CN104020227A - Alkali content determination method for steroid alkaloid produced by tissue culture method - Google Patents

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

A method for determining the alkali content of steroidal alkaloids produced by tissue culture

technical field

The invention relates to a method for determining the alkali content of steroidal alkaloids produced by tissue culture.

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 alkali content assay method that produces steroidal alkaloids with tissue culture method, utilizes ultrasonic technique, solvent consumption is relatively less, reduces cost, utilizes rotary evaporator to reduce Distill the solvent under high pressure, reduce the pressure in the system with the help of a vacuum pump, lower the boiling point of the liquid, and obtain 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 determining the alkali content of steroidal alkaloids produced by tissue culture, comprising the following steps:

(1) Configure culture medium:

a, callus medium P1, P2, P3, P4, P8 or P10,

b. Rooting medium P1, P2, P3, P4 or P10,

The P1 is MS+NAA0.2+6-BA0.2+30g/L sucrose, P2 is MS+NAA0.2+6-BA0.5+30g/L sucrose, and 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, P10 is MS+NAA1.0+6-BA2.0+30g/L sucrose;

Adjust the pH value of each of the above media to 5.8 before sterilization, add 10g/L agar, and sterilize at 1.1kg/cm2, 121°C for 20min under saturated steam;

(2) Callus culture: Take out the seedlings of Baiying tissue culture on the ultra-clean workbench, cut the cotyledon into 0.3*0.3cm square pieces or cut the hypocotyl into 1cm long pieces and inoculate them in the induced callus culture In the medium, in the incubator of temperature 22 ± 2 degrees Celsius, carry out dark cultivation, count the callus growth situation on the 30th day after inoculation;

(3) rooting culture: buds are cut off from the base, inoculated in the rooting medium, and when the 30th day after inoculation, the rooting situation is counted;

(4) Extraction and preparation of alkaloids: Wash the tissue to be tested in steps (2) and (3) and dry it at 37°C, crush it to 40 mesh, weigh 1.0g accurately, and place it in an ultrasonic cleaner Ultrasonic extraction with 50m195% ethanol aqueous solution for 30min, ultrasonic extraction for the second time in the same way, discard the filter residue, combine the second filtrate, use a rotary evaporator to distill and concentrate under reduced pressure, volatilize and recover ethanol to obtain a concentrated solution, which is dissolved in Dilute the acid solution until the pH value is 3, filter, add ammonia water to the filtrate until the pH value is 10, filter, get clear night, put it in the refrigerator for later use;

(5) Accurately weigh the sample prepared in step (4), concentrate and volatilize under reduced pressure 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;

(6) 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;

(7) Establishment of the standard curve: Precisely draw 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 let it stand at 20°C for a certain period of time for later use, using the liquid phase conditions in step (6) , 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;

(8) Determination of steroidal alkaloid content: according to the regression equation y=1185050.6095x-37397.6623 in the peak surface value recorded in step (6) and step (7), 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 measuring the alkali content of steroidal alkaloids produced by the tissue culture method of the present invention uses ultrasonic technology to shorten the extraction time and increase the extraction rate without heating, thereby improving the extraction rate of heat-sensitive alkaloids and basically having no effect on their physiological activity. The usage is relatively small, reducing the cost. 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.

The present invention produces the alkali content determination method of steroidal alkaloid by tissue culture method, comprises the following steps:

(1) Configure culture medium:

a, callus medium P1, P2, P3, P4, P8 or P10,

b. Rooting medium P1, P2, P3, P4 or P10,

Among them, 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, P10 is MS+ NAA1.0+6-BA2.0+30g/L sucrose;

Adjust the pH value of each of the above media to 5.8 before sterilization, add 10g/L agar, and sterilize at 1.1kg/cm2, 121°C for 20min under saturated steam;

(2) Callus culture: Take out the seedlings of Baiying tissue culture on the ultra-clean workbench, cut the cotyledon into 0.3*0.3cm square pieces or cut the hypocotyl into 1cm long pieces and inoculate them in the induced callus culture In the medium, in the incubator of temperature 22 ± 2 degrees Celsius, carry out dark cultivation, count the callus growth situation on the 30th day after inoculation;

(3) rooting culture: buds are cut off from the base, inoculated in the rooting medium, and when the 30th day after inoculation, the rooting situation is counted;

(4) 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 195% ethanol aqueous solution for ultrasonic extraction for 30 minutes. 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;

(5) Accurately weigh the sample prepared in step (4), concentrate and volatilize under reduced pressure 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;

(6) HPLC analysis determines the peak surface value of the sample, see 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;

(7) 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 let it stand at 20°C for a certain period of time for later use, 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;

(8) Determination of steroidal alkaloid content: according to the regression equation y=1185050.6095x-37397.6623 in the peak surface value recorded in step (6) and step (7), wherein y is the peak surface value, and x is the sample alkali content, The calculated steroidal alkaloid content, the calculation results are shown in Table 2 below.

Table 2

Sample alkaloid content

The method for measuring the alkali content of steroidal alkaloids produced by the tissue culture method of the present invention uses ultrasonic technology to shorten the extraction time and increase the extraction rate without heating, thereby improving the extraction rate of heat-sensitive alkaloids and basically having no effect on their physiological activity. The usage is relatively small, reducing the cost. 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. an alkaloid assay method producing steroidal alkaloids with tissue culture method, it is characterized in that comprising the following steps: (1) Configure culture medium: a, callus medium P1, P2, P3, P4, P8 or P10, b. Rooting medium P1, P2, P3, P4 or P10, The P1 is MS+NAA0.2+6-BA0.2+30g/L sucrose, P2 is MS+NAA0.2+6-BA0.5+30g/L sucrose, and 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, P10 is MS+NAA1.0+6-BA2.0+30g/L sucrose; Adjust the pH value of each of the above media to 5.8 before sterilization, add 10g/L agar, and sterilize at 1.1kg/cm2, 121°C for 20min under saturated steam; (2) Callus culture: Take out the seedlings of Baiying tissue culture on the ultra-clean workbench, cut the cotyledon into 0.3*0.3cm square pieces or cut the hypocotyl into 1cm long pieces and inoculate them in the induced callus culture In the medium, in the incubator of temperature 22 ± 2 degrees Celsius, carry out dark cultivation, count the callus growth situation on the 30th day after inoculation; (3) rooting culture: buds are cut off from the base, inoculated in the rooting medium, and when the 30th day after inoculation, the rooting situation is counted; (4) Extraction and preparation of alkaloids: Wash the tissue to be tested in steps (2) and (3) and dry it at 37°C, crush it to 40 mesh, weigh 1.0g accurately, and place it in an ultrasonic cleaner Ultrasonic extraction with 50m195% ethanol aqueous solution for 30min, ultrasonic extraction for the second time in the same way, discard the filter residue, combine the second filtrate, use a rotary evaporator to distill and concentrate under reduced pressure, volatilize and recover ethanol to obtain a concentrated solution, which is dissolved in Dilute the acid solution until the pH value is 3, filter, add ammonia water to the filtrate until the pH value is 10, filter, get clear night, put it in the refrigerator for later use; (5) Accurately weigh the sample prepared in step (4), concentrate and volatilize under reduced pressure 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; (6) 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; (7) Establishment of the standard curve: Precisely draw 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 let it stand at 20°C for a certain period of time for later use, using the liquid phase conditions in step (6) , 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; (8) Determination of steroidal alkaloid content: according to the regression equation y=1185050.6095x-37397.6623 in the peak surface value recorded in step (6) and step (7), wherein y is the peak surface value, and x is the sample alkali content, Calculate the steroidal alkaloid content of the sample.