CN115406986A - Quality control method and application of traditional Chinese medicine composition containing silkworm cocoon shells - Google Patents
Quality control method and application of traditional Chinese medicine composition containing silkworm cocoon shells Download PDFInfo
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Images
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N2030/042—Standards
- G01N2030/047—Standards external
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
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- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Engineering & Computer Science (AREA)
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Abstract
The invention discloses a detection method of a traditional Chinese medicine compound containing silkworm cocoon shells, which comprises the following steps: detecting a traditional Chinese medicine compound test solution and a reference solution, wherein the traditional Chinese medicine compound comprises silkworm cocoon shells, corn stigma, astragalus membranaceus and ligusticum wallichii, the reference is ferulic acid, astragaloside, serine and glycine, and the chromatographic conditions of the detection are as follows: a chromatographic column with octadecylsilane chemically bonded silica as a filler is adopted, a mobile phase A is selected from one or more of acetonitrile, methanol and tetrahydrofuran, a mobile phase B is an acid aqueous solution, an alkaline aqueous solution and/or a buffer saline aqueous solution, the flow rate is 0.6-1.5 mL/min, the column temperature is 30-50 ℃, and the detection wavelength is 200-360 nm; and obtaining component information, or component information and content information of the Chinese herbal compound according to the detection result. The method has the advantages of good linear relation, high precision, strong stability, good repeatability, high sample recovery rate, simplicity and feasibility, and is particularly suitable for quality control in industrialization of the composition.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a quality control method and application of a traditional Chinese medicine composition containing silkworm cocoon shells.
Background
Chronic Kidney Disease (CKD) is a serious disease threatening human health. At present, no good method is provided for completely treating proteinuria caused by various chronic kidney diseases. The traditional Chinese medicine considers that kidney can not store essence, essence slightly flows downwards, spleen deficiency can not take essence, clear qi sinks downwards, turbid qi flows upwards, lung can not control qi and the like, and the traditional Chinese medicine is a recognized mechanism for proteinuria in the traditional Chinese medicine at present. The pathogenesis of proteinuria is that the principal is deficiency with secondary excess, the principal is deficiency, and the pathogenic wind, damp-heat and blood stasis are combined, and they can affect each other. The traditional Chinese medicine has the advantages of treating both symptoms and root causes, improving symptoms, having small side effect, being not easy to relapse and the like in the aspect of treating proteinuria.
The project is based on the theory of traditional Chinese medicine, the traditional Chinese medicine compound preparation composed of cocoon shells, corn silk, astragalus and ligusticum wallichii, which has the functions of tonifying kidney and activating blood circulation, and clearing and resolving dampness and blood stasis, has obvious effects of eliminating edema and relieving kidney diseases, and is particularly suitable for albuminuria caused by various chronic nephritis. In the formula, astragalus root, radix astragali tonifies qi and strengthens exterior, ligusticum wallichii activates blood and removes stasis, corn stigma induces diuresis and reduces swelling, and silkworm cocoon shells tonify kidney and replenish essence. The whole prescription is combined for use, can tonify the kidney, activate the blood, clear and remove dampness and blood stasis, and has good curative effect on clinical proteinuria. The traditional Chinese medicine composition is a compound based on the guidance of the theory of traditional Chinese medicine, is an organic whole, and the exertion of the curative effect is not completely replaced by one component or a certain traditional Chinese medicine, and experimental research shows that the traditional Chinese medicine composition has the best curative effect as a whole and has obvious combined synergistic effect compared with the using effect of a single medicine. Wherein the radix astragali is dried root of Astragalus membranaceus (Fisch.) bge. Var. Mongholicus (bge.) Hsiao or Astragalus membranaceus (Fisch.) bge. Of Leguminosae, which is recorded in Chinese pharmacopoeia (2015 edition). Rhizoma Ligustici Chuanxiong is dried rhizome of Umbelliferae rhizoma Ligustici Chuanxiong (scientific name: ligusticum chuanxiong Hort) recorded in Chinese pharmacopoeia (2015 edition). Stigma Maydis is the style and stigma of Zea mays L. Many classic traditional Chinese medicine have recorded corn stigma, which is sweet and bland in flavor and mild in nature. Silkworm cocoon shells are cocoon shells of Bombyx mori l.f. an insect of family Bombyx, also known as: silkworm clothing, silkworm cocoon, cotton silk and silkworm cocoon shell. Sweet taste and warm nature. Has hemostatic, thirst quenching, toxic materials clearing away, and sore healing effects.
The research on the quality control of the Chinese herbal compound is the key of the modernization and internationalization of the Chinese herbal medicine. Effectively solves the quality control problem of the traditional Chinese medicine compound preparation and is a key link for industrialization. However, no study on the quality control of the traditional Chinese medicine composition containing silkworm cocoon shells as described in the application is available, and no method for simultaneously measuring ferulic acid, astragaloside IV, serine and glycine in the composition is available.
Disclosure of Invention
In view of the above, the present invention provides a method for detecting a compound Chinese medicine containing silkworm cocoon shells, comprising the following steps:
detecting test solution and reference solution of Chinese medicinal composition, wherein the Chinese medicinal composition comprises silkworm cocoon shell, stigma Maydis, radix astragali and rhizoma Ligustici Chuanxiong, the reference is ferulic acid, astragaloside IV, serine and glycine,
the chromatographic conditions for the detection were: a chromatographic column with octadecylsilane chemically bonded silica as a filler is adopted, a mobile phase A is selected from one or more of acetonitrile, methanol and tetrahydrofuran, a mobile phase B is an acid aqueous solution, an alkaline aqueous solution and/or a buffer saline aqueous solution, the flow rate is 0.6-1.5 mL/min, the column temperature is 30-50 ℃, and the detection wavelength is 200-360 nm;
wherein, the gradient elution procedure for detecting the astragaloside IV is as follows: 0 to 25min,15 to 25 percent of A; 25-30min, 25% -45% A,
wherein the gradient elution procedure for detecting the serine and the glycine is: 0-10 min,5% -10% A;10 to 15min,10 to 20% by weight of A; 15-30min, 20% -23% A; 30-35min, 23% -55% A,
wherein, the elution procedure for detecting the ferulic acid comprises the following steps: 0 to 40min,15% by weight A;
and obtaining component information, or component information and content information of the Chinese herbal compound according to the detection result.
Further, the information is that according to the recorded corresponding peak areas in the chromatogram of the test solution of the traditional Chinese medicine compound and the chromatogram of the reference solution, the content of one or more of the following components in the traditional Chinese medicine compound is calculated according to an external standard method: ferulic acid, astragaloside IV, serine and glycine.
Further, the preparation method of the traditional Chinese medicine compound test solution comprises the following steps: (1) preparing traditional Chinese medicine compound dry paste powder; and (2) preparing the traditional Chinese medicine compound dry paste powder into a derivatization test solution for detecting serine and glycine, a test solution for detecting ferulic acid and/or a test solution for detecting astragaloside.
Further, the preparation method of the traditional Chinese medicine compound dry paste powder comprises the following steps:
(1) Weighing the following raw material medicines in parts by weight: 25 to 35 parts of silkworm cocoon shell, 25 to 35 parts of corn stigma, 6 to 12 parts of astragalus and 12 to 18 parts of ligusticum wallichii;
(2) Weighing the astragalus, the ligusticum wallichii and the corn stigma in parts by weight, adding water, heating, extracting and filtering to obtain a first filtrate;
(3) Weighing the silkworm cocoon shells in parts by weight, adding water, heating for extraction, and filtering to obtain a second filtrate;
(4) Mixing the first filtrate and the second filtrate, and concentrating to obtain the Chinese medicinal composition; and
(5) Mixing the traditional Chinese medicine composition with one or more pharmaceutically acceptable auxiliary materials, drying, crushing and sieving to obtain the traditional Chinese medicine compound dry paste powder.
Further, the step (2) and/or the step (3) of the preparation method of the traditional Chinese medicine compound dry paste powder further comprises the step of soaking the raw material medicine in water.
Furthermore, the astragalus root, the szechuan lovage rhizome and the corn stigma are soaked in water for 30-180 min.
Furthermore, the astragalus root, the szechuan lovage rhizome and the corn stigma are soaked in water for 30-100 min.
Furthermore, the astragalus root, the szechuan lovage rhizome and the corn stigma are soaked in water for 90-100 min.
Further, the astragalus root, the ligusticum wallichii and the corn stigma are soaked in water for about 90min.
Furthermore, the time for soaking the silkworm cocoon shell in water is 30-180 min.
Furthermore, the time for soaking the silkworm cocoon shells in water is 30-90 min.
Furthermore, the silkworm cocoon shell is soaked in water for 60-90 min.
Further, the time for soaking the silkworm cocoon shell in water is about 60min.
Further, the raw materials are respectively weighed according to the following weight parts: 28 to 35 parts of silkworm cocoon shell, 28 to 35 parts of corn stigma, 8 to 12 parts of astragalus and 13 to 18 parts of ligusticum wallichii.
Further, the raw materials are respectively weighed according to the following weight parts: 28.5 to 31.5 parts of silkworm cocoon shell, 28.5 to 31.5 parts of corn stigma, 8.55 to 9.45 parts of astragalus and 14.25 to 15.75 parts of ligusticum wallichii.
Further, the raw materials are respectively weighed according to the following weight parts: 30 parts of silkworm cocoon shell, 30 parts of corn silk, 9 parts of astragalus root and 15 parts of ligusticum wallichii.
Further, the step (2) of the preparation method of the traditional 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, preferably the water is cold water, and more preferably the cold water submerges 2-5 cm of the surface of the raw material medicine;
[2] the operation of the heating extraction is repeated 2 to 4 times, for example 2 times;
[3] the heating is carried out by heating with strong fire until boiling and keeping slight boiling with slow fire;
[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;
[5] the heating extraction time is 15-180 min, preferably 30-120 min, for example about 90min;
[6] the filtration is 100-300 mesh filter cloth single layer filtration, such as 300 mesh filter cloth single layer filtration.
Further, the step (3) of the preparation method of the traditional 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, preferably the water is cold water, and more preferably the cold water submerges 2-5 cm above the surface of the raw material medicine;
[2] the operation of heating and extracting is repeated 2 to 4 times, for example, 3 times;
[3] the amount of the water is 1 to 80 times (L/kg), preferably 5 to 30 times, more preferably 12 to 16 times, for example, about 16 times;
[4] the heating extraction time is 15-180 min, preferably 30-120 min, such as about 60min;
[5] the filtration is 100-300 mesh filter cloth single layer filtration, such as 100 mesh filter cloth single layer filtration.
Further, the step (4) of the preparation method of the traditional Chinese medicine compound dry paste powder comprises any one or more of the following items [1] to [3 ]:
[1] the concentration is reduced pressure concentration;
[2] the temperature of the concentration is 60 to 90 ℃, for example about 80 ℃;
[3] the relative density of the concentrate after concentration is 1.02 to 1.35g/ml, for example 1.02 to 1.05g/ml.
Further, in the step (5) of the preparation method of the traditional Chinese medicine compound dry paste powder, the drying is spray drying, vacuum drying under reduced pressure or vacuum freeze drying.
Further, the conditions of the spray drying are: the preheating temperature of the feed liquid is 60-120 ℃, the air inlet temperature is 100-180 ℃, the sample injection speed is 5-25 rpm, the atomization pressure is 0.5-3 MPa, and the needle pressure is 0.5-3 MP.
Further, the conditions of the spray drying are: the preheating temperature of the feed liquid is 85.5-94.5 ℃, the air inlet temperature is 152-168 ℃, the sample injection speed is 19-21 rpm, the atomization pressure is 1.9-2.1 MPa, and the needle pressure is 1.9-2.1 MPa.
Further, the conditions of the spray drying are: the preheating temperature of the feed liquid is about 90 ℃, the air inlet temperature is about 160 ℃, the sampling speed is about 20rpm, the atomization pressure is about 2MPa, and the needle pressure is about 2MPa.
Further, the mixing step comprises converting the weight of the concentrated solution after concentration into the weight of theoretical dry paste powder, adding the auxiliary material according to the weight ratio of 0.05-0.25: 1 of the auxiliary material to the theoretical dry paste powder, fully dissolving and uniformly mixing.
Further, a method for preparing a derivatized test solution for detection of serine and glycine comprises: weighing a proper amount of traditional Chinese medicine compound dry paste powder, adding a certain amount of 3-8 mol/L hydrochloric acid solution, hydrolyzing at 120-200 ℃ for 0.5-1.5 hours, cooling, evaporating to dryness, adding 0.1-0.5 mol/L hydrochloric acid solution for dissolving, 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 up, standing at room temperature, adding 40-60% acetonitrile to scale, shaking up, adding a certain amount of n-hexane, shaking up, standing, taking a lower layer solution, filtering, and taking a subsequent filtrate to obtain the traditional Chinese medicine compound dry paste.
Further, the method for preparing a test solution for ferulic acid detection comprises: weighing a proper amount of Chinese herbal compound dry paste powder, adding a certain amount of 50-80% methanol, carrying out ultrasonic treatment for 20-60 minutes at the power of 300-800W and the frequency of 30-60 kHz, cooling, fixing the volume, shaking up, filtering, and taking the subsequent filtrate to obtain the Chinese herbal compound dry paste.
Further, the preparation method of the test solution for detecting astragaloside comprises the following steps: weighing a proper amount of traditional Chinese medicine compound dry paste powder, adding a certain amount of 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 reduced weight with 60-90% methanol solution containing 1-10% concentrated ammonia test solution, shaking up, filtering, precisely weighing a certain amount of subsequent filtrate, evaporating to dryness, dissolving residues with 60-90% methanol, fixing the volume, shaking up, filtering, and taking the subsequent filtrate to obtain the traditional Chinese medicine compound dry paste.
Further, the method for preparing the derivatized serine and glycine control solution comprises: weighing a proper amount of serine and glycine; adding 0.1-0.5 mol/L hydrochloric acid solution to prepare a mixed reference substance solution containing 10-200 mu g/ml of each component; sucking a proper amount of the mixed reference substance solution, 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 up, standing at room temperature, adding 40-60% acetonitrile to scale, shaking up, adding a certain amount of n-hexane, shaking up, standing, taking a lower layer solution, filtering, and taking a subsequent filtrate to obtain the derivatized reference substance solution of serine and glycine.
Further, the preparation method of the ferulic acid and/or the astragaloside IV reference solution comprises: weighing appropriate amount of ferulic acid and/or astragaloside IV; and adding 10-100% methanol water solution to prepare a reference solution containing 10-200 μ g/ml ferulic acid and/or astragaloside IV.
Further, the flow rate is 0.8 to 1.2mL/min.
Further, the column temperature is 30 to 35 ℃.
Further, the detection wavelength is 200 to 330nm.
Further, the sample size of the assay was 5 to 20. Mu.l.
Further, the Chinese medicinal compound test solution and the reference solution are filtered by a 0.22 μm or 0.45 μm microporous filter membrane before the detection.
Further, the column length of the column was 25cm, the inner diameter was 4.6mm, and the particle diameter was 5 μm.
Further, the column is an Agilent 5TC-C18 (2) column, a Waters Symmetry C18 column or an Agilent SB C18 column.
Further, the chromatographic column is an Agilent 5TC-C18 (2) chromatographic column.
Further, 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), and methanol: acetonitrile (90: 10).
Further, the aqueous acid solution, aqueous base solution and/or aqueous buffered salt solution is selected from one or more of weak acids and salts thereof, weak bases and salts thereof at different concentrations.
Further, the aqueous acid solution, aqueous base solution and/or aqueous buffer salt solution is selected from different concentrations of 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.
Further, the acid aqueous solution is 0.05% to 0.4% acid aqueous solution.
Further, the acid aqueous solution is 0.05% to 0.2% phosphoric acid or formic acid aqueous solution.
Further, the acid aqueous solution is 0.05 to 0.1 percent of phosphoric acid or formic acid aqueous solution.
Further, the concentration of the control solution of ferulic acid was about 20 μ g/ml.
Further, the concentration of the astragaloside IV control solution is about 20 μ g/ml.
Further, the concentration of the serine control solution was about 130. Mu.g/ml.
Further, the concentration of the glycine control solution was about 50. Mu.g/ml.
According to another aspect of the invention, the application of the detection method in quality detection and/or quality evaluation and/or quality control of a traditional Chinese medicine compound containing silkworm cocoon shells is provided.
The invention has the beneficial effects that:
(1) The invention establishes a quality control method of a traditional Chinese medicine composition containing silkworm cocoon shells and a method for measuring the contents of ferulic acid, astragaloside IV, serine and glycine in the application of the composition, and performs methodological verification of system adaptability, specificity, linearity, precision, repeatability and stability on detection methods of four index components to determine the feasibility of the detection methods of the four index components.
(2) The parameters related to the detection method of the invention are obtained on the basis of the research experience of the inventor, and the excellent detection effect is realized under the detection condition.
(3) The invention provides a basis for establishing a compound quality standard for preventing and/or treating proteinuria caused by various chronic kidney diseases.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the description below are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on the drawings without departing from the scope of the claimed invention.
FIG. 1 is a chromatogram for measuring the contents of serine and glycine in a Chinese medicinal composition containing silkworm cocoon shells. Wherein A is a blank solvent chromatogram; b, a chromatogram of a serine and glycine reference solution (retention time 21.827min is a serine chromatographic peak; 22.393 is a glycine chromatographic peak); a chromatogram of a C CJK-YX negative control test sample solution; and D, a chromatogram of the derivatized test solution for detecting serine and glycine (retention time 21.738min is a serine chromatographic peak; 22.369 is a glycine chromatographic peak).
FIG. 2 is a chromatogram for measuring ferulic acid content in a Chinese medicinal composition containing silkworm cocoon shells. Wherein A is a blank solvent chromatogram; b ferulic acid control solution chromatogram (retention time 22.917min is ferulic acid chromatogram peak); c CX-YX negative control sample solution chromatogram; and D, a chromatogram of the test solution for ferulic acid detection (retention time 22.718min is the ferulic acid chromatographic peak).
FIG. 3 is a chromatogram for determining astragaloside IV content in a Chinese medicinal composition containing silkworm cocoon shell. Wherein A is a blank solvent chromatogram; b astragaloside IV reference solution chromatogram (retention time 22.293min is astragaloside IV chromatogram peak); c HQ-YX negative control sample solution chromatogram; d, applying to a chromatogram of a test solution for detecting astragaloside IV (the retention time is 22.310min is the chromatographic peak of the astragaloside IV).
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, not all, embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
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.
Unless defined otherwise, all technical and scientific terms and abbreviations used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this term applies. Although any methods, conditions, materials, or materials similar or equivalent to those disclosed herein can be used in the practice of the present invention, the preferred methods, conditions, materials, or materials are described herein.
In the present invention, the term "comprising" is synonymous with "including". The terms "comprises," "comprising," "includes," "including," "has," "having," "contains," "containing," or any other variation thereof, as used herein, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises a list of 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.
As described in the background art section, no literature has been reported about a content analysis method for comprehensively analyzing chemical components of a chinese herbal compound containing silkworm cocoon shells. In order to solve the problems, the invention provides a detection method of a traditional Chinese medicine compound containing silkworm cocoon shells, which comprises the following steps:
detecting test solution and reference solution of Chinese medicinal composition, wherein the Chinese medicinal composition comprises silkworm cocoon shell, stigma Maydis, radix astragali and rhizoma Ligustici Chuanxiong, the reference is ferulic acid, astragaloside IV, serine and glycine,
the chromatographic conditions for the detection are as follows: a chromatographic column with octadecylsilane chemically bonded silica as a filler is adopted, a mobile phase A is selected from one or more of acetonitrile, methanol and tetrahydrofuran, a mobile phase B is an acid aqueous solution, an alkaline aqueous solution and/or a buffer saline aqueous solution, the flow rate is 0.6-1.5 mL/min, the column temperature is 30-50 ℃, and the detection wavelength is 200-360 nm;
wherein, the gradient elution procedure for detecting the astragaloside IV is as follows: 0-25min, 15% -25% of A; 25-30min, 25% -45% A,
wherein the gradient elution procedure for detecting the serine and the glycine is: 0-10 min,5% -10% A;10 to 15min,10 to 20% by weight A; 15-30min, 20% -23% A; 30-35min, 23% -55% A,
wherein, the elution procedure for detecting the ferulic acid comprises the following steps: 0 to 40min,15% by weight A;
and obtaining component information, or component information and content information of the Chinese herbal compound according to the detection result.
In the present invention, when ratios, equivalents, concentrations, wavelengths, temperatures, flow rates, parts by weight, or other values or parameters are expressed as ranges, preferred ranges, or ranges defined by a list of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. For example, when the range of "200 to 360" is disclosed, unless otherwise specified, the range is intended to include the endpoints thereof and all integers and fractions within the range, and the effects of the present invention can be achieved within the range disclosed above.
In a preferred embodiment, the information is that according to the recorded corresponding peak areas in the chromatogram of the test solution of the traditional Chinese medicine compound and the chromatogram of the control solution, the content of one or more of the following components in the traditional Chinese medicine compound is calculated according to an external standard method: ferulic acid, astragaloside IV, serine and glycine.
In a preferred embodiment, the preparation method of the compound traditional Chinese medicine test solution comprises the following steps: (1) preparing traditional Chinese medicine compound dry paste powder; and (2) preparing the traditional Chinese medicine compound dry paste powder into a derivatization test solution for detecting serine and glycine, a test solution for detecting ferulic acid and/or a test solution for detecting astragaloside.
In a preferred embodiment, the preparation method of the traditional Chinese medicine compound dry paste powder comprises the following steps:
(1) Weighing the following raw material medicines in parts by weight: 25 to 35 parts of silkworm cocoon shell, 25 to 35 parts of corn stigma, 6 to 12 parts of astragalus and 12 to 18 parts of ligusticum wallichii;
(2) Weighing the astragalus, the ligusticum wallichii and the corn stigma in parts by weight, adding water, heating, extracting and filtering to obtain a first filtrate;
(3) Weighing the silkworm cocoon shells in parts by weight, adding water, heating for extraction, and filtering to obtain a second filtrate;
(4) Mixing the first filtrate and the second filtrate, and concentrating to obtain the Chinese medicinal composition; and
(5) Mixing the traditional Chinese medicine composition with one or more pharmaceutically acceptable auxiliary materials, drying, crushing and sieving to obtain the traditional Chinese medicine compound dry paste powder.
In a preferred embodiment, the step (2) and/or the step (3) of the preparation method of the traditional Chinese medicine compound dry paste powder further comprises the step of soaking the raw material medicine in water.
In a preferred embodiment, the astragalus root, the ligusticum wallichii and the corn stigma are soaked in water for 30-180 min.
In a preferred embodiment, the astragalus root, the ligusticum wallichii and the corn stigma are soaked in water for 30-100 min.
In a preferred embodiment, the astragalus root, the chuanxiong rhizome and the corn stigma are soaked in water for 90-100 min.
In a preferred embodiment, the astragalus root, the ligusticum wallichii and the corn stigma are soaked in water for about 90min.
In a preferred embodiment, the silkworm cocoon shell is soaked in water for 30-180 min.
In a preferred embodiment, the silkworm cocoon shell is soaked in water for 30-90 min.
In a preferred embodiment, the silkworm cocoon shell is soaked in water for 60-90 min.
In a preferred embodiment, the silkworm cocoon shell is soaked in water for about 60min.
In the present invention, "about" refers to a value within a 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.
In a preferred embodiment, the raw materials are respectively weighed according to the following weight parts: 28 to 35 parts of silkworm cocoon shell, 28 to 35 parts of corn stigma, 8 to 12 parts of astragalus and 13 to 18 parts of ligusticum wallichii.
In a preferred embodiment, the raw materials are respectively weighed according to the following weight parts: 28.5 to 31.5 parts of silkworm cocoon shell, 28.5 to 31.5 parts of corn stigma, 8.55 to 9.45 parts of astragalus and 14.25 to 15.75 parts of ligusticum wallichii.
In a preferred embodiment, the raw materials are respectively weighed according to the following weight parts: 30 parts of silkworm cocoon shell, 30 parts of corn silk, 9 parts of astragalus root and 15 parts of ligusticum wallichii.
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.
In a preferred embodiment, the step (2) of the preparation method of the traditional 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, preferably the water is cold water, and more preferably the cold water submerges 2-5 cm of the surface of the raw material medicine;
[2] the operation of the heating extraction is repeated 2 to 4 times, for example 2 times;
[3] the heating is carried out by heating with strong fire until the water is boiled and keeping slight boiling with slow fire;
[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;
[5] the heating extraction time is 15-180 min, preferably 30-120 min, for example about 90min;
[6] the filtration is 100-300 mesh filter cloth single layer filtration, such as 300 mesh filter cloth single layer filtration.
In a preferred embodiment, the step (3) of the preparation method of the traditional 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, preferably the water is cold water, and more preferably the cold water submerges 2-5 cm of the surface of the raw material medicine;
[2] the operation of heating and extracting is repeated 2 to 4 times, for example, 3 times;
[3] the amount of the water is 1 to 80 times (L/kg), preferably 5 to 30 times, more preferably 12 to 16 times, for example, about 16 times;
[4] the heating extraction time is 15-180 min, preferably 30-120 min, such as about 60min;
[5] the filtration is 100-300 mesh filter cloth single layer filtration, such as 100 mesh filter cloth single layer filtration.
In a preferred embodiment, the step (4) of the preparation method of the traditional Chinese medicine compound dry paste powder comprises any one or more of the following items [1] to [3 ]:
[1] the concentration is reduced pressure concentration;
[2] the temperature of the concentration is 60 to 90 ℃, for example about 80 ℃;
[3] the relative density of the concentrate after concentration is 1.02 to 1.35g/ml, for example 1.02 to 1.05g/ml.
In a preferred embodiment, in the step (5) of the preparation method of the traditional Chinese medicine compound dry paste powder, the drying is spray drying, vacuum drying under reduced pressure or vacuum freeze drying.
In a preferred embodiment, the spray drying conditions are: the preheating temperature of the feed liquid is 60-120 ℃, the air inlet temperature is 100-180 ℃, the sample injection speed is 5-25 rpm, the atomization pressure is 0.5-3 MPa, and the needle pressure is 0.5-3 MP.
In a preferred embodiment, the conditions of the spray drying are: the preheating temperature of the feed liquid is 85.5-94.5 ℃, the air inlet temperature is 152-168 ℃, the sampling speed is 19-21 rpm, the atomization pressure is 1.9-2.1 MPa, and the pressure of the needle is 1.9-2.1 MPa.
In a preferred embodiment, the conditions of the spray drying are: the preheating temperature of the feed liquid is about 90 ℃, the air inlet temperature is about 160 ℃, the sampling speed is about 20rpm, the atomization pressure is about 2MPa, and the needle pressure is about 2MPa.
In a preferred embodiment, the mixing step comprises converting the weight of the concentrated solution after concentration into the weight of theoretical dry extract powder, adding the auxiliary material according to the weight ratio of 0.05-0.25: 1 of the auxiliary material to the theoretical dry extract powder, fully dissolving, and uniformly mixing.
In a preferred embodiment, a method of preparing a derivatized test solution for serine and glycine detection comprises: weighing a proper amount of traditional Chinese medicine compound dry paste powder, adding a certain amount of 3-8 mol/L hydrochloric acid solution, hydrolyzing at 120-200 ℃ for 0.5-1.5 hours, cooling, evaporating to dryness, adding 0.1-0.5 mol/L hydrochloric acid solution for dissolving, 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 up, standing at room temperature, adding 40-60% acetonitrile to scale, shaking up, adding a certain amount of n-hexane, shaking up, standing, taking a lower layer solution, filtering, and taking a subsequent filtrate to obtain the traditional Chinese medicine compound dry paste.
In a preferred embodiment, a method for preparing a test solution for ferulic acid detection comprises: weighing a proper amount of Chinese herbal compound dry paste powder, adding a certain amount of 50-80% methanol, carrying out ultrasonic treatment for 20-60 minutes at the power of 300-800W and the frequency of 30-60 kHz, cooling, fixing the volume, shaking up, filtering, and taking the subsequent filtrate to obtain the Chinese herbal compound dry paste.
In a preferred embodiment, the method for preparing a test solution for astragaloside detection comprises: weighing a proper amount of traditional Chinese medicine compound dry paste powder, adding a certain amount of 60-90% methanol solution containing 1-10% concentrated ammonia test solution, sealing, weighing, heating and refluxing for 1-3 hours, cooling, weighing again, complementing the lost weight with 60-90% methanol solution containing 1-10% concentrated ammonia test solution, shaking up, filtering, precisely weighing a certain amount of subsequent filtrate, evaporating to dryness, dissolving residues with 60-90% methanol, fixing the volume, shaking up, filtering, and taking the subsequent filtrate to obtain the traditional Chinese medicine compound dry paste.
The preparation method of the 1-10% strong ammonia water test solution comprises the step of adding water into 1-10mL of commercially available ammonia water (with the concentration of 25-28%) to dilute the ammonia water to 100mL. The commercially available aqueous ammonia may be any aqueous ammonia solution commercially available, for example, from the national pharmaceutical group chemical reagent Shanghai Co., ltd.
In a preferred embodiment, the derivatized control solutions of serine and glycine are prepared by a method comprising: weighing a proper amount of serine and glycine; adding 0.1-0.5 mol/L hydrochloric acid solution to prepare a mixed reference substance solution containing 10-200 mu g/ml of each component; sucking a proper amount of the mixed reference substance solution, 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 up, standing at room temperature, adding 40-60% acetonitrile to scale, shaking up, adding a certain amount of n-hexane, shaking up, standing, taking a lower layer solution, filtering, and taking a subsequent filtrate to obtain the derivatized reference substance solution of serine and glycine.
In a preferred embodiment, the control solution of ferulic acid and/or astragaloside is prepared by a process comprising: weighing appropriate amount of ferulic acid and/or astragaloside IV; and adding 10-100% methanol water solution to prepare a reference solution containing 10-200 μ g/ml ferulic acid and/or astragaloside IV.
In a preferred embodiment, the flow rate is 0.8 to 1.2mL/min.
As for the flow rate of the present invention, 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 are compared and screened, and as a result, 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 can all satisfy the detection requirements, but when the flow rate is 0.8-1.2 mL/min, the peak distribution is relatively uniform, the separation effect is the best, and finally 0.8-1.2 mL/min is determined as the best flow rate, and the optional flow rate is 0.7mL/min and 1.3mL/min.
In a preferred embodiment, the column temperature is from 30 to 35 ℃.
The column temperature of the invention is screened by comparing 30 ℃, 35 ℃, 40 ℃, 45 ℃ and 50 ℃, and the result shows that the column temperature can meet the detection requirements when the column temperature is 30 ℃, 35 ℃, 40 ℃, 45 ℃ and 50 ℃, but when the column temperature is 30-35 ℃, the peak distribution is more uniform, the separation effect of each main peak is the best, and finally the column temperature of 30-35 ℃ is determined to be the best column temperature, and the column temperature can be selected to be 40 ℃ and 45 ℃.
In a preferred embodiment, the detection wavelength is 200 to 330nm.
In a preferred embodiment, the sample size for the assay is between 5 and 20. Mu.l.
In a preferred embodiment, the test solution and the control solution are filtered through 0.22 μm or 0.45 μm microporous membrane before the detection.
In a preferred embodiment, the column has a column length of 25cm, an internal diameter of 4.6mm and a particle size of 5 μm.
In a preferred embodiment, the column is an Agilent 5TC-C18 (2) column, a Waters Symmetry C18 column or an Agilent SB C18 column.
In a preferred embodiment, the chromatography column is an Agilent 5TC-C18 (2) chromatography column.
According to the chromatographic column, agilent 5TC-C18 (2), waters Symmetry C18 and Agilent SB C18 are respectively screened, and the chromatographic column selected as a result can meet the detection requirement, but when the chromatographic column is Agilent 5TC-C18 (2), the peak distribution is uniform, the peak separation effect is good, and the performance parameters are optimal, and finally the Agilent 5TC-C18 (2) is determined to be the optimal chromatographic column, and the chromatographic column can be octadecylsilane chemically bonded silica gel as a filler chromatographic column (the length of the column is 25cm, the inner diameter is 4.6mm, and the particle size is 5 mu m).
In a preferred embodiment, the mobile phase a is selected from one or more of the following: 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), and methanol: acetonitrile (90: 10).
In a preferred embodiment, the aqueous acid solution, aqueous base solution and/or aqueous buffered salt solution is selected from one or more of weak acids and salts thereof, weak bases and salts thereof at different concentrations.
In a preferred embodiment, the aqueous acid solution, aqueous base solution and/or aqueous buffer salt solution is selected from different concentrations of 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 a preferred embodiment, the aqueous acid solution is a 0.05% to 0.4% aqueous acid solution.
In a preferred embodiment, the aqueous acid solution is a 0.05% to 0.2% aqueous solution of phosphoric acid or formic acid.
In a preferred embodiment, the aqueous acid solution is a 0.05% to 0.1% aqueous solution of phosphoric acid or formic acid.
In a preferred embodiment, the control solution of ferulic acid has a concentration of about 20 μ g/ml.
In a preferred embodiment, the concentration of the control solution of astragaloside is about 20 μ g/ml.
In a preferred embodiment, the concentration of the serine control solution is about 130 μ g/ml.
In a preferred embodiment, the concentration of the glycine control solution is about 50 μ g/ml.
According to another aspect of the invention, the application of the detection method in quality detection and/or quality evaluation and/or quality control of a traditional Chinese medicine compound containing silkworm cocoon shells is provided.
It should be noted that, in the present application, the embodiments and features of the embodiments may be combined with each other without conflict. The present invention will be described in detail with reference to examples.
The present invention is described in further detail below with reference to specific examples, which are not to be construed as limiting the scope of the invention as claimed herein.
Examples
Example 1 preparation of Compound Dry extract powder of Chinese medicine containing silkworm cocoon Shell
The combined decoction process of corn stigma, astragalus and ligusticum wallichii comprises the following steps: taking 90g of astragalus membranaceus, 150g of ligusticum wallichii and 300g of corn stigma, placing the astragalus membranaceus, the ligusticum wallichii and the corn stigma in a 5L round-bottom flask, adding 12 times of water, soaking for 90 minutes, heating the mixture with strong fire until the mixture is boiled, keeping the mixture slightly boiled for 90 minutes with slow fire, and filtering the mixture with 300-mesh filter cloth in a single layer manner to obtain filtrate for later use; adding 12 times of water into the filter residue, heating with strong fire to boil, keeping slight boiling with slow fire for 90 minutes, filtering with 300-mesh monolayer, and mixing the filtrates for later use.
The single-frying process of the silkworm cocoon shells comprises the following steps: taking 300g of silkworm cocoon shells, adding 16 times of water, soaking for 60 minutes, decocting for three times, each time for 1 hour, filtering, and combining the filtrates for later use.
Decocting corn stigma, radix astragali and rhizoma ligustici wallichii together, mixing with a single decoction of silkworm cocoon shells, concentrating under reduced pressure at 80 ℃ and spray drying (the specific conditions are that a concentrated solution is taken, the density of a flowing paste is 1.02-1.05 g/ml, the weight of the concentrated solution is converted into the weight of theoretical dry paste according to the paste yield, maltodextrin is added according to the ratio of 0.15: 1 of auxiliary materials to theoretical dry paste powder, stirring is carried out to fully dissolve the maltodextrin, and after uniform mixing, the preheating temperature of a feed liquid is 90 ℃, the drying parameters (the air inlet temperature is 160 ℃, the sample injection speed is 20rpm, the atomization pressure is 2MPa, and the needle introducing pressure is 2 MPa)) are adopted to prepare an extract composition 2 (ZHW, namely a traditional Chinese medicine compound dry paste powder), and 3 batch samples are prepared in parallel, wherein the batch numbers are respectively: ZHW-2-20220601, ZHW-2-20220602, ZHW-2-20220603, for use.
Example 2 preparation of negative control of Compound Dry extract powder of traditional Chinese medicine containing silkworm cocoon Shell
According to the prescription and preparation method of 'example 1', negative control extracts without cocoon shells, astragalus membranaceus and ligusticum wallichii are respectively prepared and named as: CJK-YX, HQ-YX and CX-YX for standby.
Example 3 content determination method of Compound Dry extract powder of traditional Chinese medicine containing silkworm cocoon Shell
3.1 instrumentation and materials
The instrument equipment comprises: a high performance liquid chromatograph Waters 2695 chromatographic system comprises a quaternary gradient infusion pump (Alliance 2695 type), a 120-position high-performance automatic sample injector, an original-installed inlet chromatographic column incubator, a Waters 2996 type DAD diode array detector and an Empower 3 chromatographic management system; an electronic balance: SHIMADZU AUW120D, sartorius (sartoria) BSA124S, FA2004B (shanghai blisk instruments and meters ltd) JY2002 (shanghai Pu Chun) JS6-01 (shanghai Pu Chun) JL070718R (taiwan cherry blossom); an ultrasonic cleaning machine: KQ-500DE ultrasonic Instrument Co., ltd., kunshan City; electronic constant temperature water bath: HH-6 Shanghai Power Bangxi Instrument science and technology Co., ltd; electric jacket: DZTW Tianjin Ingxing laboratory instruments Co., ltd; a chromatographic column: agilent 5TC-C18 (2) 250X 4.6mm SN: 573147/56543/547688; the methanol, the acetonitrile and the formic acid are chromatographically pure water, and the water is ultrapure water.
And (4) reference substance information: ferulic acid (110773-201915, content 99.4%, china institute for food and drug testing); astragaloside (110781-201717, content 96.9%, china institute for food and drug testing); serine (140688-202104-01, content 98%, institute of Chinese food and drug assay); glycine (140689-202006-01, 97% content, institute of food and drug testing, china).
3.2 preparation of control solutions
3.2.1 preparation and derivatization of a serine and glycine mixed control solution:
mixing and preparing a standard: taking appropriate amount of serine reference substance and glycine reference substance, accurately weighing, and adding 0.1mol/L hydrochloric acid solution to obtain mixed solution containing serine 130 μ g and glycine 50 μ g per 1 ml.
Derivatization: precisely measuring 5ml of the reference substance solution, placing the reference substance solution into a 25ml measuring flask, adding 2.5ml of acetonitrile solution of 0.1mol/L Phenyl Isothiocyanate (PITC) and 2.5ml of acetonitrile solution of 1mol/L triethylamine respectively, shaking uniformly, standing at room temperature for 1 hour, adding 50% acetonitrile to the scale, and shaking uniformly. And (3) adding 10ml of n-hexane into 10ml of the solution, shaking, standing for 10 minutes, taking the lower layer solution, and filtering to obtain a subsequent filtrate.
3.2.2 preparation of Ferulic acid reference solution
Taking a proper amount of ferulic acid reference substance, precisely weighing, placing into a brown measuring flask, and adding 70% methanol to obtain a solution containing 20 μ g per 1 ml.
3.2.3 preparation of Astragaloside IV reference solution
Accurately weighing appropriate amount of astragaloside IV control, placing into brown measuring flask, and adding 80% methanol to obtain solution containing 20 μ g per 1 ml.
3.3 preparation of test solutions
3.3.1 preparation of test solutions for serine and glycine determination and derivatization
1) Hydrolysis of a test sample: taking 2g of dry paste powder of ZHW-2 in example 1, precisely stabilizing, adding 15ml of 6mol/L hydrochloric acid solution to dissolve and transfer the dry paste powder into a penicillin bottle, hydrolyzing at 150 ℃ for 1 hour, cooling, transferring the solution into an evaporation dish, washing the penicillin bottle with 10ml of water in multiple times, adding the washing solution into the evaporation dish, drying by distillation, adding 0.1mol/L hydrochloric acid solution into residues to dissolve and transfer the residues into a 25ml measuring flask, adding 0.1mol/L hydrochloric acid solution to scale, and shaking uniformly to obtain the product.
2) Derivatization of test sample hydrolysis solution: accurately measuring 5ml of a sample solution of serine and glycine, placing the sample solution into a 25ml measuring flask, adding 2.5ml of an acetonitrile solution of 0.1mol/L Phenyl Isothiocyanate (PITC) and 2.5ml of an acetonitrile solution of 1mol/L triethylamine respectively, shaking up, standing at room temperature for 1 hour, adding 50% acetonitrile to the scale, and shaking up. And (3) adding 10ml of n-hexane into 10ml of the solution, shaking, standing for 10 minutes, taking the lower layer solution, and filtering to obtain a subsequent filtrate.
The CJK-YX negative sample solution was prepared in the same manner as the serine and glycine test samples and was used.
3.3.2 preparation of test solutions for ferulic acid assay
Taking 2g of dry paste powder of ZHW-2 in example 1, precisely and stably adding 20ml of 70% methanol, placing the mixture in a 25ml volumetric flask, carrying out ultrasonic treatment (power 500W and frequency 40 kHz) for 30 minutes, cooling, fixing the volume to 25ml, shaking up, filtering, and taking subsequent filtrate to obtain the traditional Chinese medicine.
The CX-YX negative sample is prepared according to the method for preparing the ferulic acid test solution for later use.
3.3.3 preparation of test solution for Astragaloside IV
Taking 2g of dry paste powder of ZHW-2 in 'example 1', accurately stabilizing the dry paste powder, adding 20ml of 80% methanol solution (4 ml of concentrated ammonia test solution, 80% methanol is added to 100ml, shaking up) containing 4% concentrated ammonia test solution into a conical flask with a plug, sealing the plug, weighing, heating and refluxing for 1 hour, cooling, weighing again, supplementing the lost weight with 80% methanol solution containing 4% concentrated ammonia test solution, shaking up, filtering, accurately weighing 10ml of subsequent filtrate, evaporating to dryness, dissolving residues with 80% methanol, transferring to a 5ml measuring flask, adding 80% methanol to scale, shaking up, filtering, and taking the subsequent filtrate to obtain the product.
The HQ-YX negative sample is prepared according to the same method as the preparation method of the astragaloside IV test sample solution for later use.
3.4 chromatographic Condition optimization
3.4.1 optimization of chromatographic conditions for serine and Glycine
Measuring by high performance liquid chromatography (0512 in the four-department general regulation of China pharmacopoeia 2020).
Chromatographic conditions and system applicability experiments: an Agilent 5TC-C18 chromatographic column (25 cm in column length, 4.6mm in inner diameter and 5 μm in particle size) using octadecylsilane chemically bonded silica as a filler; acetonitrile is used as a mobile phase A, and 0.1% phosphoric acid solution is used as a mobile phase B.
According to the separation degree of serine and glycine, the number of theoretical plates and the tailing factor, carrying out gradient elution optimization on chromatographic elution conditions according to the specification in the table 1; flow rate 1.0ml per minute; the column temperature was 35 ℃; the detection wavelength is 262nm. Researches show that the separation degree of serine and glycosidic acid can be more than 1.5, the theoretical plate number is more than 1000, and tailing factors are between 0.9 and 1.05 by adopting the condition (1), the effective separation of the serine and the glycosidic acid can be realized, and the requirements of pharmacopeia high performance liquid chromatography (0512 in the four-part general regulation of the 2020 edition) are met.
And (3) precisely sucking 5 mul of each of the derivatized 'under 3.2.1' reference substance solution and 'under 3.3.1' test substance solution, injecting into a liquid chromatograph, and measuring to obtain the final product.
TABLE 1 optimized comparison of gradient elution conditions for serine and glycine content determination
3.4.2 ferulic acid content chromatographic condition optimization
Measuring by high performance liquid chromatography (0512 in the four-department general regulation of China pharmacopoeia 2020). Chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a filler, and acetonitrile: 0.1% formic acid solution (15: 85) as mobile phase, elute for 40min. Gradient elution optimization was performed as specified in table 2; the flow rate was 1ml per minute; the column temperature is 30 ℃; the detection wavelength was 321nm. Researches show that the separation degree of ferulic acid can be more than 1.5, the theoretical plate number is more than 1000, and tailing factors are between 0.9 and 1.05 by adopting the condition (1), so that the ferulic acid can be effectively separated and meets the requirements of pharmacopeia high performance liquid chromatography (0512 in the four-part general regulation of 2020 edition).
During the measurement, respectively and precisely sucking 10 μ l of the reference solution under item 3.2 and the test solution under item 3.3.2, injecting into a liquid chromatograph, and measuring to obtain the final product.
TABLE 2 optimal comparison of gradient elution conditions for ferulic acid content determination
3.4.3 chromatographic Condition optimization of Astragaloside IV
Measuring by high performance liquid chromatography (0512 in the four-department general regulation of China pharmacopoeia 2020). In chromatographic conditions and system applicability tests, octadecylsilane chemically bonded silica is used as a filler, acetonitrile is used as a mobile phase A, a 0.05% formic acid solution is used as a mobile phase B, and gradient elution optimization is performed according to the specification in the table 3; the flow rate was 0.8ml per minute; the column temperature is 30 ℃; the detection wavelength is 220nm. Researches show that the separation degree of the astragaloside IV is more than 1.5 and the theoretical plate number is more than 1000 can be realized by adopting the condition (1),
tailing factors are 0.9-1.05, can be effectively separated, and meet the requirements of pharmacopeia high performance liquid chromatography (0512 in the four-part general regulation of 2020 edition in Chinese pharmacopeia).
During measurement, 20 mul of reference solution under 'item 3.2.3' and test solution under 'item 3.3.3' are respectively and precisely absorbed, injected into a liquid chromatograph, measured and calculated by an external standard two-point method logarithmic equation to obtain the product.
TABLE 3 gradient elution condition optimization for astragaloside IV assay
3.5 specialization examination
According to the chromatographic conditions of the serine and the glycine components according to the condition (1) of 3.4.1, the chromatographic conditions of the ferulic acid according to the condition (1) of 3.4.2 and the chromatographic conditions of the astragaloside according to the condition (1) of 3.4.3, respectively taking a reference substance solution, a test sample solution and a negative reference solution for injection measurement, and taking a blank solvent as a reference, the result that a negative sample does not interfere at the position of each reference substance. The results are shown in FIGS. 1, 2 and 3.
3.6 investigation of the Linear relationship
Precisely absorbing appropriate amount of each reference substance solution, and sequentially diluting to obtain solutions with series mass concentrations. The solution mass concentration was regressed on the abscissa (X) and the peak area integral value was the ordinate (Y), and a standard curve was drawn, and the results are shown in Table 4.
TABLE 4 examination result of linear relationship of four index components
| Name of ingredient | Regression equation | r | Linear range μ g/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 |
3.7 precision test
The high (H), medium (M) and low (L) mass concentration reference substance solutions are taken, sample injection is carried out for 6 times and continuous determination for 3 days under the chromatographic conditions of 3.4.1, 3.4.2 and 3.4.3 (the condition (1) of 3.4.1, the condition (1) of 3.4.2 and the condition (1) of 3.4.3) in the same period of 1d, and the peak areas RSD of ferulic acid, astragaloside, serine and glycine are determined to show that the precision of the instrument is good in the day, and the results are shown in Table 5.
TABLE 5 results of precision examination of four index ingredients
3.8 repeatability test
Four kinds of marked component test solution are prepared according to the methods under the items of '3.3.1', '3.3.2' and '3.3.3', 6 parts are paralleled, and sample injection measurement is carried out under the chromatographic conditions (3.4.1 condition (1), 3.4.2 condition (1) and 3.4.3 condition (1)) of each index component, so that the RSD of the content of the ferulic acid, the astragaloside IV, the serine and the glycine is respectively 2.44%, 2.69%, 2.37% and 3.22%, and the good repeatability of the method is proved to meet the test requirements.
3.9 stability test
Four index component test sample solutions are prepared according to the methods under the items of '3.3.1', '3.3.2' and '3.3.3', and are subjected to sample injection measurement under the chromatographic conditions of the index components (the condition (1) of 3.4.1, the condition (1) of 3.4.2 and the condition (1) of 3.4.3)) for 0, 1, 4, 6, 12, 16 and 24 hours, and the RSD of the areas of ferulic acid, astragaloside, serine and glycine are respectively 2.1%, 2.6%, 3.51% and 2.2%, which indicates that the solutions have good stability of the four index components within 24 hours.
3.10 sample application recovery test
The same batch (ZHW-2) of samples thus measured was precisely weighed, 9 parts in total were placed in a 10mL volumetric flask, and control solutions of 50%, 100%, and 150% levels were added to the flask, respectively, and subjected to sample injection measurement under the chromatographic conditions (condition (1) of 3.4.1, condition (1) of 3.4.2, and condition (1) of 3.4.3) of each index component, and the recovery rate was calculated. As a result, the average sample recovery rates of the ferulic acid, astragaloside IV, serine and glycine peak areas RSD were 101.35%, 100.22%, 101.76% and 101.50 respectively, and the RSD were 2.56%, 2.78%, 3.22% and 2.76 respectively. The results are shown in Table 6.
TABLE 6 sample recovery test for determination of contents of four index components
Example 4 content determination of Compound Dry extract powder of Chinese medicine containing silkworm cocoon Shell
Glycine, citrulline, ferulic acid, astragaloside IV and the contents thereof in ZHW-2 (ZHW-2-20220601, ZHW-2-20220602, ZHW-2-20220603) were measured by sample 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 contents were measured as shown in Table 7.
Table 7 content measurement results of compound dry extract powder of chinese herbal medicine containing silkworm cocoon shell (n = 3)
The above description is only an example of the present invention, and the common general knowledge of the known specific structures and characteristics in the schemes is not described herein. It should be noted that, for those skilled in the art, without departing from the present invention, several changes and modifications can be made, 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 practicability of the patent. The scope of the claims of the present application shall be determined by the contents of the claims, and the description of the embodiments and the like in the specification shall be used to explain the contents of the claims.
The embodiments of the present invention have been described in detail, and the principles and embodiments of the present invention are explained herein using specific examples, which are provided only to help understand the method and the core idea of the present invention. Meanwhile, those skilled in the art should also be able to make modifications or variations to the embodiments and applications of the present invention based on the idea of the present invention. In view of the above, the present disclosure should not be construed as limiting the invention.
Claims (10)
1. A detection method of a traditional Chinese medicine compound containing silkworm cocoon shells is characterized by comprising the following steps:
detecting test solution and reference solution of Chinese medicinal composition, wherein the Chinese medicinal composition comprises silkworm cocoon shell, stigma Maydis, radix astragali and rhizoma Ligustici Chuanxiong, the reference is ferulic acid, astragaloside IV, serine and glycine,
the chromatographic conditions for detection are as follows: a chromatographic column with octadecylsilane chemically bonded silica as a filler is adopted, a mobile phase A is selected from one or more of acetonitrile, methanol and tetrahydrofuran, a mobile phase B is an acid aqueous solution, an alkaline aqueous solution and/or a buffer saline aqueous solution, the flow rate is 0.6-1.5 mL/min, the column temperature is 30-50 ℃, and the detection wavelength is 200-360 nm;
wherein, the gradient elution procedure for detecting the astragaloside IV is as follows: 0 to 25min,15 to 25 percent of A;25 to 30min,25 to 45% of A,
wherein the gradient elution procedure for detecting the serine and the glycine is: 0-10min, 5% -10% of A;10 to 15min,10 to 20% by weight A; 15-30min, 20-23% of A; 30-35min, 23% -55% A,
wherein, the elution procedure for detecting the ferulic acid comprises the following steps: 0 to 40min,15% by weight A;
and acquiring component information, or component information and content information of the Chinese herbal compound according to the detection result.
2. The detection method according to claim 1, wherein the information is obtained by calculating the content of one or more of the following components in the Chinese herbal compound according to an external standard method based on the recorded chromatogram of the test solution of the Chinese herbal compound and the corresponding peak areas in the chromatogram of the control solution: ferulic acid, astragaloside IV, serine and glycine.
3. The detection method according to claim 1, wherein the preparation method of the compound traditional Chinese medicine test solution comprises the following steps: (1) preparing traditional Chinese medicine compound dry paste powder; and (2) respectively preparing the traditional Chinese medicine compound dry paste powder into a derivatization test solution for detecting serine and glycine, a test solution for detecting ferulic acid and/or a test solution for detecting astragaloside.
4. The detection method according to claim 3, wherein the preparation method of the traditional Chinese medicine compound dry paste powder comprises the following steps:
(1) Weighing the following raw material medicines in parts by weight: 25 to 35 parts of silkworm cocoon shell, 25 to 35 parts of corn stigma, 6 to 12 parts of astragalus and 12 to 18 parts of ligusticum wallichii;
(2) Weighing the astragalus, the ligusticum wallichii and the corn stigma in parts by weight, adding water, heating, extracting and filtering to obtain a first filtrate;
(3) Weighing the silkworm cocoon shells in parts by weight, adding water, heating for extraction, and filtering to obtain a second filtrate;
(4) Combining the first filtrate and the second filtrate, and concentrating to obtain the traditional Chinese medicine composition; and
(5) Mixing the traditional Chinese medicine composition with one or more pharmaceutically acceptable auxiliary materials, drying, crushing and sieving to obtain the traditional Chinese medicine compound dry paste powder.
5. The detection method according to claim 4, wherein the step (2) and/or the step (3) of the preparation method of the compound traditional Chinese medicine dry paste powder further comprises a step of soaking the raw material medicine in water;
preferably, the astragalus membranaceus, the ligusticum wallichii and the corn stigma are soaked in water for 30-180 min, more preferably 30-100 min, and still more preferably 90-100 min, for example about 90min;
preferably, the water soaking time of the silkworm cocoon shells is 30-180 min, more preferably 30-90 min, and still more preferably 60-90 min, for example about 60min;
preferably, the raw materials are respectively weighed according to the following weight part ratio: 28-35 parts of silkworm cocoon shell, 28-35 parts of corn stigma, 8-12 parts of astragalus root and 13-18 parts of ligusticum wallichii;
preferably, the raw materials are respectively weighed according to the following weight part ratio: 28.5 to 31.5 parts of silkworm cocoon shell, 28.5 to 31.5 parts of corn stigma, 8.55 to 9.45 parts of astragalus and 14.25 to 15.75 parts of ligusticum wallichii;
preferably, the raw materials are respectively weighed according to the following weight part ratio: 30 parts of silkworm cocoon shell, 30 parts of corn silk, 9 parts of astragalus root and 15 parts of ligusticum wallichii;
particularly preferably, the step (2) of the preparation method of the traditional 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, preferably the water is cold water, and more preferably the cold water submerges 2-5 cm above the surface of the raw material medicine;
[2] the operation of heating and extracting is repeated for 2 to 4 times, for example, 2 times;
[3] the heating is carried out by heating with strong fire until the water is boiled and keeping slight boiling with slow fire;
[4] the amount of the water is 1 to 80 times (L/kg), preferably 5 to 30 times, more preferably 8 to 14 times, for example, about 12 times;
[5] the time for heating and extracting is 15-180 min, preferably 30-120 min, for example about 90min;
[6] the filtration is 100-300 mesh filter cloth single-layer filtration, such as 300 mesh filter cloth single-layer filtration;
particularly preferably, the step (3) of the preparation method of the traditional 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, preferably the water is cold water, and more preferably the cold water submerges 2-5 cm above the surface of the raw material medicine;
[2] the operation of heating and extracting is repeated for 2 to 4 times, for example, 3 times;
[3] the amount of the water is 1 to 80 times (L/kg), preferably 5 to 30 times, more preferably 12 to 16 times, for example, about 16 times;
[4] the time for heating and extracting is 15-180 min, preferably 30-120 min, for example about 60min;
[5] the filtration is 100-300 mesh filter cloth single-layer filtration, for example 100 mesh filter cloth single-layer filtration;
still particularly preferably, step (4) of the preparation method of the traditional Chinese medicine compound dry paste powder comprises any one or more of the following items [1] to [3 ]:
[1] the concentration is reduced pressure concentration;
[2] the temperature of the concentration is 60 to 90 ℃, for example about 80 ℃;
[3] the relative density of the concentrate after concentration is 1.02 to 1.35g/ml, for example 1.02 to 1.05g/ml.
6. The detection method according to claim 4, wherein in the step (5) of the preparation method of the Chinese herbal compound dry paste powder, the drying is spray drying, vacuum drying under reduced pressure or vacuum freeze drying;
more preferably, the conditions of the spray drying are: the preheating temperature of the feed liquid is 60-120 ℃, the air inlet temperature is 100-180 ℃, the sample injection speed is 5-25 rpm, the atomization pressure is 0.5-3 MPa, and the needle pressure is 0.5-3 MPa;
also preferably, the conditions of the spray drying are: the preheating temperature of the feed liquid is 85.5-94.5 ℃, the air inlet temperature is 152-168 ℃, the sample injection speed is 19-21 rpm, the atomization pressure is 1.9-2.1 MPa, and the needle pressure is 1.9-2.1 MPa;
particularly preferably, the spray drying conditions are: the preheating temperature of the feed liquid is about 90 ℃, the air inlet temperature is about 160 ℃, the sampling speed is about 20rpm, the atomization pressure is about 2MPa, and the through needle pressure is about 2MPa;
particularly preferably, the step of mixing comprises converting the weight of the concentrated solution after concentration into the weight of theoretical dry extract powder, adding the auxiliary materials according to the weight ratio of 0.05-0.25: 1 of the auxiliary materials to the theoretical dry extract powder, fully dissolving and uniformly mixing.
7. The method of claim 3, wherein the derivatized test solution for detection of serine and glycine is prepared by a method comprising: weighing a proper amount of traditional Chinese medicine compound dry extract powder, adding a certain amount of 3-8 mol/L hydrochloric acid solution, hydrolyzing at 120-200 ℃ for 0.5-1.5 hours, cooling, evaporating to dryness, adding 0.1-0.5 mol/L hydrochloric acid solution for dissolving, 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 up, standing at room temperature, adding 40-60% acetonitrile to scale, shaking up, adding a certain amount of n-hexane, shaking up, standing, taking a lower layer solution, filtering, and taking a subsequent filtrate to obtain the traditional Chinese medicine compound dry extract;
preferably, the method for preparing a test solution for ferulic acid detection comprises: weighing a proper amount of Chinese herbal compound dry paste powder, adding a certain amount of 50-80% methanol, carrying out ultrasonic treatment for 20-60 minutes at the power of 300-800W and the frequency of 30-60 kHz, cooling, fixing the volume, shaking up, filtering, and taking the subsequent filtrate to obtain the Chinese herbal compound dry paste;
preferably, the preparation method of the test solution for astragaloside detection comprises the following steps: weighing a proper amount of traditional Chinese medicine compound dry paste powder, adding a certain amount of 60-90% methanol solution containing 1-10% concentrated ammonia test solution, sealing, weighing, heating and refluxing for 1-3 hours, cooling, weighing again, complementing the lost weight with 60-90% methanol solution containing 1-10% concentrated ammonia test solution, shaking up, filtering, precisely weighing a certain amount of subsequent filtrate, evaporating to dryness, dissolving residues with 60-90% methanol, fixing the volume, shaking up, filtering, and taking the subsequent filtrate to obtain the traditional Chinese medicine compound dry paste.
8. The assay of any one of claims 1 to 7, wherein the derivatized serine and glycine control solution is prepared by a method comprising: weighing a proper amount of serine and glycine; adding 0.1-0.5 mol/L hydrochloric acid solution to prepare a mixed reference substance solution containing 10-200 mu g/ml of each component; sucking a proper amount of the mixed reference substance solution, 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 up, standing at room temperature, adding 40-60% acetonitrile to scale, shaking up, adding a certain amount of n-hexane, shaking up, standing, taking a lower layer solution, filtering, taking a subsequent filtrate, and obtaining the derivatized reference substance solution of serine and glycine;
preferably, the preparation method of the control solution of ferulic acid and/or astragaloside IV comprises the following steps: weighing appropriate amount of ferulic acid and/or astragaloside IV; and adding 10-100% methanol water solution to prepare a reference solution containing 10-200 μ g/ml ferulic acid and/or astragaloside IV;
preferably, the flow rate is 0.8-1.2 mL/min, the column temperature is 30-35 ℃, and the detection wavelength is 200-330 nm;
more preferably, the sample size of the detection is 5 to 20 μ l;
more preferably, the traditional Chinese medicine compound test solution and the reference solution are filtered by a 0.22 μm or 0.45 μm microporous filter membrane before the detection;
particularly preferably, the chromatographic column has a column length of 25cm, an inner diameter of 4.6mm and a particle size of 5 μm;
even more preferably, the chromatography column is an Agilent 5TC-C18 (2) chromatography column, a Waters Symmetry C18 chromatography column or an Agilent SB C18 chromatography column;
most preferably, the chromatography column is an Agilent 5TC-C18 (2) chromatography column.
9. The detection method according to any one of claims 1 to 7, wherein 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), and methanol: acetonitrile (90: 10);
preferably, the aqueous acid solution, aqueous base solution and/or aqueous buffered salt solution is selected from one or more of different concentrations of weak acids and salts thereof, weak bases and salts thereof;
preferably, the aqueous acid solution, aqueous base solution and/or aqueous buffered salt solution is selected from different concentrations of 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;
more preferably, the aqueous acid solution is 0.05% to 0.4% aqueous acid solution;
more preferably, the acid aqueous solution is 0.05% to 0.2% phosphoric acid or formic acid aqueous solution;
more preferably, the aqueous acid solution is a 0.05% to 0.1% aqueous solution of phosphoric acid or formic acid;
still preferably, the control solution of ferulic acid has a concentration of about 20 μ g/ml;
still preferably, the concentration of the control solution of astragaloside is about 20 μ g/ml;
still preferably, the concentration of the control solution of serine is about 130 μ g/ml;
still preferably, the concentration of the glycine control solution is about 50 μ g/ml.
10. Use of the detection method according to any one of claims 1 to 9 in quality detection and/or quality evaluation and/or quality control of a traditional Chinese medicine compound comprising silkworm cocoon shells.
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Citations (2)
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|---|---|---|---|---|
| WO1997023233A1 (en) * | 1995-12-21 | 1997-07-03 | Parbold Investments N.V. | Biologically active compositions and their use |
| US20150216955A1 (en) * | 2012-03-23 | 2015-08-06 | The University Of Queensland | Immunomodulatory agent and uses therefor |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997023233A1 (en) * | 1995-12-21 | 1997-07-03 | Parbold Investments N.V. | Biologically active compositions and their use |
| US20150216955A1 (en) * | 2012-03-23 | 2015-08-06 | The University Of Queensland | Immunomodulatory agent and uses therefor |
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