CN109632978B - Poria cocos contrast extract as well as preparation method and application thereof - Google Patents
Poria cocos contrast extract as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a poria cocos contrast extract, which is derived from the following raw materials: extracting Poria coarse powder for multiple times, concentrating, drying to obtain Poria extract of different batches, and blending Poria extract of different batches to obtain Poria control extract. The poria cocos contrast extract is prepared by blending different batches of extracts, so that the consistency of different batches of poria cocos contrast extracts is ensured; and the character is stable, uniform and convenient to use. The poria cocos contrast extract is used as a contrast in the quality control of poria cocos and a medicinal material or a Chinese medicinal preparation containing active ingredients of poria cocos/poria cocos in the application, the poria cocos contrast extract can be directly used after being simply processed in the quality control process, the operation is simple and convenient, particularly when the content of multiple ingredients is measured, the preparation of a contrast extract solution is simpler and more convenient than that of a contrast solution, the qualitative identification of the medicinal material or the Chinese medicinal preparation can be carried out, and the poria cocos contrast extract can also be used for semi-quantitative or even quantitative analysis.
Description
Technical Field
The invention relates to the technical field of quality control of traditional Chinese medicines, in particular to a preparation method of a poria cocos contrast extract.
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
The invention solves the technical problem of providing a tuckahoe contrast extract which has good batch consistency, stable and uniform properties and is convenient to use, and also provides a preparation method and application of the tuckahoe contrast extract, namely, the tuckahoe contrast extract is used for quality control of tuckahoe, medicinal materials or Chinese medicinal preparations containing tuckahoe/tuckahoe active ingredients.
In a first aspect, the present invention provides a control extract of poria cocos wolf, derived from: extracting Poria crude powder of different batches for multiple times to obtain Poria extract of different batches, and blending Poria extract of different batches to obtain Poria control extract.
Preferably, the chromatogram obtained by the thin layer chromatography and/or the high performance liquid chromatography of the tuckahoe control extract is consistent with the corresponding raw material medicine.
Preferably, the chromatogram obtained by detecting the tuckahoe control extract by adopting the thin-layer chromatography and/or the high performance liquid chromatography is consistent with the tuckahoe control medicinal material.
In a second aspect, the present invention provides a method for preparing a poria cocos contrast extract, comprising the steps of:
step one, extraction: mixing coarse powder of Poria cocos medicinal material with ethanol, extracting, filtering to obtain filtrate 1 and filter residue 1, and concentrating the filtrate 1 to obtain Poria cocos dry extract;
step two, preparing the tuckahoe extract: dissolving the obtained Poria dry extract in ethanol to obtain Poria ethanol solution, adding adjuvants, drying, and sieving to obtain Poria extract;
step three, blending: and mixing different batches of poria cocos extracts to obtain the poria cocos control extract.
Preferably, the extraction, concentration and drying in steps one to three are performed by means of low-temperature treatment.
More preferably, the low temperature is 0-60 DEG C
Preferably, in the first step, the filter residue 1 is extracted for N times according to the extraction method in the first step, filtrate obtained by the N times of extraction is combined with the filtrate 1 to obtain extract, and the extract is concentrated to obtain dry poria extract; wherein the extraction times are N times, wherein N is more than or equal to 6 and more than or equal to 0, and N is an integer.
More preferably, N is 1.
Preferably, the concentration of the ethanol in the first step is 30-95%;
more preferably, the ethanol concentration of the extraction solution in the first step is 95%.
Preferably, the ratio of the weight of the poria cocos crude powder to the volume of the ethanol in the step one is 1: 5-1: 50.
More preferably, the ratio of the weight of the poria cocos crude powder to the volume of the ethanol in the step one is 1: 10.
preferably, the extraction mode in the first step is ultrasonic extraction.
Preferably, the ultrasonic extraction time in the step one is 1-60 min.
More preferably, the ultrasonic extraction time in the first step is 30 min.
Preferably, the filtration in the first step is medium-speed filter paper or 2000-mesh screen.
More preferably, the filtration in the first step is performed by using medium-speed filter paper.
Preferably, the concentration of ethanol in the second step is 30-95%.
More preferably, the concentration of ethanol in the second step is 95%.
Preferably, the weight-to-volume ratio of the poria cocos extract to the ethanol in the second step is as follows: 1: 5-1: 50.
More preferably, the weight-to-volume ratio of the poria cocos extract to the ethanol in the second step is as follows: 1: 10.
preferably, the auxiliary materials used in the second step are micro-powder silica gel and/or medicinal starch.
More preferably, the auxiliary material is micropowder silica gel.
Preferably, the second step is to add aerosil which accounts for 20 to 60 percent of the weight of the tuckahoe dry paste into the ethanol solution of the tuckahoe.
More preferably, the third step is to add aerosil 40% of the weight of tuckahoe dry paste into the ethanol solution of tuckahoe.
Preferably, the third step is drying and then filtering by a screen of 90-200 meshes.
More preferably, the drying in step three is followed by filtering through a 110 mesh screen.
Preferably, a step of detecting different batches of poria cocos extracts by thin layer chromatography and/or high performance liquid chromatography is further included between the second step and the third step.
Preferably, the third step of blending makes the spectrum of the finally obtained control extract consistent with the spectrum of the corresponding raw material medicine.
More preferably, the spectrum is obtained by thin layer chromatography and/or high performance liquid chromatography.
In the third aspect of the invention, the application of the tuckahoe contrast extract in the identification of medicinal materials or Chinese medicinal preparations or the quality control of tuckahoe or medicinal materials or Chinese medicinal preparations containing tuckahoe effective components is provided.
Preferably, the identification of the medicinal material or the Chinese medicinal preparation, or the quality control method of the tuckahoe or the medicinal material or the Chinese medicinal preparation containing tuckahoe/tuckahoe effective components comprises the following steps: detecting the Poria control extract, medicinal material or Chinese medicinal preparation by thin layer chromatography and/or high performance liquid chromatography, and comparing.
Preferably, when the poria cocos control extract and the medicinal material or the Chinese medicinal preparation are detected by thin layer chromatography and/or high performance liquid chromatography, the poria cocos control extract, the medicinal material or the Chinese medicinal preparation are required to be prepared into a solution.
Preferably, the preparation method of the tuckahoe contrast extract solution comprises the following steps: adding methanol into the Poria contrast extract, ultrasonic filtering, and collecting filtrate to obtain Poria contrast extract solution.
Preferably, the detection conditions of the thin layer chromatography are as follows:
thin-layer plate: TLC G60 precast slabs (Merck);
sample application: 10 mul, strip sample length 10 mm;
developing agent: s1: chloroform: acetonitrile: methanol 13:2.5:1 developed 5.5cm
S2: cyclohexane: ethyl acetate: methanol: formic acid 15:5:0.5:0.5 developed 8cm
Drying the thin-layer plate: placing the sample-applied thin-layer plate in a thin-layer heating plate, heating at 50 ℃ for 15min, and ensuring the drying of the thin-layer plate;
and (6) inspection: soaking in 3% ethyl acetate sulfate, heating at 105 deg.C for 1.5min, and inspecting under UV366 nm.
Preferably, the detection conditions of the high performance liquid chromatography are as follows:
chromatography apparatus: agilent HPLC-1100 HPLC with DAD detector (Agilent Technologies, USA)
A chromatographic column:
(4.6mm×250mm,5μm)；
mobile phase: a-acetonitrile, B-0.1% phosphoric acid aqueous solution;
gradient elution procedure: 0-15 min: 45% A → 55% A, 15 → 50 min: 55% A → 75% A, 50 → 60 min: 75% A → 90% A, 60 → 65min: 90% A → 100% A, 65 → 75min: 100% A.
Detection wavelength 210nm (DAD detector); the flow rate is 1 ml/min; the sample amount is 10 mul; the column temperature is 20 ℃; operating time: and (5) 75 min.
Compared with the prior art, the invention has the beneficial effects that:
the poria cocos contrast extract is prepared from 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 production places and growth environments on traditional Chinese medicine contrast medicinal materials is overcome, and the consistency of the poria cocos contrast 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, uniform and convenient to use. In the application of the poria cocos contrast extract, the poria cocos contrast extract is used as a contrast for quality control of poria cocos and a medicinal material or a Chinese medicinal preparation containing poria cocos/poria cocos active ingredients after quantitative analysis, in the quality control process, the poria cocos contrast extract can be directly used after being simply processed, the operation is simple and convenient, particularly when the content of multiple components is measured, the preparation of a contrast extract solution is simpler and more convenient than that of a contrast product solution, the poria cocos contrast extract can be qualitatively identified, and the poria cocos contrast extract can also be used for semi-quantitative or even quantitative analysis.
Drawings
FIG. 1: is a high-efficiency thin-layer chromatogram obtained by carrying out thin-layer chromatography on a tuckahoe raw material medicine by taking a tuckahoe reference medicine as a reference substance. The reference material 1 corresponds to Poria cocos, and the reference materials 2-7 correspond to Poria cocos raw material 1, Poria cocos raw material 2, Poria cocos raw material 3, Poria cocos raw material 4, Poria cocos raw material 5 and Poria cocos raw material 6 in sequence.
FIG. 2: the poria cocos contrast medicinal material is an HPLC fingerprint map overlay obtained by performing high performance liquid chromatography on a poria cocos raw medicinal material by using a reference substance, R corresponds to a common mode, 1 corresponds to the poria cocos contrast medicinal material, and 2-7 correspond to the poria cocos raw medicinal material 1, the poria cocos raw medicinal material 2, the poria cocos raw medicinal material 3, the poria cocos raw medicinal material 4, the poria cocos raw medicinal material 5 and the poria cocos raw medicinal material 6 in sequence from top to bottom.
FIG. 3: poria cocos contrast extract preparation flow chart.
FIG. 4: is a high performance thin layer chromatogram obtained by performing thin layer chromatography on commercial medicinal materials with Poria contrast extract and Poria contrast medicinal materials as reference substances. 1 is Poria control extract, 2 is Poria control medicinal material, and 3-12 are sequentially Poria 1, Poria 2, Poria 3, Poria 4, Poria 5, Poria 6, Poria 7, Poria 8, Poria 9 and Poria 10.
FIG. 5: the method is characterized in that a high-efficiency thin-layer chromatogram obtained by carrying out thin-layer chromatography on commercially available medicinal materials by taking a poria cocos contrast extract and a poria cocos contrast medicinal material as reference substances is adopted, wherein 1 corresponds to the poria cocos contrast extract, 2 corresponds to the poria cocos contrast medicinal material, and 3-12 sequentially correspond to the poria cocos test sample medicinal materials: poria cocos wolf 1, poria cocos wolf 2, poria cocos wolf 3, poria cocos wolf 4, poria cocos wolf 5, poria cocos wolf 6, poria cocos wolf 7, poria cocos wolf 8, poria cocos wolf 9 and poria cocos wolf 10.
FIG. 6: scanning the thin-layer chromatography image in the figure 5 by using ChemPattern software to obtain a scanning map, wherein the bands 1-12 correspond to the bands 1-12 in the chromatogram in the figure 5 respectively.
FIG. 7: is prepared by subjecting commercially available medicinal materials to high performance liquid chromatography to obtain HPLC fingerprint superposition chart with Poria contrast extract and Poria contrast medicinal material as reference substances, ERS corresponding to Poria contrast extract, 1 corresponding to Poria contrast medicinal material, and 2-11 from top to bottom corresponding to Poria 1, Poria 2, Poria 3, Poria 4, Poria 5, Poria 6, Poria 7, Poria 8, Poria 9 and Poria 10.
Interpretation of terms:
precisely weighing: the weighing is accurate to one thousandth of the weight.
Weighing: weighing the materials to be one percent of the weight.
Repeatedly: 1-7 times.
And (3) identification: the Poria contrast extract is used as reference substance for qualitative and quantitative analysis of Poria effective components in medicinal material or Chinese medicinal preparation
Quality control: the tuckahoe contrast extract is used as a contrast product to qualitatively and/or quantitatively analyze the tuckahoe and the medicinal materials or the Chinese medicinal preparations containing the tuckahoe/tuckahoe effective components, and whether the sample to be detected is the tuckahoe and/or the medicinal materials or the Chinese medicinal preparations containing the tuckahoe/tuckahoe effective components can be judged through analysis, so that the quality of the medicinal materials can be controlled.
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.
Instruments and materials:
poria cocos raw material medicinal materials:
6 batches of poria cocos raw medicinal materials are purchased in various national medicinal material markets: poria cocos raw material medicinal material 1, Poria cocos raw material medicinal material 2, Poria cocos raw material medicinal material 3, Poria cocos raw material medicinal material 4, Poria cocos raw material medicinal material 5 and Poria cocos raw material medicinal material 6 are identified as dry sclerotium of Poria cocos (Schww.) Wolf of Polyporaceae by professor Shebei mountain.
The poria cocos test sample medicinal materials:
10 batches of tuckahoe 1, tuckahoe 2, tuckahoe 3, tuckahoe 4, tuckahoe 5, tuckahoe 6, tuckahoe 7, tuckahoe 8, tuckahoe 9 and tuckahoe 10 are purchased in large drug stores and medicinal material markets of Guangzhou.
Tuckahoe as a reference medicinal material: the Chinese food and drug testing institute; batch number: 121117-201509.
Agilent 1100series HPLC, equipped with a DAD detector (Agilent Technologies, USA).
Acetonitrile chromatogram (Merck)
Cyclohexane, methanol, ethanol, formic acid, sulfuric acid, phosphoric acid were all analytically pure (Guangzhou chemical laboratories).
Example 1: screening of poria cocos raw medicinal material
This example provides a screening method for the raw material of a control extract of poria cocos wolf.
Analyzing and screening Poria raw material by thin layer chromatography and high performance liquid chromatography respectively with Poria reference material as reference substance.
Tuckahoe as a reference medicinal material: the Chinese food and drug testing institute; batch number: 121117-201509.
poria cocos raw material medicinal materials: purchased in various large medicinal material markets nationwide, and identified as dry sclerotium of Poriacocos (Schww.) Wolf, a fungus of Polyporaceae by professor Shebechan. 6 batches of medicinal materials are identified to meet the standard and can be used for standby.
1 thin layer chromatography
1.1 sample preparation
Preparing a poria cocos raw medicinal material solution: weighing Poria cocos raw material coarse powder, sieving with a No. 4 sieve (250 + -9.9 um, 65 meshes), precisely weighing 2g, adding 100ml of 95% methanol-containing aqueous solution, performing ultrasonic treatment for 500w30min, filtering, evaporating filtrate to dryness, adding methanol into residue to constant volume of 2ml, and sieving with a 0.22 μm filter membrane to obtain a test solution of the medicinal material; poria control medicinal solution is prepared by the same method.
1.2 thin layer chromatography detection
The detection conditions were as follows:
thin-layer plate: TLC G60 precast slabs (Merck);
sample application: 10 mul, strip sample length 10 mm;
developing agent: s1: chloroform: acetonitrile: methanol 13:2.5:1 developed 5.5 cm;
s2: cyclohexane: ethyl acetate: methanol: formic acid 15:5:0.5:0.5 developed 8 cm;
the unfolding mode is as follows: heating the sample-applied thin-layer plate on a thin-layer heating plate at 50 ℃ for 15min to ensure the drying of the thin-layer plate;
and (6) inspection: soaking in 3% ethyl acetate sulfate, heating with thin layer heating plate at 105 deg.C for 1.5min, and inspecting under UV366 nm.
The results are shown in FIG. 1, which is a thin layer chromatogram (T: 20 ℃, RH: 40%) examined under an ultraviolet lamp (366 nm). 1 corresponds to a tuckahoe contrast medicinal material, and 2-7 sequentially correspond to: poria cocos raw material medicinal material 1, poria cocos raw material medicinal material 2, poria cocos raw material medicinal material 3, poria cocos raw material medicinal material 4, poria cocos raw material medicinal material 5 and poria cocos raw material medicinal material 6. As can be seen from fig. 1, each batch of the poria cocos raw material drug and the poria cocos contrast drug can show the same spots at the same position, the similarity between each batch of the poria cocos raw material drug and the poria cocos contrast drug is high, and the raw material drugs are primarily screened as the raw material drugs of the standard contrast extract.
2 high performance liquid chromatography
2.1 sample preparation
Preparing a poria cocos raw medicinal material solution: respectively sieving Poria cocos raw material coarse powder with a fourth sieve (250 + -9.9 um, 65 mesh), weighing 1g, placing in a 15ml centrifuge tube, precisely adding 5ml of 95% methanol, performing ultrasonic treatment at 500w for 30min, taking out, cooling to room temperature, filtering with a 0.22 μm filter membrane, and collecting filtrate to obtain Poria cocos raw material solution; poria control medicinal solution is prepared by the same method.
2.2 high Performance liquid chromatography detection
The detection conditions of the high performance liquid chromatography are as follows:
chromatography apparatus: agilent HPLC-1100 HPLC with DAD detector (Agilent Technologies, USA)
A chromatographic column:
(4.6mm×250mm,5μm)；
mobile phase: a-acetonitrile, B-0.1% phosphoric acid aqueous solution;
gradient elution procedure: 0-15 min: 45% A → 55% A, 15 → 50 min: 55% A → 75% A, 50 → 60 min: 75% A → 90% A, 60 → 65min: 90% A → 100% A, 65 → 75min: 100% A.
Detection wavelength 210nm (DAD detector); the flow rate is 1 ml/min; the sample amount is 10 mul; the column temperature is 20 ℃; operating time: and (5) 75 min.
The detection method of the high performance liquid chromatography comprises the following steps: precisely sucking 10 μ l each of Poria contrast solution and Poria raw material solution, and injecting into liquid chromatograph.
And (3) detecting results by high performance liquid chromatography:
the detection result is shown as an HPLC fingerprint map overlay chart shown in figure 2, and corresponds to the following steps from top to bottom in sequence from 2 to 7: poria cocos raw material medicinal material 1, poria cocos raw material medicinal material 2, poria cocos raw material medicinal material 3, poria cocos raw material medicinal material 4, poria cocos raw material medicinal material 5 and poria cocos raw material medicinal material 6; 1 corresponds to the control material of Poria cocos.
As can be seen from FIG. 2, the fingerprints of Poria cocos raw material 1-Poria cocos raw material 6 are highly consistent, so that a common fingerprint pattern of Poria cocos raw material is established, and similarity analysis is performed on all samples. Similarity evaluation is carried out on 6 batches of poria cocos medicinal materials and a proposed common mode according to the poria cocos reference medicinal material atlas and the component and peak area of each sample by adopting an included angle cosine algorithm, and the result is shown in table 1.
TABLE 1
| Sample (I) | Degree of similarity |
| Poria cocos |
0.907 |
| Poria cocos |
0.977 |
| Poria cocos |
0.985 |
| Poria cocos |
0.988 |
| Poria cocos |
0.955 |
| Poria cocos |
0.973 |
| Poria contrast medicinal material | 0.992 |
| Common mode | 1.000 |
According to the above similarity analysis results, the similarity of 6 batches of tuckahoe raw material medicinal materials and tuckahoe reference medicinal materials is very high, and the tuckahoe raw material medicinal materials can be selected as tuckahoe standard reference extracts.
Example 2: preparation of Poria control extract
This example provides a method for preparing the control extract of Poria cocos wolf of the present invention, the flow chart of which is shown in FIG. 1.
Firstly, the method comprises the following steps: preparation of dried paste
Selecting the raw material medicinal materials which are identified in the example 1 and can be used for preparing the poria standard control extract, crushing the raw material medicinal materials into coarse powder, sieving the coarse powder with a No. four sieve (250 +/-9.9 um, 65 meshes), adding 95% ethanol with the volume of 10 times of the mass of the coarse powder (namely the solid-to-liquid ratio is 1: 10(g/ml), carrying out ultrasonic extraction (with the power of 500w and the temperature of 40 ℃) for 30min, filtering the mixture with medium-speed qualitative filter paper (a filter membrane with the diameter of 0.22 mu m), collecting filter residue 1 and filtrate 1, carrying out secondary extraction on the filter residue 1 according to the same method, combining the filtrate collected for the secondary extraction with the filtrate 1 to obtain an extracting solution, and concentrating the extracting solution at the temperature of 50 ℃ to obtain dry paste.
II, secondly: preparation of Poria extract
Dissolving the extract with 95% ethanol aqueous solution 10 times the weight of the dry extract (weight/volume ratio of 1: 10(g/ml)), adding silica gel micropowder 40% of the weight of the dry extract, recovering solvent at 60 deg.C with rotary evaporator, pulverizing, and sieving with 110 mesh sieve to obtain Poria extract.
6 batches of tuckahoe extract are prepared by using 6 batches of tuckahoe raw medicinal materials.
Thirdly, the method comprises the following steps: blending
And (3) mixing and blending the 6 batches of poria cocos extracts in the step (II) according to the proportion of 1:1:1:1:1:1 to obtain a poria cocos control extract, wherein the proportion of the corresponding raw medicinal materials of the final product is about 1: 33 (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 repeated experiments, comprehensive stability, consistency, later application and the like.
Fourthly, the method comprises the following steps: detection of
Determining the poria cocos contrast extract prepared in the third step by using a high performance liquid chromatography to obtain a fingerprint, and performing similarity detection with a common mode spectrum of the raw material medicines (the common mode spectrum of the poria cocos raw material medicines established in example 1), wherein as shown in fig. 4, ERS is the poria cocos contrast extract, R is the common mode of the poria cocos raw material medicines, the obtained similarity is 0.993, and the similarity of the common mode of the poria cocos contrast extract and the fingerprint spectrum of the poria cocos medicines is high, which indicates that the poria cocos contrast extract prepared by the method can represent the poria cocos medicines.
Example 3 analysis of the Properties of control extract of Poria cocos
1. Apparent state: the control extract of poria cocos wolf obtained in example 2 was a pale yellow-white coarse 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 tuckahoe contrast extract is 1.29 percent.
3. And (3) testing consistency: the poria cocos control extract was prepared 6 times in parallel according to the method of example 2, and it was determined that the difference in the thin layer chromatography detection pattern of the poria cocos control extract prepared each time was small. Therefore, the consistency of the tuckahoe contrast extract prepared by the preparation method of the tuckahoe contrast extract is very good.
4. And (3) stability testing:
a sample of a poria cocos control extract prepared by taking 6 different batches of poria cocos raw material medicines according to the method in example 2 is examined for indexes including properties and solubility according to relevant regulations of 'national drug standard substance development technical requirements' formulated by the Commission of the national pharmacopoeia.
The sample is placed in a clean container with an opening at 60 deg.C for 10 days, sampled on the 5 th and 10 th days, and tested according to the stability focus examination item.
The result shows that the properties of the test sample do not obviously change before and after the high-temperature test, the thin-layer chromatogram does not obviously change before and after the high-temperature test, the determination result of the dissolution rate is subjected to independent sample t-test by SPSS, the calculation result P is more than 0.05, which indicates that no significant difference exists, and the test stability of the poria cocos contrast extract is good.
Example 4 use of Poria cocos control extract
Analyzing commercially available medicinal materials (medicinal material samples) by thin layer chromatography and high performance liquid chromatography with Poria control extract and Poria control material as reference substances.
1 thin layer chromatography
1.1 sample preparation
Poria cocos control extract solution: precisely weighing 50mg of the poria cocos control extract prepared in example 2, placing the poria cocos control extract in a 2mL volumetric flask, adding 1.5mL of 95% methanol, carrying out ultrasonic treatment (500w) for 30 minutes, cooling to room temperature, fixing the volume to the scale with methanol, and filtering with a 0.22 mu m filter membrane to obtain the poria cocos control extract solution.
Poria cocos test sample medicinal material solution: weighing Poria sample crude powder (sieved with No. four sieve) 2g each, adding 100ml 95% methanol, ultrasonic treating for 30min, filtering with 0.22 μm filter membrane, evaporating filtrate, adding methanol into residue to constant volume of 2ml, and filtering with 0.22 μm filter membrane to obtain the final product; poria control medicinal solution is prepared by the same method.
1.2 thin layer chromatography detection
The detection conditions were as follows:
thin-layer plate: TLC G60 precast slabs (Merck);
sample application: 10 mul, strip sample length 10 mm;
developing agent: s1: chloroform: acetonitrile: methanol 13:2.5:1 developed 5.5cm
S2: cyclohexane: ethyl acetate: methanol: formic acid 15:5:0.5:0.5 developed 8cm
Drying the thin-layer plate: placing the sample-applied thin-layer plate in a thin-layer heating plate, heating at 50 ℃ for 15min, and ensuring the drying of the thin-layer plate;
and (6) inspection: soaking in 3% ethyl acetate sulfate, heating at 105 deg.C for 1.5min, and inspecting under UV366 nm.
The results of the detection are shown in FIG. 5, which is a thin layer chromatogram examined under UV366nm (T: 20 ℃, RH: 40%). 1 is a tuckahoe contrast extract, 2 is a tuckahoe contrast medicinal material, and 3-12 are tuckahoe test sample medicinal materials in sequence: poria cocos wolf 1, poria cocos wolf 2, poria cocos wolf 3, poria cocos wolf 4, poria cocos wolf 5, poria cocos wolf 6, poria cocos wolf 7, poria cocos wolf 8, poria cocos wolf 9 and poria cocos wolf 10.
And (4) analyzing results: as can be seen from FIG. 5, the control extract and the sample material (Poria 1-Poria 10) both showed the same spots at the same position, and the spectrum showed that the control extract and the sample material (Poria 1-Poria 10) have high consistency.
The scanning map of the thin-layer chromatography image of the image in fig. 5 can be obtained by scanning the thin-layer chromatography image through ChemPattern software of beijing kameyen, as shown in fig. 6.
2 high performance liquid chromatography
2.1 sample preparation
Poria cocos control extract solution: weighing control extract 12mg, placing in 2mL volumetric flask, adding methanol 1.5mL, treating with ultrasound 500w for 30min, cooling to room temperature, adding 95% methanol to constant volume, filtering with 0.22 μm filter membrane, and collecting filtrate to obtain Poria control extract solution.
Poria cocos test sample medicinal material solution: weighing 1g of coarse powder of a poria cocos test sample medicinal material after passing through a No. four sieve, placing the coarse powder into a 15ml centrifuge tube, precisely adding 5ml of 95% methanol, performing ultrasonic treatment at 500w for 30 minutes, taking out the coarse powder, cooling the coarse powder to room temperature, passing through a 0.22-micron filter membrane, and taking a filtrate to obtain a poria cocos test sample medicinal material solution; poria control medicinal solution is prepared by the same method.
2.2 high Performance liquid chromatography detection
The detection conditions of the high performance liquid chromatography are as follows:
chromatography apparatus: agilent HPLC-1100 HPLC with DAD detector (Agilent Technologies, USA)
A chromatographic column:
(4.6mm×250mm,5μm)；
mobile phase: a-acetonitrile, B-0.1% phosphoric acid aqueous solution;
gradient elution procedure: 0-15 min: 45% A → 55% A, 15 → 50 min: 55% A → 75% A, 50 → 60 min: 75% A → 90% A, 60 → 65min: 90% A → 100% A, 65 → 75min: 100% A.
Detection wavelength 210nm (DAD detector); the flow rate is 1 ml/min; the sample amount is 10 mul; the column temperature is 20 ℃; operating time: and (5) 75 min.
The detection method of the high performance liquid chromatography comprises the following steps: precisely sucking 10 μ l each of Poria control extract solution, Poria control solution and sample solution, and injecting into liquid chromatograph.
And (3) detecting results by high performance liquid chromatography:
the detection result is shown as an HPLC fingerprint map overlay chart shown in figure 7, and corresponds to the following steps from top to bottom in sequence from 2 to 11: poria cocos wolf 1, poria cocos wolf 2, poria cocos wolf 3, poria cocos wolf 4, poria cocos wolf 5, poria cocos wolf 6, poria cocos wolf 7, poria cocos wolf 8, poria cocos wolf 9 and poria cocos wolf 10; ERS is corresponding to Poria control extract; 1 corresponds to the control material of Poria cocos.
As can be seen from FIG. 7, the fingerprint patterns of the Poria cocos test sample medicinal materials (Poria cocos 1-Poria cocos 10) have high consistency, so that a common fingerprint pattern of the Poria cocos test sample medicinal materials is established by processing data with ChemPattern software of Beijing Kemeien company. According to the spectrum of the tuckahoe contrast extract, similarity evaluation is carried out on 10 batches of tuckahoe medicinal materials, tuckahoe contrast medicinal materials and a proposed common mode by adopting an included angle cosine algorithm according to each sample component and the peak area thereof, and the result is shown in table 2.
TABLE 2
| Sample (I) | Degree of |
| Poria cocos | |
| 1 | 0.996 |
| |
0.982 |
| |
0.973 |
| |
0.925 |
| |
0.991 |
| |
0.978 |
| |
0.960 |
| |
0.951 |
| |
0.988 |
| |
0.947 |
| Poria contrast medicinal material | 0.985 |
| Poria cocos ERS | 1.000 |
| Common mode | 0.993 |
According to the similarity analysis results, the similarity of 10 batches of tuckahoe sample medicinal materials and the fingerprint spectrum of the tuckahoe contrast extract is in the range of 0.925 to 0.996, and the similarity is very high; similarity of common fingerprint pattern of Poria control extract and Poria control medicinal material is 0.985. The similarity of the common fingerprint patterns of the tuckahoe contrast extract and the tuckahoe medicinal material is 0.993, which shows that the tuckahoe contrast extract has good consistency with the medicinal material.
According to the results of TLC and HPLC fingerprint, the chromatogram obtained by detecting the tuckahoe contrast extract by adopting the thin-layer chromatography is consistent with the corresponding raw material medicinal material, so the tuckahoe contrast extract can be applied to the qualitative identification of medicinal materials or Chinese medicinal preparations.
Claims (1)
1. A thin layer chromatography detection method for Poria cocos contrast extract solution is characterized in that,
the preparation method of the tuckahoe contrast extract solution comprises the following steps:
(1) firstly, preparing a poria cocos contrast extract:
the poria control extract is derived from: extracting Poria cocos crude powder of different batches for multiple times to obtain Poria cocos extracts of different batches, and blending the Poria cocos extracts of different batches to obtain Poria cocos control extracts;
the chromatogram obtained by adopting thin layer chromatography and high performance liquid chromatography to the Poria cocos control extract is consistent with that of the corresponding raw material medicine;
detecting the Poria control extract by thin layer chromatography and high performance liquid chromatography to obtain chromatogram consistent with Poria control medicinal material;
the preparation method of the poria cocos contrast extract comprises the following steps:
step one, extraction: adding 95% ethanol 10 times the volume of the coarse powder of Poria cocos medicinal material, performing ultrasonic extraction with ultrasonic extraction power of 500w for 30min at 40 ℃, filtering with medium-speed filter paper to obtain filtrate 1 and residue 1, extracting the residue 1 again by the same method, mixing the filtrate 1 with the filtrate 1 to obtain an extract, and concentrating the extract at 50 ℃ to obtain Poria cocos dry extract;
step two, preparing the tuckahoe extract: dissolving the obtained Poria dry extract in ethanol to obtain Poria ethanol solution, adding adjuvants, drying, and sieving to obtain Poria extract;
step three, blending: mixing different batches of Poria extract to obtain Poria control extract;
the concentration of the ethanol in the step two is 95%, and the volume ratio of the weight of the tuckahoe extract to the ethanol is 1: 10, the auxiliary material is aerosil 40 percent of the weight of the dry paste of the tuckahoe, the drying temperature is 60 ℃, and the auxiliary material is filtered by a 110-mesh screen after drying;
the step two and the step three also comprise a step of detecting poria cocos extracts of different batches by thin-layer chromatography and high performance liquid chromatography;
the third step of blending makes the finally obtained reference extract have the same spectrum as the corresponding raw material medicine;
(2) adding methanol into the poria cocos control extract, carrying out ultrasonic treatment for 30 minutes at the ultrasonic power of 500w, cooling to room temperature, filtering with a 0.22 mu m filter membrane, and taking filtrate, namely the poria cocos control extract solution;
the detection conditions of the thin layer chromatography are as follows:
thin-layer plate: TLC G60 precast slab;
sample application: 10 mul, strip sample length 10 mm;
developing agent: s1: chloroform: acetonitrile: methanol =13:2.5:1 spread 5.5 cm;
s2: cyclohexane: ethyl acetate: methanol: formic acid =15:5:0.5:0.5 spread 8 cm;
drying the thin-layer plate: heating the sample-applied thin-layer plate on a thin-layer heating plate at 50 ℃ for 15min to ensure the drying of the thin-layer plate;
and (6) inspection: soaking in 3% ethyl acetate sulfate, heating with thin layer heating plate at 105 deg.C for 1.5min, and inspecting under UV366 nm.
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| CN113514596B (en) * | 2021-04-28 | 2024-06-07 | 广州科曼生物科技有限公司 | Small polarity control extract of wolfberry fruit and its preparing process and application |
| CN113155573B (en) * | 2021-04-28 | 2022-12-13 | 广州科曼生物科技有限公司 | Fructus lycii large-polarity control extract and preparation method and application thereof |
| CN115372501B (en) * | 2022-07-27 | 2024-03-26 | 广州科曼生物科技有限公司 | Control extract of fritillaria thunbergii or fritillaria hubei, preparation method and application thereof |
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