CN108088715B - Moutan bark reference extract and preparation method and application thereof - Google Patents
Moutan bark reference extract and preparation method and application thereof Download PDFInfo
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- CN108088715B CN108088715B CN201711279755.XA CN201711279755A CN108088715B CN 108088715 B CN108088715 B CN 108088715B CN 201711279755 A CN201711279755 A CN 201711279755A CN 108088715 B CN108088715 B CN 108088715B
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
Abstract
The invention provides a moutan bark control extract, which is derived from the following raw materials: extracting the tree peony bark medicinal material powder of different batches for multiple times to obtain tree peony bark extracts of different batches, and blending the tree peony bark extracts of different batches to obtain tree peony bark reference extracts. The moutan bark control extract is prepared by blending extracts of different batches, so that the consistency of the moutan bark control extracts of different batches is ensured; and the character is stable, uniform and convenient to use. The moutan bark reference extract is used as a reference in quality control of moutan bark and medicinal materials or Chinese medicinal preparations containing moutan bark/moutan bark active ingredients, and in the quality control process, the moutan bark reference extract can be directly used after being simply processed, so that the operation is simple and convenient, qualitative identification can be carried out on the medicinal materials or the Chinese medicinal preparations, and semi-quantitative or even quantitative analysis can be carried out.
Description
Technical Field
The invention relates to the technical field of quality control of traditional Chinese medicines, in particular to a moutan bark contrast extract and application thereof.
Background
The cortex moutan is dry root bark of Paeonia suffruticosa of Ranunculaceae, has effects of clearing heat and cooling blood, promoting blood circulation and removing blood stasis, and eliminating deficiency heat, and is mainly produced in Hunan, Hubei, Anhui, Sichuan, Gansu, Shanxi, Shandong, Guizhou and other places, wherein the quality of the Fenghuang mountain of the Shang Han' ling of Anhui is the best.
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. Firstly, the 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 often has a plurality of holes so as to have the phenomenon of being insufficient. The traditional Chinese medicine reference medicinal materials are influenced by the producing area and the growth environment, 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 medicinal material components.
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, has four basic requirements (ASCS), and also has several basic conditions for the control extract: 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 methods such as thin-layer chromatography fingerprint and high performance liquid chromatography fingerprint, and semi-quantitative and quantitative analysis and detection can be further performed by using a reference extract marked by an external standard method. The traditional Chinese medicine control extract has important significance for controlling the quality of the traditional Chinese medicine. However, because the traditional Chinese medicine reference extract has the above specific requirements, no or immature moutan bark reference extract product prepared by taking moutan bark as a raw material exists in the prior art.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a moutan bark reference extract which has good batch consistency, stable and uniform properties and is convenient to use, and the invention also provides an application of the moutan bark reference extract, namely the moutan bark reference extract is used for quality control of moutan bark, medicinal materials or Chinese medicinal preparations containing moutan bark/moutan bark effective components.
The invention provides a moutan bark control extract on one hand, which is characterized by being prepared from the following raw materials in parts by weight: extracting the tree peony bark medicinal material powder of different batches for multiple times to obtain tree peony bark extracts of different batches, and blending the tree peony bark extracts of different batches to obtain tree peony bark reference extracts.
Preferably, the multi-time extraction of the moutan bark medicinal material powder of different batches is to improve the purity and/or concentration of paeoniflorin components and paeonol components contained in the moutan bark medicinal material powder, so as to obtain the moutan bark extracts of different batches.
Preferably, the content of paeoniflorin in the moutan bark control extract is more than or equal to 1.5%, and the content of paeonol is more than or equal to 4.5%.
Preferably, the cortex moutan control extract has a spectrum obtained by thin layer chromatography and/or high performance liquid chromatography consistent with that of the corresponding raw material.
Preferably, the fluorescence spots at the positions of paeoniflorin and paeonol in the chromatogram obtained by detecting the cortex moutan control extract by thin-layer chromatography and the chromatographic peaks at the positions of paeoniflorin and paeonol in the chromatogram obtained by detecting the cortex moutan control extract by high performance liquid chromatography should be respectively consistent with the corresponding chemical control or the corresponding raw material.
In another aspect, the present invention provides a method for preparing a moutan bark reference extract, comprising the steps of:
step one, extraction: respectively mixing different batches of cortex moutan powder with ethanol, performing flash extraction, and filtering to obtain filtrate 1 and residue 1;
step two, column passing: passing the concentrated and/or unconcentrated filtrate 1 through macroporous resin, collecting the extractive solution after passing through the column, and evaporating to obtain cortex moutan dry extract;
step three, preparing a tree peony bark extract: dissolving the cortex moutan dry extract in ethanol to obtain ethanol solution of cortex moutan dry extract, adding adjuvants, evaporating to dryness, and sieving to obtain cortex moutan extract;
step four, blending: blending different batches of cortex moutan extract to obtain cortex moutan reference extract.
Preferably, the filter residue 1 in the step one is extracted for N times according to the extraction method in the step one, filtrate collected by the N times of extraction is combined with the filtrate 1 to obtain an extracting solution, and the extracting solution is evaporated to dryness to obtain cortex moutan dry extract; wherein N is more than or equal to 8 and is more than or equal to 0, and N is an integer;
more preferably, said N is 3.
Preferably, the ethanol concentration of the extraction solution in the first step is 30-95%;
more preferably, the ethanol concentration of the extraction solution in the first step is 60%.
Preferably, the weight-volume ratio of the cortex moutan medicinal material powder to the ethanol in the first step is 1: 1-1: 20;
more preferably, the weight-volume ratio of the cortex moutan powder to the ethanol in the first step is 1: 5;
preferably, the flash extraction time in the first step is 1-5 min;
more preferably, the flash extraction time in the first step is 1 min;
preferably, the filtration in the first step is medium-speed filter paper or 2000-mesh screen filtration;
more preferably, the filtration in the first step is a medium speed filter paper.
Preferably, in the second step, the volume ratio of the concentrated or non-concentrated filtrate 1 to the macroporous resin is 10: 1-1: 10, and the extracting solution is evaporated to dryness to obtain cortex moutan dry extract;
more preferably, the volume ratio of the filtrate 1 to the macroporous resin in the step two is 1: 1;
more preferably, the filtrate 1 is not concentrated.
Preferably, the weight-volume ratio of the cortex moutan dry extract to the ethanol in the third step is 1: 2-1: 10;
preferably, the auxiliary materials used in the third step are micropowder silica gel and/or medicinal starch;
preferably, the third step is that 10 to 50 weight percent of superfine silica gel powder is added into the ethanol solution of the tree peony bark dry paste;
preferably, the third step is to add 40 percent of micropowder silica gel by weight into the ethanol solution of the moutan bark dry paste;
preferably, the third step is to add medicinal starch accounting for 10 to 30 percent of the weight of the cortex moutan dry paste into an ethanol solution of the cortex moutan dry paste;
preferably, the third step is to add medicinal starch with the weight of 20 percent of that of the cortex moutan dry paste into the ethanol solution of the cortex moutan dry paste;
preferably, the third step is drying and then filtering by a screen of 90-200 meshes;
preferably, the third step is drying and then filtering through a 200-mesh screen;
preferably, a step of detecting paeoniflorin and paeonol of different batches of cortex moutan radicis extracts by thin layer chromatography and/or high performance liquid chromatography is further included between the third step and the fourth step.
Preferably, the criteria formulated in step four are: the content of paeoniflorin and paeonol in the final control extract should be not less than 1.5% and not less than 4.5%.
Preferably, the cortex moutan control extract has a spectrum obtained by thin layer chromatography and/or high performance liquid chromatography consistent with that of the corresponding raw material.
Preferably, the fluorescence spots at the positions of paeoniflorin and paeonol in the chromatogram obtained by detecting the cortex moutan control extract by thin-layer chromatography and the chromatographic peaks at the positions of paeoniflorin and paeonol in the chromatogram obtained by detecting the cortex moutan control extract by high performance liquid chromatography should be respectively consistent with the corresponding chemical control or the corresponding raw material.
The invention also provides application of the moutan bark reference extract in identification of medicinal materials or Chinese medicinal preparations, or application of moutan bark or medicinal materials or Chinese medicinal preparations containing moutan bark/moutan bark effective components in quality control.
Preferably, the identification of the medicinal materials or the Chinese medicinal preparation, or the quality control method of the moutan bark and the Chinese medicinal preparation containing the moutan bark/moutan bark effective components comprises the following steps: detecting the cortex moutan control extract, medicinal material or Chinese medicinal preparation by thin layer chromatography and/or high performance liquid chromatography, and comparing and distinguishing.
Preferably, the thin layer chromatography and/or high performance liquid chromatography is used for detecting paeoniflorin and paeonol in the cortex moutan contrast extract, the crude drug or the Chinese medicinal preparation.
Preferably, when the cortex moutan reference extract, the medicinal material or the Chinese medicinal preparation is detected by thin-layer chromatography, the cortex moutan reference extract, the medicinal material or the Chinese medicinal preparation needs to be prepared into a solution and then the thin-layer chromatography is performed for detection;
the preparation method of the moutan bark contrast extract solution comprises the following steps: adding 10-500 times of ethanol into the cortex moutan control extract, performing ultrasonic treatment, shaking, and filtering with 0.12-0.32 μm filter membrane to obtain cortex moutan control extract solution.
Preferably, the moutan bark control extract is added with ethanol with the mass of 200 times of the volume of the moutan bark control extract, treated for 30 minutes by ultrasonic wave of 500w, shaken up and filtered by a filter membrane of 0.22 mu m to obtain a moutan bark control extract solution;
the preparation method of the medicinal materials or the Chinese medicinal preparation solution comprises the following steps: adding 10-150 times of ethanol, ultrasonic treating, centrifuging, and filtering the supernatant with 0.12-0.32 μm filter membrane to obtain medicinal material or Chinese medicinal preparation solution.
Preferably, the medicinal material or the Chinese medicinal preparation is added with ethanol with 100 times volume of the mass of the medicinal material or the Chinese medicinal preparation, the mixture is treated by ultrasonic for 15 minutes at 500w, centrifuged for 3 minutes at 3200 r/min, and the supernatant is filtered by a 0.22 mu m filter membrane to obtain the medicinal material or the Chinese medicinal preparation solution.
Preferably, the thin layer chromatography detection conditions are as follows:
thin-layer plate: HPTLC F254 precast slab;
sample application: 0.5 mul, strip-like sample application length 8 mm;
developing agent: toluene: ethyl acetate: methanol: water: glacial acetic acid 10:7:5:0.5: 0.1;
drying the thin-layer plate: placing the sample-applied thin-layer plate in a phosphorus pentoxide vacuum dryer for 2 hours to ensure the drying of the thin-layer plate;
inspecting by spraying 5% vanillin-10% sulphuric acid ethanol color developing agent, and inspecting under 254nm fluorescence and incandescent light.
Preferably, when the moutan bark control extract, the medicinal material or the Chinese medicinal preparation is detected by high performance liquid chromatography, the moutan bark control extract, the medicinal material or the Chinese medicinal preparation needs to be prepared into a solution and then the high performance liquid chromatography is performed for detection;
the preparation method of the moutan bark contrast extract solution comprises the following steps: precisely weighing the moutan bark control extract, adding 50% ethanol, performing ultrasonic extraction for 30 minutes, fixing the volume, preparing into a solution with the concentration of 5mg/ml, filtering with a filter membrane of 0.22 μm, and taking the subsequent filtrate as an extract control solution.
The preparation method of the medicinal materials or the Chinese medicinal preparation solution comprises the following steps: sieving the medicinal materials or Chinese medicinal preparation with a second sieve, weighing 2g, placing in 50ml conical flask with plug, adding ethanol solution 45ml precisely, refluxing in water bath for 1h, cooling, adding ethanol to 50ml, shaking, filtering with 0.22 μm filter membrane, and collecting the filtrate to obtain medicinal material or Chinese medicinal preparation solution.
Preferably, the detection conditions of the high performance liquid chromatography are as follows:
chromatography apparatus: agilent 1260 series HPLC with DAD detector (Agilent Technologies, USA)
A chromatographic column: waters Symmetry C18 column (250 mm. times.4.6 mm I.D., 5 μm);
mobile phase: a-acetonitrile, B-0.1% phosphoric acid aqueous solution;
gradient elution procedure: 0-5 min: 10% A → 15% A, 5-30 min: 15% A → 22% A, 30-60 min: 22% A → 49% A, 60-65 min: 49% A → 80% A;
0-5min：90％B→85％B，5-30min：85％B→78％B，30-60min：78％B→51％B，60-65min：51％B→20％B；
detection wavelengths 230nm, 280nm (DAD detector); the flow rate is 0.8 ml/min; the sample amount is 10 mul; column temperature 25 ℃, run time: and 65 min.
Advantageous effects
The moutan bark comparison 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 areas and growth environments on traditional Chinese medicine comparison medicinal materials is overcome, and the consistency of the moutan bark comparison extracts of different batches is ensured; and the character is stable, uniform and convenient to use. The moutan bark comparison extract is applied, after being subjected to quantitative analysis, is used as a comparison in quality control of moutan bark and medicinal materials or Chinese medicinal preparations containing moutan bark/moutan bark effective components, in the quality control process, the moutan bark comparison extract can be directly used by simple treatment, the operation is simple and convenient, particularly when the content of multiple components is measured, the preparation of a comparison extract solution is simpler and more convenient than that of a chemical comparison product solution, and not only can the medicinal materials or the Chinese medicinal preparations be qualitatively identified, but also can be used for semi-quantitative or even quantitative analysis.
Drawings
The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which,
FIG. 1 is a flow chart of preparation of a moutan bark control extract;
FIG. 2 is a high performance thin layer chromatogram obtained by thin layer chromatography of commercially available medicinal materials with reference to chemical reference and cortex moutan reference extracts, wherein the chromatographic band of reference numeral 1 sequentially corresponds to the reference products paeoniflorin, catechin, benzoyloxypaeoniflorin, benzoylpaeoniflorin and paeonol from bottom to top, 2-9 sequentially corresponds to the medicinal materials of cortex moutan 1, cortex moutan 2, cortex moutan 3, cortex moutan 4, cortex moutan 5, cortex moutan 6, cortex moutan 7 and cortex moutan 8, and 10 corresponds to the cortex moutan reference extract;
FIG. 3 is a superimposed view of HPLC fingerprint obtained by performing HPLC on commercially available medicinal materials with reference to chemical reference substance and cortex moutan reference extract, from bottom to top, wherein R corresponds to common mode, S corresponds to cortex moutan reference extract, and 1-8 correspond to cortex moutan 1, cortex moutan 2, cortex moutan 3, cortex moutan 4, cortex moutan 5, cortex moutan 6, cortex moutan 7 and cortex moutan 8, respectively;
FIG. 4 is the established fingerprint chromatogram common mode chromatogram of cortex moutan, wherein the No. 15 chromatogram peak is gallic acid, the No. 48 chromatogram peak is paeoniflorin, the No. 64 chromatogram peak is pentagalloyl glucose, the No. 99 chromatogram peak is benzoylpaeoniflorin, and the No. 107 chromatogram peak is paeonol.
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:
moutan cortex Radicis (Anhui) is used as reference extract.
A Linomat 5 thin-layer chromatography semi-automatic spotting instrument, an ATS 4 thin-layer chromatography full-automatic spotting instrument, a thin-layer chromatography double-groove developing cylinder, a Chromatogram imaging Device III chromatographic development dipping groove and a TLC visualizer thin-layer chromatography camera (CAMAG, Switzerland).
Agilent 1260 series HPLC chromatograph equipped with DAD detector (Agilent Technologies, USA).
Toluene, ethyl acetate, methanol, glacial acetic acid, phosphoric acid were all analytically pure (Guangzhou chemical laboratories).
Acetonitrile was chromatographically pure (Merck KGaA).
The marked purities of paeoniflorin and paeonol are both more than or equal to 98 percent (Chenguan Biotech, Inc., Baoji city).
Testing the medicinal materials of tree peony bark:
8 batches of cortex moutan medicinal materials are purchased in Guangzhou medicinal material market.
Example 1: preparation of cortex moutan reference extract
This example provides a method for preparing a moutan bark control extract of the present invention, and the flow chart of the method is shown in fig. 1.
Firstly, the method comprises the following steps: extraction of
Selecting cortex moutan (Anhui, genuine medicinal material), making into powder, sieving the medicinal material powder with a second sieve (850 + -29 μm, 24 meshes), adding ethanol with a volume of 3.5 times of the mass (namely the solid-to-liquid ratio is 1: 3.5(w/V)), ultrasonically extracting for 30min (power is 500w, normal temperature), filtering with medium-speed qualitative filter paper, collecting filter residue 1 and filtrate 1, extracting filter residue 1 twice according to the same method, mixing the filtrate collected twice and filtrate 1 to obtain extract, and evaporating the solvent of the extract to obtain dry extract;
II, secondly: preparation of moutan cortex extract
Dissolving the dry extract with appropriate amount of ethanol completely, adding silica gel micropowder (adjuvant) 40% of the dry extract, evaporating to dryness with rotary evaporator, pulverizing, and sieving with 110 mesh sieve to obtain cortex moutan extract.
4 batches of moutan bark extracts were prepared and tested by hplc using reference, with the test results shown in table 1:
TABLE 1
Thirdly, the method comprises the following steps: blending
And (3) mixing and blending the 4 batches of the moutan bark extracts in the step (II) according to a ratio of 1:1 to obtain a moutan bark control extract, wherein the ratio of the corresponding raw medicinal materials of the final product is about 1: 2(g/g), measured by high performance liquid chromatography using a control, wherein the measurement results of the contents of the respective components are shown in fig. 2.
TABLE 2
The blending standard is as follows: the final cortex moutan control extract contains paeoniflorin not less than 1.5% and paeonol not less than 4.5% (by wt%). The content range of each component in the blending standard is also the best data obtained by comprehensively maintaining stability, consistency and later application through repeated experiments.
Example 2 analysis of Properties of moutan cortex Radicis control extract
1. Apparent state: the moutan bark control extract obtained in example 1 was a yellow-white powder.
2. And (3) moisture determination: according to 2015 version of appendix IX G of the Chinese pharmacopoeia (reduced pressure drying). The detection result shows that the water content of the cortex moutan reference extract is 3.1%.
3. And (3) testing consistency: 14 batches of moutan bark control extracts were prepared according to the method of example 1, and the differences of the thin layer chromatography detection results for paeoniflorin and paeonol were very small among the batches, and the fluorescence spots of paeoniflorin and paeonol were both clearly displayed and distributed very uniformly; the content of paeoniflorin and paeonol is detected to be within the range of the preparation standard by high performance liquid chromatography. Therefore, the moutan bark control extract prepared by the preparation method of the moutan bark control extract has very good consistency.
4. And (3) stability testing:
4 different batches of the test sample of the moutan bark comparison extract prepared by the method of example 1 are taken, and the indexes including characters and solubility are examined according to the relevant regulations of 'national drug standard substance development technical requirements' formulated by the national pharmacopoeia commission.
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 characters 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, and no significant difference exists.
Comparative example 1
The difference from example 1 is that:
in the first step of example 1, methanol is used instead of ethanol, and the content of the finally obtained cortex moutan dry extract is obviously lower than that of example 1, so that the value is obviously much lower than the yield of ethanol extraction, and the extraction amount or times can reach the range of the preparation standard only by increasing.
Example 3
This example provides the use of the moutan bark control extract prepared in example 1.
Analyzing commercially available materials by thin layer chromatography and high performance liquid chromatography with chemical reference substance and cortex moutan reference extract as reference substances.
1 thin layer chromatography
1.1 sample preparation
Chemical control solution: accurately weighing paeoniflorin and paeonol respectively, and preparing into 1mg/ml solution with methanol respectively.
Moutan bark control extract solution: accurately weighing 0.2g of the moutan bark control extract prepared in example 1, adding 10ml of ethanol, carrying out ultrasonic treatment (500w) for 15 minutes, shaking up, and filtering with a 0.22 mu m filter membrane to obtain a moutan bark control extract solution.
The test solution of the medicinal materials: taking 1g of each medicinal material in a pharmacy, adding 50ml of ethanol, performing ultrasonic treatment (500w) for 30 minutes, taking out, centrifuging for 3 minutes (the rotating speed is 3200 revolutions per minute), and filtering supernate with a 0.22-micron filter membrane to obtain a medicinal material test solution. The medicinal materials of the pharmacy are purchased from Guangzhou and respectively: 1 part of moutan bark, 2 parts of moutan bark, 3 parts of moutan bark, 4 parts of moutan bark, 5 parts of moutan bark, 6 parts of moutan bark, 7 parts of moutan bark and 8 parts of moutan bark.
1.2 thin layer chromatography detection
The detection conditions were as follows:
thin-layer plate: HPTLC F254 precast slab (Merck);
sample application: 2. mu.l and 5. mu.l, strip spotting length 8 mm;
developing agent: toluene: ethyl acetate: methanol: water: glacial acetic acid: 10:7:5:0.5: 0.1;
the unfolding mode is as follows: adding 12ml of developing agent into a double-tank developing cylinder (20cm multiplied by 10cm), and pre-balancing for 15 minutes;
drying the thin-layer plate: placing the sample-applied thin-layer plate in a phosphorus pentoxide vacuum dryer for 2 hours to ensure the drying of the thin-layer plate;
inspecting under 254nm fluorescence, spraying 5% vanillin-10% sulphuric acid ethanol, and inspecting under incandescent light.
The results of the measurement are shown in FIG. 2, which is a thin layer chromatogram observed under incandescent light (T: 27 ℃ C., RH: 66%). The chromatographic band of reference numeral 1 sequentially corresponds to control products of paeoniflorin, catechin, benzoyloxypaeoniflorin, benzoylpaeoniflorin and paeonol from bottom to top, 2-9 sequentially corresponds to medicinal materials of cortex moutan 1, cortex moutan 2, cortex moutan 3, cortex moutan 4, cortex moutan 5, cortex moutan 6, cortex moutan 7 and cortex moutan 8, and 10 corresponds to a cortex moutan control extract.
And (4) analyzing results: as can be seen from FIG. 2, the separation of paeoniflorin, catechin, benzoyloxypaeoniflorin, benzoylpaeoniflorin, and paeonol is good, the cortex moutan contrast extract and the medicinal material (cortex moutan 1-cortex moutan 8) can show the same spot at the same position, and the cortex moutan contrast extract and the medicinal material (cortex moutan 1-cortex moutan 8) have higher consistency as seen from the graph.
2 high performance liquid chromatography
2.1 sample preparation
Chemical control solution: accurately weighing 5mg of paeoniflorin and 5mg of paeonol as reference substances, and respectively preparing into 0.5mg/ml and 1.0mg/ml solutions with methanol.
Moutan bark control extract solution: precisely weighing 0.2g of cortex moutan extract, adding 50% ethanol, performing ultrasonic treatment at 500w for 30min, diluting to constant volume, preparing into 5mg/ml solution, filtering with 0.22 μm filter membrane, and collecting filtrate as extract reference solution;
the test solution of the medicinal materials: sieving each batch of medicinal material powder with a second sieve, precisely weighing 2g each, placing into a 50ml conical flask with a plug, adding 45ml of 50% ethanol solution, refluxing in water bath for 1h, filtering to 50ml volumetric flask through rapid filter paper, cooling, adding ethanol to a constant volume of 50ml, shaking, filtering with 0.22 μm filter membrane, and collecting the filtrate to obtain medicinal material solution. All the medicinal materials are purchased from Guangzhou, and are respectively: 1 part of moutan bark, 2 parts of moutan bark, 3 parts of moutan bark, 4 parts of moutan bark, 5 parts of moutan bark, 6 parts of moutan bark, 7 parts of moutan bark and 8 parts of moutan bark.
2.2 high Performance liquid chromatography detection
The detection conditions of the high performance liquid chromatography are as follows:
chromatography apparatus: agilent 1260 series HPLC with DAD detector (Agilent Technologies, USA)
A chromatographic column: waters Symmetry C18 column (250 mm. times.4.6 mm I.D., 5 μm);
mobile phase: a-acetonitrile, B-0.1% phosphoric acid aqueous solution;
gradient elution procedure: 0-5 min: 10% A → 15% A, 5-30 min: 15% A → 22% A, 30-60 min: 22% A → 49% A, 60-65 min: 49% A → 80% A;
0-5min：90％B→85％B，5-30min：85％B→78％B，30-60min：78％B→51％B，60-65min：51％B→20％B；
detection wavelengths 230nm, 280nm (DAD detector); the flow rate is 0.8 ml/min; the sample amount is 10 mul; column temperature 25 ℃, run time: and 65 min.
The detection method of the high performance liquid chromatography comprises the following steps: respectively and precisely sucking 10 μ l of chemical reference solution, cortex moutan reference extract solution and medicinal material sample solution, and injecting into liquid chromatograph.
And (3) detecting results by high performance liquid chromatography:
measuring, and recording chromatogram to obtain HPLC fingerprint chromatogram stack diagram shown in FIG. 3, from bottom to top, wherein R corresponds to chemical reference, S corresponds to cortex moutan control extract, and 1-8 correspond to cortex moutan 1, cortex moutan 2, cortex moutan 3, cortex moutan 4, cortex moutan 5, cortex moutan 6, cortex moutan 7 and cortex moutan 8, respectively.
As can be seen from FIG. 3, the fingerprints of the moutan bark 1-moutan bark 8 medicinal materials have high consistency, so that a common fingerprint mode of the moutan bark medicinal materials is established according to a Chinese medicine chromatography fingerprint similarity evaluation system (2012.130723 version) of pharmacopoeia committee of the people's republic of China, and similarity analysis is performed on all samples. The common mode chromatogram is shown in FIG. 4, wherein the No. 15 chromatogram peak is gallic acid, the No. 48 chromatogram peak is paeoniflorin, the No. 64 chromatogram peak is pentagalloylglucose, the No. 99 chromatogram peak is benzoylpaeoniflorin, and the No. 107 chromatogram peak is paeonol.
According to a set common mode of the fingerprint of the cortex moutan radicis medicinal material, an included angle cosine algorithm is adopted to evaluate the similarity of 8 batches of cortex moutan radicis medicinal material and a comparison extract sample according to each sample component and the peak area thereof, and the result is shown in table 3.
TABLE 3
| Sample (I) | Degree of |
| Moutan bark | |
| 1 | 0.999 |
| |
0.984 |
| |
0.999 |
| |
0.990 |
| |
0.999 |
| |
0.990 |
| |
0.999 |
| |
0.935 |
| Moutan bark reference extract | 0.999 |
According to the similarity analysis results, the similarity of the fingerprint common patterns of 8 batches of medicinal materials and the cortex moutan medicinal material is within the range of 0.935-0.999, and the similarity is very high. The similarity of fingerprint spectra common mode of the moutan bark reference extract and the moutan bark medicinal material is 0.999, which shows that the moutan bark reference extract of the invention has good consistency with the medicinal material.
According to the results of TLC and HPLC fingerprints, the chromatogram obtained by detecting the moutan bark control extract by using thin layer chromatography is consistent with the corresponding raw material medicinal material, more precisely, the fluorescence spots at the positions of paeoniflorin and paeonol in the chromatogram obtained by detecting the moutan bark control extract by using thin layer chromatography and the chromatographic peaks at the positions of paeoniflorin and paeonol in the chromatogram obtained by detecting the moutan bark control extract by using high performance liquid chromatography are respectively consistent with the corresponding chemical control product or the corresponding raw material medicinal material, so that the moutan bark control extract can be applied to qualitative identification of medicinal materials or Chinese medicinal preparations, for example, qualitative analysis of paeoniflorin components of the medicinal materials or Chinese medicinal preparations.
According to HPLC determination results and statistical analysis, the established common pattern of the fingerprint of the cortex moutan radicis medicinal material has extremely high similarity with the cortex moutan radicis medicinal material, the similarity of common fingerprint patterns of the moutan bark contrast extract and the moutan bark medicinal material is extremely high, this shows that the reliability of the content determination of paeoniflorin and paeonol by using the cortex moutan contrast extract of the invention to the medicinal material or the Chinese medicinal preparation is extremely high, therefore, the moutan bark control extract of the invention can be applied to semi-quantitative identification analysis of medicinal materials or Chinese medicinal preparations, and also can be applied to the quality control of moutan bark and medicinal materials or Chinese medicinal preparations containing moutan bark/moutan bark effective components, for example, the semi-quantitative identification analysis is carried out on the paeoniflorin and paeonol components of the medicinal materials or the Chinese medicinal preparations, or semi-quantitatively detecting cortex moutan and medicinal materials or Chinese medicinal preparation containing effective components of cortex moutan/cortex moutan to control their quality.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (1)
1. A thin layer chromatography detection method for cortex moutan reference extract solution or medicinal material or Chinese medicinal preparation solution containing cortex moutan/cortex moutan effective constituent is provided,
the preparation method of the moutan bark contrast extract solution comprises the following steps:
(1) firstly, preparing a moutan bark reference extract:
the cortex moutan control extract is derived from: extracting the tree peony bark medicinal material powder of different batches for multiple times to obtain tree peony bark extracts of different batches, and blending the tree peony bark extracts of different batches to obtain tree peony bark reference extracts;
the multi-time extraction of the moutan bark medicinal material powder of different batches is to improve the purity and/or concentration of paeoniflorin components and paeonol components contained in the moutan bark medicinal material powder, so as to obtain moutan bark extracts of different batches;
the content of paeoniflorin in the cortex moutan reference extract is more than or equal to 1.5%, and the content of paeonol is more than or equal to 4.5%;
the preparation method of the moutan bark reference extract comprises the following steps:
step one, extraction: respectively mixing different batches of cortex moutan powder with ethanol, performing flash extraction, and filtering to obtain filtrate 1 and residue 1;
step two, column passing: passing the concentrated and/or unconcentrated filtrate 1 through macroporous resin, collecting the extractive solution after passing through the column, and evaporating to obtain cortex moutan dry extract;
step three, preparing a tree peony bark extract: dissolving the cortex moutan dry extract in ethanol to obtain ethanol solution of cortex moutan dry extract, adding adjuvants, evaporating to dryness, and sieving to obtain cortex moutan extract;
step four, blending: blending different batches of cortex moutan extract to obtain cortex moutan reference extract;
extracting the filter residue 1 in the step one for N times according to the extraction method in the step one, combining filtrate collected by the extraction for N times with the filtrate 1 to obtain extract, and evaporating the extract to dryness to obtain cortex moutan dry extract;
the N is 3;
the ethanol concentration of the extraction solution in the first step is 60 percent;
the weight-volume ratio of the moutan bark medicinal material powder to the ethanol in the first step is 1: 5;
the flash extraction time in the first step is 1 min;
the filtration in the first step is to use medium-speed filter paper;
the volume ratio of the filtrate 1 to the macroporous resin in the step two is 1: 1;
the filtrate 1 is not concentrated;
the weight-volume ratio of the cortex moutan dry extract to the ethanol in the third step is as follows: 1: 2-1: 10;
adding superfine silica gel powder 40% of the weight of the moutan bark dry paste into an ethanol solution of the moutan bark dry paste;
after the third step of evaporation to dryness, filtering the mixture through a 200-mesh screen;
the step three and the step four also comprise a step of detecting paeoniflorin and paeonol of different batches of cortex moutan radicis extracts by thin-layer chromatography and/or high performance liquid chromatography;
the blending standard in the fourth step is as follows: the content of paeoniflorin and paeonol in the finally obtained control extract is more than or equal to 1.5% and more than or equal to 4.5%;
(2) adding 200 times volume of ethanol into the cortex moutan control extract, treating with 500w of ultrasonic wave for 30min, shaking, and filtering with 0.22 μm filter membrane to obtain cortex moutan control extract solution;
the preparation method of the medicinal materials or the Chinese medicinal preparation solution comprises the following steps:
adding 100 times volume of ethanol into medicinal material or Chinese medicinal preparation, treating with ultrasound 500w for 15 min, centrifuging at 3200 rpm for 3 min, and filtering the supernatant with 0.22 μm filter membrane to obtain medicinal material or Chinese medicinal preparation solution;
the detection conditions of the thin layer chromatography are as follows:
thin-layer plate: HPTLC F254 precast slab;
sample application: 0.5 mul, strip-like sample application length 8 mm;
developing agent: toluene, ethyl acetate, methanol, water, glacial acetic acid 10:7:5:0.5: 0.1;
drying the thin-layer plate: placing the sample-applied thin-layer plate in a phosphorus pentoxide vacuum dryer for 2 hours to ensure the drying of the thin-layer plate;
inspecting by spraying 5% vanillin-10% sulphuric acid ethanol color developing agent, and inspecting under 254nm fluorescence and incandescent light.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101049389A (en) * | 2007-03-12 | 2007-10-10 | 张文武 | Method for controlling quality of granule preparation for treating chilly sensation type gastritis |
| CN101269182A (en) * | 2007-03-23 | 2008-09-24 | 北京亚东生物制药有限公司 | Pharmaceutical composition for treating children's wind-heat cold, preparation method and quality control method |
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| CN101269182A (en) * | 2007-03-23 | 2008-09-24 | 北京亚东生物制药有限公司 | Pharmaceutical composition for treating children's wind-heat cold, preparation method and quality control method |
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