A method for detecting total triterpenoid content in unbroken spore powder of Ganoderma lucidum

Abstract

The present invention relates to a detection method for the total triterpene content in an unbroken wall ganoderma spore powder, belongs to the field of drug analysis, and includes: weighing the unbroken wall ganoderma spore powder into a glass homogenizer, adding an extractant to break the wall; adding an extractant to the broken wall ganoderma spore powder mixed solution for ultrasonic extraction, and then high-speed low-temperature centrifugation to obtain a supernatant; concentrating the supernatant to near dryness and then re-dissolving it with n-hexane to obtain a supernatant; using anhydrous sodium sulfate and activated alumina mixed chromatography column to remove impurities in the supernatant, collecting and constant volume to obtain a sample solution; taking a certain amount of sample solution and evaporating it, adding vanillin glacial acetic acid solution and perchloric acid for color development reaction, cooling and adding glacial acetic acid to stand, filtering to obtain a solution to be tested; using ganoderic acid A as a standard sample to prepare a standard curve, and obtaining the total triterpene content in the sample by an external standard method. The present invention has high accuracy, good reproducibility, and strong applicability, and provides powerful technical support for the industrial production quality control of total triterpenes in ganoderma spore powder.

Description

Technical Field

[0001] The invention relates to a method for detecting the total triterpenoid content in unbroken wall ganoderma spore powder, and belongs to the field of drug analysis. Background Art

[0002] Ganoderma triterpenoids are a class of triterpenoid compounds found in Ganoderma lucidum. They are highly oxidized lanostane derivatives and are one of the main chemical and pharmacological components of Ganoderma lucidum. Ganoderma triterpenoids have particularly significant physiological activities, including anti-inflammatory, analgesic, anti-aging, tumor cell killing, anti-hypoxia, anti-allergic and liver function promotion. Experiments have shown that Ganoderma triterpenoids have the effect of rapidly improving immunity, which is manifested in promoting lymphocyte proliferation, improving the phagocytic ability and lethality of macrophages, NK cells and T cells, and directly and indirectly killing tumor cells. At the same time, they can activate G0 stage tumor cells that are difficult to kill with chemotherapy drugs, and kill them alone or in combination. Ganoderma triterpenoids have become a benchmark for Ganoderma lucidum tumor inhibition research.

[0003] Ganoderma lucidum spore powder is the seeds released by Ganoderma lucidum in the late stage of development. It mainly contains Ganoderma lucidum polysaccharides, triterpenes, natural organic germanium, trace element selenium, etc., and has extremely high health care effects. However, Ganoderma lucidum spore powder in its natural state is in an unbroken state, and the laboratory cannot extract effective ingredients from it using conventional extraction methods. In industrial production, special wall-breaking equipment is often used to break the wall before processing and production. However, there are few reports on how to achieve complete wall-breaking of spore powder under laboratory conditions.

[0004] The main methods for detecting total triterpenes in broken wall Ganoderma lucidum spore powder are UV-visible spectrophotometry and high performance liquid chromatography. Most of the quantitative detection by UV-visible spectrophotometry uses oleanolic acid or ursolic acid as reference substances, and uses vanillin-glacial acetic acid and perchloric acid as colorants. The principle is that perchloric acid oxidizes the phenolic hydroxyl group of triterpenes to carboxyl groups, adds a double bond, and then generates a conjugated double bond system through double bond displacement and vanillin addition, and forms a cationic salt under the action of acid to develop color. However, this method is easily interfered by fatty acids, other triterpenes and sterols, resulting in high test results and false positives. The wall-breaking treatment causes a large amount of weak polar compounds such as triglycerides, fatty acids, and sterols to dissolve, which will greatly reduce the accuracy of the measurement results of UV-visible spectrophotometry. High performance liquid chromatography can accurately determine specific triterpenoid components in Ganoderma lucidum spore powder, but there are more than 100 triterpenoids in Ganoderma lucidum. The quantification of each triterpenoid requires the purchase of expensive standards, and many standards are unavailable. At present, there are no definite chromatographic conditions that can achieve the separation of more than 100 triterpenoids, which limits the use of high performance liquid chromatography and makes it impossible to accurately and quickly meet the detection of the total amount of triterpenoids in Ganoderma lucidum. Summary of the invention

[0005] The invention aims to provide a method for detecting the total triterpenoid content in unbroken spore powder of ganoderma lucidum, which has high measurement accuracy and good reproducibility.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] A method for detecting the total triterpenoid content in unbroken spore powder of Ganoderma lucidum comprises the following steps:

[0008] (1) Sample extraction: weighing unbroken spore powder of Ganoderma lucidum into a glass homogenizer, adding an extractant to break the spores, and obtaining a mixed solution of broken spore powder of Ganoderma lucidum; adding an extractant to the mixed solution of broken spore powder of Ganoderma lucidum for ultrasonic extraction, and then centrifuging with a high-speed refrigerated centrifuge to obtain a supernatant;

[0009] (2) Sample purification: the supernatant is concentrated to near dryness, and the obtained concentrate is re-dissolved with n-hexane to obtain a supernatant; impurities in the supernatant are removed by using an anhydrous sodium sulfate and activated alumina mixed chromatography column, and the supernatant is collected and fixed to volume to obtain a sample solution;

[0010] (3) Sample detection: Take a certain amount of sample solution and evaporate it to dryness, add vanillin-glacial acetic acid solution and perchloric acid, heat in a water bath for color development, cool, add glacial acetic acid, let stand, and filter to obtain the test solution;

[0011] The standard curve solution was prepared with ganoderic acid A as the standard sample, and the standard curve solution and the sample solution were detected by high performance liquid chromatography to prepare the standard curve, and the total triterpene content in the sample was obtained by external standard method.

[0012] A further improvement of the technical solution of the present invention is that the extractant in step (1) is one of methanol, anhydrous ethanol, dichloromethane and ethyl acetate.

[0013] The technical solution of the present invention is further improved in that: the cell wall breaking treatment in step (1) is as follows: weigh 25±2 mg of unbroken spore powder of Ganoderma lucidum into a 20 mL glass homogenizer, add 2-5 mL of an extractant, and treat at a frequency of 50 times / min for 20 min under ice bath conditions;

[0014] The ultrasonic treatment is to add 50-100 mL of extractant into the mixed solution of broken wall Ganoderma lucidum spore powder for ultrasonic extraction, the ultrasonic extraction temperature is 45-55° C., the ultrasonic frequency is 25 KHz, and the ultrasonic time is 45 minutes.

[0015] A further improvement of the technical solution of the present invention is that the centrifugal temperature in step (1) is 0-5°C, the rotation speed is 10000r / min, and the time is 5min.

[0016] A further improvement of the technical solution of the present invention is that the concentration temperature of step (2) is 50-70°C and the vacuum degree is -0.8MPa.

[0017] A further improvement of the technical solution of the present invention is that the amount of n-hexane used for re-dissolution in step (2) is 10 mL.

[0018] The technical solution of the present invention is further improved in that: the preparation process of the anhydrous sodium sulfate and activated alumina chromatography column in step (2) is as follows: 100-200 mesh alumina for chromatography is calcined at 810-850° C. for 4-5 hours to prepare activated alumina for chromatography; the chromatography column has a diameter of 15 mm and a height of 300 mm, a sieve plate and a flow rate control cock are provided at the lower end, and a small amount of absorbent cotton is placed above the sieve plate; n-hexane is added to the chromatography column, 20 g of activated alumina for chromatography is added to the chromatography column, the alumina is uniformly precipitated, and a layer of anhydrous sodium sulfate with a thickness of 20 mm is then loaded;

[0019] The process of removing impurities from the upper column liquid by the chromatography column is as follows: opening the control stopcock, placing the n-hexane in the chromatography column to the upper end of the anhydrous sodium sulfate, then adding the upper column liquid from the upper end of the chromatography column, discharging the liquid into the chromatography column at a flow rate of 0.5 mL / min, then opening the control stopcock, controlling the outflow rate of the lower end to 0.5 mL / min, collecting 10 mL of liquid and discarding it; continuously adding 80 mL of n-hexane from the upper end, taking a 100 mL volumetric flask to collect all the remaining sample liquid, diluting to the scale with n-hexane, mixing well, and using the sample solution as the sample solution.

[0020] A further improvement of the technical solution of the present invention is that the specific steps of preparing the test solution in step (3) are:

[0021] Take 1 mL of the sample solution into a 10 mL colorimetric tube, place the colorimetric tube in a 60°C water bath, blow dry with nitrogen, and then cool to room temperature; then accurately add 0.4 mL of 5% vanillin glacial acetic acid solution and 1.0 mL of perchloric acid, mix well, and perform a color development reaction in a 60°C water bath; after 15 min±10 s, place the colorimetric tube in an ice bath to cool for 5 min, then accurately add 5.0 mL of glacial acetic acid, shake well, and let stand for 15-20 min, and obtain the sample solution after passing through a 0.45 μm filter membrane.

[0022] The technical solution of the present invention is further improved in that: the preparation process of the standard curve solution in the step (3) is as follows: 10 mg of the ganoderic acid A standard is taken into a 100 mL volumetric flask, dissolved with n-hexane and fixed to the scale, and mixed to obtain a stock solution; 0.00 mL, 0.10 mL, 0.20 mL, 0.40 mL, 0.80 mL, and 1.00 mL of the stock solution are accurately drawn into 10 mL colorimetric tubes respectively, each colorimetric tube is placed in a 60° C. water bath, blown dry with nitrogen, and then cooled to room temperature; 0.4 mL of 5% vanillin glacial acetic acid solution and 1.0 mL of perchloric acid are accurately added, mixed, and a color development reaction is performed in a 60° C. water bath; after 15 min±10 s, the colorimetric tube is placed in an ice bath to cool for 5 min, and then 5.0 mL of glacial acetic acid is accurately added, shaken, and allowed to stand for 15-20 min, and filtered through a 0.45 μm filter membrane to prepare standard curve test solutions of different concentrations.

[0023] A further improvement of the technical solution of the present invention is that the chromatographic conditions of the high performance liquid chromatography in step (3) are:

[0024] a) Chromatographic column: acid-resistant C18 chromatographic column, 4.6×250mm, 5μm;

[0025] b) Mobile phase conditions: acetonitrile containing 5% formic acid;

[0026] c) Flow rate: 0.5 mL / min;

[0027] d) Column temperature: 40°C;

[0028] e) Injection volume: 10 μL;

[0029] f) Detection wavelength: 550nm.

[0030] Due to the adoption of the above technical solution, the technical effects achieved by the present invention are as follows:

[0031] The invention establishes a high performance liquid chromatography detection method for the total triterpenoid content in unbroken ganoderma spore powder with high accuracy, good reproducibility and strong applicability, which provides powerful technical support for the quality control of the total triterpenoids in the industrial production of ganoderma spore powder.

[0032] The invention uses a homogenizer to break the unbroken spore powder of ganoderma lucidum, thereby effectively extracting total triterpenes from the unbroken spore powder of ganoderma lucidum.

[0033] The invention uses a high-speed refrigerated centrifuge to centrifuge and a mixed chromatography column of anhydrous sodium sulfate and neutral alumina to dehydrate, remove fat and impurities, thereby eliminating the interference of impurities and improving the accuracy and reproducibility of the detection method. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1It is a chromatogram of total triterpenes of Ganoderma lucidum detected by high performance liquid chromatography-ultraviolet light of the present invention;

[0035] Figure 2 is the standard curve of Example 1 of the present invention;

[0036] Figure 3 is the standard curve of Example 2 of the present invention;

[0037] Figure 4 is the standard curve of Example 3 of the present invention;

[0038] Figure 5 It is the standard curve of Example 4 of the present invention. DETAILED DESCRIPTION

[0039] The present invention is further described in detail below with reference to the accompanying drawings and specific embodiments:

[0040] A method for detecting the total triterpenoid content in unbroken spore powder of Ganoderma lucidum comprises the following steps:

[0041] (1) Sample extraction

[0042] Weigh 25±2mg of unbroken spore powder of Ganoderma lucidum into a 20mL glass homogenizer, add 2-5mL of an extractant, treat at a frequency of 50 times / min for 20min under ice bath conditions to obtain a mixed solution of broken spore powder of Ganoderma lucidum; add 50-100mL of an extractant to the mixed solution of broken spore powder of Ganoderma lucidum for ultrasonic extraction, the ultrasonic extraction temperature is 45-55°C, the ultrasonic frequency is 25KHz, and the ultrasonic time is 45min; then centrifuge with a high-speed refrigerated centrifuge, the centrifugation temperature is 0-5°C, the speed is 10000r / min, and the time is 5min to obtain a supernatant. The extractant is one of methanol, anhydrous ethanol, dichloromethane and ethyl acetate.

[0043] (2) Sample purification

[0044] The supernatant was concentrated to near dryness, and the obtained concentrate was redissolved with 10 mL of n-hexane to obtain a supernatant; impurities in the supernatant were removed by using an anhydrous sodium sulfate and activated alumina mixed chromatography column, and the sample solution was collected and fixed to volume to obtain a sample solution.

[0045] The concentration temperature is 50-70°C and the vacuum degree is -0.8MPa.

[0046] The preparation process of the anhydrous sodium sulfate and activated alumina chromatography column is as follows: 100-200 mesh alumina for chromatography is calcined at 810-850° C. for 4-5 hours to prepare activated alumina for chromatography; the chromatography column has a diameter of 15 mm and a height of 300 mm, and is provided with a sieve plate and a flow rate control cock at the lower end, and a small amount of absorbent cotton is placed above the sieve plate; n-hexane is added to the chromatography column, 20 g of activated alumina for chromatography is added to the chromatography column, the alumina is uniformly precipitated, and a layer of anhydrous sodium sulfate with a thickness of 20 mm is then loaded;

[0047] The process of removing impurities from the upper column liquid by the chromatography column is as follows: opening the control stopcock, placing the n-hexane in the chromatography column to the upper end of the anhydrous sodium sulfate, then adding the upper column liquid from the upper end of the chromatography column, discharging the liquid into the chromatography column at a flow rate of 0.5 mL / min, then opening the control stopcock, controlling the outflow rate of the lower end to 0.5 mL / min, collecting 10 mL of liquid and discarding it; continuously adding 80 mL of n-hexane from the upper end, taking a 100 mL volumetric flask to collect all the remaining sample liquid, diluting to the scale with n-hexane, mixing well, and using the sample solution as the sample solution.

[0048] (3) Sample testing

[0049] Take a certain amount of sample solution and evaporate it to dryness, add vanillin-glacial acetic acid solution and perchloric acid, heat in a water bath for color reaction, add glacial acetic acid after cooling, let stand, and filter to obtain the solution to be tested. The specific preparation steps are as follows:

[0050] Take 1 mL of the sample solution into a 10 mL colorimetric tube, place the colorimetric tube in a 60°C water bath, blow dry with nitrogen, and then cool to room temperature; then accurately add 0.4 mL of 5% vanillin glacial acetic acid solution and 1.0 mL of perchloric acid, mix well, and perform a color development reaction in a 60°C water bath; after 15 min±10 s, place the colorimetric tube in an ice bath to cool for 5 min, then accurately add 5.0 mL of glacial acetic acid, shake well, and let stand for 15-20 min, and obtain the sample solution after passing through a 0.45 μm filter membrane.

[0051] The standard curve solution was prepared with ganoderic acid A as the standard sample, and the standard curve solution and the sample solution were detected by high performance liquid chromatography to prepare the standard curve, and the total triterpene content in the sample was obtained by external standard method.

[0052] The preparation process of the standard curve solution is as follows: 10 mg of the ganoderic acid A standard is taken into a 100 mL volumetric flask, dissolved with n-hexane and fixed to the scale, mixed to obtain a stock solution. 0.00 mL, 0.10 mL, 0.20 mL, 0.40 mL, 0.80 mL, and 1.00 mL of the stock solution are accurately drawn into 10 mL colorimetric tubes respectively, each colorimetric tube is placed in a 60° C. water bath, blown dry with nitrogen, and then cooled to room temperature; then 0.4 mL of 5% vanillin glacial acetic acid solution and 1.0 mL of perchloric acid are accurately added, mixed, and a 60° C. water bath color reaction is performed; after 15 min ± 10 s, the colorimetric tube is placed in an ice bath to cool for 5 min, then 5.0 mL of glacial acetic acid is accurately added, shaken and allowed to stand for 15-20 min, and filtered through a 0.45 μm filter membrane to prepare standard curve test solutions of different concentrations.

[0053] The chromatographic conditions of the high performance liquid chromatography are:

[0054] a) Chromatographic column: acid-resistant C18 chromatographic column, 4.6×250mm, 5μm;

[0055] b) Mobile phase conditions: acetonitrile containing 5% formic acid;

[0056] c) Flow rate: 0.5 mL / min;

[0057] d) Column temperature: 40°C;

[0058] e) Injection volume: 10 μL;

[0059] f) Detection wavelength: 550nm.

[0060] Example 1

[0061] A method for detecting the total triterpenoid content in unbroken spore powder of Ganoderma lucidum comprises the following steps:

[0062] (1) Sample extraction

[0063] A sample of unbroken Ganoderma lucidum spore powder was taken for three parallel experiments, with the sample weights being A: 25.4 mg, B: 25.3 mg, and C: 23.8 mg respectively.

[0064] Place the unbroken spore powder sample of Ganoderma lucidum in a 20mL homogenizer, add 2mL of methanol extract; break the spores for 20min at a frequency of 50 times / min under ice bath conditions. Transfer the mixed solution to a 250mL conical flask, add 60mL of extract, the extraction temperature is 50℃, the ultrasonic frequency is 25KHz; the ultrasonic time is 45min, and the extract is obtained. The obtained extract is frozen centrifuged for 5min at a speed of 10000r / min and a centrifugal temperature of 5℃ to obtain a supernatant.

[0065] (2) Sample purification

[0066] The supernatant was transferred to a 100 mL round-bottom flask, concentrated in a 60 ° C water bath, vacuum -0.8 MPa conditions, concentrated on a rotary evaporator to a solvent content of ≤1%, to obtain a concentrated solution. The concentrated solution was redissolved with 10 mL of n-hexane to obtain a column liquid; alumina for chromatography (100-200 mesh) was taken and burned at 850 ° C for 4 hours to prepare active chromatography alumina; the chromatography column was used with a diameter of 15 mm and a height of 300 mm, with a sieve plate and a flow rate control cock at the lower end, and a small amount of absorbent cotton was placed above the sieve plate; n-hexane was added to the chromatography column, 20 g of alumina was taken and poured into the chromatography column, and anhydrous sodium sulfate with a thickness of 20 mm was added to the upper end, the control cock was opened, and n-hexane was placed at the upper end of the anhydrous sodium sulfate, and the column liquid was added from the upper end of the chromatography column, and the liquid was discharged at a flow rate of 0.5 mL / min until the sample liquid entered the chromatography column. Use a measuring cylinder to collect the liquid at the bottom, collect 10mL of liquid and discard it. Continue to add 80mL of n-hexane from the top, collect all the remaining sample liquid in a 100mL volumetric flask, dilute to the mark with n-hexane, mix well, and use this as the sample solution.

[0067] (3) Sample testing

[0068] Take 1 mL of the sample solution into a 10 mL colorimetric tube, place the colorimetric tube in a 60°C water bath, blow dry with nitrogen, and then cool to room temperature; then accurately add 0.4 mL of 5% vanillin glacial acetic acid solution and 1.0 mL of perchloric acid, mix well, and perform a color development reaction in a 60°C water bath; after 15 min±10 s, place the colorimetric tube in an ice bath to cool for 5 min, then accurately add 5.0 mL of glacial acetic acid, shake well, and let stand for 15-20 min, and obtain the sample solution after passing through a 0.45 μm filter membrane.

[0069] Preparation of standard curve: Take 10 mg of ganoderic acid A standard into a 100 mL volumetric flask, dissolve it with n-hexane and dilute to the mark, mix well to obtain a stock solution. Accurately pipette 0.00, 0.10, 0.20, 0.40, 0.80, 1.00 mL of stock solution and 1.00 mL of sample purification solution into a 10 mL colorimetric tube, place it in a 60°C water bath, blow dry with nitrogen, and cool to room temperature. Accurately add 0.4 mL of 5% vanillin glacial acetic acid solution and 1.0 mL of perchloric acid, mix well, and perform a color reaction in a 60°C water bath for 15 minutes. Quickly take it out and place it in an ice bath to cool for 5 minutes, then accurately add 5.0 mL of glacial acetic acid, shake well, and let it stand for 15 minutes. After filtering through a 0.45 μm filter, obtain a standard curve solution.

[0070] Liquid phase detection conditions: the chromatographic column selected was an acid-resistant C18 chromatographic column, 4.6×250mm, 5μm; acetonitrile containing 5% formic acid was used as the mobile phase; the flow rate was 0.5mL / min; the column temperature was 40℃; the injection volume was 10μL; the detection wavelength of the UV detector was 550nm.

[0071] High performance liquid chromatography was used to detect the standard curve solution and the sample solution to be tested, and a standard curve was prepared. The total triterpene content in the sample was obtained by external standard method and was 5.33%.

[0072] The standard curve data are shown in Table 1. Figure 2 ; According to the standard curve, the external standard method was used for quantification, and three parallel samples of unbroken Ganoderma lucidum spore powder (test numbers A, B, and C) were calculated. The analysis results are shown in Table 2.

[0073] Table 1: Standard curve data and linearity

[0074]

[0075]

[0076] Table 2: Parallel sample test results

[0077]

[0078] Example 2

[0079] A method for detecting the total triterpenoid content in unbroken spore powder of Ganoderma lucidum comprises the following steps:

[0080] (1) Sample extraction

[0081] A sample of unbroken Ganoderma lucidum spore powder was taken for three parallel experiments, with the sample weights being A: 24.8 mg, B: 25.9 mg, and C: 24.6 mg respectively.

[0082] Place the unbroken spore powder sample of Ganoderma lucidum in a 20mL homogenizer, add 2mL of anhydrous ethanol extract; break the spores for 20min at a frequency of 50 times / min under ice bath conditions. Transfer the mixed solution to a 250mL conical flask, add 60mL of extract, the extraction temperature is 50℃, the ultrasonic frequency is 25KHz; the ultrasonic time is 45min, and the extract is obtained. The obtained extract is frozen centrifuged for 5min at a speed of 10000r / min and a centrifugal temperature of 5℃ to obtain a supernatant.

[0083] (2) Sample purification

[0084] The supernatant was transferred to a 100mL round-bottom flask, concentrated in a 60°C water bath, under a vacuum degree of -0.8MPa, and concentrated on a rotary evaporator to a solvent content of ≤1% to obtain a concentrated solution. The concentrated solution was redissolved with 10mL of n-hexane to obtain an upper column liquid; alumina for chromatography (100-200 mesh) was taken and burned at 800°C for 5h to prepare active chromatography alumina; the chromatography column was used with a diameter of 15mm and a height of 300mm, with a sieve plate and a flow rate control cock at the lower end, and a small amount of absorbent cotton was placed above the sieve plate; n-hexane was added to the chromatography column, and 20g of alumina was poured into the chromatography column; anhydrous sodium sulfate with a thickness of 20mm was added to the upper end, the control cock was opened, and n-hexane was placed at the upper end of the anhydrous sodium sulfate, and the upper column liquid was added from the upper end of the chromatography column, and the liquid was discharged at a flow rate of 0.5mL / min until all the sample liquid entered the chromatography column. Use a measuring cylinder to collect the liquid at the bottom, collect 10mL of liquid and discard it. Continue to add 80mL of n-hexane from the top, collect all the remaining sample liquid in a 100mL volumetric flask, dilute to the mark with n-hexane, mix well, and use this as the sample solution.

[0085] (3) Sample testing

[0086] Take 1 mL of the sample solution into a 10 mL colorimetric tube, place the colorimetric tube in a 60°C water bath, blow dry with nitrogen, and then cool to room temperature; then accurately add 0.4 mL of 5% vanillin glacial acetic acid solution and 1.0 mL of perchloric acid, mix well, and perform a color development reaction in a 60°C water bath; after 15 min±10 s, place the colorimetric tube in an ice bath to cool for 5 min, then accurately add 5.0 mL of glacial acetic acid, shake well, and let stand for 15-20 min, and obtain the sample solution after passing through a 0.45 μm filter membrane.

[0087] Preparation of standard curve: Take 10 mg of ganoderic acid A standard into a 100 mL volumetric flask, dissolve it with n-hexane and dilute to the mark, mix well to obtain a stock solution. Accurately pipette 0.00, 0.10, 0.20, 0.40, 0.80, 1.00 mL of stock solution and 1.00 mL of sample purification solution into a 10 mL colorimetric tube, place it in a 60°C water bath, blow dry with nitrogen, and cool to room temperature. Accurately add 0.4 mL of 5% vanillin glacial acetic acid solution and 1.0 mL of perchloric acid, mix well, and perform a color reaction in a 60°C water bath for 15 minutes. Quickly take it out and place it in an ice bath to cool for 5 minutes, then accurately add 5.0 mL of glacial acetic acid, shake well, and let it stand for 15 minutes. After filtering through a 0.45 μm filter, obtain a standard curve solution.

[0088] Liquid phase detection conditions: the chromatographic column selected was an acid-resistant C18 chromatographic column, 4.6×250mm, 5μm; acetonitrile containing 5% formic acid was used as the mobile phase; the flow rate was 0.5mL / min; the column temperature was 40℃; the injection volume was 10μL; the detection wavelength of the UV detector was 550nm.

[0089] High performance liquid chromatography was used to detect the standard curve solution and the sample solution to be tested, and a standard curve was prepared. The total triterpene content in the sample was obtained by external standard method and was 5.33%.

[0090] The standard curve data are shown in Table 3, and the standard curve is shown in Figure 3 ; According to the standard curve, the external standard method was used for quantification, and three parallel samples of unbroken Ganoderma lucidum spore powder (test numbers A, B, and C) were calculated. The analysis results are shown in Table 4.

[0091] Table 3: Standard curve data

[0092] name Pipet volume mL Ganoderic acid A content μg Peak area Marking curve concentration point 1 0 0 0 Marking curve concentration point 2 0.1 10 36251 Marking curve concentration point 3 0.2 20 65254 Marking curve concentration point 4 0.4 40 130487 Marking curve concentration point 5 0.8 80 260145 Marking curve concentration point 6 1.0 100 331021

[0093] Table 4: Parallel sample test results

[0094]

[0095] Example 3

[0096] A method for detecting the total triterpenoid content in unbroken spore powder of Ganoderma lucidum comprises the following steps:

[0097] (1) Sample extraction

[0098] A sample of unbroken Ganoderma lucidum spore powder was taken for three parallel experiments, with the sample weights being A: 25.1 mg, B: 25.5 mg, and C: 24.9 mg respectively.

[0099] Place the unbroken spore powder sample of Ganoderma lucidum in a 20mL homogenizer, add 2mL of dichloromethane extract; break the spores for 20min at a frequency of 50 times / min under ice bath conditions. Transfer the mixed solution to a 250mL conical flask, add 60mL of extract, the extraction temperature is 50℃, the ultrasonic frequency is 25KHz; the ultrasonic time is 45min, and the extract is obtained. The obtained extract is frozen centrifuged for 5min at a speed of 10000r / min and a centrifugal temperature of 5℃ to obtain a supernatant.

[0100] (2) Sample purification

[0101] The supernatant was transferred to a 100 mL round-bottom flask, concentrated in a 60 ° C water bath, vacuum -0.8 MPa conditions, concentrated on a rotary evaporator to a solvent content of ≤1%, to obtain a concentrated solution. The concentrated solution was redissolved with 10 mL of n-hexane to obtain a column liquid; alumina for chromatography (100-200 mesh) was taken and burned at 820 ° C for 4.5 hours to prepare active chromatography alumina; the chromatography column was used with a diameter of 15 mm and a height of 300 mm, with a sieve plate and a flow rate control cock at the lower end, and a small amount of absorbent cotton was placed above the sieve plate; n-hexane was added to the chromatography column, 20 g of alumina was taken and poured into the chromatography column, and 20 mm thick anhydrous sodium sulfate was added to the upper end, the control cock was opened, and n-hexane was placed at the upper end of the anhydrous sodium sulfate, and the column liquid was added from the upper end of the chromatography column, and the liquid was discharged at a flow rate of 0.5 mL / min until the sample liquid entered the chromatography column. Use a measuring cylinder to collect the liquid at the bottom, collect 10mL of liquid and discard it. Continue to add 80mL of n-hexane from the top, collect all the remaining sample liquid in a 100mL volumetric flask, dilute to the mark with n-hexane, mix well, and use this as the sample solution.

[0102] (3) Sample testing

[0103] Take 1 mL of the sample solution into a 10 mL colorimetric tube, place the colorimetric tube in a 60°C water bath, blow dry with nitrogen, and then cool to room temperature; then accurately add 0.4 mL of 5% vanillin glacial acetic acid solution and 1.0 mL of perchloric acid, mix well, and perform a color development reaction in a 60°C water bath; after 15 min±10 s, place the colorimetric tube in an ice bath to cool for 5 min, then accurately add 5.0 mL of glacial acetic acid, shake well, and let stand for 15-20 min, and obtain the sample solution after passing through a 0.45 μm filter membrane.

[0104] Preparation of standard curve: Take 10 mg of ganoderic acid A standard into a 100 mL volumetric flask, dissolve it with n-hexane and dilute to the mark, mix well to obtain a stock solution. Accurately pipette 0.00, 0.10, 0.20, 0.40, 0.80, 1.00 mL of stock solution and 1.00 mL of sample purification solution into a 10 mL colorimetric tube, place it in a 60°C water bath, blow dry with nitrogen, and cool to room temperature. Accurately add 0.4 mL of 5% vanillin glacial acetic acid solution and 1.0 mL of perchloric acid, mix well, and perform a color reaction in a 60°C water bath for 15 minutes. Quickly take it out and place it in an ice bath to cool for 5 minutes, then accurately add 5.0 mL of glacial acetic acid, shake well, and let it stand for 15 minutes. After filtering through a 0.45 μm filter, obtain a standard curve solution.

[0105] Liquid phase detection conditions: the chromatographic column selected was an acid-resistant C18 chromatographic column, 4.6×250mm, 5μm; acetonitrile containing 5% formic acid was used as the mobile phase; the flow rate was 0.5mL / min; the column temperature was 40℃; the injection volume was 10μL; the detection wavelength of the UV detector was 550nm.

[0106] High performance liquid chromatography was used to detect the standard curve solution and the sample solution to be tested, and a standard curve was prepared. The total triterpene content in the sample was obtained by external standard method and was 5.23%.

[0107] The standard curve data are shown in Table 5, and the standard curve is shown in Figure 4 ; According to the standard curve, the external standard method was used for quantification, and three parallel samples of unbroken Ganoderma lucidum spore powder (test numbers A, B, and C) were calculated. The analysis results are shown in Table 6.

[0108] Table 5: Standard curve data

[0109] name Pipet volume mL Ganoderic acid A content μg Peak area Marking curve concentration point 1 0 0 0 Marking curve concentration point 2 0.1 10 35458 Marking curve concentration point 3 0.2 20 64584 Marking curve concentration point 4 0.4 40 134545 Marking curve concentration point 5 0.8 80 259872 Marking curve concentration point 6 1.0 100 334512

[0110] Table 6: Parallel sample test results

[0111]

[0112] Example 4

[0113] A method for detecting the total triterpenoid content in unbroken spore powder of Ganoderma lucidum comprises the following steps:

[0114] (1) Sample extraction

[0115] A sample of unbroken Ganoderma lucidum spore powder was taken for three parallel experiments, with the sample weights being A: 24.7 mg, B: 24.9 mg, and C: 25.1 mg respectively.

[0116] Place the unbroken spore powder sample of Ganoderma lucidum in a 20mL homogenizer, add 2mL of ethyl acetate extract; break the spores for 20min at a frequency of 50 times / min under ice bath conditions. Transfer the mixed solution to a 250mL conical flask, add 60mL of extract, the extraction temperature is 50℃, the ultrasonic frequency is 25KHz; the ultrasonic time is 45min, and the extract is obtained. The obtained extract is frozen centrifuged for 5min at a speed of 10000r / min and a centrifugal temperature of 5℃ to obtain a supernatant.

[0117] (2) Sample purification

[0118] The supernatant was transferred to a 100 mL round-bottom flask, concentrated in a 60 ° C water bath, vacuum -0.8 MPa conditions, concentrated on a rotary evaporator to a solvent content of ≤ 1%, to obtain a concentrated solution. The concentrated solution was redissolved with 10 mL of n-hexane to obtain a column liquid; alumina for chromatography (100-200 mesh) was taken and burned at 800 ° C for 5 hours to prepare active chromatography alumina; the chromatography column was used with a diameter of 15 mm and a height of 300 mm, with a sieve plate and a flow rate control cock at the lower end, and a small amount of absorbent cotton was placed above the sieve plate; n-hexane was added to the chromatography column, 20 g of alumina was poured into the chromatography column, and anhydrous sodium sulfate with a thickness of 20 mm was added to the upper end, the control cock was opened, and n-hexane was placed at the upper end of the anhydrous sodium sulfate, and the column liquid was added from the upper end of the chromatography column, and the liquid was discharged at a flow rate of 0.5 mL / min until the sample liquid entered the chromatography column. Use a measuring cylinder to collect the liquid at the bottom, collect 10mL of liquid and discard it. Continue to add 80mL of n-hexane from the top, collect all the remaining sample liquid in a 100mL volumetric flask, dilute to the mark with n-hexane, mix well, and use this as the sample solution.

[0119] (3) Sample testing

[0120] Take 1 mL of the sample solution into a 10 mL colorimetric tube, place the colorimetric tube in a 60°C water bath, blow dry with nitrogen, and then cool to room temperature; then accurately add 0.4 mL of 5% vanillin glacial acetic acid solution and 1.0 mL of perchloric acid, mix well, and perform a color development reaction in a 60°C water bath; after 15 min±10 s, place the colorimetric tube in an ice bath to cool for 5 min, then accurately add 5.0 mL of glacial acetic acid, shake well, and let stand for 15-20 min, and obtain the sample solution after passing through a 0.45 μm filter membrane.

[0121] Preparation of standard curve: Take 10 mg of ganoderic acid A standard into a 100 mL volumetric flask, dissolve it with n-hexane and dilute to the mark, mix well to obtain a stock solution. Accurately pipette 0.00, 0.10, 0.20, 0.40, 0.80, 1.00 mL of stock solution and 1.00 mL of sample purification solution into a 10 mL colorimetric tube, place it in a 60°C water bath, blow dry with nitrogen, and cool to room temperature. Accurately add 0.4 mL of 5% vanillin glacial acetic acid solution and 1.0 mL of perchloric acid, mix well, and perform a color reaction in a 60°C water bath for 15 minutes. Quickly take it out and place it in an ice bath to cool for 5 minutes, then accurately add 5.0 mL of glacial acetic acid, shake well, and let it stand for 15 minutes. After filtering through a 0.45 μm filter, obtain a standard curve solution.

[0122] Liquid phase detection conditions: the chromatographic column selected was an acid-resistant C18 chromatographic column, 4.6×250mm, 5μm; acetonitrile containing 5% formic acid was used as the mobile phase; the flow rate was 0.5mL / min; the column temperature was 40℃; the injection volume was 10μL; the detection wavelength of the UV detector was 550nm.

[0123] High performance liquid chromatography was used to detect the standard curve solution and the sample solution to be tested, and a standard curve was prepared. The total triterpene content in the sample was obtained by external standard method and was 5.26%.

[0124] The standard curve data are shown in Table 7, and the standard curve is shown in Figure 5 ; The external standard method was used for quantification according to the standard curve, and three parallel samples of unbroken Ganoderma lucidum spore powder (test numbers A, B, and C) were calculated. The analysis results are shown in Table 8.

[0125] Table 7: Standard curve data

[0126] name Pipet volume mL Ganoderic acid A content μg Peak area Marking curve concentration point 1 0 0 0 Marking curve concentration point 2 0.1 10 35458 Marking curve concentration point 3 0.2 20 64584 Marking curve concentration point 4 0.4 40 134545 Marking curve concentration point 5 0.8 80 269872 Marking curve concentration point 6 1.0 100 334512

[0127] Table 8: Parallel sample test results

[0128]

[0129]

[0130] Comparative Example 1

[0131] The difference from Example 1 is that the sample weights are 24.9 mg for A, 25.5 mg for B, and 24.5 mg for C. Alumina for chromatography (100-200 mesh) is taken and calcined at 450° C. for 12 h to prepare active alumina for chromatography.

[0132] The same standard curve as in Example 1 was used for calculation, and three parallel samples of unbroken Ganoderma lucidum spore powder (test numbers A, B, and C) were calculated. The analysis results are shown in Table 9.

[0133] Table 9: Parallel sample test results

[0134]

[0135] Comparative Example 2

[0136] The difference from Example 1 is that the sample weights are 24.1 mg for A, 23.9 mg for B, and 25.8 mg for C. No anhydrous sodium sulfate is added to the upper end, the control cock is opened, and n-hexane is placed on the upper end of the alumina.

[0137] The same standard curve as in Example 1 was used for calculation, and three parallel samples of unbroken Ganoderma lucidum spore powder (test numbers A, B, and C) were calculated. The analysis results are shown in Table 10.

[0138] Table 10: Parallel sample test results

[0139]

[0140]

[0141] Comparative Example 3

[0142] The difference from Example 1 is that the sample weights are 25.9 mg for A, 24.8 mg for B, and 26.5 mg for C. The lower end liquid is collected with a measuring cylinder, and 20 mL of liquid is collected and discarded.

[0143] The same standard curve as in Example 1 was used for calculation, and three parallel samples of unbroken Ganoderma lucidum spore powder (test numbers A, B, and C) were calculated. The analysis results are shown in Table 11.

[0144] Table 11: Parallel sample test results

[0145]

Claims

1. A method for detecting the total triterpenoid content in unbroken spore powder of Ganoderma lucidum, characterized in that: The following steps are involved: (1) Sample extraction: weighing unbroken spore powder of Ganoderma lucidum into a glass homogenizer, adding an extractant to break the spores, and obtaining a mixed solution of broken spore powder of Ganoderma lucidum, wherein the extractant is one of methanol, anhydrous ethanol, dichloromethane and ethyl acetate; adding an extractant to the mixed solution of broken spore powder of Ganoderma lucidum, performing ultrasonic treatment, and then centrifuging with a high-speed refrigerated centrifuge to obtain a supernatant; (2) Sample purification: the supernatant is concentrated to near dryness, and the concentrate is re-dissolved with n-hexane to obtain a supernatant; impurities in the supernatant are removed by a mixed chromatography column of anhydrous sodium sulfate and activated alumina, and the supernatant is collected and fixed to volume to obtain a sample solution; The preparation process of the anhydrous sodium sulfate and activated alumina chromatography column is as follows: n-hexane is added to the chromatography column, active chromatography alumina is added to the chromatography column, the alumina is uniformly precipitated, and then a layer of anhydrous sodium sulfate is added; the preparation of activated alumina: 100-200 mesh chromatography alumina is calcined at 810-850°C for 4-5h to prepare active chromatography alumina; (3) Sample detection: Take 1 mL of the sample solution and put it into a 10 mL colorimetric tube. Place the colorimetric tube in a 60°C water bath, blow dry with nitrogen, and then cool to room temperature. Then accurately add 0.4 mL of 5% vanillin glacial acetic acid solution and 1.0 mL of perchloric acid, mix well, and perform a color reaction in a 60°C water bath. After 15 min ± 10 s, place the colorimetric tube in an ice bath to cool for 5 min, then accurately add 5.0 mL of glacial acetic acid, shake well, and let stand for 15-20 min. After filtering through a 0.45 μm filter membrane, the sample solution was obtained. The standard curve solution was prepared with ganoderic acid A as the standard sample, and the standard curve solution and the sample solution were detected by high performance liquid chromatography to prepare the standard curve. The total triterpene content in the sample was obtained by external standard method. The chromatographic conditions of HPLC were: a) Chromatographic column: acid-resistant C18 column, 4.6×250mm, 5μm; b) Mobile phase conditions: acetonitrile containing 5% formic acid; c) Flow rate: 0.5 mL / min; d) Column temperature: 40°C; e) Injection volume: 10 μL; f) Detection wavelength: 550nm.

2. The method for detecting the total triterpene content in unbroken spore powder of Ganoderma lucidum according to claim 1, characterized in that: The cell wall breaking treatment in step (1) is as follows: weigh 25±2 mg of unbroken spore powder of Ganoderma lucidum into a 20 mL glass homogenizer, add 2-5 mL of an extractant, and treat at a frequency of 50 times / min for 20 min in an ice bath; The ultrasonic treatment is to add 50-100 mL of extractant into the mixed solution of broken wall Ganoderma lucidum spore powder for ultrasonic extraction, the ultrasonic extraction temperature is 45-55° C., the ultrasonic frequency is 25 KHz, and the ultrasonic time is 45 minutes.

3. The method for detecting the total triterpene content in unbroken spore powder of Ganoderma lucidum according to claim 1, characterized in that: The centrifugation temperature in step (1) is 0-5°C, the rotation speed is 10000 r / min, and the time is 5 min.

4. The method for detecting the total triterpene content in unbroken spore powder of Ganoderma lucidum according to claim 1, characterized in that: The concentration temperature of step (2) is 50-70°C and the vacuum degree is -0.8MPa.

5. The method for detecting the total triterpene content in unbroken spore powder of Ganoderma lucidum according to claim 1, characterized in that: The amount of n-hexane used for re-dissolving in step (2) is 10 mL.

6. The method for detecting the total triterpene content in unbroken spore powder of Ganoderma lucidum according to claim 5, characterized in that: Step (2) The chromatography column has a diameter of 15 mm and a height of 300 mm. The lower end is equipped with a sieve plate and a flow rate control stopcock. A small amount of absorbent cotton is placed above the sieve plate. 20 g of active chromatography alumina is added to the chromatography column, and then a layer of anhydrous sodium sulfate with a thickness of 20 mm is loaded. The process of removing impurities from the upper column liquid by the chromatography column is as follows: opening the control stopcock, placing the n-hexane in the chromatography column to the upper end of the anhydrous sodium sulfate, then adding the upper column liquid from the upper end of the chromatography column, discharging the liquid into the chromatography column at a flow rate of 0.5 mL / min, then opening the control stopcock, controlling the outflow rate of the lower end to 0.5 mL / min, collecting 10 mL of liquid and discarding it; continuously adding 80 mL of n-hexane from the upper end, taking a 100 mL volumetric flask to collect all the remaining sample liquid, diluting to the scale with n-hexane, mixing well, and using the sample solution as the sample solution.

7. The method for detecting the total triterpenoid content in unbroken spore powder of Ganoderma lucidum according to claim 1, characterized in that: The preparation process of the standard curve solution in step (3) is as follows: 10 mg of the ganoderic acid A standard is taken into a 100 mL volumetric flask, dissolved with n-hexane and diluted to the mark, and mixed to obtain a stock solution; 0.00 mL, 0.10 mL, 0.20 mL, 0.40 mL, 0.80 mL, and 1.00 mL of the stock solution are accurately pipetted into 10 mL colorimetric tubes respectively, each colorimetric tube is placed in a 60° C. water bath, blown dry with nitrogen, and then cooled to room temperature; 0.4 mL of 5% vanillin glacial acetic acid solution and 1.0 mL of perchloric acid are accurately added, mixed, and a color development reaction is performed in a 60° C. water bath; after 15 min±10 s, the colorimetric tube is placed in an ice bath to cool for 5 min, and then 5.0 mL of glacial acetic acid is accurately added, shaken, and allowed to stand for 15-20 min, and filtered through a 0.45 μm filter membrane to prepare standard curve test solutions of different concentrations.

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