CN115737696A - Radix cynanchi wilfordii extract as well as preparation method, detection method and application thereof - Google Patents
Radix cynanchi wilfordii extract as well as preparation method, detection method and application thereof Download PDFInfo
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- 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
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- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The invention relates to the field of analysis of traditional Chinese medicines, and in particular relates to a cynanchum wilfordii extract, and a preparation method, a detection method and application thereof. The preparation method of the cynanchum wilfordii extract comprises the following steps: (1) Decocting radix cynanchi wilfordii decoction pieces in water, and filtering to obtain filtrate; wherein the water decoction times are 2 times, the water with the weight 10-12 times that of cynanchum wilfordii decoction pieces is added for the first water decoction, the water with the weight 8-10 times that of cynanchum wilfordii decoction pieces is added for the second water decoction, the first water decoction time is 35-55 min, and the second water decoction time is 25-40 min; (2) Concentrating the filtrate obtained in the step (1) and drying to obtain the cynanchum wilfordii extract. The cynanchum wilfordii extract has high cream yield and high transfer rate of p-hydroxyacetophenone. The detection method for the mass content and the characteristic spectrum of the p-hydroxyacetophenone provided by the invention has the advantages of good specificity, good repeatability and good stability.
Description
Technical Field
The invention relates to the field of traditional Chinese medicine analysis, and particularly relates to a cynanchum wilfordii extract, and a preparation method, a detection method and application thereof.
Background
Radix Cynanchi Wilfordii, named as radix Cynanchi Wilfordii, radix Cynanchi auriculati, and fructus Trichosanthis, is root tuber of radix Cynanchi Wilfordii of Asclepiadaceae, and is harvested in autumn, cleaned, sliced, and sun-dried; sweet in nature and taste, slightly bitter and slightly warm in nature; the functions are mainly used for nourishing liver and kidney; strengthening bones and muscles; strengthening the spleen and stomach; and (5) detoxifying. Mainly treating deficiency of both liver and kidney; dizziness and dim eyesight; insomnia and amnesia; the beard and hair are early white; impotence; spermatorrhea; the waist and the knees are sour and climbed; failure of the spleen; abdominal fullness and distention; the lipid component is to be inactivated; diarrhea; less postpartum milk; sores and ulcers on fish mouth. Modern researches show that cynanchum wilfordii mainly contains various chemical components such as C21 steroids, acetophenones, polysaccharide, phospholipid and the like, and has the effects of promoting gastrointestinal tract movement, regulating immunity, resisting tumors, resisting aging, protecting liver and the like.
The standard decoction is prepared by standardized decoction according to a clinical decoction method, solid-liquid separation, proper concentration preparation or proper method drying according to the traditional Chinese medicine theory, and is used as a standard reference substance for judging whether the traditional Chinese medicine formula particles are basically consistent with the clinical decoction.
The Chinese medicine granule is prepared with Chinese medicine pieces as material and through extraction, low temperature concentration, instantaneous drying and other technological processes. The Chinese medicinal composition can be clinically and flexibly prepared and taken after being mixed with water, can also be used as an industrial raw material of a Chinese medicinal preparation, and provides a new way for clinical medication of Chinese medicaments. Compared with the traditional Chinese medicine decoction, the formula granule has convenient administration and small dosage,
at present, the preparation and detection methods of standard decoction and formula granules of cynanchum wilfordii are not reported in documents.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: provides a radix cynanchi wilfordii extract, a preparation method and a detection method thereof, standardizes the quality control and standard research of the traditional Chinese medicine formula granules, and realizes the integral quality control and effective supervision of the traditional Chinese medicine formula granules.
In order to achieve the purpose, the invention provides the following technical scheme:
the invention provides a preparation method of cynanchum wilfordii extract, which comprises the following steps:
(1) Decocting radix cynanchi wilfordii decoction pieces in water, and filtering to obtain filtrate; wherein the water decocting times are 2 times, 10-12 times of water by weight of cynanchum wilfordii decoction pieces is added in the first water decoction, 8-10 times of water by weight of cynanchum wilfordii decoction pieces is added in the second water decoction, the first water decoction time is 35-55 min, and the second water decoction time is 25-40 min;
(2) Concentrating the filtrate obtained in the step (1) and drying to obtain the cynanchum wilfordii extract.
Preferably, in the above preparation method, in the step (2), the temperature for concentrating the filtrate is 60 to 65 ℃; preferably, the filtrate is concentrated until the density of the concentrated solution is 1.05-1.12 g/mL.
Preferably, in the above preparation method, the cynanchum wilfordii extract has a cream yield of 7.1 to 21.1%.
Preferably, in the above preparation method, the drying includes one of freeze drying, vacuum drying, spray drying, microwave drying and infrared drying.
Preferably, in the above preparation method, the drying is freeze-drying, and the freeze-drying is divided into three stages: a. pre-freezing: the pre-freezing temperature is-55 to-50 ℃; b. primary drying: the drying temperature is minus 45 ℃ to 0 ℃; c. and (3) secondary drying for analysis: the drying temperature is 10-30 ℃; preferably, the prefreezing time is 2 to 5 hours, preferably 3 hours; the primary drying time is 35 to 45 hours, preferably 38 hours; the secondary drying time is 5 to 7 hours, preferably 6 hours; more preferably, in step (2), the degree of vacuum for primary drying is 0 to 0.2mbar.
Preferably, in the preparation method, the drying is spray drying, the air inlet temperature of the spray drying is 165-195 ℃, and the air outlet temperature of the spray drying is 100-110 ℃; preferably, the spray drying process further comprises the step of mixing the cynanchum wilfordii extract and maltodextrin; more preferably, the mixing temperature is 60 to 100 ℃; further preferably, the addition amount of the maltodextrin is 5-20% by weight of the cynanchum wilfordii decoction pieces.
The invention also provides a cynanchum wilfordii extract prepared by the preparation method; preferably, the mass content of the p-hydroxyacetophenone in the cynanchum wilfordii extract is 0.25-0.98mg/g in terms of a dry product; more preferably, the transfer rate of p-hydroxyacetophenone is 22.58 to 83.92% by weight.
The invention also provides an identification method of the cynanchum wilfordii extract, which comprises the steps of identifying the cynanchum wilfordii extract by thin-layer chromatography; the thin layer chromatography comprises the following steps: a. preparing a test solution of cynanchum wilfordii extract and a reference solution of cynanchum wilfordii; b. respectively dropping the radix cynanchi wilfordii extract test solution and radix cynanchi wilfordii reference solution on the same silica gel G thin layer plate, adding developing agent for development, taking out, air drying, spraying color-developing agent to develop spots, and detecting;
preferably, the test solution of the cynanchum wilfordii extract is prepared by the following steps of taking 1-3g of cynanchum wilfordii extract, adding water for dissolving, eluting after ion exchange to obtain eluent, evaporating to dryness to obtain residue, and dissolving the residue with methanol to obtain the cynanchum wilfordii extract; preferably, the cynanchum wilfordii reference substance solution is prepared by the following steps of taking 1-3g of cynanchum wilfordii medicinal material, adding water, heating, refluxing, filtering, eluting after ion exchange to obtain eluent, evaporating to dryness to obtain residue, and dissolving the residue with methanol to obtain the cynanchum wilfordii reference substance solution;
more preferably, the developing solvent is toluene-ethyl acetate-methanol-water, and further preferably, the volume ratio of the toluene to the ethyl acetate to the methanol to the water is 20.
The invention also provides a method for detecting the mass content of the p-hydroxyacetophenone in the cynanchum wilfordii extract, which comprises the following steps:
(1) Preparation of reference solutions
Weighing p-hydroxyacetophenone reference substance, and adding a solvent to prepare a reference substance solution;
(2) Preparation of test solution
Weighing cynanchum wilfordii extract, and adding a solvent for extraction;
(3) High performance liquid chromatography
Using octadecylsilane chemically bonded silica as a filler, using an organic solvent as a mobile phase A, using a 0.1% phosphoric acid aqueous solution as a mobile phase B, carrying out gradient elution, and sucking a reference substance solution and a test solution to inject into a high performance liquid chromatograph for analysis.
Preferably, in the detection method, the solvent in step (1) is one or more selected from the group consisting of 50% ethanol, 75% ethanol, 95% ethanol, 50% methanol, 75% methanol and methanol, and preferably 75% ethanol; preferably, the solvent in step (2) is one or more selected from 50% ethanol, 75% ethanol, 95% ethanol, 50% methanol, 75% methanol and methanol, preferably 75% ethanol.
Preferably, in the detection method, the extraction in step (2) is performed by one of reflux, shaking or ultrasound, and is preferably performed by ultrasound.
Preferably, in the detection method, the extraction time in the step (2) is 15 to 60min, preferably 60min.
Preferably, in the detection method, the organic solvent in step (3) is selected from acetonitrile or methanol, preferably acetonitrile.
Preferably, in the detection method, the conditions of the gradient elution in the step (3) are as follows: in 0-15min, the mobile phase A is 10% → 21%, the mobile phase B is 90% → 79%,15-18min, the mobile phase A is 21% → 21%, the mobile phase B is 79% → 79%,18-23min, the mobile phase A is 21% → 90%, the mobile phase B is 79% → 10%,23-27min, the mobile phase A is 90% → 10%, the mobile phase B is 10 → 90%,27-30min, the mobile phase A is 10% → 10%, and the mobile phase B is 90 → 90%.
The invention also provides a detection method of the characteristic spectrum of the cynanchum wilfordii extract, which comprises the following steps:
(1) Preparation of reference solutions
Weighing p-hydroxyacetophenone reference substance, and adding a solvent to prepare a reference substance solution;
(2) Preparation of test solution
Weighing cynanchum wilfordii extract, and adding a solvent for extraction;
(3) Ultra high performance liquid chromatography
Using octadecylsilane chemically bonded silica as filler, organic solvent as mobile phase A, and water phase as mobile phase B to perform gradient elution, and absorbing reference solution and sample solution to inject into ultra high performance liquid chromatograph for analysis.
Preferably, in the detection method, the solvent in step (1) is one or more selected from water, 50% methanol, 75% ethanol, 50% methanol, 75% methanol and methanol, preferably methanol; preferably, the solvent in step (2) is one or more selected from water, 50% methanol, 75% ethanol, 50% methanol, 75% methanol and methanol, preferably methanol.
Preferably, in the detection method, the extraction in step (2) is performed by one of reflux, shaking or ultrasound, and is preferably performed by ultrasound.
Preferably, in the detection method, the extraction time in the step (2) is 15 to 60min, preferably 30min.
Preferably, in the detection method, the organic solvent in step (3) is selected from acetonitrile or methanol, preferably methanol.
Preferably, in the detection method, the conditions of the gradient elution in the step (3) are as follows: in 0-7min, 23% → 24% of mobile phase A, 77% → 76% of mobile phase B, 7-11min, 24% → 27% of mobile phase A, 76% → 73% of mobile phase B, 11-14min, 27% → 29% of mobile phase A, 73% → 71% of mobile phase B, 14-18min, 29% → 32% of mobile phase A, 71 → 68% of mobile phase B, 18-20min, 32% → 38% of mobile phase A, 68% → 62% of mobile phase B, 20-28min, 38% → 38% of mobile phase A, 62% → 62% of mobile phase B, 28-28.1min, 38% → 23% of mobile phase A, 62% → 77% of mobile phase B, 28.1-30min, 23% → 23% of mobile phase A, and 77% → 77% of mobile phase B.
Preferably, in the above detection method, the aqueous phase in step (3) is selected from the group consisting of water, 0.1% formic acid solution, 0.05% phosphoric acid solution and 0.1% acetic acid solution, preferably 0.1% formic acid solution.
The invention also provides the application of the cynanchum wilfordii extract in preparing health-care food and traditional Chinese medicine; preferably, the traditional Chinese medicine is a standard decoction, a capsule, a tablet, a granule or a formula granule.
A standard decoction is prepared from the cynanchum wilfordii extract.
A granule is prepared by adding adjuvants into the extract of radix cynanchi wilfordii.
The invention has the beneficial effects that:
1. the invention provides a standard for the quality of the traditional Chinese medicine formula granules, and realizes the overall quality control and effective supervision of the traditional Chinese medicine formula granules.
2. The cynanchum wilfordii extract is prepared from cynanchum wilfordii medicinal materials, the paste yield of the cynanchum wilfordii extract is 7.1-21.1%, the paste yield is high, the mass content of p-hydroxyacetophenone in the cynanchum wilfordii extract is 0.25-0.98mg/g, and the transfer rate of the p-hydroxyacetophenone is 22.58-83.92%. Through 6-month accelerated stability tests, the content of p-hydroxyacetophenone and the content of water are relatively stable, the relative retention time of a characteristic map and the relative peak area are relatively stable, moisture absorption is resistant, no impurity component is generated, and the prepared cynanchum wilfordii extract is subjected to high performance liquid chromatography and ultra high performance liquid chromatography analysis.
3. The characteristic spectrum method established by the invention adopts ultra-high phase liquid chromatography, has the characteristics of simplicity, stability, high precision, good reproducibility and the like, and the obtained extract has multiple peaks of fingerprint spectrum, good peak pattern, easy identification, accuracy and reliability.
4. The method for establishing the characteristic spectrum is simple, time-saving and environment-friendly, only takes 30min for analyzing a batch of preparations, can greatly shorten the detection and analysis time, improves the production efficiency, and makes the sampling detection of large samples and the real-time monitoring of the product quality possible.
Drawings
FIG. 1 is a high performance liquid chromatogram for measuring the mass content and transfer rate of p-hydroxyacetophenone in the extract of radix cynanchi wilfordii prepared in the first measurement example;
FIG. 2 is a high performance liquid chromatogram for the specific investigation of the determination method of the mass content and transfer rate of p-hydroxyacetophenone in the cynanchum wilfordii extract;
FIG. 3 is a linear relationship investigation diagram of the determination method of the mass content and transfer rate of p-hydroxyacetophenone in radix cynanchi wilfordii extract;
FIG. 4 is a high performance liquid chromatogram of different chromatographic columns in the detection method for the mass content and transfer rate of p-hydroxyacetophenone in the extract of cynanchum wilfordii of the embodiment;
FIG. 5 is a high performance liquid chromatogram of different chromatographs in the detection method for the mass content and the transfer rate of p-hydroxyacetophenone in the extract of cynanchum wilfordii of the embodiment;
FIG. 6 is a high performance liquid chromatogram for different column temperatures in the method for detecting the mass content and transfer rate of p-hydroxyacetophenone in radix cynanchi wilfordii extract in one embodiment;
FIG. 7 is a high performance liquid chromatogram of different flow rates in the method for detecting the mass content and transfer rate of p-hydroxyacetophenone in cynanchum wilfordii extract in one embodiment;
FIG. 8 is an absorption spectrum of cynanchum wilfordii extract in the range of 190-400 nm;
FIG. 9 is an ultra-high performance liquid chromatogram of an extract from cynanchum wilfordii of embodiment, wherein the mobile phases are acetonitrile-0.05% phosphoric acid solution and methanol-0.05% phosphoric acid solution;
FIG. 10 is an ultra-high performance liquid chromatogram of a methanol-water mobile phase in a method for detecting a profile of an extract from cynanchum wilfordii according to an embodiment;
FIG. 11 is an ultra-high performance liquid chromatogram of a methanol-0.05% phosphoric acid solution as a mobile phase in a method for detecting a signature of an extract from cynanchum wilfordii according to an embodiment;
FIG. 12 is an ultra-high performance liquid chromatogram of a methanol-0.1% formic acid solution as a mobile phase in a method for detecting a signature of an extract from cynanchum wilfordii according to an embodiment;
FIG. 13 is an ultra-high performance liquid chromatogram of a methanol-0.1% acetic acid solution as a mobile phase in a method for detecting a profile of an extract from cynanchum wilfordii according to an embodiment;
FIG. 14 is an ultra high performance liquid chromatogram of different chromatographic columns in a method for detecting a signature of an extract from cynanchum wilfordii according to an embodiment;
FIG. 15 is an ultra-high performance liquid chromatogram at different column temperatures of a method for detecting a signature of an extract from cynanchum wilfordii according to an embodiment;
FIG. 16 is an ultra high performance liquid chromatogram of different flow rates in a method for detecting a profile of an extract from cynanchum wilfordii according to an embodiment;
FIG. 17 is a characteristic spectrum of the extract of cynanchum wilfordii obtained in the first example;
FIG. 18 is a chromatogram for special investigation of the radix cynanchi wilfordii feature map detection method;
FIG. 19 is an overall investigation chromatogram of the radix cynanchi wilfordii characteristic spectrum detection method;
FIG. 20 shows the measurement results of the common peak of the extract of radix cynanchi wilfordii;
FIG. 21 is a characteristic spectrum of the radix cynanchi wilfordii formula granules prepared in the fifth embodiment;
FIG. 22 shows the thin-layer chromatography identification of the modified example of the formulation of radix cynanchi wilfordii.
Detailed Description
In order to make the purpose, technical scheme and technical effect of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention is clearly and completely described. The embodiments described below are some, but not all embodiments of the invention. All other embodiments obtained by a person of ordinary skill in the art without making any creative effort in combination with the embodiments of the present invention belong to the protection scope of the present invention.
In one embodiment of the present invention, the present invention provides a method for preparing cynanchum wilfordii extract, comprising the following steps:
(1) Decocting radix cynanchi wilfordii decoction pieces in water, and filtering to obtain filtrate; wherein the water decocting times are 2 times, 10-12 times of water by weight of cynanchum wilfordii decoction pieces is added in the first water decoction, 8-10 times of water by weight of cynanchum wilfordii decoction pieces is added in the second water decoction, the first water decoction time is 35-55 min, and the second water decoction time is 25-40 min;
(2) Concentrating the filtrate obtained in the step (1) and drying to obtain the cynanchum wilfordii extract.
According to the invention, in the step (2), the filtrate is concentrated under reduced pressure, concentrated at a temperature of 60-65 ℃ to a density of 1.05-1.12g/mL, and the solid content is adjusted to 10-14%.
According to the present invention, the drying includes, but is not limited to, one of freeze drying, vacuum drying, spray drying, microwave drying and infrared drying.
In a preferred embodiment of the present invention, the drying of step (2) is freeze-drying, which is divided into three stages: a. pre-freezing: the pre-freezing temperature is-55 to-50 ℃; b. primary drying: the drying temperature is minus 45 ℃ to 0 ℃; c. and (3) secondary drying for analysis: the drying temperature is 10-30 ℃;
wherein the pre-freezing time is 2 to 5 hours, preferably 3 hours; the primary drying time is 35 to 45 hours, preferably 38 hours, and the vacuum degree of the primary drying is 0 to 0.2mbar; the secondary drying time is 5 to 7 hours, and the vacuum degree of the secondary drying is 0mbar;
in order to facilitate the intact shape and uniform color of the lyophilized powder and retain effective substances, the lyophilized powder is frozen at-45 deg.C for 2-3h, at-35 deg.C for 6-7h, at-30 deg.C for 18-20h, at-25 deg.C for 9-10h, at-20 deg.C for 2-3h, at-10 deg.C for 1-2h, and at 0 deg.C for 1-2h in one-time drying process.
In another preferred embodiment of the present invention, in step (1), the cynanchum wilfordii decoction pieces are moderately crushed and soaked for 30min before being decocted with water.
According to the invention, in step (1), the filtration is carried out while hot, and the filtration is carried out by using a 100-300 mesh screen, preferably a 100 mesh screen.
Preferably, the screen is a taylor screen (taylor corporation, usa) or a filter cloth (new chemical, llc, new county).
In another preferred embodiment of the present invention, the drying in step (2) is spray drying, which has the advantages of high drying efficiency, fast drying speed, short drying time, less damage to the effective components, uniform and fine dried powder, low water content, good solubility, etc., and is suitable for pilot plant test and commercial mass production; in addition, the control of the hot air temperature and the air outlet temperature when hot air enters during spray drying is important, the quality and the drying bulk density of the product are determined, and the inventor finds that the temperature of an air inlet of the spray drying is 165-195 ℃, the temperature of an air outlet of the spray drying is 100-110 ℃, and the product is well formed and has high quality at the temperature.
In the spray drying process, in order to prevent foreign matter mixed in the radix cynanchi wilfordii extract from blocking the pipeline, the extract is heated before drying, and the temperature of the material is kept at 60-100 ℃. In order to facilitate the drying and reduce the problems of wall sticking and caking, the auxiliary materials, such as maltodextrin, are added into the clear paste before the drying, and the addition amount of the auxiliary materials is more preferably 5-20 percent based on the weight of the cynanchum wilfordii decoction pieces added during the decoction, so the effect is better.
The invention also provides an identification method of the cynanchum wilfordii extract, which comprises the steps of identifying the cynanchum wilfordii extract by thin-layer chromatography; the thin layer chromatography comprises the following steps: a. preparing a test solution of cynanchum wilfordii extract and a reference solution of cynanchum wilfordii; b. respectively dispensing the radix cynanchi wilfordii extract test solution and radix cynanchi wilfordii reference solution on the same silica gel G thin-layer plate, adding developing agent for development, taking out, air drying, spraying color-developing agent to develop the spots, and detecting; preferably, the test solution of cynanchum wilfordii extract is prepared by the steps of taking 1-3g of cynanchum wilfordii extract, adding water for dissolving, eluting by adopting ion exchange resin such as macroporous resin column, collecting eluent, evaporating the eluent to obtain residue, and dissolving the residue by using methanol; preferably, the cynanchum wilfordii reference substance solution is prepared by the following steps of taking 1-3g of cynanchum wilfordii medicinal material, adding water, heating, refluxing, filtering, eluting by adopting ion exchange resin such as macroporous resin column, collecting eluent, evaporating the eluent to obtain residue, and dissolving the residue by using methanol; preferably, the developing solvent is toluene-ethyl acetate-methanol-water, and more preferably, the volume ratio of the toluene, ethyl acetate, methanol and water is 20.
The invention also provides a method for detecting the mass content of the p-hydroxyacetophenone in the cynanchum wilfordii extract, which comprises the following steps:
(1) Preparation of reference solutions
Weighing p-hydroxyacetophenone reference substance, and adding a solvent to prepare a reference substance solution;
(2) Preparation of test solution
Weighing cynanchum wilfordii extract, and adding a solvent for extraction;
(3) High performance liquid chromatography
Using octadecylsilane chemically bonded silica as filler, organic solvent as mobile phase A,0.1% phosphoric acid aqueous solution as mobile phase B to perform gradient elution, and absorbing reference solution and sample solution to inject into high performance liquid chromatograph for analysis.
According to the invention, when the high performance liquid chromatography is carried out, the conditions of gradient elution are as follows: in 0-15min, the content of mobile phase A is 10% → 21%, the content of mobile phase B is 90% → 79%,15-18min, the content of mobile phase A is 21% → 21%, the content of mobile phase B is 79% → 79%,18-23min, the content of mobile phase A is 21% → 90%, the content of mobile phase B is 79% → 10%,23-27min, the content of mobile phase A is 90% → 10%, the content of mobile phase B is 10 → 90%,27-30min, and the content of mobile phase A is 90% → 90%.
According to the invention, the preparation method of the reference substance solution comprises the following steps: taking a proper amount of p-hydroxyacetophenone reference substance, precisely measuring, and adding a solvent to prepare a solution containing 10 mu g of p-hydroxyacetophenone per 1 mL.
The preparation method of the test solution comprises the following steps: taking 0.1-0.15g of cynanchum wilfordii extract, precisely weighing, adding 25mL of solvent, weighing, extracting by adopting a reflux or oscillation dissolving or ultrasonic mode, preferably ultrasonic extracting, weighing again, complementing weight loss by using the solvent, shaking up, and filtering to obtain filtrate.
According to the invention, when the high performance liquid chromatography is carried out, the chromatographic column used can be Philomen SuperLu C18 (column length 250mm, inner diameter 4.6mm, particle size 5 μm), zorbax Ecl ipse Plus C18 (column length 250mm, inner diameter 4.6mm, particle size 5 μm) or Shimadzu Inerts i ODS-HL C18 (column length 250mm, inner diameter 4.6mm, particle size 5 μm); preference is given to Philomena SuperLu C18 (column length 250mm, internal diameter 4.6mm, particle size 5 μm).
According to the present invention, when the high performance liquid chromatography is performed, the column temperature of the column used is 30 to 40 ℃ and preferably 30 ℃.
According to the invention, the flow rate used for the HPLC analysis can be 0.9-1.2ml/min, preferably 1.0ml/min.
The invention also provides a detection method of the characteristic spectrum of the cynanchum wilfordii extract, which comprises the following steps:
(1) Preparation of reference solutions
Weighing p-hydroxyacetophenone reference substance, and adding a solvent to prepare a reference substance solution;
(2) Preparation of test solution
Adding solvent into radix cynanchi wilfordii extract for extraction;
(3) Ultra high performance liquid chromatography
Using octadecylsilane chemically bonded silica as filler, organic solvent as mobile phase A, and water phase as mobile phase B to perform gradient elution, and absorbing reference solution and sample solution to inject into ultra high performance liquid chromatograph for analysis.
According to the invention, the preparation method of the reference substance solution comprises the following steps: taking a proper amount of p-hydroxyacetophenone reference substance, precisely measuring, and adding a solvent to prepare a solution containing 10 microgram of p-hydroxyacetophenone per 1 mL.
The preparation method of the test solution comprises the following steps: taking 0.1-0.15g of cynanchum wilfordii extract, accurately weighing, adding 25mL of solvent, weighing, extracting by adopting a reflux or oscillation dissolving or ultrasonic mode, preferably ultrasonic extracting, weighing, complementing weight loss by using the solvent, shaking up, and filtering to obtain filtrate.
According to the present invention, when performing high performance liquid chromatography, the aqueous phase may be one of water, 0.1% formic acid solution, 0.05% phosphoric acid solution or 0.1% acetic acid solution, preferably 0.1% formic acid solution, wherein the conditions of the gradient elution are: in 0-7min, 23% → 24% of mobile phase a, 77% → 76% of mobile phase B, 7-11min, 24% → 27% of mobile phase a, 76% → 73% of mobile phase B, 11-14min, 27% → 29% of mobile phase a, 73% → 71% of mobile phase B, 14-18min, 29% → 32% of mobile phase a, 71% →68% of mobile phase B, 18-20min, 32% → 38% of mobile phase a, 68% → 62% of mobile phase B, 20-28min, 38% → 38% of mobile phase a, 62% → 62% of mobile phase B, 28-28.1min, 38% → 23% of mobile phase a, 62% → 77% of mobile phase B, 28.1-30min, 23% of mobile phase a, and 77% of mobile phase B are included.
According to the present invention, when the ultra high performance liquid chromatography is performed, the column used may be Dikma C18-A (2.1X 150mm,1.8 μm) of Dimacology Co., ltd., and ACQUITY UPLC of Watts technology Co., ltd
T3 (2.1 × 150mm,1.8 μm) or Shodex C18U 2D (2.1 × 150mm,1.9 μm) from Theobrasscience instruments, guangzhou, preferably Dikma C18-A (2.1 × 150mm,1.8 μm) from Dimackojic.
According to the present invention, when the ultra performance liquid chromatography is performed, the column temperature of the column used is 25 to 35 ℃, preferably 30 ℃.
According to the present invention, when the ultra high performance liquid chromatography is performed, the flow rate used may be 0.2 to 0.3ml/min, preferably 0.3ml/min.
The invention also provides the application of the cynanchum wilfordii extract in preparing health-care food and traditional Chinese medicine; preferably, the traditional Chinese medicine is a standard decoction, a capsule, a tablet, a granule or a formula granule.
The invention also provides a standard decoction which is prepared from the cynanchum wilfordii extract.
The invention also provides cynanchum wilfordii formula granules, which are prepared by performing dry granulation on cynanchum wilfordii extracts and pharmaceutical excipients.
Dry granulation is a method of mixing the drug and the excipients uniformly, compressing into large tablets or strips, and pulverizing into particles of the required size. The method relies on compression force to generate binding force between ions, and the preparation method comprises a heavy pressing method and a rolling method. Compared with the traditional wet granulation, the process of soft material preparation, drying and granule finishing is omitted, the process is simple, the defect of large consumption of auxiliary materials is overcome, and the drug loading capacity can be greatly improved. The related regulations of the traditional Chinese medicine formula granule require that the finished product of the traditional Chinese medicine formula granule only needs no or little auxiliary materials, so the dry granulation method is preferably selected as the granulation method of the traditional Chinese medicine formula granule. The medicinal auxiliary materials include but are not limited to maltodextrin, magnesium stearate, silicon dioxide, talcum powder or microcrystalline cellulose, can change the powder property, improve the appearance and the fluidity of materials, and are convenient to store and transport, and control the solubility, the porosity, the specific surface area and the like. Considering the comprehensive properties of particle size, fluidity, primary yield, bulk density, dissolubility and the like, the amount of the auxiliary materials added in the dry granulation process is less than or equal to 0.3 percent (based on the weight of the clear paste). Excessive use of the ingredients can cause performance deficiencies, for example, magnesium stearate can be slow to dissolve due to its hydrophobic nature.
According to the invention, in order to ensure the primary yield and the particle size of the particles, the feeding speed of the dry granulation is 70-110 r/min, the rotation speed of a roller pressing wheel is preferably 5-15 r/min, and the hydraulic pressure is 5-10 MPa.
The advantageous effects of the present invention are further illustrated by the following specific examples.
The raw materials or reagents used in the present invention are purchased from mainstream manufacturers in the market, and those who do not indicate manufacturers or concentrations are all analytical pure grade raw materials or reagents that can be obtained conventionally, and are not particularly limited as long as they can perform the intended function.
Specific techniques or conditions not specified in this example were followed as described in literature in this field or as described in the product specification.
The present invention will be described in more detail below with reference to examples and comparative examples, but the technical scope of the present invention is not limited to these examples. All percentages, parts and ratios used in the present invention are based on mass unless otherwise specified. Wherein, the raw material information and the test equipment information used in the examples and comparative examples are shown in tables 1 and 2, respectively:
TABLE 1 raw materials information used in the present invention
| Name of raw materials | Purity/batch number | Selling manufacturers |
| Radix cynanchi wilfordii decoction pieces | YP2104-1 | Guizhou province Bijie city field dam bridge town |
| Radix cynanchi wilfordii decoction pieces | YP2104-2 | Guizhou province Bijie city forest Koukun |
| Radix cynanchi wilfordii decoction pieces | YP2104-3 | Bijie town of Guizhou province |
| Radix cynanchi wilfordii decoction pieces | YP2104-4 | Dayin town of Bijie city, guizhou province |
| Radix cynanchi wilfordii decoction pieces | YP2104-5 | Guizhou Bijie poplar market cold town |
| Radix cynanchi wilfordii decoction pieces | YP2104-6 | Guizhou province, bijie city, wild corner village |
| Radix cynanchi wilfordii decoction pieces | YP2104-7 | Bijie city Hope slope of Guizhou province |
| Radix cynanchi wilfordii decoction pieces | YP2104-8 | Guizhou province Bijie city village |
| Radix cynanchi wilfordii decoction pieces | YP2104-9 | Guizhou province high slope town |
| Radix cynanchi wilfordii decoction pieces | YP2104-10 | Guizhou province high slope pond village |
| Radix cynanchi wilfordii decoction pieces | YP2104-11 | Guizhou province Longli county sheep farm |
| Radix cynanchi wilfordii decoction pieces | YP2104-12 | Guizhou Longli county dry dam |
| Radix cynanchi wilfordii decoction pieces | YP2104-13 | Yongyuncun of Longli county in Guizhou province |
| Radix cynanchi wilfordii decoction pieces | YP2104-14 | Guizhou province Longli county Qiu Jiazhuang |
| Radix cynanchi wilfordii decoction pieces | YP2104-15 | Bijie city duck pool town of Guizhou province |
| P-hydroxyacetophenone | 111897-201602 | China Institute for food and drug control |
TABLE 2 information on the experimental equipment used in the present invention
| Device name | Model number | Selling manufacturers |
| Electric ceramic pot | YMW-5 | Mother-of-pear artwork manufacturing factory in lake, city and port |
| Rotary evaporator | RE-5205A | Shanghai Yarong biochemical instrument factory |
| Rotary evaporator | YRE2000-B | GONGYI CITY YUHUA INSTRUMENT Co.,Ltd. |
| Vacuum freeze drier | TRL-0.5 | DALIAN TIANREY SCIENCE & TECHNOLOGY Co.,Ltd. |
| Super highLiquid chromatograph | WatersACQUITY/H-CLass | Vortes Tech Ltd |
| High performance liquid chromatograph | Agilent1260Infinity | Agilent Technologies Co., Ltd. |
| Numerical control ultrasonic cleaning instrument | KQ-500DB | KUNSHAN ULTRASONIC INSTRUMENTS Co.,Ltd. |
| Silica gel G thin layer plate | 10cm*10cm | SINOPHARM CHEMICAL REAGENT Co.,Ltd. |
| Multifunctional extraction and concentration unit | TS-NS-200 | SHANGHAI SUNYI EXPERIMENTAL EQUIPMENT Co.,Ltd. |
| Spray drying machine set | ZIPG-5 | CHANGZHOU HENGAN DRYING EQUIPMENT Co.,Ltd. |
| Dry granulating machine | GZL100-30L | SHIJIAZHUANG KEYUAN MACHINERY EQUIPMENT Co.,Ltd. |
The solid content of the cynanchum wilfordii concentrated solution in the following examples is measured by the following method:
referring to the determination of the content of the extract by a hot dipping method in the extract determination method 2201 in the fourth general rule of China pharmacopoeia of 2020 edition: precisely weighing 10g of the concentrated solution, placing the concentrated solution in an evaporation pan with constant weight, drying the concentrated solution by evaporation on a water bath, drying the concentrated solution for 3 hours at 105 ℃, placing the dried concentrated solution in a drier for cooling for 30min, rapidly and precisely weighing the concentrated solution, and calculating to obtain the solid content of the concentrated solution.
Example one
1. Preparation method of cynanchum wilfordii extract
(1) Taking 100g of cynanchum wilfordii decoction pieces (batch number YP 2104-1), putting in an electric ceramic pot, adding water for decocting twice, directly adding the first decoction into water which is 10 times of the weight of the cynanchum wilfordii decoction pieces, soaking for 30 minutes, boiling with strong fire (500W), keeping slight boiling with slow fire (200W) for 40 minutes, and filtering the decoction while hot through a 300-mesh screen; adding 8 times of water by weight of the cynanchum wilfordii hemsl tablets into the second decoction, heating and boiling the mixture with strong fire, keeping the mixture slightly boiled for 30 minutes with slow fire, filtering the decoction while the decoction is hot by using a 300-mesh screen, and combining the two filtrates;
(2) Transferring the filtrate into a 2000ml round-bottom flask, concentrating the filtrate to 100ml of extract at low temperature under reduced pressure by adopting a rotary evaporator (the temperature is 65 ℃, the vacuum degree is-0.080 to-0.090 MPa), and measuring the density of the extract to be 1.11 and the yield of the extract to be 14.64 percent; under magnetic stirring, subpackaging into 10ml brown penicillin bottles, wherein the subpackaging volume of each bottle is 1ml, half plugging, transferring to a vacuum freeze dryer for freeze drying after subpackaging, pre-freezing at-50 ℃ for 3 hours, then carrying out primary drying at-45 ℃, 35 ℃,30 ℃, 25 ℃,20 ℃,10 ℃ and 0 ℃ under a vacuum degree of 0.2mbar for 2 hours, 6 hours, 18 hours, 9 hours, 2 hours and 1 hour respectively, then carrying out secondary drying at 10 ℃,20 ℃ and 30 ℃ under a vacuum degree of 0mbar for 1 hour, 1 hour and 4 hours respectively, taking out, rolling an aluminum cover to obtain the cynanchum wilfordii paste extract, and the cynanchum wilfordii yield and the water mass content are calculated according to the following formula,
the paste yield = concentrate weight solid content/amount of decoction pieces fed 100%
Moisture mass content = moisture weight/sample weight 100%
2. Determination method for mass content and transfer rate of p-hydroxyacetophenone of cynanchum wilfordii hook f extract
2.1 optimization of the conditions of the detection method
2.1.1 preparation of reference solutions
Taking a proper amount of p-hydroxyacetophenone reference substance, precisely weighing, placing into a numbered 10ml volumetric flask, adding 75% ethanol to prepare a reference substance solution containing 10 microgram of p-hydroxyacetophenone per 1ml, and shaking up to obtain the p-hydroxyacetophenone reference substance.
2.1.2 preparation of test solutions
Taking about 0.14g of the cynanchum wilfordii extract prepared in the first embodiment, precisely weighing, placing in a conical flask with a plug, adding 25mL of solvent, weighing, extracting for a period of time, cooling, weighing again, supplementing the lost weight with the corresponding solvent, shaking up, filtering, taking a subsequent filtrate, taking a proper amount of the subsequent filtrate, passing through a 0.22 mu m microporous membrane, and placing in a liquid sample bottle.
2.1.3 high Performance liquid chromatography
Octadecylsilane chemically bonded silica is used as a filler (Philomen SuperLu C18, column length of 250mm, inner diameter of 4.6mm, particle size of 5.0 μm); acetonitrile is taken as a mobile phase A,0.1 percent phosphoric acid water solution is taken as a mobile phase B, and gradient elution is carried out according to the specification in the table 3; the column temperature is 30 ℃; the flow rate was 1.0ml/min and the detection wavelength was 276nm. The number of theoretical plates is not less than 10000 calculated according to the peak of p-hydroxyacetophenone. 10 μ L of the reference solution in 2.1.1 and the sample solution in 2.1.2 were respectively aspirated and injected into a high performance liquid chromatograph for analysis.
TABLE 3 elution gradient
2.1.4 the mass content of p-hydroxyacetophenone and the transfer rate of p-hydroxyacetophenone are calculated according to the following formulas:
wherein, the first and the second end of the pipe are connected with each other,
w p-hydroxyacetophenone ＝A For supplying to ×C To pair ×V For supplying to ×10 -3 /(m For supplying to ×A To pair )
w Drinking water ＝A Drinking supply ×C To pair ×V Drink supply ×10 -3 /(m Drink supply ×A To pair )
Transfer rate of p-hydroxyacetophenone = (m) Lifting device ×w P-hydroxyacetophenone )/(m Drinking water ×w Drinking water )
In the formula:
w p-hydroxyacetophenone The mass content of the p-hydroxyacetophenone in the cynanchum wilfordii extract is mg/g;
w drinking water The mass content of the p-hydroxyacetophenone in the cynanchum wilfordii decoction pieces is mg/g;
A for is to The corresponding absorption peak area of hydroxyacetophenone in the high performance liquid chromatography spectrogram of the reference substance solution;
A for supplying to -the area of the corresponding absorption peak of hydroxyacetophenone in the HPLC chromatogram of the test solution;
A drink supply The area of the corresponding absorption peak of hydroxyacetophenone in the high performance liquid chromatography spectrogram of the decoction piece test solution;
C to pair -the concentration of the reference solution control, μ g/mL;
m for supplying to Absolute dry mass of cynanchum wilfordii extract in test solution, g;
m drinking supply Absolute dry mass of cynanchum wilfordii decoction pieces in the decoction piece test sample solution, g;
V for supplying to -volume of test solution, ml;
V drinking supply Volume of the ready-to-use sample solution, ml;
m lifting device Preparing oven-dried mass, g, of the cynanchum wilfordii extract;
m drinking water Oven-dried mass of the raw material cynanchum wilfordii decoction pieces, g.
2.1.5 optimization of preparation conditions
Investigation of different extraction solvents
Taking about 0.14g of the cynanchum wilfordii extract prepared in the first embodiment, precisely weighing, respectively placing 2 samples in parallel in conical flasks with stoppers, precisely adding 25ml of water, 50% of ethanol, 75% of ethanol, 95% of ethanol, 50% of methanol, 75% of methanol and methanol in sequence, weighing, carrying out ultrasonic treatment (power 500W and frequency 40 kHz) for 30min, cooling, weighing again, complementing the weight loss with corresponding solvent, shaking up, filtering, and taking the subsequent filtrate to obtain the solution of each sample. Respectively and precisely absorbing 10 mu L of each of the reference solution and the test solution, injecting the reference solution and the test solution into a high performance liquid chromatograph, performing chromatographic analysis according to 2.1.3 chromatographic conditions, calculating the influence of different extraction solvents on the content of the reference solution and the test solution according to the method by using p-hydroxyacetophenone, and determining the optimal extraction solvent, wherein the specific results are detailed in a table 4:
TABLE 4 Effect of different extraction solvents on the content of hydroxyacetophenone in the extract of cynanchum wilfordii
As can be seen from Table 4: the different solvents have obvious influence on the content of the p-hydroxyacetophenone in the cynanchum wilfordii extract, the content and the target peak shape are comprehensively considered, and 75% ethanol is selected as the extraction solvent.
Investigation of II extraction mode
Taking about 0.14g of the cynanchum wilfordii extract prepared in the first embodiment, precisely weighing, and 3 parts in total, wherein 2 samples are parallelly placed in a cone-shaped product, respectively, precisely adding 25mL of 75% ethanol in sequence, weighing, respectively performing ultrasonic treatment (power 500W and frequency 40 kHz) for 30min, performing reflux treatment for 30min, performing shaking treatment for 30min, taking out, cooling to room temperature, supplementing with 75% ethanol, uniformly mixing, filtering, and collecting a subsequent filtrate. And (4) filtering appropriate amount of the subsequent filtrate with 0.22 μm microporous membrane, and placing in a liquid sample bottle to obtain each sample solution. Respectively and precisely sucking 10 mu L of reference solution and test solution, injecting into a high performance liquid chromatograph, performing chromatographic analysis according to 2.1.3 chromatographic conditions, calculating the influence of different extraction modes on the content of the reference solution and the test solution according to the method by using p-hydroxyacetophenone, and determining the optimal extraction mode, wherein the specific results are shown in Table 5:
TABLE 5 influence of different extraction methods on the content of p-hydroxyacetophenone in radix cynanchi wilfordii extract
As can be seen from Table 5: the influence difference of different extraction modes on the content of the p-hydroxyacetophenone in the cynanchum wilfordii extract is not obvious, the sample treatment efficiency is comprehensively considered, and ultrasonic extraction is selected for extraction.
III investigation of different extraction times
Taking about 0.14g of the cynanchum wilfordii extract prepared in the first embodiment, precisely weighing, 3 parts, respectively putting 2 samples in parallel into a cone-shaped product with a plug, sequentially and precisely adding 25mL of 75% ethanol, weighing, respectively carrying out ultrasonic treatment (power 500W and frequency 40 kHz) for 15min, 30min and 60min, taking out, cooling to room temperature, supplementing weight with 75% ethanol, uniformly mixing, filtering, and collecting filtrate. Taking a proper amount of filtrate, filtering the filtrate through a 0.22 mu m microporous filter membrane, and placing the filtrate in a liquid sample bottle to obtain each test sample solution for later use. Precisely sucking 10 μ l of each of the reference solution and the sample solution, respectively, injecting into high performance liquid chromatograph, performing chromatographic analysis according to 2.1.3 chromatographic conditions, and calculating the influence of different extraction times on the content of p-hydroxyacetophenone according to the above method. The specific results are detailed in table 6:
TABLE 6 influence of different extraction times on the content of p-hydroxyacetophenone in cynanchum wilfordii extracts
From the results in Table 6, it can be seen that: the different extraction time has no significant influence on the content of p-hydroxyacetophenone in the cynanchum wilfordii extract, the time cost and the content difference are comprehensively considered, and ultrasonic extraction is selected for 60min.
The optimized conditions are adopted to prepare the cynanchum wilfordii extract test solution and the cynanchum wilfordii decoction piece test solution, and the method comprises the following steps:
taking about 0.14g of the cynanchum wilfordii extract prepared in the first embodiment, precisely weighing, placing in a conical flask with a plug, adding 25mL of 75% ethanol, weighing, carrying out ultrasonic treatment (power 500W and frequency 40 kHz) for 60min, cooling, weighing again, supplementing the lost weight with a corresponding solvent, shaking up, filtering, taking a subsequent filtrate, taking a proper amount of the subsequent filtrate, filtering through a 0.22 mu m microporous filter membrane, and placing in a liquid sample bottle to obtain a cynanchum wilfordii extract test solution.
Preparation of Dianshan Xiao decoction piece test sample solution
Taking about 1.0g of cynanchum wilfordii decoction pieces (batch number YP 2104-1), precisely weighing, placing in a conical flask with a plug, adding 25mL of 75% ethanol, weighing, performing ultrasonic treatment (power of 500W and frequency of 40 kHz) for 60min, cooling, weighing again, supplementing the lost weight with corresponding solvent, shaking up, filtering, taking out the filtrate, filtering with a 0.22 mu m microporous filter membrane, and placing in a liquid sample bottle to obtain the cynanchum wilfordii decoction piece test solution.
According to the optimized method, the HPLC chromatogram of the cynanchum wilfordii extract prepared in the first embodiment is shown in FIG. 1, and the mass content of p-hydroxyacetophenone is 0.68mg/g and the transfer rate of p-hydroxyacetophenone is 45.61% according to the chromatogram, the calculation formula of the mass content of p-hydroxyacetophenone and the calculation formula of the transfer rate.
2.2 examination of the methodology of the detection method
2.2.1 specialization examination
Respectively and precisely sucking the test sample solution of the cynanchum wilfordii extract prepared by the optimized conditions in the step 2.1,
The results of the assay of p-hydroxyacetophenone reference solution in 2.1.1 with 10. Mu.l of 75% ethanol using the 2.1.3 chromatographic conditions described above are shown in FIG. 2.
As can be seen from FIG. 2, the assay method is specific for determining the content of p-hydroxyacetophenone in the extract of radix cynanchi wilfordii.
2.2.2 Peak purity
Respectively and precisely sucking 10 μ l of radix cynanchi wilfordii extract test solution prepared under the optimized conditions in the step 2.1 and p-hydroxyacetophenone reference solution in the step 2.1.1, and measuring according to the chromatographic conditions in the step 2.1.3 to obtain the peak purity of the target peak, wherein the results are shown in a figure 2 and a table 7.
TABLE 7 matching of standard peaks and peak purities
| Target peak | P-hydroxyacetophenone |
| Peak purity match value | 999.63 |
As can be seen from fig. 2 and table 7: the purity value of the index component is 999.63 which is more than 999, which shows that the peak purity of the index component meets the analysis requirement.
2.2.3 Linear relationship investigation
Accurately weighing appropriate amount of p-hydroxyacetophenone reference substance, placing into numbered 10ml volumetric flasks, adding 75% ethanol to obtain reference substance solution containing 0.1mg of p-hydroxyacetophenone per 1ml, shaking up to obtain reference substance stock solution, and placing in a refrigerator for use.
Taking the reference substance stock solution of the p-hydroxyacetophenone, diluting the reference substance stock solution to 50, 20, 10, 5 and 2.5 times to obtain p-hydroxyacetophenone reference substance solutions with different concentrations, carrying out on-machine detection according to 2.1.3 chromatographic conditions, and inspecting the linear range of the p-hydroxyacetophenone by taking the concentration as a horizontal coordinate and the peak area value as a vertical coordinate. The results of the linear examination are shown in FIG. 3 and Table 8.
TABLE 8 Linear examination of p-hydroxyacetophenone
| Numbering | Concentration of p-hydroxyacetophenone (μ g/ml) | Peak area value (mAU) |
| P-hydroxyacetophenone-1 | 2.00 | 116.5100 |
| P-hydroxyacetophenone-2 | 5.00 | 311.8070 |
| P-hydroxyacetophenone-3 | 10.00 | 639.4815 |
| P-hydroxyacetophenone-4 | 20.00 | 1311.266 |
| P-hydroxyacetophenone-6 | 40.00 | 2575.004 |
As can be seen from fig. 3 and table 8, the concentration of p-hydroxyacetophenone in the range of 2.00 μ g/ml to 40.00 μ g/ml has a good linear relationship with the peak area value, and the correlation coefficient r =0.9998.
2.2.4 precision investigation
Taking the sample solution of the cynanchum wilfordii extract prepared under the optimized conditions in 2.1, paralleling 6 parts, injecting 2 samples for each part, injecting 10 mu L of the sample solution, injecting the sample solution into a high performance liquid chromatograph, carrying out chromatographic analysis according to the chromatographic conditions of 2.1.3, calculating the content of p-hydroxyacetophenone according to the method, and calculating the RSD (%) value of a target peak by an external standard one-point method according to the content of p-hydroxyacetophenone, wherein the specific results are shown in Table 9.
TABLE 9 precision experimental results of the content determination method of cynanchum wilfordii extracts
As is clear from Table 9, the RSD (%) of the target peak p-hydroxyacetophenone was 0.29 and less than 2.0%, indicating that the process was accurate.
2.2.5 stability Studies
Taking the cynanchum wilfordii extract prepared in the first embodiment, preparing a test solution of the cynanchum wilfordii extract according to the optimized conditions in 2.1, performing chromatographic analysis according to 2.1.3 chromatographic conditions, injecting samples of 10 mu l in volume after 0,2,4,6,8, 12 and 24 hours respectively after the test solution is prepared, calculating the content of p-hydroxyacetophenone according to the method, calculating the RSD (%) value of the target peak content by using an external standard point method according to the content of p-hydroxyacetophenone, and particularly, the results are shown in Table 10.
TABLE 10 stability study
As is clear from Table 10, the RSD (%) of the target peak p-hydroxyacetophenone content in 24 hours was 0.45% and less than 2.0%, indicating that the solution had good stability in 24 hours.
2.2.6 repeatability Studies
Taking 6 parts of the cynanchum wilfordii extract prepared in the first embodiment, preparing a cynanchum wilfordii extract test solution according to the optimized conditions in 2.1, and injecting the sample with the volume of 10 mu l. Chromatographic analysis was performed under the chromatographic conditions of 2.1.3, the content of p-hydroxyacetophenone was calculated according to the above method, and RSD (%) value of the target peak content was calculated by the external standard one-point method based on p-hydroxyacetophenone, and the detailed results are shown in table 11.
TABLE 11 results of repeated experiments
As can be seen from Table 11, the RSD (%) of the target peak to hydroxyacetophenone content was 0.70% and less than 2.0%, indicating that the reproducibility of the method was good.
2.2.7 intermediate precision investigation
Selecting 6 experimenters, operating under two different dates and two different chromatographs (Vochchchcht H-Class Plus and Agilent HPLC 1260 II) respectively, taking the sample solution of the cynanchum wilfordii extract prepared after optimizing the conditions in 2.1, carrying out chromatographic analysis according to 2.1.3 chromatographic conditions, calculating the content of the p-hydroxyacetophenone according to the method, and calculating the RSD (%) value of the target peak content by adopting an external standard one-point method based on the p-hydroxyacetophenone in the sample solution, wherein the specific results are detailed in Table 12.
TABLE 12 measurement methods intermediate precision experimental results
As is clear from Table 12, the intermediate precision RSD (%) of the target peak to hydroxyacetophenone content was 0.68% and both were less than 2.0%, indicating that the intermediate precision of the method was good.
2.2.8 accuracy test
Taking about 0.14g of radix cynanchi wilfordii extract with known content (the content of the p-hydroxyacetophenone is 651.3381 mu g/g), precisely weighing 6 parts, sequentially and precisely adding 1ml of a p-hydroxyacetophenone reference substance (the concentration is 45.6887 mu g/ml) prepared by 75% ethanol, precisely adding 24ml of 75% ethanol, weighing, carrying out ultrasonic treatment (the power is 500W and the frequency is 40 kHz) for 60min, cooling, weighing again, supplementing the lost weight by using 75% ethanol, shaking up, filtering, taking a subsequent filtrate, taking a proper amount of the subsequent filtrate, filtering through a 0.22 mu m microfiltration membrane, and placing in a liquid sample bottle to obtain the radix cynanchi wilfordii extract test solution. Separately and precisely sucking 10 μ L of each of a p-hydroxyacetophenone reference solution and a cynanchum wilfordii extract test solution, injecting the solutions into a high performance liquid chromatograph, performing chromatographic analysis under 2.1.3 chromatographic conditions, calculating the content of the p-hydroxyacetophenone according to the above method, calculating the content of a target peak by an external standard one-point method based on the p-hydroxyacetophenone, and calculating the recovery rate and the RSD (%) value according to the following formulas, wherein the results are shown in Table 13.
TABLE 13 content determination method sample application recovery test results
As can be seen from table 13: the recovery rate of the p-hydroxyacetophenone in the cynanchum wilfordii extract is within the range of 92-97%, and the RSD (1.56%) is less than 2.0%, which shows that the accuracy of the content measurement method is good.
2.2.9 durability examination
2.2.9.1 investigation of different chromatography columns
The influence of three chromatographic columns of Feilomen, agilent and Shimadzu on the chromatographic peak shape and the separation degree of the p-hydroxyacetophenone in the cynanchum wilfordii extract is compared.
Taking the test solution of the cynanchum wilfordii extract prepared under the optimized conditions in the step 2.1, determining according to the chromatographic conditions in the step 2.1.3, counting by p-hydroxyacetophenone, and recording chromatographic data. The results of the experiment are shown in fig. 4 and table 14.
TABLE 14 Effect of different chromatographic columns on the chromatographic peak of p-hydroxyacetophenone in cynanchum wilfordii extract
As can be seen from fig. 4 and the results in table 14, the influence of different color columns on the separation degree, peak shape and peak purity of p-hydroxyacetophenone in the cynanchum wilfordii extract is different, and the three brands of chromatographic columns can achieve good separation on the target peak. According to the parameters of the experimental result, the chromatographic column Feilomen SuperLu C18 is selected for the experiment.
2.2.9.2 investigation of different chromatographs
According to the existing equipment in a laboratory, selecting a Waters ultra-performance liquid chromatograph and an Agi lens high performance liquid chromatograph, and comparing the influence of the two chromatographs on the chromatographic peak shape and the separation degree of p-hydroxyacetophenone in the cynanchum wilfordii extracts.
Taking the test solution of the cynanchum wilfordii extract prepared under the optimized conditions in the step 2.1, determining according to the chromatographic conditions in the step 2.1.3, counting by p-hydroxyacetophenone, and recording chromatographic data. The results of the experiment are shown in fig. 5 and table 15.
TABLE 15 Instrument durability examination results
Note: the Watcht H-Class Plus Instrument software specifies that purity is satisfactory for analysis when the purity angle is less than the threshold purity.
As can be seen from fig. 5 and table 15, this analysis method is superior in the durability of the chromatograph. The variation of the chromatograph can meet the requirement of system adaptability.
2.2.9.3 investigation of different column temperatures
Taking the sample solution of the cynanchum wilfordii extract prepared under the optimized conditions in the step 2.1.3, determining according to chromatographic conditions in the step 2.1.3, and comparing the influence of different column temperatures of 30 ℃, 35 ℃ and 40 ℃ on the chromatographic peak shape and the separation degree of the p-hydroxyacetophenone in the cynanchum wilfordii extract. The results of the experiment are shown in fig. 6 and table 16.
TABLE 16 detection results of different column temperatures for the content of target peak in radix cynanchi wilfordii extract
| Column temperature/. Degree.C | Index component | Degree of separation/R | Number of theoretical plate | Value of purity |
| 30 | P-hydroxyacetophenone | 3.39 | 41074 | 999.44 |
| 35 | P-hydroxyacetophenone | 2.35 | 37655 | 998.07 |
| 40 | P-hydroxyacetophenone | 2.06 | 37348 | 997.92 |
As can be seen from fig. 6 and table 16, the peak shape and separation effect of the chromatographic peak at three columns are good, the base line of the chromatogram has no drift at 30 ℃, and the peak shape has no significant difference compared with other two temperatures. The column temperature was selected to be 30 ℃ in consideration of the tolerance of the column and the time required for analysis.
2.2.9.4 investigation of different flow rates
Taking the sample solution of the cynanchum wilfordii extract prepared under the optimized conditions in 2.1, and determining according to chromatographic conditions in 2.1.3, wherein the flow rates of the mobile phase are respectively selected from 0.9ml/min, 1.0ml/min and 1.2ml/min, and comparing the influences of different flow rates of 0.9ml/min, 1.0ml/min and 1.2ml/min on the chromatographic peak shape and the separation degree of the p-hydroxyacetophenone in the cynanchum wilfordii extract. The results of the experiment are shown in fig. 7 and table 17.
TABLE 17 results of peak detection in radix cynanchi wilfordii extract at different flow rates
As can be seen from fig. 7 and table 17, the chromatographic peak shape and the separation effect were good at the three flow rates. The parameters of p-hydroxyacetophenone are better when the flow rate is 1.0ml/min, the baseline does not drift, and the peak shape difference is not obvious, so the flow rate is 1.0ml/min in the experiment.
3. Detection method of characteristic spectrum of cynanchum wilfordii extract
3.1 optimization of the conditions of the detection method
3.1.1 preparation of reference solutions
Taking a proper amount of p-hydroxyacetophenone reference substance, precisely weighing, adding methanol to prepare a reference substance solution containing 100 mu g of p-hydroxyacetophenone per 1ml, and shaking up to obtain the product.
3.1.2 preparation of test solutions
Taking about 0.15g of the cynanchum wilfordii extract prepared in the first embodiment, precisely weighing, placing in a conical flask with a plug, adding 10ml of solvent, weighing, carrying out ultrasonic treatment (with the power of 500W and the frequency of 40 kHz) for 30min, cooling, weighing again, supplementing the lost weight with the corresponding solvent, shaking up, filtering, taking out the filtrate, taking out a proper amount of subsequent filtrate, filtering through a 0.22 mu m microporous filter membrane, and placing in a liquid sample bottle.
3.1.3 ultra high performance liquid chromatography
Respectively sucking 10 mu L of reference substance solution in 3.1.1 and test substance solution in 3.1.2, injecting into an ultra-high performance liquid chromatograph, and analyzing by using octadecylsilane chemically bonded silica as filler; gradient elution was performed as specified in table 18 with the organic phase as mobile phase a and the aqueous phase as mobile phase B; the measurement is carried out at a certain column temperature and flow rate at a certain detection wavelength.
TABLE 18 gradient elution
3.1.4 optimization of the conditions of the detection method
3.1.4.1 determination of the detection wavelength
Taking about 0.15g of the cynanchum wilfordii extract prepared in the first embodiment, precisely weighing, placing in a conical flask with a plug, adding 10ml of methanol, weighing, carrying out ultrasonic treatment (power 500W and frequency 40 kHz) for 30 minutes, cooling, weighing again, supplementing the lost weight with corresponding solvent, shaking up, filtering, taking out the filtrate, taking a proper amount of the subsequent filtrate, filtering through a 0.22 mu m microporous filter membrane, placing in a liquid sample bottle to obtain a cynanchum wilfordii extract sample solution, and recording the absorption spectrum within the range of 190-400nm as shown in figure 8.
As can be seen from fig. 8, at a wavelength of 270nm, the cynanchum wilfordii extract test sample solution can detect more chromatographic peak information and less baseline noise interference, and therefore 270nm is selected as the detection wavelength.
3.1.4.2 optimization of the Mobile phase
(1) Referring to the method of 3.1.3, the column was Dikma C18-a (2.1 × 150mm,1.8 μm); the column temperature is 30 ℃, the flow rate of the mobile phase is 0.3ml/min, the preparation method of the radix cynanchi wilfordii extract test solution in 3.1.4.1 is referred to, A is an organic phase (methanol or acetonitrile), B is a 0.05% phosphoric acid aqueous solution, the gradient elution table is shown in 19, and the result is shown in figure 9.
TABLE 19 gradient elution Table
| Time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0～20 | 5→100 | 95→0 |
| 20～20.1 | 100→5 | 0→95 |
| 20.1～22 | 5 | 95 |
As can be seen from FIG. 9, the baseline of the methanol-0.05% phosphoric acid solution is relatively flat and has a good peak profile, so the methanol-0.05% phosphoric acid solution was selected for conditional exploration, and then various acids used in the mobile phase were investigated.
(2) Referring to the procedure in 3.1.3, column is Dikma C18-a (2.1 x 150mm,1.8 μm); the column temperature was 30 deg.C, the flow rate of the mobile phase was 0.3ml/min, and the results of chromatographic analysis using methanol-water, methanol-0.05% phosphoric acid, methanol-0.1% formic acid and methanol-0.1% acetic acid as the mobile phases were shown in FIGS. 10-13, respectively, referring to the preparation method in 3.1.4.1 for the cynanchum wilfordii extract sample solution.
As can be seen from FIGS. 10 to 13, the acid addition peak pattern was better than that of the non-acid addition peak pattern, and the peak pattern was the best when the mobile phase was methanol-0.1% formic acid solution, so methanol-0.1% formic acid solution was selected for elution.
3.1.4.3 different column inspection
3 columns were investigated, respectively: column 1-Dikma C18-a (2.1 x 150mm,1.8 μm); chromatographic column 2-ACQUITY UPLC
T3 (2.1 x 150mm,1.8 μm); the influence of three chromatographic columns on the appearance of peaks of the characteristic spectrum of the cynanchum wilfordii extract was examined by using a chromatographic column 3-Shodex C18U 2D (2.1 x 150mm, 1.9m), a column temperature of 30 ℃, a mobile phase flow rate of 0.3ml/min, a mobile phase A of methanol, a mobile phase B of 0.1% formic acid solution and a cynanchum wilfordii extract test solution according to the preparation method in 3.1.4.1. The results are shown in fig. 14 and table 20.
TABLE 20 results of chromatography column investigation of the Bishan Xiaobiao decoction characteristics (relative retention time)
| | Peak | 1 | Peak 2 (S) | |
|
|
| 1 | 0.755 | 1.000 | 1.277 | 1.709 | 3.291 | |
| 2 | 0.753 | 1.000 | 1.320 | 1.578 | 2.859 | |
| 3 | 0.803 | 1.000 | 1.318 | 1.610 | 2.893 |
As can be seen from fig. 14 and table 20: the elution is carried out by using an ultra-high performance liquid chromatography column of a chromatographic column 1-Dikma C18-A (2.1X 150mm,1.8 mu m), the peak type of the chromatographic peak is better, and the separation effect is best, so that the method is recommended to use the ultra-high performance liquid chromatography column with the type of Dikma C18-A (2.1X 150mm,1.8 mu m).
3.1.4.4 investigation of different column temperatures
The preparation method of the test solution of radix cynanchi wilfordii extract is 3.1.4.1, wherein the mobile phase A is methanol, the mobile phase B is 0.1% formic acid solution, and the chromatographic column is Dikma C18-A (2.1 × 150mm,1.8 μm); the flow rate of the mobile phase was 0.3ml/min, and the results of comparing the separation effect on the sample at different column temperatures of 30 ℃, 35 ℃ and 40 ℃ are shown in FIG. 15 and Table 21.
TABLE 21 column temperature examination of the extract of cynanchum wilfordii (cynanchum wilfordii) on the characteristic spectrum (relative retention time)
| | Peak | 1 | Peak 2 (S) | |
|
|
| 25℃ | 0.762 | 1.000 | 1.274 | 1.707 | 3.147 | |
| 30℃ | 0.757 | 1.000 | 1.273 | 1.706 | 3.268 | |
| 35℃ | 0.753 | 1.000 | 1.273 | 1.702 | 3.387 |
As can be seen from fig. 15 and table 21, the column temperature was selected to be 30 ℃ because the column temperature had a small influence on the peak appearance, but the separation effect was best at 30 ℃ and the peak type was good.
3.1.4.5 different flow Rate Observation
Referring to the preparation method of 3.1.4.1, the sample solution of radix cynanchi wilfordii extract is prepared by using methanol as mobile phase A,0.1% formic acid as mobile phase B, and Dikma C18-A (2.1 × 150mm,1.8 μm) as chromatographic column; the column temperature was 30 ℃ and elution was examined at different flow rates (0.2 ml/min, 0.25ml/min, 0.3 ml/min). The results are shown in FIG. 16 and Table 22.
TABLE 22 Mount Peng Xiao extract feature map flow Rate investigation results (relative Retention time)
| Flow rate of | Peak | 1 | Peak 2 (S) | |
|
|
| 0.2ml/min | 0.757 | 1.000 | 1.229 | 1.608 | 2.524 | |
| 0.25ml/min | 0.755 | 1.000 | 1.260 | 1.667 | 2.897 | |
| 0.3ml/min | 0.757 | 1.000 | 1.273 | 1.706 | 3.268 |
As can be seen from FIG. 16 and Table 22, the flow rate has a small influence on the appearance of the peak, but the peak pattern is best when the flow rate is 0.3ml/min. Therefore, 0.3ml/min was selected.
3.1.4.6 investigation of different extraction solvents
Taking about 0.14g of the cynanchum wilfordii extract prepared in the first embodiment, precisely weighing, respectively placing 2 samples in parallel in conical flasks with stoppers, precisely adding 10ml of water, 50% of ethanol, 75% of ethanol, 95% of ethanol, 50% of methanol, 75% of methanol and methanol respectively, weighing, carrying out ultrasonic treatment (power 500W and frequency 40 kHz) for 30min, cooling, weighing again, complementing the lost weight with corresponding solvent, shaking up, filtering, and taking subsequent filtrate to obtain each sample solution. Injecting according to the method in 3.1.3, wherein the chromatographic conditions are as follows: mobile phase a was methanol, mobile phase B was 0.1% formic acid solution, and column chromatography was Dikma C18-a (2.1 x 150mm,1.8 μm); the column temperature was 30 ℃ and the flow rate was 0.3ml/min, and the peak area was recorded. The results are shown in Table 23.
TABLE 23 comparison of extraction efficiency (peak area/sample weight) for different extraction modes
| Feature(s) | Water (I) | 95 |
75 |
95 | Methanol | 75 |
50 | |
| Peak | ||||||||
| 1 | 4882 | 97098 | 97967 | 85833 | 98437 | 89825 | 96378 | |
| |
229345 | 261337 | 268827 | 226231 | 273595 | 249186 | 267176 | |
| |
11629 | 17123 | 17169 | 14788 | 17489 | 16136 | 16740 | |
| |
39738 | 62989 | 65617 | 52861 | 67314 | 59867 | 63295 | |
| |
15471 | 18481 | 19632 | 15538 | 20014 | 18094 | 19412 |
As is clear from table 23, methanol was selected as the extraction solvent because the extraction solvent was methanol and the extraction efficiency was high.
The optimized conditions are adopted to determine the characteristic spectrum of the cynanchum wilfordii extract prepared in the first embodiment, and the method comprises the following steps:
preparation of test solution
Taking about 0.15g of the cynanchum wilfordii extract prepared in the first embodiment, precisely weighing, placing in a conical flask with a plug, adding 10ml of methanol, weighing, carrying out ultrasonic treatment (with the power of 500W and the frequency of 40 kHz) for 30min, cooling, weighing again, complementing the weight loss by using a corresponding solvent, shaking up, filtering, taking out the filtrate, taking a proper amount of subsequent filtrate, filtering through a 0.22 mu m microporous filter membrane, and placing in a liquid sample bottle to obtain a cynanchum wilfordii extract sample solution.
Respectively sucking 10 μ L3.1.1 reference solution and the sample solution, injecting into an ultra high performance liquid chromatograph, and analyzing with octadecylsilane chemically bonded silica as filler (Dikma C18-A,2.1 × 150mm,1.8 μm); methanol was used as the mobile phase a, and a 0.1% formic acid aqueous solution was used as the mobile phase B, and gradient elution was performed as specified in table 18; the column temperature was 30 ℃ and the flow rate was 0.3ml/min. The results are shown in FIG. 17.
As can be seen from fig. 17, there are 5 characteristic peaks in the characteristic map of the cynanchum wilfordii extract, the peak corresponding to the reference is the S peak, and the relative retention time of each characteristic peak and the S peak is calculated, wherein the relative retention time of peak 1 is 0.76, the relative retention time of peak 2 (S) is 1.00, the relative retention time of peak 3 is 1.27, the relative retention time of peak 4 is 1.71, and the relative retention time of peak 5 is 3.28.
3.2 investigation of the methodology of the detection method
3.2.1 specialization examination
Respectively sucking 1 μ l of the radix cynanchi wilfordii extract test solution prepared under the optimized conditions in 3.1 and methanol solvent, injecting into a liquid chromatograph, and measuring according to the chromatographic conditions after the optimized conditions in 3.1. The results are shown in FIG. 18.
As can be seen from FIG. 18, the solvent did not interfere with the characteristic peaks in the extract spectrum of cynanchum wilfordii.
3.2.2 Overall examination
Sucking 1 μ l of radix cynanchi wilfordii extract test solution prepared under the optimized conditions in 3.1, measuring according to the chromatographic conditions after the optimized conditions in 3.1, prolonging the elution time by one time under the condition of a flow phase ratio at a gradient end point, and showing a characteristic map as shown in figure 19.
As can be seen from fig. 19, the chromatographic condition showed no significant peak after one-fold extension of the elution time, indicating that the chromatographic condition substantially satisfied the principle of maximum information content.
3.2.3 precision investigation
Sucking the test solution of the cynanchum wilfordii extract prepared under the optimized conditions in 3.1, measuring according to the chromatographic conditions under the optimized conditions in 3.1, and repeatedly injecting samples for 6 times, wherein the injection amount is 1ul. The relative retention time of the characteristic peaks and the consistency of the relative peak areas (5 characteristic peaks were calibrated, and peak 2 was used as a reference peak) were examined, and the results are shown in table 24.
TABLE 24 results of precision of the extract profile (relative retention time)
| |
1 | 2 | 3 | 4 | 5 | 6 | Mean value of | |
| Peak | ||||||||
| 1 | 0.756 | 0.755 | 0.755 | 0.755 | 0.756 | 0.756 | 0.756 | 0.06 |
| Peak 2 (S) | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 0.00 |
| |
1.273 | 1.274 | 1.275 | 1.274 | 1.274 | 1.274 | 1.274 | 0.04 |
| |
1.703 | 1.706 | 1.706 | 1.706 | 1.706 | 1.707 | 1.706 | 0.08 |
| |
3.274 | 3.286 | 3.285 | 3.293 | 3.292 | 3.278 | 3.285 | 0.22 |
As is clear from Table 24, the RSD of each peak with respect to retention time was less than 3.0%, indicating that the precision of the instrument is good.
3.2.4 stability Studies
Taking the cynanchum wilfordii extract prepared in the first example, preparing a test solution of the cynanchum wilfordii extract according to the optimized conditions in 3.1, measuring according to the chromatographic conditions after the optimized conditions in 3.1, injecting samples in a volume of 1 mu l at 0,2,4,6,8, 12 and 24 hours after the test solution is prepared, and examining the relative retention time of characteristic peaks and the consistency of relative peak areas (calibrating 5 characteristic peaks and taking the peak 2 as a reference peak), wherein the experimental results are shown in table 25.
TABLE 25 Mount Pengshanxiao extract feature map stability results (relative retention time)
| |
1 | 2 | 3 | 4 | 5 | 6 | Mean value of | RSD/ |
| Peak | ||||||||
| 1 | 0.758 | 0.760 | 0.760 | 0.759 | 0.757 | 0.758 | 0.759 | 0.15 |
| Peak 2 (S) | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 0.00 |
| |
1.273 | 1.274 | 1.273 | 1.274 | 1.273 | 1.274 | 1.274 | 0.04 |
| |
1.707 | 1.705 | 1.704 | 1.701 | 1.703 | 1.706 | 1.704 | 0.13 |
| |
3.235 | 3.223 | 3.220 | 3.223 | 3.265 | 3.239 | 3.233 | 0.58 |
As can be seen from Table 25, the RSD of the chromatographic peak versus retention time is <3.0%, indicating that the sample solution is relatively stable.
3.2.5 repeatability Studies
Taking 6 parts of the cynanchum wilfordii extract prepared in the first example, preparing a test solution under the optimized conditions in 3.1, measuring according to the chromatographic conditions after the optimized conditions in 3.1, injecting 1 microliter of sample volume, and examining the relative retention time of characteristic peaks and the consistency of relative peak areas (calibrating 5 characteristic peaks, taking the peak 2 as a reference peak), wherein the experimental results are shown in table 26.
TABLE 26 repeatability results (relative retention time) of the extract profile of cynanchum wilfordii
| |
1 | 2 | 3 | 4 | 5 | 6 | Mean value of | |
| Peak | ||||||||
| 1 | 0.759 | 0.758 | 0.759 | 0.758 | 0.758 | 0.758 | 0.758 | 0.04 |
| Peak 2 (S) | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 0.00 |
| |
1.273 | 1.273 | 1.273 | 1.273 | 1.273 | 1.273 | 1.273 | 0.03 |
| |
1.706 | 1.708 | 1.707 | 1.708 | 1.708 | 1.707 | 1.707 | 0.05 |
| |
3.236 | 3.244 | 3.244 | 3.243 | 3.244 | 3.240 | 3.242 | 0.11 |
As can be seen from Table 26, the RSD of each peak with respect to retention time was <3.0%, indicating that the method is highly reproducible.
Example two
1. Preparation method of cynanchum wilfordii extract
(1) Taking 100g of cynanchum wilfordii decoction pieces (YP 2104-2), putting the cynanchum wilfordii decoction pieces in an electric ceramic pot, adding water for decocting twice, directly adding the first decoction into water 12 times the weight of the cynanchum wilfordii decoction pieces, soaking for 30 minutes, boiling with strong fire (500W), keeping slight boiling with slow fire (200W) for 35 minutes, and filtering the decoction while hot by using a 300-mesh screen; adding water 10 times the weight of radix cynanchi wilfordii decoction pieces, heating with strong fire, boiling, maintaining slight boiling with slow fire for 25 min, filtering the decoction with 300-mesh screen while it is hot, and mixing the filtrates;
(2) Transferring the filtrate into a 2000ml round-bottom flask, concentrating the filtrate to 100ml of extract at low temperature under reduced pressure by adopting a rotary evaporator (the temperature is 65 ℃, the vacuum degree is-0.080 to-0.090 MPa), and measuring the density of the extract to be 1.07g/ml and the extract yield to be 11.2 percent; under magnetic stirring, subpackaging into 10ml brown penicillin bottles, wherein the subpackaging volume of each bottle is 1ml, half plugging, transferring to a vacuum freeze dryer for freeze-drying after subpackaging, pre-freezing at-55 ℃ for 2 hours, then carrying out primary drying at-45 ℃, 35 ℃,30 ℃, 25 ℃,20 ℃,10 ℃ and 0 ℃ under the vacuum degree of 0.2mbar for 3 hours, 7 hours, 20 hours, 10 hours, 2 hours, 1 hour and 2 hours respectively, then carrying out secondary drying at 10 ℃,20 ℃ and 30 ℃ under the vacuum degree of 0mbar for 1 hour, 1 hour and 5 hours respectively, taking out, and rolling an aluminum cover to obtain the cynanchum wilfordii extract.
2. Detection of mass content and transfer rate of p-hydroxyacetophenone
The method in the first example is adopted to measure that the mass content of the p-hydroxyacetophenone in the cynanchum wilfordii hook f extract is 0.58mg/g, and the transfer rate of the p-hydroxyacetophenone is 38.41%.
3. Determination method of characteristic spectrum of cynanchum wilfordii extract
The profile of the cynanchum wilfordii extract was determined according to the test method shown in example one and comprised five peaks, peak 1 having a relative retention time of 0.76, peak 2 (S) having a relative retention time of 1.00, peak 3 having a relative retention time of 1.27, peak 4 having a relative retention time of 1.71 and peak 5 having a relative retention time of 3.28.
EXAMPLE III
1. Preparation method of cynanchum wilfordii hemsl extract
(1) Taking 100g of cynanchum wilfordii decoction pieces (YP 2104-2), putting the cynanchum wilfordii decoction pieces in an electric ceramic pot, adding water for decocting twice, directly adding the first decoction pieces into water 11 times of the weight of the cynanchum wilfordii decoction pieces, soaking for 30 minutes, boiling with strong fire (500W), keeping slight boiling with slow fire (200W) for 55 minutes, and filtering the decoction while the decoction is hot through a 300-mesh screen; adding water 9 times the weight of radix cynanchi wilfordii decoction pieces in the second decoction, heating with strong fire, boiling, keeping slightly boiling with slow fire for 40 minutes, filtering the decoction while hot by using a 300-mesh screen, and combining the two filtrates;
(2) Transferring the filtrate into a 2000ml round-bottom flask, concentrating the filtrate at low temperature under reduced pressure by a rotary evaporator (the temperature is 65 ℃, the vacuum degree is-0.080 to-0.090 MPa) to 100ml of extract, and measuring the density of the extract to be 1.06 and the yield of the extract to be 12.3 percent; under magnetic stirring, subpackaging into 10ml brown penicillin bottles, wherein the subpackaging volume of each bottle is 1ml, half plugging is performed, after subpackaging, transferring into a vacuum freeze dryer for freeze-drying, prefreezing is performed firstly, the prefreezing temperature is-50 ℃, the prefreezing time is 5 hours, then primary drying is performed, the drying temperature is-45 ℃, 35 ℃,30 ℃, 25 ℃,20 ℃,10 ℃ and 0 ℃, the vacuum degree is 0.2mbar, the drying time is 2 hours, 6 hours, 16 hours, 8 hours, 2 hours and 1 hour respectively, secondary drying is performed, the drying temperature is 10 ℃,20 ℃ and 30 ℃, the vacuum degree is 0mbar, the drying time is 1 hour, 1 hour and 3 hours respectively, taking out and rolling an aluminum cover to obtain the cynanchum wilfordii extract.
2. Detection of mass content and transfer rate of p-hydroxyacetophenone
The detection method in example 1 was used to determine that the mass content of p-hydroxyacetophenone was 0.52mg/g and the transfer rate of p-hydroxyacetophenone was 31.23%.
3. Determination method of characteristic spectrum of cynanchum wilfordii extract
The characterization pattern of the cynanchum wilfordii extract was determined according to the detection method in example one and comprised five peaks, respectively, with a relative retention time of 0.76 for peak 1, 1.00 for peak 2 (S), 1.27 for peak 3, 1.71 for peak 4 and 3.28 for peak 5.
Example determination of four common peaks
Determining ultra-high performance liquid chromatograms of 15 batches of cynanchum wilfordii extract samples and cynanchum wilfordii reference medicinal materials according to the detection method of the characteristic spectrum of the cynanchum wilfordii extract after optimizing the conditions in the embodiment I, wherein the preparation method of the test solution of the cynanchum wilfordii reference medicinal materials comprises the following steps: taking about 1.0g of the cynanchum wilfordii contrast medicinal material (the batch number is BT 2104-1), precisely weighing, placing in a conical flask with a plug, precisely adding 10ml of methanol, weighing, carrying out ultrasonic treatment (the power is 500W, the frequency is 40 kHz) for 30 minutes, cooling, weighing again, supplementing the lost weight with the methanol, shaking up, filtering, taking a subsequent filtrate, taking a proper amount of the subsequent filtrate, passing through a 0.22 mu m microporous membrane, and placing in a liquid sample bottle to obtain a test solution of the cynanchum wilfordii contrast medicinal material.
Results were analyzed using the "traditional Chinese medicine chromatography fingerprint similarity evaluation system (2012 edition)" recommended by the national pharmacopoeia committee, and common peaks were selected as shown in fig. 20 and table 27.
TABLE 27 results of sample measurement
As can be seen from fig. 20 and table 27, the sample chromatogram of cynanchum wilfordii shows 5 characteristic peaks, and the retention time of the 5 characteristic peaks in the sample chromatogram of the reference drug corresponds to the retention time of the sample chromatogram of cynanchum wilfordii; the peak 2 is taken as a reference peak, the relative retention time RSD values of the other 4 characteristic peaks of the characteristic spectrum of 15 batches of cynanchum wilfordii extracts are 0.05-0.22 percent and are all less than 1.0 percent, and the standard requirements of the characteristic spectrum of the cynanchum wilfordii extracts are met.
EXAMPLE five
1. Preparation method of jieshanxiao formula granules
(1) Collecting 30kg radix cynanchi wilfordii decoction pieces (YP 2104-1), placing in a multifunctional extraction and concentration machine set, decocting with water twice, adding into 12 times of water, soaking for 30min, heating to boil, keeping boiling for 55min, and filtering the decoction with 200 mesh screen while it is hot; adding 10 times of water, boiling, keeping boiling for 40min, filtering the decoction while it is hot with 200 mesh sieve, and mixing the filtrates;
(2) Transferring the filtrate into a thickener, and concentrating under reduced pressure and low temperature (temperature: 65 ℃, vacuum degree: minus 0.080-0.090 MPa) to density of 1.05g/cm3 (65 ℃) to obtain fluid extract, wherein the measured paste yield is 12%;
(3) Adding the fluid extract into a mixing tank, heating to 60 deg.C, adding 3kg maltodextrin, mixing, spray drying, controlling air inlet temperature at 165-195 deg.C and air outlet temperature at 100-110 deg.C, collecting dry extract powder, and drying without wall sticking, agglomeration or caking;
(4) Weighing 500g of the dry extract powder obtained in the step (3), crushing, sieving with a 80-mesh sieve, adding 0.5g of silicon dioxide and 0.5g of magnesium stearate, uniformly mixing, granulating by a dry method, adjusting the feeding speed to 90r/min, the rotating speed of a roller pressing wheel to be 10r/min, the hydraulic pressure to be 8MPa, the rotating speed of the pressing wheel to be 7r/min, the crushing rotating speed to be 100r/min, the final finishing rotating speed to be 80r/min, the distance between the pressing wheels to be 1mm, mixing for 30min, and the rotating speed of a mixer to be 10r/min, thus preparing the cynanchum wilfordii formula granules-1.
2. Method for detecting mass content and transfer rate of p-hydroxyacetophenone in cynanchum wilfordii prescription granule
2.1 preparation of reference solutions
Taking a proper amount of p-hydroxyacetophenone reference substance, precisely weighing, placing into a numbered 10ml volumetric flask, adding 75% ethanol to prepare a reference substance solution containing 10 microgram of p-hydroxyacetophenone per 1ml, and shaking up to obtain the p-hydroxyacetophenone.
2.2 preparation of test solutions
Taking about 0.12g of the cynanchum wilfordii formula particle prepared in the fifth embodiment, precisely weighing, placing in a conical flask with a plug, adding 25mL of 75% ethanol, weighing, carrying out ultrasonic treatment (with the power of 500W and the frequency of 40 kHz) for 60min, cooling, weighing again, complementing the weight loss by using a corresponding solvent, shaking up, filtering, taking out the filtrate, filtering through a 0.22 mu m microporous filter membrane, and placing in a liquid sample bottle to obtain a sample solution.
2.3 high Performance liquid chromatography
Octadecylsilane chemically bonded silica is used as a filler (Philomen SuperLu C18, column length of 250mm, inner diameter of 4.6mm, particle size of 5.0 μm); acetonitrile was used as mobile phase a, and 0.1% phosphoric acid aqueous solution was used as mobile phase B, and gradient elution was performed as specified in table 28; the column temperature is 30 ℃; the flow rate was 1.0ml/min and the detection wavelength was 276nm. The number of theoretical plates is not less than 10000 calculated according to the peak of p-hydroxyacetophenone. 10 μ L of the reference solution in 2.1 and the sample solution in 2.2 were respectively aspirated and injected into a high performance liquid chromatograph for analysis.
TABLE 28 elution gradient
| Time (minutes) | Mobile phase A (%) | Mobile phase B (%) |
| 0-15 | 10→21 | 90→79 |
| 15-18 | 21→21 | 79→79 |
| 18-23 | 21→90 | 79→10 |
| 23-27 | 90→10 | 10→90 |
| 27-30 | 10→10 | 90→90 |
The mass content of the p-hydroxyacetophenone measured according to the method in the first example was 0.48mg/g, and the transfer rate of the p-hydroxyacetophenone was 27.99%.
3. Detection method of characteristic spectrum of radix cynanchi wilfordii formula granules
3.1 preparation of reference solutions
Taking a proper amount of p-hydroxyacetophenone reference substance, precisely weighing, adding methanol to prepare a reference substance solution containing 100 mu g of p-hydroxyacetophenone per 1ml, and shaking up to obtain the product.
3.2 preparation of test solutions
Taking about 0.25g of the cynanchum wilfordii formula particle prepared in the fifth embodiment, precisely weighing, placing in a conical flask with a plug, precisely adding 10ml of methanol, weighing, carrying out ultrasonic treatment (with the power of 500W and the frequency of 40 kHz) for 30 minutes, cooling, weighing again, complementing the weight loss with methanol, shaking up, filtering, and taking out the filtrate to obtain the cynanchum wilfordii formula particle.
3.3 high Performance liquid chromatography
Octadecylsilane chemically bonded silica is used as a filler (the column length is 150mm, the inner diameter is 2.1mm, and the particle size is 1.8 mu m); gradient elution was performed as specified in table 43 using methanol as mobile phase a and 0.1% formic acid solution as mobile phase B; the flow rate is 0.3ml/min; the column temperature is 30 ℃; the detection wavelength was 270nm. 10 μ L of the reference solution in 3.1 and the test solution in 3.2 are respectively sucked and injected into an ultra high performance liquid chromatograph for analysis.
TABLE 29 gradient elution
| Time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0-7 | 23→24 | 77→76 |
| 7-11 | 24→27 | 76→73 |
| 11-14 | 27→29 | 73→71 |
| 14-18 | 29→32 | 71→68 |
| 18-20 | 32→38 | 68→62 |
| 20-28 | 38→38 | 62→62 |
| 28-28.1 | 38→23 | 62→77 |
| 28.1-30 | 23→23 | 77→77 |
The characteristic profile of the cynanchum wilfordii granules measured according to the above method is shown in fig. 21, which comprises five peaks, namely peak 1 having a relative retention time of 0.76, peak 2 (S) having a relative retention time of 1.00, peak 3 having a relative retention time of 1.27, peak 4 having a relative retention time of 1.71, and peak 5 having a relative retention time of 3.28.
4. Determination of angle of repose of jieshan Xiao formula granules
Measuring by adopting a fixed funnel method, taking clean coordinate paper, flatly paving the coordinate paper on a table top, fixing a funnel on an iron stand, keeping the funnel horizontal to the table top, and keeping the bottom of the funnel at a certain height (H) away from the coordinate paper; slowly pouring the test sample particles into the funnel until the tip of the particles contacts the funnel, forming a cone with the radius r by the test sample particles, and calculating the angle of repose: angle of repose = arctan (H/r)
The angle of repose of the cynanchum wilfordii formula granules was measured to be 23.1 ° according to the above method.
Example six
1. Preparation method of jieshanxiao formula granules
(1) Preparing clear paste according to the method in the fifth embodiment;
(2) Adding the above fluid extract into a mixing tank, heating to 100 deg.C, adding 6kg maltodextrin, mixing, spray drying, controlling air inlet temperature at 165-195 deg.C and air outlet temperature at 100-110 deg.C, and collecting dry extract powder; the drying process has no wall sticking phenomenon and no caking phenomenon, and is easy to dry;
(3) Weighing 500g of dry extract powder obtained in the step (3), crushing, sieving by a 80-mesh sieve, adding 1.5g of silicon dioxide and 1.5g of magnesium stearate, uniformly mixing, granulating by a dry method, adjusting the feeding speed to 90r/min, the rotating speed of a roller pressing wheel to be 10r/min, the hydraulic pressure to be 8MPa, the rotating speed of the pressing wheel to be 7r/min, the crushing rotating speed to be 100r/min, the final grading rotating speed to be 80r/min, the distance between the pressing wheels to be 1mm, mixing time to be 30min and the rotating speed of a mixer to be 10r/min.
The angle of repose was determined to be 22.8 ° according to the method in example five.
EXAMPLE seven
1. Identification of jieshanxiao prescription granule
1.1 preparation of test solutions
Selecting different batches of cynanchum wilfordii decoction pieces (batch numbers YP2104-2 and YP 2104-3), preparing cynanchum wilfordii formula particles-2 and cynanchum wilfordii formula particles-3 according to the method of the fifth embodiment, respectively weighing 3 parts of cynanchum wilfordii formula particles-1, cynanchum wilfordii formula particles-2 and cynanchum wilfordii formula particles-3, grinding, adding 10mL of water for dissolving, filtering, adding onto a macroporous resin column (D101, the outer diameter is 20 mm), sequentially eluting with 20mL of water and 20mL of 20% of ethanol, discarding the eluent, eluting with 20mL of 80% of ethanol, collecting the eluent, evaporating to dryness, collecting residues, adding 2mL of methanol for dissolving, and using the residues as a sample solution.
1.2 control solutions of radix cynanchi wilfordii
Taking 1g radix cynanchi wilfordii decoction pieces (YP 2104-1), adding 20mL of water, heating and refluxing for 30min, filtering, adding onto a macroporous resin column (D101, 20mm in outer diameter), sequentially eluting with 20mL of water and 20mL of 20% ethanol, discarding the eluate, eluting with 20mL of 80% ethanol, collecting the eluate, evaporating to dryness, collecting the residue, and dissolving with 2mL of methanol to obtain radix cynanchi wilfordii reference substance solution.
1.3 negative sample solution of Dingshan Xiao prescription granule
Weighing 1g of maltodextrin, grinding, adding 10mL of water for dissolving, filtering, adding the mixture onto a macroporous resin column (D101, the outer diameter is 20 mm), sequentially eluting with 20mL of water and 20mL of 20% ethanol, discarding the eluent, eluting with 20mL of 80% ethanol, collecting the eluent, evaporating to dryness, collecting residues, adding 2mL of methanol for dissolving, and taking the solution as a negative sample solution.
1.4 preparation of color-developing agent
Dissolving vanillin 0.5g in 98% sulfuric acid 10 mL.
1.5 thin layer chromatography conditions
The thin layer plate is a silica gel G thin layer plate;
the developing solvent is toluene-ethyl acetate-methanol-water (volume ratio is 20;
the sample amount is 10uL;
the unfolding mode is as follows: a double-groove unfolding cylinder is adopted, the unfolding distance is 8cm, and the unfolding is carried out twice;
and (6) inspection: spraying 5% vanillin sulfuric acid solution, heating to 105 deg.C to spot color, inspecting in sunlight, respectively sucking 10 μ l of each of 1.1 sample solution, 1.2 radix cynanchi wilfordii control solution and 1.3 radix cynanchi wilfordii negative sample on the same silica gel G thin layer plate, developing according to the above thin layer chromatography, taking out, air drying, spraying 5% vanillin sulfuric acid solution, heating to 105 deg.C to spot color, and inspecting in sunlight to obtain thin layer chromatogram as shown in FIG. 22.
As can be seen from fig. 22, the chromatograms of different batches of cynanchum wilfordii formula particles prepared by the method in example five all have spots with the same color at the corresponding positions of the chromatograms of the cynanchum wilfordii reference medicinal materials, which indicates that the thin-layer identification of the cynanchum wilfordii formula particles meets the specification.
Comparative example 1
The difference from example five is that, in the dry granulation, without adding silicon dioxide and magnesium stearate, the angle of repose was measured to be 25.4 ° according to the method of example five.
In summary, the method comprises the steps of decocting cynanchum wilfordii with water, filtering to obtain filtrate, concentrating and drying the filtrate to obtain cynanchum wilfordii extract, wherein the cream yield of the cynanchum wilfordii extract is 11.2-14.64%, and the mass content and transfer rate of p-hydroxyacetophenone are detected by using high performance liquid chromatography analysis on the cynanchum wilfordii extract, and the result is as follows: the mass content of the p-hydroxyacetophenone is 0.52-0.68mg/g, and the transfer rate of the p-hydroxyacetophenone is 31.23-45.61%. And the adopted detection method has high precision and good repeatability, and the stability of the test solution is good within 24 hours.
The invention adopts ultra-high performance liquid chromatography to determine the characteristic spectrum of 15 batches of cynanchum wilfordii extracts and the characteristic spectrum of a reference medicinal material, wherein the cynanchum wilfordii extracts contain 5 characteristic peaks, and the retention time of the 5 characteristic peaks in the reference substance chromatogram of the reference medicinal material corresponds to that of the 5 characteristic peaks, and respectively is that the relative retention time of a peak 1 is 0.76, the relative retention time of a peak 2 (S) is 1.00, the relative retention time of a peak 3 is 1.27, the relative retention time of a peak 4 is 1.71 and the relative retention time of a peak 5 is 3.28. The relative deviation of the relative retention time of each characteristic peak is within 1%. The detection method has high precision and good repeatability, and the stability of the test solution is good within 24 hours.
Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, while the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.
Claims (24)
1. The preparation method of the cynanchum wilfordii extract is characterized by comprising the following steps:
(1) Decocting radix cynanchi wilfordii decoction pieces in water, and filtering to obtain filtrate; wherein the water decocting times are 2 times, 10-12 times of water by weight of cynanchum wilfordii decoction pieces is added in the first water decoction, 8-10 times of water by weight of cynanchum wilfordii decoction pieces is added in the second water decoction, the first water decoction time is 35-55 min, and the second water decoction time is 25-40 min;
(2) Concentrating the filtrate obtained in the step (1) and drying to obtain the cynanchum wilfordii extract.
2. The method according to claim 1, wherein in the step (2), the temperature for concentrating the filtrate is 60 to 65 ℃; preferably, the filtrate is concentrated until the density of the concentrated solution is 1.05-1.12 g/mL.
3. The preparation method according to claim 1 or 2, wherein the cynanchum wilfordii extract has an extract yield of 7.1-21.1%.
4. The method of any one of claims 1 to 3, wherein the drying comprises one of freeze drying, vacuum drying, spray drying, microwave drying, and infrared drying.
5. The method of claim 4, wherein the drying is freeze-drying, which is divided into three stages: a. pre-freezing: the pre-freezing temperature is-55 to-50 ℃; b. primary drying: the drying temperature is minus 45 ℃ to 0 ℃; c. and (3) secondary drying for analysis: the drying temperature is 10-30 ℃; preferably, the prefreezing time is 2 to 5 hours, preferably 3 hours; the primary drying time is 35 to 45 hours, preferably 38 hours; the secondary drying time is 5 to 7 hours, preferably 6 hours; more preferably, the degree of vacuum of the primary drying is 0 to 0.2mbar.
6. The preparation method according to claim 4, wherein the drying is spray drying, the air inlet temperature of the spray drying is 165-195 ℃, and the air outlet temperature of the spray drying is 100-110 ℃; preferably, the spray drying process further comprises the step of mixing the cynanchum wilfordii extract and maltodextrin; more preferably, the mixing temperature is 60 to 100 ℃; further preferably, the addition amount of the maltodextrin is 5-20% by weight of the cynanchum wilfordii decoction pieces.
7. An cynanchum wilfordii extract, which is prepared by the preparation method of any one of claims 1 to 6; preferably, the mass content of the p-hydroxyacetophenone in the cynanchum wilfordii extract is 0.25-0.98mg/g in terms of a dry product; more preferably, the transfer rate of p-hydroxyacetophenone is 22.58 to 83.92% by weight.
8. The method for identifying cynanchum wilfordii extract according to claim 7, which comprises identifying the cynanchum wilfordii extract by thin layer chromatography; the thin layer chromatography comprises the following steps: a. preparing a test solution of cynanchum wilfordii extract and a reference solution of cynanchum wilfordii; b. respectively dropping the radix cynanchi wilfordii extract test solution and radix cynanchi wilfordii reference solution on the same silica gel G thin layer plate, adding developing agent for development, taking out, air drying, spraying color-developing agent to develop spots, and detecting;
preferably, the test solution of the cynanchum wilfordii extract is prepared by the following steps of taking 1-3g of the cynanchum wilfordii extract, adding water for dissolving, eluting after ion exchange to obtain eluent, evaporating to dryness to obtain residue, and dissolving the residue with methanol to obtain the cynanchum wilfordii extract; preferably, the cynanchum wilfordii reference substance solution is prepared by the following steps of taking 1-3g of cynanchum wilfordii medicinal material, adding water, heating, refluxing, filtering, eluting after ion exchange to obtain eluent, evaporating to dryness to obtain residue, and dissolving the residue with methanol to obtain the cynanchum wilfordii reference substance solution;
more preferably, the developing solvent is toluene-ethyl acetate-methanol-water, and further preferably, the volume ratio of the toluene to the ethyl acetate to the methanol to the water is 20.
9. The method for detecting the mass content of the p-hydroxyacetophenone in the cynanchum wilfordii extract as claimed in claim 7, which is characterized by comprising the following steps:
(1) Preparation of reference solutions
Weighing p-hydroxyacetophenone reference substance, and adding a solvent to prepare a reference substance solution;
(2) Preparation of test solution
Weighing cynanchum wilfordii extract, and adding a solvent for extraction;
(3) High performance liquid chromatography
Using octadecylsilane chemically bonded silica as a filler, using an organic solvent as a mobile phase A, using a 0.1% phosphoric acid aqueous solution as a mobile phase B, carrying out gradient elution, and sucking a reference substance solution and a test solution to inject into a high performance liquid chromatograph for analysis.
10. The detection method according to claim 9, wherein the solvent in step (1) is one or more selected from the group consisting of 50% ethanol, 75% ethanol, 95% ethanol, 50% methanol, 75% methanol, and methanol, preferably 75% ethanol; preferably, the solvent in step (2) is one or more selected from 50% ethanol, 75% ethanol, 95% ethanol, 50% methanol, 75% methanol and methanol, preferably 75% ethanol.
11. The detection method according to claim 9 or 10, wherein the extraction in step (2) is performed by one of reflux, shaking or ultrasound, preferably ultrasound.
12. The detection method according to any one of claims 9 to 11, wherein the extraction time in step (2) is 15 to 60min, preferably 60min.
13. The detection method according to any one of claims 9 to 12, wherein the organic solvent in step (3) is selected from acetonitrile or methanol, preferably acetonitrile.
14. The detection method according to any one of claims 9 to 13, wherein the conditions of the gradient elution in step (3) are: in 0-15min, the content of mobile phase A is 10% → 21%, the content of mobile phase B is 90% → 79%,15-18min, the content of mobile phase A is 21% → 21%, the content of mobile phase B is 79% → 79%,18-23min, the content of mobile phase A is 21% → 90%, the content of mobile phase B is 79% → 10%,23-27min, the content of mobile phase A is 90% → 10%, the content of mobile phase B is 10 → 90%,27-30min, the content of mobile phase A is 10% → 10%, and the content of mobile phase B is 90 → 90%.
15. The method for detecting the characteristic spectrum of the cynanchum wilfordii extract as claimed in claim 7, which comprises the steps of:
(1) Preparation of reference solutions
Weighing p-hydroxyacetophenone reference substance, and adding a solvent to prepare a reference substance solution;
(2) Preparation of test solution
Weighing cynanchum wilfordii extract, and adding a solvent for extraction;
(3) Ultra high performance liquid chromatography
Using octadecylsilane chemically bonded silica as filler, organic solvent as mobile phase A, and water phase as mobile phase B to perform gradient elution, and absorbing reference solution and sample solution to inject into ultra high performance liquid chromatograph for analysis.
16. The detection method according to claim 15, wherein the solvent in step (1) is one or more selected from the group consisting of water, 50% methanol, 75% ethanol, 50% methanol, 75% methanol and methanol, preferably methanol; preferably, the solvent in step (2) is one or more selected from water, 50% methanol, 75% ethanol, 50% methanol, 75% methanol and methanol, preferably methanol.
17. The method according to claim 15 or 16, wherein the extraction in step (2) is performed by one of reflux, shaking or ultrasound, preferably ultrasound.
18. The method according to any one of claims 15 to 17, wherein the extraction time in step (2) is 15 to 60min, preferably 30min.
19. The detection method according to any one of claims 15 to 18, wherein the organic solvent in step (3) is selected from acetonitrile or methanol, preferably methanol.
20. The detection method according to any one of claims 15 to 19, wherein the conditions of the gradient elution in step (3) are: in 0-7min, 23% → 24% of mobile phase A, 77% → 76% of mobile phase B, 7-11min, 24% → 27% of mobile phase A, 76% → 73% of mobile phase B, 11-14min, 27% → 29% of mobile phase A, 73% → 71% of mobile phase B, 14-18min, 29% → 32% of mobile phase A, 71 → 68% of mobile phase B, 18-20min, 32% → 38% of mobile phase A, 68% → 62% of mobile phase B, 20-28min, 38% → 38% of mobile phase A, 62% → 62% of mobile phase B, 28-28.1min, 38% → 23% of mobile phase A, 62% → 77% of mobile phase B, 28.1-30min, 23% → 23% of mobile phase A, and 77% → 77% of mobile phase B.
21. The detection method according to any one of claims 15 to 20, wherein the aqueous phase in step (3) is selected from the group consisting of water, a 0.1% formic acid solution, a 0.05% phosphoric acid solution and a 0.1% acetic acid solution, preferably a 0.1% formic acid solution.
22. Use of the cynanchum wilfordii extract of claim 7 for preparing health food and Chinese medicinal medicines; preferably, the traditional Chinese medicine is a standard decoction, a capsule, a tablet, a granule or a formula granule.
23. A standard decoction prepared from the cynanchum wilfordii extract of claim 7.
24. A formulation granulate, characterized in that the cynanchum wilfordii extract according to claim 7 is produced by adding adjuvants.
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