CN111135161A - Application and preparation method of perfoliate knotweed herb extract with anti-tumor activity - Google Patents
Application and preparation method of perfoliate knotweed herb extract with anti-tumor activity Download PDFInfo
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- CN111135161A CN111135161A CN201911399741.0A CN201911399741A CN111135161A CN 111135161 A CN111135161 A CN 111135161A CN 201911399741 A CN201911399741 A CN 201911399741A CN 111135161 A CN111135161 A CN 111135161A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/275—Nitriles; Isonitriles
- A61K31/277—Nitriles; Isonitriles having a ring, e.g. verapamil
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/70—Polygonaceae (Buckwheat family), e.g. spineflower or dock
- A61K36/704—Polygonum, e.g. knotweed
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/02—Antineoplastic agents specific for leukemia
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C253/00—Preparation of carboxylic acid nitriles
- C07C253/32—Separation; Purification; Stabilisation; Use of additives
- C07C253/34—Separation; Purification
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Veterinary Medicine (AREA)
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- Alternative & Traditional Medicine (AREA)
- Engineering & Computer Science (AREA)
- Oncology (AREA)
- Biotechnology (AREA)
- Botany (AREA)
- Medical Informatics (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Hematology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention relates to an application of perfoliate knotweed herb extract with anti-tumor activity in preparing anti-tumor drugs, the perfoliate knotweed herb extract is used as the only active component in the anti-tumor drugs prepared, and the component of the perfoliate knotweed herb extract is 2- (4-cyanophenyl) ethanol or (R) -1- (4-cyanophenyl) ethanol or the derivatives of the two. The preparation process of the extract comprises the following steps: 1) preparing perfoliate knotweed herb powder as a raw material; 2) performing coarse extraction by adopting ethanol; 3) pretreating the coarse fraction sample to meet the requirement of subsequent column chromatography separation; 4) carrying out at least one column chromatography separation on the coarse divided sample to obtain a subdivided sample; 5) and (4) carrying out thin-layer preparation plate separation and purification on the subdivided samples to obtain fine-divided pure products. The invention separates and extracts 2- (4-cyanophenyl) ethanol or (R) -1- (4-cyanophenyl) ethanol or the derivatives of the two from the perfoliate knotweed herb, and the further extract can be used for preparing the anti-tumor medicament, thereby improving the pharmaceutical value of the perfoliate knotweed herb and accelerating the research process of the anti-tumor medicament.
Description
Technical Field
The invention relates to the technical field of effective division and extraction of traditional Chinese medicine plants, in particular to a perfoliate knotweed herb extract and an extraction method and application thereof.
Background
Although the existing treatment modes for malignant tumors such as lung cancer, liver cancer, lymphoma and leucocyte comprise chemotherapy, radiotherapy, targeted drug therapy, immunotherapy and the like, indexes such as remission rate, long-term survival and the like are improved, a large number of patients still have the problem that the existing means cannot achieve remission or better long-term survival, and a new treatment means needs to be continuously searched. The traditional Chinese medicine for treating tumors has unique curative effects on stabilizing tumor bodies, regulating organism functions, increasing immunocompetence, improving clinical symptoms, relieving toxic and side effects of radiotherapy and chemotherapy and prolonging tumor-bearing survival time. At present, the traditional Chinese medicine in China has achieved remarkable achievements in the aspect of tumor treatment, and the research of treating tumors by the traditional Chinese medicine is a very meaningful exploration. Plants are not only one of the important sources of novel antitumor drugs, such as discovery and application of antitumor drugs such as vincristine, camptothecin, taxol and the like, but also the regulation effect of botanical drugs on the immune function of the organism is more and more concerned by people.
Perfoliate (Polygonum perfoliatum L.) alternative name: radix Seu caulis Berberidis Virgetori, herba Saxifragae, and herba Damnacanthi. Is the whole plant of herba Polygoni Cymosi of Polygonaceae. The polygonum perfoliatum is produced in a plurality of provinces of China, has rich medicine sources, and belongs to one of heat-clearing and detoxifying medicines in southern China. According to modern researches, polygonum perfoliatum has the effects of resisting virus, oxidation and bacteria, resisting inflammation, relieving cough, eliminating phlegm, protecting liver, resisting cancer and the like, and is clinically used for treating diseases such as cough, burn, herpes zoster, sting, bacillary dysentery and the like, and has better curative effect. In clinical application, the tumors of patients with terminal lymphoma, lung cancer and liver cancer, which are treated by perfoliate knotweed herb as a prescription, can be well controlled. Therefore, the inventor screens the medicines of the formula one by one to determine that the perfoliate knotweed herb is the medicine which plays the main anti-tumor effect in the formula, then separates and extracts the perfoliate knotweed herb, and screens the active ingredients step by the tumor cell inhibition test. There are studies on the separation and purification of perfoliate knotweed from which a compound is isolated: 8-carbonyl-pinoresinol (8-oxo-pinoresinol, Inc.), which was considered to have antitumor activity by in vitro cell assay (chemical composition of Periploca sepium and its biological activity Research-Xun, Master academic paper of Zhejiang university of Industrial and commercial, 2010; Wang KW, Zhu JR, Shen LQ.A new lignan with anti-tissue activity from Polygonum perfoliatum L.Natural Product Research, vol.27, vol.6, 568 and 573). There are no reports on whether other anti-tumor components or compounds exist in perfoliate knotweed herb.
Disclosure of Invention
The invention aims to provide the application of the perfoliate knotweed herb extract, and the extract has good anti-tumor activity and can be used for preparing anti-tumor drugs.
The invention also aims to provide a specific preparation method of the polygonum perfoliatum extract.
In order to achieve the purpose, the invention is realized by the following technical scheme: the perfoliate knotweed herb extract is used as the only active component in the prepared anti-tumor medicine, and the perfoliate knotweed herb extract is 2- (4-cyanophenyl) ethanol or (R) -1- (4-cyanophenyl) ethanol or the derivatives of the two.
2- (4-cyanophenyl) ethanol or (R) -1- (4-cyanophenyl) ethanol or derivatives of both are used as known compounds, generally as intermediates for synthesizing other compounds, and the biological activity of the known compounds is not independently researched, the application separates the compounds 2- (4-cyanophenyl) ethanol or (R) -1- (4-cyanophenyl) ethanol or derivatives of both from perfoliate knotweed herb, and proves that the 2- (4-cyanophenyl) ethanol or (R) -1- (4-cyanophenyl) ethanol or derivatives of both have anti-tumor effect on various tumor cell lines in vivo and in vitro through tumor cell growth inhibition, apoptosis experiments and transplantation tumor model experiments.
The preparation method of the polygonum perfoliatum extract with anti-tumor activity comprises the following steps:
(1) preparing raw materials: making herba Polygoni Perfoliati into dry powder;
(2) coarse extraction: extracting coarse fraction sample from herba Polygoni Perfoliati dry powder by ethanol extraction;
(3) pretreatment: pretreating the obtained rough separation sample to enable the rough separation sample to be subjected to subsequent column chromatography separation;
(4) carrying out at least one column chromatography separation on the pretreated dry silica gel mixed solid containing the crude sample to obtain a subdivided sample;
(5) and (4) carrying out thin-layer preparation plate separation and purification on the subdivided samples to obtain fine-divided pure products.
In order to better perform the preparation of the perfoliate knotweed herb extract with anti-tumor activity in the invention, further, in the step (2), the crude extraction process of the perfoliate knotweed herb extract is as follows: adding ethanol water solution with volume fraction of 95% into dry powder of perfoliate knotweed herb, mechanically stirring for 12-18 hours at room temperature, filtering, washing filter residue by using ethanol water solution with volume fraction of 95%, combining filter washing liquid, performing reduced pressure distillation on the filter washing liquid, and concentrating until no solvent is evaporated to obtain a blackish brown pasty crude fraction sample.
In order to better perform the preparation of the perfoliate knotweed herb extract with anti-tumor activity in the invention, further, in the step (3), the pretreatment process of the roughly divided sample is as follows: adding ethyl acetate into the crude fraction sample, heating and stirring the crude fraction sample in a hot water bath at 60 ℃ for 30 minutes, filtering the crude fraction sample while the crude fraction sample is hot, collecting filtrate, distilling the filtrate under reduced pressure, concentrating the filtrate to half of the original volume, adding 200-300 meshes of silica gel, uniformly stirring the silica gel by using a glass rod, and drying the silica gel under reduced pressure to obtain a dry silica gel mixed solid containing the crude fraction sample.
In order to better perform the preparation of the perfoliate knotweed herb extract with anti-tumor activity in the invention, further, in the step (4), a subdivided sample obtained by a pre-treated roughly divided sample is subjected to two chromatographic column separations, which are specifically as follows:
(4.1) first column chromatography separation: preparing an elution system and a silica gel column for first column chromatography separation, wherein an eluent in the elution system is petroleum ether, or ethyl acetate, or the mixture of the petroleum ether and the ethyl acetate according to a volume ratio of 1-10: 1-6, and the dosage of the eluent is 2-4L; directly adding the coarse sample and silica gel into a prepared silica gel column according to the weight ratio of 1:30, performing column chromatography separation to obtain a collection liquid, and treating the collection liquid to obtain a first subdivided sample;
(4.2) second column chromatography separation: preparing an elution system and a silica gel column for the second column chromatography separation, wherein an eluent in the elution system is ethyl acetate or petroleum ether and ethyl acetate according to a volume ratio of 1-10: 1-6 to form a mixed solution, wherein the dosage of the eluent is 1-4L; heating and dissolving the first subdivided sample by using ethyl acetate, wherein the weight ratio of the first subdivided sample to silica gel is 1: and 100, adding silica gel, uniformly stirring, drying in vacuum until no agglomeration exists, directly adding into the prepared silica gel column, performing column chromatography separation to obtain a collected liquid, and treating the collected liquid to obtain a second subdivided sample.
In order to better perform the preparation of the perfoliate knotweed herb extract with anti-tumor activity in the invention, further, the preparation process of the elution system of the first column chromatography separation in the step (4.1) is as follows: dissolving the crude fraction with 10-30 g ethyl acetate, performing TCL analysis, using petroleum ether and ethyl acetate in different proportions as developing agents, performing TLC development and sample loading condition analysis, and determining column chromatography gradient eluent to be mixed solution of petroleum ether and ethyl acetate at a volume ratio of 1:2, wherein the use amount of the eluent is 4L.
In order to better perform the preparation of the perfoliate knotweed herb extract with anti-tumor activity in the invention, further, the preparation process of the elution system of the second column chromatography separation in the step (4.2) is as follows: weighing ethyl acetate and a sample according to a weight ratio of 3:1, dissolving ethyl acetate in the ethyl acetate, subdividing the sample for the second time, performing TCL analysis, taking petroleum ether and ethyl acetate in different proportions as developing agents, and determining that column chromatography gradient eluent is ethyl acetate or a mixed solution of the petroleum ether and the ethyl acetate in a volume ratio of 1:2 or 1:6 according to TLC development and sample loading condition analysis, wherein the using amount of the mixed eluent is 2L.
In order to better prepare the perfoliate knotweed herb extract with anti-tumor activity in the invention, the preparation of silica gel columns in the first column chromatography separation in the step (4.1) and the second column chromatography separation in the step (4.2) are the same, a column with the column volume of 1.5L is selected, 500g of 200-300 mesh silica gel filler is used for filling petroleum ether into a 1L beaker, the petroleum ether is added for soaking and stirring until no bubbles exist, the column is filled by a wet method, about 2cm of petroleum ether is left at the top end after the column is filled, and the filled silica gel columns are kept stand for more than 10 hours to compact the silica gel.
In order to better prepare the perfoliate knotweed herb extract with anti-tumor activity in the invention, the collected liquid in the first column chromatography separation in the step (4.1) and the collected liquid in the second column chromatography separation in the step (4.2) are processed in the same way, and are concentrated to be dry through a rotary evaporator under reduced pressure, a little ethyl acetate is added into the residue for dissolving, the residue is transferred to a penicillin bottle for volatilizing to be dry, a sample is obtained, and the activity of the sample is detected.
In order to better prepare the perfoliate knotweed herb extract with anti-tumor activity in the invention, further, in the step (5), the finely divided sample is subjected to thin-layer preparative plate separation and purification, and the finely divided pure product is obtained by the following processes:
(5.1) determining the volume ratio of the developing solvent to be 1:1, mixing petroleum ether and ethyl acetate;
(5.2) adding ethyl acetate into the obtained second subdivided sample, stirring and dissolving to prepare a mixed solution for later use;
(5.3) preparing a silica gel plate, and drawing a straight line at the position of about 3cm away from the bottom of the silica gel plate by using a pencil ruler;
(5.4) sampling, namely sampling the mixed solution prepared in the step (5.2) by using a glass capillary until the mixed solution is just soaked, standing to volatilize the solvent, and then continuously drying by using an electric blower;
(5.5) placing the same silica gel plate into a chromatographic cylinder for unfolding, taking out the silica gel plate when the solvent reaches about 2cm of the front edge of the top end of the preparation plate, blowing dry the solvent by using electric air blowing, continuing to place the silica gel plate into the chromatographic cylinder for unfolding, taking out the silica gel plate when the solvent reaches about 2cm of the front edge of the top end of the preparation plate for the second time, blowing dry the solvent by using electric air blowing, developing under an ultraviolet lamp, and lightly scratching a color band with the largest color spot by using a pencil;
(5.6) repeating the process of the step (5.5), and performing development and purification by using a silica gel plate until all the active components are used up;
(5.7) carefully scraping all prepared silica gel plate color bands by using a scraper, placing the silica gel plates in a beaker, grinding the silica gel into powder as much as possible, adding ethyl acetate, stirring, pouring the powder into a small silica gel column with the length of 150mm and the diameter of 10mm, collecting eluent, and eluting for multiple times by using ethyl acetate until the eluent is completely eluted; mixing eluates, concentrating under reduced pressure in a rotary evaporator to dry to obtain refined pure product.
In order to better prepare the perfoliate knotweed herb extract with anti-tumor activity in the invention, further, the determination process of the developing agent in the step (5.1) is that a small amount of second subdivided sample is taken to be dissolved by ethyl acetate, the sample is spotted on a plate, the second subdivided sample is developed by petroleum ether and ethyl acetate with different proportions, and the developing agent is finally determined to be a mixed solution of the petroleum ether and the ethyl acetate with the volume ratio of 1:1 according to the separation effect.
In order to better perform the preparation of the perfoliate knotweed herb extract with anti-tumor activity in the present invention, further, the silica gel plate is made of glass, the size thereof is 150mm x 200mm, and the thickness of the silica gel thereof is 2 mm.
The invention also provides an anti-tumor medicament which comprises an oral preparation, an intravenous injection preparation, an internal spray and a prodrug prepared from the polygonum perfoliatum extract, the salt, the crystal form and the solvate thereof as the only active ingredients and pharmaceutically acceptable medicinal carriers and/or excipient auxiliary ingredients which are nontoxic and inert to human and animals. The tumor diseases which can be treated by the anti-tumor medicine comprise lung cancer, liver cancer, leukemia, lymphoma and myeloma.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) the invention separates and extracts a certain substance from the perfoliate knotweed by researching the anti-tumor activity of the perfoliate knotweed, the substance is 2- (4-cyanophenyl) ethanol or (R) -1- (4-cyanophenyl) ethanol or the derivatives of the two, and the substance separated and extracted from the perfoliate knotweed is further proved to have the anti-tumor activity by a plurality of ways of tumor cell growth inhibition, apoptosis experiments and transplantation tumor model experiments, and can be used for preparing anti-tumor drugs;
(2) the invention finds that the extract of the perfoliate knotweed herb is 2- (4-cyanophenyl) ethanol or (R) -1- (4-cyanophenyl) ethanol or derivatives of the two, and the 2- (4-cyanophenyl) ethanol or (R) -1- (4-cyanophenyl) ethanol or derivatives of the two are used as known compounds and generally used as intermediates for synthesizing other compounds, and the biological activity of the compounds is not independently researched, the invention fills the technical blind area of the application of the 2- (4-cyanophenyl) ethanol or (R) -1- (4-cyanophenyl) ethanol or derivatives of the two in the biological aspect, improves the pharmaceutical value of the perfoliate knotweed herb, accelerates the research process of antitumor drugs, the development of the anti-tumor medicine is promoted, and the positive progress significance is achieved;
(2) the invention also provides a novel method for preparing 2- (4-cyanophenyl) ethanol or (R) -1- (4-cyanophenyl) ethanol or derivatives of the two, which provides a choice of multiple ways for preparing the high-purity 2- (4-cyanophenyl) ethanol or (R) -1- (4-cyanophenyl) ethanol or derivatives of the two by separating and extracting from the perfoliate knotweed for the first time, and improves the application value of the 2- (4-cyanophenyl) ethanol or (R) -1- (4-cyanophenyl) ethanol or derivatives of the two;
(3) the invention also provides an anti-tumor medicament which takes 2- (4-cyanophenyl) ethanol or (R) -1- (4-cyanophenyl) ethanol or derivatives of the two as the only active components, the anti-tumor medicament can play a positive treatment effect on lung cancer, liver cancer, leukemia and lymphoma, and provides good news for patients suffering from tumor diseases.
Drawings
Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:
FIG. 1 is a line graph showing the dilution concentration and inhibition rate of the extract of perfoliate knotweed herb in the present invention after 36 hours of the action on human acute myeloleukemia cell-like (MOLM-13), lung cancer (H460), thyroid cancer papillary cell (BCPAP), and human hepatoma cell (HuH-7);
FIG. 2 is a line graph showing the dilution concentration and inhibition rate of the extract of perfoliate knotweed herb in the present invention after 48 hours of its action on human acute myelogenous leukemia cell-like (MOLM-13), lung cancer (H460), thyroid cancer papillary cell (BCPAP), and human hepatoma cell (HuH-7);
FIG. 3 is a flow cytogram of myeloma cell AMO.1 in a blank control group according to example 4 of the present invention;
FIG. 4 is a flow cytogram of myeloma cell AMO.1 in a control group according to example 4 of the present invention;
FIG. 5 is a flow cytogram of myeloma cell AMO.1 in experimental group 1 of example 4 of the present invention;
FIG. 6 is a flow cytogram of myeloma cell AMO.1 in experimental group 2 of example 4 of the present invention;
FIG. 7 is a flow cytogram of myeloma cell AMO.1 in experimental group 3 according to example 4 of the present invention;
FIG. 8 is a flow cytogram of myeloma cell AMO.1 in experimental group 4 of example 4 of the present invention;
FIG. 9 is a flow cytogram of myeloma cell AMO.1 in experimental group 5 of example 4 of the present invention;
FIG. 10 is a flow cytogram of myeloma cell AMO.1 in experimental group 6 of example 4 according to the present invention;
FIG. 11 is a line graph showing the effect of the extract of perfoliate knotweed on the volume of the transplanted tumor in the present invention;
FIG. 12 is a bar graph showing the effect of extract of herba Polygoni Perfoliati on the weight of transplanted tumor in the present invention.
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto, and various substitutions and alterations can be made without departing from the technical idea of the present invention as described above, according to the common technical knowledge and the conventional means in the field.
The present invention will be described in further detail with reference to the following examples for the purpose of making clear the objects, process conditions and advantages of the present invention, which are given by way of illustration only and are not intended to be limiting of the present invention.
Example 1:
the embodiment provides an application of perfoliate knotweed herb extract with antitumor activity in preparing an antitumor drug, wherein the perfoliate knotweed herb extract is used as the only active component in the prepared antitumor drug, and the component of the perfoliate knotweed herb extract is 2- (4-cyanophenyl) ethanol or (R) -1- (4-cyanophenyl) ethanol or the derivative of the two.
The principal chemical structure of the components of perfoliate knotweed herb extract is as follows:
wherein R is1、R2、R3、R4Has antitumor activity when the extract is H atom, i.e. 2- (4-cyanophenyl) ethanol or (R) -1- (4-cyanophenyl) ethanol. Other R's not affecting the antitumor activity of the compound1、R2、R3、R4The substituents can be selected.
The embodiment also provides an antitumor drug which is an oral preparation, an intravenous injection preparation, an internal spray and a prodrug prepared by using the polygonum perfoliatum extract and the salt, the crystal form and the solvate thereof as the only active ingredients and pharmaceutically acceptable medicinal carriers and/or excipient auxiliary ingredients which are nontoxic and inert to human and animals. The antitumor drug can be used for treating tumor diseases including lung cancer, liver cancer, leukemia, lymphoma, and myeloma.
Example 2:
the present embodiment provides a method for preparing a perfoliate knotweed herb extract with anti-tumor activity in the above embodiments, which comprises the following steps:
1. raw material preparation
Pulverizing dried herba Polygoni Perfoliati into powder, sieving with 80 mesh sieve
2. Extraction and pretreatment of radix Angelicae sinensis
(1) Extraction of
2Kg of perfoliate knotweed powder and 11L of 95% ethanol are added into a 20L three-necked bottle, and the mixture is mechanically stirred for about 15 hours at room temperature. Filtering, washing the filter residue with 2L 95% ethanol, and mixing the filter washing solutions. The filtrate was concentrated under reduced pressure until no solvent was distilled off, to obtain 77.25g of a brown-black paste-like crude fraction.
(2) Pretreating the coarse fraction before column chromatography
77.25g of the pasty crude sample is taken, 200mL of ethyl acetate is added, the mixture is heated and stirred in a hot water bath at 60 ℃, and the hot mixture is filtered to obtain 44g of filter residue. Concentrating the filtrate under reduced pressure to half volume, adding 80g of 200-300 mesh silica gel, uniformly stirring, and drying under reduced pressure for later use. 112.57g of dry silica gel mixed solid was weighed, wherein the sample content was about 32.57 g.
3. Separating by column chromatography
(1) The first sample column chromatography separation is carried out
(1.1) determination of elution System
Dissolving the filtrate with a small amount of ethyl acetate, and analyzing by TLC, wherein the developing solvent comprises petroleum ether and ethyl acetate in different proportions of 1: 0. 10:1, 8:1, 6:1, 2:1, 1:2, 1:4, 1:6 and 0:1, and according to TLC development and sample loading condition analysis, the ratio of petroleum ether to ethyl acetate 1: gradient elution was performed at 0(3L), 10:1(2L), 8:1(2L), 6:1(2L), 2:1(2L), 1:2(4L), 1:4(2L), 1:6(2L), and 0:1 (2L).
(1.2) silica gel column preparation
Selecting a column with the column volume of 1.5L, adding petroleum ether into a 1L beaker with silica gel filler (200-300 meshes, 500g), soaking and stirring until no bubbles exist, filling the column by a wet method, leaving about 2cm of petroleum ether at the top end after filling the column, and standing the filled silica gel column for more than 10 hours to compact the silica gel.
(1.3) sample application
The sample-containing dried silica gel powder was divided into two portions, each portion containing about 16.0g of the sample, and the ratio of the roughly divided sample to the silica gel was about 1:30 (by weight), and the divided portions were directly added to a packed silica gel column. The upper end should be leveled after the appearance, if there is the bubble, strike the sample position on the post gently, make the bubble discharge, put into the cotton wool on the chromatographic column, produce the impact to the sample when preventing to add the eluant, destroy the sample layer, influence the separation effect.
(1.4) elution and Collection
Gradient elution is carried out on the silica gel column by using a determined elution system, pure petroleum ether is eluted until the front edge of the silica gel column reaches the bottom of the column by a color circle, effective collection is carried out, and the silica gel column is collected once every 500mL, wherein the specific table is shown as the following table one:
TABLE elution for the first column chromatography separation of the samples
(1.5) grouping of the collected liquids
Concentrating each collected solution to dryness by a rotary evaporator under reduced pressure, adding a little ethyl acetate into the residue to dissolve, transferring to a penicillin bottle, and volatilizing to dryness. Performing TLC analysis on each component, performing color development analysis through an ultraviolet lamp, an iodine cylinder and phosphomolybdic acid, combining similar components, concentrating and combining No. 4-No. 18, separating out solids, and filtering to obtain solids. Obtaining IX component groups in total, wherein the V and VI components have activity, and the component groups are shown in the table two:
TABLE 2 Table of the collected liquid obtained by the first sample column chromatography
| Component number | Number of liquid collection |
| Ⅰ | 1#～3# |
| Ⅱ | 4#～18# |
| Ⅲ | 19#～23# |
| Ⅳ | 24#～25# |
| Ⅴ | 26# |
| Ⅵ | 27#～34# |
| Ⅶ | 35#～38# |
| Ⅷ | 39#～45 |
| Ⅸ | |
| 4# to 18# precipitated solid |
The remaining half of the samples were subjected to elution grouping in the same manner. The total amount of the two components is 6 g.
(2) Performing column chromatography separation on the sample for the second time
(2.1) determination of elution System
Dissolving a small amount of active sample by using ethyl acetate, and analyzing by TLC, wherein developing agents are petroleum ether and ethyl acetate in different proportions, and the proportions are respectively 1: 0. 10:1, 8:1, 6:1, 2:1, 1:2, 1:4, 1:6 and 0:1, according to TLC development analysis, the ratio of column chromatography gradient elution is determined as that petroleum ether is subjected to gradient elution with ethyl acetate 10:1(1L), 6:1(1L), 4:1(3L), 2:1(4L), 1:1(2L), 1:2(1L), 1:6(2L) and pure ethyl acetate (2L).
(2.2) silica gel column preparation
Selecting a column with the column volume of 1.5L, adding petroleum ether into a silica gel filler (200-300 meshes, 500g) in a 1L beaker, soaking and stirring until no bubbles exist, filling the column by a wet method, leaving about 2cm of petroleum ether at the top end after filling the column, and standing the filled silica gel column for more than 10 hours to compact the silica gel.
(2.3) sample application
Dissolving 6.0g of active sample in 24mL of ethyl acetate by heating, stirring silica gel, uniformly stirring, and drying in vacuum until no block is formed. The sample and silica gel ratio was about 1:100 (weight ratio) and added directly to the packed silica gel column. The upper end should be leveled after the appearance, if there is the bubble, strike the sample position on the post gently, make the bubble discharge, put into the cotton wool on the chromatographic column, produce the impact to the sample when preventing to add the eluant, destroy the sample layer, influence the separation effect.
(2.4) elution and Collection
The silica gel column was subjected to gradient elution with the established elution system, collected every 500mL, as shown in table three:
TABLE III elution for the second column chromatography separation of the samples
| Serial number | Eluent (V/V) | Dosage (L) | Collecting liquid (500 mL/portion) |
| 1 | Petroleum ether: ethyl acetate 10:1 | 1 | — |
| 2 | Petroleum ether: ethyl acetate 6:1 | 1 | — |
| 3 | Petroleum ether:ethyl acetate 4:1 | 3 | 1#～6# |
| 4 | Petroleum ether: ethyl acetate 2:1 | 4 | 7#～14# |
| 5 | Petroleum ether: ethyl acetate 1:1 | 2 | 15#～17# |
| 6 | Petroleum ether: ethyl acetate 1:2 | 2 | 18#～20# |
| 7 | Petroleum ether: ethyl acetate 1:6 | 2 | 21#～23# |
| 8 | Ethyl acetate | 2 | 24#～26# |
(2.5) grouping of the collected liquids
Concentrating each collected solution to dryness by a rotary evaporator under reduced pressure, adding a little ethyl acetate into the residue to dissolve, transferring to a penicillin bottle, and volatilizing to dryness. TLC analysis was performed for each fraction, and color analysis was performed by ultraviolet lamp, iodine cylinder and phosphomolybdic acid, and similar fractions were combined, wherein fractions (7) and (8) were active, to obtain 1.0g of an active sample. The components are grouped as in the table below.
TABLE IV Table for the second time of separating the collected liquid by column chromatography
4. Separating and purifying fine active component thin layer preparation plate
(1) Determination of a spreading agent
Taking a small amount of the component No. 7, dissolving the component with ethyl acetate, dotting the component on a plate, developing the component with petroleum ether and ethyl acetate in different proportions, and determining the petroleum ether: the best separation effect is obtained when ethyl acetate is 1:1 (V/V).
(2) Sample loading and deployment
1g of a subdivided sample subjected to two column chromatographic separations is dissolved in 10ml of ethyl acetate with stirring for further use. Pouring prepared petroleum ether into a chromatographic cylinder: ethyl acetate 1:1(V/V) developing solvent, saturated with steam in the chromatography cylinder. A silica gel plate (150mm is multiplied by 200mm, the thickness of silica gel is 2mm) is prepared by taking glass, and a straight line is drawn at the position of about 3cm of the bottom by a pencil ruler. And (3) spotting the component/ethyl acetate mixed solution by using a glass capillary until the component/ethyl acetate mixed solution is just soaked, standing until the solvent is volatilized, and then continuously drying by using an electric blower after the sample is loaded. And (3) placing the same glass preparation silica gel plate into a chromatographic cylinder for unfolding, taking out the silica gel plate when the solvent reaches about 2cm of the front edge of the top end of the preparation plate, blowing the solvent for drying by using electric air, continuing to place the preparation plate into the chromatographic cylinder for unfolding, taking out the silica gel plate when the solvent reaches about 2cm of the front edge of the top end of the preparation plate for the second time, blowing the solvent for drying by using electric air, developing under an ultraviolet lamp, and lightly marking out a color band with the largest color spot by using a pencil.
The silica gel plate prepared by glass is developed and purified by the same method until all active components are used up, and 15 silica gel plates are counted.
(3) Scraper and elution
The silica gel plate color band is carefully scraped by a scraper and is arranged inIn the beaker, silica gel was ground to a powder as much as possible. Adding ethyl acetate, stirring, pouring into small silica gel column (length 150mm,
) And (5) collecting the eluent in a 50mL single-mouth bottle, and eluting with ethyl acetate for multiple times to be clean as much as possible. Mixing eluates, concentrating under reduced pressure in rotary evaporator to dry to obtain refined pure product 15 mg.
Example 3:
this example provides the experiments of inhibitory activity of perfoliate knotweed herb extract on in vitro human tumor cell proliferation:
3.1 purpose of the experiment
The inhibition activity of the perfoliate knotweed herb extract on the proliferation of in vitro human tumor cells is detected, and the inhibition activity of the perfoliate knotweed herb extract on human acute myeloleukemia cells (MOLM-13), lung cancer cell strains (H460), thyroid cancer papillary cells (BCPAP) and liver cancer cells (Huh-7) is tested by adopting an MTT (tetramethyl azoazolate) colorimetric method. IC for inhibiting activity of perfoliate knotweed herb extract on tumor cells50(median inhibitory concentration) is shown. IC (integrated circuit)50Values can be obtained by calculating the inhibition rate of the test compound on tumor cells at a range of different concentrations.
3.2 principle of the experiment
Succinate dehydrogenase in mitochondria of living cells can reduce exogenous MTT to water-insoluble blue-purple crystalline Formazan (Formazan) and deposit in cells, while dead cells do not have this function. Dimethyl sulfoxide (DMSO) can dissolve formazan in cells, and the light absorption value of formazan is measured at 540nm or 720nm wavelength by using a microplate reader, which can indirectly reflect the number of living cells. Within a certain range of cell number, MTT crystals are formed in an amount proportional to the cell number.
3.3 Experimental procedures:
3.3.1 cell preparation
3.3.1.1 resuscitating cells
3.3.1.1.1 the water bath kettle is opened in advance to be preheated to 37 deg.C. 2-3mL of medium was placed in a 15mLBD tube;
3.3.1.1.2 taking out the cells from a refrigerator or a liquid nitrogen tank at-80 deg.C, immediately placing into a water bath, and shaking the seed-preserving tube to melt the liquid-preserving liquid as soon as possible;
3.3.1.1.3 returning to the clean bench, sucking the seed-retaining liquid into the prepared culture medium by a 1mL pipette, centrifuging at 800rpm for 5 min;
3.3.1.1.4 adding 8-10mL of culture medium into each 10cm dish, centrifuging, removing supernatant, and suspending in culture medium;
3.3.1.1.5 the next day after cell recovery, the solution is changed, and if the cells are suspended, the solution is changed by centrifugation and re-suspension.
3.3.1.2 cell passages
When the cells grow to the proper density, the passage operation is carried out, the passage density is determined according to the growth rate of the cells, and the general state of the freshly recovered cells is not stable and cannot be used for experiments immediately.
3.3.1.2.1 directly collecting the suspension cells, centrifuging at 1000rpm for 3 min;
the adherent cells need to be firstly poured out of the culture medium, washed once by PBS, added with pancreatin and put into an incubator for 30s-5 min. Then, the cells were neutralized with a medium, collected, and centrifuged at 1000rpm for 3 min.
3.3.1.2.2 the supernatant is decanted off and the cells are resuspended in culture medium and passaged at a ratio of 1:3 to 1: 8.
3.3.1.3 cell plating
3.3.1.3.1 cells were collected directly from suspension and centrifuged at 1000rpm for 3 min.
The adherent cells need to be firstly poured out of the culture medium, washed once by PBS, added with pancreatin and put into an incubator for 30s-5 min. Then, the cells were neutralized with a medium, collected, and centrifuged at 1000rpm for 3 min.
3.3.1.3.2 the cells were resuspended in culture medium and cell counted.
3.3.1.3.3 plates 96 well, adherent cells are plated at 2000 to 5000 cells per well, suspension cells are plated at 5000-20000 cells per well, 100uL of medium per well.
The side holes of the 3.3.1.3.496 well plate have strong evaporation effect and influence on cell growth, so that only the middle 60 holes are paved. Sterile culture medium or PBS and other liquid is added into the side hole.
3.3.2 cell dosing
3.3.2.1 the cells to be adhered need to be added with medicine after the cells are completely adhered, the cells are generally cultured overnight, and the medicine is added the next day. For uniform comparison of the same batch, suspension cells were also cultured overnight, with dosing the next day.
3.3.2.2 the stock solution was diluted in a 1:100 concentration ratio and added to a 96-well plate at 100uL per well. When the plates are added, 100uL of culture medium is already in the wells, and the final concentration is 2.2-70 uM.
3.3.2.3 contrast packets
Blank control wells were filled with 100uL of medium.
In addition, BLANK wells should be placed, i.e., without adding cells, only media is added.
Solvent control wells, corresponding volumes of solvent, such as DMSO, PBS, etc., are added to the volume of the dosing wells.
3.3.3 cell observations
The cells were observed daily and the inhibition rate of the cells was visually evaluated. And detecting when the scheduled time is up.
3.3.4MTT assay
MTT powder was dissolved in physiological saline to 50mg/mL and then filtered through a 0.22. mu.M filter head.
20uL of MTT solution is added into each hole, the cross is evenly shaken, and the mixture is put into an incubator to react for 1 to 4 hours.
Adherent cells were directly decanted from the medium and the residual liquid in the wells was then blotted dry on absorbent paper. The suspension cells were centrifuged at 3000rpm for 5min in a centrifuge, then the medium was carefully decanted and the residual liquid in the wells was blotted on absorbent paper.
150uL DMSO was added to each well, the mixture was shaken on a shaker for 15min, the absorbance at 570nm was measured with a microplate reader, and the OD value was recorded.
3.3.5 data calculation
Each well value is first subtracted by BLANK to participate in calculation
Survival rate is 100% of mean value of experimental wells/mean value of control wells
Inhibition rate (1-mean of experimental wells/mean of control wells) × 100%
3.3.6 results of the experiment
As shown in table five and table six:
TABLE five drugs 36 hours of cell growth inhibition
TABLE-six drug action 48 hours cell growth inhibition
According to the contents in the fifth table, the sixth table and the figures 1 and 2, the perfoliate knotweed herb extract is used as an intervention reagent, has good inhibition effect on the growth inhibition tests of human acute myeloleukemia cell-like cells (MOLM-13), lung cancer cells (H460), thyroid cancer papillary cells (BCPAP) and liver cancer cells (Huh-7), particularly has more obvious inhibition effect on cells with faster proliferation, and has the inhibition rate of more than 50 percent at 30-70 uM.
Example 4:
this example provides the effect experiment of perfoliate knotweed herb extract on apoptosis of human myeloma cell amo.1:
4.1 purpose of the experiment
The influence of the polygonum perfoliatum extract on the apoptosis of human myeloma cells AMO.1 is detected.
4.2 principle of the experiment
In normal cells, the phosphatidylserine is distributed on the inner side of a cell membrane, and when the cells are subjected to apoptosis, the cells are turned outwards to the surface of the cells. When the cells are necrosed or enter the late stage of apoptosis, the integrity of the cell membrane is destroyed, and then PI can enter the cells and is combined with cell chromosomes to generate red fluorescence.
4.3 Experimental procedures
4.3.1 Experimental groups
Blank control group (Blank group): without adding any reagent
Control group: addition of 1.2uM Adriamycin reagent
Experimental group 1: adding herba Polygoni Perfoliati extract agent with concentration of 17.5uM
Experimental group 2: adding herba Polygoni Perfoliati extract agent with concentration of 14uM
Experimental group 3: adding herba Polygoni Perfoliati extract agent with concentration of 8.75uM
Experimental group 4: adding herba Polygoni Perfoliati extract agent with concentration of 8uM
Experimental group 5: adding herba Polygoni Perfoliati extract agent with concentration of 3.5uM
Experimental group 6: adding herba Polygoni Perfoliati extract agent with concentration of 1.75uM
4.3.2 Experimental procedures
4.3.2.1 laying 6-mesh plates at a suitable density of about 1-2 x 105Individual cells/well;
4.3.2.2. culturing overnight, and waiting for cell adherence;
4.3.2.3. adding medicine according to the designed concentration, and arranging a negative control hole, a solvent control hole and a positive control hole;
4.3.2.4 after 48h, collecting cells;
4.3.2.5. diluting the 10 × binding buffer to 1 × binding buffer with ddH 20;
4.3.2.6 the cells were washed once more with 1 × binding buffer and finally resuspended with 100 uLbindingbuffer, not more than 2 × 10 cells per tube5And (4) respectively. Separating multiple cells one by one to prepare two single-staining tubes (a single-staining tube 1 and a single-staining tube 2) and a non-staining control tube;
4.3.2.7 adding 2.5uLAnnexin V into each tube, dyeing for 10-15min at normal temperature in dark place, and adding 2.5uLAnnexin V into the single dyeing tube 1;
4.3.2.8 adding 2.5uLPI or 7-AAD into each tube, mixing, dyeing for 5-10min in dark, and adding 2.5uLPI into single dyeing tube 2;
4.3.2.9 cells were washed once with 1 XBinding buffer, centrifuged at 400g for 4min, and finally resuspended in 1 XBinding buffer and flow-loaded.
4.4 results of the experiment
The Blank control group (Blank group) showed 3.48% of apoptosis rate in the cell population as shown in FIG. 3;
in the control group, as shown in fig. 4, the apoptosis rate of the cell population was 58.8%;
experimental group 1 as shown in fig. 5, the apoptosis rate of the cell population was 79.5%;
experimental group 2 as shown in fig. 6, the apoptosis rate of the cell population was 84.2%;
experimental group 6 As shown in FIG. 10, the apoptosis rate of the cell population was 5.55%.
The perfoliate knotweed herb extract is used as an intervention reagent to act on myeloma cells AMO.1, and adriamycin is used as a contrast, and the result shows that the perfoliate knotweed herb extract has obvious apoptosis promoting effect on the myeloma cells AMO.1 at 17.5uM or 14uM and the effect is better than that of 1.2uM adriamycin.
Example 5:
this example provides the effect experiment of the extract of perfoliate knotweed on the model of C26 mouse graft tumor:
5.1 purpose of the experiment
The perfoliate knotweed herb extract in the invention is verified to have printing effect on a C26 mouse transplantation tumor model.
5.2 principle of the experiment
Mice are inoculated with murine tumor cells subcutaneously to simulate the in vivo growth environment of the tumor cells. The inhibition effect of the drug on tumor cells in an in vivo environment is studied by oral administration of the drug to mice.
5.3 Experimental procedures:
5.3.1 Large female Balb/c mice, 6-8 weeks old, were purchased and acclimatized in the animal house for one week.
5.3.2 cells are simultaneously cultured in vitro and ready for inoculation by the time the cells grow to a certain amount.
5.3.3 cells were harvested at log phase, washed 3 times with PBS, and cell concentration adjusted to 1X 107one/mL into PBS
5.3.4 mice were inoculated subcutaneously in the right shoulder area with 100uL of each mouse, i.e., 1X 106Individual cell
5.3.5 mice were observed daily and tumor size was measured, approximately at day 6 post-inoculation, and the average tumor size reached around 100, the group dosing was started and the first data was recorded. The formula for calculating the tumor volume is as follows: v (mm)3) 0.5 × maximum diameter (mm) × square of minimum diameter (mm)2)
The grouping and administration modes are as follows:
solvent control group: same volume as experimental group, p.o.
Experiment group one: 0.1mg/kg, p.o.
Experiment group two: 0.2mg/kg, p.o.
Experiment group three: 0.4mg/kg, p.o.
5.3.6. Mice were dosed continuously daily and tumor size and mouse body weight were recorded every other day
5.3.7. The average tumor volume of the control group reached 2000mm3Considering the discontinuation of the administration, the mice were sacrificed uniformly the last day, the tumors were stripped off, weighed, photographed, and the mice were left with 4% paraformaldehyde for pathology.
5.3.8. And calculating the tumor inhibition rate of the medicine by using the tumor weight data. And simultaneously, the body weight change curve and the tumor growth curve of the mouse are collated.
5.4 Experimental results:
as shown in table seven:
tumor volume (mm) in mice of control group and inhibition group of Epimedium3) Influencing conditions
As shown in Table seven and FIG. 11, the tumor-transplanted C26 mice injected subcutaneously with perfoliate knotweed herb extract had significantly reduced tumor volume in vivo, as shown in FIG. 12, the tumor weight was significantly reduced when the tumors were weighed at the observation end, and it was concluded that the perfoliate knotweed herb extract of the present invention has inhibitory effect on tumor growth
While embodiments of the invention have been shown and described, it will be understood by those of ordinary skill in the art that: various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.
Claims (10)
1. The application of the perfoliate knotweed herb extract with the anti-tumor activity in preparing the anti-tumor medicine is characterized in that the perfoliate knotweed herb extract is used as the only active component in the anti-tumor medicine prepared, and the component of the perfoliate knotweed herb extract is 2- (4-cyanophenyl) ethanol or (R) -1- (4-cyanophenyl) ethanol or the derivative of the two.
2. The method for preparing perfoliate knotweed herb extract with anti-tumor activity according to claim 1, comprising the steps of:
(1) preparing raw materials: making herba Polygoni Perfoliati into dry powder;
(2) coarse extraction: extracting coarse fraction sample from herba Polygoni Perfoliati dry powder by ethanol extraction;
(3) pretreatment: pretreating the obtained rough separation sample to enable the rough separation sample to be subjected to subsequent column chromatography separation;
(4) carrying out at least one column chromatography separation on the pretreated crude sample to obtain a subdivided sample;
(5) and (4) carrying out thin-layer preparation plate separation and purification on the subdivided samples to obtain fine-divided pure products.
3. The method for preparing perfoliate knotweed herb extract with anti-tumor activity according to claim 2, wherein in the step (2), the crude extraction process of perfoliate knotweed herb extract comprises: adding ethanol water solution with volume fraction of 95% into dry powder of perfoliate knotweed herb, mechanically stirring for 12-18 hours at room temperature, filtering, washing filter residue by using ethanol water solution with volume fraction of 95%, combining filter washing liquid, performing reduced pressure distillation on the filter washing liquid, and concentrating until no solvent is evaporated to obtain a blackish brown pasty crude fraction sample.
4. The method for preparing perfoliate knotweed herb extract with anti-tumor activity according to claim 2 or 3, wherein the step (3) of pre-treating the roughly divided sample comprises: adding ethyl acetate into the crude fraction sample, heating and stirring the crude fraction sample in a hot water bath at 60 ℃ for 30 minutes, filtering the crude fraction sample while the crude fraction sample is hot, collecting filtrate, distilling the filtrate under reduced pressure, concentrating the filtrate to half of the original volume, adding 200-300 meshes of silica gel, uniformly stirring the silica gel by using a glass rod, and drying the silica gel under reduced pressure to obtain a dry silica gel mixed solid containing the crude fraction sample.
5. The method for preparing perfoliate knotweed herb extract with anti-tumor activity according to claim 2 or 3, wherein in the step (4), the finely divided sample obtained from the pre-treated coarse divided sample is subjected to two chromatographic column separations, specifically as follows:
(4.1) first column chromatography separation: preparing an elution system and a silica gel column for first column chromatography separation, wherein an eluent in the elution system is petroleum ether, or ethyl acetate, or the mixture of the petroleum ether and the ethyl acetate according to a volume ratio of 1-10: 1-6, and the dosage of the eluent is 2-4L; directly adding the coarse sample and silica gel into a prepared silica gel column according to the weight ratio of 1:30, performing column chromatography separation to obtain a collection liquid, and treating the collection liquid to obtain a first subdivided sample;
(4.2) second column chromatography separation: preparing an elution system and a silica gel column for the second column chromatography separation, wherein an eluent in the elution system is ethyl acetate or petroleum ether and ethyl acetate according to a volume ratio of 1-10: 1-6 to form a mixed solution, wherein the dosage of the eluent is 1-4L; heating and dissolving the first subdivided sample by using ethyl acetate, wherein the weight ratio of the first subdivided sample to silica gel is 1:100, adding silica gel, uniformly stirring, drying in vacuum until no agglomeration exists, directly adding into the prepared silica gel column, performing column chromatography separation to obtain a collection liquid, and treating the collection liquid to obtain a second subdivided sample;
wherein the preparation of the silica gel column in the first column chromatography separation in the step (4.1) and the preparation of the silica gel column in the second column chromatography separation in the step (4.2) are the same, the silica gel column is selected with the column volume of 1.5L, 500g of 200-300 mesh silica gel filler is used for adding petroleum ether into a 1L beaker for soaking and stirring until no bubbles exist, the column is filled by a wet method, about 2cm of petroleum ether is left at the top end after the column is filled, and the filled silica gel column is kept stand for more than 10 hours to compact the silica gel;
and (3) in the step (4.1), the collected liquid in the first column chromatography separation and the collected liquid in the second column chromatography separation in the step (4.2) are treated in the same process, the collected liquid is concentrated to be dry through a rotary evaporator under reduced pressure, a little ethyl acetate is added into the residue to be dissolved, the dissolved residue is transferred to a penicillin bottle to be volatilized to be dry, a sample is obtained, and the activity of the sample is detected.
6. The method for preparing perfoliate knotweed herb extract with anti-tumor activity according to claim 5, wherein the preparation process of the elution system for the first column chromatography separation in step (4.1) is as follows: dissolving the crude fraction with 10-30 g ethyl acetate, performing TCL analysis, using petroleum ether and ethyl acetate in different proportions as developing agents, performing TLC development and sample loading condition analysis, and determining column chromatography gradient eluent to be mixed solution of petroleum ether and ethyl acetate at a volume ratio of 1:2, wherein the use amount of the eluent is 4L.
7. The method for preparing perfoliate knotweed herb extract with anti-tumor activity according to claim 5, wherein the preparation process of the elution system for the second column chromatography separation in step (4.2) is as follows: weighing ethyl acetate and a sample according to a weight ratio of 3:1, dissolving ethyl acetate in the ethyl acetate, subdividing the sample for the second time, performing TCL analysis, taking petroleum ether and ethyl acetate in different proportions as developing agents, and determining that column chromatography gradient eluent is ethyl acetate or a mixed solution of the petroleum ether and the ethyl acetate in a volume ratio of 1:2 or 1:6 according to TLC development and sample loading condition analysis, wherein the using amount of the mixed eluent is 2L.
8. The method for preparing perfoliate knotweed herb extract with anti-tumor activity according to claim 2 or 3, wherein in the step (5), the finely divided sample is subjected to thin layer preparative plate separation and purification, and the finely divided pure product is obtained by the following processes:
(5.1) determining the volume ratio of the developing solvent to be 1:1, mixing petroleum ether and ethyl acetate; the specific determination process of the developing agent comprises the steps of dissolving 0.1g of the second subdivided sample by using ethyl acetate, dotting the sample, developing by using petroleum ether and ethyl acetate in different proportions, and finally determining the developing agent to be a mixed solution of the petroleum ether and the ethyl acetate with the volume ratio of 1:1 according to the separation effect;
(5.2) adding ethyl acetate into the obtained second subdivided sample, stirring and dissolving to prepare a mixed solution for later use;
(5.3) preparing a silica gel plate, and drawing a straight line at the position of about 3cm away from the bottom of the silica gel plate by using a pencil ruler;
(5.4) sampling, namely sampling the mixed solution prepared in the step (5.2) by using a glass capillary until the mixed solution is just soaked, standing to volatilize the solvent, and then continuously drying by using an electric blower;
(5.5) placing the well-shaped silica gel plate into a chromatographic cylinder for unfolding, taking out the silica gel plate when the ethyl acetate solution dissolved with the subdivided sample reaches about 2cm of the front edge of the top end of the preparation plate, blowing dry the solvent by electric blowing, continuing to place the silica gel plate into the chromatographic cylinder for unfolding, taking out the silica gel plate when the ethyl acetate solution dissolved with the subdivided sample reaches about 2cm of the front edge of the top end of the preparation plate for the second time, blowing dry the solvent by electric blowing, developing under an ultraviolet lamp, and lightly scratching a color band with the largest color spot by a pencil;
(5.6) repeating the process of the step (5.5), and performing development and purification by using a silica gel plate until all the active components are used up; the silica gel plate is made of glass with silica gel laid on the upper surface, the size of the silica gel plate is 150mm multiplied by 200mm, and the thickness of the silica gel plate is 2 mm;
(5.7) carefully scraping all prepared silica gel plate color bands by using a scraper, placing the silica gel plates in a beaker, grinding the silica gel into powder as much as possible, adding ethyl acetate, stirring, pouring the powder into a small silica gel column with the length of 150mm and the diameter of 10mm, collecting eluent, and eluting for multiple times by using ethyl acetate until the eluent is completely eluted; mixing eluates, concentrating under reduced pressure in a rotary evaporator to dry to obtain refined pure product.
9. An antitumor drug characterized in that the antitumor drug comprises the perfoliate knotweed herb extract as claimed in claim 1 and salts, crystal forms and solvates thereof as the only active ingredient, and the rest is pharmaceutically acceptable medicinal carriers and/or excipient auxiliary ingredients which are nontoxic and inert to human and animals, and can be prepared into oral preparations, intravenous injection preparations, internal sprays and prodrugs.
10. The antitumor drug according to claim 9, wherein the tumor diseases that the antitumor drug can treat include lung cancer, liver cancer, leukemia, lymphoma, myeloma.
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| WO2005035534A1 (en) * | 2003-10-08 | 2005-04-21 | Ono Pharmaceutical Co., Ltd. | Heterocyclic bicyclo ring and heterocyclic tricyclo ring compounds and drugs comprising the same |
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| CN103922936A (en) * | 2014-05-14 | 2014-07-16 | 上海交通大学 | Method for preparing caffeic acid ester derivatives |
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| WO2005035534A1 (en) * | 2003-10-08 | 2005-04-21 | Ono Pharmaceutical Co., Ltd. | Heterocyclic bicyclo ring and heterocyclic tricyclo ring compounds and drugs comprising the same |
| CN1906155A (en) * | 2003-12-03 | 2007-01-31 | 利奥制药有限公司 | Novel hydroxamic acid esters and pharmaceutical use thereof |
| CN103922936A (en) * | 2014-05-14 | 2014-07-16 | 上海交通大学 | Method for preparing caffeic acid ester derivatives |
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