A method for simultaneously detecting ergothioneine and ganoderic acid A in ganoderma and lentinula oral liquid
By using high-performance liquid chromatography and a JADE-PAK NH2 column, the problem of detecting ergothionein and ganoderic acid A in Ganoderma lucidum and shiitake mushroom oral liquid was solved, achieving rapid and accurate simultaneous detection.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- XIAMEN OCEAN VOCATIONAL & TECH COLLEGE
- Filing Date
- 2026-04-29
- Publication Date
- 2026-07-10
AI Technical Summary
Existing technologies cannot simultaneously and efficiently detect the content of ergothioneine and ganoderic acid A in Ganoderma lucidum and shiitake mushroom oral liquid; the detection methods are cumbersome and inefficient.
High-performance liquid chromatography (HPLC) was employed, using a JADE-PAK NH2 column, and combining specific column temperature, detection wavelength, and mobile phase composition to achieve simultaneous detection of ergothioneine and ganoderic acid A in Ganoderma lucidum and shiitake mushroom oral liquid.
The method enables accurate and rapid simultaneous determination of ergothioneine and ganoderic acid A in Ganoderma lucidum and shiitake mushroom oral liquid. The detection method is simple, specific, and reproducible, making it suitable for high-efficiency detection.
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Figure CN122361663A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the technical field of detecting active ingredients in Ganoderma lucidum and shiitake mushroom oral liquid, specifically to a method for simultaneously detecting ergothioneine and ganoderic acid A in Ganoderma lucidum and shiitake mushroom oral liquid. Background Technology
[0002] Ganoderma lucidum and shiitake mushroom oral liquid has become a popular health food and functional beverage due to its immune-regulating, antioxidant, and anti-fatigue effects. However, current products generally use crude polysaccharides as the main efficacy indicator, lacking the determination of other active ingredients in Ganoderma lucidum and shiitake mushroom oral liquid. In particular, ergothioneine and ganoderic acid A both have clear biological activities (for example, ergothioneine has free radical scavenging and antioxidant effects). Therefore, including ergothioneine and ganoderic acid A in the main efficacy indicator of Ganoderma lucidum and shiitake mushroom oral liquid will become an inevitable trend.
[0003] However, current detection methods can only determine ergothioneine and ganoderic acid A separately, which is cumbersome and inefficient. For example, ergothioneine detection typically uses HILIC or amino columns, while ganoderic acid A detection generally uses a C18 column at a detection wavelength of 254 nm. There is a lack of methods that can simultaneously detect ergothioneine and ganoderic acid A in Ganoderma lucidum and shiitake mushroom oral liquid. Summary of the Invention
[0004] The purpose of this invention is to provide a method for simultaneously detecting ergothioneine and ganoderic acid A in Ganoderma lucidum and shiitake mushroom oral liquid, aiming to improve the problem of the lack of a detection method for simultaneously detecting ergothioneine and ganoderic acid A in Ganoderma lucidum and shiitake mushroom oral liquid.
[0005] To achieve the above objectives, the present invention adopts the following technical solution: A method for simultaneously detecting ergothioneine and ganoderic acid A in Ganoderma lucidum and shiitake mushroom oral liquid includes the following steps: High performance liquid chromatography was used to detect the effects of Ganoderma lucidum and shiitake mushroom oral liquid. The high-performance liquid chromatography (HPLC) method uses a JADE-PAK NH2 column with a column temperature of 30–40 °C and an ultraviolet (UV) detector with a detection wavelength of 252–256 nm. The mobile phase comprises mobile phase A (acetonitrile solution) and mobile phase B (0.1% (v / v) aqueous acetic acid solution). The volume ratio of mobile phase A to mobile phase B is (74–76):(24–26), and the flow rate is 0.9–1.1 mL / min. -1 .
[0006] Preferably, the chromatographic column has a size of 250 mm × 4.6 mm and a diameter of 5 μm.
[0007] Preferably, the injection volume is 3 μL.
[0008] Preferably, the column temperature of the chromatographic column is 35°C.
[0009] Preferably, the detection wavelength of the detector is 254 nm.
[0010] Preferably, the flow rate of the mobile phase is 1.0 mL·min. -1 .
[0011] Preferably, the volume ratio of mobile phase A to mobile phase B is 75:25.
[0012] Preferably, the Ganoderma lucidum and shiitake mushroom oral liquid is centrifuged at 5000 r / min for 20 min, the supernatant is collected, filtered through a 0.22 μm microporous membrane, and then the Ganoderma lucidum and shiitake mushroom oral liquid is detected by high performance liquid chromatography.
[0013] By adopting the above technical solution, the present invention has the following advantages compared with the prior art: 1. This invention uses a JADE-PAK NH2 amino column for the detection of Ganoderma lucidum and shiitake mushroom oral liquid, and through optimization of chromatographic conditions, it can achieve accurate and rapid simultaneous determination of the contents of ganoderic acid A and ergothioneine in Ganoderma lucidum and shiitake mushroom oral liquid.
[0014] 2. The detection method of the present invention is simple to operate and has good separation effect. Its specificity, recovery rate, robustness and repeatability all meet the detection requirements. It is suitable for the efficient simultaneous detection of ganoderic acid A and ergothionein in Ganoderma lucidum and shiitake mushroom oral liquid. Attached Figure Description
[0015] Figure 1 The HPLC chromatograms of the reference solution and the test solution in the specificity test described in this invention are shown below. Figure 2 This is the HPLC chromatogram of ganoderic acid A and ergothionein in different stationary phases during the comparative experiment of different chromatographic columns described in this invention. Detailed Implementation
[0016] To facilitate understanding of the present invention, a more comprehensive description will be provided below. Although preferred embodiments of the invention are given herein, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed in accordance with the technical conditions described in the literature in the art or as per the product specification.
[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.
[0018] In this invention, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features.
[0019] In this invention, numerical ranges are involved. Unless otherwise specified, the numerical ranges are considered continuous and include the minimum and maximum values of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values of the range. Additionally, when multiple ranges are provided to describe features or characteristics, the ranges may be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are included.
[0020] This document only specifically discloses some numerical ranges. However, any lower limit can be combined with any upper limit to form an unspecified range; and any lower limit can be combined with other lower limits to form an unspecified range, just as any upper limit can be combined with any other upper limit to form an unspecified range. Furthermore, each individually disclosed point or single value can itself serve as a lower or upper limit and be combined with any other point or single value or with other lower or upper limits to form an unspecified range.
[0021] Unless otherwise specified, the temperature parameters in this invention can be either constant temperature processing or processing within a certain temperature range. The constant temperature processing allows temperature fluctuations within the precision range controlled by the instrument.
[0022] In the description of the invention, "multiple" means at least two, such as two, three, etc., unless otherwise explicitly specified.
[0023] Unless otherwise specified, all embodiments and optional embodiments of the present invention can be combined with each other to form new technical solutions. Unless otherwise specified, all technical features and optional technical features of the present invention can be combined with each other to form new technical solutions.
[0024] Unless otherwise specified, all steps of the present invention may be performed sequentially or randomly, but are preferably performed sequentially.
[0025] The reagents or instruments used, unless otherwise specified by the manufacturer, are all commercially available conventional products.
[0026] The instruments and materials used in this invention are as follows: JADE-PAK NH2 column (250mm×4.6mm, 5μm), LC-16 high performance liquid chromatograph equipped with SPD-16 detector (Shimadzu Corporation, Japan), TG16-WS benchtop centrifuge (Hunan Xiangyi Laboratory Instrument Development Co., Ltd.), Ganoderma lucidum and shiitake mushroom oral liquid (batch number 25031204) provided by Xiamen Shancun Biotechnology Co., Ltd., Ganoderma lucidum acid A reference standard (purity 98%) from Shanghai Yuanye Biotechnology Co., Ltd., L-(+)-ergothioneine reference standard (purity 98%) purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., acetonitrile (chromatographic grade) purchased from Guangzhou Tongyuan Chemical Technology Co., Ltd., methanol (chromatographic grade) purchased from Guangzhou Tongyuan Chemical Technology Co., Ltd., deionized water (laboratory-made), acetic acid (chromatographic grade) purchased from Guangzhou Tongyuan Chemical Technology Co., Ltd., and 0.22μm microporous filter membrane (purchased from Tianjin Jinteng Experimental Equipment Co., Ltd.). Example 1
[0027] This embodiment provides a method for simultaneously detecting ergothioneine and ganoderic acid A in Ganoderma lucidum and shiitake mushroom oral liquid. Before detection, the Ganoderma lucidum and shiitake mushroom oral liquid is centrifuged at 5000 r / min for 20 min. The supernatant is collected, filtered through a 0.22 μm microporous membrane, and then injected using high performance liquid chromatography under the following chromatographic conditions: A JADE-PAK NH2 column was used, and the chromatographic conditions were as follows: column temperature 35℃, detection wavelength 254nm, and mobile phase flow rate 1.0 mL / min. -1 The injection volume was 3 μL, and the volume ratio of mobile phase A to mobile phase B was 75:25. Mobile phase A was a chromatographic grade acetonitrile solution, and mobile phase B was a 0.1% (V / V) aqueous solution of acetic acid. Example 2
[0028] The method for simultaneous detection of ergothioneine and ganoderic acid A in Ganoderma lucidum and shiitake mushroom oral liquid provided in this embodiment differs from that in Example 1 only in that: The column temperature was 40℃, and the mobile phase flow rate was 0.9 mL / min. -1 The detection wavelength was 252 nm, and the volume ratio of mobile phase A to mobile phase B was 74:26. Mobile phase A was a chromatographic grade acetonitrile solution, and mobile phase B was a 0.1% (V / V) aqueous solution of acetic acid. Example 3
[0029] The method for simultaneous detection of ergothioneine and ganoderic acid A in Ganoderma lucidum and shiitake mushroom oral liquid provided in this embodiment differs from that in Example 1 only in that: The column temperature was 37℃, and the mobile phase flow rate was 0.95 mL / min. -1 The detection wavelength was 253 nm, and the volume ratio of mobile phase A to mobile phase B was 75:25. Mobile phase A was a chromatographic grade acetonitrile solution, and mobile phase B was a 0.1% (V / V) aqueous solution of acetic acid. Example 4
[0030] The method for simultaneous detection of ergothioneine and ganoderic acid A in Ganoderma lucidum and shiitake mushroom oral liquid provided in this embodiment differs from that in Example 1 only in that: The column temperature was 32℃, and the mobile phase flow rate was 1.05 mL / min. -1 The detection wavelength was 255 nm, and the volume ratio of mobile phase A to mobile phase B was 74:26. Mobile phase A was a chromatographic grade acetonitrile solution, and mobile phase B was a 0.1% (V / V) aqueous solution of acetic acid. Example 5
[0031] The method for simultaneous detection of ergothioneine and ganoderic acid A in Ganoderma lucidum and shiitake mushroom oral liquid provided in this embodiment differs from that in Example 1 only in that: The column temperature was 30℃, and the mobile phase flow rate was 1.1 mL / min. -1 The detection wavelength was 256 nm, and the volume ratio of mobile phase A to mobile phase B was 76:24. Mobile phase A was a chromatographic grade acetonitrile solution, and mobile phase B was a 0.1% (V / V) aqueous solution of acetic acid. Test case
[0032] Preparation of reference solution: Weigh out ergothioneine reference standard and dissolve it in deionized water to prepare a solution of 100 mg / L. -1 Ergothioneine stock solution; weigh ganoderic acid A reference standard and prepare a 100 mg / L solution with methanol. -1 The stock solution of ganoderic acid A was prepared by transferring 1 mL of ergothioneine stock solution and 5 mL of ganoderic acid A stock solution into a 50 mL volumetric flask, sonicating for 10 min, and then diluting the mixture to the 50 mL mark with deionized water to obtain a 2 mg·L⁻¹ solution. -1 Ergothioneine and 10 mg·L -1 The reference solution containing ganoderic acid A was filtered through a 0.22 μm microporous membrane before use.
[0033] Preparation of test solution: Take 20 mL of Ganoderma lucidum and shiitake mushroom oral liquid, centrifuge at 5000 r / min for 20 min, and take the supernatant to obtain the test solution. Filter the test solution through a 0.22 μm microporous membrane for later use.
[0034] Methodological validation: 1. Specificity test Take the reference solution and the test solution separately, and inject them according to the chromatographic conditions described in Example 1. Record the chromatograms of the reference solution and the test solution (e.g., ...). Figure 1 As shown), where the chromatogram Figure 1 'a' is the chromatogram of the reference solution. Figure 1 b is the chromatogram of the test solution. Chromatography Figure 1 Chromatographic peak 1 in a corresponds to 10 mg·L⁻¹ -1 Ganoderma lucidum acid A, chromatographic peak 2 corresponds to 2 mg·L -1 Ergothioneine.
[0035] The results showed that the chromatogram of the test solution... Figure 1 The chromatogram of sample b is similar to that of the reference solution. Figure 1 The chromatographic peaks of ergothioneine and ganoderic acid A with basically the same retention time, and the resolution with adjacent chromatographic peaks is greater than 1.5, indicate that the method has good specificity.
[0036] 2. Precision test Six portions of the reference solution were injected six times under the chromatographic conditions described in Example 1 to examine the changes in retention time and peak area of ganoderic acid A and ergothioneine. The results are shown in Table 1.
[0037] Table 1. Precision test results of ganoderic acid A and ergothionein.
[0038] The results showed that the RSD of the retention time of ganoderic acid A in the reference solution was 0.17%, and the RSD of the peak area was 1.41%; the RSD of the retention time of ergothioneine was 0.97%, and the RSD of the peak area was 0.17%. This indicates that the instrument used in this test has good precision.
[0039] 3. Repeatability test Six portions of the test solution were taken and injected six times according to the chromatographic conditions described in Example 1 to investigate the changes in retention time and peak area of ganoderic acid A and ergothioneine. The results are shown in Table 2.
[0040] The formulas for calculating the content of ganoderic acid A and ergothioneine in the table below are: C = x / 10 · F = x / 10, where C represents the content of ganoderic acid A or ergothioneine (mg·100mL). -1 ), where x represents the concentration (mg·L) of ganoderic acid A or ergothioneine. -1 F represents the dilution factor, which is 1 in this experiment. The concentrations of ganoderic acid A and ergothioneine can be obtained by taking the average of the peak areas of ganoderic acid A and ergothioneine in the six groups in Table 2 and substituting them into the linear equation in Table 4.
[0041] Table 2. Repeatability test results of the test solution
[0042] The results showed that the RSDs of the retention times of ganoderic acid A and ergothioneine were 0.14% and 0.04%, respectively, and the RSDs of the peak areas of ganoderic acid A and ergothioneine were 0.81% and 1.16%, respectively, indicating that the method has good reproducibility.
[0043] 4. Standard Curve Creation Take 100 mg·L -1 Ergothioneine stock solution and 100 mg·L -1 The stock solution of ganoderic acid A was continuously diluted with deionized water and injected sequentially from high to low concentrations for analysis. The stock solution of ganoderic acid A was diluted to a concentration of 3.33 mg·L⁻¹. -1 6.67 mg·L -1 10.00 mg·L -1 13.33 mg·L -1 16.67 mg·L -1 Ergothioneine stock solution was diluted to a concentration of 0.67 mg·L⁻¹. -1 1.33 mg·L -1 2.00 mg·L -1 2.67 mg·L -1 3.33 mg·L -1 .
[0044] The injection concentration y (mg·L) of the stock solution -1 Plot a standard curve with the ordinate x and the peak area x as the abscissa.
[0045] Limits of detection and limits of quantitation: The diluted stock solutions were injected sequentially from high to low concentrations for detection, and the results are shown in Table 3. A standard curve was plotted based on the peak areas and corresponding solution concentrations obtained in Table 3. The signal-to-noise ratio (S / N) was used as the indicator for evaluating the limits of quantitation and detection. A signal-to-noise ratio of 3 (S / N=3) was used as the limit of detection (DL), and a signal-to-noise ratio of 10 (S / N=3) was used as the limit of quantitation (QL). The limits of detection for ganoderic acid A and ergothioneine were found to be 0.067 mg·L⁻¹. -1 and 0.107 mg·L -1， The limit of quantitation is 0.333 mg·L. -1 and 0.400 mg·L -1 .
[0046] Table 3. Detection results of ganoderic acid A and ergothioneine.
[0047] Table 4. Linear relationship, limit of detection, and limit of quantitation of ganoderic acid A and ergothioneine.
[0048] In Table 4, y represents the injection concentration of the stock solution (mg·L⁻¹). -1 ), x represents the area of the chromatographic peak.
[0049] The results showed that ganoderic acid A ranged from 3.33 to 16.67 mg·L⁻¹. -1 Within the concentration range, ergothioneine is 0.67–3.33 mg·L⁻¹. -1 Within the concentration range, it exhibits a good linear relationship.
[0050] 5. Recovery rate test To conduct a recovery test, pipette 5 mL of the test solution into a 10 mL volumetric flask, and add 10 mg / L of 80%, 100%, and 120% of the solution at three concentration levels: low, medium, and high, respectively. -1 Ganoderma lucidum acid A and 2 mg·L -1 Three samples of ergothioneine mixed standard solution were prepared for each concentration level, for a total of nine samples. The solutions were diluted with deionized water and sonicated for 10 minutes to achieve homogeneity.
[0051] The determination was carried out under the chromatographic conditions described in Example 1, with a retention time of 20 min. The contents of ganoderic acid A and ergothioneine were calculated based on the peak area. At the same time, the recovery rates of ganoderic acid A and ergothioneine were calculated. The results are shown in Table 5.
[0052] Table 5 Results of the recovery tests of ganoderic acid A and ergothioneine.
[0053] The results showed that the recoveries of ganoderic acid A at three different concentration levels were 103–107%, with RSDs of 0.93–3.18%; the recoveries of ergothioneine at three different concentration levels were 89.6–100%, with RSDs of 1.29–2.22%, which met the requirements of the 2025 edition of the Chinese Pharmacopoeia, indicating that the method has good accuracy.
[0054] 6. Durability test The analytical method was examined by adjusting experimental parameters, specifically the flow rate of the mobile phase (±0.1 ml·min). -1 The robustness of the chromatographic system was assessed at different detection wavelengths (±2nm), column temperatures (±5℃), and mobile phase compositions (±1% organic phase). The results are shown in Table 6.
[0055] Table 6. Durability Test Results
[0056] The results show that even with minor variations in flow rate, detection wavelength, column temperature, and mobile phase composition, the system can effectively analyze ganoderic acid A, ergothioneine, and other impurities, indicating that the chromatographic system selected in this invention has good durability.
[0057] 7. Comparison test of different chromatographic columns Four chromatographic columns—Hypersil-ODS-2, Hypersil-BDS-C18, Asahipak NH2P-50 4E, and JADE-PAK NH2—were selected to investigate the peak formation of ergothioneine and ganoderic acid A. The results are as follows: Figure 2 As shown, Figure 2 In the text, 'a' represents a Hypersil-BDS-C18 column, 'b' represents a Hypersil-ODS-2 column, 'c' represents an Asahipak NH2P-50 4E column, and 'd' represents a JADE-PAK NH2 column.
[0058] Depend on Figure 2 It is known that in the Hypersil-ODS-2 and Hypersil-BDS-C18 columns, the retention times of ergothioneine and ganoderic acid A are relatively short, indicating poor retention and hindering the separation of the two components. In the Asahipak NH2P-504E column, the separation of ganoderic acid A and ergothioneine is better, but the retention of ergothioneine is poor, which is detrimental to the separation of the components from impurities in the sample. Compared with existing technologies, the JADE-PAK NH2 column used in this invention exhibits more ideal retention and separation effects. Comparative Example
[0059] Taking Example 1 as an example, the ratio of mobile phase A to mobile phase B was changed while other conditions remained the same to compare the effects of different ratios on the detection results. The results are shown in Table 7, where retention time refers to the time required from the start of injection to the chromatographic peak reaching its maximum value (apex).
[0060] Table 7. Effect of different mobile phase ratios on detection results
[0061] Taking Example 1 as an experimental example, please refer to Table 2 in the above repeatability experiment for the detection results under the condition of mobile phase A: mobile phase B being 75:25. Taking the average value of 6 groups of experiments as an example, in the experimental example, the retention time of ganoderic acid A was 9.399 min and the peak area was 25966; the retention time of ergothioneine was 12.947 min, the peak area was 10840, and the peak width was 0.351.
[0062] In Comparative Example 1, the chromatographic peak of ganoderic acid A could not be completely separated from the impurity peak (the resolution was 1.312, less than 1.5), while in Example 1, the resolution of the chromatographic peak of ganoderic acid A from the impurity peak was 1.700, indicating complete separation. In Comparative Example 2, the peak width of ergothioneine was larger, resulting in reduced sensitivity. It was confirmed that under the condition of mobile phase A:mobile phase B of 75:25, simultaneous detection of ganoderic acid A and ergothioneine yielded better results, and the detection results were reliable.
[0063] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.
Claims
1. A method for simultaneously detecting ergothioneine and ganoderic acid A in Ganoderma lucidum and shiitake mushroom oral liquid, characterized in that, Includes the following steps: High performance liquid chromatography was used to detect the effects of Ganoderma lucidum and shiitake mushroom oral liquid. The high-performance liquid chromatography (HPLC) method uses a JADE-PAK NH2 column with a column temperature of 30–40 °C and an ultraviolet (UV) detector with a detection wavelength of 252–256 nm. The mobile phase comprises mobile phase A (acetonitrile solution) and mobile phase B (0.1% (v / v) aqueous acetic acid solution). The volume ratio of mobile phase A to mobile phase B is (74–76):(24–26), and the flow rate is 0.9–1.1 mL / min. -1 .
2. The method according to claim 1, characterized in that, The chromatographic column has dimensions of 250 mm × 4.6 mm and a diameter of 5 μm.
3. The method according to claim 1, characterized in that, The injection volume was 3 μL.
4. The method according to claim 1, characterized in that, The column temperature of the chromatographic column is 35℃.
5. The method according to claim 1, characterized in that, The detector has a detection wavelength of 254 nm.
6. The method according to claim 1, characterized in that, The flow rate of the mobile phase is 1.0 mL·min. -1 .
7. The method according to claim 1, characterized in that, The volume ratio of mobile phase A to mobile phase B is 75:
25.
8. The method according to claim 1, characterized in that, The Ganoderma lucidum and Shiitake mushroom oral liquid was centrifuged at 5000 r / min for 20 min. The supernatant was collected, filtered through a 0.22 μm microporous membrane, and then analyzed by high performance liquid chromatography.