Preparation method of high-purity acacetin-7-O-glucuronide

Abstract

The invention relates to a preparation method of high-purity acacetin-7-O-glucuronide, which comprises the following steps of: crushing dry branches and leaves of a verbenaceae plant millettian tree, performing reflux extraction by using a 60-80% methanol solution, combining extracting solutions, and concentrating to obtain an extract; carrying out gel column chromatography on the extract, carrying out gradient elution by using a methanol-water system, detecting by using thin-layer chromatography (TLC), collecting eluent containing acacetin-7-O-glucuronide, merging, and concentrating under reduced pressure to obtain crude fraction of acacetin-7-O-glucuronide. And separating and purifying by using preparative high performance liquid chromatography (PHPLC), and detecting each part of collected eluent by using high performance liquid chromatography (HPLC) to obtain the acacetin-7-O-glucuronide component with the purity of more than 98%. And carrying out TLC and HPLC purity detection and content determination on the obtained product, and establishing a quality control method. The process is simple and reasonable in design, high in separation speed, short in production period, high in product purity, controllable in quality and suitable for industrial production.

Description

Technical Field

[0001] This invention relates to the technical field of extracting and separating compounds from plants, specifically to a method for extracting high-purity farnesin-7-O-glucuronide from the traditional Chinese medicine *Gnaphalium affine*. Background Technology

[0002] Chemical reference standards, also known as standard substances, are physical comparisons used in the research, testing, and control of quality standards for traditional Chinese medicine (TCM). The research on chemical reference standards for TCM is a very important part of the standardization research of TCM. TCM chemical reference standards play an extremely important role in the quality evaluation of products, especially in the quality control of drug production, and are the foundation and core of TCM quality control.

[0003] In the future, all products manufactured, whether domestically or internationally, will require high-level quality standards and advanced analytical testing technologies for development and improvement; otherwise, they will lose market share. Product quality standards and testing methods are becoming increasingly important. The international consensus is that medication standards should be "safe, effective, and of controllable quality." Pharmaceutical production should revolve around this central theme, with quality standard control at its core, and chemical reference standards playing a crucial role. However, currently, most traditional Chinese medicine (TCM) materials and their preparations lack clear chemical composition or chemical reference standards, making it impossible to elucidate the chemical basis of their effects and to conduct quality control. This hinders their acceptance by modern society and is a key constraint preventing TCM and natural medicines from entering the international pharmaceutical market. Technical barriers have created difficulties for the development of the TCM industry. Therefore, research on the chemical composition of TCM and the standardization of quality standards are essential for the modernization of TCM. This research is crucial for elucidating the material basis of TCM's effects, formulating production and processing technologies for TCM preparations, and identifying counterfeit and substandard products.

[0004] Farnesin-7-O-glucuronide is a flavonoid chemical component. It is one of the active ingredients in plants such as *Cymbidium goeringii*, *Carthamus tinctorius*, and *Buddleja officinalis*, and is also an indicator component for quality control in many plant and pharmaceutical standards. Currently, there is no corresponding national pharmaceutical standard reference material, and no systematic research on amidaline-7-O-glucuronide as a chemical reference standard for traditional Chinese medicine has been reported domestically or internationally. Referring to the technical requirements of chemical reference standards for traditional Chinese medicine (for content determination), this study investigates amidaline-7-O-glucuronide as a chemical reference standard, establishing a batch extraction process, purity and content analysis methods, and impurity testing methods for amidaline-7-O-glucuronide as a chemical reference standard. This will establish a technical standard for amidaline-7-O-glucuronide as a chemical reference standard, providing a scientific basis and guarantee for its use as a chemical reference standard for traditional Chinese medicine and for quality standard research on medicinal materials and preparations.

[0005] Acaciain-7-O-glucuronide is derived from the plant *Acacia arvense* (family Verbenaceae). Volkameria inermis The active substance isolated from L., according to publicly available literature, includes the extraction and separation process and content determination method of farnesin-7-O-glucuronide, such as: Chinese Patent 1: A method for extracting farnesin-7-O-glucuronide from *Cymbidium ensifolium*; Application Publication No.: CN 120230163 A. Chinese Patent 2: A method for extracting flavonoid components from *Cymbidium ensifolium*; Application Publication No.: CN118108775A. However, the above methods fail to meet the requirement of a purity greater than 98% for traditional Chinese medicine chemical reference standards, and cannot meet the need for high-purity farnesin-7-O-glucuronide chemical reference standards. Furthermore, although they produce farnesin-7-O-glucuronide with relatively high purity, the preparation process involves simultaneous preparation with several other similar compounds, resulting in a later peak elution time for farnesin-7-O-glucuronide and a longer preparation time. In summary, there are no reports on the quality control and evaluation of farnesin-7-O-glucuronide chemical reference standards in the existing technology. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing high-purity farnesin-7-O-glucuronide, which serves as a material basis for identifying the effects of compounds in traditional Chinese medicine and for formulating the production and processing technology of traditional Chinese medicine preparations.

[0007] From the Verbenaceae family plant, the bitter tree Volkameria inermis L. The chemical name, molecular formula, and structural formula of farnesin-7-O-glucuronide, obtained by extraction, separation, purification, and refining from the branches and leaves, are as follows: Chinese name: Acacia-7-O-glucuronide Chemical name: 5-dihydroxy-4′-methoxyflavone-7-O-glucuronide English name: acacetin 7-O-glucuronide Molecular formula: C 22 H 20 O 11 Structural formula:

[0008] The objective of this invention is achieved through the following technical solutions: The preparation method of high-purity farnesin-7-O-glucuronide specifically includes the following steps: 1) Crush the dried branches and leaves of the bitter tree, extract them by reflux with 60%-80% methanol, filter the extract, combine the filtrates, recover the methanol, and obtain the extract. 2) The extract was subjected to gel column chromatography, and the fraction containing farnesin-7-O-glucuronide was collected by gradient elution with a methanol-water system. The gradient elution conditions of the methanol-water system were as follows: elution with a 5% (v / v) aqueous methanol solution for 1-2 column volumes; elution with a 10%-20% (v / v) aqueous methanol solution for 3-5 column volumes; and elution with a 30%-50% (v / v) aqueous methanol solution for 3-5 column volumes. 3) The fractions were detected and combined by thin-layer chromatography (TLC), concentrated, and the crude fraction of farnesin-7-O-glucuronide was obtained. 4) The crude fraction of farnesin-7-O-glucuronide was separated and purified by preparative high performance liquid chromatography (PHPLC), and the farnesin-7-O-glucuronide fraction was collected. 5) Each eluent collected was analyzed by high performance liquid chromatography (HPLC) on a C-18 column. The acacia-7-O-glucuronide with the same retention time and a purity greater than 98% was combined and concentrated under reduced pressure to obtain acacia-7-O-glucuronide with a purity greater than 98%.

[0009] Preferably, in step 1), the amount of methanol added is 10-20 times the weight of the medicinal material, the methanol concentration is 70% by volume, and the reflux extraction is performed 3-5 times.

[0010] The TLC detection method in step 3) is as follows: the thin-layer plate is made of silicone GF. 254 The developing solvent system is one of the following three: 1) A solution prepared by mixing n-butanol, acetic acid, and water in a volume ratio of 4:1:5, allowing it to stand, separating the layers, and taking the upper layer; 2) A solution prepared by mixing chloroform, acetone, methanol, and acetic acid in a volume ratio of 5:1:1:1; 3) A solution prepared by mixing chloroform, ethyl acetate, methanol, and acetic acid in a volume ratio of 3:1:1:1; The spotting method is as follows: dissolve the analyte in methanol to prepare a test solution with a concentration of 100 μg / mL, and spot the solution on the same silica gel GF. 254 Different volumes of the test solution were spotted onto the plate, resulting in spotting amounts of 2 μg, 4 μg, 6 μg, 8 μg, and 10 μg, respectively. The development and inspection method was as follows: the spotted thin-layer plate was placed in a developing tank containing the selected developing solvent system, with a development distance of 8 cm. The plate was then removed, dried, and examined under a 254 nm UV lamp. (e.g.) Figure 2 (As shown) In step 4) of PHPLC, a C-18 column is used, the mobile phase is acetonitrile and an aqueous solution containing 0.1% (v / v) formic acid, the flow rate is 5-10 mL / min, the detection wavelength is 330 nm, and the column temperature is 25-35℃.

[0011] Preferably, in step 4), the mobile phase is an aqueous solution containing acetonitrile and 0.1% (v / v) formic acid in a volume ratio of 15:85 to 20:80.

[0012] The HPLC detection method in step 5) is as follows: Chromatographic conditions: column C-18, 4.6×250 mm, 5 μm; flow rate: 0.8-1.2 ml / min; injection volume: 10-20 μl; quantification using the main component self-comparison method and area normalization method; system conditions are one of the following two conditions: System conditions (1): The mobile phase is a mixed solvent consisting of methanol and a 0.1% formic acid aqueous solution in a volume ratio of 60:40, and the detection wavelength is 270 nm to 330 nm; System conditions (2): The mobile phase is a mixed solvent consisting of acetonitrile and a 0.1% formic acid aqueous solution in a volume ratio of 30:70, and the detection wavelength is 270 nm to 330 nm.

[0013] Both conditions can be used as the mobile phase. The peak time for condition (1) is 17.936 minutes, and the peak time for condition (2) is 18.429 minutes.

[0014] The present invention has the following advantages: 1. The method of the present invention is reasonably designed and the process is simple. It uses alcohol-water solvent extraction, and after one gel column chromatography, it can be prepared by PHPLC to obtain a chemical reference standard with a purity of more than 98%. The method is simple and easy to implement.

[0015] 2. The method of this invention has a fast separation speed and uses gel column chromatography to effectively remove impurities and interference, which shortens the preparation time. It can complete the preparation of 2-3 g of farnesin-7-O-glucuronide with a purity of over 98% in 1 day. The production cycle is short, suitable for industrial production, and has good application prospects.

[0016] 3. This invention uses TLC and HPLC for purity inspection, content determination and quality control to ensure product quality.

[0017] This study investigates the chemical reference standard of farnesin-7-O-glucuronide, establishing a batch extraction process, purity and content determination method, and impurity testing method for this standard. This research aims to establish a technical standard for farnesin-7-O-glucuronide, providing a scientific basis and guarantee for its use as a chemical reference standard for traditional Chinese medicine and for quality standard research on medicinal materials and preparations. The research results will provide a more complete basic chemical basis for farnesin-7-O-glucuronide, and mastering its chemical information and analytical testing techniques will facilitate the further development and utilization of related products. Furthermore, it will generate potential and immeasurable social and economic benefits for developing products unique to my country, developing high-tech, high-value-added products, and improving market competitiveness.

[0018] 4. This invention prepares a farnesin-7-O-glucuronide with a purity of over 98% from the branches and leaves of the tree of *Aristolochia debilis*, which meets the requirements of chemical reference standards. This solves the supply problem of farnesin-7-O-glucuronide chemical reference standards and provides a scientific basis and guarantee for the quality control of *Aristolochia debilis* and other medicines containing farnesin-7-O-glucuronide.

[0019] 5. Farnesin-7-O-glucuronide, as a chemical reference standard for plants, medicinal materials, and their products, is a key technology for quality control. Numerous enterprises, research institutions, and testing departments require high-purity reference standards, resulting in a large market demand. However, due to the low content of farnesin-7-O-glucuronide in medicinal materials, its extraction and separation technology is highly demanding and challenging. This invention focuses on the preparation and quality control technology of farnesin-7-O-glucuronide as a chemical standard for traditional Chinese medicine. It has produced farnesin-7-O-glucuronide with a purity of up to 100.0%, solving the problem of high-purity farnesin-7-O-glucuronide chemical reference standards. Its role in the modernization of traditional Chinese medicine is evident, possessing significant practical and academic value.

[0020] This research aims to study the quality standards of farnesin-7-O-glucuronide, establish standardized analytical testing methods, and formulate high-tech detection indicators and analytical methods for controlling the quality of farnesin-7-O-glucuronide. This will make the research more scientific and standardized, enhance international competitiveness, and create conditions for traditional Chinese medicine to enter the international market. This research has significant practical and academic value. Attached Figure Description

[0022] Figure 1 This is a process flow diagram for the preparation of high-purity farnesin-7-O-glucuronide; Figure 2 The thin-layer chromatograms are obtained from three developing systems for farnesin-7-O-glucuronide. Figure 3 This is a high-performance liquid chromatogram of farnesin-7-O-glucuronide at a wavelength of 330 nm (using the process of Example 3, with a wavelength of 330 nm selected). Figure 4 This is a high-performance liquid chromatogram of farnesin-7-O-glucuronide at a wavelength of 270 nm (using the process of Example 3, with a wavelength of 270 nm selected). Figure 5 This is a high-performance liquid chromatography (HPLC) 3D spectrum of farnesin-7-O-glucuronide; Figure 6 This is the 5-point spectrum of farnesin-7-O-glucuronide; Figure 7 This is the infrared spectrum of farnesin-7-O-glucuronide; Figure 8 This is the ultraviolet absorption spectrum of farnesin-7-O-glucuronide; Figure 9 This is the nuclear magnetic resonance spectrum of farnesin-7-O-glucuronide ( 1 H-NMR spectrum); Figure 10 This is the nuclear magnetic resonance spectrum of farnesin-7-O-glucuronide ( 13 C-NMR spectrum); Figure 11 This is the mass spectrum of farnesin-7-O-glucuronide (negative ion mode). Detailed Implementation

[0023] Unless otherwise stated, the percentage of methanol in this invention is a volume percentage, and v / v represents the volume ratio of the solution; The dried branches and leaves of the bitter gourd tree were collected from Qianjiang Village, Lianzhou Town, Hepu County, Guangxi. The weight ratio of branches to leaves was 7:3, and the moisture content was <10%.

[0024] Example 1

[0025] A method for preparing high-purity farnesin-7-O-glucuronide, the specific steps of which are as follows: 1) After crushing 5 kg of dried branches and leaves of the bitter tree, extract them five times by reflux with 15 times their weight of 65% methanol. Filter the extract, combine the filtrates, recover the methanol, and obtain the extract. 2) The extract was subjected to gel column chromatography with a methanol-water gradient elution system to collect the fraction containing farnesin-7-O-glucuronide. The methanol-water gradient elution conditions were: 5% methanol, elution for 1-2 column volumes; 10%-20% methanol, elution for 3-5 column volumes; 30-50% methanol, elution for 3-5 column volumes. 3) The fractions were analyzed and combined by TLC, and then concentrated to obtain the crude fraction of farnesin-7-O-glucuronide; 4) The crude fraction of farnesin-7-O-glucuronide was separated and purified by PHPLC, and the farnesin-7-O-glucuronide fraction was collected. 5) Each eluent collected was analyzed by HPLC on a C-18 column. Acacia 7-O-glucuronide with the same retention time and a purity greater than 98% was combined and concentrated under reduced pressure.

[0026] The TLC detection method in step 3) is as follows: A farnesin-7-O-glucuronide fraction is prepared by adding methanol to a solution containing 100 μg per 1 ml. The solution is then analyzed on the same silica gel GF... 254 On a thin-layer plate, samples were spotted at concentration gradients of 2 μg, 4 μg, 6 μg, 8 μg, and 10 μg, respectively. The developing solvent was a mixture of n-butanol, acetic acid, and water (upper layer) at a volume ratio of 4:1:5. After development, the plates were removed, dried, and examined under ultraviolet light (254 nm). In the thin-layer chromatography, all five concentration gradient spots resulted in single spots, with no impurity spots observed.

[0027] In step 4), the PHPLC used a C-18 column, a mobile phase of acetonitrile and an aqueous solution containing 0.1% (v / v) formic acid in a ratio of 15:85, a flow rate of 10 mL / min, a detection wavelength of 330 nm, and a column temperature of 25 ℃.

[0028] The HPLC detection method in step 5) is as follows: Chromatographic conditions: column C-18, 4.6×250 mm, 5 μm; flow rate: 1.0 ml / min; injection volume: 20 μl; system conditions: mobile phase is a mixed solvent of methanol and aqueous solution containing 0.1% (v / v) formic acid in a volume ratio of 60:40; detection wavelength: 270 nm; content is calculated by area normalization method, the peak of the main component (farnesin-7-O-glucuronide) shall not be less than 98.0%, and if there are impurity peaks, the sum of the areas of all impurity peaks, excluding the solvent peak, shall not exceed 2.0%.

[0029] After the experiment, farnesin-7-O-glucuronide with a purity of 99.3% was obtained.

[0030] Example 2

[0031] A method for preparing high-purity farnesin-7-O-glucuronide, the specific steps of which are as follows: 1) After crushing 5 kg of dried branches and leaves of the bitter tree, extract them four times by reflux with 10 times their weight of 70% methanol. Filter the extract, combine the filtrates, recover the methanol, and obtain the extract. 2) The extract was subjected to gel column chromatography with a methanol-water gradient elution system to collect the fraction containing farnesin-7-O-glucuronide. The methanol-water gradient elution conditions were: 5% methanol, elution for 1-2 column volumes; 10%-20% methanol, elution for 3-5 column volumes; 30-50% methanol, elution for 3-5 column volumes. 3) The fractions were analyzed and combined by TLC, and then concentrated to obtain the crude fraction of farnesin-7-O-glucuronide; 4) The crude fraction of farnesin-7-O-glucuronide was separated and purified by PHPLC, and the farnesin-7-O-glucuronide fraction was collected. 5) Each eluent collected was analyzed by HPLC on a C-18 column. The acacia-7-O-glucuronide with the same retention time and a purity greater than 98% was combined and concentrated under reduced pressure.

[0032] The TLC detection method in step 3) is as follows: Prepare a solution containing 100 μg / ml of crude farnesin-7-O-glucuronide by adding methanol. Analyze the solution on the same silica gel GF... 254 On a thin-layer plate, samples were spotted at concentration gradients of 2 μg, 4 μg, 6 μg, 8 μg, and 10 μg, respectively. The plate was developed using chloroform-ethyl acetate-methanol-acetic acid (volume ratio 3:1:1:1). After development, the plates were removed, dried, and examined under ultraviolet light (254 nm). In the thin-layer chromatography, all five concentration gradients resulted in single spots, with no impurity spots observed.

[0033] In step 4), the PHPLC used a C-18 column, the mobile phase was acetonitrile and an aqueous solution containing 0.1% (v / v) formic acid in a ratio of 18:82, the flow rate was 8 mL / min, the detection wavelength was 270 nm, and the column temperature was 30 ℃.

[0034] The HPLC detection method in step 5) is as follows: Chromatographic conditions: column C-18, 4.6×250 mm, 5 μm; flow rate: 1.2 ml / min; injection volume: 15 μl; system conditions: mobile phase is a mixed solvent of acetonitrile and 0.1% formic acid aqueous solution in a volume ratio of 25:75; detection wavelength: 330 nm; content is calculated by area normalization method, the peak of the main component (farnesin-7-O-glucuronide) shall not be less than 98.0%, and if there are impurity peaks, the sum of the areas of all impurity peaks, excluding the solvent peak, shall not exceed 2.0%.

[0035] After the experiment, farnesin-7-O-glucuronide with a purity of 98.9% was obtained.

[0036] Example 3

[0037] A method for preparing high-purity farnesin-7-O-glucuronide, the specific steps of which are as follows: 1) Crush 5 kg of dried branches and leaves of the bitter tree and extract them three times by reflux with 10 times their weight of 80% methanol. Filter the extract, combine the filtrates, recover the methanol, and obtain the extract. 2) The extract was subjected to gel column chromatography with a methanol-water gradient elution system to collect the fraction containing farnesin-7-O-glucuronide. The methanol-water gradient elution conditions were: 5% methanol, elution for 1-2 column volumes; 10%-20% methanol, elution for 3-5 column volumes; 30-50% methanol, elution for 3-5 column volumes. 3) The fractions were analyzed and combined by TLC, and then concentrated to obtain the crude fraction of farnesin-7-O-glucuronide; 4) The crude fraction of farnesin-7-O-glucuronide was separated and purified by PHPLC, and the farnesin-7-O-glucuronide fraction was collected. 5) Each eluent collected was analyzed by HPLC on a C-18 column. The acacia-7-O-glucuronide with the same retention time and a purity greater than 98% was combined and concentrated under reduced pressure.

[0038] The TLC detection method in step 3) is as follows: Prepare a solution containing 100 μg / ml of crude farnesin-7-O-glucuronide by adding methanol. Analyze the solution on the same silica gel GF... 254 On a thin-layer plate, samples were spotted at concentration gradients of 2 μg, 4 μg, 6 μg, 8 μg, and 10 μg, respectively. The plate was developed using a chloroform-acetone-methanol-acetic acid volume ratio of 5:1:1:1. After development, the plates were removed, dried, and examined under ultraviolet light (254 nm). In the thin-layer chromatography, all five concentration gradient spots resulted in single spots, with no impurity spots observed.

[0039] In step 4), the PHPLC used a C-18 column, a mobile phase of acetonitrile and an aqueous solution containing 0.1% (v / v) formic acid in a ratio of 20:80, a flow rate of 5 mL / min, a detection wavelength of 330 nm, and a column temperature of 35 ℃.

[0040] The HPLC detection method in step 5) is as follows: Chromatographic conditions: column C-18, 4.6×250 mm, 5 μm; flow rate: 1.0 ml / min; injection volume: 10 μl; system conditions: mobile phase is a mixed solvent of acetonitrile and 0.1% formic acid aqueous solution in a volume ratio of 30:70; detection wavelength is 330 nm; the content is calculated by area normalization method, and the peak of the main component (farnesin-7-O-glucuronide) shall not be less than 98.0%. If there are impurity peaks, the sum of the areas of all impurity peaks, excluding the solvent peak, shall not exceed 2.0%.

[0041] After the experiment, farnesin-7-O-glucuronide with a purity of 100% was obtained.

[0042] Example 4

[0043] A method for preparing high-purity farnesin-7-O-glucuronide, the specific steps of which are as follows: 1) After crushing 5 kg of dried branches and leaves of the bitter tree, extract them by reflux with 75% methanol at 10 times their weight 4 times. Filter the extract, combine the filtrates, recover the methanol, and obtain the extract. 2) The extract was subjected to gel column chromatography with a methanol-water gradient elution system to collect the fraction containing farnesin-7-O-glucuronide. The methanol-water gradient elution conditions were: 5% methanol, elution for 1-2 column volumes; 10%-20% methanol, elution for 3-5 column volumes; 30-50% methanol, elution for 3-5 column volumes. 3) The fractions were analyzed and combined by TLC, and then concentrated to obtain the crude fraction of farnesin-7-O-glucuronide; 4) The crude fraction of farnesin-7-O-glucuronide was separated and purified by PHPLC, and the farnesin-7-O-glucuronide fraction was collected. 5) Each eluent collected was analyzed by HPLC on a C-18 column. Acacia 7-O-glucuronide with the same retention time and a purity greater than 98% was combined and concentrated under reduced pressure.

[0044] The TLC detection method in step 3) is as follows: Prepare a solution containing 100 μg / ml of crude farnesin-7-O-glucuronide by adding methanol. Analyze the solution on the same silica gel GF... 254 On a thin-layer plate, samples were spotted at concentration gradients of 2 μg, 4 μg, 6 μg, 8 μg, and 10 μg, respectively. The plate was developed using chloroform-ethyl acetate-methanol-acetic acid (volume ratio 3:1:1:1). After development, the plates were removed, dried, and examined under ultraviolet light (254 nm). In the thin-layer chromatography, all five concentration gradients resulted in single spots, with no impurity spots observed.

[0045] In step 4), the PHPLC used a C-18 column, and the mobile phase was acetonitrile and an aqueous solution containing 0.1% (v / v) formic acid in a ratio of 15:85. The flow rate was 10 mL / min, the detection wavelength was 330 nm, and the column temperature was 25 ℃.

[0046] The HPLC detection method in step 5) is as follows: Chromatographic conditions: column C-18, 4.6×250 mm, 5 μm; flow rate: 1.0 ml / min; injection volume: 10 μl; system conditions: mobile phase is a mixed solvent of acetonitrile and 0.1% formic acid aqueous solution in a volume ratio of 30:70; detection wavelength: 265 nm; content is calculated by area normalization method, the peak of the main component (farnesin-7-O-glucuronide) shall not be less than 98.0%, and if there are impurity peaks, the sum of the areas of all impurity peaks, excluding the solvent peak, shall not exceed 2.0%.

[0047] After the experiment, farnesin-7-O-glucuronide with a purity of 98.9% was obtained.

[0048] Example 5

[0049] A method for preparing high-purity farnesin-7-O-glucuronide, the specific steps of which are as follows: 1) After crushing 5 kg of dried branches and leaves of the bitter tree, extract them three times by reflux with 20 times their weight of 60% methanol. Filter the extract, combine the filtrates, recover the methanol, and obtain the extract. 2) The extract was subjected to gel column chromatography with a methanol-water gradient elution system to collect the fraction containing farnesin-7-O-glucuronide. The methanol-water gradient elution conditions were: 5% methanol, elution for 1-2 column volumes; 10%-20% methanol, elution for 3-5 column volumes; 30-50% methanol, elution for 3-5 column volumes. 3) The fractions were analyzed and combined by TLC, and then concentrated to obtain the crude fraction of farnesin-7-O-glucuronide; 4) The crude fraction of farnesin-7-O-glucuronide was separated and purified by PHPLC, and the farnesin-7-O-glucuronide fraction was collected. 5) Each eluent collected was analyzed by HPLC on a C-18 column. Acacia 7-O-glucuronide with the same retention time and a purity greater than 98% was combined and concentrated under reduced pressure.

[0050] The TLC detection method in step 3) is as follows: Prepare a solution containing 100 μg / ml of crude farnesin-7-O-glucuronide by adding methanol. Analyze the solution on the same silica gel GF... 254On a thin-layer plate, samples were spotted at concentration gradients of 2 μg, 4 μg, 6 μg, 8 μg, and 10 μg, respectively. The developing solvent was a mixture of n-butanol, acetic acid, and water (upper layer) at a volume ratio of 4:1:5. The plates were developed, removed, dried, and examined under ultraviolet light (254 nm). In the thin-layer chromatography, all five concentration gradient spots resulted in single spots, with no impurity spots observed.

[0051] In step 4), the PHPLC used a C-18 column, and the mobile phase was acetonitrile and an aqueous solution containing 0.1% (v / v) formic acid in a ratio of 19:81. The flow rate was 6 mL / min, the detection wavelength was 330 nm, and the column temperature was 25℃.

[0052] The HPLC detection method in step 5) is as follows: Chromatographic conditions: column C-18, 4.6 × 250 mm, 5 μm; flow rate: 0.8 ml / min; injection volume: 15 μl; system conditions: mobile phase is a mixture of methanol and 0.1% formic acid aqueous solution at a volume ratio of 60:40; detection wavelength: 330 nm; content is calculated by area normalization method, and the main component (farnesin-7-O-glucuronide C) is determined. 17 H 14 The O6 peak shall not be less than 98.0%. If there are impurity peaks, the total area of ​​all impurity peaks, excluding solvent peaks, shall not exceed 2.0%.

[0053] After the experiment, farnesin-7-O-glucuronide with a purity of 99.3% was obtained.

[0054] Example 6

[0055] A method for preparing high-purity farnesin-7-O-glucuronide, the specific steps of which are as follows: 1) After crushing 5 kg of dried branches and leaves of the bitter tree, extract them three times by reflux with 75% methanol at 15 times their weight. Filter the extract, combine the filtrates, recover the methanol, and obtain the extract. 2) The extract was subjected to gel column chromatography with a methanol-water gradient elution system to collect the fraction containing farnesin-7-O-glucuronide. The methanol-water gradient elution conditions were: 5% methanol, elution for 1-2 column volumes; 10%-20% methanol, elution for 3-5 column volumes; 30-50% methanol, elution for 3-5 column volumes. 3) The fractions were analyzed and combined by TLC, and then concentrated to obtain the crude fraction of farnesin-7-O-glucuronide; 4) The crude fraction of farnesin-7-O-glucuronide was separated and purified by PHPLC, and the farnesin-7-O-glucuronide fraction was collected. 5) Each eluent collected was analyzed by HPLC on a C-18 column. Acacia 7-O-glucuronide with the same retention time and a purity greater than 98% was combined and concentrated under reduced pressure.

[0056] The TLC detection method in step 3) is as follows: Prepare a solution containing 100 μg / ml of crude farnesin-7-O-glucuronide by adding methanol. Analyze the solution on the same silica gel GF... 254 On thin-layer plates, samples were spotted at concentration gradients of 2 μg, 4 μg, 6 μg, 8 μg, and 10 μg, using chloroform-ethyl acetate-methanol-acetic acid in a volume ratio of 3:1:1:1 as the developing solvent. The plates were developed, removed, dried, and examined under ultraviolet light (254 nm). In the thin-layer chromatography, all five concentration gradients with the three developing solvent systems produced single spots, with no impurity spots observed.

[0057] In step 4), the PHPLC used a C-18 column, and the mobile phase was acetonitrile and an aqueous solution containing 0.1% (v / v) formic acid in a ratio of 20:80. The flow rate was 5 mL / min, the detection wavelength was 300 nm, and the column temperature was 25℃.

[0058] The HPLC detection method in step 5) is as follows: Chromatographic conditions: column C-18, 4.6×250mm, 5 μm; flow rate: 1.2 ml / min; injection volume: 20 μl; system conditions: mobile phase is a mixture of acetonitrile and 0.1% formic acid aqueous solution at a volume ratio of 30:70; detection wavelength: 325 nm; content is calculated by area normalization method, and the main component (farnesin-7-O-glucuronide C) is determined. 17 H 14 The O6 peak shall not be less than 98.0%. If there are impurity peaks, the total area of ​​all impurity peaks, excluding solvent peaks, shall not exceed 2.0%.

[0059] After the experiment, farnesin-7-O-glucuronide with a purity of 98.8% was obtained.

[0060] Content and purity determination

[0061] Accurately weigh an appropriate amount of the reference standard (prepared in Example 3) dried to constant weight at 105 °C, add 60% methanol solution to prepare a solution containing 100 μg per ml, and inject 20 μl (approximately equivalent to 2 μg) into the liquid chromatograph under the assay conditions. Use three mobile phase solvent systems: System ①: methanol-0.1% formic acid water (60:40), detection wavelength 330 nm; System ②: acetonitrile-0.1% formic acid water (30:70), detection wavelength 330 nm; System ③: acetonitrile-0.1% formic acid water (30:70), detection wavelength 270 nm. Chromatograms were recorded up to 2.5 times the retention time of the main component peak. The content was calculated using the area normalization method and the main component self-comparison method. The results showed that the content of the reference standard was above 98% in all systems. For impurity testing, the total area of ​​impurity peaks (excluding solvent peaks) was less than 2.0% in chromatograms recorded in different systems. The results are shown in Table 1. The chromatographic purity of the farnesin-7-O-glucuronide candidate chemical reference standard was determined to be 100 (n=3) using the self-comparison method, and the total impurity content was below 2.0%.

[0062]

[0063] Peak purity detection: Take an appropriate amount of the reference standard and, according to the mobile phase system, perform peak purity checks on a high-performance liquid chromatograph using a diode array (DAD) detector. The HPLC chromatographic peak of farnesin-7-O-glucuronide was >98%, and its chromatogram was a single peak. The three-dimensional spectrum and the 5-point spectrum completely overlapped, indicating that it was a single pure substance peak. Results are shown below. Figure 3 , Figure 4 , Figure 5 , Figure 6 .

[0064] HPLC chromatographic analysis methodology investigation

[0065] Chromatographic conditions A Waters SunFire C18 column was used, with acetonitrile-0.1% formic acid water (30:70) as the mobile phase, a detection wavelength of 330 nm, an injection volume of 20 μl, and a flow rate of 1.0 mL / min.

[0066] linear relationship Acaciatin-7-O-glucuronide candidate chemical reference standard (the product prepared in Example 3) was dried at 105℃ to constant weight. Approximately 10 mg was accurately weighed and placed in a 10 mL volumetric flask. Methanol was added to the mark, and the solution was shaken well to prepare a reference standard stock solution with a concentration of 970 μg / mL. 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, and 6 mL of the reference standard stock solution were respectively placed in 10 mL volumetric flasks, diluted with methanol, and shaken well. The solutions were injected into the liquid chromatograph under the chromatographic conditions described above, and the peak areas were measured. A standard curve was plotted with peak area as the ordinate and injection concentration as the abscissa. The regression equation was calculated as Y = 47.08236X - 1,553.36395, R = 0.99990. The acaciatin-7-O-glucuronide candidate chemical reference standard showed good linearity in the injection volume range of 97–582 μg.

[0067] Reproducibility, stability and precision Six samples of the same test sample (the product prepared in Example 3) were taken and measured according to the above method and chromatographic system. The chromatograms and peak area integral values ​​of acacia-7-O-glucuronide were recorded, and the content was calculated. The average content was 100.0%, and the RSD was 0.14%, indicating that the method has good reproducibility and can be used as a quality control method.

[0068] The same test solution was placed at room temperature for 2, 4, 6, 8, 10, 12, and 24 hours, and then injected for determination according to the method. The chromatogram and peak area integral value of farnesin-7-O-glucuronide were recorded. The results showed that RSD=0.72%, indicating that the test solution had good stability within 24 hours.

[0069] The same test solution was injected six times consecutively, and the chromatogram and peak area of ​​farnesin-7-O-glucuronide were recorded. The result RSD=0.49%, indicating that the instrument precision was good.

[0070] Durability test of the method

[0071] Three chromatographic columns from different manufacturers and brands were used to determine retention time, theoretical plate number, resolution, and impurity separation effect. Under the above analytical conditions, the farnesin-7-O-glucuronide peak achieved baseline separation from other impurity peaks, with a resolution greater than 1.5. When the theoretical plate number calculated based on the farnesin-7-O-glucuronide peak was not less than 5000, the determination requirements were met (see Table 2).

[0072]

[0073] Structural confirmation Physicochemical constants: Farnesin-7-O-glucuronide is a pale yellow granule with a melting point of 225 ℃-226 ℃ and is soluble in DMSO.

[0074] Spectral data identification Infrared absorption spectroscopy (IR) Instrument: Bruker TENSOR 27FTIR The instrument was calibrated and the IR spectrum of polystyrene films was verified in accordance with the provisions of the 2025 edition of the Chinese Pharmacopoeia.

[0075] Sample preparation method: Take an appropriate amount of sample and compress it into a potassium bromide tablet.

[0076] The infrared absorption spectrum of this product is shown below. Figure 7 Structural confirmation Physicochemical constants: Farnesin-7-O-glucuronide is a pale yellow granule with a melting point of 225 ℃-226 ℃ and is soluble in DMSO.

[0077] Spectral data identification Infrared absorption spectroscopy (IR) Instrument: Bruker TENSOR 27FTIR The instrument was calibrated and the IR spectrum of polystyrene films was verified in accordance with the provisions of the 2025 edition of the Chinese Pharmacopoeia.

[0078] Sample preparation method: Take an appropriate amount of sample and compress it into a potassium bromide tablet.

[0079] The infrared absorption spectrum of this product is shown below. Figure 7 .

[0080]

[0081] Analysis: 3388 cm -1 Stretching vibration of -OH; 2910 cm -1 Stretching vibration of :-CH2; 1731 cm -1 : Stretching vibration of COOH-; 1658 cm -1 Stretching vibration of C=O; 1605 cm -1 1497 cm -1 Aromatic ring skeletal vibration; 1246 cm -1 1180cm -1 COC; 1064 cm -1 1034 cm -1 In-plane bending vibration of aromatic ring CH; 836 cm -1 : Out-of-plane bending vibration of aromatic ring CH.

[0082] Ultraviolet absorption spectrum (UV) Instrument: Shimadzu UV-2550 ultraviolet spectrometer (Japan).

[0083] The instrument calibration and verification comply with the provisions of the 2015 edition of the Chinese Pharmacopoeia.

[0084] Solvent: Analytical grade methanol.

[0085] Test solution: Take an appropriate amount of sample and add methanol to prepare a solution containing 20 μg per 1 mL.

[0086] The UV absorption spectrum of this product is shown below. Figure 8

[0087] Analysis: The ultraviolet spectrum shows two absorption bands (band I and band II) in the ranges of 300-400 nm and 250-300 nm. Band II is red-shifted, indicating an increase in oxygen-containing substituents on ring A. There are two maximum absorption peaks at 324 nm and 269 nm. The two peaks are similar in shape and intensity, which is consistent with the UV spectral characteristics of flavonoids in methanol solution.

[0088] Nuclear magnetic resonance spectrum 1 H-NMR nuclear magnetic resonance spectroscopy Instrument: BRUKER AVANCE NEO-600 (Germany) Solvent: DMSO d 6. Internal Standard TMS This product 1 See H-NMR nuclear magnetic resonance spectrum Figure 9 .

[0089]

[0090] 13 C-NMR nuclear magnetic resonance spectroscopy Instrument: BRUKER AVANCE NEO-600 (Germany) Solvent: DMSO d 6. Internal Standard TMS This product 13 The C-NMR spectrum is shown below. Figure 10 .

[0091]

[0092] Mass spectrometry (MS) Instrument: Thermo QE FOCUS liquid chromatography-mass spectrometry system Test conditions: Mobile phase: acetonitrile-0.1% formic acid aqueous solution, 15:85; Ionization method: HESI source; negative ion mode; ionization energy: 30 eV; resolution: 70,000. The mass spectrum of this product is shown below. Figure 11 .

[0093] The measurement data are shown in List 7.

[0094]

[0095] Determination results: The quasi-molecular ion peak of this product is 459.09290 m / z [MH]. - Its MS mass number was determined to be 460, and the molecular formula of farnesin-7-O-glucuronide is C. 22 H 20 O 11 Consistent. The calculated degree of unsaturation is: Ω = (2 × 2² + 2 - 2⁰) / 2 = 13, consistent with the structure of this product. Main fragmentation pathway: The molecular ion peak of this product is m / z 459.09290 [MH]. - The fragment ions number 283.05887 [MH-176] - For the loss of 1 glucuronic acid group, C6H9O6 - The fragment ions are consistent with the structure of farnesin-7-O-glucuronide.

[0096] Based on the combined spectral data, physicochemical constants, and spectral data, this product is consistent with the structure of farnesin-7-O-glucuronide and largely consistent with literature values. Therefore, this product should be farnesin-7-O-glucuronide.

[0097] Results: The chemical reference standard of farnesin-7-O-glucuronide isolated and purified by this invention was confirmed by infrared spectroscopy, ultraviolet spectroscopy, nuclear magnetic resonance, mass spectrometry, and physicochemical detection. TLC analysis at 5 different concentrations using 3 developing systems, and HPLC analysis at 3 different mobile phases and 3 different wavelengths, showed results meeting the requirements for chemical reference standards for the determination of traditional Chinese medicine content, with a content greater than 98%.

[0098] The following is a table comparing the purity of the present invention with that of the prior art:

[0099] As can be seen, the present invention can achieve a purity of 100%, which is unattainable in the prior art.

Claims

1. A method for preparing high-purity farnesin-7-O-glucuronide, characterized in that, Includes the following steps: 1) Crush the dried branches and leaves of the bitter tree, extract them by reflux with 60%-80% methanol, filter the extract, combine the filtrates, recover the methanol, and obtain the extract. 2) The extract was subjected to gel column chromatography with a methanol-water gradient elution system. The fraction containing farnesin-7-O-glucuronide was collected. The methanol-water gradient elution conditions were as follows: elution with a 5% (v / v) methanol aqueous solution for 1-2 column volumes; elution with a 10%-20% (v / v) methanol aqueous solution for 3-5 column volumes; and elution with a 30%-50% (v / v) methanol aqueous solution for 3-5 column volumes. 3) The fractions were detected and combined by thin-layer chromatography, and then concentrated to obtain the crude fraction of farnesin-7-O-glucuronide; 4) The crude fraction of farnesin-7-O-glucuronide was separated and purified by preparative high performance liquid chromatography, and the farnesin-7-O-glucuronide fraction was collected. 5) Each eluent collected was analyzed by high performance liquid chromatography on a C-18 column. The acacia-7-O-glucuronide with the same retention time and a purity greater than 98% was combined and concentrated under reduced pressure to obtain high purity acacia-7-O-glucuronide.

2. The method for preparing high-purity farnesin-7-O-glucuronide as described in claim 1, characterized in that, In step 1), the amount of methanol added is 10-20 times the weight of the medicinal material, the methanol concentration (volume fraction) is 70%, and the reflux extraction is performed 3-5 times.

3. The method for preparing high-purity farnesin-7-O-glucuronide as described in claim 1, characterized in that, The TLC detection method in step 3) is as follows: the thin-layer plate is made of silicone GF. 254 The developing solvent system is one of the following three: 1) Mix n-butanol, acetic acid and water in a volume ratio of 4:1:5, let stand and separate the liquids, and take the upper layer of solution. 2) A solution prepared by mixing chloroform, acetone, methanol and acetic acid in a volume ratio of 5:1:1:1; 3) A solution prepared by mixing chloroform, ethyl acetate, methanol and acetic acid in a volume ratio of 3:1:1:1; The sampling method is as follows: the analyte is dissolved in methanol and prepared into a test solution with a concentration of 100 μg / mL. The solution is then sampled on the same silica gel GF. 254 On the plate, different volumes of the test solution were added to make the sample amounts 2 μg, 4 μg, 6 μg, 8 μg and 10 μg respectively; The development and inspection method is as follows: Place the spotted thin-layer plate in a developing tank containing the selected developing solvent system and develop it at a distance of 8 cm. After removing the thin-layer plate and drying it, place it under a UV lamp with a wavelength of 254 nm for inspection. The results should be: a single fluorescent spot is visible. For the three developing solvent systems and five different concentration gradient spotting, all spots are single spots, and no impurity spots are visible.

4. The method for preparing high-purity farnesin-7-O-glucuronide as described in claim 1, characterized in that, In the preparative high-performance liquid chromatography of step 4), a C-18 column is used, the mobile phase is acetonitrile and an aqueous solution containing 0.1% (v / v) formic acid, the flow rate is 5-10 mL / min, the detection wavelength is 330 nm, and the column temperature is 25-35 ℃.

5. The method for preparing high-purity farnesin-7-O-glucuronide as described in claim 4, characterized in that, The volume ratio of acetonitrile to a 0.1% formic acid aqueous solution in the mobile phase is 15:85 to 20:

80.

6. The method for preparing high-purity farnesin-7-O-glucuronide as described in claim 1, characterized in that, The HPLC detection method in step 5) is as follows: Chromatographic conditions: column C-18, 4.6×250 mm, 5 μm; flow rate: 0.8-1.2 ml / min; injection volume: 10-20 μl; quantification by area normalization; system conditions are one of the following two conditions: Condition (1): Mobile phase methanol-0.1% formic acid water, volume ratio 60:40, detection wavelength 270~330 nm; Condition (2): The mobile phase is acetonitrile-0.1% formic acid water, with a volume ratio of 30:70, and the detection wavelength is 270~330 nm.

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