CN114965729B - Method for extracting, enriching and separating alkaloid from lotus based on IS-CPR-IL-MCE - Google Patents
Method for extracting, enriching and separating alkaloid from lotus based on IS-CPR-IL-MCE Download PDFInfo
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Abstract
The invention discloses a method for extracting, enriching and separating alkaloid in lotus based on IS-CPR-IL-MCE. After the lotus and the ionic liquid are mechanically ground, the particle size is reduced, 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 Chinese medicinal extract, adding triton X-100 and ammonium sulfate, performing ultrasonic treatment, regulating pH, centrifuging to remove bubbles and phase separation, and heating in water bath to realize enrichment.
Description
Technical Field
The invention belongs to the field of extraction and enrichment preparation of alkaloids in traditional Chinese medicines, and relates to an extraction, enrichment and separation method of alkaloids in lotus based on mechanical chemical extraction (IS-CPR-IL-MCE) assisted by in-vivo cloud point reinforced ionic liquid, in particular to a method for extracting alkaloids by mechanically grinding lotus and ionic liquid, wherein the particle size IS reduced, the alkaloids are uniformly dispersed on the surface of the ionic liquid, and the alkaloids attached to the surface of the ionic liquid are rapidly dispersed in water along with hydrophilic ionic liquid after solvent water IS added; taking out the Chinese medicine extract, adding triton X-100 and ammonium sulfate, performing ultrasonic treatment, regulating pH, centrifuging to remove bubbles and phase separation, and heating in water bath to realize enrichment. The invention provides a novel method which can realize green, rapid and efficient extraction, enrichment and separation of alkaloids in lotus leaves.
Background
The cloud point extraction technology is a novel liquid-liquid extraction technology, is based on the cloud point phenomenon and solubilization of a surfactant, has the advantages of high concentration efficiency, simplicity in operation, environmental friendliness and the like, and therefore, has wide application in pre-enrichment and separation of analytes and potential application prospects in the environmental and pharmaceutical industries. Generally, aqueous solutions of surfactant micelles suddenly appear cloudy or even delaminate when heated above a certain temperature (i.e., cloud point). After standing for a period of time or centrifugation, the solution separates into two phases, including a surfactant phase and an aqueous phase, which capture the analyte by the tissue structure. Notably, at temperatures below the cloud point, the catalyst can be prepared by adding a salt (e.g., na 2 SO 4 ) To achieve and enhance micelle formation, a phenomenon known as salting-out. In addition, triton X-100 is used as a stable, non-volatile, low-toxic and cost-effective surfactant for extracting and pre-concentrating various compounds from complex matrices. This method also has some obvious disadvantages, such as long equilibration times and the need for higher temperatures to develop cloud point. However, other extraction techniques have not been reported to simplify the sample pretreatment process to achieve higher enrichment factors.
Currently, a novel pre-extraction technique for extracting bioactive substances from natural products is attracting attention, namely a mechanochemical auxiliary extraction technique (MAE). MAE has the advantages of high efficiency, energy saving, environmental protection and the like, and has wide application in the industries of food, environment, medicine and the like. The build-up of MAE is largely dependent on the development of mechanochemistry, which refers to chemical or physicochemical transformations (e.g., particle size reduction and cell wall destruction) that occur under mechanical forces. MAE is typically achieved by milling, during which the target compound reacts with a solid reagent under high mechanical pressure to convert to a water-soluble form. The solid reagents commonly used in current experiments are broadly divided into solid acid reagents (e.g. SiO 2 ) And solid alkali reagents (e.g. NaOH, na 2 CO 3 ) While less research is being done using Ionic Liquids (ILs) as MAE reagents. In fact, due to the low melting point, some ionic liquids are typically solid at room temperature and also react with the target compound during milling to convert the analyte into a water-soluble bioactive substance. Furthermore, in previous studies, MAE was usually used as a single extraction step and then combined with analytical methods, and no study has been made to combine 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 herb emergent aquatic plant of the genus Nelumbo of the family lotus, and has ornamental, cultural, edible and ecological values. Lotus leaf contains various active ingredients including alkaloids, essential oils, flavonoids and tannins. In recent years, a great deal of phytochemistry and preclinical research show that drinking lotus leaf tea can improve the activities of antioxidation, antibiosis and obesity resistance. The beneficial effects are due to the existence of lotus leaf alkaloids such as the lotus leaf alkaloid, the isoparaffinine, the lotus leaf alkaloid and the like. Researchers report a method for extracting lotus leaf, namely microwave-assisted solid phase microextraction, wherein the extraction solvent and the eluent are methanol and acetonitrile (containing 1% ammonia water) respectively. Also, researchers developed a method of extracting polysaccharide from lotus leaf, mixing the sample with 20 liters of purified water, and then extracting under reflux twice for 4 hours each. In the prior researches, the main flow methods of lotus leaf extraction comprise Soxhlet extraction, heating reflux extraction, microwave-assisted extraction and ultrasonic extraction, and have the problems of low efficiency, high organic solvent consumption, complex process and the like. Therefore, there is a need to develop an efficient and advanced lotus leaf alkaloid extraction method, which provides useful applications for human edible foods or medicines.
Disclosure of Invention
The invention aims to establish a novel, green and efficient in-vivo cloud point strengthening ionic liquid assisted mechanochemical extraction (IS-CPR-IL-MCE) technology, and 5 alkaloids in lotus leaves are simultaneously extracted, concentrated and separated by combining ultra-high performance liquid chromatography. A series of parameters affecting extraction efficiency were systematically discussed and optimized by single factor experimentation and Response Surface Method (RSM). Under the optimal condition, the effective extraction and the efficient separation of 5 alkaloids (liensinine, isoliensinine, methyl liensinine, O-desmethyl nuciferine and nuciferine) are realized.
Specifically, the sample treatment method is realized through the following technical measures:
step (1), pretreating lotus leaves, and grinding the lotus leaves into powder by a grinder;
step (2) extracting alkaloid
Carrying out mechanochemical grinding treatment on lotus leaves and ionic liquid 1-dodecyl-3-methylimidazolium 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 leaf to the ionic liquid 1-dodecyl-3-methylimidazolium 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:10mL;
preferably, the milling time is 1.0 to 10.0 minutes, more preferably 5 minutes;
preferably, the mass ratio of the lotus leaf to the ionic liquid 1-dodecyl-3-methylimidazolium bromide is 1:1, a step of;
preferably, the mass volume ratio of the ionic liquid 1-dodecyl-3-methylimidazolium bromide to the purified water is 0.25 g/10 mL;
step (3) enrichment of alkaloids
Adding a certain amount of triton X-100 (i.e. octoxynol) and ammonium sulfate into the upper layer sample solution, and carrying out ultrasonic dissolution; adjusting pH to 6.8-8.0, centrifuging to achieve phase separation and remove upper bubbles, and placing in a water bath at 30-70deg.C for a period of time to achieve enrichment; wherein the mass ratio of the triton X-100 to the lotus leaf is 6-14:5, a step of; the mass ratio of the ammonium sulfate to the lotus leaf is 4-12:1, a step of;
preferably, the pH is 7.7;
preferably, the water bath temperature is 60 ℃;
preferably, the water bath time is 10 to 30 minutes, more preferably 20 minutes;
preferably, the mass ratio of the triton X-100 to the lotus leaf is 8:5, a step of;
preferably, the mass ratio of the ammonium sulfate to the lotus leaf is 10:1, a step of;
preferably, the pH is adjusted with 1.0mol/L hydrochloric acid solution or 1.0mol/L sodium hydroxide;
step (4), separation of alkaloid
And diluting the upper layer enrichment liquid with methanol, centrifuging and filtering, and performing UHPLC analysis.
The extraction effect is shown by measuring the content of alkaloid by Agilent 1290-DAD.
The specific operation process is as follows:
the invention has the advantages that:
1. compared with the traditional method for extracting alkaloid, the method has the advantages of being green, free of pollution, capable of realizing extraction and enrichment, and the like.
2. The method has wide application range, can be used for detecting alkaloids in various medicinal materials, and has wide application potential in microanalysis of extracting alkaloids from natural medicinal materials.
The invention takes mechanochemical extraction assisted by in-vivo cloud point strengthening ionic liquid as an environment-friendly, sensitive and rapid method for determining alkaloid in traditional Chinese medicine. A series of parameters such as the dosage of ionic liquid, the dosage of surfactant, the dosage of salt, the grinding time, the pH value, the enrichment temperature and the time which influence the extraction efficiency are systematically discussed and optimized through a single factor experiment and RSM. Verification experiments of linearity, daily, daytime precision (RSD), detection Limit (LOD), quantitative Limit (LOQ), repeatability, recovery rate and the like were performed under optimal conditions. The method can be successfully applied to qualitative and quantitative analysis of alkaloids (liensinine, isoliensinine, methyl liensinine, O-desmethyl nuciferine and nuciferine) in lotus leaves.
Drawings
FIG. 1 IS a flow chart of IS-CPR-IL-MCE extraction and separation of target compounds.
Fig. 2 is a line graph for examining extraction effects of different amounts of ionic liquids.
Fig. 3 is a line graph for examining the extraction effect of different grinding times.
FIG. 4 is a line graph of the extraction effect for different surfactant amounts.
FIG. 5 is a line graph depicting the extraction effect of different salt dosages.
FIG. 6 is a line graph of examining the extraction effect of different pH.
FIG. 7 is a bar graph of the extraction effect for examining different enrichment temperatures.
FIG. 8 is a bar graph of the extraction effect for different enrichment times.
FIG. 9 is a three-dimensional response chart of the effect of ionic liquid dosage (X1, 200-300 mg), surfactant dosage (X2, 300-500 mg), pH (X3, 7.4-8.0) on extraction efficiency of target compounds. Wherein (a-1) to (a-5) are respectively three-dimensional response diagrams of different visual angles of the dosage of the ionic liquid and the dosage of the surfactant, and (b-1) to (b-5) are respectively three-dimensional response diagrams of different visual angles of the dosage of the ionic liquid and the pH, and (c-1) to (c-5) are respectively three-dimensional response diagrams of different visual angles of the dosage of the surfactant and the pH.
Fig. 10 is a chromatogram of a sample extracted from an established method of a mixed labeling solution of a target compound under otherwise identical conditions. Wherein 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 establishment method; c is the sample extracted by mechanical grinding (without cloud point enrichment); d is five mixed standard solutions.
In fig. 2 to 8 and 10, 1 is liensinine, 2 is isoliensinine, 3 is methylliensinine, 4 is O-desmethyl nuciferine, and 5 is nuciferine.
Detailed Description
As described above, in view of the shortcomings of the prior art, the present inventors have long studied and practiced in a large number of ways, and have proposed the technical solution of the present invention, which is based on at least: after the lotus and the ionic liquid are mechanically ground, the particle size is reduced, 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 Chinese medicine extract, adding triton X-100 and ammonium sulfate, performing ultrasonic treatment, regulating pH, centrifuging to remove bubbles and phase separation, and heating in water bath to realize enrichment.
The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention. In addition, the technical features of the embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.
The invention discloses a method for extracting, enriching and separating alkaloid in lotus based on in-vivo cloud point reinforced ionic liquid assisted mechanochemical extraction (IS-CPR-IL-MCE), which specifically comprises the following steps:
the sample was crushed to 120 mesh powder with a crusher. 0.25g of the powder was mixed with 0.15-0.30g of solid ionic liquid (1-dodecyl-3-methylimidazolium bromide, C) by means of a planetary ball mill 12 mimBr) is mechanically milled. Then, 10ml of pure was addedThe solution was sonicated (40 khz, 15 minutes) and centrifuged at 4000rpm for 5 minutes. Taking out 7 ml of the supernatant, adding 0.3-0.6 g of triton X-100 and 1.0-3.0 g of ammonium sulfate, regulating the pH by using 1.0mol/L hydrochloric acid solution or 1.0mol/L sodium hydroxide solution after ultrasonic treatment for 10 minutes, and centrifuging for 10 minutes to realize bubble removal and phase separation, wherein the water bath is at 30-70 ℃ for 10-30 minutes. Finally, the concentrated solution (100. Mu.l) was removed, diluted 5-fold with methanol, centrifuged at 16000rpm for 3 minutes, filtered through a microporous membrane (50 mm. Times.0.45 μm), collected and analyzed by UHPLC, and the detection wavelength was 280 nm. The above process is shown in fig. 1.
UHPLC specific conditions: chromatograph: agilent 1290UHPLC. 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; chromatographic column: agilent SB-C 18 (4.6X105 mm,5 μm); sample injection amount: 0.2 μl; flow rate: 0.4mL/min; column temperature: 280nm; detection wavelength: 30 ℃.
The following description of the present invention is further provided with reference to several preferred embodiments, but the experimental conditions and setting parameters should not be construed as limiting the basic technical scheme of the present invention. And the scope of the present invention is not limited to the following examples.
Example 1. Investigation of the Effect of Ionic liquid usage on extraction Effect
1.1 taking 4 clean ball milling tanks with the numbers of 1,2, 3 and 4, and sequentially adding 0.25g of lotus leaf powder;
1.2 adding 150, 200, 250, 300 mg of ionic liquid 1-dodecyl-3-methylimidazolium bromide respectively, and then adding 6 porcelain balls with equal weight in sequence;
1.3, symmetrically placing ball milling tanks, and grinding for 5min;
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, carrying out ultrasonic treatment for 10 minutes, pouring the mixture into a centrifuge tube, and centrifuging at 4000rpm for 5 minutes;
1.5 taking 7 ml of the supernatant, adding 0.40g of triton X-100 and 1.5 g of ammonium sulfate;
1.6 after 10 minutes of sonication, the pH was adjusted to 7.7 and centrifuged at 4000rpm for 10 minutes;
1.760 deg.C water bath for 20 minutes;
1.8 sequentially taking 0.5mL of sample solution in a centrifuge tube, diluting to 5 times by using methanol, and centrifuging at 16000rpm for 5min;
1.9 sucking the intermediate liquid and injecting the liquid phase.
The chromatographic conditions are as follows:
detection wavelength: 280nm; column temperature: 30 DEG C
The experimental results are shown in FIG. 2. Fig. 2 is a line graph of extraction effect for examining the amounts of different ionic liquids.
In the present invention, the amount of ionic liquid is chosen as the first parameter to be considered, so that under other conditions of fixation (grinding time of 5min; amount of surfactant of 0.40 g; salt content of 1.5 g; heating temperature of 60 ℃ C.; heating time of 20 min). As shown in fig. 2, with C 12 The extraction efficiency tends to be steadily improved due to the increase of the use amount of mimBr. The longer the carbon chain of the known compounds is, the stronger the nonpolar is, C according to the principle of "similar compatibility 12 mimBr has good solubility for alkaloids. In addition, hydrophilicity between the target and the ionic liquid is a major driving force for the water extraction process. The greater the amount of ionic liquid, the greater the likelihood of contact with the sample, and the likelihood of physical and chemical conversion of the target compound is simultaneously enhanced by mechanochemical treatment, which may result in 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 excessive, the mass transfer between the solution and the solute is weakened, and the extraction efficiency is unchanged or slightly reduced from 0.25g to 0.30 g. Thus, the present invention selects an ionic liquid dosage of 0.25 grams.
Example 2 investigation of the Effect of milling time on extraction
2.1, taking 4 clean ball milling tanks with the numbers of 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 porcelain balls with equal weight;
2.3, symmetrically placing ball milling tanks, and respectively grinding 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, carrying out ultrasonic treatment for 10 minutes, pouring the mixture into a centrifuge tube, and centrifuging at 4000rpm for 5 minutes;
2.5 taking 7 ml of the upper layer liquid, adding 0.40g of triton X-100 and 1.5 g of ammonium sulfate;
2.6 after 10 minutes of ultrasound, the pH was adjusted to 7.7 and centrifuged at 4000rpm for 10 minutes;
2.760 deg.C water bath for 20 minutes;
2.8 sequentially taking 0.5mL of sample solution in a centrifuge tube, diluting to 5 times by using methanol, and centrifuging for 5min at 16000 rpm;
2.9 sucking the intermediate liquid and injecting the liquid phase.
The chromatographic conditions are as follows:
detection wavelength: 280nm; column temperature: 30 DEG C
The experimental results are shown in FIG. 3. Fig. 3 is a line graph of extraction effect for examining the amounts of different ionic liquids.
In order to reduce the particle size, the contact area was enlarged and the sample was milled with the ionic liquid in a ball mill for a period of time. Under the same conditions (0.25 g of ionic liquid, 0.40g of surfactant, 1.5 g of salt, 60 ℃ C. Heating time of 20 minutes). The results (fig. 3) show that the peak areas of the five compounds increased with increasing milling time before 5 minutes, and then significantly decreased. One possible reason for the increase is that the increased milling time enhances the released target analyte to destroy the cell wall sample. Before 5 minutes, the granularity of the sample is not small enough, so that the analyte cannot be fully contacted with the ionic liquid, but the excessive grinding time can have adverse effects on the recovery of alkaloids, and the excessive collision can lead to the degradation and oxidation of target compounds. Considering 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 Effect
3.1, taking 4 clean ball milling tanks with the numbers of 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 porcelain 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, carrying out ultrasonic treatment for 10 minutes, pouring the mixture into a centrifuge tube, and centrifuging at 4000rpm for 5 minutes;
3.5 taking 7 ml of upper layer liquid, and respectively adding 0.30,0.40,0.50,0.60 g of triton X-100 and 1.5 g of ammonium sulfate;
3.6 after 10 minutes of ultrasound, the pH was adjusted to 7.7 and centrifuged at 4000rpm for 10 minutes;
3.760 deg.C water bath for 20 minutes;
3.8 sequentially taking 0.5mL of sample solution in a centrifuge tube, diluting to 5 times by using methanol, and centrifuging for 5min at 16000 rpm;
3.9 sucking the intermediate liquid and injecting the liquid phase.
The chromatographic conditions are as follows:
detection wavelength: 280nm; column temperature: 30 DEG C
The selection of proper surfactant dosage is a key step in the cloud point extraction process, so that higher extraction efficiency is met, and the sensitivity of the method cannot be influenced due to overlarge volume of an enrichment phase. Extraction was performed at different surfactant levels (0.30, 0.40,0.50,0.60 g) to find the optimal extraction conditions (fig. 4). The extraction rate of the target compound tended to rise and then fall, and reached a peak at 0.40 g. This phenomenon can be explained by the fact that when the amount is less than 0.40g, only a small amount of the compound can be extracted to the surfactant enrichment stage; when the amount of addition is greater than 0.40g, the amount of analyte entering the enrichment phase increases and the volume of the surfactant enrichment phase also increases. It is inferred from this that the decrease in the concentration of the substance is due to the fact that the rate of increase in volume is greater than the rate of increase in the substance. Thus, the peak area gradually decreases from 0.40 to 0.60 grams. In view of this, we selected as the next experiment, the amount of triton X-100 of 0.40 g.
EXAMPLE 4 examination of the Effect of salt (ammonium sulfate) usage on extraction
4.1, taking 5 clean ball milling tanks with the numbers of 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 porcelain balls with equal weight;
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, carrying out ultrasonic treatment for 10 minutes, pouring the product into a centrifuge tube, and centrifuging at 4000rpm for 5 minutes;
4.5 taking 7 ml of upper layer liquid, adding 0.40g of triton X-100, and then adding 1.0,1.5,2.0,2.5,3.0 g of ammonium sulfate respectively;
4.6 after 10 minutes of ultrasound, the pH was adjusted to 7.7 and centrifuged at 4000rpm for 10 minutes;
4.760 deg.C water bath for 20 minutes;
4.8 sequentially taking 0.5mL of sample solution in a centrifuge tube, diluting to 5 times by using methanol, and centrifuging for 5min at 16000 rpm;
4.9 sucking the intermediate liquid and injecting the liquid phase.
The chromatographic conditions are as follows:
detection wavelength: 280nm; column temperature: 30 DEG C
In the IS-CPR-IL-MCE process, salt can reduce the solubility of a target compound in an aqueous phase and increase the distribution of the target compound in an enrichment phase, so that the extraction efficiency IS affected, and the salt dosage IS an important factor to consider. The effect of this study on alkaloid extraction was evaluated at a mass loading of 1.0-3.0 grams of salt. From fig. 5, it can be seen that from 1.0 to 2.5 g, the analytical signal of the compound starts to increase with increasing salt amount, and from 2.5 to 3.0 g, the analytical signal of the compound slightly decreases. The possible reason is that the addition of salt increases the density of the aqueous phase, thereby promoting phase separation. When the salt amount is too small, the density difference between the aqueous phase and the enriched phase is not obvious and stable, and only a small amount of compound is extracted into the surfactant enriched phase after the treatment. In addition, the compounds are readily mixed with aqueous solutions. When the salt amount was increased to 2.5 grams, the density difference between the two phases was large enough to remain stable and almost all the analyte was extracted. However, at 2.5 to 3.0 g, an increase in the ionic strength of the medium results in an increase in the viscosity of the aqueous solution, resulting in a decrease in mass transfer efficiency. Thus, in the present invention, we select 2.5 grams as the most suitable salt amount.
Example 5 investigation of the influence of pH on the extraction Effect
5.1, taking 5 clean ball milling tanks with the numbers of 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 porcelain 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, carrying out ultrasonic treatment for 10 minutes, pouring the mixture into a centrifuge tube, and centrifuging at 4000rpm for 5 minutes;
5.5 taking 7 ml of the supernatant, adding 0.40g of triton X-100 and 2.5 g of ammonium sulfate;
after 5.6 sonications for 10 minutes, pH was adjusted to 6.8,7.1,7.4,7.7 and 8.0, respectively, and centrifuged at 4000rpm for 10 minutes;
5.760 deg.C water bath for 20 minutes;
5.8 sequentially taking 0.5mL of sample solution in a centrifuge tube, diluting to 5 times by using methanol, and centrifuging for 5min at 16000 rpm;
5.9 sucking the intermediate liquid and injecting the liquid phase.
The chromatographic conditions are as follows:
detection wavelength: 280nm; column temperature: 30 DEG C
It is well known that the acid-base state (pH) of an extract affects the extraction efficiency by affecting the dissociation state of the target compound. Considering that the target analyte is alkaloid, the influence of pH value on extraction efficiency is examined within the range of 6.8-8.0 (regulated by hydrochloric acid or sodium hydroxide), and the experimental parameters are that grinding time is 5min; the dosage of the ionic liquid is 0.25g; 0.40g of surfactant; the salt dosage is 1.5 g; the water bath temperature is 60 ℃; as can be seen from fig. 4, the peak area of each compound generally increases as the pH goes from 6.8 to 7.7. This phenomenon may be caused by the slightly alkaline environment to keep the analyte in a neutral molecular state, which allows for better enrichment. After addition of the lye, the partition coefficient of the compounds in the organic and aqueous phases was changed. Thereby allowing more target compound to enter the upper concentrated layer, and gradually increasing the peak area of the compound until the maximum value is reached at a pH of 7.7. However, the strong alkalinity of the solution is unfavorable for micelle formation, and the extraction recovery rate tends to be reduced within the range of 7.7-8.0. Thus, a pH of 7.7 is suitable for subsequent investigation.
Example 6 investigation of the Effect of enrichment temperature on extraction
6.1, taking 5 clean ball milling tanks with the numbers of 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 porcelain balls with equal weight;
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, carrying out ultrasonic treatment for 10 minutes, pouring the product into a centrifuge tube, and centrifuging at 4000rpm for 5 minutes;
6.5 taking 7 ml of the upper layer liquid, adding 0.40g of triton X-100 and 2.5 g of ammonium sulfate;
6.6 after 10 minutes of ultrasound, the pH was adjusted to 7.7 and centrifuged at 4000rpm for 10 minutes, respectively;
6.7 water baths at 30, 40, 50, 60, 70 ℃ for 20 minutes, respectively;
6.8, sequentially taking 0.5mL of sample solution in a centrifuge tube, diluting to 5 times by using methanol, and centrifuging for 5min at 16000 rpm;
6.9 sucking the intermediate liquid and injecting the liquid phase.
The chromatographic conditions are as follows:
detection wavelength: 280nm; column temperature: 30 DEG C
Cloud point-assisted enrichment is a continuous process performed in two immiscible solutions, which depends on the proper temperature and sufficient equilibration time. Thus, the effect of the enrichment temperature on the enrichment efficiency was investigated 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 c, indicating that the alkaloid is effectively concentrated to the surfactant stage. The reason for this may be that the solubilization of micelles increases with increasing temperature; in addition, the dehydration of nonionic surfactants at 30-60 ℃ is also enhanced, leading to micelle repulsion and aggregation. However, the temperature is too high, and the alkaloid is partially dissolved in the bottom phase, which is unfavorable for the enrichment of the alkaloid. Therefore, the present invention selects 60 ℃ as the optimal enrichment temperature.
Example 7 investigation of the Effect of enrichment time on extraction Effect
7.1, taking 5 clean ball milling tanks with the numbers of 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 porcelain balls with equal weight;
and 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, carrying out ultrasonic treatment for 10 minutes, pouring the product into a centrifuge tube, and centrifuging at 4000rpm for 5 minutes;
7.5 taking 7 ml of the upper layer liquid, adding 0.40g of triton X-100 and 2.5 g of ammonium sulfate;
after 7.6 sonications for 10 minutes, the pH was adjusted to 7.7 and centrifuged at 4000rpm for 10 minutes, respectively;
7.7 water baths 10, 15, 20, 25, 30 minutes at 60 degrees celsius, respectively;
7.8 sequentially taking 0.5mL of sample solution in a centrifuge tube, diluting to 5 times by using methanol, and centrifuging at 16000rpm for 5min;
7.9 sucking the intermediate liquid and injecting the liquid phase.
The chromatographic conditions are as follows:
detection wavelength: 280nm; column temperature: 30 DEG C
As can be seen from FIG. 8, the heating time is at least over 20min, the enriched alkaloid can reach relative stability, and the extraction rate can be reduced due to the excessive heating time. It is presumed that the extraction rate has a great relationship with the improvement of the degree of separation of two phases. After 20 minutes of heating, the mass transfer of the two phases reaches equilibrium, but as the heating time is prolonged, the volume of the enriched phase increases and the unit concentration and peak area decrease. Thus, the present invention selects 20 minutes as the optimal enrichment heating time.
FIG. 9 is a three-dimensional response chart of the effect of ionic liquid amount (X1, 200-300 mg), surfactant amount (X2, 300-500 mg), pH (X3, 7.4-8.0) on the extraction efficiency of the target compound, wherein (a-1) to (a-5) are respectively three-dimensional response charts of different viewing angles of ionic liquid amount and surfactant amount, (b-1) to (b-5) are respectively three-dimensional response charts of different viewing angles of ionic liquid amount and pH, and (c-1) to (c-5) are respectively three-dimensional response charts of different viewing angles of surfactant amount and pH.
Fig. 10 is a chromatogram of a sample extracted from an established method of a mixed labeling solution of a target compound under otherwise identical conditions. Wherein 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 establishment method; c is the sample extracted by mechanical grinding (without cloud point enrichment); d is five mixed standard solutions.
To further verify the feasibility of the method, methodological examinations were performed including intra-day precision, inter-day precision, reproducibility, and sample recovery.
Precision within the day
1, taking 3 clean 1.5mL centrifuge tubes, numbering 1,2 and 3, and respectively preparing 10 mug/mL standard solutions of three target analytes;
centrifuging at 216000rpm for 3min;
3, sucking intermediate liquid, and injecting liquid phase;
4 sample injection analysis, wherein 6 samples are injected at different time periods in the same day.
Precision of daytime
1, taking 3 clean 1.5mL centrifuge tubes, numbering 1,2 and 3, and respectively preparing 10 mug/mL standard solutions of three target analytes;
centrifuging at 216000rpm for 3min;
3, sucking intermediate liquid, and injecting liquid phase;
4 sample injection analysis, sample injection is carried out at the same time point within three days, and the sample injection is carried out 2 times a day.
Repeatability of
Referring to the following experimental procedure, 3 groups were made in parallel for investigation
1, taking 3 clean 1.5mL centrifuge tubes, numbering 1,2 and 3, and respectively preparing 10 mug/mL standard solutions of three target analytes;
centrifuging at 216000rpm for 3min;
3 sucking the intermediate liquid, injecting the intermediate liquid into the liquid phase, and analyzing the result.
Recovery rate of sample addition
Referring to the following experimental procedure, 3 groups were made in parallel for each concentration
1. 0.25g of lotus leaf powder (120 mesh) is weighed, ground with 250 mg of ion liquid for 5 minutes, 10ml of purified water is added, ultrasonic treatment is carried out for 15 minutes, after centrifugation for 5 minutes, 7 ml of supernatant is taken out, 0.40g of triton X-100 and 2.5 g of ammonium sulfate are added, after ultrasonic treatment for 10 minutes, pH is adjusted to 7.7 by sodium hydroxide solution, and centrifugation is carried out for 10 minutes, so that bubble removal and phase separation are realized, and water bath at 60 ℃ is carried out for 20 minutes. Finally, the upper concentrated solution (100 microliters) is taken out and diluted to 5 times by methanol;
2. adding a label (the content is 1 mug/mg and 10 mug/mg of mixed label), and standing at room temperature for 2 hours to enable the mixed label to uniformly react with a sample;
3. centrifuging at 16000rpm for 5min;
4. sucking the intermediate liquid and injecting the liquid phase.
The experimental 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. The extraction, enrichment and separation method of alkaloid in lotus is characterized by comprising the following steps:
step (1), pretreating lotus leaves, and grinding the lotus leaves into powder by a grinder;
step (2) extracting alkaloid
Carrying out mechanochemical grinding treatment on lotus leaves and ionic liquid 1-dodecyl-3-methylimidazolium bromide, reducing the particle size of the lotus leaves, and uniformly dispersing the lotus leaves on the surface of the ionic liquid to obtain a co-grinding 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 leaf to the ionic liquid 1-dodecyl-3-methylimidazolium 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:10mL;
step (3) enrichment of alkaloids
Adding a certain amount of triton X-100 and ammonium sulfate into the upper layer sample solution, and carrying out ultrasonic dissolution; adjusting pH to 6.8-8.0, centrifuging to achieve phase separation and remove upper bubbles, and placing in a water bath at 30-70deg.C for a period of time to achieve enrichment; wherein the mass ratio of the triton X-100 to the lotus leaf is 6-14:5, a step of; the mass ratio of the ammonium sulfate to the lotus leaf is 4-12:1, a step of;
step (4), separation of alkaloid
And diluting the upper layer enrichment liquid with methanol, centrifuging and filtering, and performing UHPLC analysis.
2. The method of claim 1, wherein the milling time of step (2) is 1.0 to 10.0 minutes.
3. The method of claim 2, wherein the milling time of step (2) is 5 minutes.
4. The method of claim 1, wherein the mass ratio of lotus leaf to ionic liquid 1-dodecyl-3-methylimidazolium bromide in step (2) is 1:1.
5. the method of claim 1, wherein the mass to volume ratio of the ionic liquid 1-dodecyl-3-methylimidazolium bromide to purified water in step (2) is 0.25 g/10 ml.
6. The method of claim 1, wherein the pH in step (3) is 7.7.
7. The method of claim 1, wherein the water bath temperature in step (3) is 60 ℃ and the water bath time is 20 minutes.
8. The method of claim 1, wherein the water bath time of step (3) is from 10 minutes 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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| WO2007110637A1 (en) * | 2006-03-28 | 2007-10-04 | Bioniqs Limited | Method for extracting target alkaloid using an ionic liquid as extracting solvent |
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| WO2007110637A1 (en) * | 2006-03-28 | 2007-10-04 | Bioniqs Limited | Method for extracting target alkaloid using an ionic liquid as extracting solvent |
| CN106153752A (en) * | 2015-04-13 | 2016-11-23 | 浙江海洋学院 | One utilizes the method for nuciferine in ionic liquid extract/HPLC detection Folium Nelumbinis |
| CN107764756A (en) * | 2017-09-11 | 2018-03-06 | 云南中烟工业有限责任公司 | A kind of method of dispersive liquid-liquid microextraction combination photometry measure nicotine |
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