Construction method and application of lithospermum erythrorhizon specific chromatogram

Abstract

The invention belongs to the field of quality detection of traditional Chinese medicinal materials, and particularly relates to a construction method and application of a specific chromatogram of lithospermum erythrorhizon. By optimizing and investigating detection wavelength, mobile phase selection, test sample preparation and specific chromatographic conditions, the method for detecting the specific chromatogram of the lithospermum medicinal material is established, and the method has the advantages of high precision, good stability, good repeatability, high accuracy and the like, and provides an effective basis for quality identification of the lithospermum medicinal material.

Description

Technical Field

[0001] The present application belongs to the field of quality inspection of traditional Chinese medicine, and specifically relates to a method for constructing a characteristic spectrum of Lithospermum officinale and its application. Background Art

[0002] Lithospermum officinale root is mainly used to cool blood, promote blood circulation, detoxify and cure rashes. It is used for blood heat and toxicity, purple and black macules, measles that do not break out, sores, eczema, and burns caused by water or fire. It is one of the commonly used Chinese medicinal materials. The quality control of Chinese medicinal materials has always been a very important link in the field of Chinese medicine. At present, the pharmacopoeia only has thin layer and high performance liquid chromatography for the detection of lithospermum officinale, and does not involve the use of characteristic spectra.

[0003] With the development of chromatography technology, the quality standards of medicinal materials include not only simple properties, identification, routine inspection and extract determination, but also the determination of HPLC fingerprints. The fingerprint of traditional Chinese medicine is a comprehensive and quantifiable identification method. It is one of the quality control modes that is in line with the characteristics of traditional Chinese medicine and evaluates the authenticity, stability and consistency of traditional Chinese medicine. Therefore, it is necessary to study the characteristic spectrum of lithospermum officinale to better monitor and detect its quality. Summary of the invention

[0004] In order to solve the above problems, a method for constructing a characteristic spectrum of Lithospermum erythrorhizon and its application were provided. By optimizing and investigating the detection wavelength, mobile phase selection, sample preparation and specific chromatographic conditions, a characteristic spectrum detection method for Lithospermum erythrorhizon was established. The method has the advantages of high precision, good stability, good repeatability and high accuracy, and provides an effective basis for the quality identification of Lithospermum erythrorhizon Chinese medicinal materials.

[0005] According to one aspect of the present application, a method for constructing a characteristic map of Lithospermum officinale is provided, comprising the following steps:

[0006] S1 Preparation of reference solution: Take L-shikonin reference substance and β,β'-dimethylacryloyl akanin reference substance, and add methanol to prepare reference solution;

[0007] S2 Preparation of test solution: Take lithospermum officinale powder, add extraction solvent to make a solution, filter after extraction, and take the filtrate as the test solution;

[0008] S3 determination: The reference solution and the test solution were respectively aspirated and injected into a liquid chromatograph for determination to construct and obtain a characteristic spectrum of Lithospermum officinale.

[0009] The measurement conditions of the liquid chromatography include:

[0010] Octadecylsilane bonded silica gel is used as filler, methanol or acetonitrile is used as mobile phase A, 0.1% phosphoric acid is used as mobile phase B, detection wavelength is 210-230nm, column temperature is 20-40℃, flow rate is 0.7-1.3ml / min;

[0011] The gradient elution program of the mobile phase was:

[0012] 0-10min, mobile phase A is 65%, mobile phase B is 35%;

[0013] 10-30 min, mobile phase A: 65% → 80%, mobile phase B: 35% → 20%;

[0014] 30-35 min, mobile phase A: 80% → 95%, mobile phase B: 20% → 5%;

[0015] 35-40 min, mobile phase A: 95%, mobile phase B: 5%;

[0016] 40-41 min, mobile phase A: 95% → 65%, mobile phase B: 5% → 35%;

[0017] 41-50min, mobile phase A is 65%, mobile phase B is 35%.

[0018] Optionally, the column temperature is 30° C., the flow rate is 1.0 ml / min, and the detection wavelength is 210 nm.

[0019] Optionally, in the step of preparing the reference solution in S1, each 1 ml of methanol contains 0.1 mg of each of the L-shikonin reference substance and the β,β'-dimethylacryloyl akanin reference substance; and / or,

[0020] In the step S2 of preparing the test solution, 0.3 g of lithospermum officinale powder is contained in every 25 ml of methanol.

[0021] Optionally, the mobile phase A is acetonitrile.

[0022] Optionally, the extraction solvent is methanol or 65-75 vol% ethanol, the extraction is ultrasonic extraction, and the time of the ultrasonic extraction is 20-40 min.

[0023] Preferably, the extraction solvent is methanol, and the ultrasonic extraction time is 30 minutes.

[0024] According to another aspect of the present application, a characteristic map of Lithospermum officinale is provided, which is established and obtained by any of the above-mentioned methods for constructing a characteristic map of Lithospermum officinale.

[0025] According to another aspect of the present application, a method for detecting Lithospermum erythrorhizon is provided, comprising the step of comparing a characteristic spectrum of the Lithospermum erythrorhizon to be tested with a characteristic spectrum of a Lithospermum erythrorhizon control; the characteristic spectrum of the Lithospermum erythrorhizon to be tested and the characteristic spectrum of the Lithospermum erythrorhizon control are constructed according to any of the above-mentioned construction methods.

[0026] According to the last aspect of the present application, a method for identifying Lithospermum erythrorhizon is provided, comprising the step of identifying Lithospermum erythrorhizon from Xinjiang and Lithospermum erythrorhizon from Inner Mongolia according to the size of the characteristic peak area in the characteristic spectrum of Lithospermum erythrorhizon, wherein the characteristic spectrum of Lithospermum erythrorhizon is constructed according to any of the above-mentioned construction methods.

[0027] Optionally, the characteristic peak area of ​​Xinjiang Lithospermum officinale is greater than the characteristic peak area of ​​Inner Mongolia Lithospermum officinale.

[0028] The beneficial effects of this application include but are not limited to:

[0029] 1. The method for constructing the characteristic spectrum of Lithospermum officinale provided in the present application, through the examination of the spectrum results at different flow rates and different column temperatures, it can be seen that this method has good durability for flow rate and column temperature conditions, and the separation degree of each characteristic peak under different flow rates and column temperatures is high.

[0030] 2. The method for constructing the characteristic spectrum of Lithospermum officinale provided in the present application not only has a good separation effect of characteristic peaks in the chromatographic results, but also takes a short time, and is suitable as a quality detection method for Lithospermum officinale materials.

[0031] 3. The method for constructing the characteristic spectrum of Lithospermum officinale provided in the present application can simultaneously detect the two components and contents of L-shikonin and β,β'-dimethylacryloyl akanin in Lithospermum officinale, and the detection method is quick and convenient. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0033] Figure 1 The chromatogram in the selection of the detection wavelength involved in this application Figure 1 : A, 210nm; B, 220nm; C, 230nm; D, 270nm;

[0034] Figure 2 The chromatogram in the selection of the detection wavelength involved in this application Figure 2 :E, 280nm; F, 510nm; G, 520nm; H, 550nm;

[0035] Figure 3 The chromatograms used in the investigation of the composition of the organic phase involved in this application are: A, methanol-water chromatogram; B, acetonitrile-water chromatogram;

[0036] Figure 4 The chromatograms used in the investigation of the aqueous phase composition involved in this application are: A, acetonitrile-0.1% phosphoric acid chromatogram; B, acetonitrile-0.1% formic acid chromatogram; C, acetonitrile-0.1% acetic acid chromatogram;

[0037] Figure 5 The chromatograms in the comparative experiment of Xinjiang Lithospermum and Inner Mongolia Lithospermum involved in this application are as follows: A, chromatogram of Xinjiang Lithospermum; B, chromatogram of Inner Mongolia Lithospermum;

[0038] Figure 6 The chromatograms in the selection of chromatographic conditions involved in this application: A, chromatogram of chromatographic condition 1; B, chromatogram of chromatographic condition 2; C, chromatogram of chromatographic condition 3;

[0039] Figure 7 Chromatograms of the durability pre-experiment involved in this application: A, normal conditions; B, 0.8 ml / min; C, 1.2 ml / min; D, 25°C; E, 35°C. DETAILED DESCRIPTION

[0040] The present application is described in detail below in conjunction with examples, but the present application is not limited to these examples. Unless otherwise specified, the raw materials and reagents in the examples of the present application are purchased through commercial channels.

[0041] The instruments and equipment used in this application include: 1) chromatographic column, model 5TC, manufactured by Agilent; 2) high performance liquid chromatograph, model 2695-2996, manufactured by Waters; 3) one-tenth balance, model BSA124S, manufactured by Sartorius; 4) ultrasonic instrument, model KQ-500DB, manufactured by Kunshan Ultrasonic Instrument.

[0042] The present application scheme is described in detail below through specific embodiments.

[0043] Example 1

[0044] Preparation of reference solution: Take appropriate amount of L-shikonin and β,β'-dimethylacryloyl akanin reference substances, accurately weigh them, add methanol to make a solution containing 0.1 mg per 1 ml.

[0045] Preparation of test solution: Take 0.3 g of lithospermum powder to be tested, accurately weigh it, put it in a stoppered conical flask, accurately measure 25 ml of methanol, ultrasonicate for 30 minutes, filter, and take the filtrate as the test solution.

[0046] Determination method: Accurately pipette 10 μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine. The chromatographic conditions are as follows: octadecylsilane bonded silica gel as filler (Aglient5TC-C18(2), 4.6mm×250mm, 5μm); acetonitrile as mobile phase A, 0.1% phosphoric acid as mobile phase B, gradient elution as specified in the table below; detection wavelength of 210nm; column temperature of 30℃; flow rate of 1.0ml per minute.

[0047] Table 1 Elution conditions

[0048] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～10 65 35 10～30 65→80 35→20 30～35 80→95 20→5 35～40 95 5 40～41 95→65 5→35 41～50 65 35

[0049] The chromatographic results are as follows Figure 6 As shown in C, the separation degree and peak purity of each peak in Example 1 meet the requirements. The small peak at 8.8 minutes is L-shikonin, and the large peak at 29.5 minutes is β, β'-dimethylacryloyl akanin. The separation degree is high and the peaks are clear, which is suitable as a quality detection method for lithospermum officinale.

[0050] Example 2 Investigation of chromatographic conditions

[0051] 1) Investigation of wavelength

[0052] Using acetonitrile-water as the mobile phase, the chromatograms were examined at wavelengths of 210nm, 220nm, 230nm, 270nm, 280nm, 510nm, 520nm, and 550nm. The results are as follows Figure 1 and Figure 2 As shown, at the detection wavelength of 210nm-230nm, the chromatographic peak combing and overall coordination are good, among which the best effect is achieved when the detection wavelength is 210nm.

[0053] 2) Mobile phase investigation

[0054] 2.1) Investigation of organic phase composition

[0055] Take about 0.3 g of lithospermum officinale powder, weigh it accurately, put it in a stoppered conical flask, accurately measure 25 ml of 70% ethanol, ultrasonicate for 30 minutes, filter, and take the filtrate as the test solution.

[0056] The mobile phases were methanol-water and acetonitrile-water, respectively, and the samples were injected under the chromatographic conditions of 5%→95% of the organic phase. Figure 3 As shown, the main chromatographic peaks in Lithospermum officinale are concentrated in the part with a higher proportion of organic phase. When methanol is used as the organic phase, the chromatographic peaks are concentrated at the end of the chromatogram with a proportion of about 90%. Therefore, if methanol is selected as the organic phase, the optimization space is small. At the same time, when acetonitrile is used as the organic phase, the separation of each peak is slightly better than methanol, so acetonitrile is selected as the organic phase.

[0057] 2.2) Investigation of water phase composition

[0058] The organic phase was acetonitrile, and the aqueous phase was 0.1% phosphoric acid, 0.1% formic acid, and 0.1% acetic acid, respectively, and the samples were injected under the same chromatographic conditions. Figure 4 As shown, after adding acid to the aqueous phase, the peak shape of each peak is greatly improved, and the separation degree and peak purity are less affected. However, the baseline is poor when 0.1% formic acid and 0.1% acetic acid are used as the aqueous phase, so 0.1% phosphoric acid is selected as the aqueous phase.

[0059] 3) Investigation of elution gradient conditions

[0060] 3.1) Elution gradient 1

[0061] Acetonitrile was used as mobile phase A, 0.1% phosphoric acid was used as mobile phase B, and the sample was injected and detected using an Agilent 5TC-C18(2) column. Gradient elution was performed according to the requirements in the following table. The detection wavelength was 210 nm. The column temperature was 30° C. and the flow rate was 1.0 ml per minute.

[0062] Table 2 Elution conditions

[0063] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～60 60→95 40→5

[0064] like Figure 6 As shown in A, the larger chromatographic peak at 30 minutes in the figure is β, β'-dimethylacryloyl akanin, an indicator component for the determination of the content of Lithospermum officinale. There is still room for improvement in the separation degree, and the peaks can be separated by trying to optimize the chromatographic gradient.

[0065] 3.2) Elution gradient 2

[0066] Acetonitrile was used as mobile phase A, 0.1% phosphoric acid was used as mobile phase B, and the sample was injected and detected using an Agilent 5TC-C18(2) column. Gradient elution was performed according to the requirements in the following table. The detection wavelength was 210 nm. The column temperature was 30° C. and the flow rate was 1.0 ml per minute.

[0067] Table 3 Elution conditions

[0068] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～10 65 35 10～40 65→80 35→20 40～41 80→95 20→5 41～50 95 5 50～51 95→65 5→35 51～60 65 35

[0069] like Figure 6 As shown in B, the separation of the peaks in the figure basically meets the requirements, and we try to shorten the running time.

[0070] 3.3) Elution gradient 3

[0071] Acetonitrile was used as mobile phase A, 0.1% phosphoric acid was used as mobile phase B, and the sample was injected and detected using an Agilent 5TC-C18(2) column. Gradient elution was performed according to the requirements in the following table. The detection wavelength was 210 nm. The column temperature was 30° C. and the flow rate was 1.0 ml per minute.

[0072] Table 4 Elution conditions

[0073] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～10 65 35 10～30 65→80 35→20 30～35 80→95 20→5 35～40 95 5 40～41 95→65 5→35 41～50 65 35

[0074] like Figure 6 As shown in C, the separation and peak purity of each peak in this embodiment basically meet the requirements. The small peak at 8.8 minutes is L-shikonin, and the large peak at 29.5 minutes is β,β'-dimethylacryloyl akanin. The peaks are clear and have good separation.

[0075] 4) Durability test

[0076] Under the conditions of Example 1, the flow rates were adjusted to 0.8 ml and 1.2 ml per minute, respectively, and the column temperatures were adjusted to 25°C and 35°C, respectively, and elution was performed at 210 nm. Figure 7 As shown in the figure, the flow rate and column temperature of this method are good.

[0077] Embodiment 3 two kinds of Lithospermum erythrorhizon are distinguished

[0078] According to the detection wavelength, mobile phase and extraction solvent determined in Example 1, Xinjiang Lithospermum and Inner Mongolia Lithospermum were taken to prepare test samples for injection, and the differences in the chromatograms of the two were compared. Figure 5 As shown, there is basically no difference in the number of chromatographic peaks between Xinjiang Lithospermum and Inner Mongolia Lithospermum, but the peak area of ​​each peak of Xinjiang Lithospermum is much larger than that of Inner Mongolia Lithospermum, so Xinjiang Lithospermum and Inner Mongolia Lithospermum can be distinguished according to the relative peak area.

[0079] Test Case 1 Methodology Verification

[0080] 1) Precision test

[0081] Take lithospermum officinale powder, and obtain a test solution according to the preparation method of the test solution in Example 1. The sample is continuously injected 5 times for determination under the chromatographic conditions of Example 1 of the present invention. The results are shown in the table below. According to the results of the relative retention time, the relative standard deviation (RSD) values ​​of the two peaks are within 5%, indicating that the characteristic spectrum detection method provided by the present invention has good precision.

[0082] Table 5 Relative retention time

[0083]

[0084] 2) Repeatability test

[0085] Take lithospermum officinale powder, and obtain 6 test solutions according to the preparation method of the test solution in Example 1. The test solutions are measured under the chromatographic conditions of Example 1. The results are shown in the table below. According to the results of measuring the relative retention time, the relative standard deviation (RSD) values ​​of the two peaks of the 6 samples are within 5%, indicating that the characteristic spectrum detection method provided by the present invention has good repeatability.

[0086] Table 6 Relative retention time

[0087]

[0088] 3) Stability test

[0089] Take lithospermum officinale powder, and obtain 1 test solution according to the preparation method of the test solution in Example 1. Under the chromatographic conditions of Example 1, the test was carried out at 0 hour, 1 hour, 2 hours, 4 hours, 6 hours, and 8 hours. The results are shown in the table below. According to the results of determining the relative retention time, the relative standard deviation (RSD) values ​​of the two peaks are within 5%, and the characteristic spectrum detection method provided by the present invention has good stability.

[0090] Table 7 Relative retention time

[0091]

[0092] In summary, the method for constructing the characteristic spectrum of Lithospermum officinale provided in the present application can simultaneously detect the two components of L-shikonin and β, β'-dimethylacryloyl akanin, and not only the characteristic peaks of L-shikonin and β, β'-dimethylacryloyl akanin in the spectrum have high separation, but also the separation time is short, the spectrum construction operation is convenient, and the method has good durability for flow rate and column temperature. Therefore, the present application scheme can be used as a method for efficient and rapid quality detection of Lithospermum officinale materials, and can improve the quality of quality monitoring of Lithospermum officinale materials in production.

[0093] The above is only the embodiment of the present application, and the protection scope of the present application is not limited by these specific embodiments, but is determined by the claims of the present application. For those skilled in the art, the present application can have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the technical ideas and principles of the present application should be included in the protection scope of the present application.

Claims

1. A method for constructing a characteristic spectrum of Lithospermum officinale, characterized in that: The following steps are involved: S1 Preparation of reference solution: Take L-shikonin reference substance and β,β'-dimethylacryloyl akanin reference substance, add methanol to prepare reference solution; S2: Prepare the test solution: Take the powder of Lithospermum officinale, add the extraction solvent to make a solution, filter after extraction, and take the filtrate as the test solution; S3 determination: The reference solution and the test solution were respectively aspirated and injected into a liquid chromatograph for determination to construct and obtain a characteristic spectrum of Lithospermum officinale. The measurement conditions of the liquid chromatography include: Octadecylsilane bonded silica gel is used as filler, methanol or acetonitrile is used as mobile phase A, 0.1% phosphoric acid is used as mobile phase B, detection wavelength is 210-230nm, column temperature is 20-40℃, flow rate is 0.7-1.3ml / min; The gradient elution program of the mobile phase was: 0-10min, mobile phase A is 65%, mobile phase B is 35%; 10-30 min, mobile phase A: 65% → 80%, mobile phase B: 35% → 20%; 30-35 min, mobile phase A: 80% → 95%, mobile phase B: 20% → 5%; 35-40 min, mobile phase A: 95%, mobile phase B: 5%; 40-41 min, mobile phase A: 95% → 65%, mobile phase B: 5% → 35%; 41-50min, mobile phase A is 65%, mobile phase B is 35%.

2. The method for constructing the characteristic spectrum of Lithospermum officinale according to claim 1, characterized in that: The column temperature was 30° C., the flow rate was 1.0 ml / min, and the detection wavelength was 210 nm.

3. The method for constructing the characteristic spectrum of Lithospermum officinale according to claim 1, characterized in that, In the step of preparing the reference solution in S1, each 1 ml of methanol contains 0.1 mg of each of the L-shikonin reference substance and the β,β'-dimethylacryloyl akanin reference substance; and / or, In the step S2 of preparing the test solution, 0.3 g of lithospermum officinale powder is contained in every 25 ml of methanol.

4. The method for constructing the characteristic spectrum of Lithospermum officinale according to claim 1, characterized in that: The mobile phase A is acetonitrile.

5. The method for constructing the characteristic spectrum of Lithospermum officinale according to claim 1, characterized in that: The extraction solvent is methanol or 65-75 vol% ethanol, the extraction is ultrasonic extraction, and the ultrasonic extraction time is 20-40 minutes.

6. The method for constructing the characteristic spectrum of Lithospermum officinale according to claim 5, characterized in that: The extraction solvent is methanol, and the ultrasonic extraction time is 30 minutes.

7. A characteristic spectrum of Lithospermum officinale, characterized in that: The method is established and obtained by constructing the characteristic spectrum of Lithospermum officinale according to any one of claims 1 to 6.

8. A method for detecting Lithospermum officinale, characterized in that: The method comprises the steps of comparing the characteristic spectrum of the lithospermum to be tested with the characteristic spectrum of the lithospermum control; The characteristic spectrum of the lithospermum to be tested and the characteristic spectrum of the lithospermum control are constructed according to the construction method described in any one of claims 1 to 6.

9. A method for identifying Lithospermum officinale, characterized in that: The method comprises the step of distinguishing Xinjiang lithospermum and Inner Mongolia lithospermum according to the size of the characteristic peak area in the lithospermum characteristic spectrum, wherein the lithospermum characteristic spectrum is constructed and obtained according to the construction method described in any one of claims 1 to 6.

10. The identification method of Lithospermum officinale according to claim 9, characterized in that: The characteristic peak area of ​​Xinjiang Lithospermum officinale is greater than the characteristic peak area of ​​Inner Mongolia Lithospermum officinale.

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