CN102018759A - Rosmarinic acid, rosmarinic acid-containing common selfheal fruit-spike active ingredient and preparation methods and application thereof to prevention and treatment of cancer postoperative metastasis - Google Patents
Rosmarinic acid, rosmarinic acid-containing common selfheal fruit-spike active ingredient and preparation methods and application thereof to prevention and treatment of cancer postoperative metastasis Download PDFInfo
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Abstract
The invention discloses rosmarinic acid, a rosmarinic acid-containing common selfheal fruit-spike active ingredient and preparation methods thereof by extraction and separation. Moreover, the invention also discloses the application of the rosmarinic acid or/and the rosmarinic acid-containing common selfheal fruit-spike active ingredient to the preparation of a medicament for preventing or treating cancer postoperative metastasis and discloses a medicinal composition which contains effective doses of the rosmarinic acid or/and the rosmarinic acid-containing common selfheal fruit-spike active ingredient for preventing or treating the cancer postoperative metastasis. The rosmarinic acid and the rosmarinic acid-containing common selfheal fruit-spike active ingredient are obtained by extraction and separation. Pharmacodynamic experiment researches indicate that the rosmarinic acid and the rosmarinic acid-containing common selfheal fruit-spike active ingredient prepared by the method have cancer metastasis resistance and can be used for preparing a medicament for preventing or treating cancer metastasis.
Description
Technical field
The invention belongs to tcm field, be specifically related to rosmarinic acid and contain the Spica Prunellae active component of rosmarinic acid and extraction separation method and its application in preparation prevention or treatment cancer postoperative metastasis medicine.
Background technology
Rosmarinic acid (Rosmarinic acid, abbreviation RosA) ((3-(3 for chemistry R (+) 2-by name, 4-dihydroxy phenyl-1-oxo-2 acrylic) oxygen base) 3,4-dihydroxy benzenes propanoic acid }, it is a kind of water solublity polyphenol compound, distribution in plant is comparatively extensive, mainly be present in Labiatae, Boraginaceae, Cucurbitaceae, Tiliaceae, in the umbelliferous various plants, Chinese patent application prospectus CN1995007A (application number 200510131129.7, open day: on July 11st, 2007) disclose the method that from the medical material that contains rosmarinic acid, prepares rosmarinic acid.
Effects such as that rosmarinic acid has is antibiotic, antiinflammatory, antiallergic, hepatoprotective, antiplatelet and thrombosis, antidepressant, anti-ultraviolet.Rosmarinic acid causes vast researcher attention gradually in the effect that anti-tumor aspect showed in recent years.Results of study such as Miyako show that rosmarinic acid has the obvious suppression effect to Hela cell, B16F10, GI
50Be respectively 75 μ g/ml and 16 μ g/ml, and to the very weak (GI of MK-1 cyto-inhibition
50Be 119 μ g/ml).Kang etc., Ahn etc., Yun-Gyoung etc. have reported that respectively rosmarinic acid suppresses the different mechanism of lymphocytic propagation.Scheckel etc. discover that thereby rosmarinic acid can suppress the COX-2 activity and can prevent and treat colon cancer.Chinese patent application prospectus CN101020637A (application number 200710067381.5, open day: on August 22nd, 2007) disclose a kind of from glabrous sarcandra herb prepare the method for rosmarinic acid and by rosmarinic acid, contain of the effect of the various medicines of rosmarinic acid total polyphenols and the preparation of rosmarinic acid derivant in anti-alimentary tract tumor (comprising malignant tumor such as stomach, pancreas, liver, esophagus, rectum, colon) and other tumors, effect experiment mainly is index with the inhibition rate of tumor growth.
Tumor treatment is a complex process, comprises anti-primary tumor growth, anti-recurrence or metastasis.The first-selected excision of present oncotherapy.With the colorectal cancer is example, lays respectively at the 3rd and the 4th of malignant tumor at China's incidence of colorectal and mortality rate.Its M ﹠ M occupies second in American-European developed country.Most humans recurs in back 2 years of operation behind the colorectal neoplasm operation.50% PATIENTS WITH LARGE BOWEL recurrence can occur or shift after radical operation, wherein local recurrence takes place 20%-30% patient, 50%-80% patient's metastasis.Only less than 15% patient single position takes place and radical cure possibility is once more arranged.The research of American Cancer Society also finds, cancer metastasis is the reason of 90% cancer patient death, and therefore stoping cancer cell metastasis is the most effective treatment cancer means.And neoplasm metastasis comprises that primary carcinoma growth, tumor vessel form, oncocyte comes off and invade substrate, enter vascular system, processes such as cancer embolus formations, secondary histoorgan located growth, metastatic carcinoma continuation diffusion, comparing with primary carcinoma has its unique characteristics.Because the transfer of tumor is the rapid process of successive multistep of a complexity, in general, the transfer of malignant tumor diffusion originates in tumor cell and passes the invasion and attack infiltration of basement membrane to its adjacent tissue.In this process, tumor cell has at least the character of three aspects to play pivotal role, be the homogeneity adhesion between tumor cell, the chemotactic motor capacity of tumor cell and the ability of tumor cell degradation of cell epimatrix or basement membrane, in the tumor cell transfer process, play important effect.This shows that the treatment or the medicine of prophylaxis of cancer postoperative metastasis should have the ability that antitumor cell adheres to, moves and soak into, and it is heavy and reduce the metastasis number effectively to suppress tumor.
Spica Prunellae is the dry fruit ear of labiate Spica Prunellae (Prunella.vulgaris L.), has and relieves inflammation or internal heat, and makes eye bright eliminating stagnation, the effect of detumescence.Modern pharmacology discovers that Spica Prunellae has multiple effects such as blood pressure lowering, blood sugar lowering, cell toxicant, antiviral, antibiotic, antioxidation, antiallergic, immunocompetence and anti-inflammatory activity.The Spica Prunellae injection that makes with the single Spica Prunellae is evident in efficacy in order to treatment cells in pleural fluids from lung cancer cases, leukemia, middle and advanced stage stomach, colorectal cancer etc. clinically.Chinese patent application prospectus CN101317883A (application number 200710041690.5, open day: on December 10th, 2008) discloses Prunella spike active site and the application in pharmaceutical compositions thereof.But do not see relevant rosmarinic acid so far or contain the combination treatment of rosmarinic acid or the report of the pharmacological action of prophylaxis of cancer postoperative metastasis.
Summary of the invention
One of the technical problem to be solved in the present invention provides a kind of Spica Prunellae active component that contains rosmarinic acid.
Two of the technical problem to be solved in the present invention provides a kind of rosmarinic acid.
Three of the technical problem to be solved in the present invention provides this preparation method that contains the Spica Prunellae active component of rosmarinic acid.
Four of the technical problem to be solved in the present invention provides the preparation method of this rosmarinic acid.
Five of the technical problem to be solved in the present invention provides above-mentioned rosmarinic acid and/or contains the application of Spica Prunellae active component in the anti-curing cancers postoperative metastasis medicine of preparation of rosmarinic acid.
Six of the technical problem to be solved in the present invention provides a kind of pharmaceutical composition of anti-curing cancers postoperative metastasis.
Chinese patent application prospectus CN101317883A (application number 200710041690.5) discloses Prunella spike active site and the application in pharmaceutical compositions thereof.The present invention is based on the research of Prunella spike active site (the HPLC collection of illustrative plates is seen Fig. 1), to the further separation and purification of Prunella spike active site, obtain containing the Spica Prunellae active component (the HPLC collection of illustrative plates is seen Fig. 2) and the rosmarinic acid (the HPLC collection of illustrative plates is seen Fig. 3) of rosmarinic acid, it has the ability that antitumor cell adheres to, moves and soak into, and can effectively suppress the heavy and minimizing metastasis number of tumor.
In one aspect of the invention, a kind of Spica Prunellae active component that contains rosmarinic acid is provided, this active component is to be raw material with the Spica Prunellae medical material, obtain by extraction separation, it mainly is made up of rosmarinic acid, Herba Rosmarini Officinalis hydrochlorate (rosmarinic acid sodium) and sodium formate, and wherein rosmarinic acid contents accounts for more than 30% (the peak area normalization method).
In another aspect of this invention, provide a kind of rosmarinic acid, this rosmarinic acid is contained the further separation and purification of Spica Prunellae active component of rosmarinic acid and is got by above-mentioned, or to Spica Prunellae separation and purification gained, its content accounts for more than 95%.
In another aspect of this invention, provide the preparation method of the Spica Prunellae active component that contains rosmarinic acid, comprised the steps:
Step (1), get Spica Prunellae medical material water boiling and extraction, the water extract filters, and adds the Different concentrations of alcohol precipitation after concentrating, and gets the clear paste of supernatant concentration to certain relative density, adds acid solution, aqueous slkali successively, and cold preservation filters, the Spica Prunellae extracting solution; Or
Get Spica Prunellae medical material water boiling and extraction, aqueous extract is used 70% ethanol elution through macroporous adsorbent resin, collects eluent, is concentrated into the clear paste of certain relative density, gets the Spica Prunellae extracting solution; Or
Get Spica Prunellae medical material water boiling and extraction, aqueous extract adds absorptive clarificant, place, and high speed centrifugation, supernatant concentration adds acid solution, aqueous slkali successively to the clear paste of certain relative density, cold preservation, sucking filtration gets the Spica Prunellae extracting solution.
Described Spica Prunellae medical material during with water boiling and extraction the weight ratio of Spica Prunellae and water be Spica Prunellae: water=1: (10～16), decocting number of times is 2～4 times;
It is 1.05～1.09 (60～65 ℃) that described aqueous extract is concentrated into relative density, put to room temperature, add 75%～95% ethanol, make that to contain the alcohol amount be 70%, cold preservation is more than 48 hours, reclaiming ethanol to solution relative density is 1.25～1.28 (80 ℃), put to room temperature, transfer with ethanol again to contain the alcohol amount, be preferably 85% to 70%～90%, cold preservation is more than 48 hours, the clear paste of supernatant concentration to 1.25～1.28 (80 ℃);
Described macroporous adsorbent resin is a non-polar resin, preferred HPD100 or D101;
Described absorptive clarificant is selected from any among chitin, chitosan, 101 fruit juice clarifiers, the ZTC1+1, ZTC-II, ZTC-III in preferred chitosan and the ZTC1+1 series;
The preferred pH value of described acid solution is the concentrated hydrochloric acid below 2,40% sodium hydroxide solution of the preferred pH value 7～8 of aqueous slkali; Cold preservation time is all more than 24 hours.
With petroleum ether, ethyl acetate, water-saturated n-butanol extraction, extracting solution: solvent is (1～3): 1 successively for step (2), Spica Prunellae extracting solution.Solvent is reclaimed at water saturated n-butanol extraction position, water-saturated n-butanol carry the position be Prunella spike active site.
Step (3), get Prunella spike active site solution, adopt the isolating method of preparation thin layer chromatography, the use developing solvent launches, the fluorescence location, divide the band dress of getting Rf value minimum silicagel column, methanol-eluted fractions reclaims solvent, residue adds the low amounts of water dissolving, is drying to obtain the Spica Prunellae active component that contains rosmarinic acid; Or
Get Prunella spike active site solution through the sephadex chromatography post, water or organic solvent are eluent, collect FeCl
3-K
3Fe (CN)
6The indicator part that is positive, solution concentration is drying to obtain the Spica Prunellae active component that contains rosmarinic acid; Or
Get Prunella spike active site solution through macroporous adsorptive resins, use certain density ethanol elution, collect eluent, evaporate to dryness, residue add the low amounts of water dissolving, are drying to obtain the Spica Prunellae active component that contains rosmarinic acid; Or
With Prunella spike active site solution drying, get the dry powder dissolve with methanol, the dress silicagel column, the eluent eluting is known collection in conjunction with the thin layer inspection and is merged same stream part or collect FeCl
3Or FeCl
3-K
3Fe (CN)
6Indicator is positive and flows part, boils off solvent, and residue adds the low amounts of water dissolving, is drying to obtain the Spica Prunellae active component that contains rosmarinic acid.
Described Prunella spike active site is prepared several arbitrarily that developing solvent that thin layer chromatography separates the Spica Prunellae active component can be in chloroform, acetone, ethyl acetate, methanol, the formic acid, the mixed liquor of preferred chloroform, ethyl acetate, formic acid, ratio is 2～6: 3～7: 1.
Described is SephdexLH-20, SephadexG10, SephadexG15 or Sephacryl S type to the further separatory polydextran gel of Prunella spike active site.
Described is non-polar resin to the further separatory macroporous adsorbent resin of Prunella spike active site, preferred HPD100 or D101; Concentration of alcohol is 30%～70%;
After finishing, step (3) adopt high performance liquid chromatography (HPLC) to detect rosmarinic acid contents in the described Spica Prunellae active component that contains rosmarinic acid, chromatographic condition is: reversed phase chromatographic column, flow velocity 1mL/min, mobile phase is the mixed solution of methanol, acetonitrile, acid, particular methanol: 0.05%～0.5% aqueous formic acid, ratio are (20～70): (30～90).Detector can be UV, DAD or ELSD.
In another aspect of this invention, provide the preparation method of rosmarinic acid, comprised the steps:
Get the Spica Prunellae water boiling and extraction, the water extract filters, and through the preparation liquid phase, the mobile phase eluting is collected the eluent that contains the rosmarinic acid single component, and evaporate to dryness, residue add the low amounts of water dissolving, are drying to obtain; Or
Get the Spica Prunellae extracting solution, through the preparation liquid phase, the mobile phase eluting is collected the eluent that contains the rosmarinic acid single component, and evaporate to dryness, residue add the low amounts of water dissolving, are drying to obtain; Or
Get Prunella spike active site solution, through the preparation liquid phase, the mobile phase eluting is collected the eluent that contains the rosmarinic acid single component, and evaporate to dryness, residue add the low amounts of water dissolving, are drying to obtain; Or
Get the Spica Prunellae active component solution that contains rosmarinic acid, through the preparation liquid phase, the mobile phase eluting is collected the eluent that contains the rosmarinic acid single component, and evaporate to dryness, residue add the low amounts of water dissolving, are drying to obtain; Or
Get the Spica Prunellae active component dry powder that contains rosmarinic acid, dissolve with methanol, last silicagel column, the eluent eluting detects collection in conjunction with thin layer and merges stream part of containing the rosmarinic acid single component or collect FeCl
3Or FeCl
3-K
3Fe (CN)
6Indicator is positive and flows part, and evaporate to dryness, residue add the low amounts of water dissolving, are drying to obtain.
Above-mentioned drying means can be methods such as vacuum drying, spray drying, lyophilization or infrared drying.
The chromatographic condition that rosmarinic acid prepares liquid phase is: anti-phase preparative column, and flow velocity 1～100mL/min, mobile phase is the mixed solution of methanol, acetonitrile, acid, particular methanol: 0.05%～0.5% aqueous formic acid, ratio are (20～70): (30～90).Detector can be UV, DAD or ELSD.
In another aspect of this invention, the application of the Spica Prunellae active component that rosmarinic acid is provided and/or has contained rosmarinic acid in the anti-curing cancers postoperative metastasis medicine of preparation.The effect of anticancer transfer of the present invention comprises lung, mammary gland, skeleton, apparatus urogenitalis, digestive tract and other cancer.
The present invention select metastasis model to isolating rosmarinic acid and the Spica Prunellae active component that contains rosmarinic acid carry out pharmacodynamic experiment in the body, studies show that rosmarinic acid and compositions thereof have extremely or the effect of the anticancer transfer of highly significant (p＜0.001, p＜0.01).The present invention separates the Spica Prunellae active component that contains rosmarinic acid of preparation and the medicine that rosmarinic acid all can be used for preparing prevention or treatment cancer metastasis.
In another aspect of this invention, provide a kind of pharmaceutical composition of anti-curing cancers postoperative metastasis, this pharmaceutical composition contains the rosmarinic acid of effective dose and/or contains the Spica Prunellae active component of rosmarinic acid, and contains conventional pharmaceutical carrier.The Spica Prunellae active component that the present invention can be rosmarinic acid both when being used to prevent or treating cancerometastasis, contain rosmarinic acid uses separately, also can be that other medicine for anti transfer of tumor (comprise antitumor effective monomer and Chinese medicine extraction effective site) clear and definite with at least a curative effect are united use, acceptable carrier be made pharmaceutical preparation in the adding pharmacy.This pharmaceutical preparation can comprise tablet, capsule, drop pill, syrup, oral liquid, injectable powder, injection etc. by oral, Sublingual, percutaneous, through muscle or intravenous route administration.Various pharmaceutical dosage form provided by the present invention all can adopt conventional preparation technique to be prepared from.The consumption of pharmaceutical composition of the present invention and the course of treatment can be done suitably to adjust according to the light and heavy degree of dosage form, patient's age, disease, the freeze-dried powder of making as the Spica Prunellae active component, be generally every day 1 time, each 1～2 (every freeze-dried powder that contains Spica Prunellae active component 7.5mg), with 1 month be a course of treatment, can use continuously 2 courses of treatment or the longer time.
Description of drawings
Fig. 1 is a Prunella spike active site HPLC collection of illustrative plates;
Fig. 2 is the Spica Prunellae active component HPLC collection of illustrative plates that contains rosmarinic acid;
Fig. 3 is a rosmarinic acid HPLC collection of illustrative plates.
Among Fig. 1-Fig. 3, HPLC (ELSD) analysis condition: Diamonsil C
18Post (200mm * 4.6mm, 5 μ m); Mobile phase is methanol: 0.1% aqueous formic acid=45: 55; Column temperature: 25 ℃; Flow velocity: 1ml/min, ELSD drift tube temperature are 40 ℃, and air pressure is 3.5-3.6bar.
Fig. 4 is that the present invention tests in the example 1 sketch map of each dosage group mice body weight during the Prunella spike active site administration;
Fig. 5 is the sketch map that the present invention tests each dosage group mice body weight during the Spica Prunellae active component administration that contains rosmarinic acid in the example 1;
Fig. 6 is that the present invention tests the sketch map of respectively organizing the mice body weight in the example 1 during the rosmarinic acid administration;
Fig. 7 is the sketch map that the present invention tests the Spica Prunellae active component anti-cell adhesive attraction that contains rosmarinic acid in the example 2, wherein, compares * * p＜0.01 with the blank group;
Fig. 8 is that the present invention tests in the example 2 the Spica Prunellae active component to the sketch map that influences of LS174-T cell locomotivity, wherein, with the blank group relatively, * * * p＜0.001;
Fig. 9 is that the present invention tests in the example 2 variable concentrations Spica Prunellae active component to the sketch map that influences of LS174-T cell locomotivity;
Figure 10 is that the present invention tests in the example 2 the Spica Prunellae active component to the sketch map that influences of LS174-T cellular infiltration ability, wherein, with the blank group relatively, * p＜0.05, * * p＜0.01.
The specific embodiment
Below in conjunction with specific embodiment, the present invention is described in further detail, but does not limit the invention.
Embodiment 1 contains the Spica Prunellae active component preparation of rosmarinic acid
Get Spica Prunellae 3kg, add 16 times of decoctings and boil three times, 1.5h for the first time, 1h for the second time, 0.5h merges three times decocting liquid for the third time, filtration is concentrated into 3000ml, by the pillar of D101 macroporous adsorbent resin is housed, with 7 times of column volume 70% ethanol elutions to FeCl
3-K
3Fe (CN)
6Reaction is negative, merge eluent, simmer down to contains the 4g crude drug for every milliliter, the Spica Prunellae extracting solution, extracting solution respectively extracts (extracting solution: extract solvent=2: 1), reject extract 5 times with petroleum ether, ethyl acetate successively, the extraction of aqueous solution reuse water-saturated n-butanol, reclaim n-butyl alcohol, Prunella spike active site solution, with the solution point sample in 20cm * 20cm silica gel G F
254On the plate, with chloroform-ethyl acetate-formic acid (5: 4: 1) is that developing solvent launches, and exhibition is taken out apart from being 13～17cm, dry, the fluorescence location is divided and is got different bands under 254nm, with the band silica gel dress post of Rf value minimum, with methanol-eluted fractions, reclaim solvent, residue adds the low amounts of water dissolving, and vacuum drying promptly gets the Spica Prunellae active component that contains rosmarinic acid.
Analyze (area normalization method) rosmarinic acid contents 32.5% through HPLC (ELSD).Analysis condition: DiamonsilC
18Post (200mm * 4.6mm, 5 μ m); Mobile phase is methanol: 0.1% aqueous formic acid=45: 55; Column temperature: 25 ℃; Flow velocity: 1ml/min, ELSD drift tube temperature are 40 ℃, and air pressure is 3.5-3.6bar, and the Spica Prunellae active component HPLC collection of illustrative plates that contains rosmarinic acid is seen Fig. 2.
Get Spica Prunellae 5kg, add 14 times of decoctings and boil secondary, for the first time 1h, 0.5h for the second time, merge decocting liquid twice, filter, be concentrated into 1.06 (60 ℃), put to room temperature, add ethanol, make that to contain alcohol amount be 70%, cold preservation is more than 48 hours, reclaiming ethanol to solution relative density is 1.28 (80 ℃), put to room temperature, transfer with ethanol to contain the alcohol amount to 85% again, cold preservation is more than 48 hours, supernatant concentration to 1.27 (80 ℃), transfer PH to 2 with concentrated hydrochloric acid, cold preservation filtered more than 24 hours, filtrate is transferred PH to 7 with 40% sodium hydroxide solution, gets the Spica Prunellae extracting solution.Extracting solution respectively extracts 5 times with petroleum ether, ethyl acetate successively, and (extracting solution: extraction solvent=3: 1), n-butyl alcohol is reclaimed in the extraction of aqueous solution reuse water-saturated n-butanol, get Prunella spike active site solution,, cross Sephadex LH-20 post active site solution, with water elution, with FeCl
3-K
3Fe (CN)
6Solution is indicator, collecting with it, reaction is glaucous eluent, merge the back recycle-water to doing, residue adds methanol eddy, filter, filtrate is reclaimed methanol to doing, and adds the low amounts of water dissolving, lyophilization promptly gets the Spica Prunellae active component that contains rosmarinic acid, analyzes (area normalization method) rosmarinic acid contents 85.0% (analysis condition is with embodiment 1) through HPLC (ELSD).
Embodiment 3 contains the Spica Prunellae active component preparation of rosmarinic acid
Get Spica Prunellae 1kg, add 12 times of decoctings and boil four times, each 1h of preceding secondary, each 0.5h of back secondary, collecting decoction filters, and is concentrated into 6000ml, add 1% chitosan-acetic acid solution 300ml, shake up, put to 12h, high speed centrifugation (4000rpm, centrifugal 10min) is got supernatant, simmer down to contains the 4g crude drug for every milliliter, transfers PH to 2 with concentrated hydrochloric acid, and cold preservation is more than 24 hours, filter, filtrate is transferred PH to 7 with 40% sodium hydroxide solution, gets the Spica Prunellae extracting solution.Extracting solution respectively extracts (extracting solution: extract solvent=3: 1) 5 times with petroleum ether, ethyl acetate successively, the reject extract, n-butyl alcohol is reclaimed in the extraction of aqueous solution reuse water-saturated n-butanol, Prunella spike active site solution.With active site solution, last HPD100 macroporous adsorbent resin is with 65% ethanol (hydrochloric acid is transferred solution PH=2～5) eluting, with FeCl
3-K
3Fe (CN)
6Solution is indicator, collecting with it, reaction is glaucous eluent, merge the back recycle-water to doing, residue adds methanol eddy, filter, filtrate is reclaimed methanol to doing, and adds the low amounts of water dissolving, lyophilization is promptly analyzed (area normalization method) rosmarinic acid contents more than 90.0% (analysis condition is with embodiment 1) through HPLC (ELSD).
Get Spica Prunellae 2kg, add 10 times of decoctings and boil three times, each 1h of preceding secondary, each 0.5h for the third time, collecting decoction filters, be concentrated into 2000ml, add 5% 101 fruit juice clarifier 800ml, stir 15min, put coldly, cold preservation 12h gets supernatant, every milliliter of solution that contains the 4g crude drug of simmer down to is transferred PH to 2 with concentrated hydrochloric acid, and cold preservation is more than 24 hours, filter, filtrate is transferred PH to 7 with 40% sodium hydroxide solution, gets the Spica Prunellae extracting solution.Extracting solution respectively extracts (extracting solution: extract solvent=1: 1) 5 times with petroleum ether, ethyl acetate successively, the reject extract, and n-butyl alcohol is reclaimed in the extraction of aqueous solution reuse water-saturated n-butanol, and lyophilization gets Prunella spike active site dry powder.Get chromatographic column, use chloroform: methanol: formic acid: water=9: 1: 0.1: 0.1 mixed solvent is as initial eluent, and wet method is adorned post; Take by weighing an amount of Prunella spike active site lyophilized powder, sample on the dry method, use chloroform then: methanol: formic acid: water=9: 1: 0.1: 0.1 mixed solvent eluent eluting, a column volume (BV) they are part collection of a stream, with FeCl
3-K
3Fe (CN)
6As indicator, merge positive reaction stream part.Reclaim solvent, residue adds the low amounts of water dissolving, and lyophilization promptly.Analyze (area normalization method) rosmarinic acid contents 92.1% (analysis condition is with embodiment 1) through HPLC (ELSD).
The preparation of embodiment 5 rosmarinic acid
Get Spica Prunellae 1kg, add 12 times of decoctings and boil secondary, each 1h at every turn, collecting decoction filters, and is concentrated into 2000ml, gets ZTC-II natural clarifying agent A component water and is mixed with 1% viscose, swelling 24h; The B component is mixed with 1% viscose, swelling 24h with 1% acetic acid.Get above-mentioned Spica Prunellae extracting solution, be heated to 80 ℃, in the ratio elder generation adding B component of 3B: 2A, the limit edged stirs, and adds component A again behind the insulation 30min in the time of 60 ℃, and the limit edged stirs, insulation 30min, cold preservation 24 hours filters, the filtrate simmer down to contains the 4g crude drug for every milliliter, transfer PH to 2 with concentrated hydrochloric acid, cold preservation filtered more than 24 hours, filtrate is transferred PH to 7 with 40% sodium hydroxide solution, gets the Spica Prunellae extracting solution.Extracting solution respectively extracts (extracting solution: extract solvent=3: 1) 6 times with petroleum ether, ethyl acetate successively, the reject extract, n-butyl alcohol is reclaimed in the extraction of aqueous solution reuse water-saturated n-butanol, Prunella spike active site solution.With active site solution through the preparation liquid phase separation, operating condition: chromatographic column: C
18Semi-preparative column (10 μ m, 12nm; 250 * 10mm); Detector: DAD; Mobile phase: methanol/0.1% formic acid (45/55, V/V) solution; Flow velocity: 1.5ml/min; Sample size: 200 μ l; Column temperature: 25 ℃; Collection contains the eluent of rosmarinic acid single component, reclaims solvent, and residue adds the low amounts of water dissolving, and lyophilization promptly.Analyze (area normalization method) rosmarinic acid contents 98.5% through HPLC (ELSD), rosmarinic acid HPLC collection of illustrative plates is seen Fig. 3 (analysis condition is with embodiment 1).
The preparation of embodiment 6 rosmarinic acid
Get Spica Prunellae 3kg, boil secondary with 13 times of decoctings, for the first time 1h, 0.5h for the second time, collecting decoction, filter, be concentrated into 1.06-1.09 (60 ℃), put to room temperature, add 85% ethanol accent and contain the alcohol amount to 70%, ethanol to 1.26 (80 ℃) is reclaimed in cold preservation 48 hours, puts to room temperature, transfer with ethanol again and contain the alcohol amount to 85%, cold preservation was reclaimed ethanol after 48 hours, was concentrated into 1.25 (80 ℃), transferred pH to 2 with concentrated hydrochloric acid, cold preservation is more than 24 hours, filter, filtrate is transferred pH to 8 with 40% sodium hydroxide solution, gets the Spica Prunellae extracting solution.Extracting solution is respectively extracted 5 times with petroleum ether, ethyl acetate successively, and (extracting solution: extraction solvent=1: 1), discard extract, n-butyl alcohol is flung in the extraction of aqueous solution reuse water-saturated n-butanol, gets active site.Get an amount of Prunella spike active site and be splined on silicagel column, use chloroform: methanol: formic acid: water=9: 1: 0.1: 0.1 mixed solvent eluting, with FeCl
3-K
3Fe (CN)
6As indicator, merge positive reaction stream part, obtain the Spica Prunellae active component.With active component solution through SephadexG10 post (200 * 10mm); Detector: UV; Mobile phase: water; Flow velocity: 10ml/min; Collection contains rosmarinic acid single component eluent, and lyophilization promptly.Analyze (area normalization method) rosmarinic acid contents 99.3% through HPLC (ELSD), rosmarinic acid HPLC collection of illustrative plates is seen Fig. 3 (analysis condition is with embodiment 1).
The preparation of embodiment 7 rosmarinic acid
Get Spica Prunellae 4kg, add 10 times of decoctings and boil three times, 1h for the first time, each 0.5h of second and third time, collecting decoction, filter, be concentrated into 1.05 (60 ℃), put to room temperature, add ethanol and make that to contain alcohol amount be 75%, cold preservation is more than 48 hours, and reclaiming ethanol to solution relative density is 1.27 (80 ℃), puts to room temperature, transfer with ethanol again and contain the alcohol amount to 85%, cold preservation is more than 48 hours, and supernatant concentration to 1.27 (80 ℃) is transferred PH to 2 with concentrated hydrochloric acid, cold preservation is more than 24 hours, filter, filtrate is transferred PH to 7 with 40% sodium hydroxide solution, gets the Spica Prunellae extracting solution.Extracting solution is through preparation liquid phase separation, chromatographic column: C
18Preparative column (10 μ m, 300 * 30mm); Detector: DAD; Mobile phase: methanol/0.1% formic acid (45/55, V/V) solution; Flow velocity: 5ml/min; Collection contains rosmarinic acid single component eluent, and lyophilization promptly.Analyze (area normalization method) rosmarinic acid contents 95.4% through HPLC (ELSD), rosmarinic acid HPLC collection of illustrative plates is seen Fig. 3 (analysis condition is with embodiment 1).
The preparation of embodiment 8 rosmarinic acid
Get the Spica Prunellae active component that contains rosmarinic acid that embodiment 1 makes, add dissolve with methanol, be splined on silicagel column, use chloroform: methanol: formic acid: water=85: 14: 0.5: 0.5 mixed solvent eluting, collection contains rosmarinic acid single component eluent, and lyophilization promptly.
The preparation of embodiment 9 rosmarinic acid
Get Spica Prunellae 3kg, add 10 times of decoctings and boil secondary, for the first time 1h, 0.5h for the second time, collecting decoction filters, filtrate respectively extracts (extracting solution: extraction solvent=1: 1), discard extract, the extraction of aqueous solution reuse water-saturated n-butanol 5 times with petroleum ether, ethyl acetate successively, evaporate to dryness, after being dissolved in water, residue is splined on reverse phase silica gel post (20 μ m, 300 * 40mm), first water eluting, the mixed liquor eluting of reuse methanol and sour water is collected and FeCl
3-K
3Fe (CN)
6The eluent that reaction is positive concentrates, and is splined on SephadexG 25 posts again, and water elution is collected and FeCl
3-K
3Fe (CN)
6The eluent that reaction is positive is through Sephadex LH-20 post (250 * 15mm); The UV detector; Chloroform: methanol (3: 7) is mobile phase; Collection contains rosmarinic acid single component eluent, and lyophilization promptly.
The preparation of embodiment 10 Spica Prunellae active component freeze-dried powders
Get the Spica Prunellae active component 7.5g that embodiment 1 makes, ascorbic acid 0.5g adds injection water 900ml and makes its dissolving, transfer PH to 6 with NaOH, filter, add the injection water to 1000ml, sterilization, cold preservation, cross 0.65 μ m filter membrane, filtrate pours in the cillin bottle, and 5ml/ props up, every freeze-dried powder that contains Spica Prunellae active component 7.5mg is made in lyophilizing.
The preparation of embodiment 11 Spica Prunellae active component capsules
Prescription:
Operation: take by weighing Spica Prunellae active component (embodiment 2 makes), microcrystalline Cellulose, lactose by recipe quantity, mix homogeneously, it is an amount of to add 25% ethanol, mixing.Set extruded velocity and round as a ball speed, mixed material is dropped into the application of sample funnel, start extruder and make cylindrical material, cylindrical material is added in the cylinder, start spheronizator, make microspheric granula, micropill is packed in the hard capsule, make the heavy 450mg of every capsules, promptly.
Below by test the example beneficial effect of the present invention is further elaborated:
Test example 1 test of pesticide effectiveness (anticancer transferance)
(1) experiment material
1 medicine
The Prunella spike active site (area normalization method rosmarinic acid contents 21.40%) that makes by the inventive method
Spica Prunellae active component of the present invention (area normalization method rosmarinic acid contents 92.1%)
Rosmarinic acid of the present invention (purity 99.3%)
Positive control drug: injection vincristine sulfate (Wanle Pharmaceutical Co Ltd, Shenzhen, the accurate word 0805V1 of traditional Chinese medicines);
2 animals
Lewis lung cancer kind Mus (Shanghai Institute of Pharmaceutical Industry pharmaceutical research chamber); C57BL/6N mice (Fudan University zoopery center); The quality certification number: Shanghai 2007-0002; Sex: male; Age in week: 6-8 week; Body weight: 20 ± 2g.
(2) experimental technique and result
1 dosage setting
Content with rosmarinic acid is that standard designs active site, active component dosage respectively, and the contained rosmarinic acid quality of its high, medium and low dosage group is equated with rosmarinic acid corresponding dosage group, specifically sees Table 1.
Each sample dosage of table 1 is provided with table
The inoculation and the administration of 2Lewis lung cancer tumor body
Lewis lung cancer kind Mus after execution and the sterilization is put into super-clean bench, choose normal tumor tissues with surgical operating instrument; The tumor tissues of choosing is placed glass homogenizer, add physiological saline solution, after the grinding, be filtered into one cell suspension through the filter screen point, every ml about 2 * 10
6Individual cell only carries out the subcutaneous vaccination of C57BL/6N mice oxter portion with 0.2ml/; Postvaccinal mice is divided into 11 groups at random, 8 every group.Inoculate back two days beginning intraperitoneal injections, once a day, continue 20 days.Body weight of weighing in per two days and record.
3 observation index and result
3.1 metastasis suppression ratio
With neutral formalin liquid the tumor piece is fixed, section, the number of the black point-like metastasis of counting pulmonary the results are shown in Table 2-table 4.
The effect of metastasis in the table 2 Prunella spike active site body
Annotate: compare * * p＜0.01 with model group; * * p＜0.001
The effect of metastasis in the table 3 Spica Prunellae active component body
Annotate: compare * * * p＜0.001 with model group
The effect of metastasis in the table 4 rosmarinic acid body
Annotate: compare * p＜0.05, * * p＜0.01 with model group; * * p＜0.001
Prunella spike active site, active component, rosmarinic acid all have extremely or the effect of the inhibition neoplasm metastasis of highly significant as can be known by experimental result.
3.2 body weight
By Fig. 4-collection of illustrative plates result shown in Figure 6 as can be known, the weight of animals difference was bigger before Prunella spike active site, the Spica Prunellae active component that contains rosmarinic acid, rosmarinic acid were extremely handled, the active site of equal rosmarinic acid contents, active component dosage group body weight weigh than rosmarinic acid dosage group, Isodose rosmarinic acid group body weight is heavy than the positive drug vincristine, illustrates that tentatively the toxicity of Prunella spike active site, Spica Prunellae active component, rosmarinic acid is littler than vincristine.
Test example 2 effect experiments
(1) contains the investigation of the Spica Prunellae active component anti-cell adhesive capacity of rosmarinic acid
1 experiment material and instrument
Human colon cancer cell strain LS174-T (Chinese science academy Shanghai RESEARCH ON CELL-BIOLOGY institute cell bank); Matrigel (Gibco Brl, grand island, NY); BSA (Invitrogen; USA); DMEM (Invitrogen; USA); DMSO (analytical pure, Shanghai chemical reagents corporation); Thermo microplate reader, Thermo incubator (Thermo company); 96 porocyte culture plates (Denmark Nunc company); MTT (packing of Sigma company); SHJ-JP superclean bench (Shanghai cleaning equipment factory); The present invention contains the Spica Prunellae active component of rosmarinic acid as the experiment medicine.
2 experimental techniques and result
In the every hole of 96 porocyte culture plates, add reconstituted basement membrane glue (Matrigel) respectively, drying at room temperature.Every hole adds 2%BSA, places 37 ℃ of cell culture incubators to hatch 1h, washes and discards with PBS.Cell, places and cultivates 45min in the incubator to contain the DMEM serum-free medium suspension cell of 0.1%BSA with experiment medicine 5,10,15,20 μ g/ml effect 24h.Discard culture fluid, wash 3 times, to remove not adherent cell with PBS.After adding MTT4h, the careful suction goes supernatant, every hole to add the dimethyl sulfoxide (DMSO) of 100 μ l, and light shaking makes the dissolving fully of purple precipitation.Be 570nm with wavelength on enzyme-linked immunosorbent assay instrument, reference wavelength is that 450nm measures optical density (OD) value.The computational methods of adhesion rate are: (blank group OD value-medicine group OD value/blank group OD value) * 100%.This experiment repeats 3 times.
During statistical analysis, relatively adopt the t check between group.
The results are shown in Figure 7, as shown in Figure 7, the adhesion that the Spica Prunellae active component can the obvious suppression cell, and along with the increase of experiment medicine dosage, the effect that suppresses cell adhesion is more and more significant.12.4 the Spica Prunellae active component of μ g/ml can reach 50% cell adhesion inhibitory action.Illustrate that the Spica Prunellae active component can significantly suppress the adhesion of cell.
(2) contain the investigation of the Spica Prunellae active component anti-cell locomotivity of rosmarinic acid
1 experiment material
Human colon cancer cell strain LS174-T (Chinese science academy Shanghai RESEARCH ON CELL-BIOLOGY institute cell bank); DMSO (analytical pure, Shanghai chemical reagents corporation); Thermo microplate reader, Thermo incubator (Thermo company); 24 porocyte culture plates (Denmark Nunc company); SHJ-JP superclean bench (Shanghai cleaning equipment factory); The present invention contains the Spica Prunellae active component of rosmarinic acid as the experiment medicine.
2 experimental techniques and result
The LS174-T cell inoculation that will be in exponential phase is put 37 ℃, 5%CO in 24 well culture plates
2And cultivate in the incubator of saturated humidity.After treating that cell grows up to monolithic, with the axis of plastics suction nozzle along the hole, trace rows dry out one, behind the PBS fine laundering, behind eyepiece micrometer measurement cut width, every hole adds two fun gi polysaccharides and handles, parallel 4 holes of every concentration, matched group adds the culture fluid of equivalent volumes, hatch 12h after, measure the cut width with eyepiece micrometer.The experiment triplicate is averaged.
Cut width after cut width-drug treating before the distance=drug treating of cell migration
During statistical analysis, relatively adopt the t check between group.
The results are shown in Figure 8 and Fig. 9.Can find out by The above results, increase along with Spica Prunellae active component concentration, the LS174-T cell is more and more littler to the distance that the cut district moves, and the Spica Prunellae active component of 10 μ g/ml can arrive 50% to the inhibition that the LS174-T cell moves, and cell moves inhibitory action can be dose dependent.
(3) contain the investigation of the Spica Prunellae active component anti-cell wetting capacity of rosmarinic acid
1 experiment material
Human colon cancer cell strain LS174-T (Shanghai RESEARCH ON CELL-BIOLOGY institute cell bank); Substrate glue (Matrigel, Gibco Brl, grand island, NY); The infiltration cup (Becton Dickinson Lab., Lincoln Park, NJ); Jim Sa (Giemsa); Thermo microplate reader, Thermo CO
2Incubator (Thermo company); SHJ-JP superclean bench (Shanghai cleaning equipment factory); The present invention contains the Spica Prunellae active component of rosmarinic acid as the experiment medicine.
2 experimental techniques and result
The substrate glue of one deck synthetic is completed on the upper strata of soaking into the cup counterdie in advance, and spending the night dries up.With behind the medicine pretreatment 12h of variable concentrations, digestion forms single cell suspension with the LS174-T cell, and is inoculated into the upper strata of soaking into cup.Cuvette is inserted in the hole of 24 orifice plates, after the following hole of 24 orifice plates adds culture fluid, put into 37 ℃ of incubators and cultivate 8h.Take out cuvette,, wipe the cell that does not soak into residual in the cup, the cell of the back side for soaking into of cup bottom with cotton rod to the culture fluid that goes in the cuvette.After the cell fixation,, count at microscopically then with Giemsa stain dyeing.
During statistical analysis, relatively adopt the t check between group.
The results are shown in Figure 10, can find out by The above results, the Spica Prunellae active component suppresses the wetting capacity of LS174-T cell in dose-dependent mode, in the visual field of equal size, the cell number that the cell number that the LS174-T cellular infiltration that the Spica Prunellae active component was handled passes the Matrigel film will be compared photograph obviously reduces, and the Spica Prunellae active component reaches 50% to the suppression ratio of tumor cell invasion during 20 μ g/ml.
Claims (18)
1. Spica Prunellae active component that contains rosmarinic acid, it is characterized in that this active component is to be raw material with the Spica Prunellae medical material, obtains by extraction separation, it mainly is made up of rosmarinic acid, Herba Rosmarini Officinalis hydrochlorate and sodium formate, and wherein rosmarinic acid contents accounts for more than 30%.
2. a rosmarinic acid is characterized in that, described rosmarinic acid contains the further separation and purification of Spica Prunellae active component of rosmarinic acid and gets by claim 1 is described, or to Spica Prunellae separation and purification gained, its content accounts for more than 95%.
3. a preparation method that contains the Spica Prunellae active component of rosmarinic acid according to claim 1 is characterized in that, comprises the steps:
(1) get Spica Prunellae medical material water boiling and extraction, the water extract filters, and adds the Different concentrations of alcohol precipitation after concentrating, and gets the clear paste of supernatant concentration to certain relative density, adds acid solution, aqueous slkali successively, and cold preservation filters, the Spica Prunellae extracting solution; Perhaps
Get Spica Prunellae medical material water boiling and extraction, aqueous extract is used ethanol elution through macroporous adsorbent resin, collects eluent, is concentrated into the clear paste of certain relative density, gets the Spica Prunellae extracting solution; Perhaps
Get Spica Prunellae medical material water boiling and extraction, aqueous extract adds absorptive clarificant, place, and high speed centrifugation, supernatant concentration adds acid solution, aqueous slkali successively to the clear paste of certain relative density, cold preservation, sucking filtration gets the Spica Prunellae extracting solution;
(2) get the Spica Prunellae extracting solution successively with petroleum ether, ethyl acetate, water saturated n-butanol extraction, solvent is reclaimed at water saturated n-butanol extraction position, get Prunella spike active site solution;
(3) get Prunella spike active site solution, adopt the isolating method of preparation thin layer chromatography, use developing solvent to launch, the band of getting the Rf value minimum silicagel column of packing into is divided, methanol-eluted fractions in fluorescence location, reclaim solvent, residue adds the low amounts of water dissolving, is drying to obtain the Spica Prunellae active component that contains rosmarinic acid; Or
Get Prunella spike active site solution through the sephadex chromatography post, water or organic solvent are eluent, collect FeCl
3-K
3Fe (CN)
6The indicator part that is positive, solution concentration is drying to obtain the Spica Prunellae active component that contains rosmarinic acid; Or
Get Prunella spike active site solution through macroporous adsorptive resins, use certain density ethanol elution, collect eluent, reclaim ethanol, residue adds the low amounts of water dissolving, is drying to obtain the Spica Prunellae active component that contains rosmarinic acid; Or
With Prunella spike active site solution drying, get the dry powder dissolve with methanol, wet method dress silicagel column, sample on the dry method, the eluent eluting is known collection in conjunction with the thin layer inspection and is merged same stream part or collect FeCl
3Or FeCl
3-K
3Fe (CN)
6Indicator is positive and flows part, reclaims solvent, and residue adds the low amounts of water dissolving, is drying to obtain the Spica Prunellae active component that contains rosmarinic acid.
4. the preparation method that contains the Spica Prunellae active component of rosmarinic acid according to claim 3, it is characterized in that, when the Spica Prunellae medical material described in the step (1) was used water boiling and extraction, the weight ratio of Spica Prunellae and water was a Spica Prunellae: water=1: (10～16), decocting number of times is 2～4 times.
5. the preparation method that contains the Spica Prunellae active component of rosmarinic acid according to claim 3, it is characterized in that, it is 1.05～1.09 that aqueous extract described in the step (1) is concentrated into relative density, put to room temperature, add ethanol, make that to contain alcohol amount be 60%～90%, be preferably 70%, cold preservation is more than 48 hours, and reclaiming ethanol to solution relative density is 1.25～1.28, puts to room temperature, add ethanol, make to contain alcohol amount to 70%～90%, cold preservation is more than 48 hours, the clear paste of supernatant concentration to 1.25～1.28.
6. the preparation method that contains the Spica Prunellae active component of rosmarinic acid according to claim 3 is characterized in that the macroporous adsorbent resin described in the step (1) is a non-polar resin.
7. the preparation method that contains the Spica Prunellae active component of rosmarinic acid according to claim 3 is characterized in that the absorptive clarificant described in the step (1) is selected from any among chitin, chitosan, 101 fruit juice clarifiers, the ZTC1+1.
8. the preparation method that contains the Spica Prunellae active component of rosmarinic acid according to claim 3 is characterized in that, the acid solution described in the step (1) is that pH value is the concentrated hydrochloric acid below 2, and described aqueous slkali is 40% sodium hydroxide solution of pH value 7～8; Described cold preservation time is all more than 24 hours.
9. the preparation method that contains the Spica Prunellae active component of rosmarinic acid according to claim 3, it is characterized in that, Spica Prunellae extracting solution described in the step (2) is successively with petroleum ether, ethyl acetate, water saturated n-butanol extraction, and the ratio of this Spica Prunellae extracting solution and this solvent is (1～3): 1.
10. the preparation method that contains the Spica Prunellae active component of rosmarinic acid according to claim 3, it is characterized in that described in the step (3) Prunella spike active site being prepared developing solvent that thin layer chromatography separates the Spica Prunellae active component is several arbitrarily in chloroform, acetone, ethyl acetate, methanol, the formic acid.
11. the preparation method that contains the Spica Prunellae active component of rosmarinic acid as claimed in claim 10 is characterized in that described developing solvent is the mixed liquor of chloroform, ethyl acetate and formic acid, mixed proportion is (2～6): (3～7): 1.
12. the preparation method that contains the Spica Prunellae active component of rosmarinic acid according to claim 3, it is characterized in that, be SephdexLH-20, SephadexG10, SephadexG15 or Sephacryl S type to the further separatory polydextran gel of Prunella spike active site described in the step (3).
13. the preparation method that contains the Spica Prunellae active component of rosmarinic acid according to claim 3 is characterized in that, is non-polar resin to the further separatory macroporous adsorbent resin of Prunella spike active site described in the step (3); Concentration of alcohol is 30%～70%.
14. the preparation method of a rosmarinic acid according to claim 2 is characterized in that, prepares by following step:
Get the Spica Prunellae water boiling and extraction, the water extract filters, and through the preparation liquid phase, the mobile phase eluting is collected the eluent that contains the rosmarinic acid single component, and evaporate to dryness, residue add the low amounts of water dissolving, are drying to obtain; Or
The Spica Prunellae extracting solution that the weighting profit requires step in 3 (1) to make, through the preparation liquid phase, the mobile phase eluting is collected the eluent that contains the rosmarinic acid single component, and evaporate to dryness, residue add the low amounts of water dissolving, are drying to obtain; Or
The Prunella spike active site solution that the weighting profit requires step in 3 (2) to make, through the preparation liquid phase, the mobile phase eluting is collected the eluent that contains the rosmarinic acid single component, and evaporate to dryness, residue add the low amounts of water dissolving, are drying to obtain; Or
The weighting profit requires the 3 Spica Prunellae active component solution that contain rosmarinic acid that make, and through the preparation liquid phase, the mobile phase eluting is collected the eluent that contains the rosmarinic acid single component, and evaporate to dryness, residue add the low amounts of water dissolving, are drying to obtain; Or
The weighting profit requires the 3 Spica Prunellae active component dry powder that contain rosmarinic acid that make, dissolve with methanol, and last silicagel column, the eluent eluting detects collection in conjunction with thin layer and merges stream part of containing the rosmarinic acid single component or collect FeCl
3Or FeCl
3-K
3Fe (CN)
6Indicator is positive and flows part, and evaporate to dryness, residue add the low amounts of water dissolving, are drying to obtain.
15. described rosmarinic acid of claim 1～2 and/or the application of Spica Prunellae active component in the anti-curing cancers postoperative metastasis medicine of preparation that contains rosmarinic acid, it is characterized in that the effect of anticancer transfer comprises the transfer of anti-lung, mammary gland, skeleton, apparatus urogenitalis, digestive tract and other cancer postoperative.
16. the pharmaceutical composition of an anti-curing cancers postoperative metastasis, it is characterized in that, this pharmaceutical composition contains the described rosmarinic acid of claim 2 and/or the described Spica Prunellae active component that contains rosmarinic acid of claim 1 of effective dose, and contains conventional pharmaceutical carrier.
17. the pharmaceutical composition of anti-curing cancers postoperative metastasis according to claim 16, it is characterized in that, this pharmaceutical composition also contains other clear and definite carcinomatosis resisting medicine things of at least a curative effect, comprises that anticancer transfer effective monomer and Chinese medicine extraction effective site unites use.
18. the pharmaceutical composition of anti-curing cancers postoperative metastasis according to claim 16 is characterized in that, described pharmaceutical composition is by oral, Sublingual, percutaneous, through muscle or intravenous route administration.
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| CN2009100579066A CN102018759B (en) | 2009-09-17 | 2009-09-17 | Rosmarinic acid, rosmarinic acid-containing common selfheal fruit-spike active ingredient and preparation methods and application thereof to prevention and treatment of cancer postoperative metastasis |
| PCT/CN2010/076929 WO2011032491A1 (en) | 2009-09-17 | 2010-09-15 | Selfheal extraction containing rosmarinic acid, its preparation method and its use for preparation of medicine for precaution or treatment of cancer metastasis after surgery |
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| CN1995007B (en) * | 2005-12-26 | 2011-12-07 | 山东绿叶天然药物研究开发有限公司 | Process for preparing rosmarinic acid |
| CN101139291A (en) * | 2006-09-06 | 2008-03-12 | 天津科技大学 | Preparing technique for separating and purifying rosmarinic acid by big-hole adsorption resin |
| CN101317883B (en) * | 2007-06-06 | 2012-01-04 | 上海中医药大学附属曙光医院 | Prunella spike active site and application of the same in preparing medicament composition |
-
2009
- 2009-09-17 CN CN2009100579066A patent/CN102018759B/en active Active
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- 2010-09-15 WO PCT/CN2010/076929 patent/WO2011032491A1/en active Application Filing
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| CN105111196A (en) * | 2015-09-06 | 2015-12-02 | 叶澄 | Highly-esterified limonin compound and medical application thereof |
| CN105106303A (en) * | 2015-09-21 | 2015-12-02 | 上海中医药大学 | Medical application of selfheal aqueous extract |
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| Publication number | Publication date |
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| CN102018759B (en) | 2012-08-22 |
| WO2011032491A1 (en) | 2011-03-24 |
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