CN110749661B - Quality control method for culturing antrodia camphorata in dish culture mode - Google Patents
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Abstract
A quality control method for culturing Antrodia camphorata by dish culture relates to triterpenes Antrodia camphorata acid compounds. Preparing a test solution; preparing a reference substance; establishing a sample HPLC fingerprint condition; investigating the adaptability of the system; examining the content of a pair of main content epimer compounds; and (3) confirming the accuracy of the method by an external standard method. The preparation method of the reference substance can obtain the reference substance with the purity of more than 98 percent. The HPLC fingerprint of the sample realizes the baseline separation of a pair of major content differential isomers. The reproducibility of the test sample preparation is good. The solution prepared from the test sample and the reference substance has good stability and can be used for content determination. The linear method of three compounds in the mass range of 0.1-4.8 mug is good in relation, and the correlation coefficient is larger than 0.9999. The quality control method has good accuracy, and the sample recovery rate is within the range of 98-104%. The quality control research of the ware culture type Antrodia camphorata solves the problems that a reference substance is difficult to obtain in a large quantity and the content can not be accurately measured.
Description
Technical Field
The invention relates to triterpenes antrodia camphorata acid compounds, in particular to a quality control method for culturing antrodia camphorata by a dish culture method.
Background
Due to the shortage of wild antrodia host antrodia camphorata wood resources and the overlong culture period, the yield of wild antrodia camphorata is too low, and therefore, research on artificially cultured antrodia camphorata needs to be increased. After comprehensively considering various culture modes, the Antrodia camphorata cultured in a dish culture mode has better market application value due to simple and convenient culture mode, moderate culture period, no need of depending on host Antrodia camphorata and the like. Moreover, the dish-culture type antrodia camphorata and the wild antrodia camphorata both contain a large amount of antrodia camphorata acid active ingredients, have the same function in pharmacological activity, and provide a foundation for market application and popularization of the dish-culture type antrodia camphorata products.
However, since these triterpenoid polypeptides exist in epimers, such as major components (25S) -Antcin K and (25R) -Antcin K, which are a pair of epimers, in numerous studies on the quality control of Antrodia camphorata cultured on a petri dish, mixed quantification of the epimers that occur in pairs commonly found in Antrodia camphorata was used ([1] Lin T Y, Chen C, en ChiS C, et al. In other reports of epimers of Chinese herbs, it was found that α -glycyrrhizic acid and β -glycyrrhizic acid are also significantly different from the main pharmacokinetic parameters of epimers converted into glycyrrhetinic acid in vivo (3 Zhang autumn, Cheng Xiao, Yi Li Xin, etc. the pharmacokinetic studies of glycyrrhizic acid epimers in rat [ J ] Zhongnan pharmacology, 2010,8(5): 350-53). The study on the excretion accumulation of 20(S/R) -protopanoxadiol in rat bile shows that the excretion of bile in R configuration is 5.4 times of that in S configuration, and the R configuration shows obvious stereoselectivity (4 Wuxiang yangmeng, Wangli, Nigli, etc. 20(S) -protopanoxadiol Octoin type epimer in rat excretion study [ J ] Chinese J.M.2014, 39(7): 1306-10). Therefore, it is necessary to quantitatively examine epimers of (25S) -Antcin K and (25R) -Antcin K, which are major components of Antrodia camphorata cultured on a dish.
Disclosure of Invention
The invention aims to provide a quality control method for culturing antrodia camphorata in a dish culture manner.
The invention solves the problems that in the culture of Antrodia camphorata in a dish culture mode, a mixed quantitative mode is mostly adopted for epimers, and the epimers are not split and quantified respectively, provides a method for measuring the content of a pair of main epimers (25S) -Antcin K and (25R) -Antcin K which are difficult to prepare in large quantity by taking a compound (25R) -Antcin C which is easy to obtain as an internal reference substance, and nuclear magnetic data of the compound is shown in nuclear magnetic data of (25S) -Antcin K and (25R) -Antcin K in a table 1 and nuclear magnetic data of (25S) -Antcin K in a table 2:
TABLE 1 Nuclear magnetic data for (25S) -Antcin K and (25R) -Antcin K
TABLE 2 Nuclear magnetic data of (25S) -Antcin K
The invention comprises the following steps:
1) preparing a test solution;
2) preparing a reference substance;
3) establishing a sample HPLC fingerprint condition;
4) investigating the adaptability of the system;
5) examining the content of a pair of main content epimer compounds;
6) and an external standard method confirms the accuracy of the method.
In step 1), the method for preparing the test article may be: accurately weighing 100.0mg of potted Antrodia camphorata, cutting into pieces of 1mm multiplied by 1mm, placing the pieces into a 10mL triangular flask with a plug, adding 10mL of chromatographic methanol, weighing, ultrasonically extracting for 30min, placing at normal temperature, supplementing the chromatographic methanol to zero weight, shaking up, filtering with 0.45 mu m, and preparing into 10mg/mL test solution;
the ratio of the pieces of Antrodia camphorata cultured in a dish to the extraction solvent can be 25: 1, wherein the pieces of Antrodia camphorata cultured in a dish are calculated by mass, and the extraction solvent is calculated by volume; the extraction solvent can be methanol; when the mass (wet weight) of the pieces of Antrodia camphorata cultured in a dish culture way is 1kg, the volume of the extraction solvent is 25L; when the mass of the pieces of antrodia camphorata cultured in a dish culture mode is 1g, the volume of the extraction solvent is 25 mL; the extracting solution for culturing the fragments of the antrodia camphorata by the dish culture method can be prepared according to the following steps: adding pieces of Antrodia camphorata cultured in a dish culture manner into methanol as an extraction solvent, stirring, soaking, filtering, and collecting filtrate; the soaking can be carried out for 3 times, and the time of each time can be 12-16 h.
In step 2), the method for preparing the reference substance may be:
(1) carrying out standing extraction on the Antrodia camphorata cultured in a dish culture manner by using methanol;
(2) concentrating the filtrate under reduced pressure, and vacuum drying to obtain crude extract;
(3) and carrying out silica gel column chromatography, reversed phase silica gel column chromatography and HPLC on the crude extract to obtain a target compound, namely a reference substance.
In the step 2) and the step (3), silica gel with 200-300 meshes of filler can be adopted in the silica gel column chromatography process; particularly, the mass ratio of silica gel with filler of 200-300 meshes to the crude extract can be 10: 1, and the ratio of the column diameter to the column height of the silica gel column can be 2: 5; particularly, in the process of silica gel column chromatography, gradient elution is carried out on a mobile phase comprising a n-hexane-ethyl acetate solution with a volume ratio of (99: 1) to (5:1) and a dichloromethane-methanol with a volume ratio of (1:0) to (0: 1), and an eluent is collected; the second chromatographic column filler can be octadecylsilane chemically bonded silica (ODS) and the like; the mass ratio of the filler octadecylsilane chemically bonded silica to the fraction can be (30-40): 1, and the ratio of the column diameter to the column height of the silica gel column is 7: 40; the mobile phase in the octadecylsilane chemically bonded silica column chromatography process is a methanol-water solution, wherein the volume ratio of methanol to water is (1: 9) - (1: 0); the type of the chromatographic column in the HPLC process can be: the welch Ultimate XB-C is 184.6 mm multiplied by 250mm, the sampling concentration is 5 mu m, the sampling concentration is 10-40 mg/mL, the sampling volume is 20-60 mu L, the mobile phase in the HPLC process is acetonitrile-water solution, 0.2% acetic acid or 0.1% phosphoric acid is added into the mobile phase, the flow rate is 4-5 mL/min, and the detection wavelength is 208nm and 254 nm.
In step 3), the method for establishing the HPLC fingerprint condition of the sample may be: the mobile phase is a three-phase system of water (A), acetonitrile (B) and methanol (C), 0.2 percent acetic acid is added for gradient elution; the specific elution conditions were: 0-30 min, 50-44% of water, 25-28% of acetonitrile, 25-29% of methanol, and the flow rate is 0.5 mL/min; 30-60 min, 36-22% of water, 38-63% of acetonitrile, 26-15% of methanol, and the flow rate is 0.5-0.7 mL/min; the injection volume is 20 mu L; the detection wavelength is 254nm, and the column temperature is 40 ℃.
In step 4), the adaptability of the investigation system mainly comprises: inspecting precision, stability, repeatability, sample recovery rate and linear range; the precision investigation steps may be: taking 0.1mg/mL (25S) -Antcin C12 μ L for continuous injection for 5 times, and calculating RSD values of compound (25S) -Antcin C peak area and retention time; the steps of the repeatability investigation may be: taking 6 parts of Antrodia camphorata cultured in a same batch of dishes, accurately weighing each part of Antrodia camphorata 100mg, preparing a test solution according to a test preparation method, analyzing by HPLC, measuring the peak areas of (25S) -Antcin K, (25R) -Antcin K and (25S) -Antcin C samples with the sample injection volume of 20 mu L, and calculating the RSD values of the peak area and the retention time; the stability investigation steps may be: taking the same part of antrodia camphorata cultured in a dish culture mode to be 100mg, precisely weighing, preparing a test sample according to a test sample preparation method, injecting samples at 0, 2,4, 8, 12 and 24 hours, measuring peak areas of (25S) -Antcin K, (25R) -Antcin K and (25S) -Antcin C, and calculating RSD values of peak area and retention time, wherein the sample injection volume is 10 mu L; the steps for linear range inspection may be: control (25S) -Antcin K (25R) -Antcin K (0.2 mg/mL) was injected in the sample volumes of 2,4, 8, 12, 16, 20 and 24. mu.L, respectively, and control (25S) -Antcin C (0.1 mg/mL) was subjected to HPLC analysis in the sample volumes of 1, 2,4, 8, 12, 16, 20, 24 and 48. mu.L.
In step 5), the method for examining the content of a pair of main content epimer compounds can be as follows:
(1) measuring the content of (25S) -Antcin C by using a standard curve method;
(2) measuring correction factors of (25S) -Antcin K and (25R) -Antcin K by taking (25S) -Antcin C as an internal reference;
(3) the content of (25S) -Antcin K and (25R) -Antcin K in the test sample was calculated using the correction factor.
In step 5), part (2), the correction factors were measured at 6 masses of 0.4,0.8,1.6,2.4,3.2,4. mu.g, and the average was calculated for the next step.
The outstanding technical effects of the invention are as follows:
1) the preparation method of the reference substance provided by the invention can obtain the reference substance with the purity of more than 98%.
2) The pair of main content differential isomers (25S) -Antcin K and (25R) -Antcin K in the HPLC fingerprint of the test sample realizes baseline separation for the first time.
3) The preparation method of the test sample adopted by the invention has good reproducibility.
4) The solution prepared by the preparation method of the test sample and the reference substance has good stability within 24 hours, and can be used for content determination.
5) The linear method for inspecting the three compounds in the mass range of 0.1-4.8 mug has good relation, and the correlation coefficient is more than 0.9999.
6) The quality control method adopted by the invention has good accuracy, and the sample adding recovery rate is within the range of 98-104%.
7) The invention carries out quality control research on the Antrodia camphorata cultured in a dish culture way, and solves the problems that a reference substance is difficult to obtain in a large quantity and the content can not be accurately measured.
8) According to the invention, the content of a pair of main content differential isomers of (25S) -Antcin K and (25R) -Antcin K is measured by using an external standard method, and the RSD value is less than 3% by comparing the measurement results of the two methods, and the result shows that the two methods have no significant difference. Therefore, the method provides a new reference for measuring the multi-component content and controlling the quality of the Antrodia camphorata cultured in a dish culture manner, and provides a reference for establishing the quality standard of the future large-scale production of the Antrodia camphorata cultured in the dish culture manner.
Drawings
FIG. 1 is a HPLC chart of a test solution for Petri dish culture of Antrodia camphorata. In fig. 1, curve 1 is: (25R) -AntcinK, curve 2 is (25S) -Antcin K, and curve 3 is (25S) -Antcin C.
FIG. 2 is an HPLC chart of compound (25S) -Antcin C.
FIG. 3 is a standard curve for compound (25S) -Antcin C. In FIG. 3, the masses are 0.1,0.2,0.4,0.8,1.6,2.4,3.2, 4.8. mu.g, respectively.
FIG. 4 is an HPLC chart of compound (25S) -Antcink.
FIG. 5 is a standard curve for compound (25S) -Antcink. In FIG. 5, the masses are 0.4,0.8,1.6,2.4,3.2, 4.8. mu.g, respectively.
FIG. 6 is an HPLC chart of compound (25R) -Antcin K.
FIG. 7 is a standard curve of compound (25R) -Antcin K. In FIG. 7, the masses are 0.4,0.8,1.6,2.4,3.2, 4.8. mu.g, respectively.
Detailed Description
The main test method and results of the invention are as follows:
1. materials and instruments
(1) Material
The Petri dish culture of Antrodia camphorata was provided and identified by the Taiwan institute of Industrial and technology, Rogine researchers, and the control products (25R) -Antcin K, (25S) -Antcin K and (25S) -Antcin C were all prepared from the Petri dish culture of Antrodia camphorata, with a purity of > 98%.
(2) The main reagents are as follows:
(3) the experimental apparatus was as follows:
2. test methods and results analysis
Example 1: preparation of control
1. Preparation of crude extract of Antrodia camphorata cultured in a dish culture manner
1) Chopping the Antrodia camphorata cultured in a dish, extracting to obtain extractive solution, wherein the extractive solution is obtained by soaking and extracting 25L methanol overnight for 3 times, and filtering to obtain filtrate;
2) vacuum drying the filtrate after vacuum concentration to obtain the crude extract of the Antrodia camphorata cultured in a dish culture manner,
2. preparation of monomeric compound by silica gel column chromatography, ODS column chromatography and HPLC
1) Dissolving the crude Antrodia camphorata cultured in a dish culture manner (104.3g) by using methanol (25mL) and dichloromethane (25mL) to obtain a methanol and dichloromethane solution of the crude Antrodia camphorata cultured in the dish culture manner, stirring the sample solution into 200g of 80-100-mesh silica gel, and volatilizing the solvent to obtain the sample.
2) Uniformly loading the sample on the top of a 200-mesh 300-mesh silica gel chromatographic column, and carrying out gradient elution by using a normal hexane-ethyl acetate solution system (99: 1, 98: 2, 97: 3, 10: 1, 5:1) and a dichloromethane-methanol system (1:0, 99: 1, 98: 2, 97: 3, 10: 1, 5:1, 0: 1), wherein each gradient elution is 1000 mL. Wherein the weight ratio of the separation silica gel to the crude extract of Antrodia camphorata cultured in a dish is 10: 1, and the ratio of the diameter of the silica gel chromatographic column to the height of the column is 2: 5; 16 fractions (Fr-1, Fr-2, Fr-3, Fr-4, Fr-5, Fr-6, Fr-7, Fr-8, Fr-9, Fr-10, Fr-11, Fr-12, Fr-13, Fr-14, Fr-15, Fr-16) were combined according to TLC analysis;
3) from the HPLC chart and the results of the weight of fractions, Fr-14 was determined as a fraction (5.2g) of the major amount-containing compound, and this fraction was subjected to ODS column chromatography;
4) dissolving the component Fr-14 with a small amount of methanol (10mL), uniformly stirring the solution into 10g of 80-100 mesh silica gel, loading a sample on the top of an ODS chromatographic column after solvent volatilization, eluting with a methanol-water system (3: 7, 5: 5, 8: 2, 1:0), and receiving by tube. Wherein the weight ratio of adsorbent ODS to component Fr-14 is 30: 1; according to octadecylsilane bonded silica thin layer plate analysis and HPLC analysis, 10 fractions (Fr-14a, Fr-14b, Fr-14c, Fr-14d, Fr-14e, Fr-14f, Fr-14g, Fr-14h, Fr-14i, Fr-14j) were combined;
5) determining Fr-14j as the main content fraction (2.1g) according to HPLC result, subjecting the fraction to HPLC, dissolving 50mg Fr-14j sample with methanol, and subjecting to HPLC preparation under the condition of adding 0.2 acetic acid in acetonitrile-water (35: 65) to obtain compounds (25S) Antcin K (10mg) and (25R) -Antcin K (9.5 mg);
6) subjecting fraction Fr-12 to silica gel column chromatography with mobile phase of dichloromethane-methanol system (50: 1, 40: 1, 30: 1, 20: 1, 0: 1), and eluting 50mL each. Wherein the weight ratio of adsorbent silica gel to fraction Fr-12 is 20: 1, and the ratio of column diameter to column height of silica gel column is 1: 12; fractions 4 (Fr-12a, Fr-12b, Fr-12c, Fr-12d) were combined according to TLC analysis.
7) Performing ODS column chromatography on fraction Fr-12b (1.1g), dissolving fraction Fr-12b with a small amount of methanol (2mL), uniformly stirring the solution in 80-100 mesh silica gel (2 g), volatilizing the solvent, loading the sample on the top of an ODS column, eluting with a methanol-water system (1: 9, 2: 8, 3: 7, 4: 6, 5: 5, 6: 4, 1:0), and receiving by tube. Wherein the weight ratio of adsorbent ODS to component Fr-12b is 40: 1; according to reverse phase silica gel plate analysis and HPLC analysis, 14 fractions (Fr-12b1, Fr-12b2, Fr-12b3, Fr-12b4, Fr-12b5, Fr-12b6, Fr-12b7, Fr-12b8, Fr-12b9, Fr-12b10, Fr-12b11, Fr-12b12, Fr-12b13, Fr-12b14) were combined;
8) according to HPLC result, Fr-12b8 is prepared by HPLC under the condition of adding 0.2 acetic acid acetonitrile-water (50: 50) to obtain compound (25S) -Antcin C (61.9mg)
Example 2: establishment of quality control method
The invention optimizes the HPLC fingerprint condition of the test article, realizes the base line separation of epimers (25R) -AntcinK and (25S) -Antcin K for the first time, performs content determination on (25S) -Antcin K and (25R) -Antcin K which are difficult to separate through the internal standard substance (25S) -Antcin C, and simultaneously uses an external standard method to prove the reliability of the result again. Lays a foundation for establishing the quality standard of the ware culture type Antrodia camphorata.
The specific operation steps are as follows:
1. analysis by HPLC conditions
The mobile phase is a three-phase system of water (A), acetonitrile (B) and methanol (C), and 0.2 percent acetic acid is added for gradient elution. The specific elution conditions were: 0-30 min, 50-44% of A, 25-28% of B, 25-29% of C, and the flow rate is 0.5 mL/min; 30-60 min, 36-22% of A, 38-63% of B, 26-15% of C, and the flow rate is 0.5-0.7 mL/min; the sample injection volume is 10 mu L; the detection wavelength is 254nm, and the column temperature is 40 ℃.
2. Preparation of control solutions
2.0mg of (25S) -Antcin K and (25R) -Antcin K were weighed precisely, dissolved in a 10mL volumetric flask with chromatographic grade methanol, mixed well, allowed to cool, and fixed to volume. A control solution of 0.2mg/mL was prepared. Accurately weighing 1mg of (5S) -Antcin C, dissolving in a 10mL volumetric flask with chromatographic grade methanol, uniformly mixing, cooling and fixing the volume. A control solution of 0.1mg/mL was prepared.
3. Preparation of test solution
Accurately weighing 100.0mg dried Antrodia camphorata, cutting into pieces of 1mm × 1mm, placing in a triangular flask with a plug, adding 10mL of chromatographic methanol, weighing, ultrasonically extracting for 30min, cooling, and supplementing for weight loss. Filtering the filtrate with 0.45 μm microporous membrane to obtain 10mg/mL sample solution.
4. Investigation of linear relationships
0.2mg/mL of reference substance (25S) -Antcin K and (25R) -Antcin K are sequentially injected, the injection volumes are respectively 2,4, 8, 12, 16, 20 and 24 mu L, meanwhile, the reference substance (25S) -Antcin C of 0.1mg/mL is subjected to HPLC analysis, the injection volumes are 1, 2,4, 8, 12, 16, 20, 24 and 48 mu L, and a standard curve is drawn by taking the sample mass as an ordinate and the time as an abscissa to obtain a corresponding equation and a related coefficient.
5. Precision survey
0.1mg/mL of (25S) -Antcin C12. mu.L was taken for 5 consecutive HPLC analyses, and RSD values of peak area and retention time were calculated.
6. Repeatability survey
6 parts of Antrodia camphorata cultured in the same batch of dishes, each part of the Antrodia camphorata is 100mg, the solution is precisely weighed, the sample solution is prepared according to the preparation method of the sample solution, HPLC analysis is carried out, the sample injection volume is 20 mu L, and the peak areas of three samples, namely (25S) -Antcin K, (25R) -Antcin K and (25S) -Antcin C, are measured. RSD values for peak area and retention time were calculated for these three compounds.
7. Stability survey
The same culture dish is taken to culture 100mg of Antrodia camphorata, the precision is determined, then the test solution is prepared according to the preparation method of the test solution, HPLC analysis is carried out for 0, 2,4, 8, 12 and 24 hours, the sample injection volume is 10 mu L, and the peak area and the RSD value of the retention time of (25S) -Antcin K, (25R) -Antcin K and (25S) -Antcin C are calculated.
8. Investigation of sample recovery
Accurately weighing 9 parts of Antrodia camphorata containing known amount of culture dish, each part is 100mg, and accurately adding the internal reference substance (25S) -Antcin C according to 80%, 100% and 120% of the content of the internal reference substance. The sample solution was prepared according to the sample solution preparation method and subjected to HPLC analysis. The average recovery of 80%, 100% and 120% was determined.
1) The content of the internal reference substance (25S) -Antcin C is calculated by an external standard method.
2) Determination of f
With (25S) -Antcin C as an internal reference, the correction factors f of (25S) -Antcin K and (25R) -Antcin K with the mass of 0.4,0.8,1.6,2.4,3.2,4 and 8 mu g are respectively measured(25S)-Antcin C/(25S)-Antcin KAnd f(25S)-Antcin C/(25R)-Antcin K。
3) The contents of (25S) -Antcin K and (25R) -Antcin K were calculated using the correction factors.
4) The content of the compounds (25S) -Antcin K and (25R) -Antcin K was determined by an external standard method and compared with the present invention.
The experimental results are as follows:
the structural formulae of the compounds (25S) -Antcin C, (25S) -Antcin K and (25R) -Antcin K are as follows:
HPLC analysis of 10mg/mL dish-cultured Antrodia camphorata in specified conditions shows that the main content differential isomer compounds (25S) -Antcin K and (25R) -Antcin K are well separated, the theoretical plate numbers of the compounds (25R) -Antcin K, (25S) -Antcin K and (25S) -Antcin C are all more than 37000, the separation degrees are all more than 3, the tailing factors are respectively 0.98, 0.98 and 1.04, and the requirements of Chinese pharmacopoeia are met.
As shown in FIG. 1, when the standard curve of (25S) -Antcin C was measured, HPLC chromatogram of the obtained compound showed a high purity, and the purity of (25S) -Antcin C reached 98%.
As shown in FIG. 2, the standard curve is recovered by measuring the standard curve for (25S) -Antcin C with the mass of the standard solution as the abscissa and the peak area as the ordinate, and the chromatographic peak area of the standard is in good linear relation with the sample amount within the mass range of 0.1-4.8. mu.g, and the equation is as follows: y is 25.715x-0.0632, correlation coefficient: r ═ 0.9999.
As shown in FIG. 3, when the standard curve of (25S) -Antcin K was measured, HPLC chromatogram of the obtained compound showed a high purity, and the purity of (25S) -Antcin K reached 98%.
As shown in FIG. 4, the standard curve is recovered by measuring the standard curve for (25S) -Antcin K, taking the mass of the standard solution as the abscissa and the peak area as the ordinate, and the chromatographic peak area of the standard substance and the sample amount have a good linear relationship within the mass range of 0.4-4.8. mu.g, and the equation is as follows: y 25.001x +0.2385, correlation coefficient: r ═ 0.9999.
As shown in FIG. 5, when the standard curve of (25R) -Antcin K was measured, the HPLC chromatogram of the obtained compound showed a higher purity. The purity of (25R) -Antcin K reaches 98%.
As shown in FIG. 6, in the measurement of the standard curve for (25R) -Antcin K, the standard curve is recovered by taking the mass of the standard solution as the abscissa and the peak area as the ordinate, and the chromatographic peak area of the standard substance and the sample amount have a good linear relationship within the mass range of 0.4-4.8. mu.g, and the equation is as follows: 24.650x-0.2886, correlation coefficient: r ═ 0.9999.
As shown in FIG. 7, the standard curve was measured for (25R) -Antcin K. The obtained compound has high purity as shown in HPLC. The purity of (25R) -Antcin K reaches 98%.
As is clear from Table 3, 0.1mg/mL of (25S) -Antcin C was taken and subjected to HPLC analysis 5 times in succession, and the RSD values calculated for the peak area and the retention time were 0.07% and 0.02%, respectively, as shown in Table 3, indicating good instrument precision.
TABLE 3 precision investigation
As can be seen from table 4, 6 portions of antrodia cinnamomea cultured in the same batch of dish culture, each 100mg, were precisely weighed, the sample solution was prepared according to the sample preparation method, and subjected to HPLC analysis, the RSD values of the calculated peak areas were 0.78%, 2.3%, and 2.19%, and the RSD values of the retention times were 0.13%, 0.12%, and 0.06%, respectively, suggesting that the method had good reproducibility.
TABLE 4 repeatability test
As can be seen from Table 5, 100mg of Antrodia camphorata cultured in the same dish is precisely weighed and then a sample is prepared according to the sample preparation method, HPLC analysis is performed at 0, 2,4, 8, 12 and 24h respectively, the RSD values of the calculated peak areas are 0.78%, 2.3% and 2.19%, and the RSD values of the retention times are 0.13%, 0.12% and 0.06%, respectively, which indicates that the sample solution is stable within 24h and can be used for content determination of (25S) -Antcin K, (25R) -Antcin K and (25S) -Antcin C.
Table 5 stability survey
As can be seen from Table 6, 9 parts of Antrodia camphorata containing known amount of culture dish were weighed out precisely, each 100mg part was added precisely (25S) -AntcinC at 80%, 100% and 120% of the content of the internal reference. The sample solution was prepared according to the sample preparation method and subjected to HPLC analysis. The average recovery rates of 80%, 100% and 120% were measured and were 10.63%, 98.78% and 99.24%, respectively, as shown in table 6, suggesting that the method was accurate.
TABLE 6 recovery survey
As can be seen from Table 7, the RSD values of the correction factors f of (25S) -Antcin C and (25S) -Antcin K and (25R) -Antcin K are respectively 0.734% and 0.537%, which are less than 3%, and the repeatability is good.
TABLE 7f calculation results
As can be seen from Table 8, the RSD of the (25S) -Antcin K and (25R) -Antcin K content calculated by the two methods is less than 3%, indicating that the results measured by the two methods are not significantly different. Therefore, the method is feasible to be applied to the quality evaluation of the multi-index components of the Antrodia camphorata cultured in a dish culture manner.
TABLE 8 comparison of the two content determination methods
The invention provides a novel method for measuring the content of a pair of epimers, namely (25S) -Antcin K and (25R) -Antcin K, which are main components in Antrodia camphorata cultured in a dish culture manner, and is used for the quality control research of the Antrodia camphorata cultured in the dish culture manner. The method mainly comprises the following steps: the method can effectively enable a pair of epimers, namely main content components (25S) -AntcinK and (25R) -Antcin K, in the dish-culture type culture antrodia camphorata to achieve baseline separation, and provides guarantee for content determination. The invention takes a compound (25S) -Anticn C which is easily obtained as an internal reference substance, measures the contents of main content components (25S) -AntcinK and (25R) -Antcin K which are difficult to obtain in large quantity, and verifies the accuracy of the result by an external standard method. Under the condition of the method, the content of a pair of epimers, namely (25S) -Antcin K and (25R) -Antcin K, which are main components of the strain can be accurately, effectively and simply determined, and the product value in the culture process of the Antrodia camphorata cultured in a dish culture manner is ensured to a certain extent.
Claims (8)
1. A method for measuring the content of epimers (25R) -Antcin K and (25S) -Antcin K in Antrodia camphorata cultured in a dish culture manner is characterized by comprising the following steps:
1) the method for preparing the test solution comprises the following steps: accurately weighing 100.0mg of potted Antrodia camphorata, cutting into pieces of 1mm multiplied by 1mm, placing the pieces into a 10mL triangular flask with a plug, adding 10mL of chromatographic methanol, weighing, ultrasonically extracting for 30min, placing at normal temperature, supplementing the chromatographic methanol to zero weight, shaking up, filtering with 0.45 mu m, and preparing into 10mg/mL test solution;
2) preparing a reference substance;
3) establishing a sample HPLC fingerprint condition, wherein the specific method comprises the following steps: the mobile phase is a three-phase system of water, acetonitrile and methanol, 0.2 percent acetic acid is added for gradient elution; the specific elution conditions were: 0-30 min, 50-44% of water, 25-28% of acetonitrile, 25-29% of methanol, and the flow rate is 0.5 mL/min; 30-60 min, 36-22% of water, 38-63% of acetonitrile, 26-15% of methanol, and the flow rate is 0.5-0.7 mL/min; the injection volume is 20 mu L; the detection wavelength is 254nm, and the column temperature is 40 ℃;
4) investigating the adaptability of the system;
5) examining the content of a pair of main content epimer compounds;
6) and an external standard method confirms the accuracy of the method.
2. The method for determining the content of epimers (25R) -Antcin K and (25S) -Antcin K in the dish-cultured Antrodia camphorata according to claim 1, wherein in the step 1), the ratio of the fragments of the dish-cultured Antrodia camphorata to the extraction solvent is 25: 1, wherein the fragments of the dish-cultured Antrodia camphorata are calculated by mass and the extraction solvent is calculated by volume; the extraction solvent is methanol; when the mass of the pieces of antrodia camphorata cultured in a dish culture mode is 1kg, the volume of the extraction solvent is 25L; when the mass of the pieces of Antrodia camphorata cultured in a dish culture mode is 1g, the volume of the extraction solvent is 25 mL.
3. The method for determining the content of epimers (25R) -Antcin K and (25S) -Antcin K in the dish-cultured Antrodia camphorata according to claim 1, wherein in the step 1), the extract of the broken pieces of the dish-cultured Antrodia camphorata is prepared according to the following steps: adding pieces of Antrodia camphorata cultured in a dish culture manner into methanol as an extraction solvent, stirring, soaking, filtering, and collecting filtrate; the soaking is carried out for 3 times, and the time of each time is 12-16 h.
4. The method for determining the content of epimers (25R) -Antcin K and (25S) -Antcin K in Antrodia camphorata cultured on a dish according to claim 1, wherein in the step 2), the method for preparing the reference substance comprises:
(1) carrying out standing extraction on the Antrodia camphorata cultured in a dish culture manner by using methanol;
(2) concentrating the filtrate under reduced pressure, and vacuum drying to obtain crude extract;
(3) and carrying out silica gel column chromatography, reversed phase silica gel column chromatography and HPLC on the crude extract to obtain a target compound, namely a reference substance.
5. The method for measuring the content of epimers (25R) -Antcin K and (25S) -Antcin K in Antrodia camphorata cultured in a dish culture manner as claimed in claim 4, wherein in the step (3), silica gel with 200-300 meshes of filler is adopted in the silica gel column chromatography process; the mass ratio of silica gel with filler of 200-300 meshes to the crude extract is 10: 1, and the ratio of the column diameter to the column height of the silica gel column is 2: 5; in the process of silica gel column chromatography, gradient elution is carried out on a mobile phase comprising a n-hexane-ethyl acetate solution with a volume ratio of (99: 1) to (5:1) and dichloromethane-methanol with a volume ratio of (1:0) to (0: 1), and an eluent is collected; the filler is octadecylsilane chemically bonded silica; the mass ratio of the octadecylsilane chemically bonded silica to the fraction is (30-40): 1, and the ratio of the column diameter to the column height of the silica gel column is 7: 40; the mobile phase in the octadecylsilane chemically bonded silica column chromatography process is a methanol-water solution, wherein the volume ratio of methanol to water is (1: 9) - (1: 0); the type of the chromatographic column in the HPLC process can be: the welch Ultimate XB-C is 184.6 mm multiplied by 250mm, the sampling concentration is 5 mu m, the sampling concentration is 10-40 mg/mL, the sampling volume is 20-60 mu L, the mobile phase in the HPLC process is acetonitrile-water solution, 0.2% acetic acid or 0.1% phosphoric acid is added into the mobile phase, the flow rate is 4-5 mL/min, and the detection wavelength is 208nm and 254 nm.
6. The method for determining the content of epimers (25R) -Antcin K and (25S) -Antcin K in Antrodia camphorata cultured in a dish culture manner as claimed in claim 1, wherein in the step 4), the adaptability of the investigation system mainly comprises: inspecting precision, stability, repeatability, sample recovery rate and linear range; the precision investigation steps are as follows: taking 0.1mg/mL (25S) -Antcin C12 μ L for continuous injection for 5 times, and calculating RSD values of compound (25S) -Antcin C peak area and retention time; the steps of the repeatability investigation are as follows: taking 6 parts of Antrodia camphorata cultured in a same batch of dishes, accurately weighing each part of Antrodia camphorata 100mg, preparing a test solution according to a test preparation method, analyzing by HPLC, measuring the peak areas of (25S) -Antcin K, (25R) -Antcin K and (25S) -Antcin C samples with the sample injection volume of 20 mu L, and calculating the RSD values of the peak area and the retention time; the stability investigation steps are: taking the same part of antrodia camphorata cultured in a dish culture mode to be 100mg, precisely weighing, preparing a test sample according to a test sample preparation method, injecting samples at 0, 2,4, 8, 12 and 24 hours, measuring peak areas of (25S) -Antcin K, (25R) -Antcin K and (25S) -Antcin C, and calculating RSD values of peak area and retention time; the linear range investigation steps are: control (25S) -Antcin K (25R) -Antcin K (0.2 mg/mL) was injected in the sample volumes of 2,4, 8, 12, 16, 20 and 24. mu.L, respectively, and control (25S) -Antcin C (0.1 mg/mL) was subjected to HPLC analysis in the sample volumes of 1, 2,4, 8, 12, 16, 20, 24 and 48. mu.L.
7. The method for determining the content of epimers (25R) -Antcin K and (25S) -Antcin K in Antrodia camphorata cultured in a dish culture according to claim 1, wherein in the step 5), the method for examining the content of a pair of main content epimer compounds comprises the following steps:
(1) measuring the content of (25S) -Antcin C by using a standard curve method;
(2) measuring correction factors of (25S) -Antcin K and (25R) -Antcin K by taking (25S) -Antcin C as an internal reference;
(3) the content of (25S) -Antcin K and (25R) -Antcin K in the test sample was calculated using the correction factor.
8. The method for determining the content of epimers (25R) -Antcin K and (25S) -Antcin K in Antrodia camphorata cultured in a dish according to claim 7, wherein in the step (2), the calibration factors are determined at 0.4,0.8,1.6,2.4,3.2,4 μ g of 6 mass, and the average value is calculated in the next step.
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