Efficient preparation method of active ingredients of semen cassiae seeds

Abstract

The invention relates to the technical field of preparation of natural products, in particular to a high-efficiency preparation method of active ingredients of semen cassiae seeds, anthraquinone compounds are purified by adopting linear gradient countercurrent chromatography, and two gradient mobile phases are adopted: n-hexane / ethyl acetate / methanol / water (5: 5: 5: 5, v / v) and (5: 5: 8: 2, v / v); n-hexane / ethyl acetate / methanol / water (5: 5: 5.5: 4.5, v / v) and (5: 5: 7: 3, v / v) are solvent systems for internal circulation separation; n-hexane / ethanol / water (1: 1: 0.2, v / v) is used as a solvent system of an off-line two-dimensional separation mode, and the separated substances are detected and analyzed through high performance liquid chromatography to obtain nine anthraquinone compounds.

Description

Technical Field

[0001] The present invention relates to the technical field of natural product preparation, and specifically, it is an efficient preparation method for the active ingredients of Cassia obtusifolia seeds. Background Art

[0002] The seeds of Cassia obtusifolia L are known as "Jue Ming Zi", which are used in traditional Chinese medicine to lower high blood pressure, relieve constipation and improve eyesight, and are included in the "Chinese Pharmacopoeia". Previous studies have shown that Cassia obtusifolia seeds have various pharmacological activities, such as anti-inflammatory, anti-diabetic, hepatoprotective and neuroprotective properties. Cassia obtusifolia seeds mainly contain fatty acids, polysaccharides, glycosides, anthraquinones and phenylpyrrolidones. Its main components are anthraquinone compounds, which have hepatoprotective, anti-diabetic, relieve non-alcoholic fatty liver and anti-Alzheimer's disease effects. Many active ingredients have been proven to be related to the functions of anthraquinone drugs. Aurantio-obtusin and chrysophanol are two anthraquinone compounds, which are used as index compounds for evaluating the quality of Cassia obtusifolia seeds in the "Chinese Pharmacopoeia". At the same time, researchers have also used various techniques such as preparative RP-HPLC, silica gel column chromatography, macroporous resin column chromatography and Sephadex LH-20 to separate and identify anthraquinone compounds in Cassia obtusifolia seeds. Due to the similar structures and low polarities of anthraquinone compounds, it is challenging to separate anthraquinone compounds from Cassia obtusifolia seeds.

[0003] High-speed counter-current chromatography (HSCCC) is a new type of liquid / liquid partition chromatography technique. Compared with traditional liquid-liquid extraction and solid-phase chromatography methods, HSCCC has the advantages of large capacity, high recovery rate, no irreversible adsorption, low risk of sample denaturation and less solvent consumption. Linear gradient counter-current chromatography has been effectively used to separate substances with high partition coefficient ( K D value) in recent years due to its advantages such as high separation efficiency and less solvent consumption. K D Natural compounds with similar ( Summary of the Invention

[0004] The present invention provides an efficient method for separating anthraquinone compounds from Cassia obtusifolia seeds by combining linear gradient counter-current chromatography with internal circulation, so as to solve the problems mentioned in the background art.

[0005] The technical solution adopted by the present invention to solve its technical problems is as follows: In the first aspect of the present invention, a method for efficiently preparing active ingredients from Cassia obtusifolia seeds is provided. The preparation method is to extract and prepare an anthraquinone crude extract from Cassia obtusifolia seeds, use a biphasic solvent composed of n-hexane, ethyl acetate, methanol, and water as the solvent system, separate and prepare the anthraquinone crude extract by high-speed countercurrent chromatography, and then perform high-performance liquid chromatography analysis on the anthraquinone fractions obtained by countercurrent chromatography separation using a linear gradient elution mode; The preparation method includes the following steps: 1) Crush the Cassia obtusifolia seeds, extract with ethanol, filter the mixture, extract with petroleum ether until colorless, adjust the pH value of the aqueous residue with hydrochloric acid, and extract the acidic aqueous residue with chloroform to obtain an anthraquinone crude extract; 2) Pump the upper phase of the solvent system of n-hexane / ethyl acetate / methanol / water into the HSCCC chromatographic column as the stationary phase, then pump mobile phase A into the chromatographic column. After reaching hydrodynamic equilibrium, the sample solution is injected into the chromatographic column for countercurrent chromatography separation through the injection loop, and the eluent is monitored at a wavelength of 280 nm; 3) Detect the separated anthraquinone fractions by high-performance liquid chromatography, with the mobile phase being acetonitrile (A) and 0.1% formic acid-water (B).

[0006] As an optimization, it is crushed through 20 - 40 meshes, the ethanol is 95% ethanol, the temperature for extraction with petroleum ether is 60 - 90°C, the pH value of the aqueous residue is adjusted to 2 with hydrochloric acid, after ultrasonic acidolysis at 40°C for 20 min, 100 mL of chloroform is used to extract the acidic aqueous residue, the chloroform extracts are combined, neutralized with water, rotary evaporated, concentrated, dried, and stored in a 4°C refrigerator.

[0007] As an optimization, when performing linear gradient countercurrent chromatography separation, the solvent system is n-hexane / ethyl acetate / methanol / water, and its volume ratio is 4 - 6:4 - 6:4 - 6:4 - 6, preferably 5:5:5:5. The lower phase is mobile phase A and the upper phase is the stationary phase; select the lower phase of n-hexane / ethyl acetate / methanol / water as mobile phase B, and its volume ratio is: 5:5:8:2. Mix 5 mL of mobile phase A and 5 mL of the stationary phase with 200 mg of Cassia obtusifolia seed extract.

[0008] As an optimization, for the inner circulation and off-line two-dimensional CCC mode, select n-hexane / ethanol / water with a volume ratio of 1:1:0.2. After reaching equilibrium, separate the lower phase and the upper phase of the biphasic solvent system. The lower phase is the mobile phase and the upper phase is the stationary phase. Dissolve approximately 50 mg of fractions A, B, and C into the corresponding solvent systems with 5 mL of the upper phase and 5 mL of the lower phase.

[0009] As an optimization, the head-tail mode is adopted. The stationary phase is pumped into the HSCCC chromatographic column at a flow rate of 15 - 25 mL / min, the rotation speed is 750 - 850 rpm, the temperature is 20 - 30 °C, and then mobile phase A is pumped into the chromatographic column at a flow rate of 1 - 5 mL / min.

[0010] As an optimization, mobile phases A and B are pumped into the chromatographic column in a gradient manner, and the gradients are respectively set as follows: 0 - 130 min, 100% A; 200 - 215 min, 50% A; 240 - 300 min, 25% A; 320 - 350 min, 0% A.

[0011] As an optimization, during the inner - cycle counter - current chromatography separation process, the linear gradient counter - current chromatography has two modes: "take - out" and "circulation". Initially, the six - way valve is placed in the "take - out" mode. The upper layer (stationary phase) is pumped into the chromatographic column for counter - current chromatography separation at a flow rate of 20 mL / min, the rotation speed is 800 rpm, the temperature is 25 °C, the lower phase (mobile phase) is pumped into the chromatographic column at a set flow rate. After reaching hydrodynamic equilibrium, the sample solution is injected into the chromatographic column for counter - current chromatography separation, and at the same time, the additional six - way valve is adjusted to the "circulation" mode.

[0012] As an optimization, the high - performance liquid chromatography elution adopts the linear gradient elution mode, and the program is as follows: 0 - 10 min, 50 - 56% B; 30 - 32 min, 42.5 - 44% B; 35 - 40 min, 1 - 5% B; 40.5 - 47 min, 50 - 56% B. The monitoring wavelength is 280 - 290 nm, preferably 280 nm, the sample injection volume is 10 µL, and the flow rate is 1.0 - 2 mL / min.

[0013] In the second aspect of the present invention, the active ingredients of cassia seeds obtained by the above - mentioned preparation method are provided. There are a total of nine kinds of the active ingredients of cassia seeds, which are: aurantioobtusin, chrysoobtusin, obtusin, meletin, emodin, rubrofusarin, cassialactone, chrysophanol, and physcion, and the specific structural formulas are as follows: The beneficial effects of the above - mentioned one or more technical solutions are as follows: The above - mentioned technical solution purifies anthraquinone compounds by linear gradient counter - current chromatography, and uses two gradient mobile phases: n - hexane / ethyl acetate / methanol / water (5:5:5:5, v / v) and (5:5:8:2, v / v); n - hexane / ethyl acetate / methanol / water (5:5:5.5:4.5, v / v) and (5:5:7:3, v / v) are the solvent systems for inner - cycle separation; n - hexane / ethanol / water (1:1:0.2, v / v) is the solvent system for the off - line two - dimensional separation mode. The separated substances are detected and analyzed by high - performance liquid chromatography, and 9 kinds of anthraquinone compounds are obtained. Description of the Drawings

[0014] Appendix Figure 1 Chemical structures of the compounds isolated from Cassia obtusifolia seeds.

[0015] Appendix Figure 2 Linear gradient countercurrent chromatography separation diagram of the crude anthraquinone extract of Cassia obtusifolia seeds: Stationary phase: upper phase of n-hexane / ethyl acetate / methanol / water (5:5:5:5, v / v). Mobile phase A: lower phase of n-hexane / ethyl acetate / methanol / water (5:5:5:5, v / v), Mobile phase B: lower phase of n-hexane / ethyl acetate / methanol / water (5:5:8:2, v / v), gradient mode: 0 - 130 min, 100% A; 200 - 215 min, 50% A; 240 - 300 min, 25% A; 320 - 350 min, 0% A. Injection volume 200 mg; detector wavelength: 280 nm; flow rate: 5 mL / min.

[0016] Appendix Figure 3 Separation diagrams of the inner circulation and off-line two-dimensional CCC modes of each component: A ( Figure 2 Peak A in): upper and lower phases with n-hexane / ethyl acetate / methanol / water (5:5:5.5:4.5, v / v) as the stationary and mobile phases; Sf value: 50%; detector wavelength: 280 nm; flow rate: 5 mL / min. B ( Figure 2 Peak B in): upper and lower phases with n-hexane / ethyl acetate / methanol / water (5:5:7:3, v / v) as the stationary and mobile phases; flow rate: 2 mL / min; Sf value: 64.9%; detector wavelength: 280 nm. C ( Figure 2 Peak C in): upper and lower phases with n-hexane / ethanol / water (1:1:0.2, v / v) as the stationary and mobile phases; flow rate: 2 mL / min; Sf value: 65.7%; detector wavelength: 280 nm.

[0017] Appendix Figure 4 High performance liquid chromatography diagrams of each monomer and the crude extract: Chromatographic column: Zafex Supperfex JX-C18 (4.6×250 mm, i.d. 5 μm); mobile phase: acetonitrile (A) and 0.1% formic acid in water (B); gradient elution: 0 - 10 min, 56% B; 30 min, 42.5% B; 40 min, 5% B; 40 - 47 min, 56%; detection wavelength: 280 nm; flow rate: 1.0 mL / min. Detailed Description of the Invention

[0018] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. The components of the embodiments of the present invention usually described and illustrated in the drawings here can be arranged and designed in various different configurations. Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the claimed present invention, but merely represents selected embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the scope of protection of the present invention.

[0019] It should be noted that similar reference numerals and letters denote similar items in the following drawings. Therefore, once an item is defined in one drawing, it does not need to be further defined and explained in subsequent drawings.

[0020] In one embodiment of the present invention, a method for efficiently preparing the active ingredients of Cassia obtusifolia seeds is provided. The preparation method is to extract and prepare an anthraquinone crude extract from Cassia obtusifolia seeds, use a biphasic solvent composed of n-hexane, ethyl acetate, methanol and water as the solvent system, separate and prepare the anthraquinone crude extract by high-speed countercurrent chromatography, and then perform high-performance liquid chromatography analysis on the fractions of anthraquinone separated by countercurrent chromatography using a linear gradient elution mode; The preparation method includes the following steps: 1) Crush the Cassia obtusifolia seeds, extract with ethanol, filter the mixture, extract with petroleum ether until colorless, adjust the pH value of the aqueous residue with hydrochloric acid, and extract the acidic aqueous residue with chloroform to obtain an anthraquinone crude extract; 2) Pump the upper phase of the solvent system of n-hexane / ethyl acetate / methanol / water as the stationary phase into the HSCCC chromatographic column, then pump the mobile phase A into the chromatographic column. After reaching hydrodynamic equilibrium, the sample solution is injected into the chromatographic column for countercurrent chromatography separation through the injection loop, and the eluent is monitored at a wavelength of 280 nm; 3) Detect the separated anthraquinone fractions by high-performance liquid chromatography, and the mobile phase is acetonitrile (A) and 0.1% formic acid-water (B).

[0021] In another specific embodiment of the present invention, it is crushed to pass through 20-40 meshes, the ethanol is 95% ethanol, the temperature for extraction with petroleum ether is 60-90°C, the pH value of the aqueous residue is adjusted to 2 with hydrochloric acid, ultrasonically acid-hydrolyzed at 40°C for 20 min, then 100 mL of chloroform is used to extract the acidic aqueous residue, the chloroform extracts are combined, neutralized with water, rotary evaporated and concentrated to dryness, and stored in a 4°C refrigerator.

[0022] In yet another specific embodiment of the present invention, when performing linear gradient countercurrent chromatography separation, the solvent system is n-hexane / ethyl acetate / methanol / water, and their volume ratio is 4 - 6:4 - 6:4 - 6:4 - 6, preferably 5:5:5:5. The lower phase is mobile phase A, and the upper phase is the stationary phase; the lower phase of n-hexane / ethyl acetate / methanol / water is selected as mobile phase B, and its volume ratio is: 5:5:8:2. 5 mL of mobile phase A and 5 mL of stationary phase are mixed with 200 mg of semen cassiae extract.

[0023] In yet another specific embodiment of the present invention, for the inner circulation and off-line two-dimensional CCC mode, n-hexane / ethanol / water with a volume ratio of 1:1:0.2 is selected. After reaching equilibrium, the lower phase and the upper phase of the biphasic solvent system are separated. The lower phase is the mobile phase, and the upper phase is the stationary phase. Approximately 50 mg of fractions A, B, and C are dissolved into the corresponding solvent system with 5 mL of the upper phase and 5 mL of the lower phase.

[0024] In yet another specific embodiment of the present invention, in the head-to-tail mode, the stationary phase is pumped into the HSCCC chromatographic column at a flow rate of 15 - 25 mL / min, the rotation speed is 750 - 850 rpm, and the temperature is 20 - 30 °C. Then, mobile phase A is pumped into the chromatographic column at a flow rate of 1 - 5 mL / min.

[0025] In yet another specific embodiment of the present invention, mobile phases A and B are pumped into the chromatographic column in a gradient manner, and the gradients are set as follows: 0 - 130 min, 100%A; 200 - 215 min, 50%A; 240 - 300 min, 25%A; 320 - 350 min, 0%A.

[0026] In yet another specific embodiment of the present invention, during the inner circulation countercurrent chromatography separation, linear gradient countercurrent chromatography has two modes: "take-out" and "circulation". Initially, the six-way valve is placed in the "take-out" mode. The upper layer (stationary phase) is pumped into the chromatographic column for countercurrent chromatography separation at a flow rate of 20 mL / min, the rotation speed is 800 rpm, and the temperature is 25 °C. The lower phase (mobile phase) is pumped into the chromatographic column at a set flow rate. After reaching hydrodynamic equilibrium, the sample solution is injected into the chromatographic column for countercurrent chromatography separation, and at the same time, the additional six-way valve is adjusted to the "circulation" mode.

[0027] In yet another specific embodiment of the present invention, high-performance liquid chromatography elution adopts a linear gradient elution mode, and the program is as follows: 0 - 10 min, 50 - 56%B; 30 - 32 min, 42.5 - 44%B; 35 - 40 min, 1 - 5%B; 40.5 - 47 min, 50 - 56%B. The monitoring wavelength is 280 - 290 nm, preferably 280 nm. The sample injection volume is 10 µL, and the flow rate is 1.0 - 2 mL / min.

[0028] In another specific embodiment of the present invention, the active ingredients of cassia seeds obtained by the above preparation method are provided. There are a total of nine kinds of active ingredients in the cassia seeds, namely: aurantio-obtusin, obtusin, obtusifolin, sennidin, emodin, rubrofusarin, cassialactone, chrysophanol, and physcion. The specific structural formulas are as follows: The present invention will be further explained and illustrated below through examples, but it does not constitute a limitation to the present invention. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. Examples

[0029] 1. Materials and methods 1.1 Preparation of crude anthraquinone extract The cassia seeds were crushed to 20 - 40 mesh, and 600 g of the powder was extracted with 95% ethanol (1500 mL, 3 × 50 min). The extracts were mixed, filtered, and dehydrated under reduced pressure at 50°C. The extract was dissolved in 500 mL of water, and petroleum ether (60 - 90°C) was used to repeatedly extract low-polarity compounds until the extract solution was almost colorless. The pH value of the aqueous residue was adjusted to 2 with hydrochloric acid. After ultrasonic acidolysis at 40°C for 20 min, 100 mL of chloroform was used to extract the acidic aqueous residue. The chloroform extracts were combined, neutralized with water, concentrated and dried by rotary evaporation, and stored in a refrigerator at 4°C. Finally, 4.3 g of crude anthraquinone extract was obtained, and the yield was 0.72%.

[0030] 1.2 Determination of partition coefficient 10 mL of solvent was prepared according to different solvent systems, shaken well, and allowed to stand for stratification. After stratification, 2 mL of each of the upper and lower phases was taken into the same test tube, about 5 mg of the sample was added, and it was dissolved by ultrasonic wave and then allowed to stand. After the upper and lower phases were completely separated, about 0.8 mL of the solution was taken from each phase into a test tube, and the test tube was placed in a centrifugal concentrator for concentration until the solvent was completely volatilized. After it was completely dried, an equal amount of methanol solution was added to the upper and lower phases for dissolution. The sample solution was filtered through a 0.22 µm nylon filter membrane and placed in a liquid phase bottle, and the peak areas of each target peak in the upper and lower phases were measured by HPLC. The peak areas of each target peak in the upper phase were recorded as AU, and the peak areas of each target peak in the lower phase were recorded as AL. Calculate the partition coefficient of the sample in different solvent systems K D .

[0031] Partition coefficient of the sample K D It is calculated according to the following formula: K D = A U / A L Among them, A U and A L respectively represent the peak areas of the target compound in the upper and lower phases.

[0032] 1.3 Preparation of biphasic solvent system and sample solution The present invention uses solvent system I, n-hexane / ethyl acetate / methanol / water (solvent system I 5:5:5:5, v / v) for linear gradient countercurrent chromatography separation. After preparation, it is shaken vigorously and left to stand for phase separation. The upper and lower phases after phase separation are placed in volumetric flasks respectively. The lower phase is the mobile phase A, and the upper phase is the stationary phase. The mobile phase B is the lower phase of n-hexane / ethyl acetate / methanol / water (solvent system II 5:5:8:2, v / v). 5 mL of the mobile phase A and 5 mL of the stationary phase are mixed with 200 mg of the semen cassiae extract.

[0033] The internal circulation and off-line two-dimensional (2D) CCC mode are carried out using a solvent system of n-hexane / ethanol / water (1:1:0.2, v / v) in different ratios. After adding the solvent system, the separating funnel is shaken gently. After reaching equilibrium, the lower and upper phases of the biphasic solvent system are separated. The lower phase is the mobile phase, and the upper phase is the stationary phase. Approximately 50 mg of fractions A, B, and C are dissolved into the corresponding solvent systems with 5 mL of the upper phase and 5 mL of the lower phase.

[0034] In the internal circulation and off-line two-dimensional countercurrent chromatography mode, solvent systems of n-hexane / ethyl acetate / methanol / water (5:5:5.5:4.5, 5:5:7:3, v / v) and n-hexane / ethanol / water (1:1:0.2, v / v) in different ratios are used for the internal circulation and off-line two-dimensional countercurrent chromatography mode. After adding the solvent system, it is shaken vigorously and left to stand for phase separation. The upper phase is the stationary phase, and the lower phase is the mobile phase. 50 mg of components A, B, and C are dissolved into the corresponding solvent systems with 5 mL of the upper phase and 5 mL of the lower phase.

[0035] 1.6 Countercurrent chromatography separation procedure When performing linear gradient countercurrent chromatography separation, the upper phase of n-hexane / ethyl acetate / methanol / water (5:5:5:5, v / v) was pumped into the HSCCC chromatographic column as the stationary phase in the head-to-tail mode at a flow rate of 20.0 mL / min, a rotation speed of 800 rpm, and a temperature of 25°C. Then, mobile phase A (flow rate 5.0 mL / min) was pumped into the chromatographic column. After reaching hydrodynamic equilibrium, the sample solution was injected into the chromatographic column for countercurrent chromatography separation through the injection loop. Mobile phases A and B were pumped into the chromatographic column in a gradient manner, and the gradients were set as follows: 0 - 130 min, 100% A; 200 - 215 min, 50% A; 240 - 300 min, 25% A; 320 - 350 min, 0% A. During the internal circulation countercurrent chromatography separation process, an additional six-port valve was used to establish a cyclic separation system. Linear gradient countercurrent chromatography has two modes: "take-out" and "circulation". Initially, the six-port valve was placed in the "take-out" mode. The upper layer (stationary phase) was pumped into the chromatographic column for countercurrent chromatography separation at a flow rate of 20 mL / min, a rotation speed of 800 rpm, and a temperature of 25°C. The lower phase (mobile phase) was pumped into the chromatographic column at a set flow rate. After reaching hydrodynamic equilibrium, the sample solution (50 mg) was injected into the chromatographic column for countercurrent chromatography separation, and at the same time, the additional six-port valve was adjusted to the "circulation" mode. The flow rate of the lower phase (mobile phase) of component A solvent system (5:5:5.5:4.5, v / v) was 5 mL / min, and the flow rate of component B solvent system (5:5:7:3, v / v) was 2 mL / min. Component C was separated by traditional countercurrent chromatography. The upper phase of the solvent system of n-hexane / ethanol / water (1:1:0.2, v / v) was pumped into the countercurrent chromatography column at a flow rate of 20 mL / min. The rotation speed was 800 rpm, and the temperature was controlled at 25°C. The lower phase was pumped into the chromatographic column at a flow rate of 2 mL / min. After reaching hydrodynamic equilibrium, the sample solution (50 mg) was injected into the chromatographic column for countercurrent chromatography separation. The eluate was monitored at a wavelength of 280 nm and collected into 10 mL test tubes according to the chromatographic peaks. The retention rate of the stationary phase was defined as the ratio of the remaining stationary phase in the chromatographic column to the total volume of the chromatographic column.

[0036] 1.7 High Performance Liquid Chromatography Analysis and Structure Identification High performance liquid chromatography detection was used. The chromatographic column was packed with C18 (4.6×250 mm, inner diameter 5 µm). The mobile phases were acetonitrile (A) and 0.1% formic acid-water (B). High performance liquid chromatography elution was carried out in a linear gradient elution mode, and the program was as follows: 0 - 10 min, 56% B; 30 min, 42.5% B; 40 min, 5% B; 40.5 - 47 min, 56% B. The monitoring wavelength was 280 nm, the sample injection volume was 10 µL, and the flow rate was 1.0 mL / min.

[0037] NMR spectra were recorded using a Bruker AV-400 spectrometer (Bruker BioSpin GmbH, Germany). TMS was used as an internal reference, and chemical shifts (δ) were expressed in ppm, while coupling constants (J) were calculated in Hz.

[0038] 2 Results 2.1 Selection of Solvent System The separation by high-speed counter-current chromatography mainly depends on a suitable two-phase solvent system. K D Whether it is between 0.5 and 2.0 is the key to evaluating the suitability of the solvent system. The separation factor (α = K 1 / K 2, K 1> K 2) greater than 1.5, K D values between 0.5 and 2.0 are all suitable. K D >2.0 may lead to too long retention time, while K D <0.5 may lead to insufficient separation.

[0039] Several two-phase solvent systems composed of n-hexane, ethyl acetate, methanol and water were selected. According to Table 2, in the solvent system of n-hexane / ethyl acetate / methanol / water (5:5:5:5, v / v), the K D values of compounds 1 and 2 were 1.18 and 1.64 respectively, which were suitable for HSCCC separation. The K D values of compounds 3 - 9 exceeded 3 ( K D >2.0), indicating that the elution time of most compounds was too long. Using the solvent system of n-hexane / ethyl acetate / methanol / water (5:5:7:3, v / v) reduced the elution intensity. The K D values of the compounds decreased. Since the K D <0.5 for compounds 1 - 3, while the K D >1.5 for the other compounds 8 and 9, this solvent system could not separate all compounds at one time. In the solvent system of n-hexane / ethyl acetate / methanol / water (5:5:8:2, v / v), the K D values of compound 8 (2.70) and compound 9 (2.33) were the largest, and the K D values of the remaining compounds were all less than 1. According to K DThe value was then determined, and an effective linear gradient countercurrent chromatography separation technique was adopted. The upper phase of n-hexane / ethyl acetate / methanol / water (5:5:5:5, v / v) was used as the stationary phase. n-hexane / ethyl acetate / methanol / water (5:5:5:5, 5:5:8:2, v / v) was used as mobile phases A and B.

[0040] Table 2 Partition coefficients of each solvent system 2.2 Optimization of separation conditions Figure 2 This is the linear gradient countercurrent chromatography separation diagram of the crude anthraquinone extract of Cassia obtusifolia seeds. As Figure 2 shown, six main peaks were successfully obtained in HPLC, among which three pure compounds, namely 1 ( Figure 4 E), 3 ( Figure 4 G), and 4 ( Figure 4 H), were obtained with yields of 27.0, 7.4, and 9.1 mg respectively. At the same time, three mixtures (peaks A, B, and C) were obtained, corresponding to Figure 4 B-C. Compound 2 was included in the mixture of peak A with a yield of 43.4 mg. Peak B was a mixture of compounds 5, 6, and 7 with a yield of 40.3 mg. Peak C was a mixture of compounds 8 and 9 with a yield of 40.6 mg. Figure 2 Peaks A, B, and C in

[0041] Figure 3 A was separated using the internal recycle countercurrent chromatography mode. Figure 2 This is the internal recycle countercurrent chromatography diagram of peak B in Figure 4 . The results showed that when the solvent system n-hexane / ethyl acetate / methanol / water (5:5:5.5:4.5, v / v) was used and the injection volume was 50 mg, compound 2 was completely separated after six cycles. The yield was 22.4 mg, and the purity detected by HPLC was 98.4% ( Figure 3 F). Figure 2 B was Figure 4 the internal recycle countercurrent chromatography diagram of peak C in Figure 4 . When the solvent system n-hexane / ethyl acetate / methanol / water (5:5:7:3, v / v) was used and the injection volume was 50 mg, compounds 5 ( Figure 4 I), 6 ( Figure 4 ), and 7 ( Figure 3 C was Figure 2 the traditional CCC mode diagram of peak C. When the solvent system n-hexane / ethanol / water (1:1:0.2, v / v) was used and the injection volume was 50 mg. Compound 8 ( Figure 4L) and 9( Figure 4 M) were effectively separated, with yields of 22.4 and 17.8 mg respectively, and the purity detected by HPLC exceeded 98%.

[0042] 2.3 Structural analysis Aurantio-obtusin (Compound 1): The molecular formula is C 17 H 14 O7, and the molecular weight is 330. 1 1H-NMR (400 MHz, DMSO) δ : 13.27 (1H, s, 8-OH), 7.78 (1H, s, H-4), 7.22 (1H, s, H-5), 3.83 (3H, s, 7-OCH3), 3.80 (3H, s, 1-OCH3), 2.29 (3H, s, 3-CH3). Obtusin (Compound 2): The molecular formula is C 19 H 18 O7, and the molecular weight is 358. 1 1H-NMR (400 MHz, DMSO) δ : 7.70 (1H, s, 4-H), 7.48 (1H, s, 5-H), 3.98 (3H, s, 7-OCH3), 3.87 (2H, s, 6-OCH3), 3.86 (3H, s, 8-OCH3), 3.81 (3H, s, 1-OCH3), 2.27 (3H, s, 3-CH3). Chryso-obtusin (Compound 3): The molecular formula is C 18 H 16 O7, and the molecular weight is 344. 1 1H-NMR (400 MHz, DMSO) δ : 7.80 (1H, s, H-4), 7.30 (1H, s, H-5), 3.99 (3H, s, 6-OCH3), 3.83 (3H, s, 7-OCH3), 3.81 (3H, s, 1-OCH3), 2.30 (3H, s, 3-CH3). Sennidin A1 (Compound 4): The molecular formula is C 16 H 12 O5, and the molecular weight is 284. 1 1H-NMR (400 MHz, DMSO) δ : 7.78 (1H, s, 4-H), 7.70 (1H, t, J= 7.9 Hz, 6-H), 7.60 (1H, d, J = 7.3 Hz,5-H), 7.26 (1H, d, J = 8.2 Hz, 7-H), 3.80 (3H, s, 1-OCH3), 2.24 (3H, s, 3-CH3). Emodin (Compound 5): The molecular formula is C 15 H 10 O5, with a molecular weight of 270. 1 1H-NMR (400 MHz, DMSO) δ : 12.08 (2H, s, 8-OH, 1-OH), 7.45 (1H, s, H-4), 7.13 (1H, s, H-2), 7.06 (1H, s, H-5), 6.51 (1H, s, H-7), 2.39 (3H, s, 3-CH3). Rubrofusarin (Compound 6): The molecular formula is C 15 H 12 O5, with a molecular weight of 272. 1 1H-NMR (400 MHz, CDCl3) δ : 15.94 (1H, s ), 9.67 (1H, s), 6.98 (1H, s, H-5), 6.58 (1H, s, H-6), 6.48 (1H, s, H-8), 6.02 (1H, s, H-2), 3.90 (3H, s, 7-OCH3), 2.40 (3H, s, 3-CH3). 13C-NMR (101 MHz, CDCl3) δ : 184.1 (C-1), 168.9 (C-3), 162.9 (C-7), 162.0 (C-10), 158.7 (C-9), 152.5 (C-4a), 140.2 (C-5a), 106.7 (C-9a), 106.5 (C-2), 102.8 (C-10a), 101.2 (C-5), 100.6 (C-8), 98.1 (C-6), 55.4 (3-CH3), 21.0 (7-OCH3). Cassia lactone (Compound 7): The molecular formula is C 15 H 12 O5, with a molecular weight of 272. 1 1H-NMR (400 MHz, CDCl3) δ: 13.54 (1H, s), 9.41 (1H, s), 6.97 (1H, s, H-5), 6.62 (1H, s, H-6), 6.54 (1H, s, H-8), 6.23 (1H, s, H-4), 3.90 (3H, s, 7-OCH3), 2.27 (3H, s, 3-CH3). 13 C-NMR (101 MHz, CDCl3) δ : 168.2 (C-1), 163.1 (C-10), 162.1 (C-7), 158.4 (C-9), 152.6 (C-3), 141.5 (C-5a), 131.8 (C-4a), 112.4 (C-5), 108.1 (C-9a), 104.6 (C-4), 101.7 (C-8), 99.0 (C-6), 97.8 (C-10a), 55.5 (7-OCH3), 19.4 (3-CH3). Chrysophanol (Compound 8): The molecular formula is C 15 H 10 O4, and the molecular weight is 254. 1 H-NMR (400 MHz, DMSO) δ : 11.95 (2H, s, 8-OH, 1-OH), 7.79 (1H, t, J J = 7.9 Hz, 6-H), 7.69 (1H, d, J J = 7.3 Hz, 5-H), 7.53 (1H, s, 4-H), 7.37 (1H, d, J J = 8.2 Hz, 7-H), 7.20 (1H, s, 2-H), 2.46 (3H, s, 3-CH3). Physcion (Compound 9): The molecular formula is C 16 H 12 O5, and the molecular weight is 284. 1 H-NMR (400 MHz, CDCl3) δ : 12.30 (1H, s, 8-OH), 12.10 (1H, s, 1-OH), 7.61 (1H, s, 4-H), 7.35 (1H, s, 2-H), 7.07 (1H, s, 5-H), 6.68 (1H, s, 7-H), 3.93 (3H, s, 6-OCH3), 2.43 (3H, s, 3-CH3). Table 1 Compounds in Cassia obtusifolia13 C-NMR data (100 MHz, DMSO- d 6) The present invention adopts the method of linear gradient countercurrent chromatography combined with an internal circulation mode to effectively separate and purify anthraquinone compounds with a relatively wide range of partition coefficients from Cassia obtusifolia seeds. A linear gradient countercurrent chromatography separation is carried out using a combined solvent system of n-hexane / ethyl acetate / methanol / water (5:5:5:5, 5:5:8:2, v / v). The internal circulation and off-line two-dimensional CCC modes are adopted to separate each component using solvent systems of n-hexane / ethyl acetate / methanol / water (5:5:5.5:4.5, 5:5:7:3, v / v) and n-hexane / ethanol / water (1:1:0.2, v / v). Through HPLC detection, nine anthraquinone compounds with a purity exceeding 98% are effectively obtained. The nine anthraquinone compounds are: aurantio-obtusin, obtusin, obtusifolin, cassiaside, emodin, rubrofusarin, obtusifolinol, chrysophanol, and physcion. The established method has obvious effects in separating several anthraquinone compounds with a wide K D value.

[0043] The above specific embodiments are only specific cases of the present invention. The patent protection scope of the present invention includes, but is not limited to, the product forms and patterns of the above specific embodiments. Any efficient preparation method of the active ingredients of Cassia obtusifolia seeds that conforms to the claims of the present invention and any appropriate changes or modifications made by those of ordinary skill in the relevant technical field shall fall within the patent protection scope of the present invention.

Claims

1. A highly efficient method for preparing active ingredients from Cassia seed, characterized in that: The preparation method comprises extracting Cassia seed to obtain a crude anthraquinone extract, using a biphasic solvent consisting of n-hexane, ethyl acetate, methanol and water as a solvent system, separating and preparing the crude anthraquinone extract by high-speed countercurrent chromatography, and then performing high-performance liquid chromatography analysis on the anthraquinone fraction obtained by countercurrent chromatography separation in a linear gradient elution mode; The preparation method comprises the following steps: The seeds of Cassia seed are crushed, extracted with ethanol, mixed and filtered, extracted with petroleum ether until colorless, the pH value of the aqueous residue is adjusted with hydrochloric acid, and the acidic aqueous residue is extracted with chloroform to obtain a crude anthraquinone extract; The upper phase of the solvent system of n-hexane / ethyl acetate / methanol / water was pumped into the HSCCC column as the stationary phase, and then the mobile phase A was pumped into the column. After reaching the hydrodynamic equilibrium, the sample solution was injected into the column of countercurrent chromatography through the injection loop, and the eluent was monitored at a wavelength of 280 nm. The separated anthraquinone fraction was detected by high performance liquid chromatography with the mobile phase consisting of acetonitrile (A) and 0.1% formic acid-water (B).

2. The preparation method according to claim 1, characterized in that The mixture was crushed to 20-40 mesh, the ethanol was 95% ethanol, the temperature of petroleum ether extraction was 60-90°C, the pH value of the aqueous residue was adjusted to 2 with hydrochloric acid, ultrasonic acid hydrolysis was performed at 40°C for 20 min, 100 mL of chloroform was used to extract the acidic water residue, the chloroform extracts were combined, neutralized with water, concentrated by rotary evaporation, and stored in a refrigerator at 4°C.

3. The preparation method according to claim 1, characterized in that: When performing linear gradient countercurrent chromatography separation, the solvent system is n-hexane / ethyl acetate / methanol / water, and the volume ratio thereof is 4-6:4-6:4-6:4-6, preferably 5:5:5:5, the lower phase is mobile phase A, and the upper phase is the stationary phase; the lower phase of n-hexane / ethyl acetate / methanol / water is selected as mobile phase B, and the upper ratio thereof is 5:5:8:2, and 5 mL of mobile phase A and 5 mL of stationary phase are mixed with 200 mg of Cassia seed extract.

4. The preparation method according to claim 1, characterized in that: For the internal circulation and offline two-dimensional CCC mode, n-hexane / ethanol / water were selected with a volume ratio of 1:1:0.

2. After reaching equilibrium, the lower phase and the upper phase of the two-phase solvent system were separated. The lower phase was the mobile phase and the upper phase was the stationary phase. About 50 mg of fractions A, B and C were dissolved in the corresponding solvent system with 5 mL of the upper phase and 5 mL of the lower phase.

5. The preparation method according to claim 3, characterized in that: Using head-to-tail mode, the stationary phase was pumped into the HSCCC column at a flow rate of 15-25 mL / min, a rotation speed of 750-850 rpm, and a temperature of 20-30°C, and then the mobile phase A was pumped into the column at a flow rate of 1-5 mL / min.

6. The preparation method according to claim 5, characterized in that: Mobile phases A and B were pumped into the chromatographic column in a gradient manner, and the gradients were set as follows: 0-130 min, 100%A; 200-215 min, 50%A; 240-300 min, 25%A; 320-350 min, 0%A.

7. The preparation method according to claim 3, characterized in that: During the internal circulation countercurrent chromatography separation process, the linear gradient countercurrent chromatography has two modes: "take-out" and "circulation". Initially, the six-way valve is placed in the "take-out" mode, and the upper layer (stationary phase) is pumped into the chromatographic column of the countercurrent chromatography separation at a flow rate of 20 mL / min, a speed of 800 rpm, and a temperature of 25°C. The lower phase (mobile phase) is pumped into the chromatographic column at a set flow rate. After reaching fluid dynamics equilibrium, the sample solution is injected into the chromatographic column of the countercurrent chromatography separation, and the additional six-way valve is adjusted to the "circulation" mode.

8. The preparation method according to claim 1, characterized in that: The HPLC elution adopted a linear gradient elution mode with the following program: 0-10 min, 50-56%B; 30-32 min, 42.5-44%B; 35-40 min, 1-5%B; 40.5-47 min, 50-56%B, the monitoring wavelength was 280-290nm, preferably 280nm, the sample volume was 10 µL, and the flow rate was 1.0-2mL / min.

9. The active ingredient of Cassia seed obtained by the preparation method according to any one of claims 1 to 8, characterized in that: There are nine active ingredients in the Cassia seed, namely: aurantin, chrysophanol, chrysophanol, chrysophanol, rhamnosin, erythromycin, cassia lactone, chrysophanol and physophanol methyl ether. The specific structural formula is as follows: 。

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