CN107422047B

CN107422047B - Method for identifying paris polyphylla

Info

Publication number: CN107422047B
Application number: CN201710189491.2A
Authority: CN
Inventors: 刘圆; 黄钟杰; 余孟杰; 张绍山
Original assignee: Southwest Minzu University
Current assignee: Southwest Minzu University
Priority date: 2017-03-27
Filing date: 2017-03-27
Publication date: 2020-05-05
Anticipated expiration: 2037-03-27
Also published as: CN107422047A

Abstract

The invention provides an identification method of paris polyphylla, which adopts liquid chromatography to detect, if the paris polyphylla contains tenuifolia dioscin, the paris polyphylla saponin VII, H and VI are not contained or the content is lower than 0.010 percent; it can be determined as paris polyphylla. The detection method is simple and accurate, has short analysis time and good stability, and can be used for identifying the paris polyphylla, the Yunnan paris polyphylla and the like.

Description

Method for identifying paris polyphylla

Technical Field

The invention relates to the field of detection of traditional Chinese medicinal materials, in particular to an identification method of paris polyphylla.

Background

The Paris polyphylla variety collected in the first part of the pharmacopoeia of the people's republic of China 2015 is dried rhizome of Paris polyphylla Smith var. yunnanensis (Franch.) hand-Mazz. or Paris polyphylla Smith var. chinensis Hara of Paris polyphylla of Liliaceae, and has the effects of clearing away heat and toxic materials, relieving swelling and pain, cooling liver and arresting convulsion. Modern researches have shown that rhizoma paridis has the effects of resisting tumor, stopping bleeding, regulating immunity, tranquilizing, relieving pain, etc., and the steroid saponin component is the main active component. In recent years, the price of the paris polyphylla is increased dramatically due to a plurality of factors such as huge market demand of the paris polyphylla, high artificial breeding technical requirement endangered by wild resources, long cultivation period and the like, and the price of the paris polyphylla is increased to 700-900 yuan/kg from 50 yuan/kg ten years ago.

Rhizoma paridis is Daliyens H.Li et V.G.Souku and rhizoma paridis Dulogensis H.Li et S.Kuritap, belonging to Liliaceae, Paris, rhizome is also used as rhizoma paridis medicinal material in Yunnan, Guizhou and other places, has bitter taste, slight cold property and small toxicity, has the effects of clearing heat and detoxicating, relieving swelling and pain, cooling liver and arresting convulsion, and is used for treating carbuncle, sore throat, venomous snake bite, traumatic injury, cold wind and convulsion, etc. At present, the standard of the Chinese pharmacopoeia or the provincial level medicinal materials of 2015 edition is not recorded. Because the demand of series medicines such as Yunnan white drug powder as a representative increasingly increases on the rhizome of the paris polyphylla, the rhizome of the paris polyphylla belongs to a plurality of plants, including the paris polyphylla and the paris polyphylla which are taken as the medicinal materials of the paris polyphylla, and whether the paris polyphylla and the paris polyphylla can be taken as the sources of the medicinal materials of the paris polyphylla and how the resources are stored, the research report of whether the market demand can be met is few, therefore, the basic research of the related varieties of the paris polyphylla in the Yunnan is necessary, and the basis is provided for the supply of.

Disclosure of Invention

The invention aims to provide a method for identifying paris polyphylla for the first time.

Specifically, the invention provides a method for identifying paris polyphylla, which adopts liquid chromatography to detect, if the paris polyphylla contains tenuiform dioscin, the paris polyphylla saponin VII, H and VI are not contained or the content of the paris polyphylla saponin is lower than 0.010 percent; it can be determined as paris polyphylla.

In the 'Chinese pharmacopoeia' published in 2015, the mass fractions of rhizoma paridis saponin I, rhizoma paridis saponin II, rhizoma paridis saponin VI and rhizoma paridis saponin VII are used as indexes for evaluating the quality of rhizoma paridis, and patent application 2016106653439 also describes that rhizoma paridis in Yunnan mainly contains rhizoma paridis saponin VII, H, II and I, and gracile dioscin is not a necessary component in rhizoma paridis in Yunnan, however, rhizoma paridis contains gracile pennogenin, S17 batches contain rhizoma paridis saponin II, and 7 batches do not contain rhizoma paridis saponin VI, rhizoma paridis saponin VII and rhizoma paridis saponin H. Therefore, compared with the paris polyphylla in Yunnan, the paris polyphylla contains the chemical components of the dioscin which is slender but does not contain the saponin VI, the saponin VII and the saponin H of the paris polyphylla, and meanwhile, the paris polyphylla and the paris polyphylla also do not have the characteristics of the paris polyphylla, so that the paris polyphylla and the paris polyphylla in Yunnan can be distinguished and separated through the components.

Of course, the method can also be used as a method for distinguishing the paris polyphylla from the paris yunnanensis, and the paris polyphylla can be distinguished from a mixed product of the paris polyphylla and the paris yunnanensis.

In one embodiment of the invention, the content of the fine dioscin is more than 0.8%. Considering error, the content of the slender dioscin is set to be more than 0.7%.

In addition, during the detection process, the paris polyphylla also contains the division of the paris saponin I, but the division exists in the paris polyphylla in Yunnan, and the division cannot be distinguished from the division of the paris polyphylla, so the division is used as a supplementary limit of the paris polyphylla. Namely, it also contains Paris saponin I.

The invention further discovers that only one of 7 samples in the paris polyphylla contains the paris saponin II and basically does not contain the paris saponin II, so the invention proposes that the identification method is defined as follows: wherein, the content of the rhizoma paridis saponin II is not contained or is less than 0.40 percent.

In a specific embodiment of the invention, the content of the paris polyphylla saponin I is more than 0.1%, and the content of the paris polyphylla saponin I is more than 0.09% in consideration of errors.

Further, the liquid chromatography is selected from UPLC (ultra performance liquid chromatography). Of course, the use of HPLC is not excluded, but chromatographic conditions need to be changed accordingly.

The chromatographic conditions of the invention can be determined by known methods, such as in patent application 2016106653439, or according to the invention: the specific operation of the UPLC is as follows:

(1) taking a dried sample to be detected, crushing, extracting by using an alcohol solvent or a water-containing alcohol solvent, combining extracting solutions, removing the solvent, re-dissolving residues by using methanol, water-containing methanol, acetonitrile or water-containing acetonitrile, and preparing a sample solution;

(2) taking a mixture of the rhizoma paridis saponins I, II, VI, VII, H and fine dioscin to prepare a reference substance solution;

(3) respectively injecting the test solution and the reference solution into an ultra-high performance liquid chromatograph, and detecting by adopting an external standard method; wherein, the chromatographic conditions are as follows:

a chromatographic column: octadecylsilane chemically bonded silica as filler

ELSD detector conditions: drift tube temperature 55 + -2 deg.C, nitrogen pressure 40 + -2 psi;

mobile phase: acetonitrile (a) -water (B) system, the gradient program is as follows:

0-1min, (5% or 10%) - (25% or 30%) A; 1 to (2.5 or 3) min, (25 percent or 30 percent) to 30 percent A; (2.5 or 3) to 4min, 30% to (35% or 40%) A; 4 to (6 or 8) min, (35 percent or 40 percent) to 40 percent A; (6 or 8) -20min, 40% -50% of A; 20-22min, 50% -70% A; 22-23min, 70% -90% A.

Wherein, in the step (1), the alcohol solvent is selected from methanol, ethanol or propanol, and the aqueous alcohol solvent is selected from aqueous methanol, aqueous ethanol or aqueous propanol; in the step (2), the solvent of the control solution is selected from methanol, aqueous methanol, acetonitrile or aqueous acetonitrile.

Wherein, in the step (3), the size and model of the chromatographic column should meet the requirements of UPLC, and in a specific embodiment of the invention, the size of the chromatographic column is 2.1 × 50mm, and the particle size of the filler is 1.8 μm.

Wherein the flow rate of the mobile phase is 0.1-0.3 mL/min^-1(ii) a The column temperature was 40. + -. 2 ℃. Of course, in the case of determination of the gradient elution conditions, satisfactory measurement results can be obtained by appropriately changing the flow rate and the column temperature, but the retention time of each peak is changed accordingly and can be determined by the peak of the control chromatogram.

In one embodiment of the present invention, the mobile phase gradient is:

0～1min，10％～30％A；1～3min，30％～30％A；3～4min，30％～40％A；4～8min，40％～40％A；8～20min，40％～50％A；20～22min，50％～70％A；22～23min，70％～90％A。

wherein, the solvent used in the "redissolution" can be selected from methanol, aqueous methanol, acetonitrile or aqueous acetonitrile. In one embodiment of the invention methanol is used as solvent. However, the flow phase should have good solubility to the target component to be detected, so that in the practical use process, the re-dissolution can be carried out by using a mobile phase solvent, namely acetonitrile or water-containing acetonitrile.

Wherein the extraction method is selected from ultrasound, soaking, percolating or refluxing. In one embodiment of the present invention, ultrasound is selected as the extraction method. Of course, no matter which extraction mode is selected, the invention is based on the premise that the detection accuracy of the target component in the sample to be detected is not influenced as much as possible, in the actual operation process, the extraction mode can be screened by the detection method, the screening experiment is a common verification experiment, and creative labor is not required.

In one embodiment of the present invention, in step (1), the solvent is removed by evaporation under reduced pressure. The purpose of the use of "reduced pressure evaporation" is to minimize the influence on the detection accuracy of the target component in the sample to be detected, and naturally, other solvent removal means such as natural evaporation, low temperature heating evaporation, and the like may be used in addition to this method. That is, the method of removing the solvent is a conventional operation method in the art, and may be selected according to the actual situation, and should not be limited to "evaporation under reduced pressure".

Drawings

FIG. 1 mixed reference chromatogram, 1, Paris saponin VII; 2. rhizoma paridis saponin H; 3. rhizoma paridis saponin VI; 4. rhizoma paridis saponin II; 5. fine dioscin; 6. paris polyphylla saponin I

FIG. 2 is a chromatogram of Paris polyphylla in the major theory, 1, Paris saponin VII; 2. rhizoma paridis saponin H; 3. paris polyphylla saponin VI

FIG. 3 chromatogram of Paris polyphylla, 1, fine dioscin; 2. paris polyphylla saponin I

FIG. 4 UPLC chromatogram of rhizoma paridis, rhizoma paridis saponin VII; 2. rhizoma paridis Saponin H

FIG. 5 UPLC chromatogram of rhizoma paridis, 1, rhizoma paridis saponin II; 2. fine dioscin; 3. rhizoma paridis saponin I

Detailed Description

Unless otherwise defined herein, technical and scientific terms used in connection with the present invention shall have the meaning commonly understood by one of ordinary skill. The meaning and scope of the terms should be clear; however, if any potential ambiguity exists, the definitions provided herein take precedence over any dictionary or extrinsic definition.

Example 1

Apparatus and materials

1.1 instruments

METTLER AE240 electronic analytical balance (Shanghai Merler-Torledo instruments, Inc.); KQ-5200E ultrasonic cleaner (40kHz, 250W, Kunshan ultrasonic Instrument Co., Ltd.); BP211D model electronic balance (Sartorius, germany); waters Acquity

H-Class ultra high performance liquid chromatograph (Waters corporation, USA).

1.2 materials and reagents

Water (drochen distilled water); methanol (pure analysis by Tandon chemical reagent factory); HPLC grade acetonitrile (Sigma company, usa).

Compound 1, paridis saponin VII (batch number: MUST-14060302, Purity: 99.03%), compound 4, paridis saponin VI (batch number: MUST-14071904, Purity: 98.67%), compound 5, paridis saponin II (batch number: MUST-15060810, Purity: 98.45%), compound 6, dioscin (batch number: MUST-15081110, Purity: 98.86%), compound 7, paridis saponin I (batch number: MUST-15061611, Purity: 98.34%) were purchased from Dowman bioscience, Inc., compound 2 (Pennogenin-3-O- β -D-glucopyranosyl (1 → 3) - [ α -L-rhamnopyranosyl (1 → 2) ] - β -D-glucopyranoside) and compound 3 (Paris saponin H) were obtained by the university of Wako university of Wakko, university of Wahua, and the subject of HPLC tests were all 98%.

The rhizoma paridis and the rhizoma paridis of the unique dragon are identified by the professor Liuyuan of national institute of medicine of southwest university and the professor Zhanghao of western medicine institute of Sichuan university, and the original plant specimen and the medicinal material sample are stored in the national institute of medicine of western university and Sichuan university in southwest. Sample information is shown in table 1.

TABLE 1

2 test method

2.1 chromatographic conditions

An ACQUITY BEH C18 column (2.1 mm. times.50 mm, 1.7 μm); ELSD detector drift tube temperature 55 deg.C, nitrogen pressure 40psi, gain 500, cooling mode; the column temperature is 40 ℃; flow rate 0.2 mL/min^-1(ii) a Sample introduction amount: 1 mu L of the solution; the mobile phase is acetonitrile (A) -water (B), and the gradient elution procedure is as follows: 10% -30% of A in 0-1 min; 1-3min, 30% -30% A; 3-4min, 30% -40% A; 4-8min, 40% -40% A; 8-20min, 40% A-50% A; 20-22min, 50% A-70% A; 22-23min, 70% A-90% A. The reference and sample are mixed as shown in FIGS. 1, 2 and 3.

2.2 preparation of control solutions

Precisely weighing 2.060mg of rhizoma paridis saponin VII, 2.100mg of rhizoma paridis saponin H, 2.060mg of rhizoma paridis saponin VI, 2.080mg of rhizoma paridis saponin II, 2.040mg of slender dioscin and 2.020mg of rhizoma paridis saponin I, respectively dissolving in methanol, filling in a 10mL volumetric flask, and filtering with 0.22 μm microporous membrane. The compound 2 is self-made by Zhanghao teacher subject group at Waysia university college of pharmacy, Sichuan university, and the quantity is not enough for quantitative analysis, but only for qualitative analysis.

2.3 preparation of test solutions

Pulverizing the dried medicinal materials into powder, sieving with a 40-mesh sieve, collecting medicinal powder 0.5g, precisely weighing, placing in a 100mL conical flask, adding methanol at a material-to-liquid ratio of 1:100(g: mL), shaking, standing for 4h, performing ultrasonic treatment for 30min, filtering, placing the filtrate in a flask, and repeating the above operation for 2 times. Combining the filtrates, recovering the solution under reduced pressure, dissolving the residue with methanol, metering to a volume of 25mL volumetric flask, and storing in a refrigerator at 4 ℃ for later use. Filtering with 0.22 μm filter membrane (before injection) for use.

2.4 methodological considerations

2.4.1 examination of the Linear relationship

Precisely sucking a proper amount of the reference substance solution under the item 2.2, diluting to 2mL, preparing mixed reference substance solutions with different dilution times, and determining according to the chromatographic condition under the item 2.1. The peak area is used as the ordinate (Y) and the sample amount (μ g) is used as the abscissa (X), and an external standard method standard curve is drawn, and the result is shown in Table 2.

TABLE 2 Linear regression equation for control

2.4.2 precision test

Taking S3 batch sample solution, continuously injecting sample for 6 times under the chromatographic condition of '2.1', and recording the relative retention time and relative peak area of rhizoma paridis saponin VII, rhizoma paridis saponin H, rhizoma paridis saponin VI, rhizoma paridis saponin II, gracillin, rhizoma paridis saponin I, and rhizoma paridis saponin V peak. The results show that the RSD of the areas of the obtained rhizoma paridis saponins VII, VI and II, the fine dioscin and I peak are respectively 1.25%, 1.33%, 1.89%, 1.67% and 1.99%, and are all less than 3%, which indicates that the precision of the instrument is good.

2.4.3 repeatability test

Taking S3 batches of test solution, preparing 6 parts of test solution in parallel under the item '2.3', measuring according to the chromatographic condition under the item '2.1', and recording the relative retention time and the relative peak area of peaks of rhizoma paridis saponin VII, rhizoma paridis saponin H, rhizoma paridis saponin VI, rhizoma paridis saponin II, slender dioscin, rhizoma paridis saponin I and rhizoma paridis saponin V. The results show that the RSD of the areas of the peaks of the paris polyphylla saponin VII, VI and II, the fine dioscin and I are respectively 1.26%, 1.21%, 1.83%, 1.27% and 1.55%, and the RSD is less than 3%, which indicates that the method has good repeatability.

2.4.4 stability test

Taking S3 batches of test solution, respectively injecting samples for determination at 0, 2, 4, 8, 12 and 24H according to the chromatographic condition under the item of 2.1, and recording the relative retention time and the relative peak area of peaks of rhizoma paridis saponin VII, rhizoma paridis saponin H, rhizoma paridis saponin VI, rhizoma paridis saponin II, slender dioscin, rhizoma paridis saponin I and rhizoma paridis saponin V. The results show that the RSD of the areas of the paris polyphylla saponin VII, VI, II, the fine dioscin and I peak are respectively 1.15%, 1.78%, 1.81%, 1.07% and 1.79%, and the RSD is less than 3%, which indicates that the stability of the test solution is good within 24 h.

2.4.5 sample recovery test

Taking 5 parts of paris polyphylla medicinal material samples with known content in S3 batches, respectively adding paris polyphylla saponin VII, VI, II, slender dioscin and I reference substances in precise weighing, preparing samples according to the steps of '2.3', fixing the volume, measuring, and calculating the recovery rate. The average sample recovery rates of rhizoma paridis saponin VII, VI and II, slender dioscin and I are respectively as follows: 98.92%, 99.44%, 102.13%, 98.44%, 99.21%, RSD 1.86%, 1.54%, 1.48%, 1.92%, 1.83%.

3 results and analysis

Precisely absorbing the sample solution prepared under the item 2.3, measuring according to the chromatographic condition under the item 2.1, and calculating the content of the paris polyphylla saponin in the sample, wherein the content result of the paris polyphylla saponin is shown in a table 2, and the content result of the paris polyphylla saponin is shown in a table 3. The chromatogram of rhizoma paridis is shown in figure 4, and the chromatogram of rhizoma paridis is shown in figure 5.

TABLE 310 contents of 6 steroidal saponins (%)

Note: "-" means none

TABLE 47 The contents (%) -of 6 steroidal saponins in Duolong Paris polyphylla samples

Note: "-" means none

The modern pharmacological research shows that the steroid saponin of the paris polyphylla is the main active component for exerting pharmacological effects of the paris polyphylla, the steroid saponin has similar structures and belongs to terminal absorption on an ultraviolet spectrum, and the sensitivity is relatively low. An Evaporative Light Scattering Detector (ELSD) is a novel universal quality detector which is applied in the last 90 th century, can generate a response signal for a compound without ultraviolet absorption in the ELSD, and has the advantages of stability, sensitivity, gradient elution analysis and the like. The experiment adopts ultra-high performance liquid chromatography combined with an evaporative light scattering detector (UPLC-ELSD) detection technology to analyze 6 steroidal saponins in the medicinal materials of the paris polyphylla and the paris polyphylla. The result shows that 6 types of rhizoma paridis saponins can be separated within 24min, the method greatly shortens the time for separating and analyzing the rhizoma paridis medicinal materials, and the precision, stability, repeatability, sample adding and recycling tests and the like of the method show that the established method is stable and reliable.

The paris polyphylla belongs to extremely endangered paris polyphylla varieties, the quality indexes of the paris polyphylla are all evaluated by the total mass fractions of paris polyphylla saponin I, paris polyphylla saponin II, paris polyphylla saponin VI and paris polyphylla saponin VII in the Chinese pharmacopoeia of the calendar, and the total mass is not less than 0.6 percent and is qualified. The test result shows that: (1) the average value of the content of the saponin of the paris polyphylla specified in pharmacopeia of 10 batches of paris polyphylla is 1.2153%, except S7 batches, other batches of paris polyphylla all reach the standard specified in the pharmacopeia; (2) rhizoma paridis contains rhizoma paridis saponin VII and rhizoma paridis saponin H; trace amount of rhizoma paridis saponin VI is detected in only S10 batches of 10 batches, and rhizoma paridis saponin I, rhizoma paridis saponin II and tenuifolin are not detected; (3) the test result is basically consistent with the content result of the paris polyphylla in the early stage of the subject group.

The paris polyphylla is only distributed in the country of the Dulongjiang river in the Yangjiang state, and the resource storage amount of the paris polyphylla is much less than that of the paris polyphylla in the Dali-Zhi province. The special trip is directed to the Yangzhou Dulongjiang county to carry out resource collection, but only 7 samples are collected. The test result shows that: (1) in 7 batches of paris polyphylla, the average mass fraction of the paris polyphylla saponin I, the paris polyphylla saponin II, the paris polyphylla saponin VI and the paris polyphylla saponin VII is 0.6872%, and only S13, S14 and S17 batches reach the standard of saponin content specified in pharmacopoeia; (2) the paris polyphylla contains paris polyphylla saponin I and slender pennogenin, the S17 batches contain paris polyphylla saponin II, and 7 batches do not contain paris polyphylla saponin VI, paris polyphylla saponin VII and paris polyphylla saponin H.

Patent application 2016106653439 states that paris polyphylla contains paris saponins VII, H, II and I, but fine dioscin is not a necessary component in paris polyphylla, and only 6 of 23 batches of medicinal materials contain the saponin with the content of 0.5 or less. Therefore, compared with the paris polyphylla, the paris polyphylla contains the chemical components of the paris polyphylla saponin I and the fine dioscin, but does not contain the paris polyphylla saponin VI, the paris polyphylla saponin VII and the paris polyphylla saponin H, and meanwhile, the paris polyphylla and the paris polyphylla also do not have the characteristics of the paris polyphylla, so that the paris polyphylla and the paris polyphylla can be distinguished and separated through the components.

Attached: content detection of paris polyphylla

Chromatographic conditions

ACQUITY

HSS C18 column (2.1 mm. times.75 mm, 1.7 μm); eThe LSD detector drift tube temperature is 55 ℃, the nitrogen pressure is 40psi, the gain is 500, and the cooling mode is adopted; the column temperature is 40 ℃; flow rate 0.2 mL/min^-1(ii) a Sample introduction amount: 2 mu L of the solution; the mobile phase was acetonitrile (a) -water (B) and the gradient elution procedure is shown in table 5.

TABLE 5 gradient elution procedure

TABLE 6 content of each component in Paris polyphylla Smith

Claims

1. A method for identifying paris polyphylla is characterized by comprising the following steps: the method is used for distinguishing the paris polyphylla from one or more of paris yunnanensis, paris polyphylla or paris polyphylla, and the method adopts liquid chromatography for detection, if the paris polyphylla contains tenuiform dioscin, the paris polyphylla saponin VII, H and VI are not contained or the content of the tenuiform dioscin the detected tenuiform is lower than 0.010 percent; then the rhizoma paridis can be judged as the single dragon;

the liquid chromatography is selected from UPLC, and the specific operation of the UPLC is as follows:

a chromatographic column: octadecylsilane chemically bonded silica is used as a filler,

mobile phase: the gradient program of the acetonitrile A-water B system is as follows:

2. the authentication method according to claim 1, wherein: wherein the content of slender dioscin is more than 0.7%.

3. The authentication method according to claim 2, wherein: wherein the content of slender dioscin is more than 0.8%.

4. The authentication method according to claim 1, wherein: wherein the composition contains rhizoma paridis saponin I.

5. The authentication method according to claim 4, wherein: the content of rhizoma paridis saponin I is above 0.09%.

6. The authentication method according to claim 5, wherein: the content of rhizoma paridis saponin I is above 0.1%.

7. The authentication method according to claim 1, wherein: wherein, the content of the rhizoma paridis saponin II is not contained or is less than 0.40 percent.

8. The authentication method according to claim 1, wherein: in the step (1), the alcohol solvent is selected from methanol, ethanol or propanol, and the aqueous alcohol solvent is selected from aqueous methanol, aqueous ethanol or aqueous propanol; in the step (2), the solvent of the control solution is selected from methanol, aqueous methanol, acetonitrile or aqueous acetonitrile.

9. The authentication method according to claim 1, wherein: in the step (3), the size of the chromatographic column is 2.1X 50mm, and the particle size of the filler is 1.8 μm.

10. The authentication method according to claim 1, wherein: the flow rate of the mobile phase is 0.1-0.3 mL/min^-1(ii) a The column temperature was 40. + -. 2 ℃.

11. The authentication method according to claim 1, wherein: the mobile phase gradient was: