CN109507310B - Fingerprint spectrum construction method and detection method of essence-nourishing jade-planting prescription - Google Patents
Fingerprint spectrum construction method and detection method of essence-nourishing jade-planting prescription Download PDFInfo
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Abstract
The invention discloses a fingerprint spectrum construction method and a detection method of a formula for nourishing essence and seed jade. The construction method comprises the following steps: preparing a test solution; preparing a reference substance solution; constructing a fingerprint spectrum: and (3) sucking the test solution and the reference solution, and injecting the solutions into a high performance liquid chromatograph for determination to obtain the fingerprint of the Yangjingdao jade formula with the common characteristic peak. On the basis of long-term experience accumulation and after a large number of experiments, the inventor selects ferulic acid, paeoniflorin, morroniside and loganin as reference substances to construct a fingerprint, so that the quality of the classic formulation of the Yangjingdao jade soup and the quality of modern preparations Yangjingdao jade fried paste and Yangjingdao jade granule can be well represented. Particularly, the fingerprint is constructed by matching and selecting proper chromatographic conditions, so that the Yangjingdao jade soup can be qualitatively and quantitatively characterized, and the Yangjingdao jade decocted extract and the Yangjingdao jade granule can be qualitatively and quantitatively characterized.
Description
Technical Field
The invention relates to the field of quality control of traditional Chinese medicine preparations, in particular to a fingerprint construction method and a detection method of a formula for nourishing essence and seeds.
Background
The Yangjingzhiyu decoction is a traditional Chinese medicine prescription, is prepared from a roll of Fu Qing Master Nuvidae, and has a prescription of 30g of prepared rehmannia root (Jiu steam), 15 g of Chinese angelica (washed by wine), 15 g of white paeony root (stir-fried with wine) and 15 g of cornus pulp (steamed), and is mainly used for treating kidney deficiency and blood deficiency, emaciation and weakness of the body and infertility.
The preparation of the Yangjingdan jade prescription sold in the market at present comprises Yangjingdan jade cream and Yangjingdan jade granules, the fingerprint spectrum and the content determination research of the Yangjingdan jade cream are carried out in the existing literature, the content determination research of the Yangjingdan jade granules is carried out, but the research only establishes a fingerprint spectrum comparison method of a plurality of batches of Yangjingdan jade creams and establishes the content determination of two components of paeoniflorin and loganin in the creams and the granules. Regarding the detection of the preparation of the essence-nourishing jade formula, no unified method standard exists for comprehensively measuring decoction, granules and decocted extract at present.
Therefore, it is highly desirable to provide a unified method for determining the recipes for breeding elite in the form of decoction, granule, decoction, etc.
Disclosure of Invention
Based on the above, the invention mainly aims to provide a method for constructing a fingerprint of a formula for cultivating the seeds of the Yangjing.
The main purpose of the invention is realized by the following technical scheme:
a construction method of a fingerprint of a formula for cultivating elite seeds comprises the following steps:
preparing a test solution: preparing the Yangjingdao jade decoction into freeze-dried powder, dissolving the freeze-dried powder in an organic solvent, and filtering to obtain a test solution;
preparation of a reference solution: dissolving ferulic acid, paeoniflorin, morroniside, and loganin reference substances in organic solvent to obtain reference substance solution;
constructing a fingerprint spectrum: and (3) sucking the test solution and the reference solution, and injecting the solutions into a high performance liquid chromatograph for determination to obtain the fingerprint of the Yangjingdao jade formula with the common characteristic peak.
In some of these embodiments, the chromatographic conditions employed for the assay comprise:
a chromatographic column: octadecylsilane chemically bonded silica is used as a filling agent;
mobile phase: acetonitrile is taken as a mobile phase A, and phosphoric acid aqueous solution with volume concentration of 0.08-0.12% is taken as a mobile phase B;
gradient elution mode: the volume percent of the mobile phase A is changed from 5% to 15% and the volume percent of the mobile phase B is changed from 95% to 85% within 0-25 min; the volume percentage of the mobile phase A is changed from 15% to 20% and the volume percentage of the mobile phase B is changed from 85% to 80% from 25min to 40 min; the volume percentage of the mobile phase A is changed from 20% to 50% and the volume percentage of the mobile phase B is changed from 80% to 50% within 40-60 min.
In some embodiments, the flow rate of the mobile phase is 0.8mL/min to 1.2 mL/min; the column temperature of the chromatographic column is 25-35 ℃; the detection wavelength adopted by the determination is 210 nm-250 nm.
In some embodiments, the organic solvent is a methanol solution with a volume concentration of 70% to 100%.
In some embodiments, 0.5g to 1g of the lyophilized powder is added to 25mL to 50mL of the organic solvent.
In some of these embodiments, the preparation of the lyophilized powder comprises:
taking prepared rehmannia root, wine-treated angelica, wine-treated white peony root and wine-treated cornus pulp decoction pieces, and mixing the prepared rehmannia root, the wine-treated angelica, the wine-treated white peony root and the wine-treated cornus pulp decoction pieces according to a weight ratio of 2: 1: 1: 1, mixing, and soaking in water for 30 minutes; decocting twice: adding water 8 times of the total weight of the decoction pieces for the first time, decocting for 1 hr, and filtering to obtain filtrate; adding water 8 times of the total weight of the decoction pieces for the second time, decocting for 1 hr, filtering, mixing the filtrate with the filtrate obtained by the first decoction, and concentrating under reduced pressure until the weight ratio of the feed amount to the concentrated solution is 1: 1-1: 1.5, adding aerosil which accounts for 7-15 percent of the weight of the clear paste, pre-freezing at-20 ℃ to-45 ℃, and then freeze-drying into powder under the conditions of-80 ℃ and less than 100Pa to obtain the freeze-dried powder.
In some embodiments, each 1mL of the control solution contains 0.6-24 μ g of ferulic acid, 2.1-84.2 μ g of paeoniflorin, 2.0-80.6 μ g of morroniside, and 2.5-101.3 μ g of loganin.
The invention also aims to provide a method for determining the essence-nourishing jade-planting prescription, which comprises the following steps:
preparing a test solution: dissolving the refined seed jade formula to be tested in an organic solvent, and filtering to obtain a test solution;
preparation of a reference solution: dissolving ferulic acid, paeoniflorin, morroniside, and loganin reference substances in organic solvent to obtain reference substance solution;
and (3) testing the test solution: sucking the test solution and the reference solution, and injecting into a high performance liquid chromatograph for determination to obtain a spectrum of the elite seed preparation to be tested; and comparing the relative retention time of each peak and/or the peak area of each peak in the atlas of the elite seed jade preparation to be tested and the fingerprint constructed by the fir, and finishing the qualitative and/or quantitative determination of the elite seed jade preparation to be tested.
The essence-nourishing seed-jade prescription provided by the invention comprises granules, extractum and decoction.
In some of these embodiments, the chromatographic conditions employed for the assay comprise:
a chromatographic column: octadecylsilane chemically bonded silica is used as a filling agent;
mobile phase: acetonitrile is taken as a mobile phase A, and phosphoric acid aqueous solution with volume concentration of 0.08-0.12% is taken as a mobile phase B;
gradient elution mode: the volume percent of the mobile phase A is changed from 5% to 15% and the volume percent of the mobile phase B is changed from 95% to 85% within 0-25 min; the volume percentage of the mobile phase A is changed from 15% to 20% and the volume percentage of the mobile phase B is changed from 85% to 80% from 25min to 40 min; the volume percentage of the mobile phase A is changed from 20% to 50% and the volume percentage of the mobile phase B is changed from 80% to 50% within 40-60 min.
In some embodiments, the flow rate of the mobile phase is 0.8mL/min to 1.2 mL/min; the column temperature of the chromatographic column is 25-35 ℃; the detection wavelength adopted by the determination is 210 nm-250 nm.
In some embodiments, the organic solvent is a methanol solution with a volume concentration of 70% to 100%.
Compared with the prior art, the invention has the following beneficial effects:
on the basis of long-term experience accumulation and after a large number of experiments, the inventor selects ferulic acid, paeoniflorin, morroniside and loganin as reference substances to construct a fingerprint, so that the quality of the classic formulation of the Yangjingdao jade soup and the quality of modern preparations Yangjingdao jade fried paste and Yangjingdao jade granule can be well represented. Particularly, the fingerprint is constructed by matching and selecting proper chromatographic conditions, so that the Yangjingdao jade soup can be qualitatively and quantitatively characterized, and the Yangjingdao jade decocted extract and the Yangjingdao jade granule can be qualitatively and quantitatively characterized.
The determination method constructed by the fingerprint spectrum can not only synchronously carry out qualitative and quantitative determination of the standard decoction of the Yangjingdao jade soup, but also be suitable for qualitative and quantitative tests of Yangjingdao jade decoction and Yangjingdao jade granules of modern preparations of the Yangjingdao jade soup, and well disclose the substance transfer of the Yangjingdao jade prescription. And the determination method has the characteristics of simplicity, convenience, stability, high precision, good reproducibility and the like.
Drawings
FIG. 1 is a fingerprint and content (specificity test) spectrum of the standard decoction of Yangjingyu in example 1; wherein, the peak 6 is morroniside, the peak 7 is loganin, the peak 11 is paeoniflorin, the peak 8 is isochlorogenic acid C, and the peak 12 is ferulic acid;
FIG. 2 is a fingerprint and content spectrum of 9 batches of elite seed standard decoction in example 1;
FIG. 3 is a comparison fingerprint of the Yangjingyu standard decoction of example 1;
FIG. 4 shows the similarity evaluation results of 9 batches of the elite seed standard decoction in example 1;
FIG. 5 is an overlay of the results of the precision test in example 1;
FIG. 6 is an overlay of the results of the repeated experiments in example 1;
FIG. 7 is a graph showing the results of the intermediate precision test-different persons in example 1;
FIG. 8 is a superimposed graph of results of intermediate precision tests on different dates in example 1;
FIG. 9 is a graph showing the results of the intermediate precision test-different instruments in example 1;
FIG. 10 is a graph showing the results of the stability test in example 1;
FIG. 11 is a standard curve of ferulic acid in example 1;
FIG. 12 is a paeoniflorin standard curve in example 1;
FIG. 13 is a morroniside standard curve of example 1;
FIG. 14 is a loganin standard curve of example 1;
FIG. 15 shows the results of the experiment in example 2;
FIG. 16 is a map result of comparative example 1;
FIG. 17 is a map result of comparative example 2;
FIG. 18 is a map result of comparative example 3;
FIG. 19 is a map result of comparative example 4;
FIG. 20 is a map result of comparative example 5;
FIG. 21 is a map result of comparative example 6;
fig. 22 is a map result of comparative example 7.
Detailed Description
In order that the invention may be more fully understood, reference will now be made to the following description. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
Example 1
1. Instrument and reagent
1.1 Instrument: an electronic balance Mettler Toledo XP205DR (d 0.01mg), Mettler Toledo MS204S (d 0.1mg), a high-frequency numerical control ultrasonic cleaner (KQ-300TD, Kunshan ultrasonic instruments Co., Ltd.) silicon UV water purifier (Millipore), a high performance liquid chromatograph (Agilent 1260), a freeze dryer (Christ, Germany, ALPHA2-4LD plus), and a rotary evaporator N-1200A (EYELA Shanghai Iran instruments Co., Ltd.).
1.2 reagent: acetonitrile and phosphoric acid are chromatographically pure, water is purified water, and other reagents are analytically pure.
1.3 reagent: paeoniflorin reference (Chinese food and drug inspection institute, 110736-201539, 110736-201741), ferulic acid reference (Chinese food and drug inspection institute, 110773-201313), loganin reference (Chinese food and drug inspection institute, 111640-201005, 111640-201707), morroniside reference (Chinese food and drug inspection institute, 111998-201501, 111998-201602), radix rehmanniae preparata, white paeony root, radix angelicae sinensis, cornus vinosus (Hubei Tianji Chinese medicine decoction pieces Co., Ltd, Shanghai medicine Co., Guangzhou City, Guangzhou Kangsheng medicine Co., Ltd and Anhui Shuyao medicine Co., Ltd), and silica gel micropowder (Huzhou Showa medicine Co., Ltd).
2. Method and results
2.1 preparation of Standard decoction
Taking 150g of prepared rehmannia root 60g, wine angelica 30g, wine white peony root 30g and wine cornus 30g, soaking the prepared rehmannia root 150g in water for 30 minutes, decocting twice, adding water 8 times of the total weight for the first time, decocting for 1 hour, filtering by a 200-mesh sieve, and keeping the filtrate for later use; adding water 8 times of the total weight for the second time, decocting for 1 hr, filtering with 200 mesh sieve, mixing the filtrate with the first filtrate, and concentrating under reduced pressure at 50 deg.C until the feed amount: the mass of the concentrated solution is 1: 1-1: 1.5 adding 8% of silica gel micropowder, prefreezing at-20 deg.C to-45 deg.C, and lyophilizing at-80 deg.C and under less than 100Pa to obtain standard decoction lyophilized powder.
Taking chemical component contents of radix rehmanniae Preparata, radix Angelicae sinensis, radix Paeoniae alba and Corni fructus as four factors, each Chinese medicinal decoction pieces has 3 batches, and taking the 3 batches as three levels, performing L9(34) And performing orthogonal test to obtain 9 batches of freeze-dried powder. The tablet factor levels are shown in table 1 and the orthogonal design tests are shown in table 2.
TABLE 1 prepared rehmannia root, Chinese angelica root, white peony root and dogwood fruit decoction pieces factor level table
Table 2 orthogonal design test table
2.2 chromatographic conditions
A chromatographic column: phenomenex C18 (4.6X 250mm, 5 μm); mobile phase: acetonitrile is taken as a mobile phase A, phosphoric acid aqueous solution with volume concentration of 0.1% is taken as a mobile phase B, and gradient elution is carried out according to the specification in the table 3; column temperature: 25 ℃; flow rate: 1.0 mL/min; detection wavelength: 240 nm; sample introduction amount: 10 μ L.
TABLE 3 mobile phase gradient elution conditions
| Time (min) | Acetonitrile (A)% | 0.1% phosphoric acid (B)% |
| 0～25 | 5～15 | 95～85 |
| 25～40 | 15～20 | 85～80 |
| 40～60 | 20～50 | 80～50 |
2.3 preparation of test solutions
Taking a proper amount of obtained powder of standard decoction freeze-dried powder, grinding, taking about 1g, precisely weighing, precisely adding 50mL of 70% methanol, sealing, weighing, carrying out ultrasonic treatment (power 300W and frequency 40kHz) for 30 minutes, taking out, cooling, weighing again, supplementing the weight loss by 70% (volume percentage) of methanol, shaking up, filtering, and taking a subsequent filtrate to obtain a test solution.
2.4 preparation of Mixed control solutions
Precisely weighing ferulic acid, paeoniflorin, morroniside and loganin reference substances respectively, mixing, adding 70% (volume percentage) methanol to obtain mixed solution containing ferulic acid 12 μ g, paeoniflorin 42.11 μ g, morroniside 40.29 μ g and loganin 50.64 μ g per 1mL, and making into mixed reference substance solution.
2.5 assay method
Respectively sucking 10 μ L of the mixed reference solution and sample solution, injecting into high performance liquid chromatograph, measuring by high performance liquid chromatography, and recording chromatogram to obtain finger print and content of standard decoction of YANGJINGFEIYU (figure 1). In FIG. 1, the 9 th peak, 10 th peak and 11 th peak are attributed to white peony root, 12 th peak to Chinese angelica root, 2 th peak, 6 th peak, 7 th peak, 8 th peak and 13 th peak to cornus fruit, and the content of the ingredient in prepared rehmannia root is very low and cannot be identified.
2.6 establishment of control fingerprint and evaluation of similarity
Comparing the 9 batches of the standard decoction of the Yangjingyao jade soup, determining 14 common peaks according to the 100% appearance rate of the common peaks, introducing the 9 batches of the standard decoction of the Yangjingyao jade soup into a 2012 version of a traditional Chinese medicine chromatogram fingerprint similarity evaluation system, generating a control fingerprint, wherein the similarity of the 9 batches of the standard decoction of the Yangjingyao jade soup is more than 0.99. See fig. 2, 3, 4.
2.79 batch of standard decoction lyophilized powder for determination of ferulic acid, paeoniflorin, morroniside and loganin content
Each batch of standard decoction samples are prepared into two parts in parallel, the content (average value) of index components such as ferulic acid, paeoniflorin, morroniside, loganin and the like in the standard decoction is calculated by an external standard two-point method in each part, and the measurement results are shown in table 4.
TABLE 49 contents of ferulic acid, paeoniflorin, morroniside, and loganin in standard decoction
3. Finger print methodology survey
3.1 specificity test
In order to examine whether the blank solvent and the auxiliary material micro silica gel have interference, a test solution, a mixed reference solution, the blank solvent (70% methanol) and the auxiliary material micro silica gel solution are respectively taken and measured according to the method under the 2.2 chromatographic condition, a chromatogram is recorded, and the measurement result is shown in figure 1.
The result shows that the auxiliary materials and the blank solvent are not interfered.
3.2 precision test
Taking 1 part of Yangjingdao jade standard decoction sample, preparing according to the method under the item of '2.3 sample solution preparation', respectively and precisely absorbing 10 mu L of the same sample solution, continuously sampling for 6 times, determining according to the method under the item of '2.2 chromatographic conditions', recording a chromatogram, and calculating the similarity according to a traditional Chinese medicine chromatogram fingerprint similarity evaluation system, wherein the similarity determination result is shown in figure 5 and table 5.
The results show that the similarity is all 1.000, and the precision of the instrument is good.
TABLE 5 evaluation table of similarity in precision test
| Number of |
1 | 2 | 3 | 4 | 5 | 6 | |
| 1 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 |
| 2 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 |
| 3 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 |
| 4 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 |
| 5 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 |
| 6 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 |
| Comparison fingerprint | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 |
3.3 repeatability test
Taking 6 parts of Yangjingdao jade standard decoction sample, preparing according to the method under the item of '2.3 sample solution preparation', respectively and precisely absorbing 10 mu L of the same sample solution, determining according to the method under the item of '2.2 chromatographic conditions', recording a chromatogram, and calculating the similarity according to a traditional Chinese medicine chromatogram fingerprint similarity evaluation system, wherein the similarity determination result is shown in figure 6 and table 6.
The results show that the similarity is 1.000, and the repeatability is good.
TABLE 6 evaluation table for similarity of repeatability tests
| Number of | Repeatability | 1 | |
|
|
|
|
|
| Repeatability | ||||||||
| 1 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | |
| |
1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | |
| |
1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | |
| |
1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | |
| |
1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | |
| |
1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | |
| Comparison fingerprint | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 |
3.4 intermediate precision
Taking the same batch of Yangjingyao jade standard decoction samples, preparing according to the method under the item of '2.3 sample solution preparation', respectively measuring by different personnel on different instruments at different time according to the method under the item of '2.2 chromatographic conditions', recording chromatogram, and calculating the similarity according to a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, wherein the similarity measurement result is shown in the figure 7, the figure 8 and the figure 9, and the table 7, the table 8 and the table 9.
The results show that the similarity is above 0.999, which indicates that the intermediate precision is good.
TABLE 7 evaluation chart of similarity between different persons
| Different persons | Liu (traditional Chinese medicine) | Displaying (Chen) | Comparison fingerprint |
| Liu (traditional Chinese medicine) | 1.000 | 0.999 | 1.000 |
| Displaying (Chen) | 0.999 | 1.000 | 1.000 |
| Comparison fingerprint | 1.000 | 1.000 | 1.000 |
TABLE 8 evaluation chart of similarity between different dates
| |
20170823 | 20170803 | |
| 20170823 | 1.000 | 0.999 | 1.000 |
| 20170803 | 0.999 | 1.000 | 1.000 |
| Comparison fingerprint | 1.000 | 1.000 | 1.000 |
TABLE 9 similarity evaluation chart for different instruments
| Different instruments | waters | agilent | Comparison fingerprint |
| waters | 1.000 | 0.997 | 0.999 |
| agilent | 0.997 | 1.000 | 0.999 |
| Comparison fingerprint | 0.999 | 0.999 | 1.000 |
3.5 stability test
Taking 1 part of Yangjingdao jade standard decoction sample, preparing according to the method under the item of '2.3 sample solution preparation', respectively measuring according to the method under the item of '2.2 chromatographic conditions' for 0, 2, 4, 8, 16, 24, 36, 48, 60 and 72 hours, recording a chromatogram, and calculating the similarity according to a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, wherein the similarity measurement result is shown in figure 10 and table 10.
The results show that the similarity is all 1.000, and the stability of the test solution is good within 72 h.
TABLE 10 stability similarity evaluation Table
4 assay methodology investigation
4.1 specificity test
Measuring under the same term of "3.1", and recording chromatogram. The results show that the blank solvent and the adjuvant micropowder silica gel solution have no chromatographic peak in the retention time corresponding to ferulic acid, paeoniflorin, morroniside and loganin.
The result shows that the blank solvent and the adjuvant micro-powder silica gel have no interference to the determination of the ferulic acid, the paeoniflorin, the morroniside and the loganin, and the determination of the content of the ferulic acid, the paeoniflorin, the morroniside and the loganin in the product by the method has specificity.
4.2 examination of the Linear relationship
Precisely weighing ferulic acid, paeoniflorin, morroniside and loganin reference substances respectively, mixing, and dissolving in 70% methanol to obtain mixed reference substance solution containing ferulic acid 12.00 μ g, paeoniflorin 42.11 μ g, morroniside 40.29 μ g and loganin 50.64 μ g per 1mL of the mixed reference substance. Precisely sucking 0.5 μ L of the above control solution2. mu.L, 5. mu.L, 8. mu.L, 10. mu.L, 12. mu.L and 15. mu.L, measured by the method under "2.2 chromatographic conditions", and the chromatogram was recorded. The standard curve was plotted with the amount of sample (mg) as abscissa and the peak area as ordinate, and is shown in tables 11, 12, 13 and 14, and fig. 11, 12, 13 and 14. The regression equation of ferulic acid is that y is 3,110.5432x +0.6280, R20.9997; the regression equation of paeoniflorin is that y is 1,104.5941x-3.7299, R20.9999; the regression equation of morroniside is 1,669.1956x +2.2166, R20.9998; the loganin regression equation is that y is 1,632.6540x +3.3200, R2＝0.9996。
The results show that the ferulic acid is 0.0060 mg-0.1800 mg, the paeoniflorin is 0.0211 mg-0.6317 mg, the morroniside is 0.0201 mg-0.6044 mg, and the loganin is 0.0253 mg-0.7596 mg.
TABLE 11 examination of the Linear relationship-Ferulic acid content
TABLE 12 examination of the Linear relationship-Paeoniflorin content
TABLE 13 examination of the Linear relationship-Morroniside content
TABLE 14 examination of the Linear relationship-loganin content
4.3 precision test
Measuring under the item of '3.2', recording chromatogram, calculating peak area, and finding out the results in tables 15, 16, 17 and 18, wherein the result shows that each component RSD is less than 2%, which indicates that the instrument precision is good.
TABLE 15 results of the precision test-Ferulic acid content
TABLE 16 results of precision tests-paeoniflorin content
TABLE 17 results of the precision test-morroniside content
TABLE 18 results of precision tests-loganin content
4.4 repeatability test
The determination is carried out under the item of '3.3', a chromatogram is recorded, the peak area is calculated, the result is shown in a table 19, and the RSD is less than 2%, which indicates that the method has good repeatability.
Table 19 repeatability test results (n ═ 6)
| Numbering | 1 | 2 | 3 | 4 | 5 | 6 | Mean value of | RSD(％) |
| Ferulic acid content (mg/g) | 0.179 | 0.184 | 0.182 | 0.186 | 0.181 | 0.183 | 0.183 | 1.33 |
| Content of paeoniflorin (mg/g) | 6.055 | 6.152 | 6.242 | 6.321 | 6.352 | 6.209 | 6.222 | 1.76 |
| Content of morroniside (mg/g) | 3.307 | 3.379 | 3.416 | 3.469 | 3.473 | 3.399 | 3.407 | 1.82 |
| Loganin content (mg/g) | 1.631 | 1.665 | 1.81 | 1.708 | 1.713 | 1.672 | 1.678 | 1.80 |
4.5 stability test
The measurement was carried out under the same conditions as in "3.4", the chromatogram was recorded, the peak area was calculated, and the measurement results are shown in Table 20. RSD of ferulic acid, paeoniflorin, morroniside and loganin is 1.43%, 0.69%, 1.28% and 0.81%, respectively, which indicates that the stability of the test solution is good within 72 hours.
TABLE 20 stability test results
4.6 accuracy test
By adopting a sample-adding recovery method, taking about 0.26g of the product powder, precisely weighing (9 parts in total), respectively and precisely adding 1mL, 2mL and 3mL (3 parts in each) of the mixed reference substance solution into conical flasks with stoppers, precisely adding 50mL of 70% methanol, sealing the conical flasks, weighing, ultrasonically treating (with the power of 250W and the frequency of 40kHz) for 30 minutes, taking out, cooling, weighing again, complementing the lost weight with 70% methanol, shaking up, filtering, precisely sucking 10 mu l of subsequent filtrate, injecting into a liquid chromatograph, and measuring to obtain the product. The results are shown in tables 21, 22, 23 and 24, the average recovery rates of ferulic acid, paeoniflorin, morroniside and loganin are 103.74%, 97.63%, 101.04% and 103.50% respectively, and the RSDs are 3.48%, 2.11%, 2.01% and 1.60% respectively, which indicates that the method has good accuracy.
TABLE 21 accuracy test results ferulic acid
TABLE 22 accuracy test results-paeoniflorin
TABLE 23 accuracy test results-morroniside
TABLE 24 accuracy test results-loganin
Example 2
In this example, the fingerprint prepared in example 1 was used to detect unqualified semen-raising jade decoction with missing raw materials.
A non-conforming sample 1 (raw material formulation of rehmanniae radix preparata: radix angelicae sinensis: cornus vinifera: 2: 1: 1), a non-conforming sample 2 (raw material formulation of rehmanniae radix preparata: radix paeoniae alba: cornus vinifera: 2: 1: 1), a non-conforming sample 3 (raw material formulation of rehmanniae radix preparata: radix angelicae sinensis: cornus vinifera: 2: 1: 1), and a non-conforming sample 4 (raw material formulation of radix angelicae sinensis: radix paeoniae alba: cornus vinifera: 1: 1) were prepared according to the method of 2.3 of example 1, a mixed control solution was prepared according to the method of 2.4 of example 1, and then the test was performed according to the chromatographic conditions of 2.2 and the method of 2.5 of example 1. The above-mentioned unqualified product is detected by the fingerprint of the above-mentioned example 1, and the chromatogram (see fig. 15) is recorded and introduced into the traditional Chinese medicine chromatogram similarity evaluation system software, and compared with the control chromatogram obtained in the example 1, the similarity is calculated, and the result is shown in the following table 25. From the results, it can be seen that the degree of similarity of the defective products was less than 0.9. The finger print in the embodiment 1 can be used as the quality control and evaluation index of the jade decoction for cultivating the seeds.
TABLE 25
Example 3
In this example, the fingerprint prepared in example 1 was used to test the Yangjingzao Yu decoction.
Preparation of a test solution: taking about 1g of the semen-nourishing jade decocted extract, precisely weighing, precisely adding 50mL of 70% methanol, sealing, weighing, ultrasonically treating (power 300W, frequency 40kHz) for 30 minutes, taking out, cooling, weighing again, supplementing the lost weight with 70% (volume percent) of methanol, shaking up, filtering, and taking the subsequent filtrate to obtain the test solution. The rest of the procedure was the same as in example 1.
The fingerprint results show that compared with the comparison fingerprint, 14 common peaks appear and the similarity is more than 0.95. The result shows that the fingerprint constructed in the example 1 can also be used for controlling and nourishing the refined seed jade decocted extract.
Example 4
In this example, the fingerprint prepared in example 1 was used to test the Yangjingdan Yu granule.
Preparation of a test solution: taking about 1g of Yangjingdao jade granules, precisely weighing, precisely adding 50mL of 70% methanol, sealing, weighing, ultrasonically treating (power 300W, frequency 40kHz) for 30 minutes, taking out, cooling, weighing again, supplementing the lost weight with 70% (volume percentage) of methanol, shaking up, filtering, and taking the subsequent filtrate to obtain the test solution. The rest of the procedure was the same as in example 1.
The fingerprint results show that compared with the comparison fingerprint, 14 common peaks appear, and the similarity is more than 0.95, which indicates that the fingerprint constructed in the embodiment 1 can also be used for quality control and fine seed corn granule cultivation.
Comparative example 1
This comparative example is a comparative example to example 1, the main differences including the different chromatographic conditions compared to example 1. The specific chromatographic conditions were as follows:
a chromatographic column: phenomenex C18 (4.6X 250mm, 5 μm); mobile phase: acetonitrile was used as a mobile phase a, and a phosphoric acid aqueous solution having a volume concentration of 0.1% was used as a mobile phase B, and the elution was carried out at equal intervals as specified in the following table (see table 26); column temperature: 25 ℃; flow rate: 1 mL/min; detection wavelength: 240 nm; sample introduction amount: 10 μ L.
TABLE 26 mobile phase isocratic elution conditions
| Time (min) | Acetonitrile (A)% | 0.1% phosphoric acid (B)% |
| 0～50 | 13 | 87 |
The fingerprint spectrum result constructed by the comparative example is shown in figure 16, wherein 1: morroniside; 2: loganin; 3: paeoniflorin; 4: ferulic acid. The results show that the morroniside interference peaks are more and difficult to separate individually.
Comparative example 2
This comparative example is a comparative example to example 1, the main differences including the different chromatographic conditions compared to example 1. The specific chromatographic conditions were as follows:
a chromatographic column: phenomenex C18 (4.6X 250mm, 5 μm); mobile phase: acetonitrile is used as a mobile phase A, a phosphoric acid aqueous solution with the volume concentration of 0.1 percent is used as a mobile phase B, and gradient elution is carried out according to the specification in the following table 27; column temperature: 25 ℃; flow rate: 1 mL/min; detection wavelength: 240 nm; sample introduction amount: 10 μ L.
The fingerprint spectrum result constructed by the comparative example is shown in figure 17, wherein 1: morroniside; 2: loganin; 3: paeoniflorin; 4: ferulic acid. The results show that the morroniside interference peaks are more and difficult to separate individually.
TABLE 27 mobile phase gradient elution conditions
| Time (min) | Acetonitrile (A)% | 0.1% phosphoric acid (B)% |
| 0～10 | 10～12 | 90～88 |
| 10～15 | 12～14 | 88～86 |
| 15～30 | 14～20 | 86～80 |
| 30～40 | 20～38 | 80～62 |
Comparative example 3
This comparative example is a comparative example to example 1, the main differences including the different chromatographic conditions compared to example 1. The specific chromatographic conditions were as follows:
a chromatographic column: phenomenex C18 (4.6X 250mm, 5 μm); mobile phase: acetonitrile is used as a mobile phase A, a phosphoric acid aqueous solution with the volume concentration of 0.1 percent is used as a mobile phase B, and gradient elution is carried out according to the specification in the following table 28; column temperature: 25 ℃; flow rate: 1 mL/min; detection wavelength: 240 nm; sample introduction amount: 10 μ L.
The fingerprint spectrum result constructed by the comparative example is shown in a figure 18, wherein 1: morroniside; 2: loganin; 3: paeoniflorin; 4: ferulic acid. The results show that the morroniside interference peaks are more and difficult to separate individually.
TABLE 28 mobile phase gradient elution conditions
| Time (min) | Acetonitrile (A)% | 0.1% phosphoric acid (B)% |
| 0～25 | 10～14 | 90～86 |
| 25～60 | 14～26 | 86～74 |
Comparative example 4
This comparative example is a comparative example to example 1, the main differences including the different chromatographic conditions compared to example 1. The specific chromatographic conditions were as follows:
a chromatographic column: phenomenex C18 (4.6X 250mm, 5 μm); mobile phase: acetonitrile is used as a mobile phase A, a phosphoric acid aqueous solution with the volume concentration of 0.1 percent is used as a mobile phase B, and gradient elution is carried out according to the specification in the following table 29; column temperature: 25 ℃; flow rate: 1 mL/min; detection wavelength: 240 nm; sample introduction amount: 10 μ L.
The fingerprint spectrum result constructed by the comparative example is shown in fig. 19, wherein 1: morroniside; 2: loganin; 3: paeoniflorin; 4: ferulic acid. The results show that the morroniside interference peaks are more and difficult to separate individually.
TABLE 29 mobile phase gradient elution conditions
Comparative example 5
This comparative example is a comparative example to example 1, the main differences including the different chromatographic conditions compared to example 1. The specific chromatographic conditions were as follows:
a chromatographic column: phenomenex C18 (4.6X 250mm, 5 μm); mobile phase: acetonitrile is used as a mobile phase A, a phosphoric acid aqueous solution with the volume concentration of 0.1 percent is used as a mobile phase B, and gradient elution is carried out according to the specification in the following table 30; column temperature: 25 ℃; flow rate: 1 mL/min; detection wavelength: 240 nm; sample introduction amount: 10 μ L.
The fingerprint spectrum result constructed by the comparative example is shown in figure 20, wherein 1: morroniside; 2: loganin; 3: paeoniflorin; 4: ferulic acid. The results show that the morroniside interference peaks are more and difficult to separate individually.
TABLE 30 mobile phase gradient elution conditions
| Time (min) | Acetonitrile (A)% | 0.1% phosphoric acid (B)% |
| 0～25 | 10～14 | 90～86 |
| 25～46 | 14～27 | 86～73 |
| 46～60 | 27～85 | 73～15 |
Comparative example 6
This comparative example is that of example 1, the main difference compared to example 1 consists in the different elution gradients used. The specific chromatographic conditions were as follows:
a chromatographic column: phenomenex C18 (4.6X 250mm, 5 μm); mobile phase: acetonitrile is used as a mobile phase A, a phosphoric acid aqueous solution with the volume concentration of 0.1 percent is used as a mobile phase B, and gradient elution is carried out according to the specification in the following table 31; column temperature: 25 ℃; flow rate: 0.8 mL/min; detection wavelength: 240 nm; sample introduction amount: 10 μ L.
The comparison example adopts the chromatographic condition to construct a fingerprint and is used for detecting the refined seed-nourishing jade formula decocted extract. The fingerprint spectrum result constructed by the comparative example is shown in figure 21, wherein, 1: morroniside; 2: loganin; 3: paeoniflorin; 4: ferulic acid; 5: acteoside. The results show that the morroniside interference peaks are more and difficult to separate individually. Meanwhile, the inventor finds that the constructed fingerprint spectrum has larger similarity difference with the spectrum of the refined seed nourishing jade square decocted extract, and the constructed fingerprint spectrum cannot be used for the quality control of the refined seed nourishing jade square decocted extract at all.
TABLE 31 conditions of mobile phase gradient elution
| Time (min) | Acetonitrile (A)% | 0.1% phosphoric acid (B)% |
| 0～25 | 10～14 | 90～86 |
| 25～46 | 14～27 | 86～73 |
| 46～50 | 27～80 | 73～20 |
Comparative example 7
Fingerprint spectra constructed by this comparative example is that of example 1, the main difference compared to example 1 consists in the different elution gradients used. The specific chromatographic conditions were as follows:
a chromatographic column: phenomenex C18 (4.6X 250mm, 5 μm); mobile phase: acetonitrile is used as a mobile phase A, a phosphoric acid aqueous solution with the volume concentration of 0.1 percent is used as a mobile phase B, and gradient elution is carried out according to the specification in the following table 32; column temperature: 25 ℃; flow rate: 1.0 mL/min; detection wavelength: 240 nm; sample introduction amount: 10 μ L.
The comparative example adopts the chromatographic condition to construct a fingerprint spectrum and is used for detecting the Yufang granules for cultivating the elite seeds. The fingerprint results constructed in this comparative example are shown in fig. 22, where 1: morroniside; 2: loganin; 3: paeoniflorin; 4: ferulic acid. The results show that the morroniside interference peaks are more and difficult to separate individually. Meanwhile, the inventor finds that the constructed fingerprint spectrum has larger similarity difference with the spectrum of the refined seed nourishing jade square granule, and the constructed fingerprint spectrum cannot be used for quality control of the refined seed nourishing jade square decocted extract at all.
TABLE 32 mobile phase gradient elution conditions
| Time (min) | Acetonitrile (A)% | 0.1% phosphoric acid (B)% |
| 0～30 | 5～25 | 95～75 |
| 30～60 | 25～80 | 75～20 |
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. A construction method of a fingerprint of a formula for cultivating elite seeds is characterized by comprising the following steps:
preparing a test solution: preparing the Yangjingdao jade decoction into freeze-dried powder, dissolving the freeze-dried powder in an organic solvent, and filtering to obtain a test solution;
preparation of a reference solution: dissolving ferulic acid, paeoniflorin, morroniside, and loganin reference substances in organic solvent to obtain reference substance solution;
constructing a fingerprint spectrum: sucking the test solution and the reference solution, and injecting into a high performance liquid chromatograph for determination to obtain a fingerprint of the Yangjingdan jade formula with a common characteristic peak;
the chromatographic conditions employed for the assay include:
a chromatographic column: octadecylsilane chemically bonded silica is used as a filling agent;
mobile phase: acetonitrile is taken as a mobile phase A, and phosphoric acid aqueous solution with volume concentration of 0.08-0.12% is taken as a mobile phase B;
gradient elution mode: the volume percent of the mobile phase A is changed from 5% to 15% and the volume percent of the mobile phase B is changed from 95% to 85% within 0-25 min; the volume percentage of the mobile phase A is changed from 15% to 20% and the volume percentage of the mobile phase B is changed from 85% to 80% from 25min to 40 min; the volume percentage of the mobile phase A is changed from 20% to 50% and the volume percentage of the mobile phase B is changed from 80% to 50% within 40-60 min.
2. The method for constructing the fingerprint of the Yangjingdao jade formula according to claim 1, wherein the Yangjingdao jade formula comprises granules, extracts and decoctions.
3. The method for constructing the fingerprint of the essence-nourishing jade formula according to claim 2, wherein the flow rate of the mobile phase is 0.8mL/min to 1.2 mL/min; the column temperature of the chromatographic column is 25-35 ℃; the detection wavelength adopted by the determination is 210 nm-250 nm.
4. The method for constructing the fingerprint of the Yangjingdan jade formula according to any one of claims 1 to 3, wherein the organic solvent is a methanol solution with a volume concentration of 70-100%.
5. The method for constructing the fingerprint of the essence-nourishing jade prescription according to any one of claims 1 to 3, wherein 0.5g to 1g of the lyophilized powder is added to 25mL to 50mL of the organic solvent.
6. The method for constructing fingerprint of Yangjingdan Yu prescription according to any one of claims 1 to 3, wherein each 1mL of the control solution contains 0.6 μ g to 24 μ g of ferulic acid, 2.1 μ g to 84.2 μ g of paeoniflorin, 2.0 μ g to 80.6 μ g of morroniside and 2.5 μ g to 101.3 μ g of loganin.
7. A method for determining a formula for breeding jade with nourishing essence is characterized by comprising the following steps:
preparing a test solution: dissolving the refined seed jade formula to be tested in an organic solvent, and filtering to obtain a test solution;
preparation of a reference solution: dissolving ferulic acid, paeoniflorin, morroniside, and loganin reference substances in organic solvent to obtain reference substance solution;
and (3) testing the test solution: sucking the test solution and the reference solution, and injecting into a high performance liquid chromatograph for determination to obtain a spectrum of the elite seed preparation to be tested; comparing the relative retention time of each peak and/or the peak area of each peak in the atlas of the elite seed jade preparation to be tested and the fingerprint constructed in the claim 1, and finishing the qualitative and/or quantitative determination of the elite seed jade preparation to be tested;
the chromatographic conditions employed for the assay include:
a chromatographic column: octadecylsilane chemically bonded silica is used as a filling agent;
mobile phase: acetonitrile is taken as a mobile phase A, and phosphoric acid aqueous solution with volume concentration of 0.08-0.12% is taken as a mobile phase B;
gradient elution mode: the volume percent of the mobile phase A is changed from 5% to 15% and the volume percent of the mobile phase B is changed from 95% to 85% within 0-25 min; the volume percentage of the mobile phase A is changed from 15% to 20% and the volume percentage of the mobile phase B is changed from 85% to 80% from 25min to 40 min; the volume percentage of the mobile phase A is changed from 20% to 50% and the volume percentage of the mobile phase B is changed from 80% to 50% within 40-60 min.
8. The method for determining the essence-nourishing jade-like prescription according to claim 7, wherein the essence-nourishing jade-like prescription comprises granules, extractum and decoction.
9. The method for determining the essence-nourishing jade-breeding prescription according to claim 8, wherein the flow rate of the mobile phase is 0.8mL/min to 1.2 mL/min; the column temperature of the chromatographic column is 25-35 ℃; the detection wavelength adopted by the determination is 210 nm-250 nm.
10. The method for determining the elite seed and jade formula according to any one of claims 7 to 9, wherein the organic solvent is a methanol solution with a volume concentration of 70% to 100%.
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