A detection method for Baiziren pill fingerprint and its application

Abstract

The present invention provides a method for detecting the fingerprint of Baiziren Pills. The present invention also provides the use of the above-mentioned method for detecting the fingerprint of Baiziren Pills in the quality detection of the ingredients in Baiziren Pills and the quality detection method thereof. The present invention further provides a method for determining the contents of 6 ingredients in the above-mentioned Baiziren Pills. The present invention further provides a method for screening the fingerprints of multiple medicinal materials in Baiziren Pills. The present invention provides a method for detecting the fingerprint of Baiziren Pills and its application, establishes a high-performance liquid fingerprint of Baiziren Pills, can quantitatively analyze the 6 chemical components in Baiziren Pills, and confirm the attribution of the chromatographic peaks of each single medicinal material in Baiziren Pills, can effectively monitor from the source of the raw materials, and strictly control the quality of the original medicinal materials.

Description

Technical Field

[0001] The present invention belongs to the technical field of detection of components of traditional Chinese medicine, and relates to a detection method of a fingerprint of Baiziren pill and an application thereof, and specifically relates to a detection method of a fingerprint of Baiziren pill and its six main active ingredients: schisandrin A, atractylodes lactone I, schisandrin A, schisandrin B, α-linolenic acid, and linoleic acid. Background Art

[0002] Our exclusive product, Baiziren Pills, is included in the Fifth Volume of the Ministry of Health of the People's Republic of China's Standardized Traditional Chinese Medicine Formulas (Standard Number: WS3-B-0962-91). It is made by sieving and blending nine finely ground Chinese medicinal ingredients: roasted Baiziren, jujube (pitted and lightly stir-fried), stir-fried Pinellia tuber, stir-fried Atractylodes macrocephala, stir-fried Schisandra chinensis, Codonopsis pilosula, ephedra root (honey-roasted), calcined oyster shell, and wheat bran (stir-fried until yellow). These ingredients are then mixed with refined honey and water to create honey-coated pills, which are then dried. These pills nourish the heart and calm the mind, harmonize the stomach, and strengthen the defensive system. They are used to treat yin deficiency and excessive fire, restless sleep, and night sweats.

[0003] Due to the complex chemical composition and multiple herbal ingredients in Baiziren Pills, there is currently a lack of accurate and effective testing methods. To effectively monitor the quality of Baiziren Pills, it is necessary to establish a sensitive, efficient, simple and rapid analytical method that can not only reveal the complex material basis of the entire formula but also quantitatively study the key active ingredients, thus serving as the primary means of quality control for Baiziren Pills. Summary of the Invention

[0004] In view of the shortcomings of the prior art mentioned above, the purpose of the present invention is to provide a method for detecting the fingerprint of Baiziren Pills and its application. By using the pretreatment and high-performance liquid chromatography method under optimized conditions, a method for detecting the fingerprint of Baiziren Pills and a high-performance liquid chromatography fingerprint of its application are established, which highlights the systematic contribution of different categories of chemical components to the fingerprint of Baiziren Pills and its application fingerprint from different aspects, thereby realizing the detection of the chemical components of the whole formula of Baiziren Pills, more comprehensively reflecting the current status of each component in Baiziren Pills, and providing a reference basis for the overall control and evaluation of the quality of Baiziren Pills.

[0005] To achieve the above-mentioned and other related purposes, the present invention provides a first aspect of a method for detecting the fingerprint of Baiziren Pills, comprising the following steps:

[0006] 1) Preparation of test solution: Dissolve the Baiziren Wan sample in solvent, extract by ultrasonication, cool, filter, and obtain the test solution by taking the filtrate;

[0007] 2) Preparation of reference solution: Schisandrin A, Atractylodes lactone I, Schisandrin A, Schisandrin B, α

[0008] - Linolenic acid and linoleic acid reference substances were added with solvent and ultrasonically dissolved, and then the volume was fixed to prepare reference substance solutions;

[0009] 3) Determination: The test solution and the reference solution are respectively determined by high performance liquid chromatography (HPLC) under the same chromatographic conditions to obtain fingerprints of the test solution and the reference solution. The fingerprints of the test solution and the reference solution are compared to identify and locate the index components in the fingerprints of the test solution, thereby obtaining the fingerprint of Baiziren Wan.

[0010] Preferably, in step 1), the baiziren pills are pulverized baiziren pills and then sieved to obtain a baiziren pill powder sample.

[0011] More preferably, the screening is through a No. 4 sieve.

[0012] Preferably, in step 1), the Baiziren pill sample is subjected to 60 Co-γ ray irradiation.

[0013] More preferably, the 60 The dose of Co-γ ray irradiation is 3-10KGy. 60 The dose of Co-γ ray irradiation is 3kGy, 6kGy, 10kGy. Most preferably, the 60 The dose of Co-γ ray irradiation was 6 kGy.

[0014] More preferably, the 60 The maximum absorbed dose of Co-γ ray irradiation is ≤6KGy.

[0015] Since Chinese medicines are mostly used as medicine in the form of roots and rhizomes, and grow in a soil environment with rich bacterial species and complex microorganisms, the medicinal materials will still carry a certain amount of microorganisms after being cleaned and cut. This is especially true for Chinese patent medicines that are directly powdered for use. The growth and reproduction of some microorganisms have a certain impact on the quality of Chinese medicinal materials and may even endanger the health and life safety of patients. The initial bacterial content of medicinal materials is required to be very strict, otherwise the bacterial content of the finished product will exceed the standard. Therefore, it is of great significance to sterilize and control Chinese medicinal materials before leaving the factory.

[0016] described 60 Co-γ ray irradiation sterilization technology refers to the use of 60The process by which gamma rays generated by Co kill microorganisms on and within materials. Irradiation with a certain dose of radiation can effectively reduce the microbial content and microbial load in traditional Chinese medicines, thereby sterilizing the herbs. Therefore, it is necessary to sterilize the raw medicinal powders used in traditional Chinese medicine preparations by irradiation to further reduce the microbial load and achieve the desired sterilization effect. Due to its pollution-free, non-destructive nature, excellent sterilization effect, room temperature operation, and suitability for large-scale production, irradiation has been widely used in recent years for sterilizing raw herbal powders.

[0017] Preferably, in step 1), the ratio of the weight (g) of the added Baiziren pill sample to the volume (mL) of the added solvent is 1:5-15.

[0018] More preferably, the ratio of the weight (g) of the added Baiziren pill sample to the volume (mL) of the added solvent is 1:10.

[0019] Preferably, in step 1) or 2), the solvent is methanol.

[0020] Preferably, in step 1), the Baiziren pill sample needs to be precisely weighed after adding the solvent.

[0021] Preferably, in step 1), the ultrasonic extraction time is 20-40 min. More preferably, the ultrasonic extraction time is 30 min.

[0022] Preferably, in step 1), the power of the ultrasonic extraction is 50-150 W, and the frequency of the ultrasonic extraction is 30-50 kHz. More preferably, the power of the ultrasonic extraction is 100 W, and the frequency of the ultrasonic extraction is 40 kHz.

[0023] Preferably, in step 1), the cooling is standing to cool.

[0024] Preferably, in step 1), the product needs to be weighed again after cooling, and the weight loss is compensated with solvent.

[0025] Preferably, in step 1), the filtration is to take the supernatant through a filtration membrane, discard the initial filtrate, and then take the subsequent filtrate.

[0026] More preferably, the filter membrane is a 0.45 μm filter membrane.

[0027] Preferably, in step 2), the reference solution is prepared by stepwise dilution.

[0028] More preferably, the reference substance stock solution used in the stepwise dilution needs to be refrigerated and kept away from light for future use.

[0029] Preferably, in step 2), the extraction time of the ultrasonic dissolution is 5-15 minutes. More preferably, the extraction time of the ultrasonic dissolution is 10 minutes.

[0030] Preferably, in step 2), the extraction power of the ultrasonic dissolution is 50-150 W, and the extraction frequency of the ultrasonic dissolution is 30-50 kHz. More preferably, the extraction power of the ultrasonic dissolution is 100 W, and the extraction frequency of the ultrasonic dissolution is 40 kHz.

[0031] Preferably, in step 2), the CAS number of the schisandrin A is 7432-28-2, the CAS number of the atractylodes lactone I is 73069-13-3, the CAS number of the schisandrin A is 61281-38-7, the CAS number of the schisandrin B is 61281-37-6, the CAS number of the α-linolenic acid is 463-40-1, and the CAS number of the linoleic acid is 60-33-3.

[0032] Preferably, in step 2), the content of schisandrin A in the reference solution ranges from 1.9766 to 253.0 μg / mL, the content of atractylodes lactone I ranges from 0.4942 to 31.625 μg / mL, the content of schisandrin A ranges from 4.0000 to 256.0 μg / mL, the content of schisandrin B ranges from 3.7950 to 242.905 μg / mL, the content of α-linolenic acid ranges from 24.619 to 393.900 μg / mL, and the content of linoleic acid ranges from 30.666 to 817.750 μg / mL.

[0033] Preferably, in step 3), the fingerprint of the test solution is compared with the fingerprint of the reference solution, and the corresponding characteristic peaks in the fingerprint of the test solution are identified by relative retention time based on the known characteristic peaks in the fingerprint of the reference solution, thereby attributing and locating the indicator components in the fingerprint of the test solution.

[0034] Preferably, in step 3), the chromatographic column used in the HPLC method is a C18 chromatographic column. More preferably, the chromatographic column used in the HPLC method is an Agilent TC-C18 chromatographic column (4.6 mm×250 mm, 5 μm) with octadecylsilane bonded silica gel as the filler.

[0035] Preferably, in step 3), the detector in the high performance liquid chromatography is a photodiode array detector (DAD).

[0036] Preferably, in step 3), in the high performance liquid chromatography method, the column temperature is 30-40°C, preferably 35°C.

[0037] Preferably, in step 3), the injection volume in the HPLC method is 5-15 μL. More preferably, the injection volume in the HPLC method is 10 μL.

[0038] Preferably, in step 3), the flow rate of the high performance liquid chromatography is 0.8-1.2 mL / min. More preferably, the flow rate of the high performance liquid chromatography is 1.0 mL / min.

[0039] Preferably, in step 3), the high performance liquid chromatography method is analyzed by wavelength switching, wherein:

[0040] When the retention time is 0-15 min, the detection wavelength is 252-256 nm, preferably 254 nm;

[0041] When the retention time is 15-23 min, the detection wavelength is 273-277 nm, preferably 275 nm;

[0042] When the retention time is 23-45 min, the detection wavelength is 203-207 nm, preferably 205 nm.

[0043] Preferably, in step 3), in the high performance liquid chromatography method, the mobile phase is methanol-0.05-0.2% formic acid aqueous solution, wherein phase A is methanol and phase B is 0.05-0.2% formic acid aqueous solution; the analysis time is 45 minutes; and gradient elution is used.

[0044] More preferably, in the high performance liquid chromatography method, the mobile phase is methanol-0.1% formic acid aqueous solution, wherein phase A is methanol and phase B is 0.1% formic acid aqueous solution; the analysis time is 45 minutes; and gradient elution is used.

[0045] The 0.05-0.2% formic acid aqueous solution is a formic acid aqueous solution with a volume percentage of 0.05-0.2%. The 0.1% formic acid aqueous solution is a formic acid aqueous solution with a volume percentage of 0.1%.

[0046] More preferably, as shown in Table 1, the specific procedure of the gradient elution is:

[0047] 0-5min, phase A:phase B volume ratio was 70:30-70:30;

[0048] 5-15 min, the volume ratio of phase A:phase B is 70:30-83:17;

[0049] 15-23 min, the volume ratio of phase A:phase B is 83:17-83:17;

[0050] 23-25 min, the volume ratio of phase A:phase B is 83:17-90:10;

[0051] 25-45min, the volume ratio of phase A:phase B is 90:10-90:10.

[0052] Table 1 Gradient elution program

[0053]

[0054] The second aspect of the present invention provides a method for detecting the fingerprint of Baiziren Pills and its use in the quality detection of the ingredients in Baiziren Pills.

[0055] The third aspect of the present invention provides a quality detection method for Baiziren Pills, comprising obtaining a fingerprint of Baiziren Pills using the aforementioned detection method for the fingerprint of Baiziren Pills, and comparing the obtained fingerprint of Baiziren Pills with a control fingerprint of Baiziren Pills obtained under the same fingerprint detection conditions for similarity.

[0056] Preferably, when comparing the measured fingerprint of Baiziren Pills with the reference fingerprint of Baiziren Pills for similarity, the comparison is performed using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 edition) software published by the State Pharmacopoeia Commission. More preferably, the similarity between the measured fingerprint of Baiziren Pills and the reference fingerprint of Baiziren Pills is greater than 0.99.

[0057] More preferably, when matching the common fingerprint peaks of the Baiziren Pill fingerprint with the control fingerprint of the Baiziren Pill, automatic full spectrum matching is performed with a time window width of 0.10 min, and the fingerprint and the control fingerprint are generated using the median method.

[0058] Preferably, the control fingerprint of Baiziren Pills is obtained under the same conditions as the detection method of the aforementioned Baiziren Pills fingerprint, and the control fingerprint of Baiziren Pills includes 17 common fingerprint peaks, with peak 10 as the reference peak (S peak, relative retention time is 1.0000), and the relative retention times of the other 16 common fingerprint peaks are peak 1 (0.2334±0.0002), peak 2 (0.3270±0.0004), peak 3 (0.4256±0.0006), peak 4 (0.4590±0.0006), peak 5 (0.5455±0.0003), peak 6 ( 0.5841±0.0002), peak 7 (0.6629±0.0003), peak 8 (0.6875±0.00023), peak 9 (0.7834±0.0094), peak 11 (1.0745±0.0002), peak 12 (1.0985±0.0002), peak 13 (1.1936±0.0003), peak 14 (1.2196±0.0007), peak 15 (1.2529±0.0004), peak 16 (1.2982±0.0007), and peak 17 (1.3792±0.0009).

[0059] The specific data of the control fingerprint of the Baiziren Pills can be found in Figure 2 .

[0060] More preferably, the control fingerprint of the Baiziren Pill is compared with the fingerprint of the reference solution, and peak No. 1 is located and determined to be the fingerprint peak of schisandrin A, peak No. 5 is the fingerprint peak of atractylodes lactone I, peak No. 6 is the fingerprint peak of schisandrin A, peak No. 8 is the fingerprint peak of schisandrin B, peak No. 10 is the fingerprint peak of α-linolenic acid, and peak No. 12 is the fingerprint peak of linoleic acid.

[0061] A fourth aspect of the present invention provides a method for determining the contents of six components in Baiziren Pills, comprising the following steps:

[0062] a) Preparation of test solution: Same as step 1) of the Baiziren Pill fingerprint detection method;

[0063] b) Preparation of reference solution: same as step 2) of the method for detecting the fingerprint of Baiziren Pills;

[0064] c) Determination: The test solution of step a) and the reference solution of step b) were respectively determined by high performance liquid chromatography using the same chromatographic conditions as in the detection method of the Baiziren Pill fingerprint, and the contents of the six components in the test solution were calculated by the external standard method.

[0065] Preferably, the external standard method refers to: respectively taking a series of different volumes of the reference solution in step b) to prepare a series of solutions with different concentrations, using a high performance liquid chromatograph for sample analysis, obtaining a linear relationship between the content and peak area of the six components in the reference solution, and plotting a corresponding standard working curve with the chromatographic peak area of each component corresponding to its corresponding content, and calculating the regression equation of each standard working curve. The test solution is then tested by a high performance liquid chromatograph, and the chromatographic peak areas of the six components in the test solution obtained are substituted into the regression equation of each standard working curve to obtain the content of the corresponding component.

[0066] More preferably, in the standard working curve, the peak area is used as the ordinate and the content of each reference solution is used as the abscissa.

[0067] A fifth aspect of the present invention provides a method for screening fingerprints of multiple medicinal materials in Baiziren Pills, comprising the following steps:

[0068] A) Preparation of single medicinal material sample solutions: Prepare any one or more of the nine medicinal material samples of Baiziren Pills, including Baiziren Semen, Pinellia Rhizome, Jujube, Schisandra Fructus, Atractylodes Macrocephala, Codonopsis Pilosula, Ephedra Root, Oyster, and Wheat Bran, according to step 1) of the Baiziren Pills fingerprint detection method to obtain at least one single medicinal material sample solution;

[0069] B) Determination: Determine the single medicinal material sample solution using high performance liquid chromatography (HPLC) under the same chromatographic conditions as in step 3) of the method for detecting the fingerprint of Baiziren Pills to obtain the fingerprint of the single medicinal material sample solution;

[0070] C) Obtaining the control fingerprint: Using the same steps as the detection method for the Baiziren Pill fingerprint, obtain the control fingerprint of the Baiziren Pill;

[0071] D) Quality testing: The fingerprint of the single medicinal material sample solution is compared with the reference fingerprint of Baiziren Wan. The corresponding characteristic peaks of the single medicinal material sample solution in the reference fingerprint of Baiziren Wan are identified by relative retention time, thereby attributing and locating the characteristic peaks in the fingerprint of the single medicinal material sample solution.

[0072] Preferably, in step A), the cypress seed kernel is the dried mature seed kernel of Platycladus orientails (L.) Franco, a plant of the Cupressaceae family. The pinellia ternata is the dried tuber of Pinellia ternata (Thunb.) Breit., a plant of the Araceae family. The jujube is the dried mature fruit of Ziziphus jujuba Mill., a plant of the Rhamnaceae family. The schisandra chinensis is the dried mature fruit of Schisandra chinensis (Turcz.) Baill., a plant of the Magnoliaceae family. The atractylodes macrocephala is the dried rhizome of Atractylodes macrocephala Koidz., a plant of the Asteraceae family. The codonopsis pilosula is the dried root of Codonopsis pilosula (Franch.) Nannf., Codonopsis pilosula Nannf. var. modesta (Nannf.) LTShen, or Codonopsis tangshen Oliv., a plant of the Campanulaceae family. The ephedra root is the dried root and rhizome of Ephedra sinica Stapf or Ephedra intermedia, both from the Ephedraceae family. The oyster is the shell of Ostrea gigas Thunberg, Ostrea talienwhanensis Crosse, or Ostrea rivularis Gould, both from the Ostreaceae family. The wheat bran is wheat bran, a byproduct of wheat flour processing, which is wheat-yellow in color and in flaky or powdery form.

[0073] Preferably, in step D), the present invention locates the attribution of characteristic peaks of the measured fingerprint of the single medicinal material sample solution and the control fingerprint of Baiziren Wan, and uses the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" 2012 version software issued by the State Pharmacopoeia Commission for analysis and processing, and confirms the attribution of the characteristic peaks of each single medicinal material of Baiziren Wan by the relative retention time of each characteristic peak on the control fingerprint of Baiziren Wan. Specific results are shown in Figure 4 and Table 2.

[0074] Table 2 The characteristic peaks of each single medicinal ingredient in Baiziren Pills

[0075]

[0076] Preferably, in step D), in the single medicinal material sample solution, the fingerprint of the Platycladi seed sample solution includes 4 common fingerprint peaks, and the 4 common fingerprint peaks are peak 10, peak 11, peak 12, and peak 13.

[0077] Preferably, in step D), in the single medicinal material sample solution, the fingerprint of the Pinellia ternata sample solution includes 5 common fingerprint peaks, and the 5 common fingerprint peaks are peak 7, peak 8, peak 10, peak 12, and peak 15.

[0078] Preferably, in step D), among the single medicinal material sample solutions, the fingerprint of the jujube sample solution includes one common fingerprint peak, and the one common fingerprint peak is Peak 9.

[0079] Preferably, in step D), in the single medicinal material sample solution, the fingerprint of the Schisandra chinensis sample solution includes 8 common fingerprint peaks, and the 8 common fingerprint peaks are peak 1, peak 2, peak 6, peak 8, peak 9, peak 10, peak 12, and peak 14.

[0080] Preferably, in step D), in the single medicinal material sample solution, the fingerprint of the Atractylodes macrocephala sample solution includes 5 common fingerprint peaks, and the 5 common fingerprint peaks are peak 3, peak 4, peak 5, peak 10, and peak 12.

[0081] Preferably, in step D), in the single medicinal material sample solution, the fingerprint of the Codonopsis pilosula sample solution includes two common fingerprint peaks, and the two common fingerprint peaks are peak 10 and peak 12.

[0082] Preferably, in step D), in the single medicinal material sample solution, the fingerprint of the wheat bran sample solution includes one common fingerprint peak, and the one common fingerprint peak is peak 12.

[0083] Preferably, in step D), the fingerprints of the ephedra root sample solution and the oyster sample solution do not include a common fingerprint peak.

[0084] Among the above common fingerprint peaks, peak 1 was determined to be the fingerprint peak of schisandrin A, peak 5 was determined to be the fingerprint peak of atractylodes lactone I, peak 6 was determined to be the fingerprint peak of schisandrin A, peak 8 was determined to be the fingerprint peak of schisandrin B, peak 10 was determined to be the fingerprint peak of α-linolenic acid, and peak 12 was determined to be the fingerprint peak of linoleic acid.

[0085] The water used in the present invention is all purified water.

[0086] As mentioned above, since Baiziren Pills are composed of nine Chinese medicinal materials and the chemical components they contain are extremely complex, the present invention provides a method for detecting the fingerprint of Baiziren Pills and its application, and establishes a quality evaluation method for Baiziren Pills that combines fingerprints with multi-component quantification, so that the advantages of qualitative and quantitative analysis complement each other.

[0087] This method uses pretreatment with optimized reaction conditions and high-performance liquid chromatography (HPLC-DAD) to establish a high-performance liquid chromatography fingerprint of Baiziren Pills, highlighting the contribution of different categories of chemical components to the fingerprint system of Baiziren Pills from different aspects, thereby realizing the detection of the chemical components of the entire formula of Baiziren Pills and being able to more comprehensively reflect the quality changes of Baiziren Pills.

[0088] The method provided by the present invention can also perform multi-component content determination of six chemical components (schisandrin A, atractylodes lactone I, schisandrin A, schisandrin B, α-linolenic acid, and linoleic acid) in Baiziren pills. The standard curves of the six components determined by this method have good linearity within their respective ranges, good repeatability, high accuracy, and high precision.

[0089] The above-mentioned fingerprint spectrum combined with the multi-component simultaneous quantitative method fills the gap in the detection method of Baiziren Pills. It has the advantages of simplicity, speed, accuracy, and good repeatability. It provides a reference basis for achieving full-process quality control of the production process to ensure safe and effective medication and controllable quality.

[0090] This method also confirmed the identity of multiple chromatographic peaks in Baiziren Pills, with the fingerprints revealing characteristic peaks for Baiziren, Pinellia, Jujube, Schisandra, Atractylodes, Codonopsis, and wheat bran. This method enables effective monitoring of raw materials at the source, strictly controlling the quality of the raw materials, and can be used for quality control during the production process, ensuring the safety and effectiveness of clinical medications. BRIEF DESCRIPTION OF THE DRAWINGS

[0091] Figure 1 Shown are chromatograms S1 and S2 of the reference solution and the test solution of Baiziren Pills in the present invention, wherein Figure S2 is the fingerprint of the reference solution, 1: Schisandrin A, 2: Atractylodes lactone I, 3: Schisandrin A, 4: Schisandrin B, 5: α-linolenic acid, 6: linoleic acid; Figure S1 is the chromatogram of the test solution.

[0092] Figure 2 The fingerprint of the control of the Baiziren Pills of the present invention is shown, wherein: 1: Schisandrin A; 5: Atractylodes lactone I; 6: Schisandrin A; 8: Schisandrin B; 10: α-linolenic acid; 12: linoleic acid.

[0093] Figure 3 The HPLC fingerprints of multiple batches of Baiziren Pill samples before and after irradiation are shown, wherein S1: 202201 (0 kGy); S2: 202201 (3 kGy); S3: 202201 (6 kGy); S4: 202201 (10 kGy); S5: 202202 (0 kGy); S6: 202202 (3 kGy); S7: 202202 (6 kGy); S8: 202202 (10 kGy); S9: 202203 (0 kGy); S10: 202203 (3 kGy); S11: 202203 (6kGy); S12: 202203 (10kGy); S13: 202204 (0kGy); S14: 202204 (3kGy); S15: 202204 (6kGy); S16: 202204 (10kGy); S17: 202205 (0kGy); S18: 202205 (3kGy); S19: 202205 (6kGy); S20: 202205 (10kGy); S21: control fingerprint of Baiziren Pills.

[0094] Figure 4 Shown is the attribution diagram of the characteristic peaks in the Baiziren Pills of the present invention, wherein S10: Baiziren Pill powder sample; S9: oyster; S8: wheat bran; S7: ephedra root; S6: Codonopsis pilosula; S5: Atractylodes macrocephala; S4: Pinellia ternata; S3: jujube; S2: Baiziren; S1: Schisandra chinensis. DETAILED DESCRIPTION

[0095] The present invention is further described below with reference to specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of protection of the present invention.

[0096] The following describes the embodiments of the present invention through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different viewpoints and applications without departing from the spirit of the present invention.

[0097] The reagents and instruments used in the following examples are as follows:

[0098] 1. Reagents

[0099] Reference substances: schisandrin A (batch number: 110857-201815; content: 99.7%), schisandrin A (batch number: 110764-201915; content: 99.5%), and atractylodes lactone I (batch number: 11975-201501; content: 99.9%) were purchased from the China Food and Drug Administration. Schisandrin B (batch number: 8366; content: 94.7%), α-linolenic acid (batch number: 111631-202006), and linoleic acid (batch number: 11622-202105) were purchased from Shanghai Standard Standard Technology Service Co., Ltd.

[0100] Samples: Baiziren pill powder, provided by Shanghai Hutchison Pharmaceutical Co., Ltd., a total of 5 batches (batch numbers are 202201, 202202, 202203, 202204 and 202205), divided into unirradiated, 3kGy, 6kGy and 10kGy irradiation doses, respectively. The information of irradiated samples and irradiated samples is shown in Table 3.

[0101] Table 3 Irradiated sample information

[0102]

[0103] Single medicinal materials: 9 medicinal materials including pine nut seeds, jujube, pinellia, atractylodes, schisandra, ephedra root, oyster, codonopsis and wheat bran were purchased from Shandong Kangyuantang Chinese Medicine Pieces Co., Ltd., powdered, passed through a 40-mesh sieve, and stored in sealed bags for later use.

[0104] Reagents: methanol and formic acid (chromatographic grade, Sinopharm Chemical Reagent Co., Ltd.), and ultrapure water were prepared using a Milli-Q ultrapure water treatment system.

[0105] 2. Instruments

[0106] An Agilent 1260 high-performance liquid chromatograph (Agilent OpenLAB CDS ChemStation workstation, G1311C quaternary pump system, G1329B standard autosampler, G1316A column oven, and G4212B diode array detector, all from Agilent, USA) was used. An AL204 1 / 10,000 electronic balance and an X205BDU 1 / 100,000 electronic balance were purchased from METTLER TOLEDO (Shanghai). A DFY-500 high-speed Chinese medicine pulverizer was purchased from Dade Traditional Chinese Medicine Machinery Co., Ltd. in Wenling, Zhejiang Province. A TDL-40B benchtop centrifuge was purchased from Shanghai Anting Scientific Instrument Factory. A DHG-9123A electric blast drying oven was purchased from Shanghai Yiheng Scientific Instrument Co., Ltd. An SB-5200 ultrasonic cleaner was purchased from Ningbo Xinzhi Biotechnology Co., Ltd., and a Mill-Q Advantage A10 ultrapure water preparation system was purchased from Millipore Shanghai Trading Co., Ltd.

[0107] Example 1

[0108] 1. Sample pretreatment

[0109] Preparation of test solution: Take 1.0 g of Baiziren Pill sample, accurately weigh it, place it in a 50 mL conical flask with a stopper, accurately add 10 mL of methanol, stopper it, weigh it, and extract it by ultrasonic (power 100 W, frequency 40 kHz) for 30 minutes. Let it stand and cool, weigh it again, make up the weight loss with methanol, filter it through a 0.45 μm filter membrane, and take the filtrate to obtain test solution 1#.

[0110] Preparation of reference solution: Accurately weigh schisandrin A, atractylodes lactone I, schisandrin A, schisandrin B, α-linolenic acid, and linoleic acid reference standards, dissolve them in methanol by ultrasonication (power 100 W, frequency 40 kHz) for 10 minutes, then dilute to volume in the same 100 mL volumetric flask, shake well, and prepare the reference stock solution.

[0111] The reference substance stock solution was then precisely measured and serially diluted with methanol to the same volume to prepare a series of reference substance solutions of varying concentrations. In these reference substance solutions, the content of schisandrin A ranged from 1.9766 to 253.0 μg / mL, the content of atractylodes lactone I ranged from 0.4942 to 31.625 μg / mL, the content of schisandrin A ranged from 4.0000 to 256.0 μg / mL, the content of schisandrin B ranged from 3.7950 to 242.905 μg / mL, the content of α-linolenic acid ranged from 24.619 to 393.900 μg / mL, and the content of linoleic acid ranged from 30.666 to 817.750 μg / mL.

[0112] 2. Chromatographic conditions

[0113] The chromatographic conditions of the HPLC method are as follows: an Agilent TC-C18 chromatographic column (4.6 mm×250 mm, 5 μm); a photodiode array detector (DAD); a column temperature of 35° C.; an injection volume of 10 μL; and a flow rate of 1.0 mL / min.

[0114] The analysis is performed using wavelength switching, where:

[0115] When the retention time was 0-15 min, the detection wavelength was 254 nm;

[0116] When the retention time was 15-23 min, the detection wavelength was 275 nm;

[0117] When the retention time was 23-45 min, the detection wavelength was 205 nm.

[0118] The mobile phase was methanol-0.1% formic acid aqueous solution, wherein phase A was methanol and phase B was 0.1% formic acid aqueous solution; the analysis time was 45 min; and the elution method was gradient elution.

[0119] As shown in Table 1, the specific procedure of the gradient elution is:

[0120] 0-5min, phase A:phase B volume ratio was 70:30-70:30;

[0121] 5-15 min, the volume ratio of phase A:phase B is 70:30-83:17;

[0122] 15-23 min, the volume ratio of phase A:phase B is 83:17-83:17;

[0123] 23-25 min, the volume ratio of phase A:phase B is 83:17-90:10;

[0124] 25-45min, the volume ratio of phase A:phase B is 90:10-90:10.

[0125] 3. Determination

[0126] The high performance liquid chromatography method under the chromatographic conditions in step 2 is used to measure the test solution 1# and the reference solution in step 1, respectively, to obtain the fingerprint of the test solution 1# and the fingerprint of the reference solution. The fingerprint of the test solution 1# is compared with the fingerprint of the reference solution. Based on the known characteristic peaks in the fingerprint of the reference solution, the corresponding characteristic peaks in the fingerprint of the test solution 1# are identified by relative retention time, thereby attributing and locating the index components in the fingerprint of the test solution 1#, and obtaining the fingerprint of Baiziren Wan.

[0127] Example 2

[0128] 1. Sample pretreatment

[0129] Preparation of test solution: Take 1.0 g of Baiziren Pills sample, accurately weigh it, place it in a 50 mL stoppered conical flask, accurately add 5 mL of methanol, stopper it, weigh it, and extract it ultrasonically (power 80 W, frequency 35 kHz) for 25 minutes. Let it stand and cool, weigh it again, make up the weight loss with methanol, filter it through a 0.45 μm filter membrane, and take the filtrate to obtain test solution 2#.

[0130] Preparation of reference solution: Accurately weigh schisandrin A, atractylodes lactone I, schisandrin A, schisandrin B, α-linolenic acid, and linoleic acid reference standards, dissolve them in methanol by ultrasonication (power 80W, frequency 35kHz) for 8 minutes, and then dilute to volume in a 100mL volumetric flask. Shake well to prepare the reference stock solution. Accurately measure the reference stock solution, dilute it stepwise with methanol, and dilute to volume to prepare a series of reference solutions of different concentrations.

[0131] In a series of reference solution with different concentrations, the concentration ranges of schisandrin A, atractylodes lactone I, schisandrin A, schisandrin B, α-linolenic acid, and linoleic acid are the same as those in step 1 of Example 1.

[0132] 2. Chromatographic conditions

[0133] The chromatographic conditions of the HPLC method are as follows: an Agilent TC-C18 chromatographic column (4.6 mm×250 mm, 5 μm); a photodiode array detector (DAD); a column temperature of 30° C.; an injection volume of 5 μL; and a flow rate of 0.8 mL / min.

[0134] The analysis is performed using wavelength switching, where:

[0135] When the retention time was 0-15 min, the detection wavelength was 253 nm;

[0136] When the retention time was 15-23 min, the detection wavelength was 274 nm;

[0137] When the retention time was 23-45 min, the detection wavelength was 204 nm.

[0138] The mobile phase was methanol-0.05% formic acid aqueous solution, wherein phase A was methanol and phase B was 0.05% formic acid aqueous solution; the analysis time was 45 min; and the elution was gradient.

[0139] The specific procedure of gradient elution is the same as step 2 in Example 1.

[0140] 3. Determination

[0141] The specific measurement process is the same as step 3 in Example 1.

[0142] Example 3

[0143] 1. Sample pretreatment

[0144] Preparation of test solution: Take 1.0 g of Baiziren Pill sample, accurately weigh it, place it in a 50 mL stoppered conical flask, accurately add 15 mL of methanol, stopper it, weigh it, and extract it by ultrasonic (power 120 W, frequency 45 kHz) for 35 minutes. Let it stand and cool, weigh it again, make up the weight loss with methanol, filter it through a 0.45 μm filter membrane, and take the filtrate to obtain the test solution 3#.

[0145] Preparation of reference solution: Accurately weigh schisandrin A, atractylodes lactone I, schisandrin A, schisandrin B, α-linolenic acid, and linoleic acid reference standards, dissolve them in methanol by ultrasonication (power 120W, frequency 45kHz) for 12 minutes, and then dilute to volume in a 100mL volumetric flask. Shake well to prepare the reference stock solution. Accurately measure the reference stock solution, dilute it stepwise with methanol, and dilute to volume to prepare a series of reference solutions of different concentrations.

[0146] In a series of reference solution with different concentrations, the concentration ranges of schisandrin A, atractylodes lactone I, schisandrin A, schisandrin B, α-linolenic acid, and linoleic acid are the same as those in step 1 of Example 1.

[0147] 2. Chromatographic conditions

[0148] The chromatographic conditions of the HPLC method are as follows: an Agilent TC-C18 chromatographic column (4.6 mm×250 mm, 5 μm); a photodiode array detector (DAD); a column temperature of 40° C.; an injection volume of 15 μL; and a flow rate of 1.2 mL / min.

[0149] When the retention time was 0-36 min, the column temperature was 20°C; when the retention time was 36-95 min, the column temperature was 35°C.

[0150] The analysis is performed using wavelength switching, where:

[0151] When the retention time was 0-15 min, the detection wavelength was 255 nm;

[0152] When the retention time was 15-23 min, the detection wavelength was 276 nm;

[0153] When the retention time was 23-45 min, the detection wavelength was 206 nm.

[0154] The mobile phase was methanol-0.15% formic acid aqueous solution, wherein phase A was methanol and phase B was 0.15% formic acid aqueous solution; the analysis time was 45 min; and the elution was gradient.

[0155] The specific procedure of gradient elution is the same as step 2 in Example 1.

[0156] 3. Determination

[0157] The specific measurement process is the same as step 3 in Example 1.

[0158] Example 4

[0159] Select 60 The baiziren pill powder before and after Co-γ ray irradiation was used as the research object for sterilization effect inspection. 10 g of irradiated baiziren pill powder was taken as the test sample after irradiation, and 10 g of unirradiated baiziren pill powder was taken as the control sample before irradiation. They were added with tryptic soytone liquid medium respectively, mixed well, and used as 1:10 test solution, and diluted with tryptic soytone liquid medium to 1:10. 2 , 1:10 3 For each dilution level, take 2 mL of the test solution and dispense an equal amount into two plates (150 mm), 1 mL into each plate, and then inject into tryptic soy peptone agar medium or Sabouraud dextrose agar medium respectively, mix well, let it stand until solidified, and invert for culture. The tryptic soy peptone agar medium plate should be cultured at 30℃-35℃ for 5 days, and the Sabouraud dextrose agar medium plate should be cultured at 20℃-25℃ for 7 days, and the growth of the colonies should be observed daily. Count the number of colonies and check 60 Total aerobic bacteria, molds, and yeasts before and after Co irradiation. A negative control experiment was performed using trypticase soytone broth in the same manner. The results are shown in Table 4.

[0160] Table 4 Bacterial content determination results of Baiziren pill powder before and after irradiation (cfu g -1 )

[0161]

[0162] Note: TAMC is the total aerobic microbial count, and TYMC is the total number of molds and yeasts.

[0163] From Table 4, we can see that with the increase of irradiation dose, the bacterial content of Baiziren pill powder decreased significantly. 60 Co-γ ray irradiation can effectively reduce the microbial level in Baiziren pills. Under the existing irradiation doses (3kGy, 6kGy and 10kGy), the microbial level of Baiziren pills can be effectively reduced. At the same time, at irradiation doses of 10kGy and below, it will not cause significant effects on the content of the six active ingredients in Baiziren pills. 60The fingerprint similarity of the Baiziren pill powder before and after Co irradiation did not change significantly (similarity ≥ 0.999). This shows that at an irradiation dose of 10 kGy or less, it will not affect the content of the six active ingredients in the Baiziren pill powder and the similarity of the fingerprint, and will not affect the quality of the preparation, which is a good opportunity for the future use of this type of medicine. 60 Co-γ ray irradiation sterilization method is provided as a reference. However, considering the potential uncertain effects of irradiation sterilization, and referring to the " 60 The "Standard for Sterilization Dose of Traditional Chinese Medicine by Co Irradiation" requires that the irradiation dose of traditional Chinese medicine raw material powder shall not exceed 6kGy. It is recommended that the irradiation dose should be reduced as much as possible under the premise of meeting the microbial limit, and the irradiation dose of 6kGy should be used for irradiation sterilization of Baiziren Pills powder to ensure the safety of Baiziren Pills.

[0164] Example 5

[0165] The detection method of the Baiziren Pill fingerprint established in the above Example 1 was used to detect 5 batches of Baiziren Pills before and after irradiation, and the fingerprint of the test solution and the fingerprint of the reference solution were obtained. The fingerprint data of the test sample were imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" 2012 version software issued by the State Pharmacopoeia Commission. Sample 202201 was used as the reference spectrum, and automatic full spectrum matching (time window width was 0.1 min) was used to generate fingerprints and reference fingerprints using the median method. The fingerprint of the test sample was compared with the reference fingerprint of the Baiziren Pill obtained under the same fingerprint detection conditions for similarity, and the similarity of the fingerprint of each batch of Baiziren Pill was calculated. The results showed that the similarity of the fingerprints of the 5 batches of Baiziren Pills was greater than 0.99, and the similarity of the fingerprints before and after irradiation was high (all ≥0.999), and there was no increase or loss of the number of peaks, indicating that the fingerprints of the 5 batches of samples were highly similar and the overall quality was relatively stable. The specific similarity is shown in Figure 3 , Table 5.

[0166] Table 5. Changes in fingerprint similarity of Baiziren pill powder before and after irradiation

[0167]

[0168] Example 6

[0169] The Baiziren Pills were tested using the Baiziren Pills fingerprint detection method established in Example 1. Based on the fingerprints of the test solution and the reference solution, 17 common characteristic peaks were identified.

[0170] The specific reference fingerprint of Baiziren Pills can be found in Figure 2 ,Depend on Figure 2It can be seen that the common peaks were determined according to the relative retention time of each chromatographic peak in the chromatogram. Among them, the proportion of the 17 common peaks in the HPLC characteristic spectrum to the total peak area reached more than 90%. These 17 common peaks were selected as characteristic fingerprint peaks. The specific data are shown in Table 6. Among them, peak 10 had a moderate elution time, good separation, and a large peak area. Therefore, peak 10 was used as the reference peak (S peak, relative retention time of 1.0000). The relative retention times of the other 16 common fingerprint peaks were peak 1 (0.2334±0.0002), peak 2 (0.3270±0.0004), peak 3 (0.4256±0.0006), peak 4 (0.4590±0.0006), peak 5 (0.5455±0.0003), peak 6 (0.5841±0.0002), peak 7 (0 0003), peak 8 (0.6875±0.00023), peak 9 (0.7834±0.0094), peak 11 (1.0745±0.0002), peak 12 (1.0985±0.0002), peak 13 (1.1936±0.0003), peak 14 (1.2196±0.0007), peak 15 (1.2529±0.0004), peak 16 (1.2982±0.0007), and peak 17 (1.3792±0.0009).

[0171] Table 6 Relative retention time of common peaks in fingerprints n=6, X±SD

[0172]

[0173] Note: Peak 10 is the positioning peak (S peak)

[0174] The control fingerprint of the above-mentioned Baiziren Pills was compared with the fingerprint of the reference solution. Figure 1 The known characteristic peaks in the fingerprint of the reference solution in the test sample were identified by relative retention time, and the corresponding characteristic peaks in the control fingerprint of Baiziren Pills were identified, and peak No. 1 was located as the fingerprint peak of schisandrin A, peak No. 5 was the fingerprint peak of atractylodes lactone I, peak No. 6 was the fingerprint peak of schisandrin A, peak No. 8 was the fingerprint peak of schisandrin B, peak No. 10 was the fingerprint peak of α-linolenic acid, and peak No. 12 was the fingerprint peak of linoleic acid.

[0175] Example 7

[0176] The method for detecting the fingerprint of Baiziren Pills in the present invention was methodologically verified, and the performance index results are as follows.

[0177] 1. Precision

[0178] Take one sample of Baiziren Pill powder from the same batch (batch number 202201, 0 kGy-before irradiation) and prepare and test it according to the detection method of Baiziren Pill fingerprint in Example 1 above. Sampling was performed continuously for 6 times, and the chromatogram was recorded. Using α-linolenic acid as the reference peak, the RSD of the relative retention time of each common peak was calculated to be <2.5%, and the RSD of the relative peak area was <3.0%, indicating that the instrument precision was good.

[0179] 2. Repeatability

[0180] Six samples of Baiziren pill powder from the same batch (batch number 202201, 0 kGy-before irradiation) were prepared and tested according to the detection method of the Baiziren pill fingerprint in Example 1 above. The chromatograms were recorded, and α-linolenic acid was used as the reference peak. The RSD of the relative retention time of each common peak was calculated to be <1.0%, and the RSD of the relative peak area was <3.0%. The results showed that the method had good repeatability.

[0181] 3. Stability

[0182] Take one sample of Baiziren Pill powder from the same batch (batch number 202201, 0 kGy-before irradiation) and prepare and test it according to the detection method of the Baiziren Pill fingerprint in Example 1 above. After preparing the test solution, place it for 0 h, 2 h, 4 h, 8 h, 12 h, and 24 h for detection, record the chromatogram, and use α-linolenic acid as the reference peak to calculate the RSD of the relative retention time of each common peak <1.0%, and the RSD of the relative peak area <3.0%. The results show that the test solution has good stability within 24 h.

[0183] Example 8

[0184] 1. Sample pretreatment

[0185] Preparation of the test solution: The preparation process of the test solution is the same as that in step 1 of Example 1.

[0186] Preparation of reference solution: The preparation process of reference solution is the same as that in step 1 of Example 1.

[0187] 2. Chromatographic conditions

[0188] The chromatographic conditions of the HPLC method are the same as those of the HPLC method in step 2 of Example 1.

[0189] 3. Determination

[0190] Using the external standard method, a series of different volumes of reference solution were pipetted to prepare a series of solutions with different concentrations. The samples were analyzed by HPLC and a standard working curve was plotted. The obtained test solution was then analyzed by HPLC and the analysis results were substituted into the standard working curve to obtain the contents of the six components in the test solution.

[0191] Specifically, a series of different volumes of reference solution were pipetted to prepare a series of solutions with different concentrations. The samples were analyzed by high performance liquid chromatography to obtain a linear relationship between the content and peak area of the six components in the reference solution. The chromatographic peak area of each component corresponded to its corresponding content, and the corresponding standard working curve was drawn. The regression equation of each standard working curve was calculated. The test solution was then tested by high performance liquid chromatography, and the chromatographic peak areas of the six components in the test solution were substituted into the regression equation of each standard working curve to obtain the content of the corresponding component.

[0192] Example 9

[0193] A series of solutions of different concentrations of the above six reference substances were prepared, and the reference substance stock solutions were accurately aspirated. The HPLC detection conditions of Example 8 were used for high performance liquid chromatography analysis. The chromatograms were recorded. The peak area (Y) was used as the ordinate and the injection volume of each reference substance (X, μg / mL) was used as the abscissa. A standard curve was drawn and a linear regression calculation was performed to obtain the regression equation, correlation coefficient, and linear range. The specific results are shown in Table 7.

[0194] As shown in Table 7, the regression equation has a good linear relationship when the sample is injected within the corresponding concentration range, and the correlation coefficient r 2 Not less than 0.9990.

[0195] Table 7 Linear relationship of 6 active ingredients

[0196]

[0197] Example 10

[0198] 1. Precision

[0199] Any reference solution prepared in Example 8 was taken and tested according to the method in Example 8. The sample was injected continuously for 6 times and the peak area was recorded. The results showed that the RSDs of the peak areas of schisandrin A, atractylodes lactone I, schisandrin A, schisandrin B, α-linolenic acid and linoleic acid were all less than 3.0%, indicating that the instrument precision was good.

[0200] 2. Repeatability

[0201] Six samples of pine nut kernel pill powder from the same batch (batch number 202201, 0 kGy-before irradiation) were taken and six test samples were prepared in parallel according to the method in Example 8 above. The peak areas were recorded. The results showed that the RSDs of the peak areas of the six components, schisandrin A, atractylodes lactone I, schisandrin A, schisandrin B, α-linolenic acid and linoleic acid, were all less than 2.5%, indicating that the method had good repeatability and high accuracy.

[0202] 3. Stability

[0203] Take one sample of the same batch of pine nuts pill powder (batch number 202201, 0 kGy-before irradiation) and prepare one test solution according to the method in Example 8 above. After being placed for 0 h, 2 h, 4 h, 8 h, 12 h, and 24 h, the peak areas were recorded. The RSDs of the peak areas of the six components, schisandrin A, atractylodes lactone I, schisandrin A, schisandrin B, α-linolenic acid, and linoleic acid, were all less than 2.2%, indicating that the test solution had good stability within 24 h.

[0204] 4. Sample recovery rate

[0205] Nine samples of Baiziren pill powder (batch number 202201, 0 kGy, non-irradiated) were accurately weighed, and 0.50 g was added to three different concentration levels of schisandrin A, atractylodes lactone I, schisandrin A, schisandrin B, α-linolenic acid, and linoleic acid reference substances. Test solutions were prepared according to step 1 of Example 8, and analyzed according to the chromatographic conditions of step 2 of Example 8. Chromatograms were recorded. The recoveries of the six active ingredients at different addition ratios were calculated, and the results are shown in Table 8. As shown in Table 8, the average recoveries of schisandrin A, atractylodes lactone I, schisandrin A, schisandrin B, α-linolenic acid and linoleic acid were 96.52%, 97.46%, 98.60%, 100.77%, 96.95% and 97.52%, respectively, all ranging from 96% to 101%; the RSDs were 1.03%, 1.22%, 2.64%, 2.71%, 1.02% and 0.52%, respectively, all less than 2.8%, indicating that the method had good accuracy.

[0206] Table 8 Sample recovery test results (n=9)

[0207]

[0208]

[0209] Example 11

[0210] Five batches of Baiziren pill powder (batch numbers: 202201, 202202, 202203, 202204, 202205) were taken and irradiated with doses of 0, 3, 6, and 10 kGy, respectively. The test solution was prepared according to step 1 in Example 8, and the samples were analyzed according to the chromatographic conditions of step 2 in Example 8. The chromatograms were recorded, and the contents of schisandrin A, atractylodes lactone I, schisandrin A, schisandrin B, α-linolenic acid, and linoleic acid in the Baiziren pill powder before and after irradiation were calculated by the external standard method. The results are shown in Table 9.

[0211] As shown in Table 9, the contents of the six compounds in the same batch of Baiziren pill powder after irradiation at 3kGy, 6kGy, and 10kGy were minimally different from those before irradiation (RSD < 2.7%). These results indicate that the three irradiation doses of 3kGy, 6kGy, and 10kGy had little effect on the contents of the six compounds in the samples tested. This method is effective in determining the contents of the six components in Baiziren pill samples, is simple to operate, has good applicability, and provides accurate and reliable results.

[0212] Table 9 Content determination results of 5 batches of Baiziren pill powder before and after irradiation

[0213]

[0214] Example 12

[0215] The sample pretreatment steps in Example 1 were used to prepare the test solution.

[0216] Powders of nine medicinal materials, namely, cypress seed, pinellia, jujube, schisandra, atractylodes, codonopsis, ephedra root, oyster, and wheat bran, were taken respectively, and the sample pretreatment steps in the above Example 1 were used to prepare nine single medicinal material sample solutions.

[0217] Adopting steps 2 and 3 of the detection method of the fingerprint of Baiziren Pills in Example 1, the test sample solution and 9 single medicinal material sample solutions were measured respectively, and the fingerprints of the test sample solution and 9 single medicinal material sample solutions were obtained respectively. The obtained fingerprints of the test sample solution and 9 single medicinal material sample solutions were imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" 2012 version software issued by the State Pharmacopoeia Committee for analysis and processing, and at the same time, the detection method of the fingerprint of Baiziren Pills in Example 1 was adopted to obtain the control fingerprint of Baiziren Pills. The fingerprints of the test sample solution and 9 single medicinal material sample solutions were compared with the control fingerprint of Baiziren Pills, and the corresponding characteristic peaks of the 9 single medicinal material sample solutions in the control fingerprint of Baiziren Pills were identified by relative retention time, so as to attribute and locate the characteristic peaks in the fingerprint of the 9 single medicinal material sample solutions. The specific results are shown in FIG. Figure 4 .

[0218] Depend on Figure 4It can be seen that among the single herbal sample solutions, the fingerprint peaks shared by the sample solutions of 9 herbs, including Platycladus Semen, Pinellia Rhizoma, Jujube, Schisandra Chinensis, Atractylodes Macrocephala, Codonopsis Pilosula, Ephedra Root, Oyster, and Wheat Bran, are shown in Table 2. Specifically, it was determined that peaks 1 (schisandrin A), 2, 6 (schisandrin A), and 14 all originated from Schisandra Chinensis; peaks 3, 4, and 5 (atractylodes lactone I) all originated from Atractylodes Macrocephala; peaks 7 and 15 all originated from Pinellia Rhizoma; peak 8 (schisandrin B) originated from Schisandra Chinensis and Pinellia Rhizoma; peak 9 originated from Schisandra Chinensis and Jujube; peak 10 (α-linolenic acid) originated from Platycladus Semen, Schisandra Chinensis, Pinellia Rhizoma, Atractylodes Macrocephala, and Codonopsis Pilosula; peaks 11 and 13 originated from Platycladus Semen; and peak 12 (linoleic acid) originated from Pinellia Rhizoma, Platycladus Semen, Schisandra Chinensis, Atractylodes Macrocephala, Codonopsis Pilosula, and wheat bran.

[0219] It can be seen that the chemical characteristic peaks of the nine medicinal materials in Baiziren Pills except ephedra root and oyster are well reflected in the fingerprint spectrum and their attribution is confirmed.

[0220] In summary, the present invention provides a method for detecting the fingerprint of Baiziren Pills and its application. An HPLC fingerprint and multi-component quantitative analysis method for Baiziren Pills were established. Seventeen common characteristic peaks were identified, the sources of the common peaks were analyzed, six components were identified, and these six chemical components (schisandrin A, atractylodes lactone I, schisandrin A, schisandrin B, α-linolenic acid, and linoleic acid) were quantitatively analyzed. Similarity analysis was used to evaluate the fingerprints of different batches of Baiziren Pills and analyze their contents. This method is stable, reliable, and has good precision and repeatability, providing a scientific experimental basis for better establishing a comprehensive quality control and evaluation system for Baiziren Pills. Therefore, the present invention overcomes the various shortcomings of the prior art and has high industrial utilization value.

[0221] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Anyone skilled in the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by one of ordinary skill in the art without departing from the spirit and technical principles disclosed herein are intended to be covered by the claims of the present invention.

Claims

1. A method for detecting the fingerprint of Baiziren Pills, comprising the following steps: 1) Preparation of test solution: Dissolve the Baiziren Wan sample in solvent, perform ultrasonic extraction, cool, filter, and obtain the test solution by taking the filtrate. 2) Preparation of reference solution: Dissolve the reference substances of schisandrin A, atractylodes lactone I, schisandrin A, schisandrin B, α-linolenic acid, and linoleic acid in solvent by ultrasonication and then dilute to volume to prepare the reference solution. 3) Determination: The test solution and the reference solution are respectively determined by high performance liquid chromatography under the same chromatographic conditions to obtain fingerprints of the test solution and the reference solution. The fingerprints of the test solution and the reference solution are compared to identify and locate the index components in the fingerprints of the test solution, thereby obtaining the fingerprint of Baiziren Wan. In steps 1) and 2), the solvent is methanol; In step 3), the chromatographic column used in the high performance liquid chromatography method is an Agilent TC-C18 chromatographic column, 4.6 mm×250 mm, 5 μm; In step 3), the chromatographic conditions of the high performance liquid chromatography method include: a mobile phase of methanol-0.05-0.2% formic acid aqueous solution, wherein phase A is methanol and phase B is 0.05-0.2% formic acid aqueous solution; an analysis time of 45 minutes; and gradient elution; The analysis is performed using wavelength switching, where: When the retention time was 0-15 min, the detection wavelength was 254 nm; When the retention time was 15-23 min, the detection wavelength was 275 nm; When the retention time was 23-45 min, the detection wavelength was 205 nm; The specific procedure of the gradient elution is: 0-5min, volume ratio of phase A:phase B was 70:30-70:30; 5-15 min, the volume ratio of phase A:phase B is 70:30-83:17; 15-23 min, the volume ratio of phase A:phase B is 83:17-83:17; 23-25 min, the volume ratio of phase A:phase B is 83:17-90:10; 25-45min, the volume ratio of phase A:phase B is 90:10-90:

10.

2. The method for detecting the fingerprint of Baiziren Pills according to claim 1, wherein: In step 1) or 2), any one or more of the following conditions are included: A1) In step 1), the Baiziren pill sample should be subjected to 60 Co-γ ray irradiation; 60 The dose of Co-γ ray irradiation is 3-10KGy; A2) In step 1), the ratio of the weight of the added Baiziren pill sample to the volume of the added solvent is 1:5-15, g / mL; A3) In step 1), the ultrasonic extraction time is 20-40 minutes; A4) In step 1) or 2), the power of the ultrasound is 50-150 W, and the frequency of the ultrasound is 30-50 kHz; A5) In step 1), the weight needs to be re-weighed after cooling, and the weight loss is compensated with solvent; A6) In step 1), the filtration is to take the supernatant through the membrane, discard the initial filtrate, and take the subsequent filtrate; A7) In step 2), the extraction time of the ultrasonic dissolution is 5-15 minutes.

3. The method for detecting the fingerprint of Baiziren Pills according to claim 1, wherein: In step 3), the chromatographic conditions of the high performance liquid chromatography method include any one or more of the following conditions: B1) The detector is a photodiode array detector; B2) Column temperature is 30-40°C; B3) The injection volume is 5-15 μL; B4) The flow rate is 0.8-1.2 mL / min.

4. Use of the method for detecting the fingerprint of Baiziren pills according to any one of claims 1 to 3 in the quality detection of ingredients in Baiziren pills.

5. A quality detection method for Baiziren pills, comprising obtaining a fingerprint of Baiziren pills using the detection method for Baiziren pills fingerprint according to any one of claims 1 to 3, and comparing the obtained fingerprint of Baiziren pills with a control fingerprint of Baiziren pills obtained under the same fingerprint detection conditions for similarity.

6. The quality inspection method of Baiziren pills according to claim 5, characterized in that: The control fingerprint of Baiziren Pills was obtained under the same conditions as the detection method of the fingerprint of Baiziren Pills according to any one of claims 1 to 3. The control fingerprint of Baiziren Pills included 17 common fingerprint peaks, with peak 10 as the reference peak S peak, the relative retention time was 1.0000, and the relative retention times of the other 16 common fingerprint peaks were peak 1 0.2334±0.0002, peak 2 0.3270±0.0004, peak 3 0.4256±0.0006, peak 4 0.4590±0.0006, peak 5 0.5455±0.0 003, peak 6 0.5841±0.0002, peak 7 0.6629±0.0003, peak 8 0.6875±0.00023, peak 9 0.7834±0.0094, peak 11 1.0745±0.0002, peak 12 1.0985±0.0002, peak 13 1.1936±0.0003, peak 14 1.2196±0.0007, peak 15 1.2529±0.0004, peak 16 1.2982±0.0007, peak 17 1.3792±0.0009.

7. A method for determining the contents of six components in Baiziren pills, comprising the following steps: a) Preparation of the test solution: the same as step 1) of the method for detecting the fingerprint of Baiziren Pills according to any one of claims 1 to 3; b) Preparation of reference solution: the same as step 2) of the method for detecting the fingerprint of Baiziren Pills according to any one of claims 1 to 3; c) Determination: Using high performance liquid chromatography under the same chromatographic conditions as in the method for detecting the fingerprint of Baiziren Pills according to any one of claims 1 to 3, respectively determine the test solution of step a) and the reference solution of step b), and calculate the contents of six components in the test solution: schisandrin A, atractylodes lactone I, schisandrin A, schisandrin B, α-linolenic acid, and linoleic acid using the external standard method.

8. A method for screening fingerprints of multiple medicinal materials in Baiziren Pills, comprising the following steps: A) Preparation of single medicinal material sample solutions: preparing any one or more of the nine medicinal material samples of Baiziren Pills, including Baiziren, Pinellia, Jujube, Schisandra, Atractylodes, Codonopsis, Ephedra, Oyster, and Wheat Bran, according to step 1) of the method for detecting the fingerprint of Baiziren Pills according to any one of claims 1 to 3, to obtain at least one single medicinal material sample solution; B) Determination: Determine the single medicinal material sample solution by high performance liquid chromatography under the same chromatographic conditions as in step 3) of the method for detecting the fingerprint of Baiziren Pills according to any one of claims 1 to 3 to obtain a fingerprint of the single medicinal material sample solution; C) Obtaining a reference fingerprint: Obtaining a reference fingerprint of Baiziren Pills using the same steps as the method for detecting the fingerprint of Baiziren Pills according to any one of claims 1 to 3; D) Quality Inspection: The fingerprint of the single medicinal material sample solution was compared with the reference fingerprint of Baiziren Pills. The corresponding characteristic peaks in the reference fingerprint of Baiziren Pills were identified by relative retention time, thereby attributing and locating the characteristic peaks in the fingerprint of the single medicinal material sample solution. The reference fingerprint of Baiziren Pills includes 17 common fingerprint peaks. Peak 1 was identified as the fingerprint peak of schisandrin A, peak 5 as the fingerprint peak of atractylodes lactone I, peak 6 as the fingerprint peak of schisandrin A, peak 8 as the fingerprint peak of schisandrin B, peak 10 as the fingerprint peak of α-linolenic acid, and peak 12 as the fingerprint peak of linoleic acid.

9. The method for screening fingerprints of multiple medicinal materials in Baiziren Pills according to claim 8, characterized in that: In step D), any one or more of the following conditions are included: C1) The fingerprint of the Semen Platycladi sample solution includes four common fingerprint peaks, which are Peak 10, Peak 11, Peak 12, and Peak 13; C2) The fingerprint of the Pinellia ternata sample solution includes 5 common fingerprint peaks, which are peak 7, peak 8, peak 10, peak 12, and peak 15; C3) The fingerprint of the jujube sample solution includes one common fingerprint peak, which is Peak 9; C4) The fingerprint of the Schisandra chinensis sample solution includes 8 common fingerprint peaks, wherein the 8 common fingerprint peaks are peak 1, peak 2, peak 6, peak 8, peak 9, peak 10, peak 12, and peak 14; C5) The fingerprint of the Atractylodes macrocephala sample solution includes 5 common fingerprint peaks, which are peak 3, peak 4, peak 5, peak 10, and peak 12; C6) The fingerprint of the Codonopsis pilosula sample solution includes two common fingerprint peaks, which are peak 10 and peak 12; C7) The fingerprint of the wheat bran sample solution includes one common fingerprint peak, which is peak 12; C8) The fingerprints of the ephedra root sample solution and the oyster sample solution do not include a common fingerprint peak.

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