A method for constructing a characteristic spectrum of a chicory water extract, a detection method and applications thereof

Abstract

The present application relates to the technical field of traditional Chinese medicine, in particular to a construction method, detection method and application of a characteristic spectrum of chicory water extract, wherein the construction method of the characteristic spectrum comprises preparing a chicory water extract test sample solution; taking the test sample solution to perform high performance liquid chromatography analysis, and constructing a characteristic spectrum of the chicory water extract; the high performance liquid chromatography analysis conditions comprise: a chromatographic column with octadecylsilane bonded silica gel as a filler; a mobile phase A is methanol, a mobile phase B is a 0.05%-0.2% formic acid aqueous solution by volume percentage, and a gradient elution program is shown in the present application. The construction method of the characteristic spectrum of the present application has good compatibility by improving the chromatographic conditions, can identify more characteristic peaks, and can mark 19 common characteristic peaks through the common characteristic peaks of multiple batches of characteristic spectra, thereby providing a better basis for comprehensive standard evaluation of the quality of the chicory water extract.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, specifically relating to a method for constructing a characteristic spectrum of chicory water extract, a detection method, and its application. Background Technology

[0002] Chicory is listed in the 2020 edition of the Chinese Pharmacopoeia, and is a commonly used medicinal herb among the Uyghur people. It refers to the dried aerial parts or roots of *Cichorium glandulosum* Boiss. et Huet or *Cichorium intybus* L., both belonging to the Asteraceae family. The aerial parts are harvested in summer and autumn, or the roots are dug up in late autumn. After removing mud and impurities, they are dried. It has the effects of clearing the liver and gallbladder, strengthening the stomach and aiding digestion, and promoting diuresis and reducing swelling. It is used to treat damp-heat jaundice, stomach pain with poor appetite, and edema with scanty urine. Besides its medicinal uses, chicory is widely used in the food industry, animal husbandry, and even the daily chemical industry. It can be used as a vegetable and coffee additive, in the development of health foods, and is also a nutrient-rich, high-quality feed. Therefore, chicory has significant application value in both medicinal and edible aspects.

[0003] Chicory commonly grows along roadsides, in grasslands, on hillsides, in gullies, fields, and wastelands. It is mainly distributed in Northeast, Northwest, and North China, as well as Shandong and Jiangxi provinces, and is found throughout Xinjiang Uygur Autonomous Region. Studies have shown that chicory contains various compounds, including terpenes, flavonoids, phenylpropanoids, phenolic acids, and sugars, and possesses hypoglycemic, anti-inflammatory, and antioxidant properties.

[0004] Fingerprint / feature spectrum, due to its holistic control concept, can comprehensively reflect the types and quantities of chemical components contained in traditional Chinese medicine and its preparations. It can identify differences between medicinal material samples and distinguish related counterfeit and adulterated products, so as to comprehensively evaluate the quality of traditional Chinese medicine.

[0005] However, current research on chicory quality standards is limited. The 2020 edition of the Chinese Pharmacopoeia only includes morphology, microscopic identification, and thin-layer chromatography for chicory medicinal materials and processed slices, lacking content determination indicators such as fingerprint / characteristic chromatograms. This makes it difficult to control its overall quality, thus requiring further improvement of quality standards. Furthermore, chicory is a multi-origin variety; both its aerial parts and roots can be used medicinally. Standardized decoctions and related preparations of chicory processed slices are made from chicory slices through water extraction, concentration, and drying. Compared to raw medicinal slices, they lose their inherent form and medicinal properties, making identification through traditional methods like morphology and odor impossible. This hinders the differentiation of different origins and parts, significantly impacting medication safety due to varying efficacy. The lack of important indicators such as morphology and identification in quality standards, coupled with the absence of effective quality standards for chicory processed decoctions, makes it difficult to effectively control and comprehensively evaluate them.

[0006] Although Chinese patent document CN 118746626 A discloses a method for constructing HPLC characteristic chromatograms of chicory slices, standard decoctions, or formulated granules, using phosphoric acid solution as mobile phase B and separating the components through a specific elution procedure, the selection of the mobile phase in HPLC research requires comprehensive consideration of multiple factors, such as the choice of chromatographic column, elution gradient, detection conditions, target component selection, separation of the target component, and peak shape. These factors must work in harmony to achieve a good extraction effect. Furthermore, phosphoric acid is a tribasic acid that can dissociate stepwise, potentially leading to more complex effects on the ionic strength and buffering capacity of the mobile phase under different pH conditions. Moreover, phosphoric acid is highly acidic, causing significant corrosion to the chromatographic column and exhibiting poor compatibility with mass spectrometry detectors. Ultimately, this method yields a limited number of common characteristic peaks, failing to comprehensively represent the components in chicory and making it difficult to accurately evaluate the overall quality of chicory. Summary of the Invention

[0007] The purpose of this invention is to address the existing technological status by improving liquid chromatography conditions and providing an elution procedure using formic acid as the mobile phase to construct a characteristic spectrum of chicory water extract. This allows for the simultaneous characterization of as many major chemical components (phenolic acids and phenylpropanoids) in chicory as possible, effectively identifying different origins and different parts. Furthermore, the same method is used to quantitatively determine multiple chemical components of chicory water extract, thereby effectively controlling the quality of chicory-related products through different means.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] First, this invention provides a method for constructing a characteristic spectrum of chicory water extract, comprising the following steps:

[0010] Preparation of a test solution for chicory aqueous extract;

[0011] The test solution was subjected to high performance liquid chromatography analysis to construct a characteristic chromatogram of chicory water extract;

[0012] The conditions for the high performance liquid chromatography analysis include: the chromatographic column is packed with octadecylsilane-bonded silica gel; mobile phase A is methanol, mobile phase B is an aqueous solution of formic acid with a volume percentage of 0.05% to 0.2%, and gradient elution is performed according to the procedure described in the table below;

[0013]

[0014] This scheme, by using formic acid as the mobile phase and combining it with optimized elution procedures, offers good compatibility, excellent separation, more stable detection, and the ability to detect more characteristic peaks.

[0015] In this invention, the chicory water extract may contain chicory raw material, chicory slices, etc., and the chicory water extract may include standard decoctions or formula granules, but is not limited thereto.

[0016] In this invention, the test solution of chicory water extract can be obtained by solvent extraction of chicory water extract.

[0017] In this invention, the test solution can be obtained by extracting a standard decoction of chicory slices with an aqueous methanol solution. The raw chicory slices used to prepare the standard decoction of chicory slices are the dried aerial parts or roots of *Cichorium glandulosum* Boiss. et Huet or *Cichorium intybus* L., belonging to the Asteraceae family; conforming to the requirements of the 2020 Chinese Pharmacopoeia.

[0018] In this invention, the standard decoction of chicory slices is prepared by the following method: Chicory slices are decocted twice with water; the decoctions are filtered and rapidly cooled, and the two decoctions are combined; the decoctions are concentrated at 60–70°C under reduced pressure (-0.05–-0.02 MPa) and then freeze-dried under vacuum. For the first decoction, 10–15 times the amount of water is added, and the slices are soaked for 20–40 minutes. After boiling over high heat, the decoction is maintained at a gentle simmer for 20–40 minutes. For the second decoction, 8–12 times the amount of water is added, and the slices are soaked for 20–40 minutes. After boiling over high heat, the decoction is maintained at a gentle simmer for 15–35 minutes. These two decoctions fully extract the characteristic components from the chicory.

[0019] In this invention, the chicory slices used to prepare the standard decoction of chicory slices are usually segments of 5 to 15 mm.

[0020] In this invention, the conditions for high performance liquid chromatography analysis also include: the detection wavelength is 190-400 nm, such as 258 nm, 280 nm, 330 nm, etc., preferably 258 nm.

[0021] In this invention, the conditions for high performance liquid chromatography analysis also include: the column temperature of the chromatographic column is 30-40℃, preferably 36-40℃, such as 36℃, 38℃, 40℃, etc.

[0022] In this invention, the conditions for high performance liquid chromatography analysis further include: mobile phase B is a formic acid aqueous solution with a volume percentage of 0.05-0.2%, such as 0.05% formic acid aqueous solution, 0.1% formic acid aqueous solution, or 0.2% formic acid aqueous solution, preferably 0.2% formic acid aqueous solution, which provides better peak shapes and separation effects.

[0023] In this invention, the conditions for high performance liquid chromatography analysis also include: the flow rate of the mobile phase is 0.8 to 1.2 mL / min, such as 0.8 mL / min, 1.0 mL / min, 1.2 mL / min, etc.

[0024] In this invention, the conditions for high performance liquid chromatography analysis also include: an injection volume of 5–15 μL, preferably 10 μL.

[0025] In this invention, the methanol-water solution used to prepare the test sample solution has a methanol volume percentage of 50% to 80%, such as 50%, 60%, 70%, 80%, etc., but is not limited thereto.

[0026] In this invention, the test solution can be prepared by ultrasound. The ultrasound power can be 200–300 W, such as 250 W; the ultrasound frequency can be 30–50 kHz, such as 40 kHz; and the ultrasound time can be 20–50 min, such as 30 min.

[0027] In this invention, the ratio of chicory decoction standard to methanol aqueous solution used to prepare the test sample solution is 0.1-0.3g:20-50mL, such as 0.1g:20mL, 0.2g:25mL, 0.3g:50mL, etc.

[0028] This invention also includes the preparation of a reference solution, which comprises a reference standard and a solvent. The reference standard comprises one or more of the following: monocaffeoyl tartaric acid, fraxin A, fraxin B, chlorogenic acid, chicoric acid, 3,5-O-dicaffeoylquinic acid, 4,5-O-dicaffeoylquinic acid, isochlorogenic acid B, and quercetin-3-O-β-D-glucuronide. The solvent is methanol.

[0029] In this embodiment of the invention, when the reference solution contains monocaffeoyl tartaric acid, the mass concentration of monocaffeoyl tartaric acid is 45-55 μg / mL, preferably 50 μg / mL.

[0030] When the reference solution contains fraxin, the mass concentration of fraxin is 5–15 μg / mL, preferably 10 μg / mL.

[0031] When the reference solution contains fraxetin, the mass concentration of fraxetin is 45–55 μg / mL, preferably 50 μg / mL.

[0032] When the reference solution contains chlorogenic acid, the mass concentration of chlorogenic acid is 45–55 μg / mL, preferably 50 μg / mL.

[0033] When the reference solution contains chicoric acid, the mass concentration of chicoric acid is 150–250 μg / mL, preferably 200 μg / mL.

[0034] When the reference solution contains 3,5-O-dicaffeoylquinic acid, the mass concentration of 3,5-O-dicaffeoylquinic acid is 45-55 μg / mL, preferably 50 μg / mL.

[0035] When the reference solution contains 4,5-O-dicaffeoylquinic acid, the mass concentration of 4,5-O-dicaffeoylquinic acid is 5-15 μg / mL, preferably 10 μg / mL.

[0036] When the reference solution contains isochlorogenic acid B, the mass concentration of isochlorogenic acid B is 45-55 μg / mL, preferably 50 μg / mL.

[0037] When the reference solution contains quercetin-3-O-β-D-glucuronide, the mass concentration of quercetin-3-O-β-D-glucuronide is 45–55 μg / mL, preferably 50 μg / mL.

[0038] In a preferred embodiment, the reference solution is a mixed reference solution of monocaffeoyl tartaric acid, fraxetin A, fraxetin B, chlorogenic acid, chicoric acid, 3,5-O-dicaffeoylquinic acid, 4,5-O-dicaffeoylquinic acid, isochlorogenic acid B, and quercetin-3-O-β-D-glucuronide, wherein the mass concentration of each reference is: caffeoyl tartaric acid 50 μg / mL, fraxetin A 10 μg / mL, fraxetin B 50 μg / mL, chlorogenic acid 50 μg / mL, chicoric acid 200 μg / mL, 3,5-O-dicaffeoylquinic acid 50 μg / mL, 4,5-O-dicaffeoylquinic acid 10 μg / mL, isochlorogenic acid B 50 μg / mL, and quercetin-3-O-β-D-glucuronide 50 μg / mL.

[0039] The preparation method of the reference solution may include the following steps: Take appropriate amounts of monocaffeoyl tartaric acid reference standard, fraxetin A reference standard, fraxetin B reference standard, chlorogenic acid reference standard, chicoric acid reference standard, 3,5-O-dicaffeoylquinic acid reference standard, 4,5-O-dicaffeoylquinic acid reference standard, isochlorogenic acid B reference standard, and quercetin-3-O-β-D-glucuronide reference standard, accurately weigh them, and add methanol to prepare a mixed solution containing 50 μg monocaffeoyl tartaric acid, 10 μg fraxetin A, 50 μg fraxetin B, 50 μg chlorogenic acid, 200 μg chicoric acid, 50 μg 3,5-O-dicaffeoylquinic acid, 10 μg 4,5-O-dicaffeoylquinic acid, 50 μg isochlorogenic acid B, and 50 μg quercetin-3-O-β-D-glucuronide per ml, as the reference solution.

[0040] The method for constructing characteristic chromatograms of chicory water extract provided by this invention involves preparing test solutions of chicory decoction standard preparations from different batches (at least two batches). These solutions are then analyzed by high-performance liquid chromatography (HPLC) to obtain chromatograms. The chromatograms are imported into a software system for evaluating the similarity of chromatographic fingerprints of traditional Chinese medicine. Common peaks are identified, and characteristic peaks with known components, good peak shape and resolution, and high purity are selected as general characteristic peaks. The method also aims to characterize as many major chemical components (phenolic acids and phenylpropanoids) as possible in chicory and use these characteristic peaks as general characteristic peaks for chicory. For example, in one embodiment of this invention, 14 different batches of chicory decoction standard preparations are used to prepare test solutions.

[0041] In this invention, the characteristic spectrum of the chicory water extract constructed according to the above construction method has a total of 19 characteristic peaks, of which peak 1 is the characteristic peak of monocaffeoyl tartaric acid, peak 2 is the characteristic peak of fraxin A, peak 4 is the characteristic peak of chlorogenic acid, peak 5 is the characteristic peak of fraxin B, peak 8 is the characteristic peak of chicoric acid, peak 10 is the characteristic peak of isochlorogenic acid B, peak 11 is the characteristic peak of 3,5-O-dicaffeoylquinic acid, peak 12 is the characteristic peak of quercetin-3-O-β-D-glucuronide, and peak 15 is the characteristic peak of 4,5-O-dicaffeoylquinic acid.

[0042] In this invention, the characteristic spectrum of the chicory water extract is preferably shown in Figure 7.

[0043] A second aspect of the present invention provides a method for detecting chicory water extract, comprising the following steps:

[0044] Preparation of a test solution for chicory aqueous extract;

[0045] The test solution was subjected to high performance liquid chromatography analysis to detect the content of the indicator components in the test solution and to establish a standard curve for the indicator components. The indicator components include one or more of the following: monocaffeoyl tartaric acid, fraxin, chlorogenic acid, fraxin B, chicoric acid, isochlorogenic acid B, 3,5-O-dicaffeoylquinic acid, quercetin-3-O-β-D-glucuronide, and 4,5-O-dicaffeoylquinic acid.

[0046] The conditions for the high performance liquid chromatography analysis include: the chromatographic column is packed with octadecylsilane-bonded silica gel; mobile phase A is methanol, mobile phase B is an aqueous solution of formic acid with a volume percentage of 0.05% to 0.2%, and gradient elution is performed according to the procedure described in the table below;

[0047]

[0048] The test solution of the chicory water extract can be prepared with reference to the test solution in the above construction method.

[0049] The conditions for high-performance liquid chromatography (HPLC) analysis can also refer to those in the above construction method, such as a detection wavelength of 190–400 nm, such as 258 nm, 280 nm, 330 nm, etc., preferably 258 nm; a column temperature of 30–40 °C, preferably 36–40 °C, such as 36 °C, 38 °C, 40 °C, etc.; mobile phase B is a 0.05–0.2% (v / v) formic acid solution, such as 0.05%, 0.1%, or 0.2%, preferably 0.2%, which provides better peak shapes and separation. The flow rate of the mobile phase can be 0.8–1.2 mL / min, such as 0.8 mL / min, 1.0 mL / min, or 1.2 mL / min. The injection volume can be 5–15 μL, preferably 10 μL.

[0050] This detection method can quantitatively or qualitatively analyze various components in chicory water extract, specifically including one or more of the following: monocaffeoyl tartaric acid, fraxetin, fraxetin B, chlorogenic acid, chicoric acid, 3,5-O-dicaffeoylquinic acid, 4,5-O-dicaffeoylquinic acid, isochlorogenic acid B, and quercetin-3-O-β-D-glucuronide.

[0051] A third aspect of the present invention provides the application of the above-described feature map construction method or detection method in any of the following:

[0052] (1) Evaluate and / or control the quality of chicory water extract;

[0053] (2) Identification of chicory water extract;

[0054] (3) Distinguishing between chicory water extract and chicory root water extract;

[0055] (4) Distinguishing between chicory water extract and hairy chicory water extract.

[0056] Specifically, the quality of chicory water extract can be evaluated and / or controlled through quantitative or qualitative detection. For example, the quality can be evaluated and / or controlled by quantitatively measuring the content of each active ingredient in each batch of samples, or by qualitatively analyzing chicory water extract samples from different parts or origins can be distinguished.

[0057] The present invention has the following beneficial effects:

[0058] (1) The present invention optimizes the program based on the separation and peak shape of multiple complex components in chicory water extract, using methanol-0.2% formic acid as the mobile phase. Methanol has moderate polarity, and its mixing ratio with water can adjust the retention time. The presence of methanol may help adjust the ionic state of the compounds, thereby affecting their retention behavior and improving peak shape. Chicory contains weakly acidic or amphoteric compounds (such as phenolic acids and flavonoid glycosides). Formic acid, by lowering the pH of the mobile phase (≈2.3), can inhibit the dissociation of carboxylic acids or phenolic hydroxyl groups, allowing them to exist in a neutral molecular form, thus enhancing their retention on the reversed-phase column (C18), thereby improving the separation of hydrophobically similar components. Nineteen characteristic peaks can be identified, with good reproducibility and accuracy.

[0059] (2) Combining the preferred detection wavelength range in this application, the use of formic acid can avoid the absorption interference of formic acid itself, thus significantly improving the response of the 19 characteristic peaks. At the same time, the methanol-0.2% formic acid system has a low viscosity, which makes it easier to achieve rapid equilibrium in gradient elution, reduce retention time drift, and ensure the reproducibility of the 19 characteristic peaks in different batch analyses.

[0060] (3) This application studies chicory water extract. Compared with the raw medicinal material, the water extract loses its inherent morphology, and its quality lies in its chemical composition rather than its appearance. Analytical methods can directly detect the content and proportion of effective components, avoiding errors caused by differences in the morphology of the raw medicinal material, thus providing a better basis for the comprehensive standard evaluation of chicory water extract quality. Furthermore, the use of ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS / MS) to assign characteristic peaks demonstrates that the constructed characteristic chromatograms can be used simultaneously for the identification of different origins and parts of chicory. The establishment of this method not only provides a basis for establishing quality standards for chicory water extract but also provides an important reference for establishing quality standards for chicory raw medicinal materials, chicory slices, chicory formulation granules, and related traditional Chinese medicine preparations. Attached Figure Description

[0061] Figure 1 Characteristic spectra of chicory decoction pieces at different detection wavelengths;

[0062] Figure 2 Full-wavelength scan (190nm~400nm) of the characteristic spectrum of standard decoction of chicory slices;

[0063] Figure 3 The results of the investigation of different mobile phase B (aqueous phase) for the characteristic chromatogram of chicory decoction pieces;

[0064] Figure 4 The results of the investigation of different elution gradients for the characteristic chromatograms of chicory decoction pieces;

[0065] Figure 5Total ion chromatogram and ultraviolet absorption chromatogram of the test solution of chicory decoction;

[0066] Figure 6 For the confirmation of characteristic peaks in the characteristic chromatogram of chicory decoction;

[0067] Figure 7(a) shows the characteristic spectrum of chicory decoction at different flow rates;

[0068] Figure 7(b) shows the characteristic spectrum of chicory decoction at different column temperatures;

[0069] Figure 8 This is a superimposed image of the characteristic spectra of 14 batches of chicory decoction pieces;

[0070] Figure 9 Characteristic spectrum of chicory decoction compared to standard decoction;

[0071] Figure 10 A specificity test diagram for the determination of chicory slices in standard decoctions;

[0072] Figure 11 Standard curves for 9 indicator components;

[0073] Figure 12 A comparison of characteristic images of different parts of chicory;

[0074] Figure 13 Comparison of characteristic maps of different chicory origins. Detailed Implementation

[0075] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with specific examples. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. Experimental methods in the following embodiments that do not specify specific conditions are performed according to conventional methods and conditions, or according to the product manual.

[0076] Example 1

[0077] A method for constructing a characteristic spectrum of a standard decoction of chicory slices, the specific implementation steps of which are as follows:

[0078] Instruments, reagents and reagents

[0079] Instruments: Waters high performance liquid chromatograph (e2695, Waters Corporation), Agilent high performance liquid chromatograph (1290, Agilent Technologies), Kromasil C18 column (4.6mm × 250mm, 5μm), analytical electronic balance of 0.001g (ME204E, Mettler Toledo), analytical electronic balance of 0.1m / million (XP26, Mettler Toledo), electric thermostatic water bath (HWS-28, Shanghai Yiheng Technology Co., Ltd.), CNC ultrasonic cleaner (KQ-500DE, Kunshan Ultrasonic Instrument Co., Ltd.), ultrapure water system (Milli-Q Direct, Merck AG).

[0080] Reagents: Ethanol (Xilong Scientific Co., Ltd.) and methanol (Xilong Scientific Co., Ltd.) were both analytical grade; Acetonitrile (Merck, Inc.), methanol (Merck, Inc.), and formic acid (Comio) for liquid chromatography were chromatographic grade, and water was ultrapure water (prepared in the laboratory).

[0081] Test reagents: monocaffeoyl tartaric acid (batch number: 112087-202101, content: 98.3%, China National Institutes for Food and Drug Control), fraxin (batch number: 110740-201806, content: 92.4%, China National Institutes for Food and Drug Control), fraxin B (batch number: 110741-202109, content: 96.0%, China National Institutes for Food and Drug Control), chlorogenic acid (batch number: 110753-202018, content: 96.1%, China National Institutes for Food and Drug Control), chicoric acid (batch number: 111752-202104, content: 99.1%, China National Institutes for Food and Drug Control), 3,5-O- Dicaffeoylquinic acid (batch number: 111782-202208, content: 95.9%, China National Institutes for Food and Drug Control), 4,5-O-dicaffeoylquinic acid (batch number: 111894-202104, content: 95.1%, China National Institutes for Food and Drug Control), isochlorogenic acid B (batch number: wkq21022206, content ≥98%, Sichuan Weikeqi Biotechnology Co., Ltd.), quercetin-3-O-β-D-glucuronide (batch number: DSDDH005501, content ≥99.52%, Chengdu Lemeitian Pharmaceutical Technology Co., Ltd.); the origin information of 14 batches of chicory, 2 batches of chicory root, and 1 batch of hairy chicory medicinal materials is shown in Table 1.

[0082] Table 1. Information on the place of origin of 17 batches of chicory medicinal materials

[0083]

[0084]

[0085] 1.1 Preparation of Standard Decoction of Chicory Slices

[0086] (1) Preparation of chicory slices: Take different batches of chicory raw materials, remove impurities, and cut into 5-15mm segments.

[0087] (2) Preparation of standard decoction of chicory slices: Take 100g of chicory slices, place them in an electric ceramic kettle, add water and decoct twice. For the first decoction, add 12 times the amount of water, soak for 30 minutes, bring to a boil over high heat (500W), then simmer over low heat (200W) for 30 minutes. Filter the decoction through a 350-mesh sieve while hot, and quickly cool the filtrate with cold water. For the second decoction, add 10 times the amount of water, bring to a boil over high heat (500W), then simmer over low heat (200W) for 25 minutes. Filter the decoction through a 350-mesh sieve while hot, and quickly cool the filtrate with cold water. Combine the two decoctions. The decoction was transferred to a 2000ml round-bottom flask and concentrated under reduced pressure at low temperature using a rotary evaporator (temperature: 65℃; vacuum degree: -0.10MPa) to 100ml. Under magnetic stirring, it was dispensed into 10ml brown vials, each with a volume of 2ml. The vials were partially stoppered, and after dispensing, they were transferred to a vacuum freeze dryer for freeze drying. The vials were then removed, and aluminum caps were screwed on to obtain the final product.

[0088] 1.2 Optimization of chromatographic conditions

[0089] 1.2.1 Investigation of Detection Wavelength

[0090] Take an appropriate amount of standard chicory decoction, grind it into a fine powder, and take about 0.2g. Prepare a test solution according to the method in section "1.4". Inject the sample for analysis under the chromatographic conditions in section "1.2.4", and record the absorption spectrum in the range of 190–400 nm. The results are as follows: Figure 1 , Figure 2 As shown, by comparing the chromatograms of different detection wavelengths, it was found that the peak response and information content were richer and the interference was smaller at a wavelength of 258 nm, and the separation was better. Therefore, 258 nm was selected as the detection wavelength.

[0091] 1.2.2 Investigation of the aqueous phase (mobile phase B)

[0092] Take an appropriate amount of standard chicory decoction, grind it into a fine powder, and take about 0.2g. Prepare a test solution according to the method in section "1.4". Inject and analyze the solution under the chromatographic conditions in section "1.2.4". Investigate the separation effect of 0.05% formic acid, 0.1% formic acid, 0.2% formic acid, and 0.1% phosphoric acid (volume percentage) as mobile phase B. The results are as follows. Figure 3 As shown, methanol, as an organic modifier, adjusts the polarity of the mobile phase, affecting the retention time of the compounds, while 0.2% formic acid adjusts the pH, improving the peak shape of acidic compounds, promoting separation, and potentially enhancing the response of the target peak. From Figure 3It was found that compared to 0.05% and 0.1% formic acid solutions, the 0.2% formic acid solution showed significant advantages in resolution and peak shape. Compared to the 0.1% phosphoric acid solution, the 0.2% formic acid solution exhibited more characteristic peaks before 38 minutes, with better peak height and resolution. Furthermore, compared to other mobile phases, when mobile phase B was 0.2% formic acid solution, the separation of each characteristic peak was better after 38 minutes, and the overall peak shape was also better. Therefore, 0.2% formic acid solution was selected as mobile phase B.

[0093] 1.2.3 Gradient Optimization

[0094] A Kromasil C18 (4.6 mm × 250 mm, 5 μm) column was used; methanol was used as mobile phase A and 0.2% formic acid solution was used as mobile phase B. Gradient elution was performed according to the specifications in Table 2. The results are as follows: Figure 4 As shown in the figure. The results show that, comparing five different gradient conditions, the separation of each chromatographic peak in the characteristic chromatogram gradually improves from gradient 1 to gradient 5, which is a progressive process. Compared with gradients 1 and 2, the elution effect of gradients 3-5 is better. Compared with gradient 5, the separation of gradient 3 is worse after 36 min and gradient 4 is worse after 50 min. In particular, one chromatographic peak in gradient 3 between 36 and 38 min is obviously not completely separated, while characteristic peaks 15-19 in gradient 4 are not completely separated after 50 min. The chromatogram obtained by gradient condition 5 has a more uniform distribution, better separation of each chromatographic peak, good peak shape, and stable baseline. Therefore, gradient 5 was finally selected as the analytical condition for the characteristic chromatogram of chicory decoction.

[0095] Table 2 Gradient Elution Table

[0096]

[0097]

[0098] 1.2.4 Determination of chromatographic conditions

[0099] A Kromasil C18 (4.6 mm × 250 mm, 5 μm) column was used; methanol was used as mobile phase A and 0.2% formic acid solution was used as mobile phase B, and gradient elution was performed according to the specifications in Table 3, i.e., gradient 5 in Table 2; the flow rate was 1.0 mL per minute; the column temperature was 38 °C; the detection wavelength was 258 nm; and the injection volume was 10 μL.

[0100] Table 3 Gradient Elution Table

[0101]

[0102] 1.3 Preparation of reference solution

[0103] Take appropriate amounts of monocaffeoyl tartaric acid reference standards, fraxetine A reference standards, fraxetine B reference standards, chlorogenic acid reference standards, chicoric acid reference standards, 3,5-O-dicaffeoylquinic acid reference standards, 4,5-O-dicaffeoylquinic acid reference standards, isochlorogenic acid B reference standards, and quercetin-3-O-β-D-glucuronide reference standards, accurately weigh them, and add methanol to prepare mixed solutions containing 50 μg of monocaffeoyl tartaric acid, 10 μg of fraxetine A reference standards, 50 μg of fraxetine B reference standards, 50 μg of chlorogenic acid, 200 μg of chicoric acid, 50 μg of 3,5-O-dicaffeoylquinic acid, 10 μg of 4,5-O-dicaffeoylquinic acid, 50 μg of isochlorogenic acid B reference standards, and 50 μg of quercetin-3-O-β-D-glucuronide reference standards per ml, as reference solutions.

[0104] 1.4 Preparation of the test solution

[0105] Take an appropriate amount of standard chicory decoction, grind it into a fine powder, take about 0.2g, weigh it accurately, place it in a stoppered conical flask, accurately add 25ml of 70% (volume percentage) methanol, weigh it, sonicate it (power 250W, frequency 40kHz) for 30 minutes, take it out, let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, and take the filtrate to obtain the product.

[0106] 1.5 Determination Method

[0107] Accurately pipette 10 μl each of the reference solution and the test solution into the high-performance liquid chromatograph and determine their properties.

[0108] 1.6 High-resolution mass spectrometry identification of characteristic peaks

[0109] (1) The ultra-high performance liquid chromatography conditions are the same as those under “1.2.4”.

[0110] (2) Mass spectrometry conditions

[0111] Table 4 Mass Spectrometry Parameters

[0112]

[0113] (3) Preparation of the test solution

[0114] Take an appropriate amount of standard decoction of chicory slices (J09), grind it into a fine powder, take about 0.2g, and prepare the test solution according to the test solution preparation method determined under "1.4".

[0115] (4) Sample determination

[0116] Accurately pipette 10 μl of the test solution and inject it into the liquid chromatography-mass spectrometry (LC-MS) instrument. Analyze the test solution under the above-described LC and MS conditions. The total ion chromatogram and UV absorption chromatogram of the test solution are shown below. Figure 5 .

[0117] (5) Results Analysis

[0118] By using precise molecular weight and fragment ion comparison analysis by mass spectrometry and matching with the Thermo Fisher mzVault standard database, nine components were identified in the characteristic spectrum of chicory decoction: monocaffeoyl tartaric acid (peak 1), fraxin A (peak 2), chlorogenic acid (peak 4), fraxin B (peak 5), chicoric acid (peak 8), isochlorogenic acid B (peak 10), 3,5-O-dicaffeoylquinic acid (peak 11), quercetin-3-O-β-D-glucuronide (peak 12), and 4,5-O-dicaffeoylquinic acid (peak 15).

[0119] 1.8 Confirmation of characteristic peaks using reference standards

[0120] (1) Chromatographic conditions

[0121] Same as under item "1.2.4"

[0122] (2) Preparation of reference solution

[0123] Same as item "1.3"

[0124] (3) Preparation of the test solution

[0125] Same as item "1.4"

[0126] (4) Determination method

[0127] Accurately pipette 10 μl each of the reference solution and the test solution into the liquid chromatography column and perform the determination.

[0128] The results show ( Figure 6 The chromatogram of the test sample showed the same chromatographic peak at the corresponding retention time as that of the reference sample, and the UV-Vis 3D absorption curve of the chromatographic peak in the test sample was basically consistent with that of the reference sample, thus confirming that characteristic peak 1 is monocaffeoyl tartaric acid, peak 2 is fraxin A, peak 4 is chlorogenic acid, peak 5 is fraxin B, peak 8 is chicoric acid, peak 10 is isochlorogenic acid B, peak 11 is 3,5-O-dicaffeoylquinic acid, peak 12 is quercetin-3-O-β-D-glucuronide, and peak 15 is 4,5-O-dicaffeoylquinic acid.

[0129] 1.9 Methodological Validation

[0130] 1.9.1 Precision Examination

[0131] Take an appropriate amount of chicory decoction standard (J09), grind it finely, and accurately weigh about 0.2g. Prepare the test solution according to the test solution preparation method under section "1.4". Inject the sample 6 times repeatedly under the chromatographic conditions determined under section "1.2.4". Use the peak corresponding to the chicoric acid reference as the reference peak S. Calculate the relative retention time of each characteristic peak and peak S, and calculate the RSD value. The results show that the RSD of the relative retention time of each characteristic peak and peak S is less than 3%, indicating that the instrument precision is good.

[0132] 1.9.2 Stability Assessment

[0133] Take an appropriate amount of chicory decoction standard (J09), grind it finely, and accurately weigh about 0.2g. Prepare the test solution according to the method for preparing the test solution under section "1.4". Inject the sample for analysis at 0, 2, 8, 12, 16, and 24 hours according to the chromatographic conditions determined under section "1.2.4". Use the peak corresponding to the chicoric acid reference as the reference peak S. Calculate the relative retention time of each characteristic peak and peak S, and calculate the RSD value. The results show that the RSD of the relative retention time of each characteristic peak and peak S is less than 3%, indicating that the test solution is stable within 24 hours.

[0134] 1.9.3 Repeatability Test

[0135] Take an appropriate amount of standard chicory decoction (J09), grind it finely, and accurately weigh about 0.2g. Prepare six parallel solutions according to the test solution preparation method in section "1.4". Inject the solutions under the chromatographic conditions determined in section "1.2.4". Use the peak corresponding to the chicoric acid reference as the reference peak S. Calculate the relative retention time of each characteristic peak and peak S, and calculate the RSD value. The results show that the RSD values ​​of the relative retention times of each characteristic peak and peak S are all less than 3%, indicating that the established method has good reproducibility.

[0136] 1.9.4 Intermediate Precision Examination

[0137] The method was performed by different analysts in different laboratories and on instruments of different brands. An appropriate amount (J09) of the same batch of chicory decoction standard was taken, finely ground, and approximately 0.2 g was accurately weighed. Six parallel aliquots were prepared according to the test solution preparation method in section "1.4". The solutions were injected and analyzed under the chromatographic conditions determined in section "1.2.4". The peak corresponding to the chicoric acid reference was used as the reference peak S. The relative retention times of each characteristic peak and peak S were calculated, and the RSD values ​​were calculated. The results showed that the RSD values ​​of the relative retention times of each characteristic peak were all less than 3%, indicating that the established method can be reproduced under different conditions and with different equipment and laboratories, demonstrating good intermediate precision.

[0138] 1.9.5 Durability Assessment

[0139] An appropriate amount (J09) of the same batch of chicory decoction standard was taken, finely ground, and approximately 0.2 g was accurately weighed. The test solution was prepared according to the method described in section "1.4". The effects of different flow rates (0.8 mL / min, 1.0 mL / min, 1.2 mL / min) (Figure 7(a)) and different column temperatures (36℃, 38℃, 40℃) (Figure 7(b)) on the characteristic chromatogram of the chicory decoction standard were compared. The results showed that, using the peak corresponding to the chicoric acid reference as reference peak S, the relative retention time RSD values ​​of each characteristic peak and peak S were all less than 3%, indicating that small changes in flow rate and column temperature had little effect on the relative retention time of each characteristic peak in the characteristic chromatogram of the chicory decoction standard.

[0140] 1.10 Establishment of Characteristic Chromatic Map of Standard Chicory Decoction

[0141] Fourteen batches of chicory decoction standard samples were collected. Test solutions were prepared according to the method described in section "1.4". 10 μl of each of the test solution and reference solution were precisely pipetted and injected under the chromatographic conditions determined in section "1.2.4". The common peaks of the characteristic chromatograms of the 14 batches of chicory decoction standard samples were identified using the "Software for Evaluating the Similarity of Chromatographic Characteristic Chromatograms of Traditional Chinese Medicine". Nineteen common peaks with known components, good peak shape and resolution, and high purity were selected as the characteristic peaks of the chicory decoction standard samples. The overlay diagram of the characteristic chromatograms of the 14 batches of chicory decoction standard samples is shown below. Figure 8 The characteristic chromatogram of chicory decoction is shown in the standard decoction. Figure 9 Using the chromatographic peak of chicoric acid, which is readily available as the reference standard, as the center, the relative retention time and relative peak area of ​​each characteristic peak and peak S were calculated, and the RSD value was calculated. The experimental results are shown in Tables 5 and 6.

[0142] Table 5. Characteristic chromatograms of standard decoctions made from 14 batches of chicory slices (relative retention time)

[0143]

[0144]

[0145] Table 6. Characteristic chromatograms (relative peak areas) of standard decoctions made from 14 batches of chicory slices.

[0146]

[0147] The results showed that, using chicoric acid as the reference peak S, the relative retention times (RSD) of each characteristic peak and peak S in the characteristic chromatograms of 14 batches of chicory decoction standard preparations ranged from 0.09% to 0.24%, less than 3%, indicating that the relative retention times of each characteristic peak were relatively stable. The average relative retention times of each characteristic peak were: 0.29 (peak 1), 0.33 (peak 2), 0.36 (peak 3), 0.50 (peak 4), 0.56 (peak 5), 0.72 (peak 6), 0.88 (peak 7), 1.09 (peak 9), 1.33 (peak 10), and 1.39 (peak 11). The peak areas were 1.44 (peak 12), 1.59 (peak 13), 1.68 (peak 14), 1.84 (peak 15), 1.91 (peak 16), 2.04 (peak 17), 2.15 (peak 18), and 2.18 (peak 19). The relative peak areas (RSD) of each characteristic peak and the S peak ranged from 22.86% to 73.46%, indicating that the relative peak areas of different batches of samples varied considerably. This suggests that the proportions of the chemical components represented by the characteristic peaks in the standard decoction of chicory slices differed significantly between different batches. Such differences may be related to various factors such as place of origin and growth years.

[0148] Example 2

[0149] A quality testing method for a standard decoction of chicory slices, the specific implementation steps of which are as follows:

[0150] 2.1 Instruments, Reagents and Test Chemicals

[0151] Same as under "Instruments, Reagents and Tests" in Example 1

[0152] 2.2 Chromatographic conditions

[0153] Same as under item "1.2.4"

[0154] 2.3 Preparation of reference solution

[0155] Accurately weigh appropriate amounts of monocaffeoyl tartaric acid reference standards, fraxetin A reference standards, fraxetin B reference standards, chlorogenic acid reference standards, chicoric acid reference standards, 3,5-O-dicaffeoylquinic acid reference standards, 4,5-O-dicaffeoylquinic acid reference standards, isochlorogenic acid B reference standards, and quercetin-3-O-β-D-glucuronide reference standards. Add methanol to prepare mixed solutions containing 50 μg monocaffeoyl tartaric acid, 10 μg fraxetin A reference standard, 50 μg fraxetin B reference standard, 50 μg chlorogenic acid reference standard, 200 μg chicoric acid reference standard, 50 μg 3,5-O-dicaffeoylquinic acid reference standard, 10 μg 4,5-O-dicaffeoylquinic acid reference standard, 50 μg isochlorogenic acid B reference standard, and 50 μg quercetin-3-O-β-D-glucuronide reference standard per 1 ml.

[0156] 2.4 Preparation of the test solution

[0157] Same as item "1.4"

[0158] 2.5 Determination Method

[0159] Same as item "1.5"

[0160] 2.6 Methodological Validation

[0161] 2.6.1 Specificity Examination

[0162] Take an appropriate amount of chicory decoction standard (J09), grind it finely, and accurately weigh about 0.2g. Prepare the test solution according to the method in section "2.4". Accurately pipette 10μl each of the test solution, the reference solution in section "2.3", and the blank solvent, and inject them for analysis according to the chromatographic conditions determined in section "2.2". The results show (see...) Figure 10 The test sample and the reference sample showed the same chromatographic peaks at the corresponding retention times, and the blank solvent did not interfere, indicating that the established method has good specificity.

[0163] 2.6.2 Linearity Examination

[0164] Preparation of reference stock solutions: Accurately weigh 5.632 mg of monocaffeoyl tartaric acid reference standard, 2.143 mg of fraxetine reference standard, 5.268 mg of chlorogenic acid reference standard, 2.089 mg of fraxetine reference standard, 9.558 mg of chicoric acid reference standard, 2.235 mg of isochlorogenic acid B reference standard, 5.347 mg of 3,5-O-dicaffeoylquinic acid reference standard, and quercetin-3-O-β-D-glucan. 5.813 mg of glucuronide reference standard and 2.417 mg of 4,5-O-dicaffeoylquinic acid reference standard were placed in a 10 ml volumetric flask and 70% methanol was added to prepare a mixed reference stock solution containing 553.6256 μg of monocaffeoyl tartaric acid, 198.0132 μg of fraxin A, 507.3084 μg of chlorogenic acid, 205.3487 μg of fraxin B, 939.5514 μg of chicoric acid, 215.9010 μg of isochlorogenic acid B, 507.9650 μg of 3,5-O-dicaffeoylquinic acid, 578.5098 μg of quercetin-3-O-β-D-glucuronide, and 229.3733 μg of 4,5-O-dicaffeoylquinic acid per ml.

[0165] Accurately pipette 0.2 ml, 0.5 ml, 1 ml, 2 ml, and 3 ml of the above-mentioned reference stock solution into 5 ml volumetric flasks, respectively. Add 70% methanol to prepare solutions containing 11.0725 μg, 55.3626 μg, 110.7251 μg, 221.4502 μg, and 332.1754 μg of monocaffeoyl tartaric acid per ml, respectively; and 3.9603 μg, 19.8013 μg, 39.6026 μg, 79.2053 μg, and 118.8079 μg of fraxin per ml, respectively. g; Contains chlorogenic acid 10.1462μg, 50.7308μg, 101.4617μg, 202.9234μg, 304.3850μg; Contains fraxetin 4.1070μg, 20.5349μg, 41.0697μg, 82.1395μg, 123.2092μg; Contains chicoric acid 18.7910μg, 93.9551μg, 187.9103μg, 375.8206μg, 563.7308μg; Contains isochlorogenic acid B 4.3180 μg, 21.5901 μg, 43.1802 μg, 86.3604 μg, 129.5406 μg; containing 10.1593 μg, 50.7965 μg, 101.5930 μg, 203.1860 μg, 304.7790 μg of 3,5-O-dicaffeoylquinic acid; containing quercetin-3-O-β-D-glucan The working solution of the mixed reference standard contained 11.5702 μg, 57.8510 μg, 115.7020 μg, 231.4039 μg, and 347.1059 μg of glucuronide; and 4.5875 μg, 22.9373 μg, 45.8747 μg, 91.7493 μg, and 137.6240 μg of 4,5-O-dicaffeoylquinic acid. Accurately pipette 10 μl each of the above mixed reference standard working solution and reference standard stock solution, and inject according to the chromatographic conditions under section "2.2". Record the peak area. Plot a standard curve with peak area as the ordinate (y) and reference standard concentration as the abscissa (x). The results are shown in Table 7. Figure 11 .

[0166] Table 7. Linear Relationships of the Nine Index Components

[0167]

[0168]

[0169] 2.6.3 Precision Test

[0170] Accurately pipette the mixed reference solution from section "2.3" and inject it six times under the chromatographic conditions described in section "2.2". Record the peak areas of the nine index components: monocaffeoyl tartaric acid, fraxin, chlorogenic acid, fraxin B, chicoric acid, isochlorogenic acid B, 3,5-O-dicaffeoylquinic acid, quercetin-3-O-β-D-glucuronide, and 4,5-O-dicaffeoylquinic acid, and calculate the RSD. The results show that the RSD values ​​of the peak areas of each index component are 1.82%, 2.55%, 1.46%, 1.72%, 0.32%, 2.05%, 1.87%, 0.76%, and 1.66%, respectively, all less than 3%, indicating good instrument precision.

[0171] 2.6.4 Repeatability Test

[0172] Take an appropriate amount (J09) of the same batch of chicory decoction standard powder, grind it into a fine powder, take about 0.2g, accurately weigh it, and prepare 6 parallel samples according to the test sample solution preparation method under section "2.4". Inject the samples for analysis according to the chromatographic conditions determined under section "2.2", and calculate the content and RSD value of the following 9 index components: monocaffeoyl tartaric acid, fraxin, chlorogenic acid, fraxin B, chicoric acid, isochlorogenic acid B, 3,5-O-dicaffeoylquinic acid, quercetin-3-O-β-D-glucuronide, and 4,5-O-dicaffeoylquinic acid. The results showed that the mean contents of monocaffeoyl tartaric acid, fraxin, chlorogenic acid, fraxin B, chicoric acid, isochlorogenic acid B, 3,5-O-dicaffeoylquinic acid, quercetin-3-O-β-D-glucuronide, and 4,5-O-dicaffeoylquinic acid were 1.307 mg / g, 0.167 mg / g, 1.384 mg / g, 0.512 mg / g, 5.961 mg / g, 0.332 mg / g, 1.573 mg / g, 1.653 mg / g, and 0.455 mg / g, respectively, with RSD values ​​of 0.52%, 2.01%, 0.74%, 1.23%, 0.15%, 1.82%, 2.33%, 2.41%, and 1.46%, respectively, all less than 3%, indicating good reproducibility of the analytical method.

[0173] 2.6.5 Stability Test

[0174] Take an appropriate amount of chicory decoction standard (J09), grind it into a fine powder, take about 0.2g, accurately weigh it, and prepare the test solution according to the test solution preparation method under section "2.4". According to the chromatographic conditions determined under section "2.2", inject the sample for analysis at 0 hours, 2 hours, 8 hours, 12 hours, 16 hours and 24 hours respectively. Determine the peak area of ​​nine index components in the test solution: monocaffeoyl tartaric acid, fraxin, chlorogenic acid, fraxin B, chicoric acid, isochlorogenic acid B, 3,5-O-dicaffeoylquinic acid, quercetin-3-O-β-D-glucuronide and 4,5-O-dicaffeoylquinic acid, and calculate the peak area RSD value. The results showed that for the same test solution, the peak area RSD values ​​of each index component were 1.69%, 2.33%, 1.06%, 0.93%, 0.41%, 2.40%, 1.63%, 1.29%, and 2.03% at 0, 2, 8, 12, 16, and 24 hours, respectively, all less than 3%, indicating that the test solution had good stability within 24 hours.

[0175] 2.6.6 Intermediate Precision Examination

[0176] The experiments were conducted by different analysts in different laboratories using instruments of different brands. An appropriate amount (J09) of the same batch of chicory decoction standard was taken, finely ground, and approximately 0.2g was accurately weighed. Six parallel samples were prepared according to the test solution preparation method in section "2.4". The samples were injected and analyzed under the chromatographic conditions determined in section "2.2". The contents and RSD values ​​of nine indicator components—monocaffeoyl tartaric acid, fraxin, chlorogenic acid, fraxin B, chicoric acid, isochlorogenic acid B, 3,5-O-dicaffeoylquinic acid, quercetin-3-O-β-D-glucuronide, and 4,5-O-dicaffeoylquinic acid—were calculated and compared with the results of the repeatability test. The results showed that the average contents of monocaffeoyl tartaric acid, fraxin, chlorogenic acid, fraxin B, chicoric acid, isochlorogenic acid B, 3,5-O-dicaffeoylquinic acid, quercetin-3-O-β-D-glucuronide, and 4,5-O-dicaffeoylquinic acid, determined by different analysts in different laboratories and on different brands of instruments, were 1.412 mg / g, 0.171 mg / g, 1.409 mg / g, 0.536 mg / g, and 6 mg / g, respectively. The concentrations were 0.32 mg / g, 0.303 mg / g, 1.598 mg / g, 1.608 mg / g, and 0.492 mg / g, with RSD values ​​of 1.86%, 1.51%, 0.74%, 1.07%, 0.49%, 2.01%, 1.89%, 1.75%, and 2.56%, respectively, all less than 3%. Compared with the repeatability results, the RSD values ​​of the nine index components were all less than 3%, indicating that the method has good intermediate precision.

[0177] 2.6.7 Sample recovery investigation

[0178] Sample recovery of reference standard mother liquor 1: Accurately weigh 6.701 mg of monocaffeoyl tartaric acid reference standard, 7.259 mg of chlorogenic acid reference standard, 2.643 mg of fraxin B reference standard, 9.558 mg of chicoric acid reference standard, 8.289 mg of 3,5-O-dicaffeoylquinic acid reference standard, 8.318 mg of quercetin-3-O-β-D-glucuronide reference standard, and 2.395 mg of 4,5-O-dicaffeoylquinic acid reference standard, and place in a 10 ml volumetric flask. In a bottle, methanol was added to prepare a mixed solution containing 658.7083 μg of monocaffeoyl tartaric acid, 699.0417 μg of chlorogenic acid, 259.8069 μg of fraxin, 939.5514 μg of chicoric acid, 787.4550 μg of 3,5-O-dicaffeoylquinic acid, 827.8074 μg of quercetin-3-O-β-D-glucuronide, and 227.2855 μg of 4,5-O-dicaffeoylquinic acid per 1 ml.

[0179] Sample recovery of reference standard mother liquor 2: Accurately weigh 2.343 mg of fraxin and 4.356 mg of isochlorogenic acid B reference standard, place them in a 25 ml volumetric flask, and add methanol to prepare a mixed solution containing 86.5973 μg of fraxin and 168.3158 μg of isochlorogenic acid B per ml.

[0180] Accurately pipette 0.5 ml, 1.0 ml, and 1.5 ml of the above-mentioned mother liquors 1 and 2 into stoppered conical flasks, respectively. Perform three parallel groups, three portions per group, for a total of nine portions. Evaporate the solvent. Then, take an appropriate amount of chicory decoction standard (J09), grind it finely, and accurately weigh approximately 0.1 g. Place this amount into the nine conical flasks mentioned above. Prepare nine portions of the test solution according to the method for preparing the test solution under section "2.4". Accurately pipette 10 μl into each portion and perform chromatographic analysis under the conditions under section "2.2". Inject the samples separately to determine the contents of monocaffeoyl tartaric acid, fraxin, chlorogenic acid, fraxin B, chicoric acid, isochlorogenic acid B, 3,5-O-dicaffeoylquinic acid, quercetin-3-O-β-D-glucuronide, and 4,5-O-dicaffeoylquinic acid in the test solution. Calculate the recovery rates for each sample. The results are shown in Table 8. The results showed that the recoveries of monocaffeoyl tartaric acid, fraxin, chlorogenic acid, fraxin B, chicoric acid, isochlorogenic acid B, 3,5-O-dicaffeoylquinic acid, quercetin-3-O-β-D-glucuronide, and 4,5-O-dicaffeoylquinic acid in the standard decoction of chicory were 92.32%–100.19%, 96.46%–103.50%, and 95.99%–100.56%, respectively. The recovery rates were 94.89%–99.27%, 96.25%–101.57%, 97.90%–103.45%, 98.80%–104.08%, 97.14%–102.69%, and 98.70%–101.67%, with RSD values ​​all less than 3%, meeting the requirements of the "Guiding Principles for Validation of Analytical Methods for Drug Quality Standards" in the 2020 edition of the Chinese Pharmacopoeia, indicating that the method had a good recovery rate.

[0181] Table 8. Results of the recovery rate of nine indicators in the standard decoction of chicory slices.

[0182]

[0183]

[0184]

[0185] 2.6.8 Durability Assessment

[0186] Take an appropriate amount (J09) of the same batch of chicory decoction standard, grind it into a fine powder, take about 0.2g, weigh it accurately, and prepare two parallel portions according to the test solution preparation method under section "2.4". Compare the effects of different flow rates (0.8ml / min, 1.0ml / min, 1.2ml / min) and different column temperatures (36℃, 38℃, 40℃) on the content of nine index components in the chicory decoction standard: monocaffeoyl tartaric acid, fraxin, chlorogenic acid, fraxin B, chicoric acid, isochlorogenic acid B, 3,5-O-dicaffeoylquinic acid, quercetin-3-O-β-D-glucuronide, and 4,5-O-dicaffeoylquinic acid. The results showed that the RSD values ​​of the contents of nine index components, namely monocaffeoyl tartaric acid, fraxin, chlorogenic acid, fraxin B, chicoric acid, isochlorogenic acid B, 3,5-O-dicaffeoylquinic acid, quercetin-3-O-β-D-glucuronide, and 4,5-O-dicaffeoylquinic acid, were all less than 3% under different flow rates and column temperatures, indicating that small changes in flow rate and column temperature had little effect on the contents of each index component.

[0187] 2.7 Determination of Chicory Slices Content in Standard Decoction Samples

[0188] Fourteen batches of chicory decoction standard samples were taken. The test solutions were prepared according to the test solution preparation method in section "2.4". The samples were injected and determined according to the chromatographic conditions in section "2.2". The contents of monocaffeoyl tartaric acid, fraxin, chlorogenic acid, fraxin B, chicoric acid, isochlorogenic acid B, 3,5-O-dicaffeoylquinic acid, quercetin-3-O-β-D-glucuronide, and 4,5-O-dicaffeoylquinic acid in the 14 batches of chicory decoction standard samples were calculated respectively. The results are shown in Table 9.

[0189] Table 9. Results of content determination of nine components in 14 batches of chicory decoction standard preparations.

[0190]

[0191]

[0192] Example 3

[0193] A comparison of the characteristic spectra of standard decoctions made from different parts of chicory slices, with the specific implementation steps as follows:

[0194] Two batches of chicory root samples were taken, and chicory root decoction standard preparations (JG01 and JG02) were prepared according to the chicory decoction standard preparation process. The test solvent was prepared according to the test solution preparation method in section "1.4", and the chromatographic conditions determined in section "1.2.4" were used for injection and determination. The characteristic chromatograms of different parts of chicory (aerial parts and roots) were compared: From... Figure 12It can be seen that the chromatographic peak information presented by the characteristic chromatogram of chicory aerial parts is more than that of chicory root. Among them, the content of peak 1 (monocaffeoyl tartaric acid), peak 2 (fraxetin), peak 7, peak 8 (chicoric acid), and peak 19 in chicory aerial parts is significantly higher than that in chicory root. In addition, peak 12 (quercetin-3-O-β-D-glucuronide), peak 15 (isochlorogenic acid B), and peak 16 are absent or present in trace amounts in chicory root.

[0195] In summary, the method established in this study can effectively control the quality of standard decoctions made from chicory slices and can identify different parts of chicory (aerial parts and roots).

[0196] Example 4

[0197] Comparison of characteristic chromatograms of standard decoctions of chicory slices from different origins, with the specific implementation steps as follows:

[0198] One batch of chicory samples was taken, and a standard decoction of chicory slices (MG01) was prepared according to the standard decoction preparation process. The test solvent was prepared according to the method specified in section "1.4," and the chromatographic conditions in section "1.2.4" were used for injection and determination. The characteristic chromatograms of different chicory origins (chicory and chicory fuciformis) were compared: From... Figure 13 It can be seen that the characteristic peaks of chicory are higher than those of wild chicory, indicating that the content of chemical components in chicory is higher than that in wild chicory. In addition, there is a peak 20 with a relatively high response in the characteristic spectrum of wild chicory.

[0199] In summary, it can be seen that the method for constructing the characteristic spectrum of chicory water extract or the method for detecting chicory water extract of the present invention has good repeatability, stability, consistency, precision and durability, and can effectively control the quality of chicory water extract, and can identify chicory from different parts (aerial parts and roots) and different origins.

[0200] The technical features of the embodiments described above can be combined arbitrarily. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as the combination of these technical features does not contradict each other, it should be considered within the scope of this specification. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this patent should be determined by the appended claims.

Claims

1. A method for constructing a characteristic spectrum of chicory water extract, characterized in that, Includes the following steps: A test solution for chicory aqueous extract is prepared; the test solution is obtained by extracting chicory aqueous extract with a methanol aqueous solution; the volume percentage of methanol in the methanol aqueous solution is 50%~80%; The test solution was subjected to high performance liquid chromatography analysis to construct a characteristic chromatogram of chicory water extract; The conditions for the high performance liquid chromatography analysis include: the chromatographic column is packed with octadecylsilane-bonded silica gel; mobile phase A is methanol, mobile phase B is a formic acid aqueous solution with a volume percentage of 0.05%~0.2%, and gradient elution is performed according to the program in the table below; The characteristic chromatogram of the chicory aqueous extract includes 19 common characteristic peaks, of which peak 1 is the characteristic peak of monocaffeoyl tartaric acid, peak 2 is the characteristic peak of fraxin A, peak 4 is the characteristic peak of chlorogenic acid, peak 5 is the characteristic peak of fraxin B, peak 8 is the characteristic peak of chicoric acid, peak 10 is the characteristic peak of isochlorogenic acid B, and peak 11 is 3,5- O -The characteristic peak of dicaffeoylquinic acid, peak 12 is quercetin-3- O - β The characteristic peak of -D-glucuronide, peak 15 is 4,5- O -The characteristic peak of dicaffeoylquinic acid.

2. The construction method as described in claim 1, characterized in that, The conditions for the high-performance liquid chromatography analysis also include: a detection wavelength of 190 nm to 400 nm; And / or, the column temperature of the chromatographic column is 30℃~40℃; And / or, the mobile phase B is an aqueous formic acid solution with a volume percentage of 0.2%; And / or, the gradient elution flow rate is 0.8 mL / min to 1.2 mL / min; And / or, the injection volume is 5 μL to 15 μL.

3. The construction method as described in claim 1, characterized in that, The extraction method includes ultrasonic extraction; And / or, the ratio of the chicory aqueous extract to the methanol aqueous solution is 0.1g~0.3g:20mL~50mL.

4. The construction method as described in claim 3, characterized in that, The power of the ultrasonic extraction is 200W~300W; And / or, the frequency of the ultrasonic extraction is 30kHz~50kHz; And / or, the ultrasonic extraction time is 20 min to 50 min.

5. The construction method as described in claim 1, characterized in that, The method also includes preparing a reference solution comprising a reference standard and a solvent, wherein the reference standard comprises monocaffeoyl tartaric acid, fraxin A, fraxin B, chlorogenic acid, chicoric acid, 3,5- O -Dicaffeoylquinic acid, 4,5- O -Dicaffeoylquinic acid, isochlorogenic acid B, quercetin-3- O - β One or more of -D-glucuronide, wherein the solvent is methanol.

6. A method for detecting chicory water extract, characterized in that, Includes the following steps: Preparation of a test solution for chicory aqueous extract; The test solution was subjected to high performance liquid chromatography analysis to detect the content of the index components in the test solution and to establish a standard curve for the index components. The preparation method of the test solution and the conditions for high-performance liquid chromatography analysis are the same as those described in any one of claims 1 to 5; The indicator components include monocaffeoyl tartaric acid, fraxin, chlorogenic acid, fraxin B, chicoric acid, isochlorogenic acid B, and 3,5- O -Dicaffeoylquinic acid, quercetin-3- O - β -D-glucuronide and 4,5- O -Dicaffeoylquinic acid.

7. The application of the construction method according to any one of claims 1 to 5 or the detection method according to claim 6 in any one of the following: (1) Evaluate and / or control the quality of chicory water extract; (2) Identification of chicory water extract; (3) Distinguishing between chicory water extract and chicory root water extract; (4) Distinguishing between chicory water extract and hairy chicory water extract.

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