CN114965779A

CN114965779A - Method for determining content of lignans in schisandra chinensis by multi-evaluation method and application

Info

Publication number: CN114965779A
Application number: CN202210599732.1A
Authority: CN
Inventors: 王隶书; 林丽娜; 程东岩; 高军; 陈昕; 王超楠; 程东红; 匡鸣
Original assignee: China Resources Sanjiu Medical and Pharmaceutical Co Ltd
Current assignee: China Resources Sanjiu Medical and Pharmaceutical Co Ltd
Priority date: 2022-05-30
Filing date: 2022-05-30
Publication date: 2022-08-30

Abstract

The invention provides a method for determining the content of lignans in schisandra chinensis by a multi-evaluation method and application thereof, wherein the method for determining the content of the lignans in schisandra chinensis by the multi-evaluation method comprises the steps of determining a schisandra chinensis test solution by high performance liquid chromatography to obtain a characteristic spectrum with characteristic peaks of the lignans, taking schisandrin as an internal reference, establishing a relative correction factor between the schisandrin and other lignans by the multi-evaluation method, and calculating the content of other lignans by the correction factor. The method has the advantages of strong practicability, simple operation, cost saving and accurate and reliable test result, and makes up the defects of the existing quality control method of the schisandra chinensis, thereby providing a new idea and reference for quality control and quality evaluation of schisandra chinensis medicinal materials and decoction pieces.

Description

Method for determining content of lignans in schisandra chinensis by multi-evaluation method and application

Technical Field

The invention relates to the field of quality control of traditional Chinese medicines, in particular to a method for determining the content of lignans in schisandra chinensis by a multi-evaluation method and application thereof.

Background

Fructus Schisandrae (known as "Schisandra chinensis"), a dried mature fruit of Schisandra chinensis (Turcz.) Baill. of Magnoliaceae, is mainly distributed in Heilongjiang, Jilin, Liaoning provinces, and has the effects of astringing, invigorating qi, promoting fluid production, invigorating kidney, and calming heart, and is commonly used for treating chronic cough, asthma, nocturnal emission, enuresis, frequent micturition, chronic diarrhea, spontaneous perspiration, night sweat, body fluid consumption, thirst, internal heat, diabetes, palpitation, insomnia, etc., and is a common traditional Chinese medicine with important therapeutic value. At present, a plurality of active ingredients are separated from schisandra chinensis, wherein lignanoid compounds are the main active ingredients for playing a role, and researches show that the lignanoid compounds have curative effects on digestion, central nervous system, cardiovascular system, endocrine system and immune system, and have various pharmacological actions such as cardiovascular protection, pressure regulation and lipid lowering, liver and enzyme protection, inflammation diminishing and cancer resistance, memory improvement, sleep improvement, immunity improvement and the like.

Due to the complexity of phytochemicals, multi-index content determination has become a consensus in quality control of plant-derived drugs. At present, more and more standards of the plant medicines at home and abroad record the multi-index content measurement. However, in the implementation process of the multi-index content measurement method, the consumption of reference substances is high, the detection operation difficulty is high, the popularization and the application of the method are severely restricted, and the method becomes a bottleneck problem restricting the quality control of medicinal plants and the industrial development thereof. The QAMS (quantitative assessment method) is a multi-index synchronous quality control method which measures the content of a representative component in the traditional Chinese medicine, calculates the content of various components to be measured in the traditional Chinese medicine according to relative correction factors and controls a calculated value and an actually measured value to meet the requirements of quantitative methodology. The method has the characteristics of standard substance saving, accurate and reliable result and the like, and is popularized and applied in the quality evaluation research of a plurality of traditional Chinese medicines.

At present, under the condition of quality standard content measurement of schisandra chinensis in 'Chinese pharmacopoeia' 2020 edition, the quality of schisandra chinensis cannot be comprehensively controlled only by taking the content of schizandrol as a detection index, so that the application of a one-measurement-multiple-evaluation method in schisandra chinensis needs to be researched, and the aim of effectively and accurately measuring the content of more lignans under the condition of less consumption of a reference substance is achieved by aiming at the defects of the existing single-component and multiple-index content measurement method.

Disclosure of Invention

Therefore, the invention provides the method for measuring the content of the lignans in the schisandra by the one-measurement-multiple-evaluation method, which can effectively and accurately measure the content of more lignans in the schisandra under the condition of less reference substance consumption, and the application thereof.

A method for determining the content of lignans in fructus Schisandrae by multi-evaluation method comprises determining fructus Schisandrae sample solution by high performance liquid chromatography to obtain characteristic spectrum with characteristic peak of lignans, taking schisandrin as internal reference, establishing relative correction factor between schisandrin and other lignans by one-evaluation method, and calculating the content of other lignans by the correction factor;

the chromatographic conditions of the high performance liquid chromatography are as follows:

and (3) chromatographic column: octadecylsilane chemically bonded silica is used as a filling agent; detection wavelength: 220-254 nm; acetonitrile is used as a mobile phase A, water is used as a mobile phase B, and elution is carried out according to the following gradient elution procedure:

the temperature of the chromatographic column is 25-35 ℃, and the flow rate is 0.8-1.2 ml/min.

The column temperature was 35 ℃ and the detection flow rate was 1 ml/min.

The specification of the chromatographic column is 4.6mm multiplied by 250mm and 5 mu m, and the detection wavelength is 250 nm;

the sample amount of the test solution is 5-20 μ l, preferably 10 μ l.

The lignanoid comprises schizandrin A, schizandrin B, schizandrin A and schizandrin B.

The preparation process of the schisandra chinensis test solution comprises the following steps: taking a schisandra chinensis test sample, adding a solvent, uniformly mixing, sealing, weighing, carrying out ultrasonic treatment, cooling, weighing again, complementing the weight loss by the solvent, uniformly shaking, and filtering to obtain a filtrate, wherein the filtrate is the test sample solution.

The power of ultrasonic treatment is 250W, and the frequency is 50 kHz; the time for the ultrasonic treatment was 30 min.

The solvent is n-hexane.

The method for determining the content of the lignanoid components in the schisandra chinensis by the one-test-multiple-evaluation method is applied to the quality control and evaluation of the schisandra chinensis.

Further, the quality grade of the schisandra chinensis is judged according to the total content of the lignanoid components detected by a one-test-multiple evaluation method; the judgment rule is as follows: the total content of lignanoid components of the first-class medicinal materials is more than or equal to 0.90 percent, and the total content of lignanoid components of the second-class medicinal materials is more than or equal to 0.70 percent.

The technical scheme of the invention has the following advantages:

1. the invention adopts high performance liquid chromatography for determination, and realizes the simultaneous determination of the contents of four lignanoid components in schisandra by using a one-determination-multiple-evaluation method. Taking widely used schizandrol A as an internal reference substance to perform one-test multiple evaluation, establishing relative correction factors between the schizandrol A and the schizandrol B, deoxyschizandrin and schisandrin B, and calculating the contents of the schizandrol B, the deoxyschizandrin and the schisandrin B in the schisandra by the correction factors; the method has the advantages of strong practicability, simple operation, cost saving and accurate and reliable test result, and makes up the defects of the existing quality control method of the schisandra chinensis, thereby providing a new idea and reference for quality control and quality evaluation of schisandra chinensis medicinal materials and decoction pieces.

2. The invention firstly carries out correlation analysis between the evaluation result of one test and more tests of the schisandra chinensis and the grade of the schisandra chinensis to find out the rule, thereby formulating a more scientific and reasonable grade standard of schisandra chinensis medicinal materials; specifically, the quality grade of the schisandra chinensis is judged according to the total content of the lignanoid components detected by a one-test-multiple evaluation method; the judgment rule is as follows: the total content of lignanoid components of the first-class medicinal materials is more than or equal to 0.90 percent, and the total content of lignanoid components of the second-class medicinal materials is more than or equal to 0.70 percent.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 shows HPLC chromatogram of mixed standard, wherein 1 is schizandrol A; 2 is schisandrin B; 3 is deoxyschizandrin; 4 is schisandrin B.

FIG. 2 shows HPLC chromatogram of fructus Schisandrae, in which FIG. 1 is schisandrin; 2 is schisandrin B; 3 is deoxyschizandrin; 4 is schisandrin B.

Detailed Description

The examples do not show the specific experimental steps or conditions, and can be performed according to the conventional experimental steps described in the literature in the field. The reagents or instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially.

The instrument comprises the following steps: LC-10AT VP high performance liquid chromatograph (Shimadzu corporation, Japan); SSI Series 1500 high performance liquid chromatograph (american scientific systems); KQ5200 desk ultrasonic cleaner (kunshan mei ultrasonic instruments ltd); DV215CD electronic balance (OHAUS corporation); CP214 electronic balance (ohaus instruments, inc.).

Reagent testing: the schisandrin A reference substance (batch No. 110857-; schizandrol B reference substance (batch number: 58546-54-6, purity ≥ 98%) is provided by WUDIHUAZHENBANZHUANGZHIBAO corporation.

Reagent: acetonitrile is chromatographically pure, water is redistilled water, and other reagents are analytically pure.

The medicinal materials are as follows: 25 batches of schisandra chinensis medicinal materials are purchased from Jilin, Heilongjiang and Liaoning Sanzhou province respectively, and are divided into No. 1-2, No. 5-6, No. 8-9, No. 11, No. 13-16, No. 18, No. 20, No. 3-4, No. 7, No. 10, No. 12, No. 17, No. 19 and No. 20-25 according to the grade standard of No. 84 article specification Standard of seventy six medicinal materials of Chinese and drug Union characters (I.E.: purple red or red brown on the surface, thick meat, shriveled grains not more than 2%, and the second II.E.: black red, dark red or light red on the surface, thinner meat, shriveled grains not more than 20%). The above Schisandra chinensis (Turcz.) Baill, a Chinese magnoliavine fruit, is identified as a dried mature fruit of Schisandra chinensis (Schisandra chinensis, Magnoliaceae).

Example 1

A method for determining the content of lignans in schisandra by a multi-evaluation method comprises the following steps:

1. chromatographic conditions

A chromatographic column: vision HT C18 HL column (250 mm. times.4.6 mm, 5 μm); mobile phase: acetonitrile (a) -water (B) gradient elution, the gradient elution procedure is given in table 1 below:

TABLE 1 procedure table for gradient elution of mobile phase

Flow rate: 0.8 ml/min ^-1 (ii) a Detection wavelength: 250 nm; column temperature: 35 ℃ is carried out.

2. Preparation of the solution

2.1 mixing control solution: accurately weighing appropriate amount of schizandrol A, schizandrol B, schizandrin A, and schizandrin B reference substances, and preparing into 0.5045, 0.1590, 0.1305, and 0.2856mg/ml solutions with methanol to obtain the final product.

2.2 test solution: taking about 0.25g of schisandra chinensis medicinal material powder (passing through a No. three sieve), precisely weighing, placing in a conical flask with a plug, precisely adding 25ml of n-hexane, weighing, carrying out ultrasonic treatment (power 250W and frequency 50kHz) for 30 minutes, cooling, weighing, adding n-hexane to supplement the loss weight, shaking up, filtering, taking 10ml of subsequent filtrate, evaporating to dryness, adding methanol to dissolve residues and transferring to a 10ml measuring flask, adding methanol to dilute to a scale, shaking up, filtering, and taking the subsequent filtrate to obtain the schisandra chinensis health care tea.

3. Acquisition of a chromatogram

Accurately sucking 10 μ l of each of the reference solution and the sample solution, injecting into high performance liquid chromatograph, and measuring; chromatograms of the test solution and the mixed control solution are shown in FIGS. 1-2.

4. Methodology investigation

4.1 investigation of the Linear relationship:

precisely sucking the mixed reference substance solutions of 0.2ml, 0.4ml, 0.8ml, 1.2ml, 1.6ml, 2.0ml and 2.4ml under the item of '2.2', respectively placing in 5ml measuring bottles, adding methanol to dilute to scale, shaking up to obtain a series of mixed reference substance solutions, and performing sample injection determination under the chromatographic condition of '1'. The peak area (a) of each component was used to perform regression processing on the amount of sample (μ g), which is the volume of sample × the concentration of the control, and the results are shown in table 2 below.

TABLE 2 examination of the Linear relationship

The linear relation between the peak area and the concentration of the reference substance detected by the chromatographic condition is proved.

4.2 precision test:

taking 10 mu l of the same test solution, continuously carrying out sample injection determination for 6 times, recording peak areas, and respectively obtaining the RSD of the peak areas of the schizandrol A, the schizandrol B, the schizandrin A and the schizandrin B which are 1.65%, 1.70%, 1.57% and 1.86% (n is 6) according to the results, thereby indicating that the precision of the instrument is good.

4.3 stability test:

taking 10 mu l of the same test solution, injecting samples and measuring at 0, 2, 4, 6, 8 and 24h after preparation, respectively, and recording peak areas, wherein the results show that the RSD of the peak areas of the schizandrol A, the schizandrol B, the schizandrin A and the schizandrin B are respectively 0.56%, 2.36%, 0.59% and 0.38% (n is 6), which indicates that the test solution has good stability in 24 h.

4.4 repeatability tests:

weighing 6 parts of the same batch of schisandra chinensis respectively, preparing a test solution according to the method under item 2.2, carrying out sample injection determination according to the chromatographic condition under item 1, recording a chromatogram, calculating the content of 4 components and RSD values thereof, and obtaining the results that the average content of schisandrin A, schisandrin B, schizandrin A and schizandrin B is respectively 0.527%, 0.148%, 0.126% and 0.340%, and the average content of RSD is respectively 1.43%, 1.81%, 0.96% and 1.97% (n is 6), which indicates that the method has good repeatability.

4.5 sample recovery test:

quantitatively adding 5ml of mixed reference substance solution (containing 1.269, 0.371, 0.305 and 0.836mg of schisandrin A, schisandrin B respectively) into fructus Schisandrae medicinal powder with known content to prepare test solution according to the method under item "2.2", performing sample injection measurement under the chromatographic condition of item "1", recording chromatogram, and calculating sample injection recovery rates and RSD values of the components, wherein the average sample injection recovery rates of schisandrin A, schisandrin B, schisandrin A and schisandrin B are respectively 100.6%, 100.8%, 100.0% and 100.4%, and the RSD values are respectively 1.64%, 1.74%, 1.49% and 1.53% (n is 6), thus indicating that the sample injection recovery rate of the method is good.

5. Establishment of correction factor

5.1 calculation of correction factor:

randomly selecting 7 batches of fructus Schisandrae test solution, taking schisandrin as internal reference, and calculating formula (f) according to relative correction factor _si ＝f _s /f _i ＝A _s C _i /A _i C _s In the formula: a. the _s Is the peak area of the internal reference substance, C _s Is the concentration of the internal reference substance, A _i Is the peak area of the component to be measured, C _i The concentration of the component to be detected), relative correction factors of the schizandrol A and the schizandrol B, the schizandrin A and the schizandrin B are respectively calculated, and the results are shown in table 3.

TABLE 33 relative correction factors for the components to be tested (n-7)

The result shows that the difference of the correction factors among different batches is small, and the method can be used for content conversion of schisandrin B, deoxyschizandrin and schisandrin B.

5.2 correction factor reproducibility study test

The following Table 4 was examined for 2 different brands of HPLC chromatographs and 4 different brands of columns (each having a specification of 250 mm. times.4.6 mm, 5 μm) versus the correction factor f _{Alcohol methanol/alcohol ethanol} 、f _{methanol/methyl-A} 、f _{Methanol methyl/ethanol} The results are shown in Table 4.

TABLE 4 Effect of different instruments, chromatography columns on relative correction factors

The result shows that different high performance liquid chromatographs and different chromatographic columns have small influence on the correction factor, and the method has good reproducibility.

5.3 locating chromatographic Peak to be measured

The stability of relative retention time values of schizandrol B, schizandrin A and schizandrin B under the conditions of 2 high performance liquid chromatographs of different brands and 4 chromatographic columns of different brands is investigated by taking schizandrol A as an internal reference, and the RSD of the schizandrol A, the schizandrin A and the schizandrin B is less than 2.0 percent, and the result shows that different instruments and different chromatographic columns have no significant influence on the relative retention time values of the schizandrol B, the schizandrin A and the schizandrin B.

6. Content determination of samples of different batches

6.11-10 sample content determination:

taking a No. 1-10 schisandra chinensis sample, preparing a test solution according to the method under the item '2.2', carrying out sample injection measurement according to the chromatographic condition under the item '1', recording peak areas, respectively adopting a one-measurement-multiple-evaluation method (QASM method) and an external standard method (ESM method) to measure the content, and comparing the obtained results, wherein the results are shown in a table 5.

TABLE 51-10 batches of Chinese magnoliavine fruit medicinal material content determination result comparison table

In the above table 5, the RSD of the content measured by the one-test-multiple-evaluation method (QASM method) and the content measured by the external standard method (ESM method) are both less than 2.0%, and the results show that there is no significant difference between the calculated value of the one-test-multiple-evaluation method and the measured value of the external standard method, thereby effectively proving the accuracy of the one-test-multiple-evaluation method in the content measurement of schizandrin b, deoxyschizandrin and schisandrin b.

Example 2

The embodiment discloses correlation analysis between lignanoid content and grade of a schisandra chinensis medicinal material and application of the correlation analysis in grade evaluation of the medicinal material, and the correlation analysis specifically comprises the following steps:

1. the content of other lignanoid components in samples No. 11-25 was calculated by the one-test-multiple evaluation method in example 1

Taking No. 11-25 schizandra chinensis samples, preparing a test solution according to the method under the item 2.2 in the example 1, carrying out sample injection measurement under the chromatographic condition under the item 1 in the example 1, recording peak areas, and measuring the content by adopting a one-measurement-multiple-evaluation method, wherein the results are shown in Table 6.

Table 611-25 batches of fructus Schisandrae chinensis medicinal material content determination results

2. Application of total content of lignanoid components in grade evaluation of schisandra chinensis medicinal material

The results of the total content of lignans in tables 5 and 6 above are compared with the grade of the medicinal material, and the comparison results are shown in table 7 below:

TABLE 7 correlation analysis of the content measurement results and grades of fructus Schisandrae chinensis

As can be seen from table 7, there is a correlation between the total content of lignans and the grade of the herbs, the sum of the four lignans in all the first-class herbs is not less than 0.90%, and the sum of the four lignans in all the second-class herbs is not less than 0.70%. Therefore, the total content of the lignanoid components can be applied to the grade evaluation of the schisandra chinensis medicinal material to make up for the defects of the original grade evaluation index (only sensory index and no internal detection index).

Meanwhile, SPSS 22.0 software is adopted to carry out correlation analysis between the schisandra chinensis medicinal material grade and the lignans content (the sum of the contents of the schizandrol A, the schizandrol B, the schizandrin A, the schizandrol B and the four lignans) in the table 7, and the result shows that the correlation exists between the schisandra chinensis medicinal material grade and the sum of the four lignans contents, the content of the schizandrol A, the content of the schizandrol B and the content of the schisandrin B (P is less than 0.05, and P is less than 0.01).

The detection results show that the detection method has strong practicability, simple operation and cost saving, makes up the defect that the content of single component schizandrol A is only determined under the content determination item of the schisandra chinensis in Chinese pharmacopoeia, can effectively detect the content of four components of the schizandrol A, the schizandrol B, the deoxyschizandrin and the schisandrin B in the schisandra chinensis, and comprehensively improves the quality detection level of the schisandra chinensis medicinal material and decoction pieces.

Meanwhile, the invention can also apply the total content of the lignans to the grade evaluation of the schisandra chinensis medicinal material, thereby providing more complete evaluation basis for raw material purchase, preparation production and clinical application of the schisandra chinensis medicinal material.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. This need not be, nor should it be exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.

Claims

1. A method for determining the content of lignans in schisandra by a multi-evaluation method is characterized in that a characteristic spectrum with characteristic peaks of the lignans is obtained by determining a schisandra sample solution by adopting a high performance liquid chromatography, schizandrol A is used as an internal reference, a relative correction factor between the schizandrol A and other lignans is established by adopting the multi-evaluation method, and the content of other lignans is calculated by the correction factor;

a chromatographic column: octadecylsilane chemically bonded silica is used as a filling agent; detection wavelength: 220-254 nm; acetonitrile is used as a mobile phase A, water is used as a mobile phase B, and elution is carried out according to the following gradient elution procedure:

2. the method for determining the content of lignans in schisandra chinensis according to claim 1, wherein the column temperature of the chromatographic column is 25-35 ℃ and the flow rate is 0.8-1.2 ml/min.

3. The method for determining the content of lignans in schisandra chinensis according to claim 2, wherein the column temperature is 35 ℃ and the flow rate is 1 ml/min.

4. The method for determining the content of lignan components in schisandra chinensis according to any one of claims 1-3, wherein the chromatographic column has a specification of 4.6mm x 250mm, 5 μm, and a detection wavelength of 250 nm;

the sample amount of the test solution is 5-20 μ l, preferably 10 μ l.

5. The method for determining the content of lignans in schisandra chinensis according to any one of claims 1 to 4, wherein the lignans comprise schizandrol A, schizandrol B, schizandrin A and schizandrin B.

6. The method for determining the content of lignans in schisandra chinensis according to any one of claims 1 to 5, wherein the schisandra chinensis test solution is prepared by the following steps: taking a schisandra chinensis test sample, adding a solvent, uniformly mixing, sealing, weighing, carrying out ultrasonic treatment, cooling, weighing again, complementing the weight loss by the solvent, uniformly shaking, and filtering to obtain a filtrate, wherein the filtrate is the schisandra chinensis test sample solution.

7. The method for determining the content of the lignan components in the schisandra chinensis by the one-test-multiple evaluation method according to claim 6, wherein the ultrasonic treatment has the power of 250W and the frequency of 50 kHz; the time for the ultrasonic treatment was 30 min.

8. The method for determining the content of lignan components in schisandra chinensis according to claim 6 or 7, wherein the solvent is n-hexane.

9. Use of the method for determining the content of lignans in schisandra chinensis according to any one of claims 1 to 8 in the quality control and evaluation of schisandra chinensis.

10. The use according to claim 9, wherein the quality grade of schisandra chinensis is judged by the detected total content of lignans; the judgment rule is as follows: the total content of lignanoid components of the first-class medicinal materials is more than or equal to 0.90 percent, and the total content of lignanoid components of the second-class medicinal materials is more than or equal to 0.70 percent.