CN110749661B - A kind of quality control method of dish culture Antrodia camphorata - Google Patents

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

A kind of quality control method of dish culture Antrodia camphorata

technical field

The invention relates to triterpenoid Antrodia camphorata acid compounds, in particular to a quality control method for dish culture Antrodia camphorata.

Background technique

Due to a series of reasons such as shortage of wild Antrodia camphorata host Antrodia camphorata and long cultivation period, the output of wild Antrodia camphorata is too low, so it is necessary to increase the research on artificially cultivated Antrodia camphorata. After comprehensively considering various cultivation methods, plate culture Antrodia camphorata has good market application value due to its simple cultivation method, moderate cultivation period and no need to rely on the host Antrodia camphorata. Moreover, both the dish cultured Antrodia camphorata and the wild Antrodia camphorata contain a large amount of antrodia cinnamomea active ingredients, which also have the same effect in terms of pharmacological activity, which provides a basis for the market application and promotion of the dish culture Antrodia camphorata products.

However, due to the presence of epimers in these triterpenes, such as the main components (25S)-Antcin K and (25R)-Antcin K are a pair of epimers. In the study of Antrodia cinnamomea, the paired epimers commonly found in Antrodia cinnamomea were mixed quantitatively ([1] Lin T Y, Chen C Y, Chien S C, et al. Metabolite profiles for Antrodia cinnamomea fruiting bodies harvested at different culture ages and from different wood substrates[J].JAgric Food Chem,2011,59(14):7626-35), and the quantitative components in HPLC fingerprints did not achieve baseline separation ([2]Y-M T.Method for HPLC anlysis of tritepenoids from antrodia campphora[J]). In the literature reports on the epimers of other traditional Chinese medicines, it was found that α-glycyrrhizic acid and β-glycyrrhizic acid also have obvious differences in the main pharmacokinetic parameters of the epimer conversion into glycyrrhetic acid in vivo. ([3] Zhang Qiufeng, Chen Xiaohui, Yi Lixin, et al. Pharmacokinetic study of glycyrrhizic acid epimers in rats [J]. Zhongnan Pharmacy, 2010, 8(5): 350-53). The study on the accumulation of 20(S/R)-protopanaxadiol in rat bile excretion showed that the biliary excretion of the R configuration was 5.4 times that of the S configuration, showing an obvious stereotype. Selective ([4] Wu Xiangmeng, Wang Li, Ni Yingying, et al. Study on the excretion of 20(S)-protopanaxadiol octilone-type epimer in rats [J]. China Journal of Traditional Chinese Medicine, 2014, 39( 7):1306-10). Therefore, it is necessary to quantitatively study the epimers of (25S)-Antcin K and (25R)-Antcin K, the main components in Antrodia camphorata.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a kind of quality control method of dish culture Antrodia camphorata.

The invention solves the problems of splitting and quantifying the epimers separately, and provides a compound (25R )-AntcinC was used as an internal reference to determine the content of a pair of main epimers (25S)-Antcin K and (25R)-Antcin K, which were difficult to prepare in large quantities. The NMR data of the compounds are shown in Table 1 (25S)-Antcin The NMR data of K and (25R)-Antcin K and the NMR data of (25S)-Antcin K in Table 2:

Table 1 NMR data of (25S)-Antcin K and (25R)-Antcin K

Table 2(25S)-Antcin K NMR data

The present invention includes the following steps:

1) Prepare the test solution;

2) Prepare a reference substance;

3) Establish the conditions for the HPLC fingerprint of the test product;

4) Investigate system adaptability;

5) investigate the content of a pair of main content epimer compounds;

6) External standard method confirms the accuracy of this method.

In step 1), the method for preparing the test sample can be: accurately weigh 100.0mg of Antrodia cinnamomea in a dish, cut into pieces of dish-cultured Antrodia camphorata of 1mm×1mm size, place in a 10mL plugged triangle Add 10 mL of chromatographic methanol to the bottle and weigh it, ultrasonically extract for 30 min, and place it at room temperature, supplement the weight loss with chromatographic methanol, shake well, and filter with 0.45 μm to prepare a 10 mg/mL test solution;

The ratio of the Antrodia cinnamomea fragments and the extraction solvent in the dish culture can be 25:1, wherein, the Antrodia cinnamomea fragments in the dish culture are calculated by mass, and the extraction solvent is calculated by volume; the extraction solvent can be methanol; When the quality (wet weight) of Antrodia camphorata cultivating in culture was 1kg, the volume of the extraction solvent was 25L; when the quality of the Antrodia cinnamomea pieces was 1g in the dish culture, the volume of the extraction solvent was 25mL; the dish culture The extract of Antrodia camphorata can be obtained according to the following steps: adding the plate cultured Antrodia camphorata pieces into the extraction solvent methanol, stirring, soaking, filtering, and collecting the filtrate; the soaking can be soaked for 3 times, and the time for each Can be 12 ~ 16h.

In step 2) in, the method for described preparation reference substance can be:

(1) The Antrodia camphorata cultured in a dish culture is statically extracted with methanol;

(2) the filtrate is concentrated under reduced pressure, and the crude extract is obtained after the vacuum drying;

(3) Silica gel column chromatography, reversed-phase silica gel column chromatography and HPLC are performed on the crude extract to obtain the target compound, that is, the reference substance.

In step 2) part (3), in the process of the silica gel column chromatography, the filler is 200-300 mesh silica gel; in particular, the filler is 200-300 mesh silica gel and the mass ratio of the crude extract can be 10:1, the ratio of the column diameter to the column height of the silica gel column can be 2:5; especially, in the process of the silica gel column chromatography, the volume ratio of the mobile phase is (99:1)～(5:1) hexane-ethyl acetate solution and dichloromethane-methanol with a volume ratio of (1:0) to (0:1), carry out gradient elution, and collect the eluent; the second chromatography column packing It can be octadecylsilane-bonded silica gel (ODS), etc.; the mass ratio of the packing octadecylsilane-bonded silica gel to the fraction can be (30-40): 1, and the column diameter of the silica gel column is equal to the column height. The ratio is 7:40; the mobile phase during the octadecylsilane-bonded silica gel column chromatography is methanol-water solution, wherein the volume ratio of methanol to water is (1:9)～(1:0); the HPLC The chromatographic column model in the process can be: welch Ultimate XB-C18 4.6×250mm, 5μm, the injection concentration is 10-40mg/mL, the injection volume is 20-60μL, and the mobile phase in the HPLC process is acetonitrile-water solution, Wherein, 0.2% acetic acid or 0.1% phosphoric acid is added to the mobile phase, the flow rate is 4-5 mL/min, and the detection wavelengths are 208 nm and 254 nm.

In step 3), the method for establishing the conditions for the HPLC fingerprint of the test product can be as follows: the mobile phase is a three-phase system of water (A), acetonitrile (B) and methanol (C), all of which are added with 0.2% acetic acid, and the gradient washes The specific elution conditions are: 0~30min, 50%~44% water, 25%~28% acetonitrile, 25%~29% methanol, flow rate 0.5mL/min; 30~60min, 36%~22% water, 38%～63% acetonitrile, 26%～15% methanol, flow rate 0.5～0.7mL/min; injection volume 20μL; detection wavelength 254nm, column temperature 40℃.

In step 4), the inspection system adaptability mainly includes: the inspection of precision, stability, repeatability, sample recovery rate and linear range; the step of the inspection of the accuracy may be: taking 0.1 mg/mL of ( 25S)-Antcin C12 μL was injected continuously for 5 times, and the RSD value of compound (25S)-Antcin C peak area and retention time was calculated; the steps of the repeatability investigation can be: taking 6 copies of Antrodia camphorata in the same batch of dish culture, Each 100 mg, accurately weighed, the test solution was prepared according to the test preparation method, HPLC analysis, the injection volume was 20 μL, and the (25S)-Antcin K, (25R)-Antcin K and (25S)-Antcin C were determined. The peak area of the sample, calculate the RSD value of the peak area and the retention time; the steps of the stability investigation can be: take the same plate culture Antrodia cinnamomea 100mg, accurately weigh and prepare the test sample according to the test sample preparation method, Samples were injected at 0, 2, 4, 8, 12, and 24 h, and the injection volume was 10 μL. The peak areas of (25S)-Antcin K, (25R)-Antcin K and (25S)-Antcin C were determined, and the peak areas and RSD value of retention time; the steps of linear range investigation can be: inject 0.2 mg/mL reference substance (25S)-Antcin K and (25R)-Antcin K sequentially, and the injection volumes are 2, 4, 8, 12, 16, 20, 24 μL, and 0.1 mg/mL reference substance (25S)-Antcin C was subjected to HPLC analysis, and the injection volume was 1, 2, 4, 8, 12, 16, 20, 24, and 48 μL.

In step 5), the method for investigating the content of a pair of main content epimer compounds may be:

(1) Determination of (25S)-Antcin C content by standard curve method;

(2) Determination of the correction factors of (25S)-Antcin K and (25R)-Antcin K with (25S)-Antcin C as an internal reference;

(3) Use the correction factor to calculate the content of (25S)-Antcin K and (25R)-Antcin K in the test sample.

In step 5) part (2), the correction factor is determined under 6 masses of 0.4, 0.8, 1.6, 2.4, 3.2, and 4 μg, and the average value is obtained for the next calculation.

The outstanding technical effect of the present invention is as follows:

1) The preparation method of the reference substance provided by the present invention can obtain the reference substance with a purity greater than 98%.

2) In the HPLC fingerprint of the test product of the present invention, the pair of main content epimers (25S)-Antcin K and (25R)-Antcin K achieved baseline separation for the first time.

3) The test sample preparation method adopted in the present invention has good reproducibility.

4) The solution prepared by the preparation method of the test substance and the reference substance adopted in the present invention has good stability within 24 hours and can be used for content determination.

5) The three compounds in the mass range of 0.1-4.8 μg investigated in the present invention have a good linear relationship, and the correlation coefficient is greater than 0.9999.

6) The quality control method adopted in the present invention has good accuracy, and the sample addition recovery rate is in the range of 98% to 104%.

7) The present invention conducts quality control research on Antrodia camphorata cultured in a dish, and solves the problems that the reference substance is difficult to obtain in large quantities and cannot be accurately measured.

8) The present invention simultaneously uses the external standard method to determine the content of a pair of main content epimers of (25S)-Antcin K and (25R)-Antcin K, and comparing the measurement results of the two methods, it is found that the RSD value is less than 3%. , the results showed no significant difference between the two. Therefore, this method provides a new reference for the determination of multi-component content and quality control methods of Antrodia cinnamomea in dish culture, and provides a reference for the establishment of quality standards for large-scale production of Antrodia cinnamomea in dish culture in the future.

Description of drawings

Fig. 1 is the HPLC chart of the test solution of Antrodia camphorata cultivated in dish culture. In Figure 1, curve 1 is: (25R)-AntcinK, curve 2 is (25S)-Antcin K, and curve 3 is (25S)-Antcin C.

Figure 2 is an HPLC chart of compound (25S)-Antcin C.

Figure 3 is a standard curve of compound (25S)-Antcin C. In Figure 3, the masses are 0.1, 0.2, 0.4, 0.8, 1.6, 2.4, 3.2, 4.8 μg, respectively.

Figure 4 is an HPLC chart of compound (25S)-AntcinK.

Figure 5 is a standard curve for compound (25S)-AntcinK. In Figure 5, the masses are 0.4, 0.8, 1.6, 2.4, 3.2, 4.8 μg, respectively.

Figure 6 is an HPLC profile of compound (25R)-Antcin K.

Figure 7 is a standard curve for compound (25R)-Antcin K. In Figure 7, the masses are 0.4, 0.8, 1.6, 2.4, 3.2, 4.8 μg, respectively.

Detailed ways

The main test methods and results of the present invention are as follows:

1. Materials and instruments

(1)Material

The dish cultured Antrodia camphorata was provided and identified by researcher Luo Jimeng of the Taiwan Institute of Industrial Technology. The reference substances (25R)-Antcin K, (25S)-Antcin K and (25S)-Antcin C were all obtained from the dish cultured Antrodia camphorata Prepared with >98% purity.

(2) The main reagents are as follows:

(3) The experimental instruments are as follows:

2. Test method and result analysis

Example 1: Preparation of reference substance

1. Preparation of the crude extract of Antrodia camphorata

1) after the dish culture Antrodia camphorata is chopped, it is subjected to extraction treatment to obtain an extract, wherein the extraction solvent is methanol 25L overnight soaking and extraction, a total of 3 times, and the filtrate is obtained after filtering;

2) the filtrate is vacuum-dried after being concentrated under reduced pressure to obtain the crude extract of Antrodia camphorata cultivated in a dish,

2. Preparation of monomer compounds by silica gel column chromatography, ODS column chromatography and HPLC

1) Dissolve the crude extract of Antrodia camphorata (104.3 g) with methanol (25 mL) and dichloromethane (25 mL) to obtain the methanol and dichloromethane solution of the crude extract of Antrodia camphorata cultivated in the dish culture, and mix the sample solution. Put it into 200g of 80-100 mesh silica gel, and get the sample after the solvent evaporates.

2) The above samples were evenly packed on the top of a 200-300 mesh silica gel chromatography column, using a n-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) for gradient elution with 1000 mL each. Among them, the weight ratio of separation silica gel and dish cultured Antrodia camphorata crude extract was 10:1, and the ratio of silica gel column diameter to column height was 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);

3) According to the HPLC chart and the fraction weight result, determine that Fr-14 is the main content compound fraction (5.2g), and carry out ODS column chromatography separation to this component;

4) Dissolve the component Fr-14 with a small amount of methanol (10 mL), stir the solution into 10 g of 80-100 mesh silica gel, and place the sample on the top of the ODS chromatography column after the solvent evaporates. The system (3:7, 5:5, 8:2, 1:0) is eluted and received in separate tubes. Among them, the weight ratio of ODS and component FR-14 is 30: 1; after analysis of the ectopane-alkane kel silicone thin layer analysis and HPLC analysis -14c, Fr-14d, Fr-14e, Fr-14f, Fr-14g, Fr-14h, Fr-14i, Fr-14j);

5) According to the HPLC results, determine that Fr-14j is the main content fraction (2.1 g), carry out HPLC on this component, and dissolve 50 mg of Fr-14j sample with methanol, add 0.2 acetic acid in acetonitrile-water (35: 65) under the conditions of HPLC preparation to obtain compounds (25S) Antcin K (10 mg) and (25R)-Antcin K (9.5 mg);

6) Silica gel column chromatography on fraction Fr-12, the mobile phase is dichloromethane-methanol system (50:1, 40:1, 30:1, 20:1, 0:1), each gradient elution is 50 mL . Among them, the weight ratio of the adsorbent silica gel to fraction Fr-12 was 20:1, and the ratio of the column diameter to the column height of the silica gel column was 1:12; according to TLC analysis, 4 fractions (Fr-12a, Fr-12a, Fr- 12b, Fr-12c, Fr-12d).

7) Perform ODS column chromatography on fraction Fr-12b (1.1 g), dissolve fraction Fr-12b with a small amount of methanol (2 mL), mix the solution evenly into 2 g of 80-100 mesh silica gel, and evaporate the solvent to dryness. The sample was loaded on top of the ODS column, eluted with methanol-water system (1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 1:0), and received in separate tubes. The weight ratio of adsorbent ODS and component Fr-12b is 40:1; 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);

8) According to the HPLC results, Fr-12b8 was prepared by HPLC under the condition of adding 0.2 acetic acid and acetonitrile-water (50:50) to obtain compound (25S)-Antcin C (61.9 mg)

Example 2: Establishment of Quality Control Method

The invention optimizes the HPLC fingerprint conditions of the test product, realizes the baseline separation of the epimers (25R)-AntcinK and (25S)-Antcin K for the first time, and separates the difficult-to-difficulties by the internal standard (25S)-Antcin C. The isolated (25S)-Antcin K and (25R)-Antcin K were assayed, and the external standard method was used to prove the reliability of the results again. It laid the foundation for the establishment of the quality standard of Antrodia camphorata cultivated in dish culture.

The specific operation steps are as follows:

1. Analysis of HPLC conditions

The mobile phase was a three-phase system of water (A), acetonitrile (B) and methanol (C), all of which were added with 0.2% acetic acid, and gradient elution was carried out. The specific elution conditions are: 0~30min, 50%~44%A, 25%~28%B, 25%~29%C, flow rate 0.5mL/min; 30~60min, 36%~22%A, 38% ～63%B, 26%～15%C, flow rate 0.5～0.7mL/min,; injection volume 10μL; detection wavelength 254nm, column temperature 40℃.

2. Preparation of reference solution

Accurately weigh 2.0 mg of (25S)-Antcin K and (25R)-Antcin K, dissolve them in a 10 mL volumetric flask with chromatography grade methanol, mix well, let cool, and make up to volume. Prepared as a reference solution of 0.2 mg/mL. Precisely weigh 1 mg of (5S)-Antcin C, dissolve it in a 10 mL volumetric flask with chromatographic grade methanol, mix well, let it cool, and make up to volume. Prepared as a reference solution of 0.1 mg/mL.

3. Preparation of the test solution

Precisely weigh 100.0 mg of dry Antrodia cinnamomea, cut into pieces of 1 mm × 1 mm and place in a stoppered conical flask, add 10 mL of chromatographic methanol, weigh and extract ultrasonically for 30 min, let cool, and make up for weightlessness. The filtrate was passed through a 0.45 μm microporous membrane to obtain a 10 mg/mL solution of the test product.

4. Examination of linear relationship

The 0.2mg/mL reference substance (25S)-Antcin K and (25R)-Antcin K were injected sequentially, and the injection volumes were 2, 4, 8, 12, 16, 20, and 24 μL, respectively. The reference substance (25S)-Antcin C was analyzed by HPLC, and the injection volume was 1, 2, 4, 8, 12, 16, 20, 24, 48 μL, and the sample mass was taken as the ordinate and the time as the abscissa. Plot and find the corresponding equations and correlation coefficients.

5. Precision inspection

Take 12 μL of 0.1 mg/mL (25S)-Antcin C for 5 consecutive HPLC analysis, and calculate the RSD value of peak area and retention time.

6. Repeat inspection

Take 6 copies of Antrodia camphorata from the same batch of dish culture, each 100 mg, accurately weighed, prepare the test solution according to the test solution preparation method, analyze by HPLC, the injection volume is 20 μL, and measure (25S)-Antcin K, Peak areas for three samples of (25R)-Antcin K and (25S)-Antcin C. Calculate the RSD values of the peak areas and retention times for these three compounds.

7. Stability inspection

Take 100 mg of Antrodia camphorata cultivated in the same dish, accurately weighed, and prepare the test solution according to the test solution preparation method, and carry out HPLC analysis at 0, 2, 4, 8, 12, and 24 hours. The injection volume is 10 μL, and the calculation RSD values of peak areas and retention times for (25S)-Antcin K, (25R)-Antcin K and (25S)-Antcin C.

8. Inspection of sample recovery rate

Accurately weigh 9 copies of Antrodia camphorata containing a known amount of dish culture, each 100 mg, and add the internal reference (25S)-Antcin C according to 80%, 100% and 120% of the content of the internal reference. Prepare the test solution according to the test solution preparation method, and carry out HPLC analysis. Average recoveries of 80%, 100% and 120% were determined.

1) Calculate the content of the internal reference (25S)-Antcin C by using the external standard method.

2) Determination of f

Using (25S)-Antcin C as an internal reference, the correction factors f _(25S) of (25S)-Antcin K and (25R)-Antcin K with masses of 0.4, 0.8, 1.6, 2.4, 3.2, 4, and 8 μg were measured, respectively. _{-AntcinC /(25S)-AntcinK and} f _{(25S)-AntcinC /(25R)-AntcinK .}

3) Calculate the content of (25S)-Antcin K and (25R)-Antcin K using a correction factor.

4) The content of compounds (25S)-Antcin K and (25R)-Antcin K was determined by external standard method and compared with the present invention.

Experimental results:

The structural formulas of compounds (25S)-Antcin C, (25S)-Antcin K and (25R)-Antcin K are as follows:

HPLC analysis of 10 mg/mL dish culture Antrodia camphorata under the specified conditions showed that the main content epimer compounds (25S)-Antcin K and (25R)-Antcin K were well separated, and the compound (25R)-Antcin K The theoretical plate numbers of , (25S)-Antcin K and (25S)-Antcin C are all greater than 37000, the resolutions are all greater than 3, and the tailing factors are 0.98, 0.98 and 1.04, respectively, all meeting the requirements of the Chinese Pharmacopoeia.

As shown in Figure 1, when the standard curve of (25S)-Antcin C was measured, the HPLC chromatogram of the obtained compound showed that the purity was high, and the purity of (25S)-Antcin C reached 98%.

As shown in Figure 2, the standard curve was measured for (25S)-Antcin C. The mass of the standard solution was taken as the abscissa and the peak area as the ordinate. The peak area has a good linear relationship with the sample amount, the equation is: y=25.715x-0.0632, and the correlation coefficient: R=0.9999.

As shown in Figure 3, when the standard curve of (25S)-Antcin K was measured, the HPLC chromatogram of the obtained compound showed that the purity was high, and the purity of (25S)-Antcin K reached 98%.

As shown in Figure 4, the standard curve was measured for (25S)-Antcin K, with the mass of the standard solution as the abscissa and the peak area as the ordinate. The peak area has a good linear relationship with the sample amount, the equation is: y=25.001x+0.2385, and the correlation coefficient: R=0.9999.

As shown in Figure 5, when the standard curve of (25R)-Antcin K was measured, the HPLC chromatogram of the obtained compound showed high purity. The purity of (25R)-Antcin K reached 98%.

As shown in Figure 6, the standard curve was measured for (25R)-Antcin K, with the mass of the standard solution as the abscissa and the peak area as the ordinate. The peak area has a good linear relationship with the sample amount, the equation is: y=24.650x-0.2886, and the correlation coefficient: R=0.9999.

As shown in Fig. 7, when the standard curve was measured for (25R)-Antcin K. The HPLC spectrum of the obtained compound showed that the purity was high. The purity of (25R)-Antcin K reached 98%.

It can be seen from Table 3 that 0.1 mg/mL of (25S)-Antcin C was used for HPLC analysis for 5 consecutive times, and the RSD values of the calculated peak area and retention time were 0.07% and 0.02%, respectively, as shown in Table 3, indicating that the instrument Precision is good.

Table 3 Precision inspection

As can be seen from Table 4, get the same batch of 6 parts of Antrodia camphorata, each 100mg, accurately weighed, prepare the test solution according to the test preparation method, carry out HPLC analysis, and the RSD value of the calculated peak area is 0.78% , 2.3% and 2.19%, and the RSD values of the retention time were 0.13%, 0.12% and 0.06%, respectively, indicating that the method had good repeatability.

Table 4 Repeated investigation

As can be seen from Table 5, take the same plate culture Antrodia cinnamomea 100mg, after accurate weighing, prepare the test product according to the test product preparation method, carry out HPLC analysis at 0, 2, 4, 8, 12, 24h respectively, calculate the peak area The RSD values were 0.78%, 2.3% and 2.19%, and the RSD values of the retention time were 0.13%, 0.12% and 0.06%, respectively, indicating that the test solution was relatively stable within 24h and could be used for (25S)-Antcin K, Determination of (25R)-Antcin K and (25S)-Antcin C.

Table 5 Stability investigation

As can be seen from Table 6, 9 parts of Antrodia camphorata containing a known amount of dish cultured cultured Antrodia camphorata were mainly used, each 100 mg, and the internal reference substance (25S)-Antcin was precisely added according to 80%, 100% and 120% of the content of the internal reference substance. C. The test solution was prepared according to the test preparation method and analyzed by HPLC. The average recoveries of 80%, 100% and 120% were determined, which were 10.63%, 98.78% and 99.24%, respectively, as shown in Table 6, indicating that the method was accurate.

Table 6 Investigation of recovery rate

It can be seen from Table 7 that with (25S)-Antcin C as the internal reference, the RSD values of the correction factors f of (25S)-Antcin K and (25R)-Antcin K are 0.734% and 0.537%, respectively, which are less than 3% and have good repeatability. .

Table 7f calculation results

It can be seen from Table 8 that the RSD of the content of (25S)-Antcin K and (25R)-Antcin K calculated by the two methods is less than 3%, indicating that there is no significant difference in the results measured by the two methods. Therefore, the application of this method in the quality evaluation of the multi-index components of Antrodia camphorata cultivated in a dish is feasible.

Table 8 Comparison of two content determination methods

The invention provides a novel method for determining the content of epimers, the main components (25S)-Antcin K and (25R)-Antcin K, in dish culture Antrodia camphorata, which is used for dish culture Antrodia camphorata quality control studies. The method mainly includes: extracting the minced plate culture Antrodia camphorata with methanol, and then taking the supernatant to carry out HPLC analysis under set conditions. The method can effectively make the main content component (25S )-Antcin K and (25R)-Antcin K this pair of epimers achieved baseline separation, which provided a guarantee for their content determination. In the present invention, the readily available compound (25S)-Anticn C is used as an internal reference to determine the contents of the main content components (25S)-AntcinK and (25R)-Antcin K, which are difficult to obtain in large quantities, and the results are verified by an external standard method. accuracy. Under the conditions of this method, the epimer content of the main components (25S)-Antcin K and (25R)-Antcin K can be determined accurately, effectively and simply, which ensures that the plate culture is to a certain extent. The value of products in the cultivation process of Antrodia camphorata.

Claims (8)

1. the assay method of epimer (25R)-Antcin K and (25S)-Antcin K in a dish cultured Antrodia camphorata, is characterized in that comprising the following steps: 1) Prepare the test solution, the specific method is: accurately weigh 100.0mg of Antrodia cinnamomea in a dish, cut into pieces of 1mm × 1mm size of Antrodia cinnamomea, and place it in a 10mL conical flask with a stopper and add 10mL of Antrodia cinnamomea. The chromatographic methanol was weighed, and after ultrasonic extraction for 30 min, it was placed at room temperature, and the weight loss was supplemented with chromatographic methanol, shaken, and filtered with 0.45 μm to prepare a 10 mg/mL test solution; 2) Prepare a reference substance; 3) Establish the conditions for the HPLC fingerprint of the test product, the specific method is: the mobile phase is a three-phase system of water, acetonitrile and methanol, all add 0.2% acetic acid, and gradient elution; the specific elution conditions are: 0 ~ 30min, 50% ~ 44% water, 25%~28% acetonitrile, 25%~29% methanol, flow rate 0.5mL/min; 30~60min, 36%~22% water, 38%~63% acetonitrile, 26%~15% methanol, flow rate 0.5～0.7mL/min; injection volume 20μL; detection wavelength 254nm, column temperature 40℃; 4) Investigate system adaptability; 5) investigate the content of a pair of main content epimer compounds; 6) The external standard method confirms the accuracy of this method. 2. the content assay method of epimer (25R)-Antcin K and (25S)-Antcin K in a kind of dish culture Antrodia camphorata as claimed in claim 1, is characterized in that in step 1) in, described The ratio of dish culture Antrodia cinnamomea fragment and extraction solvent is 25:1, wherein, dish culture Antrodia cinnamomea fragment is calculated by mass, and extraction solvent is calculated by volume; Described extraction solvent is methanol; When the mass of the fragments is 1kg, the volume of the extraction solvent is 25L; when the mass of the Antrodia camphorata fragments is 1g, the volume of the extraction solvent is 25mL. 3. the content assay method of epimer (25R)-Antcin K and (25S)-Antcin K in a kind of dish culture Antrodia camphorata as claimed in claim 1, is characterized in that in step 1) in, described The extract of dish culture Antrodia camphorata fragmentation is prepared according to the following steps: adding the dish culture Antrodia camphorata fragment into the extraction solvent methanol, stirring, soaking, filtering, and collecting the filtrate; the soaking is soaking 3 times, and each The second time is 12 ~ 16h. 4. the content assay method of epimer (25R)-Antcin K and (25S)-Antcin K in a kind of dish culture Antrodia camphorata as claimed in claim 1, is characterized in that in step 2) in, described The method of preparing the reference substance is as follows: (1) The Antrodia camphorata cultured in a dish culture is statically extracted with methanol; (2) the filtrate is concentrated under reduced pressure, and the crude extract is obtained after the vacuum drying; (3) Silica gel column chromatography, reversed-phase silica gel column chromatography and HPLC are performed on the crude extract to obtain the target compound, that is, the reference substance. 5. the content assay method of epimer (25R)-Antcin K and (25S)-Antcin K in a kind of dish culture Antrodia camphorata as claimed in claim 4, it is characterized in that in step (3), all In the process of the silica gel column chromatography, the filler is 200-300 mesh silica gel; the filler is 200-300 mesh silica gel and the mass ratio of 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; During the process of the silica gel column chromatography, the mobile phase is a n-hexane-ethyl acetate solution with a volume ratio of (99:1)～(5:1) and a volume ratio of (1:0)～(0 : 1) dichloromethane-methanol, carry out gradient elution, collect eluent; Described filler is octadecylsilane bonded silica gel; The mass ratio of described octadecylsilane bonded silica gel and fraction is ( 30～40): 1, the ratio of the column diameter to the column height of the silica gel column is 7:40; the mobile phase in the octadecylsilane-bonded silica gel column chromatography process is methanol-water solution, wherein the volume ratio of methanol to water is (1:9)～(1:0); the chromatographic column model in the HPLC process can be: welch Ultimate XB-C18 4.6×250mm, 5μm, the injection concentration is 10～40mg/mL, and the injection volume is 20-60 μL, the mobile phase in the HPLC process is acetonitrile-water solution, wherein 0.2% acetic acid or 0.1% phosphoric acid is added to the mobile phase, the flow rate is 4-5 mL/min, and the detection wavelengths are 208 nm and 254 nm. 6. the content assay method of epimer (25R)-Antcin K and (25S)-Antcin K in a kind of dish culture Antrodia camphorata as claimed in claim 1, is characterized in that in step 4) in, described Investigating system adaptability mainly includes: precision, stability, repeatability, sample addition recovery rate and linear range; the steps of precision testing are: take 0.1 mg/mL (25S)-Antcin C12 μL into the needle continuously for 5 time, calculate the RSD value of compound (25S)-Antcin C peak area and retention time; the step of the repeatability investigation is: take 6 parts of the same batch of dish culture Antrodia camphorata, each 100mg, accurately weighed, according to the supply Test preparation method Prepare the test solution, analyze by HPLC, the injection volume is 20 μL, measure the peak area of (25S)-Antcin K, (25R)-Antcin K and (25S)-Antcin C samples, calculate the peak area and retention The RSD value of time; the steps of the stability investigation are: take the same plate culture Antrodia cinnamomea 100mg, accurately weigh and prepare the test product according to the test product preparation method, at 0, 2, 4, 8, 12, 24h injection, the injection volume was 10 μL, the peak areas of (25S)-Antcin K, (25R)-AntcinK and (25S)-Antcin C were measured, and the RSD values of the peak area and retention time were calculated; the steps of the linear range investigation were as follows: : 0.2mg/mL reference substance (25S)-Antcin K and (25R)-Antcin K were injected in sequence, and the injection volumes were 2, 4, 8, 12, 16, 20, 24 μL, and 0.1 mg/mL The reference substance (25S)-Antcin C of mL was analyzed by HPLC, and the injection volume was 1, 2, 4, 8, 12, 16, 20, 24, 48 μL. 7. the assay method of epimer (25R)-Antcin K and (25S)-Antcin K in a kind of dish culture Antrodia camphorata as claimed in claim 1, is characterized in that in step 5) in, described The method for investigating the content of a pair of main content epimer compounds is: (1) Determination of (25S)-Antcin C content by standard curve method; (2) Determination of the correction factors of (25S)-Antcin K and (25R)-Antcin K with (25S)-Antcin C as an internal reference; (3) Use the correction factor to calculate the content of (25S)-Antcin K and (25R)-Antcin K in the test sample. 8. the content assay method of epimer (25R)-Antcin K and (25S)-Antcin K in a kind of dish culture Antrodia camphorata as claimed in claim 7, it is characterized in that in step (2), all The correction factor was determined under 6 masses of 0.4, 0.8, 1.6, 2.4, 3.2, and 4 μg, and the average value was obtained for the next calculation.

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