CN115684378A - Method for measuring content of morroniside and loganin in dogwood and cornel related preparations - Google Patents

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a method for measuring the content of morroniside and loganin in a dogwood and cornel related preparation, which comprises the following steps: (1) preparation of a sample solution of a dogwood and cornel related preparation, (2) preparation of a morroniside and loganin mixed reference substance solution, (3) determination of the content of the morroniside and loganin in the sample solution by an HPLC method, wherein the chromatographic conditions are as follows: c18 column, mobile phase phosphoric acid solution (a) -acetonitrile: and (3) performing gradient elution on the phosphoric acid solution (B), wherein the column temperature is 35 ℃, the flow rate is 1ml/min, the detection wavelength is 240nm, the sample injection amount is 10 mu L, and the gradient elution mode is the volume fraction of the mobile phase A: changing from 78% to 75% in 0-5 min; 5-20min from 75% to 72%; changing from 72% to 60% in 20-38 min; the preparation of the test solution in the step (1) comprises the following steps: extracting the test powder with methanol water solution as solvent, and collecting the filtrate. The method can realize good separation of chromatographic peaks, and has the advantages of good peak type, less peak interference, stable baseline and stronger method universality.

Description

Method for measuring content of morroniside and loganin in dogwood and cornel related preparation

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a method for measuring the content of morroniside and loganin in dogwood and cornel related preparations.

Background

The Corni fructus is dried mature pulp of Cornus officinalis Sieb. Et Zucc. Of Cornus of Cornaceae, fruit is collected when pericarp turns red in late autumn and early winter, and the fruit is baked with slow fire or slightly scalded in boiling water, and then kernel is removed in time and dried. Cornus officinalis is produced in Shanxi, shaanxi, gansu, shandong, jiangsu, anhui and Zhejiang provinces, is distributed in Korea and Japan, has the effects of tonifying liver and kidney, astringing and relieving depletion and the like, and is used for dizziness and tinnitus, soreness and pain of waist and knees, impotence and spermatorrhea, enuresis and frequent micturition, metrorrhagia and leukorrhagia, profuse sweat depletion, internal heat and thirst quenching.

The main chemical components of dogwood are iridoid, tannin, flavonoid and the like, wherein iridoid glycosides are the main material basis of the dogwood playing the traditional effect, and the quality of the dogwood is mostly evaluated by iridoid at present. The content of morroniside and loganin is measured and controlled under the item of dogwood in the 'Chinese pharmacopoeia' 2020 edition, in actual analysis, the defects of impurity peak interference, unstable baseline, poor method universality and the like exist, and good detection effect and strong method universality are difficult to realize on main drug effect substance components in the dogwood and related preparations thereof. Therefore, it is necessary to establish a content measuring method which has high sensitivity and accuracy, good reproducibility, practicality, simplicity and universality.

Disclosure of Invention

Aiming at the technical current situation, the invention provides an improved method for measuring the contents of morroniside and loganin in a dogwood and cornel related preparation. The invention comprises the following concrete steps:

the invention provides an improved determination method for the content of morroniside and loganin in dogwood and cornel related preparations, which comprises the following steps:

(1) Preparing a test solution of a preparation related to dogwood and cornel; (2) Preparing a morroniside and loganin mixed reference solution; (3) The HPLC method is adopted to determine the contents of the morroniside and loganin in the test solution, and the chromatographic conditions are as follows: c18 column, mobile phase phosphoric acid solution (a) -acetonitrile: performing gradient elution on the phosphoric acid solution (B), wherein the column temperature is 35 ℃, the flow rate is 1ml/min, the detection wavelength is 240nm, the sample injection amount is 10 mu L, the gradient elution mode is 0-5min, and the volume fraction of the mobile phase A is changed from 78% to 75%;5-20min, the volume fraction of the mobile phase A is changed from 75% to 72%; the volume fraction of the mobile phase A is changed from 72% to 60% in 20-38 min.

In the method of the present invention, as one embodiment, the preparation of the test solution of the cornus officinalis and cornus officinalis related preparation in step (1) comprises the following steps: taking the sample powder, extracting with methanol water solution as solvent, and collecting filtrate to obtain sample solution.

In the method of the present invention, as one embodiment, the extraction solvent in the step (1) is methanol aqueous solution, preferably 50% methanol solution.

In the method of the present invention, as one of the embodiments, the method further comprises the step (1) of extracting by sonication or refluxing, preferably sonication.

In the method of the present invention, as one embodiment, the method further comprises the step (1) of extracting the feed-liquid ratio of 0.05g to 25ml to 0.2g, preferably 0.1g.

In the method of the present invention, as one embodiment, the method further comprises the step of extracting for 30min to 90min, preferably 30min in step (1).

In the method of the present invention, as one embodiment, the preparation of the morroniside and loganin mixed control solution in the step (2) comprises the following steps: precisely weighing the dinonoside and loganin, and preparing a mixed reference substance solution by using a methanol water solution as a solvent.

In the method of the present invention, as one embodiment, the solvent in step (2) is preferably 50% methanol solution, and the mixed control solution containing 60ug of morroniside and 40ug of loganin per 1ml is prepared.

In the method of the present invention, as one of the embodiments, the method further comprises the step (3) of a mobile phase of 0.3% phosphoric acid solution (a) -acetonitrile: 0.3% phosphoric acid solution (B), preferably acetonitrile in a volume ratio of 1: 0.3% phosphoric acid solution was used as mobile phase B.

In the method of the present invention, as one of embodiments, the method further comprises:

(1) Chromatographic conditions are as follows: chromatography column Waters Xbridge C18 (4.6 x 150mm,3.5 um); mobile phase 0.3% phosphoric acid solution (a) -acetonitrile: 0.3% phosphoric acid solution (1, 3, V/V) (B) was eluted with a gradient of 35 ℃ column temperature, 1ml/min flow rate, 240nm detection wavelength, elution gradient: changing the volume fraction of the mobile phase A from 78% to 75% in 0-5 min; 5-20min, the volume fraction of the mobile phase A is changed from 75% to 72%; the volume fraction of the mobile phase A is changed from 72% to 60% in 20-38 min.

(2) Preparation of a reference solution: taking a proper amount of morroniside reference substance and loganin reference substance, precisely weighing, and adding 50% methanol to obtain mixed reference substance solution containing morroniside 60ug and loganin 40ug respectively per 1 ml;

(3) Preparing a test solution: weighing about 0.2g of the powder, precisely weighing, placing in a conical flask with a plug, precisely adding 50ml of 50% methanol, weighing, ultrasonic treating for 30min (40KHZ, 500W), cooling, weighing again, supplementing the weight loss with 50% methanol, shaking, filtering, and collecting the filtrate;

(4) The determination method comprises the following steps: precisely sucking 10 μ L of the reference solution and the sample solution, respectively, injecting into liquid chromatograph, and measuring.

According to the invention, through optimization of chromatographic conditions, investigation of a sample preparation method and methodology verification, a method for measuring the contents of morroniside and loganin in dogwood and related preparations is established, good separation of chromatographic peaks is realized, the chromatographic peak pattern is good, peak interference is less, the baseline is stable, and the method has stronger universality. Compared with the literature and legal methods, the content determination method established by the invention is more environment-friendly, scientific, economic, practical, simple and convenient, has accurate and reliable results, and can provide method basis for quality standard and quality evaluation in dogwood, alcohol cornus and preparations containing the dogwood or the alcohol cornus.

Drawings

FIG. 1 is an HPLC chromatogram of Corni fructus in example.

FIG. 2 is an HPLC chromatogram of Corni fructus in example.

FIG. 3 is the HPLC chromatogram of Liuwei Dihuang tablet in the example.

FIG. 4 is the HPLC chromatogram of the dogwood obtained by using different chromatographic columns in the examples.

FIG. 5 is the HPLC chromatogram of wine dogwood fruit obtained by using different chromatographic columns in the examples.

FIG. 6 is HPLC chromatogram of Liuwei Dihuang tablets obtained in examples by using different chromatographic columns.

FIG. 7 is a graph showing the results of peak purity in Experimental example 2.2.

FIG. 8 is an HPLC chromatogram of Cornus officinalis in a comparative example.

FIG. 9 is an HPLC chromatogram of alcohol dogwood fruit in the comparative example.

FIG. 10 is an HPLC chromatogram of Corni fructus obtained by using different chromatographic columns in the comparative example.

FIG. 11 is an HPLC chromatogram of dogwood fruit obtained by using different chromatographic columns in the comparative example.

Detailed Description

The following examples and test examples are intended to further illustrate the present invention, but are not intended to limit the effective scope of the present invention in any way.

The following examples, experimental examples or comparative examples used, the apparatus and reagents were as follows:

high performance liquid chromatography (Waters e2695, waters technologies ltd);

chromatography column Waters Xbridge C18 (4.6 x 150mm,3.5 um);

electronic balance (XSE 205DU, METTLER TOLEDO);

electronic balance (ME 204, mettler TOLEDO;

electronic balance (PL 403, METTLER TOLEDO);

morroniside (111998-201703, china institute for food and drug testing);

loganin (111640-201808, china institute for testing food and drug);

phosphoric acid (10015408, national drug group, super pure);

methanol (20200110, beijing chemical plant, analytical grade);

acetonitrile (11006830917, merck, chromatographically pure);

purified water (manufactured by MiLLI-Q).

Examples 1-13 determination of the content of morroniside and loganin in Cornus officinalis

In examples 1 to 13, the content of morroniside and loganin in different batches of dogwoods is measured by the method of the present invention;

column Waters Xbridge C18 (4.6 x 150mm,3.5 um), mobile phase 0.3% phosphoric acid solution (a) -acetonitrile: a 0.3% phosphoric acid solution (1, V/V) (B) was eluted with a gradient of 1.0mL/min; the column temperature is 30 ℃; the detection wavelength is 240nm. The elution gradient was: changing the volume fraction of the mobile phase A from 78% to 75% in 0-5 min; 5-20min, the volume fraction of the mobile phase A is changed from 75% to 72%; changing the volume fraction of the mobile phase A from 72% to 60% within 20-38 min;

preparing a reference substance solution: weighing about 0.2g of the powder, precisely weighing, placing in a conical flask with a plug, precisely adding 50ml of 50% methanol, weighing, ultrasonic treating for 30min (40KHZ, 500W), cooling, weighing again, supplementing the weight loss with 50% methanol, shaking, filtering, and collecting the filtrate;

preparing a test solution: taking a proper amount of morroniside reference substance and loganin reference substance, precisely weighing, and adding 50% methanol to obtain mixed reference substance solution containing morroniside 60ug and loganin 40ug respectively per 1 ml;

the determination method comprises the following steps: precisely sucking 10uL of each of the reference solution and the sample solution, injecting into a liquid chromatograph, and measuring;

sample preparation: the dogwood medicinal material is numbered as follows: S1S 2S 3S 4S 5S 6S 7S 8S 9S 10S 11S 12S13, the results are shown in table 1.

TABLE 1 measurement results of the content of Corni fructus

Numbering	Monoside (%)	Loganin (%)	Total amount (%)
				S1	1.59	0.78	2.37
S2	1.34	0.82	2.16
				S3	1.62	1.01	2.63
S4	0.98	0.74	1.72
				S5	1.11	0.78	1.89
S6	1.58	0.85	2.43
				S7	1.55	0.81	2.36
S8	1.61	0.84	2.45
				S9	1.42	0.83	2.25
S10	1.47	0.81	2.28
				S11	1.59	0.89	2.49
S12	1.35	0.75	2.09
				S13	1.31	0.81	2.12

The HPLC chromatogram obtained by the method for measuring the contents of morroniside and loganin in dogwood and related preparations thereof is shown in a dogwood picture 1, a dogwood wine picture 2 and a six-ingredient rehmannia tablet picture 3. Description of the drawings: morroniside (t = 12.098), loganin (t = 29.495) in fig. 1; morroniside (t = 13.682), loganin (t = 31.839) in fig. 2; morroniside (t = 11.893), loganin (t = 28.974) in fig. 3.

According to the established method for measuring the content of the morroniside and the loganin by HPLC, different chromatographic columns are adopted for analysis to measure the content of the morroniside and the loganin in the dogwood and the related preparation, and the obtained HPLC chromatogram is shown in a dogwood picture 4, a dogwood wine picture 5 and a liuwei Dihuang tablet picture 6.

Verification of Experimental examples

The following experimental examples 2.1 to 2.7 of the present invention are methodological evaluations of the content measurement method, and experiments of system adaptability, specificity, linear range, repeatability, intermediate precision, stability, accuracy, and durability were respectively performed in sequence according to determined conditions.

Experimental example 2.1 System Adaptation test

The system adaptability of the chromatographic system is examined by using a reference solution and a test solution. The results are shown in tables 2 and 3

TABLE 2 Monoloside System Adaptation results

TABLE 3 loganin system compliance results

And continuously feeding the control solution for 5 times, wherein the RSD of the peak areas of the morroniside and the loganin is less than 2.0%, the RSD of the retention time is less than 1.0%, the theoretical plate number of the morroniside and the loganin in the test solution meets the requirement, the tailing factor is less than 1.5, the separation degree is more than 1.5, and the method is feasible.

Experimental example 2.2 specificity test

Blank solvent (50% methanol), mobile phase, reference solution and test solution are injected for detection. The results are shown in FIG. 7 and Table 4

TABLE 4 Monoloside and loganin peak purity results

The result shows that the blank solvent and the flow have no interference relative to a reference substance and a sample, the chromatographic peaks of the morroniside and the loganin in the test solution have no interference, and the method has good specificity.

Experimental example 2.3 Linear Range test

Control stock solutions: precisely weighing appropriate amount of morroniside reference substance and loganin reference substance, precisely weighing, and adding 50% methanol to obtain mixed reference substance solution containing morroniside 300 μ g and loganin 200 μ g per 1 ml;

precisely sucking a proper amount of the control stock solution to prepare a series of solubility solutions, and calculating a morroniside and loganin regression equation, a regression equation correlation coefficient, an intercept on a y axis, a slope and each concentration response factor RSD.

The results are shown in tables 5 and 6

TABLE 5 loganin linearity test results

TABLE 6 Mononoside Linear test results

From the results, the correlation coefficient was greater than 0.999; the y-axis intercept is less than 2% of the peak area of 100% concentration; the RSD of each concentration response factor is less than 2.0 percent, and the linear relation is good.

Experimental example 2.4 repeatability test

Taking about 0.2g of the product powder, preparing 6 parts in parallel according to the method of example 1, and calculating the total amount of morroniside and loganin. The results are shown in Table 7

TABLE 7 content repeatability test results

The results of the morroniside, loganin and total content RSD in 6 parts of the test sample are less than 2.0 percent, which meets the requirements.

EXAMPLE 2.5 intermediate precision test

According to the reproducibility measurement method, 6 test solutions of the same batch were prepared in parallel by different analysts using different instruments on different dates and compared with the reproducibility results.

The results are shown in tables 8, 9 and 10

TABLE 8 intermediate precision test results for loganin

TABLE 9 intermediate precision test results for morroniside

TABLE 10 Total content intermediate precision test results

According to the results, the results of the content results RSD of the morroniside, the loganin and the total content in 12 samples tested by different persons, different dates and different instruments are not more than 3.0 percent, and the requirements are met.

EXAMPLE 2.6 stability test

Respectively examining the peak area change conditions of the test solution and the reference solution after the test solution and the reference solution are placed for 0h, 4h, 8h, 12h, 18h, 24h and 48h at room temperature. The results are shown in tables 11 and 12

TABLE 11 stability test results for morroniside solutions

TABLE 12 loganin solution stability test results

Compared with the initial result, the relative content of the result of each time point of the reference substance and the test solution is between 98.0 percent and 102.0 percent, which indicates that the solution stability is good.

Experimental example 2.7 accuracy test

About 0.1g of this product powder was weighed precisely and placed in 9 portions in parallel in a conical flask with a stopper, and a mixed control solution was added to prepare a test solution by the method described in example 1, and concentration levels of 50%, 100%, and 150% of the assay concentration were prepared as recovery rate test solutions, respectively. The sample was taken for measurement as in example 1. The results are shown in tables 13 and 14

TABLE 13 Monoloside content recovery test results

TABLE 14 loganin content recovery test results

The average recovery rate of morroniside is 98.79 percent, the RSD is 1.19 percent, the average recovery rate of loganin is 99.03 percent, and the RSD is 1.11 percent, which meets the requirement.

Experimental example 2.8 durability test

The tolerance of the detection method was examined when the chromatographic conditions were slightly varied (temperature. + -. 2%, concentration. + -. 0.05% of phosphoric acid, flow rate. + -. 0.2%, wavelength. + -. 2nm, different columns). The results are shown in Table 15

TABLE 15 durability test results

Under different conditions, the relative content of each index component content result of the sample solution is 95.0-105.0% compared with the normal condition, which shows that the method has good durability when the conditions such as flow rate, wavelength, different acid concentrations, different chromatographic column models and the like are changed.

Comparative example

In order to visually illustrate the innovative effect of the present invention, the following comparative experimental example adopts the method under the item of dogwood in the 'Chinese pharmacopoeia' 2020 edition to evaluate the universality of dogwood and alcohol dogwood, and the obtained HPLC chromatogram is shown in fig. 8, fig. 9, fig. 10 and fig. 11.

The pharmacopoeia method comprises the following steps: octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; acetonitrile is taken as a mobile phase A, 0.3 percent phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out according to the specification in the following table; the detection wavelength is 240nm; the column temperature was 35 ℃. The number of theoretical plates is not less than 10000 calculated according to loganin peak; the elution gradient was: 0-20min, the volume fraction of the mobile phase A is 7%; changing the volume fraction of the mobile phase A from 7% to 20% in 20-50 min;

preparing a reference solution by precisely weighing appropriate amount of morroniside reference substance and loganin reference substance, and adding 80% methanol to obtain mixed solutions each containing 50 μ g of morroniside and loganin;

preparing a test solution, namely taking about 0.2g of powder (screened by a third sieve), accurately weighing, placing the powder in a conical flask with a plug, accurately adding 25ml of 80% methanol, weighing, heating and refluxing for 1 hour, cooling, weighing again, complementing the weight loss by 80% methanol, shaking up, filtering, and taking subsequent filtrate;

the determination method comprises precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and determining.

Compared with the existing pharmacopoeia method, the method for determining the content of the dogwood disclosed by the invention realizes good separation of chromatographic peaks, and has the advantages of good chromatographic peak pattern, less peak interference, stable base line and stronger method universality.

Claims (9)

1. A method for determining the content of morroniside and loganin in a dogwood and cornel-related preparation, which is characterized in that the method comprises the following steps:

(1) Preparing a test solution of dogwood and dogwood related preparations; (2) Preparing a morroniside and loganin mixed reference solution; (3) The HPLC method is adopted to determine the contents of the morroniside and loganin in the test solution, and the chromatographic conditions are as follows: c18 column, wherein the mobile phase A is phosphoric acid solution, and the mobile phase B is acetonitrile: gradient elution is carried out on phosphoric acid solution, the column temperature is 35 ℃, the flow rate is 1ml/min, the detection wavelength is 240nm, the sample injection amount is 10 mu L, the gradient elution mode is 0-5min, and the volume fraction of the mobile phase A is changed from 78% to 75%;5-20min, and changing the volume fraction of the mobile phase A from 75% to 72%; changing the volume fraction of the mobile phase A from 72% to 60% in 20-38 min;

the preparation of the test solution of the cornel and the cornel-related preparation in the step (1) comprises the following steps: taking the sample powder, extracting with methanol water solution as solvent, and collecting filtrate to obtain sample solution.

2. The method according to claim 1, wherein the extraction solvent in step (1) is an aqueous methanol solution, preferably a 50% methanol solution.

3. The method according to claim 1, wherein the extraction mode in step (1) may be ultrasound or reflux, preferably ultrasound.

4. The method according to claim 1, wherein the extract liquid ratio in the step (1) is between 0.05g and 25ml and 0.2g.

5. The method according to claim 1, wherein the extraction time in step (1) is 30min to 90min, preferably 30min.

6. The method of claim 1, wherein the preparation of the morroniside and loganin mixed control solution in step (2) comprises the following steps: precisely weighing the obtained pennogenin and loganin, and preparing into mixed reference solution with methanol water solution as solvent.

7. The method according to claim 6, wherein the solvent in step (2) is preferably 50% methanol solution, and the mixed control solution containing morroniside 60ug and loganin 40ug per 1ml is prepared.

8. The method according to claim 1, wherein the mobile phase A in step (3) is a 0.3% phosphoric acid solution, and the mobile phase B is acetonitrile: 0.3% phosphoric acid solution, preferably acetonitrile in a volume ratio of 1: 0.3% phosphoric acid solution was used as mobile phase B.

9. The method of any one of claims 1-8, further comprising:

(1) Chromatographic conditions are as follows: chromatography column Waters Xbridge C18; the mobile phase A is 0.3% phosphoric acid solution, and the mobile phase B is acetonitrile with the volume ratio of 1: 0.3 percent phosphoric acid solution, performing gradient elution, wherein the column temperature is 35 ℃, the flow rate is 1ml/min, the detection wavelength is 240nm, and the elution gradient is as follows: changing the volume fraction of the mobile phase A from 78% to 75% in 0-5 min; 5-20min, the volume fraction of the mobile phase A is changed from 75% to 72%; changing the volume fraction of the mobile phase A from 72% to 60% within 20-38 min;

(2) Preparation of a reference solution: taking a proper amount of the morroniside reference substance and the loganin reference substance, precisely weighing, and adding 50% methanol to prepare a mixed reference substance solution containing 60ug of morroniside and 40ug of loganin in each 1 ml;

(3) Preparing a test solution: taking about 0.2g of the powder, precisely weighing, placing in a conical flask with a plug, precisely adding 50ml of 50% methanol, weighing, 40KHZ, performing ultrasonic treatment for 30 minutes under the condition of 500W, cooling, weighing again, supplementing the lost weight with 50% methanol, shaking up, filtering, and taking the subsequent filtrate to obtain the final product;

(4) And (3) determination: precisely sucking 10 μ L of the reference solution and the sample solution, respectively, injecting into liquid chromatograph, and measuring.

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