample solution to be tested respectively, and detecting them by high performance liquid chromatography, and calculating the content of biflavonoid compounds Rhusflavanone and Rhusflavone in the sample to be tested according to the peak area; The method for calculating the content (mg / g) of Rhusflavanone and Rhusflavone in the sample to be tested is:

8. The use according to claim 7, characterized in that: The HPLC conditions are ShimNex WR C18 chromatographic column with specifications of 4.6 mm×250 mm, 5 μm, detection wavelength of 300 nm, column temperature of 25° C., 0.1% glacial acetic acid as mobile phase A, methanol as mobile phase B, flow rate of 1.0 mL / min, and gradient elution; the gradient elution program is: 0-3 min, mobile phase A is 80%, mobile phase B is 20%; 3-8 min, mobile phase A decreases from 80% to 70%, mobile phase B increases from 20% to 30%; 8-10 min, mobile phase A decreases from 70% to 40%, mobile phase B increases from 20% to 30% From 10 to 25 minutes, the mobile phase A decreases from 62% to 60%, and the mobile phase B increases from 38% to 40%; from 25 to 33 minutes, the mobile phase A decreases from 60% to 47%, and the mobile phase B increases from 40% to 53%; from 33 to 35 minutes, the mobile phase A decreases from 47% to 40%, and the mobile phase B increases from 53% to 60%; from 35 to 44 minutes, the mobile phase A decreases from 40% to 30%, and the mobile phase B increases from 60% to 70%; 44-44.1min, mobile phase A increased from 30% to 80%, and mobile phase B decreased from 70% to 20%; 44.1-54min, mobile phase A was 80%, mobile phase B was 20%.

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