This is a summary of a paper published in the Chinese Medical Journal. In the article, the author proposes the basic process of separating piperine using absolute ethanol. Although we have not confirmed their argument, the separation method proposed in the article is still worthy of our reference.
Supercritical CO2 extraction process of pepper
Accurately weigh 150g of pepper powder and put it into the supercritical extraction kettle. When the temperature reaches the predetermined temperature, open the CO2 gas cylinder to supply air. When the pressure reaches the predetermined pressure, adjust the flow rate, start the extraction under constant temperature and pressure, and start timing. The extract is collected at the outlet of the separation kettle.
Determination of the content of piperine in the extract The CO2 extract of pepper is prepared according to the test solution method, the peak area of piperine is measured, and the content of piperine is calculated
Investigation of factors affecting the crystallization of piperine
Establishment of a method for determining the solubility of piperine
Weigh 5 parts of piperine reference substance each accurately, dissolve them in absolute ethanol, ethyl acetate or ether, shake in a constant temperature water bath at 25 ℃ until the dissolution equilibrium, filter with a membrane, take the supernatant and dilute to an appropriate concentration, HPLC Method to determine the concentration of piperine and the equilibrium solubility in absolute ethanol, ethyl acetate and ether.
At 0-40°C, the solubility of piperine in ethyl acetate varies the most with temperature. Experiments have shown that under the same conditions, the crystallization effect is not as good as using absolute ethanol as the crystallization solvent. Therefore, absolute ethanol is used as the solvent.
The effect of temperature on crystallization
Accurately weigh 3 parts of piperine extract, place it in a conical flask, under a constant temperature water bath at 40°C, add absolute ethanol dropwise while stirring until the extract just dissolves. Slowly lower the temperature to 0℃, place for 12h, and crystallize. According to the above operation, the influence of the temperature of 10℃ and 20℃ on the crystallization was investigated, and the content of piperine in the crystal was determined by HPLC.
The effect of solvent dosage on the crystallization of piperine
Accurately weigh 3 parts of the extract, put it in a conical flask, under a constant temperature water bath of 40℃, according to the ratio of extract to absolute ethanol
1:3, 1:4, 1:5 and 1:6 (w/v) were added dropwise absolute ethanol, while stirring, slowly cooled to 0℃, placed for 12h, crystallized. HPLC method to determine the content of piperine in the crystal, and calculate the yield of piperine,
The effect of cooling rate on crystallization
Accurately weigh 3 parts of the extract, put it in a conical flask, under a constant temperature water bath at 40°C, add anhydrous ethanol dropwise, with an amount of 1:5 (w/v), while stirring. Then the temperature was reduced to 0℃ at a cooling rate of 10/min, placed for 12h, and crystallized. According to the above operation, the effects of cooling rate of 1℃/5min and 1℃/10min on crystallization were investigated respectively. HPLC method was used to determine the content of piperine in the crystal and calculate the yield of piperine.
The effect of time on crystallization
Accurately weigh 3 parts of the extract and place it in an Erlenmeyer flask. Under a constant temperature water bath at 40°C, add absolute ethanol dropwise while stirring until the extract just dissolves. Then the temperature drops to 0℃ at a cooling rate of 1℃/min, and it is placed for 4h, 6h, 8h, 10h, 12h to crystallize. HPLC method to determine the content of piperine in the crystal, and calculate the yield of piperine.
According to the results of single factor investigation, it is preliminarily determined that the separation process of piperine in the supercritical CO2 extract of pepper is: dripping absolute ethanol in a constant temperature water bath at 40℃, the dosage is 1:5 (w/v), and then 1℃/ The cooling rate of 5min is reduced to 0℃, and it is placed at 0℃ for 4h to crystallize.
HPLC method is used to determine the content of piperine in the crystal and calculate the yield of piperine. The results: the average piperine content is about 80%, the highest can reach 90%, and the average yield of piperine is 70%.
The crystallization solvent is anhydrous ethanol, and the entrainer used in the supercritical CO2 extraction of pepper is 95% anhydrous ethanol, so the extract can be crystallized directly without the need to recover the solvent. This greatly reduces the amount of organic solvents and the cost of the entire extraction and separation process; and the cooling crystallization method has simple equipment and convenient operation. Compared with other methods, the crystallization method has the highest yield of piperine.
The obtained coarse crystals can be further refined by recrystallization, using absolute ethanol as the solvent, after repeated recrystallization, the purity can reach 99%.