Quality control method of honeysuckle medicinal material

Abstract

The application relates to the technical field of quality control of traditional Chinese medicinal materials, in particular to a quality control method of honeysuckle medicinal materials. The quality control method comprises identification, examination, content determination and other detection items of honeysuckle, wherein the identification item takes chlorogenic acid and luteolin-7-O-glucoside as quality control indexes for physical and chemical identification of thin layer chromatography, and the preparation method of the test solution and the chromatographic conditions are optimized. Compared with the prior art, the method is convenient to operate, and can more accurately identify the authenticity of the honeysuckle traditional Chinese medicinal material.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of quality control of traditional Chinese medicinal materials, and particularly relates to a quality control method of honeysuckle medicinal materials. BACKGROUND

[0002] Honeysuckle is the dried flower buds or with the initial opened flowers of Lonicera japonica Thunb. of Caprifoliaceae, has the effects of clearing heat and resolving toxicity, and dispelling wind-heat, and is used for treating carbuncle, sores, laryngalgia, erysipelas, dysentery, wind-heat cold and warm disease fever. Honeysuckle is a commonly used traditional Chinese medicinal material, and its authenticity and quality are directly related to the safety and effectiveness of clinical medication. Influenced by the production place, variety, collection time, processing method, packaging method, storage time and the like, the product quality of honeysuckle on the market is uneven, and there is a lack of reliable and quantifiable quality grading standards. Therefore, improving the quality control standards of honeysuckle medicinal materials at the present stage is beneficial to the production enterprises of Chinese patent medicines to control the quality of honeysuckle purchased, so as to improve the quality of the finished preparation products containing honeysuckle and the clinical safety and effectiveness. SUMMARY

[0003] In view of the above technical problems, the present application provides a quality control method of honeysuckle medicinal materials, which comprises identification, inspection, content determination and the like detection items of honeysuckle, and compared with the prior art, the authenticity of honeysuckle traditional Chinese medicinal materials can be more accurately identified.

[0004] In order to achieve the above application purposes, the embodiments of the present application adopt the following technical solutions:

[0005] A quality control method of honeysuckle medicinal materials, comprising thin layer chromatography identification of honeysuckle by taking chlorogenic acid and galuteolin as the quality control indexes of physicochemical identification;

[0006] The developing agent is ethyl acetate: formic acid: water with a volume ratio of 7:(2.5-5):(2.5-5);

[0007] The thin layer plate is a GF254 silica gel plate;

[0008] The color developing method is: after the thin layer plate is developed, heating at 95-105 DEG C for 2-5 min, spraying with 8-12 mg / mL diphenyl amino ethyl ester methanol solution, drying, then spraying with 48-52 mg / mL polyethylene glycol 400 methanol solution, drying, and then observing under 366 nm.

[0009] At present, the physicochemical identification of honeysuckle in Chinese Pharmacopoeia 2015 and 2020 edition takes chlorogenic acid as the quality control index, but there is also chlorogenic acid in eucommia leaf, green coffee beans, sunflower seeds, green tea, tobacco and sweet potato leaves. When the medicinal material is micronized and it is difficult to carry out property identification, the control index of chlorogenic acid cannot accurately identify honeysuckle. In order to improve the accuracy of identification, the present application takes chlorogenic acid and osmanthus together as the index component of physicochemical identification, which helps to more accurately identify whether the sample to be tested is honeysuckle.

[0010] According to the index component, the solvent of the test solution and the extraction method are investigated, and methanol is determined as the solvent, and ultrasonic extraction is carried out for 14-16 minutes. After the test solution obtained by the extraction method is developed and colored, the spots of chlorogenic acid and osmanthus glycoside can be clearly displayed.

[0011] Preferably, the quality control index further comprises neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid A, isochlorogenic acid B and isochlorogenic acid C.

[0012] Preferably, 8-12 milliliters of methanol per gram of sample powder is used for ultrasonic extraction. The ratio of sample powder and methanol is further preferably 1:10 (g:mL).

[0013] Preferably, the ultrasonic time is 15 min.

[0014] Preferably, the solid-liquid separation method is centrifugation.

[0015] Preferably, the developing agent is ethyl acetate:formic acid:water in a volume ratio of 7:5:5.

[0016] Preferably, the quality control method further comprises content determination, and the content of caffeoyl quinine acid compounds and iridoid compounds in the sample to be tested is detected by high performance liquid chromatography; the chromatographic conditions of the high performance liquid chromatography are as follows:

[0017] The chromatographic column is an octadecylsilane bonded silica gel chromatographic column.

[0018] The mobile phase A is 0.1% v / v phosphoric acid aqueous solution, and the mobile phase B is acetonitrile, and linear gradient elution is carried out, and the program of the linear gradient elution is as follows:

[0019] Time (min) Mobile phase A (%) Mobile phase B (%) 0 86 14 8 81 19 14 81 19 34 69 31 35 10 90 39.5 10 90 40 86 14 48 86 14

[0020] The flow rate is 0.65-0.75 mL / min.

[0021] The column temperature is 14-16℃.

[0022] The above linear gradient elution program indicates that the proportion of the mobile phase changes linearly between different time nodes, for example, the volume ratio of the mobile phase A linearly decreases from 86% to 81% within 0-8 min.

[0023] The chromatographic column is preferably Syncronis C18 (specification: 4.6 mm x 25 cm; 5 μm).

[0024] Preferably, the quality control method further comprises a fingerprint quality control method, wherein the fingerprint of honeysuckle is constructed by using the chromatographic conditions for the content determination, and is used for quality control; under the wavelength of 327 nm, the relative retention time of neochlorogenic acid is 0.720-0.738, the relative retention time of cryptochlorogenic acid is 1.060-1.061, the relative retention time of 3,4-2-O-caffeoylquinic acid is 2.580-0.610, the relative retention time of 3,5-2-O-caffeoylquinic acid is 2.795-2.832, and the relative retention time of 4,5-2-O-caffeoylquinic acid is 2.914-2.948; under the wavelength of 240 nm, the relative retention time of oxymatrine is 1.167-1.174, and the relative retention time of dimeric iridoid glycoside is 2.072-2.085.

[0025] Preferably, the quality control method further comprises trait identification and microscopic identification.

[0026] The trait identification and the microscopic identification should all meet the identification requirements of honeysuckle in Chinese Pharmacopoeia.

[0027] Preferably, the quality control method further comprises moisture inspection, total ash inspection and acid-insoluble ash inspection. The moisture should be less than 12%, the total ash should be less than 10.0%, and the acid-insoluble ash should be less than 3%.

[0028] The quality control method of honeysuckle medicinal materials provided by the application can accurately identify honeysuckle and detect the quality thereof, and has a positive effect on controlling the quality of purchased honeysuckle for Chinese medicine production enterprises, improving the quality of Chinese patent medicine products containing honeysuckle, and improving the clinical safety and effectiveness. BRIEF DESCRIPTION OF DRAWINGS

[0029] The application will be further described below in combination with the drawings and examples, wherein:

[0030] Figure 1 is a thin layer chromatogram obtained by using the developing agent of example 1 in example 4 of the application;

[0031] Figure 2 is a thin layer chromatogram obtained by using the developing agent of example 2 in example 4 of the application;

[0032] Figure 3 is a thin layer chromatogram at 0 h in example 5 of the application;

[0033] Figure 4 is the thin layer chromatogram of 24h in Example 5 of the present application;

[0034] Figure 5 is the thin layer chromatogram of 48h in Example 5 of the present application;

[0035] Figure 6 is the thin layer chromatogram obtained by developing on Merck thin layer plate in Example 5 of the present application;

[0036] Figure 7 is the thin layer chromatogram obtained by developing on MN thin layer plate in Example 5 of the present application;

[0037] Figure 8 is the thin layer chromatogram obtained under the condition of 20.2℃, RH 39% in Example 5 of the present application;

[0038] Figure 9 is the thin layer chromatogram obtained under the condition of 24.0℃, RH 55% in Example 5 of the present application;

[0039] Figure 10 is the thin layer chromatogram obtained under the condition of 26.7℃, RH 47% in Example 5 of the present application;

[0040] Figure 11 is the thin layer chromatogram obtained in ADC2-170409 developing instrument in Example 5 of the present application;

[0041] Figure 12 is the thin layer chromatogram obtained in ADC2-230010 developing instrument in Example 5 of the present application;

[0042] Figure 13 is the high performance thin layer chromatogram of honeysuckle obtained in Example 6 of the present application;

[0043] Figure 14 is the liquid chromatogram of caffeoylquinic acid compounds in similarity evaluation of honeysuckle in Example 8 of the present application;

[0044] Figure 15 is the liquid chromatogram of iridoid compounds in similarity evaluation of honeysuckle in Example 8 of the present application;

[0045] Figure 16 is the thin layer chromatogram of test sample solution obtained by different preparation methods in Comparative Example 1 of the present application;

[0046] Figure 17 is the thin layer chromatogram obtained by developing agent 1 in Comparative Example 2 of the present application;

[0047] Figure 18 is the thin layer chromatogram obtained by developing agent 2 in Comparative Example 2 of the present application. DETAILED DESCRIPTION

[0048] In order to make the purposes, technical solutions and advantages of the present application clearer, the present application will be further described in detail below in combination with the drawings and examples. It should be understood that the specific examples described herein are only used to explain the present application and do not limit the present application.

[0049] At present, the physicochemical identification of honeysuckle in the Chinese Pharmacopoeia 2015 edition and 2020 edition takes chlorogenic acid as the quality control index, but there is also chlorogenic acid in eucommia ulmoides leaf, green coffee beans, sunflower seeds, green tea, tobacco and sweet potato leaves. When the morphology of the medicinal material is destroyed and it is difficult to carry out property identification, the control index of chlorogenic acid cannot accurately identify honeysuckle. In order to improve the accuracy of identification, the present application takes chlorogenic acid and chrysanthemum as the index components of physicochemical identification, which helps to more accurately identify whether the sample to be tested is honeysuckle.

[0050] When the index components are multiple, the preparation of the test solution needs to comprehensively consider that the two components can be extracted and are not interfered by other components in the development process, so as to accurately carry out physicochemical identification. Therefore, the solvent and extraction method of the test solution are investigated in the present application, and it is determined that methanol or ethanol is used as the solvent and ultrasonic extraction is carried out for 14-16 minutes. The test solution obtained by the extraction method is developed by the developing agent of ethyl acetate:formic acid:water in a volume ratio of 7:(2.5-5):(2.5-5), and the chlorogenic acid and chrysanthemum spots can be clearly displayed after color development, and are not interfered by other components in honeysuckle.

[0051] In terms of content detection, the original pharmacopoeia has a single index component. The present application optimizes the content determination of chlorogenic acid and chrysanthemum glycoside in the pharmacopoeia, increases the content determination of caffeoyl quinine acid compounds and iridoid compounds, and increases the medicinal material fingerprint.

[0052] The above identification, content determination method and medicinal material fingerprint improve the quality standard of honeysuckle, can accurately identify honeysuckle and detect the quality thereof, and has a positive effect on the production enterprises of Chinese patent medicines to control the quality of honeysuckle purchased, improve the quality of the products of the prepared prescription containing honeysuckle, and the clinical safety and effectiveness.

[0053] The honeysuckle medicinal materials used in the following examples are evenly obtained from Shijiazhuang Yiling Pharmaceutical Co., Ltd., and the batch numbers are ZS-JYH-01-ZS-JYH-03, ZS-JYH-05-ZS-JYH-16;

[0054] The control samples used in the thin layer chromatography identification in the following examples are as follows:

[0055] Chlorogenic acid (China Food and Drug Inspection Institute, batch number 110753-201817), luteolin (China Food and Drug Inspection Institute, batch number 111720-201609), neochlorogenic acid (Shanghai Yuanye Biotechnology Co., Ltd., batch number P27A6F2713), cryptochlorogenic acid (Shanghai Yuanye Biotechnology Co., Ltd., batch number Z30A6B2), isochlorogenic acid A (Shanghai Yuanye Biotechnology Co., Ltd., batch number P28O7F23862), isochlorogenic acid B (Shanghai Yuanye Biotechnology Co., Ltd., batch number P25J6F1793), isochlorogenic acid C (Shanghai Yuanye Biotechnology Co., Ltd., batch number P25J6F1794), honeysuckle reference medicinal material (China Food and Drug Inspection Institute, batch number 121060-201608), and honeysuckle reference medicinal material (China Food and Drug Inspection Institute, batch number 121595-201202).

[0056] The instruments and reagents used in the thin layer chromatography identification in the following examples are:

[0057] Automatic spotter ATS4 (Kama, Switzerland), imager VISUALIZER (Kama, Switzerland), automatic unfolder ADC2 (Kama, Switzerland), thin layer automatic impregnation device III (Kama, Switzerland), thin layer heater III, 230V (Kama, Switzerland), electronic balance CPA224S (Sartorius Beijing Co., Ltd.), ultrasonic instrument (Shanghai Kedao Ultrasonic Instrument Co., Ltd.),

[0058] High-efficiency silica gel G 60F254 glass plate 20×10 cm (Merck), High-efficiency silica gel G 60F254 glass plate 20×10 cm Nano-Durasil-20UV254 (MN, Germany),

[0059] Toluene (Sinopharm Group, analytical grade, batch number 20180209), dichloromethane (Sinopharm Group, analytical grade, batch number 20160926), methanol (Sinopharm Group, analytical grade, batch number 20181011), 2-butanone (Sinopharm Group, analytical grade, batch number 20140604), ethyl acetate (Sinopharm Group, analytical grade, batch number 20180426), butyl acetate (Sinopharm Group, analytical grade, batch number 20150930), anhydrous formic acid (Aladdin, analytical grade, batch number G1826035)

[0060] The instruments and reagents used for the content determination in the following examples are:

[0061] Milli-Q Synthesis A10 ultra-pure water instrument (MILLIPORE, Bedford, MA, USA); Elma ultrasonic instrument (Elma p180H, Germany, Serial 101561035); Agilent 1260 liquid chromatography system (Agilent Technologies, Agilent 1260 Infinity I, Palo Alto CA, USA): quaternary solvent delivery system, online degassing machine, automatic sampler, temperature control module, column oven, diode array detector and chromatography workstation (Chem Station For LC 3D Systems A10.02 (1757)), see Table 1 for details of each module.

[0062] Table 1 Instrument information

[0063]

[0064] Acetonitrile (chromatographically pure, Merck, KGaA 64271 Darmstadt Germany, UN1648); methanol (chromatographically pure, Merck, KGaA 64271 Darmstadt Germany, UN1230); methanol (analytically pure, 20180119 Sinopharm Chemical Reagent Co., Ltd. (Shanghai)); phosphoric acid (chromatographically pure, Tedia, USA, LOT 911254).

[0065] The following examples use control samples for content determination:

[0066] Chlorogenic acid (Siddhant, batch number 2772), andrographolide (Siddhant, batch number 3533).

[0067] Example 1

[0068] The embodiments of the present application provide a quality control method for honeysuckle medicinal materials.

[0069] 1. Identification

[0070] 1.1 Identification of properties

[0071] The sample to be tested is in the form of a rod, slightly curved, 2-3 cm long, about 3 mm in diameter at the top and about 1.5 mm in diameter at the bottom. The surface is yellowish white or greenish white (the color gradually deepens after long storage), densely covered with short soft hair. Occasionally leafy bracts are seen. The calyx is green, with 5 lobes at the tip, and the lobes are hairy, about 2 mm long. The corolla is cylindrical, with a two-lipped tip; the stamens are 5, attached to the wall of the tube, and are yellow; the pistil is 1, and the ovary is hairless.

[0072] 1.2 Microscopic identification

[0073] The sample to be tested has the following characteristics under a NIKON NI-U upright polarizing microscope: more glandular hairs, head part conical, circular or slightly flat round, diameter 30-70-110 μm, handle part 1-5 cells, length up to 700 μm. There are two kinds of non-glandular hairs: one is thick-walled non-glandular hair, single cell, length up to 45-900 μm, diameter 15-40 μm, surface with fine warts or vesicular protrusions, some with threads; the other is thin-walled non-glandular hair, single cell, very long, curved or wrinkled, surface with fine warts. Calcium oxalate cluster crystal diameter 6-45 μm. Pollen grains are circular or triangular, with fine and dense short spines and granular engravings on the surface, with 3-pore grooves, diameter 60-90 μm.

[0074] 1.3 Physicochemical identification

[0075] Take 0.2 g of the sample to be tested powder, add 2 mL of methanol, ultrasonic treatment for 15 min, centrifuge, take the supernatant, and the test solution is obtained;

[0076] Developing agent: butyl acetate: formic acid: water (7:5:5);

[0077] Thin layer plate: high efficiency silica gel G 60F254 glass plate 20x10cm (Merck);

[0078] Development distance: 6 cm;

[0079] Sample size: 5 μl;

[0080] Coloring agent: 10 mg / mL diphenyl amino ethyl methanol solution, 50 mg / mL polyethylene glycol 400 methanol solution;

[0081] Inspection method: after the thin layer plate is developed, take it out, heat at 100°C for 3 min, spray with 10 mg / mL diphenyl amino ethyl solution, dry after spraying with 50 mg / mL polyethylene glycol 400 solution, dry, and inspect under 366 nm.

[0082] 2. Content determination

[0083] Use high performance liquid chromatography to detect caffeoyl quinine acid compounds and iridoid compounds in the sample to be tested:

[0084] Preparation of test solution: take 0.1 g of the sample to be tested powder, accurately weigh, put it in a brown volumetric flask, accurately add 10 ml of 75% v / v methanol, weigh, ultrasonic treatment for 30 min, cool to room temperature, supplement the weight lost with 75% v / v methanol, shake well, filter with 0.22 μm microporous filter membrane, discard the initial filtrate, and the test solution is obtained.

[0085] Preparation of control solution: accurately weigh a certain amount of chlorogenic acid control sample, dissolve it in a brown volumetric flask with methanol to prepare a solution containing 0.30 mg per 1 ml, namely the chlorogenic acid control solution; accurately weigh a certain amount of breviscapin A control sample, dissolve it in a brown volumetric flask with methanol to prepare a solution containing 0.05 mg per 1 ml, namely the breviscapin A control solution;

[0086] The chromatographic conditions of the high performance liquid chromatography are as follows:

[0087] Prepare the test solution with methanol as the solvent;

[0088] Chromatographic column: 4.6 mm x 25 cm; 5 μm, octadecylsilane bonded silica gel (Syncronis C18);

[0089] Mobile phase A is 0.1% v / v phosphoric acid aqueous solution, mobile phase B is acetonitrile, linear gradient elution is performed, and the procedure of the linear gradient elution is as follows:

[0090] Time (min) Mobile phase A (%) Mobile phase B (%) 0 86 14 8 81 19 14 81 19 34 69 31 35 10 90 39.5 10 90 40 86 14 48 86 14

[0091] Flow rate: 0.7 mL / min;

[0092] Detection wavelength: 327 nm (caffeoyl quinic acid compounds), 240 nm (iridoid compounds);

[0093] Column temperature: 14-16 °C

[0094] Flow rate: 0.7 mL / min

[0095] Injection volume: 2 μL.

[0096] According to the chromatographic peak area, the contents of chlorogenic acid, chlorogenic acid A and chlorogenic acid C in the test sample solution are respectively calculated by using the following formula:

[0097]

[0098] r U = peak area of the component to be tested in the sample;

[0099] r S = peak area of the control sample in the control solution;

[0100] Cs = concentration of the control sample in the control solution;

[0101] V = volume of the sample solution;

[0102] W = sample weight of honeysuckle in the sample solution (mg);

[0103] F = relative correction factor of the component to be analyzed (see Table 2).

[0104] Table 2 correction factor

[0105]

[0106]

[0107] The content of the caffeoylquinic acid compound is calculated based on the total content of chlorogenic acid, 3,5-2-O-caffeoylquinic acid and 4,5-2-O-caffeoylquinic acid, and the content in the dried product should be not less than 3.8%; the content of the iridoid glycoside compound is calculated based on the total content of swertiamin, loganin and dimeric iridoid glycoside, and the content in the dried product should be not less than 0.8%. Among them, the contents of 3,5-2-O-caffeoylquinic acid and 4,5-2-O-caffeoylquinic acid are determined based on chlorogenic acid as the reference substance, the peaks corresponding to 3,5-2-O-caffeoylquinic acid and 4,5-2-O-caffeoylquinic acid are determined according to the relative retention time in Table 2, and the contents are calculated according to the correction factor and the above formula; loganin and dimeric iridoid glycoside are determined based on swertiamin as the reference peak according to the relative retention time, and the contents of loganin and dimeric iridoid glycoside are determined according to the peak area ratio.

[0108] 3. Fingerprint

[0109] At a wavelength of 327 nm, the relative retention time of neochlorogenic acid is 0.720-0.738, the relative retention time of cryptochlorogenic acid is 1.060-1.061, the relative retention time of 3,4-2-O-caffeoylquinic acid is 2.580-0.610, the relative retention time of 3,5-2-O-caffeoylquinic acid is 2.795-2.832, and the relative retention time of 4,5-2-O-caffeoylquinic acid is 2.914-2.948, with chlorogenic acid peak as the standard peak.

[0110] At a wavelength of 240 nm, the relative retention time of loganin is 1.167-1.174, and the relative retention time of dimeric iridoid glycoside is 2.072-2.085, with swertiamin as the standard peak.

[0111] Example 2

[0112] The present application provides a physicochemical identification method in the quality control method of honeysuckle medicinal materials.

[0113] 0.2 g of sample powder was taken, 2 mL of methanol was added, ultrasonic treatment was carried out for 15 min, centrifugation was carried out, and the supernatant was taken to obtain the test solution;

[0114] Developing agent: butyl acetate: formic acid: water (7:2.5:2.5);

[0115] TLC plate: High performance silica gel G 60 F254 glass plate 20 x 10 cm (Merck);

[0116] Development distance: 6 cm;

[0117] Sample application amount: 5 μl;

[0118] Color developing agent: 10 mg / mL diphenyl amino ethyl methanol solution, 50 mg / mL polyethylene glycol 400 methanol solution

[0119] Viewing method: After the TLC plate is developed, it is taken out, heated at 100 ℃ for 3 min, sprayed with 10 mg / mL diphenyl amino ethyl solution, dried, and then sprayed with 50 mg / mL polyethylene glycol 400 solution, dried, and viewed under 366 nm.

[0120] Example 3

[0121] The embodiment of the present application provides a physicochemical identification method in a quality control method of honeysuckle medicinal materials.

[0122] 0.2 g of sample powder to be tested is taken, 2 mL of ethanol is added, ultrasonic treatment is carried out for 15 min, centrifugation is carried out, and the supernatant is taken to obtain a test solution;

[0123] Developing agent: butyl acetate: formic acid: water (7:5:5);

[0124] TLC plate: High performance silica gel G 60 F254 glass plate 20 x 10 cm (Merck);

[0125] Development distance: 6 cm;

[0126] Sample application amount: 5 μl;

[0127] Color developing agent: 10 mg / mL diphenyl amino ethyl methanol solution, 50 mg / mL polyethylene glycol 400 methanol solution

[0128] Viewing method: After the TLC plate is developed, it is taken out, heated at 100 ℃ for 3 min, sprayed with 10 mg / mL diphenyl amino ethyl solution, dried, and then sprayed with 50 mg / mL polyethylene glycol 400 solution, dried, and viewed under 366 nm.

[0129] Example 4

[0130] The embodiment provides specificity investigation of the physicochemical identification method in the quality control method.

[0131] The test sample solution and the control medicinal material solution are prepared according to the preparation method of the test sample solution in Example 1, and the control sample solution is prepared by using methanol as a solvent.

[0132] The TLC chromatography result obtained according to the developing agent, the TLC plate, the color developing agent and the viewing method in Example 1 is as follows Figure 1The thin layer chromatography results obtained by the developing agent, thin layer plate, color developing agent, and viewing method of Example 2 are shown in Figure 1. Figure 2

[0133] Figure 1 Figure 2 In Figure 1, 1 is a chlorogenic acid control, 2 is a jinyinsi glycoside control, 3 is a neochlorogenic acid control, 4 is a cryptochlorogenic acid control, 5 is an isochlorogenic acid A control, 6 is an isochlorogenic acid B control, 7 is an isochlorogenic acid C control, 8 is a honeysuckle control, 9 is a flos lonicerae control, 10 is a flos lonicerae sample, 11 is honeysuckle (ZS-JYH-01), 12 is honeysuckle (ZS-JYH-02), 13 is honeysuckle (ZS-JYH-03), 14 is honeysuckle (ZS-JYH-05), and 15 is honeysuckle (ZS-JYH-06).

[0134] In Figure 1, Figure 1 Figure 2 In the color map of Figure 1, the chlorogenic acid control is a bright blue spot at RF 0.19, the jinyinsi glycoside control is an orange spot at RF 0.23, the neochlorogenic acid control and the cryptochlorogenic acid control are blue fluorescent spots at RF 0.2 and 0.21, respectively, and the isochlorogenic acid A control is a blue fluorescent spot at RF 0.49. The isochlorogenic acid B control and the isochlorogenic acid C control overlap at RF 0.35 and are blue fluorescent spots. The honeysuckle control is a spot at RF 0.2 that is the same color and position as the chlorogenic acid spot, a weak orange spot at RF 0.09 below the honeysuckle control, a spot at RF 0.22 above the honeysuckle control that is the same color and position as the jinyinsi glycoside control, bright blue spots at RF 0.36 and 0.48 that are the same color and position as the isochlorogenic acid B control and the isochlorogenic acid A control, respectively, an orange spot at EF 0.65, and a red fluorescent spot at RF 0.86.

[0135] The flos lonicerae control does not have a jinyinsi glycoside spot of the corresponding orange color above the chlorogenic acid spot, does not have a weak orange spot below the chlorogenic acid spot, and has three blue spots at RF 0.18-0.48 that are the same color and position as the chlorogenic acid spot, the isochlorogenic acid A spot, and the isochlorogenic acid B spot, respectively, and a blue fluorescent spot at RF 0.65.

[0136] The honeysuckle sample solutions all have the same color and position as the honeysuckle control, and the spot outlines in the thin layer chromatography diagram obtained in Example 1 are clearer. The results show that the physicochemical identification method in the quality control method provided in the present application has good specificity.

[0137] Example 5

[0138] ​​​The present example provides the durability investigation of the physicochemical identification method in the above quality control method.

[0139] 1. Stability test

[0140] Prepare the test sample solution and the control medicinal material solution according to the preparation method of the test sample solution in Example 1, and prepare the control substance solution (0.5 mg / mL) using methanol as the solvent. Develop the medicinal material test sample solution, the control substance solution and the control medicinal material solution respectively at 0 h, 24 h and 48 h using the developing agent in Example 1, and develop according to the above thin layer chromatography method. The thin layer chromatograms obtained at 0 h, 24 h and 48 h are shown in Figure 3 , Figure 4 , Figure 5 respectively.

[0141] The results show that there is no obvious difference in the chromatographic profile and spot intensity of the test sample solution and the control substance solution, and the control medicinal material solution within 48 h of storage. It shows that the physicochemical identification method has good stability.

[0142] 2. Different thin layer plates

[0143] Prepare the test sample solution and the control medicinal material solution according to the preparation method of the test sample solution in Example 1, and prepare the control substance solution (0.5 mg / mL) using methanol as the solvent. Develop the medicinal material test sample solution and the control substance solution, and the control medicinal material solution respectively on Merck and MN two high-performance thin layer plates to investigate the influence of different brands of thin layer plates on the thin layer separation effect. The developing agent is the same as in Example 1. The thin layer chromatogram developed on the Merck thin layer plate is shown in Figure 6 , and the thin layer chromatogram developed on the MN thin layer plate is shown in Figure 7 .

[0144] The results show that the two thin layer plates show similar thin layer chromatographic profile spots, and the components are well separated. It shows that the physicochemical identification method has good reproducibility.

[0145] 3. Temperature and humidity influence

[0146] Prepare the test sample solution and the control medicinal material solution according to the preparation method of the test sample solution in Example 1, and prepare the control substance solution (0.5 mg / mL) using methanol as the solvent. Develop the medicinal material test sample solution and the control substance solution, and the control medicinal material solution respectively on different laboratory temperatures to investigate the thin layer chromatography system. The thin layer chromatography conditions are the same as in Example 1. The thin layer chromatogram obtained under the condition of 20.2 ℃, RH 39% is shown in Figure 8 , the thin layer chromatogram obtained under the condition of 24.0 ℃, RH 55% is shown in Figure 9 , and the thin layer chromatogram obtained under the condition of 26.7 ℃, RH 47% is shown in Figure 10 .

[0147] The results show that there is no obvious difference in the profile of the thin layer chromatogram of the control solution and the sample solution developed under different laboratory temperature conditions. It shows that the physical and chemical identification method is basically not affected by temperature and humidity under laboratory conditions.

[0148] 4. Different developing instruments

[0149] The sample solution and the control medicinal material solution were prepared according to the preparation method of the sample solution in Example 1. The control solution was prepared with methanol as the solvent (0.5 mg / mL). The sample solution, the control solution, and the control medicinal material solution were developed in different developing instruments to investigate the influence of different developing instruments on the thin layer separation effect. The thin layer chromatogram obtained in the ADC2-170409 developing instrument is shown in Figure 11 , and the thin layer chromatogram obtained in the ADC2-230010 developing instrument is shown in Figure 12 .

[0150] The results show that there is no obvious difference in the profile of the thin layer chromatogram of the control solution and the sample solution developed in different developing instruments. It shows that the separation effect of the physical and chemical identification method is basically not affected in different developing instruments.

[0151] In the above Figures 3 to 12 , 1 is chlorogenic acid control, 2 is syringin control, 3 is honeysuckle control medicinal material, 4 is honeysuckle (ZS-JYH-01), 5 is honeysuckle (ZS-JYH-02), and 6 is honeysuckle (ZS-JYH-03).

[0152] Example 6

[0153] This example provides the physical and chemical identification results of different batches of honeysuckle medicinal materials.

[0154] Take 0.2 g of sample powder, add 2 mL of methanol, and ultrasonically treat for 15 min. Centrifuge and take the supernatant to obtain the sample solution;

[0155] Developing agent: butyl acetate: formic acid: water (7:5:5);

[0156] Thin layer plate: high efficiency silica gel G 60F254 glass plate 20x10 cm (Merck);

[0157] Development distance: 6 cm;

[0158] Sample amount: 5 μl;

[0159] Color reagent: 10 mg / mL diphenyl amino ethyl methanol solution, 50 mg / mL polyethylene glycol 400 methanol solution;

[0160] Inspection method: after the thin layer plate is developed, heat for 3 min at 100°C, spray with 10 mg / mL diphenyl amino ethyl ester solution, after drying, spray with 50 mg / mL polyethylene glycol 400 solution, dry, and inspect under 366 nm.

[0161] The thin layer chromatogram of each batch of honeysuckle flower is shown in the color chart. Figure 13 Figure 13 Under the ultraviolet lamp at 366 nm, chlorogenic acid reference substance shows a bright blue spot at RF 0.19, and jinyinsi reference substance shows an orange yellow spot at RF 0.23. The honeysuckle flower reference medicinal material shows a spot at RF 0.2 which is consistent with the color and position of the chlorogenic acid spot, shows a weak orange yellow spot below it at RF 0.09, shows a spot above it at RF 0.22 which is consistent with the color and position of the jinyinsi reference substance, shows bright blue spots at RF 0.36 and 0.48 which are consistent with the color and position of isochlorogenic acid B and isochlorogenic acid A, shows an orange yellow spot at RF 0.65, and shows a red fluorescent spot at RF 0.86. The honeysuckle flower reference substance does not show the corresponding orange yellow spot of jinyinsi above the chlorogenic acid spot, does not show a weak orange yellow spot below it, shows three blue spots at RF 0.18-0.48 which are consistent with the color and position of chlorogenic acid, isochlorogenic acid A and isochlorogenic acid B, and shows a blue fluorescent spot at RF 0.65. The honeysuckle flower test sample solution shows a thin layer profile which is consistent with the color and position of the honeysuckle flower reference medicinal material.

[0162] Example 7

[0163] This example provides the content determination results of different batches of honeysuckle flower medicinal materials.

[0164] The content of caffeoylquinic acid compounds and iridoid compounds in 19 batches of honeysuckle flowers was determined according to the method for content determination in Example 1.

[0165] The determination results are as follows:

[0166] Table 3 Content of caffeoylquinic acid compounds in 19 batches of honeysuckle flowers (n=2)

[0167]

[0168]

[0169] Table 4 Content of iridoid compounds in 19 batches of honeysuckle flowers (n=2)

[0170]

[0171] ​The results showed that the content of caffeoylquinic acid compounds in 19 batches of honeysuckle was 3.8-6.431%, which met the requirements. The content of iridoid compounds in ZS-JYH-13, ZS-JYH-16 and ZS-JYH-17 batches was less than 0.80%, which was unqualified. The content of iridoid compounds in the other 16 batches was 0.80%-1.085%, which was qualified.

[0172] Example 8

[0173] This example provides the construction process of the fingerprint of honeysuckle medicinal materials.

[0174] The fingerprint was constructed by high performance liquid chromatography.

[0175] Preparation of test sample solution: 0.1 g of the sample powder to be tested was precisely weighed and placed in a brown volumetric flask. 10 ml of 75% v / v methanol was precisely added, the weight was determined, and the sample was ultrasonically treated for 30 min. After cooling to room temperature, the lost weight was made up with 75% v / v methanol, and the mixture was shaken and filtered through a 0.22 μm microporous filter. The initial filtrate was discarded, and the filtrate was obtained.

[0176] Preparation of reference solution: an appropriate amount of chlorogenic acid reference substance was precisely weighed and placed in a brown volumetric flask. Methanol solution was added for dissolution to prepare a solution containing 0.30 mg per 1 ml, and the chlorogenic acid reference solution was obtained. An appropriate amount of gambirin reference substance was precisely weighed and placed in a brown volumetric flask. Methanol solution was added for dissolution to prepare a solution containing 0.30 mg per 1 ml, and the gambirin reference solution was obtained.

[0177] The chromatographic conditions were the same as those for content determination.

[0178] Fifteen batches of honeysuckle medicinal material samples were determined, and the chromatograms were recorded.

[0179] 1. Fingerprint of caffeoylquinic acid compounds

[0180] The high performance liquid chromatograms of 15 batches of honeysuckle medicinal material samples at a wavelength of 327 nm were imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2004A edition) issued by the National Pharmacopoeia Committee in AIA format. The chromatogram of the ZS-JYH-11 batch sample was used as the reference chromatogram. The median method in the common mode generation method was used, and the time window was 0.1 min. The HPLC fingerprint superimposition chromatogram of 15 batches of honeysuckle medicinal material samples was generated, and the control fingerprint was extracted, as shown in Figure 14Peak 1 is neochlorogenic acid (8.460 min), peak 2 is chlorogenic acid (11.509 min), peak 3 is cryptochlorogenic acid (12.209 min), peak 4 is 3,4-2-O-caffeoylquinic acid (29.889 min), peak 5 is 3,5-2-O-caffeoylquinic acid (32.427 min), and peak 6 is 4,5-2-O-caffeoylquinic acid (33.826 min). Integral parameters: slope sensitivity is 5, peak width is 0.02, minimum peak area is 5, and minimum peak height is 1.

[0181] The relative retention time was calculated with peak 2 as a reference peak. There are 6 characteristic peaks in the above Lonicera japonica fingerprint, and the peak corresponding to the reference chlorogenic acid is S peak. The relative retention time of each characteristic peak and S peak was calculated, and the results are shown in Table 5-1. The similarity was calculated, and the results are shown in Table 5-2.

[0182] Table 5-1 Relative retention time of 15 batches of Lonicera japonica (at 327 nm wavelength)

[0183]

[0184]

[0185] Table 5-2 Similarity of 15 batches of Lonicera japonica (at 327 nm wavelength)

[0186]

[0187] Note: S1: ZS-JYH-11, S2: ZS-JYH-01, S3: ZS-JYH-02, S4: ZS-JYH-03, S5: ZS-JYH-05, S6: ZS-JYH-06, S7: ZS-JYH-07, S8: ZS-JYH-08, S9: ZS-JYH-09, S10: ZS-JYH-10, S11: ZS-JYH-12, S12: ZS-JYH-13, S13: ZS-JYH-14, S14: ZS-JYH-15, S15: ZS-JYH-16, R: 15 batches of generated control fingerprint.

[0188] The results show that the similarity of 15 batches of Lonicera japonica at 327 nm wavelength is all above 0.99; the relative retention time of each characteristic peak is within ±5% of the specified value of the relative retention time of each peak in the United States Pharmacopeia.

[0189] 2. Fingerprint of iridoid compounds

[0190] The high performance chromatogram AIA format of 15 batches of honeysuckle medicinal material samples at 240 nm wavelength was introduced into the "Chinese medicine chromatogram similarity evaluation system" (2004A edition) issued by the State Pharmacopoeia Commission, with the ZS-JYH-11 batch sample chromatogram as the reference chromatogram, using the median method in the common mode generation method, the time window was 0.1 min, the HPLC fingerprint superposition chromatogram of 15 batches of honeysuckle medicinal material samples was generated and the control fingerprint was extracted, see Figure 15 . Peak 1 is daphnin (13.545 min), peak 2 is oxidized loganin (15.805 min), and peak 3 is dimeric iridoid glycoside (28.133 min). Integral parameters: slope sensitivity is 5, peak width is 0.02, minimum peak area is 5, and minimum peak height is 1.

[0191] The relative retention time was calculated with peak 1 as the reference peak. The results are shown in Table 5-1. There are 3 characteristic peaks in the above honeysuckle medicinal material fingerprint, with the corresponding peak of the reference substance daphnin as the S peak, the relative retention time of each characteristic peak and the S peak was calculated, the results are shown in Table 6-1. The similarity was calculated, the results are shown in Table 6-2.

[0192] Table 6-1 Relative retention time of 15 batches of honeysuckle (at 240 nm wavelength)

[0193]

[0194]

[0195] Note: S1: ZS-JYH-11, S2: ZS-JYH-01, S3: ZS-JYH-02, S4: ZS-JYH-03, S5: ZS-JYH-05, S6: ZS-JYH-06, S7: ZS-JYH-07, S8: ZS-JYH-08, S9: ZS-JYH-09, S10: ZS-JYH-10, S11: ZS-JYH-12, S12: ZS-JYH-13, S13: ZS-JYH-14, S14: ZS-JYH-15, S15: ZS-JYH-16, R: 15 batches of control fingerprint.

[0196] The results showed that the similarity of 15 batches of honeysuckle at 240 nm wavelength was above 0.9; the relative retention time of each characteristic peak was within ±5% of the specified value of each peak in the United States Pharmacopoeia.

[0197] Comparative Example 1

[0198] This comparative example provides the results of thin layer chromatography identification of test sample solutions prepared by different solvents and different extraction methods.

[0199] The sample powder to be tested was ZS-JYH-16 batch sample.

[0200] Sample 1: 0.2 g of ZS-JYH-16 batch sample powder was added to 5 mL of methanol and left to stand for 12 hours. After centrifugation, the supernatant was taken as the test sample solution.

[0201] Sample 2: 0.2 g of ZS-JYH-16 batch sample powder was added to 2 mL of methanol and ultrasonically treated for 15 min. After centrifugation, the supernatant was taken as the test sample solution. (i.e. Example 1)

[0202] Sample 3: 0.2 g of ZS-JYH-16 batch sample powder was added to 2 mL of 70% v / v methanol and ultrasonically treated for 15 min. After centrifugation, the supernatant was taken as the test sample solution.

[0203] Sample 4: 0.2 g of ZS-JYH-16 batch sample powder was added to 2 mL of ethanol and ultrasonically treated for 15 min. After centrifugation, the supernatant was taken as the test sample solution. (i.e. Example 3)

[0204] Sample 5: 0.2 g of ZS-JYH-16 batch sample powder was added to 2 mL of toluene and ultrasonically treated for 15 min. After centrifugation, the supernatant was taken as the test sample solution.

[0205] Sample 6: 0.2 g of ZS-JYH-16 batch sample powder was added to 2 mL of ethyl acetate and ultrasonically treated for 15 min. After centrifugation, the supernatant was taken as the test sample solution.

[0206] The developing agent, thin layer plate, color developing agent and viewing method were the same as in Example 1.

[0207] The thin layer chromatogram of each sample is shown in Figure 16 After development, the sample was viewed under a UV lamp at 366 nm. The chlorogenic acid control showed a bright blue spot at RF 0.19, and the matricaria flavone control showed an orange-yellow spot at RF 0.23. The test sample solution prepared by the cold maceration method of the Chinese Pharmacopoeia showed spots of the same color as the chlorogenic acid and matricaria flavone controls at the corresponding RF positions. The extraction solvents of 70% v / v methanol, methanol and ethanol showed similar thin layer chromatograms to the existing Chinese Pharmacopoeia extraction method, and all showed spots of the same color and position as the controls. However, the test sample solution prepared with 70% v / v methanol as the solvent showed slight tailing. The toluene and ethyl acetate extraction thin layer chromatograms did not show the same spots.

[0208] Comparative Example 2

[0209] This comparative example provides the results of thin layer chromatography identification with different developing agents.

[0210] The sample powders to be tested were honeysuckle flower powders and flos lonicerae japonicae powders. The preparation method of the test solution was the same as in Example 1, that is, 0.2 g of the batch ZS-JYH-16 powder was added to 2 mL of methanol, and ultrasonic treatment was performed for 15 min, centrifugation was performed, and the supernatant was taken to obtain the test solution;

[0211] Developing agent 1: ethyl acetate: acetone: formic acid: water (20:3:1.5:1.5);

[0212] Developing agent 2: ethyl acetate: methanol: water (8:2:1).

[0213] The thin layer plate, color developing agent and viewing method were the same as in Example 1. The thin layer chromatography results of the control, flos lonicerae japonicae and honeysuckle flower are shown in Figure 17 、 Figure 18 .

[0214] Figure 17 and Figure 18 , wherein 1 is a chlorogenic acid control, 2 is a syringa glycoside control, 3 is a neochlorogenic acid control, 4 is a cryptochlorogenic acid control, 5 is an isochlorogenic acid A control, 6 is an isochlorogenic acid B control, 7 is an isochlorogenic acid C control, 8 is a honeysuckle flower control medicinal material, 9 is a flos lonicerae japonicae control medicinal material, 10 is a flos lonicerae japonicae sample, 11 is honeysuckle flower (ZS-JYH-01), 12 is honeysuckle flower (ZS-JYH-02), 13 is honeysuckle flower (ZS-JYH-03), 14 is honeysuckle flower (ZS-JYH-05), and 15 is honeysuckle flower (ZS-JYH-06).

[0215] The results show that the thin layer chromatogram obtained with developing agent 1 shows tailing spots, and the thin layer chromatogram obtained with developing agent 2 shows that syringa glycoside is not separated from other spots.

[0216] The above only describes preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement and improvement made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for controlling the quality of honeysuckle medicinal materials, characterized in that: The method comprises performing thin layer chromatography identification on honeysuckle using chlorogenic acid and luteolin as quality control indicators for physical and chemical identification; the preparation method of the test solution in the thin layer chromatography identification comprises: taking a powder of the sample to be tested, ultrasonically extracting it with methanol or ethanol for 14 to 16 minutes, separating the solid and liquid, and taking the liquid phase to obtain the test solution; The developing solvent is ethyl acetate:formic acid:water in a volume ratio of 7:(2.5~5):(2.5~5); The thin layer plate is GF254 silica gel plate; Color development method: After developing the thin layer plate, heat it at 95-105℃ for 2-5 minutes, spray it with 8-12 mg / mL diphenylaminoethyl ester methanol solution, let it dry, then spray it with 48-52 mg / mL polyethylene glycol 400 methanol solution, let it dry, and then examine it at 366 nm. The quality control method further comprises content determination, using high performance liquid chromatography to detect caffeoylquinic acid compounds and iridoid compounds in the sample to be tested; the chromatographic conditions of the high performance liquid chromatography are: Chromatographic column: octadecylsilane bonded silica gel column; Mobile phase A was 0.1% v / v phosphoric acid aqueous solution, and mobile phase B was acetonitrile, and linear gradient elution was performed. The procedure of the linear gradient elution was as follows: Flow rate: 0.65~0.75mL / min; Column temperature: 14~16℃; The detection wavelength for detecting caffeoylquinic acid compounds in the sample to be tested is 327 nm, and the detection wavelength for detecting iridoid compounds in the sample to be tested is 240 nm; The caffeoylquinic acid compound is any one or more of 3,4-2-O-caffeoylquinic acid, 3,5-2-O-caffeoylquinic acid or 4,5-2-O-caffeoylquinic acid; The iridoid compound is any one or both of oxidized strychnine and dimer iridoid glycoside.

2. The quality control method of honeysuckle medicinal material according to claim 1, characterized in that: The quality control indicators also include neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid A, isochlorogenic acid B and isochlorogenic acid C.

3. The quality control method of honeysuckle medicinal material according to claim 1, characterized in that: Each gram of the sample powder was ultrasonically extracted with 8-12 ml of methanol.

4. The quality control method of honeysuckle medicinal material according to claim 3, characterized in that: The ultrasonic time was 15 min.

5. The quality control method of honeysuckle medicinal material according to claim 1, characterized in that: The developing solvent was ethyl acetate:formic acid:water in a volume ratio of 7:5:

5.

6. The quality control method of honeysuckle medicinal material according to claim 1, characterized in that: The quality control method also includes a fingerprint quality control method; The fingerprint quality control method is high performance liquid chromatography, and the chromatographic conditions of the high performance liquid chromatography are: Chromatographic column: octadecylsilane bonded silica gel column; Mobile phase A was 0.1% v / v phosphoric acid aqueous solution, and mobile phase B was acetonitrile, and linear gradient elution was performed. The procedure of the linear gradient elution was as follows: Flow rate: 0.65~0.75mL / min; Column temperature: 14~16℃; The detection wavelength for detecting caffeoylquinic acid compounds in the sample to be tested is 327 nm, and the detection wavelength for detecting iridoid compounds in the sample to be tested is 240 nm.

7. The quality control method of honeysuckle medicinal material according to claim 6, characterized in that: At a wavelength of 327 nm, using the chlorogenic acid peak as the standard peak, the relative retention times of neochlorogenic acid were 0.720-0.738, cryptochlorogenic acid was 1.060-1.061, 3,4-2-O-caffeoylquinic acid was 2.580-0.610, 3,5-2-O-caffeoylquinic acid was 2.795-2.832, and 4,5-2-O-caffeoylquinic acid was 2.914-2.948; at a wavelength of 240 nm, using strychnine as the standard peak, the relative retention times of oxidized strychnine were 1.167-1.174, and those of dimer iridoid glycosides were 2.072-2.

085.

8. The quality control method for honeysuckle medicinal material according to any one of claims 1 to 7, characterized in that: The quality control method also includes property identification and microscopic identification.

9. The quality control method for honeysuckle medicinal material according to any one of claims 1 to 7, characterized in that: The quality control method also includes moisture inspection, total ash inspection and acid-insoluble ash inspection.

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