CN114965807A - Fructus aurantii control extract and preparation method thereof - Google Patents
Fructus aurantii control extract and preparation method thereof Download PDFInfo
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Abstract
The invention provides a preparation method of a fructus aurantii control extract, which comprises the steps of respectively carrying out low-temperature extraction, low-temperature concentration and low-temperature drying on a plurality of batches of fructus aurantii medicinal material powder for a plurality of times to obtain different batches of fructus aurantii extracts, and then blending the different batches of fructus aurantii extracts to obtain the fructus aurantii control extract. The preparation method of the fructus aurantii contrast extract is simple and convenient to operate, low in cost, good in repeatability and high in extraction rate; the preparation method for preparing the fructus aurantii control extract ensures the consistency of the fructus aurantii control extracts of different batches, and the fructus aurantii control extract has stable and uniform properties and is convenient to use; the thin-layer chromatography fingerprint and the HPLC fingerprint of the contrast extract finally obtained by adopting the preparation method of the bitter orange contrast extract are consistent with those of medicinal materials.
Description
Technical Field
The invention relates to the technical field of quality control of traditional Chinese medicines, in particular to a fructus aurantii contrast extract and a preparation method and application thereof.
Background
The quality control mode of the traditional Chinese medicine is basically along the development of natural medicinal chemistry, an analysis method for taking one or more active ingredients of the traditional Chinese medicine as targets and the concept of qualitative and quantifiable quality standard, the quality control method of foreign plant medicines is referred, the mode of chemical medicine quality control is referred, corresponding simple physicochemical identification is established by means of literature reports, and the quality standard of identification and content determination mainly based on spectrum and chromatogram is developed.
Each Chinese medicinal material is a multi-component complex, which determines the unique integrity and fuzziness of the Chinese medicinal material, and also shows that the evaluation method of taking one, two or even a plurality of components in the Chinese medicinal material as the quality of the Chinese medicinal material has great limitation. The 1990 edition of Chinese pharmacopoeia increases the thin-layer chromatography identification of reference medicinal materials, so that the identification of traditional Chinese medicines and Chinese patent medicines has great progress. At present, Chinese medicine and foreign herbal medicine pay more and more attention to the detection of multiple components or multiple components, such as the quality control of German ginkgo biloba extract. The analysis method of the fingerprint spectrum can carry out quality control on the medicinal materials on the whole, and still has great research value at present.
At present, Chinese pharmacopoeia has two kinds of reference substances, namely a chemical reference substance and a traditional Chinese medicine reference medicinal material, in the aspect of controlling the quality of medicinal materials. The chemical reference substance can be used for qualitative identification and quantitative analysis of medicinal materials, and the traditional Chinese medicine reference medicinal material can be used for microscopic identification and thin-layer identification. However, both chemical reference substances and traditional Chinese medicine reference medicinal materials have limitations in traditional Chinese medicine quality control. The limitation is shown in that chemical components in the traditional Chinese medicine are diversified, single or a plurality of compounds cannot reflect the whole appearance of the medicinal materials, and the existing standard has a plurality of holes which are sometimes generated due to the phenomenon of sub-optimal effect; secondly, the traditional Chinese medicine reference medicinal materials are influenced by the producing area and the growing environment, so that the quality consistency of each batch is difficult to ensure, and the traditional Chinese medicine reference medicinal materials can only be used for qualitative identification and cannot reflect the content of the components of the medicinal materials.
The traditional Chinese medicine control extract is an extract which is prepared from traditional Chinese medicinal materials, has stable properties and components, can be used for qualitative or quantitative analysis, and provides four basic requirements (ASCS) for the traditional Chinese medicine control extract by Mr. Shipeheng, and also provides basic conditions for the control extract: the herbal material source is reliable and representative; specificity, Specificity of the detection method used; con-static, the control extracts should remain consistent from batch to batch; stability, stable and uniform properties and convenient use. The method can be used for qualitative identification of medicinal materials by using high performance thin layer chromatography fingerprint and high performance liquid chromatography fingerprint, and the control extract standardized by external standard method can be further used for semi-quantitative and quantitative analysis and detection. The traditional Chinese medicine control extract has important significance for controlling the quality of the traditional Chinese medicine.
Disclosure of Invention
In view of the above, it is desirable to provide a fructus aurantii control extract, and a preparation method and application thereof. The invention provides a preparation method of a fructus aurantii control extract, which is simple and convenient to operate, low in cost, good in repeatability and high in extraction rate; the invention also provides the fructus aurantii control extract prepared by the preparation method of the fructus aurantii control extract, and the prepared fructus aurantii control extract has good consistency, stable and uniform properties and convenient use, and can reflect the whole appearance of medicinal materials.
The invention provides a preparation method of a fructus aurantii control extract on one hand, which comprises the following steps:
step one, ethanol extraction, wherein the ethanol extraction comprises the following steps:
a) mixing fructus Aurantii powder with ethanol, performing flash extraction, and filtering to obtain filtrate 1 and residue 1;
b) repeating the step a) on the filter residue 1 to obtain a filtrate 2 and a filter residue 2, repeating the step a) on the obtained filter residue 2, repeating the step a) by the analogy of the step b) for N times to obtain a filtrate N +1 and a filter residue N + 1;
c) mixing the filtrate 1 to (N +1) to obtain an extracting solution 1, and concentrating the extracting solution 1 at low temperature to obtain the dried bitter orange paste;
step two, preparing the bitter orange extract: dissolving the dried extract of the bitter orange obtained in the step two in ethanol to obtain ethanol solution of the dried extract of the bitter orange, adding auxiliary materials, drying at low temperature and sieving to obtain the extract of the bitter orange;
step three, blending: and blending the fructus aurantii extracts of different batches to obtain the fructus aurantii control extract.
The temperature adopted for low-temperature concentration and low-temperature drying in the preparation process is 20-60 ℃.
The different batches of fructus aurantii extract are blended, and it can be understood that at least five batches of fructus aurantii extract are blended or blended.
Preferably, in the first step, N is more than or equal to 8 and more than or equal to 0, and N is an integer.
Preferably, N in the above step one is 3.
Preferably, the weight-to-volume ratio of the shiso powder to the ethanol in the first step is 1: 5-1: 50, and the unit of the weight-to-volume ratio is g/mL.
More preferably, the weight-to-volume ratio of the fructus aurantii powder to the ethanol in the first step is 1: 10.
preferably, the concentration of ethanol in the step one is 50 to 95 percent.
More preferably, the concentration of ethanol in the first step is 70%.
Preferably, the flash extraction time in the step one is 1-3 min, and the power is 100W-3 kW.
More preferably, the flash-up time in the first step is 1min, and the power is 2000W.
Preferably, the filtration in the first step is performed by using medium-speed filter paper or 2000-mesh screen.
Preferably, the weight-to-volume ratio of the dried extract of fructus aurantii to the ethanol in the second step is 1: 5-1: 50. The unit of the weight-volume ratio is g/mL, and the concentration of the alcohol is 70%.
Preferably, the weight-volume ratio of the dried extract of fructus aurantii to ethanol in the second step is 1: 5.
Preferably, the weight of the auxiliary materials is 20-60% of the weight of the bitter orange dry paste.
Preferably, the weight of the auxiliary materials is 35% of the weight of the bitter orange dry paste.
Preferably, the auxiliary material is aerosil.
Preferably, the second step is carried out at low temperature and then filtered by a screen of 90-200 meshes.
More preferably, the low-temperature drying in the second step is performed by passing through a 110-mesh screen.
Preferably, a preparation pretreatment step of detecting different batches of fructus aurantii extract by thin layer chromatography and/or high performance liquid chromatography is further included between the second step and the fourth step.
In a second aspect, the present invention provides a control extract of fructus aurantii obtained by the above-mentioned preparation method.
Preferably, the spectrum of the fructus aurantii control extract obtained by adopting thin layer chromatography and/or high performance liquid chromatography is consistent with that of the corresponding raw material medicine.
Preferably, the spectrum of the fructus Aurantii control extract obtained by thin layer chromatography and/or high performance liquid chromatography is consistent with the spectrum of the corresponding raw material herbs at the positions of neohesperidin, naringin and nobiletin.
Preferably, the fructus aurantii control extract contains main substances including neohesperidin, naringin and nobiletin.
The third aspect of the invention provides the application of the fructus aurantii control extract in the identification of medicinal materials or Chinese medicinal preparations, or in the quality control of fructus aurantii or medicinal materials or Chinese medicinal preparations containing fructus aurantii/fructus aurantii active ingredients.
Preferably, the identification of the medicinal materials or the Chinese medicinal preparation, or the quality control method of the bitter orange or the medicinal materials or the Chinese medicinal preparation containing the effective components of the bitter orange/the bitter orange comprises the following steps: detecting the fructus Aurantii control extract, medicinal material or Chinese medicinal preparation by thin layer chromatography and/or high performance liquid chromatography, and comparing.
Preferably, the thin layer chromatography and/or high performance liquid chromatography is used for detecting main components in the fructus aurantii control extract, the medicinal materials or the Chinese medicinal preparation, wherein the main components comprise neohesperidin, naringin and nobiletin.
Preferably, when the above-mentioned fructus aurantii control extract, crude drug or traditional Chinese medicine preparation is detected by thin layer chromatography, the fructus aurantii control extract, crude drug or traditional Chinese medicine preparation needs to be prepared into a solution for detection, wherein the preparation method of the fructus aurantii control extract solution comprises: adding methanol into the fructus aurantii control extract of claim 6 to prepare a methanol solution of the fructus aurantii control extract with the concentration of 30mg/mL-80mg/mL, and filtering the solution through a filter membrane of 0.12-0.32 μm to obtain the solution of the fructus aurantii control extract, wherein the concentration of the methanol is 100%.
Preferably, the concentration of the above fructus Aurantii control extract in methanol is 50 mg/mL; the filter was 0.22 μm.
Further, the preparation method of the solution of the medicinal materials to be detected in the thin-layer chromatography comprises the following steps: weighing 0.1-2 g of a product to be detected after passing through a No. four sieve, adding 1 mL-20 mL of methanol, carrying out cold soaking for 1-30 min, carrying out ultrasonic treatment for 1-30 min, centrifuging for 1-20 min after the power is 100W-3 kW, carrying out rotation speed of 1000-5000 r/min, taking a supernatant, drying by distillation, adding 5mL of methanol into residues for dissolving, and passing through a 0.12-0.32 mu m filter membrane to obtain a solution of the product to be detected; preferably weighing 0.2g of the product to be detected, adding 10mL of methanol, cold soaking for 15min, performing ultrasonic treatment for 30min at the power of 500w, centrifuging for 10min at the rotation speed of 4000 r/min, taking the supernatant, evaporating to dryness, adding 5mL of methanol into the residue to dissolve the residue, and filtering through a 0.22 mu m filter membrane to obtain the solution of the product to be detected.
Preferably, the detection condition of the thin layer chromatography is a first detection condition or a second detection condition, and the first detection condition is:
thin-layer plate: TLC G60 precast slab;
sample application: comparison products: 10 μ l, 20 μ l, strip spotting;
developing agent:
and (3) flavonoid glycoside: chloroform-methanol-water (13: 6: 2) lower layer solution;
and (6) inspection: spraying 3% aluminum trichloride ethanol solution, heating at 105 deg.C for 5min, and inspecting under ultraviolet light (366 nm);
the second detection condition is as follows:
thin-layer plate: TLC G60 precast slab;
sample application: 2 μ l, strip spotting;
developing agent:
flavonoid aglycone: s1: toluene-ethyl acetate-formic acid-water (20: 20: 1: 1) upper solution;
s2, toluene-ethyl acetate-formic acid-water (20: 10: 1: 1) upper solution;
and (6) inspection: inspecting under ultraviolet light (366 nm);
optionally, when the above-mentioned fructus aurantii control extract, crude drug or traditional Chinese medicine preparation is detected by high performance liquid chromatography, the fructus aurantii control extract, crude drug or traditional Chinese medicine preparation needs to be prepared into a solution for detection, wherein the preparation method of the fructus aurantii control extract solution comprises: adding methanol into the fructus Aurantii control extract of claim 6 to obtain a methanol solution of the fructus Aurantii control extract with a concentration of 0.1mg/mL-1mg/mL, and filtering with a 0.12-0.32 μm filter membrane to obtain a solution of the fructus Aurantii control extract, wherein the concentration of the methanol is 100%.
Preferably, the concentration of the above fructus Aurantii control extract in methanol is 0.6 mg/mL; the filter was 0.22 μm.
Further, in the detection by the high performance liquid chromatography, the preparation method of the solution of the medicinal material to be detected comprises the following steps: and (3) precisely weighing 0.1-2 g of the product to be detected after the product to be detected passes through a No. four sieve, placing the product in a conical flask with a plug, adding 5-50 mL of methanol, heating and refluxing for 30-90 min at the temperature of 50-100 ℃, cooling, filtering, precisely weighing 10mL of subsequent filtrate, placing the subsequent filtrate in a 25mL volumetric flask, adding methanol to the scale, shaking up, passing through a 0.12-0.32 mu m filter membrane, and taking the subsequent filtrate to obtain the solution of the product to be detected. Preferably and precisely weighing 0.2g of product to be detected, placing the product into a conical flask with a plug, adding 50mL of methanol, heating and refluxing for 90min at the temperature of 80 ℃, cooling, filtering, precisely measuring 10mL of subsequent filtrate, placing the filtrate into a 25mL volumetric flask, adding methanol to scale, shaking up, and filtering through a 0.22 mu m filter membrane to obtain the solution of the product to be detected, wherein the concentration of the methanol is 100%.
Optionally, the detection conditions of the high performance liquid chromatography are as follows:
chromatography apparatus: a Vanqish ultra performance liquid chromatograph;
a detector: a DAD detector;
a chromatographic column: agilent RRHD SB C18 (2.1X 100mm,1.8 μm);
mobile phase: acetonitrile (a) -0.05% phosphoric acid solution (B) was subjected to gradient elution as specified in the following table;
detection wavelength: 320 nm; the flow rate is 0.4 ml/min; the sample volume is 2 mul; the column temperature is 30 ℃; operating time: and 20 min.
The invention has the following beneficial effects:
the preparation method of the fructus aurantii control extract adopts the steps of extracting, preparing the fructus aurantii extract and blending, and has the advantages of simple and convenient operation, low cost, good repeatability and high extraction rate. The fructus aurantii control extract prepared by the preparation method is prepared by mixing different batches of extracts, so that the defect that the quality of each batch is difficult to ensure because the traditional Chinese medicine control medicinal materials are influenced by the production place and the growth environment is overcome, and the consistency of the fructus aurantii control extracts of different batches is ensured; the low-temperature control of the whole preparation process is ensured, and the change of effective components is effectively prevented; the character is stable and uniform and is convenient to use; the thin-layer chromatography fingerprint and HPLC fingerprint of the control extract finally obtained by the preparation method of the bitter orange control extract are consistent with those of medicinal materials.
The invention also provides an identification method of the medicinal materials or the traditional Chinese medicine preparation, or a quality control method of the fructus aurantii or the medicinal materials or the traditional Chinese medicine preparation containing the effective components of the fructus aurantii/the fructus aurantii, which can carry out qualitative identification on the medicinal materials or the traditional Chinese medicine preparation.
Drawings
FIG. 1 is a thin layer chromatogram obtained by thin layer chromatography of fructus Aurantii raw material with neohesperidin and naringin as reference substances. 1 is reference substances of neohesperidin and naringin, and 2-16 correspond to the raw material medicinal materials of fructus aurantii in sequence: the medicinal composition comprises 1 raw material medicinal material of fructus aurantii, 2 raw material medicinal material of fructus aurantii, 3 raw material medicinal material of fructus aurantii, 4 raw material medicinal material of fructus aurantii, 5 raw material medicinal material of fructus aurantii, 6 raw material medicinal material of fructus aurantii, 7 raw material medicinal material of fructus aurantii, 8 raw material medicinal material of fructus aurantii, 9 raw material medicinal material of fructus aurantii, 10 raw material medicinal material of fructus aurantii, 11 raw material medicinal material of fructus aurantii, 12 raw material medicinal material of fructus aurantii, 13 raw material medicinal material of fructus aurantii, 14 raw material medicinal material of fructus aurantii and 15 raw material medicinal material of fructus aurantii.
FIG. 2 is a thin-layer chromatogram obtained by performing thin-layer chromatography on fructus Aurantii raw material with nobiletin as reference substance (II). 1 is a reference substance of nobiletin, and 2-16 correspond to the raw medicinal materials of bitter orange in sequence: the medicinal materials comprise 1 raw material medicinal material of fructus aurantii, 2 raw material medicinal material of fructus aurantii, 3 raw material medicinal material of fructus aurantii, 4 raw material medicinal material of fructus aurantii, 5 raw material medicinal material of fructus aurantii, 6 raw material medicinal material of fructus aurantii, 7 raw material medicinal material of fructus aurantii, 8 raw material medicinal material of fructus aurantii, 9 raw material medicinal material of fructus aurantii, 10 raw material medicinal material of fructus aurantii, 11 raw material medicinal material of fructus aurantii, 12 raw material medicinal material of fructus aurantii, 13 raw material medicinal material of fructus aurantii, 14 raw material medicinal material of fructus aurantii and 15 raw material medicinal material of fructus aurantii.
FIG. 3 is an overlay of HPLC fingerprints obtained by performing high performance liquid chromatography on fructus Aurantii raw material, wherein R corresponds to a common mode (common mode) of fructus Aurantii raw material, and 1-15 correspond to fructus Aurantii raw material in sequence: the medicinal materials comprise 1 bitter orange raw material medicinal material, 2 bitter orange raw material medicinal material, 3 bitter orange raw material medicinal material, 4 bitter orange raw material medicinal material, 5 bitter orange raw material medicinal material, 6 bitter orange raw material medicinal material, 7 bitter orange raw material medicinal material, 8 bitter orange raw material medicinal material, 9 bitter orange raw material medicinal material, 10 bitter orange raw material medicinal material, 11 bitter orange raw material medicinal material, 12 bitter orange raw material medicinal material, 13 bitter orange raw material medicinal material, 14 bitter orange shell material medicinal material and 15 bitter orange raw material medicinal material.
Fig. 4 is a flow chart of the present invention for preparing fructus aurantii control extract.
FIG. 5 shows that the fructus Aurantii reference extract is subjected to high performance liquid chromatography to obtain fingerprint, and similarity detection with common mode spectrum of raw material medicinal materials is carried out, ERS corresponds to fructus Aurantii reference extract, and R is common mode of raw material medicinal materials of fructus Aurantii.
Detailed Description
The following describes embodiments of the present invention in detail. The following examples are illustrative only and are not to be construed as limiting the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
The following examples use the following sources of instruments and materials:
the medicinal material to be used for preparing the control extract is fructus Aurantii.
A ThermoFishe Vanqish ultra-performance liquid chromatograph, a Vanqish DAD detector.
Methanol chromatography (Merck), aluminum trichloride (guangzhou chemical reagent plant).
The purity of the methanol not marked by the invention is more than or equal to 99.5 percent.
The 3% aluminum trichloride ethanol solution related to the invention is a mixed solution containing 3g of aluminum trichloride and 100mL of ethanol in volume.
The proportions of the developing solvent are volume ratios.
Toluene, ethyl acetate, methanol, ethanol, chloroform, glacial acetic acid, sulfuric acid, formic acid, acetone, and aluminum trichloride were all analytically pure (Guangzhou chemical laboratories).
A neohesperidin reference substance (China institute for testing and testing food and drug; batch No. 111857-201804, with the mark content being more than or equal to 99.4%);
naringin reference substance (China institute for testing and testing food and drug; lot number 110722 and 201815, the content of marked mark is more than or equal to 91.7%);
nobiletin reference substance, (Shanghai Shidande Standard technology service Co., Ltd.; batch number: 6082, mark content: 98%);
testing the medicinal material fructus aurantii:
15 batches of fructus aurantii raw medicinal materials are purchased in various Chinese medicine markets in China: the medicinal materials comprise 1 raw material medicinal material of fructus aurantii, 2 raw material medicinal material of fructus aurantii, 3 raw material medicinal material of fructus aurantii, 4 raw material medicinal material of fructus aurantii, 5 raw material medicinal material of fructus aurantii, 6 raw material medicinal material of fructus aurantii, 7 raw material medicinal material of fructus aurantii, 8 raw material medicinal material of fructus aurantii, 9 raw material medicinal material of fructus aurantii, 10 raw material medicinal material of fructus aurantii, 11 raw material medicinal material of fructus aurantii, 12 raw material medicinal material of fructus aurantii, 13 raw material medicinal material of fructus aurantii, 14 raw material medicinal material of fructus aurantii and 15 raw material medicinal material of fructus aurantii.
Example 1: screening of fructus aurantii raw material medicinal materials
This example provides a method for screening raw material herbs of the present invention fructus aurantii control extract.
Analyzing and screening fructus Aurantii raw material by high performance thin layer chromatography and high performance liquid chromatography respectively with fructus Aurantii reference material as reference substance.
The fructus Aurantii medicinal material is purchased from various major medicinal material markets in China, and is identified as dry immature fruit of Citrus aurantium L. of Rutaceae and its cultivar by the plant identification center of south China in the south China plant Garden of Chinese academy of sciences. The identified 15 batches of medicinal materials can be used for standby after meeting the standard.
1 thin layer chromatography
1.1 sample preparation
Preparation of a reference solution: weighing appropriate amount of neohesperidin and naringin as reference substances, preparing into 1mg/mL solution with methanol, and filtering with 0.22 μm filter membrane to obtain reference solution.
Raw material medicinal material solution: taking the powder of the raw material medicine of the bitter orange (1-15), sieving the powder by a fourth sieve (250 +/-9.9 mu m, 65 meshes, the same below), precisely weighing 0.2g of the powder, adding 10mL of methanol, cold soaking for 15min, carrying out ultrasonic treatment for 30min at the power of 500w, centrifuging for 10min at the rotating speed of 4000 r/min, taking the supernatant, evaporating to dryness, adding 5mL of methanol into residues to dissolve the residues, and filtering the residues by a filter membrane of 0.22 mu m to obtain the solution of the raw material medicine.
The detection conditions were as follows:
the method comprises the following steps:
thin-layer plate: TLC G60 precast slab;
sample application: 10 μ l, strip spotting;
developing agent:
and (3) flavonoid glycoside: chloroform-methanol-water (13: 6: 2) lower layer solution;
and (6) inspection: spraying 3% aluminum trichloride ethanol solution, heating at 105 deg.C for 5min, and inspecting under ultraviolet light (366 nm);
the second method comprises the following steps:
thin-layer plate: TLC G60 precast slab;
sample application: 2 μ l, strip spotting;
developing agent:
flavonoid aglycone: s1: toluene-ethyl acetate-formic acid-water (20: 20: 1: 1) upper solution;
s2, toluene-ethyl acetate-formic acid-water (20: 10: 1: 1) upper solution;
and (3) inspecting: directly inspecting under ultraviolet light (366 nm);
the detection results are shown in FIG. 1-FIG. 2, in which FIG. 1 shows that the ethanol solution of aluminum trichloride is used for color development after the first method, and the thin layer chromatogram is inspected under an ultraviolet lamp (366nm), and FIG. 2 shows that the thin layer chromatogram is inspected under ultraviolet light (366nm) by the second method. Fig. 1 and 2: reference substances of neohesperidin and naringin are adopted as 1, and 2-16 correspond to bitter orange raw material medicines in sequence: 1 of fructus aurantii raw material medicinal materials, 2 of fructus aurantii raw material medicinal materials, 3 of fructus aurantii raw material medicinal materials, 4 of fructus aurantii raw material medicinal materials, 5 of fructus aurantii raw material medicinal materials, 6 of fructus aurantii raw material medicinal materials, 7 of fructus aurantii raw material medicinal materials, 8 of fructus aurantii raw material medicinal materials, 9 of fructus aurantii raw material medicinal materials, 10 of fructus aurantii raw material medicinal materials, 11 of fructus aurantii raw material medicinal materials, 12 of fructus aurantii raw material medicinal materials, 13 of fructus aurantii raw material medicinal materials, 14 of fructus aurantii raw material medicinal materials and 15 of fructus aurantii raw material medicinal materials, wherein in the figure 2: 1 is a reference substance, namely nobiletin, and 2-16 sequentially correspond to the raw material medicaments of the bitter orange: the medicinal materials comprise 1 raw material medicinal material of fructus aurantii, 2 raw material medicinal material of fructus aurantii, 3 raw material medicinal material of fructus aurantii, 4 raw material medicinal material of fructus aurantii, 5 raw material medicinal material of fructus aurantii, 6 raw material medicinal material of fructus aurantii, 7 raw material medicinal material of fructus aurantii, 8 raw material medicinal material of fructus aurantii, 9 raw material medicinal material of fructus aurantii, 10 raw material medicinal material of fructus aurantii, 11 raw material medicinal material of fructus aurantii, 12 raw material medicinal material of fructus aurantii, 13 raw material medicinal material of fructus aurantii, 14 raw material medicinal material of fructus aurantii and 15 raw material medicinal material of fructus aurantii. As can be seen from fig. 1-2, each batch of fructus aurantii raw material can show the same spots at the same position, and is primarily screened as the raw material of fructus aurantii as the control extract.
2 high performance liquid chromatography
2.1 sample preparation
Raw material medicinal material solution: precisely weighing 0.2g of fructus aurantii raw material (1-15) powder, placing the powder in a conical flask with a plug, adding 50mL of methanol, heating and refluxing for 90min at the temperature of 80 ℃, cooling, filtering with medium-speed filter paper (30-50 mu m), precisely weighing 10mL of subsequent filtrate, placing the subsequent filtrate in a 25mL volumetric flask, adding methanol to the scale, shaking up, and filtering with a 0.22 mu m filter membrane to obtain the solution of a product to be detected, wherein the concentration of the methanol is 100%.
2.2 high Performance liquid chromatography detection
The detection conditions of the high performance liquid chromatography are as follows:
chromatography apparatus: vanqish ultra high performance liquid chromatograph;
a detector: a DAD detector;
a chromatographic column: agilent RRHD SB C18 (2.1X 100mm,1.8 μm);
mobile phase: acetonitrile (a) -0.05% phosphoric acid solution (B) with gradient elution as specified in the table below;
detection wavelength: 320 nm; the flow rate is 0.4 ml/min; the column temperature is 30 ℃; operating time: and 20 min.
The detection method of the high performance liquid chromatography comprises the following steps: respectively taking 2 μ l of exocarpium Citri Grandis control medicinal material solution and raw material medicinal material solution, and injecting into liquid chromatograph.
And (3) detecting results by high performance liquid chromatography:
measuring, and recording chromatogram to obtain UPLC fingerprint chromatogram map overlay shown in figure 3, wherein R corresponds to fructus Aurantii raw material common mode (common mode), and 1-15 correspond to fructus Aurantii raw material in sequence: the medicinal materials comprise 1 raw material medicinal material of fructus aurantii, 2 raw material medicinal material of fructus aurantii, 3 raw material medicinal material of fructus aurantii, 4 raw material medicinal material of fructus aurantii, 5 raw material medicinal material of fructus aurantii, 6 raw material medicinal material of fructus aurantii, 7 raw material medicinal material of fructus aurantii, 8 raw material medicinal material of fructus aurantii, 9 raw material medicinal material of fructus aurantii, 10 raw material medicinal material of fructus aurantii, 11 raw material medicinal material of fructus aurantii, 12 raw material medicinal material of fructus aurantii, 13 raw material medicinal material of fructus aurantii, 14 raw material medicinal material of fructus aurantii and 15 raw material medicinal material of fructus aurantii.
As can be seen from fig. 3, the fingerprint spectrums of the original medicinal material 1 to 15 of fructus aurantii have high consistency, so that a common fingerprint spectrum mode of the medicinal material of fructus aurantii is established, and similarity analysis is performed on all samples. And (3) according to the common mode map, performing similarity evaluation on 15 batches of bitter orange raw material medicines by adopting an included angle cosine algorithm according to each sample component and the peak area thereof, wherein the result is shown in table 1.
TABLE 1
According to the similarity analysis results, the similarity of 15 batches of fructus aurantii raw medicinal materials is very high, and the raw medicinal materials can be selected as pummelo peel reference extracts.
Example 2: preparation of fructus Aurantii control extract
This example provides a method for preparing a control extract of fructus Aurantii according to the present invention, and the flow chart of the method is shown in FIG. 4.
Firstly, the method comprises the following steps: extraction of
Selecting fructus Aurantii medicinal materials, making into powder, sieving the medicinal material powder with a No. four sieve, adding 70% ethanol (solid-to-liquid ratio is 1: 10(w/V, g/ml)) with the volume 10 times of the weight of the medicinal material powder, carrying out flash extraction (power 2000w, normal temperature) for 1min, filtering with medium-speed qualitative filter paper (general electric and biological technology (Hangzhou) limited, model: 99-103 and 125), collecting filter residue 1 and filtrate 1, extracting the filter residue 1 for three times again according to the same method, combining the filtrate collected for three times of re-extraction with the filtrate 1 to obtain an extracting solution, and concentrating the solvent of the extracting solution at low temperature (less than 45 ℃) to obtain the fructus Aurantii dry extract.
II, secondly: preparing fructus Aurantii extract
Dissolving the dried extract of fructus Aurantii with 70% ethanol 5 times (w/V, g/ml) of the dried extract to obtain 70% ethanol solution, adding silica gel micropowder 35% of the dried extract of fructus Aurantii (adjuvant prepared from Shanhe medicinal materials, lot No. 170115), mixing, concentrating at low temperature (below 45 deg.C) with rotary evaporator to dry, pulverizing, and sieving with 110 mesh sieve to obtain the final product.
And preparing different batches of fructus aurantii extracts from the multi-batch fructus aurantii medicinal material powder according to the operation methods from the first step to the second step.
Thirdly, the method comprises the following steps: blending
And (3) mixing and blending 15 batches of the fructus aurantii extract obtained in the step two according to the proportion of 1:1:1:1:1:1:1:1:1:1:1:1:1:1:1 to obtain a fructus aurantii control extract, wherein the proportion of the final product corresponding to the raw medicinal materials of the fructus aurantii is about 1: 2.12 (g/g).
The blending standard is as follows: the spectrum obtained by the finally obtained control extract is consistent with the spectrum of the corresponding raw material medicine, and the content range of each component in the blending standard is also the optimal data obtained by comprehensively maintaining the stability and consistency through repeated experiments and the following application and the like.
Fourthly, the method comprises the following steps: detection of
Determining the fructus Aurantii reference extract prepared in step three by high performance liquid chromatography to obtain fingerprint, and performing similarity detection with common mode spectrum of raw material medicinal materials, as shown in FIG. 5, ERS corresponds to fructus Aurantii reference extract, and R is common mode of raw material medicinal materials of fructus Aurantii. The obtained similarity is 0.998, and the similarity of the common fingerprint patterns of the fructus aurantii control extract and the fructus aurantii raw medicinal material is high, which shows that the fructus aurantii control extract can represent the fructus aurantii raw medicinal material.
Example 3: analysis of Properties of control extract of fructus Aurantii
1. Apparent state: the control extract of bitter orange obtained in example 2 was all a brown-yellow powder.
2. The moisture content was measured by the second method (baking method) in the section of moisture content determination method of the fourth part 0832 of the Chinese pharmacopoeia, 2020 edition. The detection result shows that the water content of the fructus aurantii control extract is 6.9%.
3. And (3) testing consistency: 15 batches of the bitter orange control extract were prepared according to the method of example 2, and the differences in thin layer chromatography detection profiles of the batches were determined to be small. Therefore, the fructus aurantii control extract prepared by the preparation method of the fructus aurantii control extract has very good consistency.
Claims (9)
1. The preparation method of the bitter orange control extract is characterized by comprising the following steps of:
step one, ethanol extraction, wherein the ethanol extraction comprises the following steps:
a) mixing fructus Aurantii powder with ethanol, performing flash extraction, and filtering to obtain filtrate 1 and residue 1;
b) repeating the operation of the step a) on the filter residue 1 to obtain a filtrate 2 and a filter residue 2, repeating the operation of the step a) on the obtained filter residue 2, and repeating the operation of the step a) for N times by analogy with the operation of the step b) to obtain a filtrate N +1 and a filter residue N + 1;
c) mixing the filtrate 1-N +1 to obtain an extracting solution 1, and concentrating the extracting solution 1 at low temperature to obtain the dried bitter orange paste;
step two, preparing the bitter orange extract: dissolving the dried extract of the bitter orange obtained in the step two in ethanol to obtain ethanol solution of the dried extract of the bitter orange, adding auxiliary materials, drying and sieving to obtain the extract of the bitter orange;
step three, blending: and blending the fructus aurantii extracts of different batches to obtain the fructus aurantii control extract.
2. The method according to claim 1, wherein in the first step, N is 8. gtoreq.N.gtoreq.0, and N is an integer;
optionally, the N ═ 3.
3. The preparation method according to claim 1, wherein the weight-to-volume ratio of the fructus aurantii powder to the ethanol in the first step is 1: 5-1: 50, and the unit of the weight-to-volume ratio is g/mL;
optionally, the weight-to-volume ratio of the fructus aurantii powder to the ethanol in the first step is 1: 10;
optionally, the concentration of ethanol in the first step is 50-95%;
preferably, the concentration of ethanol in the first step is 70%;
optionally, the flash-up time in the step one is 1-3 min, and the power is 100W-3 kW;
optionally, the flash-up time in the first step is 1min, and the power is 2000W;
optionally, the filtering in the first step is filtering by using medium-speed filter paper or a 2000-mesh screen;
optionally, the filtering in the first step is filtering with medium speed filter paper.
4. The preparation method according to claim 1, wherein the weight-to-volume ratio of the dried extract of fructus aurantii to ethanol in the second step is 1: 5-1: 50, the unit of the weight-to-volume ratio is g/mL, and the concentration of the ethanol is 70%;
optionally, the weight-volume ratio of the dried extract of fructus aurantii to ethanol in the second step is 1: 5;
optionally, the weight of the auxiliary materials is 20-60% of the weight of the dried bitter orange paste;
optionally, the weight of the auxiliary materials is 35% of the dry paste weight of the fructus aurantii;
optionally, the auxiliary material is micropowder silica gel;
optionally, filtering the dried product in the third step by a screen of 90-200 meshes;
optionally, the step three is dried and then screened by a 110-mesh screen.
5. The method of claim 1, further comprising a pre-blending treatment step of detecting different batches of fructus Aurantii extract by thin layer chromatography and/or high performance liquid chromatography between the second step and the third step.
6. A fructus aurantii control extract, which is obtained by the preparation method of any one of claims 1 to 5;
optionally, the fructus Aurantii control extract has a spectrum consistent with that of the corresponding raw material by thin layer chromatography and/or high performance liquid chromatography;
optionally, the spectrum of the fructus Aurantii control extract obtained by thin layer chromatography and/or high performance liquid chromatography is consistent with the spectrum of the corresponding raw material herbs at the positions of neohesperidin, naringin and nobiletin;
optionally, the fructus aurantii control extract contains main substances including neohesperidin, naringin and nobiletin.
7. The use of the fructus Aurantii control extract of claim 6 for the identification of medicinal materials or pharmaceutical preparations, or for the quality control of fructus Aurantii or medicinal materials or pharmaceutical preparations containing fructus Aurantii/effective components of fructus Aurantii.
8. The use of claim 7, wherein the identification of the medicinal material or the Chinese medicinal preparation, or the quality control method of the bitter orange or the medicinal material or the Chinese medicinal preparation containing the effective components of the bitter orange/the bitter orange comprises the following steps: detecting the fructus Aurantii reference extract, medicinal material or Chinese medicinal preparation of claim 6 by thin layer chromatography and/or high performance liquid chromatography, and comparing;
optionally, the thin layer chromatography and/or high performance liquid chromatography is used for detecting main components in the fructus aurantii control extract, the medicinal materials or the traditional Chinese medicine preparation, wherein the main components comprise neohesperidin, naringin and nobiletin;
optionally, when the fructus aurantii control extract, crude drug or traditional Chinese medicine preparation of claim 6 is detected by thin layer chromatography, the fructus aurantii control extract, crude drug or traditional Chinese medicine preparation needs to be prepared into a solution for detection, wherein the preparation method of the fructus aurantii control extract solution is as follows: adding methanol into the fructus Aurantii control extract of claim 7 to obtain a methanol solution of the fructus Aurantii control extract with a concentration of 30mg/mL-80mg/mL, and filtering with 0.12-0.32 μm filter membrane to obtain a solution of the fructus Aurantii control extract;
optionally, the concentration of the bitter orange fetal control extract in methanol is 50 mg/mL; the filter membrane is 0.22 mu m;
optionally, the detection condition of the thin layer chromatography is detection condition one or detection condition two, and the detection condition one is:
thin-layer plate: TLC G60 precast slab;
sample application: 2 μ l, strip spotting;
developing agent:
and (3) flavonoid glycoside: chloroform-methanol-water, volume ratio 13: 6: 2, lower layer solution;
and (6) inspection: spraying 3% aluminum trichloride ethanol solution, heating at 105 deg.C for 5min, and inspecting under ultraviolet light with wavelength of 366 nm;
the second detection condition is as follows:
thin-layer plate: TLC G60 precast slab;
sample application: 2 μ l, strip spotting;
developing agent:
flavonoid aglycone: s1: toluene-ethyl acetate-formic acid-water in a volume ratio of 20: 20: 1:1, upper solution;
s2, toluene-ethyl acetate-formic acid-water, wherein the volume ratio is 20: 10: 1:1, upper solution;
and (6) inspection: directly placed under ultraviolet light with the wavelength of 366nm for inspection.
9. The use of claim 8, wherein the fructus Aurantii control extract, crude drug or Chinese medicinal preparation of claim 6 is prepared into a solution for testing by high performance liquid chromatography, wherein the fructus Aurantii control extract solution is prepared by: adding methanol into the fructus Aurantii control extract of claim 6 to obtain a methanol solution of the fructus Aurantii control extract with a concentration of 0.1mg/mL-1mg/mL, and filtering with 0.12-0.32 μm filter membrane to obtain a solution of the fructus Aurantii control extract;
optionally, the concentration of the fructus aurantii control extract in methanol solution is 0.6 mg/mL; the filter membrane is 0.22 mu m;
optionally, the detection conditions of the high performance liquid chromatography are as follows:
chromatography apparatus: vanqish ultra high performance liquid chromatograph;
a detector: a DAD detector;
a chromatographic column: agilent RRHD SB C18, 2.1 × 100mm,1.8 μm;
mobile phase: acetonitrile (a) -0.05% phosphoric acid solution (B) with gradient elution as specified in the table below;
detection wavelength: 320 nm; the flow rate is 0.4 ml/min; the sample volume is 2 mul; the column temperature is 30 ℃; operating time: and (5) 20 min.
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