1. Materials and methods
1.1 Test materials
1.1.1 Test materials
Diphtheria aconite.
1.1.2 Test reagent
Aconitine reference; Anhydrous ethanol; Ammonia water.
1.1.3 Test instrument
Electronic balance; C-type glass instrument; Circulating water vacuum; Rotary evaporator; UV-1500PC ultraviolet Visible Spectrophotometer (Shanghai Meishe Instrument Co., LTD.); Electric heating constant temperature blast drying oven; Temperature control heating sleeve; Computer intelligent temperature control low temperature ultrasonic synthesis extractor.
Other instruments: beaker (5, 10, 50mL), volumetric bottle (10, 25, 50mL), pipette (2mL, 1mL), suction ball, funnel, filter paper, measuring cylinder, pipette, pipette gun.
Other instruments: beaker (5, 10, 50mL), volumetric bottle (10, 25, 50mL), pipette (2mL, 1mL), suction ball, funnel, filter paper, measuring cylinder, pipette, pipette gun.
1.2 Test method
1.2.1 Preparation of standard solution
Precision weighing 0.0109mg of reference aconitine, using anhydrous ethanol aqueous solution as solvent, prepared into 0.002mg·L-1 of reference standard reserve solution in 50mL volumetric bottle, and stored in refrigerator at 4℃.
1.2.2 Extraction of sample test solution
Precision weigh the sample coarse powder 5g of crushed diphtheria aconite and place it in a 50mL conical bottle, add 0.50mL ammonia test solution, and rinse. Then 35.0mL anhydrous ethanol was added, and ultrasonic extraction was carried out for 3 times, 0.5h each time. Filter the extract with filter paper, wash the residue with anhydrous ethanol 3 to 4 times, retain the filtrate, filter again 3 to 4 times, and then combine the filtrate. Evaporate the solvent through a water bath until it is ammonia-free. Accurately absorb the solution in a 50mL volumetric bottle, then dilute it with ethanol to the scale, shake well, and prepare the test solution. The first derivative UV spectrum was determined according to the drawing method of the standard curve, and the total alkaloid content was calculated. Each batch of samples was repeated 6 times.
1.2.3 Selection of maximum absorption fixed wavelength
Absorb 2mL filtrate into 10mL volumetric bottle, add ethanol and dilute to scale. Take an appropriate amount of the solution into the colorimetric dish, and use a visible spectrophotometer to measure the absorbance in the wavelength range of 200-400nm, and finally determine the maximum absorption wavelength of aconitine reference product (Table 1).
1.2.4 Drawing of standard curve
Transfer 0.002mg·L-1 aconitine reference solution 2, 4, 6, 8 and 10mL into a 10mL volumetric bottle with a pipette, then fill with anhydrous ethanol to the scale, shake well, and place for 10min. With the blank anhydrous ethanol solution as the reference, the absorbance was measured at the maximum absorption wavelength of 231nm by UV-visible spectrophotometer.
1.3 Item determination (determination of the total alkaloid content of the sample)
Accurately absorb 4 samples of 1.00mL diphtheria aconite solution, place them in 25.00mL volumetric bottle, and add anhydrous ethanol to 10.00mL each. Add 0.28mL of 5% sodium nitrite solution each, shake evenly, and leave for 6min; Add 0.28mL 10% aluminum nitrate solution, shake and set aside for 6min; Add 4% sodium hydroxide solution 2.00mL, dilute it with anhydrous ethanol to the scale, shake and leave for 15min. With anhydrous ethanol as the control, the absorbance was determined at 507nm by spectrophotometry. The total alkaloid content in the sample solution was calculated according to the standard curve. Calculate the concentration of total alkaloids in the solvent according to the value of absorbance.
Total alkaloid extraction rate (%) = extraction liquid concentration (mg·L-1) × dilution times × volume (mL)/weighed sample (g) ×100.
Total alkaloid extraction rate (%) = extraction liquid concentration (mg·L-1) × dilution times × volume (mL)/weighed sample (g) ×100.
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