CN101869604B - Preparation method and application of cortex meliae effective ingredients - Google Patents
Preparation method and application of cortex meliae effective ingredients Download PDFInfo
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- CN101869604B CN101869604B CN2010102147869A CN201010214786A CN101869604B CN 101869604 B CN101869604 B CN 101869604B CN 2010102147869 A CN2010102147869 A CN 2010102147869A CN 201010214786 A CN201010214786 A CN 201010214786A CN 101869604 B CN101869604 B CN 101869604B
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Abstract
The invention provides a preparation method of cortex meliae effective ingredients. The effective ingredients are obtained through the following steps: carrying out heating extraction, concentration, silicon gel post separation and elution on medical materials; concentrating and drying an elution solution; then, using the prepared liquid chromatogram for continuous separation; collecting the solution; and concentrating and drying the solution. The cortex meliae effective ingredients of the invention can be used as active components, and pharmaceutically acceptable medicine excipients or carriers are added into the effective ingredients to be prepared into preparations according to a method recorded on the pharmacy. The prepared medicine has the preparation forms such as the liquid preparation, the solid preparation, the capsule preparation or the gelatin pearl preparation, and the medication mode is oral medication or injection medication. The cortex meliae effective ingredients provided by the invention can be applied to the medicine for treating and preventing tumors. The preparation method of the invention has the advantages of simple and convenient operation and stable quality. In addition, no report about the liver-tumor-resistance activity of the cortex meliae appears in the prior art, and the invention provides the study basis for developing new cortex meliae anti-tumor medicine.
Description
Technical field
The invention belongs to Chinese medicine extract, relate in particular to the method for preparing of from Cortex Meliae, extracting active component, and the application of this active component in preparation treatment, prophylaxis of tumours medicine.
Background technology
Tumor is a kind of commonly encountered diseases, frequently-occurring disease, and wherein malignant tumor is one type of the most serious disease of present harm humans health.Be main still mainly to the treatment of malignant tumor in the industry at present, but many chemical anticarcinogenic drugs often involve normal cell when acting on target cell, cause serious side reaction with operation, radiotherapy, chemotherapy.The genetoxic of plant amedica is not obvious, and Chinese herbal medicine is having special advantages and wide application prospect aspect the anticancer mutation, and Chinese medicine also plays the effect that can not be ignored in to the auxiliary treatment of tumor.Paclitaxel promptly is the good anticancer active native compound that has that typically from plant, obtains, and has been developed as antitumor drug at present.The most serious tumor of China's hazardness is pulmonary carcinoma, nasopharyngeal carcinoma, the esophageal carcinoma, gastric cancer, colorectal cancer, hepatocarcinoma, breast carcinoma, cervical cancer, leukemia and lymphoma etc. at present.Particularly the incidence rate of hepatocarcinoma increases in recent years to some extent.Significant, the etiology of these tumors, pathogenesis and control thereof are the emphasis of China's tumor research.Cancer therapy drug and the anticancer ancillary drug of seeking high-efficiency low-toxicity are the important contents of current tumor research.
China's medicinal organism resource is very abundant, and its biological active substances is research and finds new drug guide chemicals, the natural treasure-house of developing new drug.At present; China extracts active substance from natural product, be used to be developed to treatment tumor disease, safety is good, toxicity is low new drug also seldom, from natural product, extracts active substance; Be developed to new drug, have significant application value and wide development prospect with antitumor curative effect.
Summary of the invention
The method for preparing that the purpose of this invention is to provide a kind of cortex meliae effective ingredients, realize through following steps: with adding ethyl acetate and ethanol after the Cortex Meliae pulverizing medicinal materials, heating extraction gets extracting solution; Extracting solution is condensed into extractum, it is separated, at first use low-concentration ethanol as mobile phase with the reverse phase silica gel post; Get eluent I, abandon it, change high concentration ethanol then as mobile phase; Get eluent II, with getting sample behind the eluent II concentrate drying; Continue to separate the sample that obtains with preparative liquid chromatography; The separation condition of preparative hplc: chromatographic column is a preparative column, and mobile phase is water and acetonitrile, and gradient elution is collected solution, and solution obtains active component behind concentrate drying.
Preferred cortex meliae effective ingredients method for preparing comprises the following steps: with the ethyl acetate and the ethanol that add 1: 0.8～1.2 (volume ratios) after the Cortex Meliae pulverizing medicinal materials reflux 0.8～1.2 hour; Extract 1～3 time, merging filtrate gets extracting solution, and extracting solution is condensed into extractum; (ODS) separates it with the reverse phase silica gel post, and the alcoholic solution of at first using 20%-40% (volume ratio) gets eluent I as mobile phase; Abandon it; The alcoholic solution of reuse 90%-100% (volume ratio) gets eluent II as mobile phase, with getting sample behind the eluent II concentrate drying; Continue to separate the goal object that obtains with preparative liquid chromatography; The separation condition of preparative hplc is: chromatographic column is preparative column (Zorbax SB-C18; 21.2mm * 250mm), mobile phase is water and acetonitrile, and gradient elution, flow velocity are 9～11ml/min, and column temperature is a room temperature, collects solution, and solution obtains active component behind concentrate drying.
Best cortex meliae effective ingredients method for preparing comprises the following steps: with the ethyl acetate and the ethanol that add 1: 1 (volume ratio) after the Cortex Meliae pulverizing medicinal materials reflux 1 hour; Extract 2 times, merging filtrate gets extracting solution, and extracting solution is condensed into extractum; (ODS) separates it with the reverse phase silica gel post, and the alcoholic solution of at first using 30% (volume ratio) gets eluent I as mobile phase; Abandon it; The alcoholic solution of reuse 95% (volume ratio) gets eluent II as mobile phase, with getting sample behind the eluent II concentrate drying; Continue to separate the goal object that obtains with preparative liquid chromatography; The separation condition of preparative hplc is: chromatographic column is preparative column (Zorbax SB-C18; 21.2mm * 250mm), mobile phase is water and acetonitrile, and gradient elution, flow velocity are 10ml/min, and column temperature is a room temperature.The gradient elution program is following: in the time of 0 minute, mobile phase is 80% aqueous solution and 20% acetonitrile solution; In the time of 4 minutes, mobile phase is 80% aqueous solution and 20% acetonitrile solution; In the time of 19 minutes, mobile phase is 50% aqueous solution and 50% acetonitrile solution; In the time of 54 minutes, mobile phase is 5% aqueous solution and 95% acetonitrile solution; In the time of 64 minutes, mobile phase is 5% aqueous solution and 95% acetonitrile solution.Collect solution at time period 56.0～60.0min, solution obtains active component behind concentrate drying.
A further object of the present invention provides the application of this cortex meliae effective ingredients in preparation treatment, prevention liver neoplasm medicine.Cortex meliae effective ingredients of the present invention can be used as active component, adds drug excipient or the carrier accepted on the pharmaceutics, processes preparation according to the method for putting down in writing on the pharmaceutics.The dosage form of drug prepared is liquid preparation, solid preparation, capsule or soft gelatin capsule, and administering mode is oral administration or drug administration by injection.
Cortex meliae effective ingredients of the present invention can also add other antitumor drug as one of active component, adds drug excipient or the carrier accepted on the pharmaceutics, processes preparation according to the method for putting down in writing on the pharmaceutics.
Usefulness of the present invention is: it is easy to make the cortex meliae effective ingredients method through this method, steady quality.And the active report of the anti-liver neoplasm of Cortex Meliae is not arranged in the prior art as yet, and the present invention provides certain basis for research and development Cortex Meliae anti-cancer agent.
The specific embodiment
The present invention combines following embodiment further explain flesh and blood of the present invention, and this embodiment only is used to the present invention is described and is not limitation of the present invention.
The preparation of embodiment one cortex meliae effective ingredients
With adding ethyl acetate and ethanol (1: 0.8) (volume ratio) 1500ml after the 200g Cortex Meliae pulverizing medicinal materials, heating extraction gets extracting solution 1 time, and extracting solution is condensed into extractum; With the reverse phase silica gel post it is separated, at first use 20% (volume ratio) alcoholic solution, get eluent I as mobile phase; Abandon it; Change 90% (volume ratio) alcoholic solution then as mobile phase, get eluent II, with getting sample behind the eluent II concentrate drying; Continue to separate the sample that obtains with preparative liquid chromatography, the separation condition of preparative hplc: chromatographic column is a preparative column, and mobile phase is water and acetonitrile, gradient elution.The gradient elution program is following: in the time of 0 minute, mobile phase is 80% aqueous solution and 20% acetonitrile solution; In the time of 4 minutes, mobile phase is 80% aqueous solution and 20% acetonitrile solution; In the time of 19 minutes, mobile phase is 50% aqueous solution and 50% acetonitrile solution; In the time of 54 minutes, mobile phase is 5% aqueous solution and 95% acetonitrile solution; In the time of 64 minutes, mobile phase is 5% aqueous solution and 95% acetonitrile solution.Collect solution at time period 56.0～60.0min, obtain active component behind the concentrate drying.
The preparation of embodiment two cortex meliae effective ingredients
With adding ethyl acetate and ethanol (1: 1.2) (volume ratio) 5000ml after the 500g Cortex Meliae pulverizing medicinal materials, heating extraction gets extracting solution 3 times, and extracting solution is condensed into extractum; With the reverse phase silica gel post it is separated, at first use 40% (volume ratio) alcoholic solution, get eluent I as mobile phase; Abandon it; Change 100% (volume ratio) alcoholic solution then as mobile phase, get eluent II, with getting sample behind the eluent II concentrate drying; Continue to separate the sample that obtains with preparative liquid chromatography, the separation condition of preparative hplc: chromatographic column is a preparative column, and mobile phase is water and acetonitrile, gradient elution.The gradient elution program is following: in the time of 0 minute, mobile phase is 80% aqueous solution and 20% acetonitrile solution; In the time of 4 minutes, mobile phase is 80% aqueous solution and 20% acetonitrile solution; In the time of 19 minutes, mobile phase is 50% aqueous solution and 50% acetonitrile solution; In the time of 54 minutes, mobile phase is 5% aqueous solution and 95% acetonitrile solution; In the time of 64 minutes, mobile phase is 5% aqueous solution and 95% acetonitrile solution.Collect solution at time period 56.0～60.0min, obtain active component behind the concentrate drying.
The preparation of embodiment three cortex meliae effective ingredients
With adding ethyl acetate and ethanol (1: 1) (volume ratio) 2000ml after the 250g Cortex Meliae pulverizing medicinal materials, heating extraction gets extracting solution 2 times, and extracting solution is condensed into extractum; With the reverse phase silica gel post it is separated, at first use 30% (volume ratio) alcoholic solution, get eluent I as mobile phase; Abandon it; Change 95% (volume ratio) alcoholic solution then as mobile phase, get eluent II, with getting sample behind the eluent II concentrate drying; Continue to separate the sample that obtains with preparative liquid chromatography, the separation condition of preparative hplc: chromatographic column is a preparative column, and mobile phase is water and acetonitrile, gradient elution.The gradient elution program is following: in the time of 0 minute, mobile phase is 80% aqueous solution and 20% acetonitrile solution; In the time of 4 minutes, mobile phase is 80% aqueous solution and 20% acetonitrile solution; In the time of 19 minutes, mobile phase is 50% aqueous solution and 50% acetonitrile solution; In the time of 54 minutes, mobile phase is 5% aqueous solution and 95% acetonitrile solution; In the time of 64 minutes, mobile phase is 5% aqueous solution and 95% acetonitrile solution.Collect solution at time period 56.0～60.0min, obtain active component behind the concentrate drying.
Embodiment four cortex meliae effective ingredients preparations
Get cortex meliae effective ingredients 0.5g and 10.5g Polyethylene Glycol-6000 mix homogeneously of embodiment three, heating and melting moves in the drop pill drip irrigation behind the change material, and in ℃ liquid paraffin of medicine liquid droplet to 6～8, oil removing makes 400 of drop pill.
Embodiment five cortex meliae effective ingredients preparations
Get cortex meliae effective ingredients 0.5g, glucose 4.5g, sodium thiosulfate 0.9g and the distilled water 1ml of embodiment three, behind the said components mix homogeneously, lyophilization, 500 of packing promptly get.
Embodiment six cortex meliae effective ingredients preparations
Get Lignum Dalbergiae Odoriferae oil 1.5g, join in the saturated hydroxypropyl of 13ml, stirring and dissolving filters, the filtrating cold drying, the clathrate powder of Lignum Dalbergiae Odoriferae oil and hydroxypropyl.Except that above-mentioned Lignum Dalbergiae Odoriferae oil closes the clathrate powder of hydroxypropyl, get Cortex Meliae extract 0.5g, mannitol 5.5g, calcium disodium edetate 0.9g and the distilled water 2ml of embodiment three again, behind the said components mixing, lyophilization, 300 of packing promptly get.
Embodiment seven cortex meliae effective ingredients preparations
The cortex meliae effective ingredients of getting embodiment three adds an amount of pharmaceutical carrier isotonic agent in right amount with 40 ℃ of water for injection dissolvings, and the pH value of regulator solution is 7.0-8.0; Add to the full amount of water for injection, remove thermal source, behind the mensuration pH value with the ultrafilter ultrafiltration; Use membrane filtration, after the packing, autoclaving; Check, packing promptly get.
Embodiment eight cortex meliae effective ingredients preparations
Cortex meliae effective ingredients and the Oleum Arachidis hypogaeae semen of getting embodiment three stirs in 2: 18 ratio water dissolving type rustless steel and irritates, and adds an amount of gelatin and glycerol simultaneously and mixes, and emits reuse colloid mill defibrination, and medicinal liquid is processed in the mixed liquor that grinds stirring; Under 40-50 ℃ of temperature, stir glycerol and distilled water miscible; Again with the glycerin liquid temperature to 80-90 ℃, get gelatin and add wherein to mix and stir, become glue until off-bottom; Glue was left standstill 4 hours, process offset plate with laminator and supply the pill operation to use; Above-mentioned medicinal liquid of processing and offset plate are sent into the pellet press pill, did cylinder typing in 4 hours at 22-25 ℃ of temperature canyon then, again dry 16-20 hour of 25-30 ℃ of temperature canyon; The qualified soft gelatin capsule of pick; Clean with 95% ethanol,, promptly get dry 4 hours of 25-30 ℃ of canyon.
Embodiment nine cortex meliae effective ingredients preparations
Get the cortex meliae effective ingredients of embodiment three an amount of with the microcrystalline Cellulose mix homogeneously, add 3% polyvidone alcoholic solution system soft material, mistake 18 mesh sieve system granules, 60 ℃ of dryings 1 hour, granulate, the adding Pulvis Talci is an amount of, mixing, tabletting promptly gets.
The pharmacological evaluation of embodiment ten cortex meliae effective ingredients
Negative control group: the equivalent cell of normal cultured condition
Positive controls: DOX (amycin 4 μ M)
Cell strain: HepG-2 (human hepatoma cell strain), attached cell; Culture fluid: 90%DMEM (high the sugar)+nonessential aminoacid of 10%CS+1%+100U/ml penicillin and streptomycin
Sample preparation: get embodiment three cortex meliae effective ingredients and be configured to 50 μ g/ml ,-20 ℃ of preservations with dimethyl sulfoxide (DMSO)
Testing procedure: 96 orifice plates, after drug effect (48h) finishes, to inhale and remove the supernatant culture fluid, MTT storing solution and complete culture solution were with dilution in 1: 10, and every hole adds 100 μ L.Hatch 4h in 37 ℃ of incubators.Remove the supernatant culture fluid, it is dissolving crystallized that every hole adds 150 μ L DMSO, and the 500rpm 10min that vibrates fully dissolves crystallization.550nm measures absorbance, and the result sees table 1.
Table 1
Computational methods:
Suppression ratio (%)=100-(screening group absorbance-blank group absorbance)/(negative control group absorbance-blank group absorbance) * 100
The result shows that the suppression ratio of this cortex meliae effective ingredients is 91.68%.
Claims (6)
1. the method for preparing of a cortex meliae effective ingredients, realize through following steps: with adding volume ratio after the Cortex Meliae pulverizing medicinal materials is 1: 0.8～1.2 ethyl acetate and ethanol, reflux 0.8～1.2 hour; Extract 1～3 time, merging filtrate gets extracting solution, and extracting solution is condensed into extractum; With the reverse phase silica gel post it is separated, the alcoholic solution of at first using volume ratio 20%40% gets eluent I as mobile phase; Abandon it; The reuse volume ratio be the alcoholic solution of 90%-100% as mobile phase, eluent II, with behind the eluent II concentrate drying sample; Continue to separate the goal object that obtains with preparative liquid chromatography; The separation condition of preparative hplc is: chromatographic column is preparative column Zorbax SB-C18; 21.2mm * 250mm, mobile phase is water and acetonitrile, and gradient elution, flow velocity are 9～11ml/min, and column temperature is a room temperature, after the chromatographic isolation, collects solution in 56.0～60.0min time period, and solution obtains active component behind concentrate drying.
2. the application of cortex meliae effective ingredients in preparation treatment, prevention liver neoplasm medicine for preparing according to claim 1 method.
3. the application of a kind of cortex meliae effective ingredients according to claim 2 is characterized in that, drug prepared also contains preparation allowable pharmaceutical excipients or carrier.
4. the application of a kind of cortex meliae effective ingredients according to claim 2 is characterized in that: drug prepared also contains other antitumor drug.
5. the application of a kind of cortex meliae effective ingredients according to claim 2 is characterized in that: the dosage form of said medicine is liquid preparation, solid preparation, capsule or soft gelatin capsule.
6. the application of a kind of cortex meliae effective ingredients according to claim 2 is characterized in that: the administering mode of said medicine is oral administration or drug administration by injection.
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| CN1634423A (en) * | 2004-11-11 | 2005-07-06 | 大连鑫赫化妆品有限公司 | Externally used Chinese traditional medicine preparation for treating acne and preparation method thereof |
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