Wild honeysuckle flower characteristic chromatogram and construction method and application thereof

Abstract

The invention belongs to the technical field of traditional Chinese medicinal material detection, and particularly relates to a lonicerae flos specific chromatogram, a construction method thereof, a lonicerae flos detection method and application. Chromatographic conditions and system applicability of lonicerae flos characteristic spectrum determination are verified, a pretreatment method of a sample is investigated, a test sample preparation method and the chromatographic conditions are determined, methodological verification is performed, and according to the conditions of precision, repeatability, stability, durability and the like, the content of the lonicerae flos characteristic spectrum is determined. And finally, determining the characteristic chromatograms of a plurality of batches of lonicerae flos medicinal materials and decoction pieces, so as to determine the rationality of the selected characteristic peaks. The method provided by the invention has strong specificity and is suitable for detection of lonicerae flos characteristic chromatogram determination.

Description

Technical Field

[0001] The present application belongs to the technical field of Chinese medicinal material detection, and specifically relates to a characteristic spectrum of honeysuckle and a construction method and application thereof. Background Art

[0002] Honeysuckle is cold in nature, sweet in taste, clears away heat and detoxifies, and dispels wind-heat. It is used for carbuncle, furuncle, throat paralysis, erysipelas, dysentery due to heat toxins, wind-heat cold and febrile diseases. 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 chromatography and high performance liquid chromatography for the detection of honeysuckle, 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 high-performance liquid fingerprints. The fingerprint of traditional Chinese medicine is a comprehensive and quantifiable identification method. It is one of the quality control modes that currently conforms to 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 honeysuckle to better monitor and detect its quality. Summary of the invention

[0004] In order to solve the above problems, a characteristic spectrum of Lonicera japonica and a method for constructing it and an application thereof are provided. The chromatographic conditions and system applicability for determining the characteristic spectrum of Lonicera japonica are verified, and the sample pretreatment method is investigated to determine the test sample preparation method. The applicability of the method to the detection of characteristic spectrum of Lonicera japonica medicinal materials and decoction pieces is determined based on the precision, repeatability, stability, durability and other conditions of the method. The characteristic spectrum of Lonicera japonica and a method for constructing it provided in this application can effectively improve the quality detection effect of Lonicera japonica.

[0005] According to one aspect of the present application, a method for constructing a characteristic spectrum of Lonicera japonica is provided, comprising the following steps:

[0006] S1 Preparation of reference solution: Take neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, luteolin, isochlorogenic acid A, isochlorogenic acid B, and isochlorogenic acid C reference substances, and add methanol solvent to prepare reference solution;

[0007] S2 Preparation of test solution: Take honeysuckle powder, add methanol solvent to extract, filter and take the filtrate to obtain the test solution;

[0008] S3 determination: aspirate the reference solution and the test solution respectively and inject them into the liquid chromatograph for determination to construct a characteristic spectrum of Lonicera japonica;

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

[0010] Octadecylsilane bonded silica gel was used as filler, acetonitrile was used as mobile phase A, 0.08% to 0.12% phosphoric acid solution was used as mobile phase B, the detection wavelength was 240 to 280 nm, the column temperature was 25 to 35°C, and the flow rate was 0.9 to 1.1 ml / min;

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

[0012] 0-10min, mobile phase A is 10→13%, mobile phase B is 90→87%;

[0013] 10-20 min, mobile phase A 13→20%, mobile phase B 87→80%;

[0014] 20-40 min, mobile phase A is 20→25%, mobile phase B is 80→75%;

[0015] 40-43 min, mobile phase A: 25→80%, mobile phase B: 75→20%;

[0016] 43-50 min, mobile phase A: 80%, mobile phase B: 20%;

[0017] 50-51 min, mobile phase A: 80→10%, mobile phase B: 20→90%;

[0018] 51-60min, mobile phase A is 10%, mobile phase B is 90%.

[0019] Optionally, the detection wavelength is 254 nm, the column temperature is 30° C., and the flow rate is 1.0 ml / min.

[0020] Optionally, the methanol solvent in S1 and S2 is methanol with a volume concentration of 50 to 75%.

[0021] Optionally, the methanol solvent in S1 and S2 is methanol with a volume concentration of 50%.

[0022] Optionally, the extraction process in S2 is one or more of shaking extraction, ultrasonic extraction and reflux extraction.

[0023] Optionally, the extraction process is ultrasonic extraction, and the frequency of ultrasonic extraction is 40 kHz and the time is 30 minutes.

[0024] Optionally, the characteristic spectrum of Lonicera japonica includes 7 characteristic peaks, among which characteristic peak 2 is the reference peak S peak, characteristic peak 2 is chlorogenic acid, and the relative retention times of each characteristic peak are: characteristic peak 1 is 0.647, characteristic peak 3 is 1.097, characteristic peak 4 is 2.054, characteristic peak 5 is 2.278, characteristic peak 6 is 2.401, and characteristic peak 7 is 2.662, and the relative retention time is within ±8% of the specified value.

[0025] Optionally, in the step S2 of preparing the test solution, 0.4-0.6 g of honeysuckle medicinal material powder is added to every 25 ml of methanol, and in the step S1 of preparing the reference solution, 0.08-0.12 mg each of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, luteolin, isochlorogenic acid A, isochlorogenic acid B, and isochlorogenic acid C reference substances are added to every 1 ml of methanol.

[0026] According to another aspect of the present application, a characteristic spectrum of honeysuckle is provided, wherein the characteristic spectrum of honeysuckle includes 7 characteristic peaks, with characteristic peak 2 as the reference peak S peak, characteristic peak 2 is chlorogenic acid, and the relative retention times of the characteristic peaks are: characteristic peak 1 is 0.647, characteristic peak 3 is 1.097, characteristic peak 4 is 2.054, characteristic peak 5 is 2.278, characteristic peak 6 is 2.401, characteristic peak 7 is 2.662, and the relative retention time is within ±8% of the specified value;

[0027] The characteristic peak 1 is neochlorogenic acid, the characteristic peak 2 is chlorogenic acid, the characteristic peak 3 is cryptochlorogenic acid, the characteristic peak 4 is luteolin, the characteristic peak 5 is isochlorogenic acid B, the characteristic peak 6 is isochlorogenic acid A, and the characteristic peak 7 is isochlorogenic acid C.

[0028] According to the last aspect of the present application, a method for detecting Lonicera japonica is provided, comprising the steps of comparing a characteristic spectrum of the Lonicera japonica to be detected with a control characteristic spectrum of Lonicera japonica;

[0029] The characteristic spectrum of the tested Lonicera japonica is a characteristic spectrum obtained by using the tested Lonicera japonica according to the construction method of the test solution in any of the above construction methods,

[0030] The reference characteristic spectrum of Lonicera japonica is the characteristic spectrum obtained by the construction method of the reference solution in any of the above-mentioned construction methods.

[0031] The beneficial effects of this application include but are not limited to:

[0032] 1. The method for constructing the characteristic spectrum of Lonicera japonica provided in this application has good specificity in the specificity investigation because the blank solution has no interference. The retention time of each characteristic peak in the test solution is continued to be compared with the chromatographic peak of the reference solution. Combined with the absorption curve of each characteristic peak, the attribution of each characteristic peak is determined conveniently, quickly, clearly and intuitively.

[0033] 2. The method for constructing the characteristic spectrum of Lonicera japonica provided in this application has been proved to be suitable for the detection of the characteristic spectrum of Lonicera japonica through instrument precision, injection precision, repeatability, durability, solution stability and intermediate precision tests. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] 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:

[0035] Figure 1 The chromatogram of the test sample in Example 1 involved in this application;

[0036] Figure 2 This is the chromatogram of the gradient 1 sample involved in this application;

[0037] Figure 3 This is the chromatogram of the gradient 2 sample involved in this application;

[0038] Figure 4 This is the chromatogram of the gradient 3 test sample involved in this application;

[0039] Figure 5 The wavelength comparison chromatogram involved in this application: A: 220nm; B: 254nm; C: 300nm; D: 330nm;

[0040] Figure 6 The chromatograms of the extraction solvents for the test products involved in this application are as follows: A: water; B: 25% methanol; C: 50% methanol; D: 75% methanol; E: methanol;

[0041] Figure 7 The chromatograms for the sample extraction methods involved in this application are as follows: A: shaking extraction; B: ultrasonic extraction; C: reflux extraction;

[0042] Figure 8 The chromatograms for the extraction time of the test sample involved in this application are as follows: A: 15 minutes of ultrasound; B: 30 minutes of ultrasound; C: 45 minutes of ultrasound;

[0043] Fig. 9 The chromatograms of the sample weights involved in this application are as follows: A: 0.4 g sample weight; B: 0.5 g sample weight; C: 0.6 g sample weight;

[0044] Fig.10 Chromatography for the specificity investigation involved in this application Figure 1 :A: blank; B: neochlorogenic acid; C: chlorogenic acid; D: cryptochlorogenic acid; E: olibanum;

[0045] Fig.11 Examining chromatograms for specificity of application Figure 2 :; F: isochlorogenic acid B; G: isochlorogenic acid A; H: isochlorogenic acid C; I: reference substance; J: test substance. DETAILED DESCRIPTION

[0046] The present application is described in detail below with reference to embodiments, but the present application is not limited to these embodiments.

[0047] Unless otherwise specified, the raw materials and catalysts in the examples of this application were purchased through commercial channels.

[0048] The information of instruments and equipment used in this application is as follows: Chromatograph 1: Waters 2695 chromatography system, including a quaternary gradient infusion pump (Alliance 2695), a 120-position high-performance autosampler, an imported chromatography column oven, a Waters 2996 DAD diode array UV detector, and an Empower 3 chromatography management system; Chromatograph 2: Agilent 1100 chromatography system, including a G1322A degasser, a G1311A quaternary pump, a G1313A autosampler, a G1315B DAD diode array detector, a G1316A column oven, and a ChemStation chromatography workstation; Electronic balance: Sartorius Scientific Instruments (Beijing) Co., Ltd. (BSA224S), Shanghai Instrument Co., Ltd. (FA2004B); Ultrasonic instrument: KQ-500DB CNC ultrasonic cleaner, Kunshan Ultrasonic Instrument Co., Ltd.; Electric heating jacket: DZTW, Bangsi Instrument Technology (Shanghai) Co., Ltd.

[0049] The chromatographic columns used in this application are as follows: Column 1: Agilent 5TC-C 18 (2) 5μm4.6mm×250mm, SN: 621482; Column 2: Agilent ZORBAX SB-C18 5μm4.6mm×250mm, SN: USCL109280; Column 3: Zafex Acutfex PA-C18 5μm4.6mm×250mm, SN: WX0036EL55.

[0050] The reagent information used in this application is as follows: acetonitrile is chromatographically pure, phosphoric acid and methanol are analytically pure, and water is ultrapure water; chlorogenic acid reference substance batch number: 110753-202018, purity is 96.1%, China Food and Drug Inspection Institute; neochlorogenic acid reference substance batch number: AF20030804, purity is 98%, Chengdu Aifa Biological; cryptochlorogenic acid reference substance batch number: AF21010310, purity is 98%, Chengdu Aifa Biological; isochlorogenic acid A reference substance batch number: AF20020302, purity is 98%, Chengdu Aifa Biological; isochlorogenic acid B reference substance batch number: AF20060701, purity is 98%, Chengdu Aifa Biological; isochlorogenic acid C reference substance batch number: AF20060702, purity is 98%, Chengdu Aifa Biological Product batch number: AF20121801, purity is 98%, Chengdu Aifa Biological; luteolin reference substance batch number: 111720-201820, purity is 93.5%, China Food and Drug Inspection Institutes; Honeysuckle medicinal materials and decoction pieces 1: medicinal material batch number Y15221082601, decoction piece batch number ZA15221082601, Longhui County, Shaoyang, Hunan; Honeysuckle medicinal materials and decoction pieces 2: medicinal material batch number Y15221082602, decoction piece batch number ZA15221082602, Xupu County, Huaihua, Hunan; Honeysuckle medicinal materials and decoction pieces 3: medicinal material batch number Y15221082603, decoction piece batch number ZA15221082603, Daxian County, Dazhou, Sichuan.

[0051] Honeysuckle is the dried flower bud of Lonicera hypoglauca Miq., a plant of the Caprifoliaceae family. It has the effects of clearing away heat and detoxifying, cooling and dispersing wind-heat, and is widely used in clinical practice. Studies have shown that Honeysuckle mainly includes phenolic acid, flavonoids, and triterpenoids. The flavonoids of Honeysuckle mainly include luteolin, luteolin, quercetin, rutin and other ingredients; the organic acids contained in Honeysuckle are mainly caffeoylquinic acids, such as chlorogenic acid, isochlorogenic acid, neochlorogenic acid, caffeic acid, 3,5-O-dicaffeoylquinic acid and other ingredients; the triterpenoids contained in Honeysuckle mainly include Lonicera hypoglauca saponin A, Lonicera hypoglauca saponin B, and Dipsacus saponin B and other ingredients. According to the pharmacological and pharmacodynamic components of Honeysuckle combined with the feasibility of technology, the preliminary tentative characteristic peaks are: chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, and luteolin.

[0052] In the present application, a method for determining the characteristic spectrum of Flos Lonicerae was explored. First, the characteristic peaks were tentatively determined based on the results of the component investigation, and chlorogenic acid was used as the S peak and the system adaptability of other index components was used as the evaluation index to verify the chromatographic conditions and system applicability of the characteristic spectrum of Flos Lonicerae. The sample pretreatment method was also investigated to determine the test sample preparation method. Finally, a methodological investigation was conducted on the determination method. Based on the precision, repeatability, stability, durability, etc. of the method, the applicability of the method to the detection of the characteristic spectrum of Flos Lonicerae medicinal materials and decoction pieces was determined. Furthermore, the rationality of the selected characteristic peaks was determined by determining the characteristic spectra of multiple batches of Flos Lonicerae medicinal materials and decoction pieces. It should be particularly pointed out that, since all characteristic peaks have not been fully determined during the method optimization process, chlorogenic acid is used as the S peak during the method optimization process of the embodiment. After the final method is determined, all characteristic peaks are also determined. Therefore, in order to facilitate the calculation and understanding of the relative retention time, chlorogenic acid can be used as the S peak, or other characteristic peaks can be used as the S peak and the relative retention time can be calculated. It can be understood by those skilled in the art that the method provided in the present application can use any characteristic peak as the S peak and calculate the relative retention time of other characteristic peaks based on this.

[0053] It should also be noted that in the present application, some parts are expressed as 9 characteristic peaks. It should be noted that the characteristic peaks are numbered to include the two characteristic peaks in Table 7 that are not attributed to the components, while some parts are expressed as 7 characteristic peaks. At this time, the characteristic peaks are numbered to exclude the two characteristic peaks that are not attributed to the components. Those skilled in the art can distinguish the two situations according to the specific description scenarios, and can also clearly distinguish the characteristic peaks according to the relative retention time.

[0054] Example 1

[0055] Preparation of reference solution: take neochlorogenic acid reference, chlorogenic acid reference, cryptochlorogenic acid reference, olivine glycoside reference, isochlorogenic acid A reference, isochlorogenic acid B reference, and isochlorogenic acid C reference, and add 50% methanol to make a solution containing 0.1 mg of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, and olivine glycoside per 1 ml.

[0056] Preparation of test solution: Take 0.5g of honeysuckle powder (passed through No. 4 sieve), weigh accurately, put into a stoppered conical bottle, add 25ml of 50% methanol, and seal. Ultrasonic treatment (250W, 40kHZ) for 30 minutes, take out, cool, filter, and take the filtrate to obtain the solution.

[0057] Determination method: Accurately pipette 10 μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine. The chromatographic conditions are: octadecylsilane bonded silica gel as filler (Aglient5TC-C18(2), 4.6mm×250mm, 5μm), acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, gradient elution according to the provisions in Table 1; detection wavelength is 254nm; column temperature is 30℃; flow rate is 1.0ml per minute.

[0058] Table 1 Elution gradient

[0059] Time (minutes) Mobile phase A Mobile phase B (%) 0～10 10→13 90→87 10～20 13→20 87→80 20～40 20→25 80→75 40～43 25→80 75→20 43～50 80 20 50～51 80→10 20→90 51～60 10 90

[0060] like Figure 1 As shown, this embodiment can achieve relatively ideal separation of each chromatographic peak of Lonicera japonica, and can realize the determination of major components such as neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, luteolin, isochlorogenic acid A, isochlorogenic acid B, and isochlorogenic acid C in Lonicera japonica.

[0061] exist Figure 1 In the characteristic spectrum shown, there are 9 characteristic peaks of Honeysuckle at a wavelength of 254nm, among which Peak 3 chlorogenic acid is the standard peak, and the relative retention times of the 9 peaks are: Peak 1: 0.647; Peak 2: 0.931; Peak 3: 1; Peak 4: 1.097; Peak 5: 1.572; Peak 6: 2.054; Peak 7: 2.278; Peak 8: 2.401; Peak 9: 2.662.

[0062] Example 2 Investigation of chromatographic conditions

[0063] 1. Investigation of elution gradient

[0064] 1.1 Preparation of test solution: Take 0.5g of honeysuckle powder (passed through No. 4 sieve), weigh accurately, put into a stoppered conical bottle, add 25ml of 50% methanol, and stopper. Ultrasonic treatment (250W, 40kHz) for 30 minutes, take out, cool, filter, and take the filtrate to obtain the solution.

[0065] Chromatographic conditions: Octadecylsilane bonded silica gel as filler (Aglient 5TC-C18(2), 4.6 mm×250 mm, 5 μm or a chromatographic column of equivalent performance); acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, gradient elution as specified in Table 2; detection wavelength of 254 nm; column temperature of 25°C; flow rate of 1.0 ml per minute.

[0066] Table 2 Feature spectrum gradient 1

[0067] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～90 5→90 95→10

[0068] The chromatogram results of the test product are as follows Figure 2 As shown, the chromatographic peak was completely eluted within 30 minutes, but the peak shape and separation effect of the chromatographic peak were poor.

[0069] 1.2 The difference between this embodiment and 1.1 is that the gradient elution conditions are as shown in Table 3, and the remaining steps and test conditions are the same as 1.1.

[0070] Table 3 Feature Spectrum Gradient 2

[0071] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～20 8→10 92→90 20～25 10→15 90→85 25～40 15→20 85→80 40～60 20→30 20→70

[0072] The chromatogram results of the test product are as follows Figure 3 As shown, it can be seen Figure 3 The separation effect of the chromatographic peaks was significantly improved, but the chromatographic peaks did not begin to emerge until about 20 minutes, resulting in a longer elution time.

[0073] 1.3 The difference between this embodiment and 1.1 is that the gradient elution conditions are as shown in Table 4, and the remaining steps and test conditions are the same as 1.1.

[0074] Table 4 Feature Spectrum Gradient 3

[0075] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～10 10 90 10～15 10→15 90→85 15～30 15→20 85→80 30～40 20→30 80→70 40～43 30→80 70→20 43～50 80 20 50～51 80→10 20→90 51～60 10 90

[0076] The chromatogram results of the test product are as follows Figure 4 As shown, it can be seen Figure 4 The chromatographic peaks are basically separated by baseline and have good peak shapes. Under this condition, the characteristic peaks have good separation and short elution time.

[0077] 1.4 The difference between this embodiment and 1.1 is that the column temperature is 30°C, the gradient elution conditions are as shown in Table 5, and the remaining steps and test conditions are the same as 1.1.

[0078] Table 5 Method optimization gradient

[0079] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～10 10→13 90→87 10～20 13→20 87→80 20～40 20→25 80→75 40～43 25→80 75→20 43～50 80 20 50～51 80→10 20→90 51～60 10 90

[0080] Table 6 Method optimization gradient test sample chromatographic peak performance parameters

[0081]

[0082]

[0083] The results are as follows Figure 1 As shown, from the chromatogram and performance parameters of the column test sample, it can be seen that at this time, each characteristic peak has achieved baseline separation, and the peak shape is better and the overall coordination is good. Compared with 1.3, there is no defect of no chromatographic peak between peak 5 and peak 6 within 10 minutes, and the separation effect of peak 8 and the subsequent miscellaneous peaks in this embodiment is better.

[0084] 2. Investigation of detection wavelength

[0085] The difference between this embodiment and 1.4 is that the detection wavelength is different, and the remaining steps and test conditions are the same as 1.4.

[0086] The inventors compared the full wavelength chromatograms and found that the number of chromatographic peaks was the largest at 210nm-240nm, followed by the number of chromatographic peaks at 240nm-280nm, the number of chromatographic peaks at 290nm-320nm, and the number of chromatographic peaks at 330nm and beyond was very small. The specific results of 220nm, 254nm, 300nm and 330nm are as follows: Figure 5 As shown, the results show that when the wavelength is 240-280, the chromatogram has more chromatographic peaks, the baseline is relatively stable, and the overall coordination of the chromatographic peaks is better. Among them, the best effect is achieved when the wavelength is 254nm.

[0087] Example 3 Investigation of the test product

[0088] 1.1 Investigation of the extraction solvent of the test sample

[0089] Preparation of test solution: Take about 0.5g of honeysuckle powder (passed through No. 4 sieve), weigh accurately, put into a stoppered conical bottle, add water, 25% methanol, 50% methanol, 75% methanol and 25ml methanol respectively, and seal. Ultrasonic treatment (250W, 40kHZ) for 30 minutes, take out, cool, filter, and take the filtrate to obtain the solution.

[0090] Chromatographic conditions: Same as Example 1.

[0091] The results are as follows Figure 6 As shown, the results show that when the extraction solvent is water and 25% methanol, the peak areas of peaks 6, 8 and 9 in the test sample are lower; when the extraction solvent is methanol, the peak areas of peaks 1, 3, 4, 8 and 9 in the test sample are lower; when the extraction solvent is 50% methanol and 75% methanol, there is no obvious difference in the peak area of ​​each characteristic peak, and the peak shape is also better.

[0092] 1.2 Investigation of the extraction method of the test sample

[0093] Preparation of test solution: Take about 0.5g of honeysuckle powder (passed through No. 4 sieve), weigh accurately, put into a stoppered conical bottle, add 25ml of 50% methanol accurately, and plug it tightly. Shake extraction, ultrasonic extraction and reflux extraction for 30 minutes respectively, take out, cool, filter, and take the filtrate to obtain.

[0094] Chromatographic conditions: Same as Example 1.

[0095] The results are as follows Figure 7As shown in the figure, the results show that all samples extracted by different extraction methods have tentative target peaks. Although the extraction efficiency of each target peak is different and irregular, there is no significant difference in the total peak area. In order to facilitate the experimental operation, ultrasonic extraction was selected as the extraction method for the sample.

[0096] 1.3 Investigation of the extraction time of the test sample

[0097] Preparation of test solution: Take about 0.5g of honeysuckle powder (passed through No. 4 sieve), weigh accurately, put into a stoppered conical bottle, add 25ml of 50% methanol accurately, and plug it tightly. Ultrasonic treatment (250W, 40kHZ) for 15 minutes, 30 minutes and 45 minutes respectively, take out, cool, filter, and take the filtrate to obtain the solution.

[0098] The results are as follows Figure 8 As shown, the results show that the extraction time has a certain influence on the extraction efficiency of each characteristic peak in the test sample, but there is no obvious difference in the total peak area. Considering the continuity of the experiment, the extraction time of the test sample was selected as 30 minutes.

[0099] 1.4 Inspection of sample weight

[0100] Preparation of test solution: Take about 0.4g, 0.5g and 0.6g of honeysuckle powder (passed through No. 4 sieve), weigh accurately, put into a stoppered conical bottle, add 25ml of 50% methanol accurately, and plug it. Ultrasonic treatment (250W, 40kHZ) for 30 minutes, take out, cool, filter, and take the filtrate to obtain the solution.

[0101] The results are as follows Fig. 9 As shown, the results show that there is no significant difference between the total peak area of ​​the test sample and the value of the sample weight, indicating that within the sample weight range of 0.4g to 0.6g, there is no significant effect on the total peak area of ​​the test sample.

[0102] 1.5 Determination of the test sample preparation method

[0103] According to the results of the test sample preparation method investigation, the test sample preparation method for the determination method of the characteristic spectrum of honeysuckle was determined as follows:

[0104] Preparation of test solution: Take about 0.5g of honeysuckle powder (passed through No. 4 sieve), weigh accurately, put into a stoppered conical bottle, add 25ml of 50% methanol accurately, and seal. Ultrasonic treatment (250W, 40kHZ) for 30 minutes, take out, cool, filter, and take the filtrate to obtain.

[0105] Example 4 Methodology Verification

[0106] Preparation methods of samples involved

[0107] (1) Blank solution: 50% methanol.

[0108] (2) Reference solution: Take neochlorogenic acid reference, chlorogenic acid reference, cryptochlorogenic acid reference, olivine glycoside reference, isochlorogenic acid A reference, isochlorogenic acid B reference, and isochlorogenic acid C reference, and add 50% methanol to make a solution containing 0.1 mg each of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, and olivine glycoside per ml.

[0109] (3) New chlorogenic acid reference solution: Take the new chlorogenic acid reference and add 50% methanol to prepare a solution containing 0.1 mg of new chlorogenic acid per 1 ml.

[0110] (4) Chlorogenic acid reference solution: Take the chlorogenic acid reference solution and add 50% methanol to prepare a solution containing 0.1 mg of chlorogenic acid per 1 ml.

[0111] (5) Chlorogenic acid reference solution: Take the chlorogenic acid reference solution and add 50% methanol to prepare a solution containing 0.1 mg of chlorogenic acid per 1 ml.

[0112] (6) Luteolin reference solution: Take the luteolin reference solution and add 50% methanol to prepare a solution containing 0.1 mg of luteolin per 1 ml.

[0113] (7) Isochlorogenic acid A reference solution: Take isochlorogenic acid A reference solution and add 50% methanol to prepare a solution containing 0.1 mg of isochlorogenic acid A per 1 ml.

[0114] (8) Isochlorogenic acid B reference solution: Take isochlorogenic acid B reference and add 50% methanol to prepare a solution containing 0.1 mg of isochlorogenic acid B per 1 ml.

[0115] (9) Isochlorogenic acid C reference solution: Take isochlorogenic acid C reference and add 50% methanol to prepare a solution containing 0.1 mg of isochlorogenic acid C per 1 ml.

[0116] (10) Test solution: Take about 0.5 g of powder of honeysuckle (passed through No. 4 sieve), weigh accurately, place in a stoppered conical bottle, add 25 ml of 50% methanol accurately, and stopper tightly. Ultrasonicate (250W, 40kHz) for 30 minutes, remove, cool, filter, and take the filtrate to obtain the solution.

[0117] Exclusive inspection

[0118] Accurately pipette 10 μl of each of the blank solution, reference solution and test solution, inject them into the liquid chromatograph, and record the chromatograms. The results are as follows: Fig.10 and Fig.11 As shown:

[0119] According to the above determination results, the blank solution has no interference and the method has good specificity. The retention time of each characteristic peak in the test solution is compared with that of the reference solution, and the attribution of each characteristic peak is determined in combination with the absorption curve of each characteristic peak. The results are shown in Table 7:

[0120] Table 7 Characteristic peak attribution table

[0121] Chromatographic peak Retention time Attribution to ingredients Peak 1 8.441 Neochlorogenic Acid Peak 2 12.080 \ Peak 3 13.042 Chlorogenic acid Peak 4 14.292 Cryptochlorogenic acid Peak 5 20.673 \ Peak 6 27.176 Luteolin Peak 7 30.138 Isochlorogenic acid B Peak 8 31.782 Isochlorogenic acid A Peak 9 35.209 Isochlorogenic acid C

[0122] Taking peak 3, i.e., chlorogenic acid, as the characteristic peak S, the relative retention times of other characteristic peaks were calculated. The relative retention times of the characteristic peaks were: characteristic peak 1 was 0.647, characteristic peak 2 was 0.926, characteristic peak 3 was 1, characteristic peak 4 was 1.096, characteristic peak 5 was 1.585, characteristic peak 6 was 2.084, characteristic peak 7 was 2.311, characteristic peak 8 was 2.437, and characteristic peak 9 was 2.700. The peak numbers here correspond to the reference substances in Table 7.

[0123] Instrument precision inspection

[0124] Accurately pipette 10 μl of each of the above reference solutions, perform the determination according to the established chromatographic conditions, inject the sample 6 times continuously, record the chromatogram, calculate the RSD% of the retention time and peak area of ​​each chromatographic peak, and the peak area and retention time of each chromatographic peak are shown in Table 8.

[0125] Table 8 Instrument precision investigation results

[0126]

[0127] The results showed that the RSD% value of the peak area of ​​each chromatographic peak of the reference solution injected 6 times was ≤2.0%, and the RSD% value of the retention time was ≤1.0%, indicating that the instrument had good precision.

[0128] Injection precision investigation

[0129] Accurately pipette 10 μl of each of the above test solution, measure under the established chromatographic conditions, inject 6 times continuously, record the chromatogram, and calculate the RSD% of the retention time and peak area of ​​each chromatographic peak. The peak area and retention time of each chromatographic peak are shown in Table 9:

[0130] Table 9 Injection precision investigation results

[0131]

[0132] The results showed that the peak area RSD% value of each characteristic peak of the test solution for 6 injections was ≤2.0%, and the RSD% value of the retention time was ≤1.0%, indicating that this method has good injection precision.

[0133] Repeatability test

[0134] Prepare 6 test sample solutions in parallel, accurately pipette 10 μl of each reference sample solution and 6 test sample solutions, inject into liquid chromatograph, record chromatogram, calculate relative retention time and relative peak area of ​​each characteristic peak and S peak of each test sample, and calculate RSD% for relative retention time and relative peak area. The peak area and retention time of each chromatographic peak are shown in Table 10:

[0135] Table 10 Repeatability test results

[0136]

[0137] Result analysis: The relative retention time RSD% of each characteristic peak in the 6 samples was ≤1.0%, and the relative peak area RSD% was ≤5.0%, which met the requirements and the method had good repeatability.

[0138] Intermediate precision inspection

[0139] To investigate the influence of random variation factors on precision, another analyst used different instruments to perform the determination on different dates and re-prepared 6 test solutions. According to the established determination conditions, the reference solution and the test solution were sampled separately, the chromatograms were recorded, the relative retention time and relative peak area of ​​each characteristic peak and S peak of the 6 test solutions were calculated, and the RSD% of the relative retention time and relative peak area was calculated for the relative retention time and relative peak area of ​​each characteristic peak of the 12 test solutions of 2 personnel. The content determination results of Personnel 2 are shown in Table 11, and the data analysis results of 12 groups of different personnel are shown in Table 12:

[0140] Table 11 Content determination results of personnel 2

[0141]

[0142] The results showed that the relative retention time RSD% of each characteristic peak in the six samples was ≤1.0%, and the relative peak area RSD% was ≤5.0%, which met the repeatability requirements.

[0143] Table 12 Data analysis results of 12 groups of different personnel

[0144]

[0145] Result analysis: The RSD% of the relative retention time of each characteristic peak in a total of 12 samples from two personnel was less than 5.0%, which met the requirements and the intermediate precision of this method was good.

[0146] Stability test

[0147] The test solution was stored at room temperature without being protected from light. The samples were injected at 0h, 8h, 16h, 24h, 36h, 48h, 72h, and 96h. According to the established determination conditions, the injection method was single sample single needle, the chromatogram was recorded, the relative retention time and relative peak area of ​​each characteristic peak of the test sample and the S peak were calculated, and the RSD% was calculated for the relative retention time and relative peak area. The stability test results are shown in Table 13.

[0148] Table 13 Stability test results

[0149]

[0150] Result analysis: Calculate the retention time and peak area RSD% of the test samples injected at 0h, 2h, 4h, 8h, 12h, 24h, 36h, 48h, 72h, and 96h. The RSD% of retention time is ≤1.0%, and the RSD% of peak area is ≤3.0%. The solution stability of this method is good.

[0151] Durability test

[0152] Considering that the key operations of this method are the preparation of the test solution and the operation of the HPLC, the liquid phase operation flow rate, column temperature, mobile phase ratio and the durability of chromatographic columns of different brands were investigated. The conditions are described in Table 14.

[0153] Table 14 Other durability test conditions

[0154]

[0155] According to the established determination conditions, the reference solution and the test solution were sampled separately, the chromatograms were recorded, the relative retention time and relative peak area of ​​each characteristic peak and S peak of the test sample under different conditions were calculated, and the RSD% was calculated for the relative retention time and relative peak area. The durability test results are shown in Tables 15 to 18:

[0156] Table 15 Durability-column temperature investigation results

[0157]

[0158] Table 16 Durability-flow rate investigation results

[0159]

[0160] Table 17 Durability-phosphoric acid ratio investigation results

[0161]

[0162] Table 18 Durability - Results of investigation on chromatographic columns of different brands

[0163]

[0164] The results showed that under the conditions of column temperature of 30±5℃, flow rate of 1.0±0.1ml / min, aqueous phosphoric acid ratio of 0.1±0.02%, and durability, the relative retention time RSD% of each characteristic peak was less than 5.0%, and the relative peak area RSD% was less than 10.0%, indicating that this method has good durability.

[0165] Under the durability conditions of the chromatographic column brands (ZARBAX SB-C18, Aculfex PA-C18, Agilent 5TC-C18(2)), the relative retention times of the test samples of the Aculfex PA-C18 and Agilent 5TC-C18(2) columns were within the specified value range, so the fixed chromatographic column brands were Aculfex PA-C18 and Agilent5TC-C18(2).

[0166] According to the above test results, this method has strong specificity, and the instrument precision, injection precision, repeatability, durability, solution stability and intermediate precision tests have proved that this method is suitable for the detection of the characteristic spectrum of Lonicera japonica.

[0167] 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 Lonicera japonica, characterized in that: The steps include: S1 Preparation of reference solution: Take neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, luteolin, isochlorogenic acid A, isochlorogenic acid B, and isochlorogenic acid C reference substances, and add methanol solvent to prepare reference solution; S2 Preparation of test solution: Take honeysuckle powder, add methanol solvent to extract, filter and take the filtrate to obtain the test solution; S3 determination: aspirate the reference solution and the test solution respectively and inject them into the liquid chromatograph for determination to construct a characteristic spectrum of Lonicera japonica; The measurement conditions of the liquid chromatography include: Octadecylsilane bonded silica gel was used as filler, acetonitrile was used as mobile phase A, 0.08% to 0.12% phosphoric acid solution was used as mobile phase B, the detection wavelength was 240 to 280 nm, the column temperature was 25 to 35°C, and the flow rate was 0.9 to 1.1 ml / min; The gradient elution program of the mobile phase was: 0-10min, mobile phase A is 10→13%, mobile phase B is 90→87%; 10-20 min, mobile phase A 13→20%, mobile phase B 87→80%; 20-40 min, mobile phase A is 20→25%, mobile phase B is 80→75%; 40-43 min, mobile phase A: 25→80%, mobile phase B: 75→20%; 43-50 min, mobile phase A: 80%, mobile phase B: 20%; 50-51 min, mobile phase A: 80→10%, mobile phase B: 20→90%; 51-60min, mobile phase A is 10%, mobile phase B is 90%.

2. The method for constructing the characteristic spectrum of Lonicera japonica according to claim 1, characterized in that: The detection wavelength was 254 nm, the column temperature was 30° C., and the flow rate was 1.0 ml / min.

3. The method for constructing the characteristic spectrum of Flos Lonicerae according to claim 1, characterized in that: The methanol solvent in S1 and S2 is methanol with a volume concentration of 50-75%.

4. The method for constructing the characteristic spectrum of Flos Lonicerae according to claim 3, characterized in that: The methanol solvent in S1 and S2 is methanol with a volume concentration of 50%.

5. The method for constructing the characteristic spectrum of Flos Lonicerae according to claim 1, characterized in that: The extraction process in S2 is one or more of shaking extraction, ultrasonic extraction and reflux extraction.

6. The method for constructing the characteristic spectrum of Flos Lonicerae according to claim 5, characterized in that: The extraction process is ultrasonic extraction, the frequency of ultrasonic extraction is 40kHz, and the time is 30min.

7. The method for constructing the characteristic spectrum of Flos Lonicerae according to claim 1, characterized in that: In the step of preparing the test solution in S2, 0.4-0.6 g of honeysuckle medicinal material powder is added to every 25 ml of methanol. In the step of preparing the reference solution in S1, 0.08-0.12 mg of each of the reference substances of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, luteolin, isochlorogenic acid A, isochlorogenic acid B, and isochlorogenic acid C are added to every 1 ml of methanol.

8. A method for detecting honeysuckle, characterized in that: The method comprises the steps of comparing the characteristic spectrum of the tested honeysuckle with the control characteristic spectrum of the honeysuckle; The characteristic spectrum of the tested Lonicera japonica is a characteristic spectrum obtained by using the tested Lonicera japonica in the method for constructing the test solution in the construction method according to any one of claims 1 to 7, The reference characteristic spectrum of Lonicera japonica is a characteristic spectrum obtained by the construction method of the reference solution in the construction method described in any one of claims 1 to 7.

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