CN113155572A - Pummelo peel foetus control extract and preparation method and application thereof - Google Patents
Pummelo peel foetus control extract and preparation method and application thereof Download PDFInfo
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- CN113155572A CN113155572A CN202110467614.0A CN202110467614A CN113155572A CN 113155572 A CN113155572 A CN 113155572A CN 202110467614 A CN202110467614 A CN 202110467614A CN 113155572 A CN113155572 A CN 113155572A
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- extract
- pummelo
- pummelo peel
- exocarpium citri
- citri grandis
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Abstract
The invention provides a preparation method of a pummelo peel reference extract, which comprises the steps of respectively carrying out multiple times of low-temperature extraction, low-temperature concentration and low-temperature drying on multiple batches of pummelo peel medicinal material powder to obtain different batches of pummelo peel extracts, and then blending the different batches of pummelo peel extracts to obtain the pummelo peel reference extract. The preparation method of the pummelo pee control extract is simple and convenient to operate, low in cost, good in repeatability and high in extraction rate; the preparation method for preparing the exocarpium Citri Grandis embryo control extract ensures the consistency of exocarpium Citri Grandis embryo control extracts of different batches, and has stable and uniform properties and convenient use; the thin-layer chromatography fingerprint and the HPLC fingerprint of the contrast extract finally obtained by the preparation method of the pummelo peel foetus contrast extract are consistent with those of medicinal materials.
Description
Technical Field
The invention relates to the technical field of traditional Chinese medicine quality control, in particular to a pummelo peel foetus control 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 growth 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 has four basic requirements (ASCS) for the traditional Chinese medicine control extract by Mr. schehren, which also is a control extract with the following basic conditions: the herbal material source is reliable and representative; specificity, Specificity of the detection method used; con-sistence, 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, there is a need for a control extract of exocarpium Citri Grandis embryo, and its preparation method and application. The invention provides a preparation method of a pummelo pee control extract, which is simple and convenient to operate, low in cost, good in repeatability and high in extraction rate; the invention also provides a pummelo pee control extract prepared by the preparation method of the pummelo pee control extract, and the prepared pummelo pee control extract has good consistency, stable and uniform properties and convenient use, and can reflect the full appearance of medicinal materials.
The invention provides a preparation method of a pummelo peel foetus control extract, which comprises the following steps:
step one, ethanol extraction, wherein the ethanol extraction comprises the following steps:
a) mixing exocarpium Citri Grandis embryo medicinal material powder with ethanol, flash-extracting, 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 pummelo pee dry paste;
step two, preparing the pummelo peel extract: dissolving the dried extract of the pummelo peel tires obtained in the step two in ethanol to obtain ethanol solution of the dried extract of the pummelo peel tires, adding auxiliary materials, drying at low temperature and sieving to obtain the pummelo peel tires extract;
step three, blending: blending the pummelo peel extracts of different batches to obtain the pummelo peel reference extract.
The temperature adopted for low-temperature concentration and low-temperature drying in the preparation process is 20-60 ℃.
The different batches of the pummelo peel extract are blended, and at least five batches of the pummelo peel 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-volume ratio of the exocarpium Citri Grandis powder to ethanol in the first step is 1: 5-1: 50, and the unit of the weight-volume ratio is g/mL.
More preferably, the weight volume ratio of the exocarpium citri grandis powder to the ethanol in the first step is 1: 5.
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 50%.
Preferably, the flash extraction time in the step one is 1-3 min, and the power is 100W-3 kW.
More preferably, the flashing 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-volume ratio of the pummelo peel extract dry paste 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 50%.
Preferably, the weight-volume ratio of the pummelo pee extract dry paste to the ethanol in the second step is 1: 5.
Preferably, the weight of the auxiliary materials is 20 to 60 percent of the dry extract weight of the pummelo pee.
Preferably, the weight of the auxiliary materials is 30% of the dry extract of pummelo pee.
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 pre-blending treatment step of detecting different batches of pummelo peel 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 exocarpium Citri Grandis obtained by the above preparation method.
Preferably, the obtained chromatogram of the exocarpium Citri Grandis control extract by thin layer chromatography and/or high performance liquid chromatography is consistent with that of the corresponding raw material.
Preferably, the obtained spectrum of the exocarpium Citri Grandis control extract by thin layer chromatography and/or high performance liquid chromatography is consistent with the spectrum of corresponding raw material at naringin position.
Preferably, the pummelo peel control extract contains naringin as main ingredient.
The third aspect of the invention provides the application of the pummelo pee control extract in the identification of medicinal materials or traditional Chinese medicine preparations, or in the quality control of pummelo pee or medicinal materials or traditional Chinese medicine preparations containing pummelo pee/pummelo pee effective components.
Preferably, the identification of the medicinal materials or the Chinese medicinal preparations, or the quality control method of the medicinal materials or the Chinese medicinal preparations containing the effective components of the pummelo pee or the pummelo pee/pummelo pee comprises the following steps: detecting the exocarpium Citri Grandis control extract, medicinal materials 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 control extract, the medicinal material or the Chinese medicinal preparation of the pummelo pee, wherein the main components comprise naringin.
Preferably, when the exocarpium citri grandis embryo control extract, the crude drug or the traditional Chinese medicine preparation is detected by thin layer chromatography, the exocarpium citri grandis embryo control extract, the crude drug or the traditional Chinese medicine preparation needs to be prepared into a solution for detection, wherein the preparation method of the exocarpium citri grandis embryo control extract solution comprises the following steps: adding methanol into the pummelo peel reference extract of claim 6 to prepare a pummelo peel reference extract methanol solution with the concentration of 10mg/mL-50mg/mL, and filtering the pummelo peel reference extract methanol solution with a filter membrane of 0.12-0.32 μm to obtain the pummelo peel reference extract solution, wherein the methanol concentration is 100%.
Preferably, the concentration of the methanol solution of the exocarpium Citri Grandis control extract is 40 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 1-20 mL of methanol, cold soaking for 1-30 min, performing ultrasonic treatment for 1-30 min, centrifuging for 1-20 min after the power is 100W-3 kW, rotating at the speed of 1000-5000 r/min, and filtering the supernatant through a 0.12-0.32 mu m filter membrane to obtain a solution of the product to be detected; preferably weighing 0.5g of the product to be detected, adding 5mL of methanol, cold soaking for 15min, performing ultrasonic treatment for 15min at the power of 500w, centrifuging for 10min at the rotating speed of 4000 r/min, and filtering the supernatant with 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 one or two, and the one is:
thin-layer plate: HPTLC G60 precast slab;
sample application: comparison products: 2 μ l, 5 μ l, strip spotting;
developing agent:
and (3) flavonoid glycoside: chloroform-methanol-water-glacial acetic acid (13: 4: 1: 1.5) lower layer solution;
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:
and (3) flavonoid glycoside: spraying 5% aluminum trichloride ethanol solution, immediately inspecting under ultraviolet light (366nm), spraying 5% vanillin 10% sulphuric acid ethanol mixed solution, heating at 105 deg.C until the spots are clear, and inspecting under visible light;
flavonoid aglycone: 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: ethyl acetate-acetone-glacial acetic acid-water (8: 4: 0.3: 1);
and (6) inspection: spraying 5% aluminum trichloride ethanol solution, heating at 105 deg.C for 1min, and inspecting under ultraviolet lamp (365 nm);
optionally, when the exocarpium Citri Grandis control extract, medicinal material or Chinese medicinal preparation is detected by high performance liquid chromatography, the exocarpium Citri Grandis control extract, medicinal material or Chinese medicinal preparation is required to be prepared into solution for detection, wherein the preparation method of the exocarpium Citri Grandis control extract solution comprises: adding methanol into the exocarpium Citri Grandis control extract of claim 6 to obtain a methanol solution of exocarpium Citri Grandis control extract with concentration of 2mg/mL-15mg/mL, and filtering with 0.12-0.32 μm filter membrane to obtain exocarpium Citri Grandis control extract solution with methanol concentration of 100%.
Preferably, the concentration of the methanol solution of the pummelo pee control extract is 2.5 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 10-100 mL volumetric flask, adding 5-50 mL of methanol, carrying out ultrasonic treatment for 30-60 min, keeping the power at 100W-3 kW, cooling, fixing the volume of 50% methanol to a 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 0.5g of product to be detected, placing the product into a 100mL volumetric flask, adding 50mL of methanol, carrying out ultrasonic treatment for 30min with the power of 500W, cooling, fixing the volume of 50% of methanol to the scale, shaking up, and filtering with 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%.
Preferably, the detection conditions of the high performance liquid chromatography described above are:
chromatography apparatus: ThermoFishe U3000 high performance liquid chromatograph;
a detector: a ThermoFishe DAD detector;
a chromatographic column: zorbax SB C18 (4.6X 250mm,5 μm)
Mobile phase: methanol-0.3% acetic acid solution (35: 65);
detection wavelength: 320 nm; the flow rate is 1 ml/min; the sample amount is 10 mul; the column temperature is 35 ℃; operating time: 50 min;
the invention has the following beneficial effects:
the preparation method of the exocarpium citri grandis embryo reference extract adopts the steps of extracting, preparing the exocarpium citri grandis embryo extract and blending, and has the advantages of simple and convenient operation, low cost, good repeatability and high extraction rate. The pummelo peel foetus 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 to be consistent due to the influence of the traditional Chinese medicine control medicinal materials on the producing area and the growth environment is overcome, and the consistency of the pummelo peel foetus 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 the HPLC fingerprint of the contrast extract finally obtained by the preparation method of the pummelo peel foetus contrast 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 pummelo pee or the medicinal materials or the traditional Chinese medicine preparation containing the effective components of the pummelo pee/the pummelo pee, 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 performing thin layer chromatography on exocarpium Citri Grandis raw material with naringin as reference substance. 1 is a reference product naringin, and 2-16 correspond to pummelo peel embryo raw material medicines in sequence: pummelo peel raw medicinal materials 1, pummelo peel raw medicinal materials 2, pummelo peel raw medicinal materials 3, pummelo peel raw medicinal materials 4, pummelo peel raw medicinal materials 5, pummelo peel raw medicinal materials 6, pummelo peel raw medicinal materials 7, pummelo peel raw medicinal materials 8, pummelo peel raw medicinal materials 9, pummelo peel raw medicinal materials 10, pummelo peel raw medicinal materials 11, pummelo peel raw medicinal materials 12, pummelo peel raw medicinal materials 13, pummelo peel raw medicinal materials 14 and pummelo peel raw medicinal materials 15.
FIG. 2 is a thin layer chromatogram obtained by performing thin layer chromatography on exocarpium Citri Grandis raw material with naringin as reference substance. 1 is a reference product naringin, and 2-16 correspond to pummelo peel embryo raw material medicines in sequence: pummelo peel raw medicinal materials 1, pummelo peel raw medicinal materials 2, pummelo peel raw medicinal materials 3, pummelo peel raw medicinal materials 4, pummelo peel raw medicinal materials 5, pummelo peel raw medicinal materials 6, pummelo peel raw medicinal materials 7, pummelo peel raw medicinal materials 8, pummelo peel raw medicinal materials 9, pummelo peel raw medicinal materials 10, pummelo peel raw medicinal materials 11, pummelo peel raw medicinal materials 12, pummelo peel raw medicinal materials 13, pummelo peel raw medicinal materials 14 and pummelo peel raw medicinal materials 15.
FIG. 3 is a high performance thin layer chromatogram obtained by performing thin layer chromatography on exocarpium Citri Grandis embryo raw material. 1-15 correspond to pummelo peel raw material medicinal materials in sequence: pummelo peel raw medicinal materials 1, pummelo peel raw medicinal materials 2, pummelo peel raw medicinal materials 3, pummelo peel raw medicinal materials 4, pummelo peel raw medicinal materials 5, pummelo peel raw medicinal materials 6, pummelo peel raw medicinal materials 7, pummelo peel raw medicinal materials 8, pummelo peel raw medicinal materials 9, pummelo peel raw medicinal materials 10, pummelo peel raw medicinal materials 11, pummelo peel raw medicinal materials 12, pummelo peel raw medicinal materials 13, pummelo peel raw medicinal materials 14 and pummelo peel raw medicinal materials 15.
FIG. 4 is a HPLC fingerprint chromatogram overlay obtained by performing high performance liquid chromatography on exocarpium Citri Grandis embryo raw material, wherein R corresponds to exocarpium Citri Grandis embryo raw material common mode (common mode), and 1-15 correspond to exocarpium Citri Grandis embryo raw material in sequence: pummelo peel raw medicinal materials 1, pummelo peel raw medicinal materials 2, pummelo peel raw medicinal materials 3, pummelo peel raw medicinal materials 4, pummelo peel raw medicinal materials 5, pummelo peel raw medicinal materials 6, pummelo peel raw medicinal materials 7, pummelo peel raw medicinal materials 8, pummelo peel raw medicinal materials 9, pummelo peel raw medicinal materials 10, pummelo peel raw medicinal materials 11, pummelo peel raw medicinal materials 12, pummelo peel raw medicinal materials 13, pummelo peel raw medicinal materials 14 and pummelo peel raw medicinal materials 15.
FIG. 5 is a flow chart of the preparation of control extract of exocarpium Citri Grandis of the present invention.
FIG. 6 shows that the control extract of exocarpium Citri Grandis embryo is measured by high performance liquid chromatography to obtain fingerprint, and similarity detection is performed with common mode spectrum of raw material medicinal materials, ERS corresponds to the control extract of exocarpium Citri Grandis embryo, and R is common mode of exocarpium Citri Grandis embryo raw material medicinal materials.
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 pummelo peel foetus.
ThermoFishe U3000 HPLC, ThermoFishe DAD detector.
Methanol chromatographically pure (Merck), vanillin (Macklin).
The purity of the methanol not marked by the invention is more than or equal to 99.5 percent.
The 5% vanillin 10% sulphuric acid ethanol mixed solution related by the invention is an ethanol solution containing 5% vanillin by volume percentage and 10% sulphuric acid by volume percentage.
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).
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%);
testing medicinal material exocarpium citri grandis:
15 raw material medicaments of pummelo peel embryo are purchased in Guangdong Huazhou: pummelo peel raw medicinal materials 1, pummelo peel raw medicinal materials 2, pummelo peel raw medicinal materials 3, pummelo peel raw medicinal materials 4, pummelo peel raw medicinal materials 5, pummelo peel raw medicinal materials 6, pummelo peel raw medicinal materials 7, pummelo peel raw medicinal materials 8, pummelo peel raw medicinal materials 9, pummelo peel raw medicinal materials 10, pummelo peel raw medicinal materials 11, pummelo peel raw medicinal materials 12, pummelo peel raw medicinal materials 13, pummelo peel raw medicinal materials 14 and pummelo peel raw medicinal materials 15.
Example 1: screening of pummelo peel raw material medicinal materials
The embodiment provides a screening method of raw medicinal materials of a pummelo pee control extract.
Analyzing and screening exocarpium Citri Grandis embryo raw materials by high performance thin layer chromatography and high performance liquid chromatography respectively with exocarpium Citri Grandis embryo reference material as reference substance.
Exocarpium Citri Grandis embryo medicinal material is purchased from large medicinal material markets in China, and identified as immature or near mature dry fruit of Citrus grandis (Citrus grandis 'Tomentosa') of Rutaceae by professor Shebei mountain. 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 naringin reference substance, preparing into 1mg/mL solution with methanol, and filtering with 0.22 μm filter membrane to obtain reference substance solution.
Raw material medicinal material solution: taking powder of exocarpium Citri Grandis embryo raw material (1-15), sieving with a four-mesh sieve (250 + -9.9 μm, 65 mesh, the same below), precisely weighing 0.5g each, adding 5mL of methanol, cold soaking for 15min, ultrasonic treating for 15min with power of 500w, centrifuging for 10min at rotation speed of 4000 r/min, and filtering the supernatant with 0.22 μm filter membrane to obtain the raw material solution.
The detection conditions were as follows:
the method comprises the following steps:
thin-layer plate: HPTLC G60 precast slab;
sample application: comparison products: 2 μ l, strip spotting;
developing agent:
and (3) flavonoid glycoside: chloroform-methanol-water-glacial acetic acid (13: 4: 1: 1.5) lower layer solution;
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:
and (3) flavonoid glycoside: spraying 5% aluminum trichloride ethanol solution, immediately inspecting under ultraviolet light (366nm), spraying 5% vanillin 10% sulphuric acid ethanol mixed solution, heating at 105 deg.C until the spots are clear, and inspecting under visible light;
flavonoid aglycone: 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: ethyl acetate-acetone-glacial acetic acid-water (8: 4: 0.3: 1);
and (6) inspection: spraying 5% aluminum trichloride ethanol solution, heating at 105 deg.C for 1min, and inspecting under ultraviolet lamp (366 nm);
the detection results are shown in fig. 1-3, wherein fig. 1 shows that the thin-layer chromatogram is observed under an ultraviolet lamp (366nm) after the color development of the aluminum trichloride solution in the first method, fig. 2 shows that the color development of the aluminum trichloride solution in the first method is carried out, then the color development of the mixed solution of vanillin, sulfuric acid and ethanol is carried out, and the thin-layer chromatogram is observed under visible light, and fig. 3 shows that the thin-layer chromatogram is observed under the ultraviolet light (366nm) in the second method. Fig. 1 and 2: 1 is a reference product naringin, and 2-16 correspond to pummelo peel embryo raw material medicines in sequence: pummelo peel foetus raw medicinal material 1, pummelo peel foetus raw medicinal material 2, pummelo peel foetus raw medicinal material 3, pummelo peel foetus raw medicinal material 4, pummelo peel foetus raw medicinal material 5, pummelo peel foetus raw medicinal material 6, pummelo peel foetus raw medicinal material 7, pummelo peel foetus raw medicinal material 8, pummelo peel foetus raw medicinal material 9, pummelo peel foetus raw medicinal material 10, pummelo peel foetus raw medicinal material 11, pummelo peel foetus raw medicinal material 12, pummelo peel foetus raw medicinal material 13, pummelo peel foetus raw medicinal material 14, pummelo peel foetus raw medicinal material 15, fig. 3: 1-15 correspond to pummelo peel raw material medicinal materials in sequence: pummelo peel raw medicinal materials 1, pummelo peel raw medicinal materials 2, pummelo peel raw medicinal materials 3, pummelo peel raw medicinal materials 4, pummelo peel raw medicinal materials 5, pummelo peel raw medicinal materials 6, pummelo peel raw medicinal materials 7, pummelo peel raw medicinal materials 8, pummelo peel raw medicinal materials 9, pummelo peel raw medicinal materials 10, pummelo peel raw medicinal materials 11, pummelo peel raw medicinal materials 12, pummelo peel raw medicinal materials 13, pummelo peel raw medicinal materials 14 and pummelo peel raw medicinal materials 15. As can be seen from FIGS. 1-3, the same spots can be shown in the same positions of the raw material drugs of exocarpium Citri Grandis embryo, and the raw material drugs of exocarpium Citri Grandis embryo as the control extract are primarily screened.
2 high performance liquid chromatography
2.1 sample preparation
Raw material medicinal material solution: accurately weighing 0.5g of exocarpium Citri Grandis embryo crude drug (1-15) powder, placing in a 100mL volumetric flask, adding 50mL of methanol, ultrasonically treating for 30min at a power of 500W, cooling, diluting with 50% methanol to constant volume to scale, shaking, and filtering with 0.22 μm filter membrane to obtain crude drug solution.
2.2 high Performance liquid chromatography detection
The detection conditions of the high performance liquid chromatography are as follows:
chromatography apparatus: ThermoFishe U3000 high performance liquid chromatograph;
a detector: a ThermoFishe DAD detector;
a chromatographic column: zorbax SB C18 (4.6X 250mm,5 μm);
mobile phase: methanol-0.3% acetic acid solution (35: 65);
detection wavelength: 320 nm; the flow rate is 1 ml/min; the sample amount is 10 mul; the column temperature is 35 ℃; operating time: 50 min;
the detection method of the high performance liquid chromatography comprises the following steps: respectively taking 10 μ l of exocarpium Citri Grandis control solution and raw material solution, and injecting into liquid chromatograph.
And (3) detecting results by high performance liquid chromatography:
measuring, and recording chromatogram to obtain HPLC fingerprint overlay shown in figure 4, wherein R corresponds to common mode (common mode) of exocarpium Citri Grandis crude drug, and 1-15 correspond to exocarpium Citri Grandis crude drug in sequence: pummelo peel raw medicinal materials 1, pummelo peel raw medicinal materials 2, pummelo peel raw medicinal materials 3, pummelo peel raw medicinal materials 4, pummelo peel raw medicinal materials 5, pummelo peel raw medicinal materials 6, pummelo peel raw medicinal materials 7, pummelo peel raw medicinal materials 8, pummelo peel raw medicinal materials 9, pummelo peel raw medicinal materials 10, pummelo peel raw medicinal materials 11, pummelo peel raw medicinal materials 12, pummelo peel raw medicinal materials 13, pummelo peel raw medicinal materials 14 and pummelo peel raw medicinal materials 15.
As can be seen from fig. 4, the fingerprints of the pummelo peel primary medicinal material 1-the pummelo peel primary medicinal material 15 have high consistency, so that a common fingerprint pattern of the pummelo peel medicinal materials is established, and similarity analysis is performed on all samples. And (3) according to the common mode map, adopting an included angle cosine algorithm to evaluate the similarity of 15 batches of exocarpium citri grandis raw material medicines according to the components of each sample and the peak area of the component, wherein the result is shown in table 1.
TABLE 1
According to the similarity analysis results, the similarity of the raw medicinal materials of 15 batches of pummelo pee is very high, and the raw medicinal materials can be selected as the pummelo pee control extract.
Example 2: preparation of exocarpium Citri Grandis embryo control extract
This example provides a method for preparing a control extract of exocarpium Citri Grandis according to the present invention, and the flow chart of the method is shown in FIG. 5.
Firstly, the method comprises the following steps: extraction of
Selecting pummelo peel medicinal materials, preparing the pummelo peel medicinal materials into powder, sieving the medicinal material powder by a No. four sieve, adding 50 percent ethanol (namely the solid-liquid ratio is 1: 5(w/V, g/ml)) with the mass of 5 times of the volume of the pummelo peel medicinal materials, carrying out flash extraction (the power is 2000w, the normal temperature, the specific method refers to a flash extraction method in Shenrui. flash extraction application progress in the traditional Chinese medicine [ J ] the traditional Chinese medicinal materials, 2015,38(07): 1540-.
II, secondly: preparation of exocarpium Citri Grandis extract
Dissolving exocarpium Citri Grandis extract dry extract with 50% ethanol 5 times volume (w/V, g/ml) of exocarpium Citri Grandis extract dry extract to obtain 50% ethanol solution of exocarpium Citri Grandis extract dry extract, adding silica gel micropowder 30% of exocarpium Citri Grandis extract dry extract (adjuvant drug substance in mountain river, lot No. 170115), mixing, concentrating at low temperature (less than 45 deg.C) with rotary evaporator to dry, pulverizing, and sieving with 110 mesh sieve to obtain exocarpium Citri Grandis extract.
And preparing different batches of pummelo peel extract from the multiple batches of pummelo peel 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 the 15 batches of pummelo peel extract obtained in the step (II) according to the weight ratio of 1:1:1:1:1:1: 1:1:1:1:1:1: mixing and blending according to the ratio of 1:1:1 to obtain a pummelo peel reference extract, wherein the ratio of the final product to the pummelo peel raw medicinal materials is about 1: 16.95 (g/g).
The blending standard is as follows: the spectrum obtained by the finally obtained contrast extract is consistent with the spectrum of the corresponding raw medicinal material, and the content range of each component in the blending standard is also the best data obtained by comprehensively maintaining the stability and consistency through repeated experiments and the subsequent application.
Fourthly, the method comprises the following steps: detection of
Determining the exocarpium Citri Grandis embryo control 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. 6, ERS corresponds to exocarpium Citri Grandis embryo control extract, and R is common mode of exocarpium Citri Grandis embryo raw material medicinal materials. The obtained similarity is 1, and the similarity of the common fingerprint patterns of the pummelo peel embryo reference extract and the pummelo peel embryo raw medicinal material is high, which shows that the pummelo peel embryo reference extract can represent the pummelo peel embryo raw medicinal material.
Example 3: property analysis of pummelo peel embryo control extract
1. Apparent state: the control extracts of pummelo pee obtained in example 2 were all tan powder.
2. The moisture content was measured according to the first method (1, volumetric titration method P104) in the section of the 2015 th chinese pharmacopoeia qua 0832 moisture content measurement method. The detection result shows that the water content of the pummelo peel control extract is 3.89%.
3. And (3) testing consistency: 15 batches of exocarpium Citri Grandis control extract were prepared according to the method of example 2, and each batch had little difference in TLC detection pattern. Therefore, the pummelo pee control extract prepared by the preparation method of the pummelo pee control extract has very good consistency.
Claims (9)
1. A preparation method of pummelo peel foetus control extract is characterized by comprising the following steps:
step one, ethanol extraction, wherein the ethanol extraction comprises the following steps:
a) mixing exocarpium Citri Grandis embryo medicinal material powder with ethanol, flash-extracting, 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) for N times by analogy with the step b), and obtaining 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 pummelo pee dry paste;
step two, preparing the pummelo peel extract: dissolving the dried extract of the pummelo peel tires obtained in the step two in ethanol to obtain ethanol solution of the dried extract of the pummelo peel tires, adding auxiliary materials, drying at low temperature and sieving to obtain the pummelo peel tires extract;
step three, blending: blending the pummelo peel extracts of different batches to obtain the pummelo peel reference 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 exocarpium Citri Grandis powder to 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-volume ratio of the exocarpium Citri Grandis powder to ethanol in the first step is 1: 5;
optionally, the concentration of ethanol in the first step is 50-95%;
preferably, the concentration of ethanol in the first step is 50%;
optionally, the flash extraction time in the first step 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 pummelo pee extract dry extract 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 50%;
optionally, the weight-volume ratio of the pummelo pee extract dry paste to the ethanol in the step two is 1: 5;
optionally, the weight of the auxiliary materials is 20-60% of the dry extract weight of pummelo pee;
optionally, the weight of the auxiliary materials is 30% of the dry extract weight of pummelo pee;
optionally, the auxiliary material is micropowder silica gel;
optionally, after the third step of low-temperature drying, filtering the mixture by a sieve with 90-200 meshes;
optionally, the step three low-temperature drying is followed by sieving with a 110-mesh sieve.
5. The preparation method according to claim 1, further comprising a pre-blending treatment step of detecting different batches of exocarpium Citri Grandis extract by thin layer chromatography and/or high performance liquid chromatography between the second step and the third step.
6. A pummelo pee control extract is characterized in that the pummelo pee control extract is obtained by the preparation method of any one of claims 1 to 5;
optionally, the obtained chromatogram of the exocarpium Citri Grandis embryo control extract by thin layer chromatography and/or high performance liquid chromatography is consistent with that of the corresponding raw material;
optionally, the obtained chromatogram of the exocarpium Citri Grandis embryo control extract by thin layer chromatography and/or high performance liquid chromatography is consistent with the chromatogram of corresponding raw material medicinal material at naringin position;
optionally, the pummelo peel control extract contains naringin as main ingredient.
7. Use of the exocarpium Citri Grandis control extract of claim 6 in identification of medicinal materials or Chinese medicinal preparations, or quality control of exocarpium Citri Grandis or medicinal materials or Chinese medicinal preparations containing exocarpium Citri Grandis/exocarpium Citri Grandis effective components.
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 medicinal material or the Chinese medicinal preparation in the pummelo pee or the medicinal material or the Chinese medicinal preparation containing the pummelo pee/the pummelo pee effective components comprises the following steps: detecting the pummelo peel foetus control extract, medicinal material or traditional Chinese medicine preparation of claim 6 by thin-layer chromatography and/or high performance liquid chromatography, and comparing and judging;
optionally, the thin layer chromatography and/or high performance liquid chromatography is used for detecting a main component in a control extract, a medicinal material or a Chinese medicinal preparation of pummelo pee, wherein the main component comprises naringin;
optionally, when the exocarpium Citri Grandis embryo control extract, crude drug or Chinese medicinal preparation of claim 6 is detected by thin layer chromatography, the exocarpium Citri Grandis embryo control extract, crude drug or Chinese medicinal preparation is prepared into solution for detection, wherein the preparation method of the exocarpium Citri Grandis embryo control extract solution is as follows: adding methanol into exocarpium Citri Grandis control extract of claim 7 to obtain a methanol solution of exocarpium Citri Grandis control extract with concentration of 10mg/mL-50mg/mL, and filtering with 0.12-0.32 μm filter membrane to obtain exocarpium Citri Grandis control extract solution;
optionally, the concentration of the methanol solution of the pummelo peel control extract is 40 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: HPTLC G60 precast slab;
sample application: comparison products: 2 μ l, strip spotting;
developing agent:
and (3) flavonoid glycoside: chloroform-methanol-water-glacial acetic acid (13: 4: 1: 1.5) lower layer solution;
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:
and (3) flavonoid glycoside: spraying 5% aluminum trichloride ethanol solution, immediately inspecting under ultraviolet light (366nm), spraying 5% vanillin 10% sulphuric acid ethanol mixed solution, heating at 105 deg.C until the spots are clear, and inspecting under visible light;
flavonoid aglycone: 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: ethyl acetate-acetone-glacial acetic acid-water (8: 4: 0.3: 1);
and (6) inspection: spraying 5% ethanol solution of aluminum trichloride, heating at 105 deg.C for 1min, and inspecting under ultraviolet lamp (365 nm).
9. The use of claim 8, wherein the control extract, crude drug or Chinese medicinal preparation of exocarpium Citri Grandis according to claim 6 is prepared into solution by high performance liquid chromatography, and the detection is carried out, wherein the control extract, crude drug or Chinese medicinal preparation of exocarpium Citri Grandis is prepared by: adding methanol into exocarpium Citri Grandis control extract of claim 6 to obtain exocarpium Citri Grandis control extract methanol solution with concentration of 2mg/mL-15mg/mL, and filtering with 0.12-0.32 μm filter membrane to obtain exocarpium Citri Grandis control extract solution;
optionally, the concentration of the methanol solution of the pummelo peel control extract is 2.5 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: ThermoFishe U3000 high performance liquid chromatograph;
a detector: a ThermoFishe DAD detector;
a chromatographic column: zorbax SB C18 (4.6X 250mm,5 μm);
mobile phase: methanol-0.3% acetic acid solution (35: 65);
detection wavelength: 320 nm; the flow rate is 1 ml/min; the sample amount is 10 mul; the column temperature is 35 ℃; operating time: and (5) 50 min.
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