CN1830472A - Finger print atlas determination method of Chinese medicinal material tangerine peel water extraction - Google Patents
Finger print atlas determination method of Chinese medicinal material tangerine peel water extraction Download PDFInfo
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Abstract
A fingerprint assay method for detecting the aquatic extract of tangerine peel to evaluate the quality of tangerine peel includes such steps as adding the tangerine peel in water, reflux extracting, filtering, adsorbing by macro-reticular resin column, water washing, washing with ethanol or methanol, eluting with ethanol or methanol, collecting eluting liquid, evaporating off ethanol or methanol, loading in measuring glass, adding water, and efficient liquid-phase chromatography to obtain fingerprint.
Description
Technical field
The present invention relates to the method for building up of the finger printing of Chinese crude drug Pericarpium Citri Reticulatae, the foundation of Chinese crude drug Pericarpium Citri Reticulatae water-soluble active ingredient HPLC finger printing specifically the invention still further relates to the resultant Pericarpium Citri Reticulatae medical material of method standard finger-print thus.
Background technology
Pericarpium Citri Reticulatae (Pericarpium CitriReticulatae) is the dry mature skin of rutaceae orange (Citrus reticulata Blanco) and variety thereof, has regulating qi-flowing for strengthening spleen, drying dampness to eliminate phlegm effect, the clinical fullness in the epigastrium and chest that is used for, lack of appetite and vomiting, diseases such as cough with copious phlegm.Contain volatile oil in the Pericarpium Citri Reticulatae crude drug, Nobiletin, Neosynephrine and flavones ingredient; Flavones ingredient comprises: Hesperidin, and neohesperidin, compositions such as naringenin, wherein Hesperidin is a main component.At present, to the Quality Identification of Pericarpium Citri Reticulatae and compound preparation thereof, mainly with one, two active component wherein as the quality control index that characterizes medical material quanlity, as Hesperidin (Cui Lihua etc., Chinese Pharmaceutical Affairs, 2000,14 (2), 105～106; The yellow magnificent horizontal bar in the front of a carriage used as an armrest, Jiangxi College of Traditional Chinese Medicine journal, 2004,16 (2), 66), Neosynephrine (Jia Xiaobin, Nanjing University of Traditional Chinese Medicine's journal, 1999,15 (1) 27～28; Spill beautiful bright etc., Chinese patent medicine, 1997,19 (10), 19～20), Nobiletin (Qian Shihui etc., Nanjing University of Traditional Chinese Medicine's journal, 1998,14 (4) 219～220) isoreactivity composition is as detecting index, detects and differentiates with HPLC or TLC mode.Because the method for quality control of Pericarpium Citri Reticulatae and compound preparation thereof is comprehensive inadequately, be difficult to characterize their physicochemical characteristic comprehensively, therefore, extraction process for purification, the method for quality control of Pericarpium Citri Reticulatae improved, utilize modern analytical technique to characterize the Pericarpium Citri Reticulatae medical material quanlity.
Flavonoid in the chemical constituent of Pericarpium Citri Reticulatae, Hesperidin, Neosynephrine etc. are polar components, belong to water soluble ingredient.At present, a lot of for the polarity Pharmacological action study partly of Pericarpium Citri Reticulatae, studies show that the polarity pharmacological action partly of Pericarpium Citri Reticulatae is very wide, as: Shen Mingqin, clinical pharmacy, 1997,2,32 (2), 97～100; Xu Peng, Jiangxi College of Traditional Chinese Medicine journal, 1998,10 (4), 172～173: Wang Meimei etc., China Medicine University's journal, 1998,29 (6), 462～464; And to control the effect of Pericarpium Citri Reticulatae and prescribed preparation thereof, be to be nowhere near only, must be controlled, characterize Pericarpium Citri Reticulatae medical material quanlity characteristic on the whole its material group integral body at one, two chemical constituents, at present, can characterize the research or the blank out of the overall permanence of Pericarpium Citri Reticulatae polarity part.The present invention partly extracts Pericarpium Citri Reticulatae polarity, utilizes the efficient liquid-phase chromatograph finger print atlas mode, the overall permanence of control polarity part.
Chinese medicine fingerprint is that utilization modern analytical technique (coupling of spectrum, wave spectrum, chromatograph, nuclear magnetic resonance, NMR, X-ray diffraction and various technology etc.) characterizes and described the mode of Chinese medicine chemical information with figure (image).Chinese medicine is to rely on the comprehensive medical function of its contained number of chemical composition performance, and this is the difference the most basic with chemical synthetic drug, obtains people's approval now gradually.Therefore rely on the effectiveness and the specificity of the qualitative and quantitative Chinese medicine quality evaluating method of a certain chemical constituent to be under suspicion gradually.What Chinese medicine fingerprint was set up is kind and the quantity that comprehensively reflects the contained inherent chemical constituent of Chinese medicine, and then the quality of reflection Chinese medicine, especially not clear and definite in the present stage Effective Components of Chinese Herb overwhelming majority, and the mode of employing Chinese medicine fingerprint will characterize Chinese medicine quality effectively.Finger printing is also approved by international community, in the plant medical herbs guide that U.S. FDA is formulated clearly the method for quality control (FDA.Guidance ofIndustry:Botanical Drug (Draft) .2000August) of finger printing as the compounding substances group, adopt the mode of Chinese medicine fingerprint that Chinese medicine and Chinese patent medicine are carried out quality control, help Chinese medicine and products thereof and enter the international market.
Summary of the invention
The present invention partly extracts Pericarpium Citri Reticulatae polarity, utilizes the efficient liquid-phase chromatograph finger print atlas mode, the overall permanence of control polarity part.
The objective of the invention is by appearance Pericarpium Citri Reticulatae water extract HPLC finger printing, find out a kind of method of Pericarpium Citri Reticulatae quality control, the method of the standard finger-print of Pericarpium Citri Reticulatae is set up in establishment, whereby can be with the Pericarpium Citri Reticulatae finger printing as one of index of quality control and real and fake discrimination; The present invention also provides the standard finger-print of Pericarpium Citri Reticulatae simultaneously, uses the similarity of correlation coefficient process calculated characteristics fingerprint peaks simultaneously.
For setting up the integrated control method of Pericarpium Citri Reticulatae quality of medicinal material, with State Drug Administration issued in 2000 about the Chinese medicine fingerprint specification requirement, the Pericarpium Citri Reticulatae of the different places of production and collecting time is measured, set up Pericarpium Citri Reticulatae high-efficiency liquid-phase fingerprint assay method.
The present invention implements through the following steps:
(a) preparation of need testing solution: take by weighing the Pericarpium Citri Reticulatae medical material, add water, reflux, extract,, filter, get filtrate and cross macroporous adsorptive resins, washing, ethanol or the strong polar component of methanol flush away, reuse ethanol or methanol-eluted fractions, collect ethanol or meoh eluate, water-bath volatilizes ethanol or methanol, in the dislocation measuring bottle, add water to scale, be test sample;
(b) chromatographic condition: chromatographic column is that octadecylsilane chemically bonded silica is a filler; Adopt gradient elution, mobile phase A is a glacial acetic acid aqueous solution, and Mobile phase B is a methanol; Detect wavelength: 335～350nm;
(c) measure: the accurate need testing solution of drawing injects chromatograph of liquid, according to high effective liquid chromatography for measuring, obtains finger printing.
Preferred version of the present invention can be:
Macroporous absorption fat in described (a) low pole and nonpolar macroporous adsorption resin; Ethanol is 5～15% ethanol or the strong polar component of 5～15% methanol flush awaies, and reuse 40%～90% ethanol elution is collected 40%～90% ethanol or 40%～90% meoh eluate;
Mobile phase A in described (b) is 0.5%～7% glacial acetic acid aqueous solution.
Further preferred the present invention can implement by following step: the preparation of need testing solution in the described step (a): took by weighing 24～80 mesh sieve medical material Pericarpium Citri Reticulataes, in the flask, add 8～80 times of water gagings at the bottom of the horizontalization, heating, reflux, extract, 0.5～3h, put and be chilled to 30～50 ℃, vacuum filtration adds 6～60 times of water gagings again in the medicinal residues, extract 0.25～2h, with the method operation, merge filtrate, amount is decided volume.Measure the filtrate of relative raw medicinal herbs 0.25～5g, cross AB-8 type resin column, with the washing of 3～4 times of column volumes, 5%～15% ethanol or 5%～15% methanol of 3～4 times of column volumes of reuse are washed, and discard eluent earlier, use 40%～90% ethanol or the methanol-eluted fractions of 3～4 times of column volumes at last, collect 40%～90% ethanol or 40%～90% meoh eluate, water-bath volatilizes ethanol or methanol, in dislocation 2ml～125ml measuring bottle, add pure water to scale, be the test sample sample;
Chromatographic condition described in the described step (b): detect wavelength: 336～348nm; Column temperature: 20～40 ℃;
Sample size: 5～100 μ l.Mobile phase: A is 0.5%～7% glacial acetic acid mutually, and B is methanol mutually, flow velocity: 1.0ml/min.
Further preferred again the present invention can implement by following step:
The preparation of need testing solution in the described step (a): took by weighing 24～80 mesh sieve medical material Pericarpium Citri Reticulataes, in the flask, add 20～75 times of water gagings at the bottom of the horizontalization, heating, reflux, extract, 0.6～2.5h, put and be chilled to 30～50 ℃, vacuum filtration adds 15～55 times of water gagings again in the medicinal residues, extract 0.3～1.5h, with the method operation, merge filtrate, amount is decided volume.Measure the filtrate of relative raw medicinal herbs 0.75～3g, cross AB-8 type resin column, with the washing of 3～4 times of column volumes, 6%～14% ethanol or 6%～14% methanol of 3～4 times of column volumes of reuse are washed, and discard eluent earlier, use 50%～85% ethanol or 50%～85% methanol-eluted fractions of 3～4 times of column volumes at last, collect 50%～85% ethanol or 50%～85% meoh eluate, water-bath volatilizes ethanol or methanol, in dislocation 5ml～100ml measuring bottle, add pure water to scale, be the test sample sample.
Chromatographic condition described in the described step (b): detect wavelength: 338～346nm; Column temperature: 22～38 ℃;
Sample size: 10～80 μ l; Mobile phase: A is 1%～6.5% glacial acetic acid mutually, and B is methanol mutually, flow velocity: 1.0ml/min.
Further preferred the present invention can implement by following step:
The preparation of need testing solution in the described step (a): took by weighing 24～80 mesh sieve medical material Pericarpium Citri Reticulataes, in the flask, add 30～65 times of water gagings at the bottom of the horizontalization, heating, reflux, extract, 0.75～2.0h, put and be chilled to 30～50 ℃, vacuum filtration adds 15～55 times of water gagings again in the medicinal residues, extract 0.4～1.0h, with the method operation, merge filtrate, amount is decided volume.Measure the filtrate of relative raw medicinal herbs 0.5～2g, cross AB-8 type resin column, with the washing of 3～4 times of column volumes, 8%～12% ethanol or 8%～12% methanol of 3～4 times of column volumes of reuse are washed, and discard eluent earlier, use 60%～80% ethanol or 60%～80% methanol-eluted fractions of 3～4 times of column volumes at last, collect 60%～80% ethanol or 60%～80% meoh eluate, water-bath volatilizes ethanol or methanol, in dislocation 10ml～50ml measuring bottle, add pure water to scale, be the test sample sample;
Chromatographic condition described in the described step (b): detect wavelength: 339～345nm; Column temperature: 25～35 ℃; Sample size: 15～50 μ l; Mobile phase: A is 2%～5% glacial acetic acid mutually, and B is methanol mutually, flow velocity: 1.0ml/min.
Best the present invention can implement by following step:
The preparation of need testing solution in the described step (a): precision took by weighing 24～80 mesh sieve medical material Pericarpium Citri Reticulatae 2.0g, put in the 200ml boiling flask, added 60 times of water gagings, heating, reflux, extract, 1h is put and is chilled to about 40～50 ℃, vacuum filtration, add 40 times of water gagings in the medicinal residues again, extract 0.5h,, merge filtrate with the method operation, move in the 100ml measuring bottle, be settled to scale.Get this filtrate of 50ml, cross AB-8 type resin column, detached dowel height: 50cm with 1.5～2.0ml/min flow velocity, resin height: 40cm in internal diameter: the 1.0cm, post uses the washing of 100ml more earlier with same flow velocity, 10% ethanol of reuse 100ml is washed, discard eluent, use 70% ethanol elution of 100ml at last, collect 70% ethanol elution, water-bath volatilizes ethanol, in the dislocation 25ml measuring bottle, add pure water, be the test sample sample to scale.
Chromatographic condition described in the described step (b): detect wavelength: 340nm; Column temperature: 30 ℃; Sample size: 20 μ l.Mobile phase:
A is 5% glacial acetic acid mutually, and B is methanol mutually,
Employing degree of passing eluting, the gradient elution program is as follows:
0 minute, mobile phase A was that 82% 5% glacial acetic acid solution, B are 18% methanol;
40 minutes, mobile phase A was that 76% 5% glacial acetic acid solution, B are 24% methanol;
60 minutes, mobile phase A was that 70% 5% glacial acetic acid solution, B are 30% methanol.。
Among the present invention, because of methanol has murder by poisoning to human body, select for use alcoholic solution more safer than methanol as eluant, its eluant concentration of alcohol all has effect preferably in 40%～90% scope.
Measure the finger printing of the Pericarpium Citri Reticulatae medical material water extract of 7 separate sources according to inventive method among the present invention, compare its chromatogram, " specification requirement (provisional) of Chinese medicine finger printing research " issued by National Drug Administration in 2000, demarcate fingerprint peaks with relative retention time, determining that its total peak has 9, wherein is object of reference with the Hesperidin, i.e. No. 9 peaks, formulated Pericarpium Citri Reticulatae medical material standard finger-print technical parameter, the relative retention time average of relatives value at its total peak: 1 (0.194), 2 (0.208), 3 (0.276), 4 (0.291), 5 (0.364), 6 (0.439), 7 (0.711), 8 (0.837), 9 (1); Have the relative peak area ratio range at peak and account for gross area ratio scope: 1 (0.01～0.047) (0.95%～2.05%), 2 (0.02～0.06) (0.78%～2.63%), 3 (0.011～0.051) (1.57%～2.56%), 4 (0.102～0.263) (5.81%～11.51%), 5 (0.022～0.083) (1.28%～3.62%), 6 (0.089～0.323) (5.01%～16.05%), 7 (0.231～0.395) (13.16%～19.09%), 8 (0.012～0.078) (0.67%～3.40%), 9 (1～1) (43.78%～63.15%).
The similarity of the Pericarpium Citri Reticulatae medical material characteristic fingerprint chromatographic peak of separate sources, calculate the similarity at peak according to correlation coefficient process, peak area with the peak calculates, calculate with the correlation coefficient method, its similarity scope is: .9887～0.9990 has reached the requirement (0.9～1.0) of finger printing similarity.
Advantage of the present invention is as follows:
(1) the HPLC finger printing of setting up with the Pericarpium Citri Reticulatae water extract is being represented the most of pharmacologically active of Pericarpium Citri Reticulatae, can characterize the quality of Pericarpium Citri Reticulatae medical material effectively.A large amount of Pericarpium Citri Reticulatae pharmacological experiments shows that its pharmacological action of Pericarpium Citri Reticulatae water extract is extensive, and its pharmacological action is more than volatile oil component, so just have more representativeness with the water extracting component of Pericarpium Citri Reticulatae as monitor control index.
(2) do as a wholely to treat with the whole fingerprint graph of Pericarpium Citri Reticulatae, pay attention to each front and back that constitute fingerprint characteristic peak order and mutual relation, pay attention to whole facial feature, both avoided judging the one-sidedness of Pericarpium Citri Reticulatae total quality that having reduced again was the probability of the artificial processing of requisite quality because of only measuring one, two chemical constituent.The present invention is Pericarpium Citri Reticulatae GAP base quality control method for building up for quality complete, that accurately estimate Pericarpium Citri Reticulatae provides new reference standard, and the quality control and the curative effect that improve Pericarpium Citri Reticulatae and prescribed preparation thereof contribute.
(3) the present invention have that method is easy, stable, precision is high, favorable reproducibility, the characteristics that are easy to grasp.Be reflection Pericarpium Citri Reticulatae global feature, having selected the detection wavelength for use is 340nm, detects under this wavelength, and its peak number is many, and the information of reaction is comprehensive.And be to select wavelength at the absorption maximum of a certain composition (as: Hesperidin, Neosynephrine, Nobiletin etc.) in the existing document, it can not react the information of each composition of Pericarpium Citri Reticulatae comprehensively.Because of Hesperidin is dissolved in the hot water, so before extracting liquid filtering, extracting solution keeps 30～50 ℃ of certain temperature, guarantees certain extraction ratio; In addition, sample is by behind the macroporous adsorbent resin AB-8 enriching and purifying, strong polar component such as protein, saccharide, tannin etc. have been removed, improve the purity of sample, having reduced because of sample is complicated brings influence for the chromatographic column separating effect, improve the chromatographic column post and imitate and the life-span, improved the accuracy of experiment simultaneously.
Description of drawings
Fig. 1: Pericarpium Citri Reticulatae medical material 120min chromatogram
Fig. 2: the chromatogram of 0.2mg/ml Hesperidin
Fig. 3: the characteristic peak of Pericarpium Citri Reticulatae medicinal materials fingerprint
Fig. 4: the Pericarpium Citri Reticulatae medical material water extract chromatogram of separate sources
Wherein: be followed successively by from top to bottom: adopt the 10-11 month in Hubei, adopt in Hubei November, adopt the 10-11 month in Zhejiang, adopt in the Hunan October, adopt in Zhejiang November, adopt in Zhejiang October, adopt in the Hunan November.
Embodiment
Embodiment one
(a) preparation of need testing solution: took by weighing 24～80 mesh sieve medical material Pericarpium Citri Reticulataes, and in the flask, added 8 times of water gagings at the bottom of the horizontalization, heating, reflux, extract, 3h is put and is chilled to 30～50 ℃, and vacuum filtration adds 6 times of water gagings again in the medicinal residues, extract 2h, with the method operation, merge filtrate, amount is decided volume.Measure the filtrate of relative raw medicinal herbs 5g, cross AB-8 type resin column, with the washing of 3～4 times of column volumes, 15% ethanol or 15% methanol of 3～4 times of column volumes of reuse are washed, and discard eluent earlier, use 90% ethanol or 90% methanol-eluted fractions of 3～4 times of column volumes at last, collect 90% ethanol or 90% meoh eluate, water-bath volatilizes ethanol or methanol, in the dislocation 125ml measuring bottle, add pure water to scale, be the test sample sample;
(b) chromatographic condition: detect wavelength: 340nm; Column temperature: 30 ℃; Sample size: 20 μ l.Mobile phase: A is 5% glacial acetic acid mutually, and B is methanol mutually, flow velocity: 1.0ml/rmin, and degree of passing eluting: 0 minute, mobile phase A was that 82% 5% glacial acetic acid solution, B are 18% methanol; 40 minutes, mobile phase A was that 76% 5% glacial acetic acid solution, B are 24% methanol; 60 minutes, mobile phase A was that 70% 5% glacial acetic acid solution, B are 30% methanol;
(c) measure: the accurate need testing solution of drawing injects chromatograph of liquid, according to high effective liquid chromatography for measuring, obtains the finger printing of Pericarpium Citri Reticulatae medical material water extract.
Embodiment two
(a) preparation of need testing solution: took by weighing 24～80 mesh sieve medical material Pericarpium Citri Reticulataes, and in the flask, added 80 times of water gagings at the bottom of the horizontalization, heating, reflux, extract, 0.5h is put and is chilled to 30～50 ℃, and vacuum filtration adds 60 times of water gagings again in the medicinal residues, extract 0.25h, with the method operation, merge filtrate, amount is decided volume.Measure the filtrate of relative raw medicinal herbs 0.25g, cross AB-8 type resin column, with the washing of 3～4 times of column volumes, 5% ethanol or 5% methanol of 3～4 times of column volumes of reuse are washed, and discard eluent earlier, use 40% ethanol or 40% methanol-eluted fractions of 3～4 times of column volumes at last, collect 40% ethanol or 40% meoh eluate, water-bath volatilizes ethanol or methanol, in the dislocation 2ml measuring bottle, add pure water to scale, be the test sample sample;
(b) chromatographic condition: detect wavelength: 340nm; Column temperature: 30 ℃; Sample size: 20 μ l.Mobile phase: A is 5% glacial acetic acid mutually, and B is methanol mutually, flow velocity: 1.0ml/min, and degree of passing eluting: 0 minute, mobile phase A was that 82% 5% glacial acetic acid solution, B are 18% methanol; 40 minutes, mobile phase A was that 76% 5% glacial acetic acid solution, B are 24% methanol; 60 minutes, mobile phase A was that 70% 5% glacial acetic acid solution, B are 30% methanol;
(c) measure: the accurate need testing solution of drawing injects chromatograph of liquid, according to high effective liquid chromatography for measuring, obtains the finger printing of Pericarpium Citri Reticulatae medical material water extract.
Embodiment three
(a) preparation of need testing solution: took by weighing 24～80 mesh sieve medical material Pericarpium Citri Reticulataes, and in the flask, added 20 times of water gagings at the bottom of the horizontalization, heating, reflux, extract, 2.5h is put and is chilled to 30～50 ℃, and vacuum filtration adds 15 times of water gagings again in the medicinal residues, extract 1.5h, with the method operation, merge filtrate, amount is decided volume.Measure the filtrate of relative raw medicinal herbs 3g, cross AB-8 type resin column, with the washing of 3～4 times of column volumes, 14% ethanol or 14% methanol of 3～4 times of column volumes of reuse are washed, and discard eluent earlier, use 85% ethanol or 85% methanol-eluted fractions of 3～4 times of column volumes at last, collect 85% ethanol or 85% meoh eluate, water-bath volatilizes ethanol or methanol, in the dislocation 5ml measuring bottle, add pure water to scale, be the test sample sample;
(b) chromatographic condition: detect wavelength: 340nm; Column temperature: 30 ℃; Sample size: 20 μ l.Mobile phase: A is 5% glacial acetic acid mutually, and B is methanol mutually, flow velocity: 1.0ml/min, and degree of passing eluting: 0 minute, mobile phase A was that 82% 5% glacial acetic acid solution, B are 18% methanol; 40 minutes, mobile phase A was that 76% 5% glacial acetic acid solution, B are 24% methanol; 60 minutes, mobile phase A was that 70% 5% glacial acetic acid solution, B are 30% methanol;
(c) measure: the accurate need testing solution of drawing injects chromatograph of liquid, according to high effective liquid chromatography for measuring, obtains the finger printing of Pericarpium Citri Reticulatae medical material water extract.
Embodiment four
(a) preparation of need testing solution: take by weighing the Pericarpium Citri Reticulatae medical material, cross 24～80 mesh sieves, in the flask, add 75 times of water gagings at the bottom of the horizontalization, heating, reflux, extract, 0.6h is put and is chilled to 30～50 ℃, and vacuum filtration adds 55 times of water gagings again in the medicinal residues, extract 0.3h, with the method operation, merge filtrate, amount is decided volume.Measure the filtrate of relative raw medicinal herbs 0.75g, cross AB-8 type resin column, with the washing of 3～4 times of column volumes, 6% ethanol or 6% methanol of 3～4 times of column volumes of reuse are washed, and discard eluent earlier, use 50% ethanol or 50% methanol-eluted fractions of 3～4 times of column volumes at last, collect 50% ethanol or 50% meoh eluate, water-bath volatilizes ethanol or methanol, in the dislocation 100ml measuring bottle, add pure water to scale, be the test sample sample.
(b) chromatographic condition: detect wavelength: 340nm; Column temperature: 30 ℃; Sample size: 20 μ l.Mobile phase: A is 5% glacial acetic acid mutually, and B is methanol mutually, flow velocity: 1.0ml/min, and degree of passing eluting: 0 minute, mobile phase A was that 82% 5% glacial acetic acid solution, B are 18% methanol; 40 minutes, mobile phase A was that 76% 5% glacial acetic acid solution, B are 24% methanol; 60 minutes, mobile phase A was that 70% 5% glacial acetic acid solution, B are 30% methanol.
(c) measure: the accurate need testing solution of drawing injects chromatograph of liquid, according to high effective liquid chromatography for measuring, obtains the finger printing of Pericarpium Citri Reticulatae medical material water extract.
Embodiment five
(a) preparation of need testing solution in: took by weighing 24～80 mesh sieve medical material Pericarpium Citri Reticulataes, and in the flask, added 30 times of water gagings at the bottom of the horizontalization, heating, reflux, extract, 2.0h is put and is chilled to 30～50 ℃, and vacuum filtration adds 15 times of water gagings again in the medicinal residues, extract 1.0h, with the method operation, merge filtrate, amount is decided volume.Measure the filtrate of relative raw medicinal herbs 2g, cross AB-8 type resin column, with the washing of 3～4 times of column volumes, 12% ethanol or 12% methanol of 3～4 times of column volumes of reuse are washed, and discard eluent earlier, use 80% ethanol or 80% methanol-eluted fractions of 3～4 times of column volumes at last, collect 80% ethanol or 80% meoh eluate, water-bath volatilizes ethanol or methanol, in the dislocation 10ml measuring bottle, add pure water to scale, be the test sample sample.
(b) chromatographic condition: detect wavelength: 340nm; Column temperature: 30 ℃; Sample size: 20 μ l.Mobile phase: A is 5% glacial acetic acid mutually, and B is methanol mutually, flow velocity: 1.0ml/min, and degree of passing eluting: 0 minute, mobile phase A was that 82% 5% glacial acetic acid solution, B are 18% methanol; 40 minutes, mobile phase A was that 76% 5% glacial acetic acid solution, B are 24% methanol; 60 minutes, mobile phase A was that 70% 5% glacial acetic acid solution, B are 30% methanol.
(c) measure: the accurate need testing solution of drawing injects chromatograph of liquid, according to high effective liquid chromatography for measuring, obtains the finger printing of Pericarpium Citri Reticulatae medical material water extract.
Embodiment six
(a) preparation of need testing solution in: took by weighing 24～80 mesh sieve medical material Pericarpium Citri Reticulataes, and in the flask, added 65 times of water gagings at the bottom of the horizontalization, heating, reflux, extract, 0.75h is put and is chilled to 30～50 ℃, and vacuum filtration adds 55 times of water gagings again in the medicinal residues, extract 0.4h, with the method operation, merge filtrate, amount is decided volume.Measure the filtrate of relative raw medicinal herbs 0.5g, cross AB-8 type resin column, with the washing of 3～4 times of column volumes, 8% ethanol or 8% methanol of 3～4 times of column volumes of reuse are washed, and discard eluent earlier, use 60% ethanol or 60% methanol-eluted fractions of 3～4 times of column volumes at last, collect 60% ethanol or 60% meoh eluate, water-bath volatilizes ethanol or methanol, in the dislocation 50ml measuring bottle, add pure water to scale, be the test sample sample.
(b) chromatographic condition: detect wavelength: 340nm; Column temperature: 30 ℃; Sample size: 20 μ l.Mobile phase: A is 5% glacial acetic acid mutually, and B is methanol mutually, flow velocity: 1.0ml/min, and degree of passing eluting: 0 minute, mobile phase A was that 82% 5% glacial acetic acid solution, B are 18% methanol; 40 minutes, mobile phase A was that 76% 5% glacial acetic acid solution, B are 24% methanol; 60 minutes, mobile phase A was that 70% 5% glacial acetic acid solution, B are 30% methanol.
(c) measure: the accurate need testing solution of drawing injects chromatograph of liquid, according to high effective liquid chromatography for measuring, obtains the finger printing of Pericarpium Citri Reticulatae medical material water extract.
Embodiment seven
1, instrument and reagent
1,1 instrument
U.S. Waters high performance liquid chromatograph (2996PDA diode array detector, 515 pumps, Empower work station); Waters in line Degasser AF degasser; Chinese medicine micromill (going up Haidian Chinese traditional medicine machine of a specified duration Manufacturing Co., Ltd); Universal electric furnace DL-1 (in Beijing emerging great achievement Instr Ltd.); Milli-Q ultra-pure water instrument; Mettler Toledo AB204-N analytical balance, AB-8 macroporous adsorbent resin (Nankai University).
1,2 reagents
Pericarpium Citri Reticulatae medical material (by Jiangzhong Pharmaceutical Co., Ltd.'s buying, being accredited as certified products through quality supervision portion of Jiangzhong Pharmaceutical Co., Ltd.) is for dry product, without the process of preparing Chinese medicine.Methanol (chromatographically pure); Acetic acid (analytical pure); Water (two steamings are also crossed Milli-Q ultra-pure water instrument); Hesperidin reference substance (Nat'l Pharmaceutical ﹠ Biological Products Control Institute).
2, Pericarpium Citri Reticulatae determining fingerprint pattern
2,1 Pericarpium Citri Reticulatae finger printing preparation
The pretreatment of AB-8 type resin column:
A, prior to adding ethanol or the methanol be equivalent to 0.4～0.5 times of loaded resin volume in the adsorption column, newer resin is dropped in the post. make its liquid level be higher than the about 30cm of resin bed, and soaked 24 hours.
B, with 2 times of column volume ethanol or methanol,, and soaked 4～5 hours by resin bed with the flow velocity of 2 times of column volumes per hour.Now with ethanol or methanol with the flow velocity of 2 times of column volumes per hour by resin bed, be washed till effluent and add water and be not white in color till the muddiness.And clean ethanol or methanol with same flow velocity with water.
C, with the 5%HCL solution of 2 times of column volumes,, and soaked 2～4 hours by resin bed with the flow velocity of 4～6 times of column volumes per hour, then to be washed till water outlet pH with same flow velocity be neutrality to water.
The 5%HCL solution of d, 2 times of column volumes of reuse, and soaked 2～4 hours by resin bed with the flow velocity of 4～6 times of column volumes per hour, and then to be washed till water outlet pH with same flow velocity be neutrality to water.
When carrying out the macroporous adsorbent resin Regeneration Treatment, directly with the flow velocity flushing of 95% ethanol with 4～6 times of column volumes per hour, reuse distilled water flushing resin is to balance.
The preparation of reference substance solution: precision takes by weighing the Hesperidin reference substance 10mg of vacuum drying to constant weight, put in the 25ml measuring bottle, add dissolve with methanol and be settled to scale, shake up, be mother solution, precision is measured 5ml and is placed the 10ml measuring bottle, adds methanol and is diluted to scale, shake up, promptly get (every milliliter contains Hesperidin 0.2mg).
The test sample preparation: precision took by weighing 24～80 mesh sieve medical material Pericarpium Citri Reticulatae 2.0g, put in the 200ml boiling flask, add 60 times of water gagings, straight fiery heating and refluxing extraction 1h, put and be chilled to about 40～50 ℃, vacuum filtration adds 40 times of water gagings again in the medicinal residues, extract 0.5h, operate with method, merge filtrate, move in the 100ml measuring bottle, be settled to scale.Get this filtrate of 50ml, cross AB-8 type resin column (detached dowel height: 50cm, internal diameter: 1.0cm with 1.5～2.0ml/min flow velocity, resin height in the post: 40cm), use the washing of 100ml more earlier with same flow velocity, 10% ethanol of reuse 100ml is washed, and discards eluent, use 70% ethanol elution of 100ml at last, collect 70% ethanol elution, water-bath volatilizes ethanol, in the dislocation 25ml measuring bottle, add Milli-Q water to scale, be the test sample sample.
3, chromatographic condition
Agilent ODS-C
18Analytical column (250mm * 4.6mm 5 μ m); Detect wavelength: 340nm; Column temperature: 30 ℃; Sample size: 20 μ l.Mobile phase: A is 5% glacial acetic acid mutually, and B is methanol mutually, flow velocity: 1.0ml/min gradient elution program such as following table:
| Time (min) | A:5% glacial acetic acid (%) (v/v) | B: methanol (%) (v/v) |
| 0 40 60 | 82 76 70 | 18 24 30 |
It is object of reference (S) that object of reference is selected Hesperidin for use.
Get reference substance and need testing solution 20 μ l sample introductions respectively,, write down 60 minutes chromatogram according to high effective liquid chromatography for measuring.With the relative retention time of the chromatographic peak (S) of Hesperidin and peak area is 1 to calculate relative retention time and relative peak area.
4, result
4,1 precision and stability experiment get Pericarpium Citri Reticulatae medical material need testing solution respectively 0,2,4,6, time point that 8h is different detects, and investigates the concordance of chromatographic peak relative peak area and relative retention time ratio, carry out precision simultaneously and measure.The results are shown in Table one, table two:
The relative standard deviation of table one, relative peak area
| Total peak-to-peak number | Sample injection time | ||||||
| (1)0h | (2)2h | (3)4h | (4)6h | (5)8h | Meansigma methods | RSD％ | |
| 1 2 3 4 5 6 7 8 9 | 0.024 0.049 0.035 0.207 0.056 0.323 0.274 0.055 1 | 0.027 0.052 0.034 0.205 0.057 0.314 0.279 0.058 1 | 0.025 0.049 0.031 0.205 0.057 0.316 0.27 0.055 1 | 0.025 0.05 0.033 0.206 0.057 0.319 0.269 0.053 1 | 0.024 0.049 0.032 0.206 0.059 0.323 0.265 0.059 1 | 0.025 0.05 0.033 0.206 0.057 0.319 0.271 0.056 1 | 4.9 2.64 4.79 0.42 1.96 1.27 1.97 4.37 - |
The relative standard deviation of table two, relative retention time
| Total peak-to-peak number | Sample injection time | ||||||
| (1)0h | (2)2h | (3)4h | (4)6h | (5)8h | Meansigma methods | RSD％ | |
| 1 2 3 4 5 6 7 8 9 | 0.19 0.205 0.273 0.287 0.360 0.439 0.710 0.836 1 | 0.193 0.207 0.274 0.289 0.362 0.438 0.709 0.835 1 | 0.195 0.209 0.276 0.291 0.365 0.438 0.714 0.839 1 | 0.193 0.207 0.275 0.290 0.365 0.438 0.711 0.838 1 | 0.196 0.210 0.277 0.292 0.366 0.439 0.713 0.839 1 | 0.193 0.208 0.275 0.290 0.364 0.438 0.711 0.837 1 | 1.07 1.05 0.57 0.67 0.70 0.16 0.30 0.20 - |
4,2 repeatability experiment
Precision took by weighing 5 parts in 24～65 mesh sieve Pericarpium Citri Reticulatae medical material powder, prepared sample by the test sample preparation method, respectively sample detection.The results are shown in Table three, table four.
The relative standard deviation of table three, relative peak area
| Total peak-to-peak number | Sample injection time | ||||||
| Sample one | Sample two | Sample three | Sample four | Sample five | Meansigma methods | RSD％ | |
| 1 2 3 4 5 6 7 8 9 | 0.019 0.038 0.026 0.160 0.04 0.261 0.219 0.041 1 | 0.019 0.039 0.027 0.164 0.049 0.258 0.219 0.039 1 | 0.019 0.039 0.027 0.160 0.049 0.271 0.217 0.037 1 | 0.019 0.039 0.027 0.166 0.049 0.273 0.219 0.039 1 | 0.021 0.03 0.026 0.163 0.050 0.277 0.220 0.038 1 | 0.019 0.039 0.027 0.162 0.049 0.268 0.219 0.039 1 | 5.26 1.28 2.62 1.66 2.28 3.03 0.50 3.85 - |
The relative standard deviation of table four, relative retention time
| Total peak-to-peak number | Sample injection time | ||||||
| Sample one | Sample two | Sample three | Sample four | Sample five | Meansigma methods | RSD％ | |
| 1 2 3 4 5 6 7 8 9 | 0.198 0.211 0.273 0.294 0.370 0.438 0.709 0.846 1 | 0.198 0.212 0.277 0.294 0.370 0.438 0.709 0.846 1 | 0.199 0.213 0.277 0.295 0.372 0.439 0.710 0.846 1 | 0.199 0.213 0.277 0.295 0.371 0.438 0.709 0.845 1 | 0.198 0.212 0.277 0.294 0.370 0.438 0.709 0.846 1 | 0.198 0.212 0.276 0.294 0.371 0.438 0.709 0.846 1 | 0.36 0.41 0.65 0.24 0.27 0.11 0.07 0.06 - |
5, the total peak of feature
Measure the HPLC collection of illustrative plates of its water extract of Pericarpium Citri Reticulatae medical material of 7 separate sources according to the inventive method, compare its chromatogram, demarcate fingerprint peaks by " specification requirement (provisional) of Chinese medicine finger printing research " with relative retention time, determine that its total peak has 9, wherein be object of reference, i.e. No. 9 peaks with the Hesperidin.Formulated Pericarpium Citri Reticulatae medical material standard finger-print technical parameter, its total peak relative retention time average of relatives value: 1 (0.194), 2 (0.208), 3 (0.276), 4 (0.291), 5 (0.364), 6 (0.439), 7 (0.711), 8 (0.837), 9 (1); Have peak relative peak area ratio range and account for gross area ratio scope: 1 (0.01～0.047) (0.95%～2.05%), 2 (0.02～0.06) (0.78%～2.63%), 3 (0.011～0.051) (1.57%～2.56%), 4 (0.102～0.263) (5.81%～11.51%), 5 (0.022～0.083) (1.28%～3.62%), 6 (0.089～0.323) (5.01%～16.05%), 7 (0.231～0.395) (13.16%～19.09%), 8 (0.012～0.078) (0.67%～3.40%), 9 (1～1) (43.78%～63.15%).Its concrete outcome sees Table five, table six.
The relative retention time ratio at the total peak of the Pericarpium Citri Reticulatae medical material water extract feature of table five, the different place of production and time
| Total peak-to-peak number | Relative retention time ratio | ||||||||
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | |
| 10-11 month Hunan, Hubei in November, Hubei in October, 10-11 month Hunan, Zhejiang in November, Zhejiang in October, Zhejiang 11 months | 0.195 0.195 0.196 0.192 0.192 0.194 0.196 0.194 | 0.209 0.209 0.210 0.206 0.206 0.207 0.210 0.208 | 0.277 0.276 0.278 0.274 0.274 0.274 0.277 0.276 | 0.292 0.291 0.293 0.289 0.288 0.289 0.292 0.291 | 0.366 0.366 0.367 0.362 0.361 0.363 0.366 0.364 | 0.439 0.438 0.441 0.43 0.437 0.438 0.439 0.439 | 0.713 0.712 0.713 0.711 0.708 0.709 0.713 0.71 | 0.839 0.839 0.839 0.836 0.831 0.834 0.839 0.837 | 1 1 1 1 1 1 1 1 |
The relative peak area ratio at the total peak of the Pericarpium Citri Reticulatae medical material water extract feature of table six, the different place of production and time
| Total peak-to-peak number | Relative peak area ratio | ||||||||
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | |
| 10-11 month Hunan, Hubei in November, Hubei in October, 10-11 month Hunan, Zhejiang in November, Zhejiang in October, Zhejiang 11 months | 0.047 0.028 0.030 0.021 0.016 0.010 0.024 0.025 | 0.060 0.042 0.038 0.027 0.028 0.020 0.049 0.037 | 0.051 0.046 0.048 0.034 0.031 0.011 0.032 0.036 | 0.263 0.191 0.177 0.118 0.128 0.102 0.206 0.169 | 0.083 0.056 0.051 0.030 0.045 0.022 0.059 0.049 | 0.236 0.162 0.094 0.089 0.167 0.266 0.323 0.191 | 0.395 0.364 0.359 0.231 0.253 0.265 0.265 0.304 | 0.078 0.058 0.044 0.034 0.044 0.012 0.059 0.047 | 1 1 1 1 1 1 1 1 |
6, the similarity of the Pericarpium Citri Reticulatae medical material characteristic fingerprint figure chromatographic peak of separate sources is calculated the similarity at peak according to correlation coefficient process, calculate with peak area, its similarity scope is: 0.9887～0.9990, reached the requirement (0.9～1.0) of finger printing similarity, result such as following table seven.
The similarity of the Pericarpium Citri Reticulatae medical material characteristic fingerprint chromatographic peak of table seven, the different place of production and time
| NO | Zhejiang October | Zhejiang November | The Zhejiang 10-11 month | Hunan October | Hubei November | The Hubei 10-11 month | Hunan November | Average peak area |
| 1 | 59567 | 61505 | 62402 | 66705 | 50328 | 31547 | 60965 | 56145.571 |
| 2 | 76106 | 93218 | 79505 | 85029 | 88038 | 62073 | 123907 | 86839.429 |
| 3 | 65440 | 101574 | 100365 | 105090 | 95569 | 132353 | 79439 | 97118.571 |
| 4 | 334021 | 426129 | 370034 | 367316 | 396101 | 313674 | 519450 | 389532.14 |
| 5 | 105044 | 124873 | 105454 | 94948 | 140340 | 68973 | 148387 | 112574.14 |
| 6 | 300219 | 360202 | 196563 | 276849 | 517353 | 819737 | 812317 | 469034.29 |
| 7 | 501442 | 809300 | 749782 | 718660 | 783945 | 814625 | 665948 | 720528.86 |
| 8 | 98761 | 128066 | 92664 | 104911 | 136720 | 36308 | 148770 | 106600 |
| 9 | 1270297 | 2225577 | 2086470 | 3117819 | 3099541 | 3079577 | 2515113 | 2484913.4 |
| Correlation coefficient | 0.9927 | 0.9961 | 0.9910 | 0.9942 | 0.9990 | 0.9934 | 0.9887 | 1 |
Calculating formula of similarity:
Claims (9)
1, a kind of finger print measuring method of Chinese crude drug Pericarpium Citri Reticulatae water extract, its feature comprises the steps:
(a) preparation of need testing solution: take by weighing the Chinese crude drug Pericarpium Citri Reticulatae, add water, reflux, extract,, filter, get filtrate and cross macroporous adsorptive resins, washing, ethanol or the strong polar component of methanol flush away, reuse ethanol or methanol-eluted fractions, collect eluent, water-bath volatilizes ethanol or methanol, in the dislocation measuring bottle, add water to scale, be test sample;
(b) chromatographic condition: chromatographic column is that octadecylsilane chemically bonded silica is a filler; Adopt gradient elution, mobile phase A is a glacial acetic acid aqueous solution, and Mobile phase B is a methanol; Detect wavelength: 335～350nm;
(c) measure: the accurate need testing solution of drawing injects chromatograph of liquid, according to high effective liquid chromatography for measuring, obtains finger printing.
2, be low pole and nonpolar macroporous adsorption resin according to the macroporous absorption fat in the claim 1 described (a).
3, be 5～15% ethanol or the strong polar component of 5～15% methanol flush awaies according to ethanol in the claim 1 described (a) or methanol, reuse 40%～90% ethanol or 40%～90% methanol-eluted fractions are collected 40%～90% ethanol or 40%～90% meoh eluate.
4, be 0.5%～7% glacial acetic acid aqueous solution according to the mobile phase A in the claim 1 described (b).
5, according to the preparation of need testing solution in the described step of claim 1 (a): took by weighing 24～80 mesh sieve Chinese crude drug Pericarpium Citri Reticulataes, in the flask, add 8～80 times of water gagings at the bottom of the horizontalization, heating, reflux, extract, 0.5～3h, put and be chilled to 30～50 ℃, vacuum filtration adds 6～60 times of water gagings again in the medicinal residues, extract 0.25～2h, with the method operation, merge filtrate, amount is decided volume.Measure the filtrate of relative raw medicinal herbs 0.25～5g, cross AB-8 type resin column, with the washing of 3～4 times of column volumes, 5%～15% ethanol or 5%～15% methanol of 3～4 times of column volumes of reuse are washed, and discard eluent earlier, use 40%～90% ethanol or 40%～90% methanol-eluted fractions of 3～4 times of column volumes at last, collect 40%～90% ethanol or 40%～90% meoh eluate, water-bath volatilizes ethanol or methanol, in dislocation 2ml～125ml measuring bottle, add pure water to scale, be the test sample sample;
Chromatographic condition described in the described step (b): detect wavelength: 336～348nm; Column temperature: 20～40 ℃; Sample size: 5～100 μ l; Mobile phase: A is 0.5%～7% glacial acetic acid mutually, and B is methanol mutually, flow velocity: 1.0ml/min.
6, according to the preparation of need testing solution in the described step of claim 5 (a): took by weighing 24～80 mesh sieve medical material Pericarpium Citri Reticulataes, in the flask, add 20～75 times of water gagings at the bottom of the horizontalization, heating, reflux, extract, 0.6～2.5h, put and be chilled to 30～50 ℃, vacuum filtration adds 15～55 times of water gagings again in the medicinal residues, extract 0.3～1.5h, with the method operation, merge filtrate, amount is decided volume.Measure the filtrate of relative raw medicinal herbs 0.75～3g, cross AB-8 type resin column, with the washing of 3～4 times of column volumes, 6%～14% ethanol or 6%～14% methanol of 3～4 times of column volumes of reuse are washed, and discard eluent earlier, use 50%～85% ethanol or 50%～85% methanol-eluted fractions of 3～4 times of column volumes at last, collect 50%～85% ethanol or 50%～85% meoh eluate, water-bath volatilizes ethanol or methanol, in dislocation 5ml～100ml measuring bottle, add pure water to scale, be the test sample sample.
Chromatographic condition described in the described step (b): detect wavelength: 338～346nm; Column temperature: 22～38 ℃; Sample size: 10～80 μ l.Mobile phase: A is 1%～6.5% glacial acetic acid mutually, and B is methanol mutually, flow velocity: 1.0ml/min.
7, according to the preparation of need testing solution in the described step of claim 6 (a): took by weighing 24～80 mesh sieve Chinese crude drug Pericarpium Citri Reticulataes, in the flask, add 30～65 times of water gagings at the bottom of the horizontalization, heating, reflux, extract, 0.75～2.0h, put and be chilled to 30～50 ℃, vacuum filtration adds 15～55 times of water gagings again in the medicinal residues, extract 0.4～1.0h, with the method operation, merge filtrate, amount is decided volume.Measure the filtrate of relative raw medicinal herbs 0.5～2g, cross AB-8 type resin column, with the washing of 3～4 times of column volumes, 8%～12% ethanol or 8%～12% methanol of 3～4 times of column volumes of reuse are washed, and discard eluent earlier, use 60%～80% ethanol or 60%～80% methanol-eluted fractions of 3～4 times of column volumes at last, collect 60%～80% ethanol or 60%～80% meoh eluate, water-bath volatilizes ethanol or methanol, in dislocation 10ml～50ml measuring bottle, add pure water to scale, be the test sample sample.
Chromatographic condition described in the described step (b): detect wavelength: 339～345nm; Column temperature: 25～35 ℃; Sample size: 15～50 μ l.Mobile phase: A is 2%～6% glacial acetic acid mutually, and B is methanol mutually, flow velocity: 1.0ml/min.
8, according to the preparation of need testing solution in the described step of claim 7 (a): precision took by weighing 24～80 mesh sieve Pericarpium Citri Reticulatae medical material 2.0g,, put in the 200ml boiling flask, add 60 times of water gagings, heating, reflux, extract, 1h, put and be chilled to about 40～50 ℃, vacuum filtration adds 40 times of water gagings again in the medicinal residues, extract 0.5h, operate with method, merge filtrate, move in the 100ml measuring bottle, be settled to scale.Get this filtrate of 50ml, cross AB-8 type resin column, detached dowel height: 50cm with 1.5～2.0ml/min flow velocity, resin height: 40cm in internal diameter: the 1.0cm, post uses the washing of 100ml more earlier with same flow velocity, 10% ethanol of reuse 100ml is washed, discard eluent, use 70% ethanol elution of 100ml at last, collect 70% ethanol elution, water-bath volatilizes ethanol, in the dislocation 25ml measuring bottle, add pure water, be the test sample sample to scale.
Chromatographic condition described in the described step (b): detect wavelength: 340nm; Column temperature: 30 ℃; Sample size: 20 μ l.
Mobile phase: A is 5% glacial acetic acid mutually, and B is methanol mutually.Employing degree of passing eluting, the gradient elution program is as follows:
0 minute, mobile phase A was that 82% 5% glacial acetic acid solution, B are 18% methanol;
40 minutes, mobile phase A was that 76% 5% glacial acetic acid solution, B are 24% methanol;
60 minutes, mobile phase A was that 70% 5% glacial acetic acid solution, B are 30% methanol.
9, the configuration proportion according to the mobile A solution of claim 1,4 in described is to prepare by volume.
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| CN115598255A (en) * | 2021-10-12 | 2023-01-13 | 广州白云山陈李济药厂有限公司（Cn） | Detection and classification method of dried orange peel |
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| CN115598255A (en) * | 2021-10-12 | 2023-01-13 | 广州白云山陈李济药厂有限公司（Cn） | Detection and classification method of dried orange peel |
| CN115598255B (en) * | 2021-10-12 | 2023-12-12 | 广州白云山陈李济药厂有限公司 | Method for detecting and grading dried orange peel |
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