Characteristic chromatogram construction method and marker effective component content determination method of pilose antler mushroom extract

Abstract

The present application belongs to the technical field of edible and medicinal mushroom detection, and particularly relates to a characteristic spectrum construction method of deer mushroom extract and a method for determining the content of a marker effective component. The method is detected by high performance liquid chromatography, and the characteristic spectrum and the content of the effective component ergothioneine can be determined and analyzed by one chromatographic condition. The method comprises the following steps: taking formic acid acetonitrile solution as mobile phase A, taking formic acid solution as mobile phase B, performing gradient elution through a specific elution program, obtaining 9 characteristic peaks of ergosterol and ergothioneine, and quantitatively detecting the effective component ergothioneine by an external standard method. The method is simple in operation, short in detection time, and has good specificity, repeatability, precision and stability, can fully reflect the integrity and characteristics of deer mushroom and its extract, is suitable for the characterization and quality evaluation of the whole components of deer mushroom, and provides technical support and scientific basis for the full development and utilization of deer mushroom resources.

Description

Methods for constructing characteristic spectra and determining the content of key active ingredients in deer antler mushroom extract Technical Field

[0001] This invention belongs to the field of detection technology for edible and medicinal fungi, specifically relating to a method for constructing a characteristic spectrum of deer antler mushroom extract and a method for determining the content of its key active ingredients. Background Technology

[0002] Lyophyllum decastes, scientifically known as *Lyophyllum nucifera*, gets its name from its cap, which resembles slices of deer antler, a valuable traditional Chinese medicine. It is a large fungus used for both food and medicinal purposes. Current literature reports that Lyophyllum decastes contains various bioactive substances such as polysaccharides, purines, and sterols, and possesses effects such as enhancing immunity, lowering blood sugar and lipids, antioxidation, and anti-tumor activity. It has broad application prospects in the fields of biomedicine and health food development.

[0003] Ergothioneine, a key active ingredient in velvet mushroom, possesses strong antioxidant and metal chelating activity. It can effectively prevent cardiovascular diseases, diabetes, and neurodegenerative diseases, and also has significant effects in regulating intestinal flora and antibacterial and anti-inflammatory properties.

[0004] Existing reports mostly focus on the analysis of single major categories of substances such as polysaccharides and polyphenols in *Flammulina velutipes*. For example, Luo Yu et al. used infrared spectroscopy to analyze the structural characteristics and molecular weight of polysaccharides in the fruiting body of *Flammulina velutipes* (Luo Yu. Study on efficient preparation of polysaccharides from *Flammulina velutipes* and their anti-starch digestion properties [D]. Tianjin University of Science and Technology, 2024. DOI:10.27359 / d.cnki.gtqgu.2024.000919.). Yang Bin et al. optimized the extraction process of polyphenols from *Flammulina velutipes* through response surface methodology and explored its in vitro antioxidant activity (Yang Bin, Fang Bingdong, Zheng Jun, et al. Optimization of polyphenol extraction process from *Flammulina velutipes* and its antioxidant activity [J]. Food Research and Development, 2021, 42(24):41-49.). However, a single component cannot comprehensively evaluate the overall quality of *Flammulina velutipes*. Zheng Zhongyong et al. isolated and purified 13 compounds, including ergosterol and nicotinic acid, from the fruiting bodies of *Agaricus esculentus* using silica gel chromatography and high-performance liquid chromatography (HPLC), but did not further analyze their content. Therefore, it is necessary to establish an effective evaluation method reflecting the overall quality of *Agaricus esculentus* and a quantitative detection technique for its marker active ingredients. Summary of the Invention

[0005] The purpose of this invention is to provide a method for constructing characteristic spectra of deer antler mushroom extract and a method for determining the content of its key active ingredients, as well as their applications. This method enables rapid analysis of characteristic components in deer antler mushroom while simultaneously achieving quantitative detection of the active ingredient ergothioneine, providing a reference for the quality control and overall evaluation of deer antler mushroom.

[0006] The present invention provides a method for constructing a characteristic spectrum of antler mushroom extract, comprising the following steps:

[0007] Characteristic chromatographic information of the test sample was obtained by high performance liquid chromatography.

[0008] The test sample is a test sample solution;

[0009] The chromatographic conditions for the high-performance liquid chromatography (HPLC) method include: using formic acid acetonitrile solution as mobile phase A, formic acid solution as mobile phase B, detection wavelength 190-280 nm, and elution according to the following gradient elution program:

[0010] From 0 to 7 min, mobile phase A increased from 85% to 88%, and mobile phase B increased from 15% to 12%; from 7 to 12 min, mobile phase A increased from 88% to 90%, and mobile phase B increased from 12% to 10%; from 12 to 14 min, mobile phase A increased from 90% to 56%, and mobile phase B increased from 10% to 44%; from 14 to 25 min, mobile phase A increased from 56%, and mobile phase B increased from 44% to 44%; from 25 to 40 min, mobile phase A increased from 56% to 40%, and mobile phase B increased from 44% to 60%.

[0011] Preferably, the chromatographic conditions of the high performance liquid chromatography further include: mobile phase A is a 0-0.2% (volume percentage) formic acid acetonitrile solution, and mobile phase B is a 0.05%-0.2% (volume percentage) formic acid aqueous solution; when mobile phase A is a 0% formic acid acetonitrile solution, it is actually a pure acetonitrile solution;

[0012] And / or, the column temperature of the chromatographic column is 20-30℃, such as 20℃, 25℃, 28℃, 30℃, etc.;

[0013] And / or, the flow rate is 0.55-0.6 ml / min, for example, 0.55 ml / min, 0.60 ml / min, etc.;

[0014] And / or, the detection wavelength is 260-265nm, for example, the detection wavelength is 260nm, 262nm, 265nm, etc.;

[0015] And / or, the chromatographic column is an Agilent ZORBAX NH2, with dimensions of 4.6 mm × 250 mm and a diameter of 5 μm;

[0016] And / or, the injection volume is 3-10 μl, for example, 3 μl, 5 μl, or 10 μl.

[0017] More preferably, the chromatographic conditions are: mobile phase A is a 0.1% formic acid acetonitrile solution, and mobile phase B is a 0.1% formic acid aqueous solution;

[0018] And / or, the column temperature of the chromatographic column is 25-30℃;

[0019] And / or, flow rate 0.6 ml / min;

[0020] And / or, the detection wavelength is 262nm;

[0021] And / or, the injection volume is 3-5 μl.

[0022] Most preferably, the chromatographic conditions are: column temperature of 30°C; and / or, injection volume of 3 μl; and / or, the theoretical plate number of the chromatographic peak corresponding to the ergothioneine is not less than 3000.

[0023] In this invention, the preparation method of the deer antler mushroom extract test sample is not specifically limited, and may include, but is not limited to, the following methods:

[0024] The preparation steps of the test solution include extracting the deer antler mushroom powder with an extraction solvent, filtering, and taking the filtrate.

[0025] Specifically, the preparation method of the test solution for detection by high performance liquid chromatography is as follows: Take deer antler mushroom powder, add extraction solvent and mix evenly, weigh, extract by ultrasonication, cool and then make up the lost weight, shake well, separate solid and liquid, filter the clear liquid, and take the filtrate, which is the test solution.

[0026] Furthermore, the ultrasonic extraction time is 30 minutes;

[0027] And / or, the extraction solvent is a 70% aqueous methanol solution;

[0028] And / or, the solid-liquid separation method is centrifugation;

[0029] And / or, the deer antler mushroom powder is a medium-sized powder that can pass through a No. 4 pharmacopoeia sieve after being pulverized from dried deer antler mushroom sample;

[0030] And / or, the mass / volume ratio of the mushroom powder to the extraction solvent is 1:40;

[0031] And / or, the centrifugation speed is 5000~20000 r / min, for example, 5000 r / min, 7000 r / min, 9000 r / min, 11000 r / min, 13000 r / min, 15000 r / min, 18000 r / min, 20000 r / min, etc., and the solid-liquid separation purpose of the present invention can be met within the above centrifugation speed range.

[0032] The present invention also includes a reference solution and / or a negative control solution; the preparation method of the negative control solution is the same as the preparation method of the test solution.

[0033] Preparation of reference solutions: Weigh an appropriate amount of ergosterol standard and add methanol to prepare a reference solution with a concentration of 200-300 μg / ml; weigh an appropriate amount of ergothioneine standard and add water to prepare a stock solution with a concentration of approximately 1 mg / ml, then dilute with water and 50% methanol to obtain reference solutions with a concentration of 10-150 μg / ml. Further, the ergosterol concentration is approximately 110 μg / ml and the ergothioneine concentration is approximately 10 μg / ml.

[0034] The characteristic spectrum of the test solution includes at least the characteristic peaks corresponding to ergosterol and ergothionein.

[0035] The feature map construction method obtained by the deer antler mushroom extract includes at least 9 characteristic peaks;

[0036] Furthermore, at a detection wavelength of 262nm, a total of 9 characteristic peaks were detected:

[0037] Using peak 8 as the reference peak in the characteristic spectrum, the specified values ​​for the relative retention times of each characteristic peak are as follows: peak 1: 0.18, peak 2: 0.36, peak 3: 0.38, peak 4: 0.50, peak 5: 0.52, peak 6: 0.55, peak 7: 0.95, peak 8: 1.00, peak 9: 1.60;

[0038] The relative retention time of each characteristic peak is within ±10% of the specified value.

[0039] Preferably, peak 1 is ergosterol and peak 8 is ergothionein.

[0040] This invention also provides a method for determining the content of the marker active ingredient in velvet mushroom extract, comprising the following steps: according to the aforementioned characteristic chromatogram construction method, ergothioneine reference standard and test solution are determined by HPLC, and the content of the marker component in velvet mushroom extract is calculated by external standard method.

[0041] Specifically, 10 mg of ergothioneine standard was weighed, dissolved in water, and diluted to 10 ml to obtain an ergothioneine standard stock solution. A certain amount of the ergothioneine standard stock solution was accurately transferred and serially diluted with 50% methanol solution to obtain a series of standard solutions with mass concentrations of 1.0, 2.0, 5.0, 10, and 50 mg / L. The solutions were injected and measured under the chromatographic conditions described above. An ergothioneine standard curve was established with the solution mass concentration as the abscissa and the peak area as the ordinate to obtain a linear regression equation. The content of the active ingredient was calculated based on the peak area of ​​the active ingredient in the HPLC chromatogram of the test sample and the linear regression equation.

[0042] Furthermore, in the content determination, 262nm was selected as the detection wavelength for ergothioneine.

[0043] The present invention also provides the application of the aforementioned characteristic map construction method or the aforementioned method for determining the content of the characteristic active ingredients in *Pleurotus ostreatus* extract in the quality detection, quality evaluation and / or quality control of *Pleurotus ostreatus*.

[0044] The quality testing and / or quality evaluation and / or quality control includes overall quality control and content testing of specific active ingredients, including but not limited to ergothioneine.

[0045] The technical solution of this invention has the following advantages:

[0046] 1. This invention provides a method for constructing characteristic spectra of deer antler mushroom extract, which can effectively detect characteristic components such as ergosterol and ergothionein in deer antler mushroom and its products, overcome the defects of evaluating the whole by a single component, and realize the holistic characterization of multiple chemical components in deer antler mushroom.

[0047] 2. The method provided by this invention uses ultra-high performance liquid chromatography for detection, which can simultaneously achieve characteristic chromatographic analysis and determination of effective component content under one chromatographic condition, greatly improving the analytical efficiency and detection cycle. Moreover, the sample preparation and detection conditions are simple and do not involve complicated sample pretreatment steps, which can effectively meet the requirements of quality detection and / or quality evaluation and / or quality control of *Amanita fulvidra philoxeroides*.

[0048] 3. The method provided by this invention has the advantages of high stability, high precision, and good repeatability.

[0049] 4. The method provided by this invention can be used to analyze batch-to-batch differences, origin differences, and fruiting body part differences of characteristic and effective components of *Mushroom arvensis* products, and to monitor dynamic changes during cultivation, providing scientific support for the product development of edible and medicinal fungi such as *Mushroom arvensis* and the full utilization of their medicinal value. Attached Figure Description

[0050] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0051] Figure 1 shows the characteristic spectrum of the deer antler mushroom extract in Example 1.

[0052] Figure 2 is a superimposed HPLC comparison chromatogram of the characteristic peak localization in Example 1.

[0053] Figure 3 is an overlay of HPLC chromatograms from the specificity test and the overall investigation in Example 1.

[0054] Figure 4 is an overlay of HPLC chromatograms showing the precision results in Example 1.

[0055] Figure 5 is an overlay of HPLC chromatograms showing the stability results in Example 1.

[0056] Figure 6 is an overlay of HPLC chromatograms showing the repeatability results in Example 1.

[0057] Figure 7 shows the HPLC comparison chromatograms at different wavelengths in Example 2.

[0058] Figure 8 shows the HPLC comparison chromatograms of different mobile phase systems in Example 2.

[0059] Figure 9 shows the HPLC comparison chromatograms of different manufacturers' chromatographic columns in Example 2.

[0060] Figure 10 shows the HPLC comparison chromatograms at different column temperatures in Example 2.

[0061] Figure 11 shows the HPLC comparison chromatograms at different flow rates in Example 2.

[0062] Figure 12 shows the HPLC comparison chromatograms of different injection volumes in Example 2.

[0063] Figure 13 is a standard curve of ergothionein in Example 3. Detailed Implementation

[0064] The instruments and reagents used in the following embodiments and comparative examples of this invention are as follows:

[0065] Instruments: Thermo Fisher Scientific UltiMate 3000 high performance liquid chromatograph (Thermo Fisher Scientific); METTLER TOLEDO XS105 0.0001 g electronic balance (METTLER-Toledo Instruments (Shanghai) Co., Ltd.), METTLER TOLEDO AL204 (METTLER-Toledo Instruments (Shanghai) Co., Ltd.).

[0066] Reagents: Ergosterol (batch number 15180, content calculated as 98.1%, Shanghai Shidander Standard Technical Service Co., Ltd.), Ergothioneine (batch number 21051-G250801, content calculated as 99.9%, Shanghai Shidander Standard Technical Service Co., Ltd.); Acetonitrile (chromatographic grade, Hassen Chemical, Germany); Methanol (analytical grade, Shanghai Titan Technology Co., Ltd.); Formic acid (superior grade, Jiangsu Tongsheng Chemical Reagent Co., Ltd.); Purified water (Hangzhou Wahaha Group Co., Ltd.).

[0067] Example 1

[0068] A method for constructing characteristic spectra of *Flammulina velutipes* extract, using high-performance liquid chromatography (HPLC), is disclosed. The detection process is as follows:

[0069] 1. Sample preparation

[0070] Preparation of the test solution: Pulverize the dried antler mushroom sample, take about 1g, weigh accurately, place it in a stoppered conical flask, accurately add 40ml of 70% methanol, weigh accurately, extract by sonication for 30 minutes, cool, then replenish the lost weight, shake well, centrifuge at 10000r / min, take the supernatant and filter, take the filtrate, and the test solution is obtained.

[0071] Preparation of reference solutions: Based on the solubility characteristics of each standard, take an appropriate amount of ergosterol standard, accurately weigh it, and add methanol to prepare a reference solution with a concentration of 200-300 μg / ml; take an appropriate amount of ergothioneine standard, accurately weigh it, and add water to prepare a stock solution with a concentration of 1 mg / ml, and dilute with water and 50% methanol respectively to obtain ergosterol concentration of 110 μg / ml and ergothioneine concentration of 10 μg / ml.

[0072] Preparation of negative control solution: Take blank solvent and prepare negative control solution in the same way as "Preparation of test solution" for later use.

[0073] 2. High Performance Liquid Chromatography

[0074] An Agilent ZORBAX NH2 column (250 cm long, 4.6 mm inner diameter, 5 μm particle size) was used as the chromatographic column; 0.1% formic acid acetonitrile was used as mobile phase A, and 0.1% formic acid aqueous solution was used as mobile phase B, with gradient elution performed according to the specifications in Table 1; the detection wavelength was 262 nm; the column temperature was 30 °C; and the flow rate was 0.6 mL / min. The theoretical plate number, calculated based on the characteristic peak of ergothioneine at point 8, should not be less than 5000.

[0075] Table 1 Characteristic spectrum of deer antler mushroom extract and gradient elution procedure

[0076]

[0077] Assay: Accurately pipette 3 μL each of the reference solution and the test solution. l, inject into a high-performance liquid chromatograph, and measure to obtain the characteristic chromatograms of the test solution and the reference solution.

[0078] Characteristic peaks were marked on the characteristic spectrum of the test sample solution, and nine characteristic peaks were marked, as shown in Figure 1. The relative retention times of each characteristic peak are: peak 1: 0.18, peak 2: 0.36, peak 3: 0.38, peak 4: 0.50, peak 5: 0.52, peak 6: 0.55, peak 7: 0.95, peak 8: 1.00, and peak 9: 1.60. The resolution of the above nine characteristic peaks is greater than 1.5.

[0079] The comparison results of the reference solution are shown in Figure 2. In Figure 2, S1, S2, and S3 represent ergosterol, ergothioneine, and the test solution, respectively. The comparison in Figure 2 shows that peak 1 is ergosterol and peak 8 is ergothioneine.

[0080] Among the aforementioned compounds, ergosterol is a characteristic sterol found in fungi, while ergothioneine is mainly synthesized by enzymes in the fruiting bodies of fungi. The method of this invention can effectively identify the characteristic peaks of nine compounds, particularly ergosterol and ergothioneine, demonstrating that the method for constructing the characteristic spectra disclosed in this invention can accurately and comprehensively reflect the integrity and characteristics of the intrinsic chemical composition of *Agaricus esculentus*, and can be used for the quality detection and overall evaluation of *Agaricus esculentus*.

[0081] 3. Methodological Examination

[0082] 3.1 Specificity Test and Holistic Examination

[0083] Test solution: Pulverize dried deer antler mushroom sample, take 1g, accurately weigh it, place it in a stoppered conical flask, accurately add 25ml of 70% methanol, weigh it, extract it by sonication for 30 minutes, cool it and then make up the lost weight, shake it well, centrifuge it at 10000r / min, take the supernatant and filter it, take the filtrate to obtain the test solution.

[0084] Reference solution: Take an appropriate amount of ergothioneine standard, accurately weigh it, add water to prepare a stock solution with a concentration of about 1 mg / ml, and dilute it with 50% methanol to prepare a solution containing 10 μg per ml.

[0085] Blank solvent: 70% methanol as the solvent for the test sample.

[0086] The test solution, reference solution, and blank solvent were injected into the liquid chromatograph, respectively, and the same chromatographic conditions as described in "2. High Performance Liquid Chromatography" were used for determination. The chromatograms were recorded. Simultaneously, the integrity of the test sample was examined, and the results are shown in Figure 3. In Figure 3, A: test sample solvent blank, B: reference solution, C: test sample solution, and D: integrity test.

[0087] As can be seen from the results in Figure 3, the reference peak corresponds to the corresponding position of the test solution, and the blank solvent does not interfere, indicating that the method has good specificity. The chromatographic peaks were basically collected after 40 minutes of collection, and the chromatographic conditions have reached the maximum information collection. Therefore, the collection time of 40 minutes is reasonable.

[0088] 3.2 Instrument precision test

[0089] The test solution was prepared using the same method as in "1. Sample Preparation". Under the same chromatographic conditions as in "2. High Performance Liquid Chromatography", the sample was injected 6 times consecutively, with 3 μl injected each time. The RSD% value (RSD, relative standard deviation) of the relative retention time of each characteristic peak was calculated using the retention time of the ergothioneine chromatographic peak (peak 8) as a reference. The specific measurement results are shown in Table 2 and Figure 4.

[0090] Table 2. Relative retention time results of instrument precision test (n=6)

[0091]

[0092] The results show that the relative retention times are basically consistent, and the relative retention time RSD% of each characteristic peak is less than 1.0%, indicating good instrument precision.

[0093] 3.3 Stability Test

[0094] The test solution was prepared using the same method as in "1. Sample Preparation". Under the same chromatographic conditions as in "2. High Performance Liquid Chromatography", 3 μl of sample was injected once at 0, 8, 12, 18, 22 and 24 h. The RSD% value of the relative retention time of each characteristic peak was calculated using the retention time of the ergothioneine chromatographic peak (peak 8) as a reference. The specific results are shown in Table 3 and Figure 5. In Figure 5, from bottom to top, the values ​​are 0 h, 8 h, 12 h, 18 h, 22 h and 24 h.

[0095] Table 3. Relative retention time results from stability tests

[0096]

[0097] The results showed that the relative retention times were basically consistent, and the relative retention time RSD% of each characteristic peak was less than 2.0%, indicating that the test solution had good stability within 24 hours.

[0098] 3.4 Repeatability Test

[0099] Six test solutions were prepared using the same method as in "1. Sample Preparation". Under the same chromatographic conditions as in "2. High Performance Liquid Chromatography", 3 μl was injected each time. The RSD% value of the relative retention time of each characteristic peak was calculated using the retention time of the ergothioneine chromatographic peak (peak 8) as a reference. The specific results are shown in Table 4 and Figure 6.

[0100] Table 4. Relative retention time results of repeatability tests (n=6)

[0101]

[0102] The results show that the relative retention times are basically consistent, and the relative retention time RSD% of each characteristic peak is less than 1.0%, indicating that the method has good repeatability.

[0103] Example 2

[0104] This embodiment uses different chromatographic conditions to construct the characteristic chromatograms, while other conditions are the same as in Example 1, as detailed below:

[0105] 1. Selection of detection wavelength

[0106] A DAD detector (diode array detector) was used to scan the test sample across the entire wavelength range of 190-400 nm. Chromatograms at wavelengths of 254, 260, 262, 265, and 280 nm were extracted and compared. The comparison results are shown in Figure 7. In Figure 7, A: wavelength 280 nm, B: wavelength 265 nm, C: wavelength 262 nm, D: wavelength 260 nm, and E: wavelength 254 nm.

[0107] As shown in Figure 7, the chromatogram baseline is unstable at detection wavelengths below 260 nm, and the absorption intensity of each chromatographic peak tends to weaken when the detection wavelength is greater than 265 nm. Therefore, in this invention, the detection wavelength in the range of 260-265 nm is preferred for detection.

[0108] Existing literature suggests that the optimal absorption wavelength for ergothioneine, the selected reference peak (peak 8), is 262 nm. At this wavelength, the characteristic peaks are symmetrical and have good separation. Taking all factors into consideration, 262 nm was further selected as the optimal detection wavelength.

[0109] 2. Selection of mobile phase gradient

[0110] The mobile phase gradient settings are shown in Table 5-7 below. The detection results using this mobile phase gradient are shown in Figure 8. In Figure 8, S1 is gradient 1, S2 is gradient 2, and S3 is gradient 3.

[0111] Table 5. Feature Map Gradient 1 Elution Table

[0112]

[0113] Table 6. Gradient-2 elution table of feature maps

[0114]

[0115] Table 7 Feature Map Gradient 3-Elution Table

[0116]

[0117] As shown in Figure 8, under gradient elution condition 3, the separation of each characteristic peak is good, the retention time is appropriate, the peak symmetry is good, and the theoretical plate number is high. Therefore, gradient 3 is finally selected as the best elution gradient.

[0118] 3. Selection of chromatographic column

[0119] Column 1: Merck Lichrospher NH2 10nm (4.6×250mm, 5μm);

[0120] Column 2: Agilent ZORBAX NH2 Analytical (4.6 × 250 mm, 5 μm).

[0121] The separation effect of the test solution was verified by using different chromatographic columns. The results are shown in Figure 9. In Figure 9, S1 is a Merck Lichrospher NH2 10nm column and S2 is an Agilent ZORBAX NH2 Analytical column.

[0122] As shown in Figure 9, under the same gradient elution program, when using the Agilent ZORBAX NH2 Analytical column, the chromatographic peak information is more complete, each characteristic peak can be well separated, and the peak shape is better. Therefore, the Agilent ZORBAX NH2 Analytical column was finally selected as the chromatographic column for the characteristic chromatogram.

[0123] 4. Column temperature selection

[0124] The column temperature conditions of 15℃, 20℃, 25℃ and 30℃ were screened and optimized respectively. The results are shown in Figure 10. In Figure 10, S1 is 15℃, S2 is 20℃, S3 is 25℃ and S4 is 30℃.

[0125] As shown in Figure 10, when the column temperature is below 15℃, the separation between peaks 2 and 3, and between peaks 5 and 6 is poor. Increasing the column temperature is beneficial to improving the separation of each characteristic peak. When the column temperature is 30℃, the separation of each characteristic peak is relatively good, the symmetry is good, and the influence of interference factors is small. Therefore, 30℃ is selected as the optimal column temperature condition for the characteristic spectrum.

[0126] 5. Selection of flow rate

[0127] The flow rate conditions of 0.55 ml / min, 0.60 ml / min, 0.65 ml / min, and 0.70 ml / min were screened and optimized, and the results are shown in Figure 11. In Figure 11, S1 is 0.55 ml / min, S2 is 0.60 ml / min, S3 is 0.65 ml / min, and S4 is 0.70 ml / min. As can be seen from Figure 11, the requirements of this invention can be met within the flow rate range of 0.55-0.60 ml / min. Considering the analytical efficiency, the peak shape and separation are relatively better when the flow rate is 0.60 ml / min, and it is less affected by interference factors. Therefore, the flow rate of 0.60 ml / min was finally selected as the optimal flow rate condition for the characteristic spectrum.

[0128] Selection of injection volume

[0129] The applicability of each characteristic peak system in the test solution under different injection volumes was investigated. 3 μl, 5 μl, and 10 μl of sample were injected for determination, and the results are shown in Figure 12. In Figure 12, S1 represents 3 μl, S2 represents 5 μl, and S3 represents 10 μl. Figure 12 shows that between 3 μl and 10 μl, the area of ​​each characteristic peak increases proportionally with the increase of the injection volume. When the injection volume is greater than 10 μl, the chromatographic peak symmetry is poor, the theoretical plate number decreases, and the column efficiency decreases. Therefore, 3 μl to 5 μl was selected as the optimal injection volume for the characteristic chromatogram.

[0130] Example 3

[0131] This embodiment provides a method and application for determining the content of key active ingredients in *Flammulina velutipes* and / or *Flammulina velutipes* extract. The test solution is prepared according to the method in Example 1 and the determination is performed under the same chromatographic conditions.

[0132] Preparation of standard solutions

[0133] Weigh 10 mg of ergothioneine standard, dissolve it in water and dilute to 10 ml to obtain ergothioneine standard stock solution. Accurately transfer a certain amount of ergothioneine standard stock solution and dilute it stepwise with 50% methanol solution to obtain a series of standard solutions with mass concentrations of 1.0, 2.0, 5.0, 10 and 50 mg / L. Prepare fresh before use.

[0134] Content detection

[0135] Accurately pipette 3 μL each of the series of standard solutions and the test solution. I. Inject into a high-performance liquid chromatograph (HPLC) and determine the result. A standard curve for ergothioneine was established with the mass concentration of a series of standard solutions as the x-axis and the peak area as the y-axis, yielding a linear regression equation. The content of ergothioneine was calculated based on the peak area of ​​ergothioneine in the HPLC chromatogram of the test sample and the linear regression equation.

[0136] 3. Methodological Examination

[0137] 3.1 System Adaptability

[0138] The test solution and a series of standard solutions were injected into the liquid chromatograph and analyzed under the conditions of Example 1. The results showed that ergothioneine could be baseline separated from other components, with a resolution R > 1.5 and theoretical plate numbers greater than 5000.

[0139] 3.2 Specificity

[0140] Solvent blank, test solution, and a series of standard solutions were injected into the liquid chromatograph, and the determination was performed under the conditions of Example 1. The results showed that the determination of ergothioneine content was not affected by solvent or other factors, and the peak shape had good symmetry and specificity.

[0141] 3.3 Linear Relationship and Range

[0142] Accurately weigh ergothioneine standard, dissolve in water and dilute to 10 ml to obtain ergothioneine standard stock solution. Accurately transfer a certain amount of ergothioneine standard stock solution and serially dilute with 50% methanol solution to obtain a series of standard solutions with concentrations of 52.55 μg / ml, 10.51 μg / ml, 5.25 μg / ml, 2.10 μg / ml and 1.05 μg / ml. Accurately pipette 3 μl of each solution and inject it into the liquid chromatograph. Measure at 262 nm according to the conditions of Example 1. Perform regression analysis on the peak area integral value Y of ergothioneine against the concentration X of ergothioneine reference standard and plot the standard curve. The results are shown in Table 8 and Figure 13.

[0143] Table 8. Results of Ergothionein Standard Curve Analysis

[0144]

[0145] The results showed that the regression equation for ergothioneine at injection concentrations ranging from 1.05 to 52.55 μg / ml was: Y = 240.345X - 0.033, R0 2 =1.000; the results showed that within the range of 1.05~52.55 μg / ml, the concentration X (μg / ml) of ergothioneine had a good linear relationship with the peak area Y.

[0146] 3.4 Precision

[0147] Following the preparation of the test solution in Example 1, the same test solution was injected six times consecutively into a high-performance liquid chromatograph (HPLC), and the peak area of ​​ergothioneine was recorded, and the RSD value was calculated. The results showed that the RSD% of the peak area of ​​the reference standard ergothioneine was 1.36%, indicating that the method has good precision.

[0148] 3.5 Repeatability

[0149] Following the preparation of the test solution in Example 1, six test solutions were prepared in parallel and analyzed under the chromatographic conditions of Example 1. The average content and RSD% of ergothioneine were calculated. The average content of ergothioneine in the six test solutions was found to be 1.73 μg / ml, and the RSD% was 1.27%, indicating that the content determination method has good repeatability.

[0150] 3.6 Stability

[0151] Following the preparation of the test solution in Example 1, the sample was injected at 0, 8, 12, 18, 22, and 24 hours, and analyzed under the chromatographic conditions of Example 1. The peak area of ​​ergothioneine was determined, and the RSD% value was calculated. The results showed that the RSD% of the ergothioneine peak area in the test solution was 1.95%, indicating that the test solution was basically stable within 24 hours and could meet the requirements for determination.

[0152] Based on the established characteristic chromatogram, this invention determines the content of the marker active ingredient ergothioneine. The above methodological results show that the method meets the requirements for content determination and can be used for a comprehensive evaluation of the overall quality of *Pleurotus ostreatus* and / or *Pleurotus ostreatus* extract.

[0153] 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. A method for constructing a characteristic spectrum of a deer antler mushroom extract, characterized in that, The characteristic chromatographic information of the test sample was obtained by high performance liquid chromatography; the test sample was a test sample solution; the preparation steps of the test sample solution included extracting the deer antler mushroom powder with an extraction solvent, filtering, and taking the filtrate. The extraction solvent is a 70% methanol-water solution; the chromatographic conditions of the high-performance liquid chromatography (HPLC) include: mobile phase A is 0-0.2% formic acid-acetonitrile solution, mobile phase B is 0.05%-0.2% formic acid-water solution, detection wavelength is 260-265 nm, and elution is performed according to the following gradient elution program: 0-7 min, mobile phase A 85%→88%, mobile phase B 15%→12%; 7-12 min, mobile phase A 88%→90%, mobile phase B 12%→10%; 12-14 min, mobile phase A 90%→56%, mobile phase B 10%→44%; 14-25 min, mobile phase A 56%, mobile phase B 44%; 25-40 min, mobile phase A 56%→40%, mobile phase B 44%→60%; the chromatographic column is an Agilent ZORBAX. NH2; The characteristic spectrum of the deer antler mushroom extract obtained by the characteristic spectrum construction method includes at least 9 characteristic peaks; among which, peak 1 is ergosterol and peak 8 is ergothionein.

2. The method for constructing the characteristic spectrum of the deer antler mushroom extract according to claim 1, characterized in that, The chromatographic conditions of the high-performance liquid chromatography method also include: a column temperature of 20-30℃; and / or a flow rate of 0.55-0.6 ml / min; and / or a column specification of 4.6 mm × 250 mm, 5 μm; and / or an injection volume of 3-10 μl.

3. The method for constructing the characteristic spectrum of the deer antler mushroom extract according to claim 2, characterized in that, The chromatographic conditions are as follows: mobile phase A is a 0.1% formic acid acetonitrile solution, mobile phase B is a 0.1% formic acid aqueous solution; and / or, column temperature is 25-30℃; and / or, flow rate is 0.6 ml / min; and / or, detection wavelength is 262 nm; and / or, injection volume is 3-5 μl.

4. The method for constructing the characteristic spectrum of the deer antler mushroom extract according to claim 3, characterized in that, The chromatographic conditions are as follows: column temperature 30°C; and / or, injection volume 3 μl.

5. The method for constructing the characteristic spectrum of the deer antler mushroom extract according to claim 1, characterized in that, The ultrasonic extraction time is 30 min; and / or, the solid-liquid separation method is centrifugation; and / or, the deer antler mushroom powder is medium powder that can pass through a No. 4 pharmacopoeia sieve after being pulverized from dried deer antler mushroom sample; and / or, the mass / volume ratio of the deer antler mushroom powder to the extraction solvent is 1:40; and / or, the centrifugation speed is 5000~20000 r / min.

6. The method for constructing the characteristic spectrum of the *Deer Antler Fungus* extract according to any one of claims 1-5, characterized in that, Using peak 8 as the reference peak in the characteristic spectrum, the specified values ​​for the relative retention times of each characteristic peak are as follows: peak 1: 0.18, peak 2: 0.36, peak 3: 0.38, peak 4: 0.50, peak 5: 0.52, peak 6: 0.55, peak 7: 0.95, peak 8: 1.00, and peak 9: 1.60; among them, the relative retention times of each characteristic peak are within ±10% of the specified values.

7. A method for determining the content of a key active ingredient in deer antler mushroom extract, characterized in that, The method includes the following steps: according to any one of claims 1-6, the characteristic spectrum construction method is used to determine the ergothioneine reference standard and the test solution by HPLC, and the content of the marker active ingredient in the deer antler mushroom extract is calculated by external standard method; the marker active ingredient is ergothioneine.

8. The method for determining the content of the characteristic active ingredient in deer antler mushroom extract according to claim 7, characterized in that, 262nm was chosen as the detection wavelength for ergothionein.

9. The application of the characteristic spectrum construction method according to any one of claims 1-6 or the method for determining the content of the characteristic active ingredient in the deer antler mushroom extract according to claim 7 or 8 in the quality detection, quality evaluation and / or quality control of deer antler mushroom.

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