CN106814157A - The preparation method of rascal reference extract - Google Patents
The preparation method of rascal reference extract Download PDFInfo
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- CN106814157A CN106814157A CN201710031828.7A CN201710031828A CN106814157A CN 106814157 A CN106814157 A CN 106814157A CN 201710031828 A CN201710031828 A CN 201710031828A CN 106814157 A CN106814157 A CN 106814157A
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Abstract
The invention provides a kind of preparation method of rascal reference extract, repeatedly extracted including the rascal medicinal powder to separate sources or batch, the peel extract of separate sources or batch is obtained, then the peel extract to separate sources or batch is allocated, obtain rascal reference extract.The preparation method of rascal reference extract of the invention is easy to operate, low cost, reproducible and recovery rate is high；Rascal reference extract is prepared with this preparation method, it is ensured that the uniformity of the rascal reference extract of different batches；Representative medicinal material is chosen as raw material simultaneously, and the product for obtaining is representative, and the quality of medicinal material to be detected more can be intuitively reflected as reference extract, and proterties is stable, uniform and easy to use.
Description
Technical field
The present invention relates to quality control technologies for traditional Chinese medicine field, more particularly to a kind of preparation side of rascal reference extract
Method.
Background technology
Rascal is the pericarp for drying young fruit or immature fruit of rutaceae orange and its variety.5~June collects
From the young fruit for falling, dry, practise and claim " FTUCTUS CITRI IMMATURI "；7~August harvest green fruit, on pericarp vertical profile into pintongs to base portion,
Flesh valve is eliminated, is dried, practised and claim " Sihuaqingpi ".
Rascal medicinal history is long, is the medicine that conventional soothing the liver relieving stagnant Qi, disperse accumulationsization are stagnant.Chinese Pharmacopoeia Commission's issue
In 2015 editions Chinese Pharmacopoeia quality standards, the content of the aurantiamarin of one of active ingredient specifies during to rascal as medicinal material：
(the C containing aurantiamarin in rascal medicinal material28H34O15) 5.0%, medicine materical crude slice (C containing aurantiamarin must not be less than28H34O15) 4.0% must not be less than,
(the C containing aurantiamarin in stir-baked PERICARPIUM CITRI RETICULATAE VIRIDE with vinegar28H34O15) 3.0% must not be less than.
Existing traditional Chinese medicine quality control model be substantially along the development of Natural Medicine Chemistry, it is a certain or several to Chinese medicine
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 sets up corresponding simple physics and chemistry discriminating by document report, then
Develop into the quality standard of the discriminating and assay based on spectrum, chromatogram.All it is multicomponent synthesis per Chinese medicine simply
Body, which dictates that its distinctive globality and ambiguity, also indicates that one or two using in medicinal material even several compositions as medicinal material matter
There is significant limitation in the evaluation method of amount.
1990 editions《Chinese Pharmacopoeia》Increased the indentification by TLC of control medicinal material so that the discriminating of Chinese medicine and Chinese patent drug has
Very big progress.The herbal medicine of current Chinese medicine and foreign countries increasingly payes attention to multicomponent or multi-component detection, such as German ginkgo
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.Current Chinese Pharmacopoeia has two kinds " reference substances " in terms of quality of medicinal material is controlled, and chemical reference substance is with
Medicine control medicinal material.Wherein chemical reference substance can be used for the Qualitive test of medicinal material and quantitative analysis, 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 leaks, shoddy phenomenon happens occasionally.Chinese medicine control medicinal material is influenceed by the place of production, growing environment, it is difficult to
Ensure per batch of uniform quality, and be only used for Qualitive test, it is impossible to reflect the height of medicinal ingredient content.
Chinese medicine reference extract has four basic demands (ASCS):Authenticity, crude drug source reliability, with representative
Property；Specificity, the detection method specificity for using；Con-sistency, different batches reference extract should keep one
Cause；Stability, proterties is stable, uniform, easy to use.With TLC Fingerprints and high performance liquid chromatography fingerprint
The method of collection of illustrative plates, the need for Chinese medicine reference extract can meet Qualitive test.Can also enter one by the reference extract of markization
Step carries out the even quantitative analysis of sxemiquantitative.Chinese medicine reference extract will be significant to Chinese medicine quality control.But also just
Because Chinese medicine reference extract has above-mentioned specific requirement, currently available technology does not have also or does not have the rascal control extraction of maturation
The preparation method of thing.
The content of the invention
Present invention solves the technical problem that being to provide a kind of easy to operate, low cost, reproducible and recovery rate green grass or young crops high
The preparation method of skin reference extract, the rascal reference extract batch uniformity that is prepared from this preparation method is good, proterties
It is stable, uniform and easy to use, the overall picture of medicinal material can be reflected.
The invention provides a kind of preparation method of rascal reference extract, including to separate sources or the rascal medicine of batch
Material powder is repeatedly extracted, and obtains the peel extract of separate sources or batch, then to separate sources or the rascal of batch
Extract is allocated, and obtains rascal reference extract；
In a preference, the rascal medicinal powder to separate sources or batch is repeatedly extracted, and is to contain it
The purity and/or concentration of some flavone aglycone constituents and/or flavonoid glycoside composition are improved, so as to obtain separate sources or batch
Peel extract；
In a preference, the flavone aglycone constituents are Nobiletin and tangeritin；
In a preference, the flavonoid glycoside composition is aurantiamarin.
In a preference, the preparation method of above-mentioned rascal reference extract is comprised the following steps：
Step one, slightly carry：The rascal medicinal powder for taking separate sources or batch mixes with methyl alcohol respectively, mistake after ultrasonic extraction
Filter, obtains filtrate 1 and filter residue 1, and filtrate 1 is evaporated obtains rascal crude extract；
It is step 2, refined：By gained rascal crude extract, water and suction filtration are dissolved in, obtain filter residue；By filter residue water rinse to
Water colorless, collects filtrate 2 and filter residue 2, and filtrate 2 is chromatographed with macroreticular resin；Then macroreticular resin first is rinsed with water, obtains eluent
1；Again with methanol rinses macroreticular resin, collects eluent 2；Finally eluent 2 and filter residue 2 are mixed, is evaporated, obtained rascal and refine
Thing；
Step 3, mix silica gel grinding filtering：Superfine silica gel powder is added in the refined thing of rascal, grinding filtering is obtained final product different next
Source or the peel extract of batch；
Step 4, allotment：The peel extract of separate sources or batch is allocated, rascal reference extract is obtained.
In a preference, also include to the corresponding difference of the rascal medicinal powder of separate sources or batch before step one
The screening of the raw medicinal material of source or batch；
It is described to the corresponding separate sources of the rascal medicinal powder of separate sources or batch or batch in a preference
The screening of raw medicinal material includes：By the raw medicinal material of separate sources or batch with thin-layered chromatography and/or high performance liquid chromatography
Detected；Then being obtained by thin-layered chromatography and/or high performance liquid chromatography to the raw medicinal material of separate sources or batch
To chromatogram and/or the content of measure be compared analysis, reject the abnormal bulk drug of the content of chromatogram and/or measure
The raw medicinal material that the content of material, reservation chromatogram and/or measure is consistent carries out slightly carrying for next step；
In a preference, the thin-layered chromatography and/or high performance liquid chromatography carry out detection and include to flavonoid glycoside
The detection of composition and/or flavone aglycone constituents；
In a preference, the flavone aglycone constituents are Nobiletin and tangeritin；
In a preference, the flavonoid glycoside composition is aurantiamarin.
In a preference, the filter residue 1 described in the step one is extracted according to the extracting method of step one by n times,
And merge as extract solution the filtrate that n times extract collection with filtrate 1, extract solution is evaporated and obtains rascal crude extract；Wherein 6 >=
N >=0, and N is integer；
In a preference, the N is 2.
In a preference, the rascal medicinal powder of separate sources or batch in the step one and the weighing body of methyl alcohol
Product is than being 1:8~1:50；
In a preference, the rascal medicinal powder of separate sources or batch in the step one and the weighing body of methyl alcohol
Product is than being 1：10；
In a preference, the ultrasonic time in the step one is 30~60min, and power is 100W~3kW；
In a preference, the ultrasonic time in the step one is 30min, and power is 500W；
In a preference, the filtering of the step one is with Medium speed filter paper or 2000 mesh sieve net filtrations.
In a preference, the filtrate 2 in the step 2 is chromatographed with the macroreticular resin of 1~4 times of its weight；
In a preference, the filtrate 2 in the step 2 is chromatographed with the macroreticular resin of 2 times of its weight；
In a preference, the macroreticular resin is non-polar macroporous resin；
In a preference, the aperture of the macroreticular resin is 100~1000nm；
In a preference, the macroreticular resin is D101 type macroreticular resins.
It is first to be rushed with 5~10 column volume pure water after the macroreticular resin chromatography in the step 2 in a preference
Macroreticular resin is washed, eluent 1 is collected；Macroreticular resin is rinsed with 3~10 methyl alcohol of column volume 70%~100% again, wash-out is collected
Liquid 2；
It is first to rinse macroreticular resin with 8 column volume pure water after the macroreticular resin chromatography in a preference, receives
Collection eluent 1；Macroreticular resin is rinsed with 4 methyl alcohol of column volume 100% again, eluent 2 is collected.
In a preference, the step 3 is the micro mist silicon that the 20%~50% of its weight is added in the refined thing of rascal
Glue；
In a preference, the step 3 is the superfine silica gel powder that the 40% of its weight is added in the refined thing of rascal；
It was 90~200 mesh sieve net filtrations after the step 3 grinding in a preference.
In a preference, also include with thin-layered chromatography and/or high-efficient liquid phase color between the step 3 and step 4
The step that spectrometry is detected to the flavone aglycone constituents and/or flavonoid glycoside composition of separate sources or the peel extract of batch
Suddenly；
In a preference, the flavone aglycone constituents are Nobiletin and tangeritin；
In a preference, the flavonoid glycoside composition is aurantiamarin.
In a preference, the standard allocated in the step 4 is：Orange peel in final rascal reference extract should be made
The weight percent content of glycosides maintains 16%-20%；
In a preference, the rascal reference extract is obtained using thin-layered chromatography and/or high performance liquid chromatography
The corresponding raw medicinal material of the collection of illustrative plates that arrives is consistent；
In a preference, the river in the chromatogram for obtaining is detected using thin-layered chromatography to the rascal reference extract
Fluorescence spot at hesperetin, tangeritin and aurantiamarin position and the chromatogram that is obtained using high performance liquid chromatography detection are in orange
Chromatographic peak at skin glycosides position should distinguish corresponding chemical reference substance or its corresponding raw medicinal material is consistent.
Present invention also offers a kind of medicinal material or the discrimination method of Chinese medicine preparation, or rascal or containing rascal/rascal effectively into
The medicinal material or the method for quality control of Chinese medicine preparation for dividing, by the above-mentioned rascal reference extract, the testing sample for preparing
Detected with thin-layered chromatography and/or high performance liquid chromatography, contrast differentiates；
In a preference, the thin-layered chromatography and/or high performance liquid chromatography include flavonoid glycoside and/or flavonoid glycoside
Metaclass is detected；
In a preference, the flavonoid glycoside testing conditions are：
Lamellae:Silica gel prefabricated board
Solvent：Chloroform:Methyl alcohol:Water:Glacial acetic acid=13:4:1:1.5
Inspect:5%AlCl3Ethanol develops the color, and is inspected under UV366nm
In a preference, the flavonoid glycoside metaclass testing conditions are：
Lamellae:Silica gel prefabricated board
Solvent：Solvent 1：Toluene：Ethyl acetate：Formic acid：Water=20:20:1:1
Solvent 2：Toluene：Ethyl acetate：Formic acid：Water=20:10:1:1
Inspect：Inspected under 366nmUV；
In a preference, described high performance liquid chromatography testing conditions：
Fixing phase：Chromatographic column C18 chromatographic columns；Mobile phase：A acetonitriles, B water；
Eluent gradient：
Flow velocity：0.8ml/min；Column temperature：20℃；Detector：PDAD；
Detection wavelength：280nm.
The term " Sihuaqingpi ERS " occurred in the present invention refers to Sihuaqingpi reference extract QPERS-1 or its breviary
Name QPERS-1, " FTUCTUS CITRI IMMATURI ERS " refers to FTUCTUS CITRI IMMATURI reference extract QPERS-2 or its breviary name to the term occurred in the present invention
QPERS-2。
Compared with prior art, the invention has the advantages that：
The preparation method of rascal reference extract of the invention employ slightly carry, refine, plus auxiliary material dry, grinding sieving and
The step of allotment, easy to operate, low cost, reproducible and recovery rate is high.The rascal prepared with this preparation method is compareed
Extract is overcome because Chinese medicine control medicinal material is subject to the place of production, life because being that multiple or different batches extracts carry out allotment and form
The influence of environment long, it is difficult to ensure that the defect per batch of uniform quality, extracts so as to the rascal that ensure that different batches is compareed
The uniformity of thing；Representative medicinal material is chosen as raw material simultaneously, the product for obtaining is representative, extracted as control
Thing more can intuitively reflect the quality of medicinal material to be detected；And its proterties is stable, uniform and easy to use.Using rascal of the invention
The content of aurantiamarin is in 16%~20%, and control extraction in the reference extract that the preparation method of reference extract finally gives
Thing TLC Fingerprints, HPLC finger-prints are corresponding with medicinal material consistent.Present invention also offers medicinal material or Chinese medicine preparation
Discrimination method, or rascal or medicinal material or the method for quality control of Chinese medicine preparation containing rascal/rascal active ingredient, not only can be right
Medicinal material or Chinese medicine preparation carry out Qualitive test, and can be also used for sxemiquantitative even quantitative analysis.
Brief description of the drawings
Fig. 1 is the phenotypic characteristic of medicinal material rascal A.
Fig. 2 is the phenotypic characteristic of medicinal material rascal B.
Fig. 3 is the phenotypic characteristic of medicinal material rascal C.
Fig. 4 is the phenotypic characteristic of medicinal material rascal D.
Fig. 5 is the phenotypic characteristic of medicinal material rascal E.
Fig. 6 is the phenotypic characteristic of medicinal material rascal F.
Fig. 7 is the phenotypic characteristic of medicinal material rascal G.
Fig. 8 is the phenotypic characteristic of medicinal material FTUCTUS CITRI IMMATURI H.
Fig. 9 is the phenotypic characteristic of medicinal material FTUCTUS CITRI IMMATURI I.
Figure 10 is the phenotypic characteristic of medicinal material FTUCTUS CITRI IMMATURI J.
Figure 11 is the phenotypic characteristic of the rascal 1 for being purchased from Guangzhou City pharmacy (numbering 1).
Figure 12 is the phenotypic characteristic for buying the rascal 2 in Guangzhou City pharmacy (numbering 2).
Figure 13 is the phenotypic characteristic for buying the rascal 3 in Guangzhou City pharmacy (numbering 3).
Figure 14 is the phenotypic characteristic for buying the rascal 4 in Guangzhou City pharmacy (numbering 4).
Figure 15 is the phenotypic characteristic for buying the rascal 5 in Guangzhou City pharmacy (numbering 5).
Figure 16 is the phenotypic characteristic for buying the rascal 6 in Guangzhou City pharmacy (numbering 6).
Figure 17 is the phenotypic characteristic for buying the rascal 7 in Guangzhou City pharmacy (numbering 7).
Figure 18 is the phenotypic characteristic for buying the rascal 8 in Guangzhou City pharmacy (numbering 8).
Figure 19 is the phenotypic characteristic for buying the rascal 9 in Guangzhou City pharmacy (numbering 9).
Figure 20 is the phenotypic characteristic for buying the rascal 10 in Guangzhou City pharmacy (numbering 10).
Figure 21 is rascal reference extract preparation flow figure；
Figure 22 is the rascal medicinal material TLC collection of illustrative plates for flavonoid glycoside, and band 1-10 is represented respectively：1 medicinal material rascal A；2 medicinal materials
Rascal B；3 medicinal material rascal C；4 medicinal material rascal D；5 medicinal material rascal E；6 medicinal material rascal F；7 medicinal material rascal G；8 medicinal material FTUCTUS CITRI IMMATURI H；9 medicines
Material FTUCTUS CITRI IMMATURI I；10 medicinal material FTUCTUS CITRI IMMATURI J.
Figure 23 is the rascal medicinal material TLC collection of illustrative plates for flavonoid glycoside metaclass, and band 1-10 is represented respectively：1 medicinal material rascal A；2 medicines
Material rascal B；3 medicinal material rascal C；4 medicinal material rascal D；5 medicinal material rascal E；6 medicinal material rascal F；7 medicinal material rascal G；8 medicinal material FTUCTUS CITRI IMMATURI H；9
Medicinal material FTUCTUS CITRI IMMATURI I；10 medicinal material FTUCTUS CITRI IMMATURI J.
Figure 24 is the HPLC collection of illustrative plates of rascal medicinal material, is followed successively by from top to bottom：Medicinal material rascal A, medicinal material rascal B, medicinal material rascal
C, medicinal material rascal D, medicinal material rascal E, medicinal material rascal F, medicinal material rascal G, medicinal material FTUCTUS CITRI IMMATURI H, medicinal material FTUCTUS CITRI IMMATURI I, medicinal material FTUCTUS CITRI IMMATURI
J。
Figure 25 is the peel extract TLC collection of illustrative plates for flavonoid glycoside, and band 1-9 is represented respectively：The rascal of 1 rascal B is carried
Take thing；The peel extract of 2 rascal C；The peel extract of 3 rascal D；The peel extract of 4 rascal E；The rascal of 5 rascal F is carried
Take thing；The first batch peel extract (G1) of 6 rascal G；The second lot peel extract (G2) of 7 rascal G；8 FTUCTUS CITRI IMMATURI H1
First batch peel extract (H1)；The second lot peel extract (H2) of 9 FTUCTUS CITRI IMMATURIs.S represents aurantiamarin.
Figure 26 is the peel extract TLC collection of illustrative plates for flavone aglycone detection, and band 1-9 is represented respectively：The green grass or young crops of 1 rascal B
Bark extract；The peel extract of 2 rascal C；The peel extract of 3 rascal D；The peel extract of 4 rascal E；The green grass or young crops of 5 rascal F
Bark extract；The first batch peel extract (G1) of 6 rascal G；The second lot peel extract (G2) of 7 rascal G；8 green grass or young crops
The first batch peel extract (H1) of skin H1；The second lot peel extract (H2) of 9 FTUCTUS CITRI IMMATURIs.S：It is followed successively by from top to bottom
Nobiletin, tangeritin.
Figure 27 be peel extract HPLC collection of illustrative plates, from top to bottom for：The peel extract of rascal B, the rascal of rascal C are extracted
Thing, the peel extract of rascal D, the peel extract of rascal E, the peel extract of rascal F, the first batch rascal of rascal G
It is extract (G1), the second lot peel extract (G2) of rascal G, the first batch peel extract (H1) of FTUCTUS CITRI IMMATURI H1, individual
The second lot peel extract (H2) of rascal.
Figure 28 is rascal reference extract TLC collection of illustrative plates (flavonoid glycoside detection), S：Aurantiamarin；1-2：Sihuaqingpi control is extracted
Thing QPERS-1,2 is 1 parallel control；3-4：FTUCTUS CITRI IMMATURI reference extract QPERS-2,4 is 3 parallel control.
Figure 29 is rascal reference extract TLC collection of illustrative plates (flavone aglycone detection), S：Respectively tangeritin, river are old from top to bottom
Pi Su；1-2：Sihuaqingpi reference extract QPERS-1,2 is 1 parallel control；3-4：FTUCTUS CITRI IMMATURI reference extract QPERS-
2,2 is 1 parallel control.
Figure 30 is rascal reference extract HPLC-DAD chromatograms, under：FTUCTUS CITRI IMMATURI ERS (QPERS-2)；On：Sihuaqingpi
ERS(QPERS-1)；S：Aurantiamarin.
Figure 31 is rascal reference extract and 10 batches of thin layer collection of illustrative plates of commercially available medicinal material, 1：It is from top to bottom：Aurantiamarin, new orange
Skin glycosides, aurantiin；2：Sihuaqingpi ERS (QPERS-1)；3：FTUCTUS CITRI IMMATURI ERS (QPERS-2)；4-13：The pharmacy of Guangzhou City 10
Rascal medicinal material is bought, respectively：Rascal 1, rascal 2, rascal 3, rascal 4, rascal 5, rascal 6, rascal 7, rascal 8, rascal 9 and green grass or young crops
Skin 10.
Figure 32 is rascal reference extract and 10 batches of thin layer collection of illustrative plates of commercially available medicinal material, 1：It is from top to bottom：Tangeritin, river is old
Pi Su；2：Sihuaqingpi ERS (QPERS-1)；3：FTUCTUS CITRI IMMATURI ERS (QPERS-2)；4-13：Green grass or young crops is bought by the pharmacy of Guangzhou City 10
Skin medicinal material, respectively：Rascal 1, rascal 2, rascal 3, rascal 4, rascal 5, rascal 6, rascal 7, rascal 8, rascal 9 and rascal 10.
Figure 33 be rascal reference extract and 10 batches of commercially available medicinal material HPLC-DAD collection of illustrative plates, from top to bottom for：Sihuaqingpi ERS
(QPERS-1), rascal medicinal material is bought by FTUCTUS CITRI IMMATURI ERS (QPERS-2), the pharmacy of Guangzhou City 10：Rascal 1, rascal 2, rascal 3,
Rascal 4, rascal 5, rascal 6, rascal 7, rascal 8, rascal 9 and rascal 10.
Specific embodiment
Embodiments of the invention are described below in detail.The embodiments described below is exemplary, is only used for explaining this hair
It is bright, and be not considered as limiting the invention.Unreceipted particular technique or condition in embodiment, according to text in the art
Offer described technology or condition or carried out according to product description.Agents useful for same or the unreceipted production firm person of instrument,
For can by city available from conventional products.Term as used herein "and/or" includes the listed of one or more correlations
The arbitrary and all of combination of project.
The material and source used in following examples are as follows：
It is envisaged for preparing the medicinal material (10 batches) of reference extract:Rascal A (the places of production：Guangxi), the rascal B (places of production：Hunan), it is blue or green
Skin C (the places of production：Guangxi), the rascal D (places of production：Guangxi), the rascal E (places of production：Guangxi), the rascal F (places of production：Guangxi), the rascal G (places of production：
Guangxi), the FTUCTUS CITRI IMMATURI H (places of production：Jiangxi), the FTUCTUS CITRI IMMATURI I (places of production：Jiangxi), the FTUCTUS CITRI IMMATURI J (places of production：Jiangxi).
The phenotypic characteristic of the medicinal material for being envisaged for preparing reference extract described above is shown in Fig. 1-10.
Aurantiamarin reference substance：Zhong Jian institutes, lot number 110721-201316.
Tangeritin reference substance：Baoji time bio tech ltd, lot number 20140512.
Nobiletin reference substance：Baoji time bio tech ltd, lot number 20140728.
Neohesperidin reference substance：Zhong Jian institutes, lot number 111857-201102.
Aurantiin reference substance：Zhong Jian institutes, lot number 110722-201312.
Superfine silica gel powder：Mountains and rivers medicine is auxiliary, the quasi- word F20100001 of Anhui medicine.
Test medicinal material rascal：
Rascal 1：Buy in Cai Zhilin pharmacies, numbering 1；Rascal 2：Buy in great Can woodss pharmacy, numbering 2；Rascal 3：Purchase
In Kingcon pharmacy, numbering 3；Rascal 4：Buy in Neptune occasion, numbering 4；Rascal 5：Buy in Ji Hetang, numbering 5；Rascal 6：Purchase
Buy in strong people medicine, numbering 6；Rascal 7：Buy in Renhe hall, numbering 7；Rascal 8：Buy in Tongrentang, numbering 8；Rascal 9：Purchase
Buy in two paradise, numbering 9；Rascal 10：Buy in the big pharmacy of common people, numbering 10.
The phenotypic characteristic of above-mentioned test medicinal material rascal 1-10 is shown in Figure 11-20 successively.
Embodiment 1：The preparation of rascal reference extract
The preparation method of rascal reference extract of the present invention is present embodiments provided, its method flow diagram is as shown in figure 21.
One：The screening of raw medicinal material
This step is combined using thin-layered chromatography and high performance liquid chromatography and raw medicinal material is screened.
The thin-layered chromatography screening of 1 raw medicinal material
The preparation of 1.1 test samples
The preparation of chemical reference substance solution：Take aurantiamarin, Nobiletin, tangeritin reference substance accurately weighed, first is used respectively
Alcohol is configured to the solution of 0.1mg/ml.
The preparation of medicinal material need testing solution：Take rascal A, rascal B, rascal C, rascal D, rascal E, rascal F, rascal G, individual green grass or young crops
Skin H, FTUCTUS CITRI IMMATURI I, FTUCTUS CITRI IMMATURI J medicinal powders (cross No. two sieve) each 0.5g, respectively plus methyl alcohol 5ml is in 15ml centrifuge tubes, room temperature
Immersion 15 minutes, ultrasonic (power 500w) takes out after processing 15 minutes, and 10 minutes (centrifugal force 3200crf) is centrifuged, and takes supernatant
Medicinal material need testing solution is obtained final product, or directly 0.22 μm of filter membrane excessively is not centrifuged and obtain final product medicinal material need testing solution.
1.2 thin-layer chromatographys are detected
1.2.1 it is directed to flavonoid glycoside
Testing conditions are as follows：
Lamellae：The common silica gel prefabricated boards (Merck) of TLC G60
Point sample：The μ l of chemical reference substance need testing solution point sample amount 5, the μ l of medicinal material need testing solution point sample amount 1, ribbon point
Sample；
Solvent：Chloroform:Methyl alcohol:Water:Glacial acetic acid=13:4:1:1.5 (10 DEG C, with lower leaf, are removed layer)
Launch：Solvent 10ml, pre-equilibration 15 minutes is added to launch up in double flute expansion cylinder (20cm × 10cm) side
8.5cm。
Inspect：Spray is with 5%AlCl3Ethanol solution, lamellae is put in thin layer heating plate 105 DEG C and is heated 5 minutes.It is placed in UV purple
Inspected under outer smooth lamp 366nm.
Testing result is to inspect thin-layer chromatogram (T under UV366nm as shown in figure 22:19℃,RH:55%).Label S is
Aurantiamarin reference substance, 1-10 correspond to respectively above-mentioned test sample rascal A, rascal B, rascal C, rascal D, rascal E, rascal F, rascal G,
The medicinal material of FTUCTUS CITRI IMMATURI H, FTUCTUS CITRI IMMATURI I and FTUCTUS CITRI IMMATURI J.
Interpretation of result：As shown in Figure 22,10 batches of flavonoid glycoside trace analysis of medicinal material are consistent, illustrate Huang in this 10 kinds of medicinal materials
The content of ketoside class constituents is also than more consistent.
1.2.2：For flavonoid glycoside metaclass
Lamellae and point sample with step one 1.2.1.
Remaining testing conditions is as follows：
Solvent：
Solvent 1：Toluene：Ethyl acetate：Formic acid：Water=20:20:1:1 (10 DEG C, with lower leaf, take upper strata)
Solvent 2：Toluene：Ethyl acetate：Formic acid：Water=20:10:1:1 (10 DEG C, with lower leaf, take upper strata)
Launch：First launch 8.5cm with solvent 1, then launch 8.5cm with solvent 2.Double flute expansion cylinder before launching every time
(20cm × 10cm) application 10ml solvents pre-equilibration 15 minutes, lamellae is put in thin layer heating plate 50 DEG C and is heated 15 minutes.
Inspect：Lamellae surface solvent is volatilized after expansion, is inspected under UV ultraviolet lamps 366nm, taken pictures.
Testing result is to inspect thin-layer chromatogram (T under UV366nm as shown in figure 23:19℃,RH:55%).Label S：From
Top to bottm is followed successively by tangeritin reference substance, Nobiletin reference substance, and 1-10 corresponds to above-mentioned test sample rascal A, rascal B, green grass or young crops respectively
The medicinal material of skin C, rascal D, rascal E, rascal F, rascal G, FTUCTUS CITRI IMMATURI H, FTUCTUS CITRI IMMATURI I and FTUCTUS CITRI IMMATURI J.
Interpretation of result：As shown in Figure 23, the flavone aglycone constituents of rascal A differ larger with other medicinal materials；Rascal B,
Rascal C, rascal D, rascal E, rascal F and rascal G are Sihuaqingpi, are compared with FTUCTUS CITRI IMMATURI H, FTUCTUS CITRI IMMATURI I and FTUCTUS CITRI IMMATURI J, flavones
Aglycon constituents content is more.
The high performance liquid chromatography screening of 2 raw medicinal materials
It is prepared by 2.1 test samples
The preparation of chemical reference substance solution：Take that aurantiamarin reference substance is accurately weighed, the molten of 0.1mg/ml is configured to methyl alcohol
Liquid.
The preparation of medicinal material need testing solution：Take rascal A, rascal B, rascal C, rascal D, rascal E, rascal F, rascal G, individual green grass or young crops
Skin H, FTUCTUS CITRI IMMATURI I, FTUCTUS CITRI IMMATURI J medicinal powders each 0.2g, it is accurately weighed, in putting 50ml measuring bottles, plus methyl alcohol 30ml, ultrasonic (power
500w) process 30 minutes, let cool, plus methyl alcohol shakes up to 50ml scales, middling speed qualitative filter paper filtering, precision measures filtrate 2ml,
In putting 5ml measuring bottles, plus methyl alcohol shakes up to 5ml scales, crosses 0.22 μm of filter membrane and obtains final product medicinal material need testing solution.
2.2 high performance liquid chromatography detections
High performance liquid chromatography testing conditions are as follows：
Chromatographic apparatus：Agilent HPLC1260 high performance liquid chromatographs；Detector：Agilent DAD detectors；
Chromatographic column：Zorbax SB C18(4.6×250mm,5μm；Agilent)；
Mobile phase：Mobile phase A is acetonitrile, and Mobile phase B is water；
Gradient elution program according to table 1 below is carried out：
Table 1
| Time (minute) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 5 | 95 |
| 30 | 25 | 75 |
| 55 | 95 | 5 |
| 65 | 95 | 5 |
Flow velocity：0.8ml/min；Sample size：10μl；Column temperature：20℃；Detection wavelength：280nm；Run time：65min.
High effective liquid chromatography for detecting：It is accurate respectively to draw chemical reference substance solution and medicinal material need testing solution each 10
μ l, inject liquid chromatograph.
High performance liquid chromatography testing result：
(1) determine, record chromatogram, obtain final product the rascal medicinal material HPLC collection of illustrative plates shown in Figure 24, correspond to respectively from top to bottom
State the medicinal material of rascal A, rascal B, rascal C, rascal D, rascal E, rascal F, rascal G, FTUCTUS CITRI IMMATURI H, FTUCTUS CITRI IMMATURI I and FTUCTUS CITRI IMMATURI J.
(2) rascal A, rascal B, rascal C, rascal D, rascal E, rascal F, rascal G, FTUCTUS CITRI IMMATURI H, FTUCTUS CITRI IMMATURI I and individual are calculated
The content (content is in terms of wt%) of the aurantiamarin in rascal J, it is as a result as shown in table 2 below.
Table 2
| Medicinal material assay | Content of hesperidin (%) |
| A | 4.60 |
| B | 5.69 |
| C | 6.91 |
| D | 6.46 |
| E | 6.62 |
| F | 5.69 |
| G | 6.43 |
| FTUCTUS CITRI IMMATURI H | 8.84 |
| FTUCTUS CITRI IMMATURI I | 6.22 |
| FTUCTUS CITRI IMMATURI J | 5.79 |
High performance liquid chromatography Analysis of test results：
Difference in the HPLC collection of illustrative plates of the rascal A shown in Figure 24 with other classes：Rascal A collection of illustrative plates is in 45min-50min regions
Interior chromatographic peak is seldom, substantially inconsistent with other samples, and the content of hesperidin of A is relatively low that (content of hesperidin is
4.60%, 5.0%) belong to unqualified kind according to States Pharmacopoeia specifications less than pharmacopoeial requirements, therefore rascal A is not suitable for being used as rascal
The raw medicinal material of the preparation of reference extract.Thus in the preparation of rascal reference extract from rascal B, rascal C, rascal D,
Rascal E, rascal F, rascal G, FTUCTUS CITRI IMMATURI H, FTUCTUS CITRI IMMATURI I and FTUCTUS CITRI IMMATURI J are used as the raw medicinal material for preparing rascal reference extract.
Two：Slightly carry
The selection result according to step one choose rascal B, rascal C, rascal D, rascal E, rascal F, rascal G, FTUCTUS CITRI IMMATURI H,
FTUCTUS CITRI IMMATURI I and FTUCTUS CITRI IMMATURI J totally 9 batch medicinal materials, are made powder, and medicinal powder crosses No. two sieves (850 ± 29 μm, 24 mesh), then
(i.e. solid-to-liquid ratio is 1 to add 10 times of methyl alcohol of volume of its quality：10 (w/V)), ultrasonic (power 500w, normal temperature) extracts 30min
Middling speed qualitative filter paper filtering afterwards, collects filter residue 1 and filtrate 1, and filter residue 1 is extracted secondary by same procedure again, and will be extracted again
The filtrate of secondary collection merges Ji Wei extract solution with filtrate 1, extract solution Rotary Evaporators is evaporated and obtains rascal crude extract.
Three：It is refined
Rascal crude extract is taken, filter residue is obtained with dissolved in purified water and suction filtration, filter residue rinses (blue or green to water colorless with pure water again
Skin crude extract is 1 with the mass volume ratio of pure water altogether：5, rinse 5 times altogether), filter residue 2 is collected, merge multiple filtrate and obtain
To filtrate 2, filtrate 2 is chromatographed with the D101 types macroreticular resin of its 2 times of weight, first with 8 pure washings of column volume (1100ml)
De-, eluent is discarded；Eluted with the methyl alcohol that the concentration expressed in percentage by volume of 4 column volumes (550ml) is 100% again, obtain eluent
2, then eluent 2 and filter residue 2 are mixed, it is evaporated, obtain the refined thing of rascal.
Four：Mix silica gel grinding filtering
Superfine silica gel powder is added in the refined thing of rascal, addition is the 40% of the refined thing weight of rascal, grinds and cross 110 mesh
Screen cloth, the rascal for obtaining final product rascal B, rascal C, rascal D, rascal E, rascal F, rascal G, FTUCTUS CITRI IMMATURI H, FTUCTUS CITRI IMMATURI I and FTUCTUS CITRI IMMATURI J is carried
Take thing.
1 peel extract is analyzed
1.1 thin-layer chromatographic analysis
1.1.1 sample preparation
The preparation of chemical reference substance solution：Take aurantiamarin, Nobiletin, tangeritin reference substance accurately weighed, first is used respectively
Alcohol is configured to the solution of 0.1mg/ml.
The preparation of peel extract need testing solution：What the step 4 of Example 1 was obtained take peel extract (B, C, D, E,
F, G1, G2, FTUCTUS CITRI IMMATURI H1, FTUCTUS CITRI IMMATURI H2) 9 crowdes of each 20mg, it is accurately weighed, in putting 2ml measuring bottles, plus methyl alcohol 1.5ml, ultrasonic (work(
Rate 500w) process 15 minutes, let cool, plus methyl alcohol shakes up to 2ml scales, crosses 0.22 μm of filter membrane and obtains final product peel extract solution.
Wherein peel extract G1, G2 is two peel extracts of different batches of rascal G；Peel extract-individual green grass or young crops
Skin H1, peel extract-FTUCTUS CITRI IMMATURI H2 are two peel extracts of different batches of FTUCTUS CITRI IMMATURI H, similarly hereinafter.
1.1.2 thin-layer chromatography detection
Testing conditions are as follows：
For the detection of flavonoid glycoside, lamellae, solvent, method of deploying are with 1.2.1 in the step one of embodiment 1；For
For the detection of flavonoid glycoside metaclass, lamellae, solvent, method of deploying are with 1.2.2 in the step one of embodiment 1.
Point sample：The μ l of chemical reference substance need testing solution point sample amount 5, the μ l of peel extract need testing solution point sample amount 1, band
Shape point sample.
Colour developing：For the detection of flavonoid glycoside, method with it is consistent in 1.2.1 in the step one of embodiment 1；For for Huang
The detection of ketoside metaclass, method with it is consistent in 1.2.2 in the step one of embodiment 1.
Thin-layer chromatography testing result for flavonoid glycoside is as shown in figure 25, for the testing result for flavonoid glycoside metaclass
As shown in figure 26.
1.2 high performance liquid chromatographies (HPLC) assay
1.2.1 prepared by test sample
The preparation of chemical reference substance solution：Take that aurantiamarin reference substance is accurately weighed, the molten of 0.1mg/ml is configured to methyl alcohol
Liquid.
The preparation of peel extract need testing solution：Take peel extract (B, C, D, E, F, G1, G2, FTUCTUS CITRI IMMATURI H1, individual green grass or young crops
Skin H2, FTUCTUS CITRI IMMATURI H3, Sihuaqingpi, FTUCTUS CITRI IMMATURI I1) each 25mg, it is accurately weighed, in putting 50ml measuring bottles, plus methyl alcohol 30ml, ultrasound
(power 500w) is processed 15 minutes, is let cool, plus methyl alcohol shakes up to 50ml scales, and it is molten that 0.22 μm of filter membrane of mistake obtains final product peel extract
Liquid.
Wherein peel extract-FTUCTUS CITRI IMMATURI H3 is the peel extract of FTUCTUS CITRI IMMATURI H, and unlike the step 2 of embodiment 1,
It is to add methyl alcohol to extract 7 times (3 times of the step 2 of comparative example 1), the peel extract that 7 filtrates of merging obtain.
Wherein peel extract-Sihuaqingpi is that step 2 rascal B-G methyl alcohol is extracted the dregs of a decoction after 3 times to merge continuation
Extract 4 times, obtain Sihuaqingpi extract.
Wherein peel extract-FTUCTUS CITRI IMMATURI I1 is the peel extract of FTUCTUS CITRI IMMATURI I, and unlike the step 2 of embodiment 1,
It is to add methyl alcohol to extract 7 times (3 times of the step 2 of comparative example 1), the peel extract that 7 filtrates of merging obtain.
1.1.2 high performance liquid chromatography detection
Testing conditions and assay method are with 2.2 in the step one of embodiment 1.
High performance liquid chromatography testing result：
(1) determine, record chromatogram, obtain final product the peel extract HPLC collection of illustrative plates shown in Figure 27, be respectively from top to bottom：It is blue or green
The peel extract of skin B, the peel extract of rascal C, the peel extract of rascal D, the peel extract of rascal E, rascal F
Peel extract, the first batch peel extract (G1) of rascal G, the second lot peel extract (G2) of rascal G, FTUCTUS CITRI IMMATURI
Second lot peel extract (the H2) (wherein peel extract-individual green grass or young crops of the first batch peel extract (H1) of H1, FTUCTUS CITRI IMMATURI
Skin H3, peel extract-Sihuaqingpi, peel extract-FTUCTUS CITRI IMMATURI I1 are omitted).
(2) rascal ((B, C, D, E, F, G1, G2, FTUCTUS CITRI IMMATURI H1, FTUCTUS CITRI IMMATURI H2, FTUCTUS CITRI IMMATURI H3, Sihuaqingpi, individual green grass or young crops are calculated
Skin I1) in aurantiamarin content (content is in terms of wt%), it is as a result as shown in table 3 below.
Table 3
| Numbering | Correspondence medicinal material | Content of hesperidin (%) |
| 1 | B | 12.46 |
| 2 | C | 16.63 |
| 3 | D | 16.87 |
| 4 | E | 16.00 |
| 5 | F | 13.57 |
| 6 | G1 | 8.02 |
| 7 | G2 | 8.18 |
| 8 | FTUCTUS CITRI IMMATURI H1 | 19.19 |
| 9 | FTUCTUS CITRI IMMATURI H2 | 17.60 |
The other three peel extract carries out assay using same procedure, as a result as shown in table 4：
Table 4
| Numbering | Correspondence medicinal material | Content of hesperidin (%) |
| 10 | FTUCTUS CITRI IMMATURI H3 | 39.45 |
| 11 | Sihuaqingpi | 19.20 |
| 12 | FTUCTUS CITRI IMMATURI I1 | 37.31 |
Analysis result：
1) TLC, HPLC spectrogram reappearance of the nine batches of extracts and reproducible, it is more consistent.Four flowers are found out by TLC collection of illustrative plates
Peel extract (B-G) flavone aglycone constituents are more than FTUCTUS CITRI IMMATURI extract, and both are temporarily separately prepared final ERS.
2) HPLC-DAD chromatograms show that preferably, each index components characteristic peak is obvious for each batch of peel extract similitude, but
Extract G1 and G2 content of hesperidin compared with other several for low, therefore can with peel extract-FTUCTUS CITRI IMMATURI H3 or peel extract-
FTUCTUS CITRI IMMATURI I1 is allocated.
Five：Allotment
By thin-layer chromatography TLC methods and the detection of high-efficient liquid phase chromatogram HPLC method, qualified separate sources or the rascal of batch
Extract is mixed to get rascal reference extract by a certain percentage.Allotment standard is：In should making final rascal reference extract
The content of aurantiamarin maintains 16%-20% (content is in terms of wt%).The scope of the content of the aurantiamarin in this allotment standard
It is by repeatedly testing the comprehensive optimum data for keeping stability, uniformity and application below etc. to draw repeatedly.
The concocting method of this batch rascal reference extract：Take peel extract B-G2 and respectively take 6g for totally 7 batches, and No. 10 carry
Take thing i.e. peel extract-FTUCTUS CITRI IMMATURI H3 6g to mix, obtain QPERS-1；Take No. 8 extracts i.e. peel extract-FTUCTUS CITRI IMMATURI H1
7.5g and No. 9 extract is that peel extract-FTUCTUS CITRI IMMATURI H2 11g are mixed, and obtains QPERS-2.QPERS-1 is Sihuaqingpi pair
According to extract, QPERS-2 is FTUCTUS CITRI IMMATURI reference extract.Chromatography is carried out to QPERS-1 and QPERS-2：
1 thin-layer chromatographic analysis
1.1 preparation of samples
1) chemical reference substance (CRS) solution：Take aurantiamarin, Nobiletin, tangeritin reference substance accurately weighed, first is used respectively
Alcohol is configured to the solution of 0.1mg/ml.
2) reference extract (ERS) solution：Take the appropriate accurate title of rascal reference extract 2 batches (QPERS-1, QPERS-2)
It is fixed, add methyl alcohol ultrasound (500w) to extract 30 minutes, plus methyl alcohol is configured to the solution that concentration is 10mg/ml, by 0.22 μm of filter
Film, takes filtrate as reference extract solution.
1.2 thin-layer chromatographys are detected
1.2.1 flavonoid glycoside
Lamellae:The common silica gel prefabricated boards (Merck) of TLC G60.
Point sample:Chemical reference substance solution：5 μ l, band point sample.
Reference extract solution：1 μ l, band point sample.
Solvent：Chloroform:Methyl alcohol:Water:Glacial acetic acid=13:4:1:1.5 (10 DEG C, with lower leaf, are removed layer).
Launch：Add solvent 10ml, pre-equilibration 15 minutes in double flute expansion cylinder (10cm × 10cm) side.Launch up
8.5cm。
Inspect:First spray with 5%AlCl3Ethanol solution, puts in thin layer heating plate 105 DEG C and heats 5 minutes, under UV366nm
Inspect, take pictures.Impregnated with the ethanol solution of sulfuric acid of 5% vanillic aldehyde 10% again, put in thin layer heating plate 105 DEG C and heat 5 minutes, Yu Ke
See under light and inspect, take pictures.
Testing result is as shown in figure 28.
1.2.2 flavonoid glycoside metaclass
Lamellae and point sample are with this step of the present embodiment 1.2.1.
Solvent 1：Toluene：Ethyl acetate：Formic acid：Water=20:20:1:1 (10 DEG C, with lower leaf, take upper strata)
Solvent 2：Toluene：Ethyl acetate：Formic acid：Water=20:10:1:1 (10 DEG C, with lower leaf, take upper strata)
Launch：First launch 8.5cm with solvent 1, then launch 8.5cm with solvent 2.Double flute expansion cylinder before launching every time
(10cm × 10cm) application 10ml solvents pre-equilibration 15 minutes, lamellae is put in thin layer heating plate 50 DEG C and is heated 15 minutes.
Inspect：Lamellae surface solvent is volatilized after expansion, is inspected under 366nmUV, taken pictures.
Testing result is as shown in figure 29.
The Analysis of test results of flavonoid glycoside and flavone aglycone：Rascal ERS and chemical reference substance are can be seen that from Figure 28, Figure 29
TLC chromatograms show, on Nobiletin, the corresponding position of tangeritin chemical reference substance chromatogram, rascal ERS chromatograms show same color
Fluorescence spot.
2HPLC finger-prints and assay
2.1 sample preparations
The preparation of reference substance solution：Take aurantiamarin reference substance appropriate, it is accurately weighed, it is configured to the molten of 0.1mg/ml with methyl alcohol
Liquid.
The preparation of need testing solution：Take Sihuaqingpi ERS (QPERS-1) and FTUCTUS CITRI IMMATURI ERS (QPERS-2) appropriate, it is accurate
It is weighed, add methyl alcohol ultrasound (500w) to process 30 minutes, let cool, the solution that concentration is 0.5mg/ml is configured to, cross 0.22 μm
Filter membrane, takes subsequent filtrate, obtains final product need testing solution.
2.2 high performance liquid chromatography detections
Testing conditions：
Chromatographic apparatus：Agilent HPLC1260 high performance liquid chromatographs；Detector：Agilent DAD detectors；
Chromatographic column：Zorbax SB C18(4.6×250mm,5μm；Agilent)；Mobile phase：Mobile phase A is acetonitrile, flowing
Phase B is water；
Eluent gradient is as shown in table 5.
Table 5
| Time (minute) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 5 | 95 |
| 30 | 25 | 75 |
| 55 | 95 | 5 |
| 65 | 95 | 5 |
Flow velocity：0.8ml/min；Sample size：10μl；Column temperature：20℃；Detection wavelength：280nm；Run time：65min.
High effective liquid chromatography for detecting：It is accurate respectively to draw reference solution and each 10 μ l of need testing solution, injection
Liquid chromatograph.
High performance liquid chromatography testing result：
(1) determine, record chromatogram, obtain final product the rascal medicinal material HPLC collection of illustrative plates shown in Figure 30, it is lower to represent FTUCTUS CITRI IMMATURI ERS
(QPERS-2)；On represent Sihuaqingpi ERS (QPERS-1), S represents aurantiamarin.
(2) in and calculating above-mentioned FTUCTUS CITRI IMMATURI ERS and Sihuaqingpi ERS as the chromatographic peak of the HPLC of reference substance using object of reference
Aurantiamarin content (with dry product calculate, content is in terms of wt%), it is as a result as shown in table 6 below.
Table 6
| Rascal ERS | Content of hesperidin (%) |
| QPERS1 | 16.21 |
| QPERS2 | 17.95 |
Therefore, the present embodiment is prepared Sihuaqingpi ERS (QPERS-1), FTUCTUS CITRI IMMATURI ERS (QPERS-2) through thin
Layer chromatography detects that flavonoid glycoside composition and flavone aglycone constituents meet the requirements, and its aurantiamarin is detected through high performance liquid chromatography
Content allotment critical field within, can as rascal and the Chinese medicine preparation containing rascal/rascal active ingredient reference extract.
The character analysis of the rascal reference extract of embodiment 2
1 apparent state：Rascal the reference extract QPERS-1 and QPERS-2 that embodiment 1 is obtained are yellow green xeraphium
End.
2. determination of moisture：Carried out according to 2010 editions Chinese Pharmacopoeias annex IX G (hypobaric drying method).Testing result is
QPERS1 water content is that 2.70%, QPERS2 water content is 2.30%.
3. uniformity test：According to embodiment 1 method prepare 10 batch Sihuaqingpis ERS (QPERS-1) (6 batch) and
FTUCTUS CITRI IMMATURI ERS (QPERS-2) (4 batch), after measured between each batch for flavonoid glycoside composition and flavone aglycone constituents
Thin-layer chromatography testing result difference very little, the fluorescence spot of aurantiamarin, tangeritin and Nobiletin has obvious display, and divides
Cloth is very consistent；Through high performance liquid chromatography detection, its content of hesperidin is being allocated within critical field.Therefore using the present invention
Rascal reference extract the rascal reference extract uniformity for preparing of preparation method it is very good.
4. stability test：
Take 10 the Sihuaqingpi ERS (QPERS-1) (6 batch) and FTUCTUS CITRI IMMATURI ERS (QPERS-2) (4 crowdes of different batches
It is secondary) test sample, according to Chinese Pharmacopoeia Commission work out " requirement of national drug standards material Development Techniques " relevant regulations,
Inspection target includes proterties and dissolubility.
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,
Detected by stability high spot reviews project.
Result shows, before and after hot test test sample proterties without significant change, before and after hot test thin-layer chromatogram also without
Significant change, dissolution rate measurement result carries out Independent samples t-test with SPSS, and result of calculation P ＞ 0.05 are illustrated without conspicuousness
Difference.
Comparative example 1
Difference with embodiment 1 is：To being carried out instead of methyl alcohol using 70% ethanol in the step one in embodiment 1, most
The content of the peel extract for obtaining afterwards be 1%~5% (much smaller than the present invention -8.02-19.19%), it can be seen that, its value
Low many of yield for substantially being extracted than methyl alcohol, only increase extracted amount or number of times is possible to allocate its content of hesperidin to reach
To within allotment critical field.
Embodiment 3
Present embodiments provide the application to the rascal reference extract of embodiment 1.
With thin-layered chromatography and high performance liquid chromatography respectively with chemical reference substance and rascal reference extract as reference
Material is analyzed to commercially available medicinal material.
1 thin-layer chromatography
1.1 sample preparations
Chemical reference substance solution：Precision weighs each 2.5mg of aurantiamarin, Nobiletin, tangeritin, neohesperidin, aurantiin,
25ml volumetric flasks are placed in, 20ml methyl alcohol is added, ultrasonic (500w) treatment in 15 minutes, to being completely dissolved, is let cool, and constant volume to 25ml is carved
Spend line and cross 0.22 μm of filter membrane and obtain final product.
Rascal reference extract solution：Precision weigh the rascal reference extract QPERS-1 that embodiment 1 prepares and
Each 20mg of QPERS-2, are put in 2ml measuring bottles, and methyl alcohol 1.5ml is added respectively, and ultrasonic (500w) is processed 15 minutes, let cool, plus methyl alcohol is extremely
2ml scales, shake up, and cross 0.22 μm of filter membrane and obtain final product rascal reference extract solution.
Medicinal material need testing solution：Got it filled each 0.5g of shop medicinal material, plus methyl alcohol 5ml is in 15ml centrifuge tubes, and room temperature is soaked 15 minutes,
Ultrasonic (500w) takes out after 15 minutes, centrifugation 3 minutes (rotating speed is 3200 turns per minute), takes supernatant and crosses 0.22um filter membranes and obtains final product
Medicinal material need testing solution.Pharmacy's medicinal material is bought from Guangzhou, respectively：Rascal 1, rascal 2, rascal 3, rascal 4, rascal 5, rascal 6,
Rascal 7, rascal 8, rascal 9 and rascal 10.
1.2 thin-layer chromatographys are detected
Testing conditions are as follows：
For the detection of flavonoid glycoside, condition is with 1.2.1 in the step 5 of embodiment 1；For the detection of flavonoid glycoside metaclass, bar
Part is with 1.2.2 in the step 5 of embodiment 1.
Thin-layer chromatography testing result for flavonoid glycoside is as shown in figure 31, for the testing result for flavonoid glycoside metaclass
As shown in figure 32.
Testing result：
Sihuaqingpi ERS, FTUCTUS CITRI IMMATURI ERS and chemical reference substance TLC chromatograms show, aurantiamarin, Nobiletin, tangeritin
On the corresponding position of chemical reference substance chromatogram, the fluorescence spot of the aobvious same color of Sihuaqingpi ERS, FTUCTUS CITRI IMMATURI ERS chromatograms.Four flowers
The fluorescence spot with neohesperidin, the aobvious same color in aurantiin relevant position is not found in rascal ERS and FTUCTUS CITRI IMMATURI ERS.
2 high performance liquid chromatography
It is prepared by 2.1 need testing solutions
Chemical reference substance solution：Precision weighs aurantiamarin 2.5mg, is placed in 25ml volumetric flasks, adds 20ml methyl alcohol, ultrasound
(500w) treatment in 15 minutes, to being completely dissolved, is let cool, and 0.22 μm of filter membrane of constant volume to 25ml graduation marks and mistake is obtained final product.
Rascal reference extract solution：Precision weigh the rascal reference extract QPERS-1 that embodiment 1 prepares and
Each 25mg of QPERS-2, are placed in 50ml volumetric flasks, add 30ml methyl alcohol, and ultrasonic (500w) 15 minutes lets cool, plus methanol constant volume
To 50ml scales, and 0.22 μm of filter membrane excessively obtains final product rascal reference extract solution.
Medicinal material need testing solution：The shop fine medicinal material powder of getting it filled about 0.2g, it is accurately weighed, in putting 50ml measuring bottles, plus methyl alcohol 30ml,
Ultrasonic (500w) is processed 30 minutes, is let cool, plus methyl alcohol shakes up to 50ml scales, and filtration, precision measures subsequent filtrate 2ml, puts 5ml
In measuring bottle, plus methanol constant volume shakes up to 5ml scales, crosses 0.22 μm of filter membrane and obtains final product medicinal material need testing solution.Pharmacy's medicinal material is bought certainly
Guangzhou, respectively：Rascal 1, rascal 2, rascal 3, rascal 4, rascal 5, rascal 6, rascal 7, rascal 8, rascal 9 and rascal 10.
2.2 high performance liquid chromatography detections
The 2.2 of the step of testing conditions and detection method is with embodiment 1 five.
Testing result is as shown in figure 33.
Be utilized respectively chemical reference substance and reference extract carries out assay to the content of hesperidin of medicinal material, as a result such as
Shown in table 7：
Table 7
Two kinds of RSD of measurement result are between 1.94~4.86.By two measurement results carried out with SPSS independent sample t-
Inspection, result of calculation P=0.883 ＞ 0.05 illustrate that there was no significant difference for two kinds of results of assay method.Interpretation of result：
It can be seen from result according to TLC and HPLC finger-prints, to the rascal reference extract (QPERS-1 and QPERS-
2) detect that the corresponding raw medicinal material of the chromatogram for obtaining is consistent using thin-layered chromatography, more precisely, to the rascal
Reference extract (QPERS-1 and QPERS-2) detects Nobiletin, tangeritin in the chromatogram for obtaining using thin-layered chromatography
Detect the chromatogram for obtaining at aurantiamarin position with the fluorescence spot at aurantiamarin position and using high performance liquid chromatography
The corresponding chemical reference substance of chromatographic peak difference or its corresponding raw medicinal material are consistent, therefore rascal of the invention control is extracted
Thing (QPERS-1 and QPERS-2) can be applied to the Qualitive test of medicinal material or Chinese medicine preparation, such as to medicinal material or the Huang of Chinese medicine preparation
Ketoside constituents and/or flavone aglycone constituents carry out qualitative analysis, the flavonoid glycoside metaclass into can be Nobiletin and/or
Tangeritin；The flavonoid glycoside composition can be aurantiamarin.
According to HPLC measurement results and statistical analysis, illustrate using rascal reference extract of the invention (QPERS-1 and
QPERS-2 the assay of aurantiamarin) is carried out to medicinal material and there was no significant difference using chemical reference substance, therefore green grass or young crops of the invention
Skin reference extract (QPERS-1 and QPERS-2) can be applied to the sxemiquantitative discriminatory analysis of medicinal material or Chinese medicine preparation, can also apply
In rascal and in the quality control of medicinal material or Chinese medicine preparation containing rascal/rascal active ingredient, such as to medicinal material or Chinese medicine preparation
Flavonoid glycoside composition carry out sxemiquantitative discriminatory analysis, or to rascal and medicinal material or Chinese medicine containing rascal/rascal active ingredient
Preparation carries out half-quantitative detection and then controls its quality, and the flavonoid glycoside composition can be aurantiamarin.
Claims (10)
1. a kind of preparation method of rascal reference extract, it is characterised in that including the rascal medicinal material to separate sources or batch
Powder is repeatedly extracted, and obtains the peel extract of separate sources or batch, and then the rascal to separate sources or batch is carried
Take thing to be allocated, obtain rascal reference extract；
Optional, the rascal medicinal powder to separate sources or batch is repeatedly extracted, and is the flavonoid glycoside for containing it
The purity and/or concentration of first constituents and/or flavonoid glycoside composition are improved, so as to the rascal for obtaining separate sources or batch is extracted
Thing；
Optional, the flavone aglycone constituents are Nobiletin and tangeritin；
Optional, the flavonoid glycoside composition is aurantiamarin.
2. the preparation method of rascal reference extract according to claim 1, comprises the following steps：
Step one, slightly carry：The rascal medicinal powder for taking separate sources or batch mixes with methyl alcohol respectively, is filtered after ultrasonic extraction,
Filtrate 1 and filter residue 1 are obtained, and filtrate 1 is evaporated is obtained rascal crude extract；
It is step 2, refined：By gained rascal crude extract, water and suction filtration are dissolved in, obtain filter residue；By filter residue water rinse to water without
Color, collects filtrate 2 and filter residue 2, and filtrate 2 is chromatographed with macroreticular resin；Then macroreticular resin first is rinsed with water, obtains eluent 1；Again
Macroreticular resin is rinsed with methyl alcohol, eluent 2 is collected；Finally eluent 2 and filter residue 2 are mixed, is evaporated, obtain the refined thing of rascal；
Step 3, mix silica gel grinding filtering：Add superfine silica gel powder in the refined thing of rascal, grinding filtering, obtain final product separate sources or
The peel extract of batch；
Step 4, allotment：The peel extract of separate sources or batch is allocated, rascal reference extract is obtained.
3. the preparation method of rascal reference extract according to claim 2, it is characterised in that also include before step one
Screening to the corresponding separate sources of the rascal medicinal powder of separate sources or batch or the raw medicinal material of batch；
Optional, the raw medicinal material to the corresponding separate sources of the rascal medicinal powder of separate sources or batch or batch
Screening includes：The raw medicinal material of separate sources or batch is detected with thin-layered chromatography and/or high performance liquid chromatography；So
Afterwards to the chromatogram obtained by thin-layered chromatography and/or high performance liquid chromatography of the raw medicinal material of separate sources or batch
And/or the content for determining is compared analysis, the raw medicinal material of the content exception of chromatogram and/or measure is rejected, retain chromatogram
Figure and/or the consistent raw medicinal material of the content for determining carry out the thick of next step and carry；
Optional, the thin-layered chromatography and/or high performance liquid chromatography carry out detection and include to flavonoid glycoside composition and/or Huang
The detection of ketoside unit constituents；
Optional, the flavone aglycone constituents are Nobiletin and tangeritin；
Optional, the flavonoid glycoside composition is aurantiamarin.
4. the preparation method of rascal reference extract according to claim 2, it is characterised in that described in the step one
Filter residue 1 extracted by n times according to the extracting method of step one, and n times extracted the filtrate collected merge with filtrate 1 as carrying
Liquid is taken, extract solution is evaporated and is obtained rascal crude extract；Wherein 6 >=N >=0, and N is integer；
Optional, the N is 2.
5. the preparation method of rascal reference extract according to claim 2, it is characterised in that in the step one not
It is 1 with the rascal medicinal powder of source or batch and the w/v of methyl alcohol:8~1:50；
Optional, the rascal medicinal powder of separate sources or batch in the step one is 1 with the w/v of methyl alcohol：
10；
Optional, the ultrasonic time in the step one is 30~60min, and power is 100W~3kW；
Optional, the ultrasonic time in the step one is 30min, and power is 500W；
Optional, the filtering of the step one is with Medium speed filter paper or 2000 mesh sieve net filtrations.
6. the preparation method of rascal reference extract according to claim 2, it is characterised in that the filter in the step 2
Liquid 2 is chromatographed with the macroreticular resin of 1~4 times of its weight；
Optional, the filtrate 2 in the step 2 is with the macroreticular resin chromatography of 2 times of its weight；
Optional, the macroreticular resin is non-polar macroporous resin；
Optional, the aperture of the macroreticular resin is 100~1000nm；
Optional, the macroreticular resin is D101 type macroreticular resins.
7. the preparation method of rascal reference extract according to claim 2, it is characterised in that big in the step 2
It is first to rinse macroreticular resin with 5~10 column volume pure water after the resin chromatography of hole, collects eluent 1；Again with 3~10 posts
The methyl alcohol of volume 70%~100% rinses macroreticular resin, collects eluent 2；
Optional, it is first to rinse macroreticular resin with 8 column volume pure water after the macroreticular resin chromatography, collect eluent 1；
Macroreticular resin is rinsed with 4 methyl alcohol of column volume 100% again, eluent 2 is collected.
8. the preparation method of rascal reference extract according to claim 2, it is characterised in that the step 3 is in green grass or young crops
The refined thing of skin adds the superfine silica gel powder of the 20%~50% of its weight；
Optional, the step 3 is the superfine silica gel powder that the 40% of its weight is added in the refined thing of rascal；
Optional, it was 90~200 mesh sieve net filtrations after the step 3 grinding.
Optional, also include with thin-layered chromatography and/or high performance liquid chromatography to difference between the step 3 and step 4
The step of flavone aglycone constituents and/or flavonoid glycoside composition of the peel extract of source or batch are detected；
Optional, the flavone aglycone constituents are Nobiletin and tangeritin；
Optional, the flavonoid glycoside composition is aurantiamarin.
9. the preparation method of rascal reference extract according to claim 2, it is characterised in that allocated in the step 4
Standard be：The weight percent content of aurantiamarin in final rascal reference extract should be made to maintain 16%-20%；
It is optional, the collection of illustrative plates obtained using thin-layered chromatography and/or high performance liquid chromatography to the rascal reference extract with
Its corresponding raw medicinal material is consistent；
It is optional, it is Nobiletin in the chromatogram obtained using thin-layered chromatography detection to the rascal reference extract, red
Fluorescence spot at tangerine element and aurantiamarin position and using the high performance liquid chromatography chromatogram that obtains of detection in aurantiamarin position
The chromatographic peak at place should distinguish corresponding chemical reference substance or its corresponding raw medicinal material is consistent.
10. a kind of discrimination method of medicinal material or Chinese medicine preparation, or rascal or medicinal material or Chinese medicine system containing rascal/rascal active ingredient
The method of quality control of agent, it is characterised in that the rascal reference extract for preparing claim any one of 1-9,
Testing sample is detected that contrast differentiates with thin-layered chromatography and/or high performance liquid chromatography；
Optional, the thin-layered chromatography and/or high performance liquid chromatography include that flavonoid glycoside and/or flavonoid glycoside metaclass are detected；
Optional, the flavonoid glycoside testing conditions are：
Lamellae:Silica gel prefabricated board
Solvent：Chloroform:Methyl alcohol:Water:Glacial acetic acid=13:4:1:1.5
Inspect:5%AlCl3Ethanol develops the color, and is inspected under UV366nm
Optional, the flavonoid glycoside metaclass testing conditions are：
Optional, described high performance liquid chromatography testing conditions：
Fixing phase：Chromatographic column C18 chromatographic columns；Mobile phase：A acetonitriles, B water；
Eluent gradient：
Flow velocity：0.8ml/min；Column temperature：20℃；Detector：PDAD；
Detection wavelength：280nm.
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