Method for preparing high-purity C-glycoside flavone monomer by utilizing secondary reversed-phase semi-preparative chromatography

Abstract

The invention discloses a method for preparing a high-purity C-glycoside flavone monomer by using secondary reversed-phase semi-preparative chromatography, which comprises the following steps of: performing primary elution on bamboo leaf flavone by using a C18 chromatographic column under the conditions of set column temperature, mobile phase system and elution mode, and monitoring and collecting a primary purified product of a peak corresponding to a target component; carrying out reduced pressure concentration on products of isoorientin and orientin corresponding peaks in the first purification product to remove the solvent, redissolving residues by using an organic solvent II, carrying out second elution on a sample solution obtained by filtering through a microfiltration membrane by using a C18 chromatographic column in a set column temperature, a mobile phase system and an elution mode, and collecting eluent; monitoring and collecting a secondary purified product of a peak corresponding to the target component, and performing vacuum concentration and freeze drying. The bamboo leaf flavonoid product obtained by the method has the purity of not less than 98%, the raw material utilization rate is obviously improved, the separation efficiency is high, and the purification period is greatly shortened.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of purification of bamboo leaf flavonoids, and particularly relates to a method for preparing high-purity carbon glycoside flavonoid monomers by secondary reverse phase semi-preparative chromatography. BACKGROUND

[0002] At present, the purification methods of bamboo leaf flavonoids mainly include macroporous resin adsorption, column chromatography, ultrafiltration membrane method and solvent extraction method. Among them, the macroporous resin adsorption (such as AB-8, D101 resin) is a widely used preliminary purification technology in industrialization, but it is limited by the adsorption selectivity and the resolution efficiency, and the purity of the purified product can only reach 70%-80%, and the residual polysaccharides, proteins, pigments and other impurities are difficult to completely remove, which cannot meet the quality requirements of medical grade raw materials; the traditional column chromatography (silica gel column, polyamide column) has complex operation, long time consumption, and problems such as large adsorption loss and large solvent consumption, and has low feasibility for large-scale production; the ultrafiltration membrane method is faced with the bottleneck of serious membrane pollution and limited purity improvement (the highest is only about 44%), which is difficult to realize high-purity refining. Although the high-end technologies such as supercritical fluid extraction can improve the purity, the equipment investment is large and the energy consumption is high, which is difficult to realize industrialization and popularization. Semi-preparative high performance liquid chromatography (Semi-preparative HPLC) as a purification technology between analytical and production chromatography, with its high separation degree, high selectivity and relatively moderate processing capacity, has become the preferred method for preparing high-purity natural products of gram to dozens of grams.

[0003] In the prior art, there is a lack of efficient and stable deep purification process for the preliminary purified bamboo leaf flavonoids with a purity of 70%-80%, which leads to a shortage of high-purity (≥99%) medical grade bamboo leaf flavonoids, and seriously restricts the large-scale application of bamboo leaf flavonoids in the medical field.

[0004] Therefore, it is necessary to develop a semi-preparative chromatography purification technology which can break through the limitations of the prior art and realize the efficient conversion of bamboo leaf flavonoids from 70%-80% purity to 99% medical grade purity, and has important industrialization value and clinical application significance. SUMMARY

[0005] This section aims to summarize some aspects of the embodiments of the present application and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the abstract and title of the specification to avoid obscuring the purpose of this section, abstract and title, and such simplifications or omissions cannot be used to limit the scope of the present application.

[0006] In view of the above and / or problems existing in the prior art, the present application is proposed.

[0007] Therefore, the present application aims to overcome the deficiencies in the prior art and provide a method for preparing high-purity carbon glycoside flavone monomers by using secondary reverse-phase semi-preparative chromatography.

[0008] To solve the above technical problems, the present application provides the following technical solutions: comprising, The crude bamboo leaf flavone is prepared into a sample solution with an organic solvent I, the sample solution filtered through a microporous filter is injected into a semi-preparative liquid chromatography system, a C18 chromatographic column is used for first elution under the conditions of set column temperature, mobile phase system and elution mode, and the first purification product of the target components isoswertisin, swertisin, and vitexin, and isovitexin corresponding peaks is monitored and collected; The product of the isoswertisin and swertisin corresponding peaks in the first purification product is concentrated under reduced pressure to remove the solvent, the residue is redissolved with an organic solvent II, the sample solution filtered through a microporous filter is injected into a semi-preparative liquid chromatography system, a C18 chromatographic column is used for second elution under the conditions of set column temperature, mobile phase system and elution mode, the second purification product of the target components corresponding peaks is monitored and collected, and high-purity carbon glycoside flavone monomers can be obtained by concentration under reduced pressure and freeze-drying.

[0009] As a preferred scheme of the method for preparing high-purity carbon glycoside flavone monomers by using secondary reverse-phase semi-preparative chromatography, the crude bamboo leaf flavone is prepared into a sample solution with an organic solvent I, wherein the organic solvent I is an acetonitrile aqueous solution, the concentration of the acetonitrile aqueous solution is 28-32 vol%, and the concentration of the sample solution is 1.8-2.2 mg / mL.

[0010] As a preferred scheme of the method for preparing high-purity carbon glycoside flavone monomers by using secondary reverse-phase semi-preparative chromatography, the C18 chromatographic column is used for first elution under the conditions of set column temperature, mobile phase system and elution mode, wherein the mobile phase system is composed of phase A and phase B, phase A is acetonitrile, phase B is trifluoroacetic acid aqueous solution, and the concentration of the trifluoroacetic acid aqueous solution is 0.08-0.12 wt%. The elution mode is isocratic elution mode, and when elution, phase A accounts for 18-22 vol% of the mobile phase system; the flow rate of the mobile phase system is 9-11 mL / min.

[0011] As a preferred scheme of the method for preparing high-purity carbon glycoside flavone monomers by using secondary reverse-phase semi-preparative chromatography, the duration of the first elution is 15-19 min.

[0012] As a preferred scheme of the method for preparing high-purity carbon glycoside flavone monomers by using secondary reverse-phase semi-preparative chromatography, the residue is redissolved with an organic solvent II, wherein the organic solvent II is a methanol aqueous solution, and the concentration of the methanol aqueous solution is 48-52 vol%.

[0013] As a preferred scheme of the method for preparing high-purity carbon glycoside flavone monomer by secondary reversed-phase semi-preparative chromatography, the residue is the product of the peak corresponding to isoheteroside or the product of the peaks corresponding to isoheteroside and heteroside.

[0014] As a preferred scheme of the method for preparing high-purity carbon glycoside flavone monomer by secondary reversed-phase semi-preparative chromatography, the second elution is performed by using a C18 chromatographic column under a set column temperature, a mobile phase system and an elution mode, wherein the mobile phase system is composed of phase A and phase B, phase A is methanol, and phase B is trifluoroacetic acid aqueous solution, and the concentration of the trifluoroacetic acid aqueous solution is 0.08-0.12 wt%. The elution mode is gradient elution mode, at the beginning of elution, phase A accounts for 28-32% of the mobile phase system, at the end of elution, phase A accounts for 98-100% of the mobile phase system, and the flow rate of the mobile phase system is 9-11 mL / min.

[0015] As a preferred scheme of the method for preparing high-purity carbon glycoside flavone monomer by secondary reversed-phase semi-preparative chromatography, the duration of the second elution is 23-27 min.

[0016] As a preferred scheme of the method for preparing high-purity carbon glycoside flavone monomer by secondary reversed-phase semi-preparative chromatography, the C18 chromatographic column has a column length of 240-260 mm, an inner diameter of 18-20 mm, and a particle size of 9-11 μm.

[0017] Another object of the present application is to provide high-purity carbon glycoside flavone monomer obtained by the method for preparing high-purity carbon glycoside flavone monomer by secondary reversed-phase semi-preparative chromatography.

[0018] The present application has the following advantages: (1) The purity of the bamboo leaf flavone product prepared by the present application is ≥98%, which can be used as a pharmaceutical intermediate, an injection auxiliary material and other medical-grade raw materials, breaking through the limitations of traditional food-grade (purity of 24-50%) and health product-grade (purity of 70-80%); (2) The raw material utilization rate is greatly improved: in view of the common phenomenon of precipitation during the concentration process of peak No. 1 (isoheteroside), centrifugal separation, purity detection and retention are adopted, which avoids the loss caused by discarding or repeatedly purifying high-purity precipitates, and the raw material utilization rate is improved by more than 30%, effectively reducing the production cost and reducing waste.

[0019] (3) This invention achieves efficient purification of flavonoids through a two-stage semi-preparative chromatographic purification and multi-dimensional detection and verification design, with high separation efficiency and a significantly shortened purification cycle. Moreover, the two-stage purification process design is simple, requiring no complex equipment modification. High-purity preparation can be achieved simply by optimizing the process combination and precipitation treatment method; the raw materials are readily available and suitable for large-scale production. Attached Figure Description

[0020] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 The following are liquid chromatograms of the purified solution collected in Example 1 of the present invention at various wavelengths, wherein (a) is the spectrum of peak 1 of the purified solution, (b) is the spectrum of peak 2 of the purified solution, and (c) is the spectrum of peak 3 of the purified solution.

[0022] Figure 2 The mass spectra of the purified solution collected in Example 1 of the present invention are shown in (a) and (b) are the spectra of peak 1 of the purified solution, respectively. (c) is the spectra of peak 2 of the purified solution.

[0023] Figure 3 This is the liquid chromatogram of peak 3 of the purified solution prepared in Comparative Example 1 of this invention. Detailed Implementation

[0024] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0025] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0026] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0027] The semi-preparative liquid chromatography used in the present application is a NS4201 binary semi-preparative liquid chromatography system of Jiangsu Hanbang Science and Technology Biology Co., Ltd., and the chromatographic column is a Waters SunFire Prpe C18 OBDTM column with a column length of 250 mm, an inner diameter of 19 mm, and a particle size of 10 μm.

[0028] Example 1 The present embodiment provides a method for preparing high-purity carbon glycoside flavonoid monomers by using secondary reverse phase semi-preparative chromatography, specifically: (1) First semi-preparative chromatography purification: The crude bamboo leaf flavone was prepared into a sample solution of 2 mg / mL with 30% acetonitrile aqueous solution, and the sample solution obtained by filtering through a 0.22 μm microporous filter was injected into a semi-preparative liquid chromatography system. A C18 chromatographic column was used, and the mobile phase system (A phase was acetonitrile, and B phase was 0.1% trifluoroacetic acid aqueous solution, and A phase accounted for 20% of the mobile phase system) was eluted at a flow rate of 10 mL / min under isocratic elution mode at a column temperature of 40°C. The elution time was 17 min, and the target components isoheteroside, heteroside, and vitexin, isovitexin were monitored by a dual-wavelength detector (detector detection wavelength was set to 254 nm and 330 nm, first injection amount was 0.3 mL, and subsequent injection amount was 0.5 mL), and the peak products of the target components isoheteroside, heteroside, and vitexin, isovitexin were collected, i.e. the first purification product; (2) Second semi-preparative chromatography purification: The peak product of isoheteroside and heteroside in the first purification product was concentrated under reduced pressure to remove the solvent, and it was found that the purity of the peak product of isoheteroside was high, so it was centrifuged and separated out. Only the peak product of heteroside was redissolved in 50% methanol aqueous solution, and the sample solution obtained by filtering through a 0.22 μm microporous filter was injected into a semi-preparative liquid chromatography system. A C18 chromatographic column was used, and the mobile phase system (A phase was methanol, and B phase was 0.1% trifluoroacetic acid aqueous solution, and A phase accounted for 30% of the mobile phase system and was increased to 100% in gradient) was eluted at a flow rate of 10 mL / min under gradient elution mode at a column temperature of 40°C. The elution time was 25 min, and the peak product of the target component heteroside was monitored by a dual-wavelength detector (detector detection wavelength was 254 nm and 330 nm, and injection amount was 0.4 mL), and the second purification product was collected. High-purity carbon glycoside flavonoid monomers were obtained by vacuum concentration and freeze-drying.

[0029] The purified products of the peaks of isoheteroside, heteroside, and vitexin, isovitexin collected in Example 1 were detected by HPLC together with a mixed standard sample of bamboo leaf flavone, as shown in Figure 1 , it was confirmed that peak No. 1 of the purified liquid was isoheteroside ( Figure 1 ), peak No. 2 was heteroside ( Figure 1 ), and peak No. 3 was vitexin and isovitexin ( Figure 1c)。

[0030] The mass spectrometry analysis by LC-MS liquid chromatograph-mass spectrometer confirmed that the components of the purified liquid corresponded to isohispidin ( Figure 2 a), hispidin ( Figure 2 b), vitexin and isovitexin ( Figure 2 c), and the purity of each component, wherein the content of isohispidin was 52.8wt%, the content of hispidin was 18.4wt%, and the total content of vitexin and isovitexin was 8.8wt%.

[0031] Comparative Example 1 The difference between this comparative example and Example 1 is that the concentration of the acetonitrile aqueous solution in step (1) is adjusted from 30% to 40vol% and 50vol%, respectively, and the rest of the preparation method is the same as that of Example 1.

[0032] The purified product of the peaks corresponding to isohispidin, hispidin, and vitexin and isovitexin collected in Comparative Example 1 was tested together with the mixed standard of bamboo leaf flavonoids by HPLC, as shown in Figure 3 It can be seen that the concentration of peak No. 3 is low and the signal is weak, and the requirement for flow adaptability is higher. Therefore, it can be seen that the increase of acetonitrile concentration reduces the flow polarity, which is not conducive to the separation and purification of the sample.

[0033] Comparative Example 2 The difference between this comparative example and Example 1 is that the mobile phase system in step (2) is adjusted from A phase: methanol and B phase: 0.1% trifluoroacetic acid aqueous solution to A phase: acetonitrile and B phase: 0.1% trifluoroacetic acid aqueous solution, and the rest of the preparation method is the same as that of Example 1. It is found that the separation degree and purity of the target substance do not reach the ideal state. This is because the selectivity of the mobile phase does not change essentially, which cannot break through the limitation of the first separation, and it is difficult to accurately separate the trace homologous impurities such as flavonoid aglycone isomers which are not completely removed in the first time, resulting in the failure of the purpose of "accurate refining" of secondary purification. And when acetonitrile gradient elution is used, the target peak elution interval is concentrated, which is easy to overlap with trace impurity peaks. In order to ensure the purity, more peak intervals need to be discarded, which further aggravates the loss of recovery rate.

[0034] Comparative Example 3 The difference between this comparative example and Example 1 is that step (2) is omitted and only step (1) is performed. In order to obtain 99% purity of bamboo leaf flavonoids by one-time purification, the A phase is adjusted to 15%-90% and the elution time is 40min, and the injection amount is 0.2mL, which is as follows: The crude product of bamboo leaf flavones was prepared into a sample solution of 2 mg / mL with 30% acetonitrile aqueous solution, and the sample solution filtered through a 0.22 μm microporous filter was injected into a semi-preparative liquid chromatography system, a C18 chromatographic column was used, the column temperature was 40°C, the mobile phase system (A phase was acetonitrile, B phase was 0.1% trifluoroacetic acid aqueous solution, and A phase accounted for 15% of the mobile phase system and was increased to 90% in gradient elution mode) was eluted at a flow rate of 10 mL / min, the elution time was 40 min, and the target components isoheteroside, heteroside, and vitexin, isovitexin were monitored by a dual-wavelength detector (the detector detection wavelength was set to 254 nm and 330 nm, and the injection amount was 0.2 mL), and the products collected in the peaks corresponding to the target components were carbon glycoside flavone monomers.

[0035] It was found after testing that the purity of the results did not reach the target of 99%, and the elution time was prolonged, and the elution cost was increased.

[0036] Comparative Example 4 The difference between this comparative example and Example 1 is that only the flow rate of the first elution and the second elution in steps (1) and (2) is adjusted from 3 mL / min to 5 mL / min, and the rest of the methods are the same as those in Example 1. It was found after testing that the separation effect was poor, which was due to the fact that the elution speed would affect the contact between the eluent and the solute. If the elution speed is too fast, the components in the sample solution of Example 1 cannot be fully distributed between the resin and the eluent, so that the separation degree of the elution peaks of different components is small, thereby reducing the resolution of column chromatography.

[0037] Example 2 This example provides a method for preparing high-purity carbon glycoside flavone monomers by using secondary reverse-phase semi-preparative chromatography, specifically: (1) First semi-preparative chromatography purification: the crude product of bamboo leaf flavones was prepared into a sample solution of 1.8 mg / mL with 28% acetonitrile aqueous solution, and the sample solution filtered through a 0.22 μm microporous filter was injected into a semi-preparative liquid chromatography system, a C18 chromatographic column was used, the column temperature was 40°C, the mobile phase system (A phase was acetonitrile, B phase was 0.12% trifluoroacetic acid aqueous solution, and A phase accounted for 22% of the mobile phase system) was eluted at a flow rate of 11 mL / min in isocratic elution mode, the elution time was 19 min, and the target components isoheteroside, heteroside, and vitexin, isovitexin were monitored by a dual-wavelength detector (the detector detection wavelength was set to 252 nm and 328 nm, the first injection amount was 0.3 mL, and the subsequent injection amount was 0.5 mL), and the products collected in the peaks corresponding to the target components were the first purification products; (2) Second semi-preparative chromatography purification: the product of the first purification was concentrated under reduced pressure to remove the solvent, and the results showed that the purity of the product corresponding to the isohispidin was high, so it was centrifuged and separated out. Only the product corresponding to the hispidin was redissolved with 48% methanol aqueous solution, and the sample liquid obtained by filtering through a 0.22 μm microporous filter was injected into a semi-preparative liquid chromatography system. A C18 chromatographic column was used at a column temperature of 40°C, the mobile phase system (A phase was methanol, B phase was 0.12% trifluoroacetic acid aqueous solution, A phase accounted for 28% of the mobile phase system and was increased to 98% in gradient), the flow rate was 11 mL / min, the gradient elution mode was used, the elution time was 27 min, the target component hispidin corresponding peak product was collected by a dual wavelength detector (detector detection wavelength was 252 nm and 328 nm, injection amount was 0.4 mL), and the second purification product was obtained. The high-purity carbon glycoside flavonoid monomer was obtained by vacuum concentration and freeze-drying.

[0038] Example 3 The present embodiment provides a method for preparing a high-purity carbon glycoside flavonoid monomer by using secondary reverse phase semi-preparative chromatography, specifically: (1) First semi-preparative chromatography purification: the crude bamboo leaf flavone was prepared into a 2.2 mg / mL sample liquid with 32% acetonitrile aqueous solution, and the sample liquid obtained by filtering through a 0.22 μm microporous filter was injected into a semi-preparative liquid chromatography system. A C18 chromatographic column was used at a column temperature of 40°C, the mobile phase system (A phase was acetonitrile, B phase was 0.08% trifluoroacetic acid aqueous solution, A phase accounted for 18% of the mobile phase system), the flow rate was 9 mL / min, the isocratic elution mode was used, the elution time was 15 min, the target components isohispidin, hispidin, and vitexin, isovitexin corresponding peak products were collected by a dual wavelength detector (detector detection wavelength was set to 256 nm and 332 nm, the first injection amount was 0.3 mL, and the subsequent injection amount was 0.5 mL), and the first purification product was obtained. (2) The second semi-preparative chromatography purification: the product of the first purification was concentrated under reduced pressure to remove the solvent, and it was found that the purity of the product corresponding to the isohispidin was high, so it was centrifuged and separated out. Only the product corresponding to the hispidin was redissolved with 52% methanol water solution, and the sample liquid obtained by filtering through a 0.22 μm microporous filter was injected into a semi-preparative liquid chromatography system. A C18 chromatographic column was used, the column temperature was 40 DEG C, the mobile phase system (A phase was methanol, B phase was 0.08% trifluoroacetic acid aqueous solution, A phase accounted for 32% of the mobile phase system, and was increased to 100% in gradient), the flow rate was 9 mL / min, the gradient elution mode was used, the elution time was 23 min, the target component hispidin corresponding peak product was collected by a dual wavelength detector (detector detection wavelength was 256 nm and 332 nm, injection amount was 0.4 mL), and the second purification product was obtained. After being concentrated under reduced pressure and freeze-dried, high-purity carbon glycoside flavone monomer was obtained.

[0039] The purified liquid of test examples 2-3 was found to have a purity equivalent to that of example 1.

[0040] In summary, the present application realizes efficient purification of bamboo leaf flavones through two semi-preparative chromatography purifications and multi-dimensional detection verification. In the first purification, 20% acetonitrile isocratic elution is used to accurately separate isohispidin, hispidin, vitexin and isovitexin components, and the purity is monitored in real time by a Waters high-performance liquid chromatograph (dual wavelength detection: 330 nm, 254 nm). The target fractions collected in the first purification are concentrated under reduced pressure to remove most of the mobile phase solvent, and then redissolved with 50% methanol-water solution. Concentration increases the sample concentration and reduces the injection volume, thereby obtaining a narrower starting band and improving separation efficiency. Selecting 50% methanol-water as the dissolution solvent can match the initial mobile phase conditions of gradient elution, so that the sample can be well captured by the column head when entering the chromatographic column, preventing peak broadening. In the second purification, 30%-100% methanol gradient elution is used to further remove impurities for key target components (isohispidin and hispidin), and high-purity isohispidin precipitate is separately centrifuged due to high concentration. Only the mother liquor is purified twice, which not only avoids waste of high-purity components, but also removes impurities through step-by-step purification. After freeze-drying treatment, the obtained bamboo leaf flavone monomer or enriched component has stable purity, which can meet the stringent requirements of scientific research, medicine or food field for high-purity flavones.

[0041] The first purification uses two Jiangsu Hanbang NS4201 binary semi-preparative liquid chromatography systems to run synchronously, and 220 needles of samples only need about 2000 min (about 33.3 h); the second purification only needs to prepare 140 needles for 80 mL of mother liquor, and the time consumption is about 900 min (15 h), which is nearly 1 times higher than the operation efficiency of a single device. At the same time, by fixing the column temperature (40℃), the flow rate (10 mL / min) and the clear elution program (isocratic + gradient), the increase of separation time consumption caused by parameter fluctuation is avoided, the overall purification period is greatly shortened, and the product output per unit time is improved.

[0042] The present application solves the problem of easy precipitation of the precipitate and mother liquor after the first purification by centrifugal separation, and the high concentration of No. 1 peak (isoheteroside). Through HPLC analysis, it is confirmed that the precipitate with high purity is directly retained, and only the remaining mother liquor is purified again, instead of repeatedly purifying the entire No. 1 peak (isoheteroside) component, which not only reduces unnecessary process steps and solvent consumption, but also avoids the loss of high-purity components caused by repeated processing, significantly improves the utilization rate of raw materials (crude extract of bamboo leaf flavonoids), reduces material waste in the production process, and meets the dual needs of green production and cost control.

[0043] It should be noted that the above examples are only used to illustrate the technical solutions of the present application and are not limiting. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the spirit and scope of the present application, which should be covered by the scope of the claims of the present application.

Claims

1. A method for preparing high purity carbon glycoside flavonoid monomers using semi-preparative chromatography with two reverse phases, characterized by: The bamboo leaf flavone crude product is prepared into a sample solution with an organic solvent I, the sample solution filtered through a microporous filter is injected into a semi-preparative liquid chromatography system, a C18 chromatographic column is used for first elution under a set column temperature, a mobile phase system and an elution mode, a first purification product of target components corresponding to peaks of isohyduloside, hyduloside and vitexin, isohyduloside is monitored and collected, and a second purification product of target components corresponding to peaks is monitored and collected. The product corresponding to the peak of isohyduloside and hyduloside in the first purification product is concentrated under reduced pressure to remove the solvent, the residue is redissolved with an organic solvent II, the sample solution filtered through a microporous filter is injected into a semi-preparative liquid chromatography system, a C18 chromatographic column is used for second elution under a set column temperature, a mobile phase system and an elution mode, the second purification product of target components corresponding to peaks is monitored and collected, and a high-purity carbon glycoside flavone monomer is obtained by concentration under reduced pressure and freeze-drying. In the preparation of the sample solution from the bamboo leaf flavone crude product with the organic solvent I, the organic solvent I is an acetonitrile aqueous solution, the concentration of the acetonitrile aqueous solution is 28-32 vol%, and the concentration of the sample solution is 1.8-2.2 mg / mL.

2. The method for preparing high purity carbon glycoside flavonoid monomers using secondary reverse phase semi-preparative chromatography according to claim 1, characterized in that: In the first elution with the C18 chromatographic column under the set column temperature, the mobile phase system and the elution mode, the mobile phase system is composed of phase A and phase B, phase A is acetonitrile, and phase B is a trifluoroacetic acid aqueous solution, and the concentration of the trifluoroacetic acid aqueous solution is 0.08-0.12 wt%.

3. The method for preparing high purity carbon glycoside flavonoid monomers using semi-preparative chromatography with two reverse phases as claimed in claim 1, characterized by: The elution mode is isocratic elution mode, and when elution, phase A accounts for 18-22 vol% of the mobile phase system; the flow rate of the mobile phase system is 9-11 mL / min. The duration of the first elution is 15-19 min.

4. The method for preparing high purity xanthone monomer by using secondary reverse phase semi-preparative chromatography according to claim 3, characterized in that: In the redissolution of the residue with the organic solvent II, the organic solvent II is a methanol aqueous solution, and the concentration of the methanol aqueous solution is 48-52 vol%.

5. The method for preparing high purity carbon glycoside flavonoid monomers using semi-preparative chromatography with two reverse phases as claimed in claim 1, characterized by: The residue is the product corresponding to the peak of isohyduloside or the product corresponding to the peaks of isohyduloside and hyduloside.

6. The method for preparing high purity carbon glycoside flavonoid monomers using semi-preparative chromatography with two reverse phases as claimed in claim 5, characterized by: In the second elution with the C18 chromatographic column under the set column temperature, the mobile phase system and the elution mode, the mobile phase system is composed of phase A and phase B, phase A is methanol, and phase B is a trifluoroacetic acid aqueous solution, and the concentration of the trifluoroacetic acid aqueous solution is 0.08-0.12 wt%.

7. The method for preparing high purity carbon glycoside flavonoid monomers using semi-preparative chromatography with two reverse phases as claimed in claim 1, characterized by: The elution mode is gradient elution mode, and at the beginning of elution, phase A accounts for 28-32% of the mobile phase system, at the end of elution, phase A accounts for 98-100% of the mobile phase system, and the flow rate of the mobile phase system is 9-11 mL / min. The duration of the second elution is 23-27 min.

8. The method for preparing high purity carbon glycoside flavonoid monomers using semi-preparative chromatography with two reverse phases as claimed in claim 7, characterized by: The length of the C18 chromatographic column is 240-260 mm, the inner diameter is 18-20 mm, and the particle size is 9-11 μm.

9. The method for preparing high purity carbon glycoside flavonoid monomers using semi-preparative chromatography with two reverse phases according to claim 3 or 7, characterized in that:

10. The high-purity carbon glycoside flavone monomer obtained by the method of claim 1. ​

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