CN114965729A - Extraction, enrichment and separation method of alkaloids in lotus based on IS-CPR-IL-MCE - Google Patents
Extraction, enrichment and separation method of alkaloids in lotus based on IS-CPR-IL-MCE Download PDFInfo
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Abstract
The invention discloses an IS-CPR-IL-MCE-based method for extracting, enriching and separating alkaloid in lotus. After the lotus and the ionic liquid are mechanically ground, the particle size is reduced and uniformly dispersed on the surface of the ionic liquid, and after solvent water is added, alkaloid attached to the surface of the ionic liquid is rapidly dispersed in water along with hydrophilic ionic liquid so as to realize extraction of the alkaloid; taking out the traditional Chinese medicine extract, adding triton X-100 and ammonium sulfate, performing ultrasonic treatment, adjusting pH, centrifuging to remove bubbles and separate phases, and heating in water bath to realize enrichment.
Description
Technical Field
The invention belongs to the field of extraction and enrichment preparation of alkaloid in traditional Chinese medicine, and relates to a method for extracting, enriching and separating alkaloid in lotus based on in-vivo cloud point enhanced ionic liquid assisted mechanochemical extraction (IS-CPR-IL-MCE), in particular to a method for extracting, enriching and separating alkaloid in lotus, wherein the particle size IS reduced after mechanical grinding of the lotus and ionic liquid, the particle size IS uniformly dispersed on the surface of ionic liquid, and after solvent water IS added, the alkaloid attached to the surface of the ionic liquid IS rapidly dispersed in water along with hydrophilic ionic liquid to realize extraction of the alkaloid; taking out the traditional Chinese medicine extracting solution, adding triton X-100 and g of ammonium sulfate, performing ultrasonic treatment, adjusting pH, centrifuging to remove bubbles and separate phases, and heating in water bath to realize enrichment. The invention provides a new method which is green and rapid and can simultaneously realize high-efficiency extraction, enrichment and separation of alkaloid in lotus leaves.
Background
The cloud point extraction technology is a novel liquid-liquid extraction technology, is based on the cloud point phenomenon and the solubilization of a surfactant, and has the advantages of high concentration efficiency, simplicity in operation, environmental friendliness and the like, so that the cloud point extraction technology is widely applied to pre-enrichment and separation of analytes, and has potential application prospects in the environment and the medicine industry. Generally, when heated above a certain temperature (i.e., cloud point), aqueous micellar solutions of surfactants suddenly become cloudy or even delaminated. After standing for a period of time or centrifugation, the solution separates into two phases, including a surfactant phase and an aqueous phase, where the analyte is captured by the tissue structure. It is noted that at temperatures below the cloud point, salts (e.g., Na) may be added 2 SO 4 ) To effect and enhance micelle formation, a phenomenon known as salting-out. In addition, triton X-100 is used as a stable, non-volatile, low-toxicity and cost-effective surfactant for the extraction and preconcentration of a variety of compounds from complex matrices. This method also has some significant disadvantages, such as long equilibration times and the need for higher temperatures to develop the cloud point phenomenon. However, other extraction techniques simplify the sample pretreatment process, and no report has been found on a method for achieving a higher enrichment factor.
Currently, a novel pre-extraction technique for extracting bioactive substances from natural products is receiving attention, namely Mechanochemical Assisted Extraction (MAE). The MAE has the advantages of high efficiency, energy conservation, environmental protection and the like, and has wide application in the industries of food, environment, medicine and the like. The establishment of MAE relies heavily on the development of mechanochemistry, which refers to chemical or physicochemical transformations (e.g., particle size reduction and cell wall disruption) that occur under mechanical forces. MAE is typically achieved by milling, in which the target compound is converted to a water-soluble form by reaction with a solid reagent under high mechanical pressure. The solid reagents commonly used in the present experiments are roughly classified into solid acid reagents (e.g. SiO) 2 ) And solid alkaline agents (e.g. NaOH, Na) 2 CO 3 ) And Ionic Liquids (ILs) as MAEThe study of the reagents is less. In fact, due to the low melting point, some ionic liquids are normally solid at room temperature and also react with the target compound during the milling process to convert the analyte into a water-soluble bioactive substance. Furthermore, in previous studies, MAE was usually combined with analytical methods as a single extraction step, and there has been no study of combining in vivo mechanochemical-assisted extraction with other extraction methods to achieve simultaneous extraction, high enrichment and separation of target analytes.
Lotus leaf (Nelumbo nucifera Gaertn.) is a perennial emergent aquatic plant of Nelumbo nucifera of Nelumbonaceae, and has ornamental, cultural, edible and ecological values. The lotus leaf contains a variety of active ingredients including alkaloids, essential oils, flavonoids and tannins. In recent years, a large number of phytochemistry and preclinical researches show that the antioxidant, antibacterial and anti-obesity activities of lotus leaf tea can be improved by drinking the lotus leaf tea. The beneficial effects are attributed to the existence of lotus leaf alkaloids such as liensinine, isoliensinine, and liensinine. Researchers have reported a method for extracting lotus leaves, i.e. microwave-assisted solid phase microextraction, in which the extraction solvent and the eluent are methanol and acetonitrile (containing 1% ammonia water), respectively. Also, researchers developed a method of extracting polysaccharides from lotus leaves by mixing a sample with 20 liters of purified water and then extracting twice under reflux for 4 hours each. In the previous research, the mainstream methods for extracting lotus leaves comprise Soxhlet extraction, heating reflux extraction, microwave-assisted extraction and ultrasonic extraction, and have the problems of low efficiency, large consumption of organic solvent, complex process and the like. Therefore, there is a need to develop an efficient and advanced method for extracting alkaloid from lotus leaves, which provides useful applications for human food or drugs.
Disclosure of Invention
The invention aims to establish a novel, green and efficient in-vivo cloud point enhanced ionic liquid assisted mechanochemical extraction (IS-CPR-IL-MCE) technology, and simultaneously extract, concentrate and separate 5 alkaloids in lotus leaves by combining with ultra-high performance liquid chromatography. A series of parameters affecting extraction efficiency were systematically discussed and optimized by single-factor experiments and Response Surface Method (RSM). Under the optimal condition, the effective extraction and high-efficiency separation of 5 alkaloids (liensinine, isoliensinine, neferine, O-demethylnuciferine and nuciferine) are realized.
Specifically, the method for processing the sample is realized by the following technical measures:
step (1), preprocessing lotus leaves, and grinding the lotus leaves into powder by a grinder;
step (2), extraction of alkaloid
Carrying out mechanochemical grinding treatment on lotus leaves and ionic liquid 1-dodecyl-3-methylimidazole ammonium bromide to obtain a co-ground product; adding purified water into the co-ground product, ultrasonically dissolving, centrifuging, and taking an upper-layer sample solution; wherein the mass ratio of the lotus leaves to the ionic liquid 1-dodecyl-3-methylimidazole ammonium bromide is 5: 3-6; the mass-volume ratio of the ionic liquid 1-dodecyl-3-methylimidazole ammonium bromide to the purified water is 0.15-0.30g:10 mL;
preferably, the milling time is 1.0 to 10.0 minutes, more preferably 5 minutes;
preferably, the mass ratio of the lotus leaves to the ionic liquid 1-dodecyl-3-methylimidazole ammonium bromide is 1: 1;
preferably, the mass volume ratio of the ionic liquid 1-dodecyl-3-methylimidazole ammonium bromide to the purified water is 0.25g:10 mL;
step (3), enrichment of alkaloid
Adding a certain amount of triton X-100 (namely octoxynol) and ammonium sulfate into the upper layer sample solution, and ultrasonically dissolving; adjusting pH to 6.8-8.0, centrifuging to separate and remove upper bubbles, and placing in 30-70 deg.C water bath for a period of time to achieve enrichment; wherein the mass ratio of the triton X-100 to the lotus leaves is 6-14: 5; the mass ratio of the ammonium sulfate to the lotus leaves is 4-12: 1;
preferably, the pH is 7.7;
preferably, the water bath temperature is 60 ℃;
preferably, the water bath time is 10-30 minutes, more preferably 20 min;
preferably, the mass ratio of the triton X-100 to the lotus leaves is 8: 5;
preferably, the mass ratio of the ammonium sulfate to the lotus leaves is 10: 1;
preferably, the pH is adjusted by using 1.0mol/L hydrochloric acid solution or 1.0mol/L sodium hydroxide;
step (4), separation of alkaloid
And (4) diluting the upper layer enriched solution with methanol, and performing UHPLC analysis after centrifugal filtration.
The extraction effect shows the effectiveness of the extraction method by measuring the content of alkaloid through Agilent 1290-DAD.
The specific operation process is as follows:
the invention has the advantages that:
1. compared with the traditional method for extracting the alkaloid, the method has the advantages of environmental friendliness, no pollution, realization of extraction and enrichment and the like.
2. The method has wide application range, can be used for detecting the alkaloid in various medicinal materials, and has wide application potential in microanalysis for extracting the alkaloid from natural medicinal materials.
Namely, the method takes the mechanochemical extraction assisted by the in-vivo cloud point strengthened ionic liquid as a method for measuring the alkaloid in the traditional Chinese medicine in an environment-friendly, sensitive and rapid manner. A series of parameters such as ionic liquid dosage, surfactant dosage, salt dosage, grinding time, pH, enrichment temperature, time and the like which influence the extraction efficiency are systematically discussed and optimized through a single-factor experiment and RSM. Verification experiments of linearity, daily and daytime precision (RSD%), detection Limit (LOD), quantification Limit (LOQ), repeatability, recovery rate and the like are carried out under the optimal conditions. The method can be successfully applied to qualitative and quantitative analysis of alkaloids (liensinine, isoliensinine, neferine, O-demethylnuciferine and nuciferine) in lotus leaves.
Drawings
FIG. 1 IS a scheme showing the process of IS-CPR-IL-MCE extraction and separation of target compounds.
FIG. 2 is a line graph for examining the extraction effect of different ionic liquids.
Fig. 3 is a line graph for examining the extraction effect of different grinding times.
FIG. 4 is a line graph for examining the extraction effect of different surfactant amounts.
Fig. 5 is a line graph for examining the extraction effect of different salt dosages.
FIG. 6 is a line graph for examining the effect of different pH extractions.
FIG. 7 is a bar graph for examining the extraction effect of different enrichment temperatures.
FIG. 8 is a histogram for examining the extraction effect of different enrichment times.
FIG. 9 is a three-dimensional response graph of the effects of ionic liquid dosage (X1,200-300 mg), surfactant dosage (X2,300-500 mg), pH (X3,7.4-8.0) on the extraction efficiency of target compounds. Wherein (a-1) to (a-5) are three-dimensional response graphs of different visual angles of the dosage of the ionic liquid and the dosage of the surfactant, respectively, (b-1) to (b-5) are three-dimensional response graphs of different visual angles of the dosage of the ionic liquid and the pH, and (c-1) to (c-5) are three-dimensional response graphs of different visual angles of the dosage of the surfactant and the pH.
FIG. 10 is a chromatogram of a sample extracted by the established method from a mixed standard solution of the target compound under otherwise identical conditions. Wherein, the figure a is the enrichment phase of the lotus leaf sample obtained by the establishment method; b is the water phase of the lotus leaf sample obtained by the establishing method; c is a sample extracted by a mechanical grinding method (without cloud point enrichment); d is five mixed standard solutions.
In the figures 2-8 and 10, 1 is liensinine, 2 is isoliensinine, 3 is neferine, 4 is O-demethylnuciferine, and 5 is nuciferine.
Detailed Description
As described above, in view of the deficiencies of the prior art, the present inventors have made extensive studies and extensive practices, and propose a technical solution of the present invention, which is mainly based on at least: after the lotus and the ionic liquid are mechanically ground, the particle size is reduced and the lotus and the ionic liquid are uniformly dispersed on the surface of the ionic liquid, and after solvent water is added, alkaloid attached to the surface of the ionic liquid is rapidly dispersed in water along with the hydrophilic ionic liquid so as to realize extraction of the alkaloid; taking out the traditional Chinese medicine extracting solution, adding triton X-100 and g of ammonium sulfate, performing ultrasonic treatment, adjusting pH, centrifuging to remove bubbles and separate phases, and heating in water bath to realize enrichment.
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
The invention relates to a method for extracting, enriching and separating alkaloid in lotus based on in-vivo cloud point enhanced ionic liquid assisted mechanochemical extraction (IS-CPR-IL-MCE), which specifically comprises the following steps:
the sample was pulverized into 120 mesh powder with a pulverizer. Mixing 0.25g of the powder with 0.15-0.30g of solid ionic liquid (1-dodecyl-3-methylimidazolium bromide, C) in a planetary ball mill 12 mimBr) mechanically ground. Then, 10ml of purified water was added, the solution was sonicated (40 khz, 15 min) and centrifuged at 4000rpm for 5 min. Taking out 7 ml of upper layer liquid, adding 0.3-0.6 g of triton X-100 and 1.0-3.0 g of ammonium sulfate, carrying out ultrasonic treatment for 10 minutes, adjusting the pH value by using 1.0mol/L hydrochloric acid solution or 1.0mol/L sodium hydroxide solution, centrifuging for 10 minutes to remove bubbles and separate phases, and carrying out water bath at 30-70 ℃ for 10-30 minutes. And finally, taking out the concentrated solution (100 microliters), diluting the concentrated solution to 5 times with methanol, centrifuging the solution at 16000rpm for 3 minutes, filtering the solution by using a microporous filter membrane (50mm multiplied by 0.45 mu m), collecting the solution, and performing UHPLC analysis, wherein the detection wavelength is 280 nanometers. The above process is shown in figure 1.
Specific conditions of UHPLC: chromatograph: agilent 1290 UHPLC. Mobile phase: 0.1% formic acid (a) -methanol (B); gradient: 0-2 minutes, 5% -25% B; 5-6 minutes, 25% B; 6-9 minutes, 25-45% B; 12-16 minutes, 45-62% B; 15-17 minutes, 62-100% B; a chromatographic column: agilent SB-C 18 (4.6X 150mm,5 μm); sample introduction amount: 0.2 mu L; flow rate: 0.4 mL/min; column temperature: 280 nm; detection wavelength: at 30 ℃.
The technical solutions of the present invention are further explained below with reference to several preferred embodiments, but the experimental conditions and the setting parameters should not be construed as limitations of the basic technical solutions of the present invention. And the scope of the present invention is not limited to the following examples.
Example 1 examination of the Effect of the amount of Ionic liquid on the extraction
1.1 taking 4 clean ball milling tanks, numbering 1,2, 3 and 4, and sequentially adding 0.25g of lotus leaf powder;
1.2 respectively adding 150, 200, 250 and 300 mg of ionic liquid 1-dodecyl-3-methylimidazole ammonium bromide, and then sequentially adding 6 ceramic balls with equal weight;
1.3 symmetrically placing the ball milling tanks, and grinding for 5 min;
1.4 taking out the mechanically ground product, pouring the mechanically ground product into 4 clean conical flasks in sequence, adding 10mL of deionized water, performing ultrasonic treatment for 10 minutes, pouring the mixture into a centrifuge tube, and centrifuging the mixture for 5 minutes at 4000 rpm;
1.5 taking 7 ml of supernatant, adding 0.40g of triton X-100 and 1.5 g of ammonium sulfate;
1.6, after 10 minutes of ultrasonic treatment, adjusting the pH value to 7.7, and centrifuging at 4000rpm for 10 minutes;
water bath at 1.760 deg.C for 20 min;
1.8 sequentially taking 0.5mL of sample solution in a centrifuge tube, diluting the sample solution to 5 times by using methanol, and centrifuging the sample solution for 5min at 16000 rpm;
1.9 sucking the intermediate liquid and injecting the liquid phase.
The chromatographic conditions are as follows:
detection wavelength: 280 nm; column temperature: 30 deg.C
The results of the experiment are shown in FIG. 2. FIG. 2 is a line graph for examining the extraction effect of different amounts of ionic liquids.
In the present invention, the amount of ionic liquid is selected as the first parameter to be considered, and therefore, under fixed other conditions (milling time 5 min; amount of surfactant 0.40 g; salt content 1.5 g; heating temperature 60 ℃ C.; heating time 20 min). As shown in fig. 2, with C 12 The extraction efficiency tends to be steadily improved by increasing the dose of the mimBr. It is known that the longer the carbon chain of the compound, the more nonpolar it is, and according to the principle of "similar solubility", C 12 mimBr has good solubility for alkaloids. Furthermore, the hydrophilicity between the target and the ionic liquid is the main driving force for the water extraction process. The more the amount of ionic liquid, the more likely it is to come into contact with the sample, and the more likely it is that the target compound is physically and chemically converted by mechanical forceChemotherapy is simultaneously enhanced, which may lead to improved solubility and a distribution coefficient of the target analyte in water. However, due to the existence of the viscosity of the ionic liquid, the amount of the ionic liquid is too much, the mass transfer between the solution and the solute is weakened, and the extraction efficiency is not changed or slightly reduced from 0.25g to 0.30 g. Therefore, the present invention selects an amount of ionic liquid of 0.25 g.
Example 2 examination of the Effect of grinding time on extraction
2.1 taking 4 clean ball milling tanks, numbering 1,2, 3 and 4, and sequentially adding 0.25g of lotus leaf powder;
2.2 respectively adding 250 mg of ionic liquid, and then sequentially adding 6 ceramic balls with equal weight;
2.3, symmetrically placing the ball milling tanks, and respectively milling for 1.0, 2.5, 5.0 and 10.0 minutes;
2.4 taking out the mechanically ground product, pouring the mechanically ground product into 4 clean conical flasks in sequence, adding 10mL of deionized water, performing ultrasonic treatment for 10 minutes, pouring the mixture into a centrifuge tube, and centrifuging the mixture for 5 minutes at 4000 rpm;
2.5 taking 7 ml of supernatant, adding 0.40g of triton X-100 and 1.5 g of ammonium sulfate;
2.6, after 10 minutes of ultrasonic treatment, adjusting the pH value to 7.7, and centrifuging at 4000rpm for 10 minutes;
water bath at 2.760 deg.C for 20 min;
2.8 sequentially taking 0.5mL of sample solution in a centrifuge tube, diluting the sample solution to 5 times by using methanol, and centrifuging the sample solution for 5min at 16000 rpm;
2.9 sucking the intermediate liquid and injecting the liquid phase.
The chromatographic conditions are as follows:
detection wavelength: 280 nm; column temperature: 30 deg.C
The results of the experiment are shown in FIG. 3. FIG. 3 is a line graph for examining the extraction effect of different amounts of ionic liquids.
To reduce the particle size and enlarge the contact area, the sample is ground with the ionic liquid in a ball mill for a period of time. Under the same conditions (the addition amount of the ionic liquid is 0.25g, the dosage of the surfactant is 0.40g, the dosage of the salt is 1.5 g, the heating temperature is 60 ℃, and the heating time is 20 minutes). The results (fig. 3) show that the peak areas of the five compounds increased with increasing milling time and then decreased significantly before 5 minutes. One possible reason for this increase is that the increased milling time enhances the release of the target analyte to disrupt the cell wall sample. Before 5 minutes, the sample particle size is not small enough to allow sufficient contact between the analyte and the ionic liquid, but too long a milling time can adversely affect the recovery of the alkaloid, and excessive collisions can lead to degradation and oxidation of the target compound. In view of energy saving and extraction efficiency, 5 minutes was determined as a suitable milling time.
Example 3 examination of the Effect of the amount of surfactant (Triton X-100) on the extraction
3.1 taking 4 clean ball milling tanks, numbering 1,2, 3 and 4, and sequentially adding 0.25g of lotus leaf powder;
3.2 respectively adding 250 mg of ionic liquid, and then sequentially adding 6 ceramic balls with equal weight;
3.3 symmetrically placing the ball milling tanks, and grinding for 5.0 minutes;
3.4 taking out the mechanically ground product, pouring the mechanically ground product into 4 clean conical flasks in sequence, adding 10mL of deionized water, performing ultrasonic treatment for 10 minutes, pouring the mixture into a centrifuge tube, and centrifuging the mixture for 5 minutes at 4000 rpm;
3.5 taking 7 ml of supernatant, and respectively adding 0.30g, 0.40g, 0.50 g, 0.60 g of triton X-100 and 1.5 g of ammonium sulfate;
3.6 after 10 minutes of ultrasonic treatment, adjusting the pH value to 7.7, and centrifuging at 4000rpm for 10 minutes;
water bath at 3.760 deg.C for 20 min;
3.8 sequentially taking 0.5mL of sample solution, diluting to 5 times with methanol, and centrifuging at 16000rpm for 5 min;
3.9 sucking the intermediate liquid and injecting the liquid phase.
The chromatographic conditions are as follows:
detection wavelength: 280 nm; column temperature: 30 deg.C
The selection of proper surfactant amount is a key step in the cloud point extraction process, which not only needs to meet higher extraction efficiency, but also cannot cause the sensitivity of the method to be influenced by the overlarge volume of the enriched phase. Extraction was performed at different surfactant levels (0.30, 0.40, 0.50, 0.60 g) and the optimal extraction conditions were found (figure 4). The extraction rate of the target compound tended to increase first and then decrease, and reached a peak at 0.40 g. This phenomenon can be explained by the fact that, at amounts less than 0.40g, only a small amount of compound can be extracted into the surfactant enrichment stage; when the amount is more than 0.40g, the amount of the analyte entering the enrichment stage increases, and the volume of the surfactant enrichment stage becomes large. It is concluded that the decrease in the concentration of the substance is due to the fact that the rate of increase of the volume is greater than the rate of increase of the substance. Thus, the peak area gradually decreased from 0.40 to 0.60 g. In view of this, we chose 0.40 grams of triton X-100 for the next experiment.
Example 4 examination of the Effect of salt (ammonium sulfate) amount on extraction
4.1 taking 5 clean ball milling tanks, numbering 1,2, 3, 4 and 5, and sequentially adding 0.25g of lotus leaf powder;
4.2 respectively adding 250 mg of ionic liquid, and then sequentially adding 6 ceramic balls with equal weight;
4.3 symmetrically placing the ball milling tanks, and grinding for 5.0 minutes;
4.4 taking out the mechanically ground product, pouring the mechanically ground product into 5 clean conical flasks in sequence, adding 10mL of deionized water, performing ultrasonic treatment for 10 minutes, pouring the mixture into a centrifuge tube, and centrifuging the mixture for 5 minutes at 4000 rpm;
4.5 taking 7 ml of supernatant, adding 0.40g of triton X-100, and then respectively adding 1.0, 1.5, 2.0, 2.5 and 3.0 g of ammonium sulfate;
4.6 after 10 minutes of ultrasonic treatment, adjusting the pH value to 7.7, and centrifuging at 4000rpm for 10 minutes;
water bath at 4.760 deg.C for 20 min;
4.8 sequentially taking 0.5mL of sample solution, diluting the sample solution to 5 times by using methanol, and centrifuging the sample solution for 5min at 16000 rpm;
4.9 sucking the intermediate liquid and injecting the liquid phase.
The chromatographic conditions are as follows:
detection wavelength: 280 nm; column temperature: 30 deg.C
In the IS-CPR-IL-MCE process, the salt can reduce the solubility of the target compound in the water phase and increase the distribution of the target compound in the enrichment phase, thereby influencing the extraction efficiency, and the dosage of the salt IS an important factor to be considered. In the research, the influence of the salt addition amount on the alkaloid extraction is evaluated according to the mass salt addition amount of 1.0-3.0 g. As can be seen from fig. 5, from 1.0 to 2.5 grams the analytical signal for the compound starts to increase with increasing salt amount, and from 2.5 to 3.0 grams the analytical signal for the compound slightly decreases. A possible reason is that the addition of salt increases the density of the aqueous phase, thereby promoting phase separation. When the amount of salt is too small, the difference in density between the aqueous phase and the rich phase is not significant and unstable, and only a small amount of the compound is extracted into the surfactant-rich phase after the above treatment. In addition, the compounds are readily mixed with aqueous solutions. When the amount of salt was increased to 2.5 grams, the density difference between the two phases was large enough to remain stable and almost all of the analyte was extracted. However, at 2.5 to 3.0 g, the increase in the ionic strength of the medium leads to an increase in the viscosity of the aqueous solution and thus to a decrease in the mass transfer efficiency. Thus, in the present invention, we chose 2.5 grams as the optimum salt dosage.
Example 5 examination of the influence of pH on the extraction Effect
5.1 taking 5 clean ball milling tanks, numbering 1,2, 3, 4 and 5, and sequentially adding 0.25g of lotus leaf powder;
5.2 respectively adding 250 mg of ionic liquid, and then sequentially adding 6 ceramic balls with equal weight;
5.3 symmetrically placing the ball milling tanks, and grinding for 5.0 minutes;
5.4 taking out the mechanically ground product, pouring the mechanically ground product into 5 clean conical flasks in sequence, adding 10mL of deionized water, performing ultrasonic treatment for 10 minutes, pouring the mixture into a centrifuge tube, and centrifuging the mixture for 5 minutes at 4000 rpm;
5.5 taking 7 ml of supernatant, adding 0.40g of triton X-100 and 2.5 g of ammonium sulfate;
5.6 after 10 minutes of ultrasound, adjusting the pH to 6.8, 7.1, 7.4, 7.7 and 8.0 respectively, and centrifuging at 4000rpm for 10 minutes;
water bath at 5.760 deg.C for 20 min;
5.8 sequentially taking 0.5mL of sample solution into a centrifuge tube, diluting the sample solution to 5 times by using methanol, and centrifuging the sample solution for 5min at 16000 rpm;
5.9 sucking the intermediate liquid and injecting the liquid phase.
The chromatographic conditions are as follows:
detection wavelength: 280 nm; column temperature: 30 deg.C
It is well known that the acid-base state (pH) of the extraction solution affects the extraction efficiency by affecting the dissociation state of the target compound. Considering that the target analyte is alkaloid, and investigating the influence of pH value on extraction efficiency in the range of 6.8-8.0 (adjusted by hydrochloric acid or sodium hydroxide), wherein the experimental parameter is that the grinding time is 5 min; the dosage of the ionic liquid is 0.25 g; the dosage of the surfactant is 0.40 g; the amount of salt used, 1.5 grams; the water bath temperature is 60 ℃; as can be seen from fig. 4, the peak area of each compound generally increased as the pH was from 6.8 to 7.7. This may be due to the slightly alkaline environment that keeps the analyte in a neutral molecular state that allows for better enrichment. After addition of the lye, the partition coefficients of the compounds in the organic and aqueous phases change. Thereby leading more target compound to enter the upper enrichment layer, and the peak area of the compound is gradually increased until the maximum value is reached when the pH is 7.7. However, the strong basicity of the solution is not favorable for forming micelles, and the extraction recovery rate is in a descending trend within the range of 7.7-8.0. Therefore, pH 7.7 is suitable for subsequent studies.
Example 6 examination of the Effect of enrichment temperature on extraction
6.1 taking 5 clean ball milling tanks, numbering 1,2, 3, 4 and 5, and sequentially adding 0.25g of lotus leaf powder;
6.2 respectively adding 250 mg of ionic liquid, and then sequentially adding 6 ceramic balls with equal weight;
6.3 symmetrically placing the ball milling tanks, and grinding for 5.0 minutes;
6.4 taking out the mechanically ground product, pouring the mechanically ground product into 5 clean conical flasks in sequence, adding 10mL of deionized water, performing ultrasonic treatment for 10 minutes, pouring the mixture into a centrifuge tube, and centrifuging the mixture for 5 minutes at 4000 rpm;
6.5 taking 7 ml of supernatant, adding 0.40g of triton X-100 and 2.5 g of ammonium sulfate;
6.6 after 10 minutes of ultrasound, adjusting the pH to 7.7 and centrifuging at 4000rpm for 10 minutes;
6.7 water bath at 30, 40, 50, 60 and 70 ℃ for 20 minutes;
6.8 sequentially taking 0.5mL of sample solution in a centrifuge tube, diluting the sample solution to 5 times by using methanol, and centrifuging the sample solution for 5min at 16000 rpm;
6.9 sucking the intermediate liquid and injecting the liquid phase.
The chromatographic conditions are as follows:
detection wavelength: 280 nm; column temperature: 30 deg.C
Cloud point assisted enrichment is a continuous process performed in two immiscible solutions, which relies on proper temperature and sufficient equilibration time. Therefore, the effect of the enrichment temperature on the enrichment efficiency was studied in the range of 30-70 ℃. As shown in FIG. 7, the extraction efficiency of the target analyte increases with increasing temperature in the temperature range of 30-60 ℃, indicating that the alkaloid is effectively concentrated into the surfactant stage. The reason for this may be that the solubilization of the micelles is accelerated with increasing temperature; in addition, the dehydration effect of the nonionic surfactant at 30-60 ℃ is also enhanced, so that micelle exclusion and aggregation are caused. However, the temperature is too high, and the alkaloid is dissolved in the bottom phase part, which is not beneficial to the enrichment of the alkaloid. Therefore, the invention selects 60 ℃ as the optimal enrichment temperature.
Example 7 examination of the Effect of enrichment time on extraction
7.1 taking 5 clean ball milling tanks, numbering 1,2, 3, 4 and 5, and sequentially adding 0.25g of lotus leaf powder;
7.2 respectively adding 250 mg of ionic liquid, and then sequentially adding 6 ceramic balls with equal weight;
7.3 symmetrically placing the ball milling tanks, and grinding for 5.0 minutes;
7.4 taking out the mechanically ground product, pouring the mechanically ground product into 5 clean conical flasks in sequence, adding 10mL of deionized water, performing ultrasonic treatment for 10 minutes, pouring the mixture into a centrifuge tube, and centrifuging the mixture for 5 minutes at 4000 rpm;
7.5 taking 7 ml of supernatant, adding 0.40g of triton X-100 and 2.5 g of ammonium sulfate;
7.6 after 10 minutes of ultrasound, adjusting the pH to 7.7 and centrifuging at 4000rpm for 10 minutes;
7.7 water bath at 60 ℃ for 10, 15, 20, 25 and 30 minutes respectively;
7.8 sequentially taking 0.5mL of sample solution, diluting the sample solution to 5 times by using methanol, and centrifuging the sample solution for 5min at 16000 rpm;
7.9 sucking the intermediate liquid and injecting the liquid phase.
The chromatographic conditions are as follows:
detection wavelength: 280 nm; column temperature: 30 deg.C
As can be seen from FIG. 8, when the heating time is at least 20min, the alkaloid enrichment can be relatively stable, and the extraction rate is reduced when the heating time is too long. It is presumed that the extraction rate is greatly related to the improvement of the two-phase separation degree. After heating for 20 minutes, the mass transfer of the two phases reached equilibrium, but as the heating time was extended, the volume of the enriched phase increased and the unit concentration and peak area decreased. Therefore, the present invention selects 20 minutes as the optimal enrichment heating time.
FIG. 9 is a three-dimensional response graph of the effect of the amount of ionic liquid (X1,200-300 mg), the amount of surfactant (X2,300-500 mg), and pH (X3,7.4-8.0) on the extraction efficiency of target compounds, wherein (a-1) to (a-5) are three-dimensional response graphs of the amount of ionic liquid and the amount of surfactant, respectively, and (b-1) to (b-5) are three-dimensional response graphs of the amount of ionic liquid and the pH, respectively, and (c-1) to (c-5) are three-dimensional response graphs of the amount of surfactant and the pH, respectively.
FIG. 10 is a chromatogram of a sample extracted by the established method from a mixed standard solution of the target compound under otherwise identical conditions. Wherein, the figure a is the enrichment phase of the lotus leaf sample obtained by the establishment method; b is the water phase of the lotus leaf sample obtained by the establishing method; c is a sample extracted by a mechanical grinding method (without cloud point enrichment); d is five mixed standard solutions.
To further validate the feasibility of the method, methodological studies were performed including intra-day precision, inter-day precision, reproducibility, and sample recovery.
Precision within a day
1, taking 3 clean 1.5mL centrifuge tubes, numbering 1,2 and 3, and respectively preparing standard solutions of three target analytes of 10 mu g/mL;
216000rpm for 3 min;
3 sucking the intermediate liquid and injecting the intermediate liquid into the liquid phase;
4 sample injection analysis, and 6 times of sample injection in different time periods in the same day.
Precision of day
1, taking 3 clean 1.5mL centrifuge tubes, numbering 1,2 and 3, and respectively preparing standard solutions of three target analytes of 10 mu g/mL;
centrifuging at 216000rpm for 3 min;
3 sucking the intermediate liquid and injecting the intermediate liquid into the liquid phase;
4 samples were analyzed at the same time points over three days, 2 times per day.
Repeatability of
Refer to the following experimental procedure, run 3 groups in parallel, as a survey
1, taking 3 clean 1.5mL centrifuge tubes, numbering 1,2 and 3, and respectively preparing standard solutions of three target analytes of 10 mu g/mL;
centrifuging at 216000rpm for 3 min;
3 sucking the intermediate liquid, injecting the liquid phase, and analyzing the result.
Sample recovery rate
With reference to the following experimental procedure, each concentration was run in parallel in 3 groups
1. Weighing 0.25g of lotus leaf powder (120 meshes), grinding with 250 mg of ionic liquid for 5 minutes, adding 10ml of purified water, carrying out ultrasonic treatment for 15 minutes, centrifuging for 5 minutes, taking out 7 ml of supernatant, adding 0.40g of triton X-100 and 2.5 g of ammonium sulfate, carrying out ultrasonic treatment for 10 minutes, adjusting the pH to 7.7 by using a sodium hydroxide solution, centrifuging for 10 minutes to remove bubbles and phase separation, and carrying out water bath at 60 ℃ for 20 minutes. Finally, taking out the concentrated solution (100 microliters), and diluting the concentrated solution to 5 times by using methanol;
2. adding a standard (mixed standard with the content of 1 mu g/mg and 10 mu g/mg), and standing at room temperature for 2 hours to ensure that the mixed standard and the sample uniformly react;
3. centrifuging for 5min under 16000 rpm;
4. sucking the intermediate liquid and injecting the liquid phase.
The results are summarized in tables 1-2 below:
TABLE 1
TABLE 2
The result shows that the method has good repeatability, high recovery rate and good detection accuracy.
Claims (10)
1. A method for extracting, enriching and separating alkaloid from lotus based on in-vivo cloud point enhanced ionic liquid assisted mechanochemical extraction is characterized by comprising the following steps:
step (1), preprocessing lotus leaves, and grinding the lotus leaves into powder by a grinder;
step (2), extraction of alkaloid
Carrying out mechanochemical grinding treatment on lotus leaves and ionic liquid 1-dodecyl-3-methylimidazole ammonium bromide to obtain a co-ground product; adding purified water into the co-ground product, ultrasonically dissolving, centrifuging, and taking an upper-layer sample solution; wherein the mass ratio of the lotus leaves to the ionic liquid 1-dodecyl-3-methylimidazole ammonium bromide is 5: 3-6; the mass volume ratio of the ionic liquid 1-dodecyl-3-methylimidazole ammonium bromide to the purified water is 0.15-0.30g:10 mL;
step (3), enrichment of alkaloid
Adding a certain amount of triton X-100 and ammonium sulfate into the upper layer sample solution, and performing ultrasonic dissolution; adjusting pH to 6.8-8.0, centrifuging to separate and remove upper bubbles, and placing in 30-70 deg.C water bath for a period of time to achieve enrichment; wherein the mass ratio of the triton X-100 to the lotus leaves is 6-14: 5; the mass ratio of the ammonium sulfate to the lotus leaves is 4-12: 1;
step (4), separation of alkaloid
And (4) diluting the upper layer enriched solution with methanol, and performing UHPLC analysis after centrifugal filtration.
2. The method of claim 1, wherein said milling time of step (2) is from 1.0 to 10.0 minutes.
3. The method of claim 2, wherein said milling time of step (2) is 5 minutes.
4. The method of claim 1, wherein the mass ratio of the lotus leaves to the ionic liquid 1-dodecyl-3-methylimidazolium bromide in step (2) is 1: 1.
5. the method according to claim 1, wherein the mass-to-volume ratio of the ionic liquid 1-dodecyl-3-methylimidazolium bromide to the purified water in the step (2) is 0.25g:10 mL.
6. The process according to claim 1, wherein the pH in step (3) is 7.7.
7. The method according to claim 1, wherein the temperature of the water bath in step (3) is 60 ℃ and the time of the water bath is 20 min.
8. The method of claim 1, wherein the water bath time in step (3) is 10 to 30 minutes.
9. The method of claim 1, wherein the mass ratio of triton X-100 to lotus leaf in step (3) is 8: 5.
10. the method of claim 1, wherein the mass ratio of ammonium sulfate to lotus leaf in step (3) is 10: 1.
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