CN105588885A - Salvianolic acid extract fingerprint spectrum and content measurement method of related components - Google Patents
Salvianolic acid extract fingerprint spectrum and content measurement method of related components Download PDFInfo
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Abstract
The invention relates to establishment of a salvianolic acid extract fingerprint spectrum and a content measurement method of related components. The content measurement method includes the following steps: 1) establishing the salvianolic acid extract digitized quantitative fingerprint spectrum through ultrahigh performance liquid multi-wavelength visible ultraviolet detection method; 2) recognizing peaks in the fingerprint spectrum in the step 1) with mass spectrum to determine common fingerprint peaks of ultraviolet detection; and 3) performing measurement to contents of three indicative components, which are high in content, significant in activity and definite in structure, recognized in the step 2) through high performance liquid chromatography variable-wavelength detection method.
Description
Technical field:
The present invention relates to a kind of detection method of Chinese medical extract, particularly a kind of foundation of salvia root polyphenol acid extract finger-print and the content assaying method about composition
Background technology:
The red sage root is Labiatae salvia. Have another name called: radix salviae miltiorrhizae, red, blood ginseng, clovershrub etc. The red sage root is with root hyoscine, bitter, cold nature. There is promoting blood circulation for regulating menstruation, removing blood stasis for promoting tissue regeneration, tranquilizing and allaying excitement, the cool blood function such as carbuncle, swelling and pain relieving that disappears. Be used for the treatment of the diseases such as irregular menstruation, dysmenorrhoea, postpartum stasis blocking stomachache, joint pain, neurasthenia insomnia, palpitaition, carbuncle sore tumefacting virus. The clinical proof of modern medicine, the red sage root has hemangiectasis and the effect of promoting coronary blood flow, is used for treating the diseases such as coronary heart diseases and angina pectoris, miocardial infarction, tachycardia, has significant curative effect; Also be used for treating the disease such as chronic hepatitis, early stage cirrhosis, also have good effect.
In the red sage root, contain the main active ingredient of two classes, i.e. fat-soluble tanshinone and water-soluble salvianolic acid. Modern medicine is thought; salvianolic acid (salvianolieacids) composition; can dwindle the scope of myocardial infarction; alleviate its state of an illness; myocardial ischemia in rats, reperfusion injury are had to protective effect; there are obvious inhibition platelet aggregation, anti-freezing, molten fibre simultaneously and reduce blood fat, antiatherogenic effect, thering is larger clinical medical value. This compounds mainly contains danshinolic acid, danshensu, protocatechualdehyde, caffeic acid etc., and wherein tanshin polyphenolic acid B is the main pharmacodynamics composition in red sage root water soluble ingredient.
Salvia root polyphenol acid injection is a kind of product having gone on the market, and its active material is called as " salvia root polyphenol acid ". the extraction preparation method of salvia root polyphenol acid has multiple, for example: get salvia piece, by purifying water boiling and extraction three times, each 0.5～1 hour, No. three extracts are merged, cooling, be adjusted to acidity with hydrochloric acid solution, place 4～8 hours, filter, obtain clear and bright liquid, liquid is purified by polyamide column chromatography technique, after loading, rinse by purified water, carry out wash-out with sodium bicarbonate solution, collect eluent, eluent is adjusted to acidity with hydrochloric acid solution, adopt macroporous resin column chromatography technique purifying liquid, after loading, rinse by purified water, carry out wash-out with ethanolic solution, collect eluent, eluent reduced pressure concentration is reclaimed to ethanol, the refrigeration of gained concentrate was filtered after 12～24 hours, filtrate regulates pH value to 5.3～6.0 with sodium hydroxide solution, freeze drying, obtain. again salvia root polyphenol acid extract and auxiliary material are mixed with into various preparations afterwards. for example, mix with mannitol solution, and adjust pH to 5.5～6.0, constant volume postlyophilization, prepares salvia root polyphenol acid injection.
As the active component of salvia root polyphenol acid injection, the quality standard present situation of salvia root polyphenol acid extract and preparation at present, Quality Control project is very not comprehensive, generally adopts UV-VIS spectrophotometry to measure the content of Polyphenol Acids. This method is to calculate content, specificity and the poor accuracy of Polyphenol Acids taking tanshin polyphenolic acid B as contrast. And for the Chinese medicine preparation of complicated component, only with a kind of Composition Control content's index, in lacking globality, also also inadequate for pointing out of complicated ingredient.
Therefore, point out the defects such as insufficient and quantitatively inaccurate for above-mentioned complicated ingredient. The present invention has developed the method for UPLC chromatographic fingerprinting, utilizes liquid matter to carry out peak and points out, thereby determined that 23 ultraviolets detect total fingerprint peaks. And set up HPLC content assaying method for three content pointing out out index composition large, that activity is obvious and structure is clear and definite simultaneously.
The present invention is that to characterize the quality of Chinese medicine preparation be target comprehensively, a kind of employing UPLC-PDA method (the visible uv detection method of ultra high efficiency liquid phase multi-wavelength) is provided, set up the digitlization quantitative finger print atlas of salvia root polyphenol acid extract, and uniformity and the stability of many batches of salvia root polyphenol acid extract samples are identified with level two fingerprint sizing technique, detect altogether 39 of chemical compositions, in conjunction with multi-stage ms information and reference substance, 23 of qualification root of red-rooted salvia phenolic acid compounds. Consider that UPLC is applied to the extensive use of cost and the HPLC of extensive detection, measure again the content of three index composition Rosmarinic acids, alkannic acid, tanshin polyphenolic acid B with HPLC-VWD method (high performance liquid chromatography variable wavelength detection method) simultaneously. In guaranteeing accuracy, validity, take into account again cost savings principle, be more suitable for being applied to extension and produce, the perfect quality control standard of existing Danshen injection Polyphenol Acids.
Summary of the invention:
The invention provides a kind of salvia root polyphenol acid extract finger-print and content assaying method, it is characterized in that, step is as follows:
Step 1: adopt the visible uv detection method of ultra high efficiency liquid phase multi-wavelength, set up salvia root polyphenol acid extract digitlization quantitative finger print atlas;
Step 2: utilize mass spectrum to point out the peak in finger-print in step 1, determine that ultraviolet detects total fingerprint peaks;
Step 3: three content index component content large, that activity is obvious and structure is clear and definite that adopts high performance liquid chromatography variable wavelength detection method to recognize step 2 middle finger is measured.
Further, the concrete steps of step 1 are as follows:
(1) preparation of reference substance solution: accurately weighed reference substance Sodium Danshensu, protocatechualdehyde, salvianolic acid D, salviandic acid A, danshinolic acid-LM, Rosmarinic acid, alkannic acid, tanshin polyphenolic acid B, adding ultra-pure water dissolves, be settled to Sodium Danshensu, protocatechualdehyde, salvianolic acid D, salviandic acid A, danshinolic acid-LM final concentration and be respectively 200-500 μ g/ml, Rosmarinic acid, alkannic acid final concentration are respectively 300-600 μ g/ml, and tanshin polyphenolic acid B final concentration is 500-1500 μ g/ml and get final product;
(2) preparation of need testing solution: accurately weighed 10-50 criticizes salvia root polyphenol acid extract respectively, and sample is dissolved in ultra-pure water, filter membrane after dissolving, is diluted to the solution that concentration is 1mg/mL-5mg/mL;
(3) mensuration of finger-print: the accurate need testing solution 1-5 μ L that draws, inject Ultra Performance Liquid Chromatography instrument, record chromatogram, to obtain final product;
Wherein, chromatographic condition is as follows:
Chromatographic column: UPLCC18 post or T3 post;
Mobile phase: solvent orange 2 A: water, containing 0.1%-0.5% formic acid; Solvent B: acetonitrile, containing 1%-5% methyl alcohol;
Gradient elution:
0-3min,12-18％B；3-15min,18％B；15-17min,18-20％B；17-25min,20％B；25-28min,20-23％B；28-35min,23％B；35-40min,23-40％B；40-45min,40-95％B；45-50min,95％B；
Temperature: 30-40 DEG C;
Flow velocity: 0.2-0.3ml/min;
Chromatogram spectra collection scope: 190～400nm.
Preferably, in step 1,
Chromatographic column is preferred: UPLCC18 post;
Mobile phase is preferred:
Solvent orange 2 A: water, containing 0.2% formic acid; Solvent B: acetonitrile, containing 3% methyl alcohol; Or
Solvent orange 2 A: water, containing 0.1% formic acid; Solvent B: acetonitrile, containing 2% methyl alcohol.
The chromatographic condition of UPLC of the present invention obtains through screening:
1, the selection of chromatographic column
The present invention has contrasted now widely used three kinds of different label chromatographic columns: (A) FortisC181.7μm，2.1x50mm；(B)AgilentEclipsePlusC18，1.8μm，2.1x150mm；(C)WatersHSST3C18, 1.7 μ m, 2.1x150mm. From the liquid phase contrast spectrogram of three kinds of pillars, can find out, composition to be analyzed appearance time in A type chromatographic column is more late, and separating effect is bad, considers that the cost of expanding production and the ultimate constituent points out the accuracy of result, gets rid of chromatographic column A; Then contrast the collection of illustrative plates of salvia root polyphenol acid sample in chromatographic column B and chromatographic column C, can obviously see that chromatographic column B is better compared with C separating effect, and chromatogram peak number in B figure is obviously more than C figure, is conducive to the comprehensive of composition Study. By experiment repeatedly, find that Eclipse is relatively good for the larger liposoluble ingredient separating effect of polarity, therefore EclipsePlusC18 chromatographic column has been selected in optimization of the present invention. Chromatogram is shown in Fig. 1.
2, the screening of gradient:
(1) adopt solvent orange 2 A: water, containing 0.1% formic acid, solvent B: acetonitrile, carry out system optimization, result shows that the chromatogram that polarity is larger is difficult to separate with contiguous impurity. Record as follows:
(A)0-3min:12-20％B,3-10min:20-22％B,10-22min:22％B,22-27min:22-35％B,27-29min:35％-50％B；
(B)0-3min:12-18％B,3-10min:18-22％B,10-12min:22％B,12-22min,22-25％B；22-27min:25-35％B,27-29min:35％-50％B；
(C)0-2min:12-18％B,2-5min:18-22％B,5-15min:22-25％B,15-20min:25-45％B,20-25min:45％-95％B.
Chromatogram is shown in Fig. 2.
(2) be solvent orange 2 A by Optimization of mobile phase: water, containing 0.2% formic acid, solvent B: acetonitrile, containing 2% methyl alcohol, improve obviously separating details, within 5-10 minute, separated. Further optimize, high polarity phenolic acid obtains separating (Fig. 3,13min) in 18% organic phase isocratic elution. Record as follows:
(A)0-3min:12-18％B,3-10min:18-20％B,10-20min:20％B；
(B)0-3min:12-17％B,3-20min:17％B,20-23min:17-20％B；
(C)0-3min:12-18％B,3-15min:18％B,15-17min:18-20％B,17-25min:20％B.
Chromatogram is shown in Fig. 3, Fig. 4 (Fig. 3 enlarged drawing).
3, methodological study
(1) precision test
Get need testing solution (lot number 20130202), continuous sample introduction 6 times, taking alkannic acid as reference, investigates total peak relative retention time and relative peak area respectively. The RSD of each total peak relative retention time is all less than 0.5%, and the RSD of relative peak area is all less than 3.0%, shows that instrument precision is good.
(2) repeated experiment
6 parts of sample thiefs (lot number 20130202), prepare need testing solution by method in above-mentioned steps 1, and detect, and respectively relative retention time and the relative peak area at total peak are investigated taking alkannic acid as reference. The RSD of each total peak relative retention time is all less than 0.5%, and the RSD of relative peak area is all less than 3.5%, shows that this method repeatability is good.
(3) stability test
Get need testing solution (lot number is 130301), analyze respectively at 0,2,4,8,12,24h sample introduction, taking alkannic acid as reference, respectively relative retention time and the relative peak area at total peak are investigated.
Result shows, the RSD of each total peak relative retention time is all less than 0.5%, and the RSD of relative peak area is all less than 3.0%. Show that need testing solution is stable in room temperature 24h.
Salvia root polyphenol acid extract sample UPLC chromatogram (280nm) is referring to Fig. 5.
Utilize the method for step 1 of the present invention to carry out UPLC fingerprint map analyzing to many batches of salvia root polyphenol acid extract samples, and 23 of each sample total peak ultraviolet spectras are analyzed, thereby uniformity and the stability of many batches of salvia root polyphenol acid extract samples are identified.
For the comparative analysis of different batches fingerprint similarity, can adopt the various software of selling on the market to carry out.
The present invention preferably adopts " chromatographic fingerprints of Chinese materia medica super information characteristics Digital evaluation system 4.0 " software to carry out the analysis of secondary quantitative finger print atlas to consequential signal, determine reference fingerprint, and as the similarity with reference to calculating each batch of finger-print, obtain the final finger-print quality results of many batch samples.
System fingerprint sizing technique is objective, the contribution of the each chemical substance of quantitative description to compound preparation chemical fingerprint exactly. Salvia root polyphenol acid extract UPLC fingerprint is regarded as to an entirety, chemical fingerprint quantity and distribution proportion by grand qualitative similarity (Sm) from whole monitoring salvia root polyphenol acid extract; By grand quantitative similarity
(Pm) monitoring salvia root polyphenol acid extract chemical fingerprint whole content situation; By the absolute value restriction salvia root polyphenol acid extract UPLC fingerprint meristic variation scope of sample fingerprint homogenization coefficient of alteration (α) relative deviation, wherein Sm, Pm and α press the calculating of formula method.
Table 1 system fingerprint sizing technique is divided traditional Chinese medicine quality classification standard
Adopt the system fingerprint sizing technique standard shown in table 1, with Sm, Pm and the common Identification chinese herbs medicine credit rating of α, by above-mentioned grade classification, and taking lower than quality V level as defective, uniformity and stability to many batch samples are identified, thereby quality are evaluated.
Further, the mass spectrum condition in step 2 is as follows:
Level Four bar-flight time mass spectrum (qTOF) adopts (-)-ESI ion gun, and atomization gas is high pure nitrogen, and collision gas is high-purity helium;
Sweep limits: m/z100-1200;
Collision energy: 6eV;
MSE gradient cracking energy: 20-40eV.
From salvia root polyphenol acid extract total ion current, figure can find out (Fig. 6), and it respectively becomes swarming under anion condition, all to have higher response, and corresponding with ultraviolet chromatogram.
The accurate relative molecular mass recording according to flight time mass spectrum, possible molecular composition (error < 5 × 100 is calculated in application high resolution mass spectrum data analysis (for example MassLynx mass spectral analysis software)-6), and coupled ion trap mass spectrometric each one-tenth second order ms fragment information of swarming and bibliographical information of red sage root known compound, chromatographic peak is analyzed, from test sample, identify 23 compounds that can confirm. See the following form.
Table 2 salvia root polyphenol acid extract chemical composition qualification list
Further, described in step 3, three kinds of index compositions are respectively Rosmarinic acid, alkannic acid, tanshin polyphenolic acid B.
The concrete steps that adopt high performance liquid chromatography variable wavelength detection method to measure three kinds of index composition Rosmarinic acids, alkannic acid, content of tanshin polyphenolic acid B are as follows:
(1) need testing solution preparation: get salvia root polyphenol acid extract, accurately weighed, adding initial flow phased soln and being settled to final concentration is 0.6-1.2mg/mL, shakes up, and to obtain final product;
(2) mix reference substance solution preparation: it is each appropriate that precision takes Rosmarinic acid, alkannic acid, tanshin polyphenolic acid B reference substance respectively, add initial flow and make mutually mixing reference substance solution, obtaining Rosmarinic acid mass concentration is 100-150 μ g/mL, alkannic acid mass concentration is 100-150 μ g/mL, the mixing reference substance solution that tanshin polyphenolic acid B mass concentration is 1.0-2.5mg/mL;
(3) the accurate need testing solution 1-5 μ L that draws, injects high performance liquid chromatograph, adopts external standard peak area method, measures the content of Rosmarinic acid in need testing solution, alkannic acid, tanshin polyphenolic acid B simultaneously;
Wherein, chromatographic condition is as follows:
Chromatographic column: HPLCC18 post;
Mobile phase: A phase: water, containing 0.3%～0.6% formic acid; B phase: acetonitrile, the methyl alcohol containing 2%～4% and 0.3%～0.6% formic acid;
Gradient elution: 0～40min, 20%B; 40～50min, 20-70%B;
Flow velocity: 0.9-1.1mL/min;
Column temperature: 30 DEG C-35 DEG C;
Detect wavelength: 288nm.
Wherein initial flow phase described in step (1), (2), refers to the mixed solution of 80%A mobile phase and 20%B mobile phase.
Preferably, in step 3,
Mobile phase is preferred:
A phase: water, containing 0.5% formic acid; B phase: acetonitrile, the methyl alcohol containing 2% and 0.5% formic acid; Or
A phase: water, containing 0.3% formic acid; B phase: acetonitrile, the methyl alcohol containing 3% and 0.3% formic acid;
The preferred 1mL/min of flow velocity;
Preferably 30 DEG C of column temperatures.
HPLC chromatographic condition of the present invention obtains through groping:
1, the selection of mobile phase:
In the time carrying out the groping of mobile phase, investigate the acetonitrile-water of different proportion, methanol-water, acetonitrile-trifluoroacetic acid water, acetonitrile-phosphoric acid water, acetonitrile-formic acid water and acetonitrile-methyl alcohol-trifluoroacetic acid water, acetonitrile-methyl alcohol-formic acid water, final discovery, with A phase: water (containing 0.3%～0.6% formic acid); B phase: when acetonitrile (methyl alcohol containing 2%～4% and 0.3%～0.6% formic acid) gradient elution, target peak separating effect is better, especially with A phase: water (containing 0.5% formic acid); B phase: acetonitrile (methyl alcohol containing 3% and 0.5% formic acid) separating effect is best, and main manifestations is that the micro substance of Rosmarinic acid and the right and left can reach baseline separation, and collection of illustrative plates as shown in Figure 7.
Under HPLC chromatographic condition of the present invention, the chromatographic peak of Rosmarinic acid in need testing solution, alkannic acid, tanshin polyphenolic acid B all can obtain good separation with other component peaks. As shown in Figure 8.
2, DAD detector gathers the selection of wavelength:
The absorption maximum of tanshin polyphenolic acid B is in 288nm left and right, and the absorption maximum of alkannic acid is at 250nm. Reference substance spectrogram under contrast 250,280,330,360nm, selects the absorbing wavelength of 280nm as typical fingerprint collection of illustrative plates, and 330nm is for the contrast of condition optimizing process separating degree. The different wavelength that detect are shown in Fig. 9 to the liquid chromatogram of salvia root polyphenol acid mixing reference substance separation.
3, linear relationship is investigated
(1) respectively precision measure mix reference substance solution 0.4,1,2,4,8,10ml puts in 10mL measuring bottle, is settled to mutually scale by initial flow, shakes up, and obtains series standard solution.
(2) sample introduction 10 μ L respectively, record peak area A, with peak area A to quality concentration C (μ gL-1) carry out linear regression, drawing standard curve.
Rosmarinic acid equation of linear regression A=23.787C-0.1082, R2=1.0000, the range of linearity 4.82～120.50 μ gL-1；
Alkannic acid equation of linear regression A=15.580C-1.8253, R2=1.0000, the range of linearity 4.84～121.10 μ gL-1；
Tanshin polyphenolic acid B equation of linear regression A=12.963C-5.4623, R2=1.0000, the range of linearity 60.80～1520.00 μ gL-1。
4, methodological study
(1) precision test
The accurate need testing solution of drawing is pressed above-mentioned HPLC chromatographic condition continuous sample introduction 6 times, sample size 10 μ L, the peak area of mensuration Rosmarinic acid, alkannic acid, tanshin polyphenolic acid B.
Record the RSD0.44% of Rosmarinic acid peak area; The RSD0.30% of alkannic acid peak area; The RSD0.29% of tanshin polyphenolic acid B peak area.
Result shows that the precision of instrument is good.
(2) replica test
Precision takes 6 parts, extract sample respectively, measures by given test sample preparation method and HPLC chromatographic condition, records peak area, calculates respectively the content of each mensuration composition.
Record the RSD0.86% of rosmarinic acid contents; The RSD0.70% of alkannic acid content; The RSD0.61% of content of danshinolic acid B.
Result shows that the precision of method is good.
(3) precision test in the middle of
Get with a collection of salvia root polyphenol acid, prepare need testing solution by given test sample preparation method, respectively by different personnel at same instrument, same personnel measure at different number of days sample introduction, calculate different personnel, different number of days and respectively measure the RSD of component content.
In three days, recorded the RSD0.34% of rosmarinic acid contents by same personnel; The RSD0.74% of alkannic acid content; The RSD0.37% of content of danshinolic acid B;
Recorded the RSD0.68% of rosmarinic acid contents at same instrument by three personnel; The RSD1.14% of alkannic acid content; The RSD0.86% of content of danshinolic acid B.
(4) stability test
Mixing reference substance and the need testing solution of getting new preparation, carry out chromatography at 0,3.5,7,10.5,14,18,24h by aforementioned chromatographic condition respectively.
Record the RSD1.14% that mixes Rosmarinic acid peak area in reference substance; The RSD1.09% of alkannic acid peak area; The RSD1.17% of tanshin polyphenolic acid B peak area.
The RSD0.52% of Rosmarinic acid peak area in test sample; The RSD0.70% of alkannic acid peak area; The RSD0.47% of tanshin polyphenolic acid B peak area.
Result show reference substance and sample all good at 24h internal stability.
(5) average recovery test
Prepare need testing solution by given test sample preparation method, precision measures 5ml in 10ml volumetric flask, it is appropriate that precision adds mixing reference substance solution respectively, be made into concentration and be 70%, 100%, 130% solution, 3 parts of each concentration, totally 9 parts, then with initial flow phase constant volume, as the need testing solution of basic, normal, high three concentration, calculate recovery rate and RSD value.
The average recovery rate of result Rosmarinic acid is 100.88%, RSD0.80%; The average recovery rate of alkannic acid is 103.03%, RSD0.99%; The average recovery rate of tanshin polyphenolic acid B is 101.92%, RSD1.16%. In Table 4-6.
Table 3. Rosmarinic acid average recovery
Table 4. alkannic acid average recovery
Table 5. alkannic acid average recovery
Salvia root polyphenol acid extract of the present invention, can be prepared according to arbitrary appropriate method of the prior art, can be also the preparation of arbitrary salvia root polyphenol acid.
Preferably, salvia root polyphenol acid method for preparing extractive of the present invention is: get salvia piece, by purifying water boiling and extraction three times, each 0.5～1 hour, No. three extracts are merged, cooling, be adjusted to acidity with hydrochloric acid solution, place 4～8 hours, filter, obtain clear and bright liquid, liquid is purified by polyamide column chromatography technique, after loading, rinse by purified water, carry out wash-out with sodium bicarbonate solution, collect eluent, eluent is adjusted to acidity with hydrochloric acid solution, adopt macroporous resin column chromatography technique purifying liquid, after loading, rinse by purified water, carry out wash-out with ethanolic solution, collect eluent, eluent reduced pressure concentration is reclaimed to ethanol, the refrigeration of gained concentrate was filtered after 12～24 hours, filtrate regulates pH value to 5.3～6.0 with sodium hydroxide solution, freeze drying, obtain.
Salvia root polyphenol acid extract also can be injection, and its preparation method is: getting weight ratio is salvia root polyphenol acid extract and the sweet mellow wine of 1:0.1～0.5, adds respectively the water for injection of 10～20 times of w/vs to dissolve; After being mixed, above-mentioned two kinds of solution add medical charcoal, add thermal agitation, filter, after liquid cooling, carry out ultrafiltration, filtrate is regulated to pH value to 5.5～6.0, inject water constant volume, stir, by filling to cillin bottle after above-mentioned liquid filtering with microporous membrane, freeze drying, obtains Danshen injection Polyphenol Acids.
At present, for example, in the quality standard of salvia root polyphenol acid extract, preparation (injection), study very imperfection for finger-print, consider particularity and the clinical safety of Chinese medicine preparation, it is incomplete characterizing the quality of the pharmaceutical preparations with the content of several compositions merely. This sets up UPLC chromatographic fingerprinting method, carrying out peak in conjunction with liquid matter points out, determine that 23 ultraviolets detect total fingerprint peaks, globality and the complexity of Chinese medicine preparation are fully characterized, consider simultaneously and in preparation, contain the weak composition of UV absorption, on detector is selected, selected 288 and 330 two kind of ultraviolet detect wavelength. In data processing, the present invention selects the auspicious professor of Liao Sun state to wait " chromatographic fingerprints of Chinese materia medica super information characteristics Digital evaluation system 4.0 " software of exploitation, compared with the finger-print software that this software is issued with Chinese pharmacopoeia committee, with the obvious advantage, can reflect uniformity and stability between preparation batch. This research has certain directive significance for salvia root polyphenol acid extract, the basic research of preparation medicine effective substance, quality control and clinical application thereof.
By UPLC Qualitative fingerprint+specific onset composition, (Rosmarinic acid, alkannic acid, tanshin polyphenolic acid B are three larger compositions of content in principal component in the present invention, and structure is clear and definite, wherein tanshin polyphenolic acid B is main active in Danshen injection Polyphenol Acids, there is anti-platelet aggregation, antithrombotic, anti-brain foundation of microsomal Lipid Peroxidation and the effect of removing free radical) quantitative mode, realizes the lifting of quality control standard jointly from overall and key component two aspects. Than prior art, not only comprehensive and specific quantitative quality controllability of having determined product, and due to the introducing of UPLC technology, greatly shortened the existing minute that generally adopts HPLC technology, the complexity of full product fingerprint collection of illustrative plates is characterized and be achieved. Meanwhile, select for the concrete specific composition of accusing of the combine measured mode combining with HPLC, also, fully ensureing product quality, outside qualitative properties of product, taken into account the saving of cost of determination. Testing result has good reappearance and stability, and is beneficial to industrial sector popularization, is suitable as product quality control standard and implements on a large scale.
Brief description of the drawings
The liquid chromatogram that the different chromatographic columns of Fig. 1 separate salvia root polyphenol acid extract
The liquid chromatogram of the different gradient of Fig. 2 to salvia root polyphenol acid extract sample separation (detects wavelength 330nm; Caption is ACN ratio)
The liquid chromatogram of the different gradient of Fig. 3 to salvia root polyphenol acid extract sample separation (detects wavelength 330nm; Caption is organic phase ratio)
The liquid chromatogram (Fig. 3 local amplifies, detect wavelength 330nm) of the different gradient of Fig. 4 to salvia root polyphenol acid extract sample separation
Fig. 5 salvia root polyphenol acid extract sample UPLC chromatogram (280nm)
Fig. 6 salvia root polyphenol acid extract sample qTOF-MS total ion current figure
Separate colors spectrogram (288nm) to salvia root polyphenol acid extract under the final chromatographic condition of Fig. 7
Fig. 8 reference substance and salvia root polyphenol acid sample HPLC chromatogram (1. Rosmarinic acid; 2. alkannic acid; 3. tanshin polyphenolic acid B)
Fig. 9 is different, and the wavelength that detects (is (A) 250nm to the liquid chromatogram of salvia root polyphenol acid mixing reference substance separation from top to bottom, successively; (B) 280nm; (C) 330nm; (D) 360nm.)
The UPLC/MS uv atlas overlay chart (280nm) of 1 batch of salvia root polyphenol acid extract of Figure 102
Figure 113 criticizes salvia root polyphenol acid extractive content and measures HPLC stacking chart
Detailed description of the invention
Embodiment 1
Step 1: adopt the visible uv detection method of ultra high efficiency liquid phase multi-wavelength, set up salvia root polyphenol acid extract digitlization quantitative finger print atlas;
(1) instrument and reagent: WatersUPLCsystem, series connection XevoG2qTOF mass spectrograph. Salvia root polyphenol acid extract is provided by sky Shi Lizhijiao pharmaceutcal corporation, Ltd. Acetonitrile and methyl alcohol are that chromatographically pure (Merck & Co., Inc.), formic acid are chromatographically pure (sigma), ultra-pure water. Reference substance Sodium Danshensu, protocatechualdehyde, tanshin polyphenolic acid B, Rosmarinic acid be all purchased from National Institute for Food and Drugs Control, alkannic acid (Tianjin mark's biotechnology), salvianolic acid D (Tianjin Yi Fang Science and Technology Ltd.), salviandic acid A (Nantong flies space), danshinolic acid-LM (preparation of Tian Shi power company).
(2) preparation of reference substance solution:
Take respectively the about 5mg of tanshin polyphenolic acid B reference substance; Rosmarinic acid, the each about 3mg of alkannic acid reference substance; Sodium Danshensu, protocatechualdehyde, salvianolic acid D, salviandic acid A, the each about 2mg of danshinolic acid-LM reference substance, in 5mL volumetric flask, with ultra-pure water constant volume, shake up, and (0.22 μ m), gets subsequent filtrate, to obtain final product to cross miillpore filter.
(3) preparation of need testing solution: accurately weighed 21 batches of salvia root polyphenol acid extracts respectively, sample is dissolved in ultra-pure water, is dissolved in 10ml volumetric flask, after dissolving, crosses 0.22 μ m filter membrane, is diluted to the solution that concentration is 1mg/mL;
(4) mensuration of finger-print: the accurate need testing solution 2 μ L that draw, inject Ultra Performance Liquid Chromatography instrument, record chromatogram, to obtain final product;
Wherein, chromatographic condition is as follows:
Chromatographic column: AgilentEclipsePlusC18,1.8 μ m, 2.1x150mm;
Mobile phase: solvent orange 2 A: water, containing 0.2% formic acid; Solvent B: acetonitrile, containing 3% methyl alcohol;
Gradient elution:
0-3min,12-18％B；3-15min,18％B；15-17min,18-20％B；17-25min,20％B；25-28min,20-23％B；28-35min,23％B；35-40min,23-40％B；40-45min,40-95％B；45-50min,95％B；
Temperature: 30 DEG C;
Flow velocity: 0.2ml/min;
Chromatogram spectra collection scope: 190～400nm.
Step 2: utilize mass spectrum to point out the peak in finger-print in step 1, determine that ultraviolet detects total fingerprint peaks;
Mass spectrum condition is as follows:
QTOF adopts (-)-ESI ion gun, and atomization gas is high pure nitrogen, and collision gas is high-purity helium;
Sweep limits: m/z100-1200;
Collision energy: 6eV;
MSE gradient cracking energy: 20-40eV;
21 batches of salvia root polyphenol acid extract samples are carried out to UPLC fingerprint map analyzing with above-mentioned chromatographic condition. 23 of each sample total peak ultraviolet spectrograms are analyzed. Gained uv atlas as shown in figure 10.
Utilize " chromatographic fingerprints of Chinese materia medica super information characteristics Digital evaluation system 4.0 " software to calculate the similarity of each batch of finger-print.
Adopt the system fingerprint sizing technique standard shown in table 1,21 batch samples are carried out to quality evaluation. The results are shown in Table 6:
Table 6. calculates the 2nd grade of evaluation result of Chinese medicine system fingerprint sizing technique
| Para. | Sm | P2％ | α | Grade | Quality |
| S1 | 0.966 | 63.8 | 0.015 | Ⅵ | Generally |
| S2 | 0.443 | 2.1 | 1.243 | Ⅷ | Bad |
| S3 | 0.957 | 45.3 | 0.038 | Ⅷ | Bad |
| S4 | 0.969 | 70 | 0.031 | Ⅵ | Generally |
| S5 | 0.965 | 62.2 | 0.039 | Ⅵ | Generally |
| S6 | 0.987 | 65 | 0.012 | Ⅵ | Generally |
| S7 | 0.982 | 64 | 0.035 | Ⅵ | Generally |
| S8 | 0.955 | 98.7 | 0.009 | Ⅰ | Fabulous |
| S9 | 0.944 | 96.4 | 0.015 | Ⅱ | Fine |
| S10 | 0.986 | 100.6 | 0.021 | Ⅰ | Fabulous |
| S11 | 0.992 | 104.3 | 0.007 | Ⅰ | Fabulous |
| S12 | 0.994 | 107.9 | 0.011 | Ⅱ | Fine |
| S13 | 0.992 | 109.3 | 0.019 | Ⅱ | Fine |
| S14 | 0.969 | 106.9 | 0.019 | Ⅱ | Fine |
| S15 | 0.991 | 103.7 | 0.006 | Ⅰ | Fabulous |
[0175]
| S16 | 0.992 | 106.3 | 0 | Ⅱ | Fine |
| S17 | 0.986 | 104.3 | 0.015 | Ⅰ | Fabulous |
| S18 | 0.994 | 94.3 | 0.012 | Ⅱ | Fine |
| S19 | 0.988 | 106.9 | 0.001 | Ⅱ | Fine |
| S20 | 0.973 | 94.3 | 0.031 | Ⅱ | Fine |
| S21 | 0.979 | 94.8 | 0.005 | Ⅱ | Fine |
Interpretation of result: S1-S7 batch off quality, match for production batch expired product early with these seven batches, show that the method can identify drug quality preferably, well distinguish mass discrepancy larger, production batch expired product early, the quality can be used for producing medicine is tested.
Step 3: three content index component content large, that activity is obvious and structure is clear and definite that adopts high performance liquid chromatography variable wavelength detection method to recognize step 2 middle finger is measured.
(1) instrument and reagent: Agilent1100 high performance liquid chromatograph (AgilentTechnologies, Germany), be furnished with quaternary pump, automatic sampler, column oven and VWD detector; METTLERTOLEDO electronic balance (XS type); 5 batches of salvia root polyphenol acids (Tianjin TianShiLi ZhiJiao Medicine Co., Ltd provides); Tanshin polyphenolic acid B reference substance (USP, purity 95%, lot number: F0M013); Rosmarinic acid reference substance (USP, purity 99.4%, lot number: F0M076); (sky, Tianjin scholar's power Chinese medicine provides alkannic acid reference substance, purity 95.37%, lot number: 12092801); Methyl alcohol (chromatographically pure, Merck); Acetonitrile (chromatographically pure, Merck); Ultra-pure water; Formic acid (SILVER REAGENT, Sigma-Aldrich).
(2) preparation of need testing solution: precision takes 3 crowdes of each 15mg of salvia root polyphenol acid extract, adds initial flow phased soln constant volume to 25mL, shakes up, and to obtain final product.
(3) mix the preparation of reference substance solution: it is each appropriate that precision takes Rosmarinic acid, alkannic acid, tanshin polyphenolic acid B reference substance respectively, add initial flow and make mutually mixing reference substance solution, obtaining Rosmarinic acid mass concentration is 120.50 μ g/mL, alkannic acid mass concentration is 121.10 μ g/mL, and tanshin polyphenolic acid B mass concentration is the mixing reference substance solution of 1520.00 μ g/mL.
(4) the accurate need testing solution 2 μ L that draw, inject high performance liquid chromatograph, adopt external standard peak area method, measure the content of Rosmarinic acid in need testing solution, alkannic acid, tanshin polyphenolic acid B simultaneously;
Wherein, chromatographic condition is as follows:
Chromatographic column: AgilentZORBAXEclipsePlusC18 (4.6 × 250mm, 5 μ are m);
Mobile phase: A phase: water, containing 0.5% formic acid; B phase: the methyl alcohol containing 2% and 0.5% formic acid;
Gradient elution: 0～40min, 20%B; 40～50min, 20-70%B;
Flow velocity: 1mLmin-1;
Column temperature: 30 DEG C;
Detect wavelength 288nm;
Measurement result is in table 7, and HPLC stacking chart sees Figure 11.
Each component content in table 7.3 batch salvia root polyphenol acid extract
Embodiment 2
Step 1: adopt the visible uv detection method of ultra high efficiency liquid phase multi-wavelength, set up salvia root polyphenol acid extract digitlization quantitative finger print atlas;
(1) preparation of reference substance solution:
Take respectively the about 2.5mg of tanshin polyphenolic acid B reference substance; Rosmarinic acid, the each about 1.5mg of alkannic acid reference substance; Sodium Danshensu, protocatechualdehyde, salvianolic acid D, salviandic acid A, the each about 1mg of danshinolic acid-LM reference substance, in 5mL volumetric flask, with ultra-pure water constant volume, shake up, and (0.22 μ m), gets subsequent filtrate, to obtain final product to cross miillpore filter.
(2) preparation of need testing solution: accurately weighed 10 batches of salvia root polyphenol acid extracts respectively, sample is dissolved in ultra-pure water, is dissolved in 10ml volumetric flask, after dissolving, crosses 0.22 μ m filter membrane, is diluted to the solution that concentration is 5mg/mL;
(3) mensuration of finger-print: the accurate need testing solution 1 μ L that draws, inject Ultra Performance Liquid Chromatography instrument, record chromatogram, to obtain final product;
Wherein, chromatographic condition is as follows:
Chromatographic column: WatersHSST3C18，1.7μm，2.1x150mm；
Mobile phase: solvent orange 2 A: water, containing 0.1% formic acid; Solvent B: acetonitrile, containing 2% methyl alcohol;
Gradient elution:
0-3min,12-18％B；3-15min,18％B；15-17min,18-20％B；17-25min,20％B；25-28min,20-23％B；28-35min,23％B；35-40min,23-40％B；40-45min,40-95％B；45-50min,95％B；
Temperature: 40 DEG C;
Flow velocity: 0.3ml/min;
Chromatogram spectra collection scope: 190～400nm.
Step 2: utilize mass spectrum to point out the peak in finger-print in step 1, determine that ultraviolet detects total fingerprint peaks;
Mass spectrum condition is as follows:
QTOF adopts (-)-ESI ion gun, and atomization gas is high pure nitrogen, and collision gas is high-purity helium;
Sweep limits: m/z100-1200;
Collision energy: 6eV;
MSE gradient cracking energy: 20-40eV;
10 batches of salvia root polyphenol acid extract samples are carried out to UPLC fingerprint map analyzing with above-mentioned chromatographic condition. 23 of each sample total peak ultraviolet spectrograms are analyzed.
Utilize " chromatographic fingerprints of Chinese materia medica super information characteristics Digital evaluation system 4.0 " software to calculate the similarity of each batch of finger-print.
Adopt the system fingerprint sizing technique standard shown in table 1,10 batch samples are carried out to quality evaluation.
Step 3: three content index component content large, that activity is obvious and structure is clear and definite that adopts high performance liquid chromatography variable wavelength detection method to recognize step 2 middle finger is measured.
(1) preparation of need testing solution: precision takes salvia root polyphenol acid extract, adds initial flow phased soln constant volume to 1.2mg/mL, shakes up, and to obtain final product.
(2) mix the preparation of reference substance solution: it is each appropriate that precision takes Rosmarinic acid, alkannic acid, tanshin polyphenolic acid B reference substance respectively, add initial flow and make mutually mixing reference substance solution, obtaining Rosmarinic acid mass concentration is 100 μ g/ml, alkannic acid mass concentration is 150 μ g/ml, the mixing reference substance solution that tanshin polyphenolic acid B mass concentration is 1.0mg/ml.
(4) the accurate need testing solution 5 μ L that draw, inject high performance liquid chromatograph, adopt external standard peak area method, measure the content of Rosmarinic acid in need testing solution, alkannic acid, tanshin polyphenolic acid B simultaneously;
Wherein, chromatographic condition is as follows:
Chromatographic column: AgilentZORBAXEclipsePlusC18 (4.6 × 250mm, 5 μ are m);
Mobile phase: A phase: water, containing 0.3% formic acid; B phase: the methyl alcohol containing 3% and 0.3% formic acid;
Gradient elution: 0～40min, 20%B; 40～50min, 20-70%B;
Flow velocity: 0.9mL/min;
Column temperature: 35 DEG C;
Detect wavelength 288nm;
Embodiment 3
Step 1: adopt the visible uv detection method of ultra high efficiency liquid phase multi-wavelength, set up salvia root polyphenol acid extract digitlization quantitative finger print atlas;
(1) preparation of reference substance solution:
Take respectively the about 7.5mg of tanshin polyphenolic acid B reference substance; Rosmarinic acid, the each about 2mg of alkannic acid reference substance; Sodium Danshensu, protocatechualdehyde, salvianolic acid D, salviandic acid A, the each about 2.5mg of danshinolic acid-LM reference substance, in 5mL volumetric flask, with ultra-pure water constant volume, shake up, and (0.22 μ m), gets subsequent filtrate, to obtain final product to cross miillpore filter.
(2) preparation of need testing solution: accurately weighed 50 batches of salvia root polyphenol acid extracts respectively, sample is dissolved in ultra-pure water, is dissolved in 10ml volumetric flask, after dissolving, crosses 0.22 μ m filter membrane, is diluted to the solution that concentration is 3mg/mL;
(3) mensuration of finger-print: the accurate need testing solution 5 μ L that draw, inject Ultra Performance Liquid Chromatography instrument, record chromatogram, to obtain final product;
Wherein, chromatographic condition is as follows:
Chromatographic column: AgilentEclipsePlusC18,1.8 μ m, 2.1x150mm;
Mobile phase: solvent orange 2 A: water, containing 0.5% formic acid; Solvent B: acetonitrile, containing 5% methyl alcohol;
Gradient elution:
0-3min,12-18％B；3-15min,18％B；15-17min,18-20％B；17-25min,20％B；25-28min,20-23％B；28-35min,23％B；35-40min,23-40％B；40-45min,40-95％B；45-50min,95％B；
Temperature: 35 DEG C;
Flow velocity: 0.2ml/min;
Chromatogram spectra collection scope: 190～400nm.
Step 2: utilize mass spectrum to point out the peak in finger-print in step 1, determine that ultraviolet detects total fingerprint peaks;
Mass spectrum condition is as follows:
QTOF adopts (-)-ESI ion gun, and atomization gas is high pure nitrogen, and collision gas is high-purity helium;
Sweep limits: m/z100-1200;
Collision energy: 6eV;
MSE gradient cracking energy: 20-40eV;
50 batches of salvia root polyphenol acid extract samples are carried out to UPLC fingerprint map analyzing with above-mentioned chromatographic condition. 23 of each sample total peak ultraviolet spectrograms are analyzed.
Utilize " chromatographic fingerprints of Chinese materia medica super information characteristics Digital evaluation system 4.0 " software to calculate the similarity of each batch of finger-print.
Adopt the system fingerprint sizing technique standard shown in table 1,50 batch samples are carried out to quality evaluation.
Step 3: three content index component content large, that activity is obvious and structure is clear and definite that adopts high performance liquid chromatography variable wavelength detection method to recognize step 2 middle finger is measured.
(1) preparation of need testing solution: precision takes salvia root polyphenol acid extract, adds initial flow phased soln constant volume to 1.0mg/mL, shakes up, and to obtain final product.
(2) mix the preparation of reference substance solution: it is each appropriate that precision takes Rosmarinic acid, alkannic acid, tanshin polyphenolic acid B reference substance respectively, add initial flow and make mutually mixing reference substance solution, obtaining Rosmarinic acid mass concentration is 150 μ g/ml, alkannic acid mass concentration is 100 μ g/ml, the mixing reference substance solution that tanshin polyphenolic acid B mass concentration is 2.5mg/ml.
(4) the accurate need testing solution 1 μ L that draws, injects high performance liquid chromatograph, adopts external standard peak area method, measures the content of Rosmarinic acid in need testing solution, alkannic acid, tanshin polyphenolic acid B simultaneously;
Wherein, chromatographic condition is as follows:
Chromatographic column: AgilentZORBAXEclipsePlusC18 (4.6 × 250mm, 5 μ are m);
Mobile phase: A phase: water, containing 0.6% formic acid; B phase: the methyl alcohol containing 4% and 0.6% formic acid;
Gradient elution: 0～40min, 20%B; 40～50min, 20-70%B;
Flow velocity: 1.1mL/min;
Column temperature: 30 DEG C;
Detect wavelength 288nm.
Claims (10)
1. salvia root polyphenol acid extract finger-print and a content assaying method, is characterized in that, step is as follows:
Step 1: adopt the visible uv detection method of ultra high efficiency liquid phase multi-wavelength, set up salvia root polyphenol acid extract numberWord quantitative finger print atlas;
Step 2: utilize mass spectrum to point out the peak in finger-print in step 1, determine that ultraviolet detects totalFingerprint peaks;
Step 3: three content that employing high performance liquid chromatography variable wavelength detection method is recognized step 2 middle fingerGreatly, activity index component content obvious and that structure is clear and definite is measured.
2. the method for claim 1, is characterized in that, the concrete steps of step 1 are as follows:
(1) preparation of reference substance solution: accurately weighed reference substance Sodium Danshensu, protocatechualdehyde, salvianolic acid D,Salviandic acid A, danshinolic acid-LM, Rosmarinic acid, alkannic acid, tanshin polyphenolic acid B, add ultra-pure water and dissolve, and is settled toSodium Danshensu, protocatechualdehyde, salvianolic acid D, salviandic acid A, danshinolic acid-LM final concentration are respectively200-500 μ g/ml, Rosmarinic acid, alkannic acid final concentration are respectively 300-600 μ g/mL, and tanshin polyphenolic acid B is dense eventuallyDegree, for 500-1500 μ g/mL, to obtain final product;
(2) preparation of need testing solution: accurately weighed 10-50 criticizes salvia root polyphenol acid extract respectively, and sample is moltenIn ultra-pure water, after dissolving, filter, be diluted to the solution that concentration is 1mg/mL-5mg/mL;
(3) mensuration of finger-print: the accurate need testing solution 1-5 μ L that draws, inject Ultra Performance Liquid Chromatography instrument,Record chromatogram, to obtain final product;
Wherein, chromatographic condition is as follows:
Chromatographic column: UPLCC18 post or T3 post;
Mobile phase: solvent orange 2 A: water, containing 0.1%-0.5% formic acid; Solvent B: acetonitrile, containing 1%-5% methyl alcohol;
Gradient elution:
0-3min,12-18％B；3-15min,18％B；15-17min,18-20％B；17-25min,20％B；25-28min,20-23％B；28-35min,23％B；35-40min,23-40％B；40-45min,40-95％B；45-50min,95％B；
Temperature: 30-40 DEG C;
Flow velocity: 0.2-0.3ml/min;
Chromatogram spectra collection scope: 190～400nm.
3. method as claimed in claim 2, is characterized in that, in step 1,
Chromatographic column is preferred: UPLCC18 post;
Mobile phase is preferred:
Solvent orange 2 A: water, containing 0.2% formic acid; Solvent B: acetonitrile, containing 3% methyl alcohol; Or
Solvent orange 2 A: water, containing 0.1% formic acid; Solvent B: acetonitrile, containing 2% methyl alcohol.
4. the method for claim 1, is characterized in that, the mass spectrum condition in step 2 is as follows:
Level Four bar-flight time mass spectrum adopts (-)-ESI ion gun, and atomization gas is high pure nitrogen, and collision gas is highPure helium;
Sweep limits: m/z100-1200;
Collision energy: 6eV;
MSE gradient cracking energy: 20-40eV.
5. the method for claim 1, is characterized in that, three kinds of index compositions described in step 3 respectivelyFor Rosmarinic acid, alkannic acid, tanshin polyphenolic acid B.
6. the method for claim 1, is characterized in that, the concrete steps of step 3 are as follows:
(1) need testing solution preparation: get salvia root polyphenol acid extract, accurately weighed, add initial flow and mixSeparating and be settled to final concentration is 0.6-1.2mg/mL, shakes up, and to obtain final product;
(2) mix reference substance solution preparation: precision takes Rosmarinic acid, alkannic acid, tanshin polyphenolic acid B pair respectivelyEach appropriate according to product, add initial flow and make mutually mixing reference substance solution, obtain Rosmarinic acid mass concentration and be100-150 μ g/mL, alkannic acid mass concentration is 100-150 μ g/mL, tanshin polyphenolic acid B mass concentration is1.0-2.5mg/mL mixing reference substance solution;
(3) the accurate need testing solution 1-5 μ L that draws, injects high performance liquid chromatograph, adopts external standard peak areaMethod is measured the content of Rosmarinic acid in need testing solution, alkannic acid, tanshin polyphenolic acid B simultaneously;
Wherein, chromatographic condition is as follows:
Chromatographic column: HPLCC18 post;
Mobile phase: A phase: water, containing 0.3%～0.6% formic acid; B phase: acetonitrile, containing 2%～4% methyl alcoholWith 0.3%～0.6% formic acid;
Gradient elution: 0～40min, 20%B; 40～50min, 20-70%B;
Flow velocity: 0.9-1.1mL/min;
Column temperature: 30 DEG C-35 DEG C;
Detect wavelength: 288nm;
Wherein, initial flow phase described in step (1), (2), refers to that 80%A mobile phase and 20%B flowThe mixed solution of phase.
7. method as claimed in claim 6, is characterized in that, in step 3,
Mobile phase is preferred:
A phase: water, containing 0.5% formic acid; B phase: acetonitrile, the methyl alcohol containing 2% and 0.5% formic acid; Or
A phase: water, containing 0.3% formic acid; B phase: acetonitrile, the methyl alcohol containing 3% and 0.3% formic acid;
The preferred 1mL/min of flow velocity;
Preferably 30 DEG C of column temperatures.
8. the method as described in as arbitrary in claim 1～7, wherein salvia root polyphenol acid extract, can be according to prior artIn arbitrary appropriate method be prepared, also can be prepared into arbitrary preparation.
9. method as claimed in claim 8, wherein salvia root polyphenol acid method for preparing extractive is: get salvia piece,By purifying water boiling and extraction three times, each 0.5～1 hour, No. three extracts are merged, cooling, acid-soluble with saltLiquid is adjusted to acidity, places 4～8 hours, filters, and obtains clear and bright liquid, by liquid polyamide column chromatography techniquePurifying, rinses by purified water after loading, carries out wash-out with sodium bicarbonate solution, collects eluent, by eluentBe adjusted to acidity with hydrochloric acid solution, adopt macroporous resin column chromatography technique purifying liquid, after loading, rush by purified waterWash, carry out wash-out with ethanolic solution, collect eluent, eluent reduced pressure concentration is reclaimed to ethanol, gained is concentratedLiquid cooling is hidden after 12～24 hours and is filtered, and filtrate regulates pH value to 5.3～6.0 with sodium hydroxide solution, freeze drying,Obtain.
10. method as claimed in claim 8, wherein salvia root polyphenol acid extract is injection, its preparation method is:Getting weight ratio is salvia root polyphenol acid extract and the sweet mellow wine of 1:0.1～0.5, adds respectively 10～20 times of weighing bodiesThe water for injection of long-pending ratio dissolves; After above-mentioned two kinds of solution are mixed, add medical charcoal, add thermal agitation, filter,After liquid cooling, carry out ultrafiltration, filtrate regulated to pH value to 5.5～6.0, inject water constant volume, stir,By filling to cillin bottle after above-mentioned liquid filtering with microporous membrane, freeze drying, obtains Danshen injection Polyphenol Acids.
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| CN110274962A (en) * | 2018-03-13 | 2019-09-24 | 天士力医药集团股份有限公司 | Salvia root polyphenol acid component content measuring method in a kind of QISHEN YIQI DIWAN |
| CN108982703A (en) * | 2018-08-23 | 2018-12-11 | 中国检验检疫科学研究院 | A kind of LC-MS detection method of polyphenols |
| CN108982703B (en) * | 2018-08-23 | 2021-05-14 | 中国检验检疫科学研究院 | Liquid chromatography-mass spectrometry detection method for polyphenol substances |
| CN115144485A (en) * | 2022-04-02 | 2022-10-04 | 广东省中医院（广州中医药大学第二附属医院、广州中医药大学第二临床医学院、广东省中医药科学院） | Characteristic fingerprint spectrum of traditional Chinese medicine composition for treating chronic recurrent eczema and content determination and quality control method |
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