阅读说明：本技术 一种异射干苷元的制备方法 (Preparation method of isobelamcanda rhizome aglycone ) 是由 袁崇均 陈帅 罗森 余梦瑶 许晓燕 罗恒 汤依娜 王笳 于 2021-09-18 设计创作，主要内容包括：本发明公开了异射干苷元在制备治疗抗肿瘤的药物中的用途,本发明还提供了一种异射干苷元的提取方法。本发明通过选择特定的分离纯化方法大大提高了异射干苷元的纯度和收率,降低了成本。通过药理实验证明,本发明制备得到的异射干苷元具有抗肿瘤活性,对HCT116、A549、HepG2肿瘤细胞株活性优于射干苷元,具有广阔的市场应用前景。(The invention discloses application of isobelamcandigenin in preparing a medicine for treating tumors, and also provides an extraction method of isobelamcandigenin. The invention greatly improves the purity and yield of the isobelamcanda glycoside and reduces the cost by selecting a specific separation and purification method. Pharmacological experiments prove that the isobelamcanda chinensis aglycone prepared by the method has anti-tumor activity, is superior to that of the isobelamcanda chinensis aglycone in the activity of HCT116, A549 and HepG2 tumor cell strains, and has wide market application prospect.)

1. Use of isobelamcandigenin in preparing medicine for treating tumor is provided.

2. The use according to claim 1, wherein the medicament is a medicament for the treatment of colon, lung and/or liver cancer.

3. A method for extracting isobelamcanda rhizome aglycone is characterized in that Sichuan blackberry lily rhizome medicinal materials or blackberry lily rhizome aglycone are taken as raw materials, Na is added₂CO₃Extracting with water solution, separating, and purifying to obtain isobelamcandiol.

4. The method as claimed in claim 3, wherein the rhizoma Belamcandae is extracted with Na₂CO₃The mass volume ratio of the aqueous solution is 0.5-2 g:20 ml; the blackberry lily aglycone and Na₂CO₃The mass volume ratio of the aqueous solution is 0.5-2 g:1000 ml; the Sichuan blackberry lily medicinal material is Sichuan blackberry lily medicinal material coarse powder; the Na is₂CO₃The concentration of the aqueous solution is 0.02-0.08% w/v, g/ml, preferably 0.05% w/v, g/ml; the extraction is decoction extraction or reflux extraction, preferably decoction extraction, and the time is 4 hours.

5. The extraction method according to claim 3, characterized in that the separation method is: filtering the extractive solution, adjusting pH of the filtrate to 6.5-7.5, cooling, extracting with chloroform, concentrating the chloroform extractive solution under reduced pressure, and drying to obtain crude extract;

when the raw material of the extracting solution is a Sichuan blackberry lily medicinal material, cooling and then adding petroleum ether with the same volume as the filtrate for extraction; cooling to room temperature; the chloroform was equal in volume to the filtrate.

6. The extraction method according to claim 3, wherein the purification method comprises preparative liquid chromatography purification, wet column chromatography purification or dry column chromatography purification.

7. The extraction process according to claim 6, characterized in that the preparative liquid chromatography purification is:

dissolving the crude extract in methanol, injecting into a preparative chromatographic column, eluting with mobile phase methanol-water, collecting eluate with maximum response value, concentrating under reduced pressure, and drying to obtain isobelamcandin.

8. The extraction method according to claim 7, wherein the mass-to-volume ratio of the crude extract to methanol is 5-15 mg: 1 ml; the chromatographic column filler for preparing the liquid chromatogram is octadecylsilane chemically bonded silica, the column temperature is room temperature, and the detection wavelength is 263 nm; the volume ratio of the mobile phase methanol to water is 25:75, flow rate 10 ml/min.

9. The extraction method according to claim 6, wherein the wet column chromatography purification method in step 2) is:

dissolving the crude extract in chloroform, adding 1 time w/v of 80-mesh silica gel in g/ml, stirring, drying, grinding, loading on silica gel column, gradient eluting with mobile phase chloroform-methanol, collecting eluate at each 5 times v/w of 80-mesh silica gel, collecting eluate at 1 part, mixing 20-25 parts of eluate, concentrating under reduced pressure, and drying to obtain isobelamcandan;

the silica gel column is a 200-300 mesh silica gel column packed by a chloroform wet method, the diameter of the silica gel column is 5-6 cm, and the column height is 80 cm;

the chloroform-methanol gradient elution procedure was: eluting with chloroform-methanol solutions with volume ratios of 20:1, 10:1, 6:1 and 3:1 in sequence, wherein the amount of the chloroform-methanol solution is 80 times of the volume of 80-mesh silica gel per volume ratio, and the chloroform-methanol solution is ml/g.

10. The extraction method according to claim 6, wherein the dry column chromatography purification method is:

dissolving the crude extract in chloroform, adding 1 time of w/v, g/ml of 100-mesh silica gel, uniformly stirring, drying, grinding, loading on the silica gel column, developing with chloroform-methanol as a developing agent, taking out the silica gel, cutting into 20 equal parts, combining cutting segments from 10-14 parts of the loading position, eluting with methanol, concentrating under reduced pressure, and drying to obtain isobelamcanda rhizome aglycone;

the silica gel column is a hollow glass column filled with 10-40 mu m thin-layer chromatography silica gel, the diameter of the silica gel column is 6-7 cm, and the column height is 100 cm; the volume ratio of chloroform to methanol is 10: 1.

Technical Field

The invention belongs to the field of medicine separation and purification, and particularly relates to a preparation method of isobelamcanda rhizome aglycone.

Background

Isotectorigenin, also known as irigenin (psi-tectorigenin), is an isomer of tectorigenin and has a chemical name of 5,7, 4' -trihydroxy-8-methoxyisoflavone. The structural formulas of the blackberry lily aglycone and the isoblackberry lily aglycone are as follows:

isobelamcandiol belongs to flavonoid compounds, and for a long time, the flavonoid compounds are considered to have anti-inflammatory, antioxidant, antiallergic, liver-protecting, antithrombotic, antiviral and anticancer activities, but the research reports of the isobelamcandiol are few, so the specific pharmacodynamic action of the isobelamcandiol is not clear, and the application value of the isobelamcandiol in pharmacy is unknown at present.

Patent US20080311634a1 discloses a process for the isolation of isobelamgenin from callus tissue of iris plants, which entails first culturing the callus tissue and then extracting the isobelamgenin enriched in the callus tissue by an alcohol extraction process. The method is complex and high in cost, and the high-quality isobelamcandiol can be prepared only by cooperation between the plant cultivation field and the plant extraction field, so that the method is difficult to popularize and apply in industrial production.

Disclosure of Invention

The invention provides an application of isobelamcandigenin, aiming at solving the technical problems that the pharmacodynamic action of isobelamcandigenin is undefined, and a simple, practical and large-scale method for preparing high-purity isobelamcandigenin is unavailable so far.

The invention provides an application of isobelamcanda rhizome aglycone in preparing a medicament for treating and resisting tumors.

Further, the medicament is a medicament for treating colon cancer, lung cancer and/or liver cancer.

The invention also provides an extraction method of the isobelamcanda rhizome aglycone, which takes Sichuan blackberry lily rhizome medicinal material or blackberry lily rhizome aglycone as raw material and adds Na₂CO₃Extracting with water solution, separating, and purifying to obtain isobelamcandiol.

Further, the Sichuan blackberry lily medicinal material is mixed with Na₂CO₃The mass volume ratio of the aqueous solution is 0.5-2 g:20 ml; the blackberry lily aglycone and Na₂CO₃The mass volume ratio of the aqueous solution is 0.5-2 g:1000 ml; the Sichuan blackberry lily medicinal material is Sichuan blackberry lily medicinal material coarse powder; the Na is₂CO₃The concentration of the aqueous solution is 0.02-0.08% w/v, g/ml, preferably 0.05% w/v, g/ml; the extraction is decoction extraction or reflux extraction, preferably decoction extraction, and the time is 4 hours.

Further, the separation method comprises the following steps: filtering the extractive solution, adjusting pH of the filtrate to 6.5-7.5, cooling, extracting with chloroform, concentrating the chloroform extractive solution under reduced pressure, and drying to obtain crude extract.

Furthermore, when the raw material of the extracting solution is a Sichuan blackberry lily medicinal material, cooling and then adding petroleum ether with the same volume as the filtrate for extraction; cooling to room temperature; the chloroform was equal in volume to the filtrate.

Further, the purification method comprises a preparative liquid chromatography purification method, a wet column chromatography purification method or a dry column chromatography purification method.

Further, the preparative liquid chromatography purification method comprises the following steps:

dissolving the crude extract in methanol, injecting into a preparative chromatographic column, eluting with mobile phase methanol-water, collecting eluate with maximum response value, concentrating under reduced pressure, and drying to obtain isobelamcandiol;

further, the mass-to-volume ratio of the crude extract to methanol is 5-15 mg: 1 ml; the chromatographic column filler for preparing the liquid chromatogram is octadecylsilane chemically bonded silica, the column temperature is room temperature, and the detection wavelength is 263 nm; the volume ratio of the mobile phase methanol to water is 25:75, flow rate 10 ml/min.

Further, the wet column chromatography purification method is:

dissolving the crude extract in chloroform, adding 1 time w/v of 80-mesh silica gel in g/ml, stirring, drying, grinding, loading on silica gel column, gradient eluting with mobile phase chloroform-methanol, collecting eluate at each 5 times v/w of 80-mesh silica gel, collecting eluate at 1 part, mixing 20-25 parts of eluate, concentrating under reduced pressure, and drying to obtain isobelamcandan;

the silica gel column is a 200-300 mesh silica gel column packed by a chloroform wet method, the diameter of the silica gel column is 5-6 cm, and the column height is 80 cm;

the chloroform-methanol gradient elution procedure was: eluting with chloroform-methanol solutions with volume ratios of 20:1, 10:1, 6:1 and 3:1 in sequence, wherein the amount of the chloroform-methanol solution is 80 times of the volume of 80-mesh silica gel per volume ratio, and the chloroform-methanol solution is ml/g.

Further, the dry column chromatography purification method comprises the following steps:

dissolving the crude extract in chloroform, adding 1 time of w/v, g/ml of 100-mesh silica gel, uniformly stirring, drying, grinding, loading on the silica gel column, developing with chloroform-methanol as a developing agent, taking out the silica gel, cutting into 20 equal parts, combining cutting segments from 10-14 parts of the loading position, eluting with methanol, concentrating under reduced pressure, and drying to obtain isobelamcanda rhizome aglycone;

the silica gel column is a hollow glass column filled with 10-40 mu m thin-layer chromatography silica gel, the diameter of the silica gel column is 6-7 cm, and the column height is 100 cm; the volume ratio of chloroform to methanol is 10: 1.

according to the invention, through researching the change of chemical components of a water decoction test of the Sichuan blackberry lily under the condition of simulating human intestinal tract environment (about pH 8.5), the existence of isobelamcandin and isobelamcandin is found, the isobelamcandin is separated by using a preparation liquid phase, the chemical structure of the isobelamcandin is identified, and then the isobelamcandin can be partially converted into the isobelamcandin under the condition of weak base water decoction. Meanwhile, pharmacological experiments prove that the isobelamcanda chinensis aglycone prepared by the method has anti-tumor activity, and the activity of the isobelamcanda chinensis aglycone on HCT116, A549 and HepG2 tumor cell strains is superior to that of the isobelamcanda chinensis aglycone.

The isobelamcandiol separation and purification method reduces the purification difficulty of subsequent isobelamcandiol by decocting with alkali liquor and extracting with chloroform, fully separates impurities with high polarity and low polarity in a sample by using preparative liquid chromatography, wet column chromatography and dry column chromatography, can prepare isobelamcandiol with gram-level purity of more than 99 percent in a short time (48 hours), improves the purity and yield of isobelamcandiol products, reduces the cost, is suitable for industrial production due to simple and convenient operation, and has wide market application prospect.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1 is a chromatogram of blackberry lily aglycone

FIG. 2 example 1 Isobelamcandin chromatogram (purity 97.33%)

FIG. 3 example 2 Isobelamcandin chromatogram (purity 99.71%)

FIG. 4 example 3 Isobelamcandin chromatogram (purity 99.63%)

FIG. 5 example 4 Isobelamcandin chromatogram (purity 99.29%)

FIG. 6 total ion flow diagram of rhizoma Belamcandae water decoction sample solution

FIG. 7 Total ion flow diagram of Sichuan blackberry lily weak base water decoction sample solution

FIG. 8 is a LC-MS total ion flow diagram of blackberry lily aglycone in weak base water boiling

FIG. 9 Experimental example 2 UpLC chromatogram obtained by boiling blackberry lily aglycone in weak base water

FIG. 10 Experimental example 3 Belamcanda chinensis aglycone weak base UPLC chromatogram map

FIG. 11 Experimental example 4 blackberry lily aglycone weak base water boiling UPLC chromatogram map

FIG. 12 Experimental example 5 Saurogenin weak base poaching HPLC chromatogram

FIG. 13 test example 5 MS diagram of blackberry lily aglycone water-boiled sample

FIG. 14 Infrared Spectrum of Isobelamcandin

FIG. 15 Isobelamcandiyin hydrogen spectrum

FIG. 16 Isobelamcandiol carbon spectrum

Detailed Description

The raw materials and equipment used in the specific embodiment of the invention are known products and are obtained by purchasing a commercially available product, wherein the LC-MS is American Agilent RRLC-6410 triple quatrupol, the NMR spectrometer is Bruker AV II-600 MHz, the HPLC chromatograph is American Agilent1100 liquid chromatograph, the semi-preparative liquid chromatograph is American Agilent1200, the UPLC chromatograph is American waters ultra-high performance liquid chromatograph ACQUITY, and the bottle-hanging type freeze dryer is Beijing Bo Yi kang FD-ID-80;

rhizoma Belamcandae is purchased from Limited liability company of Chinese medicinal decoction pieces in Sichuan province, rhizoma Belamcandae aglycone is prepared by self, purity is 100%, shown in figure 1), and Na₂CO₃Hydrochloric acid, petroleum ether, chloroform (analytical purity) and the like are purchased from Chengdu Kelong chemical reagent factories.

Example 1: decocting rhizoma Belamcandae with weak base, extracting with chloroform, and separating liquid phase

1. Preparation:

collecting 100g rhizoma Belamcandae coarse powder, adding 0.05% Na₂CO₃Decocting with 20 times (2L) of water solution for 2 times (each for 2 hr), filtering, mixing filtrates, adjusting pH with 1% HCl to near neutral, concentrating the filtrate to 500ml, cooling to room temperature, and sequentially extracting with 500ml petroleum ether and 500ml chloroform. Collecting chloroform extractive solution, recovering solvent at 60 deg.C under reduced pressure, and drying to obtain extract containing isobelamcandigenin. Preparing the extract into a solution with the concentration of 10mg/ml by using methanol, purifying by using a preparative liquid chromatograph, wherein the preparative column filler is octadecylsilane chemically bonded silica, the column temperature is room temperature, and the mobile phase is methanol: water (25:75) with flow rate of 10ml/min, collecting eluate with detection wavelength of 263nm in the time period of maximum response value, recovering solvent under reduced pressure at 60 deg.C, and vacuum drying under reduced pressure to obtain isobelamcandiol 10.5 mg.

2. Detection of

Detecting isobelamcandiol by UPLC (area normalization method) under the following conditions:

waters Acquity UPLC system(ii) a The column was a Waters BEH C18 column (2.1 mm. times.50 mm, 1.7 μm); the mobile phase is acetonitrile-water, and the gradient elution is carried out for 0-14 min (10: 90-22: 78) and 14-18 min (22: 78-37: 63); volume flow rate of 0.5mL.min^-1(ii) a Column temperature: 30 ℃; the detection wavelength is 263 nm; the sample size was 1. mu.L. The theoretical plate number calculated according to isobelamcandin is not lower than 4000.

3. Results

Referring to FIG. 2, it can be seen from FIG. 2 that the purity of isobelamcandin is 97.33%.

(remark: test time about 72 hours)

Example 2: decocting rhizoma Belamcandae aglycone with weak base in water, extracting with chloroform, and separating the liquid phase

1. Preparation of

Collecting 500mg rhizoma Belamcandae aglycone, adding 0.05% Na₂CO₃Decocting 500ml of water solution for 4 hours, adjusting the pH of the decoction to be nearly neutral with 1% HCl, cooling to room temperature, and extracting with 500ml of chloroform. Collecting chloroform extractive solution, recovering solvent at 60 deg.C under reduced pressure, and drying to obtain extract containing isobelamcandigenin. Preparing the extract into a solution with the concentration of 10mg/ml by using methanol, purifying by using a preparative liquid chromatograph, wherein the preparative column filler is octadecylsilane chemically bonded silica, the column temperature is room temperature, and the mobile phase is methanol: water (25:75) with flow rate of 10ml/min, collecting eluate with maximum response value, recovering solvent at 60 deg.C under reduced pressure, and vacuum drying under reduced pressure to obtain isobelamcandiol 75.4 mg.

2. Detection of

UPLC (area normalization method) is adopted to detect isobelamcandin, and the specific detection conditions are the same as those in example 1

3. Results

Referring to FIG. 3, it can be seen from FIG. 3 that the purity of isobelamcandin is 99.71%.

(remark: test time about 72 hours)

Example 3: decocting rhizoma Belamcandae aglycone with weak base in water, extracting with chloroform, and separating by wet column chromatography

1. Preparation of

Collecting 5g rhizoma Belamcandae aglycone, adding 0.05% Na₂CO₃Decocting 5000ml of water solution for 4 hr, adjusting pH of the decoction with 1% HCl to near neutral, concentrating to 1000ml, cooling to room temperature, and adding chloroform1000ml extraction. Collecting chloroform extractive solution, recovering solvent at 60 deg.C under reduced pressure, and drying to obtain extract containing isobelamcandigenin. Dissolving the extract in 10ml chloroform, stirring with 10g 80 mesh silica gel, adsorbing, drying, and grinding to obtain sample. Performing silica gel column chromatography on a sample by using a 200-300-mesh silica gel column, performing chloroform wet-process column packing (a hollow glass column, the diameter of the hollow glass column is 5-6 cm, and the column height of the hollow glass column is 80cm), performing chloroform-methanol gradient elution (20:1, 10:1, 6:1, 3:1, and V/V), collecting eluent (one part per 50 mL) with the dosage of chloroform-methanol solution of each volume ratio being 800mL, combining 20-25 parts of eluent, recovering a solvent under reduced pressure at 60 ℃, and performing vacuum reduced pressure drying to obtain 0.851g of isoquercitrin.

2. Detection of

UPLC (area normalization) is adopted to detect isobelamcandin, the specific detection conditions are the same as those in example 13, and the result is

Referring to FIG. 4, it can be seen from FIG. 4 that the purity of isobelamcandin is 99.63%.

(remark: test time about 96 hours)

Example 4: decocting rhizoma Belamcandae aglycone with weak base water, extracting with chloroform, and separating by dry column chromatography

Collecting 5g rhizoma Belamcandae aglycone, adding 0.05% Na₂CO₃Decocting 5000ml of water solution for 4 hr, adjusting pH of the decoction with 1% HCl to neutral, concentrating to 1000ml, cooling to room temperature, extracting with 1000ml of chloroform, collecting chloroform extract, recovering solvent at 60 deg.C under reduced pressure, and drying to obtain extract containing isobelamcanda rhizome. Dissolving the extract in 10ml of chloroform, adding 10g of crude silica gel (100 meshes), stirring, drying, grinding to obtain a sample, performing thin-layer chromatography on the sample by using a silica gel dry column chromatography (a hollow glass column, the diameter of which is 6-7 cm and the column height of which is 100cm) with the thickness of 10-40 mu m, and performing chromatography by using a developing agent chloroform-methanol 100: 5(V/V) unfolding, taking out thin-layer chromatography silica gel from a ventilation hood after the unfolding, cutting into 20 equal parts, combining cutting sections which are 10-14 parts away from a sample loading position, eluting with methanol, recovering the solvent under reduced pressure at 60 ℃, and drying under reduced pressure in vacuum to obtain 0.947g of isobelamcanda rhizome glycoside.

2. Detection of

UPLC (area normalization method) is adopted to detect isobelamcandin, and the specific detection conditions are the same as those in example 1

3. Results

Referring to FIG. 5, it can be seen from FIG. 5 that the purity of isobelamcandin is 99.29%.

(remark: test time about 48 hours)

The advantageous effects of the present invention are further illustrated by the following test examples

Test example 1: isomerization research of Sichuan blackberry lily medicinal material tectoridin and tectorigenin

1. Water-decocted Sichuan blackberry lily medicinal material

Weighing 100g of rhizoma Belamcandae, pulverizing, decocting with 20 times of water for 2 times, each time for 2 hours, filtering, mixing filtrates, and concentrating under reduced pressure to dryness. Dissolving 25mg of the extract powder with chromatographic methanol, fixing the volume to a 10ml measuring flask, and filtering with a 0.45 μm microporous membrane to obtain a sample solution.

LC-MS detection is carried out on rhizoma Chuanbelamcandae water decoction sample solution

Detection conditions are as follows:

a chromatographic column: agilent C₁₈Column, mobile phase:

Time	flow rate of flow	Phase B (organic phase)
			0	0.3	30
35	0.3	48
			45	0.3	70
47	0.3	70

Flow rate: 0.3 mL/min; column temperature: 30 ℃; sample introduction amount: 2 μ L.

Mass spectrum conditions: adopting an electrospray ionization source (ESI) in a positive ion mode, wherein the spray voltage is 4000V, and the source temperature is 100 ℃; the atomization gas is nitrogen, and the atomization pressure is 40 psi; the desolventizing gas is nitrogen, the temperature is 300 ℃, and the flow rate is 10L/min; the collision gas is high-purity nitrogen, and the pressure is 0.1 MPa; the drug was detected using a full scan mode (m/z 50-1000).

The total ion flow diagram is shown in FIG. 6, and the LC-MS analysis is shown in Table 1.

TABLE 1 analysis table for combined liquid chromatography and mass spectrometry of rhizoma Belamcandae water decoction sample solution

Serial number	Time to peak/min	Mass to charge ratio [ M + H]+	Mass spectrum of major fragments	Compound (I)
					1	8.652	463.20	301	Belamcandoside
2	9.467	493.10	331、118	Tectoridin B
					3	12.329	493.20	331	Tectoridin A
4	13.148	523.20	361、118	Wild tectoridin
					5	28.440	301.10		Rhizoma Belamcandae aglycone
6	29.836	331.10	118	Tectorigenin B
					7	32.465	331.10	118	Tectorigenin A
8	33.025	361.10	331、149、118	Wild tectorigenin

As can be seen from Table 1, 8 major isoflavone components in Chuanbelamcanda chinensis are detected and assigned according to the reference.

2. Decocting rhizoma Belamcandae with alkaline water

Weighing 100g of rhizoma Belamcandae, pulverizing, and adding 20 times of 0.05% Na₂CO₃Decocting in water for 2 times, each for 2 hr, filtering, mixing filtrates, adjusting pH to near neutral with 1% HCl, and concentrating under reduced pressure to dry. Dissolving 25mg of the extract powder with chromatographic methanol, fixing the volume to a 10ml measuring flask, and filtering with a 0.45 μm microporous membrane to obtain a sample solution.

And (4) performing LC-MS detection on the Sichuan blackberry lily weak base water decoction sample solution.

Detection conditions are as follows:

a chromatographic column: agilent C₁₈And (3) a column. Mobile phase:

flow rate: 0.3 mL/min; column temperature: 30 ℃; sample introduction amount: 2 μ L.

Mass spectrum conditions: adopting an electrospray ionization source (ESI) in a positive ion mode, wherein the spray voltage is 4000V, and the source temperature is 100 ℃; the atomization gas is nitrogen, and the atomization pressure is 40 psi; the desolventizing gas is nitrogen, the temperature is 300 ℃, and the flow rate is 10L/min; the collision gas is high-purity nitrogen, and the pressure is 0.1 MPa; the drug was detected using a full scan mode (m/z 50-1000).

The total ion flow diagram is shown in FIG. 7, and the LC-MS analysis is shown in Table 2.

TABLE 2 LC-MS analysis table for rhizoma Belamcandae sample solution

Table 1 compares with table 2, 8 main isoflavone components in the rhizoma belamcandae correspond one to one, but the rhizoma belamcandae weak base water decoction sample solution has 2 more peaks (i.e. peak 2, peak 10), the mass-to-charge ratio is consistent with that of belamcandin and belamcandin, so the temporary attribution is isobelamcandin and isobelamcandin. The isogenizing of the blackberry lily glycoside and the blackberry lily aglycone can be realized under the condition that the Sichuan blackberry lily is decocted by weak base water.

Test example 2: isomerization research of blackberry lily aglycone of pure blackberry lily aglycone

Weighing 5mg of pure blackberry lily aglycone in a 25ml round bottom flask, adding 0.05% Na₂CO₃10ml of aqueous solution was heated to reflux in a water bath for 2 hours, taken out and the solution was brought to near neutrality with 1% HCl. Taking 2ml of the solution, diluting the solution to a constant volume of 10ml volumetric flask by using chromatographic methanol, and filtering the solution through a 0.45 mu m microporous filter membrane to obtain a sample solution. This solution was subjected to LC-MS detection.

Detection conditions are as follows:

a chromatographic column: agilent C₁₈And (3) a column. Mobile phase:

Time	flow rate of flow	Phase B (organic phase)
			0	0.3	10
14	0.3	22
			18	0.3	37

Flow rate: 0.3 mL/min; column temperature: 30 ℃; sample introduction amount: 2 μ L.

Mass spectrum conditions: adopting an electrospray ionization source (ESI) in a positive ion mode, wherein the spray voltage is 4000V, and the source temperature is 100 ℃; the atomization gas is nitrogen, and the atomization pressure is 40 psi; the desolventizing gas is nitrogen, the temperature is 300 ℃, and the flow rate is 10L/min; the collision gas is high-purity nitrogen, and the pressure is 0.1 MPa; the drug was detected using a full scan mode (m/z 50-1000).

Performing UPLC detection (under the detection conditions of Waters Acquity UPLC system, Waters BEH C18 column (2.1mm × 50mm, 1.7 μm) as chromatographic column, acetonitrile-water as mobile phase, gradient elution for 0-6 min (10: 90-40: 60), and volume flow rate of 0.4mL.min^-1(ii) a Column temperature: 30 ℃; the detection wavelength is 263 nm; the sample size was 1. mu.L. The theoretical plate number calculated according to isobelamcandin is not lower than 4000. )

The LC-MS total ion flow diagram is shown in FIG. 8, the UPLC is shown in FIG. 9, the LC-MS sample analysis is shown in Table 3, and the UPLC sample analysis is shown in Table 4.

TABLE 3 LC-MS analysis chart of belamcanda rhizome aglycone water boiling sample

TABLE 4 UPLC analysis chart of blackberry lily aglycone weak base water boiling sample

Serial number	Retention time (min)	Peak area (%)	Compound (I)
				1	10.145	8.85	Isobelamcandigenin
2	10.938	91.15	Rhizoma Belamcandae aglycone

As can be seen from the figure and the table, the pure belamcanda chinensis aglycone is isomerized to generate isobelamcanda chinensis aglycone under the condition of decoction with weak base water, and 8.85 percent of the isobelamcanda chinensis aglycone is decomposed after 2 hours.

Test example 3: research on isomerization of blackberry lily aglycone of pure blackberry lily aglycone product

Weighing 5mg of pure blackberry lily aglycone in a 25ml round bottom flask, adding 0.05% Na₂CO₃10ml of aqueous solution was heated to reflux in a water bath for 4 hours, taken out and the solution was brought to near neutrality with 1% HCl. Taking 2ml of the solution, diluting the solution to a constant volume of 10ml volumetric flask by using chromatographic methanol, and filtering the solution through a 0.45 mu m microporous filter membrane to obtain a sample solution. The solution was subjected to UPLC detection (for chromatographic conditions see test example 2) and the chromatogram was shown in fig. 10.

As can be seen from FIG. 10, the purity of belamcandin is 0.05% Na₂CO₃Heating and refluxing the mixture in water bath for 4 hours, wherein the peak area of the isobelamcanda rhizome is 19.45%.

Test example 4: research on isomerization of blackberry lily aglycone of pure blackberry lily aglycone

Weighing 5mg of pure blackberry lily aglycone in a 25ml round bottom flask, adding 0.05% Na₂CO₃10ml of aqueous solution was heated to reflux in a water bath for 6 hours, taken out and the solution was brought to near neutrality with 1% HCl. Taking 2ml of the solution, diluting the solution to a constant volume of 10ml volumetric flask by using chromatographic methanol, and filtering the solution through a 0.45 mu m microporous filter membrane to obtain a sample solution. The solution was subjected to UPLC detection (for chromatographic conditions see test example 2) and the chromatogram was shown in fig. 11.

As can be seen from FIG. 11, the purity of belamcandin is 0.05% Na₂CO₃Heating and refluxing in water bath for 6 hr to obtain isobelamcandin with peak area of 20.80%.

As can be seen from the results of the experimental examples 2, 3 and 4, the blackberry lily glycoside pure product is extracted for 2, 4 and 6 hours by weak alkali reflux, the isoblackberry aglycone is respectively 8.85%, 19.45% and 20.80%, when the extraction time is from 4 hours to 6 hours, the increase range of the isoblackberry aglycone is not large, so the extraction time is determined to be preferably 4 hours from the cost consideration; the reflux extraction is suitable for extracting a small amount of samples, the decoction extraction is suitable for extracting a large amount of samples, and the actual industrialized mass production is considered, so that the isobelamcanda rhizome aglycone sample is finally determined to be decocted for 4 hours by the blackberry lily aglycone weak base water.

Test example 5: initial detection for determining isobelamcanda glycoside structure

Weighing 5mg of pure blackberry lily aglycone in a 25ml round bottom flask, adding 0.1% Na₂CO₃Heating and refluxing 10ml of aqueous solution (pH 9.0-10.0) in a water bath for 4 hours, taking out, adjusting the pH of the solution to be nearly neutral by using 1% HCl, adding chloroform for extraction, taking chloroform extract, recovering the solvent at 60 ℃ under reduced pressure, and drying to obtain light yellow dry loose powder. Detection by HPLC, detection conditions: the chromatographic column is Kromasil C18 column (4.6mm × 150mm, 5 μm), the mobile phase is methanol-water (30:70), and the flow rate is 1 ml/min; column temperature: 30 ℃; the detection wavelength is 282 nm; the sample amount was 10. mu.L, the purity was 100%, and MS detection was carried out, and the results are shown in FIGS. 12 and 13. The structure of the compound is preliminarily presumed to be

From this test exampleAs can be seen, belamcanda aglycone is 0.1% Na₂CO₃Heating and refluxing the mixture in a water bath for 4 hours under the condition of an aqueous solution (pH 9.0-10.0) to generate another compound instead of the isojaponin.

Test example 6: determination of isobelamcanda glycoside structure

Isobelamgenin (sample from example 4), pale yellow needle crystal, rose red in response to magnesium hydrochloride, bright yellow in response to aluminum trichloride, negative in response to Gibbs, UV lambda_max263 nm，IR、¹H NMR、¹³C NMR is shown in FIGS. 14-16, so that the compound is identified as 5,7, 4' -trihydroxy-8-methoxyisoflavone, i.e. isojaponin, and the chemical structural formula is as follows:

test example 7: isobelamcandigenin with antitumor activity

1. Purpose of the experiment

Researches on the in vitro inhibition of HCT116, A549, HepG2 and MCF7 tumor cell proliferation effects of the blackberry lily aglycone and the isoblackberry lily aglycone.

2. Test materials

2.1 cell lines

HCT116, A549 and HepG2 tumor cell lines were purchased from the cell bank of Chinese academy of sciences. Culturing in RPMI-1640 medium (containing penicillin 100U/ml and streptomycin 100. mu.g/ml) containing 10% fetal calf serum at 37 deg.C under 5% CO₂And a saturated humidity cell culture box. The passage was digested with 0.05% trypsin-0.53 mM EDTA every 2-3 d.

MCF7 tumor cell lines were purchased from the cell bank of the department of chinese academy of sciences. Culturing in RPMI-1640 medium (containing penicillin 100U/ml, streptomycin 100. mu.g/ml, recombinant human insulin 0.4U/ml) containing 10% fetal calf serum at 37 deg.C under 5% CO₂And a saturated humidity cell culture box. The passage was digested with 0.05% trypsin-0.53 mM EDTA every 4-5 days.

2.2 samples

The test samples were obtained from the institute of Chinese medicine and pharmacology, Sichuan province (example 4). Dissolved in DMSO to prepare a 100mg/ml solution/suspension, and stored at-20 ℃ for later use.

2.3 reagents

Fetal bovine serum was purchased from Bovogen, RPMI-1640 medium, MTT, trypsin, EDTA, DMSO from Sigma, and the remaining reagents were all home-made analytical grade.

3 test method

Tumor cells in logarithmic growth phase are taken respectively for experiment, HCT-116, A549 and HepG2 cells are added into a 96-well plate according to 3 multiplied by 103 cells, MCF7 cells are added into the 96-well plate according to 8 multiplied by 103 cells, each well is filled with 180 mu l, and the cells are cultured overnight. Mu.l of each sample was added to a 96-well plate at the designed concentration and incubated for 72 hours. 4h before the end of the incubation, the medium was aspirated and 100. mu.l of Earle's BSS was added. Mu.l of MTT solution (5mg/ml) was added, and after incubation for 4 hours, 100. mu.l of 10% SDS solution (prepared in 0.01M HCl) was added to each well, and the mixture was incubated overnight in a cell incubator, and the OD value was measured at 570nm using a microplate reader. The inhibition rate of each sample concentration was calculated, and the IC was calculated using the Curve Expert₅₀The value is obtained.

4 results of the test

Inhibition rate and IC of each sample on HCT-116, A549, HepG2 and MCF7 cell proliferation₅₀As shown in table 5.

As can be seen from the above table, isobelamcanda chinensis glycoside has anti-tumor activity, and is stronger than isobelamcanda chinensis glycoside for 3 kinds of human cancer cells, namely colon cancer (HCT116), lung cancer (A549) and liver cancer (HepG2), so that the isobelamcanda chinensis glycoside can be used as an anti-tumor medicament or used as a lead compound for structural modification research and the like.

In conclusion, the isobelamcandiol separation and purification method reduces the purification difficulty of the subsequent isobelamcandiol by decocting with alkali liquor and extracting with chloroform, fully separates the impurities with high polarity and low polarity in a sample by using a preparative liquid chromatography, a wet column chromatography and a dry column chromatography, can prepare the isobelamcandiol with gram-level purity of more than 99 percent in a short time, improves the purity and yield of the isobelamcandiol product, reduces the cost, is suitable for industrial production due to simple and convenient operation, and has wide market application prospect.