CN104330511B - A kind of invigorating heart is relaxed the content assaying method of ginsenoside in preparation - Google Patents
A kind of invigorating heart is relaxed the content assaying method of ginsenoside in preparation Download PDFInfo
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Abstract
The invention discloses the content assaying methods of ginsenoside in a kind of easypro preparation of invigorating heart, it is with ginsenoside Rg1、Re、Rb1, Rc reference substance be control, using methanol as mobile phase A, the high performance liquid chromatography that 0.02%~2% phosphate aqueous solution is Mobile phase B gradient elution.The present invention can simultaneously relax ginsenoside Rg in preparation to invigorating heart1、Re、Rb1, Rc ingredient content carry out the measurement of quick, accurate, high reproducibility, high-recovery.
Description
Technical field
The present invention relates to the content assaying methods of ginsenoside in a kind of easypro preparation of invigorating heart, belong to traditional Chinese medicine quality detection technique
Field.
Background technique
The easypro Chinese materia medica preparation of invigorating heart is formed with seven flavor medicines such as ginseng, Radix Astragali, Radix Salviae Miltiorrhizae, Radix Ophiopogonis, Schisandra chinensis, Rhizoma Chuanxiong, hawthorn, tool
There is the effect of Yiqi and vein recovery, activating microcirculation and removing stasis medicinal, nourishing Yin and promoting production of body fluid, be used for deficiency of both qi and yin, palpitaition knotted pulse, not easypro, pectoralgia uncomfortable in chest is come of age
Angina pectoris is shown in the person that has above-mentioned symptom, be clinically widely used at present treatment angina pectoris, myocardial ischemia, various arrhythmia cordis,
Atrial premature beats, pectoralgia and coronary disease and angina pectoris trouble etc..
Contain ginseng in the easypro Chinese materia medica preparation of invigorating heart, ginsenoside is the principle active component of ginseng, has clearly been known
There are about more than 40 for GS monomer；Content in ginseng is 4% or so.It is numerous studies have shown that its anti-tumor activity with higher,
It has no toxic side effect to normal cell, has synergistic effect with other chemotherapeutics (such as cis-platinum) use in conjunction.Wherein, Rg1: it can be quick
It relieves fatigue, improves learning and memory, delays senescence, there is stimulating central nervous system effect, inhibit platelet aggregation effect.Re has
Inhibit nervous centralis, promotes DNA, RNA synthesis, increase the effect of plasma corticosterone, expand blood vessel, there are also antifatigue effects.
Rb1: tool influences the potentiality of animal testis, also will affect the embryonic development of mouse, has the function of enhancing choline system, increases second
The synthesis and release and improvement memory effect of phatidylcholine.Rc: have the function of inhibiting cancer cell.The activity of spermatozoon can be increased
Power.It can be seen that the Rg in ginsenoside1、Re、Rb1, Rc has a highly important pharmacological action, therefore carries out to its content
Measurement has a very important significance.
HPLC measures ginsenoside Rg in invigorating heart relieving capsule1, Re, Rb1Content (Chinese experimental pharmacology of traditional Chinese medical formulae magazine, Du Jia
Newly, in September, 2010 o. 11th of volume 16,88-90 pages) one ginsenoside Rg in HPLC measurement invigorating heart relieving capsule is disclosed herein1,
Re, Rb1Content method, still, existing invigorating heart relax Chinese materia medica preparation quality determining method there is no to Ginsenoside Rc
The content of ingredient is measured, and is the quality inspection standard for further improving the easypro Chinese materia medica preparation of invigorating heart, to ensure its drug quality,
And in order to reduce the error for separately repeatedly measuring different ginseng saponin constituent contents and generating, if energy single-time measurement is a variety of
The content of ingredient can not only save time and cost, it is also possible that the test result under the conditions of same system it is more stable, can
It leans on, therefore the applicant sets about having studied while measuring ginsenoside Rg in the easypro preparation of invigorating heart1、Re、Rb1, tetra- kinds of component contents of Rc
Method.
Summary of the invention
The object of the present invention is to provide the content assaying methods of ginsenoside in a kind of easypro preparation of invigorating heart, can be simultaneously
It relaxes ginsenoside Rg in preparation to invigorating heart1、Re、Rb1, Rc ingredient content carry out quick, accurate, high reproducibility, high-recovery
Measurement.
To solve prior art problem, the present invention is adopted the following technical scheme that:
A kind of invigorating heart is relaxed the content assaying method of ginsenoside in preparation, it is with ginsenoside Rg1、Re、Rb1, Rc control
Product are control, using methanol as mobile phase A, the high performance liquid chromatography that 0.02%~2% phosphate aqueous solution is Mobile phase B gradient elution
Method.
The content assaying method of ginsenoside is according to high performance liquid chromatography in the easypro preparation of invigorating heart above-mentioned, and steps are as follows:
(1) chromatographic condition and system suitability: using octadecylsilane chemically bonded silica as filler；It is stream with methanol
Dynamic phase A, 0.02%~2% phosphate aqueous solution are Mobile phase B, gradient elution；Detection wavelength is 203nm；Flow velocity be 0.8~
1.0mL/min；Column temperature is 25~35 DEG C；Number of theoretical plate is calculated with ginsenoside Re peak is not less than 3000；
(2) preparation of reference substance solution: ginsenoside Rg is taken1, ginsenoside Re, ginsenoside Rb1, Ginsenoside Rc couple
Appropriate according to product, accurately weighed, respectively plus every 1mL is made containing ginsenoside Rg in methanol1The solution of 0.1mg, every 1mL contain ginsenoside
The solution of Re0.1mg, every 1mL Rb containing ginsenoside1The solution of the solution of 0.25mg and every 1mL 0.2mg containing Ginsenoside Rc, i.e.,
?；
(3) preparation of test solution: using following methods 1.-any one of 4.:
1. precision weighs the easypro 5~20g of preparation or its content of invigorating heart, set in 100mL stuffed conical flask, methanol is added in precision
50mL, weighed weight, ultrasound or 30~60min of reflow treatment, supply weight with methanol；Filtration, take subsequent filtrate to get；
2. precision weighs the easypro 5~20g of preparation or its content of invigorating heart, set in 100mL stuffed conical flask, methanol is added in precision
50mL, weighed weight, heating and refluxing extraction 30min~60min supply weight with methanol, filtration；Precision measures subsequent filtrate
25mL is added water and stirred, and filtration adds a small amount of water washing filter cake and container 3 times, is merged washing lotion and filtrate, is concentrated to dryness, residue adds first
Alcohol dissolution, and is transferred in 25mL measuring bottle, and methanol dilution is added to shake up, filter to scale, take subsequent filtrate to get；
3. precision weighs the easypro 5~20g of preparation or its content of invigorating heart, set in 100mL stuffed conical flask, methanol is added in precision
50mL, weighed weight are ultrasonically treated 30min~60min, supply weight with methanol, filter；Precision measures subsequent filtrate 25mL, uses
Water-saturated n-butanol extracts 3 times, each 20mL, merges n-butanol liquid, is washed 3 times, each 20mL with aqueous slkali, the concentration of n-butanol liquid
To doing, residue adds water 5mL to make to dissolve, and by the large pore resin absorption column or neutral alumina column handled well, is washed to colourless, receipts
The ethanol eluate of collection 50%~80%, is concentrated to dryness, residue adds methanol to dissolve, and is transferred in 25mL measuring bottle, adds methanol dilute
Release to scale, shake up, filter, take subsequent filtrate to get；
4. precision weighs the easypro 5~20g of preparation or its content of invigorating heart, addition petroleum ether, three chloromethanes in Soxhlet extractor is set
Alkane or both mixture is heated to reflux 3h, and the dregs of a decoction volatilize solvent, moves into 100mL stuffed conical flask together with filtration paper cylinder, and alcohol is added
Aqueous slkali 50mL, is heated to reflux 1h, lets cool, filtration, washs filter cake and container 3 times with a small amount of methanol, merges washing lotion and filtrate, dense
Be reduced to dry, residue adds water 50mL to dissolve, and the n-butanol that is saturated with water extracts 4 times, merge n-butanol liquid, respectively with ammonia solution, water,
1% potassium dihydrogen phosphate respectively washs 1 time, each 40mL, discards cleaning solution, and n-butanol liquid is evaporated, and residue adds methanol dissolution simultaneously
Move in 50mL measuring bottle, with methanol dilution to scale, shake up, filter, take subsequent filtrate to get；
(4) it measures: it is accurate respectively to draw reference substance solution and each 10 μ L of test solution, high performance liquid chromatograph is injected,
Measurement to get.
In preceding method, the actual conditions of the gradient elution are as follows: 0~5min:A:B=25:75；5~35min:A:B=
25~35:75~65；35~70min:A:B=35~50:65~50；70~80min:A:B=50~25:50~75；80~
110min:A:B=25:75.
In preceding method, the preparation method of test solution 3. described in aqueous slkali be 1% sodium hydroxide solution or ammonia
Test solution, the large pore resin absorption column are D101, AB-8 or NKA-9 resin；The preparation method of test solution 4. described in
Alcohol aqueous slkali be 2% methanolic potassium hydroxide liquid.
In preceding method, the Mobile phase B is preferably 0.2% phosphate aqueous solution.
The invigorating heart of the present invention preparation that relaxes is made of being prepared as follows: 100~150g of Rhizoma Chuanxiong and hawthorn 150~
250g, 150~250g of Radix Astragali and 150~250g of Radix Ophiopogonis are added water to cook secondary, 2.5 hours first times, and second 1.5 hours, filter
It crosses, merging filtrate, being concentrated into relative density is 1.10~1.15 (80 DEG C), 85% ethyl alcohol of one times of amount is added into concentrate,
It mixes, stands, filtration, filtrate is concentrated, spare；85% alcohol reflux is added in 100~150g of Schisandra chinensis and 250~300g of Radix Salviae Miltiorrhizae
It extracts secondary, 3 hours first times, second 1.5 hours, filtration, merging filtrate is recovered under reduced pressure ethyl alcohol and is concentrated into relative density
For 1.25~1.30 (80 DEG C), alcohol-extracted extract is obtained, it is spare；Aforementioned two kinds of medicinal extract is mixed, addition ginseng fine powder 200g (or its alcohol
Extract), it mixes, dry, pulverize, appropriate amount of auxiliary materials is added, dosage form described in any pharmacy is made, such as: tablet, capsule
The dosage forms such as agent, oral solution, buccal tablet, granule, electuary, pill, paste, powder, sublimed preparation.
In the easypro preparation of aforementioned invigorating heart in the content assaying method of ginsenoside, with ginsenoside in the easypro preparation of every gram of invigorating heart
Rgl, ginsenoside Re, ginsenoside Rb1With the total amount meter of Ginsenoside Rc, 3.4mg must not be less than.
Although ginsenoside Rg1、Re、Rb1, Rc belong to same constituents, but it is respectively had any different, suitable assay item
Part is not often identical, to measure their content simultaneously, it is necessary to be groped the terms and conditions parameter of HPLC, be tested.For
A variety of ginsenoside ingredients can be effectively separated, to mobile phase type, mobile phase ratio and condition of gradient elution
Grope, determine to be very crucial.The present invention continues to explore on the basis of the prior art, by mobile phase type, stream
Dynamic Phase Proportion, condition of gradient elution, reference substance and preparation method of test sample etc. are groped, are screened, and invigorating heart is finally established
Ginsenoside Rg in easypro preparation1、Re、Rb1, Rc high performance liquid chromatography content assaying method.It is the phase that applicant is carried out below
Close the main contents of experimental study and investigation.
One, instrument and reagent (being shown in Table 1)
1 instrument of table and reagent table
Two, method and result
1, the selection of chromatographic condition
The selection of 1.1 column temperatures
To find optimum column temperature, applicant is tested using different column temperatures, is observed as a result, the results are shown in Table 2.
2 column temperature of table tests table
As shown in Table 2, select 25~35 DEG C as column temperature.
The selection of 1.2 flow velocitys
To find optimum flow rate, applicant is tested using different in flow rate, is recorded chromatographic peak, be the results are shown in Table 3.
3 chromatographic peak record sheet of table
As shown in Table 3, setting flow velocity is 0.8~1.0mL/min, and each peak-to-peak resolution ratio is good, and chromatographic time is short, peak face
Product is moderate, and effect is best.
The selection of 1.3 mobile phases
To find optimal flow phase, applicant carries out UV detection using different mobile phases, records chromatographic peak, as a result as follows.
(1) acetonitrile-water gradient: 0~5min:A:B=25:75；5~35min:A:B=25~35:75~65；35~
70min:A:B=35~50:65~50；70~80min:A:B=50~25:50~75；80~110min:A:B=25:75.
(2) -0.02% phosphate aqueous solution gradient of acetonitrile: 0~5min:A:B=25:75；5~35min:A:B=25~35:
75~65；35~70min:A:B=35~50:65~50；70~80min:A:B=50~25:50~75；80~110min:
A:B=25:75.
(3) -0.2% phosphate aqueous solution gradient of acetonitrile: 0~5min:A:B=25:75；5~35min:A:B=25~35:
75~65；35~70min:A:B=35~50:65~50；70~80min:A:B=50~25:50~75；80~110min:
A:B=25:75.
(4) -1% phosphate aqueous solution gradient of acetonitrile: 0~5min:A:B=25:75；5~35min:A:B=25~35:75
~65；35~70min:A:B=35~50:65~50；70~80min:A:B=50~25:50~75；80~110min:A:B
=25:75.
(5) -2% phosphate aqueous solution gradient of acetonitrile: 0~5min:A:B=25:75；5~35min:A:B=25~35:75
~65；35~70min:A:B=35~50:65~50；70~80min:A:B=50~25:50~75；80~110min:A:B
=25:75.
(6) methanol-water gradient: 0~5min:A:B=25:75；5~35min:A:B=25~35:75~65；35~
70min:A:B=35~50:65~50；70~80min:A:B=50~25:50~75；80~110min:A:B=25:75.
(7) -0.02% phosphate aqueous solution gradient of methanol: 0~5min:A:B=25:75；5~35min:A:B=25~35:
75~65；35~70min:A:B=35~50:65~50；70~80min:A:B=50~25:50~75；80~110min:
A:B=25:75.
(8) -0.2% phosphate aqueous solution gradient of methanol: 0~5min:A:B=25:75；5~35min:A:B=25~35:
75~65；35~70min:A:B=35~50:65~50；70~80min:A:B=50~25:50~75；80~110min:
A:B=25:75.
(9) -1% phosphate aqueous solution gradient of methanol: 0~5min:A:B=25:75；5~35min:A:B=25~35:75
~65；35~70min:A:B=35~50:65~50；70~80min:A:B=50~25:50~75；80~110min:A:B
=25:75.
(10) -2% phosphate aqueous solution gradient of methanol: 0~5min:A:B=25:75；5~35min:A:B=25~35:75
~65；35~70min:A:B=35~50:65~50；70~80min:A:B=50~25:50~75；80~110min:A:B
=25:75.
(11) -0.2% phosphate aqueous solution gradient of methanol: 0~5min:A:B=10:90；5~40min:A:B=10~28:
90~72；40~75min:A:B=28~38:72~62；75~80min:A:B=38~10:62~90；80~110min:
A:B=10:90.
(12) -0.2% phosphate aqueous solution gradient of methanol: 0~5min:A:B=15:85；5~40min:A:B=15~30:
85~70；40~75min:A:B=30~40:70~60；75~80min:A:B=40~15:60~85；80~110min:
A:B=15:85.
(13) -0.2% phosphate aqueous solution gradient of methanol: 0~5min:A:B=12:88；5~40min:A:B=12~30:
88~70；40~75min:A:B=30~35:70~65；75~80min:A:B=35~12:65~88；80~110min:
A:B=12:88.
(14) -0.2% phosphate aqueous solution gradient of methanol: 0~8min:A:B=15:85；8~40min:A:B=15~25:
85~75；40~55min:A:B=25~35:75~65；55~80min:A:B=35~15:65~85；80~110min:
A:B=15:85.
(15) -0.2% phosphate aqueous solution gradient of methanol: 0~10min:A:B=15:85；10~45min:A:B=15~
25:85~75；45~60min:A:B=25~35:75~65；60~80min:A:B=35~15:65~85；80~
110min:A:B=15:85.
(16) -0.2% phosphate aqueous solution gradient of methanol: 0~10min:A:B=20:80；10~45min:A:B=20~
35:80~65；45~60min:A:B=35~45:65~55；60~80min:A:B=45~20:55~80；80~
110min:A:B=20:80.
As the result is shown Mobile phase B and elution requirement under the same conditions, mobile phase A with acetonitrile and methanol, and
Methanol is slightly good compared with acetonitrile effect, and because methanol prices are cheaper than acetonitrile, therefore preferable mobile phase A is methanol；It is first in mobile phase A
Under alcohol and the identical situation of elution requirement, Mobile phase B use water or 0.02%~2% phosphate aqueous solution, but with centainly it is dense
Preferable effect can be obtained in the phosphate aqueous solution substitution water of degree, and mobile phase is more preferable with -0.2% phosphate aqueous solution effect of methanol；
Using the flow visualizing of -0.2% phosphate aqueous solution of methanol, the mobile phase of different time sections different proportion is adjusted, is touched
Rope obtains methanol-phosphate aqueous solution system and uses chromatographic peak obtained by the gradient elution mode of above-mentioned (8) preferable, and separating degree accords with
It closes and requires, peak shape is preferable.
2, the preparation of reference substance solution
Take ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, Ginsenoside Rc's reference substance it is appropriate, it is accurately weighed, point
Not plus every 1mL is made containing ginsenoside Rg in methanol1The solution of 0.1mg, the solution of every 1mL 0.1mg containing ginsenoside Re, every 1mL
Rb containing ginsenoside1The solution of the solution of 0.25mg and every 1mL 0.2mg containing Ginsenoside Rc to get；
3, the preparation of test solution
Using following methods 1.-any one of 4.:
1. precision weighs the easypro 5~20g of preparation or its content of invigorating heart, set in 100mL stuffed conical flask, methanol is added in precision
50mL, weighed weight, ultrasound or 30~60min of reflow treatment, supply weight with methanol；Filtration, take subsequent filtrate to get；
2. precision weighs the easypro 5~20g of preparation or its content of invigorating heart, set in 100mL stuffed conical flask, methanol is added in precision
50mL, weighed weight, heating and refluxing extraction 30min~60min supply weight with methanol, filtration；Precision measures subsequent filtrate
25mL is added water and stirred, and filtration adds a small amount of water washing filter cake and container 3 times, is merged washing lotion and filtrate, is concentrated to dryness, residue adds first
Alcohol dissolution, and is transferred in 25mL measuring bottle, and methanol dilution is added to shake up, filter to scale, take subsequent filtrate to get；
3. precision weighs the easypro 5~20g of preparation or its content of invigorating heart, set in 100mL stuffed conical flask, methanol is added in precision
50mL, weighed weight are ultrasonically treated 30min~60min, supply weight with methanol, filter；Precision measures subsequent filtrate 25mL, uses
Water-saturated n-butanol extracts 3 times, each 20mL, merges n-butanol liquid, with aqueous slkali (such as 1% sodium hydroxide solution, ammonia solution)
It washes 3 times, each 20mL, n-butanol liquid is concentrated to dryness, and residue adds water 5mL to make to dissolve, and passes through the large pore resin absorption column handled well
(such as D101, AB-8, NKA-9 resin) or neutral alumina column are washed to colourless, the ethanol eluate of collection 50%~80%,
It is concentrated to dryness, residue adds methanol to dissolve, and is transferred in 25mL measuring bottle, adds methanol dilution to scale, shakes up, filter, take continuous filter
Liquid to get；
4. precision weighs the easypro 5~20g of preparation or its content of invigorating heart, addition petroleum ether, three chloromethanes in Soxhlet extractor is set
Alkane or both mixture is heated to reflux 3h, and the dregs of a decoction volatilize solvent, moves into 100mL stuffed conical flask together with filtration paper cylinder, and alcohol is added
Aqueous slkali 50mL (such as 2% methanolic potassium hydroxide liquid), is heated to reflux 1h, lets cool, and filtration washs filter cake and container with a small amount of methanol
3 times, merge washing lotion and filtrate, be concentrated to dryness, residue adds water 50mL to dissolve, the n-butanol being saturated with water extract 4 times (20,20,
10,10mL), merge n-butanol liquid, respectively washed 1 time, each 40mL with ammonia solution, water, 1% potassium dihydrogen phosphate respectively, abandoned
Cleaning solution is removed, n-butanol liquid is evaporated, and residue adds methanol to dissolve and moves in 50mL measuring bottle, with methanol dilution to scale, is shaken up, and is filtered
Cross, take subsequent filtrate to get.
Four kinds of preparation method of test article are compared, the results are shown in Table 4:
Table 4
| Method | Test result |
| Method one | The extraction efficiency of ginsenoside is high, but purity is slightly lower |
| Method two | The extraction efficiency of ginsenoside is high, but purity is slightly lower |
| Method three | The extraction efficiency of ginsenoside is slightly lower, but purity is high |
| Method four | The extraction efficiency of ginsenoside is slightly lower, but purity is high |
As shown in Table 4, test sample being prepared by method one and method two, step simple operations are convenient, and extraction efficiency is high, but
It is that purity is slightly lower, impurity is more；Test sample is prepared using method three and method four, complex steps, extraction efficiency is low compared with the above two,
But the ginsenoside purity is high extracted, impurity residual quantity is low, reduces the influence power of impurity in detection process, more conducively guarantees inspection
Survey the Stability and veracity of result.
4, the selection of Detection wavelength
The present inventor is by a large number of experiments repeatedly to ginsenoside Rg1、Re、Rb1, Rc reference substance solution carry out spectrum it is ultraviolet
Scanning, discovery especially have maximum absorption wavelength, therefore ginsenoside Rg in the present invention at 203nm1、Re、Rb1, Rc detect wave
Length is determined as 203nm.
5, the preparation of standard curve
It is accurate respectively to measure ginsenoside Rg1、Re、Rb1, Rc reference substance solution, concentration be respectively 0.1mg/mL,
0.1mg/mL, 0.25mg/mL, 0.2mg/mL, 5,10,15,20,25 μ L of sample introduction, measures peak area and records, with peak area respectively
It (Y) is ordinate, sample volume (X) is that abscissa draws standard curve, and regression equation and the range of linearity are shown in Table 5.
54 kinds of ginsenoside regression equations of table and related coefficient
| Component | Regression equation | Related coefficient | The range of linearity (μ g) |
| Rg1 | Y=289.59X+33.504 | 0.9999 | 0.51~2.55 |
| Re | Y=242.19X+2.560 | 0.9999 | 0.515~2.575 |
| Rb1 | Y=463.96X+53.460 | 0.9999 | 1.025~5.125 |
| Rc | Y=405.97X+46.785 | 0.9999 | 1.02~5.10 |
6, Precision Experiment
10 μ L of reference substance solution is taken, repeats sample introduction 6 times, measures ginsenoside Rg1、Re、Rb1, Rc peak area, calculate to obtain RSD
Respectively 0.47%, 0.36%, 0.51%, 0.33%, show that precision is good.
7, stability experiment
Precision draws same test solution, at room temperature respectively at 0,2,4,8,12, respectively draw 10 μ L for 24 hours, sample introduction 6 altogether
It is secondary, measure ginsenoside Rg1、Re、Rb1, Rc peak area, calculate RSD is respectively 0.64%, 0.91%, 1.36%, 0.78%,
The solution that shows that treated at room temperature for 24 hours in stablize, have good stability.
8, repeated experiment
6 parts of same test solution is taken, each 10 μ L of sample introduction measures ginsenoside Rg1、Re、Rb1, Rc peak area, calculate
RSD is respectively 0.83%, 0.76%, 1.22%, 0.74%, is shown repeated good.
9, sample recovery rate is tested
Precision weighs the sample 100mg of known content, be separately added into be equivalent to each measured object content 80%, 100%,
120% reference substance stock solution prepares each 3 parts of basic, normal, high rate of recovery sample according to method below the preparation of test solution,
It measures the content of each ginseng saponin constituent and calculates sample recovery rate (n=9), as a result ginsenoside Rg1、Re、Rb1, 4 kinds of groups of Rc
The average recovery rate divided is respectively 101.7% (RSD=1.16%), 101.3% (RSD=1.40%), 100.4% (RSD=
1.06%), 100.5% (RSD=0.76%) shows that this method accuracy is good.
10, sample size measures
Take the easypro preparation of 5 batches of invigorating hearts or its content appropriate, it is accurately weighed, respectively by the preparation of reference substance solution, for examination
Under the preparation of product solution prepared by method, and by assay under the investigation item of linear relationship, (test solution, reference substance are molten
Liquid distinguishes 10 μ L of sample introduction), calculate ginsenoside Rg1、Re、Rb1, the content of Rc and the total amount of four kinds of ginsenosides, the results are shown in Table
6。
6 sample size measurement result (n=5) of table
The present invention relaxes ginsenoside Rg in preparation to invigorating heart by high performance liquid chromatography1、Re、Rb1, Rc ingredient content
It being measured, the specificity of the method is strong, precision is high, reproducible, the rate of recovery is high, stability is high, measurement result is accurate,
Effective control drug quality is achieved the purpose that, it is ensured that the stabilization of product quality and clinical application are safely, effectively.And
And the content of four kinds of ginsenoside ingredients of single-time measurement of the present invention, can avoid because it is separated repeatedly measure different ginsenosides at
Point content and the error generated, moreover it is possible to save time and cost, and make the measurement result under the conditions of same system it is more stable, can
It leans on.
Specific embodiment
The embodiment of the present invention 1: a kind of invigorating heart is relaxed ginsenoside Rg in preparation1、Re、Rb1, Rc content assaying method, step
It is as follows:
(1) chromatographic condition and system suitability: using octadecylsilane chemically bonded silica as filler；It is stream with methanol
Dynamic phase A, 0.02% phosphate aqueous solution are Mobile phase B, gradient elution, elution requirement are as follows: 0~5min:A:B=25:75；5~
35min:A:B=25~35:75~65；35~70min:A:B=35~50:65~50；70~80min:A:B=50~25:
50~75；80~110min:A:B=25:75；Detection wavelength is 203nm, and flow velocity 0.8mL/min, column temperature is 25 DEG C, theoretical
Plate number is calculated with ginsenoside Re peak is not less than 3000；
(2) preparation of reference substance solution: ginsenoside Rg is taken1, ginsenoside Re, ginsenoside Rb1, Ginsenoside Rc couple
Appropriate according to product, accurately weighed, respectively plus every 1mL is made containing ginsenoside Rg in methanol1The solution of 0.1mg, every 1mL contain ginsenoside
The solution of Re0.1mg, every 1mL Rb containing ginsenoside1The solution of the solution of 0.25mg and every 1mL 0.2mg containing Ginsenoside Rc, i.e.,
?；
(3) preparation of test solution: precision weighs the easypro 5~20g of preparation or its content of invigorating heart, sets 100mL tool plug cone
In shape bottle, methanol 50mL, weighed weight, ultrasound or 30~60min of reflow treatment is added in precision, supplies weight with methanol；Filtration,
Take subsequent filtrate to get；
(4) it measures: it is accurate respectively to draw reference substance solution and each 10 μ L of test solution, high performance liquid chromatograph is injected,
Measurement to get.
The embodiment of the present invention 2: a kind of invigorating heart is relaxed ginsenoside Rg in preparation1、Re、Rb1, Rc content assaying method, step
It is as follows:
(1) chromatographic condition and system suitability: using octadecylsilane chemically bonded silica as filler；It is stream with methanol
Dynamic phase A, 0.2% phosphate aqueous solution are Mobile phase B, gradient elution, elution requirement are as follows: 0~5min:A:B=25:75；5~
35min:A:B=25~35:75~65；35~70min:A:B=35~50:65~50；70~80min:A:B=50~25:
50~75；80~110min:A:B=25:75；Detection wavelength is 203nm, and flow velocity 0.9mL/min, column temperature is 30 DEG C, theoretical
Plate number is calculated with ginsenoside Re peak is not less than 3000；
(2) preparation of reference substance solution: ginsenoside Rg is taken1, ginsenoside Re, ginsenoside Rb1, Ginsenoside Rc couple
Appropriate according to product, accurately weighed, respectively plus every 1mL is made containing ginsenoside Rg in methanol1The solution of 0.1mg, every 1mL contain ginsenoside
The solution of Re0.1mg, every 1mL Rb containing ginsenoside1The solution of the solution of 0.25mg and every 1mL 0.2mg containing Ginsenoside Rc, i.e.,
?；
(3) preparation of test solution: precision weighs the easypro 5~20g of preparation or its content of invigorating heart, sets 100mL tool plug cone
In shape bottle, methanol 50mL, weighed weight is added in precision, and heating and refluxing extraction 30min~60min supplies weight with methanol, is filtered
It crosses；Precision measures subsequent filtrate 25mL, adds water and stirs, and filters, and adds a small amount of water washing filter cake and container 3 times, merges washing lotion and filtrate,
It is concentrated to dryness, residue adds methanol to dissolve, and is transferred in 25mL measuring bottle, adds methanol dilution to scale, shakes up, filter, take continuous filter
Liquid to get；
(4) it measures: it is accurate respectively to draw reference substance solution and each 10 μ L of test solution, high performance liquid chromatograph is injected,
Measurement to get.
The embodiment of the present invention 3: a kind of invigorating heart is relaxed ginsenoside Rg in preparation1、Re、Rb1, Rc content assaying method, step
It is as follows:
(1) chromatographic condition and system suitability: using octadecylsilane chemically bonded silica as filler；It is stream with methanol
Dynamic phase A, 1% phosphate aqueous solution are Mobile phase B, gradient elution, elution requirement are as follows: 0~5min:A:B=25:75；5~35min:
A:B=25~35:75~65；35~70min:A:B=35~50:65~50；70~80min:A:B=50~25:50~75；
80~110min:A:B=25:75；Detection wavelength is 203nm, and flow velocity 1.0mL/min, column temperature is 35 DEG C, and number of theoretical plate is with people
Join the peak saponin(e Re to calculate not less than 3000；
(2) preparation of reference substance solution: ginsenoside Rg is taken1, ginsenoside Re, ginsenoside Rb1, Ginsenoside Rc couple
Appropriate according to product, accurately weighed, respectively plus every 1mL is made containing ginsenoside Rg in methanol1The solution of 0.1mg, every 1mL contain ginsenoside
The solution of Re0.1mg, every 1mL Rb containing ginsenoside1The solution of the solution of 0.25mg and every 1mL 0.2mg containing Ginsenoside Rc, i.e.,
?；
(3) preparation of test solution: precision weighs the easypro 5~20g of preparation or its content of invigorating heart, sets 100mL tool plug cone
In shape bottle, methanol 50mL is added in precision, and weighed weight is ultrasonically treated 30min~60min, supplies weight with methanol, is filtered；Essence
Close measurement subsequent filtrate 25mL is saturated with water extracting n-butyl alcohol 3 times, each 20mL, merges n-butanol liquid, with aqueous slkali (ammonia solution)
It washes 3 times, each 20mL, n-butanol liquid is concentrated to dryness, and residue adds water 5mL to make to dissolve, and passes through the large pore resin absorption column handled well
(D101), be washed to it is colourless, collect 50%~80% ethanol eluate, be concentrated to dryness, residue adds methanol to dissolve, and is transferred to
In 25mL measuring bottle, methanol dilution is added to shake up to scale, filtered, take subsequent filtrate to get；
(4) it measures: it is accurate respectively to draw reference substance solution and each 10 μ L of test solution, high performance liquid chromatograph is injected,
Measurement to get.
The embodiment of the present invention 4: a kind of invigorating heart is relaxed ginsenoside Rg in preparation1、Re、Rb1, Rc content assaying method, step
It is as follows:
(1) chromatographic condition and system suitability: using octadecylsilane chemically bonded silica as filler；It is stream with methanol
Dynamic phase A, 2% phosphate aqueous solution are Mobile phase B, gradient elution, elution requirement are as follows: 0~5min:A:B=25:75；5~35min:
A:B=25~35:75~65；35~70min:A:B=35~50:65~50；70~80min:A:B=50~25:50~75；
80~110min:A:B=25:75；Detection wavelength is 203nm, and flow velocity 1.0mL/min, column temperature is 35 DEG C, and number of theoretical plate is with people
Join the peak saponin(e Re to calculate not less than 3000；
(2) preparation of reference substance solution: ginsenoside Rg is taken1, ginsenoside Re, ginsenoside Rb1, Ginsenoside Rc couple
Appropriate according to product, accurately weighed, respectively plus every 1mL is made containing ginsenoside Rg in methanol1The solution of 0.1mg, every 1mL contain ginsenoside
The solution of Re0.1mg, every 1mL Rb containing ginsenoside1The solution of the solution of 0.25mg and every 1mL 0.2mg containing Ginsenoside Rc, i.e.,
?；
(3) preparation of test solution: precision weighs the easypro 5~20g of preparation or its content of invigorating heart, sets in Soxhlet extractor
Petroleum ether, chloroform or both mixture is added and is heated to reflux 3h, the dregs of a decoction volatilize solvent, move into 100mL tool together with filtration paper cylinder
It fills in conical flask, alcohol aqueous slkali (2% methanolic potassium hydroxide liquid) 50mL is added, is heated to reflux 1h, lets cool, filter, with a small amount of first
Alcohol washs filter cake and container 3 times, merges washing lotion and filtrate, is concentrated to dryness, and residue adds water 50mL to dissolve, the n-butanol being saturated with water
It extracts 4 times, merges n-butanol liquid, respectively washed 1 time with ammonia solution, water, 1% potassium dihydrogen phosphate respectively, each 40mL is discarded
Cleaning solution, n-butanol liquid are evaporated, and residue adds methanol to dissolve and moves in 50mL measuring bottle, with methanol dilution to scale, are shaken up, filter
Cross, take subsequent filtrate to get；
(4) it measures: it is accurate respectively to draw reference substance solution and each 10 μ L of test solution, high performance liquid chromatograph is injected,
Measurement to get.
The embodiment of the present invention 5: a kind of invigorating heart is relaxed ginsenoside Rg in preparation1、Re、Rb1, Rc content assaying method, step
It is as follows:
In addition to " condition of gradient elution are as follows: 0~10min:A:B=15:85；10~45min:A:B=15~25:85~75；
45~60min:A:B=25~35:75~65；60~80min:A:B=35~15:65~85；80~110min:A:B=15:
85 " outside, remaining is the same as embodiment 2.
The embodiment of the present invention 6: a kind of invigorating heart is relaxed ginsenoside Rg in preparation1、Re、Rb1, Rc content assaying method, step
It is as follows:
In addition to " condition of gradient elution are as follows: 0~10min:A:B=20:80；10~45min:A:B=20~35:80~65；
45~60min:A:B=35~45:65~55；60~80min:A:B=45~20:55~80；80~110min:A:B=20:
80 " outside, remaining is the same as embodiment 2.
The embodiment of the present invention 7: a kind of invigorating heart is relaxed the content assaying method of ginsenosides Rg1, Re and Rb1 in preparation, Rc, step
It is rapid as follows:
In addition to " passing through the large pore resin absorption column (AB-8) handled well, washing in the preparation of test solution in step (3)
It is extremely colourless " outside, remaining is the same as embodiment 3.
The embodiment of the present invention 8: a kind of invigorating heart is relaxed ginsenoside Rg in preparation1、Re、Rb1, Rc content assaying method, step
It is as follows:
In addition to " passing through the large pore resin absorption column (NKA-9) handled well, water in the preparation of test solution in step (3)
It is washed till colourless " outside, remaining is the same as embodiment 3.
The embodiment of the present invention 9: a kind of invigorating heart is relaxed ginsenoside Rg in preparation1、Re、Rb1, Rc content assaying method, step
It is as follows:
In addition to " being washed 3 times with aqueous slkali (1% sodium hydroxide solution) " in the preparation of test solution in step (3) and
" by the neutral alumina column handled well, being washed to colourless " outside, remaining is the same as embodiment 3.
The embodiment of the present invention 10: a kind of invigorating heart is relaxed ginsenoside Rg in preparation1、Re、Rb1, Rc content assaying method, step
It is rapid as follows:
In addition to " condition of gradient elution are as follows: 0~5min:A:B=10:90；5~40min:A:B=10~28:90~72；40
~75min:A:B=28~38:72~62；75~80min:A:B=38~10:62~90；80~110min:A:B=10:
90 " outside, remaining is the same as embodiment 2.
Consistent assay result can be obtained in any combination of all conditions parameter in above-described embodiment.
Claims (2)
1. the content assaying method of ginsenoside in a kind of easypro preparation of invigorating heart, it is characterised in that: it is with ginsenoside Rg1、Re、
Rb1, Rc reference substance be control, be the efficient of Mobile phase B gradient elution by mobile phase A, 0.02% ~ 2% phosphate aqueous solution of methanol
Liquid chromatography is measured in accordance with the following steps:
(1) chromatographic condition and system suitability: using octadecylsilane chemically bonded silica as filler；Using methanol as mobile phase
A, 0.02% ~ 2% phosphate aqueous solution are Mobile phase B, gradient elution；Detection wavelength is 203nm, and flow velocity is 0.8 ~ 1.0mL/min, column
Temperature is 25 ~ 35 DEG C, and number of theoretical plate is calculated with ginsenoside Re peak is not less than 3000；The actual conditions of the gradient elution are as follows: 0 ~
5min:A:B=25:75；5 ~ 35min:A:B=25 ~ 35:75 ~ 65；35 ~ 70min:A:B=35 ~ 50:65 ~ 50；70 ~ 80min:A:B=
50 ~ 25:50 ~ 75；80 ~ 110min:A:B=25:75；
(2) preparation of reference substance solution: ginsenoside Rg is taken1, ginsenoside Re, ginsenoside Rb1, Ginsenoside Rc's reference substance
In right amount, accurately weighed, respectively plus every 1mL is made containing ginsenoside Rg in methanol1The solution of 0.1mg, every 1mL contain ginsenoside
The solution of Re0.1mg, every 1mL Rb containing ginsenoside1The solution of the solution of 0.25mg and every 1mL 0.2mg containing Ginsenoside Rc, i.e.,
?；
(3) preparation of test solution: using following methods 1.-any one of 2.:
1. precision weighs the easypro 5 ~ 20g of preparation or its content of invigorating heart, to set in 100mL stuffed conical flask, methanol 50mL is added in precision,
Weighed weight, ultrasound or 30 ~ 60min of reflow treatment, supply weight with methanol；Filtration, take subsequent filtrate to get；
2. precision weighs the easypro 5 ~ 20g of preparation or its content of invigorating heart, to set in 100mL stuffed conical flask, methanol 50mL is added in precision,
Weighed weight, heating and refluxing extraction 30min ~ 60min supply weight with methanol, filtration；Precision measures subsequent filtrate 25mL, adds water
Stirring, filtration add a small amount of water washing filter cake and container 3 times, merge washing lotion and filtrate, are concentrated to dryness, and residue adds methanol to dissolve, and
Be transferred in 25mL measuring bottle, methanol dilution added to shake up to scale, filter, take subsequent filtrate to get；
(4) measure: difference is accurate to draw reference substance solution and each 10 μ L of test solution, injects high performance liquid chromatograph, measures,
To obtain the final product.
2. the content assaying method of ginsenoside in the easypro preparation of invigorating heart according to claim 1, which is characterized in that the stream
Dynamic phase B is 0.2% phosphate aqueous solution.
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| CN104807905A (en) * | 2015-05-04 | 2015-07-29 | 中国人民解放军第三七一医院 | Method for determining contents of ginsenosides Rg1 and Re in Xinnaoning tablet |
| CN106226435B (en) * | 2016-08-29 | 2018-11-30 | 江苏康缘药业股份有限公司 | A kind of fingerprint atlas detection method of Yixinshu tablet saponin component |
| CN110187022A (en) * | 2019-05-21 | 2019-08-30 | 贵州联盛药业有限公司 | The measuring method of ginsenoside Re's content in one seed ginseng times Guchang capsule |
| CN110082460B (en) * | 2019-06-03 | 2022-05-20 | 国药集团精方（安徽）药业股份有限公司 | Quality detection method of Jingshu granules |
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