Fingerprint of panax quinquefolium extract and construction method thereof

Abstract

This application relates to the field of quality control of traditional Chinese medicine extracts, specifically disclosing a fingerprint chromatogram of American ginseng extract and its construction method. The invention employs a silica gel column with gradient elution using a phosphoric acid aqueous solution and acetonitrile as the mobile phase, achieving well-separated and highly characteristic chromatograms. Based on this method, a standard fingerprint chromatogram containing at least 29 common characteristic peaks was constructed by analyzing the aqueous and alcoholic extracts of American ginseng separately. This standard chromatogram comprehensively reflects the overall chemical characteristics of American ginseng extracts obtained from different extraction solvents, achieving a unified and objective evaluation of products from different extraction processes. This solves the technical problem in the prior art of horizontally controlling the quality of American ginseng extracts due to different extraction methods.

Description

Technical Field

[0001] This application relates to the field of quality control of traditional Chinese medicine extracts, and more specifically, it relates to a fingerprint spectrum of American ginseng extract and its construction method. Background Technology

[0002] American ginseng is a precious traditional Chinese medicine widely used in health care and medical fields. Its active ingredients are mainly various ginsenosides (such as ginsenosides Rg1, Re, Rb1, etc.). To ensure the stable quality, efficacy, and safety of American ginseng-related products, it is crucial to establish a scientific, comprehensive, and objective quality control method.

[0003] Traditional quality control methods typically employ qualitative and quantitative analysis of one or a few known indicator components. However, traditional Chinese medicine and its extracts constitute complex chemical systems, and their efficacy is often the result of the synergistic effects of multiple components. Relying solely on a few indicator components for quality control makes it difficult to comprehensively reflect the overall chemical characteristics and intrinsic quality consistency of the product. This can lead to situations where different batches meet the requirements for the content of indicator components, but other components show significant differences, thus affecting the efficacy and safety of the final product.

[0004] Fingerprint spectroscopy is an effective means to solve the above problems. It obtains a spectrum characterizing the overall chemical properties of a sample by analyzing it under specific conditions. By comparing the similarity of fingerprint spectra, the authenticity, stability, and consistency of the sample can be comprehensively evaluated. Currently, although fingerprint spectroscopy is widely used in the quality control of various traditional Chinese medicinal materials, a unified, specific, and well-separated fingerprint spectroscopy detection method is still lacking for American ginseng extract, especially for extracts obtained through different extraction methods.

[0005] Therefore, there is an urgent need in this field for a fingerprint chromatographic detection method that is highly specific, has good chromatographic peak separation, stable baseline, and can comprehensively reflect the overall characteristics of the chemical components of American ginseng extract, in order to fill the gap in the existing technology and provide a more scientific and comprehensive technical basis for the quality control of American ginseng extract. Summary of the Invention

[0006] To address the aforementioned issues, this application provides a fingerprint spectrum of American ginseng extract and its construction method.

[0007] This application provides the following technical solution:

[0008] In a first aspect, this application provides a fingerprint spectrum of an American ginseng extract, the fingerprint spectrum containing at least 29 common characteristic peaks, the fingerprint spectrum being determined by the following method:

[0009] American ginseng was extracted by heating and reflux with water or an ethanol solution of a certain concentration to prepare American ginseng extract and obtain the test solution.

[0010] The test solution was subjected to high performance liquid chromatography (HPLC) to obtain HPLC chromatograms of different extracts;

[0011] The high-performance liquid chromatograms of different extracts were evaluated for fingerprint similarity to obtain standard fingerprints;

[0012] The chromatographic conditions for the high-performance liquid chromatography (HPLC) detection include:

[0013] Chromatographic column: A chromatographic column packed with octadecylsilane-bonded silica gel;

[0014] Mobile phase: Phase A is an aqueous solution containing phosphoric acid, and Phase B is acetonitrile, for gradient elution;

[0015] Detection wavelength: 200nm-400nm;

[0016] Column temperature: 20℃-40℃;

[0017] Injection volume: 5-15 μL;

[0018] Flow rate: 0.8-1.2 mL / min.

[0019] Furthermore, the process parameters for the above-mentioned heating reflux extraction are: material-to-liquid ratio 1:8-12, extraction time 0.8-1.2h / time, extraction times 2-3 times, and no soaking before extraction.

[0020] Furthermore, the gradient elution procedure described above is as follows:

[0021] From 0 to 15 minutes, phase A decreased from 90% to 85%, and phase B decreased from 10% to 15%.

[0022] Over 15-35 minutes, phase A decreased from 85% to 76%, and phase B decreased from 15% to 24%.

[0023] Over 35-47 minutes, phase A decreased from 76% to 73%, and phase B decreased from 24% to 27%.

[0024] Over 47-70 minutes, phase A decreased from 73% to 63%, and phase B decreased from 27% to 37%.

[0025] Over 70-110 minutes, phase A decreased from 63% to 25%, and phase B decreased from 37% to 75%.

[0026] 110-130 min, Phase A changes from 25% to 0%, Phase B changes from 75% to 100%;

[0027] 130-132 min, phase A changes from 0% to 90%, phase B changes from 100% to 10%.

[0028] Furthermore, the chromatographic peaks of the above-mentioned common characteristic peaks are referenced by the chromatographic peaks of ginsenoside Rg1, ginsenoside Re and ginsenoside Rb1, and the relative retention times of each characteristic peak are within ±2.0% of the specified value.

[0029] Furthermore, the retention times of the aforementioned common characteristic peaks under high-performance liquid chromatography (HPLC) conditions are as follows: 6.3 min, 6.9 min, 8.2 min, 8.7 min, 11.1 min, 12.0 min, 12.4 min, 13.7 min, 14.0 min, 17.5 min, 20.7 min, 23.3 min, 34.5 min, 40.8 min, 43.4 min, 43.9 min, 66.2 min, 67.5 min, 68.0 min, 68.8 min, 70.2 min, 71.6 min, 73.5 min, 74.9 min, 75.6 min, 99.5 min, 103.8 min, and 113.5 min, with an allowable deviation of ±0.5 min for each retention time. Furthermore, phase A is a phosphoric acid aqueous solution with a volume fraction of 0.05-0.2%.

[0030] Furthermore, the chromatographic conditions for the above high-performance liquid chromatography detection are as follows:

[0031] Mobile phase: Phase A is a 0.1% (v / v) aqueous solution of phosphoric acid, and Phase B is acetonitrile;

[0032] Flow rate: 1.0 mL / min;

[0033] Column temperature: 30℃;

[0034] Injection volume: 10 μL;

[0035] Detection wavelength: 210nm.

[0036] Secondly, this application provides a method for constructing a fingerprint spectrum of American ginseng extract, comprising the following steps:

[0037] American ginseng and its extract were subjected to ultrasonic extraction with 30-80 vol% alcohol solution to obtain the test solution.

[0038] The test solution was analyzed under the above-mentioned high-performance liquid chromatography conditions to obtain high-performance liquid chromatograms of different extracts;

[0039] The high-performance liquid chromatograms of different extracts are evaluated for fingerprint similarity to obtain the standard fingerprint chromatogram.

[0040] In summary, this application has the following beneficial effects:

[0041] 1. This application breaks through the technical barrier that makes it difficult to make horizontal comparisons of traditional methods due to different extraction solvents. It provides a unified and comparable quality evaluation standard for American ginseng extracts produced by different processes such as water extraction or alcohol extraction, and realizes consistent quality control of American ginseng extracts from multiple sources and processes.

[0042] 2. The standard fingerprint spectrum obtained in this application is rich in information and highly characteristic. It can not only characterize a few known saponins, but also comprehensively reflect the types and relative contents of many unknown components in American ginseng extract. Compared with traditional methods that only detect 1-2 indicator components, this invention can more comprehensively and realistically reflect the overall chemical characteristics of the product, effectively avoid the quality risk of overgeneralization, and ensure the stability of the intrinsic quality of different extracts.

[0043] 3. This application quantifies and evaluates complex chemical composition information using a single, clear numerical indicator: "similarity." This transforms quality control from subjective judgment relying on expert experience to objective evaluation based on data and software, significantly improving the scientific rigor, consistency, and efficiency of the evaluation. Enterprises or testing institutions can use this as a standard to quickly and accurately control the quality of American ginseng extract. Attached Figure Description

[0044] Figure 1 These are liquid chromatograms of American ginseng extracts extracted with different solvents in this application;

[0045] Figure 2 This is a matching spectrum of American ginseng extracts extracted with different solvents and American ginseng raw materials in this application;

[0046] Figure 3 These are the results of the fingerprint spectrum similarity evaluation of different American ginseng extracts. Detailed Implementation

[0047] The embodiments of the present invention will be described in detail below with reference to the examples. However, those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention. Specific conditions not specified in the examples shall be carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0048] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.

[0049] Example

[0050] This embodiment provides a method for determining the fingerprint spectrum of American ginseng extract and establishing its standard fingerprint spectrum. The specific steps are as follows:

[0051] 1. Experimental Materials and Equipment

[0052] Materials: American ginseng, a Chinese medicinal herb, identified as the dried root of Panax quinquefolius L., a plant of the Araliaceae family.

[0053] Reference standards: Ginsenoside Rg1 (purity: 99.63%), Ginsenoside Re (purity: 98.6%), and Ginsenoside Rb1 (purity: 99.41%), purchased from a professional standard material production unit.

[0054] Reagents: Acetonitrile (chromatographic grade), phosphoric acid (chromatographic grade), ethanol (analytical grade), methanol (chromatographic grade), and ultrapure water.

[0055] Instruments: High performance liquid chromatograph (equipped with DAD detector or ultraviolet detector), electronic analytical balance, ultrasonic cleaner, reflux extraction device, rotary evaporator.

[0056] 2. Preparation of the test solution

[0057] American ginseng was pulverized and passed through a No. 3 sieve. Approximately 280g of the powder was accurately weighed and extracted under reflux using different extraction solvents. Specific process parameters are shown in Table 1. After extraction, the two extracts were combined, filtered, and the filtrate was concentrated under reduced pressure at 50℃ to a density of 1.1-1.2. The filtrate was then spray-dried, pulverized, and sieved to obtain five American ginseng extracts (numbered S2-S6). Raw American ginseng was used as a control, and the pulverized extract was used as sample S1.

[0058] Table 1. Process parameters for hot reflux extraction of American ginseng

[0059]

[0060] 3. Preparation of reference solution

[0061] Accurately weigh appropriate amounts of ginsenoside Rg1, Re, and Rb1 reference standards, dissolve and dilute them in methanol to prepare single reference standard stock solutions with concentrations of 0.5660 mg / mL, 0.5800 mg / mL, and 0.4840 mg / mL, respectively. These can be diluted to form mixed reference standard solutions as needed.

[0062] 4. Chromatographic conditions

[0063] Column: wondasil C 18 (250mm × 4.6mm, 5.0μm);

[0064] Mobile phase: Phase A is 0.1% phosphoric acid aqueous solution (v / v), and Phase B is acetonitrile;

[0065] Flow rate: 1 ml / min;

[0066] Column temperature: 30℃;

[0067] Injection volume: 10 μl;

[0068] Detection wavelength: 210nm;

[0069] Gradient elution procedure: as shown in Table 2.

[0070] Table 2. Gradient elution procedure

[0071]

[0072] 5. Measurement:

[0073] Take 5.0g of the above-mentioned American ginseng medicinal material, and 5g of the equivalent raw medicinal material of American ginseng water extract, American ginseng 30% ethanol extract, American ginseng 50% alcohol extract, American ginseng 70% ethanol extract and American ginseng 90% ethanol extract. Add 25mL of 50% ethanol to each, sonicate for 30min, shake well, filter, and determine according to the above chromatographic conditions. The main components are controlled to be ginsenoside Rg1, ginsenoside Re and ginsenoside Rb1.

[0074] 6. Fingerprint mapping and similarity evaluation

[0075] High performance liquid chromatography (HPLC) results of different American ginseng extracts are as follows: Figure 1 As shown in the liquid chromatography chromatograms of American ginseng raw material, water extract, 30% ethanol extract, 50% ethanol extract, 70% ethanol extract, and 90% ethanol extract, the extraction rates of the main components ginsenosides Rg1, Rb1, and Re are 70% ethanol > 50% ethanol > 30% ethanol ≈ water extraction > 90% ethanol extraction. A total of 29 common peaks were found in the liquid chromatography peaks of the American ginsenosides obtained from the above-mentioned medicinal materials using different extraction methods. Among them, peaks 15, 16, and 17 represent ginsenoside Rg1, ginsenoside Re, and ginsenoside Rb1, respectively.

[0076] The HPLC chromatogram data files of the six American ginseng extracts (S1-S6) were imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" software for further comparison of the chromatographic results of American ginseng raw material and American ginseng extracts obtained through different methods, generating a control fingerprint (R). The results are as follows: Figure 2 As shown.

[0077] The software calculated the similarity between the liquid chromatography spectra of six American ginseng extract samples (S1-S6) and the generated control fingerprint spectra (R). The results are as follows: Figure 3 As shown, the similarity of different extract samples was above 0.974, indicating good correlation. This suggests that the fingerprint spectra of ginsenosides obtained by different extraction methods are similar, and the intrinsic quality of ginseng extracts obtained by different extraction methods did not change significantly.

[0078] The generated reference fingerprint chromatogram (R) is the standard fingerprint chromatogram of this invention. This chromatogram identifies 29 common characteristic peaks, and the relative retention times of these characteristic peaks are referenced to the chromatographic peaks of ginsenoside Rg1, ginsenoside Re, and ginsenoside Rb1. The relative retention times of each common peak are within ±2.0% of the specified value.

[0079] The absolute retention times (average values) of each common peak are approximately 6.3 min, 6.9 min, 8.2 min, 8.7 min, 11.1 min, 12.0 min, 12.4 min, 13.7 min, 14.0 min, 17.5 min, 20.7 min, 23.3 min, 34.5 min, 40.8 min, 43.4 min (ginsenoside Rg1), 43.9 min (ginsenoside Re), 66.2 min (ginsenoside Rb1), 67.5 min, 68.0 min, 68.8 min, 70.2 min, 71.6 min, 73.5 min, 74.9 min, 75.6 min, 99.5 min, 103.8 min, and 113.5 min, respectively. The allowable deviation for each retention time is ±0.5 min.

[0080] Specifically, the absolute retention times of the common peaks of each American ginseng extract are shown in Table 3:

[0081] Table 3. Chromatographic data of common peaks in American ginseng extract

[0082]

[0083] The results of this embodiment show that, using the chromatographic conditions described in this invention (C18 column, 0.1% phosphoric acid water-acetonitrile gradient elution, detection wavelength 210 nm, etc.), the fingerprint chromatograms obtained for detecting the water extract and alcohol extract of American ginseng have extremely high similarity to the standard fingerprint chromatogram (R) (average similarity > 0.97).

[0084] This fully demonstrates that the fingerprint spectroscopy determination method and standard fingerprint spectrum established in this invention can be applied simultaneously to the quality control of both American ginseng water extract and ethanol extracts of a certain concentration (such as 30%-90% ethanol extract), achieving a unified and objective evaluation of products from different extraction processes. It solves the technical problem mentioned in the background art of difficulty in horizontal quality control due to different extraction methods. For the quality evaluation of the American ginseng extract sample to be tested, only a similarity comparison with the standard fingerprint spectrum (R) of this application is required. Furthermore, this also further illustrates that using this fingerprint spectroscopy technology can provide a rapid and accurate quality standard for the quality control of American ginseng extract.

[0085] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A fingerprint spectrum of an American ginseng extract, characterized in that, The fingerprint spectrum contains at least 29 common characteristic peaks, and the fingerprint spectrum is determined by the following method: American ginseng was extracted by heating and reflux with water or an ethanol solution of a certain concentration to prepare American ginseng extract and obtain the test solution. The test solution was subjected to high performance liquid chromatography (HPLC) to obtain HPLC chromatograms of different extracts; The high-performance liquid chromatograms of different extracts were evaluated for fingerprint similarity to obtain standard fingerprints; The chromatographic conditions for the high-performance liquid chromatography (HPLC) detection include: Chromatographic column: A chromatographic column packed with octadecylsilane-bonded silica gel; Mobile phase: Phase A is an aqueous solution containing phosphoric acid, and Phase B is acetonitrile, for gradient elution; Detection wavelength: 200nm-400nm; Column temperature: 20℃-40℃; Injection volume: 5-15 μL; Flow rate: 0.8-1.2 mL / min.

2. The fingerprint spectrum of the American ginseng extract according to claim 1, characterized in that, The process parameters for the heating reflux extraction are: material-to-liquid ratio 1:8-12, extraction time 0.8-1.2 h / time, and extraction times 2-3 times.

3. The fingerprint spectrum of the American ginseng extract according to claim 1, characterized in that, The gradient elution procedure is as follows: From 0 to 15 minutes, phase A decreased from 90% to 85%, and phase B decreased from 10% to 15%. Over 15-35 minutes, phase A decreased from 85% to 76%, and phase B decreased from 15% to 24%. Over 35-47 minutes, phase A decreased from 76% to 73%, and phase B decreased from 24% to 27%. Over 47-70 minutes, phase A decreased from 73% to 63%, and phase B decreased from 27% to 37%. Over 70-110 minutes, phase A decreased from 63% to 25%, and phase B decreased from 37% to 75%. 110-130 min, Phase A changes from 25% to 0%, Phase B changes from 75% to 100%; 130-132 min, phase A changes from 0% to 90%, phase B changes from 100% to 10%.

4. The fingerprint spectrum of the American ginseng extract according to claim 3, characterized in that, The common characteristic peaks are referenced by the chromatographic peaks of ginsenoside Rg1, ginsenoside Re and ginsenoside Rb1, and the relative retention times of each characteristic peak are within ±2.0% of the specified value.

5. The fingerprint spectrum of the American ginseng extract according to claim 3, characterized in that, The retention times of the common characteristic peaks under high performance liquid chromatography (HPLC) conditions were 6.3 min, 6.9 min, 8.2 min, 8.7 min, 11.1 min, 12.0 min, 12.4 min, 13.7 min, 14.0 min, 17.5 min, 20.7 min, 23.3 min, 34.5 min, 40.8 min, 43.4 min, 43.9 min, 66.2 min, 67.5 min, 68.0 min, 68.8 min, 70.2 min, 71.6 min, 73.5 min, 74.9 min, 75.6 min, 99.5 min, 103.8 min, and 113.5 min, respectively, with an allowable deviation of ±0.5 min for each retention time.

6. The fingerprint spectrum of the American ginseng extract according to claim 1, characterized in that, Phase A is a phosphoric acid aqueous solution with a volume fraction of 0.05-0.2%.

7. The fingerprint spectrum of the American ginseng extract according to claim 1, characterized in that, The chromatographic conditions for the high-performance liquid chromatography detection are as follows: Mobile phase: Phase A is a 0.1% (v / v) aqueous solution of phosphoric acid, and Phase B is acetonitrile; Flow rate: 1.0 mL / min; Column temperature: 30℃; Injection volume: 10 μL; Detection wavelength: 210nm.

8. A method for constructing a fingerprint spectrum of American ginseng extract, characterized in that, Includes the following steps: American ginseng and its extract were subjected to ultrasonic extraction with 30-80 vol% alcohol solution to obtain the test solution. The test solution was detected under the high performance liquid chromatography conditions as described in any one of claims 1-7 to obtain high performance liquid chromatograms of different extracts; The high-performance liquid chromatograms of different extracts are evaluated for fingerprint similarity to obtain the standard fingerprint chromatogram.

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