HK1072939B - Method for extracting antineoplastic components from bupleurum scorzonerifolium - Google Patents

Description

Preparation method of anti-malignant tumor bupleurum falcatum extract

Technical Field

The present invention relates to a plant extract for treating Cell Proliferative diseases (Cell Proliferative disorders) extracted from natural substances and an extraction method thereof, particularly to a southern Bupleurum extract which is separated from southern Bupleurum (Bupleurum scorzonerifolium) and uses heterocyclic compounds with gamma-Butyrolactone (gamma-Butyrolactone) core as main active ingredients for treating cancer cells such as Human liver cancer (Hepatoma), Lung cancer (Lung cancer), Ovarian cancer (Ovarian cancer), Malignant brain glioma (Human Malignant glioma) and colorectal cancer (Colorcalccer) and an extraction method thereof.

Background

Cancer is a cell proliferative disease and has been the first cause of death. According to the 1993 statistics of Boring et al, about fifty-two-thousand people die of Cancer in the united states every year, and as an example of the first Breast Cancer (Breast Cancer) of female Cancer, a few have become the major killer in women between 40 and 55 years of age. With the increasing environmental pollution, patients with Solid cancers (Solid tumors) such as Ovarian cancer (Ovarian carcinosoma), lung cancer and liver cancer, and skin cancer are also increasing. According to the research of Fitzpatrick et al in 1986, the number of cancer patients is six times more than that in 1945, and it is obvious that the cancer detection and treatment are not slow.

According to the current cancer research, the Cell aging (Senescence), Replication (Replication) and Division (Division) of eukaryotic cells (Eukaryocyte) are regulated following the Cell Cycle (Cell Cycle) pattern. When the Cell is replicating, the DNA content of the Chromosome (Chromosome) carrying the gene is doubled from 2N to 4N to generate two 2N Daughter cells (Daughter Cell) after Mitosis (Mitosis). Blackburn et al, 1995, showed that during eukaryotic cell division, the ends of chromosomes repeat a fixed sequence called "telomeres" (Telomeres). Taking human cells as an example, telomeres at the ends of human chromosomes will have the 5' -TTAGGG sequence fixed repeatedly. From the current research results, it is known that "telomeres" are related to the regulation of the Cell cycle time (Cell Clock), and as the number of mitosis increases, the telomeres gradually become shorter, and when the telomeres shorten to a certain length, the telomeres covered by the ends of chromosomes easily adhere to each other to cause abnormal chromosome pairing, even Cell death (Cell death).

Feng et al found that in 1995, in actively dividing cells such as embryonic cells (Germ-line cells), Stem cells (Stem cells) and Tumor cells (Tumor cells), a Ribonucleoprotein Complex (ribosomal Complex) called "Telomerase" was attached to telomeres at the ends of chromosomes of these cells, and the action of Telomerase was to maintain the length of telomeres and prevent them from undergoing multiple mitoses and becoming shorter, so that the presence of Telomerase could help the cells to jump out of the Cell cycle and go to immortalization.

If further TRAP (A) is addedTelomerase Repeat Amplification PIn the test of chromosome Telomerase Activity (Telomerase Activity), the fact that the Activity of the Telomerase is very high in tumor cells or incompletely differentiated cell lines (such as embryonic cells, stem cells and the like) is found, but the Telomerase Activity is almost absent in normal somatic cells except bone marrow cells and germ cells; furthermore, Kim and Broccoli et al, 1994 and 1995, have also demonstrated that telomerase plays an important role in the linkage mechanism (apoptosis cassette) that assists tumor cells in controlling programmed cell death by evading, and therefore, taking telomerase activity as the target for anticancer drug detection can effectively replace the conventional chemical target to accurately detect the drug poisoning condition on tumor cells, so that the detection of telomerase activity has become a highly specific (Specificity) indicator for testing the therapeutic efficacy of anticancer drugs.

On the other hand, according to clinical treatment experience for years in the Chinese medical field, five or sixty Chinese medicinal materials which are clinically considered to have anticancer efficacy are found to have obvious treatment effect on malignant tumors through TRAP activity screening. Meanwhile, compared with the existing anticancer drugs, the traditional Chinese medicine is found to cause mild side effects (such as reduction of the number of leucocytes, Cachexia (Cachexia) and the like) of patients, so that active ingredients with tumor inhibition effects are extracted from the traditional Chinese medicine, and the traditional Chinese medicine is very likely to be used as a screening source of novel anticancer drugs.

In addition, taking the chemotherapeutic agent (Paclitaxel, trade name TAXOL) widely used today for the treatment of metastatic solid cancer as an example, TAXOL was first extracted from Pacific Yew Tree (Pacific Yew Tree) and has the formula: (formula C)₄₇H₅₁NO₁₄Molecular weight 854, centered on a two-part class (Diterpen) structure)

Paclitaxel (Paclitaxel) is mainly used for treating cancers such as Ovarian Cancer (Ovarian Cancer), Metastatic Breast Cancer (metastic Breast Cancer), Lung Cancer (Lung Cancer) and Melanoma (Melanoma), and its action mechanism of poisoning tumor cells is to inhibit the Depolymerization of microtubules (Microtubule) by Paclitaxel binding to β -microtubules (β -tubulin) in the Cytoskeleton (Cytoskeleton) at a ratio of 1: 1, thereby blocking Mitosis (Mitosis) completion to activate tumor cell apoptosis (bladosklonny et al, 1995). Therefore, at the beginning of the treatment of ovarian cancer with taxoid (Taxane) drugs, tumor cells can be effectively killed, and the two-year survival rate of patients is improved by about 15%.

However, as the course of treatment is prolonged, it is clinically found that tumor cells gradually develop resistance to paclitaxel. In particular, it has been discovered from recent studies that some paclitaxel-resistant tumor cell lines are significantly different from conventional tumor cells in the Expression (Expression) and electrophoretic mobility (electrophoretic mobility) of β microtubules. According to the research of Rao et al in 1995, it is known that these tumor cells (such as human lung cancer cell line AT-12) change the six Subunit configurations (Subunit Configuration) constituting the beta microtubules, so that paclitaxel cannot be bound to the beta microtubules but is excluded by the cells as foreign matter (Ion-pumping), resulting in the gradual development of resistance of the tumor cells to paclitaxel and the gradual failure of the tumor cells in the late phase of chemotherapy.

In addition, the clinical effect of treating patients with drug resistance to paclitaxel with other existing anticancer drugs, such as 5-fluoropyrimidinedione (5-Fluorouracil), Epothilone (Epothilone), Cisplatin (Cisdiammine dichloratin, commonly known as cissplatin), methylbenzyl hydrazine (procazine), and Cyclophosphamide (cyclophosphsphamide), is not good, and it has been found from the cell experiment that the current anticancer drugs are difficult to inhibit the proliferation of paclitaxel-resistant tumor cell Line (Taxol-resistance tumor cell Line), however, when the drug administration dose is increased, for example, the paclitaxel dose is increased to 300 mg/kg of white rats, the Cytotoxicity (Cytotoxicity) generated by paclitaxel is very strong, and the normal cell Necrosis (Necrosis) is often caused after the drug administration.

Therefore, in order to enhance the poisoning effect of cancer cells in the later stage of chemotherapy, anticancer drugs capable of effectively inhibiting drug-resistant tumor cell lines of paclitaxel are developed from traditional Chinese medicinal materials with clinical anticancer efficacy, and become urgent needs in the medical field.

Disclosure of Invention

To overcome the above-mentioned drawbacks of the prior art, the main object of the present invention is to provide a compound extracted from Bupleurum scorzonerifolium Willd, which has a core heterocyclic compound of gamma-Butyrolactone (gamma-Butyrolactone) or a pharmaceutically acceptable salt, ester, ketone and derivatives thereof for inhibiting Taxol-resistant Tumor Cell Line (Taxol-resistance Tumor Cell Line), and an extraction method for extracting the heterocyclic compounds and derivatives thereof.

Another objective of the invention is to provide a method for extracting bupleurum lactone (Chaihulactone) Analogues (Analogues) and derivatives thereof, or pharmaceutically acceptable salts, esters, ketones and derivatives thereof, which have tumor cell inhibitory effect and are separated from crude drugs of Bupleurum falcatum, and which are novel compounds such as Isochaihulactone.

Still another object of the present invention is to provide a method for extracting a heterocyclic compound having a gamma-Butyrolactone (gamma-Butyrolactone) core, or a pharmaceutically acceptable salt, ester, ketone thereof, and derivatives thereof, which are isolated from crude drugs of bupleuri radix and are capable of inhibiting human liver cancer, ovarian cancer, malignant brain tumor, lung cancer, and colorectal cancer.

It is still another object of the present invention to provide an extract of bupleurum longiradiatum which has a high specific poisoning effect on paclitaxel resistant tumor cell lines without affecting normal liver and kidney functions, and a method for extracting the same, wherein the extract further comprises a heterocyclic compound having γ -Butyrolactone (γ -Butyrolactone) as a core, or pharmaceutically acceptable salts, esters, ketones and derivatives thereof. In order to accomplish the object of the present invention, the present invention provides a method for extracting Bupleurum falcatum for isolating an active ingredient for combating cell proliferative diseases from Bupleurum falcatum (Bupleurum scorzonerifolium), the method comprising:

extracting bupleuri radix to obtain lignanoid mixture, wherein the lignanoid mixture contains at least one active component capable of inhibiting tumor proliferation; and

separating the active component for inhibiting tumor proliferation to obtain single type of extract of bupleuri radix.

In the method, the step of extracting the lignan mixture from bupleuri radix comprises performing the following steps with first, second, third and fourth solutions having different polarities:

dissolving pulverized bupleuri radix with the first solution, and separating to obtain first extract and residue of bupleuri radix;

dissolving the residue with the second solution, and separating to obtain a second extract of bupleuri radix;

extracting the first extract of the bupleurum falcatum by using a third solution, and removing alcohols in an extracted water layer; and

the alcohol-removed extracted aqueous layer is extracted with a fourth solution and concentrated.

In other words, the tumor suppressor and the method for extracting the same according to the present invention include the extract of Bupleurum falcatum (Bupleurum scorzonerifolium Willd.), the extract of Bupleurum falcatum for treating lung cancer, ovarian cancer, liver cancer, human malignant glioma or colorectal cancer, and the tumor suppressor, or pharmaceutically acceptable salts, esters, ketones or derivatives thereof, further separated from the Crude extract of Bupleurum falcatum (Crude Extracts).

The tumor inhibiting component of the bupleurum falcatum extract mainly comprises a heterocyclic compound which takes gamma-Butyrolactone (gamma-Butyrolactone) as a core and takes carbon-2 (5) as Z configuration or E configuration, or pharmaceutically acceptable salts, esters, ketones and derivatives thereof, as represented by a general formula (I):

formula (I)

Wherein X is N, O, S, Se;

a and B are respectively selected from substituent groups with the following formula:

wherein, R1, R2, R3, R4 and R5 are respectively selected from hydrogen atoms, halogen atoms, hydroxyl groups, sulfhydryl groups, amino groups, alkoxy groups and nitro groups.

Meanwhile, the formula (I) also comprises a heterocyclic compound which is shown as a general formula (II) and a general formula (III), is disclosed for the first time and is respectively named as bupleurum lactone (Chaihulactone), isobupleurum lactone (Isochaihulactone) and analogues thereof. The said bupleurum lactone, isobupleurum lactone and the said bupleurum lactone analogue and its derivative belong to Lignan (Lignan) existing in Bupleurum falcatum. Comparing the formula (I), the formula (II) and the formula (III), the bupleurum lactone and the isobupleurum lactone are heterocyclic compounds which take a gamma-butyrolactone structure as a core and have Z configuration or E configuration of carbon 2- (5).

Formula (II)

Wherein R represents an alkoxy group.

Formula (III)

Wherein R represents a hydrogen atom, an alkoxy group or an aryl group.

In other words, the present invention relates to a γ -butyrolactone compound having the structure shown below:

wherein X ═ O; a is

The B substituent is selected from the following substituent structures:

further, the gamma-butyrolactone compound heterocyclic compound is in Z configuration at carbon 2 (5).

Further, the gamma-butyrolactone compound heterocyclic compound is in an E configuration at carbon 2 (5).

In addition, the present invention relates to an extract of Bupleurum falcatum including the gamma-butyrolactone compound of the present invention as an active ingredient.

Further, the present invention relates to a pharmaceutical composition for treating cell proliferative diseases, comprising the gamma-butyrolactone compound of the present invention as an active ingredient. Wherein the cell proliferative disorder comprises human liver cancer, human ovarian cancer, human malignant glioma, human lung cancer, human colorectal cancer, and the cell proliferative disorder is resistant to a taxane therapeutic agent, preferably paclitaxel. Furthermore, the active component for inhibiting cell proliferation in the composition is a G2/M arresting agent for regulating cell cycle, or a microtubule stabilizing agent for promoting the aggregation of cytoskeleton beta-microtubules.

The invention also relates to an extraction method for separating the bupleurum falcatum extract, which comprises the following steps:

dissolving pulverized bupleuri radix in acetone solution, and separating to obtain acetone extract and residue of bupleuri radix;

dissolving the residue with methanol solution, and separating to obtain methanol extract of bupleuri radix;

extracting the acetone extract of Bupleurum falcatum with 95% methanol water solution, and removing alcohols in the extracted water layer; and

the alcohol-removed aqueous layer was extracted with chloroform solution and concentrated.

The experimental results of the embodiment of the invention show that the extracts of Bupleurum falcatum containing heterocyclic compounds such as gamma-butyrolactone structure and the like can be effectively inhibited from proliferating by separating the extracts of Bupleurum falcatum with different solvents (such as acetone, methanol aqueous solution and the like), and the tumor inhibition effect of the acetone crude extract of Bupleurum falcatum is the best. Furthermore, the acetone crude extract of Bupleurum falcatum Linn is further separated by Chromatography (Chromatography), and the molecular structure of each concentrate is analyzed, so as to obtain the novel heterocyclic compounds of Bupleurolide, IsoBupleurolide, Bupleurolide related analogues and derivatives thereof.

According to the experimental results of the preferred embodiment of the present invention, the bupleurum lactone (Chaihulactone), isobupleurum lactone (isochaihulacetone), bupleurum lactone analogue and their derivatives separated from the extract of bupleurum falcatum are added into human lung cancer, liver cancer, malignant brain glioma and large intestine cancer Cell Line, and the Telomerase Activity (Telomerase Activity) of the Tumor Cell Line is obviously reduced by about 5 times compared with that of the control group without drug addition, especially the Telomerase Activity of the Taxol-resistance Tumor Cell Line can generate obvious poisoning effect after the bupleurum falcatum extract is added, which shows that the extract of bupleurum falcatum provided by the present invention really contains Tumor inhibiting components, and the Tumor inhibiting components comprise heterocyclic compounds with gamma-butyrolactone structure as core and Z configuration or E configuration at carbon 2(5) of butyrolactone core, or pharmaceutically acceptable salts thereof, The ester, ketone and derivatives thereof, wherein the newly discovered bupleurum lactone (Chaihulactone), isobupleurum lactone (Isochaihulactone) and derivatives thereof have the most obvious tumor poisoning effect, therefore, the invention uses heterocyclic compounds including isobupleurum lactone as the main active ingredient of tumor inhibiting substances (Antineoplastic Agent).

The extraction method comprises two stages, wherein the first stage is to separate the crude drug of Bupleurum falcatum Linne from different solvents with different polarities (Polarity) to obtain different extraction layers, and the second stage is to separate a specific extraction layer by Chromatography (Chromatography) to obtain a single type of Bupleurum falcatum Linne extract, the method for extracting tumor inhibition components from the crude drug of Bupleurum falcatum Linne is gradually explained as follows:

soaking in Acetone (Acetone), stirring, repeatedly extracting and concentrating to obtain Bupleurum Acetone Crude Extract (BS-A layer), extracting the residue with methanol to obtain Bupleurum methanol Crude Extract (BS-M layer), and extracting the residue with water to obtain Bupleurum aqueous Extract (BS-W layer);

dissolving the BS-A layer in methanol water solution, extracting with n-Hexane (n-Hexane), and separating to obtain n-Hexane layer (BS-H layer for short) of bupleuri radix and methanol water layer of bupleuri radix;

removing methanol from methanol water layer of bupleuri radix, and adding Chloroform (Chloroform, CHCl)₃) Repeatedly extracting and concentrating;

separating the chloroform extract of Bupleurum falcatum by Chromatography (Chromatography), collecting methanol/dichloromethane Elution layers (Elution) with different concentrations, and concentrating;

the methanol/dichloromethane elution layers of different concentrations were each chromatographically separated and purified to give a single type of compound.

The bupleurum acetone crude extract (BS-A), the bupleurum methanol crude extract (BS-M), the 5% methanol/dichloromethane elution layer, the chromatography collection and the like obtained by the method are added into A tumor cell line for culture, and obvious tumor poisoning effect is found, and Mass spectrA (Mass spectrA) and Nuclear Magnetic Resonance spectrA (Nuclear Magnetic Resonance spectrA) are used for identifying the compound structure with tumor inhibition activity in bupleurum, and the compounds are also shown to have gammA-butyrolactone structures, so that the bupleurum extracted by the extraction method can be proved, and the tumor inhibition components in the traditional Chinese medicinal materials can be effectively reserved.

In addition, TRAPASSAY is used for detecting the activity inhibition situation of Telomerase (Telomerase) after a tumor cell line is added into the southern bupleurum extract, and the result shows that the southern bupleurum acetone crude extract can effectively inhibit the Telomerase activity of human lung cancer cell line A549 and the expression of hTERT information ribonucleic acid, and shows that the southern bupleurum acetone crude extract possibly has highly specific poisoning effect on cancer cells.

In particular, the crude extract of Bupleurum acetone containing heterocyclic compounds centered on gamma-butyrolactone, as well as Bupleurone analogs and derivatives thereof isolated from the Bupleurum falcatum extract, are added to a paclitaxel-resistant Tumor Cell Line (Taxol-resistance Tumor Cell Line), such as A549-T12 (a human lung cancer Cell Line resistant to paclitaxel), and it has been found that gamma-butyrolactone core heterocyclic compounds and derivatives thereof in the Bupleurum falcatum extraction service induce Apoptosis (Apoptosis) in the paclitaxel-resistant Tumor Cell Line; further examining the action mechanism of the extract of Bupleurum falcatum Linn from the results of Flow cytometry (Flow cytometry) and Western blotting (Western Blot), it was also found that the extract of Bupleurum falcatum Linn induces the cells to produce a large amount of tumor suppressor protein (tumor suppressor) such as p21 and p53, and arrests the tumor cells in the post-mitotic (high G2/M ratio) state of the spun-lobe Polymerization (spin Polymerization) after the tumor cell line is added. Therefore, from the viewpoint of Cell Regulation (Cell Regulation), the acetone crude extract of kadsura longipedunculata and the heterocyclic compound containing therein with γ -Butyrolactone as the core can be regarded as a Microtubule Stabilizing Agent (Microtubule Stabilizing Agent) for inhibiting mitosis, which has the similar action mechanism as Paclitaxel (Paclitaxel), and both of them have the efficacy of promoting Microtubule Polymerization (Microtubule Polymerization), so that the proliferating tumor cells are arrested at the G2/M stage and become ineffective cells (Junk cells) to cause apoptosis.

Drawings

FIG. 1 is A schematic diagram showing the variation of survival rate of tumor cells and dosage of bupleuri radix after performing cytotoxicity test (MTT Assay) on acetone crude extract (BS-A), methanol crude extract (BS-M) and water crude extract (BS-W) of bupleuri radix obtained by separating bupleuri radix by the extraction method of the present invention;

FIGS. 2A to 2D are schematic diagrams showing the cell cycle peaks of various Bupleurum falcatum extracts against tumor cell lines after the acetone crude extract (BS-A), methanol crude extract (BS-M) and water layer crude extract (BS-W) of Bupleurum falcatum obtained by separating Bupleurum falcatum by the extraction method of the present invention are added to the lung cancer cell line A549 and detected by flow cytometry;

FIGS. 3A to 3C are graphs showing quadrant graphs of the apoptosis of Annexin V-FLOUS cells measured by flow cytometry after human lung cancer cell line A549 was cultured continuously for 48 hours without drug addition, with the addition of 20. mu.M isobupleurum lactone and 60. mu.g/ml of bupleurum acetone extract (the horizontal axis represents the fluorescence intensity of tumor cells conjugated with Annexin V-FLOUS antibody, and the vertical axis represents the fluorescence intensity of tumor cells conjugated with PI antibody);

FIG. 4 is A graph showing the cell cycle change analysis of the human lung cancer cell line A549 in the absence of drug (control group), the addition of the acetone crude extract of Bupleurum falcatum (BS-A), the eighth component of the extract of Bupleurum falcatum (BS-8), and paclitaxel by flow cytometry;

FIG. 5 is A Western Blot (Western Blot) showing the measurement of Tumor Suppressor protein (Tumor suprA) p21 and p53 levels in human lung cancer cell line A549 after one or three days of non-drug addition (C), addition of acetone extract of Bupleurum falcatum (BS-A), methanol extract of Bupleurum falcatum (BS-M), aqueous extract of Bupleurum falcatum (BS-W), eighth component of Bupleurum falcatum extract (BS1), third component of Bupleurum falcatum extract (BS 2);

FIG. 6 is a schematic diagram showing Western blot method of first-type α -microtubules and fifth-type β -microtubules in the testing of tumor cell cytoskeleton after the addition of human lung cancer cell line A549 without drug (C), the addition of the acetone extract of Bupleurum falcatum for 12 hours, 24 hours and 48 hours;

FIG. 7 is a schematic diagram showing Western blotting of the beta-microtubules of tumor cells tested in lysis (expressed as S) or particle (expressed as P) after the addition of the human lung cancer cell line A549 in the absence of drug (A549), paclitaxel, and Vinca alkaloid;

FIG. 8 is a photograph of conjugated Microscope (Confocal Microscope) fluorescence-labeled sections showing that spindle silks are elongated after the addition of the Bupleurum falcatum extract to the human lung cancer cell line A549;

FIGS. 9A to 9D are graphs showing Annexin V-FLOUS apoptosis quadrant (horizontal axis represents fluorescence intensity of tumor cells conjugated with Annexin V-FLOUS antibody, and vertical axis represents fluorescence intensity of tumor cells conjugated with PI antibody) of human lung cancer paclitaxel resistant cell line A549-T12 after adding crude extract of Bupleurum falcatum acetone (BS-A), eighth component of Bupleurum falcatum extract (BS-8), and fifteenth component of Bupleurum falcatum extract (BS-15) for 24 hours without adding drugs;

FIGS. 10A-10C are schematic diagrams showing the cytotoxicity test (MTTAssay) of tumor cells of human lung carcinomA paclitaxel resistant cell line AT-12 during 24 hours and 48 hours after the addition of the acetone extract of Bupleurum falcatum (BS-A), the eighth component of Bupleurum falcatum extract (BS-8) and the fifteenth component of Bupleurum falcatum extract (BS-15);

FIG. 11A is a schematic drawing showing a nude mouse subcutaneous implantation of A549 cell line (control group), a tissue section magnified 100 times and treated by hematoxylin and Eosin Stain (Haematoxylin and Eosin Stain, H & E Stain);

FIG. 11B is a schematic diagram showing a histological section showing hemorrhagic necrosis of large tumor tissues after five consecutive days of subcutaneous implantation of the A549 cell line in nude mice, intraperitoneal injection of 500 mg/kg of the acetone extract of Bupleurum falcatum, 100-fold enlargement of the tumor on the seventh day and hematoxylin and Eosin Stain (H & E Stain) treatment;

FIG. 12A is a schematic drawing showing a tissue section of nude mice subcutaneously implanted with A549-T12 cell line (control group), at 100-fold magnification, and treated with hematoxylin and Eosin Stain (Haematoxylin and Eosin Stain, H & E Stain);

FIG. 12B shows the subcutaneous implantation of the A549-T12 cell line in nude mice, five consecutive days of intraperitoneal injection of 400 mg/kg Bupleurum falcatum acetone extract, large pieces of tissue fibrosis and only a few remaining tumor cells after tumor treatment on day seven with 100-fold magnification and hematoxylin and Eosin Stain (H & E Stain);

FIG. 13A is A magnified partial photograph showing the diameter of subcutaneous tumors in nude mice treated with intraperitoneal injection of 400 mg/kg of BS-A five consecutive days after the tumor formation by subcutaneous implantation of A549 cell line, compared to the control group without drug administration after the seventh day;

FIG. 13B is a graph showing the in vivo status of 500 mg/kg body weight of a crude extract of Bupleurum falcatum acetone injected intraperitoneally into experimental animals, comparing the relative tumor volume and the treatment days for the non-drug-added control group and the drug-added treatment group;

FIG. 14 is a graph showing the biochemical indicator enzyme changes in the functions of the pancreas, liver, heart, kidney and hematopoietic tissues during the administration of 400 mg/kg body weight extract of Bupleurum falcatum to conscious mice for 72 hours;

FIG. 15 is a schematic diagram showing the change of platelets, leukocytes and lymphocytes during the administration of 400 mg/kg body weight of Bupleurum falcatum extract intravenously to conscious mice for 72 hours;

FIG. 16 is a graph showing the changes in cardiac activity, systolic pressure, diastolic pressure and mean pressure during 72 hours of intravenous administration of 400 mg/kg body weight extract of Bupleurum falcatum to conscious mice; and

FIG. 17 is a histological section of liver cells and kidney cells of conscious mice after intraperitoneal injection of 300 mg/kg body weight of extract of Bupleurum falcatum into conscious mice for five consecutive days.

Detailed Description

The following description is provided for the purpose of illustrating the embodiments of the present invention by way of specific examples, and other advantages and effects of the present invention will become apparent to those skilled in the art from the disclosure herein. The invention is capable of other and different embodiments and its several details are capable of modification in various other respects, all without departing from the spirit and scope of the present invention.

The invention at least comprises three parts:

an extraction method of Bupleurum falcatum (Bupleurum scorzonerifolium Willd) crude drug to extract natural extract with inhibitory effect on human liver cancer cell line J5, ovarian cancer cell line OVCAR-3, malignant glioma cell line DBTRG-05MD, lung cancer cell line A549 and large intestine rectal cancer cell line HT 29.

Secondly, according to the extraction method, the heterocyclic compounds which take gamma-butyrolactone as the core and have Z configuration or E configuration at the carbon 2(5) position are separated from the extract of the bupleurum falcatum, and pharmaceutically acceptable salts, esters, ketones and similar derivatives of the heterocyclic compounds are formed. Wherein the heterocyclic compound further comprises a novel compound: bupleurum lactone (Chaihulactone), isobupleurum lactone (Isochaihulactone), and analogs and derivatives thereof related to bupleurum lactone.

And the third part is that the coarse extract of Bupleurum acetone with gamma-butyrolactone core heterocyclic compound, Bupleurone, isoBupleurone, Bupleurone analogue and their derivatives, or their pharmaceutically acceptable salts, esters and ketones are added into the tumor cell line to test the inhibition effect of each Bupleurum extract on the tumor cell line in vivo (in vivo) and in vitro (in vitro) environments, and then the influence of the Bupleurum extract on the normal cells and tumor cells of the organism is further evaluated by using animal models, so that the inhibition effect of the Bupleurum extract on liver cancer, ovarian cancer, lung cancer, malignant brain tumor or colorectal cancer can be tested in vivo.

According to the preferred embodiment of the present invention, Bupleurum scorzonerifolium is pulverized and soaked in Acetone (Acetone), and after four times of repeated stirring, extraction and concentration, the crude extract (BS-A layer) of Bupleurum scorzonerifolium Acetone is obtained, and the residue is extracted with methanolObtaining methanol crude extract of Bupleurum falcatum Linne (BS-M layer for short), and extracting the residue with water to obtain water extract of Bupleurum falcatum Linne (BS-W layer for short); dissolving the acetone crude extract of Bupleurum falcatum Linn in methanol water solution, performing n-Hexane (n-Hexane) crude extraction, and separating to obtain a n-Hexane layer (BS-H layer for short) of Bupleurum falcatum Linn and a methanol water layer of Bupleurum falcatum Linn; then, the methanolic aqueous layer of Bupleurum falcatum was stripped of methanol and washed with Chloroform (Chloroform, CHCl)₃) Repeatedly extracting and concentrating to obtain chloroform extract of Bupleurum falcatum L; then, separating the chloroform extract of Bupleurum falcatum by chromatography (chromatography-graphics), and collecting methanol/dichloromethane Elution layers (Elution) with different concentrations respectively for concentration; finally, the methanol/dichloromethane eluted layer is separated and purified by Chromatography (e.g., Silica Gel Chromatography, preparative high performance liquid Chromatography (preparative HPLC), etc.) to obtain a single type of compound.

The molecular weight and structure of each single compound are identified by Mass Spectrum (Mass Spectrum) and Nuclear Magnetic resonance Spectrum (Nuclear Magnetic resonance Spectrum) of the single compound separated by chromatography, and the heterocyclic compound shown in the following table can be obtained:

the extraction layers of Bupleurum falcatum and the heterocyclic compounds separated from the acetone crude extract of Bupleurum falcatum are respectively applied with drug screening, so that the tumor inhibiting components contained in the crude drug of Bupleurum falcatum mainly remain in the acetone crude extract of Bupleurum falcatum and the methanol water layer of Bupleurum falcatum, and the n-hexane layer of Bupleurum falcatum and the water layer of Bupleurum falcatum separated after chloroform treatment have almost no tumor inhibiting effect. Further purification by Chromatography, such as low pressure liquid Chromatography or High Performance Liquid Chromatography (HPLC), can be performed by collecting the eluate fractions in fractions for concentration to obtain individual compounds of the above-mentioned classes.

However, from the results of drug screening according to the preferred embodiment of the present invention, it was found that the third component and the eighth component among the above-mentioned components separated by chromatography have the most significant effect of killing tumors in human liver cancer, ovarian cancer, lung cancer, malignant brain tumor and colorectal cancer cell line. Furthermore, further analyzing the main representative molecular structures of the third and eighth components with tumor effect, it can be seen that the main characteristics of the molecules of the effective cancer-suppressing component in the extract of Bupleurum falcatum are shown in the general formula (I), and the effective cancer-suppressing component has a gamma-Butyrolactone (gamma-Butyrolactone) core, and uses the heterocyclic compound having a gamma-Butyrolactone structure and having a Z-configuration or an E-configuration at the carbon-2 (5) position, or the pharmaceutically acceptable salts, esters, ketones and derivatives thereof as the main active component for suppressing tumor proliferation.

Formula (I)

Wherein X is N, O, S, Se;

a and B are respectively selected from substituent groups with the following formula:

wherein, R1, R2, R3, R4 and R5 are respectively selected from hydrogen atoms, halogen atoms, hydroxyl groups, sulfhydryl groups, amino groups, alkoxy groups and nitro groups.

If the molecular structures of the third, eighth, fourteenth and fifteenth components in the purified compound are further analyzed, it can be seen that the extract of bupleurum falcatum includes a class of novel heterocyclic compounds which are first disclosed and named as bupleurum lactone (chaihulalactone), isochalihulalactone (Isochaihulactone) and analogs or derivatives related to the bupleurum lactone, such as chaihunaphenone (chaihunaphenone), etc. Wherein, the bupleurum lactone and the isobupleurum lactone both take gamma-butyrolactone as a carbon skeleton core, and form a heterocyclic compound with Z configuration at the carbon-2 (5) position, and the general formulas of the bupleurum lactone analogue and the isobupleurum lactone analogue are respectively shown as a formula (II) and a formula (III):

formula (II)

Wherein R represents an alkoxy group.

Formula (III)

Wherein R represents a hydrogen atom, an alkoxy group or an aryl group.

The new heterocyclic compound bupleurum lactone, isobupleurum lactone, and the analogs related to bupleurum lactone and derivatives thereof belong to Lignan (Lignan) existing in dried Bupleurum falcatum nakai product, because the crude extract of Bupleurum falcatum nakai acetone and the Isochaihulactone (Isochaihulactone) of the eighth component in various Bupleurum falcatum extracts have the best tumor inhibition effect during drug screening, the following preferred embodiments use the crude extract layer and the eighth component of Bupleurum falcatum nakai acetone as the extract of Bupleurum falcatum nakai containing gamma-butyrolactone, and the active index components of the new compound Bupleurum falcatum naeus "Bupleurum falcatum analogs and derivatives thereof" respectively, so as to inhibit the efficacy index components of human liver cancer, ovarian cancer, lung cancer, malignant brain glioma and colorectal cancer.

The indications of (gamma-Butyrolactone) core heterocyclic compounds and bupleurum lactone analogs were used to examine the tumor inhibition of Bupleurum falcatum extract in cell lines and in vivo.

However, from the tissue section results of the preferred embodiment of the present invention, it is clear that acetone extract of Bupleurum falcatum has been shown to effectively reduce the Tumor Volume (Tumor Volume), so that the Tumor cell nucleus is cracked and the lymph ball infiltrates to cause large necrosis of the Tumor tissue. Furthermore, as a result of animal toxicity tests, it is also known that after administering the extract of kadsura longipedunculata to mammals, functional indicators of organs in vivo, such as lipolytic enzyme (Lipase), Amylase (Amylase), Creatinine Kinase (Creatinine Kinase), Lactate Dehydrogenase (Lactate Dehydrogenase), GOT, BUN, etc., do not differ significantly before and after administering the extract of kadsura longipedunculata, but telomerase activity at tumor sites is significantly reduced after administering the extract of kadsura longipedunculata, showing that administration of the crude extract of kadsura longipedunculata acetone and the novel compound "bupleurum lactone, isobupleurum lactone, and bupleurum lactone-related analogs and derivatives thereof" to mammals can produce highly specific toxicity for human liver cancer, ovarian cancer, lung cancer, malignant brain tumor and colorectal cancer without harming normal liver and kidney functions.

On the other hand, the crude extract of Bupleurum acetone containing gamma-butyrolactone heterocyclic compounds and the eighth component isobupleurolide (which is an indicator of Bupleurolide, Bupleurolide analogues and derivatives thereof) isolated from the Bupleurum falcatum extract were administered to a paclitaxel-resistant tumor cell line (e.g., human lung cancer cell line AT-12) and tumor suppression was observed. The result shows that the effective concentration (about 1.2 micrograms/ml) of analogs such as bupleurum lactone, isobupleurum lactone and derivatives thereof acting on the telomerase activity for inhibiting the taxol-resistant tumor cell line is far less than the effective concentration (about 60 micrograms/ml) of acetone extract of bupleurum chinense after the separation and purification steps, the analogs such as bupleurum lactone, isobupleurum lactone, bupleurum lactone related analogs and derivatives thereof are the most main tumor inhibition active components in bupleurum chinense, and the tumor inhibition effect of the bupleurum chinense extract of crude drug type is more obvious after the crude drug type bupleurum chinense is separated by the extraction method of the invention.

Therefore, in view of the mechanism of action of the drugs in the extract of Bupleurum falcatum, particularly the acetone crude extract of Bupleurum falcatum, Bupleurone, IsoBupleurone and their related analogs and derivatives, the extract of Bupleurum falcatum is similar to paclitaxel and is a Microtubule Stabilizing Agent (Microtubule Stabilizing Agent) for inhibiting mitosis, however, the action sites of the two drugs on beta microtubules may not be the same.

In conclusion, the novel compounds 'Bupleurolide, isoBupleurolide, Bupleurolide analogues and derivatives thereof' separated from Bupleurum falcatum have high possibility of becoming a new source of anti-cancer drugs in the development of new drugs.

The following examples further illustrate aspects of the present invention in detail, but are not intended to limit the scope of the present invention in any way.

The invention will now be described in detail with reference to the accompanying drawings: (1) the preferred embodiments of the present invention are only used for showing the preferred embodiments of the present invention, but not for limiting the scope of the present invention, and the pharmaceutical composition, dosage form and synthetic manner of the bupleuri radix extract and its active ingredients are adjusted according to the actual implementation when they are prepared into pharmaceutically acceptable salts, esters, ketones and analogs.

Example 1: method for extracting antitumor active component from Bupleurum falcatum Linne

The Bupleurum scorzonerifolium Willd is green, single-leaf, and multiple-umbellate inflorescence Bupleurum scorzonerifolium Willd, drying and pulverizing, soaking 6 kg of Bupleurum scorzonerifolium powder in 20L acetone at room temperature, stirring for four hours, concentrating, filtering and repeatedly performing coarse extraction for four times to obtain A crude extract of Bupleurum scorzonerifolium acetone (BS-A), extracting the residue with methanol to obtain A methanol extract of Bupleurum scorzonerifolium (BS-M layer), extracting the residue with water to obtain A water layer extract of Bupleurum scorzonerifolium (BS-W layer), dissolving the crude extract of Bupleurum scorzonerifolium acetone in 95% methanol water solution, extracting with n-Hexane (n-Hexane) for three times, separating A n-Hexane layer of Bupleurum scorzonerifolium (BS-H) and A methanol water layer of Bupleurum scorzonerifolium, adding 500 ml of distilled water to remove methanol in Bupleurum scorzonerifolium and water layer, adding Chloroform (Chloroform) into methanol water layer of bupleuri radix to extract, performing Chloroform extraction for three times, separating Chloroform layer and water layer, mixing, and concentrating to obtain Chloroform extract (BS-C).

The acetone crude extract (BS-A), methanol crude extract (BS-M), n-hexane extract (BS-H), chloroform extract (BS-C) and water extract (BS-W) of Bupleurum falcatum were tested by MTTA ssay to determine the drug toxicity of the extracts collected from different extraction layers to the lung cancer cell line A549, and the results are shown in FIG. 1. However, in order to separate the components with tumor inhibiting effect from the bupleurum falcatum extract, the invention further uses Chromatography (Chromatography) to elute and separate the bupleurum falcatum chloroform extract.

About 100 g of chloroform extract of Bupleurum falcatum was separated by Silica Gel Chromatography (Silica Gel Chromatography) with 5% methanol/dichloromethane, 10% methanol/dichloromethane, 20% methanol/dichloromethane and methanol Elution (Elution) to give 27.5 g of 5% methanol/dichloromethane layer extract, 14.04 g of 10% methanol/dichloromethane layer extract, 10.96 g of 20% methanol/dichloromethane layer extract and 7.25 g of methanol layer extract, wherein the tumor suppressor was mainly retained in the 5% methanol/dichloromethane layer extract. Then, the 5% methanol/dichloromethane extract is separated by a silica gel column Chromatography, or preparative High Performance Liquid Chromatography (HPLC), or Medium Pressure Liquid Chromatography (Medium Pressure Liquid Chromatography), or Lobar, and the third, eighth, fourteenth and fifteenth components having tumor-inhibiting effects are concentrated and collected.

Example 2: structure of tumor inhibiting active component of bupleurum root

The molecular weight and structure of each compound of the third, eighth, fourteenth and fifteenth main representatives isolated from the acetone extract of Bupleurum falcatum according to the above extraction method are defined by mass Spectrum (MassSpectrum) and Nuclear Magnetic Resonance Spectrum (NMR), respectively, and the results are shown in the following table:

table 1: separated compound with tumor inhibition effect in acetone extract of bupleurum falcatum

Molecular structure integration and induction show that the carbon skeletons of the extract of bupleurum chinense having the effect of inhibiting tumor cell lines of human liver cancer, ovarian cancer, lung cancer, malignant glioma and colorectal cancer are Heterocyclic Compounds (Heterocyclic Compounds) which take gamma-butyrolactone as the core and have Z configuration or E configuration at carbon 2 (5). WhileFurthermore, the cytotoxicity test results show that the poisoning effect of the third component and the eighth component on the lung cancer cell line is better than that of other components, so that the third component and the eighth component are crystallized and then subjected to hydrogen nuclear magnetic resonance spectrum (b)¹H-NMR) and carbon 13 NMR spectrum (C¹³C-NMR) to obtain novel compounds designated as "chaihulalactone" and "Isochaihulactone" (i.e.: chaihulalactone), respectively, as shown in the general formula (I) and the general formula (II):

formula (I)

Wherein R represents an alkoxy group.

Formula (II)

Wherein R represents a hydrogen atom or an alkoxy group.

(white needle-like crystals, melting point 137 ℃ -]_D ²⁵-29.0°(c0.5，CHCl3)；IR(KBr)v_maxcm^-1：1745，1635，1581，1335，1153；UV(CHCl3)λ_maxnm(logε)：247(4.08)，298(4.17)，327(4.08))

Then, if the structure of other compounds with tumor-inhibiting effect, such as the representative molecules of the first, second, eleventh and fifteenth components and related analogs or derivatives thereof, is further analyzed and compared with the existing drug database. The comparison result shows that the pharmaceutical compositions with tumor poisoning effect in the extract of bupleurum falcatum are heterocyclic compounds containing gamma-Butyrolactone (gamma-Butyrolactone) core structure and having Z configuration or E configuration at carbon-2 (5), or pharmaceutically acceptable salts, esters, ketones and derivatives thereof. The general formula of the heterocyclic compound is shown as the formula (I):

formula (I)

Wherein X is N, O, S, Se;

a and B are respectively selected from substituent groups with the following formula:

wherein, R1, R2, R3, R4 and R5 are respectively selected from hydrogen atoms, halogen atoms, hydroxyl groups, sulfhydryl groups, amino groups, alkoxy groups and nitro groups; and the substituent further comprises the following substituent structure:

moreover, the cytotoxicity test results show that the poisoning effect of the eighth component isobupleurum lactone on lung cancer cell lines is obviously better than that of other bupleurum lactone analogues, therefore, the following examples included in the invention all use the acetone extract of southern bupleurum (BS-A) and the isobupleurum lactone of the eighth component (BS- (8)) as indicators for evaluating the efficacy of the tumor inhibiting component of southern bupleurum, "gammA-butyrolactone core, carbon 2(5) heterocyclic compound in Z configuration", and novel compounds, "bupleurum lactone, isobupleurum lactone, bupleurum lactone analogues and derivatives thereof".

Example 3: extract of Bupleurum falcatum Linne for cellEffect of Proliferation (Cell Proliferation)

Acetone crude extract (BS-A), methanol crude extract (BS-M), water crude extract (BS-W) and eighth component (BS- (8)) of Bupleurum falcatum are added into culture medium of human liver cancer cell line, ovarian cancer cell line, lung cancer cell line, malignant gliomA and colorectal cancer cell line, and the inhibition of each tumor cell line is observed within 7 days. In this example, taking lung cancer Cell line A549 and colorectal cancer Cell line HT-29 as examples, when each extract was administered for three days, the decrease in Tumor Cell number (Tumor Cell Counts) by adding 60 mg of the crude extract of Bupleurum longiradiatum acetone was similar to the decrease in Tumor Cell number by adding 600 mg of the crude extract of Bupleurum longiradiatum methanol, indicating that the content of Tumor-inhibiting components in BS-M layer was much lower than that in BS-A layer, and the substances with Tumor-inhibiting effect in Bupleurum longiradiatum remained mainly in the acetone extract of Bupleurum longiradiatum.

Furthermore, the cell proliferation changes of the human lung cancer cell line a549 before and after the addition of each bupleuri radix extract were compared, and the results thereof were examined by Flow Cytometry (Flow Cytometry). As shown in FIGS. 2A to 2D, the horizontal axis represents the chromosome number of the tumor cells conjugated with the antibody, and the vertical axis represents the FITC fluorescence intensity, it is clear from the results of the graphs that the lung cancer cell line A549 is mainly distributed at the G0/G1 before the medicine is added, but when the acetone extract of Bupleurum falcatum (BS-A) and the methanol extract of Bupleurum falcatum (BS-M) are added into the tumor cells, the tumor cells are obviously stagnated at the G2/M stage, and particularly, the efficacy of the acetone extract of Bupleurum falcatum is most remarkable. On the other hand, from the PI staining results of flow cytometry, it was also found that the number of chromosomes at the G0/G1 stage was decreased but the number of chromosomes (2N, 4N, etc.) at the G2/M stage was greatly increased after the addition of the extract of Kadsura longipedunculata, and the phenomenon exhibited similar results in the human liver cancer cell line, ovarian cancer cell line, lung cancer cell line and large intestine rectal cancer cell line, indicating that the tumor suppressor turnover of the extract of Kadsura pedunculata was most likely to be associated with the G2/M Arrest (G2/M Arrest).

Example 4: extract of Bupleurum falcatum and Apoptosis (Apoptosis)

To further confirm whether the extract of Bupleurum falcatum has the effect of inducing Apoptosis (Apoptosis) in the tumor cell line, the preferred embodiment of the present invention uses flow cytometry, Reverse Transcription polymerase chain Reaction (RT-PCR) and Western blotting (Western Blot) to examine the change of the tumor cells in the cell cycle and regulatory proteins p21 and p53 after the tumor cell line is added with the eighth component of the extract of Bupleurum falcatum and acetone extract of Bupleurum falcatum.

Taking the lung cancer cell line A549 as an example, after an unformed A549 cell line, an A549 cell line added with 20M isobupleurum lactone and an A549 cell line added with 60 micrograms/ml acetone extract of Bupleurum falcatum were continuously cultured for 48 hours, the staining results between Annexin V-FLOUS and PI were examined by using a Flow cytometer (Flow Cytometry). In the above, the horizontal axis represents the fluorescence intensity of the tumor cell conjugated to the Annexin V-flo antibody, and the vertical axis represents the fluorescence intensity of the tumor cell conjugated to the PI antibody, as shown in fig. 3A to 3C, the number of cells bound to Annexin V (indicating cell membrane eversion and Apoptosis indicator) was only 3.8% after 48 hours of culture in the control group (indicating the non-medicated a549 tumor cell line), whereas the number of tumor cells bound to Annexin V was found to be increased to 32.7% after 48 hours of culture in the a549 lung cancer cell line after adding isobupleurolide or crude extract of kakkonene, indicating that the tumor cells can induce Apoptosis (Apoptosis) under the action of the extract of kakkonen.

Therefore, as can be seen from the cell cycle results in FIG. 4 and the Western blotting results in FIG. 5, the action mechanism of the isocorychophralactone in the acetone extract and the extract of bupleurum chinense to Tumor cells is similar to that of paclitaxel, and both of them are arrested in the mitotic maturation phase (G2/M), and the crude extract of bupleurum chinense (BS-A) and the analogs of bupleurum chinense and their derivatives (BS-3 or BS-8) greatly increase the production of Tumor Suppressor protein (Tumor pressor) p21 and p53 to block Cyclin D and Cyclin E, thereby blocking the cells from entering G0/G1 phase and arresting the Tumor cells in G2/M phase.

Therefore, from the above results, it can be seen that the acetone extract of kadsura longipedunculata, the bupleurum lactone analogue containing isobupleurum lactone and the derivatives thereof have the effect of promoting tumor cell arrest at the stage of G2/M, so as to induce tumor cell to enter Apoptosis (Apoptosis), and the above phenomena occur in human liver cancer, lung cancer, ovarian cancer, malignant brain tumor and colorectal cancer cell lines, which shows that the acetone extract of kadsura longipedunculata has the effect of inhibiting human liver cancer, ovarian cancer, lung cancer, malignant brain tumor and colorectal cancer proliferation, and the action mechanism thereof is probably similar to that of paclitaxel, and is a G2/M arresting agent.

If the Western blotting method is used to detect the change of Cytoskeleton (Cytoskeleton) after the administration of acetone extract and the eighth component of the extract of Bupleurum falcatum to the tumor cell line. As shown in fig. 6, when the lung cancer cell line a549 was added to the acetone extract of kadsura longipedunculata for 12 hours, 24 hours and 48 hours, the first-type α -microtubules in the cytoskeleton did not change significantly, but gradually decreased in the fifth-type β -microtubules, and compared with the presence of aggregation state of β -microtubules before and after the addition of the acetone extract of kadsura longipedunculata, it can be seen from the results of fig. 7 that after the eighth component of the acetone extract of kadsura longipedunculata, the protein band of the Soluble form (β -microtubules in the unpolymerized state, denoted by S in the figure) disappeared, and the protein band of the particle form (β -microtubules after polymerization, denoted by P in the figure) increased, indicating that the acetone extract of kadsura longipedunculata has the function of causing aggregation of β -microtubules.

Furthermore, the movement of the spindle yarn in the cytoskeleton after the fluorescent microtubule antibody was attached to the microtubules was further examined by conjugate Microscope (Confocal Microscope). The results are shown in fig. 8, when the tumor cells are added with the extract of bupleurum chinense nakai, the beta-microtubules generate aggregation phenomenon, which causes the continuous elongation of spindle yarn, and further prevents the 2-fold or multi-fold chromosome from moving to the two poles of the cells, so that the tumor cells cannot be divided into two parts. Thus, tumor cells not only fail to effectively perform mitosis, but even cause apoptosis of null cells (Junk cells) as 2N, 4N chromosomes are continuously accumulated.

Example 5: bupleurum falcatum extract for Taxol resistant tumor cell line (Taxol) Influence of Resistance Tumor Cell Line)

Because the existing drugs have no satisfactory poisoning effect on the paclitaxel-resistant tumor cells in the later period of chemotherapy, and the Flow Cytometry results also discover that the acetone crude extract of bupleurum chinense, the isobupleurum lactone analogues and the derivatives thereof have similar tumor inhibition mechanisms to paclitaxel on human liver cancer, ovarian cancer, malignant brain cancer, lung cancer and colorectal cancer, the invention takes the bupleurum chinense extract as a new drug screening source and further inspects the poisoning effect of the bupleurum chinense extract on paclitaxel-resistant cell lines.

In the following, the present invention will take an example of A paclitaxel-resistant lung cancer cell line A549-T12 generated by culturing A human lung cancer cell line A549, to test the effect of the acetone extract of kadsurA longipedunculatA (BS-A), the eighth component and the fifteenth component of the extract of kadsurA pedunculatA (BS-8, BS-15), each representing bupleurum lactone and similar derivatives related to bupleurum lactone), on A549-T12 cell line, and to evaluate the poisoning effect of the extract of kadsurA pedunculatA on paclitaxel-resistant tumor cell lines by using flow cytometry, drug toxicity test and tissue section.

As shown in FIGS. 9A to 9D, the changes between Annexin V-FLOUS and PI were compared between control groups (non-dosed A549-T12) and A549-T12 supplemented with 100nM paclitaxel, 30. mu.g/ml BS-A, 8. mu.g/ml BS-8 and 8. mu.g/ml BS-15 in A flow cytometer. The horizontal axis represents the fluorescence intensity of the tumor cells to which Annexin V-FLOUS antibody has been conjugated, while the vertical axis represents the fluorescence intensity of tumor cells conjugated with PI antibody, the result shows that after 48 hours of culture of the control group (referring to the non-drug-added paclitaxel-resistant tumor cell line A549-T12), the number of cells combined with Annexin V is only 6.8%, then the A549-T12 cell line is respectively added with isobupleurum lactone, bupleurum lactone derivative or bupleurum acetone crude extract, and cultured for 48 hours, the amount of the tumor cells combined with Annexin V is greatly increased by 30.6 percent, 23.1 percent and 24 percent, which shows that the capability of the isobupleurum lactone for inducing the apoptosis of the taxol-resistant cell line is better than that of other bupleurum lactone derivatives, moreover, the acetone extract of the bupleurum falcatum has a more obvious effect of promoting the tumor cells to generate withering of cells than the state of the crude extract after being purified.

Furthermore, from the results of cytotoxicity tests, when the crude extract of Bupleurum acetone (BS-A), Bupleurolide analogue (BS-8) and Bupleurolide derivative (BS-15) were added to the paclitaxel-resistant lung cancer cell line for 48 hours, as shown in FIGS. 10A to 10C, the survival rate of tumor cells was significantly reduced, and the effect of the purified Bupleurolide analogue on killing tumor cells was much smaller than that of acetone extract in terms of the half-lethal dose (IC50 or ED50) of the cells or animals. The bupleurum lactone analogue and the derivative thereof containing bupleurum lactone and isobupleurum lactone are shown to be main active substances for inhibiting tumor components in acetone crude extract of bupleurum, and the separated bupleurum extract only needs extremely low dose (1.5 micrograms/ml) to cause tumor cell apoptosis.

Example 6: evaluation of cancer suppressing Effect of extract of Bupleurum falcatum Linne in vivo (in vivo)

For example, tissue sections were further examined for the inhibition of the A549 cell line and the paclitaxel-resistant tumor cell (A549-T12) in vivo (in vivo) by using Bupleurum falcatum extract. As shown in fig. 11A, fig. 11B and fig. 12A and fig. 12B, when the necrosis of tumor tissue before and after treatment with BS-A was compared from the results of tissue sections by hematoxylin and Eosin Stain (haemataxylin and Eosin Stain, H & E Stain) at 100-fold magnification, it was found that tumor cells were lysed at the nuclear part, the lymphocytes infiltrated and hemorrhagic necrosis of large pieces of tumor cells occurred after administration of more than 300 mg/kg of BS-A to animals; further shown in FIG. 12B is that only A few tumor cells remained after BS-A treatment. Meanwhile, from the comparison of the subcutaneous tumor tissue (A549 cell line) in FIG. 13A, it is also shown that the subcutaneous tumor volume of the animals is significantly reduced by 77% (the tumor diameter is reduced from 13 microns to 3 microns) by the administration of 500 mg/kg of the extract of Bupleurum falcatum, and the tumor growth rate is also significantly slowed down after the tumor is treated with the extract of Bupleurum falcatum in terms of the relative tumor volume, as shown in FIG. 13B. Therefore, it can be known from tissue slices and subcutaneous tumor tissues that the extract of Bupleurum falcatum has a significant poisoning effect on tumor tissues in vivo and can control the expansion rate of tumor volume.

Example 7: effect of Bupleurum falcatum extract on cytotoxicity and animal toxicity

The experimental results of the invention detect that the acetone crude extract of bupleurum falcatum (60 micrograms/ml) has significant inhibitory effect on human liver cancer, ovarian cancer, lung cancer, malignant cerebroma and colorectal cancer cell lines, and in tissue section and subcutaneous tumor experiments, the acetone crude extract of bupleurum falcatum can poison tumor tissues in vivo (in vivo). Therefore, in order to further measure the toxicity of Bupleurum falcatum L.extract to other normal cells or organs while performing tumor poisoning, and to evaluate the possibility of Bupleurum falcatum L.extract as a new drug source, the present invention takes conscious mice as an example to show the change of biochemical indicator enzymes in each organ of mice after administering 400 micrograms/kg of Bupleurum falcatum L.extract.

As shown in fig. 14 and 15, after the injection of 400 μ g/kg acetone extract of bupleuri radix was intravenously administered to awake mice for 72 hours in vivo, the injection of the acetone extract of bupleuri radix did not poison the digestive system, circulatory system, metabolic system, hematopoietic function and germ cells in vivo, in various biochemical values such as pancreatic function index Lipase (Lipase), Amylase (Amylase), liver function index enzyme Glucose Oxidase (GOT), glucose phosphate converting enzyme (GPT), cardiac function index Lactate Dehydrogenase (LDH), Creatinine Kinase (CK), renal function index Creatinine (Creatinine), serum Urea Nitrogen (Blood Urea nitrate, BUN), and the like, as well as cardiac activity, diastolic pressure, systolic pressure, platelets and leukocytes. In addition, the liver and kidney tissue sections shown in FIG. 15 can be administered continuously five days into the abdominal cavity with 300 μ g of the extract of Bupleurum falcatum in each kilogram, and the normal liver cells and kidney cells will not be destroyed. On the other hand, since the acetone extract of Bupleurum falcatum or the isobupleurum lactone has obvious inhibition effect on the activity of Telomerase (Telomerase) of tumor cells of living animals, the above results clearly show that the Bupleurum falcatum extract does not affect the normal cell function or organ operation after being administered to the living body, but has highly specific poisoning effect on the tumor cells including the paclitaxel drug-resistant cell line, so that the Bupleurum falcatum extract or the pharmaceutically acceptable salts, esters, ketones and derivatives thereof can be used as the therapeutic drug, thereby effectively solving the problem that the existing drug has poor poisoning effect on the paclitaxel drug-resistant tumor cells, and providing a new source of antitumor drug for patients with gradually ineffective paclitaxel in the later period of chemotherapy.

Conclusion

In summary, the bupleurum falcatum extract obtained by the extraction method of the present invention is an acetone extract of bupleurum falcatum containing heterocyclic compounds with gamma-butyrolactone structure and carbon 2(5) in Z configuration or E configuration as shown in the general formula (I), and bupleurum lactone analogs and derivatives thereof, or pharmaceutically acceptable salts, esters, ketones and derivatives thereof, which are separated from the acetone extract of bupleurum falcatum and are represented by isobupleurum lactone, as tumor inhibiting active ingredients for treating human liver cancer, ovarian cancer, lung cancer, malignant brain tumor and colorectal cancer.

Formula (I)

Wherein X is N, O, S, Se;

a and B are respectively selected from substituent groups with the following formula:

wherein, R1, R2, R3, R4 and R5 are respectively selected from hydrogen atoms, halogen atoms, hydroxyl groups, sulfhydryl groups, amino groups, alkoxy groups and nitro groups.

However, according to the preferred embodiment of the present invention, the isobupleurum lactone obtained by separating and purifying the extract of bupleurum chinense has better tumor inhibition effect than the crude extract of acetone, and after the extract of bupleurum chinense is administered, it has significant effects in inhibiting tumor telomerase activity, reducing relative tumor volume, inhibiting tumor cell proliferation and promoting tumor cell withering, and can highly specifically poison tumor cells without affecting the normal digestive system, circulatory system, metabolic system, hematopoietic system and genitourinary system of the organism, no matter the general tumor cells or paclitaxel-resistant tumor cells.

Furthermore, from the viewpoint of cell regulation, the acetone extract of bupleurum chinense is a Microtubule Stabilizing Agent (microtubuling Agent) which promotes the aggregation of beta-microtubules of cytoskeleton and leads the elongated spindle silks not to be pulled to the two poles to cause apoptosis, and the action mechanism of the acetone extract is similar to that of paclitaxel, so that the mitosis of tumor cells can be promoted to be arrested in the G2/M phase, and the effect of controlling the proliferation of the tumor cells is achieved.

Claims (15)

1. A gamma-butyrolactone compound having the structure shown below:

wherein X is 0; a is

The B substituent is selected from the following substituent structures:

2. the γ -butyrolactone compound of claim 1, wherein the heterocyclic compound is in the Z configuration at carbon 2 (5).

3. The γ -butyrolactone compound of claim 1, wherein the heterocyclic compound is in the E configuration at carbon 2 (5).

4. An extract of bupleuri radix comprising the gamma-butyrolactone compound according to any one of claims 1 to 3 as an active ingredient.

5. A pharmaceutical composition for treating a cell proliferative disease, comprising the γ -butyrolactone compound according to any one of claims 1 to 3 as an active ingredient.

6. The pharmaceutical composition of claim 5, wherein the cell proliferative disorder is liver cancer in humans.

7. The pharmaceutical composition of claim 5, wherein the cell proliferative disorder is human ovarian cancer.

8. The pharmaceutical composition of claim 5, wherein the cell proliferative disorder is human malignant glioma.

9. The pharmaceutical composition of claim 5, wherein the cell proliferative disorder is human lung cancer.

10. The pharmaceutical composition of claim 5, wherein the cell proliferative disorder is human colorectal cancer.

11. The pharmaceutical composition of claim 5, wherein the cell proliferative disorder is resistant to a taxane therapeutic agent.

12. The pharmaceutical composition of claim 11, wherein the taxane therapeutic agent is paclitaxel.

13. The pharmaceutical composition of claim 5, wherein the active ingredient that inhibits cell proliferation is a G2/M arresting agent that regulates the cell cycle.

14. The pharmaceutical composition of claim 5, wherein the active ingredient that inhibits cell proliferation is a microtubule stabilizing agent that promotes aggregation of β -microtubules of the cytoskeleton.

15. An extraction process for isolating the extract of Bupleurum falcatum of claim 4 from Bupleurum falcatum, said extraction process comprising:

dissolving pulverized bupleuri radix in acetone solution, and separating to obtain acetone extract and residue of bupleuri radix;

dissolving the residue with methanol solution, and separating to obtain methanol extract of bupleuri radix;

extracting the acetone extract of Bupleurum falcatum with 95% methanol water solution, and removing alcohols in the extracted water layer; and

the alcohol-removed aqueous layer was extracted with chloroform solution and concentrated.