CN104020227A - Alkali content determination method for steroid alkaloid produced by tissue culture method - Google Patents
Alkali content determination method for steroid alkaloid produced by tissue culture method Download PDFInfo
- Publication number
- CN104020227A CN104020227A CN201410216205.3A CN201410216205A CN104020227A CN 104020227 A CN104020227 A CN 104020227A CN 201410216205 A CN201410216205 A CN 201410216205A CN 104020227 A CN104020227 A CN 104020227A
- Authority
- CN
- China
- Prior art keywords
- alkali content
- sucrose
- alkaloid
- sample
- steroid alkaloid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Medicines Containing Plant Substances (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Steroid Compounds (AREA)
Abstract
The invention relates to an alkali content determination method for steroid alkaloid produced by tissue culture method. The alkali content determination method comprises the following steps of (1) preparing culture media; (2) carrying out callus culture; (3) carrying out rooting culture; (4) extracting and preparing the alkaloid; (5) weighing a sample prepared by the step (4) precisely, concentrating and volatilizing by reduced pressure distillation to recover ethanol, preparing a solution by using 5 mL of glacial acetic acid and standing in a refrigerator for a certain time for use; (6) analyzing and determining a peak area value by using a high performance liquid chromatography; (7) establishing a standard curve; and (8) determining the steroid alkaloid content: calculating the steroid alkaloid content of the sample according to the peak area value determined by the step (6) and a regression equation of the step (7). The alkali content determination method takes use of an ultrasonic technique, is relatively small in solvent usage amount, reduces cost, takes use of a rotary evaporator to distill the solvent under the reduced pressure, reduces pressure in the system by virtue of a vacuum pump, reduces a boiling point of the liquid, and obtains an alkaloid concentrated solution at a relatively low temperature.
Description
Technical field
The present invention relates to a kind of alkali content assay method of producing steroid alkaloid with tissue culture method.
Background technology
The alkaloidal method of tradition extraction has decocting method, infusion process, circumfluence method and percolation, and operation is simple, can extract more effective constituent, but in leaching process, exist the problems such as energy consumption is large, effective constituent loss is large, impurity is more, efficiency is lower.Development along with physics, for the problem existing in traditional leaching process, some new technologies are applied in alkaloid extracting technique, the invigoration effect of new technology or fluid extract under supercriticality, greatly improved extraction efficiency, reduced process energy consumption, because its significant advantage becomes study hotspot.Sun Zhiliang etc. explore and extract the proper method of solanum lyratum alkaloid: alkaloid and alkaloid salt thereof can be dissolved in methyl alcohol or ethanol, but methyl alcohol toxicity is very large, should adopt 95% alcohol reflux to extract, then decompression and solvent recovery obtains alkaloid crude product, alkaloid crude product is dissolved in to sour water, with lipophilicity organic solvent, removes water-fast oil-soluble impurities, then sour water alkalization makes alkaloid free, with chloroform extraction, reclaim chloroform and obtain Fat-soluble alkaloids.Remaining alkaline water mother liquor need, with the larger organic solvent extracting n-butyl alcohol of polarity, reclaim normal butyl alcohol and obtain water-soluble alkaloid.
Summary of the invention
Above-mentioned deficiency for prior art, the object of this invention is to provide a kind of alkali content assay method of producing steroid alkaloid with tissue culture method, utilize ultrasonic technique, solvent use amount is relatively less, reduce costs, utilize rotary evaporator decompression distillation solvent, by means of vacuum pump, reduce system internal pressure, the boiling point that reduces liquid, obtains alkaloid concentrate under lower temperature.
For achieving the above object, the present invention is achieved by the following technical solutions:
An alkali content assay method of producing steroid alkaloid with tissue culture method, comprises the following steps:
(1) configuration nutrient culture media:
A, callus nutrient culture media P1, P2, P3, P4, P8 or P10,
B, root media P1, P2, P3, P4 or P10,
Described P1 is MS+NAA0.2+6-BA0.2+30g/L sucrose, P2 is MS+NAA0.2+6-BA0.5+30g/L sucrose, P3 is MS+NAA0.2+6-BA0.8+30g/L sucrose, P4 is MS+NAA0.2+6-BA1.0+30g/L sucrose, P8 is MS+NAA1.5+6-BA2.0+30g/L sucrose, and P10 is MS+NAA1.0+6-BA2.0+30g/L sucrose;
Above-mentioned each nutrient culture media adjust pH before sterilizing is 5.8, adds 10g/L agar, and at 1.1kg/cm2, sterilizing 20min under 121 ℃ of saturated vapors;
(2) callus is cultivated: on superclean bench, take out bittersweet group and cultivate sub-seedling, cotyledon is cut into the square small pieces of 0.3*0.3cm or hypocotyl is cut into the long segment of 1cm and be inoculated in evoked callus nutrient culture media, in the incubator of 22 ± 2 degrees Celsius of temperature, secretly cultivate the 30th day statistics callus growth situation after inoculation;
(3) culture of rootage: bud is cut from base portion, be inoculated in root media,, in the time of latter the 30th day the situation of taking root is added up to inoculation;
(4) alkaloidal extraction and preparation: at the tissue to be measured in step (2) and (3) is cleaned to 37 ℃, dry, and be crushed to 40 orders, accurately take 1.0g, be placed in the ultrasonic extraction of 50m195% ethanol water 30min for ultrasonic cleaner, with the ultrasonic extraction of same method for the second time, through filter residue, discard, merge secondary filtrate, utilize rotary evaporator decompression distillation concentrated, ethanol is reclaimed in volatilization, obtain concentrate, it is 3 that concentrate is dissolved in dilute acid soln to pH value, filters, it is 10 that filtrate adds ammoniacal liquor to pH value, filter, obtain the stillness of night, put in refrigerator standby;
(5) precision takes the sample of preparation in step (4), and ethanol is reclaimed in the concentrated volatilization of decompression distillation, with 5mL glacial acetic acid, is mixed with solution, standby in the standing regular hour of refrigerator;
(6) efficient liquid phase chromatographic analysis is measured peak face amount: described liquid phase chromatogram condition is as follows:
Chromatographic column: Cosmosil5C18AR-II (250 * 4.6mm);
Mobile phase: acetonitrile-potassium dihydrogen phosphate (75:25);
Detect wavelength: 205nm; Flow velocity: 1.0mL/min;
Sample size 10 μ L, column temperature: 25 ℃;
(7) foundation of typical curve: the accurate reference substance 0.2 of drawing, 0.4, 0.6, 0.8, 1.0mg is settled to 5mL with glacial acetic acid, standby 20 ℃ of standing regular hours, with the liquid-phase condition in step (6), high efficiency liquid phase chromatographic analysis method is measured the absorbance of each concentration reference substance, measured data acquisition is calculated to the regression equation of typical curve: y=1185050.6095x-37397.6623 with return law of the straight line, wherein y is peak face amount, x is sample alkali content, correlation coefficient r=0.9960 wherein, r is the correlativity between sample size and two groups of data of absorbance, conform to langbobier law,
(8) mensuration of steroid alkaloid content: according to the regression equation y=1185050.6095x-37397.6623 in the peak face amount recording in step (6) and step (7), wherein y is peak face amount, x is sample alkali content, calculates the steroid alkaloid content of sample.
Beneficial effect of the present invention is as follows:
The present invention produces the alkali content assay method of steroid alkaloid with tissue culture method, utilize ultrasonic technique to shorten extraction time, improve extraction ratio, and without heating, improved the alkaloidal extraction ratio of thermal sensitivity and its physiologically active has not been had to impact substantially, solvent use amount is relatively less, reduces costs.Utilize rotary evaporator decompression distillation solvent, by means of vacuum pump, reduce system internal pressure, reduce the boiling point of liquid, under lower temperature, obtain alkaloid concentrate.High efficiency liquid phase chromatographic analysis method has height, highly sensitive, velocity of separation is fast, applied widely, favorable reproducibility, the advantage such as easy to operate.
Embodiment
Below in conjunction with specific embodiment, the present invention is further illustrated, but protection scope of the present invention is not limited to this.
The present invention produces the alkali content assay method of steroid alkaloid with tissue culture method, comprise the following steps:
(1) configuration nutrient culture media:
A, callus nutrient culture media P1, P2, P3, P4, P8 or P10,
B, root media P1, P2, P3, P4 or P10,
Wherein P1 is MS+NAA0.2+6-BA0.2+30g/L sucrose, P2 is MS+NAA0.2+6-BA0.5+30g/L sucrose, P3 is MS+NAA0.2+6-BA0.8+30g/L sucrose, P4 is MS+NAA0.2+6-BA1.0+30g/L sucrose, P8 is MS+NAA1.5+6-BA2.0+30g/L sucrose, and P10 is MS+NAA1.0+6-BA2.0+30g/L sucrose;
Above-mentioned each nutrient culture media adjust pH before sterilizing is 5.8, adds 10g/L agar, and at 1.1kg/cm2, sterilizing 20min under 121 ℃ of saturated vapors;
(2) callus is cultivated: on superclean bench, take out bittersweet group and cultivate sub-seedling, cotyledon is cut into the square small pieces of 0.3*0.3cm or hypocotyl is cut into the long segment of 1cm and be inoculated in evoked callus nutrient culture media, in the incubator of 22 ± 2 degrees Celsius of temperature, secretly cultivate the 30th day statistics callus growth situation after inoculation;
(3) culture of rootage: bud is cut from base portion, be inoculated in root media,, in the time of latter the 30th day the situation of taking root is added up to inoculation;
(4) alkaloidal extraction and preparation: at organ to be measured is cleaned to 37 ℃, dry, and be crushed to 40 orders, accurately take 1.0g, be placed in the ultrasonic extraction of 50m195% ethanol water 30min for ultrasonic cleaner, with the ultrasonic extraction of same method for the second time, through filter residue, discard, merge secondary filtrate, utilize rotary evaporator decompression distillation concentrated, ethanol is reclaimed in volatilization, obtain concentrate, it is 3 that concentrate is dissolved in dilute acid soln to pH value, filters, it is 10 that filtrate adds ammoniacal liquor to pH value, filter, obtain the stillness of night, put in refrigerator standby;
(5) precision takes the sample of preparation in step (4), and ethanol is reclaimed in the concentrated volatilization of decompression distillation, with 5mL glacial acetic acid, is mixed with solution, standby in the standing regular hour of refrigerator;
(6) the peak face amount of efficient liquid phase chromatographic analysis working sample, in Table 1:
Table 1
| ? | 1 | 2 | 3 | 4 | 5 | 6 |
| One secondary peak face amount | 806484.6250 | 348708.125 | 740635.1880 | 225073.3590 | 630558.3130 | 1013165.5630 |
| Two secondary peak face amounts | 770891.1880 | 323335.719 | 721808.2500 | 228938.8440 | 625633.8130 | 1101731.1250 |
| A sample alkali content | 0.7121 | 0.3258 | 0.6565 | 0.2215 | 0.5637 | 0.8865 |
| Secondary sample alkali content | 0.6821 | 0.3044 | 0.6407 | 0.2247 | 0.5595 | 0.9612 |
| Mean value | 0.6971 | 0.3151 | 0.6486 | 0.2231 | 0.5616 | 0.9239 |
Liquid phase chromatogram condition is as follows:
Chromatographic column: Cosmosil5C18AR-II (250 * 4.6mm);
Mobile phase: acetonitrile-potassium dihydrogen phosphate (75:25);
Detect wavelength: 205nm; Flow velocity: 1.0mL/min;
Sample size 10 μ L, column temperature: 25 ℃;
(7) foundation of typical curve: the accurate reference substance 0.2 of drawing, 0.4, 0.6, 0.8, 1.0mg is settled to 5mL with glacial acetic acid, standby 20 ℃ of standing regular hours, with the liquid-phase condition in step (3), high efficiency liquid phase chromatographic analysis method is measured the absorbance of each concentration reference substance, measured data acquisition is calculated to the regression equation of typical curve: y=1185050.6095x-37397.6623 with return law of the straight line, wherein y is peak face amount, x is sample alkali content, correlation coefficient r=0.9960 wherein, r is the correlativity between sample size and two groups of data of absorbance, conform to langbobier law,
(8) mensuration of steroid alkaloid content: according to the regression equation y=1185050.6095x-37397.6623 in the peak face amount recording in step (6) and step (7), wherein y is peak face amount, x is sample alkali content, the steroid alkaloid content calculating, result of calculation sees the following form 2.
Table 2
Sample alkaloid
The present invention produces the alkali content assay method of steroid alkaloid with tissue culture method, utilize ultrasonic technique to shorten extraction time, improve extraction ratio, and without heating, improved the alkaloidal extraction ratio of thermal sensitivity and its physiologically active has not been had to impact substantially, solvent use amount is relatively less, reduces costs.Utilize rotary evaporator decompression distillation solvent, by means of vacuum pump, reduce system internal pressure, reduce the boiling point of liquid, under lower temperature, obtain alkaloid concentrate.High efficiency liquid phase chromatographic analysis method has height, highly sensitive, velocity of separation is fast, applied widely, favorable reproducibility, the advantage such as easy to operate.
Above-described embodiment is only for the inventive concept of the present invention of explaining, but not restriction to rights protection of the present invention, allly utilizes this design to carry out the change of unsubstantiality to the present invention, all should fall into protection scope of the present invention.
Claims (1)
1. with tissue culture method, produce an alkali content assay method for steroid alkaloid, it is characterized in that comprising the following steps:
(1) configuration nutrient culture media:
A, callus nutrient culture media P1, P2, P3, P4, P8 or P10,
B, root media P1, P2, P3, P4 or P10,
Described P1 is MS+NAA0.2+6-BA0.2+30g/L sucrose, P2 is MS+NAA0.2+6-BA0.5+30g/L sucrose, P3 is MS+NAA0.2+6-BA0.8+30g/L sucrose, P4 is MS+NAA0.2+6-BA1.0+30g/L sucrose, P8 is MS+NAA1.5+6-BA2.0+30g/L sucrose, and P10 is MS+NAA1.0+6-BA2.0+30g/L sucrose;
Above-mentioned each nutrient culture media adjust pH before sterilizing is 5.8, adds 10g/L agar, and at 1.1kg/cm2, sterilizing 20min under 121 ℃ of saturated vapors;
(2) callus is cultivated: on superclean bench, take out bittersweet group and cultivate sub-seedling, cotyledon is cut into the square small pieces of 0.3*0.3cm or hypocotyl is cut into the long segment of 1cm and be inoculated in evoked callus nutrient culture media, in the incubator of 22 ± 2 degrees Celsius of temperature, secretly cultivate the 30th day statistics callus growth situation after inoculation;
(3) culture of rootage: bud is cut from base portion, be inoculated in root media,, in the time of latter the 30th day the situation of taking root is added up to inoculation;
(4) alkaloidal extraction and preparation: at the tissue to be measured in step (2) and (3) is cleaned to 37 ℃, dry, and be crushed to 40 orders, accurately take 1.0g, be placed in the ultrasonic extraction of 50m195% ethanol water 30min for ultrasonic cleaner, with the ultrasonic extraction of same method for the second time, through filter residue, discard, merge secondary filtrate, utilize rotary evaporator decompression distillation concentrated, ethanol is reclaimed in volatilization, obtain concentrate, it is 3 that concentrate is dissolved in dilute acid soln to pH value, filters, it is 10 that filtrate adds ammoniacal liquor to pH value, filter, obtain the stillness of night, put in refrigerator standby;
(5) precision takes the sample of preparation in step (4), and ethanol is reclaimed in the concentrated volatilization of decompression distillation, with 5mL glacial acetic acid, is mixed with solution, standby in the standing regular hour of refrigerator;
(6) efficient liquid phase chromatographic analysis is measured peak face amount: described liquid phase chromatogram condition is as follows:
Chromatographic column: Cosmosil5C18AR-II (250 * 4.6mm);
Mobile phase: acetonitrile-potassium dihydrogen phosphate (75:25);
Detect wavelength: 205nm; Flow velocity: 1.0mL/min;
Sample size 10 μ L, column temperature: 25 ℃;
(7) foundation of typical curve: the accurate reference substance 0.2 of drawing, 0.4, 0.6, 0.8, 1.0mg is settled to 5mL with glacial acetic acid, standby 20 ℃ of standing regular hours, with the liquid-phase condition in step (6), high efficiency liquid phase chromatographic analysis method is measured the absorbance of each concentration reference substance, measured data acquisition is calculated to the regression equation of typical curve: y=1185050.6095x-37397.6623 with return law of the straight line, wherein y is peak face amount, x is sample alkali content, correlation coefficient r=0.9960 wherein, r is the correlativity between sample size and two groups of data of absorbance, conform to langbobier law,
(8) mensuration of steroid alkaloid content: according to the regression equation y=1185050.6095x-37397.6623 in the peak face amount recording in step (6) and step (7), wherein y is peak face amount, x is sample alkali content, calculates the steroid alkaloid content of sample.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410216205.3A CN104020227B (en) | 2014-05-21 | 2014-05-21 | A kind of tissue culture method produces the alkali content assay method of steroid alkaloid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410216205.3A CN104020227B (en) | 2014-05-21 | 2014-05-21 | A kind of tissue culture method produces the alkali content assay method of steroid alkaloid |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104020227A true CN104020227A (en) | 2014-09-03 |
| CN104020227B CN104020227B (en) | 2015-08-19 |
Family
ID=51437098
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410216205.3A Expired - Fee Related CN104020227B (en) | 2014-05-21 | 2014-05-21 | A kind of tissue culture method produces the alkali content assay method of steroid alkaloid |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104020227B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1434123A (en) * | 2002-01-25 | 2003-08-06 | 北京达科豪科技有限公司 | Method for producing tripterygium alkaloids by plant suspension cultivation cell |
| CN1634295A (en) * | 2004-09-27 | 2005-07-06 | 方芳 | Preparation method for climbing nightshade extract |
| CN101168732A (en) * | 2007-08-24 | 2008-04-30 | 清华大学 | Method for producing Vinca rosea alkaloid |
| WO2010114567A1 (en) * | 2009-04-03 | 2010-10-07 | Dianaplantsciences, Inc. | Production and extraction of procyanidins from plant cell cultures |
-
2014
- 2014-05-21 CN CN201410216205.3A patent/CN104020227B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1434123A (en) * | 2002-01-25 | 2003-08-06 | 北京达科豪科技有限公司 | Method for producing tripterygium alkaloids by plant suspension cultivation cell |
| CN1634295A (en) * | 2004-09-27 | 2005-07-06 | 方芳 | Preparation method for climbing nightshade extract |
| CN101168732A (en) * | 2007-08-24 | 2008-04-30 | 清华大学 | Method for producing Vinca rosea alkaloid |
| WO2010114567A1 (en) * | 2009-04-03 | 2010-10-07 | Dianaplantsciences, Inc. | Production and extraction of procyanidins from plant cell cultures |
Non-Patent Citations (6)
| Title |
|---|
| A. EMKE AND U. EILERT: "Steroidal alkaloids in tissue cultures and regenerated plants of Solanum dulcamara", 《PLANT CELL REPORTS》, vol. 5, 31 December 1986 (1986-12-31), pages 31 - 34 * |
| CHUNG-IO KUO ET AL.: "Effects of Auxins on the Production of Steroidal Alkaloids in Rapidly Proliferating Tissue and Cell Cultures of Solanum lyratum", 《PHYTOCHEMICAL ANALYSIS》, vol. 23, 19 October 2011 (2011-10-19), pages 400 - 404 * |
| 孙立新 等: "HPLC法测定中药白英中薯蓣皂苷元的含量", 《沈阳药科大学学报》, vol. 24, no. 1, 31 January 2007 (2007-01-31), pages 29 - 31 * |
| 石伟等: "白英中总皂苷测定及提取工艺的选择", 《哈尔滨商业大学学报（自然科学版）》, vol. 26, no. 6, 31 December 2010 (2010-12-31) * |
| 聂振朋 等: "颠茄的组织培养与快速繁殖", 《江苏农业科学》, no. 1, 31 January 2006 (2006-01-31), pages 106 - 107 * |
| 蔡淑娴: "白英（Solanum Lyratum）糖苷甾体生物碱提取、纯化、检测技术与灭螺效果研究", 《中国优秀硕士学位论文全文数据库》, no. 06, 15 October 2005 (2005-10-15), pages 13 - 15 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104020227B (en) | 2015-08-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101735231B (en) | Method for extracting purified dendrobine from dendrobium stem | |
| CN103755623B (en) | The acid ethanol solution that a kind of response phase method is optimized extracts the method for 1-Deoxynojirimycin in Mulberry Leaves | |
| CN102965290B (en) | Pichia kudriavzevii ZJPH0802 and application thereof in preparation of curcumin derivatives | |
| CN103342668A (en) | Simple method for extracting natural taurine from abalone viscera | |
| CN103694209A (en) | Marine fungus-derived acetophenone compounds and preparation method and application thereof | |
| CN105334301A (en) | Pyrrolo quinoline quinone (PQQ) disodium salt impurity separation and purification method | |
| CN104311525A (en) | Use of marine fungus-derived Azaphilone compound in preparation of antituberculosis drugs | |
| CN104311526B (en) | The dinaphthalene analog derivative in thalassiomycetes source and preparation method thereof and the application in tuberculosis | |
| CN104020227B (en) | A kind of tissue culture method produces the alkali content assay method of steroid alkaloid | |
| CN101880269B (en) | Diterpene monomers and method for separating and preparing diterpene monomers from Clerodendron cyrtophyllum Turcz | |
| CN103772460A (en) | Extraction process of cordycepin (active substance) from silkworm pupa cordyceps militaris | |
| CN101298449A (en) | Novel preparation of myricetin | |
| CN104650173A (en) | Preparation method of tenuifolin through extraction from polygala tenuifolia | |
| CN109111495B (en) | Method for reducing content of related substances in mono-ammonium glycyrrhizinate | |
| CN102250174A (en) | Method for extracting jasminin from winter jasmine leaves | |
| CN104593443A (en) | Preparation method of agilawood chromone component | |
| CN103739648A (en) | Preparation method for mussaendoside U | |
| CN103323551A (en) | Method for detecting content of medlar acid | |
| CN105734005A (en) | Tissue culture method for promoting liquorice adventive root glycyrrhizic acid accumulation by means of fungal elicitors | |
| CN102199079A (en) | Phenol compound and preparation method and application thereof | |
| CN102670634A (en) | C-glycosylflavones composition, preparation method and application thereof | |
| CN104020228A (en) | Determination method for steroid alkaloid content | |
| CN102127089B (en) | Method for extracting camptothecin | |
| CN105477029A (en) | Preparation method of evening primrose seed flavone | |
| CN104357525A (en) | Method for producing glycyrrhetinic acid by using microbial fermentation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20150819 Termination date: 20180521 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |