A ganoderma lucidum spore powder and a method for determining the content of ganoderma triterpenes in raw materials thereof

Abstract

The application discloses a ganoderma spore powder and a method for determining the content of ganoderma triterpenes in the raw material of the ganoderma spore powder, and has the steps that the ganoderma spore powder sample is ultrasonic-assisted extracted, chloroform is used as an extraction solvent, and a total triterpene crude extract is obtained; the crude extract is separated and purified through an ADS-8 macroporous adsorption resin column, a gradient elution mode is used to remove impurities, 70% ethanol is used for elution and enrichment of target ganoderma triterpene components; the eluent is concentrated to dryness under reduced pressure, dissolved accurately with methanol, filtered through an organic phase filter membrane to prepare a test sample solution; a ganoderma acid C2 reference substance solution is prepared under the same conditions, detection is carried out under specific column and mobile phase conditions through a high performance liquid chromatography system, and the content of ganoderma triterpenes in the sample is calculated through an external standard method. The method has the advantages of reasonable design, clear extraction and purification process parameters, easy standardized operation, moderate detection cost, short cycle, reliable results and the like, and has good industrial application prospect and popularization value.

Description

Technical Field

[0001] This invention belongs to the field of natural product extraction and analytical chemistry, and specifically relates to a method for determining the content of Ganoderma triterpenes in Ganoderma lucidum spore powder and its raw materials. Background Technology

[0002] Reishi mushroom, a traditional and precious Chinese medicine, has been used in my country for thousands of years and is known as the "immortal herb." Reishi triterpenoids are one of the core active components in reishi mushroom, possessing various pharmacological effects such as anti-tumor, liver protection and detoxification, anti-inflammation, and antiviral activity. Their content directly determines the quality and market value of reishi mushroom and its processed products.

[0003] With the development of the health industry, Ganoderma lucidum spore powder, as a high-value-added raw material for health products, is experiencing increasing market demand. However, current methods for determining the content of Ganoderma lucidum triterpenes in Ganoderma lucidum spore powder still have many shortcomings. Traditional analytical methods mostly employ spectrophotometry, which, although simple to operate, has poor specificity and is easily interfered with by coexisting components such as polysaccharides and proteins, leading to low accuracy of the results. While some high-performance liquid chromatography (HPLC) methods have certain separation capabilities, the sample pretreatment process is often cumbersome, with low extraction efficiency and a lack of effective purification methods. The matrix effect is significant, affecting the precision and accuracy of quantification. Furthermore, existing technologies often use ganoderic acid A as a reference standard for quantification, but the content of ganoderic acid A is extremely low or even absent in some Ganoderma lucidum varieties, making it difficult to fully reflect the true level of total triterpenes in the sample.

[0004] Therefore, establishing a high-performance liquid chromatography (HPLC) method that is simple to operate, highly sensitive, reproducible, and provides accurate and reliable results is of great significance for scientifically evaluating the quality of Ganoderma lucidum spore powder, regulating market order, and promoting the healthy development of the Ganoderma lucidum industry. Summary of the Invention

[0005] In view of the problems existing in the prior art, the purpose of this invention is to provide a method for determining the content of Ganoderma triterpenes in Ganoderma lucidum spore powder and its raw materials.

[0006] This invention is specifically implemented through the following technical solutions: A method for determining the content of Ganoderma triterpenes in Ganoderma lucidum spore powder and its raw materials, comprising the following steps: 1) Chloroform was used as the extraction solvent, and ultrasonic-assisted extraction technology was used to extract Ganoderma lucidum spore powder samples to obtain crude chloroform extract rich in triterpenes; 2) Concentrate the triterpenoid-rich crude chloroform extract obtained in step 1) until there is no chloroform odor, filter it into a lipid column, and then elute it with 70% ethanol solution, collecting the eluent; 3) After concentrating the eluent collected in step 2) under reduced pressure to dryness, redissolve it with methanol and filter it through an organic phase filter membrane to obtain the test solution; 4) Prepare standard solutions of ganoderic acid C2 with the same matrix to obtain a series of standard working solutions of different concentrations; 5) Using a high-performance liquid chromatograph, under the set specific chromatographic column and mobile phase system, the test solution and standard working solution were detected respectively, and the chromatograms were recorded; 6) Plot a standard curve with the concentration of the standard working solution as the x-axis and the peak area as the y-axis. Substitute the peak area of ​​ganoderic acid C2 in the test solution into the standard curve to calculate the concentration of ganoderic acid C2 in the test solution. Then calculate the content of ganoderic triterpenes in the sample according to the formula.

[0007] Further, in step 1), the ultrasonic-assisted extraction is specifically as follows: first, add 60 mL of chloroform and ultrasonically extract for 30 min, then filter and add another 40 mL of chloroform to the residue, ultrasonically extract for 30 min, and then filter and combine the two filtrates.

[0008] Further, in step 2), the loading flow rate for the ADS-8 macroporous adsorption resin column is 1 mL / min, and the elution flow rate for the 70% ethanol solution is 8 mL / min.

[0009] Furthermore, the high-performance liquid chromatography conditions in step 5) are as follows: Chromatographic column: 4.6 mm × 250 mm, 5 μm reversed-phase high-performance liquid chromatography column; column temperature: 30℃; detection wavelength: 254 nm; flow rate: 1.0 mL / min; injection volume: 10 μL; mobile phase: phase A is acetonitrile (chromatographic grade), phase B is methanol (chromatographic grade), phase C is 0.01% acetic acid aqueous solution, gradient elution conditions; gradient elution program: At 0 min, A: 20%, B: 20%, C: 60%; At 30 minutes, A: 30%, B: 20%, C: 50%; At 35 minutes, A: 38%, B: 20%, C: 42%; At 40 minutes, A: 20%, B: 20%, C: 60%; At 45 minutes, A: 20%, B: 20%, C: 60%.

[0010] Furthermore, the formula for calculating the content of Ganoderma triterpenes in the sample in step 6) is: ; In the formula: X—content of Ganoderma triterpenes in Ganoderma lucidum spore powder (calculated as Ganoderma lucidum acid C2), % C—The concentration of ganoderic acid C2 in the test solution, calculated from the standard curve, in μg / mL; V1 — The volume of methanol dissolved after the eluent is concentrated, 1 mL; m—The weight of the Ganoderma lucidum spore powder sample, in grams; 10 6 —Unit conversion factor.

[0011] Based on repeated experiments, this invention discovered that chloroform, as a moderately polar organic solvent, has a solubility property that highly matches the physicochemical properties of Ganoderma lucidum triterpenoids, effectively dissolving and releasing the target components within cells. Simultaneously, chloroform exhibits extremely low solubility for a large number of water-soluble macromolecular impurities such as polysaccharides and proteins in Ganoderma lucidum spore powder, thus achieving "refining from crude substances" at the extraction source and significantly reducing the burden on subsequent purification steps.

[0012] Ultrasonic-assisted extraction utilizes the strong mechanical vibrations generated by cavitation to effectively break down the tough double-layered chitinous walls of Ganoderma lucidum spore powder, disrupting the cell structure and making intracellular triterpenoids more readily soluble. Compared to traditional stirring, ultrasound provides a more uniform energy distribution, accelerating the diffusion rate of solute molecules and thus significantly improving extraction efficiency. Ganoderma lucidum spore powder has a dense structure, and a single ultrasound treatment often fails to completely extract deeply bound triterpenoids. Through repeated ultrasonic extraction, a continuous concentration gradient can be established, driving the extraction process in the positive direction and ensuring that the target components are thoroughly and completely extracted, avoiding underestimation of content due to incomplete extraction.

[0013] This invention employs a chromatographic column packed with ADS-8 macroporous adsorption resin for separation and purification. Macroporous adsorption resin is a purified pharmaceutical-grade non-polar adsorption resin, exhibiting significant advantages and wide application in the separation and purification of active ingredients from traditional Chinese medicine. It primarily relies on a hydrophobic adsorption mechanism to selectively adsorb ganoderic acid, separating it from water-soluble impurities. Among these, ADS-8 resin demonstrates excellent performance in the extraction of natural products due to its high adsorption capacity, easy elution, and good kinetic properties.

[0014] Through repeated experiments, this invention has found that using an ADS-8 macroporous adsorption resin-filled chromatographic column for separation and purification, treating the loading solution with 40% ethanol at pH=3 and then eluting with 70% ethanol results in a higher recovery rate and a purer test solution, which is beneficial for high-performance liquid chromatography (HPLC) detection of ganoderic acid C2 content.

[0015] This invention uses ganoderic acid C2 as a reference for quantitative analysis. Ganoderic acid C2 is a unique component in Ganoderma lucidum spore powder and its content is relatively stable. Compared with the use of non-specific references such as oleanolic acid in traditional methods, it can more accurately reflect the actual content of triterpenoid components in Ganoderma lucidum spore powder, effectively avoiding detection bias caused by improper selection of references, and improving the specificity and accuracy of quantification.

[0016] The beneficial effects of this invention are that, due to the thorough removal of impurities during the pretreatment process, the target components are highly enriched, resulting in a stable baseline and good resolution of the characteristic peaks of Ganoderma lucidum triterpenes during high-performance liquid chromatography (HPLC). This not only improves the detection sensitivity (lower detection limit) but also ensures the reproducibility of multiple parallel determinations, with a small RSD (relative standard deviation) value in the experimental data, meeting the stringent requirements for trace component analysis.

[0017] The present invention features a rationally designed procedure, clearly defined extraction and purification process parameters, and is easy to standardize. This method is not only suitable for quality control of Ganoderma lucidum spore powder raw materials, but can also be widely applied to the determination of total triterpenoid content in deep-processed products such as Ganoderma lucidum spore oil and Ganoderma lucidum capsules. It has advantages such as moderate detection cost, short cycle time, and reliable results, and has good industrial application prospects and promotional value. Attached Figure Description

[0018] Figure 1 The chromatograms are of the reference standards ganoderic acid C2 and ganoderic acid A in Example 1; Figure 2 A comparison chart showing the content of ganoderic acid C2 and ganoderic acid A obtained by eluting with ethanol solutions of different concentrations in Comparative Example 2. Detailed Implementation

[0019] The present invention will be further described below with reference to specific examples.

[0020] The measurement procedure of this invention is as follows: The sample was extracted using ultrasound-assisted extraction with chloroform as solvent to obtain a crude extract rich in triterpenes. The crude extract was then loaded onto an ADS-8 macroporous adsorption resin-filled chromatographic column, and impurities were effectively removed through a gradient elution program.

[0021] The contents of ganoderic acid A and ganoderic acid C2 in the samples were determined using reference standards, and the results of the two reference standards were compared.

[0022] The contents of ganoderic acid A and ganoderic acid C2 were compared by selectively eluting and enriching the target triterpenoid components with ethanol solutions of different concentrations.

[0023] The collected eluent was concentrated under reduced pressure to remove the solvent, then precisely redissolved in methanol, and filtered through an organic microporous membrane to prepare the final sample solution. Simultaneously, a ganoderic acid reference solution with the same matrix was prepared. Finally, the sample solution and reference solution were introduced together into a high-performance liquid chromatograph (HPLC) system for separation and detection under a specific column and mobile phase. Based on the external standard method, the content of ganoderic triterpenes in the sample was accurately calculated.

[0024] Example 1: A high-performance liquid chromatography method for determining the content of Ganoderma triterpenes in Ganoderma lucidum spore powder raw material. Main reagents: 1) Organic solvents: chloroform, anhydrous ethanol, methanol, acetonitrile; 2) Ethanol solution: 70% ethanol solution (volume ratio); 3) Acetic acid aqueous solution: 0.01% (measure 0.1 mL of acetic acid, dilute to 1000 mL with ultrapure water, and shake well). 4) Reference material: Ganoderma lucidum acid C2 reference standard (purity ≥98%).

[0025] Main materials: 1) Macroporous adsorption resin: ADS-8 type (Nankai Hecheng); 2) Filter membrane and sieve: 0.22μm organic phase microporous filter membrane, No. 3 pharmaceutical sieve, qualitative filter paper; 3) Chromatography column consumables: Chromatography column with an inner diameter of 3cm and a column height of 60cm.

[0026] Main equipment and instruments: 1) High-performance liquid chromatograph: equipped with an ultraviolet detector (UV) and a chromatography workstation 2) Electronic analytical balance: accuracy 0.0001g 3) Sample pretreatment equipment: ultrasonic cleaner (power ≥ 200W, frequency 40kHz), forced-air drying oven (accuracy ±1℃), and Chinese herbal medicine pulverizer. 4) Chromatography equipment: Chromatography column with an inner diameter of 3cm and a height of 60cm. 5) Glassware: stoppered conical flasks (50mL, 250mL), volumetric flasks (1mL, 5mL, 10mL, 50mL, 100mL), pipettes / graduated pipettes (calibrated), separatory funnels (100mL, 250mL), chromatographic bottles 6) Others: Vortex oscillator, dryer.

[0027] Sample preparation: Take Ganoderma lucidum spore powder sample, mix it thoroughly and evenly, then reduce it to no less than 50g using the quartering method, and remove impurities; pulverize it with a pulverizer, pass it through a No. 3 sieve, place the pulverized sample in a forced-air drying oven, dry it at 70℃ for 6h, and then seal it in a desiccator for later use.

[0028] Ultrasonic extraction of chloroform: Accurately weigh 2.0 g of crude sample powder (accurate to 0.0001 g) and place it in a 250 mL stoppered conical flask. Add 60 mL of chloroform and seal the stopper. Place the conical flask in an ultrasonic cleaner, set the power to 200 W and the frequency to 40 kHz, and ultrasonically extract for 30 min. After extraction, remove the flask and let it stand and cool to room temperature. Filter the flask with qualitative filter paper and collect the filtrate in a 100 mL volumetric flask. Add 40 mL of chloroform to the residue again and repeat the above ultrasonic extraction, cooling, and filtration operations. Combine the two filtrates and dilute to 100 mL with chloroform. Shake well to obtain crude chloroform extract.

[0029] Pretreatment of ADS-8 macroporous adsorption resin: Pack ADS-8 macroporous adsorption resin into a 3cm×60cm chromatography column, add 40% ethanol solution with pH=3 and soak for 24h; after soaking, rinse the resin column with 40% ethanol solution with pH=3 at a flow rate of 8mL / min until the pH of the effluent is consistent with that of the eluent, and set aside for use.

[0030] Preparation of loading solution: Transfer the crude chloroform extract obtained above to an evaporation flask of a rotary evaporator and concentrate under reduced pressure at 60°C until there is no chloroform odor (no oily residue on the flask wall and no liquid dripping); after cooling to room temperature, add 50 mL of anhydrous ethanol to the evaporation flask and sonicate in a 60°C constant temperature water bath for 20 min; filter with qualitative filter paper, transfer the filtrate to a 100 mL volumetric flask, and dilute to the mark with 40% ethanol solution at pH=3, shake well, and obtain the loading solution.

[0031] Dynamic adsorption and gradient elution: Pour the sample solution into the injection port of the chromatography column, and load the sample at a flow rate of 1 mL / min. After loading, let it stand for 30 min to allow the resin to fully adsorb the target component. Elute with 500 mL of 70% ethanol solution at an elution flow rate of 8 mL / min and collect the eluent.

[0032] Eluent preparation: Transfer the 70% ethanol target eluent collected above to an evaporation flask of a rotary evaporator and concentrate to dryness under reduced pressure at 60°C; after cooling to room temperature, accurately add 1 mL of methanol to the evaporation flask and vortex for 1-2 min until the concentrate is completely dissolved; filter the solution through a 0.22 μm organic phase microporous membrane into a clean chromatographic bottle, collect the filtrate as the test solution for later use.

[0033] High performance liquid chromatography detection: 1) Preparation of reference standard Ganoderic acid C2 stock solution (1.0 mg / mL): Accurately weigh 10.0 mg (accurate to 0.0001 g) of ganoderic acid C2 reference standard and place it in a 10 mL volumetric flask. Add an appropriate amount of methanol, sonicate to dissolve, and then dilute to the mark with methanol. Shake well and store in a sealed container at 4°C. Shelf life is 7 days. Accurately measure 0.1 mL, 0.2 mL, 0.5 mL, 1.0 mL, and 2.0 mL of ganoderic acid C2 stock solution into five 10 mL volumetric flasks respectively. Dilute to the mark with methanol, vortex well, and prepare a series of standard working solutions of different concentrations. Prepare and use immediately.

[0034] 2) Preparation of blank solution Accurately measure 1 mL of methanol and filter it through a 0.22 μm organic phase microporous membrane to obtain a blank solution.

[0035] 3) Chromatographic conditions Chromatographic column: 4.6 mm × 250 mm, 5 μm reversed-phase high-performance liquid chromatography column; column temperature: 30 ℃; detection wavelength: 254 nm; flow rate: 1.0 mL / min; injection volume: 10 μL; mobile phase: phase A is acetonitrile (chromatographic grade), phase B is methanol (chromatographic grade), phase C is 0.01% acetic acid aqueous solution, and the gradient elution program is shown in Table 1.

[0036] Table 1

[0037] Inject blank solution, a series of standard working solutions, and the test solution sequentially, and record the chromatograms, such as... Figure 1 As shown.

[0038] 4) Plotting the standard curve The series of standard working solutions were injected sequentially under chromatographic conditions, and the peak area of ​​ganoderic acid C2 corresponding to each concentration was recorded. A standard curve was plotted using a chromatography workstation or data analysis software, with the concentration of ganoderic acid C2 (μg / mL) as the x-axis and the peak area of ​​ganoderic acid C2 as the y-axis. The regression equation and correlation coefficient were calculated. The standard curve gradient data are shown in Table 2.

[0039] Table 2

[0040] 5) Sample determination According to the chromatographic conditions, inject blank solution and test solution sequentially, and record the chromatogram; read the peak area of ​​ganoderic acid C2 in the test solution and calculate the peak area ratio between the two; substitute the ratio into the standard curve regression equation to calculate the concentration of ganoderic acid C2 in the test solution; use the peak area ratio for calculation.

[0041] 6) Calculation of Ganoderma lucidum acid C2 content The total triterpenoid content (calculated as ganoderic acid C2) in Ganoderma lucidum spore powder is calculated according to formula (1):

[0042] In the formula: X—content of Ganoderma triterpenes in Ganoderma lucidum spore powder (calculated as Ganoderma lucidum acid C2), % C—The concentration of ganoderic acid C2 in the test solution, calculated from the standard curve, in μg / mL; V1 — The volume of methanol dissolved after the eluent is concentrated, 1 mL; m—The weight of the Ganoderma lucidum spore powder sample, in grams; 10 6 —Unit conversion factor.

[0043] According to the linear equation y = 7.5309x - 1.8015, R 2 = 1 gives the concentration of ganoderic acid C2 in the sample. Based on the above formula, the total triterpenoid content of the sample (calculated as ganoderic acid C2) is 2.06 × 10⁻⁶. -4 %.

[0044] Comparative Example 1 The difference from Example 1 is that ganoderic acid A was used as a reference standard for the determination of total triterpenoid content, and the standard curve gradient is shown in Table 3.

[0045] Table 3

[0046] The other steps were the same as in Example 1, and the total triterpenoid content of the sample (calculated as ganoderic acid C2) was measured to be 9.19 × 10⁻⁶. -5 It can be observed that in the method for determining the content of Ganoderma lucidum triterpenoids, the use of ganoderic acid C2 as a reference standard in this invention shows a significant advantage compared to using ganoderic acid A as a reference standard. Experimental data shows that the results obtained using ganoderic acid A as a reference standard are lower, suggesting that it may have insufficient response under certain detection conditions. Conversely, using ganoderic acid C2 as a reference standard yields more reliable and higher numerical results, demonstrating its higher sensitivity, better linearity, or stronger representativeness in this detection system. Therefore, this invention, by selecting ganoderic acid C2... As a reference standard, it effectively improves the accuracy and reproducibility of Ganoderma lucidum triterpenoid content determination, and solves the technical problem of content underestimation caused by improper selection of reference standards in the existing technology, which is a significant advancement.

[0047] Comparative Example 2 This invention, in its method for determining the content of triterpenoids in Ganoderma lucidum, involves in-depth research and optimization of the concentration of the elution solvent. The experiment involved eluting Ganoderma lucidum samples with ethanol solutions of different concentrations and determining the concentrations of ganoderic acid A and ganoderic acid C2. The measured concentrations of ganoderic acid A and ganoderic acid C2 are shown below. Figure 2 The results show that the elution effect is optimal when the ethanol concentration is 70%, and the measured concentrations of ganoderic acid A and ganoderic acid C2 are significantly higher than under other concentration conditions. This proves that the 70% ethanol solution can achieve the most ideal balance between desorption and dissolution in this extraction system, effectively improving the elution rate and detection response value of the target components. Compared with existing technologies that do not specify or use other concentrations of ethanol for elution, this invention significantly improves the detection efficiency and accuracy of Ganoderma lucidum triterpenoids by limiting the ethanol concentration to 70%, solving the technical problems of incomplete elution at low concentrations or significant interference from high-concentration solvents.

Claims

1. A method for determining the content of Ganoderma lucidum triterpenes in Ganoderma lucidum spore powder and its raw materials, characterized in that, Includes the following steps: 1) Chloroform was used as the extraction solvent, and ultrasonic-assisted extraction technology was used to extract Ganoderma lucidum spore powder samples to obtain crude chloroform extract rich in triterpenes; 2) Concentrate the triterpenoid-rich crude chloroform extract obtained in step 1) until there is no chloroform odor, filter it into a lipid column, and then elute it with 70% ethanol solution, collecting the eluent; 3) After concentrating the eluent collected in step 2) under reduced pressure to dryness, redissolve it with methanol and filter it through an organic phase filter membrane to obtain the test solution; 4) Prepare standard solutions of ganoderic acid C2 with the same matrix to obtain a series of standard working solutions of different concentrations; 5) Using a high-performance liquid chromatograph, under the set specific chromatographic column and mobile phase system, the test solution and standard working solution were detected respectively, and the chromatograms were recorded; 6) Plot a standard curve with the concentration of the standard working solution as the x-axis and the peak area as the y-axis. Substitute the peak area of ​​ganoderic acid C2 in the test solution into the standard curve to calculate the concentration of ganoderic acid C2 in the test solution. Then calculate the content of ganoderic triterpenes in the sample according to the formula.

2. The method for determining the content of Ganoderma lucidum triterpenes in Ganoderma lucidum spore powder and its raw materials as described in claim 1, characterized in that, In step 1), the ultrasonic-assisted extraction is as follows: first, add 60 mL of chloroform and ultrasonically extract for 30 min. After filtration, add another 40 mL of chloroform to the residue and ultrasonically extract for 30 min. Then, filter and combine the two filtrates.

3. The method for determining the content of Ganoderma lucidum triterpenes in Ganoderma lucidum spore powder and its raw materials as described in claim 1, characterized in that, Step 2) The loading flow rate for the ADS-8 macroporous adsorption resin column is 1 mL / min, and the elution flow rate for the 70% ethanol solution is 8 mL / min.

4. The method for determining the content of Ganoderma lucidum triterpenes in Ganoderma lucidum spore powder and its raw materials as described in claim 1, characterized in that, The high-performance liquid chromatography (HPLC) conditions in step 5) are as follows: Chromatographic column: 4.6 mm × 250 mm, 5 μm reversed-phase high-performance liquid chromatography column; column temperature: 30℃; detection wavelength: 254 nm; flow rate: 1.0 mL / min; injection volume: 10 μL; mobile phase: phase A is acetonitrile (chromatographic grade), phase B is methanol (chromatographic grade), phase C is 0.01% acetic acid aqueous solution, gradient elution conditions; gradient elution program: At 0 min, A: 20%, B: 20%, C: 60%; At 30 minutes, A: 30%, B: 20%, C: 50%; At 35 minutes, A: 38%, B: 20%, C: 42%; At 40 minutes, A: 20%, B: 20%, C: 60%; At 45 minutes, A: 20%, B: 20%, C: 60%.

5. The method for determining the content of Ganoderma lucidum triterpenes in Ganoderma lucidum spore powder and its raw materials as described in claim 1, characterized in that, The formula for calculating the content of Ganoderma triterpenes in the sample in step 6) is: ； In the formula: X—content of Ganoderma triterpenes in Ganoderma lucidum spore powder (calculated as Ganoderma lucidum acid C2), % C—The concentration of ganoderic acid C2 in the test solution, calculated from the standard curve, in μg / mL; V1 — The volume of methanol dissolved after the eluent is concentrated, 1 mL; m—The weight of the Ganoderma lucidum spore powder sample, in grams; 10 6 —Unit conversion factor.

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