Method for detecting content of triterpenes in ganoderma lucidum spore powder

Abstract

The invention relates to the technical field of detection of the content of triterpenes in ganoderma lucidum spore powder, and discloses a method for detecting the content of triterpenes in ganoderma lucidum spore powder, which comprises the following steps: a, sample preparation: weighing a ganoderma lucidum spore powder sample, and adding chloroform as a to-be-detected sample solution; b, preparing an ursolic acid ethyl acetate solution, adding vanillic aldehyde glacial acetic acid and perchloric acid solution, adding glacial acetic acid, determining absorbance, and drawing a standard curve; c, measuring a to-be-measured sample solution, evaporating the to-be-measured sample solution in an evaporating dish, cooling, adding a vanillin glacial acetic acid solution to dissolve residues, adding perchloric acid, and heating; and d, adding glacial acetic acid to measure absorbance, and calculating the concentration of total triterpenes in the sample according to the standard curve. According to the method, the ganoderma lucidum spore powder sample and the reagent are selected according to fixed values, so that the phenomenon that the detection results are greatly different due to the fact that the substance selection interval is too large is avoided, the uniformity of the detection method is standardized, and the problem that the detection results are greatly different due to non-uniform operation steps in the prior art is solved.

Description

Technical Field

[0001] The invention relates to the technical field of triterpene content detection in ganoderma lucidum spore powder, in particular to a method for detecting triterpene content in ganoderma lucidum spore powder. Background Art

[0002] Ganoderma lucidum spore powder is the seed of Ganoderma lucidum, which is the spores released by Ganoderma lucidum during its maturity. Spore powder is rich in a variety of bioactive substances, especially triterpenoids. Triterpenoids have many pharmacological effects, including anti-tumor, anti-inflammatory, and immune regulation. Therefore, the triterpenoid content in Ganoderma lucidum spore powder directly affects its efficacy and nutritional value.

[0003] Due to the various potential benefits of triterpenoids to human health, the quality evaluation of Ganoderma lucidum spore powder often takes triterpenoid content as the core indicator. Therefore, accurate detection of triterpenoid content in Ganoderma lucidum spore powder helps to determine its medicinal value and quality, and is conducive to product quality control and standardization. At the same time, in commercial applications, the level of triterpenoid content is also related to the market value of the product. Therefore, establishing a scientific and accurate triterpenoid content detection method is of great significance to improving the overall quality of Ganoderma lucidum spore powder products.

[0004] The detection steps of the prior art are:

[0005] 1. Take a certain amount of mixed sample (except soft capsules), accurately weigh (the amount of total triterpenes in the sample is about 0.5-5 mg), put it in a 50mL volumetric flask, add about 30mL of chloroform (3.1.2), ultrasonically treat for 30min, let cool, add chloroform to the scale, shake well, centrifuge, and take the supernatant for later use;

[0006] 2. Oil products: Accurately weigh an appropriate amount of the mixed sample (the amount of total triterpenes in the sample is about 0.5-5 mg), place it in a 100 mL volumetric flask, dissolve it with ethyl acetate (3.1.1) and dilute to the scale, and shake well;

[0007] 3. Soft capsule preparation: Take a certain amount of the mixed clean capsule contents, accurately weigh (the amount of total triterpenes in the sample is about 0.5-5 mg), put it in a 100 mL volumetric flask, add about 60 mL of ethyl acetate (3.1.1), ultrasonically treat for 30 min, cool, add ethyl acetate to the scale, and shake well;

[0008] 4. Preparation of standard curve: Accurately pipette 0.00, 0.10, 0.20, 0.40, 0.80, and 1.00 mL of ursolic acid standard solution (3.3) into an evaporating dish, evaporate to dryness in a 60°C water bath, and after cooling, accurately add 0.4 mL of 5% vanillin glacial acetic acid solution (3.4), rotate the evaporating dish to dissolve the residue, accurately add 1.0 mL of perchloric acid (3.1.4), mix well, transfer 10 mL into a stoppered colorimetric tube and stopper it, heat in a 60°C water bath for 15 min, take out, cool in an ice bath, accurately add 5.0 mL of glacial acetic acid and shake well, and after 15 min, measure the absorbance at a wavelength of 548 nm with a 1 cm stoppered colorimetric tube cell, and draw a standard curve with absorbance as the ordinate and concentration as the abscissa; then, obtain the concentration of total triterpenes in the test solution according to the standard curve.

[0009] However, in the detection process of this method step, the numerical values ​​are mostly selected as range values. Therefore, in actual measurement, there will be a certain range difference in the amount of raw materials and reagents used, resulting in incomplete uniformity of the operating steps for each test, and ultimately leading to inconsistent test results. Summary of the invention

[0010] In view of the shortcomings of the prior art, the present invention provides a method for detecting the triterpene content in Ganoderma lucidum spore powder, which solves the problem in the prior art that most of the selected numerical values ​​are range values, and there will be certain interval differences in the amount of raw materials and reagents used in actual measurements, resulting in incomplete uniformity of the operating steps for each detection, and ultimately leading to inconsistent test results.

[0011] To achieve the above object, the present invention is implemented by the following technical scheme: a method for detecting triterpenoid content in Ganoderma lucidum spore powder, comprising the following steps:

[0012] a. Sample preparation: weigh a sample of Ganoderma lucidum spore powder containing 0.5-5 mg triterpenes, place it in a 50 mL volumetric flask, add 30 mL of chloroform, treat it with an ultrasonic cleaner for 30 min, cool it, and use chloroform to make up to 50 mL; after centrifugation, take the supernatant as the sample solution to be tested; wherein weigh a sample of Ganoderma lucidum spore powder containing 5 mg triterpenes;

[0013] b. Prepare 100 μg / mL ethyl acetate solution of ursolic acid, accurately measure 0.00mL, 0.10mL, 0.20mL, 0.40mL, 0.80mL, 1.00mL, 1.20mL, 1.40mL, 1.60mL, 1.80mL and 2.00mL standard solution and evaporate to dryness respectively, add 0.6mL vanillin glacial acetic acid solution after cooling, add 1.3mL perchloric acid after dissolving, place in a 60℃ water bath and heat for 15min, transfer the solution into a stoppered colorimetric tube after cooling, add 6.0mL glacial acetic acid, let stand for 15min, measure the absorbance at a wavelength of 548nm, and draw a standard curve with absorbance as the Y-axis and concentration as the X-axis;

[0014] c. Take 1.0 mL of the sample solution to be tested and place it in an evaporating dish. Place it in a 60°C water bath and evaporate it for 5 hours at a fan speed of 0.4 m / s. After cooling, add 0.6 mL of vanillin glacial acetic acid solution to dissolve the residue, then add 1.3 mL of perchloric acid and heat it in a 60°C water bath for 15 minutes.

[0015] d. After taking out the solution obtained in c, immediately cool it in an ice bath, add 6.0 mL of glacial acetic acid, let it stand for 15 minutes, measure the absorbance, and calculate the total triterpene concentration in the sample according to the standard curve.

[0016] Preferably, the amount of vanillin glacial acetic acid solution is 0.55-0.65 mL, the amount of perchloric acid is 1.2-1.4 mL, and during the centrifugation operation in step a, the centrifugal speed is 2900-3100 rpm and the centrifugal time is 15-16 min.

[0017] Preferably, when performing absorbance measurement in step d, a cuvette is used and the consistency of the photometric measurement is maintained.

[0018] Preferably, the temperature of the water bath in steps b and c is controlled at 60°C±1°C, and after adding perchloric acid in step c, heating is continued at 60°C±1°C for 15 minutes.

[0019] Preferably, the total triterpene content in the sample is calculated using the following formula:

[0020]

[0021] Among them, X i is the total triterpenoid content in the sample, expressed in g / 100g, C i is the mass of triterpenoids in the sample solution obtained from the standard curve, in μg, m is the mass of the sample, in g, V 1 V is the volume of the extract in mL. 2 is the volume of sample used for determination, in mL;

[0022] To ensure the accuracy of the results, three independent measurements were performed under the same conditions in step d, and the average value was calculated to ensure that the relative deviation of the two measurement results did not exceed 10% of the arithmetic mean, and the results of the three independent measurements were set as follows: A 2 and A 3

[0023] Then calculate the average The formula is:

[0024]

[0025] The relative deviation A of each measured value was then calculated using the following formula i , and ensure that the relative deviation A i Not more than 10%:

[0026]

[0027] Here, i is the number of measurements.

[0028] Preferably, the fan is placed 20-30 cm obliquely above the evaporating dish, and the airflow output by the fan maintains a horizontal flow direction so that the air evenly covers the surface of the evaporating dish.

[0029] The present invention provides a method for detecting the triterpene content in ganoderma lucidum spore powder. The method has the following beneficial effects:

[0030] 1. The present invention achieves the purpose of avoiding too large a material selection interval and causing too large a difference in test results by adopting a fixed value to select Ganoderma lucidum spore powder samples and reagents. Therefore, it is conducive to standardizing the uniformity of the detection method and solves the problem in the prior art that the test results are greatly different due to the non-uniform operation steps.

[0031] 2. The present invention achieves the goal of improving the uniformity and stability of the sample evaporation process by using a fixed wind speed evaporation auxiliary technology. Compared with the technical solution in the prior art where unstable wind speed during the evaporation process may lead to sample loss, the present invention solves the shortcomings of low evaporation efficiency and unstable results. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is a flow chart of the detection method of the present invention; DETAILED DESCRIPTION

[0033] The following will be combined with the drawings in the specification of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] Please see attached Figure 1 The present invention provides a method for detecting triterpenoid content in Ganoderma lucidum spore powder, comprising the following steps:

[0035] a. Sample preparation: weigh a sample of Ganoderma lucidum spore powder containing 0.5-5 mg triterpenes, place it in a 50 mL volumetric flask, add 30 mL of chloroform, treat it with an ultrasonic cleaner for 30 min, cool it, and use chloroform to make up to 50 mL; after centrifugation, take the supernatant as the sample solution to be tested;

[0036] b. Prepare 100 μg / mL ethyl acetate solution of ursolic acid, accurately measure 0.00mL, 0.10mL, 0.20mL, 0.40mL, 0.80mL, 1.00mL, 1.20mL, 1.40mL, 1.60mL, 1.80mL and 2.00mL standard solution and evaporate to dryness respectively, add 0.6mL vanillin glacial acetic acid solution after cooling, add 1.3mL perchloric acid after dissolving, place in a 60℃ water bath and heat for 15min, transfer the solution into a stoppered colorimetric tube after cooling, add 6.0mL glacial acetic acid, let stand for 15min, measure the absorbance at a wavelength of 548nm, and draw a standard curve with absorbance as the Y-axis and concentration as the X-axis;

[0037] c. Take 1.0 mL of the sample solution to be tested and place it in an evaporating dish. Place it in a 60°C water bath and evaporate it for 5 hours at a fan speed of 0.4 m / s. After cooling, add 0.6 mL of vanillin glacial acetic acid solution to dissolve the residue, then add 1.3 mL of perchloric acid and heat it in a 60°C water bath for 15 minutes.

[0038] d. After taking out the solution obtained in c, immediately cool it in an ice bath, add 6.0 mL of glacial acetic acid, let it stand for 15 minutes, measure the absorbance, and calculate the total triterpene concentration in the sample according to the standard curve.

[0039] The amount of vanillin glacial acetic acid solution used is 0.55-0.65 mL, the amount of perchloric acid used is 1.2-1.4 mL, and during the centrifugal operation in step a, the centrifugal speed is 2900-3100 rpm, and the centrifugal time is 15-16 min.

[0040] When performing absorbance measurement in step d, a cuvette is used and the consistency of the photometric measurement is maintained.

[0041] The temperature of the water bath in steps b and c is controlled at 60°C ± 1°C, and after adding perchloric acid in step c, heating is continued at 60°C ± 1°C for 15 minutes.

[0042] The total triterpene content in the sample was calculated using the following formula:

[0043]

[0044] Among them, X i is the total triterpenoid content in the sample, expressed in g / 100g, C i is the mass of triterpenoids in the sample solution obtained from the standard curve, in μg, m is the mass of the sample, in g, V 1 V is the volume of the extract in mL. 2 is the volume of sample used for determination, in mL;

[0045] To ensure the accuracy of the results, three independent measurements were performed under the same conditions in step d, and the average value was calculated to ensure that the relative deviation of the two measurement results did not exceed 10% of the arithmetic mean, and the results of the three independent measurements were set as follows: A 2 and A 3

[0046] Then calculate the average The formula is:

[0047]

[0048] The relative deviation A of each measured value was then calculated using the following formula i , and ensure that the relative deviation A i Not more than 10%:

[0049]

[0050] Here, i is the number of measurements.

[0051] The fan is placed 20-30cm above the evaporating dish, and the airflow output by the fan is kept horizontal so that the air evenly covers the surface of the evaporating dish.

[0052] Comparative Experimental Design

[0053] Experimental steps:

[0054] 1. Five groups of examples were prepared, and 3 mg, 4 mg, 5 mg, 6 mg and 7 mg of Ganoderma lucidum spore powder samples were selected respectively;

[0055] 2. Determine the true triterpene content of the five groups of samples in the laboratory;

[0056] 3. The triterpene contents of five groups of samples were determined respectively using the prior art solution and the technical solution of the present invention, and a table was established to compare the differences in the determination results between the prior art solution and the technical solution of the present invention.

[0057] Embodiment 1

[0058] Experimental group (technical solution of the present invention)

[0059] Sample: 3mg Ganoderma lucidum spore powder sample

[0060] Testing steps:

[0061] Sample preparation: weigh a sample of Ganoderma lucidum spore powder containing 3 mg of triterpenes, place it in a 50 mL volumetric flask, add 30 mL of chloroform, treat it with an ultrasonic cleaner for 30 minutes, cool it, and dilute it to 50 mL with chloroform; centrifuge at 3000 rpm for 15 min, and take the supernatant as the sample solution to be tested;

[0062] Preparation of standard curve: prepare 100 μg / mL ursolic acid ethyl acetate solution, accurately measure the standard solution (0.00-2.00 mL), evaporate to dryness, add 0.6 mL of vanillin glacial acetic acid solution, add 1.3 mL of perchloric acid after dissolving, heat in a 60°C water bath for 15 minutes, cool and transfer to a stoppered colorimetric tube, add 6.0 mL of glacial acetic acid, let stand for 15 minutes, measure the absorbance at a wavelength of 548 nm, and draw a standard curve;

[0063] Sample determination: Measure 1.0 mL of the sample solution to be tested and place it in an evaporating dish, place in a 60°C water bath, wind speed 0.4 m / s, stopper the test tube and evaporate for 5 hours. After cooling, add 0.6 mL of vanillin glacial acetic acid solution to dissolve the residue, add 1.3 mL of perchloric acid, and heat in a 60°C water bath for 15 minutes;

[0064] Absorbance determination: After cooling in an ice bath, transfer to a cuvette, add 6.0 mL of glacial acetic acid, let stand for 15 minutes, measure the absorbance, and calculate the total triterpene concentration in the sample based on the standard curve.

[0065] Control group (prior art assay)

[0066] Sample: 3mg Ganoderma lucidum spore powder sample

[0067] Testing steps:

[0068] Sample preparation: According to the existing technical requirements, triterpenes were extracted with chloroform, ultrasonically treated, evaporated to dryness, and then vanillin glacial acetic acid solution and perchloric acid were added;

[0069] Standard curve: Prepare a standard curve according to the existing technical method;

[0070] Determination: Determine the absorbance and calculate the total triterpene content;

[0071] Embodiment 2

[0072] Experimental group (technical solution of the present invention)

[0073] Sample: 4mg Ganoderma lucidum spore powder sample

[0074] Detection steps: (same as the experimental group in Example 1)

[0075] Control group (prior art assay)

[0076] Sample: 4mg Ganoderma lucidum spore powder sample

[0077] Detection steps: (same as the control group in Example 1)

[0078] Embodiment 3

[0079] Experimental group (technical solution of the present invention)

[0080] Sample: 5mg Ganoderma lucidum spore powder sample

[0081] Detection steps: (same as the experimental group in Example 1) Control group (existing technology measurement scheme)

[0082] Sample: 5 mg Ganoderma lucidum spore powder Sample detection steps: (same as the control group in Example 1) Example 4

[0083] Experimental group (technical solution of the present invention)

[0084] Sample: 6 mg Ganoderma lucidum spore powder Sample detection steps: (same as the experimental group in Example 1) Control group (existing technology measurement scheme)

[0085] Sample: 6 mg Ganoderma lucidum spore powder Sample detection steps: (same as the control group in Example 1) Example 5

[0086] Experimental group (technical solution of the present invention)

[0087] Sample: 7 mg Ganoderma lucidum spore powder Sample detection steps: (same as the experimental group in Example 1) Control group (existing technology measurement scheme)

[0088] Sample: 7 mg Ganoderma lucidum spore powder Sample testing steps: (same as the control group in Example 1) Test results comparison table

[0089]

[0090]

[0091] Comparative analysis and verification

[0092] 1. Measurement accuracy

[0093] From the data, it can be seen that the difference between the experimental group measurement results and the actual triterpene content is very small. The experimental group measurement results in all embodiments are close to the actual triterpene content, and the difference is maintained between 0.01 and 0.02. In contrast, the measurement results of the control group have a larger deviation, with a difference between 0.20 and 0.40.

[0094] The specific analysis is as follows:

[0095] Accuracy of experimental group: The difference range of the experimental group is small, which shows that the determination result of this scheme is almost consistent with the actual content and can accurately reflect the triterpene content in the sample. This shows that the scheme of the present invention has a significant advantage in determination accuracy.

[0096] The accuracy of the control group is insufficient: the results of the control group are relatively low, with a wide range of deviations, especially in samples with higher contents (Examples 4 and 5), with deviations of 0.40 and 0.38. This shows that the prior art has large errors and is not accurate enough when measuring high contents.

[0097] 2. Consistency of measurement results

[0098] The results of the experimental group showed high consistency in samples with different triterpene contents, and the difference was always kept between 0.01 and 0.02. This consistency shows that the technical solution of the present invention can provide stable measurement results under different sample conditions:

[0099] Consistency of experimental groups: The assay differences of both low-content (3 mg) and high-content (7 mg) samples in the experimental groups were kept within a relatively low range, which reflects the stability of the scheme of the present invention in the detection and is not affected by changes in sample concentration.

[0100] Poor consistency of the control group: The difference in the determination of the control group under different sample contents varies greatly, and the error gradually increases with the increase of sample content. This lack of consistency makes it difficult for existing technical means to provide reliable determination results, especially in samples with higher content, where the error is significant and lacks universality.

[0101] 3. Operational complexity comparison

[0102] From the perspective of operational complexity, the solution of the present invention has been optimized in multiple key steps, such as fixed value operation and standardized wind speed control, which effectively reduces the influence of human factors and simplifies the operational process:

[0103] Simplicity of experimental group: In the scheme of the present invention, by fixing key operating parameters (such as the amount of vanillin glacial acetic acid solution, wind speed control, etc.), human errors in the operating steps are reduced, making the experimental process simpler and easier, and reducing the operating difficulty of the experimenter. This optimization makes the detection method more stable and avoids unnecessary variables.

[0104] The control group operation is complicated: the existing technical means lack constant value control of key parameters and are easily affected by changes in experimental conditions, especially the lack of standardized operations in the extraction, evaporation and dissolution steps, which leads to the instability of the measurement results. The operation process is relatively cumbersome and easily affected by the technical level of the operator.

[0105] Summarize

[0106] Through the above comparative analysis, the significant advantages of the solution of the present invention can be verified:

[0107] Accuracy: The test results of the experimental group are close to the actual content and have high accuracy.

[0108] Consistency: The experimental groups had small differences in the determination of samples with different concentrations, showing high consistency.

[0109] Simplicity: The solution of the present invention optimizes the operation process, reduces complexity and improves detection efficiency.

[0110] In general, the technical solution of the present invention is significantly superior to existing technical means in terms of measurement accuracy, result consistency and ease of operation, can provide more reliable triterpene content detection results, and is suitable for promotion and application.

[0111] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for detecting triterpenoid content in Ganoderma lucidum spore powder, characterized in that: The following steps are involved: a. Sample preparation: weigh a sample of Ganoderma lucidum spore powder containing 0.5-5 mg triterpenes, place it in a 50 mL volumetric flask, add 30 mL of chloroform, treat it with an ultrasonic cleaner for 30 min, cool it, and use chloroform to make up to 50 mL; after centrifugation, take the supernatant as the sample solution to be tested; b. Prepare 100 μg / mL ethyl acetate solution of ursolic acid, accurately measure 0.00mL, 0.10mL, 0.20mL, 0.40mL, 0.80mL, 1.00mL, 1.20mL, 1.40mL, 1.60mL, 1.80mL and 2.00mL standard solution and evaporate to dryness respectively, add 0.6mL vanillin glacial acetic acid solution after cooling, add 1.3mL perchloric acid after dissolving, place in a 60℃ water bath and heat for 15min, transfer the solution into a stoppered colorimetric tube after cooling, add 6.0mL glacial acetic acid, let stand for 15min, measure the absorbance at a wavelength of 548nm, and draw a standard curve with absorbance as the Y-axis and concentration as the X-axis; c. Take 1.0 mL of the sample solution to be tested and place it in an evaporating dish. Place it in a 60°C water bath and evaporate it for 5 hours at a fan speed of 0.4 m / s. After cooling, add 0.6 mL of vanillin glacial acetic acid solution to dissolve the residue, then add 1.3 mL of perchloric acid and heat it in a 60°C water bath for 15 minutes. d. After taking out the solution obtained in c, immediately cool it in an ice bath, add 6.0 mL of glacial acetic acid, let it stand for 15 minutes, measure the absorbance, and calculate the total triterpene concentration in the sample according to the standard curve.

2. A method for detecting triterpenoid content in Ganoderma lucidum spore powder according to claim 1, characterized in that: The amount of vanillin glacial acetic acid solution used is 0.55-0.65 mL, the amount of perchloric acid used is 1.2-1.4 mL, and during the centrifugal operation in step a, the centrifugal speed is 2900-3100 rpm, and the centrifugal time is 15-16 min.

3. A method for detecting triterpenoid content in Ganoderma lucidum spore powder according to claim 1, characterized in that: When performing absorbance measurement in step d, a cuvette is used and the consistency of the photometric measurement is maintained.

4. A method for detecting triterpenoid content in Ganoderma lucidum spore powder according to claim 1, characterized in that: The temperature of the water bath in steps b and c is controlled at 60°C ± 1°C, and after adding perchloric acid in step c, heating is continued at 60°C ± 1°C for 15 minutes.

5. A method for detecting triterpenoid content in Ganoderma lucidum spore powder according to claim 1, characterized in that: The total triterpene content in the sample was calculated using the following formula: Among them, X i is the total triterpenoid content in the sample, expressed in g / 100g, C i is the mass of triterpenes in the sample solution obtained from the standard curve, in μg, m is the mass of the sample, in g, V1 is the volume of the extract, in mL, and V2 is the volume of the sample used for determination, in mL; To ensure the accuracy of the results, three independent measurements were performed under the same conditions in step d, and the average value was calculated to ensure that the relative deviation of the two measurement results did not exceed 10% of the arithmetic mean, and the results of the three independent measurements were set as follows: A2 and A3 Then calculate the average The formula is: The relative deviation A of each measured value was then calculated using the following formula i , and ensure that the relative deviation A i Not more than 10%: Here, i is the number of measurements.

6. A method for detecting triterpenoid content in Ganoderma lucidum spore powder according to claim 1, characterized in that: The fan is placed 20-30cm above the evaporating dish, and the airflow output by the fan is kept horizontal so that the air evenly covers the surface of the evaporating dish.

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