CN108037222A - Radix Paeoniae Alba reference extract and its preparation method and application - Google Patents
Radix Paeoniae Alba reference extract and its preparation method and application Download PDFInfo
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- CN108037222A CN108037222A CN201711277332.4A CN201711277332A CN108037222A CN 108037222 A CN108037222 A CN 108037222A CN 201711277332 A CN201711277332 A CN 201711277332A CN 108037222 A CN108037222 A CN 108037222A
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- G—PHYSICS
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Abstract
The present invention provides a kind of Radix Paeoniae Alba reference extract, it is derived from：The white Peony Root powder of different batches is repeatedly extracted, obtains the radix paeoniae alba extraction of different batches, then the radix paeoniae alba extraction of different batches is allocated, obtains Radix Paeoniae Alba reference extract.The Radix Paeoniae Alba reference extract of the present invention ensure that the uniformity of the Radix Paeoniae Alba reference extract of different batches because being that the extract of different batches carries out allotment and forms；And its character is stable, uniform and easy to use.The Radix Paeoniae Alba reference extract is upper in application, it is as in quality control of the control for Radix Paeoniae Alba and medicinal material or Chinese medicine preparation containing Radix Paeoniae Alba/effective component of paeonia lactiflora, in this quality control procedure, it is that Radix Paeoniae Alba reference extract progress simple process can be used directly, it is easy to operate, can not only Qualitive test be carried out to medicinal material or Chinese medicine preparation, but also can be also used for sxemiquantitative even quantitative analysis.
Description
Technical field
The present invention relates to quality control technologies for traditional Chinese medicine field, more particularly to a kind of Radix Paeoniae Alba reference extract and its preparation side
Method and application.
Background technology
Radix Paeoniae Alba is Ranunculaceae Radix Paeoniae Alba platymiscium, is the dry root of cohosh Chinese herbaceous peony, is a kind of smooth liver and relieve pain, blood-nourishing tune
Chinese medicine through, astringing YIN to stop sweating.
Existing traditional Chinese medicine quality control model is substantially the development along Natural Medicine Chemistry, a certain to Chinese medicine or several
Analysis method and the not only design of difinite quality but also the quality standard that can quantify of the active ingredient for target, with reference to the matter of external autonomic drug
Amount control method, uses for reference the pattern of chemicals quality control, and establishes corresponding simple physics and chemistry by document report and differentiate, then
Develop into the discriminating based on spectrum, chromatography and the quality standard of assay.All it is multicomponent synthesis per Chinese medicine simply
Body, which dictates that its distinctive globality and ambiguity, also indicate that one or two using in medicinal material even several components are used as medicinal material matter
There are significant limitation for the evaluation method of amount.
1990 editions《Chinese Pharmacopoeia》Add the indentification by TLC of control medicinal material so that the discriminating of Chinese medicine and Chinese patent drug has
Very big progress.Chinese medicine and external herbal medicine increasingly pay attention to multicomponent or multi-component detection, such as German ginkgo at present
The quality control of leaf extract.The analysis method of finger-print, can carry out quality control, currently still to medicinal material on the whole
There is very big researching value.Chinese Pharmacopoeia has two kinds " reference substances " in terms of quality of medicinal material is controlled at present, and chemical reference substance is with
Medicine control medicinal material.Wherein chemical reference substance can be used for Qualitive test and the quantitative analysis of medicinal material, and Chinese medicine control medicinal material is then used to show
Micro- discriminating and thin layer differentiate.However, chemical reference substance and Chinese medicine control medicinal material have its limitation in traditional Chinese medicine quality control.It is first
First, chemical composition variation in Chinese medicine, single or several compounds can not reflect the overall picture of medicinal material, and existing standard is past
Toward there is many loopholes, shoddy phenomenon happens occasionally.Chinese medicine control medicinal material is influenced be subject to the place of production, growing environment, it is difficult to
Ensure per batch of uniform quality, and be only used for Qualitive test, can not reflect the height of medicinal ingredient content.
Chinese medicine reference extract is the character and stable components prepared with Chinese medicine, can be used for qualitative or quantitative analysis
Extract, have four basic demands (ASCS), and reference extract should possess several primary conditions：Authenticity,
Crude drug source is reliable, representative；Specificity, the detection method specificity used；Con-sistency, difference batch
Secondary reference extract should be consistent；Stability, character are stable, uniform, easy to use.With TLC Fingerprints
And the methods of high-efficiency liquid-phase fingerprint, it can be used for the Qualitive test of medicinal material, by the reference extract of external standard method mark,
Sxemiquantitative and quantitative analysis detection can further be carried out.Chinese medicine reference extract will have Chinese medicine quality control important meaning
Justice.But also just due to Chinese medicine reference extract have it is above-mentioned it is specific require, currently available technology does not have also or does not have maturation
Pass through the Radix Paeoniae Alba reference extract product being prepared using white Peony Root as raw material.
The content of the invention
The technical problem to be solved by the present invention is to provide a kind of batch uniformity is good, character is stable, uniform and easy to use
Radix Paeoniae Alba reference extract, will the Radix Paeoniae Alba reference extract present invention also offers the application of the Radix Paeoniae Alba reference extract
For Radix Paeoniae Alba and in the quality control of medicinal material or Chinese medicine preparation containing Radix Paeoniae Alba/effective component of paeonia lactiflora.
One aspect of the present invention provides a kind of Radix Paeoniae Alba reference extract, it is characterised in that it is derived from：To different batches
White Peony Root powder is repeatedly extracted, and obtains the radix paeoniae alba extraction of different batches, then to the radix paeoniae alba extraction of different batches
Allocated, obtain Radix Paeoniae Alba reference extract.
Preferably, the white Peony Root powder to different batches is repeatedly extracted, and is the peony lactone for containing it
Methods of glycosides, the purity of Chinese herbaceous peony methods of glycosides and benzoylpaeoniflorin constituents and/or concentration improve, so as to obtain different batches
Radix paeoniae alba extraction.
Preferably, in the Radix Paeoniae Alba reference extract albiflorin, Paeoniflorin and benzoylpaeoniflorin content >=
5.0%.
Preferably, the figure for using thin-layered chromatography and/or high performance liquid chromatography to obtain to the Radix Paeoniae Alba reference extract
It is consistent to compose corresponding raw medicinal material.
Preferably, the peony lactone in the chromatogram for using thin-layered chromatography to detect the Radix Paeoniae Alba reference extract
The chromatogram that fluorescence spot and use high performance liquid chromatography at glycosides, Paeoniflorin and benzoylpaeoniflorin detect is in Chinese herbaceous peony
Chromatographic peak at lactone glycoside, Paeoniflorin and benzoylpaeoniflorin position should distinguish corresponding chemical reference substance or its is corresponding
Raw medicinal material is consistent.
Another aspect of the present invention provides a kind of preparation method of Radix Paeoniae Alba reference extract, comprises the following steps：
Step 1: extraction：Take the white Peony Root powder of different batches to be mixed respectively with ethanol, filter, obtain after homogenate extraction
To filtrate 1 and filter residue 1, and filtrate 1 is evaporated to obtain Radix Paeoniae Alba dry cream；
Step 2: prepare radix paeoniae alba extraction：By gained Radix Paeoniae Alba dry cream, ethanol is dissolved in, the ethanol for obtaining Radix Paeoniae Alba dry cream is molten
Liquid, adds auxiliary material, is evaporated sieving and obtains radix paeoniae alba extraction；
Step 3: allotment：The radix paeoniae alba extraction of different batches is allocated, obtains Radix Paeoniae Alba reference extract.
Preferably, the filter residue 1 described in the step 1 is extracted according to the extracting method of step 1 by n times, and by n times
The filtrate that extraction is collected merges Ji Wei extracting solution with filtrate 1, and extracting solution is evaporated to obtain Radix Paeoniae Alba dry cream；Wherein 6 >=N >=0, and N
For integer；
It is highly preferred that the N is 3.
Preferably, the extraction solution ethanol concentration described in the step 1 is 30%~95%；
It is highly preferred that it is 50% that solution ethanol concentration is extracted in the step 1.
Preferably, the w/v of the white Peony Root powder in the step 1 and ethanol is 1:5~1:45；
It is highly preferred that the w/v of the white Peony Root powder and ethanol in the step 1 is 1：10；
Preferably, the homogenate extraction time in the step 1 is 1~5min；
It is highly preferred that the homogenate extraction time in the step 1 is 1min；
Preferably, the filtering in the step 1 is with Medium speed filter paper or 2000 mesh sieve net filtrations；
It is highly preferred that the filtering in the step 1 is to use Medium speed filter paper.
Preferably, the w/v of Radix Paeoniae Alba dry cream and ethanol is 1 in the step 2:2~1:10；
Preferably, auxiliary material used is superfine silica gel powder and/or medical starch in the step 2；
Preferably, the auxiliary material is superfine silica gel powder；
Preferably, the step 2 is that 10%~50% micro mist of its weight is added in the ethanol solution of Radix Paeoniae Alba dry cream
Silica gel；
Preferably, the step 2 is that 40% superfine silica gel powder of its weight is added in the ethanol solution of Radix Paeoniae Alba dry cream；
Preferably, it was 90~200 mesh sieve net filtrations after the step 2 is evaporated；
Preferably, it was 200 mesh sieve net filtrations after the step 2 is evaporated；
Preferably, further included between the step 2 and step 3 with thin-layered chromatography and/or high performance liquid chromatography pair
The step of albiflorin, Paeoniflorin and the benzoylpaeoniflorin of the radix paeoniae alba extraction of different batches are detected.
Preferably, the standard allocated in the step 3 is：It should make albiflorin in the reference extract that finally obtains
>=2.0%, content >=0.2% of Paeoniflorin >=7.5%, benzoylpaeoniflorin.
Preferably, the figure for using thin-layered chromatography and/or high performance liquid chromatography to obtain to the Radix Paeoniae Alba reference extract
It is consistent to compose corresponding raw medicinal material.
Preferably, the peony lactone in the chromatogram for using thin-layered chromatography to detect the Radix Paeoniae Alba reference extract
The chromatogram that fluorescence spot and use high performance liquid chromatography at glycosides, Paeoniflorin and benzoylpaeoniflorin position detect exists
Chromatographic peak at albiflorin, Paeoniflorin and benzoylpaeoniflorin position should distinguish corresponding chemical reference substance or its is right
The raw medicinal material answered is consistent.
Another aspect of the invention provides a kind of discriminating of Radix Paeoniae Alba reference extract in medicinal material or Chinese medicine preparation, or Radix Paeoniae Alba or
Medicinal material containing Radix Paeoniae Alba/effective component of paeonia lactiflora or the application in the quality control of Chinese medicine preparation.
Preferably, the discriminating of the medicinal material or Chinese medicine preparation, or Radix Paeoniae Alba and the Chinese medicine preparation containing Radix Paeoniae Alba/effective component of paeonia lactiflora
The method of quality control be：By Radix Paeoniae Alba reference extract, medicinal material or the Chinese medicine preparation with thin-layered chromatography and/or efficiently
Liquid chromatography is detected, and contrast differentiates.
Preferably, the thin-layered chromatography and/or high performance liquid chromatography are to Radix Paeoniae Alba reference extract, medicinal material or Chinese medicine
Albiflorin, Paeoniflorin and benzoylpaeoniflorin in preparation are detected.
Preferably, when Radix Paeoniae Alba reference extract, medicinal material or the Chinese medicine preparation are detected with thin-layered chromatography,
Need Radix Paeoniae Alba reference extract, medicinal material or Chinese medicine preparation being configured to solution to carry out thin-layered chromatography detection again；
The preparation method of wherein Radix Paeoniae Alba reference extract solution is：The Radix Paeoniae Alba reference extract is added into its quality
The ethanol of 10-100 times of volume, is ultrasonically treated, shakes up, and crosses 0.12-0.32 μm of filter membrane up to Radix Paeoniae Alba reference extract solution.
Preferably, the Radix Paeoniae Alba reference extract is added to the ethanol of its 20 times of volume of quality, ultrasonic 500w processing 30
Minute, shake up, cross 0.22 μm of filter membrane up to Radix Paeoniae Alba reference extract solution；
The preparation method of its Chinese medicine or Chinese medicine preparation solution is：Get it filled material or Chinese medicine preparation adds 10-100 times of its quality
The ethanol of volume, is ultrasonically treated, is settled in 2~10ml volumetric flasks, centrifuges, takes supernatant to cross 0.12-0.32 μm of filter membrane to obtain the final product
Medicinal material or Chinese medicine preparation solution.
Preferably, getting it filled material or Chinese medicine preparation adds the ethanol of its 100 times of volume of quality, ultrasonic 500w is handled 15 minutes,
It is settled in 2ml volumetric flasks, 3200 revs/min centrifuge 3 minutes, take supernatant to cross 0.22um filter membranes up to medicinal material or Chinese medicine preparation
Solution.
Preferably, the thin-layered chromatography testing conditions are：
Lamellae：HPTLC G60 prefabricated boards；
Point sample：2 μ l, the long 8mm of ribbon point sample；
Solvent：Chloroform：Ethyl acetate：Methanol：Formic acid=30:5:10:1；
The drying of lamellae：Lamellae after point sample be placed in phosphorus pentoxide vacuum desiccator 2 it is small when, ensure lamellae
It is dry；
Inspect:Spray is placed under incandescence and is inspected with -10% sulfuric acid ethanol color developing agent of 5% vanillic aldehyde.
Preferably, examined when by Radix Paeoniae Alba reference extract, medicinal material or the Chinese medicine preparation with high performance liquid chromatography
, it is necessary to which Radix Paeoniae Alba reference extract, medicinal material or Chinese medicine preparation are configured to solution carries out high performance liquid chromatography detection again during survey；
The preparation method of wherein Radix Paeoniae Alba reference extract solution is：Precision weigh the Radix Paeoniae Alba reference extract 30mg~
300mg, is configured to the solution that concentration is 0.6~60mg/ml with ethanol, crosses the filter membrane of 0.12-0.32 μm of filter membrane, take subsequent filtrate,
Up to Radix Paeoniae Alba reference extract solution.
Preferably, accurate to weigh the Radix Paeoniae Alba reference extract 30mg, it is 0.6mg/ml's to be configured to concentration with ethanol
Solution, crosses the filter membrane of 0.22 μm of filter membrane, subsequent filtrate is taken, up to Radix Paeoniae Alba reference extract solution；
The preparation method of its Chinese medicine or Chinese medicine preparation solution is：Get it filled material or Chinese medicine preparation cross No. two sieves after precision weigh
2g~20g, puts in 50ml~250ml conical flask with cover, adds ethanol solution 45ml~240ml, water-bath reflux or ultrasound 500w
After handling 30min, let cool, by Medium speed filter paper or 2000 mesh sieve net filtrations into 50ml or 250ml volumetric flasks, add ethanol to quarter
Spend at 50ml~250ml, shake up, cross 0.12-0.32 μm of filter membrane, take subsequent filtrate up to medicinal material or Chinese medicine preparation solution.
Preferably, get it filled material or Chinese medicine preparation cross No. two sieves after precision weigh 2g, put in 50ml conical flask with cover, add second
After alcoholic solution 45ml, ultrasonic 500w processing 30min, let cool, filtered by Medium speed filter paper into 50ml volumetric flasks, add ethanol to quarter
Spend at 50ml, shake up, cross 0.22 μm of filter membrane, take subsequent filtrate up to medicinal material or Chinese medicine preparation solution.
Preferably, the high performance liquid chromatography testing conditions：
Chromatographic apparatus：Agilent 1100series high performance liquid chromatographs, equipped with DAD detectors (U.S., Agilent
Technologies)
Chromatographic column：Waters Symmetry C18column (250mm × 4.6mm I.D, 5 μm)；
Mobile phase：A- acetonitriles, B-0.1% phosphate aqueous solutions；
Gradient elution program：0-5min：10%A → 15%A, 5-30min：15%A → 22%A, 30-60min：22%A
→ 49%A, 60-65min：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 wavelength 230nm (DAD detectors)；Flow velocity 0.8ml/min；10 μ l of sample size；25 DEG C of column temperature, run time：
65min。
Beneficial effect
The Radix Paeoniae Alba reference extract of the present invention is overcome because of Chinese medicine pair because being that the extract of different batches carries out allotment and forms
Influenced according to medicinal material be subject to the place of production, growing environment, it is difficult to ensure that the defects of every batch of uniform quality, so as to ensure that different batches
The uniformity of secondary Radix Paeoniae Alba reference extract；And its character is stable, uniform and easy to use.The Radix Paeoniae Alba reference extract exists
It is by Radix Paeoniae Alba reference extract after quantitative analysis, as control for Radix Paeoniae Alba and containing Radix Paeoniae Alba/effective component of paeonia lactiflora using upper
Medicinal material or Chinese medicine preparation quality control in, be simply to be located Radix Paeoniae Alba reference extract in this quality control procedure
Reason can be used directly, and easy to operate, especially in multicomponent assay, the preparation of reference extract solution is more molten than reference substance
The preparation of liquid is easier, not only can carry out Qualitive test to medicinal material or Chinese medicine preparation, but also can be also used for sxemiquantitative very
To quantitative analysis.
Brief description of the drawings
From detailed description below in conjunction with the accompanying drawings, it will be more clearly understood the present invention above-mentioned and other purposes,
Feature and other advantages, wherein,
Fig. 1 is Radix Paeoniae Alba reference extract preparation flow figure；
Fig. 2 is to be directed to carry out thin-layer chromatography to commercially available medicinal material as reference substance using chemical reference substance and Radix Paeoniae Alba reference extract
The high performance thin layer chromatography figure that method obtains, the wherein chromatographic band of label S are corresponding in turn to as reference substance albiflorin, Chinese herbaceous peony from lower to upper
Medicine glycosides, catechin, benzoylpaeoniflorin, 8 correspond to Radix Paeoniae Alba reference extract, 1-7 be corresponding in turn to Radix Paeoniae Alba 1, Radix Paeoniae Alba 2, Radix Paeoniae Alba 3,
Radix Paeoniae Alba 4, Radix Paeoniae Alba 5, the medicinal material of Radix Paeoniae Alba 6 and Radix Paeoniae Alba 7；
Fig. 3 is to be directed to carry out efficient liquid phase to commercially available medicinal material as reference substance using chemical reference substance and Radix Paeoniae Alba reference extract
The HPLC finger-print stacking charts that chromatography obtains, from top to bottom 1-7 correspond to Radix Paeoniae Alba 1, Radix Paeoniae Alba 2, Radix Paeoniae Alba 3, Radix Paeoniae Alba 4, white respectively
Chinese herbaceous peony 5, Radix Paeoniae Alba 6 and Radix Paeoniae Alba 7, S correspond to Radix Paeoniae Alba reference extract, and R corresponds to chemical reference substance；
Fig. 4 is the finger-print common pattern collection of illustrative plates for the white Peony Root established, wherein No. 7 chromatographic peaks correspond to peony lactone
Glycosides, No. 8 chromatographic peaks correspond to Paeoniflorin, and No. 11 chromatographic peaks correspond to Penta-O-galloyl-D-glucopyranose, and No. 12 chromatographic peaks correspond to benzene
Formyl Paeoniflorin.
Embodiment
The embodiment of the present invention is described below in detail.The embodiments described below is exemplary, and is only used for explaining this hair
It is bright, and be not considered as limiting the invention.Particular technique or condition are not specified in embodiment, according to text in the art
Offer described technology or condition or carried out according to product description.Reagents or instruments used without specified manufacturer,
For can be with conventional products that are commercially available.Term as used herein "and/or" includes one or more relevant listed
The arbitrary and all combination of project.
The instrument used in following embodiments and the source of material are as follows：
It is envisaged for preparing the medicinal material of reference extract:Radix Paeoniae Alba (Anhui).
The semi-automatic point sample instrument of 5 thin-layer chromatographys of Linomat, the full-automatic point sample instrument of 4 thin-layer chromatographys of ATS, thin-layer chromatography double flute
Expansion cylinder, Chromatogram Immersion Device III chromatography colour developing dipping tanks and TLC visualizer thin layers
Chromatography video camera (Switzerland, CAMAG).
Agilent 1100series high performance liquid chromatographs, equipped with DAD detectors (U.S., Agilent
Technologies)。
Chloroform, ethyl acetate, methanol, formic acid, phosphoric acid are to analyze pure (Guangzhou Chemical Reagent Factory).
Acetonitrile is chromatographically pure (Merck KGaA).
Albiflorin, Paeoniflorin, benzoylpaeoniflorin, >=98%, (Baoji time biotechnology has sign purity
Limit company).
Test medicinal material Radix Paeoniae Alba：
7 batches of buyings of white Peony Root are in the major pharmacy in Guangzhou and medicinal material market, Radix Paeoniae Alba 1~6：Guangzhou The Qingping Free Market；Radix Paeoniae Alba 7：
Cai Zhilin；
Embodiment 1：The preparation of Radix Paeoniae Alba reference extract
The preparation method of Radix Paeoniae Alba reference extract of the present invention is present embodiments provided, its method flow diagram is as shown in Figure 1.
One：Extraction
Choose white Peony Root (Anhui, genunie medicinal materials), be made powder, medicinal powder cross No. two sieves (850 ± 29 μm, 24
Mesh), then add its 10 times of volume of quality ethanol (i.e. solid-to-liquid ratio be 1：10 (w/V)), ultrasonic (power 500w, room temperature) extraction
Middling speed qualitative filter paper filters after 30min, collects filter residue 1 and filtrate 1, filter residue 1 extracted again by same procedure it is secondary, and will again
The filtrate for extracting secondary collection merges Ji Wei extracting solution with filtrate 1, and extracting solution solvent evaporated is got dry extract；
Two：Prepare radix paeoniae alba extraction
After dry cream is dissolved completely with ethanol in proper amount, then add 50% superfine silica gel powder (mountains and rivers medicine is auxiliary) of dry cream quality, use
Rotary Evaporators are evaporated, and be crushed 110 mesh sieves, are obtained radix paeoniae alba extraction.
3 batches of radix paeoniae alba extractions are prepared, high effective liquid chromatography for measuring are utilized by reference substance, testing result is as shown in table 1：
Table 1
Three：Allotment
The radix paeoniae alba extraction of 3 batches of step 2 is pressed 1:The mixing of 1 ratio blends to obtain Radix Paeoniae Alba reference extract, finally
It is about 1 that product, which corresponds to crude drug ratio,：3.5 (g/g), high effective liquid chromatography for measuring is utilized by reference substance, wherein respectively into
The measurement result for the content divided is as shown in Figure 2.
Table 2
Allotment standard is：It should make content >=2.0% of albiflorin in final Radix Paeoniae Alba reference extract, Paeoniflorin >=
7.5%, benzoylpaeoniflorin >=0.2% (content is in terms of wt%).The scope of the content of each component in this allotment standard is also
By the repeatedly comprehensive optimum data for keeping stability, uniformity and application below etc. to draw of experiment repeatedly.
The character analysis of 2 Radix Paeoniae Alba reference extract of embodiment
1. apparent state：The Radix Paeoniae Alba reference extract that embodiment 1 obtains is white powder.
2. determination of moisture：Carried out according to 2015 editions Chinese Pharmacopoeia annex IX G (hypobaric drying method).Testing result is Radix Paeoniae Alba
Reference extract water content is 3.5%.
3. uniformity test：3 batch Radix Paeoniae Alba reference extracts are prepared according to the method for embodiment 1, after measured between each batch
For albiflorin, Paeoniflorin, the thin-layer chromatography testing result difference very little of benzoylpaeoniflorin, albiflorin, Chinese herbaceous peony
Medicine glycosides, the fluorescence spot of benzoylpaeoniflorin have obvious display, and are distributed very consistent；Through high performance liquid chromatography detection its
Albiflorin, Paeoniflorin, benzoylpaeoniflorin content are within allotment critical field.Therefore the Radix Paeoniae Alba pair of the present invention is utilized
The Radix Paeoniae Alba reference extract uniformity being prepared according to the preparation method of extract is very good.
4. stability test：
The test sample of the Radix Paeoniae Alba reference extract of the method preparation according to embodiment 1 of 3 different batches is taken, according to country
The relevant regulations of " requirements of national drug standards material Development Techniques " that pharmacopoeia commission works out, inspection target include character and molten
Xie Xing.
Test sample opening is put in suitable clean container, is placed 10 days at a temperature of 60 DEG C, is sampled in the 5th day and the 10th day,
It is detected by stability high spot reviews project.
The results show that before and after hot test test sample character without significant change, before and after hot test thin-layer chromatogram also without
Significant change, dissolution rate measurement result carry out Independent samples t-test with SPSS, and result of calculation P ＞ 0.05, illustrate no conspicuousness
Difference.
Comparative example 1
With embodiment 1 difference lies in：
To using methanol to be carried out instead of ethanol in the step one in embodiment 1, the content of the Radix Paeoniae Alba dry cream finally obtained is bright
It is aobvious to be less than embodiment 1, it can be seen that, yield of its value substantially than ethanol extraction is much lower, only increases extracted amount or number
Be possible to reach within allotment critical field.
Embodiment 3
Present embodiments provide the application for the Radix Paeoniae Alba reference extract that embodiment 1 is prepared.
With thin-layered chromatography and high performance liquid chromatography respectively using chemical reference substance and Radix Paeoniae Alba reference extract as reference
Material analyzes commercially available medicinal material.
1 thin-layer chromatography
1.1 sample preparation
Chemical reference substance solution：Precision weighs albiflorin, Paeoniflorin, benzoylpaeoniflorin, is configured to methanol
The solution of 1mg/ml.
Radix Paeoniae Alba reference extract solution：Precision weighs the Radix Paeoniae Alba reference extract 0.2g that embodiment 1 is prepared, and adds second
Alcohol 1ml, ultrasonic (500w) are handled 15 minutes, shaken up, and add ethanol to be settled to 2ml volumetric flasks, cross 0.22 μm of filter membrane up to Radix Paeoniae Alba pair
According to extract solution.
Medicinal material test solution：Get it filled each 1g of shop medicinal material, adds ethanol 50ml, and ultrasonic (500w) takes out after 30 minutes, from
The heart 3 minutes (rotating speed is 3200 turns per minute), takes supernatant to cross 0.22um filter membranes up to medicinal material test solution.Medicinal material is bought certainly
Guangzhou, is respectively：Radix Paeoniae Alba 1, Radix Paeoniae Alba 2, Radix Paeoniae Alba 3, Radix Paeoniae Alba 4, Radix Paeoniae Alba 5, Radix Paeoniae Alba 6 and Radix Paeoniae Alba 7.
1.2 thin-layer chromatographys detect
Testing conditions are as follows：
Lamellae：HPTLC G60 prefabricated boards (Merck)；
Point sample：6 μ l, the long 8mm of ribbon point sample；
Solvent：Chloroform：Ethyl acetate：Methanol：Formic acid=30:5:10:1；
Expansion mode：Add solvent 12ml, common pre-equilibration 15 minutes in side in double flute expansion cylinder (20cm × 10cm)；
The drying of lamellae：Lamellae after point sample be placed in phosphorus pentoxide vacuum desiccator 2 it is small when, ensure the drying of lamellae；
Inspect:Spray is placed under incandescence and is inspected with -10% sulfuric acid ethanol of 5% vanillic aldehyde.
Testing result under incandescence as shown in Fig. 2, to inspect thin-layer chromatogram (T：21 DEG C, RH：35%).The color of label S
Bands of a spectrum are arranged from lower to upper to be corresponding in turn to as reference substance Paeoniflorin, catechin, benzoylpaeoniflorin, and 1-7 is corresponding in turn to Radix Paeoniae Alba 1, white
Chinese herbaceous peony 2, Radix Paeoniae Alba 3, Radix Paeoniae Alba 4, Radix Paeoniae Alba 5, Radix Paeoniae Alba 6 and the medicinal material of Radix Paeoniae Alba 7,8 correspond to Radix Paeoniae Alba reference extract.
Interpretation of result：As shown in Figure 2, albiflorin, Paeoniflorin, catechin, benzoylpaeoniflorin separation situation are preferable,
Radix Paeoniae Alba reference extract and medicinal material (Radix Paeoniae Alba 1- Radix Paeoniae Albas 7) can show identical spot in same position, find out from collection of illustrative plates white
Chinese herbaceous peony reference extract has high consistency with medicinal material (Radix Paeoniae Alba 1- Radix Paeoniae Albas 7).
2 high performance liquid chromatography
2.1 sample preparation
Chemical reference substance solution：It is appropriate that precision weighs chemical reference substance Paeoniflorin, adds methanol dissolving, 0.1mg/ml is made
Solution；Appropriate benzoylpaeoniflorin, adds absolute ethyl alcohol dissolving, 1.0mg/ml is made；Appropriate albiflorin, adds anhydrous
Ethanol dissolves, and 0.05mg/ml is made, to obtain the final product.
Radix Paeoniae Alba reference extract solution：30mg extracts and ERS are weighed respectively, are placed in 50nl volumetric flasks, are separately added into
50% ethanol shakes up, solution takes subsequent filtrate, up to Radix Paeoniae Alba reference extract solution through 0.22 μm of membrane filtration to scale；
Medicinal material test solution：Each pharmacy's medicinal powder crosses No. two sieves, and precision weighs 0.1g, is placed in 50ml volumetric flasks,
50% ethanol of about 35ml is separately added into, carries out supersound process 30min, adds 50% ethanol to be shaken up, solution is through 0.22 μm to scale
Membrane filtration, to obtain the final product.Pharmacy's medicinal material is bought from Guangzhou, is respectively：Radix Paeoniae Alba 1, Radix Paeoniae Alba 2, Radix Paeoniae Alba 3, Radix Paeoniae Alba 4, Radix Paeoniae Alba 5,6 and of Radix Paeoniae Alba
Radix Paeoniae Alba 7.
2.2 high performance liquid chromatography detection
High performance liquid chromatography testing conditions are as follows：
Chromatographic apparatus：Agilent 1100series high performance liquid chromatographs, equipped with DAD detectors (U.S., Agilent
Technologies)
Chromatographic column：Waters Symmetry C18column (250mm × 4.6mm I.D, 5 μm)；
Mobile phase：A- acetonitriles, B-0.1% phosphate aqueous solutions；
Gradient elution program：0-5min：10%A → 15%A, 5-30min：15%A → 22%A, 30-60min：22%A
→ 49%A, 60-65min：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 wavelength 230nm (DAD detectors)；Flow velocity 0.8ml/min；10 μ l of sample size；25 DEG C of column temperature, run time：
65min。
High effective liquid chromatography for detecting：It is accurate respectively to draw chemical reference substance solution, Radix Paeoniae Alba reference extract solution
With each 10 μ l of medicinal material test solution, liquid chromatograph is injected.
High performance liquid chromatography testing result：
Measure, records chromatogram, and up to the HPLC finger-print stacking charts shown in Fig. 3,1-7 is corresponding white respectively from top to bottom
Chinese herbaceous peony 1, Radix Paeoniae Alba 2, Radix Paeoniae Alba 3, Radix Paeoniae Alba 4, Radix Paeoniae Alba 5, Radix Paeoniae Alba 6 and Radix Paeoniae Alba 7, S correspond to Radix Paeoniae Alba reference extract, and R corresponds to common pattern.
As seen from Figure 3, the finger-print of 7 medicinal material of Radix Paeoniae Alba 1- Radix Paeoniae Albas has high consistency, thus in accordance with China
Pharmacopoeia commission of people's republic similarity evaluation (2012.130723 version) establishes RADIX PAEONIAE ALBA
The finger-print common pattern of material, and similarity analysis is carried out to all samples.Common pattern collection of illustrative plates is as shown in figure 4, wherein No. 7
Chromatographic peak corresponds to albiflorin, and No. 8 chromatographic peaks correspond to Paeoniflorin, and No. 44 chromatographic peaks correspond to five nutgall acyl grapes
Sugar, No. 12 chromatographic peaks correspond to benzoylpaeoniflorin.
According to set white Peony Root finger-print common pattern, using included angle cosine algorithm according to each sample component and its
Peak area carries out similarity evaluation to 7 batches of white Peony Roots and reference extract sample, and the results are shown in Table 3.
Table 3
| Sample | Similarity |
| Radix Paeoniae Alba 1 | 0.999 |
| Radix Paeoniae Alba 2 | 0.999 |
| Radix Paeoniae Alba 3 | 0.999 |
| Radix Paeoniae Alba 4 | 0.999 |
| Radix Paeoniae Alba 5 | 0.999 |
| Radix Paeoniae Alba 6 | 0.999 |
| Radix Paeoniae Alba 7 | 0.999 |
| Radix Paeoniae Alba reference extract | 1.000 |
According to above similarity analysis as a result, the similarity of 7 batches of medicinal materials and white Peony Root finger-print common pattern is impartial
In 0.999, similarity is very high.The similarity of Radix Paeoniae Alba reference extract and white Peony Root finger-print common pattern is 0.999, table
The uniformity of bright Radix Paeoniae Alba reference extract of the invention and medicinal material is good.
According to the result of TLC and HPLC finger-prints, the Radix Paeoniae Alba reference extract is examined using thin-layered chromatography
The corresponding raw medicinal material of the chromatogram that measures is consistent, more precisely, thin layer is used to the Radix Paeoniae Alba reference extract
Fluorescence spot at albiflorin, Paeoniflorin and benzoylpaeoniflorin position in the chromatogram that chromatography detects and adopt
With chromatography of the chromatogram that high performance liquid chromatography detects at albiflorin, Paeoniflorin and benzoylpaeoniflorin position
Corresponding chemical reference substance is distinguished at peak or its corresponding raw medicinal material is consistent, therefore the Radix Paeoniae Alba reference extract of the present invention can
Applied to medicinal material or the Qualitive test of Chinese medicine preparation, such as the albiflorin to medicinal material or Chinese medicine preparation, Paeoniflorin and benzene first
Acyl Paeoniflorin component carries out qualitative analysis.
According to HPLC measurement results and statistical analysis, the white Peony Root finger-print common pattern and white Peony Root of foundation
Similarity it is high, and the present invention Radix Paeoniae Alba reference extract and white Peony Root finger-print common pattern similarity pole
Height, this explanation carry out albiflorin, Paeoniflorin and benzene using the Radix Paeoniae Alba reference extract of the present invention to medicinal material or Chinese medicine preparation
The assay reliability of formyl Paeoniflorin is high, therefore the Radix Paeoniae Alba reference extract of the present invention can be applied to medicinal material or Chinese medicine system
The sxemiquantitative discriminatory analysis of agent, can also be applied to Radix Paeoniae Alba and the quality of the medicinal material containing Radix Paeoniae Alba/effective component of paeonia lactiflora or Chinese medicine preparation
In control, such as the albiflorin to medicinal material or Chinese medicine preparation, Paeoniflorin and benzoylpaeoniflorin component carry out sxemiquantitative mirror
Do not analyze, or to Radix Paeoniae Alba and the medicinal material containing Radix Paeoniae Alba/effective component of paeonia lactiflora or Chinese medicine preparation carry out half-quantitative detection and then control
Its quality.
The foregoing is merely the better embodiment of the present invention, it is not intended to limit the invention, it is all the present invention's
All any modification, equivalent and improvement made within spirit and principle etc., should all be included in the protection scope of the present invention.
Claims (10)
1. a kind of Radix Paeoniae Alba reference extract, it is characterised in that it is derived from：The white Peony Root powder of different batches is carried out repeatedly
Extraction, obtains the radix paeoniae alba extraction of different batches, then the radix paeoniae alba extraction of different batches is allocated, and obtains Radix Paeoniae Alba control
Extract；
Optional, the white Peony Root powder to different batches is repeatedly extracted, and is the peony lactone glycoside for containing it
Component, the purity of Chinese herbaceous peony methods of glycosides and benzoylpaeoniflorin constituents and/or concentration improve, so as to obtain the white of different batches
Chinese herbaceous peony extract；
Optional, content >=5.0% of albiflorin, Paeoniflorin and benzoylpaeoniflorin in the Radix Paeoniae Alba reference extract；
It is optional, to the Radix Paeoniae Alba reference extract using the collection of illustrative plates that thin-layered chromatography and/or high performance liquid chromatography obtain with
Its corresponding raw medicinal material is consistent；
It is optional, albiflorin in the chromatogram detected to the Radix Paeoniae Alba reference extract using thin-layered chromatography,
The chromatogram that fluorescence spot and use high performance liquid chromatography at Paeoniflorin and benzoylpaeoniflorin detect is in Chinese herbaceous peony
Chromatographic peak at ester glycosides, Paeoniflorin and benzoylpaeoniflorin position should distinguish corresponding chemical reference substance or its corresponding original
Expect that medicinal material is consistent.
2. a kind of preparation method of the Radix Paeoniae Alba reference extract described in claim 1, comprises the following steps：
Step 1: extraction：Take the white Peony Root powder of different batches to be mixed respectively with ethanol, filter, filtered after homogenate extraction
Liquid 1 and filter residue 1, and filtrate 1 is evaporated to obtain Radix Paeoniae Alba dry cream；
Step 2: prepare radix paeoniae alba extraction：By gained Radix Paeoniae Alba dry cream, ethanol is dissolved in, obtains the ethanol solution of Radix Paeoniae Alba dry cream, then
Auxiliary material is added, sieving is evaporated and obtains radix paeoniae alba extraction；
Step 3: allotment：The radix paeoniae alba extraction of different batches is allocated, obtains Radix Paeoniae Alba reference extract.
3. the preparation method of Radix Paeoniae Alba reference extract according to claim 2, it is characterised in that described in the step 1
Filter residue 1 extracted according to the extracting method of step 1 by n times, and the filtrate that n times extraction is collected merges with filtrate 1 as carrying
Liquid is taken, extracting solution is evaporated to obtain Radix Paeoniae Alba dry cream；Wherein 6 >=N >=0, and N is integer；
Optional, the N is 3.
4. the preparation method of Radix Paeoniae Alba reference extract according to claim 2, it is characterised in that described in the step 1
Extraction solution ethanol concentration be 30%~95%；
Optional, it is 50% that solution ethanol concentration is extracted in the step 1.
5. the preparation method of Radix Paeoniae Alba reference extract according to claim 3, it is characterised in that white in the step 1
The w/v of Chinese herbaceous peony medicinal powder and ethanol is 1:5~1:45；
Optional, the w/v of white Peony Root powder and ethanol in the step 1 is 1：10；
Optional, the homogenate extraction time in the step 1 is 1~5min；
Optional, the homogenate extraction time in the step 1 is 1min；
Optional, the filtering in the step 1 is with Medium speed filter paper or 2000 mesh sieve net filtrations；
Optional, the filtering in the step 1 is to use Medium speed filter paper.
6. the preparation method of Radix Paeoniae Alba reference extract according to claim 3, it is characterised in that Radix Paeoniae Alba in the step 2
The w/v of dry cream and ethanol：1:2~1:10；
Optional, auxiliary material used is superfine silica gel powder and/or medical starch in the step 2；
Optional, the auxiliary material is superfine silica gel powder；
Optional, the step 2 is its weight is added in the ethanol solution of Radix Paeoniae Alba dry cream 10%~50% micro mist silicon
Glue；
Optional, the step 2 is that 40% superfine silica gel powder of its weight is added in the ethanol solution of Radix Paeoniae Alba dry cream；
It is optional, the step 2 be evaporated after be 90~200 mesh sieve net filtrations；
It is optional, the step 2 be evaporated after be 200 mesh sieve net filtrations；
Optional, further included between the step 2 and step 3 with thin-layered chromatography and/or high performance liquid chromatography to difference
The step of albiflorin, Paeoniflorin and the benzoylpaeoniflorin of the radix paeoniae alba extraction of batch are detected.
7. the preparation method of Radix Paeoniae Alba reference extract according to claim 2, it is characterised in that allocated in the step 3
Standard be：It should make albiflorin >=2.0%, Paeoniflorin >=7.5%, benzoyl Chinese herbaceous peony in the reference extract that finally obtains
Content >=0.2% of glycosides；
It is optional, to the Radix Paeoniae Alba reference extract using the collection of illustrative plates that thin-layered chromatography and/or high performance liquid chromatography obtain with
Its corresponding raw medicinal material is consistent；
It is optional, albiflorin in the chromatogram detected to the Radix Paeoniae Alba reference extract using thin-layered chromatography,
The chromatogram that fluorescence spot and use high performance liquid chromatography at Paeoniflorin and benzoylpaeoniflorin position detect is in Chinese herbaceous peony
Chromatographic peak at medicine lactone glycoside, Paeoniflorin and benzoylpaeoniflorin position should distinguish corresponding chemical reference substance or its correspondence
Raw medicinal material it is consistent.
8. Radix Paeoniae Alba reference extract described in claim 1 is in the discriminating of medicinal material or Chinese medicine preparation, or Radix Paeoniae Alba or containing Radix Paeoniae Alba/Radix Paeoniae Alba
Application in the quality control of the medicinal material or Chinese medicine preparation of active ingredient；
Optional, the discriminating of the medicinal material or Chinese medicine preparation, or the matter of Radix Paeoniae Alba and the Chinese medicine preparation containing Radix Paeoniae Alba/effective component of paeonia lactiflora
Measuring the method controlled is：By Radix Paeoniae Alba reference extract, medicinal material or the Chinese medicine preparation described in claim 1 with thin-layered chromatography and/
Or high performance liquid chromatography is detected, contrast differentiates；
Optional, the thin-layered chromatography and/or high performance liquid chromatography are to Radix Paeoniae Alba reference extract, medicinal material or Chinese medicine preparation
In albiflorin, Paeoniflorin and benzoylpaeoniflorin be detected.
9. application according to claim 8, it is characterised in that when by the Radix Paeoniae Alba reference extract described in claim 1, medicinal material or
, it is necessary to which Radix Paeoniae Alba reference extract, medicinal material or Chinese medicine preparation are configured to solution when Chinese medicine preparation is detected with thin-layered chromatography
Thin-layered chromatography detection is carried out again；
The preparation method of wherein Radix Paeoniae Alba reference extract solution is：Radix Paeoniae Alba reference extract described in claim 1 is added it
The ethanol of 10-100 times of volume of quality, is ultrasonically treated, shakes up, and crosses 0.12-0.32 μm of filter membrane up to Radix Paeoniae Alba reference extract solution；
It is optional, the Radix Paeoniae Alba reference extract described in claim 1 adds to the ethanol of its 20 times of volume of quality, at ultrasonic 500w
Reason 30 minutes, shakes up, and crosses 0.22 μm of filter membrane up to Radix Paeoniae Alba reference extract solution；
The preparation method of its Chinese medicine or Chinese medicine preparation solution is：Get it filled material or Chinese medicine preparation adds 10-100 times of volume of its quality
Ethanol, be ultrasonically treated, be settled in 2~10ml volumetric flasks, centrifuge, take supernatant to cross 0.12-0.32 μm of filter membrane up to medicinal material
Or Chinese medicine preparation solution；
It is optional, get it filled material or Chinese medicine preparation adds the ethanol of its 100 times of volume of quality, ultrasonic 500w is handled 15 minutes, constant volume
Into 2ml volumetric flasks, 3200 revs/min centrifuge 3 minutes, take supernatant to cross 0.22um filter membranes molten up to medicinal material or Chinese medicine preparation
Liquid；
Optional, the thin-layered chromatography testing conditions are：
Lamellae：HPTLC G60 prefabricated boards；
Point sample：2 μ l, the long 8mm of ribbon point sample；
Solvent：Chloroform：Ethyl acetate：Methanol：Formic acid=30:5:10:1；
The drying of lamellae：Lamellae after point sample be placed in phosphorus pentoxide vacuum desiccator 2 it is small when, ensure the dry of lamellae
It is dry；
Inspect:Spray is placed under incandescence and is inspected with -10% sulfuric acid ethanol color developing agent of 5% vanillic aldehyde.
10. application according to claim 8, it is characterised in that when by Radix Paeoniae Alba reference extract, the medicinal material described in claim 1
Or Chinese medicine preparation when being detected with high performance liquid chromatography, it is necessary to which Radix Paeoniae Alba reference extract, medicinal material or Chinese medicine preparation are prepared
High performance liquid chromatography detection is carried out again into solution；
The preparation method of wherein Radix Paeoniae Alba reference extract solution is：Precision weighs the Radix Paeoniae Alba reference extract described in claim 1
30mg~300mg, is configured to the solution that concentration is 0.6~60mg/ml with ethanol, crosses the filter membrane of 0.12-0.32 μm of filter membrane, take continuous
Filtrate, up to Radix Paeoniae Alba reference extract solution；
Optional, precision weighs the Radix Paeoniae Alba reference extract 30mg described in claim 1, and it is 0.6mg/ to be configured to concentration with ethanol
The solution of ml, crosses the filter membrane of 0.22 μm of filter membrane, subsequent filtrate is taken, up to Radix Paeoniae Alba reference extract solution；
The preparation method of its Chinese medicine or Chinese medicine preparation solution is：Get it filled material or Chinese medicine preparation cross precision after No. two sieves weigh 2g~
20g, puts in 50ml~250ml conical flask with cover, adds ethanol solution 45ml~240ml, water-bath reflux or ultrasound 500w processing
After 30min, let cool, by Medium speed filter paper or 2000 mesh sieve net filtrations into 50ml or 250ml volumetric flasks, add ethanol to scale
At 50ml~250ml, shake up, cross 0.12-0.32 μm of filter membrane, take subsequent filtrate up to medicinal material or Chinese medicine preparation solution；
It is optional, get it filled material or Chinese medicine preparation cross No. two sieves after precision weigh 2g, put in 50ml conical flask with cover, it is molten to add ethanol
After liquid 45ml, ultrasonic 500w processing 30min, let cool, filtered by Medium speed filter paper into 50ml volumetric flasks, add ethanol to scale
At 50ml, shake up, cross 0.22 μm of filter membrane, take subsequent filtrate up to medicinal material or Chinese medicine preparation solution；
Optional, described high performance liquid chromatography testing conditions：
Chromatographic apparatus：1100 series high performance liquid chromatographs of Agilent, equipped with DAD detectors (U.S., Agilent
Technologies)
Chromatographic column：Waters Symmetry C18 column (250mm × 4.6mm I.D, 5 μm)；
Mobile phase：A- acetonitriles, B-0.1% phosphate aqueous solutions；
Gradient elution program：0-5min：10%A → 15%A, 5-30min：15%A → 22%A, 30-60min：22%A →
49%A, 60-65min：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 wavelength 230nm (DAD detectors)；Flow velocity 0.8ml/min；10 μ l of sample size；25 DEG C of column temperature, run time：
65min。
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| CN113155573A (en) * | 2021-04-28 | 2021-07-23 | 广州科曼生物科技有限公司 | Fructus lycii large-polarity control extract and preparation method and application thereof |
| CN113514596A (en) * | 2021-04-28 | 2021-10-19 | 广州科曼生物科技有限公司 | Small and medium polarity control extract of wolfberry fruit and preparation method and application thereof |
| 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 |
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| CN109596754A (en) * | 2018-07-05 | 2019-04-09 | 广州卡马生物科技有限公司 | A kind of Radix Salviae Miltiorrhizae reference extract and its application |
| CN109596755A (en) * | 2018-07-05 | 2019-04-09 | 广州卡马生物科技有限公司 | A kind of Radix Angelicae Sinensis reference extract and its application |
| CN113155573A (en) * | 2021-04-28 | 2021-07-23 | 广州科曼生物科技有限公司 | Fructus lycii large-polarity control extract and preparation method and application thereof |
| CN113514596A (en) * | 2021-04-28 | 2021-10-19 | 广州科曼生物科技有限公司 | Small and medium polarity control extract of wolfberry fruit and preparation method and application thereof |
| 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 |
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