阅读说明：本技术 川射干水提药渣中射干苷和鸢尾黄素的分离方法 (Method for separating tectoridin and tectorigenin from rhizoma Belamcandae water extraction residues ) 是由 李宁 王丽 胡星园 韩丽娟 杨学慧 杨晓彬 周建苗 于 2021-08-06 设计创作，主要内容包括：本发明公开了一种川射干水提药渣中射干苷和鸢尾黄素的分离方法,包括如下步骤：将川射干水提药渣提取物上羟丙基葡聚糖凝胶色谱柱,洗脱羟丙基葡聚糖凝胶色谱柱,收集洗脱液,分别得到射干苷粗品和鸢尾黄素粗品；其中,羟丙基葡聚糖凝胶色谱柱的径高比小于等于1:25。本发明能够从川射干水提药渣中同时分离出射干苷和鸢尾黄素。(The invention discloses a method for separating tectoridin and tectorigenin from rhizoma belamcandae water extraction residues, which comprises the following steps: loading the extract of the water extraction residue of the rhizoma Belamcandae into a hydroxypropyl sephadex chromatographic column, eluting the hydroxypropyl sephadex chromatographic column, and collecting the eluent to respectively obtain a crude product of the tectoridin and a crude product of tectorigenin; wherein the diameter-height ratio of the hydroxypropyl dextran gel chromatographic column is less than or equal to 1: 25. The method can simultaneously separate out the tectoridin and tectorigenin from the water extraction dregs of the Sichuan blackberry lily.)

1. A method for separating tectoridin and tectorigenin from rhizoma Belamcandae water extraction residues is characterized by comprising the following steps:

loading the extract of the water extraction residue of the rhizoma Belamcandae into a hydroxypropyl sephadex chromatographic column, eluting the hydroxypropyl sephadex chromatographic column, and collecting the eluent to respectively obtain a crude product of the tectoridin and a crude product of tectorigenin;

wherein the diameter-height ratio of the hydroxypropyl dextran gel chromatographic column is less than or equal to 1: 25.

2. The separation method of claim 1, wherein the hydroxypropyl Sephadex chromatographic column is a Sephadex LH-20 chromatographic column.

3. The separation method according to claim 1, wherein the mobile phase of the eluting hydroxypropyl sephadex chromatographic column is selected from one or more of methanol-chloroform, acetone-water, methanol-water.

4. The separation method of claim 1, wherein the hydroxypropyl dextran gel chromatographic column is eluted by isocratic elution.

5. The separation process according to claim 1, wherein the amount of the mobile phase is 4 to 15 times the column volume.

6. The separation method according to any one of claims 1 to 5, wherein the preparation method of the rhizoma Belamcandae water extraction residue extract comprises the following steps:

extracting the rhizoma ligustici wallichii water extraction dregs by adopting a solvent, filtering an extracting solution, and recovering the solvent to obtain a rhizoma ligustici wallichii water extraction dregs extract; the solvent is one or more selected from methanol, ethanol, 60-95 vol% methanol water solution and 60-95 vol% ethanol water solution; the dosage of the solvent is 2-20 times of the mass of the rhizoma ligustici wallichii water extraction dregs.

7. The separation method according to claim 6, wherein the extraction method is one of soaking extraction, ultrasonic extraction or heating reflux extraction, and the extraction time is 0.4-24 h.

8. The separation method according to claim 6, wherein the content of tectoridin in the rhizoma Belamcandae water extraction residue is 0.5-2.5 wt%, and the content of tectorigenin is 0.5-2 wt%.

9. The separation method according to any one of claims 1 to 5, further comprising a step of purifying crude tectoridin:

separating the crude product of belamcanda chinensis glycoside by liquid chromatography, collecting the solution of the chromatographic section of belamcanda chinensis glycoside, and recovering the solvent to obtain the belamcanda chinensis glycoside;

the chromatographic conditions are selected from one of the following conditions:

(A1) a chromatographic column: c₁₈A chromatographic column; mobile phase: methanol A and water B; gradient elution procedure: 0-30 min, 15-30 vol% A;

(B1) a chromatographic column: c₁₈A chromatographic column; mobile phase: acetonitrile A and water B; isocratic elution procedure: 15 vol% A;

(C1) a chromatographic column: c₁₈A chromatographic column; mobile phase: methanol A and water B; isocratic elution procedure: 25 vol% A.

10. The separation process according to any one of claims 1 to 5, further comprising a step of purifying the crude tectorigenin:

separating the crude tectorigenin product by liquid chromatography, collecting tectorigenin chromatographic fraction solution, and recovering solvent to obtain tectorigenin;

the chromatographic conditions are selected from one of the following conditions:

(A2) a chromatographic column: c₁₈A chromatographic column; mobile phase: methanol A and water B; gradient elution procedure: 0-30 min, 40-60 vol% A;

(B2) a chromatographic column: c₁₈A chromatographic column; mobile phase: acetonitrile A and water B; isocratic elution procedure: 40 vol% A;

(C2) a chromatographic column: c₁₈A chromatographic column; mobile phase: methanol A and water B; isocratic elution procedure: 50 vol% A.

Technical Field

The invention relates to a method for separating tectoridin and tectorigenin from rhizoma belamcandae water extraction residues.

Background

The traditional Chinese medicine formula particle is a particle prepared from single traditional Chinese medicine decoction pieces. Under the guidance of the traditional Chinese medicine theory, the Chinese medicinal composition is taken by patients after being formulated according to the clinical prescription of the traditional Chinese medicine. The regulations in technical requirements for quality control and standard formulation of Chinese medicinal granule (solicited comments) published by the State drug administration: the preparation of the traditional Chinese medicine formula granule is basically consistent with the traditional decoction except the molding process, namely, the preparation is produced by taking water as a solvent for extraction, and carrying out solid-liquid separation, concentration, drying, granule molding and other processes by a physical method. The effective components of the Chinese medicinal granule should be substantially identical to those of decoction pieces of Chinese medicinal materials.

The evaluation indexes of the extraction process and the quality of the traditional Chinese medicine formula granules are 'basically consistent with the traditional decoction', namely: the quality attributes of the paste yield, the content and transfer rate of effective (or index) components, the characteristic map and the like are basically consistent with those of the traditional decoction, but the paste yield, the content and transfer rate of the effective (or index) components are not maximized. The quality standard of the traditional Chinese medicine formula granules stipulates the upper and lower limits of the cream yield, and in the production process, the extraction rate is too high, so that the range is broken through, and the product is unqualified. In addition, the traditional Chinese medicine formula granules are extracted by taking water as a solvent, and the extraction rate of components with low polarity and poor water solubility is lower. Therefore, certain components are inevitably left in the dregs after the extraction of the traditional Chinese medicine formula particles. This aspect results in a waste of medicinal material. On the other hand, in order to prevent the dregs of a decoction from flowing into the market again, enterprises need to destroy the dregs of a decoction, and the process and the cost investment are increased. Therefore, the comprehensive utilization of the medicine dregs is a necessary ring for developing the medicine recycling economy, building the resource-saving and environment-friendly society in China.

Rhizoma Belamcandae is dried rhizome of Iris tectorum (Iris tectorum Maxim.) of Iridaceae. Bitter and cold. It enters lung meridian. Has effects of clearing away heat and toxic materials, eliminating phlegm, and relieving sore throat. Can be used for treating stagnation of heat-toxin and phlegm-fire, sore throat, excessive phlegm and saliva, cough and asthma. The tectoridin and tectorigenin are effective components and index components of rhizoma Belamcandae, have important physiological activity to human body, and are common chemical reference substances in traditional Chinese medicine research and analysis test, so that the preparation of tectoridin and tectorigenin has important application value. At present, no method for simultaneously separating and preparing tectoridin and tectorigenin from rhizoma belamcandae water extraction residues is reported.

Disclosure of Invention

In view of the above, the invention provides a method for separating tectorigenin and tectorigenin from rhizoma belamcandae water extraction residues, which can simultaneously separate the tectorigenin and tectorigenin from the rhizoma belamcandae water extraction residues. Furthermore, the method has simple process, and high purity tectorigenin and tectorigenin can be obtained.

The invention provides a method for separating tectoridin and tectorigenin from rhizoma belamcandae water extraction dregs, which comprises the following steps:

loading the extract of the water extraction residue of the rhizoma Belamcandae into a hydroxypropyl sephadex chromatographic column, eluting the hydroxypropyl sephadex chromatographic column, and collecting the eluent to respectively obtain a crude product of the tectoridin and a crude product of tectorigenin;

wherein the diameter-height ratio of the hydroxypropyl dextran gel chromatographic column is less than or equal to 1: 25.

In the invention, the rhizoma ligustici wallichii water extraction dregs refer to dregs obtained by extracting rhizoma ligustici wallichii medicinal materials with water, for example, dregs obtained by extracting the rhizoma ligustici wallichii medicinal materials with water when the rhizoma ligustici wallichii formula particles are produced.

According to the separation method of the present invention, preferably, the hydroxypropyl Sephadex chromatographic column is a Sephadex LH-20 chromatographic column. The hydroxypropyl dextran gel chromatographic column is easy to generate dead adsorption with complex components of traditional Chinese medicines to denature and discard, is usually used for finely dividing components, and is less used for roughly dividing extracts. The applicant unexpectedly found that tectoridin and tectorigenin in the rhizoma ligustici wallichii water extraction residue extract can be simultaneously separated by adopting hydroxypropyl glucan gel chromatography.

In the invention, the diameter-height ratio of the hydroxypropyl glucan gel chromatographic column is less than or equal to 1: 25; preferably, the diameter-height ratio of the hydroxypropyl glucan gel chromatographic column is 1: 50-200; more preferably, the diameter-height ratio of the hydroxypropyl glucan gel chromatographic column is 1: 90-200. According to one embodiment of the invention, the diameter-height ratio of the hydroxypropyl dextran gel chromatographic column is 1: 100-150. Therefore, the tectoridin and tectorigenin in the rhizoma ligustici wallichii water extraction residue extract can be completely separated, and the separation time can be shortened.

According to the separation method of the present invention, preferably, the hydroxypropyl dextran gel chromatographic column is eluted by isocratic elution. The method adopts isocratic elution to completely separate the tectoridin and tectorigenin in the rhizoma belamcandae water extraction residue extract, so that the operation is easier.

According to the separation method of the invention, preferably, the mobile phase for eluting the hydroxypropyl glucan gel chromatographic column is selected from one or more of methanol-chloroform, acetone, methanol and methanol-water. The volume ratio of methanol to chloroform in the methanol-chloroform can be 0.5-2: 1; preferably 1: 1. The volume fraction of methanol in the methanol-water can be 50-90 vol%; preferably 75 vol%. According to one embodiment of the invention, the mobile phase is methanol. This enables better separation of tectoridin and tectorigenin and easy recovery of the solvent.

According to the separation method of the present invention, preferably, the amount of the mobile phase is 4 to 15 times of the column volume. More preferably, the amount of the mobile phase is 5 to 8 times the column volume. Therefore, the tectoridin and tectorigenin components can be completely separated, the dosage of the mobile phase can be reduced, and the recovery is easy.

According to the separation method, the rhizoma ligustici wallichii water extraction residue extract is an extract obtained by extracting the rhizoma ligustici wallichii water extraction residue with an organic solvent. The rhizoma ligustici wallichii water extraction residue extract can be prepared by the following method:

extracting the rhizoma ligustici wallichii water extraction dregs by adopting a solvent, filtering an extracting solution, and recovering the solvent to obtain a rhizoma ligustici wallichii water extraction dregs extract; the solvent is one or more selected from methanol, ethanol, 60-95 vol% methanol water solution and 60-95 vol% ethanol water solution; the dosage of the solvent is 2-20 times of the mass of the rhizoma ligustici wallichii water extraction dregs.

In the present invention, the solvent is preferably a 60 to 95 vol% ethanol aqueous solution. According to one embodiment of the present invention, the solvent is 70 vol% aqueous ethanol, thereby sufficiently extracting tectoridin and tectorigenin.

In the present invention, the extraction manner may be one selected from immersion extraction, ultrasonic extraction, or heating reflux extraction. Preferably, the extraction mode is ultrasonic extraction or heating reflux extraction. This enables a higher extraction rate.

The extraction time of heating reflux extraction or ultrasonic extraction can be 0.5-5 h; preferably 0.5-2 h. The dosage of the solvent can be 2-20 times of the mass of the rhizoma ligustici wallichii water extraction dregs; preferably 4 to 6 times.

The extraction time of soaking and extraction can be 8-24 h; preferably 16-24 h. The dosage of the solvent can be 5-20 times of the mass of the rhizoma ligustici wallichii water extraction dregs; preferably 8 to 12 times.

According to the separation method provided by the invention, preferably, the extraction mode is one of soaking extraction, ultrasonic extraction or heating reflux extraction, and the extraction time is 0.4-24 h.

According to the separation method provided by the invention, preferably, the content of tectoridin in the rhizoma Belamcandae dregs is 0.5-2.5 wt%, and the content of tectorigenin is 0.5-2 wt%. More preferably, the content of the Belamcandin in the Sichuan blackberry lily dregs is 1-2 wt%, and the content of the tectorigenin is 0.5-1.5 wt%.

In the separation method of the invention, the method can also comprise a step of refining crude tectoridin and/or crude tectorigenin.

The refining of the crude blackberry lily glycoside can comprise the following steps: and (3) separating the crude product of the belamcandin by adopting liquid chromatography, collecting the solution of the chromatographic section of the belamcandin, and recovering the solvent to obtain the belamcandin.

According to one embodiment of the invention, the chromatographic conditions for refining the crude blackberry lily glycoside are as follows: a chromatographic column: c₁₈Chromatography column (9.4 mm. times.250 mm, 5 μm); mobile phase: methanol A and water B; gradient elution procedure: 0-30 min, 15-30 vol% A, flow rate of 2mL/min, and detection wavelength of 265 nm.

According to another embodiment of the invention, the chromatographic conditions for refining the crude blackberry lily glycoside are as follows: a chromatographic column: c₁₈Chromatography column (30 mm. times.250 mm, 10 μm); mobile phase: acetonitrile A and water B; isocratic elution processThe method comprises the following steps: 15 vol% A, flow rate 10mL/min, detection wavelength 265 nm.

According to another embodiment of the invention, the chromatographic conditions for refining the crude blackberry lily glycoside are as follows: a chromatographic column: c₁₈Chromatography column (50 mm. times.250 mm, 10 μm); mobile phase: methanol A and water B; isocratic elution procedure: 25 vol% A, flow rate 30mL/min, detection wavelength 265 nm.

The refining of the crude tectorigenin can comprise the following steps: separating the crude tectorigenin product by liquid chromatography, collecting tectorigenin chromatographic fraction solution, and recovering solvent to obtain tectorigenin.

According to one embodiment of the invention, the chromatographic conditions for refining the crude tectorigenin are as follows: a chromatographic column: c₁₈Chromatography column (9.4 mm. times.250 mm, 5 μm); mobile phase: methanol A and water B; gradient elution procedure: 0-30 min, 40-60 vol% A, flow rate of 2mL/min, and detection wavelength of 265 nm.

According to another embodiment of the present invention, the chromatographic conditions for refining crude tectorigenin are as follows: a chromatographic column: c₁₈Chromatography column (30 mm. times.250 mm, 10 μm); mobile phase: acetonitrile A and water B; isocratic elution procedure: 40 vol% A, flow rate 10mL/min, detection wavelength 265 nm.

According to another embodiment of the invention, the chromatographic conditions for refining the crude tectorigenin are as follows: a chromatographic column: c₁₈Chromatography column (50 mm. times.250 mm, 10 μm); mobile phase: methanol A and water B; isocratic elution procedure: 50 vol% A, flow rate 30mL/min, detection wavelength 265 nm.

The separation method according to the present invention preferably further comprises the steps of:

separating the crude product of belamcanda chinensis glycoside by liquid chromatography, collecting the solution of the chromatographic section of belamcanda chinensis glycoside, and recovering the solvent to obtain the belamcanda chinensis glycoside;

the chromatographic conditions are selected from one of the following conditions:

(A) a chromatographic column: c₁₈A chromatographic column; mobile phase: methanol A and water B; gradient elution procedure: 0-30 min, 15-30 vol% A;

(B) a chromatographic column: c₁₈A chromatographic column; mobile phase: acetonitrile A and water B; isocratic elution procedure: 15 vol% A;

(C) a chromatographic column: c₁₈A chromatographic column; mobile phase: methanol A and water B; isocratic elution procedure: 25 vol% A.

The separation method according to the present invention preferably further comprises the steps of:

separating the crude tectorigenin product by liquid chromatography, collecting tectorigenin chromatographic fraction solution, and recovering solvent to obtain tectorigenin;

the chromatographic conditions are selected from one of the following conditions:

(A) a chromatographic column: c₁₈A chromatographic column; mobile phase: methanol A and water B; gradient elution procedure: 0-30 min, 40-60 vol% A;

(B) a chromatographic column: c₁₈A chromatographic column; mobile phase: acetonitrile A and water B; isocratic elution procedure: 40 vol% A;

(C) a chromatographic column: c₁₈A chromatographic column; mobile phase: methanol A and water B; isocratic elution procedure: 50 vol% A.

The applicant finds that the tectoridin and tectorigenin still exist in the water extraction dregs of the blackberry lily, and the blackberry lily glycoside and tectorigenin are wasted because the water extraction dregs of the blackberry lily are generally directly discarded as wastes. The method adopts the hydroxypropyl glucan gel chromatographic column to simultaneously separate out the residual tectoridin and tectorigenin in the rhizoma ligustici wallichii water extract dregs, changes waste into valuable, improves the resource utilization rate, and has environmental benefit and economic benefit. The method has the advantages of simple and convenient process, few steps, short time consumption, easy control and high separation efficiency. Further, the method of the invention can prepare high-purity tectoridin and tectorigenin. The method is easy to scale up, and gram-grade tectorigenin and tectorigenin monomer compounds can be prepared.

Detailed Description

The instrument is described below:

agilent1260 high performance liquid chromatograph, purchased from Agilent technologies, Inc. LC-20AT high performance liquid chromatograph, purchased from Shimadzu corporation.

B5510E-DTH ultrasonic extractor, purchased from BINEUTRAL ULTRASONIC, Inc.

The following raw materials are introduced:

the rhizoma Belamcandae water extraction residue is the residue after rhizoma Belamcandae formula granule is produced by Kangmei pharmaceutical industry Gmbycis, namely the residue after rhizoma Belamcandae water extraction; the tectoridin reference substance is purchased from the Chinese food and drug testing research institute; tectorigenin control was purchased from the chinese food and drug testing institute.

The chromatographic solvent was chromatographically pure, the water was ultrapure water (made by Milli-Q ultrapure water machine available from Millipore corporation, USA), and the other reagents were analytically pure.

The determination method of the dry extract yield comprises the following steps: drying the water extraction residue extract of the rhizoma ligustici wallichii franchet, drying for 3 hours at 105 ℃, cooling for 30 minutes in a drier, and quickly and precisely weighing. And calculating the dry extract yield according to the weight of the dry product of the rhizoma ligustici wallichii water extract herb residue which is extracted and fed at this time.

Detecting the content of tectoridin and tectorigenin in the residue of rhizoma Belamcandae water extract

10 batches of rhizoma Belamcandae water extraction dregs after rhizoma Belamcandae formula granules are produced by Kangmei pharmaceutical industry Gmbycis, are taken respectively, and the labels are 1 to 10 in sequence. And respectively preparing the rhizoma ligustici wallichii franchet herb residues of the batches 1 to 10 into a test solution. The preparation method of the test solution comprises the following steps: taking a proper amount of rhizoma ligustici wallichii water extract dregs, grinding, taking 1.0g, placing in a conical flask with a plug, precisely adding 50ml of 70 vol% ethanol, weighing, carrying out ultrasonic treatment (power 250W and frequency 40kHz) for 30 minutes, cooling, weighing again, complementing the weight loss by 70 vol% ethanol, shaking up, filtering, and taking the subsequent filtrate to obtain the traditional Chinese medicine composition.

Preparing reference substance solution from tectorigenin and tectorigenin. The control solutions were prepared as follows: adding 70 vol% ethanol into the reference substance to obtain stock solution containing 200 μ g of reference substance per 1ml, mixing, and diluting to obtain reference substance solutions with a series of concentrations.

Chromatographic conditions are as follows: a chromatographic column: TC-C₁₈Chromatography column (4.6 mm. times.250 mm, 5 μm); mobile phase: acetonitrile A and 0.2 vol% phosphoric acid solution B; gradient elution: 0-25 min, 18 vol% A; 25-35 min, 18-36 vol% A; 35-60 min 36 vol% A); flow rate: 1 mL/min; the column temperature was 40 ℃; detection wavelengthAnd 265 nm.

The determination method comprises the following steps: precisely sucking the reference solution and the sample solution, respectively, injecting into LC-20AT high performance liquid chromatograph, and measuring.

And (3) measuring results: the content of the blackberry lily glycosides in the blackberry lily water extraction medicine dregs of the batches 1 to 10 is respectively as follows according to the dry product: 1.51 wt%, 1.62 wt%, 1.46 wt%, 1.57 wt%, 1.69 wt%, 1.63 wt%, 1.58 wt%, 1.72 wt%, 1.45 wt%, 1.39 wt%. The content of tectorigenin in the rhizoma ligustici wallichii water extraction medicine dregs of the batches 1 to 10 is respectively as follows according to the dry product: 0.97 wt%, 1.05 wt%, 1.06 wt%, 0.88 wt%, 0.94 wt%, 1.14 wt%, 0.97 wt%, 1.00 wt%, 1.17 wt%, 0.93 wt%.

Therefore, the rhizoma Belamcandae water extraction dregs still contain high content of tectoridin and tectorigenin.

Belamcandin and tectorigenin are extracted and separated from the rhizoma Belamcandae water extraction dregs of the batch 1 as raw materials.

Preparation examples 1 to 14

Extracting 100g of rhizoma Belamcandae water extraction residue with solvent, and extracting; filtering the extracting solution, and recovering the solvent to obtain the extract of the rhizoma ligustici wallichii water extraction dregs. The specific parameters are shown in Table 1, and the dry extract yield is shown in Table 1.

TABLE 1

As shown in Table 1, methanol aqueous solution, ethanol, and ethanol aqueous solution can be used for extracting rhizoma Belamcandae water extraction residue, and 70 vol% ethanol aqueous solution has the best extraction effect. The dry extract yield is increased along with the increase of the solvent dosage, when the dosage of the solvent reaches 5 times of the mass of the rhizoma belamcandae water extraction dregs, the increase of the dry extract yield tends to be gentle, and the dosage of the solvent is preferably 5 times of the mass of the rhizoma belamcandae water extraction dregs in consideration of later-stage solvent recovery and cost. The method of heating reflux or ultrasonic extraction can obtain higher dry extract yield.

Preparation example 15

Taking 5kg of rhizoma Belamcandae water extraction residues, adding 25kg of 70 vol% ethanol water solution, and heating and reflux extracting for 1 h; filtering the extractive solution, and recovering solvent to obtain rhizoma Belamcandae water extraction residue extract 0.4 kg.

Examples 1 to 8 and comparative example 1

Sephadex LH-20 chromatographic column (Sephadex LH-20 filler 900g), the diameter height ratio is shown in Table 2, use mobile phase to swell Sephadex LH-20 filler, pack into 9 columns each containing 100g of filler. Taking 18g of the rhizoma ligustici wallichii water extraction decoction dreg extract obtained in the preparation example 15, dissolving the rhizoma ligustici wallichii water extraction decoction dreg extract in a mobile phase, loading the sample, wherein the sample loading amount of each column is 2g (based on the rhizoma ligustici wallichii water extraction decoction dreg extract), eluting at equal degrees by the mobile phase, eluting for 6 column volumes in total, collecting eluent, visually observing the separation condition of a colored band, detecting components containing the tectoridin and tectorigenin by TLC, and combining to obtain a crude product of the tectoridin and a crude product of the tectorigenin.

TABLE 2

The radial-height ratio is too high, the tectoridin and tectorigenin cannot be completely separated, the separation degree of the tectoridin and tectorigenin is increased along with the reduction of the radial-height ratio, but the separation time is increased along with the overlong column length, and the radial-height ratio is preferably 1: 50-200. The methanol system, chloroform methanol 1:1 system, acetone system and 75 vol% methanol system can completely separate the components of tectoridin and tectorigenin, and the separation degree is the best in the methanol system. Chloroform methanol 1:1 system, a toxic organic solvent chloroform is required. The acetone system Sephadex LH-20 has low swelling coefficient, the column volume is reduced, and the sample loading is reduced. The solubility of the 75 vol% methanol system sample was poor, the sample volume increased, and the resolution decreased.

Comparative example 2

And (2) taking 5g of the extract of the rhizoma belamcandae water extraction residue, loading the extract on a silica gel column (silica gel filler 100g, the diameter-height ratio is 1:10), performing gradient elution by using chloroform-methanol 10:1, chloroform-methanol 5:1, chloroform-methanol 2:1 and chloroform-methanol 1:1 in sequence, performing gradient elution by using 4 column volumes each, collecting eluent, detecting components containing the tectoridin and tectorigenin by using TLC (thin layer chromatography), and combining to obtain a crude product of the tectoridin and a crude product of the tectorigenin. The results show that the tectoridin and tectorigenin components are crossed and not completely separated.

The trajaponin and tectorigenin are seriously smeared on silica gel filler, and toxic organic solvents such as chloroform and the like are required for a silica gel column.

Comparative example 3

Extracting 5g rhizoma Belamcandae with water, and adding C₁₈Chromatographic column (C)₁₈100g of filler, the diameter-height ratio of 1:10), performing gradient elution with methanol-water 2:8, methanol-water 3:7, methanol-water 4:6, methanol-water 5:5, methanol-water 6:4 and methanol in sequence, performing gradient elution with 3 column volumes each, collecting eluent, detecting components containing tectoridin and tectorigenin by TLC, and combining to obtain crude tectoridin and crude tectorigenin. The tectoridin and tectorigenin components can be completely separated. Separation time: over 36 h.

Although C is adopted₁₈The column can completely separate the components of the belamcandin and the tectorigenin, but gradient elution is needed, 18 column volumes are needed to be eluted, the operation is more complicated, a large amount of water-containing solvent needs to be recovered in the operation process, the experimental period is long, the energy consumption is larger, and the effect is not as good as that of a Sephadex LH-20 chromatographic column.

Example 9

Filtering the tectoridin reference substance, and subjecting to Agilent1260 high performance liquid chromatograph to determine tectoridin retention time and peak shape. The crude tectoridin obtained in example 3 was filtered and applied to an Agilent1260 hplc chromatograph, and according to the retention time and peak shape of the tectoridin control, the tectoridin chromatographic peak was collected for solvent preparation and solvent was recovered to obtain 200mg of tectoridin (white powder).

Chromatographic conditions of the belamcandin reference substance and the crude product of the belamcandin are as follows: a chromatographic column: c₁₈Chromatography column (9.4 mm. times.250 mm, 5 μm); mobile phase: methanol A and water B; gradient elution procedure: 0-30 min, 15-30 vol% A; flow rate: 2 mL/min; detection wavelength: 265 nm.

Filtering tectorigenin reference substance, and subjecting to Agilent1260 high performance liquid chromatograph to determine tectorigenin retention time and peak shape. The crude tectorigenin obtained in example 3 was filtered, subjected to Agilent1260 hplc, and the tectorigenin spectrum peak fragments were collected in a targeted manner to prepare a solvent based on the retention time and peak shape of the tectorigenin control, and the solvent was recovered to obtain 200mg of tectorigenin (white powder).

The chromatographic conditions of the tectorigenin reference substance and the crude tectorigenin product are as follows: a chromatographic column: c₁₈Chromatography column (9.4 mm. times.250 mm, 5 μm); mobile phase: methanol A and water B; gradient elution procedure: 0-30 min, 40-60 vol% A; flow rate: 2 mL/min; detection wavelength: 265 nm.

Example 10

Filtering the tectoridin reference substance, and subjecting to Agilent1260 high performance liquid chromatograph to determine tectoridin retention time and peak shape. The crude tectoridin obtained in example 3 was filtered and applied to an Agilent1260 hplc chromatograph, and according to the retention time and peak shape of the tectoridin control, a tectoridin chromatographic peak was collected for solvent preparation, and the solvent was recovered to obtain tectoridin 1.0g (white powder).

Chromatographic conditions of the belamcandin reference substance and the crude product of the belamcandin are as follows: a chromatographic column: c₁₈Chromatography column (30 mm. times.250 mm, 10 μm); mobile phase: acetonitrile A and water B; isocratic elution procedure: 15 vol% A; flow rate: 10 mL/min; detection wavelength: 265 nm.

Filtering tectorigenin reference substance, and subjecting to Agilent1260 high performance liquid chromatograph to determine tectorigenin retention time and peak shape. The crude tectorigenin obtained in example 3 was filtered, subjected to Agilent1260 hplc, and the tectorigenin spectrum peak fragments were collected in a targeted manner to prepare a solvent based on the retention time and peak shape of the tectorigenin control, and the solvent was recovered to obtain 1.0g of tectorigenin (white powder).

The chromatographic conditions of the tectorigenin reference substance and the crude tectorigenin product are as follows: a chromatographic column: c₁₈Chromatography column (30 mm. times.250 mm, 5 μm); mobile phase: acetonitrile A and water B; isocratic elution procedure: 40 vol% A; flow rate: 10 mL/min; detection wavelength: 265 nm.

Example 11

Filtering the tectoridin reference substance, and subjecting to Agilent1260 high performance liquid chromatograph to determine tectoridin retention time and peak shape. The crude tectoridin obtained in example 3 was filtered and applied to an Agilent1260 hplc chromatograph, and according to the retention time and peak shape of the tectoridin control, a tectoridin chromatographic peak was collected for solvent preparation and solvent was recovered to obtain tectoridin 5.0g (white powder).

Chromatographic conditions of the belamcandin reference substance and the crude product of the belamcandin are as follows: a chromatographic column: c₁₈Chromatography column (50 mm. times.250 mm, 10 μm); mobile phase: methanol A and water B; isocratic elution procedure: 25 vol% A; flow rate: 30 mL/min; detection wavelength: 265 nm.

Filtering tectorigenin reference substance, and subjecting to Agilent1260 high performance liquid chromatograph to determine tectorigenin retention time and peak shape. The crude tectorigenin obtained in example 3 was filtered, subjected to Agilent1260 hplc, and the tectorigenin spectrum peak fragments were collected in a targeted manner to prepare a solvent based on the retention time and peak shape of the tectorigenin control, and the solvent was recovered to obtain 5.0g of tectorigenin (white powder).

The chromatographic conditions of the tectorigenin reference substance and the crude tectorigenin product are as follows: a chromatographic column: c₁₈Chromatography column (50 mm. times.250 mm, 10 μm); mobile phase: methanol A and water B; isocratic elution procedure: 50 vol% A; flow rate: 30 mL/min; detection wavelength: 265 nm.

Examples of the experiments

1. Structure validation

Performing nuclear magnetic resonance experiments on the tectorigenin and tectorigenin obtained in examples 9-11, wherein the obtained spectrogram data is as follows:

blackberry lily glycosides:¹³C-NMR(125MHz,DMSO-d₆)δ:180.8(C-4),157.5(C-4′),156.6(C-7),154.6(C-2),152.9(C-9),152.5(C-5),132.4(C-6),130.2(C-2′6′),121.1(C-1′),121.1(C-3),115.1(C-3′5′),106.5(C-10),100.1(C-1″),94.0(C-8),77.3(C-5″),76.7(C-4″),73.1(C-3″),69.6(C-2″),60.6(C-6″),60.3(6-CH₃). The data are consistent with the literature reports (Liuwei, Li Lujun, Li Yu, etc., preparation method of belamcandin in Chuanbelamcanda, Chinese herbal medicine 2006,37(2): 209-210.).

Tectorigenin:¹³C-NMR(125MHz,DMSO-d₆)δ:180.6(C-4),157.7(C-4′),157.5(C-7),154.2(C-2),153.3(C-9),152.8(C-5),131.5(C-6),130.2(C-2′,6′),121.9(C-1′),121.2(C-3),115.1(C-3′,5′),104.9(C-10),93.9(C-8),60.0(6-OCH₃). The data are consistent with the literature reports (Qieyingukan, Gaoyuibai, Xuebuixia, etc.. chemical composition research of blackberry lily. China pharmaceutical journal, 2006,41(15): 1133-1135.).

2. Purity detection

The purity of the crude tectoridin and tectorigenin in example 3 and the purity of tectorigenin and tectorigenin in examples 9-11 were determined.

Preparation of a test solution: precisely weighing the sample, adding 70 vol% ethanol to prepare stock solution containing 200 μ g of sample per 1ml, and diluting to obtain test solution.

Chromatographic conditions are as follows: a chromatographic column: TC-C₁₈Chromatography column (4.6 mm. times.250 mm, 5 μm); mobile phase: acetonitrile A and 0.2 vol% phosphoric acid solution B; gradient elution: 0-25 min, 18 vol% A; 25-35 min, 18-36 vol% A; 35-60 min 36 vol% A); flow rate: 1 ml/min; the column temperature was 40 ℃; the detection wavelength was 265 nm.

The detection method comprises the following steps: precisely sucking a sample solution to be tested, injecting the sample solution into an LC-20AT high performance liquid chromatograph to obtain a liquid chromatogram, and measuring the purity of the sample by a peak area normalization method, wherein the obtained result is shown in Table 3.

TABLE 3

The present invention is not limited to the above-described embodiments, and any variations, modifications, and substitutions which may occur to those skilled in the art may be made without departing from the spirit of the invention.