CN111307966A - HPLC (high Performance liquid chromatography) determination method for triterpenoid components in ganoderma lucidum spore powder and product thereof - Google Patents
HPLC (high Performance liquid chromatography) determination method for triterpenoid components in ganoderma lucidum spore powder and product thereof Download PDFInfo
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G01N30/02—Column chromatography
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- G01N30/02—Column chromatography
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Abstract
The invention establishes an analytical determination method for main triterpenoid components in ganoderma lucidum spore powder and products thereof by using a high performance liquid chromatograph. The method relates to optimization of sample pretreatment conditions, selection of chromatographic columns and optimization of chromatographic conditions, and meanwhile, methodology verification is carried out on the established method. The HPLC determination method for the triterpenoid components in the ganoderma lucidum spore powder and the products thereof eliminates the interference of weak-polarity fat-soluble components such as fatty acid, sterol and the like, breaks through the limitation of the spectrophotometry method for determining the triterpenoid components in the spore powder and the products thereof, and has high accuracy and strong specificity.
Description
Technical Field
The invention relates to the field of pharmaceutical analysis, in particular to an HPLC (high performance liquid chromatography) determination method for triterpenoid components in ganoderma lucidum spore powder and a product thereof.
Background
Ganoderma is fungus of Ganoderma of Polyporaceae of Agaricaceae of Basidiomycota, and has high medicinal value, and chemical components of Ganoderma are complex, including polysaccharide, triterpene, sterol, alkaloid, amino acid, fatty acid, etc. The ganoderma triterpene has various effects of resisting tumor and HIV-1 type virus, protecting liver, detoxifying, improving memory and the like, and is one of important indexes for measuring the quality of ganoderma products. Ganoderma spore powder is germ cell ejected from Ganoderma Pleurotus in fruiting body development and maturation stage, and contains various chemical components such as fatty acids, triterpenic acids, polysaccharides, etc.
In recent years, with the development of spore powder collection and wall breaking technology, the pharmacological action of ganoderma lucidum spore powder has been widely accepted by consumers, but the marked effective components are not clear and lack legal standards, and the active component triterpene or polysaccharide in ganoderma lucidum fruiting bodies is commonly used as a quality control index for spore powder and related products and is measured by a spectrophotometer method. However, the spore powder and the spore oil contain a large amount of weak polar fat-soluble components such as oleic acid, linoleic acid, ergosterol and the like, and can react with vanillin perchloric acid in a color manner, so that the triterpene measurement result is far higher than the true value. The content of triterpenoids in the wall-broken spore powder is 2.94-3.43% and the content of triterpenoids in the spore oil is higher than 20% as measured by a spectrophotometry method. The research of the high performance liquid chromatography finds that the content of ganoderic acid components in the commercial ganoderma lucidum spore powder is very low, and some ganoderma lucidum acid components can not be detected. The differences in the results of the two main-stream detection methods make it controversial whether the ganoderma lucidum spore powder contains triterpenes or not and the specific content of triterpenes.
On the basis of the existing research, the method eliminates the interference of weak polar fat-soluble components such as fatty acid, sterol and the like, optimizes the pretreatment method and the analysis conditions of the ganoderma lucidum spore powder, establishes an HPLC (high performance liquid chromatography) method suitable for analyzing and determining the triterpenoid components in the ganoderma lucidum spore powder and the products thereof, improves the accuracy and reliability of the result, reduces the loss of a chromatographic column and the pollution of a detector, and provides a scientific basis for evaluating the quality of the ganoderma lucidum spore powder and the products thereof.
Disclosure of Invention
The invention aims to eliminate the interference of weak-polarity fat-soluble components such as fatty acid, sterol and the like, break through the limitation that the result of a spectrophotometry method for measuring triterpenoid components in spore powder and products thereof is high, and provide the method for detecting the triterpenoid components in the ganoderma spore powder and the products thereof, which has high accuracy, strong specificity and wide application range.
The invention relates to an HPLC (high performance liquid chromatography) determination method for triterpenoid components in ganoderma lucidum spore powder and a product thereof, which comprises the following steps: 40% ethanol water solution is used as an extraction solvent, the extract is concentrated by 50 times, and the content of triterpenes in ganoderma lucidum spore powder and products thereof is measured by high performance liquid chromatography.
The invention relates to an HPLC (high performance liquid chromatography) determination method for triterpenoid components in ganoderma lucidum spore powder and a product thereof, which comprises the following specific operation processes:
(1) the method comprises the steps of pretreatment of ganoderma spore powder and products thereof, adding a solvent into the ganoderma spore powder or related products for extraction to obtain an extract, centrifuging, re-dissolving supernate with ethanol after vacuum drying, and filtering to obtain a sample solution to be detected;
(2) a high performance liquid chromatography detection step, wherein the high performance liquid chromatography analysis is carried out on the sample solution to be detected;
(3) the index components are triterpenes components: ganoderic acid I, ganoderic acid C2, ganoderic acid C6, ganoderic acid G, ganoderic acid B, ganoderic acid A, ganoderic acid E, ganoderic acid A, ganoderic acid D, and ganoderic acid F;
specifically, the ganoderma lucidum spore powder and the product thereof comprise non-wall-broken spore powder, wall-broken spore powder and ganoderma lucidum spore oil products.
More specifically, the invention relates to a pretreatment step of ganoderma lucidum spore powder and a product thereof, and the extraction method comprises the following steps: the extraction solvent is one or more of n-hexane, petroleum ether, ethyl acetate, ethanol and methanol or water solutions with different concentrations; the extraction method comprises ultrasonic extraction and heating oscillation extraction, preferably ultrasonic extraction; the extraction time is 10-90min, preferably 30 min; the ratio of material to liquid is 1: 10-1: 50, wherein the spore powder sample is preferably 1: the spore oil sample is preferably 1:30, of a nitrogen-containing gas; the leaching times include one leaching or repeated leaching.
The use of the extraction solvent comprises the following steps: the extraction solvent is one of n-hexane, petroleum ether, ethyl acetate, ethanol and methanol, preferably ethanol; continuously optimizing the extraction solvent, wherein the extraction solvent is ethanol water solution with different concentrations, and the ethanol water solution with 20-50 percent of concentration, preferably 40 percent of concentration is used for extraction.
The treatment of the leach liquor comprises the following steps: centrifuging the leaching solution at 4000r/min for 15min, collecting supernatant, vacuum concentrating and drying at 50 deg.C, re-dissolving the concentrate with ethanol at concentration ratio of 10-100 times, preferably 50 times, and filtering the solution with 0.22 μm microporous organic phase membrane.
Further, the HPLC method for measuring triterpenes in ganoderma lucidum spore powder and products thereof comprises the following steps:
the chromatographic column is a reversed phase C18 chromatographic column, and the component of the mobile phase solution is acetonitrile-water gradient elution, wherein the concentration of glacial acetic acid in water is 0.00-0.02%, preferably 0.01%; the column temperature is 20-40 ℃, preferably 30 ℃; the flow rate is 0.8-1.2ml/min, preferably 1.0 ml/min; the detection wavelength is 250-260nm, preferably 252 nm; the injection volume is 5-50. mu.L, preferably 10. mu.L. The chromatographic column is Agilent ZORBAX SB-C18(4.6 mm. times.250 mm, 5 μm) chromatographic column, Waters CORTECS-C18(150 mm. times.4.6 mm, 2.7 μm) chromatographic column or Omni Bond Hubbele-C18 (4.6 mm. times.250 mm, 5 μm) chromatographic column.
More specifically, the elution procedure for the mobile phase was: mobile phase (a) acetonitrile + (B) 0.01% acetic acid water (v/v) 100% (v/v), 0-5min, 22% (a); 5-10min, 22-35% (A); 10-30min, 35-40% (A); 30-32min, 40-42% (A); 32-37min, 42-45% (A); 37-45min, 45-65% (A); 45-55min, 65-100% (A); 55-60min, 100% (A).
Further, the HPLC method for measuring triterpenoid in ganoderma lucidum spore powder and products thereof further comprises the following specific steps:
1) preparing a series of standard solutions: preparing a mixed standard solution of 13 triterpenoids with proper amounts of the 13 triterpenoids by using methanol, and sequentially diluting the mixed standard solution into a series of concentration gradients by using the methanol for later use;
2) characterization of each triterpene component: performing HPLC analysis on each triterpene single component and the mixed standard solution, and determining the character according to the retention time and the ultraviolet spectrum of each component;
3) drawing a standard curve: taking the series of standard substance solutions in the step 1), determining according to the chromatographic conditions of the invention, and drawing a standard curve by using the concentration (X, mu g/mL) of each component solution to the chromatographic peak area (Y) to obtain a regression equation.
Furthermore, the HPLC determination method for the triterpenoid in the ganoderma lucidum spore powder and the product thereof is characterized in that the HPLC method and the spectrophotometry method are used for determining the triterpenoid in the ganoderma lucidum spore powder and the product thereof, and the difference of the results of the two methods is compared. Further verifies that the spectrophotometry method can lead the determination result of triterpenes in the spore powder and the spore oil to be seriously higher and show false positive, and the content of triterpenes in the ganoderma lucidum spore powder and related products is more objective and accurate by the HPLC method.
Drawings
FIG. 1 HPLC chart of samples of different concentration ratios
FIG. 2 HPLC chromatogram comparison of wall-broken and non-wall-broken spore powder
FIG. 3 HPLC profiles of different extraction solvents (left) and different concentrations of ethanol (right)
FIG. 4 HPLC profiles of spore powder sample and triterpene control
In fig. 4, 1 to 13 represent: 1. ganoderic acid I; 2. ganoderic acid C; 3. ganoderic acid C2; 4. ganoderic acid C6; 5. ganoderic acid G; 6. ganoderic acid B; 7. ganoderic acid A; 8. ganoderic acid A; 9. ganoderic acid E; 10. ganoderma lucidum acid A; 11. ganoderic acid D; 12. ganoderic acid D; 13. ganoderic acid F
TABLE 1 gradient elution procedure
TABLE 2 methodological data for different triterpene compositions
TABLE 3 composition of triterpene in non-wall-broken Ganoderma spore powder (n 2. mu.g/g)
TABLE 4 composition of triterpene in sporoderm-broken Ganoderma spore powder (n 2. mu.g/g)
TABLE 5 composition of triterpenes in Ganoderma spore oil (n 2. mu.g/g)
TABLE 6 spectrophotometric determination of Total triterpene content (%)
Detailed Description
The present invention is further illustrated by the following figures and the following examples, which are illustrative only and not limiting, and the scope of the invention is not to be limited thereby, and all equivalent changes and modifications that can be made within the scope of the invention are intended to be covered thereby.
The invention relates to an instrument and a reagent for an HPLC (high performance liquid chromatography) determination method of triterpenoid components in ganoderma lucidum spore powder and a product thereof
1. The main apparatus is as follows:
waters1525 hplc, Waters2489 uv detector, Waters corporation, usa; omnibond Hubble C18(4.6 mm. times.250 mm, 5 μm) column, Tianjin ohm Nigene science and technology, Inc.; KQ-2200E ultrasonic cleaner (working power 100W, frequency 40kHz), ultrasonic instruments Inc. of Kunshan city; MS105DU electronic analytical balance, Metter Toledo, switzerland; a testable nitrogen purge instrument, shanghai geon leap electronics ltd; rotary evaporator, BUCHI company.
2. The main reagents are as follows:
absolute ethyl alcohol, methanol, ethyl acetate, normal hexane, petroleum ether and glacial acetic acid are all analytical pure reagents of chemical reagents Limited of national drug group; the high performance liquid phase uses acetonitrile as the chromatographic purity of Dikma company in America; the water is ultrapure water and is prepared by an ELGA PureLabultra pure water instrument; comparison products: the ganoderic acid I, ganoderic acid C2, ganoderic acid C6, ganoderic acid G, ganoderic acid B, ganoderic acid A, ganoderic acid E, ganoderic acid A, ganoderic acid D and ganoderic acid F are obtained by separation and purification in the laboratory, and the purity of each component is over 95% by HPLC detection.
Example 1 selection of concentration ratio of Ganoderma spore powder and its product
Preparation of sample solution: weighing a proper amount of wall-broken ganoderma lucidum spore powder, and mixing the materials according to a material-liquid ratio of 1:10 adding ethanol, extracting with ultrasound for 30min, centrifuging the extractive solution at 4000r/min for 15min, collecting supernatant, vacuum concentrating and drying at 50 deg.C, re-dissolving the concentrate with ethanol at concentration ratio of 10 times, 25 times, 50 times and 100 times, respectively, and filtering the solution with 0.22 μm microporous organic phase membrane to obtain test solution.
Through high performance liquid chromatography analysis (the map is shown in figure 1), the peak shape of the triterpenoid in the map is obvious and in the quantitative analysis range after the extracting solution is concentrated by more than or equal to 50 times, so that the concentration ratio of the leaching solution in the sample pretreatment is preferably 50 times.
Example 2 comparison of triterpene composition in spore powder treated by wall breaking and not wall breaking
Preparation of sample solution: respectively weighing appropriate amount of wall-broken and non-wall-broken ganoderma lucidum spore powder according to a material-liquid ratio of 1:10 adding ethanol, ultrasonic extracting for 30min, centrifuging the extractive solution at 4000r/min for 15min, collecting supernatant, vacuum concentrating and drying at 50 deg.C, re-dissolving the concentrate with ethanol at a concentration ratio of 50 times, and filtering the solution with 0.22 μm microporous organic phase membrane to obtain test solution.
By high performance liquid chromatography (the chromatogram is shown in figure 2), the results show that the HPLC chromatogram in the wall-broken and non-wall-broken spore powders has similar composition and content in the medium polarity chromatogram peak of 10-22 min, while in the weak polarity chromatogram peak of 24-34 min, the species and content of the wall-broken spore powder are obviously more than those of the non-wall-broken spore powder. The literature reports that triterpenes separated and purified from the wall-broken ganoderma lucidum spore powder are mainly medium-grade triterpenic acid compounds, and compared with a standard product, the triterpenes are mainly contained in peaks of 10-22 min. In addition, the wall breaking treatment enables a large amount of low-polarity compounds such as triglyceride, fatty acid, sterol and the like to be dissolved out, the qualitative and quantitative analysis of the low-polarity triterpenoid can be interfered, and the existing determination is difficult due to the fact that the number of interference substances is large and the separation difficulty is large. Therefore, the invention only analyzes and measures the medium polarity triterpenic acid compounds in the spore powder.
the oil content of the wall-broken spore powder is not lower than 20%, so that a great amount of spore oil is separated out after the wall-broken spore powder extracting solution is concentrated, the concentration and the constant volume of a sample solution are influenced, and a great amount of grease can pollute a chromatographic column and a detector, and the determination and analysis of triterpenoid components in the wall-broken spore powder are influenced. Therefore, the influence of most of the spore oil in the pretreatment of the sample was removed, and the extraction solvent was examined.
1) Selection of extraction solvent
Wall-broken spore powder is taken as a material, n-hexane, petroleum ether, ethyl acetate, ethanol and methanol are respectively selected as extraction solvents, and the material-liquid ratio is 1:10 extracting spore powder, centrifuging the leaching solution at 4000r/min for 15min, collecting supernatant, vacuum concentrating and drying at 50 deg.C, re-dissolving the concentrate with ethanol at a concentration ratio of 50 times, and filtering the solution with 0.22 μm microporous organic phase filter membrane to obtain test solution.
As a result of comparison of HPLC analysis (fig. 3, left), it was found that the extraction rate of medium polar triterpenes was the highest and the elution of fats and oils was relatively small when ethanol and methanol were used as extractants; when ethyl acetate is used as an extractant, a large amount of triterpenes and grease are dissolved out; when n-hexane and petroleum ether are used as extraction agents, the elution amount of oil is the most, and triterpene components are hardly detected. Considering the extraction rate of triterpenoids, the oil extraction amount and the experimental safety comprehensively, ethanol is a more suitable extraction solvent for measuring the triterpenoids in the spore powder.
2) Optimization of ethanol concentration of extraction solvent
In order to reduce the dissolution of the spore oil as much as possible, the extraction scheme is further optimized on the basis of taking ethanol as an extraction solvent. Taking wall-broken spore powder as a material, respectively selecting 20%, 30%, 40%, 50% ethanol aqueous solution and absolute ethanol as extracting agents for extraction, centrifuging the extract liquid for 15min at 4000r/min, taking supernatant, performing vacuum concentration and drying at 50 ℃, redissolving the concentrate with ethanol according to 50 times concentration ratio, filtering the solution with a 0.22-micron microporous organic phase filter membrane, and performing HPLC analysis comparison (right side in figure 3). On the premise of ensuring the extraction rate of the triterpenoids, the 40% ethanol reduces the dissolution of the oil to the maximum extent, and finally the 40% ethanol is selected as the best extraction solvent.
Example 4 optimization of high performance liquid chromatography conditions
Preparation of a test solution: wall-broken spore powder is taken as a material, 40% ethanol water solution is taken as an extraction solvent, and the ratio of material to liquid is 1:10 extracting spore powder, centrifuging the leaching solution at 4000r/min for 15min, collecting supernatant, vacuum concentrating and drying at 50 deg.C, re-dissolving the concentrate with ethanol at a concentration ratio of 50 times, and filtering the solution with 0.22 μm microporous organic phase filter membrane to obtain test solution.
Injecting the test solution into a high performance liquid chromatograph for detection, and finally determining the optimal chromatographic conditions by comparing chromatograms under different chromatographic conditions as follows: an Omni Bond Hubble-C18(4.6 mm. times.250 mm, 5 μm) chromatographic column was selected, and gradient elution was carried out using (A) acetonitrile + (B) 0.01% acetic acid (v/v) ═ 100% (by volume) as a mobile phase (see Table 1), at a flow rate of 1.0mL/min, a detection wavelength of 252nm, a column temperature of 30 ℃ and a sample volume of 10 μ L. Under the chromatographic condition, the 13 triterpene components achieve better separation degree, the retention time is moderate, the chromatographic reproducibility is high, and the chromatogram of each triterpene standard product and spore powder sample is shown in figure 4.
Example 5 methodological verification of HPLC determination method of Ganoderma spore powder and triterpene component in product thereof
(1) Preparation of a test solution:
taking a spore powder sample, taking a 40% ethanol water solution as an extraction solvent (the spore powder sample without wall breaking can be directly taken by absolute ethanol as the extraction solvent because no interference of grease exists), carrying out ultrasonic extraction for 30min according to the material-liquid ratio of 1:10, cooling to room temperature, shaking up, centrifuging at 4000r/min for 15min, taking supernatant, concentrating, spin-drying, fixing the volume by ethanol according to the concentration ratio of 50 times, and passing through a microporous organic phase filter membrane of 0.22 mu m to obtain a test solution.
(2) Preparing a reference substance solution:
accurately weighing a standard product dried to constant weight: ganoderic acid I, ganoderic acid C2, ganoderic acid C6, ganoderic acid G, ganoderic acid B, ganoderic acid A, ganoderic acid E, ganoderic acid A, ganoderic acid D, ganoderic acid F, and adding methanol to obtain 1mg/mL standard stock solution. Precisely sucking a proper amount of each standard substance stock solution to prepare a mixed standard substance solution, shaking up, and diluting into 6 series of mixed standard substance solutions for later use.
(3) Methodology validation
1) The linear relationship is: and (3) taking the series of concentration standard substance solutions in the step (2), sequentially carrying out sample injection analysis according to the optimized chromatographic conditions, and recording the peak area. And (3) drawing a standard curve by taking the concentration of each reference substance as an abscissa (X) and the peak area as an ordinate (Y) to obtain a linear regression equation and a linear range of each triterpene component. The results are shown in Table 2, and the correlation coefficients of the 13 triterpenoids in the linear range are all larger than 0.999, and the linear relationship is good.
2) And (3) precision test: and (3) continuously sampling the same mixed reference substance solution obtained in the step (2) for 6 times, calculating the R.S.D. of each triterpene peak area measured each time, and inspecting the precision of the instrument, wherein the result is shown in a table 2, and each R.S.D. is less than 5%, which indicates that the precision of the instrument is good.
3) And (3) repeatability test: and (2) precisely weighing 6 parts of the same spore powder sample, preparing a sample solution in parallel according to the method in the step (1), calculating the content of each triterpene measured by each sample, calculating the R.S.D., and investigating the repeatability of the method, wherein the result is shown in a table 2, and each R.S.D. is less than 5%, which indicates that the method has good repeatability.
4) And (3) stability test: taking the same sample solution, respectively extracting for 0, 2, 4, 6, 8, 12 and 24 hours, then carrying out sample determination, calculating the peak area R.S.D. of each triterpene measured each time, and inspecting the stability of the sample, wherein the result is shown in Table 2, and each R.S.D. is less than 5%, which indicates that the stability of the sample is good for 24 hours.
EXAMPLE 6 HPLC method for determination of triterpene content in spore powder sample
Taking a spore powder sample, taking a 40% ethanol water solution as an extraction solvent (the spore powder sample without wall breaking can be directly taken by absolute ethanol as the extraction solvent because no interference of grease exists), carrying out ultrasonic extraction for 30min according to the material-liquid ratio of 1:10, cooling to room temperature, shaking up, centrifuging at 4000r/min for 15min, taking supernatant, concentrating, spin-drying, fixing the volume by ethanol according to the concentration ratio of 50 times, and passing through a microporous organic phase filter membrane of 0.22 mu m to obtain a test solution. And sequentially injecting and analyzing each sample solution according to the optimized chromatographic conditions, calculating the content of triterpenes in each spore powder sample by an external standard curve method, and showing the results in tables 3 and 4, wherein the results show that the content of triterpenes in the spore powder is in a micro-scale and the elution amount of triterpenes has no correlation with whether the wall is broken or not.
Example 7 HPLC determination of triterpene content in spore oil product
Taking a spore oil sample, taking 40% ethanol water solution as an extraction solvent, carrying out ultrasonic extraction for 30min according to the material-liquid ratio of 1:30, cooling to room temperature, shaking up, centrifuging for 15min at 4000r/min, taking supernatant, concentrating, spin-drying, fixing the volume by using ethanol according to 50 times of concentration ratio, and filtering through a microporous organic phase filter membrane of 0.22 mu m to obtain a sample solution. The sample solutions are sequentially subjected to sample injection analysis according to optimized chromatographic conditions, the content of triterpenoids in each spore oil sample is calculated by an external standard curve method, and the result is shown in table 5, which shows that the total content of triterpenoids in the spore oil is less than 50.0 mu g/g, further verifies that the spectrophotometry method can lead the determination result of triterpenoids in the spore powder and the spore oil to be seriously higher and show false positive, and the content of triterpenoids in the ganoderma spore powder and related products is determined more objectively and accurately by an HPLC method.
Example 8 spectrophotometric assay of content of triterpenes in Ganoderma spore powder and related products
(1) Drawing of standard curve
Taking a proper amount of oleanolic acid reference substance, precisely weighing, and adding methanol to prepare a reference substance solution of 1 mg/mL. Precisely measuring the reference substance solutions of 0 μ L, 20 μ L, 40 μ L, 60 μ L, 80 μ L, 100 μ L and 120 μ L, respectively placing into 20mL test tubes with plugs, placing the test tubes into a boiling water bath to volatilize the solvent, cooling, precisely adding 0.2mL of 5% vanillin glacial acetic acid solution and 1.0mL of perchloric acid, mixing uniformly, keeping the temperature in the 60 ℃ water bath for 20min, taking out, adding 5mL glacial acetic acid into the ice water bath for 3min, shaking uniformly, and measuring the absorbance at 550 nm. The absorbance value is plotted as ordinate and the oleanolic acid concentration (μ g/mL) is plotted as abscissa to obtain a standard curve.
(2) Preparation of test solution
Taking 200mg of spore powder sample, precisely weighing, placing in a 50mL centrifuge tube, adding 20mL 95% ethanol, performing ultrasonic extraction for 60min, and filtering to obtain a spore powder test sample solution.
Taking 10mg of a spore oil sample, precisely weighing, placing in a 50mL centrifuge tube, adding 10mL 95% ethanol, performing ultrasonic extraction for 60min, and filtering to obtain a sample solution of the spore oil.
(3) And sample measurement
Precisely measuring 0.2mL of test solution, placing in a 20mL test tube with a plug, measuring the absorbance value according to the method of drawing the standard curve in the step (1), and calculating the content of the total triterpenes in each sample by contrasting the standard curve of the oleanolic acid (see Table 6).
TABLE 1 gradient elution procedure
TABLE 2 methodological data for different triterpene compositions
TABLE 3 composition of triterpene in non-wall-broken Ganoderma spore powder (n 2. mu.g/g)
TABLE 4 composition of triterpene in sporoderm-broken Ganoderma spore powder (n 2. mu.g/g)
TABLE 5 composition of triterpenes in Ganoderma spore oil (n 2. mu.g/g)
TABLE 6 spectrophotometric determination of Total triterpene content (%)
Claims (10)
1. An HPLC determination method for triterpene components in ganoderma lucidum spore powder and products thereof is characterized by comprising the following steps: taking 40% ethanol water solution as extraction solvent, concentrating for multiple times, and measuring the content of triterpenes in Ganoderma spore powder and its product by high performance liquid chromatography.
2. The HPLC determination method for triterpene components in ganoderma lucidum spore powder and products thereof according to claim 1, which is characterized in that the specific operation process comprises the following steps:
(1) the method comprises the steps of pretreatment of ganoderma spore powder and products thereof, adding a solvent into the ganoderma spore powder or related products for extraction to obtain an extract, centrifuging, re-dissolving supernate with ethanol after vacuum drying, and filtering to obtain a sample solution to be detected;
(2) a high performance liquid chromatography detection step, wherein the high performance liquid chromatography analysis is carried out on the sample solution to be detected;
(3) the index components are triterpenes components: ganoderic acid I, ganoderic acid C2, ganoderic acid C6, ganoderic acid G, ganoderic acid B, ganoderic acid A, ganoderic acid E, ganoderic acid A, ganoderic acid D, and ganoderic acid F.
3. The HPLC method for measuring triterpene components in ganoderma lucidum spore powder and products thereof according to claims 1-2, wherein the ganoderma lucidum spore powder and products thereof comprise non-wall-broken spore powder, wall-broken spore powder and ganoderma lucidum spore oil products.
4. The HPLC method for measuring triterpene components in ganoderma lucidum spore powder and products thereof according to claims 1-2, wherein the step (1) of pretreating ganoderma lucidum spore powder and products thereof comprises the following steps: the extraction solvent is one or more of n-hexane, petroleum ether, ethyl acetate, ethanol and methanol or water solutions with different concentrations; the extraction method comprises ultrasonic extraction and heating oscillation extraction, preferably ultrasonic extraction; the extraction time is 10-90min, preferably 30 min; the ratio of material to liquid is 1: 10-1: 50, wherein the spore powder sample is preferably 1: the spore oil sample is preferably 1:30, of a nitrogen-containing gas; the leaching times include one leaching or repeated leaching.
5. The HPLC method for measuring triterpene component in Ganoderma spore powder and its product according to claims 1-2, wherein the step (1) of pretreating Ganoderma spore powder and its product comprises the following steps: the extraction solvent is one of n-hexane, petroleum ether, ethyl acetate, ethanol and methanol, preferably ethanol; continuously optimizing the extraction solvent, wherein the extraction solvent is ethanol water solution with different concentrations, and the ethanol water solution with 20-50 percent of concentration, preferably 40 percent of concentration is used for extraction.
6. The HPLC method for measuring triterpene components in ganoderma lucidum spore powder and products thereof according to claims 1-2, wherein the step (1) of pretreating ganoderma lucidum spore powder and products thereof comprises the following steps: centrifuging the leaching solution at 4000r/min for 15min, collecting supernatant, vacuum concentrating and drying at 50 deg.C, re-dissolving the concentrate with ethanol at concentration ratio of 10-100 times, preferably 50 times, and filtering the solution with 0.22 μm microporous organic phase membrane.
7. The HPLC method for measuring triterpene components in Ganoderma spore powder and its product according to claims 1-2, wherein the step (2) high performance liquid chromatography comprises the following steps:
the chromatographic column is a reversed phase C18 chromatographic column, and the component of the mobile phase solution is acetonitrile-water gradient elution, wherein the concentration of glacial acetic acid in water is 0.00-0.02%, preferably 0.01%; the column temperature is 20-40 ℃, preferably 30 ℃; the flow rate is 0.8-1.2ml/min, preferably 1.0 ml/min; the detection wavelength is 250-260nm, preferably 252 nm; the injection volume is 5-50. mu.L, preferably 10. mu.L.
8. The HPLC method for measuring triterpene components in ganoderma lucidum spore powder and products thereof according to claim 7, which is characterized in that: the chromatographic columns used were an Agilent ZORBAX SB-C18(4.6 mm. times.250 mm, 5 μm) column, a Waters CORTECS-C18(150 mm. times.4.6 mm, 2.7 μm) column or an Omni Bond Hubbele-C18 (4.6 mm. times.250 mm, 5 μm) column.
9. The HPLC method for measuring triterpene components in ganoderma lucidum spore powder and products thereof according to claim 7, which is characterized in that: the elution procedure for the mobile phase was: mobile phase (a) acetonitrile + (B) 0.01% acetic acid water (v/v) 100% (v/v), 0-5min, 22% (a); 5-10min, 22-35% (A); 10-30min, 35-40% (A); 30-32min, 40-42% (A); 32-37min, 42-45% (A); 37-45min, 45-65% (A); 45-55min, 65-100% (A); 55-60min, 100% (A).
10. The HPLC method for measuring triterpene components in Ganoderma lucidum spore powder and its products according to claims 1-2, wherein the steps (2) - (3) high performance liquid chromatography further comprises the following steps:
1) preparing a series of standard solutions: preparing a triterpene mixed standard solution containing proper amounts of the 13 triterpene compounds by using methanol, and sequentially diluting the triterpene mixed standard solution into a series of concentration gradients by using the methanol for later use;
2) characterization of each triterpene component: performing HPLC analysis on each triterpene single component and the mixed standard solution, and determining the character according to the retention time and the ultraviolet spectrum of each component;
3) drawing a standard curve: taking the series of standard substance solutions in the step 1), determining according to the chromatographic conditions of the invention, and drawing a standard curve by using the concentration (X, mu g/mL) of each component solution to the chromatographic peak area (Y) to obtain a regression equation; or;
the method comprises the steps of measuring triterpenoid components in ganoderma spore powder and products thereof by an HPLC method and a spectrophotometry method, and comparing the difference of results of the two methods. Further verifies that the spectrophotometry method can lead the determination result of triterpenes in the spore powder and the spore oil to be seriously higher and show false positive, and the content of triterpenes in the ganoderma lucidum spore powder and related products is more objective and accurate by the HPLC method.
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| CN111829979A (en) * | 2020-07-20 | 2020-10-27 | 中国科学院合肥物质科学研究院 | Method for quantitatively determining total triterpenoids in ganoderma lucidum fruiting body based on NIR (near infrared) spectrum |
| CN112326854A (en) * | 2020-11-06 | 2021-02-05 | 上海市农业科学院 | Method for simultaneously detecting six triterpene compounds in Ganoderma applanatum fruiting body |
| CN112345681A (en) * | 2020-11-06 | 2021-02-09 | 上海市农业科学院 | Method for simultaneously detecting eight ganoderic acids in ganoderma lucidum mycelia |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111829979A (en) * | 2020-07-20 | 2020-10-27 | 中国科学院合肥物质科学研究院 | Method for quantitatively determining total triterpenoids in ganoderma lucidum fruiting body based on NIR (near infrared) spectrum |
| CN111829979B (en) * | 2020-07-20 | 2023-09-12 | 中国科学院合肥物质科学研究院 | Method for quantitatively determining total triterpenes in ganoderma lucidum fruiting bodies based on NIR (near infrared spectroscopy) |
| CN112326854A (en) * | 2020-11-06 | 2021-02-05 | 上海市农业科学院 | Method for simultaneously detecting six triterpene compounds in Ganoderma applanatum fruiting body |
| CN112345681A (en) * | 2020-11-06 | 2021-02-09 | 上海市农业科学院 | Method for simultaneously detecting eight ganoderic acids in ganoderma lucidum mycelia |
| CN112326854B (en) * | 2020-11-06 | 2022-08-12 | 上海市农业科学院 | Method for simultaneously detecting six triterpene compounds in Ganoderma applanatum fruiting body |
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