Ligustrum lucidum ait fingerprint spectrum construction method and application thereof

Abstract

The embodiment of the invention provides a glossy privet fruit fingerprint spectrum construction method and application thereof. The method comprises the following steps: detecting a test solution of glossy privet fruit by adopting ultra-high performance liquid chromatography; in the step of high performance liquid chromatography detection, the chromatographic conditions further include that the column temperature is 30 DEG C, the flow velocity is 0.3 mL / min, and the detection wavelength is 210 nm. According to the scheme, the glossy privet fruit fingerprint spectrum is constructed, specnuezhenide and salidroside in the glossy privet fruit medicinal material can be quantitatively analyzed by adopting UPLC chromatographic conditions, the index ligustroside G13 closely related to the maturity degree of the glossy privet fruit is screened out, then the quality of the glossy privet fruit is detected, and the maturity degree of the glossy privet fruit medicinal material is further determined. Compared with a manual judgment method in the prior art, the method provided by the invention has the advantages that the determination result is more reliable, the operation is simple and convenient, and a scientific basis is provided for the revision of Chinese Pharmacopoeia 2020 edition.

Description

Technical Field

[0001] The embodiments of the present application relate to the technical field of quality detection of traditional Chinese medicines, and in particular to a method for constructing a fingerprint spectrum of Ligustrum lucidum and its application, and further disclose the method for constructing the fingerprint spectrum and a method for quality detection of Ligustrum lucidum. Background Art

[0002] Ligustrum lucidum Ait., a plant of the Oleaceae family, is widely distributed in my country and is mainly produced in Zhejiang, Jiangsu, Henan, Guangxi, Sichuan, Shaanxi and other places. It likes a warm and humid climate, is light-loving and shade-tolerant, and is not very cold-resistant. It often grows in the mountains or is introduced into gardens as a greening plant. Ligustrum lucidum fruit is the dried mature fruit of Ligustrum lucidum. It was first recorded in the "Shennong Bencao Jing" and was listed as a top-grade product. It has a history of more than 2,000 years of application. It is mild in nature, sweet and bitter in taste [2], and belongs to the lung, liver, and kidney meridians. It has the effect of nourishing the liver and kidneys, improving eyesight and darkening hair. It is used to treat symptoms such as dizziness, tinnitus, blurred vision, back and knee pain, premature graying of hair, etc., and is often used to strengthen the body.

[0003] The Chinese Pharmacopoeia (2020 edition) stipulates that the fruit of Ligustrum lucidum is harvested in winter when it is ripe, and its appearance is oval, elliptical or kidney-shaped. The survey found that the Ligustrum lucidum slices circulating in the medicinal material market are green, brown and black, and their shapes are kidney-shaped (chicken waist-shaped), oval or oval. The processed Ligustrum lucidum wines are all black, but the shapes and sizes vary greatly. These phenomena indicate that Ligustrum lucidum is harvested and flows into the market when it is immature. The confusion in the actual harvest period leads to large differences in the quality of the medicinal material and its processed products. The reason is that Ligustrum lucidum has a wide distribution range, and there are differences in phenological periods in different places. The statutory standards stipulate a wide range of picking times, and the degree of maturity of the fruits varies.

[0004] The 2000 edition of the Chinese Pharmacopoeia added an item for determining the content of Ligustrum lucidum fruit, with oleanolic acid as the indicator component. After the 2010 edition, the more specific privet glycoside was used as the indicator component of Ligustrum lucidum fruit and its processed liquor. The 2020 edition added salidroside as the indicator for determining the content of Ligustrum lucidum fruit, thereby reflecting the changes in the composition of Ligustrum lucidum fruit after processing. The current medicinal material standards only use individual components as indicators and set limit requirements. The relationship between the indicator components and the maturity of Ligustrum lucidum fruit is currently not accurately stated, which makes it difficult to ensure the quality of Ligustrum lucidum fruit and its clinical medicinal effects in practical applications. Summary of the Invention

[0005] In view of this, the embodiments of the present application provide a method for constructing a fingerprint map of Ligustrum lucidum and its application to at least partially solve the above problems.

[0006] According to the first aspect of the embodiment of the present application, a method for constructing a fingerprint spectrum of Ligustrum lucidum is provided, the method comprising: 1. A method for constructing a fingerprint spectrum of Ligustrum lucidum, characterized in that it comprises: a step of detecting a test solution of Ligustrum lucidum by ultra-high performance liquid chromatography; in the high performance liquid chromatography detection step, the chromatographic conditions further comprise: a column temperature of 30°C, a flow rate of 0.3mL / min, and a detection wavelength of 210nm.

[0007] Preferably, the ultra-high performance liquid chromatography comprises: octadecylsilane bonded silica gel as filler, 0.1% phosphoric acid solution as mobile phase A, acetonitrile as mobile phase B,

[0008] Perform gradient elution as follows:

[0009] 0-3.5min, A:B is 98%:2%;

[0010] 3.5-10 min, A:B 98%:2% → 91%:9%;

[0011] 10-30 min, A:B 91%:9% → 87%:13%;

[0012] 30 to 70 minutes later, the ratio of A to B was 87%:13% → 10%:90%.

[0013] Preferably, the preparation method of the test solution comprises the following steps: taking a Ligustrum lucidum control medicinal material, using methanol to treat the powder of the Ligustrum lucidum control medicinal material through a No. 3 sieve, adding an organic solvent and ultrasonically treating it, filtering and collecting the filtrate.

[0014] Preferably, the amount of the solvent is 10 mL of organic solvent per 1.0 g of the Ligustrum lucidum medicinal material powder.

[0015] Preferably, the organic solvent comprises methanol;

[0016] The power of the ultrasonic treatment step was 500 W and the frequency was 40 kHz.

[0017] Preferably, the extraction time of the ultrasonic treatment is 30 minutes.

[0018] This solution also discloses a Ligustrum lucidum fingerprint map and / or a reference fingerprint map, and the Ligustrum lucidum fingerprint map or the reference fingerprint map is constructed by executing the method described in the first aspect.

[0019] This solution also discloses the method for constructing the Ligustrum lucidum fingerprint map and / or the application of the Ligustrum lucidum fingerprint map and / or the control fingerprint map in the field of Ligustrum lucidum quality detection.

[0020] Preferably, the maturity of Ligustrum lucidum fruit is determined according to the content of Ligustrum lucidum G13.

[0021] This scheme constructs a fingerprint of Ligustrum lucidum fruit and uses UPLC chromatographic conditions to quantitatively analyze the two components in Ligustrum lucidum fruit, screen out the indicator Ligustrum lucidum glycoside G13, which is closely related to the maturity of Ligustrum lucidum fruit, and then test the quality of Ligustrum lucidum fruit to further determine the maturity of Ligustrum lucidum fruit. Compared with the existing method relying on manual judgment, the measurement results of this scheme are more reliable and easy to operate, providing a scientific basis for the revision of the 2020 edition of the "Chinese Pharmacopoeia". BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments recorded in the embodiments of the present application. For ordinary technicians in this field, other drawings can also be obtained based on these drawings.

[0023] Figure 1 This is an overlapping chromatogram of the fingerprints of 32 batches of Ligustrum lucidum medicinal materials in Example 1 of the present invention;

[0024] Figure 2 This is the characteristic spectrum of 32 batches of Ligustrum lucidum medicinal materials in Example 1 of the present invention;

[0025] Figure 3 The chromatogram of the reference solution in Example 9 of Example 1 of the present invention is shown in FIG.

[0026] Figure 4 This is a PCA score graph of 27 characteristic peaks of Ligustrum lucidum medicinal material in Example 1 of the present invention;

[0027] Figure 5 is the VIP value of OPLS-DA of 27 characteristic peaks of Ligustrum lucidum medicinal material in Example 1 of the present invention;

[0028] Figure 6 Graph showing the deviations between the 27 characteristic peak variables of the Ligustrum lucidum medicinal material and the model average value in Example 1 of the present invention;

[0029] Figure 7 This is a specificity investigation graph of the characteristic graph of Ligustrum lucidum medicinal material in Example 1 of the present invention;

[0030] Figure 8 This is a heat map of the distribution of the content (%) of ligustrin in Ligustrum lucidum fruit in six regions of Shaanxi Province in different months in Example 2 of the present invention;

[0031] Figure 9 This is a bar graph showing the distribution of the content (%) of ligustrin in Ligustrum lucidum fruits in six regions of Shaanxi Province in different months in Example 2 of the present invention;

[0032] Figure 10 This is a heat map of the distribution of the content (%) of Ligustrum lucidum G13 in six regions of Shaanxi Province in different months in Example 2 of the present invention;

[0033] Figure 11 This is a bar graph showing the distribution of the content (%) of Ligustrum lucidum G13 in the fruits of Ligustrum lucidum in six regions of Shaanxi Province in different months in Example 2 of the present invention; DETAILED DESCRIPTION

[0034] In order to enable those skilled in the art to better understand the technical solutions in the embodiments of the present application, the technical solutions in the embodiments of the present application will be clearly and in detail described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the embodiments of the present application, all other embodiments obtained by ordinary technicians in this field should fall within the scope of protection of the embodiments of the present application.

[0035] It should be understood that the various steps described in the method embodiments of the present disclosure may be performed in different orders and / or in parallel. In addition, the method embodiments may include additional steps and / or omit the steps shown. The scope of the present disclosure is not limited in this respect.

[0036] The term "including" and its variations used in this document are open inclusions, that is, "including but not limited to". The term "based on" means "based at least in part on". The term "one embodiment" means "at least one embodiment"; the term "another embodiment" means "at least one other embodiment"; the term "some embodiments" means "at least some embodiments". The relevant definitions of other terms will be given in the description below. It should be noted that the concepts of "first", "second", etc. mentioned in this disclosure are only used to distinguish different devices, modules or units, and are not used to limit the order or interdependence of the functions performed by these devices, modules or units.

[0037] It should be noted that the modifiers "one" and "plurality" mentioned in this disclosure are illustrative and non-restrictive. Those skilled in the art will understand that unless the context clearly indicates otherwise, they should be understood as "one or more." The names of the messages or information exchanged between multiple devices in the embodiments of this disclosure are for illustrative purposes only and are not intended to limit the scope of these messages or information.

[0038] The specific implementation of the embodiment of the present application is further explained below with reference to the accompanying drawings.

[0039] The samples tested in the following examples and comparative examples are some commercially available Ligustrum lucidum fruits and Ligustrum lucidum fruits from different months. The preparation methods and experimental instruments are shown below.

[0040] The instruments and reagents used in the embodiments of the present invention are as follows:

[0041] Instruments and Equipment

[0042] Chromatograph 1: Agilent 1290 high performance liquid chromatograph;

[0043] Fully automatic ceramic kettle: Amoi Technology Co., Ltd., 2L;

[0044] Centrifuge: Hitachi;

[0045] Mixer: VORTEX 3000 vortex mixer from WIGGENS LLC;

[0046] Electronic balance: XP205, ME204 (Mettler-Toledo Instruments (Shanghai) Co., Ltd.);

[0047] Ultrasonic cleaner: Milli-Q ultrapure water system (Millipore, MA, USA) from Kunshan Ultrasonic Instrument Co., Ltd.

[0048] Chromatographic column: Thermo Hypersil Aq column, 100 mm × 2.1 mm, 1.9 μm;

[0049] Reagents and test drugs

[0050] Methanol, acetonitrile, and formic acid were of chromatographic grade, and water was ultrapure water; the remaining reagents were of analytical grade.

[0051] Reference substance: Salidroside (batch number: 111975-201501, purity 99.9%), purchased from China Food and Drug Administration;

[0052] Tyrosol (batch number: 111676-200602, purity 99.7%) was purchased from the China Food and Drug Administration.

[0053] Ligustrum lucidum (batch number: 111926-201906, purity 95.0%) was purchased from the China Food and Drug Administration;

[0054] Oleanolic acid (batch number: 110709-202109, purity 95.8%) was purchased from the China Food and Drug Administration.

[0055] Ursolic acid (batch number: 110742-201823, purity 99.9%) was purchased from the China Food and Drug Administration.

[0056] Echinacoside (batch number: DSTDS003801, purity 98%) was purchased from Chengdu Desit Biotechnology Co., Ltd.

[0057] Ligustrum lucidum G13 (batch number: DSTDN005201, purity 98%) was purchased from Chengdu Desite Biotechnology Co., Ltd.

[0058] Neoligustrin (batch number: DST220223-198, purity 98%) was purchased from Chengdu Desite Biotechnology Co., Ltd.

[0059] Ligustrum lucidum (batch number: DSTDO001201, purity 98%) was purchased from Chengdu Desit Biotechnology Co., Ltd.

[0060] Luteolin (batch number: PRF20030901, purity 98%) was purchased from Chengdu PRF Technology Development Co., Ltd.

[0061] Example 1

[0062] This example is a method for establishing a UPLC characteristic spectrum of Ligustrum lucidum medicinal material.

[0063] Ligustrum lucidum medicinal materials: A total of 32 batches of Ligustrum lucidum medicinal materials that comply with the requirements of the 2020 edition of the "Chinese Pharmacopoeia" were collected from production areas such as Anhui, Zhejiang, Jiangsu, Sichuan, Shandong, Guangxi, Henan and Shaanxi.

[0064] 1. Chromatographic conditions and system suitability test:

[0065] C18 column: Thermo Hypersil Aq (100 mm × 2.1 mm, 1.9 μm); mobile phase: 0.1% phosphoric acid solution (A) and acetonitrile (B); gradient elution program: 0–3.5 min, 2% B; 3.5–10 min, 2%–9% B; 10–30 min, 9%–13% B; 30–70 min, 13%–90% B. Flow rate: 0.3 mL / min; wavelength: 210 nm; injection volume: 2 μL; column temperature: 30°C.

[0066] 2. Preparation of reference solution:

[0067] Accurately weigh appropriate amounts of salidroside, tyrosol, echinacoside, ligustrin, luteolin, ligustrin G13, oleoyl ligustrin, oleanolic acid, and ursolic acid reference substances, accurately weigh them, and add methanol to prepare a mixed reference solution containing 100 μg of each of the above 9 compounds per 1 mL.

[0068] 3. Preparation of test solution:

[0069] Grind the Ligustrum lucidum fruit and pass it through a 50-mesh sieve (No. 3 sieve) to prepare the sample. Accurately weigh 1.0 g of the powdered sample and place it in a stoppered conical flask. Accurately add 10 mL of methanol and weigh the sample. Ultrasonic extraction (40 kHz, 500 W) for 30 minutes was performed, and the weight was re-weighed. The weight loss was compensated with methanol. Transfer the sample to a centrifuge tube and centrifuge at 5000 rpm for 5 minutes. Filter the supernatant through a 0.22 μm filter.

[0070] 4. Determination:

[0071] Accurately pipette 1 μL of the reference solution and the test solution respectively, inject them into the ultra-high performance liquid chromatography instrument, and measure the obtained chromatogram.

[0072] 5. Measurement results:

[0073] The UPLC superposition chromatograms of 32 batches of Ligustrum lucidum samples were established by using the "Chinese Herbal Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 edition). Figure 1 The spectrum of sample number S37 was used as the reference spectrum, the median method was used, the time window width was set to 0.1min, the spectrum was automatically peak matched and multi-point correction was performed to generate the fingerprint spectrum (see Figure 2 ), a total of 27 common peaks were identified, and compared with the mixed control spectrum ( Figure 3 ) comparison, pointed out 9 common peaks, confirming that peak 3 is salidroside, peak 4 is tyrosol, peak 9 is echinacoside, peak 12 is tereliglucosidoside, peak 15 is luteolin, peak 17 is ligustrin G13, peak 19 is oleoyl ligustrin, peak 26 is oleanolic acid, and peak 27 is ursolic acid. Taking peak 12 tereliglucosidoside as the reference peak S, reference data for the characteristic spectrum analysis method of Ligustrum lucidum medicinal material was formulated. From the perspective of the characteristic spectrum unique to Ligustrum lucidum medicinal material, characteristic peaks were selected, and the relative retention time and relative peak area of ​​each characteristic peak were calculated to form the overall picture of the characteristic spectrum of Ligustrum lucidum medicinal material. Specifically, taking peak 12 tereliglucosidoside as the reference peak, the relative retention time and relative peak area of ​​the remaining characteristic peaks were calculated. The experimental results are shown in Tables 1 and 2.

[0074] Using the reference fingerprint as a reference, an overall similarity evaluation was conducted on 32 batches of Ligustrum lucidum fruit samples. The results showed that the majority of Ligustrum lucidum fruit samples had similarities greater than 0.9 to the reference fingerprint. Samples S5 (Xiaoxian County, Suzhou, Anhui Province), S17 (Cangxi County-1, Guangyuan City, Sichuan Province), S18 (Cangxi County-2, Guangyuan City, Sichuan Province), S23 (Xuecheng District, Zaozhuang City, Shandong Province), S28 (Lingbao City, Sanmenxia City, Henan Province), and S29 (Dengfeng City, Zhengzhou City, Henan Province) had similarities less than 0.9, with similarities of 0.883, 0.627, 0.498, 0.725, 0.839, and 0.897, respectively. S5, S23, S28, and S29 showed some, but not significant, differences in quality from materials from other origins. S17 and S18 showed significant differences from materials from other origins, likely due to factors such as the growth environment and collection time. The similarity evaluation results for the 32 batches of Ligustrum lucidum fruit samples are shown in Table 3.

[0075] Table 1-3 Relative retention time of characteristic spectra of 2 batches of Ligustrum lucidum medicinal materials

[0076]

[0077] Table 2-3 Relative peak areas of characteristic spectra of 2 batches of Ligustrum lucidum medicinal materials

[0078]

[0079] Table 3-32 Results of Similarity Evaluation of Spectra of Ligustrum lucidum Herbs

[0080]

[0081] The 27 common peak data were subjected to principal component analysis (PCA) and partial orthogonal least squares discriminant analysis (OPLS-DA). Figure 4 The PCA score scatter plot shows that the p[1] score of the principal component is 0.501, the p[2] score is 0.295, and the total score of the two principal components is 0.796, which can represent most of the data information. The compounds with the largest differences are peak 12 (privet glycoside), peak 17 (privet glycoside G13), and peak 13.

[0082] The OPLS-DA model was used to analyze the differences in common peaks, and compounds with large differences in Ligustrum lucidum from different origins were screened according to VIP values. The results are shown in Tables 4 and Figure 5 , the VIP values ​​> 1 include peak 12 (privet glycoside), peak 17 (privet glycoside G13), peak 13, peak 4 (tyrosol), peak 26 (oleanolic acid), peak 3 (salidroside) and peak 1. Figure 6 The degree to which 27 variables deviate from the model mean is shown, and the results show that peak 12 (privet glycoside) and peak 17 (privet glycoside G13) deviate more than 3 times the standard deviation.

[0083] Table 4 VIP scores of 27 compounds

[0084]

[0085] In summary, 27 common peaks were identified in the 32 batches of Ligustrum lucidum (Ligustrum lucidum) with well-resolved and relatively stable characteristic spectra. Similarities ranged from 0.498 to 1.0. Ligustrum lucidum (Ligustrum lucidum) from different origins exhibited similar composition, but the content of each component varied significantly. This may be due to factors such as the growth environment and collection time, suggesting that the quality of the medicinal material is related to factors such as impurities, harvesting period, processing, and storage methods. Of the 27 common peaks, nine were identified: salidroside, tyrosol, echinacoside, tereligrucoside, luteolin, ligustrin G13, oleoylligustrin, oleanolic acid, and ursolic acid. Combined PCA and OPLS-DA analysis revealed that the most variable components in commercially available Ligustrum lucidum (Ligustrum lucidum) were tereligrucoside and ligustrin G13.

[0086] 6. Methodological Validation

[0087] (1) Specificity test

[0088] Take blank solvent and reference solution and test under the above liquid chromatography conditions, inject 1 μL each time and record the chromatogram. Figure 7 As shown, there is no chromatographic peak in the blank solvent chromatogram at the retention time position corresponding to the characteristic peak, indicating that the extraction solvent has no interference with the determination of each characteristic peak and has good specificity.

[0089] (2) Precision test

[0090] The same test solution was injected six times under the above HPLC conditions, with 1 μL injected each time. The peak of terglycin was designated as the S peak, and the RSD values ​​of the relative retention time and relative peak area of ​​each characteristic peak were calculated. The RSDs of the relative retention time and relative peak area of ​​each characteristic peak were less than 2%, and less than 3%, respectively. These results indicate that the instrument has good precision.

[0091] (3) Repeatability test

[0092] Six sample solutions were prepared from the same batch according to the sample preparation method. 2 μL of sample was injected each time under the above-described HPLC conditions. The peak of terglycin was designated as the S peak, and the relative standard deviations (RSDs) of the relative retention times and relative peak areas of each characteristic peak were calculated. The RSDs for the relative retention times and relative peak areas of each characteristic peak were all less than 1.0%, and less than 5.0%. The results demonstrate that this analytical method has excellent reproducibility.

[0093] (4) Stability test

[0094] The test solution was placed at room temperature and tested under the above-mentioned liquid chromatography conditions at 0 hours, 2 hours, 4 hours, 8 hours, 12 hours, and 24 hours. 1 μL was injected each time, and the peak of terephthaloside was taken as the S peak. The relative retention time and relative peak area RSD values ​​of each characteristic peak were calculated. The RSD of the relative retention time of each characteristic peak was less than 2.2%, and the RSD of the relative peak area was less than 5.0%. The results showed that the stability of the test solution was good within 24 hours.

[0095] Example 2

[0096] This example is the determination and analysis of the contents of privetside and privetside G13 in Ligustrum lucidum medicinal materials in different months.

[0097] Investigations have found that the pieces of Ligustrum lucidum circulated on the medicinal material market are green, brown and black, and have kidney-shaped (chicken kidney-shaped), oval or elliptical shapes. These phenomena indicate that Ligustrum lucidum is harvested and flows into the market when it is immature, the actual harvest period is chaotic, and the quality of the medicinal materials varies greatly. According to the fingerprint analysis results of the commercially available Ligustrum lucidum in Example 1, it was found that the components with the greatest difference were privetside and privetside G13. The present invention uses these two components as content determination indicators to evaluate Ligustrum lucidum medicinal materials in different months.

[0098] Chromatographic conditions and system suitability test

[0099] C18 chromatographic column: Thermo Hypersil Aq (100 mm × 2.1 mm, 1.9 μm); 0.1% phosphoric acid solution as mobile phase A, acetonitrile as mobile phase B, gradient elution program: 0-3.5 min, 2% B; 3.5-

[0100] 10 min, 2%-9% B; 10-30 min, 9%-13% B; 30-70 min, 13%-90% B. Flow rate: 0.3 mL / min; wavelength: 210 nm; column temperature: 30°C. The theoretical plate number calculated based on the ligustrin peak should be no less than 30,000.

[0101] Preparation of reference solution:

[0102] Accurately weigh about 10 mg each of the special ligustrin, ligustrin G13 and new ligustrin reference substances into a 10 mL volumetric flask, add appropriate amount of methanol and ultrasonically dissolve them, and dilute to the scale with methanol to obtain the reference substance stock solution (the concentration of special ligustrin is 1.013 mg / mL, the concentration of ligustrin G13 is 0.987 mg / mL, and the concentration of new ligustrin is 1.008 mg / mL).

[0103] Preparation of test solution

[0104] The preparation of the test solution is the same as in Example 1.

[0105] Determination

[0106] Accurately pipette 2 μL of the reference solution and the test solution respectively, and inject them into the ultra-high performance liquid chromatograph Agilent 1290 high performance liquid chromatograph to obtain the product.

[0107] Measurement results

[0108] Ligustrum lucidum fruit samples were collected from July to December in six regions of Shaanxi Province (Yanta District, Xi'an, Beilin District, Xi'an, Lantian County, Xi'an, Hanbin District, Ankang, Linwei District, Weinan, and Qindu District, Xianyang). 500 g of samples were collected from three plants with similar locations and growth conditions in each region each month. The samples were mixed, cleaned, and dried in the shade as one batch of samples. A total of 36 batches of samples were collected from the six regions over six months. The results of the determination of privetside and privetside G13 contents are shown in Table 5.

[0109] Table 5 Percentage results of ligustrin and ligustrin G13 in Ligustrum lucidum samples

[0110]

[0111] Note: ND means not detected

[0112] The content range of ligustrin is low in July and August. In September, the content is 0.854% to 0.951%, with an average of 0.884%. In October, the content is 0.768% to 0.862%, with an average of 0.823%. In November, the content is 0.721% to 0.815%, with an average of 0.789%. In December, the content is 0.725% to 0.808%, with an average of 0.773%. See the data distribution. Figure 8 (Content distribution heat map) and Figure 9 (Content distribution bar graph). The results showed that the content of tereligrosin was low in July and August, peaked in September, and remained high in November and December. This means that the content of tereligrosin reached a high level in September when Ligustrum lucidum fruit was still immature, and therefore cannot be used as an indicator of maturity.

[0113] The content of ligustrin G13 ranged from 0.094% to 0.328% in September, with an average of 0.183%. In October, it ranged from 0.306% to 0.782%, with an average of 0.602%. In November, it ranged from 0.77% to 1.187%, with an average of 0.940%. In December, it ranged from 0.726% to 1.003%, with an average of 0.868%. Data distribution is shown in Figure 2. Figure 10 (Content distribution heat map) and Figure 11 (Content distribution bar graph) The results showed that privet glycoside G13 was not detected in July and August, the content was low in September samples, and the content was high in November and December samples, which can be used as a maturity indicator.

[0114] Methodological validation

[0115] (1) Detection limit and quantification limit

[0116] An appropriate amount of the standard solution was diluted and injected into the liquid chromatography system for analysis. The concentration at a chromatographic peak signal-to-noise ratio of 3:1 was the detection limit, and the concentration at a signal-to-noise ratio of 10:1 was the quantification limit. The results showed that the detection limit for terglucosidoside was 1.013 μg / mL, with a quantification limit of 2.76 μg / mL; the detection limit for glucosidoside G13 was 0.987 μg / mL, with a quantification limit of 3.343 μg / mL.

[0117] (2) Precision test

[0118] Accurately pipette the reference stock solution, dilute it 10-fold, and inject it six times continuously. Record the chromatogram and data. Record the peak areas and calculate the relative standard deviation (RSD). The results show an RSD of 0.42% for the chromatographic peak of ligustrin and 0.32% for the chromatographic peak of ligustrin G13, indicating good instrument precision.

[0119] (3) Repeatability test

[0120] Take 6 samples of Ligustrum lucidum fruit, each about 1.0 g, accurately weighed, and prepare 6 test solutions in parallel. Sampling and determination were performed separately, and the percentage content of the components to be tested was determined separately, and the RSD was calculated. The results showed that the RSD of the content of privet glycoside was 1.12%, and the RSD of the content of privet glycoside G13 was 0.95%, indicating that the experimental method was stable and reliable.

[0121] (4) Stability test

[0122] The test solution under the repeatability test was placed at room temperature for 1, 2, 4, 6, 12, and 24 hours, then injected and measured. The peak area of ​​the chromatographic peak was recorded and the RSD was calculated. The results showed that the RSD of the chromatographic peak of privet glycoside was 0.58%, and the RSD of the chromatographic peak of privet glycoside G13 was 0.64%, indicating that the test solution had good stability within 24 hours.

[0123] (5) Linear relationship and range

[0124] Accurately pipette an appropriate amount of the mixed reference stock solution and dilute it by 2, 5, 10, 50, and 100 times to prepare a series of reference solutions of different concentrations. Inject 2 μL of each solution and record the peak area of ​​the chromatographic peak. Draw a standard curve with concentration X as the horizontal axis and peak area Y as the vertical axis.

[0125] Table 6 Linear relationship and range

[0126]

[0127] (6) Accuracy test

[0128] Accurately weigh 9 samples of the same batch of Ligustrum lucidum fruit, each about 0.5 g, accurately weighed, and added with appropriate amounts of reference solution (low, medium and high concentrations) to prepare the test solution. The solution was determined according to the given chromatographic conditions. The recovery results are shown in Table 7.

[0129] Table 7 Recovery rate test of spiked Ligustrum lucidum samples

[0130]

[0131] This scheme constructs a fingerprint of Ligustrum lucidum fruit and uses UPLC chromatographic conditions to quantitatively analyze the two components in Ligustrum lucidum fruit, screen out the indicator Ligustrum lucidum glycoside G13, which is closely related to the maturity of Ligustrum lucidum fruit, and then test the quality of Ligustrum lucidum fruit to further determine the maturity of Ligustrum lucidum fruit. Compared with the existing method relying on manual judgment, the measurement results of this scheme are more reliable and easy to operate, providing a scientific basis for the revision of the 2020 edition of the "Chinese Pharmacopoeia".

Claims

1. A method for constructing a fingerprint of Ligustrum lucidum, characterized in that: include: The steps of using ultra-high performance liquid chromatography to detect the test solution of Ligustrum lucidum; In the high performance liquid chromatography detection step, the chromatographic conditions also include: column temperature of 30° C., flow rate of 0.3 mL / min, and detection wavelength of 210 nm.

2. The method according to claim 1, characterized in that The ultra-high performance liquid chromatography comprises: octadecylsilane bonded silica gel as filler, 0.1% phosphoric acid solution as mobile phase A, acetonitrile as mobile phase B, Perform gradient elution as follows: 0-3.5min, A:B is 98%:2%; 3.5-10 min, A:B 98%:2% → 91%:9%; 10-30 min, A:B 91%:9% → 87%:13%; 30 to 70 minutes later, the ratio of A to B was 87%:13% → 10%:90%.

3. The method according to claim 1 or 2, characterized in that The preparation method of the test solution comprises the following steps: taking a Ligustrum lucidum control medicinal material, using methanol to process the powder of the Ligustrum lucidum control medicinal material through a No. 3 sieve, adding an organic solvent and performing ultrasonic treatment, filtering and collecting the filtrate.

4. The method according to claim 3, characterized in that The amount of the solvent used is 10 mL of organic solvent per 1.0 g of the Ligustrum lucidum medicinal material powder.

5. The method according to any one of claims 2 to 4, characterized in that: The organic solvent includes methanol; The power of the ultrasonic treatment step was 500 W and the frequency was 40 kHz.

6. The method according to claim 5, characterized in that The extraction time of the ultrasonic treatment is 30 minutes.

7. A fingerprint of Ligustrum lucidum and / or a control fingerprint, characterized in that: The Ligustrum lucidum fingerprint or control fingerprint is constructed by the method described in any one of claims 1-6.

8. The method for constructing a Ligustrum lucidum fingerprint according to any one of claims 1 to 6 and / or the application of the Ligustrum lucidum fingerprint and / or control fingerprint according to claim 7 in the field of Ligustrum lucidum quality detection, characterized in that: The Ligustrum lucidum fingerprint is used to detect the content of Ligustrum lucidum glycoside G13 in the target Ligustrum lucidum fruit.

9. An application as claimed in claim 8, characterized in that: The maturity of Ligustrum lucidum fruit is determined according to the content of Ligustrum lucidum G13.

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