Process for purifying aurantio-obtusin and application of aurantio-obtusin

Abstract

The purification process comprises the following steps: (1) crushing; (2) crude extraction; (3) silica gel acidification; (4) silica gel chloropropylation; then carrying out gradient elution by using an eluent to obtain an aurantio-obtusin crude product; and (7) purification and crystallization: purifying the crude product of the aurantio-obtusin, and crystallizing to separate out aurantio-obtusin crystals. According to the present invention, the process steps are simple and easy to perform, the time consumption is short, the solvent consumption is low, the separation purity is high, the yield is high, the purity of the obtained aurantio-obtusin can achieve more than 99.9%, the yield can achieve 73%, the requirements of scientific research on the aurantio-obtusin purity can be met, and the method is suitable for large-scale production.

Description

Technical Field

[0001] This invention relates to the field of extraction of effective substances from traditional Chinese medicine, and in particular to a process for purifying cassia seed extract and its application. Background Technology

[0002] Cassia seed, the dried, mature seed of the legume *Cassia tora* or *Cassia obtusifolia*, is a traditional Chinese medicine with a long history. It was first recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica) and is now listed by the Ministry of Health as one of the medicinal and edible herbs, possessing high medicinal and edible value and widely used in the medical and health food fields. Modern pharmacological research shows that the chemical components of cassia seed are the material basis for its various pharmacological activities (such as clearing heat and improving eyesight, moistening the intestines and relieving constipation, lowering blood pressure, lowering blood lipids, anti-oxidation, liver protection, anti-diabetic effects, and neuroprotection), showing broad application prospects in the treatment of eye diseases, constipation, hypertension, hyperlipidemia, and diabetes.

[0003] Orange-yellow cassia extract is a natural plant extract with the chemical formula C. 17 H 14 O7, a yellow needle-shaped anthraquinone compound, is the exclusive active ingredient of cassia seed. It possesses various pharmacological effects, including anti-inflammatory, antioxidant, anticoagulant, and antihypertensive properties, and has broad application prospects in functional foods and pharmaceuticals. In recent years, with the continuous growth in market demand for natural products, the green and efficient preparation technology of high-purity cassia seed extract has become a research hotspot in the industry.

[0004] Existing extraction and purification technologies for cassia seed extract mainly cover traditional methods such as solvent extraction, column chromatography, and crystallization, as well as novel technologies such as supercritical extraction and microwave-assisted extraction. For example, CN101967090A uses gasoline extraction, ultrasonic extraction with ethanol or methanol solvents, Soxhlet reflux extraction with ethyl acetate, followed by elution with a polyamide chromatography column, and finally recrystallization to obtain cassia aurantium. Although the equipment is simple, it suffers from low purity (98.62%) and long processing time. CN111228325A integrates supercritical CO2 extraction and high-pressure differential low-temperature continuous extraction technologies to achieve efficient extraction of multiple active ingredients from cassia seeds. This technology has significant advantages in extraction efficiency, component retention, and environmental friendliness. However, this invention extracts a mixture of multiple components and suffers from disadvantages such as expensive equipment, high operating costs, and complex processes. CN113214070A uses microwave pretreatment combined with silica gel column chromatography. Although its application in laxative health products is clear, this invention uses 300-mesh silica gel isocratic elution, which has shortcomings in actual operation such as easy sample loss, high solvent consumption, and low separation efficiency.

[0005] Comprehensive analysis shows that existing extraction technologies generally have one or more of the following shortcomings: ① The purity of the final product is difficult to exceed 99.9%; ② High energy consumption and high production costs. Summary of the Invention

[0006] This invention aims to overcome the limitations of existing technologies and provide a silica gel column chromatography method for effectively improving the separation of cassia aurantium, as well as its crystallization and preparation process. This process offers advantages such as high product purity (≥99.9%), simple operation, and low energy consumption.

[0007] The technical solution adopted by this invention to solve its technical problem is: A purification process for cassia seed extract, the steps of which are as follows: (1) Grinding: After drying the cassia seed medicinal material in an oven, grind it into coarse powder and sieve it for later use; (2) Crude extraction: The crude powder was heated and refluxed with 90% ethanol twice, filtered, and the filtrates were combined and the solvent was recovered under reduced pressure to obtain a concentrated solution. (3) Silica gel acidification: Weigh silica gel and add it to hydrochloric acid solution. Stir at room temperature for 8 hours and then filter. Wash the filter cake repeatedly with ultrapure water until the pH of the filtrate is 7. Dry the filter cake at 80℃ for 12 hours to obtain acidified silica gel. (4) Chloropropylation of silica gel: Take acidified silica gel, add anhydrous toluene, and add 3-chloropropyltrimethoxysilane under nitrogen protection. React for 20-30 hours. After the reaction is completed, cool to room temperature, filter, wash, and dry to obtain chloropropyl silica gel. (5) Phosphorylation of silica gel: Chloropropyl silica gel was added to DMF and anhydrous sodium phosphate and reacted under nitrogen protection for 24 h. After filtration and washing, it was treated with hydrochloric acid solution for 4 h, washed with water until neutral, and dried to obtain modified silica gel. (6) Silica gel column chromatography: The concentrate from step (2) was subjected to chromatographic column chromatography with chloropropyl-phosphate bonded silica gel packing; then, gradient elution with elution buffer was used to obtain crude cassia aurantiae. (7) Purification and crystallization: The crude cassia seed extract is purified and crystallized to obtain cassia seed extract crystals.

[0008] Moreover, the heating and reflux treatment in step (2) is as follows: Cassia seed powder and 90% ethanol solution are mixed at a material-to-liquid ratio of 1:6, heated and refluxed twice at 90 °C for 1 hour each time, filtered, and the filtrates are combined and the solvent is recovered under reduced pressure to obtain a concentrated solution for later use.

[0009] Moreover, in step (6), the column diameter is 2 cm and the diameter-to-height ratio is 1:7-1:12.

[0010] Furthermore, the high-performance liquid chromatography parameters in step (6) are: C 18 The chromatographic column was 4.6 mm × 250 mm, 5 μm; the mobile phase was gradient elution with acetonitrile B pump and 0.1% phosphate A pump, and the elution program is shown in Table 1; the flow rate was 1 mL / min; the detection wavelength was 285 nm; the injection volume was 10 μL; and the column temperature was 30 ℃.

[0011] Moreover, the hydrochloric acid concentration in step (3) is 10% by volume.

[0012] Moreover, in step (4), the acidified silica gel is 3-chloropropyltrimethoxysilane in a weight ratio of 2-5:0.5-1.25.

[0013] Moreover, in step (5), the weight ratio of chloropropyl silica gel to sodium phosphate is 2-5:0.5-1.25.

[0014] Furthermore, step (5) is a gradient elution method as follows: eluent A: petroleum ether-acetone = 9:1; eluent B: petroleum ether-acetone = 8:2; eluent C: petroleum ether-acetone = 7:3. The eluents are collected by gradient elution with a volume ratio of A, B, and C of 1:3:3.

[0015] Furthermore, the purification and crystallization method in step (7) is as follows: the crude cassia aurantium is dissolved in a crystallization solvent and heated to 30-80 ℃; when crystals are precipitated by cooling crystallization, the temperature is lowered to room temperature or below 0 ℃ for crystallization; the crystals are vacuum dried at 40-60 ℃ to constant weight to obtain cassia aurantium. The crystallization solvent is selected from one or more of the following solvents: acetone, methanol, ethanol, acetonitrile, ethyl acetate, and chloroform.

[0016] Furthermore, the purification method in step (7) involves separating and purifying the column chromatography product using preparative chromatography, with the following chromatographic parameters: C 18 The chromatographic column was 20 mm × 250 mm, 10 μm; the mobile phase was gradient elution with acetonitrile B pump-0.1% phosphate A pump; the flow rate of the preparative chromatography was 5-20 mL / min; the injection volume of the preparative chromatography was 1-4 mL; and the orange-yellow cassia seed extract was obtained after crystallization.

[0017] The advantages and positive effects of this invention are as follows: This invention utilizes a core process of silica gel activation-acidification-amination to prepare the packing material. The operation is safe and under mild conditions, enabling efficient and selective separation of anthraquinone compounds such as cassia aurantium, with stable product bonding and excellent performance. The use of petroleum ether-acetone gradient elution reduces the possibility of cross-contamination, optimizes separation accuracy, shortens separation time, and reduces solvent consumption. Preparative liquid chromatography (HPLC) is employed for separation and purification, resulting in high resolution, high product purity, and flexible scale-up.

[0018] The process of this invention is relatively simple and easy to implement, with short time consumption, low solvent consumption, high separation purity, and high yield. The purity of the obtained cassia aurantium reaches over 99.9%, and the yield can reach 73%, which can meet the purity requirements of scientific research for cassia aurantium and is suitable for large-scale production. Attached Figure Description

[0019] Figure 1 The standard curve for cassia seed extract.

[0020] Figure 2 This is a liquid chromatogram of the crude extract.

[0021] Figure 3 This is a liquid chromatogram of the column chromatography product.

[0022] Figure 4 This is a liquid chromatogram obtained by recrystallization of the column chromatography product.

[0023] Figure 5 This is a liquid chromatogram obtained after the column chromatography product is purified by preparative liquid chromatography.

[0024] Figure 6 This is a picture of the actual product, Orange Cassia Seed Extract. Detailed Implementation

[0025] The embodiments of the present invention are described in detail below. It should be noted that these embodiments are descriptive and not limiting, and should not be used to limit the scope of protection of the present invention.

[0026] This invention relates to a purification process for cassia aurantium, which uses a special modified silica gel to improve the separation efficiency of silica gel, thereby separating and purifying the weakly acidic substance cassia aurantium from cassia seeds, and obtaining high-purity cassia aurantium by crystallization or preparative liquid chromatography.

[0027] This invention relates to a purification process for cassia seed extract, the steps of which are as follows: (1) Grinding: After drying the cassia seed medicinal material in an oven, grind it into coarse powder and sieve it for later use; (2) Crude extraction: The crude powder was heated and refluxed with 90% ethanol twice, filtered, and the filtrates were combined and the solvent was recovered under reduced pressure to obtain a concentrated solution. (3) Silica gel acidification: Weigh silica gel and add it to 10% hydrochloric acid solution. Stir at room temperature for 8 hours and then filter. Wash the filter cake repeatedly with ultrapure water until the pH of the filtrate is 7. Dry the filter cake at 80℃ for 12 hours to obtain acidified silica gel. (4) Chloropropylation of silica gel: Take 20g of acidified silica gel, add 100 mL of anhydrous toluene, add 5 mL of 3-chloropropyltrimethoxysilane under nitrogen protection, reflux at 80 °C for 24 h, filter, wash the filter cake with anhydrous toluene and methanol, and dry the filter cake to obtain chloropropyl silica gel. (5) Phosphorylation of silica gel: 20 g of chloropropyl silica gel was added to 100 mL of DMF and 5 g of anhydrous sodium phosphate and reacted under nitrogen protection for 24 h. After filtration and washing, 0.1 M hydrochloric acid solution was added and treated for 4 h. After washing with water until neutral, the modified silica gel was obtained. (6) Silica gel column chromatography: Prepare 50-100 mL eluent A (petroleum ether-acetone = 9:1), 100-200 mL eluent B (petroleum ether-acetone = 8:2), and 100-200 mL eluent C (petroleum ether-acetone = 7:3). Weigh 12-20 g of silica gel and pack it into a column using a dry method. Mix the silica gel and the prepared sample at a ratio of 3:1 in a rotary evaporator, dry thoroughly, and load the sample using a dry method. Elute with a gradient of solutions A, B, and C, collect the eluent, and monitor the content of cassia aurantiacus in the eluent by high performance liquid chromatography. Separate the eluent fraction with higher content by rotary evaporation under reduced pressure to recover the organic solvent and obtain the column chromatography product. (7) Purification: Two purification processes were used to purify cassia seed extract; Method 1: The column chromatography product is crystallized by cooling, and the crystals are vacuum dried at 40-60 °C to constant weight to obtain cassia aurantium.

[0028] Method 2: The column chromatography product is separated and purified by preparative chromatography to obtain cassia aurantium.

[0029] The heating and reflux treatment in step (2) is as follows: Cassia seed powder and 90% ethanol solution are mixed at a material-to-liquid ratio of 1:6, heated and refluxed twice at 90 °C for 1 hour each time, filtered, and the filtrates are combined and the solvent is recovered under reduced pressure to obtain a concentrated solution for later use.

[0030] In step (6), the column diameter is 2 cm and the diameter-to-height ratio is 1:7-1:12.

[0031] The high-performance liquid chromatography parameters in step (6) are: C 18 The chromatographic column was 4.6 mm × 250 mm, 5 μm; the mobile phase was gradient elution with acetonitrile B pump and 0.1% phosphate A pump, and the elution program is shown in Table 1; the flow rate was 1 mL / min; the detection wavelength was 285 nm; the injection volume was 10 μL; and the column temperature was 30 ℃.

[0032] In step (7), in method 1, the crude product is dissolved in a crystallization solvent by means of ultrasound or heating, and the heating temperature is 30-80 ℃; when crystals are precipitated by cooling crystallization, the temperature is lowered to room temperature or below 0 ℃; the crystallization solvent is selected from one or more of the following solvents: acetone, methanol, ethanol, acetonitrile, ethyl acetate, chloroform.

[0033] In step (7), the chromatographic parameters prepared in method 2 are: C 18 The chromatographic column was 20 mm × 250 mm, 10 μm; the mobile phase was gradient elution with acetonitrile B pump and 0.1% phosphate A pump, and the elution program is shown in Table 2. The flow rate for preparative chromatography was 5-20 mL / min, and the injection volume for preparative chromatography was 1-4 mL.

[0034] Example 1 A silica gel column chromatography method for cassia aurantium includes the following steps: (1) Grinding: After drying the cassia seed medicinal material in an oven, grind it into coarse powder and sieve it for later use; (2) Crude extraction: The crude powder was heated and refluxed with 90% ethanol twice, filtered, and the filtrates were combined and the solvent was recovered under reduced pressure to obtain a concentrated solution; (3) Acidification of silica gel: Weigh silica gel and add it to 10% hydrochloric acid solution. Stir at room temperature for 8 hours and then filter. Wash repeatedly with ultrapure water until the pH of the filtrate is 7. Dry the filter cake at 80℃ for 12 hours to obtain acidified silica gel. (4) Chloropropylation of silica gel: Take 20g of acidified silica gel, add 100 mL of anhydrous toluene, add 5 mL of 3-chloropropyltrimethoxysilane under nitrogen protection, reflux at 80℃ for 24h, filter, wash the filter cake with anhydrous toluene and methanol, and dry the filter cake to obtain chloropropyl silica gel. (5) Phosphorylation of silica gel: 20 g of chloropropyl silica gel was added to 100 mL of DMF and 5 g of anhydrous sodium phosphate and reacted under nitrogen protection for 24 h. After filtration and washing, 0.1 M hydrochloric acid solution was added and treated for 4 h. After washing with water until neutral, the modified silica gel was obtained. (6) Silica gel column chromatography: Prepare 50 mL eluent A (petroleum ether-acetone = 9:1), 150 mL eluent B (petroleum ether-acetone = 8:2), and 150 mL eluent C (petroleum ether-acetone = 7:3). Weigh 18 g of silica gel and pack it into a column using a dry method. Mix the silica gel with the prepared sample at a ratio of 3:1 in a rotary evaporator, dry thoroughly, and load the sample using a dry method. Elute with a gradient of solutions A, B, and C, collect the eluent, and monitor the content of cassia aurantiacus in the eluent by high performance liquid chromatography (HPLC). The eluent fraction with higher content is then evaporated under reduced pressure to recover the organic solvent and obtain the column chromatography product. The purity is 95.3% as determined by HPLC.

[0035] Example 2 A silica gel column chromatography method for cassia aurantium includes the following steps: (1) Grinding: After drying the cassia seed medicinal material in an oven, grind it into coarse powder and sieve it for later use; (2) Crude extraction: The crude powder was heated and refluxed with 90% ethanol twice, filtered, and the filtrates were combined and the solvent was recovered under reduced pressure to obtain a concentrated solution; (3) Silica gel column chromatography gradient: Prepare 50 mL eluent A (petroleum ether-acetone = 9:1); 150 mL eluent B (petroleum ether-acetone = 8:2); and 100 mL eluent C (petroleum ether-acetone = 7:3). Weigh 18 g of 200-300 mesh silica gel and pack it into the column using a dry method. Mix the silica gel with the prepared sample at a ratio of 3:1 in a rotary evaporator, dry thoroughly, and load the sample using a dry method. Elute with solutions A, B, and C respectively, collect the eluent, monitor the content of cassia aurantium in the eluent by high performance liquid chromatography, and extract the eluent fraction with higher content. After rotary evaporation under reduced pressure to recover the organic solvent, the column chromatography product is obtained. The purity is 93.0% as determined by HPLC.

[0036] Example 3 A silica gel column chromatography method for cassia aurantium includes the following steps: (1) Grinding: After drying the cassia seed medicinal material in an oven, grind it into coarse powder and sieve it for later use; (2) Crude extraction: The crude powder was heated and refluxed with 90% ethanol twice, filtered, and the filtrates were combined and the solvent was recovered under reduced pressure to obtain a concentrated solution; (3) Isocratic Silica Gel Column Chromatography: Prepare 200 mL of eluent (petroleum ether-acetone = 8:2). Weigh 20 g of the modified silica gel from step (5) in Example 1, pack it into a column using a dry method, mix the silica gel and the prepared sample at a ratio of 3:1 in a rotary evaporator, dry thoroughly, and load the sample using a dry method. Elute isocratically with the eluent, collect the eluent, monitor the content of cassia aurantiacus in the eluent by high performance liquid chromatography, and extract the eluent fraction with higher content. After rotary evaporation under reduced pressure to recover the organic solvent, the column chromatography product is obtained. The purity is 94.2% as determined by HPLC.

[0037] Example 4 A purification process for cassia seed extract includes the following steps: (1) Grinding: After drying the cassia seed medicinal material in an oven, grind it into coarse powder and sieve it for later use; (2) Crude extraction: The crude powder was heated and refluxed with 90% ethanol twice, filtered, and the filtrates were combined and the solvent was recovered under reduced pressure to obtain a concentrated solution; (3) Silica gel column chromatography gradient: Prepare 50 mL eluent A: (petroleum ether-acetone = 9:1); 150 mL eluent B: (petroleum ether-acetone = 8:2); 150 mL eluent C: (petroleum ether-acetone = 7:3). Weigh 18 g of the modified silica gel from step (5) in Example 1, pack it into a column using a dry method, mix the silica gel and the prepared sample at a ratio of 3:1 in a rotary evaporator, dry it thoroughly, and load the sample using a dry method. Elute with a gradient of solutions A, B, and C respectively, collect the eluent, monitor the content of cassia aurantiacus in the eluent by high performance liquid chromatography, and extract the eluent fraction with higher content by rotary evaporation under reduced pressure to recover the organic solvent and obtain the column chromatography product; (4) The obtained column chromatography product was dissolved in acetone by heating and then cooled to crystallize, and orange cassia seed with a purity of 99.92% was obtained with a yield of 68.7%.

[0038] Example 5 A purification process for cassia seed extract includes the following steps: (1) Grinding: After drying the cassia seed medicinal material in an oven, grind it into coarse powder and sieve it for later use; (2) Crude extraction: The crude powder was heated and refluxed with 90% ethanol twice, filtered, and the filtrates were combined and the solvent was recovered under reduced pressure to obtain a concentrated solution; (3) Silica gel column chromatography gradient: Prepare 100 mL eluent A: (petroleum ether-acetone = 9:1); 100 mL eluent B: (petroleum ether-acetone = 8:2); 100 mL eluent C: (petroleum ether-acetone = 7:3). Weigh 18 g of the modified silica gel from step (5) in Example 1, pack it into a column using a dry method, mix the silica gel and the prepared sample at a ratio of 3:1 in a rotary evaporator, dry it thoroughly, and load the sample using a dry method. Elute with a gradient of solutions A, B, and C respectively, collect the eluent, monitor the content of cassia aurantiacus in the eluent by high performance liquid chromatography, and extract the eluent fraction with higher content by rotary evaporation under reduced pressure to recover the organic solvent and obtain the column chromatography product; (4) The obtained column chromatography product was dissolved in ethanol by heating and then cooled to crystallize, and orange cassia seed with a purity of 99.16% was obtained, with a yield of 51.5%.

[0039] Example 6 A purification process for cassia seed extract includes the following steps: (1) Grinding: After drying the cassia seed medicinal material in an oven, grind it into coarse powder and sieve it for later use; (2) Crude extraction: The crude powder was heated and refluxed with 90% ethanol twice, filtered, and the filtrates were combined and the solvent was recovered under reduced pressure to obtain a concentrated solution; (3) Silica gel column chromatography gradient: Prepare 100 mL eluent A: (petroleum ether-acetone = 9:1); 100 mL eluent B: (petroleum ether-acetone = 8:2); 100 mL eluent C: (petroleum ether-acetone = 7:3). Weigh 18 g of the modified silica gel from step (5) in Example 1, pack it into a column using a dry method, mix the silica gel and the prepared sample at a ratio of 3:1 in a rotary evaporator, dry it thoroughly, and load the sample using a dry method. Elute with a gradient of solutions A, B, and C respectively, collect the eluent, monitor the content of cassia aurantiacus in the eluent by high performance liquid chromatography, and extract the eluent fraction with higher content by rotary evaporation under reduced pressure to recover the organic solvent and obtain the column chromatography product; (4) Preparative chromatography: The obtained column chromatography product was dissolved in methanol and then separated and purified in a preparative high performance liquid chromatograph. The mobile phase was set as acetonitrile (pump B) - 0.1% phosphoric acid water (pump A) = 40:60 isocratic elution. The sample loading volume was 4.0 mL, the flow rate was set as 20 mL / min, the detection wavelength was 285 nm, and the fraction was collected. (5) The collected fraction was dried by rotary evaporation to obtain Cassia tora extract with a purity of 98.78% and a yield of 69.28%.

[0040] Example 7 A purification process for cassia seed extract includes the following steps: (1) Grinding: After drying the cassia seed medicinal material in an oven, grind it into coarse powder and sieve it for later use; (2) Crude extraction: The crude powder was heated and refluxed with 90% ethanol twice, filtered, and the filtrates were combined and the solvent was recovered under reduced pressure to obtain a concentrated solution; (3) Silica gel column chromatography gradient: Prepare 100 mL eluent A: (petroleum ether-acetone = 9:1); 100 mL eluent B: (petroleum ether-acetone = 8:2); 100 mL eluent C: (petroleum ether-acetone = 7:3). Weigh 18 g of the modified silica gel from step (5) in Example 1, pack it into a column using a dry method, mix the silica gel and the prepared sample at a ratio of 3:1 in a rotary evaporator, dry it thoroughly, and load the sample using a dry method. Elute with a gradient of solutions A, B, and C respectively, collect the eluent, monitor the content of cassia aurantiacus in the eluent by high performance liquid chromatography, and extract the eluent fraction with higher content by rotary evaporation under reduced pressure to recover the organic solvent and obtain the column chromatography product; (4) Preparative chromatography: The obtained column chromatography product was dissolved in methanol and then separated and purified in a preparative high performance liquid chromatograph. The mobile phase was set as acetonitrile (pump B) - 0.1% phosphoric acid water (pump A) gradient elution. The sample loading volume was 2.0 mL, the flow rate was set as 15 mL / min, the detection wavelength was 285 nm, and the fraction was collected. (5) The collected fraction was dried by rotary evaporation to obtain Cassia tora extract with a purity of 99.96% and a yield of 73.7%.

[0041] The main performance indicators of the cassia tora column chromatography product obtained by the method of this invention are as follows: ① It appears as a yellow powder with no turbidity; ②The content of cassia aurantiacus in the crystallized column chromatography product was tested, and the purity was above 99.9%; ③ After preparative chromatography, the content of cassia a-orange in the column chromatography product was tested, and the purity was found to be above 99.95%.

[0042] Table 1. Analytical Liquid Mobile Phase Gradient Settings

[0043] Table 2. Preparative liquid mobile phase gradient settings

[0044] Although embodiments of the invention have been disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, variations, and modifications are possible without departing from the spirit and scope of the invention and the appended claims. Therefore, the scope of the invention is not limited to the contents disclosed in the embodiments.

Claims

1. A purification process for cassia seed extract, characterized in that: The steps are as follows: (1) Grinding: After drying the cassia seed medicinal material in an oven, grind it into coarse powder and sieve it for later use; (2) Crude extraction: The crude powder was heated and refluxed with 90% ethanol twice, filtered, and the filtrates were combined and the solvent was recovered under reduced pressure to obtain a concentrated solution. (3) Silica gel acidification: Weigh silica gel and add it to hydrochloric acid solution. Stir at room temperature for 8 hours and then filter. Wash the filter cake repeatedly with ultrapure water until the pH of the filtrate is 7. Dry the filter cake at 80℃ for 12 hours to obtain acidified silica gel. (4) Chloropropylation of silica gel: Take acidified silica gel, add anhydrous toluene, and add 3-chloropropyltrimethoxysilane under nitrogen protection. React for 20-30 hours. After the reaction is completed, cool to room temperature, filter, wash, and dry to obtain chloropropyl silica gel. (5) Phosphorylation of silica gel: Chloropropyl silica gel was added to DMF and anhydrous sodium phosphate and reacted under nitrogen protection for 24 h. After filtration and washing, it was treated with hydrochloric acid solution for 4 h, washed with water until neutral, and dried to obtain modified silica gel. (6) Silica gel column chromatography: The concentrate from step (2) was subjected to chromatographic column chromatography with chloropropyl-phosphate bonded silica gel packing; then, gradient elution with elution buffer was used to obtain crude cassia aurantiae. (7) Purification and crystallization: The crude cassia seed extract is purified and crystallized to obtain cassia seed extract crystals.

2. The purification process of cassia seed extract according to claim 1, characterized in that: The heating and reflux treatment in step (2) is as follows: Cassia seed powder and 90% ethanol solution are mixed at a material-to-liquid ratio of 1:6, heated and refluxed twice at 90 °C for 1 hour each time, filtered, and the filtrates are combined and the solvent is recovered under reduced pressure to obtain a concentrated solution for later use.

3. The purification process of cassia seed extract according to claim 1, characterized in that: In step (6), the column diameter is 2cm and the diameter-to-height ratio is 1:7-1:

12.

4. The purification process of cassia seed extract according to claim 1, characterized in that: The high-performance liquid chromatography parameters in step (6) are: C 18 The chromatographic column was 4.6 mm × 250 mm, 5 μm; the mobile phase was gradient elution with acetonitrile B pump and 0.1% phosphate A pump, and the elution program is shown in Table 1; the flow rate was 1 mL / min; the detection wavelength was 285 nm; the injection volume was 10 μL; and the column temperature was 30 ℃.

5. The purification process of cassia seed extract according to claim 1, characterized in that: In step (3), the concentration of hydrochloric acid is 10% by volume.

6. The purification process of cassia seed extract according to claim 1, characterized in that: In step (4), the acidified silica gel is 3-chloropropyltrimethoxysilane in a weight ratio of 2-5:0.5-1.

25.

7. The purification process of cassia seed extract according to claim 1, characterized in that: In step (5), the weight ratio of chloropropyl silica gel to sodium phosphate is 2-5:0.5-1.

25.

8. The purification process of cassia seed extract according to claim 1, characterized in that: The gradient elution method in step (5) is as follows: eluent A: petroleum ether-acetone = 9:1; eluent B: petroleum ether-acetone = 8:2; eluent C: petroleum ether-acetone = 7:

3. Eluents are eluted with a gradient of A, B, and C in a volume ratio of 1:3:3, and the eluents are collected.

9. The purification process of cassia seed extract according to claim 1, characterized in that: The purification and crystallization method in step (7) is as follows: the crude cassia aurantium is dissolved in a crystallization solvent and heated to 30-80 ℃; when crystals are precipitated by cooling crystallization, the temperature is lowered to room temperature or below 0 ℃ for crystallization; the crystals are vacuum dried at 40-60 ℃ to constant weight to obtain cassia aurantium. The crystallization solvent is selected from one or more of the following solvents: acetone, methanol, ethanol, acetonitrile, ethyl acetate, and chloroform.

10. The purification process of cassia seed extract according to claim 1, characterized in that: The purification method in step (7) involves separating and purifying the column chromatography product using preparative chromatography. The purification chromatographic parameters are: C 18 The chromatographic column was 20 mm × 250 mm, 10 μm; the mobile phase was gradient elution with acetonitrile B pump-0.1% phosphate A pump; the flow rate of the preparative chromatography was 5-20 mL / min; the injection volume of the preparative chromatography was 1-4 mL; and the orange-yellow cassia seed extract was obtained after crystallization.

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