CN104020228A - Determination method for steroid alkaloid content - Google Patents
Determination method for steroid alkaloid content Download PDFInfo
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- CN104020228A CN104020228A CN201410216741.3A CN201410216741A CN104020228A CN 104020228 A CN104020228 A CN 104020228A CN 201410216741 A CN201410216741 A CN 201410216741A CN 104020228 A CN104020228 A CN 104020228A
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Abstract
The invention relates to a determination method for steroid alkaloid content. The determination method comprises the following steps of (1) extracting and preparing alkaloid; (2) weighing a sample prepared by the step (1), 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; (3) analyzing and measuring a peak area value by using a high performance liquid chromatography; (4) establishing a standard curve: determining absorbance of reference samples in various concentrations by using the high performance liquid chromatography and calculating a regression equation of the standard curve by using a linear regression method according to the measured data; (5) determining the steroid alkaloid content: calculating the steroid alkaloid content of the sample according to the peak area value measured by the step (3) and the regression equation of the step (4), wherein the regression equation is as follows: y=1185050.6095x-37397.6623. The 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 assay method of steroid alkaloid content.
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 assay method that the object of this invention is to provide a kind of steroid alkaloid content, 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 assay method for steroid alkaloid content, comprises the following steps:
(1) 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;
(2) precision takes the sample of preparation in step (1), 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;
(3) 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 ℃;
(4) 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 absorbance, x is sample size, correlation coefficient r=0.9960 wherein, r is the correlativity between sample size and two groups of data of absorbance, conform to langbobier law,
(5) mensuration of steroid alkaloid content: according to the regression equation y=1185050.6095x-37397.6623 in the peak face amount recording in step (3) and step (4), 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 assay method of steroid alkaloid content of the present invention, 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 is not 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.
Embodiment 1--6
The assay method of steroid alkaloid content of the present invention, comprises the following steps:
(1) 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; This step (1) is prepared respectively 6 groups, is numbered 1-6;
(2) precision takes 6 groups of samples of preparation in step (1), 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;
(3) the peak face amount that efficient liquid phase chromatographic analysis is measured 6 groups of samples is 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 ℃;
(4) 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,
(5) mensuration of steroid alkaloid content: according to the regression equation y=1185050.6095x-37397.6623 in the peak face amount recording in step (3) and step (4), wherein y is absorbance, x is sample size, the steroid alkaloid content that calculates 6 groups of samples, result of calculation sees the following form 2.
Table 2
Sample alkaloid
The assay method of steroid alkaloid content of the present invention, 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 is not 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. an assay method for steroid alkaloid content, is characterized in that comprising the following steps:
(1) 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;
(2) precision takes the sample of preparation in step (1), 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;
(3) 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 ℃;
(4) 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 absorbance, x is sample size, correlation coefficient r=0.9960 wherein, r is the correlativity between sample size and two groups of data of absorbance, conform to langbobier law,
(5) mensuration of steroid alkaloid content: according to the regression equation y=1185050.6095x-37397.6623 in the peak face amount recording in step (3) and step (4), wherein y is peak face amount, x is sample alkali content, calculates the steroid alkaloid content of sample.
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Application publication date: 20140903 |