A method for determining the content of triterpene acids in poria cocos and application thereof

Abstract

The present application relates to a kind of triterpenoid acid compound content determination method and application in Poria cocos, belong to the field of traditional Chinese medicine quality analysis and control.The determination method includes using liquid chromatography (high performance liquid chromatography, HPLC) to detect Poria cocos test solution;Wherein, triterpenoid acid compound includes 6 alpha-hydroxy poria acid C, Poria cocos new acid B, dehydrogenated tuma acid, tuma acid, Poria cocos new acid A, 3-epidehydrogen tuma acid, poria acid C, 3-epidehydrogen tuma acid, dehydrogenated tuma acid, Poria cocos acid, Songling new acid, dehydrogenated zishikuan acid, zishikuan acid.The present application establishes suitable HPLC analysis method of Poria cocos triterpenoid acid compound, selects Poria cocos acid as internal reference, establishes one measurement multiple evaluation method for simultaneously determining the content of 13 kinds of Poria cocos triterpenoid acid in Poria cocos, saves detection cost and time, and provides reference for the quality control of Poria cocos.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of traditional Chinese medicine quality analysis and control, and particularly relates to a determination method and application of triterpenoid acid content in Poria cocos. BACKGROUND

[0002] Poria cocos is the dried sclerotium of Wolfiporia hoelen (Fr.) Y.C. Dai & V. Papp of the family of Hymenochaetaceae, mainly produced in Sichuan, Anhui, Yunnan, Hunan, Hubei and other places, and is usually dug in autumn. It has the effects of promoting water excretion, invigorating the spleen and stomach, and calming the heart and soothing the nerves, and is clinically used for treating edema, oliguria, dizziness, anorexia, diarrhea and other symptoms. The main active components of Poria cocos are triterpenoids and polysaccharides. At present, more than 100 triterpenoids have been isolated from Poria cocos, and it has been confirmed that Poria cocos triterpenoids have the pharmacological effects of invigorating the spleen, diuresis, anti-inflammation and anti-tumor.

[0003] The action characteristics of traditional Chinese medicine with multiple components and multiple effects make it difficult to evaluate the quality of medicinal materials by determining the content of a single component. That is, due to the complexity of the components of traditional Chinese medicine, the determination of a single component cannot comprehensively reflect the quality of medicinal materials. Therefore, the content determination method based on multi-component and multi-index analysis has become a generally recognized quality evaluation method. The related technology discloses a determination method and application of triterpenoid acid content in Poria cocos. The method detects by liquid chromatography, selects pachymic acid as a reference, and establishes a one-measurement-multiple-evaluation method for simultaneously determining the contents of pachymic acid B, dehydroxytumulosic acid, pachymic acid A, dehydroxytumulosic acid, pachymic acid and pachymic acid in Poria cocos. It can be seen that the method in the related technology can detect a limited number of triterpenoid acids, and cannot comprehensively reflect the quality of medicinal materials. Moreover, there is no control standard for Poria cocos [content determination] in the Chinese Pharmacopoeia.

[0004] Therefore, it is necessary to establish a determination method for simultaneously determining the contents of more kinds of Poria cocos triterpenoid acids in Poria cocos, for detecting the main triterpenoid components in Poria cocos, and providing a basis for the quality control of Poria cocos medicinal materials. SUMMARY

[0005] The present application aims to at least solve one of the technical problems existing in the prior art. To this end, the present application provides a determination method of triterpenoid acid content in Poria cocos, which realizes the simultaneous determination of 13 main triterpenoid acids in Poria cocos, and has low determination cost and high determination efficiency.

[0006] The present application also provides the application of the above determination method in the quality control of Poria cocos and its derivative products.

[0007] In one aspect of the present application, a determination method of triterpenoid acid content in Poria cocos is provided, which comprises detecting a Poria cocos test solution by liquid chromatography;

[0008] The triterpenoid acid compounds include 6a-hydroxypachymic acid C, pachymic acid new B, dehydrotrametenolic acid, trammolic acid, pachymic acid new A, 3-epidehydrotrametenolic acid, pachymic acid C, 3-epidehydrotrametenolic acid, dehydrotrametenolic acid, pachymic acid, pachymic acid new A, dehydrotrametenolic acid, trammolic acid.

[0009] The detection condition of the liquid chromatography includes:

[0010] The mobile phase A is a phosphoric acid aqueous solution with a mass fraction of 0.05% to 0.4%;

[0011] The mobile phase B is acetonitrile;

[0012] The elution mode is gradient elution;

[0013] The process of the gradient elution is as follows:

[0014] 0 min to 20 min, the volume fraction of the mobile phase B is increased from 48% to 52% to 52% to 55%;

[0015] 20 min to 39 min, the volume fraction of the mobile phase B is kept as 52% to 55%;

[0016] 39 min to 43 min, the volume fraction of the mobile phase B is increased from 52% to 55% to 60% to 64%;

[0017] 43 min to 47 min, the volume fraction of the mobile phase B is kept as 60% to 64%;

[0018] 47 min to 70 min, the volume fraction of the mobile phase B is increased from 60% to 64% to 90% to 94%;

[0019] 70 min to 80 min, the volume fraction of the mobile phase B is kept as 90% to 94%.

[0020] According to some embodiments of the present application, the test sample solution is prepared by the following steps: mixing the Poria cocos sample with methanol, then performing solid-liquid separation, and collecting the filtrate.

[0021] According to some embodiments of the present application, the mass-volume ratio of the Poria cocos sample and methanol is 1 g: 20 mL to 30 mL.

[0022] The sample obtained by the above method can be directly used for HPLC analysis, and the content of the target component is in the linear range.

[0023] The present application uses methanol as an extractant, and realizes efficient extraction of triterpenoid acid compounds in the Poria cocos sample.

[0024] According to some embodiments of the present application, the Poria cocos sample includes natural Poria cocos sample and artificially cultivated / fermented Poria cocos sample. According to some embodiments of the present application, the Poria cocos sample includes Poria cocos skin and its derivative products, Poria cocos rubrum and its derivative products, Poria cocos albus and its derivative products, Poria cocos shen and its derivative products. According to some embodiments of the present application, the Poria cocos sample includes the product obtained by fermentation of Poria cocos fungus.

[0025] According to some embodiments of the present application, the particle size of the Poria cocos sample is 40-100 mesh.

[0026] According to some embodiments of the present application, after mixing the Poria cocos sample with methanol, the mixture is subjected to ultrasonic treatment and then solid-liquid separation; wherein the power of the ultrasonic treatment is 200W-250W, the frequency of the ultrasonic treatment is 38kHz-42kHz, and the time of the ultrasonic treatment is 30min-40min.

[0027] According to some embodiments of the present application, the method of solid-liquid separation is microfiltration membrane filtration. The pore size of the microfiltration membrane is 0.2μm-0.25μm.

[0028] According to some embodiments of the present application, when the Poria cocos sample is Poria cocos skin and / or Poria cocos rubrum, the test sample solution is prepared by the following steps:

[0029] After crushing the Poria cocos sample and passing through a 40-100 mesh sieve, the sample is mixed with methanol, subjected to ultrasonic treatment, and then filtered by a 0.22μm microfiltration membrane.

[0030] According to some embodiments of the present application, when the Poria cocos sample is Poria cocos albus and / or Poria cocos shen, the test sample solution is prepared by the following steps:

[0031] After crushing the Poria cocos sample and passing through a 40-100 mesh sieve, the sample is mixed with methanol, subjected to ultrasonic treatment, and then filtered by a 0.22μm microfiltration membrane.

[0032] According to some embodiments of the present application, the mobile phase A is 0.2%-0.4% phosphoric acid aqueous solution.

[0033] According to some embodiments of the present application, the process of gradient elution is as follows:

[0034] 0min-20min, the volume fraction of mobile phase B is increased from 50% to 53%;

[0035] 20min-39min, the volume fraction of mobile phase B is kept at 53%;

[0036] 39min~43min, the volume fraction of mobile phase B is raised from 53% to 62%;

[0037] 43min~47min, the volume fraction of mobile phase B is kept at 62%;

[0038] 47min~70min, the volume fraction of mobile phase B is raised from 62% to 92%;

[0039] 70min~80min, the volume fraction of mobile phase B is kept at 92%.

[0040] The above gradient elution procedure is adopted to ensure the detection of multiple components in the test solution with different physicochemical properties and polarities and to ensure the separation degree between peaks, so as to ensure the accuracy of component content and comprehensiveness of components to be detected.

[0041] According to some embodiments of the present application, the detection condition of the liquid chromatography comprises using a variable wavelength procedure for detection.

[0042] 0min~35min, the detection wavelength is 240nm~245nm;

[0043] 35min~42min, the detection wavelength is 200nm~205nm;

[0044] 42min~65min, the detection wavelength is 240nm~245nm;

[0045] 65min~67min, the detection wavelength is 200nm~205nm;

[0046] 67min~76min, the detection wavelength is 240nm~245nm;

[0047] 76min~78min, the detection wavelength is 200nm~205nm;

[0048] 78min~80min, the detection wavelength is 240nm~245nm.

[0049] At the common point of the above intervals, it is the time for switching the wavelength. For example, at 35min, the detection wavelength is adjusted from 240nm~245nm to 200nm~205nm; and so on.

[0050] According to some preferred embodiments of the present application, the variable wavelength procedure is:

[0051] 0min~35min, the detection wavelength is 242nm;

[0052] 35min~42min, the detection wavelength is 203nm;

[0053] 42min~65min, detection wavelength is 242nm;

[0054] 65min~67min, detection wavelength is 203nm;

[0055] 67min~76min, detection wavelength is 242nm;

[0056] 76min~78min, detection wavelength is 203nm;

[0057] 78min~80min, detection wavelength is 242nm.

[0058] The wavelength detection program is adopted in the application, so that the wavelength of the instrument is set near the maximum absorption wavelength of the component to be detected, the signal sensitivity is improved, and interference is reduced; and multiple substances can be detected simultaneously, so that the spectrum is more complete and beautiful.

[0059] At the common point of the above intervals, it is the time of the conversion wavelength. For example, the detection wavelength is adjusted from 242nm to 203nm at 35min; and the like.

[0060] According to some embodiments of the application, the chromatographic column of the liquid chromatography is an octadecylsilane bonded silica chromatographic column; preferably, the chromatographic column is an Agilent XDB-C18 chromatographic column with a specification of 250mm*4.6mm, 5um.

[0061] The Agilent XDB-C18 chromatographic column is adopted in the application, so that the separation degree of the triterpenoid acid compound in the test sample solution can be improved.

[0062] According to some embodiments of the application, the column temperature of the liquid chromatography is 20~30℃.

[0063] According to some embodiments of the application, the flow rate of the mobile phase B of the liquid chromatography is 0.8mL / min~1.2mL / min.

[0064] According to some embodiments of the application, the injection volume of the liquid chromatography is 1~50um.

[0065] The column temperature, volume flow rate and the like will affect the peak time of each Pachymaran, and under the temperature and volume flow rate conditions of the application, the separation degree of the chromatographic peaks of each component to be detected on the HPLC chromatogram is good, and the baseline is stable.

[0066] According to some embodiments of the application, the content of the triterpenoid acid compound in the test sample solution of the Pachyman is calculated by using the external standard method or the quantitative analysis method of multiple components in one sample.

[0067] According to some embodiments of the present application, when the method is measured by the method of measuring multiple indicators, the method further comprises: detecting the control solution containing the internal reference by the liquid chromatography method, calculating the content of the internal reference in the Poria cocos test solution according to the detection result; and calculating the content of the triterpenoid acid compound in the Poria cocos test solution according to the relative correction factor;

[0068] The internal reference includes any one of 6α-hydroxypachymic acid C, pachymic acid B, dehydroxylopodic acid, lopodic acid, pachymic acid A, 3-epidehydroxylopodic acid, pachymic acid C, 3-epidehydroxylopodic acid, dehydroxylopodic acid, pachymic acid, pachymic acid, dehydroxylopodic acid, and isodonolic acid.

[0069] According to some embodiments of the present application, the content of 6α-hydroxypachymic acid C, pachymic acid B, dehydroxylopodic acid, lopodic acid, pachymic acid A, 3-epidehydroxylopodic acid, pachymic acid C, 3-epidehydroxylopodic acid, dehydroxylopodic acid, pachymic acid, dehydroxylopodic acid, and isodonolic acid in the test solution is calculated by taking the pachymic acid as the internal reference.

[0070] The calculation formula is as follows:

[0071] W m =W k ×A m / (A k ×f km );

[0072] Wherein, A k is the peak area of the internal reference; W k is the mass of the internal reference, in mg or μg; A m is the peak area of the target m, and W m is the mass of the target m, in mg or μg; f km is the correction factor.

[0073] According to some embodiments of the present application, the relative correction factor is calculated by the following method:

[0074] The mixed control solution of 6α-hydroxypachymic acid C, pachymic acid B, dehydroxylopodic acid, lopodic acid, pachymic acid A, 3-epidehydroxylopodic acid, pachymic acid C, 3-epidehydroxylopodic acid, dehydroxylopodic acid, pachymic acid, pachymic acid, dehydroxylopodic acid, and isodonolic acid is detected by the high performance liquid chromatography method, and the relative correction factors of 6α-hydroxypachymic acid C, pachymic acid B, dehydroxylopodic acid, lopodic acid, pachymic acid A, 3-epidehydroxylopodic acid, pachymic acid C, 3-epidehydroxylopodic acid, dehydroxylopodic acid, pachymic acid, pachymic acid, dehydroxylopodic acid, and isodonolic acid are calculated according to the detection results, and the calculation formula is as follows:

[0075] The relative correction factor f = f i / fs = (A i / W i ) / (A s / W s );

[0076] wherein A s represents the chromatographic peak area of the internal standard control, W s represents the mass concentration of the internal standard control; A i represents the chromatographic peak area of the test control, and W i represents the mass concentration of the test control.

[0077] According to some embodiments of the present application, the concentration of each control in the mixed control solution is 0.01-1 mg·mL -1 .

[0078] According to some embodiments of the present application, the solvent of the mixed control solution is methanol.

[0079] According to some embodiments of the present application, with pachymic acid as the internal standard, the relative correction factors of 6α-hydroxypachymic acid C, pachymic acid B, dehydrotrametenolic acid, trametenolic acid, pachymic acid A, 3-epidehydrotrametenolic acid, pachymic acid C, 3-epidehydrotrametenolic acid, dehydrotrametenolic acid, pachymanolic acid, dehydrotrachylobanolic acid, and trachylobanolic acid are 0.43-0.47, 1.30-1.41, 3.87-4.04, 0.91-0.96, 2.46-2.65, 0.87-0.92, 3.92-4.15, 0.36-0.40, 2.62-2.68, 1.49-1.59, 1.43-1.48, and 0.58-0.64, respectively. By controlling the relative correction factors within the above ranges, the present application realizes effective detection of 6α-hydroxypachymic acid C, pachymic acid B, dehydrotrametenolic acid, trametenolic acid, pachymic acid A, 3-epidehydrotrametenolic acid, pachymic acid C, 3-epidehydrotrametenolic acid, dehydrotrametenolic acid, pachymic acid, pachymanolic acid, dehydrotrachylobanolic acid, and trachylobanolic acid.

[0080] According to some embodiments of the present application, the relative retention times of 6α-hydroxypachymic acid C, pachymic acid B, dehydrotrametenolic acid, trametenolic acid, pachymic acid A, 3-epidehydrotrametenolic acid, pachymic acid C, 3-epidehydrotrametenolic acid, dehydrotrametenolic acid, pachymic acid, pachymanolic acid, dehydrotrachylobanolic acid, and trachylobanolic acid are 0.17-0.19, 0.41-0.43, 0.48-0.50, 0.54-0.56, 0.57-0.60, 0.71-0.72, 0.73-0.75, 0.77-0.78, 0.97-0.98, 1.00, 1.07-1.09, 1.12-1.14, and 1.15-1.18, respectively, when the liquid chromatography method is used to detect a pachyman test solution.

[0081] By controlling the relative retention time in the above range, the present application realizes effective separation and detection of 6α-hydroxypachymic acid C, pachymic acid B, dehydrotrametenolic acid, trametenolic acid, pachymic acid A, 3-epidehydrotrametenolic acid, pachymic acid C, 3-epidehydrotrametenolic acid, dehydrotrametenolic acid, pachymic acid, pachymic acid A, dehydrophomolic acid, phomolic acid.

[0082] In a second aspect of the present application, the above-mentioned determination method is applied in quality control of Poria cocos and its derivative products.

[0083] According to some embodiments of the present application, the derivative products include at least one of Poria cocos paste, Poria cocos biscuit and Poria cocos crisp.

[0084] Beneficial effects:

[0085] By adjusting the chromatographic column, gradient elution program and variable wavelength detection program, the present application realizes separation of 13 main Poria cocos triterpene acids including 6α-hydroxypachymic acid C, pachymic acid B, dehydrotrametenolic acid, trametenolic acid, pachymic acid A, 3-epidehydrotrametenolic acid, pachymic acid C, 3-epidehydrotrametenolic acid, dehydrotrametenolic acid, pachymic acid, pachymic acid A, dehydrophomolic acid and phomolic acid. Then, by using the QAMS technology, the relatively easy-to-obtain pachymic acid is selected as the internal reference, the relative correction factors of the internal reference pachymic acid and the remaining 12 kinds of triterpene acid compounds to be detected, i.e. 6α-hydroxypachymic acid C, pachymic acid B, dehydrotrametenolic acid, trametenolic acid, pachymic acid A, 3-epidehydrotrametenolic acid, pachymic acid C, 3-epidehydrotrametenolic acid, dehydrotrametenolic acid, pachymic acid A, dehydrophomolic acid and phomolic acid, are established, so that the contents of the other 12 kinds of triterpene acid components can be calculated by only determining the content of pachymic acid and using the correction factors. The method is sensitive, efficient and accurate, avoids multiple detection operations, saves detection cost and time, provides a reference for quality control of Poria cocos multi-components, and can more comprehensively reflect the quality of medicinal materials.

[0086] Other features and advantages of the present application will be set forth in the following description, and in part will become apparent to those skilled in the art from the description, or can be learned by practice of the present application.

[0087] Terms and definitions:

[0088] The "quantitative analysis by multiple components simultaneously" (QAMS) method herein refers to a method for determining the concentration of a plurality of components in a sample by using the internal function relationship and proportion relationship of the effective components of traditional Chinese medicines, and by determining only one component (a reference substance) to realize the simultaneous determination of a plurality of components (a reference substance is difficult to obtain or is difficult to supply).

[0089] The "external standard method" (ESM) herein refers to a method for preparing a reference sample by adding a certain amount of a reference substance (a reference substance) to a blank solvent according to a gradient, and performing sample treatment and detection on the reference sample and an unknown sample in parallel. BRIEF DESCRIPTION OF DRAWINGS

[0090] The above and / or additional aspects and advantages of the present application will become apparent and be readily understood from the following description, taken in conjunction with the following drawings, in which:

[0091] Figure 1 is an HPLC chromatogram of 13 mixed reference substances (A), Poria cocos peel (B), Radicis Poriae Cum Morindae Extract (C), Poria cocos (D) and Poria cocos (E) samples according to the method of Example 1 of the present application, wherein 1-13 are 1-6α-hydroxy-pachymic acid C; 2-pachymic acid B; 3-dehydro-tumulosic acid; 4-tumulosic acid; 5-pachymic acid A; 6-3-epidehydro-pachymic acid; 7-pachymic acid C; 8-3-epidehydro-tumulosic acid; 9-dehydro-pachymic acid; 10-pachymic acid; 11-pachymic acid; 12-dehydro-fargesic acid; and 13-fargesic acid, respectively.

[0092] Figure 2 is an HPLC chromatogram of 6 mixed reference substances (A), Poria cocos peel (B), Radicis Poriae Cum Morindae Extract (C), Poria cocos (D), Poria cocos (E) samples according to the method of Comparative Example 1 of the present application, wherein 1-6 are 1-pachymic acid B; 2-dehydro-tumulosic acid; 3-pachymic acid A; 4-dehydro-pachymic acid; 5-pachymic acid; and 6-pachymic acid, respectively. DETAILED DESCRIPTION

[0093] The concept and technical effects of the present application will be described below in conjunction with examples to make the purpose, features and effects of the present application clear, complete and fully understood. Obviously, the described examples are only some of the examples of the present application, but not all the examples. Based on the examples of the present application, other examples obtained by those skilled in the art without creative labor are within the protection scope of the present application.

[0094] The words "preferably," "more preferably," and the like serve to define parameters that are desirable, but not necessarily essential, to certain embodiments of the invention. In some instances, other embodiments can be preferred, in the same or other instances. Furthermore, the use of the words "preferably" or "more preferably" to describe one or more preferred embodiments does not imply that other embodiments are not useful, nor does it serve to exclude other embodiments from the scope of the invention.

[0095] When a numerical range is disclosed herein, the range is to be construed as continuous, and to include each and every value and sub-range within the range. Further, where a range is provided, unless otherwise specified, the range is intended to include both the minimum and maximum values, and every value between the minimum and maximum values. Also, it is specifically intended that the description set forth herein includes all available sub-ranges of each element being described. In other words, unless otherwise indicated, the description set forth herein is to be construed to include all sub-ranges possible for described elements.

[0096] Unless otherwise indicated, conventional conditions or manufacturer's recommended conditions were used in the examples. Unless otherwise indicated, the reagents or instruments used were conventional products available commercially.

[0097] The instruments used in the specific embodiments of the present invention are as follows:

[0098] Agilent 1200 high performance liquid chromatograph; Agilent 1260 high performance liquid chromatograph; EX224ZH electronic analytical balance (OHAUS Corporation); JS-40 ultrasonic cleaner (Changzhou Hongze Experimental Technology Co., Ltd.); Agilent 5TC-C18(2) chromatographic column (250 x 4.6 mm, 5 μm); Agilent XDB-C18 chromatographic column (250 x 4.6 mm, 5 μm).

[0099] The reagents used in the specific embodiments of the present invention are as follows:

[0100] Reference Pachymic acid B (batch number 140723, CAS number: 137551-39-4), Pachymic acid A (batch number 140314, CAS number: 137551-38-3), Dehydro Pachymic acid (batch number 140903, CAS number: 77012-31-8), Pachymic acid (batch number 140902, CAS number: 29220-16-4), Pachymic acid (batch number 140212, CAS number: 29070-92-6) were purchased from Chengdu Keluoma Biological Technology Co., Ltd., and the mass fraction was all ≥98%; Dehydro Tumulosic acid (batch number AF21032705, mass fraction ≥98%, CAS number: 6754-16-1) was purchased from Chengdu Efa Biological Technology Co., Ltd.; 6α-hydroxy Polyporenic acid C (CRN0225, CAS number: 24513-63-1), Tumulosic acid (CRN0325, CAS number: 508-24-7), 3-epidehydro Pachymic acid (CRN0245, CAS number: 168293-15-0), Polyporenic acid C (CRN0251, CAS number: 465-18-9), 3-epidehydro Tumulosic acid (CRN0307, CAS number: 167775-54-4), Echinocystic acid (CRN0398, CAS number: 560-66-7), Dehydro Echinocystic acid (CRN0486, CAS number: 6879-05-6) were all purchased from Hubei Caiyuan Biological Technology Co., Ltd., and the mass fraction was all ≥98%. After the method was established, only one reference (i.e. Pachymic acid) was needed to be purchased, and the contents of 13 kinds of Pachymic triterpene acids could be detected at the same time, which significantly reduced the consumption of reference, saved reagents and analysis time, and overcame the difficulty of inconsistent maximum absorption wavelength of Pachymic triterpene, which led to difficulty in peak under the same chromatographic conditions.

[0101] Acetonitrile was chromatographically pure, water was pure water from Nongfu Spring, and the rest of the reagents were analytical pure.

[0102] The Pachymic medicinal material samples were as shown in Table 1.

[0103] Table 1 Pachymic medicinal material sample source

[0104]

[0105]

[0106] Example 1

[0107] The present embodiment provides a method for determining the content of triterpene acid compounds in Pachymic, comprising the following steps:

[0108] S1, preparation of test solution:

[0109] Take Poria cocos sample powder 2.0 g (pass through 60 mesh sieve), accurately weigh, put into 100 mL dry conical flask with plug, add methanol 50 mL, tightly plug, weigh, ultrasonic (power 240 W, frequency 40 kHz) treatment for 30 min, cool, make up the weight loss with methanol, filter, Poria cocos skin and Radicis Poriae Cocos sample filtrate is filtered through 0.22 μm microporous filter membrane, take the filtrate, and Poria cocos skin and Radicis Poriae Cocos test solution is obtained.

[0110] Take Poria cocos sample powder 2.0 g (pass through 60 mesh sieve), accurately weigh, put into 100 mL dry conical flask with plug, add methanol 50 mL, tightly plug, weigh, ultrasonic (power 240 W, frequency 40 kHz) treatment for 30 min, cool, make up the weight loss with methanol, filter, Poria cocos sample filtrate is filtered through 0.22 μm microporous filter membrane, take the filtrate, and Poria cocos test solution is obtained.

[0111] S2, Preparation of mixed reference solution:

[0112] Accurately weigh a certain amount of each Poria cocos triterpene acid reference substance, dissolve in methanol to prepare a mixed reference solution of 6α-hydroxypolypodosic acid C, poriasol B, dehydrotrametenolic acid, trammolic acid, poriasol A, 3-epidehydrotrametenolic acid, polypodosic acid C, 3-epidehydrotrametenolic acid, dehydrotrametenolic acid, poria acid, pachymic acid, dehydrotrichosanic acid and trichosanic acid with concentrations of 0.0360, 0.0520, 0.0317, 0.1140, 0.0700, 0.0580, 0.0180, 0.1300, 0.0450, 0.1900, 0.1462, 0.1260, 0.0700 mg·mL respectively. Store in refrigerator at 4 ℃ for standby use. -1

[0113] S3, Liquid chromatography detection:

[0114] The Poria cocos skin test solution, white Poria cocos test solution, Radicis Poriae Cocos test solution, poriasol test solution prepared in step S1 and the mixed reference solution prepared in S2 are detected by liquid chromatography.

[0115] The chromatographic conditions are as follows:

[0116] Chromatograph: Agilent 1200 high performance liquid chromatograph;

[0117] Chromatographic column: Agilent XDB-C18 chromatographic column (250×4.6 mm, 5 μm);

[0118] Mobile phase A: 0.3% phosphoric acid aqueous solution;

[0119] Mobile phase B: acetonitrile; ​

[0120] Gradient elution procedure:

[0121] 0-20 min, the volume fraction of mobile phase B was raised from 50% to 53%;

[0122] 20-39 min, the volume fraction of mobile phase B was kept at 53%;

[0123] 39-43 min, the volume fraction of mobile phase B was raised from 53% to 62%;

[0124] 43-47 min, the volume fraction of mobile phase B was kept at 62%;

[0125] 47-70 min, the volume fraction of mobile phase B was raised from 62% to 92%;

[0126] 70-80 min, the volume fraction of mobile phase B was kept at 92%;

[0127] Variable wavelength procedure detection:

[0128] 0 min-35 min, the detection wavelength was 242 nm;

[0129] 35 min-42 min, the detection wavelength was 203 nm;

[0130] 42 min-65 min, the detection wavelength was 242 nm;

[0131] 65 min-67 min, the detection wavelength was 203 nm;

[0132] 67 min-76 min, the detection wavelength was 242 nm;

[0133] 76 min-78 min, the detection wavelength was 203 nm;

[0134] 78 min-80 min, the detection wavelength was 242 nm;

[0135] Injection volume: 10 μL;

[0136] Column temperature: 25 °C;

[0137] Volume flow rate 1.0 mL·min -1 .

[0138] The HPLC chromatograms of the Poria cocos periderm test sample solution, white Poria cocos test sample solution, red Poria cocos test sample solution, Poria cocos-like test sample solution and mixed reference substance solution in this example are shown in Figures 1-5, respectively. Figure 1. Peaks 1-13 are 1-6α-hydroxypachymic acid C; 2-pachymic acid B; 3-dehydrotramatic acid; 4-tramatic acid; 5-pachymic acid A; 6-3-epidehydrotramatic acid; 7-pachymic acid C; 8-3-epidehydrotramatic acid; 9-dehydropachymic acid; 10-pachymic acid; 11-pachymanic acid; 12-dehydroechinocystic acid; and 13-echinocystic acid, respectively. As can be seen, the detection method of the present embodiment has obtained a complete and beautiful liquid chromatogram with a stable baseline and symmetrical peak shape of each component, and has realized effective separation of the above-mentioned 13 kinds of triterpene acids in each test solution.

[0139] Example 2

[0140] The present embodiment is a methodology investigation of the determination method in Example 1.

[0141] (1) Linear relationship investigation

[0142] A mixed control solution of 6α-hydroxypachymic acid C, pachymic acid B, dehydrotramatic acid, tramatic acid, pachymic acid A, 3-epidehydrotramatic acid, pachymic acid C, 3-epidehydrotramatic acid, dehydropachymic acid, pachymic acid, pachymanic acid, dehydroechinocystic acid, and echinocystic acid was taken, and 1 μL, 10 μL, 20 μL, 30 μL, 40 μL, and 50 μL of the sample were respectively determined according to the chromatographic conditions in Example 1, and the chromatographic peak areas of each control were recorded. The regression equation and linear range of each control were calculated by taking the sample amount X (μg) as the abscissa and the peak area Y as the ordinate. The results showed that the linear relationship of the 13 components was good, as shown in Table 2.

[0143] Table 2 Linear regression equation of 13 kinds of pachyman triterpene acid components

[0144]

[0145] (2) Precision investigation

[0146] 10 μL of the same test solution (No. S1) (prepared according to the preparation method of the test solution of pachyman skin in Example 1) was precisely taken and continuously injected for 6 times, and the peak areas of the 13 kinds of triterpene acid compounds were recorded according to the chromatographic conditions in Example 1, and the RSD value was calculated. The RSD values of the peak areas of 6α-hydroxypachymic acid C, pachymic acid B, dehydrotramatic acid, tramatic acid, pachymic acid A, 3-epidehydrotramatic acid, pachymic acid C, 3-epidehydrotramatic acid, dehydropachymic acid, pachymic acid, pachymanic acid, dehydroechinocystic acid, and echinocystic acid were 1.16%, 1.96%, 0.66%, 2.76%, 0.50%, 0.76%, 1.62%, 2.14%, 1.24%, 2.43%, 0.60%, 0.72%, and 2.27%, respectively, and the RSD values were all less than 3%, indicating that the precision of the instrument was good.

[0147] (3) Repeatability

[0148] Precisely take the Poria cocos medicinal material numbered S1, prepare 6 test sample solutions in parallel according to the Poria cocos test sample solution preparation method in Example 1, and inject 10 μL of each sample into the instrument for analysis according to the chromatographic conditions in Example 1, and record the chromatogram. Take pachymic acid as the reference peak (S peak), and record the relative retention time and relative peak area of each chromatographic peak and the pachymic acid chromatographic peak. The RSDs of the relative retention times of 6α-hydroxypachymic acid C, pachymic acid B, dehydrotaerangic acid, taerangic acid, pachymic acid A, 3-epidehydrotaerangic acid, pachymic acid C, 3-epidehydrotaerangic acid, dehydropachymic acid, pachymic acid A, dehydroechinocystic acid, and echinocystic acid are 1.49%, 2.52%, 2.21%, 2.11%, 2.18%, 0.64%, 0.68%, 0.59%, 0.02%, 0.04%, 0.07%, and 0.17%, respectively; the RSDs of the relative peak areas are 1.89%, 2.34%, 2.51%, 2.42%, 2.36%, 2.41%, 1.37%, 2.68%, 1.07%, 1.60%, 1.83%, and 1.29%, respectively; the RSDs of the relative retention times and the relative peak areas are all <3%, indicating that the method has good repeatability.

[0149] (4) Stability

[0150] Precisely take 10 μL of the same test sample solution (numbered S1) (prepared according to the Poria cocos test sample solution preparation method in Example 1), and inject the sample into the instrument for analysis at 0, 2, 4, 8, 12, and 24 h after preparation, respectively, record the peak area at each determination time point, and calculate the RSD value. The results show that the relative average deviation RSDs of the peak areas of 6α-hydroxypachymic acid C, pachymic acid B, dehydrotaerangic acid, taerangic acid, pachymic acid A, 3-epidehydrotaerangic acid, pachymic acid C, 3-epidehydrotaerangic acid, dehydropachymic acid, pachymic acid, pachymic acid A, dehydroechinocystic acid, and echinocystic acid are all <3%, and are 2.06%, 2.45%, 2.24%, 2.96%, 1.92%, 1.97%, 1.06%, 0.65%, 2.44%, 1.07%, 1.93%, 1.57%, and 1.79%, respectively, indicating that the test sample solution is stable within 24 h.

[0151] (5) Investigation of sample addition recovery rate

[0152] A certain amount of sample with known content (numbered S1) was accurately weighed, and 6 sample solutions were prepared in parallel by halving the amount of sample solution prepared according to the preparation method of the Poria cocos test sample solution in Example 1, and a certain amount of 13 kinds of reference substances were added by mass ratio of 1:1 for injection analysis, and the average sample addition recovery rates of the 13 kinds of components were calculated. The average sample addition recovery rates of 6a-hydroxypolypodiopic acid C, pachymic acid B, dehydrotrametenolic acid, trammolic acid, pachymic acid A, 3-epidehydrotrametenolic acid, polypodiopic acid C, 3-epidehydrotrametenolic acid, dehydrotrametenolic acid, pachymic acid, pachymic acid, dehydrotrachylobinic acid and trachylobinic acid were 99.77%, 102.98%, 104.22%, 101.23%, 104.43%, 101.02%, 103.99%, 101.35%, 102.59%, 102.46%, 104.20%, 104.60% and 104.40% respectively, and the average sample addition recovery rates were between 95% and 105%; RSDs were 2.84%, 0.77%, 0.47%, 1.31%, 0.22%, 2.27%, 0.38%, 1.03%, 1.12%, 2.88%, 0.16%, 0.20%, 0.27% respectively, all less than 3.0%. It is shown that the accuracy of the method is good.

[0153] (6) Establishment of relative correction factor (f)

[0154] Under the HPLC chromatographic conditions established in Example 1, 1 μL, 5 μL, 10 μL, 20 μL, 25 μL, 30 μL, 40 μL and 50 μL of the mixed reference substance solution prepared in Example 1 were injected respectively, and the peak areas of the 13 kinds of triterpene acid components were recorded. Pachymic acid was used as an internal reference to calculate the f of the tested components and the internal reference. According to the formula f = f i / f s = (A i / W i ) / (A s / W s )(A i is the peak area of the tested component, W i is the content of the tested component, A s is the peak area of the internal reference, and W s is the content of the internal reference). The results are shown in Table 3. When pachymic acid is used as an internal reference, the RSDs of f of 6a-hydroxypolypodiopic acid C, pachymic acid B, dehydrotrametenolic acid, trammolic acid, pachymic acid A, 3-epidehydrotrametenolic acid, polypodiopic acid C, 3-epidehydrotrametenolic acid, dehydrotrametenolic acid, pachymic acid, dehydrotrachylobinic acid and trachylobinic acid are all less than 3%.

[0155] Table 3 f of 13 kinds of triterpene acids

[0156]

[0157]

[0158] (7) Evaluation of robustness of the relative correction factor

[0159] (a) Effect of different flow rates on f

[0160] The mixed reference solution prepared in Example 1 was taken to investigate the effect of different volume flow rates (0.8, 1.0, 1.2 mL·min -1 ) on the f values of the 13 triterpene acids in Poria cocos (except for the flow rate, other parameters not listed refer to the chromatographic detection conditions in Example 1). The f values of 6α-hydroxypachymic acid C, pachymic acid B, dehydroechinocystic acid, echinocystic acid, pachymic acid A, 3-epidehydroechinocystic acid, pachymic acid C, 3-epidehydroechinocystic acid, dehydropachymic acid, pachymic acid A, dehydroechinocystic acid, echinocystic acid were calculated to be 0.4687, 1.3398, 3.9187, 0.9341, 2.4628, 0.8559, 3.9991, 0.3499, 2.6669, 1.5023, 1.4369, and 0.6261, respectively. The f values of each component were well reproducible, with RSD values less than 3%, indicating that different volume flow rates had no significant effect on the f values of each component. The results are shown in Table 4.

[0161] Table 4 Effect of different flow rates on the f values of each component

[0162]

[0163]

[0164] (b) Effect of different column temperatures on f

[0165] The mixed reference solution prepared in Example 1 was taken to investigate the effect of different column temperatures (20, 25, 30℃) on the f values of the 13 triterpene acids in Poria cocos (except for the column temperature, other parameters not listed refer to the chromatographic detection conditions in Example 1). The results showed that the RSD values of the f values of 6α-hydroxypachymic acid C, pachymic acid B, dehydroechinocystic acid, echinocystic acid, pachymic acid A, 3-epidehydroechinocystic acid, pachymic acid C, 3-epidehydroechinocystic acid, dehydropachymic acid, pachymic acid A, dehydroechinocystic acid, and echinocystic acid were all less than 3%, indicating that the column temperature had no significant effect on the f values of each component. The results are shown in Table 5.

[0166] Table 5 Effect of different column temperatures on the f values of each component

[0167]

[0168]

[0169] (c) Effect of different instruments on f values

[0170] Take the mixed reference solution prepared in Example 1, and investigate the influence of different instruments (Wayeal 3100 and Agilent 1200) on the 13 kinds of f of Poria cocos triterpene acids (except for the instruments, other parameters not listed are referred to the chromatographic detection conditions in Example 1). The results show that the RSD of each component f is less than 5%, indicating that different instruments have no significant influence on the value of each component f, and the results are shown in Table 6.

[0171] Table 6 Influence of different instruments on the value of each component f

[0172]

[0173] (8) Chromatographic peak positioning parameter investigation

[0174] The reproducibility of two parameters, relative retention value or retention time difference, at different column temperatures (20, 25, 30℃) and different operators (A, B, C, D) was investigated respectively, and the parameter with smaller RSD value was taken as the peak positioning basis (relative retention value refers to the ratio of the retention time between each component to be tested and the internal reference (S). Retention time difference refers to the difference between the retention time of each component to be tested and the internal reference (S); other parameters not listed are referred to the chromatographic conditions in Example 1, and the sample solution is the mixed reference solution prepared in Example 1). The investigation results show that the retention time difference between the other 12 kinds of Poria cocos triterpene acid components (6α-hydroxypachymic acid C, pachymic acid B, dehydrothamolic acid, thamolic acid, pachymic acid A, 3-epidehydrothamolic acid, pachymic acid C, 3-epidehydrothamolic acid, dehydropachymic acid, pachymic acid A, dehydroechinocystic acid, echinocystic acid) and the internal reference pachymic acid fluctuates greatly, while the relative retention time value fluctuates less, and the RSD is less than 3% (Table 7). Therefore, the relative retention time value is used for chromatographic peak positioning.

[0175] Table 7 Relative retention time (t) and retention time difference (Δt)

[0176]

[0177]

[0178] (9) Comparison of QAMS method and ESM determination results:

[0179] Take each batch of Poria cocos medicinal materials purchased in Table 1, prepare the test sample solution according to the test sample solution preparation method in Example 1, and inject and determine according to the chromatographic conditions in Example 1, and record the peak area of 13 kinds of triterpene acid components. According to the standard curve, the contents of 13 triterpene acid components are calculated by external standard method (ESM); then taking pachymic acid as the reference, and according to the average value of each component to be tested and the internal reference f, the quantitative calculation formula of one measurement multiple evaluation method W m = W k × A m / (Ak Xf km (A k is the peak area of the internal reference; W k is the mass of the internal reference, in mg or μg; A m is the peak area of the target m, W m is the mass of the target m, in mg or μg; f km is a correction factor) to calculate the content of each triterpenoid acid component. The measured value of ESM is paired with the calculated value of QAMS for t-test, and the result shows P>0.05, indicating that there is no significant difference between the determination results of the two methods, suggesting that the one measurement multiple evaluation method established in the present application is more accurate and reliable, as shown in Table 8.

[0180] Table 8 Contents of 13 kinds of triterpenoid acid compounds in 11 batches of Poria cocos samples determined by ESM and QAMS

[0181]

[0182]

[0183]

[0184] Comparative Example 1

[0185] Comparative Example 1 provides a method for determining the content of triterpenoid acid compounds in Poria cocos, which is carried out according to Example 1, with the difference being that the chromatographic conditions for liquid chromatography detection are as follows:

[0186] Chromatographic column: Agilent 5TC-C18(2) (250x4.6mm, 5μm) chromatographic column;

[0187] Mobile phase A: 0.3% phosphoric acid aqueous solution by mass fraction;

[0188] Mobile phase B: acetonitrile;

[0189] Gradient elution program:

[0190] 0-18 min, the volume fraction of mobile phase B is increased from 50% to 60%;

[0191] 18-28 min, the volume fraction of mobile phase B is kept at 60%;

[0192] 28-58 min, the volume fraction of mobile phase B is increased from 60% to 92%;

[0193] 58-68 min, the volume fraction of mobile phase B is kept at 92%;

[0194] Variable wavelength program detection:

[0195] The detection wavelength was 242 nm, ranging from 0 min to 45.0 min.

[0196] The detection wavelength was 203 nm, lasting from 45.0 min to 46.8 min.

[0197] The detection time was 46.8 min to 48.8 min, with a detection wavelength of 242 nm.

[0198] The detection time was 48.8 min to 54.0 min, with a detection wavelength of 203 nm.

[0199] The detection wavelength was 242 nm, lasting from 54.0 min to 57.0 min.

[0200] The detection wavelength was 203 nm, ranging from 57.0 min to 64.0 min.

[0201] The detection wavelength was 242nm, and the time ranged from 64.0 min to 68.0 min.

[0202] Injection volume: 10 μL;

[0203] Column temperature: 25℃;

[0204] Volumetric flow rate: 1.0 mL·min -1 .

[0205] The HPLC chromatograms of the Poria cocos peel test solution, white Poria cocos test solution, red Poria cocos test solution, Poria cocos sclerotium test solution, and mixed reference solution in Comparative Example 1 are shown in [Figure 1]. Figure 2 .Depend on Figure 2 It can be seen that the method in Comparative Example 1 is not applicable to the separation of the 13 triterpenic acid components in this invention, and cannot achieve complete elution of all 13 triterpenic acid components.

[0206] The inventors discovered that, under the conditions of Comparative Example 1, tomolic acid and pachymonic acid A, 3-epidehydropachymonic acid and pachymonic acid C overlapped, and could not be completely separated even after continuously adjusting the elution gradient. The inventors then adjusted the chromatographic column, examining multiple columns and finding peak overlap issues in all of them. Finally, they determined that the Agilent XDB-C18 column (250×4.6mm, 5μm) was the optimal column for completely separating the 13 peaks. Different chromatographic columns result in different peak times for each component; therefore, the wavelength variation program needs to be adaptively adjusted based on the column selection.

[0207] In general, the present application establishes a variable wavelength-gradient elution HPLC method for 13 accurate and reliable triterpene acids (6alpha-hydroxypolypodosic acid C, pachymic acid B, dehydrodammarenolic acid, dammarenolic acid, pachymic acid A, 3-epidehydrodammarenolic acid, polypodosic acid C, 3-epidehydrodammarenolic acid, dehydrodammarenolic acid, pachymic acid, pachymic acid, dehydroechinocystic acid, echinocystic acid) in Poria cocos; on this basis, pachymic acid, a representative triterpene acid component of Poria cocos, which is relatively easy to obtain and stable in properties, is selected as an internal reference, and the relative correction factor of the internal reference and each component to be measured is established; the contents of 13 triterpene acids in 11 batches of commercially available medicinal parts (Poria cocos skin, Poria cocos rubra, Poria cocos alba, Poria cocos) of Poria cocos are determined, and the accuracy of the QAMS determination results is verified by the traditional ESM method determination results, which provides a reference for the comprehensive quality evaluation of Poria cocos medicinal materials and saves the detection components and time.

[0208] The embodiments of the present application are described in detail above in combination with the drawings, but the present application is not limited to the above embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the purpose of the present application. In addition, the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

Claims

1. A method for determining the content of triterpenoid acids in Poria cocos, characterized in that: The test solution of the Poria cocos is detected by using liquid chromatography; ​ The triterpenoid acid compounds include 6α-hydroxypachymic acid C, pachymic acid B, dehydroxydehydrotirucallenic acid, dehydroxytumulosic acid, pachymic acid A, 3-epidehydroxydehydrotirucallenic acid, pachymic acid C, 3-epidehydroxydehydrotirucallenic acid, dehydroxydehydrotirucallenic acid, pachymic acid, pachymic acid A, dehydroxydehydrotirucallenic acid, and dehydroxydehydrotirucallenic acid. The detection conditions of the liquid chromatography include: The mobile phase A is 0.05% to 0.4% phosphoric acid aqueous solution; and the mobile phase B is acetonitrile. The elution mode is gradient elution, and the process of the gradient elution is as follows: 0 min to 20 min, the volume fraction of the mobile phase B is increased from 50% to 53%; 20 min to 39 min, the volume fraction of the mobile phase B is kept at 53%; 39 min to 43 min, the volume fraction of the mobile phase B is increased from 53% to 62%; 43 min to 47 min, the volume fraction of the mobile phase B is kept at 62%; 47 min to 70 min, the volume fraction of the mobile phase B is increased from 62% to 92%; 70 min to 80 min, the volume fraction of the mobile phase B is kept at 92%; The detection conditions of the liquid chromatography include a variable wavelength program: 0 min to 35 min, the detection wavelength is 240 nm to 245 nm; 35 min to 42 min, the detection wavelength is 200 nm to 205 nm; 42 min to 65 min, the detection wavelength is 240 nm to 245 nm; 65 min to 67 min, the detection wavelength is 200 nm to 205 nm; 67 min to 76 min, the detection wavelength is 240 nm to 245 nm; 76 min to 78 min, the detection wavelength is 200 nm to 205 nm; 78 min to 80 min, the detection wavelength is 240 nm to 245 nm; The chromatographic column is an Agilent XDB-C18 chromatographic column with a specification of 250 mm*4.6 mm*5 μm. The test solution is prepared by the following steps: mixing the Poria cocos sample with methanol, performing solid-liquid separation, and collecting the filtrate.

2. The assay method according to claim 1, characterized by The detection conditions of the liquid chromatography further include: The column temperature is 20°C to 30°C; And / or, the injection volume is 1 μL to 50 μL.

3. The assay method according to claim 1, characterized by, The mass-volume ratio of the Poria cocos sample and methanol is 1 g:20 mL to 30 mL.

4. The assay method according to claim 1, characterized by, The content of the triterpenoid acid compounds in the test solution of the Poria cocos is calculated by using an external standard method or a quantitative analysis method.

5. The assay method according to claim 4, characterized in that, When the quantitative analysis method is used, the control solution containing an internal reference is detected by using the liquid chromatography, the content of the internal reference in the test solution of the Poria cocos is calculated according to the detection result, and the content of the triterpenoid acid compounds in the test solution of the Poria cocos is calculated according to the relative correction factor. The internal reference includes any one of 6α-hydroxypachymic acid C, pachymic acid B, dehydroxydehydrotirucallenic acid, dehydroxytumulosic acid, pachymic acid A, 3-epidehydroxydehydrotirucallenic acid, pachymic acid C, 3-epidehydroxydehydrotirucallenic acid, dehydroxydehydrotirucallenic acid, pachymic acid, pachymic acid A, dehydroxydehydrotirucallenic acid, and dehydroxydehydrotirucallenic acid.

6. The assay method according to claim 5, characterized in that, The relative correction factors of 6α-hydroxypachymic acid C, pachymic acid B, dehydro-tumulosic acid, tumulosic acid, pachymic acid A, 3-epidehydro-pachymic acid, pachymic acid C, 3-epidehydro-tumulosic acid, dehydro-pachymic acid, pachymic acid, pachymic acid A, dehydro-fargesic acid, fargesic acid are 0.43-0.47, 1.30-1.41, 3.87-4.04, 0.91-0.96, 2.46-2.65, 0.87-0.92, 3.92-4.15, 0.36-0.40, 2.62-2.68, 1.49-1.59, 1.43-1.48 and 0.58-0.64, respectively, with pachymic acid as the internal reference.

7. The assay method according to claim 5, characterized by The relative retention times of 6α-hydroxypachymic acid C, pachymic acid B, dehydro-tumulosic acid, tumulosic acid, pachymic acid A, 3-epidehydro-pachymic acid, pachymic acid C, 3-epidehydro-tumulosic acid, dehydro-pachymic acid, pachymic acid, pachymic acid A, dehydro-fargesic acid, fargesic acid are 0.17-0.19, 0.41-0.43, 0.48-0.50, 0.54-0.56, 0.57-0.60, 0.71-0.72, 0.73-0.75, 0.77-0.78, 0.97-0.98, 1.00, 1.07-1.09, 1.12-1.14, 1.15-1.18, respectively.

8. Use of the assay method according to any one of claims 1 to 7 in the quality control of Poria cocos.

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