CN115201393B - Quality detection method of rhizoma polygonati and semen euryales soup - Google Patents
Quality detection method of rhizoma polygonati and semen euryales soup Download PDFInfo
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Abstract
The application discloses a quality detection method of polygonatum sibiricum gorgon fruit soup, which utilizes liquid chromatography to measure fingerprint spectrum of polygonatum sibiricum gorgon fruit soup and content of paeoniflorin, albiflorin and gallic acid, and the average value of the content of paeoniflorin, albiflorin and gallic acid in a polygonatum sibiricum gorgon fruit soup sample is measured to be 0.119%, 0.126% and 0.039% respectively. According to the quality detection method of the Polygonatum sibiricum Gordon euryale seed soup, disclosed by the application, the quality of the Polygonatum sibiricum Gordon euryale seed soup is assessed through multiple-aspect measurement, a feasible quality standard of the Polygonatum sibiricum and Gordon euryale seed soup can be established, and effective control of the quality of the Polygonatum sibiricum Gordon euryale seed soup is achieved.
Description
Technical Field
The application relates to the technical field of quality control of traditional Chinese medicinal materials, in particular to a quality detection method of rhizoma polygonati and semen euryales soup.
Background
The modern medicine needs to have three characteristics of stability, uniformity, safety and effectiveness, and the Chinese patent medicine is difficult to compare with western medicines in the aspects, so that the detection is more needed by adopting various means, and the reliability and the stability of the detection result are ensured. The rhizoma polygonati and semen euryales soup is derived from cold Bai Zhifang of clinical theraphy of traditional Chinese medicine, has the efficacy of tonifying spleen yin and is mainly used for treating spleen yin deficiency syndrome. It comprises rhizoma Polygonati, semen euryales, rhizoma Dioscoreae, radix Paeoniae alba, fructus Jujubae, radix Pseudostellariae and herba Eupatorii. At present, a systematic quality detection method for the rhizoma polygonati and semen euryales soup is not formed yet, and the traditional detection means are only adopted to detect the rhizoma polygonati and semen euryales soup, so that the quality control requirement of a traditional Chinese medicine formula cannot be met. Therefore, it is necessary to establish a quality detection method of rhizoma polygonati and semen euryales soup for controlling the quality of medicinal materials.
Disclosure of Invention
The quality detection method for the Polygonatum sibiricum Gordon euryale seed soup aims at solving the defects of the prior art, and is capable of controlling the quality of the Polygonatum sibiricum Gordon euryale seed soup better, representing the quality of a medicine and improving the stability of the medicine.
In order to achieve the above purpose, the present application adopts the following technical scheme:
measuring the Shang Zhiwen spectrum of rhizoma Polygonati and the content of paeoniflorin, albiflorin and gallic acid, and determining the fingerprint spectrum and the content of paeoniflorin, albiflorin and gallic acid by liquid chromatography;
the fingerprint determination by liquid chromatography comprises: performing liquid chromatograph analysis, taking solutions prepared by the taste-deficient negative samples as reference solutions, taking solutions prepared by paeoniflorin, albiflorin and gallic acid reference substances as reference solutions, taking solutions prepared by Polygonatum sibiricum and semen euryales soup samples as test solutions, respectively precisely sucking the reference solutions, the reference solutions and the test solutions, respectively injecting the reference solutions, the reference solutions and the test solutions into the liquid chromatograph, and measuring to obtain the extract; wherein the chromatographic conditions adopted are that: thermo Hypersil GOLD C18 column 18; mobile phase: using methanol as a mobile phase A and 0.1% formic acid aqueous solution as a mobile phase B, and performing gradient elution according to the specification of a table a;
table a gradient elution procedure
| Time (min) | Mobile phase a (%) | Mobile phase B (%) |
| 0→3 | 2 | 98 |
| 3→9 | 2→16 | 98→84 |
| 9→13 | 16→18 | 84→82 |
| 13→14 | 18→30 | 82→70 |
| 14→19 | 30→34 | 70→66 |
| 19→22 | 34→45 | 66→55 |
| 22→24 | 45→90 | 55→10 |
Flow rate: 0.3mL/min; column temperature: 30 ℃; sample injection amount: 10. Mu.L; detection wavelength: 280nm.
In one embodiment, the fingerprint determination by liquid chromatography further comprises the steps of:
(1) Preparation of reference solution a: weighing appropriate amount of rhizoma Polygonati, semen euryales, rhizoma Dioscoreae, radix Paeoniae alba, fructus Jujubae, radix Pseudostellariae, and herba Eupatorii, pulverizing into coarse powder, and sieving with 40 mesh sieve; precisely weighing each single medicine decoction piece powder according to the proportion of the rhizoma polygonati and semen euryales soup, and respectively preparing taste-lacking negative samples without rhizoma polygonati, semen euryales, chinese yam, white paeony root, chinese date, radix pseudostellariae or herba eupatorii by taking 3g as the total amount of the prescription; precisely weighing 1.0g of the taste-deficient negative sample, placing in a conical bottle with a plug, precisely adding 50mL of boiling water, weighing the mass, performing ultrasonic extraction for 30min, standing to room temperature, weighing the mass again, supplementing the lost mass with boiling water, shaking, and preserving in dark at 4 ℃; filtering with 0.22 μm needle filter before sample injection, and collecting filtrate;
(2) Preparing a reference substance solution a: respectively weighing appropriate amounts of paeoniflorin, albiflorin and gallic acid reference substances, precisely weighing, placing into the same volumetric flask, adding methanol for dissolving, and fixing volume to scale to obtain paeoniflorin, albiflorin and gallic acid with mass concentration ratio of 0.5:0.311:0.697 of control solution a;
(3) Preparing a test sample solution a: weighing appropriate amount of rhizoma Polygonati, semen euryales, rhizoma Dioscoreae, radix Paeoniae alba, fructus Jujubae, radix Pseudostellariae, and herba Eupatorii, pulverizing into coarse powder, and sieving with 40 mesh sieve; precisely weighing each single medicine decoction piece powder according to the proportion of rhizoma polygonati and semen euryales soup, and uniformly mixing to obtain rhizoma polygonati and semen euryales Shang Yangpin; accurately weighing Shang Yangpin 1.0.0 g of the rhizoma polygonati and semen euryales, placing in a conical bottle with a plug, accurately adding 50mL of boiling water, weighing the mass, performing ultrasonic extraction for 30min, standing to room temperature, weighing the mass again, supplementing the lost mass with boiling water, shaking, and storing in a dark place at 4 ℃; filtering with 0.22 μm needle filter before sampling, and collecting the filtrate.
In one embodiment, the content of paeoniflorin, albiflorin and gallic acid measured by liquid chromatography comprises:
performing liquid chromatograph analysis, taking a solution prepared from paeoniflorin, albiflorin and gallic acid reference substances as a reference substance solution b, taking a solution prepared from a polygonatum gordon euryale seed soup sample as a test substance solution b, precisely sucking the reference substance solution b and the test substance solution b respectively, injecting the reference substance solution b and the test substance solution b into the liquid chromatograph respectively, and measuring to obtain the extract; wherein the chromatographic conditions adopted are that: thermo Hypersil GOLD C18 column 18; mobile phase: methanol is taken as a mobile phase A, 0.1% formic acid aqueous solution is taken as a mobile phase B, and gradient elution is carried out according to a specified rule; flow rate: 0.3mL/min; column temperature: 30 ℃; sample injection amount: 10. Mu.L; detection wavelength: 280nm.
In one embodiment, the measuring paeoniflorin, albiflorin and gallic acid content by liquid chromatography further comprises the following steps:
(1) Preparing a reference substance solution b: precisely weighing paeoniflorin 5.00mg, albiflorin 3.11mg and gallic acid 6.97mg, adding methanol solution, and fixing volume in a 10mL volumetric flask to obtain reference solution b;
(2) Preparing a test sample solution b: accurately weighing Shang Yangpin 1.0.0 g of rhizoma Polygonati and semen euryales, placing in a conical bottle with a plug, accurately adding 50mL of boiling water, weighing the mass, performing ultrasonic extraction for 30min, standing to room temperature, weighing the mass again, supplementing the lost mass with boiling water, shaking, and preserving in dark at 4deg.C; filtering with 0.22 μm needle filter before sampling, and collecting the filtrate.
In one embodiment, the quality detection method of the rhizoma polygonati gorgon fruit soup further comprises cluster analysis, principal component analysis and orthorhombic partial least square method discriminant analysis of the rhizoma polygonati gorgon fruit Shang Zhiwen map.
In one embodiment, the content of gallic acid, paeoniflorin and paeoniflorin in the Polygonatum sibiricum Gordon euryale seed soup sample is calculated by an external standard one-point method.
In one embodiment, the fingerprint of the rhizoma polygonati gorgon fruit soup is determined by fingerprint measurement of the rhizoma polygonati gorgon fruit soup, and 19 common peaks are determined to form the fingerprint of the rhizoma polygonati gorgon fruit soup.
In one embodiment, the fingerprint of rhizoma Polygonati and semen euryales decoction is compared with the fingerprint of the reference solution, and it is determined that peak 8 is gallic acid, peak 18 is albiflorin, and peak 19 is paeoniflorin.
In one embodiment, comparing the fingerprint of rhizoma Polygonati semen euryales decoction with the fingerprint of each taste-deficient negative sample, and attributing the 19 common peaks to herba Eupatorii, wherein peak 1 and peak 18 are attributed to herba Eupatorii; peak 2 and peak 11 are ascribed to white peony root and eupatorium; peak 5 is attributed to white peony root, eupatorium and yam; peak 6 and Peak 8 are ascribed to Dioscorea opposita; peak 7 belongs to Polygonatum sibiricum; peak 10 is attributed to Eupatorium and Dioscorea opposita; peak 15 and peak 19 are ascribed to white peony root.
In one embodiment, a plurality of batches of rhizoma polygonati gorgon euryale Shang Zhiwen spectrum data are sequentially imported into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, one batch of rhizoma polygonati gorgon euryale Shang Zhiwen spectrum is selected as a reference fingerprint, and a comparison fingerprint is generated by a multi-point correction and an average method.
The beneficial effects of this application are:
the quality of the Polygonatum sibiricum Gordon euryale seed soup is evaluated by researching the fingerprint spectrum of the Polygonatum sibiricum and the content measurement of paeoniflorin, albiflorin and gallic acid and measuring the content in multiple aspects, a solid foundation is laid for the stable quality of the product, a feasible quality standard of the Polygonatum sibiricum and Gordon euryale seed soup can be established, the effective control of the quality of the Polygonatum sibiricum and Gordon euryale seed soup is realized, and the chromatographic condition of the Polygonatum sibiricum and Gordon euryale seed soup is adopted for liquid phase analysis, so that a chromatogram with better and clearer separation degree can be obtained. In the fingerprint of the polygonatum sibiricum Gorgon fruit Shang Yangpin of a plurality of batches, the RSD of the relative retention time of each common peak is 0.023-2.11%, which shows that the chemical composition consistency is better; RSD of the relative peak area of each common peak is 1.23% -2.79%, which shows that the contents of certain common chemical components of the Polygonatum sibiricum and Gordon euryale seed soup samples in each batch are different; the similarity results of the Shang Yangpin fingerprint of the polygonatum sibiricum and the reference fingerprint of each batch are not lower than 0.885, which shows that the chemical composition consistency of the polygonatum sibiricum and the gorgon euryale soup of each batch is better; the average value of paeoniflorin, albiflorin and gallic acid content in each batch of polygonatum sibiricum gordon euryale seed soup samples is 0.119%, 0.126% and 0.039% respectively. The finger print and content determination method of the polygonatum sibiricum gorgon fruit Shang Yaocai established by the application is good in repeatability and strong in specificity, and can be used for quality evaluation of polygonatum sibiricum gorgon fruit soup medicinal materials and related preparations.
Drawings
Fig. 1 is a fingerprint of 15 batches of rhizoma polygonati Gordon euryales Shang Yangpin and a control fingerprint in the Shang Zhiwen spectrum measurement of rhizoma polygonati Gordon euryales in the application; wherein S1-S15 respectively represent fingerprint patterns of 15 batches of rhizoma polygonati and gorgon euryale seeds Shang Yangpin, and R represents a comparison fingerprint pattern.
Fig. 2 is a graph of similarity evaluation between the fingerprints of 15 batches of polygonatum sibiricum gorgon fruit Shang Yangpin and the reference fingerprint in the Shang Zhiwen spectrum measurement of polygonatum sibiricum.
Fig. 3 is a graph of a mixed control of the traditional Chinese medicine glycoside, the paeoniflorin and the gallic acid, which is determined by the graph of the polygonatum sibiricum gorgon fruit Shang Zhiwen.
Fig. 4 is a fingerprint of a polygonatum gorgon fruit Shang Yangpin in the Shang Zhiwen spectrum measurement of polygonatum gorgon fruit.
FIG. 5 is a graph comparing the flavor-deficient negative sample with the Polygonatum sibiricum Gordon euryale seed soup sample in the Polygonatum sibiricum Gordon euryale seed Shang Zhiwen graph determination of the present application; wherein S1-S7 respectively represent a white peony root negative sample, a Chinese date negative sample, a rhizoma polygonati negative sample, a herba eupatorii negative sample, a semen euryales negative sample, a Chinese yam negative sample and a radix pseudostellariae negative sample in sequence, and S8 represents rhizoma polygonati semen euryales Shang Yangpin.
Fig. 6 is a cluster analysis tree diagram of 15 batches of polygonatum gorgon fruit Shang Yangpin in the present application.
Fig. 7 is a principal component analysis chart of 15 batches of polygonatum sibiricum gorgon fruit soup samples in the present application.
FIG. 8 is a graph showing the result of the substitution test of OPLS-DA model of 15 batches of Polygonatum sibiricum Gordon euryales Shang Yangpin.
FIG. 9 is an OPLS-DA score of lot 15 of Polygonatum sibiricum Gordon euryales Shang Yangpin of the present application.
Fig. 10 is a PCA load diagram of 19 common peaks of a finger print of polygonatum sibiricum gorgon fruit Shang Yangpin in the application; wherein, p1 to p19 represent common peaks 1 to 19.
Fig. 11 is a graph of VIP values of 19 common peak areas of the finger print Shang Yangpin of polygonatum sibiricum.
FIG. 12 is a chromatogram of comparative example 1.
Fig. 13 is a chromatogram of comparative example 2.
FIG. 14 is a chromatogram of comparative example 3.
Detailed Description
For the purposes of making the objects, technical solutions and advantages of the present application more apparent, the present application will be further described in detail below with reference to the accompanying drawings.
Example 1
1. Fingerprint measurement
1.1 liquid chromatography
(1) Preparing a reference solution: weighing appropriate amount of rhizoma Polygonati, semen euryales, rhizoma Dioscoreae, radix Paeoniae alba, fructus Jujubae, radix Pseudostellariae, and herba Eupatorii, pulverizing into coarse powder, and sieving with 40 mesh sieve; precisely weighing each single medicine decoction piece powder according to the proportion of the rhizoma polygonati and semen euryales soup, and respectively preparing taste-lacking negative samples without rhizoma polygonati, semen euryales, chinese yam, white paeony root, chinese date, radix pseudostellariae or herba eupatorii by taking 3g as the total amount of the prescription; precisely weighing 1.0g of the taste-deficient negative sample, placing in a conical bottle with a plug, precisely adding 50mL of boiling water, weighing the mass, performing ultrasonic extraction for 30min, standing to room temperature, weighing the mass again, supplementing the lost mass with boiling water, shaking, and preserving in dark at 4 ℃; filtering by a 0.22 mu m needle filter before sample injection, and taking a subsequent filtrate to obtain seven odor-deficiency negative sample reference substance solutions;
(2) Preparing a reference substance solution: precisely weighing paeoniflorin 5.00mg, albiflorin 3.11mg and gallic acid 6.97mg, adding methanol solution, and fixing volume in 10mL volumetric flask to obtain reference solution;
(3) Preparing a test solution: weighing appropriate amount of rhizoma Polygonati, semen euryales, rhizoma Dioscoreae, radix Paeoniae alba, fructus Jujubae, radix Pseudostellariae, and herba Eupatorii, pulverizing into coarse powder, and sieving with 40 mesh sieve; precisely weighing each single medicine decoction piece powder according to the proportion of rhizoma polygonati and semen euryales soup, and uniformly mixing to obtain rhizoma polygonati and semen euryales Shang Yangpin; accurately weighing Shang Yangpin 1.0.0 g of the rhizoma polygonati and semen euryales, placing in a conical bottle with a plug, accurately adding 50mL of boiling water, weighing the mass, performing ultrasonic extraction for 30min, standing to room temperature, weighing the mass again, supplementing the lost mass with boiling water, shaking, and storing in a dark place at 4 ℃; filtering with 0.22 μm needle filter before sampling, and collecting the filtrate.
Chromatographic conditions:
chromatographic column: thermo Hypersil GOLD C18 column (2.1 mm. Times.100 mm,1.9 μm); mobile phase: gradient elution was performed with methanol as mobile phase a and 0.1% formic acid aqueous solution as mobile phase B, as specified in table 1;
TABLE 1 gradient elution procedure
| Time (min) | Mobile phase a (%) | Mobile phase B (%) |
| 0→3 | 2 | 98 |
| 3→9 | 2→16 | 98→84 |
| 9→13 | 16→18 | 84→82 |
| 13→14 | 18→30 | 82→70 |
| 14→19 | 30→34 | 70→66 |
| 19→22 | 34→45 | 66→55 |
| 22→24 | 45→90 | 55→10 |
Flow rate: 0.3mL/min; column temperature: 30 ℃; sample injection amount: 10. Mu.L; detection wavelength: 280nm.
Assay: respectively precisely sucking 10 μl of reference solution, reference solution and sample solution, respectively, injecting into liquid chromatograph, and recording chromatogram. The obtained 15 batches of polygonatum sibiricum gorgon fruit decoction chromatogram data are sequentially imported into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition), a sample S1 is taken as a reference fingerprint, an average method (time window width=0.10) is adopted to generate a comparison fingerprint, and automatic matching is carried out after multi-point correction, so that the result is shown in fig. 1. The results show that the RSD of each common peak versus retention time is less than 1.0%. And (3) performing similarity evaluation on the high-performance liquid chromatograms of 15 batches of polygonatum sibiricum gorgon fruit soup samples and the control fingerprint, wherein the similarity of the S1-S15 samples and the control fingerprint is higher than 0.85, as shown in figure 2, the chemical compositions of the polygonatum sibiricum gorgon fruit Shang Yangpin in different batches are basically the same, and the difference is small.
RSD of the relative retention time of the common peaks of 15 batches of rhizoma polygonati and gorgon fruit soup is 0.023-2.11%, which shows that the chemical composition consistency is better; the RSD of the relative peak area of each common peak is 1.23-2.79%, which suggests that the content of certain common chemical components of 15 batches of rhizoma polygonati gordon euryale seed soup is different.
1.2 analytical method validation
Repeatability test: taking a proper amount of sample of rhizoma polygonati and gorgon fruit soup (S15), preparing six sample solutions in parallel according to the method under 1.1, and carrying out sample injection measurement according to the chromatographic condition under 1.1. The relative retention time and relative peak area of each common peak were calculated using paeoniflorin chromatographic peak as reference peak (S). The results showed that the RSD of each common peak was less than 2.00% relative to the retention time and less than 3.00% relative to the peak area, indicating good reproducibility of the method.
Precision test: taking a proper amount of sample of rhizoma polygonati and gorgon fruit soup (S1), preparing a sample solution according to the method of 1.1, continuously injecting sample for six times according to the chromatographic condition of 1.1, and recording a chromatogram. The relative retention time and the relative peak area of each common peak are calculated by taking paeoniflorin chromatographic peaks (which are stable in chromatographic peak, good in separation degree and moderate in retention time and peak area) as reference peaks (S). The results showed that the RSD of each common peak was less than 2.11% relative to the retention time and less than 2.79% relative to the peak area, indicating good precision of the instrument used.
Stability test: taking a proper amount of sample of rhizoma polygonati and gorgon fruit soup (S15), preparing test solution according to the method under 1.1, respectively placing for 0, 3, 6, 9, 12, 24 and 48 hours at room temperature, and carrying out sample injection measurement according to the chromatographic condition under 1.1. The relative retention time and relative peak area of each common peak were calculated using paeoniflorin chromatographic peak as reference peak (S). The results show that the RSD of each common peak relative retention time is less than 2.00% and the RSD of the relative peak area is less than 3.00%, which shows that the stability of the test sample solution is good when the test sample solution is placed at room temperature for 48 hours.
1.3 assignment and assignment of common peaks of finger prints
Taking sample solution of rhizoma Polygonati semen euryales soup and reference substance solution, and introducing under chromatographic conditions of 1.1 item, wherein the chromatogram of reference substance solution is shown in figure 3, and the chromatogram of rhizoma Polygonati semen euryales soup sample solution is shown in figure 4. Through retention time and graph comparison analysis, the peak No. 8 is gallic acid, the peak No. 18 is paeoniflorin, and the peak No. 19 is paeoniflorin.
Seven kinds of taste-lacking negative sample reference solutions and rhizoma polygonati gordon euryale seed soup test solution are subjected to sample injection measurement according to chromatographic conditions under the condition of 1.1, and a chromatogram is recorded, and the result is shown in figure 5. Comparing the chromatograms of the rhizoma polygonati semen euryales Shang Yangpin and seven taste-lacking negative samples, and carrying out medicinal material attribution on 19 common peaks. Wherein peak 1 and peak 18 are attributed to eupatorium; peak 2 and peak 11 are ascribed to white peony root and eupatorium; peak 5 is attributed to white peony root, eupatorium and yam; peak 6 and Peak 8 are ascribed to Dioscorea opposita; peak 7 belongs to Polygonatum sibiricum; peak 10 is attributed to Eupatorium and Dioscorea opposita; peak 15 and peak 19 are ascribed to white peony root.
2. Content determination
2.1 test methods
The chromatographic peaks of the rhizoma polygonati Gordon euryale Shang Zhiwen are identified, and the chromatographic peaks of the rhizoma polygonati Gordon euryale Shang Zhiwen are gallic acid, paeoniflorin and paeoniflorin respectively at numbers 8, 18 and 19. These three components were therefore selected for content determination.
Measuring the content of gallic acid, paeoniflorin and paeoniflorin by adopting a liquid chromatography, taking a solution prepared from gallic acid, paeoniflorin and paeoniflorin reference substances as a reference substance solution, taking a solution prepared from a polygonatum sibiricum gordon euryale seed soup sample as a test substance solution, respectively precisely sucking the reference substance solution and the test substance solution, respectively injecting the reference substance solution and the test substance solution into a liquid chromatograph, and measuring; wherein the chromatographic conditions adopted are that: thermo Hypersil GOLD C18 column (2.1 mm. Times.100 mm,1.9 μm); mobile phase: gradient elution was performed with methanol as mobile phase a and 0.1% formic acid aqueous solution as mobile phase B, as specified in table 1;
flow rate: 0.3mL/min; column temperature: 30 ℃; sample injection amount: 10. Mu.L; detection wavelength: 280nm.
Preparing a reference substance solution: precisely weighing paeoniflorin 5.00mg, albiflorin 3.11mg and gallic acid 6.97mg, adding methanol solution, and fixing volume in 10mL volumetric flask to obtain reference solution.
Preparing a test solution: accurately weighing Shang Yangpin 1.0.0 g of rhizoma Polygonati and semen euryales, placing in a conical bottle with a plug, accurately adding 50mL of boiling water, weighing the mass, performing ultrasonic extraction for 30min, standing to room temperature, weighing the mass again, supplementing the lost mass with boiling water, shaking, and preserving in dark at 4deg.C; filtering with 0.22 μm needle filter before sampling, and collecting the filtrate.
Assay: precisely sucking 10 μl of each of the control solution and the sample solution, and measuring with a liquid chromatograph.
2.2 determination of the content of the Chinese herbal medicine in the Polygonatum sibiricum Gordon euryale seed decoction
According to the proposed content determination method, the content of gallic acid, albiflorin and paeoniflorin in 15 batches of polygonatum sibiricum gordon euryale seed soup samples is calculated by adopting an external standard one-point method, and the result is shown in Table 2.
Table 2 determination of the main component content (n=2)
| Numbering device | Paeoniflorin (mg/g) | Albiflorin (mg/g) | Gallic acid (mg/g) |
| S1 | 0.116 | 0.119 | 0.041 |
| S2 | 0.120 | 0.127 | 0.036 |
| S3 | 0.122 | 0.126 | 0.033 |
| S4 | 0.115 | 0.133 | 0.045 |
| S5 | 0.118 | 0.125 | 0.035 |
| S6 | 0.119 | 0.134 | 0.037 |
| S7 | 0.114 | 0.118 | 0.039 |
| S8 | 0.123 | 0.123 | 0.042 |
| S9 | 0.124 | 0.126 | 0.043 |
| S11 | 0.115 | 0.129 | 0.031 |
| S12 | 0.123 | 0.117 | 0.047 |
| S13 | 0.119 | 0.135 | 0.039 |
| S14 | 0.118 | 0.124 | 0.032 |
| S15 | 0.120 | 0.128 | 0.046 |
2.3 linear relationship investigation
Precisely weighing paeoniflorin 5.00mg, albiflorin 3.11mg and gallic acid 6.97mg, adding methanol solution, and fixing volume in 10mL volumetric flask to obtain mixed reference solution. And precisely sucking 5, 10, 15, 20, 25 and 30 mu L of the mixed reference substance solution, respectively injecting samples, and measuring according to the chromatographic condition under 1.1 item. And (3) carrying out linear regression by taking gallic acid, paeoniflorin and paeoniflorin sample injection amount as an abscissa (X, mg) and peak area as an ordinate (Y) to obtain a regression equation.
The regression equation of paeoniflorin is Y= 90716904.99X-6750.79178, R 2 ＝0.9999；
The regression equation of the paeoniflorin is Y= 90716904.99X-6750.79178, R 2 ＝0.9997；
The regression equation of gallic acid is Y= 2725712543X-322861.3857, R 2 ＝0.999131197。
The results show that the three components have good linear relation with peak area in the corresponding mass range.
2.4 precision test
Taking about 5g of a Polygonatum sibiricum Gorgon fruit decoction sample (S1), precisely weighing, preparing a sample solution according to the method under the condition of 1.1, continuously injecting sample for six times according to the chromatographic condition under the condition of 1.1, and recording the chromatographic peak area. RSD of peak areas of gallic acid, paeoniflorin and paeoniflorin are calculated to be 0.92%, 0.99% and 0.87%, respectively, which shows that the instrument precision is good.
2.5 stability test
Precisely weighing about 5g of rhizoma polygonati and gorgon euryale seed decoction sample (S1), preparing a test solution according to the method under 1.1, respectively sampling the sample at 0, 3, 6, 9, 12 and 24 hours after the preparation of the test solution according to the method under item chromatography, and recording the chromatographic peak area. The RSD of the peak areas of the components of gallic acid, paeoniflorin and paeoniflorin are calculated to be 1.1%, 0.99% and 0.97%, respectively, which shows that the stability of the sample solution is good within 24 hours.
2.6 repeatability test
Taking about 5g of a Polygonatum sibiricum Gordon euryale seed decoction sample (S1), precisely weighing, respectively preparing six sample solutions in parallel according to the method under 1.1, sampling according to the chromatographic method under 1.1, recording chromatographic peak area, and calculating according to an external standard one-point method. As a result, the average mass fractions of paeoniflorin, albiflorin and gallic acid are respectively 0.119mg/g, 0.126mg/g and 0.039mg/g, and RSD are respectively 1.2%, 1.9% and 2.1%, which shows that the method has good repeatability.
2.7 sample recovery test
About 1g of a polygonatum sibiricum gorgon fruit decoction medicine sample (S1) is taken, a sample solution is prepared according to the method under the condition of 1.1 according to the ratio of the reference substance quantity to the content of about 1:1 in the sample, six sample solutions are prepared in parallel, the measurement is carried out according to the chromatographic condition under the condition of 1.1, the peak area is recorded, and the recovery rate is calculated. The results show that the average sample recovery rates of gallic acid, albiflorin and paeoniflorin are respectively 101.0%, 101.1%,100.9% and RSD are respectively 2.1% and 3.5%, which indicates that the method has good accuracy.
3. Statistical analysis
3.1 Cluster Analysis (CA)
The cluster analysis is an unsupervised pattern recognition method, and samples with similar characteristics can be returned to the same class. The common peak area of 15 batches of polygonatum sibiricum gorgon fruit soup map data is used as a variable, SPSS26.0 software is used, a Walder connection method (namely a dispersion square sum method) is adopted, and a square Euclidean distance is used as a measure for carrying out clustering analysis. As shown in fig. 6, when the distance is 5, 15 batches of the sealwort gorgon fruit soup can be grouped into two types, wherein S3, S5, S13, S12, S4, S6, S11, S8 are classified into one type, S7, S10, S9, S15, S1, S14, S2 are classified into one type, which means that the groups S3, S5, S13, S12, S4, S6, S11, S8 are different from the groups S7, S10, S9, S15, S1, S14, S2 of the sealwort gorgon fruit soup.
3.2 principal component analysis (principal component analysis, PCA)
PCA is the most common data analysis method in multivariate statistical analysis, which can simplify a large amount of relevant data through a "dimension reduction" process, describe samples with uncorrelated variables representing data features, and analyze according to the data features in an "unsupervised" mode. In order to better and objectively reflect the difference between the batches of the Polygonatum sibiricum Gordon euryale seed soup, the peak area result of the common peak of 15 batches of Polygonatum sibiricum Gordon euryale seed soup samples is imported into SIMCA14.1 software for main component analysis. Four variables were extracted, the cumulative variance contribution was 87.067%, suggesting that the model was better predictive. The first two main components are extracted to be used as PCA score chart of 15 batches of rhizoma polygonati and gorgon fruit soup medicinal materials, and the PCA score chart is shown in figure 7. The results show that 15 batches of rhizoma polygonati and gorgon fruit soup are classified into two types, and mutually prove with the clustering analysis result: s3, S5, S13, S12, S4, S6, S11 and S8 are the same types, S7, S10, S9, S15, S1, S14 and S2 are the same types, and certain differences exist among different batches of polygonatum sibiricum gorgon fruit soup.
3.3 orthogonal partial least squares discriminant analysis (OPLS-DA)
OPLS-DA is a supervised pattern recognition method that can be used to find variables that cause inter-sample variability. Therefore, on the basis of PCA analysis, in order to further analyze the differences among the samples of the Polygonatum sibiricum and Gorgon fruit decoction of different sources, 19 common peak areas of the finger print of 15 batches of Polygonatum sibiricum and Gorgon fruit decoction medicine materials are taken as variables, SIMCA14.1 software is adopted to carry out OPLS-DA analysis, and the analysis result shows that parameters R of the OPLS-DA model are calculated 2 X is 0.751, R 2 Y is 0.984, Q 2 And the model stability and prediction capability are better as shown by 0.921. Is thatFurther verifying the validity of the model, and performing 200 substitution tests on the model by using SIMCA14.1 software, wherein the test results are shown in FIG. 8, and all Q 2 Are all at R 2 Under, and Q 2 The intersection point of the regression line of (c) and the y axis is at the negative half axis, which indicates that the model is effective. As can be seen from the OPLS-DA score chart shown in fig. 9, 15 batches of samples were well divided into two classes. As shown in fig. 10, the farther the point in the OPLS-DA load diagram is from the origin, the larger the corresponding variable weight value is, and it is known that there are peak 2, peak 7, peak 9, peak 10, peak 11, peak 13, peak 14, peak 16, and peak 19, which have a large influence on the first main component, and there are peak 1, peak 3, peak 4, peak 5, peak 8, and peak 12, which have a large influence on the second main component. As shown in fig. 11, the marker components causing the difference of the sealwort gorgon fruit soup are screened out by VIP values, and 5 components with larger contributions are marked out by taking VIP values larger than 1.4 as screening criteria, and the components are sequentially numbered 8, 4, 19, 18 and 5 chromatographic peaks. The comparison of the reference substances shows that the peak 8 is gallic acid, the peak 18 is paeoniflorin, the peak 19 is paeoniflorin, the content of the three main components is measured, and the chemometric analysis result shows that the selection of the three measurement indexes is reasonable.
The Chinese medicinal materials have complex components, are influenced by different growth environments, growth periods, harvesting periods, processing methods and the like, and have great differences in internal quality. The method can make up for the defect that the average method is easily influenced by individual abnormal samples by adopting a median method when the control fingerprint is generated, so that the generated control fingerprint is more representative. Based on the early-stage experiment, the method predicts and analyzes the quality markers of the Polygonatum sibiricum Red gorgon fruit soup from the aspects of chemical component specificity, traditional efficacy, chemical component testability and the like, selects the markers closely related to the quality characteristics of the traditional Chinese medicines, establishes a Polygonatum sibiricum Red gorgon fruit Shang Zhiwen map, determines 19 sharing peaks, and identifies three chemical components of gallic acid, paeoniflorin and albiflorin in, wherein the average value of the contents of paeoniflorin, albiflorin and gallic acid in 15 batches of Polygonatum sibiricum Red gorgon fruit soup samples is 0.119%, 0.126% and 0.039%, respectively. RSD of the relative retention time of the common peaks of 15 batches of rhizoma polygonati and gorgon fruit soup is 0.023-2.11%, which shows that the chemical composition consistency is better; the RSD of the relative peak area of each common peak is 1.23-2.79%, which suggests that the content of certain common chemical components of 15 batches of rhizoma polygonati gordon euryale seed soup is different. The similarity result of the sample patterns of each batch and the comparison patterns is not lower than 0.885, which shows that the chemical composition consistency of the Polygonatum sibiricum gorgon fruit soup of each batch is better.
And (3) carrying out CA, PCA and OPLS-DA analysis on the fingerprint spectrum of the rhizoma polygonati gorgon fruit decoction, wherein the common peak area shows that the quality of each batch of rhizoma polygonati gorgon fruit decoction is basically similar. Furthermore, chromatographic peaks 8, 4, 19, 18 and 5 are marker components affecting the difference of the ingredients of the Polygonatum sibiricum Gordon euryale seed decoction, so that it is reasonable to select the chromatographic peaks 8, 18 and 19 for quantitative determination. The finger print and content determination method of the polygonatum sibiricum gorgon fruit Shang Yaocai established by the application is good in repeatability and strong in specificity, and can be used for quality evaluation of polygonatum sibiricum gorgon fruit soup medicinal materials and related preparations.
Comparative example 1
The comparative example differs from example 1 in that the chromatographic conditions used for the fingerprint measurement are different, specifically in that the gradient elution is performed using the specifications of table 3 instead of table 1, and the other chromatographic conditions are the same.
TABLE 3 gradient elution procedure
| Time (min) | Methanol (%) | 0.1% formic acid water (%) |
| 0→5 | 1 | 98 |
| 5→10 | 2→16 | 98→84 |
| 10→15 | 16→18 | 84→82 |
| 15→20 | 18→30 | 82→70 |
| 20→25 | 30→34 | 70→66 |
| 25→30 | 34→45 | 66→55 |
The chromatogram is shown in fig. 12, and the result shows that: the individual peaks were not uniformly separated and had a leading and trailing peak.
Comparative example 2
The comparative example differs from example 1 in that the chromatographic conditions used for the fingerprint measurement are different, specifically in that the gradient elution is performed using the specifications of table 4 instead of table 1, and the other chromatographic conditions are the same.
TABLE 4 gradient elution procedure
| Time (min) | Methanol (%) | 0.1% formic acid water (%) |
| 0→3 | 4 | 96 |
| 3→9 | 4→20 | 96→80 |
| 9→13 | 20→24 | 80→76 |
| 13→14 | 24→30 | 76→70 |
| 14→19 | 30→35 | 70→65 |
| 19→22 | 35→50 | 65→50 |
| 22→24 | 50→90 | 50→10 |
The chromatogram is shown in fig. 13, and the result shows that: the front yield is low and front and tail peaks appear.
Comparative example 3
The comparative example differs from example 1 in that the chromatographic conditions used for the fingerprint measurement are different, specifically in that acetonitrile-0.1% formic acid is used as the mobile phase, and other chromatographic conditions are the same.
The chromatogram is shown in FIG. 14, and the separation degree of each peak is uneven.
Those of ordinary skill in the art will appreciate that: the discussion of any of the embodiments above is merely exemplary and is not intended to imply that the scope of the present application is limited to such examples; combinations of features of the above embodiments or in different embodiments are also possible within the spirit of the application, steps may be implemented in any order, and there are many other variations of the different aspects of one or more embodiments described above which are not provided in detail for the sake of brevity.
One or more embodiments herein are intended to embrace all such alternatives, modifications and variations that fall within the broad scope of the present application. Any omissions, modifications, equivalents, improvements, and the like, which are within the spirit and principles of the one or more embodiments in the present application, are therefore intended to be included within the scope of the present application.
Claims (7)
1. A quality detection method of rhizoma polygonati and semen euryales soup is characterized by comprising the steps of measuring a rhizoma polygonati and semen euryales Shang Zhiwen map, measuring contents of paeoniflorin, albiflorin and gallic acid, and measuring the rhizoma polygonati and semen euryales Shang Zhiwen map, the contents of paeoniflorin, albiflorin and gallic acid by adopting a liquid chromatography;
the method for measuring the finger print of the Polygonatum sibiricum Gordon euryale seed decoction by adopting the liquid chromatography comprises the following steps: performing liquid chromatograph analysis, taking solutions prepared by the taste-deficient negative samples as reference solutions, taking solutions prepared by paeoniflorin, albiflorin and gallic acid reference substances as reference solutions, taking solutions prepared by Polygonatum sibiricum and semen euryales soup samples as test solutions, respectively precisely sucking the reference solutions, the reference solutions and the test solutions, respectively injecting the reference solutions, the reference solutions and the test solutions into the liquid chromatograph, and measuring to obtain the extract; wherein the chromatographic conditions adopted are that: thermo Hypersil GOLD C18 column, 2.1mm×100mm,1.9 μm, mobile phase: gradient elution was performed with methanol as mobile phase a and 0.1% formic acid aqueous solution as mobile phase B, as specified in table 1;
TABLE 1 gradient elution procedure
Flow rate: 0.3mL/min; column temperature: 30 ℃; sample injection amount: 10. Mu.L; detection wavelength: 280nm;
wherein, the fingerprint spectrum is measured by adopting the liquid chromatography method, and the method further comprises the following steps:
(1) Preparation of reference solution a: weighing appropriate amount of rhizoma Polygonati, semen euryales, rhizoma Dioscoreae, radix Paeoniae alba, fructus Jujubae, radix Pseudostellariae, and herba Eupatorii, pulverizing into coarse powder, and sieving with 40 mesh sieve; precisely weighing each single medicine decoction piece powder according to the proportion of the rhizoma polygonati and semen euryales soup, and respectively preparing taste-lacking negative samples without rhizoma polygonati, semen euryales, chinese yam, white paeony root, chinese date, radix pseudostellariae or herba eupatorii by taking 3g as the total amount of the prescription; precisely weighing 1.0g of the taste-deficient negative sample, placing in a conical bottle with a plug, precisely adding 50mL of boiling water, weighing the mass, performing ultrasonic extraction for 30min, standing to room temperature, weighing the mass again, supplementing the lost mass with boiling water, shaking, and preserving in dark at 4 ℃; filtering with 0.22 μm needle filter before sample injection, and collecting filtrate;
(2) Preparing a reference substance solution a: precisely weighing paeoniflorin 5.00mg, albiflorin 3.11mg and gallic acid 6.97mg, adding methanol solution, and fixing volume in 10mL volumetric flask to obtain reference solution;
(3) Preparing a test sample solution a: weighing appropriate amount of rhizoma Polygonati, semen euryales, rhizoma Dioscoreae, radix Paeoniae alba, fructus Jujubae, radix Pseudostellariae, and herba Eupatorii, pulverizing into coarse powder, and sieving with 40 mesh sieve; precisely weighing each single medicine decoction piece powder according to the proportion of rhizoma polygonati and semen euryales soup, and uniformly mixing to obtain rhizoma polygonati and semen euryales Shang Yangpin; accurately weighing Shang Yangpin 1.0.0 g of the rhizoma polygonati and semen euryales, placing in a conical bottle with a plug, accurately adding 50mL of boiling water, weighing the mass, performing ultrasonic extraction for 30min, standing to room temperature, weighing the mass again, supplementing the lost mass with boiling water, shaking, and storing in a dark place at 4 ℃; filtering with 0.22 μm needle filter before sample injection, and collecting filtrate;
(4) Precisely sucking 10 μl of each of the reference solution a, the reference solution a and the sample solution a, respectively, injecting into a liquid chromatograph, and recording chromatograms;
wherein, the content of paeoniflorin, albiflorin and gallic acid measured by liquid chromatography comprises:
(1) Preparing a reference substance solution b: precisely weighing paeoniflorin 5.00mg, albiflorin 3.11mg and gallic acid 6.97mg, adding methanol solution, and fixing volume in a 10mL volumetric flask to obtain reference solution b;
(2) Preparing a test sample solution b: accurately weighing Shang Yangpin 1.0.0 g of rhizoma Polygonati and semen euryales, placing in a conical bottle with a plug, accurately adding 50mL of boiling water, weighing the mass, performing ultrasonic extraction for 30min, standing to room temperature, weighing the mass again, supplementing the lost mass with boiling water, shaking, and preserving in dark at 4deg.C; filtering with 0.22 μm needle filter before sample injection, and collecting filtrate;
(3) Performing liquid chromatograph analysis, taking a solution prepared from paeoniflorin, albiflorin and gallic acid reference substances as a reference substance solution b, taking a solution prepared from a polygonatum gordon euryale seed soup sample as a test substance solution b, precisely sucking the reference substance solution b and the test substance solution b respectively, injecting the reference substance solution b and the test substance solution b into the liquid chromatograph respectively, and measuring to obtain the extract; the chromatographic conditions adopted are that the chromatographic column: thermo Hypersil GOLD C18 column, 2.1mm×100mm,1.9 μm; mobile phase: using methanol as a mobile phase A and 0.1% formic acid aqueous solution as a mobile phase B, and performing gradient elution according to the specification of the table 1; flow rate: 0.3mL/min; column temperature: 30 ℃; sample injection amount: 10. Mu.L; detection wavelength: 280nm.
2. The method for detecting the quality of the rhizoma polygonati and semen euryales soup according to claim 1, further comprising performing cluster analysis, principal component analysis and orthorhombic least square discriminant analysis on a rhizoma polygonati and semen euryales Shang Zhiwen map.
3. The quality inspection method of rhizoma polygonati gordon euryale seed soup according to claim 1, wherein the content of gallic acid, albiflorin and paeoniflorin in the rhizoma polygonati gordon seed soup sample is calculated by an external standard one-point method.
4. The method for detecting the quality of the rhizoma polygonati and semen euryales soup according to claim 1, wherein the fingerprint of the rhizoma polygonati and semen euryales soup is formed by determining 19 common peaks through fingerprint measurement of the rhizoma polygonati and semen euryales soup.
5. The method for detecting the quality of the rhizoma polygonati gorgon fruit decoction according to claim 4, wherein the fingerprint of the rhizoma polygonati gorgon fruit decoction is compared with the fingerprint of a reference solution, and the peak 8 is gallic acid, the peak 18 is paeoniflorin, and the peak 19 is paeoniflorin.
6. The quality detection method of rhizoma polygonati gorgon fruit soup according to claim 4, wherein the fingerprint of rhizoma polygonati gorgon fruit soup is compared with the fingerprint of each taste-lacking negative sample, and the 19 common peaks are subjected to medicinal material attribution, and peak 1 and peak 18 are attributed to eupatorium; peak 2 and peak 11 are ascribed to white peony root and eupatorium; peak 5 is attributed to white peony root, eupatorium and yam; peak 6 and Peak 8 are ascribed to Dioscorea opposita; peak 7 belongs to Polygonatum sibiricum; peak 10 is attributed to Eupatorium and Dioscorea opposita; peak 15 and peak 19 are ascribed to white peony root.
7. The quality detection method of rhizoma polygonati and semen euryales soup according to claim 1, wherein a plurality of batches of rhizoma polygonati and semen euryales Shang Zhiwen map data are sequentially imported into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, one batch of rhizoma polygonati and semen euryales Shang Zhiwen maps are selected as reference fingerprints, and a comparison fingerprint is generated by a multi-point correction and an average method.
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