CN113759043B - Characteristic spectrum of motherwort fruit and construction method thereof, method for measuring content of stachydrine hydrochloride, motherwort fruit formula granules and preparation method thereof - Google Patents
Characteristic spectrum of motherwort fruit and construction method thereof, method for measuring content of stachydrine hydrochloride, motherwort fruit formula granules and preparation method thereof Download PDFInfo
- Publication number
- CN113759043B CN113759043B CN202111039967.7A CN202111039967A CN113759043B CN 113759043 B CN113759043 B CN 113759043B CN 202111039967 A CN202111039967 A CN 202111039967A CN 113759043 B CN113759043 B CN 113759043B
- Authority
- CN
- China
- Prior art keywords
- solution
- motherwort fruit
- motherwort
- stachydrine hydrochloride
- peak
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/53—Lamiaceae or Labiatae (Mint family), e.g. thyme, rosemary or lavender
- A61K36/533—Leonurus (motherwort)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
- A61K9/1605—Excipients; Inactive ingredients
- A61K9/1629—Organic macromolecular compounds
- A61K9/1652—Polysaccharides, e.g. alginate, cellulose derivatives; Cyclodextrin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
- A61K9/1682—Processes
-
- 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
- G01N30/50—Conditioning of the sorbent material or stationary liquid
- G01N30/52—Physical parameters
- G01N30/54—Temperature
-
- 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
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Physics & Mathematics (AREA)
- Engineering & Computer Science (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Medicinal Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Alternative & Traditional Medicine (AREA)
- Mycology (AREA)
- Microbiology (AREA)
- Medical Informatics (AREA)
- Botany (AREA)
- Biotechnology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a characteristic spectrum of motherwort fruit, a construction method of the characteristic spectrum, a stachydrine hydrochloride content measuring method, motherwort fruit formula particles and a preparation method of the motherwort fruit formula particles. By controlling the gradient program, the characteristic spectrum of the motherwort fruit can be constructed by a specific method, and the characteristic spectrum is reliable, stable and good in repeatability, so that a more scientific, reasonable and detailed basis is provided for quality control of the motherwort fruit. The construction method changes the peak time by adjusting the flow rate, shortens the operation time, is easy to operate and realize, can fully reflect the information of characteristic peaks of the motherwort fruits, displays the chemical component characteristics of the motherwort fruits, keeps good separation degree and peak shape between main components, can comprehensively evaluate the motherwort fruits of different production places and the quality thereof, and is quick and effective.
Description
Technical Field
The invention belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a characteristic spectrum of motherwort fruit and a construction method thereof, a stachydrine hydrochloride content measuring method, motherwort fruit formula particles and a preparation method thereof; more particularly, the invention relates to a characteristic spectrum of motherwort fruit formula particles, decoction pieces, freeze-dried powder or medicinal materials and a construction method thereof, a method for measuring the content of threonine hydrochloride in motherwort fruit, the motherwort fruit formula particles and a preparation method thereof.
Background
The motherwort fruit is dried mature fruit of Leonurus japonicus Houtt. Of Labiatae, has the efficacies of mainly activating blood and regulating menstruation, and clearing liver and improving vision, is a commonly used gynecological traditional Chinese medicine, and is distributed in most regions of the country. In autumn, the overground part of the fruit is harvested when the fruit is ripe, the fruit is dried in the sun, and impurities are removed. According to the reports of the existing documents, the analysis research on the composition of the motherwort fruit by some students at present shows that the motherwort fruit mainly contains chemical components such as alkaloid, fatty oil, flavone, volatile oil and the like, and is rich in various amino acids, mineral elements and the like. Wherein, the stachydrine hydrochloride is monomer alkaloid with high content and strong activity, and has the effects of promoting blood circulation, regulating menstruation, inducing diuresis, relieving swelling, contracting uterus and the like. The content of stachydrine hydrochloride in motherwort fruit medicinal materials is specified in 'Chinese pharmacopoeia' of 2020 edition, but the sample preparation method is complex and has large energy loss; in addition, the method for evaluating the quality of motherwort fruit in the prior art needs to be further improved, and the motherwort fruit with different yields and the quality thereof cannot be comprehensively evaluated.
Disclosure of Invention
Therefore, the invention provides a characteristic spectrum of motherwort fruit formula particles, decoction pieces, freeze-dried powder or medicinal materials, a construction method thereof and a method for measuring the content of threonine hydrochloride in the motherwort fruit, and aims to overcome the defects that the prior art cannot comprehensively evaluate the quality of the motherwort fruit in different production places, and the method for measuring the content of threonine hydrochloride in the motherwort fruit is complex and has large energy consumption.
Furthermore, the invention also provides a basis for preparing the motherwort fruit formula particles meeting the quality standard in the field, and further provides the motherwort fruit formula particles and the preparation method thereof.
Therefore, the invention provides the following technical scheme.
The invention provides a method for constructing a characteristic spectrum of motherwort fruit formula particles, decoction pieces, freeze-dried powder or medicinal materials, which comprises the following steps,
(1) Preparing a motherwort fruit sample solution;
(2) Performing high performance liquid chromatography detection on a motherwort fruit sample solution, taking a potassium dihydrogen phosphate solution as a mobile phase, and performing gradient elution, wherein the gradient elution procedure comprises the following steps: 0-16min, and the flow rate of the mobile phase is 0.6-0.8ml/min;16-17min, the flow rate is 0.6-0.8 → 0.9-1.1ml/min;17-41min, and the flow rate of the mobile phase is 0.9-1.0ml/min;41-41.1min, the flow rate of the mobile phase is 0.9-1.0 → 0.8ml/min;41.1min, and the flow rate of the mobile phase is 0.8ml/min.
Preferably, an ion exchange method among high performance liquid chromatography is used.
The chromatographic conditions of the high performance liquid chromatography further comprise: the detection wavelength is 190-194nm, and the column temperature is 28-32 ℃;
preferably, the potassium dihydrogen phosphate solution further comprises triethylamine and phosphoric acid;
preferably, the volume fraction of triethylamine in the potassium dihydrogen phosphate solution is 0.04-0.07%, and the volume fraction of phosphoric acid is 0.12-0.1%;
the concentration of the potassium dihydrogen phosphate solution is 13-16mmol/L;
0-16min, and the flow rate of the mobile phase is 0.8ml/min;16-17min, the flow rate of the mobile phase is 0.8-1.0ml/min;17-41min, and the flow rate of the mobile phase is 1.0ml/min;41-41.1min, and the flow rate of the mobile phase is 1.0-0.8ml/min;41.1min, and the flow rate of the mobile phase is 0.8ml/min.
The construction method also comprises a step of preparing a reference substance by adopting stachydrine hydrochloride, and a step of detecting a reference substance solution according to the construction method of claim 1 or 2 to obtain a reference substance characteristic map;
preferably, the preparation method of the reference solution comprises the following steps: taking stachydrine hydrochloride reference substance, and adding solvent to make into stachydrine hydrochloride 0.1-0.3mg per 1 ml.
The preparation method of the motherwort fruit test sample solution comprises the steps of taking a test sample, adding a solvent, and extracting to obtain a test sample solution;
preferably, the solvent for extraction is at least one of aqueous ethanol, methanol, ethyl acetate and water;
the extraction mode is one of ultrasonic, shaking and refluxing;
the extraction time is not less than 20min, preferably 20-40min.
When the sample to be detected is freeze-dried powder, decoction pieces or medicinal materials, the preparation method of the test solution specifically comprises the following steps: taking the test sample powder, precisely weighing, adding an ethanol-containing aqueous solution, extracting, and filtering to obtain a subsequent filtrate, namely the test sample solution. Wherein the extraction method is preferably ultrasonic extraction.
When the sample to be tested is a formula particle, the preparation method of the test solution specifically comprises the following steps: taking a test sample, precisely weighing, adding water, extracting, and filtering to obtain a subsequent filtrate, namely a test sample solution. Wherein the extraction method is preferably ultrasonic extraction.
The invention provides a motherwort fruit characteristic spectrum obtained by the construction method.
The invention also provides a motherwort fruit characteristic spectrum, wherein the characteristic spectrum at least has 5 characteristic peaks;
taking the No. 3 peak as a reference peak, the relative retention time of each characteristic peak is within +/-10% of a specified value, and the specified value is as follows: 0.585 (peak 1), 0.613 (peak 2), 1.077 (peak 4), and 1.288 (peak 5).
The invention provides a method for measuring the content of threonine hydrochloride in motherwort fruit, which comprises the following steps,
(1) Preparing a stachydrine hydrochloride reference product solution and a motherwort fruit test product solution;
(2) Determining the characteristic spectrums of the leonurus fruit reference product and the leonurus fruit test product according to the construction method of the leonurus fruit characteristic spectrum, and obtaining the content of the stachydrine hydrochloride in the leonurus fruit according to the China pharmacopoeia 2020 edition rule 0512.
In addition, the invention provides a preparation method of motherwort fruit formula granules, which comprises the following steps,
(1) Extracting fructus Leonuri or decoction pieces to obtain fructus Leonuri extractive solution;
(2) Concentrating fructus Leonuri extractive solution to obtain fructus Leonuri concentrated solution with relative density of 1.05-1.1;
(3) Preheating the motherwort fruit concentrated solution, performing spray drying to obtain spray dried powder, and granulating to obtain the motherwort fruit formula granules.
The extraction rate of the motherwort fruit extracting solution is 4-8%;
preferably, the concentration temperature is 50-80 ℃, and the concentration time is not higher than 24h;
more preferably, the inlet air temperature for spray drying is 170-185 ℃.
The extraction comprises the steps of extracting a motherwort fruit medicinal material or decoction pieces by a water decoction method to obtain a motherwort fruit extracting solution;
preferably, the motherwort fruit extract is obtained by decocting motherwort fruit medicinal material or decoction pieces with water for at least two times;
more preferably, 9-13 times of water is added into fructus Leonuri or decoction pieces, boiling extraction is carried out for 60-90min, and repeated at least twice to obtain fructus Leonuri extractive solution.
In the spray drying process, adding a first auxiliary material into the concentrated solution, and performing spray drying, wherein the addition amount of the first auxiliary material is 0-3% of the weight of the decoction pieces.
And adding a second auxiliary material into the spray-dried powder, and granulating to obtain a formula granule, wherein the adding amount of the second auxiliary material is 0-3% of the amount of the decoction pieces.
Further, the invention provides the motherwort fruit formula particle prepared by the method.
The technical scheme of the invention has the following advantages:
1. the method for constructing the characteristic spectrum of the motherwort fruit comprises the following steps of (1) preparing a motherwort fruit test product solution; (2) Detecting the motherwort fruit sample solution by adopting a high performance liquid chromatography, taking a potassium dihydrogen phosphate solution as a mobile phase, and performing gradient elution, wherein the gradient elution procedure comprises the following steps: 0-16min, and the flow rate of the mobile phase is 0.6-0.8ml/min;16-17min, the flow rate is 0.6-0.8 → 0.9-1.1ml/min;17-41min, and the flow rate of the mobile phase is 0.9-1.0ml/min;41-41.1min, the flow rate of the mobile phase is 0.9-1.0 → 0.8ml/min;41.1min, and the flow rate of the mobile phase is 0.8ml/min. The construction method provided by the invention can be used for constructing the motherwort fruit characteristic spectrum which is reliable, stable and good in repeatability, and provides a more scientific, reasonable and detailed basis for the quality control of the motherwort fruit. The construction method changes the peak time by adjusting the flow rate, shortens the operation time, is easy to operate and realize, can fully reflect the information of characteristic peaks of the motherwort fruits, displays the chemical component characteristics of the motherwort fruits, keeps good separation degree and peak shape between main components, can comprehensively evaluate the motherwort fruits of different production places and the quality thereof, and is quick and effective.
2. The method for measuring the content of the threonine hydrochloride in the motherwort fruit provided by the invention is wider in application range, higher in accuracy and stronger in applicability.
3. According to the preparation method of the motherwort fruit formula particles, provided by the invention, the effective ingredients in the motherwort fruit decoction pieces can be converted into the formula particles, so that the loss is reduced, and the powder yield of the prepared formula particles is high by optimizing the extraction process.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a feature spectrum of 17 batches of motherwort fruit freeze-dried powder in example 1 of the present invention;
FIG. 2 is a control profile in example 1 of the present invention;
FIG. 3 is a characteristic map obtained by the gradient elution procedure A of section 1.1 in Experimental example 1 of the present invention;
FIG. 4 is a characteristic map obtained by the gradient elution program B of section 1.1 in Experimental example 1 of the present invention;
FIG. 5 is a characteristic map obtained by the gradient elution procedure C of section 1.1 in Experimental example 1 of the present invention;
FIG. 6 is a feature map of section 2.1 of the investigation of specificity in Experimental example 2 of the present invention;
FIG. 7 is a feature map of the invention in Experimental example 2, section 2.1 for specificity study;
FIG. 8 is a feature map of 2.2 sections of delay test in Experimental example 2 of the present invention;
FIG. 9 is a linear graph of section 2.7 in Experimental example 2 of the present invention.
Detailed Description
The following examples are provided to further understand the present invention, not to limit the scope of the present invention, but to provide the best mode, not to limit the content and the protection scope of the present invention, and any product similar or similar to the present invention, which is obtained by combining the present invention with other prior art features, falls within the protection scope of the present invention.
The examples do not show the specific experimental steps or conditions, and can be performed according to the conventional experimental steps described in the literature in the field. The reagents or instruments used are conventional reagent products which are commercially available, and manufacturers are not indicated.
Reagent and apparatus
Reagent: potassium dihydrogen phosphate: the top grade is pure; phosphoric acid: the chromatogram is pure; triethylamine: carrying out chromatographic purification; acetonitrile: carrying out chromatographic purification; methanol: the chromatogram is pure; ethanol: analyzing and purifying; the water is distilled water of Drech.
The instrument comprises the following steps: ML204T electronic balance (Mettler Tollido), XY2000-2C electronic balance (Changzhou lucky electronic devices, inc.), MSA125P-1CE-DI electronic balance (Sadous scientific instruments, beijing, inc.), waters Acquity Arc high performance liquid chromatograph.
A chromatographic column:
SCX (4.6X 250 mm) chromatography column
Reagent preparation: stachydrine hydrochloride reference substances (batch number: 110712-201614, china institute for food and drug testing).
The motherwort fruit freeze-dried powder comprises the following batch numbers: YP190801-015000-01; YP190801-473500-02, YP190907-024300-03; YP190915-236000-04; YP190915-473000-05; YP190915-236000-06; YP190915-237200-07; YP190915-237200-08; YP190915-237200-09; YP190915-237200-10; YP190915-237200-11; YP190915-237200-12; YP190927-014100-14; YP190927-014100-15; YP190927-014100-16; YP190927-014100-17; YP190927-014100-18; corresponding in turn to S1-S17.
The motherwort fruit decoction piece batch number and the product place are 190801-015000-01 respectively, and the Bayankee muscle cell-the city of inner Mongolia autonomous area riverside; 190801-473500-02, new county of south-Yang city of Henan province; 190907-024300-03, and inner Mongolia autonomous region Rifeng City Aohan flag; 190915-236000-04, anhui province, mazhou city; 190915-473000-05, south-yang city of Henan province; 190915-236000-06, anhui province, mazhou city; 190915-237200-07, anhui Huo Shanshi; 190915-236000-08, anhui province, mazhou city; 190915-130000-09, liaoning province; 190915-230000-10, anhui; 190915-230000-11, anhui; 190915-130000-12, jilin province; 190927-014100-14, inner Mongolia autonomous region Berthold's right flag; 190927-474750-15, nan Yang city, tu Bai county, henan province; 190927-471600-16, in the city of Luoyang, henan province; 190927-152300-17, hailon city, suizhihua city, heilongjiang province; 190927-629000-18, tuning City, sichuan province, in the mountain area.
Example 1
The embodiment provides a method for constructing a characteristic spectrum of motherwort fruit freeze-dried powder, which comprises the following steps,
(1) Preparation of control solutions: taking stachydrine hydrochloride, adding 70% ethanol to prepare a reference substance solution containing 0.2mg of stachydrine hydrochloride per 1 ml;
preparation of a test solution: collecting 0.1g of fructus Leonuri lyophilized powder, grinding, precisely weighing, placing in a conical flask, precisely adding 10ml of 70% ethanol water solution, sealing, weighing, ultrasonically treating at 40kHz and 500W for 30min, cooling, weighing again, supplementing with 70% ethanol water solution, shaking, filtering with 0.22 μm microporous membrane, and collecting filtrate.
(2) Respectively and precisely sucking 10 mu L of test solution and reference solution, injecting into a high performance liquid chromatograph, and measuring under the chromatographic conditions: to be provided with
SCX (4.6X 250mm,5 μm) column, 15mmol/L potassium dihydrogen phosphate solution (containing 0.06% triethylamine and 0.14% phosphoric acid) as mobile phase, gradient elution: 0-16min, and the flow rate of the mobile phase is 0.8ml/min;16-17min, flowThe phase flow rate is 0.8-1.0ml/min;17-41min, and the flow rate of the mobile phase is 1.0ml/min;41-41.1min, and the flow rate of the mobile phase is 1.0-0.8ml/min;41.1min, and the flow rate of the mobile phase is 0.8ml/min; the column temperature is 30 ℃; the detection wavelength was 192nm.
The embodiment also provides a method for measuring the content of stachydrine hydrochloride in the motherwort fruit freeze-dried powder, which comprises the following steps,
the characteristic spectrums of the stachydrine hydrochloride reference solution and the sample solution are obtained according to the method, the content of the stachydrine hydrochloride in the motherwort fruit is obtained according to the Chinese pharmacopoeia 2020 edition general rule 0512, and the calculation formula is as follows:
wherein, W Test article Representing the peak area of stachydrine hydrochloride in the characteristic spectrum of the sample, C Reference substance Represents the concentration of the stachydrine hydrochloride reference solution, V Reference substance Represents the volume of the stachydrine hydrochloride control solution, W Reference substance Peak area, C, representing the stachydrine hydrochloride control Test article Represents the concentration of the test solution, V Test article Represents the volume of the stachydrine hydrochloride control solution.
Example 2
The embodiment provides a method for constructing a characteristic spectrum of motherwort fruit freeze-dried powder, which comprises the following steps,
(1) Preparation of control solutions: taking stachydrine hydrochloride, adding 70% ethanol to prepare a reference substance solution containing 0.23mg of stachydrine hydrochloride per 1 ml;
preparing a test solution: collecting 0.1g of fructus Leonuri lyophilized powder, grinding, precisely weighing, placing in a conical flask, precisely adding 10ml of 70% ethanol water solution, sealing, weighing, ultrasonically treating at 40kHz and 500W for 30min, cooling, weighing again, supplementing with 70% ethanol water solution, shaking, filtering with 0.22 μm microporous membrane, and collecting filtrate.
(2) Respectively and precisely sucking 10 μ L of sample solution and reference solution, and injecting into high performance liquidChromatograph, determine, chromatographic conditions are: to be provided with
SCX (4.6X 250 mm) column, 15mmol/L potassium dihydrogen phosphate solution (containing 0.06% triethylamine and 0.14% phosphoric acid) as mobile phase, gradient elution: 0-16min, and the flow rate of the mobile phase is 0.8ml/min;16-17min, the flow rate of the mobile phase is 0.8-1.0ml/min;17-41min, and the flow rate of the mobile phase is 1.0ml/min;41-41.1min, and the flow rate of the mobile phase is 1.0-0.8ml/min;41.1min, and the flow rate of the mobile phase is 0.8ml/min; the column temperature was 28 ℃; the detection wavelength was 190nm.
Example 3
The embodiment provides a method for constructing a characteristic spectrum of motherwort fruit decoction pieces, which comprises the following steps of,
(1) Preparation of control solutions: taking stachydrine hydrochloride, adding 70% ethanol to prepare a reference substance solution containing 0.2mg of stachydrine hydrochloride per 1 ml;
preparation of a test solution: weighing 1g of motherwort fruit decoction piece powder, precisely weighing, placing in a conical flask, precisely adding 10ml of 70% ethanol aqueous solution, sealing, weighing, ultrasonically treating at a power of 500W and a frequency of 40kHz for 30min, cooling, weighing again, supplementing the weight loss by 70% ethanol aqueous solution, shaking uniformly, filtering through a 0.22 mu m microporous membrane, and taking the filtrate to obtain the traditional Chinese medicine composition.
(2) Respectively and precisely sucking 10 mu L of test solution and reference solution, injecting into a high performance liquid chromatograph, and measuring under the chromatographic conditions: to be provided with
SCX (4.6X 250mm,5 μm) column, 15mmol/L potassium dihydrogen phosphate solution (containing 0.06% triethylamine and 0.14% phosphoric acid) as mobile phase, gradient elution: 0-16min, and the flow rate of the mobile phase is 0.8ml/min;16-17min, the flow rate of the mobile phase is 0.8-1.0ml/min;17-41min, and the flow rate of the mobile phase is 1.0ml/min;41-41.1min, and the flow rate of the mobile phase is 1.0-0.8ml/min;41.1min, and the flow rate of the mobile phase is 0.8ml/min; the column temperature is 30 ℃; the detection wavelength was 192nm.
The embodiment also provides a method for measuring the content of stachydrine hydrochloride in the motherwort fruit decoction pieces, which comprises the following steps,
the characteristic spectrums of the stachydrine hydrochloride reference substance solution and the test substance solution are obtained according to the method, the content of the stachydrine hydrochloride in the motherwort fruit is obtained according to the China pharmacopoeia 2020 edition convention 0512, and the calculation formula is as follows:
wherein, W Test article Representing the peak area of stachydrine hydrochloride in the characteristic spectrum of the sample, C Reference substance Represents the concentration of the stachydrine hydrochloride reference solution, V Reference substance Represents the volume of the stachydrine hydrochloride control solution, W Reference substance Represents the peak area of the stachydrine hydrochloride control, C Test article Represents the concentration of the test solution, V Test article Represents the volume of the stachydrine hydrochloride control solution.
Example 4
The embodiment provides a method for constructing a characteristic spectrum of motherwort fruit formula particles, which comprises the following steps,
(1) Preparation of control solutions: taking stachydrine hydrochloride, adding 70% ethanol to prepare a reference substance solution containing 0.2mg of stachydrine hydrochloride per 1 ml;
preparation of a test solution: collecting 0.1g fructus Leonuri granule, grinding, precisely weighing, placing in a conical flask, precisely adding 10ml water, sealing, weighing, ultrasonically treating at 40kHz and 500W for 30min, cooling, weighing again, adding 70% ethanol water solution to reduce weight, shaking, filtering with 0.22 μm microporous membrane, and collecting filtrate.
(2) Respectively and precisely sucking 10 mu L of test solution and reference solution, injecting into a high performance liquid chromatograph, and measuring under the chromatographic conditions: to be provided with
SCX (4.6X 250mm,5 μm) column, 15mmol/L potassium dihydrogen phosphate solution (containing 0.06% triethylamine and 0.14% phosphoric acid) as mobile phase, gradient elution: 0-16min, flowThe phase flow rate is 0.8ml/min;16-17min, the flow rate of the mobile phase is 0.8-1.0ml/min;17-41min, and the flow rate of the mobile phase is 1.0ml/min;41-41.1min, and the flow rate of the mobile phase is 1.0-0.8ml/min;41.1min, and the flow rate of the mobile phase is 0.8ml/min; the column temperature is 30 ℃; the detection wavelength was 192nm.
The embodiment also provides a method for measuring the content of stachydrine hydrochloride in the motherwort fruit formula particles, which comprises the following steps,
the characteristic spectrums of the stachydrine hydrochloride reference solution and the sample solution are obtained according to the method, the content of the stachydrine hydrochloride in the motherwort fruit is obtained according to the Chinese pharmacopoeia 2020 edition general rule 0512, and the calculation formula is as follows:
wherein, W Test article Representing the peak area of stachydrine hydrochloride in the characteristic spectrum of the sample, C Reference substance Represents the concentration of the stachydrine hydrochloride reference solution, V Reference substance Represents the volume of the stachydrine hydrochloride control solution, W Reference substance Peak area, C, representing the stachydrine hydrochloride control Test article Represents the concentration of the test solution, V Test article Represents the volume of the stachydrine hydrochloride control solution.
Experimental example 1 creation of characteristic map
Preparation of control solutions: taking stachydrine hydrochloride, adding 70% ethanol to prepare a reference substance solution containing 0.2mg of stachydrine hydrochloride per 1 ml;
preparation of a test solution: collecting 0.1g of fructus Leonuri lyophilized powder, grinding, precisely weighing, placing in a conical flask, precisely adding 10ml of 70% ethanol water solution, sealing, weighing, ultrasonically treating at 40kHz and 500W for 30min, cooling, weighing again, supplementing with 70% ethanol water solution, shaking, filtering with 0.22 μm microporous membrane, and collecting filtrate.
Precisely sucking 10 μ L of each of the test solution and the reference solution, injecting into a high performance liquid chromatograph, and measuring under the chromatographic conditions: to be provided with
SCX (4.6X 250 mm) column, 15mmol/L potassium dihydrogen phosphate solution (containing 0.06% triethylamine and 0.14% phosphoric acid) as mobile phase, gradient elution: 0-16min, and the flow rate of the mobile phase is 0.8ml/min;16-17min, the flow rate of the mobile phase is 0.8-1.0ml/min;17-41min, and the flow rate of the mobile phase is 1.0ml/min;41-41.1min, and the flow rate of the mobile phase is 1.0-0.8ml/min;41.1min, and the flow rate of the mobile phase is 0.8ml/min; the column temperature is 30 ℃; the detection wavelength was 192nm.
Obtaining the characteristic spectrum of the motherwort fruit freeze-dried powder and the reference product solution of the stachydrine hydrochloride, and selecting 5 characteristic peaks according to the principles of good stability, separation degree and peak shape of relative retention time.
The characteristic map has 5 characteristic peaks, the No. 3 peak is taken as a reference peak, the relative retention time of each characteristic peak is within +/-10% of a specified value, and the specified value is as follows: 0.585 (peak 1), 0.613 (peak 2), 1.077 (peak 4), 1.288 (peak 5); wherein, the peak No. 3 is stachydrine hydrochloride.
The method comprises the steps of obtaining a characteristic spectrum of 17 batches of motherwort fruit freeze-dried powder (S1-S17) according to the method and obtaining the content of stachydrine hydrochloride according to the method of embodiment 1, analyzing by using software of a traditional Chinese medicine chromatography fingerprint spectrum similarity evaluation system (2012 edition), automatically matching by taking a No. 3 peak as a reference peak after multi-point correction to obtain 17 batches of sample superposition spectrums and comparison characteristic spectrums thereof, and determining 5 common peaks, wherein S1-S17 are sequentially corresponding to each other from bottom to top in the graph 1 as shown in figures 1 and 2, the similarity evaluation result is shown in a table 1, and the similarity value of 17 batches of motherwort fruit test products is between 0.981 and 1.000. The content of stachydrine hydrochloride in 17 batches of motherwort fruit freeze-dried powder is shown in table 2, unit mg/g represents the mass of the stachydrine hydrochloride contained in each 1g of motherwort fruit freeze-dried powder.
TABLE 1 motherwort fruit batch similarity evaluation results
TABLE 2 stachydrine hydrochloride content in motherwort fruit lyophilized powder batch
| Batch number | Stachydrine hydrochloride content mg/g |
| 190801-015000-01 | 33.4 |
| 190801-473500-02 | 20.3 |
| 190907-024300-03 | 31.2 |
| 190915-236000-04 | 27.7 |
| 190915-473000-05 | 29.6 |
| 190915-236000-06 | 20.1 |
| 190915-237200-07 | 29.9 |
| 190915-237200-08 | 32.7 |
| 190915-237200-09 | 21.3 |
| 190915-237200-10 | 42.8 |
| 190915-237200-11 | 27.2 |
| 190915-237200-12 | 15.1 |
| 190927-014100-14 | 20.4 |
| 190927-014100-15 | 20.1 |
| 190927-014100-16 | 28.2 |
| 190927-014100-17 | 34.3 |
| 190927-014100-18 | 24.7 |
1.1 examination of gradient elution procedure
4 parts of the same sample solution is prepared, and the other parts are the same as the experimental example 1 except for different gradient elution procedures, and the characteristic spectrum of the sample is obtained through detection.
A. The gradient elution procedure was: the detection result is shown in figure 3 by isocratic elution with 15mmol/L potassium dihydrogen phosphate solution (containing triethylamine with volume fraction of 0.06% and phosphoric acid with volume fraction of 0.14%) as mobile phase.
B. The gradient elution procedure was: gradient elution is carried out by taking 15mmol/L potassium dihydrogen phosphate solution (containing 0.06% triethylamine and 0.14% phosphoric acid) as mobile phase A and acetonitrile as mobile phase B; the flow rate is 0.8mL/min, and the column temperature is 30 ℃; wherein, the gradient elution procedure is as follows: 0-22min, the mobile phase A is 100%, and the mobile phase B is 0%;22-25min, the mobile phase A is 100-90%, and the mobile phase B is 0-10%;25-40min, 90% for mobile phase A and 10% for mobile phase B, the results are shown in FIG. 4.
C. The gradient elution procedure was: taking 15mmol/L potassium dihydrogen phosphate solution (containing 0.06% triethylamine and 0.14% phosphoric acid) as mobile phase A, and gradient eluting; the flow rate is 0.8mL/min; wherein, the gradient elution procedure is as follows: 0-16min, the flow rate is 0.8ml/min;16-17min, and the flow rate is 0.8-1.0ml/min;17-42min, with flow rate of 1ml/min;42-43min, with flow rate of 1.0-0.8ml/min;43-45min, flow rate of 0.8ml/min, and results are shown in FIG. 5.
D. The gradient elution procedure was the same as in example 1.
The characteristic spectrum obtained by the gradient elution program A has a larger solvent peak in 2-6min and a large peak in 48-50 min; the gradient elution procedure B found that the addition of mobile phase B (acetonitrile) caused a large shift in chromatographic peak retention time, which was not feasible; the chromatographic peak of the characteristic spectrum obtained under the chromatographic condition C has a good separation effect, no chromatographic peak appears after 41min, and the chromatographic peak obtained through the gradient elution procedure D has a good separation degree effect, rich information and high reproducibility.
Experimental example 2 methodological examination
2.1 specificity
Taking 70% ethanol water solution as a blank sample, sampling stachydrine hydrochloride reference substance solution, and sampling by adopting the method of the embodiment 1 to obtain the characteristic maps of the blank sample and the reference substance solution, and referring to the figures 6 and 7, and discharging the interference of the ethanol water solution.
2.2 retardation
The test solution was injected as in example 1 and subjected to the delayed test, as shown in FIG. 8, and no other peak appeared after 40min.
2.3 repeatability
Motherwort fruit lyophilized powder (batch No. 190907-024300-03) 6 parts is taken to prepare a test solution according to the method in example 1, the characteristic spectrum and the stachydrine hydrochloride content of the test solution are determined by the method in example 1, the No. 3 peak is taken as a reference peak, the relative retention time and the relative peak area of each characteristic peak are calculated and shown in tables 3 and 4, and the stachydrine hydrochloride content of the test solution is shown in table 5. The results in tables 3-4 show that the relative retention time RSD of 5 characteristic peaks is 0.0% -0.1%, and the RSD of the relative peak area is 0.0% -4.7%, which indicates that the repeatability of the characteristic spectrum is good; the results in table 5 show that the RSD value of the stachydrine hydrochloride measurement result is 2.2%, which meets the requirement of the fourth part 9101 (drug quality standard analysis method verification guideline) of 2020 edition in the "chinese pharmacopoeia" on the repeatability of the method.
TABLE 3 Retention time and relative Retention time of the individual characteristic peaks
TABLE 4 Peak area and relative Peak area of each characteristic Peak
TABLE 5 stachydrine hydrochloride repeatability test results
2.4 intermediate precision
Different analysts perform intermediate precision tests at different times by using another Waters ultra high performance liquid chromatograph (2998 detector), prepare 6 parts of test sample solution in parallel according to the method of example 1, measure according to the chromatographic conditions, calculate the relative retention time and the relative peak area of each common peak in the characteristic map by using the peak 3 (stachydrine hydrochloride) as the reference peak (S), see tables 6 and 7, and the results show that the relative retention time RSD of 5 identification peaks is 0.0-0.1%, and the relative peak area RSD is 0.0-6.4%, which indicates that the precision of the characteristic map is better. Comparing the samples under the intermediate precision term with the samples under the repeatability term, see tables 8 and 9, the relative retention time RSD is in the range of 0.0-2.2%, and the relative peak area RSD is in the range of 0.0-29.2%, which shows that the intermediate precision of the characteristic spectrum is better.
TABLE 6 retention time and relative retention time of characteristic peak of intermediate precision of fructus Leonuri
TABLE 7 peak area and relative peak area of characteristic peak of intermediate precision of fructus Leonuri
TABLE 8 repeatability and intermediate precision relative retention time
TABLE 9 repeatability and intermediate precision relative peak areas
Intermediate precision tests were performed by different analysts at different times using another waters high performance liquid chromatograph (PDA detector). Motherwort fruit lyophilized powder (batch number: 190907-024300-03) 6 parts is taken, 6 parts of test sample solution is prepared in parallel according to the method of example 1, the stachydrine hydrochloride content in the test sample solution is determined according to the method of example 1, the result is shown in table 10, the RSD value of the stachydrine hydrochloride content is 2.1%, the RSD of the intermediate precision is 2.8%, the RSD accords with the regulation of the four parts 9101 (drug quality standard analysis method verification guide principle) in the 2020 edition of Chinese pharmacopoeia, and the stability of the method is good.
TABLE 10 results of intermediate precision experiments
2.5 stability
Taking a test sample solution (the preparation method is the same as that of example 1), and determining a characteristic spectrum and the peak area of stachydrine hydrochloride according to the method of example 1 at 0 hour, 2 hour, 4 hour, 6 hour, 8 hour, 10 hour, 12 hour, 24 hour, 36 hour and 48 hour respectively, wherein the relative retention time and the relative peak area of each characteristic peak in the characteristic spectrum are shown in tables 11 and 12; meanwhile, the reference solution of stachydrine hydrochloride is taken to measure the peak areas of the reference solution of stachydrine hydrochloride in the steps of 0 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 24 hours, 36 hours and 48 hours according to the method in the example 1, and the peak areas of the stachydrine hydrochloride in the test solution and the reference solution are shown in the table 13.
TABLE 11 retention time and relative retention time of the individual characteristic peaks
TABLE 12 peak area and relative peak area of each characteristic peak
TABLE 13 Peak areas of stachydrine hydrochloride in test solution and control at different times
| Time | Peak area of stachydrine hydrochloride in test sample | Peak area of control |
| 0h | 931110 | 598977 |
| 2h | 932025 | 595367 |
| 4h | 939630 | 599855 |
| 6h | 939247 | 600298 |
| 8h | 920596 | 600171 |
| 12h | 940278 | 601613 |
| 24h | 939054 | 584945 |
| 36h | 932449 | 584666 |
| 48h | 942427 | 590494 |
| RSD | 0.7％ | 1.3％ |
In tables 11 and 12, by examining the solution stability for 48 hours, the relative retention time RSD of the characteristic peak of the test solution is in the range of 0.1-0.6%, and the relative peak area RSD% is in the range of 0.0-2.9%. In Table 13, the RSD value of the peak area of the stachydrine hydrochloride reference substance within 48h is 1.3%, and the RSD value of the peak area of the stachydrine hydrochloride peak in the test substance is 0.7%, which indicates that the test substance solution is stable within 48 h.
2.6 accuracy
Preparation of control solutions: precisely weighing appropriate amount of stachydrine hydrochloride reference substance, precisely weighing, and respectively adding 70% ethanol to obtain reference substance solution A containing 0.201mg of stachydrine hydrochloride per 1ml, reference substance solution B containing 0.1419mg of stachydrine hydrochloride per 1ml, and reference substance solution C containing 0.060mg of stachydrine hydrochloride per 1 ml.
Preparation of sample solution: precisely weighing 9 parts of motherwort fruit freeze-dried powder, wherein each part of motherwort fruit freeze-dried powder is about 0.05g, precisely adding 10ml of reference solutions A-C and 3 parts of reference solutions A-C into a test sample respectively to obtain 9 parts of sample solutions, detecting according to the method of the embodiment 1, and calculating the recovery rate according to the following formula, wherein the results are shown in a table 10;
wherein the measured value refers to the content of stachydrine hydrochloride in the sample solution; the added amount of the control is the amount of the control in the control solution added when the sample solution is prepared.
The recovery rate range of the stachydrine hydrochloride measured by the recovery rate test in the table 14 is 92.03-100.88%, the average recovery rate is 97.69%, and the method conforms to the regulation of the four parts 9101 (drug quality standard analytical method verification guiding principle) in the 2020 edition of Chinese pharmacopoeia, and shows that the established content determination method has good accuracy.
TABLE 14 recovery test results
2.7 Linear survey
Accurately weighing a stachydrine hydrochloride reference substance 6.993mg in a 10ml volumetric flask, adding 70% ethanol water solution to dissolve the stachydrine hydrochloride reference substance and fixing the volume to a scale, shaking up to obtain a stachydrine hydrochloride reference substance solution A, respectively taking 0.5ml, 1ml, 2ml and 4ml from the stachydrine hydrochloride reference substance solution A, placing the stachydrine hydrochloride reference substance solution A in the 10ml volumetric flask, fixing the volume to a scale line by using 70% ethanol water solution, and shaking up to obtain four stachydrine hydrochloride reference substance solutions B-E with different concentrations; respectively filtering the stachydrine hydrochloride reference substance solutions A-E with 0.22 μm microporous filter membrane, taking the subsequent filtrate, injecting sample with the amount of 10 μ L according to the method to obtain the characteristic map of the stachydrine hydrochloride reference substance solution, injecting each reference substance solution twice, performing linear regression by using the least square method with the average value of the peak areas of the two stachydrine hydrochloride characteristic peaks as the ordinate and the concentration of the stachydrine hydrochloride reference substance solution as the abscissa, see table 15 and figure 9, to obtain the solution with the regression equation of Y =3479617.3X-11034.0, r =1.0 and the linear range of 0.034965-0.6993 mg. Ml -1 。
TABLE 15 concentration of stachydrine hydrochloride control solution and characteristic peak area of stachydrine hydrochloride
| Standard curve | A | B | C | D | E |
| Peak area 1 | 2422006 | 103036 | 237082 | 476499 | 961743 |
| |
2421204 | 105136 | 238480 | 477258 | 963753 |
| Mean value of | 2421605 | 104086 | 237781 | 476879 | 962748 |
| Concentration mg.ml -1 | 0.6993 | 0.034965 | 0.06993 | 0.13986 | 0.27972 |
Example 5
The embodiment provides a preparation method of motherwort fruit formula granules, which comprises the following steps,
(1) Collecting fructus Leonuri decoction pieces, adding 12 times of water into the first decoction, boiling and extracting for 60min, adding 10 times of water into the second decoction, boiling and extracting for 60min, filtering the medicinal liquid, and mixing to obtain fructus Leonuri extractive solution.
(2) Concentrating the obtained fructus Leonuri extractive solution at 65 deg.C to obtain concentrated solution with relative density of 1.10 (measured at 60 deg.C), and refrigerating for no more than 24 hr.
(3) Preheating the obtained concentrated solution to 60 deg.C, adding dextrin as first adjuvant in an amount of 2% of the decoction pieces, spray drying at 175 deg.C, and collecting spray dried powder; adding a second auxiliary material into the spray-dried powder, wherein the adding amount is 2% of the amount of the decoction pieces, uniformly mixing, performing dry granulation, and controlling the compression roller gap of a granulator to be 0.1-1.3 mm; the rotating speed of the compression roller is 20rpm; the pressure is 60-90 bar, granules are made and granulated by a 16-mesh sieve and a 60-mesh sieve, and the medicinal composite membrane is packaged to obtain a finished product.
Experimental example 3
3.1 determination of the ratio of the extracted liquid of motherwort fruit
Taking 17 batches of motherwort fruit decoction pieces, each 200g, adding 8 times of water into each decoction piece, soaking for 30min, heating to boil, decocting for 30min, filtering while hot by using 150-mesh filter cloth, decocting for 20min while boiling with 6 times of water, filtering while hot by using 150-mesh filter cloth, combining the two filtrates, cooling the liquid medicine in water bath to room temperature to obtain a motherwort fruit extracting solution, and weighing the total weight of the extracting solution liquid medicine. Weighing 1/10 of the weight of the medicinal liquid, placing in an evaporation dish (taking parallel sample), evaporating in water bath, drying at 105 deg.C for 5 hr, cooling in a desiccator for 30min, and preciselyWeighing (M) 1 ) Drying at 105 deg.C for 1 hr, cooling in a desiccator for 30min, weighing to constant weight, and precisely weighing (M) 2 And the unit is g) the extraction rate of the motherwort fruit extract is calculated according to the formula (1).
In the preparation period, W is the mass of the decoction pieces, and the unit is g; n is the total mass of the motherwort fruit extracting solution, and the unit is g; m is 1/10 of the total mass of the motherwort fruit extracting solution, and the unit is g; m 0 The mass of the evaporating dish is given in g.
Concentrating the rest fructus Leonuri extractive solution at 65 deg.C under reduced pressure until the ratio of fluid extract to decoction pieces is about 1:1, transferring to lyophilizing tray, vacuum lyophilizing (at-35 deg.C, specifically drying at-35 deg.C for 4h, drying at-30 deg.C for 4h, drying at-20 deg.C for 4h, drying at-10 deg.C for 4h, drying at 0 deg.C for 3h, drying at 10 deg.C for 3h, drying at 20 deg.C for 3h, drying at 30 deg.C for 10h, and drying at 35 deg.C for 10h, and vacuum degree of 10 Pa), collecting lyophilized powder, preparing fructus Leonuri sample solution according to the method of example 1, measuring the content of stachydrine hydrochloride in the sample solution, calculating stachydrine hydrochloride transfer rate according to formula (2), and calculating the stachydrine hydrochloride transfer rate of each batch of fructus Leonuri decoction pieces, see Table 16.
TABLE 16 transfer rate of stachydrine hydrochloride
According to the above experiment results, the average motherwort fruit yield of 17 batches is + -10% as the range of the motherwort fruit standard decoction yield, namely 4.2% -7.8%.
3.2 examination of extraction Process parameters
The water adding amount for extraction is investigated by adopting an orthogonal test,The extraction times and time affect the extraction rate and the stachydrine hydrochloride content. Performing L by orthogonal experiment with water addition amount, extraction frequency and extraction time as indexes 9 (3 4 ) And performing orthogonal experiment to determine the optimal extraction process condition. The specific experimental steps comprise that 9 parts of motherwort fruit decoction pieces (190907-024300-03) are taken, 200g of each part of motherwort fruit decoction pieces are placed in a round bottom flask, the motherwort fruit decoction pieces are respectively extracted according to the parameters in the table 13, the extracting solution is filtered through 150-mesh filter cloth, the filtrates are combined, the extracting solution is cooled to room temperature in water bath, one tenth of the extracting rate is taken to measure the paste rate, the rest extracting solution is decompressed and concentrated at 65 ℃ until the ratio of the clear paste to the decoction pieces is about 1:1, the extracting solution is transferred to a freeze-drying tray, vacuum freezing is carried out (the parameters are the same as 3.1), the freeze-dried powder is collected, the stachydrine hydrochloride content is measured according to the embodiment 1, and the stachydrine hydrochloride transfer rate is calculated according to the formula 2. The factor levels are shown in Table 17, the experimental protocol is shown in Table 18, and the experimental results are shown in Table 19.
TABLE 17 orthogonal experiment factor horizon
Note: the water addition amount for the second and third extractions is A-2,A, which represents the multiple of the water addition amount for the first extraction.
Table 18 orthogonal experimental protocol table
TABLE 19 orthogonal Experimental results Table
| Test No. | Percentage of cream discharged (%) | Hydrochloric acidStachydrine content (mg/g) | Transfer Rate (%) |
| 1 | 4.4 | 37.5 | 56.9 |
| 2 | 7.1 | 33.1 | 81 |
| 3 | 8.7 | 29.9 | 89.7 |
| 4 | 5.6 | 35.4 | 68.4 |
| 5 | 6.4 | 32.1 | 70.8 |
| 6 | 7.3 | 34.0 | 85.6 |
| 7 | 5.9 | 33.7 | 68.6 |
| 8 | 6.7 | 33.9 | 78.30 |
| 9 | 8.5 | 32.7 | 95.8 |
Note: the content of stachydrine hydrochloride in the decoction pieces is =2.9mg/g.
The results of the extraction rate and the stachydrine hydrochloride transfer rate in the table 14 are input into Design-expert10.0.4.0 for processing, the results of the motherwort fruit extract are referred to, various factors such as the loss in the comprehensive production process are taken as target values for comprehensive evaluation, and the extraction process combination closest to the standard decoction is preferably as follows: extracting for 2 times, decocting with 12 times of water, boiling and extracting for 1 hr; decocting twice with 10 times of water, boiling and extracting for 1 hour.
3.3 investigation of concentration temperature
200g of motherwort fruit decoction pieces (190907-024300-03), adding 8 times of water into the decoction pieces in the first decoction, soaking for 30min, then decocting for 30min, adding 6 times of water into the decoction pieces in the second decoction, decocting for 20min, filtering the extracting solution by using 150-mesh filter cloth, combining the filtrates, cooling to room temperature in a water bath, weighing the total mass of the extracting solution, dividing the extracting solution into 42 equal parts, wherein each part is about 45ml, totaling 42 samples, placing the 42 samples in a centrifuge tube, sealing, taking 2 of the samples as 0h samples (the content in 0h in table 20 is the average value of the two samples), heating the remaining 40 samples in a water bath to 50 ℃, 60 ℃, 70 ℃ and 80 ℃, placing 10 samples at each temperature, respectively taking 2 samples at different temperatures under 3h, 6h, 9h, 12h and 24h, placing the samples at room temperature, supplementing weight, obtaining 42 leonurus fruit concentration temperature samples, and determining according to the method of example 1 to obtain the content of stachydrine hydrochloride, which is shown in table 20.
TABLE 20 stachydrine hydrochloride content (mg/g) in the different samples
| Temperature of |
50 |
60℃ | 70℃ | 80℃ |
| 0h | 37.48 | 37.48 | 37.48 | 37.48 |
| 3h | 36.76 | 37.03 | 36.94 | 37.32 |
| 6h | 36.98 | 37.29 | 37.27 | 37.84 |
| 9h | 37.19 | 37.41 | 37.66 | 38.39 |
| 12h | 38.19 | 38.40 | 38.25 | 38.56 |
| 24h | 37.34 | 37.57 | 38.57 | 39.32 |
The experimental result shows that the content of the stachydrine hydrochloride has smaller change amplitude within 24 hours of being heated under different conditions of 50-80 ℃, and can be considered to be basically kept unchanged; the content of stachydrine hydrochloride tends to increase or decrease at 24h, so the concentration time is recommended to be controlled within 24h.
3.4 inspection of spray drying parameters
Collecting fructus Leonuri decoction pieces 3000g, adding water 8 times the decoction pieces into the first decoction, soaking for 30min, boiling and decocting for 30min, adding water 6 times the decoction pieces into the second decoction, boiling and decocting for 20min, filtering the extractive solution with 150 mesh filter cloth, mixing filtrates, concentrating the filtrate at 50 deg.C under reduced pressure to certain density, weighing the concentrated solution, and measuring the solid content. In the production process of the motherwort fruit formula particles, the optimal spray drying parameters are determined by inspecting parameters such as air inlet temperature, addition of the first auxiliary material and the like in a spray drying mode.
Experimental group 1: concentrating the concentrated solution until the relative density is 1.1, adding no first adjuvant, spray drying at air inlet temperature of 170 deg.C with fan set at 35 and peristaltic pump speed of 20rpm, and spray drying to obtain spray dried powder.
Experimental group 2: concentrating the concentrated solution to relative density of 1.1, adding a first adjuvant (dextrin) into the concentrated solution, wherein the dextrin is 2% of the decoction pieces, the air inlet temperature of spray drying is 170 deg.C, the fan setting is 35, and the peristaltic pump speed is 20rpm, to obtain spray dried powder.
Experimental group 3: concentrating the concentrated solution to relative density of 1.1, adding 2% dextrin into the concentrated solution, spray drying at air inlet temperature of 180 deg.C, setting fan at 35, and peristaltic pump at 20rpm to obtain spray dried powder.
Experimental group 4: concentrating the concentrated solution until the relative density is 1.05, adding 2% of dextrin into the concentrated solution, setting the air inlet temperature of spray drying at 180 ℃, setting a fan at 35 and setting the speed of a peristaltic pump at 20rpm to obtain spray drying powder.
Experimental group 5: concentrating the concentrated solution until the relative density is 1.05, adding 4% of dextrin into the concentrated solution, setting the air inlet temperature of spray drying at 180 ℃, setting a fan at 35 and setting the speed of a peristaltic pump at 20rpm to obtain spray drying powder.
The powder collection rate of the spray-dried powder obtained in experimental groups 1 to 5 was calculated, and the moisture and the dissolution rate in the dried powder were measured. Meanwhile, a test solution was prepared according to the method of example 1, and the content of stachydrine hydrochloride was measured, and the results are shown in Table 21.
Table 21 motherwort fruit spray drying process investigation result
| Experimental group | Percentage of collected pollen (%) | Stachydrine hydrochloride content (mg/g) | Phenomenon of spray drying |
| 1 | 79.62 | 18.6 | The tower sticking phenomenon occurs at the bottom of the |
| 2 | 82.17 | 18.3 | No wall sticking, good powder fluidity |
| 3 | 84.47 | 24.4 | Has slight tower adhesion phenomenon, and the powder is easy to collect |
| 4 | 87.19 | 18.1 | Has slight tower adhesion and |
| 5 | 90.10 | 11.9 | The phenomena of tower adhesion and powder leakage are reduced, and the collection is easy |
The experimental groups 2 and 3 show that the powder yield is not obviously different when the air inlet temperature is 170 ℃ and 180 ℃; the experimental groups can see that the powder yield is not obviously different when the density is 1.10 and 1.05; in experimental groups 4 and 5, the powder yield is only improved by 2.91% when 4% of auxiliary materials are added, the principle of low auxiliary materials is followed, and 2% of auxiliary materials are added in the historical process, so that the proportion of 2% of auxiliary materials is still maintained.
In summary, when preparing motherwort fruit formula particles, the air inlet temperature of spray drying is 170-185 ℃, the relative density of the concentrated solution is 1.05-1.1, and the addition amount of the first auxiliary material in the spray drying step is 2-4%, preferably 2%.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the spirit or scope of the invention.
Claims (15)
1. A method for constructing a characteristic spectrum of motherwort fruit formula particles, decoction pieces, freeze-dried powder or medicinal materials is characterized by comprising the following steps of,
(1) Preparing a motherwort fruit test solution;
(2) Detecting motherwort fruit test solution by adopting a high performance liquid chromatography, wherein the chromatographic columns are Waters SPHERISORB SCX;
15mmol/L potassium dihydrogen phosphate solution is used as a mobile phase, the potassium dihydrogen phosphate solution contains 0.06 percent of triethylamine and 0.14 percent of phosphoric acid, and the gradient elution is as follows: 0-16min, and the flow rate of the mobile phase is 0.8ml/min;16-17min, the flow rate of the mobile phase is 0.8 → 1.0ml/min;17-41min, and the flow rate of the mobile phase is 1.0ml/min;41-41.1min, the flow rate of the mobile phase is 1.0 → 0.8ml/min;41.1min, and the flow rate of the mobile phase is 0.8ml/min;
in preparing the test solution, the solvent used for extraction is at least one of ethanol water solution, methanol, ethyl acetate and water.
2. The method for constructing according to claim 1, wherein the chromatographic conditions of the high performance liquid chromatography further comprise: the detection wavelength is 190-194nm, the column temperature is 28-32 ℃, and the sample injection amount is 8-12 mul.
3. The construction method according to claim 1 or 2, characterized in that the construction method further comprises a step of preparing a reference substance by using stachydrine hydrochloride, and a step of detecting a reference substance solution according to the construction method of claim 1 or 2 to obtain a reference substance characteristic map.
4. The construction method according to claim 3, wherein the preparation method of the control solution comprises the following steps: taking stachydrine hydrochloride reference substance, and adding solvent to make into stachydrine hydrochloride 0.1-0.3mg per 1 ml.
5. The method according to claim 1 or 2, wherein the fructus Leonuri sample solution is prepared by collecting a sample, adding solvent, and extracting to obtain a sample solution;
the extraction mode is one of ultrasonic, shaking and refluxing;
the extraction time is not less than 20min.
6. The construction method according to claim 5, wherein the extraction time is 20-40min.
7. The construction method according to claim 1 or 2, wherein the feature map has at least 5 feature peaks;
taking the No. 3 peak as a reference peak, the relative retention time of each characteristic peak is within +/-10% of a specified value, and the specified values of the No. 1 peak, the No. 2 peak, the No. 4 peak and the No. 5 peak are respectively as follows: 0.585, 0.613, 1.077, 1.288.
8. The method according to claim 1 or 2, wherein the method for determining the content of threonine hydrochloride in fructus Leonuri comprises the steps of,
(1) Preparing a stachydrine hydrochloride reference solution and a motherwort fruit test solution;
(2) The characteristic spectrums of the stachydrine hydrochloride reference product and the motherwort fruit test product are determined according to the construction method, and the content of the stachydrine hydrochloride in the motherwort fruit is obtained according to the China pharmacopoeia 2020 edition rule 0512.
9. The method of claim 1 or 2, wherein the method of preparing the motherwort fruit formula comprises the steps of,
(1) Extracting motherwort fruit medicinal materials or decoction pieces to obtain a motherwort fruit extracting solution;
(2) Concentrating the fructus Leonuri extractive solution to obtain fructus Leonuri concentrated solution with relative density of 1.05-1.1;
(3) Preheating the motherwort fruit concentrated solution, performing spray drying to obtain spray dried powder, and granulating to obtain the motherwort fruit formula granules.
10. The method according to claim 9, wherein the motherwort fruit extract has a ratio of 4-8%.
11. The construction method according to claim 10, wherein the concentration temperature is 50-80 ℃ and the concentration time is not higher than 24h.
12. The method of construction according to claim 9, wherein the temperature of the inlet air for spray drying is 170-185 ℃.
13. The method according to claim 9, wherein the extracting comprises extracting fructus Leonuri or decoction pieces with water to obtain fructus Leonuri extractive solution.
14. The method according to claim 12, wherein the fructus Leonuri extract is obtained by decocting fructus Leonuri or decoction pieces with water at least twice.
15. The method according to claim 12, wherein the fructus Leonuri extractive solution is obtained by adding 9-13 times of water into fructus Leonuri or decoction pieces, boiling and extracting for 60-90min, and repeating for at least two times.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111039967.7A CN113759043B (en) | 2021-09-06 | 2021-09-06 | Characteristic spectrum of motherwort fruit and construction method thereof, method for measuring content of stachydrine hydrochloride, motherwort fruit formula granules and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111039967.7A CN113759043B (en) | 2021-09-06 | 2021-09-06 | Characteristic spectrum of motherwort fruit and construction method thereof, method for measuring content of stachydrine hydrochloride, motherwort fruit formula granules and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113759043A CN113759043A (en) | 2021-12-07 |
| CN113759043B true CN113759043B (en) | 2023-03-21 |
Family
ID=78793196
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111039967.7A Active CN113759043B (en) | 2021-09-06 | 2021-09-06 | Characteristic spectrum of motherwort fruit and construction method thereof, method for measuring content of stachydrine hydrochloride, motherwort fruit formula granules and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113759043B (en) |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1973855B (en) * | 2006-10-16 | 2011-12-14 | 成都第一药物研究所有限公司 | Motherwort injection |
| CN111272905B (en) * | 2020-03-26 | 2022-03-11 | 广东一方制药有限公司 | Characteristic spectrum construction method and detection method of motherwort fruit medicinal material |
| CN112946134A (en) * | 2021-02-04 | 2021-06-11 | 成都第一制药有限公司 | HPLC fingerprint spectrum of motherwort injection and establishing method thereof |
-
2021
- 2021-09-06 CN CN202111039967.7A patent/CN113759043B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN113759043A (en) | 2021-12-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107149623B (en) | Content determination method of traditional Chinese medicine composition | |
| CN110632208B (en) | Detection method for main components of traditional Chinese medicine composition for clearing lung, eliminating phlegm, relieving cough and asthma | |
| CN113759045B (en) | Characteristic spectrum of notopterygium root decoction pieces or formula granules, construction method of characteristic spectrum, notopterygium root formula granules, preparation method of notopterygium root formula granules and quality control method of notopterygium root formula granules | |
| CN112611816B (en) | Hawthorn standard reference substance and preparation method, detection method and application thereof | |
| CN102309531B (en) | Detection method of American ginseng fingerprint | |
| CN113759043B (en) | Characteristic spectrum of motherwort fruit and construction method thereof, method for measuring content of stachydrine hydrochloride, motherwort fruit formula granules and preparation method thereof | |
| CN110638990B (en) | Extraction process of cassia twig, peony and rhizoma anemarrhenae prescription preparation extract | |
| CN115778997B (en) | Preparation method and quality detection method of astragalus root and burdock granules | |
| CN113759038B (en) | Trachelospermi caulis characteristic map, construction method and quality control method thereof, trachelospermi caulis formula granules and preparation method thereof | |
| CN113759057B (en) | Characteristic spectrum of allium macrostemon white water extract and preparation thereof and construction method thereof | |
| CN113759006B (en) | Quality control method of radix curcumae longae formula granules | |
| CN113759011B (en) | Method for establishing characteristic spectrum of starwort root and preparation thereof | |
| CN111965269A (en) | Method for measuring oleanolic acid content and ursolic acid content in patrinia heterophylla by using ultra-high performance liquid chromatography | |
| CN109342579B (en) | HPLC fingerprint detection method for traditional Chinese medicine for relaxing bowel | |
| CN113866334A (en) | Method for establishing fingerprint of pinellia ternate heart-fire purging decoction | |
| CN113092655A (en) | Method for detecting effective components in aconite decoction by high performance liquid chromatography | |
| CN109061024B (en) | Construction method and application of standard fingerprint spectrum of infant spleen-supporting granules | |
| CN113252826A (en) | HPLC fingerprint detection method of hypnotic brain-nourishing oral liquid | |
| CN105353065A (en) | Establishing method of HPLC (high-performance liquid chromatography) fingerprint spectrum of lychee seeds, standard fingerprint spectrum obtained with method and application of standard fingerprint spectrum | |
| CN115887525B (en) | Preparation method, detection method and characteristic map construction method of agastache rugosa extract | |
| CN113533563B (en) | Method for simultaneously detecting contents of four components of liver-soothing, stomach-harmonizing and pain-relieving traditional Chinese medicine | |
| CN116773702A (en) | Preparation method and detection method of salvia yunnanensis extract | |
| CN115950971A (en) | Characteristic map establishing and detecting method for Dan e fukang decoction cream | |
| CN115308331B (en) | Method for measuring content of 5 ingredients in oldenlandia standard decoction freeze-dried powder or formula granules by adopting one-measurement-multiple-evaluation method | |
| CN115436524B (en) | Method for detecting ginsenoside Rg1, re and Rb1 in decoction for warming meridians and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |