Preparation method of sugar-free compound acanthopanax granules as well as fingerprint spectrum and multi-component content determination method of sugar-free compound acanthopanax granules

Abstract

The invention provides a preparation method of sugar-free compound acanthopanax granules. The invention also provides a sugar-free compound acanthopanax granule and a detection method of the fingerprint spectrum of the sugar-free compound acanthopanax granule. The invention further provides a construction method of the standard fingerprint spectrum of the sugar-free compound acanthopanax granules. The invention also provides a quality detection method of the fingerprint spectrum of the sugar-free compound acanthopanax granules. The invention further provides a method for detecting the content of eight components in the sugar-free compound acanthopanax granules. According to the preparation method of the sugar-free compound acanthopanax granules as well as the fingerprint spectrum and the multi-component content determination method of the sugar-free compound acanthopanax granules, the sugar-free compound acanthopanax granules can be prepared, the high performance liquid fingerprint spectrum of the sugar-free compound acanthopanax granules is established, index components can be identified, the content can be determined, and the quality of the granules is improved. The quality of the sugar-free compound acanthopanax granules is favorably controlled.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of traditional Chinese medicine preparation, quality control and detection of its components, and relates to a preparation method of sugar-free compound acanthopanax senticosus granules, a fingerprint spectrum thereof and a multi-component content determination method, in particular to a preparation method of sugar-free compound acanthopanax senticosus granules, a detection method of a fingerprint spectrum of sugar-free compound acanthopanax senticosus granules, a construction method of a standard fingerprint spectrum of sugar-free compound acanthopanax senticosus granules, a quality detection method of a fingerprint spectrum of sugar-free compound acanthopanax senticosus granules and a high-performance liquid chromatography detection method of index components in sugar-free compound acanthopanax senticosus granules. BACKGROUND

[0002] Compound acanthopanax senticosus granules (referring to Chinese invention patent publication No. CN118787697A) is a proprietary product developed by Heilongjiang Quanle Pharmaceutical Co., Ltd., which is composed of 925-1000 parts of acanthopanax senticosus and 25-100 parts of schisandra chinensis. It has remarkable curative effect on anxiety and depression, and its anti-anxiety and anti-depression efficacy is better than that of western medicine fluoxetine hydrochloride dispersible tablets, and has no obvious toxic and side effects. However, due to the presence of sucrose in the auxiliary material, it has limitations for people who need to control blood sugar.

[0003] The auxiliary material of the existing compound acanthopanax senticosus granules accounts for 95%, and the granulation method of wet granulation is adopted. In order to ensure the molding rate, stability and solubility of the preparation, there are great challenges in the investigation of the auxiliary material prescription and the granulation process. At present, there is no sugar-free granule auxiliary material with similar auxiliary material ratio for reference.

[0004] In addition, the combination of fingerprint spectrum and multi-component quantification realizes the "double-dimensional" evaluation of traditional Chinese medicine quality control: the fingerprint spectrum reflects the distribution rule of component groups through the overall chromatographic characteristics, ensuring the batch consistency and authenticity identification of the product; the multi-component quantification accurately determines the content of key active ingredients, ensuring the stability of the pharmacodynamic material basis. The combination of the two can not only reflect the overall component distribution of the compound, but also meet the needs of modern drug supervision for quantitative indicators.

[0005] At present, Yang Wengweng published "Compound Acanthopanax senticosus Granules HPLC fingerprint and multi-component content determination research", which uses HPLC method to establish the fingerprint of compound Acanthopanax senticosus granules, and determines the content of 6 components such as syringin, acanthopanax saponin E, isofraxidin, schisandrol A, schisandrin A, schisandrin B. At the same time, the patent CN202211720108.9 discloses a method for simultaneously determining the content of syringin, acanthopanax saponin E, isofraxidin, schisandrol A, schisandrol B, schisantherin A, schisantherin B. But the above two methods have long detection time (90-125 minutes), and only detect and quantify part of the components of Acanthopanax senticosus and Schisandra chinensis, without involving organic acid components such as chlorogenic acid and cryptochlorogenic acid. In fact, in addition to the lignan glycosides such as syringin and acanthopanax saponin E contained in Acanthopanax senticosus and the biphenyl cyclooctene lignans such as schisandrol A contained in Schisandra chinensis as the main effective material basis, the sugar-free compound Acanthopanax senticosus granules also contain organic acids such as chlorogenic acid. In the plant secondary metabolic pathway, chlorogenic acid, as an intermediate product of phenylpropanoid and phenolic compound biosynthesis pathway, provides structural precursors (substrate sources) and regulation signals for glycosylation and modification of phenylpropanoid glycosides such as syringin, and is an important bridge in the phenylpropanoid network of compound Acanthopanax senticosus. In addition, chlorogenic acid components also have good anti-depression and anti-anxiety effects, and have quantitative significance in the content of compound Acanthopanax senticosus. Therefore, it is necessary to combine the three components in the form of fingerprint and multi-component quantification for overall characterization, and provide quality control basis for compound Acanthopanax senticosus preparation. SUMMARY

[0006] In view of the above-mentioned shortcomings of the prior art, the purpose of the present application is to provide a preparation method of sugar-free compound Acanthopanax senticosus granules and its fingerprint and multi-component content determination method, which can prepare sugar-free compound Acanthopanax senticosus granules, and establish a detection method of sugar-free compound Acanthopanax senticosus granules fingerprint and its application of high performance liquid chromatography fingerprint, realize the detection of multi-components in sugar-free compound Acanthopanax senticosus granules, and have important significance for the quality stability of sugar-free compound Acanthopanax senticosus granule products.

[0007] To achieve the above-mentioned purposes and other related purposes, the first aspect of the present application provides a preparation method of sugar-free compound Acanthopanax senticosus granules, which comprises the following steps:

[0008] S1) The binder is divided into a first part and a second part, the Acanthopanax extract and the Schisandra chinensis extract are added to the first part of the binder and mixed uniformly to obtain a liquid medicine mixture;

[0009] S2) The flavoring agent, the first filler, the absorbent and the second filler are mixed in turn and then added to the liquid medicine mixture for mixing, and then the second part of the binder is added to prepare wet granules, which are dried and sized to obtain the required sugar-free compound Acanthopanax senticosus granules.

[0010] The second aspect of the present application provides a sugar-free compound acanthopanax senticosus granule prepared by the above method.

[0011] The third aspect of the present application provides a method for detecting the fingerprint spectrum of the sugar-free compound acanthopanax senticosus granule, comprising the following steps:

[0012] 1) Preparation of the test sample solution: the sugar-free compound acanthopanax senticosus granule sample is added into the first solvent, ultrasonically extracted, cooled, filtered, and the filtrate is taken as the test sample solution;

[0013] 2) Preparation of the mixed control sample solution: at least one control sample of acanthopanoside E, isofraxidin, syringa, chlorogenic acid, cryptochlorogenic acid, neochlorogenic acid, schisantherin A, protocatechuic acid, protocatechuic aldehyde, schisantherin B, schisantherin E, deoxyschizandrin is dissolved in the first solvent, and schisantherin F is dissolved in the second solvent, and the obtained single standard control sample stock solution is mixed, dissolved in the first solvent, and then diluted to obtain the mixed control sample solution;

[0014] 3) Determination: the test sample solution and the mixed control sample solution are determined by high performance liquid chromatography (HPLC) under the same chromatographic conditions, the fingerprint spectrum of the test sample solution and the fingerprint spectrum of the mixed control sample solution are obtained, the fingerprint spectrum of the test sample solution is compared with the fingerprint spectrum of the mixed control sample solution, and the index components in the fingerprint spectrum of the test sample solution are attributed and positioned, so as to obtain the fingerprint spectrum of the sugar-free compound acanthopanax senticosus granule.

[0015] The fourth aspect of the present application provides a method for constructing the standard fingerprint spectrum of the sugar-free compound acanthopanax senticosus granule, comprising: detecting multiple batches of sugar-free compound acanthopanax senticosus granule samples by the above method for detecting the fingerprint spectrum of the sugar-free compound acanthopanax senticosus granule, obtaining the fingerprint spectrum of the multiple batches of sugar-free compound acanthopanax senticosus granule samples, generating a common mode control spectrum, taking the chromatographic peaks existing in the spectrum as the common characteristic peaks, determining the relative retention time of the common characteristic peaks, the ratio of the area of each common characteristic peak to the total peak area, and according to the relative retention time, attributing and positioning the index components in the fingerprint spectrum of the sugar-free compound acanthopanax senticosus granule, and establishing the standard fingerprint spectrum of the sugar-free compound acanthopanax senticosus granule.

[0016] The fifth aspect of the present application provides a quality detection method for the fingerprint spectrum of the sugar-free compound acanthopanax senticosus granule, comprising: obtaining the fingerprint spectrum of the sugar-free compound acanthopanax senticosus granule by the above method for detecting the fingerprint spectrum of the sugar-free compound acanthopanax senticosus granule, and comparing the similarity with the standard fingerprint spectrum of the sugar-free compound acanthopanax senticosus granule obtained by the above method for constructing the standard fingerprint spectrum of the sugar-free compound acanthopanax senticosus granule.

[0017] The sixth aspect of the present application provides a method for detecting the content of eight components in the sugar-free compound acanthopanax senticosus granules, comprising the following steps:

[0018] A) Preparation of test sample solution: same as step 1) of the detection method of the sugar-free compound acanthopanax senticosus granules fingerprint;

[0019] B) Preparation of mixed control solution: at least one control of acanthopanax senticosus glycoside E, isofraxidin, syringa, chlorogenic acid, cryptochlorogenic acid, neochlorogenic acid, schisantherin A was dissolved in the first solvent and then diluted to volume, and schisandrin B was dissolved in the second solvent and then diluted to volume. The mixed control solution was obtained by mixing the sample of each single control stock solution, dissolving in the first solvent and then diluting to volume.

[0020] C) Determination: using high performance liquid chromatography with the same chromatographic conditions as the detection method of the sugar-free compound acanthopanax senticosus granules fingerprint, the test sample solution of step A) and the mixed control solution of step B) were determined respectively, and the content of eight components in the test sample solution was calculated by external standard method.

[0021] As described above, the present application provides a preparation method of sugar-free compound acanthopanax senticosus granules, its fingerprint and a multi-component content determination method, which has the following beneficial effects:

[0022] (1) The present application optimizes the prescription, changes the type of excipients and optimizes the ratio without changing the specifications of the preparation, and develops a sugar-free compound acanthopanax senticosus granule that meets the requirements of the pharmacopoeia and is suitable for mass production, which can further expand the clinical application population.

[0023] (2) The present application establishes the fingerprint of the sugar-free compound acanthopanax senticosus granule by high performance liquid chromatography, with a total of 19 common peaks, and 13 characteristic peaks are qualitatively identified, which reflects the contribution of different categories of chemical components to the fingerprint of the sugar-free compound acanthopanax senticosus granule from different aspects, thereby realizing the detection of the whole chemical components of the sugar-free compound acanthopanax senticosus granule, and comprehensively reflecting the quality change of the sugar-free compound acanthopanax senticosus granule.

[0024] (3) The present application also establishes a simultaneous quantitative detection method for cryptochlorogenic acid, chlorogenic acid, syringa, neochlorogenic acid, isofraxidin, acanthopanax senticosus glycoside E, schisantherin A and schisandrin B in the sugar-free compound acanthopanax senticosus granule, which can effectively detect multiple index components in the sugar-free compound acanthopanax senticosus granule. The standard curve of the eight components measured by the method has good linear relationship in the respective range, good repeatability, high accuracy and high precision.

[0025] (4) The present application combines quantitative method with fingerprint method, further shortens the detection time (about 60 minutes), greatly saves the economic cost and time cost of preparation detection, has important significance for the quality stability of compound acanthopanax granules, can improve the quality control and market competitiveness. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 The standard fingerprint spectrum of the sugar-free compound acanthopanax granules of the present application is shown.

[0027] Figure 2 The HPLC superimposed spectrum of the standard fingerprint spectrum of the mixed reference substance and the sugar-free compound acanthopanax granules of the present application is shown, wherein HB is the mixed reference substance, R is the standard fingerprint spectrum; (13) indicates 13 identifiable characteristic peaks.

[0028] Figure 3 The HPLC superimposed fingerprint spectrum of 15 batches of sugar-free compound acanthopanax granule samples of the present application is shown, wherein R is the standard fingerprint spectrum; S1-S15 are 15 batches of samples, (19) indicates 19 common characteristic peaks. DETAILED DESCRIPTION

[0029] The present application inventors develop a preparation method of sugar-free compound acanthopanax granules and a fingerprint spectrum and multi-component content determination method thereof, which are specifically described as follows.

[0030] The present application provides a preparation method of sugar-free compound acanthopanax granules in the first aspect, which comprises the following steps:

[0031] S1) The binder is divided into a binder first part and a binder second part, the acanthopanax extract and the schisandra chinensis extract are added into the binder first part and mixed uniformly to obtain a medicinal liquid mixture;

[0032] S2) The flavoring agent, the first filler, the absorbent and the second filler are sequentially mixed and then added into the medicinal liquid mixture for mixing, and then the binder second part is added to prepare wet granules, which are dried, sized and then obtained as the required sugar-free compound acanthopanax granules.

[0033] In step S1), the binder is an ethanol aqueous solution with a volume percentage of 5-50%, preferably an ethanol aqueous solution with a volume percentage of 5-30%, and more preferably an ethanol aqueous solution with a volume percentage of 10%.

[0034] In step S1), the volume ratio of the binder first part to the binder second part is 0-30:0-50.

[0035] In step S1), the extract of acanthopanax is commercially available and can be purchased from the market, specifically the extract of acanthopanax produced by Heilongjiang Quanle Pharmaceutical Co., Ltd. Alternatively, the extract of acanthopanax can be prepared according to the preparation method of the extract of acanthopanax in the first part of Chinese Pharmacopoeia, specifically, 1000 g of acanthopanax is crushed into coarse powder, decocted twice for 3 hours each time, the decoction is combined and filtered, and the filtrate is concentrated into 50 g of extract (water extract), which is obtained.

[0036] In step S1), the extract of schisandra is commercially available and can be purchased from the market, specifically the extract of schisandra produced by Heilongjiang Quanle Pharmaceutical Co., Ltd. Alternatively, the extract of schisandra can be prepared, specifically, schisandra is added with 1 times the amount of 30% ethanol aqueous solution to reflux extract twice, the first time for 4 h and the second time for 3 h, the extract is combined, filtered through a 100-mesh filter, ethanol is recovered, and the extract is concentrated into an extract with a relative density of 1.25-1.35 (80°C); then the extract is diluted with 30% ethanol aqueous solution to 1 ml of extract corresponding to 1 g of schisandra, allowed to stand, filtered through a 200-mesh filter, allowed to stand at room temperature for 12 h or more, loaded into a 50 mL centrifuge tube, centrifuged at 2000 rpm for 10 min, and the supernatant is taken, which is the extract of schisandra.

[0037] In step S1), the ratio of the mass g of the extract of acanthopanax to the volume mL of the extract of schisandra is 1:2.

[0038] In step S1), the ratio of the mass g of the extract of acanthopanax to the volume mL of the first part of the binder is 5-10:0-30, preferably 5-10:10-30, and more preferably 5-10:30.

[0039] In step S2), the flavoring agent is selected from one of aspartame, sodium cyclamate, or stevioside, and is preferably aspartame.

[0040] In step S2), the absorbent is soluble starch.

[0041] In step S2), the first filler is malt dextrin.

[0042] In step S2), the second filler is corn dextrin.

[0043] In step S2), the ratio of the mass of the extract of acanthopanax to the mass of the flavoring agent, the first filler, and the absorbent is 50-100:1-3:90-110:250-300, and is preferably 50-100:2:100:250.

[0044] In step S2), the ratio of the mass of the first filler to the mass of the second filler is maintained at 1:5-7, and is preferably 1:6.

[0045] In step S2), the drying temperature is 40-60°C, preferably 60°C.

[0046] In step S2), the drying is performed in an electric heating air drying oven.

[0047] In step S2), the drying time is 0.5-2h, preferably 1.5h.

[0048] In step S2), the addition of the second part of the binder adjusts the stickiness.

[0049] In step S2), the wet granulation, granulation and sizing are all conventional wet granulation, granulation and sizing processes in the art.

[0050] The second aspect of the present application provides a sugar-free compound acanthopanax senticosus granule prepared by the above method.

[0051] The third aspect of the present application provides a method for detecting the fingerprint spectrum of a sugar-free compound acanthopanax senticosus granule, comprising the following steps:

[0052] 1) Preparation of test sample solution: the sugar-free compound acanthopanax senticosus granule sample is added to the first solvent, ultrasonically extracted, cooled, filtered, and the filtrate is taken as the test sample solution;

[0053] 2) Preparation of mixed control solution: at least one control substance of acanthopanax senticosus glycoside E, isofraxidin, syringic glycoside, chlorogenic acid, cryptochlorogenic acid, neochlorogenic acid, schisantherin A, protocatechuic acid, protocatechuic aldehyde, schisantherin B, schisantherin C, deoxyschizandrin is added to the first solvent to dissolve and dilute, and then schizandrin B is added to the second solvent to dissolve and dilute, the obtained single standard control substance stock solution is sampled and mixed, and the first solvent is added to dissolve and dilute, to obtain the mixed control solution;

[0054] 3) Determination: the test sample solution and the mixed control solution are determined by high performance liquid chromatography (HPLC) under the same chromatographic conditions, the fingerprint spectrum of the test sample solution and the fingerprint spectrum of the mixed control solution are obtained, the fingerprint spectrum of the test sample solution is compared with the fingerprint spectrum of the mixed control solution, the index components in the fingerprint spectrum of the test sample solution are attributed and positioned, and thus the fingerprint spectrum of the sugar-free compound acanthopanax senticosus granule is obtained.

[0055] In step 1), the sugar-free compound acanthopanax senticosus granule sample is precisely weighed before being added to the first solvent.

[0056] In step 1) or 2), the first solvent is 50% methanol aqueous solution.

[0057] In step 1), the ratio of the mass g of the sample of the sugar-free compound Shu-Jia-Gui granules to the volume mL of the first solvent is 1:15-35, preferably 1:25.

[0058] In step 1), the sample of the sugar-free compound Shu-Jia-Gui granules needs to be precisely weighed after being added with the first solvent.

[0059] In step 1), the time of the ultrasonic extraction is 10-35 min, preferably 30 min.

[0060] In step 1), the power of the ultrasonic extraction is 350-500 W, and the frequency of the ultrasonic extraction is 40-60 kHz. In an embodiment, the power of the ultrasonic extraction is 500 W, and the frequency of the ultrasonic extraction is 50 kHz.

[0061] In step 1), the cooling is at room temperature. The room temperature is 20-30℃.

[0062] In step 1), the sample needs to be precisely weighed again after the cooling, and the weight loss needs to be made up with the first solvent.

[0063] In step 1), the filtration is to take the supernatant to filter membrane. In an embodiment, the filter membrane is 0.22 μm microporous filter membrane.

[0064] In step 1), the continued filtrate is the filtrate collected after the initial filtrate is discarded.

[0065] In step 2), the CAS number of Shu-Jia-Gui E is 39432-56-9, the CAS number of isofraxidin is 486-21-5, the CAS number of syringin is 118-34-3, the CAS number of chlorogenic acid is 327-97-9, the CAS number of cryptochlorogenic acid is 905-99-7, the CAS number of neochlorogenic acid is 906-33-2, the CAS number of schisanol A is 7432-28-2, the CAS number of protocatechuic acid is 99-50-3, the CAS number of protocatechuic aldehyde is 139-85-5, the CAS number of schisanol B is 58546-54-6, the CAS number of schisantherin A is 58546-56-8, the CAS number of deoxyschizandrin is 61281-38-7, and the CAS number of γ-schisandrin is 61281-37-6.

[0066] In step 2), the second solvent is methanol.

[0067] In step 3), the fingerprint spectrum of the test sample solution is compared with the fingerprint spectrum of the mixed control sample solution. According to the known characteristic peaks in the fingerprint spectrum of the mixed control sample solution, the corresponding characteristic peaks in the fingerprint spectrum of the test sample solution are identified by the relative retention time, so as to attribute and locate the index components in the fingerprint spectrum of the test sample solution.

[0068] In step 3), the chromatographic column in the high performance liquid chromatography is C 18 Chromatographic column.

[0069] In an embodiment, the chromatographic column is ZORBAX Eclipse XDB-C 18 Chromatographic column (250 μm x 4.6 mm, 5 μm) with octadecylsilane bonded silica gel as the filler, produced by Agilent Corporation of the United States.

[0070] In step 3), the detector in the high performance liquid chromatography is an ultraviolet-visible light detector (UV) or a photodiode array detector (DAD).

[0071] In step 3), the column temperature in the high performance liquid chromatography is 20-30°C, preferably 25°C.

[0072] In step 3), the injection volume in the high performance liquid chromatography is 10-30 μL, preferably 20 μL.

[0073] In step 3), the flow rate in the high performance liquid chromatography is 0.9-1.1 mL / min, preferably 1.0 mL / min.

[0074] In step 3), the detection wavelength in the high performance liquid chromatography is 203-240 nm, preferably 220 nm.

[0075] In step 3), the mobile phase in the high performance liquid chromatography is acetonitrile-0.05-0.15% phosphoric acid aqueous solution; wherein phase A is acetonitrile; phase B is 0.05-0.15% phosphoric acid aqueous solution, preferably 0.1% phosphoric acid aqueous solution; the analysis time is 60 min; and gradient elution. The 0.05-0.15% phosphoric acid aqueous solution is a 0.05-0.15% phosphoric acid aqueous solution by volume percentage. The 0.1% phosphoric acid aqueous solution is a 0.1% phosphoric acid aqueous solution by volume percentage.

[0076] In an embodiment, the specific procedure of the gradient elution is as shown in Table 1.

[0077] 0-25 min, volume ratio of phase A to phase B: 5:95-11:89;

[0078] 25-26 min, volume ratio of phase A to phase B: 11:89-15:85;

[0079] 26-35min, A phase: B phase volume ratio of 15:85-15:85;

[0080] 35-39min, A phase: B phase volume ratio of 15:85-17:83;

[0081] 39-40min, A phase: B phase volume ratio of 17:83-65:35;

[0082] 40-60min, A phase: B phase volume ratio of 65:35-82:18.

[0083] Table 1

[0084]

[0085] The fourth aspect of the present application provides a method for constructing a standard fingerprint spectrum of sugar-free compound acanthopanax senticosus granules, comprising: detecting a plurality of batches of sugar-free compound acanthopanax senticosus granule samples by using the detection method of the fingerprint spectrum of the sugar-free compound acanthopanax senticosus granules, obtaining the fingerprint spectrum of the plurality of batches of sugar-free compound acanthopanax senticosus granule samples, generating a common mode control spectrum, taking the chromatographic peaks existing in the spectrum as common characteristic peaks, determining the relative retention time of the common characteristic peaks, the ratio of the area of each common characteristic peak to the total peak area, and according to the relative retention time, attributing and positioning the index components in the fingerprint spectrum of the sugar-free compound acanthopanax senticosus granules, and establishing the standard fingerprint spectrum of the sugar-free compound acanthopanax senticosus granules.

[0086] In the above method, the detection batch of the sugar-free compound acanthopanax senticosus granule sample is 15 batches.

[0087] In the above method, in the common mode control spectrum, the similarity of the fingerprint spectra of the plurality of batches of sugar-free compound acanthopanax senticosus granule samples is compared by using the 2012 version software of the Chinese Pharmacopoeia Commission's "Traditional Chinese Medicine Chromatographic Fingerprint Spectrum Similarity Evaluation System".

[0088] In an embodiment, the similarity of the fingerprint spectra of the plurality of batches of sugar-free compound acanthopanax senticosus granule samples is ≥0.950.

[0089] In the above method, the standard fingerprint spectrum of the sugar-free compound acanthopanax senticosus granules is as shown in Figure 1As shown in the figure, the retention time of each common characteristic peak is taken as the relative retention time, 19 common characteristic peaks are included, peak 7 is taken as the reference peak S peak, the retention time is 1.000, and the relative retention times of the other 18 common characteristic peaks are as follows: peak 1 is 0.102-0.124, preferably 0.113; peak 2 is 0.108-0.132, preferably 0.120; peak 3 is 0.142-0.174, preferably 0.158; peak 4 is 0.489-0.598, preferably 0.544; peak 5 is 0.605-0.740, preferably 0.673; peak 6 is 0.759-0.927, preferably 0.843; peak 8 is 0.961-1.174, preferably 1.067; peak 9 is 1.088-1.330, preferably 1.209; peak 10 is 1.317-1.609, preferably 1.463; peak 11 is 1.512-1.848, preferably 1.680; peak 12 is 1.725-2.108, preferably 1.916; peak 13 is 1.849-2.259, preferably 2.054; peak 14 is 1.976-2.415, preferably 2.195; peak 15 is 2.007-2.453, preferably 2.230; peak 16 is 2.057-2.514, preferably 2.285; peak 17 is 2.153-2.631, preferably 2.392; peak 18 is 2.395-2.927, preferably 2.661; and peak 19 is 2.515-3.073, preferably 2.794.

[0090] In an embodiment, in the standard fingerprint spectrum of the sugar-free compound acanthopanax senticosus granules, 13 identifiable characteristic peaks are determined: peak 4 is protocatechuic acid, peak 5 is neochlorogenic acid, peak 6 is protocatechualdehyde, peak 7 is syringin, peak 8 is chlorogenic acid, peak 9 is cryptochlorogenic acid, peak 11 is acanthopanax saponin E, peak 12 is isofraxidin, peak 14 is schisantherin A, peak 15 is schisantherin B, peak 17 is schisantherin ester A, peak 18 is deoxyschizandrin, and peak 19 is schisantherin B.

[0091] The fifth aspect of the present application provides a quality detection method for the sugar-free compound acanthopanax senticosus granule fingerprint spectrum, which comprises: obtaining the sugar-free compound acanthopanax senticosus granule fingerprint spectrum by using the detection method for the sugar-free compound acanthopanax senticosus granule fingerprint spectrum, and comparing the similarity with the standard fingerprint spectrum of the sugar-free compound acanthopanax senticosus granule obtained by using the construction method for the standard fingerprint spectrum of the sugar-free compound acanthopanax senticosus granule.

[0092] In the above method, when the similarity comparison is performed, the software of the Chinese Medicine Chromatographic Fingerprint Spectrum Similarity Evaluation System 2012 version published by the State Pharmacopoeia Commission is used. Preferably, the similarity is ≥0.950.

[0093] The sixth aspect of the present application provides a method for detecting the content of eight components in sugar-free compound acanthopanax granules, comprising the following steps:

[0094] A) Preparation of test sample solution: same as step 1) of the method for detecting the fingerprint of sugar-free compound acanthopanax granules;

[0095] B) Preparation of mixed control solution: at least one control of acanthopanax senticosus glycoside E, isofraxidin, syringa, chlorogenic acid, cryptochlorogenic acid, neochlorogenic acid, and schisantherin A is dissolved in the first solvent and then diluted to volume, and then schisandrin B is dissolved in the second solvent and then diluted to volume, and then the obtained sample control stock solution is sampled and mixed, dissolved in the first solvent and then diluted to volume, to obtain the mixed control solution;

[0096] C) Determination: using high performance liquid chromatography with the same chromatographic conditions as the method for detecting the fingerprint of sugar-free compound acanthopanax granules, the test sample solution of step A) and the mixed control solution of step B) are determined respectively, and the content of eight components in the test sample solution is calculated by external standard method.

[0097] In step B), each single standard control stock solution needs to be stored at 4℃ for standby.

[0098] In step B), the concentration of acanthopanax senticosus glycoside E in each single standard control stock solution is 1.256 mg / mL, the concentration of isofraxidin is 1.276 mg / mL, the concentration of syringa is 1.146 mg / mL, the concentration of chlorogenic acid is 0.843 mg / mL, the concentration of cryptochlorogenic acid is 1.461 mg / mL, the concentration of neochlorogenic acid is 1.356 mg / mL, the concentration of schisantherin A is 0.858 mg / mL, and the concentration of schisandrin B is 0.984 mg / mL.

[0099] In step B), the concentration of acanthopanax senticosus glycoside E in the mixed control solution is 1.93-61.73 μg / mL, the concentration of isofraxidin is 0.40-12.71 μg / mL, the concentration of syringa is 3.51-112.31 μg / mL, the concentration of chlorogenic acid is 2.62-83.79 μg / mL, the concentration of cryptochlorogenic acid is 2.25-72.03 μg / mL, the concentration of neochlorogenic acid is 2.11-67.60 μg / mL, the concentration of schisantherin A is 0.66-21.26 μg / mL, and the concentration of schisandrin B is 0.76-24.21 μg / mL.

[0100] The sugar-free compound acanthopanax granules of the present application contain multiple types of components, including lignans (such as acanthopanax glycoside E, schisandrin A, etc.), phenylpropanoid glycosides (such as syringin), coumarins (such as isofraxidin), and organic acids (such as chlorogenic acid, cryptochlorogenic acid, etc.). The chemical properties of these components are quite different, but they all have certain anti-anxiety and anti-depression effects. For example, syringin can inhibit the activation of NF-κB, reduce the release of pro-inflammatory cytokines such as TNF-α and IL-1β, and inhibit neuroinflammatory response to play an anti-depression role. Chlorogenic acid, cryptochlorogenic acid, and the like can inhibit the NF-κB signaling pathway, activate the Nrf2 pathway, and thus play a role in reducing neuroinflammation, inhibiting neuronal apoptosis, and improving hippocampal synaptic plasticity to play an anti-anxiety and anti-depression role and improve memory behavior. Schisandrin, such as schisandrin A, schisandrin B, and schisantherin B, can significantly reduce the increase in hormone levels such as norepinephrine, dopamine, serotonin, and corticosterone caused by restraint stress in mice, thereby playing an anti-anxiety role. As can be seen, the multiple components contained in the compound acanthopanax have certain anti-depression and anti-anxiety effects. Determining the above components can effectively control the quality of the sugar-free compound acanthopanax granules.

[0101] In step C), the external standard method refers to: respectively taking a series of different volumes of the mixed control solution in step B) to prepare a series of solutions with different concentrations, using a high-performance liquid chromatograph for sample analysis to obtain the linear relationship between the content of the eight components in the mixed control solution and the peak area, and drawing the corresponding standard working curve to calculate the regression equation of each standard working curve. Then, the test sample solution is detected by a high-performance liquid chromatograph, and the chromatographic peak area of each of the eight components in the test sample solution is substituted into the regression equation of each standard working curve to obtain the content of the corresponding component.

[0102] In an embodiment, in the standard working curve, the peak area is used as the ordinate, and the content of the mixed control solution is used as the abscissa.

[0103] In the present application, the water used is pure water.

[0104] The present application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and not to limit the protection scope of the present application.

[0105] The embodiments of the present application are described below through specific examples. Those skilled in the art can easily understand other advantages and effects of the present application from the disclosure of the present specification. The present application can also be implemented or applied through other different specific embodiments, and various modifications or changes can be made to the details in the present specification based on different viewpoints and applications without departing from the spirit of the present application.

[0106] The reagents and instruments used in the following examples are as follows:

[0107] 1. Reagents

[0108] Acanthopanax extract (Heilongjiang Quanle Pharmaceutical Co., Ltd., batch numbers JG23111101, 20221204), Schisandra extract (Heilongjiang Quanle Pharmaceutical Co., Ltd., batch numbers 20241001, 20230209, 20241122, 250527X, 250527S, 250527W), soluble starch (Xi'an Tianzheng Pharmaceutical Auxiliary Material Co., Ltd., batch number 240404, pharmaceutical grade), maltodextrin (Xi'an Jinxing Pharmaceutical Auxiliary Material Co., Ltd., 2024022152, pharmaceutical grade), corn dextrin (Xi'an Jinxing Pharmaceutical Auxiliary Material Co., Ltd., batch number 202409041, pharmaceutical grade), aspartame (103420230201, Hunan Er Kang Pharmaceutical Co., Ltd.), sodium cyclamate (20220403, Hunan Huari Pharmaceutical Co., Ltd.), steviol glycoside (106420221203, Hunan Er Kang Pharmaceutical Co., Ltd.), purified drinking water (Hangzhou Wahaha Group Co., Ltd.), edible ethanol (95%, Henan Hanyong Alcohol Co., Ltd., batch number 24083106, food grade).

[0109] Anhydrous ethanol (batch number 20231010; AR grade; National Pharmaceutical Group Chemical Reagent Co., Ltd.), acetonitrile (batch number I1291707330; HPLC grade; Merck KGaA, Germany), phosphoric acid (batch number 20220823; AR grade; National Pharmaceutical Group Chemical Reagent Co., Ltd.). The reference substances are shown in Table 2 below.

[0110] Table 2

[0111]

[0112] 2. Instruments

[0113] Electric heating air drying oven (Shanghai Yiheng Scientific Instrument Co., Ltd.); ultrasonic cleaner (SK8210HP; Shanghai Kedo Instrument Co., Ltd.); high performance liquid chromatograph (1260 Infinity II; DAD / ELSD detector; Agilent, USA).

[0114] Example 1 Preparation of sugar-free compound Acanthopanax granules

[0115] Take the extract of acanthopanax senticosus 5g and the extract of schisandra chinensis 2.5mL, add 30mL of 10% ethanol, mix well, get the mixture of medicinal liquid. Mix 0.2g aspartame, 10g malt dextrin, 25g soluble starch, 60g corn dextrin in turn, add the mixture of medicinal liquid, mix well, add 0mL of 10% ethanol, make wet granules, granulate, dry at 60℃ for 1.5h, make 100g, size, package, get sugar-free compound acanthopanax senticosus granules sample 1.

[0116] Example 2 Preparation of sugar-free compound acanthopanax senticosus granules

[0117] Take the extract of acanthopanax senticosus 10g and the extract of schisandra chinensis 5mL, add 30mL of 10% ethanol, mix well, get the mixture of medicinal liquid. Mix 0.2g aspartame, 10g malt dextrin, 25g soluble starch, 60g corn dextrin in turn, add the mixture of medicinal liquid, mix well, add 0mL of 10% ethanol, make wet granules, granulate, dry at 60℃ for 1.5h, make 100g, size, package, get sugar-free compound acanthopanax senticosus granules sample 2.

[0118] Example 3 Preparation of sugar-free compound acanthopanax senticosus granules

[0119] Take the extract of acanthopanax senticosus 5g and the extract of schisandra chinensis 2.5mL, add 20mL of 10% ethanol, mix well, get the mixture of medicinal liquid. Mix 0.3g aspartame, 10g malt dextrin, 26g soluble starch, 60g corn dextrin in turn, add the mixture of medicinal liquid, mix well, add 10mL of 30% ethanol, make wet granules, granulate, dry at 60℃ for 1.5h, make 100g, size, package, get sugar-free compound acanthopanax senticosus granules sample 3.

[0120] Example 4 Preparation of sugar-free compound acanthopanax senticosus granules

[0121] Take the extract of acanthopanax senticosus 5g and the extract of schisandra chinensis 2.5mL, add 0mL of 30% ethanol, mix well, get the mixture of medicinal liquid. Mix 0.2g aspartame, 10g malt dextrin, 28g soluble starch, 60g corn dextrin in turn, add the mixture of medicinal liquid, mix well, add 50mL of 30% ethanol, make wet granules, granulate, dry at 55℃ for 2h, make 100g, size, package, get sugar-free compound acanthopanax senticosus granules sample 4.

[0122] Example 5 Preparation of sugar-free compound acanthopanax senticosus granules

[0123] Take the extract of acanthopanax senticosus 10 g and the extract of schisandra chinensis 5 mL, add 10 mL of 5% ethanol, mix well to obtain a mixture of medicinal liquid. Mix 0.25 g of aspartame, 10 g of malt dextrin, 30 g of soluble starch, and 65 g of corn dextrin, then add the mixture of medicinal liquid, mix well, add 20 mL of 5% ethanol, make wet granules, granulate, dry at 50°C for 1 h, make 100 g, sieve, and pack to obtain a sample of sugar-free compound acanthopanax granules 5.

[0124] Investigation of the amount of absorbent in Comparative Example 1

[0125] Take the extract of acanthopanax senticosus 5 g and the extract of schisandra chinensis 2.5 mL, and soluble starch as the absorbent, according to the preparation process and the mass ratio of raw materials in Example 1, investigate the use of 40 g, 35 g, 30 g, 25 g, and 20 g of soluble starch to absorb the medicinal liquid, observe the properties of the soft material, and optimize the ratio of the absorbent. The experimental results are shown in Table 3. As shown in Table 3, the performance of 25-30 g of soluble starch is better.

[0126] Table 3

[0127] Absorbent mass Soft material properties 40g Light color, too low humidity, more dry powder, no wall sticking phenomenon, mixed with filler more evenly, loose after wetting, not easy to granulation 35g Light color, too low humidity, more dry powder, no wall sticking phenomenon, mixed with filler more evenly, loose after wetting, not easy to granulation 30g Medium color, lower humidity, lower viscosity, no wall sticking phenomenon, mixed with filler more evenly, easier to granulation after wetting 25g Medium color, moderate humidity, moderate viscosity, no wall sticking phenomenon, mixed with filler more evenly, easy to granulation after wetting 20g Dark color, too large humidity, large viscosity, serious wall sticking phenomenon, mixed with filler unevenly, not easy to granulation after wetting

[0128] Investigation of the type of binder in Comparative Example 2

[0129] Take the extract of acanthopanax senticosus 5 g and the extract of schisandra chinensis 2.5 mL, and according to the preparation process and the mass ratio of raw materials in Example 1, investigate the use of water, 5% ethanol, 10% ethanol, 50% ethanol, and 90% ethanol as binders for granulation, observe the effects of different binders on the granulation rate and properties of the granules, and repeat the experiment twice. The results are shown in Table 5. As shown in Table 5, the granules prepared with water as the binder have too large particle size and hardness and obvious color spots; the granules prepared with 90% ethanol as the binder have uniform color but too small particle size and hardness, and are prone to loose; the granules prepared with 5-50% ethanol as the binder have better particle size and hardness and are not prone to loose, but have a small amount of color spots; among them, the granules prepared with 10% ethanol as the binder have the best particle size and hardness and no color spots. Overall, 5-50% ethanol solution can be used as the binder, but the granules prepared with 10% ethanol solution as the binder have uniform color and size and moderate hardness.

[0130] Table 4

[0131] Binder Granule properties 90% ethanol Light color, uniform color, small particle size, too low hardness, more fine powder, easy to loose 50％ Dark color, more uniform color, small particle size, lower hardness, not easy to loose 10% ethanol Dark color, uniform color, small particle size, moderate hardness, not easy to loose 5％ Dark color, more uniform color, large particle size, larger hardness, not easy to loose Water Dark color, uneven color, large particle size, too large hardness, not easy to loose

[0132] Investigation of the ratio of fillers in Comparative Example 3

[0133] Take 5 g of ZHIZIYAOWU extract and 2.5 mL of WUWEIZI extract, mix 30 g, 20 g, 10 g of malt dextrin with 40 g, 50 g, 60 g of corn dextrin respectively according to the preparation process and raw material mass ratio of Example 1, mix with soft material, add appropriate amount of 10% ethanol as liquid binder, granulate, measure the molding rate, dissolution rate, moisture absorption rate, angle of repose, 2 times in parallel, the results are shown in Table 5. As can be seen from Table 5, when the ratio of corn dextrin to malt dextrin is 6:1, the granules have a molding rate of more than 90%, and have good fluidity, and the granules have moderate hardness, moderate soft material adhesion and easy granulation.

[0134] Table 5

[0135]

[0136] The above molding rate is calculated according to the second method (double sieve method) in the particle size test method in the fourth part of the 2020 edition of Chinese Pharmacopoeia under the granules item, the particles that can pass through No. 1 sieve (10 mesh) but cannot pass through No. 5 sieve (80 mesh) are qualified, the formula is molding rate = (qualified particle mass / total particle mass) x 100%

[0137] The above dissolution rate is calculated according to the 2020 edition of Chinese Pharmacopoeia, accurately weigh 2 g of qualified particles, add 50 mL of hot water and stir for 5 min, centrifuge, place the precipitate after centrifugation in an already constant weight evaporating dish, dry in a water bath until constant weight at 105℃, weigh the mass, calculate the dissolution rate, the formula is dissolution rate = (1-precipitate mass / particle mass) x 100%

[0138] The above moisture absorption rate is to place the weighing bottle in a glass dryer (relative humidity 75%) containing NaCl supersaturated solution, and equilibrate at 25℃ for 24 h. Weigh an appropriate amount of sample and dry at 105℃ until constant weight, take 2 g of dried sample and place it in a weighing bottle in the dryer until constant weight, weigh the mass after 24 h, calculate the moisture absorption rate of the particles, the formula is moisture absorption rate = (moisture absorption after particle mass-moisture absorption before particle mass) / moisture absorption before particle mass x 100%.

[0139] The above angle of repose refers to the maximum angle formed by the free slope of the powder accumulation layer and the horizontal plane, the smaller the angle of repose, the better the flowability of the particles. It is generally considered that when θ≤30°, the flowability is good, and when θ≤40°, the flowability can meet the needs of the production process. In this study, the BT-1001 intelligent powder property tester was used to test the angle of repose of the particles by the falling method (fixed drop method).

[0140] Example 6 Detection method of fingerprint (DAD)

[0141] 1. Sample pretreatment

[0142] Accurately weigh 1 g of the sample of the sugar-free compound acanthopanax granules prepared in Example 1, accurately weigh, accurately add 25 mL of 50% methanol, tightly seal, weigh, ultrasonically treat (500 W, 50 kHz) for 30 minutes, cool, re-weigh, make up the weight loss with 50% methanol, shake well, filter the supernatant through a 0.22 μm microporous filter, and take the filtrate to obtain a test sample solution 1 # .

[0143] Accurately weigh the reference substances of acanthopanoside E, isofraxidin, syringidin, chlorogenic acid, cryptosin, neochlorogenic acid, schisantherin, protocatechuic acid, protocatechuic aldehyde, schisantherin B, schisantherin A, and deoxyschizandrin, dilute and make up to volume with 50% methanol, accurately weigh the reference substance of schisanhenin, dilute and make up to volume with methanol, take samples of the obtained reference substance solutions of each single marker to mix, dissolve by adding 50% methanol, and make up to volume to obtain a mixed reference substance solution.

[0144] 2. Chromatographic conditions

[0145] The chromatographic conditions of the high performance liquid chromatography are as follows: a chromatographic column: ZORBAX Eclipse XDB-C 18 a chromatographic column (250 μm x 4.6 mm, 5 μm) with octadecylsilane bonded silica gel as the filler; a detector: a photodiode array detector (DAD); a column temperature: 25 °C; an injection volume: 20 μL; a flow rate: 1.0 ml / min; a detection wavelength: 220 nm; a mobile phase: acetonitrile-0.1% phosphoric acid aqueous solution, wherein phase A is acetonitrile and phase B is 0.1% phosphoric acid aqueous solution; an analysis time: 60 min; and gradient elution.

[0146] The specific procedure of the gradient elution is as follows:

[0147] 0-25 min, phase A: phase B in a volume ratio of 5:95-11:89;

[0148] 25-26 min, phase A: phase B in a volume ratio of 11:89-15:85;

[0149] 26-35 min, phase A: phase B in a volume ratio of 15:85-15:85;

[0150] 35-39 min, phase A: phase B in a volume ratio of 15:85-17:83;

[0151] 39-40 min, phase A: phase B in a volume ratio of 17:83-65:35;

[0152] 40-60 min, phase A: phase B in a volume ratio of 65:35-82:18.

[0153] 3. Determination

[0154] High-performance liquid chromatography (HPLC) under the chromatographic conditions described in step 2 above was used to determine the test solution 1 in step 1 above. # Mix the reference solution to obtain the test solution 1. # The fingerprint chromatograms of the test sample solution and the fingerprint chromatograms of the mixed reference solution. The test sample solution 1... # The fingerprint spectrum of the test solution should be compared with that of the mixed reference solution. Based on the known characteristic peaks in the fingerprint spectrum of the mixed reference solution, the test solution 1 should be identified by its relative retention time. # The corresponding characteristic peaks in the fingerprint spectrum, thereby identifying the test sample solution 1 # The index components in the fingerprint spectrum were assigned and located to obtain the fingerprint spectrum of sugar-free compound Acanthopanax senticosus granules.

[0155] Example 7: Fingerprint Spectrum Detection Method (UV)

[0156] 1. Sample pretreatment

[0157] Accurately weigh 1g of the sugar-free compound Acanthopanax senticosus granules prepared in Example 1, accurately add 30mL of 48% methanol, seal tightly, weigh again, sonicate (230W, 55kHz) for 25 minutes, cool, weigh again, replenish the lost weight with 48% methanol, shake well, filter the supernatant through a 0.22μm microporous membrane, and collect the filtrate to obtain the test solution 2. # .

[0158] Weigh out the reference standards of Acanthopanax senticosin E, isozymidine, syringin, chlorogenic acid, cryptochlorogenic acid, neochlorogenic acid, schisandrol A, protocatechuic acid, protocatechuic aldehyde, schisandrol B, schisandrin A, and schisandrin A. Dilute with 48% methanol aqueous solution and bring to volume. Weigh out the reference standard of schisandrin B. Dilute with methanol and bring to volume. Take samples of the stock solutions of each single standard reference standard, mix them, dissolve them in 48% methanol aqueous solution, and bring to volume to obtain the mixed reference standard solution.

[0159] 2. Chromatographic conditions

[0160] In the chromatographic conditions of high performance liquid chromatography, the detector is an ultraviolet-visible detector (UV), and other detection conditions are the same as in step 2 of Example 6.

[0161] 3. Measurement

[0162] High-performance liquid chromatography (HPLC) under the chromatographic conditions described in step 2 above was used to determine the test solution 2 in step 1 above. # 1. Mix the reference solution to obtain the test solution 2. # The fingerprint spectrum of the mixed reference solution. Wherein, the test solution 2 #The fingerprint spectrum of the test solution should be compared with that of the mixed reference solution. Based on the known characteristic peaks in the fingerprint spectrum of the mixed reference solution, the relative retention time should be used to identify the test solution 2. # The corresponding characteristic peaks in the fingerprint spectrum, thereby identifying the test sample solution 2 # The index components in the fingerprint spectrum were assigned and located to obtain the fingerprint spectrum of sugar-free compound Acanthopanax senticosus granules.

[0163] Example 8: Establishment of Standard Fingerprint Spectrum (Similarity Evaluation, Peak Count)

[0164] The detection method for the fingerprint spectrum of sugar-free compound Acanthopanax senticosus granules established in Example 6 was used to detect 15 batches of sugar-free compound Acanthopanax senticosus granules prepared in Example 1 (S1-S15), obtaining fingerprint spectra of the test solution and the mixed reference solution. Specifically, the fingerprint spectra of the test solution and the mixed reference solution were generated by importing them into the 2012 version of the "Similarity Evaluation System for Chromatographic Fingerprint Spectra of Traditional Chinese Medicine" software published by the National Pharmacopoeia Commission. The fingerprint spectra of the test solution and the mixed reference solution were then compared for similarity with the standard fingerprint spectra of sugar-free compound Acanthopanax senticosus granules obtained under the same fingerprint spectrum detection conditions. The similarity of the fingerprint spectra of each batch of sugar-free compound Acanthopanax senticosus granules was calculated, and the results are shown in […]. Figure 3 .

[0165] like Figure 3 As shown, the similarity of the fingerprint spectra of the 15 batches of sugar-free compound Acanthopanax senticosus granules was greater than 0.950, specifically 0.976, 0.963, 0.978, 0.974, 0.981, 0.982, 0.980, 0.968, 0.959, 0.970, 0.982, 0.980, 0.986, 0.986, and 0.988, respectively. There was no increase or loss in the number of peaks, indicating that the fingerprint spectra of the 15 batches of samples had high similarity, the preparation process was stable, and the quality was relatively uniform. Among them, peak 7 has a moderate retention time, good separation, and stable peak area, so it was selected as the reference peak (S) with a retention time of 1.000. The relative retention times of the other 18 common characteristic peaks are as follows: peak 1 is 0.113; peak 2 is 0.120; peak 3 is 0.158; peak 4 is 0.544; peak 5 is 0.673; peak 6 is 0.843; peak 7 is 1. .000; Peak 8 is 1.067; Peak 9 is 1.209; Peak 10 is 1.463; Peak 11 is 1.680; Peak 12 is 1.916; Peak 13 is 2.054; Peak 14 is 2.195; Peak 15 is 2.230; Peak 16 is 2.285; Peak 17 is 2.392; Peak 18 is 2.661; Peak 19 is 2.794. Establish as follows Figure 1 The standard fingerprint spectrum of the sugar-free compound Acanthopanax senticosus granules.

[0166] Establishment of standard fingerprint (peak identification) of Example 9

[0167] The standard fingerprint of the sugar-free compound acanthopanax senticosus granules obtained according to Example 8 was compared with the fingerprint of the mixed reference substance solution. According to the known characteristic peaks in the fingerprint of the mixed reference substance solution, the corresponding characteristic peaks in the standard fingerprint of the sugar-free compound acanthopanax senticosus granules were identified by relative retention time, as shown in Table 1, and 13 identifiable characteristic peaks were located and determined: Peak No. 4 was protocatechuic acid, Peak No. 5 was neochlorogenic acid, Peak No. 6 was protocatechualdehyde, Peak No. 7 was syringin, Peak No. 8 was chlorogenic acid, Peak No. 9 was cryptochlorogenic acid, Peak No. 11 was acanthopanax saponin E, Peak No. 12 was isofraxidin, Peak No. 14 was schisanol A, Peak No. 15 was schisanol B, Peak No. 17 was schisantherin A, Peak No. 18 was schisantherin B, and Peak No. 19 was schisantherin C. Figure 2

[0168] Methodological investigation of fingerprint of Example 10

[0169] 1. Precision

[0170] The same batch of sugar-free compound acanthopanax senticosus granules prepared in Example 1 was taken, and the test sample solution was prepared according to the detection method of the sugar-free compound acanthopanax senticosus granule fingerprint in Example 6 above. After 6 consecutive injections, the retention time and peak area RSD of each component were measured to be less than 1.28%, indicating that the precision of the instrument was good. The obtained chromatograms were input into the similarity evaluation software for similarity comparison, and the similarity of each chromatogram was calculated with the reference fingerprint (S1). The similarity of each chromatogram was greater than 0.99, and the RSD was less than 0.1%, indicating that the precision of the method was good.

[0171] 2. Stability

[0172] The same batch of sugar-free compound acanthopanax senticosus granules prepared in Example 1 was taken, and the test sample solution was prepared according to the detection method of the sugar-free compound acanthopanax senticosus granule fingerprint in Example 6 above. After 6 consecutive injections, the retention time and peak area RSD of each component were measured to be less than 1.28%, indicating that the precision of the instrument was good. The obtained chromatograms were input into the similarity evaluation software for similarity comparison, and the similarity of each chromatogram was calculated with the reference fingerprint (S1). The similarity of each chromatogram was greater than 0.99, and the RSD was less than 0.1%, indicating that the precision of the method was good.

[0173] 3. Repeatability

[0174] ​Take the same batch of sample 1 g of sugar-free compound acanthopanax granules prepared in Example 1, a total of 6, according to the above-mentioned detection method of sugar-free compound acanthopanax granules fingerprint in Example 6, parallel preparation of 6 test solution after detection, the obtained chromatogram into the similarity evaluation software for similarity comparison, with reference to the fingerprint (S1) to calculate the similarity of each chromatogram, the results show that the similarity of each chromatogram is greater than 0.99, and the RSD is less than 0.1%, the repeatability of the method is good.

[0175] Example 4 Investigation of durability

[0176] The sugar-free compound acanthopanax granules sample prepared in Example 1 was detected according to the liquid chromatography detection conditions of Example 6, wherein the detection wavelengths were 210 nm and 220 nm, respectively. The specific detection results are shown in Table 6. As can be seen from Table 6, the 210 nm detection wavelength cannot quantitatively determine cryptochlorogenic acid, while the 220 nm detection wavelength can ensure more components to be detected. Therefore, the detection wavelength is set to 220 nm.

[0177] Table 6

[0178]

[0179] Example 11 Content determination

[0180] 1. Sample pretreatment

[0181] Preparation of test solution: the preparation process of test solution in step 1 of Example 6 is the same, and test solution 1* is obtained.

[0182] Preparation of mixed reference solution: take acanthopanax saponin E, isofraxidin, syringin, chlorogenic acid, cryptochlorogenic acid, neochlorogenic acid and schizandrin A in appropriate amounts, accurately weigh and determine, respectively, in 10 mL volumetric flask, dilute and constant volume with 50% methanol water solution, then take schizandrin B accurately, weigh and determine in 10 mL volumetric flask, dilute and constant volume with methanol, obtain each single standard reference solution. In each single standard reference solution, the concentration of acanthopanax saponin E is 1.256 mg / mL, the concentration of isofraxidin is 1.276 mg / mL, the concentration of syringin is 1.146 mg / mL, the concentration of chlorogenic acid is 0.843 mg / mL, the concentration of cryptochlorogenic acid is 1.461 mg / mL, the concentration of neochlorogenic acid is 1.356 mg / mL, the concentration of schizandrin A is 0.858 mg / mL, and the concentration of schizandrin B is 0.984 mg / mL. Each single standard reference solution needs to be stored at 4℃ in a sealed state for standby.

[0183] The mixed reference solution was prepared by sampling and mixing each single reference solution, dissolving in 50% methanol solution and then diluting to volume. In the mixed reference solution, the concentration of eleutheroside E was 1.93-61.73 μg / mL, the concentration of isofraxidin was 0.40-12.71 μg / mL, the concentration of syringin was 3.51-112.31 μg / mL, the concentration of chlorogenic acid was 2.62-83.79 μg / mL, the concentration of cryptochlorogenic acid was 2.25-72.03 μg / mL, the concentration of neochlorogenic acid was 2.11-67.60 μg / mL, the concentration of schizandrin A was 0.66-21.26 μg / mL, and the concentration of schizandrin B was 0.76-24.21 μg / mL.

[0184] 2. Chromatographic conditions

[0185] The chromatographic conditions of the high performance liquid chromatography were the same as those of the high performance liquid chromatography in step 2 in Example 6.

[0186] 3. Determination

[0187] The external standard method was used, a series of different volumes of the mixed reference solution was taken, and the high performance liquid chromatograph was used for sample analysis to draw a standard working curve. The obtained test sample solution was analyzed by the high performance liquid chromatograph, and the analysis results were substituted into the standard working curve to obtain the content of the eight components in the test sample solution.

[0188] Specifically, a series of different volumes of the mixed reference solution was taken, the high performance liquid chromatograph was used for sample analysis to obtain the linear relationship between the content of the eight components in the reference solution and the peak area, and the chromatographic peak area of each component was substituted into the corresponding content to draw the corresponding standard working curve, and the regression equation of each standard working curve was calculated. The test sample solution was detected by the high performance liquid chromatograph, the chromatographic peak area of the eight components in the obtained test sample solution was substituted into the regression equation of each standard working curve, and the content of the corresponding component was obtained.

[0189] Example 12 Content determination

[0190] 1. Sample pretreatment

[0191] Preparation of the test sample solution: the preparation process of the test sample solution was the same as that in step 1 in Example 7 to obtain test sample solution 2*.

[0192] Preparation of the mixed reference solution: the preparation process of the mixed reference solution was the same as that in step 1 in Example 11.

[0193] 2. Chromatographic conditions

[0194] The chromatographic conditions of the high performance liquid chromatography are the same as those of the high performance liquid chromatography in step 2 in Example 7.

[0195] 3. Assay

[0196] The determination process is the same as that in step 3 in Example 11.

[0197] Investigation of content determination methodology of Example 13

[0198] The single standard substance stock solutions in step 1 in Example 11 were precisely measured, diluted with 50% methanol aqueous solution to become a series of mixed control substance solutions with different mass concentrations, and determined by sample injection under the chromatographic conditions in step 2 in Example 11. The chromatograms were recorded. The standard curve was plotted with the mass concentration of the control substance as the abscissa (X) and the peak area as the ordinate (Y), and linear regression calculation was performed to obtain the regression equation, the correlation coefficient and the linear range. The specific results are shown in Table 7. As shown in Table 7, the correlation coefficients r of oleanolic acid, chlorogenic acid, syringin, neochlorogenic acid, isofraxidin, eleutheroside E, schisandrin A and schisandrin B are 0.9998, 0.9997, 0.9998, 0.9998, 0.9998, 0.9999, 0.9998 and 0.9998, respectively, all of which are not less than 0.9998, indicating that each component has a good linear relationship within its respective range.

[0199] Table 7

[0200]

[0201] Investigation of content determination methodology of Example 14

[0202] 1. Precision

[0203] The mixed control substance solution was prepared according to the method in step 1 in Example 11, and determined by continuous sample injection under the chromatographic conditions in step 2 in Example 11 for 6 times. The specific results are shown in Table 8. As shown in Table 8, the RSDs of the contents of oleanolic acid, chlorogenic acid, syringin, neochlorogenic acid, isofraxidin, eleutheroside E, schisandrin A and schisandrin B in the high, medium and low concentration control substance solutions are all less than 0.60%, indicating that the precision of the instrument is good.

[0204] Table 8

[0205]

[0206] 2. Stability

[0207] The mixed reference solution was prepared according to the method in step 1 of Example 11 above, and was placed for 0, 4, 8, 12, 16, and 24 hours, respectively, and was injected for determination according to the chromatographic conditions in step 2 of Example 11 above. The results are shown in Table 9. As shown in Table 9, the RSDs of the contents of cryptochlorogenic acid, chlorogenic acid, syringin, neochlorogenic acid, isofraxidin, eleutheroside E, schisantherin, and schisantherin B were all less than 2.86%, indicating that the test sample was stable within 24 hours.

[0208] Table 9

[0209]

[0210] 3. Reproducibility

[0211] The same batch of compound schisandra granules sample prepared in Example 1 was taken, 1 g in total and 6 portions, and 6 test sample solutions were prepared in parallel according to the method in step 1 of Example 11 above. The test sample solutions were injected for determination according to the chromatographic conditions in step 2 of Example 11 above. The results are shown in Table 10. As shown in Table 10, the RSDs of the contents of cryptochlorogenic acid, chlorogenic acid, syringin, neochlorogenic acid, isofraxidin, eleutheroside E, schisantherin, and schisantherin B were all less than 1.68%, indicating that the method was reproducible.

[0212] Table 10

[0213]

[0214] 4. Recovery rate

[0215] The known content of sugar-free compound schisandra granules sample was taken, 0.5 g in total and 8 portions, and reference substances corresponding to 50%, 100%, and 150% of the contents of cryptochlorogenic acid, chlorogenic acid, neochlorogenic acid, schisantherin, schisantherin B, syringin, isofraxidin, and eleutheroside E in the test sample were added, respectively. Nine test sample solutions were prepared in parallel according to the method in step 1 of Example 11 above. The test sample solutions were injected for determination according to the chromatographic conditions in step 2 of Example 11 above. The results are shown in Table 11. As shown in Table 11, the recovery rate ranges of cryptochlorogenic acid, chlorogenic acid, syringin, neochlorogenic acid, isofraxidin, eleutheroside E, schisantherin, and schisantherin B were all qualified, and the RSDs were all less than 2.6%, indicating that the method was accurate.

[0216] Table 11

[0217]

[0218]

[0219] In summary, the application provides a preparation method of a sugar-free compound acanthopanax granule, a fingerprint spectrum and a multi-component content determination method, which can prepare the sugar-free compound acanthopanax granule, establish the high performance liquid chromatography fingerprint spectrum of the sugar-free compound acanthopanax granule, identify and determine the content of the index components, and be beneficial to control the quality of the sugar-free compound acanthopanax granule. Therefore, the application overcomes various shortcomings in the prior art and has a high industrial utilization value.

[0220] The above examples only exemplarily illustrate the principles and effects of the application, and are not used to limit the application. Any person skilled in the art can modify or change the above examples without departing from the spirit and scope of the application. Therefore, all equivalent modifications or changes completed by those skilled in the art without departing from the spirit and technical thought disclosed by the application should be covered by the claims of the application.

Claims

1. A method for preparing sugar-free compound Acanthopanax senticosus granules, comprising the following steps: S1) Divide the adhesive into a first part and a second part. Add Acanthopanax senticosus extract and Schisandra chinensis fluid extract to the first part of the adhesive and mix well to obtain a mixture of medicinal liquids. S2) After mixing the flavoring agent, first filler, absorbent and second filler in sequence, add them to the drug liquid mixture and mix. Then add the second part of the binder to make wet granules, dry and granulate to obtain the desired sugar-free compound Acanthopanax senticosus granules.

2. The preparation method of sugar-free compound Acanthopanax senticosus granules according to claim 1, characterized in that, Includes one or more of the following conditions: S11) In step S1), the adhesive is an aqueous ethanol solution with a volume percentage of 5-50%; S12) In step S1), the volume ratio of the first part of the adhesive to the second part of the adhesive is 0-30:0-50; S13) In step S1), the ratio of the mass of Acanthopanax senticosus extract added to the volume of Schisandra chinensis extract added is 1:2, g / mL; S14) In step S1), the ratio of the mass of Acanthopanax senticosus extract added to the volume of the first part of the binder added is 5-10:0-30, g / mL. S21) In step S2), the flavoring agent is selected from one of aspartame, sodium cyclolatate or steviol glycoside; S22) In step S2), the first filler is maltodextrin; S23) In step S2), the absorbent is soluble starch; S24) In step S2), the second filler is corn dextrin; S25) In step S2), the mass ratio of the Acanthopanax senticosus extract to the flavoring agent, the first filler, and the absorbent is 50-100:1-3:90-110:250-300; S26) In step S2), the mass ratio of the first filler to the second filler is maintained at 1:5-7; S27) In step S2), the drying temperature is 40-60°C; S28) In step S2), the drying time is 0.5-2 hours.

3. A sugar-free compound Acanthopanax senticosus granule, prepared by the method described in any one of claims 1-2.

4. A method for detecting the fingerprint spectrum of sugar-free compound Acanthopanax senticosus granules, comprising the following steps: 1) Preparation of test solution: Add sugar-free compound Acanthopanax senticosus granules to the first solvent, extract by ultrasonication, cool, filter, and take the filtrate to obtain the test solution. 2) Preparation of mixed reference solution: At least one of the following reference standards, namely eleutheroside E, isozymidine, syringin, chlorogenic acid, cryptochlorogenic acid, neochlorogenic acid, schisandrol A, protocatechuic acid, protocatechuic aldehyde, schisandrol B, schisandrin A, and schisandrin A, is dissolved in the first solvent and then diluted to volume. Schisandrin B is dissolved in the second solvent and then diluted to volume. Samples of the obtained single-standard reference solutions are mixed, dissolved in the first solvent, and then diluted to volume to obtain the mixed reference solution. 3) Determination: The test solution and the mixed reference solution were determined by high performance liquid chromatography under the same chromatographic conditions to obtain the fingerprint chromatograms of the test solution and the mixed reference solution. The fingerprint chromatograms of the test solution and the mixed reference solution were compared to determine the index components in the fingerprint chromatogram of the test solution, thereby obtaining the fingerprint chromatogram of sugar-free compound Acanthopanax senticosus granules.

5. The method for detecting the fingerprint spectrum of sugar-free compound Acanthopanax senticosus granules according to claim 4, characterized in that, Includes one or more of the following conditions: A1) In step 1) or 2), the first solvent is a 50% aqueous methanol solution; A2) In step 1), the ratio of the mass of the sugar-free compound Acanthopanax senticosus granules added to the volume of the first solvent added is 1:15-35, g / mL; A3) In step 1), the ultrasonic extraction time is 10-35 min; A4) In step 1), the power of the ultrasonic extraction is 350-500W, and the frequency of the ultrasonic extraction is 40-60kHz; A5) In step 1), the filtration is a supernatant filtration membrane; preferably, the filtration membrane is a 0.22μm microporous membrane; A6) In step 2), the second solvent is methanol; A7) In step 3), the chromatographic column in the high-performance liquid chromatography is C18. 18 Chromatographic column; preferably, the chromatographic column is a ZORBAX Eclipse XDB-C. 18 Chromatographic column; A8) In step 3), the detector in the high performance liquid chromatography is an ultraviolet-visible detector or a photodiode array detector; A9) In step 3), the column temperature in the high-performance liquid chromatography is 20-30℃; A10) In step 3), the injection volume in the high-performance liquid chromatography is 10-30 μL; A11) In step 3), the flow rate in the high-performance liquid chromatography is 0.9-1.1 mL / min; A12) In step 3), the detection wavelength in the high-performance liquid chromatography is 203-240 nm; A13) In step 3), the mobile phase in the high performance liquid chromatography is acetonitrile-0.05-0.15% phosphoric acid aqueous solution; wherein, phase A is acetonitrile; phase B is 0.05-0.15% phosphoric acid aqueous solution; the analysis time is 60 min; gradient elution.

6. The method for detecting the fingerprint spectrum of sugar-free compound Acanthopanax senticosus granules according to claim 5, characterized in that, In item A13), the specific procedure for gradient elution is as follows: 0-25 min, the volume ratio of phase A: phase B is 5:95-11:89; 25-26 min, the volume ratio of phase A to phase B is 11:89-15:85; 26-35 min, the volume ratio of phase A to phase B is 15:85-15:85; 35-39 min, the volume ratio of phase A to phase B is 15:85-17:83; 39-40 min, the volume ratio of phase A to phase B is 17:83-65:35; For 40-60 minutes, the volume ratio of phase A to phase B is 65:35-82:

18.

7. A method for constructing a standard fingerprint spectrum of sugar-free compound Acanthopanax senticosus granules, comprising: By employing the detection method for the fingerprint spectrum of sugar-free compound Acanthopanax senticosus granules according to any one of claims 4-6, multiple batches of sugar-free compound Acanthopanax senticosus granule samples were detected to obtain fingerprint spectra of multiple batches of sugar-free compound Acanthopanax senticosus granule samples and generate common pattern control spectra. The chromatographic peaks present in all spectra were taken as common characteristic peaks. The relative retention time of the common characteristic peaks and the ratio of the area of ​​each common characteristic peak to the total peak area were determined. Based on the relative retention time, the index components in the fingerprint spectrum of sugar-free compound Acanthopanax senticosus granules were assigned and located, and a standard fingerprint spectrum of sugar-free compound Acanthopanax senticosus granules was established.

8. The method for constructing the standard fingerprint spectrum of sugar-free compound Acanthopanax senticosus granules according to claim 7, characterized in that, The standard fingerprint spectrum of the sugar-free compound Acanthopanax senticosus granules uses the ratio of the retention time of each common characteristic peak to the retention time of the reference peak as the relative retention time. It includes 19 common characteristic peaks, with peak 7 as the reference peak (S peak) and a retention time of 1.

000. The relative retention times of the other 18 common characteristic peaks are as follows: peak 1: 0.102–0.124; peak 2: 0.108–0.132; peak 3: 0.142–0.174; peak 4:

0. Peak 489–0.598; Peak 5: 0.605–0.740; Peak 6: 0.759–0.927; Peak 8: 0.961–1.174; Peak 9: 1.088–1.330; Peak 10: 1.317–1.609; Peak 11: 1.512–1.848; Peak 12: 1.725–2.108; Peak 13: 1.849–2.259; Peak 14: 1.976–2.415; Peak 15 ranges from 2.007 to 2.453; Peak 16 ranges from 2.057 to 2.514; Peak 17 ranges from 2.153 to 2.631; Peak 18 ranges from 2.395 to 2.927; and Peak 19 ranges from 2.515 to 3.

073.

9. A quality detection method for the fingerprint spectrum of sugar-free compound Acanthopanax senticosus granules, comprising: The fingerprint spectrum of sugar-free compound Acanthopanax senticosus granules was obtained by using the detection method of the fingerprint spectrum of sugar-free compound Acanthopanax senticosus granules according to any one of claims 4-6, and the similarity was compared with the standard fingerprint spectrum of sugar-free compound Acanthopanax senticosus granules obtained by using the construction method of the standard fingerprint spectrum of sugar-free compound Acanthopanax senticosus granules according to any one of claims 7-8.

10. A method for detecting the content of eight components in sugar-free compound Acanthopanax senticosus granules, comprising the following steps: A) Preparation of the test solution: Same as step 1) of the detection method for the fingerprint spectrum of sugar-free compound Acanthopanax senticosus granules according to any one of claims 4-6; B) Preparation of mixed reference solution: Add at least one reference standard from the following sources to the first solvent to dissolve and dilute to volume: add eleutheroside E, isozytin, syringin, chlorogenic acid, cryptochlorogenic acid, neochlorogenic acid, and schisandrol A. Add schisandrin B to the second solvent to dissolve and dilute to volume. Take samples of the obtained single-standard reference solutions, mix them, add the first solvent to dissolve and dilute to volume to obtain the mixed reference solution. C) Determination: High performance liquid chromatography with the same chromatographic conditions as in the detection method of fingerprint spectrum of sugar-free compound Acanthopanax senticosus granules according to any one of claims 4-6 was used to determine the test solution in step A) and the mixed reference solution in step B), and the content of 8 components in the test solution was calculated by external standard method.

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