阅读说明：本技术 异喹啉生物碱及其制备方法和应用 (Isoquinoline alkaloid and preparation method and application thereof ) 是由 肖晗 杨渊 徐佳 高雯琪 龚红建 于 2021-08-23 设计创作，主要内容包括：本发明涉及医药技术领域,具体公开了异喹啉生物碱及其制备方法和应用。异喹啉类生物碱化合物,具有如下式Ⅰ所示结构通式。异喹啉类生物碱化合物的制备方法,包括下述步骤：取干燥的青风藤原药材、粉碎、溶媒提取、合并提取液、用旋转蒸发仪减压除去溶媒、得青风藤提取物；用不同极性的溶媒对青风藤提取物进行萃取、合并萃取液、用旋转蒸发仪减压除去溶媒、得不同极性的萃取物；用柱色谱法对不同极性的萃取物进行分离纯化。异喹啉类生物碱化合物在制备抗肿瘤制剂中的应用。提供新的异喹啉类生物碱化合物,开拓了其在肿瘤治疗中的应用,给出了一些新的肿瘤治疗药物；还进一步提供了通过青风藤来提取异喹啉类生物碱化合物的方法。(The invention relates to the technical field of medicines, and particularly discloses isoquinoline alkaloid and a preparation method and application thereof. Isoquinoline alkaloid compound has a structural general formula shown in the following formula I. The preparation method of the isoquinoline alkaloid compound comprises the following steps: pulverizing dried caulis Sinomenii, extracting with solvent, mixing extractive solutions, and removing solvent under reduced pressure with rotary evaporator to obtain caulis Sinomenii extract; extracting caulis Sinomenii extract with different polar solvents, mixing extractive solutions, and removing solvent under reduced pressure with rotary evaporator to obtain different polar extracts; separating and purifying the extracts with different polarities by column chromatography. Application of isoquinoline alkaloid compound in preparing antitumor preparation is provided. Provides a new isoquinoline alkaloid compound, develops the application of the compound in tumor treatment and provides some new tumor treatment medicines; further provides a method for extracting the isoquinoline alkaloid compound by using the caulis sinomenii.)

1. The isoquinoline alkaloid compound is characterized by having a structural general formula as shown in a formula I:

wherein R1, R2, R3 and R4 are respectively one of the following

-OH，

-O(CH₂)_nR ', n are natural numbers, R' is a halogenated group or-CH₃，

R5 is-H or-OH.

2. According to claim 1The isoquinoline alkaloid compound is characterized in that R' is-CH₃(ii) a And 0. ltoreq. n.ltoreq.10, preferably 0. ltoreq. n.ltoreq.5.

3. The process for producing an isoquinoline alkaloid compound of claim 2, comprising the steps of:

extraction: pulverizing dried caulis Sinomenii, extracting with solvent, mixing extractive solutions, and removing solvent under reduced pressure with rotary evaporator to obtain caulis Sinomenii extract;

and (3) extraction: extracting caulis Sinomenii extract with different polar solvents, mixing extractive solutions, and removing solvent under reduced pressure with rotary evaporator to obtain different polar extracts;

and (3) purification: separating and purifying the extracts with different polarities by column chromatography.

4. The method of claim 3, wherein the solvent in the extraction step and the extraction step comprises at least one of water, absolute ethanol, ethanol with a water volume ratio of 5-70%, absolute methanol, methanol with a water volume ratio of 5-70%, cyclohexane, petroleum ether, ethyl acetate, acetone with a water volume ratio of 5-70%, n-butanol, and carbon dioxide supercritical fluid; and/or

The extraction method in the extraction step is at least any one of percolation, immersion, decoction, reflux extraction, continuous reflux extraction and supercritical fluid extraction.

5. The method for preparing an isoquinoline alkaloid compound according to claim 3, wherein the column chromatography in the purification step comprises at least one of macroporous resin column chromatography, silica gel column chromatography, sephadex column chromatography, reverse phase column chromatography, high performance liquid chromatography;

preferably, the first and second liquid crystal materials are,

the macroporous resin column chromatography comprises the following steps: the eluent is a mixed solution of methanol and water, and the elution method comprises the step of gradient elution of the mixed solution and the water in a ratio of 5: 5-10: 0 in sequence;

the silica gel column chromatography comprises the following steps: the particle size of the silica gel is 100-200 meshes, the eluent is a mixed solution of acetone and ethyl acetate, and the elution method comprises the steps of sequentially carrying out gradient elution according to the ratio of the acetone to the ethyl acetate of 5: 1-1: 1;

sephadex column chromatography: the eluent is a mixed solution of trichloromethane and methanol, and the elution method comprises gradient elution of the trichloromethane and the methanol in a ratio of 2: 1-1: 2 in sequence;

reversed phase column chromatography: the eluent is a mixed solution of methanol and water, and the elution method comprises the following steps of gradient elution of the methanol and the water in a ratio of 7: 3-10: 0;

high performance liquid chromatography: the eluent is a mixed solution of methanol or acetonitrile, water, phosphoric acid and triethylamine or diethylamine, and the proportion of the four components is (4-6): (0-6): (0-0.01): (0.05-1), and gradient elution is carried out in sequence.

6. The method for preparing isoquinoline alkaloid compounds according to claim 5, wherein the high performance liquid chromatography step is any one of the following steps:

a. the eluent of the high performance liquid chromatography is a mixed solution of methanol, water, phosphoric acid and triethylamine, the elution method is that the ratio of the four is 4:6:0.01: 0.01-9: 1:0.01:0.01, gradient elution is carried out in sequence,

b. the eluent of the high performance liquid chromatography is a mixed solution of acetonitrile, water and diethylamine, the elution method is that the ratio of the three is 6:4: 0.005-10: 0:0.005, gradient elution is carried out in sequence,

c. the eluent of the high performance liquid chromatography is a mixed solution of acetonitrile, water and triethylamine, the elution method is that the ratio of the three is 5:5: 0.01-9: 1:0.01, gradient elution is carried out in sequence,

d. the eluent of the high performance liquid chromatography is a mixed solution of acetonitrile, water, phosphoric acid and triethylamine, and the elution method comprises the following steps of carrying out gradient elution in sequence according to the ratio of 5:5:0.01: 0.01-10: 0:0.01: 0.01.

7. The use of the isoquinoline alkaloid compounds of claim 1 in the preparation of an anti-tumor formulation.

8. The use according to claim 7, wherein the isoquinoline alkaloid compound is any one of

The isoquinoline alkaloid compound is 1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1- [ [ (1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolyl) -tolyloxy ] benzyl ] -isoquinoline, preferably, the tumor is lung cancer or breast cancer,

1,2,3, 4-tetrahydro-1- [ [ 4-hydroxy-3- [4- [ (1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolinyl) methyl ] phenoxy ] phenyl ] methyl ] -7-methoxy-2-methyl-6-isoquinoline, preferably, the tumor is breast or lung cancer,

1,2,3, 4-tetrahydro-1- [ [ 4-hydroxy-3- [4- [ (1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolinyl) methyl ] phenoxy ] phenyl ] methyl ] -6-methoxy-2-methyl-7-isoquinoline, preferably, the tumor is liver cancer or breast cancer,

4- [ [1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolinyl ] methyl ] -2- [4- [ [1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolinyl ] methyl ] phenoxy ] phenol, preferably, the tumour is liver cancer or breast cancer.

9. A drug for inhibiting the proliferation of tumor cells, which comprises the isoquinoline alkaloid compound of claim 1; preferably, the isoquinoline alkaloid compound is at least one of the following compounds

The isoquinoline alkaloid compound is 1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1- [ [ (1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolyl) -tolyloxy ] benzyl ] -isoquinoline,

1,2,3, 4-tetrahydro-1- [ [ 4-hydroxy-3- [4- [ (1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolinyl) methyl ] phenoxy ] phenyl ] methyl ] -7-methoxy-2-methyl-6-isoquinoline,

1,2,3, 4-tetrahydro-1- [ [ 4-hydroxy-3- [4- [ (1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolinyl) methyl ] phenoxy ] phenyl ] methyl ] -6-methoxy-2-methyl-7-isoquinoline,

4- [ [1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolinyl ] methyl ] -2- [4- [ [1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolinyl ] methyl ] phenoxy ] phenol.

An antitumor agent characterized by containing an isoquinoline alkaloid compound according to claim 1;

preferably, the first and second liquid crystal materials are,

the isoquinoline alkaloid compound is 1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1- [ [ (1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolyl) -tolyloxy ] benzyl ] -isoquinoline, and the tumor is lung cancer or breast cancer;

the isoquinoline alkaloid compound is 1,2,3, 4-tetrahydro-1- [ [ 4-hydroxy-3- [4- [ (1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolinyl) methyl ] phenoxy ] phenyl ] methyl ] -7-methoxy-2-methyl-6-isoquinoline, and the tumor is breast cancer or lung cancer;

the isoquinoline alkaloid compound is 1,2,3, 4-tetrahydro-1- [ [ 4-hydroxy-3- [4- [ (1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolinyl) methyl ] phenoxy ] phenyl ] methyl ] -6-methoxy-2-methyl-7-isoquinoline, and the tumor is liver cancer or breast cancer;

the isoquinoline alkaloid compound is 4- [ [1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolinyl ] methyl ] -2- [4- [ [1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolinyl ] methyl ] phenoxy ] phenol, and the tumor is liver cancer or breast cancer.

Technical Field

The invention relates to the technical field of medicines, and particularly discloses isoquinoline alkaloid and a preparation method and application thereof.

Background

According to the records of 'Chinese pharmacopoeia' 2020 edition: sinomenii (Sinomenii Caulis) is dried rattan of Sinomenium acutum (Thunb.) rehd. et wils. and Sinomenium acutum (Thunb.) rehd. et wils. of Sinomenium genus of Menispermaceae family. Qingfengteng is mild in nature and bitter and pungent in taste. It enters liver and spleen meridians. Has the functions of dispelling wind-damp, dredging channels and collaterals and promoting urination. Can be used for treating rheumatalgia, joint swelling, paralysis and pruritus. Modern pharmacological research finds that the caulis sinomenii has the effects of relieving pain, calming, relieving cough, resisting inflammation, reducing blood pressure, inhibiting immunity, resisting arrhythmia, resisting myocardial ischemia, protecting reperfusion injury, blocking transmission of ganglia and neuromuscular, releasing histamine, reducing contractility of cardiac muscle and inhibiting autonomy induced by epinephrine; has exciting effect on gastrointestinal tract, but also has slight adverse side effect on gastrointestinal tract. The research on the chemical components of the orientvine is found that the orientvine mainly contains various alkaloids such as sinomenine, acutiffantrine, stephanine, N-demethylacutiffantrine, coculine and the like, and components such as racemic eugenol, acetyl oleanolic acid and the like. At present, scholars at home and abroad have less research on the anti-tumor effect of isoquinoline alkaloids in caulis sinomenii.

Malignant tumor is a global significant problem threatening the health of human beings at present, and according to the statistics of the world health organization, the total incidence rate of the malignant tumor is on the increasing trend year by year in the world. The active prevention and treatment of a few malignant tumors have been controlled, for example, the rising trend of the incidence rate of cervical cancer in developed areas in Europe and America has been suppressed, however, the incidence rate of breast cancer has been continuously rising since the last 70 s of the past century. The latest Chinese cancer statistical data show that the incidence of breast cancer in China is in the 1 st position of female malignant tumor in tumor registration area. Although recent studies on breast cancer have achieved considerable success, and diagnostic strategies and methods have also made significant progress, about one third of breast cancer patients still die from metastatic breast cancer or its treatment complications. Therefore, the search and development of a new high-efficiency low-toxicity therapeutic drug for breast cancer is an important problem to be solved urgently in the research and development of new anti-tumor drugs in China.

Disclosure of Invention

Aiming at the problems, the invention mainly aims to solve the defects of the existing isoquinoline alkaloid compounds and make up the application blank of part of the isoquinoline alkaloid compounds; meanwhile, the further research is carried out on the extraction of isoquinoline alkaloid compounds from the caulis sinomenii.

In order to solve the problems, the invention adopts the following technical scheme:

isoquinoline alkaloid compound has the following structural general formula I:

wherein R1, R2, R3 and R4 are respectively one of the following

-OH，

-O (CH2) nR ', n is a natural number, R' is halo or-CH 3;

r5 is-H or-OH.

In some forms, R' is-CH₃(ii) a 0. ltoreq. n.ltoreq.10, preferably 0. ltoreq. n.ltoreq.5.

The preparation method of the isoquinoline alkaloid compound comprises the following steps:

extraction: pulverizing dried caulis Sinomenii, extracting with solvent, mixing extractive solutions, and removing solvent under reduced pressure with rotary evaporator to obtain caulis Sinomenii extract;

and (3) extraction: extracting caulis Sinomenii extract with different polar solvents, mixing extractive solutions, and removing solvent under reduced pressure with rotary evaporator to obtain different polar extracts;

and (3) purification: separating and purifying the extracts with different polarities by column chromatography.

In some embodiments, the solvent in the extracting step and the extracting step respectively comprises at least one of water, absolute ethyl alcohol, ethanol with a water volume ratio of 5-70%, absolute methanol, methanol with a water volume ratio of 5-70%, cyclohexane, petroleum ether, ethyl acetate, acetone with a water volume ratio of 5-70%, n-butanol, and carbon dioxide supercritical fluid.

In some embodiments, the extraction method in the extraction step is at least one of percolation, maceration, decoction, reflux extraction, continuous reflux extraction, and supercritical fluid extraction.

In some embodiments, the column chromatography in the purification step includes at least one of macroporous resin column chromatography, silica gel column chromatography, sephadex column chromatography, reverse phase column chromatography, and high performance liquid chromatography;

preferably, the first and second liquid crystal materials are,

the macroporous resin column chromatography comprises the following steps: the eluent is a mixed solution of methanol and water, and the elution method comprises the step of gradient elution of the mixed solution and the water in a ratio of 5: 5-10: 0 in sequence;

the silica gel column chromatography comprises the following steps: the particle size of the silica gel is 100-200 meshes, the eluent is a mixed solution of acetone and ethyl acetate, and the elution method comprises the steps of sequentially carrying out gradient elution according to the ratio of the acetone to the ethyl acetate of 5: 1-1: 1;

sephadex column chromatography: the eluent is a mixed solution of trichloromethane and methanol, and the elution method comprises gradient elution of the trichloromethane and the methanol in a ratio of 2: 1-1: 2 in sequence;

reversed phase column chromatography: the eluent is a mixed solution of methanol and water, and the elution method comprises the step of gradient elution of the mixed solution and the water according to the ratio of 7: 3-10: 0;

high performance liquid chromatography: the eluent is a mixed solution of methanol or acetonitrile, water, phosphoric acid and triethylamine or diethylamine, and the elution method comprises the following four components in proportion (4-6): (0-6): (0-0.01): (0.05-1), and gradient elution is carried out in sequence. Wherein, in the four components, one of methanol or acetonitrile and one of triethylamine or diethylamine; in some cases, diethylamine is not used simultaneously with methanol.

In some embodiments, the high performance liquid chromatography step is any one of:

a. the eluent of the high performance liquid chromatography is a mixed solution of methanol, water, phosphoric acid and triethylamine, the elution method is that the ratio of the four is 4:6:0.01: 0.01-9: 1:0.01:0.01, gradient elution is carried out in sequence,

b. the eluent of the high performance liquid chromatography is a mixed solution of acetonitrile, water and diethylamine, the elution method is that the ratio of the three is 6:4: 0.005-10: 0:0.005, gradient elution is carried out in sequence,

c. the eluent of the high performance liquid chromatography is a mixed solution of acetonitrile, water and triethylamine, the elution method is that the ratio of the three is 5:5: 0.01-9: 1:0.01, gradient elution is carried out in sequence,

d. the eluent of the high performance liquid chromatography is a mixed solution of acetonitrile, water, phosphoric acid and triethylamine, and the elution method comprises the following steps of carrying out gradient elution in sequence according to the ratio of 5:5:0.01: 0.01-10: 0:0.01: 0.01.

Application of isoquinoline alkaloid compound in preparing antitumor preparation is provided.

In some embodiments, the isoquinoline alkaloid compound is any one of the following compounds

The isoquinoline alkaloid compound is 1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1- [ [ (1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolyl) -tolyloxy ] benzyl ] -isoquinoline, preferably, the compound is mainly applied to the preparation of medicaments for preventing and treating lung cancer or breast cancer,

1,2,3, 4-tetrahydro-1- [ [ 4-hydroxy-3- [4- [ (1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolinyl) methyl ] phenoxy ] phenyl ] methyl ] -7-methoxy-2-methyl-6-isoquinoline, preferably, the compound is mainly applied to the preparation of medicaments for preventing and treating breast cancer or lung cancer,

1,2,3, 4-tetrahydro-1- [ [ 4-hydroxy-3- [4- [ (1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolinyl) methyl ] phenoxy ] phenyl ] methyl ] -6-methoxy-2-methyl-7-isoquinoline, preferably, the compound is mainly applied to the preparation of medicaments for preventing and treating liver cancer or breast cancer,

4- [ [1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolinyl ] methyl ] -2- [4- [ [1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolinyl ] methyl ] phenoxy ] phenol, preferably, the compound is mainly applied to the preparation of medicines for preventing and treating liver cancer or breast cancer.

A drug for inhibiting tumor cell proliferation, which contains the isoquinoline alkaloid compound; preferably, the isoquinoline alkaloid compound is at least one of the following compounds

The isoquinoline alkaloid compound is 1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1- [ [ (1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolyl) -tolyloxy ] benzyl ] -isoquinoline,

1,2,3, 4-tetrahydro-1- [ [ 4-hydroxy-3- [4- [ (1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolinyl) methyl ] phenoxy ] phenyl ] methyl ] -7-methoxy-2-methyl-6-isoquinoline,

1,2,3, 4-tetrahydro-1- [ [ 4-hydroxy-3- [4- [ (1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolinyl) methyl ] phenoxy ] phenyl ] methyl ] -6-methoxy-2-methyl-7-isoquinoline,

4- [ [1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolinyl ] methyl ] -2- [4- [ [1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolinyl ] methyl ] phenoxy ] phenol.

An antitumor agent comprising the isoquinoline alkaloid compound;

preferably, the first and second liquid crystal materials are,

the isoquinoline alkaloid compound is 1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1- [ [ (1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolyl) -tolyloxy ] benzyl ] -isoquinoline, and the tumor is lung cancer or breast cancer;

the isoquinoline alkaloid compound is 1,2,3, 4-tetrahydro-1- [ [ 4-hydroxy-3- [4- [ (1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolinyl) methyl ] phenoxy ] phenyl ] methyl ] -7-methoxy-2-methyl-6-isoquinoline, and the tumor is breast cancer or lung cancer;

the isoquinoline alkaloid compound is 1,2,3, 4-tetrahydro-1- [ [ 4-hydroxy-3- [4- [ (1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolinyl) methyl ] phenoxy ] phenyl ] methyl ] -6-methoxy-2-methyl-7-isoquinoline, and the tumor is liver cancer or breast cancer;

the isoquinoline alkaloid compound is 4- [ [1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolinyl ] methyl ] -2- [4- [ [1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolinyl ] methyl ] phenoxy ] phenol, and the tumor is liver cancer or breast cancer.

The invention has the beneficial effects that:

provides a new isoquinoline alkaloid compound, simultaneously develops the application of the compound in tumor treatment, provides a plurality of new tumor treatment modes, and particularly provides a new inhibiting medicament for partial cancers; further provides a method for extracting the isoquinoline alkaloid compound by using the caulis sinomenii.

Drawings

FIG. 1 is a nuclear magnetic hydrogen spectrum and a high performance liquid chromatogram of the compound of example 1;

FIG. 2 is a nuclear magnetic hydrogen spectrum and a high performance liquid chromatogram of the compound of example 2;

FIG. 3 is a nuclear magnetic hydrogen spectrum and a high performance liquid chromatogram of the compound of example 3;

FIG. 4 is a nuclear magnetic hydrogen spectrum and a high performance liquid chromatogram of the compound of example 4;

FIG. 5 shows a general structure of isoquinoline alkaloid compounds.

Detailed Description

The invention is further illustrated below:

the isoquinoline alkaloid compound has the following structural general formula:

wherein R1, R2, R3 and R4 are respectively one of the following

-OH，

-O (CH2) nR ', n is a natural number, R' is halo or-CH 3;

r5 is-H or-OH.

In some embodiments, R 'is-CH 3, 0. ltoreq. n.ltoreq.10, preferably 0. ltoreq. n.ltoreq.5, and when R' is halo, 1. ltoreq. n.

The preparation method of the isoquinoline alkaloid compound comprises the following steps:

extraction: pulverizing dried caulis Sinomenii, extracting with solvent, mixing extractive solutions, and removing solvent under reduced pressure with rotary evaporator to obtain caulis Sinomenii extract;

and (3) extraction: extracting caulis Sinomenii extract with different polar solvents, mixing extractive solutions, and removing solvent under reduced pressure with rotary evaporator to obtain different polar extracts;

and (3) purification: separating and purifying the extracts with different polarities by column chromatography.

In some embodiments, the solvent in the extracting step and the extracting step respectively comprises at least one of water, absolute ethyl alcohol, ethanol with a water volume ratio of 5-70%, absolute methanol, methanol with a water volume ratio of 5-70%, cyclohexane, petroleum ether, ethyl acetate, acetone with a water volume ratio of 5-70%, n-butanol, and carbon dioxide supercritical fluid.

In some embodiments, the extraction method in the extraction step is at least one of percolation, maceration, decoction, reflux extraction, continuous reflux extraction, and supercritical fluid extraction.

In some embodiments, the column chromatography in the purification step includes at least one of macroporous resin column chromatography, silica gel column chromatography, sephadex column chromatography, reverse phase column chromatography, and high performance liquid chromatography;

preferably, the first and second liquid crystal materials are,

the macroporous resin column chromatography comprises the following steps: the eluent is a mixed solution of methanol and water, and the elution method comprises the step of gradient elution of the mixed solution and the water in a ratio of 5: 5-10: 0 in sequence;

the silica gel column chromatography comprises the following steps: the particle size of the silica gel is 100-200 meshes, the eluent is a mixed solution of acetone and ethyl acetate, and the elution method comprises the steps of sequentially carrying out gradient elution according to the ratio of the acetone to the ethyl acetate of 5: 1-1: 1;

sephadex column chromatography: the eluent is a mixed solution of trichloromethane and methanol, and the elution method comprises gradient elution of the trichloromethane and the methanol in a ratio of 2: 1-1: 2 in sequence;

reversed phase column chromatography: the eluent is a mixed solution of methanol and water, and the elution method comprises the step of gradient elution of the mixed solution and the water according to the ratio of 7: 3-10: 0;

high performance liquid chromatography: the eluent is a mixed solution of methanol or acetonitrile, water, phosphoric acid and triethylamine or diethylamine, and the elution method comprises the following four components in proportion (4-6): (0-6): (0-0.01): (0.05-1), and gradient elution is carried out in sequence. Wherein, methanol or acetonitrile is selected, and triethylamine or diethylamine is selected from four components of methanol or acetonitrile, water, phosphoric acid and triethylamine or diethylamine; in some cases, diethylamine is not used simultaneously with methanol.

In some embodiments, the high performance liquid chromatography step is any one of:

a. when the extracted isoquinoline alkaloid compound is mainly 1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1- [ [ (1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolinyl) -tolyloxy ] benzyl ] -isoquinoline: the eluent of the high performance liquid chromatography is a mixed solution of methanol, water, phosphoric acid and triethylamine, the elution method is that the ratio of the four is 4:6:0.01: 0.01-9: 1:0.01:0.01, gradient elution is carried out in sequence, the gradient interval is not particularly limited, and the aim is to extract higher-purity isoquinoline alkaloids;

b. when the extracted isoquinoline alkaloid compound is mainly 1,2,3, 4-tetrahydro-1- [ [ 4-hydroxy-3- [4- [ (1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolinyl) methyl ] phenoxy ] phenyl ] methyl ] -7-methoxy-2-methyl-6-isoquinoline: the eluent of the high performance liquid chromatography is a mixed solution of acetonitrile, water and diethylamine, the ratio of the eluent to the acetonitrile to the water to the diethylamine is 6:4: 0.005-10: 0:0.005, gradient elution is carried out in sequence, the gradient interval is not particularly limited, and the higher purity isoquinoline alkaloids can be extracted;

c. when the extracted isoquinoline alkaloid compound is mainly 1,2,3, 4-tetrahydro-1- [ [ 4-hydroxy-3- [4- [ (1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolinyl) methyl ] phenoxy ] phenyl ] methyl ] -6-methoxy-2-methyl-7-isoquinoline: the eluent of the high performance liquid chromatography is a mixed solution of acetonitrile, water and triethylamine, the elution method is that the ratio of the acetonitrile to the water to the triethylamine is 5:5: 0.01-9: 1:0.01, the gradient interval is not particularly limited, and the higher purity isoquinoline alkaloid can be extracted;

d. when the extracted isoquinoline alkaloid compound is mainly 4- [ [1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolinyl ] methyl ] -2- [4- [ [1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolinyl ] methyl ] phenoxy ] phenol: the eluent of the high performance liquid chromatography is a mixed solution of acetonitrile, water, phosphoric acid and triethylamine, the elution method is that the ratio of the acetonitrile to the water to the phosphoric acid to the triethylamine is 5:5:0.01: 0.01-10: 0:0.01:0.01, gradient elution is carried out in sequence, the gradient interval is not particularly limited, and the aim is to extract higher-purity isoquinoline alkaloids.

Application of isoquinoline alkaloid compound in preparing antitumor preparation is provided.

In some embodiments, the isoquinoline alkaloid compound is any one of the following compounds

The isoquinoline alkaloid compound is 1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1- [ [ (1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolyl) -tolyloxy ] benzyl ] -isoquinoline, preferably, the tumor is lung cancer or breast cancer,

1,2,3, 4-tetrahydro-1- [ [ 4-hydroxy-3- [4- [ (1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolinyl) methyl ] phenoxy ] phenyl ] methyl ] -7-methoxy-2-methyl-6-isoquinoline, preferably, the tumor is breast or lung cancer,

1,2,3, 4-tetrahydro-1- [ [ 4-hydroxy-3- [4- [ (1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolinyl) methyl ] phenoxy ] phenyl ] methyl ] -6-methoxy-2-methyl-7-isoquinoline, preferably, the tumor is liver cancer or breast cancer,

4- [ [1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolinyl ] methyl ] -2- [4- [ [1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolinyl ] methyl ] phenoxy ] phenol, preferably, the tumour is liver cancer or breast cancer.

A drug for inhibiting tumor cell proliferation, which contains the isoquinoline alkaloid compound; preferably, the isoquinoline alkaloid compound is at least one of the following compounds

The isoquinoline alkaloid compound is 1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1- [ [ (1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolyl) -tolyloxy ] benzyl ] -isoquinoline,

1,2,3, 4-tetrahydro-1- [ [ 4-hydroxy-3- [4- [ (1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolinyl) methyl ] phenoxy ] phenyl ] methyl ] -7-methoxy-2-methyl-6-isoquinoline,

1,2,3, 4-tetrahydro-1- [ [ 4-hydroxy-3- [4- [ (1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolinyl) methyl ] phenoxy ] phenyl ] methyl ] -6-methoxy-2-methyl-7-isoquinoline,

4- [ [1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolinyl ] methyl ] -2- [4- [ [1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolinyl ] methyl ] phenoxy ] phenol.

The above-mentioned "at least one of the following" means one or more of the following.

An antitumor agent comprising the isoquinoline alkaloid compound;

in some of the following specific cases:

the isoquinoline alkaloid compound is 1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1- [ [ (1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolyl) -tolyloxy ] benzyl ] -isoquinoline, and the tumor is lung cancer or breast cancer; namely, the compound is contained in the medicine for treating lung cancer or breast cancer.

The isoquinoline alkaloid compound is 1,2,3, 4-tetrahydro-1- [ [ 4-hydroxy-3- [4- [ (1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolinyl) methyl ] phenoxy ] phenyl ] methyl ] -7-methoxy-2-methyl-6-isoquinoline, and the tumor is breast cancer or lung cancer; namely, the compound is contained in the medicine for treating breast cancer or lung cancer.

The isoquinoline alkaloid compound is 1,2,3, 4-tetrahydro-1- [ [ 4-hydroxy-3- [4- [ (1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolinyl) methyl ] phenoxy ] phenyl ] methyl ] -6-methoxy-2-methyl-7-isoquinoline, and the tumor is liver cancer or breast cancer; namely, the compound is contained in the medicine for treating liver cancer or breast cancer.

The isoquinoline alkaloid compound is 4- [ [1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolinyl ] methyl ] -2- [4- [ [1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolinyl ] methyl ] phenoxy ] phenol, and the tumor is liver cancer; namely, the compound is contained in the medicine for treating liver cancer or breast cancer.

Example 1

This example provides a method for the preparation of compound 1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1- [ [ (1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolinyl) -tolyloxy ] benzyl ] -isoquinoline and methods and results for the determination of its anti-tumor activity.

The chemical structural formula is as follows:

the extraction, separation and purification steps are as follows:

s1, extraction: pulverizing caulis Sinomenii, soaking in 10% ethanol, extracting under reflux for 3 times (2 hr each time), filtering, collecting filtrate, and concentrating under reduced pressure to obtain concentrated solution;

s2, extraction: dissolving the concentrated solution in 0.5-1.5 times of water, sequentially extracting with petroleum ether, ethyl acetate and n-butyl alcohol with the same volume, collecting the extraction solutions with different polarities, and concentrating under reduced pressure to obtain a concentrated solution;

s3, purification: the ethyl acetate extract obtained in S2 is subjected to repeated purification by macroporous resin column chromatography, silica gel column chromatography, sephadex column chromatography, reverse phase column chromatography, or the like in this order.

In the macroporous column chromatography in the S3, the eluent is a mixed solution of methanol and water, and the elution method is that the two are sequentially subjected to gradient elution according to the ratio of 5: 5-10: 0;

the particle size of silica gel in the silica gel column chromatography in the S3 is 100-200 meshes, the eluent is a mixed solution of acetone and ethyl acetate, and the elution method is that the two are sequentially subjected to gradient elution according to the ratio of 5: 1-1: 1;

the eluent of the sephadex column chromatography in the S3 is a mixed solution of chloroform and methanol, and the elution method is that the chloroform and the methanol are sequentially subjected to gradient elution according to the ratio of 2: 1-1: 2;

the eluent of the reversed-phase column chromatography in the S3 is a mixed solution of methanol and water, and the elution method is that gradient elution is carried out sequentially according to the ratio of 7: 3-10: 0;

the eluent of the high performance liquid chromatography in the S3 is a mixed solution of methanol, water, phosphoric acid and triethylamine, and the elution method is that the four eluent are in a ratio of 4:6:0.01: 0.01-9: 1:0.01:0.01, and gradient elution is carried out sequentially.

One specific mode is as follows:

sample dissolving solvent: 80% methanol water;

the elution method comprises the following steps: 75% methanol + 25% water + 0.1% phosphoric acid + 0.1% triethylamine, elution duration: for 20min, gradually increasing the methanol ratio of 75% methanol and 25% water to 100% methanol, and carrying out elution for a period of time: 40 min;

flow rate: 1.0 mL/min;

a chromatographic column: chromacore 120C 18, 5 μm, 4.6 × 250 mm;

column temperature: at 40 ℃.

To prepare 1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1- [ [ (1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolyl) -tolyloxy ] benzyl ] -isoquinoline, and the product detection refers to figure 1. The target product can be prepared within the range of the proportioning scheme.

The in vitro anti-tumor activity test method comprises the following steps: the tumor cells were cultured in a culture medium containing 10% calf serum, penicillin and streptomycin, and cultured at 37 ℃ in a 5% CO2 incubator. And taking cells in logarithmic growth phase for proliferation inhibition experiment. MTT method is used to detect proliferation inhibition of the extract on 5 kinds of tumor cells (human breast cancer cell MDA-MB-231, human cervical cancer cell HeLa, human liver cancer cell HepG2, human prostate cancer cell PC-3 and human lung cancer cell A549), and cell activity data are shown in Table 1-1.

TABLE 1-1 data Table of Compound 1-cell viability%

The concentration of the extract is used for plotting the growth inhibition rate of the tumor cells, so that a dose-effect curve can be obtained, and the inhibition rate is calculated. The results of in vitro antitumor activity experiments: see tables 1-2.

Tables 1 to 2: inhibition rate of 1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1- [ [ (1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolinyl) -tolyloxy ] benzyl ] -isoquinoline on 5 tumor cells

Example 2

This example provides a method for the preparation of the compound 1,2,3, 4-tetrahydro-1- [ [ 4-hydroxy-3- [4- [ (1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolinyl) methyl ] phenoxy ] phenyl ] methyl ] -7-methoxy-2-methyl-6-isoquinoline and methods for the determination of its anti-tumor activity and the results.

The chemical structural formula is as follows:

the extraction, separation and purification steps are as follows:

s1, extraction: pulverizing caulis Sinomenii, soaking in 10% ethanol, extracting under reflux for 3 times (2 hr each time), filtering, collecting filtrate, and concentrating under reduced pressure to obtain concentrated solution;

s2, extraction: dissolving the concentrated solution in 0.5-1.5 times of water, sequentially extracting with petroleum ether, ethyl acetate and n-butyl alcohol with the same volume, collecting the extraction solutions with different polarities, and concentrating under reduced pressure to obtain a concentrated solution;

s3, purification: the ethyl acetate extract obtained in S2 is subjected to repeated purification by macroporous resin column chromatography, silica gel column chromatography, sephadex column chromatography, reverse phase column chromatography, or the like in this order.

In the macroporous column chromatography in the S3, the eluent is a mixed solution of methanol and water, and the elution method is that the two are sequentially subjected to gradient elution according to the ratio of 5: 5-10: 0;

the particle size of silica gel in the silica gel column chromatography in the S3 is 100-200 meshes, the eluent is a mixed solution of acetone and ethyl acetate, and the elution method is that the two are sequentially subjected to gradient elution according to the ratio of 5: 1-1: 1;

the eluent of the sephadex column chromatography in the S3 is a mixed solution of chloroform and methanol, and the elution method is that the chloroform and the methanol are sequentially subjected to gradient elution according to the ratio of 2: 1-1: 2;

the eluent of the reversed-phase column chromatography in the S3 is a mixed solution of methanol and water, and the elution method is that gradient elution is carried out sequentially according to the ratio of 7: 3-10: 0;

the eluent of the high performance liquid chromatography in the S3 is a mixed solution of acetonitrile, water and diethylamine, and the elution method is that the ratio of the three is 6:4: 0.005-10: 0:0.005, and gradient elution is carried out in sequence.

One specific mode is as follows:

sample dissolving solvent: 70% acetonitrile water;

eluent: 65% acetonitrile + 35% water + 0.05% diethylamine, elution duration: 15min, gradually increasing the proportion of acetonitrile to 100% acetonitrile by 65% acetonitrile and 35% water, wherein the elution time is as follows: 30 min;

flow rate: 0.8 mL/min;

a chromatographic column: einrichrom ODS BP, 5 μm, 250 × 4.6 mm;

column temperature: at 40 ℃.

To prepare 1,2,3, 4-tetrahydro-1- [ [ 4-hydroxy-3- [4- [ (1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolinyl) methyl ] phenoxy ] phenyl ] methyl ] -7-methoxy-2-methyl-6-isoquinoline, and the product detection refers to a figure 2. The target product can be prepared within the range of the proportioning scheme.

The in vitro anti-tumor activity test method comprises the following steps: the tumor cells were cultured in a culture medium containing 10% calf serum, penicillin and streptomycin, and cultured at 37 ℃ in a 5% CO2 incubator. And taking cells in logarithmic growth phase for proliferation inhibition experiment. MTT method is used to detect proliferation inhibition of the extract on 5 kinds of tumor cells (human breast cancer cell MDA-MB-231, human cervical cancer cell HeLa, human liver cancer cell HepG2, human prostate cancer cell PC-3 and human lung cancer cell A549), and cell activity data are shown in Table 2-1.

TABLE 2-1 data Table of Compound 2-cell viability%

The concentration of the extract is used for plotting the growth inhibition rate of the tumor cells, so that a dose-effect curve can be obtained, and the inhibition rate is calculated. The results of in vitro antitumor activity experiments: see Table 2-2.

Tables 2 to 2: inhibition rate of 1,2,3, 4-tetrahydro-1- [ [ 4-hydroxy-3- [4- [ (1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolinyl) methyl ] phenoxy ] phenyl ] methyl ] -7-methoxy-2-methyl-6-isoquinoline against 5 tumor cells

Example 3

This example provides a method for the preparation of the compound 1,2,3, 4-tetrahydro-1- [ [ 4-hydroxy-3- [4- [ (1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolinyl) methyl ] phenoxy ] phenyl ] methyl ] -6-methoxy-2-methyl-7-isoquinoline and methods for the determination of its anti-tumor activity and the results.

The chemical structural formula is as follows:

the extraction, separation and purification steps are as follows:

s1, extraction: pulverizing caulis Sinomenii, soaking in 10% ethanol, extracting under reflux for 3 times (2 hr each time), filtering, collecting filtrate, and concentrating under reduced pressure to obtain concentrated solution;

s2, extraction: dissolving the concentrated solution in 0.5-1.5 times of water, sequentially extracting with petroleum ether, ethyl acetate and n-butyl alcohol with the same volume, collecting the extraction solutions with different polarities, and concentrating under reduced pressure to obtain a concentrated solution;

s3, purification: the ethyl acetate extract obtained in S2 is subjected to repeated purification by macroporous resin column chromatography, silica gel column chromatography, sephadex column chromatography, reverse phase column chromatography, or the like in this order.

In the macroporous column chromatography in the S3, the eluent is a mixed solution of methanol and water, and the elution method is that the two are sequentially subjected to gradient elution according to the ratio of 5: 5-10: 0;

the particle size of silica gel in the silica gel column chromatography in the S3 is 100-200 meshes, the eluent is a mixed solution of acetone and ethyl acetate, and the elution method is that the two are sequentially subjected to gradient elution according to the ratio of 5: 1-1: 1;

the eluent of the sephadex column chromatography in the S3 is a mixed solution of chloroform and methanol, and the elution method is that the chloroform and the methanol are sequentially subjected to gradient elution according to the ratio of 2: 1-1: 2;

the eluent of the reversed-phase column chromatography in the S3 is a mixed solution of methanol and water, and the elution method is that gradient elution is carried out sequentially according to the ratio of 7: 3-10: 0;

the eluent of the high performance liquid chromatography in the S3 is a mixed solution of acetonitrile, water and triethylamine, the elution method is that the ratio of the three is 5:5: 0.01-9: 1:0.01, and gradient elution is carried out sequentially.

One specific mode is as follows:

sample dissolving solvent: 90% methanol water;

eluent: 50% of acetonitrile, 50% of water and 0.1% of triethylamine, wherein the elution time is as follows: 20min, gradually increasing the proportion of acetonitrile to 90% of acetonitrile and 10% of water by using 50% of acetonitrile and 50% of water, wherein the elution time is as follows: 40 min;

flow rate: 0.8 mL/min;

a chromatographic column: nano-micro UniSil 5-100C 18 Ultra, 250 × 4.6mm Column;

column temperature: at 40 ℃.

To obtain 1,2,3, 4-tetrahydro-1- [ [ 4-hydroxy-3- [4- [ (1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolinyl) methyl ] phenoxy ] phenyl ] methyl ] -6-methoxy-2-methyl-7-isoquinoline, and the product detection refers to figure 3. The target product can be prepared within the range of the proportioning scheme.

The in vitro anti-tumor activity test method comprises the following steps: the tumor cells were cultured in a culture medium containing 10% calf serum, penicillin and streptomycin, and cultured at 37 ℃ in a 5% CO2 incubator. And taking cells in logarithmic growth phase for proliferation inhibition experiment. MTT method is used to detect proliferation inhibition of the extract on 5 kinds of tumor cells (human breast cancer cell MDA-MB-231, human cervical cancer cell HeLa, human liver cancer cell HepG2, human prostate cancer cell PC-3 and human lung cancer cell A549), and cell activity data are shown in Table 3-1.

Table 3-1: data sheet for Compound 3-cell viability [% ]

The concentration of the extract is used for plotting the growth inhibition rate of the tumor cells, so that a dose-effect curve can be obtained, and the inhibition rate is calculated. The results of in vitro antitumor activity experiments: see Table 3-2.

Tables 3-2: inhibition rate of 1,2,3, 4-tetrahydro-1- [ [ 4-hydroxy-3- [4- [ (1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolinyl) methyl ] phenoxy ] phenyl ] methyl ] -6-methoxy-2-methyl-7-isoquinoline against 5 tumor cells

Example 4

This example provides a method for the preparation of the compound 4- [ [1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolinyl ] methyl ] -2- [4- [ [1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolinyl ] methyl ] phenoxy ] phenol and a method for the determination of its anti-tumor activity and results therefrom.

The chemical structural formula is as follows:

the extraction, separation and purification steps are as follows:

s1, extraction: pulverizing caulis Sinomenii, soaking in 5% ethanol, extracting under reflux for 3 times (2 hr each time), filtering, collecting filtrate, and concentrating under reduced pressure to obtain concentrated solution;

s2, extraction: dissolving the concentrated solution in 0.5-1.5 times of water, sequentially extracting with petroleum ether, ethyl acetate and n-butyl alcohol with the same volume, collecting the extraction solutions with different polarities, and concentrating under reduced pressure to obtain a concentrated solution;

s3, purification: the ethyl acetate extract obtained in S2 is subjected to repeated purification by macroporous resin column chromatography, silica gel column chromatography, sephadex column chromatography, reverse phase column chromatography, or the like in this order.

In the macroporous column chromatography in the S3, the eluent is a mixed solution of methanol and water, and the elution method is that the two are sequentially subjected to gradient elution according to the ratio of 5: 5-10: 0;

the particle size of silica gel in the silica gel column chromatography in the S3 is 100-200 meshes, the eluent is a mixed solution of acetone and ethyl acetate, and the elution method is that the two are sequentially subjected to gradient elution according to the ratio of 5: 1-1: 1;

the eluent of the sephadex column chromatography in the S3 is a mixed solution of chloroform and methanol, and the elution method is that the chloroform and the methanol are sequentially subjected to gradient elution according to the ratio of 2: 1-1: 2;

the eluent of the reversed-phase column chromatography in the S3 is a mixed solution of methanol and water, and the elution method is that gradient elution is carried out sequentially according to the ratio of 7: 3-10: 0;

the eluent of the high performance liquid chromatography in the S3 is a mixed solution of acetonitrile, water, phosphoric acid and triethylamine, and the elution method comprises the following steps of carrying out gradient elution in sequence according to the ratio of 5:5:0.01: 0.01-10: 0:0.01: 0.01.

One specific mode is as follows:

sample dissolving solvent: 50% methanol water;

eluent: 45% of acetonitrile, 55% of water, 0.1% of phosphoric acid and 0.1% of triethylamine, wherein the elution time is as follows: 20min, gradually increasing the proportion of acetonitrile to 95% of acetonitrile and 5% of water by 45% of acetonitrile and 55% of water, wherein the elution time is as follows: 40 min;

flow rate: 1.0 mL/min;

a chromatographic column: MinXi Teeh CG-C185 mic, 250 × 4.6 mm;

column temperature: at 40 ℃.

Preparing 4- [ [1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolyl ] methyl ] -2- [4- [ [1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolyl ] methyl ] phenoxy ] phenol, and referring to a figure 4 for product detection. The target product can be prepared within the range of the proportioning scheme.

The in vitro anti-tumor activity test method comprises the following steps: the tumor cells were cultured in a culture medium containing 10% calf serum, penicillin and streptomycin, and cultured at 37 ℃ in a 5% CO2 incubator. And taking cells in logarithmic growth phase for proliferation inhibition experiment. MTT method is used to detect proliferation inhibition of the extract on 5 kinds of tumor cells (human breast cancer cell MDA-MB-231, human cervical cancer cell HeLa, human liver cancer cell HepG2, human prostate cancer cell PC-3 and human lung cancer cell A549), and cell activity data are shown in Table 4-1.

TABLE 4-1 data Table of Compound 4-cell viability%

The concentration of the extract is used for plotting the growth inhibition rate of the tumor cells, so that a dose-effect curve can be obtained, and the inhibition rate is calculated. The results of in vitro antitumor activity experiments: see Table 4-2.

Tables 4-2: inhibition rate of 4- [ [1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolinyl ] methyl ] -2- [4- [ [1,2,3, 4-tetrahydro-6, 7-dimethoxy-2-methyl-1-isoquinolinyl ] methyl ] phenoxy ] phenol on 5 tumor cells

The isoquinoline alkaloid compound also has other structural general formulas, and when R1-R2 in the formula I are all-OCH 2CH2CH3, R3-R4 are all-OCH 2CH3, as shown in the formula II, the isoquinoline alkaloid compound also has corresponding inhibition effect on tumor cells. Wherein, the carbon chain length of R1-R4 also varies and has similar properties, and all the characters are within the protection scope of the invention.

Meanwhile, in the formula I, R1 and R2 are both-OCH 2CH2CH3, R3 is-OCH 2CH 2Cl, and R4 is-OCH 2CH3, as shown in the formula III, the compound also has corresponding inhibition effect on tumor cells. The compounds can be prepared from the compounds of formula II by substitution, wherein only one position is substituted, and the substitution of other positions is referred to the existing data.

The use of the compounds of formula I or their prospective homologs and substitutes is intended to be within the scope of the invention, provided that they do not interfere with the inhibitory effect on tumor cells.

Experimental results show that the isoquinoline alkaloid extracted from caulis sinomenii provided by the invention has strong anti-tumor activity and important economic effect. The isoquinoline alkaloids can be used alone or in combination, and can be used in the dosage forms of tablet, capsule, granule, suppository, pill, ointment, cream, spray, aerosol, gel, powder, syrup, liniment, plastics, tincture, patch, emplastrum, oral solution, oral suspension, oral emulsion, membrane, lotion, rinse, medicated wine, plaster, and tea.

It will be apparent to those skilled in the art that various modifications may be made to the above embodiments without departing from the general spirit and concept of the invention. All falling within the scope of protection of the present invention. The protection scheme of the invention is subject to the appended claims.