Fingerprint spectrum of gold essence mixture and method for determining multi-component content

Abstract

The application discloses a kind of gold essence mixture fingerprint and the determination method of multi-component content, with same chromatographic condition can realize the qualitative analysis of 10 components in gold essence mixture, and can be carried out to the quantitative of four key components such as calyx rutinoside, ellagic acid, hyperoside and calyx rutinoside.The separation effect of each characteristic peak in the fingerprint established by the method is good, a total of 33 common peaks are included, the result is reliable, objective, and has good precision, repeatability, specificity and stability.The method constructed by the application can comprehensively reflect the main chemical component characteristics of gold essence mixture, effectively solve the technical problems that its quality is difficult to be comprehensively controlled, and provide overall quality control basis for the establishment of the quality standard of the preparation.

Description

Technical Field

[0001] This invention belongs to the field of quality control of traditional Chinese medicine, specifically relating to a fingerprint spectrum of Jinjing compound and a method for determining the content of multiple components. Background Technology

[0002] The Jin Jing He Ji (Golden Essence Compound) of this invention is composed of sixteen medicinal herbs: deer antler powder, dodder seed, astragalus, dipsacus root, wolfberry fruit, plantain seed, psoralea seed, raspberry, polygonatum rhizome, poria cocos, rehmannia root, north ginseng, prepared rehmannia root, south ginseng, charred hawthorn fruit, and malt. It has the effects of tonifying essence and strengthening the foundation, warming and tonifying kidney yang, and is clinically applicable to oligospermia and asthenospermia caused by kidney essence deficiency. While Jin Jing He Ji, a formula based on years of clinical experience at Kunshan Traditional Chinese Medicine Hospital, has proven effective, its complex formulation and diverse chemical components make systematic evaluation difficult using existing quality control methods. The lack of quality standards not only restricts the in-depth explanation of its pharmacodynamic material basis but also seriously hinders the subsequent development of this preparation.

[0003] Traditional Chinese medicine (TCM) fingerprinting is an analytical technique capable of comprehensively characterizing the chemical composition and relative abundance of TCM and its preparations, enabling holistic evaluation and control of drug quality. High-performance liquid chromatography (HPLC) plays a crucial role, not only achieving quantitative control of known components but also effectively addressing the core challenge of the complexity of TCM components—providing reliable characteristic spectra even for numerous unidentified chemical components, thus enabling effective quality identification and consistency evaluation. This comprehensive understanding of both the "known" and "unknown" components in complex systems provides a key basis for the quality consistency assessment and identification of TCM.

[0004] Currently, research on the quality of Jinjing Heji itself is relatively limited, and a systematic and complete quality control system has not yet been formed. There are only some literature studies on related medicinal ingredients, which may be of reference value for the construction of quality standards for Jinjing Heji. For example, the literature "Research on Quality Evaluation of Wuzi Yanzong Pill Based on Fingerprint Spectrum, Content Determination and Chemical Pattern Recognition [J]. Journal of Pharmaceutical Analysis, 2023, 43(01): 103-112" used high performance liquid chromatography with acetonitrile-0.05% phosphoric acid aqueous solution as the mobile phase and gradient elution to determine the content of hyperoside and verbascoside in the common medicinal ingredients Cuscuta chinensis and Plantago asiatica. However, there was no clear characterization for the same medicinal pair Lycium barbarum and Rubus idaeus, indicating incomplete content determination and lack of chemical component directionality. The literature "Research Progress and Quality Marker (Q-Marker) Prediction Analysis of Zuogui Pill [J]. Chinese Journal of Traditional Chinese Medicine, 2025, 1-16" conducted a prediction analysis of the quality markers of Zuogui Pill. Lycium barbarum polysaccharide, hyperoside, verbascoside and rehmannia glutinosa in the common medicinal pair Lycium barbarum, Cuscuta chinensis and Rehmannia glutinosa were identified as the key quality markers of the formula. The literature lacks fingerprint chromatogram determination; the literature "Study on high performance liquid chromatography fingerprint of Gushen Pill [J]. Journal of Hubei University of Medicine, 2022, 41(04): 338-343" uses HPLC gradient elution-dual wavelength detection method to determine the fingerprint chromatogram of Gushen Pill and confirms four known chromatographic peaks as: chlorogenic acid, paeoniflorin, stilbene glycoside, and icariin, but does not identify and quantify the common medicinal ingredients Astragalus membranaceus, Cuscuta chinensis, Lycium barbarum, Rehmannia glutinosa, and Rubus idaeus; the literature "Evaluation of Rubus idaeus quality based on UPLC-Q-TOF-MS / MS method, HPLC fingerprint chromatogram, and content determination [J]. Chinese Traditional and Herbal Drugs, 2025, 47(04): 1077-1084" uses UPLC to establish a fingerprint chromatogram method for Rubus idaeus and determines the content of nine substances including chlorogenic acid, ferulic acid, ellagic acid, and isoquercitrin, but UPLC detection has high requirements for instruments and operators and has high operating costs. While the above methods have laid the foundation for the systematic construction of quality standards for Jinjing compound in terms of methodological design, component definition, and quality evaluation dimensions, they require different analytical methods, are complex and cumbersome to operate, are time-consuming, and have low efficiency. Summary of the Invention

[0005] In view of the limitations of existing related technologies and the current lack of quality control methods for Jinjing compound preparations, this invention aims to establish a quality evaluation method based on high-performance liquid chromatography (HPLC) fingerprinting combined with multi-component content determination. This method can achieve qualitative analysis of 10 components in Jinjing compound preparations under the same chromatographic conditions, and accurately quantify four key components: verbascoside, ellagic acid, hyperoside, and verbascoside. This effectively solves the quality control challenges caused by the complex chemical composition and overlapping chromatographic peaks of this preparation. This method provides a comprehensive and reliable scientific basis for the quality consistency evaluation and standardization of Jinjing compound preparations.

[0006] The present invention achieves the above-mentioned objectives by adopting the following technical solution: The first aspect of the present invention provides a fingerprint spectrum of a gold essence compound and a method for determining the content of multiple components.

[0007] Furthermore, the method includes the following steps: (1) Preparation of reference solution: Take the reference standards of verbascoside, ellagic acid, hyperoside and verbascoside, dissolve them in solvent and make up to volume to obtain the solution; (2) Preparation of test solution: Take the gold essence mixture sample, add solvent to extract, filter, and take the filtrate to obtain the solution; (3) Preparation of single herbal medicine sample solution: Take a single herbal medicine sample, add solvent to extract, filter, and take the filtrate to obtain the solution; (4) Preparation of negative test solution: Take each negative sample, add solvent to extract, filter, and take the filtrate to obtain the solution; (5) Fingerprint chromatogram establishment: High performance liquid chromatography was used to inject the reference solution from step (1), the test solution from step (2), the single herb test solution from step (3), and the negative test solution from step (4) into the high performance liquid chromatograph in sequence, record the chromatogram, and construct the fingerprint chromatogram of Jinjing compound. (6) Content determination: Using the same chromatographic conditions as in step (5), take the reference solution from step (1) and the test solution from step (2), inject them into the high performance liquid chromatograph, record the chromatogram, and calculate the content of the four components, verbascoside, ellagic acid, hyperoside and verbascoside, in the test solution using the external standard method.

[0008] Furthermore, in step (5), the chromatographic conditions of the high performance liquid chromatography method are as follows: mobile phase A is 0.1% phosphoric acid aqueous solution, mobile phase B is acetonitrile, and gradient elution is performed. Optionally, the elution gradient of the high-performance liquid chromatography method in step (5) is as follows: 0 ~ 10 min, 5% B; 10 ~ 15 min, 5% → 8% B; 15 ~ 20 min, 8% → 8% B; 20 ~ 22 min, 8% → 10% B; 22 ~ 40 min, 10% → 10% B; 40 ~ 55 min, 10% → 13% B; 55 ~ 65 min, 13% → 15% B; 65 ~ 75 min, 15% → 15% B; 75 ~ 95 min, 15% → 19% B; 95 ~ 105 min, 19% → 23% B; 105 ~ 107 min, 23% → 32% B; 107 ~ 108 min, 32% → 32% B. Optionally, the chromatographic column for the high performance liquid chromatography method described in step (5) is a Kromasil 100-5-C18 (4.6 mm × 250 mm, 5 μm). Optionally, the chromatographic conditions for the high performance liquid chromatography method described in step (5) further include: a flow rate of 0.6 ~ 1.0 mL / min; Optionally, the chromatographic conditions for the high performance liquid chromatography method described in step (5) further include: a column temperature of 15 ~ 25℃; Optionally, the chromatographic conditions of the high performance liquid chromatography method in step (5) further include: a detection wavelength of 210 ~ 300 nm; Optionally, the chromatographic conditions for the high performance liquid chromatography method described in step (5) further include: an injection volume of 5 ~ 40 μL; Optionally, the solvent used in steps (1)-(4) is a 60%~80% methanol aqueous solution; Optionally, the extraction method described in steps (2)-(4) is ultrasonic extraction, heating reflux extraction or cold soaking. Optionally, the ultrasonic extraction method uses an ultrasonic power of 400-800 W, an ultrasonic frequency of 30-50 kHz, and an ultrasonic time of 10-50 min.

[0009] Furthermore, the chromatographic conditions for the high performance liquid chromatography method described in step (5) also include: a flow rate of 0.8 mL / min; Optionally, the chromatographic conditions for the high performance liquid chromatography method described in step (5) further include: a column temperature of 20°C; Optionally, the chromatographic conditions for the high performance liquid chromatography method described in step (5) further include: a detection wavelength of 254 nm; Optionally, the chromatographic conditions for the high performance liquid chromatography method described in step (5) further include: an injection volume of 20 μL; Optionally, the solvent used in steps (1)-(4) is an 80% methanol aqueous solution; Optionally, the extraction method described in steps (2)-(4) is ultrasonic extraction; Optionally, the ultrasonic extraction method uses an ultrasonic power of 760 W, an ultrasonic frequency of 40 kHz, and an ultrasonic time of 30 min. Optionally, the filtration described in steps (2)-(4) is filtration using a 0.22 μm microporous membrane.

[0010] Furthermore, the fingerprint mapping construction described in step (5) includes the following steps: 1) Ten batches of test sample solutions were injected sequentially to obtain chromatograms of ten batches of test samples. The chromatograms of the ten batches of test samples were then imported into the Chinese medicine chromatographic fingerprint similarity evaluation system, and 33 common peaks were identified and a reference fingerprint chromatogram of Jinjing compound was generated. 2) Compare the chromatograms of the common peaks with those of the single herbal medicine test sample, negative test sample, and reference sample. Assign each common peak and identify the chemical components of some common peaks. Peak 8 is identified as 5-hydroxymethylfurfural, peak 10 as genipin glycoside, peak 11 as protocatechuic acid, peak 18 as chlorogenic acid, peak 20 as vanillic acid, peak 25 as ferulic acid, peak 26 as verbascoside, peak 27 as ellagic acid, peak 28 as hyperoside, and peak 29 as verbascoside. 3) The quality of Jinjing compound was evaluated using the aforementioned reference fingerprint chromatogram, and the similarity was calculated according to the Chinese herbal chromatographic fingerprint chromatogram similarity evaluation system; Optionally, the similarity between the fingerprint chromatograms of each batch of test samples and the control fingerprint chromatograms shall not be less than 0.97.

[0011] In some embodiments, the single-herb medicinal sample refers to a concentrated extract of each of 16 single-herb medicinal slices weighed according to the prescription ratio, decocted and concentrated using the same preparation process as Jin Jing He Ji. In a specific embodiment of the present invention, the single-herb medicinal sample refers to a concentrated extract of each of the following sixteen single-herb medicinal slices weighed according to the Jin Jing He Ji prescription ratio: deer antler powder, dodder seed, astragalus, dipsacus root, wolfberry fruit, plantain seed, psoralea seed, raspberry, polygonatum rhizome, poria cocos, rehmannia root, north ginseng, prepared rehmannia root, south ginseng, charred hawthorn fruit, and malt. These slices are then processed individually using the same preparation process (such as decoction and concentration) as Jin Jing He Ji, resulting in concentrated extracts of each single herb. The purpose of this sample preparation is for subsequent analysis of chromatographic peaks in the chromatogram to clarify the source of the medicinal materials for each common peak in the fingerprint spectrum of Jin Jing He Ji.

[0012] In some embodiments, the negative sample refers to a negative concentrate obtained by decocting and concentrating the medicinal slices lacking certain herbs according to a specific process. In a specific embodiment of the present invention, the negative sample refers to a series of negative control concentrates obtained by preparing simulated formulas lacking one or more of the herbs in the Jin Jing He Ji prescription, and then decocting and concentrating them using the same preparation process as Jin Jing He Ji. Specifically, a series of negative samples are prepared, each lacking any one of the sixteen herbs, such as deer antler powder, dodder seed, and astragalus. The chromatograms of this series of negative samples are compared with the chromatogram of the complete Jin Jing He Ji prescription to verify and confirm whether the common peaks (especially the attributed and identified characteristic peaks) truly originate from the corresponding missing herbs, thereby ensuring the specificity and accuracy of the fingerprint chromatogram feature identification.

[0013] In some embodiments, the preparation of the reference solution may further include concentration and linearity verification steps. In practice, not only are the four reference standards (vernaculone glucoside, ellagic acid, hyperoside, and verbascoside) accurately weighed and dissolved in 80% methanol to a final volume, but a series of mixed reference solution solutions with gradient concentrations can also be prepared. These solutions are sequentially injected into a high-performance liquid chromatograph (HPLC), and a standard curve is plotted with the concentration of each component on the x-axis and the corresponding peak area on the y-axis, followed by linear regression calculation. This step aims to obtain the standard curve equation (e.g., Y=aX+b) for subsequent content quantification calculations and to verify its linearity within a specified concentration range (typically requiring a correlation coefficient R). 2 >0.999), thereby ensuring the accuracy, linearity, and reliability of subsequent content determination results.

[0014] In some implementation schemes, the attribution and identification of the 33 common peaks are analyzed using liquid chromatography-mass spectrometry (LC-MS). To more accurately and comprehensively identify the chemical structures of the characteristic chromatographic peaks, ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) can be used to analyze the gold-containing compound sample solution under the same or optimized gradient elution conditions. The precise molecular weight (primary mass spectrometry) and characteristic fragment ion information (secondary mass spectrometry) of each common peak are obtained by high-resolution mass spectrometry and compared with theoretical mass spectrometry data of relevant chemical components from reference standards or related literature and databases. This method can verify the 10 identified characteristic peaks and holds promise for structural deduction or preliminary identification of other unknown common peaks.

[0015] In some implementations, the method underwent comprehensive methodological evaluation. Precision validation involved injecting the same test solution six times consecutively and calculating the relative standard deviation (RSD) of the relative retention time and relative peak area (with ellagic acid as the reference peak) of each common peak. The results showed RSD values ​​of less than 1% and 3%, respectively. Repeatability validation involved preparing six parallel samples from the same batch and calculating the RSDs of relevant parameters, which also met the requirements. Stability validation showed that the test solution was stable within 48 hours. The assay section also validated precision, repeatability, linearity, range, and recovery. The average recovery rate ranged from 96.56% to 99.24%, with RSD values ​​all less than 2%, demonstrating the accuracy, stability, and reliability of the method for quantitative analysis.

[0016] In some implementations, the method was applied to the quality evaluation of multiple batches of actual samples, yielding quantitative results. This method was applied to the analysis of 10 different batches of Jinjing compound samples. Fingerprint similarity evaluation results showed that the similarity between the fingerprint spectra of the 10 batches and the control fingerprint spectra was greater than 0.97, indicating good consistency and stability among the batches. Simultaneously, the content determination results provided the specific content (mg / mL) of the four target components in each batch of samples and calculated their average range. For example, the contents of verbascoside, ellagic acid, hyperoside, and verbascoside were approximately 0.90±0.08, 1.52±0.23, 1.03±0.10, and 1.02±0.08 mg / mL, respectively. These data provide specific and objective quantitative basis for establishing quality standards for Jinjing compound (such as setting content limits) and conducting batch-to-batch quality comparisons.

[0017] A second aspect of the present invention provides a method for determining the content of four components in a gold essence compound.

[0018] Furthermore, the method includes the following steps: ① Preparation of reference solution: Same as step (1) in the method for determining the fingerprint spectrum and multi-component content of Jinjing compound described in the first aspect of the present invention; ② Preparation of the test solution: The same as step (2) in the method for determining the fingerprint spectrum and multi-component content of the gold essence compound described in the first aspect of the present invention; ③ Content determination: The same as step (6) in the method for determining the fingerprint spectrum and multi-component content of the gold essence compound described in the first aspect of the present invention.

[0019] In some embodiments, the content determination employs the external standard method, and the linear range is clearly defined. The external standard method described in step ③ is based on an established standard curve. In specific operations, a series of mixed reference solutions of four components (vernacoside, ellagic acid, hyperoside, and verbascoside) with known gradient concentrations need to be prepared in advance. These solutions are then injected and analyzed under the same chromatographic conditions to obtain peak area-concentration standard curves for each component, and their linearity is verified. In specific embodiments of the present invention, the linear ranges for each component are: vernacoside 0.7268 ~ 11.6280 μg / mL, ellagic acid 1.0545 ~ 16.8720 μg / mL, hyperoside 0.5209 ~ 8.3336 μg / mL, and verbascoside 1.2429 ~ 19.8870 μg / mL. The content of the actual sample is calculated by substituting its chromatographic peak area into the corresponding standard curve regression equation.

[0020] A third aspect of the present invention provides a method for establishing a fingerprint spectrum of a gold essence compound.

[0021] Furthermore, the method includes the following steps: (A) Preparation of reference solution: Same as step (1) in the method for determining the fingerprint spectrum and multi-component content of Jinjing compound according to the first aspect of the present invention; (B) Preparation of test solution: Same as step (2) in the method for determining the fingerprint spectrum and multi-component content of gold essence compound described in the first aspect of the present invention; (C) Preparation of single-herb decoction piece test solution: the same as step (3) in the method for determining the fingerprint spectrum and multi-component content of Jinjing compound described in the first aspect of the present invention; (D) Preparation of negative test solution: Same as step (4) in the method for determining the fingerprint spectrum and multi-component content of Jinjing compound described in the first aspect of the present invention; (E) Fingerprint spectrum establishment: Same as step (5) in the method for determining the fingerprint spectrum and multi-component content of the gold essence compound described in the first aspect of the present invention; Optionally, the fingerprint pattern establishment in step (E) includes the following steps: A) Ten batches of test sample solutions were injected sequentially to obtain chromatograms of ten batches of test samples. The chromatograms of the ten batches of test samples were then introduced into the Chinese medicine chromatographic fingerprint similarity evaluation system. 33 common peaks were identified and a reference fingerprint spectrum of Jinjing compound was generated, which is the fingerprint spectrum of Jinjing compound composed of 33 common peaks. B) Compare the chromatograms of the common peaks with those of the single herbal medicine test sample, negative test sample, and reference sample. Assign each common peak and identify the chemical components of some common peaks. Peak 8 is identified as 5-hydroxymethylfurfural, peak 10 as genipin glycoside, peak 11 as protocatechuic acid, peak 18 as chlorogenic acid, peak 20 as vanillic acid, peak 25 as ferulic acid, peak 26 as verbascoside, peak 27 as ellagic acid, peak 28 as hyperoside, and peak 29 as verbascoside.

[0022] In some implementations, the generation of the reference fingerprint is based on a common pattern from multiple batches of representative samples. In step A), to construct a reliable reference fingerprint for Jinjing compound, this invention selects at least 10 different batches of representative Jinjing compound samples, prepares test solutions according to a predetermined method, and performs chromatographic analysis. The chromatograms of the obtained 10 batches of samples are superimposed and compared. Automatic matching and manual calibration are performed using professional software such as the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine," ultimately identifying 33 common chromatographic peaks that consistently appear in different batches of samples. The relative retention times and relative peak areas of these common peaks constitute a "common pattern" or "reference fingerprint," which serves as a unified standard for subsequent quality evaluation.

[0023] In some implementations, the process of establishing the fingerprint spectrum also includes a systematic analysis of the attribution and identification of common peaks. Step B) above details the chromatogram analysis process. First, attribution analysis: The chromatogram of the complete Jin Jing He Ji formula is carefully compared with the chromatograms of each single herb sample and the negative (flavorless) sample to clarify which of the sixteen herbs the 33 common peaks originate from, revealing their source. Secondly, component identification: By accurately comparing the retention time and UV spectrum (if any) of the complete chromatogram with the chromatogram of the known reference solution under the same chromatographic conditions, the specific chemical components corresponding to 10 characteristic chromatographic peaks were successfully identified. These are 5-hydroxymethylfurfural at peak 8, genipinic acid at peak 10, protocatechuic acid at peak 11, chlorogenic acid at peak 18, vanillic acid at peak 20, ferulic acid at peak 25, verbascoside at peak 26, ellagic acid at peak 27, hyperoside at peak 28, and verbascoside at peak 29. This gives the fingerprint spectrum a clear chemical substance orientation.

[0024] In some implementation schemes, the established fingerprint spectroscopy has undergone methodological validation to ensure its applicability. In a specific implementation scheme of this invention, to demonstrate that the established fingerprint spectroscopy method is scientific, reliable, and stable, its key parameters have been validated. For example, precision tests (six consecutive injections of the same test solution, with RSD of relative retention time of common peaks <1% and RSD of relative peak area <3%), repeatability tests (six samples from the same batch prepared and analyzed independently, with consistent results), and stability tests (the test solution remained stable within 48 hours) were conducted. These validation results indicate that the method has good reproducibility and can be reliably used for the acquisition and comparison of fingerprint spectroscopy for Jinjing compound, providing a methodological basis for subsequent quality evaluation.

[0025] In some implementations, the fingerprint spectrum of the 33 common peaks can be used for visual comparison and preliminary quality screening. The final generated reference fingerprint spectrum of Jinjing compound is a standardized chromatographic profile containing 33 common peaks. In practical applications, the chromatogram of the Jinjing compound sample to be tested can be visually superimposed and compared with this reference fingerprint spectrum to observe whether the peak shape, number of peaks, and relative positions (retention times) of each peak are consistent. Furthermore, similarity evaluation software (such as the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" (2012 version)) can be used for quantitative calculation. In a specific implementation of the present invention, based on the analysis of 10 batches of qualified samples, an acceptance standard of not less than 0.97 was established. This method can quickly and comprehensively determine the consistency between the sample to be tested and the qualified product, and is an effective tool for identifying authenticity and controlling intra-batch and inter-batch quality stability.

[0026] A fourth aspect of the present invention provides a fingerprint spectrum of a gold essence compound.

[0027] Furthermore, the fingerprint spectrum of the gold essence compound is constructed using the method for establishing the fingerprint spectrum of the gold essence compound according to the third aspect of the present invention.

[0028] In some implementations, the fingerprint spectrum of the Jinjing compound is a standardized chromatographic profile containing 33 common peaks, which can serve as a benchmark for quality evaluation. This fingerprint spectrum is generated by analyzing the chromatograms of at least 10 representative batches of Jinjing compound samples according to the method described in the third aspect of the present invention, and then processing them using the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System". It is characterized by 33 stable common chromatographic peaks, whose relative retention times and relative peak areas (based on a reference peak, such as ellagic acid) constitute the digital characteristics of the spectrum. This spectrum is not a simple image, but a digital standard containing the aforementioned quantitative characteristic information, which can be used for automatic computer comparison. It serves as an objective and unified technical benchmark for subsequent quality similarity evaluation, authenticity identification, and batch consistency monitoring.

[0029] In some embodiments, the fingerprint of the Jinjing mixture has a clear chemical substance orientation, and 10 characteristic peaks have been accurately identified. This fingerprint is not only an overall "chemical barcode", but also assigns and identifies the chemical components of the key chromatographic peaks.

[0030] In the specific embodiments of the present invention, 10 out of 33 common peaks have been successfully identified as specific known chemical components, including peak No. 8 (5-hydroxymethylfurfural), peak No. 10 (geniposide acid), peak No. 11 (protocatechuic acid), peak No. 18 (chlorogenic acid), peak No. 20 (vanillic acid), peak No. 25 (ferulic acid), peak No. 26 (calycosin-7-O-β-D-glucoside), peak No. 27 (ellagic acid), peak No. 28 (hyperoside), and peak No. 29 (verbascoside). This makes the fingerprint go beyond simple pattern comparison and become a bridge connecting macroscopic chromatographic features and microscopic chemical components, providing a powerful technical tool for deeply understanding the quality attributes of the Jinjing mixture from the material basis level and monitoring the changes of key components.

[0031] The fifth aspect of the present invention provides a method for evaluating the quality of the Jinjing mixture.

[0032] Furthermore, the method includes the following steps: Take a test sample of the Jinjing mixture, obtain the fingerprint corresponding to the test sample of the Jinjing mixture under the same fingerprint detection conditions as in the first aspect of the present invention, and compare the fingerprint corresponding to the test sample with the fingerprint of the Jinjing mixture described in the fourth aspect of the present invention; Optionally, use the Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints for similarity comparison; Optionally, if the similarity is greater than 0.97, the test sample of the Jinjing mixture is a qualified product.

[0033] In some embodiments, the quality evaluation method is a quantitative discrimination process based on fingerprint similarity calculation. The core of this method is to digitally compare the fingerprint of the test sample with the reference fingerprint of the Jinjing mixture established in the fourth aspect of the present invention as the "gold standard". In actual operation, usually import the chromatographic data of the two into the professional "Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints", and this system will automatically match the chromatographic peaks of the two maps and calculate a similarity index. This index is a value between 0 and 1, which comprehensively considers the matching degree of multiple dimensions such as the number of common peaks, retention time, and peak area ratio, thereby transforming the overall similarity of complex chromatograms into a simple and objective quantitative index, greatly improving the efficiency and consistency of quality discrimination.

[0034] In some implementations, the "similarity greater than 0.97" qualification standard is established based on statistical analysis of multiple batches of qualified products. In a specific implementation of the present invention, the results of evaluating the fingerprint similarity of 10 batches of qualified Jinjing compound samples showed that the similarity values ​​of each batch of samples and the control fingerprint were all above 0.97, with most concentrated above 0.99. Therefore, a similarity greater than 0.97 was set as the quality qualification threshold. This standard means that qualified and consistently high-quality Jinjing compound products must maintain a high degree of consistency with the control standard in terms of chemical composition and relative proportions. This threshold can be used not only to determine whether a single product is qualified, but also to monitor quality fluctuations between different production batches, serving as a key quantitative basis for ensuring the uniformity of drug quality.

[0035] In some implementations, the quality evaluation method can be used in conjunction with the content determination method described in the second aspect of the invention. This method, together with the content determination method for the four components provided in the second aspect, constitutes a complete and comprehensive quality control system. In practice, fingerprint similarity evaluation and quantitative analysis of the four components can be performed simultaneously on the same sample. The final quality conclusion requires a comprehensive assessment of two aspects: first, the fingerprint similarity must be greater than 0.97 to ensure the integrity, correctness, and batch consistency of its overall chemical characteristics; second, the content determination results of the four indicator components (verrucoside, ellagic acid, hyperoside, and verbascoside) should fall within a reasonable range established based on qualified batches. Only when both standards are met simultaneously can the gold essence compound sample be determined to meet the quality requirements in both "overall chemical pattern" and "key component content," thereby achieving more comprehensive and reliable control over product quality.

[0036] The sixth aspect of the present invention provides a system for implementing the method for determining the fingerprint spectrum and multi-component content of the gold essence compound described in the first aspect of the present invention.

[0037] Furthermore, the system includes: Sample processing unit: used to prepare the test solution in the method for determining the fingerprint spectrum and multi-component content of the gold essence compound as described in the first aspect of the present invention; High performance liquid chromatograph: used to analyze the test solution according to the chromatographic conditions described in the fingerprint spectrum and multi-component content determination method of Jinjing compound as described in the first aspect of the present invention, and obtain a chromatogram; A data processing unit is used to receive and process the chromatogram to construct a fingerprint and / or calculate the content of the target component.

[0038] In some embodiments, the sample processing unit includes a precision pipette, a volumetric container, an ultrasonic treatment device, a high-speed centrifuge, and a microporous filter. Specifically, this unit is used to perform the test solution preparation process: First, 1 mL of the gold-containing compound sample is measured using a precision pipette and placed in a 50 mL volumetric flask; then, 80% methanol aqueous solution is added to make up to volume and the flask is sealed; next, the volumetric flask is placed in an ultrasonic cleaner with a power of 760 W and a frequency of 40 kHz for 30 minutes; after ultrasonication, the solution is centrifuged at 12000 r / min for 10 minutes; finally, the supernatant is filtered using a microporous membrane with a pore size of 0.22 μm, and the resulting filtrate is the test solution that meets the injection requirements. The integration of this unit ensures the standardization, automation, and high reproducibility of the sample pretreatment process.

[0039] In some embodiments, the high-performance liquid chromatograph is configured with specific hardware components and chromatographic method parameters. The core of the instrument includes a high-pressure pump, an autosampler, a column oven, a detector, and a data processing workstation. The method parameters are set as follows: a Kromasil 100-5-C18 (4.6 mm × 250 mm, 5 μm) column is used; mobile phase A is 0.1% aqueous phosphoric acid solution, mobile phase B is acetonitrile, and elution is performed according to the gradient program described in the first aspect of the invention; the column temperature is kept constant at 20°C; the flow rate is set to 0.8 mL / min; the detection wavelength of the detector (e.g., a DAD detector) is set to 254 nm; and the injection volume of the autosampler is 20 μL.

[0040] In some implementations, the data processing unit includes fingerprinting software and a content calculation module. This unit receives raw chromatographic data files exported from a high-performance liquid chromatograph (HPLC) workstation. Its fingerprinting function can import, overlay, automatically match, and calibrate chromatograms from multiple batches (e.g., 10 batches) of samples, identifying and confirming 33 common peaks through an algorithm, ultimately generating a reference fingerprint (common mode). Its content calculation module operates based on the external standard method: first, it establishes standard curves for four components—vernaculone glucoside, ellagic acid, hyperoside, and verbascoside—using data from a mixed reference solution; then, it automatically identifies and integrates the peak areas of the corresponding components in the chromatogram of the sample to be tested, substitutes them into their respective standard curve equations, and automatically calculates the specific content (mg / mL) of each component in the sample.

[0041] In some implementations, the system further integrates quality assessment and report generation functions. The data processing unit's functions are not limited to chromatogram construction and content calculation; it also integrates a data analysis module. This module can automatically calculate the similarity between the fingerprint chromatogram of the sample to be tested and the control fingerprint chromatogram stored in the system (e.g., by calling the core algorithm of the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System"), and directly provide a judgment conclusion of "similarity greater than 0.97, qualified" or "similarity less than 0.97, unqualified." Simultaneously, the system can integrate the fingerprint chromatogram similarity results, the quantitative determination results of the four components, and the corresponding judgment criteria (such as content limits) to automatically generate a structured test report or quality assessment certificate, achieving full automation and intelligence from sample injection to the issuance of a complete report.

[0042] The seventh aspect of the present invention provides for any of the following applications: (1) The fingerprint spectrum of the gold essence compound and the method for determining the content of multiple components described in the first aspect of the present invention or the application of the fingerprint spectrum of the gold essence compound in the quality control of the gold essence compound generation process in the fourth aspect of the present invention; (2) The fingerprint spectrum of the gold essence compound and the method for determining the content of multiple components described in the first aspect of the present invention or the application of the fingerprint spectrum of the gold essence compound described in the fourth aspect of the present invention in the quality evaluation of the finished gold essence compound; (3) The fingerprint spectrum of the gold essence compound and the method for determining the content of multiple components described in the first aspect of the present invention or the application of the fingerprint spectrum of the gold essence compound described in the fourth aspect of the present invention in the batch stability analysis of the gold essence compound; (4) The fingerprint spectrum of Jinjing compound and the method for determining the content of multiple components described in the first aspect of the present invention or the application of the fingerprint spectrum of Jinjing compound described in the fourth aspect of the present invention in the identification of the authenticity of Jinjing compound; (5) The fingerprint spectrum of Jinjing compound and the method for determining the content of multiple components as described in the first aspect of the present invention or the application of the fingerprint spectrum of Jinjing compound in the determination of the content of components in Jinjing compound as described in the fourth aspect of the present invention.

[0043] In some implementation schemes, the application of the method or fingerprint spectrum of Jinjing compound in batch stability analysis of Jinjing compound is specifically manifested in the sampling at different time points (such as 0, 1, 3, 6 months, etc.) during accelerated and long-term stability tests of the drug, and the fingerprint spectrum and the content of four key components are simultaneously determined using the method described in the first aspect of the present invention; by systematically comparing the similarity change trend of the fingerprint spectrum of the samples at each time point with the initial (0 month) control fingerprint spectrum, the stability of its chemical composition can be assessed macroscopically; at the same time, by tracking the specific decay or fluctuation curves of the content of the four indicator components (verrucoside, ellagic acid, hyperoside, and verbascoside), the degradation kinetics of the key active ingredients can be quantified microscopically, thereby providing objective and comprehensive data support for scientifically determining the shelf life and storage conditions of Jinjing compound.

[0044] In some implementation schemes, the application of the method or fingerprint spectrum of Jinjing compound in the identification of the authenticity of Jinjing compound is specifically manifested in using the established characteristic fingerprint spectrum containing 33 common peaks (10 of which have been identified) as a "chemical barcode" or standard template, and comparing the overall similarity of the chromatogram of the sample to be identified with that of the standard fingerprint spectrum; if the similarity between the fingerprint spectrum of the sample to be tested and the standard fingerprint spectrum is significantly lower than the qualified threshold of 0.97, or if the number of chromatographic peaks, relative retention time, peak shape profile, etc., are significantly different from the standard spectrum (especially the absence or serious deviation of the identified key characteristic peaks), then the sample can be effectively determined to be a counterfeit, inferior, or substitute product that does not conform to the standard process product, thereby achieving rapid and objective identification.

[0045] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows: (1) The content detection method provided by the present invention has been verified and the precision, repeatability, stability, linearity and recovery rate all meet the requirements. The detection results are accurate and reliable. It can simultaneously quantify the content of four chemical components in Jinjing compound, which is of great significance for improving the testing efficiency, quality control and standard evaluation of the preparation. (2) This invention organically combines the overall characterization of fingerprint spectra with the precise quantitative analysis of multiple index components to construct a comprehensive and efficient quality control method system. This system can comprehensively evaluate drug quality from both macroscopic and microscopic levels, providing sufficient scientific basis for the scientific formulation and improvement of the quality standards of Jinjing Compound. Attached Figure Description

[0046] Figure 1 Chromatograms at different column temperatures; Figure 2 Chromatograms at different flow rates; Figure 3The high performance liquid chromatogram (A) of 10 batches of Jinjing compound and the reference fingerprint chromatogram of Jinjing compound (B, from left to right in the figure are common peaks 1 to 33). Figure 4 Chromatograms of the gold essence compound and individual herbal decoction pieces of the present invention; Figure 5 Chromatograms of the gold-containing compound and various negative test samples of the present invention; Figure 6 The chromatogram of the mixture of the gold essence compound and the designated component of the present invention is shown in the following: peak 8 is 5-hydroxymethylfurfural, peak 10 is genipin glycoside, peak 11 is protocatechuic acid, peak 18 is chlorogenic acid, peak 20 is vanillic acid, peak 25 is ferulic acid, peak 26 is verrucoside glucoside, peak 27 is ellagic acid, peak 28 is hyperoside, and peak 29 is verrucoside. Figure 7 The specificity chromatogram for content determination in this invention is as follows: A is blank solvent, B is the test solution of Jinjing compound, and C is the mixed solution of quantitative components (from left to right in the figure, they are verbascoside, ellagic acid, hyperoside and verbascoside). Detailed Implementation

[0047] The present invention will be further illustrated below with reference to specific embodiments. These specific embodiments are for illustrative purposes only and should not be construed as limiting the invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention. The scope of the invention is defined by the claims and their equivalents. The experimental consumables, reagents, and raw materials used in this invention are readily available to those skilled in the art and, unless otherwise specified, can be obtained commercially. Experimental methods not specifying specific conditions in this invention are generally performed under conventional conditions or according to the manufacturer's recommended conditions.

[0048] The instruments and reagents used in this invention are as follows: Instruments: LC-2050C 3D high performance liquid chromatograph (Shimadzu Corporation); MS204S analytical balance (d=0.0001 g, Mettler AG, Switzerland); T1000 analytical balance (d=0.1 g, Changshu Shuangjie Test Instrument Factory); LC-UC-150 ultrasonic cleaner (Shanghai Lichen Bangxi Instrument Technology Co., Ltd.)

[0049] Reagents: Acetonitrile and phosphoric acid were of chromatographic grade, and all other reagents were of analytical grade.

[0050] Reference substances: Calycosin-7-O-β-D-glucoside (batch number 111920-202308, content 96.9%), Ellagic acid (batch number 111959-201903, content 88.8%), Hyperoside (batch number 111521-202310, content 94.7%), Acteoside (batch number 111530-202315, content 97.6%). The reference substances were all purchased from the National Institutes for Food and Drug Control.

[0051] Samples: Multiple batches of Jinjing mixture were provided by Kunshan Traditional Chinese Medicine Hospital. The sample information is shown in Table 1. The Jinjing mixture is composed of sixteen herbs, including Cornu Cervi Degelatinatum, Cuscuta chinensis, Astragalus membranaceus, Dipsacus asperoides, Lycium barbarum, Plantago asiatica, Astragalus complanatus, Rubus idaeus, Polygonatum sibiricum, Poria cocos, Rehmannia glutinosa, Glehnia littoralis, Rehmannia glutinosa preparata, Adenophora tetraphylla, Hawthorn Fruit (processed) and Fructus Hordei Germinatus. It has the effects of tonifying essence and consolidating the root, warming and tonifying the kidney yang. Clinically, it is applicable to oligoasthenospermia caused by kidney essence deficiency syndrome. It is a clinical experience formula of Kunshan Traditional Chinese Medicine Hospital for many years. Those skilled in the art can obtain the Jinjing mixture and its specific prescription ratio based on the above disclosed medicinal flavor composition and specific source.

[0052]

[0053] Example 1 Optimization of column temperature 1. Preparation of test solution: Precisely measure 1 mL of Jinjing mixture, place it in a 50 mL volumetric flask, add 80% methanol to volume, tightly stopper, ultrasonically treat for 30 min, cool, shake well, centrifuge at 12000 r / min for 10 min, filter through a 0.22 μm microporous membrane, and take the subsequent filtrate to obtain the test solution.

[0054] 2. Chromatographic conditions Chromatographic column: Kromasil 100-5-C18 (4.6 mm×250 mm, 5 μm); Mobile phase: 0 - 10 min, 5% B; 10 - 15 min, 5% → 8% B; 15 - 20 min, 8% → 8% B; 20 - 22 min, 8% → 10% B; 22 - 40 min, 10% → 10% B; 40 - 55 min, 10% → 13% B; 55 - 65 min, 13% → 15% B; 65 - 75 min, 15% → 15% B; 75 - 95 min, 15% → 19% B; 95 - 105 min, 19% → 23% B; 105 - 107 min, 23% → 32% B; 107 - 108 min, 32% → 32% B; Injection volume: 20 μL; Flow rate: 0.8 mL / min; Detection wavelength: 254 nm.

[0055] In the high-performance liquid chromatography (HPLC) chromatographic conditions described above, mobile phase A is a 0.1% aqueous phosphoric acid solution, and mobile phase B is acetonitrile, with gradient elution.

[0056] 3. Measurement Take the test solution, use the above chromatographic conditions, select 15, 20 and 25℃ as column temperatures, inject the sample and determine the chromatogram.

[0057] The results are as follows Figure 1 As shown, all common peaks can be well separated and have good peak shapes under different column temperatures. A column temperature of 20℃ is preferred for the determination.

[0058] Example 2: Flow Rate Optimization 1. Preparation of the test solution: as shown in Example 1.

[0059] 2. Chromatographic conditions Column: Kromasil 100-5-C18 (4.6 mm × 250 mm, 5 μm); Mobile phase: 0 ~ 10 min, 5% B; 10 ~ 15 min, 5%→8% B; 15 ~ 20 min, 8%→8% B; 20 ~ 22 min, 8%→10% B; 22 ~ 40 min, 10%→10% B; 40 ~ 55 min, 10%→13% B; 55 ~ 65 min, 13%→15% B; 65 ~ 75 min, 15%→15% B; 75 ~ 95 min, 15%→19% B; 95 ~ 105 min, 19%→23% B; 105 ~ 107 min, 23%→32% B; 107 ~ 108 min, 32%→32% B; Injection volume: 20 μL; Detection wavelength: 254 nm; Column temperature: 20℃.

[0060] In the high-performance liquid chromatography (HPLC) chromatographic conditions described above, mobile phase A is a 0.1% aqueous phosphoric acid solution, and mobile phase B is acetonitrile, with gradient elution.

[0061] 3. Measurement Take the test solution, and use the above chromatographic conditions, selecting flow rates of 0.6, 0.8, and 1.0 mL / min respectively, to inject and determine the chromatogram.

[0062] The results are as follows Figure 2 As shown, all common peaks were well separated and exhibited good peak shapes under different flow rate conditions. A flow rate of 0.8 mL / min was preferred for the determination.

[0063] Example 3: Construction of fingerprint map 1. Preparation of reference solution and test solution 1.1 Reference solution Take appropriate amounts of verbascoside, ellagic acid, hyperoside and verbascoside respectively, weigh them accurately, and add methanol to prepare a mixed reference solution containing approximately 11.63 μg of verbascoside, 16.87 μg of ellagic acid, 8.33 μg of hyperoside and 19.89 μg of verbascoside per mL.

[0064] 1.2 Test solution Accurately measure 1 mL of different batches of gold essence mixture, place it in a 50 mL volumetric flask, add 80% methanol to make up to volume, seal tightly, sonicate for 30 min, cool, shake well, centrifuge at 12000 r / min for 10 min, filter through a 0.22 μm microporous membrane, and collect the filtrate to obtain the test solution.

[0065] 1.3 Negative test solution Weigh out the missing medicinal ingredients according to the prescription ratio, decoct and concentrate them according to the preparation process of Jin Jing He Ji to obtain each negative concentration solution. Accurately measure 1 mL of each negative concentration solution, place it in a 50 mL volumetric flask, add 80% methanol to make up to volume, stopper tightly, sonicate for 30 min, cool, shake well, centrifuge at 12000 r / min for 10 min, filter through a 0.22 μm microporous membrane, and collect the filtrate to obtain the negative test solution.

[0066] 1.4 Single-herb medicinal slices test solution According to the prescription ratio, 16 single-herb medicinal slices were weighed, decocted and concentrated according to the preparation process of Jin Jing He Ji, and concentrated to obtain concentrated liquid of each medicinal slice. 1 mL of each concentrated liquid was accurately measured and placed in a 50 mL volumetric flask. 80% methanol was added to make up to volume, the flask was sealed, and the mixture was sonicated for 30 min. After cooling and shaking, the mixture was centrifuged at 12000 r / min for 10 min and filtered through a 0.22 μm microporous membrane. The filtrate was collected to obtain the single-herb medicinal slice test solution.

[0067] 2. Chromatographic conditions Kromasil 100-5-C18 (4.6 mm × 250 mm, 5 μm); Mobile phase: 0 ~ 10 min, 5% B; 10 ~ 15 min, 5%→8% B; 15 ~ 20 min, 8%→8% B; 20 ~ 22 min, 8%→10% B; 22 ~ 40 min, 10%→10% B; 40 ~ 55 min, 10%→13% B; 55 ~ 65 min, 13%→15% B; 65 ~ 75 min, 15%→15% B; 75 ~ 95 min, 15%→19% B; 95 ~ 105 min, 19%→23% B; 105 ~ 107 min, 23%→32% B; 107 ~ 108 min, 32%→32% B; Injection volume: 20 μL; Flow rate: 0.8 mL / min; Detection wavelength: 254 nm; Column temperature: 20℃.

[0068] In the high-performance liquid chromatography (HPLC) chromatographic conditions described above, mobile phase A is a 0.1% aqueous phosphoric acid solution, and mobile phase B is acetonitrile, with gradient elution.

[0069] 3. Measurement The reference standard and the test sample from Example 3 were injected and analyzed under the chromatographic conditions described above, and the chromatograms were recorded.

[0070] 4. Determination of common peaks Ten batches of Jinjing compound were taken, and test solutions were prepared according to the above method and analyzed. The resulting high-performance liquid chromatograms (HPLC) of the ten batches of Jinjing compound were overlaid, as shown in the figure below. Figure 3 As shown in A, the high performance liquid chromatography fingerprint spectra of the 10 batches of Jinjing compound obtained above were compared with those of the "Similarity Evaluation System for Chromatographic Fingerprint Spectra of Traditional Chinese Medicine" (2012 edition) to identify 33 common peaks and obtain a common pattern (reference fingerprint spectra). Figure 3 B is the comparative fingerprint spectrum of the gold essence compound of the present invention. Figure 3 From left to right, the common peaks are 1 to 33.

[0071] 5. Attribution and identification of common peaks By comparing with reference solution and single-herb decoction pieces (test sample) Figure 4 ) and negative test samples ( Figure 5 The comparison yields the following peak attribution and identification: Upon identification, peak 8 was 5-hydroxymethylfurfural, belonging to Lycium barbarum, Rehmannia glutinosa (raw and processed), and Crataegus pinnatifida (roasted); peak 10 was genipinic acid, belonging to Plantago asiatica and Rehmannia glutinosa (processed); peak 11 was protocatechuic acid, belonging to Rubus idaeus and Crataegus pinnatifida; peak 18 was chlorogenic acid, belonging to Cuscuta chinensis, Dipsacus asper, and Crataegus pinnatifida; peak 20 was vanillic acid, belonging to Lycium barbarum and Plantago asiatica; peak 25 was ferulic acid, belonging to malt; peak 26 was verbascoside, belonging to Astragalus membranaceus; peak 27 was ellagic acid, belonging to Rubus idaeus; peak 28 was hyperoside, belonging to Cuscuta chinensis; and peak 29 was verbascoside, belonging to Rehmannia glutinosa (raw) and Plantago asiatica.

[0072] The remaining peaks are as follows: peaks 1, 2, 3, 4, 6, 16, 19, 23, 24, and 31 belong to Cuscuta chinensis; peaks 1, 2, 6, 7, 17, 19, and 22 belong to Lycium barbarum; peaks 1, 2, 3, 4, 15, and 32 belong to Astragalus membranaceus; peaks 1, 2, 6, and 15 belong to Polygonatum sibiricum; peaks 1, 3, 7, 12, 14, 16, 17, 21, 23, and 24 belong to Rubus idaeus; peaks 1, 2, 3, and 15 belong to Plantago asiatica; and peaks 1, 2, 15, 22, 31, 32, and 33 belong to... According to Dipsacus asper, peaks 1, 2, 12, 16, 23, 30, 32, and 33 belong to Astragalus complanatus; peaks 1, 2, 4, 5, 6, 7, 9, 12, 13, and 30 belong to Rehmannia glutinosa (processed); peaks 1, 2, 3, 4, 9, and 30 belong to Rehmannia glutinosa (raw); peaks 1 and 2 belong to Poria cocos; peaks 1, 2, 3, and 15 belong to Adenophora stricta; peaks 1, 2, 3, 4, 6, 15, and 16 belong to Adenophora stricta (southern); peaks 1, 2, 6, 7, 9, and 12 belong to Crataegus pinnatifida (processed); and peaks 1, 2, 3, and 15 belong to Hordeum vulgare (malt).

[0073] 6. Methodological Research 6.1 Precision Take a sample of Jinjing compound, prepare a test solution according to the above method, and inject it 6 times consecutively under the above chromatographic conditions. Detect the fingerprint spectrum, record the chromatogram, and calculate the RSD value of the relative retention time and relative peak area of ​​each common peak using ellagic acid as the reference peak.

[0074] Results: After six consecutive injections of the gold-containing compound, the RSD values ​​of the relative retention times of all common peaks were less than 1%, and the RSD values ​​of the relative peak areas were all less than 3%, indicating good precision.

[0075] 6.2 Repeatability Six portions of the same batch of gold essence compound were accurately weighed and prepared according to the method described in Example 4, Test Solution Preparation. The samples were injected separately, the fingerprint spectrum was detected, and the chromatogram was recorded. Using ellagic acid as the reference peak, the RSD values ​​of the relative retention time and relative peak area of ​​each common peak were calculated.

[0076] Results: The RSD values ​​of the relative retention times of the common peaks in all six test solutions were less than 1.0%, and the RSD values ​​of the relative peak areas were all less than 3.0%, indicating good repeatability.

[0077] 6.3 Solution stability Accurately weigh the gold essence mixture sample and operate according to the method under the test solution preparation section of Example 3. Inject the sample at 0, 2, 4, 6, 8, 12, 24 and 48 h respectively, detect the fingerprint spectrum, record the chromatogram, and calculate the RSD value of the relative retention time and relative peak area of ​​each common peak using ellagic acid as the reference peak.

[0078] Results: Within 48 h, the RSD values ​​of the relative retention times of all common peaks were less than 1.0%, and the RSD values ​​of the relative peak areas were less than 6.0%. This indicates that the test solution has good stability within 48 h.

[0079] In summary, the methodological validation results show that the fingerprint spectrum detection method established in this invention has good repeatability, meets the requirements of instrument precision, and exhibits good stability of the test solution within 48 hours. It can be used to more comprehensively and reliably control the quality of Jinjing compound.

[0080] 7. Similarity results of fingerprint spectrum of Jinjing compound Ten batches of Jinjing compound were prepared according to the test solution preparation method, and the samples were injected and measured under the above chromatographic conditions. The chromatograms were recorded and imported into the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" (2012 version) for similarity calculation. The similarity results of the fingerprint chromatograms of each batch of test samples and the control fingerprint chromatograms are shown in Table 2.

[0081]

[0082] Results: The similarity between the fingerprint chromatograms of each batch of samples and the control fingerprint chromatograms was greater than 0.97, indicating good similarity.

[0083] Example 4: Determination of the content of index components in Jinjing Mixture 1. Preparation of reference solution and test solution 1.1 Mixed reference solution Take appropriate amounts of verbascoside, ellagic acid, hyperoside and verbascoside respectively, weigh them accurately, and add methanol to prepare a mixed reference solution containing approximately 11.63 μg of verbascoside, 16.87 μg of ellagic acid, 8.33 μg of hyperoside and 19.89 μg of verbascoside per mL.

[0084] 1.2 Test solution The same method as the preparation of the test solution in Example 3.

[0085] 2. Chromatographic conditions The chromatographic conditions are the same as in Example 3.

[0086] 3. Measurement The reference standard and the test sample from Example 4 were injected and analyzed under the chromatographic conditions described above, and the chromatograms were recorded.

[0087] 4. Methodological Research 4.1 Specificity Take 80% methanol blank solvent, mixed reference solution and test solution respectively, inject them into high performance liquid chromatograph, determine and record the chromatograms (e.g. Figure 7 As shown in the figure, the blank solvent did not interfere with the detection of each component, indicating that the method has good specificity.

[0088] 4.2 Linearity and Range Vernixone glucoside reference standards were prepared at concentrations of 0.7268, 1.4535, 2.9070, 5.8140, and 11.6280 μg / mL, respectively, and injected under the chromatographic conditions described above. A standard curve was plotted with the concentration of the reference standard as the abscissa (X) and the peak area as the ordinate (Y), and regression calculations were performed. The results showed that vernixone glucoside exhibited good linearity. The regression equation is shown in Table 3.

[0089]

[0090] Ellagic acid reference standards at concentrations of 1.0545, 2.1090, 4.2180, 8.4360, and 16.8720 μg / mL were injected under the chromatographic conditions described above. A standard curve was plotted with the concentration of the reference standard as the abscissa (X) and the peak area as the ordinate (Y), and regression calculations were performed. The results showed that ellagic acid exhibited good linearity. The regression equation is shown in Table 4.

[0091]

[0092] Hypericin reference standards at concentrations of 0.5209, 1.0417, 2.0834, 4.1668, and 8.3336 μg / mL were injected under the chromatographic conditions described above. A standard curve was plotted with the concentration of the reference standard as the abscissa (X) and the peak area as the ordinate (Y), and regression calculations were performed. The results showed that hypericin exhibited good linearity. The regression equation is shown in Table 5.

[0093]

[0094] Verbascoside reference standards at concentrations of 1.2429, 2.4859, 4.9718, 9.9435, and 19.8870 μg / mL were injected under the chromatographic conditions described above. A standard curve was plotted with the concentration of the reference standard as the abscissa (X) and the peak area as the ordinate (Y), and regression calculations were performed. The results showed that verbascoside exhibited good linearity. The regression equation is shown in Table 6.

[0095]

[0096] 4.3 Precision Reference solutions of verbascoside, ellagic acid, hyperoside, and verbascoside were injected six times consecutively according to the above method. The chromatograms were recorded, and the RSD values ​​of the peak areas of verbascoside, ellagic acid, hyperoside, and verbascoside were calculated to be 0.57%, 0.44%, 0.79%, and 1.11%, respectively. This indicates that the method has good precision.

[0097] 4.4 Repeatability Test Six portions of the same batch of Jinjing compound were prepared according to the test solution preparation method. The samples were then injected and analyzed under the chromatographic conditions described above. The RSD values ​​of the contents of verbascoside, ellagic acid, hyperoside, and verbascoside were calculated to be 0.32%, 1.21%, 0.98%, and 0.88%, respectively. This indicates that the method has good repeatability.

[0098] 4.5 Recovery Test Six portions of the Jinjing compound were prepared by adding appropriate amounts of each reference standard to each sample, following the method for preparing the test solution. The solutions were then injected and analyzed under the chromatographic conditions described above. The average recoveries of verbascoside, ellagic acid, hyperoside, and verbascoside were calculated to be 97.63%, 98.25%, 96.56%, and 99.24%, respectively, with RSD values ​​of 1.69%, 1.45%, 1.27%, and 1.78%, respectively. This indicates that the method has good accuracy.

[0099] 5. Results of content determination of Jinjing compound Ten batches of Jinjing compound were prepared according to the test solution preparation method, and the samples were injected and determined under the above chromatographic conditions. The contents of four components, namely, verbascoside, ellagic acid, hyperoside and verbascoside, in the samples were calculated. The results are shown in Table 7.

[0100]

[0101] In summary, this invention establishes a fingerprinting and multi-component content determination method for Jinjing compound based on high-performance liquid chromatography (HPLC). This method can simultaneously perform qualitative analysis of 10 components in Jinjing compound and quantitative analysis of four key components: verbascoside, ellagic acid, hyperoside, and verbascoside. This method enables a comprehensive quality evaluation of Jinjing compound, providing a scientific and effective basis for its quality control.

Claims

1. A method for determining the fingerprint spectrum and multi-component content of a gold essence compound, characterized in that, The method includes the following steps: (1) Preparation of reference solution: Take the reference standards of verbascoside, ellagic acid, hyperoside and verbascoside, dissolve them in solvent and make up to volume to obtain the solution; (2) Preparation of test solution: Take the gold essence mixture sample, add solvent to extract, filter, and take the filtrate to obtain the solution; (3) Preparation of single herbal medicine sample solution: Take a single herbal medicine sample, add solvent to extract, filter, and take the filtrate to obtain the solution; (4) Preparation of negative test solution: Take each negative sample, add solvent to extract, filter, and take the filtrate to obtain the solution; (5) Fingerprint chromatogram establishment: High performance liquid chromatography was used to inject the reference solution from step (1), the test solution from step (2), the single herb test solution from step (3), and the negative test solution from step (4) into the high performance liquid chromatograph in sequence, record the chromatogram, and construct the fingerprint chromatogram of Jinjing compound. (6) Content determination: Using the same chromatographic conditions as in step (5), take the reference solution from step (1) and the test solution from step (2), inject them into the high performance liquid chromatograph, record the chromatogram, and calculate the content of the four components, verbascoside, ellagic acid, hyperoside and verbascoside, in the test solution using the external standard method.

2. The method for determining the fingerprint spectrum and multi-component content of the gold essence compound according to claim 1, characterized in that, In step (5), the chromatographic conditions of the high performance liquid chromatography method are as follows: mobile phase A is 0.1% phosphoric acid aqueous solution, mobile phase B is acetonitrile, and gradient elution is performed. Optionally, the elution gradient of the high-performance liquid chromatography method in step (5) is as follows: 0 ~ 10 min, 5% B; 10 ~ 15 min, 5% → 8% B; 15 ~ 20 min, 8% → 8% B; 20 ~ 22 min, 8% → 10% B; 22 ~ 40 min, 10% → 10% B; 40 ~ 55 min, 10% → 13% B; 55 ~ 65 min, 13% → 15% B; 65 ~ 75 min, 15% → 15% B; 75 ~ 95 min, 15% → 19% B; 95 ~ 105 min, 19% → 23% B; 105 ~ 107 min, 23% → 32% B; 107 ~ 108 min, 32% → 32% B. Optionally, the chromatographic column for the high performance liquid chromatography method described in step (5) is a Kromasil 100-5-C18 (4.6 mm × 250 mm, 5 μm). Optionally, the chromatographic conditions for the high performance liquid chromatography method described in step (5) further include: a flow rate of 0.6 ~ 1.0 mL / min; Optionally, the chromatographic conditions for the high performance liquid chromatography method described in step (5) further include: a column temperature of 15 ~ 25℃; Optionally, the chromatographic conditions for the high performance liquid chromatography method described in step (5) further include: a detection wavelength of 210 ~ 300 nm; Optionally, the chromatographic conditions for the high performance liquid chromatography method described in step (5) further include: an injection volume of 5 ~ 40 μL; Optionally, the solvent used in steps (1)-(4) is a 60%~80% methanol aqueous solution; Optionally, the extraction method described in steps (2)-(4) is ultrasonic extraction, heating reflux extraction or cold soaking. Optionally, the ultrasonic extraction method uses an ultrasonic power of 400-800 W, an ultrasonic frequency of 30-50 kHz, and an ultrasonic time of 10-50 min.

3. The method for determining the fingerprint spectrum and multi-component content of the gold essence compound according to claim 2, characterized in that, The chromatographic conditions for high performance liquid chromatography described in step (5) also include: a flow rate of 0.8 mL / min; Optionally, the chromatographic conditions for the high performance liquid chromatography method described in step (5) further include: a column temperature of 20°C; Optionally, the chromatographic conditions for the high performance liquid chromatography method described in step (5) further include: a detection wavelength of 254 nm; Optionally, the chromatographic conditions for the high performance liquid chromatography method described in step (5) further include: an injection volume of 20 μL; Optionally, the solvent used in steps (1)-(4) is an 80% methanol aqueous solution; Optionally, the extraction method described in steps (2)-(4) is ultrasonic extraction; Optionally, the ultrasonic extraction method uses an ultrasonic power of 760 W, an ultrasonic frequency of 40 kHz, and an ultrasonic time of 30 min. Optionally, the filtration described in steps (2)-(4) is filtration using a 0.22 μm microporous membrane.

4. The method for determining the fingerprint spectrum and multi-component content of the gold essence compound according to claim 1, characterized in that, The fingerprint mapping construction described in step (5) includes the following steps: 1) Ten batches of test sample solutions were injected sequentially to obtain chromatograms of ten batches of test samples. The chromatograms of the ten batches of test samples were then imported into the Chinese medicine chromatographic fingerprint similarity evaluation system, and 33 common peaks were identified and a reference fingerprint chromatogram of Jinjing compound was generated. 2) Compare the chromatograms of the common peaks with those of the single herbal medicine test sample, negative test sample, and reference sample. Assign each common peak and identify the chemical components of some common peaks. Peak 8 is identified as 5-hydroxymethylfurfural, peak 10 as genipin glycoside, peak 11 as protocatechuic acid, peak 18 as chlorogenic acid, peak 20 as vanillic acid, peak 25 as ferulic acid, peak 26 as verbascoside, peak 27 as ellagic acid, peak 28 as hyperoside, and peak 29 as verbascoside. 3) The quality of Jinjing compound was evaluated using the aforementioned reference fingerprint chromatogram, and the similarity was calculated according to the Chinese herbal chromatographic fingerprint chromatogram similarity evaluation system; Optionally, the similarity between the fingerprint chromatograms of each batch of test samples and the control fingerprint chromatograms shall not be less than 0.

97.

5. A method for determining the content of four components in a gold essence compound, characterized in that, The method includes the following steps: ① Preparation of reference solution: Same as step (1) in the method for determining the fingerprint spectrum and multi-component content of Jinjing compound according to any one of claims 1-4; ② Preparation of the test solution: The same as step (2) in the method for determining the fingerprint spectrum and multi-component content of the gold essence compound according to any one of claims 1-4; ③ Content determination: The same as step (6) in the method for determining the fingerprint spectrum and multi-component content of the gold essence compound as described in any one of claims 1-4.

6. A method for establishing a fingerprint spectrum of a gold essence compound, characterized in that, The method includes the following steps: (A) Preparation of reference solution: Same as step (1) in the method for determining the fingerprint spectrum and multi-component content of Jinjing compound according to any one of claims 1-4; (B) Preparation of the test solution: the same as step (2) in the method for determining the fingerprint spectrum and multi-component content of the gold essence compound according to any one of claims 1-4; (C) Preparation of single-herb decoction piece test solution: the same as step (3) in the method for determining the fingerprint spectrum and multi-component content of Jinjing compound according to any one of claims 1-4; (D) Preparation of negative test solution: Same as step (4) in the method for determining the fingerprint spectrum and multi-component content of Jinjing compound according to any one of claims 1-4; (E) Fingerprint spectrum establishment: the same as step (5) in the method for determining the fingerprint spectrum and multi-component content of the gold essence compound according to any one of claims 1-4; Optionally, the fingerprint pattern establishment in step (E) includes the following steps: A) Ten batches of test sample solutions were injected sequentially to obtain chromatograms of ten batches of test samples. The chromatograms of the ten batches of test samples were then introduced into the Chinese medicine chromatographic fingerprint similarity evaluation system. 33 common peaks were identified and a reference fingerprint spectrum of Jinjing compound was generated, which is the fingerprint spectrum of Jinjing compound composed of 33 common peaks. B) Compare the chromatograms of the common peaks with those of the single herbal medicine test sample, negative test sample, and reference sample. Assign each common peak and identify the chemical components of some common peaks. Peak 8 is identified as 5-hydroxymethylfurfural, peak 10 as genipin glycoside, peak 11 as protocatechuic acid, peak 18 as chlorogenic acid, peak 20 as vanillic acid, peak 25 as ferulic acid, peak 26 as verbascoside, peak 27 as ellagic acid, peak 28 as hyperoside, and peak 29 as verbascoside.

7. A fingerprint spectrum of a gold essence compound, characterized in that, The fingerprint spectrum of the gold essence compound is constructed according to the method for establishing the fingerprint spectrum of the gold essence compound as described in claim 6.

8. A method for evaluating the quality of a gold essence compound, characterized in that, The method includes the following steps: Take a sample of gold essence compound to be tested, and obtain the fingerprint spectrum corresponding to the sample of gold essence compound under the same fingerprint spectrum detection conditions as any one of claims 1-4. Compare the similarity between the fingerprint spectrum corresponding to the sample to be tested and the fingerprint spectrum of gold essence compound as described in claim 7. Optionally, a similarity comparison can be performed using a chromatographic fingerprint similarity evaluation system for traditional Chinese medicine. Optionally, if the similarity is greater than 0.97, the sample of Jinjing compound is considered qualified.

9. A system for implementing the method for determining the fingerprint spectrum and multi-component content of the gold essence compound according to any one of claims 1-4, characterized in that, The system includes: Sample processing unit: used to prepare the test solution in the method for determining the fingerprint spectrum and multi-component content of the gold essence compound as described in any one of claims 1-4; High performance liquid chromatograph: used to analyze the test solution according to the chromatographic conditions described in the fingerprint spectrum and multi-component content determination method of Jinjing compound as described in any one of claims 1-4, and obtain a chromatogram; A data processing unit is used to receive and process the chromatogram to construct a fingerprint and / or calculate the content of the target component.

10. Applied to any of the following aspects: (1) The fingerprint spectrum of the gold essence compound and the method for determining the content of multiple components as described in any one of claims 1-4, or the application of the fingerprint spectrum of the gold essence compound as described in claim 7 in the quality control of the gold essence compound production process; (2) The fingerprint spectrum of the gold essence compound and the method for determining the content of multiple components as described in any one of claims 1-4, or the application of the fingerprint spectrum of the gold essence compound as described in claim 7 in the quality evaluation of the finished gold essence compound; (3) The fingerprint spectrum of the gold essence compound and the method for determining the content of multiple components as described in any one of claims 1-4, or the application of the fingerprint spectrum of the gold essence compound as described in claim 7 in the batch stability analysis of the gold essence compound; (4) The fingerprint spectrum of Jinjing compound and the method for determining the content of multiple components as described in any one of claims 1-4, or the application of the fingerprint spectrum of Jinjing compound as described in claim 7 in the identification of the authenticity of Jinjing compound; (5) The fingerprint spectrum of Jinjing compound and the method for determining the content of multiple components as described in any one of claims 1-4, or the application of the fingerprint spectrum of Jinjing compound as described in claim 7 in the determination of the content of components in Jinjing compound.

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