CN102908340B - Isolicoflavonol-containing antitumor drug and application thereof - Google Patents
Isolicoflavonol-containing antitumor drug and application thereof Download PDFInfo
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Abstract
The invention provides an isolicoflavonol-containing antitumor drug, wherein suspension type injections, tablets and microcapsules are processed and manufactured by taking isolicoflavonol as the active ingredient and combining with pharmaceutically acceptable auxiliary materials and carriers. The antitumor drug is used for preventing and treating breast cancer, ovarian cancer, cervical cancer and colon cancer and usual gastrointestinal cancer, lung cancer, kidney cancer, bladder cancer, prostate cancer, skin cancer, head and neck cancer, brain tumor or leukemia. Confirmed by pharmacological experiments and antitumor effect comparing evaluation, the isolicoflavonol coming from traditional Chinese medicine liquorice has the function of inhibiting the generation of new blood vessels and is capable of achieving the purpose of resisting tumors through inhibiting the generation of the new blood vessels of the tumors. The invention provides an industrial preparation method of the isolicoflavonol serving as a medicinal material, comprising extraction, separation, grain size specification and other concrete process requirements.
Description
Technical field
The invention belongs to pharmaceutical field, specifically a kind of antitumor drug containing Isolicoflavonol. and application thereof, relate to the preparation method of active component Isolicoflavonol. and preparing the application in antitumor drug.
Background technology
Angiogenesis is considered to the absolute necessary requirement of the tumor growth more than 1-2mm.In the tumor being less than this limit, oxygen and nutrient are by spreading to cell.But when reaching certain size, each tumor will rely on angiogenesis in order to continued growth.Tumorigenic cell in low oxygen area reacts by stimulating VEGF (VEGF) to generate, and this causes the activation of quiescent endothelial cells to stimulate new vascularization.In addition, undertaken by ras signal transduction path without any the VEGF in the tumor region of angiogenesis.Hybridization in situ experiment has confirmed that VEGF mRNA is by strong upregulation in various human tumor, and these tumors comprise pulmonary carcinoma, breast carcinoma, gastrointestinal cancer, ovarian cancer and angiosarcoma and several intracranial tumor.
Therefore, solid tumor can carry out effective prevention by VEGF formation inhibitor, because the generation of these tumors all depends on angiogenesis, supports that it grows necessary blood vessel to be formed.These solid tumors comprise the cancer of sense of organization lymphoma, brain, genitourinary tract, lymphsystem, stomach, larynx and pulmonary, comprise adenocarcinoma of lung and small cell lung cancer.
Growth, the Infiltration and metastasis of entity tumor all depend on angiogenesis.And nearest research shows, angiogenesis and regulatory factor thereof and leukemic generation, differentiation, prognosis are closely related, are that the anti-angiogenic therapy of target site is equally applicable to leukemic treatment with angiogenesis.
So, the therapeutic strategy " dying of hunger tumor " by Tumor suppression angiogenic growth, be not only applicable to solid tumor, also can be applicable to leukemia, this Therapeutic Method compared with existing chemotherapeutics, have side effect little, harmless to human normal cell, can not cause chemotherapy syndrome, can the distinguishing feature such as metastasis and extension of remarkable Tumor suppression.
Exploitation for anti-angiogenic agent is used for the prevention and therapy of tumor, has become the focus pursued of Ge great drugmaker of the world.The Tumor suppression angiogenic growth agent of chemosynthesis, enters clinical significant obstacle because inevitable toxicity becomes; Comparatively speaking, if the anti-angiogenic agent of natural origin found can be screened, and be used as active group and prepare cancer therapy drug, huge exploitation advantages and market prospect widely will be had.But; the active component extract with anti-cancer function of current natural origin is still in the Test Study stage; disclosed report is only only limitted to component separating and the test of laboratory, not have discovery about can the report of active component of natural origin of large-scale production.Have impact on the antitumor drug industrialized developing containing the active component of natural origin and effect thereof.
Summary of the invention
The object of the invention is the anti-angiogenic agent active component Isolicoflavonol. developing and provide a kind of natural origin, and provide a kind of can suitability for industrialized production, be suitable for the preparation method of Isolicoflavonol. improving bioavailability, to promote its application in field of antineoplastic medicaments.
The present inventor has carried out a large amount of screening and pharmacological evaluation and antitumous effect comparative evaluation to natural medicinal ingredients, final confirmation derives from the Isolicoflavonol. in glycyrrhiza uralensis fisch, tumor vascular endothelium somatomedin is generated there is significant inhibitory action, can reach antitumor object by the generation of Tumor suppression new vessels, be a kind of natural origin active medicine component compound having the antitumor drug of Development volue.
Isolicoflavonol. (isolicoflavonol) is a kind of isoflavonoid, is yellow needles, is insoluble in water, is dissolved in the solvent that polarity is little, as ether, chloroform etc.; Be present in the plurality of Chinese such as the root of glycyrrhizic legume, this makes industrialization extraction, pharmacological evaluation, toxicity test, clinical research and utilizes Isolicoflavonol. to develop new drug to become possibility.
Isolicoflavonol. structural formula is as follows:
R in formula
1=R
2=R
4=R
7=OH, R3=R
5=H, R
6=-CH
2cH=C (CH
3)
2
According to the literature, the flavone compound in Radix Glycyrrhizae, can be divided into following a few class: flavone or flavonols, chalcones, osajin, isoflavanone class etc.In screening study and pharmacological evaluation process, the present inventor surprisingly finds, the structure and energy of flavonoid drugs exists dependency, so flavonoid drugs becomes the emphasis that the present inventor pays close attention to.The flavonols composition contained in bibliographical information Radix Glycyrrhizae is two kinds of Isolicoflavonol .s and Licoflavonol. mainly.The difference of the two is Isolicoflavonol. R
3=H, R
6=-CH
2cH=C (CH
3)
2; And Licoflavonol. R
3=-CH
2cH=C (CH
3)
2, R
6=H.The larger difference of spatial configuration of molecules is caused at the iso-amylene chain of molecular long axis end.As everyone knows, the structure that the physiologically active of compound is through receptor in medicine and body or target spot produces, and the space structure of compound or conformation are the key factors determining pharmaceutically active.Inventor has carried out a large amount of pharmacological evaluation contrasts to two kinds of flavonol, and pleasantly surprised discovery, Isolicoflavonol. has the significant active function suppressing new vessels to generate, and Be very effective is better than Licoflavonol..
The present inventor, in a series of pharmacology pathological study, confirms Isolicoflavonol. further and has and suppress the effect of new vessels systematic function, can the growth of Tumor suppression and transfer by the generation of Tumor suppression new vessels, thus reaches antitumor object.This is a kind of newfound anticancer mechanism, from reported in the past all different.
Term " tumor " means tumor proliferative disorders herein, such as solid tumor disease, liquid tumor diseases.
Term used herein " solid tumor disease " is breast carcinoma, ovarian cancer, cervical cancer, colon cancer and common gastrointestinal cancer, pulmonary carcinoma especially, and such as small cell lung cancer and nonsmall-cell lung cancer, renal carcinoma, bladder cancer, carcinoma of prostate, skin carcinoma are as melanoma, head and neck cancer or cerebroma.
Term used herein " liquid tumor diseases " especially leukemia.
The inhibitory action to tumor that isoliquiritigenin alcoholic compound of the present invention relates to, by a series of test effect Data Comparison of the present inventor, provides conclusive evidence.
The test data of representative instance disclosed in this invention, proves: Isolicoflavonol. obviously can suppress the growth of mice S180 tumor further; Obviously can suppress the growth of Mice Bearing Lewis Lung Cancer, and effectively suppress the transfer of pulmonary carcinoma focus; Prove further in the test of Brachydanio rerio, the antitumor action of Isolicoflavonol., can VEGF be suppressed with it thus the generation of Tumor suppression new vessels is relevant; Isolicoflavonol. obviously can suppress the generation of Brachydanio rerio new vessels, and under same experiment condition, its analog-Licoflavonol., only has faint vascular study effect, compared with blank, does not show significant difference.
The extraction process comparative maturity of flavone, but the preparation technology of acquisition high-purity flavonol monomeric compound is also remarkable, what reason to be in Radix Glycyrrhizae at present that only flavones ingredient finds just has tens kinds, the similarity of molecular structure, result in the difficulty of separation and purification, need through purifies and separates repeatedly according to the conventional separation means of laboratory, generally can only obtain several milligrams of samples, can only be used for detecting and test, can not amplify and be applied to production, the loaded down with trivial details isolation technics of laboratory, does not have the value of suitability for industrialized production.
The present invention needs solution technical problem to be to provide a kind of method of extraction purification Isolicoflavonol. from liquorice root, to overcome the defect that current laboratory research cannot amplify production, to carry out the purification Isolicoflavonol. product of scale.The present invention is according to the molecular structure feature of Isolicoflavonol., and not containing glycosyl in its molecule, containing hydroxyl, B ring structure causes polarity and other flavone to have difference to a certain degree.The present invention is exactly the construction features utilizing Isolicoflavonol.; devise the method that polyamide column-silicagel column-polydextran gel combination chromatograph carries out separation and purification; successfully achieve the scale operation being separated from Radix Glycyrrhizae and obtaining isoliquiritigenin alcohol monomer, the response rate reaches 75%.
In the separation process of polyamide column, the flavonoid glycoside that polarity is larger becomes branch to be first eluted, and the flavone aglycone not containing glycosyl just can be eluted subsequently.Containing causing different polarity in molecule, according to this feature, flavone and saponin constituent separate by silica gel column chromatography.And hydroxypropyl polydextran gel can identify the different spaces structure that the B ring in flavone structure is formed, flavone component close with other structures for Isolicoflavonol. is separated.
Pass through the inventive method, the stable extract that Isolicoflavonol. content is not less than 3% can be obtained through extraction, defat purification, through further polyamide column-silicagel column-polydextran gel tandem compound chromatographic isolation preparation, the Isolicoflavonol. of purity >=95% can be obtained.
The industrialized process for preparing of the main active Isolicoflavonol. of antitumor drug of the present invention is specific as follows:
1) extract
Extracting liquorice root, drying and crushing, extracts in a heated state with medical material dry weight 4-10 polar solvent doubly at every turn, extracts 2-3 time.Described pulverizing medicinal materials granularity is below 80 orders; Described polar solvent is the mixed solvent of alcohols (being generally methanol, ethanol, propanol or isopropyl alcohol) or alcohol water: the heating-up temperature of described leaching process is 50-80 DEG C; In described mixed extraction solvent, shared by alcohol, volume ratio is 60%-95%.
2) defat
Above-mentioned extract is concentrated, recycling design, obtain extractum, add 5-15 alcohol-water mixture doubly and make dispersant, add equal-volume petroleum ether, ether, alkanes (normal hexane, pentane or normal heptane) carry out extraction defat, extract 2-4 time.Described alcohol-water mixture is the mixture of alcohol (being generally methanol, ethanol, propanol or isopropyl alcohol) and water, and relative volume ratio is 5-20%.
3) polyamide column is separated
Polyamide column top is arrived by the alcohol aqueous phase after above-mentioned defat, adopt and carry out eluting with the alcohol water mixed solvent of loading medicinal liquid same concentrations, be eluted to colourless, flavonoid glycoside impurity constituents eluting is removed, then carry out eluting by the alcohol water mixtures of eluents of 40-60%, collect eluent.The applied sample amount of described column chromatography is 2-6 times of column volume, and loading flow velocity is 3-5 times of column volume/per hour; Described alcohol-water mixture is the mixture of alcohol (being generally methanol, ethanol, propanol or isopropyl alcohol) and water.
4) silicagel column is separated
Upper step eluent is concentrated, recycling design, silicagel column is adopted to be separated, each applied sample amount is the 1/300-1/50 of silica gel weight, adopt the mixed solvent eluting that halogenated hydrocarbons (chloroform, dichloromethane) and alcohol (being generally methanol, ethanol, propanol or isopropyl alcohol) form, collect eluent, recycling design obtains yellow solid.Described mixed solvent ratio is 3:1 ~ 5:1.
5) sephadex column is separated
Polydextran gel dress post (being generally hydroxypropyl polydextran gel), blade diameter length ratio 10:1, obtains yellow eluate upper prop by upper step, adopts alcohol (being generally methanol, ethanol, propanol or isopropyl alcohol) eluting, receives first colour band, recycling design, concentrated, dry.
The Isolicoflavonol. obtained adopts the method test of high performance liquid chromatography, and result shows, Isolicoflavonol. content is more than 95%.
But the lower problem of the inherent bioavailability of flavones ingredient becomes another obstacle that Isolicoflavonol. is applied to clinical treating disease.The chemical structure characteristic of flavones ingredient, cause its water solublity and fat-soluble all poor, the clinical practice of a lot of flavonoid medicine is all limited to this problem of drug absorption, and drug absorption difference then bioavailability is low, so the performance of drug effect is also restricted.
With regard to the Isolicoflavonol. product that the method for routine obtains, owing to usually there is partial over saturation phenomenon in crystallization process, cause Isolicoflavonol. crystal size uneven, particle size distribution is wide, and crystal poor fluidity, specific volume is high.
The change of medicament powder will cause the difference of medicine essence.The present invention is after Isolicoflavonol. crystallize, add micronization processes process, and through Experimental Comparison, determine to control Isolicoflavonol. micropowder diameter of particle median at≤100 μm, bioavailability can be improved, increase drug absorption, and the hot-spot that conventional method can be avoided to cause is cohered, decomposing phenomenon.Because the distribution of Isolicoflavonol. grain size of micropowder is concentrated, narrow particle size distribution, reaches good medicinal effects requirement, and what make Isolicoflavonol. prepare medicine as active component may become reality.
The present invention adopts jet-impingement technology or micronizing to carry out micronization processes to extracting obtained Isolicoflavonol., obtains, micronization Isolicoflavonol. product that mean diameter is controlled.
Production technology of the present invention is with short production cycle, safety, and cost is lower, and yield is high, is particularly suitable for industrialization and produces in a large number.In view of Isolicoflavonol. has the effect of strong suppression new vessels, production technology of the present invention industrialization from Radix Glycyrrhizae is prepared Isolicoflavonol. in a large number and will be had broad prospects.
Just based on the further investigation of the mechanism of action to Isolicoflavonol. inhibiting angiogenesis and the exploitation to Isolicoflavonol. scale extracting method; the present inventor confirms further; with isoliquiritigenin alcoholic compound for main anti-tumor active component; the exploitation having carried out corresponding antitumor drug product is feasible, can provide a kind of antitumor drug with new antitumoral mechanism for pharmaceutical field.
Antitumor drug containing Isolicoflavonol. of the present invention, it is the main active of growth and transfer using Isolicoflavonol. as Tumor suppression, be processed into various types of pharmaceutical formulation with pharmaceutically acceptable adjuvant, carrier combinations, comprise suspension type injection, tablet or capsule etc.
In antitumor drug of the present invention, active component Isolicoflavonol., accounts for 10% ~ 99% of medicine gross weight.
The active component of suspension type injection of antitumor drug of the present invention and the composed as follows of adjuvant: according to weight ratio, Isolicoflavonol. 1 part, vitamin B
12 ~ 2.5 parts, 1.8 ~ 2.5 parts, sodium chloride, sodium alginate 2.0 ~ 2.6 parts, Polyoxyethylene Sorbitan Monooleate 0.8 ~ 1.3 part, uses water for injection suspendible, makes suspension type injection.
The tablet of antitumor drug of the present invention or capsule take Isolicoflavonol. as effective ingredient, the conventional tablet formed with the filler in pharmaceutics meaning, disintegrating agent or capsule; Or the slow releasing tablet that forms of Isolicoflavonol. and filler and hydroxypropyl methylcellulose or capsule; The wherein optional lactose of filler, microcrystalline Cellulose, dextrin, starch, calcium phosphate; Disintegrating agent can select hyprolose, carboxymethyl starch sodium, polyvinylpolypyrrolidone, cross-linking sodium carboxymethyl cellulose; Also optionally binding agent, lubricant or wetting agent is added.
Described good pharmaceutical composition compositing formula is as follows: according to weight ratio, Isolicoflavonol. 1 part: lactose or microcrystalline Cellulose 0.05 ~ 0.2: hyprolose or carboxymethyl starch sodium 0.05 ~ 0.2; Or, Isolicoflavonol. 1 part: lactose or microcrystalline Cellulose 0.03 ~ 0.08: hydroxypropyl methylcellulose K4M 0.15 ~ 0.4.
Described preferred pharmaceutical composition compositing formula is as follows: according to weight ratio, Isolicoflavonol. 1 part: lactose or microcrystalline Cellulose 0.08 ~ 0.12: hyprolose or carboxymethyl starch sodium 0.08 ~ 0.12; Or, Isolicoflavonol. 1 part: lactose or microcrystalline Cellulose 0.05 ~ 0.07: hydroxypropyl methylcellulose K4M 0.2 ~ 0.3.
The compounds of this invention or the pharmaceutical composition containing it can administrations in a unit, and route of administration can be intestinal or non-bowel, as oral, muscle, subcutaneous, nasal cavity, oral mucosa, skin, peritoneum or rectum etc.
The compounds of this invention or the route of administration containing its pharmaceutical composition can be drug administration by injection.Injection comprises intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection and acupoint injection therapy etc.
The dosage of the compounds of this invention pharmaceutical composition depends on many factors, such as to prevent or the character of disease therapy and the order of severity, the sex of patient or animal, age, body weight, individual reaction, route of administration administration number of times, therapeutic purposes, therefore therapeutic dose of the present invention can have change in a big way, can according to the unit formulation dosage in the compounds of this invention compositions, in addition suitable adjustment, to reach the requirement of its treatment effective dose, complete prevention of the present invention or therapeutic purposes.Suitable dosage ranges every day of the compounds of this invention elects again 0.1 ~ 50mg/kg body weight as, is more preferably 50mg ~ 1000mg/ days/people.Above-mentioned dosage can single dose form or be divided into several, and such as two, three, four dosage forms for administration, this is limited to administration doctor clinical experience and comprises the dosage regimen simultaneously using other treatment means.
detailed description of the invention:
Essence for a better understanding of the present invention, illustrates its novelty teabag in pharmaceutical field by the pharmacological testing of Isolicoflavonol. and result below.
embodiment 1:
Radix Glycyrrhizae sample 5kg, pulverizes, and 10 times amount 95 % ethanol 50 DEG C extract 3 times, each 4 h.Extracting liquid filtering, concentrating under reduced pressure recycling design, dilute with the appropriate amount of ethanol of 15 times amount 20 %, after defat with petroleum ether, upper polyamide column is separated, applied sample amount is 6BV, loading flow velocity is 3BV/h, colourless to effluent by 20% methanol-eluted fractions, use 40% methanol-eluted fractions afterwards instead, collect the eluent of 3 times of column volume to 6 times column volumes, after concentrated removal solvent, through silica gel column chromatography, chloroform-methanol (3: 1, v/ v) carry out eluting, eluent is separated through Sephadex L H-20 gel chromatographic columns again, methanol-eluted fractions, collect first color band, collect eluent, concentrated, crystallize, micronization processes, obtain the Isolicoflavonol. of diameter of particle median≤100 μm, purity 98.0%, the response rate 75%.
embodiment 2:
Radix Glycyrrhizae sample 8kg, pulverizes, and 5 times amount 75% ethanol 80 DEG C extract 3 times, each 2 h.Extracting liquid filtering, concentrating under reduced pressure recycling design, dilute with the appropriate amount of ethanol of 5 times amount 10 %, after defat with petroleum ether, upper polyamide column is separated, applied sample amount is 4BV, loading flow velocity is 4BV/h, colourless to effluent by 10% methanol-eluted fractions, use 50% methanol-eluted fractions afterwards instead, collect the eluent of 3 times of column volume to 6 times column volumes, after concentrated removal solvent, through silica gel column chromatography, dichloromethane-ethanol (4:1, v/ v) carry out eluting, eluent is separated through Sephadex L H-20 gel chromatographic columns again, methanol-eluted fractions, collect first color band, collect eluent, concentrated, crystallize, micronization processes, obtain the Isolicoflavonol. of diameter of particle median≤100 μm, purity 95.1%, the response rate 76.2%.
embodiment 3:
Radix Glycyrrhizae sample 10kg, pulverizes, and 8 times amount 60% ethanol 70 DEG C extract 3 times, each 2 h.Extracting liquid filtering, concentrating under reduced pressure recycling design, dilute with the appropriate amount of ethanol of 10 times amount 5 %, after defat with petroleum ether, upper polyamide column is separated, applied sample amount is 2BV, loading flow velocity is 5BV/h, colourless to effluent by 10% methanol-eluted fractions, use 60% methanol-eluted fractions afterwards instead, collect the eluent of 3 times of column volume to 6 times column volumes, after concentrated removal solvent, through silica gel column chromatography, methylene chloride-methanol (5:1, v/ v) carry out eluting, eluent is separated through Sephadex L H-20 gel chromatographic columns again, ethanol elution, collect first color band, collect eluent, concentrated, crystallize micronization processes, obtain the Isolicoflavonol. of diameter of particle median≤100 μm, purity 96.7%, the response rate 75.9%.
embodiment 4
The change of medicament powder particle diameter will cause the difference of medicine essence.In an experiment of the character of research micronization Isolicoflavonol. product of the present invention, we observed the difference of the effect caused by the difference of grain size of micropowder.After the Isolicoflavonol. micropowder process zebra fish model using different particle diameters, observe the consistent effect to the generation of Brachydanio rerio intersegmental blood vessel, result shows, when particle diameter≤100 μm, inhibit activities shows the difference (p<0.05) of significance
Table 1 variable concentrations Isolicoflavonol. is to the inhibitory action of zebrafish embryo intersegmental blood vessel
embodiment 5: example 2 Isolicoflavonol. of the present invention is to the observation of mice S180 tumor-inhibiting action
Aseptic aspiration lotus S
180mouse ascites, puts counted under microscope by cell suspension, and adjustment cell number is 2.5 × 10
7/ ml and 2.0 × 10
7/ ml, in mice right fore armpit subcutaneous vaccination tumor liquid 0.2ml/ only.Next day random packet, Isolicoflavonol. micropowder with do not carry out micronizedly being set to 2 groups, positive controls (FT
207110mg/kg) and saline control group.Gastric infusion 9 days all continuously, every Mus gavage every day volume is 0.5 ml.Saline control group gavage is to same volume normal saline.Within after last administration 24 hours, put to death animal, dissect tumor block and claim tumor to weigh and body weight, calculate tumour inhibiting rate.
The results are shown in Table 1, result display micronization Isolicoflavonol. is to mice S
180have stronger tumor-inhibiting action (p<0.05), effect is suitable with positive controls (suppression ratio is 53.2%).
Table 2 Isolicoflavonol. is to mice S
180the inhibitory action that sarcoma generates
Note: *, compared with the control;
embodiment 6:example 2 Isolicoflavonol. of the present invention is on the impact of Mice Bearing Lewis Lung Cancer spontaneity transfer
Lewis lung cancer cell strain makes oncocyte suspension, and trypan blue counting is about 1 × 10
7/ mL oncocyte.The right front axil of sterilization mice, gets 0.2 mL (about 1 × 10
6-2 × 10
6individual oncocyte) be inoculated in subcutaneous.Go down to posterity 3 times.Aseptically peel off Lewis lung cancer mice with tumor subcutaneous tumors block, choose well-grown tumor tissue, shred, claim quality, homogenate is made with cell homogeniser, 1:3 adds normal saline and makes oncocyte suspension by volume, be inoculated in C57BL/6J mice right fore axillary fossa subcutaneous, every 0.2mL (about 1 × 10
6~ 2 × 10
6individual oncocyte).Next day random packet, Isolicoflavonol. micropowder with do not carry out micronizedly being set to 2 groups, positive controls (FT
207110mg/kg) and saline control group.Gastric infusion 9 days all continuously, every Mus gavage every day volume is 0.5 ml.Saline control group gavage is to same volume normal saline.Within after last administration 24 hours, put to death animal, dissect tumor block and claim tumor to weigh and body weight.Calculate tumour inhibiting rate and the focus rate of transform.
On the spontaneous metastasis model of Mice Bearing Lewis Lung Cancer, abdominal cavity is administered to and gives Isolicoflavonol., result Isolicoflavonol. can obviously suppress the weight of Lewis lung cancer tumor to increase (p<0.05), and reducing the number of mice (rate of transform) occurring Lung metastases, the number of Lung metastases also obviously reduces simultaneously.The results are shown in Table 2
The inhibitory action that table 3 Isolicoflavonol. shifts Mice Bearing Lewis Lung Cancer
, * p<0.05, compared with the control
embodiment 7:the inhibitory action that Isolicoflavonol. of the present invention generates new vessels
When being TG(VEGFR2:GFP) blood vessel fluorescent transgenic Brachydanio rerio development of fertilized ova 24h, under the mirror of stereoscopic aobvious position, selecting normal zebrafish embryo, move in 96 well culture plates, 1 piece, every hole.Add certain volume medicine in advance in sample hole, every concentration 32 hole, then supplement volume to 300 μ l with nutrition water, contrast as embryotrophy water.When development of fertilized ova 72h, the growing state of fluorescence microscope Brachydanio rerio body intersegmental blood vessel, calculates suppression ratio, and observes embryo or prelarva death condition.
The vascular development of zebrafish embryo is extremely similar to the mankind, and the component of its back major blood vessel is columns arrangement, easily observes, and is the best model that observation medicine affects neovascularization growth.The new vessels occurred when the growth at tumor initial stage or focus transfer can be evaluated with zebra fish model.Found that Isolicoflavonol. all obviously can suppress the generation (see table 3) of Brachydanio rerio new vessels, be Isolicoflavonol. compound antitumor of the present invention effect, provide evidence.Under same experiment condition, its analog-Licoflavonol., only has faint vascular study effect, compared with blank, does not show significant difference.
Table 4 variable concentrations Isolicoflavonol. is to the inhibitory action of zebrafish embryo intersegmental blood vessel
Note: * has significant difference (P<0.05) compared with matched group; * has extremely significant difference (P<0.001) compared with matched group
Claims (4)
1. be used as an industrial production process for antitumor drug main active Isolicoflavonol., it is characterized in that concrete preparation method is as follows:
1) extract
Extracting liquorice root, drying and crushing, extracts in a heated state with medical material dry weight 4-10 polar solvent doubly at every turn, extracts 2-3 time;
Described pulverizing medicinal materials granularity is below 80 orders; Described polar solvent is alcohols or alcohol water mixed solvent; The heating-up temperature of described leaching process is 50-80 DEG C; In described mixed solvent, volume ratio shared by alcohols is 60%-95%;
2) defat
Above-mentioned extract is concentrated, recycling design, obtain extractum, add 5-15 alcohol-water mixture doubly and make dispersant, add equal-volume petroleum ether, ether, normal hexane, pentane or normal heptane and carry out extraction defat, extract 2-4 time; Described alcohol-water mixture is the mixture of methanol, ethanol, propanol or isopropyl alcohol and water, and relative volume ratio is 5-20%;
3) polyamide column is separated
Polyamide column top is arrived by the alcohol aqueous phase after above-mentioned defat, adopt and carry out eluting with the alcohol water mixed solvent of loading medicinal liquid same concentrations, be eluted to colourless, flavonoid glycoside impurity constituents eluting is removed, then carry out eluting by the alcohol water mixtures of eluents of 40-60%, collect eluent; The applied sample amount of described column chromatography is 2-6 times of column volume, and loading flow velocity is 3-5 times of column volume/per hour; Described alcohol-water mixture is the mixture of methanol, ethanol, propanol or isopropyl alcohol and water;
4) silicagel column is separated
Concentrated by upper step eluent, recycling design, adopt silicagel column to be separated, each applied sample amount is the 1/300-1/50 of silica gel weight, adopts the mixed solvent eluting that halogenated hydrocarbons and alcohol form, and collect eluent, recycling design obtains yellow eluate; Described halogenated hydrocarbons is chloroform or dichloromethane, and described alcohol is methanol, ethanol, propanol or isopropyl alcohol; In described mixed solvent, halogenated hydrocarbons and alcohol ratio are 3:1 ~ 5:1;
5) sephadex column is separated
With hydroxypropyl polydextran gel dress post, blade diameter length ratio 10:1, obtains yellow eluate upper prop by upper step, adopts alcohols eluting, receives first colour band, recycling design, concentrate drying; Described alcohols is methanol, ethanol, propanol or isopropyl alcohol;
6) micronization processes, controls Isolicoflavonol. micropowder diameter of particle median≤100 μm.
2. the industrial production process being used as the Isolicoflavonol. of antitumor drug main active as claimed in claim 1, is characterized in that concrete preparation method is as follows:
Radix Glycyrrhizae sample 5kg, pulverizes, and 10 times amount 95 % ethanol 50 DEG C extract 3 times, each 4 h;
Extracting liquid filtering, concentrating under reduced pressure recycling design, dilute with the appropriate amount of ethanol of 15 times amount 20 %, after defat with petroleum ether, upper polyamide column is separated, applied sample amount is 6BV, loading flow velocity is 3BV/h, colourless to effluent by 20% methanol-eluted fractions, use 40% methanol-eluted fractions afterwards instead, collect the eluent of 3 times of column volume to 6 times column volumes, after concentrated removal solvent, through silica gel column chromatography, eluting is carried out with the chloroform-methanol that volume ratio is 3: 1, eluent is separated through Sephadex L H-20 gel chromatographic columns again, methanol-eluted fractions, collect first color band, collect eluent, concentrated, crystallize, micronization processes, obtain the Isolicoflavonol. of diameter of particle median≤100 μm.
3. the industrial production process being used as the Isolicoflavonol. of antitumor drug main active as claimed in claim 1, is characterized in that concrete preparation method is as follows:
Radix Glycyrrhizae sample 8kg, pulverizes, and 5 times amount 75% ethanol 80 DEG C extract 3 times, each 2 h;
Extracting liquid filtering, concentrating under reduced pressure recycling design, dilute with the appropriate amount of ethanol of 5 times amount 10 %, after defat with petroleum ether, upper polyamide column is separated, applied sample amount is 4BV, loading flow velocity is 4BV/h, colourless to effluent by 10% methanol-eluted fractions, use 50% methanol-eluted fractions afterwards instead, collect the eluent of 3 times of column volume to 6 times column volumes, after concentrated removal solvent, through silica gel column chromatography, eluting is carried out with the dichloromethane-ethanol that volume ratio is 4:1, eluent is separated through Sephadex L H-20 gel chromatographic columns again, methanol-eluted fractions, collect first color band, collect eluent, concentrated, crystallize, micronization processes, obtain the Isolicoflavonol. of diameter of particle median≤100 μm.
4. the industrial production process being used as the Isolicoflavonol. of antitumor drug main active as claimed in claim 1, is characterized in that concrete preparation method is as follows:
Radix Glycyrrhizae sample 10kg, pulverizes, and 8 times amount 60% ethanol 70 DEG C extract 3 times, each 2 h;
Extracting liquid filtering, concentrating under reduced pressure recycling design, dilute with the appropriate amount of ethanol of 10 times amount 5 %, after defat with petroleum ether, upper polyamide column is separated, applied sample amount is 2BV, loading flow velocity is 5BV/h, colourless to effluent by 10% methanol-eluted fractions, use 60% methanol-eluted fractions afterwards instead, collect the eluent of 3 times of column volume to 6 times column volumes, after concentrated removal solvent, through silica gel column chromatography, eluting is carried out with the methylene chloride-methanol that volume ratio is 5:1, eluent is separated through Sephadex L H-20 gel chromatographic columns again, ethanol elution, collect first color band, collect eluent, concentrated, crystallize, micronization processes, obtain diameter of particle median at the Isolicoflavonol. of≤100 μm.
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| CN112047916A (en) * | 2020-09-21 | 2020-12-08 | 苏州昊帆生物股份有限公司 | Method for synthesizing isolicoflavonol |
| CN112851615B (en) * | 2021-01-21 | 2023-04-07 | 厦门大学 | Preparation of isopentenyl flavone and application of isopentenyl flavone as medicine for treating cervical cancer |
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| Title |
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| 倪刚.石菖蒲、华桑化学成分及生物活性研究.《中国博士学位论文全文数据库(医药卫生科技辑)》.2010,第2010年卷(第9期),61,84,95-98. * |
| 朱大元等.甘草化学成分的研究-异甘草黄酮醇及甘草香豆素的结构.《化学学报》.1984,第42卷1080-1084. * |
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