CN113960233A - Method for determining content of 8 types of rhizoma paridis saponins in rhizoma paridis Yunnanensis - Google Patents
Method for determining content of 8 types of rhizoma paridis saponins in rhizoma paridis Yunnanensis Download PDFInfo
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Abstract
The invention provides a method for determining the content of 8 types of rhizoma paridis saponins in rhizoma paridis Yunnanensis, which comprises the following steps: preparing a reference substance solution by using a methanol solution; extracting a sample by using a methanol solvent, carrying out ultrasonic treatment on the obtained sample, standing the obtained sample solution overnight, and taking supernate to be used as the sample solution by using membrane filtration; and respectively loading the reference solution and the test solution to a high performance liquid chromatograph for determination to obtain chromatograms. The content of rhizoma paridis saponin I, II, III, H, V, VI, VII and slender dioscin in rhizoma paridis is calculated by external standard method. The method is simple, is convenient to operate, can accurately and quickly realize the determination of the contents of the paris polyphylla saponins I, II, III, H, V, VI, VII and fine dioscin in the paris polyphylla rhizome, and can provide a theoretical basis for further and comprehensively evaluating the quality of the paris polyphylla medicinal material.
Description
Technical Field
The invention relates to a method for measuring the content of active ingredients in traditional Chinese medicines, in particular to a method for measuring the content of 8 paridis saponins in paris polyphylla rhizomes.
Background
Paris polyphylla var. yunnanensis (Franch.) hand-Mazz. is a plant used for perennial crude drugs in Paris genus of Liliaceae family. The part of the medicine is dry rhizome, and has the effects of clearing away heat and toxic material, relieving swelling and pain, cooling liver and arresting convulsion. Modern researches show that the main chemical components of the paris polyphylla are steroidal saponins, triterpenes, flavonoids and the like, can treat various diseases, has remarkable antibacterial and anticancer cell effects, hemostasis and detumescence effects and the like, and is a raw material of more than 40 common Chinese patent medicines such as Yunnan white drug powder, Gongxuening capsules, Jinfukang oral liquid and the like.
The traditional Chinese medicinal materials have very complex components, many impurities and large interference, and have great influence on sample preparation and component extraction technologies. The main effective component in rhizoma paridis Yunnanensis is steroid saponin, and the method for extracting saponin components adopts ultrasonic extraction method in addition to decocting method and ethanol reflux extraction method. Ultrasonic extraction, also known as ultrasonic extraction and ultrasonic-assisted extraction, is based on the principle that the frequency and speed of molecular motion of a substance and the penetrating power of a solvent are increased by utilizing multiple functions of strong cavitation effect, mechanical vibration, disturbance effect, high acceleration, emulsification, diffusion, crushing, stirring and the like generated by ultrasonic radiation, so that target components are accelerated to enter the solvent. Compared with extraction methods such as a decoction method, a heating reflux method and the like, the ultrasonic extraction can greatly improve the extraction efficiency, save the solvent and avoid the influence of high temperature on the extracted components.
In 'Chinese pharmacopoeia' of 2020 edition, the total content of three saponins, namely, paris polyphylla saponin I, saponin II and saponin VII is only used as an index component for evaluating the quality of paris polyphylla, but the saponins VI, the saponins H, the saponins III, the saponins V and the tenuiform dioscin are also one of the main active components of paris polyphylla. Although scholars carry out quantitative analysis on active ingredients in the Yunnan manyleaf paris rhizome, aiming at 4 paridis saponins collected in 2015 edition and 2020 edition of Chinese pharmacopoeia, the invention provides a method for simultaneously determining contents of paridis saponins I, II, III, H, V, VI, VII and dioscin tencel in the Yunnan manyleaf paris rhizome in order to more comprehensively evaluate the quality of Yunnan manyleaf paris rhizome medicinal materials and further provide reference for resource development of the Yunnan manyleaf paris rhizome.
Disclosure of Invention
The invention aims to solve the technical problem of providing a high-efficiency liquid phase detection method for the paris polyphylla saponin I, II, III, H, V, VI, VII and tenuous dioscin in paris polyphylla, which is simple and quick, has good separation effect and high accuracy.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
a method for measuring the content of 8 paridis saponins in paris polyphylla rhizome comprises the following steps:
(1) preparing a reference substance from rhizoma paridis saponin I, II, III, H, V, VI, VII and slender dioscin standard substance;
(2) preparation of a test solution: crushing a sample, sieving the crushed sample by a third pharmacopoeia sieve, drying the crushed sample in an oven to a constant amount, precisely weighing medicinal material powder into a conical flask with a plug, adding methanol, weighing, performing ultrasonic treatment, standing and cooling the sample after ultrasonic treatment, weighing again, complementing the weight loss by methanol, shaking up the sample, standing the sample overnight, taking supernatant, and filtering the supernatant by a microporous filter membrane to obtain subsequent filtrate;
(3) respectively loading the reference solution and the sample solution onto a high performance liquid chromatography column, eluting after loading, and recording a chromatogram;
(4) and calculating the concentrations of the rhizoma paridis saponins I, II, III, H, V, VI, VII and slender dioscin in the test sample by adopting an external standard method according to the concentration of the reference solution and the chromatographic peak area thereof.
Further, the preparation of the test solution in the step (3) comprises the following steps: crushing a sample, sieving the crushed sample by a third pharmacopoeia sieve, drying the crushed sample in a 45 ℃ oven to a constant amount, precisely weighing 0.5g of medicinal powder into a conical flask with a plug, adding 5mL of methanol, weighing the weighed powder, carrying out ultrasonic treatment at 55 ℃ and 70kHz for 60min, carrying out ultrasonic treatment, standing the powder for cooling, weighing the powder again, complementing the weight loss by methanol, shaking the powder evenly, standing the powder overnight, taking supernate, and filtering the supernate by a 0.22 mu m microporous filter membrane to obtain a subsequent filtrate.
Further, the high performance liquid chromatograph used in step (3) is an Agilent model 1260 high performance liquid chromatograph.
Further, the high performance liquid chromatography column in the step (3) is an Agilent ZORBAX SB-Aq column with the specification: 250 mm. times.4.6 mm5 μm.
Further, in the step (3), the chromatographic mobile phase A is pure water, the chromatographic mobile phase B is chromatographic grade acetonitrile, and the gradient elution method is 0-10 min and is 35-40% of the chromatographic mobile phase B; 10-22 min, 40% -42% of B; 22-23 min, 42% -40% B; 23-38 min, 40% B; 38-45 min, 40-47% of B.
Further, the flow rate of the chromatographic mobile phase in the step (3) is 0.7 mL/min.
Further, the column temperature of the high performance liquid chromatography in the step (3) is 25 ℃.
Further, the loading volume of the HPLC apparatus in the step (3) was 10. mu.L.
Further, the step (3) adopts a variable wavelength scanning ultraviolet detector.
Further, the chromatogram recorded in step (3) was recorded at a detection wavelength of 203 nm.
Compared with the prior art, the invention has the following beneficial effects:
the high performance liquid detection method for the contents of the paridis saponins I, II, III, H, V, VI, VII and the slender dioscin can quickly and efficiently detect the 8 paridis saponins, and the separation degree can reach more than 1.5, while the detection method of the prior art is difficult to realize the effective separation of the paridis saponins and related substances; the HPLC spectrum baseline of the detection method is stable and does not drift; the detection time of the invention is short, and the high performance liquid detection process can be completed only by 45 min. The detection efficiency is greatly improved; the method is used for measuring the contents of the rhizoma paridis saponin I, II, III, H, V, VI, VII and the slender dioscin, has good linear relation, and is respectively R20.9999, 0.9996, 0.9996, 0.9996, 0.9995, 0.9997, 0.9995, 0.9999, the reproducibility is high, and the instrument has good precision and accuracy, and provides theoretical basis for further comprehensively evaluating the quality of the Yunnan manyleaf Paris rhizome medicinal material.
Drawings
FIG. 1 is a high performance liquid chromatogram of Paris polyphylla.
In the figure: A. a control chromatogram; s1 rhizome chromatogram map; and C, S2 rhizome chromatogram. 1. Rhizoma paridis saponin VII; 2. rhizoma paridis saponin H; 3. rhizoma paridis saponin VI; 4. rhizoma paridis saponin II; 5. rhizoma paridis saponin III; 6. fine dioscin; 7. rhizoma paridis saponin I; 8. rhizoma paridis saponin V.
FIG. 2 is a linear plot of Paris Saponin I.
FIG. 3 is a linear plot of Paris Saponin II.
FIG. 4 is a linear plot of Paris Saponin III.
Fig. 5 is a linear graph of polyphyllin H.
Figure 6 is a linear plot of polyphyllin V.
FIG. 7 is a linear plot of Paris Saponin VI.
Fig. 8 is a linear plot of polyphyllin VII.
Fig. 9 is a linear graph of fine dioscin.
Detailed Description
The technical solution of the present invention will be described clearly and completely below, and it is obvious that the described embodiments are some embodiments of the present invention.
The inventor of the application obtains a high performance liquid detection method for contents of I, II, III, H, V, VI, VII and fine dioscin of the traditional Chinese medicine paris polyphylla through extensive research and a large number of experiments, the method provides the method which can effectively separate the I, II, III, H, V, VI, VII and fine dioscin of the traditional Chinese medicine paris polyphylla, and can realize good separation effect on the paris polyphylla saponin which is difficult to effectively separate in the prior art, and the separation degree R can reach more than 1.5.
The chromatographic conditions were as follows:
the chromatographic column adopts an Agilent ZORBAX SB-Aq column (250mm multiplied by 4.6mm,5 μm);
the detector adopts a VWD detector;
the mobile phase comprises a mobile phase A and a mobile phase B; mobile phase a term is pure water and B term is chromatographic grade acetonitrile (merck, germany);
gradient elution is adopted, the method is 0-10 min, and B accounts for 35% -40%; 10-22 min, 40% -42% of B; 22-23 min, 42% -40% B; 23-38 min, 40% B; 38-45 min, 40-47% of B.
In a preferred embodiment of the invention, the column length of the chromatography column is 250 mm.
In a preferred embodiment of the present invention, the flow rate is 0.5 to 1.0mL/min, and more preferably 0.7 mL/min.
In a preferred embodiment of the present invention, the preferred detection wavelength is 203 nm.
In a preferred embodiment of the present invention, the column temperature is controlled to be 25 to 30 ℃, and more preferably 25 ℃.
In a preferred embodiment of the invention, the sample size is 10. mu.L.
The technical solutions of the present invention will be described in detail and fully with reference to the following specific embodiments, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
The experimental conditions in the examples of the invention are as follows:
the instrument comprises the following steps: agilent 1260 type high performance liquid chromatograph
A chromatographic column: agilent ZORBAX SB-Aq column (250 mm. times.4.6 mm,5 μm)
Preparing a reference substance solution: accurately weighing rhizoma paridis saponin I, II, III, H, V, VI, VII and reference substance of slender dioscin, placing into a 5mL measuring flask, adding methanol to dissolve and fix volume, preparing reference substance mother liquor with mass concentrations of 0.672, 1.47, 0.488, 0.21, 0.448, 1.062, 0.452 and 0.448mg/mL respectively, diluting step by step, and storing in a refrigerator at 4 deg.C for later use.
Preparation of a test solution: crushing a sample, sieving the crushed sample by a third sieve in pharmacopoeia, drying the crushed sample in a 45 ℃ oven to a constant amount, precisely weighing 0.5g of medicinal powder into a conical flask with a plug, adding 5mL of methanol, weighing, placing the weighed sample at 55 ℃ and 70kHz for ultrasonic treatment for 60min, placing the sample after ultrasonic treatment for cooling, weighing again, complementing the weight loss by methanol, shaking the sample evenly, placing the sample overnight, taking supernate, filtering the supernate by a 0.22 mu m microporous filter membrane, and taking the subsequent filtrate to obtain the medicinal powder.
Detection wavelength: 203 nm;
column temperature: 25 ℃;
mobile phase: mobile phase A + mobile phase B is 100%;
gradient elution: (ii) a Mobile phase a term is pure water and B term is chromatographic grade acetonitrile (merck, germany); the method is 0-10 min, 35-40% B; 10-22 min, 40% -42% of B; 22-23 min, 42% -40% B; 23-38 min, 40% B; 38-45 min, 40-47% of B.
Example 1
Mobile phase a term is pure water and B term is chromatographic grade acetonitrile (merck, germany); the method comprises the following steps: 0-10 min, 35-40% of B; 10-22 min, 40% -42% of B; 22-23 min, 42% -40% B; 23-38 min, 40% B; 38-45 min, 40-47% of B. The flow rate is 0.7 mL/min; the column temperature was 25 ℃; the detection wavelength is 203 nm;
according to the method, three needles are used for sample injection, HPLC (high performance liquid chromatography) spectra are shown in figure 1, a baseline is stable, the repeatability is good, all peaks can be produced within 32min, the retention time of the paris polyphylla saponin I, II, III, H, V, VI, VII and the tenuous dioscin is 26.0653-26.2368, 22.1747-22.3881, 23.7549-23.8939, 15.1416-15.1970, 30.5553-30.7707, 16.3185-16.4107 and 13.5819-13.6422 respectively, the separation degree is greater than 1.5, and the result is good.
Example 2
Mobile phase a term is pure water and B term is chromatographic grade acetonitrile (merck, germany); 0-10 min, 35-40% of B; 10-26 min, 40% -43% B; 26-31 min, 43% -44% of B; 31-41 min, 44% -47% of B; 41-46 min, 47-60% of B. The flow rate is 0.8 mL/min; the detection wavelength is 250 nm;
the baseline is stable under the method, and no target peak exists.
Example 3
Mobile phase a term is pure water and B term is chromatographic grade acetonitrile (merck, germany); the method comprises the following steps: 0-31 min, 35% -48% B; 31-38 min, 48% -60% B; the flow rate is 0.8 mL/min; the column temperature is 30 ℃; the detection wavelength is 203 nm;
the method has unstable baseline, more miscellaneous peaks around the target peak, no separation of rhizoma paridis saponin H and VI, and fine dioscin and I, retention time of 19.121, 19.729, 26.211 and 26.088, separation degree of 0.89 and 1.05, and incomplete separation of four peaks.
Example 4
Mobile phase a term is pure water and B term is chromatographic grade acetonitrile (merck, germany); the method comprises the following steps: 0-31 min, 35% -48% B; 31-38 min, 48% -60% B; the flow rate is 0.6 mL/min; the column temperature is 30 ℃; the detection wavelength is 203 nm;
the method has stable baseline, but the rhizoma paridis saponin H and VI, the slender dioscin and I are not separated, the retention time is about 22.892, 23.644, 31.205 and 31.999 respectively, the separation degree is 0.96 and 0.89 respectively, and the four peaks are not completely separated.
Example 5
Mobile phase a term is pure water and B term is chromatographic grade acetonitrile (merck, germany); the method comprises the following steps: 0-9 min, 35-40% of B; 9-25 min, 40% -43% B; 25-40 min, 43% -44% B; for 40-45 min, 44% -47% of B; the flow rate is 0.6 mL/min; the column temperature was 21 ℃; the detection wavelength is 203 nm;
the method has stable baseline, completely separated rhizoma paridis saponin H and rhizoma paridis saponin VI, retention time of 17.498 and 21.775, separation degree of 3.42 and 2.14, and good result. However, the number of the peaks is large in the sample within 0-16 minutes, the fine dioscin and the paris polyphylla saponin I cannot be separated, the retention time is about 28.863 and about 30.028, the separation degree is 1.33 and 0.91, and the two peaks are not completely separated.
Example 6
Mobile phase a term is pure water and B term is chromatographic grade acetonitrile (merck, germany); the method comprises the following steps: 0-10 min, 35-40% of B; 10-22 min, 40% -44% B; 22-40 min, 42% -41% of B; the flow rate is 0.7 mL/min; the column temperature is 30 ℃; the detection wavelength is 203 nm;
the baseline is not stable, the peak shape is not good, and the target peak has a trailing phenomenon. The paris polyphylla saponin H and the paris polyphylla saponin VI can be completely separated, the retention time is respectively about 21.200 and 22.147, the separation degree is respectively 2.77 and 1.53, and the result shows good. But the slender dioscin and the rhizoma paridis saponin I cannot be separated, the retention time is about 32.125 and 33.377 respectively, the separation degree is 1.58 and 0.95 respectively, and the two peaks are not completely separated.
Example 7
Determination of contents of rhizoma paridis saponin I, II, III, H, V, VI, VII and fine dioscin in rhizoma paridis
Based on the detection method, the inventor also provides a high performance liquid chromatography method for determining the contents of the paridis saponins I, II, III, H, V, VI, VII and slender dioscin in the traditional Chinese medicine paris polyphylla, which comprises the following steps:
(1) preparing a reference substance solution and a test solution: accurately weighing rhizoma paridis saponin I, II, III, H, V, VI, VII and reference substance of slender dioscin, placing into a 5mL measuring flask, adding methanol to dissolve and fix volume, preparing reference substance mother liquor with mass concentrations of 0.672, 1.47, 0.488, 0.21, 0.448, 1.062, 0.452 and 0.448mg/mL respectively, diluting step by step, and storing in a refrigerator at 4 deg.C for later use. Crushing a sample, sieving the crushed sample by a third sieve in pharmacopoeia, drying the crushed sample in a 45 ℃ oven to a constant weight, precisely weighing 0.5g of medicinal powder into a conical bottle with a plug, adding 5mL of methanol, weighing the weighed sample, carrying out ultrasonic treatment at 60 ℃ and 70kHz for 60min, carrying out ultrasonic treatment, standing the sample for cooling, weighing the sample again, complementing the weight loss by methanol, shaking the sample evenly, standing the sample overnight, taking supernate, filtering the supernate by a 0.22 mu m microporous filter membrane, and taking the subsequent filtrate to obtain the traditional Chinese medicine composition. The information of the sources of 4 paris polyphylla samples is shown in the following table.
(2) Chromatographic conditions
The chromatographic column adopts an Agilent ZORBAX SB-Aq column (250mm multiplied by 4.6mm,5 μm);
the detector adopts a variable wavelength scanning ultraviolet detector (VWD);
the mobile phase comprises a mobile phase A and a mobile phase B; the mobile phase A is pure water; mobile phase B was chromatographic grade acetonitrile (merck, germany);
gradient elution was used, the gradient elution procedure was performed as follows: 0-10 min, 35-40% of B; 10-22 min, 40% -42% of B; 22-23 min, 42% -40% B; 23-38 min, 40% B; 38-45 min, 40-47% of B.
(3) Measurement method
And sequentially injecting the 8 reference substance solutions and the sample solution according to the chromatographic conditions, recording a chromatogram, and preparing a linear correlation working curve (shown in figures 2-9 respectively) according to the peak areas and concentrations of the paris polyphylla saponin I, II, III, H, V, VI, VII and the dioscin tenuinea of the 8 reference substance solutions. The area and concentration of the paris polyphylla saponin I peak are shown in the following table.
| # | Concentration (mg/ml) | Peak area |
| 1 | 0.0067 | 27.90267 |
| 2 | 0.0672 | 313.5295 |
| 3 | 0.1344 | 634.5012 |
| 4 | 0.2016 | 928.7526 |
| 5 | 0.2688 | 1234.174 |
| 6 | 0.4704 | 2199.337 |
| 7 | 0.6048 | 2799.15 |
| 8 | 0.672 | 3120.279 |
Paris saponin II peak areas and concentrations are shown in the following table.
| # | Concentration (mg/ml) | Peak area |
| 1 | 0.0147 | 33.04824 |
| 2 | 0.147 | 590.8375 |
| 3 | 0.294 | 1187.025 |
| 4 | 0.588 | 2303.143 |
| 5 | 0.882 | 3359.981 |
| 6 | 1.176 | 4599.645 |
| 7 | 1.323 | 5177.23 |
| 8 | 1.47 | 5802.411 |
Paris saponin III peak areas and concentrations are shown in the following table.
| # | Concentration (mg/ml) | Peak area |
| 1 | 0.00488 | 16.81091 |
| 2 | 0.0488 | 198.4776 |
| 3 | 0.0976 | 414.7701 |
| 4 | 0.1464 | 619.3733 |
| 5 | 0.1952 | 827.7834 |
| 6 | 0.2928 | 1211.232 |
| 7 | 0.3904 | 1658.805 |
| 8 | 0.488 | 2109.542 |
Paris saponin H peak area and concentration are shown in the following table.
| # | Concentration (mg/ml) | Peak area |
| 1 | 0.0021 | 6.018176 |
| 2 | 0.021 | 87.10094 |
| 3 | 0.042 | 187.8084 |
| 4 | 0.063 | 279.6311 |
| 5 | 0.126 | 554.7289 |
| 6 | 0.168 | 754.8375 |
| 7 | 0.189 | 860.3638 |
| 8 | 0.21 | 964.8698 |
Paris saponin V peak areas and concentrations are shown in the following table.
| # | Concentration (mg/ml) | Peak area |
| 1 | 0.0045 | 25.19404 |
| 2 | 0.0448 | 293.8121 |
| 3 | 0.1344 | 860.8287 |
| 4 | 0.1792 | 1126.145 |
| 5 | 0.2688 | 1647.424 |
| 6 | 0.3584 | 2277.653 |
| 7 | 0.4032 | 2554.569 |
| 8 | 0.448 | 2866.862 |
Paris saponin VI peak areas and concentrations are shown in the following table.
| # | Concentration (mg/ml) | Peak area |
| 1 | 0.01062 | 43.1666 |
| 2 | 0.1062 | 559.6124 |
| 3 | 0.2124 | 1172.979 |
| 4 | 0.3186 | 1729.273 |
| 5 | 0.6372 | 3361.131 |
| 6 | 0.8496 | 4557.859 |
| 7 | 0.9558 | 5175.44 |
| 8 | 1.062 | 5794.206 |
Paris saponin VII peak areas and concentrations are shown in the following table.
| # | Concentration (mg/ml) | Peak area |
| 1 | 0.0045 | 21.52237 |
| 2 | 0.0452 | 159.9554 |
| 3 | 0.0904 | 326.9059 |
| 4 | 0.1808 | 643.3827 |
| 5 | 0.2712 | 939.9485 |
| 6 | 0.3616 | 1285.955 |
| 7 | 0.4068 | 1454.681 |
| 8 | 0.452 | 1634.983 |
The area and concentration of the fine dioscin peak are shown in the table below.
| # | Concentration (mg/ml) | Peak area |
| 1 | 0.00448 | 16.78507 |
| 2 | 0.0448 | 189.8018 |
| 3 | 0.0896 | 393.6531 |
| 4 | 0.1344 | 590.5269 |
| 5 | 0.1792 | 785.7259 |
| 6 | 0.3136 | 1407.51 |
| 7 | 0.4032 | 1796.339 |
| 8 | 0.448 | 2001.725 |
As can be seen from FIGS. 2-9, the linear relationship of the working curves is good, R2Is more than 0.999; substituting into the area of the rhizoma paridis saponin peak of the test sample to calculate and obtain the concentration of the test sample solution, and comparing the measured concentration of the test sample solution with the constant volume concentration of the test sample solution and the dilution times during sample injection to calculate and obtain the content of the rhizoma paridis saponin in the test sample solution.
The high performance liquid detection method for the contents of the paridis saponins I, II, III, H, V, VI, VII and the slender dioscin can quickly and efficiently detect the 8 paridis saponins, and the separation degree can reach more than 1.5, while the detection method of the prior art is difficult to realize the effective separation of the paridis saponins and related substances; the HPLC spectrum baseline of the detection method is stable and does not drift; the detection time of the invention is short, and the high performance liquid detection can be completed in only 45minAnd (6) carrying out the process. The detection efficiency is greatly improved; the method is used for measuring the contents of the rhizoma paridis saponin I, II, III, H, V, VI, VII and the slender dioscin, has good linear relation, and is respectively R20.9999, 0.9996, 0.9996, 0.9996, 0.9995, 0.9997, 0.9995, 0.9999, the reproducibility is high, and the instrument has good precision and accuracy, and provides theoretical basis for further comprehensively evaluating the quality of the Yunnan manyleaf Paris rhizome medicinal material.
In summary, the above embodiments and drawings are only preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and any modifications, equivalent substitutions, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (10)
1. A method for measuring the content of 8 paridis saponins in paris polyphylla rhizomes is characterized by comprising the following steps:
(1) preparing a reference substance from rhizoma paridis saponin I, II, III, H, V, VI, VII and slender dioscin standard substance;
(2) preparation of a test solution: crushing a sample, sieving the crushed sample by a third pharmacopoeia sieve, drying the crushed sample in an oven to a constant amount, precisely weighing medicinal material powder into a conical flask with a plug, adding methanol, weighing, performing ultrasonic treatment, standing and cooling the sample after ultrasonic treatment, weighing again, complementing the weight loss by methanol, shaking up the sample, standing the sample overnight, taking supernatant, and filtering the supernatant by a microporous filter membrane to obtain subsequent filtrate;
(3) respectively loading the reference solution and the sample solution onto a high performance liquid chromatography column, eluting after loading, and recording a chromatogram;
(4) and calculating the concentrations of the rhizoma paridis saponins I, II, III, H, V, VI, VII and slender dioscin in the test sample by adopting an external standard method according to the concentration of the reference solution and the chromatographic peak area thereof.
2. The method for determining the content of 8 paridis saponins in the rhizome of Paris polyphylla Smith according to claim 1, wherein the preparation of the test solution in the step (3) comprises the following steps: crushing a sample, sieving the crushed sample by a third pharmacopoeia sieve, drying the crushed sample in a 45 ℃ oven to a constant amount, precisely weighing 0.5g of medicinal powder into a conical flask with a plug, adding 5mL of methanol, weighing the weighed powder, carrying out ultrasonic treatment at 55 ℃ and 70kHz for 60min, carrying out ultrasonic treatment, standing the powder for cooling, weighing the powder again, complementing the weight loss by methanol, shaking the powder evenly, standing the powder overnight, taking supernate, and filtering the supernate by a 0.22 mu m microporous filter membrane to obtain a subsequent filtrate.
3. The method for determining the content of 8 paridis saponins in the rhizome of Paris polyphylla Smith as claimed in claim 1, wherein the high performance liquid chromatography used in step (3) is Agilent 1260 type high performance liquid chromatography.
4. The method for determining the content of 8 paridis saponins in the rhizome of Paris polyphylla Smith according to claim 1, wherein the high performance liquid chromatography column in step (3) is an Agilent ZORBAX SB-Aq column with specification: 250 mm. times.4.6 mm5 μm.
5. The method for determining the content of 8 paridis saponins in rhizoma paridis Yunnanensis according to claim 1, characterized in that, in the step (3), the chromatographic mobile phase A is pure water, the chromatographic mobile phase B is chromatographic grade acetonitrile, the gradient elution method is 0-10 min, and the content of B is 35-40%; 10-22 min, 40% -42% of B; 22-23 min, 42% -40% B; 23-38 min, 40% B; 38-45 min, 40-47% of B.
6. The method for determining the content of 8 paridis saponins in rhizoma paridis Yunnanensis according to claim 1, characterized in that, the flow rate of chromatographic mobile phase in step (3) is 0.7 mL/min.
7. The method for determining the content of 8 paridis saponins in Yunnan Paris polyphylla rhizome as claimed in claim 1, wherein the column temperature of the high performance liquid chromatography in the step (3) is 25 ℃.
8. The method for determining the content of 8 paridis saponins in Paris polyphylla rhizome according to claim 1, wherein the sample loading volume of the HPLC in step (3) is 10 μ L.
9. The method for determining the content of 8 paridis saponins in the rhizome of Paris polyphylla Smith according to claim 1, wherein in step (3), a variable wavelength scanning ultraviolet detector is adopted.
10. The method for determining the content of 8 paridis saponins in the rhizome of Paris polyphylla Smith according to claim 1, wherein the chromatogram recorded in step (3) is recorded at a detection wavelength of 203 nm.
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| CN106872616A (en) * | 2017-03-27 | 2017-06-20 | 西南民族大学 | The method for distinguishing Dali Paris polyphylla and Yunnan Rhizoma Paridis |
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| CN108008042A (en) * | 2017-12-07 | 2018-05-08 | 贵州圣济堂制药有限公司 | A kind of content assaying method of four kinds of saponin components of Paris polyphylla detoxification tincture preparation |
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