CN113759009B - Method for constructing characteristic spectrum of equisetum hiemale and preparation thereof - Google Patents
Method for constructing characteristic spectrum of equisetum hiemale and preparation thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of drug detection, and particularly relates to a construction method of a characteristic spectrum of a horsetail and a preparation thereof. The method adopts high performance liquid chromatography to measure a sample to be detected of the equisetum hiemale, so as to obtain a characteristic map of the equisetum hiemale. The method uses the mixed solution of methanol and hydrochloric acid as a solvent for preparing the test solution, greatly simplifies the preparation method of the test solution, has simple and convenient operation, shortens the preparation time of the test solution, establishes the optimal elution condition and shortens the detection time of the characteristic spectrum of the equisetum hiemale and the preparation thereof; the method for constructing the characteristic spectrum of the equisetum hiemale and the preparation thereof has the advantages of good peak shape and separation degree of the characteristic peak, good repeatability of the characteristic peak, and better separation efficiency, peak capacity and sensitivity. The method has the advantages of short analysis time, high working efficiency, simplicity, stability, high precision, good repeatability, science and reliability.
Description
Technical Field
The invention belongs to the technical field of drug detection, and particularly relates to a method for constructing a characteristic spectrum of equisetum hiemale and a preparation thereof.
Background
The horsetail is one of the traditional Chinese medicines, is the dry overground part of Equisetum hyemale L. of Equisetum, and has light smell, sweet and light taste, slight astringent taste and sand feeling when being chewed; sweet, bitter and neutral in nature; it enters lung and liver meridians. According to traditional medicine, the horsetail has the effects of dispelling wind and heat, improving eyesight and removing nebula, is used for treating conjunctival congestion due to wind heat, epiphora induced by wind and nebula generated by eyes, and is a variety collected from the calendar of pharmacopoeia of the people's republic of China.
At present, the forms of administration of the horsetail on the market comprise horsetail decoction pieces, horsetail standard decoction, horsetail formula granules, horsetail-containing compound medicines and the like. The lyophilized powder is prepared by decocting herba Equiseti hiemalis decoction pieces in water twice, filtering while hot, mixing filtrates, concentrating under reduced pressure, and lyophilizing. The herba Equiseti hiemalis granule is prepared by decocting herba Equiseti hiemalis decoction pieces in water twice, filtering, mixing filtrates, concentrating under reduced pressure, spray drying, adding adjuvants, and granulating.
In recent years, medical workers at home and abroad research horsetails in many aspects, only a liquid phase analysis method of HPLC is adopted to stipulate and limit the content of kaempferide in horsetails in standard identification of horsetails under the item of the national pharmacopoeia 2015 edition, the preparation process of a test sample is complicated, the measured components are single, the exertion of the drug effect of traditional Chinese medicine is the result of the interaction of various components according to the theory of traditional Chinese medicine, and the quality control of only one component is limited. Traditional Chinese medicines and preparations are all multi-component complex systems, which cannot comprehensively reflect the types and the amounts of various chemical components in the horsetail products, and cannot describe and evaluate the overall quality of the horsetail products.
' HPLC fingerprint and quality evaluation research of Equisetum hiemale (Equisetum hiemale) published by Wangxia et al, fingerprint analysis is carried out on Equisetum hiemale, but the method has longer analysis time and low working efficiency, the preparation of a test solution is more complicated, the separation degree of the obtained spectrum is poor, and the method is not suitable for detection of other preparation types.
The method discloses a method for measuring the fingerprint spectrum of an aqueous extract of equisetum hiemale under the standard item of the equisetum hiemale medicinal materials, which totally identifies 3 characteristic peaks, and defines the relative retention time of other two characteristic peaks by taking kaempferol-3-beta-sophoroside as a reference peak. Although the method can shorten the preparation method of the equisetum hiemale test solution, the obtained fingerprint has less characteristic peak information, relatively weaker specificity for basic expression of the integral substances of the equisetum hiemale, poor separation degree of the characteristic peaks, and long time consumption for measuring the characteristic spectrum of the equisetum hiemale, and cannot well meet the requirements of quick detection and product quality control of the equisetum hiemale sample.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is to overcome the defects that the determination of the characteristic spectrum of the horsetail and the preparation thereof in the prior art is long in time consumption and low in efficiency, the preparation method of the test solution is complex, the preparation time of the test solution cannot be considered, the separation degree of the characteristic spectrum is good, and the like, so that the construction method of the characteristic spectrum of the horsetail and the preparation thereof is provided.
Therefore, the invention provides the following technical scheme.
The invention provides a method for constructing a characteristic map of a horsetail and a preparation thereof, which comprises the following steps,
preparation of a test solution: taking a sample to be detected of the horsetail, adding a mixed solvent of methanol and a hydrochloric acid solution, extracting, filtering, and taking a subsequent filtrate to obtain the horsetail extract;
the kaempferide is used as a reference substance of a reference substance, and a characteristic spectrum of the equisetum hiemale and the preparation thereof is obtained by adopting a high performance liquid chromatography.
The chromatographic conditions of the high performance liquid chromatography are as follows: using an octadecylsilane bonded silica gel column as a chromatographic column; using acetonitrile as mobile phase A, using formic acid with volume fraction of 0.05-0.4% as mobile phase B, gradient eluting at flow rate of 0.2-0.4ml/min, detection wavelength of 305-320nm, and column temperature of 33-36 deg.C;
the procedure for gradient elution includes: 0-3.2min, mobile phase A: the volume ratio of the mobile phase B is 5-19%:95-81%;3.2-10min, mobile phase A: the volume ratio of the mobile phase B is 19-25%:81-75 percent; 10-16min, mobile phase A: the volume ratio of the mobile phase B is 25-74%:75-26%;16-18min, mobile phase A: the volume ratio of the mobile phase B is 74-100%:26 to 0 percent.
The preparation method of the reference substance solution of the reference substance comprises the following steps: adding kaempferol into alcohol solvent to obtain reference substance solution of reference substance;
the preparation method of the reference solution of the reference medicinal materials comprises the following steps: taking a herba Equiseti hiemalis reference medicinal material, adding a mixed solvent of 75% methanol and hydrochloric acid solution by volume fraction, extracting for 0.7-1.3h, filtering, and taking filtrate to obtain a reference substance solution of the reference medicinal material;
preparation of a test solution: taking a sample to be detected of the horsetail, adding a mixed solvent of 75% methanol and hydrochloric acid solution by volume fraction, extracting for 0.7-1.3h, filtering, and taking a subsequent filtrate to obtain the horsetail extract.
The characteristic map of the equisetum hiemale and the preparation thereof comprises 8 characteristic peaks, wherein the No. 8 peak is taken as a reference peak, the relative retention time of other characteristic peaks is within +/-10% of a specified value, and the specified values of the relative retention time are respectively as follows:
peak No. 1 had a relative retention time of 0.12; peak No. 2 had a relative retention time of 0.24; peak No. 3 had a relative retention time of 0.44; peak No. 4 had a relative retention time of 0.55; peak 5 had a relative retention time of 0.60; peak No. 6 had a relative retention time of 0.81; peak 7 had a relative retention time of 0.86;
wherein, the peak No. 8 is a kaempferol peak.
The volume ratio of the methanol to the hydrochloric acid is (3-5): 1;
the mass fraction of the hydrochloric acid solution is 36-38%;
the concentration of the test solution is 2.5-25mg/ml.
When the sample to be detected of the horsetail is a horsetail decoction piece or a horsetail medicinal material, the concentration of the test solution is 18-25mg/ml;
when the sample to be tested is lyophilized powder of herba Equiseti hiemalis standard decoction or herba Equiseti hiemalis granule, the concentration of the test solution is 2.5-18mg/ml.
The preparation method of the test solution comprises the specific steps of taking a sample to be tested of the horsetail, grinding, precisely weighing, adding a mixed solvent of 75% methanol and hydrochloric acid solution by volume fraction, heating and refluxing for extraction for 1h, filtering, and taking a subsequent filtrate to obtain the test solution;
the reference substance solution of the reference substance is prepared by precisely weighing kaempferol and adding methanol with volume fraction of 75%;
the reference solution of the reference medicinal material is prepared by grinding herba Equiseti hiemalis, precisely weighing, adding mixed solvent of 75% methanol and hydrochloric acid solution, reflux-extracting under heating for 1 hr, filtering, and collecting filtrate.
The sample to be detected of the horsetail is a horsetail decoction piece, a horsetail medicinal material, horsetail standard decoction freeze-dried powder or horsetail granules.
The concentration of the reference substance solution of the reference substance is 18-23 mug/ml;
the concentration of the reference solution of the reference medicinal material is 15-25mg/ml.
The chromatographic conditions are as follows: using an octadecylsilane bonded silica gel column as a chromatographic column; acetonitrile is used as a mobile phase A, formic acid with volume fraction of 0.1% is used as a mobile phase B, gradient elution is carried out, the flow rate is 0.3ml/min, the detection wavelength is 310nm, and the column temperature is 35 ℃.
The technical scheme of the invention has the following advantages:
1. the method for constructing the characteristic spectrum of the equisetum hiemale and the preparation thereof adopts the high performance liquid chromatography to measure a sample to be detected of the equisetum hiemale to obtain the characteristic spectrum of the equisetum hiemale. The method uses the mixed solution of methanol and hydrochloric acid as a solvent for preparing the test solution, greatly simplifies the preparation method of the test solution, is simple and convenient to operate, can simultaneously realize the extraction and hydrolysis of active ingredients in the horsetail by adding the mixed solvent of methanol and hydrochloric acid as an extraction solvent, and obviously shortens the preparation time of the test solution; the method for constructing the characteristic spectrum of the equisetum hiemale and the preparation thereof has the advantages that the obtained characteristic peak has good peak shape and separation degree, good repeatability, better separation efficiency, peak capacity and sensitivity; the method has the advantages of short analysis time, high working efficiency, simplicity, stability, high precision and good repeatability, is scientific and reliable, can be suitable for different samples to be detected of the equisetum hiemale, and can be used for quickly realizing the quality control of the equisetum hiemale, wherein the samples to be detected of the equisetum hiemale can be medicinal materials, decoction pieces, standard decoction freeze-dried powder or granules and the like.
According to the method, the hydrochloric acid is added in the preparation of the test solution, so that the glycoside component is hydrolyzed and converted into aglycone, the characteristic peak number of the characteristic spectrum obtained by using the test solution is large, the peak response value and the separation effect are good, the specificity of the equisetum hiemale can be better represented, the quality of the equisetum hiemale can be integrally evaluated and described, and the quality of the equisetum hiemale can be effectively controlled.
2. According to the construction method of the characteristic spectrum of the equisetum hiemale and the preparation thereof, the extraction time of the test solution can be shortened to 1h, the elution gradient time can be shortened to within 20min, the analysis time is greatly shortened, the working efficiency is improved, and the labor and material costs are saved.
The characteristic spectrum obtained by the invention takes the No. 8 peak as a reference peak, the No. 8 peak is identified as a kaempferide peak through a reference substance, the characteristic spectrum is a representative peak of aglycone active ingredients in the equisetum hiemale, the separation is good, the area is moderate, and the accuracy of the quality control of the equisetum hiemale can be realized in a qualitative and quantitative mode by taking the characteristic spectrum as the reference. The 8 characteristic peaks determined in the characteristic map are stable common peaks of 18 equisetum hiemale in different producing areas and batches, the accuracy of the method applied to the aspect of quality control of the equisetum hiemale can be well guaranteed, the 8 characteristic peaks are good in peak shape, the peak separation effect and the peak symmetry are good, the retention time and the relative retention time of the characteristic peaks can be well controlled, and the reproducibility and the applicability of the method are greatly improved.
The method establishes the optimal elution condition, shortens the detection time of the characteristic spectrum of the equisetum hiemalis and the preparation thereof, can realize good separation of 8 characteristic peaks by the elution gradient, and has the advantages of stable baseline of the obtained characteristic spectrum, small noise interference, good peak shape of the characteristic peak, high separation degree, short analysis time and high separation efficiency.
The construction method of the characteristic map of the equisetum hiemale and the preparation thereof provided by the invention is simple and convenient, the time consumption is short, and the determination result is accurate.
3. The construction method of the characteristic spectrum of the equisetum hiemale and the preparation thereof can meet the requirements of high speed, high separation degree and high sensitivity.
The method can better represent the characteristic peaks of the integral substance components in the equisetum hiemale by controlling the chromatographic conditions, thereby realizing the quality control of equisetum hiemale products according to the relative retention time of each characteristic peak.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the prior art descriptions will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a characteristic spectrum of 18 batches of equisetum hiemale standard decoction freeze-dried powder in example 1 of the present invention;
FIG. 2 is a characteristic spectrum of a kaempferol reference substance in example 1 of the present invention;
FIG. 3 is a comparison feature map generated from 18 batches of equisetum hiemale standard decoction lyophilized powder in example 1 of the present invention;
FIG. 4 is a full wavelength scanning three-dimensional chromatogram of Experimental example 1 of the present invention;
FIG. 5 is a characteristic map of experimental group 1 in example 2 of the present invention;
FIG. 6 is a characteristic map of control group 1 in Experimental example 2 of the present invention;
FIG. 7 is a characteristic map of control group 2 in Experimental example 2 of the invention;
FIG. 8 is a characteristic map of a control group 3 in Experimental example 2 of the present invention;
FIG. 9 is a characteristic map obtained for the experimental group 1 and the control group 1 in Experimental example 3 of the present invention;
FIG. 10 is a characteristic diagram of test solutions of different concentrations in Experimental example 4 of the present invention;
FIGS. 11 and 12 are characteristic maps of sample solutions obtained by different extraction solvents in Experimental example 5 of the present invention;
FIG. 13 is a characteristic diagram of a sample solution prepared by extracting a solvent and hydrochloric acid at different volume ratios in Experimental example 5 of the present invention;
FIG. 14 is a characteristic map of samples of different equisetum hyemale in Experimental example 6 of the present invention.
Detailed Description
The following examples are provided to further understand the present invention, not to limit the scope of the present invention, but to provide the best mode, not to limit the content and the protection scope of the present invention, and any product similar or similar to the present invention, which is obtained by combining the present invention with other prior art features, falls within the protection scope of the present invention.
The examples do not indicate specific experimental procedures or conditions, and can be performed according to the procedures or conditions of the conventional experimental procedures described in the literature in the field. The reagents or instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially.
The invention relates to a reagent and an instrument:
reagent preparation:
a control medicinal material of horsetail (batch No. 180310, oakuchi biotechnology, beijing century, inc.) is marked as R;
kaempferol (batch No. 110861-201611, purity 95.5%, china institute for food and drug testing);
the preparation method of the lyophilized powder of the standard decoction of equisetum hiemale comprises the following steps: soaking herba Equiseti hiemalis decoction pieces in casserole for 30min, adding water 14 times the amount of the decoction pieces, boiling with strong fire, decocting with slow fire for 20min, filtering, and cooling; adding water 10 times the amount of decoction pieces, boiling with strong fire, decocting with slow fire for 15min, filtering, and cooling; mixing filtrates, concentrating under reduced pressure, and freeze drying. The invention uses 18 batches of horsetail standard decoction freeze-dried powder, and the batch number and the production area of each batch of horsetail standard decoction freeze-dried powder are as follows:
the preparation method of the equisetum hiemale formula granules comprises the following steps: taking herba Equiseti hiemalis decoction pieces, adding 16 times of water into the decoction piece for the first time, decocting for 40min, filtering with 150 mesh filter cloth while hot, adding 14 times of water into the second time, decocting for 40min, filtering, mixing filtrates, concentrating the filtrate into fluid extract, drying, adding appropriate amount of adjuvant, mixing, granulating, and packaging; the production place of the equisetum hiemale decoction pieces for preparing the equisetum hiemale formula granules is Xifeng county in Tieling City of Liaoning province.
Producing area of the equisetum hiemale decoction pieces: xifeng county, tieling City, liaoning province;
the production area of the equisetum hiemale medicinal materials: xifeng county, tieling City, liaoning province.
Reagent:
methanol (national pharmaceutical group chemical reagent Co., ltd., batch No.: 20190715) was analytically pure;
hydrochloric acid (national drug group chemical reagent, inc., batch number: 20190404), analytically pure;
acetonitrile (Merck, batch number: JB 094230), chromatographically pure;
the water is distilled water (drochen).
The instrument comprises:
Waters ACQUITY
an H-Class ultra-high performance liquid chromatograph, a PDA Detector, a TUV Detector and an Empower 3 chromatographic workstation;
XP26 electronic balance (mettler-toledo) and BSA124S electronic balance (sidos scientific instruments (beijing) ltd);
KQ-500DB ultrasonic cleaner (ultrasonic instruments, inc., kunshan).
Example 1
The embodiment provides a method for constructing a characteristic map of equisetum hiemale and a preparation thereof, which comprises the following steps,
(1) Preparation of control reference solutions: precisely weighing 4mg kaempferol, adding 200ml of 75% methanol with volume fraction, and making into reference substance solution of kaempferol reference substance of 20 μ g/ml;
(2) Preparation of reference solution of reference drug: taking 0.6g of a horsetail reference drug, placing the horsetail reference drug in a conical flask with a plug, adding 30ml (24 ml of 75% methanol and 6ml of hydrochloric acid) of a mixed solvent of 75% methanol and hydrochloric acid aqueous solution in volume fraction, weighing, heating and refluxing for 1h, taking out, cooling, weighing again, complementing the weight loss by using the mixed solvent of 75% methanol and hydrochloric acid in volume fraction, shaking up, and filtering to obtain a reference drug solution;
(3) Preparing a test solution: taking 0.1g of a to-be-detected sample of the horsetail standard decoction freeze-dried powder, placing the to-be-detected sample in a conical flask with a plug, adding 30ml (24 ml of 75% methanol and 6ml of hydrochloric acid) of a mixed solvent of 75% methanol and hydrochloric acid aqueous solution by volume fraction, weighing the weight, heating and refluxing for 1h, taking out, cooling, weighing the weight again, complementing the loss weight by using the mixed solvent of 75% methanol and hydrochloric acid by volume fraction, shaking up, and filtering the filtrate to obtain a test sample solution, wherein the concentration of the test sample solution is 3.33mg/ml;
(4) Chromatographic conditions and system applicability test: according to high performance liquid chromatography, octadecylsilane bonded silica gel column is used as chromatographic column (Waters ACQUITY column)
BEH Shield RP18, column length of 100mm, inner diameter of 2.1mm, particle size of 1.7 μm); taking acetonitrile as a mobile phase A and formic acid with volume fraction of 0.1% as a mobile phase B, and carrying out gradient elution, wherein the gradient elution procedure is shown in table 1, the flow rate is 0.3ml/min, the detection wavelength is 310nm, the column temperature is 35 ℃, and the number of theoretical plates is not lower than 8000;
(5) And (3) determination: respectively sucking 2 μ L of reference substance solution of reference substance, 1 μ L of reference substance solution of reference medicinal material and 1 μ L of test solution, injecting into liquid chromatograph, and measuring to obtain characteristic spectrum.
TABLE 1 gradient elution procedure
| Time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0-3.2 | 5-19 | 95-81 |
| 3.2-10 | 19-25 | 81-75 |
| 10-16 | 25-74 | 75-26 |
| 16-18 | 74-100 | 26-0 |
| 18-20 | 100-5 | 0-95 |
(6) Identification of characteristic peaks
Taking 18 batches of lyophilized powder of the equisetum hiemale standard decoction, preparing to obtain 18 batches of lyophilized powder of the equisetum hiemale standard decoction test solution according to the method in the step (3), determining according to the analysis conditions in the step (4) and the step (5) to obtain the characteristic spectrum of the lyophilized powder of the 18 batches of the equisetum hiemale standard decoction, as shown in figure 1, wherein the sample volume of the test solution is 1 mu L, in fig. 1, R is a characteristic spectrum of a reference substance of a horsetail, 8 stably existing chromatographic peaks shared by the reference substance of the reference substance are selected as characteristic peaks by comparing the characteristic spectrum with the reference substance of the reference substance, and the characteristic peaks are respectively marked as a peak 1, a peak 2, a peak 3, a peak 4, a peak 5, a peak 6, a peak 7 and a peak 8. The characteristic spectrum of the reference solution of the kaempferol reference substance is shown in FIG. 2, and the comparison shows that the No. 8 peak is a kaempferol peak, the No. 8 peak is a reference peak, the relative retention time and RSD are calculated, and the results are shown in Table 2.
TABLE 2 relative retention time of 18 batches of lyophilized powder of standard decoction of Equisetum hiemale
As is clear from table 2 and fig. 1, the relative retention times of the 8 characteristic peaks are small in difference, and the average value of the relative retention times is selected as the measurement value, and the Relative Retention Times (RRT) of the 8 characteristic peaks are: RRT of peak No. 1 is 0.12, RRT of peak No. 2 is 0.24, RRT of peak No. 3 is 0.44, RRT of peak No. 4 is 0.55, RRT of peak No. 5 is 0.60, RRT of peak No. 6 is 0.81, RRT of peak No. 7 is 0.86, tolerance: 10% of the total weight of the composition.
Leading the characteristic spectrums of 18 batches of horsetail standard decoction freeze-dried powder and a reference medicinal material into a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2012.1 version), and obtaining the similarity of the characteristic spectrums of the 18 batches of horsetail standard decoction freeze-dried powder and the reference medicinal material characteristic spectrums, wherein the similarity is respectively 0.972, 0.958, 0.979, 0.976, 0.956, 0.983, 1.000, 0.996, 1.000, 0.999, 1.000, 0.988, 0.985, 0.997, 0.993, 0.984 and 0.998, which indicates that the contact degree of the characteristic spectrums of the 18 batches of horsetail standard freeze-dried powder and the reference medicinal material is higher. The characteristic spectrum of 18 batches of the horsetail standard decoction freeze-dried powder is shown in figure 1, the 18 batches of the horsetail standard decoction freeze-dried powder is compared with a characteristic spectrum (shown in figure 3) generated by similarity software, the similarity result and the spectrum in figure 1 show that the 18 batches of the horsetail standard decoction have good similarity, 8 characteristic peaks are stable, and the construction method is used for well realizing the control on the quality of the horsetail.
Experimental example 1 investigation of wavelength
Taking a sample (batch number is S1) to be detected of a equisetum hiemale, preparing a sample solution according to the method in the step (3) in the embodiment 1, and then carrying out full-wavelength scanning on the sample solution at 210-400nm to obtain a full-wavelength scanned three-dimensional chromatogram, as shown in figure 4, the result shows that the peak at 310nm is rich, the peak shape is good and superior to other wavelengths, so 310nm is selected as the detection wavelength.
Experimental example 2 examination of elution gradient procedure
Taking 4 parts of the same batch of sample (batch number is S1) to be detected of the equisetum hiemale, preparing the sample solution according to the method in the step (3) of the embodiment 1, and measuring the sample volume of the experimental group 1 according to the chromatographic conditions in the step (4) to obtain 1 muL of sample.
TABLE 3 elution gradient procedure for control 1
The control group 3 is different from the experimental group 1 only in that the mobile phase and the elution method are different, and the control group 3 is measured according to the isocratic elution method in "chinese pharmacopoeia" 2015 edition, and the mobile phase is acetonitrile-0.4% phosphoric acid solution (50.
The characteristic maps of the control group 1, the control group 2 and the control group 3 are shown in FIGS. 6-8, and the characteristic map of the experimental group 1 is shown in FIG. 5; as can be known from the characteristic spectrums of the control group 1 and the control group 2, the separation degree of the obtained chromatographic peak is better, the characteristic peak information is richer, and the system pressure is more stable by taking acetonitrile as the mobile phase A; as can be seen from the characteristic maps of the experimental group 1 and the control group 1, the baseline of the characteristic map obtained from the experimental group 1 is more stable, and the separation degree and symmetry of chromatographic peaks are better.
The characteristic spectrums of the control group 3 and the experimental group 1 are known, the control group 3 mainly focuses on the separation of single chromatographic peaks of kaempferide, only one characteristic peak is arranged in the characteristic spectrum, the information content in the characteristic spectrum is less, 8 characteristic peaks of the characteristic spectrum obtained under the elution gradient of the experimental group 1 are stable, the information content is more abundant, the types and the quantity of chemical components of reacted equisetum hiemale are more, and the quality control of equisetum hiemale products is facilitated.
Experimental example 3 examination of method for preparing test solution
Taking the same batch of samples (with the batch number of S1) to be detected of the equisetum hiemale, and taking 2 parts in total; experimental group 1 a sample solution was prepared according to the method in step (3) of example 1, and then measured according to the chromatographic conditions in step (4) of example 1 with a sample volume of 1 μ L, to obtain a characteristic map, see fig. 9; the control group 1 is prepared according to a method of 'Chinese pharmacopoeia' 2015 edition, and the specific steps comprise that 0.75g of a horsetail sample is precisely weighed, the horsetail sample is placed in a conical flask with a plug, 50ml of 75% methanol is precisely added, the plug is sealed, the weight is weighed, heating reflux is carried out for 1 hour, cooling is carried out, the weight is weighed again, the loss weight is complemented with 75% methanol, shaking is carried out uniformly, filtering is carried out, 20ml of subsequent filtrate is obtained by precise amount, 5ml of hydrochloric acid is added, the mixture is placed in a water bath for heating hydrolysis for 1 hour, cooling is carried out, the subsequent filtrate is transferred to a 50ml measuring flask, 75% methanol is added to the scale, shaking is carried out uniformly, filtering is carried out, and the subsequent filtrate is obtained; then, a characteristic map was obtained by measuring the sample volume of 1. Mu.L according to the chromatographic conditions in the step (4) of example 1, see FIG. 9.
As can be seen from fig. 9, the separation degree, the peak shape, the symmetry factor, the baseline of the characteristic peak obtained in the experimental group 1 and the control group 1 have little difference, but the preparation method provided by the experimental group 1 shortens the time for preparing the test solution, and further shortens the time for constructing the characteristic map.
Experimental example 4 examination of concentration of test solution
Taking 7 parts of the same batch of sample (batch number is S1) to be tested of the equisetum hiemale, preparing the sample solutions with different concentrations according to the method in the step (3) of the embodiment 1, wherein the sample solutions are respectively 16mg/ml, 12mg/ml, 10mg/ml, 8mg/ml, 4mg/ml, 3.33mg/ml and 2.5mg/ml, then determining according to the chromatographic conditions in the step (4) of the embodiment 1, and the sample injection amount is 1 mu L to obtain a characteristic spectrum, as shown in figure 10, the result shows that the theoretical plate numbers and the separation degrees of the characteristic spectrums obtained by the sample solutions with different concentrations are not different.
Experimental example 5 examination of solvent in preparation of test solution
Examination of extraction solvent
Taking 9 parts of the same batch of samples (batch number is S1) to be detected of the equisetum hiemale, preparing a sample solution according to the method in the step (3) of the embodiment 1 in the experimental group 1, and then determining according to the chromatographic conditions in the step (4) to obtain a characteristic spectrum, wherein the sample injection amount is 1 mu L. The control 1 is different from the experimental 1 only in that 75% by volume of methanol aqueous solution is changed to methanol solvent at the time of preparation of the test solution.
The control group 2 is different from the experimental group 1 only in that a 75% methanol aqueous solution by volume fraction was changed to a 50% methanol aqueous solution by volume fraction at the time of preparation of the test solution.
The control group 3 is different from the experimental group 1 only in that a 75% by volume methanol aqueous solution is changed to a 30% by volume methanol aqueous solution at the time of preparation of the test solution.
The control group 4 is different from the experimental group 1 only in that a 75% volume fraction of methanol aqueous solution was changed to water at the time of preparation of the test solution.
The control group 5 is different from the experimental group 1 only in that a test solution is prepared by replacing 75% by volume of an aqueous methanol solution with ethanol.
The control 6 is different from the experimental group 1 only in that 75% by volume of methanol aqueous solution is changed to 75% by volume of ethanol at the time of preparation of the test solution.
The control group 7 is different from the experimental group 1 only in that 75% by volume of methanol aqueous solution was changed to 50% by volume of ethanol at the time of preparation of the test solution.
The control group 8 is different from the experimental group 1 only in that 75% by volume of methanol aqueous solution is changed to 30% by volume of ethanol at the time of preparation of the test solution.
The characteristic maps of the experimental group 1 and the control groups 1-8 are shown in fig. 11 and fig. 12, when ethanol is used as a solvent, the peak shapes of the No. 4 peak and the No. 5 peak are poor, and the No. 1 peak is forked; when water is used as a solvent, the characteristic map has peak deletion; the peak shape of the chromatographic peak of 75% methanol in different proportions of methanol is superior to that of other proportions of methanol, so that 75% of methanol is preferably used as the extraction solvent.
Examination of the ratio of extraction solvent to hydrochloric acid
Taking 3 parts of a same batch of samples (batch number is S1) to be detected of the equisetum hiemale, and preparing a sample solution according to the method in the step (3) in the embodiment 1, wherein the difference is that the volume ratio of 75% methanol to hydrochloric acid in volume fraction is different, and the volume ratio of 75% methanol to hydrochloric acid in the experimental group 1 is 3; the volume ratio of 75% methanol to hydrochloric acid in volume fraction in experimental group 2 is 4; the volume ratio of 75% methanol to hydrochloric acid in volume fraction in experimental group 3 is 5; then, the sample solutions prepared in the experimental groups 1 to 3 were measured under the chromatographic conditions in the step (4) of example 1 at a sample volume of 1. Mu.L to obtain a characteristic map, as shown in FIG. 13; as can be seen from fig. 13, the volume ratio of the volume fraction of 75% methanol and hydrochloric acid has little influence on the characteristic map.
Experimental example 6 examination of test sample of Equisetum hiemale
Preparing a test solution from different equisetum samples to be tested, which comprises the following steps:
experimental group 1: taking 0.6g of equisetum hiemale, putting the equisetum hiemale in a conical flask with a plug, adding 30ml (24 ml of 75% methanol and 6ml of hydrochloric acid) of a mixed solvent of 75% methanol and hydrochloric acid in volume fraction, weighing, heating and refluxing for 1h, taking out, cooling, weighing again, complementing the weight loss by using the mixed solvent of 75% methanol and hydrochloric acid in volume fraction, shaking up, and filtering to obtain a sample solution.
Experimental group 2: taking 0.6g of equisetum hiemale decoction pieces, placing the equisetum hiemale decoction pieces in a conical flask with a plug, adding 30ml of a mixed solvent of 75% methanol and hydrochloric acid (24 ml of 75% methanol and 6ml of hydrochloric acid) by volume fraction, weighing, heating and refluxing for 1h, taking out, cooling, weighing again, complementing the weight loss by the mixed solvent of 75% methanol and hydrochloric acid by volume fraction, shaking up, and filtering to obtain a sample solution.
Experimental group 3: taking 0.1g of lyophilized powder of a equisetum hiemale standard decoction, placing the lyophilized powder in a conical flask with a plug, adding 30ml (24 ml of 75% methanol and 6ml of hydrochloric acid) of a mixed solvent of 75% methanol and hydrochloric acid in volume fraction, weighing, heating and refluxing for 1h, taking out, cooling, weighing again, complementing the weight loss by using the mixed solvent of 75% methanol and hydrochloric acid in volume fraction, shaking up, and filtering to obtain a test solution.
Experimental group 4: taking 0.1g of equisetum hiemale formula particles, placing the particles in a conical flask with a plug, adding 30ml of mixed solvent of 75% methanol and hydrochloric acid in volume fraction (24 ml of 75% methanol and 6ml of hydrochloric acid), weighing, heating and refluxing for 1h, taking out, cooling, weighing again, complementing the weight loss by using the mixed solvent of 75% methanol and hydrochloric acid in volume fraction, shaking up, and filtering to obtain a sample solution.
Then, the test solutions prepared in experimental groups 1 to 4 were measured under the chromatographic conditions in step (4) of example 1 in a sample amount of 1. Mu.L to obtain a characteristic map, as shown in FIG. 14.
As can be seen from fig. 14, the construction method provided by the present invention can be applied to horsetail herbs, horsetail decoction pieces, horsetail standard decoction freeze-dried powder and horsetail formula granules.
Experimental example 7 methodological examination
(1) Precision experiment
Repeatability survey
Taking a same batch of sample (batch number is S1) to be detected of the equisetum hiemale, preparing 6 parts of sample solution to be detected according to the method in the step (3) in the embodiment 1, then determining according to the chromatographic conditions in the step (4), obtaining a characteristic spectrum with the sample amount of 1 muL, taking the peak No. 8 as a reference peak, calculating the relative retention time and the relative peak area, and calculating the RSD, wherein the results are shown in tables 4 and 5.
TABLE 4 relative Retention time Table of characteristic peaks
TABLE 5 relative peak area table of characteristic peaks
As can be seen from tables 4 and 5, the RSD of the characteristic peak of the characteristic spectrum is in the range of 0.02% -0.14%, and the RSD of the relative peak area is in the range of 0.7% -3.34%, which indicates that the repeatability of the characteristic spectrum is better.
Survey of instrument precision
Taking a same batch of samples (batch number is S1) to be detected of the equisetum hiemale, preparing a sample solution according to the method in the step (3) of the embodiment 1, measuring the sample solution by the chromatographic condition in the step (4), measuring the sample injection amount by 1 mu L, measuring the 6 samples by continuous sample injection to obtain a characteristic spectrum, calculating relative retention time and relative peak area by taking a peak 8 as a reference peak, and calculating RSD (red fluorescence), wherein the results are shown in tables 6 and 7.
TABLE 6 relative Retention time Table of characteristic peaks
TABLE 7 relative peak area table of characteristic peaks
The result shows that the relative retention time RSD of 8 characteristic peaks of the characteristic spectrum obtained by continuous sample injection for 6 times is within the range of 0.01-0.0.6%, and the relative peak area RSD is within the range of 0.11-0.44%, which indicates that the precision of the instrument meets the requirement.
(2) Examination of stability of test solution
The sample solution was prepared according to the method in step (3) of example 1, left to stand at room temperature, measured according to the chromatographic conditions in step (4) of example 1 at 0, 2, 4, 6, 8, 10, 12, and 24 hours, and the amount of sample was 1. Mu.L, and then characteristic peaks were analyzed to obtain relative retention time and relative peak area, and the results are shown in tables 8 and 9.
TABLE 8 relative Retention time
TABLE 9 relative peak area of characteristic peaks
From tables 8 and 9, it can be seen that, through the investigation of 24-hour solution stability, the relative retention time RSD of each characteristic peak is in the range of 0.16% to 1.61%, but the relative peak areas RSD of the peak 1 and the peak 5 are greater than 5%, and the RSD of the peak area of each characteristic peak is in the range of 0.78% to 4.5% within 12 hours, so that the method for constructing the horsetail according to the present invention needs to complete the detection within 12 hours after the preparation of the test solution.
In conclusion, the construction method of the equisetum hiemale characteristic spectrum provided by the invention has the advantages of good stability, high precision, good repeatability, good peak shape of the obtained characteristic peak and high separation degree; meanwhile, the construction method of the characteristic map of the equisetum hiemale provided by the invention can also greatly shorten the analysis time and improve the efficiency.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the spirit or scope of the invention.
Claims (9)
1. A method for constructing a characteristic map of a horsetail and a preparation thereof is characterized by comprising the following steps,
preparing a test solution: taking a sample to be detected of the horsetail, adding a mixed solvent of methanol and hydrochloric acid solution, extracting, filtering, and taking a subsequent filtrate to obtain the horsetail extract;
taking kaempferide as a reference substance of a reference substance, and obtaining a characteristic map of the equisetum hiemale and a preparation thereof by adopting a high performance liquid chromatography;
the chromatographic conditions of the high performance liquid chromatography are as follows: the chromatographic column is Waters ACQUITY UPLC BEHShield RP18, the column length is 100mm, the inner diameter is 2.1mm, the particle size is 1.7 mu m, acetonitrile is used as a mobile phase A, formic acid with the volume fraction of 0.05-0.4% is used as a mobile phase B, gradient elution is carried out, and the gradient elution program comprises the following steps: 0-3.2min, mobile phase A: the volume ratio of the mobile phase B is 5-19%:95 to 81 percent; 3.2-10min, mobile phase A: the volume ratio of the mobile phase B is 19-25%:81-75 percent; 10-16min, mobile phase A: the volume ratio of the mobile phase B is 25-74%:75-26%;16-18min, mobile phase A: the volume ratio of the mobile phase B is 74-100%:26 to 0 percent.
2. The construction method according to claim 1, wherein the chromatographic conditions of the high performance liquid chromatography are as follows: the flow rate is 0.2-0.4ml/min, the detection wavelength is 305-320nm, and the column temperature is 33-36 ℃.
3. The construction method according to claim 1 or 2, wherein the preparation method of the reference substance solution comprises: adding kaempferol into alcohol solvent to obtain reference substance solution of reference substance;
the preparation method of the reference solution of the reference medicinal materials comprises the following steps: taking a herba Equiseti hiemalis reference medicinal material, adding a mixed solvent of 75% methanol and hydrochloric acid solution by volume fraction, extracting for 0.7-1.3h, filtering, and taking filtrate to obtain a reference substance solution of the reference medicinal material;
preparation of a test solution: taking a sample to be detected of the horsetail, adding a mixed solvent of 75% methanol and hydrochloric acid solution by volume fraction, extracting for 0.7-1.3h, filtering, and taking a subsequent filtrate to obtain the horsetail extract.
4. The construction method according to claim 1 or 2, wherein the characteristic map of the equisetum hiemale and the preparation thereof comprises 8 characteristic peaks, and the relative retention time of the other characteristic peaks is within ± 10% of the specified value by taking the peak No. 8 as a reference peak, and the specified values of the relative retention time are respectively:
peak No. 1 had a relative retention time of 0.12; peak No. 2 had a relative retention time of 0.24; peak No. 3 had a relative retention time of 0.44; peak No. 4 had a relative retention time of 0.55; peak 5 had a relative retention time of 0.60; peak No. 6 had a relative retention time of 0.81; peak 7 had a relative retention time of 0.86;
wherein, the peak No. 8 is a kaempferol peak.
5. Construction process according to claim 1 or 2, characterized in that the volume ratio of methanol to hydrochloric acid is (3-5): 1;
the mass fraction of the hydrochloric acid solution is 36-38%;
the concentration of the test solution is 2.5-25mg/ml.
6. The construction method according to claim 1 or 2, wherein the preparation of the sample solution comprises the steps of taking a sample to be tested from equisetum hiemale, grinding, precisely weighing, adding a mixed solvent of 75% methanol and hydrochloric acid solution by volume fraction, heating and refluxing for 1h, filtering, and taking a subsequent filtrate;
the reference substance solution of the reference substance is prepared by precisely weighing kaempferol, and adding 75% methanol by volume;
the reference solution of the reference medicinal material is prepared by grinding herba Equiseti hiemalis, precisely weighing, adding mixed solvent of 75% methanol and hydrochloric acid solution, reflux-extracting under heating for 1 hr, filtering, and collecting filtrate.
7. The construction method according to claim 1 or 2, wherein the sample to be tested is a decoction piece of herba Equiseti hiemalis, a crude drug of herba Equiseti hiemalis, a lyophilized powder of standard decoction of herba Equiseti hiemalis, or a granule of herba Equiseti hiemalis.
8. The construction method according to claim 1 or 2, wherein the concentration of the reference solution of the control substance is 18-23 μ g/ml;
the concentration of the reference solution of the reference medicinal material is 15-25mg/ml.
9. The construction method according to claim 1 or 2, wherein the chromatographic conditions are: the flow rate was 0.3ml/min, the detection wavelength was 310nm, and the column temperature was 35 ℃.
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