CN112067705A - High performance liquid detection method for detecting alkaloid content in lotus plumule - Google Patents
High performance liquid detection method for detecting alkaloid content in lotus plumule Download PDFInfo
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Abstract
The invention relates to the technical field of lotus plumule content determination, in particular to a high performance liquid detection method for detecting the content of alkaloid in lotus plumule, which comprises the following steps: firstly, preparing a series of reference substance solutions with a certain concentration from a proper amount of liensinine, isoliensinine and neferine reference substances respectively, and then performing high performance liquid detection to determine a detection limit and a quantification limit; step two, preparing the alcohol extract of the lotus plumule into a sample solution to be tested, carrying out high performance liquid detection under the chromatographic conditions that a chromatographic column is an extended-C18 chromatographic column, the flow rate is 1ml/min, the detection wavelength is 282nm, the column temperature is 28-32 ℃, and the sample injection amount is 20 mu L, and calculating the alkaloid content of the lotus plumule through the peak area. The high performance liquid detection method for detecting the content of the lotus plumule alkaloid has the advantages of specificity, linear range, accuracy, stability and good repeatability, can be used for quantitative analysis, and has good durability.
Description
Technical Field
The invention relates to the technical field of lotus plumule content determination, in particular to a high performance liquid detection method for detecting the content of alkaloid in lotus plumule.
Background
Lotus plumule is a herbal medicine recorded in Chinese pharmacopoeia, and is prepared from green radicle (plumula Nelumbinis) of mature seed of Nelumbo nucifera of Nymphaeaceae, by taking out and sun drying.
Lotus plumule is bitter in taste, but has excellent effects of lowering blood pressure and removing fat. Different name Job's tears (Erya), bitter Job's tears (Ben Cao Tu Jing), lotus seed and Job's tears (gang mu), lotus nut (Ben Cao Xin). Is green embryo of mature seed of Nymphaeaceae plant Nelumbo nucifera Gaertn. Mainly produced in Hunan, Hubei, Jiangxi, Fujian, Jiangsu, Zhejiang, etc. Bitter in property and cold in nature. Clear heart fire, clear heat, stop bleeding, astringe essence. For restlessness, thirst, hematemesis, spermatorrhea, and mild qi of the five viscera.
The lotus plumule is a green plumule in a mature lotus seed kernel, is usually drunk as tea for folk, has the functions of clearing heart fire and stopping spermatorrhea, and is most suitable for insomnia patients with heart-kidney imbalance and yin deficiency and fire excess. In clinical application, the lotus plumule is suitable for people with mild insomnia, and can be used for stabilizing and controlling if the lotus plumule does not take effect, but the lotus plumule is harmful to the body after being taken for a long time. In addition, insomnia patients should develop good living habits, such as more walking after dinner, more exercise in common and the like, which are good for recovering sleep.
The lotus plumule mainly contains various active ingredients such as alkaloid, flavone and polysaccharide. The plumula Nelumbinis alkaloid mainly comprises liensinine, isoliensinine, neferine and plumula Nelumbinis quaternary ammonium base, and has pharmacological effects of lowering blood pressure, resisting arrhythmia, resisting platelet aggregation, and resisting cancer. Therefore, the establishment of a method for accurately detecting the content of the lotus plumule alkaloid has very important significance.
Disclosure of Invention
In view of the above disadvantages of the prior art, an object of the present invention is to provide a high performance liquid detection method for detecting the content of alkaloid in lotus plumule, which is used to solve the problem of inaccurate measurement of the content of alkaloid in lotus plumule in the prior art, and the provided high performance liquid detection method for detecting the content of alkaloid in lotus plumule has the advantages of specificity, linear range, accuracy, stability and good repeatability, can be used for quantitative analysis, and has good durability.
In order to attain the above and other related objects,
the invention provides a high performance liquid detection method for detecting the content of alkaloid in lotus plumule, which comprises the following steps:
respectively preparing a proper amount of a liensinine reference substance, an isoliensinine reference substance and a neferine reference substance into a series of liensinine reference substance solutions with concentration, an isoliensinine reference substance solution and a neferine reference substance solution, wherein the volume ratio of an extend-C18 chromatographic column, water and triethylamine as a mobile phase A is 1000: (0.9-1.1) the volume ratio of the mixed solution and the mobile phase B is 1: (0.99-1.01) respectively measuring a liensinine reference substance solution, an isoliensinine reference substance solution and a neferine reference substance solution with different concentrations under the chromatographic conditions that the flow rate is 1ml/min, the detection wavelength is 282nm, the column temperature is 28-32 ℃ and the sample injection amount is 20 mu L, carrying out high performance liquid phase detection, wherein the detection limit is set when S/N is approximately equal to 3, and the quantification limit is set when S/N is approximately equal to 10;
step two, preparing the lotus plumule alcohol extract into a series of lotus plumule alkaloid test sample solutions with concentration, wherein the volume ratio of water to triethylamine in a chromatographic column of an extended-C18 chromatographic column and a mobile phase A is 1000: (0.9-1.1) the volume ratio of the mixed solution and the mobile phase B is 1: (0.99-1.01), performing high performance liquid detection on the mixed solution under the chromatographic conditions of flow rate of 1ml/min, detection wavelength of 282nm, column temperature of 28-32 ℃ and sample amount of 20 mu L, and calculating the content of the lotus plumule alkaloid in the sample solution through peak area.
In an embodiment of the present invention, the volume ratio of the mobile phase a and the mobile phase B in the elution process in the first step and the second step is specifically shown in table 2:
| time/min | Mobile phase A/%) | Mobile phase B/%) |
| 0 | 90 | 10 |
| 5 | 80 | 20 |
| 20 | 50 | 50 |
| 25 | 20 | 80 |
| 35 | 20 | 80 |
Table 2 volume ratio of mobile phase a and mobile phase B.
In an embodiment of the present invention, the mobile phase a in the first step and the second step is water and triethylamine in a volume ratio of 1000: 1, and the mobile phase B is a mixed solution of acetonitrile and methanol in a volume ratio of 1: 1.
In an embodiment of the invention, the column temperature of the chromatographic column in the first step and the second step is 30 ℃, the length of the chromatographic column is 250mm, the inner diameter of the chromatographic column is 4.6mm, and the diameter of the filler particles of the chromatographic column is 5 μm.
In an embodiment of the present invention, the preparation process of the alcohol extract of lotus plumule in the second step is as follows: crushing a lotus plumule sample, sieving the crushed lotus plumule sample with a 20-mesh sieve, placing 499-501 mg of lotus plumule powder into a 25mL volumetric flask, adding 70-100% by mass of methanol, performing ultrasonic treatment for 25-35 min, cooling to room temperature, standing for at least 30min, metering the volume to 25mL by using 70-100% by mass of methanol, filtering by using a 0.45-micron microporous filter membrane, and taking a subsequent filtrate to obtain the lotus plumule alcohol extract.
In an embodiment of the present invention, the preparation process of the alcohol extract of lotus plumule in the second step is as follows: crushing a lotus plumule sample, sieving the crushed lotus plumule sample with a 20-mesh sieve, placing 500mg of lotus plumule powder in a 25mL volumetric flask, adding methanol, performing ultrasonic treatment for 30min, cooling to room temperature, standing for 30min, metering volume to 25mL by using methanol, filtering by using a 0.45-micron microporous filter membrane, and taking a subsequent filtrate, namely the lotus plumule alcohol extract.
In an embodiment of the present invention, the preparation process of the alcohol extract of lotus plumule in the second step is as follows: taking 100 parts by weight of lotus plumule, performing reflux extraction for 2-3 times by using 50-95% ethanol with the mass of 4-6 times that of the lotus plumule for 1-2 hours each time, filtering, combining filtrates, concentrating under reduced pressure to 1.5-2.5 times that of the lotus plumule to obtain an alcohol extract, filling 100 parts by weight of 732 type cation exchange resin into a glass column with the inner diameter of 60mm, adding the alcohol extract, fully adsorbing the 732 type cation exchange resin for 2-3 hours, washing off water-soluble impurities by using 10-15 BV of distilled water at the flow rate of 4-6 BV/h, eluting by using 10-15 BV of 95% ethanol at the flow rate of 2-3 BV/h, eluting by using 10-15 BV of 95% ethanol containing 5% of ammonia water at the flow rate of 2-3 BV/h, collecting eluent, concentrating and drying to obtain the alcohol extract of the lotus plumule.
In an embodiment of the present invention, the preparation process of the alcohol extract of lotus plumule in the second step is as follows: extracting plumula Nelumbinis 100 weight parts with 95% ethanol 6 times of plumula Nelumbinis mass under reflux for 3 times, each time for 1 hr, filtering, mixing filtrates, concentrating under reduced pressure to 2.0 times of plumula Nelumbinis mass to obtain ethanol extractive solution, loading 732 type cation exchange resin 100 weight parts into glass column with inner diameter of 60mm, adding ethanol extractive solution, adsorbing 732 type cation exchange resin for 3 hr, washing off water soluble impurities with 15BV distilled water at flow rate of 6 BV/hr, eluting with 15BV 95% ethanol at flow rate of 3 BV/hr, eluting with 15BV 95% ethanol containing ammonia 5% at flow rate of 3 BV/hr, collecting eluate, concentrating, and drying to obtain alcoholic extract of plumula Nelumbinis
As described above, the high performance liquid detection method for detecting the content of the alkaloid in the lotus plumule has the following beneficial effects: under the chromatographic condition, the liensinine, isoliensinine and neferine are respectivelyThe separation degrees of the peaks are all larger than 1.5, so that the peaks can be effectively separated and have good specificity; the quantitative limits of the liensinine, the isoliensinine and the neferine are 91.3 mu g, 124.7 mu g and 269.3 mu g respectively, and the result is good; the detection limits of the liensinine, the isoliensinine and the neferine are 47.1 mu g, 62.3 mu g and 135.2 mu g respectively, and the result is good; three main alkaloid components were in the respective sample ranges, (liensinine (0.0879mg to 1.465mg), isoliensinine (0.0997mg to 1.6623mg) and neferine (0.2127mg to 3.5446mg)), the linear relationship between peak area and content was good, and the correlation coefficient R for each substance was good2Are all larger than 0.998; through the results of 6 times of parallel operations, the contents RSD of the three components in each sample are 0.23%, 0.50% and 0.10%, which are both less than 2%, and the repeatability is good; the control mixed solution of the three components is placed for 12 hours at room temperature, the peak area and the content of the control mixed solution are not obviously changed, and the solution stability is good; the sample recovery rates of liensinine, isoliensinine and neferine are all within the range of 80.0-120.0%, the RSD values of the recovery rates among concentration levels are all less than 5%, and the high performance liquid phase determination method is good in accuracy; by changing the column temperature with a different column, the measurement result RSD was found to be less than 2%, and the durability was good.
Drawings
FIG. 1 is a full wavelength scan;
FIG. 2 is a chromatogram of methanol/0.1% triethylamine as eluent;
FIG. 3 is a chromatogram of acetonitrile/0.1% triethylamine as eluent;
FIG. 4 is a typical chromatogram;
FIG. 5 is a negative sample solution HPLC plot;
FIG. 6 is a HPLC chart of the test solution;
FIG. 7 is an HPLC chart of a control solution;
FIG. 8 is a limit of quantitation of liensinine, isoliensinine and neferine (LOQ) chromatogram;
FIG. 9 is a LOD (LOD) chromatogram of liensinine, isoliensinine and neferine;
FIG. 10 is a graph showing the linear relationship between liensinine;
FIG. 11 is a linear relationship diagram of isoliensinine;
FIG. 12 is a graph of the linear relationship of neferine;
FIG. 13 is a graph comparing the results of twenty-four sample measurements.
Detailed Description
The following description of the embodiments of the present invention is provided for illustrative purposes, and other advantages and effects of the present invention will become apparent to those skilled in the art from the present disclosure.
Aiming at three main active alkaloids in the lotus plumule, namely liensinine, isoliensinine and neferine, a corresponding method for measuring the content of the active alkaloids is explored and optimized, and the portions of specificity, linear range, accuracy, repeatability, intermediate precision, quantitative limit, detection limit, solution stability, method durability, system applicability and the like of the method are examined and verified respectively. 1. HPLC detection of lotus plumule alkaloid content
1.1 instruments and reagents
1.1.1 instruments
High performance liquid chromatograph: agilent 1100
A chromatographic column: agilent Extend-C18 (4.6X 250mm,5 μm)
Analytical balance: one-ten-thousandth balance Mettler-ToledoAX205
Ultrasonic instrument KQ3200E ultrasonic instrument from Kunshan city
Disintegrator lingdan LD-750A
1.1.2 reagents
| Reagent and drug name | Rank of | Manufacturer of the product |
| Water (W) | Distilled water | Watsons |
| Acetonitrile | HPLC grade | Adamas-beta |
| Triethylamine | Analytical grade | Chinese medicine |
| Methanol | HPLC grade | LiChrosolv |
| Lotus seed plumule alkali | - | Self-made |
| Isoliensinine | - | Self-made |
| Neferine | - | Self-made |
TABLE 3 reagents used in the Experimental procedures
The content of the liensinine, isoliensinine and neferine samples is over 99.5 percent after content calibration, and the liensinine, isoliensinine and neferine samples can be used as a secondary reference substance.
1.2 HPLC optimization experiment of plumula Nelumbinis alkaloid
1.2.1 selection of detection wavelength
Three alkaloids, namely liensinine, isoliensinine and neferine, are taken and scanned within the wavelength range of 200-400 nm, the full-wavelength scanning is shown in a full-wavelength scanning map as shown in figure 1, the full-wavelength scanning of the three components is similar, the maximum absorption is respectively found at 215nm and 282nm, and 282nm is selected as the detection wavelength in order to reduce the interference caused by terminal absorption.
3.3.2 Mobile phase
When methanol or acetonitrile and water are used as mobile phases for direct sample injection, the peak shape tailing of the chemical component peak of the lotus plumule is found, because the main component is alkaloid, 0.1 percent of triethylamine is added as a tail scavenging agent, the peak shape can be improved and the tailing can be inhibited.
The peak still had a tail when methanol/0.1% triethylamine was used as eluent, while the peak had a front when acetonitrile/0.1% triethylamine was used as eluent. Methanol/0.1% triethylamine as eluent is shown in figure 2 and acetonitrile/0.1% triethylamine as eluent is shown in figure 3.
Therefore, by combining the two, the abnormal peak shape can be well prevented by using methanol/acetonitrile/0.1% triethylamine as an eluent.
When a gradient of 10% -50% is adopted, the components with the retention time of 5-20 minutes can be partially separated, and the components with the retention time of 25-30 minutes have slow peak emergence;
when a gradient of 10-60% is adopted, the peak appearance of the component with the retention time of 25-30 minutes is not obviously improved compared with the peak appearance of the component with the retention time of the gradient;
when a gradient of 10-80% is adopted, the component with the retention time of 25-30 minutes is completely separated from the peak;
thus, the former gradient set at 10-50% allows for a better separation of components with retention times of 5-20 minutes, while the latter gradient is set at 80% allowing for a faster separation of components with retention times of 25-30 minutes.
3.3.3 chromatographic conditions
A chromatographic column: agilent extended-C18 (4.6X 250mm,5 μm); mobile phase: water-triethylamine (1000: 1)/acetonitrile-methanol (1: 1); the flow rate is 1 ml/min; detection wavelength: 282 nm; the column temperature is 30 ℃, and under the chromatographic condition, the theoretical plate numbers of the liensinine, isoliensinine and neferine are all more than 5000. It was found that the measurement effect was good under the chromatographic conditions. The separation degrees of the three components are all larger than 1.5, and the separation degrees meet the requirements according to the measurement and calculation results.
TABLE 4 chromatographic conditions for typical chromatograms
A typical chromatogram obtained under the chromatographic conditions is shown in FIG. 4, under the chromatographic conditions, the theoretical plate numbers of the three components of liensinine, isoliensinine and neferine are 307510,283372 and 189676 respectively, the separation degree is-/10.91/7.79, and the separation degrees are all more than 1.5.
1.3 method for extracting alkaloid from lotus plumule
According to literature reports, the chemical structures of the three alkaloids are not very stable and are easy to generate oxidation reaction, the structures are damaged in the heating reflux extraction process, and the extraction efficiency is lower than that of ultrasonic extraction, so that the experiment decides that the ultrasonic extraction is the first choice, but the ultrasonic extraction is not the only choice, and the cation resin can be used for separation, purification and extraction.
The cation resin separation, purification and extraction are as follows: taking 100 parts by weight of lotus plumule, performing reflux extraction for 2-3 times by using 50-95% ethanol with the mass of 4-6 times that of the lotus plumule for 1-2 hours each time, filtering, combining filtrates, concentrating under reduced pressure to 1.5-2.5 times that of the lotus plumule to obtain an alcohol extract, filling 100 parts by weight of 732 type cation exchange resin into a glass column with the inner diameter of 60mm, adding the alcohol extract, fully adsorbing the 732 type cation exchange resin for 2-3 hours, washing off water-soluble impurities by using 10-15 BV of distilled water at the flow rate of 4-6 BV/h, eluting by using 10-15 BV of 95% ethanol at the flow rate of 2-3 BV/h, eluting by using 10-15 BV of 95% ethanol containing 5% of ammonia water at the flow rate of 2-3 BV/h, collecting eluent, concentrating and drying to obtain the alcohol extract of the lotus plumule.
1.3.1 selection of extraction solvent
The lotus plumule of the same batch is 200g, crushed and sieved by a 20-mesh sieve. 500mg of the powder was taken and placed in a 25mL volumetric flask, and methanol, 70% aqueous methanol solution, 50% aqueous methanol solution, 30% aqueous methanol solution and 10% aqueous methanol solution were added, respectively. Performing ultrasonic treatment for 30min, standing at room temperature for 30min, and metering to 25 mL. Filtering with 0.45 micrometer microporous membrane, and collecting filtrate as sample solution.
TABLE 5 examination of extraction solvent
As a result: the comparison shows that under the same chromatographic conditions, pure methanol is used as an extraction solvent, and the chromatographic peak areas of three main peaks in the lotus plumule are larger than the chromatographic peak areas of the extraction result of a mixed solution of pure methanol and water, so that the selection of methanol as the extraction solvent is more suitable from the judgment of the extraction effect.
1.3.2 examination of extraction time
Taking 500mg of lotus plumule powder of the same batch into a 25mL volumetric flask, adding a methanol solution, and respectively placing in an ultrasonic instrument for 5min, 15min, 35min, 45min and 60 min. Standing at room temperature for 30min, and metering to 25 mL. Filtering with 0.45 micrometer microporous membrane, and collecting filtrate as sample solution.
TABLE 6 extraction of time finding results
Experiments show that when the ultrasonic time is 30min, the chromatographic peak area of the liensinine is maximized, and when the time is continuously prolonged, the peak area is reduced to a certain extent. The extraction rate of isoliensinine and neferine is slightly reduced after the ultrasonic treatment time is prolonged, so that three effective components can be maximally reserved by selecting 30min for ultrasonic treatment time.
As a result: the comparison shows that the crushed lotus plumule sample is extracted most completely by taking methanol as an extraction solvent and ultrasonic time of 30min, and the content of the three components is the maximum. When the time is prolonged, the effect is rather reduced. Therefore, it is suitable to select 30min as the extraction time.
1.3.3 method for preparing test solution
In order to extract the three effective components of the lotus plumule to the maximum degree, the extraction conditions selected in the experiment are methanol as an extraction solvent, and the ultrasonic treatment is carried out for 30 min.
The lotus plumule of 200g is taken, rapidly crushed and sieved by a 20-mesh sieve. Putting 500mg of the powder into a 25mL volumetric flask, respectively adding methanol, performing ultrasonic treatment for 30min, standing at room temperature for 30min, and metering to 25 mL. Filtering with 0.45 micrometer microporous membrane, and collecting filtrate as sample solution.
1.4 verification of Alkaloids of Lotus plumule analysis methodology
1.4.1 chromatographic conditions
Table 7 chromatographic conditions validated by analytical methodology
1.4.2 specificity
Precisely weighing 17.31mg of liensinine, 25.28mg of isoliensinine and 27.56mg of neferine as reference substances, respectively placing the reference substances into different 25mL volumetric flasks, adding methanol for ultrasonic dissolution, and fixing the volume to the scale. And respectively sucking 1mL of the liensinine alkali solution, 1mL of the isoliensinine and 2mL to 10mL of the neferine into volumetric flasks by using a pipette, uniformly mixing, and fixing the volume to the scale. As a mixed solution of three controls. The HPLC chart of the negative sample solution is shown in FIG. 5, that of the test sample solution is shown in FIG. 6, that of the control sample solution is shown in FIG. 7, and the retention time of the plumula Nelumbinis alkaloids is shown in Table 8.
| Retention time (min) | Degree of separation | Number of theoretical plate | |
| Lotus seed plumule alkali | 26.785 | -- | 307510 |
| Isoliensinine | 29.028 | 10.91 | 283372 |
| Neferine | 30.979 | 7.79 | 189676 |
TABLE 8 Lotus plumule alkaloid retention time
As a result: under the chromatographic condition, the solvent blank is free from interference, the separation degree of other impurity peaks and three components to be detected is far more than 1.5, and the method has good specificity.
1.4.3 detection and quantitation limits
Taking a proper amount of liensinine, isoliensinine and neferine reference substances to prepare solutions with a series of concentrations, respectively measuring the solutions, injecting 20 mu L of injection sample for analysis, and taking the detection Limit (LOD) when S/N is approximately equal to 3;
taking a proper amount of liensinine, isoliensinine and neferine reference substances to prepare solutions with a series of concentrations, respectively measuring the solutions, injecting 20 mu L of sample for analysis, and taking the sample as a quantitative Limit (LOQ) when S/N is approximately equal to 10;
| LOD(μg) | LOQ(μg) | |
| lotus seed plumule alkali | 47.1 | 91.3 |
| Isoliensinine | 62.3 | 124.7 |
| Neferine | 135.2 | 269.3 |
TABLE 9 quantitation Limit (LOQ) and detection Limit (LOD) results for liensinine, isoliensinine and neferine
FIG. 8 is a limit of quantitation (LOQ) spectrum of liensinine, isoliensinine and neferine; FIG. 9 shows the LOD spectra of liensinine, isoliensinine and neferine. The results show that the detection limit and the quantification limit are far lower than the concentration of the test sample, and the results are good.
1.4.4 Linear Range inspection
Precisely measuring 5mL of the mixed solution of the reference substance, placing the mixed solution into a 10mL measuring flask, adding the diluent to dilute to a scale, shaking up, and obtaining a series of solutions to be measured by a stepwise dilution method.
Injecting 20 μ L of each sample solution with different concentrations into a chromatograph, injecting sample 1 time per concentration at 282nm wavelength, taking the sample amount (μ g) of each component as abscissa, taking the chromatographic peak area of the three components obtained under the same chromatographic condition as ordinate, performing linear regression respectively, and calculating respective correlation coefficient. The results are as follows:
| plumula Nelumbinis alkali sample amount (mg) | 0.0879 | 0.1758 | 0.3663 | 0.7327 | 1.4654 |
| Peak area | 65.4 | 130.1 | 271.6 | 545.2 | 1078.2 |
| Isoliensinine sample amount (mg) | 0.0997 | 0.1995 | 0.4156 | 0.8311 | 1.6623 |
| Peak area | 85.5 | 174.1 | 365.5 | 734.0 | 1470.4 |
| Mesostigmine sample size (mg) | 0.2127 | 0.4254 | 0.8862 | 1.7723 | 3.5446 |
| Peak area | 182.1 | 365.4 | 764.7 | 1531.3 | 3061.4 |
TABLE 10 results of the Linear relationship experiment
FIG. 10 is a linear relationship of liensinine, regression equation of liensinine: y 735.74x +1.9491, correlation coefficient: r21.0000; FIG. 11 is a linear relationship of isoliensinine, regression equation: 886.27x-2.7646, correlation coefficient: r21.0000; FIG. 12 is a linear relationship of neferine, regression equation: 864.27x-1.5460, correlation coefficient: r21.0000. As a result: within the above range, the chromatographic contents of liensinine (0.0879mg to 1.4654mg), isoliensinine (0.0997mg to 1.6623mg) and neferine (0.2127mg to 3.5446mg) have a good linear relationship with the amount of sample.
1.4.5 accuracy
Preparing a reference substance solution: precisely weighing 29.05mg of liensinine, 21.15mg of isoliensinine and 52.10mg of neferine serving as reference substances, respectively placing the reference substances into different 5mL volumetric flasks, adding methanol for ultrasonic dissolution, and fixing the volume to the scale. And respectively sucking 1mL of the liensinine alkali solution, 1mL of the isoliensinine and 2mL to 10mL of the neferine into volumetric flasks by using a pipette, uniformly mixing, and fixing the volume to the scale. As a mixed solution of three controls.
Recovery was determined using standard addition methods. Precisely weighing about 250mg of lotus plumule sample, totally 6 parts, respectively placing into 25mL conical bottles with stoppers, respectively adding 1mL of reference solution prepared according to the above method into each part, precisely weighing, preparing a sample solution according to the preparation method of the sample, injecting 20 muL of sample under the condition of content measurement chromatography, recording peak area, calculating recovery rate by using an external standard method, and obtaining the results shown in tables 11, 12 and 13.
Percent recovery [ (C-A)/B [ (] 100%) ]
Wherein A is the content of the target substance contained in the test sample, B is the amount of the added reference substance, and C is the measured value;
TABLE 11 liensinine sample recovery rate measurement results
TABLE 12 isoliensinine sample recovery rate measurement results
TABLE 13 measurement results of the sample recovery of neferine
As a result: from the average recovery of the three components and their RSD, RSD < 3% indicates good accuracy.
1.4.6 repeatability test
The lotus plumule of 200g is taken, rapidly crushed and sieved by a 20-mesh sieve. Putting 500mg of the powder into a 25mL volumetric flask, respectively adding methanol, performing ultrasonic treatment for 30min, standing at room temperature for 30min, and metering to 25 mL. Filtering with 0.45 micrometer microporous membrane, and collecting filtrate as sample solution. Preparing 6 parts in parallel, precisely measuring 20 mu L of sample injection and analyzing, recording peak area and calculating content. Calculating the content of three alkaloid components to obtain RSD. The results are shown in Table 14.
TABLE 14 results of repeated experiments
1.4.7 stability test
Taking lotus plumule reference substance solution, preparing sample solution according to the method under the content determination item in the text, respectively determining peak areas within 0,2, 4, 6, 8 and 12 hours, and the result is shown in Table 15.
| Sample introduction time (h) | 0 | 2 | 4 | 6 | 8 | 12 | RSD％ |
| Area of plumula Nelumbinis alkali peak | 1074.0 | 1073.5 | 1075.9 | 1079.2 | 1083.6 | 1080.1 | 0.37 |
| Isoliensinine peak area | 1470.3 | 1470.2 | 1473.3 | 1473.6 | 1475.7 | 1469.8 | 0.16 |
| Area of neferine peak | 3055.3 | 3054.6 | 3062.2 | 3068.2 | 3077.7 | 3071.4 | 0.30 |
TABLE 15 results of stability experiments
As a result: the change of the chromatographic peak area is very small and does not have a decreasing trend after the liensinine, isoliensinine and neferine are kept stand for 0,2, 4, 6, 8 and 12 hours in a methanol solution at room temperature. Three components RSD < 2%, indicating that the samples were stable within 0-12 hours.
1.4.8 durability
(1) Comparison of different chromatography columns
Table 16 results of different chromatographic column tests
(2) Comparison of different column temperatures
TABLE 17 test results at different column temperatures
As a result: the analysis method can also be used for measuring the content of liensinine, isoliensinine and neferine in the lotus plumule sample when the temperature of different chromatographic columns and the same chromatographic column fluctuates within the range.
1.4.9 summary of analytical method validation
The HPLC method is established and optimized for three main alkaloid components in the lotus plumule, namely liensinine, isoliensinine and neferine, so that the three components can be well separated, and peaks of other components with low content can be reduced and overlapped as much as possible by the method. Has reference significance for follow-up research.
The high performance liquid chromatography detection method for detecting the content of the lotus plumule alkaloid has the advantages of specificity, linear range, accuracy, stability and good repeatability, can be used for quantitative analysis, and has good durability.
The results of the methodological validation of the above items are shown in table 18:
table 18 method verification results
2. On-site investigation of main active alkali components of lotus plumule
2.1 materials of the experiment
Lotus plumule samples purchased from lotus seed producing areas. Respectively selected from Jiangsu Suqian, Jiangxi Guangchang, Hunan quan, Anhui Huangshan, Hunan Yueyang, Guangxi Guigang, Zhejiang Lishui, Zhejiang Hangzhou, Shandong Jinan, Shandong Jinning, Zhejiang Wuyi, Zhejiang Jianshan, Hubei Honghu, Fujian Jianyang, Shandong Weishan lake, Henan Yang, Hubei Jindian, Hunan Qidong, Jiangsu Hongze lake, Jiangxi Shicheng, Zhejiang Longyou, Chongqing Shizhu, Fujiangning and Anhui Tuhu 24 producing areas, and all the materials are lotus seed crude cores which are not processed.
2.2 Experimental methods
And (3) taking 200g of lotus plumule of the same batch, quickly crushing, and sieving by a 20-mesh sieve. Putting 500mg of the powder into a 25mL volumetric flask, respectively adding methanol, performing ultrasonic treatment for 30min, standing at room temperature for 30min, and then fixing the volume to 25 mL. Filtering with 0.45 micrometer microporous membrane, and collecting filtrate as sample solution.
With the above-identified chromatographic method, 20 μ L of the sample solution was separately injected, and its peak area was determined by external standard method, and the respective contents of the three components were calculated.
2.3 results and conclusions of the experiment
Purchased from: jiangsu Suqian, Jiangxi Guangchang, Hunan quan, Anhui Huangshan, Hunan Yueyang, Guangxi Guigang, Zhejiang Lishui, Zhejiang Hangzhou, Shandong Jinan, Shandong Jining, Zhejiang Wuyi, Zhejiang Jianshan, Hubei Honghu, Fujian Jianyang, Shandong Weishan lake, Henan Yang, Hubei Jindian, Hunan Qidong, Jiangsu Hongze lake, Jiangxi Shicheng, Zhejiang Longyou, Chongqing Shi, Fujiangjiangning, Anhui Genghu lake, 24-production-place lotus plumes. The test results are shown in table 19 and fig. 13.
| Numbering | Producing area | Liensinine (mg) | Isoliensinine (mg) | Neferine (mg) | Total alkaloid (mg) |
| 1 | Persistent branches of Jiangsu | 0.395 | 1.319 | 4.668 | 6.382 |
| 2 | Guangchang in Jiangxi province | 1.001 | 1.476 | 4.457 | 6.934 |
| 3 | Hunan pond | 0.446 | 1.433 | 3.956 | 5.835 |
| 4 | Huangshan mountain of Anhui province | 0.425 | 1.127 | 3.985 | 5.537 |
| 5 | Yueyang lake | 0.527 | 1.390 | 4.605 | 6.522 |
| 6 | Guangxi noble harbor | 0.360 | 1.117 | 3.576 | 5.053 |
| 7 | Zhejiang Lishui | 0.441 | 1.448 | 4.512 | 6.401 |
| 8 | Hangzhou Zhejiang province | 0.580 | 0.861 | 3.291 | 4.732 |
| 9 | Shandong Jinan | 0.399 | 1.241 | 4.194 | 5.834 |
| 10 | Shandong Jining (Chinese character of Shandong) | 0.614 | 1.397 | 4.313 | 6.324 |
| 11 | Wuyi Zhejiang province | 1.271 | 1.227 | 4.592 | 7.09 |
| 12 | Jiang mountain of Zhejiang river | 0.312 | 1.638 | 3.351 | 5.301 |
| 13 | Flood lake in North Hu lake | 0.771 | 1.427 | 4.173 | 6.371 |
| 14 | Fujian Jianyang | 0.376 | 2.053 | 3.450 | 5.879 |
| 15 | Weishan lake of Shandong province | 0.573 | 0.997 | 3.811 | 5.381 |
| 16 | South of Henan Yangyang | 0.397 | 1.257 | 4.038 | 5.692 |
| 17 | Classical of Hubei province | 1.161 | 1.556 | 6.372 | 9.089 |
| 18 | Hunan Qidong | 0.782 | 1.137 | 6.639 | 8.558 |
| 19 | Hongze lake of Jiangsu | 0.255 | 1.495 | 5.962 | 7.712 |
| 20 | Jiangxi stone city | 0.773 | 1.755 | 4.844 | 7.372 |
| 21 | Dragon tour of Zhejiang | 0.371 | 2.753 | 2.686 | 5.81 |
| 22 | Chongqing stone congratulation | 0.510 | 1.262 | 4.239 | 6.011 |
| 23 | Anhui turnip lake | 0.394 | 1.580 | 4.086 | 6.06 |
| 24 | Fujian Jianning medicine | 1.071 | 0.720 | 3.603 | 5.394 |
TABLE 19 Twenty-four sample measurements
Wherein the content is alkaloid content (calculated by milligram) corresponding to 500mg lotus plumule powder, and the total alkaloid is calculated according to the sum of the three alkaloid contents.
As can be seen from the measurement results, the average value of the liensinine content from the 24 production places is 0.592mg/500mg, the isoliensinine content is 1.403mg/500mg, the neferine content is 4.308mg/500mg, and the total alkaloid of the three contents is 6.303mg/500 mg.
From the chemical structures of the three alkaloids, the structures of liensinine, isoliensinine and neferine are similar, and the main pharmacological actions of the liensinine and the isoliensinine are also similar. Therefore, the total alkaloid selected from the three components is more reasonable. The sum of neferine and three alkaloids in the lotus plumule sample from golden classic in Hubei is relatively high.
The content of the main alkaloid in the lotus plumule samples of different producing areas is measured by an HPLC quantitative method, and the difference of the three active ingredients in the lotus plumules of different producing areas can be found to be very large. From the data, it can be seen that the content of neferine is much higher than that of the other two alkaloids, which can be several times higher, and should be related to the relative stability of the structure.
In conclusion, the high performance liquid detection method for detecting the content of the lotus plumule alkaloid provided by the invention has the advantages of specificity, linear range, accuracy, stability and good repeatability, can be used for quantitative analysis, and is good in durability. Therefore, the invention effectively overcomes various defects in the prior art and has high industrial utilization value.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.
Claims (8)
1. A high performance liquid detection method for detecting the content of alkaloid in lotus plumule is characterized by comprising the following steps:
respectively preparing a proper amount of a liensinine reference substance, an isoliensinine reference substance and a neferine reference substance into a series of liensinine reference substance solutions with concentration, an isoliensinine reference substance solution and a neferine reference substance solution, wherein the volume ratio of an extend-C18 chromatographic column, water and triethylamine as a mobile phase A is 1000: (0.9-1.1) the volume ratio of the mixed solution and the mobile phase B is 1: (0.99-1.01) respectively measuring a liensinine reference substance solution, an isoliensinine reference substance solution and a neferine reference substance solution with different concentrations under the chromatographic conditions that the flow rate is 1ml/min, the detection wavelength is 282nm, the column temperature is 28-32 ℃ and the sample injection amount is 20 mu L, carrying out high performance liquid phase detection, wherein the detection limit is set when S/N is approximately equal to 3, and the quantification limit is set when S/N is approximately equal to 10;
step two, preparing the ethanol extract of the lotus plumule into a sample solution to be tested, wherein the volume ratio of water to triethylamine in a chromatographic column of an extended-C18 chromatographic column and a mobile phase A is 1000: (0.9-1.1) the volume ratio of the mixed solution and the mobile phase B is 1: (0.99-1.01), performing high performance liquid detection on the mixed solution under the chromatographic conditions of a flow rate of 1ml/min, a detection wavelength of 282nm, a column temperature of 28-32 ℃ and a sample injection amount of 20 mu L, and calculating the content of liensinine, isoliensinine and neferine in the sample solution through peak areas.
2. The high performance liquid chromatography detection method for detecting the content of the alkaloids in the lotus plumule as claimed in claim 1, wherein the detection method comprises the following steps: the volume ratio of the mobile phase A and the mobile phase B in the elution process in the first step and the second step is specifically shown in Table 1:
Table 1 volume ratio of mobile phase a and mobile phase B.
3. The high performance liquid chromatography detection method for detecting the content of the alkaloids in the lotus plumule as claimed in claim 1 or 2, wherein the detection method comprises the following steps: in the first step and the second step, the mobile phase A is water and triethylamine, and the volume ratio of the water to the triethylamine is 1000: 1, and the mobile phase B is a mixed solution of acetonitrile and methanol in a volume ratio of 1: 1.
4. The high performance liquid chromatography detection method for detecting the content of the alkaloids in the lotus plumule as claimed in claim 1 or 2, wherein the detection method comprises the following steps: the column temperature of the chromatographic column in the first step and the second step is 30 ℃, the length of the chromatographic column is 250mm, the inner diameter of the chromatographic column is 4.6mm, and the diameter of the chromatographic column packing particles is 5 mu m.
5. The high performance liquid chromatography detection method for detecting the content of the alkaloids in the lotus plumule as claimed in claim 1 or 2, wherein the detection method comprises the following steps: the preparation process of the alcohol extract of lotus plumule in the second step is as follows: crushing a lotus plumule sample, sieving the crushed lotus plumule sample with a 20-mesh sieve, placing 499-501 mg of lotus plumule powder into a 25mL volumetric flask, adding 70-100% by mass of methanol, performing ultrasonic treatment for 25-35 min, cooling to room temperature, standing for at least 30min, metering the volume to 25mL by using 70-100% by mass of methanol, filtering by using a 0.45-micron microporous filter membrane, and taking a subsequent filtrate to obtain the lotus plumule alcohol extract.
6. The high performance liquid chromatography detection method for detecting the content of the alkaloids in the lotus plumule as claimed in claim 5, wherein the detection method comprises the following steps: the preparation process of the alcohol extract of lotus plumule in the second step is as follows: crushing a lotus plumule sample, sieving the crushed lotus plumule sample with a 20-mesh sieve, placing 500mg of lotus plumule powder in a 25mL volumetric flask, adding methanol, performing ultrasonic treatment for 30min, cooling to room temperature, standing for 30min, metering volume to 25mL by using methanol, filtering by using a 0.45-micron microporous filter membrane, and taking a subsequent filtrate, namely the lotus plumule alcohol extract.
7. The high performance liquid chromatography detection method for detecting the content of the alkaloids in the lotus plumule as claimed in claim 1 or 2, wherein the detection method comprises the following steps: the preparation process of the alcohol extract of lotus plumule in the second step is as follows: taking 100 parts by weight of lotus plumule, performing reflux extraction for 2-3 times by using 50-95% ethanol with the mass of 4-6 times that of the lotus plumule for 1-2 hours each time, filtering, combining filtrates, concentrating under reduced pressure to 1.5-2.5 times that of the lotus plumule to obtain an alcohol extract, filling 100 parts by weight of 732 type cation exchange resin into a glass column with the inner diameter of 60mm, adding the alcohol extract, fully adsorbing the 732 type cation exchange resin for 2-3 hours, washing off water-soluble impurities by using 10-15 BV of distilled water at the flow rate of 4-6 BV/h, eluting by using 10-15 BV of 95% ethanol at the flow rate of 2-3 BV/h, eluting by using 10-15 BV of 95% ethanol containing 5% of ammonia water at the flow rate of 2-3 BV/h, collecting eluent, concentrating and drying to obtain the alcohol extract of the lotus plumule.
8. The high performance liquid chromatography detection method for detecting the content of the alkaloids in the lotus plumule as claimed in claim 7, wherein the detection method comprises the following steps: the preparation process of the alcohol extract of lotus plumule in the second step is as follows: taking 100 parts by weight of lotus plumule, carrying out reflux extraction for 3 times by using 95% ethanol with the mass 6 times that of the lotus plumule, each time for 1 hour, filtering, combining filtrates, concentrating under reduced pressure to 2.0 times that of the lotus plumule to obtain an alcohol extract, filling 100 parts by weight of 732 type cation exchange resin into a glass column with the inner diameter of 60mm, adding the alcohol extract, fully adsorbing the 732 type cation exchange resin for 3 hours, washing off water-soluble impurities by using 15BV of distilled water at the flow rate of 6BV/h, eluting by using 15BV of 95% ethanol at the flow rate of 3BV/h, eluting by using 15BV of 95% ethanol containing 5% of ammonia water at the flow rate of 3BV/h, collecting an eluent, concentrating and drying to obtain the alcohol extract of the lotus plumule.
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