Method for determining content of triterpenic acid compounds in poria cocos and application

Abstract

The invention relates to a method for determining the content of triterpenic acid compounds in poria cocos and application, and belongs to the field of quality analysis and control of traditional Chinese medicines. The determination method comprises the following steps: detecting a poria cocos test sample solution by adopting a high performance liquid chromatography (HPLC) method, and detecting the poria cocos test sample solution by adopting a high performance liquid chromatography (HPLC) method, wherein the triterpenic acid compounds comprise 6 alpha-hydroxy polyporus acid C, poria cocos neoic acid B, dehydrotumuloic acid, tumuloic acid, poria cocos neoic acid A, 3-epidehydrotumuloic acid, polyporus acid C, 3-epidehydrotumuloic acid, dehydrotumuloic acid, pachymic acid, Songling neoic acid, dehydroorifice acid and orifice acid. According to the present invention, the appropriate HPLC analysis method of the poria cocos triterpenic acid compound is established, the pachymic acid is selected as the internal reference substance, and the quantitative analysis of multiple components by single marker for simultaneously determining the content of 13 poria cocos triterpenic acids in the poria cocos is established, such that the detection cost and the detection time are saved, and the reference is provided for the quality control of the poria cocos.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine quality analysis and control, and specifically relates to a method for determining the content of triterpene acid compounds in Poria cocos and an application thereof. Background Art

[0002] Poria cocos is the dried sclerotium of the Polyporaceae fungus Wolfiporia hoelen (Fr.) YCDai & V.Papp. It is mainly produced in Sichuan, Anhui, Yunnan, Hunan, Hubei and other places. It is mostly dug in autumn. It has the effects of diuresis, invigorating the spleen and stomach, and calming the mind. It is clinically used to treat symptoms such as edema, oliguria, phlegm and fluid, palpitations, spleen deficiency, poor appetite, loose stools and diarrhea. The main active ingredients of Poria cocos are Poria cocos triterpenoids and Poria cocos polysaccharide compounds. At present, more than 100 triterpenoid compounds have been isolated from Poria cocos, and Poria cocos triterpenoids have also been proven to have pharmacological effects such as invigorating the spleen, diuresis, anti-inflammatory, and anti-tumor.

[0003] The multi-component and multi-functional characteristics of traditional Chinese medicine 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 traditional Chinese medicine components, the determination of a single component cannot fully reflect the quality of medicinal materials; therefore, the content determination method based on multi-component and multi-index analysis has become the currently generally recognized quality evaluation method. The related art discloses a method for determining the content of triterpenic acid compounds in Poria cocos and its application, which is detected by liquid chromatography, and pachymic acid is selected as a reference, and a one-test-multiple-evaluation method is established for simultaneously determining the content of pachymic acid B, dehydrogenated turmeric acid, pachymic acid A, dehydrogenated pachymic acid, pachymic acid, and pine ...

[0004] Therefore, it is necessary to establish a method for simultaneously determining the contents of more types of Poria triterpenoid acids in Poria cocos, which can be used to detect the main Poria triterpenoid components in Poria cocos and provide a basis for the quality control of Poria cocos medicinal materials. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention provides a method for determining the content of triterpenic acid compounds in Poria cocos, which achieves the simultaneous determination of 13 major triterpenic acid compounds in Poria cocos, and has low determination cost and high determination efficiency.

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

[0007] One aspect of the present invention provides a method for determining the content of triterpenoid acid compounds in Poria cocos, comprising detecting a Poria cocos test solution by liquid chromatography;

[0008] Among them, the triterpene acid compounds include 6α-hydroxypolyporic acid C, tuckahoe acid B, dehydrotumoric acid, tumoric acid, tuckahoe acid A, 3-epi-dehydrotuckahoe acid, polyporic acid C, 3-epi-dehydrotumoric acid, dehydrotuckahoe acid, tuckahoe acid, pine acid, dehydroporous acid, porous acid;

[0009] The detection conditions of the liquid chromatography method include:

[0010] Mobile phase A: 0.05% to 0.4% phosphoric acid aqueous solution;

[0011] Mobile phase B: acetonitrile;

[0012] Elution method: gradient elution;

[0013] The process of gradient elution is:

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

[0015] From 20 to 39 minutes, the volume fraction of mobile phase B was maintained at 52% to 55%;

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

[0017] From 43 to 47 minutes, the volume fraction of mobile phase B was maintained at 60% to 64%;

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

[0019] From 70 min to 80 min, the volume fraction of mobile phase B was maintained at 90% to 94%.

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

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

[0022] The samples treated by the above method can be directly used for HPLC analysis, and the contents of target components are all within the linear range.

[0023] The invention adopts methanol as an extractant to achieve efficient extraction of triterpene acid compounds in a Poria cocos sample.

[0024] According to some embodiments of the present invention, the Poria cocos sample includes a natural Poria cocos sample and a Poria cocos sample obtained by artificial cultivation / fermentation. According to some embodiments of the present invention, the Poria cocos sample includes Poria cocos peel and its derivatives, red Poria cocos and its derivatives, white Poria cocos and its derivatives, Poria cocos and its derivatives. According to some embodiments of the present invention, the Poria cocos sample includes a product obtained by fermentation and culture of Poria cocos.

[0025] According to some embodiments of the present invention, the particle size of the Poria cocos sample is sieved through a 40-100 mesh sieve.

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

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

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

[0029] The Poria cocos sample is crushed and passed through a 40-100 mesh sieve, and then mixed with methanol after being sieved. After ultrasonic treatment, the mixture is filtered through a 0.22 μm microporous filter membrane to obtain the product.

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

[0031] The Poria cocos sample is crushed and passed through a 40-100 mesh sieve, and then mixed with methanol after being sieved. After ultrasonic treatment, it is dissolved again after rotary evaporation, and filtered through a 0.22 μm microporous filter membrane to obtain the product.

[0032] According to some embodiments of the present invention, the mobile phase A is a phosphoric acid aqueous solution with a mass fraction of 0.2% to 0.4%.

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

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

[0035] From 20 min to 39 min, the volume fraction of mobile phase B was maintained at 53%;

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

[0037] From 43 to 47 min, the volume fraction of mobile phase B was maintained at 62%;

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

[0039] From 70 min to 80 min, the volume fraction of mobile phase B was maintained at 92%.

[0040] The above-mentioned gradient elution procedure is adopted to ensure that multiple components with large differences in physical and chemical properties and polarity in the test solution are detected and the separation between peaks is guaranteed, so as to ensure the accuracy of the component content and the comprehensiveness of the components to be detected.

[0041] According to some embodiments of the present invention, the detection conditions of the liquid chromatography method include detection using a variable wavelength program:

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

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

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

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

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

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

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

[0049] The common point in the above range is the moment to switch the wavelength. For example, at 35 minutes, the detection wavelength is adjusted from 240nm to 245nm to 200nm to 205nm; and so on.

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

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

[0052] 35min~42min, 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 present invention adopts a variable wavelength detection program to set the instrument wavelength near the maximum absorption wavelength of the component to be detected, thereby improving signal sensitivity and reducing interference; it can also detect multiple substances at the same time, making the spectrum more complete and beautiful.

[0059] The common point in the above interval is the moment to switch the wavelength. For example, at 35 minutes, the detection wavelength is adjusted from 242nm to 203nm; and so on.

[0060] According to some embodiments of the present invention, the chromatographic column of the liquid chromatography method is an octadecylsilane bonded silica gel chromatographic column; preferably, the chromatographic column is an Agilent XDB-C18 chromatographic column with a specification of 250 mm×4.6 mm, 5 μm.

[0061] The present invention adopts an Agilent XDB-C18 chromatographic column, which can improve the separation degree of triterpenic acid compounds in a test solution.

[0062] According to some embodiments of the present invention, the column temperature of the liquid chromatography is 20°C to 30°C.

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

[0064] According to some embodiments of the present invention, the injection volume of the liquid chromatography method is 1 μL to 50 μL.

[0065] Column temperature, volume flow rate, etc. will affect the elution time of each Poria triterpene acid. The HPLC chromatogram obtained under the temperature and volume flow conditions of the present invention has good chromatographic peak separation of each component to be measured and a stable baseline.

[0066] According to some embodiments of the present invention, the content of triterpenoid acid compounds in the Poria cocos test solution is measured by an external standard method or a one-measurement-multiple-evaluation method.

[0067] According to some embodiments of the present invention, when the one-measurement-multiple-evaluation method is used for measurement, it also includes: using the liquid chromatography method to detect the reference solution containing the internal reference substance, and calculating the content of the internal reference substance in the Poria cocos test solution according to the detection result; calculating the content of triterpene acid compounds in the Poria cocos test solution according to the relative correction factor;

[0068] Among them, the internal reference includes any one of 6α-hydroxypolyporus acid C, tuckahoe acid B, dehydrotumoric acid, tumoric acid, tuckahoe acid A, 3-epi-dehydrotuckahoe acid, tuckahoe acid C, 3-epi-dehydrotumoric acid, dehydrotuckahoe acid, tuckahoe acid, pine ling acid, dehydroporous acid, and porous acid.

[0069] According to some embodiments of the present invention, the pachymic acid is used as an internal reference to calculate the contents of 6α-hydroxypolysaccharide C, pachymic acid B, dehydroturmoic acid, turmoic acid, pachymic acid A, 3-epi-dehydropachymic acid, pachymic acid C, 3-epi-dehydroturmoic acid, dehydropachymic acid, pine ling acid, dehydroporous acid, and porous acid in the test solution;

[0070] The calculation formula is as follows:

[0071] W m =W k ×A m / (A k ×f km );

[0072] Among them, A k is the peak area of ​​the internal reference; W k is the mass of the internal reference substance, in mg or μg; A m is the peak area of ​​target m, W m is the mass of the target object m, in mg or μg; f km is the correction factor.

[0073] According to some embodiments of the present invention, the relative correction factor is calculated in the following manner:

[0074] The high performance liquid chromatography method is used to detect the mixed reference solution of 6α-hydroxypolyporic acid C, Poria cocos acid B, dehydrothiazol acid, thiazol acid, Poria cocos acid A, 3-epi-dehydro-pachymic acid, Polyporic acid C, 3-epi-dehydrothiazol acid, dehydro-pachymic acid, Poria cocos acid, Pinus polyporus acid, dehydro-tetrasodium acid, and tetrasodium acid, and the relative correction factors of 6α-hydroxypolyporic acid C, Poria cocos acid B, dehydrothiazol acid, thiazol acid, Poria cocos acid A, 3-epi-dehydro-pachymic acid, Polyporic acid C, 3-epi-dehydrothiazol acid, dehydro-pachymic acid, Pinus polyporus acid, dehydro-tetrasodium acid, and tetrasodium acid are calculated according to the test results, and the calculation formula is as follows;

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

[0076] Among them, A s Represents the chromatographic peak area of ​​the internal reference substance, W s Represents the mass concentration of the internal reference substance; A i Represents the chromatographic peak area of ​​the analyte reference substance, W i Represents the mass concentration of the test substance.

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

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

[0079] According to some embodiments of the present invention, with pachymic acid as an internal reference, the relative correction factors of 6α-hydroxypolyporic acid C, pachymic acid B, dehydrotumoric acid, tumoric acid, pachymic acid A, 3-epi-dehydropachymic acid, pachymic acid C, 3-epi-dehydrotumoric acid, dehydropachymic acid, pine ling acid, dehydroporous acid, and pine ling 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. The present invention realizes effective detection of 6α-hydroxypolyporic acid C, tuckahoe acid B, dehydrotumoric acid, tumoric acid, tuckahoe acid A, 3-epi-dehydrotuckahoe acid, tuckahoe acid C, 3-epi-dehydrotumoric acid, dehydrotuckahoe acid, tuckahoe acid, tuckahoe acid, dehydroporous acid, and porous acid by controlling the relative correction factor within the above range.

[0080] According to some embodiments of the present invention, the liquid chromatography method is used to detect the Poria test solution, and the relative retention times of 6α-hydroxypolyporic acid C, Poria acid B, dehydrotumoric acid, tumoric acid, Poria acid A, 3-epi-dehydropolyporic acid, Poria acid C, 3-epi-dehydrotumoric acid, dehydroporic acid, Poria acid, pine acid, dehydroporous acid, and pine 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.

[0081] The present invention realizes the effective separation and detection of 6α-hydroxypolyporic acid C, tuckahoe acid B, dehydrotumoric acid, tumoric acid, tuckahoe acid A, 3-epi-dehydrotuckahoe acid, polyporic acid C, 3-epi-dehydrotumoric acid, dehydrotuckahoe acid, tuckahoe acid, tuckahoe acid, dehydroporous acid, and porous acid by controlling the relative retention time within the above range.

[0082] The second aspect of the present invention provides the application of the above-mentioned determination method in the quality control of Poria cocos and its derivative products.

[0083] According to some embodiments of the present invention, the derivative product includes at least one of Poria cocos paste, Poria cocos biscuits, and Poria cocos crisps.

[0084] Beneficial effects:

[0085] The present invention realizes the separation of 13 main tuckahoe triterpene acids including 6α-hydroxypolyporic acid C, tuckahoe acid B, dehydrotumoric acid, tumoric acid, tuckahoe acid A, 3-epi-dehydrotuckahoe acid, tuckahoe acid C, 3-epi-dehydrotumoric acid, dehydrotuckahoe acid, tuckahoe acid, tuckahoe acid, dehydrotetrasomic acid, and tetrasomic acid by adjusting the chromatographic column, the gradient elution program, and the variable wavelength detection program. Then, the one-test-multiple-evaluation (QAMS) technology was used to select the relatively easy-to-obtain pachymic acid as the internal reference, and the relative correction factors of the internal reference pachymic acid and the other 12 triterpenic acid compounds to be tested, 6α-hydroxypolysaccharide C, pachymic acid B, dehydroturmoic acid, turmoic acid, pachymic acid A, 3-epidehydropachymic acid, pachymic acid C, 3-epidehydroturmoic acid, dehydropachymic acid, pine ling acid, dehydroporous acid, and porous acid, were established. Only the content of pachymic acid was measured, and the correction factors were used to calculate the content of the other 12 triterpenic acid components. This method is sensitive, efficient, and accurate, avoids multiple detection operations, saves detection costs and time, provides a reference for the quality control of multiple components of Poria cocos, and can more comprehensively reflect the quality of medicinal materials.

[0086] Other features and advantages of the present invention will be set forth in the description which follows, and in part will be apparent from the description, or may be learned by practice of the present invention.

[0087] Terms and Definitions:

[0088] The "Quantitative Analysis by Multiple Components Simultaneously" (QAMS) method in this article refers to the use of the intrinsic functional relationship and proportional relationship of the effective components of traditional Chinese medicine to measure only one component (the reference material is available) to achieve the simultaneous determination of multiple components (the reference material is difficult to obtain or supply). Standard samples of different concentrations are injected, and a standard curve is drawn with the peak area as the value, so as to deduce the quantitative method of the concentration of the measured component in the unknown sample.

[0089] In this article, "external standard method" (ESM) refers to adding a certain amount of standard substance (reference substance) to a blank solvent according to a gradient to prepare a control sample, and performing sample processing and detection in parallel with the unknown sample. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0091] Figure 1 It is the HPLC chromatogram of 13 kinds of mixed reference substances (A), Poria peel (B), red Poria (C), white Poria (D) and Poria sample (E) under the method of Example 1 of the present invention, wherein 1 to 13 are: 1-6α-hydroxypolysaccharide C; 2-polysaccharide B; 3-dehydrotrisaccharide; 4-trisaccharide; 5-polysaccharide A; 6-3-epi-dehydropolysaccharide; 7-polysaccharide C; 8-3-epi-dehydrotrisaccharide; 9-dehydropolysaccharide; 10-polysaccharide; 11-pinene acid; 12-dehydroporous acid; 13-porous acid;

[0092] Figure 2 It is an HPLC chromatogram of 6 mixed reference substances (A), Poria peel (B), red Poria (C), white Poria (D), and Poria (E) samples under the method of comparative example 1 of the present invention, wherein 1 to 6 are respectively: 1-Poria acid B; 2-dehydropyruvic acid; 3-Poria acid A; 4-dehydropyruvic acid; 5-Poria acid; 6-pyruvic acid. DETAILED DESCRIPTION

[0093] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0094] The words "preferably", "more preferably", etc. in the present invention refer to embodiments of the present invention that may provide certain beneficial effects in certain circumstances. However, other embodiments may also be preferred under the same circumstances or other circumstances. In addition, the description of one or more preferred embodiments does not imply that other embodiments are not applicable, nor is it intended to exclude other embodiments from the scope of the present invention.

[0095] When a numerical range is disclosed herein, the above range is considered to be continuous and includes the minimum and maximum values ​​of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0096] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0097] The instruments used in the specific implementation 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); JS-40 ultrasonic cleaner (Changzhou Hongze Experimental Technology Co., Ltd.); Agilent 5TC-C18 (2) chromatographic column (250×4.6mm, 5μm); Agilent XDB-C18 chromatographic column (250×4.6mm, 5μm).

[0099] The reagents selected in the specific implementation manner of the present invention are as follows:

[0100] Reference substances: tuckahoe acid B (batch number 140723, CAS number: 137551-39-4), tuckahoe acid A (batch number 140314, CAS number: 137551-38-3), dehydrotuckahoe acid (batch number 140903, CAS number: 77012-31-8), pine ling acid (batch number 140902, CAS number: 29220-16-4), tuckahoe acid (batch number 140212, CAS number: 29070-92-6) were purchased from Chengdu Kroma Biotechnology Co., Ltd., with mass fractions of ≥ 98%; dehydrotuckahoe acid (batch number AF21032705, mass fraction ≥ 98%, CAS number: 6754-16-1)) was purchased from Chengdu Aifa Biotechnology Co., Ltd.; 6α-Hydroxypolycormic acid C (CRN0225, CAS No.: 24513-63-1), tachymosic acid (CRN0325, CAS No.: 508-24-7), 3-epi-dehydropachymosic acid (CRN0245, CAS No.: 168293-15-0), polycormic acid C (CRN0251, CAS No.: 465-18-9), 3-epi-dehydrotachymosic acid (CRN0307, ​​CAS No.: 167775-54-4), scutellaria baicalensis (CRN0398, CAS No.: 560-66-7), and dehydroscutellaria baicalensis (CRN0486, CAS No.: 6879-05-6) were all purchased from Hubei Cuiyuan Biotechnology Co., Ltd., with mass fractions of all ≥ 98%. After the establishment of this method, only one reference substance (i.e., Pachymaranth acid) needs to be purchased to simultaneously detect the contents of 13 types of Pachymaranth triterpenoid acids, which significantly reduces the consumption of reference substances, saves reagents and analysis time, and overcomes the problem of difficulty in peaking under the same chromatographic conditions due to inconsistent maximum absorption wavelengths of Pachymaranth triterpenoids.

[0101] Acetonitrile was of chromatographic grade, water was Yibao purified water, and the other reagents were of analytical grade.

[0102] The sources of Poria cocos samples are shown in Table 1.

[0103] Table 1 Sources of Poria cocos medicinal material samples

[0104]

[0105]

[0106] Example 1

[0107] This embodiment provides a method for determining the content of triterpenoid compounds in Poria cocos, comprising the following steps:

[0108] S1. Preparation of test solution:

[0109] Take 2.0 g of Poria cocos sample powder (pass through a 60-mesh sieve), accurately weigh it, place it in a 100-mL dry stoppered conical flask, add 50 mL of methanol, stopper it, weigh it, treat it with ultrasound (power 240 W, frequency 40 kHz) for 30 min, cool it, make up the lost mass with methanol, filter it, filter the filtrate of Poria cocos peel and red Poria cocos samples through a 0.22 μm microporous filter membrane, and take the subsequent filtrate to obtain the Poria cocos peel and red Poria cocos test sample solutions.

[0110] Take 2.0g of Poria sample powder (pass through a 60-mesh sieve), accurately weigh it, place it in a 100mL dry stoppered conical flask, add 50mL of methanol, stopper it, weigh it, treat it with ultrasound (power 240W, frequency 40kHz) for 30min, cool it, make up the lost mass with methanol, filter it, and first rotary evaporate the filtrate of the white Poria and Poria sample to dryness and then make up the volume to 2mL with methanol, and then filter it through a 0.22μm microporous filter membrane, and take the filtrate to obtain the white Poria and Poria test sample solution.

[0111] S2. Preparation of mixed reference solution:

[0112] Accurately weigh an appropriate amount of each Poria triterpenoid acid reference substance, add methanol to dissolve and prepare 6α-hydroxypolysaccharide C, Poria acid B, dehydrotrisaccharide, trisaccharide, Poria acid A, 3-epi-dehydropolysaccharide, Poria acid C, 3-epi-dehydrotrisaccharide, dehydroporia acid, Poria acid, pine acid, dehydroporous acid and porous 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 and 0.0700 mg·mL -1 The mixed reference solution was stored in a refrigerator at 4°C for future use.

[0113] S3, Liquid chromatography detection:

[0114] Liquid chromatography is used to detect the Poria peel test solution, white Poria test solution, red Poria test solution, Poria test solution prepared in step S1 and the mixed reference solution prepared in S2.

[0115] The chromatographic conditions were as follows:

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

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

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

[0119] Mobile phase B: acetonitrile;

[0120] Gradient elution program:

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

[0122] From 20 to 39 min, the volume fraction of mobile phase B was maintained at 53%;

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

[0124] From 43 to 47 min, the volume fraction of mobile phase B was maintained at 62%;

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

[0126] From 70 to 80 min, the volume fraction of mobile phase B was maintained at 92%;

[0127] Variable wavelength program detection:

[0128] 0min~35min, detection wavelength is 242nm;

[0129] 35min~42min, detection wavelength is 203nm;

[0130] 42min~65min, detection wavelength is 242nm;

[0131] 65min~67min, detection wavelength is 203nm;

[0132] 67min~76min, detection wavelength is 242nm;

[0133] 76min~78min, detection wavelength is 203nm;

[0134] 78min~80min, detection wavelength is 242nm;

[0135] Injection volume: 10 μL;

[0136] Column temperature: 25°C;

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

[0138] The HPLC spectra of the Poria peel test solution, white Poria test solution, red Poria test solution, Poria test solution and mixed reference solution in this example are shown in Figure 1. Among them, peaks 1 to 13 are: 1-6α-hydroxypolyporic acid C; 2-poria acid B; 3-dehydrotriterpenic acid; 4-triterpenic acid; 5-poria acid A; 6-3-epi-dehydropolyporic acid; 7-poria acid C; 8-3-epi-dehydrotriterpenic acid; 9-dehydropolyporic acid; 10-poria acid; 11-pinene acid; 12-dehydrotetraterpenic acid; 13-tetraterpenic acid. It can be seen that by adopting the detection method of this embodiment, a complete and beautiful liquid chromatogram with a stable spectrum baseline and symmetrical peak shapes of each component is obtained, and the above 13 triterpenic acids in each test solution are effectively separated.

[0139] Example 2

[0140] This example is a methodological investigation of the determination method in Example 1.

[0141] (1) Linear relationship investigation

[0142] Take 6α-hydroxypolysaccharide C, tuckahoe acid B, dehydrogenated turmeric acid, turmeric acid, tuckahoe acid A, 3-dehydro-tuckahoe acid, tuckahoe acid C, 3-dehydro-tuckahoe acid, dehydrogenated tuckahoe acid, tuckahoe acid, pine ling acid, dehydrogenated porphyric acid, porphyric acid mixed reference solution, respectively with 1 μ L, 10 μ L, 20 μ L, 30 μ L, 40 μ L, 50 μ L injection determination, according to the chromatographic conditions in Example 1, record the chromatographic peak area of ​​each reference substance. With each reference substance injection amount X (μ g) as the abscissa, peak area Y as the ordinate, draw a standard curve and perform linear regression calculation, and obtain the regression equation and linear range of 13 components. The result shows that the linear relationship of 13 components is good, as shown in Table 2.

[0143] Table 2 Linear regression equations of triterpenoid acid components of 13 species of Poria cocos

[0144]

[0145] (2) Precision inspection

[0146] Accurately pipette 10 μL of the same test solution (No. S1) (prepared with reference to the preparation method of the Poria peel test solution in Example 1), inject 6 times continuously, refer to the chromatographic conditions in Example 1, record the peak areas of 13 triterpenic acid compounds, and calculate the RSD value. The calculated RSD values ​​of the peak areas of 13 triterpenic acids, including 6α-hydroxypolyporic acid C, tuckahoe acid B, dehydrotumoric acid, tumoric acid, tuckahoe acid A, 3-epidehydrotuckahoe acid, polyporic acid C, 3-epidehydrotumoric acid, dehydrotuckahoe acid, tuckahoe acid, piniporic acid, dehydroporous acid and porous 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. The RSD values ​​were all less than 3%, indicating that the precision of the instrument was good.

[0147] (3) Repeatability study

[0148] Accurately weigh the Poria cocos medicinal material numbered S1, refer to the preparation method of the Poria cocos peel test solution in Example 1, prepare 6 test solutions in parallel, refer to the chromatographic conditions in Example 1, inject 10 μL of sample analysis respectively, and record the chromatogram. Take Pachymic acid as the reference peak (S peak), 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-hydroxypolyporic acid C, Poria cocos acid B, dehydrotrimoxanic acid, trimoxanic acid, Poria cocos acid A, 3-epi-dehydropolyporic acid, Poria cocos acid C, 3-epi-dehydrotrimoxanic acid, dehydropolyporic acid, pine cocos acid, dehydrotetrasomic acid and tetrasomic acid were calculated to be 1.49%, 2.52%, 2.21%, 2.11%, 2.18%, 0.64%, 0.68%, 0.59%, 0.66%, 0.70%, 0.81%, 0.90%, 0.91%, 0.92%, 0.93%, 0.94%, 0.96%, 0.97%, 0.98%, 0.9 ... .02%, 0.04%, 0.07% and 0.17%; the RSDs of relative peak areas were 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 RSD values ​​of relative retention time and relative peak area were both <3%, indicating that the method had good repeatability.

[0149] (4) Stability test

[0150] Accurately pipette 10 μL of the same test solution (No. S1) (prepared by referring to the preparation method of the Poria peel test solution in Example 1), and inject and analyze at 0, 2, 4, 8, 12, and 24 h after preparation, record the peak area at each measurement time point, and calculate the RSD value. The results showed that the relative average deviations (RSDs) of the peak areas of 6α-hydroxypolyporic acid C, tuckahoe acid B, dehydrotumoric acid, tumoric acid, tuckahoe acid A, 3-epidehydrotuckahoe acid, polyporic acid C, 3-epidehydrotumoric acid, dehydrotuckahoe acid, tuckahoe acid, tuckahoe acid, dehydrotetrasomic acid and tetrasomic acid were all <3%, which were 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 solution was stable within 24 hours.

[0151] (5) Sample recovery rate investigation

[0152] Accurately weigh an appropriate amount of sample with known content (numbered as S1), refer to the method for preparing the Poria peel test solution in Example 1, halve the sample amount and prepare 6 test solutions in parallel, and add a certain amount of 13 reference substances at a mass ratio of 1:1 for sample analysis, and calculate the average sample recovery rate of the 13 components. The average sample recovery rates of 6α-hydroxypolyporic acid C, Poria acid B, dehydrotrimoxanic acid, trimoxanic acid, Poria acid A, 3-epi-dehydropolyporic acid, Poria acid C, 3-epi-dehydrotrimoxanic acid, dehydropolyporic acid, Poria acid, pine acid, dehydroporous acid and pine acid were calculated to be 99.77%, 102.98%, 104.22%, 101.23%, 104.43%, 101.02%, 103.99%, 101.3% respectively. The average recovery rates were between 95% and 105%, and the 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% and 0.27%, respectively, all less than 3.0%, indicating that the method has good accuracy.

[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 solution prepared in Example 1 were injected respectively, and the peak areas of the 13 triterpenic acid components were recorded. Pachymic acid was used as an internal reference, and the f between the test component and the internal reference was calculated. 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 component to be measured, W i is the content of the component to be tested, A s is the peak area of ​​the internal reference, W s The results are shown in Table 3. When pachymic acid was used as the internal reference, the RSDs of f of 6α-hydroxypolysaccharide C, pachymic acid B, dehydroturbid acid, turbid acid, pachymic acid A, 3-epi-dehydropachymic acid, pachymic acid C, 3-epi-dehydroturbid acid, dehydropachymic acid, pine ling acid, dehydrostomated acid and stomated acid were all less than 3%.

[0155] Table 3 f of 13 triterpene acids

[0156]

[0157]

[0158] (7) Durability evaluation of relative correction factors

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

[0160] The mixed reference solution prepared in Example 1 was taken and different volume flow rates (0.8, 1.0, 1.2 mL·min -1 ) on the influence of 13 kinds of Poria triterpenoid acids f (except flow rate, other parameters not listed are all referred to the chromatographic detection conditions in Example 1). The f of 6α-hydroxypolysaccharide C, Poria acid B, dehydrotriterpenic acid, triterpenic acid, Poria acid A, 3-epi-dehydropolysaccharide, Poria acid C, 3-epi-dehydrotriterpenic acid, dehydropolysaccharide, pine acid, dehydroporous acid, and porous 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 repeatability between each component is good, and the RSD value is less than 3%, indicating that different volume flow rates have no significant effect on the f of each component. The results are shown in Table 4.

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

[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°C) on the f of 13 kinds of Poria triterpenoid acids (except column temperature, all other parameters not listed refer to the chromatographic detection conditions in Example 1). The results showed that the RSD values ​​of f of 6α-hydroxypolyporic acid C, Poria new acid B, dehydroturbid acid, turbid acid, Poria new acid A, 3-epi-dehydropolyporic acid, Poria acid C, 3-epi-dehydroturbid acid, dehydropolyporic acid, pine new acid, dehydroporous acid, and porous acid were all less than 3%, indicating that the column temperature had no significant effect on the f value of each component. The results are shown in Table 5.

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

[0167]

[0168]

[0169] (c) The influence of different instruments on f-value

[0170] The mixed reference solution prepared in Example 1 was used to investigate the effects of different instruments (Wayeal 3100 and Agilent 1200) on 13 kinds of Poria triterpenoid acids f (except for the instrument, all other parameters not listed refer to the chromatographic detection conditions in Example 1). The results showed that the RSD of each component f was less than 5%, indicating that different instruments had no significant effect on the f value of each component. The results are shown in Table 6.

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

[0172]

[0173] (8) Investigation of chromatographic peak positioning parameters

[0174] The reproducibility of two parameters, relative retention value or retention time difference, at different column temperatures (20, 25, 30°C) and different operators (A, B, C, D) was investigated respectively, and the parameter with the smallest RSD value was used as the basis for peak positioning after calculation (relative retention value refers to the ratio of the retention time between each component to be measured and the internal reference (S). The retention time difference refers to the difference in retention time between each component to be measured and the internal reference (S); other parameters not listed refer to the chromatographic conditions in Example 1, and the injection solution is the mixed reference solution prepared in Example 1). The results of the investigation showed that the retention time differences between the other 12 triterpene acids of Poria cocos (6α-hydroxypolysaccharide C, polysaccharide B, dehydrotriterpene acid, triterpene acid, polysaccharide A, 3-epi-dehydropolysaccharide, polysaccharide C, 3-epi-dehydrotriterpene acid, dehydropolysaccharide, pine polysaccharide, dehydroporous acid, porous acid) and the internal reference polysaccharide fluctuated greatly, while the relative retention time values ​​fluctuated less, with RSDs of less than 3% (Table 7). Therefore, the relative retention time values ​​were used to locate the chromatographic peaks.

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

[0176]

[0177]

[0178] (9) Comparison of the results between QAMS and ESM:

[0179] Take each batch of Poria cocos medicinal materials purchased in Table 1, prepare the test solution according to the test solution preparation method in Example 1, and measure the peak areas of 13 triterpenic acid components according to the chromatographic conditions in Example 1. Use the external standard method (ESM) to calculate the contents of 13 triterpenic acid components according to the standard curve; then use Pachycolic acid as a reference, and use the measured f average values ​​of each component to be tested and the internal reference to quantitatively calculate the formula W by the one-measurement-multiple-evaluation method. m =W k ×A m / (Ak ×f km )(A k is the peak area of ​​the internal reference; W k is the mass of the internal reference substance, in mg or μg; A m is the peak area of ​​target m, W m is the mass of the target object m, in mg or μg; f km The content of each triterpenic acid component was calculated by using the correction factor (where α is the correction factor). The ESM measured value and the QAMS calculated value were subjected to a paired t-test, and the result showed that P>0.05, indicating that there was no significant difference between the results of the two methods, indicating that the one-measurement-multiple-evaluation method established by the present invention is more accurate and reliable, as shown in Table 8.

[0180] Table 8 ESM and QAMS determination of 13 triterpenoid compounds in 11 batches of Poria samples

[0181]

[0182]

[0183]

[0184] Comparative Example 1

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

[0186] Chromatographic column: Agilent 5TC-C18(2) (250×4.6mm, 5μm) column;

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

[0188] Mobile phase B: acetonitrile;

[0189] Gradient elution program:

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

[0191] From 18 to 28 min, the volume fraction of mobile phase B was maintained at 60%;

[0192] From 28 to 58 min, the volume fraction of mobile phase B was kept increasing from 60% to 92%;

[0193] From 58 to 68 min, the volume fraction of mobile phase B was maintained at 92%;

[0194] Variable wavelength program detection:

[0195] 0min~45.0min, detection wavelength is 242nm;

[0196] 45.0min~46.8min, detection wavelength is 203nm;

[0197] 46.8min~48.8min, detection wavelength is 242nm;

[0198] 48.8min~54.0min, detection wavelength is 203nm;

[0199] 54.0min~57.0min, detection wavelength is 242nm;

[0200] 57.0min~64.0min, detection wavelength is 203nm;

[0201] 64.0min~68.0min, detection wavelength is 242nm.

[0202] Injection volume: 10 μL;

[0203] Column temperature: 25°C;

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

[0205] The HPLC spectra of the Poria peel test solution, white Poria test solution, red Poria test solution, Poria test solution and mixed reference solution in Comparative Example 1 are shown in Figure 2 .Depend on Figure 2 It can be seen that the method in Comparative Example 1 is not suitable for the separation of 13 triterpenic acid components in the present invention, and it is impossible to achieve complete peak separation of all 13 triterpenic acid components.

[0206] The inventor found that under the conditions of Comparative Example 1, turmeric acid and tuckahoe acid A, 3-epi-dehydrotuckahoe acid and policolic acid C overlapped with each other, and could not be completely separated after continuously adjusting the elution gradient. The inventor then adjusted the chromatographic column, and found that there was a problem of peak overlap in multiple chromatographic columns. Finally, the chromatographic column Agilent XDB-C18 chromatographic column (250×4.6mm, 5μm) was determined to be the best chromatographic column that can completely separate the 13 peaks. Different chromatographic columns have different peak elution times for each component, so the variable wavelength program needs to be adaptively adjusted based on the selection of the chromatographic column.

[0207] In general, the present invention establishes an accurate and reliable variable wavelength-gradient elution HPLC method for 13 kinds of triterpenoid acid components of Poria cocos (6α-hydroxypolyporic acid C, poria cocos new acid B, dehydrothiazic acid, thiazic acid, poria cocos new acid A, 3-epi-dehydroporia cocos acid, poria cocos acid C, 3-epi-dehydrothiazic acid, dehydroporia cocos acid, poria cocos acid, pine cocos new acid, dehydroporous acid, porous acid); on this basis, poria cocos acid, which is relatively easy to obtain and has stable properties, is selected as an internal reference among the representative triterpenoid acid components of Poria cocos, and a relative correction factor between the internal reference and each component to be tested is established; the contents of 13 triterpenoid acids of Poria cocos in 11 batches of commercially available Poria cocos decoction pieces from different medicinal parts (Poria cocos peel, red Poria cocos, white Poria cocos, and Poria cocos) from different origins 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 detection components and time. The method provides a reference for the quantitative evaluation of triterpenoid components in Poria cocos, saving detection cost and time; in addition, the method of the invention has good linear relationship, high precision, good repeatability and good stability.

[0208] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A method for determining the content of triterpenoid compounds in Poria cocos, characterized in that: It includes testing the Poria cocos test solution by liquid chromatography; Among them, the triterpene acid compounds include 6α-hydroxypolyporic acid C, tuckahoe acid B, dehydrotumoric acid, tumoric acid, tuckahoe acid A, 3-epi-dehydrotuckahoe acid, polyporic acid C, 3-epi-dehydrotumoric acid, dehydrotuckahoe acid, tuckahoe acid, pine acid, dehydroporous acid, porous acid; The detection conditions of the liquid chromatography method include: Mobile phase A: 0.05% to 0.4% phosphoric acid aqueous solution; Mobile phase B: acetonitrile; Elution method: gradient elution; the process of gradient elution is: From 0 min to 20 min, the volume fraction of mobile phase B increased from 48% to 52% to 52% to 55%; From 20 to 39 minutes, the volume fraction of mobile phase B was maintained at 52% to 55%; From 39 to 43 minutes, the volume fraction of mobile phase B increased from 52% to 55% to 60% to 64%; From 43 to 47 minutes, the volume fraction of mobile phase B was maintained at 60% to 64%; From 47 to 70 minutes, the volume fraction of mobile phase B increased from 60% to 64% to 90% to 94%; From 70 min to 80 min, the volume fraction of mobile phase B was maintained at 90% to 94%.

2. The measuring method according to claim 1, characterized in that The detection conditions of the liquid chromatography method include a variable wavelength program: 0min~35min, detection wavelength is 240nm~245nm; 35min~42min, detection wavelength is 200nm~205nm; 42min~65min, detection wavelength is 240nm~245nm; 65min~67min, detection wavelength is 200nm~205nm; 67min~76min, detection wavelength is 240nm~245nm; 76min~78min, detection wavelength is 200nm~205nm; 78min~80min, detection wavelength is 240nm~245nm.

3. The measuring method according to claim 1, characterized in that The detection conditions of the liquid chromatography method also include: Chromatographic column: octadecylsilane bonded silica gel chromatographic column; and / or, column temperature: 20°C to 30°C; and / or, flow rate of mobile phase B: 0.8 mL / min to 1.2 mL / min; And / or, injection volume: 1μL~50μL.

4. The measuring method according to claim 3, characterized in that The chromatographic column is an Agilent XDB-C18 chromatographic column.

5. The measuring method according to claim 1, characterized in that The test solution is prepared by the following steps: After the Poria cocos sample is mixed with methanol, the solid and liquid are separated, and the filtrate is collected to obtain; Preferably, the mass volume ratio of the Poria cocos sample and methanol is 1 g: 20 mL to 30 mL.

6. The measuring method according to claim 1, characterized in that The content of triterpenoid acid compounds in the Poria cocos test solution is measured by an external standard method or a one-measurement-multiple-evaluation method.

7. The measuring method according to claim 6, characterized in that When the one-measurement-multiple-evaluation method is used for measurement, the method further includes: using the liquid chromatography method to detect the reference solution containing the internal reference substance, and calculating the content of the internal reference substance in the Poria cocos test solution according to the detection result; calculating the content of the triterpene acid compound in the Poria cocos test solution according to the relative correction factor; Among them, the internal reference includes any one of 6α-hydroxypolyporus acid C, tuckahoe acid B, dehydrotumoric acid, tumoric acid, tuckahoe acid A, 3-epi-dehydrotuckahoe acid, tuckahoe acid C, 3-epi-dehydrotumoric acid, dehydrotuckahoe acid, tuckahoe acid, pine ling acid, dehydroporous acid, and porous acid.

8. The measuring method according to claim 7, characterized in that Taking pachymic acid as the internal reference, the relative correction factors of 6α-hydroxypolyporic acid C, pachymic acid B, dehydrotumoric acid, tumoric acid, pachymic acid A, 3-epi-dehydropachymic acid, pachymic acid C, 3-epi-dehydrotumoric acid, dehydropachymic acid, pine acid, dehydroporous acid and pine 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.

9. The measuring method according to claim 7, characterized in that The relative retention times of 6α-hydroxypolyporic acid C, tuckahoe acid B, dehydrotumoric acid, tumoric acid, tuckahoe acid A, 3-epidehydrotuckahoe acid, polyporic acid C, 3-epidehydrotumoric acid, dehydrotuckahoe acid, tuckahoe acid, tuckahoe acid, dehydrotetrasomic acid, and tetrasomic acid were 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.

10. Use of the determination method according to any one of claims 1 to 9 in the quality control of Poria cocos and its derivative products.

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