CN104020228A - Determination method for steroid alkaloid content - Google Patents

Abstract

The invention relates to a method for measuring the content of steroidal alkaloids, comprising the following steps: (1) extracting and preparing the alkaloids; (2) weighing the samples prepared in the step (1), distilling under reduced pressure, concentrating and volatilizing to recover ethanol, Prepare solution with 5mL glacial acetic acid, stand in the refrigerator for a certain period of time for subsequent use; (3) HPLC analysis determines the peak surface value (4) establishment of standard curve: HPLC method measures the absorbance of each concentration reference substance, Measured data adopt linear regression method to calculate the regression equation of standard curve; (5) mensuration of steroidal alkaloid content: according to the regression equation y in the peak surface value recorded in step (3) and step (4) =1185050.6095x-37397.6623 to calculate the steroidal alkaloid content of the sample. The invention utilizes the ultrasonic technology, the amount of solvent used is relatively small, the cost is reduced, the solvent is distilled under reduced pressure by a rotary evaporator, the internal pressure of the system is reduced by means of a vacuum pump, the boiling point of the liquid is lowered, and the concentrated alkaloid liquid is obtained at a relatively low temperature.

Description

A kind of assay method of steroidal alkaloid content

technical field

The invention relates to a method for determining the content of steroidal alkaloids.

Background technique

The traditional methods for extracting alkaloids include decoction, dipping, reflux and percolation, which are easy to operate and can extract more active ingredients. Many, low efficiency and other issues. With the development of physical science, in view of the problems existing in the traditional extraction process, some new technologies are applied to the alkaloid extraction process. The strengthening effect of the new technology or the extraction of the fluid in a supercritical state greatly improves the extraction efficiency and reduces the Process energy consumption has become a research hotspot because of its significant advantages. Sun Zhiliang et al. have explored a more suitable method for extracting the alkaloids of the white hair vine: alkaloids and their alkaloid salts can be dissolved in methanol or ethanol, but methanol is very toxic, and 95% ethanol should be used for reflux extraction, and then the solvent is recovered under reduced pressure To obtain crude alkaloids, dissolve the crude alkaloids in acidic water, use a lipophilic organic solvent to remove fat-soluble impurities that are insoluble in water, alkalinize the acidic water to free the alkaloids, extract with chloroform, and recover the chloroform to obtain fat-soluble alkaloids. The remaining alkaline jellyfish solution needs to be extracted with a polar organic solvent n-butanol, and the n-butanol is recovered to obtain a water-soluble alkaloid.

Contents of the invention

For the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide a kind of assay method of steroid alkaloid content, utilize ultrasonic technology, solvent consumption is relatively less, reduce cost, utilize rotary evaporator to distill solvent under reduced pressure, by means of The vacuum pump reduces the pressure in the system, lowers the boiling point of the liquid, and obtains the alkaloid concentrate at a lower temperature.

To achieve the above object, the present invention is achieved through the following technical solutions:

A method for assaying steroidal alkaloid content, comprising the steps of:

(1) Extraction and preparation of alkaloids: Wash and dry the organ to be tested at 37°C, crush it to 40 meshes, weigh 1.0 g accurately, place it in an ultrasonic cleaner, and use 50 ml of 95% ethanol aqueous solution for ultrasonic extraction for 30 min. The same method is used for ultrasonic extraction for the second time, and the filter residue is discarded, and the second filtrate is combined, concentrated by distillation under reduced pressure with a rotary evaporator, and the ethanol is recovered by volatilization to obtain a concentrated solution. The concentrated solution is dissolved in a dilute acid solution until the pH value is 3, and filtered , add ammonia water to the filtrate until the pH value is 10, filter to obtain a clear night, and put it in the refrigerator for subsequent use;

(2) Accurately weigh the sample prepared in step (1), distill under reduced pressure, concentrate and volatilize to reclaim ethanol, prepare a solution with 5mL glacial acetic acid, and leave it in the refrigerator for a certain period of time for subsequent use;

(3) HPLC analysis and determination of peak face value: the described liquid chromatography conditions are as follows:

Chromatographic column: Cosmosil5C18AR-II (250×4.6mm);

Mobile phase: acetonitrile - potassium dihydrogen phosphate (75:25);

Detection wavelength: 205nm; flow rate: 1.0mL/min;

Injection volume: 10 μL, column temperature: 25°C;

(4) Establishment of the standard curve: Precisely absorb 0.2, 0.4, 0.6, 0.8, 1.0 mg of the reference substance and dilute it to 5 mL with glacial acetic acid, and set it aside at 20°C for a certain period of time, using the liquid phase conditions in step (3) , the absorbance of each concentration reference substance is measured by high performance liquid chromatography, and the linear regression method is used to calculate the regression equation of the standard curve to the measured data: y=1185050.6095x-37397.6623, where y is the absorbance, and x is the sample content, Among them, the correlation coefficient r=0.9960, r is the correlation between the two sets of data of sample content and absorbance, which is consistent with Lambert-Beer's law;

(5) mensuration of steroidal alkaloid content: according to the regression equation y=1185050.6095x-37397.6623 in the peak surface value recorded in step (3) and step (4), wherein y is the peak surface value, and x is the sample alkali content, Calculate the steroidal alkaloid content of the sample.

The beneficial effects of the present invention are as follows:

The method for determining the content of steroidal alkaloids of the present invention uses ultrasonic technology to shorten the extraction time and increase the extraction rate without heating, which improves the extraction rate of heat-sensitive alkaloids and basically has no effect on its physiological activity, and the amount of solvent used is relatively small. cut costs. A rotary evaporator is used to distill the solvent under reduced pressure, and a vacuum pump is used to reduce the internal pressure of the system, lower the boiling point of the liquid, and obtain the concentrated alkaloid liquid at a lower temperature. High-performance liquid chromatography has the advantages of high performance, high sensitivity, fast separation speed, wide application range, good reproducibility, and convenient operation.

Detailed ways

The present invention will be further described below in conjunction with specific examples, but the protection scope of the present invention is not limited thereto.

Example 1--6

The assay method of steroidal alkaloid content of the present invention comprises the following steps:

(1) Extraction and preparation of alkaloids: Wash and dry the organ to be tested at 37°C, crush it to 40 meshes, weigh 1.0 g accurately, place it in an ultrasonic cleaner, and use 50 ml of 95% ethanol aqueous solution for ultrasonic extraction for 30 min. The same method is used for ultrasonic extraction for the second time, and the filter residue is discarded, and the second filtrate is combined, concentrated by distillation under reduced pressure with a rotary evaporator, and the ethanol is recovered by volatilization to obtain a concentrated solution. The concentrated solution is dissolved in a dilute acid solution until the pH value is 3, and filtered , add ammonia water to the filtrate to a pH value of 10, filter to obtain a clear night, and put it in the refrigerator for subsequent use; this step (1) prepares 6 groups respectively, numbered 1-6;

(2) Accurately weigh the 6 groups of samples prepared in step (1), distill under reduced pressure, concentrate and volatilize to reclaim ethanol, prepare a solution with 5mL glacial acetic acid, and put it in the refrigerator for a certain period of time for subsequent use;

(3) HPLC analysis determines the peak surface values of 6 groups of samples in Table 1:

Table 1

1 2 3 4 5 6 primary peak face value 806484.6250 348708.125 740635.1880 225073.3590 630558.3130 1013165.5630 Secondary Peak Face Value 770891.1880 323335.719 721808.2500 228938.8440 625633.8130 1101731.1250 Primary 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 average value 0.6971 0.3151 0.6486 0.2231 0.5616 0.9239

The liquid chromatography conditions are as follows:

Chromatographic column: Cosmosil5C18AR-II (250×4.6mm);

Mobile phase: acetonitrile - potassium dihydrogen phosphate (75:25);

Detection wavelength: 205nm; flow rate: 1.0mL/min;

Injection volume: 10 μL, column temperature: 25°C;

(4) Establishment of the standard curve: Precisely absorb 0.2, 0.4, 0.6, 0.8, 1.0 mg of the reference substance and dilute it to 5 mL with glacial acetic acid, and set it aside at 20°C for a certain period of time, using the liquid phase conditions in step (3) , the absorbance of each concentration reference substance is measured by high performance liquid chromatography, and the linear regression method is used to calculate the regression equation of the standard curve for the measured data: y=1185050.6095x-37397.6623, wherein y is the peak surface value, and x is the sample base Content, wherein the correlation coefficient r=0.9960, r is the correlation between the sample content and the absorbance two groups of data, consistent with Lambert-Beer's law;

(5) Determination of steroidal alkaloid content: according to the regression equation y=1185050.6095x-37397.6623 in the peak surface value recorded in step (3) and step (4), wherein y is the absorbance, x is the sample content, calculates The steroidal alkaloid contents of the 6 groups of samples are calculated in Table 2 below.

Table 2

Sample alkaloid content

The method for determining the content of steroidal alkaloids of the present invention uses ultrasonic technology to shorten the extraction time and increase the extraction rate without heating, which improves the extraction rate of heat-sensitive alkaloids and basically has no effect on its physiological activity, and the amount of solvent used is relatively small. cut costs. A rotary evaporator is used to distill the solvent under reduced pressure, and a vacuum pump is used to reduce the internal pressure of the system, lower the boiling point of the liquid, and obtain the concentrated alkaloid liquid at a lower temperature. High-performance liquid chromatography has the advantages of high performance, high sensitivity, fast separation speed, wide application range, good reproducibility, and convenient operation.

The above-mentioned embodiments are only used to explain the inventive concept of the present invention, but not to limit the protection of the rights of the present invention. Any insubstantial changes made to the present invention by using this concept should fall within the scope of protection of the present invention.

Claims (1)

1. a method for assaying steroidal alkaloid content, is characterized in that comprising the following steps: (1) Extraction and preparation of alkaloids: Wash and dry the organ to be tested at 37°C, crush it to 40 meshes, weigh 1.0 g accurately, place it in an ultrasonic cleaner, and use 50 ml of 95% ethanol aqueous solution for ultrasonic extraction for 30 min. The same method is used for ultrasonic extraction for the second time, and the filter residue is discarded, and the second filtrate is combined, concentrated by distillation under reduced pressure with a rotary evaporator, and the ethanol is recovered by volatilization to obtain a concentrated solution. The concentrated solution is dissolved in a dilute acid solution until the pH value is 3, and filtered , add ammonia water to the filtrate until the pH value is 10, filter to obtain a clear night, and put it in the refrigerator for subsequent use; (2) Accurately weigh the sample prepared in step (1), distill under reduced pressure, concentrate and volatilize to reclaim ethanol, prepare a solution with 5mL glacial acetic acid, and leave it in the refrigerator for a certain period of time for subsequent use; (3) HPLC analysis and determination of peak face value: the described liquid chromatography conditions are as follows: Chromatographic column: Cosmosil5C18AR-II (250×4.6mm); Mobile phase: acetonitrile - potassium dihydrogen phosphate (75:25); Detection wavelength: 205nm; flow rate: 1.0mL/min; Injection volume: 10 μL, column temperature: 25°C; (4) Establishment of the standard curve: Precisely absorb 0.2, 0.4, 0.6, 0.8, 1.0 mg of the reference substance and dilute it to 5 mL with glacial acetic acid, and set it aside at 20°C for a certain period of time, using the liquid phase conditions in step (3) , the absorbance of each concentration reference substance is measured by high performance liquid chromatography, and the linear regression method is used to calculate the regression equation of the standard curve to the measured data: y=1185050.6095x-37397.6623, where y is the absorbance, and x is the sample content, Among them, the correlation coefficient r=0.9960, r is the correlation between the two sets of data of sample content and absorbance, which is consistent with Lambert-Beer's law; (5) mensuration of steroidal alkaloid content: according to the regression equation y=1185050.6095x-37397.6623 in the peak surface value recorded in step (3) and step (4), wherein y is the peak surface value, and x is the sample alkali content, Calculate the steroidal alkaloid content of the sample.