Quality control method of a traditional Chinese medicine composition containing silkworm cocoon shells and application thereof

Abstract

The invention discloses a detection method for a Chinese medicine compound containing silkworm cocoon shells, which comprises the following steps: taking a test solution and a reference solution of the Chinese medicine compound for detection, wherein the Chinese medicine compound comprises silkworm cocoon shells, corn silk, astragalus and chuanxiong, the reference substances are ferulic acid, astragaloside IV, serine and glycine, and the chromatographic conditions of the detection are: using a chromatographic column with octadecylsilane bonded silica gel as filler, mobile phase A is selected from one or more of acetonitrile, methanol and tetrahydrofuran, mobile phase B is an acid aqueous solution, an alkaline aqueous solution and / or a buffered saline solution, the flow rate is 0.6-1.5 mL / min, the column temperature is 30-50° C., and the detection wavelength is 200-360 nm; according to the detection results, the component information of the Chinese medicine compound, or the component information and content information, is obtained. The method has a good linear relationship, high precision, strong stability, good repeatability, high sample recovery rate, is simple and easy to operate, and is particularly suitable for quality control in the industrialization of the composition.

Description

Technical Field

[0001] The invention relates to the technical field of medicines, and in particular to a quality control method for a traditional Chinese medicine composition comprising silkworm cocoon shells and an application thereof. Background Art

[0002] Chronic Kidney Disease (CKD) is a major disease that threatens human health. At present, there is no good way to completely treat proteinuria caused by various chronic kidney diseases. Traditional Chinese medicine believes that the kidney cannot store essence, the essence is deposited downward, the spleen is weak and cannot retain essence, the clear qi sinks, the turbid qi rises, and the lungs cannot control the qi. These are the recognized mechanisms of proteinuria in traditional Chinese medicine. The pathogenesis of proteinuria is deficiency of the root and excess of the symptoms, with deficiency of the root as the main factor, and exogenous wind evil, damp heat and blood stasis, etc., which can affect each other. Traditional Chinese medicine has the advantages of treating both the root and the symptoms, improving symptoms, less side effects and less recurrence in the treatment of proteinuria.

[0003] According to the theory of traditional Chinese medicine, the Chinese medicine compound preparation composed of silkworm cocoon shell, corn silk, astragalus and Chuanxiong has the effects of tonifying the kidney, activating blood circulation, clearing dampness and blood stasis, eliminating edema and relieving kidney diseases, and is particularly suitable for proteinuria caused by various chronic nephritis. In the prescription, astragalus invigorates qi and consolidates the exterior, Chuanxiong activates blood circulation and removes blood stasis, corn silk promotes diuresis and reduces swelling, and silkworm cocoon shell invigorates the kidney and replenishes essence. The whole prescription can tonify the kidney, activate blood circulation, clear dampness and blood stasis, and has a good effect on clinical proteinuria. This prescription is a compound prescription guided by the theory of traditional Chinese medicine. It is an organic whole. Its efficacy cannot be completely replaced by a single component or a certain Chinese medicine. Experimental studies have shown that this prescription has the best efficacy as a whole, and has a significant combined synergistic effect compared with the use of a single medicine. Among them, Huangqi is the dried root of Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao or Astragalus membranaceus (Fisch.) Bge. of the Leguminosae family as recorded in the Chinese Pharmacopoeia (2015 edition). Chuanxiong is the dried rhizome of Chuanxiong (scientific name: Ligusticum chuanxiong hort) of the Apiaceae family as recorded in the Chinese Pharmacopoeia (2015 edition). Corn silk is the style and stigma of Zea mays L. of the Gramineae family. Corn silk is recorded in many Chinese medicine books. It is sweet, light and neutral in nature. Silkworm cocoon shell is the cocoon shell of the Bombyx mori L. insect of the Bombycidae family. It is also known as silkworm clothing, cocoon yellow, cotton silkworm and silkworm cocoon shell. It tastes sweet and is warm in nature. It has the effects of stopping bleeding, quenching thirst, detoxifying and curing sores.

[0004] The research on the quality control of Chinese medicine compound is the key to the modernization and internationalization of Chinese medicine. Effectively solving the quality control problem of Chinese medicine compound preparations is a key link in its industrialization. However, there is no research on the quality control of the Chinese medicine composition containing silkworm cocoon shells as described in the present application, nor is there a method for simultaneously determining ferulic acid, astragaloside IV, serine and glycine in the composition. Summary of the invention

[0005] In view of this, the present invention provides a method for detecting a traditional Chinese medicine compound containing silkworm cocoon shells, comprising the following steps:

[0006] A test solution and a reference solution of a Chinese herbal compound were tested, wherein the Chinese herbal compound includes silkworm cocoon shell, corn silk, astragalus and ligusticum chuanxiong, and the reference solution includes ferulic acid, astragaloside IV, serine and glycine.

[0007] The chromatographic conditions of the detection are: using a chromatographic column with octadecylsilane bonded silica as filler, mobile phase A is selected from one or more of acetonitrile, methanol and tetrahydrofuran, mobile phase B is an acid aqueous solution, an alkaline aqueous solution and / or a buffered saline solution, the flow rate is 0.6 to 1.5 mL / min, the column temperature is 30 to 50°C, and the detection wavelength is 200 to 360 nm;

[0008] The gradient elution procedure for detecting the astragaloside IV is as follows: 0-25 min, 15%-25% A; 25-30 min, 25%-45% A;

[0009] The gradient elution program for detecting the serine and glycine is: 0-10 min, 5%-10% A; 10-15 min, 10%-20% A; 15-30 min, 20%-23% A; 30-35 min, 23%-55% A,

[0010] The elution procedure for detecting the ferulic acid is: 0-40 min, 15% A;

[0011] According to the test results, the ingredient information, or the ingredient information and content information of the Chinese medicine compound is obtained.

[0012] Furthermore, the information is calculated according to the external standard method based on the corresponding peak areas in the recorded chromatogram of the test solution of the Chinese medicine compound and the chromatogram of the reference solution, to determine the content of one or more of the following ingredients in the Chinese medicine compound: ferulic acid, astragaloside I, serine and glycine.

[0013] Furthermore, the method for preparing the Chinese medicine compound test solution comprises: (1) preparing the Chinese medicine compound dry paste powder; and (2) preparing the Chinese medicine compound dry paste powder into a derivatized test solution for serine and glycine detection, a test solution for ferulic acid detection and / or a test solution for astragaloside IV detection.

[0014] Furthermore, the preparation method of the Chinese medicine compound dry paste powder comprises the following steps:

[0015] (1) Weigh the following raw materials according to the following weight ratio: 25-35 parts of silkworm cocoon shell, 25-35 parts of corn silk, 6-12 parts of astragalus, and 12-18 parts of chuanxiong;

[0016] (2) Weighing the above-mentioned weight portions of astragalus, chuanxiong and corn silk, adding water, heating and extracting, filtering, and obtaining a first filtrate;

[0017] (3) weighing the above-mentioned cocoon shells by weight, adding water, heating and extracting, and filtering to obtain a second filtrate;

[0018] (4) combining the first filtrate and the second filtrate, and concentrating them to obtain the Chinese medicine composition; and

[0019] (5) mixing the Chinese medicine composition with one or more pharmaceutically acceptable excipients, drying, crushing and sieving to obtain the Chinese medicine compound dry paste powder.

[0020] Furthermore, step (2) and / or step (3) of the method for preparing the traditional Chinese medicine compound dry paste powder further comprises the step of soaking the raw material medicine in water.

[0021] Furthermore, the astragalus, chuanxiong and corn silk are soaked in water for 30 to 180 minutes.

[0022] Furthermore, the astragalus, chuanxiong and corn silk are soaked in water for 30 to 100 minutes.

[0023] Furthermore, the astragalus, chuanxiong and corn silk are soaked in water for 90 to 100 minutes.

[0024] Furthermore, the astragalus, chuanxiong and corn silk are soaked in water for about 90 minutes.

[0025] Furthermore, the silk cocoon shells are soaked in water for 30 to 180 minutes.

[0026] Furthermore, the silk cocoon shells are soaked in water for 30 to 90 minutes.

[0027] Furthermore, the silk cocoon shells are soaked in water for 60 to 90 minutes.

[0028] Furthermore, the silk cocoon shells are soaked in water for about 60 minutes.

[0029] Furthermore, the raw materials are weighed according to the following weight ratios: 28-35 parts of silkworm cocoon shells, 28-35 parts of corn silk, 8-12 parts of astragalus, and 13-18 parts of ligusticum chuanxiong.

[0030] Furthermore, the raw materials are weighed according to the following weight ratios: 28.5-31.5 parts of silkworm cocoon shells, 28.5-31.5 parts of corn silk, 8.55-9.45 parts of astragalus, and 14.25-15.75 parts of ligusticum chuanxiong.

[0031] Furthermore, the raw materials were weighed according to the following weight ratios: about 30 parts of silkworm cocoon shells, about 30 parts of corn silk, about 9 parts of astragalus, and about 15 parts of chuanxiong.

[0032] Furthermore, step (2) of the method for preparing the Chinese medicine compound dry paste powder comprises any one or more of the following items [1] to [6]:

[0033] [1] The water is distilled water or deionized water, preferably cold water, more preferably the cold water covers the surface of the API by 2 to 5 cm;

[0034] [2] The heating extraction operation is repeated 2 to 4 times, for example 2 times;

[0035] [3] The heating is carried out by heating over high heat until boiling and then heating over low heat to maintain a slight boil;

[0036] [4] The amount of water used is 1 to 80 times (L / kg), preferably 5 to 30 times, more preferably 8 to 14 times, for example about 12 times;

[0037] [5] The heating extraction time is 15 to 180 minutes, preferably 30 to 120 minutes, for example about 90 minutes;

[0038] [6] The filtration is performed using a single layer of filter cloth with a mesh size of 100 to 300, for example, a single layer of filter cloth with a mesh size of 300.

[0039] Furthermore, step (3) of the method for preparing the Chinese medicine compound dry paste powder comprises any one or more of the following items [1] to [5]:

[0040] [1] The water is distilled water or deionized water, preferably cold water, more preferably the cold water covers the surface of the API by 2 to 5 cm;

[0041] [2] The heating extraction operation is repeated 2 to 4 times, for example 3 times;

[0042] [3] The amount of water used is 1 to 80 times (L / kg), preferably 5 to 30 times, more preferably 12 to 16 times, for example about 16 times;

[0043] [4] The heating extraction time is 15 to 180 minutes, preferably 30 to 120 minutes, for example about 60 minutes;

[0044] [5] The filtration is performed by using a single layer of filter cloth with a mesh size of 100 to 300, for example, a single layer of filter cloth with a mesh size of 100.

[0045] Furthermore, step (4) of the method for preparing the Chinese medicine compound dry paste powder comprises any one or more of the following items [1] to [3]:

[0046] [1] The concentration is concentration under reduced pressure;

[0047] [2] The concentration temperature is 60 to 90°C, for example about 80°C;

[0048] [3] The relative density of the concentrated solution after the concentration is 1.02 to 1.35 g / ml, for example, 1.02 to 1.05 g / ml.

[0049] Furthermore, in step (5) of the method for preparing the traditional Chinese medicine compound dry paste powder, the drying is spray drying, reduced pressure vacuum drying or vacuum freeze drying.

[0050] Furthermore, the spray drying conditions are as follows: the preheating temperature of the feed liquid is 60-120°C, the air inlet temperature is 100-180°C, the injection speed is 5-25rpm, the atomization pressure is 0.5-3MPa, and the needle pressure is 0.5-3MPa.

[0051] Furthermore, the spray drying conditions are as follows: the preheating temperature of the feed liquid is 85.5-94.5°C, the inlet air temperature is 152-168°C, the injection speed is 19-21rpm, the atomization pressure is 1.9-2.1 MPa, and the needle pressure is 1.9-2.1 MPa.

[0052] Furthermore, the spray drying conditions are as follows: the preheating temperature of the feed liquid is about 90°C, the air inlet temperature is about 160°C, the injection speed is about 20rpm, the atomization pressure is about 2MPa, and the needle pressure is about 2Mpa.

[0053] Furthermore, the mixing step includes converting the weight of the concentrated liquid into the weight of theoretical dry paste powder, adding the auxiliary material at a weight ratio of 0.05 to 0.25:1 to the theoretical dry paste powder, fully dissolving and mixing evenly.

[0054] Furthermore, the preparation method of the derivatized test solution for serine and glycine detection includes: weighing an appropriate amount of a Chinese medicine compound dry paste powder, adding a certain amount of 3-8 mol / L hydrochloric acid solution, hydrolyzing at 120-200° C. for 0.5-1.5 hours, cooling, evaporating to dryness, adding 0.1-0.5 mol / L hydrochloric acid solution to dissolve, then adding a certain amount of 0.1-0.5 mol / L acetonitrile solution of phenyl isothiocyanate and 1-5 mol / L acetonitrile solution of triethylamine, shaking well, leaving at room temperature, adding 40-60% acetonitrile to the scale, shaking well, adding a certain amount of n-hexane, shaking, leaving, taking the lower layer of solution, filtering, and taking the filtrate to obtain.

[0055] Furthermore, the preparation method of the test solution for ferulic acid detection includes: weighing an appropriate amount of Chinese herbal compound dry paste powder, adding a certain amount of 50-80% methanol, ultrasonically treating at a power of 300-800W and a frequency of 30-60kHz for 20-60 minutes, cooling, fixing the volume, shaking well, filtering, and taking the filtrate to obtain.

[0056] Furthermore, the preparation method of the test solution for the detection of astragaloside IV includes: weighing an appropriate amount of a Chinese medicine compound dry paste powder, adding a certain amount of a 60-90% methanol solution containing 1-10% concentrated ammonia test solution, sealing, weighing, heating and refluxing for 1-3 hours, cooling, weighing again, making up the lost weight with a 60-90% methanol solution containing 1-10% concentrated ammonia test solution, shaking, filtering, accurately measuring a certain amount of subsequent filtrate, evaporating to dryness, dissolving the residue with 60-90% methanol, fixing the volume, shaking, filtering, and taking the subsequent filtrate.

[0057] Furthermore, the preparation method of the serine and glycine derivatization reference solution comprises: weighing appropriate amounts of serine and glycine; adding 0.1-0.5 mol / L hydrochloric acid solution to prepare a mixed reference solution containing each component at a concentration of 10-200 μg / ml; drawing an appropriate amount of the mixed reference solution, adding a certain amount of 0.1-0.5 mol / L phenyl isothiocyanate acetonitrile solution and 1-5 mol / L triethylamine acetonitrile solution, shaking well, leaving at room temperature, adding 40-60% acetonitrile to the scale, shaking well, adding a certain amount of n-hexane, shaking, leaving, taking the lower layer solution, filtering, taking the filtrate, and obtaining the serine and glycine derivatization reference solution.

[0058] Furthermore, the preparation method of the reference solution of ferulic acid and / or astragaloside IV comprises: weighing an appropriate amount of ferulic acid and / or astragaloside IV; and adding a methanol aqueous solution with a volume percentage concentration of 10% to 100% to prepare a reference solution of ferulic acid and / or astragaloside IV containing each component at a concentration of 10 to 200 μg / ml.

[0059] Furthermore, the flow rate is 0.8 to 1.2 mL / min.

[0060] Furthermore, the column temperature is 30-35°C.

[0061] Furthermore, the detection wavelength is 200-330 nm.

[0062] Furthermore, the injection volume of the test is 5 to 20 μl.

[0063] Furthermore, the Chinese herbal compound test solution and the reference solution are filtered with a 0.22 μm or 0.45 μm microporous filter membrane before the test.

[0064] Furthermore, the chromatographic column has a length of 25 cm, an inner diameter of 4.6 mm, and a particle size of 5 μm.

[0065] Furthermore, the chromatographic column is an Agilent 5TC-C18(2) chromatographic column, a Waters Symmetry C18 chromatographic column or an Agilent SB C18 chromatographic column.

[0066] Furthermore, the chromatographic column is an Agilent 5TC-C18(2) chromatographic column.

[0067] Furthermore, the mobile phase A is selected from one or more of the following: methanol, acetonitrile, methanol:acetonitrile (10:90), methanol:acetonitrile (20:80), methanol:acetonitrile (30:70), methanol:acetonitrile (40:60), methanol:acetonitrile (50:50), methanol:acetonitrile (60:40), methanol:acetonitrile (70:30), methanol:acetonitrile (80:20), methanol:acetonitrile (90:10).

[0068] Furthermore, the acid aqueous solution, alkali aqueous solution and / or buffered saline solution is selected from one or more of weak acids and their salts, weak bases and their salts of different concentrations.

[0069] Further, the acid aqueous solution, alkali aqueous solution and / or buffered saline solution is selected from formic acid, acetic acid, phosphoric acid, trifluoroacetic acid, formic acid and ammonium formate, acetic acid and sodium acetate, acetic acid and ammonium acetate, disodium hydrogen phosphate and sodium dihydrogen phosphate, disodium hydrogen phosphate and potassium dihydrogen phosphate, disodium hydrogen phosphate and citric acid, citric acid and sodium citrate, glycine and hydrochloric acid, or phthalic acid and hydrochloric acid in different concentrations.

[0070] Furthermore, the acid aqueous solution is a 0.05% to 0.4% acid aqueous solution.

[0071] Furthermore, the acid aqueous solution is a 0.05% to 0.2% phosphoric acid or formic acid aqueous solution.

[0072] Furthermore, the acid aqueous solution is a 0.05% to 0.1% phosphoric acid or formic acid aqueous solution.

[0073] Furthermore, the concentration of the ferulic acid reference solution is about 20 μg / ml.

[0074] Furthermore, the concentration of the astragaloside IV reference solution is about 20 μg / ml.

[0075] Furthermore, the concentration of the serine reference solution is about 130 μg / ml.

[0076] Furthermore, the concentration of the glycine reference solution is about 50 μg / ml.

[0077] According to another aspect of the present invention, there is provided a use of the above detection method in quality detection and / or quality evaluation and / or quality control of a traditional Chinese medicine compound containing silkworm cocoon shells.

[0078] Beneficial effects of the present invention:

[0079] (1) The present invention establishes a quality control method for a traditional Chinese medicine composition comprising silkworm cocoon shells and a method for determining the contents of ferulic acid, astragaloside I, serine and glycine in its application, and conducts methodological verification of the detection methods of the four index components in terms of system adaptability, specificity, linearity, precision, repeatability and stability to determine the feasibility of the detection methods of the four index components. The results show that the method has good linear relationship, high precision, strong stability, good repeatability, high sample recovery rate, is simple and easy to operate, and is particularly suitable for quality control in the industrialization of the composition.

[0080] (2) The parameters involved in the detection method of the present invention are obtained based on the inventor's research experience, and excellent detection results are achieved under the detection conditions.

[0081] (3) The present invention provides a basis for the formulation of quality standards for compound prescriptions for preventing and / or treating proteinuria caused by various chronic kidney diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0082] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without exceeding the scope of protection required by the present invention.

[0083] Figure 1The chromatograms are for the determination of serine and glycine content in a traditional Chinese medicine compound containing silkworm cocoon shells. A is a blank solvent chromatogram; B is a chromatogram of serine and glycine reference solution (retention time 21.827min is the serine chromatogram peak; 22.393 is the glycine chromatogram peak); C is a chromatogram of the CJK-YX negative control test solution; D is a chromatogram of the derivatized test solution for serine and glycine detection (retention time 21.738min is the serine chromatogram peak; 22.369 is the glycine chromatogram peak).

[0084] Figure 2 The chromatograms are for the determination of ferulic acid content in a traditional Chinese medicine compound containing silkworm cocoon shells. A is a blank solvent chromatogram; B is a chromatogram of a ferulic acid reference solution (retention time 22.917 min is the ferulic acid chromatogram peak); C is a chromatogram of a CX-YX negative control test solution; and D is a chromatogram of a test solution for ferulic acid detection (retention time 22.718 min is the ferulic acid chromatogram peak).

[0085] Figure 3 The chromatograms are for the determination of astragaloside IV in a traditional Chinese medicine compound containing silkworm cocoon shells. A is a blank solvent chromatogram; B is a chromatogram of an astragaloside IV reference solution (the retention time 22.293 min is the astragaloside IV chromatogram peak); CHQ-YX negative control test solution chromatogram; D is a chromatogram of a test solution for astragaloside IV detection (the retention time 22.310 min is the astragaloside IV chromatogram peak). DETAILED DESCRIPTION

[0086] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0087] As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0088] Unless otherwise indicated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by those of ordinary skill in the field of the invention or the field in which the terms are used. Although any methods, conditions, substances or materials similar to or equivalent to those disclosed herein may be used in the practice of the present invention, preferred methods, conditions, substances or materials are described herein.

[0089] In the present invention, the term "comprising" is synonymous with "including". As used herein, the terms "comprising", "including", "having", "containing" or any other variation thereof are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus comprising the listed elements is not necessarily limited to only those elements, but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0090] As described in the background technology section, there is no literature report on a method for comprehensively analyzing the chemical components of a traditional Chinese medicine compound containing silkworm cocoon shells. In order to solve the above problem, the present invention provides a method for detecting a traditional Chinese medicine compound containing silkworm cocoon shells, which comprises the following steps:

[0091] A test solution and a reference solution of a Chinese herbal compound were tested, wherein the Chinese herbal compound includes silkworm cocoon shell, corn silk, astragalus and ligusticum chuanxiong, and the reference solution includes ferulic acid, astragaloside IV, serine and glycine.

[0092] The chromatographic conditions of the detection are: using a chromatographic column with octadecylsilane bonded silica as filler, mobile phase A is selected from one or more of acetonitrile, methanol and tetrahydrofuran, mobile phase B is an acid aqueous solution, an alkaline aqueous solution and / or a buffered saline solution, the flow rate is 0.6 to 1.5 mL / min, the column temperature is 30 to 50°C, and the detection wavelength is 200 to 360 nm;

[0093] The gradient elution procedure for detecting the astragaloside IV is as follows: 0-25 min, 15%-25% A; 25-30 min, 25%-45% A;

[0094] The gradient elution program for detecting the serine and glycine is: 0-10 min, 5%-10% A; 10-15 min, 10%-20% A; 15-30 min, 20%-23% A; 30-35 min, 23%-55% A,

[0095] The elution procedure for detecting the ferulic acid is: 0-40 min, 15% A;

[0096] According to the test results, the ingredient information, or the ingredient information and content information of the Chinese medicine compound is obtained.

[0097] In the present invention, when a ratio, equivalent, concentration, wavelength, temperature, flow rate, weight portion or other value or parameter is expressed as a range, a preferred range, or a range defined by a series of upper preferred values ​​and lower preferred values, it should be understood as specifically disclosing all ranges formed by any pairing of any range upper limit or preferred value with any range lower limit or preferred value, regardless of whether the range is disclosed separately. For example, when a range of "200 to 360" is disclosed, unless otherwise specified, the range is intended to include its end value and all integers and fractions within the range, and the effects of the present invention can be achieved within the above disclosed range.

[0098] In a preferred embodiment, the information is calculated according to the external standard method based on the corresponding peak areas in the recorded chromatogram of the test solution of the Chinese medicine compound and the chromatogram of the reference solution, and the content of one or more of the following ingredients in the Chinese medicine compound: ferulic acid, astragaloside I, serine and glycine.

[0099] In a preferred embodiment, the method for preparing the Chinese medicine compound test solution comprises: (1) preparing the Chinese medicine compound dry paste powder; and (2) preparing the Chinese medicine compound dry paste powder into a derivatized test solution for serine and glycine detection, a test solution for ferulic acid detection and / or a test solution for astragaloside IV detection.

[0100] In a preferred embodiment, the method for preparing the Chinese medicine compound dry paste powder comprises the following steps:

[0101] (1) Weigh the following raw materials according to the following weight ratio: 25-35 parts of silkworm cocoon shell, 25-35 parts of corn silk, 6-12 parts of astragalus, and 12-18 parts of chuanxiong;

[0102] (2) Weighing the above-mentioned weight portions of astragalus, chuanxiong and corn silk, adding water, heating and extracting, filtering, and obtaining a first filtrate;

[0103] (3) weighing the above-mentioned cocoon shells by weight, adding water, heating and extracting, and filtering to obtain a second filtrate;

[0104] (4) combining the first filtrate and the second filtrate, and concentrating them to obtain the Chinese medicine composition; and

[0105] (5) mixing the Chinese medicine composition with one or more pharmaceutically acceptable excipients, drying, crushing and sieving to obtain the Chinese medicine compound dry paste powder.

[0106] In a preferred embodiment, step (2) and / or step (3) of the method for preparing the traditional Chinese medicine compound dry paste powder further comprises the step of soaking the raw material in water.

[0107] In a preferred embodiment, the astragalus, chuanxiong and corn silk are soaked in water for 30 to 180 minutes.

[0108] In a preferred embodiment, the astragalus, chuanxiong and corn silk are soaked in water for 30 to 100 minutes.

[0109] In a preferred embodiment, the astragalus, chuanxiong and corn silk are soaked in water for 90 to 100 minutes.

[0110] In a preferred embodiment, the time for soaking the astragalus, chuanxiong and corn silk in water is about 90 minutes.

[0111] In a preferred embodiment, the silkworm cocoon shells are soaked in water for 30 to 180 minutes.

[0112] In a preferred embodiment, the cocoon shells are soaked in water for 30 to 90 minutes.

[0113] In a preferred embodiment, the cocoon shells are soaked in water for 60 to 90 minutes.

[0114] In a preferred embodiment, the silk cocoon shells are soaked in water for about 60 minutes.

[0115] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 90" includes ±5% of 90, i.e., from 85.5 to 94.5; "about 60" includes ±5% of 60, i.e., from 57 to 63.

[0116] In a preferred embodiment, the raw materials are weighed according to the following weight ratios: 28-35 parts of silkworm cocoon shells, 28-35 parts of corn silk, 8-12 parts of astragalus, and 13-18 parts of ligusticum chuanxiong.

[0117] In a preferred embodiment, the raw materials are weighed according to the following weight ratios: 28.5-31.5 parts of silkworm cocoon shells, 28.5-31.5 parts of corn silk, 8.55-9.45 parts of astragalus, and 14.25-15.75 parts of ligusticum chuanxiong.

[0118] In a preferred embodiment, the raw materials are weighed according to the following weight ratios: about 30 parts of silkworm cocoon shells, about 30 parts of corn silk, about 9 parts of astragalus, and about 15 parts of ligusticum chuanxiong.

[0119] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 30" includes ±5% of 30, i.e., from 28.5 to 31.5; "about 9" includes ±5% of 9, i.e., from 8.55 to 9.45; "about 15" includes ±5% of 15, i.e., from 14.25 to 15.75.

[0120] In a preferred embodiment, step (2) of the method for preparing the Chinese medicine compound dry paste powder comprises any one or more of the following items [1] to [6]:

[0121] [1] The water is distilled water or deionized water, preferably cold water, more preferably the cold water covers the surface of the API by 2 to 5 cm;

[0122] [2] The heating extraction operation is repeated 2 to 4 times, for example 2 times;

[0123] [3] The heating is carried out by heating over high heat until boiling and then heating over low heat to maintain a slight boil;

[0124] [4] The amount of water used is 1 to 80 times (L / kg), preferably 5 to 30 times, more preferably 8 to 14 times, for example about 12 times;

[0125] [5] The heating extraction time is 15 to 180 minutes, preferably 30 to 120 minutes, for example about 90 minutes;

[0126] [6] The filtration is performed using a single layer of filter cloth with a mesh size of 100 to 300, for example, a single layer of filter cloth with a mesh size of 300.

[0127] In a preferred embodiment, step (3) of the method for preparing the Chinese medicine compound dry paste powder comprises any one or more of the following items [1] to [5]:

[0128] [1] The water is distilled water or deionized water, preferably cold water, more preferably the cold water covers the surface of the API by 2 to 5 cm;

[0129] [2] The heating extraction operation is repeated 2 to 4 times, for example 3 times;

[0130] [3] The amount of water used is 1 to 80 times (L / kg), preferably 5 to 30 times, more preferably 12 to 16 times, for example about 16 times;

[0131] [4] The heating extraction time is 15 to 180 minutes, preferably 30 to 120 minutes, for example about 60 minutes;

[0132] [5] The filtration is performed by using a single layer of filter cloth with a mesh size of 100 to 300, for example, a single layer of filter cloth with a mesh size of 100.

[0133] In a preferred embodiment, step (4) of the method for preparing the Chinese medicine compound dry paste powder comprises any one or more of the following items [1] to [3]:

[0134] [1] The concentration is concentration under reduced pressure;

[0135] [2] The concentration temperature is 60 to 90°C, for example about 80°C;

[0136] [3] The relative density of the concentrated solution after the concentration is 1.02 to 1.35 g / ml, for example, 1.02 to 1.05 g / ml.

[0137] In a preferred embodiment, in step (5) of the method for preparing the traditional Chinese medicine compound dry paste powder, the drying is spray drying, reduced pressure vacuum drying or vacuum freeze drying.

[0138] In a preferred embodiment, the spray drying conditions are: liquid preheating temperature is 60-120°C, air inlet temperature is 100-180°C, injection speed is 5-25rpm, atomization pressure is 0.5-3MPa, and needle pressure is 0.5-3MPa.

[0139] In a preferred embodiment, the spray drying conditions are: the preheating temperature of the liquid is 85.5-94.5°C, the inlet air temperature is 152-168°C, the injection speed is 19-21rpm, the atomization pressure is 1.9-2.1MPa, and the needle pressure is 1.9-2.1MPa.

[0140] In a preferred embodiment, the spray drying conditions are: the preheating temperature of the feed liquid is about 90°C, the inlet air temperature is about 160°C, the injection speed is about 20rpm, the atomization pressure is about 2 MPa, and the needle pressure is about 2Mpa.

[0141] In a preferred embodiment, the mixing step includes converting the weight of the concentrated liquid into the weight of a theoretical dry paste powder, adding the auxiliary material at a weight ratio of the auxiliary material to the theoretical dry paste powder of 0.05 to 0.25:1, fully dissolving, and mixing evenly.

[0142] In a preferred embodiment, the preparation method of the derivatized test solution for serine and glycine detection includes: weighing an appropriate amount of a Chinese medicine compound dry paste powder, adding a certain amount of 3-8 mol / L hydrochloric acid solution, hydrolyzing at 120-200°C for 0.5-1.5 hours, cooling, evaporating to dryness, adding 0.1-0.5 mol / L hydrochloric acid solution to dissolve, then adding a certain amount of 0.1-0.5 mol / L acetonitrile solution of phenyl isothiocyanate and 1-5 mol / L acetonitrile solution of triethylamine, shaking, leaving at room temperature, adding 40-60% acetonitrile to the scale, shaking, adding a certain amount of n-hexane, shaking, leaving, taking the lower layer of solution, filtering, and taking the filtrate.

[0143] In a preferred embodiment, the preparation method of the test solution for ferulic acid detection comprises: weighing an appropriate amount of a Chinese medicine compound dry paste powder, adding a certain amount of 50-80% methanol, ultrasonically treating at a power of 300-800 W and a frequency of 30-60 kHz for 20-60 minutes, cooling, fixing the volume, shaking well, filtering, and taking the filtrate to obtain.

[0144] In a preferred embodiment, the preparation method of the test solution for the detection of astragaloside IV comprises: weighing an appropriate amount of a dry paste powder of a traditional Chinese medicine compound, adding a certain amount of a 60-90% methanol solution containing 1-10% concentrated ammonia test solution, sealing, weighing, heating and refluxing for 1-3 hours, cooling, weighing again, making up the lost weight with a 60-90% methanol solution containing 1-10% concentrated ammonia test solution, shaking, filtering, accurately measuring a certain amount of subsequent filtrate, evaporating to dryness, dissolving the residue with 60-90% methanol, fixing the volume, shaking, filtering, and taking the subsequent filtrate.

[0145] The 1-10% concentrated ammonia solution of the present invention is prepared by taking 1-10 mL of commercially available ammonia solution (concentration 25% to 28%) and diluting it to 100 mL with water. The commercially available ammonia solution can be any ammonia solution sold on the market, for example, from Sinopharm Group Chemical Reagent Shanghai Co., Ltd.

[0146] In a preferred embodiment, the preparation method of the serine and glycine derivatized reference solution comprises: weighing appropriate amounts of serine and glycine; adding 0.1-0.5 mol / L hydrochloric acid solution to prepare a mixed reference solution containing each component at a concentration of 10-200 μg / ml; drawing an appropriate amount of the mixed reference solution, adding a certain amount of 0.1-0.5 mol / L phenyl isothiocyanate acetonitrile solution and 1-5 mol / L triethylamine acetonitrile solution, shaking well, leaving at room temperature, adding 40-60% acetonitrile to the scale, shaking well, adding a certain amount of n-hexane, shaking, leaving, taking the lower layer solution, filtering, taking the filtrate, and obtaining the serine and glycine derivatized reference solution.

[0147] In a preferred embodiment, the preparation method of the reference solution of ferulic acid and / or astragaloside IV comprises: weighing an appropriate amount of ferulic acid and / or astragaloside IV; and adding a methanol aqueous solution with a volume percentage concentration of 10% to 100% to prepare a reference solution of ferulic acid and / or astragaloside IV containing each component at a concentration of 10 to 200 μg / ml.

[0148] In a preferred embodiment, the flow rate is 0.8-1.2 mL / min.

[0149] Regarding the flow rate of the present invention, 0.6ml / min, 0.7ml / min, 0.8ml / min, 0.9ml / min, 1.0ml / min, 1.1ml / min, 1.2ml / min, 1.3ml / min, 1.4ml / min, and 1.5ml / min were compared for screening, and the results showed that the flow rates of 0.6ml / min, 0.7ml / min, 0.8ml / min, 0.9ml / min, 1.0ml / min, 1.1ml / min, 1.2ml / min, 1.3ml / min, 1.4ml / min, and 1.5ml / min could meet the detection requirements, but when the flow rate was 0.8-1.2mL / min, the peak distribution was more uniform and the separation effect was the best. Finally, 0.8-1.2mL / min was determined to be the optimal flow rate, and the optional flow rates were 0.7ml / min and 1.3ml / min.

[0150] In a preferred embodiment, the column temperature is 30-35°C.

[0151] Regarding the column temperature of the present invention, 30°C, 35°C, 40°C, 45°C and 50°C were compared for screening. The results showed that column temperatures of 30°C, 35°C, 40°C, 45°C and 50°C all met the detection requirements, but when the column temperature was 30-35°C, the peak distribution was more uniform, and the separation effect of the main peaks was the best. Finally, 30-35°C was determined to be the optimal column temperature, and the optional column temperature was 40°C and 45°C.

[0152] In a preferred embodiment, the detection wavelength is 200-330 nm.

[0153] In a preferred embodiment, the injection volume for the detection is 5-20 μl.

[0154] In a preferred embodiment, the Chinese herbal compound test solution and the reference solution are filtered with a 0.22 μm or 0.45 μm microporous filter membrane before the detection.

[0155] In a preferred embodiment, the chromatographic column has a length of 25 cm, an inner diameter of 4.6 mm, and a particle size of 5 μm.

[0156] In a preferred embodiment, the chromatographic column is an Agilent 5TC-C18(2) chromatographic column, a Waters Symmetry C18 chromatographic column or an Agilent SB C18 chromatographic column.

[0157] In a preferred embodiment, the chromatographic column is an Agilent 5TC-C18(2) chromatographic column.

[0158] Regarding the chromatographic column of the present invention, Agilent 5TC-C18(2), Waters Symmetry C18, and Agilent SB C18 were compared and screened. The results showed that the selected chromatographic columns all met the detection requirements. However, when the chromatographic column was Agilent5TC-C18(2), the peak distribution was more uniform, the peak separation effect was better, and the performance parameters were optimal. Finally, Agilent 5TC-C18(2) was determined to be the best chromatographic column. The optional chromatographic column was octadecylsilane bonded silica gel as the filler chromatography (column length 25 cm, inner diameter 4.6 mm, particle size 5 μm).

[0159] In a preferred embodiment, the mobile phase A is selected from one or more of the following: methanol, acetonitrile, methanol:acetonitrile (10:90), methanol:acetonitrile (20:80), methanol:acetonitrile (30:70), methanol:acetonitrile (40:60), methanol:acetonitrile (50:50), methanol:acetonitrile (60:40), methanol:acetonitrile (70:30), methanol:acetonitrile (80:20), methanol:acetonitrile (90:10).

[0160] In a preferred embodiment, the acid aqueous solution, base aqueous solution and / or buffered saline solution is selected from one or more of weak acids and their salts, weak bases and their salts of different concentrations.

[0161] In a preferred embodiment, the acid aqueous solution, alkali aqueous solution and / or buffered saline solution is selected from formic acid, acetic acid, phosphoric acid, trifluoroacetic acid, formic acid and ammonium formate, acetic acid and sodium acetate, acetic acid and ammonium acetate, disodium hydrogen phosphate and sodium dihydrogen phosphate, disodium hydrogen phosphate and potassium dihydrogen phosphate, disodium hydrogen phosphate and citric acid, citric acid and sodium citrate, glycine and hydrochloric acid, or phthalic acid and hydrochloric acid in different concentrations.

[0162] In a preferred embodiment, the acid aqueous solution is a 0.05% to 0.4% acid aqueous solution.

[0163] In a preferred embodiment, the acid aqueous solution is a 0.05% to 0.2% phosphoric acid or formic acid aqueous solution.

[0164] In a preferred embodiment, the acid aqueous solution is a 0.05% to 0.1% phosphoric acid or formic acid aqueous solution.

[0165] In a preferred embodiment, the concentration of the ferulic acid reference solution is about 20 μg / ml.

[0166] In a preferred embodiment, the concentration of the astragaloside IV reference solution is about 20 μg / ml.

[0167] In a preferred embodiment, the concentration of the serine control solution is about 130 μg / ml.

[0168] In a preferred embodiment, the concentration of the glycine control solution is about 50 μg / ml.

[0169] According to another aspect of the present invention, there is provided a use of the above detection method in quality detection and / or quality evaluation and / or quality control of a traditional Chinese medicine compound containing silkworm cocoon shells.

[0170] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0171] The present invention is further described in detail below in conjunction with specific embodiments. These embodiments should not be construed as limiting the scope of protection claimed in this application.

[0172] Example

[0173] Example 1 Preparation of Traditional Chinese Medicine Compound Dry Paste Powder Containing Silkworm Cocoon Shell

[0174] The decoction process of corn silk, astragalus and Chuanxiong is as follows: take 90g of astragalus, 150g of Chuanxiong, and 300g of corn silk, put them in a 5L round-bottom flask, add 12 times the amount of water, soak for 90 minutes, heat to boiling over high heat, keep it slightly boiling for 90 minutes over low heat, filter through a single layer of 300-mesh filter cloth, and set the filtrate aside; add 12 times the amount of water to the residue, heat to boiling over high heat, keep it slightly boiling for 90 minutes over low heat, filter through a single layer of 300-mesh filter cloth while hot, combine the filtrates, and set aside.

[0175] The single decoction process of silkworm cocoon shell is as follows: take 300g of silkworm cocoon shell, add 16 times of water, soak for 60 minutes, decoct three times, each time for 1 hour, filter, combine the filtrate and set aside.

[0176] Corn silk, astragalus and chuanxiong were decocted together, and then mixed with silkworm cocoon shell decoction, and then concentrated under reduced pressure at 80°C and spray-dried (specific conditions were: taking the concentrate, the flow paste density was 1.02-1.05 g / ml, and the weight of the concentrate was converted into the weight of the theoretical dry paste according to the paste yield, and then maltodextrin was added at a ratio of auxiliary materials to theoretical dry paste powder of 0.15:1, and the maltodextrin was stirred to fully dissolve and mix evenly, and the preheating temperature of the liquid was 90°C, and the drying parameters (inlet air temperature was 160°C, injection speed was 20 rpm, atomization pressure was 2 MPa, and needle pressure was 2 MPa)) to prepare extract composition 2 (ZHW-2, i.e., Chinese medicine compound dry paste powder), and 3 batch samples were prepared in parallel, with batch numbers: ZHW-2-20220601, ZHW-2-20220602, and ZHW-2-20220603, for standby use.

[0177] Example 2 Preparation of negative control of Chinese medicinal compound dry paste powder containing silkworm cocoon shells

[0178] According to the prescription and preparation method of "Example 1", negative control extracts without silkworm cocoon shell, astragalus and chuanxiong were prepared and named as: CJK-YX, HQ-YX and CX-YX, respectively, for later use.

[0179] Example 3 Study on the content determination method of Chinese medicine compound dry paste powder containing silkworm cocoon shell

[0180] 3.1 Instruments, Equipment and Materials

[0181] Instruments and equipment: High performance liquid chromatograph Waters 2695 chromatography system, including quaternary gradient infusion pump (Alliance 2695), 120-position high performance autosampler, imported column oven, Waters 2996 DAD diode array detector, Empower 3 chromatography management system; electronic balance: SHIMADZU (Shimadzu) AUW120D, Sartorius (Sartorius) BSA124S, FA2004B (Shanghai Youke Instrument Co., Ltd.) JY2002 (Shanghai Puchun) JS6-01 (Shanghai Puchun) JL070718R (Taiwan Sakura); ultrasonic cleaning machine: KQ-500DE Kunshan Ultrasonic Instrument Co., Ltd.; electronic constant temperature water bath: HH-6 Shanghai Lichen Bangxi Instrument Technology Co., Ltd.; electric heating jacket: DZTW Tianjin Gongxing Laboratory Instrument Co., Ltd.; chromatographic column: Agilent 5TC-C18 (2) 250×4.6mm SN: 573147 / 563543 / 547688; methanol, acetonitrile and formic acid were of chromatographic grade, and water was ultrapure water.

[0182] Reference substance information: Ferulic acid (110773-201915, content is 99.4%, China Food and Drug Inspection Institute); Astragaloside I (110781-201717, content is 96.9%, China Food and Drug Inspection Institute); Serine (140688-202104-01, content is 98%, China Food and Drug Inspection Institute); Glycine (140689-202006-01, content is 97%, China Food and Drug Inspection Institute).

[0183] 3.2 Preparation of reference solution

[0184] 3.2.1 Preparation and derivatization of serine and glycine mixed reference solution:

[0185] Preparation of mixed standard: Take appropriate amount of serine reference substance and glycine reference substance, weigh accurately, add 0.1 mol / L hydrochloric acid solution to make a mixed solution containing 130 μg of serine and 50 μg of glycine per 1 ml.

[0186] Derivatization: Accurately measure 5 ml of the above reference solution, place it in a 25 ml volumetric flask, add 2.5 ml of 0.1 mol / L phenyl isothiocyanate (PITC) in acetonitrile solution, 2.5 ml of 1 mol / L triethylamine in acetonitrile solution, shake well, leave it at room temperature for 1 hour, add 50% acetonitrile to the mark, shake well. Take 10 ml, add 10 ml of n-hexane, shake, leave it for 10 minutes, take the lower layer solution, filter, and take the filtrate to obtain.

[0187] 3.2.2 Preparation of Ferulic Acid Reference Solution

[0188] Take an appropriate amount of ferulic acid reference substance, weigh it accurately, place it in a brown volumetric flask, and add 70% methanol to make a solution containing 20 μg per 1 ml.

[0189] 3.2.3 Preparation of Astragaloside IV Reference Solution

[0190] Take an appropriate amount of Astragaloside IV reference substance, weigh it accurately, place it in a brown volumetric bottle, add 80% methanol to make a solution containing 20 μg per 1 ml.

[0191] 3.3 Preparation of test solution

[0192] 3.3.1 Preparation and derivatization of test solutions for serine and glycine determination

[0193] 1) Hydrolysis of the test sample: Take 2g of the dry paste powder of ZHW-2 in "Example 1", which is precise and stable, add 15ml of 6mol / L hydrochloric acid solution to dissolve it and transfer it to a vial, hydrolyze it at 150°C for 1 hour, cool it, transfer it to an evaporating dish, wash the vial with 10ml of water in batches, add the washing liquid to the evaporating dish, evaporate it to dryness, add 0.1mol / L hydrochloric acid solution to dissolve the residue and transfer it to a 25ml volumetric flask, add 0.1mol / L hydrochloric acid solution to the scale, shake well, and obtain.

[0194] 2) Derivatization of the hydrolyzed solution of the test sample: Accurately measure 5 ml of the test solution of serine and glycine, place in a 25 ml volumetric flask, add 2.5 ml of 0.1 mol / L phenyl isothiocyanate (PITC) in acetonitrile solution, 2.5 ml of 1 mol / L triethylamine in acetonitrile solution, shake well, leave at room temperature for 1 hour, add 50% acetonitrile to the scale, shake well. Take 10 ml, add 10 ml of n-hexane, shake, leave for 10 minutes, take the lower layer solution, filter, and take the filtrate to obtain.

[0195] CJK-YX negative sample solution was prepared in the same way as the serine and glycine test samples and set aside.

[0196] 3.3.2 Preparation of test solution for ferulic acid determination

[0197] Take 2g of the dry powder of ZHW-2 in "Example 1", accurately stabilize it, add 20ml of 70% methanol, place it in a 25ml volumetric flask, ultrasonically treat it (power 500W, frequency 40kHz) for 30 minutes, cool it, make up to 25ml, shake it well, filter it, and take the filtrate to obtain the product.

[0198] CX-YX negative samples were prepared in the same way as the ferulic acid test solution and set aside.

[0199] 3.3.3 Preparation of test solution for astragaloside IV

[0200] Take 2g of the dry paste powder of ZHW-2 in "Example 1", accurately stabilize it, put it into a stoppered conical flask, accurately add 20ml of 80% methanol solution containing 4% concentrated ammonia test solution (take 4ml of concentrated ammonia test solution, add 80% methanol to 100ml, shake well), stopper it, weigh it, heat it under reflux for 1 hour, cool it, weigh it again, make up the lost weight with 80% methanol solution containing 4% concentrated ammonia test solution, shake well, filter it, accurately measure 10ml of the filtrate, evaporate it to dryness, dissolve the residue with 80% methanol, transfer it to a 5ml volumetric flask, add 80% methanol to the scale, shake well, filter it, and take the filtrate.

[0201] HQ-YX negative sample was prepared in the same way as the astragaloside IV test solution and set aside.

[0202] 3.4 Optimization of chromatographic conditions

[0203] 3.4.1 Optimization of chromatographic conditions for serine and glycine

[0204] Determined by high performance liquid chromatography (General Rules 0512 of Part IV of the 2020 edition of the Chinese Pharmacopoeia).

[0205] Chromatographic conditions and system suitability experiments: Agilent 5TC-C18 chromatographic column (column length 25 cm, inner diameter 4.6 mm, particle size 5 μm) with octadecylsilane bonded silica as filler; acetonitrile as mobile phase A, and 0.1% phosphoric acid solution as mobile phase B.

[0206] According to the separation degree, theoretical plate number and tailing factor of serine and glycine, the chromatographic elution conditions were optimized by gradient elution according to the provisions in Table 1; the flow rate was 1.0 ml per minute; the column temperature was 35 °C; and the detection wavelength was 262 nm. The study found that the separation degree of serine and glycine>1.5, the theoretical plate number>1000, and the tailing factor between 0.9-1.05 can be achieved by using condition ①, which can effectively separate serine and glycine, and meet the requirements of the pharmacopoeia high performance liquid chromatography (General Rules 0512 of Part IV of the 2020 edition of the Chinese Pharmacopoeia).

[0207] During the determination, accurately pipette 5 μl of the reference solution in “3.2.1” and the test solution in “3.3.1” after derivatization, inject into the liquid chromatograph, and determine to obtain the result.

[0208] Table 1 Comparison of optimization of gradient elution conditions for determination of serine and glycine content

[0209]

[0210]

[0211] 3.4.2 Optimization of chromatographic conditions for ferulic acid

[0212] Determined according to high performance liquid chromatography (General Rules 0512 of Part IV of the 2020 edition of the Chinese Pharmacopoeia). Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel is used as the filler, acetonitrile: 0.1% formic acid solution (15:85) is used as the mobile phase, and the elution is 40 minutes. Gradient elution was optimized according to the provisions in Table 2; the flow rate was 1 ml per minute; the column temperature was 30°C; the detection wavelength was 321 nm. The study found that the use of condition ① can achieve a separation degree of ferulic acid >1.5, a theoretical plate number >1000, and a tailing factor between 0.9-1.05, which can be effectively separated and meets the requirements of the pharmacopoeia high performance liquid chromatography (General Rules 0512 of Part IV of the 2020 edition of the Chinese Pharmacopoeia).

[0213] During the determination, accurately pipette 10 μl of the reference solution under “3.2.2” and the test solution under “3.3.2” respectively, inject into the liquid chromatograph, and determine to obtain the result.

[0214] Table 2 Comparison of optimization of gradient elution conditions for determination of ferulic acid content

[0215]

[0216]

[0217] 3.4.3 Optimization of Chromatographic Conditions for Astragaloside IV

[0218] Determined according to high performance liquid chromatography (General Rules 0512 of Part IV of the 2020 edition of the Chinese Pharmacopoeia). The chromatographic conditions and system suitability test used octadecylsilane bonded silica as the filler, acetonitrile as the mobile phase A, and 0.05% formic acid solution as the mobile phase B. The gradient elution was optimized according to the provisions in Table 3; the flow rate was 0.8 ml per minute; the column temperature was 30°C; and the detection wavelength was 220 nm. The study found that the use of condition ① can achieve a separation degree of astragaloside I>1.5, a theoretical plate number>1000,

[0219] The tailing factor is between 0.9-1.05, which can be effectively separated and meets the requirements of the pharmacopoeia high performance liquid chromatography (General Chapter 0512 of Part IV of the 2020 edition of the Chinese Pharmacopoeia).

[0220] During the determination, accurately pipette 20 μl of the reference solution under "3.2.3" and the test solution under "3.3.3", inject them into the liquid chromatograph, determine, and calculate with the external standard two-point method logarithmic equation to obtain the result.

[0221] Table 3 Optimization of gradient elution conditions for determination of astragaloside IV

[0222]

[0223] 3.5 Specificity inspection

[0224] According to the above chromatographic conditions of serine and glycine components according to the chromatographic conditions of condition ① of "3.4.1", ferulic acid according to the chromatographic conditions of condition ① of "3.4.2", and astragaloside IV according to the chromatographic conditions of condition ① of "3.4.3", the reference solution, test solution, and negative control solution were respectively sampled and measured, and the blank solvent was used as the control. The results showed that the negative sample had no interference at the position of each reference substance. The results are as follows Figure 1 , Figure 2 and Figure 3 shown.

[0225] 3.6 Investigation of linear relationships

[0226] Accurately pipette appropriate amount of each reference solution and dilute it in sequence to obtain a series of mass concentration solutions. Perform regression with the mass concentration of the solution as the horizontal axis (X) and the peak area integral value as the vertical axis (Y) to draw a standard curve. The results are shown in Table 4.

[0227] Table 4 Results of the linear relationship investigation of the four index components

[0228] Ingredient name Regression equation r Linear range μg / mL Ferulic acid Y=12.11X–1.29 0.9999 0.42～54.13 Astragaloside IV Y = 17.11X – 3.67 0.9999 0.08～32.24 Glycine Y=23.4X+2.1 0.9998 2.24～100.4 Serine Y=19.77X+2.33 0.9999 1.25～80.77

[0229] 3.7 Precision test

[0230] Take high (H), medium (M) and low (L) mass concentration reference solution, and inject 6 times under the chromatographic conditions of "3.4.1", "3.4.2" and "3.4.3" (condition ① of 3.4.1, condition ① of 3.4.2 and condition ① of 3.4.3) within the same day, and measure continuously for 3 days. The peak area RSD of ferulic acid, astragaloside I, serine and glycine was measured, indicating that the intra-day precision of the instrument was good. The results are shown in Table 5.

[0231] Table 5 Precision test results of four index components

[0232]

[0233]

[0234] 3.8 Repeatability test

[0235] Four characteristic component test solutions were prepared according to the methods under "3.3.1", "3.3.2" and "3.3.3" respectively, and 6 parallel samples were injected and measured under the chromatographic conditions of each indicator component (condition ① of 3.4.1, condition ① of 3.4.2 and condition ① of 3.4.3). The RSDs of ferulic acid, astragaloside IV, serine and glycine were 2.44%, 2.69%, 2.37% and 3.22% respectively, indicating that the method had good repeatability and met the test requirements.

[0236] 3.9 Stability test

[0237] The test solutions of four indicator components were prepared according to the methods under "3.3.1", "3.3.2" and "3.3.3" respectively, and the samples were injected and measured under the chromatographic conditions of each indicator component (condition ① of 3.4.1, condition ① of 3.4.2 and condition ① of 3.4.3) at 0, 1, 4, 6, 12, 16 and 24 h. The RSDs of the peak areas of ferulic acid, astragaloside I, serine and glycine were 2.1%, 2.6%, 3.51% and 2.2%, respectively, indicating that the four indicator components of the solution had good stability within 24 h.

[0238] 3.10 Sample recovery test

[0239] Accurately weigh the same batch of samples (ZHW-2) that have been measured, a total of 9 parts, placed in a 10mL volumetric flask, added 50%, 100%, 150% level of reference solution, respectively, and injected and measured under the chromatographic conditions of each index component (condition ① of 3.4.1, condition ① of 3.4.2 and condition ① of 3.4.3), and calculated the recovery rate. As a result, the average sample recovery rates of ferulic acid, astragaloside IV, serine and glycine peak area RSD were 101.35%, 100.22%, 101.76%, and 101.50, respectively, and the RSDs were 2.56%, 2.78%, 3.22%, and 2.76&, respectively. The results are shown in Table 6.

[0240] Table 6 Sample recovery test for determination of four index components

[0241]

[0242]

[0243]

[0244] Example 4 Determination of the content of Chinese medicinal compound dry paste powder containing silkworm cocoon shells

[0245] The contents of glycine, citrulline, ferulic acid and astragaloside I in ZHW-2 (ZHW-2-20220601, ZHW-2-20220602, and ZHW-2-20220603) were measured by injection under chromatographic conditions (condition ① of 3.4.1, condition ① of 3.4.2, and condition ① of 3.4.3), and the measured contents are shown in Table 7.

[0246] Table 7 Determination results of the content of Chinese medicine compound dry paste powder containing silkworm cocoon shell (n=3)

[0247]

[0248]

[0249] The above is only an embodiment of the present invention, and the common knowledge such as the known specific structure and characteristics in the scheme is not described in detail here. It should be pointed out that for those skilled in the art, several modifications and improvements can be made without departing from the present invention, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

[0250] The embodiments of the present invention are described in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core idea. At the same time, changes or deformations made by those skilled in the art based on the ideas of the present invention, the specific implementation methods and application scope of the present invention, all belong to the scope of protection of the present invention. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A method for detecting a traditional Chinese medicine compound containing silkworm cocoon shells, characterized in that: The steps include: A test solution and a reference solution of a Chinese medicine compound are taken for testing, wherein the Chinese medicine compound comprises silkworm cocoon shell, corn silk, astragalus and ligusticum chuanxiong, and the reference solution comprises ferulic acid, astragaloside IV, serine and glycine. The method for preparing the Chinese medicine compound test solution comprises: (1) preparing a Chinese medicine compound dry paste powder; and (2) preparing the Chinese medicine compound dry paste powder into a derivatized test solution for serine and glycine detection, a test solution for ferulic acid detection, and / or a test solution for astragaloside IV detection; The preparation method of the derivatized test solution for detecting serine and glycine comprises: weighing an appropriate amount of a Chinese medicine compound dry paste powder, adding a certain amount of a 3-8 mol / L hydrochloric acid solution, hydrolyzing at 120-200° C. for 0.5-1.5 hours, cooling, evaporating to dryness, adding a 0.1-0.5 mol / L hydrochloric acid solution to dissolve, then adding a certain amount of a 0.1-0.5 mol / L acetonitrile solution of phenyl isothiocyanate and a 1-5 mol / L acetonitrile solution of triethylamine, shaking well, leaving at room temperature, adding 40-60% acetonitrile to the scale, shaking well, adding a certain amount of n-hexane, shaking, leaving, taking the lower layer solution, filtering, and taking the filtrate to obtain; The preparation method of the test solution for ferulic acid detection comprises: weighing an appropriate amount of a Chinese medicine compound dry paste powder, adding a certain amount of 50-80% methanol, performing ultrasonic treatment at a power of 300-800W and a frequency of 30-60kHz for 20-60 minutes, cooling, fixing the volume, shaking, filtering, and taking the filtrate to obtain; The preparation method of the test solution for detecting astragaloside IV comprises: weighing an appropriate amount of a Chinese medicine compound dry paste powder, adding a certain amount of a 60-90% methanol solution containing 1-10% concentrated ammonia test solution, sealing, weighing, heating and refluxing for 1-3 hours, cooling, weighing again, supplementing the lost weight with a 60-90% methanol solution containing 1-10% concentrated ammonia test solution, shaking, filtering, accurately measuring a certain amount of subsequent filtrate, evaporating to dryness, dissolving the residue with 60-90% methanol, fixing the volume, shaking, filtering, and taking the subsequent filtrate to obtain; The chromatographic conditions of the detection are: a chromatographic column with octadecylsilane bonded silica as filler is used, the mobile phase A for the detection of serine and glycine is acetonitrile, the mobile phase B is 0.1% phosphoric acid aqueous solution, the mobile phase A for the detection of ferulic acid is acetonitrile, the mobile phase B is 0.1% formic acid aqueous solution, the mobile phase A for the detection of astragaloside IV is acetonitrile, the mobile phase B is 0.05% formic acid aqueous solution, the flow rate is 0.6-1.5 mL / min, the column temperature is 30-50° C., the detection wavelength of serine and glycine is 262 nm, the detection wavelength of ferulic acid is 321 nm, and the detection wavelength of astragaloside IV is 220 nm; Wherein, the gradient elution program for detecting the astragaloside IV is: 0-25 min, 15%-25% A; 25-30 min, 25%-45% A, Wherein, the gradient elution program for detecting the serine and the glycine is: 0-10 min, 5%-10% A; 10-15 min, 10%-20% A; 15-30 min, 20%-23% A; 30-35 min, 23%-55% A, Wherein, the elution procedure for detecting the ferulic acid is: 0-40 min, 15% A; According to the test results, the ingredient information, or the ingredient information and content information of the Chinese medicine compound is obtained.

2. The detection method according to claim 1, characterized in that: The information is calculated according to the corresponding peak areas in the recorded chromatogram of the test solution of the Chinese medicine compound and the chromatogram of the reference solution, using the external standard method to calculate the content of one or more of the following ingredients in the Chinese medicine compound: ferulic acid, astragaloside I, serine and glycine.

3. The detection method according to claim 1, characterized in that: The preparation method of the Chinese medicine compound dry paste powder comprises the following steps: (1) Weigh the raw materials according to the following weight ratio: 25-35 parts of silkworm cocoon shell, 25-35 parts of corn silk, 6-12 parts of astragalus, and 12-18 parts of chuanxiong; (2) Weighing the above-mentioned weight portions of astragalus, chuanxiong and corn silk, adding water, heating and extracting, filtering, and obtaining a first filtrate; (3) weighing the above-mentioned cocoon shells by weight, adding water, heating and extracting, and filtering to obtain a second filtrate; (4) combining the first filtrate and the second filtrate, and concentrating them to obtain a Chinese medicine composition; and (5) Mixing the Chinese medicine composition with one or more pharmaceutically acceptable excipients, drying, crushing and sieving to obtain the Chinese medicine compound dry paste powder.

4. The detection method according to claim 3, characterized in that: Step (2) and / or step (3) of the method for preparing the traditional Chinese medicine compound dry paste powder further comprises the step of soaking the raw material medicine in water.

5. The detection method according to claim 4, characterized in that: In step (2), the astragalus, chuanxiong and corn silk are soaked in water for 30 to 180 minutes.

6. The detection method according to claim 5, characterized in that: The astragalus, chuanxiong and corn silk are soaked in water for 30 to 100 minutes.

7. The detection method according to claim 6, characterized in that: The astragalus, chuanxiong and corn silk are soaked in water for 90 to 100 minutes.

8. The detection method according to claim 7, characterized in that: The time for soaking the astragalus, chuanxiong and corn silk in water is about 90 minutes.

9. The detection method according to claim 4, characterized in that: In step (3), the silkworm cocoon shells are soaked in water for 30 to 180 minutes.

10. The detection method according to claim 9, characterized in that: The cocoon shells are soaked in water for 30 to 90 minutes.

11. The detection method according to claim 10, characterized in that: The cocoon shells are soaked in water for 60 to 90 minutes.

12. The detection method according to claim 11, characterized in that: The cocoon shells are soaked in water for about 60 minutes.

13. The detection method according to claim 3, characterized in that: Weigh the raw materials according to the following weight ratio: 28-35 parts of silkworm cocoon shells, 28-35 parts of corn silk, 8-12 parts of astragalus and 13-18 parts of chuanxiong.

14. The detection method according to claim 13, characterized in that: The raw materials were weighed according to the following weight ratios: 28.5-31.5 parts of silkworm cocoon shells, 28.5-31.5 parts of corn silk, 8.55-9.45 parts of astragalus, and 14.25-15.75 parts of chuanxiong.

15. The detection method according to claim 14, characterized in that: The raw materials were weighed according to the following weight ratio: about 30 parts of silkworm cocoon shells, about 30 parts of corn silk, about 9 parts of astragalus, and about 15 parts of chuanxiong.

16. The detection method according to claim 3, characterized in that: Step (2) of the method for preparing the Chinese medicine compound dry paste powder comprises any one or more of the following items [1] to [6]: [1] The water is distilled water or deionized water; [2] The heating extraction operation is repeated 2 to 4 times; [3] The heating is performed by heating with high heat until boiling and heating with low heat to maintain a slight boil; [4] The amount of water used is 1 to 80 times, in L / kg; [5] The heating extraction time is 15 to 180 minutes; [6] The filtration is performed by using a single layer of filter cloth with a mesh size of 100 to 300.

17. The detection method according to claim 16, characterized in that: In step [1], the water is cold water.

18. The detection method according to claim 17, characterized in that: The cold water covers the surface of the API by 2 to 5 cm.

19. The detection method according to claim 16, characterized in that: In step [2], the heating extraction operation is repeated twice.

20. The detection method according to claim 16, characterized in that: In step [4], the amount of water used is 5 to 30 times the amount, in L / kg.

21. The detection method according to claim 20, characterized in that: The amount of water used is 8 to 14 times the amount, and the unit is L / kg.

22. The detection method according to claim 21, characterized in that: The amount of water used is about 12 times the amount, and the unit is L / kg.

23. The detection method according to claim 16, characterized in that: In step [5], the heating extraction time is 30 to 120 minutes.

24. The detection method according to claim 23, characterized in that: The heating extraction time is about 90 minutes.

25. The detection method according to claim 16, characterized in that: In step [6], the filtration is performed by single-layer filtration using a 300-mesh filter cloth.

26. The detection method according to claim 3, characterized in that: Step (3) of the method for preparing the Chinese medicine compound dry paste powder comprises any one or more of the following items [1] to [5]: [1] The water is distilled water or deionized water; [2] The heating extraction operation is repeated 2 to 4 times; [3] The amount of water used is 1 to 80 times, in L / kg; [4] The heating extraction time is 15 to 180 minutes; [5] The filtration is a single-layer filtration using a 100-300 mesh filter cloth.

27. The detection method according to claim 26, characterized in that: In step [1], the water is cold water.

28. The detection method according to claim 27, characterized in that: The cold water covers the surface of the API by 2 to 5 cm.

29. The detection method according to claim 26, characterized in that: In step [2], the heating extraction operation is repeated 3 times.

30. The detection method according to claim 26, characterized in that: In step [3], the amount of water used is 5 to 30 times the amount, in units of L / kg.

31. The detection method according to claim 30, characterized in that: The amount of water used is 12 to 16 times the amount, in L / kg.

32. The detection method according to claim 31, characterized in that: The amount of water used is about 16 times the amount, and the unit is L / kg.

33. The detection method according to claim 26, characterized in that: In step [4], the heating extraction time is 30 to 120 minutes.

34. The detection method according to claim 33, characterized in that: The heating extraction time is about 60 minutes.

35. The detection method according to claim 26, characterized in that: In step [5], the filtration is performed by single-layer filtration using a 100-mesh filter cloth.

36. The detection method according to claim 3, characterized in that: Step (4) of the method for preparing the Chinese medicine compound dry paste powder comprises any one or more of the following items [1] to [3]: [1] The concentration is concentration under reduced pressure; [2] The concentration temperature is 60 to 90° C.; [3] The relative density of the concentrated solution after concentration is 1.02 to 1.35 g / ml.

37. The detection method according to claim 36, characterized in that: In step [2], the concentration temperature is about 80°C.

38. The detection method according to claim 36, characterized in that: In step [3], the relative density of the concentrated solution after concentration is 1.02 to 1.05 g / ml.

39. The detection method according to claim 3, characterized in that: In step (5) of the method for preparing the traditional Chinese medicine compound dry paste powder, the drying is spray drying, reduced pressure vacuum drying or vacuum freeze drying.

40. The detection method according to claim 39, characterized in that The spray drying conditions are as follows: the preheating temperature of the feed liquid is 60-120°C, the air inlet temperature is 100-180°C, the injection speed is 5-25rpm, the atomization pressure is 0.5-3MPa, and the needle pressure is 0.5-3MPa.

41. The detection method according to claim 40, characterized in that The spray drying conditions are as follows: the preheating temperature of the feed liquid is 85.5-94.5°C, the air inlet temperature is 152-168°C, the injection speed is 19-21rpm, the atomization pressure is 1.9-2.1MPa, and the needle pressure is 1.9-2.1MPa.

42. The detection method according to claim 41, characterized in that The spray drying conditions are as follows: the preheating temperature of the feed liquid is about 90° C., the air inlet temperature is about 160° C., the injection speed is about 20 rpm, the atomization pressure is about 2 MPa, and the needle pressure is about 2 MPa.

43. The detection method according to claim 3, characterized in that In step (5) of the method for preparing the traditional Chinese medicine compound dry paste powder, the mixing step includes converting the weight of the concentrated liquid into the weight of the theoretical dry paste powder, and then adding the auxiliary material at a weight ratio of the auxiliary material to the theoretical dry paste powder of 0.05 to 0.25:1, fully dissolving, and mixing evenly.

44. The detection method according to any one of claims 1 to 43, characterized in that The preparation method of the serine and glycine derivatized reference solution comprises: weighing appropriate amounts of serine and glycine; adding 0.1-0.5 mol / L hydrochloric acid solution to prepare a mixed reference solution containing each component at a concentration of 10-200 μg / ml; drawing an appropriate amount of the mixed reference solution, adding a certain amount of 0.1-0.5 mol / L phenyl isothiocyanate acetonitrile solution and 1-5 mol / L triethylamine acetonitrile solution, shaking well, leaving at room temperature, adding 40-60% acetonitrile to the scale, shaking well, adding a certain amount of n-hexane, shaking, leaving, taking the lower layer solution, filtering, taking the filtrate, and obtaining the serine and glycine derivatized reference solution.

45. The detection method according to any one of claims 1 to 43, characterized in that The preparation method of the reference solution of ferulic acid and / or astragaloside IV comprises: weighing an appropriate amount of ferulic acid and / or astragaloside IV; and adding a methanol aqueous solution with a volume percentage concentration of 10% to 100% to prepare a reference solution of ferulic acid and / or astragaloside IV containing each component at a concentration of 10 to 200 μg / ml.

46. ​​The detection method according to claim 1, characterized in that The flow rate is 0.8-1.2 mL / min, and the column temperature is 30-35°C.

47. The detection method according to claim 1, characterized in that The injection volume of the detection is 5 to 20 μl.

48. The detection method according to claim 1, characterized in that The Chinese medicine compound test solution and the reference solution are filtered with a 0.22 μm or 0.45 μm microporous filter membrane before the detection.

49. The detection method according to claim 1, characterized in that The chromatographic column has a length of 25 cm, an inner diameter of 4.6 mm, and a particle size of 5 μm.

50. The detection method according to claim 1, characterized in that The chromatographic column is an Agilent 5TC-C18 (2) chromatographic column, a Waters Symmetry C18 chromatographic column or an Agilent SB C18 chromatographic column.

51. The detection method according to claim 50, characterized in that The chromatographic column is an Agilent 5TC-C18(2) chromatographic column.

52. The detection method according to any one of claims 1 to 43, characterized in that The concentration of the ferulic acid reference solution is about 20 μg / ml.

53. The detection method according to any one of claims 1 to 43, characterized in that The concentration of the astragaloside IV reference solution is about 20 μg / ml.

54. The detection method according to any one of claims 1 to 43, characterized in that The concentration of the serine control solution is about 130 μg / ml.

55. The detection method according to any one of claims 1 to 43, characterized in that The concentration of the glycine control solution is about 50 μg / ml.

56. Use of the detection method according to any one of claims 1 to 55 in quality detection and / or quality evaluation and / or quality control of a traditional Chinese medicine compound containing silkworm cocoon shells.

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