A method for simultaneously determining the contents of six active ingredients in Hericium erinaceus extract by HPLC

Abstract

This invention discloses an HPLC method for simultaneously determining the content of six active ingredients in Hericium erinaceus extract, belonging to the field of natural compound detection and analysis technology. The detection steps of this invention include: preparing a mixed standard solution using Hericium erinaceus ketone E, Hericium erinaceus ketone C, Hericium erinaceus ketone D, Hericium erinaceus D, ergosterol, and Hericium erinaceus C as reference standards; analyzing the mixed standard solution using high-performance liquid chromatography (HPLC); plotting standard curves for the six active ingredients with the concentration of the mixed standard solution as the abscissa and the peak area as the ordinate; determining the linear regression equation; and calculating the content of each active ingredient in the sample solution. The HPLC method for simultaneously determining six active ingredients in Hericium erinaceus extract established in this invention has good selectivity, high sensitivity, accuracy, and reliability, providing a new analytical method and basis for the quality analysis and quality control of Hericium erinaceus extract.

Description

Technical Field

[0001] This invention belongs to the field of natural compound detection and analysis technology, specifically relating to an HPLC (high performance liquid chromatography) method for simultaneously determining the content of six active ingredients in Hericium erinaceus extract. Background Technology

[0002] Hericium erinaceus, also known as monkey head mushroom, belongs to the phylum Basidiomycota and family Hericaceae. Both its fruiting body and mycelium have significant medicinal value. Modern pharmacological studies have shown that Hericium erinaceus contains various active ingredients such as polysaccharides, small molecule peptides, terpenes, and phenols, exhibiting protective effects on the gastrointestinal mucosa, anti-inflammatory properties, antioxidant effects, immune regulation, and neuroprotective effects. It is noteworthy that although crude extracts or decoctions of Hericium erinaceus have shown significant efficacy in clinical applications, the content of its small molecule active substances is extremely low, and the mushrooms exhibit high structural diversity and similar physicochemical properties, making it difficult to obtain standard samples.

[0003] Due to a lack of standard samples, research on the efficacy mechanisms of small molecule components and drug quality control remain bottlenecks, severely hindering the in-depth development and international application of Hericium erinaceus. The entire Hericium erinaceus industry also lacks standardized testing and analytical methods. Hericium erinaceus ketones, as important active ingredients in Hericium erinaceus, show potential in neuroprotection and cognitive enhancement. Therefore, quantitative research on the small molecule active ingredients in Hericium erinaceus extracts to improve the utilization rate of Hericium erinaceus ketones is extremely meaningful. Summary of the Invention

[0004] Objective of the invention: To address the shortcomings of existing methods for quantitative analysis of small molecule active ingredients in Hericium erinaceus, this invention provides an HPLC method for simultaneously determining the content of six active ingredients in Hericium erinaceus extract. The HPLC method provided by this invention features high selectivity, high sensitivity, accuracy, and reliability, effectively solving the problem of quantitative analysis of small molecule components in Hericium erinaceus caused by the scarcity of standard samples. This provides reliable technical support for the quality control, pharmacodynamic material basis research, and subsequent in-depth development of Hericium erinaceus pharmaceuticals.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows: an HPLC detection method for simultaneously determining the content of six active ingredients in Hericium erinaceus extract, comprising the following steps:

[0006] (1) Prepare mixed standard solutions of hericenone E, hericenone C, hericenone D, hericene D, ergosterol, and hericene C at different concentrations. Analyze them using high performance liquid chromatography. Plot the standard curves of hericenone E, hericenone C, hericenone D, hericene D, ergosterol, and hericene C with the concentration of the single standard as the x-axis and the corresponding peak area as the y-axis, and determine the linear regression equations.

[0007] (2) Prepare a Hericium erinaceus extract solution and analyze it using high performance liquid chromatography to determine the content of the extract. six Peak area of ​​each active ingredient;

[0008] (3) Based on the peak area determined in step (2), compare it with the standard curve and regression equation in step (1) to calculate the contents of Hericium ergium ketone E, Hericium ergium ketone C, Hericium ergium ketone D, Hericium ergium styracin D, ergosterol and Hericium ergium styracin C in the sample solution.

[0009] Preferably, the mixed standard solution curve has five concentration gradients, with the latter four gradients obtained by diluting the first gradient. The concentrations of each component in the five concentrations of the mixed standard solution are as follows:

[0010]

[0011] In step (1), the solvent for mixing the standard is methanol.

[0012] In steps (1) and (2), the detection wavelength for analysis using a high-performance liquid chromatograph is 290-310 nm.

[0013] Furthermore, in steps (1) and (2), the detection wavelength for analysis using a high-performance liquid chromatograph is 295 nm.

[0014] Furthermore, the detection wavelength for Hericium erinaceus ketone E, Hericium erinaceus ketone C, Hericium erinaceus ketone D, Hericium erinaceus D, ergosterol, and Hericium erinaceus C is 295 nm.

[0015] In steps (1) and (2), the chromatographic conditions for analysis using a high-performance liquid chromatograph include: a Waters Symmetry C18 column as the stationary phase, with a size of 4.6×250mm and a particle size of 3.5μm, and a column temperature of 25-30℃.

[0016] In steps (1) and (2), the chromatographic conditions for analysis using a high-performance liquid chromatograph include: mobile phase A is a 0.1-0.3% formic acid aqueous solution; mobile phase B is a pure methanol solution; and a specific gradient elution program is used.

[0017] The gradient elution procedure is as follows:

[0018]

[0019] The flow rates of mobile phase A and mobile phase B are 0.5-0.7 mL / min.

[0020] In steps (1) and (2), the injection volume of high performance liquid chromatography is 2-5 μL.

[0021] Furthermore, in steps (1) and (2), the chromatographic conditions for analysis using high performance liquid chromatography include: a Waters Symmetry C18 column as the stationary phase, with dimensions of 4.6 × 250 mm and a particle size of 3.5 μm; mobile phase A being 0.1% formic acid in water; mobile phase B being a pure methanol solution; a flow rate of 0.6 mL / min; an injection volume of 5 μL; a column temperature of 30 °C; and gradient elution.

[0022] The preparation method of the Hericium erinaceus extract is as follows: add an appropriate amount of alcohol solution to dried Hericium erinaceus powder, reflux extract at 40-80℃ for 4-8 hours, and take the extracted solution for analysis.

[0023] As a preferred method, the dried Hericium erinaceus powder was extracted at a material-to-liquid ratio of 1:10 (g / mL). The crude extract contained Hericium erinaceus ketone E, Hericium erinaceus ketone C, Hericium erinaceus ketone D, Hericium erinaceus D, ergosterol, and Hericium erinaceus C at contents of 0.27 mg / g, 1.71 mg / g, 0.56 mg / g, 0.26 mg / g, 3.61 mg / g, and 0.31 mg / g, respectively.

[0024] The alcohol solution is a 50-90% aqueous ethanol solution.

[0025] This invention proposes for the first time an HPLC method for simultaneously determining the content of six active ingredients in Hericium erinaceus extract. The main design and improvement of this invention are as follows:

[0026] 1. Optimized HPLC chromatographic conditions:

[0027] (1) Selection of chromatographic column: Waters Symmetry C18 column (4.6×250mm, particle size 3.5μm) was used. This stationary phase has good separation efficiency and stability and is suitable for the analysis of complex natural products.

[0028] (2) Optimization of mobile phase: Gradient elution was performed using 0.1% formic acid water (mobile phase A) and pure methanol (mobile phase B). The addition of formic acid adjusted the pH of the mobile phase, which improved the peak shape symmetry and resolution.

[0029] (3) Gradient elution program: The ratio of the mobile phase was controlled by a fine time gradient (0-60 minutes) to achieve baseline separation of the six components.

[0030] (4) Detection conditions: The detection wavelength was set to 295 nm, based on the maximum absorption of the six components at this wavelength to ensure high sensitivity; other parameters such as flow rate (0.6 mL / min), injection volume (5 μL) and column temperature (30 °C) were optimized to balance analysis speed and resolution.

[0031] 2. Extraction process optimization:

[0032] (1) Extraction solvent: By comparing 50%, 70% and 90% ethanol, 90% ethanol was determined to be the best extraction solvent, which can maximize the dissolution of active ingredients.

[0033] (2) Extraction temperature and time: Optimized to reflux extraction at 40℃, 60℃ and 80℃ for 4 hours to ensure extraction efficiency while avoiding component degradation.

[0034] 3. Methodological validation:

[0035] (1) Linearity and sensitivity: The standard curves of the six components showed good linearity (R² ≥ 0.9997), and the limits of detection (LOD) and quantitation (LOQ) were both low (e.g., the LOD of Hericium erinaceus E was 0.277 μg / mL and the LOQ was 0.833 μg / mL), indicating that the method was highly sensitive.

[0036] (2) Precision and stability: The RSD of the precision experiment is < 2%, and the RSD of the stability experiment is < 2% within 24 hours, which proves that the method has good repeatability and the solution is stable.

[0037] (3) Specificity and recovery: The chromatogram showed good separation of each component (the separation of adjacent peaks was greater than 1.5), and the recovery rate was between 80% and 120% (RSD < 2%), ensuring accuracy and reliability.

[0038] The measurement method designed in this invention has the following advantages:

[0039] 1. A single injection can simultaneously quantify six active ingredients, significantly improving analytical efficiency. Traditional methods have not yet achieved the simultaneous detection of these six active ingredients.

[0040] 2. This method exhibits high sensitivity and accuracy. By optimizing the conditions, the method can detect components down to the μg / mL level, with excellent recovery and linearity, making it suitable for quality control.

[0041] 3. This method is practical and reliable. It has undergone comprehensive validation (linearity, precision, stability, etc.) and can be directly applied to the quality control of Hericium erinaceus extract, providing a standardized analytical basis for the industry.

[0042] The key focus of this invention is the simultaneous determination of multiple components and optimization of system conditions. By integrating chromatographic separation, extraction processes, and methodological validation, it achieves optimization of the entire process from sample pretreatment to instrumental analysis. Specifically: 1. It not only focuses on HPLC chromatographic conditions but also simultaneously optimizes extraction solvents, temperature, and time to ensure the applicability of the method to actual samples. 2. Addressing the problems of insufficient standards for small molecule components in Hericium erinaceus, poor resolution, and low detection efficiency in existing technologies, a detection scheme for the simultaneous detection of multiple components is designed, avoiding errors and resource waste caused by multiple detections. 3. Optimal parameters are determined through numerous comparative experiments (such as different gradient programs, mobile phase additives, and extraction conditions) to ensure the scientific reliability of the method, as detailed in Examples 1-4.

[0043] This invention is an HPLC chromatographic method based on gradient elution and mobile phase optimization, specifically including: 1. Fine design of the gradient elution program: The time program ranges from 0 to 60 minutes, and the complete separation of six components with similar physicochemical properties is achieved through multi-stage adjustments of the mobile phase ratio. 2. Addition of 0.1% formic acid to the mobile phase: This design significantly improves peak shape symmetry and retention time, avoiding column damage caused by the use of strong acids. 3. Synergistic selection of the chromatographic column and detection wavelength: A C18 column and a 295nm detection wavelength are used, with customized matching based on the hydrophobicity and UV absorption characteristics of the active ingredients in Hericium erinaceus.

[0044] This invention establishes for the first time an HPLC method for the simultaneous detection of six specific active components in Hericium erinaceus. The key design feature of this invention, "using 0.1% formic acid in mobile phase A combined with a specific gradient elution procedure (Table 2)," is a novel approach. 0.1% formic acid significantly shortens retention time and improves peak symmetry, while traditional methods often use ultrapure water or phosphate buffer, leading to peak tailing or insufficient separation. Compared to existing technologies, this method solves the co-elution problem: specificity experiments show no interference from the six active components, achieving baseline separation. It improves detection efficiency: existing methods often require separate detection or complex pretreatment. This invention, by simultaneously determining six components in a single injection, shortens the analysis time to within 60 minutes, significantly improving efficiency. It establishes a standardized method: existing technologies lack unified standards for Hericium erinaceus detection. This invention provides complete validation data (linearity, precision, recovery, etc.), enabling the method to be promoted as an industry standard.

[0045] This invention employs high-performance liquid chromatography (HPLC) technology. By systematically optimizing key parameters such as chromatographic column, mobile phase composition, gradient elution program, column temperature, and detection wavelength, an analytical method has been established that can simultaneously, rapidly, and accurately separate and quantify seven key active ingredients (including but not limited to specific hericium erinaceus ketones, terpenes, and phenolic substances) in Hericium erinaceus extract.

[0046] Beneficial effects: Compared with the prior art, the method of the present invention has the following significant advantages:

[0047] (1) High selectivity: The established chromatographic conditions can effectively separate the six target components from the complex matrix in the extract. The peaks of each component are well-shaped and the separation degree meets the requirements of quantitative analysis, avoiding interference from co-elution peaks.

[0048] (2) High sensitivity: The method has low limit of detection (LOD) and limit of quantitation (LOQ), and can accurately detect trace amounts of small molecule active ingredients in Hericium erinaceus extract. The peak area RSD (%) of the seven components is less than 20%, and the signal-to-noise ratio is greater than or equal to 3.

[0049] (3) Accurate and reliable: The method has been verified and has shown excellent performance in terms of linear range, specificity, precision (intra-day and inter-day), stability, repeatability and recovery rate. The results are accurate, reliable and highly reproducible.

[0050] (4) High efficiency and practicality: This method has a short analysis time and is easy to operate. It is suitable for routine quality inspection and control of Hericium erinaceus raw materials, intermediates and finished products, and has good prospects for promotion and application.

[0051] (5) Through the implementation of this invention, the content of key active ingredients of Hericium erinaceus can be accurately controlled, laying a solid analytical technology foundation for the standardized production, activity evaluation and international market development of Hericium erinaceus-related products.

[0052] The HPLC detection method established in this invention for simultaneously determining six active ingredients in Hericium erinaceus extract has good selectivity, high sensitivity, accuracy and reliability, providing a new analytical method and basis for the quality analysis and quality control of Hericium erinaceus extract. Attached Figure Description

[0053] Figure 1 This is the standard curve of Hericium erinaceus ketone E.

[0054] Figure 2 This is the standard curve of Hericium erinaceus ketone C.

[0055] Figure 3 This is the standard curve for Hericium erinaceus ketone D.

[0056] Figure 4This is the standard curve for Hericium erinaceus D.

[0057] Figure 5 This is the ergosterol standard curve.

[0058] Figure 6 This is the standard curve for Hericium erinaceus C.

[0059] Figure 7 This is the chromatogram of mixed standard solution M when the chromatographic column is Waters Symmetry C18 (250 mm × 4.6 mm, 3.5 μm).

[0060] Figure 8 This is the chromatogram of Hericium erinaceus test sample solution N when the chromatographic column is Waters Symmetry C18 (250 mm × 4.6 mm, 3.5 μm).

[0061] Figure 9 This is the chromatogram of Hericium erinaceus test solution N when the chromatographic column is an Agilent Eclipse Plus C18 (250 mm × 4.6 mm, 5 μm).

[0062] Figure 10 This is the chromatogram of Hericium erinaceus test solution N when the chromatographic column is Waters Hypersil Gold AQ (250 mm × 4.6 mm, 5 μm). Detailed Implementation

[0063] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0064] Example 1

[0065] An HPLC method for simultaneously determining the content of six active ingredients in Hericium erinaceus extract includes the following steps:

[0066] (1) Prepare mixed standard solutions of hericenone E, hericenone C, hericenone D, hericene D, ergosterol, and hericene C at different concentrations. Analyze them using high performance liquid chromatography. Plot the standard curves of hericenone E, hericenone C, hericenone D, hericene D, ergosterol, and hericene C with the concentration of the single standard as the x-axis and the corresponding peak area as the y-axis, and determine the linear regression equations.

[0067] (2) Prepare Hericium erinaceus extract solution and analyze it using high performance liquid chromatography to determine the peak areas of six active ingredients in the extract;

[0068] (3) Based on the peak area determined in step (2), compare it with the standard curve and regression equation in step (1) to calculate the contents of Hericium ergium ketone E, Hericium ergium ketone C, Hericium ergium ketone D, Hericium ergium styracin D, ergosterol and Hericium ergium styracin C in the sample solution.

[0069] An investigation of different gradient elution procedures for extracts of Hericium erinaceus.

[0070] In this study, reference stock solutions of Hericium erinaceus E, C, D, D, ergosterol, and C were prepared separately using methanol. These stock solutions were then diluted with methanol to prepare reference standard positioning solutions and mixed reference solutions. The concentrations of Hericium erinaceus E, C, D, D, D, ergosterol, and C in the mixed reference solution were 0.100 mg / mL, 0.200 mg / mL, 0.100 mg / mL, 0.400 mg / mL, 0.080 mg / mL, and 0.060 mg / mL, respectively. Subsequently, the solutions were sequentially diluted to different concentrations and filtered through a 0.22 μm membrane. A standard curve was plotted with peak area on the ordinate and standard concentration on the abscissa, as detailed in Example 7.

[0071] The preparation method of Hericium erinaceus extract was as follows: 50 mL of 90% alcohol solution was added to 5 g of dried Hericium erinaceus powder, and the mixture was refluxed at 60 °C for 4 h. When exploring the chromatographic method, a Waters Symmetry C18 column (4.6 × 250 mm, particle size 3.5 μm) was used. Ultrapure water was used as mobile phase A, and methanol as mobile phase B. Elution was performed according to the mobile phase gradient in Table 1. The detection wavelength was 295 nm, the flow rate was 0.6 mL / min, the column temperature was 30 °C, and the injection volume was 5 μL. The experimental results are shown in Table 1.

[0072] Table 1 shows the separation effects of different gradient elution programs on six active ingredients from Hericium erinaceus.

[0073]

[0074] As shown in Table 1, the optimization of the mobile phase ratio mainly involves adjusting the mobile phase ratio within the 5-28 minute timeframe. Originally, mobile phase A decreased from 90% to 0% within 5-28 minutes. After optimization, mobile phase B increased from 10% to 100%. Mobile phase A rapidly decreased from 90% to 2% and remained stable, while mobile phase B rapidly increased to 98%. This adjustment resulted in a more refined chromatographic separation process and effectively improved the separation effect. Based on this, an exploratory experiment was conducted to investigate the effect of different additives in the gradient eluent on the peak elution. The chromatographic conditions used were: a Waters Symmetry C18 column, gradient elution of mobile phases A and B according to the ratios shown in Table 1, a detection wavelength of 295 nm, a flow rate of 0.6 mL / min, and an injection volume of 5 μL. The experimental results are shown in Table 2.

[0075] Table 2 shows the separation effect of different mobile phase additives on six active components of Hericium erinaceus.

[0076]

[0077] Experiments were conducted to explore mobile phase additives. According to the experimental results in Table 2, adding 0.1% formic acid and 0.2% formic acid to mobile phase A both yielded excellent peak shape effects. However, the pH of 0.2% formic acid solution is close to the lower limit of silica gel tolerance, which increases the risk of long-term use. Based on this, the optimal gradient elution scheme was obtained: Mobile phase: 0.1% formic acid solution (A) - methanol (B), gradient elution (0-5 min, 90% A, 10% B; 5-10 min, 90% → 2% A, 10 → 98% B; 10-28 min, 2% A, 98% B; 28-29 min, 2%~0% A, 98%~100%; 29-56 min, 0% A, 100% B; 56-58 min, 0%~90% A, 100%~10% B; 58-60 min, 90% A, 10% B).

[0078] Example 2

[0079] Experiments on the separation performance of Hericium erinaceus extract samples using different chromatographic columns

[0080] To further optimize the separation effect of Hericium erinaceus extract samples, the influence of different chromatographic columns on their separation performance was investigated. Comparative analysis was conducted using the optimal chromatographic conditions described in Example 1. Specific chromatographic conditions were as follows: mobile phase A was ultrapure water containing 0.1% formic acid, and mobile phase B was methanol; the gradient elution program was set as follows: 0–5 min, 90% A, 10% B; 5–10 min, 90% → 2% A, 10 → 98% B; 10–28 min, 2% A, 98% B; 28–29 min, 2%–0% A, 98%–100%; 29–56 min, 0% A, 100% B; 56–58 min, 0%–90% A, 100%–10% B; 58–60 min, 90% A, 10% B. The detection wavelength was 295 nm, the flow rate was 0.6 mL / min, and the injection volume was 5 μL.

[0081] Three chromatographic columns were selected for comparison: Agilent Eclipse Plus C18 (250 mm × 4.6 mm, 5 μm), Waters Hypersil Gold AQ (250 mm × 4.6 mm, 5 μm), and Waters Symmetry C18 (250 mm × 4.6 mm, 3.5 μm) used in Example 1. Figure 8 , Figure 9 and Figure 10 The images show the chromatograms of Hericium erinaceus test solutions obtained using Waters Symmetry C18 (250 mm × 4.6 mm, 3.5 μm), Agilent Eclipse Plus C18 (250 mm × 4.6 mm, 5 μm), and Waters Hypersil Gold AQ (250 mm × 4.6 mm, 5 μm) columns. The chromatograms show that the Waters Symmetry C18 (250 mm × 4.6 mm, 3.5 μm) column produced more elution peaks and demonstrated better resolution.

[0082] Example 3

[0083] Experiment on the concentration of alcohol solvent for extracting Hericium erinaceus samples

[0084] Dilute ethanol (50%), 70%, and 90% ethanol were used as extraction solvents. Using the same batch of sample, 50 mL of ethanol of different concentrations was added to 5 g of Hericium erinaceus powder, and extraction was performed at 60°C for 4 h. Analysis was conducted according to the optimal chromatographic scheme obtained in Example 1. The results showed that 90% ethanol was the best extraction solvent, with more significant peak height. The peak shape and resolution met the system adaptability requirements. 90% ethanol was chosen as the extraction solvent for the content determination of this product. The results are shown in Table 3.

[0085] Table 3 shows the peak areas of six active components from Hericium erinaceus extracted with different concentrations of ethanol solvent.

[0086]

[0087] Example 4

[0088] Experiment on the extraction temperature of Hericium erinaceus samples

[0089] To investigate whether temperature affects the content of six active ingredients in Hericium erinaceus in this extraction method, reflux extraction with 90% ethanol at 40℃, 60℃, and 80℃ was performed. The same batch of Hericium erinaceus powder was used for content determination according to the optimal chromatographic scheme obtained in Example 1. The results showed that the Hericium erinaceus sample extracted best by reflux in a water bath at 60℃. Therefore, 60℃ was selected as the heating temperature for the heat extraction method. The experimental results are shown in Table 4.

[0090] Table 4 shows the peak areas of six active ingredients in Hericium erinaceus at different extraction temperatures.

[0091]

[0092] Example 5

[0093] Experiment on the extraction time of Hericium erinaceus samples

[0094] Using the same batch of Hericium erinaceus samples, extraction times of 4h, 6h, and 8h were performed during the experiment. The optimal elution protocol obtained in Example 1 was followed to investigate whether this condition affected the determination of the content of six active ingredients in Hericium erinaceus. The results showed that extraction times of 4h, 6h, and 8h did not significantly differ in the results. Considering the actual situation, the 4h extraction time was adopted.

[0095] Example 6

[0096] The peak times of six active ingredient standards were simultaneously determined using HPLC, including:

[0097] (1) Prepare reference stock solutions of Hericium erinaceus E, Hericium erinaceus C, Hericium erinaceus D, Hericium erinaceus D, Ergosterol, and Hericium erinaceus C respectively with methanol. Dilute them with methanol to prepare reference positioning solutions and mixed reference solutions. The concentrations of Hericium erinaceus E, Hericium erinaceus C, Hericium erinaceus D, Hericium erinaceus D, Ergosterol, and Hericium erinaceus C in the mixed reference solution are 0.100 mg / mL, 0.200 mg / mL, 0.100 mg / mL, 0.400 mg / mL, 0.080 mg / mL, and 0.060 mg / mL, respectively.

[0098] (2) The mixed standard solution was analyzed by HPLC. The chromatographic conditions were as follows: stationary phase: Waters Symmetry C18 column, size 4.6 × 250 mm, particle size 3.5 μm; mobile phase A: 0.1% formic acid aqueous solution; mobile phase B: pure methanol; flow rate: 0.6 mL / min; detection wavelength: 295 nm; injection volume: 5 μL; column temperature: 30 °C. The mixed standard solution was subjected to gradient elution, and the elution program is shown in Table 5.

[0099] Table 5 Gradient elution program

[0100]

[0101] The elution times of each active ingredient are shown in Table 6.

[0102] Table 6 Peak times of each active ingredient

[0103]

[0104] Example 7

[0105] Method validation for simultaneous determination of six active components in Hericium erinaceus extract by HPLC.

[0106] (1) Examination of linear relationships

[0107] Stock solutions of reference standards for Hericium erinaceus E, C, D, D, ergosterol, and C were prepared separately with methanol. These were then diluted with methanol to prepare standard positioning solutions and mixed standard solutions. The concentrations of Hericium erinaceus E, C, D, D, D, ergosterol, and C in the mixed standard solution were 0.100 mg / mL, 0.200 mg / mL, 0.100 mg / mL, 0.400 mg / mL, 0.080 mg / mL, and 0.060 mg / mL, respectively. The solutions were then diluted sequentially to different concentrations, filtered through a 0.22 μm membrane, and a standard curve was plotted using the optimal chromatographic scheme obtained in Example 1, with peak area as the ordinate and standard concentration as the abscissa. The regression equation for the standard curve is as follows:

[0108] Hericium erinaceus ketone E: y = 7759.7x - 4758.4, R 2 =1.0000

[0109] Hericium erinaceus ketone C: y = 20165x - 17889, R 2 =1.0000

[0110] Hericium erinaceus ketone D: y = 17379x - 18922, R 2 =0.9997

[0111] Hericin D: y = 9837.1x - 5397.9, R 2 =0.9999

[0112] Ergosterol: y = 4970.5x - 12919, R 2 =1.0000

[0113] Hericin C: y = 7972.3x - 5003.5, R 2 =1.0000

[0114] This indicates that the linear relationship of each active substance is good, and their standard curves are shown in the figure below. Figure 1-6 .

[0115] (2) Specificity experiment

[0116] Accurately pipette the mixed standard solution M and the test solution N separately, and perform the determination according to the optimal chromatographic conditions in Example 1. Record the chromatograms as shown below. Figure 7-8 As shown ( Figure 7 and 8 Peaks 1-6 in the image correspond to Hericium erinacetate E, Hericium erinacetate C, Hericium erinacetate D, Hericium erinol D, and Hericium erinol C, respectively. Figure 8 As can be seen, Hericium ergium ketone E, Hericium ergium ketone C, Hericium ergium ketone D, Hericium ergium styracin D, Ergosterol, and Hericium ergium styracin C in the test solution all exhibited good chromatographic behavior, with no adjacent peaks having a resolution of less than 1.5, indicating good specificity.

[0117] (3) Precision test

[0118] Mixed standard solution M was injected six times, with an injection volume of 5 μL. The chromatographic conditions were determined according to the optimal conditions described in Example 1. Peak areas were recorded, and RSD (relative standard deviation) was calculated. The experimental results showed that the peak area RSD (%) of Hericium erinacetate E, Hericium erinacetate C, Hericium erinacetate D, Hericium erinol D, ergosterol, and Hericium erinol C were all less than 2.0%, indicating good instrument precision.

[0119] Table 7 Precision Experiment Results

[0120]

[0121] (4) Stability test

[0122] Take mixed standard solution M and inject it at 0, 3, 6, 9, 12, and 24 hours (numbered 1-6 in sequence). Measure the peak area and calculate the RSD according to the optimal chromatographic conditions in Example 1.

[0123] As shown in Table 8, the peak area RSD (%) of Hericium ergium ketone E, Hericium ergium ketone C, Hericium ergium ketone D, Hericium ergium styracin D, Ergosterol, and Hericium ergium styracin C is less than 2.0%, indicating that the solution is stable within 24 hours and can meet the determination requirements.

[0124] Table 8. Stability test results

[0125]

[0126] (5) Limit of detection and limit of quantitation tests

[0127] The limits of quantitation (LOQ) and detection (LOD) were determined using the signal-to-noise ratio (SNR) method. The reference standard was serially diluted according to the optimal chromatographic conditions described in Example 1. The measured signal was compared with the baseline noise, and the lowest concentration that could be accurately quantified was calculated. The concentration with a SNR of approximately 10:1 was designated as the LQ. In six repeated determinations, the peak area RSD (%) of Hericium ergium ketone E, Hericium ergium ketone C, Hericium ergium ketone D, Hericium ergium styracin D, Ergosterol, and Hericium ergium styracin C were all less than 10%, and the SNR was greater than or equal to 10. The measured signal was compared with the baseline noise, and the lowest detectable concentration was calculated. The concentration with a SNR of approximately 3:1 was designated as the LQ. The results are shown in Table 9. In three repeated determinations, the peak area RSD (%) of all seven components was less than 20%, and the SNR was greater than or equal to 3.

[0128] Table 9 Results of Limit of Detection and Limit of Quantification

[0129]

[0130] (6) Spiking recovery test

[0131] Precisely pipette a mixed standard solution of high, medium, and low concentrations of Hericium erinaceus ketone E, Hericium erinaceus ketone C, Hericium erinaceus ketone D, Hericium erinol D, and Hericium erinol C into a solution containing Hericium erinaceus extract of known concentrations. Inject 5 μL of the solution and determine the peak area under the optimal chromatographic conditions described in Example 1. Calculate the recovery rate and RSD. As shown in Table 10, the recoveries were all within the range of 80%-120%, and the RSDs were all less than 2.0%, indicating that the method has good recovery.

[0132] Table 10 Results of the recovery experiment

[0133]

[0134] (7) Repeatable experiments

[0135] To investigate the stability and reproducibility of the method under standard experimental conditions, a repeatability test was conducted. Following the optimal chromatographic conditions described in Example 1, three parallel test solutions were prepared from the same batch of Hericium erinaceus samples using the "Test Solution Preparation" method. These solutions were then sequentially injected and analyzed by the same operator on a single day using the same high-performance liquid chromatograph. The peak areas of Hericium erinaceus ketone E, Hericium erinaceus ketone C, Hericium erinaceus ketone D, Hericium erinaceus extract D, ergosterol, and Hericium erinaceus extract C were recorded, and the relative standard deviation (RSD) of each component's content was calculated.

[0136] Table 11 shows that the RSD (n=3) of the contents of the six active ingredients were all between 0.95% and 1.98%, indicating that the method has good repeatability and can meet the requirements of routine analysis and detection.

[0137] Table 11 Results of Repeatability Experiments

[0138]

[0139] Example 8

[0140] Six active components in Hericium erinaceus extract were simultaneously determined by HPLC.

[0141] Six active substances in the ethanol extract of Hericium erinaceus were simultaneously determined by HPLC, including:

[0142] (1) Take 5g of Hericium erinaceus powder and add 50mL of 90% ethanol. Reflux at 60℃ for 4h to extract. Obtain Hericium erinaceus ethanol extract.

[0143] (2) After centrifugation, the supernatant was collected, filtered through a 0.22 μm membrane, and analyzed using high performance liquid chromatography (HPLC). The peak areas corresponding to the sample solution and the mixed standard were measured separately. The determination was performed under the optimal chromatographic conditions in Example 1, and the results are shown in Table 12.

[0144] Table 12 Results of Active Ingredient Determination in Example 3

[0145]

[0146] Note: Quantification is based on the respective standard, and the unit is mg / g.

[0147] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. An HPLC method for simultaneously determining the content of six active ingredients in Hericium erinaceus extract, characterized in that, Includes the following steps: (1) Prepare mixed standard solutions of hericenone E, hericenone C, hericenone D, hericene D, ergosterol, and hericene C at different concentrations. Analyze them using high performance liquid chromatography. Plot the standard curves of hericenone E, hericenone C, hericenone D, hericene D, ergosterol, and hericene C with the concentration of the single standard as the x-axis and the corresponding peak area as the y-axis, and determine the linear regression equations. (2) Prepare Hericium erinaceus extract solution and analyze it using high performance liquid chromatography to determine the peak areas of six active ingredients in the extract; (3) Based on the peak area determined in step (2), compare it with the standard curve and regression equation in step (1) to calculate the contents of Hericium ergium ketone E, Hericium ergium ketone C, Hericium ergium ketone D, Hericium ergium styracin D, ergosterol and Hericium ergium styracin C in the sample solution.

2. The HPLC detection method for simultaneously determining the content of six active ingredients in Hericium erinaceus extract according to claim 1, characterized in that, In step (1), the solvent for mixing the standards is methanol.

3. The HPLC detection method for simultaneously determining the content of six active ingredients in Hericium erinaceus extract according to claim 1, characterized in that, In steps (1) and (2), the detection wavelength for analysis using a high-performance liquid chromatograph is 290-310 nm.

4. The HPLC detection method for simultaneously determining the content of six active ingredients in Hericium erinaceus extract according to claim 1, characterized in that, In steps (1) and (2), the chromatographic conditions for analysis using a high-performance liquid chromatograph include: a Waters Symmetry C18 column as the stationary phase, with dimensions of 4.6 × 250 mm and a particle size of 3.5 μm, and a column temperature of 25-30 °C.

5. The HPLC method for simultaneously determining the content of six active ingredients in Hericium erinaceus extract according to claim 1, characterized in that, In steps (1) and (2), the chromatographic conditions for analysis using a high-performance liquid chromatograph include: mobile phase A is a 0.1-0.3% formic acid aqueous solution; mobile phase B is a pure methanol solution.

6. The HPLC method for simultaneously determining the content of six active ingredients in Hericium erinaceus extract according to claim 5, characterized in that, The preferred gradient elution procedure is as follows: 。 7. The HPLC method for simultaneously determining the content of six active ingredients in Hericium erinaceus extract according to claim 6, characterized in that, The flow rates of mobile phase A and mobile phase B are 0.5-0.7 mL / min.

8. The HPLC method for simultaneously determining the content of six active ingredients in Hericium erinaceus extract according to claim 1, characterized in that, In steps (1) and (2), the injection volume for analysis using a high-performance liquid chromatograph is 2-5 μL.

9. The HPLC method for simultaneously determining the content of six active ingredients in Hericium erinaceus extract according to claim 1, characterized in that, The preparation method of the Hericium erinaceus extract is as follows: add an alcohol solution to dried Hericium erinaceus powder, reflux extract at 40-80℃ for 4-8 hours, and take the extracted solution for analysis.

10. The HPLC method for simultaneously determining the content of six active ingredients in Hericium erinaceus extract according to claim 9, characterized in that, The alcohol solution is a 50-90% aqueous ethanol solution.

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