A method for constructing a compound fingerprint of chicory and its application

Abstract

The present disclosure discloses a method for constructing a chicory poria complex fingerprint, including (1) preparing a sample solution of a chicory poria complex, (2) preparing a mixed control solution using chlorogenic acid, 6'-O-β-D-glucosylgentiopicroside, swerthiamarin, gentiopicroside, swelloside, 3-O-caffeoylshikimic acid, neoastilbin, astilbin, rutin, isoastilbin, isochlorogenic acid C, quercitrin, kaempferol-3-O-rutinoside, and narcissine, and (3) injecting the sample solution and the mixed control solution into a high-performance liquid chromatograph, respectively, and performing a similarity analysis on the resulting chromatograms to obtain a chicory poria complex fingerprint. The present disclosure also provides a chicory poria complex fingerprint constructed by the construction method and its application to identifying and / or evaluating the quality of chicory poria complexes. The disclosed chicory poria compound fingerprint can comprehensively, objectively, and accurately detect and evaluate the quality of chicory poria compound, thereby ensuring safe and effective medication for patients.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims priority to Chinese Patent Application No. 202311014248.9, filed with the China Patent Office on August 11, 2023, entitled "Method for constructing a chicory compound fingerprint and its fingerprint and application," the entire contents of which are incorporated herein by reference.

[0002] (Technical field) The present disclosure relates to the field of traditional Chinese medicine detection technology, and in particular to a method for constructing a chicory compound fingerprint and its fingerprint and application. [Background technology]

[0003] Chicory-Poria Cocos Formula is a highly condensed and comprehensive summary of the effectiveness of Professor Zhang Bing, a renowned and experienced Chinese herbalist, in treating gouty arthritis and acute and chronic gouty arthritis, and it focuses on a medication strategy that treats metabolic diseases in an integrated manner. Although the symptoms of gout appear in the joints, Professor Zhang Bing believes that the root cause is an abnormality in the body's uric acid metabolism, which is also triggered by external factors. Therefore, Professor Zhang believes that treatment should involve regulating uric acid metabolism by strengthening the spleen and clearing dampness, and intervening in the deposition of uric acid salts by transforming and clearing turbidity. Chicory-Poria Cocos Compound is composed of chicory, Dobutsu Rhizome, Jingbao, Poria Cocos, and Sophora Rhizome. Chicory and Dobutsu Rhizome are principal compounds, serving to strengthen the spleen, dehumidify, and turbidify the body, smoothing joint circulation and regulating uric acid metabolism. Poria Cocos and Jingbao are subordinate compounds, serving to eliminate wind and dampness, relieve pain, and alleviate swelling associated with gouty inflammation. Sophora Rhizome is an auxiliary compound, serving to clear heat, cool blood, and reduce inflammation and pain. Bibliographical and network pharmacology studies have shown that this compound contains multiple chemical components that can affect the production, excretion, and inflammatory pathways of uric acid. Its multi-component, multi-target, and multi-efficacy advantages for prevention and treatment make it a comprehensive anti-gout drug that meets modern understanding of the pathology of gout.

[0004] Currently, research on chicory-poria compound is focused on its clinical application and pharmacological efficacy, but there is a lack of effective methods to thoroughly analyze the influence of differences in the inherent medicinal components on the quality of chicory-poria compound.

[0005] Traditional Chinese medicine fingerprinting is an effective method for identifying traditional Chinese medicine products and evaluating their quality. For example, "HPLC Fingerprint Construction and Mode Recognition Study of Chicory and Hairy Chicory (Cichorium glandulosum Boiss. et Huet)" (Chinese Journal of Traditional Chinese Medicine, Vol. 38, No. 9, pp. 1401-1405) studied the construction method of HPLC fingerprints of chicory and hairy chicory and the application of the obtained HPLC fingerprints. However, traditional Chinese medicine compounds have complex ingredients, and the fingerprint construction method for single-ingredient traditional Chinese medicines in this document cannot be applied to traditional Chinese medicine compounds. Furthermore, in this document, chicory and hairy chicory are extracted using an organic solvent, methanol, with ultrasonic waves, which is inconsistent with clinical practice.

[0006] Therefore, establishing an ultra-high performance liquid chromatography fingerprint feature print for chicory-Poria cocos compound, analyzing it with a traditional Chinese medicine chromatography feature print similarity evaluation system, and establishing a control fingerprint to provide a basis for quality control of chicory-Poria cocos compound is currently an urgent task that needs to be solved. Summary of the Invention [Problem to be solved by the invention]

[0007] The present disclosure aims to provide a method for constructing a chicory-poor lecithin compound fingerprint, and the fingerprint and its applications, so that the quality of the chicory-poor lecithin compound can be comprehensively, objectively, and accurately evaluated. [Means for solving the problem]

[0008] The present disclosure provides: Step (1) of preparing a chicory-poria complex into a sample solution; (2) preparing a mixed control solution using at least two control substances selected from the group consisting of chlorogenic acid, 6'-O-β-D-glucosylgentiopicroside, swerthiamarin, gentiopicroside, swelloside, 3-O-caffeoylshikimic acid, neoastilbin, astilbin, rutin, isoastilbin, isochlorogenic acid C, quercitrin, kaempferol-3-O-rutinoside, and narcissin; and (3) injecting the sample solution and the mixed control solution into a high performance liquid chromatograph, respectively, and performing a similarity analysis on the obtained chromatograms to obtain the chicory poyleurope compound fingerprint.

[0009] Optionally, in step (1), the chicory-Poria cocos complex extract powder or extract powder is extracted with methanol, and the resulting supernatant is filtered to obtain a filtrate, which is used as the sample solution; Preferably, in step (1), the chicory-poria complex extract powder or extract powder is extracted using 70% methanol, and the resulting supernatant is filtered through a 0.22 μm microfiltration membrane to obtain a filtrate, which is used as the sample solution.

[0010] Optionally, in step (2), a mixed control solution is prepared using chlorogenic acid, 6'-O-β-D-glucosylgentiopicroside, swelltiamarin, gentiopicroside, swelloside, 3-O-caffeoylshikimic acid, neoastilbin, astilbin, rutin, isoastilbin, isochlorogenic acid C, quercitrin, kaempferol-3-O-rutinoside, and narcissin.

[0011] Alternatively, in step (2), the control products are mixed and then dissolved in methanol to obtain the mixed control product solution, or the control products are formulated into respective control product solutions using methanol and then mixed to obtain the mixed control product solution; Preferably, in step (2), the control products are mixed in equal proportions and then dissolved in 70% methanol to obtain the mixed control product solution with a content of 1 μg / ml for each control product, or the control products are formulated into 100 μg / ml control product solutions using 70% methanol, and then mixed to obtain the mixed control product solution with a content of 1 μg / ml for each control product.

[0012] Optionally, in step (3), the high performance liquid chromatography column is an ACCQUITY UPLC HSS T3 column, with methanol as mobile phase A and 0.1% formic acid aqueous solution as mobile phase B, the flow rate is 0.3 mL / min, the column temperature is 30°C, the injection volume is 2 μL, and the detection wavelength is 300 nm; Preferably, the high performance liquid chromatography column is an ACCQUITY UPLC HSS T3 column with a dimension of 2.1 x 100 mm and 1.8 μm.

[0013] Optionally, in step (3), the elution conditions of the high performance liquid chromatography are: During the 0-2 min period, the volume fraction of mobile phase A increased from 5% to 10%, and the volume fraction of mobile phase B decreased from 95% to 90%. During the 2-16 min period, the volume fraction of mobile phase A increased from 10% to 40%, and the volume fraction of mobile phase B decreased from 90% to 60%. During the 16-26 min period, the volume fraction of mobile phase A increased from 40% to 54%, and the volume fraction of mobile phase B decreased from 60% to 46%. During 26-26.5 min, the volume fraction of mobile phase A increased from 54% to 95%, and the volume fraction of mobile phase B decreased from 46% to 5%. 26.5-27.5 min, the volume fraction of mobile phase A was 95% and the volume fraction of mobile phase B was 5%; During 27.5-28 min, the volume fraction of mobile phase A decreased from 95% to 5%, and the volume fraction of mobile phase B increased from 5% to 95%.

[0014] The present disclosure further provides a chicory poria complex fingerprint constructed by the above method, comprising 15 fingerprint peaks, wherein the second peak has a relative retention time of 9.64 minutes and corresponds to chlorogenic acid, the third peak has a relative retention time of 10.57 minutes and corresponds to 6'-O-β-D-glucosylgentiopicroside, the fifth peak has a relative retention time of 11.51 minutes and corresponds to gentiopicroside, the eighth peak has a relative retention time of 16.73 minutes and corresponds to neoastilbin, the ninth peak has a relative retention time of 17.16 minutes and corresponds to astilbin, the tenth peak has a relative retention time of 18.50 minutes and corresponds to rutin, and the eleventh peak has a relative retention time of 18.90 minutes and corresponds to isoastilbin.

[0015] The present disclosure further provides an application of the above chicory poria compound fingerprint to distinguishing and / or evaluating the quality of chicory poria compound.

[0016] The present disclosure further provides a method for identifying and / or evaluating the quality of a chicory-poor lecithin compound, comprising comparing a high performance liquid chromatography graph of a to-be-measured chicory-poor lecithin compound with the above-described chicory-poor lecithin compound fingerprint, and identifying those having a similarity of greater than 0.9 as acceptable. [Effects of the Invention]

[0017] From the above technical solutions, it can be determined that the chicory compound fingerprint construction method and its fingerprint and application disclosed herein have at least the following beneficial effects:

[0018] In this disclosure, based on the structural characteristics of the active ingredients contained in chicory poria compound (including flavones, organic acids, iridoid glycosides, etc.), chromatographic analysis conditions such as mobile phase composition, gradient elution step, detection wavelength, column, and column temperature were determined through experiments. Verification through multiple experiments revealed that the chicory poria compound fingerprint constructed using the disclosed method can comprehensively, objectively, and accurately detect and evaluate the quality of chicory poria compound, which is of great significance for ensuring safe and effective medication for patients.

[0019] This paper presents a more comprehensive fingerprint of the complex chicory-poor cocoon compound by ultra-high performance liquid chromatography (UHPLC), which complements the fingerprint studies of the single-ingredient herbal ingredients chicory and hairy chicory. Furthermore, this paper also extracts the chicory-poor cocoon compound by a decoction method, which is closer to clinical use. [Brief explanation of the drawings]

[0020] [Figure 1] FIG. 1 shows the retention times of common fingerprint peaks for 10 lots of chicory / Poria cocos complex in Example 1 of the present disclosure. [Figure 2] FIG. 2 shows the control fingerprints of 10 lots of chicory-poor-coconut compound in Example 1 of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0021] In order to fully understand the objectives, features and effects of the present disclosure, the present disclosure will be described in detail with reference to the following specific embodiments. All parts of the method of the present disclosure other than the following content utilize methods or devices commonly known in the art. Unless otherwise stated, all technical and scientific terms used in the present disclosure have the meanings commonly understood by those skilled in the art.

[0022] In this disclosure, the term "chicory-Poria columbine compound" refers to a herbal composition prepared using chicory, Poria columbine, Dobutsu Rhizome, Sophora Root, and Jewel of the Rose. Preferably, the herbal composition is prepared from 6-18 parts by weight of chicory, 5-15 parts by weight of Poria columbine, 10-60 parts by weight of Dobutsu Rhizome, 5-10 parts by weight of Sophora Root, and 3-10 parts by weight of Jewel of the Rose; more preferably, the herbal composition is prepared from 6-10 parts by weight of chicory, 10-15 parts by weight of Poria columbine, 20-40 parts by weight of Dobutsu Rhizome, 5-10 parts by weight of Sophora Root, and 3-10 parts by weight of Jewel of the Rose; and most preferably, the herbal composition is prepared from 9 parts by weight of chicory, 10 parts by weight of Poria columbine, 30 parts by weight of Dobutsu Rhizome, 6 parts by weight of Sophora Root, and 9 parts by weight of Jewel of the Rose. Specifically, reference may be made to the herbal composition described in the Chinese Patent Application for Invention with application number 202210821181.9, which is incorporated herein by reference in its entirety.

[0023] Chicory-Poria cocos compound contains multiple chemical components that can act on the production, excretion, and inflammatory pathways of uric acid, and has the advantages of multi-component, multi-target, and multi-efficacy prevention and treatment. However, currently, traditional Chinese herbal compound formulations generally only have one or two index components for content control, and quality control is single with few indexes. Therefore, traditional evaluation methods are unable to comprehensively reflect the quality status of multi-component, multi-target chicory-poria cocos compound compositions, which have a large variety of drugs.

[0024] To address these issues, the inventors of the present disclosure have applied HPLC fingerprint technology for traditional Chinese medicines to the evaluation of the quality of chicory-poria columbine compound, and have proposed a method for constructing a fingerprint for chicory-poria columbine compound by optimizing and complementing the chromatographic analysis conditions of the HPLC fingerprint technology, such as the mobile phase composition, gradient elution process, detection wavelength, column, and column temperature, based on the active ingredients and structural characteristics of the chicory-poria columbine compound itself, and screening matching control products.

[0025] In this study, instead of traditional high-performance liquid chromatography, ultra-high-performance liquid chromatography was used to study the fingerprint of chicory-Poria cocos compound, which allowed all active ingredients in the compound to be well separated and detected synchronously, with a large number of peaks appearing and the peaks appearing stably.

[0026] In a first aspect, the present disclosure provides a method for constructing a chicory poria compound fingerprint, the method comprising: (1) preparing a chicory poria compound into a sample solution; (2) preparing a mixed control solution; and (3) injecting the sample solution and the mixed control solution into a high-performance liquid chromatograph and performing a similarity analysis on the obtained chromatogram to obtain a chicory poria compound fingerprint. The method for constructing a chicory poria compound fingerprint of the present disclosure will be described in detail below with reference to specific embodiments.

[0027] (1) Prepare a chicory-poria complex as a sample solution. In the present disclosure, the sample solution may be prepared using a chicory / Poria cocos complex extract powder or extract powder.

[0028] In one preferred embodiment, a sample solution is prepared using chicory-poor cocoon compound extract powder. Specifically, chicory-poor cocoon compound extract powder is precisely weighed and placed in a stoppered Erlenmeyer flask. 70% methanol is added for extraction. The extract is then cooled to room temperature and reweighed. The lost weight is replenished with 70% methanol solution. The supernatant is then filtered through a membrane filter, and the filtrate is collected to obtain a chicory-poor cocoon compound sample solution. The preparation method for chicory-poor cocoon compound extract powder can refer to related technical solutions in the prior art, and the present disclosure does not specifically limit the scope of the present disclosure.

[0029] In another preferred embodiment, the sample solution is prepared using chicory-poria complex extract powder, which is precisely weighed and placed in a stoppered Erlenmeyer flask, diluted to 10 mL with 70% methanol (v / v), diluted to 6 mg / mL, shaken, ultrasonicated for 30 minutes, and filtered. The continuous filtrate is then filtered through a 0.22 μm microfiltration membrane to obtain the sample solution.

[0030] Here, the preparation method of chicory-poor cosme compound extract powder can refer to the relevant technical solutions in the prior art. For illustrative purposes, chicory-poor cosme compound extract powder can be prepared as follows: Chicory, Ginger, Sophora Root, Poria Cosme, and Dobrichia Root slices are accurately weighed according to the proportions of chicory-poor cosme compound, and 12 times the amount of water is added. The mixture is steeped for 30 minutes and then heated to decoction. After boiling, the mixture is decoctioned for 1.5 hours and filtered. The extraction is repeated three times, and the resulting decoctions are combined and concentrated under reduced pressure in a rotary evaporator to a herbal content of 1 g / mL to obtain a composite decoction concentrate. The concentrate is placed in an 80°C water bath and steamed until it loses its fluidity. It is then placed in a vacuum drying box and dried. The drying temperature is maintained at 80°C for 7 days. The concentrate is then removed, allowed to cool to room temperature, and crushed to obtain a compound extract powder.

[0031] (2) Prepare a mixed control solution. Based on the characteristics of the active ingredients of the chicory-poria complex, the present disclosure provides a control product containing at least one compound selected from the group consisting of chlorogenic acid, 6'-O-β-D-glucosylgentiopicroside, swerthiamarin, gentiopicroside, sweroside, 3-O-caffeoylshikimic acid, neoastilbin, astilbin, rutin, isoastilbin, isochlorogenic acid C, quercitrin, kaempferol-3-O-rutinoside, and narcissin. At least two substances are selected, and preferably, the present disclosure prepares a mixed control solution using chlorogenic acid, 6'-O-β-D-glucosylgentiopicroside, swerthiamarin, gentiopicroside, swelloside, 3-O-caffeoylshikimic acid, neoastilbin, astilbin, rutin, isoastilbin, isochlorogenic acid C, quercitrin, kaempferol-3-O-rutinoside, and narcissine as controls.

[0032] In the present disclosure, each control product may be mixed and then dissolved in methanol to obtain a mixed control product solution, or each control product may be formulated into a control product solution using methanol and then mixed to obtain a mixed control product solution.

[0033] In one preferred embodiment, each of the reference products, chlorogenic acid, 6'-O-β-D-glucosylgentiopicroside, swelltiamarin, gentiopicroside, swelloside, 3-O-caffeoylshikimic acid, neoastilbin, astilbin, rutin, isoastilbin, isochlorogenic acid C, quercitrin, kaempferol-3-O-rutinoside, and narcissine, is precisely weighed and placed in a volumetric flask. 70% methanol is added to dissolve, the mixture is adjusted to the specified volume, shaken evenly, and filtered through a microfiltration membrane to obtain a mixed reference product solution.

[0034] In another preferred embodiment, each of the following reference samples is precisely weighed and dissolved in 70% methanol to produce a solution containing approximately 100 μg per ml (i.e., 100 μg / ml reference solution). 1 ml of each reference solution is precisely weighed and placed in a 100 ml volumetric flask. 70% methanol is added up to the fill mark, and the mixture is shaken to obtain a mixed reference solution containing 1 μg of each reference sample per ml. This results in a 1 μg / ml mixture of each reference sample.

[0035] (3) The sample solution and the mixed control solution are injected into a high-performance liquid chromatograph, and a similarity analysis is performed on the obtained chromatogram to obtain a chicory / Poria cocos complex fingerprint. In the present disclosure, the chromatography conditions used are as follows: an ACCQUITY UPLC HSS T3 column is used, and its specifications are preferably an inner diameter of 2.1 mm, a length of 100 mm, and a particle size of 1.8 μm; methanol is used as mobile phase A, and 0.1% aqueous formic acid solution is used as mobile phase B; the flow rate is 0.3 mL / min; the column temperature is 30°C; the injection volume is 2 μL; and the detection wavelength is 300 nm.

[0036] In this disclosure, gradient elution is performed according to the following table: [Table 1]

[0037] The chromatogram obtained by high-performance liquid chromatography was introduced into the Chinese herbal medicine chromatographic fingerprint similarity evaluation system (2012.130723 version) to obtain the chicory poria complex fingerprint.

[0038] In addition, various substances related to the construction method of the present disclosure, such as herbal medicines and control products, can all be purchased on the market.

[0039] In a second aspect, the present disclosure provides a chicory poria compound fingerprint comprising 15 fingerprint peaks.

[0040] Referring to the numbers in FIG. 2, the key peaks of the chicory poria complex fingerprint of the present disclosure include the 2nd, 3rd, 5th, 8th, 9th, 10th, 11th, etc. fingerprint peaks. Here, the second peak has a relative retention time of 9.64 minutes and corresponds to chlorogenic acid, the third peak has a relative retention time of 10.57 minutes and corresponds to 6'-O-β-D-glucosylgentiopicroside, the fifth peak has a relative retention time of 11.51 minutes and corresponds to gentiopicroside, the eighth peak has a relative retention time of 16.73 minutes and corresponds to neoastilbin, the ninth peak has a relative retention time of 17.16 minutes and corresponds to astilbin, the tenth peak has a relative retention time of 18.50 minutes and corresponds to rutin, and the eleventh peak has a relative retention time of 18.90 minutes and corresponds to isoastilbin.

[0041] Because the chicory po rye compound fingerprint of the present disclosure can be used for comprehensive, objective, and accurate detection and evaluation of the quality of chicory po rye compound, in a third aspect, the present disclosure provides a method for distinguishing and / or evaluating the quality of chicory po rye compound, comprising obtaining a high-performance liquid chromatography graph of the chicory po rye compound to be measured using the above-mentioned chromatography conditions and gradient elution process, comparing the high-performance liquid chromatography graph with the chicory po rye compound fingerprint of the present disclosure, and deeming the chicory po rye compound to be an acceptable product if the similarity is greater than 0.9.

[0042] (Example) The present disclosure will be further described below in the form of examples, but the present disclosure is not limited to the scope of the above examples. In the following examples, the experimental methods without specific conditions are selected according to the usual methods and conditions or according to the product instructions.

[0043] Example 1: Establishment and application of chicory and poria complex control fingerprints 1. Equipment, reagents, and chemicals Waters ACQUITY UPLC H-CLASS ultra-high performance liquid chromatography, ACCQUITY UPLC HSS T3 column, ultrasonic cleaner model KQ-5200E (KUNSHAN ULTRASONIC INSTR CO LTD, power 250W, frequency 40kHz), electronic balance model MTW120 (SHENZHEN MEIFU ELECTRONICS CO LTD), and grinder (BEAR ELECTRIC CO LTD).

[0044] Methanol, formic acid (chromatographic purity, Fisher, USA), distilled water (AS Watson Group (Hong Kong) Ltd.), and pure water were provided by the laboratory.

[0045] Ten lots of sections of five types of medicinal ingredients from chicory-Poria cocos compounds were collected from all over China, and their origins and lots are shown in Tables 2-1 and 2-2.

[0046] [Table 2-1]

[0047] [Table 2-2]

[0048] 2. Construction of Chicory Compound Fingerprint (1) Preparation of sample solution Accurately weigh out each piece of lots S1-S10 in Tables 2-1 and 2-2 using 9g of chicory, 10g of Poria cocos, 30g of Dobukuri rhubarb, 6g of Sophora japonica, and 9g of Jingyou to obtain 10 lots of samples. The following procedure is carried out for each of these 10 lots of samples.

[0049] Add 12 times the amount of water, steep for 30 minutes, heat and decoction. After boiling, decoction for 1.5 hours, and filter. Extraction is repeated three times, and the decoction liquid is combined and concentrated under reduced pressure in a rotary evaporator to a medicinal herb content of 1g / mL to obtain a composite decoction concentrate. The composite concentrate is placed in an 80°C water bath and steamed until it loses its fluidity, then placed in a vacuum drying box to dry, maintaining the drying temperature at 80°C, and dried for 7 consecutive days. It is then removed, left to cool to room temperature, and crushed to obtain a composite extract powder. The extract powder was precisely weighed and placed in a stoppered Erlenmeyer flask, and the volume was adjusted to 10 mL with 70% methanol. The solution was diluted to 6 mg / mL, shaken, ultrasonicated for 30 minutes, and filtered. The continuous filtrate was collected and filtered through a 0.22 μm microfiltration membrane to obtain the sample solution.

[0050] (2) Preparation of mixed control solution Weigh out an accurate amount of each of the following reference products: chlorogenic acid, 6'-O-β-D-glucosylgentiopicroside, swertiamarin, gentiopicroside, swelloside, 3-O-caffeoylshikimic acid, neoastilbin, astilbin, rutin, isoastilbin, isochlorogenic acid C, quercitrin, kaempferol-3-O-rutinoside, and narcissine. Dissolve in 70% methanol to prepare a solution containing approximately 100 μg per ml. Accurately measure 1 ml of each of the above reference solutions into the same 100 ml volumetric flask. Add 70% methanol up to the line. Shake evenly to obtain a mixed reference solution (containing 1 μg of each reference product per ml).

[0051] (3) Measurement of Chicory and Poria Coccine Fingerprints by Ultra High Performance Liquid Chromatography The 10 lots of chicory-Poria cocos complex sample solutions prepared in step (1) and the mixed control solution prepared in step (2) were each injected into a high-performance liquid chromatograph.

[0052] Chromatography conditions: The column used was an ACCQUITY UPLC HSS T3 column (inner diameter 2.1 mm, length 100 mm, particle size 1.8 μm). The mobile phase A was methanol and the mobile phase B was 0.1% formic acid aqueous solution. The flow rate was 0.3 mL / min, the column temperature was 30°C, the injection volume was 2 μL, and the detection wavelength was 300 nm. Gradient elution was performed according to the table below.

[0053] [Table 3]

[0054] The fingerprints of the obtained 10 batches of chicory and poria compound sample solutions were derived and input into the Chinese herbal medicine chromatography fingerprint similarity evaluation system (2012.130723 version). Chromatographic peaks that were present in all 10 batches of chicory and poria compound chromatography graphs and had high abundance were selected as common peaks. A control fingerprint of chicory and poria compound was generated using the median calculation method, and the relative retention time and relative peak area of ​​each common peak were calculated. The control fingerprint contained 15 common fingerprint peaks (the discrimination degree of the fingerprints of the 10 batches of sample solutions was all greater than 0.90).

[0055] The retention times of the 15 common fingerprint peaks are shown in FIG. 1, and the control fingerprints obtained in this example are shown in FIG.

[0056] Example 2: Methodological Considerations 1. Accuracy experiment The same batch of chicory and poria compound powder was precisely sampled and prepared according to the method in step (1) of Example 1 above to obtain a sample solution. Injection was repeated six times according to the chromatographic conditions in step (3) of Example 1 above, and the RSD values ​​of the peak areas of common peaks 1, 2, 3, 5, 6, 7, 8, and 10 were calculated. All were less than 4.0%, and the RSDs of the retention times were all less than 1.0%, proving the precision of the instrument.

[0057] 2.Reproducibility test The same batch of chicory and poria compound powder was precisely sampled and prepared in parallel according to the method in step (1) of Example 1 above, to obtain six portions of sample solution. Six portions were injected according to the chromatographic conditions in step (3) of Example 1 above, and the RSD values ​​of the peak areas of common peaks 1, 2, 3, 5, 6, 7, 8, and 10 were calculated. All of them were less than 3.5%, and the RSDs of the retention times were all less than 1.0%, proving the good reproducibility of this method.

[0058] 3. Stability testing The same batch of chicory and poria compound powder was precisely sampled and prepared according to the method in step (1) of Example 1 above to obtain a sample solution. Following the chromatographic conditions in step (3) of Example 1 above, the sample solution was injected and analyzed at 0, 1, 2, 5, 12, and 24 hours after sampling. The RSD values ​​of the peak areas of common peaks 1, 2, 3, 5, 6, 7, 8, and 10 were calculated, and all were less than 3.5%, and the RSDs of the retention times were all less than 1.0%, proving the sample solution to have good stability within 24 hours.

[0059] Based on the above research, this disclosure confirms that by using fingerprint technology to establish a UHPLC fingerprint of chicory-poria compound, the basis of medicinal substances in complex traditional Chinese medicine decoction systems can be quickly and intuitively identified, and by evaluating the quality differences between samples from each batch while referring to chemical identification modes such as grey relational analysis and partial least squares analysis, it can provide reference for the study of important quality attributes of chicory-poria compound and provide valuable reference for the subsequent preparation process, quality control, and product safety and stability evaluation of related formulations.

[0060] Finally, it should be noted that the above are merely specific examples of the present disclosure, and that modifications and variations to the present disclosure are obvious to those skilled in the art. All of these modifications and variations that fall within the scope of the claims of the present disclosure and their equivalent technical scope should be considered to be within the scope of protection of the present disclosure.

[0061] (Addendum) (Appendix 1) Step (1) of preparing a chicory-poria complex into a sample solution; (2) preparing a mixed control solution using at least two control substances selected from the group consisting of chlorogenic acid, 6'-O-β-D-glucosylgentiopicroside, swerthiamarin, gentiopicroside, swelloside, 3-O-caffeoylshikimic acid, neoastilbin, astilbin, rutin, isoastilbin, isochlorogenic acid C, quercitrin, kaempferol-3-O-rutinoside, and narcissin; and (3) injecting the sample solution and the mixed control solution into a high-performance liquid chromatograph, respectively, and performing a similarity analysis on the resulting chromatography graph to obtain a chicory poria compound fingerprint.

[0062] (Appendix 2) In step (1), the chicory-Poria cocos complex extract powder or extract powder is extracted with methanol, and the resulting supernatant is filtered to obtain a filtrate, which is used as the sample solution; Preferably, in step (1), the chicory-poria complex extract powder or extract powder is extracted using 70% methanol, and the resulting supernatant is filtered through a 0.22 μm microfiltration membrane to obtain a filtrate, which is used as the sample solution.

[0063] (Appendix 3) The construction method described in Appendix 1, characterized in that in step (2), a mixed control solution is prepared using chlorogenic acid, 6'-O-β-D-glucosylgentiopicroside, swelltiamarin, gentiopicroside, swelloside, 3-O-caffeoylshikimic acid, neoastilbin, astilbin, rutin, isoastilbin, isochlorogenic acid C, quercitrin, kaempferol-3-O-rutinoside, and narcissin.

[0064] (Appendix 4) In step (2), the control products are mixed and then dissolved in methanol to obtain the mixed control product solution, or the control products are formulated into respective control product solutions using methanol and then mixed to obtain the mixed control product solution; Preferably, in step (2), the control products are mixed in equal proportions and then dissolved in 70% methanol to obtain the mixed control product solution having a content of 1 μg / ml for each control product, or the control products are formulated into a 100 μg / ml control product solution using 70% methanol, and then mixed to obtain the mixed control product solution having a content of 1 μg / ml for each control product.

[0065] (Appendix 5) In step (3), the high performance liquid chromatography column used was an ACCQUITY UPLC HSS T3 column, with methanol as mobile phase A and 0.1% formic acid aqueous solution as mobile phase B, the flow rate was 0.3 mL / min, the column temperature was 30°C, the injection volume was 2 μL, and the detection wavelength was 300 nm. Preferably, the high performance liquid chromatography column is an ACCQUITY UPLC HSS T3 column with a 2.1 x 100 mm, 1.8 μm specification, according to the construction method described in Appendix 1.

[0066] (Appendix 6) In step (3), the elution conditions of the high performance liquid chromatography are as follows: During the 0-2 min period, the volume fraction of mobile phase A increased from 5% to 10%, and the volume fraction of mobile phase B decreased from 95% to 90%. During the 2-16 min period, the volume fraction of mobile phase A increased from 10% to 40%, and the volume fraction of mobile phase B decreased from 90% to 60%. During the 16-26 min period, the volume fraction of mobile phase A increased from 40% to 54%, and the volume fraction of mobile phase B decreased from 60% to 46%. During 26-26.5 min, the volume fraction of mobile phase A increased from 54% to 95%, and the volume fraction of mobile phase B decreased from 46% to 5%. 26.5-27.5 min, the volume fraction of mobile phase A was 95% and the volume fraction of mobile phase B was 5%; 6. The method of claim 5, wherein the volume fraction of mobile phase A is decreased from 95% to 5% and the volume fraction of mobile phase B is increased from 5% to 95% over a period of 27.5-28 minutes.

[0067] (Appendix 7) 7. A chicory complex fingerprint constructed by the method of any one of claims 1 to 6, comprising 15 fingerprint peaks.

[0068] (Appendix 8) 8. The chicory poria compound fingerprint of claim 7, wherein the second peak has a relative retention time of 9.64 minutes and is identified as chlorogenic acid; the third peak has a relative retention time of 10.57 minutes and is identified as 6'-O-β-D-glucosyl gentiopicroside; the fifth peak has a relative retention time of 11.51 minutes and is identified as gentiopicroside; the eighth peak has a relative retention time of 16.73 minutes and is identified as neoastilbin; the ninth peak has a relative retention time of 17.16 minutes and is identified astilbin; the tenth peak has a relative retention time of 18.50 minutes and is identified as rutin; and the eleventh peak has a relative retention time of 18.90 minutes and is identified as isoastilbin.

[0069] (Appendix 9) 1. Use of the chicory poria compound fingerprints described in Appendix 7 or Appendix 8 for the identification and / or assessment of the quality of chicory poria compound.

[0070] (Appendix 10) A method for distinguishing and / or evaluating the quality of chicory / poor lecithin compound, comprising comparing a high performance liquid chromatography graph of a chicory / poor lecithin compound to be measured with the chicory / poor lecithin compound fingerprint described in Appendix 7 or Appendix 8, and classifying those with a similarity of greater than 0.9 as passing.

Claims

1. Step (1) of preparing a chicory / poria complex into a sample solution; (2) preparing a mixed control solution using at least two control products selected from the group consisting of chlorogenic acid, 6'-O-β-D-glucosylgentiopicroside, swerthiamarin, gentiopicroside, swelloside, 3-O-caffeoylshikimic acid, neoastilbin, astilbin, rutin, isoastilbin, isochlorogenic acid C, quercitrin, kaempferol-3-O-rutinoside, and narcissine; A method for constructing a chicory poria compound fingerprint, comprising step (3) injecting the sample solution and the mixed control solution into a high-performance liquid chromatograph, performing a similarity analysis on the obtained chromatography graph, and obtaining a chicory poria compound fingerprint.

2. In step (1), the chicory / Poria cocos complex extract powder or extract powder is extracted with methanol, and the resulting supernatant is filtered to obtain a filtrate, which is used as the sample solution; Preferably, in step (1), the chicory / Poria cocos complex extract powder or extract powder is extracted with 70% methanol, and the resulting supernatant is filtered through a 0.22 μm microfiltration membrane to obtain a filtrate, which is used as the sample solution.

3. The method of claim 1, wherein in step (2), a mixed control solution is prepared using chlorogenic acid, 6'-O-β-D-glucosylgentiopicroside, swelltiamarin, gentiopicroside, swelloside, 3-O-caffeoylshikimic acid, neoastilbin, astilbin, rutin, isoastilbin, isochlorogenic acid C, quercitrin, kaempferol-3-O-rutinoside, and narcissin.

4. In step (2), the control products are mixed and then dissolved in methanol to obtain the mixed control product solution, or the control products are formulated into control product solutions using methanol, and then the mixed control product solution is obtained; Preferably, in step (2), the control products are mixed in equal proportions and then dissolved in 70% methanol to obtain the mixed control product solution with a content of 1 μg / ml for each control product, or the control products are formulated into 100 μg / ml control product solutions using 70% methanol, and then mixed to obtain the mixed control product solution with a content of 1 μg / ml for each control product.

5. In step (3), the high performance liquid chromatography column used was an ACCQUITY UPLC HSS T3 column, with methanol as mobile phase A and 0.1% aqueous formic acid solution as mobile phase B, at a flow rate of 0.3 mL / min, a column temperature of 30°C, an injection volume of 2 μL, and a detection wavelength of 300 nm; The method for constructing a column according to claim 1, wherein the high performance liquid chromatography column is preferably an ACCQUITY UPLC HSS T3 column with a dimension of 2.1 x 100 mm and a thickness of 1.8 μm.

6. In step (3), the elution conditions of the high performance liquid chromatography are as follows: During 0-2 minutes, the volume fraction of mobile phase A increased from 5% to 10%, and the volume fraction of mobile phase B decreased from 95% to 90%. During 2-16 minutes, the volume fraction of mobile phase A increased from 10% to 40%, and the volume fraction of mobile phase B decreased from 90% to 60%. During 16-26 minutes, the volume fraction of mobile phase A increased from 40% to 54%, and the volume fraction of mobile phase B decreased from 60% to 46%. During 26-26.5 minutes, the volume fraction of mobile phase A increased from 54% to 95%, and the volume fraction of mobile phase B decreased from 46% to 5%. 26.5-27.5 min, the volume fraction of mobile phase A was 95% and the volume fraction of mobile phase B was 5%, 6. The method of claim 5, wherein the volume fraction of mobile phase A is decreased from 95% to 5% and the volume fraction of mobile phase B is increased from 5% to 95% during 27.5-28 minutes.

7. A Chicory Poria complex fingerprint constructed by the method of any one of claims 1 to 6, comprising 15 fingerprint peaks.

8. The chicory Poria Coccinea compound fingerprint of claim 7, wherein the second peak has a relative retention time of 9.64 minutes and corresponds to a chemical substance called chlorogenic acid, the third peak has a relative retention time of 10.57 minutes and corresponds to a chemical substance called 6'-O-β-D-glucosyl gentiopicroside, the fifth peak has a relative retention time of 11.51 minutes and corresponds to a chemical substance called gentiopicroside, the eighth peak has a relative retention time of 16.73 minutes and corresponds to a chemical substance called neoastilbin, the ninth peak has a relative retention time of 17.16 minutes and corresponds to a chemical substance called astilbin, the tenth peak has a relative retention time of 18.50 minutes and corresponds to a chemical substance called rutin, and the eleventh peak has a relative retention time of 18.90 minutes and corresponds to a chemical substance called isoastilbin.

9. 9. The application of the chicory poria compound fingerprint according to claim 7 or 8 to the identification and / or evaluation of the quality of the chicory poria compound.

10. A method for distinguishing and / or evaluating the quality of a chicory / poor lecithin compound, comprising comparing a high-performance liquid chromatography graph of a chicory / poor lecithin compound to be measured with the chicory / poor lecithin compound fingerprint described in claim 7 or 8, and classifying those having a similarity of greater than 0.9 as passing.

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