CN107490594A - A kind of hydrogen nuclear magnetic resonance detection method of composite salvia dropping extract of bolus - Google Patents
A kind of hydrogen nuclear magnetic resonance detection method of composite salvia dropping extract of bolus Download PDFInfo
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Abstract
The present invention provides a kind of quantitative analysis method of main component in composite salvia dropping extract of bolus based on hydrogen nuclear magnetic resonance technology, the analysis method can identify 20 compounds in extract, wherein 6 kinds of phenolic acid compound, 3 kinds of saponins, 4 kinds of carbohydrate, 3 kinds of amino acids, 4 kinds of organic acid, meanwhile ginsenoside Rg1 therein, ginsenoside Rb1, danshensu, tanshin polyphenolic acid B, the content of 6 kinds of main components of Rosmarinic acid and protocatechualdehyde can be determined.
Description
Technical field
Main component quantifies in a kind of composite salvia dropping extract of bolus based on hydrogen nuclear magnetic resonance technology of present invention offer
Analysis method, the analysis method can identify 20 compounds in extract, wherein 6 kinds of phenolic acid compound, saponin(e
3 kinds of class, 4 kinds of carbohydrate, 3 kinds of amino acids, 4 kinds of organic acid, meanwhile, ginsenoside Rg1 therein, ginsenoside can be determined
Rb1, danshensu, tanshin polyphenolic acid B, the content of 6 kinds of main components of Rosmarinic acid and protocatechualdehyde, can be realized to compound danshen dripping pills
Comprehensive, the quick quality evaluation of extract.
Background technology
Establish strict quality standards in Chinese drugs and create modern Chinese herbal medicine industrial production Quality Control Technology, it is ensured that tcm product
Safely, effectively and stably, be the advanced subject in modernization of cmm field, and the great section that China's medical industry faces
Skill innovates task.Therefore, it is total quality shape that is more comprehensive, quickly and accurately reflecting compound danshen dripping pills in production process
Condition, it is necessary to further upgrading is done to existing standard using nuclear magnetic resonance (NMR) technology, to improve in tcm product production
Quality control level, it is ensured that the stability of product quality.Nuclear magnetic resonance, refer to the atomic nucleus that nuclear magnetic moment is not zero, in external magnetic field
In the presence of, Zeeman splitting, the physical process of the radio-frequency radiation of a certain specific frequency of RESONANCE ABSORPTION occur for nuclear spin energy level.Mesh
Before, Japanese Pharmacopoeia has included the quality evaluation that NMR analysis methods are used for Chinese medicine, Chinese prescription pharmaceutical preparation.
More comprehensively, quickly and accurately to reflect the total quality shape of compound Salviae Miltiorrhizae extract (medicinal extract) in production process
Condition, the present invention are established for the non-of compound danshen dripping pills quality of production control using quantitative proton magnetic (qHNMR) technology first
Chromatographic quantitative analysis method, " macro qualitative analysis " of product intermediate can be both carried out, can also be to wherein main component-danshensu, original
Catechu aldehyde and tanshin polyphenolic acid B carry out accurate quantitative analysis, to improve the quality control level in production, it is ensured that product matter
The stability of amount.
Quantitative hydrogen nuclear magnetic resonance method (quantitative 1H nuclear magnetic resonance, qHNMR), i.e.,
A kind of method of quantitative analysis is carried out to compound using hydrogen nuclear magnetic resonance technology.
Compound danshen dripping pills is made up of the red sage root, pseudo-ginseng, the taste Chinese medicine of borneol 3, on sale in the market, commercially available compound Danshen Root
The main contents of dripping pill specification are as follows：
【Major function】It is promoting blood circulation and removing blood stasis, regulating qi-flowing for relieving pain.The obstruction of qi in the chest symptoms include uncomfortable in chest, pareordia thorn for caused by energy stagnation and blood stasis
Pain coronary disease and angina pectoris is shown in above-mentioned patient.
【Main component】Compound danshen dripping pills main component：The red sage root, pseudo-ginseng, borneol.
【Packing specification】Film-coating dripping pill weight 27mg, 180 balls/bottle per ball.
【Usage and dosage】Oral or sublingual administration, 10 balls, 3 times a day, 4 weeks are a course for the treatment of；Or follow the doctor's advice.
Also disclose that its preparation method such as in relevant document：
Formula：The red sage root 82.87%, pseudo-ginseng 16.21%, borneol 0.92%.
Preparation method：
(1) the red sage root, pseudo-ginseng are extracted 2 times with the boiling boiling of 8 times of weight, 2 hours for the first time, second 1 hour, mixing extraction
Liquid.
(2) extract solution filtering and concentrating.
(3) with extract and borneol together as active constituents of medicine, matrix is polyethylene glycol, and rate of charge is：Extract medicine
Thing：Matrix=1：5.5, borneol is added, atoleine is condensing agent, 90 DEG C of medicine temperature, 1.5 hours material time, condensate liquid
1 DEG C of temperature, drip and be prepared into dripping pill away from 7cm, drop speed 35 drop per minute.
Compound danshen dripping pills needs to prepare the red sage root, the extract of pseudo-ginseng, in order to carry the red sage root, pseudo-ginseng in preparation process
Thing is taken to carry out quality control, it is necessary to find effectively, quickly, accurate authentication method, the present invention is studied this, and is found
A kind of method detects to it, methods described can reach to greatest extent above-mentioned requirements for control drug quality provide according to
According to.
The content of the invention
The present invention provides a kind of hydrogen nuclear magnetic resonance detection method of Chinese medicine compound prescription Danshen Root dropping ball extract, and methods described includes
Following steps：
Step 1, the preparation of working solution
Internal standard compound 3- (trimethyl silyl) malonic acid-d4 sodium salts are weighed, working solution is made with the dissolving of deuterated methanol solution；
Step 2, the preparation of standard solution
Weigh danshensu, tanshin polyphenolic acid B, Rosmarinic acid, protocatechualdehyde, ginsenoside Rg1, ginsenoside Rb1's standard items use
Standard solution is made in the dissolving of deuterated methanol solution；
Step 3, the preparation of need testing solution
Composite salvia dropping extract of bolus dry powder is weighed, adds working solution, by extracting isolated need testing solution；
Step 4, determine
By standard solution and need testing solution injection NMR spectrometer with superconducting magnet, NMR spectrum figure is obtained；
Step 5, according in gained NMR spectrum figure with danshensu, tanshin polyphenolic acid B, Rosmarinic acid, protocatechualdehyde, ginseng
Proton peak peak area corresponding to saponin(e Rg1, ginsenoside Rb1, calculate the content of each component；
Wherein, the proton magnetic tester condition that NMR spectrometer with superconducting magnet described in step 4 is set is as follows：
Using the 1H NMR spectras of 25-35 ° of pulse sequence acquisition sample solution, measurement temperature 290-310K, observing frequency
590-610MHz, spectrum width 12330-12340Hz, sampled data points 65530-65540, scanning times 8-64 times, time delay
0.9-1.1s, acquisition time 2.6560-2.6570s, gain acceptance in 120-180, calibrated with TSP signals；
Wherein, step 5 application below equation calculates the content of each component：
Wherein PX is the concentration of determinand, and PTSP is internal standard TSP concentration；AX is the peak face of the middle proton peak of determinand
Product, ATSP are the peak area of internal standard TSP proton peak；MX is the molecular weight of determinand, and MTSP is internal standard TSP molecular weight；NX
For the proton number of the generation peak area of determinand, NTSP is the proton number of peak area produced by internal standard TSP.
Preferably, the hydrogen nuclear magnetic resonance detection method of composite salvia dropping extract of bolus of the invention is as follows:
Step 1, the preparation of working solution
Accurately weighed 3- (trimethyl silyl) malonic acid-d4 sodium salt internal standard compounds are appropriate, with deuterated methanol solution constant volume, system
Contain the storage of 0.48-0.52mg 3- (trimethyl silyl) malonic acid-d4 sodium salts into every 0.8-1.2mL deuterated methanols solution
Standby liquid, then the deuterated methanol work containing 0.0098-0.012mg/mL3- (trimethyl silyl) malonic acid-d4 sodium salts is made
Liquid；
Step 2, the preparation of standard solution
Accurately weighed danshensu, tanshin polyphenolic acid B, Rosmarinic acid, protocatechualdehyde, ginsenoside Rg1, ginsenoside Rb1 respectively
Appropriate standard items, with deuterated methanol working solution constant volume, hybrid standard product solution I is made, by the doubling dilution of hybrid standard product solution I
Obtain hybrid standard product solution II, III, IV, V, the concentration of wherein tanshin polyphenolic acid B is respectively 0.05-0.10mg/mL, 0.10-
0.20mg/mL, 0.20-0.40mg/mL, 0.50-0.70mg/mL, 0.80-1.20mg/mL；The concentration of danshensu is respectively
0.10-0.20mg/mL, 0.20-0.40mg/mL, 0.50-0.70mg/mL, 0.80-1.20mg/mL, 1.80-2.20mg/mL；Fan
Repeatedly the concentration of fragrant acid is respectively 0.05-0.10mg/mL, 0.10-0.20mg/mL, 0.20-0.40mg/mL, 0.50-0.70mg/
ML, 0.80-1.0mg/mL；The concentration of protocatechualdehyde is respectively 0.05-0.10mg/mL, 0.10-0.20mg/mL, 0.20-
0.40mg/mL, 0.50-0.70mg/mL, 0.80-1.20mg/mL；Ginsenoside Rg1 0.10-0.30mg/mL, 0.40-
0.60mg/mL, 0.70-0.90mg/mL, 1.20-1.80mg/mL, 2.50-3.50mg/mL；Ginsenoside Rb1 0.10-
0.20mg/mL, 0.30-0.40mg/mL, 0.50-0.60mg/mL, 1.0-1.20mg/mL, 2.0-2.50mg/mL；
Step 3, the preparation of need testing solution
Dripping pill extract medicinal extract is freeze-dried, obtains the dried powder of dripping pill extract, accurately weighed extract is dried
Powder 5-100mg, 300-2000 μ L deuterated methanol working solutions are separately added into, ultrasonic 8-12min, centrifuge 13000-15000rpm,
8-12min, it is transferred in 4-6mm nuclear magnetic tubes and is used as need testing solution；
Step 4, determine
By standard solution and need testing solution injection NMR spectrometer with superconducting magnet, NMR spectrum figure is obtained；
Step 5, according to standard NMR spectrum figure calculate need testing solution in danshensu, tanshin polyphenolic acid B, Rosmarinic acid,
Protocatechualdehyde, ginsenoside Rg1, the content of ginsenoside Rb1；
Wherein, the proton magnetic tester condition that NMR spectrometer with superconducting magnet described in step 4 is set is as follows：
The 1H NMR spectras of sample solution, measurement temperature 290-310K, observing frequency are gathered using 30 ° of pulse train zg30
590-610MHz, spectrum width 12330-12340Hz, sampled data points 65530-65540, scanning times 16-64 times, time delay
1.0000s, acquisition time 2.6564s, gain acceptance in 120-130, calibrated with TSP signals.
It is further preferred that the hydrogen nuclear magnetic resonance detection method of the composite salvia dropping extract of bolus of the present invention is as follows:
Step 1, the preparation of working solution：Accurately weighed 3- (trimethyl silyl) malonic acid-d4 sodium salt internal standard compounds are appropriate, with
Deuterated methanol solution constant volume, it is made in every 0.8-1.2mL deuterated methanols solution and contains 0.48-0.52mg 3- (trimethyl first silicon
Alkane) malonic acid-d4 sodium salts storing solution, then be made containing 0.0098-0.012mg/mL3- (trimethyl silyl) malonic acid-d4
The deuterated methanol working solution of sodium salt；
Step 2, the preparation of standard solution：Respectively accurately weighed danshensu, tanshin polyphenolic acid B, Rosmarinic acid, protocatechualdehyde,
Ginsenoside Rg1, ginsenoside Rb1's standard items are appropriate, and with deuterated methanol working solution constant volume, hybrid standard product solution I is made, will
The doubling dilution of hybrid standard product solution I obtains hybrid standard product solution II, and III, IV, V, the concentration of wherein tanshin polyphenolic acid B is respectively
0.05-0.10mg/mL, 0.10-0.20mg/mL, 0.20-0.40mg/mL, 0.50-0.70mg/mL, 0.80-1.20mg/mL；It is red
The concentration of ginseng element is respectively 0.10-0.20mg/mL, 0.20-0.40mg/mL, 0.50-0.70mg/mL, 0.80-1.20mg/mL,
1.80-2.20mg/mL；The concentration of Rosmarinic acid is respectively 0.05-0.10mg/mL, 0.10-0.20mg/mL, 0.20-0.40mg/
ML, 0.50-0.70mg/mL, 0.80-1.0mg/mL；The concentration of protocatechualdehyde is respectively 0.05-0.10mg/mL, 0.10-
0.20mg/mL, 0.20-0.40mg/mL, 0.50-0.70mg/mL, 0.80-1.20mg/mL；Ginsenoside Rg1 0.10-
0.30mg/mL, 0.40-0.60mg/mL, 0.70-0.90mg/mL, 1.20-1.80mg/mL, 2.50-3.50mg/mL；Ginseng soap
Glycosides Rb1 0.10-0.20mg/mL, 0.30-0.40mg/mL, 0.50-0.60mg/mL, 1.0-1.20mg/mL, 2.0-2.50mg/
mL；
Step 3, the preparation of need testing solution：Dripping pill extract medicinal extract is freeze-dried, obtains the drying of dripping pill extract
Powder, accurately weighed extract dried powder 10-20mg, it is separately added into 500-600 μ L deuterated methanol working solutions, ultrasonic 8-
12min, 13000-15000rpm, 8-12min are centrifuged, is transferred in 4-6mm nuclear magnetic tubes and is used as need testing solution；
Step 4, determine：By standard solution and need testing solution injection NMR spectrometer with superconducting magnet, obtain nuclear-magnetism and be total to
Vibration wave spectrogram；
Step 5, according to standard NMR spectrum figure calculate need testing solution in danshensu, tanshin polyphenolic acid B, Rosmarinic acid,
Protocatechualdehyde, ginsenoside Rg1, the content of ginsenoside Rb1；
Wherein, the proton magnetic tester condition that NMR spectrometer with superconducting magnet described in step 4 is set is as follows：
The 1H NMR spectras of sample solution, measurement temperature 298K, observing frequency 590- are gathered using 30 ° of pulse train zg30
610MHz, spectrum width 12330-12340Hz, sampled data points 65530-65540, scanning times 16-32 times, time delay
1.0000s, acquisition time 2.6564s, gain acceptance in 120-130, calibrated with TSP signals.
Still more preferably, the hydrogen nuclear magnetic resonance detection method of composite salvia dropping extract of bolus of the invention is as follows:
Step 1, the preparation of working solution
Accurately weighed 3- (trimethyl silyl) malonic acid-d4 sodium salt internal standard compounds are appropriate, with deuterated methanol solution constant volume, system
Contain the storage of 0.48-0.52mg 3- (trimethyl silyl) malonic acid-d4 sodium salts into every 0.8-1.2mL deuterated methanols solution
Standby liquid, then the deuterated methanol work containing 0.0098-0.012mg/mL3- (trimethyl silyl) malonic acid-d4 sodium salts is made
Liquid；
Step 2, the preparation of standard solution
Accurately weighed danshensu, tanshin polyphenolic acid B, Rosmarinic acid, protocatechualdehyde, ginsenoside Rg1, ginsenoside Rb1 respectively
Appropriate standard items, with deuterated methanol working solution constant volume, hybrid standard product solution I is made, by the doubling dilution of hybrid standard product solution I
Obtain hybrid standard product solution II, III, IV, V, the concentration of wherein tanshin polyphenolic acid B is respectively 0.05mg/mL, 0.10mg/mL,
0.25mg/mL, 0.50mg/mL, 1.0mg/mL；The concentration of danshensu is respectively 0.10mg/mL, 0.25mg/mL, 0.50mg/mL,
1.0mg/mL, 2.0mg/mL；The concentration of Rosmarinic acid is respectively 0.05mg/mL, 0.10mg/mL, 0.20mg/mL, 0.50mg/
ML, 0.80mg/mL；The concentration of protocatechualdehyde is respectively 0.05mg/mL, 0.10mg/mL, 0.25mg/mL, 0.50mg/mL,
1.0mg/mL；Ginsenoside Rg1 0.2mg/mL, 0.4mg/mL, 0.8mg/mL, 1.5mg/mL, 3.0mg/mL；Ginsenoside Rb1
0.15mg/mL, 0.30mg/mL, 0.60mg/mL, 1.20mg/mL, 2.50mg/mL；
Step 3, the preparation of need testing solution
Dripping pill extract medicinal extract is freeze-dried, obtains the dried powder of dripping pill extract, accurately weighed extract is dried
Powder 8-12mg, 480-520 μ L deuterated methanol working solutions are separately added into, ultrasonic 8-12min, centrifuge 13000-15000rpm, 8-
12min, it is transferred in 4-6mm nuclear magnetic tubes and is used as need testing solution；
Step 4, determine
By standard solution and need testing solution injection NMR spectrometer with superconducting magnet, NMR spectrum figure is obtained；
Step 5, according to standard NMR spectrum figure calculate need testing solution in danshensu, tanshin polyphenolic acid B, Rosmarinic acid,
Protocatechualdehyde, ginsenoside Rg1, the content of ginsenoside Rb1；
Wherein, the proton magnetic tester condition that NMR spectrometer with superconducting magnet described in step 4 is set is as follows：
The 1H NMR spectras of sample solution, measurement temperature 298K, observing frequency are gathered using 30 ° of pulse train zg30
600.23MHz, spectrum width 12335.5Hz, sampled data points 65536, scanning times 16 or 32 times, time delay 1.0000s, collection
Time 2.6564s, gain acceptance in 128, calibrated with TSP signals.
Most preferably, the hydrogen nuclear magnetic resonance detection method of composite salvia dropping extract of bolus of the invention is as follows:
Step 1, the preparation of working solution
Accurately weighed 3- (trimethyl silyl) malonic acid-d4 sodium salts (TSP) appropriate internal standard compound, is determined with deuterated methanol solution
Hold, the storing solution for containing 0.50mg3- (trimethyl silyl) malonic acid-d4 sodium salts in every 1mL deuterated methanols solution is made, then
The deuterated methanol working solution containing 0.01mg/mL3- (trimethyl silyl) malonic acid-d4 sodium salts is made；
Step 2, the preparation of standard solution
Accurately weighed danshensu, tanshin polyphenolic acid B, Rosmarinic acid, protocatechualdehyde, ginsenoside Rg1, ginsenoside Rb1 respectively
Appropriate standard items, with deuterated methanol working solution constant volume, hybrid standard product solution I is made, by the doubling dilution of hybrid standard product solution I
Obtain hybrid standard product solution II, III, IV, V, the concentration of wherein tanshin polyphenolic acid B is respectively 0.0675mg/mL, 0.135mg/mL,
0.270mg/mL, 0.540mg/mL, 1.08mg/mL；The concentration of danshensu is respectively 0.122mg/mL, 0.244mg/mL,
0.488mg/mL, 0.975mg/mL, 1.95mg/mL；The concentration of Rosmarinic acid is respectively 0.0550mg/mL, 0.110mg/mL,
0.220mg/mL, 0.440mg/mL, 0.880mg/mL；The concentration of protocatechualdehyde is respectively 0.0625mg/mL, 0.125mg/mL,
0.250mg/mL, 0.500mg/mL, 1.000mg/mL；Ginsenoside Rg1 0.186mg/mL, 0.373mg/mL, 0.745mg/
ML, 1.49mg/mL, 2.98mg/mL；Ginsenoside Rb1 0.153mg/mL, 0.305mg/mL, 0.610mg/mL, 1.26mg/
ML, 2.52mg/mL；
Step 3, the preparation of need testing solution
Dripping pill extract medicinal extract is freeze-dried, obtains the dried powder of dripping pill extract, accurately weighed extract is dried
Powder 10mg, 500 μ L deuterated methanol working solutions are separately added into, ultrasonic 10min, 14000rpm, 10min is centrifuged, is transferred to 5mm cores
Need testing solution is used as in magnetic tube；
Step 4, determine
By standard solution and need testing solution injection NMR spectrometer with superconducting magnet, NMR spectrum figure is obtained；
Step 5, according to standard NMR spectrum figure calculate need testing solution in danshensu, tanshin polyphenolic acid B, Rosmarinic acid,
Protocatechualdehyde, ginsenoside Rg1, the content of ginsenoside Rb1；
Wherein, the proton magnetic tester condition that NMR spectrometer with superconducting magnet described in step 4 is set is as follows：
The 1H NMR spectras of sample solution, measurement temperature 298K, observing frequency are gathered using 30 ° of pulse train zg30
600.23MHz, spectrum width 12335.5Hz, sampled data points 65536, scanning times 16 times, time delay 1.0000s, acquisition time
2.6564s, gain acceptance in 128, calibrated with TSP signals.
It is the explanation to title term of the present invention below：
Composite salvia dropping extract of bolus：The water extract of the red sage root and pseudo-ginseng, it is the intermediate extract of compound danshen dripping pills
3- (trimethyl silyl) malonic acid-d4 sodium salts (TSP)：Commonly used in hydrogen nuclear magnetic resonance analysis
A kind of internal standard compound, for calibrating chemical shift, quantitative analysis
Deuterated methanol (CD3OD)：A kind of conventional deuterated reagent
NMR spectrometer with superconducting magnet：A kind of new nuclear magnetic resonance chemical analyser, it is using made of high temperature superconducting materia
Coil produces high field intensity stabilizing magnetic field, can greatly reduce noise, improves signal to noise ratio, can also increase the dynamic range of test
The present invention obtains by screening, and screening process is as follows：
1. instrument and reagent
The 600MHz NMR spectrometer with superconducting magnet of BRUKER AVANCE III, proton excitation frequency 600.23MHz, configuration
BBFO positives observe broadband probe；5mm standard nuclear-magnetisms sample cell (NORELL companies of the U.S.)；METTLER TOLEDO XP6 balances
(METTLER companies of Switzerland)；Waters ACQUITY series Ultra Performance Liquid Chromatography instrument (Waters Co., Ltds)；TGL-16C
Desk centrifuge (Anting Scientific Instrument Factory, Shanghai).
Deuterated methanol (CD3OD, deuterated degree>99.8%, Sigma-Aldrich)；3- (trimethyl silyl)
Malonic acid-d4Sodium salt (TSP, deuterated degree>98%, Sigma-Aldrich)；Butanedioic acid (5g, MKBK2580V, the U.S.
Sigma-Aldrich companies), formic acid (5g, MKBP2956, Sigma-Aldrich), glucose (500g,
WXBB6986V, Sigma-Aldrich), sucrose (250g, 090M02112V, Sigma-Aldrich),
Pyroglutamic acid (50mg, 100785-200701, Nat'l Pharmaceutical ＆ Biological Products Control Institute), protocatechualdehyde (50mg, 110810-
200506, Nat'l Pharmaceutical ＆ Biological Products Control Institute), danshensu (200mg, 76822-21-4, in new medicine company), Rosmarinic acid
(20mg, 5FXZ-PXXT, National Institute for Food and Drugs Control), proline (50mg, CKVN-MMXW, the inspection of Chinese food medicine
Determine research institute), tanshin polyphenolic acid B (20mg, HCF0-9JE3, National Institute for Food and Drugs Control), ginsenoside Rg1 (20mg,
NSB7-26L4, National Institute for Food and Drugs Control), ginsenoside Rb1 (20mg, AB120G, the limited public affairs of the side of Tianjin one science and technology
Department), ginsenoside Rd (20mg, AW014G001, the side Science and Technology Ltd. of Tianjin one)；20 batches of compound danshen dripping pills extract leachings
Cream (being free of borneol) is provided by Tianjin day scholar's power modern times Resources Co., Ltd, and lot number is as shown in table 1 below；Acetonitrile, methanol are color
Compose pure (Sigma companies)；Formic acid is chromatographically pure (Tianjin great Mao chemical reagent factories)；Ultra-pure water is by laboratory Milli Q pure water
Mechanism obtains.
The experiment numbers and product batch number of 1 20 batches of compound danshen dripping pills extracts of table
2. experimental method
The preparation of 2.1 working solutions
Accurately weighed TSP internal standard compounds are appropriate, with CD3OD solution constant volumes, every 1mL CD are made3Contain 0.50mg in OD solution
TSP storing solution, then the CD containing 0.01mg/mLTSP is made3OD working solutions.
The preparation of 2.2 standard solutions
Accurately weighed danshensu, tanshin polyphenolic acid B, Rosmarinic acid, protocatechualdehyde, ginsenoside Rg1, ginsenoside Rb1 respectively
Appropriate standard items, with CD3OD working solution constant volumes, are made standard solution.
The preparation of 2.3 need testing solutions
Dripping pill extract medicinal extract is freeze-dried, obtains the dried powder of three kinds of extracts, accurately weighed extract is dried
Powder 10mg, it is separately added into 500 μ LCD3OD working solutions, ultrasonic 10min, centrifuge (14000rpm, 10min), be transferred to 5mm cores
Need testing solution is used as in magnetic tube.
2.4 1H NMR test conditions
Sample solution is gathered using 30 ° of pulse trains (zg30)1H NMR spectras.Measurement temperature 298K, observing frequency
600.23MHz, spectrum width 12335.5Hz, sampled data points 65536, scanning times 16 times (setting 8~64 times, preferably 64 times), prolong
Slow time 1.0000s, acquisition time 2.6564s, gain acceptance in 128.Calibrated with TSP signals.
2.5 cubage formula
1The low proton signal peak of high resolution, plyability quantifying for corresponding compound is selected in H NMR finger-prints
Peak, the content of each compound is calculated using below equation：
Wherein PXFor the concentration of determinand, PTSPFor internal standard TSP concentration；AXFor the peak area of the middle proton peak of determinand,
ATSPFor the peak area of internal standard TSP proton peak；MXFor the molecular weight of determinand, MTSPFor internal standard TSP molecular weight；NXTo be to be measured
The proton number of the generation peak area of thing, NTSPFor the proton number of peak area produced by internal standard TSP.
2.6 methodological study
It is 2.6.1 linear
Precision measures each standard solution and is placed in right amount in 5mL volumetric flasks, uses CD3OD working solution constant volumes, mixing mark is made
Quasi- product solution I, the doubling dilution of hybrid standard product solution I is obtained into hybrid standard product solution II, III, IV, V (wherein tanshin polyphenolic acid B
Concentration be respectively 0.0675mg/mL, 0.135mg/mL, 0.270mg/mL, 0.540mg/mL, 1.08mg/mL；Danshensu it is dense
Degree is respectively 0.122mg/mL, 0.244mg/mL, 0.488mg/mL, 0.975mg/mL, 1.95mg/mL；The concentration of Rosmarinic acid
Respectively 0.0550mg/mL, 0.110mg/mL, 0.220mg/mL, 0.440mg/mL, 0.880mg/mL；The concentration of protocatechualdehyde
Respectively 0.0625mg/mL, 0.125mg/mL, 0.250mg/mL, 0.500mg/mL, 1.000mg/mL；Ginsenoside Rg1
0.186mg/mL, 0.373mg/mL, 0.745mg/mL, 1.49mg/mL, 2.98mg/mL；Ginsenoside Rb1 0.153mg/mL,
0.305mg/mL, 0.610mg/mL, 1.26mg/mL, 2.52mg/mL) respectively take 500 μ L to be placed in nuclear magnetic tube, by 2.4 lower conditions
Measure.Record danshensu, Rosmarinic acid, protocatechualdehyde, tanshin polyphenolic acid B, the integral area of Ginsenoside Rg1 and Rb1,
Draw standard curve.
2.6.2 precision
Dripping pill extract need testing solution is taken, respectively by 2.4 lower condition METHOD FOR CONTINUOUS DETERMINATIONs 6 times, integral area is recorded, calculates
The RSD at sample amounts peak and internal standard peak integral area ratio.
It is 2.6.3 repeated
Precision weighs each 6 parts, every part of about 10mg of dripping pill extract powder respectively, by 2.3 lower section legal system available test products, presses
2.4 lower conditions are measured, and record integral area, and the content of compound in sample is calculated according to the formula under 2.5.
2.6.4 stability test
Dripping pill extract need testing solution is taken, determines (room temperature placement) in 0,2,4,8,10,12h sample introduction respectively, record product
Facet is accumulated, and calculates the RSD at sample amounts peak and internal standard peak integral area ratio.
2.6.5 average recovery
Take 30 parts of the dripping pill extract powder that content has been predicted under 2.6.3 items, every part of about 5mg is accurately weighed.It is accurate respectively
Add standard solution (danshensu 0.1900mg, tanshin polyphenolic acid B 0.0466mg, Rosmarinic acid 0.0266mg, protocatechualdehyde
0.0269mg, ginsenoside Rg1 0.2083mg, ginsenoside Rb1 0.1500mg), test sample is prepared by 2.3 lower conditions,
It is measured by 2.4 lower conditions, calculates the rate of recovery.
2.6.6 sample determines
The dripping pill extract (3 parts/batches) of 20 lot numbers is taken, test sample is prepared by 2.3 lower conditions, by 2.4 lower conditions
It is measured.
3. result is with discussing
3.1 1H NMR finger-prints parse and signal peak ownership
Dripping pill extract typical case1H NMR finger-prints are as shown in Figure 1.Proton signal peak be mainly distributed on δ 0.5-9.5 it
Between, it is divided into three characteristic areas：δ 0.5-3.0 are the diagnostic protons of saponins, amino acids, aliphatic acid and organic acid composition
Signaling zone, δ 3.0-5.5 are the proton signal area of glycosyl, and δ 5.5-9.5 are aromatic signaling zone.
According to the chemical shift at proton signal peak, the relevant information that point situation is split in coupling and a variety of two-dimensional maps provide, bag
The carbon spectrum of dripping pill extract is included,1H-1H COSY, TOCSY, HSQC, HMBC collection of illustrative plates (referring to Fig. 2), reflects altogether from dripping pill extract
Make 20 compounds, wherein 6 kinds of (danshensu, salviandic acid A, tanshin polyphenolic acid B, Rosmarinic acid, alkannic acid, originals of phenolic acid compound
Catechu aldehyde), 3 kinds of saponins (ginsenoside Rg1, ginsenoside Rb1, life saponin(e Rd), carbohydrate 4 kinds of (glucose, sucrose, honey
Trisaccharide, arabinose), 3 kinds of amino acids (proline, glutamic acid, pyroglutamic acid), 4 kinds of organic acid (formic acid, acetic acid, the third two
Acid, butanedioic acid), each compound1The detailed attaching information of H-NMR finger-prints is referring to table 2.
The dripping pill extract of table 21The signals assignment of 20 compounds in H-NMR collection of illustrative plates
aMltiplicity:singlet(s),doublet(d),triplet(t),doublet of doublets
(dd),quintet(q),multiplet(m).
The selection at the quantitative peak of 3.2 compounds
The choosing at quantitative peak is illustrated by taking the main component danshensu in dripping pill, Ginsenoside Rg1 and Rb1 as an example
Select.According to dripping pill mixture1H-1H COSY and hsqc spectrum can determine δ 6.70 (d, 2.0Hz), δ 6.62 (d, 8.0Hz), δ
6.58 (dd, 8.0,2.0Hz) are 3 H on danshensu phenyl ring.By adding danshensu standard items into compound dripping pill extract
Method, compare1The change of H-NMR collection of illustrative plates, further determine that the diagnostic protons signal peak of danshensu.Due on danshensu phenyl ring
H-2 and H-5 is in compound dripping pill extract1Interference in H-NMR collection of illustrative plates by other proton peaks is more apparent, and what H-6 was disturbed
Degree is smaller and identification is higher, therefore selected quantitative peaks of the δ 6.58 (dd, J=8.0,2.0Hz, H-6) as danshensu.According to
Dripping pill mixture hsqc spectrum can determine δ 0.95 (s), 0.99 (s), 1.00 (s), 1.10 (s), 1.32 (s), 1.34 (s), 1.62
(s), 1.67 (s) is the proton signal peak of the angular methyl of ginsenoside Rg1；δ0.85(s),0.91(s),1.00(s),1.06
(s), 1.36 (s), 1.62 (s), 1.68 (s) are the methyl proton signal peak of ginsenoside Rb1.Due to both ginsenosides
Methyl proton signal peak compares concentration and interfered with each other, so we add comparison method using standard control, i.e., to compound dripping pill
Ginsenoside Rg1 and Rb1 standard items are separately added into extract, passes through and compares1The change of H NMR spectras, it is determined that
The quantitative peak of two kinds of ginsenosides, as a result shows δ 0.95 (s), δ 0.85 (s) is respectively as ginsenoside Rg1 and ginsenoside
Rb1 quantitative peak.
3.3 methodological study results
Select tanshin polyphenolic acid B, danshensu, the rosemary in compound dripping pill extract with fine resolution proton signal peak
Totally 6 compounds carry out assay analysis for acid, protocatechualdehyde, Ginsenoside Rg1 and Rb1, and main right1H-
The linear of NMR methods, stability, precision, repeatability and average recovery are investigated respectively.Compound dripping pill extract sample
This methodological study result is listed in detail in table 3 and table 4.
Due to organic compound proton in nuclear magnetic resonance response all same, therefore1H-NMR quantitative analysis methods are led to
The range of linearity need not often be investigated.The linear verification result of compound dripping pill extract displays that：Tanshin polyphenolic acid B, danshensu, rosemary
Acid, protocatechualdehyde, Ginsenoside Rg1 and Rb1 have good linear relationship, Fig. 36 really in test scope
The standard curve of kind compound.
In compound dripping pill extract sample 6 kinds of Contents of Main Components test precision RSD values scopes be 0.08%~
2.97%, stability RSD values scope is 1.26%~3.43%, and repeated RSD values scope is 0.26%~3.23%.Mainly into
Point danshensu, tanshin polyphenolic acid B, Rosmarinic acid, protocatechualdehyde, the average recovery of Ginsenoside Rg1 and Rb1 are
84.20%~110.75%.The above results show1H-NMR methods have good precision, stability, repeatability and accurate
Degree.
6 kinds of Contents of Main Components methods for measuring investigate result in the dripping pill extract of table 3
The sample-adding recovery (n=6) of 6 kinds of main components in the dripping pill extract of table 4
3.4 assays are analyzed with multivariate process quality control
For 6 kinds of main compound assays the results detailed in Table 5, related spectrogram refers to Fig. 4 in 20 batches of dripping pill extract samples.
The qHNMR assay results of 6 kinds of main components in 5 20 batches of compound dripping pill extract samples of table
4 beneficial effects
The present invention establishes the non-chromatogram for compound danshen dripping pills production process quality control using qHNMR technologies first
Quantitative analysis method, " macro qualitative analysis " of product intermediate can be both carried out, can also be simultaneously to wherein main component danshensu, former youngster
Tea aldehyde, tanshin polyphenolic acid B, Rosmarinic acid, ginsenoside Rg1 and Rb1 carry out accurate quantitative analysis, and this method is in sample analysis speed
Had a clear superiority on degree (each sample analysis time only 2min).
Brief description of the drawings
1H-NMR finger-prints (a. total figures of Fig. 1 composite salvia dropping extract of bolus；B. saponins, amino acids, aliphatic acid
With the diagnostic protons signaling zone of organic acid composition；C. the proton signal area of glycosyl；D. aromatic signaling zone)
Fig. 2-1 dripping pill extract carbon spectrograms
Fig. 2-2 dripping pill extract DEPT spectrograms
Fig. 2-3 dripping pill extract 1H-1H COSY spectrograms
Fig. 2-4 dripping pill extract TOCSY spectrograms
Fig. 2-5 dripping pill extracts HSQC schemes
Fig. 2-6 dripping pill extracts HMBC schemes
Fig. 3 tanshin polyphenolic acid Bs, danshensu, Rosmarinic acid, protocatechualdehyde, the standard curve of Ginsenoside Rg1 and Rb1
The 1H NMR spectras of 20 batches of dripping pill extracts of Fig. 4
Embodiment
Embodiment 1
Step 1：The preparation of working solution
Accurately weighed 3- (trimethyl silyl) malonic acid-d4Sodium salt (TSP) appropriate internal standard compound, with deuterated methanol (CD3OD)
Solution constant volume, every 1mL CD are made3Storing solution containing 0.48mg TSP in OD solution, then be made containing 0.012mg/mLTSP
CD3OD working solutions.
Step 2：The preparation of standard solution
Accurately weighed danshensu, tanshin polyphenolic acid B, Rosmarinic acid, protocatechualdehyde, ginsenoside Rg1, ginsenoside Rb1 respectively
Appropriate standard items, with CD3OD working solution constant volumes, are made standard solution.
Step 3：The preparation of need testing solution
Composite salvia dropping extract of bolus medicinal extract is freeze-dried, obtains the dried powder of three kinds of extracts, it is accurately weighed each
Each 5mg of extract dried powder, it is separately added into 300 μ L CD3OD working solutions, ultrasonic 8min, centrifuge 13000rpm, 8min, transfer
Need testing solution is used as into 5mm nuclear magnetic tubes.
Step 4：NMR spectrometer with superconducting magnet is injected, calculates the content of each compound.
Proton magnetic tester condition is as follows：
Sample solution is gathered using 30 ° of pulse trains (zg30)1H NMR spectras.Measurement temperature 310K, observing frequency
609.23MHz, spectrum width 12338.5Hz, sampled data points 65530, scanning times 8 times, time delay 0.9s, acquisition time
2.6564s gain acceptance in 180.Calibrated with TSP signals.
Embodiment 2
Detection method, step are as follows:
Step 1, the preparation of working solution
Accurately weighed 3- (trimethyl silyl) malonic acid-d4 sodium salts (TSP) appropriate internal standard compound, is determined with deuterated methanol solution
Hold, the storing solution for containing 0.50mg3- (trimethyl silyl) malonic acid-d4 sodium salts in every 1mL deuterated methanols solution is made, then
The deuterated methanol working solution containing 0.01mg/mL3- (trimethyl silyl) malonic acid-d4 sodium salts is made；
Step 2, the preparation of standard solution
Accurately weighed danshensu, tanshin polyphenolic acid B, Rosmarinic acid, protocatechualdehyde, ginsenoside Rg1, ginsenoside Rb1 respectively
Appropriate standard items, with deuterated methanol working solution constant volume, hybrid standard product solution I is made, by the doubling dilution of hybrid standard product solution I
Obtain hybrid standard product solution II, III, IV, V, the concentration of wherein tanshin polyphenolic acid B is respectively 0.0675mg/mL, 0.135mg/mL,
0.270mg/mL, 0.540mg/mL, 1.08mg/mL；The concentration of danshensu is respectively 0.122mg/mL, 0.244mg/mL,
0.488mg/mL, 0.975mg/mL, 1.95mg/mL；The concentration of Rosmarinic acid is respectively 0.0550mg/mL, 0.110mg/mL,
0.220mg/mL, 0.440mg/mL, 0.880mg/mL；The concentration of protocatechualdehyde is respectively 0.0625mg/mL, 0.125mg/mL,
0.250mg/mL, 0.500mg/mL, 1.000mg/mL；Ginsenoside Rg1 0.186mg/mL, 0.373mg/mL, 0.745mg/
ML, 1.49mg/mL, 2.98mg/mL；Ginsenoside Rb1 0.153mg/mL, 0.305mg/mL, 0.610mg/mL, 1.26mg/
ML, 2.52mg/mL；
Step 3, the preparation of need testing solution
Dripping pill extract medicinal extract is freeze-dried, obtains the dried powder of dripping pill extract, accurately weighed extract is dried
Powder 5mg, 500 μ L deuterated methanol working solutions are separately added into, ultrasonic 10min, 14000rpm, 10min is centrifuged, is transferred to 5mm cores
Need testing solution is used as in magnetic tube；
Step 4, determine
By standard solution and need testing solution injection NMR spectrometer with superconducting magnet, NMR spectrum figure is obtained；
Step 5, according to standard NMR spectrum figure calculate need testing solution in danshensu, tanshin polyphenolic acid B, Rosmarinic acid,
Protocatechualdehyde, ginsenoside Rg1, the content of ginsenoside Rb1；
Wherein, the proton magnetic tester condition that NMR spectrometer with superconducting magnet described in step 4 is set is as follows：
The 1H NMR spectras of sample solution, measurement temperature 298K, observing frequency are gathered using 30 ° of pulse train zg30
600.23MHz, spectrum width 12335.5Hz, sampled data points 65536, scanning times 16 times, time delay 1.0000s, acquisition time
2.6564s, gain acceptance in 128, calibrated with TSP signals.
Embodiment 3
Detection method, step are as follows:
Step 1, the preparation of working solution
Accurately weighed 3- (trimethyl silyl) malonic acid-d4 sodium salt internal standard compounds are appropriate, with deuterated methanol solution constant volume, system
Contain the storing solution of 0.52mg 3- (trimethyl silyl) malonic acid-d4 sodium salts into every 0.8mL deuterated methanols solution, then make
Into the deuterated methanol working solution containing 0.0098mg/mL3- (trimethyl silyl) malonic acid-d4 sodium salts；
Step 2, the preparation of standard solution
Accurately weighed danshensu, tanshin polyphenolic acid B, Rosmarinic acid, protocatechualdehyde, ginsenoside Rg1, ginsenoside Rb1 respectively
Appropriate standard items, with deuterated methanol working solution constant volume, hybrid standard product solution I is made, by the doubling dilution of hybrid standard product solution I
Obtain hybrid standard product solution II, III, IV, V, the concentration of wherein tanshin polyphenolic acid B is respectively 0.05mg/mL, 0.10mg/mL,
0.25mg/mL, 0.50mg/mL, 1.0mg/mL；The concentration of danshensu is respectively 0.10mg/mL, 0.25mg/mL, 0.50mg/mL,
1.0mg/mL, 2.0mg/mL；The concentration of Rosmarinic acid is respectively 0.05mg/mL, 0.10mg/mL, 0.20mg/mL, 0.50mg/
ML, 0.80mg/mL；The concentration of protocatechualdehyde is respectively 0.05mg/mL, 0.10mg/mL, 0.25mg/mL, 0.50mg/mL,
1.0mg/mL；Ginsenoside Rg1 0.2mg/mL, 0.4mg/mL, 0.8mg/mL, 1.5mg/mL, 3.0mg/mL；Ginsenoside Rb1
0.15mg/mL, 0.30mg/mL, 0.60mg/mL, 1.20mg/mL, 2.50mg/mL；
Step 3, the preparation of need testing solution：Dripping pill extract medicinal extract is freeze-dried, obtains the drying of dripping pill extract
Powder, accurately weighed extract dried powder 50mg, it is separately added into 520 μ L deuterated methanol working solutions, ultrasonic 12min, centrifugation
15000rpm, 12min, it is transferred in 6mm nuclear magnetic tubes and is used as need testing solution；
Step 4, determine：By standard solution and need testing solution injection NMR spectrometer with superconducting magnet, obtain nuclear-magnetism and be total to
Vibration wave spectrogram；
Step 5, according to standard NMR spectrum figure calculate need testing solution in danshensu, tanshin polyphenolic acid B, Rosmarinic acid,
Protocatechualdehyde, ginsenoside Rg1, the content of ginsenoside Rb1；
Wherein, the proton magnetic tester condition that NMR spectrometer with superconducting magnet described in step 4 is set is as follows：
Using the 1H NMR spectras of 35 ° of pulse sequence acquisition sample solutions, measurement temperature 310K, observing frequency 610MHz,
Spectrum width 12340Hz, sampled data points 65540, scanning times 24 times, time delay 1.1s, acquisition time 2.6570s, receive increasing
Benefit 130, calibrated with TSP signals.
Embodiment 4
Step 1, the preparation of working solution
Accurately weighed 3- (trimethyl silyl) malonic acid-d4 sodium salt internal standard compounds are appropriate, with deuterated methanol solution constant volume, system
Contain the storing solution of 0.5mg 3- (trimethyl silyl) malonic acid-d4 sodium salts into every 1.2mL deuterated methanols solution, then be made
Deuterated methanol working solution containing 0.012mg/mL3- (trimethyl silyl) malonic acid-d4 sodium salts；
Step 2, the preparation of standard solution
Accurately weighed danshensu, tanshin polyphenolic acid B, Rosmarinic acid, protocatechualdehyde, ginsenoside Rg1, ginsenoside Rb1 respectively
Appropriate standard items, with deuterated methanol working solution constant volume, hybrid standard product solution I is made, by the doubling dilution of hybrid standard product solution I
Obtain hybrid standard product solution II, III, IV, V, the concentration of wherein tanshin polyphenolic acid B is respectively 0.05mg/mL, 0.10mg/mL,
0.25mg/mL, 0.50mg/mL, 1.0mg/mL；The concentration of danshensu is respectively 0.10mg/mL, 0.25mg/mL, 0.50mg/mL,
1.0mg/mL, 2.0mg/mL；The concentration of Rosmarinic acid is respectively 0.05mg/mL, 0.10mg/mL, 0.20mg/mL, 0.50mg/
ML, 0.80mg/mL；The concentration of protocatechualdehyde is respectively 0.05mg/mL, 0.10mg/mL, 0.25mg/mL, 0.50mg/mL,
1.0mg/mL；Ginsenoside Rg1 0.2mg/mL, 0.4mg/mL, 0.8mg/mL, 1.5mg/mL, 3.0mg/mL；Ginsenoside Rb1
0.15mg/mL, 0.30mg/mL, 0.60mg/mL, 1.20mg/mL, 2.50mg/mL；
Step 3, the preparation of need testing solution
Dripping pill extract medicinal extract is freeze-dried, obtains the dried powder of dripping pill extract, accurately weighed extract is dried
Powder 100mg, 2000 μ L deuterated methanol working solutions are separately added into, ultrasonic 10min, 14000rpm, 10min is centrifuged, is transferred to 5mm
Need testing solution is used as in nuclear magnetic tube；
Step 4, determine
By standard solution and need testing solution injection NMR spectrometer with superconducting magnet, NMR spectrum figure is obtained；
Step 5, according to standard NMR spectrum figure calculate need testing solution in danshensu, tanshin polyphenolic acid B, Rosmarinic acid,
Protocatechualdehyde, ginsenoside Rg1, the content of ginsenoside Rb1；
Wherein, the proton magnetic tester condition that NMR spectrometer with superconducting magnet described in step 4 is set is as follows：
Using the 1H NMR spectras of 30 ° of pulse sequence acquisition sample solutions, measurement temperature 298K, observing frequency 600MHz,
Spectrum width 12335.5Hz, sampled data points 65530, scanning times 32 times, time delay 0.9s, acquisition time 2.6564s, receive
Gain 128, calibrated with TSP signals.
Embodiment 5
Step 1, the preparation of working solution
Accurately weighed 3- (trimethyl silyl) malonic acid-d4 sodium salts (TSP) appropriate internal standard compound, is determined with deuterated methanol solution
Hold, the storing solution for containing 0.50mg3- (trimethyl silyl) malonic acid-d4 sodium salts in every 1mL deuterated methanols solution is made, then
The deuterated methanol working solution containing 0.01mg/mL3- (trimethyl silyl) malonic acid-d4 sodium salts is made；
Step 2, the preparation of standard solution
Accurately weighed danshensu, tanshin polyphenolic acid B, Rosmarinic acid, protocatechualdehyde, ginsenoside Rg1, ginsenoside Rb1 respectively
Appropriate standard items, with deuterated methanol working solution constant volume, hybrid standard product solution I is made, by the doubling dilution of hybrid standard product solution I
Obtain hybrid standard product solution II, III, IV, V, the concentration of wherein tanshin polyphenolic acid B is respectively 0.0675mg/mL, 0.135mg/mL,
0.270mg/mL, 0.540mg/mL, 1.08mg/mL；The concentration of danshensu is respectively 0.122mg/mL, 0.244mg/mL,
0.488mg/mL, 0.975mg/mL, 1.95mg/mL；The concentration of Rosmarinic acid is respectively 0.0550mg/mL, 0.110mg/mL,
0.220mg/mL, 0.440mg/mL, 0.880mg/mL；The concentration of protocatechualdehyde is respectively 0.0625mg/mL, 0.125mg/mL,
0.250mg/mL, 0.500mg/mL, 1.000mg/mL；Ginsenoside Rg1 0.186mg/mL, 0.373mg/mL, 0.745mg/
ML, 1.49mg/mL, 2.98mg/mL；Ginsenoside Rb1 0.153mg/mL, 0.305mg/mL, 0.610mg/mL, 1.26mg/
ML, 2.52mg/mL；
Step 3, the preparation of need testing solution
Dripping pill extract medicinal extract is freeze-dried, obtains the dried powder of dripping pill extract, accurately weighed extract is dried
Powder 5mg, 500 μ L deuterated methanol working solutions are separately added into, ultrasonic 10min, 14000rpm, 10min is centrifuged, is transferred to 5mm cores
Need testing solution is used as in magnetic tube；
Step 4, determine
By standard solution and need testing solution injection NMR spectrometer with superconducting magnet, NMR spectrum figure is obtained；
Step 5, according to standard NMR spectrum figure calculate need testing solution in danshensu, tanshin polyphenolic acid B, Rosmarinic acid,
Protocatechualdehyde, ginsenoside Rg1, the content of ginsenoside Rb1；
Wherein, the proton magnetic tester condition that NMR spectrometer with superconducting magnet described in step 4 is set is as follows：
The 1H NMR spectras of sample solution, measurement temperature 298K, observing frequency are gathered using 30 ° of pulse train zg30
600.23MHz, spectrum width 12334Hz, sampled data points 65538, scanning times 64 times, time delay 1.0000s, acquisition time
2.6570s, gain acceptance in 130, calibrated with TSP signals.
Embodiment 6
Step 1, the preparation of working solution
Accurately weighed 3- (trimethyl silyl) malonic acid-d4 sodium salts (TSP) appropriate internal standard compound, is determined with deuterated methanol solution
Hold, the storing solution for containing 0.50mg3- (trimethyl silyl) malonic acid-d4 sodium salts in every 1mL deuterated methanols solution is made, then
The deuterated methanol working solution containing 0.01mg/mL3- (trimethyl silyl) malonic acid-d4 sodium salts is made；
Step 2, the preparation of standard solution
Accurately weighed danshensu, tanshin polyphenolic acid B, Rosmarinic acid, protocatechualdehyde, ginsenoside Rg1, ginsenoside Rb1 respectively
Appropriate standard items, with deuterated methanol working solution constant volume, hybrid standard product solution I is made, by the doubling dilution of hybrid standard product solution I
Obtain hybrid standard product solution II, III, IV, V, the concentration of wherein tanshin polyphenolic acid B is respectively 0.05mg/mL, 0.10mg/mL,
0.25mg/mL, 0.50mg/mL, 1.0mg/mL；The concentration of danshensu is respectively 0.10mg/mL, 0.25mg/mL, 0.50mg/mL,
1.0mg/mL, 2.0mg/mL；The concentration of Rosmarinic acid is respectively 0.05mg/mL, 0.10mg/mL, 0.20mg/mL, 0.50mg/
ML, 0.80mg/mL；The concentration of protocatechualdehyde is respectively 0.0625mg/mL, 0.125mg/mL, 0.250mg/mL, 0.500mg/
ML, 1.000mg/mL；Ginsenoside Rg1 0.186mg/mL, 0.373mg/mL, 0.745mg/mL, 1.49mg/mL, 2.98mg/
mL；Ginsenoside Rb1 0.153mg/mL, 0.305mg/mL, 0.610mg/mL, 1.26mg/mL, 2.52mg/mL；
Step 3, the preparation of need testing solution
Dripping pill extract medicinal extract is freeze-dried, obtains the dried powder of dripping pill extract, accurately weighed extract is dried
Powder 50mg, 1000 μ L deuterated methanol working solutions are separately added into, ultrasonic 10min, 15000rpm, 10min is centrifuged, is transferred to 5mm
Need testing solution is used as in nuclear magnetic tube；
Step 4, determine
By standard solution and need testing solution injection NMR spectrometer with superconducting magnet, NMR spectrum figure is obtained；
Step 5, according to standard NMR spectrum figure calculate need testing solution in danshensu, tanshin polyphenolic acid B, Rosmarinic acid,
Protocatechualdehyde, ginsenoside Rg1, the content of ginsenoside Rb1；
Wherein, the proton magnetic tester condition that NMR spectrometer with superconducting magnet described in step 4 is set is as follows：
The 1H NMR spectras of sample solution, measurement temperature 298K, observing frequency are gathered using 30 ° of pulse train zg30
600.23MHz, spectrum width 12335.5Hz, sampled data points 65536, scanning times 16 times, time delay 1.0000s, acquisition time
2.6564s, gain acceptance in 128, calibrated with TSP signals.
Claims (5)
1. a kind of hydrogen nuclear magnetic resonance detection method of Chinese medicine compound prescription Danshen Root dropping ball extract, methods described comprise the following steps：
Step 1, the preparation of working solution：Internal standard compound 3- (trimethyl silyl) malonic acid-d4 sodium salts are weighed, with deuterated methanol solution
Working solution is made in dissolving；
Step 2, the preparation of standard solution：Weigh danshensu, tanshin polyphenolic acid B, Rosmarinic acid, protocatechualdehyde, ginsenoside Rg1,
Standard solution is made in the dissolving of ginsenoside Rb1's standard items deuterated methanol solution；
Step 3, the preparation of need testing solution：Composite salvia dropping extract of bolus dry powder is weighed, adds working solution, is separated by extraction
Obtain need testing solution；
Step 4, determine：By standard solution and need testing solution injection NMR spectrometer with superconducting magnet, nuclear magnetic resonance ripple is obtained
Spectrogram；
Step 5, according in gained NMR spectrum figure with danshensu, tanshin polyphenolic acid B, Rosmarinic acid, protocatechualdehyde, ginsenoside
Proton peak peak area corresponding to Rg1, ginsenoside Rb1, calculate the content of each component；
Wherein, the proton magnetic tester condition that NMR spectrometer with superconducting magnet described in step 4 is set is as follows：
Using the 1H NMR spectras of 25-35 ° of pulse sequence acquisition sample solution, measurement temperature 290-310K, observing frequency 590-
610MHz, spectrum width 12330-12340Hz, sampled data points 65530-65540, scanning times 8-64 times, time delay 0.9-
1.1s, acquisition time 2.6560-2.6570s, gain acceptance in 120-180, calibrated with TSP signals；
Wherein, step 5 application below equation calculates the content of each component：
<mrow>
<msub>
<mi>P</mi>
<mi>X</mi>
</msub>
<mo>=</mo>
<mfrac>
<msub>
<mi>A</mi>
<mi>X</mi>
</msub>
<msub>
<mi>A</mi>
<mrow>
<mi>T</mi>
<mi>S</mi>
<mi>P</mi>
</mrow>
</msub>
</mfrac>
<mfrac>
<msub>
<mi>N</mi>
<mrow>
<mi>T</mi>
<mi>S</mi>
<mi>P</mi>
</mrow>
</msub>
<msub>
<mi>N</mi>
<mi>X</mi>
</msub>
</mfrac>
<mfrac>
<msub>
<mi>M</mi>
<mi>X</mi>
</msub>
<msub>
<mi>M</mi>
<mrow>
<mi>T</mi>
<mi>S</mi>
<mi>P</mi>
</mrow>
</msub>
</mfrac>
<msub>
<mi>P</mi>
<mrow>
<mi>T</mi>
<mi>S</mi>
<mi>P</mi>
</mrow>
</msub>
</mrow>
Wherein PX is the concentration of determinand, and PTSP is internal standard TSP concentration；AX is the peak area of the middle proton peak of determinand,
ATSP is the peak area of internal standard TSP proton peak；MX is the molecular weight of determinand, and MTSP is internal standard TSP molecular weight；NX is to treat
The proton number of the generation peak area of thing is surveyed, NTSP is the proton number of peak area produced by internal standard TSP.
2. detection method according to claim 1, it is characterised in that step is as follows:
Step 1, the preparation of working solution：Accurately weighed 3- (trimethyl silyl) malonic acid-d4 sodium salt internal standard compounds are appropriate, with deuterated
Methanol solution constant volume, it is made in every 0.8-1.2mL deuterated methanols solution and contains 0.48-0.52mg 3- (trimethyl silyl) third
The storing solution of diacid-d4 sodium salts, then be made containing 0.0098-0.012mg/mL3- (trimethyl silyl) malonic acid-d4 sodium salts
Deuterated methanol working solution；
Step 2, the preparation of standard solution：Accurately weighed danshensu, tanshin polyphenolic acid B, Rosmarinic acid, protocatechualdehyde, ginseng respectively
Saponin(e Rg1, ginsenoside Rb1's standard items are appropriate, with deuterated methanol working solution constant volume, hybrid standard product solution I are made, will mix
The doubling dilution of standard solution I obtains hybrid standard product solution II, and III, IV, V, the concentration of wherein tanshin polyphenolic acid B is respectively 0.05-
0.10mg/mL, 0.10-0.20mg/mL, 0.20-0.40mg/mL, 0.50-0.70mg/mL, 0.80-1.20mg/mL；Danshensu
Concentration be respectively 0.10-0.20mg/mL, 0.20-0.40mg/mL, 0.50-0.70mg/mL, 0.80-1.20mg/mL, 1.80-
2.20mg/mL；The concentration of Rosmarinic acid is respectively 0.05-0.10mg/mL, 0.10-0.20mg/mL, 0.20-0.40mg/mL,
0.50-0.70mg/mL, 0.80-1.0mg/mL；The concentration of protocatechualdehyde is respectively 0.05-0.10mg/mL, 0.10-0.20mg/
ML, 0.20-0.40mg/mL, 0.50-0.70mg/mL, 0.80-1.20mg/mL；Ginsenoside Rg1 0.10-0.30mg/mL,
0.40-0.60mg/mL, 0.70-0.90mg/mL, 1.20-1.80mg/mL, 2.50-3.50mg/mL；Ginsenoside Rb1 0.10-
0.20mg/mL, 0.30-0.40mg/mL, 0.50-0.60mg/mL, 1.0-1.20mg/mL, 2.0-2.50mg/mL；
Step 3, the preparation of need testing solution：Dripping pill extract medicinal extract is freeze-dried, obtains the dried powder of dripping pill extract,
Accurately weighed extract dried powder 5-100mg, is separately added into 300-2000 μ L deuterated methanol working solutions, ultrasonic 8-12min, from
The heart 13000-15000rpm, 8-12min, it is transferred in 4-6mm nuclear magnetic tubes and is used as need testing solution；
Step 4, determine：By standard solution and need testing solution injection NMR spectrometer with superconducting magnet, nuclear magnetic resonance ripple is obtained
Spectrogram；
Step 5, danshensu, tanshin polyphenolic acid B, Rosmarinic acid, former youngster in need testing solution are calculated according to standard NMR spectrum figure
Tea aldehyde, ginsenoside Rg1, the content of ginsenoside Rb1；
Wherein, the proton magnetic tester condition that NMR spectrometer with superconducting magnet described in step 4 is set is as follows：
The 1H NMR spectras of sample solution, measurement temperature 290-310K, observing frequency 590- are gathered using 30 ° of pulse train zg30
610MHz, spectrum width 12330-12340Hz, sampled data points 65530-65540, scanning times 16-64 times, time delay
1.0000s, acquisition time 2.6564s, gain acceptance in 120-130, calibrated with TSP signals.
3. detection method according to claim 1, it is characterised in that step is as follows:
Step 1, the preparation of working solution：Accurately weighed 3- (trimethyl silyl) malonic acid-d4 sodium salt internal standard compounds are appropriate, with deuterated
Methanol solution constant volume, it is made in every 0.8-1.2mL deuterated methanols solution and contains 0.48-0.52mg 3- (trimethyl silyl) third
The storing solution of diacid-d4 sodium salts, then be made containing 0.0098-0.012mg/mL3- (trimethyl silyl) malonic acid-d4 sodium salts
Deuterated methanol working solution；
Step 2, the preparation of standard solution：Accurately weighed danshensu, tanshin polyphenolic acid B, Rosmarinic acid, protocatechualdehyde, ginseng respectively
Saponin(e Rg1, ginsenoside Rb1's standard items are appropriate, with deuterated methanol working solution constant volume, hybrid standard product solution I are made, will mix
The doubling dilution of standard solution I obtains hybrid standard product solution II, and III, IV, V, the concentration of wherein tanshin polyphenolic acid B is respectively 0.05-
0.10mg/mL, 0.10-0.20mg/mL, 0.20-0.40mg/mL, 0.50-0.70mg/mL, 0.80-1.20mg/mL；Danshensu
Concentration be respectively 0.10-0.20mg/mL, 0.20-0.40mg/mL, 0.50-0.70mg/mL, 0.80-1.20mg/mL, 1.80-
2.20mg/mL；The concentration of Rosmarinic acid is respectively 0.05-0.10mg/mL, 0.10-0.20mg/mL, 0.20-0.40mg/mL,
0.50-0.70mg/mL, 0.80-1.0mg/mL；The concentration of protocatechualdehyde is respectively 0.05-0.10mg/mL, 0.10-0.20mg/
ML, 0.20-0.40mg/mL, 0.50-0.70mg/mL, 0.80-1.20mg/mL；Ginsenoside Rg1 0.10-0.30mg/mL,
0.40-0.60mg/mL, 0.70-0.90mg/mL, 1.20-1.80mg/mL, 2.50-3.50mg/mL；Ginsenoside Rb1 0.10-
0.20mg/mL, 0.30-0.40mg/mL, 0.50-0.60mg/mL, 1.0-1.20mg/mL, 2.0-2.50mg/mL；
Step 3, the preparation of need testing solution：Dripping pill extract medicinal extract is freeze-dried, obtains the dried powder of dripping pill extract,
Accurately weighed extract dried powder 10-20mg, is separately added into 500-600 μ L deuterated methanol working solutions, ultrasonic 8-12min, from
The heart 13000-15000rpm, 8-12min, it is transferred in 4-6mm nuclear magnetic tubes and is used as need testing solution；
Step 4, determine：By standard solution and need testing solution injection NMR spectrometer with superconducting magnet, nuclear magnetic resonance ripple is obtained
Spectrogram；
Step 5, danshensu, tanshin polyphenolic acid B, Rosmarinic acid, former youngster in need testing solution are calculated according to standard NMR spectrum figure
Tea aldehyde, ginsenoside Rg1, the content of ginsenoside Rb1；
Wherein, the proton magnetic tester condition that NMR spectrometer with superconducting magnet described in step 4 is set is as follows：
The 1H NMR spectras of sample solution, measurement temperature 298K, observing frequency 590- are gathered using 30 ° of pulse train zg30
610MHz, spectrum width 12330-12340Hz, sampled data points 65530-65540, scanning times 16-32 times, time delay
1.0000s, acquisition time 2.6564s, gain acceptance in 120-130, calibrated with TSP signals.
4. detection method according to claim 1, it is characterised in that step is as follows:
Step 1, the preparation of working solution：Accurately weighed 3- (trimethyl silyl) malonic acid-d4 sodium salt internal standard compounds are appropriate, with deuterated
Methanol solution constant volume, it is made in every 0.8-1.2mL deuterated methanols solution and contains 0.48-0.52mg 3- (trimethyl silyl) third
The storing solution of diacid-d4 sodium salts, then be made containing 0.0098-0.012mg/mL3- (trimethyl silyl) malonic acid-d4 sodium salts
Deuterated methanol working solution；
Step 2, the preparation of standard solution：Accurately weighed danshensu, tanshin polyphenolic acid B, Rosmarinic acid, protocatechualdehyde, ginseng respectively
Saponin(e Rg1, ginsenoside Rb1's standard items are appropriate, with deuterated methanol working solution constant volume, hybrid standard product solution I are made, will mix
The doubling dilution of standard solution I obtains hybrid standard product solution II, and III, IV, V, the concentration of wherein tanshin polyphenolic acid B is respectively
0.05mg/mL, 0.10mg/mL, 0.25mg/mL, 0.50mg/mL, 1.0mg/mL；The concentration of danshensu is respectively 0.10mg/mL,
0.25mg/mL, 0.50mg/mL, 1.0mg/mL, 2.0mg/mL；The concentration of Rosmarinic acid is respectively 0.05mg/mL, 0.10mg/
ML, 0.20mg/mL, 0.50mg/mL, 0.80mg/mL；The concentration of protocatechualdehyde is respectively 0.05mg/mL, 0.10mg/mL,
0.25mg/mL, 0.50mg/mL, 1.0mg/mL；Ginsenoside Rg1 0.2mg/mL, 0.4mg/mL, 0.8mg/mL, 1.5mg/mL,
3.0mg/mL；Ginsenoside Rb1 0.15mg/mL, 0.30mg/mL, 0.60mg/mL, 1.20mg/mL, 2.50mg/mL；
Step 3, the preparation of need testing solution：Dripping pill extract medicinal extract is freeze-dried, obtains the dried powder of dripping pill extract,
Accurately weighed extract dried powder 8-12mg, it is separately added into 480-520 μ L deuterated methanol working solutions, ultrasonic 8-12min, centrifugation
13000-15000rpm, 8-12min, it is transferred in 4-6mm nuclear magnetic tubes and is used as need testing solution；
Step 4, determine：By standard solution and need testing solution injection NMR spectrometer with superconducting magnet, nuclear magnetic resonance ripple is obtained
Spectrogram；
Step 5, danshensu, tanshin polyphenolic acid B, Rosmarinic acid, former youngster in need testing solution are calculated according to standard NMR spectrum figure
Tea aldehyde, ginsenoside Rg1, the content of ginsenoside Rb1；
Wherein, the proton magnetic tester condition that NMR spectrometer with superconducting magnet described in step 4 is set is as follows：
The 1H NMR spectras of sample solution, measurement temperature 298K, observing frequency are gathered using 30 ° of pulse train zg30
600.23MHz, spectrum width 12335.5Hz, sampled data points 65536, scanning times 16 or 32 times, time delay 1.0000s, collection
Time 2.6564s, gain acceptance in 128, calibrated with TSP signals.
5. detection method according to claim 1, it is characterised in that step is as follows:
Step 1, the preparation of working solution：Accurately weighed 3- (trimethyl silyl) malonic acid-d4 sodium salts (TSP) appropriate internal standard compound,
With deuterated methanol solution constant volume, it is made in every 1mL deuterated methanols solution and contains 0.50mg3- (trimethyl silyl) malonic acid-d4
The storing solution of sodium salt, then the deuterated methanol work containing 0.01mg/mL3- (trimethyl silyl) malonic acid-d4 sodium salts is made
Liquid；
Step 2, the preparation of standard solution：Accurately weighed danshensu, tanshin polyphenolic acid B, Rosmarinic acid, protocatechualdehyde, ginseng respectively
Saponin(e Rg1, ginsenoside Rb1's standard items are appropriate, with deuterated methanol working solution constant volume, hybrid standard product solution I are made, will mix
The doubling dilution of standard solution I obtains hybrid standard product solution II, and III, IV, V, the concentration of wherein tanshin polyphenolic acid B is respectively
0.0675mg/mL, 0.135mg/mL, 0.270mg/mL, 0.540mg/mL, 1.08mg/mL；The concentration of danshensu is respectively
0.122mg/mL, 0.244mg/mL, 0.488mg/mL, 0.975mg/mL, 1.95mg/mL；The concentration of Rosmarinic acid is respectively
0.0550mg/mL, 0.110mg/mL, 0.220mg/mL, 0.440mg/mL, 0.880mg/mL；The concentration of protocatechualdehyde is respectively
0.0625mg/mL, 0.125mg/mL, 0.250mg/mL, 0.500mg/mL, 1.000mg/mL；Ginsenoside Rg1 0.186mg/
ML, 0.373mg/mL, 0.745mg/mL, 1.49mg/mL, 2.98mg/mL；Ginsenoside Rb1 0.153mg/mL, 0.305mg/
ML, 0.610mg/mL, 1.26mg/mL, 2.52mg/mL；
Step 3, the preparation of need testing solution：Dripping pill extract medicinal extract is freeze-dried, obtains the dried powder of dripping pill extract,
Accurately weighed extract dried powder 10mg, it is separately added into 500 μ L deuterated methanol working solutions, ultrasonic 10min, centrifugation
14000rpm, 10min, it is transferred in 5mm nuclear magnetic tubes and is used as need testing solution；
Step 4, determine：By standard solution and need testing solution injection NMR spectrometer with superconducting magnet, nuclear magnetic resonance ripple is obtained
Spectrogram；
Step 5, danshensu, tanshin polyphenolic acid B, Rosmarinic acid, former youngster in need testing solution are calculated according to standard NMR spectrum figure
Tea aldehyde, ginsenoside Rg1, the content of ginsenoside Rb1；
Wherein, the proton magnetic tester condition that NMR spectrometer with superconducting magnet described in step 4 is set is as follows：
The 1H NMR spectras of sample solution, measurement temperature 298K, observing frequency are gathered using 30 ° of pulse train zg30
600.23MHz, spectrum width 12335.5Hz, sampled data points 65536, scanning times 16 times, time delay 1.0000s, acquisition time
2.6564s, gain acceptance in 128, calibrated with TSP signals.
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Cited By (3)
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| CN108152319A (en) * | 2018-02-07 | 2018-06-12 | 山东省分析测试中心 | A kind of method that ginsenoside Rd's reference substance content is measured based on proton magnetic quantitative analysis tech |
| CN108398452A (en) * | 2018-02-07 | 2018-08-14 | 山东省分析测试中心 | A method of ginsenoside Re's reference substance content is measured based on proton magnetic quantitative analysis tech |
| CN113237913A (en) * | 2021-03-31 | 2021-08-10 | 正大青春宝药业有限公司 | Salvia miltiorrhiza injection and process intermediate thereof1H NMR one-test-multiple-evaluation method |
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| CN102160872A (en) * | 2010-02-23 | 2011-08-24 | 天津天士力制药股份有限公司 | Compound salvia dropping pill capsules |
| CN104297277A (en) * | 2013-07-15 | 2015-01-21 | 天士力制药集团股份有限公司 | 1H-NMR fingerprint spectrum of compound red sage root dropping pill |
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| CN108398452A (en) * | 2018-02-07 | 2018-08-14 | 山东省分析测试中心 | A method of ginsenoside Re's reference substance content is measured based on proton magnetic quantitative analysis tech |
| CN113237913A (en) * | 2021-03-31 | 2021-08-10 | 正大青春宝药业有限公司 | Salvia miltiorrhiza injection and process intermediate thereof1H NMR one-test-multiple-evaluation method |
| CN113237913B (en) * | 2021-03-31 | 2023-10-13 | 正大青春宝药业有限公司 | Red sage root injection and its technological intermediate 1 H NMR one-measurement-multiple-evaluation method |
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