CN105125700A - Method for preparing radix zanthoxyli common anti-tumor component through multi-solvent method extraction and macroporous resin method enrichment - Google Patents
Method for preparing radix zanthoxyli common anti-tumor component through multi-solvent method extraction and macroporous resin method enrichment Download PDFInfo
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Abstract
The invention discloses a method for preparing radix zanthoxyli common anti-tumor component through multi-solvent method extraction and macroporous resin method enrichment. The method comprises the following steps: 1, grinding medicinal materials; 2, carrying out enzymolysis pretreatment; 3, carrying out extraction and enrichment by adopting neutral high-concentration ethanol; 4, carrying out extraction and enrichment by adopting acid low-concentration ethanol; 5, carrying out extraction and enrichment by adopting acid high-concentration ethanol; and 6, mixing, and thus obtaining the radix zanthoxyli common anti-tumor component mixture. Through measurement by adopting the MTT method, the result proves that the mixture has the median inhibitory concentration of 0.04-0.06 mg/mL for HepG-2 hepatoma carcinoma cells, and has the median inhibitory concentration of 0.06-0.08 mg/mL for hela cervical cancer cells. According to the method, the solvents with different acid-base properties and polarities are adopted for carrying out extraction sequentially, and the extracts are enriched by adopting a macroporous resin column, so that the damage to part of the anti-tumor components by acid is avoided, and the anti-tumor components with different polarities are extracted to the greatest degree; the process is simple and convenient, the overall yield of the common anti-tumor component achieves the maximum, the total curative effect achieves the maximum, the potential of being developed and prepared into an excellent anti-tumor medicine is great, and the market prospect is good.
Description
Technical field
The invention belongs to medicine and field of health care products, specifically a kind of method of multi-solvent method extraction and the common antineoplastic component of Amberlyst process enrichment Radix Zanthoxyli.
Background technology
The dry root of Radix Zanthoxyli system Rutaceae xanthoxylum Radix Zanthoxyli Zanthoxylumnitidum (Roxb.) DC., have another name called climing green pepper, Radix Zanthoxyli, two dorsal stylets etc., have dispel the wind, dredging collateral, detumescence, pain relieving effect, cure mainly rheumatic ostalgia, traumatic injury, stomachache, toothache, sore throat, scrofula, venom and the various disease conditions such as soup, burning hot wound, be recorded in Shennong's Herbal with the name of climing green pepper the earliest, " Chinese Pharmacopoeia " 2010 editions records.Radix Zanthoxyli contains number of chemical composition, comprise nitidine chloride, nitidine, chelerythrine, ethoxychelerythrine, 6-methoxyl group-5,6-dihydrochelerythrine, Sanguinarine, α-allocryptopine .beta.-fagarine, magnoflorine, dictamine, precipice green pepper determines alkali, green pepper alkali, liriodendrin, Herba Macleayae Cordatae alcohol alkali and De Kalin alkali etc. are stung in oxidation; Flavones ingredient has Hesperidin, diosmin, vitexin etc.; Lignans has crystallization-8, L-sesamin, L-asarinin, L-syringaresinol etc.; Also have coumarin, terpenoid, quinones, steroidal and organic acid etc.Large quantity research shows, nitidine chloride, nitidine, chelerythrine, 6-methoxyl group-5,6-dihydrochelerythrine, ethoxychelerythrine, Sanguinarine, allocryptopine, precipice green pepper determine alkali, different precipice green pepper determines alkali and sesamin etc. anti-tumor activity.Nitidine, nitidine chloride and allocryptopine carry out inhibition tumor cell propagation by suppressing topoisomerase I or II, nitidine chloride and sesamin carry out inhibition tumor cell propagation by controlling cell cycle, nitidine chloride, chelerythrine and different precipice green pepper determine alkali can inducing apoptosis of tumour cell, nitidine chloride reversible tumor cell multidrug resistance.Therefore, in Radix Zanthoxyli, Multiple components plays antitumor action in the different phase of tumor cell proliferation and apoptosis, and they can play combined effect at anti-tumor aspect.In Chinese medicine, Multiple components plays the key values of overall biological effect Chinese medicine disease just jointly.Therefore, the Multiple components with common antitumor action fully extracted simultaneously, can be antineoplastic agent exploitation, prepare and prepare.
In existing Radix Zanthoxyli Study on extraction, the goal in research that there is no document is that common antineoplastic component is fully extracted simultaneously.Approximate technique has, and Lei Xinchao is with 80% alcohol reflux Radix Zanthoxyli 3 times, and at every turn with 14 times amount solvent refluxing 1h, nitidine chloride, ethoxychelerythrine, L-Semen Sesami have certain productive rate; Lu Shihui cellulase, pectase (zymolyte mass ratio is 1:250) be (30 DEG C) pretreatment Radix Zanthoxyli medicinal powder 30min in pH5 buffer under room temperature, use 12,4 and 3 times amount 60% ethanol (containing 0.5% hydrochloric acid) Soakage extraction 3 times more respectively, each 2h, total alkaloids extraction ratio 85.22%; Some other Radix Zanthoxyli extraction processes are still had to deliver.All these techniques all have the following disadvantages:
First, common antineoplastic Multiple components structure is different, its the suitableeest Extraction solvent is different, nitidine chloride, nitidine, allocryptopine, 6-methoxyl group-5,6-dihydrochelerythrine, ethoxychelerythrine and sesamin etc. should extract with high concentration ethanol, and chelerythrine and Sanguinarine etc. should extract with low-concentration ethanol, repeatedly extract they can not be extracted all fully by same solvent.
The second, Extraction solvent is acid adding not, then antineoplastic component productive rate is low; Extraction solvent acid adding, the extraction of the antineoplastic components such as nitidine chloride, chelerythrine, Sanguinarine and sesamin can be promoted, but the structure of the antineoplastic components such as 6-methoxyl group-5,6-dihydrochelerythrine, ethoxychelerythrine and allocryptopine is destroyed.
Summary of the invention
The object of the invention is, in order to overcome the deficiencies in the prior art, providing the method for the extraction of a kind of multi-solvent method and the common antineoplastic component of Amberlyst process enrichment Radix Zanthoxyli.The method is fully extracted and the common antineoplastic component of enrichment Radix Zanthoxyli by a kind of simple and easy, economic method, simultaneously protect antineoplastic component do not go to pot, for antineoplastic agent exploitation, preparation prepare.
The technical scheme that the present invention solves the problems of the technologies described above is as follows:
A method for the extraction of multi-solvent method and the common antineoplastic component of Amberlyst process enrichment Radix Zanthoxyli, comprises the following steps:
1. pulverizing medicinal materials: Radix Zanthoxyli medical material cleans, cut into slices, dry, be crushed to particle diameter≤0.5 ~ 2mm;
2. enzymolysis pretreatment: add its quality 3 ~ 6 times amount pH4.5 ~ 6.0 Acetic acid-sodium acetate buffer in medical material, with cellulase, pectase compound enzyme pretreatment 0.5 ~ 1.5h at temperature >=30 DEG C; The mass ratio of described compound enzyme cellulase, pectase and medical material is 1:200 ~ 1:300;
3. neutral high-strength ethanol extraction and enrichment: carry out first time through the pretreated medical material of enzymolysis with the neutral high-strength ethanol ultrasonic method of its quality 10 times ~ 12 times, circumfluence method or infusion process and extract, filter, obtain filtrate 1 and filtering residue 1, after filtrate 1 decompression recycling ethanol, upper macroporous resin column, then use ethanol elution again with pure water cleaning, obtain eluent 1; The volumetric concentration of described neutral high-strength ethanol is 60% ~ 80%;
4. acid low-concentration ethanol extracts and enrichment: filtering residue 1 carries out second time with the acid low-concentration ethanol ultrasonic method of its quality 4 ~ 8 times, circumfluence method or infusion process and extracts, filters, obtain filtrate 2 and filtering residue 2, after filtrate 2 decompression recycling ethanol, upper macroporous resin column, then use ethanol elution again with pure water cleaning, obtain eluent 2; Described acid low-concentration ethanol is that to add mass concentration be the hydrochloric acid of 0.5% ~ 1% or the low-concentration ethanol of sulphuric acid, and its volumetric concentration is 30% ~ 50%;
5. acidic high-strength ethanol extraction and enrichment: filtering residue 2 carries out third time with the acidic high-strength ethanol ultrasonic method of its quality 4 ~ 6 times, circumfluence method or infusion process again and extracts, filters, obtain filtrate 3, after filtrate 3 decompression recycling ethanol, upper macroporous resin column, then use ethanol elution again with pure water cleaning, obtain eluent 3; Described acidic high-strength ethanol is that to add mass concentration be the hydrochloric acid of 0.5% ~ 1% or the high concentration ethanol of sulphuric acid, and its volumetric concentration is 60% ~ 80%;
6. merge: by eluent 1, after eluent 2 and eluent 3 merge, decompression recycling ethanol obtains concentrated solution, concentrated solution drying, pulverize, obtain the common antineoplastic component mixture of Radix Zanthoxyli, mixture accounts for the quality of medicinal material 5% ~ 10% of step (1), wherein nitidine chloride, chelerythrine, 6-methoxyl group-5, 6-dihydrochelerythrine, ethoxychelerythrine, Sanguinarine, the common antineoplastic component such as allocryptopine and sesamin accounts for mixture quality 30% ~ 40%, recording mixture to the half-inhibition concentration of HepG-2 hepatoma carcinoma cell with mtt assay is 0.04 ~ 0.06mg/mL, be 0.06 ~ 0.08mg/mL to the half-inhibition concentration of Hela cervical cancer cell.
Above-mentioned first time extracts, second time is extracted and third time extracts 4 ~ 12 times that each Extraction solvent amount is quality of medicinal material; The time of described extracting method: ultrasonic method ultrasonic time is 10 ~ 30min, circumfluence method return time is 20 ~ 40min, the impregnation time is 2 ~ 24h; Described ultrasonic method power is 250W, and frequency is 40KHz; Described circumfluence method bath temperature is 90 ~ 95 DEG C; Described infusion process is flooded under normal temperature and pressure.
Above-mentioned macroporous resin model is D-101 or HPD-722, and eluting is 60% ~ 80% ethanol by 3 ~ 6 times of bed volume volumetric concentrations.
Described mtt assay is: take the common antineoplastic component mixture of Radix Zanthoxyli appropriate, degerming with 0.22 μm of filtering with microporous membrane after dissolve with ethanol, gets filtrate in right amount, adds RPMI-1640 culture medium, prepare serial variable concentrations Radix Zanthoxyli medicinal liquid.Collect HepG-2 hepatoma carcinoma cell, the Hela cervical cancer cell of exponential phase, after PBS washing, RPMI-1640 culture medium re-suspended cell, adjustment cell concentration 5 × 10
4/ mL, is inoculated in 96 orifice plates with every hole 100 μ L, dosing after cultivation 24h.The every hole of experimental port adds Radix Zanthoxyli medicinal liquid 100 μ L, and negative control hole adds 100 μ LRPMI-1640 culture medium, and each concentration establishes 5 multiple holes, in 37.5 DEG C, 5%CO
2after cultivating 48h under full wet condition, careful suction abandons culture supernatant in hole, and every hole washes 1 time with PBS, then supernatant suction is abandoned.Every hole adds the complete RPMI-1640 culture fluid of 100 μ L and 10 μ LMTT liquid (5mg/mL), carefully supernatant is sucked after continuing to cultivate 4h, add DMSO150 μ L/ hole, plate shaker vibration 5min, abundant dissolving blue-purple granule, measure OD value by microplate reader with 570nm, 630nm wavelength, calculate cell proliferation inhibition rate.Suppression ratio=(negative control group OD value-experimental group OD value)/negative control group OD value × 100%.NOSA2.30 software BLiss method calculation of half inhibitory concentration.
Advantage of the present invention is:
1, the present invention first uses neutral high-strength ethanol extraction, and the antineoplastic components such as protection 6-methoxyl group-5,6-dihydrochelerythrine, ethoxychelerythrine and allocryptopine exempt from acid and destroy.
2, extract with acid low-concentration ethanol again, improve the productive rate of the antineoplastic component such as chelerythrine and Sanguinarine.
3, finally use acidic high-strength ethanol extraction, improve the productive rate of the antineoplastic components such as nitidine, nitidine chloride and sesamin.
4, multi-solvent extraction method of the present invention and Amberlyst process cross coupled, exempt filtrate neutralization procedure, technique is easier, the gross production rate of common antineoplastic component is maximized, total effects maximizes, and develop, be prepared into having a high potential of excellent antineoplastic agent, market prospect is good.
Detailed description of the invention
Below in conjunction with embodiment, the present invention is described in more detail.But it should be noted that, process conditions described in example are not equal to the scope of patent requirements of the present invention protection.
Embodiment 1
A method for the extraction of multi-solvent method and the common antineoplastic component of Amberlyst process enrichment Radix Zanthoxyli, comprises the following steps:
1, pulverizing medicinal materials: Radix Zanthoxyli medical material cleans, cut into slices, dry, be crushed to particle diameter≤1mm;
2, enzymolysis pretreatment: add its quality 4 times amount pH6.0 Acetic acid-sodium acetate buffer in medical material, with cellulase, pectase compound enzyme pretreatment 0.5h at temperature >=30 DEG C; The mass ratio of described compound enzyme cellulase, pectase and medical material is 1:200;
3, neutral high-strength ethanol extraction and enrichment: be that 80% ethanol infusion process extracts 24h, filtration with its quality 10 times amount neutral body volume concentrations through the pretreated medical material of enzymolysis, obtain filtrate 1 and filtering residue 1, after filtrate 1 decompression recycling ethanol, upper HPD-722 macroporous resin column, then be 80% ethanol elution by 3 times of bed volume volumetric concentrations again with pure water cleaning, obtain eluent 1;
4, acid low-concentration ethanol extracts and enrichment: the volumetric concentration that filtering residue 1 contains 1% sulphuric acid with 6 times amount is that 50% ethanol infusion process extracts 24h, filtration, obtain filtrate 2 and filtering residue 2, after filtrate 2 decompression recycling ethanol, upper HPD-722 macroporous resin column, then be 60% ethanol elution by 4 times of bed volume volumetric concentrations again with pure water cleaning, obtain eluent 2;
5, acidic high-strength ethanol extraction and enrichment: the volumetric concentration that filtering residue 2 contains 1% sulphuric acid with 6 times amount is again that 80% ethanol infusion process extracts 24h, filtration, obtain filtrate 3, after filtrate 3 decompression recycling ethanol, upper HPD-722 macroporous resin column, then be 80% ethanol elution by 3 times of bed volume volumetric concentrations again with pure water cleaning, obtain eluent 3;
6, merge: by eluent 1, after eluent 2 and eluent 3 merge, decompression recycling ethanol obtains concentrated solution, concentrated solution drying, pulverize, obtain the common antineoplastic component mixture of Radix Zanthoxyli, mixture accounts for the quality of medicinal material 5.6% of step 1, wherein nitidine chloride, chelerythrine, 6-methoxyl group-5, 6-dihydrochelerythrine, ethoxychelerythrine, Sanguinarine, the common antineoplastic component such as allocryptopine and sesamin accounts for mixture quality 35%, recording mixture to the half-inhibition concentration of HepG-2 hepatoma carcinoma cell with mtt assay is 0.05mg/mL, be 0.07mg/mL to the half-inhibition concentration of Hela cervical cancer cell.
Described mtt assay is: take the common antineoplastic component mixture of Radix Zanthoxyli appropriate, degerming with 0.22 μm of filtering with microporous membrane after dissolve with ethanol, gets filtrate in right amount, adds RPMI-1640 culture medium, prepare serial variable concentrations Radix Zanthoxyli medicinal liquid.Collect HepG-2 hepatoma carcinoma cell, the Hela cervical cancer cell of exponential phase, after PBS washing, RPMI-1640 culture medium re-suspended cell, adjustment cell concentration 5 × 10
4/ mL, is inoculated in 96 orifice plates with every hole 100 μ L, dosing after cultivation 24h.The every hole of experimental port adds Radix Zanthoxyli medicinal liquid 100 μ L, and negative control hole adds 100 μ LRPMI-1640 culture medium, and each concentration establishes 5 multiple holes, in 37.5 DEG C, 5%CO
2after cultivating 48h under full wet condition, careful suction abandons culture supernatant in hole, and every hole washes 1 time with PBS, then supernatant suction is abandoned.Every hole adds the complete RPMI-1640 culture fluid of 100 μ L and 10 μ LMTT liquid (5mg/mL), carefully supernatant is sucked after continuing to cultivate 4h, add DMSO150 μ L/ hole, plate shaker vibration 5min, abundant dissolving blue-purple granule, measure OD value by microplate reader with 570nm, 630nm wavelength, calculate cell proliferation inhibition rate.Suppression ratio=(negative control group OD value-experimental group OD value)/negative control group OD value × 100%.NOSA2.30 software BLiss method calculation of half inhibitory concentration.
Embodiment 2
A method for the extraction of multi-solvent method and the common antineoplastic component of Amberlyst process enrichment Radix Zanthoxyli, comprises the following steps:
1, pulverizing medicinal materials: Radix Zanthoxyli medical material cleans, cut into slices, dry, be crushed to particle diameter≤2mm;
2, enzymolysis pretreatment: add its quality 6 times amount pH6.0 Acetic acid-sodium acetate buffer in medical material, with cellulase, pectase compound enzyme pretreatment 1.5h at temperature >=30 DEG C; The mass ratio of described compound enzyme cellulase, pectase and medical material is 1:300;
3, neutral high-strength ethanol extraction and enrichment: be that 60% ethanol infusion process extracts 2h, filtration with its quality 8 times amount neutral body volume concentrations through the pretreated medical material of enzymolysis, obtain filtrate 1 and filtering residue 1, after filtrate 1 decompression recycling ethanol, upper HPD-722 macroporous resin column, then be 60% ethanol elution by 6 times of bed volume volumetric concentrations again with pure water cleaning, obtain eluent 1;
4, acid low-concentration ethanol extracts and enrichment: filtering residue 1 extracts 2h, filtration with 5 times amount containing 30% ethanol infusion process of 0.5% sulphuric acid, obtain filtrate 2 and filtering residue 2, after filtrate 2 decompression recycling ethanol, upper HPD-722 macroporous resin column, then be 60% ethanol elution by 4 times of bed volume volumetric concentrations again with pure water cleaning, obtain eluent 2;
5, acidic high-strength ethanol extraction and enrichment: the volumetric concentration that filtering residue 2 contains 0.5% sulphuric acid with 5 times amount is again that 60% ethanol infusion process extracts 2h, filtration, obtain filtrate 3, after filtrate 3 decompression recycling ethanol, upper HPD-722 macroporous resin column, then be 60% ethanol elution by 4 times of bed volume volumetric concentrations again with pure water cleaning, obtain eluent 3;
6, merge: by eluent 1, after eluent 2 and eluent 3 merge, decompression recycling ethanol obtains concentrated solution, concentrated solution drying, pulverize, obtain the common antineoplastic component mixture of Radix Zanthoxyli, mixture accounts for the quality of medicinal material 5.0% of step 1, wherein nitidine chloride, chelerythrine, 6-methoxyl group-5, 6-dihydrochelerythrine, ethoxychelerythrine, Sanguinarine, the common antineoplastic component such as allocryptopine and sesamin accounts for mixture quality 40%, recording mixture to the half-inhibition concentration of HepG-2 hepatoma carcinoma cell with mtt assay is 0.04mg/mL, be 0.06mg/mL to the half-inhibition concentration of Hela cervical cancer cell.
Described mtt assay is with embodiment 1.
Embodiment 3
A method for the extraction of multi-solvent method and the common antineoplastic component of Amberlyst process enrichment Radix Zanthoxyli, comprises the following steps:
1, pulverizing medicinal materials: Radix Zanthoxyli medical material cleans, cut into slices, dry, be crushed to particle diameter≤2mm;
2, enzymolysis pretreatment: add its quality 3 times amount pH5.0 Acetic acid-sodium acetate buffer in medical material, with cellulase, pectase compound enzyme pretreatment 1h at temperature >=30 DEG C; The mass ratio of described compound enzyme cellulase, pectase and medical material is 1:250;
3, neutral high-strength ethanol extraction and enrichment: be that 70% ethanol is in 95 DEG C of water-bath reflux, extract, 20min, filtration with its quality 8 times amount neutral body volume concentrations through the pretreated medical material of enzymolysis, obtain filtrate 1 and filtering residue 1, after filtrate 1 decompression recycling ethanol, upper D-101 macroporous resin column, then be 80% ethanol elution by 4 times of bed volume volumetric concentrations again with pure water cleaning, obtain eluent 1;
4, acid low-concentration ethanol extracts and enrichment: the volumetric concentration that filtering residue 1 contains 0.5% hydrochloric acid with 4 times amount is that 40% ethanol is in 95 DEG C of water-bath reflux, extract, 20min, filtration, obtain filtrate 2 and filtering residue 2, after filtrate 2 decompression recycling ethanol, upper D-101 macroporous resin column, then be 70% ethanol elution by 3 times of bed volume volumetric concentrations again with pure water cleaning, obtain eluent 2;
5, acidic high-strength ethanol extraction and enrichment: the volumetric concentration that filtering residue 2 contains 0.5% hydrochloric acid with 4 times amount is again that 70% ethanol is in 95 DEG C of water-bath reflux, extract, 20min, filtration, obtain filtrate 3, after filtrate 3 decompression recycling ethanol, upper D-101 macroporous resin column, then be 80% ethanol elution by 3 times of bed volume volumetric concentrations again with pure water cleaning, obtain eluent 3;
6, merge: by eluent 1, after eluent 2 and eluent 3 merge, decompression recycling ethanol obtains concentrated solution, concentrated solution drying, pulverize, obtain the common antineoplastic component mixture of Radix Zanthoxyli, mixture accounts for the quality of medicinal material 9.0% of step 1, wherein nitidine chloride, chelerythrine, 6-methoxyl group-5, 6-dihydrochelerythrine, ethoxychelerythrine, Sanguinarine, the common antineoplastic component such as allocryptopine and sesamin accounts for mixture quality 31%, recording mixture to the half-inhibition concentration of HepG-2 hepatoma carcinoma cell with mtt assay is 0.06mg/mL, be 0.08mg/mL to the half-inhibition concentration of Hela cervical cancer cell.
Described mtt assay is with embodiment 1.
Embodiment 4
A method for the extraction of multi-solvent method and the common antineoplastic component of Amberlyst process enrichment Radix Zanthoxyli, comprises the following steps:
1, pulverizing medicinal materials: Radix Zanthoxyli medical material cleans, cut into slices, dry, be crushed to particle diameter≤1mm;
2, enzymolysis pretreatment: add its quality 3 times amount pH4.5 Acetic acid-sodium acetate buffer in medical material, with cellulase, pectase compound enzyme pretreatment 1h at temperature >=30 DEG C; The mass ratio of described compound enzyme cellulase, pectase and medical material is 1:200;
3, neutral high-strength ethanol extraction and enrichment: be that 70% ethanol is in 90 DEG C of water-bath reflux, extract, 40min, filtration with its quality 12 times amount neutral body volume concentrations through the pretreated medical material of enzymolysis, obtain filtrate 1 and filtering residue 1, after filtrate 1 decompression recycling ethanol, upper D-101 macroporous resin column, then be 80% ethanol elution by 4 times of bed volume volumetric concentrations again with pure water cleaning, obtain eluent 1;
4, acid low-concentration ethanol extracts and enrichment: the volumetric concentration that filtering residue 1 contains 1% hydrochloric acid with 6 times amount is that 40% ethanol is in 90 DEG C of water-bath reflux, extract, 40min, filtration, obtain filtrate 2 and filtering residue 2, after filtrate 2 decompression recycling ethanol, upper D-101 macroporous resin column, then use 4 times of bed volume 60% ethanol elutions again with pure water cleaning, obtain eluent 2;
5, acidic high-strength ethanol extraction and enrichment: the volumetric concentration that filtering residue 2 contains 1% hydrochloric acid with 6 times amount is again that 70% ethanol is in 90 DEG C of water-bath reflux, extract, 40min, filtration, obtain filtrate 3, after filtrate 3 decompression recycling ethanol, upper D-101 macroporous resin column, then be 80% ethanol elution by 3 times of bed volume volumetric concentrations again with pure water cleaning, obtain eluent 3;
6, merge: by eluent 1, after eluent 2 and eluent 3 merge, decompression recycling ethanol obtains concentrated solution, concentrated solution drying, pulverize, obtain the common antineoplastic component mixture of Radix Zanthoxyli, mixture accounts for the quality of medicinal material 10% of step 1, wherein nitidine chloride, chelerythrine, 6-methoxyl group-5, 6-dihydrochelerythrine, ethoxychelerythrine, Sanguinarine, the common antineoplastic component such as allocryptopine and sesamin accounts for mixture quality 30%, recording mixture to the half-inhibition concentration of HepG-2 hepatoma carcinoma cell with mtt assay is 0.06mg/mL, be 0.08mg/mL to the half-inhibition concentration of Hela cervical cancer cell.
Described mtt assay is with embodiment 1.
Embodiment 5
A method for the extraction of multi-solvent method and the common antineoplastic component of Amberlyst process enrichment Radix Zanthoxyli, comprises the following steps:
1, pulverizing medicinal materials: Radix Zanthoxyli medical material cleans, cut into slices, dry, be crushed to particle diameter≤0.5mm;
2, enzymolysis pretreatment: add its quality 4 times amount pH4.5 Acetic acid-sodium acetate buffer in medical material, with cellulase, pectase compound enzyme pretreatment 1h at temperature >=30 DEG C; The mass ratio of described compound enzyme cellulase, pectase and medical material is 1:200;
3, neutral high-strength ethanol extraction and enrichment: be 60% ethanol ultrasonic method (power 250W with its quality 12 times amount neutral body volume concentrations through the pretreated medical material of enzymolysis, frequency 40KHz) extract 10min, filtration, obtain filtrate 1 and filtering residue 1, after filtrate 1 decompression recycling ethanol, upper D-101 macroporous resin column, then be 70% ethanol elution by 5 times of bed volume volumetric concentrations again with pure water cleaning, obtain eluent 1;
4, acid low-concentration ethanol extracts and enrichment: the volumetric concentration that filtering residue 1 contains 0.5% hydrochloric acid with 8 times amount is 30% ethanol ultrasonic method (power 250W, frequency 40KHz) extract 10min, filtration, obtain filtrate 2 and filtering residue 2, after filtrate 2 decompression recycling ethanol, upper D-101 macroporous resin column, then be 60% ethanol elution by 4 times of bed volume volumetric concentrations again with pure water cleaning, obtain eluent 2;
5, acidic high-strength ethanol extraction and enrichment: the volumetric concentration that filtering residue 2 contains 0.5% hydrochloric acid with 6 times amount is again 60% ethanol ultrasonic method (power 250W, frequency 40KHz) extract 10min, filtration, obtain filtrate 3, after filtrate 3 decompression recycling ethanol, upper D-101 macroporous resin column, then be 70% ethanol elution by 3 times of bed volume volumetric concentrations again with pure water cleaning, obtain eluent 3;
6, merge: by eluent 1, after eluent 2 and eluent 3 merge, decompression recycling ethanol obtains concentrated solution, concentrated solution drying, pulverize, obtain the common antineoplastic component mixture of Radix Zanthoxyli, mixture accounts for the quality of medicinal material 7.6% of step 1, wherein nitidine chloride, chelerythrine, 6-methoxyl group-5, 6-dihydrochelerythrine, ethoxychelerythrine, Sanguinarine, the common antineoplastic component such as allocryptopine and sesamin accounts for mixture quality 33%, recording mixture to the half-inhibition concentration of HepG-2 hepatoma carcinoma cell with mtt assay is 0.05mg/mL, be 0.07mg/mL to the half-inhibition concentration of Hela cervical cancer cell.
Described mtt assay is with embodiment 1.
Embodiment 6
A method for the extraction of multi-solvent method and the common antineoplastic component of Amberlyst process enrichment Radix Zanthoxyli, comprises the following steps:
1, pulverizing medicinal materials: Radix Zanthoxyli medical material cleans, cut into slices, dry, be crushed to particle diameter≤0.5mm;
2, enzymolysis pretreatment: add its quality 3 times amount pH5.0 Acetic acid-sodium acetate buffer in medical material, with cellulase, pectase compound enzyme pretreatment 0.5h at temperature >=30 DEG C; The mass ratio of described compound enzyme cellulase, pectase and medical material is 1:200;
3, neutral high-strength ethanol extraction and enrichment: be 60% ethanol ultrasonic method (power 250W with its quality 10 times amount neutral body volume concentrations through the pretreated medical material of enzymolysis, frequency 40KHz) extract 30min, filtration, obtain filtrate 1 and filtering residue 1, after filtrate 1 decompression recycling ethanol, upper D-101 macroporous resin column, then be 70% ethanol elution by 5 times of bed volume volumetric concentrations again with pure water cleaning, obtain eluent 1;
4, acid low-concentration ethanol extracts and enrichment: the volumetric concentration that filtering residue 1 contains 0.5% hydrochloric acid with 7 times amount is 30% ethanol ultrasonic method (power 250W, frequency 40KHz) extract 30min, filtration, obtain filtrate 2 and filtering residue 2, after filtrate 2 decompression recycling ethanol, upper D-101 macroporous resin column, then use 4 times of bed volume 60% ethanol elutions again with pure water cleaning, obtain eluent 2;
5, acidic high-strength ethanol extraction and enrichment: the volumetric concentration that filtering residue 2 contains 0.5% hydrochloric acid with 5 times amount is again 60% ethanol ultrasonic method (power 250W, frequency 40KHz) extract 30min, filtration, obtain filtrate 3, after filtrate 3 decompression recycling ethanol, upper D-101 macroporous resin column, then be 70% ethanol elution by 3 times of bed volume volumetric concentrations again with pure water cleaning, obtain eluent 3;
6, merge: by eluent 1, after eluent 2 and eluent 3 merge, decompression recycling ethanol obtains concentrated solution, concentrated solution drying, pulverize, obtain the common antineoplastic component mixture of Radix Zanthoxyli, mixture accounts for the quality of medicinal material 7.5% of step 1, wherein nitidine chloride, chelerythrine, 6-methoxyl group-5, 6-dihydrochelerythrine, ethoxychelerythrine, Sanguinarine, the common antineoplastic component such as allocryptopine and sesamin accounts for mixture quality 31%, recording mixture to the half-inhibition concentration of HepG-2 hepatoma carcinoma cell with mtt assay is 0.06mg/mL, be 0.08mg/mL to the half-inhibition concentration of Hela cervical cancer cell.
Described mtt assay is with embodiment 1.
Claims (3)
1. a method for the extraction of multi-solvent method and the common antineoplastic component of Amberlyst process enrichment Radix Zanthoxyli, comprises the following steps:
(1) pulverizing medicinal materials: Radix Zanthoxyli medical material cleans, cut into slices, dry, be crushed to particle diameter≤0.5 ~ 2mm;
(2) enzymolysis pretreatment: add its quality 3 ~ 6 times amount pH4.5 ~ 6.0 Acetic acid-sodium acetate buffer in medical material, with cellulase, pectase compound enzyme pretreatment 0.5 ~ 1.5h at temperature >=30 DEG C; The mass ratio of described compound enzyme cellulase, pectase and medical material is 1:200 ~ 1:300;
(3) neutral high-strength ethanol extraction and enrichment: carry out first time through the pretreated medical material of enzymolysis with the neutral high-strength ethanol ultrasonic method of its quality 10 times ~ 12 times, circumfluence method or infusion process and extract, filter, obtain filtrate 1 and filtering residue 1, after filtrate 1 decompression recycling ethanol, upper macroporous resin column, then use ethanol elution again with pure water cleaning, obtain eluent 1; The volumetric concentration of described neutral high-strength ethanol is 60% ~ 80%;
(4) acid low-concentration ethanol extracts and enrichment: filtering residue 1 carries out second time with the acid low-concentration ethanol ultrasonic method of its quality 4 ~ 8 times, circumfluence method or infusion process and extracts, filters, obtain filtrate 2 and filtering residue 2, after filtrate 2 decompression recycling ethanol, upper macroporous resin column, then use ethanol elution again with pure water cleaning, obtain eluent 2; Described acid low-concentration ethanol is that to add mass concentration be the hydrochloric acid of 0.5% ~ 1% or the low-concentration ethanol of sulphuric acid, and its volumetric concentration is 30% ~ 50%;
(5) acidic high-strength ethanol extraction and enrichment: filtering residue 2 carries out third time with the acidic high-strength ethanol ultrasonic method of its quality 4 ~ 6 times, circumfluence method or infusion process again and extracts, filters, obtain filtrate 3, after filtrate 3 decompression recycling ethanol, upper macroporous resin column, then use ethanol elution again with pure water cleaning, obtain eluent 3; Described acidic high-strength ethanol is that to add mass concentration be the hydrochloric acid of 0.5% ~ 1% or the high concentration ethanol of sulphuric acid, and its volumetric concentration is 60% ~ 80%;
(6) merge: by eluent 1, after eluent 2 and eluent 3 merge, decompression recycling ethanol obtains concentrated solution, concentrated solution drying, pulverize, obtain the common antineoplastic component mixture of Radix Zanthoxyli, mixture accounts for the quality of medicinal material 5% ~ 10% of step (1), wherein nitidine chloride, chelerythrine, 6-methoxyl group-5, 6-dihydrochelerythrine, ethoxychelerythrine, Sanguinarine, the common antineoplastic component such as allocryptopine and sesamin accounts for mixture quality 30% ~ 40%, recording mixture to the half-inhibition concentration of HepG-2 hepatoma carcinoma cell with mtt assay is 0.04 ~ 0.06mg/mL, be 0.06 ~ 0.08mg/mL to the half-inhibition concentration of Hela cervical cancer cell.
2. the method for a kind of multi-solvent method extraction according to claim 1 and the common antineoplastic component of Amberlyst process enrichment Radix Zanthoxyli, it is characterized in that, described first time extracts, second time extracts and third time extracts 4 ~ 12 times that each Extraction solvent amount is quality of medicinal material; The time of described extracting method: ultrasonic method ultrasonic time is 10 ~ 30min, circumfluence method return time is 20 ~ 40min, the impregnation time is 2 ~ 24h; Described ultrasonic method power is 250W, and frequency is 40KHz; Described circumfluence method bath temperature is 90 ~ 95 DEG C; Described infusion process is flooded under normal temperature and pressure.
3. the method for a kind of multi-solvent method extraction according to claim 1 and the common antineoplastic component of Amberlyst process enrichment Radix Zanthoxyli, it is characterized in that: described macroporous resin model is D-101 or HPD-722, eluting is 60% ~ 80% ethanol by 3 ~ 6 times of bed volume volumetric concentrations.
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